U.S. patent application number 10/544986 was filed with the patent office on 2006-11-16 for novel virus vector.
This patent application is currently assigned to Fuso Pharmaceutical Industries, Ltd.. Invention is credited to Takao Hayakawa, Koichi Kawasaki, Mitsuko Maeda, Tadanori Mayumi, Hiroyuki Mizuguchi, Shinsaku Nakagawa, Yasuo Tsutsumi.
Application Number | 20060258005 10/544986 |
Document ID | / |
Family ID | 32866406 |
Filed Date | 2006-11-16 |
United States Patent
Application |
20060258005 |
Kind Code |
A1 |
Mayumi; Tadanori ; et
al. |
November 16, 2006 |
Novel virus vector
Abstract
There is provided a virus vector characterized in that a
water-soluble polymer is directly or indirectly linked to a surface
of a virus particle and a heterogeneous peptide having an affinity
for integrin present on a surface of a target cell is linked to the
water-soluble polymer.
Inventors: |
Mayumi; Tadanori; (Kobe-shi,
JP) ; Nakagawa; Shinsaku; (Yao-shi, JP) ;
Tsutsumi; Yasuo; (Toyono-gun, JP) ; Kawasaki;
Koichi; (Kobe-shi, JP) ; Maeda; Mitsuko;
(Akashi-shi, JP) ; Hayakawa; Takao; (Setagaya-ku,
JP) ; Mizuguchi; Hiroyuki; (Mino-shi, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
Fuso Pharmaceutical Industries,
Ltd.
Osaka-shi
JP
|
Family ID: |
32866406 |
Appl. No.: |
10/544986 |
Filed: |
February 17, 2004 |
PCT Filed: |
February 17, 2004 |
PCT NO: |
PCT/JP04/01739 |
371 Date: |
May 18, 2006 |
Current U.S.
Class: |
435/456 ;
435/235.1 |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 2710/10343 20130101; C12N 15/86 20130101; C12N 2810/405
20130101 |
Class at
Publication: |
435/456 ;
435/235.1 |
International
Class: |
C12N 15/86 20060101
C12N015/86; C12N 7/00 20060101 C12N007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 17, 2003 |
JP |
2003-038780 |
Claims
1. A virus vector, characterized in that a water-soluble polymer is
directly or indirectly linked to a surface of a virus particle and
a heterogeneous peptide having an affinity for integrin present on
a surface of a target cell is linked to the water-soluble
polymer.
2. The virus vector according to claim 1, wherein said
water-soluble polymer is linked to the surface of the virus
particle via a linker amino acid and a linker.
3. The virus vector according to claim 1, wherein said virus is
adenovirus.
4. The virus vector according to claim 1, wherein said
water-soluble polymer is polyethylene glycol or a derivative
thereof.
5. The virus vector according to claim 4, wherein said polyethylene
glycol has a molecular weight of 3000-4000.
6. The virus vector according to claim 2, wherein said linker amino
acid is cysteine and said linker is one having a linking ability to
a thiol group and an amino group.
7. The virus vector according to claim 6, wherein said linker is
N-(6-maleimidocaproyloxy) succinimide (EMCS).
8. The virus vector according to claim 1, wherein said integrin is
.alpha.V .beta.3 or .alpha.V.beta.5.
9. The virus vector according to claim 1, wherein said
heterogeneous peptide has a sequence containing
arginine(R)-glycine(G)-aspartic acid(D).
10. The virus vector according to claim 9, wherein said
heterogeneous peptide has a sequence containing one or more of
.beta.-alanine.
11. The virus vector according to claim 9, wherein said
heterogeneous peptide contains lysine (K) and is branched via said
lysine.
12. The virus vector according to claim 11, wherein said
heterogeneous peptide has an amino acid sequence:
tyrosine(Y)-glycine(G)-glycine(G)-arginine(R)-glycine(G)-aspartic
acid(D)-threonine(T)-proline(P)-.beta.-alanine(X)-lysine(K)-.beta.-alanin-
e(X)-proline(P)-threonine(T)-aspartic
acid(D)-glycine(G)-arginine(R)-glycine(G)-glycine(G)-tyrosine(Y).
13. A method of gene transfer comprising using the virus vector as
defined in claim 1.
14. A method of production of a virus vector comprising steps of:
a) linking a linker amino acid to one terminal of a water-soluble
polymer to obtain a water-soluble polymer-linker amino acid; b)
linking a heterogeneous peptide having an affinity for integrin to
the water-soluble polymer of the water-soluble polymer-linker amino
acid to obtain a heterogeneous peptide-water-soluble polymer-linker
amino acid; c) linking a linker to the linker amino acid of the
obtained heterogeneous peptide-water soluble polymer-linker amino
acid; and d) linking the heterogeneous peptide-water-soluble
polymer-linker amino acid with a virus via the linker.
15. The method of production of a virus vector according to claim
14, wherein the steps a) and b) are conducted while the linker
amino acid is linked to a resin (Resin) and, thereafter, the steps
c) and d) are conducted after the produced heterogeneous
peptide-water soluble polymer-linker amino acid is cut from the
resin.
16. The method of production of a virus vector according to claim
14, wherein said water-soluble polymer is polyethylene glycol or a
derivative thereof.
17. The method of production of a virus vector according to claim
14, wherein said linker amino acid is cysteine and said linker is
one having a linking ability to a thiol group and an amino
group.
18. The virus vector according to claim 2, wherein said virus is
adenovirus.
19. The virus vector according to claim 18, wherein said
water-soluble polymer is polyethylene glycol or a derivative
thereof.
20. The virus vector according to claim 19, wherein said
polyethylene glycol has a molecular weight of 3000-4000.
21. The virus vector according to claim 20, wherein said linker
amino acid is cysteine and said linker is one having a linking
ability to a thiol group and an amino group.
22. The virus vector according to claim 21, wherein said linker is
N-(6-maleimidocaproyloxy) succinimide (EMCS).
23. The virus vector according to claim 22, wherein said integrin
is .alpha.V .beta.3 or .alpha.V.beta.5.
24. The virus vector according to claim 23, wherein said
heterogeneous peptide has a sequence containing
arginine(R)-glycine(G)-aspartic acid(D).
25. The virus vector according to claim 24, wherein said
heterogeneous peptide has a sequence containing one or more of
.beta.-alanine.
26. The virus vector according to claim 25, wherein said
heterogeneous peptide contains lysine (K) and is branched via said
lysine.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a virus vector having a
structure, in which a water-soluble polymer is linked to a surface
of a virus particle and a heterogeneous peptide having an affinity
for integrin is linked to the water-soluble polymer.
BACKGROUND OF THE INVENTION
[0002] At present, an adenovirus vector, an adenovirus-associated
virus vector, a retrovirus vector, a liposome and the like are
known as a vector used for gene transfer. Among them, the
adenovirus vector is frequently used, because it has an advantage
such as 1) a high gene transfer and gene expression efficiency, 2)
a possibility of gene transfer into a stationary phase cell or
other many kinds of cells, 3) an availability of direct gene
transfer into a tissue in vivo, 4) a possibility of gene transfer
of a relatively large heterogeneous gene, 5) facile production of a
high titer vector, and 6) a low possibility of causing cytotoxic
events.
[0003] Furthermore, following steps are known as an infection mode
of adenovirus: a fiber projected from a surface of a virus particle
binds to an adenovirus receptor CAR (coxackie-adenovirus receptor)
present on a surface of a cell to be infected, then a penton base
(having five sequences of arginine(R)-glycine(G)-aspartic acid(D))
present on the surface of the virus particle binds to integrin
(.alpha.V.beta.3, .alpha.V.beta.5) present on the surface of the
cell and, thereby, the virus particle is endocytosed into the cell,
establishing infection of adenovirus (see, T. J. Wickham et al.,
Cell, Vol. 73, pp. 309-319, (1993)).
[0004] However, in the case that adenovirus is used as a vector for
gene transfer, there are problems that 1) an inflammatory reaction
is caused in an individual depending upon a dose, due to its high
immunogenicity, 2) a half-life in blood is short, 3) accumulation
in live is high, leading to a risk of hepatopathy, 4) a gene
transfer efficiency is low for a low CAR-expressing cell (e.g.,
respiratory tract epithelial cells, smooth muscle cells, skeletal
muscle cells, T cells, hematopoietic stem cells, dendritic cells
etc.), and 5) antigenecity is high, leading to easy attack by a
neutralizing antibody or a phagocyte, resulting in a low gene
transfer efficiency.
[0005] Although a method of topically administrating the adenovirus
vector, a genetic-engineering method of deleting an antigenic
portion of adenovirus, a genetic-engineering method of modifying an
adenovirus genome, and the like have been tried in order to solve
above problems, not all problems were solved.
[0006] On the other hand, there have recently been reported an
adenovirus vector in which polyethylene glycol (PEG) is linked to a
surface of a virus particle (hereinafter, referred to as
"PEG-adenovirus vector")(see, JP-A-2001-521381) for prevention of a
lowered gene transfer efficiency due to actions of the neutralizing
antibody and the phagocyte to a virus and for enhancement of
stability in blood; an adenovirus vector in which a peptide motif
having a basic sequence of arginine(R)-glycine(G)-aspartic acid(D)
which is known to bind to integrin present on a surface of a target
cell (hereinafter, referred to as "RGD motif") is integrated in a
knob at a viral fiber tip using a genetic-engineering technique
(hereinafter, referred to as "fiber-modified adenovirus
vector")(see, H. Mizuguchi et al., Gene Ther., Vol. 8, pp. 730-735
(2001)); an adenovirus vector in which a peptide having a
specificity for respiratory tract epithelial cells (sss. 17
peptide, SDQLASPYSHPR) is added to an outermost portion of PEG of
the PEG-adenovirus vector as described above (hereinafter, referred
to as "respiratory tract epithelial cell specific
peptide-PEG-adenovirus vector)(see, H. Romanczuk et al., Human Gene
Therapy, Vol. 10, pp. 2615-2626 (1999)).
