Note: Descriptions are shown in the official language in which they were submitted.
CA 02401357 2002-08-27
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DESCRIPTION
TISSUE DEGRADATION INHIBITING AGENT
TECHNICAL FIELD
The present invention relates to a tissue degradation inhibiting agent which
comprises a substance inhibiting the binding of parathyroid hormone-related
peptide (PTHrP)
to its receptor.
BACKGROUND ART
Parathyroid hormone-related peptide (hereinafter, PTHrP) is a protein produced
by
a tumor and is a major causative substance of humoral hypercalcemia of
malignancy.
PTHrP induces humoral hypercalcemia of malignancy (hereinafter referred to as
"HHM") by
promoting bone resorption and renal tubular calcium reabsorption. Currently,
calcitonin and
bisphosphonate having an inhibiting agenty action on bone resorption are used
to treat HHM.
However, since the progression of HHM is so rapid as to significantly
deteriorate the QOL
(Quality of Life) of patients with terminal cancer, development of a more
effective therapeutic
agent on the basis of any cause of the disease has been awaited.
An antibody against parathyroid hormone-related peptide (hereinafter referred
to as
an "anti-PTHrP antibody") is superior to bisphosphonate because the anti-PTHrP
antibody
produces an effect for HHM immediately after administration, while the
bisphosphonate need
days to produce an effect. Further, the anti-PTHrP antibody is also useful as
a therapeutic
agent for cachexia seen in patients with terminal cancer (Japanese Patent
Application Laying-
Open (kokai) No. 11-80025).
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Cancer cachexia is one of the paraneoplastic syndromes, which are
characterized by
a sharp body weight loss, and is often found in many patients with advanced
cancer. The
body weight loss has been thought to be caused by, for example, a reduced
dietary intake due
to loss of appetite. Development of cancer cachexia not only largely affects
survival time,
but also significantly deteriorates the QOL of patients, and adversely
affects, for example,
treatment with a chemotherapeutic agent. Accordingly, controlling cachexia is
of extreme
importance as a part of cancer treatment.
It is known that HHM is induced in a model rat transplanted with a human lung
cancer strain LC-6. In this model rat, blood calcium level is improved using a
bisphosphonate, which is the existing HHM therapeutic agent, or the like.
However, administration of the bisphosphonate to a HHM model rat showing a
symptom of cachexia, such as a sharp decrease in body weight, results in only
a slight
recovery in the body weight.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a tissue degradation
inhibiting agent
which comprises, as an active ingredient, a substance inhibiting the binding
of PTHrP to its
receptor.
As a result of thorough studies to address the above-mentioned problems, the
present
inventors have completed the present invention on the basis of the finding
that a substance
inhibiting the binding of parathyroid hormone-related peptide to its receptor
can inhibit tissue
degradation.
Specifically, the present invention is a tissue degradation inhibiting agent
which
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CA 02401357 2002-08-27
comprises, as an active ingredient, a substance inhibiting the binding of
parathyroid hormone-
related peptide to its receptor. Examples of the substance include an
antagonist against the
parathyroid hormone-related peptide receptor, an anti-parathyroid hormone-
related peptide
antibody (for example, a humanized or chimeric monoclonal antibody), and a
fragment and/or
a modified form of the antibody. An example of a humanized antibody is a
humanized #23-
57-137-1 antibody or the like. Further, an example of a tissue is a muscle
tissue or adipose
tissue. Examples of the above tissue degradation include those caused by
cancer cachexia,
septicemia, severe injury or muscular dystrophy. The inhibiting agent is also
effective for
patients having, for example, a blood cytokine level (at least one of those
selected from the
group consisting of, for example, IL-6, G-CSF, IL-11 and LIF) higher than the
normal level.
In particular, the inhibiting agent of the present invention is effective,
even in the presence of
inflammatory cytokines, such as IL-6, IL-11 and LIF, which are believed to
involve tissue
degradation.
A detailed description of the present invention will now be given.
This specification includes part or all of the contents disclosed in the
specification
and drawings of Japanese Patent Application No. 2000-52414, of which the
present
application claims priority.
The present invention is a tissue degradation inhibiting agent which
comprises, as an
active ingredient, a substance inhibiting the binding of parathyroid hormone-
related peptide
(PTHrP) to its receptor (PTHrP receptor).
The present inventors have surmised that the anti-PTHrP antibody may play some
role in weight fluctuation based on findings that the antibody has a drug
effect of normalizing
calcium level as well as of recovering weight. That is, the present inventors
have postulated
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CA 02401357 2002-08-27
that body weight loss resulting from cachexia is caused by distruction of
tissues. Thus, the
present inventors have completed the present invention on the basis of the
finding that the
anti-PTHrP antibody inhibits disruption of tissues.
In the present specification, the term "PTHrP receptor" refers to, for
example, a
receptor binding to PTHrP described in Japanese Patent Application Laying-Open
(kohyo) No.
6-506598. The term "PTHrP receptor" can refer to a PTHrP receptor which may be
or not
present on a target organ (for example, bone or kidney).
The term "a substance inhibiting the binding of PTHrP to the PTHrP receptor"
refers to either one of, or both (1) a substance which inhibits the binding of
PTHrP to the
PTHrP receptor by binding of itself to PTHrP (for example, an anti-PTHrP
antibody), and (2)
a substance which inhibits the binding of PTHrP to the PTHrP receptor by
binding of itself to
the PTHrP receptor [for example, an antagonist against the PTHrP receptor
(which is also
referred to as a PTHrP antagonist), specifically, PTHrP peptide having
substitution and/or
deletion of at least one amino acid, and a partial sequence of PTHrP peptide,
etc.].
The anti-PTHrP antibody used in the present invention is not limited based on
its
origin, type (monoclonal, polyclonal) and form, so far as the anti-PTHrP
antibody has an
inhibitory effect on tissue degradation by binding to PTHrP.
Examples of the anti-PTHrP antibody include a humanized antibody, a human
antibody (W096/33735), a chimeric antibody (Japanese Patent Application Laying-
Open
(kokai) No. 4-228089), and a mouse antibody (for example, #23-57-137-1
antibody produced
by hybridoma #23-57-137-1). Note that the antibody may be a polyclonal
antibody, but a
monoclonal antibody is preferable.
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Examples of the PTHrP antagonist include a polypeptide and a low molecular
compound, but are not limited thereto. Specific examples of a substance
binding
antagonistically to the PTHrP receptor against PTHrP include polypeptides
having PTHrP
antagonist activity described in Japanese Patent Application Laying-Open
(kokai) No. 7-
165790, Japanese Patent Application Laying-Open (kohyo) No. 5-509098, Peptides
(The
United States) 1995, 16 (6) 1031-1037, and Biochemistry (The United States)
Apr. 28, 1992,
31 (16) 4026-4033. Moreover, the PTHrP antagonist according to the present
invention also
includes the polypeptide which has deletion, substitution, addition and/or
insertion of at least
one amino acid and has an equivalent PTHrP antagonist activity.
The present invention is illustrated below with an anti-PTHrP antibody as an
example of "a substance inhibiting the binding of PTHrP to the PTI3rP
receptor".
1. Anti-PTHrP antibody
The anti-PTHrP antibody used in the present invention can be produced by any
known method as a polyclonal or monoclonal antibody. Preferably, the anti-
PTHrP antibody
used in the present invention is a monoclonal antibody derived from,
particularly, a mammal.
The mammal-derived monoclonal antibody includes those produced by a hybridoma
and
those produced by the host transformed with the expression vector carrying the
gene cording
the antibody which is constructed using genetic engineering techniques. The
antibody can
bind to PTHrP to inhibit binding of PTHrP to the PTH/PTHrP receptor, thereby
blocking the
signal transduction of PTHrP and consequently inhibiting the biological
activity of PTHrP
A specific example of such the antibody is #23-57-137-1 antibody which is
produced
by a hybridoma clone #23-57-137-1, or the like.
The hybridoma clone #23-57-137-1 was designated as "mouse-mouse hybridoma
#23-57-137-1" and deposited under the terms of the Budapest Treaty on August
15, 1996 with
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the International Patent Organism Depositary (IPOD), National Institute of
Advanced
Industrial Science and Technology, Japan (1-l, Higashi 1-chome, Tsukuba-shi,
Ibaraki, Japan)
under the accession No. FERM BP-5631.
2. Antibody-producing hybridoma
A monoclonal antibody-producing hybridoma can be produced as follows. Namely,
in this process PTHrP is used as a sensitizing antigen in accordance with a
standard
immunization method. The resulting immunocytes are fused to known parent cells
by a
standard cell fusion method, and monoclonal antibody-producing cells are
screened from the
fused cells by a standard screening method.
First, human PTHrP, which is used as a sensitizing antigen for producing the
antibody, is prepared by expressing the PTHrP gene/amino acid sequence
disclosed in Suva, L.
J. et al., Science (1987) 237, 893. That is, the gene encoding PTHrP is
inserted into a known
expression vector system, and a suitable host cell is transformed with the
expression vector.
The target PTHrP protein is then purified from the transformed host cell or
from the culture
supernatant of the transformed host cell by any known method.
Then, the purified PTHrP protein is used as a sensitizing antigen.
Alternatively, the
N-terminal 34 peptides of PTHrP may be chemically synthesized and used as the
sensitizing
antigen.
The mammal to be immunized with the sensitizing antigen is not particularly
limited.
However, the mammal is preferably selected taking into consideration
compatibility with the
parent cell used for cell fusion. Generally, a rodent (e.g., mouse, rat,
hamster), rabbit,
monkey or the like may be used.
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The immunization of the animal with the sensitizing antigen can be performed
in
accordance with any known method. For example, a general technique is
performed by
injecting the sensitizing antigen to a mammal intraperitoneally or
subcutaneously.
Specifically, the sensitizing antigen is diluted with or suspended in an
appropriate amount of
phosphate-buffered saline (PBS), physiological saline or the like. The
resulting dilution or
suspension is then mixed with an appropriate amount of a common adjuvant
(e.g., Freund's
complete adjuvant), if necessary. The mixture is emulsified and administered
to a mammal
several times at intervals of 4 to 21 days. For the immunization with the
sensitizing antigen,
a suitable Garner may be used together with it.
After the immunization, the serum antibody level is checked. When the serum
antibody level is confirmed to have reached a desired level, immunocytes are
isolated from
the mammal and then subjected to cell fusion. A particularly preferable
immunocyte is a
spleen cell.
The parent cell used for fusion with the above immunocyte (i.e., the
counterpart in
the cell fusion with the immunocyte) is a myeloma cell derived from a mammal.
The
myeloma cell preferably used herein is of any known cell line, and, for
example, P3
(P3x63Ag8.653) (J. Immnol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics
in
Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler, G. and Milstein,
C., Eur. J.
Immunol. (1976) 6, 511-519), MPC-11 (Margulies, D. H. et al., Cell (1976) 8,
405-415),
SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, S.
F. et al., J.
Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I. S. J. Exp. Med. (1978)
148, 313-
323) or 8210 (Galfre, G. et al., Nature (1979) 277, 131-133).
Cell fusion of the above immunocyte to the myeloma cell is basically performed
in
accordance with any known method, such as the method of Milstein et al.
(Kohler, G. and
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Milstein, C., Methods Enzymol. (1981) 73, 3-46).
More specifically, the cell fusion is performed, for example, in a
conventional
nutrient culture medium in the presence of a cell fusion promoter. The cell
fusion
promoteing reagent may be polyethylene glycol (PEG), a Sendai virus
(Hemagglutinating
Virus of Japan; HVJ), or the like. If desired, for the purpose of improving
the fusion
efficiency, an auxilliary agent such as dimethyl sulfoxide may be used for
incorporation.
The ratio between the immunocytes and the myeloma cells used for the cell
fusion
may be optionally set. For example, the immunocytes are used preferably in the
number of
1-10 times larger than the myeloma cells. The culture medium used for the cell
fusion is, for
example, RPMI 1640 medium or MEM medium, which is suitable for the growth of
the
above-mentioned myeloma cell lines, or another medium normally used for the
culture of
such cell lines. Further, a serum supplement, such as feral calf serum (FCS),
may be added
to the culture medium.
The cell fusion is performed by fully mixing given amounts of the immunocytes
and
the myeloma cells in the above culture medium, adding a PEG solution (e.g.,
mean molecular
weight: about 1000-6000) (which has been preheated to about 37°C) to
the mixture usually to
a concentration of 30-60% (w/v), and then mixing the resulting solution,
thereby producing
the desired fusion cells (hybridomas). Subsequently, an appropriate culture
medium is added
to the culture solution successively, and the solution is centrifuged to
remove the supernatant.
This procedure is repeated several times to remove substances such as the cell
fusion
promoting agent that are undesirable for the growth of the hybridomas, from
the culture
medium.
The thus obtained hybridomas can be selected by culturing in a standard
selective
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medium, such as Hypoxanthine-Aminopterin-Thymidine (HAT) medium. The culturing
of
the hybridomas in HAT medium is performed for a period of time long enough to
cause the
death of the cells other than the desired hybridomas (i.e., cells that fail to
fuse), usually a
period of several days to several weeks. Subsequently, a standard limiting
dilution method is
performed for screening and mono-cloning of the hybridomas that are producing
the desired
antibody.
In addition to preparing the hybridomas by immunizing a non-human mammal with
the antigen as described above, a human lymphocyte may be sensitized with
PTHrP in vitro,
followed by fusion of the sensitized lymphocyte to a desired human-derived
myeloma cell
capable of infinite growth, thereby producing a human antibody having a
binding activity
against PTHrP (Japanese Patent Publication No. 1-59878). Alternatively, a
human antibody
against PTHrP may be prepared by administering PTHrP as an antigen to a
transgenic animal
that has the entire repertories of human antibody genes to produce an anti-
PTHrP antibody-
producing cell, and then immortalizing the cells, thus producing the human
antibody from the
immortalized cell (International Patent Publication Nos. WO 94/25585, WO
93/12227, WO
92/03918 and WO 94/02602).
The monoclonal antibody-producing hybridoma prepared as described above can be
subcultured in a standard culture medium and stored under liquid nitrogen for
a long period.
Examples of a method that may be employed to obtain a monoclonal antibody from
the hybridoma include a method that involves culturing the hybridoma in
accordance with a
standard technique and collecting the monoclonal antibody from the culture
supernatant, or
that involves administering the hybridoma to a mammal compatible with the
hybridoma to
grow the hybridoma in the mammal and collecting the hybridoma from the ascites
of the
mammal. The former method is suitable for producing the antibody in high
purity, while the
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latter method is suitable for producing the antibody in a large amount.
3. Recombinant antibody
In the present invention, a recombinant-type monoclonal antibody may be used,
which can be produced by cloning an antibody gene from the hybridoma,
integrating the
antibody gene into a suitable vector, introducing the vector into a host, and
then producing the
antibody from the host according to a standard genetic recombination technique
(see, for
example, Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-775, 1990).
Specifically, mRNA encoding a variable (V) region of an anti-PTHrP antibody is
isolated from the anti-PTHrP antibody-producing hybridoma. The mRNA is
isolated by
preparing total RNA by any known method, such as a guanidium
ultracentrifugation method
(Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299) or an AGPC method
(Chomczynski, P et al., Anal. Biochem. (1987) 162, 156-159), and then
producing the desired
mRNA from the total RNA using an mRNA Purification Kit (Pharmacia) or the
like.
Alternatively, the mRNA may also be prepared directly using a QuickPrep mRNA
Purification Kit (Pharmacia).
Next, cDNA for the antibody V-region is synthesized from the mRNA with a
reverse
transcriptase. The synthesis of the cDNA is performed using an AMV Reverse
Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) or the
like. The
cDNA may also be synthesized and amplified by, for example, the 5'-RACE method
(Frohman, M.A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002;
Belyavsky, A. et al.,
Nucleic Acids Res. (1989) 17, 2919-2932) using a 5'-Ampli FINDER RACE Kit
(CLONETECH) in combination with the PCR method.
A DNA fragment of interest is purified from the resulting PCR product and then
CA 02401357 2002-08-27
ligated to a vector DNA to obtain a recombinant vector. The recombinant vector
is
introduced into E. coli or the like, and then the resulting colony is
selected, thereby preparing
the desired recombinant vector. The nucleotide sequence of the DNA of interest
is
confirmed by a known method, for example, a dideoxynucleotide chain
termination method.
Once DNA encoding the anti-PTHrP antibody V-region is obtained, the DNA is
integrated into an expression vector containing DNA encoding a desired
antibody constant
(C) region.
For the production of the target anti-PTHrP antibody used in the present
invention,
the antibody gene is integrated into an expression vector so that the antibody
gene can be
expressed under the control of expression control regions (e.g., enhancer and
promoter). A
host cell is transformed with the expression vector to express the antibody.
To express the antibody gene, DNA encoding the heavy (H) chain and DNA
encoding the light (L) chain of the antibody may be integrated separately into
expression
vectors, and then a host cell is co-transformed with the resulting recombinant
expression
vectors. Alternatively, both the DNA encoding the H-chain and the DNA encoding
the L-
chain of the antibody may be integrated together into a single expression
vector, and then a
host cell may be transformed with the resulting recombinant expression vector
(WO
94/11523).
To produce the recombinant antibody, besides the above-mentioned host cells, a
transgenic animal may also be used as a host. For example, the antibody gene
is inserted
into a gene encoding a protein inherently produced in the milk of an animal
(e.g., goat a -
casein) to obtain a fusion gene. A DNA fragment containing the antibody gene-
introduced
fusion gene is injected into an embryo of a goat, and the embryo is then
introduced into a
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CA 02401357 2002-08-27
female goat. The female goat having the embryo bears a transgenic goat. The
antibody of
interest is secreted in the milk from the transgenic goat or a progeny
thereof. For increasing
the amount of the desired antibody-containing milk from the transgenic goat,
an appropriate
hormone may be administered to the transgenic goat (Ebert, K.M. et al.,
Bio/Technology
(1994) 12, 699-702).
4. Modified antibody
In the present invention, for the purpose of, for example, reducing the
heteroantigenicity for a human body, an artificially modified genetic
recombinant antibody
may be used, such as a chimeric antibody or a humanized antibody. These
modified
antibodies can be prepared by the following known methods.
A chimeric antibody useful in the present invention can be prepared by
ligating the
DNA encoding the antibody V-region prepared as set forth above to a DNA
encoding a
human antibody C-region, integrating the ligation product into an expression
vector, and
introducing the resulting recombinant expression vector into a host to produce
the chimeric
antibody.
A humanized antibody is also referred to as a "reshaped human antibody", in
which
the complementarity determining regions (CDRs) of an antibody of a non-human
mammal
(e.g., a mouse) are grafted to those of a human antibody. The general genetic
recombination
procedures for producing such humanized antibody are also known (EP 125023; WO
96/02576).
Specifically, a DNA sequence which has been designed to have mouse antibody
CDRs ligated through framework regions (FRs) of a human antibody is amplified
by the PCR
method using as primers several oligonucleotides which have been prepared to
have regions
12
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overlapping the terminal regions of both the CDRs and the FRs. The resulting
DNA is
ligated to DNA encoding human antibody C-region, and the ligation product is
integrated into
an expression vector. The resulting recombinant expression vector is
introduced into a host,
thereby producing the humanized antibody (EP 239400, WO 96/02576).
The FRs of the human antibody ligated through the CDRs are selected so that
the
CDRs can form a suitable antigen binding site. If necessary, an amino acids)
in the FRs of
the antibody V-region may be replaced so that the CDRs of the reshaped human
antibody can
form a suitable antigen binding site (Sato, K. et al., Cancer Res. (1993) 53,
851-856).
The C-region of the chimeric or humanized antibody may be any human antibody
C-region, such as C Y 1, C Y 2, C ?' 3 or C Y 4 for the H-chain, and C r~ or C
~l for the L-
chain. The human antibody C-region may be modified for improving the antibody
or the
stable production of the antibody.
The chimeric antibody is composed of V-regions derived from a non-human
mammalian antibody and C-regions derived from a human antibody. The humanized
antibody is composed of CDRs derived from a non-human mammalian antibody and
FRs and
C-regions derived from a human antibody. The humanized antibody is useful as
an active
ingredient for the inhibiting agent of the present invention, because the
antigenicity of the
antibody against a human body is reduced.
A specific example of the humanized antibody usable in the present invention
is
humanized #23-57-137-1 antibody, in which the CDRs are derived from mouse-
derived #23-
57-137-1 antibody, the L-chain is composed of the CDRs ligated through three
FRs (FR1,
FR2 and FR3) derived from human antibody HSU 03868 (GEN-BANK, Deftos, M. et
al.,
Scand. J. Immunol., 39, 95-103, 1994) and a FR fragment (FR4) derived from
human
13
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antibody S25755 (NBRF-PDB), and the H-chain is composed of the CDRs ligated
through
FRs derived from human antibody S31679 (NBRF-PDB, Cuisinier, AM et al., Eur.
J.
Immunol. 23, 110-118, 1993) in which a part of the amino acid residues in the
FRs is
substituted so that the reshaped humanized antibody can exhibit an antigen-
binding activity.
The E. coli strain JM 109 (hMBC 1 HcDNA/pUC 19) having a plasmid containing
DNA encoding the H-chain and the E. coli strain JM 109 (hMBC 1 Lq ~l /pUC 19)
having a
plasmid containing DNA encoding the L-chain of the humanized #23-57-137-1
antibody were
deposited under the terms of the Budapest Treaty on August 15, 1996 with the
International
Patent Organism Depositary (IPOD), National Institute of Advanced Industrial
Science and
Technology, Japan (1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) under
the accession
Nos. FERM BP-5629 and FERM BP-5630, respectively.
5. Modified Antibody
The antibody used in the present invention may be a fragment of an antibody or
a
modified form of the fragment, as long as it binds to PTHrP to inhibit the
activity thereof.
Examples of such a fragment of an antibody include Fab, F(ab')2, Fv, or a
single chain Fv
(scFv) composed of a H-chain Fv fragment and a L-chain Fv fragment linked
together
through a suitable linker. Specifically, such antibody fragments can be
produced by cleaving
the antibody with an enzyme (e.g., papain, pepsin) into antibody fragments, or
by constructing
a gene encoding the antibody fragment and inserting the gene into an
expression vector and
introducing the resulting recombinant expression vector into a suitable host
cell, thereby
expressing the antibody fragment (see, for example, Co, M. S., et al., J.
Immunol. (1994), 152,
2968-2976; Better, M. & Horwitz, A. H., Methods in Enzymology (1989), 178, 476-
496,
Academic Press, Inc.; Plueckthun, A. & Skerra, A., Methods in Enzymology
(1989) 178, 476-
496, Academic Press, Inc.; Lamoyi, E., Methods in Enzymology (1989) 121, 652-
663;
Rousseaux, J. et al., Methods in Enzymology ( 1989) 121, 663-669; and Bird, R.
E. et al.,
14
CA 02401357 2002-08-27
TIBTECH (1991) 9, 132-137).
An scFv can be produced by linking the H-chain V-region to the L-chain V-
region
of antibodies through a linker, preferably a peptide linker (Huston, J. S. et
al., Proc. Natl.
Acad. Sci. USA (1988) 85, 5879-5883). The H-chain V-region and the L-chain V-
region in
the scFv may be derived from any one of the antibodies described herein. The
peptide linker
which binds the V-regions may be, for example, any single chain peptide of 12-
19 amino acid
residues.
DNA encoding the scFv can be prepared by amplifying in PCR two template
sequences which are the entire sequences or partial sequences of DNA encoding
the H-chain
or the H-chain V-region and DNA encoding the L-chain or the L-chain V-region
of the
antibody, wherein the partial sequence is corresponding to a desired amino
acid sequence
region encoded in the DNAs using two primer pairs that are located on the
terminal sides of
the template sequences respectively; and subsequently further amplifying both
the resulting
amplified products and DNA encoding the peptide linker as template sequence
using primer
pairs which is designed to be enabled to ligate the terminal ends of the
peptide linker to the H-
chain and the L-chain respectively, during amplification.
Once the DNA encoding the scFv is prepared, an expression vector carrying the
DNA and a host transformed with the expression vector can be prepared by
standard methods.
The scFv can be produced from the transformed host by a standard method.
The fragments of the antibody may be produced by preparing genes and
expressing
the genes in suitable hosts as described above. The antibody fragments are
also
encompassed in the "antibody" of the present invention.
CA 02401357 2002-08-27
As a modified form of the above-mentioned antibodies, for example, an anti-
PTHrP
antibody conjugated to any molecule (e.g., polyethylene glycol (PEG)) may also
be used.
Such modified antibodies are also encompassed in the "antibody" of the present
invention.
The modified antibodies can be prepared by chemical modification of the
antibodies. The
chemical modification techniques suitable for this purpose have already been
established in
the art.
6. Expression and production of recombinant antibody or modified antibody
The antibody gene constructed as described above can be expressed and obtained
by known methods. For the expression in a mammalian cell, a conventional
useful promoter,
the antibody gene to be expressed and a poly(A) signal (located downstream of
the 3' end of
the antibody gene) may be operably linked and expressed. For example, as a
promoter/enhancer system, a human cytomegalovirus immediate early
promoter/enhancer
system may be used.
Other promoter/enhancer systems usable in the expression of the antibody used
in
the present invention include those derived from virus promoters (e.g.,
retrovirus, polyoma
virus, adenovirus and sinuan virus 40 (SV40)) and those derived from mammalian
cells (e.g.,
human elongation factor 1 a (HEFT cx ).
When an SV40 promoter/enhancer system is used, the gene expression may be
performed readily by the method of Mulligan et al. (Nature (1979) 277, 108).
When a HEF1
a promoter/enhancer system is used, the gene expression may be performed
readily by the
method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322).
