U.S. patent application number 10/133912 was filed with the patent office on 2002-11-07 for tcf mutant.
This patent application is currently assigned to Daiichi Pharmaceutical Co., Ltd.. Invention is credited to Goto, Masaaki, Higashio, Kanji, Kinosaki, Masahiko, Kobayashi, Fumie, Murakami, Akihiko, Ueda, Masatsugu, Yamaguchi, Kyoji, Yamashita, Yasushi.
Application Number | 20020165358 10/133912 |
Document ID | / |
Family ID | 18312917 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020165358 |
Kind Code |
A1 |
Kinosaki, Masahiko ; et
al. |
November 7, 2002 |
TCF mutant
Abstract
The present invention relates to TCF mutant having a novel amino
acid sequence which is obtained by mutagenesis of one or more amino
acid between N-terminus and the first kringle of the amino acid
sequence of native TCF and has lowered affinity to heparin and/or
elevated biological activity. The present TCF mutant is prepared by
gene manipulation of TCF. The TCF mutants of the present invention
have proliferative activity and/or growth stimulative activity in
hepatocyte and beneficial as a therapeutic agent for various
hepatic diseases and an antitumor agent.
Inventors: |
Kinosaki, Masahiko;
(Tochigi, JP) ; Yamaguchi, Kyoji; (Saitama,
JP) ; Kobayashi, Fumie; (Tochigi, JP) ; Goto,
Masaaki; (Tochigi, JP) ; Murakami, Akihiko;
(Tochigi, JP) ; Ueda, Masatsugu; (Saitama, JP)
; Higashio, Kanji; (Saitama, JP) ; Yamashita,
Yasushi; (Tochigi, JP) |
Correspondence
Address: |
DARBY & DARBY P.C.
Post Office Box 5257
New York
NY
10150-5257
US
|
Assignee: |
Daiichi Pharmaceutical Co.,
Ltd.
|
Family ID: |
18312917 |
Appl. No.: |
10/133912 |
Filed: |
April 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10133912 |
Apr 25, 2002 |
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08700519 |
Aug 26, 1996 |
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6399744 |
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08700519 |
Aug 26, 1996 |
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PCT/JP95/02708 |
Dec 27, 1995 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
C07K 14/4753 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
530/350 ;
514/12 |
International
Class: |
C07K 014/705; A61K
038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 27, 1994 |
JP |
337885(1994) |
Claims
1. A TCF mutant which is obtained by mutagenesis of more than one
amino acid residue at the position from N-terminus to the first
kringle of the amino acid sequence of native TCF and has lowered
affinity to heparin and/or elevated biological activity.
2. The TCF mutant according to claim 1, wherein Arg2-Lys-Arg-Arg5
of native TCF is mutagenized to Ala-Ala-Ala-Ala.
3. The TCF mutant according to claim 1, wherein
Lys27-Ile-Lys-Thr-Lys-Lys3- 2 of native TCF is mutagenized to
Ala-Ile-Ala-Thr-Ala-Ala.
4. The TCF mutant according to claim 2 or 3, wherein proliferative
activity thereof per unit amount of protein in hepatocyte is more
than 10 folds than that of native TCF.
5. The TCF mutant according to claim 2 or 3, wherein proliferative
activity thereof per unit amount of protein in kidney epithelial
cell is more than 2 folds than that of native TCF.
6. The TCF mutant according to claim 2 or 3, wherein proliferative
activity thereof per unit amount of protein in bone marrow cell is
1/2-{fraction (1/20)} of that of native TCF.
7. The TCF mutant according to claim 1, wherein Lys54 of native TCF
is mutagenized to Ala.
8. The TCF mutant according to claim 1, wherein
Argl32-Gly-Lys-Aspl35 of native TCF is mutagenized to
Ala-Gly-Ala-Ala.
9. The TCF mutant according to claim 1, wherein Argl42 of native
TCF is mutagenized to Ala.
10. The TCF mutant according to claim 1, wherein Arg42 of native
TCF is mutagenized to Ala.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to TCF mutants comprising a
novel amino acid sequence, more specifically, TCF mutants which are
obtained by mutagenesis of one or more amino acid in the sequence
from N-terminus to the first kringle of native TCF and show lowered
affinity to heparin and/or elevated biological activity. The TCF
mutants of the present invention which show proliferative activity
and growth stimulative activity in hepatocyte are beneficial for
treatment of various hepatic diseases and as an antitumor
agent.
BACKGROUND OF THE INVENTION
[0002] Tumor cytotoxic factor (TCF-II) produced in human fibroblast
cells is a novel antitumor substance different from any antitumor
proteins so far reported. The present inventors have succeeded in
cloning of cDNA coding for the protein of the present invention,
determined the total amino acid sequence thereof and confirmed
usefulness thereof (WO90/10651). The molecular weight of TCF was
78,000.+-.2,000, or 74,000.+-.2,000 according to the results of SDS
electrophoresis under non-reducing conditions, while the results
under reducing conditions indicated A-chain of
52,000.+-.2,000,common band, B-chain of 30,000.+-.2,000 and/or
C-chain of 26,000.+-.2,000. TCF is a protein which has a high
affinity to heparin or heparin-like substance and shows high
antitumor activity against tumor cells and proliferative activity
to normal cells. Further, it was confirmed that it belongs to a
wide variety of family of HGF, a growth factor for hepatocyte.