[0007] However, in the PEG-adenovirus vector as described above,
the binding between the virus particle and CAR is inhibited by PEG
and, thereby, there arises a problem that the gene transfer
efficiency in a CAR-expressing cell is lowered (see, C. R.
O'riordan et al., Human Gene Therapy, Vol. 10, pp. 1349-1358
(1999)). Moreover, due to such the lowered gene transfer
efficiency, there is also a possibility that the PEG-adenovirus
vector administered to the tissue is not endocytosed into the
target cell, and transferred by a blood stream and accumulated in
liver to cause a hepatopathy.
[0008] Moreover, in the fiber-modified adenovirus vector as
described above, since the RGD motif is merely inserted into the
fiber of virus and, thereby, the virus vector has a similar
antigenecity to that of a normal adenovirus vector, there is
problem that the gene transfer efficiency is lowered due to the
actions of the neutralizing antibody and the phagocyte.
[0009] Furthermore, in the respiratory tract epithelial cell
specific peptide-PEG-adenovirus vector as described above, it can
transfer a gene only into the respiratory tract epithelial cell,
and a substance on a surface of the respiratory tract epithelial
cell which is a target of the sss. 17 peptide has not been
elucidated. Moreover, it is suggested that all twelve amino acid
residues of the sss. 17 peptide are necessary for the binding of
the vector to the respiratory tract epithelial cell in view of
Table 1 and FIG. 2 in H. Romanczuk et al., but, generally, there is
a high possibility that a peptide consisting of as long as twelve
amino acid residues exhibits the immunogenicity for an administered
living body. Accordingly, in vivo administration of the virus
vector comprising the sss.17 peptide is problematic.
[0010] Furthermore, in the respiratory tract epithelial cell
specific peptide-PEG-adenovirus vector as described above, there
are various problems also in its method of production That is, the
vector is produced by adding cysteine having an active SH group to
a terminal of the sss.17 peptide to synthesize an sss.17 peptide
derivative, and, separately, linking divalent hetero-reactive PEG,
which has a group reactive with a lysine residue on a surface of
adenovirus and a group reactive with the active SH group of the
sss. 17 peptide derivative as described above respectively at each
terminal of PEG, to adenovirus (PEG-adenovirus) and, thereafter,
linking the sss. 17 peptide derivative as described above to the
PEG-adenovirus. But, in this vector production, there were problems
that the sss. 17 peptide derivatives having the active SH group are
inter-molecularly cross-linked each other during the reaction and,
thereby, they become impossible to be linked to PEG, that an
original useful ability of adenovirus itself is lowered because the
virus is exposed to a two-stage reaction system, and the like.
[0011] Therefore, development of the virus vector in which these
problems or defects of the previously-used virus vectors are solved
has been desired.
[0012] An object of the present invention is to provide a virus
vector in which each of defects of the previously-used virus
vectors is overcome while advantages thereof as described above are
maintained.
[0013] In particular, an object of the present invention is to
provide a virus vector in which each of defects of the
PEG-adenovirus vector, the fiber-modified adenovirus vector and the
respiratory tract epithelial cell specific peptide-PEG-adenovirus
vector is overcome while the advantages thereof as described above
are maintained. That is, an object of the present invention is to
produce a virus vector while 1) an immunogenicity of the adenovirus
vector is lowered to avoid an inflammatory reaction for an
individual, 2) an antigenecity of the adenovirus vector is lowered
to avoid an attack of a neutralizing antibody and a phagocyte, 3) a
problem of a lowered gene transfer efficiency in the PEG-adenovirus
vector is improved, 4) a stability of the fiber-modified adenovirus
vector in blood is further enhanced, the immunogenicity of the
virus vector is improved, the attack of the neutralizing antibody
and the phagocyte is avoided, 5) a range of a target cell into
which the respiratory tract epithelial cell specific
peptide-PEG-adenovirus vector can transfer a gene is broaden, 6)
production of the virus vector is made easy and efficient, 7) an
advantageous ability of an original adenovirus itself is
maintained, and the like.
SUMMARY OF THE INVENTION
[0014] The inventors have intensively studied in order to solve the
problems as described above and, as the result, found that a virus
vector having a structure, in which a water-soluble polymer is
linked to a surface of a virus particle and a heterogeneous peptide
having an affinity for integrin present on a surface of a cell is
linked to the water-soluble polymer, can solve the problems of a
conventional virus vector for gene transfer and is useful for
maintaining the advantages of the conventional virus vector, which
resulted in completion of the present invention.
[0015] That is, in the first aspect, the present invention
provides;
[0016] 1) a virus vector in which a water-soluble polymer is
directly or indirectly linked to a surface of a virus particle and
a heterogeneous peptide having an affinity for integrin present on
a surface of a target cell is linked to the water-soluble
polymer;
[0017] 2) the virus vector according to 1), wherein said
water-soluble polymer is linked to a surface of the virus particle
via a linker amino acid and a linker;
[0018] 3) the virus vector according to 1) or 2), wherein said
virus is adenovirus;
[0019] 4) the virus vector according to any one of 1) to 3),
wherein said water-soluble polymer is polyethylene glycol or a
derivative thereof;
[0020] 5) the virus vector according to 4), wherein said
polyethylene glycol has a molecular weight of 3000-4000;
[0021] 6) the virus vector according to any one of 2) to 5),
wherein said linker amino acid is cysteine and said linker is one
having a linking ability to a thiol group and an amino group;
[0022] 7) the virus vector according to 6), wherein said linker is
N-(6-maleimidocaproyloxy)succinimide (EMCS);
[0023] 8) the virus vector according to any one of 1) to 7),
wherein said integrin is .alpha.V.beta.3 or .alpha.V.beta.5;
[0024] 9) the virus vector according to any one of 1) to 8),
wherein said heterogeneous peptide has a sequence containing
arginine(R)-glycine(G)-aspartic acid(D);
[0025] 10) the virus vector according to 9), wherein said
heterogeneous peptide has a sequence containing one or more of
.beta.-alanine;
[0026] 11) the virus vector according to 9) or 10), wherein said
heterogeneous peptide contains lysine (K) and is branched via said
lysine;
[0027] 12) the virus vector according to 11), wherein said
heterogeneous peptide has an amino acid sequence:
tyrosine(Y)-glycine(G)-glycine(G)-arginine(R)-glycine(G)-aspartic
acid(D)-threonine(T)-proline(P)-.beta.-alanine(X)-lysine(K)-.beta.-alanin-
e(X)-proline(P)-threonine(T)-aspartic
acid(D)-glycine(G)-arginine(R)-glycine(G)-glycine(G)-tyrosine(Y).
[0028] Also, in the second aspect, the present invention
provides:
[0029] 13) a method of gene transfer comprising using the virus
vector as defined in any one of 1) to 12).
[0030] Furthermore, in the third aspect, the present invention
provides:
[0031] 14) a method of production of a virus vector comprising
steps of:
a) linking a linker amino acid to one terminal of a water-soluble
polymer to obtain a water-soluble polymer-linker amino acid;
b) linking a heterogeneous peptide having an affinity for integrin
to the water-soluble polymer of the water-soluble polymer-linker
amino acid to obtain a heterogeneous peptide-water soluble
polymer-linker amino acid;
c) linking a linker to the linker amino acid of the obtained
heterogeneous peptide-water soluble polymer-linker amino acid;
and
d) linking the heterogeneous peptide-water soluble polymer-linker
amino acid with virus via the linker;
[0032] 15) the method of production of a virus vector according to
claim 14), wherein the steps a) and b) are conducted while the
linker amino acid is linked to a resin (Resin) and, thereafter, the
steps c) and d) are conducted after the produced heterogeneous
peptide-water soluble polymer-linker amino acid is cut from the
resin;
[0033] 16) the method of production of a virus vector according to
14) or 15), wherein said water-soluble polymer is polyethylene
glycol or a derivative thereof; and
[0034] 17) the method of production of a virus vector according to
any one of 14) to 16), wherein said linker amino acid is cysteine
and said linker is one having a linking ability to a thiol group
and an amino group.
[0035] According to the present invention, there is provided a
vector for gene transfer, which has a low immunogenicity for an
administered living body, which has a relatively long half-time in
blood since it is hardly attacked by a neutralizing antibody or a
phagocyte due to its low antigenecity, which can transfer a gene
into a cell in a high efficiency and, thereby, which has a low
accumulative property in liver, which can be produced easily and
efficiently without lowering an advantageous ability of an original
virus, and which is stable even after it has been repeatedly thawed
and frozen after long term storage at low temperature, as well as a
method of gene transfer.
[0036] A phrase used herein "a surface of a virus particle" refers
to all portions of hexon and penton bases which constitute a virus
coat (capsid), and a fiber and a knob projected from the penton
base.
[0037] A term used herein "heterogeneous peptide" refers to a
peptide which is artificially added to the virus vector.
