Note: Descriptions are shown in the official language in which they were submitted.
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MONOCLONAL ANTIBODIES Sl'tCl~lC TO ENDOTHELIAL CELL
CADHERINS AND USES THEREOF
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BACKGROUND OF THE INVENT~ON
Endothelial cells constitute an important i"Le- race lining the internal vascular
surface and regulating the p~ss~ge of plasma proteins and circulating cells fromblood to tissues. (Caveda et al., J. Clin. Invest. 98~4): 886-893, August, 1~96).
Endothelial permeability is regulated by intercellular junctions. These junctions
are complex structures formed by transl I ~embl ~ne adhesive molecules, such as
cadherins, linked to a network of cytoplas~ic and cytoskeletal proteins.
Adhesive molecules regulate leukocyte extravasation, endothelial cell growth,
and permeability. (Dejana, E. et al, Review: Endothelial Cell-to-Cell Jul 11tiOI IS,
FAS~B J., 9:910-918 (1995). Cadherins are adhesive glycoproteins that mediate
- homotypic cell-to-cell adhesion, are calcium-dependent, and protease-sensitive.
All cell types that form solid tissues express sorrle mernbers of the cadherin
family and each member displays a hornophilic binding specificity. Members of
the cadherin superfamily share a comrnon basic structure. The common
structures of cadherins include an N-terminal extracellular domain that
determines binding specihcity; a hydrophobic transmembrane dornain; and a C-
terminal cytoplasmic domain. The C-terminal cytoplasmic domain, which is
highly conserved among the superfarnily mernbers, interacts with the
cytoskeleton through catenins and other ,~, ut~ s. Some cadherins, however,
lack a cytoplasmic domain. The most il l~pOI lal1l biological role of cadherins is to
support homotypic cell aggregation and segregation, which during
embryogenesis promote the formation of defined tissues and organs. (Brevario,
F., et al., Arterioscler. Thromb. Vasc. Biol. 15:1229-1239 (19~5)).
Despite their similar biochemical properties, each cadherin is characterized by a
different spatiotemporal pattern of expression and cell binding specificity. For
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example, in humans, E-cadherin, or uvomorulin, is essentially found in epitheliaand in subsets of neurons. N-cadherin is expressed in the nervous system and
in skeletal and cardiac muscles, and P-cadherin exhibits a wides,uread
distribution. (Takeichi, M., Annu. Rev. Biochem. 59:237-252 (1990)). Vascular
endothelial cadherins (VE-cadherins) are endothelial-specific cadherins strictlylocalized at intercellular junctions of essentially all types of endothelium.
(Brevario, F., et al. 1995) VE-cadherins are so farthe only cadherins
consistently organized at interendothelial adherence junctions. VE-cadherins areconstitutive components of all types of endothelia, have adhesive properties,
restrict endothelial permeability, and mediate hornotypic cell adhesion. (Caveda~t ~ Th~3 amino acid se~uence of one such VE~cadherin, identified in this
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Unlike most endothelial markers, VE-cadherins are not found in blood cells or inhematopoietic precursors. The observation that VE-cadherins are constitutively
expressed by the endothelium of most organs and tissues suggests that their
biologic properties are required for the early assembly and integrity of blood
vessels. (Breier, G. et al., Blood, 87(2)L630~41 (1996)).
Blood vessels are formed by v~-~cl 'ogenesis, a process during which a primary
capillary plexus is formed that is remodeled either by fusion or regression, andangiogenesis (also called neovascularization), a process in which vasculature isformed by new vessels sprouting from preexisting vessels and invading the
developing organ. (Breier et al. 1996) Angiogenesis is an important process in
the menstrual cycle in the endometrium, in pregnancy, and during neonatal
growth. Angiogenesis is also important in wound healing and in the
pathogenesis of a large variety of ciinical diseases including tissue inflammation,
arthritis, tumor growth, diabetic retinopathy, and macular degeneration by
neovascula~ ion of the retina. These clinical manifestations ~CSoci~cd with
angiogenesis are referred to as angiogenic diseases. (Folkman et al., Science1
235:442447 (1987). Ar Iyioyel ~esis is generally absent in healthy adult or mature
tissues, although it does occur in wound healing and in the corpous luteum
growth cycle. See, for example, Moses et al., Science, 248:1408-1410 (1990).
Angiogenesis is required for tumor proliferation because tumors need an
adequate blood per~usion to obtain nutrients. Inhibition of angiogenesis by
limiting vessel growth or selectively destroying proliferating endothelium wouldbe a useful therapy for restricting tumor growth. Various methods of inhibiting
angiogenesis have been proposed: (1) inhibition of the release of "angiogenic
molecules" such as basic-FGF (basicfibroblast growth factor); (2) neutralizationof angiogenic molecules, such as basic-FGF by the use of anti-basic -FGF
antibodies; and (3) il ll l,bilio~ 1 of ~ndoLhelial cell response to angiogenic stimuli.
This latter strategy has received attention, and Folkman et al., Cancer Biology,3:89-96 (1992), have described several endothelial cell response inhibitors,
including coilagenase inhibitor, an yiOS~ iC steroids, fungal-derived angiogenesis
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- inhibitors, platelet factor 4, thrombospondin, arthritis drugs such as ~-penicillamine and gold thiomalate, vitamin D3 analogs, alpha-interFeron, and
others that might be used to inhibit angiogenesis. For additional proposed
inhibitors of angiogenesis, see Blood et al., Bioch. Biophys. Acta., 1032:89-8
(1~90), Moses et al., Science 248:1408-1410 (1990~, Ingber et al., Lab. Invest.,5g:44-51 (1988~, and U.S. Patent Nos. 5,092,885; 5,112,946; 5,192,744; and
~,202,352. Other new inhibitors of angiogenesis include angiostatin and
endostatin (O'Reilly et al., Cell, 88(2) 277-28~). None of the inhibitors of
angiogenesis described in these references are targeted at inhibition of
cadherins.
An object of this invention is to provide membrane markers of proliferating
endothelial cells, i.e., VE-cadherins, which are useful in quanti~ying the degree of
angiogenesis, and thus as diagnostic tools in evaluating the invasive state and
other properties of a tumor. A further object of this invention is to provide
antibodies against VE-cadherins, which participate in angiogenesis. Another
object of this invention is to use such antibodies against VE-cadherins to inhibit
angiogenesis to treat or prevent angiogenic dise~ses, such as tumor
angiogenesis, rheumatoid a, lhl ilis, diabetic retinopathy and psoriasis. Such
antibodies against VE-cadherins can also be useful as diagnostic tools to
evaluate the invasive state and properties of a tumor.
SUMMARY OF THE INVENTION
The present invention provides a glycosylated or unglycosylated protein
comprising an amino-acid sequence shown in SEQ ID NO:1 or a homologous
sequence having at least 70% homology to the sequence shown in SEQ ID
NO:1.
The present invention provides monoclonal antibodies which specifically bind to
VE-cadherin molecules and modify their activity.
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Further, the invention provides a method of modifying VE-cadherin activity in
endothelial cells co"~prisil Ig contacting the cells with a monoclonal antibody of
the invention.
The invention also provides a method of i~ IhiLJilil Ig angiogenesis in a mammalcomprising administering an effective amount of any one of the antibodies of theinvention to the mammal. In addition, the invention provides a method of
ir Ihibilil Ig tumor growth in a 1 ~ lal "" ~al comprising administering an effective
amount of any one of the antibodies of the invention to the mammal.
The invention also provides a pharmaceutical composition comprising any one of
the antibodies of the invention and a pharmaceutically acceptable carrier.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides new transrnembrane cadherin proteins located
at cell-to-cell junctions in endothelial cells. In this specification, these
cadherin proteins, found on vascular endothelial cells, are called VE-
cadherins. One such VE-cadherin protein is VE-cadherin-1 (also known as
cadherin-~, as well as VE-cadherin), whose amino acid sequence is presented
in Lampugnani, M. et al., J.Cell Biol. 118:1511-1522 (1992). A second VE-
cadherin provided by this invention is called in this specification
protocadherin-4 (pcdh-4); alternatively, pcdh-4 is called VE-cadherin-2. The
VE-cadherins of the invention promote cell-to-cell hornotypic adhesion and its
expression is upregulated in proliferating endothelial cells in comparison to
resting cells. The VE-cadherin proteins of this invention are preferably of
human origin, but may also be found in other animals such as mice, rats,
pigs, monkeys, sheep and goats.
The present invention provides a pcdh~ protein in glycosylated or
unglycosylated form comprising an amino-acid sequence selected from the
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- sequence SEQ ID NO: 1 and homologous sequences having at least 70%
homology to the sequence S~Q ID NO: 1. The percentage of homology may
for instance be at least 75%, 80% or as high as 85%, or even higher such as
90% or 95~~0, especially if the homologous sequence originates from a
transmembrane protein of the same or closely related species. However, it is
anticipated that proteins which have at least 70 % homology to the amino-
acid sequence SEQ ID NO: 1 will share both diagnostic and medical
properties to such a high degree that they can be used for the various
applications of the present invention. Among such proteins may be included
both naturally occurring analogs and variants of the same protein from the
same or from different species as well as synthetic or recombinant
equivalents of these proteins.
The synthetically or recombinantly produced proteins of the invention function
as competitors in cell-cell adhesion processes at cell-to-cell junctions.
The DNA of the invention can be any DNA that encodes the protein of the
invention. Such DNA can be genomic or synthetic.
Another aspect of the invention is directed to a cDNA sequence coding for a
protein of the present invention. A specific embodiment of this aspect of the
invention is the cDNA sequence of SEQ ID NO: 2 coding for the protein
having the amino-acid sequence of SEQ ID N0: 1. The cDNA molecules of
the invention may be used in gene therapy. For example, they may be used
as oncosuppressors by transfection in carcinoma cells lacking this molecule.
