Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ALTERNATIVELY TARGETED ADENOVIRUS
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an alternately targeted adenovirus and
includes
S methods for producing and purifying such viruses as well as protein
modifications
mediating alternate targeting.
BACKGROUND OF THE INVENTION
Adenoviral infection begins with the attachment of the virion to the target
cell.
l 0 The adenovirus attaches to two cellular surface proteins, both of which
must be present
for the virus to infect the target cell (Wickham et al., Cell, 73, 309-19
(1993)). Wild-type
adenovirus first binds the cell surface by means of a cellular adenovirai
receptor (AR).
One such AR is the recently-identified coxsackievirus and adenovirus receptor
(CAR)
(Bergelson et al., Science, 275, 1320-23 (1997); Tanako et al., Proc. Nat.
Acad. Sci.,
IS U.S.A., 94, 3352-56 (1997)); the MHC class I receptor also is an AR (Hong
et al., EMBO
J., 16(9), 2294-06 ( 1997)). After attachment to an AR, the virus attaches to
av integrins, a
family of a heterodimeric cell-surface receptors mediating interaction with
the
extracellular matrix and playing important roles in cell signaling (Hynes,
Cell, 69, 11-25
( 1992)).
20 Following attachment to the cell surface, infection proceeds by receptor-
mediated
internalization of the virus into endocytotic vesicles (Svensson et al., J.
Virol., S1, 687-94
( 1984); Chardonnet et al., Virology, 40, 462-77 ( 1970)). Within the cell,
virions are
disassembled (Greber et al., Cell, 7S, 477-86 (1993)), the endosome disrupted
(Fitzgerald
et al., Cell, 32, 607-17 (1983)), and the viral particles transported to the
nucleus via the
2S nuclear pore complex (Dales et al., Virology, S6, 465-83 (1973)).
The adenoviral virion is a non-enveloped icosahedron about 65-80 nm in
diameter
(Home et al., J. Mol. Biol., 1, 84-86 (1959)). The adenoviral capsid comprises
252
capsomeres -- 240 hexons and 12 pentons (Ginsberg et al., Virology, 28, 782-83
(1966)).
The hexons and pentons are derived from three viral proteins (Maizel et al.,
Virolog~, 36,
30 115-25 (1968); Weber et al., Virology, 76, 709-24 (1977)). The hexon
comprises three
identical proteins of 967 amino acids each, namely polypeptide II (Roberts et
al., Science,
232, 1148-51 ( 1986)). The penton contains a base, which is bound to the
capsid, and a
fiber, which is non-covalently bound to and projects from, the penton base.
Proteins IX,
VI, and IIIa also are present in the adenoviral coat and are thought to
stabilize the viral
3S capsid (Stewart et al., Cell, 67, 145-54 (1991 ); Stewart et al., EMBO J.,
12(7), 2589-99
( 1993)).
The penton base is highly conserved among serotypes of adenovirus and (except
for the enteric adenovirus Ad40) has five RGD tripeptide motifs (Neumann et
al., Gene,
69, 153-57 (1988)). In adenovirus, the RGD tripeptides apparently mediate
adenoviral
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binding to a" integrins and endocytosis of the virion (Wickham et al. (1993),
supra; Bai et
al., J. Virol., 67, 5198-3205 (1993)).
The adenoviral fiber is a homotrimer of the adenoviral polypeptide IV (Devaux
et
al., J. Molec. Biol., 215, 567-88 (1990)). Structurally, the fiber has three
discrete
domains. The amino-terminal tail domain attaches non-covalently to the penton
base. A
relatively long shaft domain comprising a variable number of repeating 15
amino acid
residues forming ~i-sheets extends outward from the vertices of the viral
particle (Yeh et
al., Virus Res., 33, 179-98 { 1991 )). Lastly, roughly 200 amino-acid residues
at the
carboxy-terminal form the knob domain. Functionally, the knob mediates primary
viral
binding to the cellular AR and fiber trimerization (Henry et al., J. Virol.,
68(8), 5239-46
( 1994)). Hence, the trimerization domain of a fiber is a ligand for a cell-
surface receptor
native for the adenoviral serotype. The trimerization domain also appears
necessary for
the tail of the fiber to properly associate with the penton base (Novelli et
al., Virology,
185, 365-76 (1991)). In addition to recognizing cell ARs and binding the
penton base, the
1~ fiber protein contributes to serotype integrity and mediates nuclear
localization.
Fiber proteins from different adenoviral serotypes differ considerably. For
example, the number of 15 amino-acid ~i-sheet repeats differs between
adenoviral
serotypes (Green et al., EMBO J., 2, 1357-65 (1983)). Moreover, the knob
regions from
the closely related Ad2 and Ad5 serotypes are only 63% similar at the amino
acid level
(Chroboczek et al., Virology, 186, 280-85 (1992)), and Ad2 and Ad3 fiber knobs
are only
20% identical (Signas et al., J. Yirol., 53, 672-78 (1985)). In contrast, the
penton base
sequences are 99% identical. Despite these differences in the knob region, a
sequence
comparison of even the Ad2 and Ad3 fiber genes demonstrates distinct regions
of
conservation, most of which are also conserved among the other human
adenoviral fiber
genes.
A number of factors present the adenovirus as an attractive vector choice for
use in
a variety of gene transfer applications (e.g., cellular protein production,
therapy, academic
study, etc.). For example, the adenovirus is a superior expression vector.
Recombinant
adenovirus can be produced in high titers (e.g., about 10'3 viral
particles/ml), and
adenoviral vectors can transfer genetic material to non-replicating, as well
as replicating,
cells (in contrast with retroviral vectors). The adenoviral genome can be
manipulated to
carry a large amount of exogenous DNA (up to about 7.5 kb), and the adenoviral
capsid
can potentiate the transfer of even longer sequences (Curiel et al., Hum. Gene
Ther., 3,
147-54 ( 1992)). Additionally, several features suggest that adenoviruses
represent a safe
choice for gene transfer, a particular concern for therapeutic applications.
For example,
adenoviruses do not integrate into the host cell chromosome, thus minimizing
the
likelihood that an adenoviral vector will interfere with normal cell function.
Moreover,
adenoviral infection does not correlate with human malignancy, and
recombination of the
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adenoviral genome is rare. Due to these advantages, clinicians have employed
adenoviral
vectors safely as a human vaccine and for gene therapy for many years.
Based on the popularity of adenoviral vectors, efforts have been made to
increase
the ability of adenovirus to enter certain cells, e.g., those few cells it
does not infect, an
approach referred to as "targeting" (see, e.g., International Patent
Application WO
95/26412 (Curiel et al.), International Patent Application WO 94/10323
(Spooner et al.),
U.S. Patent 5,543,328 (McClelland et al.), International Patent Application WO
94/24299
(Gotten et al.)). Of course, while the ability to target adenoviruses to
certain cell types is
an important goal, far more desirable is an adenovirus which infects only a
desired cell
type, an approach referred to as "exclusive targeting." However, to
exclusively target a
virus, its native affinity for host cell ARs must first be abrogated,
producing a
recombinant adenovirus incapable of productively infecting the full set of
natural
adenoviral target cells. Efforts aimed at abrogating native adenoviral cell
affinity have
focused logically on changing the fiber knob. These efforts have proven
disappointing,
IS largely because they fail to preserve the important fiber protein functions
of stable
trimerization and penton base binding (Spooner et al., supra). Moreover,
replacement of
the fiber knob with a cell-surface ligand (McClelland et al., supra) produces
a virus only
suitable for infecting a cell type having that ligand. Such a strategy
produces a virus
having many of the same targeting problems associated with wild-type
adenoviruses (in
which fiber trimerization and cellular tropism are mediated by the same
protein domain),
thus decreasing the flexibility of the vector. Moreover, due to the necessity
of having a
host cell, and the integral connection between the fiber trimization and
targeting
functions, obtaining a mutant virus with substituted targeting is difficult.
For example,
removing the fiber knob and replacing it with a non-trimerizing ligand (e.g.,
McClelland
et al., supra) results in a virus lacking appreciable fiber protein. As such,
there is
currently a need for an adenoviral fiber having reduced affinity for natural
ARs but
retaining fiber trimerization and penton base-binding function.
While exclusive adenoviral targeting requires reducing native cellular
tropism, the
abrogation of natural targeting also reduces the ability of the virus to
infect cell lines
normally employed for its propagation (e.g., 293 cells) (see Curiel et ai.,
supra). One
published attempt at surmounting this barrier fortuitously employed a cell
line expressing
the relevant cell surface binding site (McClelland et al., supra), and thus
did not address
this central concern. However, many cell lines do not express important
cellular
receptors. Moreover, many available cell lines expressing potentially useful
cell surface
binding sites are inadequate for production of recombinant adenoviruses,
especially
viruses useful for clinical application (e.g., cell lines harboring and
expressing the
essential adenoviral immediate early genes from the E1, E2, and/or E4 regions
of the
genome). There is thus a need for a cell line, and a means of producing a cell
line, which
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can propagate and package a recombinant adenovirus substantially incapable of
productively infecting cells via native ARs.
Typical protocols for purifying viral vectors from packaging cell lysates
involve
centrifuging the viruses through a CsClz gradient one or more times. While
such methods
adequately isolate viruses, they generally require considerable material
(CsCl2) and are
therefore relatively inefficient. Moreover, such protocols are not readily
amenable to high
throughput application, presenting a significant barrier to economic
development of viral
vectors on a commercial scale. Other methods involving column purification do
not bind
the viruses specifically (Shabram et al., Hum. Gene Ther., 8, 453 (1997);
Huyghe et al.,
Hum. Gene Ther., 6, 1403 (1995)), often resulting in an unacceptable amount of
contaminants compared to the purity obtainable in affinity purification of
other materials.
Thus, there is a need for an efficient method of purifying and isolating
recombinant viral
vectors.
In many applications involving in vivo delivery of viral vectors, it is
desirable to
contain infection (and gene delivery) to the tissue of interest. Por example,
the threat of
systemic infection and delivery of a biologically active gene represents a
significant
concern to gene therapy applications. Moreover, ectopic expression of a
transgene would
spoil many experimental applications. While, in theory, host blood cells can
express
proteins mediating the clearing of foreign substances, such as adenoviruses
(News and
Comment, Science, 275, 744-45 (1997)), engineering such cells and producing
them in
the host are difficult and intrusive. Moreover, while antibodies directed
against the
adenoviral hexon can inactivate the virus (Toogood et al., J. Gen. Virol., 73,
1429-35
( 1992)), efficient protocols for delivering a sufficient quantity of anti-
hexon antisera to
the gene transfer recipient in time to reduce or prevent ectopic viral
infection have not
been forthcoming, and such a strategy can actually interfere with gene
transfer protocols
by blocking infection in desired tissues. Thus, there is a need for a method
of inactivating
recombinant viral vectors leaving the desired locus of delivery within a host
animal.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a trimer comprising three monomers, each having
an amino terminus of an adenoviral fiber protein and each having a
trimerization domain.
The trimer exhibits reduced affinity for a native substrate than a native
adenoviral fiber
trimer. The present invention further provides an adenovirus incorporating the
trimer of
the present invention. The present invention also provides a cell line
expressing a non-
3~ native cell-surface receptor to which an adenovirus having a ligand for the
receptor binds,
and a method of propagating an adenovirus using the cell line.
The present invention also provides a method of purifying an adenovirus having
a
ligand for a substrate from a composition comprising the adenovirus. The
method
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to selectively bind the substrate. Subsequently, the composition not bound to
the
substrate is separated from the substrate, after which the bound adenovirus is
eluted from
the substrate.
The present invention further provides a method of inactivating an adenovirus
S having a ligand recognizing a blood- or lymph-borne substrate by exposing
the virus to
the substrate. Within the blood or lymph, the ligand binds its substrate,
thereby adsorbing
the free virus from the blood or Lymph.
Additionally, the present invention provides a chimeric blocking protein
comprising a substrate for an adenovirus fiber, and a method of interfering
with
IO adenoviral receptor binding by incubating an adenovirus with such chimeric
blocking
protein in a solution such that the chimeric blocking protein binds the fiber.
The present invention is useful in a variety of applications, in vitro and in
vivo,
such as therapy, for example, as a vector for delivering a therapeutic gene to
a cell with
minimal ectopic infection. Specifically, the present invention permits more
efficient
15 production and construction of safer vectors for gene therapy applications.
The present
invention is also useful as a research tool by providing methods and reagents
for the study
of adenoviral attachment and infection of cells and in a method of assaying
receptor-
ligand interaction. Similarly, the recombinant fzber protein trimers can be
used in
receptor-ligand assays and as adhesion proteins in vitro or in vivo.
Additionally, the
20 present invention provides reagents and methods permitting biologists to
investigate the
cell biology of viral growth and infection. Thus, the vectors of the present
invention are
highly useful in biological research.
BRIEF DESCRIPTION OF THE FIGURES
25 Figures lA and 1B depict the three-dimensional structure of an adenoviral
knob
protein (serotype 5). Figure lA is a ribbon diagram representing ~3-sheets and
the loops
interconnecting the sheets. Figure 1 B is a filled-in diagram taking into
account the
relative sizes of the amino acid residues.
Figure 2 is a sequence comparison between adenoviral serotypes.
30 Figures 3A-3C depict vectors for creating recombinant adenoviral fiber
trimers
having non-native trimerization domains. Figure 3A depicts
pAcT5S7GCNTS.PS.LS.X.
Figure 3B depicts pAcTSsigDel.TS.PS.LS. Figure 3C depicts
pAcT5S7sigDel.TS.PS.LS.
Figure 4 depicts pAcTSsigDel.GFP.TS.PS.LS, a vector containing a gene encoding
a fiber-sigDel-GFP chimera.
35 Figures SA-SD depict vectors useful for the construction of recombinant
adenovirus vectors containing fiber trimers having non-native trimerization
domains.
Figure SA depicts pAS pGS HAAV. Figure SB depicts pAS pGS pK7. Figure SC
depicts
pAS TSS7sigDelpGS.HAAV. Figure SD depicts pAST5S7sigDel.GFP.pGS.pK7.
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Figures 6A-6D represent vectors used in the construction of fiber trimers
having
non-native trimerization domains. Figure 6A represents pAcPig4KN. Figure 6B
represents pAcPigKN D363E. Figure 6C depicts pAcPigKN N437D. Figure 6D depicts
pAcPig4KN(FLAG).
Figures 7A-7B represent vectors employed in creating a fiber trimer having a
non-
native trimerization domain. Figure 7A depicts PNS FSF2K. Figure 7B depicts
pNS
Pig4.SS.
Figures 8A-8C represent vectors useful for creating an adenoviral vector
having a
chimeric fiber trimer comprising a mutant NADC-1 knob lacking native receptor-
binding
ability and containing a functional non-native ligand. Figure 8A depicts
pAcPig4KN
D363E N437D. Figure 8B depicts pAcPig4KN D363E N437D HAAV. Figure 8C
depicts pNS Pig4 D363E N437D HAAV SS.
Figures 9A-9B represent vectors useful for creating chimeric blocking proteins
of
the present invention able to interfere with native adenoviral receptor
binding. Figure 9A
IS depicts pACSG2-SCAR. Figure 9B depicts pACSG2-SCAR-HAAV.
Figures l0A-l OB represent vectors useful for creating chimeric blocking
proteins
able to form trimers interfering with native adenoviral receptor binding.
Figure l0A
depicts pAcSG2sCAR.sigDel. Figure lOB depicts pAcSG2-sCARsigDel (HAAV).
Figures 11A-11E depict vectors useful for creating construction of adenovirus
vectors having specific non-native Iigands. Figure 1 lA depicts pBSSpGS.
Figure 11B
depicts pBSS pGS (RKKK)2. Figure 11C depicts pNSF5F2K(RKKK)2. Figure i 1D
depicts pBSSpGS (FLAG). Figure I lE depicts pNS FSF2K(FLAG).
Figures 12A-12E represent vectors useful for creating a cell line expressing a
non-
native cell surface binding site substrate. Figure 12A depicts pHOOK3. Figure
12B
depicts pRC/CMVp-Puro. Figure 12C pScHAHK. Figure 12D depicts pNSE4GLP.
Figures I3A-13D represent vectors useful for creating a fiber-expressing cell
line
for the production of targeted adenovirus particles. Figure 13A depicts pCR2.1-
TOPO+fiber. Figure 13B depicts pKSII Fiber. Figure 13C depicts pSMTZeo-DBP.
Figure 13D depicts pSMTZeo-Fiber.
Figure 14 depicts pAdE1(Z)E3/E4(B), a plasmid useful for the construction of
targeted adenovirus particles having genomes encoding chimeric fibers.
Figures 15A-15E illustrate the locations of mutations within adenoviral knobs
which interfere with ligand binding. Figure 15A is a top view, Figure 15B a
side view,
and Figure 15C a bottom view of the knob illustrating the location of the 3D9
mutation.
Figure 15D is a top view, Figure 15E a side view, and Figure 15F a bottom view
of the
knob illustrating the locations of the CD loop mutation, the FG loop mutation,
and the IJ
mutation.
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Figure 16 depicts a vector useful for the construction of a recombinant
adenovirus
containing a short-shafted fiber and a mutant fiber knob exhibiting reduced
affinity for its
native receptor.
Figures 17A-17B depict vectors useful for constructing a cell line able to
replicate
adenoviruses lacking native cell-binding function (but targeted for a pseudo-
receptor).
Figure 17A depicts pCANTABSE(HA). Figure 17B depicts pScFGHA.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
An adenovirus is any virus of the genera Mastadenoviridae or Aviaadenoviridae,
and can be of any serotype within those genera. Adenoviral stocks that can be
employed
as a source of adenovirus or adenovirus coat protein such as penton base
and/or fiber
protein can be amplified from the adenovirus serotypes currently available
from
American Type Culture Collection (ATCC, Rockville, MD), or from any other
source.
IS A ligand is any species selectively binding an identifiable substrate.
Native refers to a protein or property of an unmodified virus or cell. Thus, a
non-
native protein can be a modified or mutated protein differing from its native
homologue
within the virus or cell. Alternatively, a non-native protein can be a protein
having no
native homologue within the virus or cell.
?0 An AR refers to an adenoviral receptor. In particular, an AR is a ligand
binding
the mastadenoviral knob.
A first species is selectively bound to a substrate if it binds the substrate
with
greater affinity than a second species. The first species is not selectively
bound if binds
the substrate with the same or lesser affinity than the second species, even
if the first
25 species binds with some affinity.
Trimers
The present invention provides a trimer comprising three monomers (e.g., at
least
a portion of each of three adenoviral fiber monomers), each having an amino
terminus
30 derived from an adenoviral fiber protein and each having a trimerization
domain. The
inventive trimer exhibits reduced affinity for a native substrate, such as an
antibody, a
cellular binding cite, etc. (i.e., native to the serotype from which the
shaft, and particularly
the amino-terminus, is drawn) as compared to a native adenoviral fiber trimer.
The trimer
can be a homotrimer or a heterotrimer of different fiber monomers. Any
modification of
35 the monomeric units reducing the affinity of the resulting trimer for its
native cell surface
binding site (i.e., a native AR) is within the scope of the invention.
Preferably, the
reduction in affinity is a substantial reduction in affinity (such as at least
an order of
magnitude, and preferably more) relative to the unmodified corresponding
fiber.
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As mentioned, where a trimerization domain is itself a ligand for a native
cell
surface binding site, trimers possessing such trimerization domains present
some of the
same problems for targeting as native adenoviral fiber trimerization domains.
Therefore,
the trimerization domain of a monomer incorporated into the trimer of the
invention
preferably is not a ligand for the CAR or MHC-1 cell surface domains, or
antibodies
recognizing the fiber. Most preferably, the non-native trimerization domain is
not a
ligand for any native mammalian cell-surface binding site, whether the site is
an AR or
other cell surface binding site. As is discussed herein, adenoviruses
incorporating such
trimers exhibit reduced ability to appreciably infect their native host cells,
and can serve
as efficient source vectors for engineering selectively targeted vectors.
Therefore, while
the trimerization domain preferably is not a ligand for a cell surface binding
site, the
entire trimer can be such a ligand (by virtue of a non-native ligand as
discussed herein).
Moreover, the trimerization domain can be a ligand for a substrate other than
a native cell
surface binding site, as such trimerization-ligands do not present the same
concern for cell
IS targeting as do trimerization domains which are ligands for cell surface
binding sites.
Thus, for example, the non-native trimerization domain can be a ligand for a
substrate on
an affinity column, on a blood-borne molecule, or even on a cell surface when
it is not a
native cell-surface binding site (e.g., on a cell engineered to express a
substrate cell
surface protein not native to the unmodified cell type).
A monomer for inclusion into a trimer can be all or a part of a native
adenoviral
fiber monomeric protein. For example, a modified monomer can lack a sizable
number of
residues, or even identifiable domains, as herein described. For example, a
monomer can
lack the native knob domain; it can lack one or more native shaft ~-sheet
repeats, or it can
be otherwise truncated. Thus, a monomer can have any desired modification so
long as it
trimerizes. Furthermore, a monomer preferably is not modified appreciably at
the amino
terminus (e.g., the amino-terminus of a monomer preferably consists
essentially of the
native fiber amino-terminus) to ensure that the resultant trimer interacts
properly with the
penton base. Hence, the present invention also provides a composition of
matter
comprising a trimer of the present invention and an adenoviral penton base.
Preferably,
the trimer and the penton base associate much in the same manner as wild-type
fibers and
penton bases. Of course, while the trimer comprises modified fiber monomers,
the penton
base can also be modified, for example, to include a non-native ligand, for
example as is
described in U.S. Patent 5,559,099.
Mutant Knobs
A fiber monomer for incorporation into the trimer of the present invention has
a
trimerization domain which binds a native mammalian AR (i.e., an AR native for
the
adenoviral serotype of interest) with less affinity than a native adenoviral
fiber. Trimers
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incorporating such monomers preferably are not ligands for their native
cellular binding
sites. The monomers can be modified in any manner suitable for reducing the
affinity of
the fiber for native AR while permitting the monomers to trimerize. For
example, in one
embodiment, the trimerization domain is a modified adenoviral fiber knob
domain lacking
a native receptor-binding amino acid. Any native amino-acid residue mediating
or
assisting in the interaction between the knob and a native cellular AR is a
suitable amino
acid for mutation or deletion from the monomer. Moreover, the knob domain can
lack
any number of such native receptor-binding amino acids, so long as, in the
aggregate, the
monomers associate to form a trimer of the present invention.
Native amino acid residues for modification or deletion can be selected by any
method. For example, the sequences from different adenoviral serotypes can be
compared to deduce conserved residues likely to mediate AR-binding.
