U.S. patent application number 11/183599 was filed with the patent office on 2006-02-16 for purified polypeptides having il-13 receptor activity.
This patent application is currently assigned to sanofi-aventis. Invention is credited to Daniel Caput, Pascual Ferrara, Patrick Laurent, Natalio Vita.
Application Number | 20060035856 11/183599 |
Document ID | / |
Family ID | 9485198 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060035856 |
Kind Code |
A1 |
Caput; Daniel ; et
al. |
February 16, 2006 |
Purified polypeptides having IL-13 receptor activity
Abstract
This invention relates to monoclonal and polyclonal antibodies
capable of specifically recognizing IL-13 receptor .alpha. and/or
IL13 receptor .beta. and uses thereof.
Inventors: |
Caput; Daniel;
(Avignolet-Lauragais, FR) ; Ferrara; Pascual;
(Avignolet-Lauragais, FR) ; Laurent; Patrick;
(Auterive, FR) ; Vita; Natalio; (Montgiscard,
FR) |
Correspondence
Address: |
ROSS J. OEHLER;AVENTIS PHARMACEUTICALS INC.
ROUTE 202-206
MAIL CODE: D303A
BRIDGEWATER
NJ
08807
US
|
Assignee: |
sanofi-aventis
Paris
FR
|
Family ID: |
9485198 |
Appl. No.: |
11/183599 |
Filed: |
July 15, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09077817 |
Sep 14, 1998 |
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PCT/FR96/01756 |
Nov 7, 1996 |
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11183599 |
Jul 15, 2005 |
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Current U.S.
Class: |
514/44R ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
A61P 37/00 20180101;
A61K 38/00 20130101; A61P 43/00 20180101; A61P 31/12 20180101; A61P
29/00 20180101; A61P 35/00 20180101; A61P 37/02 20180101; C07K
14/7155 20130101; A61P 37/08 20180101 |
Class at
Publication: |
514/044 ;
530/350; 536/023.5; 435/069.1; 435/320.1; 435/325 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/04 20060101 C07H021/04; C07K 14/715 20060101
C07K014/715 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 6, 1995 |
FR |
95/14424 |
Claims
1-43. (canceled)
44. An antibody that specifically binds to a protein selected from
the group consisting of: (a) a protein consisting of amino acid
residues of SEQ ID NO:4; (b) a protein consisting of a biologically
active amino acid sequence derived from SEQ ID NO:4 or a
biologically active amino acid sequence fragment of SEQ ID NO: 4
wherein said biologically active amino acid sequence is selected
from the group consisting of (i) participating in the transduction
of the signal specifically produced by IL-13 at the cell membrane
and (ii) interacting with the receptor specific for IL-4 to form a
complex capable of binding IL-4 and IL-13; (c) a protein consisting
of the extracellular domain of SEQ ID NO:4; (d) a protein
consisting of the soluble amino acid residues of SEQ ID NO:4; and
(d) a protein comprising SEQ ID NO:4.
45. The antibody of claim 44 which is polyclonal.
46. The antibody of claim 44 which is monoclonal.
47. The antibody of claim 44 which is selected from the group
consisting of: (a) a chimeric antibody; (b) a Fab fragment; (c) a
humanized antibody; and (d) a F(ab').sub.2 fragment.
48. Process for diagnosis of pathologies correlated with an
abnormal expression of IL-13 receptor in a biological sample,
comprising of the steps of: (a) contacting said biological sample
with at least one antibody according to claim 44 under conditions
allowing the formation of specific immunological complexes between
the IL-13 receptor and said antibody and (b) detecting said
immunological complexes which are formed and comparing to a normal
control sample.
49. A kit for in vitro diagnosis for measuring the level of
expression of the IL-13 receptor in a biological sample comprising:
(a) at least one antibody specific for the IL-13 receptor according
to claim 44, optionally attached onto a support, (c) means for
revealing the formation of specific IL-13 receptor/antibody
complexes and quantifying these complexes.
50. An antibody that specifically binds to a protein selected from
the group consisting of: (a) a protein consisting of amino acid
residues of SEQ ID NO:2; (b) a protein consisting of a biologically
active amino acid sequence derived from SEQ ID NO:2 or a
biologically active amino acid sequence fragment of SEQ ID NO: 2
wherein said biologically active amino acid sequence is selected
from the group consisting of (i) participating in the transduction
of the signal specifically produced by IL-13 at the cell membrane
and (ii) interacting with the receptor specific for IL-4 to form a
complex capable of binding IL-4 and IL-13; (c) a protein consisting
of the extracellular domain of SEQ ID NO:2; (d) a protein
consisting of the soluble amino acid residues of SEQ ID NO:2; and
(d) a protein comprising SEQ ID NO:2.
51. The antibody of claim 50 which is polyclonal.
52. The antibody of claim 50 which is monoclonal.
53. The antibody of claim 50 which is selected from the group
consisting of: (a) a chimeric antibody; (b) a Fab fragment; (c) a
humanized antibody; and (d) a F(ab').sub.2 fragment.
54. A process for diagnosis of pathologies correlated with an
abnormal expression of IL-13 receptor in a biological sample,
comprising the steps of: (d) contacting said biological sample with
at least one antibody according to claims 44 or 50 under conditions
allowing the formation of specific immunological complexes between
the IL-13 receptor and said antibody and (e) detecting said
immunological complexes which are formed and comparing to a normal
control sample.
55. A kit for in vitro diagnosis that measures the level of
expression of the IL-13 receptor in a biological sample comprising:
(a) at least one antibody specific for IL-13 receptor according to
claim 44 or 50, optionally attached onto a support, and (b) means
for revealing the formation of specific IL-13 receptor/antibody
complexes and quantifying said complexes.
Description
[0001] The present invention relates to purified polypeptides
having a receptor activity specific for interleukin-13 (IL-13), to
their biologically active fragments and to the corresponding
nucleic acid sequences and to their applications.
[0002] IL-13 is a recently identified (1, 2) cytokine of 112 amino
acids secreted by the activated T lymphocytes, the B lymphocytes
and the mastocytes after activation.
[0003] By virtue of its numerous biological properties shared with
IL-4, IL-13 has been described as an IL-4-like cytokine. Its
activities are indeed similar to those of IL-4 on the B cells
(3-5), the monocytes (6-10) and other non-haematopoietic cells
(11-12). On the other hand, contrary to IL-4, it would not exert a
specific effect on resting or activated T cells (13).
[0004] Various biological activities of IL-13 on the
monocytes/macrophages, the B lymphocytes and certain haematopoietic
precursors have been described in detail by A. J. Minty, as well as
in review articles on IL-13 (see for example 14). Several data
indicate, in addition, that this cytokine has a pleiotropic effect
on other cell types. These non-haematopoietic cells which are
directly affected by IL-13 are endothelial and microglial cells,
keratinocytes and kidney and colon carcinomas.
[0005] The anti-inflammatory and immunoregulatory activities of
IL-13 may be useful, for example, in the treatment of autoimmune,
tumour and viral pathologies.
[0006] An exploitation of these biological properties at the
clinical level requires, however, a perfect knowledge of the
signals and mechanisms via which these effects are exerted, so as
to be able to control and modulate them in the relevant
pathologies.
[0007] One of the stages in the analysis of the signal transmitted
by a biological molecule within a cell consists in identifying its
membrane receptor. The research studies carried out to this end on
the IL-13 receptor have shown that IL-13 and IL-4 had a common
receptor, or at the very least some of the components of a common
receptor complex, as well as common signal transduction elements
(15-18). This receptor is present at the surface of various cell
types, in a variable number according to the cell type considered.
The comparative distribution of the IL-13 and IL-4 receptors has
been indicated by A. J. Minty (14).
[0008] Kondo et al. (19) have described the structure of a receptor
having a high affinity for IL-4. This receptor is a dimer, formed
by the association of a glycoprotein of 140 kDa (IL-4R) and of the
y chain of the IL-2 receptor (.gamma.c). IL-4 can bind to the
glycoprotein subunit of 140 kDa (IL-4R or gp 140) with a high
affinity (Kd between 50 and 100 pM) (15). However, this affinity is
increased by a factor of 2 to 3 when the .gamma.c chain is
associated with gp 140. This association is, in addition, necessary
for the transmission of certain signals mediated by IL-4 (19,
20).
[0009] Cross-competition experiments for binding either of IL-13 or
of IL-4 have demonstrated that IL-4 can normally prevent the
binding of IL-13, whereas IL-13 can generally only partially
prevent the binding of IL4 to its receptor (17, 21) and does not
attach to any of the two subunits of the IL-4 receptor or to the
complex formed by their association. On the basis of these
observations, the authors of the present invention have assumed
that the receptor specific for IL-13 consisted of the receptor
complex IL-4 associated with another IL-13 binding component
(IL-13R.beta.).
[0010] Research studies carried out on an erythro-leukemic cell
line capable of proliferating in response to IL-13 and IL-4 (TF-1
line) allowed them to show that these two cytokines produced
similar intracellular events after attachment to their receptor
(18). In parallel, cross-linking experiments allowed them to show
that gp 140 could form heterodimers either with the .gamma.chain,
or with a new subunit, of a molecular weight of 55 to 70 kDa (17,
21).
[0011] Moreover, research studies recently carried out on a mouse
embryonic stem cell line have made it possible to isolate the
genomic DNA and the cDNA encoding a polypeptide of 424 amino acid
residues (IL-13R.alpha.), suggesting that the IL-13 receptor shared
with the IL-4 receptor a common chain 80 as to constitute a
high-affinity receptor (22, 23), that is to say has an affinity
whose constant Kd is situated between values of between about 10 pM
and 100 pM (a low-affinity receptor having a constant Kd situated
between the values of between 2 nM and 10 nM).
[0012] Given the importance, at the medical level, of the fine
understanding of the phenomena of regulation of IL-4 and of IL-13,
and in particular of the possibility of being able to separate and
control separately the effects produced by either of these two
cytokines, the authors of the present invention were interested on
the one hand, in the characterization of a polypeptide specifically
binding IL-13 with a high affinity and, on the other hand, in the
characterization of another polypeptide which, alone, specifically
binds IL-13 with a low affinity and which, if it is associated with
the IL-4 receptor, constitutes a high-affinity receptor for
IL-13.
[0013] These authors have now identified a human carcinoma cell
line expressing the IL-13 specific receptor in a quantity greater
than other known human renal carcinoma lines (21), and have now
carried out the cloning of the primary subunit responsible for the
attachment of IL-13 to the IL-4/IL-13 receptor, called
IL-13R.beta., as well as the cloning of the common chain shared by
the IL-13 receptor and the IL-4 receptor in order to constitute a
high-affinity receptor which allows cross-competition between the 2
cytokines, called IL-13R.alpha.. The present invention therefore
relates to purified polypeptides specifically linking IL-13.
[0014] More particularly, the subject of the invention is purified
polypeptides whose amino acid sequences correspond to that of a
receptor specific for IL-13 (IL-13R.beta. and IL-13R.alpha.), or
biologically active fragments thereof.
[0015] The subject of the invention is also isolated DNA sequences
encoding the said polypeptides or their biologically active
fragments.
[0016] It relates, in addition, to the expression vectors
containing at least one of the nucleotide sequences defined above,
and the host cells transfected with these expression vectors under
conditions allowing the replication and/or expression of one of the
said nucleotide sequences.
[0017] The methods for producing recombinant IL-13R.beta. and
IL-13R.alpha. or their biological active fragments by the
transfected host cells are also part of the invention.
[0018] The invention also comprises pharmaceutical compositions
comprising IL-13R.beta. and/or IL-13R.alpha. or biologically active
fragments thereof for the regulation of the immunological and
inflammatory mechanisms produced by IL-13. It relates, in addition,
to a method for the identification of agents capable of modulating
the activity of IL-13R.beta. and/or IL-13R.alpha., and the use of
IL-R13.beta. and/or IL-13R.alpha. or of fragments thereof for
screening these agents as well as for the manufacture of new
products capable of modulating the activity of the IL-13
receptor.
[0019] The invention also comprises antibodies or derivatives of
antibodies specific for IL-13R.beta. and/or IL-13R.alpha..
[0020] Finally, it relates to a method of therapeutic treatment for
modulating the immunological reactions mediated by IL-13,
comprising the administration, to a patient, of IL-13R.beta. and/or
IL-13R.alpha. or of one of their biologically active fragments or
of a compound capable of specifically modulating the activity of
this receptor, in combination with a pharmaceutically acceptable
vehicle.
