Note: Descriptions are shown in the official language in which they were submitted.
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CAML-BINDING PEPTIDES
FIELD OF THE INVENTION
This application relates to modulation of apoptosis and immune response.
BACKGROUND OF THE INVENTION
Viral infection is a cellular injury, and can result in the induction of
programmed
cell death (apoptosis) of the host cell. Many viruses, particularly persistent
DNA viruses
modify the apoptotic response of a cell to allow continued virus replication.
Apoptosis
can be induced by the members of the TNF receptor super-family such as Fas
(APO-1 or
CD95) and p55 Tumor Necrosis Factor Receptor (p55 TNFR) as well as the death
domain-containing receptors 3, 4 and 5 (DR3, DR4 and DRS, respectively). The
intracellular factors responsible for death of the cell are highly conserved
across species
and are the target of viral inhibitors of apoptosis.
It appears that proteins belonging to very different classes of virus have
evolved to
block the same cellular apoptotic event. This convergent evolution is
evidenced by the
classification of viral inhibitors of apoptosis. For example, adenovirus E1B
SSK, SV40
Large T antigen and human papilloma virus E6 inhibit p53-mediated lysis. The
cellular
survival factor Bcl-2 is mimicked by adenovirus E1B 19K, Epstein-Barr virus
BHRF1
and African swine fever virus LMWS-HL. Members of the Interleukin 1b
Converting
Enzymes (ICE)-family of terminal proteolytic enzymes, also known as caspases,
are
blocked by baculovirus p35 and crmA, the cowpox serpin protein. The adenovirus
25' E3/10.4K and E3/14.SK proteins downregulate surface Fas, while the
Inhibitors of
Apoptosis (IAP) family of baculovirus and mammalian homologues interact with
the
TNF-a receptor associated factors (TRAFs) therefore blocking the signalling
cascade that
leads to the recruitment of caspases. The activation of FADD-like interleukin-
lbeta-
converting enzyme (FLICE), also known as caspase-8, through Fas is blocked by
viral-
FLICE-inhibitory proteins (vFLIPs), found in the genomes of various types of
herpesvirus
and by the E3/14.7K of adenovirus.
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Adenovirus (Ad) is a very common human pathogen that results in persistent
infections of the respiratory or gastrointestinal tract. Persistent infections
stem from an
elaborate evasion of the host defense mechanisms. The adenovirus genes
responsible for
immune evasion map to the Early 3 (E3) region of the Ad genome. The
persistence, ease
of infection and weak pathogenesis have made adenovirus suitable as vectors
for gene
therapy. Currently, Ad gene transfer vectors are the most efficient technique
available for
in vivo gene transduction. In the case of Ad vectors the genetic makeup of the
original
vectors was designed to accommodate large fragments of DNA for the transduced
gene,
to the expense of areas of the adenoviral genome that were considered
dispensable. The
E3 region was one of the first areas to be replaced.
The 6.7K protein encoded by the E3 region (E3/6.7K) does not have any
significant homology to any other known proteins. It is well conserved between
group C
Ad2 and Ad5 adenovirus and between group B Ad3, Ad7 and Ad35 adenovirus. The
Ad2
E3/6.7I~ protein (Wilson-Rawls et al., (1990) Virology 178:204-212) has been
shown to
1S be an integral membrane protein localized to the endoplasmic xeticulum (ER)
(Wilson-
Rawls and Wold, (1993) Virology 195:6-15). The protein is present in two
forms, one
unglycosylated with an apparent molecular weight of BkDa and one glycosylated
with an
apparent weight of l4kDa. The protein, though targeted to the ER, does not
have a
cleavable signal sequence, but it has a hydrophobic central region that could
act as a
signal anchor (Wilson-Rawls et a1.,(1994) Virology 201:66-76).
The major impediment for the success of Ad vectors as well as all the other
gene
transfer technologies is the unexpectedly strong immune response to cells
infected by a
modified Adenovirus. The strong immune response to modified Ad vectors appears
to be
mediated by the circulating cytokine Tumor Necrosis Factor (TNF) a and by the
innate
immune response. The negative effects of an immune response might be
alleviated by
implementing immunomodulatory proteins that allow the vector and the
transduced cells
to survive the immune response.
The evasion of immune response is also a central impediment to the
establishment
of successful transplant technology as well as the treatment of autoimmune and
neurodegenerative diseases. Apoptosis of the affected organ is often the
result of
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neurodegenerative inflammatory disease. Factors that prevent apoptosis could
lead to
better therapies for these conditions.
Cell culture reactor expression systems are typically limited only by the
ability of
cells to grow and produce proteins of interest. As cells grow, they reach
densities where
protein production stops and producer cells undergo apoptosis in response to
factors that
are currently poorly characterized. There is potential for improving protein
yield by
avoiding the apoptotic response of cells grown in culture by including an
antiapoptotic
protein in the makeup of the cell.
CAML (also known as "calcium-signal modulating cyclophilin ligand"; United
States Patent No's. 5,523,227 and 5,969,102) is located in cytoplasmic
vesicles and
regulates Caz+ influx by modulating intracellular Caa+ release. CAML affects
calcineurin
in response to an extrinsic signal and has been shown to be important in
lymphocyte
activation. CAML is involved in activation of transcription factors in
lymphocytes,
including T-cell transcription factor (NF-AT) and is important in modulation
of immune
response or modulation of apoptosis.
A lymphocyte surface receptor that binds CAML has been discovered and the
human and marine sequences have been published. This protein is termed
"transmembrane activator and CAML interactor" (TACI) and has been the focus of
much
attention in view of the interaction of TACI with CAML and its role in
lymphocyte
activation, particularly in B-cells (see: United States Patent No. 5,969,102;
Von Bulow,
G. & Bram, R.J. (1997) Science, 278:138-141; Von Bulow, G., et al. (2000),
Mammalian
Genome, 11:628-632; Xia, X., et al. (2000), J. Exp. Med., 192:137-143; and,
Ware, C.F.
(2000), J. Exp. Med., 192:F35-F37). TACI is reported to have a N-terminal
extracellular
domain, a centrally located transmembrane domain, and a C-terminal
intracellular
domain. Investigation of sequence homology with respect to TACI only revealed
homology between portions of the N-terminal domain of TACI (representing a
TNFR NGFR repeat motif in the regions of amino acids 33-104 of human TACI),and
a
number of growth factor receptors (United States Patent No. 5,969,102). In the
293
amino acid human sequence of TACI, the transmembrane domain is thought to
reside at
about amino acids 167-186. First reports with respect to binding of TACI to
CAML
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indicated that the CAML-binding region is in the C-terminus domain of TACI,
since
CAML-binding did not occur with amino acids 1-168 of human TACI (United States
Patent No. 5,969,102; and, Von Bulow, G. & Bram, R.J. (1997) [supra]). Results
published more recently seem to show that amino acids 1-212 or 1-233 of human
TACI
are capable of binding to CAML (Xia, X.Z., et al. [supra]).
SUMMARY OF THE INVENTION
It has now been discovered that adenovirus E3/6.7L prevents inflammation,
apoptosis and the cellular damage response following viral infection. The
presence of the
E3/6.7K protein correlates with reduced inflammatory response in the lungs of
virally
infected mice. Transfected cells that express the E3/6.7K protein are now
shown to be
protected against apoptosis induced by TNF-a. TNF-a induced release of
arachidonic
acid is significantly. reduced in cells expressing transfected E3/6.7K. Efflux
of calcium
ions from the ER is also reduced in the presence of E3/6.7K. Therefore, the
mechanism-
of action of E3/6.7K does involve maintenance of calcium ion homeostasis. The
release
of calcium ions from the ER is known to be important for the generation of
mediators of
inflammation, apoptosis and of the cellular damage response.
E3/6.7K has no sequence homology to any of the previously described inhibitors
of apoptosis. E3/6.7K therefore represents a member of a new class of
modulators of
apoptosis (particularly in lymphocytes) and the immune response, and which are
useful as
modulators of inflammation. This new class of modulators act through
alteration of Ca2+
influx and thus may be inhibiting or promoting in their effects.
By this invention, it is now known that E3/6.7K binds to CAML and inhibits
Caa+
influx, thus resulting in inhibition of apoptosis and inflammation. Binding by
E316.7K
fragments to full length CAML has been determined by immunoprecipitation and
by
yeast two-hybrid assays.
Examples of portions of E3/6.7K capable of interacting with CAMh are a 26
amino acid domain situated at amino acids 32 to 57 of Ad2 and amino acids 34
to 59 of
AdS, and in a corresponding 26 amino acid tract of Ad3. Such E3/6.7K domains
are
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collectively termed herein "6.7-effector domain" (SED). Surprisingly, part of
this domain
shares homology with the putative transmembrane domain of TACK contrary to the
previous published reports with respect to TACI binding to CAML. Examples of
the
CAML-binding domains in TACI which correspond to SED, include domains of about
28
amino acids in length found from about amino acids 163-189 of human TACI and
from
about amino acids 126-152 or 153 of mouse TACI.