[0038] Moreover, the term used herein "integrin" refers to a
non-covalently linked transmembrane protein having a binding
ability with an extracellular matrix, consisting of an
.alpha.-subunit and a .beta.-subunit. As integrin, there are known
.alpha.4.beta., .alpha.5.beta.1, .alpha.8.beta.1, .alpha.V.beta.1,
.alpha.V.beta.3, .alpha.V.beta.6, .alpha.IIb.beta.3 and the like as
one having an affinity for an extracellular matrix component,
fibronectin, .alpha.1.beta.1, .alpha.2.beta.1, .alpha.3.beta.1,
.alpha.6.beta.1, .alpha.7.beta.1, .alpha.6.beta.4, .alpha.V.beta.8
and the like as one having an affinity for laminin,
.alpha.8.beta.1, .alpha.V.beta.1, .alpha.V.beta.3, .alpha.V.beta.5,
.alpha.V.beta.8, .alpha.IIb.beta.3 and the like as one having an
affinity for vitronectin, .alpha.1.beta.1, .alpha.2.beta.1,
.alpha.V.beta.8 and the like as one having an affinity for
collagen, .alpha.4.beta.1, .alpha.4.beta.7 and the like as one
having an affinity for VCAM-1, .alpha.8.beta.1, .alpha.9.beta.1,
.alpha.V.beta.6 and the like as one having an affinity for
tenascin-C, .alpha.4.beta.1, .alpha.V .beta.3 and the like as one
having an affinity for thrombospondin, .alpha.L.beta.2,
.alpha.M.beta.2, .alpha.D.beta.2 and the like as one having an
affinity for ICAM-1, .alpha.V.beta.3 and the like as one having an
affinity for osteopontin, .alpha.V .beta.3, .alpha.IIb.beta.3,
.alpha.M.beta.2, .alpha.X.beta.2 and the like as one having an
affinity for fibrinogen, .alpha.E.beta.7 and the like as one having
an affinity for E-cadherin, .alpha.V.beta.3, .alpha.IIb.beta.3 and
the like as one having an affinity for a von Willebrand factor, and
all of them are included in integrin of the present invention.
[0039] Furthermore, when an amino acid sequence is described in the
specification, it is represented by a conventional single-letter or
three-letters code.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 is a graph showing a result of a comparative
experiment of a gene expression efficiency of the control
adenovirus vector, the PEG-adenovirus vector, the fiber-modified
adenovirus vector and the RGD-PEG-adenovirus vector, with an A549
human lung adenocarcinoma cell (ATCC: CCL-185, FIG. 1A) and a mouse
melanoma B16BL6 cell (Tohoku University, Institute of Development,
Aging and Cancer: TKG0598, FIG. 1B).
[0041] FIG. 2 is a graph showing a result of a comparative
experiment of a gene expression efficiency of the control
adenovirus vector, the PEG-adenovirus vector, the fiber-modified
adenovirus vector and the RGD-PEG-adenovirus vector under the
presence of a neutralizing antibody, with the A549 human lung
adenocarcinoma cell (FIG. 2A) and the mouse melanoma B16BL6 cell
(FIG. 2B).
[0042] FIG. 3 is a graph showing a gene expression efficiency of
the control adenovirus vector, the PEG-adenovirus vector, the
fiber-modified adenovirus vector and the RGD-PEG-adenovirus vector,
immediately after production, and after they have been repeatedly
thawed and frozen after long term storage at low temperature, with
the A549 human lung adenocarcinoma cell (FIG. 3A) and the mouse
melanoma B16BL6 cell (FIG. 3B).
DETAILED DESCRIPTION OF THE INVENTION
[0043] A virus which can be used in a virus vector of the present
invention, is preferably adenovirus, retrovirus,
adenovirus-associated virus or the like, and is particularly
preferably adenovirus, but it is not limited thereto so long as it
is known to be used in a virus vector for gene transfer. Moreover,
adenovirus which is properly modified by a genetic-engineering
technique may be used in a virus vector for gene transfer. For
example, adenovirus in which an E1 gene region or an E3 gene region
of an adenovirus genome has been deleted, adenovirus in which a
heterogeneous gene has been inserted into the deleted region, and
the like may be used for production of the virus vector.
[0044] A water-soluble polymer which may be used in the virus
vector of the present invention, is preferably polyalkylene glycol
such as polyethylene glycol, polypropylene glycol and the like
having a molecular weight of 30004000, or a polyvinyl copolymer
such as styrene-maleic acid copolymer, polyvinyl pyrrolidone,
polyvinyl alcohol and the like, and is particularly preferably
polyethylene glycol. Moreover, the water-soluble polymer includes a
derivative in which a terminal of the water-soluble polymer is
protected and/or activated. The water-soluble polymer having a
protected terminal is suitable for peptide synthesis, and one
protected with Fmoc (9-fluorenylmethoxycarbonyl), t-Boc
(tert-butoxycarbonyl) or the like is preferably used (for example,
Shearwater Catalog Nos. 1P2Z0F02, 2Z530F02). Moreover, the
water-soluble polymer having an activated terminal includes one
having an active group which links with a part of an amino acid
(amino, carboxyl, thio group and the like).
[0045] A heterogeneous peptide having an affinity for integrin
which may be used in the virus vector of the present invention is
preferably a peptide containing an RGD, LDV or REDV motif present
in fibronectin, an RYVVLPR, LGTIPG PDSGR, YIGSR, LRE, IKVAV,
RNIAEIIKDI or RGD motif present in laminin, an RGD motif present in
vitronectin, an RGD motif present in collagen, an RGD motif present
in thrombin, a GPRP or RGD motif present in fibrinogen, as well as
a peptide containing an EILDV, KQAGDV or DEGA motif which is
reported to have an affinity for integrin.
[0046] Among the motifs having an affinity for integrin as
described above, the peptide containing a fibronectin motif (RGD,
LDV, REDV), a laminin motif (RYVVLPR, LGTIPG, PDSGR, YIGSR, LRE,
IKVAV, RNIAEIIKDI, RGD) or a vitronectin motif (RGD) is
particularly preferable.
[0047] Moreover, a peptide having an affinity for .alpha.4.beta.1,
.alpha.5.beta.1, .alpha.8.beta.1, .alpha.V.beta.1, .alpha.V
.beta.3, .alpha.V.beta.6, .alpha.IIb.beta.3 (integrin having an
affinity for fibronectin), .alpha.1.beta.1, .alpha.2.beta.1,
.alpha.3.beta.1, .alpha.6.beta.1, .alpha.7.beta.1, .alpha.6.beta.4,
.alpha.V.beta.8 (integrin having an affinity for laminin),
.alpha.8.beta.1, .alpha.V.beta.1, .alpha.V.beta.3, .alpha.V.beta.5,
.alpha.V.beta.8, .alpha.IIb.beta.3 (integrin having an affinity for
vitronectin) may be suitably used as the heterogeneous peptide
having an affinity for integrin of the present invention. As the
peptide, one containing a known peptide motif (for example,
.alpha.5.beta.1: RGD, .alpha.2.beta.1: DEGA, .alpha.4.beta.1 or
.alpha.4.beta.7: EILDV, .alpha.6.beta.1: RGD, YIGSR or IKVAV) may
be used, or it may be obtained by searching a peptide having an
affinity for integrin as described above by a phage display
method.
[0048] Moreover, a peptide containing a peptide having an affinity
for .alpha.V .beta.3 or .alpha.V.beta.5 (for example, RGD, LDV,
REDV, GPRP and the like) may be also suitably used as the
heterogeneous peptide having an affinity for integrin of the
present invention, since adenovirus is reported to bind to CAR and,
subsequently, to .alpha.V .beta.3 or .alpha.V.beta.5.
[0049] Moreover, a peptide derivative in which an amino acid or the
like is properly added to either or both of terminals of the amino
acid sequence of the peptide having an affinity for integrin as
described above, may be used in the present invention so long as it
does not exhibit a significant antigenecity.
[0050] The virus vector of the present invention may be produced
from the virus, the water-soluble polymer and the heterogeneous
peptide as described above as an essential component, but the
number of linkage, a linkage portion or linkage manner of each
component between the virus particle, the water-soluble polymer and
the heterogeneous peptide are not particularly limited so long as
an effect of the invention is not deteriorated, and they may be
properly increased, decreased or changed according to a method as
described below or the known per se.
[0051] Briefly, the virus vector of the present invention may be
produced by the steps of: (i) linking a linker amino acid to one
terminal of a water-soluble polymer to obtain a water-soluble
polymer-linker amino acid; (ii) synthesizing a heterogeneous
peptide having an affinity for integrin in a stepwise manner from
its carboxyl terminal, at another terminal of the water-soluble
polymer of the water-soluble polymer-linker amino acid to obtain a
heterogeneous peptide-water-soluble polymer-linker amino acid;
(iii) linking a linker to the linker amino acid of the obtained
heterogeneous peptide-water-soluble polymer-linker amino acid; and
(iv) linking the heterogeneous peptide-water-soluble polymer-linker
amino acid with a virus via the linker. More briefly, the
water-soluble polymer may be polyethylene glycol (PEG), the linker
amino acid may be cysteine, and the linker may be one having a
linking ability to a thiol group and an amino group.