[0021] In the description of the invention below, the following
definitions are used: [0022] polypeptide specifically binding IL-13
with a high affinity (IL-13R.beta.): a polypeptide comprising the
amino acid sequence SEQ ID No. 2 or any biologically active
fragment or derivative thereof; [0023] polypeptide which, alone,
specifically binds IL-13 with a low affinity and which, if it is
associated with the IL-4 receptor, constitutes a high-affinity
receptor (IL-13R.alpha.) : a polypeptide comprising the amino acid
sequence SEQ ID NO 4 or any biologically active fragment or
derivative thereof; [0024] biologically active: capable of binding
specifically to IL-13 and/or of participating in the transduction
of the signal specifically produced by IL-13 at the level of the
cell membrane, and/or capable of interacting with the receptor
specific for IL-4 (IL-4R/gp 140) so as to form a complex capable of
binding IL-4 and IL-13, and/or which is recognized by antibodies
specific to the polypeptide of sequence SEQ ID No. 2 and/or of
sequence SEQ ID No. 4, and/or capable of inducing antibodies which
recognize the polypeptide of sequence SEQ ID No. 2 and/or of
sequence SEQ ID No. 4; [0025] derivative: any polypeptide which is
a variant of the polypeptide of sequence SEQ ID No. 2 and/or of
sequence SEQ ID No. 4, or any molecule resulting from a
modification of a genetic and/or chemicai nature of the sequence
SEQ ID No. 2 or of sequence SEQ ID No. 4, that is to say which is
obtained by mutation, deletion, addition, substitution and/or
chemical modification of one or of a limited number of amino acids,
as well as any isoform sequence, that is to say a sequence which is
identical to the sequence SEQ ID No. 2 or to the sequence SEQ ID
No. 4, to one of their fragments or to one of their modified
sequences, containing one or more amino acids in the D enantiomer
form, the said variant, modified or isoform sequences having
conserved at least one of the properties which make them
biologically active.
[0026] The subject of the present invention is a purified
polypeptide comprising an amino acid sequence chosen from: [0027]
a) the sequence SEQ ID No. 2 or the sequence SEQ ID No. 4, [0028]
b) any biologically active sequence derived from SEQ ID No. 2 or
SEQ ID No. 4, according to the definition given above.
[0029] The manufacture of derivatives may have various objectives,
including in particular that of increasing the affinity of the
receptor for IL-13, that of modulating the cross-competition
between IL-13 and IL-4, that of enhancing their levels of
production, of increasing their resistance to proteases, of
modifying their biological activity or of conferring new
pharmaceutical and/or biological properties on them.
[0030] Among biologically active variants of the polypeptides as
defined above, the fragments produced by alternate splicing of the
transcripts (messenger RNAs) of the gene encoding one of the amino
acid sequences described above are preferred.
[0031] In an advantageous variant, the 8 C-terminal amino acids of
the polypeptide of sequence SEQ ID No. 2 are substituted by the
following 6 amino acids: VRCVTL.
[0032] According to another advantageous aspect, the invention
relates to a soluble form of IL-13R.beta., called IL-13R.beta.s,
comprising especially the extracelluar domain of the polypeptide of
sequence SEQ ID No. 2 stretching up to residue 343 and preferably
up to residue 337 as well as a soluble form of IL-13R.alpha.,
called IL-13R.beta.s, comprising especially the extracelluar domain
of the polypeptide of sequence SEQ ID No. 4 stretching up to
residue 343 and preferably up to the residues between 336 and
342.
[0033] The polypeptide which comprises the sequence SEQ ID No. 2 or
the sequence SEQ ID No. 4 represents a specific embodiment of the
invention. As will emerge in the examples, this polypeptide may be
expressed at the surface of human cells so as to form a functional
IL-13 receptor and/or combine with the IL-4 receptor so as to form,
with the .gamma.chain of the IL-2 receptor, the receptor complex
common to IL-4 and IL-13.
[0034] The subject of the present invention is also an isolated
nucleic acid sequence, chosen from: [0035] a) the sequence SEQ ID
No. 1, [0036] b) the sequence SEQ ID No. 3, [0037] c) the nucleic
acid sequences capable of hybridizing to the sequence SEQ ID No. 1
or to the sequence SEQ ID No. 3, or to their complementary
sequences and encoding polypeptides having an IL-13 receptor
activity, or allowing the reconstitution of a receptor having a
high affinity for IL-13 and IL-4, [0038] d) the nucleic acid
sequences derived from the sequences a) and b) and c) because of
the degeneracy of the genetic code.
[0039] More particularly, the subject of the invention is a
sequence encoding the soluble part of IL-13R.beta. or of
IL-13R.alpha. and any variant produced by alternate splicing of the
transcripts of IL-13R.beta. or of IL-13R.alpha., conserving at
least one of the biological properties described.
[0040] A preferred embodiment is represented by a nucleic acid
sequence comprising or consisting of the stretch of nucleotides
stretching from nucleotide No. 1 up to nucleotide 1081, and
preferably up to nucleotide 1063 on the sequence SEQ ID No. 1.
[0041] Another preferred embodiment is represented by a nucleic
acid sequence comprising or consisting of the stretch of
nuclootides stretching from nucleotide No. 1 up to nucleotide No.
1059, and preferably up to the nucleotides between numbers 1041 and
1056 on the sequence SEQ ID No. 3.
[0042] Advantageously, the nucleic acid sequence according to the
invention in a sequence encoding a protein corresponding to the
mature form of IL-13R.beta. or of IL-13R.alpha., this mature
protein being the result of the release of the signal peptide.
[0043] The various nucleotide sequences of the invention may be of
artificial origin or otherwise. They may be DNA or RNA sequences
obtained by screening sequence libraries by means of probes
produced on the basis of the sequence SEQ ID No. 1 or of the
sequence SEQ ID No. 3. Such libraries may be prepared by
conventional molecular biology techniques known to persons skilled
in the art.
[0044] The nucleotide sequences according to the invention may also
be prepared by chemical synthesis or alternatively by a combination
of methods including chemical or enzymatic modification of
sequences obtained by screening of the libraries.
[0045] These nucleotide sequences allow the preparation of
nucleotide probes encoding a polypeptide according to the invention
or a biologically active fragment thereof. The appropriate
hybridization conditions correspond to the temperature and ionic
strength conditions customarily used by persons skilled in the art,
preferably to temperature conditions of between T.sub.m-5.degree.
C. and T.sub.m-30.degree. C. and still more preferably, to
temperature conditions between T.sub.m-5.degree. C. and
T.sub.m-10.degree. C. (high stringency), T.sub.m being the melting
temperature, defined as the temperature at which 50% of the
base-paired strands separate. Such probes are also part of the
invention. They may be used as a IN VITRO diagnostic tool for the
detection, by hybridization experiments, of transcripts specific
for the polypeptides of the invention in biological samples or for
the detection of aberrant syntheses or of genetic abnormalities
resulting from a polymorphism, from mutations or from a poor
splicing.
[0046] The probes of the invention comprise at least 10
nucleotides, and comprise at most the entire nucleotide sequence
SEQ ID No. 1 or the entire nucleotide sequence SEQ ID No. 3 or
their complementary strand.
[0047] Among the shortest probes, that is to say of about 10 to 15
nucleotides, the appropriate hybridization conditions correspond to
the temperature and ionic strength conditions customarily used by
persons skilled in the art.
[0048] Preferably, the probes of the invention are labelled prior
to their use. For that, several techniques are within the
capability of persons skilled in the art, such as for example
fluorescent, radioactive, chemiluminescent or enzymatic
labelling.
[0049] The IN VITRO diagnostic methods in which these nucleotide
probes are used for the detection of aberrant syntheses or of
genetic abnormalities, such as the loss of heterozygosity and
genetic rearrangement, at the level of the nucleic sequences
encoding an IL-13 receptor polypeptide or a biologically active
fragment, are included in the present invention. Such a type of
method comprises: [0050] bringing a nucleotide probe of the
invention into contact with a biological sample under conditions
allowing the formation of a hybridization complex between the said
probe and the above-mentioned nucleotide sequence, optionally after
a preliminary step of amplification of the abovementioned
nucleotide sequence; [0051] detection of the hybridization complex
which may be formed; [0052] optionally, sequencing the nucleotide
sequence forming the hybridization complex with the probe of the
invention.
[0053] The cDNA probes of the invention may, in addition, be
advantageously used for the detection of chromosomal
abnormalities.
[0054] The nucleotide sequences of the invention are also useful
for the manufacture and the use of sense and/or antisense
oligonucleotide primers for sequencing reactions or for specific
amplification reactions according to the so-called PCR (polymerase
chain reaction) technique or any other variant thereof.
[0055] The nucleotide sequences according to the invention have,
moreover, uses in the therapeutic field for the preparation of
antisonse sequences which are capable of hybridizing specifically
with a nucleic acid sequence, including a messenger RNA, and may be
used in gene therapy. The subject of the invention is thus
antisense sequences capable of inhibiting, at least partially, the
production of IL-13 receptor polypeptides as defined above. Such
sequences advantageously consist of those which constitute the
reading frame encoding IL-13R.beta. or IL-13R.alpha. at the level
of the transcript.
[0056] They may be more particularly used in the treatment of
allergies and of inflammation.
[0057] The nucleotide sequences according to the invention may,
moreover, be used for the production of recombinant polypeptides,
aso defined above., having an IL-13 receptor activity.
[0058] These polypeptides may be produced from the nucleotide
sequences defined above, according to techniques for the production
of recombinant products known to persons skilled in the art. In
this case, the nucleotide sequence used is placed under the control
of signals allowing its expression in a cellular host. The cellular
host used may be chosen from prokaryotic systems, such as bacteria,
or from eukaryotic systems, such as yeasts, insect cells, CHO cells
(chinese hamster ovary cells) or any other system which is
advantageously available commercially. A cellular host preferred
for the expression of the polypeptides of the invention consists of
the fibroblast line COS-7 or COS-3.
[0059] The signals controlling the expression of the polypeptides,
such as the promoters, the activators or the terminal sequences,
are chosen according to the cellular host used. To this end, the
nucleotide sequences according to the invention may be inserted
into autonomously replicating vectors within the chosen host, or
integrative vectors of the chosen host. Such vectors will be
prepared according to the methods commonly used by persons skilled
in the art, and the resulting clones may be introduced into an
appropriate host by standard methods, such as for example
electroporation.
[0060] The expression vectors containing at least one of the
nucleotide sequences defined above are also part of the present
invention.
[0061] In the case of the COS-7 or COS-3 cells, the transfection
may be carried out using the vector pSE-1, as described in
(17).
[0062] The invention relates, in addition, to the host cells
transfected by these expression vectors. These cells may be
obtained by the introduction, into host cells, of a nucleotide
sequence inserted into a vector as defined above, followed by the
culture of the said cells under conditions allowing the replication
and/or expression of the transfected nucleotide sequence.
[0063] These cells may be used in a method for the production of a
recombinant polypeptide of sequence SEQ ID No. 2 or SEQ ID No. 4 or
a derivative, which method is itself included in the present
invention and is characterized in that the transfected cells are
cultured under conditions allowing the expression of a recombinant
polypeptide of sequence SEQ ID No. 2 or SEQ ID No. 4, or a
derivative, and in that the said recombinant polypeptide is
recovered.
[0064] The purification processes used are known to persons skilled
in the art. The recombinant polypeptide may be purified from cell
lysates and extracts, from the culture supernatant, by methods used
individually or in combination, such as fractionation,
chromatographic methods, immunoaffinity techniques using specific
mono- or polyclonal antibodies.
[0065] The mono- or polyclonal antibodies capable of specifically
recognizing IL-13R.beta. and/or IL-13R.alpha. according to the
definition given above are also part of the invention. Polyclonal
antibodies may be obtained from the serum of an animal immunized
against IL-13R.beta. and/or IL-13R.alpha. according to the usual
procedures.
[0066] The monoclonal antibodies may be obtained according to the
conventional hybridoma culture method described by Kohler and
Milstein (Nature, 1975, 256, 495-497).
[0067] Advantageous antibodies are antibodies directed against the
extracelluar domain of IL-13R.beta. and/or IL-13R.alpha..
[0068] The antibodies according to the invention are, for example,
chimeric antibodies, humanized antibodies, Fab and F(ab')2
fragments. They may also exist in the form of labelled antibodies
or immunoconjugates. For example, they may be associated with a
toxin, such as the diphtheria toxin or with a radioactive product.
These immunotoxins may in this case constitute therapeutic agents
which may be used for the treatment of certain pathologies
involving an overexpression of IL-13R.beta. and/or
IL-13R.alpha..
[0069] The antibodies of the invention, in particular the
monoclonal antibodies, may also be used for the immunocytochemical
analyses of the IL-13 receptors on specific tissue sections, for
example by immunofluorescence or by gold or peroxidase
labelling.
[0070] They may be advantageously used in any situation where the
expression of IL-13R.beta. and/or IL-13R.alpha. needs to be
observed, such as for example an abnormal over-expression or the
monitoring of the regulation of membrane expression.