With knowledge of the CAML-binding domain of TACI and E3/6.7K, a CAML
binding motif has been determined and has been employed to identify known
proteins
. which share this motif, thereby making such proteins available for use in
CAML-binding.
One aspect of the invention involves the provision of and use of CAML-binding
motifs that may be used to confer CAML-binding function on a ligand or other
moiety
intended to bind to CAML. Such motifs may in themselves modulate CAML function
or
may be used to facilitate CAML modulation by permitting binding of a CAML
modulating ligand or moiety to CAML. The motifs may also be used to permit
binding of
moieties wluch are not intended to modulate CAML function (such as a CAML
specific
labeling moieties).
In alternative embodiments, the CAML-binding motifs may comprise sequences
of 26, 27 or 28 amino acids, such as follows:
LALLCLRVAACCTHVCTYCQLFKRWG (from Ad2 E3/6.7K) SEQ ID NO:1;
LTLLCLRLAACCVHICIYCQLFKRWG (from Ad5 E3/6.7K) SEQ ID N0:2;
VLILCYLYTPCCAYLVILCCWFI~KWG (from Ad3 E3/1.6K) SEQ ID NO:3;
VYSTLGLCLCAVLCCFLVAVACFLKKR (from human TACI) SEQ ID N0:4;
LYCTLGVCLCAIFCCFLVALASFLRRRG (from marine TACI) SEQ ID NO:S.
In alternative embodiments, the CAML-binding motif of this invention may
comprise a sequence having any one of the following consensus sequences,
wherein: a
"x" represents any amino acid with the number of amino acids (or range of
possible
numbers of amino acids) being indicated by a bracketed number or numbers
following
"x"; and, single letter amino acid abbreviations within square brackets
represent
alternative amino acids at a single position:
(a) C-C-x(2)-[FILV]-[ACV]-x(2)-[CS]-x(3)-[KR]-[KR];
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(b) C-C-x(2)-[ILV]-[ACV]-x(2)-[CS]-x(3)-[KR]-[KR];
(c) C-x(3,5)-C-C-x(2)-[FILV]-[ACV]-x(2)-[CS]-x(3)-[KR]-[KR];
(d) L-x(2,3)-C-x(4,5)-C-C-x(2)-[ILV]-[ACV]-x(2)-[CS]-x(3)-[KR]-[KR]; and,
(e) L-x(1,2)-Cx(5)-C-C-x(2)-[ILV][ACV]-x(2)-[CS]-x(3)-[KR]-[KR].
In alternative embodiments, the CAML-binding motifs of the invention may be
derived from or used as part of known proteins or fragments of known proteins,
including
those set out in the following list in which the accession number of the
database reference
is followed by the name and source of the protein, together with
identification of the
amino acid sequence and its position within the protein corresponding to
consensus
sequence (b) above.
[1] SWISS-PROT: E316 ADE03 P11320
Early E3 16 KDA glycoprotein
Human adenovirus type 3
129-142 CCAYLVILCCWFKK; SEQ ID N0:6
[2] SWISS-PROT: SY61 DISOM P24505
Synaptotagmin A (synaptic vesicle protein O-P65-A)
Discopyge ommata (Electric ray)
79-92 CCFCICKKCLLKKK; SEQ ID NO:7
[3] SWISS-PROT: SY62 DISOM P24506
Synaptotagmin B (synaptic vesicle protein O-P65-B)
Discopyge ommata (Electric ray)
96-109 C CLCICKKCCCKKK; SEQ ID NO:B
[4] SWISS-PROT: SYT1 BOVIN P48018
Synaptotagmin I (P65)
Bos taurus (bovine)
75-88 CCFCICKKCLFKKK; SEQ ID N0:9
[5] SWISS-PROT: SYT1 CHICK P47191
Synaptotagmin I (P65)
Gallus gallus (chicken)
77-90 CCFCLCKKCLFKKK; SEQ ID NO:10
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[6] SWISS-PROT: SYTl HUMAN P21579
Synaptotagmin I (P65)
Homo Sapiens (human)
75-88 CCFCICKKCLFKKK; SEQ ID NO:11
[7] SWISS-PROT: SYT1 MOUSE P46096
Synaptotagmin I (P65)
Mus musculus (mouse)
74-87 CCFCVCKKCLFKKK; SEQ ID N0:12
[8] SWISS-PROT: SYT1 RAT P21707
Synaptotagmin I (P65)
Rattus norvegicus (rat)
74-87 CCFCVCKKCLFKKK; SEQ ID N0:13
[9] SWISS-PROT: SYT2 MOUSE P46097
Synaptotagmin II
Mus musculus (mouse)
82-95 CCFCICKKCCCKKK; SEQ ID N0:14
[10] SWISS-PROT: SYT2 RAT P29101
Synaptotagmin II
Rattus norvegicus (rat)
82-95 CCFCICKKCCCKKI~; SEQ ID NO:15
[11] TrEMBL: 014836 014836
Transmembrane activator and CAML interactor
Homo Sapiens (human)
176-189 CCFLVAVACFLI~KR; SEQ ID~N0:16 (or,,amino acids 139-152
of marine TACI: CCFLVALASFLRRR; SEQ ID N0:17)
[12] TrEMBL: Q64830 Q64830
URF E3A 7.1K
Human adenovirus type.5
44-57 CCVHICIYCQLFKR; SEQ ID N0:18
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[13] TrEMBL: Q9Q8F7 Q9Q8F7
M147R
Myxoma virus
126-139 CCTGLASVCKYTKK; SEQ ID N0:19
S Peptides of this invention may comprise an amino acid sequence corresponding
to
the above-described CAML-binding motifs combined with additional amino acids
selected in order for the peptide to affect CAML function.
Peptides of this invention will have a minimum of about 14 amino acids. While
no specific maximum length of peptides (including proteins comprising
peptides) of this
invention is contemplated and may comprise (for example) up to about 300 amino
acids,
peptides of this invention selected or employed for purposes of CAML-binding
will
typically have a maximum length of about 100, preferably less than 100 amino
acids.
Preferably, peptides of this invention will have a number of amino acids
selected from a
number from 14-60, more preferably from 18-60, more preferably from 20-60,
more
preferably from 25-60, more preferably from 25-S0. For example, peptides of
this
invention may consist of from about 20 to about 75 amino acids; or, from about
25 to
about 60 amino acids; or, from about 26 to about 40 amino acids; or, from
about 26 to
about 35 amino acids; or, from about 26 to about 30 amino acids. Any
combination of
these examples of minimum and maximum lengths may be employed.
This invention does not include the use of previously identified, native (e.g.
full
length) human and marine TACI proteins or known fragments of such proteins for
CAML-binding, CAML modulation, or modulation of apoptosis or immune response.
However, this invention does include peptides and the use of peptides derived
from TACI
as described herein. Such TACT derived proteins and fragments may comprise or
consist
of the 27 and 28 amino acid CAML-binding peptides from human and marine TACI
described above and the CAML-binding motifs comprising amino acids 176-189 of
human TACI or amino acids 139-152 of marine TACI as described above.
This invention also does not include compositions of matter consisting only or
essentially of native E3/6.7K protein, native human and marine TACI, known
proteins
identified at items [2]-[10], [12] and [13] above or known fragments of these
proteins.
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However, this invention includes compositions of matter consisting of the CAML-
binding
peptides, sequences and motifs of this invention, as defined herein.
This invention includes an isolated CAML-binding peptide comprising a sequence
of amino acids defined by the motif
C-C-x(2)-[FILV]-[ACV]-x(2)-[CS]-x(3)-[KR]-[KR], wherein: x represents any
amino acid; a bracketed numeral represents the number of or a range of numbers
of any
amino acid represented by x; a single letter represents a specific amino acid
identified by
standard single letter amino acid code; and, a bracketed set of two or more
single letters
represents alternate amino acids at a single position; providing that the
ligand does not
comprise more than 100 contiguous amino acids of native TACI.
This invention provides for methods for targeted binding of a moiety or ligand
to
CAML, including for modulation of immune response and for modulation of
apoptosis, .
which methods make use of the CAML-binding peptides of this invention.
This invention provides a method for binding a ligand to CAML, comprising
combining CAML with a ligand, the ligand comprising a peptide defined by the
amino
acid motif
C-C-x(2)-[FILV]-[ACV]-x(2)-[CS]-x(3)-[KR]-[KR], wherein: x represents any
amino acid; a bracketed numeral represents the number of or a range of numbers
of any
amino acid represented by x; a single letter other than x represents a
specific amino acid
identified by standard single letter amino acid code; and, a bracketed set of
two or more
single letters represents alternate specific amino acids at a single position;
providing that
the ligand does not comprise more than 100 contiguous amino acids of native
TACI.
This invention also provides nucleic acids and nucleic acid vectors encoding
the
CAML-binding peptides of this invention, for use in treatment (such as gene
therapy or to
minimize transplant rejection), and for use in recombinant expression of the
peptides of
this invention or proteins comprising the peptides of this invention. Also
included are
cells comprising nucleic acids and vectors according to this invention.
