U.S. patent application number 11/262356 was filed with the patent office on 2006-04-13 for fusion polypeptide suitable as a cytotoxin.
Invention is credited to Jurg Nuesch, Jean Rommelaere.
Application Number | 20060078970 11/262356 |
Document ID | / |
Family ID | 32981795 |
Filed Date | 2006-04-13 |
United States Patent
Application |
20060078970 |
Kind Code |
A1 |
Nuesch; Jurg ; et
al. |
April 13, 2006 |
Fusion polypeptide suitable as a cytotoxin
Abstract
The present invention relates to fusion polypeptides including
(a) a binding site for a cytoskeleton component and (b.sub.1) an
effector protein or the catalytic domain thereof or (b.sub.2) a
binding site for the effector protein, and nucleic acid sequences
encoding the fusion polypeptides. Moreover, various therapeutic
uses of the fusion polypeptides are described, e.g., the treatment
of diseases associated with the presence of an aberrant cell
population, preferably cancer or AIDS.
Inventors: |
Nuesch; Jurg; (Heidelberg,
DE) ; Rommelaere; Jean; (Heidelberg, DE) |
Correspondence
Address: |
INTELLECTUAL PROPERTY / TECHNOLOGY LAW
PO BOX 14329
RESEARCH TRIANGLE PARK
NC
27709
US
|
Family ID: |
32981795 |
Appl. No.: |
11/262356 |
Filed: |
October 28, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/EP04/04477 |
Apr 28, 2004 |
|
|
|
11262356 |
Oct 28, 2005 |
|
|
|
Current U.S.
Class: |
435/69.7 ;
435/194; 435/320.1; 435/325; 536/23.2 |
Current CPC
Class: |
A61P 35/04 20180101;
C12N 9/1205 20130101; C12N 2750/14322 20130101; A61P 31/12
20180101; C07K 14/005 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
435/069.7 ;
435/194; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12N 9/12 20060101
C12N009/12; C07H 21/04 20060101 C07H021/04; C12P 21/04 20060101
C12P021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 2003 |
EP |
03009952.7 |
Claims
1. A fusion polypeptide comprising (a) a binding site for a
cytoskeleton component and (b.sub.1) an effector protein or the
catalytic domain therof or (b.sub.2) a binding site for said
effector protein.
2. The fusion polypeptide of claim 1, wherein both parts (a) and
(b) of the fusion polypeptide are linked by a peptide linker.
3. The fusion polypeptide of claim 2, wherein said peptide linker
is EGFP (SEQ ID NO: 1).
4. The fusion polypeptide of claim 1, wherein said cytoskeleton
component is tropomyosin, gelsolin or tubulin.
5. The fusion polypeptide of claim 1, wherein said binding site is
derived from parvovirus NS1.
6. The fusion polypeptide of claim 5, wherein said binding site
comprises the amino acid sequence from positions 235 to 358 of
NS1.
7. The fusion polypeptide of claim 1, wherein said effector protein
is a protein kinase.
8. The fusion polypeptide of claim 7, wherein said protein kinase
is casein kinase II (CKII.alpha.).
9. The fusion polypeptide of claim 7, wherein said binding site for
casein kinase II (CKII.alpha.) comprises the amino acid sequence
DLEPDEELED (SEQ ID NO: 2).
10. The fusion polypeptide of claim 1, wherein in parts (a) and/or
(b) modifications are present required for inducing and/or
enhancing morphological changes of the host cell.
11. A nucleic acid sequence encoding the fusion polypeptide of
claim 1.
12. A recombinant vector containing the nucleic acid sequence of
claim 11.
13. The recombinant vector of claim 12, wherein the nucleic acid
sequence is operatively linked to regulatory elements allowing
transcription and synthesis of a translatable RNA in prokaryotic
and/or eukaryotic host cells.
14. A recombinant host cell which contains the recombinant vector
of claim 12.