For the expression in E. coli, a conventional useful promoter, a signal
sequence for
secreting an antibody and the antibody gene to be expressed may be operably
linked. As
16
CA 02401357 2002-08-27
such a promoter, lacz promoter or araB promoter may be used. When lacz
promoter is used,
the gene expression may be performed by the method of Ward et al. (Nature
(1098) 341, 544-
546; FASEB J. (1992) 6, 2422-2427). When araB promoter is used, the gene
expression
may be performed by the method of Better et al. (Better et al., Science (1988)
240, 1041-
1043).
Regarding the signal sequence for secretion of the antibody, when the antibody
of
interest is intended to be secreted in a periplasmic space of the E. coli,
pelB signal sequence
(Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379) may be used. The antibody
secreted into the
periplasmic space is isolated and then refolded so that the antibody takes an
appropriate
configuration for use.
Regarding the replication origin, those derived from viruses (e.g., SV40,
polyoma
virus, adenovirus, and bovine papilloma virus (BPV)) or the like may be used.
To increase
the gene copy number in the host cell system, the expression vector may
further contain a
selective marker gene, such as an aminoglycoside phosphotransferase (APH)
gene, a
thymidine kinase (TK) gene, an E. coli xanthine-guanine
phosphoribosyltransferase (Ecogpt)
gene and a dihydrofolate reductase (dhfr) gene.
For the production of the antibody used in the present invention, any
expression
system, such as eukaryotic and prokaryotic cell systems, may be used. Examples
of the
eukaryotic cell include established cell lines of animals (e.g., mammals,
insects, molds and
fungi, yeast). Examples of the prokaryotic cell include bacterial cells such
as E. coli cells.
It is preferable that the antibody used in the present invention be expressed
in a mammalian
cell, such as a CHO, COS, myeloma, BHK, Vero or HeLa cell.
Next, the transformed host cell is cultured in vitro or in vivo to produce the
antibody
17
CA 02401357 2002-08-27
of interest. The culturing of the host cell may be performed by any known
method. For
example, the culture medium usable herein may be DMEM, MEM, RPMI 1640 or IMDM
medium, and the culture medium may contain a serum supplement, such as fetal
calf serum
(FCS).
7. Isolation and purification of antibody
The antibody expressed and produced as described above may be isolated from
the
cells or the host animal and purified to uniformity. The isolation and
purification of the
antibody used in the present invention may be performed on an affinity column.
For
instance, examples of a protein A column include Hyper D, POROS and Sepharose
F.F.
(Pharmacia). The method is not particularly limited and other methods
conventionally used
for the isolation and purification of a normal protein may also be employed.
For example,
various chromatographs using columns other than the above-mentioned affinity
column,
filtration, ultrafiltration, salting out and dialysis may be appropriately
used singly or in
combination to isolate and purify the antibody of interest (Antibodies A
Laboratory Manual.
Ed. Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).
8. Determination of the activities of the antibody
The determination of the antigen-binding activity (Antibodies A Laboratory
Manual,
Ed. Harlow, David Lane, Cold Spring Harbor Laboratory, 1988) or the inhibiting
agenty
activity against binding of a ligand receptor (Harada, A. et al.,
International Immunology
(1993) 5, 681-690) of the antibody used in the present invention may be
performed by any
known methods.
As the method for the determination of the antigen-binding activity of the
anti-
PTHrP antibody used in the present invention, ELISA (enzyme-linked
immunosorbent assay),
EIA (enzyme immunoassay), RIA (radioimmunoassay) or a fluorescent antibody
method may
18
CA 02401357 2002-08-27
be employed. For example, when enzyme immunoassay is employed, a sample
containing
the anti-PTHrP antibody (e.g., a culture supernatant of anti-PTHrP antibody-
producing cells,
or the anti-PTHrP antibody in a purified form) is added to a plate on which
PTHrP (1-34) is
previously coated. A secondary antibody labeled with an enzyme (e.g., alkaline
phosphatase) is further added to the plate. The plate is incubated and washed.
A substrate
for the enzyme (e.g., p-nitrophenylphosphoric acid) is added to the plate, and
the absorbance
is measured to evaluate the antigen-binding activity of the antibody.
To confirm the activity of the antibody used in the present invention, a
neutralizing
activity of the anti-PTHrP antibody is determined.
9. Routes for administration and pharmaceutical preparations
The tissue degradation inhibiting agent containing the anti-PTHrP antibody of
the
present invention as an active ingredient may be administered orally or
parenterally, but
preferably parenterally. Specifically, the agent may be administered in a
transpulmonary
dosage form (e.g., which is administered with the help of a device such as a
nebulizer), a
transnasal dosage form, a transdermal dosage form (e.g., ointment, cream), an
injection
dosage form, or the like. Examples of an injection dosage form include those
for an
intravenous injection such as a drip, an intramuscular injection, an
intraperitoneal injection
and a subcutaneous injection and may be administered systematically or
locally. The route
of administration may be appropriately selected depending on the age and the
symptoms of a
patient. An effective single dose may be selected within the range from 0.001
to 1,000 mg
per kg of body weight. Alternatively, the dose per patient may be selected
within the range
from 0.01 to 100,000 mg/body. However, the dosage of the inhibiting agent
comprising the
anti-PTHrP antibody of the present invention is not particularly limited to
these ranges.
Examples of diseases for which the inhibiting agent of the present invention
is
19
CA 02401357 2002-08-27
administered include, but are not limited to, cancer cachexia, septicemia,
external injury (e.g.,
severe and moderate injuries), muscular dystrophy and the like. The inhibiting
agent of the
present invention may also be administered a patient with two or more of the
above diseases
or the above diseases complicated with other diseases. Further, the inhibiting
agent, for
example, may also be administered to a patient with a blood level higher than
the normal level
with respect to at least one of interleukin-6 (IL-6), granulocyte colony-
stimulating factor (G-
CSF), IL-11, leukemia inhibitory factor (LIF) and ai acid glycoprotein (al -
AG). The term
"normal level" in the present invention means the mean value of the blood
levels of 10 to 100
healthy persons, or means the commonly published clinical test values. For
example, the
clinical test values range from 0.2 to 4.6 pg/mL for IL-6, 7.7 to 38.9 pg/mL
for G-CSF, less
than 31.3 pg/mL for IL-11, and 42 to 93 mg/dl for al -AG.
Regarding the timing of administration of the inhibiting agent of the present
invention,
the inhibiting agent may be administered either before or after the
development of the clinical
symptoms of the above diseases, or may be administered at a time when body
weight loss is
predicted.
The inhibiting agent comprising the anti-PTHrP antibody of the present
invention as
an active ingredient may be formulated by any standard method (Remington's
Pharmaceutical
Science, latest edition, Mark Publishing Company, Easton, USA). The
formulation may
further comprise pharmaceutically acceptable carriers and additives.
Examples of such carriers and pharmaceutical additives include water,
pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol,
polyvinyl
pyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, poly(sodium
acrylate),
sodium arginate, water soluble dextran, sodium carboxymethyl starch, pectin,
methyl
cellulose, ethyl cellulose, xanthane gum, gum arabic, casein, agar,
polyethylene glycol,
CA 02401357 2002-08-27
diglycerin, glycerin, propylene glycol, vaseline, paraffin, stearyl alcohol,
stearic acid, human
serum albumin (HSA), mannitol, sorbitol, lactose, and surfactants acceptable
as
pharmaceutical additives.
In the practical use, the additive is appropriately selected from, but is not
limited to,
the above members, either singly or in combination, depending on the employed
dosage form
of the inhibiting agent of the present invention. For example, for use as an
injection
formulation, the purified anti-PTHrP antibody is dissolved in a solvent (e.g.,
physiological
saline, a buffer, or a glucose solution) and then an adsorption-preventing
agent (e.g., Tween
80, Tween 20, a gelatin, or human serum albumin) is added thereto. The
inhibiting agent of
the present invention may also be in a reconstitutable freeze-dried form,
which is dissolved
before use. For the formulation of the freeze-dried dosage form, an excipient
such as a sugar
alcohol (e.g., mannitol, grape sugar) or a sugar may be incorporated.
BRIEF DESCRIPTION OF DRAWINGS
Fig. 1 shows the effect of the humanized antibody on body weight and blood
ionized calcium level in OCC-1-JCK transplanted mice.
Fig. 2 shows the effect of the humanized antibody on body weight and blood
ionized calcium level in Colon 26 transplanted mice.
Fig. 3 shows the effect of the humanized antibody on body weight and blood
ionized calcium level in LC-6-JCK transplanted rats.
Fig. 4 shows time course in leukocyte count in LC-6-JCK transplanted rats.
BEST MODE FOR CARRYING OUT THE INVENTION
21
CA 02401357 2002-08-27
The present invention will now be further described by means of examples and
reference examples. However, the examples are provided for illustrative
purposes only, and
are not intended to limit the technical scope of the invention.
[EXAMPLE 1) Test (1) Inhibition of Tissue degradation
1. Preparation of humanized anti-PTHrP antibody
In this example, a humanized anti-PTHrP antibody against PTHrP (1-34) was used
as
an example of the anti-PTHrP antibody.
The humanized anti-PTHrP antibody was prepared by genetic recombination
techniques as described in Reference Example 4 (L-chain version q). The
obtained
humanized anti-PTHrP antibody (hereinafter referred to as "humanized
antibody") was added
to 20 mmol/L citric acid solution, and 1 mol/L Tris solution was added
thereto, and then the
solution is adjusted to pH 6.6 (concentration: 6.34 mg/mL). The solution was
stored at or
below -70°~C . Antibodies to be used herein were confirmed to be
intended antibodies by
SDS-PAGE for its molecular weight and by binding assay using ELISA for its
binding ability.
After thawing the above antibody solution, the solution was diluted to 1 mg/mL
using
Dulbecco's PBS (-) (NISSUI PHARMACEUTICAL CO., LTD., code. 05913, hereinafter,
"PBS") under sterile condition.
2. Experimental Animal
Mice used herein were generally and widely used BALB/cAnNCrj mice (for
transplantation with Colon 26) or BALB/cAJcl-nu mice (for transplantation with
OCC-1-
JCK) transplantable with mouse colon cancer strain Colon 26 or human oral
cavity cancer
strain OCC-1-JCK. 30 male mice were used for each experiment. 6 week-old mice
were
selected to be transplanted with tumor cells. In addition, BALB/cAnNCrj mice
were
22
CA 02401357 2002-08-27
purchased from CHARLES RIVER JAPAN, INC., and BALB/cAJcl-nu mice from CLEA
JAPAN, INC.
After 30 purchased mice were acclimatized for about 1 week, 7 mice were
randomly
chosen, and put in a normal group to which tumors were not transplanted.
Tumors were
transplanted to the remaining 23 mice. On day 7 after transplantation, the
tumors were
confirmed to "take." 20 mice in which tumors took were selected. The mice were
divided,
mice per group, into a group administered with PBS and a group administered
with the
humanized antibody of the present invention, while balancing between the
groups in body
weight as an index.
The rearing conditions
for mice were
as follows.
Room's temperature: 24 2C
Relative humidity : 55 10%
Ventilation frequency: 10 to 30 times/hour
Lighting duration : 14 hours
Rearing cage : M-4 cage (215x320x 130 mm, made of polycarbonate,
provided with bedding)
Rearing density : 5 - 7 mice/cage
Feed and feeding CE-2 (CLEA JAPAN, INC.), free feeding
method:
Water and supply tap water, free intake of water with a 200
method: mL water supply bottle
3. Experimental method
3-1. Preparation of disease model
Tumor strains used: Colon 26 (Mouse colon cancer strain), OCC-1-JCK (human
oral
cavity cancer strain)
23
CA 02401357 2002-08-27
Tumor strain source: Colon 26 (Chemotherapy Center, Japanese Foundation for
Cancer Research), OCC-1-JCK (Central Institute for Experimental Animals)
Subculture of tumor strain: Colon 26 was subcultured in vivo every 2 weeks
using
BALB/cAnNCrj. OCC-1-JCK was subcultured in vivo every 3 weeks using BALB/cAJcl-
nu. Colon 26 and OCC-1-JCK transplanted mouse models were prepared as follows.
Specifically, on a day of transplantation with tumor cells, the mice having in
vivo subcultured
tumor were sacrificed by cervical-vertebral dislocation. Then, the tumor was
excised to
prepare a 3 mm block, and then transplanted subcutaneously to a mouse. The
tumors were
allowed to "take" in the transplanted mice, thereby establishing model mice.
3-2. Measurement of blood ionized calcium level (blood iCa level)
Immediately after collecting approximately 60 p1 of blood from the eye pit,
the blood
iCa level was measured with 643Ca++/pH analyzer (Chiron).
3-3. Measurement of serum biochemical level (glucose, triglyceride and
cholesterol
levels)
Under anesthesia, approximately 1 mL of blood was collected from the
descending
aorta into a Separapid tube (SEKISUI CHEMICAL Co., Ltd.), and then allowed to
stand at
room temperature for 30 min or more. The serum was separated by centrifugation
(3,000
rpm, 20 min, HTTACHI, OSPR-22), and then frozen and stored at or below -
20°C .
The serum biochemical levels (glucose, triglyceride and cholesterol levels)
were
measured using an autoanalyzer (HITACHI, type 7170, Autoanalyzer).
3-4. Administration of humanized antibody
Humanized antibodies were administered intravenously (i.v.) at a dose of 0.1
24
CA 02401357 2002-08-27
mg/mouse (a humanized antibody-administered group). Further, Dulbecco's PBS (-
) was
administered in a PBS administered group. Administration was performed twice
in total, on
days 7 and 10 after transplantation. No substance was administered in the
normal group,
which had had undergone no tumor transplantation.
Table 1 summarizes the mouse group configuration and the administration
schedule.
Table 1. Group configuration
Group Administration
Abbreviated Substance
Number Administration
of Number
of
soup administered
name Dose days
of adminis-
group
animals
trations
PBS
1 administered10 PBS O.lmL/mouse
ou
Humanized Days 7 2
and 10
0.1 mg/mouse
antibody- Humanized
administered10 antibody (PBS solution
0.1 mL/mouse)
ou
_
3 Normal- 7 none
group
The drug e~cacy test is summarized as follows.
Parameters tested: body weight, adipose tissue (periphery of orchis) weight,
muscle
tissue (gastrocnemius muscle) weight, serum glucose level, serum triglyceride
level, serum
cholesterol level, blood iCa level, and systemic condition.
For OCC-1-JCK transplanted mice, body weight was measured on days 7, 10 and 13
after tumor transplantation; blood iCa level was measured on days ? and 13;
and other
parameters were measured on day 13.
For Colon 26 transplanted mice, body weight was measured on days 7, 10 and 14
after
CA 02401357 2002-08-27
tumor transplantation; blood iCa level was measured on days 7 and 14; and
other parameters
were measured on day 14.
Evaluation of drug efficacy: The inhibiting agenty effect of administration of
the
humanized antibody on the decrease in body weight, adipose tissue weight and
muscle tissue
weight was studied. Further, effects on recovery of blood iCa level, serum
glucose level,
serum triglyceride level and serum cholesterol level to levels close to normal
levels were also
studied.
3-S. Examination of effect of humanized antibody
Groups subjected to comparison: PBS-administered group and humanized antibody-
administered group
Hypothesis verified: Compared to the PBS-administered group, the humanized
antibody-administered group shows changes in body weight, adipose tissue
weight, muscle
tissue weight, blood iCa level, serum glucose level, serum triglyceride level,
and serum
cholesterol level.
Statistical analysis method employed: Body weight, adipose tissue weight,
muscle
tissue weight, blood iCa level, serum glucose level, serum triglyceride level,
and serum
cholesterol level were tested by t-test for the normal group and the PBS-
administered group.
For the parameters showing significant differences as tested by t-test, we
further determined if
significant differences exist between the PBS-administered group and the
humanized
antibody-administered group by employing t-test. The t-test is performed at 5%
two-sided
significance level. SAS was used for the analysis.
4. Result
a6
CA 02401357 2002-08-27
4-1. Effect on OCC-1-3CK transplanted mice
( 1 ) Effect on body weight, blood iCa level and systemic condition (Fig. 1 )
Body weight: Compared to the normal group, the PBS-administered group showed a
significant decrease in body weight (on days 12 and 13 after tumor
transplantation, * : p<0.05
compared to the normal group, tested by t-test). Compared to the PBS-
administered group,
the humanized antibody-administered group showed a significantly inhibited
body weight loss
(#: p<0.05 compared to PBS-administered group, tested by t-test), and body
weight was
maintained to the same degree as that in the normal group (body weight (g) on
day 13 after
transplantation: 24.64 ~ 0.61 in normal group, 19.37 ~ 0.59 in PBS-
administered group, 24.11
~ 0.47 in humanized antibody-administered group) (Indicated by mean ~ SE. The
same
indication applies hereinafter.).
Blood iCa level: A significant increase was observed in the PBS-administered
group
compared to the normal group (on day 13 after tumor transplantation (*)).
Compared to the
PBS-administered group, the humanized antibody-administered group showed a
significantly
inhibited increase in blood iCa level (#) [blood iCa level (mmol/L) on day 13
after
transplantation: 1.36 ~ 0.01 in normal group, 2.73 ~ 0.10 in PBS-administered
group, 1.62 ~
0.01 in humanized antibody-administered group].
Systemic condition: Compared to the normal group, a deteriorated systemic
condition was visually confirmed in the PBS-administered group. In the
humanized
antibody-administered group, an improvement in the deteriorated systemic
condition was
found.
(2) Effect on adipose tissue (periphery of orchis) weight and muscle tissue
(gastrocnemius muscle) weight (Table 2)
27
CA 02401357 2002-08-27
W .-.
0 ~
b
y ~ N o
O O~
V1
07!
I
N
b~ ~ ~ N
04 N M ~~ v1 tn
'" b ~ ~ O '-': ~ N
O ~' O ~ DO .-""- N t~
dp ~ d'
~
N
U
D
N ran yp
r Ov
N
N
~..
H " ~ +I N +I N +I
~
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.
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'
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z ~ 0
~ 0
.. ,
x
CA 02401357 2002-08-27
Adipose tissue weight: Compared to the normal group, a significant decrease
was
found in the PBS-administered group (*). Compared to the PBS-administered
group, the
decrease was significantly inhibited in the humanized antibody-administered
group (#).
Muscle tissue weight: Compared to the normal group, a significant decrease was
observed in the PBS-administered group (*). Compared to the PBS-administered
group, the
decrease was significantly inhibited in the humanized antibody-administered
group (#).
(3) Effect on serum biochemical levels (glucose, triglyceride, and cholesterol
levels)
(Table 2)
Significance decreases in glucose, triglyceride and cholesterol levels were
observed in
the PBS-administered group, compared to the normal group (*). Compared to the
PBS-
administered group, the humanized antibody-administered group showed the
tendency to
inhibit decreases of such levels, but showed a significant improvement only in
triglyceride
level (#).
4-2. Effect on Colon 26 transplanted mice
(1) Effect on body weight, blood iCa level and systemic condition (Fig. 2)
Body weight: Compared to the normal group, a significant decrease in body
weight
was observed in the PBS-administered group (on and after day 10 after tumor
transplantation,
* : p<0.05 compared to the normal group, tested by t-test). Compared to the
PBS-
administered group, the body weight loss was significantly inhibited in the
humanized
antibody-administered group (#: p<0.05 compared to the PBS-administered group,
tested by t-
test). Body weight (g) on day 14 after transplantation was 26.78 ~ 0.38 in the
normal group,
19.04 ~ 0.35 in the PBS-administered group, and 21.51 ~ 0.32 in the humanized
antibody-
administered group.
29
CA 02401357 2002-08-27
Blood iCa level: A significant increase was observed in the PBS-administered
group
compared to the normal group (on day 14 after tumor transplantation (*)).
Compared to the
PBS-administered group, the increase in blood iCa level was significantly
inhibited in the
humanized antibody-administered group (#). Blood iCa level (mmol/L) on day 14
after
transplantation was 1.34 ~ 0.01 in the normal group, 1.94 ~ 0.09 in the PBS-
administered
group, and 1.54 ~ 0.03 in the humanized antibody-administered group.
Systemic condition: Compared to the normal group, a deteriorated systemic
condition was visually confirmed in the PBS-administered group. In the
humanized
antibody-administered group, such a deteriorated systemic condition was
somewhat improved.
(2) Effect on adipose tissue (periphery of orchis) weight and muscle tissue
(gastrocnemius muscle) weight (Table 3)
CA 02401357 2002-08-27
r.
a,
c i o~
ov
°~ +I °' +I °° +I
b
~ oo c~ W
_>, o~ N ~ oo
°° ~ ~ +~ '~ +I '~ +I
a,
_~ a4 N O \O O~ t~ M
+~ ~' +~
U
M
~ 00 ~ ~ O O~
N ~ ~ pv ~' ~ OOv ~
H ~ c " N +I N +I ''' +I
O
cd
00
U
N
s' N
'
h a oo
~D
~t N ~O V1
o
o
a.7~ ~ 0~0 p ~ ~ ..O
~
' +I ~ '~ +I
+I
d a
..
0
b
o
~"o
.
~
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a,
o 0 0
~ o ~ ~ ~ _
'n -
o o ~ o
b ~ b
C7 ~ c~
.O O O
z
0 0
O
V
x
31
CA 02401357 2002-08-27
Adipose tissue weight: Compared to the normal group, the PBS-administered
group
showed a significant decrease (*). Compared to the PBS-administered group, the
decrease
was significantly inhibited in the humanized antibody-administered group (#).
Muscle tissue weight: Compared to the normal group, the PBS-administered group
showed a significant decrease (*). Compared to the PBS-administered group, the
decrease
was significantly inhibited in the humanized antibody-administered group (#).
(3) Effect on serum biochemical levels (glucose, triglyceride, and cholesterol
levels)
(Table 3)
Compared to the normal group, the PBS-administered group showed a significant
decrease in serum glucose level (*), which was not improved by administration
with the
humanized antibody. The PBS administered group showed a significant decrease
in serum
triglyceride level, compared to the normal group; and the humanized antibody-
administered
group showed an inhibited decrease, which was not a significant inhibition, in
serum
triglyceride level. The serum cholesterol level did not change in the PBS-
administered and
the humanized antibody-administered groups, and also the normal group.
4-3. Conclusion
Cancer cachexia is a clinical syndrome which causes in cancer patients a
significant
body weight loss due to loss of appetite, hypermetabolism or the like. The
serum
biochemical characteristics of cancer cachexia include low glucose level and
hyperlipidemia.
Some tumors are known to induce cancer cachexia in a tumor transplantation
model.
Typical tumor strains include SEKI melanoma (human melanoma), Colon 26 (mouse
colon
cancer cell line), and OCC-1-JCK (human oral cavity cancer cell line).
Transplantation of
such a tumor into a nude mouse results in a significant body weight loss.
32
CA 02401357 2002-08-27
On the other hand, a significant body weight loss similar to that in cancer
cachexia is
also observed in a HHM model. PTHrP has been determined as a causative agent
for HHM
(L.J. Suva, et al, Science 237, 893-896, 1987).
Accordingly, the present inventors prepared cancer cachexia models
transplanted with
either of 2 tumor strains, human oral cavity cancer cell line OCC-1-JCK and
mouse colon
cancer cell line Colon 26, and examined the effect of humanized antibodies in
the models.
It was showed that the body weight (and further the adipose tissue weight and
the
muscle tissue weight) started to decrease markedly after transplantation, on
day 10 in OCC-1-
JCK transplanted mice, or on day 7 in Colon 26 transplanted mice. Both types
of the
transplanted mice also showed in a low glucose state, which was a clinical
feature of cancer
cachexia. These models exhibited hypercalcemia, suggesting a complication of
cancer
cachexia with HHM.
When humanized antibodies were administered to the above 2 types of model
mice, a
significant improvement effect was observed not only in body weight and blood
iCa level, but
also in adipose tissue weight and muscle tissue weight. Moreover, an
improvement effect on
low triglyceride level was observed in OCC-1-JCK transplanted mice. That is,
OCC-1-JCK
mice showed the tendency, though not significant, to improve low glucose
level.
Thus, in the present invention, the humanized anti-PTHrP antibody is useful as
a
therapeutic agent or a preventive agent for tissue degradation associated with
various diseases,
such as cancer cachexia, because the antibody exhibited a tissue degradation
inhibitory effect
on adipose tissue and muscle tissue.
(Example 2] Test (2) Inhibition of tissue degradation
33
CA 02401357 2002-08-27
1. Humanized anti-PTHrP antibody
Humanized anti-PTHrP antibodies used herein were those prepared in Example 1.
2. Experimental animal
Rats used herein were nude rats F344/N Jcl-rnu (CLEA JAPAN, INC)
transplantable
with a human tumor cell line and capable of stably producing a HHM model. 80
rats at 5
weeks old were purchased, and then acclimatized for about 1 week. Next, SS
rats randomly
chosen therefrom were transplanted with tumors. The rats were euthanized by
ether
inhalation.
The rearing conditions for rats were as follows.
Room's temperature : 24 ~ 2°C
Relative humidity : 55 ~ 10%
Ventilation frequency : 10 to 30 times/hour
Lighting time : 5:00 to 19:00
Rearing cage and rearing density: 8 to 11 rats were reared per cage (a
stainless wire mesh
lifting cage with 660 x 310 x 200 mm in dimension) during the acclimatization
period and the
period ranging from tumor transplantation to the start of the experiment.
During the
experiment, one rat was reared per polycarbonate cage (265 x 425 x 160 mm in
dimension).
Feed and feeding method: CE-2 (CLEA JAPAN, INC.), free feeding
Water and supply method: tap water, free intake of water
Routine management: according to management criteria for rearing experimental
animals
(edited by the Experimental Animal Care Committee of Chugai Pharmaceutical
Co., Ltd.)
3. Experimental method
3-1. Preparation of disease model
34
CA 02401357 2002-08-27
Tumor strains used: LC-6-JCK (human lung large cell carcinoma strain)
Tumor strain source: Purchased from Central Institute for Experimental Animals
Subculture of tumor strain: Subculturing was performed by transplanting
subcutaneously a tumor block and growing the tumor for an in vivo tumor strain
into mice
(BALB/cA Jcl-nu; CLEA JAPAN, INC.). Subculturing was performed every 3 to 4
weeks.