This invention provides methods for effecting targeted binding of a ligand or
other
moiety to CAML, including for inhibiting or inducing apoptosis or treatment of
inflammation comprising treating the cell, a mammal comprising the cell, or a
tissue
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comprising the cell, with a medicament comprising a CAML-binding peptide of
this
invention. The treating may comprise administering a nucleic acid encoding a
peptide of
this invention whereby the peptide is expressed in the cell. The administering
may be by
a viral vector comprising the nucleic acid, with the proviso that if the
vector is adenovirus
or myxoma virus, the nucleic acid is other than a naturally occurring nucleic
acid from E3
of adenovirus, or the nucleic acid is under the transcription control of a
promotor not
found in the selected virus.
The methods of this invention may be employed for treatment of a mammalian
patient suffering from a degenerative (e.g. neurodegenerative) disease, an
immunodeficiency, or an inflammatory disease by modulation of immune response
or
apoptosis.
This invention also provides for methods of modulating immune response or
programmed cell death in a tissue or cell population in a patient comprising:
(a)
withdrawing tissue or a cell from the patient, (b) . treating the tissue or
cells with an
apoptosis modulating moiety comprising a CAML-binding peptide of this
invention; and
(c) returning the treated tissue or cells to the patient.
This invention also provides medicaments, pharmaceutical compositions and
medical devices comprising a CAML-binding peptide of this invention, a Garner
suitable
for facilitating delivery of the peptide to a cell and optionally other active
components
such as a moiety intended to affect CAML activity. Also provided is a nucleic
acid
comprising a non-naturally occurring adenovirus E3 nucleic acid capable of
encoding a
CAML-binding peptide of this invention.
This invention also provides recombinant virus comprising a nucleic acid
encoding a CAML-binding peptide of this invention with the proviso that if the
virus is
adenovirus or myxoma virus, the nucleic acid is other than a naturally
occurring
adenovirus E3 nucleic acid or myxoma virus nucleic acid; or, the nucleic acid
is under the
transcriptional control of a promoter not from the selected virus.
This invention .also provides: the use of a CAML-binding peptide of this
invention, a nucleic acid encoding said peptide or a vector comprising said
nucleic acid,
for treatment, including for inducing or inhibiting apoptosis, for modulating
an immune
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or inflammatory response; and, the use of a CAML-binding peptide of this
invention, a
nucleic acid encoding said peptide or a vector comprising said nucleic acid
for the
preparation of a medicament, pharmaceutical composition or implantable medical
device
or material, for such uses.
This invention also provides an assay for selecting an agent capable of
modulating
activity of CAML which comprises: combining a CAML-binding peptide with a
sample
suspected of comprising such an agent or with a putative agent; and,
determining whether
such CAML activity is modulated. Determining whether CAML activity is
modulated
may be done by known means for assessing CAML activity.
Combining of CAML and a CAML-binding peptide may occur in vitro (e.g. in a
cell extract) or may be done in vivo, as in a cell that is a cell that is
rescued from apoptosis
or an immune response. The combining in an assay of this invention may include
a
coupling of a peptide of this invention to an agent or putative CAML
modulating agent.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 A chart showing alignment of a nucleic acid sequence which is
capable of encoding a E3/6.7K protein corresponding to that of Adenovirus
serotype Ad2
wild-type(Wt.) (SEQ ID N0:20); and the polymerase chain reaction (PCR) nucleic
acid
product expected (SEQ ID NO: 21) when a forward primer (FP - SEQ ID NO: 22)
and a
reverse primer (RP - SEQ ID NO: 23) are used to amplify the wild-type
sequence(Wt.).
Start codons are underlined. The nucleic acids shown in bold in the forward
primer (FP)
represent a modification to provide a Kozak consensus sequence. The nucleic
acids shown
in bold in the reverse primer (RP) is a modified stop codon to enhance
translation.
Figure 2 A chart showing alignment of E3/6.7K amino acid sequences from
the Ad2 (SEQ ID NO: 24) and Ad5 (SEQ ID NO: 25) Adenovirus serotypes. The Ad2
E3/6.7K amino acid sequence is 61 amino acids in length and the Ad5 E3/6.7K
amino
acid sequence is 63 amino acids in length.
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DETAILED DESCRIPTION OF THE INVENTION
As used herein for description of this invention, the terms E3/6.7K protein,
peptide or polypeptide includes a protein or fragment thereof encoded by a
nucleic acid
as depicted in Figure f or an Ad2 or Ad5 adenovirus serotype protein or
fragment thereof
as depicted in Figure 2. The Ad5 protein shown in Figure 2 is about 7.1K.
Modulation of apoptosis, including inhibition of apoptosis or rescue of a cell
from
apoptosis may be determined by various methods known in the art, including
assays
which directly measure apoptosis or which measure the activity of TNF-a, such
as those
described herein.
Recombinant Expression and Gene Therapy Methods. The isolated nucleic
acid molecule depicted in Figure 1, a nucleic acid molecule encoding a CAML-
binding
peptide as defined herein or a nucleic acid molecule complementary to those
described
above, may be incorporated into a vector suitable for expression in a host
cell or to act as ..
a transformation vector, such as to introduce the nucleic acid into cells of a
mammal to be
treated. Suitable vectors for such purposes include retroviruses and
adenoviruses.
Techniques for the formation of the transfection vector comprising a CAML-
binding peptide encoding nucleic acid molecule are well-known in the art, and
are
generally described in "Working Toward Human Gene Therapy," Chapter 28 in
Recombinant DNA, 2nd Ed , Watson, J.D. ci al., eds., New York: Scientific
American
Books, pp. 567-581 (1992), and in the references cited therein. Various
promoters may
be used to enhance expression in host cells or gene expression in specific
tissues. For
example, in neuronal tissue the neuron-specific enolase promoter (Ad-NSE) and
in .
Lymphocytes the lck promoter could be used for gene therapy methods.
Organ Transplant Methods. CAML-binding peptides have potential uses in
tissue and organ transplantation, for example, to render them less susceptible
to apoptosis.
In particular, they can be used to genetically modify endothelial or other
mammalian cells
to render them capable of expressing a protein which binds to and is designed
to
specifically inhibit apoptosis (e.g. as induced by TNF-a) in transfected
cells. Peptides of
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this invention may also be used in the transplantation of genetically modified
cells, or
tissue or organs comprising such cells, capable of expressing the inhibiting
protein; it -
most particularly is directed to methods of transplanting modified xenog~neic
or
allogeneic cells, tissue or organs; recombinant genes, proteins and vectors
for
accomplishing same; and the cells, tissue or organs, as well as non-human
transgenic or
somatic recombinant animals, so modified.
Appropriate methods of inserting foreign cells or nucleic acids into animal
tissue
include microinjection, embryonic stem (ES) cell manipulation,
electroporation, cell gun,
transfection-k, transduction, retroviral infection, etc. Nucleic acids can be
inserted into
germ cells (e.g. fertilized ova) to produce transgenic non-human animals
bearing the gene,
which is°then passed on to offspring. Nucleic acids can also be
inserted into somatic/body
cells to provide somatic recombinants, from whom the gene is not passed on to
offspring. ,
Transcription of DNA may be made subject to an inducible promoter, so that
expression
of a recombinant protein can be delayed for a suitable period of time prior to
grafting.
DNA may be inserted into a particular locus, e.g. the thrombomodulin or P-
selectin locus_
and the construct introduced into embryonic stem (ES) cells, with the
resulting progeny
expressing the construct in vascular endothelium. Retroviral vectors, and in
particular
replication-defective retroviral vectors lacking one or more of the gag, pol,
and env
sequences required for retroviral replication, are well-known to the art and
may be used to
transform endothelial cells. The ability of adenoviruses to attach to cells at
low ambient
temperatures is an advantage in the transplant setting which, can facilitate
gene transfer
during cold preservation.
Alternative means of targeted nucleic acid delivery comprise DNA-protein
conjugates, liposomes, etc.
Cells or cell populations can be treated in accordance with the present
invention
in vivo or ifz vitro. For example, for purposes of ifa vivo treatments, p65RHD
vectors can
be inserted by direct infection of cells, tissues or organs in situ. For
example, the vessels
of an organ such as a kidney can be temporarily clamped off from the blood
circulation,
and the blood vessels perfused with a solution comprising a transmissible
vector construct
containing a CAML-binding peptide for a time sufficient for the gene to be
inserted into
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cells of the organ; and on removal of the clamps, blood flow can then be
restored to the
organ and its normal functioning resumed.
Cell modification can be carried out ex vivo. Cell populations can be removed
from the donor or patient, genetically modified by insertion of a vector, and
then
implanted into the patient or a syngeneic or allogeneic recipient. For
example, an organ
can be removed from a donor, subjected ex vivo to the perfusion step described
above, and
the organ can be re-grafted into the donor or implanted into a different
recipient of the
same or different species.
