U.S. patent application number 10/441729 was filed with the patent office on 2003-12-11 for novel human thrombopoietin mutein.
This patent application is currently assigned to Daewoong Pharmaceutical Co., Ltd.. Invention is credited to Ahn, Hyea Kyung, Chang, Woo Ik, Chung, Joo Young, Ju, Sang Myoung, Koh, Yeo Wook, Lim, Seung Wook, Park, Ji Soo, Park, Sang Kyu, Park, Seung Kook.
Application Number | 20030228666 10/441729 |
Document ID | / |
Family ID | 29715925 |
Filed Date | 2003-12-11 |
United States Patent
Application |
20030228666 |
Kind Code |
A1 |
Chung, Joo Young ; et
al. |
December 11, 2003 |
Novel human thrombopoietin mutein
Abstract
The present invention relates to novel human thrombopoietin (;
hTPO) derivatives, and to process of preparation thereof.
Particularly, sugar chains are introduced into native hTPO by
substituting amino acids such as asparagine for amino acids at
specific positions in native hTPO, preparing novel hTPO derivatives
with high activities enhancing the platelet production in vivo.
Therefore, the novel hTPO derivatives of this invention may be
useful for the treatment of thrombocytopenia associated with
anticancer therapy or the transplantation of bone marrow.
Inventors: |
Chung, Joo Young;
(Sungnam-si, KR) ; Park, Sang Kyu; (Seoul, KR)
; Ju, Sang Myoung; (Sungnam-si, KR) ; Ahn, Hyea
Kyung; (Sungnam-si, KR) ; Lim, Seung Wook;
(Sungnam-si, KR) ; Chang, Woo Ik; (Koonpo-si,
KR) ; Park, Seung Kook; (Sungnam-si, KR) ;
Koh, Yeo Wook; (Sungnam-si, KR) ; Park, Ji Soo;
(Seoul, KR) |
Correspondence
Address: |
Attention of Karen S. Canady
Gates & Cooper LLP
Howard Hughes Center
6701 Center Drive West, Suite 1050
Los Angeles
CA
90045
US
|
Assignee: |
Daewoong Pharmaceutical Co.,
Ltd.
|
Family ID: |
29715925 |
Appl. No.: |
10/441729 |
Filed: |
May 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10441729 |
May 20, 2003 |
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09720410 |
Dec 21, 2000 |
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09720410 |
Dec 21, 2000 |
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PCT/KR99/00347 |
Jun 30, 1999 |
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Current U.S.
Class: |
435/69.4 ;
435/320.1; 435/325; 530/397; 530/399; 536/23.5 |
Current CPC
Class: |
C07K 14/524
20130101 |
Class at
Publication: |
435/69.4 ;
435/320.1; 435/325; 530/399; 536/23.5; 530/397 |
International
Class: |
C12P 021/02; C12N
005/06; C07K 014/575; C07H 021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 30, 1998 |
KR |
1998-25935 |
Jun 29, 1999 |
KR |
1999-25143 |
Claims
What is claimed is
1. Human thrombopoietin derivative which derived from human
thrombopoietin (hTPO) described by SEQ ID NO: 30; which has at
least one additional N-linked glycosylation site; and which is
selected from the group comprising: [Asn.sup.108] hTPO;
[Asn.sup.117] hTPO; [Asn.sup.147] hTPO; [Asn.sup.153] hTPO;
[Asn.sup.164] hTPO; [Asn.sup.193] hTPO; [Asn.sup.117, Asn.sup.147]
hTPO; [Asn.sup.117, Asn.sup.164] hTPO; [Asn.sup.108, Asn.sup.147]
hTPO; [Asn.sup.108, Asn.sup.164] hTPO; [Asn.sup.147, Asn.sup.164]
hTPO; [Asn.sup.117, Asn.sup.147, Asn.sup.164] hTPO; [Asn.sup.108,
Asn.sup.147, Asn.sup.164] hTPO; [Asn.sup.108, Asn.sup.117,
Asn.sup.164] hTPO; [Asn.sup.157, Asn.sup.164] hTPO; [Asn.sup.162,
Ser.sup.164] hTPO; [Asn.sup.162, Thr.sup.164] hTPO; [Asn.sup.153,
Ser.sup.155, Asn.sup.164] hTPO; [Asn.sup.153, Thr.sup.155,
Asn.sup.164] hTPO; [Asn.sup.159, Ser.sup.161, Asn.sup.164] hTPO;
[Asn.sup.159 , Thr.sup.161Asn.sup.164] hTPO; [Asn.sup.166,
Ser.sup.168] hTPO; [Asn.sup.166 , Thr.sup.168] hTPO; and
[Asn.sup.164, Asn.sup.168] hTPO.
2. The human thrombopoietin derivative of claim 1 which is
[Asn.sup.164] hTPO, [Asn.sup.193] hTPO, [Asn.sup.108, Asn.sup.117,
Asn.sup.164] hTPO, or [Asn.sup.157, Asn.sup.164] hTPO.
3. Recombinant gene encoding human thrombopoietin derivative of
claim 1.
4. Recombinant gene encoding human thrombopoietin derivative of
claim 2.
5. Eukaryotic expression vector containing the recombinant gene of
claim 3.
6. The eukaryotic expression vector of claim 5 which is p40433,
p40434, p40449, p40458, pD40433, pD40434, pD40449, or pD40458.
7. Mammalian cell line CHO K-1/p40433 (Accession NO: KCTC 0495BP)
transfected with the expression vector p40433 of claim 6.
8. Mammalian cell line CHO dhfr-/pD40434 (Accession NO: KCTC
0630BP) transfected with the expression vector pD40434 of claim
6.
9. Mammalian cell line CHO dhfr-/pD40449 (Accession NO: KCTC
0631BP) transfected with the expression vector pD40449 of claim
6.
10. Mammalian cell line CHO dhfr-/pD40458 (Accession NO: KCTC
0632BP) transfected with the expression vector pD40458 of claim
6.
11. Process of preparing the human thrombopoietin derivative of
claim 1 wherein a mammalian cell line containing the recombinant
gene of claim 3 is used to obtain the human thrombopoietin
derivative of claim 1.
12. Pharmaceutical composition containing the human thrombopoietin
derivative of claim 1 which is used for the treatment of
thrombocytopenia.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/720,410, filed Dec. 21, 2000, the entire contents of
which are incorporated herein by reference, and which is a national
stage filing of PCT KR99/00347, filed Jun. 30, 1999, which claims
priority to Korean patent application numbers KR 1999-25143, filed
Jun. 29, 1999, and KR 1998-25935, filed Jun. 30, 1998.
FIELD OF THE INVENTION
[0002] The present invention relates to novel human thrombopoietin
(; hTPO) derivatives with high activities enhancing the platelet
production in vivo, and to process of preparation thereof.
[0003] Particularly, this invention relates to novel hTPO
derivatives wherein sugar chains are introduced by substituting
amino acids such as asparagine for amino acids at specific
positions in native hTPO; to nucleotide sequences encoding the hTPO
derivatives; to expression vectors containing the nucleotide
sequences; to process of construction thereof; to cell lines
transformed with the vectors; and to process of preparing the hTPO
derivatives thereby.
BACKGROUND
[0004] Thrombocytopenia is the disease of platelet deficiency
caused by anticancer therapy, bone marrow graft and so on. In the
process of anticancer therapy or bone marrow graft, megakaryocyte
colony forming cells, the platelet precursor cells in bone marrow,
are disrupted, and this leads to platelet deficiency.
Thrombocytopenia patient is subject to bleeding in response to a
light trauma, and more serious patient becomes bleeding without
trauma. Bleeding is often fatal in this case since the blood is not
stanched at all.
[0005] The current therapy for thrombocytopenia is nothing but the
platelet transfusion. However, several problems and side effects
are associated with this therapy, such as insufficient donors,
transfusion-meditated infection with e.g. HIV (human
immunodeficiency virus) and hepatitis viruses, the elicitation of
immune response, and so on.
[0006] Platelet is a component of blood, originated from
megakaryocyte precursor cells, and plays a role in the suppression
of bleeding. Thrombopoietin (hereafter, referred to as "TPO"), a
glycoprotein synthesized and secreted in liver or kidney, regulates
the platelet level in blood. TPO accelerates the proliferation and
differentiation of the megakaryocyte precursor cells, which is
followed by the platelet production (Lok et al., Nature, 369:
565-568, 1994; De savage et al., Nature, 369: 533-568, 1994).
[0007] Since a gene encoding TPO was isolated first from human in
1994 (Lok et al., Nature, 369: 565-568, 1994; De savage et al.,
Nature, 369: 533-568, 1994; Miyazaki et al., Experimental hematol.,
22: 838, 1994; WO 95/18858), clinical approaches for
thrombocytopenia have been based on the function of human TPO
(hereinafter, referred to as "hTPO"), that is, the regulation of
the platelet level.
