U.S. patent application number 12/147924 was filed with the patent office on 2009-03-19 for receptor-mediated uptake of an extracellular bcl-xl fusion protein inhibits apoptosis.
This patent application is currently assigned to The Government of the United States of America as represented by the Secretary of the Department of. Invention is credited to R. John Collier, Liu Xiuhuai, Richard J. Youle.
Application Number | 20090075882 12/147924 |
Document ID | / |
Family ID | 32302120 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090075882 |
Kind Code |
A1 |
Youle; Richard J. ; et
al. |
March 19, 2009 |
RECEPTOR-MEDIATED UPTAKE OF AN EXTRACELLULAR BCL-XL FUSION PROTEIN
INHIBITS APOPTOSIS
Abstract
Apoptosis-modifying fusion polypeptides, and the corresponding
nucleic acid molecules, are disclosed. Pharmaceutical compositions
comprising these polypeptides, and the use of these polypeptides to
modify apoptosis, are also provided.
Inventors: |
Youle; Richard J.; (Chevy
Chase, MD) ; Xiuhuai; Liu; (Rockville, MD) ;
Collier; R. John; (Wellesley, MA) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 S.W. SALMON STREET, SUITE #1600
PORTLAND
OR
97204-2988
US
|
Assignee: |
The Government of the United States
of America as represented by the Secretary of the Department
of
Health and Human Services, National Institute of Health
|
Family ID: |
32302120 |
Appl. No.: |
12/147924 |
Filed: |
June 27, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11692112 |
Mar 27, 2007 |
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12147924 |
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10792517 |
Mar 2, 2004 |
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11692112 |
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09639245 |
Aug 15, 2000 |
6737511 |
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10792517 |
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60149220 |
Aug 16, 1999 |
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Current U.S.
Class: |
514/1.1 ;
435/375 |
Current CPC
Class: |
C07K 2319/01 20130101;
A61K 39/00 20130101; Y02A 50/466 20180101; C07K 14/34 20130101;
Y02A 50/30 20180101; C07K 2319/00 20130101; C07K 14/4747 20130101;
A61P 43/00 20180101; A61K 38/00 20130101 |
Class at
Publication: |
514/12 ;
435/375 |
International
Class: |
A61K 38/00 20060101
A61K038/00; C12N 5/06 20060101 C12N005/06; A61P 43/00 20060101
A61P043/00 |
Claims
1. A method for inhibiting apoptosis in a target cell, comprising
contacting the target cell with an amount of a functional
apoptosis-inhibiting fusion protein sufficient to inhibit apoptosis
in the target cell, wherein the fusion protein comprises: a first
domain capable of inhibiting apoptosis in the target cell; and a
second domain capable of specifically targeting the fusion protein
to the target cell, and wherein the fusion protein integrates into
or otherwise crosses a cellular membrane of the target cell upon
binding.
2. The method of claim 1, wherein the protein is administered in
the form of a pharmaceutical composition.
3. The method of claim 1, wherein the target cell is a neuron.
4. The protein of claim 3, wherein the neuron is a retinal ganglion
cell.
5. The method of claim 1, wherein the first domain of the fusion
protein is a Bcl-2 family protein, or a variant or fragment thereof
that inhibits apoptosis in the target cell to which the protein is
exposed, and wherein the amount is sufficient to inhibit
apoptosis.
6. The method of claim 5, wherein the first domain of the fusion
protein is Bcl-x.sub.L, or a variant or fragment thereof that
inhibits apoptosis in the target cell to which the protein is
exposed, and wherein the amount is sufficient to inhibit
apoptosis.
7. The method of claim 1, wherein the fusion protein comprises an
amino acid sequence selected from the group consisting of: (a) the
amino acid sequence shown in SEQ ID NO: 2 (Bcl-x.sub.L-DTR); (b)
the amino acid sequence shown in SEQ ID NO: 8
(LF.sub.n-Bcl-x.sub.L); and (c) amino acid sequences that differ
from those specified in (a) or (b) by one or more conservative
amino acid substitutions, but which retain targeting and
apoptosis-inhibiting abilities.
8. The method of claim 1, wherein the target cell is in vivo in a
subject.
9. The method of claim 1, wherein the target cell is in vivo in a
subject and the fusion protein is administered to the subject after
transient ischemic neuronal injury.
10. The method of claim 9, wherein the transient ischemic neuronal
injury comprises a spinal cord injury.
11. The method of claim 9, wherein the protein is administered in
the form of a pharmaceutical composition.
12. The method of claim 9, further comprising the step of
co-administering an agent selected from the group consisting of a
chemotherapeutic agent, an anti-inflammatory agent, an anti-viral
agent, and an antibiotic agent.
13. The method of claim 1, wherein the second domain comprises a
receptor-binding domain derived from a bacterial toxin, a
monoclonal antibody, a growth factor, or a cytokine.
14. The method of claim 13, wherein the second binding domain
comprises a receptor-binding domain derived from diphtheria toxin
or anthrax toxin.
15. The method of claim 13, wherein the second binding domain
comprises a receptor-binding domain derived from epidermal growth
factor
16. The method of claim 13, wherein the receptor-binding domain
comprises diphtheria toxin receptor binding domain, or a variant or
fragment thereof that targets the fusion protein to the target cell
to which the protein is exposed.
17. The method of claim 14, wherein the second domain further
comprises a translocation domain of diphtheria toxin.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This is a divisional of co-pending U.S. patent application
Ser. No. 11/692,112, filed Mar. 27, 2007; which is a continuation
of U.S. patent application Ser. No. 10/792,517, filed Mar. 2, 2004,
now abandoned; which is a divisional of U.S. patent application
Ser. No. 09/639,245, filed Aug. 15, 2000, issued as U.S. Pat. No.
6,737,511, on May 18, 2004; which claims the benefit of U.S.
Provisional Application No. 60/149,220, filed Aug. 16, 1999. All of
these listed patent documents are incorporated by reference in
their entirety.
FIELD
[0002] This invention relates to modification of the apoptotic
response of target cells, for instance target cells in a subject.
More specifically, it relates to apoptosis-modifying fusion
proteins with at least two domains, one of which targets the fusion
protein to a target cell, and another of which modifies an
apoptotic response of the target cell.
BACKGROUND
[0003] Tissue and cell homeostasis in multicellular organisms is
largely influenced by apoptosis, the phenomenon of programmed cell
death by which an intra- or extra-cellular trigger causes a cell to
activate a biochemical "suicide" pathway. Morphological indicia of
apoptosis include membrane blebbing, chromatin condensation and
fragmentation, and formation of apoptotic bodies, all of which take
place relatively early in the process of programmed cell death.
Degradation of genomic DNA during apoptosis results in formation of
characteristic, nucleosome sized DNA fragments; this degradation
produces a diagnostic 180 bp laddering pattern when analyzed by gel
electrophoresis. A later step in the apoptotic process is
degradation of the plasma membrane, rendering apoptotic cells leaky
to various dyes (e.g., trypan blue and propidium iodide). Apoptotic
cells are usually engulfed and destroyed early in the death
process; thus, apoptosis tends not to be associated with
inflammation caused by cytoplasm leakage, as is found in
necrosis.
[0004] Various in vivo triggers can induce apoptosis; the
paradigmatic trigger is a shortage of one or more necessary growth
factors. Apoptosis plays a significant role in development of the
neural system (reviewed in Cowan et al., Science 225:1258-1265,
1984; Davies, Development 101:185-208, 1987; Oppenheim, Annu. Rev.
Neurosci. 14:453-501, 1991) and lymphoid system (reviewed in
Blackman et al., Science 248:1335-1341, 1990; Rothenberg, Adv.
Immunol. 51:85-214, 1992) of vertebrates. System development occurs
through selective apoptotic extinction of certain cell
populations.
[0005] In spite of much study, the molecular mechanisms of
apoptosis are not fully elucidated. It does appear, however, that
different apoptosis inducers may trigger different apoptotic
pathways. For instance, certain pathways are
transcription-dependent, in that apoptosis requires the synthesis
of new proteins after stimulation by, for instance, withdrawal of
growth factors. Staurosporine, a non-specific kinase inhibitor, in
contrast, stimulates a transcription-independent pathway.
Transcription dependent and independent pathways appear to share
downstream components, including the ICE family of proteases
(caspases). See Rubin, British Med. Bulle., 53:617-631, 1997, for a
review of apoptosis in neurons; More general reviews include
Ashkenazi and Dixit, Science 281:1305-1308; Thornberry and
Lazebnik, Science 281:1312-1316; and Adams and Cory, Science
281:1322-1326.
[0006] Apoptosis is recognized as a gene-directed event, controlled
by a complex set of interacting gene products that inhibit or
enhance apoptosis (Williams and Smith, Cell 74:777-779, 1993;
reviewed in White, Genes Dev. 10:1-15, 1996). Extensive effort is
currently underway to identify and characterize the genes involved
in this process. The first protein characterized as influencing
apoptosis was Bcl-2 (Cleary et al., Cell 47:19-28, 1986; Tsujimoto
and Croce, Proc. Natl. Acad. Sci. USA 83:5214-5218, 1986). Since
its discovery, several Bcl-2-related proteins (the Bcl-2 family of
proteins) have been identified as being involved in regulation of
apoptosis (White, Genes Dev. 10:1-15, 1996; Yang et al., Cell
80:285-291, 1995). One such is Bcl-x, which is expressed in two
different forms, long (Bcl-x.sub.L) and short (Bcl-x.sub.S) (Boise
et al., Cell 74:597-608, 1993).
[0007] Bcl-x.sub.L and certain other members of the Bcl-2 family
are, like Bcl-2 itself, powerful inhibitors of cell death (the
"anti-death" Bcl-2 family members). Genetic overexpression of Bcl-2
has been shown to block apoptosis in the nervous system of
transgenic mice (Chen et al., Nature 385:434-439, 1997; Henkart,
Immunity 4:195-201, 1996; Lippincott-Schwartz et al., Cell
67:601-616, 1991; Hunziker et al., Cell 67:617-627, 1991; Krajewski
et al., Cancer Research 53:4701-4714, 1993; Martinou et al., Neuron
13:1017-1030, 1994).
[0008] Other members of the Bcl-2 protein family, including
Bcl-x.sub.S, Bad and Bax, are potent enhancers of apoptosis and
therefore toxic to cells ("pro-death" Bcl-2 family members). Though
the mechanism of apoptosis induction by these proteins remains
unknown, it has been suggested that Bad binding to Bcl-x.sub.L may
promote cell death (Yang et al., Cell 80:285-291, 1995; Zha et al.,
J Biol. Chem 272:24101-24104, 1997) and that phosphorylation of Bad
may prevent its binding to Bcl-x.sub.L, thereby blocking cell death
(Zha et al., J Biol. Chem. 272:24101-24104, 1997; Zha et al., Cell
87:619-628, 1996).
[0009] In addition to its involvement in neuronal and lymphoid
system development and overall cell population homeostasis,
apoptosis also plays a substantial role in cell death that occurs
in conjunction with various disease and injury conditions. For
instance, apoptosis is involved in the damage caused by
neurodegenerative disorders, including Alzheimer's disease
(Barinaga, Science 281:1303-1304), Huntington's disease, and
spinal-muscular atrophy. There is also a substantial apoptotic
component to the neuronal damage caused during stroke episodes
(reviewed in Rubin, British Med. Bulle., 53(3):617-631, 1997; and
Barinaga, Science 281:1302-1303), and transient ischemic neuronal
injury, as in spinal cord injury. It would be of great benefit to
prevent undesired apoptosis in various disease and injury
situations.
[0010] Treatment with standard apoptosis inhibitory molecules, for
instance peptide-type caspase inhibitors (e.g., DEVD-type), though
useful for laboratory experiments where microinjection can be
employed, has proven unsatisfactory for clinical work due to low
membrane permeability of these inhibitors. Transfection of cells
with various native proteins, including members of the Bcl-2 family
of regulatory proteins, has dual disadvantages. First, transfection
is usually not cell-specific, and thus may disrupt apoptotic
processes non-specifically in all cells. Second, transfection tends
to provide long term alterations in the apoptotic process, in that
once a transgene is integrated and functional in the genome of
target cells, it may be difficult to turn off. Especially in
instances of stroke episodes or transient ischemic neuronal injury,
it would be more advantageous to be able to apply apoptosis
regulation for short periods of time. Therefore, there is still a
strong need to develop pharmaceutical agents that overcome these
disadvantages.
[0011] Cancer and other hyper-proliferative cell conditions can be
viewed as inappropriate escape from appropriate cell death. As
such, it would be advantageous to be able to enhance apoptosis in
certain of these cells to stop unregulated or undesired growth.
Various attempts have been made to selectively eliminate cancerous
cells through the use of targeted immunotoxins (genetic or
biochemical fusions between a toxic molecule, for instance a
bacterial toxin, and a targeting domain derived, typically from an
antibody molecule).
[0012] One bacterial toxin that has been employed in attempts to
kill cancerous cells is diphtheria toxin (DT). Diphtheria toxin has
three structurally and functionally distinct domains: (1) a cell
surface receptor binding domain (DTR), (2) a translocation domain
(DTT) that allows passage of the active domain across the cell
membrane, and (3) the A (enzymatically active) chain that, upon
delivery to a cell, ADP-ribosylates elongation factor 2 and thereby
inactivates translation. Altering the receptor specificity of the
diphtheria toxin has been used to generate toxins that may
selectively kill cancer cells in vitro (Thorpe et al., Nature
271:752-755, 1978) and in man (Laske et al., Nature Medicine
3:1362-1368, 1997). Promising though they might have seemed, these
and similar hybrid immunotoxins have proven to be substantially
less effective at targeted cell death than the toxins from which
they were generated. This is perhaps due to difficulties in
translocation of the fusion protein into the target cell
(Columbatti et al., J. Biol. Chem. 261:3030-3035, 1986). In
addition, in vivo results have been particularly poor using such
hybrid constructs (Fulton et al., Fed. Proc. 461:1507, 1987).
[0013] It is to biological molecules that overcome deficiencies in
the prior art that the present invention is directed.
SUMMARY OF THE DISCLOSURE
[0014] Disclosed herein are apoptosis-modifying fusion proteins
constructed by fusing a protein, or an apoptosis-modifying fragment
or variant thereof, from the Bcl-2 protein family with a
cell-binding, targeting domain such as one derived from a bacterial
toxin. Using this approach, apoptosis-modifying fusion proteins can
be delivered effectively throughout the body and targeted to select
tissues and cells. In certain embodiments, fusing various
cell-binding domains to Bcl-2 family proteins (such as Bcl-x.sub.L
or Bad) allows targeting to specific subsets of cells in vivo,
permitting treatment and/or prevention of the cell-death related
consequences of various diseases and injuries. The delivery of
other Bcl-2 homologues to the cell permits regulation of cell
viability either positively (using anti-death Bcl-2 family
members), or negatively (using pro-death members of the Bcl-2
family).
[0015] The apoptosis-modifying fusion proteins disclosed herein
have specifiable cell-targeting and apoptosis-modifying activities.
Thus, they may be used clinically to treat various disease and
injury conditions, through inhibition or enhancement of an
apoptotic cellular response. For instance, apoptosis-inhibiting
fusion proteins are beneficial to minimize or prevent apoptotic
damage that can be caused by neurodegenerative disorders (e.g.,
Alzheimer's disease, Huntington's disease, spinal-muscular
atrophy), stroke episodes, and transient ischemic neuronal injury
(e.g., spinal cord injury). The apoptosis-enhancing fusion proteins
n can be used to inhibit cell growth, for instance uncontrolled
cellular proliferation.
[0016] Accordingly, a first embodiment is a functional
apoptosis-modifying fusion protein capable of binding a target
cell, having a first domain capable of modifying apoptosis in the
target cell, and a second domain capable of specifically targeting
the fusion protein to the target cell. This fusion protein further
integrates into or otherwise crosses a cellular membrane of the
target cell upon binding to that cell.
[0017] Certain embodiments will also include a linker between these
two domains. This linker will usually be at least 5 amino acids
long, for example between 5 and 100 amino acids in length, and may
for instance include the amino acid sequence shown in SEQ ID NO: 6.
Appropriate linkers may be 6, 7, or 8 amino acids in length, and so
forth, including linkers of about 10, 20, 30, 40 or 50 amino acids
long.
[0018] The apoptosis modifying fusion proteins may also include a
third domain from one of the two original proteins, or from a third
protein. This third domain may improve the fusion protein's ability
to be integrated into or otherwise cross a cellular membrane of the
target cell. An example of such a third domain is the translocation
region (domain or sub-domain) of diphtheria toxin.
[0019] Target cells for the fusion proteins disclosed herein
include, but are not limited to, neurons, lymphocytes, stem cells,
epithelial cells, cancer cells, neoplasm cells, and others,
including other hyper-proliferative cells. The target cell chosen
will depend on what disease or injury condition the fusion protein
is intended to treat.
[0020] Receptor-binding domains may be derived from various
cell-type specific binding proteins, including for instance
bacterial toxins (e.g., diphtheria toxin or anthrax toxin), growth
factors (e.g., epidermal growth factor), monoclonal antibodies, or
single-chain antibodies derived from antibody genes. Further,
variants or fragments of such proteins may also be used, where
these fragments or variants maintain the ability to target the
fusion protein to the appropriate target cell.
[0021] Further specific embodiments employ essentially the entire
Bcl-x.sub.L protein as the apoptosis-modifying domain of the fusion
protein, or variants or fragments thereof that maintain the ability
to inhibit apoptosis in a target cell to which the protein is
exposed. Examples of such proteins are fusion proteins made of the
Bcl-x.sub.L protein, functionally linked to the diphtheria toxin
receptor binding domain through a peptide linker of about six amino
acids. One such protein is Bcl-x.sub.L-DTR, which consists of
Bcl-x.sub.L and DTR, without the translocation domain of diphtheria
toxin. The nucleotide sequence of this fusion protein is shown in
SEQ ID NO: 1, and the corresponding amino acid sequence in SEQ ID
NOs: 1 and 2.