A further aspect of the invention is directed to a structural gene coding for a
protein of the present invention or a peptide derived from the protein. The
structural gene may be used in the production of a protein or peptide of the
in~lention. The flanking regions, such as promoter or leader sequences, are
preferably chosen with regard to the expression system to be used to
promote good production. Further, the codons used in the structural gene
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may be selected with regard to the codons most frequently used by the
selected expression host, in order to optimize the expression yield. For
instance, if yeast is selected as the expression host, the codons may be
optimized for yeast. The specific example of a structural gene of the
invention is the protein coding region of a cDNA of the invention, namely the
structural gene having the nucleotide sequence SEQ ID NO: 3 coding for the
protein having the amino-acid sequence SEQ ID NO: 1.
The present invention is also directed to a recombinant protein or peptide
expressed by a structural gene or a fragment of a gene provided by the
invention.
The invention is further directed to a modifier of the homophilic binding of VE-cadherins at cell-to-cell junctions. The term "modifier" is to be interpreted
broadly and comprises both inhibitors and activators of the binding of the VE-
cadherins. In one embodiment, the modifiers of the invention either prevent
or promote binding of pcdh-4 molecules at cell-to-cell junctions. In other
embodiments, the modifiers of the invention either prevent or promote binding
of VE-cadherin-1 (cadherin-~;) or pcdh4 ~VE-cadherin-2~ molecules at cell-to-
cell junctions.
A modifier of the invention rnay be any ligand to the protein of the invention, or
any ligand, which binds to the protein and has the ability to prevent or promotethe homophilic binding of VE-cadherin proteins. For example, the modifier of
the invention may have a structure which is complementary to a VE-cadherin
protein of the invention or a part of the protein. Such a modifier of the
invention may bind to a VE-cadherin protein of the invention. In a preferred
embodiment of this aspect of the invention the modifier is selected from the
group consisting of antibodies specifically binding to the protein according to
the invention and inhibiting or inducing or promoting the homophilic binding of
said protein, and homophilic-binding-inhibiting or -inducing proteins, peptides,peptidomimetics and organic molecule-ligands derived from the amino-acid
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sequence of the protein according to the invention. The invention also
inciudes antisense oligonucleotides based on the cDNA sequence encoding
the proteins of the invention, which may be used in cancer therapy as
modifiers of angiogenesis.
The present invention provides antibodies that bind specifically to a VE-cadherin
protein molecule of the invention or to a part of the VE-cadherin. The antibodies
of the invention may be polyclonal, but ~rt3r~,~bly are monoclonal and prere~ably
bind to the extracellular domain of a VE-ca-ll ,e, i" molecule. The VE-cadherin
molecule may be any cadherin molecule that is an endothelial-specific cadherin
localized at intercellular junctions of essentially all types of endothelium, and that
has adhesive properties, restrict endothelial perrneability, and mediate homotypic
cell adhesion. In one embodiment of the invention, the VE-cadherin molecule is
called VE-cadherin-1, previously described as cadherin-5 or VE-cadherin, whose
amino acid se~uence is presented in Lampugnani, M. et al., J.Cell Biol.
118:1~1 1-1522 (1992)). In another embodiment of the invention, the VE-cadherin
is pcdh~, which is altematively named VE-cadherin-2.
The antibodies of the invention modify the activity of a VE-cadherin molecule.
One way of modifying such activity is by inter~ering with or preventing cell-to-cell
binding of the VE-cadherin's extracellular binding domain. Another way of
modifying such activity is by inducing or pr~"l.,lirlg such cell-to-cell binding. In
one embodiment of this invention, the antibodies of the invention will either
prevent or promote homophilic binding of VE-cadherin molecules at cell-to-cell
Junctions. Accordil l~ly, modification of VE-cadherin activity encompasses both
inhibition of and activation of VE-cadherin activity.
- UTILITY
A. Modifying VE-cadherin activity
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- In vivo:
Modification of VE-cadherin activity in a sample of endothelial cells may be
performed in vivo. Such modification occurs when a modiher of the invention,
preferabiy an antibody, is contacted with a VE-cadherin upon administering the
modifier to a mammal.
Additional modifiers of the invention include, but are not limited to, peptides,peptidometics, and srnail molecules.
Methods of administration to a mammal include, for example, oral, intravenous,
intraperitoneal, subcutaneous, or intramuscular adrninistration.
This in vivo method is useful for inl liiJiliny a. ~y:o~Jellesis in a mammal. The in vivo
neutralization method is a useful therapeutic rnethod such as for preventing or
inhibiting angiogenesis associated with pathologicdl conditions such as tumor
growth in a mammal. Accordingly, the modifiers, and more specifically, the
antibodies, of the invention are anti-angiogenic immunotherapeutic agents.
The methods of il lhibilil lg angiogenesis and of inhibiting pathological conditions
such as tumor growth in a mammal co",,~r~ies adnninistering an effective amount
of any one of the invention's antibodies to a rnamrnal or directly to a tumor within
the "~" ,n ,al. The mamrnal is preferably human. This method is effective for
treating subjects with carcinomas or SdlCOIll~S, preferably highly vascuiar tumors
such as Kaposi's sarcoma, CNS neoplasrns ~capillary heman~i~ bl~tomas,
meningiomas and cereb~ al metastases), melanornas, gastrointestinal and renal
s~,.;ol"as, rhabdomyosarcoma, glioiJld:,loll,a, preferably glioblastoma multiforma,
and leiomyosa, cor"a.
A cocktail of at least two monoclonal antibodies of the invention provides an
especially efficient treatment for il 1l liiJiLi~ Iy angio~enesis and thus the growth of
tumor cells Any number of antibodies that is effective may be used, the upper
--tO--
~ --. = = .
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Iimit is d~:le~ ed by cost; preferably 10, more preferably 6, and most preferably
not higher than 4.
The combined treatrrlent of one or more of the antibodies of the invention with an
anti-neoplastic or anti-chemotherapeutic drug such as doxorubicin, cisplatin or
taxol provides an efficient treatment for inhibiting the growth of tumor cells. In
one embodiment, the pharmz~cel Itic~l composition comprises the antibody and
carrier with an anti-chemotherapeutic drug attached thereto.
Preventing or inhibiting angiogenesis is also useful to treat non-neoplastic
angiogenic pathologic conditions such as neovascular glaucoma, proliferative
retinopathy including proliferative diabetic retinopathy, macular degeneration,
hemangiomas, angiuribrol"as, and psoriasis.
In addition to prevention or inhibition of angiogenesis, other applications of the
modifiers of the invention include the prevention or inhibition of leukocyte
infiltration, tumor cell metastasis, or endothelial permeability. r-urther
applications include using the modifiers as vaccines and for making endothelial
junctions more permeable to antigens, thus indicating use of the modifiers for
treatment or prevention of acute and chronic inflammatory diseases, organ
transplantation, myocardial ischemia, atherosclerosis, cancer, diabetic
retinopathy, psoriasis, rheumatoid arthritis, and intestinal infection.
A further application of the invention is that the antibodies of the invention may
be labeled and used for detecting early endothelial cell damage in vivo.
Additionally, the labeled antibodies can be used to detect and/or isolate cells that
express the VE-cadherin molecules both in vivo and in vitro. Standard methods
for labeling and using labeled antibodies are know in the art, such as standard
blot and ELlSA formats. These ro""~Ls are normaily based on incubating an
antibody with a sample suspected of conlai~ g the protein and detecting the
presence of a complex between the antibody and the protein. The antibody is
labeled either before, during, or after the incubation step. The protein is
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preferably immobilized prior to detection. Immobili~Lion may be accomplished
by directly binding the protein to a solid surface, such as a microtiter well, or by
binding the protein to immobilized anli~oc;;es. (R.H. Kenneth, "Enzyme-Linked
Antibody Assay with Cells Attached to Polyvinyl Chloride Plates" in Kenneth et al,
Monoclonal Antibodies, Plenum Press, N.Y., page 376 (1981).)
In Vitro:
The invention provides a method of modifying VE-cadherin activity in a sample ofendothelial cells comprising contacting the sample with an antibody of the
invention before, simultaneously with, or after, adding VE-cadherin to the cell
sample.
BA Using the Antiboclies of the Invention to Isolate and Purify the VE-Cadherins and VE-Ca~ll,~,i,~ E~ ssir~ Cells
The antibodies of the present invention may be used to isolate and purify VE-
cadherins, and cells exp~essing VE-cadherins, using conventional methods such
as affinity chromatography (Dean, P. D.G et al., Affinity C~ " on ,aLography: A
Practical Approach, IRL Press, Arlington, VA (1985)). Other methods well known
in the art include magnetic separation with an~ibody-coated magnetic beads,
"panning" with an antibody attached to a solid matrix, and flow cytometry.
The source of VE-cadherins is typically vascular endothelial cells, which express
VE-cadherins. Suitable sources of vascular endothelial cells are blood vessels.
The VE-cadherins may be used as starting material to produce other materials,
such as DNA encoding the cadherins, or as antigen for making additional
monoclonal and polyclonal antibodies that recognize and bind to the VE-cadherin
or other related antigens on endothelial cells.
Modifiers of VE-cadherin-1, particularly rronoclonal antibodies made against
VE-cadherin-1, can bind to the extracellular domain of the protein. In some
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cases the antibodies bind between amino acid residues 343 and 351. These
antibodies block angiogenesis. The specific amino acid sequence to which
these antibodies bind is TIDLRYMSP.
