U.S. patent application number 13/641027 was filed with the patent office on 2013-03-07 for hexon isolated from simian adenovirus serotype 19, hypervariable region thereof and chimeric adenovirus using the same.
This patent application is currently assigned to MOGAM BIOTECHNOLOGY RESEARCH INSTITUTE. The applicant listed for this patent is Eui-Cheol Jo, Daekyung Koh, Hong-Kyu Lee, Kyuhyun Lee, Seongtae Yun. Invention is credited to Eui-Cheol Jo, Daekyung Koh, Hong-Kyu Lee, Kyuhyun Lee, Seongtae Yun.
Application Number | 20130058897 13/641027 |
Document ID | / |
Family ID | 44798828 |
Filed Date | 2013-03-07 |
United States Patent
Application |
20130058897 |
Kind Code |
A1 |
Lee; Kyuhyun ; et
al. |
March 7, 2013 |
HEXON ISOLATED FROM SIMIAN ADENOVIRUS SEROTYPE 19, HYPERVARIABLE
REGION THEREOF AND CHIMERIC ADENOVIRUS USING THE SAME
Abstract
Novel hexon isolated from simian adenovirus serotype 19 encoded
in the polynucleotide defined as SEQ ID NO: 3, hepervariable region
thereof, chimeric adenovirus comprising the same, and therapeutic
use thereof provides a solution to the problem of safety and
effective systemic treatment for developing gene therapeutic agents
using adenovirus.
Inventors: |
Lee; Kyuhyun; (Yongin-si,
KR) ; Yun; Seongtae; (Yongin-si, KR) ; Koh;
Daekyung; (Yongin-si, KR) ; Lee; Hong-Kyu;
(Yongin-si, KR) ; Jo; Eui-Cheol; (Yongin-si,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lee; Kyuhyun
Yun; Seongtae
Koh; Daekyung
Lee; Hong-Kyu
Jo; Eui-Cheol |
Yongin-si
Yongin-si
Yongin-si
Yongin-si
Yongin-si |
|
KR
KR
KR
KR
KR |
|
|
Assignee: |
MOGAM BIOTECHNOLOGY RESEARCH
INSTITUTE
Yongin-si, Gyeonggi-do
KR
|
Family ID: |
44798828 |
Appl. No.: |
13/641027 |
Filed: |
April 14, 2010 |
PCT Filed: |
April 14, 2010 |
PCT NO: |
PCT/KR2010/002298 |
371 Date: |
November 12, 2012 |
Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/366; 435/456; 530/350; 536/23.72 |
Current CPC
Class: |
A61K 39/001182 20180801;
C12N 15/86 20130101; A61K 39/0011 20130101; A61K 39/001151
20180801; C12N 2810/6018 20130101; C07K 14/005 20130101; A61P 35/00
20180101; C12N 2710/10345 20130101; A61K 39/001128 20180801; C12N
2710/10342 20130101; C12N 2710/10321 20130101; C12N 2710/10332
20130101; C12N 2710/10322 20130101; C12N 2710/10343 20130101; A61K
39/001129 20180801; A61K 2039/5256 20130101; C12N 7/00
20130101 |
Class at
Publication: |
424/93.2 ;
530/350; 536/23.72; 435/235.1; 435/456; 435/366 |
International
Class: |
A61K 35/76 20060101
A61K035/76; C12N 15/34 20060101 C12N015/34; C12N 5/10 20060101
C12N005/10; C12N 15/861 20060101 C12N015/861; A61P 35/00 20060101
A61P035/00; C07K 14/075 20060101 C07K014/075; C12N 7/01 20060101
C12N007/01 |
Claims
1. A hexon isolated from simian adenovirus serotype 19 having the
amino acid sequence of SEQ ID NO: 16.
2. A DNA encoding the hexon of claim 1.
3. The DNA of claim 2 having the nucleotide sequence of SEQ ID NO:
3.
4. A hypervariable region (HVR) having the amino acid sequence of
SEQ ID NO: 21 isolated from simian adenovirus serotype 19.
5. A DNA encoding the HVR of claim 4.
6. The DNA of claim 5 having the nucleotide sequence of SEQ ID NO:
20.
7. A chimeric adenovirus having a normative amino acid sequence in
the hexon by the substitution of the hexon protein having the amino
acid sequence of SEQ ID NO: 16, or seven (7) or more consecutive
residues therefrom.
8. The chimeric adenovirus of claim 7, wherein the normative amino
acid sequence is the HVR having the amino acid sequence of SEQ ID
NO: 21.
9. The chimeric adenovirus of claim 7, wherein the normative amino
acid sequence is a fragment of the HVR selected from the group
consisting of: amino acid residues 11 to 41 of SEQ ID NO: 21; amino
acid residues 46 to 52 of SEQ ID NO: 21; amino acid residues 69 to
78 of SEQ ID NO: 21; amino acid residues 106 to 119 of SEQ ID NO:
21; amino acid residues 126 to 138 of SEQ ID NO: 21; amino acid
residues 160 to 173 of SEQ ID NO: 21; and amino acid residues 275
to 303 of SEQ ID NO: 21.
10. The chimeric adenovirus of claim 7, which is a human
adenovirus.
11. The chimeric adenovirus of claim 10, wherein the human
adenovirus is selected from the group consisting of human
adenovirus serotypes 2, 3, 5, 11, 24, 26, 30, 34, 35, 36, 41, 48,
49, and 50.
12. The chimeric adenovirus of claim 7, wherein the chimeric
adenovirus further comprises a therapeutic transgene.
13. The chimeric adenovirus of claim 12, wherein the therapeutic
transgene is selected from the group consisting of tumor suppressor
gene, antigenic gene, cytotoxic gene, cytostatic gene, suicide
gene, anti-angiogenic gene, and immune-modulatory gene.
14. The chimeric adenovirus of claim 13, wherein the tumor
suppressor gene is selected from the group consisting of p53 gene,
APC gene, DPC-4/Smad-4 gene, BRCA-1 gene, BRCA-2 gene, WT-1 gene,
retinoblastoma gene, MMAC-1 gene, adenomatous polyposis coil
protein, DCC (deleted in colorectal cancer) gene, MMSC-2 gene, NF-1
gene, NF-2 gene, MTS1 gene, CDK4 gene, and VHL gene; the antigenic
gene is carcinoembryonic antigen (CEA), CD3, CD133, CD44, or p53;
the cytotoxic gene is selected from the group consisting of genes
coding Pseudomonas exotoxin, ricin toxin, and diphtheria toxin; the
cytostatic gene is selected from the group consisting of p21,
retinoblastoma gene, E2F-Rb fusion protein gene, a gene coding
cyclin-dependent kinase inhibitor, and growth arrest specific
homeobox (GAX) gene; the suicide gene is selected from the group
consisting of genes coding herpes simplex virus thymidine kinase,
varicella thymidine kinase, cytosine deaminase, purine nucleoside
phosphorylase, beta-lactanase, carboxypeptidase G2, cytochrome
P450-2B1, nitroreductase, beta-glucuronidase and TRAIL (TNF related
apoptosis-inducing ligand); the anti-angiogenic gene is selected
from the group consisting of genes coding vascular endothelial
growth factor (VEGF), soluble VEGF receptor, angiostatin,
endostatin, and apolipoprotein (a) kringle domain (LK8); and the
immune-modulatory gene is selected from the group consisting of
genes coding CD16, CTLA-4, IL24 and GM-CSF.
15. A composition comprising the chimeric adenovirus of claim
7.
16. A method for delivering a therapeutic transgene to a mammalian
cell comprising introducing into said cell the chimeric adenovirus
of claim 12.
17. A method for treating cancers comprising administering into a
subject the chimeric adenovirus of claim 12.
18. A method for preparing an adenoviral vector for gene therapy
comprising substituting seven (7) or more amino acid residues in
the hexon of a human adenovirus with seven (7) or more residues of
the hexon protein having the amino acid sequence of SEQ ID NO:
16.
19. An isolated host cell comprising the chimeric adenovirus of
claim 7.
20. The isolated host cell of claim 19, wherein the isolated host
cell is a human cell.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel hexon isolated from
simian adenovirus serotype 19 ("SAd19"), hypervariable region
("HVR") thereof, chimeric adenovirus using the same, and a
therapeutic use thereof.
BACKGROUND OF THE INVENTION
[0002] Adenovirus belongs to the family Adenoviridae first isolated
in 1953. Human adenoviruses are categorized into six (6) subgenera
(A through F) based on the genome similarity, oncogenecity, and
blood coagulation characteristics. Adenoviruses infect most
non-divided cells such as muscle, lung, brain, and cardiac cells,
and its molecular biological characteristics are well known in the
art. Its genome is composed of a linear, double-stranded DNA of 35
kb and its replication in the host cell depends on viral protein,
E1A.
[0003] Above characteristics of adenovirus can be exploited by
using a nonreplicative vector having E1A deleted therefrom. Since
the development of HEK293 cell line in which the adenoviral E1 gene
is inserted, the adenovirus vector system has been used in numerous
studies, which has led to the development of virotherapeutics that
utilize cytotoxicity of the host cell. Oncorine.RTM., which is the
oncolytic virotherapeutics commercialized in China in 2005, is an
E1B55-defective adenovirus for selectively inducing apoptotic cell
death in p53-defective tumor.
[0004] In the development of selective replicative adenovirus
therapeutic agents, selective expression of E1A protein is most
important, and there have been suggested many cases regarding
possible tumor-selective adenovirus gene therapeutic agents using
tumor selective expression promoter. Most of the tumor-selective
adenoviruses are prepared using the commonly-found human adenovirus
serotype 5 ("HAd5"). It has been reported that human has high
levels of adenovirus neutralizing antibodies, since HAd5 occupies
80% of prevalence (Appaiahgari, M. B., et al. (2007) Clinical and
Vaccine Immunol. 14, 1053-1055). These neutralizing antibodies
against adenoviral capsid protein influence the efficacy and
toxicity of the adenovirus systemically administered (Chen, Y., et
al. (2000) Hum. Gene Ther. 11, 1553-1567).
[0005] Viral capsid consists of three (3) kinds of proteins, i.e.,
hexon, fiber, and penton, and comprises a capsomere having a
symmetric icosahedron structure consisting of 240 hexons and 12
pentons. Each penton binds a protruded trimeric fiber of
70.about.100 nm. When infected by adenovirus, the trimeric fiber
attaches to Coxackie Adenovirus Receptor (CAR) on the surface
membrane of host cell in the process of adenovirus infection. The
RGD region of penton binds to integrin, which leads to viral
absorption and penetration into the host cell.
[0006] It has been reported that Loop 1 (L1) and Loop 2 (L2) of a
hexon protein are exposed on the outside of the viral capsomere
structure. L1 and L2 respectively contain six (6) hypervariable
regions (HVRs), i.e., HVR-1 to HVR-6 within the 132.sup.nd to
320.sup.th amino acids and seventh HVR (HVR-7) within the
408.sup.th to 459.sup.th amino acids of the hexon protein.
[0007] The adenoviruses provide an elegant and efficient means of
transferring therapeutic genes into cells. However, one problem
encountered with the use of adenoviral vectors for gene transfer in
vivo is the generation of antibodies to antigenic epitopes on
adenoviral capsid proteins.
[0008] When adenovirus is administered to human body, neutralizing
antibodies against hexon proteins are formed, and such antibodies
mostly target the dominant HVR regions. It is also known that the
antibodies reduce the efficiency of viral replication by way of
inhibiting the infection of host cells (Wohlfart, C. (1988) J.
Virol. 62, 2321-2328, Toogood, C. I. A., et al. (1992) J. Gen.
Virol. 73, 1429-1435. Sumida, S. M., et al. (2005) J. Immunol. 174,
7179-7185).
[0009] The problems caused by the preponderance of human
neutralizing antibodies against HAd5 in human must be overcome when
administering adenovirus using a viral gene delivery vector and
viral therapeutic agent. In addition to the above mentioned
problems associated with the neutralizing antibodies, it has been
reported that adenoviruses infect the liver when exposing
systemically. In this connection, adenovirus has been reported to
have hepatotropism, and when adenoviruses are administered via an
intravenous route, 90% thereof is transferred to the liver within
24 hours (Worgall, S., et al. (1997) Hum. Gene Ther. 8, 37-44). Due
to such hepatoselectivity of the adenovirus, in 1999, young
patient, Jessie Gelsinger, under a clinical trial using gene
therapeutic adenovirus agents succumbed due to acute
hepatotoxicity. Thus, dose of adenovirus has been restricted to an
amount that does not exceed 1.times.10.sup.13 vp since then.
Therefore, hepatotoxicity is generally considered as a
dose-limiting factor in nonclinical/clinical trials for many gene
therapeutic agents using adenovirus (Alemany, R. et al. (2001) Gene
Ther., 8(17), 1347-1353; Christ, M., et al. (2000) Hum. Gene Ther.,
11(3), 415-427; Lieber, A., et al. (1997) J. Virol., 7(11),
8798-8807). Such liver selectivity is a major problem in achieving
efficient cure by systemic administration of an adenoviral
therapeutic agent (Worgall, S., et al. (1997) Hum. Gene Ther., 8,
37-44).
[0010] In this regard, Waddington et al. have recently reported
that Gla domain, blood coagulating factor, combines with hexon
protein of adenovirus in blood, which facilitates adenovirus
transfer to the liver (Waddington, S. N., et al. (2008) Cell, 132,
397-409). It has been speculated that HVR-3, HVR-5 or HVR-7 of
hexon can combine with blood coagulating factor, Gla domain
(Kalyuzhniy, O., et al. (2008) Proc. Nat'l Acd. Sci. 105,
5483-5488). The HVR varies depending on the serotype of adenovirus,
and it is not clear yet what is the crucial factor for binding
affinity to blood coagulating factor. It has been reported that the
maximum tolerated dose of adenovirus can be raised tenfold by way
of inserting a specific protein such as RGD, RFP, and BAP (Biotin
Acceptor Peptide) to significantly weaken binding affinity to blood
coagulating factor and to reduce the hepatotropism (Shashkova, E.
V., et al. (2009) Mol. Ther. 17, 2121-2130).