Alternatively or in
combination, the sequence can be mapped onto a three dimensional
representation of~the
protein (such as the crystal structure) to deduce those residues most likely
responsible for
AR binding. These analyses can be aided by resorting to any common algorithm
or
program for deducing protein structural functional interaction. Alternatively,
random
mutations can be introduced into a cloned adenoviral fiber expression
cassette. One
method of introducing random mutations into a protein is via the Taq
polymerase. For
example, a clone encoding the fiber knob (see, e.g., SEQ ID N0:9; Roelvink et
al., J.
Virol., 70, 7614-21 (1996)) can serve as a template for PCR amplification of
the
adenoviral fiber knob, or a portion thereof. By varying the concentration of
divalent
cations in the PCR reaction, the error rate of the transcripts can be largely
predetermined
(see, e.g., Weiss et al., J. Virol., 71, 4385-94 (1997); Zhou et al., Nucl.
Acid. Res., 19,
6052 (1991)). The PCR products then can be subcloned back into the template
vector to
replace the sequence within the fiber coding sequence employed as a source for
the PCR
reaction, thus generating a library of fibers, some of which will harbor
mutations which
diminish native AR binding while retaining the ability to trimerize.
A monomer lacking one or more amino acids, as herein described, can optionally
comprise a non-native residue (e.g., several non-native amino acids) in
addition to or in
place of the missing native amino acid(s); of course, alternatively, the
native amino
acids) can simply be deleted from the knob. Preferably, the amino-acid is
substituted
with another non-native amino acid to preserve topology and, especially,
trimerization.
Moreover, if substituted, the replacement amino acid preferably confers novel
qualities to
the monomer. For example, to maximally ablate binding to the native AR, a
native amino
acid can be substituted with a residue (or a plurality of residues) having a
different charge.
Such a substitution maximally interferes with the electrostatic interaction
between native
adenoviral knob domains and cellular ARs. Similarly, a native amino acid can
be
substituted with a heavier residue (or a plurality of residues) where
possible. Heavier
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residues have longer side-chains; hence, such a substitution maximally
interferes with the
steric interaction between native adenoviral knob domains and cellular ARs.
Non-native Trimerization Domains
5 In another embodiment, the trimer includes modified monomers which are
chimeric adenoviral fiber polypeptides. A suitable chimeric monomer lacks all
or a
portion of the trimerization domain native to the source adenoviral serotype.
The
trimerization domain of such a monomer can be deleted from the virus, or the
trimerization domain can be ablated by inserting or substituting non-native
amino acids
1 D into the domain. Of course, a monomer lacking the native trimerization
domain can also
lack the entire native knob. Because the native trimerization domain is a
ligand for a
native AR, a trimer of chimeric adenoviral fiber monomers lacking the native
trimerization domain binds its native AR with less affinity than the native
adenoviral
fiber.
IS For the chimeric monomers to form a trimer of the present invention, they
must
incorporate a replacement (i.e., non-native) trimerization domain. To
maximally promote
the targeting of the virus, preferably the non-native trimerization domain is
not a ligand
for a mammalian cell-surface receptor, or any cell-surface receptor. Any
domain able to
form homotrimers is a suitable trimerization domain for inclusion into the
trimers of the
present invention, and several are known in the art. For example, a chimeric
monomer
can include the trimerization domain from the heat shock factor (HSF) protein
of K. lactis
(Sorger and Nelson, Cell, 59, 807 ( 1989)), trout axonal dynein (Garber et
al., EMBO J., 8,
1727 (1989)), parainfluenza virus hemagglutanin protein (Coelingh et al.,
Virology, 162,
137 (1988)), the sigma 1 protein of reovirus type 1 (Strong et al., Virology,
184, 12
( 1991 )), or other suitable trimer. Alternatively, a chimeric monomer can
include a
modified leucine-zipper motif. Leucine zippers comprise heptad repeats of
Ieucines,
which mediate dimerization. However, replacement of one or more leucine with
isoleucine results in stable trimerization of the domains. An example of such
a modified
leucine zipper motif is the 32 amino acid GCN4p-II trimer (Harbury et al.,
Science, 262,
1401 (1993)).
Of these trimerization domains, the reovirus sigma 1 trimerization domain is
preferred. This protein contains 17 alpha helical heptad repeats, reminiscent
of the
coiled-coil trimer structure of the aforementioned mutant isoleucine zipper
domains
(Harbury et al., Nature, 371, 80-83 (1994)). Fiber chimeras containing the
sigma 1
domain can thus protrude farther from the virus than corresponding chimeras
containing
shorter trimerization domains. An advantage of the reovirus sigma 1
trimerization domain
over a mutant leucine-zipper (e.g., GCN4) domain is that the sigma 1 domain is
22 nm
long (Fraser et al., J. Yirol., 64, 2990-3000 (1990)) whereas GCN4 domain is
only 5 nm
CA 02291323 1999-11-24
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11
long (Harbury et al., supra). An additional advantage to employing the
reovirus sigma 1
attachment protein is that, unlike the adenoviral shaft protein, it exhibits
intrinsic
trimerization propensity (Leone et al., V irolo~, 182, 336-45 ( 1991 )). As
fiber length
appears to increase the efficiency and specificity of adenoviral-cell
attachment (Roelvink
et al., J. Virol., 70, 7614-21 ( 1996)), longer fibers possible with the sigma
1 domain are
preferred to other chimeric fibers.
A chimeric monomer can alternatively include a knob domain from another
adenoviral serotype. For example, the trimerization domain can be replaced
with a
mutated knob from an adenoviral serotype capable of productive infection
within the host
species (e.g., a mutant knob of Ad3 containing a mutation in the HI loop).
Alternatively,
it can be replaced with a knob from a serotype not capable of productive
infection within
the host species. For example, the fiber knob of a mammalian adenoviral
serotype can be
replaced with a knob from an avian serotype. While the avian knob mediates
trimerization of the fiber proteins, it is likely unable to recognize a
mammalian AR;
IS hence, such chimeric fibers lack the native ability to bind the native host
AR. Similarly,
the fiber knob of one mammalian adenoviral serotype can be replaced with a
knob from
another mammalian serotype. In this regard, a modified or unmodified knob from
the
porcine adenovirus NADC-1 fiber is a preferred domain, as the NADC-1 is well
characterized. The NADC-1 knob has identifiable ligands, e.g., galectin (which
binds
galactose), and LDZ and RGD peptides, (which bind integrins) (see, e.g.,
Hirabayashi et
al., J. Biol. Chem., 266, 13648-53 (1991)). Thus, chimeric human adenoviral
fibers
having NADC-1 knobs with such mutations can form trimers and associate with
the
penton base, but they bind native cell-surface receptors with reduced
affinity.
The non-native trimerization domain can be ligated to the native fiber monomer
at
any suitable site, so long as the monomers can trimerize properly (i.e., be
capable of
interacting with an adenoviral penton base). For example, the domain can be
inserted into
the native knob to disrupt knob topology. Alternatively, the trimerization
domain can be
inserted after any of the 15 amino acid shaft repeats, preferably after the
7'", 15'", or 22d
repeats to mimic native adenoviral shaft size. Where the non-native
trimerization domain
is inserted into the adenoviral shaft, it can form the carboxy-terminus of the
chimeric
protein, or it can be inserted into the middle of the amino acid sequence.
Moreover, any
number of trimerization domains can be so inserted into the fiber monomer, so
long as the
resulting trimer properly associates with the penton base.
Blocking Domain
Another suitable chimeric monomer has a novel domain blocking the ligand for
the native host AR. The blocking domain is any peptide which can be tightly
bound to
the native ligand. (See, e.g., Hong et al., EMBO J., 16, 2294-2306 (1997)). In
other
CA 02291323 1999-11-24
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12
words, the blocking domain is a substrate to which the (native or modified)
fiber
monomer ligand selectively binds. Desirably, the ligand-substrate interaction
occurs at
least immediately upon viral production and effectively continues until the
fiber trimer is
destroyed. Because the native ligand binds the blocking domain, the ligand is
incapable
of binding its native substrate on cell surfaces. Because the native
trimerization domain is
a ligand for a native AR, trimers of chimeric adenoviral fiber monomers having
such
blocking domains bind the native AR with less affinity than a native
adenoviral fiber.
The blocking domain can be at any position on the adenovirus to bind the
native
ligand without appreciably affecting trimerization or penton base interaction.
For
example, the blocking domain can be appended to the above-referenced ~i-sheets
or loops,
either by fusion within the reading frame, by covalent post-translational
modification, etc.
Alternatively, the blocking domain can be appended to another portion of the
monomer,
such as the shaft. The blocking domain can also include a linker or spacer
polypeptide to
afford an opportunity for the blocking domain to interact with the native
ligand. If the
I S blocking domain is attached via such a spacer, the spacer can include a
protease
recognition site for subsequent cleavage, as described herein.
Preparation
The monomers for inclusion into the trimers of the present invention can be
produced by any suitable method. For example, the mutant fiber protein can be
synthesized using standard direct peptide synthesizing techniques (e.g., as
summarized in
Bodanszky, Principles of Peptide Synthesis (Springer-Verlag, Heidelberg:
1984)), such as
via solid-phase synthesis (see, e.g., Merrifield, J. Am. Chem. Soc., 85, 2149-
54 (1963);
Barany et al., Int. J. Peptide Protein Res., 30, ?OS-739 (1987); and U.S.
Patent
5,424,398). Alternatively, site-specific mutations (such as replacing the knob
with a non-
native trimerization domain, removing, replacing, or mutating the AR-binding
residues, or
adding a blocking domain, as herein described) can be introduced into the
monomer by
ligating into an expression vector a synthesized oligonucleotide comprising
the modified
site. Alternatively, a plasmid, oligonucleotide, or other vector encoding the
desired
mutation can be recombined with the adenoviral genome or with an expression
vector
encoding the monomer to introduce the desired mutation. Oligonucleotide-
directed site-
specific mutagenesis procedures also are appropriate(e.g., Walder et al.,
Gene, 42, 133
(1986); Bauer et al., Gene, 37, 73 (1985); Craik, Biotechniques, 12-19 (i995);
U.S.
Patents 4,518,584 and 4,737,462). However engineered, the DNA fragment
encoding the
modified monomer can be subcloned into an appropriate vector using well known
molecular genetic techniques. The fragment is then transcribed and the peptide
subsequently translated in vitro within a host cell. Any appropriate
expression vector
(e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual (Elsevior, NY:
1985)) and
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13
corresponding suitable host cells can be employed for production of
recombinant
peptides. Expression hosts include, but are not limited to, bacterial species,
mammalian
or insect host cell systems including baculovirus systems (e.g., Luckow et
al.,
BiolTechnolog~, b, 47 ( 1988)), and established cell lines such 293, COS-7, C
127, 3T3,
CHO, HeLa, BHK, etc. An especially preferred expression system for preparing
modified
fibers of the invention is a baculovirus expression system (Wickham et al., J.
Virol., 70,
6831-38 (1995)) as it allows the production of high levels of recombinant
proteins. Of
course, the choice of expression host has ramifications for the type of
peptide produced,
primarily due to post-translational modification.
l0 Once produced, the monomers are assayed for fiber protein activity.
Specifically,
the ability of the monomers to form trimers, interact with the penton base,
and interact
with native ARs is assayed. Any suitable assay can be employed to measure
these
parameters. For example, as improperly folded monomers are generally insoluble
(Scopes, "Protein Purification" (3d Ed., 1994), Chapter 9, p. 270-82 (Springer-
Verlag,
IS New York)), one assay for trimerization is whether the recombinant fiber is
soluble.
Determining solubility of the fiber is aided if an amount of radioactive amino-
acid is
incorporated into the protein during synthesis. Lysate from the host cell
expressing the
recombinant fiber protein can be centrifuged, and the supernatant and pellet
can be
assayed via a scintillation counter or by Western analysis. Subsequently, the
proteins
20 within the pellet and the supernatant are separated (e.g., on an SDS-PAGE
gel) to isolate
the fiber protein for further assay. Comparison of the amount of radioactivity
in the fiber
protein isolated from the pellet vis-a-vis the fiber protein isolated from the
supernatant
indicates whether the mutant protein is soluble. Alternatively, trimerization
can be
assayed by using a monoclonal antibody recognizing only the amino portion of
the
25 trimeric form of the fiber (e.g., via immunoprecipitation, Western
blotting, etc.). One
such antibody is described in International Patent Application WO 95/26412,
and others
are known in the art. A third measure of trimerization is the ability of the
recombinant
fiber to form a complex with the penton base (Novelli and Boulanger, Virology,
185,
1189 (1995)), as only fiber trimers can so interact. This propensity can be
assayed by co-
30 immunoprecipitation, gel mobility-shift assays, SDS-PAGE (boiled samples
run as
monomers, otherwise, they run as larger proteins), etc. A fourth measure of
trirnerization
is to detect the difference in molecular weight of a trimer as opposed to a
monomer. For
example, a boiled and denatured trimer will run as a lower molecular weight
than a non-
denatured stable trimer (Hong and Angler, J. Virol., 70, 7071-78 (1996)).
35 A trimeric recombinant fiber must also be assayed for its ability to bind
native
ARs. Any suitable assay that can detect this is sufficient for use in the
present invention.
A preferred assay involves exposing cells expressing a native AR (e.g., 293
cells) to the
recombinant fiber trimers under standard conditions of infection.
Subsequently, the cells
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14
are exposed to native adenoviruses, and the ability of the viruses to bind the
cells is
monitored. Monitoring can be by autoradiography (e.g., employing radioactive
viruses),
immunocytochemistry, or by measuring the level of infection or gene delivery
(e.g., using
a reporter gene). In contrast with native trimers which reduce or
substantially eliminate
subsequent viral binding to the 293 cells, those trimers not substantially
reducing the
ability of native adenoviruses to subsequently bind the cells are trimers of
the present
invention. The reduction of interference with subsequent viral binding
indicates that the
trimer is itself not a ligand for its native mammalian AR, or at least binds
with reduced
affinity.
Alternatively, a vector including a sequence encoding a mutated fiber (or a
library
of putative mutated fibers, such as described herein) can be introduced into a
suitable host
cell strain to express the fiber protein. For high-efficiency screening,
preferably the host
cells are bacteria. Where bacteria are employed as host cells, mutants can be
identified by
assaying the ability to bind the soluble CAR protein. For example, a replica
of the
IS bacterial plate (e.g., on a nitrocellulose filter lift) can be cultured in
a suitable medium to
induce expression from the vector. Subsequently, the filter is exposed to a
solution
suitable for lysing the bacteria adhering to it, and the probed with a
radiolabled CAR
protein. Preferably, the filter is first "blocked" with a high protein
solution to minimize
nonspecific adherence of the CAR probe to the filter. After the hybridization,
the filter is
exposed to film to identify colonies expressing fiber proteins that bind the
CAR. Those
colonies not hybridizing to the radiolabeled CAR probe (or binding with
reduced affinity
as indicated by weaker signal) potentially express fiber monomers of the
present
invention. Because a reduction in CAR-binding could be due to either selective
ablation
of the ligand or structural modification affecting trimerization, mutant
fibers identified as
non-CAR binding by such a bacterial library screen must be assayed for the
ability to
trimerize, as described above.
Blocking Proteins
As an alternate means for reducing native viral tropism, the present invention
provides a chimeric blocking protein comprising a substrate for an adenovirus
fiber. The
chimeric blocking protein can include any suitable domain having a substrate
recognized
by the ligand on the adenoviral fiber. For example, for interfering with the
receptor-
binding of a wild-type adenovirus, the chimeric blocking protein can comprise
the
extracellular domain of the CAR cell-surface protein (Bergelson et al.,
Science, 275,
1320-23 (1997); Tomko et al., Proc. Nat. Acad. Sci. U.S.A., 94, 3352-56
(1997)), the
extracellular domain for the MHC class I receptor (Hong et al., EMBO J.,
16(9), 2294-06
( 1997)), or other similar extracellular substrate domain for an AR. Moreover,
for
interfering with the substrate-binding of recombinant adenoviruses, such as
adenoviruses
CA 02291323 1999-11-24
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1$
having chimeric fiber trimers as described herein, the blocking protein can
comprise a
substrate recognized by a ligand present on the trimer. While, as mentioned,
the chimeric
blocking protein can comprise domains from cell-surface proteins, typically it
is not itself
a cell-surface protein. Instead, the chimeric blocking protein is preferably a
free soluble
protein able to interact with an adenovirus in solution.
A chimeric blocking protein of the present invention affords a method of
interfering with adenoviral receptor-binding by incubating an adenovirus with
the
chimeric blocking protein in a solution such that the chimeric blocking
protein binds the
ligand present on the adenoviral fiber. The virus and the chimeric blocking
protein can be
l0 incubated for any length of time, and under any suitable conditions, to
promote the ligand
on the fiber to bind the substrate on the chimeric blocking protein. The
parameters of
time, temperature, and solution chemistry suitable for promoting selective
binding
between the fiber ligand and the chimeric blocking protein substrate can vary
according to
the affinity with which the ligand selectively binds the substrate. Generally,
where known
IS ligand-substrate systems are employed, these parameters are also known.
Where novel
ligand-substrate systems are employed, however, the binding conditions can, in
large
measure, be predetermined as discussed herein (e.g., by employing such
conditions when
screening the protein library for the novel ligand-substrate interaction).
However,
preferably the concentration of the chimeric blocking proteins is sufficient
to saturate the
20 cell-surface ligands present on the fibers of the adenovirus during the
incubation.
In addition to including a domain having a substrate recognized by the ligand
on
an adenoviral fiber, a chimeric blocking protein also can have other domains.
For
example, the protein can include domains to promote secretion (see, e.g.,
Suter et al.,
EMBO, J., 10, 2395-2400 (1991); Beutler et al., J. Neurochem., 64, 475-81
(1995)), thus
25 aiding in the collection of free chimeric blocking proteins from cells
producing the
protein. Additionally, the chimeric blocking protein preferably further
includes a ligand
domain (i.e., a ligand in addition to the substrate for the viral knob), such
as those ligands
described herein. The presence of a ligand on the chimeric blocking protein,
notably
peptide tags and other similar sequences, facilitates purification and
identification of the
30 chimeric blocking protein after production. A more preferred ligand is one
recognizing a
cell surface binding site or other substrate, as discussed herein. Such
blocking proteins
function as "bi-specific" molecules for altering adenoviral receptor binding.
For example,
where a chimeric blocking protein includes a ligand for a cell-surface binding
site, the
blocking protein is able to effect selective targeting of the adenovirus by
interfering with
35 fiber-mediated receptor binding while directing novel targeting through the
Iigand present
on the chimeric blocking protein. Thus, the present invention provides a
method of
directing adenoviral targeting by incubating an adenovirus with a chimeric
blocking
protein having a ligand recognizing a substrate present on a cell surface
binding site in a
CA 02291323 1999-11-24
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16
solution such that the chimeric blocking protein binds the adenoviral fiber to
form a
complex, and thereafter exposing the complex to a cell having a substrate for
the ligand.
In addition to including a domain having a substrate recognized by the ligand
on
an adenoviral fiber (and possibly a non-adenoviral ligand domain), the
chimeric blocking
protein also can include a trimerization domain, such as those trimerization
domains
discussed herein. The presence of such trimerization domains permits the
chimeric
blocking protein monomers to trimerize. While, as monomers, the chimeric
blocking
proteins can saturate the ligands present on the fibers, such bonds are, of
course, subject
to dissociation at a certain rate depending on the kinetics of the Iigand-
substrate
interaction. However, because the probability that all three Iigand/substrate
bonds
between a trimeric fiber and the trimeric blocking protein will be severed at
the same time
is significantly less than the probability that any one such bond will be
broken, a trimeric
blocking protein more easily saturates the available ligands present on the
fiber. In effect,
the trimeric structure effectively holds each substrate against the fiber knob
ligand,
thereby increasing the likelihood that each Iigand is blocked.
The chimeric blocking proteins can be produced by any suitable method, such as
by direct protein synthesis, cellular production, in vitro translation or
other method known
in the art. Many suitable methods for producing proteins are described
elsewhere herein
and are otherwise known in the art.
Viruses
The present invention provides an adenovirus incorporating the recombinant
fiber
trimers of the present invention. The adenovirus of the present invention does
not infect
its native host cell via the native AR as readily as the wild-type serotype,
due to the
above-mentioned reduction in affinity of the fiber trimers present in the
viral coat (e.g.,
via replacement of the trimerization domain with a non-ligand trimerization
domain,
selective mutation of the responsible residues, or incorporation of a blocking
domain, as
herein described). Thus, the adenovirus preferably incorporates a non-
adenoviral ligand
to facilitate its propagation, isolation and/or targeting.
The virus can include any suitable ligand (e.g., a peptide specifically
binding to a
substrate). For example, for targeting the adenovirus to a cell type other
than that
naturally infected (or a group of cell types other than the natural range or
set of host
cells), the ligand can bind a cell surface binding site (e.g., any site
present on the surface
of a cell with which the adenovirus can interact to bind the cell and thereby
promote cell
entry) other than its native AR or even any native AR. A cell surface binding
site can be
any suitable type of molecule, but typically is a protein (including a
modified protein}, a
carbohydrate, a glycoprotein, a proteoglycan, a lipid, a mucin molecule or
mucoprotein,
or other similar molecule. Examples of potential cell surface binding sites
include, but
CA 02291323 1999-11-24
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17
are not limited to: heparin and chondroitin sulfate moieties found on
glycosaminoglycans;
siaiic acid moieties found on mucins, glycoproteins, and gangliosides; common
carbohydrate molecules found in membrane glycoproteins, including mannose,
N-acetyl-galactosamine, N-acetyl-glucosamine, fucose, and galactose;
glycoproteins such
as ICAM-1, VCAM, selectins (e.g., E-selectin, P-selectin, L-selectin, etc.),
and integrin
molecules; and tumor-specific antigens present on cancerous cells, such as,
for instance,
MUC-1 tumor-specific epitopes. The protein can thus be expressed in a narrow
class of
cell types (e.g., cardiac muscle, skeletal muscle, smooth muscle, etc.) or
expressed within
a broader group encompassing several cell types.
In other embodiments (e.g., to facilitate purification or propagation within a
specific engineered cell type), the non-native ligand can bind a compound
other than a
natural cell-surface protein. Thus, the ligand can bind blood- and/or lymph-
borne
proteins (e.g., albumin), synthetic peptide sequences such as polyamino acids
(e.g.,
polylisine, polyhistadine, etc.), artificial peptide sequences (e.g., FLAG SEQ
ID N0:16},
I S and RGD peptide fragments (Pasqualini et al., J. Cell. Biol., 130, 1189 (
1995)).
Alternatively, the ligand can bind non-peptide substrates, such as plastic
(e.g., Adey et al.,
Gene, 156, 27 (1995)), biotin (Saggio et al., Biochem. J., 293, 613 (1993)), a
DNA
sequence (Cheng et al., Gene, 171, 1, (1996); Krook et al., Biochem. Biophys.,
Res.