[0071] The invention therefore also relates to a process for the IN
VITRO diagnosis of pathologies correlated with an abnormal
expression of IL-13R.beta. and/or of IL-13R.alpha., in biological
samples capable of containing IL-13R.beta. and/or IL-13R.alpha.
expressed at an abnormal level, characterized in that at least one
antibody of the invention is brought into contact with the said
biological sample, under conditions allowing the possible formation
of specific immunological complexes between IL-13R.beta. and/or of
IL-13R.alpha. and the said antibody(ies) and in that the specific
immunological complexes which may be formed are detected.
[0072] The invention also relates to a kit for the IN VITRO
diagnosis of an abnormal expression of IL-13R.beta. and/or of
IL-13R.alpha. in a biological sample and/or for measuring the level
of expression of the IL-13 receptor in the said sample comprising:
[0073] at least one antibody specific for IL-13R.beta. and/or
IL-13R.alpha., optionally attached onto a support, [0074] means for
revealing the formation of specific antigen/antibody complexes
between IL-13R.beta. and/or IL-13R.alpha. and the said
antibody(ies) and/or means for quantifying these complexes.
[0075] Another subject of the invention relates to a method for the
identification and/or isolation of ligands specific for
IL-13R.beta. and/or IL-13R.alpha. or agents capable of modulating
its activity, characterized in that a compound or a mixture
containing various compounds, optionally nonidentified, is brought
into contact with cells expressing at their surface IL-13R.beta.
and/or IL-13R.alpha., under conditions allowing interaction between
the IL-13 receptor and the said compound, in the case where the
latter would have an affinity for the receptor, and in that the
compounds bound to IL-13R.beta. and/or IL-l3R.alpha., or those
capable of modulating the biological activity thereof, are detected
and/or isolated.
[0076] In a specific embodiment, this method of the invention is
adapted to the identification and/or isolation of agonists and of
antagonists of IL-13 for its IL-13R.beta. and/or IL-13R.alpha.
receptor.
[0077] The invention also comprises pharmaceutical compositions
comprising, as active ingredient, a polypeptide corresponding to
the preceding definitions, preferably in a soluble form, combined
with a pharmaceutically acceptable vehicle.
[0078] Such a polypeptide may indeed act in competition with
IL-13R.beta. and/or IL-13R.alpha. expressed at the cell surface,
and thereby constitute an antagonist specific for the binding of
IL-13 to its receptor, which may be advantageously used for the
synthesis of a medicinal product intended for modulating the
reactions mediated by IL-13 in pathological situations.
[0079] Finally, the invention comprises a method for the
therapeutic treatment of conditions linked to inmunological
reactions mediated by IL-13, comprising the administration to a
patient of IL-13R.beta. and/or IL-13R.alpha. (or of one of their
biologically active fragments), or of a compound capable of
specifically modulating the biological activity thereof, in
combination with a pharmaceutically acceptable vehicle.
[0080] Other characteristics and advantages of the invention will
emerge in the rest of the description with the examples and the
figures, of which the legends are represented below.
LEGEND TO THE FIGURES
[0081] FIG. 1: characterization of the han IL-13R.beta. receptor
present in Caki-1 cells. [0082] a) Scatchard analysis (inset) of
the saturation curve of IL-13 labelled with [.sup.125I]; [0083] b)
binding of [.sup.125I] [Phe43]-IL-13-GlyTyrGlyTyr in the presence
of increasing concentrations of unlabelled IL-13 (.) and of IL-4
(o); [0084] c) cross-linking experiments using radioactive IL-13 in
the absence (lane a) and in the presence of a one hundred times
excess of unlabelled IL-13 (lane b) or of IL-4 (lane c); [0085] d)
inhibition of the secretion of IL-6 induced by IL-13 and IL-4 in
the presence of a monoclonal antibody specific for the IL-4R chain
and the IL-4 antagonist Y124DIL-4.
[0086] FIG. 2: Nucleotide sequence of the cDNA of IL-13R.beta., and
comparison of the protein sequences of IL-5R and IL-13R.beta..
[0087] a) nucleotide sequence of the cDNA of IL-13R.beta.. The
amino acids corresponding to the deduced signal peptide of the
nucleic sequence are indicated in italics and those corresponding
to the transmembrane domain are indicated in bold characters. The
potential N-glycosylation sites (Asn-X-Ser/Thr) are underlined;
[0088] b) alignment of the amino acids of the IL-13R.beta. and
IL-5R sequences. The protein sequences of IL-13R and IL-5R are
aligned as described above (24). The cysteine residues and the
WSXWS motif which are characteristic of this family of receptors
are boxed.
[0089] FIG. 3: patterns of expression of the IL-13R.beta. mRNA.
[0090] The RNA was prepared from the following cells: Caki-1 (lane
a), A431 (lane b), TF-1 (lane c), U937 (lane d), Jurkat (line e)
and IM9 (lane f).
[0091] FIG. 4: characterization of the recombinant IL-13R.beta.
receptor for IL-13. The COS-7 cells are transfected with
IL-13R.beta. CDNA and used for: [0092] a) studies for the binding
of radiolabelled IL-13 (inset) by Scatchard analysis of the
saturation curve; [0093] b) cross-linking experiments using
radiolabelled IL-13 in the absence (lane a) and in the presence of
a one hundred times excess of unlabelled IL-13 (lane b); [0094]
c-d) cotransfection experiments using cloned IL-13R.beta., IL-4R
(gp140) and the .gamma.c chain followed by the binding of
radiolabelled IL-13 (c) or of IL-4 (d). The black and white columns
represent the specific binding of IL-13 and of IL-4
respectively.
[0095] FIG. 5: inhibition of the binding of IL-13 to IL-13R.beta.
by the soluble form of the receptor (IL-13R.beta.s) in transient
expression. [0096] The expression of IL-13R.beta.s in the
supernatant of the cells transfected with 2034 is tested by
inhibition of the binding of IL-13 on cells transfected with
IL-13R.beta. (2036). The supernatants are tested in the crude state
by diluting them one half in the iodinated ligand. [0097] 2036 NSB:
nonspecific binding in the presence of an excess of unlabelled
IL-13. [0098] 2036 BT: total binding on cells transfected with 2036
2036+sgt 2034: binding to cells transfected with 2036 in the
presence of supernatant of cells transfected with 2034. [0099]
2036+sgt pSE1: control
[0100] FIG. 6: inhibition of the binding of IL-13 to IL-13R.beta.
by the soluble form of the receptor (IL-13R.beta.s) on stable
lines. [0101] T2036-22: total binding on the clone IL-13R.beta.
(2036-22) in the absence of supernatant of clone secreting
IL-13R.beta.s (reference 100%) [0102] 2034-4 [0103] 2034-6 [0104]
2034-19 4 clones IL-13R.beta.s [0105] 2034-21 [0106] 1274-20: in
the presence of supernatant of CHO cells not expressing
IL-13R.beta.s (control).
[0107] FIG. 7: nucleotide sequence of the IL-13R.alpha. cDNA and
comparison of the protein sequences of human IL-13R.alpha. and of
murine IL-13R.alpha.. [0108] a) Nucleotide sequence of the
IL-13R.alpha. cDNA. The amino acids corresponding to the signal
peptide deduced from the nucleic sequence are underlined with a
dotted line and those corresponding to the transmembrane domain are
underlined with a double line. The potential N-glycosylation sites
(Asn-X-Ser/Thr) are boxed. [0109] b) Alignment of the amino acids
of human IL-l3R.alpha. and of murine IL-13R.alpha.. The protein
sequences of human IL-13R.alpha. and of murine IL-13R.alpha. are
aligned as described above (24). The cysteine residues and the
motif WSXWS which are characteristic of this family of receptors
are boxed.
[0110] FIG. 8: characterization of the recombinant IL-13R.alpha.
receptor for IL-13. [0111] The CHO or COS-3 cells transfected with
the IL-13R.alpha. and/or IL-4R cDNA and used for: [0112] a) studies
of the binding of iodine-125 labelled IL-13 by Scatchard analysis
of the saturation curve with CHO cells transfected with
IL-13R.beta. cDNA (Figure A), transfected with IL-13R.beta. cDNA
and IL-4R cDNA (Figure B), transfected with IL-13R.alpha. cDNA
(Figure C) and transfected with IL-13R.alpha. cDNA and IL-4R cDNA
(Figure D), [0113] b) competition experiments of binding of
[.sup.125I]-IL-13 on CHO cells transfected with IL-13R.beta. cDNA
(Figure E), transfected with IL-13R.beta. cDNA and IL-4R cDNA
(Figure F), transfected with IL-13R.alpha. cDNA (Figure G) and
transfected with IL-13R.alpha. cDNA and IL-4R cDNA (Figure H). The
white and shaded columns represent respectively the specific
binding of radiolabelled IL-13 in the presence of an excess (1,000
times more) of IL-13 or IL-4, the black columns represent total
binding.
[0114] FIG. 9: comparison of the electrophoretic mobility in EMSA
of cellular extracts expressing the receptor for IL-4 alone
(CHO-4), the receptor.for IL-13R.alpha. alone (CHO-13) or the
combined receptors IL-13R.alpha. and IL-4R (CHO-4-13) after
activation of the CHO cells in the presence of IL-4 or IL-13 (4 or
13), c representing the nonactivated control.
MATERIALS AND METHODS
Binding And Cross-Linking Experiments
[0115] The binding and cross-linking experiments are carried out as
described for [.sup.125] [Phe43]-IL-13-GlyTyrGlyTyr (17).
Induction of the Secretion of IL-6
[0116] The Caki-1 cells (ATCC HTB46) are placed in 24-well plates
at a density of 5.times.10.sup.4 cells/well and after 3 days of
culture, confluent monolayers are washed three times with DMEM
medium without foetal calf serum. The stimulation of the Caki-1
cells is carried out with 30 ng/ml of IL-4 or of IL-13 in the
absence or in the presence of Y124DIL-4 or of an anti-gp140
monoclonal antibody. The quantity of IL-6 released into the culture
medium after incubating for 24 hours is measured by an ELISA
technique (Innotest, France).
Isolation And Analysis of the Human IL-13R.beta. cDNA
[0117] Total RNA was extracted from the Caki-1 cells as described
above (25). The poly(A) RNA is isolated from the total RNAS with
magnetic beads coated with oligo(dT).sub.25 (Dynal). A cDNA library
containing 2.times.10.sup.5 clones was constructed using the
primer-adaptor procedure (26) and the vector pSE-1 (27). The
cloning strategy for the expression which was used has been
previously described (17).
Preparation of Human IL-13R.beta. cDNA
[0118] The RNA samples are copied with reverse transcriptase and
subjected to PCR (polymerase chain reaction) using a sense primer
corresponding to the sequence +52 to +71 and an antisense primer
corresponding to +489 to 470 (the numbering is made on the basis of
the cDNA sequence shown in FIG. 2). The PCR-amplified products are
hybridized with a probe complementary to sequences +445 to +461 of
the cDNA. The size markers are indicated on the left of the
figure.
Isolation And Analysis of the Human IL-13R.alpha. cDNA
[0119] 1) Preparation of the murine IL-13R.alpha. probe
[0120] a) Culture of the B9 cells (28)
[0121] The B9 cells are cultured in RPMI medium (Gibco)
supplemented with 10% foetal calf serum and 50 .mu.g/ml of
gentamycin.
[0122] b) Preparation of the RNA of the B9 cells.
[0123] The cells are washed twice with PBS buffer (phosphate
buffered saline, reference 04104040-GIBCO-BRL). After
centrifugation for 10 min at 1000 rpm, the cellular pellet is
suspended in the lysis buffer of the following composition: 4M
guanidine-thiocyanate; 25 mM sodium citrate pH 7; 0.5% sarcosyl;
0.1 M .beta.2-mercapto-ethanol.
[0124] The suspension is sonicated using an Ultraturax sonicator
No. 231256 (JANKE and KUNDEL) at the maximum power for one minute.
Sodium acetate pH 4 is added to 0.2 M. The solution is extracted
with one volume of a phenol/chloroform mixture (v/v: 5/1).
[0125] The RNA contained in the aqueous phase is precipitated at
-20.degree. C. with the aid of one volume of isopropanol. The
pellet is resuspended in the lysis buffer. The solution is again
extracted withia phenol/chloroform mixture and the RNA is
precipitated with isopropanol. After washing the pellet with 70%
and then 100% ethanol, the RNA is resuspended in water.