[0008] Three different approaches are proceeded in order to improve
the activity of native hTPO.
[0009] Glycoprotein hTPO is expressed in cells as an inactive
precursor comprising 353 amino acids, and the cleavage of signal
peptide (21 amino acids) leads to the secretion of active hTPO
protein (332 amino acids) out of the cells. The amino acid sequence
of hTPO is divided into two regions. The N-terminal region
comprising 151 amino acids contains catalytic site, and shows high
similarity to that of erythropoietin (; EPO) The other region,
C-terminal region is presumed to have a key role in the
extracellular secretion and in vivo stability of hTPO.
[0010] The first method for modifying native hTPO relates to the
deletion of the C-terminal region or the addition of new amino
acids to the deleted hTPO.
[0011] In support of this approach, Amgen INC. developed various
hTPO derivatives such as hTPO.sub.151 (consisting of amino acids
1-151), hTPO.sub.174 (consisting of amino acids 1-174) and the
hTPO.sub.163 supplemented with methionine-lysine in its N-terminal.
However, these derivatives proved to show lower hTPO activity in
vivo than native hTPO, although their activities were maintained in
vitro (WO 95/26746, WO 95/25498).
[0012] In addition, Genentech INC. prepared from E. coli a
recombinant hTPO.sub.153 derivative having an N-terminal methionine
(WO 95/18858). Kirin produced diverse hTPO derivatives with
C-terminal deletion and hTPO.sub.163 derivatives with substitution,
deletion, or insertion at a specific amino acid residue (WO
95/21920). Other hTPO derivatives with C-terminal deletion were
provided by Zymogenetics INC. (WO 95/21920; WO 96/17062) and G. D.
Searl (WO 96/23888). These derivatives, however, failed to show
higher activity of platelet production in vivo than native
hTPO.
[0013] The second method is associated with the conjugation of
polyethyleneglycol (; PEG) with hTPO fragment, which is exampled by
hTPO.sub.163-PEG of Amgen INC. (WO 95/26746).
[0014] The derivatives according to this method, however, have
critical handicaps such as poor stability and safety, since they do
not contain C-terminal region that is important for the stability
of hTPO and since immune response may be elicited by the shift of
their folding structures. Moreover, the qualities of products may
be uneven because PEG is not so conjugated at a uniform
proportion.
[0015] The third method exploits the glycosylation of hTPO, which
may increase the hTPO activity.
[0016] Amgen INC. performed a mutagenesis where a specific
nucleotide in cDNA encoding hTPO was substituted to bear amino acid
sequence "Asn-X-Ser/Thr" (where X is any amino acid but proline).
The mutated gene was used to prepare hTPO derivatives with
C-terminal deletion, which comprised 174 amino acids and into which
one or more N-linked glycosylation sites are produced (WO
96/25498).
[0017] Korea Research Institute of Biology and Biotechnology
(KRIBB) produced a hTPO derivative where one sugar chain is
incorporated into the intact native hTPO (Park et al., J. Biol.
Chem., 273: 256-261, 1998), distinctive from the Amgen's partial
hTPO derivatives.
[0018] However, all these derivatives did not show significantly
higher levels of hTPO activity.
[0019] As described above, although various strategies have been
employed to develop hTPO derivatives with enhanced biological
activity, all failed to obtain the derivatives with higher in vivo
hTPO activities than native hTPO.
[0020] Generally, numerous proteins exist as proteins adorned by
oligosaccharide chains in specific position, i.e. glycoproteins.
Two types of glycosylation have been found. In O-linked
glycosylation, sugar chain is attached to the hydroxyl group of
Ser/Thr residue in the glycoprotein. In N-linked glycosylation,
sugar chain is attached to the amide group of "Asn-X-Ser/Thr" (X is
any amino acid but proline).
[0021] The sugar chain in a glycoprotein exert various effects on
the physical, chemical and biological properties such as protein
stability and secretion, especially on the biological activity in
vivo and pharmacokinetic properties (Jenkins et al., Nature
Biotechnological., 14: 975-981, 1996; Liu et al., Act. TIBTECH.,
10: 114-120, 1992).
[0022] These effects are exemplified by human interferon-.gamma.
and glucose transport protein, where amino acid substitution at
proper glycosylation site gave rise to the striking decrease in the
hTPO activity, suggesting that N-linked sugar chain may have
significant effects on the activity of the glycoprotein (Sareneva
et al., Biochem. J. 303: 831-840, 1994; Asano et al., FEBS, 324:
258-261, 1993).
[0023] However, the introduction of additional sugar chains is not
always accompanied with an increase in the catalytic activity of
the glycoprotein, as described in the precedent art of Amgen INC.
and KRIBB (WO 96/25498; Park et al., J. Biol. Chem., 273: 256-261,
1993). Although additional sugar chains were introduced into these
hTPO derivatives, the biological activities of the glycoproteins
were rather reduced when compared with native hTPO. According to
this observation, it is not the number of sugar chains but the
specific glycosylation site that is crucial for elevating its
catalytic activity.
[0024] We, the inventors of this invention, have prepared various
hTPO derivatives and examined their activities. This invention is
performed by disclosing that several hTPO derivatives such as
derivative wherein Asn is substituted for Arg.sup.164; derivative
wherein Asn is substituted for Thr.sup.193; derivative wherein Asn
is substituted for Pro.sup.157 and Arg.sup.164; and derivative
wherein Asn is substituted for Leu.sup.108 Arg.sup.117 and
Arg.sup.164 produce the remarkably higher levels of platelets than
native hTPO does, which is not ever observed in the current hTPO
derivatives.
SUMMARY OF THE INVENTION
[0025] It is an object of this invention to provide novel hTPO
derivatives that show the higher activities enhancing. the platelet
production in vivo than native hTPO does.
[0026] In accordance with the present invention, the foregoing
objects and advantages are readily obtained.
[0027] The present invention provides novel hTPO derivatives with
higher activity inducing the platelet production in vivo.
Additional sugar chains are introduced into said hTPO derivatives
through substituting amino acids such as asparagine for amino acids
at specific positions in native hTPO.
[0028] This invention also provides genes encoding said hTPO
derivatives.
[0029] In addition, this invention provides process of preparing
said hTPO derivatives, comprising the step wherein said genes are
inserted into appropriate vector; the step wherein a host cell is
transfected with said vector; and the step wherein the transfected
cells are cultured in appropriate medium.
[0030] Further features of the present invention will appear
hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 depicts PCR-based mutagenesis wherein the cDNAs
encoding hTPO derivatives are produced, where
[0032] 1: primer described by SEQ ID NO: 1;
[0033] 2: primer described by SEQ ID NO: 2;
[0034] N: N-primer;
[0035] C: C-primer;
[0036] S: signal sequence.
[0037] FIG. 2 depicts the process of linking the mutated genes to
pBlueBac4 vector.
[0038] FIG. 3 depicts the process of constructing animal expression
vectors that the mutated cDNAs are subcloned in pCDT vector.
[0039] FIG. 4 presents the result of cell proliferation assay where
the activity of M-07e cell proliferation is measured in the
presence of hTPO derivatives expressed in animal cells.
[0040] FIG. 5 presents the in vivo activity of native hTPO, which
is determined by measuring the number of platelets in mouse blood
after treatment with various doses of native hTPO.
[0041] FIG. 6. presents the in vivo activities of various hTPO
derivatives, which are determined by measuring the number of
platelets in mouse blood after treatment with hTPO derivatives (36
.mu.g/kg) expressed in animal cells.
[0042] FIGS. 7a and 7b present the in vivo activities of various
hTPO derivatives, which are determined by measuring the number of
platelets in mouse blood after treatment with hTPO derivatives (10
.mu.g/kg) expressed in animal cells.
[0043] FIG. 8 depicts the process of constructing the dhfr
expression vectors that contain a gene encoding native hTPO or hTPO
derivatives.
[0044] FIG. 9 presents the result of SDS-PAGE and silver staining
with the various fractions obtained in the purification of a hTPO
derivative, where
[0045] lane M: size marker;
[0046] lane 1: culture supernatant;
[0047] lane 2: CM-ion exchange affinity column elutes;
[0048] lane 3: phenylsephrose column elutes;
[0049] lane 4: hydroxyapatite column elutes;
[0050] lane 5: Q cartridge column elutes.
[0051] FIG. 10 presents the in vivo activities of native hPO and
various hTPO derivatives, which are determined by measuring the
number of platelets in mouse blood after treatment with native hTPO
or purified hTPO derivatives (10 .mu.g/kg).