Therefore, since TCF is not only an antitumor factor, but also a
growth factor for hepatocytes, it is known that it is beneficial
for liver regeneration after hepatectomy.
[0003] Many researches have been carried out from the aspects of
structure-function relationship of hepatocyte growth factor(HGF) so
far. About 20 species of deletion mutants and about 50 species of
point mutants have been reported so far (K. Matsumoto, et. al.,
Biochem. Biophys. Res. Comm., vol. 181, pp691-699 (1991); G.
Hartmann, et. al. Proc. Natl. Acad. Sci. USA, vol. 89,
pp11574-11587 -(1992); N. A. Lokker, et. al., EMBO J. vol. 11,
pp2503-2510 (1992); M. Okigaki et. al., Biochemistry, vol. 31, pp
9555-9561 (1992); N. A. Lokker, et. al. Protein Engineering, vol.
7, pp895-903 (1994)), however, any mutant which clearly shows an
elevated biological activity is not obtained at present. Half-life
of TCF in vivo is known to be extremely short, about 2 minutes.
Therefore, it is anticipated that a comparatively large amount of
the protein should be administered for treatment of various
diseases. It is conceivable that the dosage level of TCF
administered will be reduced by elevation of biological activity
thereof or by prolongation of the half-life thereof in vivo. Though
it was described on TCF mutants with prolonged half-life in patent
publication WO94/14845, any TCF mutant with elevated biological
activity is not obtained at present, like HGF described above.
[0004] Therefore, the present inventors have investigated to obtain
a TCF mutant which shows elevated biological activity or
prolongation of half-life in vivo. More specifically, the present
inventors have carried out research to obtain the above-mentioned
mutant with elevated biological activity or with prolonged
half-life in vivo which is different from native TCF with respect
to amino acid sequence by altering the DNA sequence coding for the
amino acid sequence of native TCF and expressing DNA thereof.
Accordingly, an object of the present invention is to provide a TCF
mutant with elevated biological activity or with prolonged
half-life in vivo due to lowered affinity to heparin.
[0005] The present inventors have eagerly investigated on the above
object and obtained novel TCF mutants which have amino acid
sequences different from that of TCF mutant found prior to the
present invention and show elevated biological activity and/or
lowered affinity to heparin. The present invention provides TCF
mutants which show more than 10 folds of specific activity
(biological activity per unit amount of protein) and/or lowered
affinity to heparin. These are the first mutants with extremely
elevated biological activity by mutagenizing the amino acid
sequence of native TCF.
SUMMARY OF THE INVENTION
[0006] An object of the present invention is to provide a TCF
mutant with lowered affinity to heparin and/or with elevated
biological activity which is obtained by mutagenesis of one or more
amino acid residue(s) in the amino acid sequence from N-terminus to
the first kringle of native TCF.
BRIEF DESCRIPTION OF DRAWINGS
[0007] FIG. 1 shows SDS electrophoresis profiles of purified TCF
and TCF mutants of the present invention
[0008] FIG. 2 shows proliferative action of purified TCF and TCF
mutants of the present invention in hepatocyte. The relative
activity (%) of vertical axis is represented as the ratio of
proliferative activity of each sample based on that of 10 ng/ml TCF
as 100%.
[0009] FIG. 3 shows comparison of proliferative action in
hepatocytes between purified mutant RKRR2AAAA and TCF.
[0010] FIG. 4 shows comparison of proliferative action in
hepatocytes between purified mutant KIKTKK27AIATAA and TCF.
[0011] FIG. 5 shows comparison of proliferative action in kidney
epithelial cells among purified mutant RKRR2AAAA, mutant
KIKTKK27AIATAA and TCF.
[0012] FIG. 6 shows comparison of proliferative action in bone
marrow cells among purified mutant RKRR2AAAA, mutant KIKTKK27AIATAA
and TCF.
[0013] FIG. 7 shows dose effects of purified TCF, mutant RKRR2AAAA
and mutant KIKTKK27AIATAA on the serum level of total protein in
rats.
[0014] FIG. 8 shows dose effects of purified TCF, mutant RKRR2AAAA
and mutant KIKTKK27AIATAA on the serum level of HDL-cholesterol in
rats.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
[0015] By comparing properties of native protein and a mutant
obtained by mutagenesis at some portion of the amino acid sequence
of the protein, function of that portion can be estimated. In the
case of a protein whose structure is not clearly known, it is often
used to substitute an amino acid, such as Ala, which will not
affect the steric structure for a polar amino acid supposed to be
on the surface of a protein to prevent a structural change of the
protein due to the mutagenesis. To site-specifically change one
amino-acid sequence of a protein into another, cDNA with
site-specific mutations can be prepared by PCR (polymerase chain
reaction) method using cDNA coding for native TCF as template and
synthetic oligonucleotides coding for the other amino acids. cDNA
obtained as described above can be inserted into a vector having an
appropriate expression promotor (cytomegalovirus (CMV), SR.alpha.