15. The recombinant host cell of claim 14, which is a mammalian
cell, a bacterial cell, an insect cell or a yeast cell.
16. A non-human transgenic animal comprising the nucleic acid
molecule of claim 11.
17. An antibody that binds specifically to the fusion polypeptide
of claim 1.
18. The antibody of claim 17 which is detectably labelled.
19. The antibody of claim 18, wherein the label is a radioisotope,
a bioluminescent compound, a chemiluminescent compound, a
fluorescent compound, a metal chelate, or an enzyme.
20. A pharmaceutical composition containing the fusion polypeptide
of claim 1 or a nucleic acid sequence that encodes for such fusion
polypeptide.
21. A method for the treatment of a disease associated with the
presence of an aberrant cell population, the method comprising
administering a therapeutic amount of the composition of claim
20.
22. The method according to claim 21, wherein said disease is
cancer or AIDS.
23. A fusion polypeptide comprising (a) a tropomyosin binding
fragment of Minuter virus of Mice (MVM) non-structural protein
(NS)-1, and (b.sub.1) catalytic domain of casein kinase II-alpha
(CKII.alpha.) or (b.sub.2) a binding site for (CKII.alpha.).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation in Part application of
and claims priority to PCT International Application No:
PCT/EP2004/004477 filed on Apr. 28, 2004, which in turn claims
priority to European Patent Application No. 03009952.7 filed on
Apr. 30, 2003, the contents of which are incorporatated herein for
reference for all purposes.
BACKGROUND OF THE INVENTION
[0002] 1. Field of Technology
[0003] The present invention relates to fusion polypeptides
comprising (a) a binding site for a cytoskeleton component and
(b.sub.1) an effector protein or the catalytic domain thereof or
(b.sub.2) a binding site for said effector protein, and nucleic
acid sequences encoding said fusion polypeptides. Furthermore, the
present invention relates to various therapeutic uses of said
fusion polypeptides, e.g., the treatment of diseases associated
with the presence of an aberrant cell population, preferably cancer
or AIDS.
[0004] 2. Discussion of Related Art
[0005] For many reasons it is desirable to generate and use toxins
that preferentially kill neoplastically transformed cells. In the
past, this has been achieved with chemical compounds (cytotoxins,
cytostatica), which with more or less specificity enabled a
successful cancer treatment after surgery. Besides selectivity, a
main problem of such compounds consists in the side effects but
also in the lack of proficient targeting of the substance, which
leads to the requirement for relatively high doses. One way to
circumvent this problem is thought to be brought about by the use
of targeted genetics using recombinant viruses to bring genetic
elements into the tumors leading to an onsite expression of the
toxin. Autonomous parvoviruses such as KRV, MVM or H-1 have been
shown to preferentially propagate in and to kill neoplastically
transformed cells. In addition, they consist of a class of viruses
that, despite causing viremia in their infected host, mostly
produce an apathogenic infection. For these reasons, autonomous
parvoviruses are thought to be excellent tools for cancer gene
therapy. Particular interests are focused on recombinant vectors
maintaining their natural oncotropism, as well as their oncolytic
and oncosuppressive potential. However, so far, little is known
about the nature of the oncosuppressive potential of parvoviruses
(which is independent of the parvoviral replicon) and, accordingly,
the therapeutic use of said viruses, e.g., incorporated in
heterologous systems such as recombinant adenoviruses or Measles
viruses, for targeted gene therapy, e.g. cancer therapy is still in
its infancy.
SUMMARY OF THE INVENTION
[0006] Thus, the technical problem underlying the present invention
is to provide parvovirus based means for gene therapy, in
particular for targeted cancer therapy, which overcome the
disadvantages of the prior art therapeutic methods, e.g., as
regards side effects and lack of specificity.