[0052] FIG. 11 presents the result of SDS-PAGE and western blot
analysis with the purified native hTPO and hTPO derivatives,
where
[0053] lane M: size marker;
[0054] lane 1: native hTPO;
[0055] lane 2: hTPO derivative 40433;
[0056] lane 3: hTPO derivative 40434;
[0057] lane 4: hTPO derivative 40449;
[0058] lane 5: hTPO derivative 40458;
[0059] FIGS. 12a and 12b present the result of Western blot
analysis, in which the thrombin-digestion pattern of native hTPO
(FIG. 12a) or its derivative 40433 (FIG. 12b) is shown according to
the time after digestion, where
[0060] lane M: size marker;
[0061] lane 1: Before digestion;
[0062] lane 2: 30 minutes after digestion;
[0063] lane 3: 1 hour;
[0064] lane 4: 2 hours;
[0065] lane 5: 3 hours;
[0066] lane 6: 4 hours;
[0067] lane 7: 6 hours.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0068] Hereinafter, the present invention is described in
detail.
[0069] The present invention provides novel hTPO derivatives with
enhanced activity inducing the platelet production in vivo.
Additional sugar chains are introduced into said hTPO derivatives
through substituting amino acids such as asparagine for amino acids
at specific positions in native hTPO.
[0070] To develop novel hTPO derivatives with enhanced activity
inducing the platelet production in vivo, a variety of hTPO
derivatives were prepared, into which one or more sugar chains are
introduced through substituting one or more amino acids at specific
positions in a hTPO protein. In result, N-linked glycosylation site
"Asn-X-Ser/Thr" (where X is any amino acid but proline) is created
at the specific positions.
[0071] In a preferred embodiment, site-specific mutagenesis using
overlap PCR (Cheng et al., Proc. Natl. Acad. Soc. USA, 91: 5695,
1994) was employed to produce the genes encoding hTPO derivatives
with specific amino acids substituted at specific position (see
FIG. 1).
[0072] First, the following primer pairs containing mutated
sequences were synthesized chemically. These oligonucleotide primer
pairs contain the nucleotide sequences corresponding to the mutated
amino acid residues, and extend to the 5' or 3' neighboring
sequence to the mutated region in hTPO cDNA.
1TABLE 1 Primer pairs for site-specific mutagenesis Derivative
primer SEQ ID NO: Nucleotide sequence 40429 29-N 3 5'-GCTGT GGTGT
TGCCC TGTGG-3' 29-C 4 5'-ACAGG GCAAC ACCAC AGCTC-3' 40430 30-N 5
5'-GGGTT CCGTT TAAAC TCTGC AG-3' 30-C 6 5'-CTGCA GAGTT TAAAC GGAAC
CCAG-3' 40431 31-N 7 5'-AGAGG GTGGA ATTCC CTACA AGCA-3' 31-C 8
5'-TGCTT GTAGG GAATT CCACC CTCT-3' 40432 32-N 9 5'-GGGCC CGGTT
GACGC AGA-3' 32-C 10 5'-TCTGC GTCAA CCGGG CCC-3' 40433 33-N 11
5'-GGACT AGAGA CGTGT TGCTG GGGAC-3' 33-C 12 5'-GTCCC CAGCA ACACG
TCTCT AGTCC-3' 40434 34-N 13 5'-GAAGC CCAGA TCCGT TAGTT CTGGC-3'
34-C 14 5'-GCCAG AACTA ACGGA TCTGG GCTTC-3' 40458 58-N 15 5'-AGCTG
TGGTG TTTGG GGCCC GC-3' 58-C 16 5'-GCGGG CCCCA AACAC CACAG CT-3'
33-N 11 5'-GGACT AGAGA CGTGT TGCTG GGGAC-3' 33-C 12 5'-GTCCC CAGCA
ACACG TCTCT AGTCC-3' 40459 59-N 17 5'-CTAGA GAGGT GCTGT TGACA GCTGT
G-3' 59-C 18 5'-CACAG CTGTC AACAG CAGCA CCTCT CTAG-3' 40460 60-N 19
5'-GGTGG GTGGG GTCCG GTTGA CGCAG AGG-3' 60-C 20 5'-CCTCT GCGTC
AACCG GACCC CACCC ACC-3' 33-N 11 5'-GGACT AGAGA CGTGT TGCTG
GGGAC-3' 33-C 12 5'-GTCCC CAGCA ACACG TCTCT AGTCC-3' 40461 61-N 21
5'-TCTGC TGGGG GAAGC GTTGG TGGGT GG-3' 61-C 22 5'-CCACC CACCA ACGCT
TCCCC CAGCA GA-3' 33-N 11 5'-GGACT AGAGA CGTGT TGCTG GGGAC-3' 33-C
12 5'-GTCCC CAGGA ACACG TCTCT AGTCC-3' 40462 62-N 23 5'-CAGTG TGAGG
GTTAG ATTGG TTCTG CTG-3' 62-C 24 5'-CAGCA GAACC AATCT AACCC TCACA
CTG-3' 40463 63-N 25 5'-CAGTG TGAGG TTTAG AGAGG TT-3' 63-C 26
5'-AACCT CTCTA AACCT CACAC TG-3' 33-N 11 5'-GGACT AGAGA CGTGT TGCTG
GGGAC-3' 33-C 12 5'-GTCCC CAGCA ACACG TCTCT AGTCC-3'
[0073] Overlap PCR was performed wherein the established vector
pBlue404 (KOREA APPLICATION NO. 97-7512) containing hTPO cDNA was
employed as a template. On the one hand, the oligonucleotide (SEQ
ID NO: 1) encoding hTPO signal peptide and one of oligonucleotides
(N-primer series in Table 1) encoding mutated sequences were
employed as PCR primers. On the other hand, the oligonucleotide
(SEQ ID NO: 2) containing hTPO C-terminal ORF and stop codon and
one of oligonucleotides (C-primer series in Table 1) encoding
mutated sequences were employed as PCR primers.
[0074] The overlap PCR products contain the DNA sequences covering
from N-terminal signal sequence to mutated region and the DNA
sequences covering from mutated region to C-terminal region,
respectively.
[0075] To obtain the full-length hTPO cDNA sequence containing the
target site for amino acid substitution, PCR was done where the two
overlap PCR products were employed as a template and two
oligonucleotides (SEQ ID NO: 1 and NO: 2) were employed as PCR
primers.
[0076] Through aforesaid processes, 1078-bp full-length cDNA
sequences encoding hTPO derivatives were prepared, which contained
a variety of mutated sequences (see FIG. 1).
[0077] In a further embodiment, vectors containing the cDNAs for
hTPO derivatives were constructed in order to obtain the expression
vectors containing the cDNAs and finally to produce the cell lines
transfected with the expression vectors.
[0078] Particularly, the established vector pBlueBac4 and each cDNA
encoding hTPO derivative were digested with BglII and EcoRI
restriction enzymes, respectively. Then the two DNA fragments were
linked with T4 DNA ligase to construct vectors containing the hTPO
derivative cDNA (see FIG. 2).
[0079] The resulting vectors are illustrated by Table 2, which
gives the names of the vectors, the mutated sequences encoding hTPO
derivatives, and the amino acid residues modified in accordance
with the mutation.
[0080] The amino acid sequences of hTPO derivatives of this
invention are represented by a method where they are described with
the amino acid residue substituted and a specified position in the
amino acid sequence of native hTPO (SEQ ID NO: 30). For instance, a
hTPO derivative of this invention, 40430, may be also referred to
as "[Asn.sup.108] hTPO" corresponding to the amino acid sequence
described by SEQ ID NO: 30, except for asparagine substituted for
the amino acid residue 108.