(Mole. Cell. Biol. vol. 8, No.1, pp466-472 (1988) and Japanese
Published Unexamined Patent Application 277489 (1989) and
transfected into eukaryotic cells, such as mammalian cells. By
culturing these cells, objective TCF mutants can be prepared from
the culture broth.
[0016] Many TCF mutants can be constructed by introducing mutations
at different sites or residues. In the present invention, 6 mutants
were prepared. These mutants are specified by enumerating the amino
acid sequence before mutagenesis, the number of amino acid at
N-terminus of mutagenized portion and changed amino acid sequence
after mutagenesis by one letter code of amino acid. For example, if
the whole sequence of Arg-Lys-Arg-Arg at the second position from
N-terminus is replaced with Ala, the mutant is represented as
RKRR2AAAA. For another example, mutant whose original sequence
Lys-Ile-lys-Thr-Lys-lys at 27th position from N-terminus is
replaced with Ala-Ile-Ala-Thr-Ala-Ala is represented as
KIKTKK27AIATAA.
[0017] The present invention will be explained in detail by
describing examples. However, these are only exemplified and the
scope of the invention will not be limited by these examples.
EXAMPLE 1
[0018] Site-specific mutation was introduced by the method
described below using the 6.3 kb TCF expression plasmid obtained by
the method described in WO92/01053. E. coli comprising this plasmid
was deposited as FERM BP-3479.
[0019] I. Preparation of Template Plasmid pcD TCF001
[0020] According to the method below, a mutation was introduced at
PstI cleavage site of nucleotide number 34 to change to a
nucleotide sequence which could not be cleaved. PCR was carried out
using 8 ng of plasmid pUC TCF (plasmid in which SalI/SphI fragment
of TCF cDNA was inserted into plasmid pUC18) as a template in the
presence of a combination of mutagenized primer Pst01 (Seq.Id.No.1)
and a nonmutagenized primer TCF415 R (Seq.Id.No.2), and in the
presence of a combination of mutagenized primer P002 (Seq.Id.No.3)
and a non-mutagenized primer TCFSal-77 (Seq.Id.No.4).
[0021] After the primers were removed from the reaction mixture by
molecular sieving with microcon 100 (Amicon), the products were
mixed. And the second PCR was carried out using primer TCFSal-77
and TCF415R. The obtained product was digested by restriction
enzymes BstPI and PstI. By using a ligation kit (Takara-shuzo), the
fragment was ligated with the largest BstPI-PstI fragment of pUC
TCF BstPI/PstI prepared beforehand. E.coli DH5.alpha. was
transformed by using a part of the ligation reaction mixture.
Transformed E.coli DH5.alpha. was cultured in L broth containing 50
.mu.g/ml ampicillin and an objective plasmid was selected from
ampicillin resistant colonies. This plasmid was digested by
restriction enzymes SalI and SphI, mixed with new pcDNAI (in which
multi-cloning site of pcDNAI was mutagenized and there was a
HindIII-SalI-BamHI-SphI-NotI cloning site) SalI/SphI large fragment
prepared beforehand and inserted by using a ligation kit. Using the
reaction mixture, E.coli MC1061/P3 (Invitrogen) was transformed.
Transformed E.coli MC1061/P3 was cultured in L broth containing 50
.mu.g/ml ampicillin and 7.5 .mu.g/ml tetracyclin.
[0022] Plasmid DNAs were prepared from obtained
ampicillin-tetracyclin resistant colonies and the nucleotide
sequence thereof were determined by a DNA sequencer (Perkin-Elmer).
Plasmid pcD TCF001 having an objective structure was obtained and
TCF mutants were prepared by using the obtained plasmid.
[0023] II. Construction of an Expression Vector for TCF Mutants and
Preparation of Transformed E.coli.
[0024] i. Construction of RKRR2AAAA Expression Vector and
Preparation of Transformed E.coli.
[0025] An expression vector for cDNA coding for RKRR2AAAA was
constructed by 2 steps of PCR. In the first step, a combination of
mutagenized primer 2RKRRF (Seq.Id.No.5) and non-mutagenized primer
TCF977 R (Seq.Id.No.6) and a combination of mutagenized primer
2RKRR R (Seq.Id.No.7) and non-mutagenized primer TCFSal-77
(Seq.Id.No.4) were used.