Preferably, DNA encoding a peptide of this invention will be placed under the
control of a constitutive or inducible promoter. An advantage of employing an
inducible
promoter for transplantation purposes is that the desired high level
transcription/expression of the active gene/protein can be delayed for a
.suitable period of
time before grafting. For example, transcription can be obtained on demand in
response
to a predetermined stimulus, such as, e.g. the presence of tetracycline in the
cellular
environment. An example of a tetracycline-inducible promoter which is suitable
for use
in the invention is disclosed by Furte et al., PEAS US 91 (1994) 9302-9306.
Alternatively, a promoter system where transcription is initiated by the
withdrawal of
tetracycline is described by Gossen and Bujard, PEAS URSA 90 (I992) 5547-51.
Peptide preparation, expression and administration. A peptide according to
the invention or a derivative thereof such as a chimeric peptide (including
proteins)
comprising a CAML-binding peptide may be administered as a pharmaceutical
composition which may be formulated according to various methods. For example,
such
a formulation may be a solution or suspension. However, as is well known,
peptides can
also be formulated for therapeutic administration as tablets, pills, capsules,
sustained
release formulations or powders. The preparation of therapeutic compositions
which '
comprise polypeptides as active ingredients is well understood in the art.
Typically, such
compositions are prepared in injectable form, e.g. as liquid solutions or
suspensions.
Peptides to be used according to this invention may be synthesized using
standard
techniques such as those described in Bodansky, M. Principles of Peptide
Synthesis
(1993) Springer Verlag, Berlin. Automated peptide synthesizers are
commercially
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WO 02/46231 PCT/CA01/01769
available (e.g. Advanced ChemTech Model 396; Milligen/Biosearch 9600).
Peptides may
be purified by high pressure liquid chromatography and analyzed by mass
spectrometry.
One or more modifying groups may be attached to such a peptide by standard
methods,
for example by modification of amino, carboxyl, hydroxyl or other suitable
reactive
S groups on an amino acid side chain or. at either terminus of a peptide (e.g.
Greene, T.W.
and Wuts, P.G.M. Protective Groups in Organic Synthesis (1991) John Wyley &
Sons
Inc., New York). Peptides may also be prepared according to standard
recombinant
techniques using a nucleic acid molecule encoding the peptide. A nucleotide
sequence
encoding a desired peptide may be determined pursuant to the genetic code and
an
oligonucleotide having this sequence may be synthesized by standard DNA
synthesis
methods (e.g. using automated DNA synthesizer) or by deriving such DNA from a
natural
gene or cDNA using standard molecular biology techniques such as site-directed
mutagenesis, polymerase chain reaction, and/or restriction enzyme digestion.
For
example, production of recombinant adenovirus and TACI proteins is lrnown in
the art,
including from literature described herein.
To facilitate expression of a peptide in a host cell by recombinant
techniques,
nucleic acids according to this invention may be incorporated into a
recombinant vector.
Accordingly, this invention also provides such vectors comprising the nucleic
acid
molecules of this invention. As used herein, the term "vector" refers to a
nucleic acid
molecule capable of transporting another nucleic acid to which it has been
linked:
Vectors may include circular double stranded plasmids and viral vectors.
Certain vectors
are capable of autonomous replication in a host cell such as vectors of
bacterial origin and
episomal mammalian vectors. .Other vectors such as non-episomal mammalian
vectors
may be integrated into the genome of a host cell upon introduction into the
host cell and
thereby may be replicated along with the host cell genome. Certain vectors may
be
capable of directing the expression of genes to which they have been
operatively linked
and are referred to as expression vectors.
A nucleotide sequence encoding a peptide of or to be used in this invention
may
be operatively linked to one or more regulatory sequences selected on the
basis of the host
cells to be used for expression. This means that the sequences encoding the
peptide are '
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linked to a regulatory sequence in a manner that allows for expression of the
peptide.
Such regulatory sequences may include promoters, enhancers, polyadenylation
signals
and other expression control elements such as are described in Goddel; Gene
Expression
Technology: Methods in Enzymology 185 (1990) Academic Press, San Diego,
California. Regulatory sequences may direct constituative expression in many
types of
host cells or may direct expression only in certain tissues or cells.
Regulatory elements
may direct expression in a regulatable manner such as only in the presence of
an inducing
agent. Suitable expression vectors for adenovirus proteins and for TACI are
known in the
art, including references referred to herein.
Peptides, polypeptides and proteins to be used according to this invention may
comprise sequences of amino acids not derived from the natural source for a
dedicated
CAML-binding peptide employed (e.g. fusion or chimeric protein). For example,
such
proteins may comprise a peptide of this invention fused to a peptide that
facilitates
transfer across a cell membrane or fused to a peptide that affects or
facilitates modulation
of CAML activity. Also included in this invention axe derivatives of peptides
of this..
invention, including derivatives intended to enhance the immunogenicity,
biological
activity, or pharmacokinetic properties of the peptide or protein comprising
the peptide.
Further, peptides of this invention may be modified by labeling or by coupling
to another
agent intended to facilitate detection or recovery of the peptide or its
binding partners,
including CAML. Examples of such labeling include coupling to an enzyme or a
detectable label such as a metallic, radioactive, or fluorescent element.
Examples of
modification to affect pharmacokinetic properties include modification of N or
C termini
(e.g. to include an amide group or a D-amino acid) to reduce the ability of a
peptide to act
as a substrate for a carboxypeptidase or a aminopeptidase, or myristoylation
to improve
accessibility to a cell interior.
Examples of suitable parenteral administration include intravenous,
subcutaneous
and intramuscular routes. Intravenous administration can be used to obtain
acute
regulation of peals plasma concentrations of the drug as might be needed for
example to
treat acute episodes of airway hyper-responsiveness. Improved half life and
targeting of
the drug to the airway epithelia may be aided by entrapment of the drug in
liposomes. It
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may be possible to improve the selectivity of liposomal targeting to the
airways by
incorporation of ligands into the outside of the liposomes that bind to airway-
specific -
macromolecules. Alternatively intramuscular or subcutaneous depot injection
with or
without encapsulation of the drug into degradable microspheres e.g. comprising
poly
(DL-lactide-co-glycolide) may be used to obtain prolonged sustained drug
release as may
be necessary to suppress the development of airway hyper-responsiveness. For
improved
convenience of the dosage form it may be possible to use an i.p. implanted
reservoir and
septum such as the Percuseal system available from Pharmacia. Improved
convenience
and patient compliance may also be achieved by the use of either injector pens
(e.g. the
Novo Pin or Q-pen) or needle-free jet injectors (e.g. frorn Bioject, Mediject
or Becton
Dickinson). Prolonged zero-order or other precisely controlled release such as
pulsatile
release can also be achieved as needed using implantable pumps. Examples
include the
subcutaneously implanted osmotic pumps available from ALZA, such as the ALZET
osmotic pump.
Nasal delivery may be achieved by incorporation of the protein drug into
bioadhesive particulate carriers (<200 wrn) such as those comprising
cellulose,
polyacrylate or polycarbophil, in conjunction with suitable absorption
enhancers such as
phospholipids or acylcarnitines. Available systems include those developed by
DanBiosys and Scios Nova.
Oral delivery may be achieved by incorporation of a drug into enteric coated
capsules designed to release the drug into the colon where digestive protease
activity is
low. Examples include the OROS-CT/Osmet.TM, and PULSINCAP.TM. systems, from
ALZA and Scherer Drug Delivery Systems respectively. Other systems use azo-
crosslinked polymers that are degraded by colon specific bacterial
azoreductases, or pH
sensitive polyacrylate polymers that are activated by the rise in pH at the
colon. The
above systems may be used in conjunction with a wide range of available
absorption
enhancers.
Targeted delivery of high doses of a drug to the site of airway hyper-
responsiveness can be directly achieved by pulmonary delivery (see: McElvaney,
et al., J.
Clin. Invest., 90, 1296-1301 (1992); and Vogelmeier et al., J. Appl. Physiol.,
69, 1~43-
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1848 (1990). The lower airway epithelia are highly permeable to wide range of
proteins
of molecular sizes up to 20 kDa (e.g. granulocyte colony stimulating factor).
It is possible
to spray dry proteins in suitable carriers such as mannitol, sucrose or
lactose. Micron-
sized particles may be delivered to the distal alveolar surface using dry
powder inhalers
S similar in principle to those designed by Inhale, Dura, Fisons (Spinhaler),
Glaxo
(Rotahaler) or Astra (Turbohaler) propellant-based metered dose inhalers.
Solution
formulations with or without liposomes may be delivered using ultrasonic
nebulizers.
Examples of inflammation caused by implants or surgical procedures, the
treatment of which may make use of this invention include: restenosis, senile
calcific
aortic stenosis, balloon angioplasty induced inflammation, intimal hyperplasia
(a cause of
vascular restenosis), atherosclerosis, benign prostate hyperplasia,
hysteroscopically
delivered fallopian tube fertilization and sterilization aided therapies,
inflammation
caused by catheters, inflammation caused by the use of mesh or other implants
for hernia
repair, inflammation caused by the use of mesh in the surgical repair of
rectocoele and
rectal prolapses, urological stress, incontinence, inflammation caused by
surgical uterine
suspensions, inflammation caused by tapes, staples and sutures, and
inflammation
resulting from vascular grafts (including peripheral, coronary artery and
bypass grafts).