[0052] In a detailed description, a known peptide synthesis method
(for example, a solid phase method and the like) may be used for
synthesis of the heterogeneous peptide having an affinity for
integrin present on a surface of the target cell. In the
heterogeneous peptide of the present invention, in addition to an
amino acid sequence having an affinity for integrin present on a
surface of the target cell, other amino acids may be used as a
spacer. In particular, one to several amino acids as a spacer, such
as .beta.-alanine, are preferably inserted between the amino acid
sequence having an affinity for integrin and PEG in order to expand
a distance therebetween.
[0053] More particularly, the sequence: TABLE-US-00001
RGD-.beta.Ala-PEG, RGD-.beta.Ala-.beta.Ala-PEG,
RGDTP-.beta.Ala-PEG, YGGRGDTP-.beta.Ala-PEG,
or the like are preferable.
[0054] In the case that addition of two or more of the
heterogeneous peptides to the water-soluble polymer is desired, one
or more of amino acids having two amino groups may be contained in
the spacer to make the heterogeneous peptide have a branched
structure. Thereby, a binding ability of the virus vector of the
present invention to integrin present on a surface of the target
cell is enhanced. In particular, as described in the working
example of the present specification, the heterogeneous peptide is
preferably made to have a branched structure by inclusion of lysine
between the amino acid sequence having an affinity for integrin and
PEG Moreover, more preferably, one to several amino acids, such as
.beta.-alanine, are inserted between the amino acid sequence having
an affinity for integrin and lysine in order to expand the distance
therebetween.
[0055] More particularly, the heterogeneous peptide preferably has
a branched structure such as ##STR1## or the like.
[0056] In order to link the water-soluble polymer, to which the
heterogeneous peptide having an affinity for integrin present on a
surface of the target cell is linked, to a surface of the virus
particle, although they may be directly linked, they are preferably
linked by adding at least one amino acid (hereinafter, referred to
as "linker amino acid") which can link with a divalent linker
(preferably, a divalent hetero-reactive linker) to one terminal of
the water-soluble polymer, to link the linker amino acid with the
divalent linker and, thereafter, linking one terminal of the
divalent linker with a surface of the virus particle. That is, the
linker amino acid and the divalent linker are preferably placed
between the water-soluble polymer and a surface of the virus
particle. This method is effective in the case that the
heterogeneous peptide having an affinity for integrin present on a
surface of the target cell contains an acidic amino acid. This is
because although activation of the terminal of the water-soluble
polymer, to which the heterogeneous peptide having an affinity for
integrin present on a surface of the target cell is linked, is
necessary for directly linking it to a surface of the virus
particle, the acidic amino acid is also activated and, thereby, an
original property thereof is changed in the case that the
heterogeneous peptide contains an acidic amino acid. Accordingly,
in the case that the heterogeneous peptide having an affinity for
integrin present on a surface of the target cell contains the
acidic amino acid, the water-soluble polymer to which the
heterogeneous peptide is linked is preferably linked to a surface
of the virus particle with the linker amino acid and the divalent
linker, without activation of the water-soluble polymer.
[0057] As the linker amino acid, an amino acid such as cysteine
having a thiol group, lysine which is a basic amino acid, alanine
which is a neutral amino acid, aspartic acid which is an acidic
amino acid, or other amino acids may be used, but preferably it
contains at least one cysteine.
[0058] An active group must be added to the terminal of the
water-soluble polymer such that only the water-soluble polymer is
linked to the linker amino acid. The active group to be linked with
an amino group of the amino acid includes an N-hydroxysuccinimide
group, a succinimidyl group, a carboxyl group, an aldehyde group, a
benztriazole group, and the like. The active group to be linked
with a carboxyl group of the amino acid includes an amino group,
and the like. The active group to be linked with a thiol group of
the amino acid includes a maleimide group, a vinylsulfone group,
and the like. Among them, a derivative having the active group
which links with the amino group of the amino acid is preferably
used, and a derivative having the N-hydroxysuccinimide group or the
succinimidyl group at the terminal thereof is particularly
preferable.
[0059] If necessary, one to several amino acids may be inserted as
a spacer between the linker and the water-soluble polymer. In
particular, as described in the working example of the present
specification, .beta.-alanine is preferably inserted.
[0060] Next, the linker amino acid as described above, and the
divalent linker which can link to the amino, carboxyl, thiol group
or the like are preferably used in order to link the water soluble
polymer, to which the heterogeneous peptide having an affinity for
integrin present on a surface of the target cell is linked, to a
surface of the virus particle. In particular, because the amino
group present on a surface of the virus particle is preferable as a
target for linking to a surface of the virus particle, at least,
the linker having a linking ability to the amino group must be
used. Moreover, in the case that cysteine is used as the linker
amino acid, at least, the linker having a linking ability to the
thiol group must be used.
[0061] The divalent linker includes those having a linking ability
to the thiol group and the amino group (in general, it has a
maleimide, N-hydroxysuccinimide or succinimidyl group in a
molecule), for example, EMCS (N-(6-maleimidocaproyloxy)
succinimide), GMBS (N-(4-maleimidobutyryloxy) succinimide), MBS
(m-maleimidobenzyl-N-hydroxysuccinimide ester), SATA
(N-succinimidyl S-acetylthioacetate), SMCC (succinimidyl
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate), SMPB
(succinimidyl 4-p-maleimidophenyl butyrate), SPDP (N-succinimidyl
3-(2-pyridylthio)propionate), Sulfo-GMBS
(N-(.gamma.-maleimidobutyloxy) sulfosuccinimide ester),
Sulfo-LC-SPDP (sulfosuccinimidyl 6-(3'-(2-pyridyldithio)
propionamide)hexanoate), Sulfo-MBS
(m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester), Sulfo-SMCC
(sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate),
Sulfo-SMPB (sulfosuccinimidyl 4-(p-maleimidophenyl)-butyrate), and
Sulfo-SBED
(sulfosuccinimidyl(2-6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido)
ethyl-1,3'-dithiopropionate), which are commercially available from
Techno Chemical Corporation, and the like. Moreover,
N-succinimidyl-N-maleimido acetate,
N-succinimidyl4-(N-maleimido)butyrate, N-succimidyl-6-(N-maleimido)
hexanoate, N-succinimidyl-m-(N-maleimido)benzoate,
N-succinimidyl-m-(N-maleimido)benzoate, and the like may be used.
Moreover, the divalent linker includes one having a linking ability
to two amino groups (in general, it has an N-hydroxysuccinimide or
succinimidyl group in a molecule), for example, BS3
(bis(sulfosuccinimidyl) suberate), DMP (dimethyl suberunudate), DMS
(dimethyl suberimidate), DSG (disuccinimidyl glutarate), DSP
(Loman's Reagent), DSS (disuccinimidyl suberate), DTSSP
(3,3'-dithiobis(sulfosuccinimidyl propionate)), EGS (ethyleneglycol
bis(succinimidyl succinate)), and Sulfo-EGS (ethylene glycol
bis(succinimidyl succinate)), which are commercially available from
Techno Chemical Corporation, and the like. Moreover, the divalent
linker includes one having a linking ability to an amino group and
a carboxyl group, for example, EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), which is
commercially available from Techno Chemical Corporation, and the
like. Moreover, the divalent linker includes one having a linking
ability to two thiol groups (in general, it has a maleimide group
in a molecule), for example, BMH (bis-maleimidohexane) commercially
available from Techno Chemical Corporation. Among them, the
divalent linker, which is used for linking the water-soluble
polymer, to which the heterogeneous peptide having an affinity for
integrin present on a surface of the target cell is linked, to a
surface of the virus particle, is preferably one having a linking
ability to a thiol group and an amino group (in general, it has a
maleimide, N-hydroxysuccinimide, or succinimidyl group in a
molecule).
[0062] In a preferable embodiment, at first, a linker amino acid is
linked to a resin (formation of a linker amino acid-resin), then
the water-soluble polymer is linked to the linker amino acid
(formation of a water-soluble polymer-linker amino acid-resin) and,
thereafter, the heterogeneous peptide having an affinity for
integrin present on a surface of the target cell is linked to the
water-soluble polymer (formation of a heterogeneous peptide having
an affinity for integrin-water-soluble polymer-linker amino
acid-resin). Next, the heterogeneous peptide having an affinity for
integrin-water-soluble polymer-linker amino acid is cut from the
resin to obtain the heterogeneous peptide having an affinity for
integrin-water-soluble polymer-linker amino acid. Thereafter, the
divalent linker is linked to the linker amino acid (formation of
the heterogeneous peptide having an affinity for
integrin-water-soluble polymer-linker amino acid-divalent linker).
Finally, this is linked to a surface of the virus particle
(formation of a heterogeneous peptide having an affinity for
integrin-water-soluble polymer-linker amino acid-divalent
linker-virus particle). Such production of the virus vector of the
present invention may be conducted according to the known peptide
synthesis method.
[0063] In a more preferable embodiment, the linker amino acid
contains cysteine, and the divalent linker has a linking ability to
a thiol group and an amino group (in general, it has a maleimide,
N-hydroxysuccinimide or succinimidyl group in a molecule). Although
this method is particularly effective in the case that the
heterogeneous peptide having an affinity for integrin present on a
surface of the target cell contains an acidic amino acid, it is
also applicable even if the heterogeneous peptide contains no
acidic amino acid.
[0064] Furthermore, the heterogeneous peptide sequence to be linked
to the water-soluble polymer preferably has a chain length as short
as possible, in view of lowering an immunogenicity for the living
body as the virus vector as described above. Moreover, one or more
of the heterogeneous peptides may be contained in the virus vector
in view of enhancing an affinity of the virus vector for the target
cell. A chain length and the number of the heterogeneous peptide
may be properly adjusted in view of the effects of the virus
vector. Moreover, one or more kinds of motifs having an affinity
for integrin may be contained in the heterogeneous peptide.