[0022] Another such example is LF.sub.n-Bcl-x.sub.L, which includes
the amino terminal portion (residues 1-255) of mature anthrax
lethal factor (LF), coupled to residues 1-209 of Bcl-x.sub.L. The
nucleotide sequence of this fusion protein is shown in SEQ ID NO:
7, and the corresponding amino acid sequence in SEQ ID NOs: 7 and
8.
[0023] Also encompassed are fusion proteins wherein the
apoptosis-modifying domain is an apoptosis-enhancing domain. Such
domains include the various pro-death members of the Bcl-2 family
of proteins, for instance Bad, and variants or fragments thereof
that enhance apoptosis in a target cell. A specific appropriate
variant of the Bad protein has an amino acid other than serine at
amino acid position 112 and/or position 136, to provide
constitutively reduced phosphorylation.
[0024] Thus, one specific embodiment is a functional
apoptosis-enhancing fusion protein capable of binding a target
cell, comprising the Bad protein and the diphtheria toxin
translocation and receptor binding domains, functionally linked to
each other. The Bad protein of this embodiment can also contain a
mutation(s) at position 112 and/or 136 to change the serine residue
to some other amino acid, to reduce phosphorylation of the protein.
One such protein is Bad-DTTR; the nucleotide sequence of this
protein is shown in SEQ ID NO: 3, and the corresponding amino acid
sequence in SEQ ID NOs: 3 and 4.
[0025] Also disclosed herein are nucleic acid molecules encoding
apoptosis-modifying fusion proteins, for instance the nucleic acid
sequences in SEQ ID NOs: 1, 3, and 7, and nucleic acid sequences
having at least 90% sequence identity to these sequences, for
instance those encoding for proteins containing one or more
conservative amino acid substitutions. Other nucleic acid sequences
may have 95% or 98% sequence identity with SEQ ID NO: 1, 3, or 7.
Also encompassed are recombinant nucleic acid molecules in which
such a nucleic acid sequence is operably linked to a promoter,
vectors containing such a molecule, and transgenic cells comprising
such a molecule.
[0026] Methods also are provided for producing functional
recombinant apoptosis-modifying fusion proteins capable of binding
to a target cell, integrating into or otherwise translocating
across the cell membrane, and modifying an apoptotic response of
the target cell. Such a protein can be produced in a prokaryotic or
eukaryotic cell, for instance by transforming or transfecting such
a cell with a recombinant nucleic acid molecule comprising a
sequence which encodes a disclosed bispecific fusion protein.
Appropriate eukaryotic cells include yeast, algae, plant or animal
cells. Such transformed cells can then be cultured under conditions
that cause production of the fusion protein, which is then
recovered through protein purification means. The protein can
include a molecular tag, such as a six histidine (hexa-his) tag, to
facilitate its recovery.
[0027] Protein analogs, derivatives, or mimetics of the disclosed
proteins, which retain the ability to target to appropriate target
cells and modify apoptosis in those cells, are also encompassed in
embodiments.
[0028] Compositions containing these apoptosis modifying fusion
proteins, and analogs, derivatives, or mimetics of these proteins,
are further aspects of this disclosure. Such compositions may
further contain a pharmaceutically acceptable carrier, various
other medical or therapeutic agents, and/or additional apoptosis
modifying substances.
[0029] Methods for modifying apoptosis in a target cell are also
encompassed, wherein a sufficient amount of a fusion protein of the
current disclosure to modify apoptosis in the target cell is
contacted with a target cell. Modification of apoptosis can be by
either inhibition or enhancement of an apoptotic response of the
target cell. The fusion protein can be administered to the target
cell in the form of a pharmaceutical composition, and can further
be administered with various medical or therapeutic agents, and/or
additional apoptosis modifying substances. Such agents may include,
for instance, chemotherapeutic, anti-inflammatory, anti-viral, and
antibiotic agents.
[0030] Bcl-x.sub.L-DTR, LF.sub.n-Bcl-x.sub.L, or related fusion
proteins can be used to inhibit apoptosis in a target cell by
contacting the target cell with an amount of this protein
sufficient to inhibit apoptosis. Alternatively, Bad-DTTR or related
fusion proteins can be used to enhance apoptosis in a target cell
by contacting the target cell with an amount of this protein
sufficient to enhance apoptosis.
[0031] A specific aspect disclosed herein is the method of reducing
apoptosis in a subject after transient ischemic neuronal injury,
for instance a spinal cord injury, comprising administering to the
subject a therapeutically effective amount of an
apoptosis-inhibiting protein according to this disclosure. Examples
of such fusion proteins include Bcl-x.sub.L-DTR and
LF.sub.n-Bcl-x.sub.L. These proteins can be administered in the
form of a pharmaceutical composition, and can be co-administered
with various medical or therapeutic agents, and/or additional
apoptosis modifying substances.
[0032] The foregoing and other features and advantages of the
invention will become more apparent from the following detailed
description of several embodiments, which proceeds with reference
to the accompanying figures and tables.
BRIEF DESCRIPTION OF THE FIGURES
[0033] FIG. 1 shows the construction, production and bioactivity of
Bcl-x.sub.L-DTR and Bcl-x.sub.L transfected into HeLa cells. FIG.
1A is a schematic representation of construction of
Bcl-x.sub.L-DTR. FIG. 1B is a Western blot of the lysates of HeLa
cells transiently transfected with Bcl-x.sub.L (lane b) and
Bcl-x.sub.L-DTR (lane c). Lane a contains untransfected cells as a
control. A small amount of endogeneous Bcl-x.sub.L is present in
lanes a and c. FIG. 1C is a graph that shows transient transfection
of Bcl-x.sub.L (.largecircle.) and Bcl-x.sub.L-DTR (.diamond.)
genes into HeLa cells inhibits apoptotic cell death induced by the
addition of STS. Apoptosis in control cells transfected with the
vector (pcDNA3) vector is shown for comparison (.quadrature.).
[0034] FIG. 2 is a graph that shows the results of a diphtheria
toxin receptor competitive binding assay. Cold competitor proteins
[native DT (.DELTA.), Bcl-x.sub.L-DTR (.tangle-solidup.),
Bcl-x.sub.L (.largecircle.), and DTR ( )] were used to displace
I.sup.125 labeled diphtheria toxin (DT) tracer, and the amount of
bound, labeled tracer was measured. Native DT and the fusion
protein Bcl-x.sub.L-DTR compete for DT receptor binding in the
nanomolar concentration range.
[0035] FIG. 3 depicts the results of several experiments that
demonstrate the apoptosis-inhibiting character of the fusion
construct Bcl-x.sub.L-DTR. Panel A is a graph of a time course of
apoptosis induced by staurosporine (STS). Cells were treated with
0.1 .mu.M STS (.largecircle.), 0.1 .mu.M STS plus 4.8 .mu.M
Bcl-x.sub.L-DTR protein medium (.DELTA.), or 20 .mu.l of PBS
(.quadrature.). Results are presented as the average number of
apoptotic cells per field (magnification 160.times.). For each
point, at least 5 fields were counted in each of at least 3 wells.
FIG. 3B is a SDS-PAGE gel that shows that Bcl-x.sub.L-DTR prevents
PARP cleavage. Lane a contains control HeLa cells not incubated
with STS (uninduced cells); Lane b, HeLa cells treated with STS
plus 1 .mu.M Bcl-x.sub.L-DTR protein; Lane c, HeLa cells treated
with STS plus 1.48 .mu.M Bcl-x.sub.L-DTR protein; and Lane d, HeLa
cells treated with STS and no fusion protein.
[0036] FIG. 4 shows that Bcl-x.sub.L-DTR inhibits of apoptosis
induced by .gamma.-radiation, but not that induced by .alpha.-Fas
antibody. FIG. 4A is a graph showing that the addition of
Bcl-x.sub.L-DTR prior to irradiation of Jurkat cells reduces
apoptotic death in response to .gamma.-radiation. Control cells
were not irradiated and not treated with Bcl-x.sub.L-DTR. FIG. 4B
is a graph that shows that, in Jurkat cells, Bcl-x.sub.L-DTR had
little inhibitory effect on apoptosis induced by anti-Fas antibody.
Control cells were treated with PBS and no anti-Fas antibody.
[0037] FIG. 5 shows that Bcl-x.sub.L-DTR inhibits apoptosis induced
by poliovirus.
[0038] FIG. 6 is a graph showing the time course of viability of
cells treated with Bad-DTTR.
[0039] FIG. 7 shows the results of experiments that demonstrate
that Bad-DTTR combined with STS triggers massive cell death. FIG.
7A is a graph quantifying cell death after treatment of U251 MG
cells with various combinations of STS and Bad-DTTR. Apoptosis is
most enhanced when cells are treated with 0.1 .mu.M STS plus 0.65
.mu.M Bad-DTTR, and cells begin to die about 12 hours after
treatment. In the experiment depicted in FIG. 7B, the use of 1
.mu.M STS in combination with various concentrations of Bad-DTTR
cause an earlier onset of apoptosis in U251 MG cells. Key:
.quadrature.=PBS; .diamond.=0.1 .mu.M STS; .largecircle.=0.65 .mu.M
Bad-DTTR; .DELTA.=0.065 .mu.M Bad-DTTR; =0.1 .mu.M STS+0.65 .mu.M
Bad-DTTR; .crclbar.=0.1 .mu.M STS+0.065 .mu.M Bad-DTTR.
[0040] FIG. 8 is a schematic diagram of the chimera
LF.sub.n-Bcl-x.sub.L. The fusion gene, LF.sub.n-Bcl-x.sub.L, was
inserted into the vector, pET15b, yielding a histidine tag sequence
at the N terminus of the LF.sub.n-Bcl-x.sub.L gene.
[0041] FIG. 9 is a graph showing the time course of apoptosis
induced by STS in J774 cells, with or without LF.sub.n-Bcl-x.sub.L
protein. J774 cells at 3.times.10.sup.4/cm.sup.2 were treated with
0.1 .mu.M staurosporine alone, 0.1 .mu.M staurosporine along with
LF.sub.n-Bcl-x.sub.L (28 .mu.g/ml) plus PA (33 .mu.g/ml), or with
PBS alone. The apoptotic and living cells were stained with Hoechst
33342 and counted at the indicated times, and the data were
calculated as reported (Liu et al., Proc. Natl. Acad. Sci. USA 96:
9563-9567, 1999).
[0042] FIG. 10 is a bar graph showing the effect of
LF.sub.n-Bcl-x.sub.L against J774 treated with STS. J774 cells at
10.sup.4/cm.sup.2 were treated with PBS, 0.1 .mu.M staurosporine
alone, 0.1 .mu.M staurosporine along with LF.sub.n (28 .mu.g/ml),
0.1 .mu.M staurosporine along with Bcl-x.sub.L (28 .mu.g/ml), 0.1
.mu.M staurosporine along with LF.sub.n-Bcl-x.sub.L (28 .mu.g/ml),
0.1 .mu.M staurosporine along with LF.sub.n-Bcl-x.sub.L (28
.mu.g/ml) plus PA (33 .mu.g/ml), 0.1 .mu.M staurosporine along with
PA (33 .mu.g/ml) and 0.1 .mu.M staurosporine along with LF.sub.n
(28 .mu.g/ml) plus PA (33 .mu.g/ml). The apoptotic and living cells
were stained with Hoechst 33342 48 hours later and counted, and the
data were calculated as for FIG. 9.
[0043] FIG. 11 is a bar graph showing the effect of
LF.sub.n-Bcl-x.sub.L against Jurkat cells treated with STS. Jurkat
cells at 10.sup.5/ml were treated with 0.1 .mu.M staurosporine
alone, 0.1 .mu.M staurosporine along with LF.sub.n-Bcl-x.sub.L (28
.mu.g/ml) plus PA (33 .mu.g/ml) or with PBS. The apoptotic and
living cells were stained with Hoechst 33342 21 hours later and
counted, and the data were calculated as for FIG. 9.
[0044] FIG. 12 is a bar graph showing that the fusion protein
LF.sub.n-Bcl-x.sub.L prevents apoptosis by in neonatal rat retinal
ganglion cells 24 hours after optic nerve section. The apoptotic
and living cells in retinal ganglion layers were counted 24 hours
after optic nerve section immediately followed by the injection of
PBS or the indicated protein(s). The percentage of apoptotic cells
versus total retinal ganglion cells per retina is represented.
SEQUENCE LISTING
[0045] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids. Only one strand of each nucleic acid sequence is shown, but
the complementary strand is understood as included by any reference
to the displayed strand.
[0046] SEQ ID NO: 1 shows the DNA coding sequence and corresponding
amino acid sequence of Bcl-x.sub.L-DTR.
[0047] SEQ ID NO: 2 shows the amino acid sequence of
Bcl-x.sub.L-DTR.
[0048] SEQ ID NO: 3 shows the DNA coding sequence and corresponding
amino acid sequence of Bad-DTTR.
[0049] SEQ ID NO: 4 shows the amino acid sequence of Bad-DTTR.
[0050] SEQ ID NO: 5 shows the nucleotide sequence of the linker
used to link Bcl-x.sub.L to DTR in the fusion construct
Bcl-x.sub.L-DTR.
[0051] SEQ ID NO: 6 shows the amino acid sequence of the linker
used to link Bcl-x.sub.L to DTR to form Bcl-x.sub.L-DTR.
[0052] SEQ ID NO: 7 shows the DNA coding sequence and corresponding
amino acid sequence of LF.sub.n-Bcl-x.sub.L.
[0053] SEQ ID NO: 8 shows the amino acid sequence of
LF.sub.n-Bcl-x.sub.L.
DETAILED DESCRIPTION OF THE INVENTION
I. Abbreviations and Definitions
[0054] A. Abbreviations
[0055] DT: diphtheria toxin
[0056] DTR: diphtheria toxin receptor binding domain
[0057] DTT: diphtheria toxin translocation domain
[0058] DTTR: diphtheria toxin translocation and receptor binding
domains
[0059] E. coli: Escherichia coli
[0060] EF: anthrax edema factor
[0061] LF: anthrax lethal factor
[0062] LF.sub.n: first 255 residues of anthrax lethal factor
[0063] moi: multiplicity of infection
[0064] PA: anthrax protective antigen
[0065] PCR: polymerase chain reaction
[0066] RE: restriction endonuclease
[0067] SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel
electrophoresis
[0068] STS: staurosporine
[0069] TdT: terminal deoxyribonucleotidyl transferase
[0070] TUNEL: TdT-dependent dUTP-biotin nick end labeling
[0071] B. Definitions
[0072] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Lewin, Genes V published by Oxford
University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.,
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8). The nomenclature for DNA bases and the three-letter
code for amino acid residues, as set forth at 37 CFR .sctn. 1.822,
are used herein.
[0073] In order to facilitate review of the various embodiments of
the invention, the following definitions of terms are provided.
These definitions are not intended to limit such terms to a scope
narrower than would be known to a person of ordinary skill in the
field.
[0074] Animal: Living multi-cellular vertebrate organisms, a
category that includes, for example, mammals and birds. The term
mammal includes both human and non-human mammals. Similarly, the
term "subject" includes both human and veterinary subjects.
[0075] Apoptosis-modifying ability: A protein has
apoptosis-modifying ability if it is capable of modifying apoptosis
in a cell. This ability is usually measurable, either in vivo or in
vitro, using any one of myriad apoptosis assays. The art is replete
with methods for measuring apoptosis. Appropriate techniques
include dye exclusion (e.g. Hoechst dye No. 33342), assaying for
caspase activity, and TUNEL-staining. The specific ability of a
fusion protein to modify the apoptotic response of a cell to
various apoptosis-inducing stimuli can be determined by running
standard apoptosis assays in the absence of or presence of various
concentrations of the fusion proteins. The results of the assay are
then compared, and can be reported for instance by presenting the
percentage of apoptosis that occurs in the presence of the fusion
protein.
[0076] The invention also includes analogs, derivatives or mimetics
of the disclosed fusion proteins, and which have
apoptosis-modifying ability. Such molecules can be screened for
apoptosis-modifying ability by assaying a protein similar to the
disclosed fusion protein, in that it has one or more conservative
amino acid substitutions or short in-frame deletions or insertions,
or analogs, derivatives or mimetics thereof, and determining
whether the similar protein, analog, derivative or mimetic provides
modification of apoptosis in a desired target cell. The
apoptosis-modifying ability and target cell binding affinity of
these derivative compounds can be measured by any known means,
including those discussed in this application.
[0077] Apoptosis-modifying fusion protein: Proteins that have at
least two domains fused together, at least one domain comprising a
cell binding region capable of targeting the fusion protein to a
target cell (the targeting or cell-binding domain), and at least
one domain capable of modifying apoptosis in the target cell (the
apoptosis-modifying domain). The apoptosis-modifying fusion
proteins of the current invention are further characterized by
their ability to integrate into or otherwise cross a cellular
membrane of the target cell when delivered extracellularly. An
apoptosis-modifying fusion protein is considered functional if it
targets to the correct target cell, and modifies an apoptotic
response of that cell.
[0078] In general, the two domains of the disclosed fusions are
genetically fused together, in that nucleic acid molecules that
encode each protein domain are functionally linked together, for
instance directly or through the use of a linker oligonucleotide,
thereby producing a single fusion-encoding nucleic acid molecule.
The translated product of such a fusion-encoding nucleic acid
molecule is the apoptosis-modifying fusion protein.