Commun., 204, 849 (1994)), streptavidin (Geibel et al., Biochemistry, 34,
15430 (1995),
Katz, Biochemistry, 34, 15421 (1995)), nitrostreptavidin (Balass et al., Anal.
Biochem.,
243, 264 (1996)), heparin (Wickham et al., Nature Biotechnol., 14, 1570-73
(1996)),
cationic supports, metals such as nickel and zinc (e.g., Rebar et al.,
Science, 263, 671
( 1994); Qui et al., Biochemistry, 33, 8319 (1994)), or other potential
substrates.
Examples of suitable ligands and their substrates for use in the method of the
invention
include, but are not limited to: CR2 receptor binding the amino acid residue
attachment
sequences, CD4 receptor recognizing the V3 loop of HIV gp120, transferrin
receptor and
its ligand (transferrin), low density lipoprotein receptor and its ligand, the
ICAM-1
receptor on epithelial and endothelial cells in lung and its ligand, linear or
cyclic peptide
ligands for streptavidin or nitrostreptavidin (Katz, Biochemistry, 34, 15421
(1995)),
galactin sequences that bind lactose, galactose and other galactose-containing
compounds,
and asialoglycoproteins that recognize deglycosylated protein ligands.
Moreover,
additional ligands and their binding sites preferably include (but are not
limited to) short
(e.g., 6 amino acid or less) linear stretches of amino acids recognized by
integrins, as well
as polyamino acid sequences such as polylysine, polyarginine, etc. Inserting
multiple
lysines and/or arginines provides for recognition of heparin and DNA. Also, a
ligand can
comprise a commonly employed peptide tag (e.g., short amino acid sequences
known to
be recognized by available antisera) such as sequences from glutathione-S-
transferase
(GST) from Shistosoma manosi, thioredoxin (3-galactosidase, or maltose binding
protein
CA 02291323 1999-11-24
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18
(MPB) from E. coli., human alkaline phosphatase, the FLAG octapeptide (SEQ ID
N0:16), hemagluttinin {HA) (Wickham et al., 1996, supra), polyoma virus
peptides, the
SV40 large T antigen peptide, BPV peptides, the hepatitis C virus core and
envelope E2
peptides and single chain antibodies recognizing them (Chan, ,l. Gen. Virol.,
77, 2531
( 1996)), the c-myc peptide, adenoviral penton base epitopes (Stuart et al.,
EMBO J., 16,
1189-98 ( 1997)), epitopes present in the E2 envelope of the hepatitis C virus
SEQ ID
N0:17, SEQ ID N0:18 (see, e.g., Chan et al., 1996, supra), and other commonly
employed tags. A preferred substrate for a tag ligand is an antibody directed
against it, a
derivative of such an antibody (e.g., a FAB fragment, Single Chain antibody
(ScAb)), or
I D other suitable substrate.
As mentioned, a suitable ligand can be specific for any desired substrate,
such as
those recited herein or otherwise known in the art. However, adenovirai
vectors can also
be engineered to include novel ligands by first assaying for the ability of a
peptide to
interact with a given substrate. Generally, a random or semirandom peptide
library
JS containing potential ligands can be produced, which is essentially a
library within an
expression vector system. Such a library can be screened by exposing the
expressed
proteins (i.e., the putative ligands) to a desired substrate. Positive
selective binding of a
species within the library to the substrate indicates a ligand for that
substrate, at least
under the conditions of the assay. For screening such a peptide library, any
assay able to
20 detect interactions between proteins and substrates is appropriate, and
many are known in
the art. However, one preferred assay for screening a protein library is the
phage display
system, which employs bacteriophage expressing the library (e.g., Koivunen et
al.,
BiolTechnology, 13, 265-70 (1995); Yanofsky et al., Proc. Nat. Acad. Sci.
U.S.A., 93,
7381-86 (1996); Barry et al., Nature Med., 2(3), 299-305 (1996)). Binding of
the phage
25 to the substrate is assayed by exposing the phage to the substrate, rinsing
the substrate,
and selecting for phage remaining bound to the substrate. Subsequently,
limiting dilution
of the phage can identify individual clones expressing the putative ligand. Of
course, the
insert present in such clones can be sequenced to determine the identity of
the ligand.
Phage display is preferred for identifying potential ligands because it best
mimics
30 viral interaction with the microenvironment. Notably, phage display is an
extraceliular
system (as is the initial stage of viral infection); moreover, phage display
incorporates an
actual virus (phage) presenting the actual potential ligand. Phage display
also offers
significantly more flexibility than other protein binding assays (especially
intracellular
assays). Notably, phage display not only identifies proteins (Iigands) binding
to a
35 particular substrate, but it identifies those which bind under predefined
conditions. Thus,
the use of phage display can identify ligands useful for incorporation into an
adenovirus
to facilitate purification under largely predefined conditions. For example,
the phage
display library can be screened by exposure to a particular plastic, resin, or
other desired
CA 02291323 1999-11-24
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19
substrate used in an affinity column. Phage expressing peptides that either
bind the
substrate or that are eluted from the substrate under a specific condition or
range of
conditions (e.g., high or low salt, pH, temperature, etc.), but do not so bind
or elute under
other conditions, can be readily identified. Thereafter, adenovirus
incorporating the
.S Iigand can be purified by expositing it to the substrate under like
conditions, as discussed
herein.
Once a given ligand is identified, it can be incorporated into any location of
the
virus capable of interacting with a substrate (i.e., the viral surface). For
example, the
ligand can be incorporated into the fiber, the penton base, the hexon, or
other suitable
location. Where the ligand is attached to the fiber protein, preferably it
does not disturb
the interaction between viral proteins or monomers. Thus, the ligand
preferably is not
itself an oligomerization domain, as such can adversely interact with the
trimerization
domain as discussed above. Moreover, the ligand preferably does not replace a
portion of
the fiber protein, as such perturbance can adversely affect trimerization and
interaction
IS with the penton. Rather, the ligand preferably is added to the fiber
protein, and is
incorporated in such a manner as to be readily exposed to the substrate (e.g.,
at the
carboxy-terminus of the protein, attached to a residue facing the substrate,
positioned on a
peptide spacer to contact the substrate, etc.) to maximally present the ligand
to the
substrate. Where the ligand is attached to or replaces a portion of the
penton, preferably it
is within the hypervariable regions to ensure that it contacts the substrate.
Furthermore,
where the iigand is attached to the penton, preferably, the recombinant fiber
is truncated
or short (e.g., from 0 to about 10 shaft repeats) to maximally present the
ligand to the
substrate (see, e.g., U.S. Patent 5,559,099 (Wickham et al.)). Where the
ligand is attached
to the hexon, preferably it is within a hypervariable region (Miksza et al.,
J. Virol., 70(3),
1836-44 ( 1996)).
When engineered into an adenoviral protein (or blocking protein), the Iigand
can
comprise a portion of the native sequence in part and a portion of the non-
native sequence
in part. Similarly, the sequences (either native and/or nonnative) that
comprise the ligand
in the protein need not necessarily be contiguous in the chain of amino acids
that
comprise the protein. In other words, the ligand can be generated by the
particular
conformation of the protein, e.g., through folding of the protein in such a
way as to bring
contiguous and/or noncontiguous sequences into mutual proximity. Of course an
adenovirus of the present invention (or a blocking protein) can comprise
multiple ligands,
each binding to a different substrate. For example, a virus can comprise a
first ligand
permitting affinity purification as described herein, a second ligand that
selectively binds
a cell-surface site as described herein, and/or a third ligand for
inactivating the virus, also
as described herein.
CA 02291323 1999-11-24
WO 98/54346 PCT/US98/11024
The protein including the Iigand can include other non-native elements as
well.
For example, a non-native, unique protease site also can be inserted into the
amino acid
sequence. The protease site preferably does not affect fiber trimerization or
substrate
specificity of the fiber ligand. Many such protease sites are known in the
art. For
S example, thrombin recognizes and cleaves at a known amino acid sequence
(Stenflo et al.,
J. Biol. Chem., 257, 12280-90 (1982)). The presence of such a protease
recognition
sequence facilitates purification of the virus in some protocols, as discussed
herein. The
protein can be engineered to include the ligand by any suitable method, such
as those
methods described above for introducing mutations into proteins.
10 In addition to the trimer and the ligand, a virus of the present invention
can include
one or more non-native passenger genes as well. A "passenger gene"" can be any
suitable
gene, and desirably is either a therapeutic gene (i.e., a nucleic acid
sequence encoding a
product that effects a biological, preferably a therapeutic, response either
at the cellular
level or systemically), or a reporter gene (i.e., a nucleic acid sequence
which encodes a
15 product that, in some fashion, can be detected in a cell). Preferably a
passenger gene is
capable of being expressed in a cell into which the vector has been
internalized.
Preferably the passenger gene exerts its effect at the level of RNA or
protein. For
instance, a protein encoded by a transferred therapeutic gene can be employed
in the
treatment of an inherited disease, such as, e.g., the cystic fibrosis
transmembrane
20 conductance regulator cDNA for the treatment of cystic fibrosis.
Alternatively, the
protein encoded by the therapeutic gene can exert its therapeutic effect by
effecting cell
death. For instance, expression of the gene in itself can lead to cell
killing, as with
expression of the diphtheria toxin. Alternatively, a gene, or the expression
of the gene,
can render cells selectively sensitive to the killing action of certain drugs,
e.g., expression
of the HSV thymidine kinase gene renders cells sensitive to antiviral
compounds
including aciclovir, ganciclovir, and FIAU (1-(2-deoxy-2-fluoro-(3-D-
arabinofuranosil)-5-
iodouracil). Moreover, the therapeutic gene can exert its effect at the level
of RNA, for
instance, by encoding an antisense message or ribozyme, a protein which
affects splicing
or 3' processing (e.g., polyadenylation), or a protein affecting the level of
expression of
another gene within the cell (i.e., where gene expression is broadly
considered to include
all steps from initiation of transcription through production of a processed
protein),
perhaps, among other things, by mediating an altered rate of mRNA
accumulation, an
alteration of mRNA transport, andlor a change in post-transcriptional
regulation. Of
course, where it is desired to employ gene transfer technology to deliver a
given
passenger gene, its sequence will be known in the art.
The altered protein (e.g., the trimer or the coat protein having the Iigand)
and the
passenger gene (where present) can be incorporated into the adenovirus by any
suitable
method, many of which are known in the art. As mentioned herein, the protein
is
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21
preferably identified by assaying products produced in high volume from genes
within
expression vectors (e.g., baculovirus vectors). The genes from the vectors
harboring the
desired mutation can be readily subcloned into plasmids, which are then
transfected into
suitable packaging cells (e.g., 293 cells). Transfected cells are then
incubated with
adenoviruses under conditions suitable for infection. At some frequency within
the cells,
homologous recombination between the vector and the virus will produce an
adenoviral
genome harboring the desired mutation.
Adenoviruses of the present invention can be either replication competent or
replication deficient. Preferably, the adenoviral vector comprises a genome
with at least
one modification therein, rendering the virus replication deficient (see,
e.g., International
Patent Application WO 95/34671). The modification to the adenoviral genome
includes,
but is not limited to, addition of a DNA segment, rearrangement of a DNA
segment,
deletion of a DNA segment, replacement of a DNA segment, or introduction of a
DNA
lesion. A DNA segment can be as small as one nucleotide and as large as the
adenoviral
genome (e.g., about 36 kb) or, alternately, can equal the maximum amount which
can be
packaged into an adenoviral virion (i.e., about 38 kb). Preferred
modifications to the
adenoviral genome include modifications in the E1, E2, E3, and/or E4 regions.
An
adenovirus also preferably can be a cointegrate, i.e., a ligation of
adenoviral genomic
sequences with other sequences, such as other virus, phage, or plasmid
sequences.
The adenovirus of the present invention has many qualities which render it an
attractive choice for use in gene transfer, as well as other, applications.
For example, the
adenovirus does not infect its native host cells as readily as does wild-type
adenovirus,
due to the mutant fiber trimers (e.g., selective mutation of residues
responsible for AR
binding, replacement of the trimerization domain, or addition of a blocking
domain, as
herein described). Furthermore, the adenovirus has at least one non-native
ligand specific
for a substrate which facilitates viral propagation, targeting, purification,
and/or
inactivation as discussed herein. For ease in cloning, the ligands and the
trimerization
domains preferably are separate domains, thus permitting the virus to be
easily be
reengineered to incorporate different ligands without perturbing fiber
trirnerization.
Alternatively, if the fiber trimer incorporates a mutated fiber knob, the
ligand can be
incorporated into the knob, as herein described.
Of course, for delivery into a host (such as an animal), a virus of the
present
invention can be incorporated into a suitable carrier. As such, the present
invention
provides a composition comprising an adenovirus of the present invention and a
pharmacologically acceptable carrier. Any suitable preparation is within the
scope of the
invention, the exact formulation, of course, depends on the nature of the
desired
application (e.g., cell type, mode of administration, etc.), many suitable
preparations are
set forth in U.S. Patent 5,559,099 (Wickham et al.).
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22
Cell Line
As mentioned herein, an adenovirus of the present invention does not readily
infect
its native host cell via the native AR because its ability to bind ARs is
significantly
attenuated (due to the incorporation of the chimeric trimers of the present
invention).
Therefore, the invention provides a cell line able to propagate the inventive
adenovirus.
Preferably, the cell line can support viral growth for at least about 10
passages (e.g., about
15 passages), and more preferably for at least about 20 passages (e.g., about
25 passages),
or even 30 or more passages.
For example, the adenoviruses can be first grown in a packaging cell line
which
expresses a native fiber protein gene. The resultant viral particles are
therefore likely to
contain both native fibers encoded by the complementing cell line and non-
native fibers
encoded by the adenoviral genome (such as those fibers described herein);
hence a
population of such resultant viruses will contain both fiber types. Such
particles will be
able to bind and enter packaging cell lines via the native fiber more
efficiently than
particles which lack native fiber molecules. Thus, the employment of such a
fiber-
encoding cell line permits adenovirus genomes encoding chimeric, targeted
adenovirus
fibers to be grown and amplified to suitably high titers. The resultant
"mixed" stocks of
adenovirus produced from the cell lines encoding the native fiber molecule
will contain
both native and chimeric adenovirus fiber molecules; however, the particles
contain
genomes encoding only the chimeric adenovirus fiber. Thus, to produce a pure
stock of
adenoviruses having only the chimeric adenovirus fiber molecules, the "mixed"
stock is
used to infect a packaging cell line which does not produce native fiber (such
as 293 for
EI-deleted viruses). The resultant adenoviruses contain only the fiber
molecules encoded
by the genomes (i.e., the chimeric fiber molecules).
Similar fiber-complementing cell lines have been produced and used to grow
mutant adenovirus lacking the fiber gene. However, the production rates of
these cell
lines have generally not been great enough to produce adenovirus titers of the
fiber-
deleted adenovirus comparable to those of fiber-expressing adenovirus
particles. The
lower titers produced by such mutants can be improved by temporally regulating
the
expression of the native fiber to more fully complement the mutant adenovirus
genome.
One strategy to produce such an improved cell line is to use of an inducible
promoter,
(e.g., the metailothionine promoter), to permit fiber production to be
controlled and
activated once the cells are infected with adenovirus. Alternatively, an
efficient mRNA
splice site introduced into the fiber gene in the complementing cell line
improves the level
of fiber protein production in the cell line.
When the adenovirus is engineered to contain a ligand specific for a given
cell
surface binding site, any cell line expressing that receptor and capable of
supporting
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23
adenoviral growth is a suitable host cell line. However, because many ligands
do not bind
cell surface binding sites (especially the novel ligands discussed herein), a
cell line can be
engineered to express the substrate for the ligand.
The present invention provides a cell line expressing a non-native cell-
surface
biding site to which an adenovirus (or a bi-specific blocking protein) having
a ligand for
the receptor binds. Any cell line capable of supporting adenoviral growth is a
suitable
cell line for use in the present invention. Where the adenovirus lacks genes
essential for
viral replication, preferably the cell line expresses complementing levels of
the gene
products. As 293 cells are superior for supporting adenoviral growth,
preferably the cell
line of the present invention is derived from 293 cells.
The non-native cell surface binding site is a substrate molecule, such as
those
described herein, to which an adenovirus (or a bi-specific blocking protein)
having a
ligand selectively binding that substrate can bind the cell and thereby
promote cell entry.
Where the ligand is on the adenovirus, the binding site can recognize a non-
native ligand
I S incorporated into the adenoviral coat or a ligand native to a virus. For
example, where the
non-native viral ligand is a tag peptide, the binding site can be a single
chain antibody
(ScAb) receptor recognizing the tag. Alternatively, the ScAb can recognize an
epitope
present in a region of a mutated fiber knob (where present), or even an
epitope present on
a native adenoviral coat protein, (e.g., on the fiber, penton, hexon, etc.).
Alternatively,
where the non-native ligand recognizes a cell-surface substrate (e.g.,
membrane-bound
protein), the binding site can comprise that substrate. Where the substrate
binding side is
native to a cell-surface receptor, the cell line can express a mutant receptor
with decreased
ability to interact with the cellular signal transduction pathway (e.g., a
truncated receptor,
such as NMDA, (Li, et al., Nat. Biotech., 14, 989 (1996)), attenuated ability
to act as an
ion channel, or other modification. Infection via such modified proteins
minimizes the
secondary effects of viral infection on host-cell metabolism by reducing the
activation of
intracellular messaging pathways and their various response elements. In
short, the
choice of binding site depends to a large extent on the nature of the
adenovirus in
question. However, to promote specificity of the cell type for the virus, the
binding site
preferably is not a native mammalian AR. Moreover, the binding site must be
expressed
on the surface of the cell to be accessible to the virus. Hence, where the
binding site is a
protein, it preferably has leader sequence and a membrane tethering sequence
(see, e.g.,
Davitz et al., J. Exp. Med. 163, 1150 (1986)). to promote proper integration
into the
membrane.
The cell line can be produced by any standard method. For example, a vector
(e.g., an oligonucleotide, plasmid, viral, or other vector) containing a gene
encoding the
non-native receptor can be introduced into source cell line by standard means.
Preferably,
the vector also encodes an agent permitting the cells harboring it to be
selected (e.g., the
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24
vector can encode resistance to antibiotics which kill cells not harboring the
plasmid). At
some frequency, the vector will recombine with the cell genome to produce a
transformed
cell line expressing the binding site.
Method of Propagation
In connection with the cell line expressing a non-native adenoviral cell-
surface
binding site, the present invention provides a method of propagating the
inventive
adenovirus. The inventive method involves infecting the cell with an
adenovirus having a
non-native ligand selectively binding to the receptor, incubating the cells,
and recovering
l0 the adenoviruses produced within the cells. Adenoviruses recovered from the
cells can be
propagated again (e.g., amplified) to produce viral stocks of very high titer.
The ligand
on the adenovirus can be any ligand, such as those discussed herein. The cells
of the
present invention are infected by the virus at any suitable m.o.i. to promote
efficient
infection of the cell line (e.g., from about 1 m.o.i. to about 10 m.o.i.). The
conditions of
15 cell culture largely depend on the nature of the host cell. However, it is
within the skill of
the art to select culture conditions suitable for a given cell type. Viruses
are recovered
from the cells by standard means, such as by cell lysis. Thereafter they can
be purified by
standard methods or the method of the present invention.
20 Method of Purifying
As mentioned, the substrate for the ligand engineered into the adenovirus need
not
be present on the surface of a cell. For example, the substrate can be located
on a support,
e.g., an inanimate support such as plastic, glass, metal, resin, or other
material commonly
employed in chromatographic or affinity separation. Examples of such supports
include
25 metals, natural polymeric carbohydrates and their synthetically modified,
cross-linked or
substituted derivatives, such as agar, agarose, cross-linked alginic acid,
substituted and
cross-linked guar gums, cellulose esters, especially with nitric acid and
carboxylic acids,
mixed cellulose esters, and cellulose ethers; natural polymers containing
nitrogen, such as
proteins and derivatives, including cross-linked or modified gelatins; natural
hydrocarbon
30 polymers, such as latex and rubber; synthetic polymers which may be
prepared with
suitably porous structures, such as vinyl polymers, including polyethylene,
polypropylene,
polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed
derivatives,
polyacrylamides, polymethacrylates, copolymers and terpolymers of the above
polycondensates, such as polyesters, polyamides, and other polymers, such as
35 polyurethanes or polyepoxides; porous inorganic materials such as sulfates
or carbonates
of alkaline earth metals and magnesium, including barium sulfate, calcium
sulfate,
calcium carbonate, silicates of alkali and alkaline earth metals, aluminum and
magnesium;
and aluminum or silicon oxides or hydrates, such as clays, alumina, talc,
kaolin, zeolite,
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silica gel, or glass (these materials may be used as filters with the above
polymeric
materials); and mixtures or copolymers of the above classes, such as graft
copolymers
other material commonly employed in chromatographic or affinity separation.
Such
supports can be fashioned into beads, films, sheets, plates, etc., or coated
onto, bonded,
.S laminated, or otherwise joined to appropriate inert carriers, such as
paper, glass,
polymeric films, fabrics, etc.
The presence of a substrate for a ligand on the surface of an adenovirus of
the
present invention permits adenoviruses to be readily purified with high
affinity and
fidelity. Accordingly, the present invention provides a method of purifying an
adenovirus
10 having a ligand for a substrate from a composition comprising the
adenovirus. The
method involves exposing the composition to the substrate under conditions to
promote
the ligand present on the adenovirus to selectively bind the substrate.
Subsequently, the
composition (e.g., at least a significant portion of the composition) not
selectively binding
the substrate is removed from the substrate, after which the adenovirus bound
to the
IS substrate is eluted from the substrate. Using this method, an adenovirus
having a ligand
can be purified from a variety of compositions (e.g., solutions, dispersions,
suspensions,
gels, etc.). While adenoviruses can be present in a variety of compositions, a
common
composition containing adenoviruses is a cell lysate, such as produced from a
packaging
cell during adenoviral propagation.
20 Generally, the substrate is bound to a support, as previously described.
Fusing
desired ligand-substrates to a suitable support material is known in the art,
and the present
invention contemplates any suitable method for engineering a support having
the
substrate. Indeed, as mentioned, the substrate can itself be such a plastic,
glass, metal,
resin, etc. Any method of exposing the composition containing the adenovirus
to the
25 substrate is suitable for use in the present inventive method. For example,
the
composition can be passed through a column comprising the support onto which
the
substrate is bound. Of course, the composition also can be mixed with a slurry
of such a
support (e.g., beads or other preparation comprising the support-bound
substrate), placed
into a container (e.g., a tube, the well of a dish, etc.) which has been
coated with the
substrate, or otherwise exposed to the substrate.