[0126] c) Preparation of the Complementary DNA
[0127] The cDNA is prepared from 5 .mu.g of total RNA using a poly
T12 primer. The total RNA is incubated in a volume of 30 .mu.l of
buffer: 50 mM Tris-HCl pH 8.3, 6 MM MgCl.sub.2, 10 mM DTT, 40 mM
KCl, containing 0.5 mM of each of the deoxynucleotide triphosphates
and 30 units of Rnasin (Promega), for one hour at 37.degree. C.,
and then for 10 minutes at 50.degree. C., and then for a further 10
minutes at 37.degree. C., with 200 units of the reverse
transciptaze enzyme Rnase H (Gibco-BRL reference 8064A). The
reaction is stopped by heating for 10 minutes at 65.degree. C.
[0128] d) Specific Amplification of A Mouse IL-13R.alpha. cDNA
Fragment By the PCR Technique
[0129] The polymerization is carried out with 6 .mu.l of cDNA in 50
.mu.l final volume with the buffer of the following composition: 10
mM Tris-HCl pH 8.3, 2.5 mM MgCl.sub.2, 50 mM KCl, 4 dNTP 0. 2 mM, 2
.mu.g/ml of each of the two nucleic primers and 2.5 U of TAQ DNA
polymerase (Beckman). The pairs of primers were chosen on the
sequence published by Hilton (22).
[0130] Sense primer: nucleotide 249 to 268 TABLE-US-00001 5'
AGAGGAATTACCCCTGGATG 3'
[0131] Antisense primer: nucleotide 1256 to 1275 TABLE-US-00002 5'
TCAAGGAGCTGCTTTCTTCA 3'
[0132] The reaction is carried out for 30 cycles of 1 minute at
94.degree. C., 1 minute at 58.degree. C., 4 minutes at 72.degree.
C., followed by a final cycle of 10 minutes at 72.degree. C.
[0133] e) Purification of the PCR Amplification Product
[0134] After running on a 1% agarose gel (Sigma) in TAE buffer (40
mM, Tris-HCl, 1mM EDTA pH 7.9) for 1 hour at 100 volts, the gel is
stained in the presence of 1 .mu.g/ml of ethidium bromide in the
same buffer. The band corresponding to the amplification product
(cDNA fragment of 1027 base pairs (bp) of IL-13R.alpha.) is
extracted using a Glass Max kit (Gibco).
[0135] f) Preparation of the Probe
[0136] 25 ng of the purified cDNA fragment of 1027 bp corresponding
to the mouse IL-13R.alpha. receptor are labelled with phosphorus-32
with the BRL Random Primers DNA labelling systems kit at a specific
activity of 2.4.times.10.sup.9 dpm/.mu.g; alternatively, 100 ng are
labelled by nick translation using the Boeringher kit at a specific
activity of 4.times.10.sup.8 dpm/.mu.g.
[0137] 2) Isolation And Analysis of the Human IL-13R.alpha.
cDNA
[0138] a) Preparation of the Total RNA
[0139] The total RNA was extracted from Caki-1 cells as described
above in paragraph 1b.
[0140] b) Purification of the messenger RNA (polyA+ fraction).
[0141] The purification of the polyA+ fraction of the RNA is
carried out using the DYNAL oligo (dT).sub.25 Dynabeads kit
(reference 610.05) following the procedure recommended by the
manufacturer. The principle is based on the use of
superparamagnetic polystyrene beads onto which a poly(dT).sub.25
oligonucleotide is attached. The polyA+ fraction is hybridized with
the oligo(dT).sub.25 oligonucleotide coupled to the beads which are
trapped on a magnetic support.
[0142] c) Northern Blot
[0143] 5 .mu.g of polyA+ messenger RNA are loaded on a 1% agarose,
8% formaldehyde denaturing gel in MOPS buffer (10mM pH 7.4, 0.5 mM
EDTA). After migration and transfer onto an N+ Hybond membrane
(Amersham) in a 20.times.SSC buffer, the RNA is fixed by heating in
an oven at 80.degree. C. under vacuum. The membrane is then
prehybridized for 2 hours at 42.degree. C. in the following buffer:
1 M NaCl, 30% formamide; 1% SDS, 5.times. Denhart's; 100 .mu.g/ml
of salmon sperm DNA. After 2 hours of prehybridization, the
membrane is hybridized in the same buffer with a concentration of
mouse IL-13R.alpha. probe prepared by random priming of
2.5.times.10.sup.6 dpm/ml, for 16 hours. The membrane is then
washed twice for 30 minutes in 2.times.SSC buffer 0.1% SDS at room
temperature for 2 hours at 50.degree. C. in the same buffer. After
4 days of exposure in a cassette (Molecular Dynamics), the Northern
blot is analysed with an Instant Imager (Molecular Dynamics). A
predominant transcript of 4200 bp and a doublet of 1500 bp and 2000
bp are detected in the Caki-1 cells, U373 and U937.
Characterization of the Properties of the Human IL-13R.alpha. And
IL-13R.beta.
[0144] The COS-7 or CHO cells are transfected in Petri dishes as
described above (17). 24 hours later, the cells are trypsinized and
cultured in 24-well plates at a density of 8.times.10.sup.4
cells/well. After culturing for 48 hours at 37.degree. C., the
cells are used for the binding experiments (assays carried out in
triplicate show a variation of less than 10%) with iodinated IL-13
as described (17). For the transfection, the COS-7 or CHO cells
were transfected in 25-cm.sup.2 plates using 0.6 mg of various
plasmids. After 24 hours, the cell monolayers are trypsinized and
cultured in 12-well plates at 8.times.10.sup.4 cells/well. Three
days later, the binding and competition experiments are carried out
with labelled IL-13 and with unlabelled IL-13 and/or IL-4. The
results are representative of at least three experiments conducted
independently.
Comparison of Electrophoretic Mobilities In EMSA of the Nuclear
Extracts of the Cells Expressing the Human IL-13R.alpha. and/or
IL-4R
[0145] 2.times.10.sup.6 CHO cells are plated onto 10 cm Petri
dishes. 24 hours later, the cells are transfected with 6 .mu.g of
plasmid DNA (34). After 48 hours, the cells are incubated at
37.degree. C. for 30 minutes in 3 ml of medium with or without
IL-13 or IL-4 at a concentration of 100 ng per ml. The cells are
then rinsed twice with a PBS-0.5 mM EDTA buffer and then harvested
in 1.2 ml of PBS. The cells are then centrifuged and the cellular
extracts prepared as described in (35). The EMSAs are then carried
out as described in (36) with 10 to 20 .mu.g of cellular extracts
and with an oligonucleotide probe radiolabelled with .sup.32p
(50,000-100,000 cpm), a probe corresponding to the C.epsilon.
element of the human C.epsilon. promoter (37). The oligonucleotide
probe synthesized has the following sequence: TABLE-US-00003
5'-GATCCACTTCCCAAGAACAGA-3'.
EXAMPLES
Example 1
Analysis of the Expression of Human IL-13R.beta. At the Surface of
Caki-1 Cells
[0146] It was recently discovered that human renal carcinoma cells
expressed, in addition to the receptors shared by IL-4 and IL-13, a
large excess of specific IL-13 receptors (21). On the basis of
these results, a sample of human carcinoma cell lines was studied
for the attachment of IL-13 as described above (17). A specific
line, Caki-1 (ATCC HTB46), which expresses a particularly large
number of binding sites for IL-13, was analysed in greater detail.
The Scatchard curves obtained from saturation experiments show the
presence of binding sites with a Kd of 446.+-.50 pM and a capacity
of 7.2.times.10.sup.4 receptors/cell (FIG. 1a). In competition
experiments, unlabelled IL-13 completely displaces labelled IL-13
in a dose-dependent manner, whereas IL-4 displaces with a high
affinity about 10% of the labelled IL-13. Higher concentrations of
IL-4 (greater than 100 nM) do not displace the remaining 90% of
bound IL-13 (FIG. 1b).
[0147] These results are in agreement with the existence of two
sites, one shared by the two cytokines, the other specific for
IL-13. The experiments on cross-linking by affinity for IL-13 show
a complex of about 70 kDa, which coincides with the complex
observed in similar cross-linking experiments with IL-13 in various
cell types (17, 21). Labelled IL-13 is completely displaced from
the complex by IL-13 but not by IL-4, which in in agreement with
the competition experiments (FIG. 1c).
Example 2
Analysis of the Secretion of IL-6 Induced By IL-4 Or IL-13.
[0148] The authors of the invention analysed the secretion induced
by IL-4 or IL-13 on Caki-1 cells. The two cytokines induce the
secretion of similar levels of IL-6, and the secretion is inhibited
by antibodies specific for the .alpha. chain of IL-4R and by the
antagonist Y124DIL-4 (FIG. 1d). This suggests that the receptors
shared by the two cytokines in the Caki-1 cells are responsible for
the induction of the secretion of IL-6. Similar results are
observed when the phosphorylation of the protein complex IRS1/4PS
(18) induced by IL-4 and IL-13 is analysed in the presence or in
the absence of anti-IL-4R antibodies and of IL-4 antagonist.
[0149] These results, taken as a whole, suggest that the receptor
complex IL-4/IL-13 expressed in the Caki cells is identical to that
which was previously described and that the protein binding IL-13
(IL-13R.beta. ) which is over-expressed is a component of the
receptor responsible for the recognition of IL-13 in a functional
complex which includes IL-4R. These cells were therefore used as
source of messenger RNA for the cloning of this IL-13 binding
entity.
Example 3
Cloning of the Primary Subunit of the IL-13 Receptor (IL-13R.beta.
)
[0150] The strategy for the cloning and expression which was used
has been previously described (17). A cDNA library containing
2.times.10.sup.5 recombinant clones was constructed (26) using
Caki-1 cells. The library was divided into batches of 1000 cDNAs in
which the DNA of each batch, in plasmid form, was introduced into
COS-7 cells (29). The binding of labelled IL-13 to the transfected
COS-7 cells makes it possible to identify the batches of clones
encoding an IL-13 receptor. The positive batches were distributed
out and rescreened until a single clone capable of carrying out the
synthesis of a cell surface protein capable of binding IL-13 is
identified. Two independent IL-13R.beta. cDNAs were finally
isolated. The complete nucleotide sequence of the IL-13R.beta. cDNA
and the amino acid sequence deduced therefrom are shown in FIG. 2a.
The cDNA has a length of 1298 bases excluding the poly-A tail and
has a short 3' untranslated region of 106 bases. A canonical AATAAA
polyadenylation signal is in the expected place. The open reading
frame between nucleotides 53 and 1192 defines a polypeptide of 380
amino acids. The sequence encodes a membrane protein with a
potential signal peptide, a single transmembrane domain and a short
intracytoplasmic tail.
[0151] Four potential N-glycosylation sites are located in the
extracelluar region. It is important to note that two consensus
motifs considered as signatures of the type II family of cytokine
receptors (30) are also present, the first being derived from an
N-terminal disulphide bridge loop structure, the second being the
WSXWSA type motif located at the C-terminal end of the
extracellular region. The very short cytoplasmic sequence might
explain why it is only the receptor complex shared by IL-4 and by
IL-13 in the Caki cells which transduces a signal in the cell.
[0152] Alignment studies demonstrate homologies with the human
IL-5R .alpha. chain (51% similarity and 27% identity, FIG. 2b) and,
to a lesser extent, with the prolactin receptor. It is interesting
to note that the IL-5R complex consists of an a chain which binds
IL-5 but which needs another protein, the .beta. chain shared with
the IL-3 and GM-CSF receptors, to form a high-affinity receptor
which is capable of transducing a signal (31).
Example 4
Detection of the Human IL-13R.beta. Messenger RNAs In Various Cell
Lines
[0153] Surprisingly, in the Caki-1 cells, similar quantities of
messenger RNAs for IL-13R.beta. and IL-4R are detected by Northern
analyses although a large excess of IL-13R.beta. is expressed. This
observation suggests that there is a greater translation of this
mRNA compared with the IL-4R transcript and explains the lack of
detection of the IL-13R.beta. mRNA in the cell lines expressing a
small number of IL-13 binding sites. RT-PCR analyses (FIG. 3) show
that the transcript found in the Caki-1 cells is also present at
lower levels in the keratinocytic line A431, the premyeloid cells
TF-1, the premocytic cells U937 and the cell line B IM9. No
transcript was detected in the Jurkat T cell line or in the pre-B
NALM6 cell line. These results are in agreement with the IL-13
binding studies on these same lines previously described by the
authors of the present invention (17), and with the known
biological targets of IL-13.
Example 5
Binding Analyses Carried Out On COS-7 Cells Transfected With Human
IL-13R.beta. cDNA
[0154] The COS-7 cells transfected with the isolated CDNA encoding
IL-13R.beta. specifically bind labelled IL-13. The Scatchard
analysis of the saturation curve shows a single component site with
a Kd value of 250.+-.30 pM and a maximum binding capacity of
5.6.times.10.sup.5 receptors/cell (FIG. 4a).