In such cases, treatment may include provision of peptides or nucleic acids of
this
invention on an implantable medical device including beads, tape, mesh, gauze,
membranes, appliances such as stems, and the like. Methods for associating
(e.g. coating) ;
of peptides or nucleic acids with such devices or materials are known,
including those
described in: WO 98116268, WO 00/23123, US 6,140,128, US 4,610,692, US
6,117,456,
US 4,946,686, Tarr, E.R. (1997) Biomed Sci Instrum, 33:143-8, and Abrams, L.
(1994)
Biomed Sci Instrum, 30:169-74.
Percutaneous transluminal coronary angioplasty (PTCA) is widely used as the
primary treatment modality in many patients with coronary artery disease, to
reduce
obstruction and improving coronary flow. However, restenosis often results
with
significant morbidity and frequently necessitates further interventions such
as repeat
angioplasty or coronary bypass surgery.
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Tlae present invention includes therapeutic methods comprising the
administration
of a therapeutic agent comprising or in addition to a CAML-binding protein of
this
invention to a procedurally traumatized mammalian vessel (e.g., by an
angioplasty
procedure). Preferably, the therapeutic agent is an E3/6.7K or TACI derived
peptide or
polynucleotide of this invention. Preferred therapeutics in the practice of
the present
invention, include, for example The SED domain of E316.7K or analogs or
derivatives
thereof. The administration of a therapeutic agent of the invention is
effective to
biologically stmt the vessel, inhibit or reduce vascular remodeling of the
vessel, inhibit or
reduce vascular smooth muscle cell proliferation, or any , combination
thereof. The
administration of the therapeutic agent preferably is carried out during the
procedure,
which traumatizes the vessel, e.g., during the angioplasty or other vascular
surgical
procedure. The invention also provides therapeutic compositions and dosage
forms
adapted for use in the present method, as well as kits containing them.
Thus, this invention includes methods for biologically stenting a traumatized
mammalian blood vessel. The method comprises administering to. the blood
vessel an
effective amount of a therapeutic agent to biologically stmt the vessel. As
used herein,
"biological stenting" .means the fixation of the vascular lumen in a dilated
state near its
maximal systolic diameter, e.g., the diameter achieved following balloon
dilation and
maintained by systolic pressure. The method may comprise the administration of
an
effective amount of an E3/6.7K peptide or polynucleotide into the blood
vessel.
Preferably, the peptide or polynucleotide is dispersed in a pharmaceutically
acceptable
liquid carrier, viral vector or liposome mediated gene delivery preferably
administered
locally via a catheter. Th peptide or polynucleotide may be dispersed in a
pharmaceutically acceptable liquid earner at about 0.001 to about 25 mu.g per
ml of
aqueous vehicle. Preferably, a portion of the amount administered penetrates
to at least
about 6 to 9 cell layers of the inner tunica media of the vessel and so is
effective to
biologically stmt the vessel.
The invention also includes therapeutic methods comprising inhibiting
diminution
of vessel lumen diameter by administering to a traumatized vessel of a mammal
an
effective amount of a CAML-binding peptide. For example, an E3/6.7K or TACI
derived
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peptide or polynucleotide is administered via an implantable device, wherein
the
implantable device is not a catheter, which has a first and a second expansile
member, i.e., -
balloons, which are disposed on opposite sides of the vessel area to be
treated in order to
isolate the portion of the vessel to be treated prior to peptide or
polynucleotide
administration. Preferably, the isolated portion of the vessel is not washed
to remove
blood prior to peptide or polynucleotide administration ("bloodless
angioplasty").
"Isolated," as used above, does not mean occlusive contact of the actual
treatment area by
the catheter balloon, which is preferred in the practice of the present
invention.
The invention further includes methods for inhibiting or reducing diminution
in
vessel lumen volume in a traumatized mammalian blood vessel. The method may
comprise administering to the blood vessel of a mammal an effective amount of
peptide
or polynucleotide of this invention, wherein the peptide or polynucleotide is
in sustained ,
release dosage form. Preferably, the peptide or polynucleotide is administered
in situ, by
means of an implantable device, wherein the peptide or polynucleotide is
releasably
embedded in, coated on, or embedded in and coated on, the implantable device.
A..
crystalline peptide may be releasably embedded in, or dispersed in, a
adventitial wrap,
e.g., a silicone membrane.
The invention further includes therapeutic methods comprising administering to
a
traumatized mammalian blood vessel a sustained release dosage form comprising
microparticles or nanoparticles comprising a peptide or polynucleotide of this
invention
or analogs thereof. For example, a sustained release dosage form comprising a
SED
peptide is preferably administered via an implantable device, which is not a
catheter used
to perform bloodless angioplasty. The amount administered will be effective to
inhibit or
reduce diminution in vessel lumen area of the mammalian blood vessel. The
sustained
release dosage form preferably comprises microparticles of 4 to about 50
microns in
diameter. The sustained release dosage form can also preferably comprise about
2 to
about 50, and more preferably greater than 3 and less than 10, microns in
diameter. For
nanoparticles, preferred sizes include about 10 to about 5000, more preferably
about 20 to
about 500, and more preferably about 50 to about 200, nanometers.
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Also included are methods comprising administering to a mammalian blood vessel
a dosage form of peptide of this invention or an analog thereof in a non-
liquid vehicle or
matrix effective inhibit or reduce diminution in vessel lumen area of the
mammalian
blood vessel. Preferably the dosage form is a substantially solid dosage form.
As used
S herein, "solid form" does not include microparticles, nanoparticles, and the
like. The non-
liquid vehicle or matrix preferably includes, but is not limited to, a gel,
paste, gauze or a
membrane, which comprises the peptide.
Also included is a kit comprising, preferably separately packaged, at least
one
implantable device adapted for the ih situ delivery of at least one peptide or
. 10 polynucleotide of this invention to a site in the lumen of a traumatized
mammalian vessel
and at least one unit dosage form of a therapeutic agent comprising the
peptide or
polynucleotide in a liquid vehicle adapted for delivery by said device. The
administration
of at least a portion of the unit dosage form to the traumatized vessel is
intended to be
effective to biologically stent the vessel, inhibit or reduce the vascular
remodeling of the
1S vessel, inhibit or reduce vascular smooth muscle cell proliferation, or any
combination
thereof.
Further included is a kit comprising, preferably separately packaged, an
implantable device adapted for the delivery of at least one therapeutic agent
to a site in the
lumen of a traumatized mammalian vessel and a unit dosage form comprising at
least one
20 peptide or polynucleotide of this invention, wherein the administration of
at least a
portion of the unit dosage form is effective to cause CAML-binding and actions
of the
therapeutic agent to inhibit or reduce diminution in vessel lumen diameter of
the vessel.
The device could be a catheter, having a first and a second expansile member,
which are
disposed on opposite sides of the region to be treated so as to isolate a
portion of the
2S vessel to be treated. Alternatively, the isolated portion of the vessel is
not washed to
remove blood prior to administration. .
The invention also ,includes pharmaceutical compositions suitable for
administration by means of an implantable device. The composition may comprise
an
amount of a SED peptide or analog thereof effective to inhibit or reduce
stenosis or
30 restenosis of a mammalian vessel traumatized by a surgical procedure and a
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pharmaceutically acceptable non-liquid release matrix for said SED peptide.
Preferably,
the release matrix comprises a gel, paste, gauze or membrane.
The invention also includes therapeutic devices. One such embodiment comprises
a therapeutic shunt comprising an amount of a peptide or polynucleotide of
this invention
effective to facilitate inhibition of stenosis or reduce restenosis following
placement of the
therapeutic shunt as a result of CAML interaction. Another embodiment of the
invention
comprises therapeutic artificial graft comprising an amount of a SED peptide
or analog
thereof to inhibit stenosis or reduce restenosis following placement of the
graft. Yet
another embodiment of the invention comprises a therapeutic adventitial wrap
comprising
an amount of a SED peptide or polynucleotide effective to inhibit stenosis or
reduce
restenosis following placement of the wrap.
The amount of a pharmaceutical composition according to this invention to be
employed will depend on the recipient and the condition being treated. The
requisite
amount may be determined without undue experimentation by protocols known to
hose
skilled in the art. Alternatively, the requisite amount may be calculated,
based on a
deternvnation of the amount of tryptase which, must be inhibited in order to
treat the
condition.
~~rer~mT Fc
Virus strains and tissue culture. Wild Type Ad5 (AdSwt) was obtained from
the American Type Culture Collection (Rockville, Maryland, USA) and d1739,
E316.7K-deleted viral mutant (d1739) was according to Brady et al. (1992), J.
Virol.
66:5914-5923. These two adenovirus group C viruses share a great degree of
similarity,
but differ in the expression of E3/6.7K protein, which is deleted in d1739.