[0079] Apoptosis-modifying fusion proteins can be labeled according
to how they influence apoptosis in the target cell. For instance,
an apoptosis-modifying fusion protein according to the current
invention that inhibits apoptosis in the target cell can be
referred to as an apoptosis-inhibiting fusion protein (e.g.,
Bcl-x.sub.L-DTR and LF.sub.n-Bcl-XL). Likewise, if the fusion
protein enhances apoptosis in the target cell, it can be referred
to as an apoptosis-enhancing fusion protein (e.g., Bad-DTTR).
Specific apoptosis-modifying fusion proteins are usually named for
the proteins from which domains are taken to form the fusion, or
from the domains actually used. For instance, "Bcl-x.sub.L-DTR"
(SEQ ID NOs: 1 and 2) consists of the entire Bcl-x.sub.L protein
fused in frame to the receptor-binding domain of diphtheria toxin
(DTR) via a short linker.
[0080] A Bcl-2 protein: A Bcl-2 protein is a protein from the Bcl-2
family of proteins and includes those proteins related to Bcl-2 by
sequence homology, which affect apoptosis. By way of example, the
family includes Bcl-2, Bcl-x (both the long and short forms), Bax,
and Bad. Additional members of the Bcl-2 family of proteins are
known (Adams and Cory, Science 281:1322-1326, 1998).
[0081] Molecules that are derived from proteins of the Bcl-2 family
include fragments of such proteins (e.g., fragments of Bcl-x.sub.L
or Bad), generated either by chemical (e.g., enzymatic) digestion
or genetic engineering means. Such fragments may comprise nearly
all of the native protein, with one or a few amino acids being
genetically or chemically removed from the amino or carboxy
terminal end of the protein, or genetically removed from an
internal region of the sequence.
[0082] Derived molecules, or derived from: The term "X-derived
molecules" or "derived from X," where X is a protein also
encompasses analogs (non-protein organic molecules), derivatives
(chemically functionalized protein molecules obtained starting with
the disclosed protein sequences) or mimetics (three-dimensionally
similar chemicals) of the native protein structure, as well as
proteins sequence variants or genetic alleles, that maintain
biological functionality. Where the derived molecule is used as the
targeting domain of an apoptosis-modifying fusion protein, the
biological functionality maintained is the ability to target to
fusion protein to the desired target cell. Likewise, where the
derived molecule is used as the apoptosis-modifying domain of the
fusion, the functionality maintained is the ability to affect
apoptosis in the target cell. Each of these functionalities can be
measured in various ways, including specific protein binding and
apoptosis assays, respectively.
[0083] Injectable composition: A pharmaceutically acceptable fluid
composition comprising at least one active ingredient, e.g., an
apoptosis-modifying fusion protein. The active ingredient is
usually dissolved or suspended in a physiologically acceptable
carrier, and the composition can additionally comprise minor
amounts of one or more non-toxic auxiliary substances, such as
emulsifying agents, preservatives, and pH buffering agents and the
like. Such injectable compositions that are useful for use with the
fusion proteins of this invention are conventional; appropriate
formulations are well known in the art.
[0084] Isolated: An "isolated" biological component (such as a
nucleic acid molecule, protein or organelle) has been substantially
separated or purified away from other biological components in the
cell of the organism in which the component naturally occurs, i.e.,
other chromosomal and extra-chromosomal DNA and RNA, proteins and
organelles. Nucleic acids and proteins that have been "isolated"
include nucleic acids and proteins purified by standard
purification methods. The term also embraces nucleic acids and
proteins prepared by recombinant expression in a host cell as well
as chemically synthesized nucleic acids.
[0085] Linker: A peptide, usually between two and 150 amino acid
residues in length, which serves to join two protein domains in a
multi-domain fusion protein. Peptide linkers are generally encoded
for by a corresponding oligonucleotide linker. This can be
genetically fused, in frame, between the nucleotides that encode
the domains of a fusion protein.
[0086] Oligonucleotide: A linear polynucleotide sequence of between
six and 300 nucleotide bases in length.
[0087] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary
to join two protein-coding regions, in the same reading frame.
[0088] Parenteral: Administered outside of the intestine, e.g., not
via the alimentary tract. Generally, parenteral formulations are
those that will be administered through any possible mode except
ingestion. This term especially refers to injections, whether
administered intravenously, intrathecally, intramuscularly,
intraperitoneally, or subcutaneously, and various surface
applications including intranasal, intradermal, and topical
application, for instance.
[0089] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful in this invention are conventional.
Martin, Remington's Pharmaceutical Sciences, published by Mack
Publishing Co., Easton, Pa., 15th Edition, 1975, describes
compositions and formulations suitable for pharmaceutical delivery
of the fusion proteins herein disclosed.
[0090] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0091] Purified: The term purified does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified fusion protein preparation is one in which the
fusion protein is more enriched than the protein is in its
generative environment, for instance within a cell or in a
biochemical reaction chamber. Preferably, a preparation of fusion
protein is purified such that the fusion protein represents at
least 50% of the total protein content of the preparation. More
purified preparations will have fusion protein that represents at
least 60%, 70%, 80% or 90% of the total protein content.
[0092] Recombinant: A recombinant nucleic acid molecule is one that
has a sequence that is not naturally occurring or has a sequence
that is made by an artificial combination of two otherwise
separated segments of sequence. This artificial combination can be
accomplished by chemical synthesis or, more commonly, by the
artificial manipulation of isolated segments of nucleic acids,
e.g., by genetic engineering techniques.
[0093] Similarly, a recombinant protein is one encoded for by a
recombinant nucleic acid molecule.
[0094] Sequence identity: The similarity between two nucleic acid
sequences, or two amino acid sequences is expressed in terms of the
similarity between the sequences, otherwise referred to as sequence
identity. Sequence identity is frequently measured in terms of
percentage identity (or similarity or homology); the higher the
percentage, the more similar the two sequences are. Homologs of the
apoptosis-modifying fusion protein will possess a relatively high
degree of sequence identity when aligned using standard methods.
For instance, encoding sequences encompassed in the current
invention include those that share about 90% sequence identity with
SEQ ID NO: 1 and NO: 3.
[0095] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981;
Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and
Lipman, PNAS. USA 85:2444, 1988; Higgins and Sharp, Gene,
73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet
et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al., Comp.
Appls Biosci. 8:155-65, 1992; and Pearson et al., Meth. Mol. Biol.
24:307-31, 1994. Altschul et al., Nature Genet. 6:119-29, 1994,
presents a detailed consideration of sequence alignment methods and
homology calculations.
[0096] The alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17,
1989) or LFASTA (Pearson and Lipman, PNAS. USA 85:2444, 1988) may
be used to perform sequence comparisons (Internet Program.COPYRGT.
1996, W. R. Pearson and the University of Virginia, "fasta20u63"
version 2.0u63, release date December 1996). ALIGN compares entire
sequences against one another, while LFASTA compares regions of
local similarity. These alignment tools and their respective
tutorials are available on the Internet at the NCSA web-site.
[0097] For comparisons of amino acid sequences of greater than
about 30 amino acids, the Blast 2 sequences function is employed
using the default BLOSUM62 matrix set to default parameters, (gap
existence cost of 11, and a per residue gap cost of 1). When
aligning short peptides (fewer than around 30 amino acids), the
alignment should be performed using the Blast 2 sequences function,
employing the PAM30 matrix set to default parameters (open gap 9,
extension gap 1 penalties). Proteins with even greater similarity
to the reference sequences will show increasing percentage
identities when assessed by this method, such as at least 90%, at
least 92%, at least 94%, at least 95%, at least 97%, at least 98%,
or at least 99% sequence identity.
[0098] An alternative indication that two nucleic acid molecules
are closely related is that the two molecules hybridize to each
other under stringent conditions. Stringent conditions are
sequence-dependent and are different under different environmental
parameters. Generally, stringent conditions are selected to be
about 5.degree. C. to 20.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Conditions for nucleic
acid hybridization and calculation of stringencies can be found in
Sambrook et al., In Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, N.Y., 1989; and Tijssen, Laboratory Techniques in
Biochemistry and Molecular Biology Part I, Ch. 2, Elsevier, New
York, 1993. Nucleic acid molecules that hybridize to the disclosed
apoptosis-modifying fusion protein sequences under stringent
conditions will typically hybridize to a probe (based on the entire
fusion protein encoding sequence, an entire domain, or other
selected portions of the encoding sequence) under wash conditions
of 0.2.times.SSC, 0.1% SDS at 65.degree. C.
[0099] Nucleic acid sequences that do not show a high degree of
identity may nevertheless encode similar amino acid sequences, due
to the degeneracy of the genetic code. It is understood that
changes in nucleic acid sequence can be made using this degeneracy
to produce multiple nucleic acid sequences that encode
substantially the same protein.
[0100] Specific binding agent: An agent that binds substantially
only to a defined target. Thus a Bcl-x.sub.L-DTR-specific binding
agent binds substantially only the Bcl-x.sub.L-DTR protein in a
specific preparation. As used herein, the term
"Bcl-x.sub.L-DTR-specific binding agent" includes Bcl-x.sub.L-DTR
antibodies and other agents that bind substantially only to a
Bcl-x.sub.L-DTR protein in that preparation.
[0101] Anti-Bcl-x.sub.L-DTR antibodies may be produced using
standard procedures described in a number of texts, including
Harlow and Lane (Using Antibodies, A Laboratory Manual, CSHL, New
York, 1999, ISBN 0-87969-544-7). The determination that a
particular agent binds substantially only to Bcl-x.sub.L-DTR
protein may readily be made by using or adapting routine
procedures. One suitable in vitro assay makes use of the Western
blotting procedure (described in many standard texts, including
Harlow and Lane, 1999). Western blotting may be used to determine
that a given protein binding agent, such as an anti-Bcl-x.sub.L-DTR
monoclonal antibody, binds substantially only to the
Bcl-x.sub.L-DTR protein.
[0102] Alternately, because the disclosed apoptosis-modifying
proteins are fusion proteins, they can be detected using antibodies
to one or the protein domains used in their construction. For
instance, fusions containing Bcl-x.sub.L can be detected using the
monoclonal antibody 2H12 (Hsu and Youle, J. Biol. Chem.
272:13829-13834, 1997; now available from Neo Markers, Union City,
Calif., clone #2H121-3) or other professionally available antibody
preparations, for instance, polyclonal anti-Bcl-x.sub.L/x.sub.S
#06-851 from Upstate Biotechnology, Lake Placid, N.Y.; polyclonal
rabbit anti-Bcl-x.sub.L #65189E from PharMingen, San Diego, Calif.;
and rabbit polyclonal (#B22630-050/B22630-150) or mouse monoclonal
(B61220-050/B61220-150) anti-Bcl-x.sub.L from Transduction
Laboratories, Lexington, Ky.). Antibodies that recognize diphtheria
toxin are, for instance, available from the Centers for Disease
Control, Atlanta, Ga.
[0103] Shorter fragments of antibodies can also serve as specific
binding agents. For instance, FAbs, Fvs, and single-chain Fvs
(SCFvs) that bind to Bcl-x.sub.L-DTR would be
Bcl-x.sub.L-DTR-specific binding agents.
[0104] Target cell binding affinity: The physical interaction
between a target cell and an apoptosis-modifying fusion protein as
disclosed in this invention can be examined by various methods.
Alternatively, the ability of fusion protein to compete for binding
to its target cell with either native targeting domain or antibody
that recognizes the targeting domain binding site on the target
cell can be measured. This allows the calculation of relative
binding affinities through standard techniques.
[0105] Therapeutically effective amount of an apoptosis-modifying
fusion protein: A quantity of apoptosis-modifying fusion protein
sufficient to achieve a desired effect in a subject being treated.
For instance, this can be the amount necessary to measurably
inhibit or enhance apoptosis in a target cell.
[0106] An effective amount of apoptosis-modifying fusion protein
may be administered in a single dose, or in several doses, for
example daily, during a course of treatment. However, the effective
amount of fusion protein will be dependent on the fusion protein
applied, the subject being treated, the severity and type of the
affliction, and the manner of administration of the fusion protein.
For example, a therapeutically effective amount of fusion protein
can vary from about 0.01 mg/kg body weight to about 1 g/kg body
weight.
[0107] The fusion proteins disclosed in the present invention have
equal application in medical and veterinary settings. Therefore,
the general term "subject being treated" is understood to include
all animals (e.g., humans, apes, dogs, cats, horses, and cows), and
particularly mammals, that are or may suffer from a chronic or
acute condition or injury that causes apoptosis, or a lack thereof,
susceptible to modification using molecules of the current
invention.
[0108] Transformed: A transformed cell is a cell into which has
been introduced a nucleic acid molecule by molecular biology
techniques. As used herein, the term transformation encompasses all
techniques by which a nucleic acid molecule might be introduced
into such a cell, including transfection with viral vectors,
transformation with plasmid vectors, and introduction of naked DNA
by electroporation, lipofection, and particle gun acceleration.
[0109] Transgenic cell: A transgenic cell is one that has been
transformed with a recombinant nucleic acid molecule.
[0110] Vector: A nucleic acid molecule as introduced into a host
cell, thereby producing a transformed host cell. A vector may
include nucleic acid sequences that permit it to replicate in a
host cell, such as an origin of replication. A vector may also
include one or more selectable marker genes and other genetic
elements known in the art.
II. Construction, Expression, and Purification of
Apoptosis-Modifying Fusion Proteins
[0111] A. Selection of Component Domains.
[0112] This invention provides generally an apoptosis-modifying
fusion protein that binds to a target cell, translocates across or
otherwise integrates into the membrane(s) of the target cell, and
modifies an apoptotic response of the target cell. As such, any
target cell in which it is desirous to modify (either inhibit or
enhance) apoptosis is an appropriate target for a bispecific fusion
protein. The choice of appropriate protein binding domain for
incorporation into the disclosed apoptosis-modifying fusion protein
will be dictated by the target cell or cell population chosen.
Examples of targeting domains include, for instance, nontoxic cell
binding domains or components of bacterial toxins (such as
diphtheria toxin or anthrax toxin), growth factors (such as
epidermal growth factor), monoclonal antibodies, cytokines, and so
forth, as well as targeting competent variants and fragments
thereof.
[0113] The choice of appropriate Bcl-2 family member-derived
apoptosis-modifying domain will depend on the manner in which the
target cell's response to apoptosis is to be modified. Where
apoptosis is to be inhibited by the resultant fusion protein,
anti-death members of the Bcl-2 protein family are appropriate
sources for apoptosis-modifying domains. One such fusion protein is
Bcl-x.sub.L-DTR, which employs the long form of Bcl-x, Bcl-x.sub.L,
as the apoptosis-modifying domain. Alternately, where enhancement
of apoptosis is desired, pro-death members of the Bcl-2 family of
proteins will be appropriate. For instance, Bad-DTTR employs the
pro-death protein Bad as its apoptosis-modifying domain.
[0114] Translocation of the apoptosis-modifying fusion protein into
the target cell is important. A translocation domain may be
included in the fusion protein as a separate, third domain. This
could be supplied from a third protein, unrelated to the
cell-binding and apoptosis-modifying domains, or be a translocation
domain of one of these proteins (e.g., the diphtheria toxin
translocation (DTT) domain used in Bad-DTTR). The DTT domain
contains several hydrophobic and amphipathic alpha helices and,
after insertion into cell membranes, creates voltage dependent ion
channels (Kagan et al., Proc Natl Acad Sci USA 78:4950-4954, 1981;
Donovan et al., Proc Natl Acad Sci USA 78:172-176, 1981).
[0115] Alternately, the translocation function can be provided
through the use of a cell-binding domain or apoptosis-modifying
domain that confers the additional functionality of membrane
translocation or integration. This is true in Bcl-x.sub.L-DTR,
wherein Bcl-x.sub.L provides both the apoptosis-modifying ability
and translocation into the cell.
[0116] B. Assembly
[0117] The construction of fusion proteins from domains of known
proteins is well known. In general, a nucleic acid molecule that
encodes the desired protein domains are joined using standard
genetic engineering techniques to create a single, operably linked
fusion oligonucleotide. Appropriate molecular biological techniques
may be found in Sambrook et al., In Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, N.Y., 1989. Specific examples of
genetically engineered multi-domain proteins, including those
joined by various linkers, can be found in the following patent
documents:
[0118] U.S. Pat. No. 5,834,209 to Korsmeyer;
[0119] U.S. Pat. No. 5,821,082 to Chinnadurai;
[0120] U.S. Pat. No. 5,696,237 to FitzGerald et al.;
[0121] U.S. Pat. No. 5,668,255 to Murphy;
[0122] U.S. Pat. No. 5,587,455 to Berger et al.;
[0123] WO 98/17682 to Korsmeyer; and
[0124] WO 98/12328 to Home et al.
[0125] It will usually be convenient to generate various control
molecules for comparison to an apoptosis-modifying fusion protein,
in order to measure the specificity of the apoptosis modification
provided by each fusion protein. Appropriate control molecules may
include one or more of the native proteins used in construction of
the fusion, or fragments or mutants thereof.
[0126] C. Expression
[0127] One skilled in the art will understand that there are myriad
ways to express a recombinant protein such that it can subsequently
be purified. In general, an expression vector carrying the nucleic
acid sequence that encodes the desired protein will be transformed
into a microorganism for expression. Such microorganisms can be
prokaryotic (bacteria) or eukaryotic (e.g., yeast). One appropriate
species of bacteria is Escherichia coli (E. coli), which has been
used extensively as a laboratory experimental expression system. A
eukaryotic expression system will be preferred where the protein of
interest requires eukaryote-specific post-translational
modifications such as glycosylation. Also, protein can be expressed
using a viral (e.g., vaccinia) based expression system.
[0128] Protein can also be expressed in animal cell tissue culture,
and such a system will be appropriate where animal-specific protein
modifications are desirable or required in the recombinant
protein.