[0026] Four nano grams of pcD TCF001 was used as a template in both
reactions. After the reactions, both reaction mixtures were admixed
and purified with microcon 100. One twentieth of the mixture was
used as template in the second PCR. TCFSal-77 and TCF977 R were
used as primers. The reaction mixture was purified with microcon
100 and digested by restriction enzymes BstPI and EcoRV. By using
the ligation kit, the fragment was inserted into the large fragment
of an SR.alpha.-containing TCF expression vector cleaved by BstPI
and EcoRV beforehand. E.coli DH5.alpha. was transformed with the
ligation reaction mixture and an objective clone was obtained from
the obtained ampicillin resistant cells by the same method as
described before. Plasmid DNA was prepared from the obtained clone
and the DNA sequence thereof was determined by the DNA sequencer
(Perkin-Elmer). And this plasmid was cleaved by restriction enzymes
EcoRV and BstPI and inserted into the fragment of pUC TCF digested
by restriction enzymes EcoRV and BstPI beforehand, followed by
transformation of E.coli DH5.alpha. therewith. E.coli comprising
this plasmid was deposited as pUC TCF2 at National Institute of
Bioscience and Human Technology on Nov. 10, 1994 and has a deposit
number FERM P-14624.
[0027] ii. Construction of KIKTKK27AIATAA Expression Vector and
Preparation of Transformed E.coli.
[0028] An expression plasmid for CDNA coding for KIKTKK27AIATAA
mutant was constructed by 2 steps of PCR. In the first PCR, a
combination of a mutagenized primer 27KIKTKK F (Seq.Id.No.8) and
non-mutagenized primer TCF977 R (Seq.Id.No.6) and a combination of
mutagenized primer 27KIKTKK R (Seq.Id.No.9) and non-mutagenized
primer TCFSal-77 (Seq.Id.No.4) were used. Four ng of pcD TCF001 was
used as a template in both reactions. After the reactions, both
reaction mixtures were admixed and purified with microcon 100. One
twentieth of the mixture was used as template in the second PCR.
TCFSal-77 and TCF977 R were used as primers.
[0029] The reaction mixture was purified with microcon 100 and
digested by restriction enzymes BstPI and EcoRV. By using a
ligation kit, the fragment was inserted into the large fragment of
the SR-.alpha.-containing TCF expression vector cleaved by BstPI
and EcoRV beforehand. E.coli DH5.alpha. was transformed with the
ligation reaction mixture and an objective clone was obtained from
the obtained ampicillin resistant cells by the same method as
described before. Plasmid DNA was prepared from the obtained clone
and the DNA sequence thereof was determined by DNA sequencer. And
this plasmid was cleaved by restriction enzymes EcoRV and BstPI and
incorporated into a fragment of pUC TCF by digested restriction
enzymes EcoRV and BstPI, followed by transformation of E.coli
DH5.alpha. therewith. E.coli comprising this plasmid was deposited
at National Institute of Bioscience and Human-Technology Nov. 10,
1994 and has the deposit number FERM P-14623.
[0030] iii. Construction of K54A Expression Vector and Preparation
of Transformed E.coli.
[0031] An expression plasmid for cDNA coding for K54A mutant was
constructed by 2 steps of PCR. In the first PCR, a combination of
mutagenized primer 54K F (Seq.Id.No.10) and non-mutagenized primer
TCF 977 R (Seq.Id.No.6) and a combination of mutagenized primer 54K
R (Seq.Id.No.11) and non-mutagenized primer TCFSal-77 (Seq.Id.No.4)
were used. Four ng of pcD TCF001 was used as a template in both
reactions. After the reactions, both reaction mixtures were admixed
and purified with microcon 100.
[0032] One twentieth of the mixture was used as template in the
second PCR. TCFSal-77 and TCF 977 R were used as primers. The
reaction product was purified with microcone 100 and digested by
restriction enzymes BstPI and EcoRV. By using a ligation kit, the
fragment was inserted into the large fragment of the
SR.alpha.-containing TCF expression vector cleaved by BstPI and
EcoRV beforehand. E.coli DH5.alpha. was transformed with the
ligation reaction mixture and an objective clone was obtained from
the obtained ampicillin resistant cells by the same method as
described before. Plasmid DNA was prepared from the obtained clone
and the DNA sequence thereof was determined by DNA sequencer.
[0033] iv. Construction of RGKD132AGAA Expression Vector and
Preparation of Transformed E.coli.
[0034] An expression plasmide for cDNA coding for RGKD132AGAA
mutant was constructed by 2 steps of PCR. In the first PCR, a
combination of mutagenized primer 132RGKD F (Seq.ID.No.12) and
non-mutagenized primer TCF977R (Seq.ID.No.6) and a combination of
mutagenized primer 132RGKD R (Seq.ID.No.13) and primer TCF Sal-77
(Seq.ID.No.4) were used. Four ng of pcD TCF001 was used as a
template in both reactions. After the reaction was through, both
reaction mixtures were admixed and purified with microcon 100.
[0035] One twentieth of the mixture was used as template in the
second PCR. TCFSal-77 and TCF977 R were used as primers. The
reaction product was purified with microcon 100 and digested by
restriction enzymes BstPI and EcoRV. By using a ligation kit, the
fragment was inserted into the large fragment of the SRa-containing
TCF expression vector cleaved by BstPI and EcoRV beforehand. E.coli
DH5.alpha. was transformed with the ligation reaction mixture and
an objective clone was obtained from the obtained ampicillin
resistant cell lines. Plasmid DNA was prepared from the obtained
clone in the same way as described before and the base sequence
thereof was determined by DNA sequencer.