Both viral
serotypes Were propagated in monolayer culture of A549 cells grown in Minimal
Essential Media (Gibco BRL Life Technologies Inc., Gaithersburg, Maryland,
USA)
supplemented with 10% Fetal Calf Serum (FCS). Two to five days after
inoculation with
AdS, cells were freeze/ thawed twice, sonicated for 30s three times and
centrifuged at
SOOxg for 5 min. The supernatant was collected and its viral titer determined
by plaque
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assays on A549 monolayers grown on six well plates. Titers ranged from 10$ to
10~
plaque-forming units (pfu)/ml. Control inoculum was prepared from uninfected
A549
cells treated in an identical manner to the infected cells. .
Inoculation of airway ducts and viral plaque assays. Two groups of 24 mice
were anaesthetized with Halothane. One group of mice were infected
intranasally with
l0~pfu of AdSwt in 60 ~,1 of culture media while the other group of mice was
infected
intranasally with l0~pfu of d1739 in 60 p,1 of culture media. Six animals were
infected
with sterile.culture media alone. Six animals from each of the two groups were
sacrificed
with an overdose of Halothane 2 hours, 1, 3 and 7 days post infection (p.i.).
Two sham
infected animals were sacrificed on days 1, 3 and 7 days p.i.. The left lung
was removed
and frozen in liquid nitrogen for use in viral plaque assays. The right dung
was inflated
with 4% paraformaldehyde in PBS pH7.4 (0.149 M NaCI, 0.012 M Na2HP04, 0.004M
KH2P04) and embedded in paraffin.
Viral titer. Viral plaque assays were used to quantitate the amount of
replicating
virus in mouse lungs. Approximately 200mg of lung was homogenized on ice in
1m1.
sterile MEM with a polytron. The homogenate was spun for 2 min at 10,000g
while the
supernatant was removed and stored at -70°C. The lung homogenate
supernatant titer was
determined by plaque assay on A549 cell monolayer cultures grown in MEM/10%FBS
on
six well plates using decimal dilutions from 10-1 to 10-6 in MEM from the
supernatant of
animal lung homogenate from all time groups. Each well was inoculated with 500
p,1 of
diluted supernatant and virus was allowed to adsorb onto the monolayer of A549
cells for
1h. at 37°C An agarose overlay (0.9% agarose, MEM, 2% FCS, and 0.001
neutral red at
37°C) was applied after adsorption. Plaques were counted after 10-14
days and
normalized to lung mass and expressed as (log pfu/g lung tissue).
Histologic scoring. Four ~m sections of paraffin embedded lung tissue were
mounted on glass slides and stained with hematoxylin and eosin. An independent
observer scored the airway mucosal, airway adventitia and the vascular
adventitia for
inflammation. The histopathologic grades were 0 - no inflammation, 1 - mild
inflammation. 2 - moderate inflammation, 3 - severe inflammation for each
feature. The
scores for each feature were summed to give a total inflammatory score with
maximum
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being 9 for each animal. A mean inflammatory score was calculated for each
animal by
dividing the total score by 3. The mean and standard deviation was calculated
for each
experimental group.
Statistical analysis. Comparisons between the two virus were made for viral
titer, inflammatory score and time using a 2-way ANOVA. The level of
significance was
p<0.05.
Plasmid constructs. cDNA for E3/6.7K was obtained by amplifying by PCR the
region coding for the E3/6.7K ORF from a vector carrying the Ad2 E3 region
using SEQ
ID N0:22 and 23 as primers (Figure 1). The PCR product was cloned in the Xhol
site of
the BPV based cDNA expression vector pBCMGSneo (Karasuyama and Melchers,
(1988), Eur. J. Invmunol. 18:97-104) and sequenced to ensure accuracy.
To isolate the cDNA for E3/6.7K, the primers and template DNA purified from
HEK-293 cells infected with Ad2 and AdS, 24hr. post infection were used. The
reaction
cocktail contained template DNA, O.S~.M of each primer forward and reverse,
250~,M of
each nucleotide, SU of Pfu polymerase (Canadian Life Technologies, 2270
Industrial
St., Burlington , Ontario) in 1X Pfu Buffer. ' The reaction conditions are:
melting of
double stranded DNA at 95°C for 30sec., followed by annealing at
57°C for 30 sec.,
followed by a 30 sec. ramp to 72 and continued elongation for an additional
30sec. 30
cycles of the above PCR reaction produced sufficient DNA in most cases. The
newly
generated cDNA for E3/6.7K contained modifications (highlighted in bold in
Figure 1)
which are not found in naturally occurring E3 nucleic acid. Both modifications
enhance
translation initiation at the start site of E3/6.7K and provide for increased
production of
the protein in a transformed cell. ,The forward primer provides the start site
of E3/6.7K
with an optimal upstream Kozak consensus sequence. The reverse primer was
modified
to replace the naturally occurring TGA-Stop codon with an Ochre-Stop codon
(TAA).
The latter modification eliminates the start site of E3119K, which overlaps
with the
sequence of E3/6.7K in the naturally occurring E3 nucleic acid and decreases
translation
efficiency of E3/6.7K from the natural sequence.
pBD-6.7, a bait vector containing E316.7, was constructed by subcloning full
length E3/6.7K cDNA in frame into the C terminus of the GAL4 DNA binding
domain of
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vector pGBKT7 (CLONTECH). pAD-CAML was constructed by subcloning full length
CAML in frame into the C terminus of the GAL4 activation domain of the prey
vector
pGADT7 (CLONTECH). pAD-CAML(201) was constructed by subcloning amino acids
1 to 201 of CAML in frame into the C terminus of the GAL4 activation domain of
the
S prey vector pGADT7. The fidelity of the constructs was confirmed by
sequencing.
Yeast Transformation and Selection. pBD-6.7 and pAD-CAML or pAD-
CAML(201) were transformed together into Saccharomyces cerevisiae AH109 using
the
Lithium Acetate Yeast Transformation procedure as described by Gietz, D., et
al. (1992)
Nucleic Acids Res., 20:1425. Control cotransformation of pBD-53 / pAD-T and
pBD-
Lam / pAD-T were also done. pBD-53 l pAD-T encode fusions between the GAL4
DNA-BD and AD and marine p53 and SV40 large T-antigen, respectively. p53 and
large
T-antigen interact in a yeast two-hybrid assay. pBD-Lam encodes a fusion of
the DNA-
BD with human lamin C and provides a negative control for an interaction with
pAD-T.
For yeast selection, pBD containing the TRPl gene was selected on synthetic
drop out
media lacking tryptophan (SD-T). pAD contains the LEII2 gene and was selected
for on
SD-L (leucine). When selecting for protein interaction AH109 was grown on
leucine/-
tryptophan/-histidine deficient/X-a-Gal supplemented media (SD-LTHX) or
adenine/-
histidine/-leucine/-tryptophan deficient /X-oc-Gal supplemented media (SD-
AHLTX). The
transformed yeast can only grow on SD-LTHX or SD-AHLTX if a protein
interaction
20- occurs. Yeast transformed with pBD-6.7 and pAD-CAML or pBD-6.7 and pAD-
CAML(201) were able to grow on both SD-LTHX or SD-AHLTX indicating that the
reporter enzymes necessary to grow on deficient media are synthesized which
indicates
that the proteins E3/6.7K and CAML interact.
Generation of stable U937 cell Iines expressing E3/6.7K. U937 human
histiocytic lymphoma cells obtained from ATCC (CRL 1593) were maintained in
RPMI
1640, 10% FCS, 2mM L-glutamine, lOmM HEPES, 100U/ml penicillin and 100~g/ml
streptomycin in an atmosphere of 5% C02 and 100% humidity. Cells were
transfected
with the appropriate construct by using the DMRIE-C cationic lipid reagent
available
from Life Technologies using the manufacturer's protocol. Transfected cells
were
maintained in medium containing geneticin G-418 sulphate at a final
concentration of
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WO 02/46231 PCT/CA01/01769
800p.g1ml. Media and supplements were purchased from Life Technologies.
Subclones
of the transfected cell lines were generated by serial dilution and examined
for expression
of E3/6.7K by Northern Blotting. The expression of E3/6.7K mRNA was very
similar in
all the clones examined. All the G-418 resistant cells that survived the
selection
procedure were pooled and used for the in vitro assays, in order to avoid
clonal variations
known to arise in U937 cells.