[0007] The solution to said technical problem is achieved by
providing the embodiments characterized in the claims. The
experiments resulting in the present invention were based on the
observations that the main component involved in cell killing and
hence, oncolytic activity of autonomous parvoviruses consists of
NS1, the major nonstructural protein, which plays a key role during
replication of progeny virus particles. The characterization of NS1
revealed a multifunctional protein endowed with a variety of
enzymatic and regulatory functions, which have to act in a
coordinated manner during a productive infection. Particularly, it
was shown that the cytotoxic functions of NS1 were dependent upon
the integrity of (PKC) phosphorylation sites, which led to the
dissection (at least in part) of NS1 replicative functions from its
cytotoxic activities by site-directed mutagenesis. In order to
design new eventually more efficient and specific toxins it is
important to understand the mechanisms for the NS1 induced
selective killing and in consequence to eliminate undesired
eventually even contraproductive functions of the polypeptide.
[0008] With these perspectives, new (desirably small)
polypeptides/compounds with distinct features were designed on the
basis of NS1 induced cell killing, independent on the replicative
functions of the polypeptide. By analyzing the cytotoxic functions
of NS1, it was shown that rather distinct regions of the
polypeptide are important for cell killing than its enzymatic
activities. These findings led to the conclusion that distinct
regions of NS1 might specifically interact with cellular partner
proteins and that the cytotoxic activity(ies) consist of a
multiprotein complex assembled through NS1 rather than a catalytic
activity of the viral protein alone. In addition, mutagenesis at
conserved consensus PKC phosphorylation sites led to obliteration
of the toxic activities of NS1, suggesting a strong regulation of
this NS1 property. Initial studies leading to the present invention
implied that such regulation of NS1 toxicity not only occurs in a
timely coordinated manner by phosphorylation through distinct
kinases, but also by the compartimentalization of the cell, leading
to the conclusion that NS1 targeting to the site of action is an
additional feature of regulation.
[0009] Two partner proteins could be identified which bind with
high affinity to wild type NS1, however, lack affinity to
site-directed NS1 mutants deficient for cytotoxicity. Thus, casein
kinase II.alpha. binding to the region around S473 and tropomyosine
binding to the region around T363 of NS1 could be identified.
Analyses of NS1 impact to the host cell have presented multiple
effects, which could lead to cytostasis and/or induction of
cytolysis. Some of them might only be side effects of NS1
replicative functions, whereas others are induced specifically to
release progeny virus particles from infected cells. The latter NS1
activities are of particular interest to design new drugs. Besides
cell cycle arrest in S-phase of infected cells, expression of NS1
protein alone leads to dramatic morphological changes (cell
shrinking), manifested by a disorganization of the cytoskeleton in
susceptible cells. More detailed analyses have shown that after MVM
infection of A9 cells predominantly tropomyosin and vimentin
filaments are affected, while microtubles remain unaffected,
indicating a selective impact of the NS1 protein to the host cell.
In part, the dynamics of these cytoskeleton filaments seem to be
under regulation of PKC, which in turn are disregulated upon MVM
infection. More importantly, however, tropomyosin filments are
affected directly by complex formation of NS1 with CKII.alpha..
These investigations led to the assumption that tropomyosin, a
cytoskeleton component that is subject to alterations upon
transformation, is targeted by NS1/CKII.alpha. leading to cell
death and eventually cytolysis. In particular, while tropomyosin 2
(TM2) is genuine target of CKII.alpha. (the holoenzyme existing in
eukaryotic cells), the high affinity interaction with NS1 forming
the NS1/CKII.alpha. complex does not recognize TM2 as a substrate
anymore but is able to phosphorylate TM5 an alternative tropomyosin
isoform. In consequence the tropomyosin filament structure becomes
altered in the presence of the viral protein.