The parameters of time, temperature, and solution chemistry necessary to
promote
selective binding can vary according to the affinity with which the ligand
selectively
binds the substrate. Generally, where known ligand-substrate systems are
employed,
these parameters are also known. Where novel ligand-substrate systems are
employed,
however, the binding conditions can, in large measure, be predetermined as
discussed
herein (e.g., by employing such conditions when screening the protein library
for the
novel ligand-substrate interaction). Preferably, the conditions for selective
binding do not
permit selective binding of other constituents of the composition to the
substrate. Where
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26
other constituents do not selectively bind the substrate, a significant amount
of the
adenovirus can be removed from the composition by association with the
substrate.
After the selective binding step, the adenoviral-deprived composition is
removed
from the presence of the substrate (e.g., selectively eluted). Any suitable
method for so
removing the adenoviral-deprived composition from the substrate can be
employed,
provided the adenovirus remains selectively bound to the substrate. In other
words, the
conditions employed for removing the adenoviral-deprived composition from the
substrate generally are insufficient to elute the adenovirus from the
substrate. The method
of removing the adenoviral-deprived composition is largely a function of the
type of
substrate and support. For example, the adenoviral-deprived composition can be
removed
from a column comprising the substrate by rinsing the column with several
volumes of a
suitable solution. Moreover, the adenoviral-deprived composition can be
removed from a
slurry of the support containing the substrate by repeated centrifugation,
resuspension in a
suitable solution, and recentrifugation. Alternatively, where the support is a
magnetic
IS material, it can be physically removed from the solution by exposing the
vessel containing
the solution to a magnet and rinsing the magnetic support. Moreover, where the
substrate
is bound to a dish or a well, the dish can simply be rinsed with several
volumes of a
suitable solution.
After the adenoviral-deprived composition has been removed from the substrate,
the adenovirus is eluted from the substrate. Any method for separating the
adenovirus
from the substrate is suitable for use in the present inventive method. In
many
applications, the adenovirus can be liberated by exposing the support-
adenovirus complex
to an elution solution incompatible with the ligand-substrate bond. The
parameters of
time, temperature, and solution chemistry necessary to promote selective
elution of the
virus from the support can vary according to the affinity with which the
ligand selectively
binds the substrate. Generally, where known ligand-substrate systems are
employed,
these parameters are also known. Where novel ligand-substrate systems are
employed,
however, the elution conditions can, in large measure, be predetermined, for
example, by
adjusting the conditions when screening a protein library, as discussed
herein.
Additionally, where the ligand is incorporated into the adenovirus on a spacer
or other
peptide, as described, the spacer can include a peptidase recognition sequence
or other
specific cleavage motif. Adenoviruses containing such a cleavage sequence can
be
liberated from the support by exposing the support to an agent effecting the
cleavage,
such as an endoprotease or other agent. While the cleavage method severs the
ligand
from the adenovirus, in many applications this is preferred. For example, the
ligand for
purifying the virus might interfere with a second ligand for targeting the
virus to a
particular cell type. Removal of the purifying ligand thus permits the
isolated adenovirus
to more readily infect the cell type of interest.
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77
While any suitable binding or elution conditions can be employed, a practical
limit
is set by the ability of the adenovirus to survive the conditions. However, as
adenoviruses
are able to withstand a wide variety of environmental variation, such as high
salt, high
osmolality, and basic conditions, the present method can be employed under a
wide range
of conditions. In any event, such conditions are known to those of skill in
the art.
The inventive method for purifying adenoviruses need not remove all of the
virus
from the solution, or even a majority of the virus. Indeed. in many
applications, the
amount of virus present in the initial composition can saturate the amount of
substrate
present on the support. Moreover, while the ligand on the adenovirus
selectively binds
the substrate, such selective binding can be of any affinity. As such, a
substantial amount
of substrate can not bind available ligands in the separation step. Therefore,
to obtain as
much adenovirus from the initial composition as possible, the adenoviral-
depleted
composition removed from the support, as herein described, can be subjected to
successive rounds of purification, and the viruses obtained from each round
can be
IS combined into a single stock. Similarly, while other constituents of the
initial
composition preferably do not selectively bind the resin, the complete absence
of
erroneous binding is not common, at least in early rounds of purification. The
presence of
background levels of erroneous binding necessarily results in some
contamination of the
initial viral stock obtained. To reduce or substantially eliminate such
background
contamination, the viral stock can be subjected to successive rounds of
purification until
the background level of contaminants approaches zero. As such, the present
inventive
method provides an economical, efficient, and reliable means of purifying
adenoviruses
having known ligands. Moreover, the use of slurries and columns is common in
industrial
applications, rendering the present method amenable to high throughput, or
commercial-
scale application.
Method of Infecting a Celt
As mentioned, the non-native ligand present on the virus of the present
invention
(or on the virus/blocking protein complex) can recognize a substrate present
within a cell
surface binding site. Therefore, the present invention provides a method of
infecting a
cell having a cell surface binding site including a substrate for the non-
native ligand. The
method involves contacting the cell with the adenovirus such that the non-
native ligand of
the adenovirus (or on the virus/blocking protein complex) binds the particular
cell surface
binding site and thereby effects entry of the adenovirus. Because the viruses
of the
present invention incorporate fiber trimers having reduced ability to bind
native
mammalian ARs, the adenovirus is internalized into the cell primarily due to
the non-
native ligand. As such, the present inventive method effects selective
targeting of the
virus comprising the ligand to a cell type expressing a binding site
comprising the
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28
substrate for that ligand without significant infection of cells via native
mammalian ARs.
In the case where the ligand is on the penton base (such as a modified or
unmodified
penton base), the virus is internalized via the ligand on the penton.
Any cell expressing a cell surface binding site including a substrate for the
ligand
can be selectively targeted in accordance with the present invention. A cell
can be present
as a single entity, or can be part of a larger collection of cells, such as a
cell culture (either
mixed or pure), a tumor, a tissue (e.g., epithelial, muscle, or other tissue),
an organ, an
organ system (e.g., circulatory system, respiratory system, gastrointestinal
system, urinary
system, nervous system, integumentary system or other organ system), or even
an entire
organism (e.g., a human). Preferably, the cells being targeted are selected
from the group
consisting of heart, blood vessel, smooth muscle, skeletal muscle, lung,
liver, gallbladder,
urinary bladder, and eye cells.
The method for infecting a cell ideally is carried out wherein the adenovirus
includes a passenger gene, such as those vectors herein described. Where the
adenovirus
of the present invention includes a passenger gene, the method permits the
adenovirus to
serve as a vector for introducing that gene into a targeted cell. Once
internalized, the
passenger gene is expressed within the cell. Thus, the vectors and methods of
the present
invention provide useful tools for introducing a passenger gene into a
selected class of
cells without significantly providing the gene to cells ubiquitously or
ectopicly.
Method of Inactivating a Virus
As mentioned, the non-native ligand present on the virus of the present
invention
can recognize substrate present within blood or lymphatic fluid (such as a
ligand present
on a free blood-borne protein, a protein present on erythrocytes, etc.).
Therefore, the
present invention provides a method of inactivating an adenovirus having a
ligand
recognizing a blood- or lymph-borne substrate by exposing the virus to the
substrate.
Within the blood or lymph, the Iigand selectively binds its substrate, thereby
adsorbing
the free virus from the fluid. Preferably, the substrate is present within a
large
macromolecule (e.g., albumin) or on the surface of erythrocytes (which lack
transcription
machinery required to propagate viruses). Of course, a ligand for inactivating
the virus
can be present at any location on the viral coat (Fender et al., Virology,
214, 110 (1995)).
However, as antibodies recognizing and/or neutralizing adenoviruses primarily
bind
epitopes on the hexon (Gahery-Segard et al., Eur. J. Immunol., 27, 653
(1997)), non-
native ligands for inactivation of the virus preferably are incorporated into
the hexon, as
herein described.
By providing a means of effectively inactivating adenoviruses, the method
assists
in confining the viral infection to a desired locus (tissue, cell type, etc.).
Specifically, the
method effectively inactivates an individual virus by tethering it to the
substrate, thereby
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29
reducing its ability to contact (and therefore enter) a cell. Even where a
virus so adsorbed
does contact a cell, it is significantly less likely to be internalized due to
the presence of
the particle having the substrate. Due to the aggregation of these effects,
the inventive
method effectively inactivates a viral stock (outside of the desired locus of
infection) by
dramatically reducing its effective free titer.
The inventive method for inactivating the virus complements the other
embodiments of the present invention. For example, as stated, the viruses of
the present
invention incorporate fiber trimers having reduced affinity for native
mammalian ARs,
thereby substantially reducing the likelihood that the virus will infect cell
types other than
l0 the desired cell type. Moreover, the viruses of the present invention can
include ligands
specific for a substrate present on a cell surface binding site, permitting
the virus to be
targeted to a predetermined cell type. While those two qualities effect
selective targeting,
and thereby significantly attenuate ectopic infection, viruses also having a
ligand
recognizing a blood- or lymph- borne substrate are much less likely to even
contact an
IS ectopic tissue by reason of the effective reduction of viral titer.
While it is believed that one of skill in the art is fully able to practice
the invention
after reading the foregoing description, the following examples further
illustrate some of
its features. As these examples are included for purely illustrative purposes,
they should
not be construed to limit the scope of the invention in any respect. The
procedures
20 employed in these examples, such as affinity chromatography, Southern
blots, PCR, DNA
sequencing, vector construction (including DNA extraction, isolation,
restriction
digestion, ligation, etc.), cell culture (including antibiotic selection),
transfection of cells,
protein assays (Western blotting, immunoprecipitation, immunofluorescence),
etc., are
techniques routinely performed by those of skill in the art (see generally
Sambrook et al.,
25 Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring
Harbor, NY ( 1989)). Accordingly, in the interest of brevity, experimental
protocols are
not discussed in detail.
EXAMPLE 1
30 This example describes two different fiber trimers having non-native
trimerization
domains, each of which interacts properly with the adenoviral penton base.
Specifically,
the fiber chimeras incorporate the reovirus sigma 1 trimerization domain.
Two chimeras were constructed, TSS7sigDel and TSsigDel. TSsigDel contained
only the Ad5 fiber tail (TS) fused to sigDel without any Ad fiber shaft
sequence.
35 TSS7sigDel contained the tail plus the first 7 (3-sheet repeats of the Ad
shaft (S7) fused to
sigDel. The DNA and respective amino acid sequences of these two clones are
set forth
at SEQ ID NO: I and SEQ ID N0:2.
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The sigDel region of the reovirus sigma 1 gene was amplified via PCR and
cloned
into the vector, pAcT5S7GCNTS.PS.LS.X (Fig. 3A), to create the baculovirus
transfer
vector, pAcTSsigDel.TS.PS.LS (Fig. 3B). This vector encodes the Ad5 fiber tail
fused to
the N-terminal trimerization domain of reovirus type 3 sigma 1 protein
followed by a
5 FLAG epitope near the C-terminus. At the C-terminus of the gene, the vector
also
contains multiple restriction sites to facilitate the cloning of targeting and
purification
sequences into the gene.
The second vector, pAcT5S7sigDel.TS.PS.LS (Fig. 3C), was created by cutting
the above PCR product with the restriction enzymes NheI and BamHI and cloning
this
10 fragment into the vector, pAcT5S7GCNTS.PS.LS.X (Fig. 3A), also cut with
NheI and
BamHI. The resultant vector encodes a protein containing the tail and first
seven ~3-sheet
shaft repeats of Ad5 fiber fused to sigDel, followed by a FLAG epitope.
Recombinant baculovirus clones encoding each of the fiber chimeras were then
generated by standard means using each of the above plasmids. The resultant
baculovirus
IS clones were used to produce recombinant proteins in Tn5 insect cells. To
compare the
sigDel trimerization domain with the GCN domain, another baculovirus was
constructed
from the initial plasmid, pAcT5S7GCNTS.PS.LS.X (Fig. 3A), which contained the
GCN
trimerization domain in place of the sigDel trimerization domain.
The baculovirus-infected cells were pelleted at 3 days post infection. The
cell
20 pellet was resuspended in PBS plus protease inhibitors and freeze-thawed
three times to
release the soluble intracellular proteins. The cell debris were then pelleted
by
centrifugation at high speed and the cleared cell lysate was removed. The
pellet was then
resuspended in the same volume of PBS as previously.
Pellet and lysate samples were then run on an 0.1% SDS, 12.5% polyacrylamide
25 gel and transferred to nitrocellulose for Western analysis using anti-FLAG
M2 MAb
(Kodak). These results demonstrated that over 90% of each of the proteins,
TSS7GCN.TS.PS.LS, TSsigDel.TS.PS.LS and TSS7sigDel.TS.PS.LS were soluble in
the
lysate.
The proteins were further assayed for their ability to form trimers. To test
for
30 chimera trimerization, the lysates from each sample were either boiled or
not boiled prior
to running the samples on a 0.1% SDS, 12.5% polyacrylamide gel. Western
analysis of
the boiled samples showed that the boiled samples migrated at molecular
weights
corresponding to the size of the monomeric protein, whereas the unboiled
proteins
containing the sigDel trimerization domains migrated at molecular weights
commensurate
with a trimer. The unboiled TSS7GCN.TS.PS.LS protein also migrated as a
trimer;
however, a significant portion (over half) of the unboiled sample migrated as
a monomer.
Similar analyses of wild type fiber and sigma 1 protein have shown that these
proteins
migrate completely as trimers when not boiled and as monomers when boiled.
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That the vast majority of the proteins were soluble in the lysate (as opposed
to the
pellet) strongly suggests that they were correctly folded. Moreover, the
migration of the
unboiled samples demonstrates that sigDel-containing chimeras are soluble
trimers and
that the sigDel domain functions better than GCN by forming more stable
trimeric fiber
chimeras.
To test for the ability of the trimers to complex properly with adenoviral
penton
base protein, recombinant penton base is mixed in solution with the
TSS7GCN.TS.PS.LS,
TSsigDel.TS.PS.LS and TSS7sigDel.TS.PS.LS trimeric fiber proteins. The
resultant
penton base/fiber chimera complex is then immunoprecipitated with anti-penton
base
l0 antibody coupled to protein A-agarose. The precipitated sample is then run
on an SDS-
PAGE gel and evaluated by Western analysis as described above using the FLAG
antibody. Binding of the FLAG antibody indicates that the fiber chimera
containing the
FLAG epitope complexes with the penton base in solution.
15 EXAMPLE 2
This example demonstrates the ability of the fiber-sigDel chimeras to
incorporate
exogenous protein domains larger than peptide tags.
The sequence encoding a modified version of the green fluorescent protein was
amplified by PCR using the primers containing restriction sites to allow
efficient cloning
20 into the fiber-sigDel chimera plasmids described above in Example 1.
Cloning of the
GFP sequence in the proper orientation into the SpeI site of
pAcTSsigDel.TS.PS.LS (Fig.
3B) yields the plasmid, pAcTSsigDel.GFP.TS.PS.LS (Fig. 4), encoding a fiber-
sigDel-
GFP chimera. The DNA and amino acid sequence of this clone is set forth as SEQ
ID
N0:3.
25 This piasmid was then used to produce recombinant protein using the
baculovirus
expression system, as described above. The solubility of the chimeric fiber
proteins
(indicative of correct folding) and the ability of the resultant proteins to
bind penton base
(indicative of trimerization) was confirmed as discussed above. Production of
soluble,
trimeric protein containing the GFP domains indicates that large, functional
protein
30 domains can be incorporated into the fiber-sigDel chimeras as easily as can
be the smaller
peptide tags. The results predict that such chimeras could also incorporate
ligands, such
as ScAbs, without significantly interfering with protein function.
EXAMPLE 3
35 This example describes the construction of recombinant adenovirus vectors
containing fiber trimers having non-native trimerization domains.
The NdeI to BamHI fragment is excised from pAcT5S7sigDel.TS.PS.LS (Fig.
3C), to replace the corresponding fragments in pAS pGS HAAV (Fig. SA), and pAS
pGS
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32
pK7 (Fig. SB), to produce the final transfer vectors pAS TSS7sigDelpGS.HAAV
(Fig.
SC) and pAST5S7sigDelpGS.pK7 (Fig. SD), respectively. The vectors encode the
fiber-
sigDel chimera containing either the RGD or pK7 binding domains at their C-
terminus for
binding to an a~ integrin and heparin sulfate-containing receptors that are
expressed by
293 cells.
These vectors are then linearized and then transfected 293 cells had been
preincubated with the E1, E3, E4-deleted adenovirus AdCMVZ.I lA (GenVec, Inc.,
Rockville, MD) prior to transfection with the plasmids. Recombination of
the,E4+ pNS
plasmid with the E4-deleted vector results in the rescue of an El-, E3-, E4+
vector
l0 capable of replication in 293 cells. The infected/transfected cells are
harvested after 5
days and lysed to release virions. The lysate is then used to infect freshly
plated cells and
to further plaque-purify the recombinant viruses. Plaques cross-contaminated
with the
original AdCMVZ.I lA stain blue when plagued in medium containing X-glu
substrate.
White plaques (indicating viable vector) are then amplified to produce pure
virus stocks
of the recombinant adenovirus.
EXAMPLE 4
This example describes the production of targeted adenovirus particles having
genomes encoding chimeric fibers. The chimeric fibers represent the Ad5 fiber
tail and
20 seven shaft repeats fused to the sigDel trimerization domain from reovirus
followed by a
high affinity RGD sequence for binding av integrins.
The plasmid, pAS TSS7sigDel.HAAV (Fig. SC), is cut with the restriction enzyme
DrdI, and the large fragment containing all the adenovirus sequences is
isolated and
purified. This fragment is then electroporated into BJ5183 bacterial cells
along with a
25 linearized plasmid, containing the majority of Ad genome prior to the fiber
gene with a
small overlap of identical sequence with the pAS TSS7sigDel.HAAV plasmid. Upon
recombination of the two pieces of DNA, a new plasmid is produced in the
bacterial cells
through homologous recombination. This plasmid encodes a modified adenovirus
genome
that is capable of replicating in the appropriate complementing mammalian cell
line (E 1
30 and fiber-complementing). The plasmid DNA from selected colonies is
isolated and
confirmed to be the correct plasmid by restriction analysis. This plasmid DNA
is then
used to transform DHSa bacterial cells in order to obtain adequate amounts of
DNA for
transfection into the fiber-complementing cell line.
One microgram of the plasmid is cut with the appropriate restriction enzyme
and
35 transfected into a fiber-complementing cell line, such as the cell line
described above. At
0-4 days post-transfection, the cells are induced with zinc, and 1-5 days
later the cells are
lysed. The lysate is passaged onto fresh fiber-complementing cells. This
passage and lysis
cycle is repeated until a cytopathic effect develops in the cells. During the
cycle, the LacZ
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activity of the cell lysate is also followed, as it should increase as the
recombinant vector
is amplified. Once an adequate titer of the "mixed" stock is obtained, a final
passage onto
non-fiber-complementing cells is made to produce a targeted virus lacking a
native fiber
protein. The resulting virus is then assayed for its ability to bind and enter
cells via the
interaction of its high affinity RGD sequence with av integrins.
EXAMPLE 5
This example describes four different fiber trimers having non-native
trimerization
domains. Specifically, the exemplified fiber trimers are chimeras
incorporating the knob
1 D portion of the NADC-1 fiber, a porcine adenoidal strain. The exemplified
trimers, thus,
contain known receptor-binding motifs (i.e., a galectin motif and an RGD
motif).
Furthermore, exemplified trimers incorporate mutations known to reduce the
affinity of
each of the receptor-binding motifs. Finally, this example describes the
incorporation of a
non-native ligand (FLAG) into an exposed loop of a non-native trimer.
Using PCR, the knob of the NADC-1 fiber gene was amplified from a plasmid
containing the full length gene. The PCR product was then cloned into a
baculovirus
expression plasmid to produce a plasmid which encoded the NADC-1 knob plus an
N-
terminal polyhistidine tag (the Pig4KN protein) for purification and detection
by Western
analysis using an anti-polyhistidine antibody. The DNA and amino acid
sequences of this
clone are set forth at SEQ ID N0:4.
The resultant plasmid, pAcPig4KN (Fig. 6A), was then mutated by site-directed
mutagenesis using the two oligonucleotide primer pairs PigD363Es (SEQ ID
NO:10) and
PigD363Ea {SEQ ID NO:11), and PigN437Ds (SEQ ID N0:12) and PigN437Da (SEQ ID
N0:13). The former pair of primers was used to produce the plasmid pAcPigKN
D363E
(Fig. 6B), in which the DNA sequence encoding the RGD integrin binding motif
(a.a.
361-363 in the native fiber protein) was mutated to the non-functional
sequence RGE.
The second pair of primers was used to produce the plasmid pAcPigKN N437D
(FIG.
6C), in which the DNA sequence encoding the native amino acid N (a.a. 437) was
mutated to a D. This mutation has been previously shown to abrogate the
binding of
another galectin protein to its ligand, galactose (Hirabayashi et al., J.
Biol. Chem., 266,
23648-53 ( 1991 }).
A final baculovirus plasmid was constructed to demonstrate the feasibility of
incorporating a novel binding motif into an exposed loop on the NADC-1 knob.
Hydrophobicity analysis of the NADC-1 knob protein revealed that the protein
sequence
immediately prior to the RGD motif was likely to be an exposed loop that would
be
capable of incorporating additional amino acid sequences (e.g., polypeptide
domains) for
the purpose of targeting or purification. Therefore, the plasmid,
pAcPig4KN(FLAG)
(Fig. 6D), was produced using complementary overlapping oligonucleotides,
which
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34
encoded the FLAG binding domain. The oligonucleotides were annealed and cloned
into
the plasmid pAcPig4KN (Fig. 11A), which contained a unique, native restriction
site,
AvrII, just prior to sequence encoding the RGD domain.
The four baculovirus transfer plasmids described above carrying NADC-1 knob
genes were used to express recombinant protein in insect cells using the
baculovirus
expression system. Tn5 insect cells were infected with the recombinant
baculovirus
clones derived from the plasmids. After three days the cells were pelleted and
freeze-
thawed three times in PBS plus protease inhibitors to release the soluble
intracellular
protein. The debris were pelleted and the cleared lysate was decanted. The
remaining
pellet was resuspended in PBS.
Lysate and pellet samples were then evaluated by SDS-PAGE and Western
analysis to determine whether the recombinant knob proteins were soluble.
Western
analysis revealed that the majority of all four knob proteins were present in
the cell lysate,
indicating that they were soluble and correctly folded. These results
demonstrate that
IS neither the point mutations introduced into the receptor-binding domains
nor the FLAG
binding sequence inserted into an exposed loop adversely affected knob folding
and
solubility.
To investigate whether the chimeric trimers having the NADC-1 knob-FLAG
domains can interact with the FLAG antibody, cell lysates are
immunoprecipitated using
anti-FLAG M2 antibody and then blotted. Western analysis will demonstrate that
the
NADC-1 knob containing the FLAG epitope is precipitated by the anti-FLAG
antibody.