2TABLE 2 The substituted amino acid and nucleotide sequences in the
vectors containing hTPO derivative cDNAs Vectors substituted amino
acid Mutated nucleotide pBlue29 R.sup.117.fwdarw.N.sup.117
AGG.fwdarw.AAC pBlue30 L.sup.108.fwdarw.N.sup.108 CTT.fwdarw.AAT
pBlue31 G.sup.146G.sup.147.fwdarw.G.sup.146N.sup.147
GGAGGG.fwdarw.GGGAAT pBlue32 R.sup.153.fwdarw.N.sup.- 153
AGG.fwdarw.AAC pBlue33 R.sup.164T.sup.165.fwdarw.N-
.sup.164T.sup.165 AGAACC.fwdarw.AACACG pBlue34
T.sup.193G.sup.194.fwdarw.N.sup.193G.sup.194 ACTGGT.fwdarw.AACGGA
pBlue58 p.sup.157.fwdarw.N.sup.157 CCC.fwdarw.AAC
R.sup.164T.sup.165.fwdarw.N.sup.164T.sup.165 AGAACC.fwdarw.AACACG
pBlue59 R.sup.162,R.sup.164.fwdarw.N- .sup.162,S.sup.164 CCC,
AGA.fwdarw.AAC, AGC pBlue60
R.sup.153,A.sup.155.fwdarw.N.sup.153,T.sup.155 AGG, GCC.fwdarw.AAC,
ACC R.sup.164T.sup.165.fwdarw.N.sup.164T.sup.165
AGAACC.fwdarw.AACACG pBlue61 T.sup.159,V.sup.161.fwdarw.N-
.sup.159,S.sup.161 ACA, GTC.fwdarw.AAC, TCC
R.sup.164T.sup.165.fwdarw.N.sup.164T.sup.165 AGAACC.fwdarw.AACACG
pBlue62 S.sup.166,v.sup.168.fwdarw.N.sup.166,T.sup.168 TCT,
GTC.fwdarw.AAT, ACC pBlue63 R.sup.164T.sup.165.fwdarw.N.s-
up.164T.sup.165 AGAACC.fwdarw.AACACG V.sup.168.fwdarw.N.sup.168
GTC.fwdarw.AAC
[0081] In another preferred embodiment, the expression vectors,
which contain the hTPO derivative cDNA sequences, were constructed
in order to be introduced into an animal cells.
[0082] Specifically, pCDT vector was prepared through the insertion
of native hTPO cDNA into the established vector pCDNA3.1. The pCDT
and the vectors containing hTPO derivative genes, such as pBlue29,
pBlue30, pBlue31, pBlue32, pBlue33, pBlue34, pBlue58, pBlue59,
pBlue60, pBlue61, pBlue62 and pBlue63 were digested with NheI and
EcoRI enzymes. Then, these fragments were ligated with T4 DNA
ligase to obtain animal expression vector containing each hTPO
derivative gene (see FIG. 3 and Table 3).
3TABLE 3 The substituted amino acid and nucleotide sequences in
animal expression vectors containing hTPO derivative CDNAs. Ex-
press- ion vector Mutated amino acid Mutated base p40429
R.sup.117.fwdarw.N.sup.117 AGG.fwdarw.AAC p40430
L.sup.108.fwdarw.N.sup.108 CTT.fwdarw.AAT p40431
G.sup.146G.sup.147.fwdarw.G.sup.146N.sup.147 GGAGGG.fwdarw.GGGAAT
p40432 R.sup.153.fwdarw.N.sup.153 AGG.fwdarw.AAC p40433
R.sup.164T.sup.165.fwdarw.N.sup.164T.sup.165 AGAACC.fwdarw.AACACG
p40434 T.sup.193G.sup.194.fwdarw.- N.sup.193G.sup.194
ACTGGT.fwdarw.AACGGA p40435 p40429 +p40431 p40436 p40429 +p40433
p40437 p40430 +p40431 p40438 p40430 +p40433 p40439 p40431 +p40433
p40446 p40429 +p40431 +p40433 p40447 p40430 +p40431 +p40433 p40449
p40429 +p40430 +p40433 p40458 p.sup.157 CCC.fwdarw.AAC
R.sup.164T.sup.165.fwdarw- .N.sup.164T.sup.165 AGAACC.fwdarw.AACACG
p40459 R.sup.162,R.sup.164.fwdarw.N.sup.162,S.sup.164 CCC,
AGA.fwdarw.AAC, AGC p40460
R.sup.153,A.sup.155.fwdarw.N.sup.153,T.sup.155 AGG, GCC.fwdarw.AAC,
ACC R.sup.164T.sup.165.fwdarw.N- .sup.164T.sup.165
AGAACC.fwdarw.AACACG p40461
T.sup.159,V.sup.161.fwdarw.N.sup.159,S.sup.161 ACA, GTC.fwdarw.AAC,
TCC R.sup.164T.sup.165.fwdarw.N.sup.164T.sup.165
AGAACC.fwdarw.AACACG p40462 R.sup.166,V.sup.168.fwdarw-
.N.sup.166,T.sup.168 AGAACC.fwdarw.AACACG p40463
R.sup.164T.sup.165.fwdarw.N.sup.164T.sup.165 AGAACC.fwdarw.AACACG
V.sup.168.fwdarw.N.sup.168 GTC.fwdarw.AAC
[0083] The scope of this invention includes not only DNA sequences
of Table 3 but also other DNA sequences corresponding to the amino
acid sequences of Table 3 , based on the degeneracy of genetic
code. In other words, all of DNA sequences encoding hTPO
derivatives that contain the modified amino acids of Table 3 may be
employed as a mutant hTPO gene.
[0084] For example, a hTPO derivative, which may be prepared from
an expression vector p40433, includes a polypeptide [Asn.sup.164]
hTPO, which may be encoded not only by DNA sequence of SEQ ID NO:
31 but also by degenerate DNA sequences.
[0085] To confirm the insertion of mutated sequences into the
vector, the DNA sequencing of PCR products may be employed.
Alternatively, if the overlap PCR primers are designed to contain a
new restriction site or to delete a wild-type restriction site, the
restriction map of the vector may be used to examine mutagenesis.
If an expression vector p40433, for example, has a mutated sequence
ACACGT in place of wild-type sequence GAACCT, AflIII restriction
site will be created in p40433. Thus, the digestion of p40433 with
AflIII can be used to confirm the introduction of mutated
sequence.
[0086] In a preferred embodiment, hTPO derivatives with two or more
amino acid modifications were produced using said expression
vectors in order to attach additional sugar chains to the modified
amino acid of native hTPO.
[0087] Particularly, two kinds of said expression vectors were
digested with appropriate restriction enzymes, and then the
resulting fragments were subcloned in said pCDT vector to construct
expression vectors which contain the hTPO derivative genes with two
or three regions modified. For example, an expression vector p40429
was digested with NheI and BspMI enzymes to obtain a DNA fragment
involved in the amino acid substitution
Arg.sup.117.fwdarw.Asn.sup.117 In addition, an expression vector
p40431 was digested with BspMI and Bsu36I enzymes to obtain a DNA
fragment involved in the amino acid substitution
Gly.sup.147.fwdarw.Asn.sup.147. The resulting two DNA fragments
were inserted into the BspMI-Bsu36I site of the pCDT vector,
constructing an expression vector p40435 that contained a DNA
sequence encoding hTPO with two amino acid substitutions,
Arg.sup.117.fwdarw.Asn.sup.117 and Gly.sup.147.fwdarw.Asn.sup.147.
In accordance with this procedure, expression vectors such as
p40436, p40437, p40438, p40439, p40446, p40447, p40448, and p40449
were constructed (see Table 3).
[0088] In a further preferred embodiment, animal cell transformants
expressing each hTPO derivative was prepared.
[0089] Particularly, said expression vectors were transfected to
animal cell line CHO/K-1 through the lipofectamin method, preparing
animal cell line expressing each hTPO derivative.
[0090] According to the name of the expression vector introduced,
the transfected lines were designated CHO K-1/p40429, CHO
K-1/p40430, CHO K-1/p40431, CHO K-1/p40432 etc., and CHO K-1/p40433
was deposited in Korean Collection for Type Cultures (; KCTC) on
Jun. 17, 1998 (Accesion NO: KCTC 0495BP).
[0091] In another preferred embodiment, hTPO derivatives were
prepared, by culturing animal cell lines transfected with the
expression vector of this invention.
[0092] Particularly, the transfected lines were subcultured in a
serum-containing medium on large scale, and then transferred to a
secretion medium. Cultured medium was concentrated and dialyzed to
obtain hTPO derivatives.
[0093] A hTPO derivative isolated from CHO K-1/p40433 is
polypeptide [Asn.sup.164] hTPO where asparagine is substituted for
Arginine.sup.164 in native hTPO sequence.
[0094] A hTPO derivative isolated from CHO K-1/p40434 is
polypeptide [Asn.sup.193] hTPO where Asn is substituted for
threonine.sup.193 in native hTPO sequence.
[0095] A hTPO derivative isolated from CHO K-1/p40449 is
polypeptide [Asn.sup.108, Asn.sup.117, Asn.sup.164] hTPO where
asparagine is substituted for leucine.sup.108, arginine.sup.117 and
arginine.sup.164 in native hTPO sequence.
[0096] A hTPO derivative isolated from CHO K-1/p40458 is
polypeptide [Asn.sup.157, Asn.sup.164] hTPO where asparagine is
substituted for proline.sup.157 and arginine.sup.164 in native hTPO
sequence.
[0097] In accordance with the names of expression vectors, the hTPO
derivatives expressed in the animal cells were designated 40429 to
40439, 40446, 40447, 40449, and 40458 to 40463, respectively. Their
in vitro activities were estimated by measuring proliferation of
megakaryocyte leukemia cell line.