[0010] As previously mentioned, instead of its own enzymatic
functions, NS1 induced cytolysis is rather dependent on the
formation of a (multi)protein complex assembled by the viral
protein through protein/protein interactions with (pre-existing)
cellular polypeptides. Such protein complexes could have entirely
different catalytic activities from the purified NS1 protein as
characterized in extensive investigations. Particularly the NS1
interaction with the catalytic subunit of casein kinase II proposes
a variety of new options. In fact, it could be shown that casein
kinase II alters the substrate specificity in the presence of an
NS1-oligomer, using for instance empty MVM capsids as substrate,
which remain unaffected by recombinant highly active
CKII.alpha./.beta. complex. This observation leads to the
conclusion that novel cellular targets can be phosphorylated and
regulated through this NS1/CKII.alpha. complex leading to cell
death and cytolysis of MVM infected susceptible cells.
[0011] In addition to the interaction with a kinase, MVM NS1 has
been shown to bind to tropomyosin as well. In fibroblasts
Tropomyosin filaments are associated with filamentous actin. These
filaments can be composed of different isoforms (Tropomyosin 1 to
5), which share large homologies in the primary structure. As part
of the cytoskeleton, tropomyosin is also responsible for the
intracellular organization and despite there is little known so far
about the impact of tropomyosin for the organization of signaling
pathways, in analogy to microtubules they could serve as scaffold
proteins anchoring larger regulatory complexes at distinct
locations within the cytoplasm. With this background, the NS1
interaction with this cytoskeleton component might on the one hand
be important to target the (cytotoxic) NS1/CKII.alpha. complex to a
distinct location within an infected cell, on the other hand
tropomyosin itself might be a target for regulation by this "novel
kinase". Interestingly, recently evidence was obtained that
tropomyosin is differentially phosphorylated by CKII.alpha./.beta.
compared to NS1/CKII.alpha. in vitro and using cell lines
overexpressing mutant CKII.alpha. a different phosphorylation
pattern of tropomyosin in infected cells was observed. Moreover,
these cell lines showed a certain resistance towards MVM induced
alterations of the cytoskeleton and in consequence cell killing
compared to the parental A9 fibroblasts.
[0012] In regard to these results, it was proposed that NS1
mediated targeting of CKII.alpha. to tropomyosin leads to dramatic
morphological alterations of the host cell and eventually cell
death, which could be a prerequisite for MVM induced cell lysis. To
proof this hypothesis, constructs were generated which are able to
target wild type of endogenous CKII.alpha. to tropomyosin. These
constructs were tested for their impact on cell survival and it was
found that specific cytotoxicity could be induced by NS1 targeting
of CKII.alpha. to tropomyosin, i.e. that these constructs are
suitable for targeted gene therapy, preferably targeted cancer
therapy. The findings of these experiments also suggest that NS1
(or parts of it) mediates CKII kinase activity within the cell by
either one, (i) targeting the catalytic enzyme within the cell to
appropriate subcellular compartments and (ii) mediating the
substrate specificity of this enzyme.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows constructs to test putative oncotoxins for
their effects on host cells
[0014] FIG. 2 shows results of toxicity assays and colony formation
inhibition assays performed with the constructs described in
Example 1.
[0015] FIGS. 3 to 5 illustrate the potential properties of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The peptide of the invention is a fusion polypeptide
combining a binding site for a cytoskeleton component (e.g.
tropomyosin) (i.e. a targeting site), an effector protein, e.g. a
protein kinase, or a binding site for an effector protein, e.g., a
protein kinase (e.g. casein kinase 11; CKII.alpha.). Since both
elements can be very short (e.g., CKII.sub.B consists only of a
dekapeptide), the two elements can be spaced by a "stufferpeptide"
(e.g., enhanced green fluorescent protein; EGFP (SEQ ID NO: 1) to
enhance accessibility of both sites and stability of the toxin. The
peptide can be applied as a genetic element or a compound and
could/should harbor desired regulatory elements such as signals for
post-translational modifications (e.g. phosphorylation sites)
altering the specificity, additional signaling peptides (e.g. for
secretion), and/or degradation signals controlling the stability.