C. Monitoring Levels of VE-Ca.ll ,~ In Vifro or In Vivo
The antibodies of the invention rnay be used to monitor levels of VE-cadherin invitro or in vivo in biological samples using standard assays and methods known
in the art. Some examples of biological samples include solid tissues, such
as vascular tissue. Standard assays involve, for exarnple, labeling the
antibodies and conducting standard immunoassays, such as enzyme linked
immunosorbent assays (ELISA) and radioimrnunoassays, as is well known in
the art. A pre~erred embodiment of the invention is a diagnostic kit
comprising as a diagnostic reagent an antibody according to the invention or
a modifier according to the invention. The actual diagnostic method, such as
ELISA, to be used will determine any additional components in the kit.
The invention also provides transgenic anirnals or cells overexpressing or
lacking a VE-cadherin protein. Transgenic animals carrying null mutation of
pcdh-4 created by standard techniques, such as the knock-in and knock-out
methods. Some examples of transgenic anirnals are those described by
Hogan, B., et. al., 1994. These transgenic anirnals may be used as in vivo
models for screening replacing, activating rnolecules for VE-cadherins such as
pcdh-4, and for providing the therapeutic potential of such cadherins in gene
therapy in medicine. Transgenic animals ~ay be engineered to overexpress by
using promoters selected from NSE, Thy 1, PDGFB, VE cadherin, Willebrand
factor, and transomodulin. Such transgenic anirnals can be used for screening
in vivo for the therapeutic use of modifiers of VE-cadherin hornophilic binding.Transgenic VE-cadherin cells may be used for in vit~o testing purposes.
PREPARATION OF ANTIBODIES
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The polyclonal and monoclonal antibodies of the invention that specifically bindto the VE-cadherins may be produced by methods known in the art. These
methods include the immunological rnethod described by Kohler and Milstein in
Nature 256l 495~97 ~1975) and Campbell in "Monoclonal Antibody Technology,
The Production and Characterization of Rodent and Human Hybridomas" in
Burdon et al., Eds., Laboratory Techniques in Biochemistry and Molecular
Biology, Volume 13, ElsevierScience Publishers, A~sler~am (1985); aswell as
by the recombinant DNA method described by Huse et al in Science 246, 1275-
1281 (1989).
Such antibody techniques include imrnunizing an animal, preferably a mouse,
with an amount of a VE-cadherin molecule to cause an immune response. The
spleen of an immunized animal, which demonstrates a proper antibody titre, is
removed and a fused with an immortal cell line such as a myeloma cell line. The
resultant hybridoma line is then screened for antibody producing cells; said cells
are then clonally isolated.
The antibody may be prepared in any mammal, including mice, rats, rabbits,
goats and humans. The antibody may be a mernber of one of the following
immunoglobulin classes: IgG, IgM, I~A, IgD, or IgE, and the sl Ihcl~ses thereof,and preferably is an IgG antibody.
Functional Equivalents of Antibodies
The invention also includes functional equivalents of the antibodies described in
this specification. Functional equivalents have binding characteristics
comparable to those of the antibodies, and include, for example, chimerized,
humanized and single chain antibodies as well as fragments thereof. Diabodies
may also be functional equivalents of the antibodies of this invention. Methods of
producing sucn functional equivalents are disclosed in PCT Application No. WO
-14-
-
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93/21319, European Patent Application No. EPO 239,400; PCT Application Wo
89/09622; European Patent Application No. 338,745; and European Patent
Application EPO 332,4~4.
Functional equivalents include polypeptides with amino acid sequences
substantially the same as the amino acid sequence of the variable or
hypervariable regions of the antibodies of the invention. "Substantially the same"
amino acid sequence is defined herein as a sequence with at least 70% percent
homology to an amino acid sequence of an antibody of the invention, as
determined by the FASTA search method in accordance with Pearson and
Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988).
Chimerized antibodies preferably have constant regions derived substantially or
exclusively from human antibody constant regions and variable regions derived
substantially or exclusively from the sequence of the variable region ~rom a
mammal other than a human.
~umanized antibodies are commonly created by transplanting the antigen
binding segments, known as complementarity determining regions (CDRs), from
rodent antibodies into human antibodies. (Carter and Merchant, Current
Opinions in Biotechnology (8):449-454, 1997.) Humanized antibodies pl~fel~Lly
have co, ~ n~ regions and variable regions other than the hypervariable region
derived substantially or exclusively from the corresponding human antibody
regions and complementarity deterrnining regions (CDRs) derived substantially
or exclusively from a mammal other than a human. The extent to which an
antibody is subs~nLially or exclusively modified can be deler,l,ined by standardmethods for optimizing the humanization methodology.
Suitable mammals other than a human include any mammal from which
monoclonal antibodies may be made, such as a rabbit, rat, mouse, horse, goat,
or primate.
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Single chain antibodies or Fv fragments (scFv) are polypeptides which consist ofthe variable (V) region of the heavy chain of the antibody linked to the variable
(V~ region of the light chain with or without an i, ILe, connecting linker. Thiscomprises the entire antibody combining site, and is the rninimal antibody binding
site. These chains may be produced in bacteria.
Functional equivalents further include fragments o~ antibodies that have the
samé or binding characteristics comparable to those of the whole antibody. Such
r, ~yl 1 ~ may contain one or both Fab r, a~" ~el ll~ or the F(ab')2 fragment.
~, ~rer~bly the antibody ~ragments contain all six complementarity determining
regions of the whole antibody, although r,~y, ne~ containing fewer than ail of
such regions, such as three, four or five CDRs, may also be functional.
Diabodies are examples of additional functional equivalents. A diabody is an
antibody fragment which has two antigen binding sites and can be a bivalent or
bispecific ~ragment. Bispecific diabodies are l~elerodimers of two 'crossover'
scFv fragments in which the variable light and variable heavy domains of the twoantibodies are present on dirr3renl polypeptide chains. (Carter and Merchant,
Current Opinions in Biotechnology ~8):449~54, 1997.)
Further, the functional equivalents rnay be or may combine members of any one
of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the
subclasses thereof.
intracellularly expressed antibodies, referred to as "intrabodies" an be designed
to bind and inactivate target molecules inside cells. Thegenes encoding can
be expressed intracellularly. The specific and high-affinity binding properties of
antibodies, combined with their ability to be stably expressed in precise
intracellular locations inside mammalian cells, provides a molecules for gene
therapy applications. (Marasco, W., Gene Ther (4) 1, p1 1-5, 1997).
dlion of VE-Cadherin Immunoqens
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The VE-cadherins of the invention may be used as immunogens against which
an antibody can be raised, particularly the antibodies of the invention.
Alternatively, antibodies can be generated using as immunogens both synthetic
peptides and VE-cadherin fragments. Such fragments and synthetic peptides
are provided by the VE-cadherin amino acid sequences provided herein and by,
for example, Lampugnani, M. et al., J.Cell Biol. 1 18:151 1-1522 (1992)).
As a further alternative, DNA encoding a VE-cadherin, such as a cDNA or a
fragment thereof1 may be cloned and expressed and the resulting polypeptide
recovered and used as an immunogen to raise an antibody of the invention. In
order to prepare the VE-cadherins against which the antibodies are made,
nucleic acid molecules that encode the VE-cadherins of the invention, or portions
thereof, especially the extracellular portions thereof, may be inserted into known
vectors for expression in host cells using standard recombinant DNA techniques.
Standard recombinant DNA techniques are described in Sambrook et al.,
- "Molecular Cloning," Second Edition, Cold Spring Harbor Laboratory Press
(1987) and by Ausubel et al. (Eds) "Current Protocols in Molecular Biology,"
Green Publishing Associates/Wiley-lnterscience, NewYork (1990).
A suitable source of cells containing nucleic acid molecules that express the VE-
cadherin includes vascular endothelial cells.
Total RNA or mRNA is prepared by standard procedures from endothelial tissue,
or alternatively, from isolated endothelial cells. Standard methods may be used
for Isolation of endothelial cells.
The total RNA or mRNA is used to direct cDNA synthesis. Standard methods for
isolating RNA and synthesizing cDNA are provided in slal ~dard manuals of
molecular biology such as, for example, in Sambrook et al., "Molecular Cloning,"Second Edition, Cold Spring Harbor Laboratory Press (1987) and in ~usubel et
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al., (Eds), "Current Protocols in Molecular Biology," Greene AssociateslWiley
Interscience, New York (1990).
The cDNA of the VE-cadherin may be amplified by known methods. For example,
the cDNA may be used as a template for arnplification by polymerase chain
reaction (PCR); see Saiki et al., Science, 239, 487 (1988~ or Mullis et al., U.S.
patent 4,683,195. The sequences of the oligonucleotide primers for the PCR
amplification are derived from the sequences of mouse and human VE-cadherin
respectively. The oligonucleotides are synthesized by methods known in the art.
Suitable methods include those described by Caruthers in Science 230, 281-285
(~985).
In order to isolate the entire protein-coding regions for the VE-cadherins, the
upstream PCR oligonucleotide prirner is complementary to the sequence at the 5'
end, preferably encompassing the ATG start codon and at least ~-10 nucleotides
upstream of the start codon. The downstream PCR oligonucleotide primer is
complementary to the sequence at the 3' end of the desired DNA sequence. The
desired DNA sequence pl~r~rably encodes the entire extracellular portion of the
VE-cadherin, and optionally encodes all or part of the transmembrane region,
andlor all or part of the intracellular region, including the stop codon. A mixture
of upstream and downstream oligonucleotides are used in the PCR amplification.
The conditions are optimized for each particular primer pair according to
standard procedures. The PCR product is analyzed by ele~;L~ ~pt)oresis for
cDNA having the correct size, cor~ esponding to the sequence between the
tJI ;mel ~.
Alternatively, the coding region may be amplified in two or more ove, la~pi, Ig
fragments. The overlapping fragments are designed to include a restriction site
perrnitting the assembly of the intact cDNA from the fragments.