[0155] The affinity of the recombinant receptor is in good
agreement with the Kd value of 446 pM for IL-13R.beta. in the
Caki-1 cells and for what has been described in several other cells
(17). Consequently, in spite of a sequence homology with the a
chain of IL-5R, the cloned receptor behaves differently since it
does not need a second chain to reconstitute a high affinity
binding site.
[0156] It is interesting to note that the protein binding IL-15
recently described likewise has the characteristic of binding IL-15
with a high affinity, in the absence of the other two components of
the IL-15R complex (32).
[0157] In competition experiments, IL-13 is capable of inhibiting
the binding of labelled IL-13 to the cloned receptor, with an
inhibitory constant (Ki) of 1.5.+-.0.5 nM, whereas IL-4 does not
inhibit the binding. The pharmacology of the cloned receptor is
therefore similar to that of the IL-13R.beta. present in Caki-1
cells. Cross-linking experiments show a radiolabelled band of 70
kDa. This band has the same mobility as that observed in the Caki
cells as well as in other cells (17). This complex most probably
corresponds to the 60-70 kDa band observed in addition to the IL-4R
140 kDa band in cross-linking experiments carried out with labelled
IL-4. This could also suggest that a strong interaction exists
between the two proteins in the functional receptor complex. The
authors of the present invention therefore checked if IL-13R.beta.
and IL-4R interact in the cell membrane to reconstitute a receptor
which allows cross-competition between the two cytokines. The
results of a coexpression experiment are shown in FIGS. 4c and
d.
[0158] It appears clearly that the expression of the two receptors,
either separately or simultaneously, results in a large number of
receptors which specifically recognize either of the two cytokines.
However, when they are expressed together, a small number of
receptors (5 to 10%) is capable of recognizing the two cytokines.
The cotransfection of the .gamma.c chain with IL-4R and
IL-13R.beta. does not bring about an increase in the number of
shared binding sites. These results suggest that the IL-13R.beta.
and IL-4R chains can interact with each other in the cell membrane
to reconstitute a receptor for which IL-13 and IL-4 may be in
competition. The low percentage of reconstituted receptors is an
argument in favour of the presence of another protein
(IL-13R.alpha.) in limiting amounts in the COS cells which is
necessary for the reconstitution of the receptor complex to which
IL-13 and IL-4 bind competitively.
[0159] The results obtained in the transfection experiments with
the .gamma.c chain demonstrate that this protein is not the
limiting factor which was previously suggested (15). This
conclusion is also supported by the absence of .gamma.c messenger
RNA in the Caki-1 cells (21).
[0160] Another possible reason which explains the low number of
reconstituted receptors is the existence of an incorrect
stoichiometry of the two proteins in the cell membrane. However,
cotransfections using different relative quantities of IL-4R and
IL-13R.beta. do not show a major difference in the number of
reconstituted receptors. The possibility that another IL-13R with a
greater capacity to interact with IL-4R exists was confirmed in
mice (22) and in man by the isolation of the IL-13R.alpha. cDNA
(cf. EXAMPLE 7). It should be noted that the expression of .gamma.c
enhances the binding of IL-4 as previously described (19) but
reduces the binding of IL-13, suggesting a complex interaction
between the different chains.
Example 6
Study of the Inhibition of the Binding of IL-13 To Its Membrane
Receptor By A Receptor In Soluble Form
[0161] The results in transient expression (FIG. 5) or on stable
lines (FIG. 6) are described.
[0162] The two cDNA sequences encoding IL-13R.beta. and
IL-13R.beta.s are inserted into the vector p7055 in place of the
IL-2 cDNA (33). The resulting plasmids are called 2036 and 2034
respectively.
[0163] a) Transient Expression
[0164] The CEO cells are inoculated into 12-well plates at
3.times.10.sup.5 cells/well and transfected the next day by the
DEAE-Dextran method as for the COS cells, either with the plasmid
2036 or 2034, or with the empty plasmid pSE-1 as control.
[0165] The cells are cultured for three days so as to allow
accumulation of IL-13R.beta.s in the supernatant of the cells
transfected with the plasmid 2034 and good expression of
IL-13R.beta. in the membrane of the cells transfected with the
plasmid 2036.
[0166] The supernatant of the cells transfected with IL-13R.beta.s
(2034) or the negative control (empty pSE-1) is then collected and
the cells transfected with IL-13R.beta. are used to study the
inhibition of the binding of IL-13.
[0167] The binding of IL-13 to the surface of the CHO cells
expressing IL-13R.beta. (2036) is measured in the presence or
otherwise of these crude supernatants diluted one half with the
radioligand or in the presence of an excess of nonradiolabelled
IL-13 (NSB). The binding is carried out on whole cells in a final
volume of 500 ml with 300 pM of radioligand, in triplicate.
[0168] b) Stable Lines
[0169] Two stable transformed CHO lines are obtained by
transfection with the coding sequences of the complete IL-13R.beta.
(polypeptide of 380 residues) or of the IL-13R.beta. in soluble
form (IL-13R.beta.s, truncated polypeptide corresponding to
residues 1 to 337 of IL-13R.beta.). These sequences are inserted
into the vector p7055.
[0170] The CHO-DHFR.sup.- cells are transfected with the plasmids
2036 (IL-13R.beta. ) and 2034 (IL-13R.beta.s) and the recombinant
clones selected as previously described (33).
[0171] One of the clones CHO-IL-13R.beta. (CHO 2036) obtained,
having 2 to 5.times.10.sup.5 sites per cell, is inoculated into a
12-well plate at a density of 10.sup.5 cells per well and the cells
are used two days later for binding experiments in the presence or
otherwise of IL-13R.beta. s.
[0172] For that, the CHO-IL-13R.beta.s (CHO 2034) clones are
inoculated into 6 cm dishes, in triplicate, at 5.times.10.sup.5
cells per dish. After 3 days of accumulation in the culture medium,
the medium (5 ml per dish) is collected for the IL-13 binding
inhibition studies on IL-13R.beta. of the CHO 2036 clone. In the
same manner, the supernatant of CHO cells not expressing the
soluble IL-13R.beta. is collected.
[0173] The binding of IL-13 at the surface of the CHO 2036-22 clone
is measured in the presence or otherwise of these crude
supernatants diluted one half with the radioligand, or in the
presence of an excess of nonradio-labelled IL-13 (NSB). The binding
is carried out in triplicate, on whole cells, in a volume of 500 ml
with 300 pM of radioligand.
[0174] The histograms of FIGS. 5 and 6 represent the inhibition of
the binding of IL-13 on IL-13R.beta. by IL-13R.beta.s. Inhibition
of the binding of IL-13 to its receptor can be observed on several
clones.
Example 7
Cloning of the Human IL-13R.alpha. Receptor
[0175] a) Preparation of the cDNA Library From PolyA+ Messenger
RNAs of Caki-1 Cells
[0176] Starting with 0.5 .mu.g of polyA+ messenger RNA,
single-stranded complementary DNA labelled with [.sup.32P]dCTP (the
complementary DNA obtained has a specific activity of 3000 dpm/ng)
is prepared with the synthetic primer having the following sequence
(comprising a BamHI site): TABLE-US-00004 5'
<GATCCGGGCCCTTTTTTTTTTTT <3' (SEQ ID NO.10)
[0177] in a volume of 30 .mu.l of the following buffer:. 50 mM
Tris-HCl pH 8.3, 6mM MgCl.sub.2, 10 mM DTT, 40 mM KCl, containing
0.5 mM of each of the deoxynucleic tri-phosphates, 30 .mu.Ci of
[.alpha..sup.32P]dCTP and 30 U of Rnasin (Promega). After
incubating for 1 hour at 37.degree. C., and then for 10 minutes at
50.degree. C. and then for a further 10 minutes at 37.degree. C.,
with 200 units of the reverse transcriptase enzyme Rnase H
(Gibco-BRL), 4 .mu.l of EDTA are added. The RNA template is then
degraded by adding 6 .mu.l of a 2 N NaOH solution and incubating
for 5 minutes at 65.degree. C.
[0178] To remove the synthetic primer, the complementary DNA is
purified on a 1 ml Sephacryl S400 column (Pharmacia), equilibrated
in TE buffer. The first two radioactive fractions are combined and
precipitated with a 1/10 volume of a 10 M ammonium acetate solution
and 2.5 volumes of ethanol, this after extraction with chloroform.
The CDNA is then extended in 5' by adding a dG homopolymeric tail
with 20 units of terminal transferase enzyme (Pharmacia 27073001).
Next, incubation is performed in 20 .mu.l of buffer having the
following composition: 30 mM Tris-HCl pH 7.6: 1 mM cobalt chloride;
140 mM cacodylic acid; 0.1 mM DTT; 1 mM dGTP, for 15 minutes at
37.degree. C., and then 2 .mu.l of 0.5 M EDTA are added. A further
treatment with sodium hydroxide is carried out without heating,
followed by repurification on an S400 column, extraction with
chloroform and precipitation with ethanol. The pellet is dissolved
in 33 .mu.l of TE buffer. The next stage consists in pairing the
cloning vector pT7T3-18 through which a homopolymeric dC tail has
been added beforehand after cutting with Pst1, the cDNA and the
adaptor. The CDNA (33 .mu.l) is brought into contact with 75 ng of
vector pT7/T3-18 (5 .mu.l), 120 ng of adaptor (1 .mu.l) of the
following sequence (comprising an Apa1 site), TABLE-US-00005
5'AAAAAAAAAAAAAGGGCCCG 3'
10 .mu.l of a 200 mM NaCl solution, and the mixture is incubated
for 5 minutes at 65.degree. C. and then the reaction mixture is
allowed to cool to room temperature. The next stage consists in
ligating the cloning vector and the single-stranded cDNA in a
reaction volume of 100 .mu.l with 32.5 units of the enzyme T4 phage
DNA ligase (Pharmacia) overnight at 15.degree. C. in a buffer
having the composition: 50 mM Tris-HCl pH 7.5; 10 mM MgCl.sub.2, 1
mM ATP. The proteins are then removed by extraction with phenol
followed by extraction with chloroform and then a 1/10 volume of a
10 mM ammonium acetate solution and 2.5 volumes of ethanol are
added. The mixture is centrifuged, the pellet is taken up in the
buffer having the composition: 33 mM Tris-acetate pH 7.9, 62.5 mM
potassium acetate, 1 mM magnesium acetate and 1 mM DTT; the second
cDNA strand is synthesized in a volume of 30 .mu.l with 30 units of
the enzyme T4 phage DNA polymerase (Pharmacia) and a mixture of 1
mM of the four deoxynucleotide triphosphates as well as two units
of the protein of the T4 phage gene 32 (Pharmacia) for one hour at
37.degree. C. The mixture is extracted with phenol and traces are
removed by depositing on a P10 column (Biogel P10-200-400
mesh--reference 15011050--Biorad).
[0179] The last stage consists in transforming E. Coli MC 1061
cells by electroporation of the recombinant DNA using a Biorad Gene
Pulser apparatus used at 2.5 kV under the conditions recommended by
the manufacturer, and then the bacteria are cultured for one hour
in LB medium having the composition: bactotryptone 10 g/l; yeast
extract 5 g/l; NaCl 10 g/l.
[0180] The number of independent clones obtained is determined by
plating a 1/1000 dilution from the transformation after a one hour
incubation on a dish of LB medium supplemented with 1.5% agar (w/v)
and with 100 .mu.g/ml of ampicillin called, in what follows, LB
agar medium.
[0181] The number of independent clones obtained is 1 million.
[0182] b) Screening of the cDNA Library
[0183] The entire library was plated on agar medium (Petri dishes
150 mm in diameter) coated with Biodyne A membranes (PALL reference
BNNG 132). After leaving overnight at 37.degree. C., the clones are
transferred by contact onto new membranes. The latter are treated
by placing them on Wathman 3 MM paper impregnated with the
following solutions: 0.5 N NaOH, 1.5 M NaCl for 5 minutes and then
0.5 M Tris-HCl pH 8, 1.5 M NaCl for 5 minutes. After treatment with
proteinase K in the following buffer, 10 mM Tris-HCl pH8, 10 mM
EDTA, 50 mM NaCl, 0.1% SDS, 100 .mu.g/ml proteinase K for 30
minutes at 37.degree. C., the membranes are thoroughly washed in
2.times.SSC buffer (sodium citrate-NaCl), and then dried in an oven
under vacuum at 80.degree. C. for 20 minutes.