Preparation of model: Mice having the subcultured tumor strains were
sacrificed by
cervical-vertebral dislocation. Then, the tumor was excised from the mice to
prepare a 3
mm block, and then transplanted subcutaneously to a rat, thereby preparing a
model rat.
Transplantation was performed for 55 rats randomly chosen from the 80 rats.
The remaining
25 rats were untreated.
Criteria for model establishment: A rat individual having a visible
subcutaneous
growth of the transplanted tumor and showing higher blood iCa level than that
of a normal
group was determined as a HHM model rat. In this experiment, a rat showing an
iCa level
of 1.80 mmol/L, or more was used.
Grouping method: Blood iCa level was measured on day 49 after tumor
transplantation, so as to confirm whether or not a HHM model had been
established. HHM
models were classified using the blood iCa level as an index. 24 rats were
randomly chosen
therefrom and divided evenly into 3 groups (8 rats/group): a saline-
administered group, a
humanized antibody-administered group, and a tumor-excised group. Further, 8
rats were
randomly chosen from the 25 untreated rats and grouped as a normal group.
3-2. Measurement of blood ionized calcium level (blood iCa level)
CA 02401357 2002-08-27
Blood was collected via the tail vein, and then immediately measured using a
calcium
analyzer (automatic pH/Ca++ analyzer Chiron 634, Bayer Medical).
3-3. Collection of sera and measurement of biochemical levels (glucose,
triglyceride,
cholesterol and free fatty acid levels), cytokines (human IL-6, human G-CSF,
human IL-11,
and human LIF) and rat a~-AG
Blood was collected from a rat under anesthesia. Blood collected from the
descending aorta into a Separapid tube (SEKISUI CHEMICAL Co., Ltd.) was
allowed to
stand at room temperature for 30 min or more. The serum was separated by
centrifugation
(3000 rpm, 20 min, HITACHI, OSPR-22), and then frozen and stored at or below -
20~ .
Serum biochemical levels (glucose, triglyceride, cholesterol and free fatty
acid levels)
were measured using an autoanalyzer (HITACHI, type 7170, autoanalyzer).
Cytokines (human IL-6, human G-CSF, human IL-11 and human LIF) and rat al-AG
were respectively measured using a commercially available EIA kit, Quantikine~
human IL-6
(R&D systems), Quantikine° human G-CSF (R&D systems), Quantikine~ human
IL-11
(R&D systems), LIF/HILDA EASIAiK2 (BIOSOURCE), and Panatest~ A series rat al-
AG
(Panafarm Laboratories).
3-4. Measurement of leukocyte count
20 p1 of blood was collected via the tail vein, and then immediately suspended
in a cell
pack (PL30L). Then, the leukocyte count was measured using Sysmex Microcell
counter F-
800.
3-5. Measurement of adipose tissue weight and muscle tissue weight
Adipose tissues (periphery of orchis) and muscle tissues (gastrocnemius
muscle) were
36
CA 02401357 2002-08-27
excised, and then the weights were measured using 5HIMAZU LIBROR EB-330H with
SHIMAZU EP-50 printer.
3-6. Administration of humanized antibody
A dose of 3.0 mg/kg of humanized antibodies was administered intravenously
(i.v.) (a
humanized antibody-administered group). Physiological saline was intravenously
administered to a negative control group (a saline-administered group). The
administered
solutions were used at a dose of 0.1 mL was administered per 100g of body
weight. Nothing
was administered to the normal group that had not been transplanted with
tumors.
Excision of tumor:
The skin at the root portion of the tumor was tied with a vinyl string (Tie
band: a band
for an sterile bag) so as to be enclosed by the string (a tumor-excised
group). A few hours
later and on the next day, the string was stretched and tied again. On the
next day, the tumors
were confirmed as being necrotized.
Test schedule:
On day 49 after tumor transplantation, body weight, blood iCa level and
leukocyte
count were measured. Based on the measurement results, grouping was performed.
Then,
for the saline-administered group and the humanized antibody-administered
group, saline or
humanized antibodies were administered, respectively. For the tumor-excised
group, tumors
were excised therefrom. On days 1, 4, 7 and 10 after administration, body
weight, blood iCa
level and leukocyte count were measured. On day 10 after administration,
adipose tissue
weight and muscle tissue weight were measured, blood and serum were collected,
and then
serum biochemical levels and cytokines were measured.
3-7. Method for statistical analysis
37
CA 02401357 2002-08-27
An unpaired t-test was performed for the differences in the mean values of the
normal
group and the saline-administered group on each day of measurement. In this
test, first, an
F-test was performed for variances. When an F-value was 5 % or more, Student's
t-test for
equal variance was performed, and when an F-value was 5 % or less, Welch's t-
test for
unequal variance was performed at 5 % two-sided significance level. Next,
Dunnett's
multiple comparison test was performed at 5 % two-sided significance level,
for the
differences in the mean values on each day of measurement between the saline-
administered
group, and the humanized antibody-administered group or the tumor-excised
group. SAS
Ver. 6.12 was used for the statistical analysis.
4. Result
4-1. Effect on body weight and blood iCa level (Fig. 3)
Body weight (Fig. 3, upper panel): Throughout the days of measurement, the
saline-
administered group (-~-) showed significant decreases in body weight compared
to the
normal group (-1-) (p<0.05 compared to the normal group, measured by t-test).
Compared
to the saline-administered group, the body weight started to significantly
increase in the
humanized antibody-administered group (-~-) from day 4 after administration,
and showed
significant differences up to days 7 and 10 (#: p<0.05 compared to the saline-
administered
group, measured by Dunnett's multiple comparison test). The tumor-excised
group (-~-)
showed an equivalent recovery from body weight loss, and showed a significant
difference on
days 4, 7 and 10 after administration compared to the saline-administered
group (#).
Blood iCa level (Fig. 3, lower panel): Throughout the days of measurement, the
saline-
administered group showed a significantly higher blood iCa level compaired to
the normal
group. Compared to the saline-adnunistered group, the humanized antibody-
administered
group showed a significantly inhibited increase in blood iCa level on days 1,
4, 7 and 10 after
administration (#). The tumor-excised group also showed a significantly
inhibited increase
38
CA 02401357 2002-08-27
in blood iCa level compared to the saline-administered group on days l, 4, 7
and 10 after
administration (#).
4-2: Effect on adipose tissue (periphery of orchis) weight, and muscle tissue
(gastrocnemius muscle) weight (Table 4)
Adipose tissue weight: The saline-administered group showed a significant
decrease
compared to the normal group (*). Compared to the saline-administered group,
the decrease
was significantly inhibited in the humanized antibody-administered group and
the tumor-
excised group (#).
Muscle tissue weight: The saline-administered group showed a significant
decrease
compared to the normal group (*). Compared to the saline-administered group,
the decrease
was significantly inhibited in the humanized antibody-administered group and
the tumor-
excised group (#).
4-3. Effect on serum biochemical levels (glucose, triglyceride, cholesterol
and free
fatty acid levels) (Table 4)
Compared to the normal group, the saline-administered group showed significant
decreases in glucose and free fatty acid levels, and an increase in
cholesterol level (*).
Compared to the saline-administered group, the humanized antibody-administered
group
showed a significant decrease in cholesterol level (#); however, none of the
remaining
parameters were significantly improved. The tumor-excised group showed
improving
effects on glucose and cholesterol levels (#).
39
CA 02401357 2002-08-27
b
o
i W ~ a\ ~ O
O .-.
b '# ~ N x ~ Os
N
N ~ ~? ~D C) M ~
~O l~ <t
c4
U
N
'O ~O
N b N ~ l~ ~ I~ ~ d;
N +I '''' +I N +I r' +I
00 V7 N l'~
O b O~~MM~~O~O
U
b~ ~ N v_~ N ~ ~N ~ N
Q.
N 09
U
N
c
U '"'O p~ V1 V7 ~ 00 V ~p O G'
~ ~ ~ ~
H ~ ~ ~ +I +I r' +I ~
+I
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N a)
n O
U p
O
N p 4
.b ~O
O p ~ ~ ~ Ov y N
~
p ~ 0 M ~
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_
N N +~ ~ +I rr v'~ v~
+I +I C
Y O
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" w N A
'b
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C~.
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~0 C
O au.7 ~ ~ G
y by ~ O N N N N
O b
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O
c
f.~w ~ ~p 't7 O U c
C
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. O v~
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U ~ o
~ i ~ ~ v
O
O ctt O b~1 by
z ~ ~ ~ z w w
H Q Q
N .. ..
~k
O
x
CA 02401357 2002-08-27
4-4. Effect on cytokines (hIL-6, hG-CSF, hIL-11 and hLIF) and ral-AG
(Table 5)
Compared to the normal group, the saline-administered group showed significant
increases in hIL-6, hG-CSF, hIL-11 and hLIF (*). The saline-administered group
also
showed an increase in ray-AG (al acid glycoprotein) which is induced by
inflammatory
cytokines (for example, hIL-6, hIL-11 and LIF). The humanized antibody-
administered
group showed no reducing effect compared to that of the saline-administered
group.
Compared to the saline-administered group, the tumor-excised group showed
decreases in
hIL-6, hG-CSF, hIL-11 and hLIF in addition to a decrease in ray-AG which is
induced by
inflammatory cytokines, such as hIL-6, hIL-11 and LIF.
41
CA 02401357 2002-08-27
~t ~G ~ r
+I Q ~ ~ ~ ~ ~ O ~ ~ N
v7 N
cY~C ~ O ~'~ ~ +~
_w _
0,.., O~ ~ ~ ~ ~ M
Q.
,~ ~ N N 0~1 x ~ 00
_ M 00 Op
~ aUM N
w
00 .--n
'~ U ~ oo ~. o, o o
.a:C7 ~ d; vi ~-i ~ ~ ~ p
N p
cV '~ ,-. c.j
H ~ " M +I ~~ +I ~ +I d' +i
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'b
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A.
n
O fn .b
~ 00 ~ p ~ t3
i
.. ~ O O .
.i O ~
O O O ~
. M w
.
' ~ +I M +I ~ +I +I
~ ~ b ~
a, cd
s ~ N
~
O ~ ~ G
b ' N
A i
O O .a
~ '-'
~J _
.~ U .b
c
p N ~ L~ C~,
Q ~
,
~D '~" O C~.' ~ O .a0-n
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~ . ~ ~ . O
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1
~ O
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O WO ~ ~ ~U Uf", ~
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s..~.,' 00 ~, 00 ' ~ ~ N O N ,-,
~ U f~ 00 b0
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'U W
.D '
cd ~ O z p
Z N ~ b V
~ v O G 4;
p U
~
c'~ ~ H +:
on
..
a Q a
x .. ..
42
CA 02401357 2002-08-27
4-5. Effect on leukocyte (Fig. 4)
Compared to the normal group, the saline-administered group showed a
significant
increase in the leukocyte count throughout the days of measurement (p<0.05
compared to the
normal group, measured by t-test). The humanized antibody-administered group
showed a
significant decrease in the leukocyte count on day 4 after administration
compared to the
saline-administered group (# significance level: 5%, measured by Dunnett's
multiple
comparison), but showed no difference on other days of measurement. On days 1,
4, 7 and
after administration, the tumor-excised group showed significant decreases
(#).
#: There was a significant difference compared to the saline-administered
group.
5. Conclusion
The present inventors prepared a cancer cachexia model rat transplanted with
the
human lung large cell carcinoma strain LC-6-JCK, and studied the effect of the
humanized
antibody by comparing the model with the tumor-excised model.
After tumor transplantation, LC-6-JCK-transplanted rats developed
hypercalcemia
with marked body weight loss, thereby establishing a model developing cancer
cachexia
together with HHM. The model also showed the clinical features of cancer
cachexia, such
as decreased adipose tissue weight and muscle tissue weight, and lowered
glucose level, in
addition to production of cytokines derived from tumors and a protein induced
therefrom, and
increased leukocyte count.
When the humanized antibody was administered to the above model rat,
significant
improving effects were observed not only in body weight and blood iCa level,
but also in
adipose tissue weight and in muscle tissue weight. Moreover, a tendency to
improve low
glucose level, though not a significant effect, was observed. However, no
inhibiting agenty
effect was observed on production of cytokines derived from tumors and the
protein induced
therefrom, or on leukocytes.
43
CA 02401357 2002-08-27
When tumors were excised, significant improving effects at the level
equivalent to that
resulting from administration of the antibody were observed on body weight,
blood iCa level,
adipose tissue weight and muscle tissue weight. Improving effects on low
glucose level and
on high cholesterol level were observed. Inhibiting agenty effects were also
observed on
production of cytokines derived from tumors and the protein induced therefrom.
Further, an
inhibiting agenty effect was observed on an increase in leukocyte count.
In the present invention, the humanized anti-PTHrP antibody exhibited an
inhibiting
agenty effect on tissue degradation of the adipose tissue and the muscle
tissue, suggesting that
the antibody is useful as a therapeutic agent for tissue degradation
associated with various
diseases, such as cancer cachexia. Based on the fact that production of
cytokines derived
from tumors was not inhibited by the administration of the antibody, the
present inventors
have found that even in a state of a high blood level of cytokines, such as IL-
6, G-CSF, IL-11,
LIF and al-AG, administration of the substance of the present invention that
inhibits the
binding of PTHrP to its receptor can inhibit tissue degradation associated
with various
diseases. Particularly, the present inventors have found that administration
of the substance
of the present invention that inhibits the binding of PTHrP to its receptor
can inhibit tissue
degradation associated with various diseases, even in the presence of
inflammatory cytokines
at high concentration, such as IL-6, IL-11 and LIF, which are believed to
involve tissue
degradation (for example, as described in Clinical Science, 89, 431-439,
1995).
[REFERENCE EXAMPLE 1 ]
Preparation of hybridomas producing anti-PTHrP (1-34) mouse monoclonal
antibody
Hybridomas capable of producing a monoclonal antibody against human PTHrP (1-
34) (SEQ ID NO: 75), #23-57-154 and #23-57-137-1, were prepared as follows
(see Sato, K.
et al., J. Bone Miner. Res. 8, 849-860, 1993). The amino acid sequence of the
human PTHrP
(1-34) is shown in SEQ ID N0:75.
44
CA 02401357 2002-08-27
For use as an immunogen, PTHrP (1-34) (Peninsula) was conjugated with a
carrier
protein thyroglobulin using carbodiimide (Dojinn). The thycloglobulin-
conjugated PTHrP
(1-34) was dialyzed to obtain a solution having a protein concentration of 2
~.g/ml. The
resulting solution was mixed with Freund's adjuvant (Difco) at a mixing ratio
of 1:1 to give an
emulsion. This emulsion was injected to 16 female BALB/C mice 11 times
subcutaneously
at the back or intraperitoneally at a dose level of 100 gg/mouse for each
injection, thereby
immunizing the mice. For the priming immunization, Freund's complete adjuvant
was used;
while for the boosting immunization, Freund's incomplete adjuvant was used.
Each of the immunized mice was determined for its antibody titer in the serum
in
the following manner. That is, each of the mice was blood-drawn via its tail
vein, and the
anti-serum is separated from the blood. The anti-serum was diluted with a RIA
buffer and
mixed with lzsI-labeled PTHrP (1-34) to determine binding activity. The mice
that were
confirmed to have a sufficiently increased titer were injected with PTHrP (1-
34) without a
carrier protein intraperitoneally at a dose level of 50 g.g/mouse for the
final immunization.
Three days after the final immunization, the mouse is sacrificed and the
spleen was
removed therefrom. The spleen cells were subjected to cell fusion with mouse
myeloma cell
line P3x63Ag8U.1 in accordance with a conventional known method using 50%
polyethylene
glycol 4000. The fused cells thus prepared were seeded to each well of eighty-
five 96-well
plates at a density of 2 x 104iwe11. Hybridomas were screened in HAT medium as
follows.
The screening of hybridomas was performed by determining the presence of
PTHrP-recognition antibodies in the culture supernatant of the wells in which
cell growth had
been observed in HAT medium, by solid phase RIA method. The hybridomas were
collected
from the wells in which binding ability to the PTHrP-recognition antibodies
had been
CA 02401357 2002-08-27
confirmed. The hybridomas thus obtained was suspended into RPMI-1640 medium
containing 15% FCS supplemented with OPI-supplement (Sigma), followed by
unification of
the hybridomas by limiting dilution method. Thus, two types of hybridoma
clones, #23-57-
154 and #23-57-137-l, could be obtained, both which had a high binding ability
to PTHrP (1-
34).
Hybridoma clone #23-57-137-1 was designated as "mouse-mouse hybridoma #23-
57-137-1 ", and has been deposited under the terms of the Budapest Treaty on
August 15, 1996
at the International Patent Organism Depositary (IPOD), National Institute of
Advanced
Industrial Science and Technology, Japan (1-1, Higashi 1-chome, Tsukuba-shi,
Ibaraki, Japan)
under the accession No. FERM BP-5631.
[REFERENCE EXAMPLE 2]
Cloning of DNAs encoding V-regions of mouse monoclonal antibody against human
PTHrP
(1-34)
Cloning of DNAs encoding the V-regions of a mouse monoclonal antibody against
human PTHrP (1-34), #23-57-137-1, was performed in the following manner.
( 1 ) Preparation of mRNA
mRNA from hybridoma #23-57-137-1 was prepared using Quick Prep mRNA
Purification Kit (Pharmacia Biotech). That is, cells of hybridoma #23-57-137-1
were fully
homogenized with an extraction buffer, and mRNA was isolated and purified
therefrom on an
oligo(dT)-Cellulose Spun Column in accordance with the instructions included
in the kit. The
resulting solution was subjected to ethanol precipitation to obtain the mRNA
as a precipitate.
The mRNA precipitate was dissolved in an elution buffer.
(2) Production and amplification of cDNA far gene encoding mouse H-chain V-
region
46
CA 02401357 2002-08-27
(i) Cloning of cDNA for #23-57-137-1 antibody H-chain V-region
A gene encoding H-chain V-region of the mouse monoclonal antibody against
human PTHrP was cloned by 5'-RACE method (Frohman, M. A. et al., Proc. Natl.
Acad. Sci.
USA, 85, 8998-9002, 1988; Belyavsky, A. et al., Nucleic Acids Res. 17, 2919-
2932, 1989).
The 5'-RACE method was performed using 5'-Ampli FINDER RACE Kit (CLONETECH) in
accordance with the instructions included in the kit. In this method, the
primer used for
synthesis of cDNA was MHC2 primer (SEQ ID NO: 1) which is capable of
hybridizing to
mouse H-chain C-region. The above-prepared mRNA (about 2 fig), which was a
template
for the cDNA synthesis, was mixed with MHC2 primer (10 pmoles). The resulting
mixture
was reacted with a reverse transcriptase at 52~C for 30 minuets to effect the
reverse
transcription of the mRNA into cDNA.
The resulting reaction solution was added with 6N NaOH to hydrolyze any RNA
remaining therein (at 65~ for 30 min.) and then subjected to ethanol
precipitation to isolate
and purify the cDNA as a precipitate. The purified cDNA was ligated to Ampli
FINDER
Anchor (SEQ ID NO: 42) at the 5' end by reacting with T4 RNA ligase at 37~C
for 6 hours
and additionally at room temperature for 16 hours. As the primers for
amplification of the
cDNA by PCR method, Anchor primer (SEQ ID NO: 2) and MHC-G 1 primer (SEQ ID
NO:
3) (S.T. Jones, et al., Biotechnology, 9, 88, 1991) were used.
The PCR solution comprised (per 50 p1) 10 mM Tris-HCl (pH 8.3), 50 mM KCI,
0.25 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl2, 2.5 units of TaKaRa Taq
(Takara Shuzo Co., Ltd.), 10 pmoles Anchor primer, and 1 p1 of the reaction
mixture of the
cDNA to which MHC-G1 primer and Ampli FINDER Anchor primer had been ligated,
over
which mineral oil (50 ~1) was layered. The PCR was performed on Thermal Cycler
Model
480J (Perkin Elmer) for 30 cycles under the conditions: 94~ for 45 sec.; 60~
for 45 sec.;
and 72°C for 2 min.
47
CA 02401357 2002-08-27
,. "
(ii) Cloning of cDNA for #23-57-137-1 antibody L-chain V-region
A gene encoding L-chain V-region of the mouse monoclonal antibody against
human PTHrP was cloned by 5'-RACE method (Frohman, M. A. et al., Proc. Natl.
Acad. Sci.
USA, 85, 8998-9002, 1988; Belyavsky, A. et al., Nucleic Acids Res. 17, 2919-
2932, 1989).
The 5'-RACE method was performed using 5'-Ampli Finder RACE Kit (CLONETECH) in
accordance with the instructions included in the kit. In this method, oligo-dT
primer was
used as the primer for synthesizing cDNA. The above-prepared mRNA (about 2
fig), which
was a template for the cDNA synthesis, was mixed with oligo-dT primer. The
resulting
mixture was reacted with a reverse transcriptase at 52°C for 30 min. to
effect the reverse
transcription of the mRNA into cDNA. The resulting reaction solution was added
with 6N
NaOH to hydrolyze any RNA remaining therein (at 65°C for 30 min.). The
resulting
solution was subjected to ethanol precipitation to isolate and purified the
cDNA as a
precipitate. The cDNA thus synthesized was ligated to Ampli FINDER Anchor at
the 5' end
by reacting with T4 RNA ligase at 37~ for 6 hours and additionally at room
temperature for
16 hours.
A PCR primer MLC (SEQ ID NO: 4) was designed based on the conserved
sequence of mouse L-chain ~ chain C-region and then synthesized using 394
DNA/RNA
Synthesizer (ABI). The PCR solution comprised (per 100 g.1) 10 mM Tris-HCl (pH
8.3), 50
mM KCI, 0.25 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl2, 2.5 units of
AmpliTaq (PERKIN ELMER), 50 pmoles of Anchor primer (SEQ ID NO: 2), and 1 ~1
of the
reaction mixture of the cDNA to which MLC (SEQ m NO: 4) and Ampli FINDER
Anchor
were ligated, over which mineral oil (50 ~,l) was layered. The PCR reaction
was performed
on Thermal Cycler Model 4803 (Perkin Elmer) for 35 cycles under the
conditions: 94°C for
45 sec.; 60~ for 45 sec.; and 72°C for 2 min.
48
CA 02401357 2002-08-27
(3) Purification and fragmentation of PCR products
Each of the DNA fragments amplified by PCR method described above was
separated by agarose gel electrophoresis on a 3% Nu Sieve GTG agarose (FMC
Bio.
Products). For each of the H-chain V-region and the L-chain V-region, an
agarose gel
segment containing a DNA fragment of about 550 by was excised from the gel.
Each of the
gel segments was subjected to purification of the DNA fragment of interest
using
GENECLEAN II Kit (8I0101) in accordance with the instructions included in the
kit. The
purified DNA was precipitated with ethanol, and the DNA precipitate was
dissolved in 20 p,1
of a solution containing 10 mM Tris-HCl(pH 7.4) and 1 mM EDTA. An aliquot (1
p1) of the
DNA solution was digested with a restriction enzyme XmaI (New England Biolabs)
at 37°C
for 1 hour and further digested with a restriction enzyme EcoRI (Takara 5huzo
Co., Ltd.) at
37~ for 1 hour. The digestion solution was extracted with phenol and
chloroform and then
precipitated with ethanol to collect the DNA.
In this manner, two DNA fragments containing a gene encoding mouse H-chain V-
region and a gene encoding mouse L-chain V-region, respectively, were
obtained, both which
had an EcoRI recognition sequence on the 5' end and an XmaI recognition
sequence on the 3'
end.
The EcoRI-XmaI DNA fragments containing a gene encoding mouse H-chain V-
region and a gene encoding mouse L-chain V-region, respectively, were
separately ligated to
pUC 19 vector that had been digested with EcoRI and XmaI at 16~C for 1 hour
using DNA
Ligation Kit ver.2 (Takara Shuzo Co., Ltd.) in accordance with the
instructions included in the
kit. An aliquot (10 p1) of the ligation mixture was added to 100 p.1 of a
solution containing
competent cells of E. coli, JM 109 (Nippon Gene Co., Ltd.). The cell mixture
was allowed
to stand on ice for 15 min., at 42~ for 1 min. and additionally for 1 min. on
ice. The
resulting cell mixture was added with 300 p1 of SOC medium (Molecular Cloning:
A
49
CA 02401357 2002-08-27
Laboratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press,
1989) and then
incubated at 37°C for 30 min. The resulting cell solution was plated on
LB agar medium or
2xYT agar medium (Molecular Cloning: A Laboratory Manual, Sambrook, et al.,
Cold Spring
Harbor Laboratory Press, 1989) containing either 100 or 50 pg/ml of
ampicillin, 0.1 mM of
IPTG and 20 ~g/ml of X-gal, and then incubated at 37°C overnight. In
this manner, E. coli
transformants were prepared.
The transformants were cultured at 37~ overnight in 2 ml of LB or 2xYT medium
containing either 100 or 50 pg/ml of ampicillin. The cell fraction was applied
to Plasmid
Extracter PI-100( (Kurabo Industries, Ltd.) or QIAprep Spin Plasmid Kit
(QIAGEN) to give a
plasmid DNA. The plasmid DNA was sequenced as follows.
(4) Sequencing of genes encoding mouse antibody V-regions
The nucleotide sequence of the cDNA coding region carried on the plasmid was
determined in DNA Sequencer 373A (ABI; Perkin-Elmer) using Dye Terminator
Cycle
Sequencing Kit (Perkin-Elmer). M13 Primer M4 (Takara Shuzo Co., Ltd.) (SEQ 117
NO: 5)
and M13 Primer RV (Takara Shuzo Co., Ltd.) (SEQ 117 NO: 6) were used as the
primers for
sequencing, and the nucleotide sequence was confirmed in the both directions.