-18-
CA 022459S6 1998-08-11
W 098/25946 PCT~US97/20006
The DNA encoding the VE-cadherins may also be replicated in a wide variety of
cloning vectors in a wide variety of host cells. The host cell may be prokaryotic
or eukaryotic.
The vector into which the DNA is spliced rnay c;ol ",, ise segments ofchromosomal, non-chromosomal and synthetic DNA sequences. Some suitable
prokaryotic cloning vectors include plasmids from E. coli, such as colE1, PcR
pE3R322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include
derivatives of phage DNA such as M13 and otherfilamentous single-stranded
DNA phages.
C~c~.l ession and Isolation of VE-Cadherin Immunoqens
DNA encoding the VE-cadherins of the invention are inserted into a suitable
expression vector and expressed in a suitable prokaryotic or eucaryotic host.
The DNA inserted into a host rnay encode the entire extracellular portion o~ theVE-cadherin, or a soluble ~ragment of the extracellular portion of the VE-
cadherin. The extracellular portion of the VE-cadherin encoded by the DNA is
optionally attached at eitherl or both, the 5' end or the 3' end to additional amino
acid sequences. The additional amino acid sequence may be attached to the
VE-cadherin extracellular region in nature, such as the leader sequence, the
transmembrane region and/or the intracellular region of the VE-cadherin. The
additional amino acid sequences may also be sequences not attached to the VE-
cadherin in nature. Prerel~bly, such additional amino acid sequences serve a
particular purpose, such as to irnprove expression levels, secretion, solubility, or
immunogenicity.
Vectors for expressing proteins in bacteria, especially E coli, are known. Such
vectors include the PATH vectors described by Dieckmann and Tzagoloff in J.
Biol. Chem. 260, 1513-1520 (1985). These vectors contain DNA sequences that
encode anthranilate synthetase (TrpE) followed by a polylinker at the carboxy
terminus. Other expression vector systems are based on beta-gal~c~oskl~se
_19_
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WO 98/25g46 PCT/US97/20006
(pEX); lambda PL; maltose binding protein (pMAL~; and glutathione S-transferase
(pGST) -see Gene 67, 31 (1988) and Peptide Research 3, 167 (1990).
Vectors useful in yeast are available. A suitable example is the 2,u plasmid.
Suitable vectors for use in mammalian cells are also known. Such vectors
include well-known derivatives of SV-40, adenovirus, retrovirus-derived DNA
sequences and shuttle vectors derived from combination of functional
Irlarlllilalian vectors, such as those described above, and functional plasmids and
phage DNA.
Further eukaryotic expression vectors are known in the art (e.g., P.J. Southern
and P. Berg, J. Moi. Appl. Genet.1, 327-341 (1982); S. Subramani et al, Mol.
Cell. Biol. 1, 854-864 (1981); R.J. Kaufmann and P.A. Sharp, "Amplification And
Expression Of Sequences Cotransfected with A Modular Dihydrofolate
Reductase Complementary DNA Gene," J. Mol. Biol.159, 601 ~21 (1982); R.J.
Kaufmann and P.A. Sharp, Mol. Cell. Biol.159, 601-664 (1982); S.l. Scahill et al,
"Expression And Characterization Of The Product Of A Human Immune
Interferon DNA Gene In Chinese Hamster Ovary Cells," Proc. Natl. Acad. Sci.
USA 80, 4654-4659 (1983); G. Urlaub and L.A. Chasin, Proc. Natl. Acad. Sci.
USA77, 42164220, (1980).
The expression vectors useful in the present invention contain at least one
expression control sequence that is operatively linked to the DNA sequence or
fragment to be expressed. The control sequence is inserted in the vector in
order to control and to regulate the expression of the cloned DNA sequence.
Examples of useful expression control sequences are the lac system, the trp
system, the tac system, the trc system, maior operator and promoter regions of
phage lambda, the control region of fd coat protein, the glycolytic promoters ofyeast, e.g., the ,uro" ,o~er for 3-phosphoglycerate kinase, the promoters of yeast
acid phosphatase, e.g., Pho5, the ,ulullloters of the yeast alpha-mating factors,
and promoters derived from polyoma, adenovirus, retrovirus, and simian virus,
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CA 02245956 1998-08-11
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e.g., the early and late promoters or SV40, and other sequences known to
control the expression of genes of prokaryotic or eukaryotic cells and their
viruses or combinations thereof.
Vectors containing the receptor-encoding DNA and control signals are inserted
~ into a host cell for expression of the r~ceplor. Some useful expression host cells
include well-known prokaryotic and eukaryotic cells. Sorne suitable prokaryotic
hosts include, for example, E. col!, such as E. coli SG-936, E coli HS 101, E. coli
W3110, E. coli X1776, E. coli X2282, E. coli Dtll, and E. coli MRCI,
Pseudomonas. Bacillus. such as Bacillus subtilis, and st,~Ptc)l~ ces. Suitable
eukaryotic cells include yeast and other fungi, insect, animal cells, such as COS
cells and CHO cells, human cells and plant cells in tissue culture.
~ollowing expression in a host cell maintained in a suitable medium, the VE-
cadherins may be isolated from the medium, and purified by methods known in
the art. If the VE-cadherins are not secreted into the culture rnedium, the hostcells are Iysed prior to isolation and purification.
The antibodies of the invention rnay also be prepared frorn VE-cadherins
expressed by endothelial cells, or altematively a cell into which the DNA
encoding a VE-cadherin has been 1, a~ l~r~-:led, such as 3T3 cells.
_
CA 0224~9~6 1998-08-11
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EXAMPLES
The Examples which follow are set forth to aid in understanding the invention but
are not intended tO1 and should not be construed to, limit its scope in any way.The Examples do not include detailed descriptions of conventionai methods
employed in the construction of vectors and plasmids, the insertion of genes
encoding polypeptides into such vectors and plasmids or the introduction of
plasmids into hosts. Such methods are well known to those of ordinary skill in
the art and are described in numerous publications including Sarrlbrook, J.,
Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual,
2nd edition, Cold Spring Harbor Laboratory Press.
Identification of the cDNA of pcdh-4
Librarv Screenin~, DNA sequencinq.
A Igt10 library from P4-P8 postnatal mouse brain capillary was screened as
previously described (Breviario et al. 1992) using a 130 bp cDNA probe
obtained by means of RT-PCR. RT-PCR was carried out using, as primers,
degenerated oligonucleotides (Sano et al. 1993) and a cDNA preparation
from endothelioma 1 15V cells (Garlanda et al. 1991). Plaques showing a
strong positive hybridization signal were screened three times to obtain a
single clone. Phage inserts were rescued in pBluescript vector and
sequenced by use of the dideoxynucleotide chain termination method.
Generation of recombinant f.d~ ,e~ and Production of polyclonal
antibodies
Recombinant fragments and polyclonal antibodies were produced in the
laboratory using Qiaexpressionist Kit, Qiagen. The cDNA corresponding to
EC1 (aa 74)-EC3 (recombinant fragment Extra 1 ) and to EC1 -EC4
(recombinant fragment Extra 2) of pcdh-4 were prepared by PCR and
subcloned into the BamHI-Hindlll site of the expression vector pQE30 in the
--22--
CA 0224S9~6 1998-08-11
: W 098/25946 PCT~US~7/20006
correct reading frame. The plasmid DNAs were then introduced into M15
(pREP4) cells by a single-step transformation method. The fusion proteins
were induced by the addition of IPTG and were purified from the extract by
Ni-NTA resin affinity chromatography, as described by the manufacturer
(Qiaexpressionist Kit, Qiagen).
Polyclonal antibodies against pcdh-4 were produced in rabbits by injecting
0.5 mg of the fusion protein in Freund's complete adjuvant at three
subcutaneous sites. Subsequent injections were in Freund's incomplete
adjuvant with 0.~ mg of the fusion protein. The resultant antibodies were
purified with a protein A column.
Constructs and Trans~ections
Constructs: Preparation and transfection procedure were performed
according to Breviario et al. 1995. Briefly, the mouse pcdh4 cDNA cloned in
pBluescript vector was cut with EcoRI, and the insert was subcloned into the
pECE eucaryotic expression vector to give the pECE-pcdh-4 construct. The
construct was checked for correct orientation by sequence analysis.
CH0 cells were plated at 3-4x1 o6 cells per 100 mm petri dish in DMEM with
10% FCS. After 24 hrs cells were transfected by calcium phosphate
precipitation with 20 ,ug pECE-pcdh-4 and 2 ~g pSV2neo plasmid. Medium
was replaced by fresh medium 24 hours later and maintained for further 48
hrs. Then cells were detached and plated at 1X106 per 100 mm petri dish and
cultured in selective medium with 600 ,ug/ml G418 (Geneticin, GIBC0).
Resistant colonies were isolated and tested for pcdh-4 antigen expression by
immunofluorescence staining and immunoprecipitation analysis. Positive cells
were cloned by limiting dilution and expanded for further studies.
Localization of Pcdh-4 at intercellular iunctiions
Immunofluorescence microscopv
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~ells were seeded on glass coverslips and grown to confluence in D-MEM
medium containing 1C~% fetal calf serum before immunofluorescence staining.
Cells were fixed with MeOH for 4 min. and processed for indirect
immunofluorescence microscopy as previously described in detail by
Lampugnani et al. (1992~. Briefly, incubation with the primary antibody
(Extra1 or Extra 2 and others) was followed by rhodamine-conjugated
secondary antibody (Dakopatts) with several washes with 0.1% BSA in PBS
between the various steps. Coverslips were then mounted in Mowiol 4-88
(Calbiochem). A Zeiss Axiophot microscope was used for observation and
image recording on Kodak TMax P3200 films.