[0011] A number of functions of the hexon protein are now known,
and many studies to modify hexon proteins in order to overcome the
problems of hepatotoxicity and anti-adenoviral immunity are
currently being conducted. There are four strategies for modifying
hexon protein: 1) replacing the hexon gene with the corresponding
hexon gene of other adenovirus serotype, 2) inserting a peptide
into HVR, 3) replacing the gene encoding HVR of a hexon protein
with the corresponding gene encoding HVR of other adenovirus
serotype, and 4) removing the region from the HVR that binds the
blood coagulating factor and neutralizing antibody. Up to date, the
method of inserting a peptide into the HVR and the method of
replacing the gene encoding HVR with the corresponding gene
encoding HVR of other adenovirus serotype are carried out for hexon
modification. Among above mentioned four strategies, complete hexon
substitution is most apparent method to change viral
immunogenicity. However, a method to achieve complete hexon
exchange for modifying hexon protein has the problem of
deteriorated productivity due to the fact that subtle structural
differences in binding hexon to penton and fiber induce instability
of the adenoviral capsid structure (Roberts, D. M., et al. (2006)
Nature, 441, p 239-243; Youil, R., et al. (2002) Hum. Gene. Ther.
13, p 311-320; Shashkova, E., et al. (2009) Mol. Ther. 17,
2121-2130).
[0012] Further, intense studies for the modification of capsid
protein using serotypes of heterogenous adenovirus as well as those
of human adenovirus are in progress. It has been reported that the
prevalence rate of neutralizing antibodies against chimpanzee
adenoviruses pan 5, 6, 7 and 9, classified as simian adenovirus
serotypes 22 to 25, respectively, is less than 6%, and therefore, a
simian adenoviral vector system including chimpanzee adenovirus can
be useful as a gene therapeutic vector (Roy, S. et al. (2004) Hum.
Gene Ther. 15, p 519-530). International Patent Publication Nos. WO
2006/040330 and WO 2002/083902 teach the use of the fiber or hexon
protein of human serotypes 11, 24, 26, 30, 34, 35, 48, 49, and 50
for suppressing immune response caused by neutralizing antibodies
in the recombinant chimeric adenovirus where the adenoviral knob
domain binding to the CAR or a hexon protein is substituted with
those of other serotypes. Regarding simian adenovirus serotype,
International Publication No. WO 2005/001103 discloses a chimeric
adenovirus using simian adenovirus serotype 18.
[0013] However, there exists a strong need to develop an adenovirus
having lower immunogenicity and lower toxicity. Thus, the present
inventors have identified a novel SAd19 hexon gene isolated from
baboon excrements and have found that it is highly capable of
evading the neutralizing antibodies against HAd5 and it exhibits a
low toxicity.
SUMMARY OF THE INVENTION
[0014] Accordingly, it is an object of the present invention to
provide a novel hexon protein and a DNA encoding the same for use
in the preparation of a chimeric adenovirus.
[0015] It is another object of the present invention to provide a
chimeric adenovirus comprising the novel hexon protein.
[0016] It is a further object of the present invention to provide a
composition comprising the chimeric adenovirus.
[0017] It is a still further object of the present invention to
provide a method of gene therapy employing the chimeric
adenovirus.
[0018] In accordance with one aspect of the present invention,
there is provided a hexon isolated from SAd19 and a DNA encoding
the hexon.
[0019] In accordance with another aspect of the present invention,
there is provided a HVR of the hexon isolated from SAd19 and a DNA
encoding the HVR.
[0020] In accordance with a further aspect of the present
invention, there is provided a chimeric adenovirus having a
normative amino acid sequence in the hexon by the substitution of
the hexon protein isolated from SAd19, or seven (7) or more
consecutive residues therefrom.
[0021] In accordance with a still further aspect of the present
invention, there is provided a composition comprising the chimeric
adenovirus of the present invention.
[0022] In accordance with a still further aspect of the present
invention, there is provided a method for delivering a therapeutic
transgene to a mammalian cell comprising introducing into said cell
the chimeric adenovirus of the present invention.
[0023] In accordance with a still further aspect of the present
invention, there is provided a method for treating cancers
comprising administering into a subject the chimeric adenovirus of
the present invention.
[0024] In accordance with a still further aspect of the present
invention, there is provided a method for preparing an adenoviral
vector for gene therapy comprising substituting seven (7) or more
amino acid residues in the hexon of a human adenovirus with seven
(7) or more residues of the hexon protein of the present
invention.
[0025] In accordance with a still further aspect of the present
invention, there is provided an isolated host cell comprising the
chimeric adenovirus of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The above and other objects and features of the present
invention will become apparent from the following description of
the invention, when taken in conjunction with the accompanying
drawings, which respectively show:
[0027] FIG. 1: amino acid sequences of hexon proteins from human
adenovirus (HAd) serotypes 5 (A) and 41 (B), and SAd19;
[0028] FIG. 2: cleavage maps of pHex-SAd19 Hexon vector (A), pAd
H5/S19.sub.--8DS vector (B), pAd328 H5/S19_DS vector (C), and
pAd328 H5/S19_DS .DELTA.19k (D);
[0029] FIG. 3: the results of western blot analysis showing the
expression level of tumor-specific EIA proportional to infected
units of Ad H5.sub.--8DS and Ad H5/S19.sub.--8DS;
[0030] FIG. 4: SPR results for measuring the binding affinity
between human blood coagulating factor X and Ad H5/S19.sub.--8DS
(A) and the western blot result for human blood coagulating factor
and adenoviral fiber protein after co-immunoprecipitation of human
blood coagulating factor IX or X with either Ad H5.sub.--8DS, Ad
H5/S19.sub.--8DS, Ad328 H5/S19_DS or Ad328 H5/S19_DS .DELTA.19k
(B-D);
[0031] FIG. 5: results of agarose gel electrophoresis showing the
PCR-amplified DNA bands of adenoviral fiber gene in respective
organs after intravenous injection of Ad H5.sub.--8DS and Ad
H5/S19.sub.--8DS;
[0032] FIG. 6: Western blot analysis results demonstrating the
infection-evading ability of chimeric adenoviruses having hexon of
simian adenovirus serotype 19, Ad H5/S19.sub.--8DS (A), Ad328
H5/S19_DS and Ad328 H5/S19_DS .DELTA.19k (B), by employing
neutralizing antibodies against HAd5;
[0033] FIG. 7: an immunostaining photograph and bar graph showing
the infection-evading ability of chimeric adenoviruses having hexon
of SAd19, Ad H5/S19.sub.--8DS (A), Ad328 H5/S19_DS and Ad328
H5/S19_DS.DELTA.19k (B), by employing neutralizing antibodies
against HAd5;
[0034] FIG. 8: a graph showing the expression pattern of LK8 gene
in blood transferred by Ad H5/S19.sub.--8DS and Ad H5.sub.--8DS
which are administered to Syrian hamster immunized by HAd5;
[0035] FIG. 9: a photograph of each organ excised from autopsied
mice into which Ad H5.sub.--8DS and Ad H5/S19.sub.--8DS are
intravenously administered dose-dependently;
[0036] FIG. 10: blood analysis data of the mice into which Ad
H5.sub.--8DS is intravenously administered;
[0037] FIG. 11: blood analysis data of the mice into which Ad
H5/S19.sub.--8DS is intravenously administered;
[0038] FIG. 12: a tumor-growth curve in the animal model for H460
non-small cell lung cancer after intravenous injection of Ad
H5/S19.sub.--8DS;
[0039] FIG. 13: a lung photograph of the animal model for H2172
orthotopic lung cancer autopsied after injection of tumor-specific
chimeric adenovirus, Ad H5/S19.sub.--8DS depending on the
immunization against HAd5; and
[0040] FIG. 14: the results of analysing the volume, weight, the
number and the size of the tumors on the lungs of FIG. 13.
DETAILED DESCRIPTION OF THE INVENTION
[0041] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention belongs. Further,
all documents mentioned herein are incorporated by reference in
their entireties.
[0042] The term "adenovirus" as used herein refers to a
non-enveloped icosahedral double-stranded DNA virus having about a
linear genome of about 36 kb.
[0043] The term "chimeric adenovirus" as used herein refers to an
adenovirus whose nucleic acid sequence is comprised of the nucleic
acid sequences of at least two of the adenoviral serotypes.
[0044] As used herein, "substitution" results from the replacement
of one or more polynucleotides or amino acids by different
polynulceotides or amino acids, respectively.
[0045] The term "hypervariable region" or "HVR" as used herein
means a variable domain, whose sequence is hypervariable, forming
structurally limited loop.
[0046] The term "normative amino acid sequence" as used herein
means any amino acid sequence that is not found in the native hexon
protein of a given serotype of adenovirus, and is introduced into
the hexon protein at the level of gene expression (i.e., by
production of a nucleic acid sequence that encodes the normative
amino acid sequence).
[0047] The term "therapeutic transgene" as used herein refers to a
polynucleotide that is introduced into a cell and is capable of
being translated and/or expressed under appropriate conditions and
confers a desired property to a cell into which it was introduced,
or otherwise leads to a desired therapeutic outcome.
[0048] The term "vector" as used herein refers to a vehicle for
gene transfer as that term is understood by those skilled in the
art, and includes viruses, plasmids, and the like.
[0049] The term "neutralizing antibody" as used herein means an
antibody being able to inhibit infectivity of (i.e., cell entry) or
gene expression commanded by an adenovirus. The neutralizing
antibody may be an antibody that either is purified from or is
present in serum.
[0050] The present invention is described in detail
hereinafter.
[0051] In the present invention, there is provided a hexon isolated
from SAd19, a HVR thereof and DNAs encoding the hexon.
[0052] The SAd19 according to the present invention may be provided
by the isolation from the baboon excrements and is classified as
subgroup F. The hexon of SAd19 has an amino acid sequence of SEQ ID
NO: 16, which has 85% homology with that of the human adenovirus
serotype 41 ("HAd41") hexon and 76% homology with that of HAd5
hexon. Further, the nucleotide sequence encoding the hexon of SAd19
has 76% homology with that of the HAd41 hexon and 70% homology with
that of the HAd5 hexon. Preferably, the hexon of SAd19 according to
the present invention has the DNA sequence of SEQ ID NO: 3.
[0053] The hexon of SAd19 or seven (7) or more residues therefrom
may be incorporated into various types of adenoviruses by the
substitution in order to provide a chimeric adenovirus.
Accordingly, there is provided a chimeric adenovirus having a
normative amino acid sequence in the hexon by the substitution of
the hexon protein of SAd19 of the present invention, or one or more
residues therefrom.
[0054] Preferably, the chimeric adenovirus may have a normative
amino acid sequence in the hexon by the substitution of seven (7)
or more residues from the hexon having the amino acid sequence of
SEQ ID NO: 16. The seven (7) or more residues from the hexon of
SAd19 may be HVR. The HVR may have the amino acid sequence of SEQ
ID NO: 21 and may be encoded by the nucleotide sequence of SEQ ID
NO: 20. More preferably, the chimeric adenovirus may have a
normative amino acid sequence in the hexon by the substitution of
the fragment of the HVR, i.e., HVR-1 to -7, which correspond to the
amino acid residues 11 to 41, 46 to 52, 69 to 78, 106 to 119, 126
to 138, 160 to 173, and 275 to 303 of SEQ ID NO: 21,
respectively.
[0055] The chimeric adenovirus having the hexon of SAd19 or seven
(7) or more residues therefrom shows little immune inhibition by
neutralizing antibodies and hepatotoxicity.
[0056] Various types of the adenovirus, preferably human adenovirus
serotype, more preferably, human adenovirus serotypes 5, 11, 24,
26, 30, 34, 35, 48, 49, and 50 may be useful for the preparation of
the present chimeric adenovirus. Tumor-specific,
replication-competent adenovirus or tumor-specific,
replication-restricted adenovirus may be useful as the adenovirus
therapeutic agent. The chimeric adenovirus of the present invention
has a normative amino acid sequence so as to overcome problems of
immune response and hepatotoxicity.
[0057] In particular, the normative amino acid sequence according
to the present invention is prepared by substituting a hexon region
of a wild-type adenovirus with that of SAd19. Optimally the
resultant normative amino acid sequence is such that seven (7) or
more of the existing epitopes for neutralizing antibodies directed
against the corresponding wild-type adenovirus hexon protein have
been rendered non-immunogenic.
[0058] According to the present invention, the chimeric adenovirus
comprises hexon modification of seven (7) or more amino acids, and
such hexon modification is made in seven (7) or more regions.
[0059] The most preferable chimeric adenovirus may be Ad
H5/S19.sub.--8DS, which is prepared by transduction of the
adenoviral vector pAd H5/S19.sub.--8DS into human lung
adenocarcinoma epithelial cell line, A549, thereby substituting
human adenoviral hexon with the hexon of SAd19.
[0060] In a preferred embodiment of the present invention, the
chimeric adenovirus of the present invention may further contain
therapeutic transgene. The non-limiting examples of such
therapeutic transgene include tumor suppressor gene, antigenic
gene, cytotoxic gene, cytostatic gene, suicide gene,
anti-angiogenic gene and immune-modulatory gene.
[0061] Concretely, the "tumor suppressor gene" is a nucleotide
sequence, the expression of which in the target cell is capable of
suppressing the neoplastic phenotype and/or inducing apoptosis.
Examples of the tumor suppressor gene include p53-gene, APC-gene,
DPC-4/Smad4 gene, BRCA-1 gene, BRCA-2 gene, WT-1 gene,
retinoblastoma gene (Lee et al., Nature, 1987, 329,642), MMAC-1
gene, adenomatous polyposis coil protein (U.S. Pat. No. 5,783,666),
DCC (deleted in colorectal cancer) gene, MMSC-2 gene,
nasopharyngeal cancer suppressor gene located on chromosome 3p21.3
(Cheng et al., Proc. Nat. Acad. Sci., 1998, 95, 3042-3047), MTS1
gene, CDK4 gene, NF-1 gene, NF-2 gene, VHL gene, etc.
[0062] The "antigenic gene" is a nucleotide sequence, the
expression of which in the target cells results in the production
of a cell surface antigenic protein capable of recognition by the
immune system. In the example of this antigenic gene, the
carcinoembryonic antigen (CEA), CD3, CD133, CD44, and p53
(International Publication No. WO94/02167) are included. For easy
recognization of the immune system, the antigenic gene may be
combined with the MHC type I antigen.