[0184] c) Prehybridization And Hybridization of the Membranes
[0185] The membranes are then prehybridized for 2 hours at
42.degree. C. in the following buffer: 1 M NaCl; 30% formamide; 1%
SDS; 5.times. Denhart's 100 .mu.g/ml of salmon sperm DNA. After 2
hours of prehybridization, the membranes are hybridized in the same
buffer with a concentration of mouse IL-13R.alpha. probe prepared
by nick translation of 2.5.times.10.sup.6dpm/ml, for 16 hours. The
membranes are washed for twice 30 minutes in 2.times.SSC, 0.1% SDS
buffer at room temperature and then 2 hours at 50.degree. C. in the
same buffer. After overnight exposure at -80.degree. C. in the
presence of a Kodak X-OMAT film, several positive clones are
detected.
[0186] d) Sequencing of A Human IL-13R.alpha. Clone And Analysis of
the Sequence
[0187] The sequence is obtained using the Applied Biosystem kit
(reference 401628). The complete nucleic sequence of the
IL-13R.alpha. cDNA and the amino acid sequence deduced therefrom
are shown in FIG. 7. The cDNA is 3999 bases long excluding the
poly-A tail and has a long untranslated 3' region of 2145
bases.
[0188] A canonical polyadenylation signal exists at the expected
place. The open reading frame between nucleotides 34 and 1851
defines a polypeptide of 427 amino acids. The sequence encodes a
membrane protein with a potential signal peptide and a single
transmembrane domain and a short intracytoplasmic region.
[0189] 10 potential glycosylation sites are located in the
extracelluar region. It is important to note that two consensus
motifs considered as signatures of the type II family of cytokine
receptors are also present, the first being derived from an
N-terminal disulphide bridge loop structure, the second being the
WSXWS type motif located at the C-terminal end of the extracelluar
region.
Example 8
Binding Analyses carried Out On COS-3 Or CHO Cells Transfected With
Human IL-13R.alpha. cDNA
[0190] The CHO cells transfected with the isolated cDNA encoding
IL-13R.alpha. specifically bind labelled IL-13. The Scatchard
analysis of the saturation curve shows a single component site with
a Kd value of 4.5.+-.0.4nM and a maximum binding capacity of 26000
receptors/cell (FIGS. 8C and 8G).
[0191] The results of coexpression experiments are shown in FIGS.
8D and 8H.
[0192] Analysis of the results of FIG. 8C shows that IL-13R.alpha.
is well expressed in the clone 2036 of the CHO cells. It can be
noted that IL-4R displaces 60% of the binding of IL-13 in the CHO
cells cotransfected with IL-4R and IL-13R.alpha. cDNA (FIG. 8H) but
taking into account a Kd of 7.5 nM for IL-13R.alpha., there would
be 10 times as many IL-13R.alpha. sites as IL-4R sites.
[0193] The CHO-hIL4R cells (human IL-4R) expressing hIL-4R which
are transfected with the cDNA encoding hIL-13R.alpha. specifically
bind labelled IL-13.
[0194] The Scatchard analysis of the saturation curve shows clearly
2 component sites, one of high affinity with a Kd value of
23.+-.8.9 pM and a maximum binding capacity of 28000 sites/cell and
the other of low affinity with a Kd value of 4.2.+-.1.4 nM and a
maximum binding capacity of 150000 sites/cell (FIG. 8D).
[0195] The second site characterized has the same affinity as
hIL-13R.alpha. (human IL-13R.alpha.) expressed alone and
corresponds to the nonassociated IL-13R.alpha. chains because they
are expressed in a larger quantity than hIL-4R.
[0196] These high-affinity receptors reconstituted in the presence
of the 2 hIL-13R.alpha. and hIL-4R chains are capable of
recognizing the 2 cytokines (FIGS. 8D and 8H). This is even clearer
on the COS/pSE1 cells coexpressing the 2 hIL-13R.alpha. and hIL-4R
chains in a comparable quantity where IL-4 displaces all the
binding IL-13.
[0197] The affinity of the recombinant human IL-13R.alpha. is
comparable to that described for the mouse IL-13R.alpha. receptor
(2-10nM) (ref. 22).
[0198] In contrast to the hIL-13R.beta. chain previously described,
human IL-l3R.alpha. does not constitute, on its own, a
high-affinity binding site.
[0199] IL-13R.alpha. and IL-4R therefore interact in the cell
membrane to reconstitute a high-affinity receptor.
Example 9
Activation of the STAT Proteins By IL-13 and IL-4 In the CRO Cells
Coexpressing hIL-13R.alpha. And hIL-4R
[0200] In human PBMC cells, hIL-4 and IL-13 activate 2 tyrosine
kinases of the janus family, Jak1 and Jak2 which phosphorylate a
latent transcription factor, STAT6. This activated factor enters
the nucleus and binds to specific elements in the promoters of the
genes regulated by IL-4.
[0201] We chose the C.epsilon. element of the human C.epsilon.
promoter as probe in an electrophoretic mobility switch assay
(EMSA) to demonstrate the activation by IL-13 of a binding factor
similar to STAT6.
[0202] The nuclear extracts of the CHO cells, expressing IL-13R
alone, IL-4R alone, or the 2 chains together, stimulated with
100ng/ml of IL-13 or IL-4 for 30 min at 37.degree. C., are
incubated with the radiolabelled C.epsilon. element.
[0203] The nuclear extracts of the cells coexpressing
hIL-13R.alpha. and hIL-4R form a complex having the same mobility
in EMSA whether the cells are induced with IL-4 or IL-13 (cf. FIG.
9). On the other hand, with the cells expressing either chain
alone, no complex is detected.
[0204] In the CHO cells expressing hIL-13R.alpha. and
hIL-4R.alpha., IL-13 and IL-4 therefore initiate the same
signalling cascade.
[0205] The cloning of IL-13R.beta. and IL-13R.alpha. described here
makes it possible to improve the knowledge of the factors involved
in the responses specifically induced by IL-13 compared with the
responses induced by IL-4. It makes it possible, in addition, to
have a tool for studying the regulation of the expression of the
receptor under normal and pathological conditions where IL-13 plays
a key role.
[0206] Moreover, the availability of cDNA makes it possible to
facilitate the cloning of other proteins necessary for the
reconstitution of an I1-4/IL-13 receptor complex and is also useful
for the manufacture or the rational modelling of new medicinal
products capable of being specific antagonists of the activities of
IL-13.
REFERENCES
[0207] 1. Minty, A. et al., Nature, 1993, 362, 248-250. [0208] 2.
McKenzie, A. N. et al., Proc. Natl. Acad. Sci. U.S.A, 1993, 90,
3735-3739. [0209] 3. Defrance, T. et al., J. Exp. Med., 1994, 179,
135-143. [0210] 4. Punnonen, J. et al., Proc. Natl. Acad. Sci.
(USA), 1993, 90, 3730-3734. [0211] 5. Fior, R. et al., Eur.
Cytokine Network, 1994, 5, 593-600. [0212] 6. Muzio, M. R. F. et
al., Blood, 1994, 83, 1738-1743. [0213] 7. De Waal Malefyt, R. et
al., J. Immunol, 1993, 151, 6370-6381. [0214] 8. Doyle, A. et al.,
Eur. J. Immunol. 1994, 24, 1441-1445. [0215] 9. Montaner, L. J. et
al., J. Exp. Med., 1993, 178, 743-747. [0216] 10. Sozzani, P. et
al., J. Biol. Chem., 1995, 270, 5084-5088. [0217] 11. Herbert, J.
M. et al., Febs Lett., 1993, 328, 268-270. [0218] 12. Derocq, J. N.
et al., Febs Lett. 1994, 343, 32-36. [0219] 13. Zurawski, G. et
al., Immunol. Today, 1994, 15, 19-26. [0220] 14. Interleukin-13 for
Cytokines in Health and Disease. Eds D. G. Remick and J. S. Frie,
Marcel Decker, New York 1996. [0221] 15. Zurawaki S. M. et al.,
Embo Journal, 1993, 12, 2663-2670. [0222] 16. Aversa, G. et al., J.
Exp. Med., 1993, 178, 2213-2218. [0223] 17. Vita, N. et al., Biol.
Chem., 1995, 270, 3512-3517. [0224] 18. Lefort, S. et al., Febs
Lett., 1995, 366, 122-126. [0225] 19. Kondo, M. et al., Science,
1993, 262, 1874-1883. [0226] 20. Russell, S. M. et al., Science,
1993, 262, 1880-1883. [0227] 21. Obiri, N. et al., J. Biol. Chem.,
1995, 270, 8797-8804. [0228] 22. Hilton, D. J. et al., Proc. Natl.
Acad. Sci. USA, 1996, 93, 497-501. [0229] 23. Callard, R. E. et
al., Immunology Today, 1996, 17, 3 108-110. [0230] 24. Devereux, J.
et al., Nucleic Acids Res., 1984, 12, 387-395. [0231] 25.
Chomczynaki, P. et al., N. Anal. Biochem., 1987, 162, 156-159.
[0232] 26. Caput, D. et al., Proc. Natl. Acad. Sci. USA, 1986, 83,
1670-1674. [0233] 27. Minty, A. et al., Eur. Cytokine Network,
1993, 4, 99-110 [0234] 28. Labit Le Bouteiller, C. et al., J. of
Immunol. Methods, 1995, 181, 1, 29-36. [0235] 29. Seed, B. et al.,
Proc. Natl. Acad. Sci. USA, 1987, 84, 3365-3369. [0236] 30. Bazan,
J. F. et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 6934-6938.
[0237] 31. Honjo, T. et al., Current Opinion in Cell Biology, 1991,
1, 201-203. [0238] 32. Giri, J. G. et al., Embo Journal, 1993, 14,
3654-3663. [0239] 33. Miloux, B. et al., Gene, 1994, 149, 341-344.
[0240] 34. Sampayrac, L. M. et al., PNAS USA, 1981, 78, 7575-7578.
[0241] 35. Jiang, S-W et al., Nucleic Acid Res., 1995, 23,
3607-3608. [0242] 36. Kohler, I. et al., FEBS Letters, 1994, 345,
187-192. [0243] 37. Seidel, H. M. et al., PNAS USA, 1995, 92,
3041-3045.