[0098] In result, derivatives such as 40429, 40430, 40432, 40433,
40434, 40437, 40438, 40439, and the like showed higher levels of
biological activity than native hTPO did. No significant
relationship between the numbers of additional sugar chains and the
in vitro activities was observed, since activities were increased
or decreased regardless of the number of sugar chains introduced
(see FIG. 4).
[0099] In a preferred embodiment, hTPO derivatives were
administered to mouse and then platelet levels were measured in
order to investigate the in vivo biological activities of the hTPO
derivatives.
[0100] In detail, 8-week-old mice were divided into 4.about.5
groups according to their weight and then a predetermined
concentration of hTPO was subcutaneously administered to mice.
After administration, blood was collected from peripheral vessels
of the mice, and platelet levels in blood were measured. While most
of derivatives were found to show lower platelet levels than native
hTPO did, derivatives 40433, 40434, 40449 and 40458 produced
platelets at similar or higher efficiencies (see FIGS. 6, 7a, or
7b).
[0101] These results suggested that hTPO activity in vivo is
dependent not on the number of introduced sugar chains but on the
specific position of sugar chains. That is, in order to increase
the in vivo activity of hTPO, sugar chains should be introduced
into specific positions in hTPO, such as amino acid 164, amino acid
193, and so on.
[0102] Most notably, platelet levels in 40433-treated group were
higher than in native hTPO-treated group, for 2 days from day 3 or
4 after administration, demonstrating that 40433 can be used as a
therapeutic agent of thrombocytopenia. The maximum platelet levels
in 40433-treated mice were observed on day 5 after administration,
reaching 134% of native hTPO activity on day 5, and more than 180%
in total.
[0103] In another aspect of this invention, in vivo hTPO activities
were investigated in purified hTPO derivatives that had produced
same or higher platelet levels than native hTPO. To do this, dhfr
expression vectors containing the hTPO derivative genes were
constructed, and the resulting vectors were used to prepare cell
lines expressing hTPO genes efficiently.
[0104] Particularly, BamHI linker was connected to the PvuII-SphI
fragment of pSV2-dhfr vector containing dfhr gene. This 1710-bp DNA
fragment containing dhfr gene was inserted into pCDT to prepare
dhfr expression vector pDCT containing native hTPO gene. Then, the
hTPO derivative genes were inserted into pDCT in place of native
hTPO gene, constructing dhfr expression vectors pD40433, pD40434,
pD40449, and pD40458 (see FIG. 8).
[0105] The dhfr expression vectors containing hTPO derivative genes
can be readily amplified in the genome of the transfected
eukaryotic cells by subculturing the cells. In a preferred
embodiment, these vectors were transfected into animal cell line
CHO/dhfr(-). The novel transfected cell lines were designated CHO
dhfr-/pD40433, CHO dhfr-/pD40434, CHO dhfr-/pD40449, and CHO
dhfr-/pD40458, respectively. CHO dhfr-/pD40434, CHO dhfr-/pD40449,
and CHO dhfr-/pD40458 were deposited in Korean Collection for Type
Cultures (; KCTC) on Jun. 8, 1999 (Accession NO: KCTC 0630BP, KCTC
0631BP, KCTC 0632BP, respectiviely). Other dhfr vectors containing
hTPO derivative genes and its transfected cell lines may be
obtained according to the said procedure.
[0106] The transfected cell lines can be cultured on large scale,
and hTPO derivatives can be purified in accordance with the
established methods. Various column chromatography procedures may
be employed to purify hTPO derivatives from cell lines that are
transfected with dhfr expression vectors containing said hTPO
derivative genes. In a preferred embodiment, CM ion-exchange
affinity column, phenyl sepharose column, hydroxylapatite column,
and so on were employed (see FIG. 9).
[0107] To evaluate the in vivo biological activities of the
purified hTPO derivatives, platelet levels were measured according
to said process, after the derivatives were administered to mice.
In 40433-,40434-, 40449- and 40458-treated groups, the platelet
yields reached 177%, 191%, 126% and 179% of native hTPO-treated
group, respectively, for 10 days since the administration (see FIG.
10).
[0108] To confirm the introduction of additional sugar chains into
hTPO derivatives, SDS-PAGE and subsequent Western blot analysis
were performed with the purified native hTPO and hTPO derivatives.
In result, the molecular weights of derivatives 40433 and 40434
were larger than that of native hTPO. The molecular weights of
40458 with two additional sugar chains and 40449 with three ones
were proportionally increased, depending on the number of sugar
chains (see FIG. 11).
[0109] To examine the stability of hTPO derivatives, native hTPO
and a derivative 40433 were treated with thrombin, and then the
protein bands in Western blot were observed in accordance with the
digestion time. In result, 40433 was more stable against digestion
with thrombin than native hTPO (see FIG. 12). Thus, it was
suggested that increased stability due to glycosylation might
contribute to the elevation of in vivo hTPO activity.
[0110] The pharmaceutical composition containing the hTPO
derivatives of this invention may be prepared in a conventional
process, and may be formulated alone or in combination with
pharmaceutically acceptable carriers, forming agents, diluents and
so on. The composition may be used in the formulation of powders,
granules, tablets, capsules, injections, and the like.
[0111] Particularly, it may be employed in combination with water,
phosphate buffer, extroso solution, albumin solution, antioxidants,
dextrin and the like. Preferably, it may be administered
intravenously or subcutaneously.
[0112] The hTPO derivatives may be administered in still less dose
than native hTPO, for example, in a dosage range of about
0.01.about.1000 .mu.g/kg/day.
[0113] The hTPO derivatives of this invention may be used for the
treatment of thrombocytopenia caused by various conditions.
[0114] For instance, it may be useful for the treatment of
thrombocytopenia caused by administration of anticancer agents,
radiotherapy, bone marrow graft, hepatitis, liver cirrhosis etc. To
treat these diseases, the hTPO derivatives may be administered in
combination with anticancer agents such as Adriamycin and
Cisplatin, and hematopoietic cytokines such as IL-3, MCSF, SCF and
EPO.
EXAMPLES
[0115] Practical and presently preferred embodiments of the present
invention are illustrative as shown in the following Examples.
[0116] However, it will be appreciated that those skilled in the
art, on consideration of this disclosure, may make modifications
and improvements within the spirit and scope of the present
invention.
Example 1
The PCR-amplification of cDNAs Encoding hTPO Derivatives
[0117] To induce site-specific mutagenesis in the gene encoding
native hTPO, 12 pairs of oligonucleotides shown in Table 1 were
prepared, which contained the specific nucleotide sequences
corresponding to the mutated amino acid residues.
[0118] The established vector pBlue404 (KOREA PATENT APPLICATION
NO. 97-7512) containing hTPO cDNA was employed as a template on
which hTPO gene would be amplified.
[0119] In detail, PCR was carried out, employing 50 ng of pBlue404
as a template. As primers, oligonucleotide (SEQ ID NO: 1)
containing the hTPO signal sequence and one of antisense
oligonucleotides containing the mutated sequences (N-primers in
Table 1) were used. The PCR reactions were performed in 100 .mu.l
total volume containing 4 .mu.l of the primer solution (40
pmol/.mu.l) and 1 .mu.l of Pfu (; Pyrococcus furiosus) polymerase
(2.5 u/.mu.l; Stratagene, Cat. No. 600153). Thermocycle in the PCR
was as follows: 90 sec at 94.degree. C. for pre-denatuation; 35
amplification cycles comprising 40 sec at 94.degree. C. for
denaturation, 60 sec at 55.degree. C. for annealing and 120 sec at
72.degree. C. for elongation; and 5 min at 72.degree. C. for
post-elongation.
[0120] Another PCR was performed in accordance with above reaction.
As PCR primers, oligonucleotide (SEQ ID NO: 2) containing hTPO
C-terminal ORF and stop codon, and one of sense oligonucleotides
containing the mutated sequences (C-primers in Table 1) were
employed.
[0121] Obtained in the PCR were DNA fragments covering from
N-terminal hTPO signal sequence to mutated sequence, and DNA
fragments from the mutated sequence to hTPO C-terminal.
[0122] The PCR products were brought to 1% agarose gel
electrophoresis, and then the DNA bands of interest were cut with a
razor and eluted in 50 .mu.l of tertiary distilled water with QIAEX
II kit (Qiagen, Cat No. 20021).