The plasmid containing a gene encoding mouse H-chain V-region derived from
hybridoma #23-57-137-1 was designated as "MBC1H04", and the plasmid containing
a gene
encoding mouse L-chain V-region derived from hybridoma #23-57-137-1 was
designated as
"MBC 1 L24". The nucleotide sequences (including the corresponding amino acids
sequences) of the gene encoding the mouse #23-57-137-1 antibody-derived H-
chain V-region
in plasmid MBC1H04 and the gene encoding the mouse #23-57-137-1 antibody-
derived L-
chain V-region in plasmid MBC1H24 were shown in SEQ. ID Nos: 57 and 65,
respectively.
The amino acid sequences of the polypeptides for the H-chain V-region and the
L-chain V-
CA 02401357 2002-08-27
region were shown in SEQ. ID NOs: 46 and 45, respectively.
The E. coli strain containing the above plasmid MBC1H04 and the E. coli strain
containing the above plasmid MBC1L24 were designated as "Escherichia coli
JM109
(MBC 1H04)" and "Escherichia coli JM 109 (MBC 1L24)", respectively. These E.
coli strains
have been deposited under the terms of the Budapest Treaty at the
International Patent
Organism Depositary (IPOD), National Institute of Advanced Industrial Science
and
Technology, Japan (1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) on
August 15, 1996,
under the Accession No. FERM BP-5628 for Escherichia coli JM109 (MBC1H04) and
FERM
BP-5627 for Escherichia coli JM 109 (MBC 1 L24), respectively.
(5) Determination of CDRs of mouse monoclonal antibody #23-57-137-1 against
human
PTHrP
The H-chain V-region and the L-chain V-region have general structures similar
to
each other, each of which has four framework regions (FRs) linked through
three
hypervariable regions (i.e., complementarity determining regions; CDRs). The
amino acid
sequences of the FRs are relatively well conserved, while the amino acid
sequence of the
CDRs have an extremely high variability (Kabat, E.A. et al., "Sequence of
Proteins of
Immunological Interest", US Dept. Health and Human Services, 1983).
In view of these facts, the homology in amino acid between the V-regions of
the
mouse monoclonal antibody against human PTHrP was determined with reference to
the
database of amino acid sequences of antibodies established by Kabat et al.
Thus, the CDRs
of the V-regions were determined as shown in Table 6.
The amino acid sequences of CDRs 1-3 in the L-chain V-region are shown in SEQ
ID Nos: 59 to 61, respectively; and the amino acid sequences of CDRs 1-3 in
the H-chain V-
51
CA 02401357 2002-08-27
a
region are shown in SEQ ID Nos: 62 to 64, respectively.
Table 6
V-region SEQ ID NO. CDR 1 CDR2 CDR3
H-chain V-region57 31-35 50-66 99-107
L-chain V-region65 23-34 50-60 93-105
[REFERENCE EXAMPLE 3J Construction of Chimeric Antibody
(1) Construction of chimeric antibody H-chain
(i) Construction of H-chain V-region
To ligate to an expression vector carrying a genomic DNA of human H-chain C-
region C r 1, the cloned DNA encoding mouse H-chain V-region was modified by
PCR
method. A backward primer MBC 1-S 1 (SEQ ID NO: 7) was designed to hybridize
to a
DNA sequence encoding the 5' region of the leader sequence of the V-region and
to have both
a Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol., 196, 947-950,
1987) and a
HindIII-recognition sequence. A forward primer MBC1-a (SEQ D7 NO: 8) was
designed to
hybridize to a DNA sequence encoding the 3' region of the J region and to have
both a donor
splice sequence and a BamHI-recognition sequence. The PCR reaction was
performed using
TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) and a buffer appended thereto. The PCR
solution
comprised (per 50 ~l) 0.07 ~,g of plasmid MBC 1 H04 as a template DNA, 50
pmoles of
MBC 1-a and 50 pmoles of MBC 1-S 1 as primers, 2.5U of TaKaRa Ex Taq and 0.25
mM
dNTPs in the buffer, over which 50 ~,l of mineral oil was layered. The PCR was
run for 30
cycles under the conditions: 9490 for 1 min.; 55~ for 1 min.; 72~ for 2 min.
The DNA
fragments thus amplified by the PCR method were separated by agarose gel
electrophoresis
on a 3% Nu Sieve GTG Agarose (FMC Bio. Products).
52
CA 02401357 2002-08-27
Then, an agarose gel segment containing a DNA fragment of 437 by was excised,
and the DNA fragment was purified therefrom using GENECLEAN II Kit (BIO101) in
accordance with the instructions included in the kit. The purified DNA was
collected by
ethanol precipitation, and then dissolved in 20 p1 of a solution containing 10
mM Tris-HCl
(pH 7.4) and 1 mM EDTA. An aliquot (1 p1) of the resulting DNA solution was
digested
with restriction enzymes BamHI and HindIII (Takara Shuzo Co., Ltd.) at
37°C for 1 hour.
The digestion solution was extracted with phenol and chloroform and then
precipitated with
ethanol to collect the DNA of interest.
The obtained HindIII-BamHI DNA fragment, which containing a gene encoding the
mouse H-chain V-region, was subcloned into pUC 19 vector that had been
digested with
HindIII and BamHI. The resulting plasmid was sequenced on DNA Sequencer 373A
(Perkin-Elmer) using M 13 Primer M4 and M 13 Primer RV as primers and Dye
Terminator
Cycle Sequencing Kit (Perkin-Elmer). As a result, a plasmid which carried a
gene of correct
nucleotide sequence encoding the mouse H-chain V-region derived from hybridoma
#23-57-
137-1 and had a HindIII-recognition sequence and a Kozak sequence on its 5'
region and a
BamHI-recognition sequence on its 3' region was obtained, which was designated
as
"MBC lHIpUC 19".
(ii) Construction of H-chain V-region for preparation of cDNA-type of mouse-
human
chimeric H-chain
To ligate to cDNA of the human H-chain C-region C r l, the DNA encoding the
mouse H-chain V-region constructed as described above was modified by PCR
method. A
backward primer MBC1HVS2 (SEQ ID NO: 9) for the V-region was designed to cause
the
replacement of the second amino acid (asparagine) of the sequence encoding the
front part of
the leader sequence of the H-chain V-region by glycine and to have a Kozak
consensus
sequence (Kozak, M. et al., J. Mol. Biol., 196, 947-950, 1987) and HindIII-
and EcoRI-
53
CA 02401357 2002-08-27
recognition sequences. A forward primer MBC1HVR2 (SEQ ID NO: 10) for the H-
chain V-
region was designed to hybridize to a DNA sequence encoding the 3' region of
the J region, to
encoding the 5' region of the C-region and to have ApaI- and SmaI-recognition
sequences.
The PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.)
and a buffer appended thereto. The PCR solution comprised (per 50 p1) 0.6 p.g
of plasmid
MBC1H/pUCl9 as a template DNA, 50 pmoles of MBC1HVS2 and SO pmoles of
MBC1HVR2 as primers, 2.5U of TaKaRa Ex Taq and 0.25 mM of dNTPs in the buffer,
over
which 50 ~1 of mineral oil was layered. The PCR reaction was run for 30 cycles
under the
conditions: 94°C for 1 min.; 55°C for 1 min.; 72~ for 1 min. The
DNA fragments
amplified by the PCR reaction were separated by agarose gel electrophoresis on
a 1 % Sea
Kem GTG Agarose (FMC Bio. Products). Then, an agarose gel segment containing a
DNA
fragment of 456 by was excised and the DNA fragment was purified therefrom
using
GENECLEAN II Kit (BIO101) in accordance with the instructions included in the
kit. The
purified DNA was precipitated with ethanol and then dissolved in 20 ~.l of a
solution
containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA.
The resulting DNA solution ( 1 p,g) was digested with restriction enzymes
EcoRI
and SmaI (Takara Shuzo Co., Ltd.) at 37°C for 1 hour. The digestion
solution was extracted
with phenol and chloroform and then precipitated with ethanol to collect the
DNA. The
obtained EcoRI-SmaI DNA fragment, which containing a gene encoding the mouse H-
chain
V-region, was subcloned into pUCl9 vector that had been digested with EcoRI
and SmaI.
The resulting plasmid was sequenced on DNA Sequencer 373A (Perkin-Elmer) using
M 13
Primer M4 and M 13 Primer RV, and Dye Terminator Cycle Sequencing Kit (Perkin-
Elmer).
As a result, a plasmid which contained a gene of correct nucleotide sequence
encoding mouse
H-chain V-region derived from hybridoma #23-57-137-1 and had EcoRI- and
HindIII-
recognition sequences and a Kozak sequence on its 5' region and ApaI- and SmaI-
recognition
54
CA 02401357 2002-08-27
sequences on its 3' region was obtained, which was designated as "MBC 1Hv/pUC
19".
(iii) Construction of expression vector for chimeric antibody H-chain
cDNA containing the DNA for human antibody H-chain C-region C ?' 1 was
prepared as follows. mRNA was prepared from a CHO cell into which both an
expression
vector DHFR-DE-RVh-PM-1-f (see WO 92/19759) encoding the genomic DNAs of
humanized PM 1 antibody H-chain V-region and human antibody H-chain C-region
IgG 1 (N.
Takahashi et al., Cell 29, 671-679, 1982) and an expression vector RV1-PMIa
(see WO
92/19759) encoding the genomic DNAs of humanized PMl antibody L-chain V-region
and
human antibody L-chain r~ chain C-region had been introduced. Using the mRNA,
cDNA
containing the humanized PM 1 antibody H-chain V-region and the human antibody
C-region
C ?' 1 was cloned by RT-PCR method, and then subcloned into plasmid pUC 19 at
the HindIII-
BamHI site. After sequencing, a plasmid which had the correct nucleotide
sequence was
obtained, which was designated as "pRVh-PMlf cDNA".
An expression vector DHFR- D E-RVh-PM-1-f in which both a HindIII site located
between SV40 promoter and a DHFR gene and an EcoRI site located between EF-1 a
promoter and a humanized PM 1 antibody H-chain V-region gene had been deleted,
was
prepared for the construction of an expression vector for cDNA containing the
humanized
PM 1 antibody H-chain V-region gene and the human antibody C-region C r 1
gene.
The plasmid obtained (pRVh-PMlf cDNA) was digested with BamHI, blunt-ended
with Klenow fragment, and further digested with HindIII, thereby obtaining a
blunt-ended
HindIII-BamHI fragment. The blunt-ended HindIII-BamHI fragment was ligated to
the
above-mentioned HindIII site- and EcoRI site-deleted expression vector DHFR- O
E-RVh-
PM1-f that had been digested with HindIII and BamHI. Thus, an expression
vector RVh-
PM 1 f cDNA was constructed which contained cDNA encoding the humanized PM 1
antibody
CA 02401357 2002-08-27
H-chain V-region and the human antibody C-region C 'Y 1.
The expression vector RVh-PM 1 f cDNA containing the cDNA encoding the
humanized PM 1 antibody H-chain V-region and the human antibody C-region C Y 1
was
digested with ApaI and BamHI, and a DNA fragment containing the H-chain C-
region was
collected therefrom. The resulting DNA fragment was introduced into the
plasmid
MBC 1Hv/pUC 19 that had been digested with ApaI and BamHI. The plasmid thus
prepared
was designated as "MBC IHcDNA/pUC 19". This plasmid contained cDNA encoding
the
mouse antibody H-chain V-region and the human antibody C-region C Y 1, and had
EcoRI-
and HindIII-recognition sequences on its 5' region and a BamHI-recognition
sequence on its
3' region.
The plasmid MBC 1 HcDNA/pUC 19 was digested with EcoRI and BamHI to give a
DNA fragment comprising a nucleotide sequence encoding the chimeric antibody H-
chain.
The resulting DNA fragment was introduced into an expression vector pCOS 1
that had been
digested with EcoRI and BamHI, thereby giving an expression vector for the
chimeric
antibody, which was designated as "MBC 1 HcDNA/pCOS 1 ". Here, the expression
vector
pCOSl was constructed using HEF-PMh-g Y 1 (see WO 92/19759) by deleting
therefrom an
antibody genes by digestion with EcoRI and SmaI, and then ligating it to EcoRI-
NotI-BamHI
Adaptor (Takara Shuzo Co., Ltd.)
For preparing a plasmid for the expression in a CHO cell, the plasmid
MBCIHcDNA/pUCl9 was digested with EcoRI and BamHI to obtain a DNA fragment
containing a gene for the chimeric antibody H-chain. The DNA fragment was then
introduced into an expression plasmid pCH01 that had been digested with EcoRI
and BamHI
to give an expression plasmid for the chimeric antibody, which was designated
as
"MBCIHcDNA/pCH01". Here, the expression vector pCH01 was constructed using
56
CA 02401357 2002-08-27
DHFR-DE-rvH-PMl-f (see WO 92/19759) by deleting therefrom an antibody gene by
digestion with EcoRI and SmaI, and then ligating it to EcoRI-NotI-BamHI
Adaptor (Takara
Shuzo Co., Ltd.)
(2) Construction of human L-chain C-region
(i) Preparation of cloning vector
To construct pUC 19 vector containing a gene for human L-chain C-region, a
HindIII site-deleted pUC 19 vector was prepared. pUC 19 vector (2 pg) was
digested in 20 ~l
of a reaction solution containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 1 mM
DTT, 100
mM KCI, 8 U of HindIII (Takara Shuzo Co., Ltd.) at 37~ for 1 hour. The
resulting
digestion solution was extracted with phenol and chloroform, and then
subjected to ethanol
precipitation to collect the DNA of interest.
The DNA collected was reacted in 50 ~,l of a reaction solution containing 50
mM
Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 100 mM NaCI, 0.5 mM dNTPs and 6U of
Klenow fragment (GIBCO BRL) at room temperature for 20 min., thereby rendering
the
terminal ends of the DNA blunt. This reaction mixture was extracted with
phenol and
chloroform and then subjected to ethanol precipitation to collect the vector
DNA.
The vector DNA thus collected was reacted in 10 ~.1 of a reaction solution
containing 50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 5 % (v/v)
polyethylene glycol-8000 and 0.5 U of T4 DNA ligase (GIBCO BRL) at 16~ for 2
hours, to
cause self ligation of the vector DNA. The reaction solution (5 ~1) was added
to 100 ~,1 of a
solution containing competent cells of E. coli, JM109 (Nippon Gene Co., Ltd.),
and the
resulting solution was allowed to stand on ice for 30 min., at 42~ for 1 min.,
and
additionally on ice for 1 min. SOC culture medium (500 p1) was added to the
reaction
solution and then incubated at 37~ for 1 hour. The resulting solution was
plated on 2xYT
57
CA 02401357 2002-08-27
agar medium (containing 50 ~g/ml of ampicillin) on which X-gal and IPTG had
been applied
(Molecular Cloning: A Laboratory Manual, Sambrook, et al., Cold Spring Harbor
Laboratory
Press, 1989), and then cultured at 37°C overnight, thereby obtaining a
transformant.
The transformant was cultured in 2xYT medium (20 ml) containing ampicillin (50
pg/ml) at 37~ overnight. From the cell fraction of the culture medium, a
plasmid DNA
was isolated and purified using Plasmid Mini Kit (QIAGEN) in accordance with
the
instructions included in the kit. The purified plasmid was digested with
HindIII. The
plasmid that was confirmed to have a HindIII site-deletion was designated as
"pUC 19 D
HindIII" .
(ii) Construction of DNA encoding human L-chain ~1 chain C-region
Human antibody L-chain ~l chain C-region is known to have at least four
isotypes
including Mcg+Ke+Oz-, Mcg-Ke Oz-, Mcg~Ke-Oz+ and Mcg-Ke+Oz- (P. Dariavach, et
al.,
Proc. Natl. Acad. Sci. USA, 84, 9074-9078, 1987). A search was made for a
human
antibody L-chain ~l chain C-region homologous to the #23-57-137-1 mouse L-
chain ~l
chain C-region from the EMBL database. As a result, it was found that the
isotype
Mcg+Ke+Oz~ of the human antibody L-chain ~l chain (Accession No. X57819) (P
Dariavach, et al., Proc. Natl. Acad. Sci. USA, 84, 9074-9078, 1987) showed the
highest
degree of homology to the #23-57-137-1 mouse L-chain ~1 chain C-region, with a
64.4%
homology in terms of amino acid sequence and a 73.4% homology in terms of
nucleotide
sequence.
Then, a gene encoding the human antibody L-chain ~ chain C-region was
constructed by PCR method. The primers for the PCR were synthesized using 394
DNA/RNA
Synthesizer (ABI). The synthesized primers were as follows: HLAMBI (SEQ ID NO:
11)
and HLAMB3 (SEQ ID NO: 13), both having a sense DNA sequence; and HLAMB2 (SEQ
58
CA 02401357 2002-08-27
ID NO: 12) and HLAMB4 (SEQ ID NO: 14), both having an antisense DNA sequence;
each
primer containing a complementary sequence of 20-23 by on the both terminal
ends.
External primers HLAMBS (SEQ ID NO: 15) and HLAMBR (SEQ ID NO: 16) had
sequences homologous to the primers HLAMB 1 and HLAMB4, respectively. HLAMBS
contained EcoRI-, HindIII- and BInI-recognition sequences, and HLAMBR
contained an
EcoRI-recognition sequence. In the first-round PCR reaction, the reactions
between
HLAMB 1 and HLAMB2 and between HLAMB3 and HLAMB4 were performed. After the
reactions were completed, both of the resulting PCR products were mixed in
equivalent
quantities, and then assembled in the second-round PCR reaction. The reaction
solution was
added with the external primers HLAMBS and HLAMBR. This reaction mixture was
subjected to the third-round PCR reaction to amplify the full length DNA.
Each PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.)
in accordance with the instructions included in the kit. In the first-round
PCR reaction, 100
p,1 of either a reaction solution containing 5 pmoles of HLAMB1, 0.5 pmole of
HLAMB2 and
SU of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) or a reaction solution containing
0.5 pmole of
HLAMB3, 5 pmoles of HLAMB4 and SU of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.)
was
used, over which 50 p1 of mineral oil was layered. The PCR reaction was run
for 5 cycles
under the conditions: 94°C for 1 min., 60~ for 1 min. and 72°C
for 1 min.
In the second-round PCR reaction, a mixture of both the reaction solutions (50
~1
each) was used, over which 50 ~l of mineral oil was layered. The PCR reaction
was run for
3 cycles under the conditions: 94~ for 1 min., 60~ for 1 min. and 72~ for 1
min.
In the third-round PCR reaction, the reaction solution to which the external
primers
HLAMBS and HLAMBR (50 pmoles each) were added was used. The PCR reaction was
59
CA 02401357 2002-08-27
run for 30 cycles under the conditions: 94~ for 1 min., 60~ for 1 min. and 72~
for 1 min.
The DNA fragment obtained by the third-round PCR reaction was subjected to
electrophoresis on a 3 % low-melting agarose gel (NuSieve GTG Agarose, FMC),
and
separated and purified from the gel using GENECLEAN II Kit (BIO 101 ) in
accordance with
the instructions included in the kit.
The obtained DNA fragment was digested in a reaction solution (20 p1)
containing
50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 100 mM NaCI and 8U of EcoRI
(Takara Shuzo Co., Ltd.) at 37°C for 1 hour. The digestion solution was
extracted with
phenol and chloroform, and the DNA was collected therefrom by the ethanol
precipitation.
The DNA was dissolved in a solution (8 p1) containing 10 mM Tris-HCl (pH 7.4)
and 1 mM
EDTA.
The above-prepared plasmid pUC 19 D HindIII (0.8 fig) was digested with EcoRI
in
the same manner as set forth above. The digestion solution was subjected to
phenoUchloroform extraction and then ethanol precipitation, thereby giving a
digested
plasmid pUCl9 0 HindIII. The digested plasmid was reacted in a reaction
solution (50 p1)
containing 50 mM Tris-HCl (pH 9.0), 1 mM MgCl2 and alkaline phosphatase (E.
coli C75;
Takara Shuzo Co., Ltd.) at 37~ for 30 min. to dephosphorylate (i.e., BAP-
treat) the plasmid.
The reaction solution was subjected to phenol/chloroform extraction, and the
DNA was
collected therefrom by ethanol precipitation. The DNA thus obtained was
dissolved in a
solution (10 ~,1) containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA.
The BAP-treated plasmid pUC 19 D HindIII ( 1 p1) was ligated to the above-
obtained PCR product (4 p1) using DNA Ligation Kit Ver.2 (Takara Shuzo Co.,
Ltd.). The
resulting plasmid was introduced into a competent cell of E. coli, JM 109, to
give a
CA 02401357 2002-08-27
transformant. The transformant was cultured overnight in 2xYT medium (2 ml)
containing
50 ~g/ml of ampicillin. From the cell fraction, the plasmid was isolated using
QIAprep Spin
Plasmid Kit (QIAGEN).
The obtained plasmid was sequenced for the cloned DNA part. The sequencing
was performed on 373A DNA Sequencer (ABI) using M13 Primer M4 and M13 Primer
RV
(Takara Shuzo Co., Ltd.). As a result, it was found that the cloned DNA had a
12-by
deletion therein. The plasmid was designated as "C ~l 0 /pUC 19". Then, for
making up for
the deleted part, primers HCLMS (SEQ ID NO: 17) and HCLMR (SEQ ID NO: 18) were
newly synthesized, and a DNA of correct sequence was reconstructed using these
primers by
PCR method.
In the first-round PCR reaction, the plasmid C ~l D /pUC 19 having the DNA
deletion therein was used as a template, and the reaction was performed with
each of the
primer sets of HLAMBS and HCLMS and HCLMS and HLAMB4. The PCR products were
purified separately. In the second-round PCR reaction, the PCR products were
assembled
together. In the third-round PCR reaction, the reaction product of the second-
round PCR
reaction was added with external primers HLAMBS and HLAMB4 and amplified to
give the
full length DNA.
In the first-round PCR reaction, a reaction solution ( 100 p1) containing 0.1
pg of
C ~l 0 /pUC 19 as a template, either 50 pmoles of each of the primers HLAMBS
and HCLMR
or 50 pmoles of each of the primers HCLMS and HLAMB4, and SU of TaKaRa Ex Taq
(Takara Shuzo Co., Ltd.) was used, over which 50 p1 of mineral oil was
layered. The PCR
reaction was run for 30 cycles under the conditions: 94~ for 1 min., 60~ for 1
min. and 72
°C for 1 min.
61
CA 02401357 2002-08-27
The PCR products of the first-round PCR reaction, HLAMBS-HCLMR (236 bp)
and HCLMS-HLAMB4 (147 bp), were subjected to electrophoresis separately on a
3% low-
melting agarose gel to isolate the DNA fragments. The DNA fragments were
collected and
purified from the gels using GENECLEAN II Kit (BIO101). In the second-round
PCR
reaction, 20 p1 of a reaction solution containing 40 ng of each of the
purified DNA fragments
and 1U of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) was used, over which 25 p1 of
mineral oil
was layered. The PCR reaction was run for 5 cycles under the conditions: 94~C
for 1 min.,
60°C for 1 min. and 72°C for 1 min.
In the third-round PCR reaction, 100 p1 of a reaction solution containing 2
p,1 of the
reaction solution obtained by the second-round PCR reaction, 50 pmoles of each
of external
primers HLAMBS and HLAMB4 and SU of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) was
used, over which 50 p1 of mineral oil was layered. The PCR reaction was run
for 30 cycles
under the conditions: 94~ for 1 min., 60°rC for 1 min. and 72~ for 1
min., thereby
obtaining a DNA fragment of 357 by (the third PCR product). The DNA fragment
was
subjected to electrophoresis on a 3% low-melting agarose gel to isolate the
DNA fragment.
The resulting DNA fragment was collected and purified using GENECLEAN Kit
(BIO101).
An aliquot (0.1 pg) of the DNA fragment thus obtained was digested with EcoRI,
and then subcloned into plasmid pUC 19 D HindIII that had been BAP-treated.
The resulting
plasmid was introduced into a competent cell of E. coli, 3M 109, to form a
transformant. The
transformant was cultured overnight in 2 ml of 2xYT medium containing 50 pg/ml
of
ampicillin. From the cell fraction, the plasmid was isolated and purified
using QIAprep Spin
Plasmid Kit (QIAGEN).
The purified plasmid was sequenced on 373A DNA Sequencer (ABI) using M 13
Primer M4 and M 13 Primer RV (Takara Shuzo Co., Ltd.). The plasmid that was
confirmed
62
CA 02401357 2002-08-27
to have the correct nucleotide sequence without any deletion was designated as
"C ~1 /pUC 19".
(iii) Construction of gene encoding human L-chain ~ chain C-region
A DNA fragment encoding the L-chain ~c chain C-region was cloned from
plasmid HEF-PMIk-gk (WO 92/19759) by PCR method. A forward primer HKAPS (SEQ
ID NO: 19) was designed to contain EcoRI-, HindIII and BInI-recognition
sequences, and a
backward primer HKAPA (SEQ 117 NO: 20) was designed to contain an EcoRI-
recognition
sequence. These primers were synthesized on 394 DNA/RNA Synthesizer (ABI).
A PCR reaction was performed using 100 p1 of a reaction solution containing
0.1 pg
of plasmid HEF-PMIk-gk as a template, 50 pmoles of each of primers HKAPS and
HKAPA
and SU of TaKaRa Ex Taq (Takara Shuzo Co., Ltd.), over which 50 p,1 of mineral
oil was
layered. The PCR reaction was run for 30 cycles under the conditions:
94°C for 1 min., 60
°C for 1 min. and 72°C for 1 min., thereby giving a PCR product
of 360 bp. The DNA
fragment was isolated and purified by electrophoresis on a 3 % low-melting
agarose, and then
collected and purified using GENECLEAN II Kit (BIO101).
The thus obtained DNA fragment was digested with EcoRI, and then cloned into
plasmid pUC 19 (HindIII that had been BAP-treated. The resulting plasmid was
introduced
into a competent cell of E. coli, JM 109, to form a transformant. The
transformant was
cultured overnight in 2 ml of 2xYT medium containing 50 pg/ml of ampicillin.