[0065] A measurement of a modification ratio of the water-soluble
polymer in the virus vector of the present invention produced as
described above may be conducted by measuring the number of amino
groups remaining on the water-soluble polymer-adenovirus vector
according to a fluorescamine method (A. Croyle Maria et al., Human
Gene Therapy, Vol. 11, pp. 1713-1722, (2000)). According to this
measurement, those skilled in the art can properly determine an
optimal modification ratio of the water-soluble polymer in view of
the gene transfer efficiency, and can apply it to the present
invention. More particularly, to a 0.42 mg/mL solution of
fluorescamine in dioxane (trade name Fluram, manufactured by
Fluka), a three-fold volume of 5.times.10.sup.11 particles/mL of
the water-soluble polymer-adenovirus vector is added, and the
mixture is vigorously stirred. After it is incubated at room
temperature for 10 minutes, fluorescence (excitation wavelength Ex:
392 nm, emission wavelength Em: 480 nm) is measured. A standard
curve is produced with a non-modified adenovirus vector, and the
modification ratio of the water-soluble polymer of the
water-soluble polymer-adenovirus vector is calculated.
[0066] A measurement of a particle diameter of the water-soluble
polymer-adenovirus vector may be conducted with ZETASIZER 3000HS
(manufactured by Malvem Instruments). Similar to the modification
ratio as described above, those skilled in the art can properly
determine an optimal particle diameter of the virus vector in view
of the gene transfer efficiency, can select the water-soluble
polymer having an optimal molecular weight, and can apply it to the
present invention. Moreover, the present inventors have confirmed
that the modification ratio of the water-soluble polymer of the
water-soluble polymer-adenovirus vector and an average particle
diameter measured with ZETASIZER are increased correlatively with
an amount of the water-soluble polymer added and the number of
addition times. Accordingly, modification of the water-soluble
polymer of the adenovirus vector can be controlled by an amount of
the water-soluble polymer to be added and the number of addition
times.
[0067] A measurement of the number of the virus particles may be
conducted according to a method of Maizel et al. (J. V Jr. Maizel
et al., Virology, Vol. 36, pp. 115-125, (1968)), and those skilled
in the art can properly determine the number of the viruses to be
used upon gene transfer. That is, a suitable amount of a purified
virus solution is collected and dissolved in 1% SDS/PBS(-), and an
absorbance at OD 260 nm thereof is measured with a spectrophotomer.
The number of the virus particles is calculated in terms of
1.1.times.10.sup.12 particles/OD.sub.260.
[0068] An individual, which is a subject in the case that the virus
vector of the present invention is used for a therapeutic purpose,
includes, for example, human, mouse, rat, hamster, guinea pig and
the like. Moreover, an administration site, which is a subject of
the virus vector of the present invention, includes brain, liver,
kidney, spleen, prostate, small intestine, large intestine, lung,
bronchial tube, skin, esophagus, stomach, duodenum, skeletal muscle
and the like. Moreover, a cell, which is a subject of the virus
vector of the present invention, includes cells derived from a
living body (epithelial cells, muscle cells, cerebral nerve cells
and the like), cancer cells, cultured cells and the like. Moreover,
a cell, which is a subject in the case that the virus vector of the
present invention is used in vitro, includes A594 cells, B16BL6
cells, HepG2 cells, COS1 cells, CHO cells and the like. Moreover, a
cell, which is a subject in the case that the virus vector of the
present invention is used ex vivo, includes T cells, B cells,
hematopoietic stem cells, embryonic stem cells and the like.
[0069] Although integrin is expressed or exhibits a physiological
function at various sites, at least, the following integrin appears
to be expressed or to exhibit a physiological function at following
sites: .alpha.1.beta.1 at axons and lymphocytes, .alpha.2.beta.1 at
platelets and cancer cells, .alpha.3.beta.1 at kidney, lung and
cancer cells, .alpha.4.beta.1 at lymphocytes, monocytes and
acidophiles, .alpha.5.beta.1 at various cells, .alpha.6.beta.1 at
epithelial cells, axons and cancer cells, .alpha.7.beta.1 at
skeletal muscle, .alpha.8.beta.1 at kidney and nerve cells,
.alpha.9.beta.1 at tracheal epithelium, .alpha.V.beta.1 at various
cells and cancer cells, .alpha.V .beta.3 at blood vessel and bone,
.alpha.V.beta.5 at blood vessel and epithelium, .alpha.V.beta.6 at
epithelium, .alpha.V.beta.8 at axons, .alpha.4.beta.7 at
lymphocytes, .alpha.6.beta.4 at epithelial cells, .alpha.L.beta.2
at leukocytes, .alpha.M.beta.2 at neutrophils and monocytes,
.alpha.X.beta.2 at monocytes and granulocytes, .alpha.D.beta.2 at
foam cells, .alpha.IIb.beta.3 at platelets, .alpha.E.beta.7 at
lymphocytes, but is not limited thereto. In general,
.alpha.9.beta.1 is expressed at tracheal epithelium, and a .beta.2
chain is expressed on a surface of leukocytes. .alpha.L.beta.2 is
expressed on LFA-1 (lymphocyte function-associated protein), and
.alpha.M.beta.2 is expressed on Mac-1 (surface protein of
macrophage). Moreover, a .beta.3 chain is expressed on various
cells including platelets.
[0070] The heterogeneous peptide having an affinity for integrin
which is expressed site-specifically or in the cells as described
above is obtained (for example, it is obtained by screening
according to a phage display method or the like), and is
transferred specifically into cells or sites expressing integrin as
a target by applying the resultant heterogeneous peptide to the
virus vector of the present invention.
[0071] In the case that the virus vector of the present invention
is used in vivo, an administration route thereof may be property
selected from topical, intravenous, mucosal, intramuscular, and
oral administration to tissues or organs, or the like.
[0072] Furthermore, a therapeutic gene may be integrated in the
virus vector of the present invention, such as a p53 gene (for
inducing apoptosis in cancer cells), a thymidine kinase gene (for
inducing apoptosis in cancer cells), an adenosine deaminase (ADA)
gene (for adenosine deaminase deficiency), and the like.
[0073] In order to isolate the RGD-PEG-adenovirus vector of the
present invention from a mixture of adenovirus, RGD-PEG and the
RGD-PEG-adenovirus vector, for example, centrifugation with a CsCl
gradient may be used. Moreover, it may be isolated by dialysis, or
by a combination of ultracentrifugation with the CsCl gradient and
dialysis. More particularly, a Spectrum/Pro CE (cellulose ester)
Sterile Dispo Dialyzer (manufactured by SPCTRUM, molecular weight
cutoff (MWCO); 300,000) may be used for isolation of the
RGD-PEG-adenovirus vector. Furthermore, a validity of dialysis may
be confirmed by examining whether the FITC-dextran which has a
greater molecular weight than that of PEG or RGD-PEG can pass
through a dialysis membrane.
EXAMPLE
Example 1
Comparison of Gene Transfer Efficiency of Various Adenovirus
Vectors
[0074] In order to examine a gene transfer efficiency of the
adenovirus vector of the present invention, various adenovirus
vectors were produced and compared. That is, as illustrated in the
following steps 1)-4), i) an adenovirus vector as a control, ii) a
PEG-adenovirus vector, iii) a fiber-modified adenovirus vector, and
iv) an adenovirus vector of the present invention were
produced.
[0075] 1) Production of Control Adenovirus Vector
[0076] A virus vector produced by Mizuguchi et al. was used as a
control adenovirus vector. In the control adenovirus vector, E1 and
E3 regions of adenovirus have been deleted, and a luciferase gene
has been integrated in a deleted E1 region.
[0077] In order to propagate, isolate and purify the control
adenovirus vector, at first, the virus was added together with a
Dulbecco's Modified Eagle's medium (DMEM, manufactured by Sigma)
with 5% fetal bovine serum to infect 293 cells. After about 2 to 3
days, the 293 cells on which CPE (cytopathic effect) was confirmed
were collected together with a culture supernatant, and were
centrifuged at 3000 rpm for 5 minutes. Then, obtained cells were
suspended in a small amount of a culture medium, and broken by
repeatedly freezing and thawing the culture four-times to liberate
the virus into a solution. Thereafter, the solution was centrifuged
at 3000 rpm for 5 minutes, and a supernatant was collected as a
crude virus lysate (CVL). A CsCl (specific gravity 1.25/TD solution
[750 mM NaCl, 50 mM KCl, 250 mM Tris, 10 mM Na.sub.2HPO.sub.4,
pH=7.4] was poured into an SW 41 tube, and CsCl (specific gravity
1.40/TD solution) was laid under it to prepare a gradient. Then,
the collected crude virus lysate was superposed on the gradient,
and centrifuged at 35000 rpm for one hour at 18.degree. C. with an
SW 41 rotor (manufactured by Beckman)(primary centrifugation).
Thereafter, a lower white band generated in the tube was collected
(primary purification). Thereafter, CsCl (specific gravity 1.34/TD
solution) was poured into the SW 41 tube, the virus solution
obtained in the primary purification was superposed on CsCl, and it
was centrifuged with the SW 41 rotor at 35000 rpm for 18 hours at
18.degree. C. as described above (secondary centrifugation).