[0036] v. Construction of R142A Expression Vector and Preparation
of Transformed E.coli
[0037] An expression plasmid for cDNA coding for R142A mutant was
constructed by 2 steps of PCR. In the first PCR, a combination of
mutagenized primer 142R F (Seq.ID.No.14) and non-mutagenzed primer
TCF977 R (Seq.ID.No.6) and a combination of mutagenized primer 142R
R (Seq.ID.No.15) and TCFSal-77 (Seq.ID.No.4) were used. Four ng of
pcD TCF was used as template in both reactions. After the reaction
was through, both reaction mixtures were admixed and purified with
microcon 100.
[0038] Then, one twentieth of the mixture was used as template in
the second PCR. The reaction mixture was purified with microcon 100
and digested by restriction enzymes BstPI and EcoRV. By using a
ligation kit, the fragment was inserted into the large fragment of
the SR.alpha.-containing TCF expression vector cleaved by BstPI and
EcoRV beforehand. E.coli DH5.alpha. was transformed with the
ligation reaction mixture and an objective clone was obtained from
the obtained ampicillin resistant cell lines in the same way as
described before. The plasmid DNA was prepared from the obtained
clone and the DNA sequence thereof was determined by DNA
sequencer.
[0039] vi. Construction of R42A Expression Vector and Preparation
of Transformed E.coli.
[0040] An expression plasmid for cDNA coding for R42A mutant was
constructed by 2 steps of PCR. In the first PCR, a combination of
mutagenized primer 42R F (Seq.ID.No.16) and non-mutagenized primer
TCF977 R (Seq.ID.No.6) and a combination of mutagenized primer 42R
R (Seq.ID. No.17) and TCFSal-77 (Seq.ID.No.4) were used. Four ng of
pcD TCF001 was used as template in the both reactions.
[0041] After the reaction was through, the both reaction mixtures
were admixed and purified with microcon 100. One twentieth of the
mixture was used as template in the second PCR. TCFSal-77 and
TCF977 R were used as primers. The reaction mixture was purified
with microcon 100 and was digested by restriction enzyme
BstPI/EcoRV. By using a ligation kit, the fragment was inserted
into the large fragment of the SR.alpha.-containing TCF expression
vector cleaved by BstPI and EcoRV beforehand. E.coli DH5.alpha. was
transformed with the ligation reaction mixture and an objective
clone was obtained from ampicillin resistant cell lines in the same
way as described before. The plasmid DNA was prepared from the
obtained clone and the DNA sequence thereof was determined by DNA
sequencer.
[0042] III. Preparation and Purification of Expression Plasmids for
TCF Mutants
[0043] Six species of transformed E.coli comprising the above
expression plasmids were cultured in L broth (400ml) containing 50
.mu.g/ml ampicillin in a shaking incubator at 37.degree. C.
overnight, wherein Spectinomycin (Sigma) was added up to a final
concentration of 0.3 mg/ml when OD600 of cultured broth became 1.0.
According to the method of Maniatis (Molecular cloning 2nd ed.
ppl.21-1.52 (1989), Cold Spring Harbor Laboratory), plasmid DNA was
isolated by alkaline SDS method and 6 species of TCF mutant
expression plasmids were purified by cesium density gradient
centrifugation method.
[0044] IV. Transfection of TCF Mutant Expression Plasmid into
Animal Cell.
[0045] All the mutant expression plasmids were transfected into
Chinese Hamster Ovary (CHO) Cells. CHO cells (2.times.10.sup.6)
were suspended in 0.8 ml IMDM medium (Gibco) containing 10% fetal
calf serum (FCS) (Gibco), in which a solution of 200 .mu.g of
expression vector and 10 .mu.g of Blasticidin resistant gene
expression plasmid pSV2 bsr (Funakoshi) dissolved beforehand in 25
.mu.l of TE (10 mM Tris-HCl (pH8.0)-1 mM EDTA) was further
suspended. This suspension received electroporation under the
conditions of 330V and 960 .mu.F. After leaving it at room
temperature for 10 minutes, it was suspended in 10 ml of IMDM
containing 10% FCS medium and cultured at 37.degree. C. in a
CO.sup.2 incubator (5% CO.sup.2) for 2 days. Two days after, the
supernatant was collected and the amount of the expressed TCF
mutant was analyzed by enzyme immunoassay (EIA) (N. Shima, et. al.,
Gastro-enterologia Japonica, Vol. 26, No. 4. pp477-482 (1991))
using anti-TCF monoclonal antibody. It was used as a sample for
assaying biological activity. The cells were harvested from the
bottom of flasks by trypsin (Gibco) treatment and the number of
viable cells was counted. About 10,000 cells/well were placed in
96-well plates(Nunc) and cultured in 200 .mu.l/well of IMDM medium
containing 10% FCS and 5 .mu.g/ml Blastcidine for 2-3 weeks. 2-3
weeks after, 50 .mu.l aliquot was taken from each well and
investigated on the expression of TCF mutant by EIA. Cell clones
expressing the TCF mutants were grown in 12-well plates and 25
cm.sup.2 flasks. The cell lines producing TCF mutant were
established from CHO cells by the above operation.