From the
cell fraction, the plasmid was purified using QIAprep Spin Plasmid Kit
(QIAGEN).
The purified plasmid was sequenced on 373A DNA Sequencer (ABI) using M13
Primer M4 and M 13 Primer RV (Takara Shuzo Co., Ltd.). The plasmid that was
confirmed
to have the correct nucleotide sequence was designated as "C ~ /pUC 19".
63
CA 02401357 2002-08-27
(3) Construction of chimeric antibody L-chain expression vector
An expression vector for the chimeric #23-57-137-1 antibody L-chain was
constructed. A gene encoding #23-57-137-1 L-chain V-region was ligated to the
HindIII-
BInI site (located just in front of the human antibody C-region) of each of
the plasmids C ~1
/pUC 19 and C r~ /pUC 19, thereby obtaining pUC 19 vectors that contained the
DNAs
encoding the chimeric #23-57-137-1 antibody L-chain V-region and either of the
L-chain
chain C-region or the L-chain ~ region C-region, respectively. Each of the
resulting
vectors was then digested with EcoRI to separate the gene for the chimeric
antibody L-chain.
The gene was subcloned into HEF expression vector.
That is, a DNA fragment encoding #23-57-137-1 antibody L-chain V-region was
cloned from plasmid MBC1L24 by PCR method. Primers used in the PCR method were
separately synthesized using 394 DNA/RNA Synthesizer (ABI). A backward primer
MBCCHL1 (SEQ ID NO: 21) was designed to contain a HindIII-recognition sequence
and a
Kozak sequence (Kozak, M. et al., J. Mol. Biol. 196, 947-950, 1987), and a
forward primer
MBCCHL3 (SEQ D7 NO: 22) was designed to contain BgIII- and RcoRI-recognition
sequences.
The PCR reaction was performed using 100 ~,l of a reaction solution containing
10
mM Tris-HCl (pH 8.3), 50 mM KCI, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.1 ~,g MBC 1
L24, SO
pmoles of each of primers MBCCHL1 and MBCCHL3 and 1 w1 of AmpliTaq (PERKIN
ELMER), over which 50 ~,l of mineral oil was layered. The PCR reaction was run
for 30
cycles under the conditions: 94°~ for 45 sec., 60'~ for 45 sec. and 72~
for 2 min.
A PCR product of 444 by was electrophoresed on a 3 % low-melting agarose gel,
and collected and purified using GENECLEAN II Kit (BIO101). The purified PCR
product
was dissolved in 20 ~.1 of a solution containing 10 mM Tris-HCl (pH 7.4) and 1
mM EDTA.
64
CA 02401357 2002-08-27
The PCR product ( 1 ~,1) was digested in 20 ~.1 of a reaction solution
containing 10 mM Tris-
HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 50 mM NaCI, 8U of HindIII (Takara Shuzo
Co.,
Ltd.) and 8U of EcoRI (Takara Shuzo Co., Ltd.) at 37~C for 1 hour. The
digestion solution
was subjected to phenol/chloroform extraction, and the DNA of interest was
collected
therefrom by ethanol precipitation. The DNA was dissolved in 8 p1 of a
solution containing
mM Tris-HCl (pH 7.4) and 1 mM EDTA.
In the same manner, plasmid pUCl9 (1 ~.g) was digested with HindIII and EcoRI,
and subjected to phenol/chloroform extraction and then ethanol precipitation.
The obtained
digested plasmid was BAP-treated with alkaline phosphatase (E. coli C75;
Takara Shuzo Co.,
Ltd.). The resulting reaction solution was extracted with phenol and
chloroform, and the
DNA was collected therefrom by ethanol precipitation. The DNA was dissolved in
10 ~1 of
a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA.
The BAP-treated plasmid pUCl9 (1 w1) was ligated to the above-obtained PCR
product (4 ~l) using DNA Ligation Kit Ver. 2 (Takara Shuzo Co., Ltd.). The
resulting
plasmid was introduced into a competent cell of E. coli, JM109 (Nippon Gene
Co., Ltd.), in
the same manner as set forth above, to form a transformant. The transformant
was plated on
2xYT agar medium containing 50 ~,g/ml of ampicillin and cultured at
37°C overnight. The
resulting transformant was then cultured at 37°C overnight in 2 ml of
2xYT medium
containing 50 ~g/ml of ampicillin. From the cell fraction, the plasmid was
purified using
QIAprep Spin Plasmid Kit (QIAGEN). After determining the nucleotide sequence,
the
plasmid that was confirmed to have the correct nucleotide sequence was
designated as
"CHL/pUC 19".
Each of plasmids C ~ /pUC 19 and C ~c /pUC 19 ( 1 ~.g each) was digested in 20
p1 of
a reaction solution containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 1 mM DTT,
100 mM
CA 02401357 2002-08-27
The transient expression of the chimeric antibodies was performed using each
of the
combinations of plasmids MBC 1 HcDNA/pCOS 1 and MBC 1 L ( ~ )/neo and plasmids
MBC 1 HcDNA/pCOS 1 and MBC 1 L( r~ )/neo, by co-tansfecting a COS-7 cell with
the
plasmids by electroporation using Gene Pulser (Bio Rad). That is, the plasmids
(10 pg each)
were added to a COS-7 cell suspension (0.8 ml; 1 x 107 cells/ml) in PBS(-).
The resulting
solution was applied with pulses at an electrostatic capacity of 1,SOOV and 2
~,F to cause
electroporation. After 10 min. of recovery period at room temperature, the
electroporated
cells were suspended in DMEM medium (GIBCO) containing 2% Ultra Low IgG fetal
calf
serum (GIBCO), and then cultured using a 10-cm culture dish in a C02
incubator. After
culturing for 72 hours, a culture supernatant was collected and centrifuged to
remove cell
debris, and was provided for use as a sample for the subsequent ELISA.
In this procedure, the purification of the chimeric antibody from the COS-7
cell
culture supernatant was performed using AffiGel Protein A MAPSII Kit (Bio Rad)
in
accordance with the instructions included in the kit.
(5) ELISA
(i) Determination of antibody concentration
An ELISA plate for determining antibody concentration was prepared as follows.
Each well of a 96-well ELISA plate (Maxisorp, NLJNC) was coated with 100 p1 of
a coating
buffer (0.1 M NaHC03, 0.02% NaN3) supplemented with 1 pg/ml of goat anti-human
IgG
antibody (TAGO), and then blocked with 200 p,1 of a dilution buffer [50 mM
Tris-HCI, 1 mM
MgCl2, 0.1 M NaCI, 0.05% Tween 20, 0.02% NaN3, 1% bovine serum albumin (BSA);
pH
7.2]. Each well of the plate was added with each of the serial dilutions of
the COS-7 cell
culture supernatant in which each of the chimeric antibodies had been
expressed, or added
with each of the serial dilutions of each of the chimeric antibodies per se in
a purified form.
68
CA 02401357 2002-08-27
The plate was incubated at room temperature for 1 hour and washed with PBS-
Tween 20.
Each well of the plate was then added with 100 ~1 of a solution of alkaline
phosphatase-
conjugated goat anti-human IgG antibodies (TAGO). After the plate was
incubated at room
temperature for 1 hour and washed with PBS-Tween 20, each well was added with
1 mg/ml of
a substrate solution ("Sigma 104", p-nitrophenylphosphoric acid, SIGMA). The
solution
was measured on its absorbance at 405 nm using Microplate Reader (Bio Rad) to
determine
the antibody concentration. In this determination, Hu IgGI ~l Purified (The
Binding Site)
was used as the standard substance.
(ii) Determination of antigen-binding ability
An ELISA plate for the determination of antigen-binding ability was prepared
as
follows. Each well of a 96-well ELISA plate was coated with 100 p1 of a
coating buffer
supplemented with 1 ~.g/ml of human PTHrP (1-34) (Peptide Research Institute),
and then
blocked with 200 p1 of a dilution buffer. Each well was added with each of the
serial
dilutions of the COS-7 cell culture supernatant in which each of the chimeric
antibodies had
been expressed, or added with each of the serial dilutions of each of the
chimeric antibodies
per se in a purified form. After the plate was incubated at room temperature
and washed
with PBS-Tween 20, each well of the plate was added with 100 ~,1 of a solution
of alkaline
phosphatase-conjugated goat anti-human IgG antibodies (TAGO). After the plate
was
incubated at room temperature and washed with PBS-Tween 20, each well of the
plate was
added with 1 mg/ml of a substrate solution ("Sigma 104", p-
nitrophenylphosphoric acid,
SIGMA). The solution was measured on its absorbance at 405 nm using Microplate
Reader
(Bio Rad).
As a result, it was found that the chimeric antibodies had an ability to bind
to
human PTHrP (1-34) and the cloned mouse antibody V-regions had the correct
structures
(FIG. 5). It was also found that there was no difference in the ability to
bind to PTHrP (1-
d9
CA 02401357 2002-08-27
34) between the chimeric antibody with L-chain ~l chain C-region and the
chimeric
antibody with L-chain ~c chain C-region. Therefore, the humanized antibody L-
chain ~l
chain was used for construction of the L-chain C-region of the humanized
antibody.
(6) Establishment of CHO cell line capable of stable production of chimeric
antibodies
To establish a cell line capable of producing the chimeric antibodies stably,
the
above-prepared expression plasmids were introduced into CHO cells (DXB11).
For the establishment of a cell line capable of producing the chimeric
antibodies
stably, either of the following combinations of the expression plasmids for
CHO cell was
used: MBC IHcDNA/pCH01 and MBC 1 L( ~1 )/neo; and MBC 1 HcDNA/pCH01 and MBC 1
L(
r~ )/neo. A CHO cell was co-transfected with the plasmids by electroporation
using Gene
Pulser (Bio Rad) as follows. The expression vectors were separately cleaved
with a
restriction enzyme PvuI to give linear DNAs. The resulting DNAs were extracted
with
phenol and chloroform and collected by precipitation with ethanol. The plasmid
DNAs thus
prepared were subjected to electroporation. That is, each of the plasmid DNAs
(10 g,g each)
was added to 0.8 ml of a cell suspension of CHO cells in PBS(-) (1x10'
cells/ml). The
resulting solution was applied with pulses at an electrostatic capacity of
1,SOOV and 25 p.F.
After 10 min. of recovery period at room temperature, the electroporated cells
were
suspended in MEM- cx medium (GIBCO) containing 10% fetal calf serum (GIBCO).
The
resulting suspension was cultured using three 96-well plates (Falcon) in a C02
incubator.
On the day following the culturing being started, the medium was replaced by a
selective
medium [ribonucleoside- or deoxyribonucleoside-free MEM- c~ medium (GIBCO)
containing 10% fetal calf serum (GIBCO) and 500 mg/ml of GENETICIN
(G418Sulfate;
GIBCO)]. From the culture medium, cells into which the antibody gene was
introduced
were selected. The selective medium is replaced by a fresh one. About two
weeks after the
medium replacement, the cells were observed under a microscope. When a
satisfactory cell
CA 02401357 2002-08-27
growth was observed, the amount of the antibodies produced was determined by
ELISA as set
forth above. Among the cells, those cells which produced a larger amount of
antibodies
were screened.
Then, the culturing of the established cell line capable of stable production
of the
antibodies was scaled up in a roller bottle using ribonucleoside- or
deoxyribonucleoside-free
MEM medium containing 2% Ultra Low IgG fetal calf serum. On day 3 and day 4 of
the
culturing, the culture supernatant was collected and then filtered on a 0.2-wm
filter (Millipore)
to remove cell debris therefrom.
Purification of the chimeric antibodies from the CHO cell culture supernatant
was
performed using POROS Protein A Column (PerSeptive Biosystems) on ConSep LC
100
(Millipore) in accordance with the instructions included in the kit. The
purified chimeric
antibodies were provided for use as samples for the determination of
neutralizing activity and
for the examination of therapeutic efficacy in hypercalcemic model animals.
The
concentration and the antigen-binding activity of the purified chimeric
antibodies were
determined using the same ELISA system as set forth above.
[REFERENCE EXAMPLE 4] Construction of humanized antibody
(1) Construction of humanized antibody H-chain
(i) Construction of humanized H-chain V-region
A humanized #23-57-137-1 antibody H-chain was produced by CDR-grafting
technique by means of PCR method. For the production of a humanized #23-57-137-
1
antibody H-chain (version "a") having FRs derived from human antibody S31679
(NBRF-
PDB; Cuisinier, A. M. et al., Eur. J. Immunol., 23, 110-118, 1993), the
following six PCR
primers were used: CDR-grafting primers: MBC1HGP1 (SEQ ID NO: 23) and MBC1HGP3
(SEQ ID NO: 24) (both containing a sense DNA sequence) and MBC1HGP2 (SEQ ID
NO:
71
CA 02401357 2002-08-27
25) and MBC1HGP4 (SEQ ID NO: 26) (both containing an antisense DNA sequence),
all of
which containing a 15-21 by complementary sequence on both terminal ends
thereof; and
external primers: MBC1HVS1 (SEQ ID NO: 27) and MBC1HVR1 (SEQ ID NO: 28) having
a homology to the CDR-grafting primers MBC1HGP1 and MBC1HGP4, respectively.
The CDR-grafting primers MBC1HGP1, MBC1HGP2, MBC1HGP3 and
MBC1HGP4 were separated on an urea-denatured polyacrylamide gel (Molecular
Cloning: A
Laboratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press,
1989), and
extracted therefrom by crush-and-soak method (Molecular Cloning: A Laboratory
Manual,
Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989) in the following
manner.
Each of the CDR-grafting primers (1 nmole) was separated on a 6% denatured
polyacrylamide gel to give DNA fragments. From the resulting DNA fragments, a
DNA
fragment having a desired length was identified on a silica gel thin plate by
irradiation of UV
ray and then collected therefrom by crush-and-soak method. The resulting DNA
was
dissolved in 20 p1 of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM
EDTA. The
PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.). The
PCR
reaction solution (100 ~.1) comprised 1 p1 of each of the above-mentioned CDR-
grafting
primers MBC1HGP1, MBC1HGP2, MBC1HGP3 and MBC1HGP4, 0.25 mM dNTPs and
2.5U of TaKaRa Ex Taq in the buffer. The PCR reaction was run for 5 cycles
under the
conditions: 94~C for 1 min., 55~ for 1 min. and 72~C for 1 min. The resulting
reaction
solution was added with the external primers MBC1HVS1 and MBC1HVR1 (50 pmoles
each). Using this reaction mixture, the PCR reaction was run for additional 30
cycles under
the same conditions. The DNA fragment thus amplified was separated by agarose
gel
electrophoresis on a 4% Nu Sieve GTG agarose (FMC Bio. Products).
An agarose segment containing a DNA fragment of 421 by was excised, and the
n
CA 02401357 2002-08-27
DNA fragment was purified therefrom using GENECLEANII Kit (BIO101) in
accordance
with the instructions included in the kit. The DNA fragment thus purified was
precipitated
with ethanol and then dissolved in 20 ~.l of a solution containing 10 mM Tris-
HCI (pH 7.4)
and 1 mM EDTA. The resulting PCR reaction mixture was used for subcloning of
the DNA
fragment into plasmid pUC 19 that had been digested with BamHI and HindIII,
and
subsequently the nucleotide sequence of the resulting plasmid was determined.
A plasmid
having the correct nucleotide sequence was designated as "hMBCHv/pUCl9".
(ii) Construction of H-chain V-region of Humanized H-chain cDNA
To ligate to cDNA for humanized H-chain C-region C r 1, the DNA for the
humanized H-chain V-region constructed in the above step was modified by PCR
method.
For the PCR method, a backward primer MBC1HVS2 was designed to hybridize to
the
sequence encoding the 5' region of the leader sequence for the V-region and to
have a Kozak
consensus sequence (Kozak et al., J. Mol. Biol. 196, 947-950, 1987) and
HindIII- and EcoRI-
recognition sequences; and a forward primer MBC 1HVR2 was designed to
hybridize to both
the DNA sequence encoding the 3' region of the J region and the DNA sequence
encoding the
5' region of the C-region and to have ApaI- and SmaI-recognition sequences.
The PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.)
and a buffer appended thereto. The PCR reaction solution comprised 0.4 pg of
hMBCHv/pUCl9 as a DNA template, 50 pmoles of each of MBC1HVS2 and MBC1HVR2 as
primers, 2.5U of TaKaRa Ex Taq and 0.25 mM dNTPs in the buffer. The PCR
reaction was
run for 30 cycles under the conditions: 94~C for 1 min., 55~ for 1 min. and
72~ for 1 min.
The DNA fragment thus amplified was separated by agarose gel electrophoresis
on a 3% Nu
Sieve GTG agarose (FMC Bio. Products).
A gel segment containing a DNA fragment of 456 by was excised, and the DNA
73
CA 02401357 2002-08-27
fragment was purified therefrom using GENECLEANII Kit (BIO101) in accordance
with the
instructions included in the kit. The DNA fragment thus purified was
precipitated with
ethanol and then dissolved in 20 ~l of a solution containing 10 mM Tris-HCl
(pH 7.4) and 1
mM EDTA. The PCR reaction solution thus obtained was used for subcloning of
the DNA
fragment into plasmid pUC 19 that had been digested with EcoRI and SmaI, and
then the
resulting plasmid was sequenced. As a result, a plasmid was obtained which
contained a
DNA encoding mouse H-chain V-region derived from hybridoma #23-57-137-1 and
also
contained EcoRI- and HindIII-recognition sequences and a Kozak sequence on the
5' region
and ApaI- and SmaI-recognition sequences on the 3' region, which was
designated as
"hMBC 1 Hv/pUC 19".
(2) Construction of expression vector for humanized antibody H-chain
Plasmid RVh-PM 1 f cDNA carrying a cDNA sequence for hPM 1 antibody H-chain
was digested with ApaI and BamHI to give a DNA fragment containing a DNA
fragment
containing a DNA encoding the H-chain C-region. The DNA fragment was
introduced into
plasmid hMBC 1Hv/pUC 19 that had been digested with ApaI and BamHI. The
obtained
plasmid was designated as "hMBC IHcDNA/pUC 19". This plasmid contained both a
DNA
encoding the humanized #23-57-137-1 antibody H-chain V-region and a DNA
encoding the
human H-chain C-region C Y 1 and had EcoRI- and HindIII-recognition sequences
on the 5'
region and a BamHI-recognition sequence on the 3' region. The nucleotide
sequence and the
corresponding amino acid sequence of the humanized H-chain version "a" carried
on the
plasmid hMBCIHcDNA/pUCl9 are shown in SEQ ID NO: 58 and SEQ ID NO: 56,
respectively.
The plasmid hMBC 1 HcDNA/pUC 19 was digested with EcoRI and BamHI to give a
DNA fragment containing a DNA encoding the H-chain. The DNA fragment was
introduced into expression plasmid pCOS 1 that had been digested with EcoRI
and BamHI.
74
CA 02401357 2002-08-27
As a result, an expression plasmid for a humanized antibody was obtained,
which was
designated as "hMBCIHcDNA/pCOSI".
To produce a plasmid used for expression in a CHO cell, plasmid
hMBC lHcDNA/pUC 19 was digested with EcoRI and BamHI to give a DNA fragment
containing a DNA encoding the H-chain. The DNA fragment was introduced into
expression vector pCH01 that had been digested with EcoRI and BamHI. As a
result, an
expression plasmid for the humanized antibody was obtained, which was
designated as
"hMBC 1 HcDNA/pCH01 ".
(3) Construction of L-chain hybrid V-region
(i) Preparation of FR1,2/FR3,4 hybrid antibody
A gene for the FR hybrid L-chain having both FRs from a humanized antibody and
FRs from a mouse (chimeric) antibody was constructed, and evaluated each
region for the
humanization. In this step, a hybrid antibody having FRl and FR2 both derived
from a
human antibody and FR3 and FR4 both derived from a mouse antibody was prepared
by
utilizing the AflII restriction site located on CDR2.
Plasmids MBC 1 L( ~ )/neo and hMBC 1 L( ~l )/neo ( 10 g.g each) were
separately
digested in 100 p1 of a reaction solution containing 10 mM Tris-HCl (pH 7.5),
10 mM MgCl2,
1 mM DTT, 50 mM NaCI, 0.01 % (w/v) of BSA and 10 U of AflII (Takara Shuzo Co.,
Ltd.) at
37~ for 1 hour. The reaction solutions were subjected to electrophoresis on a
2% low-
melting agarose gel, thereby giving DNA fragments of 6282 by (referred to as
"c1" ) and 1022
by (referred to as "c2") from the plasmid MBC1L( ~l )/neo or DNA fragments of
6282 by
(referred to as "hl" ) and 1022 by (referred to as "h2") from the plasmid
hMBCIL( ~ )/neo.
These DNA fragments were collected and purified from the gels using
GENECLEANII Kit
(BIO101).
CA 02401357 2002-08-27
Each of the c 1 and h 1 fragments ( 1 pg each) was BAP-treated. The DNA
fragment was extracted with phenol and chloroform, collected by ethanol
precipitation, and
then dissolved in 10 p1 of a solution containing 10 mM Tris-HCl (pH 7.4) and 1
mM EDTA.
The BAP-treated c1 and hl DNA fragments (1 p1 each) were ligated to the h2 and
c2 DNA fragments (4 ~,l each), respectively, (at 4~ overnight). Each of the
ligation
products was introduced into a competent cell of E. coli, JM 109, to form a
transformant.
The transformant was cultured in 2 ml of 2xYT medium containing 50 pg/ml of
ampicillin.
From the cell fraction, the plasmid was purified using QIAprep Spin Plasmid
Kit (QIAGEN).
The purified plasmid was digested in 20 ~1 of a reaction solution containing
10 mM
Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, and either 2U of ApaLI (Takara Shuzo
Co.,
Ltd.) or 8U of BamHI (Takara Shuzo Co., Ltd.) and HindIII (Takara Shuzo Co.,
Ltd.) at 37°C
for 1 hour. It was expected that if the cl-h2 was ligated correctly, this
digestion reaction
would give fragments of 5560/1246/498 by (by the ApaLI digestion) or fragments
of
7134/269 by (by the BamHI/HindIII digestion). Based on this expectation, the
desired
plasmids were identified.
The expression vector encoding the human FR1,2/mouse FR3,4 hybrid antibody L-
chain was designated as "h/mMBCIL(~ )/neo". On the other hand, since a clone
for the hl-
c 1 could not be obtained, recombination on a pUC vector was performed and
then the
resulting recombinant product was cloned into a HEF vector. In this procedure,
plasmid
hMBC 1 La ~ /pUC 19, which contained DNA encoding a humanized antibody L-chain
V-
region without any amino acid replacements, and plasmid hMBCILd ~1 /pUCl9,
which
contained a DNA encoding a humanized antibody L-chain V-region with an amino
acid
replacement at the 91-position amino acid tyrosine in FR3 (i.e., the 87th
amino acid in
76
CA 02401357 2002-08-27
accordance with The Kabat's prescription) by isoleucine, were used as
templates.
Plasmids MBC 1 L( ~l )/pUC 19, hMBC 1 La ~1 /pUC 19 and hMBC 1 Ld ~l /pUC 19 (
10
~,1 each) were separately digested in 30 p.1 of a reaction solution containing
10 mM Tris-HCl
(pH 7.5), 10 mM MgCl2, 1 mM DTT, 50 mM NaCI, 0.01 % (w/v) of BSA, 16U of
HindIII and
4U of AflII at 37~ for 1 hour. The reaction solutions were separately
subjected to
electrophoresis on a 2% low-melting agarose gel, thereby giving a DNA fragment
of 215 by
from plasmid MBC1L( ~ )/pUCl9 (referred to as "c2"') and a DNA fragment of
3218 by from
each of plasmids hMBC 1 La ~1 /pUC 19 and hMBC 1 Ld ~ /pUC 19 (referred to as
"ha 1 "' and
"hdl"', respectively). These DNA fragments were collected and purified using
GENECLEANII Kit (BIO101).
Each of the ha 1' and hd 1' fragments was ligated to the c2' fragment and then
introduced into a competent cell of E. coli, JM 109, to form a transformant.
The
transformant was cultured in 2 ml of 2xYT medium containing 50 g,g/ml of
ampicillin.
From the cell fraction, the plasmid was purified using QIAprep Spin Plasmid
Kit (QIAGEN).
The plasmids thus prepared were designated as "m/hMBC 1 La ~ /pUC 19" for the
ha 1'
fragment-containing plasmid and "m/hMBC 1 Ld ~1 /pUC 19" for the hd 1'
fragment-containing
plasmid.
Each of the obtained plasmids m/hMBC 1 La ~l /pUC 19 and m/hMBC 1 Ld ~1 /pUC
19
was digested with EcoRI. The DNA fragment of 743 by was electrophoresed on a
2% low-
melting agarose gel, and then collected and purified therefrom using
GENECLEANII Kit
(BIO 101 ). The resulting DNA fragment was dissolved in 20 g,1 of a solution
containing 10
mM Tris-HCl (pH 7.4) and 1 mM EDTA.
Each of the DNA fragments (4 p1 each) was ligated to the above-obtained BAP-
77
CA 02401357 2002-08-27
treated HEF vector (1 p1). The ligation product was introduced into a
competent cell of E.
coli, JM 109, to form a transformant. The transformant was cultured in 2 ml of
2xYT
medium containing 50 pg/ml of ampicillin. From the cell fraction, the plasmid
was purified
using QIAprep Spin Plasmid Kit (QIAGEN).
Each of the purified plasmids was digested in 20 p1 of a reaction solution
containing
20 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 1 mM DTT, 100 mM KCI, 8U of HindIII
(Takara
Shuzo Co., Ltd.) and 2U of PvuI (Takara Shuzo Co., Ltd.) at 37~ for 1 hour. It
was
expected that if the DNA fragment was inserted in the plasmid in a correct
orientation, this
digestion would give digestion fragments of 5104/2195 bp, whereas if the DNA
fragment is
inserted in the plasmid in the reverse orientation, this digestion would give
digestion
fragments of 4378/2926 bp. The plasmid DNA was identified based on the
expectation.