[0063] The "cytotoxic gene" is a nucleotide sequence exhibiting the
toxic effect when expressed in cells. Examples of the cytotoxic
gene include nucleotide sequences coding Pseudomonas exotoxin,
ricin toxin, diphtheria toxin, etc.
[0064] The "cytostatic gene" is a nucleotide sequence inducing cell
cycle arrest when expressed in cells. The non-limiting examples of
the cytostatic gene include p21, retinoblastoma gene, E2F-Rb fusion
protein gene, genes coding cyclin-dependent kinase inhibitor (for
example, p16, p15, p18, and p19), growth arrest specific homeobox
(GAX) gene (International Patent Publication Nos. WO97/16459 and WO
96/30385), etc.
[0065] The "suicide gene" is a nucleotide sequence inducing cell
death through apoptosis when expressed in the cell. The
non-limiting examples of the suicide gene encompasses genes coding
herpes simplex virus thymidine kinase, varicella thymidine kinase,
cytosine deaminase, purine nucleoside phosphorylase,
beta-lactanase, carboxypeptidase G2, cytochrome P450-2B1,
nitroreductase, beta-glucuronidase, TRAIL (TNF related
apoptosis-inducing ligand), etc.
[0066] The "anti-angiogenic gene" is a nucleotide sequence, the
expression of which results in the extracellular secretion of
anti-angiogenic factors. The anti-angiogenic factor encompasses
angiostatin, inhibitor of vascular endothelial growth factor (VEGF)
and placental growth factor (P1GF) such as soluble VEGFR1 (sFLT-1)
(PNAS (USA), 1998, 95, 8795-800), endostatin, and apolipoprotein
(a) kringle domain (LK8). The preferred anti-angiogenic gene is a
gene encoding LK8. LK8 directly influence the vascular endothelial
cell to induce apoptosis and inhibit the migration of the
epithelial cell (Kim J S et al., J. Biol. Chem., (2003) 278:29000).
In particular, it has been reported that adenovirus-mediated
expression of LK8 suppresses hepatocellular carcinoma growth in
mice (Lee K. et al., Hepatology (2006) 43:1063). Therefore, it is
expected that oncolytic effect of adenovirus may be improved by the
introduction of LK8.
[0067] The "immune-modulatory gene" is a nucleotide sequence, which
modulates humoral and cellular immune response, when expressed in
the cell. The non-limiting examples of the immuno-modulatory gene
encompass genes coding CD16, CTLA-4, IL24, GM-CSF, etc.
[0068] The therapeutic transgene may be inserted into the inventive
chimeric adenovirus by various DNA recombinant technologies known
in the art.
[0069] The present invention also provides the use of the inventive
chimeric adenoviruses for the inhibition of tumor cell growth, as
well as for the preparation of adenoviral vectors to deliver
therapeutic transgene useful in the treatment of tumor and other
disease. Specifically, there are provided a method for delivering a
therapeutic transgene to mammalian cell comprising introducing said
cell the chimeric adenovirus of the present invention; a method for
treating cancers comprising administering said adenovirus into a
subject; and a method for preparing an adenoviral vector for gene
therapy comprising substituting seven (7) or more amino acid
residues in the hexon of a human adenovirus with seven (7) or more
residues of the SAd19 hexon protein.
[0070] In the present invention, there is also provided a
composition comprising the chimeric adenovirus of the present
invention. The composition of the present invention is useful for
gene therapy or viral therapy, preferably for cancer treatment.
[0071] The compositions of the present invention may be formulated
so as to provide various formulations together with
pharmaceutically acceptable carrier and/or excipient. Thus, the
formulations may be in the form of a solution in oil or water
medium, suspension or emulsion, extract, powder, granule, tablet or
capsule.
[0072] For oral formulation, various preparation methods including
those specifically designed for adenovirus release may be used, for
example, by employing Eudragit or timeclock release system (Lubeck
et al., Proc. Natl. Acad. Sci. USA, 86(17), 6763-6767 (1989); and
Chourasia and Jain, J. Pharm. Pharm. Sci., 6(1), 33-66 (2003)).
[0073] As mentioned above, the chimeric adenovirus may be
transferred via any gene transfer systems known in the art. A lot
of gene transfer system such as those disclosed in [Rolland (1998)
Crit. Rev. Therap. Drug Carrier Systems 15: 143-198] and the
references cited therein are well known in the art. Accordingly,
the composition of the present invention may be formulated suitable
for these gene transfer system.
[0074] The composition of the present invention may comprise any
pharmaceutically acceptable carriers known in the art. Examples of
suitable carriers are water, salt water, alcohol, lipid, wax,
buffer solution, solid carrier such as mannitol, lactose, starches,
magnesium stearate, sodium saccharin, talc, cellulose, glucose,
sucrose, and magnesium carbonate, or biodegradable microsphere
(e.g., polylactate polyglycolate).
[0075] The composition of the present invention may be provided in
the form of single dose or multi-dose container such as sealed
ampule or vial. Preferably, such container may be sealed so as to
conserve aseptic condition of pharmaceutical formulations before
using. In general, the formulation may be preserved as suspension,
fluid, and emlusion in oil or aqueous vehicle. Further, the
pharmaceutical formulation may be preserved under freeze drying
conditions.
[0076] The chimeric adenovirus and the compositions comprising the
same may be administered with site-specific injection or
intravenous injection. Site-specific injection includes, for
example, intraperitoneal injection, intrapleural injection,
intrathecal injection, intraarterial injection, intratumoral
injection or local application. Such administering methods may be
also readily applied to the combination of the treatment utilizing
adenoviral vector and the treatment for other target diseases. The
preferred method is intravenous injection.
[0077] It should be understood that the suitable amount of the
active ingredient actually administered ought to be determined in
light of various relevant factors including the condition to be
treated, the age and weight of the individual patient, food,
administration time, excretion rate, the severity of the patient's
symptom and reaction susceptibility; and, therefore, the above dose
should not be intended to limit the scope of the invention in any
way. Generally, the composition of the present invention contains
1.times.10.sup.7 to 1.times.10.sup.13 pfu/ml of the present
chimeric adenovirus, and the present chimeric adenovirus may be
injected in amount of 1.times.10.sup.11 pfu once a week for 3 to 5
weeks.
[0078] The composition of the present invention may be used as the
single therapy. But it may be combined with other anti-tumor
protocols, such as conventional chemotherapy or radiation therapy
for treating cancer. The chemotherapy drug which can be used with
composition of the present invention encompasses paclitaxel,
cisplatin, carboplatin, procarbazine, mechlorethamine,
cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan,
nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin,
plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum,
5-fluorouracil, vincristin, vinblastin and methotrexate. The
radiation therapy which can be used with the composition of the
present invention may be X-ray irradiation and y-ray irradiation,
etc.
[0079] The chimeric adenovirus of the present invention hardly
renders liver transduction since it does not interact with blood
coagulation factors, and exhibits low hepatotoxicity. Accordingly,
the high dose of the present chimeric adenovirus may be
administered to a subject due to its low risk of immune response
and hepatotoxicity and, therefore, the present chimeric adenovirus
is useful for safe and efficient gene therapy and viral
therapy.
[0080] The following examples are intended to illustrate the
present invention without limiting its scope.
Example 1
Identification of Hexon Gene of SAd19
[0081] A hexon gene was amplified from SAd19 using PCR, cloned into
a pGEM-T easy vector, and sequenced with an ABI automatic DNA
sequencer.
<1-1> Identification of Hexon Gene
[0082] The genome of SAd19 was isolated using a DNeasy Tissue Kit
(QIAGEN, Germany) and used as a template for the amplification of a
hexon gene via PCR with a primer set of SEQ ID NOs: 1 and 2. The
amplified hexon gene was purified by Wizard.RTM. SV Gel and PCR
Clean-Up system (Promega, WI, USA) and inserted into a pGEM-T easy
vector (Promega, WI, USA) with the aid of T4 DNA ligase (Roche,
Switzerland). The resulting recombinant vector was named pGEM-SAd19
Hexon vector. After transformation of E. coli cells with the
vector, the vector DNAs were extracted from 10 clones of the
transformants and sequenced by employing an ABI automatic DNA
sequencer. Among the 10 base sequences was selected the one which
had the highest sequence homology with others, thereby determining
the nucleotide sequence of the hexon gene of SAd19 (SEQ ID NO:
3).
<1-2> Comparison with Base and Amino Acid Sequences of Human
Adenovirus Serotypes
[0083] The base and amino acid sequences of the hexon gene of SAd19
were compared with those of 51 human serotype adenoviruses. The
hexon of SAd19, belonging to subgroup F, was found to be most
similar to that of HAd41, with 85% homology in amino acid sequence
therebetween. It showed an amino acid homology of 76% with the
hexon of HAd5. Further, the hexon of SAd19 was found to share
nucleotide sequence homologies of 76% and 70% with those of HAd41
and HAd5, respectively (FIG. 1).
Example 2
Preparation of Chimeric Adenovirus with Hexon of SAd19 Substituted
Therein
[0084] A shuttle vector for exchanging a hexon gene was constructed
and named pHex vector, which is carrying left- and right-extended
regions of hexon gene for homologous recombination. The hexon gene
of SAd19 was cloned into unique restriction site of pHex vector,
locating between left- and right-extended arms of hexon to afford a
recombinant vector, named pHex-SAd19 Hexon. SphI-linearized
pHex-SAd19 Hexon was subject to homologous recombination with
AsiSI-linearized pAd H5.sub.--8DS in BJ5183 (Stratagene, CA, USA)
to give a SAd19 Hexon-carrying recombinant vector, named pAd
H5/S19.sub.--8DS. After being linearized with Pad, the pAd
H5/S19.sub.--8DS was transfected into A549 cells to generate novel
chimeric adenovirus with the hexon of SAd19 anchored therein (Ad
H5/S19.sub.--8DS).
<2-1> Construction of Shuttle Vector
[0085] A shuttle vector suitable for the substitution of a hexon
gene through homologous recombination was constructed. In this
regard, an about 1 kb-long region upstream of the 5' end of the
hexon gene of HAd5 was amplified by PCR using a set of the primers
of SEQ ID NOs: 4 and 5. The PCR product thus obtained was named
Hexon L, and then inserted into pCR2.1 Topo vector (Invitrogen, CA,
USA). A DNA sequence analysis allowed the selection of a clone free
of mutation, which was named pCR2.1-Hexon L. Separately, an about 1
kb-long region downstream of the 3' end of the hexon gene of HAd5
was amplified by PCR using a set of the primers of SEQ ID NOs: 6
and 7. The PCR product thus obtained was named Hexon R, and then
inserted into pCR2.1 Topo vector (Invitrogen, CA, USA). A
mutation-free clone, as analyzed by DNA sequencing, was named
pCR2.1-Hexon R.
[0086] After being excised from pCR2.1-Hexon L by use of both XhoI
and EcoRI, Hexon L was inserted into pENTR2B (Invitrogen, CA, USA),
which was previously cut with both SalI and EcoRI, to give a
recombinant vector, pENTR2B-Hexon L. pCR2.1-Hexon R was digested
with HindIII, followed by treatment with a Klenow fragment to make
blunt ends. Digestion with EcoRI excised Hexon R from the
blunt-ended pCR2.1-Hexon R. This Hexon R was inserted into an
Blunted XbaI and EcoRI site of the pENTR2B-Hexon L vector. The
resulting recombinant vector was named pHex. PCR was performed in
the presence of pfu polymerase (Stratagene, CA, USA) using a set of
the primers of SEQ ID NOs: 1 and 2, with pGEM-SAd19 Hexon serving
as a template. The Mfe-1-resticted hexon gene of SAd19 was cloned
into EcoRI site of pHex vector. The resulting recombinant plasmid,
which was found to have the hexon of SAd19 in a correct position as
analyzed by DNA sequencing, was named pHex-SAd19 Hexon (FIG.
2A).
<2-2> Substitution with the Hexon of SAd19 through Homologous
Recombination
[0087] In order to prepare a chimeric adenovirus recombined with
the hexon of SAd19, first, pENTR2B vector (Invitrogen, CA) was
treated with EcoRI to remove ccdB region therefrom. The
pAAV-CMV_LK8_UN vector, previously constructed by the present
inventors (see PCT Publication No. WO2009/102085), was treated with
KpnI/BglII to give a CMV_LK8 fragment (later blunt ended) which was
then inserted into a KpnI/XhoI site (later blunt ended) of the
EcoRI-treated pENTR2B to give a recombinant plasmid
pENTR-CMV_LK8.
[0088] In order to construct plasmid vector carrying tumor specific
expression unit of E1A gene, the proximal promoter region of DNMT-1
(DNA (cytosine-5)-methyltransferase) gene (DS promoter) was
amplified from human genomic DNA and cloned into pCR2.1-TOPO vector
(Invitrogen, CA) to give a recombinant plasmid pCR-DS. Gene
amplification was conducted in the presence of Ex-Taq polymerase
(Takara, Japan) by PCR using human genomic DNA as PCR template and
a set of the primers of SEQ ID NO: 8 (5'-CTT CTC GCT GCT TTA TCC
CCA TC-3') and SEQ ID NO: 9 (5'-CTC GGA GGC TTC AGC AGA CGC-3'),
which binds to both ends of proximal promoter region of DNMT-1
gene. Starting from denaturation at 94.degree. C. for 5 min, PCR
was performed with 30 cycles of denaturing at 94.degree. C. for 30
sec, annealing at 56.degree. C. for 30 sec and extension at
72.degree. C. for 1 min, followed by extension at 72.degree. C. for
additional 3 min.
[0089] A DNA fragment excised from pCR-DS by SacI and XhoI was
inserted ClaI and SalI sites in front of mutant E1A gene in the
p.DELTA.E1Sp1B-E2F-1 Rb7.DELTA.19k vector (Kim, J., et al. (2007)
Hum. Gene Ther. 18, p 773-786; mE1A, Korean Patent No. 746122;
.DELTA.E1B19K, Korean Patent No. 432953) to construct a recombinant
plasmid pSP72-DS_mE1A_.DELTA.E1B19K. A fragment resulting from the
treatment of pSP72-DS_mE1A_.DELTA.E1B19K with BamHI was inserted
into a BglII site of pENTR-CMV_LK8 to afford a shuttle vector,
named pENTR-CMV_LK8-DS_mE1A_.DELTA.E1B19K.