Results
Pcdh-4 distributes selectively at cell-cell contacts in cultured mouse
endothelial cells and in transfectant cells.
Localization of pcdlh-4 in endothelial cells
Immunohistochemistry
Immunohistochemistry was performed according to Lampugnani et al. 1992.Tissue fragments were embedded in OCT compound (Ames Division), snap
frozen in liquid nitrogen and stored at -80~C until sectioning. Cryostat
sections were fixed in acetone for 10 min. at RT and were immunostained
with the polyclonal antibodies Extra 1 or Extra 2 using avidin-biotin
peroxidase complex technique. Sections were preincubated with horse serum
to prevent non-specific binding, and then incubated with an optimal dilution of
the primary antibody (1/50) for 30 min. The slides were sequentially
incubated with biotin-conjugated horse anti-rabbit lg antibodies followed by
avidin-biotin peroxidase complex. Each incubation step lasted 30 min. with 5
min. TBS washes between each step. The sections were finally incubated
with 0.03% H202 and 0.06% 3,3' DAB for 3-5 min. Slides were then washed
for 5 min. in running water, counterstained with hematoxilin for 5 min., and
mounted in Canada balsam.
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Results
Staining of different tissues indicate that pcdh4 antibodies stain endothelial
cells of the microvasculature, with higher staining intensity in proliferating
vessels of tumors. Extra 1 also cross reacts with human tissues.
The Polvclonal Ab Extra ~ can be used to detect Pcdh-4 in ELISA assay
ELISA assav
Microtiter wells of confluent cells were washed three times with DMEM+2.5%
horse serum +0.01% sodium azide and incubated for 1 h at 37~C with 100
~I/well of rabbit anti-pcdh-4 serum, diluted 1/100 in PBS+2.5% horse serum.
After incubation, cells were washed three times with PBS+2.5% horse serum
(washing bufler). Then, cells were fixed with glutaraldehyde 0.025% in
washing buffer for 5 min., washed two times and incubated for 1 h with
washing buffer. Fixation was required to prevent cells detaching from the
culture wells during the following washes. After rinsing in washing buffer,
cells were incubated with peroxidase conjugated anti-rabbit IgG (diluted
1/500 SIGMA) for 1h at RT. After incubation and three washes, 100 ~l
chromogen substrate was added. Absorbance values were read at 490 nm.
In each experiment, the binding of peroxidase conjugated anti-rabbit IgG to
the cells in the presence of non-immune serum was evaluated. This value,
considered as background was subtracted from each measurement.
Results
In ELISA the Ab Extra 1 was able to detect pcdh-4 protein in endothelial cells
(such as H5V from heart microvasculature; bEnd from brain
microvasculature) and pcdh-4 transfectant cells, while it gave negative values
using cells which do not express pcdh-4 such as CH0 parental and L929
fibroblast.
The results are presented in Table 1.
CA 02245956 1998-08-11
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Table 1. Detection of pcdh-4 on endothelial cells and transfectants in ELISA
Cell types OD x 1000
H5V 305.3 + 12*
bEnd 300.2 i 18*
CHO-pcdh-4 380.5 + 10*
CHO-parental 118.6 i 6
L929 148.4 + 5
Non-immune serum was used as dilution 1/100. Absorbance values are
means ~ SD of five replicates of a typical experiment out of four performed.
~Pc0.01 in comparison to CHO-parental by analysis of variance and
Duncan's test.
The ab i~xtra 1 can be used to detect proliferatinq endothelial cells
ELISA assay was used to detect pcdh-4 in subconfluent proliferating
endothelial celis 1.2x104 cellslcm2 in comparison to non-proliferating
confluent cells 1.2 x105 cells/cm2.
Results
Pcdh-4 expression is higher in endothelial cells in growth than in cells at
confluence.
Results are presented in Table 2.
- --26--
CA 02245956 1998-08-11
: WO 98/259'16 PCT/US97/20006
-Table 2. Modulation of the expression of pcdh-4 at different stages of growth
H5V growth stage OD x 1000
Subconfluents (1.2x104 cells/cm2) 389.3 + 2*
Confluents (1.2 x 1Q5 cellslcm2) 243.2 + 4
Values are means + SD of five replicates of a typical experiment out of four
performed. *P<0.01 by Student's test.
Protocadherin 4 mediates hornotvpiic adhesion between cells
Cell A~re~ation
The procedure to measure cell aggregation is extensively described in
Breviario et al. (1995). Briefly, confluent control cells, CHO transfected with
the empty pECE and pSV2neo plasmids, and confluent pcdh-4-transfectants
were washed several times with Ca~ and Mg~ -PBS. Then, 0.01% trypsin
in Hank's balanced salt solution (HBSS) with 25rnM HEPI~S, 10 mM Ca~+
and 5 mM Mg++ was added and rnaintained on the cells for the shortest time
interval before the appearance of intercellular retraction. Cells were
completely detached by vigorous shaking of the flasks. Trypsin was
neutralized by adding DMEM with 10% FCS and 0.1% soybean trypsin
inhibitor. The cell suspensions were centrifuged and resuspended in HBSS
without Ca+~ and Mg~+ and then centrifuged and resuspended in 1% BSA in
HBSS Ca++ and Mg~ free at a concentration of 4x105 ml. Cell suspensions
(0.5 ml per well) were seeded in a 24-well plate previously coated with 1%
BSA to prevent cell adhesion, and treaLIllel lls. 5 mM CaCI2, 5mM ~GTA, 50
~Lglml rabbit pcdh-4 purified antiserum (Extra 1), rabbit non-immune serum,
were added to start aggregation. Controls without calciurn addition wer
always run in parallel. Incubation was for 90 min. at 37~C on a rotating
plafform (80 rpm). The reaction was stopped with 5% glutaraldehyde. The
initial number of particles (NtO) and the nunlber of particles at 90 min. (Nt90)
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CA 02245956 1998-08-11
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were counted using a ZM Couiter Counter. Aggregation was quantified by
use of the formula (NtO-Nt90)/NtO x 100.
For those experiments where anactin cytoskeleton disrupting agent was
used, cytochalasin D was added at 1 llg/ml after the first centrifugation, and
the cells were incubated at 37~C for 30 min. and processed as described.
Results
Only pcdh4 transfectants showed siylliric~lll calcium-dependent
aggregation.
The aggregation capacity was lost when EGTA was added to the CHO-pcdh-
4 suspension. The rabbit purified antiserum, generated using the
recombinant fragment Extra 1, was able to neutralize the aggregation
capacity of CHO-pcdh-4 cells. Cytochalasin D did not affect aggregation
indicating that the formation of aggregates does not require an intact actin
cytoskeleton.
The results are presented in Table 3.
Table 3. Effect of pcdh~ transfection on cell aggregation capacity.
Transfectant cells % Aggregation
CHO-parental 10 ~ 2.0
CHO-pcdh4 12 ~ 4.0
CHO-pcdh-4+Ca 5mM 50 ~: 8.2*
CHO-pcdh4-Ca 5mM+EGTA 5 mM 15 ~ 5.4
CHO-pcdh4+Ca 5mM+Ab (Extra1 ) 22 + 7.0
CHO-pcdh-4-Ca 5mM+non-immune serum 42 + 6.5
CHO-pcdh-4+Ca 5mM+Cyt D 55 + 4.1~
Values are means + SD from triplicates of a typical experirnent out of three
perFormed. *P<0.01 by analysis of variance and Duncan s test in comparison
to CHO parental.
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- WO 98/2S946 PCT/US97/20006
Pcdh4 bindin~ was homoPhilic
Aggregation assays were performed after mixing parental CHO cells and
pcdh-4 transfectants. To distin~uish the two cell types, parental cells were
~ ~ labeled with 5 ?llg/ml of the fluorescent dye calcein in HBSS for 10 min. at
37~C, immediately after the first centrifugation and processed as described
above. Aggregates were examined by fluorescence microscopy. The results
showed that aggregation is essentially homophilic: only pcdh-4 transfectants
were present in the aggregates whereas control cells remained mostly single.
Pcdh-4 promote homotvpic cell adhesion
Cell Adhesion
Cells in monolayers were obtained by culturing control cells and CHO-pcdh-4
transfectants (5x1 031well at the seeding) in 96-well plates for 4 - 5 days to
confluence. Cells to be used in suspension were labeled 1h with 51Cr
(1 ,~LCi/10~ cells). Detachment was as described above for the cell aggregation
assay. Labeled cell suspensions (4x104 cells in 100 ,ul DMEM with 10% FCS
for each well) were added on the top of adherent cells (from which culture
medium had been removed with no rinsing). Incubation was for 30 min. at
37~C. Non-adherent cells were removed by three washes with Ca++ and
Mg++-PBS containin~ 10% FCS. The well content was then solubilized with 1
M NaOH/0.1% SDS (50 ?11) and counted for radioactivity.
Results
CHO-pcdh-4 significantly adhered to homologous pcdh-4 transfectant
monolayer? whereas parental cells adhered poorly to the transfectants.
The results are presented in Table 4.
Table 4. Effect on pcdh-4 transfection on cell adhesion
--29--
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Cell layer Cell suspension Number of adhered cells
CH0-parental CH0-pcdh4 4770 + 950*
CH0-pcdh-4 CH0-pcdh4 14800 i 1800
Values are means + SD of five replicates of a typical experiment out of two
performed. *P<0.01 by Student's test.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Tamas Bartfai Konsulting AB
(B) STREET: Brinken 3
(C) CITY: Stocksund
(E) COUNTRY: Sweden
(F) POSTAL CODE (ZIP): 182 74
(ii) TITLE OF INVENTION: A component of intercellular junctions in the endothelium.