The plasmids thus obtained were expression vectors encoding mouse FR1,2/human
FR3,4
hybrid antibody L-chain, which were designated as expression vectors "m/hMBC 1
La ~l /neo"
and "m/hMBC 1 Ld ~ / neo", respectively.
(ii) Preparation of FR1/FR2 hybrid antibody
An FR1/FR2 hybrid antibody was prepared in the same manner as set forth above
utilizing a SnaBI restriction site located on CDR1.
Plasmids MBC1L( ~1 )/neo and h/mMBCIL( ~ )/neo (10 pg each) were separately
digested in 20 p,1 of a reaction solution containing 10 mM Tris-HCl (pH 7.9),
10 mM MgCl2,
1 mM DTT, 50 mM NaCI, 0.01 % (w/v) of BSA and 6U of SnaBI (Takara Shuzo Co.,
Ltd.) at
37~ for 1 hour. The resulting reaction solutions were further digested in 50
~1 of a reaction
solution containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 1 mM DTT, 100 mM
KCI,
0.01 % (w/v) of BSA and 6U of PvuI at 37°~ for 1 hour.
78
CA 02401357 2002-08-27
The resulting reaction solutions were separately subjected to electrophoresis
on a
1.5% low-melting agarose gel, thereby giving DNA fragments of 4955 by (ml) and
2349 by
(m2) from the plasmid MBC 1 L( ~ )/neo and DNA fragments of 4955 by (hm 1 )
and 2349 by
(hm2) from the plasmid h/mMBC 1L( ~l )/neo. These DNA fragments were collected
and
purified from the gels using GENECLEANII Kit (BIO101). Each of the DNA
fragments
obtained was dissolved in 40 p1 of a solution containing 10 mM Tris-HCI (pH
7.4) and 1 mM
EDTA.
The m 1 and hm 1 fragments ( 1 ~,1 each) were ligated to the hm2 and m2
fragments
(4 ~,1 each), respectively. Each of the resulting ligation products was
introduced into a
competent cell of E. coli, JM 109, to form a transformant. The transformant
obtained was
cultured in 2 ml of 2xYT medium containing 50 ~g/ml of ampicillin. From the
cell fraction,
the plasmid was purified using QIAprep Spin Plasmid Kit QIAGEN).
Each of the purified plasmids was digested in 20 ~l of a reaction solution
containing
mM Tris-HCI (pH 7.5), 10 mM MgClz, 1 mM DTT and either 8U of ApaI (Takara
Shuzo
Co., Ltd.) or 2U of ApaLI (Takara Shuzo Co., Ltd.) at 37~C for 1 hour.
It was expected that if the fragments were ligated correctly, the digestion
reaction
would give a fragment of 7304 by (by the ApaI digestion) or fragments of
5560/1246/498 by
(by the ApaLI digestion) for ml-hm2, and would give fragments of 6538/766 by
(by the ApaI
digestion) or fragments of 3535/2025/1246/498 by (by the ApaLI digestion) for
hml-m2.
Based on this expectation, the plasmids were identified. As a result, an
expression vector
encoding a human FRl/mouse FR2,3,4 hybrid antibody L-chain (designated as
"hmmMBCIL( ~ )/neo") and an expression vector encoding a mouse FR1/human
FR2/mouse
FR3,4 hybrid antibody L-chain (designated as "mhmMBC 1 L( ~1 )/neo") were
obtained.
79
CA 02401357 2002-08-27
(4) Construction of humanized antibody L-chain
A humanized #23-57-137-1 antibody L-chain was prepared by CDR-grafting
technique by means of PCR method. For the preparation of a humanized #23-57-
137-1
antibody L-chain (version "a") that contained FR1, FR2 and FR3 derived from
human
antibody HSU03868 (GEN-BANK, Deftos M. et al., Scand. J. Immunol., 39, 95-103,
1994)
and FR4 derived from human antibody 525755 (NBRF-PDB), six PCR primers were
used.
The six primers were as follows: CDR-grafting primers MBC1LGP1 (SEQ 1T7 NO:
29) and MBC1LGP3 (SEQ 117 NO: 30), both having a sense DNA sequence, CDR-
grafting
primers MBC1LGP2 (SEQ ID NO: 31) and MBC1LGP4 (SEQ ID NO: 32), both having an
antisense DNA sequence, all of which had a 15-21 by complementary sequence on
the both
terminal ends; and external primers MBC1LVS1 (SEQ ID NO: 33) and MBC1LVR1 (SEQ
ID
NO: 34) having a homology tv the CDR-grafting primers MBC 1 LGP 1 and MBC 1
LGP4,
respectively.
The CDR-grafting primers MBC1LGP1, MBC1LGP2, MBC1LGP3 and
MBC1LGP4 were separated on a urea-denatured polyacrylamide gel (Molecular
Cloning: A
Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989)
and
extracted therefrom by crush-and-soak method (Molecular Cloning: A Laboratory
Manual,
Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).
Each of the CDR-grafting primers (1 nmole each) was separated on a 6%
denatured
polyacrylamide gel. The identification of the DNA fragment of a desired length
was
performed on a silica gel thin plate by irradiation of UV ray. The desired DNA
fragment
was collected from the gel by crush-and-soak method. The collected DNA
fragment was
dissolved in 20 p1 of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM
EDTA.
CA 02401357 2002-08-27
The PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.)
and a buffer appended thereto. The PCR reaction solution comprised (per 100
p1) 1 ~.1 of
each of the CDR-grafting primers MBC 1 LGP l, MBC 1 LGP2, MBC 1 LGP3 and MBC 1
LGP4,
0.25 mM dNTPs, 2.5U of TaKaRa Ex Taq in the buffer. The PCR reaction was run
for 5
cycles under the conditions: 94°C for 1 min., 55~ for 1 min. and 72~
for 1 min. The
resulting reaction mixture was added with 50 pmoles of each of the external
primers
MBC1LVS1 and MBC1LVR1. Using this reaction nuxture, the PCR reaction was run
for
additional 30 cycles under the same conditions. The DNA fragment thus
amplified was
separated by agarose gel electrophoresis on a 3 % Nu Sieve GTG agarose (FMC
Bio.
Products).
An agarose segment containing a DNA fragment of 421 by was excised, and the
DNA fragment was purified therefrom using GENECLEANII Kit (BIO 101 ) in
accordance
with the instructions included in the kit. The PCR reaction mixture thus
obtained was used
for subcloning of the DNA fragment into plasmid pUC 19 that had been digested
with BamHI
and HindIII. The resulting plasmid was sequenced. The plasmid thus prepared
was
designated as "hMBCL/pUC 19". In this plasmid, however, the 104-position amino
acid
(corresponding to the 96th amino acid in accordance with the Kabat's
prescription) of CDR4
was replaced by arginine. For the correction of this amino acid to tyrosine, a
correction
primer MBC1LGP10R (SEQ ID NO: 35) was designed and synthesized. The PCR
reaction
was performed using TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) and a buffer
appended thereto.
The PCR reaction solution comprised (per 100 ~l) 0.6 ~,g of the plasmid
hMBCL/pUC 19 as a
template DNA, 50 pmoles of each of the primers MBC1LUS1 and MBC1LGP10R, 2.5U
of
TaKaRa Ex Taq (Takara Shuzo Co., Ltd.) and 0.25 mM dNTPs in the buffer, over
which
mineral oil (50 L~.1) was layered. The PCR reaction was run for 30 cycles
under the
conditions: 94~ for 1 min., 55~ for 1 min. and 72~ for 1 min. The DNA fragment
thus
amplified was separated by agarose gel electrophoresis on a 3% Nu Sieve GTG
agarose (FMC
81
CA 02401357 2002-08-27
Bio. Products).
A gel segment containing a DNA fragment of 421 by was excised, and the DNA
fragment was purified therefrom using GENECLEANII Kit (BIO101) in accordance
with the
instructions included in the kit. The PCR reaction mixture thus prepared was
used for
subcloning of the DNA fragment into plasmid pUC 19 that had been digested with
BamHI and
HindIII.
The plasrnid was sequenced using M 13 Primer M4 and M 13 Primer RV. As a
result, it was confirmed that the plasmid had the correct sequence. The
plasmid was then
digested with HindIII and BInI, and a DNA fragment of 416 by was separated by
electrophoresis on a 1 % agarose gel. The DNA fragment was purified using
GENECLEANII Kit (BIO101) in accordance with the instructions included in the
kit, and
then introduced into plasmid C ~ /pUC 19 that had been digested with HindIII
and BInI. The
resulting plasmid was designated as "hMBC 1 La ~ /pUC 19". This plasmid was
digested with
EcoRI to give a DNA fragment encoding humanized L-chain. The DNA fragment was
introduced into plasmid pCOS 1 so that the initiation codon for the humanized
L-chain was
located downstream to the EFl c~ promoter. The plasmid thus obtained was
designated as
"hMBC 1La ~l /pCOS 1". The DNA sequence (including the corresponding amino
acid
sequence) of the humanized L-chain version "a" is shown in SEQ ID NO: 66. The
amino
acid sequence of the version "a" is also shown in SEQ ID NO: 47.
A humanized L-chain version "b" was prepared using mutagenesis by PCR method.
The version "b" was designed such that the 43-position amino acid glycine
(corresponding to
the 43th amino acid in accordance with the Kabat's prescription) was replaced
by proline and
the 49-position amino acid lysine (corresponding to the 49th amino acid
accordance with the
Kabat's prescription) by aspartic acid in the version "a". The PCR reaction
was performed
82
CA 02401357 2002-08-27
using plasmid hMBC 1 La ~l /pUC 19 as a template and a mutagenic primer MBC 1
LGPSR
(SEQ ID NO: 36) and a primer MBC 1 LV S 1. The DNA fragment obtained was
digested
with BamHI and HindIII, and the digestion fragment was subcloned into the
BamHI-HindIII
site of pUC 19. After sequencing, the plasmid was digested with HindIII and Af
l II, and the
resulting digestion fragment was ligated to plasmid hMBC 1 La ~ /pUC 19 that
had been
digested with HindIII and AfIII.
The thus obtained plasmid was designated as "hMBCILb ~ /pUCl9". This
plasmid was digested with EcoRI to give a DNA fragment containing a DNA
encoding the
humanized L-chain. The DNA fragment was introduced into plasmid pCOS 1 such
that the
initiation colon for the humanized L-chain was located downstream to the EF1
cx promoter.
The plasmid thus obtained was designated as "hMBC 1Lb ~. /pCOS 1".
A humanized L-chain version "c" was prepared using mutagenesis by PCR method.
The version "c" was designed such that the 84-position amino acid serine
(corresponding to
the 80th amino acid in accordance with the Kabat's prescription) was replaced
by proline.
The PCR reaction was performed using plasmid hMBC 1 La ~1 /pUC 19 as a
template and a
mutagenic primer MBC 1 LGP6S (SEQ ID NO: 37) and a primer M 13 Primer RV The
DNA
fragment obtained was digested with BamHI and HindIII and then subcloned into
pUC 19 that
had been digested with BamHI and HindIII.
After sequencing, the plasmid was digested with BstPI and Aor5lHI, and the
resulting DNA fragment was ligated to plasmid hMBC 1La ~l /pUC 19 that had
been digested
with BstPI and Aor5lHI. The plasmid thus obtained was designated as "hMBCILc ~
/pUC 19". This plasmid was digested with EcoRI to give a DNA fragment
containing a
DNA encoding the humanized L-chain. The fragment was introduced into the EcoRI
site of
plasmid pCOS 1 such that the initiation colon for the humanized L-chain was
located
83
CA 02401357 2002-08-27
downstream to the EF1 cx promoter. The plasmid thus obtained was designated as
"hMBC 1 Lc ~1 /pCOS 1 ".
Humanized L-chain versions "d", "e" and "f' were also prepared using
mutagenesis
by PCR method. The versions "d", "e" and "f' were designed such that the 91-
position
amino acid tyrosine (corresponding to the 87th amino acid in accordance with
the Kabat's
prescription) was replaced by isoleucine in the versions "a", "b" and "c",
respectively. For
each of the versions "d", "e" and "f', a PCR reaction was performed using each
of plasmid
hMBC 1La ~1 /pCOS 1 (for version "d"), hMBC 1 Lb ~l /pCOS 1 (for version "e")
and hMBC 1Lc
~/pCOSl (for version "f'), respectively, as a template, a mutagenic primer
MBC1LGP11R
(SEQ D7 NO: 38) and a primer M-S 1 (SEQ ID NO: 44). The DNA fragment thus
obtained
was digested with BamHI and HindIII and then subcloned into pUC 19 that had
been digested
with BamHI and HindIII. After sequencing, the plasmid was digested with
HindIII and BInI,
and the resulting digestion fragment was ligated to plasmid C ~1 /pUC 19 that
had been
digested with HindIII and BInI.
The thus obtained plasmids were respectively designated as "hMBC 1 Ld ~. /pUC
19"
(for version "d"), "hMBC 1 Le ~ /pUC 19" (for version "e") and "hMBC 1 Lf ~l
/pUC 19" (for
version "f'). Each of these plasmids was digested with EcoRI to give a DNA
fragment
containing a DNA encoding the humanized L-chain. The DNA fragment was
introduced
into the EcoRI site of plasmid pCOS 1 such that the initiation codon for the
humanized L-
chain was located downstream to the EF 1 a promoter of the plasmid. The
plasmids thus
obtained were respectively designated as "hMBC 1Ld ~l /pCOS 1" (for version
"d"),
"hMBC 1Le ~ /pCOS 1" (for version "e") and "hMBC 1Lf ~1 /pCOS 1" (for version
"f').
Humanized L-chain versions "g" and "h" were also prepared using mutagenesis by
PCR method. The versions "g" and "h" were designed such that the 36-position
amino acid
84
CA 02401357 2002-08-27
histidine (corresponding to the 36th amino acid in accordance with the Kabat's
prescription)
was replaced by tyrosine in the versions "a" and "d", respectively. The PCR
reaction was
performed using a mutagenic primer MBC1LGP9R (SEQ ID NO: 39), M13 Primer RV
and
plasmid hMBC 1 La ~1 /pUC 19 as a template. An additional PCR was performed
using the
PCR product thus obtained and M 13 Primer M4 as primers and plasmid hMBC 1 La
~ /pUC 19
as a template. The DNA fragment obtained was digested with HindIII and BInI
and then
subcloned into plasmid C ~1 /pUC 19 that had been digested with HindIII and
BInI. Using
this plasmid as a template, a PCR reaction was performed using primers
MBC1LGP13R
(SEQ ID NO: 40) and MBC 1 LV S 1. The PCR fragment obtained was digested with
ApaI
and HindIII and then introduced into either of plasmids hMBC 1 La ~ /pUC 19
and hMBC 1 Ld
~ /pUC 19 that had been digested with ApaI and HindIII. The plasmids obtained
were
sequenced. Plasmids that were confirmed to contain the correct sequence were
designated as
"hMBC 1 Lg ~1 /pUC 19" (for version "g") and "hMBC 1 Lh ~1 /pUC 19" (for
version "h"). Each
of these plasmids was digested with EcoRI to give a DNA fragment containing a
DNA
encoding the humanized L-chain. The DNA fragment was introduced into the EcoRI
site of
plasmid pCOS 1 such that the initiation codon for the humanized L-chain was
located
downstream to the EF1 c~ promoter. The plasmids thus obtained were
respectively
designated as "hMBCILg~1/pCOSI" (for version "g") and "hMBCILh~1/pCOSl" (for
version "h").
Humanized L-chain versions "i", "j ", "k", "1", "m", "n" and "o" were also
prepared
using mutagenesis by PCR method. The PCR reaction was performed using plasmid
hMBCILa~1 /pUCl9 as a template and a mutagenic primer MBC1LGP14S (SEQ ID NO:
41)
and a primer V 1 RV ( ~ ) (SEQ ID NO: 43). The resulting DNA fragment was
digested with
ApaI and BInI and then subcloned into plasmid hMBC 1 Lg ~1 /pUC 19 that had
been digested
with ApaI and BInI. The obtained plasmid was sequenced, and the clone into
which the
mutation for each version was introduced was selected. The thus obtained
plasmid was
g5
CA 02401357 2002-08-27
designated as "hMBC 1 Lx ~l /pUC 19 (x=i, j, k, l, m, n or o)". This plasmid
was digested with
EcoRI to give a DNA fragment containing a DNA encoding the humanized L-chain.
The
DNA fragment was introduced into the EcoRI site of plasmid pCOS 1 such that
the initiation
codon for the humanized L-chain was located downstream of the EF1 c~ promoter.
The thus
obtained plasrnid was designated as "hMBCILx ~. /pCOSl" (x = i, j, k, l, m, n
or o). The
DNA sequences (including the corresponding amino acid sequences) of the
versions "j ", "1",
"m" and "o" are shown in SEQ ID NOs: 67, 68, 69 and 70, respectively. The
amino acid
sequences of these versions are also shown in SEQ ID Nos: 48, 49, 50 and 51,
respectively.
Humanized L-chain versions "p", "q", "r", "s" and "t" were designed such that
the
87-position amino acid (tyrosine) was replaced by isoleucine in the versions
"i", "j", "m", "1"
and "o", respectively. These versions were prepared utilizing an Aor5lMI
restriction site on
FR3 and replacing that site of each of the versions "i", "j", "m", "1" or "o"
by that site of the
version "h". That is, an Aor5lHI restriction fragment (514 bp) containing
CDR3, a part of
FR3 and the entire FR4 were removed from an expression plasmid hMBCILx ~1
/pCOS 1 (x =
i, j, m, l or o). To the removed site, an Aor5lHI restriction fragment (514
bp) in the
expression plasmid hMBC 1 Lh ~ /pCOS, which containing CDR3 and a part of FR3
and the
entire FR4, was ligated, so that the 91-position amino acid tyrosine
(corresponding to the 87th
amino acid in accordance with the Kabat's prescription) was replaced by
isoleucine. The
resulting plasmid was sequenced. A clone of each of the versions "i", "j", "m"
"1" and "o" in
which 91-position amino acid tyrosine (corresponding to the 87th amino acid in
accordance
with the Kabat's prescription) was replaced by isoleucine was selected. These
modified
versions respectively corresponding to the versions "i", "j ", "m" "1" and "o"
were designated
as versions "p", "q", "s", "r" and "t", respectively. The obtained plasmid was
designated as
"hMBC 1 Lx ~1 /pCOS 1 (x =p, q, s, r or t). The DNA sequences (including the
corresponding
amino acids) of the versions "q", "r", "s" and "t" are shown in SEQ ID Nos:
71, 72, 73 and 74,
respectively. The amino acid sequences of these versions are also shown in SEQ
ID Nos: 52,
86
CA 02401357 2002-08-27
53, 54 and 55, respectively.
Plasmid hMBC 1 Lq ~ /pCOS 1 was digested with HindIII and EcoRI and then
subcloned into plasmid pUC 19 that had been digested with HindIII and EcoRI.
The plasmid
thus obtained was designated as "hMBC 1Lq ~l /pUC 19.
The positions of the replaced amino acids in the individual versions of the
humanized L-chain are shown in Table 7 below.
8'7
CA 02401357 2002-08-27
Table 7
Versions 36 43 45 47 49 80 87
a
P D
c P
D I
P I
g
I
i Y K
D
k Y K V
D
m D
n Y V
o D
P
q Y K D I
r Y D I
s D
t D I
In Table 7, capital letters represent the following amino acids: Y: tyrosine;
P: proline; K:
lysine, V: valine; D: aspartic acid; and I: isoleucine.
E. coli strains each containing plasmids hMBC 1 HcDNA/pUC 19 and hMBC 1 Lq ~
/pUC 19 were designated as "Escherichia coli JM 109 (hMBC 1 HcDNA/pUC 19)" and
"Escherichia coli JM 109 (hMBC 1 Lq ~ /pUC 19)", respectively, which have been
deposited
under the terms of Budapest Treaty at the International Patent Organism
Depositary (IPOD),
National Institute of Advanced Industrial Science and Technology, Japan (1-1,
Higashi 1-
chome, Tsukuba-shi, Ibaraki, Japan) on August 15, 1996, under the accession
No. FERM BP-
5629 for Escherichia coli JM109 (hMBCIHcDNA/pUCl9), and FERM BP-5630 for
Escherichia coli JM 109 (hMBC 1 Lq ~ /pUC 19).
(5) Transfection into COS-7 cell
CA 02401357 2002-08-27
For the evaluation of the antigen-binding activity and the neutralizing
activity of the
hybrid antibodies and the humanized #23-57-137-1 antibodies, the above-
prepared expression
plasmids were expressed transiently in COS-7 cells. For the transient
expression of the L-
chain hybrid antibodies, each of the following combinations of plasmids were
co-transfected
into a COS-7 cell by electroporation using Gene Pulser (Bio Rad):
hMBCIHcDNA/pCOSl
and h/mMBC 1L( ~1 )/neo; hMBC lHcDNA/pCOS 1 and m/hMBC 1La ~l /neo;
hMBC lHcDNA/pCOS 1 and m/hMBC 1 Ld ~ /neo; hMBC lHcDNA/pCOS 1 and
hmmMBC 1 L( ~1 )/neo; and hMBC 1 HcDNA/pCOS 1 and mhmMBC 1 L( ~. )/neo. That
is, a
cell suspension (0.8 ml) of COS-7 cells in PBS(-) (1x10' cells/ml) was added
with each
combination of the plasmid DNAs ( 10 pg each). The resulting solution was
applied with
pulses at an electrostatic capacity of 1,500V and 25 gF. After 10 min. of
recovery period at
room temperature, the electroporated cells were suspended in DMEM medium
containing 2%
Ultra Low IgG fetal calf serum (GIBCO), and then cultured using a 10-cm
culture dish in a
C02 incubator. After culturing for 72 hours, a culture supernatant was
collected and
centrifuged to remove cell debris. The solutions thus prepared were provided
for use in the
ELISA below.
For the transient expression of the humanized #23-57-137-1 antibodies,
plasmids of
hMBC lHcDNA/pCOS 1 and hMBC 1 Lx ~l /pCOS 1 (x = a-t) were co-transfected into
a COS-7
cell using Gene Pulser (Bio Rad) in the same manner as described for the above
hybrid
antibodies. The culture supernatants were prepared and provided for use in the
ELISA
below.
The purification of the hybrid antibodies and the humanized antibodies from
the
COS-7 cell culture supernatants was performed using AffiGel Protein A MAPSII
Kit (Bio
Rad) in accordance with the instructions included in the kit.
89
CA 02401357 2002-08-27
(6) ELISA
(i) Determination of antibody concentration
An ELISA plate for determining antibody concentration was prepared as follows.
Each well of a 96-well ELISA plate (Maxisorp, NUNC) was coated with 100 p1 of
a coating
buffer (0.1 M NaHC03, 0.02% NaN3) containing 1 pg/ml of goat anti-human IgG
antibody
(TAGO) and then blocked with 200 ~c 1 of a dilution buffer [50 mM Tris-HCI, 1
mM MgCl2,
0.1 M NaCI, 0.05% Tween 20, 0.02% NaN3, 1% bovine serum albumin (BSA); pH
7.2].
Each of the wells was added with each of the serial dilutions of the COS cell
culture
supernatant in which each of the hybrid antibodies and the humanized
antibodies was
expressed, or added with each of the serial dilutions of each of the hybrid
antibodies and
humanized antibodies in a purified form. The plate was incubated at room
temperature for 1
hour and washed with PBS-Tween 20. Subsequently, each of the wells was added
with 100
p1 of alkaline phosphatase-conjugated goat anti-human IgG antibody (TAGO). The
plate
was incubated at room temperature for 1 hour and washed with PBS-Tween 20.
Subsequently, each of the wells was added with 1 mg/ml of a substrate solution
("Sigma 104",
p-nitrophenylphosphoric acid, SIGMA). The solution in each well was measured
on its
absorbance at 405 nm using Microplate Reader (Bio Rad) to determine the
antibody
concentration. In this determination, Hu IgGI ~ Purified (The Binding Site)
was used as
the standard substance.
(ii) Determination of antigen-binding ability
An ELISA plate for determining antigen-binding ability was prepared as
follows.
Each well of a 96-well ELISA plate (Maxisorp, NUNC) was coated with 100 p,1 of
a coating
buffer containing 1 p,g/ml of human PTHrP (1-34) and then blocked with 200 p.1
of a dilution
buffer. Subsequently, each well was added with each of the serial dilutions of
the COS-7
cell culture supernatant in which each of the hybrid antibodies and humanized
antibodies was
expressed, or added with each of the serial dilutions of each of the hybrid
antibodies and
CA 02401357 2002-08-27
humanized antibodies in a purified form. The plate was incubated at room
temperature and
washed with PBS-Tween 20. Subsequently, each well was added with 100 ~.l of
alkaline
phosphatase-conjugated goat anti-human IgG antibody (TAGO). The plate was
incubated at
room temperature and washed with PBS-Tween 20. Subsequently, each well was
added with
1 mg/ml of a substrate solution ("Sigma 104", p-nitrophenylphosphoric acid,
SIGMA). The
solution was measured on its absorbance at 405 nm using Microplate Reader (Bio
Rad).
(7) Confirmation of activities
(i) Evaluation of humanized H-chain
It was found that an antibody having both a humanized H-chain version "a" and
a
chimeric L-chain exhibited the same level of PTHrP-binding activity as that of
a chimeric
antibody. This result suggests that the version "a" achieves the humanization
of the H-chain
V-region in the degree enough to evaluate the humanization. Therefore, the
humanized H-
chain version "a" was provided for use as a humanized antibody H-chain in the
following
experiments.