[0090] To a mixture of 100 ng of pAd-PL Dest (Invitrogen, CA, USA)
and 500 ng of pENTR-CMV_LK8-DS_mE1A_.DELTA.E1B19K was added 16
.mu.L of clonase I reaction buffer (Invitrogen, CA, USA). The
reaction mixture was incubated at room temperature for 1 hr with 2
.mu.L of clonase I and then at 37.degree. C. for 10 min with 2
.mu.L of proteinase K (2 .mu.g/.mu.L). 10 .mu.L of the resulting
reaction mixture was taken and used to transform E. coli DH5.alpha.
competent cells which were then spread over ampicillin plates.
Plasmid DNA extracted from colony positive in the restriction
mapping was identified by DNA sequencing analysis as an adenovirus
vector of interest, and then named pAd H5-8DS.
[0091] In order to substitute the hexon gene of pAd H5.sub.--8DS
with SAd19 Hexon, first, 500 ng of pHex-SAd19 Hexon and 50 ng of
pAd H5.sub.--8DS were linearized respectively with SphI and AsiSI
and mixed together before transformation into E. coli BJ5183 by
electroporation. Homologous recombination was screened by PCR using
a set of the primers of SEQ ID NO: 10 (5'-ATG CGC AAG GTG TAG
CCA-3') and SEQ ID NO: 11 (5'-AGC GTG CTG GCC AGC GTG-3'), which
were designed to examine homologous recombination. Starting from
denaturation at 94.degree. C. for 5 min, PCR was performed with 30
cycles of denaturation at 94.degree. C. for 30 sec, annealing at
55.degree. C. for 40 sec and extension at 72.degree. C. for 1.5
min, followed by extension at 72.degree. C. for an additional 3
min. The positively screened colonies were subjected to secondary
screening by PCR using a set of the primers of SEQ ID NO: 12
(5'-CCC GTT ACA TAA CTT ACG-3') (CMV sense primer) and SEQ ID NO:
13 (5'-TTA TGG CCT GGG GCG TTT ACA G-3') (E1A antisense primer),
which were designed to amplify a region comprising both LK8 and E1A
genes. Starting from denaturation at 94.degree. C. for 5 min, the
PCR was performed with 30 cycles of denaturation at 94.degree. C.
for 30 sec, annealing at 55.degree. C. for 40 sec and extension at
72.degree. C. for 30 sec, followed by extension at 72.degree. C.
for an additional 5 min. DNA was isolated from the clones which
were positively screened by the two PCRs and was amplified in E.
coli DH5.alpha.. A clone which was coincident in all the cutting
patterns with EcoRI, SpeI, XbaI and PshAI was secured and named pAd
H5/S19.sub.--8DS (FIG. 2B). DNA sequencing again identified that
pAd H5/S19.sub.--8DS contained the hexon of SAd19.
[0092] A DNA fragment containing DS promoter was excised from
pCR-DS by EcoRI digestion and cloned into the EcoRI site of
phRL-null vector (Promega, WI, USA) to generate phRL-DS. A fragment
resulting from the treatment of phRL-DS with SalI and PstI was
inserted into the SalI and PstI sites of pE1.2 (O.D.260 Inc. Boise,
Id. US) shuttle vector, generating pE1.2-DS. The DNA fragment of
1.7 kb size excised from pE1.2-DS by AlwNI digestion was cloned to
SfiI site of pAd328 (O.D.260 Inc. Boise, Id. US) by enzyme-ligation
method using E. coli XL1-blue electro-competent cells. Plasmid DNA
extracted from colony growing on LB plate supplemented with
ampicillin and kanamycin was secured by coincidence in all the
cutting patterns with EcoRI, SpeI, XbaI and PshAI and named
pAd328-DS adenoviral vector (FIG. 2C). The adenovirus E1 gene was
amplified from genomic DNA of HAd5 and cloned to pGEM-T easy vector
(Promega, WI, USA) to give a recombinant plasmid pGEMT-Ad E1. The
pGEMT-Ad E1 was treated very shortly with EcoNI after BssHI
digestion to remove a E1B19k region therefrom. E1B19k-deleted
pGEMT-Ad E1 was named to pGEMT-Ad E1.DELTA.19k. In order to
substitute the E1 gene of pAd328-DS with E1B.DELTA.19k-deleted E1
gene, first, 100 ng of pAd328-DS and 1 .mu.g of SphI-linearized
pGEMT-Ad E1.DELTA.19k were mixed together before transformation
into E. coli BJ5183 by electroporation. Homologous recombination
was screened by digestion with HindIII, SphI and EcoRV. A clone
which was coincident in all the cutting patterns was secured and
named pAd328-DS.DELTA.19k. According to above procedure described
to make pAd H5/S19 8DS plasmid, pAd328 H5/S19_DS and pAd328
H5/S19_DS.DELTA.19k were generated by homologous recombination
between pHex-SAd19 Hexon and pAd328_DS or pAd328-DS.DELTA.19k (FIG.
2D).
<2-3> Preparation of Chimeric Adenovirus Recombined with the
Hexon of SAd19
[0093] The plasmid pAd H5/S19.sub.--8DS was linearized with PacI
and transfected into A549 cells. 14 Days after the transfection,
the cells were observed to undergo cytopathy, and thus collected
along with the medium. In order to completely separate the virus
therefrom, first, the cells were frozen and thawed three times,
followed by centrifugation. The supernatant containing the virus
was subjected to two rounds of plaque purification to give pure
virus, named Ad H5/S19.sub.--8DS virus. Ad H5/S19.sub.--8DS virus
was identified as chimeric adenovirus having the hexon gene of
SAd19 as analyzed by sequencing of their genomic DNA. Ad328
H5/S19_DS and Ad328 H5/S19_DS.DELTA.19k virus were prepared as the
above-mentioned procedure for the generation of Ad
H5/S19.sub.--8DS.
<2-4> Production and Purification of Chimeric Adenovirus
Having Hexon of SAd19
[0094] A549 lung cancer cells were grown at 80% confluency in 30
culture dishes of 150 mm-size and then infected with Ad
H5/S19.sub.--8DS virus at MOI of 20. After incubation at 37.degree.
C. for two days, the cells were harvested by centrifugation at
12,000.times.g for 10 min and then suspended in 10 mL of lysis
buffer (0.5M Tris, pH8.0, 1 mM MgCl.sub.2). Three rounds of
freezing and thawing lysed the cells, followed by refrigerated
centrifugation at 12,000.times.g for 10 min to remove cell debris.
In order to prepare a discontinuous CsCl gradient, 8 mL of a CsCl
solution having a specific gravity of 1.4 was placed into an
ultracentrifuge tube (Beckman, CA, USA) and covered with 6 mL of a
CsCl solution having a specific gravity of 1.2 in such a manner as
to keep the boundary therebetween definitely. A virus sample
filtered through a 0.22 .mu.m filter was loaded onto the CsCl
1.4/1.2 gradient so as not to dishevel the boundary, and the tube
was weight balanced with 10 mM Tris-HCl (pH7.9). The tube was
applied to a SW28 rotor and ultra-centrifuged at 23,000 rpm for 90
min under a refrigerated condition to form a virus band.
[0095] The virus thus separated was again purified using a
continuous CsCl gradient ultracentrifugation process. For this, 8
mL of a CsCl solution having a specific gravity of 1.4 was placed
in an ultracentrifuge tube (Beckman, CA, USA) and then covered with
6 mL of a CsCl solution having a specific gravity of 1.2 in such a
manner as to keep a definite boundary therebetween. Using a
gradient station (Biocomp, Canada), a continuous gradient was
formed in the tube. The virus sample obtained by the discontinuous
CsCl gradient ultracentrifugation was diluted with one volume of 10
mM Tris-HCl (pH7.9) and loaded into the CsCl 1.4/1.2 gradient tube
so as not to blur the boundary therebetween. After being weight
balanced with 10 mM Tris-HCl (pH7.9), the tube was
ultra-centrifuged at 23,000 rpm for 90 min under a refrigerated
condition to form a virus band. It was dialyzed three times with
PBSG buffer (PBS containing 10% Glycerol), while exchanging the
buffer with fresh one every 6 hrs.
Example 3
In Vitro Study of Chimeric Adenovirus Having the Hexon of SAd19
[0096] Ad H5/S19.sub.--8DS virus was in vitro assayed for
selectivity for lung cancer by comparing expression levels of E1A
and LK8 in the lung cancer cell line A549 and the normal cell line
MRCS both of which were infected at various MOIs with the virus. In
addition, Ad H5/S19.sub.--8DS virus was measured for affinity for
the coagulation factors which mediate blood-circulating virus to
liver.
<3-1> MOI-dependent E1A Expression
[0097] A549 lung cancer cells and MRCS normal cells, which were
both grown at about 80% confluency in 6-well plates, were infected
with Ad H5/S19.sub.--8DS virus at an MOI of 100, 25, 10 and 1.
After incubation at 37.degree. C. for 24 hrs, the cells were
harvested by centrifugation at 3,000 rpm for 5 min, suspended in
1.times.SDS-PAGE buffer (50 mM Tris(pH6.8), 2% SDS, 100 mM
dithiothreitol, 0.1% bromophenol blue, 10% Glycerol), heated at
100.degree. C. for 5 min in a water bath, and centrifuged at 10,000
rpm for 2 min. The resulting supernatant was elctrophoresed at 20
mA for about 2 hrs on 4.about.12% SDS-PAGE gel (Invitrogen, CA,
USA). The protein bands separated on the gel were transferred for
about 90 min onto a PVDF membrane using a transfer unit
(Invitrogen, CA, USA) in Tris-Glycine buffer (39 mM Glycine, 48 mM
Tris, 0.037% SDS, 20% methanol) with an electric field of 300 mA
applied thereto. The membrane was blocked at room temperature for
one hour with a TBS Blocking solution (Thermo Scientific, IL, USA).
A mouse anti-E1A monoclonal antibody (BD Pharmingen, CA, USA),
serving as a primary antibody, was 1:3,000 diluted with 5% skim
milk/1.times.TBST buffer, probed at room temperature for one hour
and washed for six times each for 5 min with 1.times.TBST buffer.
Anti-mouse HRP (KPL, MA, USA), serving as a secondary antibody, was
1:5,000 diluted with 5% skim milk/1.times.TBST buffer, probed for
30 min, washed six times each for 5 min with 1.times.TBST buffer,
and reacted with a color developing agent (ECL, Amersham, UK) to
visualize an E1A protein band. The expression level of E1A was
increased in a MOI-dependent manner, being 100-fold higher in A549
cancer cells than in MRC-5 normal cells (FIG. 3).
<3-2> Affinity for Coagulation Factor X
[0098] Ad H5/S19.sub.--8DS was analyzed for affinity for
coagulation factor X using an SPR (Surface Plasmon Resonance)
method. Purified coagulation factor X (HCX-0050, Haematological
Technologies Inc., VT, USA) was applied to a CS5 sensor chip
(Biacore, Sweden). Each of the purified Ad H5.sub.--8DS and Ad
H5/S19.sub.--8DS viruses was diluted at concentrations of
3.0.times.10.sup.11 VP/mL, 1.5.times.10.sup.11 VP/mL and
0.75.times.10.sup.11 VP/mL in HBSP buffer (10 mM HEPES pH7.4, 150
mM NaCl, 0.005% Tween 20) containing 5 mM CaCl.sub.2. HBSEP (10 mM
HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20) was used as
regeneration buffer for detaching virus from the chip. While the
virus solution was passed over the coagulation factor X-immobilized
CM5 sensor chip at a flow rate of 30 .mu.L/min, RU values were
recorded. At the three different concentrations, the adenovirus
serotype 5 Ad H5.sub.--8DS showed SPR signals for immobilized
factors of 300, 150 and 70RU, respectively, whereas Ad
H5/S19.sub.--8DS, the chimeric adenovirus having the hexon of
SAd19, showed very weak or little affinity for coagulation factor X
as the SPR signals were found to be within 5RU at all the three
concentrations (FIG. 4A). Ad328 H5/S19_DS and Ad328 H5/S19_DS
.DELTA.19k were also assayed for affinity for coagulation factor X
by an immunoprecipitation method as described in <3-3> below
using coagulation factor X protein instead of factor IX (FIG.
4D).
<3-3> Affinity for Coagulation Factor IX
[0099] Ad H5/S19.sub.--8DS was assayed for affinity for coagulation
factor IX using an immunoprecipitation method. To 1 mL of PBS in a
tube was added 1.times.10.sup.11 VP of Ad H5.sub.--8DS or Ad
H5/S19.sub.--8DS, together with 10 .mu.g of purified coagulation
factor IX (HCIX-0040, Haematological Technologies Inc., VT, USA), 5
.mu.g of a goat anti-coagulation factor IX antibody and 50 .mu.L of
sepharose-protein G (50% slurry), followed by incubation at
4.degree. C. for 2 hrs in an orbital shaker. After centrifugation
at 5,000 rpm for 5 min, the sepharose-protein G pellet thus
obtained was washed three times with PBS, suspended in 100 .mu.L of
1.times.SDS-PAGE sample buffer (50 mM Tris(pH6.8), 2% SDS, 100 mM
dithiothreitol, 0.1% bromophenol blue, 10% Glycerol), and heated
for 5 min. The suspension was centrifuged and the resulting
supernatant was electrophoresed at 20 mA for about 2 hrs on 4-12%
SDS-PAGE gel. The proteins thus separated were transferred for
about 90 min onto a PVDF membrane in a transfer unit containing
Tris-Glycine buffer (39 mM Glycine, 48 mM Tris, 0.037% SDS, 20%
methanol) to which 300 mA was applied. The membrane was blocked at
room temperature for one hour with a TBS Blocking solution (Thermo
Scientific, IL, USA). A mouse anti-HAd5 fiber monoclonal antibody
(NeoMarkers, CA, USA), serving as a primary antibody, was 1:3,000
diluted with 5% skim milk/1.times.TBST buffer, probed at room
temperature for one hour and washed six times with each for 5 min
1.times.TBST buffer. Anti-mouse HRP (KPL, MA, USA), serving as a
secondary antibody, was 1:5,000 diluted with 5% skim
milk/1.times.TBST buffer, probed for 30 min, washed six times each
for 5 min with 1.times.TBST buffer, and reacted with a color
developing agent (ECL, Amersham, UK) to visualize a fiber band. The
blots on the PVDF membrane were immersed for one hour in
Restore.TM. Western Blot Stripping Buffer (Thermo Scientific, IL,
USA) with shaking in an orbital shaker, washed three times each for
10 min with 1.times.TBST buffer, and blocked at room temperature
for one hour with a TBS Blocking solution. A sheep anti-coagulation
factor IX antibody (Affinity Biologicals, Canada), used as a
primary antibody, was 1:3,000 diluted with 5% skim
milk/1.times.TBST buffer, probed at room temperature for one hour,
and washed six times each for 5 min with 1.times.TBST buffer.