Thus, the NADC-1-fiber trimers are soluble, and each is capable of interacting
with the
anti-FLAG M2 monoclonal antibody.
EXAMPLE 6
This example describes the synthesis of recombinant Ad5-based vector
containing
an NADC-1 (porcine adenovirus) fiber knob.
Using PCR, the knob of the NADC-1 fiber gene was amplified from a plasmid
containing the full length gene. The PCR product was then cloned into the
plasmid PNS
FSF2K (Fig. 7A) to produce the plasmid, pNS Pig4.SS (Fig. 7B) which encodes
the first 7
(3-repeats of the Ad5 shaft fused to the NADC-1 knob. The DNA and amino-acid
sequences of this clone are set forth at SEQ ID NO:S.
The pNS Pig4.SS plasmid was then used to create a recombinant adenovirus
vector. The plasmid was transfected into 293 cells which had been infected
with an
adenovirus vector lacking the E4 region. Homologous recombination between the
plasmid and the vector produced an E4-containing, replication competent vector
having
the chimeric NADC-1 fiber. The recombinant virus was then plaque purified on
293
cells. Preincubation of Ramos cells (which do not express av integrins but do
express
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receptors for the fiber protein of adenovirus) with recombinant NADC-1 knob
blocked the
transduction of these cells by the AdZ.PigSS vector, demonstrating that the
vector
contains a functional NADC knob. The results indicate that chimeric NADC-1
fiber can
be correctly synthesized and incorporated into viable virus particles.
5
EXAMPLE 7
This example describes an Ad5-based adenoviral vector having a chimeric fiber
trimer comprising a mutant NADC-I knob with attenuated receptor-binding
ability and
containing a functional non-native ligand.
10 The ApaI to BamHI fragment containing the N-D mutation in pAcPig4KN N437D
(Fig. 6C) is cloned into the plasmid pAcPig4KN D363E (Fig. 6B) containing the
RGD-
RGE mutation to create the plasmid pAcPig4KN D363E N437D (Fig. 8A) containing
both mutations in the NADC-1 knob gene. Overlapping, complementary
oligonucleotide
primers encoding the high affinity a,, integrin binding domain, are thereafter
cloned into
15 the native AvrII site to produce the plasmid pAcPig4KN D363E N437D HAAV
(Fig.
8B). The mutated NADC-I gene fragment EcoRI to BamHI is then cloned into the
plasmid pNSPig4.SS (Fig. 7B) to create the plasmid, pNS Pig4 D363E N437D HAAV
SS
(Fig. 8C). This plasmid is then used to create a recombinant adenovirus vector
containing
the mutated and a" integrin-targeted NADC-1 knob as described above.
20 The ability of the double mutation in the NADC-1 knob to block binding to
the
native cell surface binding sites (galectin and integrin) is confirmed via
competition
assays. Moreover, the ability of the resultant virus to target cell-surface a"
integrin is
confirmed using 293 cells, as discussed above.
25 EXAMPLE 8
This example describes two chimeric blocking proteins able to interfere with
native adenoviral receptor binding. In particular, the blocking protein each
include a
domain having a substrate for the native adenovirus fiber, namely the
extracellular
domain of the CAR.
30 The extracellular domain of CAR was amplified from the CAR gene (Bergelson
et
al., supra; Tomko et al., supra) via PCR. The PCR product was then cloned into
a
baculovirus expression vector to create the plasmid pACSG2-sCAR (Fig. 9A). The
soluble CAR protein {SCAR) also contained a FLAG epitope for purification and
for
detection by Western analysis. The DNA and amino acid sequences of this sCAR
clone
35 are set forth at SEQ ID NO:b.
Western analysis of SCAR produced in insect cells using a baculovirus clone
containing SCAR revealed that the protein was secreted from the cell and that
some of the
protein was retained within the cell.
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To assess whether the SCAR protein retains the function of the native CAR,
radiolabeled adenovirus type 2 were preincubated in a solution containing
various
concentrations of sCAR and then exposed to 293 cells. The data demonstrated
that
increasing concentrations of sCAR blocked virus binding to 293 cells. This
result
demonstrated that the soluble sCAR protein retains the structure and function
of the native
extracellular domain of CAR. Moreover, these results demonstrate that
preincubation
with SCAR can ablate native adenoviral receptor binding via the CAR-binding
ligand on
the adenovirus fiber.
A second sCAR-containing chimera was produced in which DNA sequence
encoding an RGD targeting motif was cloned into an Spel site following the C-
terminal
end of sCAR using complementary, overlapping primers. The chimeric gene
retained the
FLAG epitope on the C-terminus. The resultant plasmid, SG2-sCAR-HAAV (Fig.
9B),
was used to produce recombinant sCAR.RGD protein as was done for sCAR protein
described above. The DNA sequence of this clone is set forth at SEQ ID N0:7.
The sCAR.RGD protein was synthesized and secreted from insect cells similarly
to
the sCAR protein. To assess whether the sCAR.RGD protein retains the function
of the
native CAR, radiolabeled adenovirus type 2 were preincubated in a solution
containing
various concentrations of sCAR.RGD and then exposed to Ramos cells, which do
not
express av integrins but do express receptors for the fiber protein of
adenovirus.
Preincubation of radiolabeled adenovirus type 2 with either sCAR or sCAR.RGD
blocked
virus binding to Ramos cells. This result demonstrates that the sCAR domain
present in
the sCAR.RGD protein is functional.
To assess whether the sCAR.RGD protein retains the function of the native RGD
domain, cell adhesion studies were conducted. Both sCAR.RGD, and sCAR were
immobilized onto tissue culture plastic plates, which were subsequently
contacted with
293 cells (which express a,, integrin). After the cells were incubated on the
coated plates,
the plates were rinsed, and the number of cells remaining in contact with the
plates were
assayed. The results showed that cells adhered to plates coated with sCAR.RGD,
while
they did not adhere to plates coated with SCAR or control plates,
demonstrating that the
RGD motif present in the sCAR.RGD protein is functional.
EXAMPLE 9
This example demonstrates the inventive method of directing adenoviral
targeting
using a chimeric blocking protein having a ligand for a cell surface binding
site.
An adenovirus vector carrying a IacZ reporter gene is preincubated with either
the
sCAR.RGD protein or the sCAR protein, described above in Example 8. The
resultant
complexes are then exposed to either Ramos cells (which express fiber receptor
(CAR)
but lack a" integrins) or HuVEC cells (which express both CAR and a,,
integrins) under
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37
conditions suitable for viral infection. Subsequently; the cells are assayed
for lacZ
expression, the level of which will correlate to the degree to which the
viruses infect the
cells. The results will demonstrate that both sCAR and sCAR.RGD effectively
block
adenovirus transduction of Ramos cells whereas sCAR, but not sCAR.RGD, blocks
S adenovirus transduction of HuVEC cells, indicating that the Ad/sCAR.RGD
complex is
targeted to av integrins while avoiding adenoviral-mediated gene delivery to
cells via
CAR.
EXAMPLE 10
This example describes two chimeric blocking proteins able to form trimers
interfering with native adenoviral receptor binding. In particular, the
blocking proteins
each include a domain having a substrate for the native adenovirus fiber,
namely the
extracellular domain of the CAR, and a trimerization domain, namely the sigDel
trimerization domain of the Sigma-1 reovirus protein.
IS The sigDel trimerization domain of the Sigma-1 reovirus protein is
amplified by
PCR, and the resultant PCR product is cloned into the pAcSG2-SCAR plasmid
(Figure
9A). The resultant plasmid, pAcSG2sCAR.sigDel (Fig. l0A) contains a gene
chimera
encoding the extracellular domain of CAR, a spacer region, the trimerization
domain from
sigma 1 protein of reovirus, and a FLAG binding domain. An SpeI restriction
site
following the trimerization domain allows for the convenient cloning of
targeting
domains, such as the high affinity RGD motif which binds a,, integrins. The
DNA and
amino acid sequences of this clone are set forth at SEQ ID N0:8.
PAcsCAR.sigDel was used to make baculovirus. Western analysis of boiled and
unboiled cell lysates from baculovirus-infected cells showed that the unboiled
chimeric
sCAR.sigDel migrated as a trimer.
A second sCAR-containing chimera is produced in which DNA sequence encoding
an RGD targeting motif is cloned into an SpeI site following the C-terminal
end of
sCAR.sigDel using complementary, overlapping primers. The resultant plasmid,
pAcSG2-sCARsigDel (HAAV) (Fig. lOB), encodes a chimera having the
extracellular
domain of CAR, a spacer region, the trimerization domain from sigma 1 protein
of
reovirus, and the high affinity RGD motif which binds a" integrins.
The pAcSG2sCAR.sigDel and pAcSG2-sCARsigDel.RGD (HAAV) plasmids
were used to produce recombinant baculovirus which are used to produce the
recombinant
chimeric protein in insect cells by standard means. Western analysis of boiled
and
unboiled cell lysates from bacculovirus-infected cells demonstrated that the
unboiled
sCAR.sigDel protein migrated as a trimer.
To assess the ability of the trimeric sCAR.sigDel and sCARsigDel.RGD proteins
to block adenoviral infection, an adenovirus vector carrying a IacZ reporter
gene is
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38
preincubated with either the sCAR.sigDel or the sCARsigDel.RGD trimer or the
sCAR
monomeric protein. Several concentrations are employed to generate dose-
response data.
The resultant complexes are then exposed to 293 cells under conditions
suitable for viral
infection. Subsequently, the cells are assayed for lacZ expression, the level
of which will
correlate to the degree to which the viruses infect the cells. The results
will demonstrate
that the trimeric sCAR.sigDel and sCARsigDel.RGD proteins are more potent in
blocking
adenovirus binding to via the sCAR protein cells than the sCAR monomers.
EXAMPLE 11
This example demonstrates the inventive method of directing adenoviral
targeting
using a trimeric blocking protein having a Iigand for a cell surface binding
site.
An adenovirus vector carrying a lacZ reporter gene is preincubated with either
sCAR.sigDel, sCARsigDel.RGD, or sCAR described above. Similarly, the
adenovirus
can be preincubated with a blocking protein isolated, for example, by phage
display. The
IS resultant complexes are then exposed to either Ramos cells or HuVEC cells
under
conditions suitable for viral infection. Subsequently, the cells are assayed
for IacZ
expression, the level of which will correlate to the degree to which the
viruses infect the
cells. The results will demonstrate that, while such proteins will effectively
block
adenovirus transduction of Ramos cells, the trimers are more potent in
blocking
adenovirus binding than the sCAR monomers. Moreover, both sCAR and
sCAR.sigDel,
will block adenovirus transduction of HuVEC cells; however, sCARsigDel.RGD
will not
effectively block adenovirus transduction of HuVEC cells. Such results
strongly suggests
that the Ad sCARsigDel.RGD complex is targeted to a" integrins while avoiding
adenoviral-mediated gene delivery to cells via CAR.
EXAMPLE 12
This example describes the construction and evaluation of mutated fiber knobs
each having reduced affinities for native substrates, particularly monoclonal
antibodies
raised against the native fiber knob.
Using site-directed mutagenesis, separate mutations were introduced into the
full
length Ad5 fiber gene in a baculoviral vector. The resultant plasmids were
then used to
generate recombinant bacuioviral clones.
Each of the mutants, plus a native Ad5 fiber control, were used to produce
protein
in infected insect cells. Three days post infection, the cells were harvested
and lysed.
Western analysis using polyclonal antisera recognizing the Ad5 fiber revealed
the
presence of high amounts of fiber protein in lysates from cells infected with
each of the
vectors. In cells infected with five of the mutant clones (see table 1 ) (as
well as the native
fiber gene), the signal was predominantly in the soluble portion of the
lysates, indicating
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39
that the protein encoded by each mutant was correctlyThe sequences of
folded. the wild-
type Ad5 fiber is set forth a SEQ ID N0:9. The aminof SEQ ID N0:9 changed
acids o by
each of these mutations is indicated in Table 1.
' Table 1
Mutations Monoclonal Antibodies
2C9 4B8 3D9 2E5
CD Loop (449 SGTVQ-GSGSG) - - + +
IJ Loop (559 GSHN-GSGS) - ~ + +
FG Loop (507 SHGKTA-GSGSGS) - - + +
IO T533S/T353S (535 TIT-SIS) + - + +
K506R (506 K-R) + + - +
C-Term Addition + + + +
Native Ad5 Fiber + + + +
Boiled Ad5 Fiber - - - -
IS
Using Western slot-blot analysis, each of the five soluble mutant fiber
proteins, the
native Ad5 fiber, and a denatured Ad5 fiber were screened against a panel of
four
monoclonal antibodies raised against the fiber knob. The signals were detected
by
chemiluminescence and the strength of signals of each band compared. The
results of this
20 assay are set forth in Table 1.
That none of the antibodies recognizes the denatured fiber demonstrates that
each
binds only correctly folded, trimeric fibers. Furthermore, that none of the
mutants
exhibited reduced affinity for the 2E5 antibody confirmed that each of the
mutant fibers
was, indeed, trimeric.
25 The K506R mutation significantly reduced the affinity of the resultant
fiber for the
3D9 antibody without affecting the affinity for any of the other antibodies.
The location
of this mutation within the fiber knob is indicated in Figs. ISA-ISC.
Mutations in the CD, IJ, or FG loops, in which 4-6 amino acids were replaced
by
altering serines and glycines, significantly reduced the affinity of the
resultant mutant
30 trimers for the 2C9 antibody. Moreover, the double mutant T533S/T535S also
reduced
the affinity of the mutant knob for the 4B8 antibody. The location of each of
these
mutations within the fiber knob are indicated in Figs. 15D-15F.
These results indicate that the trimeric fiber knobs having reduced affinity
for
' native substrates can be generated. A similar screening protocol can be used
to identify
35 mutants having reduced affinity for cellular receptors. For example, a
soluble form of
sCAR having a FLAG epitope (or other tag), such as described above, can be
used as a
probe in place of the monoclonal antibodies described above. The blots are
then screened
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with anti-FLAG monoclonal antibodies to detect mutations interfering with
fiber-CAR
binding.
EXAMPLE 13
5 This example describes the construction of a recombinant adenovirus
containing a
short-shafted fiber (e.g., 8 shaft repeats) and a mutant fiber(5) knob having
reduced
affinity for its native receptor (i.e., CAR). Such a fiber permits targeting
via a ligand
expressed in the penton base.
Using standard recombination techniques, a deletion is introduced into the
10 sequence encoding the fiber shaft. For example, a portion of the mutant
fiber knob from
the 22d shaft repeat until the end of the coding sequence and containing the
K506R
mutation (see Example 12) is amplified by PCR from SEQ ID N0:8. The resultant
product is used to create the pAS TSS7FSK(R506K) plasmid (Fig. 16). The
plasmid,
thus, contains a gene encoding a short-shafted fiber with reduced affinity for
a native
I S substrate (the 3D9 antibody). An adenovirus having such a short-shafted
fiber will be
able to bind to cells via the RGD ligand on the penton base. Of course, a
similar strategy
can be used to create adenoviral vectors having short-shafted fibers with
reduced affinity
for the CAR.
20 EXAMPLE 14
This example demonstrates the construction of adenovirus vectors having
specific
non-native ligands that can be used to purify the vector via affinity
chromatography.
The base vector pNSF5F2K (Figure 8A) contains a gene which encodes a
chimeric fiber having the shaft of the Ad5 fiber and the knob of the Ad2 fiber
protein.
25 The Ad2 fiber gene contains an SpeI restriction site in the region of the
knob which
encodes the flexible, exposed HI loop of the fiber knob. This SpeI restriction
site was
used to insert sequences which encode the FLAG peptide SEQ ID N0:16 or a
DNA/heparin-binding ligand (SEQ ID NO:15).
The base vector pBSSpGS (Figure 11A) encodes a C-terminal 12 amino acid
30 extension (SEQ ID N0:14). The codons encoding the TS also are a unique SpeI
site that
was used to insert sequences which encode the FLAG peptide (SEQ ID N0:16) or
the
DNA/heparin-binding polypeptide (SEQ ID NO:15) as described below.
Transfer plasmids (pBSS pGS (RKKK)2 (Figure 11B) and pNSF5F2K(RKKK)2
(Figure 11 C)) for introducing the DNA/heparin-binding ligand into the
adenoviral
35 genome were created using overlapping oligonucleotides. Sense and antisense
oligonucleotides were mixed in equimoiar ratios and cloned into the SpeI site
of pBSS
pGS (Fig. 1 lA) or pNS FSF2K (Fig. 8A) to create the transfer plasmids.
Sequencing in
CA 02291323 1999-11-24
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41
both directions across the region of the inserts verified that the clones
contained the
appropriate sequence.
Similarly, transfer plasmids pBSSpGS (FLAG) (Figure I 1D) and
pNSFSF2K(FLAG) (Figure 1 lE) for introducing the FLAG ligand (SEQ ID N0:16)
into
S the adenoviral genome were created. Sequencing in both directions across the
region of
the inserts verified that the clones contained the appropriate sequence.
The plasmid DNA from the four transfer vectors were linearized with SaII,
purified and transfected using calcium phosphate into 293 cells which had been
preincubated for 1 h with the E1, E3, E4-deleted adenovirus AdCMVZ.11A
(GenVec,
IO Inc., Rockville, MD) a multiplicity of 1 ffu per cell. Recombination of the
E4+ pNS
plasmid with the E4-deleted vector resulted in the rescue of an El-, E3-, E4+
vector
capable of replication in 293 cells. The resultant vectors, AdZ.F2K(RKKIC)2,
AdZ.F2K(FLAG), AdZ.F(RKKK)2 and AdZ.F(FLAG), were isolated in two successive
rounds of plaguing on 293 cells.
15 Each vector was verified to contain the correct insert by sequencing PCR
products
derived from virus DNA template using primers spanning the region of the
insert DNA.
Restriction analysis of Ad DNA from each of the viruses showed that the
viruses were
pure and contained the BamHI restriction site unique to the correctly
constructed virus.
20 EXAMPLE 15
This example demonstrates that an adenoviral vector having a non-native ligand
can bind a support conjugated to a substrate for that ligand.
T'he vector AdZ.PK was constructed similarly to the vectors described above;
the
virus has a fiber protein containing polylysines. AdZ.PK was assayed to
determine
25 whether the virus could bind a support having a substrate for polylisine,
heparin. 50 ml of
heparin-agarose beads (SIGMA) were added to 1.0 ml of phosphate buffers
containing
150, 300, 500 and 1000 mM NaCi, respectively. 6600 cpm of either AdZ or AdZ.PK
were then added to the saline buffers containing the heparin-agarose beads and
rocked for
60 min. The beads were then washed three times with a buffer of equal salinity
to the
30 incubation buffer ( 150, 300, 500, and 1000 mM NaCI, respectively). The
bead-associated
cpm were then measured and showed the preferential binding of AdZ.PK over AdZ
at
150, 300, and 500 mM NaCI. However, at 1000 mM NaCI the binding of AdZ.PK to
the
beads was much lower and approximately equal to the background binding
observed for
AdZ.
35 These results demonstrate that the AdZ.PK vector binds a heparin-linked
support
material and that binding is ablated by high salt concentration. Therefore,
such a support
can be used to purify the modified vector by first binding the virus to the
support at low
salt conditions and then eluting the vector at high salt conditions.
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42
EXAMPLE 16
This example demonstrates that an adenoviral vector having a non-native ligand
can be purified on a column comprising substrate for that ligand.
20 175 cm2 tissue culture flasks containing 293 packaging cell lines are
infected at
an m.o.i. of 5 with one of the three vectors: AdZ.PK, AdZ.F2K(RKKK)2 or
AdZ.F(RKKK)2 described above. The cells are then incubated for 2 days, after
which
any remaining adherent cells are then dislodged from the plastic. The removed
cells are
centrifuged at 3,000 g to form a pellet, the culture medium removed, and the
pellet gently
washed 2 times with PBS. The cells are then resuspended in a total volume of 5
ml PBS
containing 10 mM MgCl2.
The resuspended cells are then freeze-thawed 3 times to release the virus, and
the
cell debris is then centrifuged at 15,000 g for 15 min. The supernatant is
passed over a 3
ml column containing heparin-linked agarose beads. The column is then washed
with 30
IS ml of PBS followed by elution of the virus from the column by a salt step
gradient. To
elute the virus, 3 ml volumes of buffers containing successively larger
concentrations of
NaCI (in 100 mM steps) are successively passed over the column, and 1 ml
elution
volumes are collected (3 ml 200 mM NaCI; 3 ml 300 mM NaCI; 3 ml 400 mM NaCI;
up
to 2000 mM NaCI).
The fractions, including the runthrough and wash fractions, are then evaluated
for
adenovirus coat proteins by Western blot, for active virus particles by lacZ
transduction
levels of A549 cells or by plaque assay, and for overall purity by analytical
high
performance liquid chromatography (HPLC) as previously described (Shabram, et
al,
1997, Hum. Gene Ther. 8, 453-46; Huyghe, et al, 1995, Hum. Gene Ther., 6: 1403-
1416;
Shabram et al, WO 96/27677). The overall purity of the fractions determined to
contain
peak adenovirus concentrations is evaluated by running the fractions on HPLC
and
comparing the profile to a pre-column fraction and a highly purified
adenovirus
preparation (prepared by 3 successive rounds of purification on CsCI
gradients).
EXAMPLE 17
This example describes the production of a pseudo-receptor for constructing a
cell
line able to replicate adenoviruses lacking native cell-binding function (but
targeted for
the pseudo-receptor). Specifically, the exemplary pseudo-receptor includes a
binding
domain from a single-chain antibody (ScFv).
First a vector expressing the ScFv from pHOOK3 (Figure 12A) (Invitrogen),
which encodes a ScFv synthesized with a murine Ig signal peptide. The ScFv has
an N-
terminal HA epitope tag, and its C-terminus is linked to a pair of myc
epitopes followed
by the PDGF receptor transmembrane anchor. An expression cassette including
this
CA 02291323 1999-11-24
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43
construct was cloned into plasmid pRC/CMVp-Puro (Fig. 12B) to create the
pScHAHK
plasmid (Fig. 12C). This plasmid has cloning sites for inserting genes after
the CMV
promoter and unique AgeI and XbaI sites for the addition of cytoplasmic
sequences at the
C-terminus of the gene.
To demonstrate cell-surface expression of the ScFv pseudo-receptor, either the
pNSE4GLP plasmid alone (Figure 12D), which carries a green fluorescent protein
gene
for detection of tranfectants, or in combination with pSeHAHK, were
transfected into 293
cells. One day post transfection, the pScHAHK-exhibited surface
imrnunofluorescence
using an antibody directed to the HA epitope, demonstrating proper surface
expression of
I D the pseudo-receptor.