[0129] The expression vector can include a sequence encoding a
synthesis targeting peptide, positioned in such a way as to be
fused to the coding sequence of the apoptosis-modifying fusion
protein. This allows the apoptosis-modifying fusion protein to be
targeted to specific sub-cellular or extra-cellular locations.
Various appropriate prokaryotic and eukaryotic targeting peptides,
and nucleic acid molecules encoding such, are well known to one of
ordinary skill in the art. In a prokaryotic expression system, a
signal sequence can be used to secrete the newly synthesized
protein. In a eukaryotic expression system, the targeting peptide
would specify targeting of the hybrid protein to one or more
specific sub-cellular compartments, or to be secreted from the
cell, depending on which peptide is chosen. Through the use of a
eukaryotic secretion signal sequence, the apoptosis-modifying
fusion protein can be expressed in a transgenic animal (for
instance a cow, pig, or sheep) in such a manner that the protein is
secreted into the milk of the animal.
[0130] Vectors suitable for stable transformation of culturable
cells are also well known. Typically, such vectors include a
multiple-cloning site suitable for inserting a cloned nucleic acid
molecule, such that it will be under the transcriptional control of
5' and 3' regulatory sequences. In addition, transformation vectors
include one or more selectable markers; for bacterial
transformation this is often an antibiotic resistance gene. Such
transformation vectors typically also contain a promoter regulatory
region (e.g., a regulatory region controlling inducible or
constitutive expression), a transcription initiation start site, a
ribosome binding site, an RNA processing signal, and a
transcription termination site, each functionally arranged in
relation to the multiple-cloning site. For production of large
amounts of recombinant proteins, an inducible promoter is
preferred. This permits selective production of the recombinant
protein, and allows both higher levels of production than
constitutive promoters, and enables the production of recombinant
proteins that may be toxic to the expressing cell if expressed
constitutively.
[0131] In addition to these general guidelines, protein
expression/purification kits are produced commercially. See, for
instance, the QIAexpress.TM. expression system from QIAGEN
(Chatsworth, Calif.) and various expression systems provided by
INVITROGEN (Carlsbad, Calif.). Depending on the details provided by
the manufactures, such kits can be used for production and
purification of the disclosed apoptosis-modifying fusion
proteins.
[0132] D. Purification
[0133] One skilled in the art will understand that there are myriad
ways to purify recombinant polypeptides, and such typical methods
of protein purification may be used to purify the disclosed
apoptosis-modifying fusion proteins. Such methods include, for
instance, protein chromatographic methods including ion exchange,
gel filtration, HPLC, monoclonal antibody affinity chromatography
and isolation of insoluble protein inclusion bodies after over
production. In addition, purification affinity-tags, for instance a
six-histidine sequence, may be recombinantly fused to the protein
and used to facilitate polypeptide purification. A specific
proteolytic site, for instance a thrombin-specific digestion site,
can be engineered into the protein between the tag and the fusion
itself to facilitate removal of the tag after purification.
[0134] Commercially produced protein expression/purification kits
provide tailored protocols for the purification of proteins made
using each system. See, for instance, the QIAexpress.TM. expression
system from QIAGEN (Chatsworth, Calif.) and various expression
systems provided by INVITROGEN (Carlsbad, Calif.). Where a
commercial kit is employed to produce a bispecific fusion protein,
the manufacturer's purification protocol is a preferred protocol
for purification of that protein. For instance, proteins expressed
with an amino-terminal hexa-histidine tag can be purified by
binding to nickel-nitrilotriacetic acid (Ni-NTA) metal affinity
chromatography matrix (The QIAexpressionist, QIAGEN, 1997).
[0135] Alternately, the binding specificities of the
cell-binding/targeting domain of the disclosed apoptosis-modifying
protein may be exploited to facilitate specific purification of the
proteins. A preferred method of performing such specific
purification would be column chromatography using column resin to
which the target cell surface receptor, or an appropriate epitope
or fragment or domain of the target molecule, has been
attached.
[0136] If the apoptosis-modifying fusion protein is produced in a
secreted form, e.g. secreted into the milk of a transgenic animal,
purification will be from the secreted fluid. Alternately,
purification may be unnecessary if it is appropriate to apply the
fusion protein directly to the subject in the secreted fluid (e.g.
milk).
III. Variation of a Bispecific Fusion Protein
[0137] A. Sequence Variants
[0138] The binding and apoptosis-modifying characteristics of the
apoptosis-modifying fusion proteins disclosed herein lies not in
the precise amino acid sequence, but rather in the
three-dimensional structure inherent in the amino acid sequences
encoded by the DNA sequences. It is possible to recreate the
functional characteristics of any of these proteins or protein
domains of this invention by recreating the three-dimensional
structure, without necessarily recreating the exact amino acid
sequence. This can be achieved by designing a nucleic acid sequence
that encodes for the three-dimensional structure, but which
differs, for instance by reason of the redundancy of the genetic
code. Similarly, the DNA sequence may also be varied, while still
producing a functional apoptosis-modifying fusion protein.
[0139] Variant apoptosis-modifying fusion proteins include proteins
that differ in amino acid sequence from the disclosed sequence but
that share structurally significant sequence homology with any of
the provided proteins. Variation can occur in any single domain of
the fusion protein (e.g., the binding or apoptosis-modifying
domain, or, where appropriate, the linker). Variation can also
occur in more than one of such domains in any particular variant
protein. Such variants may be produced by manipulating the
nucleotide sequence of, for instance, a Bcl-x.sub.L-encoding
sequence, using standard procedures, such as site-directed
mutagenesis or PCR. The simplest modifications involve the
substitution of one or more amino acids for amino acids having
similar biochemical properties. These so-called conservative
substitutions are likely to have minimal impact on the activity of
the resultant protein, especially when made outside of the binding
site or active site of the respective domain. The regions or
sub-domains of DTR that are essential to targeted cell binding are
known in the art (see, Choe et al., Nature 357:216-222, 1992;
Parker and Pattus, TIBS 18:391-395, 1993). Regions or sub-domains
of Bcl-2 proteins responsible for apoptosis modification are under
intense study; much of this work is reviewed in Adams and Cory,
Science 281:1322-1326.
[0140] Table 1 shows amino acids that may be substituted for an
original amino acid in a protein, and which are regarded as
conservative substitutions.
TABLE-US-00001 TABLE 1 Original Residue Conservative Substitutions
Ala ser Arg lys Asn gln; his Asp glu Cys ser Gln asn Glu asp Gly
pro His asn; gln Ile leu; val Leu ile; val Lys arg; gln; glu Met
leu; ile Phe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val
ile; leu
[0141] More substantial changes in protein structure may be
obtained by selecting amino acid substitutions that are less
conservative than those listed in Table 1. Such changes include
changing residues that differ more significantly in their effect on
maintaining polypeptide backbone structure (e.g., sheet or helical
conformation) near the substitution, charge or hydrophobicity of
the molecule at the target site, or bulk of a specific side chain.
The following substitutions are generally expected to produce the
greatest changes in protein properties: (a) a hydrophilic residue
(e.g., seryl or threonyl) is substituted for (or by) a hydrophobic
residue (e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl);
(b) a cysteine or proline is substituted for (or by) any other
residue; (c) a residue having an electropositive side chain (e.g.,
lysyl, arginyl, or histadyl) is substituted for (or by) an
electronegative residue (e.g., glutamyl or aspartyl); or (d) a
residue having a bulky side chain (e.g., phenylalanine) is
substituted for (or by) one lacking a side chain (e.g.,
glycine).
[0142] Variant binding domain, apoptosis-modifying domain, or
fusion protein-encoding sequences may be produced by standard DNA
mutagenesis techniques, for example, M13 primer mutagenesis.
Details of these techniques are provided in Sambrook et al., In
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,
1989, Ch. 15. By the use of such techniques, variants may be
created which differ in minor ways from the apoptosis-modifying
fusion protein-encoding sequences disclosed. DNA molecules and
nucleotide sequences which are derivatives of those specifically
disclosed herein and that differ from those disclosed by the
deletion, addition, or substitution of nucleotides while still
encoding a protein that binds to a target cell, translocates or
otherwise integrates into the target cell membrane(s), and thereby
modifies an apoptotic response in the target cell, are comprehended
by this invention. In their most simple form, such variants may
differ from the disclosed sequences by alteration of the coding
region to fit the codon usage bias of the particular organism into
which the molecule is to be introduced.
[0143] Alternatively, the coding region may be altered by taking
advantage of the degeneracy of the genetic code to alter the coding
sequence such that, while the nucleotide sequence is substantially
altered, it nevertheless encodes a protein having an amino acid
sequence substantially similar to the disclosed fusion sequences.
For example, the 57th amino acid residue of the Bcl-x.sub.L-DTR
protein is alanine. The nucleotide codon triplet GCC encodes this
alanine residue. Because of the degeneracy of the genetic code,
three other nucleotide codon triplets--(GCG, GCT and GCA)--also
code for alanine. Thus, the nucleotide sequence of the disclosed
Bcl-x.sub.L-DTR encoding sequence could be changed at this position
to any of these three alternative codons without affecting the
amino acid composition or characteristics of the encoded protein.
Based upon the degeneracy of the genetic code, variant DNA
molecules may be derived from the cDNA and gene sequences disclosed
herein using standard DNA mutagenesis techniques as described
above, or by synthesis of DNA sequences. Thus, this invention also
encompasses nucleic acid sequences which encode an
apoptosis-modifying fusion protein, but which vary from the
disclosed nucleic acid sequences by virtue of the degeneracy of the
genetic code. Apoptosis assays, including those discussed herein,
can be used to determine the ability of the resultant variant
protein to modify apoptosis.
[0144] B. Peptide Modifications
[0145] The present invention includes biologically active molecules
that mimic the action of the apoptosis-modifying fusion proteins of
the present invention, and specifically modify apoptosis in a
target cell. The proteins of the invention include synthetic
versions of naturally-occurring proteins described herein, as well
as analogues (non-peptide organic molecules), derivatives
(chemically functionalized protein molecules obtained starting with
the disclosed peptide sequences) and variants (homologs) of these
proteins that specifically bind to a chosen target cell and modify
apoptosis in that target cell. Each protein of the invention is
comprised of a sequence of amino acids, which may be either L-
and/or D-amino acids, naturally occurring and otherwise.
[0146] Proteins may be modified by a variety of chemical techniques
to produce derivatives having essentially the same activity as the
unmodified proteins, and optionally having other desirable
properties. For example, carboxylic acid groups of the protein,
whether carboxyl-terminal or side chain, may be provided in the
form of a salt of a pharmaceutically-acceptable cation or
esterified to form a C.sub.1-C.sub.16 ester, or converted to an
amide of formula NR.sub.1R.sub.2 wherein R.sub.1 and R.sub.2 are
each independently H or C.sub.1-C.sub.16 alkyl, or combined to form
a heterocyclic ring, such as a 5- or 6-membered ring. Amino groups
of the protein, whether amino-terminal or side chain, may be in the
form of a pharmaceutically-acceptable acid addition salt, such as
the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric
and other organic salts, or may be modified to C.sub.1-C.sub.16
alkyl or dialkyl amino or further converted to an amide.
[0147] Hydroxyl groups of the protein side chains may be converted
to C.sub.1-C.sub.16 alkoxy or to a C.sub.1-C.sub.16 ester using
well-recognized techniques. Phenyl and phenolic rings of the
protein side chains may be substituted with one or more halogen
atoms, such as fluorine, chlorine, bromine or iodine, or with
C.sub.1-C.sub.16 alkyl, C.sub.1-C.sub.16 alkoxy, carboxylic acids
and esters thereof, or amides of such carboxylic acids. Methylene
groups of the protein side chains can be extended to homologous
C.sub.2-C.sub.4 alkylenes. Thiols can be protected with any one of
a number of well-recognized protecting groups, such as acetamide
groups. Those skilled in the art will also recognize methods for
introducing cyclic structures into the proteins of this invention
to select and provide conformational constraints to the structure
that result in enhanced stability.
[0148] Peptidomimetic and organomimetic embodiments are also within
the scope of the present invention, whereby the three-dimensional
arrangement of the chemical constituents of such peptido- and
organomimetics mimic the three-dimensional arrangement of the
protein backbone and component amino acid side chains in the
apoptosis-modifying fusion protein, resulting in such peptido- and
organomimetics of the proteins of this invention having measurable
or enhanced neutralizing ability. For computer modeling
applications, a pharmacophore is an idealized, three-dimensional
definition of the structural requirements for biological activity.
Peptido- and organomimetics can be designed to fit each
pharmacophore with current computer modeling software (using
computer assisted drug design or CADD). See Walters,
Computer-Assisted Modeling of Drugs, in Klegerman & Groves
(eds.), Pharmaceutical Biotechnology, Interpharm Press: Buffalo
Grove, Ill., 165-174, 1993; and Munson (ed.) Principles of
Pharmacology, Ch. 102, 1995, for descriptions of techniques used in
CADD. Also included within the scope of the invention are mimetics
prepared using such techniques that produce apoptosis-modifying
fusion proteins.
IV. Activity of Fusion Proteins
[0149] Because the apoptosis modifying fusion proteins provided in
this invention are at least bi-functional, having one domain
required for cell targeting and another for modification of
apoptosis in the target cell, there are at least two activities for
each fusion protein. These include the affinity of the fusion
protein for a specific target cell, class of target cells, tissue
type, etc., (the binding ability), and the ability of the targeted
fusion to effect apoptosis in the targeted cell (the
apoptosis-modifying ability). Various techniques can be used to
measure each of these activities.
[0150] A. Fusion Protein Affinity for Target Cells Fusion protein
affinity for the target cell, or to a specific cell surface
protein, can be determined using various techniques known in the
art. One common method is a competitive binding assay (Greenfield
et al., Science 238:536-539, 1987). In a competitive binding assay,
radiolabeled receptor binding protein, or a derivative or fragment
thereof, is exposed to the target native cell in the presence of
one or varying concentrations of cold fusion protein and other
competitive proteins being assayed. The amount of bound, labeled
binding protein can be measured through standard techniques to
determine the relative cell-binding affinity of the fusion.
[0151] B. Apoptosis Inhibition or Enhancement
[0152] Several in vitro systems are used to study the process of
apoptosis. These include growth factor deprivation in culture,
treatment of cells with staurosporine (a non-specific protein
kinase inhibitor), application of .gamma.-radiation, and infection
by viruses. Apoptosis as stimulated by any signal can be examined
or measured in a variety of ways. Detection of morphological
indicia of apoptosis (e.g., membrane blebbing, chromatin
condensation and fragmentation, and formation of apoptotic bodies)
can provide qualitative information. More quantitative techniques
include TUNEL staining, measurement of DNA laddering, measurement
of known caspase substrate degradation (e.g., PARP; Taylor et al.,
J. Neurochem. 68:1598-605, 1997) and counting dying cells, which
have become susceptible to dye uptake. Many companies (e.g.,
Trevigen, Gaithersburg, Md.; and R&D Systems, Minneapolis,
Minn.) also supply kits useful for the measurement of apoptosis by
various methods; many of these kits can be used to measure the
effect of disclosed apoptosis-modifying fusion proteins on
apoptosis in a variety of cell types.
[0153] By way of example, the following techniques can be used to
measure the modification of apoptosis caused in a target cell after
it is contacted with an apoptosis-modifying fusion protein of the
present invention.
[0154] TUNEL staining: Terminal end-labeling of broken DNA
fragments with labeled nucleotides; the reaction is catalyzed by
terminal nucleotide transferase (TdT). Various kits are available
for measurement of TUNEL staining, including the TdT in situ
TUNEL-based Kit (R&D Systems, Minneapolis, Minn.).
[0155] Measurement of Caspase Activity: Another common system for
measuring the amount of apoptosis occurring in an in vitro cell
system is to measure the poly-ADP ribose Polymerase (PARP) cleavage
after treatment of the cells with various stimulators of apoptosis.
PARP is a known substrate for a caspase (CPP-32) involved in the
apoptotic kinase cascade. This technique can be carried out using
essentially the following protocol. HeLa cells are plated in growth
media (e.g., EMEM containing 10% FBS at 2.times.10.sup.5 cells/ml)
and treated with one or more concentrations of an
apoptosis-modifying fusion protein according to the current
invention. The appropriate concentration for each fusion protein
will depend on various factors, including the fusion protein in
question, target cell, and apoptosis stimulator employed.
Appropriate concentrations may include, for instance, about 0.5
.mu.M to about 3 .mu.M final. It may be beneficial to treat the
target cells multiple times with the fusion protein, usually after
a period of incubation ranging from one to several hours. For
instance, cells can be exposed to the fusion protein a second time
about fifteen hours after the original treatment. Usually the same
concentration(s) of fusion protein is used in the second
treatment.
[0156] Apoptosis is induced immediately after the last treatment of
the target cells with apoptosis modifying fusion protein. The
method of application of the apoptosis stimulus, amount applied,
appropriate incubation time with the inducer, etc., will be
specific to the type of apoptosis induction used (e.g.,
staurosporine, .gamma.-radiation, virions, caspase inhibitor,
etc.). Such details are in general well known to those of ordinary
skill in the art. After an appropriate incubation period, cell
lysates are prepared from the treated target cells, and aliquots
loaded onto SDS-PAGE for analysis. The resultant gels can be
examined using any of various well-known techniques, for instance
by performing a Western analysis immunoblotted with anti-PARP
polyclonal antibody (Boehringer Mannheim GmbH, Germany), developed
with enhanced chemiluminescence.
[0157] Known inhibitors of apoptotic pathways, for instance caspase
inhibitors, can be used to compare the effectiveness of
apoptosis-modifying fusion proteins of this invention. Appropriate
inhibitors include viral caspase inhibitors like crmA and
baculovirus p35, and peptide-type caspase inhibitors including
zVAD-fmk, YVAD- and DEVD-type inhibitors. See Rubin, British Med.