Labelling, immunoprecipitation and Western Blotting of proteins from
transfected cells. U937 cells transfected with vector or with vector carrying
E316.7K
were grown in suspension until they were growing exponentially. 10$ cells were
harvested, washed and intracellular pools of cysteine and methionine were
depleted by
incubation in prewarmed methionine/cysteine-free essential media without FCS
for one
hour at 37°C at a concentration of 5x106 cells/ml. A total of 2x10
cells were labelled for
one hour in prewarmed methionine/cysteine-free media containing O.SmCi/ml
[35S]-
Cysteine and 0.2 mCi/ml (Amersham) [35S]-Methionine (Amersham) at a
concentration
of 5x106 cells /ml. Cells were washed and then lysed on ice in freshly made
lysis buffer
containing 1% TritonX-100, 1% BSA (bovine serum albumin), 1mM iodoacetamide,
1mM PMSF, 2.STILT/ml aprotinin, O.OlM Tris pH8.0, 0.14M NaCl. Samples were
counted by TCA precipitation and approx. l0~cpm of each sample was precleared
O/N
using protein A-Sepharose CL-4B, the supernatant was immunoprecipitated using
a
polyclonal rabbit antiserum raised against the C-terminal portion of E3/6.7K
and protein
A-Sepharose. The pellet was denatured in SDS/sample buffer and loaded on a
Tricine-
SDS PAGE gel, 16.5%T, 3%C separating gel with a 10%T, 3%C spacer gel (Schagger
and von Jagow, 1987). Alternatively, cell lysate equivalent to 105 cells was
denatured in
SDS-PAGE loading buffer and loaded on 10% glycine SDS-PAGE gel system,
separated
and blotted onto a Trnmobilon-P PVDF membrane (Millipore) and probed with
cPLA2
rabbit polyclonal antiserum (Cayman Chemical). The signal was detected via
horse
radish peroxidase-conjugated, goat antirabbit antiserum and by
chemiluminescence using
the ECL kit (Biorad).
Arachidonic acid release assays. Cells were grown at low density in 10%
Hyclone FCS, RPMI 1640, 2mM L-glutamine, lOmM HEPES for several days then
-2G-
CA 02430948 2003-06-05
WO 02/46231 PCT/CA01/01769
harvested and washed twice in PBS, 1% BSA. Approximately SxlO~ cells (5x105
cells/ml) were labelled for 20hrs in same media as above supplemented with 0.4
~,Cilml
[3H] arachidonic acid [5,6,8,9,11,12,14,15-3H(N)] (O.lmCi/ml stock; New
England
Nuclear). Cells were washed twice in RPMI 1640, 0.2%BSA and incubated for one
hour
in the wash media in order to minimize the spontaneous release of [3H]
arachidonic acid.
The number of cells was normalized in all cell lines and 400w1 of cell
suspension was
aliquoted in each well of a 24 well plate containing 100p1 of treatment media
(2x105
cells/well corresponding to 1.4x103 countslwell). The assay was set up in
triplicate and
the cells were stimulated either with media alone or with 20ng/ml human rTNF-a
(2000U/ml) (Boehringer, Mannheim), or with 10~,g/ml cycloheximide or with a
combination of 20ng1m1 TNF-a and 10~g/ml cycloheximide. After 20 hours of
treatment
the cells were centrifuged and 100w1 of supernatant out of SOOp,I total was
mixed with
3m1 scintillation fluid and counted. For each cell line three samples were
lysed in lysis
buffer and the lysate was used to determine the total counts of incorporated
[3H]
1 S Arachidonic Acid. The counts per minute of released [3H] arachidonic acid
were
expressed as a percentage of the average of total incorporated [3H]
arachidonic acid.
Annexin V-FAGS apoptosis assay. Annexin V-FITC (PharMingen) was used to
determine the binding of Annexin V to externalized phosphatidyl serine. The
protocol
followed was based on the manufacturers Annexin V-FITC staining protocol.
Cells were
grown at low density in 10% Hyclone FCS, RPMI 1640', 2mM L-glutamine, lOmM
HEPES for several days then Sx106 cells were harvested and washed twice in
PBS. Cells
resuspended in above media were treated for 7 hours with media alone or with
100ng/ml
(10,000 U/ml) human rTNF-a or with 200 p.g/ml cycloheximide or with a
combination of
100 ng/ml TNF-a and 200 ~g/ml cycloheximide. The cells were resuspended at
1x106
cells/ml in lxBinding Buffer (lOmMHepes/NaOH, pH7.4, 140mM NaCl, 2.SmM CaCl2).
1X105 cells (100p.1 of above suspension) were combined with Spl of Annexin V-
FITC.
One sample of cells was not stained and used to set up the baseline
fluorescence. The
cells were examined with a fluorescence-activated cell sorter (FAGS) on a
Beckton
Dickson FACS Analyzer.
7_
CA 02430948 2003-06-05
WO 02/46231 PCT/CA01/01769
Production of Ad vectors for gene tlierapy. The SVS backbone previously
described (Chen (1997) PNAS) has been successfully used to transduce i~a vivo
the -
dystrophin gene. The baclcbone lacks the E1 and E2 region. Without these two
regions
the SVS Ad vector is replication defective and therefore safer to use as well
as it elicits a
reduced inflammatory response. The cDNA encoding E3/6.7K under the control of
the
actin promoter and the CMV enhancer was added to SVS and used to rescue a new
vector
called SVS-6.7, which will incorporate E3/6.7K as an immunomodulatory protein.
Creation of Producer Cells resistant to apoptosis. Creation of hybridoma,
Chinese hamster ovary (CHO) or insect cells that are resistant to apoptosis
follow the
same procedure as the transfection of U937 cells outlined above except at the
end of the
selection in G-418, the cells are sorted or clonally expanded in order to
screen for the
expression of the protein of interest.
E3/6.7K Results in more Persistent Viral Titers and a reduction of the
inflammatory response. The presence of E3/6.7K results in more persistent
viral titers
during the course of infection by comparing mice infected with the E3/6.7K
deletion virus
(41739) to mice infected with the wild type virus (AdSwt). The titers of 41739
were
significantly higher than Ad5 wild type one day after inoculation (p<0:001).
Over time,
the titers of 41739 decreased as the virus was cleared (p<0.001). In contrast
AdSwt titers
did not change significantly over the 7 day experimental period. The rapid
reduction of
41739 over the seven day period is attributed to a strong host response due to
the increased
inflammation in the absence of E3/6.7K. Inflammation of the perivascular
region of the
blood vessels and the adventitia of the airways was greater in animals
infected with 41739
than in animals infected with AdSwt over the seven days experimental period
(p=0.025).
There was also a significant increase in inflammation from day three to day
seven for both
types of viruses (p=0.029).
TNF-a Mediated Arachidonic Acid Release Is Reduced in the Presence of
E316.7K. E3/6.7K can affect the cellular response to inflammatory cytokines. A
U937
cell line was transfected with the cDNA 'for E3/6.7K and expression of E3/6.7K
was
confirmed using immunoprecipitation with a polyclonal rabbit antiserum raised
against an
E3/6.7K C-terminal derived peptide and SDS-PAGE electrophoresis. The U937
cells
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CA 02430948 2003-06-05
WO 02/46231 PCT/CA01/01769
transfected with E3/6.7K cDNA (U937-E3/6.7K) decreased [3H] arachidonic acid
release
by 50% when compared with U937 cells transfected with vector alone (LT937neor)
when
stimulated with TNF-a. When the stimulus was increased by the addition of TNF-
a and
cycloheximide (CHX), a protein synthesis inhibitor synergistic with TNF-a,
U937-
E3/6.7K were still able to reduce the release of [3H] arachidonic acid by 60%
when
compared to U937neor. The presence of E316.7K reduces the levels of inducible
release
of [3H] arachidonic acid during TNF-a stimulation.
Apoptosis Induced by TNF-a is Reduced in the Presence of E3/6.7K. TNF-a
induced apoptosis was assayed by measuring by measuring the externalization of
phosphatidyl serine using FITC labelled Annexin V according to Martin et al.,
(1995), J.
Exp. Med. 182:1545-1556. Cells expressing E3/6.7K show a 55% reduction in
percentage of apoptotic cells compared with U937neor following stimulation
with TNF-
a. The U937-E3/6.7K cells show a 65% reduction in apoptosis compared to
U937neor
following an augmented stimulation with a combination of TNF-a and CHX. The
presence of E316.7K decreased the apoptotic response in U937 cells upon
stimulation
with TNF-a or a combination of TNF-a and CHX.
In the Presence of E3/6.7K, cPLA2 Is Cleaved to a 78kDa Form Following
TNF-a Induction. The expression of cPLA2 in U937 cells following induction
with
TNF-a was assayed. The cPLA2 antiserum recognized two forms of the enzyme: one
larger form of approximately 110kDa; and a second form of 78kda. There was a
difference between U937neor cells and U937-E316.7K with regards to the ratio
of the
110kDa versus the 78kDa forms of cPLA2. While TNF-a does not seem to alter
this ratio
in U937neor (cells-where the predominant form migrates as a 110kDa protein) in
U937-
E3/6.7 K cells following induction with TNF-a the most predominant form of
cPLA2 is
78kDa. The antisera was raised against a peptide corresponding to residues 443-
462 of
the cPLA2 sequence, therefore the only fragment detected by immunoblotting
following
cleavage is the 78kDa fragment corresponding to the 1-522 amino acid sequence
of
cPLA2 as isolated from U937 cells according to Sharp et al. (1991), J. Biol.
Chem.,
266:14850.