To demonstrate that the expressed pseudo-receptor is functional, transfected
cells
were exposed to magnetic CAPTURE-TEC beads conjugated with antigens recognized
by
the ScFv. Following incubation, the beads were collected in the bottom of a
tube using a
magnet, washed, and transferred to a culture dish. The culture dishes were
then viewed
IS under a fluorescence microscope to identify GFP-expressing cells. No
staining was
observed from cells transfected only with pNSE4GLP alone, indicating that
these cells
did not bind the beads. However, cells transfected with pNSE4GLP and pScHAHK
were
observed in the wells. This result demonstrates that the doubly transfected
cells bound to
the beads.
EXAMPLE 18
This example describes the production of a pseudo-receptor for constructing a
cell
line able to replicate adenoviruses lacking native coil-binding function (but
targeted for
the pseudo-receptor). Specifically, the exemplary pseudo-receptor includes a
binding
domain from a single-chain antibody recognizing HA.
Anti-HA ScFv was constructed as an N-Term-VL-VH fusion protein. RT-PCR
was performed on RNA obtained from hybridomas producing HA antibodies using
primers specific for x- or y2(3- and C-terminus of the VL and VH genes (see
Gilliland et
al., Tissue Antigens, 47, 1-20 ( 1996)). After sequencing the resulting PCR
products,
specific oligonucleotides were designed to amplify the VL-VH fusion in a
second round
of PCR. The final PCR product was cloned to create the pCANTABSE(HA) plasmid
(Fig. 17A) for production of anti HA ScFv in E. toll. The expressed protein
has a C-
terminal E peptide for detection of binding to HA-tagged penton base via
Western
analysis of ELISA assay. Upon transformation of bacterial cells with the
pCANTABSE(HA) plasmid, Western analysis using an antibody recognizing the E
peptide revealed a protein of the expected size.
To determine whether the anti-HA ScFv was functional, it was used in protein A
immunoprecipitation assays using adenoviral coat proteins (recombinant penton
base)
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44
containing the HA epitope. The anti-HA ScFv was able to precipitate HA-
containing
penton base proteins. These results indicate the successful construction of
the
extracellular portion of a pseudo-receptor for binding an adenovirus having a
non-native
Iigand (i.e., HA).
To create an entire anti-HA pseudo-receptor, the anti-HA ScFv was cloned into
the
pSCHAHK plasmid in which the HA had been removed to create the pScFGHA plasmid
(Fig. 17B). This plasmid will produce an anti-HA pseudo-receptor able to bind
recombinant adenoviruses having the HA epitope, similar to adenoviruses
described
above having FLAG epitopes.
EXAMPLE 19
This example describes the creation of a fiber-expressing cell line for the
production of targeted adenovirus particles. The complementing cell line
produces a fiber
protein with or without additional complementary genes from the adenovirus
genome.
IS The entire adenovirus type 2 fiber gene was amplified from adenovirus type
2
DNA by PCR. The resultant product was cloned into the pCR2.1-TOPO plasmid
(Invitrogen) to make the plasmid pCR2.1-TOPO+fiber (Fig. 13A). T'he fiber2
gene was
then excised from the pCR2.1-TOPO+fiber plasmid with the restriction enzymes
BamHI
and EagI, and it was then subcloned into the plasmid, pKSII (Stratagene), to
construct the
plasmid pKSII Fiber (Fig. 13B). The fiber2 gene was then excised from the
pKSII Fiber
plasmid using the restriction enzymes KpnI and EagI, and it was then cloned
into the
plasmid, pSMTZeo-DBP (Fig. 13C). The resultant plasmid, pSMTZeo-Fiber {Fig.
13D),
encoded the entire fiber2 gene under control of the metallothionine promoter.
This
construct also placed an efficient mRNA splice site before the fiber gene to
enhance fiber
protein synthesis following induction. The pSMTZeo-Fiber plasmid also contains
a Zeo
resistance marker to allow selection of cell lines on the antibiotic zeocin.
To produce the cell line, the pSMTZeo-Fiber plasmid is transfected into 293
cells
(or some other cell line) with or without additional adenovirus complementing
functions.
Individual zeocin-resistant cell colonies are then amplified by standard means
and tested
for fiber2 production (e.g., by Western analysis using an anti-fiber2
antibody) before and
after induction with zinc, which activates the metallothionine promoter.
Selected fiber-
expressing clones are then tested for the ability to plaque and/or complement
the growth
of adenoviruses containing mutated fibers. Clones that adequately complement
mutated
fibers are suitable for amplifying and growing adenovirus particles having
genomes
encoding mutant fiber genes.
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All references cited herein are hereby incorporated by reference to the same
extent
as if each reference were individually and specifically indicated to be
incorporated by
reference and were set forth in its entirety herein.
' While this invention has been described with an emphasis on preferred
5 embodiments, it will be obvious to those of ordinary skill in the art that
variations of the
preferred embodiments can be used and that it is intended that the invention
can be
practiced otherwise than as specifically described herein. Accordingly, this
invention
includes all modifications encompassed within the spirit and scope of the
invention as
defined by the following claims.
CA 02291323 1999-11-24
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46
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: GENVEC, INC.
(B) STREET: 12111 PARKLAWN DRIVE
(C) CITY: ROCKVILLE
(D) STATE: MD
(E) COUNTRY: US
(F) POSTAL CODE (ZIP): 20852
(G) TELEPHONE: (301)816-0396
(H) TELEFAX: (301)816-0085
(A) NAME: WICKHAM, THOMAS J.
(B) STREET: 2106 HUTCHISON GROVE COURT
(C) CITY: FALLS CHURCH
(D) STATE: VA
(E) COUNTRY: US
(F) POSTAL CODE (ZIP): 22043
(A) NAME: KOVESDI, IMRE
(B) STREET: 7713 WARBLER LANE
(C) CITY: ROCKVILLE
(D) STATE: MD
(E) COUNTRY: US
(F) POSTAL CODE (ZIP): 20855
(A) NAME: ROELVINK, PETRUS W.
(B) STREET: 17502 GALLAGHER WAY
(C) CITY: OLNEY
(D) STATE: MD
(E) COUNTRY: US
(F) POSTAL CODE (ZIP): 20832
(A) NAME: EINFELD, DAVID
(B) STREET: 17502 GALLAGHER WAY
(C) CITY: OLNEY
(D) STATE: MD
(E) COUNTRY: US
(F) POSTAL CODE (ZIP): 20832
(A) NAME: BROUGH, DOUGLAS E.
(B) STREET: 3900 SHALLOWBROOK LANE
(C) CITY: OLNEY
(D) STATE: MD
(E) COUNTRY: US
(F) POSTAL CODE (ZIP): 20832
(A) NAME: LIZONOVA, ALENA
(B) STREET: 5329 RANDOLPH ROAD
(C) CITY: ROCKVILLE
(D) STATE: MD
(E) COUNTRY: US
(F) POSTAL CODE (ZIP): 20852
(A) NAME: YONEHIRO, GRANT
(B) STREET: 9395 KENTBURY DRIVE
(C) CITY: BETHESDA
(D) STATE: MD
(E) COUNTRY: US
(F) POSTAL CODE (ZIP): 20814
(ii) TITLE OF INVENTION: ALTERNATIVELY TARGETED ADENOVIRUS
(iii) NUMBER OF SEQUENCES: 18
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WO 98/54346 PCT/US98/11024
47
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
{C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60-047849
(B) FILING DATE: 28-MAY-1997
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60-071668
(B) FILING DATE: 16-JAN-1998
(2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 960 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..957
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
ATGAAG CGCGCAAGACCG TCTGAAGAT ACCTTCAAC CCCGTGTATCCA 48
MetLys ArgAlaArgPro SerGluAsp ThrPheAsn ProValTyrPro
1 5 10 15
TATGAC ACGGAAACCGGT CCTCCAACT GTGCCTTTT CTTACTCCTCCC 96
TyrAsp ThrGluThrGly ProProThr ValProPhe LeuThrProPro
20 25 30
TTTGTA TCCCCCAATGGG TTTCAAGAG AGTCCCCCC GGGGTACTCTCT 149
PheVal SerProAsnGly PheGlnGlu SerProPro GlyValLeuSer
35 40 45
TTGCGC CTATCCGAACCT CTAGTTACC TCCAATGGC ATGCTTGCGCTC 192
LeuArg LeuSerGluPro LeuValThr SerAsnGly MetLeuAlaLeu
50 55 60
AAAATG GGCAACGGCCTC TCTCTGGAC GAGGCCGGC AACCTTACCTCC 240
LysMet GlyAsnGlyLeu SerLeuAsp GluAlaGly AsnLeuThrSer
65 70 75 80
CAAAAT GTAACCACTGTG AGCCCACCT CTCAAAAAA ACCAAGTCAAAC 288
GlnAsn ValThrThrVal SerPro.ProLeuLysLys ThrLysSerAsn
85 90 95
ATAAAC CTGGAAATATCT GCACCCCTC ACAGTTACC TCAGAAGCCCTA 336
IleAsn LeuGluIleSer AlaProLeu ThrValThr SerGluAlaLeu
100 105 110
ACTGTG GCTGCCGCCGCA CCTCTAATG GTCGCGGGC AACACACTCACC 389
ThrVal AlaAlaAlaAla ProLeuMet ValAlaGly AsnThrLeuThr
115 120 I25
ATGCAA TCACAGGCCCCG CTAACCGTG CACGACTCC AAACTTAGCATT 432
MetGln SerGlnAlaPro LeuThrVal HisAspSer LysLeu5erIle
130 135 140
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48
GCCACCCAA GGA CTC ACAGTGTCAGAAGGA CTAGCA TCAAGG 480
CCC AAG .
AlaThrGln Gly Leu ThrValSerGluGly LysLeuAla SerArg
Pro
145 150 155 160
GTCTCGGCG CTC AAG ACGTCTCAAATACAC TCTGATACT ATCCTC 528
GAG
ValSerAla Leu Lys ThrSerGlnIleHis SerAspThr IleLeu
Glu
165 170 175
CGGATCACC CAG CTC GATGATGCAAACAAA CGAATCATC GCTCTT 576
GGA
ArgIleThr Gln Leu AspAspAlaAsnLys ArgileIle AlaLeu
Gly
180 185 190
GAGCAAAGT CGG GAC TTGGTTGCATCAGTC AGTGATGCT CAACTT 624
GAT
GluGlnSer Arg Asp LeuValAlaSerVal SerAspAla GlnLeu
Asp
195 200 205
GCAATCTCC AGA GAA AGCTCTATCGGAGCC CTCCAAACA GTTGTC 672
TTG
AlaIleSer Arg Glu SerSerIleGlyAla LeuGlnThr ValVal
Leu
210 215 220
AATGGACTT GAT AGT GTTACCCAGTTGGGT GCTCGAGTG GGACAA 720
TCG
AsnGlyLeu Asp Ser ValThrGlnLeuGly AlaArgVal GlyGln
Ser
225 230 235 240
CTTGAGACA GGA GCA GACGTACGCGTTGAT CACGACAAT CTCGTT 768
CTT
LeuGluThr Gly Ala AspValArgValAsp HisAspAsn LeuVal
Leu
245 250 255
GCGAGAGTG GAT GCA GAACGTAACATTGGA TCATTGACC ACTGAG 816
ACT
AlaArgVal Asp Ala GluArgAsnIleGly SerLeuThr ThrGlu
Thr
260 265 270
CTATCAACT CTG TTA CGAGTAACATCCATA CAAGCGGAT TTCGAA 864
ACG
LeuSerThr Leu Leu ArgValThrSerIle GlnAlaAsp PheGlu
Thr
275 280 285
TCTAGGGGA TCC GGC ACTAGTGGCGGCGAC TACAAGGAC GACGAC 912
GGC
SerArgGly Ser Gly ThrSerGlyGlyAsp TyrLysAsp AspAsp
Gly
290 295 300
GACAAGGGC CCT GGC GCCCGCCGCGCCTCC CTTGGCTCT AGATAA 960
AGG
AspLysGly Pro Gly AlaArgArgAlaSer LeuGlySer Arg
Arg
305 310 315
(2)INFORMATION SEQ ID :
FOR N0:2
(i ) SEQUENCEHARACTER ISTICS:
C
(A) LENGT H: 33 asepairs
6 b
(B) TYPE: nuc leicacid
(C) STRAN DEDNESS:unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE DNA(genomic)
TYPE:
(ix)
FEATURE:
(A) NAME/ KEY:CDS
{B) LOCATION: 1..630
(xi) ON:SEQID
SEQUENCE N0:2:
DESCRIPTI
ATG TCTGAA 98
AAG GAT
CGC ACC
GCA TTC
AGA AAC
CCG CCC
GTG
TAT
CCA
Met SerGlu
Lys Asp
Arg Thr
Ala Phe
Arg Asn
Pro Pro
Val
Tyr
Pro
325 330 335
TAT 96
GAC
ACG
GAA
ACC
GGT
CCT
CCA
ACT
GTG
CCT
TTT
CTT
ACT
CCT
CCC
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49
Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro
340 345. 350
TTTGTATCC CCCAATGGG TTTCAAGAGAGT CCCCCCGGG GGAGGGCTA 144
PheValSer ProAsnGly PheGlnGluSer ProProGly GlyGlyLeu
355 360 365
GCATCAAGG GTCTCGGCG CTCGAGAAGACG TCTCAAATA CACTCTGAT 192
AlaSerArg ValSerAla LeuGluLysThr SerGlnIle HisSerAsp
370 375 380
ACTATCCTC CGGATCACC CAGGGACTCGAT GATGCAAAC AAACGAATC 240
ThrIleLeu ArgIleThr GlnGlyLeuAsp AspAlaAsn LysArgIle
385 390 395 400
ATCGCTCTT GAGCAAAGT CGGGATGACTTG GTTGCATCA GTCAGTGAT 288
IleAlaLeu GluGlnSer ArgAspAspLeu ValAlaSer ValSerAsp
405 410 415
GCTCAACTT GCAATCTCC AGATTGGAAAGC TCTATCGGA GCCCTCCAA 336
AlaGlnLeu AlaIleSer ArgLeuGluSer SerIleGly AlaLeuGln
420 425 430
ACAGTTGTC AATGGACTT GATTCGAGTGTT ACCCAGTTG GGTGCTCGA 3B4
ThrValVal AsnGlyLeu AspSerSerVa1 ThrGlnLeu GlyAlaArg
435 440 445
GTGGGACAA CTTGAGACA GGACTTGCAGAC GTACGCGTT GATCACGAC 432
ValGlyGln LeuGluThr GlyLeuAlaAsp ValArgVal AspHisAsp
450 455 460
AATCTCGTT GCGAGAGTG GATACTGCAGAA CGTAACATT GGATCATTG 980
AsnLeuVal AlaArgVal AspThrAlaGlu ArgAsnIle GlySerLeu
465 470 475 480
ACCACTGAG CTATCAACT CTGACGTTACGA GTAACATCC ATACAAGCG 528
ThrThrGlu LeuSerThr LeuThrLeuArg ValThrSer IleGlnAla
485 490 495
GATTTCGAA TCTAGGGGA TCCGGCGGCACT AGTGGCGGC GACTACAAG 576
AspPheGlu SerArgGly SerGlyGlyThr SerGlyGly AspTyrLys
500 505 510
GACGACGAC GACAAGGGC CCTAGGGGCGCC CGCCGCGCC TCCCTTGGC 629
AspAspAsp AspLysGly ProArgGlyAla ArgArgAla SerLeuGly
515 520 525
TCTAGATAA 633
SerArg
530
(2)INFORMATION FORSEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1704 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..1701
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
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ATGAAGCGCGCA TCT GAT CCC GTG 48
AGA GAA ACC TAT
CCG TTC CCA
AAC
MetLysArgAla Ser Thr Pro Val Pro
Arg Glu Phe Tyr
Pro Asp Asn
215 220 225
TATGACACGGAA CCT CCAACTGTG TTTCTT ACTCCTCCC 96
ACC CCT
GGT
TyrAspThrGlu Thr Pro ProThrVal ProPheLeu ThrProPro
Gly
230 235 240
TTTGTATCCCCC AATGGGTTT CAAGAGAGT CCCCCCGGG GTACTCTCT 144
PheValSerPro AsnGlyPhe GlnGluSer ProProGly ValLeuSer
295 250 255
TTGCGCCTATCC GAACCTCTA GTTACCTCC AATGGCATG CTTGCGCTC 192
LeuArgLeuSer GluProLeu ValThrSer AsnGlyMet LeuAlaLeu
260 265 270 275
AAAATGGGCAAC GGCCTCTCT CTGGACGAG GCCGGCAAC CTTACCTCC 240
LysMetGlyAsn GlyLeuSer LeuAspGlu AlaGlyAsn LeuThrSer
280 285 290
CAAAATGTAACC ACTGTGAGC CCACCTCTC AAAAAAACC AAGTCAAAC 288
GlnAsnValThr ThrValSer ProProLeu LysLysThr LysSerAsn
295 300 305
ATAAACCTGGAA ATATCTGCA CCCCTCACA GTTACCTCA GAAGCCCTA 336
IleAsnLeuGlu IleSerAla ProLeuThr ValThrSer GluAlaLeu
310 315 320
ACTGTGGCTGCC GCCGCACCT CTAATGGTC GCGGGCAAC ACACTCACC 384
ThrValAlaAla AlaAlaPro LeuMetVal AlaGlyAsn ThrLeuThr
325 330 335
ATGCAATCACAG GCCCCGCTA ACCGTGCAC GACTCCAAA CTTAGCATT 432
MetGlnSerGln AlaProLeu ThrValHis AspSerLys LeuSerIle
340 345 350 355
GCCACCCAAGGA CCCCTCACA GTGTCAGAA GGAAAGCTA GCATCAAGG 480
AlaThrGlnGly ProLeuThr ValSerGlu GlyLysLeu AlaSerArg
360 365 370
GTCTCGGCGCTC GAGAAGACG TCTCAAATA CACTCTGAT ACTATCCTC 528
ValSerAlaLeu GluLysThr SerGlnIle HisSerAsp ThrIleLeu
375 380 385
CGGATCACCCAG GGACTCGAT GATGCAAAC AAACGAATC ATCGCTCTT 576
ArgIleThrGln GlyLeuAsp AspAlaAsn LysArgIle IleAlaLeu
390 395 400
GAGCAAAGTCGG GATGACTTG GTTGCATCA GTCAGTGAT GCTCAACTT 629
GluGlnSerArg AspAspLeu ValAlaSer ValSerAsp AlaGlnLeu
405 410 415
GCAATCTCCAGA TTGGAA TCTATCGGA GCCCTCCAA ACAGTTGTC 672
AGC
AlaIleSerArg LeuGluSer SerIleGly AlaLeuGln ThrValVal
420 925 430 435
AATGGA GAT TCGAGTGTT ACCCAGTTG GGTGCTCGA GGACAA 720
CTT GTG
AsnGly SerSerVal ThrGlnLeu GlyAlaArg ValGlyGln
Leu
Asp
440 445 450
CTT CTTGCA GTA GTT GATCACGAC CTCGTT 768
GAG GAC CGC AAT
ACA
GGA
Leu LeuAla ValArg AspHisAsp LeuVal
Glu Asp Val Asn
Thr
Gly
455 460 465
GCG ACTGCA GGA ACTGAG 816
AGA GAA TCA
GTG CGT TTG
GAT AAC ACC
ATT
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51
Ala Arg Val Asp Thr Ala Glu Arg Asn Ile Gly Ser Leu Thr Thr Glu
470 475 480
CTATCAACT CTGACGTTACGA GTAACATCC ATACAAGCG GATTTCGAA 869
LeuSerThr LeuThrLeuArg ValThrSer IleGlnAla AspPheGlu