Anti-sheep HRP (KPL, MA, USA), serving as a secondary antibody, was
1:5,000 diluted with 5% skim milk/1.times.TBST buffer, probed for
30 min, washed for 5 min six times with 1.times.TBST buffer, and
reacted with a color developing agent (ECL, Amersham, UK) to
visualize a coagulation factor IX band (FIG. 4B). Ad328 H5/S19_DS
and Ad328 H5/S19_DS .DELTA.19k were also assayed for affinity for
coagulation factor IX using an immunoprecipitation method as
described above (FIG. 4C).
Example 4
In Vivo Study of Chimeric Adenovirus Having the Hexon of SAd19
<4-1> Biodistribution
[0100] Ad H5/S19.sub.--8DS was intravenously injected in a dose of
3.times.10.sup.10 VP into Balb/c normal mice and nude mice, both 6
weeks old. Two days after injection, DNA was isolated from the
brain, the liver, the lung, the heart, the thymus, the spleen, the
ovary, the uterus and blood using a DNeasy Blood and Tissue Kit
(QIAGEN, Germany). The isolated DNA was quantitatively analyzed
using an OD spectrophotometer and used in an amount of 200 ng as a
template for PCR. The E4 region of adenovirus was amplified by PCR
using a set of primers of SEQ ID NO: 14 (5'-ACT CGA GCA CGT TGT GCA
TTG TCA-3') and SEQ ID NO: 15 (5'-TGT CGA CTA GTT TTC TTA AAA
TGG-3'). Starting from denaturation at 94.degree. C. for 5 min, PCR
was performed with 30 cycles of denaturation at 94.degree. C. for
30 sec, annealing at 55.degree. C. for 40 sec and extension at
72.degree. C. for 1 min, followed by extension at 72.degree. C. for
additional 3 min. The PCR products were run on 1% agarose gel in
the presence of an electric field to analyze the distributions of
adenovirus by organs. When injected, the HAd5, i.e., Ad
H5.sub.--8DS, was most highly observed in the liver and the lung,
and partially detected in the heart and the spleen. In contrast,
the chimeric adenovirus having the hexon of SAd19, Ad
H5/S19.sub.--8DS virus, was mostly detected in the heart and the
spleen, and only a part was detected in the liver and the lung
(FIG. 5). This distribution pattern is quite different from the
biodistribution of adenovirus serotype 5, which is known to be
transduced into the liver upon intravenous injection and induce
hepatotoxicity upon injection in large doses. That is, Ad
H5/S19.sub.--8DS virus was transduced in a very small amount into
the liver, as inferred from the observation of Example <3-2>
that the virus showed little affinity for coagulation factor X.
Example 5
In Vitro Assay for Ability of Chimeric Adenovirus Having the Hexon
of SAd19 to Evade Immune Recognition of HAd5 Neutralizing
Antibody
<5-1> Examination of the Effect of Anti-HAd5-Positive Plasma
on Transduction of Ad H5/S19.sub.--8DS and Ad328 H5/S19_DS by E1A
Expression Pattern
[0101] Wild-type HAd5 was intramuscularly injected at a dose of
1.times.10.sup.11 VP into the hind legs of mice and two weeks
thereafter, the same dose of the virus was injected again to boost
an immune response. Sera were separated from the blood taken from
all of the virus-injected mice and then measured for
anti-adenovirus antibody levels. In this regard, 1/25, 1/100, and
1/1,000 dilutions of the sera were added to adenovirus-coated ELISA
plates, incubated for one hour, and washed three times with PBST
(PBS containing 0.1% Tween20), followed by incubation for one hour
with a 1/5,000 dilution of an HRP-conjugated anti-mouse IgG
antibody. After the plates were washed five times with PBST (PBS
containing 0.1% Tween20), a color was developed by reaction for 30
min with a TMB substrate and termination with 1 M phosphoric acid.
The plates were measured for absorbance. They were regarded as
inducing a positive immune response when the absorbance thereof was
50% or higher as compared with that of a positive control, prepared
by reacting with a 1/1000 dilution of an anti-adenovirus antibody
(AbD Serotec, NC, USA). A549 cells, grown at 90% confluency in
6-well plates, were infected at MOI of 25 with Ad H5/S19.sub.--8DS
or Ad328 H5/S19_DS one hr before being incubated at room
temperature with the mouse plasma which were determined to be of
positive immune response. After incubation at 37.degree. C. for 24
hrs, the cells were harvested by centrifugation at 3,000 rpm for 5
min, suspended in 1.times.SDS-PAGE sample buffer (50 mM Tris
(pH6.8), 2% SDS, 100 mM dithiothreitol, 0.1% bromophenol blue, 10%
glycerol), heated at 100.degree. C. for 5 min in a water bath, and
centrifuged at 10,000 rpm for 2 min. The clarified supernatant was
run for about 2 hrs on 4-12% SDS-PAGE gel in the presence of 20 mA
using an electrophoresis kit (Novex). The separated proteins were
transferred for 90 min onto a PVDF membrane in a transfer unit
(Novex) containing Tris-Glycine buffer (39 mM Glycine, 48 mM Tris,
0.037% SDS, 20% methanol) with an electric field of 300 mA applied
thereinto. The membrane was blocked at room temperature for one
hour with a TBS Blocking solution (Thermo Scientific, IL, USA). The
membrane was incubated at room temperature for one hour with a
1:3,000 dilution of mouse anti-E1A monoclonal antibody (BD
Pharmingen, CA, USA), a primary antibody, in 5% skim
milk/1.times.TBST buffer and then washed six times each for 5 min
with 1.times.TBST buffer. Incubation with a 1:5,000 dilution of
anti-mouse HRP (KPL, MA, USA) as a secondary antibody in 5% skim
milk/1.times.TBST buffer for 30 min was followed by washing six
times each for 5 min with 1.times.TBST buffer. An E1A protein band
was visualized with a color developing reagent (ECL, Amersham, UK).
Whereas the transduction of Ad H5.sub.--8DS was inhibited by the
anti-HAd5 antibody-positive plasma, there were no effects of the
anti-HAd5 antibody-positive plasma on the transduction of the
chimeric adenovirus Ad H5/S19.sub.--8DS (FIG. 6A), Ad328 H5/S19_DS
and Ad328 H5/S19_DS .DELTA.19k (FIG. 6B).
<5-2> Examination of the Effect of Anti-HAd5-Positive Plasma
on H5/S19.sub.--8DS Transduction by Immunostaining
[0102] After being incubated at room temperature for one hour with
the mouse plasmas which were determined to be of positive immune
response, 90% confluently cultured A549 cells were infected with Ad
H5/S19.sub.--8DS or Ad H5/S19_DS at an MOI of 25, followed by
incubation at 37.degree. C. for two days. The cells were fixed with
cold methanol and blocked for one hour with a protein-free T20
(TBS) blocking buffer (Thermo Scientific, IL, USA). The cells were
incubated for one hour with a dilution (1/3,000) of a mouse
anti-E1A monoclonal antibody (BD Pharmingen, CA, USA) in PBST
buffer and washed six times each for 5 min with PBST buffer. Then,
the cells were again treated for one hour with a dilution (1/5,000)
of a HRP-conjugated anti-mouse secondary antibody in PBST buffer,
washed six times each for 5 min with PBST buffer, and reacted with
a DAB solution to produce a color. The anti-HAd5 antibody-positive
plasma was found to inhibit the transduction of Ad H5.sub.--8DS,
but to have no influence on the transduction of the chimeric
adenovirus Ad H5/S19.sub.--8DS (FIG. 7A), Ad328 H5/S19_DS and Ad
H5/S19_DS.DELTA.19k (FIG. 7B).
<5-3> In Vivo Examination into Ability of Chimeric Adenovirus
Having the Hexon of SAd19 to Evade Immune Recognition of HAd5
Neutralizing Antibody
[0103] Wild-type HAd5 was intramuscularly injected at a dose of
1.times.10.sup.11 VP into the hind legs of hamster and, two weeks
thereafter, which the same dose of the virus was injected again to
induce an immune response. Sera were separated from the blood taken
from all of the virus-injected hamsters and then measured for
anti-adenovirus antibody level therein. In this regard, 1/25,
1/100, and 1/1,000 dilutions of the sera were added to
adenovirus-coated ELISA plates, incubated for one hour, and washed
three times with PBST (PBS containing 0.1% Tween20), followed by
incubation for one hour with a 1/5,000 dilution of an
HRP-conjugated anti-hamster IgG antibody. After the plates were
washed five times with PBST (PBS containing 0.1% Tween20), a color
was developed by reaction for 30 min with a TMB substrate and
termination with 1 M phosphoric acid. The plates were measured for
absorbance. They were regarded as inducing a positive immune
response when the absorbance thereof was 50% or higher as compared
with that of a positive control, prepared by reacting with a
1/1,000 dilution of an anti-adenovirus antibody. The immunized
hamsters into which HAd5, i.e., Ad H5.sub.--8DS was intravenously
injected at a dose of 1.times.10.sup.11 VP was found to be too low
in blood LK8 level to detect. In contrast, the intravenous
injection of Ad H5/S19.sub.--8DS at a dose of 1.times.10.sup.11 VP
ensured a blood LK8 level of 200 ng/mL or higher, and the
expression amount was maintained at a level of 200 ng/mL or higher
for 28 days, indicating that the chimeric adenovirus having the
hexon of SAd19 is not inhibited on their transduction by the
anti-human adenovirus serotype 5 neutralizing antibody (FIG.
8).
Example 6
Toxicity of Chimeric Adenovirus Having the Hexon of SAd19
[0104] The hexon of SAd19 was studied for toxicity. To this end,
the virus was intravenously injected at doses of 1.times.10.sup.11
VP, 5.times.10.sup.10 VP, 1.times.10.sup.10 VP, 5.times.10.sup.9
VP, and 1.times.10.sup.9 VP, respectively, into five groups of five
Balb/c mice which were weighed every other day. At week three and
six after the injection, bloods and sera were taken from the mice
and analyzed for leukocyte, erythrocyte, platelet and hemoglobin
levels, hematocrit, MCV (Mean Corpuscular Volume), MCH (Mean
Corpuscular Hemoglobin Concentration), MCHC (Mean Corpuscular
Hemoglobin Concentration) and differential leucocyte count. An
examination was made of levels of albumin, total protein, SGPT
(ALT), SGOT (AST) and ALP for liver function tests, creatinine and
BUN for kidney function tests, creatinine kinase for muscle tests,
and total cholesterol and glucose for metabolism tests. Of 5 mice
in the group which was injected at a dose of 1.times.10.sup.11 VP
with the human adenovirus serotype 5 virus Ad H5.sub.--8DS, four
were dead on day 5 while the remaining one was heavily sick. They
were found to suffer from hepatocirrhosis by an autopsy, with no
abnormality in the other organs. All the other groups were observed
to be normal, as examined by autopsy (FIG. 9).
<6-1> Blood Test
[0105] The HAd5, i.e., Ad H5.sub.--8DS was intravenously injected
at doses of 1.times.10.sup.11 VP, 5.times.10.sup.10 VP,
1.times.10.sup.10 VP, 5.times.10.sup.9 VP, and 1.times.10.sup.9 VP.
On day 5, all of the mice injected with 1.times.10.sup.11 VP of the
virus were dead. On the other hand, the other groups showed no
observations of abnormal toxicity. There were no statistical
differences in hematological and blood biochemistry tests between
the test groups and the negative control group injected with PBS
(FIG. 10). Abnormal toxicity observations were found in none of the
groups to which the chimeric adenovirus having the hexon of SAd19,
Ad H5/S19.sub.--8DS, was injected at doses of 1.times.10.sup.11 VP,
5.times.10.sup.10 VP, 1.times.10.sup.10 VP, 5.times.10.sup.9 VP and
1.times.10.sup.9 VP. Also, no statistical differences in
hematological and blood biochemistry tests were found between the
test groups and the PBS-injected negative control group (FIG. 11).
Hepatotoxicity detected in the group injected with a maximum dose
of HAd5 was not observed in the corresponding group injected with
the chimeric adenovirus having the hexon of SAd19.
<6-2> Biopsy
[0106] The brain, the heart, the liver, the lung, the kidney, the
spleen, the uterus and the ovary were excised from the injected
mice, fixed with 3.7% neutral formalin, embedded in paraffin,
sliced, and stained with H&E. No abnormalities of organs were
found in all of the test groups except the group injected with
1.times.10.sup.11 VP of human adenovirus serotype. Liver biopsy
results showed massive hepatic necrosis in the 1.times.10.sup.11
VP-injected group, which was thought to induce acute hepatic
failure of which the mice died. This was a general observation of
adenoviral hepatotoxicity. As for the chimeric adenovirus having
the hexon of SAd19, it did not hepatotoxicity even when injected at
a dose of 1.times.10.sup.11 VP, indicating that the hexon of SAd19
may be a promising solution to the hepatotoxicity problem caused
when HAd5 is used.