[0046] V. Large Scale Cultivation of TCF Mutant Producing Cells
[0047] Mutant producing cells were harvested from 75 cm.sup.2
flasks by trypsin treatment when it became confluent and those
cells were transferred into 10 225-cm.sup.2 flasks containing 100
ml of the medium and cultured for a week. Then the cultured
supernatant was collected. By repeating this operation once or
twice, 1-21 of the cultured broth was obtained.
[0048] VI. Purification of the TCF mutants
[0049] It was purified by 3 steps as described below.
[0050] i. Heparin-Sepharose CL-6B
[0051] Precipitates were removed from one-two litter of cultured
medium of CHO cells expressing each TCF mutants by centrifugation
(2,000 rpm.times.10 min.) of the medium and filtrating the
supernatant through a 0.45 .mu.m filter (German Science). TCF
mutant was adsorbed at 4 ml/min. on a heparin-Sepharose CL-6B
column (25 mm.times.120 mm, pharmacia) equilibrated with 10 mM
Tris-HCl (pH 7.5) containing 0.3M NaCl and 0.01% Tween 20. The
column was washed with about 500 ml of equilibration buffer and the
TCF mutant was eluted by 10 mM Tris-HCl (pH 7.5) containing 2M NaCl
and 0.01% Tween 20. The eluted solution was fractionated to 4 ml
each by a fraction collector and the fractions having absorption at
280 nm were collected.
[0052] ii. Mono S FPLC
[0053] The fraction containing TCF mutant eluted with 2M NaCl was
dialyzed against 10 mM phosphate buffer (pH 7.0) containing 0.15M
NaCl, followed by centrifugation (12,000 rpm.times.90 min.) to
remove precipitate. The supernatant containing TCF mutant was
passed through on a Mono S column (5 mm.times.50 mm, Pharmacia)
equilibrated with 10 mM phosphate buffer (pH 7.0) containing 0.15 M
NaCl and 0.01% Tween 20 at flow rate of 1 ml/min. for TCF mutant to
be adsorbed thereon. After the column was washed with about 30 ml
of equilibration buffer, TCF mutant was eluted ,by changing the
flow rate to 0.5 ml/min, with a linear gradient of NaCl up to 1.0 M
for 60 min.. The eluted solution was fractionated to 5 ml each by a
fraction collector and fractions containing TCF mutant was analyzed
by absorption at 280 nm and EIA and collected.
[0054] iii. Heparin 5-PW FPLC
[0055] To the fraction containing TCF mutant obtained using Mono S
column chromatography, 2-fold amount of 10 mM Tris-HCl (pH 7.5)
containing 0.01% Tween 20 was added. The solution was passed
through a Heparin 5-PW column (5 mm.times.75 mm TOSOH) 1 ml/min.
equilibrated with 10 mM Tris-HCl (pH 7.5) containing 0.3M NaCl and
0.01% Tween 20 for TCF mutant to be absorbed thereon. By changing
the flow rate to 0.5 ml/min., TCF mutant was eluted with a linear
gradient of NaCl up to 2.0 M for 60 min.
[0056] The eluted solution was fractionated to 5 ml each by a
fraction collector. The fraction containing TCF mutant was analyzed
by 280 nm absorption and EIA and collected. Obtained TCF mutant
solution was dialyzed against PBS containing 0.01% of Tween 20
(TPBS) so as to be the final purified product. The amount of
protein in the final purified product was determined by Lowry
method. The amino acid sequence of TCF mutant RKRR2AAAA and that of
mutant KIKTKK27 were represented in Seq.ID.No.18 and in
Seq.ID.No.19 respectively.
[0057] VII. SDS-polyacrylamide Gel Electrophoresis of Purified TCF
Mutant
[0058] Purified TCF mutant (200 ng) was applied on SDS
polyacrylamide gel electrophoresis. Schematic representation of
electrophoresis of TCF mutant RKRR2AAAA and KIKTKKK27AIATAA, which
exhibited 10-fold increase in biological activity as described
below, and native TCF was shown in FIG. 1. Both of the results
under reducing conditions(in the presence of
.beta.-mercaptoethanol) and non-reducing conditions (in the absence
of .beta.-mercaptoethanol) did not show any difference among the
three. In addition, there was no band but those to be expected from
the structure of both TCF mutants.