Bulle., 53:617-631, 1997.
V. Incorporation of Apoptosis-Modifying Fusion Proteins into
Pharmaceutical Compositions
[0158] Pharmaceutical compositions that comprise at least one
apoptosis modifying fusion protein as described herein as an active
ingredient will normally be formulated with an appropriate solid or
liquid carrier, depending upon the particular mode of
administration chosen. The pharmaceutically acceptable carriers and
excipients useful in this invention are conventional. For instance,
parenteral formulations usually comprise injectable fluids that are
pharmaceutically and physiologically acceptable fluid vehicles such
as water, physiological saline, other balanced salt solutions,
aqueous dextrose, glycerol or the like. Excipients that can be
included are, for instance, other proteins, such as human serum
albumin or plasma preparations. If desired, the pharmaceutical
composition to be administered may also contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0159] One or more other medicinal and pharmaceutical agents, for
instance chemotherapeutic, anti-inflammatory, anti-viral or
antibiotic agents, also may be included.
[0160] The dosage form of the pharmaceutical composition will be
determined by the mode of administration chosen. For instance, in
addition to injectable fluids, topical and oral formulations can be
employed. Topical preparations can include eye drops, ointments,
sprays and the like. Oral formulations may be liquid (e.g., syrups,
solutions or suspensions), or solid (e.g., powders, pills, tablets,
or capsules). For solid compositions, conventional non-toxic solid
carriers can include pharmaceutical grades of mannitol, lactose,
starch, or magnesium stearate. Actual methods of preparing such
dosage forms are known, or will be apparent, to those skilled in
the art.
[0161] The pharmaceutical compositions that comprise apoptosis
modifying fusion protein will preferably be formulated in unit
dosage form, suitable for individual administration of precise
dosages. One possible unit dosage contains approximately 100 .mu.g
of protein. The amount of active compound administered will be
dependent on the subject being treated, the severity of the
affliction, and the manner of administration, and is best left to
the judgment of the prescribing clinician. Within these bounds, the
formulation to be administered will contain a quantity of the
active component(s) in an amount effective to achieve the desired
effect in the subject being treated. Ideally, a sufficient amount
of the protein is administered to achieve tissue a concentration at
the site of action that is at least as great as in vitro
concentrations that have been shown to be effective.
VI. Clinical Use of Apoptosis-Modifying Fusion Proteins
[0162] The targeted apoptosis-regulating activity exhibited by the
disclosed fusion proteins makes these fusions useful for treating
neurodegenerative diseases, transient ischemic injuries, and
unregulated cell growth (as may for instance be found in tumors and
various cancers).
[0163] The apoptosis-modifying fusion proteins of this invention
may be administered to humans, or other animals on whose cells they
are effective, in various manners such as topically, orally,
intravenously, intramuscularly, intraperitoneally, intranasally,
intradermally, intrathecally, and subcutaneously. Administration of
apoptosis-modifying fusion protein composition is indicated for
patients with a neurodegenerative disease, suffering from stroke
episodes or transient ischemic injury, or experiencing uncontrolled
or unwanted cell growth, such as malignancies or neoplasms. More
generally, treatment is appropriate for any condition in which it
would be beneficial to alter (either inhibit or enhance) an
apoptotic response of a subject's target cells. The particular mode
of administration and the dosage regimen will be selected by the
attending clinician, taking into account the particulars of the
case (e.g., the patient, the disease, and the disease-state
involved). By way of example, when apoptosis is being generally
inhibited over the short term, for instance after transient
ischemic neuronal injury, it may be advantageous to administer
relatively large doses of fusion protein repeatedly for a few days.
In contrast, if apoptosis is being enhanced in specific cell types,
for instance in hyper-proliferative cells, it may be of greater
benefit to apply a relatively small dose of fusion protein
repeatedly, e.g., daily, weekly, or monthly, over a much longer
period of treatment.
[0164] In addition to their individual use, apoptosis-modifying
fusion proteins as disclosed in the current invention may be
combined with various therapeutic agents. For instance, an
apoptosis-enhancing fusion protein such as Bad-DTTR may be combined
with or used in association with other chemotherapeutic or
chemopreventive agents for providing therapy against neoplasms or
other hyper-proliferative cellular growth conditions. Various such
anti-cancer agents are well known to those of ordinary skill in the
art. Apoptosis-modifying fusion proteins according to this
invention also can be supplied in the form of kits; the
construction of kits appropriate for therapeutically active
proteins known.
EXAMPLE 1
Construction of Functional Apoptosis-Modifying Fusion Proteins
A. Bcl-x.sub.L-DTR
[0165] The human Bcl-x.sub.L gene from codon 1 through 233
(provided by Dr. Craig Thompson) and the diphtheria toxin gene from
codon 384 through 535 (receptor binding domain, DTR), containing
mutations in codons 508 and 525, were amplified by PCR so that the
DT mutation at codon 525 was mutated to the wild-type by the PCR
primer. The two PCR products, Bcl-x.sub.L1-233 and DT384-535 (DTR),
were digested with NdeI/NotI and NotI/XhoI restriction enzymes,
respectively. Bcl-x.sub.L was fused to the 5' end of the DTR gene
with a linker (GCG TAT TCT GCG GCC GCG, SEQ ID NO: 5) to encode for
Ala Tyr Ser Ala Ala Ala (SEQ ID NO: 6) between the two peptide
domains. The two digested fragments were ligated into the
prokaryotic expression vector pET16b (Novagen, Inc., Madison, Wis.)
cut with NdeI and XhoI (FIG. 1A). The codon 508 of DTR was mutated
to the wild-type form (Phe.fwdarw.Ser) and the first three
nucleotides (CAT) of NdeI were deleted by double-stranded,
site-directed mutagenesis. FIG. 1A shows a schematic representation
of the resultant apoptosis-modifying fusion protein,
Bcl-x.sub.L-DTR.
[0166] As controls, human Bcl-x.sub.L (codons 1-233) and DTR
(codons 384-535 of DT) genes were separately subcloned into pET16b
vectors through NdeI and XhoI sites. The histidine tag and Factor
Xa digestion site sequences from the expression vector were
upstream of Bcl-x.sub.L, DTR and Bcl-x.sub.L-DTR coding sequences.
All three expression constructs were verified by sequencing.
[0167] For expression in eukaryotic cells, Bcl-x.sub.L-DTR and
Bcl-x.sub.L gene constructs were inserted in the eukaryotic vector
pcDNA3 (Invitrogen, Carlsbad, Calif.) and the constructs verified
by sequencing.
B. Bad-DTTR
[0168] The full-length mouse Bad gene with two Ser.fwdarw.Ala
mutations at codons 112 and 136 (Schendel et al., Proc. Natl. Acad.
Sci. USA 94:5113-5118, 1997), and the diphtheria toxin gene from
codons 194 through 535 (translocation and receptor-binding domains,
DTTR, without the catalytic domain) were amplified by PCR. The two
PCR products, Bad and DT194-535 (DTTR), were used as templates to
directly fuse the Bad gene to the 5' end of DTTR gene by a second
round of PCR. The Bad-DTTR gene fragment was digested with NdeI and
XhoI and ligated into the prokaryotic expression vector pET16b
(Novagen, Inc., Madison, Wis.) digested with NdeI and XhoI. The
histidine tag and Factor Xa digestion site sequences from the
expression vector were upstream of the Bad-DTTR coding sequence.
The expression construct was verified by sequencing.
EXAMPLE 2
Expression and Purification of Functional Apoptosis-Modifying
Fusion Proteins
A. Prokaryotic Expression
[0169] To produce proteins for extracellular addition to cells, the
Bcl-x.sub.L gene, the DTR domain gene and the Bcl-x.sub.L-DTR
fusion gene were cloned into pET16b. E. coli BL21(DE3) strain was
used to express Bcl-x.sub.L-DTR, Bad-DTTR, Bcl-x.sub.L and DTR,
with addition of 1 mM IPTG when the OD260 reached 0.5-0.7. After
two hours incubation and lysis by French press the inclusion bodies
were collected and dissolved in 6M guanidine-HCl.
B. Eukaryotic Expression
[0170] Transfection of HeLa cells with the fusion constructs was
performed as reported previously (Wolter et al., J Cell Biol
139:1281-1292, 1997). HeLa cells were harvested and lysed in 1 ml
buffer containing 100 .mu.g/ml leupeptin 20 hours after
transfection, centrifuged to remove cell debris, and 15 .mu.l
aliquots of the supernatant loaded onto 10-20% SDS-PAGE. The
plasmid encoded proteins were visualized by immunoblotting with
anti-Bcl-x.sub.L monoclonal antibody (2H12, Trevigen, Gaithersburg,
Md.) and developed using enhanced chemiluminescence (Amersham Inc.,
Arlington Heights, Ill.). Results are shown in FIG. 1B.
C. Purification
[0171] Histidine tag binding resin (Novagen, Inc., Madison, Wis.)
was used to purify Bcl-x.sub.L-DTR, Bad-DTTR, Bcl-x.sub.L, and DTR.
Proteins were refolded by dialysis against, or dilution into, 100
mM Tris-Acetate (pH 8.0)/0.5 M arginine, concentrated with
PEG15,000-20,000 and dialyzed against PBS. This yielded protein
purified to greater than 90% homogeneity. The four proteins were
subjected to 10-20% SDS-PAGE, visualized by immunoblotting with
either anti-Bcl-x.sub.L monoclonal (2H12) or horse anti-DT
polyclonal antibodies (Centers for Disease Control, Atlanta, Ga.)
and developed as above. They were of the expected molecular weight
on SDS PAGE and of the expected immunoreactivity to antibodies
against Bcl-x.sub.L or DT on Western blots.
EXAMPLE 3
Assays for Measuring Fusion Protein Binding to, and Translocation
into, Target Cells
A. Competitive Binding Assay
[0172] Protein binding to the diphtheria toxin receptor was
performed as previously reported (Greenfield et al., Science
238:536-539, 1987) with the following modifications. DT was
radiolabeled with I.sup.125 using iodobeads (Pierce Chem. Co.,
Rockford, Ill.) as described by the manufacturer. Cos-7 cells,
grown to confluency in 12 well costar plates, were analyzed for
receptor binding and competition by incubation for three hours on
ice. Results are reported in FIG. 2. Cold competitor proteins,
native DT (.DELTA.), Bcl-x.sub.L-DTR (.tangle-solidup.),
Bcl-x.sub.L (.largecircle.), and DTR ( ), were used to displace
I.sup.125 labeled DT tracer.
[0173] Native DT and Bcl-x.sub.L-DTR compete for DT receptor
binding in the nanomolar concentration range. DT and the
Bcl-x.sub.L-DTR fusion protein competed for I.sup.125-DT binding to
its receptor to a similar extent although the affinity of the
fusion was three times lower than that of native DT (FIG. 2).
Neither the Bcl-x.sub.L domain alone nor the DTR domain alone was
able to compete for DT receptor binding. The more complete protein
(Bcl-x.sub.L-DTR), where Bcl-x.sub.L is substituted for the DT
translocation domain, folded such that DT receptor binding activity
was retained whereas the isolated binding domain (DTR) did not.
Addition of the DT A chain domain to the N-terminus of
Bcl-x.sub.L-DTR further increased the affinity of the chimera to
the DT receptor.
B. Assays for Effective Transport of the Fusion Protein into the
Target Cell
[0174] Diphtheria toxin is endocytosed by cells and reaches low pH
intracellular compartments. The low pH triggers a conformational
change in the translocation domain, which allows this domain to
insert into membranes and form channels. The toxicity of DT is
blocked by lysosomotropic agents such as chloroquine, which
increase the pH of intracellular compartments. Chloroquine at a
concentration that blocks diphtheria toxin toxicity (10 .mu.M) did
not block the activity of Bcl-x.sub.L-DTR to inhibit
poliovirus-induced cell death. Thus, the mechanism of membrane
interaction of Bcl-x.sub.L-DTR differs to some extent from that of
DT. However, brefeldin A, an inhibitor of vesicle traffic between
the ER and the Golgi apparatus (Lippincott-Schwartz et al., Cell
67:601-616, 1991; Hunziker et al., Cell 67:617-627, 1991), does
block the anti-apoptosis activity of Bcl-x.sub.L-DTR (Table 3).
These results indicate that Bcl-x.sub.L-DTR must be endocytosed and
suggest that Bcl-x.sub.L-DTR must reach the Golgi apparatus or the
ER to prevent cell death. The subcellular location from which
native Bcl-2 family members regulate apoptosis is currently under
scrutiny (Hunziker et al., Cell 67:617-627, 1991). Several
intracellular membrane locations, including the ER, appear able to
mediate Bcl-2 family regulation of cell death (Krajewski et al.,
Cancer Res. 53:4701-4714, 1993). Bcl-x.sub.L-DTR may reach the ER
to translocate into the cell cytosol or perhaps Bcl-x.sub.L-DTR,
when bound closely to a membrane, can insert into that membrane and
inhibit apoptosis in the membrane-intercalated form.
EXAMPLE 4
Measurement of Bcl-x.sub.L-DTR Apoptosis-Inhibiting Activity
[0175] A. Apoptosis Inhibition after Transient Cell
Transfection
[0176] To demonstrate that Bcl-x.sub.L-DTR is effective at
inhibiting apoptosis when expressed from within the target cell,
this construct and the control construct containing Bcl-x.sub.L
were transiently transfected into HeLa cells. Assay of apoptosis
inhibition after transient transfection was performed as reported
previously (Wolter et al., J. Cell Biol. 139:1281-1292, 1997). The
Bcl-x.sub.L-DTR fusion gene blocked apoptosis after transient
transfection into HeLa cells (FIG. 1C) to an extent similar to that
of the Bcl-x.sub.L gene after C-terminal tail truncation (Wolter et
al., J Cell Biol 139:1281-1292, 1997).
B. Inhibition of STS-Induced Apoptosis by Extracellular Treatment
with Bcl-x.sub.L-DTR
[0177] Hoechst dye no. 33342 staining: The effectiveness of
extracellular delivery of Bcl-x.sub.L or the Bcl-x.sub.L-DTR fusion
protein for inhibiting the rate of cell death by apoptosis was
examined as follows. Cos-7 cells at 3.times.10.sup.4 cells/cm.sup.2
in 100 .mu.l DMEM with 10% FBS were incubated with 0.1 .mu.M STS
(.largecircle.), 0.1 .mu.M STS plus 4.8 .mu.M Bcl-x.sub.L-DTR
protein added to the medium (.DELTA.) or 20 .mu.l of PBS
(.largecircle.). Apoptotic cells were quantified by staining with
Hoechst dye no. 33342. Results in FIG. 3A are presented as the
average number of cells per field (magnification 160.times.). For
each point, at least 5 fields were counted in each of at least 3
wells. Bcl-x.sub.L-DTR dramatically decreased the rate of apoptosis
in Cos-7 cells. Six different preparations of Bcl-x.sub.L-DTR were
found to have activity and the apoptosis prevention activity was
stable for at least 5 months when Bcl-x.sub.L-DTR was stored at
4.degree. C. Addition of Bcl-x.sub.L-DTR minutes before the
addition of STS blocked more than 70% of Cos-7 cell death after 6
hours and more than 50% of cell death after 12 hours of STS
exposure (FIG. 3A).
[0178] Jurkat, HeLa and U251 cells were also protected from
STS-induced apoptosis by Bcl-x.sub.L-DTR (Table 2). Bcl-x.sub.L
protein added to Cos-7 cells, however, did not alter the extent of
cell death induced by STS. A nontoxic DT mutant able to bind the DT
receptor, CRM197, also had no effect on apoptosis induced by STS.
To further test the role of DT receptor binding in apoptosis
inhibition, cells expressing DT receptors were compared with cells
lacking DT receptors. Mouse and rat cells are thousands of times
less sensitive to DT than human or monkey cell lines due to a lack
of the DT receptor (Pappenheimer The Harvey Lectures 76:45-73,
1982). Comparing human, monkey, mouse and rat cell lines revealed
that those cells lacking the DT receptor, WEHI-7.1 and 9L, were
insensitive to apoptosis protection by Bcl-x.sub.L-DTR (Table 2).
The sensitivity of the six cell lines to DT toxicity, thought to
reflect DT receptor levels, correlated with sensitivity to
apoptosis prevention by Bcl-x.sub.L-DTR (Table 2).
[0179] The magnitude of apoptosis inhibition by extracellular
Bcl-x.sub.L-DTR (FIG. 3A, Table 2) was similar to that found by
transfection of the fusion gene into cells (FIG. 1C). Although
fusion to the C-terminus of Bcl-x.sub.L inhibited bioactivity
relative to native Bcl-x.sub.L after transfection (FIG. 1C), a very
substantial prevention of cell death was obtained at both the gene
level and the protein level (FIG. 3A). Thus the delivery of
Bcl-x.sub.L-DTR is efficient and apoptosis can be prevented by
delivery of Bcl-x.sub.L from the outside of cells.
[0180] Measurement of caspase activity: To confirm the results of
cell death measurements by Hoechst staining and trypan blue dye
exclusion, we examined caspase-induced cleavage of poly-ADP ribose
polymerase (PARP). HeLa cells were plated in EMEM containing 10%
FBS at 2.times.10.sup.5 cells/ml and treated with two different
preparations of Bcl-x.sub.L-DTR at 1.48 .mu.M or 1 .mu.M. Fifteen
hours later, cells were treated again with Bcl-x.sub.L-DTR at 1.48
.mu.M or 1 .mu.M. Immediately after the second treatment, 0.8 .mu.M
STS was added. Three hours later, cell lysates were made and
aliquots were loaded onto SDS-PAGE, immunoblotted with anti-PARP
polyclonal antibody (Boehringer Mannheim GmbH, Germany) and
developed with enhanced chemiluminescence. Lane a contains control
HeLa cells not incubated with STS (uninduced cells); Lane b, HeLa
cells treated with STS plus 1 .mu.M Bcl-x.sub.L-DTR protein; Lane
c, HeLa cells treated with STS plus 1.48 .mu.M Bcl-x.sub.L-DTR
protein; and Lane d, HeLa cells treated with STS and no fusion
protein. HeLa cells incubated with Bcl-x.sub.L-DTR showed
significantly less cleavage of PARP after apoptosis induction with
STS (FIG. 3B).