Example 7
Anti-Tumor Activity of Chimeric Adenovirus Having the Hexon of
SAd19 in Animal Tumor Model
<7-1> Tumor-Selective, Anti-Tumor Activity of Chimeric
Adenovirus on Human Lung Cancer Xenograft Animal Model
[0107] The non-small cell lung cancer (NSCLC) cell line NCI-H460
was subcutaneously injected at a dose of 5.times.10.sup.6 cells
into the right flank of immune-deficient Balb/c nude mice to form
tumors 50.about.100 mm.sup.3 in size. The mice were randomly
divided into four groups of five mice. Mice of control groups were
intravenously administered with either replication defective
adenovirus carrying LK8 gene, Ad-LK8, at a dose of 1.times.10.sup.9
pfu or PBS, three times at regular intervals of two days. As for
test groups, they were intravenously injected with Ad
H5/S19.sub.--8DS in doses of 1.times.10.sup.9 pfu and
2.times.10.sup.8 pfu, three times at regular intervals. The tumors
were measured for size every two or three days to plot tumor growth
curves. On Day 24 after the first injection of virus, the group
injected at a dose of 1.times.10.sup.9 pfu with Ad H5/S19.sub.--8DS
exhibited a 74% higher tumor growth inhibition rate than the
PBS-administered group and a 64% higher rate than the
Ad-LK8-administered group. On the other hand, the group injected at
a dose of 2.times.10.sup.8 pfu with Ad H5/S19.sub.--8DS showed a
61% higher tumor growth inhibition rate than the PBS-administered
group, and a 48% higher rate than the Ad-LK8-administered group. A
2-way RM ANOVA test revealed statistical significances of both the
groups injected with Ad H5/S19.sub.--8DS over the PBS-administered
group from 17 days after the injection (Day 17 after the injection,
P<0.05; Days 21 and 24 after the injection, P<0.01) (FIG.
12).
<7-2> Tumor-Selective, Anti-Tumor Activity of Chimeric
Adenovirus on Human Lung Cancer Orthotopic Animal Model Immunized
with HAd5
[0108] Balb/c nude mice were immunized with HAd5 by two rounds of
intramuscular injection at a dose of 1.times.10.sup.10 VP for each
round at regular intervals of two weeks into the hind leg thereof.
Blood taken from the mice was found to contain anti-HAd5 antibody.
The NSCLC cell line NCI-H2172 was inoculated at a dose of
1.times.10.sup.6 cells into the tail vein. The naeve and immunized
mice were received triple intravenous injections of either Ad-LK8,
Ad H5/S19.sub.--8DS or Ad H5.sub.--8DS on day 7, 9 and 11 after
tumor inculation at an injection dose of 1.times.10.sup.9 pfu. At
Week 6, the lung was excised from the mice (FIG. 13). The tumors
generated in the lung were counted and divided into groups by size:
x.ltoreq.0.5 cm, 0.5 cm.ltoreq.x.ltoreq.0.7 cm, and 0.7
cm<x.ltoreq.1 cm, which were respectively given 5, 7 and 10
points. The tumor generated in the lung was found to count, on
average, 18.6 in the PBS-administered, negative control group, and
11.2 in the Ad-LK8-administered group. As for the groups
administered with Ad H5/S19.sub.--8DS, they were found to have 5.6
and 5.4 tumors, on average, in the lung when immunized or not
immunized with the HAd5, respectively. On the other hand, mice in
the groups administered with Ad H5.sub.--8DS were found to have
10.8 and 6.2 tumors, on average, in the lungs of immunized or naive
mice. Therefore, Ad H5/S19.sub.--8DS showed almost the same titers
irrespective of immunization, but Ad H5.sub.--8DS showed efficacy
decreased significantly by anti-HAd5 antibody. Mean lung volumes
were measured to be 274 mm.sup.3 in normal mice, 570 mm.sup.3 in
the PBS-administered group, and 480 mm.sup.3 in the
Ad-LK8-administered group. However, the mouse groups injected with
Ad H5/S19.sub.--8DS were found to have 370 mm.sup.3 lungs and 360
mm.sup.3 lungs, respectively, when immunized or non-immunized.
There was no significant difference in lung volume between them. In
the groups administered with Ad H5.sub.--8DS, mean lung volumes
were 410 mm.sup.3 and 350 mm.sup.3, respectively, when immunized or
non-immunized. Mean weights of the lungs were measured to be 170 mg
in the normal mice, 376 mg in the PBS-administered group and 278 mg
in the Ad-LK8-administered group. When injected with Ad
H5/S19.sub.--8DS, mean lung weight of both the immunized or
non-immunized mouse groups was measured to be 218 mg, while mean
lung weights of Ad H5.sub.--8DS-administered groups were 240 mg for
immunized and 230 mg for non-immunized. Turning to the scores
gained according to tumor sizes, they were measured to be, on
average, 107.8 in the PBS-administered group and 60.8 in the
Ad-LK8-administered group. When injected with Ad H5/S19.sub.--8DS,
the immunized group and the non-immunized group gained 28 and 27
points, respectively. On the other hand, the immunized group and
non-immunized group gained 57.2 and 32.6, respectively, when
injected with Ad H5.sub.--8DS (FIG. 14). Taken together, these data
indicate that Ad H5/S19.sub.--8DS, irrespective of immunization
with HAd5, can exert anti-tumor activity even when intravenously
injected.
[0109] While the invention has been described with respect to the
above specific embodiments, it should be recognized that various
modifications and changes may be made to the invention by those
skilled in the art which also fall within the scope of the
invention as defined by the appended claims.
Sequence CWU 1
1
21126DNAArtificial SequenceHexon F primer (Mfe I) 1ggcaattgat
ggccacccca tcgatg 26226DNAArtificial SequenceHexon R primer (Mfe I)
2ggcaattgtt aggtggtggc gttgcc 2632799DNASimian Adenovirus Serotype
19 3atggccaccc catcgatgat gccgcagtgg tcttacatgc acatcgccgg
gcaggacgcc 60tcggagtacc tgagccccgg cctcgtgcag tttgcccgcg ccaccgacac
ctacttcagc 120ttgggaaaca agtttagaaa ccccaccgtg gcccccacgc
acgatgtgac cacggaccgc 180tcgcagagac tgaccctgcg ctttgtgccc
gtagaccggg aggacaccgc ttactcgtac 240aaagtgcgct tcaccctagc
agtaggggac aaccgtgtgt tggacatggc cagtacctat 300tttgacatcc
ggggcacgct agaccgcggt cccagcttca agccttattc tggcacagct
360tacaacgcgc tggcccctaa gggcgctccg aatgcttgtc agtggacaac
cacgaatggg 420ggtaacaaaa ctaattcatt tgctcaggcc ccagtaatcg
gcctaagtat tgacgccacc 480aacgggctaa aagtagggga ggagatacct
gccactggag gggcaaatac gcccgtgtac 540gccgacaaaa cattccagcc
tgaacctcaa gtaggagaaa caaaatggaa ttctaaccct 600actgagaatg
cagctggaag aattttaaag ccaaacacac ctatgcagcc ctgctacgga
660tcgtacgctc gaccaacaaa cgaaaaagga ggacaggcaa agctagttac
taacggtcaa 720gacaatcaaa caacgccaga cgttagttta aactttttta
ctactgcgtc agaaaccaca 780acattcacgc cgaaagttgt tctgtatagc
gaaaacgtca acttggaagc tccagatacg 840catctagtat acaagccaga
cggcactgac ggaatcacca acgccgaaac tctcttagga 900cttcagtcag
ctccgaacag accaaattac attggttttc gagataactt tataggccta
960atgtattaca actccactgg aaatatgggg gttctggccg gacaggcttc
gcaattaaac 1020gcagtggttg atttgcaaga cagaaacaca gaattgtcat
accaacttat gctggatgcc 1080ctgggagacc gcagtaggta cttctccatg
tggaatcagg ctgtggacag ctatgatcct 1140gatgttagga taatagaaaa
ccatggcgta gaagacgaat tgcctaacta ctgctttcca 1200cttaatgcgc
aaggtgtagc caacacttac cagggcgtta aaaatggctc gggaaactgg
1260tcgaaagaca ctaacgttgg cacggcaaat gaaatcggga taggtaacat
ttttgctttc 1320gaaattaatc tagctgccaa cttgtggcga agttttcttt
actccaatgt ggccttgtac 1380ctgcccgacg cttacaaatt aacccctgac
aacattacgc ttccagacaa caaaaacacc 1440tacgagtata taaacggccg
cgtggctgcg cccgcctctc tagacaccta cgttaacatt 1500ggagcgcgct
ggtctcccga cccgatggat aacgttaacc cctttaacca tcaccgcaac
1560gcgggtttgc gctatcgctc tatgctactg ggcaacggcc gctacgttcc
ttttcacata 1620caagtgcccc aaaaattttt tgccattaaa aacctgctgc
tccttccggg ctcctacacc 1680tacgagtgga attttaggaa ggatgtaaac
atgattttgc agagcacact tggtaacgac 1740ctacgggttg acggggcgag
cgttaggttt gatagcatca acctgtacgc aaattttttc 1800cccatggcgc
ataacacagc gtccacgctg gaagccatgc tgcgtaatga cacaaacgac
1860cagtctttta acgactacct ctgcgccgcc aacatgcttt accctattcc
ggccaatgcc 1920actagtgttc caatttccat tccctctcgc aattgggcag
ctttccgcgg ctggagcttc 1980acgcgactta aaactcggga aacgccttcc
cttggatctg gatttgaccc ttactttgtg 2040tactccggtt ccatccccta
cctggatggc accttttatc taaaccatac ttttaaaaag 2100gtgtccatta
tgttcgactc ctcagtaagc tggcctggca acgaccgcct cctgactcct
2160aatgaattcg agattaagcg gtcggtggac ggagaaggct acaacgtggc
ccaaagcaat 2220atgacaaaag attggttttt aattcaaatg ttaagtcact
acaacatcgg ttatcaggga 2280ttctacgttc cagaatccta caaggacaga
atgtactctt ttttcagaaa cttccaacct 2340atgagccgac aggtggtgga
tcctgtaaat tacacaaact acaaggaagt tacattgccg 2400tatcagcata
ataattcagg ctttgtgggg tacatgggtc ctaccatgag agagggtcag
2460gcctacccag ctaactaccc ttacccgcta ataggcaaaa cggcagtacc
cagcctcacc 2520cagaaaaagt tcctgtgcga cagggtgatg tggagaattc
ccttttctag caactttatg 2580tctatggggg ctctgaccga cctggggcag
aacatgctgt atgccaactc cgctcatgcc 2640ttggacatga cttttgaggt
ggatcccatg gatgagccca cgcttcttta tgttttgttc 2700gaagtcttcg
acgtggtgcg cattcaccag ccgcaccgcg gcgtcatcga ggccgtctac
2760ctgcgcacgc ctttctctgc cggcaacgcc accacctaa 2799424DNAArtificial
SequenceHexonL F primer (XhoI) 4ctcgaggtcg caccgtcgca tgcg
24524DNAArtificial SequenceHexonL R primer (EcoRI) 5gaattccttg
gaaagcgggc gcgc 24624DNAArtificial SequenceHexonR F primer (EcoRI)
6gaattcagaa gcaagcaaca tcaa 24724DNAArtificial SequenceHexonR R
primer (HindIII) 7aagcttctga tagtgttcca gtgc 24823DNAArtificial
SequenceDS promoter F primer 8cttctcgctg ctttatcccc atc
23921DNAArtificial SequenceDS Promoter R primer 9ctcggaggct
tcagcagacg c 211018DNAArtificial SequenceHexon HR F primer
10atgcgcaagg tgtagcca 181118DNAArtificial SequenceHexon HR R primer
11agcgtgctgg ccagcgtg 181218DNAArtificial SequenceCMV sense primer
12cccgttacat aacttacg 181322DNAArtificial SequenceE1A antisense
primer 13ttatggcctg gggcgtttac ag 221424DNAArtificial SequenceE4
sense primer 14actcgagcac gttgtgcatt gtca 241524DNAArtificial
SequenceE4 antisense primer 15tgtcgactag ttttcttaaa atgg
2416932PRTArtificial Sequencesynthetic construct of SAd19 Hexon
16Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ala1
5 10 15 Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe
Ala 20 25 30 Arg Ala Thr Asp Thr Tyr Phe Ser Leu Gly Asn Lys Phe
Arg Asn Pro 35 40 45 Thr Val Ala Pro Thr His Asp Val Thr Thr Asp
Arg Ser Gln Arg Leu 50 55 60 Thr Leu Arg Phe Val Pro Val Asp Arg
Glu Asp Thr Ala Tyr Ser Tyr65 70 75 80 Lys Val Arg Phe Thr Leu Ala
Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95 Ala Ser Thr Tyr Phe
Asp Ile Arg Gly Thr Leu Asp Arg Gly Pro Ser 100 105 110 Phe Lys Pro
Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125 Ala