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CA 02430948 2003-06-05
WO 02/46231 PCT/CA01/01769
CAML-Binding witli SED. The yeast two hybrid system takes advantage of the
GAL4 transcriptional activator which consists of two separable a~ld
functionally essential
domains: a DNA binding domain and a domain that activates transcription. A
bait gene
(fragments of the E3/6.7K gene) was expressed as a fusion to the GAL4 DNA-
binding
domain (BD), while a prey gene (CAML) was expressed as a fusion to the GAL4
activation domain (AD). When bait and prey fusion proteins interact, the BD
and AD are
brought into proximity, thus activating transcription of reporter genes. Using
this
methodology, it is demonstrated that E3/6.7 K interacts with full length human
and mouse
CAML and more particularly, with the same domain of CAML that TACI has been
previously shown to interact (the 1-201 residue domain of CAML).
Although various aspects of the present invention have been described in
detail, it
will be apparent that changes and modification of those aspects described
herein will fall
within the scope of the appended claims. It will be readily apparent to one of
ordinary
skill in the relevant arts that other suitable modifications and adaptations
to the methods
and applications described herein are obvious and may be made without
departing from
the scope of the invention or any embodiment thereof. All publications and
patent
documents referred to herein are incorporated by reference.
-30-
CA 02430948 2003-06-05
WO 02/46231 PCT/CA01/01769
SEQUENCE LISTING
<110> MOISE, Alexandru R.
JEFFERIES, Wilfred A.
VITALIS, Timothy Z.
GRANT, Jason R.
The University of British Columbia
<120> CAML Binding Peptides
<130> 80021-329
<150> CA 2,325,610
<151> 2000-12-07
<150> CA 2,335,411
<151> 2001-03-02
<150> US 60/316,254
<151> 2001-09-04
<160> 25
<270> Patentln Ver. 2.0
<210> 1
<211> 26
<212> PRT
<213> Adenovirus serotype Ad2
<400> 1
Leu Ala Leu Leu Cys Leu Arg Val Ala Ala Cys Cys Thr His Val Cys
1 5 10 15
Thr Tyr Cys Gln Leu Phe Lys Arg Trp Gly
20 25
<210> 2
<211> 26
<212> PRT
<213> Adenovirus serotype Ad5
<400> 2
Leu Thr Leu Leu Cys Leu Arg Leu Ala Ala Cys Cys Val His Ile Cys
1 5 10 15
Ile Tyr Cys Gln Leu Phe Lys'Arg Trp Gly
20 25
<210> 3
<2l1> 26
<212> PRT
<213> Adenovirus serotype Ad3
<400> 3
Val Leu Ile Leu Cys Tyr Leu Tyr Thr Pro Cys Cys Ala Tyr Leu Val
1 5 10 15
- 1 -
CA 02430948 2003-06-05
WO 02/46231 PCT/CA01/01769
Tle Leu Cys Cys Trp Phe Lys Lys Trp Gly
2p 25
<210> 4
<211> 27
<212> PRT
<213> Homo Sapiens
<400> 4
Val Tyr Ser Thr Leu Gly Leu Cys Leu Cys Ala Val Leu Cys Cys Phe
1 5 10 15
Leu Val Ala Val Ala Cys Phe Leu Lys Lys Arg
20 25
<210> 5
<211> 28
<212> PRT
<213> Mus musculus
<400> 5
Leu Tyr Cys Thr Leu Gly Val Cys Leu Cys Ala Ile Phe Cys Cys Phe
1 5 10 15
Leu Val Ala Leu Ala Ser Phe Leu Arg Arg Arg Gly
20 25
<210> 6
<211> 14
<212> PRT
<213> Human adenovirus type 3
<400> 6
Cys Cys Ala Tyr Leu Val Ile Leu Cys Cys Trp Phe Lys Lys
1 5 10
<210> 7
<211> 14
<212> PRT
<213> Discopyge ommata
<400> 7
Cys Cys Phe Cys Ile Cys Lys Lys Cys Leu Leu Lys Lys Lys
1 5 10
<210> 8
<211> 14
<212> PRT
<213> Discopyge ommata
<400> 8
Cys Cys Leu Cys Ile Cys Lys Lys Cys Cys Cys Lys Lys Lys
1 5 10
<210> 9
- 2 -
CA 02430948 2003-06-05
WO 02/46231 PCT/CA01/01769
<211> 14
<212> PRT
<213> Bos taurus
<400> 9
Cys Cys Phe Cys Ile Cys Lys Lys Cys Leu Phe Lys Lys Lys
1 5 10
<210> 10
<211> 14
<212> PRT
<213> Gallus gallus
<400> 10
Cys Cys Phe Cys Leu Cys Lys Lys Cys Leu Phe Lys Lys Lys
1 ' 5 10
<210> 11
<211> 14
<212> PRT
<213> Homo sapiens
<400> 11
Cys Cys Phe Cys Ile Cys Lys Lys Cys Leu Phe Lys Lys Lys
1 5 10
<210> 12
<211> 14
<212> PRT
<213> Mus musculus
<400> 12
Cys Cys Phe Cys Val Cys Lys Lys Cys Leu Phe Lys Lys Lys
1 5 10
<210> 13
<211> 14
<212> PRT
<213> Rattus norvegicus
<400> 13
Cys Cys Phe Cys Val Cys Lys Lys Cys Leu Phe Lys Lys Lys
1 5 10
<210> 14
<211> 14
<212> PRT
<213> Mus musculus
<400> 14
Cys Cys Phe Cys Ile Cys Lys Lys Cys Cys Cys Lys Lys Lys
1 5 . 10
<210> 15
<211> 14
- 3 -
CA 02430948 2003-06-05
WO 02/46231 PCT/CA01/01769
<212> PRT
<2l3> Rattus norvegicus
<400> 15
Cys Cys Phe Cys Ile Cys Lys Lys Cys Cys Cys Lys Lys Lys
l 5 10
<210> 16
<211> 14
<212> PRT
<213> Homo Sapiens
<400> 16
Cys Cys Phe Leu Val Ala Val Ala Cys Phe Leu Lys Lys Arg
1 5 10
<210> 17
<211> 14
<212> PRT
<213> Mus musculus
<400> 17
Cys Cys Phe Leu Val Ala Leu Ala Ser Phe Leu Arg Arg Arg
1 5 10
<210> 18
<211> 14
<212> PRT
<213> Human adenovirus type 5
<400> 18
Cys Cys Val His Ile Cys Ile Tyr Cys Gln Leu Phe Lys Arg
l 5 10
<210> 19
<211> 14
<212> PRT
<213> Myxoma virus
<400> 19
Cys Cys Thr Gly Leu Ala Ser Val Cys Lys Tyr Thr Lys Lys
1 5 10
<210> 20
<211> 238
<212> DNA
<213> Adenovirus Serotype, Ad2 Wild-Type
<400> 20
taagtatatg agcaattcaa gtaactctac aagcttgtct aatttttctg gaattggggt 60
cggggttatc cttactcttg taattctgtt tattcttata ctagcacttc tgtgccttag 120
ggttgccgcc tgctgcacgc acgtttgtac ctattgtcag ctttttaaac gctgggggca 180
acatccaaga tgaggtacat gattttaggc ttgctcgccc ttgcggcagt ctgcagcg 238
<210> 21
<211> 201
- 4 -
CA 02430948 2003-06-05
WO 02/46231 PCT/CA01/01769
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Polymerase
Chain Reaction (PCR) Nucleic Acid Product
<400> 21
acaccaccat gagcaattca agtaactcta caagcttgtc taatttttct ggaattgggg 60
tcggggttat ccttactctt gtaattctgt ttattcttat actagcactt ctgtgcctta 120
gggttgccgc ctgctgcacg cacgtttgta cctattgtca gctttttaaa cgctgggggc 180
aacatccaag ataagggaat t 201
<210> 22
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Forward Primer
<400> 22
accaccatga gcaattcaag taactc 26
<210> 23
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Reverse Primer
<400> 23
ccttatcttg gatgttgccc ccag 24
<210> 24
<211> 61
<212> PRT
<213> Adenovirus, Ad2 E3/6.7K
<400> 24
Met Ser Asn Ser Ser Asn Ser Thr Ser Leu Ser Asn Phe Ser Gly Ile
1 5 10 15
Gly Val Gly Val Ile Leu Thr Leu Val Ile Leu Phe Ile Leu Ile Leu
20 25 30
Ala Leu Leu Cys Leu Arg Val Ala Ala Cys Cys Thr His Val Cys Thr
35 40 45
Tyr Cys Gln Leu Phe Lys Arg Trp Gly Gln His Pro Arg
50 55 60
<210> 25
<211> 63
<212> PRT
<213> Adenovirus, Ad5 E3/6.7K-
<400> 25
Met Asn Asn Ser Ser Asn Ser Thr Gly Tyr Ser Asn Ser Gly Phe Ser
1 5 10 15
- 5 -
CA 02430948 2003-06-05
WO 02/46231 PCT/CA01/01769
Arg Ile Gly ~Tal Gly Val Ile Leu Cys Leu Val Ile Leu Phe Ile Leu
20 25 30
Ile Leu Thr Leu Leu Cys Leu Arg Leu Ala Ala Cys Cys Val His Ile
35 40 45
Cys Thr Tyr Cys Gln Leu Phe Lys Arg Trp Gly Arg His Pro Arg
50 55 60
- 6 -