EXAMPLE 2
[0059] Affinity of TCF and TCF Mutant to Heparin
[0060] I. Heparin-Sepharose CL-6B
[0061] Precipitates were removed from the cultured medium of CHO
cells expressing each TCF mutant by centrifugation (1,200
g.times.10 min.) of the medium and by filtrating the supernatant
through a 0.22 m filter. The filtrated supernatant was charged on a
heparin-Sepharose CL-6B column (5 mm.times.5 mm; Pharmacia)
equilibrated with TPBS for TCF mutant to be adsorbed thereon. After
washing with 3 ml TPBS, TCF mutant was eluted with 1 ml of TPBS
containing 0.2-0.3M NaCl, increasing the salt concentration
stepwise. The concentration of TCF mutant in the eluate was
analyzed by EIA and the salt concentration of the eluate was
defined as affinity of mutant to heparin.
[0062] II. Heparin 5-PW FPLC
[0063] The cultured broth of CHO cells expressing each TCF mutant
(30-60 ml) was centrifuged (1,000 g.times.10 min.), passed through
0.22 .mu.m filter to remove precipitate and applied on a Heparin
5-PW column equilibrated with 20 mM Tris-HCl buffer solution
containing 0.01% Tween 20 at a flow rate of 1.0 ml/min. for TCF
mutant to be adsorbed. After washing the column with about 20 ml of
equilibration buffer solution and changing the flow rate to 0.5
ml/min., TCF mutant was eluted with a linear gradient of NaCl up to
1.5 M for 45 minutes. Fractions of 0.5 ml each were taken by a
fraction collector and the concentration of TCF mutant in each
fraction was quantified by EIA and the salt concentration of the
elution was defined as affinity of mutant to heparin.
[0064] The results of determination of affinity of these TCF mutant
to heparin are shown in table 1. The elution concentration of NaCl
from heparin-Sepharose represents the concentration at which TCF
mutant is eluted in the maximum amount. The relative ratio of
elution concentration is defined as (the elution concentration of
NaCl of mutant TCF/that of native TCF). And n.d. means "not
determined". In the examination with heparin-Sepharose, RKRR2AAAA,
KIKTKK27AIATAA, and R42A exhibited significantly lowered affinity
to heparin. Further, in the examination with heparin 5-PW, it was
observed that affinity of the mutants to heparin was lowered to
around 70% of that of native TCF.
1 TABLE 1 Heparin- Sepharose Heparin 5-PW Relative Elution Elution
Ratio of Concentration Concentration Elution of NaCl (M) of NaCl
(M) concentration TCF 0.9 1.14 1.00 RKRR2AAAA 0.6 0.78 0.68
KIKTKK27AIATAA 0.6 0.82 0.72 R42A 0.7 0.84 0.74 K54A 0.9 1.10 0.96
RGKD132AGAA 0.9 n.d. n.d. R142A 0.9 n.d. n.d.
EXAMPLE 3
[0065] Proliferative Activity of TCF and TCF Mutants on Hepatocyte
in vitro
[0066] Proliferative activity was investigated by the following
method: According to the method of Segren (Method in cell biology,
Vol. 13, p29 (1976) Academic Press, New York), hepatocyte was
isolated from Wister rats (about 200 g of body weight). The cells
(1.0.times.10.sup.4/50 .mu.l/well) were placed into the wells of
96-well plates (Falcon) and cultured at 37.degree. C. overnight
using Williams E medium (Flow Laboratory)containing 10% fetal calf
serum and 10 .mu.M dexamethasone (hereinafter, abbreviated as base
medium). After 24 hours, 10 .mu.l of base medium containing TCF or
TCF mutant was added to each well. The plates were incubated at
37.degree. C. for another 22 hours. After 22 hours,
.sup.3H-thymidine (Amersham) was added thereto so as to be 1
.mu.Ci/well, keeping the culture another 2 hours. After then, the
cells were washed twice with PBS and harvested by treatment of 0.5%
trypsin followed by collection of the cells in a glass filter by
cell harvester. The radio activity incorporated in each well was
measured by Matrix 96 (Packard) as the amount of DNA synthesis. The
results are shown in FIG. 2. Mutant K54A, RGKD132AGAA and R142A had
1.4-fold, 2.0-fold and 1.6-fold, respectively, higher biological
activity than native TCF at a TCF antigen concentration of 2.5
ng/ml. Further each mutant which had lowered affinity to heparin
was determined by Lowery method. Then the biological activity was
compared with regard to the protein concentration exhibiting 50% of
maximum proliferative activity (ED50) (FIG. 3 and 4).
[0067] As the results, 2 species of protein, that is, RKRR2AAAA and
KIKTKK27AIATAA, exhibited more than 10 folds of biological activity
per unit amount of protein comparing with that of native TCF.