C. Inhibition of .gamma.-Radiation-Induced Apoptosis by
Extracellular Treatment with Bcl-x.sub.L-DTR
[0181] Radiation is a potent inducer of apoptosis in many
hematopoetic cell types. The ability of Bcl-x.sub.L-DTR to prevent
radiation-induced apoptosis was examined in the human T cell line,
Jurkat. When added to the media (serum-free RPMI-1640 medium with
insulin and transferrin) of Jurkat cells plated at 10.sup.5
cells/ml a few minutes prior to induction of apoptosis by 10 gray
.gamma.-radiation, Bcl-x.sub.L-DTR (4.63 .mu.M) blocked almost half
of the ensuing cell death (FIG. 4A). Apoptotic cells were counted
using Hoechst dye no. 33342. Control cells were not irradiated and
not treated with Bcl-x.sub.L-DTR.
[0182] In a clonogenic assay measuring long term survival, Jurkat
cells showed more than a 3-fold greater survival when
Bcl-x.sub.L-DTR was added to the media immediately prior to 5 gray
.gamma.-radiation.
D. Inhibition of Anti-Fas-Induced Apoptosis by Extracellular
Treatment with Bcl-x.sub.L-DTR
[0183] Jurkat cells are also sensitive to apoptosis induced by
antibody binding to the Fas/APO-1/CD95 receptor. The Fas pathway of
apoptosis is one of the few pathways shown to be less sensitive or
insensitive to apoptosis protection by Bcl-2 and Bcl-x.sub.L (Boise
& Thompson, Proc. Natl. Acad. Sci. USA, 94:3759-3764, 1997;
Memon et al., J. Immunol. 155:4644-4652, 1995) and contrasts with
radiation-induced apoptosis in this regard. Jurkat cells were
plated at 10.sup.5 cells/ml in serum-free RPMI-1640 medium with
insulin and transferrin, and treated with 100 ng/ml anti-Fas
antibody (CH11, Upstate Biotechnology, Lake Placid, N.Y.) minutes
after addition of Bcl-x.sub.L-DTR to a concentration 4.68 .mu.M.
Control cells were treated with PBS and no anti-Fas antibody. Fas
antigen-induced apoptosis (measured by counting dying cells using
Hoechst dye no. 33342) showed very little inhibition by
Bcl-x.sub.L-DTR, although there was a statistically significant
decrease in apoptosis between 2 and 4 hours in some experiments
(FIG. 4B). The degree of protection of different apoptosis pathways
by extracellular Bcl-x.sub.L-DTR corresponded with that seen by
transfection with the Bcl-x.sub.L gene.
E. Inhibition of Poliovirus-Induced Apoptosis by Extracellular
Treatment with Bcl-x.sub.L-DTR
[0184] Viruses induce a powerful apoptosis response in certain
cells and prevention of this apoptosis may have therapeutic utility
(Hardwick, Adv. Pharm. 41:295-336, 1997). Poliovirus-induced
apoptosis of HeLa cells was also examined for sensitivity to
extracellular Bcl-x.sub.L-DTR, a system where inhibition of cell
death by transfection with the Bcl-x.sub.L gene has been
demonstrated (Castelli et al., J Exp. Med. 186:967-972, 1997).
Adding Bcl-x.sub.L-DTR 30 minutes after infection of cells with low
titers (MOI of 1 pfu/cell) of poliovirus (FIG. 5) or with
moderately high titers (MOI of 20 pfu/cell) of poliovirus prevented
more than half of the cell death for up to 24 hours. Addition of
extracellular Bcl-x.sub.L or the DTR domain proteins alone had no
affect on poliovirus-induced apoptosis.
F. Competition of Apoptosis Inhibition
[0185] Caspase inhibitors block many pathways of apoptosis and are
being explored for pharmacologic potential to inhibit cell death
(Chen et al., Nature 385:434-439, 1997). zVAD-fmk and Boc-D-fmk are
powerful, broad specificity caspase inhibitors that block many
apoptosis pathways (Henkart, Immunity 4:195-201, 1996). Apoptosis
inhibition activity of zVAD-fmk and Boc-D-fmk was compared with
that of Bcl-x.sub.L-DTR. HeLa cells were plated at a density of
1.times.10.sup.5 cells/well in EMEM containing 10% FBS and
antibiotics, infected with poliovirus at an MOI of 1 pfu/cell as
reported previously (Castelli et al., J Exp Med 186:967-972, 1997)
and immediately treated with negative control peptide zFA-fmk at 20
.mu.M, Bcl-x.sub.L-DTR at 0.48 .mu.M, or peptides zVAD-fmk or
Boc-D-fmk at 20 .mu.M. Cell viability was assessed by trypan blue
dye exclusion 24 hours following addition of virus. zFA-fmk,
zVAD-fmk and Boc-D-fmk were from Enzyme Systems Products, Dublin,
Calif.
[0186] Bcl-x.sub.L-DTR at 0.48 .mu.M blocked cell death to a
greater extent than either zVAD-fmk or Boc-D-fmk at 20 .mu.M (FIG.
5). Bcl-x.sub.L-DTR showed a strong inhibition of a potent and
pathologically important apoptosis pathway. Interestingly,
Bcl-x.sub.L appears to act at an early step in the cell death
pathway when intervention can permit long term viability of cells,
whereas caspase inhibitors appear to work relatively more
downstream in the apoptosis pathway (Chinnaiyan et al., J Biol Chem
271:4573-4576, 1996; Xiang et al., Proc. Natl. Acad. Sci. USA
93:14559-14563, 1996; Miller et al., J. Cell Biol 139:205-217,
1997).
EXAMPLE 5
Measurement of Bad-DTTR Apoptosis-Enhancing Activity
[0187] A. Stimulation of Apoptosis by Extracellular Treatment with
Bad-DTTR
[0188] To determine the effectiveness of the fusion protein
Bad-DTTR at triggering apoptosis, cell survival after exposure to
Bad-DTTR was examined. U251 MG cells at 3.times.10.sup.4
cells/cm.sup.2 in 100 .mu.l DMEM with 10% FBS were incubated with
0.65 .mu.M Bad-DTTR protein added to the medium or 20 .mu.l of PBS.
Total and apoptotic cells were quantified by staining with Hoechst
dye no. 33342. Results are presented in FIG. 6 as the average
number of cells per field (magnification 160.times.). Bad-DTTR
decreases cell viability 12 hours after treatment.
B. Enhancement of STS-Triggered Apoptosis by Extracellular
Treatment with Bad-DTTR
[0189] To examine the ability of Bad-DTTR to enhance apoptosis
triggered by STS, cell survival was determined after exposure to
various concentrations of STS, in combination with various
combinations of Bad-DTTR. U251 MG cells at 3.times.10.sup.4
cells/cm.sup.2 in 100 .mu.l DMEM with 10% FBS were treated with
PBS, 0.1 .mu.M STS, 0.65 .mu.M Bad-DTTR, 0.065 .mu.M Bad-DTTR, 0.1
.mu.M STS plus 0.65 .mu.M Bad-DTTR and 0.1 .mu.M STS plus 0.065
.mu.M Bad-DTTR. Apoptotic death cells were quantified at different
times by staining with Hoechst dye no. 33342. Results are presented
as the average number of cells per field (magnification
160.times.). Apoptosis is most enhanced when cells are treated with
0.1 .mu.M STS plus 0.65 .mu.M Bad-DTTR, and cells begin to die
about 12 hours after treatment.
[0190] U251 MG cells at 3.times.10.sup.4 cells/cm.sup.2 in 100
.mu.l DMEM with 10% FBS were treated with PBS, 1 .mu.M STS, 0.65
.mu.M Bad-DTTR, 0.065 .mu.M Bad-DTTR, 1 .mu.M STS plus 0.65 .mu.M
Bad-DTTR and 1 .mu.M STS plus 0.065 .mu.M Bad-DTTR. Apoptotic cells
were quantified and presented as above. The combination of 1 .mu.M
STS and Bad-DTTR at various concentrations causes an earlier onset
of apoptosis in U251 MG cells.
EXAMPLE 6
LF.sub.n-Bcl-x.sub.L Inhibits Neuron, Macrophage, and Lymphocyte
Apoptosis
[0191] Anthrax toxin includes three components: lethal factor (LF),
edema factor (EF) and protective antigen (PA) (Leppla, Anthrax
toxin. In Handbook of Natural Toxins, Moss et al., Eds., Dekker,
New York, Vol. 8, pp. 543-572, 1995). PA binds simultaneously to LF
and to a cell surface receptor existing on the cells of almost all
species including rodents (Leppla, 1995; Friedlander, J. Biol.
Chem. 261:7123-7126, 1986), and transports LF into cells where LF
causes toxic effects. PA alone, however, is not toxic. It has been
found that the first 255 residues (LF.sub.n) of LF, which
constitute the PA-binding domain and are not toxic to cells, are
sufficient for delivery of heterologous peptides to the cytosol.
Cytotoxins have been fused to LF.sub.n (Leppla, 1995; Arora et al.,
J Biol. Chem. 269:26165-26171, 1994; Milne et al., Mol. Microbiol.
15: 661-666, 1995). Administration of a fusion protein containing
LF.sub.n and the gp120 envelope glycoprotein of HIV-1 along with PA
to antigen-presenting cells sensitized them to cytolysis by
cytotoxic T-lymphocytes (CTL) specific to gp120 (Goletz et al.,
Proc Natl Acad Sci USA 94:12059-12064, 1997). In vivo,
LF.sub.n-fused to CTL epitopes injected along with PA has been
shown to stimulate a CTL response against the antigens in mice
(Ballard et al., Proc. Natl. Acad. Sci. USA 93: 12531-12534, 1996;
Ballard et al, Infect. Immun. 66:615-619, 1998; Ballard et al.,
Infect. Immun. 66:4696-4699, 1998; Doling et al., Infect. Immun.
67: 3290-3296, 1999).
[0192] To inhibit neuron apoptosis, another protein delivery system
was engineered by fusing a nontoxic domain of anthrax toxin to
Bcl-x.sub.L, to create the LF.sub.n-Bcl-x.sub.L chimeric fusion
protein. Macrophage and lymphocyte death in culture, and neuron
death in vivo in a retinal ganglion cell model of apoptosis induced
by axotomy, can be prevented by application of this fusion
protein.
A. Construction of LF.sub.n-Bcl-x.sub.L in a Prokaryotic Expression
Plasmid
[0193] The coding sequence for lethal factor (LF) from codons 34 to
288 (LF.sub.n) (Bragg et al., Gene 81:45-54, 1989), which is the
amino-terminal domain (residues 1-255) of mature LF (Leppla, 1995),
was amplified using PCR with the template of pET15b/LF.sub.n (Milne
et al., Mol. Microbiol. 15: 661-666, 1995). The gene of human
Bcl-x.sub.L from Codons 1 to 209 (Bcl-x.sub.L(1-209)) (Boise et
al., Cell 74: 597-608, 1993) was amplified by PCR. Then the
LF.sub.n encoding sequence was fused to the 5' end of
Bcl-x.sub.L(1-209) encoding sequence by a second round of PCR. A
stop codon was introduced immediately after Codon 209 of
Bcl-x.sub.L. The fused DNA fragment, LF.sub.n-Bcl-x.sub.L, was cut
with NdeI and Xho I, and inserted into prokaryotic expression
vector pET15b cut with Nde I and Xho I (FIG. 8). A histidine tag
and thrombin cleavage site were linked to the N-terminal of
LF.sub.n-Bcl-x.sub.L. Similarly, the Bcl-x.sub.L gene from codons 1
to 209 was also genetically inserted into pET15b at the sites of
Nde I and Xho I. All the constructs were verified by DNA
sequencing.
B. Construction of Eukaryotic Expression Plasmids, Transfection,
Western Blotting and Biologic Activity Assay
[0194] The sequences encoding LF.sub.n-Bcl-x.sub.L, Bcl-x.sub.L
from codons 1 to 209, and full-length Bcl-x.sub.L, were separately
engineered into eukaryotic expression vector pcDNA3.1+ and verified
by DNA sequencing. Cos-7 cells were co-transfected with plasmid
EGFP-C3 and one of the three plasmids as reported (Keith et al., J
Cell Biol 139: 1281-1292, 1997). The cells were treated with 0.1
.mu.M staurosporine (STS) 12 hours later. The dead and living cells
were counted with Hoechst 33342 at different times after STS
treatment (Liu et al., Proc Natl Acad Sci USA 96: 9563-9567, 1999;
Keith et al., J Cell Biol 139: 1281-1292, 1997). The cells were
harvested and lysed 20 hours after transfection, and aliquots were
loaded onto SDS/10-20% PAGE gels. The plasmid-encoded proteins were
visualized by immunoblotting with anti-Bcl-x.sub.L mAb (Trevigen,
Gaithersburg, Md.) and developed by using enhanced
chemiluminescence (Amersham Pharmacia).
C. Protein Expression, Purification, SDS-Page and Western
Blotting
[0195] The proteins LF.sub.n, LF.sub.n-Bcl-x.sub.L and Bcl-x.sub.L
from codons 1 to 209 were individually expressed in E. coli BL21
(DE3) (Novagen, Inc.) and purified with a His-Tag binding
purification kit (Novagen, Inc.). The transformed BL21(DE3) was
cultured at 37.degree. C. in LB medium until the OD600 reached
0.5-0.8, and treated with 1 mM IPTG, and then cultured for 3 more
hours. The cells was pelleted, suspended in 1.times.His-Tag binding
buffer with 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM
aprotinin and 1 mM leupeptin, and disrupted with French Press. The
cytosol was separated from cell debris and undisrupted cells by
centrifugation at 20,000.times.g for 30 minutes and loaded on the
His.cndot.Tag binding column. The eluted proteins were dialyzed
against 1.times.PBS and sterilized with 0.22-um filter. Protective
antigen (PA) was purified as reported (Milne et al., Mol.
Microbiol. 15: 661-666, 1995). The proteins were run on SDS-PAGE
gels, and stained with Coomassie Blue or visualized by
immunoblotting with anti-Bcl-x.sub.L antibody, and developed as
above.
D. J744 Macrophage-Like Cell Culture, Treatment and Apoptosis
Assay
[0196] J744 macrophage-like cells at 10.sup.5/ml were placed in
96-well plates (100 .mu.l per well), and cultured overnight in RPMI
1640 with 10% FCS. The cells were treated with PBS, 0.1 .mu.M
staurosporine alone or 0.1 .mu.M staurosporine along with the
different combinations of the proteins LF.sub.n-Bcl-x.sub.L (28
.mu.g/ml), PA (33 .mu.g/ml), LF.sub.n (28 .mu.g/ml) and Bcl-x.sub.L
(28 .mu.g/ml). The apoptotic and living cells were counted with
Hoechst dye no. 33342 as reported (Liu et al., Proc Natl Acad Sci
USA 96: 9563-9567, 1999).
E. Optic Nerve Section and Intra-Ocular Protein Injection
[0197] The P0 pups of Fisher 344 rat strain were used for the
present study. P0 is defined as the day of birth. The intracranial
lesion of unilateral optic nerve was performed as reported (Rabachi
et al., J Neurosci. 14: 5292-301, 1994). Briefly, a P0 pup was
anesthetized by hypothermia. Under a dissecting microscope, an
incision over the right eye was cut and a piece of bone flipped up.
The right optic nerve was sectioned after suctioning the overlying
cerebral cortex. The section site of optic nerve is about 3 mm away
from the eyeball. A piece of gelfoam was put in the hole, and the
flipped bone replaced, and the incision repaired with
SUPERGLUE.TM.. Immediately after the operation, seven, ten and four
mice were respectively treated with administration of PBS,
LF.sub.n-Bcl-x.sub.L (0.65 .mu.g) plus PA (0.35 .mu.g) and PA (0.35
.mu.g) in a volume of 350 nanoliters (nl) per eye through ora
serrata into the posterior chamber of the right eyes by using a
micro-injector with a pulled micropipette. The pups were warmed up
with a light lamp until the recovery, and then sent back to the
mother. Four pups from the same litters, which were not operated
and not treated, were used for normal control.
F. Histology
[0198] About 24 hours after sectioning of the optic nerve, the
right eyes were removed under deep anesthesia with sodium
pentobarbital, fixed in 4% paraformaldehyde for approximately 30
hours, embedded in paraffin and cut at 6 .mu.m. The eyes taken from
the normal pups in the same litters were processed in the same way
to serve as controls. The sections were rehydrated, stained with
0.2% cresyl violet, dehydrated, and mounted with DPX mountant. The
number of pyknotic cells and the number of living cells were
counted by the use of 40.times. objective in the entire retinal
ganglion cell layer of three sections per retina. The pyknotic
cells were identified as reported (Rabachi et al., J. Neurosci. 14:
5292-301, 1994). The values were presented as the percentage of
pyknotic cells versus total cells per retina (FIG. 12).