Thereafter, the lower white band generated in the tube by the
secondary centrifugation was collected (secondary purification).
The virus solution obtained was placed into a dialysis tube, and
the tube was placed into a phosphate buffered saline (PBS)(-) to
subject to dialysis at 4.degree. C. with stirring. A dialysis
solution was exchanged 3 times every 1 hour and, finally, dialysis
was performed with PBS(-) with 10% glycerol for longer than 2
hours. The dialyzed virus solution was stored at 80.degree. C.
until an initiation of experiment, and it was used as a control
adenovirus vector. Above procedures were performed aseptically.
[0078] 2) Production of PEG-Adenovirus Vector
[0079] For production of the PEG-adenovirus vector, the control
adenovirus vector produced in the above step 1) and, thereupon,
methoxy polyethylene glycol-succinimidyl propionate: (mPEG-SPA,
molecular weight 5,000, manufactured by Shearwater, Catalog No.:
2M4M0D01) was used.
[0080] That is, an amount of the mPEG-SPA which corresponds to
100-fold moles relative to a primary amine present on a coat
protein (hexon, penton base, fiber) of one particle adenovirus
vector was added to 1.times.10.sup.12 particles/ml of the control
adenovirus vector, and they were reacted at 37.degree. C. for 15
minutes with stirring at 300 rpm to link the adenovirus vector to
the mPEG-SPA, which was further reacted for 30 minutes under the
same condition to complete the reaction. Consequently, an
adenovirus vector with PEG linked thereto (PEG-adenovirus vector)
was obtained.
[0081] 3) Production of Fiber-Modified Adenovirus Vector
[0082] As a fiber-modified adenovirus vector, in which a part of an
amino acid sequence present in a knob of an adenovirus fiber is
modified to a peptide, arginine(R)-glycine(G)-aspartic acid(D), by
a genetic-engineering method, a virus vector produced by Mizuguchi
et al., was used (see, H. Mizuguchi et al., Gene Ther., Vol. 8, pp.
730-735 (2001)). In the fiber-modified adenovirus vector, the E1
and E3 regions of the adenovirus vector have been deleted, and the
luciferase gene has been integrated in the deleted E1 region. The
fiber-modified adenovirus vector has a characteristic that it can
also bind to a non CAR-expressing cell via integrin, and a gene can
be efficiently transferred thereinto. Furthermore, propagation,
isolation and purification of the fiber-modified adenovirus vector
were conducted as described in the above step 1) (see, H. Mizuguchi
et al., Gene Ther., Vol. 8, pp. 730-735 (2001)).
[0083] A vector plasmid pAdHM15 having Csp451 and Cla I restriction
sites at a gene sequence portion encoding an HI loop of a fiber
portion, was cut with both restriction enzymes, and then a
synthetic oligo DNA corresponding to the sequence,
arginine(R)-glycine(G)-aspartic acid(D), was introduced therein by
in vitro ligation. Thereafter, the luciferase gene was inserted
into the deleted E1 region. The resulting plasmid was cut with Pac
I and transfected into the 293 cells to obtain a
luciferase-expressing adenovirus vector having an
arginine(R)-glycine(G)-aspartic acid(D) sequence in the fiber
portion.
[0084] 4) Production of Adenovirus Vector (RGD-PEG-Adenovirus
Vector)
[0085] In one embodiment of the present invention, a method of
production of the adenovirus vector will be illustrated with
referring to a following reaction chart.
[0086] 4-1) Synthesis of Fmoc-K(Fmoc)-PEG-.beta.AC(Trt)-Amide Resin
(compound d)
[0087] In order to synthesize
(Ac-YGGRGDTP.beta.A).sub.2K-PEG-.beta.AC-amide (compound f)
containing three amino acids, arginine(R)-glycine(G)-aspartic
acid(D), having an affinity for integrin, at first,
Fmoc-K(Fmoc)-PEG-.beta.AC(Trt)-Amide Resin (see, Reaction Chart as
described below, compound d) was synthesized. In this synthesis,
cysteine (Cys) to be used as the linker amino acid is important for
linking with a maleimide entity which is a divalent hetero-reactive
linker specifically linking to a SH group. Moreover, .beta.-alanine
(.beta.Ala) present between the linker amino acid and PEG was used
as a spacer for facilitating the reaction. Lysine (Lys) having two
amino groups was introduced for linking two RGD sequences per one
PEG molecule, for the purpose of enhancing an affinity of the virus
vector for integrin.
[0088] The synthesis was conducted with Fmoc as a protecting group
according to a solid phase method. That is, 1.5 g of Fmoc-Amide
Resin (functional group content 0.66 mmol/g)(corresponding to 1.0
mmol)(manufactured by Applied Biosystems) was weighed and placed
into a propylene reaction vessel (manufactured by Kokusan Chemical
Co., Ltd.). The reaction vessel was set on a shaker (manufactured
by IKA Co., Ltd., VIBRAX VXR), and dichloromethane (DCM) was added
thereto to swell the resin. The Fmoc group was removed with 20%
piperidine/DMF (N,N-dimethylformamide), and washed with DMF. A
carboxylic acid of Fmoc-Cys(Trt)-OH was activated with 1 mol/L
DIPC/DMF (DIPC=diisopropylcarbodiimide) and 1 mol/L HOBt/DMF
(HOBt=N-hydroxybenzotriazole) and, thereafter, this was added to
the resin to condense them (compound a). Thereafter, a condensation
reaction was proceeded by repeating the procedure of removal of the
Fmoc protecting group on a solid phase (deprotection), that is,
freeing of an amino group DMF washing.fwdarw.a reaction of a
Fmoc-amino acid derivative and a freed amino group by an active
ester of HOBt with a condensation reagent suitable for each step
(coupling).fwdarw.DMF washing. Similarly, removal of the Fmoc group
(deprotection) with piperidine and condensation with
Fmoc-.beta.Ala-OH (compound b) were conducted, followed by
deprotection to obtain H-.beta.Ala-Cys(Trt)-Amide Resin. Then, a
reaction with the Fmoc-PEG-NHS (Fmoc-PEG-NHS, manufactured by
Shearwater, Catalog No. 1P2Z0F02, molecular weight 3400) was
conducted with 0.45 mol/L HBTU/HOBt/DMF
(HBTU=2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate) as a condensation reagent under the presence
of DIEA (diisopropylethylamine), since the reaction progresses very
slowly only with this labile Fmoc-PEG-NHS (compound c). After
deprotection with piperidine, the reaction with Fmoc-Lys(Fmoc)-OH
was conducted with 1 mol/L DIPC/DMF and 1 mol/L HOBt/DMF as
described above to obtain
Fmoc-Lys(Fmoc)-PEG-.beta.Ala-Cys(Trt)-Amide Resin (compound d
(PEG-Resin)).
[0089] 4-2) Synthesis of
[Ac-Y(Bu.sup.t)GGR(Pmc)GD(OBu.sup.t)T(Bu.sup.t)P.beta.A].sub.2K-PEG-.beta-
.AC(Trt)-Amide Resin (compound e)
[0090] Next, a peptide elongation of
Fmoc-Lys(Fmoc)-PEG-.beta.Ala-Cys(Trt)-Amide Resin (compound d)
produced in the step 4-1) of Example 1 was conducted by repeating
deprotection and condensation on a peptide synthesizer (Model ABI
433A, synthesis program: FastMoc0.25.OMEGA. MonPrevPk) using
sequentially Fmoc-.beta.Ala-OH, Fmoc-Pro-OH, Fmoc-Thr(Bu.sup.t)-OH,
Fmoc-Asp(OBu.sup.t)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pmc)-OH, and
Fmoc-Tyr(Bu.sup.t)-OH. In this synthesis, .beta.Ala between RGD and
PEG was used as a spacer for facilitating the reaction.
[0091] Consequently,
(Fmoc-Y(Bu.sup.t)GGR(Pmc)GD(OBu.sup.t)T(Bu.sup.t)P.beta.A).sub.2K-PEG-.be-
ta.AC(Trt)-Amide Resin was synthesized. Then, a free N-terminal
amino group of the heterogeneous peptide containing the RGD
sequence was blocked by acetylation, such that EMCS described in
the following step 4-4) of Example 1 reacts only with a primary
amine present on a surface of the adenovirus particle. That is, the
free amino group was acetylated by the reaction with acetic
anhydride under the presence of DIEA after deprotection to obtain
[Ac-Y(Bu.sup.t)GGR(Pmc)GD(OBu.sup.t)T(Bu.sup.t)P.beta.A].sub.2
K-PEG-.beta.AC(Trt)-Amide Resin (compound e (RGD-PEG-Resin)).