[0123] To obtain full-length hTPO cDNAs encoding mutated hTPO, PCR
in 100 .mu.l final volume was performed, where two series of PCR
products (10 ng, respectively) were employed as templates and two
oligonucleotides (SEQ ID NO: 1 and NO: 2) as primers. Thermocycle
in the PCR was as follows: 90 sec at 94.degree. C. for
pre-denatuation; 35 amplification cycles comprising 40 sec at
94.degree. C. for denaturation, 60 sec at 58.degree. C. for
annealing and 120 sec at 72.degree. C. for elongation; and 5 min at
72.degree. C. for post-elongation.
[0124] The PCR products were brought to 1% agarose gel
electrophoresis, and then the 1078-bp DNA bands were eluted in 30
.mu.l of tertiary distilled water in accordance with said
procedure.
[0125] To prepare hTPO genes containing two or more regions of
mutated DNA sequences, four pairs of primers (the primers 58-N and
58-C, 60-N and 60-C, 61-N and 61-C, 63-N and 63-C) were used in
PCR. The full-length cDNAs containing mutated sequence were
prepared in accordance with said procedure, and then again brought
to site-specific mutagenesis procedure where a primer pair 33-N and
33-C was used.
[0126] The modified amino acid and nucleotide sequences in the
resulting cDNAs were shown in Table 2.
Example 2
The Construction of Mammalian Expression Vectors Containing hTPO
Derivative cDNAs and Their Expression in CHO Cells
[0127] (2-1) Construction of Transfer Vectors
[0128] The genes encoding hTPO derivatives, which was prepared in
Example 1, were subcloned in a commercially available vector
pBlueBac4 (Invitrogen, Cat. No. V1995-20), as follows.
[0129] The PCR products corresponding to each hTPO derivative were
digested with BglII and EcoRI enzymes at 37.degree. C. for 3 hours,
and then 1068-bp DNA fragment was isolated from the reaction
mixture by 1% agarose gel electrophoresis. The 4771-bp DNA fragment
was also obtained from pBlueBac4 vector digested with BglII and
EcoRI enzymes.
[0130] To subclone cDNAs encoding hTPO derivatives in the pBlueBac4
vector, two DNA fragments in a molar ratio of cDNA to vector DNA
fragment 4:1 were ligated by incubating them with T4 DNA ligase
(NEB, Cat. No. 202S) at 16.degree. C. for 16 hours. Then, the
ligation mixtures were used to transform E. coli TOP10F' strain
(Invitrogen, Cat. No. C3030-03) with the resulting transfer
vectors. Electroporation method established already was employed to
obtain the E. coli transformants. After these transformants were
cultured in 50 ml of LB medium (10 g Trypton, 5 g Yeast extract, 10
g NaCl in one. liter of water) at 37.degree. C. for 18 hours, the
transfer vectors were obtained from the cultures with Wizard
Midiprep kit (Promega, Cat. No. A7640).
[0131] These transfer vectors containing hTPO derivative genes were
designated pBlue29, pBlue30, pBlue31, pBlue32, pBlue33, pBlue34,
pBlue58, pBlue59, pBlue60, pBlue61, pBlue62, and pBlue63,
respectively (see FIG. 2 ).
[0132] (2-2) Construction of Animal Expression Vectors
[0133] To construct recombinant animal expression vectors
containing hTPO derivative genes, PCDT was employed which was
prepared by inserting wild-type hTPO gene into a commercially
available vector pCDNA3.1 (Invitrogen, Cat. No. 790-20).
[0134] Particularly, 5 .mu.g of pCDT vector was digested with EcoRI
and NheI enzymes at 37.degree. C. for 3 hours, and then 4958-bp DNA
fragment was isolated from the reaction mixture by running on 1%
agarose gel. The transfer vectors of Example 2-1 were digested with
EcoRI and NheI enzymes, and then 1087-bp DNA fragment was also
isolated from each restriction mixture.
[0135] To subclone cDNA fragments encoding various hTPO derivatives
in the pCDT vector, two DNA fragments were mixed to 3:1 molar ratio
and incubated with T4 DNA ligase (NEB, Cat No. 202S) at 16.degree.
C. for 18 hours. Then, the ligation mixtures were employed to
transform E. coli TOP10F' strain (Invitrogen, Cat. No. C3030-03)
with the resulting expression vectors. Electroporation method
established already was employed to obtain the E. coli
transformants (see FIG. 3). After these transformants were cultured
in 50 ml of LB medium at 37.degree. C. for 18 hours, the expression
vectors were obtained from the cultures with Wizard Midiprep kit
(Promega, Cat No. A7640) . The animal expression vectors containing
hTPO derivative genes were designated p40429, p40430, p40431,
p40432, p40433, p40434, p40458, p40459, p40460, p40461, p40462, and
p40463, respectively (see FIG. 3). The isolated plasmid DNA was
digested with NheI, EcoRI, BamHI and Bsu36I enzymes to verify the
insertion of the cDNAs. The mutation in the expression vectors was
confirmed through restriction mapping and sequencing. The
expression vectors were quantified by DNA electorophoresis
according to Sambrook et al. (Sambrook et al., Molecular cloning--A
laboratory manual, 2nd Ed., Cold spring harbor laboratory press,
1987) and used to transfect CHO/K-1 cell line.
[0136] (2-3) Expression of hTPO Derivative Genes in CHO Cells
[0137] The transfection procedure was carried out according to
lipofectamin (Gibco-BRL, Cat. No. 18324012) method. On the day
before transfection, CHO/K-1 cells (ATCC CCL-61) were loaded on
6-well microtiter plates at the density of 2.times.10.sup.5
cells/well. After 24 hours, the cells were once washed with
CHO-S-SFM II medium (Gibco-BRL, Cat. No. 12052-098) and 0.8 ml of
fresh medium was added to the cells. Meanwhile, 12 .mu.g of each
expression vector was added to 600 .mu.l of CHO-S-SFM II medium and
then mixed with 600 .mu.l of CHO-S-SFM II medium containing 36
.mu.l of lipofectamin. After the mixture was incubated at room
temperature for 30 min, 200-.mu.l aliquots of the mixture per one
well were added into the cells in 6-well plates. Then the cells
were incubated at 37.degree. C. for 5 hours in an atmosphere of 5%
CO.sub.2. After the addition of 1 ml of medium containing 10% FBS
(Gibco-BRL, Cat. No. 16000-036) to the cells, they were further
cultured at 37.degree. C. for 24 hours in an atmosphere of 5%
CO.sub.2. The medium in the plates was replaced with Ham F-12
(Gibco-BRL, Cat. No. 11059) containing 10% FBS, and then the cells
were further cultured at 37.degree. C. for 72 hours in an
atmosphere of 5% CO.sub.2 to prepare a culture for transient
expression.
[0138] In addition, after the cells in Ham F-12 medium were
cultured for 48 hours, cells in one well of 6-well plates were
transferred to medium containing 500 .mu.g/ml of zeocin (Gibco-BRL,
Cat. No. R25001) in 100-mm dishes. After the cells were cultured
for 7.about.10 days, zeocin-resistant colonies were identified
through microscope. Cloning cylinder (Bellco, Cat. No. 2090-01010)
was used to isolate more than 12 colonies per one hTPO derivative.
Gene expression levels were determined by ELISA kit for hTPO
(R&D, Cat. No. DTP00), and thereby the cell lines showing the
highest expression levels were selected.
Example 3
The Construction of Mammalian Expression Vectors Containing hTPO
Derivative cDNAs with Two or more Modified Regions, and Their
Expression in CHO Cells
[0139] To produce hTPO derivatives where two or more modified amino
acid regions, mammalian expression vectors of Example 2 were
exploited.
[0140] In order to construct p40435, the expression vector p40429
was digested with NheI and BspMI enzymes to isolate 494-bp DNA
fragment encoding a substituted amino acid (Arg.sup.117 to
Asn.sup.117). Another expression vector p40431 was cut with BspMI
and Bsu36I enzymes to isolate 355-bp DNA fragment encoding a
substituted amino acid (Gly.sup.147 to Asn.sup.147) . Additionally,
animal expression vector pCDT containing hTPO cDNA was digested
with NheI and Bsu36I enzymes. The fragments of p40429 and p40431
were inserted into the fragment of pCDT to construct animal
expression vector p40435, which contains cDNA encoding the hTPO
derivative with two modified regions (Arg.sup.117 to Asn.sup.117
and Gly.sup.147 to Asn.sup.147).
[0141] Another expression vector p40436 is associated with two
amino acid substitutions (Arg .sup.117 to Asn .sup.117 and
Arg.sup.164 to Asn.sup.164) and was prepared by inserting the
494-bp fragment of p40429 and 593-bp BspMI-EcoRI fragment of p40433
into pCDT.
[0142] Expression vectors such as p40437, p40438, and p40439, were
prepared in accordance with the above procedure, where two DNA
fragments encoding substituted amino acids were isolated from the
corresponding vector and inserted into the expression vector pCDT
(see Table 3).