(iii) NUMBER OF SEQUENCES: 3
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTFM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version ~1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1180 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DFSCRIPTION: SEQ ID NO: 1:
Met Met Leu Leu Leu Pro Phe Leu Leu Gly Leu Leu Giy Pro Gly Ser
--31--
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WO 98/25946 PCT/US97/20006
Tyr Leu Phe lle Ser Gly Asp Cys Gln Glu Val Ala Thr Val Met Val
Lys Phe Gln Val Thr Glu Glu Val Pro Ser Gly Thr Val lle Gly Lys
Leu Ser Gln Glu Leu Arg Val Glu Glu Arg Arg Gly Lys Ala Gly Asp
~5 60
Ala Phe Gln lle Leu Gln Leu Pro Gln Ala Leu Pro Val Gln Met Asn
Ser Glu Asp Gly Leu Leu Ser Thr Ser Ser Arg Leu Asp Arg Glu Lys
Leu Cys Arg Gln Glu Asp Pro Cys Leu Val Ser Phe Asp Val Leu Ala
100 105 110
Thr Gly Ala Ser Ala Leu lle His Val Glu lle Gln Val Leu Asp lle
115 120 125
Asn Asp His Gln Pro Gln Phe Pro Lys Asp Glu Gln Glu Leu Glu lle
130 135 140
Ser Glu Ser Ala Ser Leu His Thr Arg lle Pro Leu Asp Arg Ala Leu
145 150 155 160
Asp Gln Asp Thr Gly Pro Asn Ser Leu Tyr Ser ryr Ser Leu Ser Pro
165 170 175
Ser Glu His Phe Ala Leu Asp Val lle Val Gly Pro Asp Glu Thr Lys
180 185 190
His Ala Glu Leu Val Val Val Lys Glu Leu Asp Arg Glu Leu His Ser
195 200 205
Tyr Phe Asp Leu Val Leu Thr Ala Tyr Asp Asn Gly Asn Pro Pro Lys
210 215 220
--32--
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WO 98125946 PCT/US97/20006
Ser Gly lle Ser Val Val Lys Val Asn Val Leu Asp Ser Asn Asp Asn
225 230 235 240
Ser Pro Val Phe Ala Glu Ser Ser Leu Ala Leu Glu lle Pro Glu Asp
245 250 255
Thr Val Pro Gly Thr Leu Leu lle Asn Leu Thr Ala Thr Asp Pro Asp
260 265 270
Gln Gly Pro Asn Gly Glu Val Glu Phe Phe Phe Gly Lys His Val Ser
275 280 285
Pro Glu Val Met Asn Thr Phe Gly lle Asp Ala Lys Thr Gly Gln lle
290 295 300
lle Leu Arg Gln Ala Leu Asp Tyr Glu Lys Asn Pro Ala Tyr Glu Val
305 310 315 320
Asp Val Gln Ala Arg Asp Leu Gly Pro Asn Ser lle Pro Gly His Cys
325 330 335
Lys Val Leu lle Lys Val Leu Asp Val Asn Asp Asn Ala Pro Ser lle
340 345 350
Leu lle Thr Trp Ala Ser Gln Thr Ser Leu Val Ser Glu Asp Leu Pro
355 360 365
Arg Asp Ser Phe lle Ala Leu Val Ser Ala Asn Asp Leu Asp Ser Gly
370 375 380
Asn Asn Gly Leu Val His Cys Trp Leu Asn Gln Glu Leu Gly His Phe
385 390 395 400
Arg Leu Lys Arg Thr Asn Gly Asn Thr Tyr Met Leu Leu Thr Asn Ala
405 410 41 ~
.
Thr Leu Asp Arg Glu Gln Trp Pro lle Tyr Thr Leu Thr Val Phe Ala
420 425 430
Gln Asp Gln Gly Pro Gln Pro Leu Ser Ala Glu Lys Glu Leu Gln lle
CA 0224~9~6 l998-08-ll
: WO 98/25946 PCT~US97/2~006
435 440 445
Gln Val Ser Asp Val Asn Asp Asn Ala Pro Val Phe Glu Lys Ser Arg
450 455 460
Tyr Glu Val Ser Thr Trp Glu Asn Asn Pro Pro Ser Leu His Leu lle
465 470 475 480
Thr Leu Lys Ala His Asp Ala Asp Leu Gly Ser Asn Gly Lys Val Ser
485 490 495
Tyr Arg lle Lys Asp Ser Pro Val Ser His Leu Val lle lle Asp Phe
500 505 510
Glu Thr Gly Glu Val Thr Ala Gln Arg Ser Leu Asp Tyr Glu Gln Met
515 520 525
Ala Gly Phe Glu Phe Gln Val lle Ala Glu Asp Arg Gly Gln Pro Gln
530 535 540
Leu Ala Ser Ser lle Ser Val Trp Val Ser Leu Leu Asp Ala Asn Asp
545 550 555 560
Asn Ala Pro Glu Val lle Gln Pro Val Leu Ser Glu Gly Lys Ala Thr
565 570 575
Leu Ser Val Leu Val Asn Ala Ser Thr Gly His Leu Leu Leu Pro lle
580 585 590
Glu Asn Pro Ser Gly Met Asp Pro Ala Gly Thr Gly lle Pro Pro Lys
595 600 605
Ala Thr His Ser Pro Trp Ser Phe Leu Leu Leu Thr lle Val Ala Arg
610 615 620
Asp Ala Asp Ser Gly Ala Asn Gly Glu Leu Phe Tyr Ser lle Gln Ser
625 630 635 640
Gly Asn Asp Ala His Leu Phe Phe Leu Ser Pro Ser Leu Gly Gln Leu
645 650 655
- 34 -
CA 0224~9~6 1998-08-11
WO 98/25946 PCT/US97/20006
.
Phe lle Asn Val Thr Asn Ala Ser Ser Leu lle Gly Ser Gln Trp Asp
- 660 665 670
Leu Gly lle Val Val Glu Asp Gln Gly Ser Pro Ser Leu Gln Thr Gln
675 680 685
Val Ser Leu Lys Val Val Phe Val Thr Ser Val Asp His Leu Arg Asp
690 695 700
Ser Ala His Glu Pro Giy Val Leu Ser Thr Pro Ala Leu Ala Leu lle
705 710 715 7Z0
Cys Leu Ala Val Leu Leu Ala lle Phe Gly Leu Leu Leu Ala Leu Phe
725 730 735
Val Ser lle Cys Arg Thr Glu Arg Lys Asp Asn Arg Ala Tyr Asn Cys
740 745 750
Arg Glu Ala Glu Ser Ser Tyr Arg His Gln Pro Lys Arg Pro Gln Lys
755 760 765
His lle Gln Lys Ala Asp lle His Leu Val Pro Val Leu Arg Ala His
770 775 780
Glu Asn Glu Thr Asp Glu Val Arg Pro Ser His Lys Asp Thr Ser Lys
785 790 795 800
Glu Thr Leu Met Glu Ala Gly Trp Asp Ser Cys Leu Glu Ala Pro Phe
805 810 815
His Leu Thr Pro Thr Leu Tyr Arg Thr Leu Arg Asn Gln Gly Asn Gln
820 825 830
Gly Glu Leu Ala Glu Ser Gln Glu Val Leu Gln Asp Thr Phe Asn Phe
835 840 845
Leu Phe Asn His Pro Arg Gln Arg Asn Ala Ser Arg Glu Asn Leu Asn
850 855 860
CA 0224~9~6 l998-08-ll
WO 98/25946 PCT/US97/20006
- Leu Pro Glu Ser Pro Pro Ala Val Arg Gln Pro Leu Leu Arg Pro Leu
865 870 875 880
Lys Val Pro Gly Ser Pro lle Ala Arg Ala Thr Gly Asp Gln Asp Lys
885 890 895
Glu Glu Ala Pro Gln Ser Pro Pro Ala Ser Ser Ala Thr Leu Arg Arg
900 905 910
Gln Arg Asn Phe Asn Gly Lys Val Ser Pro Arg Gly Glu Ser Gly Pro
915 920 925
His Gln lle Leu Arg Ser Leu Val Arg Leu Ser Val Ala Ala Phe Ala
930 935 940
Glu Arg Asn Pro Val Glu Glu Pro Ala Gly Asp Ser Pro Pro Val Gln
945 950 955 960
Gln lle Ser Gln Leu Leu Ser Leu Leu His Gln Gly Gln Phe Gln Pro
965 970 975
Lys Pro Asn His Arg Gly Asn Lys Tyr Leu Ala Lys Pro Gly Gly Ser
980 985 990
Ser Arg Gly Thr lle Pro Asp Thr Glu Gly Leu Val Gly Leu Lys Pro
995 1000 1005
Ser Gly Gln Ala Glu Pro Asp Leu Glu Glu Gly Pro Pro Ser Pro Glu
1010 1015 1020
Glu Asp Leu Ser Val Lys Arg Leu Leu Glu Glu Glu Leu Ser Ser Leu
1025 1030 1035 1040
Leu Asp Pro Asn Thr Gly Leu Ala Leu Asp Lys Leu Ser Pro Pro Asp
1045 1050 1055
Pro Ala Trp Met Ala Arg Leu Ser Leu Pro Leu Thr Thr Asn Tyr Arg
1060 1065 1070
Asp Asn Leu Ser Ser Pro Asp Ala Thr Thr Ser Glu Glu Pro Arg Thr
--36 --
CA 0224~9~6 l998-08-ll
: W0 98/25946 PCT~US97/20006
1075 1080 1085
Phe Gln Thr Phe Gly Lys Thr Val Gly Pro Gly Pro Glu Leu Ser Pro
1090 10~5 1100
.