G. Results
[0199] The PA protein from the Anthrax bacillus binds cell
receptors and can mediate the delivery of the anthrax LF protein to
the cell cytosol where LF effects toxicity to cells. The N-terminal
domain of LF binds to PA. When exogenous peptides are fused to the
N-terminal domain of LF (LF.sub.n), they can be delivered to the
cell cytosol by PA. Deletion of the C-terminal region of LF
prevents toxicity to cells. To deliver Bcl-x.sub.L to cells, the
N-terminal 255 amino acids of LF were fused to Bcl-x.sub.L without
including the C-terminal 24 hydrophobic amino acids of Bcl-x.sub.L,
as shown schematically in FIG. 8. The nucleotide and amino acid
sequences of the fusion protein, LF.sub.n-Bcl-x.sub.L, are shown in
SEQ ID NOs: 7 and 8. The fusion protein was expressed in E. coli
and purified to near homogeneity.
[0200] The bioactivity of the LF.sub.n-Bcl-x.sub.L was explored in
J774 cells in tissue culture. LF.sub.n-Bcl-x.sub.L, at 28
micrograms per ml plus PA at 33 micrograms per ml was added to the
media of cells at the time of apoptosis induction with 0.1 .mu.M
staurosporine (STS). Cells treated with staurosporine alone died by
apoptosis over the following 36 hours as shown in FIG. 9. When the
cells were treated with LF.sub.n-Bcl-x.sub.L plus PA, most of the
cell death was inhibited.
[0201] Controls were performed to explore the requirements for
apoptosis inhibition. FIG. 10 shows data demonstrating that J774
cells treated with LF.sub.n alone, Bcl-x.sub.L alone,
LF.sub.n-Bcl-x.sub.L without PA, and PA without
LF.sub.n-Bcl-x.sub.L were not protected from apoptosis induced by
staurosporine, whereas LF.sub.n-Bcl-x.sub.L plus PA prevented more
than half of the cell death. Jurkat cells were also protected from
apoptosis by LF.sub.n-Bcl-x.sub.L plus PA (FIG. 11).
[0202] This new strategy to block cell death was explored in an in
vivo model of neuron apoptosis. Retinal ganglion cells were
axotomized and immediately afterwards a mixture containing 0.35
.mu.g of PA and 0.65 .mu.g of LF.sub.n-Bcl-x.sub.L was injected
into the eye. Control mice were either not axotomized, axotomized
and injected with PBS, or axotomized and injected with PA alone.
Mice were sacrificed 24 hours later, and the eyes examined
histologically. An increase in pyknotic cells, i.e., apoptotic
cells (Rabachi et al., J Neurosci. 14: 5292-301, 1994), occurs in
the ganglion layer 24 hours after axotomy. However, when eyes are
injected with LF.sub.n-Bcl-x.sub.L and PA, much of the cell death
is inhibited. PA alone did not prevent cell death. To quantitate
the extent of cell death, the number of living and pyknotic cells
in three entire ganglion layers in one eye from each of 4-10 mice
was counted. The quantified results are shown in FIG. 12.
LF.sub.n-Bcl-x.sub.L inhibited more than half of the cell death due
to neuron axotomy in vivo.
[0203] In view of the many possible embodiments to which the
principles of our invention may be applied, it should be recognized
that the illustrated embodiments are only preferred examples of the
invention, and should not be taken as limitations on its scope.
Rather, the scope of the invention is defined by the following
claims. We therefore claim as our invention all that comes within
the scope and spirit of these claims.
TABLE-US-00002 TABLE 2 Inhibition of Apoptosis by Bcl-x.sub.L-DTR
Concentration of Time of STS Apoptosis Apoptosis Bcl-x.sub.L-DTR
Treatment Prevention DT Cell line inducer (.mu.M) (Hrs) (%*)
IO.sub.50 (M) Cos-7 0.1 .mu.M STS 4.8 12 58.4 10.sup.-12-10.sup.-11
(monkey kidney) U251 0.1 .mu.M STS 4.68 16 57.5
10.sup.-12-10.sup.-11 (human glioma) HeLa 0.2 .mu.M STS 2.17 10
32.4 10.sup.-12-10.sup.-11 (human cervical Ca) Jurkat 0.1 .mu.M STS
4.68 12 21.2 10.sup.-9 (human T leukemia) 9L 0.1 .mu.M STS 4.68 12
-5.4 >10.sup.-7 (rat gliosarcoma) WEH7.1 0.1 .mu.M STS 4.68 12
0.5 >10.sup.-7 (mouse T lymphoma) *Apoptotic cells were counted
with Hoechst dye no. 33342 and the percent prevention from
apoptosis was calculated as 1 - (number of apoptotic cells with STS
and Bcl-x.sub.L-DTR - number of apoptotic cells without STS and
Bcl-x.sub.L-DTR)/(number of apoptotic cells with STS - number of
apoptotic cells without STS and Bcl-x.sub.L-DTR) except for the
non-adherent Jurkat and WEHI7.1 cells which were counted by trypan
blue dye exclusion and % apoptosis prevention calculated as (number
of living cells with STS and Bcl-x.sub.L-DTR - number of living
cells with STS)/(number of living cells without STS and
Bcl-x.sub.L-DTR).
TABLE-US-00003 TABLE 3 Brefeldin A prevents Bcl-x.sub.L-DTR
blockade of apoptosis 0.1 .mu.M STS + PBS 0.1 .mu.M STS 2.24 .mu.M
Bcl-x.sub.L-DTR Bcl-x.sub.L-DTR Cell 1 24 11 56% protection death
(%) 2 .mu.M 0.1 .mu.M 0.1 .mu.M STS + brefeldin STS +2 .mu.M 2
.mu.M brefeldin A + Bcl-x.sub.L-DTR + A brefeldin A 2.24 .mu.M
Bcl-x.sub.L-DTR brefeldin A Cell 2 35 32 9% protection death (%)
Apoptotic cells were counted with Hoechst dye no. 33342 14 hours
after addition of STS and/or brefeldin A minutes after
Bcl-x.sub.L-DTR was added to Cos-7 cells. The protection percentage
was calculated as 1 - (number of apoptotic cells with STS and
Bcl-x.sub.L-DTR - number of apoptotic cells without STS and
Bcl-x.sub.L-DTR)/(number of apoptotic cells with STS - number of
apoptotic cells without STS and Bcl-x.sub.L-DTR).
Sequence CWU 1
1
811236DNAArtificial SequenceDescription of Artificial Sequence
genetic fusion 1atg ggc cat cat cat cat cat cat cat cat cat cac agc
agc ggc cat 48Met Gly His His His His His His His His His His Ser
Ser Gly His1 5 10 15atc gaa ggt cgt atg tct cag agc aac cgg gag ctg
gtg gtt gac ttt 96Ile Glu Gly Arg Met Ser Gln Ser Asn Arg Glu Leu
Val Val Asp Phe20 25 30ctc tcc tac aag ctt tcc cag aaa gga tac agc
tgg agt cag ttt agt 144Leu Ser Tyr Lys Leu Ser Gln Lys Gly Tyr Ser
Trp Ser Gln Phe Ser35 40 45gat gtg gaa gag aac agg act gag gcc cca
gaa ggg act gaa tcg gag 192Asp Val Glu Glu Asn Arg Thr Glu Ala Pro
Glu Gly Thr Glu Ser Glu50 55 60atg gag acc ccc agt gcc atc aat ggc
aac cca tcc tgg cac ctg gca 240Met Glu Thr Pro Ser Ala Ile Asn Gly
Asn Pro Ser Trp His Leu Ala65 70 75 80gac agc ccc gcg gtg aat gga
gcc act gcg cac agc agc agt ttg gat 288Asp Ser Pro Ala Val Asn Gly
Ala Thr Ala His Ser Ser Ser Leu Asp85 90 95gcc cgg gag gtg atc ccc
atg gca gca gta aag caa gcg ctg agg gag 336Ala Arg Glu Val Ile Pro
Met Ala Ala Val Lys Gln Ala Leu Arg Glu100 105 110gca ggc gac gag
ttt gaa ctg cgg tac cgg cgg gca ttc agt gac ctg 384Ala Gly Asp Glu
Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu115 120 125aca tcc
cag ctc cac atc acc cca ggg aca gca tat cag agc ttt gaa 432Thr Ser
Gln Leu His Ile Thr Pro Gly Thr Ala Tyr Gln Ser Phe Glu130 135
140cag gta gtg aat gaa ctc ttc cgg gat ggg gta aac tgg ggt cgc att
480Gln Val Val Asn Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg
Ile145 150 155 160gtg gcc ttt ttc tcc ttc ggc ggg gca ctg tgc gtg
gaa agc gta gac 528Val Ala Phe Phe Ser Phe Gly Gly Ala Leu Cys Val
Glu Ser Val Asp165 170 175aag gag atg cag gta ttg gtg agt cgg atc
gca gct tgg atg gcc act 576Lys Glu Met Gln Val Leu Val Ser Arg Ile
Ala Ala Trp Met Ala Thr180 185 190tac ctg aat gac cac cta gag cct
tgg atc cag gag aac ggc ggc tgg 624Tyr Leu Asn Asp His Leu Glu Pro
Trp Ile Gln Glu Asn Gly Gly Trp195 200 205gat act ttt gtg gaa ctc
tat ggg aac aat gca gca gcc gag agc cga 672Asp Thr Phe Val Glu Leu
Tyr Gly Asn Asn Ala Ala Ala Glu Ser Arg210 215 220aag ggc cag gaa
cgc ttc aac cgc tgg ttc ctg acg ggc atg act gtg 720Lys Gly Gln Glu
Arg Phe Asn Arg Trp Phe Leu Thr Gly Met Thr Val225 230 235 240gcc
ggc gtg gtt ctg ctg ggc tca ctc ttc agt cgg aaa gcg tat tct 768Ala
Gly Val Val Leu Leu Gly Ser Leu Phe Ser Arg Lys Ala Tyr Ser245 250
255gcg gcc gcg cat aaa acg caa cca ttt ctt cat gac ggg tat gct gtc
816Ala Ala Ala His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala
Val260 265 270agt tgg aac act gtt gaa gat tcg ata atc cga act ggt
ttt caa ggg 864Ser Trp Asn Thr Val Glu Asp Ser Ile Ile Arg Thr Gly
Phe Gln Gly275 280 285gag agt ggg cac gac ata aaa att act gct gaa
aat acc ccg ctt cca 912Glu Ser Gly His Asp Ile Lys Ile Thr Ala Glu
Asn Thr Pro Leu Pro290 295 300atc gcg ggt gtc cta cta ccg act att
cct gga aag ctg gac gtt aat 960Ile Ala Gly Val Leu Leu Pro Thr Ile
Pro Gly Lys Leu Asp Val Asn305 310 315 320aag tcc aag act cat att
tcc gta aat ggt cgg aaa ata agg atg cgt 1008Lys Ser Lys Thr His Ile
Ser Val Asn Gly Arg Lys Ile Arg Met Arg325 330 335tgc aga gct ata
gac ggt gat gta act ttt tgt cgc cct aaa tct cct 1056Cys Arg Ala Ile
Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro340 345 350gtt tat
gtt ggt aat ggt gtg cat gcg aat ctt cac gtg gca ttt cac 1104Val Tyr
Val Gly Asn Gly Val His Ala Asn Leu His Val Ala Phe His355 360
365aga agc agc tcg gag aaa att cat tct aat gaa att tcg tcg gat tcc
1152Arg Ser Ser Ser Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp
Ser370 375 380ata ggc gtt ctt ggg tac cag aaa aca gta gat cac acc
aag gtt aat 1200Ile Gly Val Leu Gly Tyr Gln Lys Thr Val Asp His Thr
Lys Val Asn385 390 395 400tct aag cta tcg cta ttt ttt gaa atc aaa
agc tga 1236Ser Lys Leu Ser Leu Phe Phe Glu Ile Lys Ser405
4102411PRTArtificial SequenceDescription of Artificial Sequence
genetic fusion 2Met Gly His His His His His His His His His His Ser
Ser Gly His1 5 10 15Ile Glu Gly Arg Met Ser Gln Ser Asn Arg Glu Leu
Val Val Asp Phe20 25 30Leu Ser Tyr Lys Leu Ser Gln Lys Gly Tyr Ser
Trp Ser Gln Phe Ser35 40 45Asp Val Glu Glu Asn Arg Thr Glu Ala Pro
Glu Gly Thr Glu Ser Glu50 55 60Met Glu Thr Pro Ser Ala Ile Asn Gly
Asn Pro Ser Trp His Leu Ala65 70 75 80Asp Ser Pro Ala Val Asn Gly
Ala Thr Ala His Ser Ser Ser Leu Asp85 90 95Ala Arg Glu Val Ile Pro
Met Ala Ala Val Lys Gln Ala Leu Arg Glu100 105 110Ala Gly Asp Glu
Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu115 120 125Thr Ser
Gln Leu His Ile Thr Pro Gly Thr Ala Tyr Gln Ser Phe Glu130 135
140Gln Val Val Asn Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg
Ile145 150 155 160Val Ala Phe Phe Ser Phe Gly Gly Ala Leu Cys Val
Glu Ser Val Asp165 170 175Lys Glu Met Gln Val Leu Val Ser Arg Ile
Ala Ala Trp Met Ala Thr180 185 190Tyr Leu Asn Asp His Leu Glu Pro
Trp Ile Gln Glu Asn Gly Gly Trp195 200 205Asp Thr Phe Val Glu Leu
Tyr Gly Asn Asn Ala Ala Ala Glu Ser Arg210 215 220Lys Gly Gln Glu
Arg Phe Asn Arg Trp Phe Leu Thr Gly Met Thr Val225 230 235 240Ala
Gly Val Val Leu Leu Gly Ser Leu Phe Ser Arg Lys Ala Tyr Ser245 250
255Ala Ala Ala His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala
Val260 265 270Ser Trp Asn Thr Val Glu Asp Ser Ile Ile Arg Thr Gly
Phe Gln Gly275 280 285Glu Ser Gly His Asp Ile Lys Ile Thr Ala Glu
Asn Thr Pro Leu Pro290 295 300Ile Ala Gly Val Leu Leu Pro Thr Ile
Pro Gly Lys Leu Asp Val Asn305 310 315 320Lys Ser Lys Thr His Ile
Ser Val Asn Gly Arg Lys Ile Arg Met Arg325 330 335Cys Arg Ala Ile
Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro340 345 350Val Tyr
Val Gly Asn Gly Val His Ala Asn Leu His Val Ala Phe His355 360
365Arg Ser Ser Ser Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp
Ser370 375 380Ile Gly Val Leu Gly Tyr Gln Lys Thr Val Asp His Thr
Lys Val Asn385 390 395 400Ser Lys Leu Ser Leu Phe Phe Glu Ile Lys
Ser405 41031704DNAArtificial SequenceDescription of Artificial
Sequence genetic fusion 3atg ggc cat cat cat cat cat cat cat cat
cat cac agc agc ggc cat 48Met Gly His His His His His His His His
His His Ser Ser Gly His1 5 10 15atc gaa ggt cgt cat atg gga acc cca
aag cag ccc tcg ctg gct cct 96Ile Glu Gly Arg His Met Gly Thr Pro
Lys Gln Pro Ser Leu Ala Pro20 25 30gca cac gcc cta ggc ttg agg aag
tcc gat ccc gga atc cgg agc ctg 144Ala His Ala Leu Gly Leu Arg Lys
Ser Asp Pro Gly Ile Arg Ser Leu35 40 45ggg agc gac gcg gga gga agg
cgg tgg aga cca gca gcc cag agt atg 192Gly Ser Asp Ala Gly Gly Arg
Arg Trp Arg Pro Ala Ala Gln Ser Met50 55 60ttc cag atc cca gag ttt
gag ccg agt gag cag gaa gac gct agt gct 240Phe Gln Ile Pro Glu Phe
Glu Pro Ser Glu Gln Glu Asp Ala Ser Ala65 70 75 80aca gat agg ggc
ctg ggc cct agc ctc act gag gac cag cca ggt ccc 288Thr Asp Arg Gly
Leu Gly Pro Ser Leu Thr Glu Asp Gln Pro Gly Pro85 90 95tac ctg gcc
cca ggt ctc ctg ggg agc aac att cat cag cag gga cgg 336Tyr Leu Ala
Pro Gly Leu Leu Gly Ser Asn Ile His Gln Gln Gly Arg100 105 110gca
gcc acc aac agt cat cat gga ggc gca ggg gct atg gag act cgg 384Ala
Ala Thr Asn Ser His His Gly Gly Ala Gly Ala Met Glu Thr Arg115 120
125agt cgc cac agt gcg tac cca gcg ggg acc gag gag gat gaa ggg atg
432Ser Arg His Ser Ala Tyr Pro Ala Gly Thr Glu Glu Asp Glu Gly
Met130 135 140gag gag gag ctt agc cct ttt cga gga cgc tcg cgt gcg
gct ccc ccc 480Glu Glu Glu Leu Ser Pro Phe Arg Gly Arg Ser Arg Ala
Ala Pro Pro145 150 155 160aat ctc tgg gca gcg cag cgc tac ggc cgt
gag ctc cga agg atg agc 528Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg
Glu Leu Arg Arg Met Ser165 170 175gat gag ttt gag ggt tcc ttc aag
gga ctt cct cgc cca aag agc gca 576Asp Glu Phe Glu Gly Ser Phe Lys
Gly Leu Pro Arg Pro Lys Ser Ala180 185 190ggc act gca aca cag atg
cga caa agc gcc ggc tgg acg cgc att atc 624Gly Thr Ala Thr Gln Met