[0143] Other expression vectors such as p40446, p40447, or p40449,
were prepared according to a procedure where three DNA fragments
encoding substituted amino acids were isolated from the
corresponding vector and inserted into PCDT (see Table 3).
[0144] These eight vectors obtained here were transfected into
CHO/K-1 cells in 6-well plates. According to the procedure of
Example 2, cultures for transient expression were prepared, and
zeocin-resistant colonies were isolated, respectively.
Example 4
Estimation of In Vitro Activities of hTPO Derivatives: M-07e Cell
Proliferation Assay
[0145] To prepare hTPO derivatives, the transfected cell lines of
Example 2 and 3 were cultured in Cell Factory (Nunc, Cat. No.
170009) on 10-liter scale. Each transfected cells (5.times.10.sup.4
cells/ml) were transferred into Cell Factory containing Ham F-12
medium supplemented with 10% FBS. Cultured for 72 hours, the cells
were washed once with PBS and then cultured in ExCell medium (JRH,
Cat. No. 14311-10L). After the cells were further cultured at
37.degree. C. for 96 hours in an atmosphere of 5% CO.sub.2,
supernatants were obtained from the culture. The supernatants were
concentrated first with pelicon membrane (Millipore, Cat. No.
42PEL60) and second with minitan membrane (Millipore, Cat. No.
80EL004). After concentration, each sample was brought to dialysis
in 1.times. TNT buffer (10 mM Tris, 0.15 M NaCl, 0.01% Tween 20, pH
7.4) at 4.degree. C. for 30 hours, followed by third concentration
with Ultrafree (Millipore, Cat. No. UFV2BGC10). The samples were
quantified with ELISA kit three times.
[0146] Megakaryocyte leukemia cell line M-07e was maintained in
RPMI1640 medium (Gibco-BRL, Cat. No. 22400-089) supplemented with
GM-CSF (100 u/ml) and 10% FBS.
[0147] To estimate activity, assay medium (RPM1640 supplemented
with 5% FBS) was prepared, and M-07e cells were harvested by
centrifugation, then washed with RPM1640 three times. The cells
were resuspended in the assay medium, adjusted to 8.times.10.sup.4
cells/ml in T-75 flask, and cultured for 24 hours in an atmosphere
of 5% CO.sub.2. Again, the cells were harvested and adjusted to
1.times.10.sup.5 cells/ml. 100 .mu.l aliquots of the cell
suspension were added to 96-well plates. Eight-step concentrations
(100.0.about.0.78125 ng/ml) of standard material (rhTPO, 25 .mu.g)
were prepared by serial dilution with RPMI1640 medium, and CHO
cell-derived native hTPO was employed as control. Total 11 species
of hTPO derivatives (from 40429 to 40439) were prepared at the
concentration of 1.5625, 6.25 and 25 ng/ml. A 100-.mu.l aliquot of
each sample per well was added, adjusting to 200 .mu.l/well. After
incubated for 20 hours in an atmosphere of 5% CO.sub.2, the cells
were fed with 1 .mu.Ci (37 kBq) of .sup.3H-Thymidine and further
incubated for 4 hours. Then, cells were harvested using cell
harvester equipped with a glass fiber filter, which was washed with
PBS seven times.
[0148] The filters in which cells were harvested were put in
counting vials one by one, and .sup.3H-radioactivities emitted from
each sample were measured with a liquid scitilation counter.
Riasmart software was used to calculate the half-maximal
concentration of standards, contol and samples.
[0149] All derivatives showed similar patterns of activities
stimulating M-07e cell proliferation. At the concentration of 25
ng/ml, 8 species of derivatives 40429, 40430, 40432, 40433, 40434,
40437, 40438 and 40439 showed similar or higher activities than
native hTPO did, their activities amounting to 117, 135, 120, 131,
97, 121, 166 and 133% of native hTPO activity (see FIG. 4).
Example 5
In Vivo Activities of hTPO Derivatives Isolated from CHO Fells
[0150] In vivo hTPO assay was carried out where platelet levels
were determined in the mice treated with various hTPO derivatives
of this invention, and FIGS. 6, 7a and 7b give the results. 7-week
female Balb/c mice (Charles River, Japan) were adapted in a
conditioning room (24.+-.1.degree. C., 55% R. H., lighting for 12
hours, from 7:00 a.m. to 7:00 p.m.) for a week. The 8-week mice
were brought to the assay and kept in the domestication room during
the test.
[0151] The mice were randomly divided into groups comprising 5 mice
on the basis of weights. The groups were specified as groups
treated with medium only, treated with native hTPO, treated with
each hTPO derivative of this invention, or not treated,
respectively.
[0152] Various hTPO derivatives (36 .mu.g/kg or 10 .mu.g/kg) were
subcutaneously administered to the mice in single injection, and
the blood samples of mice were collected everyday from day 0 (the
day of injection) to day 10. Samples were collected from abdominal
vena cava within 24 hours after administration. Whole blood in
EDTA-treated tube was set on automatic hemocytometer (Cell dyn
3500, Abbott), by which platelet levels in samples were measured.
The results were presented in `mean.+-.standard error`.
[0153] On day 3, native hTPO stimulated an increase in platelet
level. The platelet level reached a maximum on day 5 and came to
normal level on day 10. All derivatives were found to stimulate an
increase in platelet level, and derivatives 40433, 40434, 40449 and
40458 produced equal or higher platelet levels than native hTPO
did. Especially, 40433 showed approximately 34% higher maximal in
vivo activity of platelet production on day 5 than native hTPO, and
80% or more in total.
Comparative Example 1
In Vivo Activity of Native hTPO
[0154] FIG. 5 shows the platelet level in a mouse that was treated
with native hTPO derived from animal cells. 7-week female Balb/c
mice (Charles River, Japan) were adapted in a conditioning room
(24.+-.1.degree. C., 55% R. H., lighting for 12 hours, from 7:00
a.m. to 7:00 p.m.) for a week. The 8-week mice were brought to the
assay and kept in the domestication room during the test.
[0155] The mice were randomly divided into groups comprising 5 mice
on the basis of weights. The groups were specified as groups
treated with medium only, treated with native hTPO, or not treated,
respectively. Various concentrations (1, 5 and 10 .mu.g/kg) of
native hTPO were subcutaneously administered in single injection,
and the blood samples of mice were harvested on day 4, 8 and 10
(where day 1 is the day of injection). Sample was harvested from
abdominal vena cava within 24 hours after administration. Whole
blood in EDTA-treated tube was set on automatic hemocytometer (Cell
dyn 3500, Abbott), by which platelet levels in samples were
measured. The results were presented in `mean.+-. standard error`.
Native hTPO stimulated an increase in platelet level from day 4.
The platelet level reached a maximum on day 8 and came down to 80%
of the maximal value on day 10.
Example 6
Construction of dhfr Expression Vectors Containing hTPO Derivative
cDNAs, and Selection of Mammalian Cell Lines Expressing them
[0156] (6-1) Construction of dhfr Expression Vectors Containing
hTPO Derivative cDNAs
[0157] According to the result of Example 5, dhfr expression
vectors were constructed, which corresponding to the derivatives
40433, 40434, 40449 and 40458.
[0158] At first, BamHI linker was inserted into pSV-dhfr (ATCC
37146) containing dhfr gene. To prepare BamHI linker, two
oligonucleotides (SEQ ID NO: 27 and NO: 28) were phosphorylated and
then annealed to hybridize with each other. Particularly, T4
polynucleotide kinase (NEB, Cat. No. 201S) was used in the
phosphorylation reaction at 37.degree. C. for 3 hours. In the
annealing reaction, the equimolar oligonucleotides were mixed and
placed at 94.degree. C. for 2 min, then the mixture was stepwisely
cooled down from 65.degree. C. to 37.degree. C. with the
temperature decreased by 0.2.degree. C. per 30 sec. The vector
pSV2-dhfr was restricted with PvuII and SphI enzymes, then the
BamHI linker was connected with the fragment of pSV2-dhfr. The
resulting vector was digested with BamHI enzyme in order to prepare
the 1710-bp fragment containing dhfr gene.
[0159] After the expression vector PCDT containing wild-type hTPO
gene was digested with BglII enzyme, the 1710-bp fragment were
inserted into the pCDT. The resulting dhfr expression vector
expressing native hTPO was designated pDCT (see FIG. 8).
[0160] To dhfr expression vectors corresponding to 5 derivatives,
two oligonucleotides (SEQ ID NO: 29 and NO: 2) were employed as PCR
primers. Except for primers, the PCR was performed under the same
condition as in Example 1. Amplified DNA sequences encoding hTPO
derivatives were cut with KpnI and EcoRI enzymes, and then inserted
into the KpnI-EcoRI site of the pDCT vector. The resulting vectors
were designated pD40433, pD40434, pD40449 and pD40458,
respectively.