Thr Gly Thr Arg Leu Ala Ser Thr Phe Val Ser Glu Met Ser Ser Leu
1105 1110 1115 1120
Leu Glu Met Leu Leu Gly Gln His Thr Val Pro Val Glu Ala Ala Ser
1125 1130 1135
Ala Ala Leu Arg Ar~ Leu Ser Val Cys Gly Arg Thr Leu Ser Leu Asp
1140 1145 1150
Leu Ala Thr Ser Gly Ala Ser Ala Ser Glu Ala Gln Gly Arg Lys Lys
1155 1160 1165
Ala Ala Glu Ser Arg Leu Gly Cys Gly Arg Asn Leu
1170 1175 1180
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3868 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: Z:
GCGGCCGCTGTCAAGTCTCTCTTAGACCGGTACTTGCCCATCACTGCTAA GTGGACCAGC 60
TGGTGCTGTG GCGGAGCAGG AA~ ll~CAGCAATTTATCTGTTCTGGG ACCTCTCACT 120
- 37 -
CA 0224~9~6 l998-08-ll
- WO 98/2~;946 PCT/US97120006
TGTCACGGAG ATGGCCTTGG TTGGGAATCC TGCTCCTTAC AGCATCTTCT AATTATGGGA 180
CAGAGTTGTA ACAGCAGGTC TGAAGTGGGA GATCCAGGCT GACCAAGCCA TTCAGGAACT 240
ACTGGGGACA AGCTCTGCCT TTGAAAAACT CCAGTCCAGC CTACCTGCCG GTAAGCATGA 300
TGCTACTTCT GCCATTCCTG CTAGGGCTCT TAGGGCCAGG AAGCTACTTG TTCATTTCAG 360
GGGATTGTCA GGAGGTGGCC ACTGTCATGG TGAAATTCCA AGTGACAGAG GAAGTGCCGT 420
CTGGCACGGT GATAGGGAAA CTGTCCCAAG AACTAAGAGT GGAGGAGAGG CGTGGGAAGG
480
CAGGAGATGC CTTCCAGATT CTGCAGCTGC CTCAGGCACT GCCGGTTCAG ATGAACTCTG 540
AGGACGGCCT GCTCAGCACT TCCAGCCGGC TGGATCGGGA GAAGCTATGT CGGCAGGAAG
600
ATCCCTGTCT GGTGTCATTT GACGTGCTTG CCACAGGGGC GTCTGCTCTA ATTCATGTGG 660
AGATTCAGGT GCTAGACATC AATGACCACC AGCCACAGTT rCCCAAAGAC GAGCAGGAAC 720
TGGAAATCTC AGAGAGTGCC TCTCTGCACA CACGAATCCC CTTGGACAGA GCTCTTGACC 780
AAGACACGGG TCCTAACAGC TTATATTCCT ACTCCCTGTC TCCCAGTGAA CACTTTGCCC 840
TGGATGTTAT TGTGGGCCCT GATGAGACCA MCATGCAGA GCTTGTGGTG GTGAAGGAGT 900
TGGACAGGGA ACTCCACTCA TA l l l l GATC TGGTGCTGAC CGCCTATGAC AATGGGAATC 960
CCCCTAAGTC AGGAATCAGC GTGGTCAAGG TCMTGTCCT GGACTCCAAT GACAATAGTC 1020
CAGTG l l I GC TGAGAGTTCA CTAGCACTAG AAATCCCAGA AGACACTGTT CCTGGTACTC 1080
TTCTCATAAA CCTGACTGCT ACAGATCCCG ACCAAGGACC CAATGGGGAG GTAGAGTTCT 1140
TCTTTGGCAA GCATGTGTCC CCAGAGGTGA TGAACACCTT TGGCATAGAT GCCAAGACAG 1200
GCCAGATCAT TCTGCGCCAA GCCCTAGATT ACGAGAAGM CCCTGCCTAT GAGGTGGATG 1260
TCCAGGCAAG GGA l l l GGGT CCCAATTCCA TCCCAGGCCA TTGCAAAGTT CTTATCAAAG 1320
TTCTGGATGT CAATGACAAT GCCCCAAGCA TCCTCATCAC GTGGGCCTCC CAGACGTCGC 1380
CA 022459~6 1998-08-11
W 098/25946 PCT~US97/20006
TGGTGTCAGA AGATCTTCCC AGGGATAGCT TCATTGCCCT TGTCAGTGCG AATGACTTGG 1440
ACTCAGGAAA CAACGGTCTC GTCCACTGTT GGCTGAATCA AGAGCTGGGC CACTTCAGAC 1500
TGAMAGGAC TAACGGCAAC ACGTACATGC TGCTCACCAA TGCCACACTG GACAGAGAGC 1560
AGTGGCCCAT ATATACTCTC ACTGTGmG CCCAAGACCA AGGACCCCAG CCCTTATCAG 1620
CTGAGAAGGA GCTCCAAATT CAGGTTAGTG ATGTCMTGA CAATGCCCCT c ~ l l I GAGA 1680
AGAGCCGGTA CGAGGTCTCC ACTTGGGAAA ATAACCCACC CTCTCTTCAC CTCATCACGC 1740
TCAAAGCGCA TGATGCTGAC TTGGGCAGTA ATGGAAAAGT GTCATACCGT ATCAAGGACT 1800
CCCCCGmC TCACTTAGTC ATTATTGACT TTGAAACAGG AGAAGTCACT GCTCAGAGGT 1860
CACTGGACTA TGAACAGATG GCAGGCmG AGTTCCAGGT GATAGCAGAG GACAGAGGGC 1920
AACCCCAGCT CGCATCCAGC ATCTCGGTGT GGGTTAGCCT CTTGGATGCC AATGATAATG 1980
CCCCAGAAGT GATTCAGCCT GTGCTCAGTG AAGGCAAAGC CACCCmCG GTGCTTGTAA 2040
ATGCCTCCAC GGGCCACCTT CTGTTGCCCATTGAGAATCC CAGTGGCATG GATCCAGCAG 2100
GTACTGGTAT ACCACCAAAG GCTACCCACA GCCCCTGGTC TTTCCTTTTG TrAACAATCG 2160
TGGCTAGGGA TGCAGACTCG GGGGCCAATG GGGAACTCTT CTACAGCATT CAAAGTGGGA 2220
ATGATGCTCA TCTCTTTTTC CTCAGCCCTT CCTTGGGGCA GCTATTCATT AATGTCACCA 2280
ATGCCAGCAG CCTCATCGGG AGTCAGTGGG ACCTGGGGAT AGTGGTAGAG GACCAGGGCA
2340
GCCCCTCCTT GCAGACCCAA GTTTCATTGA AGGTCGTGTT TGTCACCAGT GTGGACCACC 2400
TAAGGGATTC TGCTCATGAG CCCGGAGTTC TGAGCACACC AGCACTGGCT TTGATCTGCC 2460
TGGCTGTACT GCTGGCCATC TTTGGATTGC TCTrAGCCCT GTTCGTGTCC ATCTGCAGGA 2520
CAGAGAGAAA GGATAATAGG GCCTACAACT GTCGAGAAGC TGAGTCGTCA TACCGCCACC 2580
AGCCCAAGAG GCCCCAGAAA CAcATTcAGA AGGCAGATAT CCACCTGGTG CCTGTGCTTA 2640
GGGCCCACGA GAATGAGACT GATGAAGTCA GGCCATCTCA CAAGGATACC AGCAAGGAGA 2700
.