Pro Asn Ala Cys Gln Trp Thr Thr Thr Asn Gly Gly Asn Lys Thr 130 135
140 Asn Ser Phe Ala Gln Ala Pro Val Ile Gly Leu Ser Ile Asp Ala
Thr145 150 155 160 Asn Gly Leu Lys Val Gly Glu Glu Ile Pro Ala Thr
Gly Gly Ala Asn 165 170 175 Thr Pro Val Tyr Ala Asp Lys Thr Phe Gln
Pro Glu Pro Gln Val Gly 180 185 190 Glu Thr Lys Trp Asn Ser Asn Pro
Thr Glu Asn Ala Ala Gly Arg Ile 195 200 205 Leu Lys Pro Asn Thr Pro
Met Gln Pro Cys Tyr Gly Ser Tyr Ala Arg 210 215 220 Pro Thr Asn Glu
Lys Gly Gly Gln Ala Lys Leu Val Thr Asn Gly Gln225 230 235 240 Asp
Asn Gln Thr Thr Pro Asp Val Ser Leu Asn Phe Phe Thr Thr Ala 245 250
255 Ser Glu Thr Thr Thr Phe Thr Pro Lys Val Val Leu Tyr Ser Glu Asn
260 265 270 Val Asn Leu Glu Ala Pro Asp Thr His Leu Val Tyr Lys Pro
Asp Gly 275 280 285 Thr Asp Gly Ile Thr Asn Ala Glu Thr Leu Leu Gly
Leu Gln Ser Ala 290 295 300 Pro Asn Arg Pro Asn Tyr Ile Gly Phe Arg
Asp Asn Phe Ile Gly Leu305 310 315 320 Met Tyr Tyr Asn Ser Thr Gly
Asn Met Gly Val Leu Ala Gly Gln Ala 325 330 335 Ser Gln Leu Asn Ala
Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu 340 345 350 Ser Tyr Gln
Leu Met Leu Asp Ala Leu Gly Asp Arg Ser Arg Tyr Phe 355 360 365 Ser
Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile 370 375
380 Ile Glu Asn His Gly Val Glu Asp Glu Leu Pro Asn Tyr Cys Phe
Pro385 390 395 400 Leu Asn Ala Gln Gly Val Ala Asn Thr Tyr Gln Gly
Val Lys Asn Gly 405 410 415 Ser Gly Asn Trp Ser Lys Asp Thr Asn Val
Gly Thr Ala Asn Glu Ile 420 425 430 Gly Ile Gly Asn Ile Phe Ala Phe
Glu Ile Asn Leu Ala Ala Asn Leu 435 440 445 Trp Arg Ser Phe Leu Tyr
Ser Asn Val Ala Leu Tyr Leu Pro Asp Ala 450 455 460 Tyr Lys Leu Thr
Pro Asp Asn Ile Thr Leu Pro Asp Asn Lys Asn Thr465 470 475 480 Tyr
Glu Tyr Ile Asn Gly Arg Val Ala Ala Pro Ala Ser Leu Asp Thr 485 490
495 Tyr Val Asn Ile Gly Ala Arg Trp Ser Pro Asp Pro Met Asp Asn Val
500 505 510 Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg Tyr Arg
Ser Met 515 520 525 Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His Ile
Gln Val Pro Gln 530 535 540 Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu
Leu Pro Gly Ser Tyr Thr545 550 555 560 Tyr Glu Trp Asn Phe Arg Lys
Asp Val Asn Met Ile Leu Gln Ser Thr 565 570 575 Leu Gly Asn Asp Leu
Arg Val Asp Gly Ala Ser Val Arg Phe Asp Ser 580 585 590 Ile Asn Leu
Tyr Ala Asn Phe Phe Pro Met Ala His Asn Thr Ala Ser 595 600 605 Thr
Leu Glu Ala Met Leu Arg Asn Asp Thr Asn Asp Gln Ser Phe Asn 610 615
620 Asp Tyr Leu Cys Ala Ala Asn Met Leu Tyr Pro Ile Pro Ala Asn
Ala625 630 635 640 Thr Ser Val Pro Ile Ser Ile Pro Ser Arg Asn Trp
Ala Ala Phe Arg 645 650 655 Gly Trp Ser Phe Thr Arg Leu Lys Thr Arg
Glu Thr Pro Ser Leu Gly 660 665 670 Ser Gly Phe Asp Pro Tyr Phe Val
Tyr Ser Gly Ser Ile Pro Tyr Leu 675 680 685 Asp Gly Thr Phe Tyr Leu
Asn His Thr Phe Lys Lys Val Ser Ile Met 690 695 700 Phe Asp Ser Ser
Val Ser Trp Pro Gly Asn Asp Arg Leu Leu Thr Pro705 710 715 720 Asn
Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu Gly Tyr Asn Val 725 730
735 Ala Gln Ser Asn Met Thr Lys Asp Trp Phe Leu Ile Gln Met Leu Ser
740 745 750 His Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Val Pro Glu Ser
Tyr Lys 755 760 765 Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro
Met Ser Arg Gln 770 775 780 Val Val Asp Pro Val Asn Tyr Thr Asn Tyr
Lys Glu Val Thr Leu Pro785 790 795 800 Tyr Gln His Asn Asn Ser Gly
Phe Val Gly Tyr Met Gly Pro Thr Met 805 810 815 Arg Glu Gly Gln Ala
Tyr Pro Ala Asn Tyr Pro Tyr Pro Leu Ile Gly 820 825 830 Lys Thr Ala
Val Pro Ser Leu Thr Gln Lys Lys Phe Leu Cys Asp Arg 835 840 845 Val
Met Trp Arg Ile Pro Phe Ser Ser Asn Phe Met Ser Met Gly Ala 850 855
860 Leu Thr Asp Leu Gly Gln Asn Met Leu Tyr Ala Asn Ser Ala His
Ala865 870 875 880 Leu Asp Met Thr Phe Glu Val Asp Pro Met Asp Glu
Pro Thr Leu Leu 885 890 895 Tyr Val Leu Phe Glu Val Phe Asp Val Val
Arg Ile His Gln Pro His 900 905 910 Arg Gly Val Ile Glu Ala Val Tyr
Leu Arg Thr Pro Phe Ser Ala Gly 915 920 925 Asn Ala Thr Thr 930
17952PRTArtificial SequenceSynthetic construct of HAd5 Hexon 17Met
Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser1 5 10
15 Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala
20 25 30 Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg
Asn Pro 35 40 45 Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg
Ser Gln Arg Leu 50 55 60 Thr Leu Arg Phe Ile Pro Val Asp Arg Glu
Asp Thr Ala Tyr Ser Tyr65 70 75 80 Lys Ala Arg Phe Thr Leu Ala Val
Gly Asp Asn Arg Val Leu Asp Met 85 90 95 Ala Ser Thr Tyr Phe Asp
Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110 Phe Lys Pro Tyr
Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125 Ala Pro
Asn Pro Cys Glu Trp Asp Glu Ala Ala Thr Ala Leu Glu Ile 130 135 140
Asn Leu Glu Glu Glu Asp Asp Asp Asn Glu Asp Glu Val Asp Glu Gln145
150 155 160 Ala Glu Gln Gln Lys Thr His Val Phe Gly Gln Ala Pro Tyr
Ser Gly 165 170 175 Ile Asn Ile Thr Lys Glu Gly Ile Gln Ile Gly Val
Glu Gly Gln Thr 180 185 190 Pro Lys Tyr Ala Asp Lys Thr Phe Gln Pro
Glu Pro Gln Ile Gly Glu 195 200 205 Ser Gln Trp Tyr Glu Thr Glu Ile
Asn His Ala Ala Gly Arg Val Leu 210 215 220 Lys Lys Thr Thr Pro Met
Lys Pro Cys Tyr Gly Ser Tyr Ala Lys Pro225 230 235 240 Thr Asn Glu
Asn Gly Gly Gln Gly Ile Leu Val Lys Gln Gln Asn Gly 245 250 255 Lys
Leu Glu Ser Gln Val Glu Met Gln Phe Phe Ser Thr Thr Glu Ala 260 265
270 Thr Ala Gly Asn Gly Asp Asn Leu Thr Pro Lys Val Val Leu Tyr Ser
275 280 285 Glu Asp Val Asp Ile Glu Thr Pro Asp Thr His Ile Ser Tyr
Met Pro 290 295 300 Thr Ile Lys Glu Gly Asn Ser Arg Glu Leu Met Gly
Gln Gln Ser Met305 310 315 320 Pro Asn Arg Pro Asn Tyr Ile Ala Phe
Arg Asp Asn Phe Ile Gly Leu 325 330 335 Met Tyr Tyr Asn Ser Thr Gly
Asn Met Gly Val Leu Ala Gly Gln Ala 340 345 350 Ser Gln Leu Asn Ala
Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu 355 360 365 Ser Tyr Gln
Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe 370 375 380 Ser
Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile385 390
395 400 Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe
Pro 405 410 415 Leu Gly Gly Val Ile Asn Thr Glu Thr Leu Thr Lys Val
Lys Pro Lys 420 425 430 Thr Gly Gln Glu Asn Gly Trp Glu Lys Asp Ala
Thr Glu Phe Ser Asp 435 440 445 Lys Asn Glu Ile Arg Val Gly Asn Asn
Phe Ala Met Glu Ile Asn Leu 450 455 460 Asn Ala Asn Leu Trp Arg Asn
Phe Leu Tyr Ser Asn Ile Ala Leu Tyr465 470 475 480 Leu Pro Asp Lys
Leu Lys Tyr Ser Pro Ser Asn Val Lys Ile Ser Asp 485 490 495 Asn Pro
Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly 500 505 510
Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr 515
520 525 Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu
Arg 530 535 540 Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro
Phe His Ile545 550 555 560 Gln Val Pro Gln Lys Phe Phe Ala Ile Lys
Asn Leu Leu Leu Leu Pro 565 570 575 Gly Ser Tyr Thr Tyr Glu Trp Asn
Phe Arg Lys Asp Val Asn Met Val 580 585 590 Leu Gln Ser Ser Leu Gly
Asn Asp Leu Arg Val Asp Gly Ala Ser Ile 595 600 605 Lys Phe Asp Ser
Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala His 610 615 620 Asn Thr
Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn Asp625 630 635
640 Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu Tyr Pro Ile
645 650 655 Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg
Asn Trp 660 665 670 Ala Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys
Thr Lys Glu Thr 675 680 685 Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr
Tyr Thr Tyr Ser Gly Ser 690 695 700 Ile Pro Tyr Leu Asp Gly Thr Phe
Tyr Leu Asn His Thr Phe Lys Lys705 710 715 720 Val Ala Ile Thr Phe
Asp Ser Ser Val Ser Trp Pro Gly Asn Asp Arg 725 730 735 Leu Leu Thr
Pro Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu 740 745 750 Gly
Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp Phe Leu Val 755 760
765 Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Ile Pro
770 775 780 Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe
Gln Pro785 790 795 800 Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr
Lys Asp Tyr
Gln Gln 805 810 815 Val Gly Ile Leu His Gln His Asn Asn Ser Gly Phe
Val Gly Tyr Leu 820 825 830 Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr
Pro Ala Asn Phe Pro Tyr 835 840 845 Pro Leu Ile Gly Lys Thr Ala Val
Asp Ser Ile Thr Gln Lys Lys Phe 850 855 860 Leu Cys Asp Arg Thr Leu
Trp Arg Ile Pro Phe Ser Ser Asn Phe Met865 870 875 880 Ser Met Gly
Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn 885 890 895 Ser
Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro Met Asp Glu 900 905
910 Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val
915 920 925 His Arg Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg
Thr Pro 930 935 940 Phe Ser Ala Gly Asn Ala Thr Thr945 950
18925PRTArtificial SequenceSynthetic construct of HAd41 Hexon 18Met
Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ala1 5 10
15 Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala
20 25 30 Arg Ala Thr Asp Thr Tyr Phe Ser Leu Gly Asn Lys Phe Arg
Asn Pro 35 40 45 Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg
Ser Gln Arg Leu 50 55 60 Thr Leu Arg Phe Val Pro Val Asp Arg Glu
Asp Thr Ala Tyr Ser Tyr65 70 75 80 Lys Val Arg Phe Thr Leu Ala Val
Gly Asp Asn Arg Val Leu Asp Met 85 90 95 Ala Ser Thr Tyr Phe Asp
Ile Arg Gly Val Leu Asp Arg Gly Pro Ser 100 105 110 Phe Lys Pro Tyr
Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Thr 115 120 125 Ala Pro
Asn Pro Cys Glu Trp Lys Asp Asn Asn Lys Ile Lys Val Arg 130 135 140
Gly Gln Ala Pro Phe Ile Gly Thr Asn Ile Asn Lys Asp Asn Gly Ile145
150 155 160 Gln Ile Gly Thr Asp Thr Thr Asn Gln Pro Ile Tyr Ala Asp
Lys Thr 165 170 175 Tyr Gln Pro Glu Pro Gln Val Gly Gln Thr Gln Trp
Asn Ser Glu Val 180 185 190 Gly Ala Ala Gln Lys Val Ala Gly Arg Val
Leu Lys Asp Thr Thr Pro 195 200 205 Met Leu Pro Cys Tyr Gly Ser Tyr
Ala Lys Pro Thr Asn Glu Lys Gly 210 215 220 Gly Gln Ala Ser Leu Ile
Thr Asn Gly Thr Asp Gln Thr Leu Thr Ser225 230 235 240 Asp Val Asn
Leu Gln Phe Phe Ala Leu Pro Ser Thr Pro Asn Glu Pro 245 250 255 Lys
Ala Val Leu Tyr Ala Glu Asn Val Ser Ile Glu Ala Pro Asp Thr 260 265
270 His Leu Val Tyr Lys Pro Asp Val Ala Gln Gly Thr Ile Ser Ser Ala
275 280 285 Asp Leu Leu Thr Gln Gln Ala Ala Pro Asn Arg Pro Asn Tyr
Ile Gly 290 295 300 Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn
Ser Thr Gly Asn305 310 315 320 Met Gly Val Leu Ala Gly Gln Ala Ser
Gln Leu Asn Ala Val Val Asp 325 330 335 Leu Gln Asp Arg Asn Thr Glu