[0161] (6-2) Transfection into CHO/dhfr(-) Cell Line and Gene
Amplification
[0162] The dhfr expression vectors of Example 6-1 were transfected
into animal cell line CHO/dhfr(-) (ATCC CRL-9096) according to the
transfection procedure of Example 2. IMDM medium (Gibco-BRL, Cat.
No. 12200-036) was used for the transfection, and IMDM medium
supplemented with 10% dialyzed FBS (Gibco-BRL, Cat. No. 26300-061)
for subsequent culture.
[0163] To select transformed line, the cells were added to 96-well
microtiter plates (1.times.10.sup.3 cells/well) in 48 hours after
transfection, and cultured for 10-14 days in medium containing 500
.mu.g/ml zeocin. Zeocin-resistant colonies were isolated, and the
10-20 cell lines producing higher expression levels were selected
by ELISA quantification.
[0164] The selected cell lines were subcultured in medium
containing 20 nM MTX (Methotrexate, Sigma, Cat. No. M8407) to
amplify hTPO gene. In detail, the cells-were cultured in T-25 flask
until flask was saturated with the cells. One-fifth of the
saturated cells were subcultured, then {fraction (1/10)} and
{fraction (1/15)}, successively. Amplification finished when T-25
flask was saturated with cells in 3-4 days after the {fraction
(1/15)} subculture. Cell lines producing highest expression levels
were selected by ELISA from amplified cell lines in 20 nM MTX. The
cell lines were used to prepare samples for in vivo hTPO assay.
Example 7
Expression of Native hTPO and Derivatives Thereof in CHO/dhfr(-)
Cells, and Their Purification
[0165] To prepare native hTPO and derivatives thereof, the cell
lines of Example 6 were cultured in Cell Factory (Nunc, Cat. No.
170069) on 4-liter scale. Each cell line (5.times.10.sup.4
cells/ml) was transferred into Cell Factory containing IMDM medium
supplemented with 10% FBS. Cultured for 72 hours, the cells were
washed once with PBS and then cultured in DMEM/Ham F-12 medium.
After the cells were further cultured at 37.degree. C. for 96 hours
in an atmosphere of 5% CO.sub.2, supernatants obtained from the
culture were brought to purification steps.
[0166] After XK26/20 column (Amersham-pharmacia, Cat. No.
18-1000-72) was filled with 50 ml -of CM Affi-Gel blue resin
(Bio-Rad, Cat. No. 153-7304), the column was washed with buffer A
(10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4) overnight.
4-liter of the culture supernatants was loaded and-passed through
the column with the flow rate of 5 ml/min, and was monitored by
spectrophotometry at UV wavelength 280 nm. After the whole -culture
supernatant was distributed throughout the column, the column was
washed with buffer B (10 mM sodium phosphate, 2 M urea, pH 7.4)
until the UV absorption dropped to basal level. Bound proteins
including hTPO were eluted with buffer C (10 mM sodium phosphate, 2
M urea, 1 M sodium chloride, pH 7.4), and this fraction was applied
to subsequent phenylsepharose column chromatography. XK26/20 column
was filled with 50 ml of phenylsepharose CL4B resin (Sigma, Cat.
No. P7892) and then washed with buffer C overnight. The fraction
eluted from CM Affi-Gel blue column was applied to the
pheylsepharose column with flow rate of 3 ml/min and monitored by
spectrophotometry at UV wavelength 280 nm. After the whole culture
supernatant was distributed throughout the column, the column was
washed with buffer C until the UV absorption dropped to basal
level. Proteins bound to resin were eluted with buffer B and this
fraction was applied to subsequent hydroxylapatite column
chromatography. XK16/20 column (Amersham-pharmacia, Cat. No.
.18-8773-01) was filled with 10 ml of hydroxylapatite resin
(Bio-Rad, Cat. No. 130-0420) and washed with buffer D (10 mM sodium
phosphate, 2 M urea, pH 6.8) overnight. The fraction eluted from
the pheylsepharose column was adjusted to pH 6.8 with 5 N HCl and
then applied to hydroxylapatite column with flow rate of 3 ml/min.
Since hTPO is not bound to hyroxylapatite resin, the unbound
fraction was reserved. The column was washed with buffer D until
the UV absorption dropped to basal level. Then, impure proteins
bound to resin were eluted with buffer E (0.5 M sodium phosphate, 2
M urea, pH 6.8). The obtained hTPO fraction was concentrated to
10-ml volume using Econo-Pac Q cartridge (Bio-Rad, Cat. No.
732-0021), and then dialyzed in 10 mM sodium phosphate for 24 hours
to eliminate salts and urea. Each fraction in the purification
steps was visualized through SDS-PAGE and silver staining (see FIG.
9), where Silver-stain Plus kit (Bio-Rad, Cat. No. 161-0449) was
used in accordance with the manufacturer's instruction.
[0167] In vivo hTPO assay was performed with the purified hTPO
derivatives (dose: 10 .mu.g/kg) according to the method of Example
5. All derivatives were found not only to stimulate an increase in
platelet level, but also to produce higher platelet levels than
native hTPO did. Particularly, 40433, 40434, 40449, and 40458
showed 77%, 91%, 26%, and 79% higher activities for total 10 days
after administration than native hTPO, respectively (see FIG.
10).
Example 8
Characterization of hTPO Derivatives: Verifying the Introduction of
Sugar Chains and Examining the Stability of hTPO Derivatives
[0168] To investigate whether additional sugar chains were
introduced. into the hTPO derivatives, SDS-PAGE and Western blot
analysis was performed. If sugar chains are introduced, the
molecular weights of hTPO derivatives will be heavier than that of
native hTPO.
[0169] Purified native hTPO and derivatives thereof were loaded
into -wells in 10.about.20% gradient tricine polyacrylamide gel
(Novex, Cat. No. EC66252), which was run at a voltage of 10 V/cm.
After electrophoresis, the proteins fractionated on the gel were
transferred onto a nitrocellulose filter. The filter was incubated
for 1 hour in TBS (pH 7.5) containing 5% non-fat dried milk, and
then further incubated for 18 hours with goat anti-hTPO polyclonal
antibody (R&D system, Cat. No. AB-288-NA) diluted in TBS
(1:1000). The filter was subsequently incubated for 2 hours with a
seconday antibody, alkaline phosphatase-conjugated anti-goat IgG
(Sigma, Cat. No. A4187) diluted in TBS (1:10000). The coloring
substrate BCIP/NBT (Sigma, Cat. No. B5655) was used for detecting
hTPO band. In result, molecular weights of purified hTPO
derivatives were heavier than that of native hTPO, depending on the
number of sugar chains introduced (FIG. 11).
[0170] To evaluate the stability of hTPO derivatives, native hTPO
and a hTPO derivative 40433 were digested with Thrombin, and then
the time-dependent digestion patterns were observed. The hTPO
derivative (50 .mu.g/ml) was treated with Thrombin (5 units/ml,
Sigma, Cat. No. T6759) at 37.degree. C. for 0.5, 1, 2, 3, 4, or 6
hours. Then, SDS-PAGE and Western blot analysis was performed to
observe the digestion patterns. Native hTPO was strikingly degraded
in 30 min after treatment with Thrombin, while the derivative 40433
was digested in 4 hours (see FIG. 12). This result verified that
the derivative 40433 is more stable than native hTPO, which can be
explained from the sugar chain introduced.
INDUSTRIAL APPLICABILITY
[0171] As shown above, the hTPO derivatives of this invention
induce the production of platelet precursor cells in vivo, and thus
are useful for the treatment of thrombocytopenia associated with
anticancer therapies or bone marrow graft. Especially, the hTPO
derivatives 40433, 40434, 40449 and 40458 show significantly higher
efficacy inducing platelet production than native hTPO, providing
various advantages. Since low dose of hTPO derivatives shows
similar efficacy to native hTPO, small dose of hTPO can be
infrequently administered to the patients suffering from
thrombocytopenia. Therefore, use of derviatives of this invention
will reduce the cost of treating the disease and will elevate the
welfare of patients as well as the safety of the drug, with the
inclusion of impure proteins excluded, owing to the small dose
used.
[0172] Those skilled in the art will appreciate that the
conceptions and specific embodiments disclosed in the foregoing
description may be readily utilized as a basis for modifying or
designing other embodiments for carrying-out the same purposes of
the present invention. Those skilled in the art will also
appreciate that such equivalent embodiments do not depart from the
spirit and scope of the invention as set forth in the appended
claims.
* * * * *