CA 0224~9~6 l998-08-ll
WO 98/25946 PCT/US97/20006
CACTGATGGA GGCAGGCTGG GACTCTTGCC TGGAGGCCCC CTTCCACCTC ACACCAACCC 2760
TATACAGGAC CCTGCGTAAC CAAGGCAACC AGGGAGAACT GGCAGAGAGC CAGGAGGTAC
2820
TGCAGGACAC CTTCAACTTT CTCTTl AACC ATCCCAGGCA GAGGAATGCC TCCCGGGAGA 2880
ACCTAAACCT TCCTGAGTCC CCACCTGCTG TACGCCAACC ACTCTTAAGG CCTCTGAAGG 2940
TGCCTGGTAG CCCCATAGCG AGGGCGACTG GAGACCAAGA CAAGGAGGAG GCCCCACAGA
3000
GCCCACCAGC GTCCTCTGCA ACCCTAAGAC GACAGCGGAA TTTCAATGGC AAAGTGTCTC 3060
CTAGAGGAGA GTCCGGTCCT CATCAGATTC TGAGGAGCCT GGTTAGGCTC TCTGTGGCTG 3120
CTTTTGCGGA ACGGAACCCG GTGGAGGAGC CTGCTGGGGA CTCTCCTCCT GTCCAGCAAA 3180
TCTCCCAGCT GCTGTCCTTG CTGCACCAGG GCCAATTCCA GCCCAAACCA AACCACCGAG 3240
GAAATAAATA CTTGGCCAAG CCCGGCGGCA GCAGCAGGGG TACCATCCCA GACACAGAGG
3300
GCCTTGTAGG CCTCAAGCCT AGTGGCCAAG CAGAACCTGA CCTGGAAGAA GGGCCCCCGA
3360
GCCCGGAGGA GGACCTTTCT GTAAAGCGAC TTCTAGAAGA AGAGCTGTCG AGCCTGTTGG 3420
ACCCTAATAC AGGTCTAGCC CTGGACAAGC TGAGTCCGCC TGACCCAGCC TGGATGGCGA 3480
GATTGTCATT GCCCCTCACC ACCAATTATC GAGACAACTT GTCTTCCCCC GATGCTACAA 3540
CATCAGAGGA ACCGAGAACC TTCCAGACAT TCGGCAAGAC AGTTGGACCG GGACCCGAGC 3600
TGAGCCCAAC AGGCACGCGC CTGGCCAGCA CTTTCGTCTC GGAGATGAGC TCTCTGCTGG 3660
AAATGTTGTT GGGGCAGCAC ACGGTACCAG TGGAAGCTGC GTCCGCGGCT TTGCGGAGGC
3720
TCTCGGTGTG CGGGAGGACC CTCAGTCTAG ACCTAGCCAC CAGTGGGGCT TCAGCTTCAG 3780
AAGCACAGGG TAGAAAGAAG GCAGCTGAGA GCAGACTTGG CTGTGGCAGG AATCTATGAA 3840
-- 40 --
CA 0224S9~6 1998-08-11
W098/25946 PCT~US97t20006
CATGTTTGGT TGGGATGTGT TTGGATCC 3868
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3540 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
ATGATGCTAC TTCTGCCATT CCTGCTAGGG CTCTTAGGGC CAGGAAGCTA CTTGTTCATT 60
TCAGGGGATT GTCAGGAGGT GGCCACTGTC ATGGTGAAAT TCCAAGTGAC AGAGGAAGTG 120
CCGTCTGGCA CGGTGATAGG GAAACTGTCC CAAGAACTAA GAGTGGAGGA GAGGCGTGGG
180
AAGGCAGGAG ATGCCTTCCA GATTCTGCAG CTGCCTCAGG CACTGCCGGT TCAGATGAAC 240
TCTGAGGACG GCCTGCTCAG CACTTCCAGC CGGCTGGATC GGGAGAAGCT ATGTCGGCAG 300
GAAGATCCCTGTCTGGTGTCAI I IGACGTGCTTGCCACAGGGGCGTCTGCTCTAATTCAT 360
GTGGAGATTC AGGTGCTAGA CATCAATGAC CACCAGCCAC AGTTTCCCAA AGACGAGCAG 420
GAACTGGAAA TCTCAGAGAG TGCCTCTCTG CACACACGAA TCCCCTTGGA CAGAGCTCTT 480
GACCAAGACA CGGGTCCTAA CAGCTTATAT TCCTACTCCC TGTCTCCCAG TGAACAC I I 1 540
GCCCTGGATG TTATTGTGGG CCCTGATGAG ACCAAACATG CAGAGCTTGT GGTGGTGAAG 6Q0
GAGTTGGACA GGGAACTCCA CTCATATTTT GATCTGGTGC TGACCGCCTA TGACAATGGG 660
AATCCCCCTA AGTCAGGAAT CAGCGTGGTC AAGGTCAATG TCCTGGACTC CAATGACAAT 720
AGTCCAGTGT TTGCTGAGAG TTCACTAGCA CTAGAAATCC CAGAAGACAC TGTTCCTGGT 780
-- 41 --
CA 0224~9~6 l998-08-ll
WO 98/25g46 PCT/US97/20006
ACTCTTCTCA TAAACCTGAC TGCTACAGAT CCCGACCAAG GACCCAATGG GGAGGTAGAG 840
I 11; l l C; I I I G GCAAGCATGT GTCCCCAGAG GTGATGAACA CC I I I GGCAT AGATGCCAAG 900
ACAGGCCAGA TCATTCTGCG CCAAGCCCTA GATTACGAGA AGAACCCTGC CTATGAGGTG 960
GATGTCCAGG CAAGGGAI I I GGGTCCCAAT TCCATCCCAG GCCATTGCAA AGTTCTTATC 1020
AAAGTTCTGG ATGTCAATGA CAATGCCCCA AGCATCCTCA TCACGTGGGC CTCCCAGACG 1080
TCGCTGGTGT CAGAAGATCT TCCCAGGGAT AGCTTCATTG CCCTTGTCAG TGCGAATGAC 1140
TTGGACTCAG GAAACAACGG TCTCGTCCAC TGTTGGCTGA ATCAAGAGCT GGGCCACTTC 1200
AGACTGAAAA GGACTAACGG CAACACGTAC ATGCTGCTCA CCAATGCCAC ACTGGACAGA 1260
GAGCAGTGGC CCATATATAC TCTCACTGTG l l l GCCCAAG ACCAAGGACC CCAGCCCTTA 1320
TCAGCTGAGA AGGAGCTCCA AATTCAGGTT AGTGATGTCA ATGACAATGC CCCTGTGl i 1 1380
GAGAAGAGCC GGTACGAGGT CTCCACTTGG GAAAATAACC CACCCTCTCT TCACCTCATC 1440
ACGCTCAAAG CGCATGATGC TGACTTGGGC AGTAATGGAA AAGTGTCATA CCGTATCAAG 1500
GACTCCCCCG l l l CTCACTT AGTCATTATT GAC l l l GAAA CAGGAGAAGT CACTGCTCAG 1560
AGGTCACTGG ACTATGAACA GATGGCAGGC l l l GAGTTCC AGGTGATAGC AGAGGACAGA 1620
GGGCAACCCC AGCTCGCATC CAGCATCTCG GTGTGGGTTA GCCTCTTGGA TGCCAATGAT 1680
AATGCCCCAG AAGTGATTCA GCCTGTGCTC AGTGAAGGCA AAGCCACCCT TTCGGTGCTT 1740
GTAAATGCCT CCACGGGCCA CCT I c; I ~ CCCATTGAGA ATCCCAGTGG CATGGATCCA 1800
GCAGGTACTG GTATACCACC AAAGGCTACC CACAGCCCCT G(i I ~; l l I ~;CT I I I GTTAACA 1860
ATCGTGGCTA GGGATGCAGA CTCGGGGGCC AATGGGGAAC TCTTCTACAG CATTCAAAGT 1920
GGGAATGATG CTCATCTCTT I I I CCTCAGC CCTTCCTTGG GGCAGCTATT CATTAATGTC 1980
ACCAATGCCA GCAGCCTCAT CGGGAGTCAG TGGGACCTGG GGATAGTGGT AGAGGACCAG
2040
GGCGCCCCTC CTTGCAGACC CAAG l l l CAT TGAAGGTCGT GTTTGTCACC AGTGTGGACC 2100
-- 4Z --
CA 0224~9~6 l998-08-ll
- WO 98/25946 PCT/USg7/20006
ACCTMGGGA TTCTGCTCAT GAGCCCGGAG TTCTGAGCAC ACCAGCACTG GC l l l GATCT 2160
GCCTGGCTGT ACTGCTGGCC ATCTTTGGAT TGCTCTTAGC CCTGTTCGTG TCCATCTGCA 2220
GGACAGAGAG AAAGGATAAT AGGGCCTACA ACTGTCGAGA AGCTGAGTCG TCATACCGCC Z280
ACCAGCCCAA GAGGCCCCAG AAACACATTC AGAAGGCAGA TATCCACCTG GTGCCTGTGC 2340
TTAGGGCCCA CGAGAATGAG ACTGATGAAG TCAGGCCATC TCACAAGGAT ACCAGCAAGG 2400
AGACACTGAT GGAGGCAGGC TGGGACTCTT GCCTGGAGGC CCCCTTCCAC CTCACACCAA 2460
CCTATACAGG ACCCTGCGTA ACCAAGGCAA CCAGGGAGAA CTGGCAGAGA GCCAGGAGGT
2520
ATGCAGGACA CCTTCAACTT TCTCTTTAAC CATCCCAGGC AGAGGAATGC CTCCCGGGAG 2580
AACCTAAACC TTCCTGAGTC CCCACCTGCT GTACGCCAAC CACTCTTAAG GCCTCTGAAG 2640
GTGCCTGGTA GCCCCATAGC GAGGGCGACT GGAGACCAAG ACAAGGAGGA GGCCCCACAG
2700
AGCCCACCAG CGTCCTCTGC AACCCTAAGA CGACAGCGGA ATTTCAATGG CAAAGTGTCT 2760
CCTAGAGGAG AGTCCGGTCC TCATCAGATT CTGAGGAGCC TGGTTAGGCT CTCTGTGGCT 2820
GC l l l TGCGG AACGGAACCC GGTGGAGGAG CCTGCTGGGG ACTCTCCTCC TGTCCAGCAA
2880
ATCTCCCAGC TGCTGTCCTT GCTGCACCAG GGCCAATTCC AGCCCAAACC AAACCACCGA 2940
GGAAATAAAT ACTTGGCCAA GCCCGGCGGC AGCAGCAGGG GTACCATCCC AGACACAGAG
3000
GGCCTTGTAG GCCTCAAGCC TAGTGGCCAA GCAGAACCTG ACCTGGAAGA AGGGCCCCCG
3060
AGCCCGGAGG AGGACCTTTC TGTAAAGCGA CTTCTAGAAG AAGAGCTGTC GAGCCTGTTG 3120
GACCCTAATA CAGGTCTAGC CCTGGACAAG CTGAGTCCGC CTGACCCAGC CTGGATGGCG 3180
AGATTGTCAT TGCCCCTCAC CACCAATTAT CGAGACAACT TGTCTTCCCC CGATGCTACA 3240
CA 0224~9~6 1998-08-11
WO 98/25946 PCT/US97/20006
ACATCAGAGG AACCGAGAAC CTTCCAGACA TTCGGCAAGA CAGTTGGACC GGGACCCGAG 3300
CTGAGCCCAA CAGGCACGCG CCTGGCCAGC AC I I I CGTCT CGGAGATGAG (; I C I C; I GCTG 3360
GAAATGTTGT TGGGGCAGCA CACGGTACCA GTGGAAGCTG CGTCCGCGGC 111 GCGGAGG
3420
CTCTCGGTGT GCGGGAGGAC CCTCAGTCTA GACCTAGCCA CCAGTGGGGC TTCAGCTTCA 3480
GAAGCACAGG GTAGAAAGAA GGCAGCTGAG AGCAGACTTG GCTGTGGCAG GAATCTATGA
3540
-- q4 --
-