Arg Gln Ser Ala Gly Trp Thr Arg Ile Ile195 200 205cag tcc tgg tgg
gat cga aac ttg ggc aaa gga ggc tcc acc ccc tcc 672Gln Ser Trp Trp
Asp Arg Asn Leu Gly Lys Gly Gly Ser Thr Pro Ser210 215 220cag tca
gta ggt agc tca ttg tca tgc ata aat ctt gat tgg gat gtc 720Gln Ser
Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val225 230 235
240ata agg gat aaa act aag aca aag ata gag tct ttg aaa gag cat ggc
768Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His
Gly245 250 255cct atc aaa aat aaa atg agc gaa agt ccc aat aaa aca
gta tct gag 816Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr
Val Ser Glu260 265 270gaa aaa gct aaa caa tac cta gaa gaa ttt cat
caa acg gca tta gag 864Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His
Gln Thr Ala Leu Glu275 280 285cat cct gaa ttg tca gaa ctt aaa acc
gtt act ggg acc aat cct gta 912His Pro Glu Leu Ser Glu Leu Lys Thr
Val Thr Gly Thr Asn Pro Val290 295 300ttc gct ggg gct aac tat gcg
gcg tgg gca gta aac gtt gcg caa gtt 960Phe Ala Gly Ala Asn Tyr Ala
Ala Trp Ala Val Asn Val Ala Gln Val305 310 315 320atc gat agc gaa
aca gct gat aat ttg gaa aag aca act gct gct ctt 1008Ile Asp Ser Glu
Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu325 330 335tcg ata
ctt cct ggt atc ggt agc gta atg ggc att gca gac ggt gcc 1056Ser Ile
Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala340 345
350gtt cac cac aat aca gaa gag ata gtg gca caa tca ata gct tta tcg
1104Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu
Ser355 360 365tct tta atg gtt gct caa gct att cca ttg gta gga gag
cta gtt gat 1152Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu
Leu Val Asp370 375 380att ggt ttc gct gca tat aat ttt gta gag agt
att atc aat tta ttt 1200Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser
Ile Ile Asn Leu Phe385 390 395 400caa gta gtt cat aat tcg tat aat
cgt ccc gcg tat tct ccg ggg cat 1248Gln Val Val His Asn Ser Tyr Asn
Arg Pro Ala Tyr Ser Pro Gly His405 410 415aaa acg caa cca ttt ctt
cat gac ggg tat gct gtc agt tgg aac act 1296Lys Thr Gln Pro Phe Leu
His Asp Gly Tyr Ala Val Ser Trp Asn Thr420 425 430gtt gaa gat tcg
ata atc cga act ggt ttt caa ggg gag agt ggg cac 1344Val Glu Asp Ser
Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His435 440 445gac ata
aaa att act gct gaa aat acc ccg ctt cca atc gcg ggt gtc 1392Asp Ile
Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val450 455
460cta cta ccg act att cct gga aag ctg gac gtt aat aag tcc aag act
1440Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys
Thr465 470 475 480cat att tcc gta aat ggt cgg aaa ata agg atg cgt
tgc aga gct ata 1488His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg
Cys Arg Ala Ile485 490 495gac ggt gat gta act ttt tgt cgc cct aaa
tct cct gtt tat gtt ggt 1536Asp Gly Asp Val Thr Phe Cys Arg Pro Lys
Ser Pro Val Tyr Val Gly500 505 510aat ggt gtg cat gcg aat ctt cac
gtg gca ttt cac aga agc agc tcg 1584Asn Gly Val His Ala Asn Leu His
Val Ala Phe His Arg Ser Ser Ser515 520 525gag aaa att cat tct aat
gaa att tcg tcg gat tcc ata ggc gtt ctt 1632Glu Lys Ile His Ser Asn
Glu Ile Ser Ser Asp Ser Ile Gly Val Leu530 535 540ggg tac cag aaa
aca gta gat cac acc aag gtt aat tct aag cta tcg 1680Gly Tyr Gln Lys
Thr Val Asp His Thr Lys Val Asn Ser Lys Leu Ser545 550 555 560cta
ttt ttt gaa atc aaa agc tga 1704Leu Phe Phe Glu Ile Lys
Ser5654567PRTArtificial SequenceDescription of Artificial Sequence
genetic fusion 4Met Gly His His His His His His His His His His Ser
Ser Gly His1 5 10 15Ile Glu Gly Arg His Met Gly Thr Pro Lys Gln Pro
Ser Leu Ala Pro20 25 30Ala His Ala Leu Gly Leu Arg Lys Ser Asp Pro
Gly Ile Arg Ser Leu35 40 45Gly Ser Asp Ala Gly Gly Arg Arg Trp Arg
Pro Ala Ala Gln Ser Met50 55 60Phe Gln Ile Pro Glu Phe Glu Pro Ser
Glu Gln Glu Asp Ala Ser Ala65 70 75 80Thr Asp Arg Gly Leu Gly Pro
Ser Leu Thr Glu Asp Gln Pro Gly Pro85 90 95Tyr Leu Ala Pro Gly Leu
Leu Gly Ser Asn Ile His Gln Gln Gly Arg100 105 110Ala Ala Thr Asn
Ser His His Gly Gly Ala Gly Ala Met Glu Thr Arg115 120 125Ser Arg
His Ser Ala Tyr Pro Ala Gly Thr Glu Glu Asp Glu Gly Met130 135
140Glu Glu Glu Leu Ser Pro Phe Arg Gly Arg Ser Arg Ala Ala Pro
Pro145 150 155 160Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg Glu Leu
Arg Arg Met Ser165 170 175Asp Glu Phe Glu Gly Ser Phe Lys Gly Leu
Pro Arg Pro Lys Ser Ala180 185 190Gly Thr Ala Thr Gln Met Arg Gln
Ser Ala Gly Trp Thr Arg Ile Ile195 200 205Gln Ser Trp Trp Asp Arg
Asn Leu Gly Lys Gly Gly Ser Thr Pro Ser210 215 220Gln Ser Val Gly
Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val225 230 235 240Ile
Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly245 250
255Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser
Glu260 265 270Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr
Ala Leu Glu275 280 285His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr
Gly Thr Asn Pro Val290 295 300Phe Ala Gly Ala Asn Tyr Ala Ala Trp
Ala Val Asn Val Ala Gln Val305 310 315 320Ile Asp Ser Glu Thr Ala
Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu325 330 335Ser Ile Leu Pro
Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala340 345 350Val His
His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser355 360
365Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val
Asp370 375 380Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile
Asn Leu Phe385 390 395 400Gln Val Val His Asn Ser Tyr Asn Arg Pro
Ala Tyr Ser Pro Gly His405 410 415Lys Thr Gln Pro Phe Leu His Asp
Gly Tyr Ala Val Ser Trp Asn Thr420 425 430Val Glu Asp Ser Ile Ile
Arg Thr Gly Phe Gln Gly Glu Ser Gly His435 440 445Asp Ile Lys Ile
Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val450 455 460Leu Leu
Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys Thr465 470 475
480His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala
Ile485 490 495Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro Val
Tyr Val Gly500 505 510Asn Gly Val His Ala Asn Leu His Val Ala Phe
His Arg Ser Ser Ser515 520 525Glu Lys Ile His Ser Asn Glu Ile Ser
Ser Asp Ser Ile Gly Val Leu530 535 540Gly Tyr Gln Lys Thr Val Asp
His Thr Lys Val Asn Ser Lys Leu Ser545 550 555 560Leu Phe Phe Glu
Ile Lys Ser565518DNAArtificial
SequenceDescription of Artificial Sequence oligonucleotide linker
5gcgtattctg cggccgcg 1866PRTArtificial SequenceDescription of
Artificial Sequence oligopeptide linker 6Ala Tyr Ser Ala Ala Ala1
571455DNAArtificial SequenceDescription of Artificial Sequence
genetic fusion 7atg ggc agc agc cat cat cat cat cat cac agc agc ggc
ctg gtg ccg 48Met Gly Ser Ser His His His His His His Ser Ser Gly
Leu Val Pro1 5 10 15cgc ggc agc cat atg gcg ggc ggt cat ggt gat gta
ggt atg cac gta 96Arg Gly Ser His Met Ala Gly Gly His Gly Asp Val
Gly Met His Val20 25 30aaa gag aaa gag aaa aat aaa gat gag aat aag
aga aaa gat gaa gaa 144Lys Glu Lys Glu Lys Asn Lys Asp Glu Asn Lys
Arg Lys Asp Glu Glu35 40 45cga aat aaa aca cag gaa gag cat tta aag
gaa atc atg aaa cac att 192Arg Asn Lys Thr Gln Glu Glu His Leu Lys
Glu Ile Met Lys His Ile50 55 60gta aaa ata gaa gta aaa ggg gag gaa
gct gtt aaa aaa gag gca gca 240Val Lys Ile Glu Val Lys Gly Glu Glu
Ala Val Lys Lys Glu Ala Ala65 70 75 80gaa aag cta ctt gag aaa gta
cca tct gat gtt tta gag atg tat aaa 288Glu Lys Leu Leu Glu Lys Val
Pro Ser Asp Val Leu Glu Met Tyr Lys85 90 95gca att gga gga aag ata
tat att gtg gat ggt gat att aca aaa cat 336Ala Ile Gly Gly Lys Ile
Tyr Ile Val Asp Gly Asp Ile Thr Lys His100 105 110ata tct tta gaa
gca tta tct gaa gat aag aaa aaa ata aaa gac att 384Ile Ser Leu Glu
Ala Leu Ser Glu Asp Lys Lys Lys Ile Lys Asp Ile115 120 125tat ggg
aaa gat gct tta tta cat gaa cat tat gta tat gca aaa gaa 432Tyr Gly
Lys Asp Ala Leu Leu His Glu His Tyr Val Tyr Ala Lys Glu130 135
140gga tat gaa ccc gta ctt gta atc caa tct tcg gaa gat tat gta gaa
480Gly Tyr Glu Pro Val Leu Val Ile Gln Ser Ser Glu Asp Tyr Val
Glu145 150 155 160aat act gaa aag gca ctg aac gtt tat tat gaa ata
ggt aag ata tta 528Asn Thr Glu Lys Ala Leu Asn Val Tyr Tyr Glu Ile
Gly Lys Ile Leu165 170 175tca agg gat att tta agt aaa att aat caa
cca tat cag aaa ttt tta 576Ser Arg Asp Ile Leu Ser Lys Ile Asn Gln
Pro Tyr Gln Lys Phe Leu180 185 190gat gta tta aat acc att aaa aat
gca tct gat tca gat gga caa gat 624Asp Val Leu Asn Thr Ile Lys Asn
Ala Ser Asp Ser Asp Gly Gln Asp195 200 205ctt tta ttt act aat cag
ctt aag gaa cat ccc aca gac ttt tct gta 672Leu Leu Phe Thr Asn Gln
Leu Lys Glu His Pro Thr Asp Phe Ser Val210 215 220gaa ttc ttg gaa
caa aat agc aat gag gta caa gaa gta ttt gcg aaa 720Glu Phe Leu Glu
Gln Asn Ser Asn Glu Val Gln Glu Val Phe Ala Lys225 230 235 240gct
ttt gca tat tat atc gag cca cag cat cgt gat gtt tta cag ctt 768Ala
Phe Ala Tyr Tyr Ile Glu Pro Gln His Arg Asp Val Leu Gln Leu245 250
255tat gca ccg gaa gct ttt aat tac atg gat aaa ttt aac gaa caa gaa
816Tyr Ala Pro Glu Ala Phe Asn Tyr Met Asp Lys Phe Asn Glu Gln
Glu260 265 270ata aat cta tcc atg tct cag agc aac cgg gag ctg gtg
gtt gac ttt 864Ile Asn Leu Ser Met Ser Gln Ser Asn Arg Glu Leu Val
Val Asp Phe275 280 285ctc tcc tac aag ctt tcc cag aaa gga tac agc
tgg agt cag ttt agt 912Leu Ser Tyr Lys Leu Ser Gln Lys Gly Tyr Ser
Trp Ser Gln Phe Ser290 295 300gat gtg gaa gag aac agg act gag gcc
cca gaa ggg act gaa tcg gag 960Asp Val Glu Glu Asn Arg Thr Glu Ala
Pro Glu Gly Thr Glu Ser Glu305 310 315 320atg gag acc ccc agt gcc
atc aat ggc aac cca tcc tgg cac ctg gca 1008Met Glu Thr Pro Ser Ala
Ile Asn Gly Asn Pro Ser Trp His Leu Ala325 330 335gac agc ccc gcg
gtg aat gga gcc act gcg cac agc agc agt ttg gat 1056Asp Ser Pro Ala
Val Asn Gly Ala Thr Ala His Ser Ser Ser Leu Asp340 345 350gcc cgg
gag gtg atc ccc atg gca gca gta aag caa gcg ctg agg gag 1104Ala Arg
Glu Val Ile Pro Met Ala Ala Val Lys Gln Ala Leu Arg Glu355 360
365gca ggc gac gag ttt gaa ctg cgg tac cgg cgg gca ttc agt gac ctg
1152Ala Gly Asp Glu Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp
Leu370 375 380aca tcc cag ctc cac atc acc cca ggg aca gca tat cag
agc ttt gaa 1200Thr Ser Gln Leu His Ile Thr Pro Gly Thr Ala Tyr Gln
Ser Phe Glu385 390 395 400cag gta gtg aat gaa ctc ttc cgg gat ggg
gta aac tgg ggt cgc att 1248Gln Val Val Asn Glu Leu Phe Arg Asp Gly
Val Asn Trp Gly Arg Ile405 410 415gtg gcc ttt ttc tcc ttc ggc ggg
gca ctg tgc gtg gaa agc gta gac 1296Val Ala Phe Phe Ser Phe Gly Gly
Ala Leu Cys Val Glu Ser Val Asp420 425 430aag gag atg cag gta ttg
gtg agt cgg atc gca gct tgg atg gcc act 1344Lys Glu Met Gln Val Leu
Val Ser Arg Ile Ala Ala Trp Met Ala Thr435 440 445tac ctg aat gac
cac cta gag cct tgg atc cag gag aac ggc ggc tgg 1392Tyr Leu Asn Asp
His Leu Glu Pro Trp Ile Gln Glu Asn Gly Gly Trp450 455 460gat act
ttt gtg gaa ctc tat ggg aac aat gca gca gcc gag agc cga 1440Asp Thr
Phe Val Glu Leu Tyr Gly Asn Asn Ala Ala Ala Glu Ser Arg465 470 475
480aag ggc cag gaa cgc 1455Lys Gly Gln Glu Arg4858485PRTArtificial
SequenceDescription of Artificial Sequence genetic fusion 8Met Gly
Ser Ser His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg
Gly Ser His Met Ala Gly Gly His Gly Asp Val Gly Met His Val20 25
30Lys Glu Lys Glu Lys Asn Lys Asp Glu Asn Lys Arg Lys Asp Glu Glu35
40 45Arg Asn Lys Thr Gln Glu Glu His Leu Lys Glu Ile Met Lys His
Ile50 55 60Val Lys Ile Glu Val Lys Gly Glu Glu Ala Val Lys Lys Glu
Ala Ala65 70 75 80Glu Lys Leu Leu Glu Lys Val Pro Ser Asp Val Leu
Glu Met Tyr Lys85 90 95Ala Ile Gly Gly Lys Ile Tyr Ile Val Asp Gly
Asp Ile Thr Lys His100 105 110Ile Ser Leu Glu Ala Leu Ser Glu Asp
Lys Lys Lys Ile Lys Asp Ile115 120 125Tyr Gly Lys Asp Ala Leu Leu
His Glu His Tyr Val Tyr Ala Lys Glu130 135 140Gly Tyr Glu Pro Val
Leu Val Ile Gln Ser Ser Glu Asp Tyr Val Glu145 150 155 160Asn Thr
Glu Lys Ala Leu Asn Val Tyr Tyr Glu Ile Gly Lys Ile Leu165 170
175Ser Arg Asp Ile Leu Ser Lys Ile Asn Gln Pro Tyr Gln Lys Phe
Leu180 185 190Asp Val Leu Asn Thr Ile Lys Asn Ala Ser Asp Ser Asp
Gly Gln Asp195 200 205Leu Leu Phe Thr Asn Gln Leu Lys Glu His Pro
Thr Asp Phe Ser Val210 215 220Glu Phe Leu Glu Gln Asn Ser Asn Glu
Val Gln Glu Val Phe Ala Lys225 230 235 240Ala Phe Ala Tyr Tyr Ile
Glu Pro Gln His Arg Asp Val Leu Gln Leu245 250 255Tyr Ala Pro Glu
Ala Phe Asn Tyr Met Asp Lys Phe Asn Glu Gln Glu260 265 270Ile Asn
Leu Ser Met Ser Gln Ser Asn Arg Glu Leu Val Val Asp Phe275 280
285Leu Ser Tyr Lys Leu Ser Gln Lys Gly Tyr Ser Trp Ser Gln Phe
Ser290 295 300Asp Val Glu Glu Asn Arg Thr Glu Ala Pro Glu Gly Thr
Glu Ser Glu305 310 315 320Met Glu Thr Pro Ser Ala Ile Asn Gly Asn
Pro Ser Trp His Leu Ala325 330 335Asp Ser Pro Ala Val Asn Gly Ala
Thr Ala His Ser Ser Ser Leu Asp340 345 350Ala Arg Glu Val Ile Pro
Met Ala Ala Val Lys Gln Ala Leu Arg Glu355 360 365Ala Gly Asp Glu
Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu370 375 380Thr Ser
Gln Leu His Ile Thr Pro Gly Thr Ala Tyr Gln Ser Phe Glu385 390 395
400Gln Val Val Asn Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg
Ile405 410 415Val Ala Phe Phe Ser Phe Gly Gly Ala Leu Cys Val Glu
Ser Val Asp420 425 430Lys Glu Met Gln Val Leu Val Ser Arg Ile Ala
Ala Trp Met Ala Thr435 440 445Tyr Leu Asn Asp His Leu Glu Pro Trp
Ile Gln Glu Asn Gly Gly Trp450 455 460Asp Thr Phe Val Glu Leu Tyr
Gly Asn Asn Ala Ala Ala Glu Ser Arg465 470 475 480Lys Gly Gln Glu
Arg485
* * * * *