EXAMPLE 4
[0068] Proliferative Activity of TCF and TCF Mutant in Kidney
Epithelial Cells
[0069] Proliferative activity in kidney epithelial cell was
determined by the following method:
[0070] OK cells derived from kidney epithelial cell line of
American Opossum were placed into each well of 96-well plates so as
to be 1.0.times.10.sup.4/100 .mu.l/well and cultured in DMEM medium
containing 10% fetal calf serum at 37.degree. C. overnight. After
then, each well was washed 2-3 times with DMEM medium containing no
serum. The medium in each well was replaced with DMEM medium
containing no serum and the culture was kept at 37.degree. C. for
another 2 days. Then, the medium in each well was again replaced
with 50 .mu.l of fresh DMEM medium containing no serum and, with 50
.mu.l of addition of TCF or TCF mutant diluted with DMED medium
containing 0.2% bovine serum albumin, the culture was kept for
another 24 hours. After 24 hours, H-thymidine was added thereto so
as to be 1 .mu.Ci/well and the culture was kept for another 2
hours. Then, cells were washed with PBS twice and the cells were
harvested by treatment of 0.5% trypsin, followed by collection of
the cells in a glassfilter by a cell harvester. The radio activity
incorporated in each well was measured by Matrix 96 and determined
as the amount of DNA synthesis. The results were exhibited in FIG.
5. As the results, it was observed that biological activities per
unit amount of protein of RKRR2AAAA and KIKTKK27AIATAA in kidney
epithelial cell increased more than 2 folds comparing with that of
native TCF.
EXAMPLE 5
[0071] Proliferative Activity of TCF and TCF Mutant in Bone Marrow
Cell in vitro Proliferative activity in bone marrow cell was
determined by the following method: NFS-60 cells which are from a
mouse bone marrow cell line were placed into each well of 96
well-plate so as to be 5.0.times.10.sup.4 cells/50 .mu.l/well in
RPMI medium containing 10% fetal calf serum and, with addition of
50 .mu.l of TCF or TCF mutant diluted with the medium, cultured at
37.degree. C. for 24 hours. After 24 hours, 10 .mu.l of 5 mg/ml MTT
(Sigma) was added to each well and the culture was kept for another
4 hours. Then, 100 .mu.l of 10% SDS/10 mM ammonium chloride was
added to each well and it was left at room temperature overnight.
After that, optical absorbance at 590 nm was measured by
Immunoreader NJ-2000 (Intermed) as proliferative activity.
[0072] The results were exhibited in FIG. 6. As the results, it was
observed that biological activities per unit amount of protein of
RKRR2AAAA and KIKTKK27AIATAA in bone marrow cell decrease to
1/2-{fraction (1/20)} of that of native TCF.
EXAMPLE 6
[0073] In vivo Biological Activity of TCF and TCF Mutants
[0074] In vivo Biological activity was assayed by the following
method: TCF or TCF mutant dissolved in PBS containing 0.01% Tween
20 was intravenously administered through tail (2 ml/kg.times.2
times/day) in 6 weeks old male Wister rats for 4 days. At the next
day to the final administration, blood samples were taken from
caudal vena cava under ether anesthesia and serum thereof were
collected by centrifugation (3000 rpm.times.10 min.) and, in the
case of plasma, immediately after sampling blood, sodium citrate
(the final concentration was 0.38%) was added thereto followed by
centrifugation(3000 rpm.times.10 min.) to give plasma. After serum
or plasma obtained was preserved in a freezer kept at -30.degree.
C., serum level of total protein, albumin, unsaturated iron binding
capacity, total cholesterol, free cholesterol, HDL-cholesterol and
phospholipid were analyzed by serum autoanalyzer (Hitachi 7150
Autoanalyzer) and plasma level of prothrombin time and fibrinogen
were analyzed by Auto blood coagulation analyzer KC40 (Amerung).
For these analysis, the following analyzing kits were used:
[0075] Total protein: Autosera.sup.TR TP, Albumin: Autosera.sup.TR
ALB, Unsaturated iron-binding capacity: Clinimate UIBC, Total
cholesterol: Autosera.sup.TR CHO-2, Free cholesterol:
Autosera.sup.TR F-CHO-2, HDL-cholesterol: HDL-C.multidot.2
"DAIICHI", Phospholipid: Autosera.sup.TR PL-2, (All the above kits
were products of Daiichi-Pure Chemicals Co., Ltd.) Prothrombin
time: Orthobrain thromboplastin (Ortho Diagnostic System Inc.),
Fibrinogen: Sun assay Fib (Nitto Boseki Co., Ltd.). As typical
examples, dose effects thereof on serum level of total protein and
on serum level of HDL-cholesterol were exemplified in FIG. 7 and
FIG. 8 respectively.
[0076] According to the results of statistical analysis of parallel
line assey, with respect to increase of total protein, RKRR2AAAA
exhibited 2.12 folds of specific activity and KIKIKTKK27AIATAA
exhibited 1.37 folds of specific activity, comparing to that of
native one. Further, with respect to increase HDL-cholesterol,
RKRR2AAAA exhibited 1.66 folds of specific activity and
KIKTKK27AIATAA exhibited 1.62 folds of specific activity, comparing
to that of native one.
[0077] Industrial Availabilities
[0078] The present invention is to provide a novel TCF mutant. The
TCF mutant of the present invention has proliferative activity and
growth stimulative activity in hepatocyte and beneficial for
treatment of various hepatic diseases and as an antitumor agent.
Sequence CWU 1
1
* * * * *