[0092] 4-3) Isolation and Purification of
[Ac-Y(Bu.sup.t)GGR(Pmc)GD(OBu.sup.t)T(Bu.sup.t)P.beta.A].sub.2K-PEG-.beta-
.AC(Trt)-Amide (compound f (RGD-PEG))
[0093] A sequence
[Ac-Y(Bu.sup.t)GGR(Pmc)GD(OBu.sup.t)T(Bu.sup.t)P.beta.A].sub.2K-PEG-.beta-
.AC(Trt)-Amide (compound f (RGD-PEG)) in
[Ac-Y(Bu.sup.t)GGR(Pmc)GD(OBu.sup.t)T(Bu.sup.t)P.beta.A].sub.2K-PEG-.beta-
.AC(Trt)-Amide Resin (compound e (RGD-PEG-Resin)) obtained in the
step 4-2) of Example 1 was isolated from Resin. That is, a crude
compound f (compound f (RGD-PEG)) was obtained by cutting RGD-PEG
from Resin by a treatment with a cocktail consisting of 90:5:5 of
trifluoroacetic acid: TIPS (triisopropyl silane): water, and
distilling off a solven, followed by freeze-drying. Then, in order
to conduct purification of the crude compound f, 20 mg of this
sample was dissolved in 1 mL of 10% acetonitril/ultra-purified
water, the solution was centrifuged, and a supernatant was
subjected to HPLC (preparative column: DAISOPAK SP-120-5-ODS-B,
20.times.250 mm, flow rate: 10 mL/min., mobile phase A: 0.01%
trifluoroacetic acid/acetonitrile, mobile phase B: 0.01%
trifluoroacetic acid/ultra-purified water, gradient: linear
gradient from A/B=1/9 to A/B=7/3 over 60 minutes). Fractions at a
retention time of 45 to 60 minutes were collected and combined
together, and the solvent was distilled off on an evaporator. A
residue was freeze-dried to obtain a pure compound f (compound f
(RGD-PEG)).
[0094] 4-4) Cross-linking of Compound f and EMCS
(N-(6-maleimidocaproyloxy) succinimide)
[0095] In order to link the pure compound f (RGD-PEG) obtained in
the step 4-3) of Example 1 to a surface of the virus particle, the
pure compound f (RGD-PEG) was modified with EMCS, a divalent
hetero-reactive reagent which can cross-link with an amino group
and an SH group. Here, it is known that EMCS has a maleimide group
and an N-hydroxysuccinimide active ester at its both terminals, and
that the active ester reacts with an amino group and the maleimide
group selectively reacts with an SH group.
[0096] At first, the pure compound f (RGD-PEG) was dissolved in a
10 mmol/L sodium phosphate buffer (pH 6.0), and an EMCS solution in
dimethyl sulfoxide (DMSO) was added dropwise thereto. After the
reaction at room temperature for 30 minutes, a 10 mmol/L sodium
phosphate buffer (pH 7.4) was added to the solution, and it was
frozen and stored until next use. Thus, the compound g (RDG-PEG)
modified with EMCS was obtained.
[0097] 4-5) Linking of RDG-PEG and Adenovirus Vector
[0098] The compound g (RDG-PEG) obtained in the step 4-4) of
Example 1 was linked to the adenovirus vector produced in the step
1) of Example 1. That is, an amount of the compound g (RDG-PEG)
which corresponds to 250-fold moles relative to the primary amine
present on the coat protein (hexon, penton base, fiber) of the
adenovirus vector was added to 1.times.10.sup.12 particles/ml of
the adenovirus vector, and they were reacted at 37.degree. C. for
15 minutes with stirring at 300 rpm to link the adenovirus vector
to RDG-PEG, which was further reacted for 30 minutes under the same
condition to complete the reaction. Consequently, an adenovirus
vector to which the compound g is linked (RGD-PEG-adenovirus
vector) was obtained. ##STR2##
[0099] 5) Comparative Experiment of Gene Expression Efficiency
[0100] A comparative experiment of gene expression efficiencies of
the control adenovirus vector, the PEG-adenovirus vector, the
fiber-modified adenovirus vector and the RGD-PEG-adenovirus vector,
which were produced in the steps 1) to 4) of Example 1 as described
above, was performed.
[0101] The experiment was performed with an A549 human lung
adenocarcinoma cell (ATCC: CCL-185, FIG. 1A) which is a high
CAR-expressing cell and a mouse melanoma B16BL6 cell (Tohoku
University, Institute of Development, Aging and Cancer TKG0598,
FIG. 1B) which is a low CAR-expressing cell. The A549 cell was
subcultured to subconfluent in DMEM with 10% fetal bovine serum,
which was used in the experiment. Moreover, the B16BL6 cell was
subcultured to subconfluent in an Eagle's minimum essential medium
(MEM, manufactured by Sigma) with 7.5% fetal bovine serum, which
was used in the experiment.
[0102] The A549 cell and the B16BL6 cell were seeded onto a 48-well
plate at 2.times.10.sup.4 cells/0.5 mL/well and cultured for 24
hours. Each vector prepared in the medium of each cell was added to
the well at 300, 1000, 3000, or 10000 particles/cell/0.5 mL, and
cultured at 37.degree. C. for 24 hours under saturated vapor
pressure (5% CO.sub.2).
[0103] Moreover, a measurement of a luciferase activity as an
indicator of a gene expression efficiency of the virus vector was
conducted using Luciferase Assay System (manufactured by Promega)
and Microlumat Plus LB96 (manufactured by Perkin Elmer) after the
cells were digested in 100 .mu.L of Luciferase Cell Culture Lysis
Reagent (manufactured by Promega). An activity was expressed as a
Luciferase activity (RLU (relative light unit)/well).
[0104] The results thereof are shown in FIG. 1 as a change in the
luciferase activity when the number of the virus particles per each
cell was changed. The RGD-PEG-adenovirus vector shows several
hundred times higher gene expression in the A549 cell, the high
CAR-expressing cell, than that of the PEG-adenovirus vector, and
shows a similar degree of gene expression to that of the control
adenovirus vector. Moreover, the RGD-PEG-adenovirus vector shows
hundred times or more higher gene expression than that of the
control adenovirus vector in the B16BL6 cell, the low
CAR-expressing cell, in which a gene transfer efficiency of the
control adenovirus vector is low. Furthermore, the
RGD-PEG-adenovirus vector shows a similar degree of gene expression
to that of the fiber-modified adenovirus vector, which has enabled
the gene to be highly expressed even in the low CAR-expressing cell
by inserting the RGD sequence into the fiber projected from a
surface of the virus particle.
[0105] From above matters, it was found that the virus vector
adsorbs onto the target cell to efficiently transfer the gene and
the transferred gene can be efficiently expressed in the target
cell, by adding the heterogeneous peptide having an affinity for
integrin including RGD to PEG of the PEG-adenovirus vector.
[0106] It is believed that the fiber-modified adenovirus vector has
approximately the same virus particle diameter and mass as those of
the control adenovirus vector, since RGD has been integrated into
its fiber. On the other hand, it may be said that the
PEG-adenovirus vector has significantly greater virus particle
diameter and mass as compared with those of the fiber-modified and
control adenovirus vectors. Also, it may be said that the
RGD-PEG-adenovirus vector of the present invention has further
greater virus particle diameter and mass as compared with those of
the PEG-adenovirus vector which contains PEG having the molecule
weight of 2000, to such an extent that the RGD-PEG-adenovirus
vector of the present invention contains PEG of the molecular
weight of 3400 and the heterogeneous peptide having an affinity for
integrin, RGD, has been integrated therein. In general, in the
virus vector having a large structure like the RGD-PEG-adenovirus
vector of the present invention, it is expected that a binding
ability of the virus vector to integrin becomes weak even if it
contains a peptide having an affinity for integrin in the structure
and, thereby, expected that a gene transfer efficiency is lowered
than that of the fiber-modified adenovirus vector. However, in view
of the result of this working example, it is believed that the
RGD-PEG-adenovirus vector having such the large structure maintains
a high affinity for integrin, because it can efficiently transfer a
gene as effectively as the fiber-modified adenovirus vector. It is
a completely unexpected result that a similar gene transfer
efficiency to that of the fiber-modified adenovirus vector is shown
by addition of only three amino acids,
arginine(R)-glycine(G)-aspartic acid(D), to the PEG-adenovirus
vector.
[0107] From this result, it is demonstrated that even a large
structured virus vector to which many water-soluble polymers have
been linked can efficiently transfer a gene by linking the peptide
having an affinity for integrin to the water-soluble polymer.
[0108] Moreover, since it is known that adenovirus binds to CAR
present on a cell surface and, thereafter, RGD present in the
penton base of the virus binds to .alpha.V .beta.3 and
.alpha.V.beta.5 of the cell, it is believed that integrin,
particularly .alpha.V .beta.3 and .alpha.V.beta.5 are involved also
in a binding mechanism of the RGD-PEG-adenovirus vector of the
present invention.
Example 2
Comparative Experiment of Gene Expression Efficiency of Various
Adenovirus Vectors Under the Presence of Neutralizing Antibody
[0109] A comparative experiment of a gene transfer efficiency under
the presence of a neutralizing antibody was performed for the
control adenovirus vector, the PEG-adenovirus vector, the
fiber-modified adenovirus vector and the RGD-PEG-adenovirus vector,
which were produced in the steps 1)-4) of Example 1.
[0110] The experiment was performed with the A549 human lung
adenocarcinoma cell (FIG. 2A) which is a high CAR-expressing cell
and the mouse melanoma B16BL6 cell (FIG. 2B) which is a low
CAR-expressing cell. The A549 cell was subcultured to subconfluent
in DMEM with 10% fetal bovine serum, which was used in the
experiment. Moreover, the B16BL6 cell was subcultured to
subconfluent in the Eagle's minimum essential medium (MEM,
manufactured by Sigma) with 7.5% fetal bovine serum, which was used
in the experiment.
[0111] The A540 cell and the B16BL6 cell were seeded onto the
48-well plate at 1.times.10.sup.4 cells/0.5 mL/well and cultured
for 24 hours. Each vector prepared in the medium of each cell was
added to the well at 1000 particles/cell/0.5 mL under the presence
or absence of ICR mouse serum and cultured at 37.degree. C. for 24
hours under the saturated vapor pressure (5% CO.sub.2).