(ii) Activity of hybrid antibodies
(ii-a) FR1,2/FR3,4 hybrid antibody
When the L-chain was h/mMBC 1L( ~ ), no antigen-binding activity was observed.
In contrast, when the L-chain was either m/hMBC 1 La ~ or m/hMBC 1 Ld ~1, the
same level
of antigen-binding activity as that of the chimeric #23-57-137-1 antibody was
observed (FIG.
7). These results suggest that FR3 and FR4 have no problem as humanized
antibodies but
FR1 and FR2 contain amino acid residues) that need to be replaced.
(ii-b) FR1/FRZ hybrid antibody
When the L-chain was mhmMBCIL ( ~l ), no antigen-binding activity was
observed.
In contrast, when the L-chain was hmmMBC 1 L( ~ ), the same level of antigen-
binding
91
CA 02401357 2002-08-27
activity as that of the chimeric #23-57-137-1 antibody was observed (FIG. 8).
These results
suggest that FR 1 has no problem as a humanized antibody but FR2 contains
amino acid
residues) that need to be replaced.
(iii) Activity of humanized antibodies
The antigen-binding activity of the humanized antibodies having the L-chain
versions "a" to "t", respectively, were determined. As a result, it was found
that the
humanized antibodies having the L-chain versions "j ", "1" "m", "o", "q", "r",
"s" and "t"
exhibited the same levels of PTHrP-binding activity as that of the chimeric
antibody.
(8) Establishment of CHO cell line capable of stable production of antibody
For establishing a cell line capable of stable production of humanized
antibodies,
each of the above-prepared expression plasmids was introduced into a CHO cell
(DXB11).
That is, the establishment of a cell line capable of stable production of a
humanized
antibody was performed using each of the following combinations of plasmids as
expression
vectors for a CHO cell; hMBCIHcDNA/pCH01 and hMBCILm~/pCOSl;
hMBCIHcDNA/pCH01 andhMBCILq~1/pCOSl; andhMBCIHcDNA/pCH01 and
hMBC 1 Lr ~ /pCOS 1. The plasmids were co-transfected into a CHO cell by
electroporation
using Gene Pulser (Bio Rad). Subsequently, the expression vectors were
separately cleaved
with restriction enzyme PvuI to give linear DNA fragments. The resulting DNA
fragments
were extracted with phenol and chloroform and then precipitated with ethanol.
The DNA
fragments thus prepared were used in the subsequent electroporation. That is,
the plasmid
DNA fragments (10 pg each) were added to 0.8 ml of a cell suspension of CHO
cells in PBS(-
( 1 x 107 cells/ml). The resulting solution was applied with pulses at an
electrostatic capacity
of 1,500V and 25 ~.F. After 10 min. of recovery period at room temperature,
the cells thus
treated were suspended in MEM- c~ medium (GIBCO) containing 10% fetal calf
serum
92
CA 02401357 2002-08-27
(GIBCO), and then cultured in a C02 incubator using 96-well plates (Falcon).
On the day
following the culturing being started, the medium was replaced by
ribonucleoside- or
deoxyribonucleoside-free MEM- a selective medium containing 10% fetal calf
serum
(GIBCO) and 500 mg/ml of GENETICIN (G418Sulfate; GIBCO). From the culture
medium, cells into which the antibody gene was introduced were selected. The
culture
medium was replaced by a fresh one. About two weeks after the medium
replacement, the
cells were observed microscopically. When a satisfactory cell growth was
observed, the
amount of the antibodies produced was determined by conventional ELISA for
determination
of antibody concentration as set forth above. Among the cells, those cells
which produced a
larger amount of antibodies were screened.
The culturing of the established cell line capable of stable production of
antibodies
was scaled up in a roller bottle using a ribonucleoside- or
deoxyribonucleoside-free MEM- cx
medium containing 2% Ultra Low IgG fetal calf serum. On each of day 3 and day
4 of the
culturing, the culture supernatant was collected and filtered on a 0.2-pm
filter (Millipore) to
remove cell debris therefrom. The purification of the humanized antibodies
from the culture
supernatant of the CHO cells was performed using POROS Protein A Column
(PerSeptive
Biosystems) on ConSep LC 100 (Millipore) in accordance with the appended
instructions.
The humanized antibodies were provided for use in the determination of
neutralizing activity
and examination of pharmacological efficacy in hypercalcemic model animals.
The
concentration and the antigen-binding activity of the purified humanized
antibodies were
determined by the ELISA system as set forth above.
[REFERENCE EXAMPLE 5] Determination of neutralizing activity
The determination of neutralizing activity of the mouse antibodies, the
chimeric
antibodies and the humanized antibodies was performed using rat myeloma cell
line
ROS 17/2.8-5 cells. The ROS 17/2.8-5 cells were cultured in Ham'S F-12 medium
(GIBCO)
93
CA 02401357 2002-08-27
containing 10% fetal calf serum (GIBCO) in a C02 incubator. The ROS17/2.8-5
cells were
seeded into each well of a 96-well plate at a density of 104 cells/100 pl/well
and cultured for
one day. After the culturing was completed, the culture medium was replaced by
Ham'S F-
12 medium (GIBCO) containing 4 mM Hydrocortisone and 10% fetal calf serum.
After
culturing for three to four days, the cultured cells were washed with 260 ~,1
of Ham'S F-12
medium (GIBCO), and then added with 80 p1 of Ham's F-12 medium containing 1 mM
isobutyl-1-methyl xanthine (IBMX, SIGMA), 10% fetal calf serum and 10 mM
HEPES.
The resulting mixture was incubated at 37~ for 30 min.
The culture mediums of the mouse antibodies, the chimeric antibodies and the
humanized antibodies to be tested for neutralizing activity were previously
diluted serially in
the following dilution series: [10 pg/ml, 3.3 wg/ml, 1.1 g,g/ml and 0.37
pg/ml], [10 pglml, 2
pg/ml, 0.5 pg/ml and 0.01 pg/ml] and [ 10 pg/ml, 5 ~,g/ml, 1.25 pg/ml, 0.63
~.g/ml and 0.31
~g/ml]. Each of the diluted antibody sample solutions was mixed with an
equivalent amount
of 4 ng/ml of PTHrP (1-34). The resulting mixed solution (80 ~,l) was added to
each well.
In each well, the final concentration of each antibody became a quarter of the
above-
mentioned concentration of the antibody, and accordingly the concentration of
PTHrP (1-34)
became 1 ng/ml. After the treatment at room temperature for 10 min., the
culture
supernatant was removed and the residue was washed with PBS three times.
Subsequently,
cAMP in the cells was extracted with 100 p1 of a 0.3% HCl-95% ethanol and then
evaporated
using a water jet aspirator to remove the HCl-ethanol. The residue was
dissolved in 120 p1
of EIA buffer appended to cAMP EIA Kit (CAYMAN CHEMICAL'S) to extract the CAMP
therefrom. The cAMP was determined using cAMP EIA Kit (CAYMAN CHEMICAL'S) in
accordance with the instructions included in the kit. As a result, it was
found that, among
the humanized antibodies having the same levels of antigen-binding activity as
that of the
chimeric antibody, those antibodies having L-chain versions "q", "r", "s" and
"t" (in which the
91-position tyrosine was replaced by isoleucine) exhibited the similar
neutralizing activity to
9~
CA 02401357 2002-08-27
that of the chimeric antibody, and that antibody having a L-chain version "q"
exhibited the
strongest neutralizing activity.
All publications, patents and patent applications cited herein are
incorporated herein
by reference in their entirety.
Industrial Applicability
The present invention provides a tissue degradation inhibiting agent which
comprises
as an active ingredient a substance inhibiting the binding of a parathyroid
hormone-related
peptide to its receptor. The inhibiting agent of the present invention is
useful as a
pharmaceutical composition for inhibiting body weight loss associated
cancerous cachexia.
Sequence
Listing
Free Text
SEQ ID NO: Synthesized
1 DNA
SEQ ID NO: Synthesized
2 DNA
SEQ ID NO: Synthesized
3 DNA
SEQ 117 NO: Synthesized
4 DNA
SEQ ID NO: Synthesized
DNA
SEQ ll~ NO: Synthesized
6 DNA
SEQ ID NO: Synthesized
7 DNA
SEQ ID NO: Synthesized
8 DNA
SEQ ID NO: Synthesized
9 DNA
SEQ ID NO: Synthesized
DNA
SEQ ID NO: Synthesized
11 DNA
SEQ ID NO: Synthesized
12 DNA
SEQ ID NO: Synthesized
13 DNA
SEQ ID NO: Synthesized
14 DNA
CA 02401357 2002-08-27
SEQ m NO: Synthesized
15 DNA
SEQ m NO: Synthesized
16 DNA
SEQ m NO: Synthesized
17 DNA
SEQ m NO: Synthesized
18 DNA
SEQ a7 NO: Synthesized
19 DNA
SEQ m NO: Synthesized
20 DNA
SEQ m NO: Synthesized
21 DNA
SEQ m NO: Synthesized
22 DNA
SEQ ~ NO: Synthesized
23 DNA
SEQ m NO: Synthesized
24 DNA
SEQ m NO: Synthesized
25 DNA
SEQ m NO: Synthesized
26 DNA
SEQ m NO: Synthesized
27 DNA
SEQ m NO: Synthesized
28 DNA
SEQ m NO: Synthesized
29 DNA
SEQ n7 NO: Synthesized
30 DNA
SEQ m NO: Synthesized
31 DNA
SEQ m NO: Synthesized
32 DNA
SEQ m NO: Synthesized
33 DNA
SEQ m NO: Synthesized
34 DNA
SEQ m NO: Synthesized
35 DNA
SEQ m NO: Synthesized
36 DNA
SEQ m NO: Synthesized
37 DNA
SEQ m NO: Synthesized
38 DNA
SEQ m NO: Synthesized
39 DNA
SEQ m NO: Synthesized
40 DNA
SEQ m NO: Synthesized
41 DNA
96
CA 02401357 2002-08-27
r
SEQ B7 NO: 42 Synthesized DNA
SEQ m NO: 43 Synthesized DNA
SEQ m NO: 44 Synthesized DNA
97
r
CA 02401357 2002-08-27
SEQUENCE LISTING
<110~ CHUGAI PHARMACEUTICAL CO., LTD.
<120~ Tissue degradation inhibiting agent
<130~ PH-1015-PCT
<140~ PCT/JP00/05886
<141~ 2000-08-30
<150> JP2000-52414
<151> 2000-02-28
<160~ 75
<170~ PatentIn Ver. 2.0
<210~ 1
<211~ 20
<212~ DNA
<213> Artificial SeQuence
<220>
<223~ Synthetic DNA
<400~ 1
1/49
CA 02401357 2002-08-27
aaatagccct tgaccaggca 20
<210> 2
<211~ 38
<212~ DNA
<213> Artificial Sequence
<220~
<223~ Synthetic DNA
<400~ 2
ctggttcggc ccacctctga aggttccaga atcgatag 38
<210~ 3
<211~ 28
<212~ DNA
<213~ Artificial Sequence
<220~
<223~ Synthetic DNA
<400~ 3
ggatcccggg ccagtggata gacagatg 28
<210~ 4
<211~ 29
<212~ DNA
<213~ Artificial Sequence
2/49
CA 02401357 2002-08-27
<220~
<223~ Synthetic DNA
<400~ 4
ggatcccggg tcagrggaag gtggraaca 29
<210~ 5
<211~ 17
<212~ DNA
<213~ Artificial Sequence
<220~
<223> Synthetic DNA
<400~ 5
gttttcccag tcacgac 17
<210~ 6
<211~ 17
<212~ DNA
<213> Artificial Sequence
<220~
<223~ Synthetic DNA
<400~ 6
caggaaacag ctatgac 17
3/49
CA 02401357 2002-08-27
<210~ 7
<211~ 31
<212~ DNA
<213~ Artificial Sequence
<220~
<223~ Synthetic DNA
<400~ 7
gtctaagctt ccaccatgaa acttcgggct c 31
<210~ 8
<211~ 30
<212~ DNA
<213~ Artificial Sequence
<220>
<223~ Synthetic DNA
<400~ 8
tgttggatcc ctgcagagac agtgaccaga 30
<210~ 9
<211~ 36
<212~ DNA
<213~ Artificial Sequence
4/49
CA 02401357 2002-08-27
<220~
<223~ Synthetic DNA
<400~ 9
gtctgaattc aagcttccac catggggttt gggctg 36
<210~ 10
<211~ 41
<212~ DNA
<213~ Artificial SeQUence
<220~
<223~ Synthetic DNA
<400~ 10
tttcccgggc ccttggtgga ggctgaggag acggtgacca g 41
<210~ 11
<211> 109
<212~ DNA
<213> Artificial SeQUence
<220~
<223~ Synthetic DNA
<400~ 11
gtctgaattc aagcttagta cttggccagc ccaaggccaa ccccacggtc accctgttcc 60
cgccctcctc tgaggagctc caagccaaca aggccacact agtgtgtct 109
5/49
CA 02401357 2002-08-27
<210~ 12
<211~ 110
<212~ DNA
<213~ Artificial Sequence
<220~
<223> Synthetic DNA
<400> 12
ggtttggtgg tctccactcc cgccttgacg gggctgccat ctgccttcca ggccactgtc 60
acagctcccg ggtagaagtc actgatcaga cacactagtg tggccttgtt 110
<210~ 13
<211> 98
<212> DNA
<213~ Artificial Sequence
<220~
<223~ Synthetic DNA
<400~ 13
ggagtggaga ccaccaaacc ctccaaacag agcaacaaca agtacgcggc cagcagctac 60
ctgagcctga cgcccgagca gtggaagtcc cacagaag 98
<210~ 14
<211~ 106
<212~ DNA
6/49
CA 02401357 2002-08-27
<213~ Artificial Sequence
<220~
<223> Synthetic DNA
<400~ 14
tgttgaattc ttactatgaa cattctgtag gggccactgt cttctccacg gtgctccctt 60
catgcgtgac ctggcagctg tagcttctgt gggacttcca ctgctc 106
<210~ 15
<211~ 43
<212> DNA
<213~ Artificial Sequence
<220>
<223~ Synthetic DNA
<400~ 15
gtctgaattc aagcttagta cttggccagc ccaaggccaa ccc 43
<210~ 16
<211~ 20
<212~ DNA
<213~ Artificial Sequence
<220~
<223~ Synthetic DNA
7/49
CA 02401357 2002-08-27
<400~ 16
tgttgaattc ttactatgaa 20
<210~ 17
<211~ 39
<212~ DNA
<213~ Artificial SeQuence
<220>
<223~ Synthetic DNA
<400~ 17
caacaagtac gcggccagca gctacctgag cctgacgcc 39
<210> 18
<211> 39
<212~ DNA
<213~ Artificial SeQuence
<220~
<223~ Synthetic DNA
<400~ 18
gtagctgctg gccgcgtact tgttgttgct ctgtttgga 39
<210~ 19
<211~ 46
<212~ DNA
8/49
CA 02401357 2002-08-27
<213~ Artificial SeQUence
<220>
<223~ Synthetic DNA
<400~ 19
gtctgaattc aagcttagtc ctaggtcgaa ctgtggctgc accatc 46
<210> 20
<211> 34
<212> DNA
<213> Artificial Sequence
<220~
<223> Synthetic DNA
<400~ 20
tgttgaattc ttactaacac tctcccctgt tgaa 34
<210~ 21
<211~ 35
<212~ DNA
<213~ Artificial SeQUence
<220~
<223> Synthetic DNA
<400~ 21
9/49
CA 02401357 2002-08-27
gtctaagctt ccaccatggc ctggactcct ctctt 35
<210~ 22
<211~ 48
<212~ DNA
<213~ Artificial Sequence
<220~
<223~ Synthetic DNA
<400~ 22
tgttgaattc agatctaact acttacctag gacagtgacc ttggtccc 48
<210~ 23
<211> 128
<212> DNA
<213~ Artificial Se4uence
<220~
<223> Synthetic DNA
<400~ 23
gtctaagctt ccaccatggg gtttgggctg agctgggttt tcctcgttgc tcttttaaga 60
ggtgtccagt gtcaggtgca gctggtggag tctgggggag gcgtggtcca gcctgggagg 120
tccctgag 128
<210~ 24
<211~ 125
10/49
CA 02401357 2002-08-27
<212~ DNA
<213~ Artificial SeQuence
<220~
<223~ Synthetic DNA
<400~ 24
accattagta gtggtggtag ttacacctac tatccagaca gtgtgaaggg gcgattcacc 60
atctccagag acaattccaa gaacacgctg tatctgcaaa tgaacagcct gagagctgag 120
gacac
125
<210> 25
<211~ 132
<212~ DNA
<213~ Artificial Sequence
<220~
<223~ Synthetic DNA
<400~ 25
ctaccaccac tactaatggt tgccacccac tccagcccct tgcctggagc ctggcggacc 60
caagacatgc catagctact gaaggtgaat ccagaggctg cacaggagag tctcagggac 120
ctcccaggct gg 132
<210~ 26
<211~ 110
<212~ DNA
<2I3~ Artificial SeQUence
11/49
CA 02401357 2002-08-27
<220~
<223~ Synthetic DNA
<400~ 26
tgttggatcc ctgaggagac ggtgaccagg gttccctggc cccagtaagc aaagtaagtc 60
atagtagtct gtctcgcaca gtaatacaca gccgtgtcct cagctctcag 110
<210> 27
<211~ 30
<212~ DNA
<213~ Artificial SeQuence
<220~
<223~ Synthetic DNA
<400~ 27
gtctaagctt ccaccatggg gtttgggctg 30
<210~ 28
<211~ 30
<212~ DNA
<213~ Artificial SeQuence
<220~
<223~ Synthetic DNA
<400~ 28
12/49
CA 02401357 2002-08-27
tgttggatcc ctgaggagac ggtgaccagg 30
<210~ 29
<211~ 133
<212~ DNA
<213~ Artificial Sequence
<220>
<223~ Synthetic DNA
<400~ 29
acaaagcttc caccatggcc tggactcctc tcttcttctt ctttgttctt cattgctcag 60
gttctttctc ccagcttgtg ctgactcaat cgccctctgc ctctgcctcc ctgggagcct 120
cggtcaagct cac 133
<210~ 30
<211~ 118
<212~ DNA
<213> Artificial Sequence
<220~
<223~ Synthetic DNA
<400~ 30
agcaagatgg aagccacagc acaggtgatg ggattcctga tcgcttctca ggctccagct 60
ctggggctga gcgctacctc accatctcca gcctccagtc tgaggatgag gctgacta 118
<210~ 31
13/49
CA 02401357 2002-08-27
<211> 128
<212~ DNA
<213~ Artificial Sequence
<220~
<223~ Synthetic DNA
<400~ 31
ctgtggcttc catcttgctt aagtttcatc aagtaccgag ggcccttctc tggctgctgc 60
tgatgccatt caatggtgta cgtactgtgc tgactactca aggtgcaggt gagcttgacc 120
128
gaggctcc
<210~ 32
<211~ 114
<212> DNA
<213> Artificial Sequence
<220>
<223~ Synthetic DNA
<400~ 32
cttggatccg ggctgaccta ggacggtcag tttggtccct ccgccgaaca ccctcacaaa 60
ttgttcctta attgtatcac ccacaccaca gtaatagtca gcctcatcct caga 114
<210~ 33
<211> 17
<212~ DNA
<213~ Artificial Sequence
14/49
CA 02401357 2002-08-27
<220~
<223~ Synthetic DNA
<400~ 33
acaaagcttc caccatg 17
<210> 34
<211~ 19
<212~ DNA
<213> Artificial SeQuence
<2Z0~
<223~ Synthetic DNA
<400~ 34
cttggatccg ggctgacct 19
<210> 35
<211~ 75
<212~ DNA
<213> Artificial SeQUence
<220~
<223~ Synthetic DNA
<400~ 35
cttggatccg ggctgaccta ggacggtcag tttggtccct ccgccgaaca cgtacacaaa 60
15/49
CA 02401357 2002-08-27
ttgttcctta attgt 75
<210> 36
<211~ 43
<212~ DNA
<213~ Artificial Sequence
<220~
<223> Synthetic DNA
<400~ 36
aaaggatcct taagatccat caagtaccga gggggcttct ctg 43
<210~ 37
<211~ 46
<212> DNA
<213~ Artificial Sequence
<220~
<223~ Synthetic DNA
<400~ 37
acaaagctta gcgctacctc accatctcca gcctccagcc tgagga 46
<210~ 38
<211~ 111
<212> DNA
<213~ Artificial Sequence
16/49
CA 02401357 2002-08-27
<220~
<223~ Synthetic DNA
<400~ 38
cttggatccg ggctgaccta ggacggtcag tttggtccct ccgccgaaca cgtacacaaa 60
ttgttcctta attgtatcac ccacaccaca gatatagtca gcctcatcct c 111
<210> 39
<211~ 42
<212~ DNA
<213~ Artificial Sequence
<220>
<223> Synthetic DNA
<400> 39
cttctctggc tgctgctgat accattcaat ggtgtacgta ct 42
<210> 40
<211~ 26
<212~ DNA
<213~ Artificial Sequence
<220~
<223> Synthetic DNA
<400~ 40
17/49
CA 02401357 2002-08-27
cgagggccct tctctggctg ctgctg 26
<210~ 41
<211~ 35
<212~ DNA
<213~ Artificial Sequence
<220~
<223~ Synthetic DNA
<400~ 41
gagaagggcc ctargtacst gatgrawctt aagca 35
<210~ 42
<211~ 35
<212~ DNA
<213~ Artificial SeQUence
<220~
<223~ Synthetic DNA
<400~ 42
cacgaattca ctatcgattc tggaaccttc agagg 35
<210~ 43
<211> 18
<212> DNA
<213~ Artificial Sequence
18/49
CA 02401357 2002-08-27
<220~
<223~ Synthetic DNA
<400~ 43
ggcttggagc tcctcaga 18
<210~ 44
<211~ 20
<212~ DNA
<213> Artificial Sequence
<220~
<223~ Synthetic DNA
<400~ 44
gacagtggtt caaagttttt 20
<210~ 45
<211~ 118
<212~ PRT
<213~ Mus musculus
<400~ 45
Gln Leu Val Leu Thr Gln Ser Ser Ser Ala Ser Phe Ser Leu Gly Ala
1 5 10 15
Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr
20 25 30
19/49
CA 02401357 2002-08-27
Ile Glu Trp Tyr Gln Gln Gln Pro Leu Lys Pro Pro Lys Tyr Val Met
35 40 45
Asp Leu Lvs Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp
50 55 60
Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Ser Ile Ser
65 70 75 80
Asn Ile Gln Pro Glu Asp Glu Ala Met Tyr Ile Cys Gly Val Gly Asp
85 90 95
Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Val
100 105 110
Thr Val Leu Gly Gln Pro
115
<210~ 46
<211~ 118
<212~ PRT
<213~ Mus musculus
<400> 46
Glu Val Leu ValGlu Ser Gly AspLeu Val ProGly
Gln Gly Lys Gly
1 5 10 15
Ser Leu Leu SerCys Ala Ser GlyPhe Thr SerSer
Lys Ala Phe Tyr
20 25 30
Gly Met Trp IleArg Gln Pro AspLys Arg GluTrp
Ser Thr Leu Val
35 40 45
Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
20/49
CA 02401357 2002-08-27
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys
85 90 95
Ala Arg Gln Thr Thr Met Thr Tyr Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210~ 47
<211~ 116
<212~ PRT
<213~ Homo sapiens
<400~ 47
Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala
1 5 10 15
Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr
20 25 30
Ile Glu Trp His Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Leu Met
35 40 45
Lys Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp
50 55 60
Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp
85 90 95
Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu
100 105 110
21/49
CA 02401357 2002-08-27
Thr Val Leu Gly
115
<210~ 48
<211~ 118
<2I2~ PRT
<213~ Homo Sapiens
<400~ 48
Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala
1 5 10 15
Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr
20 25 30
Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys Tyr Leu Met
35 40 45
Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp
50 55 60
Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp
85 90 95
Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu
100 105 I10
Thr Val Leu Gly Gln Pro
115
<210~ 49
22/49
CA 02401357 2002-08-27
<211~118
<212~PRT
<213~Homo Sapiens
<400~ 49
Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala
1 5 10 15
Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr
20 25 30
Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys Tyr Val Met
35 40 45
Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp
50 55 60
Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp
85 90 95
Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu
100 105 110
Thr Val Leu Gly Gln Pro
115
<210>50
<211~118
<212~PRT
<213~Homo sapiens
<400~ 50
23/49
CA 02401357 2002-08-27
Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala
1 5 10 15
Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr
20 25 30
Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Leu Met