Leu Ser Tyr Gln Leu Met Leu Asp Ala 340 345 350 Leu Gly Asp Arg Ser
Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp 355 360 365 Ser Tyr Asp
Pro Asp Val Arg Ile Ile Glu Asn His Gly Val Glu Asp 370 375 380 Glu
Leu Pro Asn Tyr Cys Phe Pro Leu Gly Gly Ser Ala Ala Thr Asp385 390
395 400 Thr Tyr Ser Gly Ile Lys Ala Asn Gly Gln Thr Trp Thr Ala Asp
Asp 405 410 415 Asn Tyr Ala Asp Arg Gly Ala Glu Ile Glu Ser Gly Asn
Ile Phe Ala 420 425 430 Met Glu Ile Asn Leu Ala Ala Asn Leu Trp Arg
Ser Phe Leu Tyr Ser 435 440 445 Asn Val Ala Leu Tyr Leu Pro Asp Ser
Tyr Lys Ile Thr Pro Asp Asn 450 455 460 Ile Thr Leu Pro Glu Asn Lys
Asn Thr Tyr Ala Tyr Met Asn Gly Arg465 470 475 480 Val Ala Val Pro
Ser Ala Leu Asp Thr Tyr Val Asn Ile Gly Ala Arg 485 490 495 Trp Ser
Pro Asp Pro Met Asp Asn Val Asn Pro Phe Asn His His Arg 500 505 510
Asn Ala Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr 515
520 525 Val Pro Phe His Ile Gln Val Pro Gln Lys Phe Phe Ala Ile Lys
Asn 530 535 540 Leu Leu Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn
Phe Arg Lys545 550 555 560 Asp Val Asn Met Ile Leu Gln Ser Ser Leu
Gly Asn Asp Leu Arg Val 565 570 575 Asp Gly Ala Ser Val Arg Phe Asp
Ser Ile Asn Leu Tyr Ala Asn Phe 580 585 590 Phe Pro Met Ala His Asn
Thr Ala Ser Thr Leu Glu Ala Met Leu Arg 595 600 605 Asn Asp Thr Asn
Asp Gln Ser Phe Asn Asp Tyr Leu Cys Ala Ala Asn 610 615 620 Met Leu
Tyr Pro Ile Pro Ser Asn Ala Thr Ser Val Pro Ile Ser Ile625 630 635
640 Pro Ser Arg Asn Trp Ala Ala Phe Arg Gly Trp Ser Phe Thr Arg Leu
645 650 655 Lys Thr Lys Glu Thr Pro Ser Leu Gly Ser Gly Phe Asp Pro
Tyr Phe 660 665 670 Thr Tyr Ser Gly Ser Val Pro Tyr Leu Asp Gly Thr
Phe Tyr Leu Asn 675 680 685 His Thr Phe Lys Lys Val Ser Ile Met Phe
Asp Ser Ser Val Ser Trp 690 695 700 Pro Gly Asn Asp Arg Leu Leu Thr
Pro Asn Glu Phe Glu Ile Lys Arg705 710 715 720 Thr Val Asp Gly Glu
Gly Tyr Asn Val Ala Gln Cys Asn Met Thr Lys 725 730 735 Asp Trp Phe
Leu Ile Gln Met Leu Ser His Tyr Asn Ile Gly Tyr Gln 740 745 750 Gly
Phe Tyr Val Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe 755 760
765 Arg Asn Phe Gln Pro Met Ser Arg Gln Val Val Asn Thr Thr Thr Tyr
770 775 780 Lys Glu Tyr Gln Asn Val Thr Leu Pro Phe Gln His Asn Asn
Ser Gly785 790 795 800 Phe Val Gly Tyr Met Gly Pro Thr Met Arg Glu
Gly Gln Ala Tyr Pro 805 810 815 Ala Asn Tyr Pro Tyr Pro Leu Ile Gly
Gln Thr Ala Val Pro Ser Leu 820 825 830 Thr Gln Lys Lys Phe Leu Cys
Asp Arg Thr Met Trp Arg Ile Pro Phe 835 840 845 Ser Ser Asn Phe Met
Ser Met Gly Ala Leu Thr Asp Leu Gly Gln Asn 850 855 860 Met Leu Tyr
Ala Asn Ser Ala His Ala Leu Asp Met Thr Phe Glu Val865 870 875 880
Asp Pro Met Asp Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe 885
890 895 Asp Val Val Arg Ile His Gln Pro His Arg Gly Val Ile Glu Ala
Val 900 905 910 Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr
915 920 92519964PRTArtificial SequenceConsensus sequence among Ad5,
Ad41 and SAd19 Hexons 19Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser
Tyr Met His Ile Ala1 5 10 15 Gly Gln Asp Ala Ser Glu Tyr Leu Ser
Pro Gly Leu Val Gln Phe Ala 20 25 30 Arg Ala Thr Asp Thr Tyr Phe
Ser Leu Xaa Asn Lys Phe Arg Asn Pro 35 40 45 Thr Val Ala Pro Thr
His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60 Thr Leu Arg
Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr65 70 75 80 Lys
Xaa Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90
95 Ala Ser Thr Tyr Phe Asp Ile Arg Gly Xaa Leu Asp Arg Gly Pro Ser
100 105 110 Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro
Lys Xaa 115 120 125 Ala Pro Asn Xaa Cys Xaa Trp Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 130 135 140 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa145 150 155 160 Xaa Xaa Xaa Xaa Lys Xaa Xaa
Xaa Xaa Ala Gln Ala Pro Xaa Xaa Gly 165 170 175 Xaa Xaa Ile Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa
Xaa Xaa Asn Xaa Pro Xaa Tyr Ala Asp Lys Thr Phe Gln 195 200 205 Pro
Glu Pro Gln Ile Gly Xaa Ser Xaa Trp Xaa Xaa Xaa Xaa Xaa Xaa 210 215
220 Xaa Xaa Xaa Xaa Ala Gly Arg Ile Leu Lys Xaa Xaa Thr Pro Met
Xaa225 230 235 240 Pro Cys Tyr Gly Ser Tyr Ala Lys Pro Thr Asn Glu
Xaa Gly Gly Gln 245 250 255 Ala Xaa Leu Ile Xaa Asn Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Val 260 265 270 Xaa Leu Asn Phe Phe Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280 285 Xaa Xaa Xaa Pro Lys Xaa
Val Leu Tyr Ala Glu Xaa Val Xaa Ile Glu 290 295 300 Xaa Pro Asp Thr
His Ile Xaa Tyr Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa305 310 315 320 Xaa
Xaa Ala Xaa Xaa Leu Leu Xaa Xaa Gln Ala Xaa Pro Asn Arg Pro 325 330
335 Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn
340 345 350 Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala Ser Gln
Leu Asn 355 360 365 Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu
Ser Tyr Gln Leu 370 375 380 Leu Leu Asp Ala Ile Gly Asp Arg Ser Arg
Tyr Phe Ser Met Trp Asn385 390 395 400 Gln Ala Val Asp Ser Tyr Asp
Pro Asp Val Arg Ile Ile Glu Asn His 405 410 415 Gly Xaa Glu Asp Glu
Leu Pro Asn Tyr Cys Phe Pro Leu Xaa Ala Xaa 420 425 430 Xaa Xaa Xaa
Xaa Thr Xaa Xaa Xaa Ile Lys Xaa Xaa Xaa Xaa Xaa Xaa 435 440 445 Xaa
Xaa Trp Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Ile 450 455
460 Xaa Xaa Gly Asn Xaa Phe Ala Xaa Glu Ile Asn Leu Xaa Ala Asn
Leu465 470 475 480 Trp Arg Xaa Phe Leu Tyr Ser Asn Ile Ala Leu Tyr
Leu Pro Asp Xaa 485 490 495 Xaa Lys Xaa Ser Pro Xaa Asn Ile Xaa Ile
Xaa Asp Asn Xaa Asn Thr 500 505 510 Tyr Xaa Tyr Ile Asn Xaa Arg Val
Xaa Xaa Pro Ala Xaa Leu Asp Xaa 515 520 525 Tyr Ile Asn Ile Gly Ala
Arg Trp Ser Xaa Asp Xaa Met Asp Asn Val 530 535 540 Asn Pro Phe Asn
His His Arg Asn Ala Gly Leu Arg Tyr Arg Ser Met545 550 555 560 Leu
Leu Gly Asn Gly Arg Tyr Val Pro Phe His Ile Gln Val Pro Gln 565 570
575 Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro Gly Ser Tyr Thr
580 585 590 Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Ile Leu Gln
Ser Ser 595 600 605 Leu Gly Asn Asp Leu Arg Val Asp Gly Ala Ser Ile
Lys Phe Asp Ser 610 615 620 Ile Xaa Leu Tyr Ala Xaa Phe Phe Pro Met
Ala His Asn Thr Ala Ser625 630 635 640 Thr Leu Glu Ala Met Leu Arg
Asn Asp Thr Asn Asp Gln Ser Phe Asn 645 650 655 Asp Tyr Leu Xaa Ala
Ala Asn Met Leu Tyr Pro Ile Pro Ala Asn Ala 660 665 670 Thr Xaa Val
Pro Ile Ser Ile Pro Ser Arg Asn Trp Ala Ala Phe Arg 675 680 685 Gly
Trp Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr Pro Ser Leu Gly 690 695
700 Ser Gly Phe Asp Pro Tyr Phe Xaa Tyr Ser Gly Ser Ile Pro Tyr
Leu705 710 715 720 Asp Gly Thr Phe Tyr Leu Asn His Thr Phe Lys Lys
Val Ala Ile Xaa 725 730 735 Phe Asp Ser Ser Val Ser Trp Pro Gly Asn
Asp Arg Leu Leu Thr Pro 740 745 750 Asn Glu Phe Glu Ile Lys Arg Ser
Val Asp Gly Glu Gly Tyr Asn Val 755 760 765 Ala Gln Xaa Asn Met Thr
Lys Asp Trp Phe Leu Ile Gln Met Leu Ala 770 775 780 Xaa Tyr Asn Ile
Gly Tyr Gln Gly Phe Tyr Ile Pro Glu Ser Tyr Lys785 790 795 800 Asp
Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro Met Ser Arg Gln 805 810
815 Val Val Xaa Xaa Xaa Xaa Tyr Xaa Xaa Tyr Xaa Xaa Val Xaa Ile Xaa
820 825 830 Xaa Gln His Asn Asn Ser Gly Phe Val Gly Tyr Leu Ala Pro
Thr Met 835 840 845 Arg Glu Gly Gln Ala Tyr Pro Ala Asn Phe Pro Tyr
Pro Leu Ile Gly 850 855 860 Xaa Thr Ala Val Xaa Ser Ile Thr Gln Lys
Lys Phe Leu Cys Asp Arg865 870 875 880 Xaa Leu Trp Arg Ile Pro Phe
Ser Ser Asn Phe Met Ser Met Gly Ala 885 890 895 Leu Thr Asp Leu Gly
Gln Asn Leu Leu Tyr Ala Asn Ser Ala His Ala 900 905 910 Leu Asp Met
Thr Phe Glu Val Asp Pro Met Asp Glu Pro Thr Leu Leu 915 920 925 Tyr
Val Leu Phe Glu Val Phe Asp Val Val Arg Ile His Xaa Pro His 930 935
940 Arg Gly Val Ile Glu Xaa Val Tyr Leu Arg Thr Pro Phe Ser Ala
Gly945 950 955 960 Asn Ala Thr Thr20942DNAArtificial
SequenceSequence of hypervariable region of SAd19 Hexon
20ggcgctccga atgcttgtca gtggacaacc acgaatgggg gtaacaaaac taattcattt
60gctcaggccc cagtaatcgg cctaagtatt gacgccacca acgggctaaa agtaggggag
120gagatacctg ccactggagg ggcaaatacg cccgtgtacg ccgacaaaac
attccagcct 180gaacctcaag taggagaaac aaaatggaat tctaacccta
ctgagaatgc agctggaaga 240attttaaagc caaacacacc tatgcagccc
tgctacggat cgtacgctcg accaacaaac 300gaaaaaggag gacaggcaaa
gctagttact aacggtcaag acaatcaaac aacgccagac 360gttagtttaa
acttttttac tactgcgtca gaaaccacaa cattcacgcc gaaagttgtt
420ctgtatagcg aaaacgtcaa cttggaagct ccagatacgc atctagtata
caagccagac 480ggcactgacg gaatcaccaa cgccgaaact ctcttaggac
ttcagtcagc tccgaacaga 540ccaaattaca ttggttttcg agataacttt
ataggcctaa tgtattacaa ctccactgga 600aatatggggg ttctggccgg
acaggcttcg caattaaacg cagtggttga tttgcaagac 660agaaacacag
aattgtcata ccaacttatg ctggatgccc tgggagaccg cagtaggtac
720ttctccatgt ggaatcaggc tgtggacagc tatgatcctg atgttaggat
aatagaaaac 780catggcgtag aagacgaatt gcctaactac tgctttccac
ttaatgcgca aggtgtagcc 840aacacttacc agggcgttaa aaatggctcg
ggaaactggt cgaaagacac taacgttggc 900acggcaaatg aaatcgggat
aggtaacatt tttgctttcg aa 94221314PRTArtificial SequenceSequence of
hypervariable region of SAd19 Hexon 21Gly Ala Pro Asn Ala Cys Gln
Trp Thr Thr Thr Asn Gly Gly Asn Lys1 5 10 15 Thr Asn Ser Phe Ala
Gln Ala Pro Val Ile Gly Leu Ser Ile Asp Ala 20 25 30 Thr Asn Gly
Leu Lys Val Gly Glu Glu Ile Pro Ala Thr Gly Gly Ala 35 40 45 Asn
Thr Pro Val Tyr Ala Asp Lys Thr Phe Gln Pro Glu Pro Gln Val 50 55
60 Gly Glu Thr Lys Trp Asn Ser Asn Pro Thr Glu Asn Ala Ala Gly
Arg65 70 75 80 Ile Leu Lys Pro Asn Thr Pro Met Gln Pro Cys Tyr Gly
Ser Tyr Ala 85 90 95 Arg Pro Thr Asn Glu Lys Gly Gly Gln Ala Lys
Leu Val Thr Asn Gly 100 105 110 Gln Asp Asn Gln Thr Thr Pro Asp Val
Ser Leu Asn Phe Phe Thr Thr 115 120 125 Ala Ser Glu Thr Thr Thr Phe
Thr Pro Lys Val Val Leu Tyr Ser Glu 130 135 140 Asn Val Asn Leu Glu
Ala Pro Asp Thr His Leu Val Tyr Lys Pro Asp145 150 155 160 Gly Thr
Asp Gly Ile Thr Asn Ala Glu Thr Leu Leu Gly Leu Gln Ser 165 170 175
Ala Pro Asn Arg Pro Asn Tyr Ile Gly Phe Arg Asp Asn Phe Ile Gly 180
185 190 Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly
Gln 195 200 205 Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg
Asn Thr Glu 210 215 220 Leu Ser Tyr Gln Leu Met Leu Asp Ala Leu Gly
Asp
Arg Ser Arg Tyr225 230 235 240 Phe Ser Met Trp Asn Gln Ala Val Asp
Ser Tyr Asp Pro Asp Val Arg 245 250 255 Ile Ile Glu Asn His Gly Val
Glu Asp Glu Leu Pro Asn Tyr Cys Phe 260 265 270 Pro Leu Asn Ala Gln
Gly Val Ala Asn Thr Tyr Gln Gly Val Lys Asn 275 280 285 Gly Ser Gly
Asn Trp Ser Lys Asp Thr Asn Val Gly Thr Ala Asn Glu 290 295 300 Ile
Gly Ile Gly Asn Ile Phe Ala Phe Glu305 310
* * * * *