Furthermore, a measurement of a luciferase activity was performed
according to the procedure in the step 5) of Example 1. Moreover,
ICR mouse serum was prepared by administering the control
adenovirus vector to an ICR mouse at about 10.sup.10
particles/mouse three times.
[0112] The results thereof are shown in FIG. 2 as a change in the
luciferase activity when a dilution rate of anti-adenovirus serum
was changed. The RGD-PEG-adenovirus maintains far higher gene
expression in both of the high CAR-expressing cell and the low
CAR-expressing cell under the presence of an anti-adenovirus vector
antibody than that of the control adenovirus vector and the
fiber-modified adenovirus vector. For example, in the A549 cell,
the degrees of gene expression of the control adenovirus vector and
the fiber-modified adenovirus vector are lowered to 24% and 42%
under the presence of antiserum diluted to 1/10000, respectively,
as a degree of their gene expression under the absence of the
antibody is deemed as 100%. On the contrary, the RGD-PEG-adenovirus
vector maintains 100% of gene expression.
[0113] In the B16BL6 cell, the degrees of gene expression of the
control adenovirus vector, the PEG-adenovirus vector and the
fiber-modified adenovirus vector are significantly lowered under
the presence of antiserum diluted to 1/10000 and 1/3000. On the
contrary, the RGD-PEG-adenovirus vector maintains far higher gene
expression than that of other vectors. Therefore, it was elucidated
that the RGD-PEG-adenovirus vector is less affected by the
neutralizing antibody, and can easily transfer a gene into the
target cell, as compared with the other virus vectors.
[0114] In view of above matters, it was found that the
RGD-PEG-adenovirus vector of the present invention may be a
superior vector for gene transfer to the fiber-modified adenovirus
vector, and that the virus vector may attach to the target cell
while it maintains advantages of PEG to subsequently attain
efficient gene transfer and gene expression, by addition of the
heterogeneous peptide having an affinity for integrin, the RGD
sequence, to the virus vector via PEG
[0115] Therefore, it was found that the PEG-adenovirus vector, to
which the heterogeneous peptide having an affinity for integrin
such as RGD has been added, has not only both of a high gene
transfer ability and a high gene expression ability like the
fiber-modified adenovirus vector, but also an antibody evading
ability possessed by the PEG-adenovirus vector.
Example 3
Comparative Experiment of Gene Expression Efficiency of Various
Adenovirus Vectors which Have Been Repeatedly Thawed and Frozen
after One Month Storage at -80.degree. C.
[0116] Each of the control adenovirus vector, the PEG-adenovirus
vector, the fiber-modified adenovirus vector and the
RGD-PEG-adenovirus vector, which were produced in the steps 1)-4)
of Example 1, was stored in a phosphate buffered saline (PBS) at
-80.degree. C. for one month. Thereafter, each of the adenovirus
vectors was subjected to a step of repeatedly thawing at room
temperature and freezing at -80.degree. C. five times and,
thereafter, a comparative experiment of a gene expression
efficiency of each virus vector was performed.
[0117] The experiment was performed with the A549 human lung
adenocarcinoma cell (FIG. 3A) which is a high CAR-expressing cell,
and the mouse melanoma B16BL6 cell (FIG. 3B) which is a low
CAR-expressing cell. The A549 cell was subcultured to subconfluent
in DMEM with 10% fetal bovine serum, which was used in the
experiment. Moreover, the B16BL6 cell was subcultured to
subconfluent in the Eagle's minimum essential medium (MEM,
manufactured by Sigma) with 7.5% fetal bovine serum, which was used
in the experiment.
[0118] The A549 cell and the B16BL6 cell were seeded onto the
48-well plate at 2.times.10.sup.4 cells/0.5 mL/well and cultured
for 24 hours. Each vector prepared in the medium of each cell was
added to the well at 3000 particles/cell/0.5 mL and cultured at
37.degree. C. for 24 hours under the saturated vapor pressure (5%
CO.sub.2).
[0119] Furthermore, a measurement of a luciferase activity as an
indicator of a gene expression efficiency of the adenovirus vector
was performed according to the procedure in the step 5) of Example
1.
[0120] The results thereof are shown in FIG. 3 as gene expression
of each adenovirus vector immediately after production and after
repeatedly thawing and freezing after long term storage at low
temperature.
[0121] At first, it was confirmed that the PEG-adenovirus vector
immediately after production has a lower gene expression efficiency
in the A549 cell as compared with other virus vectors as in the
step 5) of Example 1. Moreover, the control adenovirus vector, the
PEG-adenovirus vector, the fiber-modified adenovirus vector and the
RGD-PEG-adenovirus vector which had been repeatedly thawed and
frozen five times after one month storage maintained an
approximately similar degree of gene expression to that upon
production.
[0122] Next, it was confirmed that the RGD-PEG-adenovirus vector
immediately after production has about 100 times higher gene
expression in the B16BL6 cell than that of the control adenovirus
vector as in the step 5) of Example 1, and has a similar degree of
gene expression to that of the fiber-modified adenovirus vector.
Moreover, the RGD-PEG-adenovirus vector maintained an approximately
similar degree of gene expression to that upon production, even
after it had been repeatedly thawed and frozen five times after one
month storage.
[0123] In view of above matters, it may be said that gene
expression of the RGD-PEG-adenovirus vector of the present
invention is adequately maintained even after it has been
repeatedly thawed and frozen after long term storage at low
temperature, and that it has a high stability.
[0124] In general, in the case that a large structure such as
RGD-PEG of the present invention is added to a surface of the
adenovirus vector, it is expected that a stability of the resulting
vector is lowered because RGD and the adenovirus vector are linked
via a linker, cysteine, PEG, lysine, and .beta.-alanine.
Nonetheless, in view of the results in the working examples, the
RGD-PEG-adenovirus vector of the present invention maintained an
excellent gene expression efficacy even after it had been
repeatedly thawed and frozen after long term storage at low
temperature. It was an unexpected result that a stability of the
adenovirus vector having a large structure such as RGD-PEG is
adequately maintained even after it had been repeatedly thawed and
frozen after long term storage at low temperature.
[0125] According to the present invention, there is provided a
vector for gene transfer, which has a low immunogenicity for a
living body to be administered, which has a relatively long
half-time in blood since it is less attacked by a neutralizing
antibody or a phagocyte due to its low antigenecity, which can
transfer a gene into a broad range of a cell in a high efficiency
and, thereby, which has a low accumulative property in liver, which
can be prepared easily and efficiently without lowering an
advantageous ability of an original virus, and which is stable even
after it has been repeatedly thawed and frozen after long term
storage at low temperature, as well as a method of gene
transfer.
Sequence Listing Free Text
SEQ ID NO: 1
Peptide motif having integrin-binding activity.
SEQ ID NO: 2
Peptide motif having integrin-binding activity.
SEQ ID NO: 3
Peptide motif having integrin-binding activity.
SEQ ID NO: 4
Peptide motif having integrin-binding activity.
SEQ ID NO: 5
Peptide motif having integrin-binding activity.
SEQ ID NO: 6
Peptide motif having integrin-binding activity.
SEQ ID NO: 7
Peptide motif having integrin-binding activity.
SEQ ID NO: 8
Peptide motif having integrin-binding activity.
SEQ ID NO: 9
Peptide motif having integrin-binding activity.
SEQ ID NO: 10
Peptide motif having integrin-binding activity.
SEQ ID NO: 11
Peptide motif having integrin-binding activity.
SEQ ID NO: 12
Designed peptide containing peptide motif having integrin-binding
activity.
SEQ ID NO: 13
Designed peptide containing peptide motif having integrin-binding
activity.
Sequence CWU 1
1
13 1 4 PRT Artificial synthetic 1 Arg Glu Asp Val 1 2 7 PRT
Artificial synthetic 2 Arg Tyr Val Val Leu Pro Arg 1 5 3 6 PRT
Artificial synthetic 3 Leu Gly Thr Ile Pro Gly 1 5 4 5 PRT
Artificial synthetic 4 Pro Asp Ser Gly Arg 1 5 5 5 PRT Artificial
synthetic 5 Tyr Ile Gly Ser Arg 1 5 6 5 PRT Artificial synthetic 6
Ile Lys Val Ala Val 1 5 7 10 PRT Artificial synthetic 7 Arg Asn Ile
Ala Glu Ile Ile Lys Asp Ile 1 5 10 8 4 PRT Artificial synthetic 8
Gly Pro Arg Pro 1 9 5 PRT Artificial synthetic 9 Glu Ile Leu Asp
Val 1 5 10 6 PRT Artificial synthetic 10 Lys Gln Ala Gly Asp Val 1
5 11 4 PRT Artificial synthetic 11 Asp Glu Gly Ala 1 12 9 PRT
Artificial Designed peptide containing peptide motif having
integrin-binding activity. The N-terminal amino acid residue is
acetylated. 12 Tyr Gly Gly Arg Gly Asp Thr Pro Xaa 1 5 13 19 PRT
Artificial Designed peptide containing peptide motif having
integrin-binding activity. The N-terminal amino acid residue is
acetylated. 13 Tyr Gly Gly Arg Gly Asp Thr Pro Xaa Lys Xaa Pro Thr
Asp Gly Arg 1 5 10 15 Gly Gly Tyr
* * * * *