Sequence CWU 1
1
15 1 1298 DNA homo sapiens 1 ggtgcctgtc ggcggggaga gaggcaatat
caaggtttta aatctcggag aaatggcttt 60 cgtttgcttg gctatcggat
gcttatatac ctttctgata agcacaacat ttggctgtac 120 ttcatcttca
gacaccgaga taaaagttaa ccctcctcag gattttgaga tagtggatcc 180
cggatactta ggttatctct atttgcaatg gcaaccccca ctgtctctgg atcattttaa
240 ggaatgcaca gtggaatatg aactaaaata ccgaaacatt ggtagtgaaa
catggaagac 300 catcattact aagaatctac attacaaaga tgggtttgat
cttaacaagg gcattgaagc 360 gaagatacac acgcttttac catggcaatg
cacaaatgga tcagaagttc aaagttcctg 420 ggcagaaact acttattgga
tatcaccaca aggaattcca gaaactaaag ttcaggatat 480 ggattgcgta
tattacaatt ggcaatattt actctgttct tggaaacctg gcataggtgt 540
acttcttgat accaattaca acttgtttta ctggtatgag ggcttggatc atgcattaca
600 gtgtgttgat tacatcaagg ctgatggaca aaatatagga tgcagatttc
cctatttgga 660 ggcatcagac tataaagatt tctatatttg tgttaatgga
tcatcagaga acaagcctat 720 cagatccagt tatttcactt ttcagcttca
aaatatagtt aaacctttgc cgccagtcta 780 tcttactttt actcgggaga
gttcatgtga aattaagctg aaatggagca tacctttggg 840 acctattcca
gcaaggtgtt ttgattatga aattgagatc agagaagatg atactacctt 900
ggtgactgct acagttgaaa atgaaacata caccttgaaa acaacaaatg aaacccgaca
960 attatgcttt gtagtaagaa gcaaagtgaa tatttattgc tcagatgacg
gaatttggag 1020 tgagtggagt gataaacaat gctgggaagg tgaagaccta
tcgaagaaaa ctttgctacg 1080 tttctggcta ccatttggtt tcatcttaat
attagttata tttgtaaccg gtctgctttt 1140 gcgtaagcca aacacctacc
caaaaatgat tccagaattt ttctgtgata catgaagact 1200 ttccatatca
agagacatgg tattgactca acagtttcca gtcatggcca aatgttcaat 1260
atgagtctca ataaactgaa tttttcttgc gaatgttg 1298 2 380 PRT Homo
sapiens 2 Met Ala Phe Val Cys Leu Ala Ile Gly Cys Leu Tyr Thr Phe
Leu Ile 1 5 10 15 Ser Thr Thr Phe Gly Cys Thr Ser Ser Ser Asp Thr
Glu Ile Lys Val 20 25 30 Asn Pro Pro Gln Asp Phe Glu Ile Val Asp
Pro Gly Tyr Leu Gly Tyr 35 40 45 Leu Tyr Leu Gln Trp Gln Pro Pro
Leu Ser Leu Asp His Phe Lys Glu 50 55 60 Cys Thr Val Glu Tyr Glu
Leu Lys Tyr Arg Asn Ile Gly Ser Glu Thr 65 70 75 80 Trp Lys Thr Ile
Ile Thr Lys Asn Leu His Tyr Lys Asp Gly Phe Asp 85 90 95 Leu Asn
Lys Gly Ile Glu Ala Lys Ile His Thr Leu Leu Pro Trp Gln 100 105 110
Cys Thr Asn Gly Ser Glu Val Gln Ser Ser Trp Ala Glu Thr Thr Tyr 115
120 125 Trp Ile Ser Pro Gln Gly Ile Pro Glu Thr Lys Val Gln Asp Met
Asp 130 135 140 Cys Val Tyr Tyr Asn Trp Gln Tyr Leu Leu Cys Ser Trp
Lys Pro Gly 145 150 155 160 Ile Gly Val Leu Leu Asp Thr Asn Tyr Asn
Leu Phe Tyr Trp Tyr Glu 165 170 175 Gly Leu Asp His Ala Leu Gln Cys
Val Asp Tyr Ile Lys Ala Asp Gly 180 185 190 Gln Asn Ile Gly Cys Arg
Phe Pro Tyr Leu Glu Ala Ser Asp Tyr Lys 195 200 205 Asp Phe Tyr Ile
Cys Val Asn Gly Ser Ser Glu Asn Lys Pro Ile Arg 210 215 220 Ser Ser
Tyr Phe Thr Phe Gln Leu Gln Asn Ile Val Lys Pro Leu Pro 225 230 235
240 Pro Val Tyr Leu Thr Phe Thr Arg Glu Ser Ser Cys Glu Ile Lys Leu
245 250 255 Lys Trp Ser Ile Pro Leu Gly Pro Ile Pro Ala Arg Cys Phe
Asp Tyr 260 265 270 Glu Ile Glu Ile Arg Glu Asp Asp Thr Thr Leu Val
Thr Ala Thr Val 275 280 285 Glu Asn Glu Thr Tyr Thr Leu Lys Thr Thr
Asn Glu Thr Arg Gln Leu 290 295 300 Cys Phe Val Val Arg Ser Lys Val
Asn Ile Tyr Cys Ser Asp Asp Gly 305 310 315 320 Ile Trp Ser Glu Trp
Ser Asp Lys Gln Cys Trp Glu Gly Glu Asp Leu 325 330 335 Ser Lys Lys
Thr Leu Leu Arg Phe Trp Leu Pro Phe Gly Phe Ile Leu 340 345 350 Ile
Leu Val Ile Phe Val Thr Gly Leu Leu Leu Arg Lys Pro Asn Thr 355 360
365 Tyr Pro Lys Met Ile Pro Glu Phe Phe Cys Asp Thr 370 375 380 3
4009 DNA Homo sapiens 3 tcagcccggc cgggctccga ggcgagaggc tgcatggagt
ggccggcgcg gctctgcggg 60 ctgtgggcgc tgctgctctg cgccggcggc
gggggcgggg gcgggggcgc cgcgcctacg 120 gaaactcagc cacctgtgac
aaatttgagt gtctctgttg aaaacctctg cacagtaata 180 tggacatgga
atccacccga gggagccagc tcaaattgta gtctatggta ttttagtcat 240
tttggcgaca aacaagataa gaaaatagct ccggaaactc gtcgttcaat agaagtaccc
300 ctgaatgaga ggatttgtct gcaagtgggg tcccagtgta gcaccaatga
gagtgagaag 360 cctagcattt tggttgaaaa atgcatctca cccccagaag
gtgatcctga gtctgctgtg 420 actgagcttc aatgcatttg gcacaacctg
agctacatga agtgttcttg gctccctgga 480 aggaatacca gtcccgacac
taactatact ctctactatt ggcacagaag cctggaaaaa 540 attcatcaat
gtgaaaacat ctttagagaa ggccaatact ttggttgttc ctttgatctg 600
accaaagtga aggattccag ttttgaacaa cacagtgtcc aaataatggt caaggataat
660 gcaggaaaaa ttaaaccatc cttcaatata gtgcctttaa cttcccgtgt
gaaacctgat 720 cctccacata ttaaaaacct ctccttccac aatgatgacc
tatatgtgca atgggagaat 780 ccacagaatt ttattagcag atgcctattt
tatgaagtag aagtcaataa cagccaaact 840 gagacacata atgttttcta
cgtccaagag gctaaatgtg agaatccaga atttgagaga 900 aatgtggaga
atacatcttg tttcatggtc cctggtgttc ttcctgatac tttgaacaca 960
gtcagaataa gagtcaaaac aaataagtta tgctatgagg atgacaaact ctggagtaat
1020 tggagccaag aaatgagtat aggtaagaag cgcaattcca cactctacat
aaccatgtta 1080 ctcattgttc cagtcatcgt cgcaggtgca atcatagtac
tcctgcttta cctaaaaagg 1140 ctcaagatta ttatattccc tccaattcct
gatcctggca agatttttaa agaaatgttt 1200 ggagaccaga atgatgatac
tctgcactgg aagaagtacg acatctatga gaagcaaacc 1260 aaggaggaaa
ccgactctgt agtgctgata gaaaacctga agaaagcctc tcagtgatgg 1320
agataattta tttttacctt cactgtgacc ttgagaagat tcttcccatt ctccatttgt
1380 tatctgggaa cttattaaat ggaaactgaa actactgcac catttaaaaa
caggcagctc 1440 ataagagcca caggtcttta tgttgagtcg cgcaccgaaa
aactaaaaat aatgggcgct 1500 ttggagaaga gtgtggagtc attctcattg
aattataaaa gccagcaggc ttcaaactag 1560 gggacaaagc aaaaagtgat
gatagtggtg gagttaatct tatcaagagt tgtgacaact 1620 tcctgaggga
tctatacttg ctttgtgttc tttgtgtcaa catgaacaaa ttttatttgt 1680
aggggaactc atttggggtg caaatgctaa tgtcaaactt gagtcacaaa gaacatgtag
1740 aaaacaaaat ggataaaatc tgatatgtat tgtttgggat cctattgaac
catgtttgtg 1800 gctattaaaa ctcttttaac agtctgggct gggtccggtg
gctcacgcct gtaatcccag 1860 caatttggga gtccgaggcg ggcggatcac
tcgaggtcag gagttccaga ccagcctgac 1920 caaaatggtg aaacctcctc
tctactaaaa ctacaaaaat taactgggtg tggtggcgcg 1980 tgcctgtaat
cccagctact cgggaagctg aggcaggtga attgtttgaa cctgggaggt 2040
ggaggttgca gtgagcagag atcacaccac tgcactctag cctgggtgac agagcaagac
2100 tctgtctaaa aaacaaaaca aaacaaaaca aaacaaaaaa acctcttaat
attctggagt 2160 catcattccc ttcgacagca ttttcctctg ctttgaaagc
cccagaaatc agtgttggcc 2220 atgatgacaa ctacagaaaa accagaggca
gcttctttgc caagaccttt caaagccatt 2280 ttaggctgtt aggggcagtg
gaggtagaat gactccttgg gtattagagt ttcaaccatg 2340 aagtctctaa
caatgtattt tcttcacctc tgctactcaa gtagcattta ctgtgtcttt 2400
ggtttgtgct aggcccccgg gtgtgaagca cagacccctt ccaggggttt acagtctatt
2460 tgagactcct cagttcttgc cacttttttt tttaatctcc accagtcatt
tttcagacct 2520 tttaactcct caattccaac actgatttcc ccttttgcat
tctccctcct tcccttcctt 2580 gtagcctttt gactttcatt ggaaattagg
atgtaaatct gctcaggaga cctggaggag 2640 cagaggataa ttagcatctc
aggttaagtg tgagtaatct gagaaacaat gactaattct 2700 tgcatatttt
gtaacttcca tgtgagggtt ttcagcattg atatttgtgc attttctaaa 2760
cagagatgag gtggtatctt cacgtagaac attggtattc gcttgagaaa aaaagaatag
2820 ttgaacctat ttctctttct ttacaagatg ggtccaggat tcctcttttc
tctgccataa 2880 atgattaatt aaatagcttt tgtgtcttac attggtagcc
agccagccaa ggctctgttt 2940 atgcttttgg ggggcatata ttgggttcca
ttctcaccta tccacacaac atatccgtat 3000 atatcccctc tactcttact
tcccccaaat ttaaagaagt atgggaaatg agaggcattt 3060 cccccacccc
atttctctcc tcacacacag actcatatta ctggtaggaa cttgagaact 3120
ttatttccaa gttgttcaaa catttaccaa tcatattaat acaatgatgc tatttgcaat
3180 tcctgctcct aggggagggg agataagaaa ccctcactct ctacaggttt
gggtacaagt 3240 ggcaacctgc ttccatggcc gtgtagaagc atggtgccct
ggcttctctg aggaagctgg 3300 ggttcatgac aatggcagat gtaaagttat
tcttgaagtc agattgaggc tgggagacag 3360 ccgtagtaga tgttctactt
tgttctgctg ttctctagaa agaatatttg gttttcctgt 3420 ataggaatga
gattaattcc tttccaggta ttttataatt ctgggaagca aaacccatgc 3480
ctccccctag ccatttttac tgttatccta tttagatggc catgaagagg atgctgtgaa
3540 attcccaaca aacattgatg ctgacagtca tgcagtctgg gagtggggaa
gtgatctttt 3600 gttcccatcc tcttctttta gcagtaaaat agctgaggga
aaagggaggg aaaaggaagt 3660 tatgggaata cctgtggtgg ttgtgatccc
taggtcttgg gagctcttgg aggtgtctgt 3720 atcagtggat ttcccatccc
ctgtgggaaa ttagtaggct catttactgt tttaggtcta 3780 gcctatgtgg
attttttcct aacataccta agcaaaccca gtgtcaggat ggtaattctt 3840
attctttcgt tcagttaagt ttttcccttc atctgggcac tgaagggata tgtgaaacaa
3900 tgttaacatt tttggtagtc ttcaaccagg gattgtttct gtttaacttc
ttataggaaa 3960 gcttgagtaa aataaatatt gtctttttgt atgtcaccca
aaaaaaaaa 4009 4 427 PRT Homo sapiens 4 Met Glu Trp Pro Ala Arg Leu
Cys Gly Leu Trp Ala Leu Leu Leu Cys 1 5 10 15 Ala Gly Gly Gly Gly
Gly Gly Gly Gly Ala Ala Pro Thr Glu Thr Gln 20 25 30 Pro Pro Val
Thr Asn Leu Ser Val Ser Val Glu Asn Leu Cys Thr Val 35 40 45 Ile