35 40 45
Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp
50 55 60
Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp
85 90 95
Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu
100 105 110
Thr Val Leu Gly Gln Pro
115
<210~51
<211>118
<212~PRT
<213~Homo sapiens
<400~ 51
Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala
1 5 10 15
Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr
20 25 30
Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Val Met
24/49
CA 02401357 2002-08-27
35 40 45
Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp
50 55 60
Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp
85 90 95
Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu
100 105 110
Thr Val Leu Gly Gln Pro
115
<210~ 52
<211~ 118
<212~ PRT
<213~ Homo sapiens
<400~ 52
Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala
1 5 10 15
Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr
20 25 30
Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys Tyr Leu Met
35 40 45
Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp
50 55 60
Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser
65 70 75 80
25/49
CA 02401357 2002-08-27
Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp
85 90 95
Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu
100 105 110
Thr Val Leu Gly Gln Pro
115
<210> 53
<211~ 118
<212> PRT
<213~ Homo Sapiens
<400> 53
Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala
1 5 10 15
Ser Va1 Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr
20 25 30
Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Leu Met
35 40 45
Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp
50 55 60
Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp
85 90 95
Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu
100 105 110
Thr Val Leu Gly Gln Pro
26/49
CA 02401357 2002-08-27
115
<210~ 54
<211~ 118
<212~ PRT
<213~ Homo sapiens
<400> 54
Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala
1 5 10 15
Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr
20 25 30
Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys Tyr Val Met
35 40 45
Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp
50 55 60
Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp
85 90 95
Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu
100 105 110
Thr Val Leu Gly Gln Pro
115
<210> 55
<211~ 118
<212~ PRT
27/49
CA 02401357 2002-08-27
<213> Homo sapiens
<400~ 55
Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala
1 5 10 15
Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr Tyr Thr
20 25 30
Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Val Met
35 40 45
Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly Ile Pro Asp
50 55 60
Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Ile Cys Gly Val Gly Asp
85 90 95
Thr Ile Lys Glu Gln Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu
100 105 110
Thr Val Leu Gly Gln Pro
115
<210~ 56
<211~ 118
<212~ PRT
<213~ Homo Sapiens
<400~ 56
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
28/49
CA 02401357 2002-08-27
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gln Thr Thr Met Thr Tyr Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210~ 57
<211~ 411
<212~ DNA
<213~ Mus musculus
<220~
<221~ CDS
<222~ ( 1) . . (411)
<220~
<221> mat_peptide
<222~ (58) . . (411)
29/49
CA 02401357 2002-08-27
<400~
57
atgaac ttcggg ctcagc ttgatt ttcctt gcc ctc:att ttaaaa ggt 48
MetAsn PheGly LeuSer LeuIle PheLeu Ala LeuIle LeuLys Gly
-15 -10 -5
gtccag tgtgag gtgcaa ctggtg gagtct ggg ggagac ttagtg aag 96
ValGln CysGlu ValGln LeuVal GluSer Gly GlyAsp LeuVal Lys
-1 1 5 10
cctgga gggtcc ctgaaa ctctcc tgtgca gcc tctgga ttcact ttc 144
ProGly GlySer LeuLys LeuSer CysAla Ala SerGly PheThr Phe
15 20 25
agtagc tatggc atgtct tggatt cgccag act ccagac aagagg ctg 192
SerSer TyrGly MetSer TrpIle ArgGln Thr ProAsp LysArg Leu
30 35 40 45
gagtgg gtcgca accatt agtagt ggtggt agt tacacc tactat cca 240
GluTrp ValAla ThrIle SerSer GlyGly Ser TyrThr TyrTyr Pro
50 55 60
gacagt gtgaag gggcga ttcacc atctcc aga gacaat gccaag aac 288
AspSer ValLys GlyArg PheThr IleSer Arg AspAsn AlaLys Asn
65 70 75
acccta tacctg caaatg agcagt ctgaag tct gaggac acagcc atg 336
ThrLeu TyrLeu GlnMet SerSer LeuLys Ser GluAsp ThrAla Met
80 85 90
ttttac tgtgca agacag actact atgact tac tttget tactgg ggc 384
PheTyr CysAla ArgGln ThrThr MetThr Tyr PheAla TyrTrp Gly
95 100 105
caaggg actctg gtcact gtctct gca 411
GlnGly ThrLeu ValThr ValSer Ala
110 115
30/49
CA 02401357 2002-08-27
<210~ 58
<211~ 411
<212~ DNA
<213~ Homo sapiens
<220~
<221~ CDS
<222~ (1) . . (411)
<220~
<221~ mat_peptide
<222~ (58) . . (411)
<400~
58
atgggg tttggg ctgagc tgggtt ttcctc gtt getctt ttaaga ggt 48
MetGly PheGly LeuSer TrpVal PheLeu Val AlaLeu LeuArg Gly
-15 -10 -5
gtccag tgtcag gtgcag ctggtg gagtct ggg ggaggc gtggtc cag 96
ValGln CysGln ValGln LeuVal GluSer Gly GlyGly ValVal Gln
-1 1 5 10
cctggg aggtcc ctgaga ctctcc tgtgca gcc tctgga ttcacc ttc 144
ProGly ArgSer LeuArg LeuSer CysAla Ala SerGly PheThr Phe
15 20 25
agtagc tatggc atgtct tgggtc cgccag get ccaggc aagggg ctg 192
SerSer TyrGly MetSer TrpVal ArgGln Ala ProGly LysGly Leu
30 35 40 45
gagtgg gtggca accatt agtagt ggtggt agt tacacc tactat cca 240
31 /49
CA 02401357 2002-08-27
Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro
50 55 60
gac agt gtg aag ggg cga ttc acc atc tcc aga gac aat tcc aag aac 288
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
65 70 75
acg ctg tat ctg caa atg aac agc ctg aga get gag gac acg get gtg 336
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
80 85 90
tat tac tgt gcg aga cag act act atg act tac ttt get tac tgg ggc 384
Tyr Tyr Cys Ala Arg Gln Thr Thr Met Thr Tyr Phe Ala Tyr Trp Gly
95 100 105
cag gga acc ctg gtc acc gtc tcc tca 411
Gln Gly Thr Leu Val Thr Val Ser Ser
110 115
<210> 59
<211~ 11
<212~ PRT
<213~ Homo sapiens
<400~ 59
Lys Ala Ser Gln Asp Val Asn Thr Ala Val Ala
1 5 10
<210~ 60
<211> 7
<212~ PRT
<213~ Homo sapiens
32/49
CA 02401357 2002-08-27
<400~ 60
Ser Ala Ser Asn Arg Tyr Thr
1 5
<210~ 61
<211> 9
<212~ PRT
<213~ Homo Sapiens
<400~ 61
Gln Gln His Tyr Ser Thr Pro Phe Thr
1 5
<210~ 62
<211~ 5
<212~ PRT
<213~ Homo sapiens
<400~ 62
Pro Tyr Trp Met Gln
1 5
<210~ 63
<211~ 16
<212> PRT
<213~ Homo sapiens
33/49
cag gga acc ctg gtc ac
CA 02401357 2002-08-27
<400~ 63
Ser Ile Phe Gly Asp Gly Asp Thr Arg Tyr Ser Gln Lys Phe Lys Gly
1 5 10 15
<210~ 64
<211~ 11
<212> PRT
<213~ Homo sapiens
<400~ 64
Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr
1 5 10
<210~ 65
<211~ 411
<212~ DNA
<213~ Mus musculus
<220>
<221~ CDS
<222~ (1) . . (411)
<220~
<221~ mat_peptide
<222~ (58) . . (411)
<400~ 65
atg gcc tgg act cct ctc ttc ttc ttc ttt gtt ctt cat tgc tca ggt 48
34/49
CA 02401357 2002-08-27
Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser Gly
-15 -10 -5
tctttc tcccaa cttgtg ctcact cagtca tcttca gcc tctttc tcc 96
SerPhe SerGln LeuVal LeuThr GlnSer SerSer Ala SerPhe Ser
-1 I 5 10
ctggga gcctca gcaaaa ctcacg tgcacc ttgagt agt cagcac agt 144
LeuGly AlaSer AlaLys LeuThr CysThr LeuSer Ser GlnHis Ser
15 20 25
acgtac accatt gaatgg tatcag caacag ccactc aag cctcct aag 192
ThrTyr ThrIle GluTrp TyrGln GlnGln ProLeu Lys ProPro Lys
30 35 40 45
tatgtg atggat cttaag caagat ggaagc cacagc aca ggtgat ggg 240
TyrVal MetAsp LeuLys GlnAsp GlySer HisSer Thr GlyAsp Gly
50 55 60
attcct gatcgc ttctct ggatcc agctct ggtget gat cgctac ctt 288
IlePro AspArg PheSer GlySer SerSer GlyAla Asp ArgTyr Leu
65 70 75
agcatt tccaac atccag ccagaa gatgaa gcaatg tac atctgt ggt 336
SerIle SerAsn IleGln ProGlu AspGlu AlaMet Tyr IleCys Gly
80 85 90
gtgggt gataca attaag gaacaa tttgtg tatgtt ttc ggcggt ggg 384
ValGly AspThr IleLys GluGln PheVal TyrVal Phe GlyGly Gly
95 100 105
accaag gtcact gtccta ggtcag ccc 411
ThrLys ValThr ValLeu GlyGln Pro
1I0 115
<210~
66
35/49
CA 02401357 2002-08-27
<211~411
<212~DNA
<213~Homo sapiens
<220>
<221~ CDS
<222~ (1).. (411)
<220~
<221~ mat_peptide
<222~ (58) . . (411)
<400>
66
atggcc tggact cctctc ttcttc ttcttt gttctt cat tgctca ggt 48
MetAla TrpThr ProLeu PhePhe PhePhe ValLeu His CysSer Gly
-15 -10 -5
tctttc tcccag cttgtg ctgact caatcg ccctct gcc tctgcc tcc 96
SerPhe SerGln LeuVal LeuThr GlnSer ProSer Ala SerAla Ser
-1 1 5 10
ctggga gcctcg gtcaag ctcacc tgcacc ttgagt agt cagcac agt 144
LeuGly AlaSer ValLys LeuThr CysThr LeuSer Ser GlnHis Ser
15 20 25
acgtac accatt gaatgg catcag cagcag ccagag aag ggccct cgg 192
ThrTyr ThrIle GluTrp HisGln GlnGln ProGlu Lys GlyPro Arg
30 35 40 45
tacttg atgaaa cttaag caagat ggaagc cacagc aca ggtgat ggg 240
TyrLeu MetLys LeuLys GlnAsp GlySer HisSer Thr GlyAsp Gly
50 55 60
36/49
CA 02401357 2002-08-27
attcct gatcgc ttctca ggctcc agc tctggg getgag cgctac ctc 288
IlePro AspArg PheSer GlySer Ser SerGly AlaGlu ArgTyr Leu
65 70 75
accatc tccagc ctccag tctgag gat gagget gactat tactgt ggt 336
ThrIle SerSer LeuGln SerGlu Asp GluAla AspTyr TyrCys Gly
80 85 90
gtgggt gataca attaag gaacaa ttt gtgtac gtgttc ggcgga ggg 384
ValGly AspThr IleLys GluGln Phe ValTyr ValPhe GlyGly Gly
g5 100 105
accaaa ctgacc gtccta ggtcag ccc 411
ThrLys LeuThr ValLeu GlyGln Pro
110 115
<210~ 67
<211~ 411
<212~ DNA
<213> Homo Sapiens
<220~
<221~ CDS
<222~ (1) . . (411)
<220~
<221> mat_peptide
<222~ (58) . . (411)
<400~ 67
atg gcc tgg act cct ctc ttc ttc ttc ttt gtt ctt cat tgc tca ggt 48
37/49
CA 02401357 2002-08-27
MetAla Trp ThrProLeu PhePhe PhePhe Val LeuHis CysSer Gly
-15 -10 -5
tctttc tcc cagcttgtg ctgact caatcg ccc tctgcc tctgcc tcc 96
SerPhe Ser GlnLeuVal LeuThr GlnSer Pro SerAla SerAla Ser
-1 1 5 10
ctggga gcc tcggtcaag ctcacc tgcacc ttg agtagt cagcac agt 144
LeuGly Ala SerValLys LeuThr CysThr Leu SerSer GlnHis Ser
15 20 25
acgtac acc attgaatgg tatcag cagcag cca gagaag ggccct aag 192
ThrTyr Thr IleGluTrp TyrGln GlnGln Pro GluLys GlyPro Lys
30 35 40 45
tacctg atg gatcttaag caagat ggaagc cac agcaca ggtgat ggg 240
TyrLeu Met AspLeuLys GlnAsp GlySer His SerThr GlyAsp Gly
50 55 60
attcct gat cgcttctca ggctcc agctct ggg getgag cgctac ctc 288
IlePro Asp ArgPheSer GlySer SerSer Gly AlaGlu ArgTyr Leu
65 70 75
accatc tcc agcctccag tctgag gatgag get gactat tactgt ggt 336
ThrIle Ser SerLeuGln SerGlu AspGlu Ala AspTyr TyrCys Gly
80 85 90
gtgggt gat acaattaag gaacaa tttgtg tac gtgttc ggcgga ggg 384
ValGly Asp ThrIleLys GluGln PheVal Tyr ValPhe GlyGly Gly
95 100 105
accaaa ctg accgtccta ggccag ccc 411
ThrLys Leu ThrValLeu GlyGln Pro
110 115
<210~ 8
6
38/49
CA 02401357 2002-08-27
<211~411
<212~DNA
<213~Homo sapiens
<220~
<221~ CDS
<222~ (1) . . (411)
<220>
<221~ mat_peptide
<222~ (58) . . (411)
<400~ 68
atg gcc tgg act cct ctc ttc ttc ttc ttt gtt ctt cat tgc tca ggt 48
Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser Gly
-15 -10 -5
tct ttc tcc cag ctt gtg ctg act caa tcg ccc tct gcc tct gcc tcc 96
Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser
-1 1 5 10
ctg gga gcc tcg gtc aag ctc acc tgc acc ttg agt agt cag cac agt 144
Leu Gly Ala Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser
15 20 25
acg tac acc att gaa tgg tat cag cag cag cca gag aag ggc cct aag 192
Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Glu Lys Gly Pro Lys
30 35 40 45
tac gtg atg gat ctt aag caa gat gga agc cac agc aca ggt gat ggg 240
Tyr Val Met Asp Leu Lys Gln Asp Gly Ser His Ser Thr Gly Asp Gly
50 55 60
39/49
CA 02401357 2002-08-27
attcct gatcgc ttctca ggctcc agc tctggg getgagcgc tac ctc 288
IlePro AspArg PheSer GlySer Ser SerGly AlaGluArg Tyr Leu
65 70 75
accatc tceage etccag tctgag gat gagget gactattac tgt ggt 336
ThrIle SerSer LeuGln SerGlu Asp GluAla AspTyrTyr Cys Gly
80 85 90
gtgggt gataca attaag gaacaa ttt gtgtac gtgttcggc gga ggg 384
ValGly AspThr IleLys GluGln Phe ValTyr ValPheGly Gly Gly
95 100 105
accaaa ctgacc gtccta ggccag ccc 411
ThrLys LeuThr ValLeu GlyGln Pro
110 115
<210~ 69
<211~ 411
<212~ DNA
<213~ Homo sapiens
<220>
<221~ CDS
<222~ (1) . . (411)
<220~
<221~ mat_peptide
<222~ (58) . . (4111
<400~ 69
atg gcc tgg act cct ctc ttc ttc ttc ttt gtt ctt cat tgc tca ggt 48
40/49
CA 02401357 2002-08-27
Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser Gly
-15 -10 -5
tct ttc tcc cag ctt gtg ctg act caa tcg ccc tct gcc tct gcc tcc 96
Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser
-1 1 5 10
ctggga gcctcg gtcaag ctcacc tgcacc ttgagt agt cagcac agt 144
LeuGly AlaSer ValLys LeuThr CysThr LeuSer Ser GlnHis Ser
15 20 25
acgtac accatt gaatgg tatcag cagcag ccagag aag ggccct agg 192
ThrTyr ThrIle GluTrp TyrGln GlnGln ProGlu Lys GlyPro Arg
30 35 40 45
tacctg atggat cttaag caagat ggaagc cacagc aca ggtgat ggg 240
TyrLeu MetAsp LeuLys GlnAsp GlySer HisSer Thr GlyAsp Gly
50 55 60
attcct gatcgc ttctca ggctcc agctct gggget gag cgctac ctc 288
IlePro AspArg PheSer GlySer SerSer GlyAla Glu ArgTyr Leu
65 70 75
accatc tccagc ctccag tctgag gatgag getgac tat tactgt ggt 336
ThrIle SerSer LeuGln SerGlu AspGlu AlaAsp Tyr TyrCys Gly
80 85 90
gtgggt gataca attaag gaacaa tttgtg tacgtg ttc ggcgga ggg 384
ValGly AspThr IleLys GluGln PheVal TyrVal Phe GlyGly Gly
95 100 105
accaaa ctgacc gtccta ggccag ccc 411
ThrLys LeuThr ValLeu GlyGln Pro
110 115
<210~ 70
41/49
CA 02401357 2002-08-27
<211~411
<212~DNA
<213~Homo sapiens
<220~
<221~ CDS
<222> ( 1) . . (411)
<220~
<221~ mat_peptide
<222> (58) . . (411)
<400~
70
atggcc tggact cctctc ttcttc ttcttt gttctt cat tgctca ggt 48
MetAla TrpThr ProLeu PhePhe PhePhe ValLeu His CysSer Gly
-15 -10 -5
tctttc tcccag cttgtg ctgact caatcg ccctct gcc tctgcc tcc 96
SerPhe SerGln LeuVal LeuThr GlnSer ProSer Ala SerAla Ser
-1 1 5 10
ctggga gcctcg gtcaag ctcacc tgcacc ttgagt agt cagcac agt 144
LeuGly AlaSer ValLys LeuThr CysThr LeuSer Ser GlnHis Ser
15 20 25
acgtac accatt gaatgg tatcag cagcag ccagag aag ggccct agg 192
ThrTyr ThrIle GluTrp TyrGln GlnGln ProGlu Lys GlyPro Arg
30 35 40 45
tacgtg atggat cttaag caagat ggaagc cacagc aca ggtgat ggg 240
TyrVal MetAsp LeuLys GlnAsp GlySer HisSer Thr GlyAsp Gly
50 55 60
42/49
CA 02401357 2002-08-27
attcctgat cgc ttctca ggctcc agctct gggget gagcgc tac ctc 288
IleProAsp Arg PheSer GlySer SerSer GlyAla GluArg Tyr Leu
65 70 75
accatctcc ~agcctccag tctgag gatgag getgac tattac tgt ggt 336
ThrIleSer Ser LeuGln SerGlu AspGlu AlaAsp TyrTyr Cys Gly
80 85 90
gtgggtgat aca attaag gaacaa tttgtg tacgtg ttcggc gga ggg 384
ValGlyAsp Thr IleLys GluGln PheVal TyrVal PheGly Gly Gly
95 100 105
accaaactg acc gtccta ggccag ccc 411
ThrLysLeu Thr ValLeu GlyGln Pro
110 115
<210~71
<211~411
<212~DNA
<213~Homo sapiens
<220~
<221> CDS
<222~ ( 1) . . (411)
<220~
<221~ mat_peptide
<222~ (58) . . (411)
<400~ 71
atg gcc tgg act cct ctc ttc ttc ttc ttt gtt ctt cat tgc tca ggt 48
43/49
CA 02401357 2002-08-27
Met Ala TrpThr ProLeu PhePhe PhePhe ValLeu His CysSer Gly
-15 -10 -5
tct ttc tcccag cttgtg ctgact caatcg ccctci gcc tctgcc tcc 96
Ser Phe SerGln LeuVal LeuThr GlnSer ProSer Ala SerAla Ser
-1 1 5 10
ctg gga gcctcg gtcaag ctcacc tgcacc ttgagt agt cagcac agt 144
Leu Gly AlaSer ValLys LeuThr CysThr LeuSer Ser GlnHis Ser
15 20 25
acg tac accatt gaatgg tatcag cagcag ccagag aag ggccct aag 192
Thr Tyr ThrIle GluTrp TyrGln GlnGln ProGlu Lys GlyPro Lys
30 35 40 45
tac ctg atggat cttaag caagat ggaagc cacagc aca ggtgat ggg 240
Tyr Leu MetAsp LeuLys GlnAsp GlySer HisSer Thr GlyAsp Gly
50 55 60
att cct gatcgc ttctca ggctcc agctct gggget gag cgctac ctc 288
Ile Pro AspArg PheSer GlySer SerSer GlyAla Glu ArgTyr Leu
65 70 75
acc atc tccagc ctccag tctgag gatgag getgac tat atctgt ggt 336
Thr Ile SerSer LeuGln SerGlu AspGlu AlaAsp Tyr IleCys Gly
80 85 90
gtg ggt gataca attaag gaacaa tttgtg tacgtg ttc ggcgga ggg 384
Val Gly AspThr IleLys GluGln PheVal TyrVal Phe GlyGly Gly
95 100 105
acc aaa ctgacc gtccta ggccag ccc 411
Thr Lys LeuThr ValLeu GlyGln Pro
110 115
<210~
72
44/49
CA 02401357 2002-08-27
<211~411
<212~DNA
<213~Homo sapiens
<220>
<221~ CDS
<222~ (1) . . (411)
<220~
<221> mat_peptide
<222~ (58) . . (411)
<400~
72
atggcc tgg actcct ctcttcttc ttc tttgtt cttcat tgctca ggt 48
MetAla Trp ThrPro LeuPhePhe Phe PheVal LeuHis CysSer Gly
-15 -10 -5
tctttc tcc cagctt gtgctgact caa tcgccc tctgcc tctgcc tcc 96
SerPhe Ser GlnLeu ValLeuThr Gln SerPro SerAla SerAla Ser
-1 1 5 10
ctggga gcc tcggtc aagctcacc tgc accttg agtagt cagcac agt 144
LeuGly Ala SerVal LysLeuThr Cys ThrLeu SerSer GlnHis Ser
15 20 25
acgtac acc attgaa tggtatcag cag cagcca gagaag ggccct agg 192
ThrTyr Thr IleGlu TrpTyrGln Gln GlnPro GluLys GlyPro Arg
30 35 40 45
tacctg atg gatctt aagcaagat gga agccac agcaca ggtgat ggg 240
TyrLeu Met AspLeu LysGlnAsp Gly SerHis SerThr GlyAsp Gly
50 55 60
45/49
~r ,
CA 02401357 2002-08-27
attcct gatcgc ttctca ggc tccagc tctggg gct:gag cgctac ctc 288
IlePro AspArg PheSer Gly SerSer SerGly AlaGlu ArgTyr Leu
65 70 75
accatc tccagc ctccag tct gaggat gagget gactat atctgt ggt 336
ThrIle SerSer LeuGln Ser GluAsp GluAla AspTyr IleCys Gly
80 85 90
gtgggt gataca attaag gaa caattt gtgtac gtgttc ggcgga ggg 384
ValGly AspThr IleLys Glu GlnPhe ValTyr ValPhe GlyGly Gly
95 100 105
accaaa ctgacc gtccta ggc cagccc 411
ThrLys LeuThr ValLeu Gly GlnPro
110 115
<210> 73
<211~ 411
<212> DNA
<213~ Homo sapiens
<220~
<221~ CDS
<222~ (1) . . (411)
<220~
<221~ mat_peptide
<222~ (58) . . (411)
<400~ 73
atg gcc tgg act cct ctc ttc ttc ttc ttt gtt ctt cat tgc tca ggt 48
46/49
CA 02401357 2002-08-27
Met Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser Gly
-15 -10 -5
tct ttc tcc cag ctt gtg ctg act caa tcg ccc tct gcc tct gcc tcc 96
Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser
-1 1 5 10
ctggga gcctcg gtcaag ctcacc tgcacc ttgagt agtcag cacagt 144
LeuGly AlaSer ValLys LeuThr CysThr LeuSer SerGln HisSer
15 20 25
acgtac accatt gaatgg tatcag cagcag ccagag aagggc cctaag 192
ThrTyr ThrIle GluTrp TyrGln GlnGln ProGlu LysGly ProLys
30 35 40 45
tacgtg atggat cttaag caagat ggaagc cacagc acaggt gatggg 240
TyrVal MetAsp LeuLys GlnAsp GlySer HisSer ThrGly AspGly
50 55 60
attcct gatcgc ttctca ggctcc agctct gggget gagcgc tacctc 288
IlePro AspArg PheSer GlySer SerSer GlyAla GluArg TyrLeu
65 70 75
accatc tccagc ctccag tctgag gatgag getgac tatatc tgtggt 336
ThrIle SerSer LeuGln SerGlu AspGlu AlaAsp TyrIle CysGly
80 85 90
gtgggt gataca attaag gaacaa tttgtg tacgtg ttcggc ggaggg 384
ValGly AspThr IleLys GluGln PheVal TyrVal PheGly GlyGly
95 100 105
accaaa ctgacc gtccta ggccag ccc 411
ThrLys LeuThr ValLeu GlyGln Pro
110 115
<210~ 74
47/49
CA 02401357 2002-08-27
<211~411
<212>DNA
<213~Homo sapiens
<220~
<221~ CDS
<222~ (1) . . (411)
<220>
<221~ mat_peptide
<222~ (58) . . (41 I)
<400>
74
atggcc tggact cctctc ttcttc ttcttt gttctt cattgc tca ggt 48
MetAla TrpThr ProLeu PhePhe PhePhe ValLeu HisCys Ser Gly
-15 -10 -5
tctttc tcccag cttgtg ctgact caatcg ccctct gcctct gcc tcc 96
SerPhe SerGln LeuVal LeuThr GlnSer ProSer AlaSer Ala Ser
-1 1 5 10
ctggga gcctcg gtcaag ctcacc tgcacc ttgagt agtcag cac agt 144
LeuGly AlaSer ValLys LeuThr CysThr LeuSer SerGln His Ser
15 20 25
acgtac accatt gaatgg tatcag cagcag ccagag aagggc cct agg 192
ThrTyr ThrIle GluTrp TyrGln GlnGln ProGlu LysGly Pro Arg
30 35 40 45
tacgtg atggat cttaag caagat ggaagc cacagc acaggt gat ggg 240
TyrVal MetAsp LeuLys GlnAsp GlySer HisSer ThrGly Asp Gly
50 55 60
48/49
r~
CA 02401357 2002-08-27
attcctgat cgcttc tca ggctcc agctct gggget gagcgc tacctc 288
IleProAsp ArgPhe Ser GlySer SerSer GlyAla GluArg TyrLeu
65 70 75
accatctcc agcctc cag tctgag gatgag getgac tatatc tgtggt 336
ThrIleSer SerLeu Gln SerGlu AspGlu AlaAsp TyrIle CysGly
80 85 90
gtgggtgat acaatt aag gaacaa tttgtg tacgtg ttcggc ggaggg 384
ValGlyAsp ThrIle Lys GluGln PheVal TyrVal PheGly GlyGly
95 100 105
accaaactg accgtc cta ggccag ccc 411
ThrLysLeu ThrVal Leu GlyGln Pro
110 115
<210~ 75
<211~ 34
<212> PRT
<213> Homo sapiens
<400~ 75
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln
1 5 10 15
Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His
20 25 30
Thr Ala
49/49