485 490 495
TCTAGGGGA TCCGGCGGCACT AGAGGAGGT GGAATGAGC AAGGGCGAG 912
SerArgGly SerGlyGlyThr ArgGlyGly GlyMetSer LysGlyGlu
500 505 510 515
GAACTGTTC ACTGGCGTGGTC CCAATTCTC GTGGAACTG GATGGCGAT 960
GluLeuPhe ThrGlyValVal ProIleLeu ValGluLeu AspGlyAsp
520 525 530
GTGAATGGG CACAAATTTTCT GTCAGCGGA GAGGGTGAA GGTGATGCC 1008
ValAsnGly HisLysPheSer ValSerGly GluGlyGlu GlyAspAla
535 540 545
ACATACGGA AAGCTCACCCTG AAATTCATC TGCACCACT GGAAAGCTC 1056
ThrTyrGly LysLeuThrLeu LysPheIle CysThrThr GlyLysLeu
550 555 560
CCTGTGCCA TGGCCAACACTG GTCACTACC TTCACCTAT GGCGTGCAG 1104
ProValPro TrpProThrLeu ValThrThr PheThrTyr GlyValGln
565 570 575
TGCTTTTCC AGATACCCAGAC CATATGAAG CAGCATGAC TTTTTCAAG 1152
CysPheSer ArgTyrProAsp HisMetLys GlnHisAsp PhePheLys
580 585 590 595
AGCGCCATG CCCGAGGGCTAT GTGCAGGAG AGAACCATC TTTTTCAAA 1200
SerAlaMet ProGluGlyTyr ValGlnGlu ArgThrTle PhePheLys
600 605 610
GATGACGGG AACTACAAGACC CGCGCTGAA GTCAAGTTC GAAGGTGAC 1298
AspAspGly AsnTyrLysThr ArgAlaGlu ValLysPhe GluGlyAsp
615 620 625
ACCCTGGTG AATAGAATCGAG TTGAAGGGC ATTGACTTT AAGGAAGAT 1296
ThrLeuVal AsnArgIleGlu LeuLysGly IleAspPhe LysGluAsp
630 635 640
GGAAACATT CTCGGCCACAAG CTGGAATAC AACTATAAC TCCCACAAT 1394
GlyAsnIle LeuGlyHisLys LeuGluTyr AsnTyrAsn SerHisAsn
695 650 655
GTGTACATC ATGGCCGACAAG CAAAAGAAT GGCATCAAG GTCAACTTC 1392
ValTyrIle MetAlaAspLys GlnLysAsn GlyIleLys ValAsnPhe
660 665 670 675
AAGATCAGA CACAACATTGAG GATGGATCC GTGCAGCTG GCCGACCAT 1490
LysIleArg HisAsnIleGlu AspGlySer ValGlnLeu AlaAspHis
680 685 690
TATCAACAG AACACTCCAATC GGCGACGGC CCTGTGCTC CTCCCAGAC 1488
TyrGlnGln AsnThrProIle GlyAspGly ProValLeu LeuProAsp
695 700 705
AACCATTAC CTGTCCACCCAG TCTGCCCTG TCTAAAGAT CCCAACGAA 1536
AsnHisTyr LeuSerThrGln SerAlaLeu SerLysAsp ProAsnGlu
710 715 720
AAGAGAGAC CACATGGTCCTG CTGGAGTTT GTGACCGCT GCTGGGATC 1584
LysArgAsp HisMetValLeu LeuGluPhe ValThrAla AlaGlyIle
725 730 735
CA 02291323 1999-11-24
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ACA CAT GGC ATG GAC GAG CTG TAC AAG GGT GGA GGT AGA TCT ACT AGT 1632
Thr His Gly Met Asp Glu Leu Tyr Lys Gly Gly Gly Arg Ser Thr Ser
740 745 750 755
GGC GGC GAC TAC AAG GAC GAC GAC GAC AAG GGC CCT AGG GGC GCC CGC 1680
Gly Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Pro Arg Gly Ala Arg
760 765 770
CGC GCC TCC CTT GGC TCT AGA TAA 1704
Arg Ala Ser Leu Gly Ser Arg
775
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1830 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A} NAME/KEY: CDS
(B) LOCATION:1..1827
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
ATGAGAGGA TCTCACCATCAC CATCACCAT GGCGAAGATGGA GCTTTG 48
MetArgGly SerHisHisHis HisHisHis GlyGluAspGly AlaLeu
570 575 580
TCCCTGACA AAAACCTTAGTC TATCCCACC CTGTGGACGGGG CCTGCT 96
SerLeuThr LysThrLeuVal TyrProThr LeuTrpThrGly ProAla
585 590 595 600
CCCGAGGCC AACGTCACCTTC TCGGGGGAG AATTCCCCATCT GGCATT 149
ProGluAla AsnValThrPhe SerGlyGlu AsnSerProSer GlyIle
605 610 615
CTCAGACTG TGTCTCAGCAGA ACCGGGGGC ACGGTCATTGGC ACCCTG 192
LeuArgLeu CysLeuSerArg ThrGlyGly ThrValIleGly ThrLeu
620 625 630
TCTGTACAA GGTAGCCTCACG AACCCCAGT ACCGGTCAGACC CTGGGC 290
SerValGln GlySerLeuThr AsnProSer ThrGlyGlnThr LeuGly
635 640 645
ATGAACCTT TACTTTGACGCA GACGGCAAT GTGCTGTCTGAG AGCAAC 288
MetAsnLeu TyrPheAspAla AspGlyAsn ValLeuSerGlu SerAsn
650 655 660
CTCGTCCGA GGGTCCTGGGGA ATGAAAGAC CAAGATACCCTG GTGACT 336
LeuValArg GlySerTrpGly MetLysAsp GlnAspThrLeu ValThr
665 670 675 680
CCCATTGCC AATGGGCAGTAC CTGATGCCC AACCTCACTGCA TACCCT 384
ProIleAla AsnGlyGlnTyr LeuMetPro AsnLeuThrAla TyrPro
685 690 695
CGCCTCATA CAGACCCTAACT TCCAGCTAC ATTTACACACAA GCGCAC 432
ArgLeuIle GlnThrLeuThr SerSerTyr IieTyrThrGln AlaHis
700 705 710
CTTGACCAC AATAACAGTGTG GTGGACATC AAGATAGGGCTC AACACA 480
CA 02291323 1999-11-24
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53
Leu Asp His Asn Asn Ser Val Val Asp Ile Lys Ile Gly Leu Asn Thr
715 720 725
GAC CTGAGG CCCACTGCG GCCTACGGC CTAAGCTTT ACCATGACCTTC 528
Asp LeuArg ProThrAla AlaTyrGly LeuSerPhe ThrMetThrPhe
730 735 740
ACT AACTCT CCCCCCACC TCATTTGGT ACCGACCTG GTGCAATTTGGC 576
Thr AsnSer ProProThr SerPheGly ThrAspLeu ValGlnPheGly
745 750 755 760
TAC CTGGGT CAGGATAGC TCCCCCTCC TTCCTGAGA GAACTTCCCCTT 624
Tyr LeuGly GlnAspSer SerProSer PheLeuArg GluLeuProLeu
765 770 775
GCA TCCGAG GCGGGCTAC TTTGGCAAA CTGGCAGCT GCCTCTGAGGAA 672
Ala SerGlu AlaGlyTyr PheGlyLys LeuAlaAla AlaSerGluGlu
780 785 790
ATG CCAGCC CCTCCTGAG GCCCAGACG CAGGACCAA GCAGCTGAGGAG 720
Met ProAla ProProGlu AlaGlnThr GlnAspGln AlaAlaGluGlu
795 800 805
CCC CCGGCT CCTGCTGAG GCTGAGGCC CCCGCTCCT GCTGAGGCTGAG 768
Pro ProAla ProAlaGlu AlaGluAla ProAlaPro AlaGluAlaGlu
810 815 820
GCT GAGGCT GAACCGCCC CGAAAACCC CCTAGGGGT GACCTGGCCGCC 816
Ala GluAla GluProPro ArgLysPro ProArgGly AspLeuAlaAla
825 830 835 840
CTA TACAAT AGGGTCCAC AGCGACACC CGCGCAGAG GACACACCAACC 864
Leu TyrAsn ArgValHis SerAspThr ArgAlaGlu AspThrProThr
845 850 855
AGC CCCGAG TTGGTCACA ACCTTGCCA GACCCCTTT GTCCTCCCCCTA 912
Ser ProGlu LeuValThr ThrLeuPro AspProPhe ValLeuProLeu
860 865 870
CCC GACGGA GTCCCAACC GGTGCGAGC ATTGTGTTG GAAGGTACCCTC 960
Pro AspGly ValProThr GlyAlaSer IleValLeu GluGlyThrLeu
875 880 885
ACA CCCTCC GCTGTGTTT TTTACCCTG GATCTGGTG ACCGGGCCCGCC 1008
Thr ProSer AlaValPhe PheThrLeu AspLeuVal ThrGlyProAla
890 B95 900
AGT CTGGCG CTGCACTTT AACGTGCGC CTCCCACTG GAAGGCGAAAAG 1056
Ser LeuAla LeuHisPhe AsnValArg LeuProLeu GluGlyGluLys
905 910 915 920
CAC ATTGTG TGCAACTCC AGAGAGGGT AGCAGCAAC TGGGGCGAAGAA 1104
His IleVal CysAsnSer ArgGluGly SerSerAsn TrpGlyGluGlu
925 930 935
GTA AGACCG CAGGAGTTC CCCTTTGAA AGGGAAAAG CCATTCGTCCTG 1152
Val ArgPro GlnGluPhe ProPheGlu ArgGluLys ProPheValLeu
940 945 950
GTC ATTGTC ATCCAAAGT GACACATAC CAGATCACT GTGAACGGGAAG 1200
Val IleVal IleGlnSer AspThrTyr GlnIleThr ValAsnGlyLys
955 960 965
CCT CTGGTG GATTTTCCA CAGAGACTA CAGGGCATT ACCCGTGCCTCC 1248
Pro LeuVal AspPhePro GlnArgLeu GlnGlyIle ThrArgAlaSer
970 975 980
CA 02291323 1999-11-24
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54
CTA TCCGGA GAC CTT TTT TTGACAATG TACCCACCC GGA 1296
GTG ACC
CGG
Leu SerGly Asp Leu PheThr LeuThrMet TyrProPro Gly
Val Arg
985 990 995 1000
GAC CCCCGT CCC ACA TTGTTA CCCCCCGCA GCTCCCCTG GAC 1344
ACC CCA-
Asp ProArg Pro Thr LeuLeu ProProAla AlaProLeu Asp
Thr Pro
1005 1010 1015
GTA ATCCCA GAT GCC GTGCTC CTGCCCACC GGACTGACG CCT 1392
TAT AAT
Val IlePro Asp Ala ValLeu LeuProThr GlyLeuThr Pro
Tyr Asn
1020 1025 1030
AGA ACACTC CTC ACC ACGGGA CCCACGCCC CTCGCCGAA TTT 1440
GTC ACC
Arg ThrLeu Leu Thr ThrGly ProThrPro LeuAlaGlu Phe
Val Thr
1035 1040 1045
TTT ATTGTG AAT CTG TACGAT CACTATGAT TCCAAAAAT GTG 1488
GTC TTA
Phe IleVal Asn Leu TyrAsp HisTyrAsp SerLysAsn Val
Val Leu
1050 1055 1060
GCC CTCCAC TTT AAT GGCTTC TCTGACAGC AAAGGCCAC ATC 1536
GTC ACC
Ala LeuHis Phe Asn GlyPhe SerAspSer LysGlyHis Ile
Val Thr
1065 1070 1075 1080
GCC TGCAAT GCC AGA AATGGC TGGGGAAGT GAAATCACA GTG 1584
ATG ACA
Ala CysAsn Ala Arg AsnGly TrpGlySer GluIleThr Val
Met Thr
1085 1090 1095
TCT GATTTC CCC TTT AGGGGA CCCTTCACT CTGCAGATT CTC 1632
CAA AAA
Ser AspPhe Pro Phe ArgGly ProPheThr LeuGlnIle Leu
Gln Lys
1100 1105 1110
ACC AGAGAG GCA GAC CAAGTC GTAGATAAA CAACCTTTA ACC 1680
TTC CTC
Thr ArgGlu A1a Asp GlnVal ValAspLys GlnProLeu Thr
Phe Leu
1115 1120 1125
CAG TTTCAA TAC AGG AAGGAA GACCAAATC AAATATGTA CAC 1728
CTG CTG
Gln PheGln Tyr Arg LysGlu AspGlnIle LysTyrVal His
Leu Leu
1130 1135 114 0
ATG TTTGGC CAT GTT CAAACC CTGGAACAC CAAGTGCCA GAT 1776
GTG CAC
Met PheGly His Val GlnThr LeuGluHis GlnValPro Asp
Val His
114 5 1150 1155 1160
ACT CCAGTT TTT TCT GCGGGA TCGAAAGTT TACCCTCAG ATA 1824
ACT GTT
Thr ProVal Phe Ser AlaGly SerLysVal TyrProGln Ile
Thr Val
1165 1170 217 5
CTG TAG 1830
Leu
{2) INFORMATION ID N0:5:
FOR
SEQ
(i ) SEQUENCE ISTICS:
CHARACTER
(A) LENGTH: 253base rs
2 pai
(B) TYPE: leicacid
nuc
(C) STRANDEDNESS:unknown
(D) TOPOLOGY:unknown
(ii) DNA
MOLECULE (genomic)
TYPE:
(ix)
FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION:1..2250
CA 02291323 1999-11-24
WO 98/54346 PCT/US98/11024
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:5:
ATG AAG CGCGCA CCG TCTGAA ACCTTCAACCCC GTGTAT CCA 48
AGA GAT
Met Lys ArgAla Pro SerGlu ThrPheAsnPro ValTyr Pro
Arg Asp
615 620 625
TAT GAC ACGGAA GGT CCTCCA GTGCCTTTTCTT ACTCCT CCC 96
ACC ACT
Tyr Asp ThrGlu Gly ProPro ValProPheLeu ThrPro Pro
Thr Thr
630 635 640
' TTT GTA TCCCCC GGG TTTCAA AGTCCCCCTGGG GTACTC TCT 144
AAT GAG
Phe Val SerPro Gly PheGln SerProProGly ValLeu Ser
Asn Glu
645 650 655
TTGCGC CTATCCGAACCT CTAGTTACCTCC AATGGCATG CTTGCGCTC 192
LeuArg LeuSerGluPro LeuValThrSer AsnGlyMet LeuAlaLeu
660 665 670
AAAATG GGCAACGGCCTC TCTCTGGACGAG GCCGGCAAC CTTACCTCC 240
LysMet GlyAsnGlyLeu SerLeuAspGlu AlaGlyAsn LeuThrSer
675 680 685 690
CAAAAT GTAACCACTGTG AGCCCACCTCTC AAAAAAACC AAGTCAAAC 288
GlnAsn ValThrThrVal SerProProLeu LysLysThr LysSerAsn
695 700 705
ATAAAC CTGGAAATATCT GCACCCCTCACA GTTACCTCA GAAGCCCTA 336
IleAsn LeuGluIleSer AlaProLeuThr ValThrSer GluAlaLeu
710 715 720
ACTGTG GCTGCCGCCGCA CCTCTAATGGTC GCGGGCAAC ACACTCACC 384
ThrVal AlaAlaAlaAla ProLeuMetVal AlaGlyAsn ThrLeuThr
725 730 735
ATGCAA TCACAGGCCCCG CTAACCGTGCAC GACTCCAAA CTTAGCATT 432
MetGln SerGlnAlaPro LeuThrValHis AspSerLys LeuSerIle
740 795 750
GCCACC CAAGGACCCCTC ACAGTGTCAGAA GGAAAGCTA GCCCTGACA 480
AlaThr GlnGlyProLeu ThrValSerGlu GlyLysLeu AlaLeuThr
755 760 765 770
AAAACC TTAGTCTATCCC ACCCTGTGGACG GGGCCTGCT CCCGAGGCC 528
LysThr LeuValTyrPro ThrLeuTrpThr GlyProAla ProGluAla
775 780 785
AACGTC ACCTTCTCGGGG GAGAATTCCCCA TCTGGCATT CTCAGACTG 576
AsnVal ThrPheSerGly GluAsnSerPro SerGlyIle LeuArgLeu
790 795 800
TGTCTC AGCAGAACCGGG GGCACGGTCATT GGCACCCTG TCTGTACAA 624
CysLeu SerArgThrGly GlyThrValIle GlyThrLeu SerValGln
805 810 815
GGTAGC CTCACGAACCCC AGTACCGGTCAG ACCCTGGGC ATGAACCTT 672
GlySer LeuThrAsnPro SerThrGlyGln ThrLeuGly MetAsnLeu
820 825 830
TACTTT GACGCAGACGGC AATGTGCTGTCT GAGAGCAAC CTCGTCCGA 720
TyrPhe AspAlaAspGly AsnValLeuSer GluSerAsn LeuValArg
835 840 845 850
GGGTCC TGGGGAATGAAA GACCAAGATACC CTGGTGACT CCCATTGCC 768
GlySer TrpGlyMetLys AspGlnAspThr LeuValThr ProIleAla
855 860 865
CA 02291323 1999-11-24
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AAT TAC CTGATGCCC CTCACT TACCCT CTCATA 816
GGG AAC GCA CGC
CAG
Asn Tyr LeuMetPro LeuThr TyrPro LeuIle
Gly Asn Ala Arg
Gln
870 875 880
CAG CTAACT TCCAGCTAC TACACA CAAGCGCAC CTTGACCAC 864
ACC ATT
Gln ThrLeuThr SerSerTyr IleTyrThr GlnAlaHis LeuAspHis
885 890 895
AAT AACAGTGTG GTGGACATC AAGATAGGG CTCAACACA GACCTGAGG 912
Asn AsnSerVal ValAspIle LysIleGly LeuAsnThr AspLeuArg
900 ~ 905 910
CCC ACTGCGGCC TACGGCCTA AGCTTTACC ATGACCTTC ACTAACTCT 960
Pro ThrAlaAla TyrGlyLeu SerPheThr MetThrPhe ThrAsnSer
915 920 925 930
CCC CCCACCTCA TTTGGTACC GACCTGGTG CAATTTGGC TACCTGGGT 1008
Pro ProThrSer PheGlyThr AspLeuVal GlnPheGly TyrLeuGly
935 940 945
CAG GATAGCTCC CCCTCCTTC CTGAGAGAA CTTCCCCTT GCATCCGAG 1056
Gln Asp5erSer ProSerPhe LeuArgGlu LeuProLeu AlaSerGlu
950 955 960
GCG GGCTACTTT GGCAAACTG GCAGCTGCC TCTGAGGAA ATGCCAGCC 1104
Ala GlyTyrPhe GlyLysLeu AlaAlaAla SerGluGlu MetProAla
965 970 975
CCT CCTGAGGCC CAGACGCAG GACCAAGCA GCTGAGGAG CCCCCGGCT 1152
Pro ProGluAla GlnThrGln AspGlnAla AlaGluGlu ProProAla
980 985 990
CCT GCTGAGGCT GAGGCCCCC GCTCCTGCT GAGGCTGAG GCTGAGGCT 1200
Pro AlaGluAla GluAlaPro AlaProP.laGluAlaGlu AlaGluAla
995 1000 1005 1010
GAA CCGCCCCGA AAACCCCCT AGGGGTGAC CTGGCCGCC CTATACAAT 1248
Glu ProProArg LysProPro ArgGlyAsp LeuAlaAla LeuTyrAsn
1015 1020 1025
AGG GTCCACAGC GACACCCGC GCAGAGGAC ACACCAACC AGCCCCGAG 1296
Arg ValHisSer AspThrArg AlaGluAsp ThrProThr SerProGlu
1030 1035 1040
TTG GTCACAACC TTGCCAGAC CCCTTTGTC CTCCCCCTA CCCGACGGA 1344
Leu ValThrThr LeuProAsp ProPheVal LeuProLeu ProAspGly
1045 1050 1055
GTC CCAACCGGT GCGAGCATT GTGTTGGAA GGTACCCTC ACACCCTCC 1392
Val ProThrGly AlaSerIle ValLeuGlu GlyThrLeu ThrProSer
1060 1065 1070
GCT GTGTTTTTT ACCCTGGAT CTGGTGACC GGGCCCGCC AGTCTGGCG 1440
Ala ValPhePhe ThrLeuAsp LeuValThr GlyProAla SerLeuAla
107 5 1080 1085 1090
CTG CACTTTAAC GTGCGCCTC CCA GAA GGCGAAAAG CACATTGTG 1488
CTG
Leu HisPheAsn ValArgLeu ProLeuGlu GlyGluLys HisIleVal
1095 110 0 1105
TGC AACTCCAGA GGTAGC AGC TGG GGCGAA GTAAGA 1536
GAG AAC GAA CCG
Cys AsnSerArg GluGly Ser GlyGluGlu ValArgPro
Ser Asn
Trp
111 0 1115 1120
CAG GAGTTCCCC GAA GAA TTC GTCATTGTC 1584
TTT AGG AAG GTC
CCA CTG
Gln GluPhePro Glu Glu Phe ValIle
Phe Arg Lys Val Val
Pro Leu
CA 02291323 1999-11-24
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1125 1130 1135
ATC CAA AGT GAC ACA TAC CAG ATC ACT GTG AAC GGG AAG CCT CTG GTG 1632
Ile Gln Ser Asp Thr Tyr Gln Ile Thr Val Asn Gly Lys Pro Leu Val
1140 1195 1150
GAT TTT CCA CAG AGA CTA CAG GGC ATT ACC CGT GCC TCC CTA TCC GGA 1680
Asp Phe Pro Gln Arg Leu Gln Gly Ile Thr Arg Ala Ser Leu Ser Gly
1155 1160 1165 1170
GAC CTT GTG TTT ACC CGG TTG ACA ATG TAC CCA CCC GGA GAC CCC CGT 1728
Asp Leu Val Phe Thr Arg Leu Thr Met Tyr Pro Pro Gly Asp Pro Arg
1175 1180 1185
CCC ACAACC TTACCA CCCCCC GCTCCCCTGGAC GTAATCCCA 1776
TTG GCA
Pro ThrThr LeuPro ProPro AlaProLeuAsp ValIlePro
Leu Ala
1190 1195 1200
GAT GCCTAT CTCAAT CTGCCC GGACTGACGCCT AGAACACTC 1824
GTG ACC
Asp AlaTyr LeuAsn LeuPro GlyLeuThrPro ArgThrLeu
Val Thr
1205 1210 1215
CTC ACCGTC GGAACC CCCACG CTCGCCGAATTT TTTATTGTG 1872
ACG CCC
Leu ThrVa1 GlyThr ProThr LeuAlaGluPhe PheIleVal
Thr Pro
1220 1225 1230
AAT CTGGTC GATTTA CACTAT TCCAAAAATGTG GCCCTCCAC 1920
TAC GAT
Asn LeuVal AspLeu HisTyr SerLysAsnVal AlaLeuHis
Tyr Asp
1235 1240 1245 1250
TTT AATGTC TTCACC TCTGAC AAAGGCCACATC GCCTGCAAT 1968
GGC AGC
Phe AsnVal PheThr SerAsp LysGlyHisIle AlaCysAsn
Gly Ser
1255 1260 1265
GCC AGAATG GGCACA TGGGGA GAAATCACAGTG TCTGATTTC 2016
AAT AGT
Ala ArgMet GlyThr TrpGly GluIleThrVal SerAspPhe
Asn Ser
1270 1275 1280
CCC TTTCAA GGAAAA CCCTTC CTGCAGATTCTC ACCAGAGAG 2064
AGG ACT
Pro PheGln GlyLys ProPhe LeuGlnIleLeu ThrArgGlu
Arg Thr
1285 1290 1295
GCA GACTTC GTCCTC GTAGAT CAACCTTTAACC CAGTTTCAA 2112
CAA AAA
Ala AspPhe ValLeu ValAsp GlnProLeuThr GlnPheGln
Gln Lys
1300 1305 1310
TAC AGGCTG GAACTG GACCAA AAATATGTACAC ATGTTTGGC 2160
AAG ATC
Tyr ArgLeu GluLeu AspGln LysTyrValHis MetPheGly
Lys Ile
1315 1320 1325 1330
CAT GTTGTG ACCCAC CTGGAA CAAGTGCCAGAT ACTCCAGTT 2208
CAA CAC
His ValVal ThrHis LeuGlu GlnValProAsp ThrProVal
Gln His
1335 1390 1345
TTT TCTACT GGAGTT TCGAAA TACCCTCAGATA CTGTAG 2253
GCG GTT
Phe SerThr GlyVal SerLys TyrProGlnIle Leu
Ala Val
1350 1355 1360
(2) INFORMATION FORSEQ ID N0:6:
(i ) SEQUENCE
CHARACTERISTICS:
(A) ase
LENGTH: pairs
795
b
(B) nucleic acid
TYPE:
(C) unknown
STRANDEDNESS:
(D) unknown
TOPOLOGY:
CA 02291323 1999-11-24
WO 98/54346 PCT/US98/11024
58
(ii)MOLECULE DNA (genomic)
TYPE:
(ix)FEATURE:
(A ) CDS
NAME/KEY:
(B ) CATION:1..792
LO
(xi)SEQUENCE SCRIPTION: EQ D :6:
DE S I N0
ATGGCGCTCCTG CTGTGCTTC GTGCTCCTG TGCGGAGTA GTGGATTTC 48
MetAlaLeuLeu LeuCysPhe ValLeuLeu CysGlyVal ValAspPhe
755 760 765
GCCAGAAGTTTG AGTATCACT ACTCCTGAA GAGATGATT GAAAAAGCC 96
AlaArgSerLeu SerIleThr ThrProGlu GluMetIle GluLysAla
770 775 780
AAAGGGGAAACT GCCTATCTG CCGTGCAAA TTTACGCTT AGTCCCGAA 144
LysGlyGluThr AlaTyrLeu ProCysLys PheThrLeu SerProGlu
785 790 795
GACCAGGGACCG CTGGACATC GAGTGGCTG ATATCACCA GCTGATAAT 192
AspGlnGlyPro LeuAspIle GluTrpLeu IleSerPro AlaAspAsn
800 805 810 815
CAGAAGGTGGAT CAAGTGATT ATTTTATAT TCTGGAGAC AAAATTTAT 240
GlnLysValAsp GlnValIle IleLeuTyr SerGlyAsp LysIleTyr
820 825 830
GATGACTACTAT CCAGATCTG AAAGGCCGA GTACATTTT ACGAGTAAT 288
AspAspTyrTyr ProAspLeu LysGlyArg ValHisPhe ThrSerAsn
835 840 845
GATCTCAAATCT GGTGATGCA TCAATAAAT GTAACGAAT TTACAACTG 336
AspLeuLysSer GlyAspAla SerIleAsn ValThrAsn LeuGlnLeu
850 855 860
TCAGATATTGGC ACATATCAG TGCAAAGTG AAAAAAGCT CCTGGTGTT 389
SerAspIleGly ThrTyrGln CysLysVal LysLysAla ProGlyVal
865 870 875
GCAAATAAGAAG ATTCATCTG GTAGTTCTT GTTAAGCCT TCAGGTGCG 432
AlaAsnLysLys IleHisLeu ValVaILeu ValLysPro SerGlyAla
880 885 890 895
AGATGTTACGTT GATGGATCT GAAGAAATT GGAAGTGAC TTTAAGATA 480
ArgCysTyrVal AspGlySer GluGluIle GlySerAsp PheLysIle
900 905 910
AAATGTGAACCA AAAGAAGGT TCACTTCCA TTACAGTAT GAGTGGCAA 528
LysCysGluPro LysGluGly SerLeuPro LeuGlnTyr GluTrpGln
915 920 925
AAATTGTCTGAC TCACAGAAA ATGCCCACT TCATGGTTA GCAGAAATG 576
LysLeuSerAsp SerGlnLys MetProThr SerTrpLeu AlaGluMet
930 935 940
ACTTCATCTGTT ATATCTGTA AAAAATGCC TCTTCTGAG TACTCTGGG 624
ThrSerSerVal IleSerVal LysAsnAla SerSerGlu TyrSerGly
945 950 955
ACATACAGCTGT ACAGTCAGA AACAGAGTG GGCTCTGAT CAGTGCCTG 672
ThrTyrSerCys ThrValArg AsnArgVal GlySerAsp GlnCysLeu
960 965 970 975
TTGCGTCTA GTTGTCCCT CCTTCAAAT AAAGCTGGA TCTGGATCC 720
AAC
LeuArgLeuAsn ValValPro ProSerAsn LysAlaGly SerGlySer
CA 02291323 1999-11-24
WO 98/5434b PCT/US98/11024
59
980 985 990
GGC TCAGGG TCTACTAGT GGGGCC CAG CCG GCC CTG CAG GCG GCC GCA
768
Gly SerGly SerThrSer GlyAla Gln Pro Ala Leu Gln Ala Ala Ala
995 1000 1005
GAC TATAAA GATGACGAC GATAAG TGA 795
Asp TyrLys AspAspAsp AspLys
1010 1015
(2) INFORMATION FORSEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 839 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..831
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
ATGGCGCTC CTGCTGTGC TTCGTGCTCCTG TGCGGAGTA GTGGATTTC 48
MetAlaLeu LeuLeuCys PheValLeuLeu CysGlyVal ValAspPhe
270 275 280
GCCAGAAGT TTGAGTATC ACTACTCCTGAA GAGATGATT GAAAAAGCC 96
AlaArgSer LeuSerIle ThrThrProGlu GluMetIle GluLysAla
285 290 295
AAAGGGGAA ACTGCCTAT CTGCCGTGCAAA TTTACGCTT AGTCCCGAA 149
LysGlyGlu ThrAlaTyr LeuProCysLys PheThrLeu SerProGlu
300 305 310
GACCAGGGA CCGCTGGAC ATCGAGTGGCTG ATATCACCA GCTGATAAT 192
AspGlnGly ProLeuAsp IleGluTrpLeu IleSerPro AlaAspAsn
315 320 325
CAGAAGGTG GATCAAGTG ATTATTTTATAT TCTGGAGAC AAAATTTAT 240
GlnLysVal AspGlnVal IleIleLeuTyr SexGlyAsp LysIleTyr
330 335 340 395
GATGACTAC TATCCAGAT CTGAAAGGCCGA GTACATTTT ACGAGTAAT 288
AspAspTyr TyrProAsp LeuLysGlyArg ValHisPhe ThrSerAsn
350 355 360
GATCTCAAA TCTGGTGAT GCATCAATAAAT GTAACGAAT TTACAACTG 336
AspLeuLys SerGlyAsp AlaSerIleAsn ValThrAsn LeuGlnLeu
365 370 375
TCAGATATT GGCACATAT CAGTGCAAAGTG AAAAAAGCT CCTGGTGTT 384
SerAspIle GlyThrTyr GlnCysLysVal LysLysAla ProGlyVal
380 385 390
GCAAATAAG AAGATTCAT CTGGTAGTTCTT GTTAAGCCT TCAGGTGCG 432
AlaAsnLys LysIleHis LeuValValLeu ValLysPro SerGlyAla
395 400 905
AGATGTTAC GTTGATGGA TCTGAAGAAATT GGAAGTGAC TTTAAGATA 480
ArgCysTyr ValAspGly SerGluGluIle GlySerAsp PheLysIle
910 915 420 425
CA 02291323 1999-11-24
WO 98/54346 PCT/US98/11024
AAATGTGAA CCA GAA GGTTCACTTCCA TTACAGTAT GAGTGGCAA 528
AAA
LysCysGlu ProLysGlu GlySerLeuPro LeuGlnTyr GluTrpGln
430 435 440
AAATTGTCT GACTCACAG AAAATGCCCACT TCATGGTTA GCAGAAATG 576
LysLeuSer AspSerGln LysMetProThr SerTrpLeu AlaGluMet
445 450 455
ACTTCATCT GTTATATCT GTAAAAAATGCC TCTTCTGAG TACTCTGGG 624
ThrSerSer ValIleSer ValLysAsnAla SerSerGlu TyrSerGly
460 465 470
ACATACAGC TGTACAGTC AGAAACAGAGTG GGCTCTGAT CAGTGCCTG 672
ThrTyrSer CysThrVal ArgAsnArgVal GlySerAsp GlnCysLeu
475 480 485
TTGCGTCTA AACGTTGTC CCTCCTTCAAAT AAAGCTGGA TCTGGATCC 720
LeuArgLeu AsnValVal ProProSerAsn LysAlaGly SerGlySer
490 495 500 505
GGCTCAGGG TCTACTAGA GCCTGCGACTGT CGCGGCGAT TGTTTTTGC 768
GlySerGly SerThrArg AlaCysAspCys ArgGlyAsp CysPheCys
510 515 520
GGTACTAGT GGGGCCCAG CCGGCCCTGCAG GCGGCCGCA GACTATAAA 816
GlyThrSer GlyAlaGln ProAlaLeuGln AlaAlaAla AspTyrLys
525 530 535
GATGACGAC GATAAGTGA 834
AspAspAsp AspLys
540
(2)INFORMATION FORSEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1194 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..1191
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:8:
ATGGCGCTC CTGCTGTGC TTCGTGCTCCTG TGCGGAGTA GTGGATTTC 4B
MetAlaLeu LeuLeuCys PheValLeuLeu CysGlyVal Va1AspPhe
280 285 290
GCCAGAAGT TTGAGTATC ACTACTCCTGAA GAGATGATT GAAAAAGCC 96
AlaArgSer LeuSerIle ThrThrProGlu GluMetIle GluLysAla
295 300 305 310
AAAGGGGAA ACTGCCTAT CTGCCGTGCAAA TTTACGCTT AGTCCCGAA 144
LysGlyGlu ThrAlaTyr LeuProCysLys PheThrLeu SerProGlu
315 320 325
GACCAGGGA CCGCTGGAC ATCGAGTGGCTG ATATCACCA GCTGATAAT 192
AspGlnGly ProLeuAsp IleGluTrpLeu IleSerPro AlaAspAsn
330 335 340
CAGAAGGTG GATCAAGTG ATTATTTTATAT TCTGGAGAC AAAATTTAT 240
G1nLysVal AspGlnVal IleIleLeuTyr SerGlyAsp LysIleTyr
CA 02291323 1999-11-24
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61
345 350 355
GAT GAC TAC TAT CCA GAT CTG AAA GGC CGA GTA CAT TTT ACG AGT AAT 288
Asp Asp Tyr Tyr Pro Asp Leu Lys Gly Arg Val His Phe Thr Ser Asn
360 365 370
GAT CTC AAA TCT GGT GAT GCA TCA ATA AAT GTA ACG AAT TTA CAA CTG 336
Asp Leu Lys Ser Gly Asp Ala Ser Ile Asn Val Thr Asn Leu Gln Leu
375 380 385 390
TCA GAT ATT GGC ACA TAT CAG TGC AAA GTG AAA AAA GCT CCT GGT GTT 384
Ser Asp Ile Gly Thr Tyr Gln Cys Lys Val Lys Lys Ala Pro Gly Val
395 400 405
GCA AAGAAGATT CATCTGGTAGTT CTTGTTAAG CCTTCAGGT GCG 932
AAT
AlaAsn LysLysIle HisLeuValVal LeuValLys ProSerGly Ala
410 415 420
AGATGT TACGTTGAT GGATCTGAAGAA ATTGGAAGT GACTTTAAG ATA 480
ArgCys TyrValAsp GlySerGluGlu IleGlySer AspPheLys Ile
425 430 435
AAATGT GAACCAAAA GAAGGTTCACTT CCATTACAG TATGAGTGG CAA 528
LysCys GluProLys GluGlySerLeu ProLeuGln TyrGluTrp Gln
440 445 950
AAATTG TCTGACTCA CAGAAAATGCCC ACTTCATGG TTAGCAGAA ATG 576
LysLeu SerAspSer GlnLysMetPro ThrSerTrp LeuAlaGlu Met
455 460 465 970
ACTTCA TCTGTTATA TCTGTAAAAAAT GCCTCTTCT GAGTACTCT GGG 624
ThrSer SerValIle SerValLysAsn AlaSerSer GluTyrSer Gly
475 480 985
ACATAC AGCTGTACA GTCAGAAACAGA GTGGGCTCT GATCAGTGC CTG 672
ThrTyr SerCysThr ValArgAsnArg ValGlySer AspGlnCys Leu
490 495 500
TTGCGT CTAAACGTT GTCCCTCCTTCA AATAAAGCT GGATCTGGA TCC 720
LeuArg LeuAsnVal ValProProSer AsnLysAla GlySerGly Ser
505 510 515
GGCTCA GGGTCTACT AGAGGAGGTGGT GCATCAAGG GTCTCGGCG CTC 768
GlySer GlySerThr ArgGlyGlyGly AlaSerArg ValSerAla Leu
520 525 530
GAGAAG ACGTCTCAA ATACACTCTGAT ACTATCCTC CGGATCACC CAG 816
GluLys ThrSerGln IleHisSerAsp ThrIleLeu ArgIleThr Gln
535 540 545 550
GGACTC GATGATGCA AACAAACGAATC ATCGCTCTT GAGCAAAGT CGG 864
GlyLeu AspAspAla AsnLysArgIle IleAlaLeu GluGlnSer Arg
555 560 565
GATGAC TTGGTTGCA TCAGTCAGTGAT GCTCAACTT GCAATCTCC AGA 912
AspAsp LeuValAla SerValSerAsp AlaGlnLeu AlaIleSer Arg
570 575 580
TTGGAA AGCTCTATC GGAGCCCTCCAA ACAGTTGTC AATGGACTT GAT 960
LeuGlu SerSerIle GlyAlaLeuGln ThrValVal AsnGlyLeu Asp
585 590 595
TCGAGT GTTACCCAG TTGGGTGCTCGA GTGGGACAA CTTGAGACA GGA 1008
SerSer ValThrGln LeuGlyAlaArg ValGlyGln LeuGluThr Gly
600 605 610
CA 02291323 1999-11-24
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62
CTTGCAGACGTA CGCGTTGAT CACGACAAT CTCGTTGCGAGA GTGGAT 1056
LeuAlaAspVal ArgValAsp HisAspAsn LeuValAlaArg ValAsp
615 620 625 630
ACTGCAGAACGT AACATTGGA TCATTGACC ACTGAGCTATCA ACTCTG 1104
ThrAlaGluArg AsnIleGly SerLeuThr ThrGluLeuSer ThrLeu
635 640 645
ACGTTACGAGTA ACATCCATA CAAGCGGAT TTCGAATCTAGG ACTAGT 1152
ThrLeuArgVal ThrSerIle GlnAlaAsp PheGluSerArg ThrSer
650 655 660
ATGCAGGCGGCC GCAGACTAT AAAGATGAC GACGATAAGTGA 1194
MetGlnAlaAla AlaAspTyr LysAspAsp AspAspLys
665 670 675
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1793 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..1743
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
ATGAAGCGC GCAAGACCG TCTGAAGAT ACCTTCAACCCC GTGTATCCA 48
MetLysArg AlaArgPro SerGluAsp ThrPheAsnPro ValTyrPro
1 5 10 15
TATGACACG GAAACCGGT CCTCCAACT GTGCCTTTTCTT ACTCCTCCC 96
TyrAspThr GluThrGly ProProThr ValProPheLeu ThrProPro
20 25 30
TTTGTATCC CCCAATGGG TTTCAAGAG AGTCCCCCTGGG GTACTCTCT 144
PheValSer ProAsnGly PheGlnGlu SerProProGly ValLeuSer
35 40 45
TTGCGCCTA TCCGAACCT CTAGTTACC TCCAATGGCATG CTTGCGCTC 192
LeuArgLeu SerGluPro LeuValThr SerAsnGlyMet LeuAlaLeu
50 55 60
AAAATGGGC AACGGCCTC TCTCTGGAC GAGGCCGGCAAC CTTACCTCC 240
LysMetGly AsnGlyLeu SerLeuAsp GluAlaGlyAsn LeuThrSer
65 70 75 80
CAAAATGTA ACCACTGTG AGCCCACCT CTCAAAAAAACC AAGTCAAAC 288
GlnAsnVal ThrThrVal SerProPro LeuLysLysThr LysSerAsn
85 90 95
ATAAACCTG GAAATATCT GCACCCCTC ACAGTTACCTCA GAAGCCCTA 336
IleAsnLeu GluIleSer AlaProLeu ThrValThrSer GluAlaLeu
100 105 110
ACTGTGGCT GCCGCCGCA CCTCTAATG GTCGCGGGCAAC ACACTCACC 384
ThrValAla AlaAlaAla ProLeuMet ValAlaGlyAsn ThrLeuThr
115 120 125
ATGCAATCA CAGGCCCCG CTAACCGTG CACGACTCCAAA CTTAGCATT 432
MetGlnSer GlnAlaPro LeuThrVal HisAspSerLys LeuSerIle
CA 02291323 1999-11-24
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63
130 135 140
GCC ACC CAA GGA CCC CTC ACA GTG TCA GAA GGA AAG CTA GCC CTG CAA 980
Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln
145 150 155 160
ACA TCA GGC CCC CTC ACC ACC ACC GAT AGC AGT ACC CTT ACT ATC ACT 528
Thr Ser Gly Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr
165 170 175
GCCTCA CCCCCTCTAACT ACTGCCACT GGTAGCTTG GGCATTGACTTG 576
AlaSer ProProLeuThr ThrAlaThr GlySerLeu GlyIleAspLeu
180 185 190
AAAGAG CCCATTTATACA CAAAATGGA AAACTAGGA CTAAAGTACGGG 629
LysGlu ProIleTyrThr GlnAsnGly LysLeuGly LeuLysTyrGly
195 200 205
GCTCCT TTGCATGTAACA GACGACCTA AACACTTTG ACCGTAGCAACT 672
AlaPro LeuHisValThr AspAspLeu AsnThrLeu ThrValAlaThr
210 215 220
GGTCCA GGTGTGACTATT AATAATACT TCCTTGCAA ACTAAAGTTACT 720
GlyPro GlyValThrIle AsnAsnThr SerLeuGln ThrLysValThr
225 230 235 240
GGAGCC TTGGGTTTTGAT TCACAAGGC AATATGCAA CTTAATGTAGCA 768
GlyAla LeuGlyPheAsp SerGlnGly AsnMetGln LeuAsnValAla
295 250 255
GGAGGA CTAAGGATTGAT TCTCAAAAC AGACGCCTT ATACTTGATGTT 816
GlyGly LeuArgIleAsp SerGlnAsn ArgArgLeu IleLeuAspVal
260 265 270
AGTTAT CCGTTTGATGCT CAAAACCAA CTAAATCTA AGACTAGGACAG 869
SerTyr ProPheAspAla GlnAsnGln LeuAsnLeu ArgLeuGlyGln
275 280 285
GGCCCT CTTTTTATAAAC TCAGCCCAC AACTTGGAT ATTAACTACAAC 912
GlyPro LeuPheIleAsn SerAlaHis AsnLeuAsp IleAsnTyrAsn
290 295 300
AAAGGC CTTTACTTGTTT ACAGCTTCA AACAATTCC AAAAAGCTTGAG 960
LysGly LeuTyrLeuPhe ThrAlaSer AsnAsnSer LysLysLeuGlu
305 310 315 320
GTTAAC CTAAGCACTGCC AAGGGGTTG ATGTTTGAC GCTACAGCCATA 1008
ValAsn LeuSerThrAla LysGlyLeu MetPheAsp AlaThrAlaIle
325 330 335
GCCATT AATGCAGGAGAT GGGCTTGAA TTTGGTTCA CCTAATGCACCA 1056
AlaIle AsnAlaGlyAsp GlyLeuGlu PheGlySer ProAsnAlaPro
340 395 350
AACACA AATCCCCTCAAA ACAAAAATT GGCCATGGC CTAGAATTTGAT 1104
AsnThr AsnProLeuLys ThrLysIle GlyHisGly LeuGluPheAsp
355 360 365
TCAAAC AAGGCTATGGTT CCTAAACTA GGAACTGGC CTTAGTTTTGAC 1152
SerAsn LysAlaMetVal ProLysLeu GlyThrGly LeuSerPheAsp
370 375 380
AGCACA GGTGCCATTACA GTAGGAAAC AAAAATAAT GATAAGCTAACT 1200
SerThr GlyAlaIleThr ValGlyAsn LysAsnAsn AspLysLeuThr
385 390 395 400
CA 02291323 1999-11-24
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64
TTGTGG ACC ACA CCA CCA TCT AACTGTAGA CTA GCA GAG 1248
GCT CCT AAT
LeuTrp Thr Thr Pro Pro Ser AsnCysArg LeuAsnAla Glu
Ala Pro
405 410 415
AAAGAT GCT AAA CTC TTG GTC ACAAAATGT GGCAGTCAA ATA 1296
ACT TTA
LysAsp Ala Lys Leu Leu Val ThrLysCys GlySerGln Ile
Thr Leu
420 425 430
CTTGCT ACA GTT TCA TTG GCT AAAGGCAGT TTGGCTCCA ATA 1344
GTT GTT
LeuAla Thr Val Ser Leu Ala LysGlySer LeuAlaPro Ile
Val Val
435 440 445
TCTGGA ACA GTT CAA GCT CAT ATTATAAGA TTTGACGAA AAT 1392
AGT CTT
SerGly Thr Val Gln Ala His IleIleArg PheAspGlu Asn
Ser Leu
450 455 460
GGAGTG CTA CTA AAC TCC TTC GACCCAGAA TATTGGAAC TTT 1440
AAT CTG
GlyVal Leu Leu Asn Ser Phe AspProGlu TyrTrpAsn Phe
Asn Leu
465 470 475 480
AGAAAT GGA GAT CTT GAA GGC GCCTATACA AACGCTGTT GGA 1488
ACT ACA
ArgAsn Gly Asp Leu Glu Gly AlaTyrThr AsnAlaVal Gly
Thr Thr
485 490 495
TTTATG CCT AAC CTA GCT TAT AAATCTCAC GGTAAAACT GCC 1536
TCA CCA
PheMet Pro Asn Leu Ala Tyr LysSerHis GlyLysThr Ala
Ser Pro
500 505 510
AAAAGT AAC ATT GTC CAA GTT TTAAACGGA GACAAAACT AAA 1589
AGT TAC
LysSer Asn Ile Val Gln Val LeuAsnGly AspLysThr Lys
Ser Tyr
515 520 525
CCTGTA ACA CTA ACC ACA CTA GGTACACAG GAAACAGGA GAC 1632
ATT AAC
ProVal Thr Leu Thr Thr Leu GlyThrGln GluThrGly Asp
Ile Asn
530 535 540
ACAACT CCA AGT GCA TCT ATG TTTTCATGG GACTGGTCT GGC 1680
TAC TCA
ThrThr Pro Ser Ala Ser Met PheSerTrp AspTrpSer Gly
Tyr Ser
545 550 555 560
CACAAC TAC ATT AAT ATA TTT ACATCCTCT TACACTTTT TCA 1728
GAA GCC
HisAsn Tyr Ile Asn Ile Phe ThrSerSer TyrThrPhe Ser
Glu Ala
565 570 575
TACATT GCC CAA GAA 1793
TyrIle Ala Gln Glu
580
(2)INFORMATION ID N0:10:
FOR
SEQ
(i)
SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 6 base
3 pairs
(B) TYPE: leic acid
nuc
(C) STRANDEDNESS: single
(D) TOPOLOGY:unknown
(ii) DNA (genomic)
MOLECULE
TYPE:
(xi) ID
SEQUENCE N0:10:
DESCRIPTION:
SEQ
TGCATGCATA 36
CTAGTCCTAG
ATTCGAAATC
CGCTTG
(2)INFORMATION
FOR
SEQ
ID
N0:11:
(i)
SEQUENCE
CHARACTERISTICS:
(A) LENGTH:
38 base pairs
CA 02291323 1999-11-24
WO 98/54346 PCT/US98/11024
(H) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
. (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GCTCTAGAGG AGGTGGTGCA TCAAGGGTCT CGGCGCTC 38
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
CCGGATCCCT ACAGTATCTG AGGGTAAAC 29
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
GGGCACCATG GCGAAGATGG AGCTTTGTCC C 31
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Thr Ser
1 5 10
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Arg Lys Lys Lys Arg Lys Lys Lys
CA 02291323 1999-11-24
WO 98/54346 PCT/US98/11024
66
1 5
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Pro Lys Ala Arg Arg Pro Ala Gly Arg Thr Trp Ala Gln Pro
1 5 10
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Arg Pro Ile Asp Asp Phe Asp Gln Gly Trp Gly Pro Ile Thr Tyr
1 5 10 15