Trp Thr Trp Asn Pro Pro Glu Gly Ala Ser Ser Asn Cys Ser Leu 50 55
60 Trp Tyr Phe Ser His Phe Gly Asp Lys Gln Asp Lys Lys Ile Ala Pro
65 70 75 80 Glu Thr Arg Arg Ser Ile Glu Val Pro Leu Asn Glu Arg Ile
Cys Leu 85 90 95 Gln Val Gly Ser Gln Cys Ser Thr Asn Glu Ser Glu
Lys Pro Ser Ile 100 105 110 Leu Val Glu Lys Cys Ile Ser Pro Pro Glu
Gly Asp Pro Glu Ser Ala 115 120 125 Val Thr Glu Leu Gln Cys Ile Trp
His Asn Leu Ser Tyr Met Lys Cys 130 135 140 Ser Trp Leu Pro Gly Arg
Asn Thr Ser Pro Asp Thr Asn Tyr Thr Leu 145 150 155 160 Tyr Tyr Trp
His Arg Ser Leu Glu Lys Ile His Gln Cys Glu Asn Ile 165 170 175 Phe
Arg Glu Gly Gln Tyr Phe Gly Cys Ser Phe Asp Leu Thr Lys Val 180 185
190 Lys Asp Ser Ser Phe Glu Gln His Ser Val Gln Ile Met Val Lys Asp
195 200 205 Asn Ala Gly Lys Ile Lys Pro Ser Phe Asn Ile Val Pro Leu
Thr Ser 210 215 220 Arg Val Lys Pro Asp Pro Pro His Ile Lys Asn Leu
Ser Phe His Asn 225 230 235 240 Asp Asp Leu Tyr Val Gln Trp Glu Asn
Pro Gln Asn Phe Ile Ser Arg 245 250 255 Cys Leu Phe Tyr Glu Val Glu
Val Asn Asn Ser Gln Thr Glu Thr His 260 265 270 Asn Val Phe Tyr Val
Gln Glu Ala Lys Cys Glu Asn Pro Glu Phe Glu 275 280 285 Arg Asn Val
Glu Asn Thr Ser Cys Phe Met Val Pro Gly Val Leu Pro 290 295 300 Asp
Thr Leu Asn Thr Val Arg Ile Arg Val Lys Thr Asn Lys Leu Cys 305 310
315 320 Tyr Glu Asp Asp Lys Leu Trp Ser Asn Trp Ser Gln Glu Met Ser
Ile 325 330 335 Gly Lys Lys Arg Asn Ser Thr Leu Tyr Ile Thr Met Leu
Leu Ile Val 340 345 350 Pro Val Ile Val Ala Gly Ala Ile Ile Val Leu
Leu Leu Tyr Leu Lys 355 360 365 Arg Leu Lys Ile Ile Ile Phe Pro Pro
Ile Pro Asp Pro Gly Lys Ile 370 375 380 Phe Lys Glu Met Phe Gly Asp
Gln Asn Asp Asp Thr Leu His Trp Lys 385 390 395 400 Lys Tyr Asp Ile
Tyr Glu Lys Gln Thr Lys Glu Glu Thr Asp Ser Val 405 410 415 Val Leu
Ile Glu Asn Leu Lys Lys Ala Ser Gln 420 425 5 420 PRT Homo sapiens
5 Met Ile Ile Val Ala His Val Leu Leu Ile Leu Leu Gly Ala Thr Glu 1
5 10 15 Ile Leu Gln Ala Asp Leu Leu Pro Asp Glu Lys Ile Ser Leu Leu
Pro 20 25 30 Pro Val Asn Phe Thr Ile Lys Val Thr Gly Leu Ala Gln
Val Leu Leu 35 40 45 Gln Trp Lys Pro Asn Pro Asp Gln Glu Gln Arg
Asn Val Asn Leu Glu 50 55 60 Tyr Gln Val Lys Ile Asn Ala Pro Lys
Glu Asp Asp Tyr Glu Thr Arg 65 70 75 80 Ile Thr Glu Ser Lys Cys Val
Thr Ile Leu His Lys Gly Phe Ser Ala 85 90 95 Ser Val Arg Thr Ile
Leu Gln Asn Asp His Ser Leu Leu Ala Ser Ser 100 105 110 Trp Ala Ser
Ala Glu Leu His Ala Pro Pro Gly Ser Pro Gly Thr Ser 115 120 125 Ile
Val Asn Leu Thr Cys Thr Thr Asn Thr Thr Glu Asp Asn Tyr Ser 130 135
140 Arg Leu Arg Ser Tyr Gln Val Ser Leu His Cys Thr Trp Leu Val Gly
145 150 155 160 Thr Asp Ala Pro Glu Asp Thr Gln Tyr Phe Leu Tyr Tyr
Arg Tyr Gly 165 170 175 Ser Trp Thr Glu Glu Cys Gln Glu Tyr Ser Lys
Asp Thr Leu Gly Arg 180 185 190 Asn Ile Ala Cys Trp Phe Pro Arg Thr
Phe Ile Leu Ser Lys Gly Arg 195 200 205 Asp Trp Leu Ser Val Leu Val
Asn Gly Ser Ser Lys His Ser Ala Ile 210 215 220 Arg Pro Phe Asp Gln
Leu Phe Ala Leu His Ala Ile Asp Gln Ile Asn 225 230 235 240 Pro Pro
Leu Asn Val Thr Ala Glu Ile Glu Gly Thr Arg Leu Ser Ile 245 250 255
Gln Trp Glu Lys Pro Val Ser Ala Phe Pro Ile His Cys Phe Asp Tyr 260
265 270 Glu Val Lys Ile His Asn Thr Arg Asn Gly Tyr Leu Gln Ile Glu
Lys 275 280 285 Leu Met Thr Asn Ala Phe Ile Ser Ile Ile Asp Asp Leu
Ser Lys Tyr 290 295 300 Asp Val Gln Val Arg Ala Ala Val Ser Ser Met
Cys Arg Glu Ala Gly 305 310 315 320 Leu Trp Ser Glu Trp Ser Gln Pro
Ile Tyr Val Gly Asn Asp Glu His 325 330 335 Lys Pro Leu Arg Glu Trp
Phe Val Ile Val Ile Met Ala Thr Ile Cys 340 345 350 Phe Ile Leu Leu
Ile Leu Ser Leu Ile Cys Lys Ile Cys His Leu Trp 355 360 365 Ile Lys
Leu Phe Pro Pro Ile Pro Ala Pro Lys Ser Asn Ile Lys Asp 370 375 380
Leu Phe Val Thr Thr Asn Tyr Glu Lys Ala Gly Ser Ser Glu Thr Glu 385
390 395 400 Ile Glu Val Ile Cys Tyr Ile Glu Lys Pro Gly Val Glu Thr
Leu Glu 405 410 415 Asp Ser Val Phe 420 6 424 PRT Mus musculus 6
Met Ala Arg Pro Ala Leu Leu Gly Glu Leu Leu Val Leu Leu Leu Trp 1 5
10 15 Thr Ala Thr Val Gly Gln Val Ala Ala Ala Thr Glu Val Gln Pro
Pro 20 25 30 Val Thr Asn Leu Ser Val Ser Val Glu Asn Leu Cys Thr
Ile Ile Trp 35 40 45 Thr Trp Ser Pro Pro Glu Gly Ala Ser Pro Asn
Cys Thr Leu Arg Tyr 50 55 60 Phe Ser His Phe Asp Asp Gln Gln Asp
Lys Lys Ile Ala Pro Glu Thr 65 70 75 80 His Arg Lys Glu Glu Leu Pro
Leu Asp Glu Lys Ile Cys Leu Gln Val 85 90 95 Gly Ser Gln Cys Ser
Ala Asn Glu Ser Glu Lys Pro Ser Pro Leu Val 100 105 110 Lys Lys Cys
Ile Ser Pro Pro Glu Gly Asp Arg Glu Ser Ala Val Thr 115 120 125 Glu
Leu Lys Cys Ile Trp His Asn Leu Ser Tyr Met Lys Cys Ser Trp 130 135
140 Leu Pro Gly Arg Asn Thr Ser Pro Asp Thr His Tyr Thr Leu Tyr Tyr
145 150 155 160 Trp Tyr Ser Ser Leu Glu Lys Ser Arg Gln Cys Glu Asn
Ile Tyr Arg 165 170 175 Glu Gly Gln His Ile Ala Cys Ser Phe Lys Leu
Thr Lys Val Glu Pro 180 185 190 Ser Phe Glu His Gln Asn Val Gln Ile
Met Val Lys Asp Asn Ala Gly 195 200 205 Lys Ile Arg Pro Ser Cys Lys
Ile Val Ser Leu Thr Ser Tyr Val Lys 210 215 220 Pro Asp Pro Pro His
Ile Lys His Leu Leu Leu Lys Asn Gly Ala Leu 225 230 235 240 Leu Val
Gln Trp Lys Asn Pro Gln Asn Phe Arg Ser Arg Cys Leu Thr 245 250 255
Tyr Glu Val Glu Val Asn Asn Thr Gln Thr Asp Arg His Asn Ile Leu 260
265 270 Glu Val Glu Glu Asp Lys Cys Gln Asn Ser Glu Ser Asp Arg Asn
Met 275 280 285 Glu Gly Thr Ser Cys Phe Gln Leu Pro Gly Val Leu Ala
Asp Ala Val 290 295 300 Tyr Thr Val Arg Val Arg Val Lys Thr Asn Lys
Leu Cys Phe Asp Asp 305 310 315 320 Asn Lys Leu Trp Ser Asp Trp Ser
Glu Ala Gln Ser Ile Gly Lys Glu 325 330
335 Gln Asn Ser Thr Phe Tyr Thr Thr Met Leu Leu Thr Ile Pro Val Phe
340 345 350 Val Ala Val Ala Val Ile Ile Leu Leu Phe Tyr Leu Lys Arg
Leu Lys 355 360 365 Ile Ile Ile Phe Pro Pro Ile Pro Asp Pro Gly Lys
Ile Phe Lys Glu 370 375 380 Met Phe Gly Asp Gln Asn Asp Asp Thr Leu
His Trp Lys Lys Tyr Asp 385 390 395 400 Ile Tyr Glu Lys Gln Ser Lys
Glu Glu Thr Asp Ser Val Val Leu Ile 405 410 415 Glu Asn Leu Lys Lys
Ala Ala Pro 420 7 20 DNA Artificial primer 7 agaggaatta cccctggatg
20 8 20 DNA Artificial anti-sense primer 8 tcaaggagct gctttcttca 20
9 21 DNA Artificial primer 9 gatccacttc ccaagaacag a 21 10 23 DNA
Artificial primer 10 gatccgggcc cttttttttt ttt 23 11 6 PRT
Artificial variant sequence substituting 8 c-termial amino acids of
SEQ ID 2 11 Val Arg Cys Val Thr Leu 1 5 12 378 PRT Artificial SEQ
ID NO. 2 substitution 8-C terminal amino acids with VRCTL 12 Met
Ala Phe Val Cys Leu Ala Ile Gly Cys Leu Tyr Thr Phe Leu Ile 1 5 10
15 Ser Thr Thr Phe Gly Cys Thr Ser Ser Ser Asp Thr Glu Ile Lys Val
20 25 30 Asn Pro Pro Gln Asp Phe Glu Ile Val Asp Pro Gly Tyr Leu
Gly Tyr 35 40 45 Leu Tyr Leu Gln Trp Gln Pro Pro Leu Ser Leu Asp
His Phe Lys Glu 50 55 60 Cys Thr Val Glu Tyr Glu Leu Lys Tyr Arg
Asn Ile Gly Ser Glu Thr 65 70 75 80 Trp Lys Thr Ile Ile Thr Lys Asn
Leu His Tyr Lys Asp Gly Phe Asp 85 90 95 Leu Asn Lys Gly Ile Glu
Ala Lys Ile His Thr Leu Leu Pro Trp Gln 100 105 110 Cys Thr Asn Gly
Ser Glu Val Gln Ser Ser Trp Ala Glu Thr Thr Tyr 115 120 125 Trp Ile
Ser Pro Gln Gly Ile Pro Glu Thr Lys Val Gln Asp Met Asp 130 135 140
Cys Val Tyr Tyr Asn Trp Gln Tyr Leu Leu Cys Ser Trp Lys Pro Gly 145
150 155 160 Ile Gly Val Leu Leu Asp Thr Asn Tyr Asn Leu Phe Tyr Trp
Tyr Glu 165 170 175 Gly Leu Asp His Ala Leu Gln Cys Val Asp Tyr Ile
Lys Ala Asp Gly 180 185 190 Gln Asn Ile Gly Cys Arg Phe Pro Tyr Leu
Glu Ala Ser Asp Tyr Lys 195 200 205 Asp Phe Tyr Ile Cys Val Asn Gly
Ser Ser Glu Asn Lys Pro Ile Arg 210 215 220 Ser Ser Tyr Phe Thr Phe
Gln Leu Gln Asn Ile Val Lys Pro Leu Pro 225 230 235 240 Pro Val Tyr
Leu Thr Phe Thr Arg Glu Ser Ser Cys Glu Ile Lys Leu 245 250 255 Lys
Trp Ser Ile Pro Leu Gly Pro Ile Pro Ala Arg Cys Phe Asp Tyr 260 265
270 Glu Ile Glu Ile Arg Glu Asp Asp Thr Thr Leu Val Thr Ala Thr Val
275 280 285 Glu Asn Glu Thr Tyr Thr Leu Lys Thr Thr Asn Glu Thr Arg
Gln Leu 290 295 300 Cys Phe Val Val Arg Ser Lys Val Asn Ile Tyr Cys
Ser Asp Asp Gly 305 310 315 320 Ile Trp Ser Glu Trp Ser Asp Lys Gln
Cys Trp Glu Gly Glu Asp Leu 325 330 335 Ser Lys Lys Thr Leu Leu Arg
Phe Trp Leu Pro Phe Gly Phe Ile Leu 340 345 350 Ile Leu Val Ile Phe
Val Thr Gly Leu Leu Leu Arg Lys Pro Asn Thr 355 360 365 Tyr Pro Lys
Met Val Arg Cys Val Thr Leu 370 375 13 5 PRT Artificial motif
characteristic of family of SEQ ID NO. 2 and SEQ ID NO 4. 13 Trp
Ser Xaa Trp Ser 1 5 14 6 DNA Artificial polyadenylation signal 14
aataaa 6 15 20 DNA Artificial primer 15 aaaaaaaaaa aaagggcccg
20
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