In order to increase specificity, the nucleic acid sequence
encoding the fusion polypeptide of the invention can be placed
under control of constitutive or inducible (including tissue
specific) promoters and packaged into recombinant particles
harboring targeting signals for appropriate cell/tissue
specificity.
[0017] Thus, in a first embodiment, the present invention relates
to a fusion polypeptide comprising (a) a binding site for a
cytoskeleton component and (b.sub.1) an effector protein or the
catalytic domain thereof or (b.sub.2) a binding site for said
effector protein.
[0018] Parts (a) and (b) may comprise wild type sequences as well
as sequences differing from the wild type sequences, e.g. by
deletion(s), substitution(s) and/or addition(s) of amino acids.
Such differences may result in peptides exhibiting improved or new
biological activities, e.g., an improved binding of a cytoskeleton
component.
[0019] Both parts (a) and (b) are preferably linked by covalent
bond. Alternatively, other non-covalent interactions between the
two elements (a) and (b) are possible. The orientation between the
two elements (a) and (b) is interchangeable. Preferably, part (a)
forms the N-terminal part of the fusion polypeptide.
[0020] In a preferred embodiment, both parts (a) and (b) of said
fusion protein are linked by a suitable (poly)peptide linker, e.g.,
ensuring (i) that parts (a) and (b) can interact with their
partners and/or (ii) that the fusion polypeptide has sufficient
stability. Suitable (poly)peptide linkers are known to the person
skilled in the art and, can very in length considerable. Examples
of suitable (poly)peptide linkers are EGFP, or other inert soluble
proteins such as croE, which are generally used to stabilize
peptides for immunization purposes. Alternatively, short random
polypeptide stretches can be sufficient to separate the two
elements (a) and (b).
[0021] Dramatic effects of targeting the catalytic subunit of
casein kinase II, CKII.alpha. to tropomyosin could be shown; see
Example 2. Similarly, since other cytoskeleton and/or nuclear
filaments are subject for regulation of their polymerization and
depolymerization properties, it is possible to design other fusion
polypeptides (toxins) analogous to the fusion polypeptide of
Example 1 containing alternative effector proteins or binding sites
thereof (e.g. other protein kinases, phosphatases, enzymes exerting
other post-translational modifications), which are able to
destabilize or stabilize the cytoskeleton or nuclear lamina
according to the desired effects. This effectors could either be
targeted to tropomyosin or alternatively to other cytoskeleton
filaments, lamins and/or regulatory components involved in
polymerization/depolymerization of the filaments, such as cofilin
or profilin. The latter seems to be involved in the polymerization
activity of tropomyosin and is subject to regulation through
PKC.lamda., which is subject to regulation by MVM NS1 protein. Both
effects, the targeting of CKII.alpha. to tropomyosin leading to
restructuring of tropomyosin and the reduction of polymerization
activity through PKC.lamda./profilin through NS1 are able to damage
the host cell and in consequence inducing cell death. Therefore, it
can be expected that the principle of the present invention is
useful for inducing destruction (or reorganization) of the
cytoskeleton by targeting alternative components (cf. FIG. 5).
[0022] Thus, cytoskeleton components useful for binding by the
fusion polypeptide of the invention comprise cytoskeleton filaments
like tropomyosin, actin, microtubules, intermediate filaments etc.,
lamins or regulatory proteins involved in cytoskeleton dynamics
(e.g., polymerization/depolymerization), e.g., cofilin or profilin)
with tropomyosin being preferred. Recent investigations have shown
that tropomyosin is not the only (cellular) target of CKII.alpha.
that is subject to differential phosphorylation in the presence of
NS1. Thus, particularly gelsolin was identified, an actin severing
protein (modulator of actin filaments) whose phosphorylation
pattern is altered by the complex formation of NS1 with
CKII.alpha., and tubulin. Moreover, it was found that viral NS2
proteins might be further candidates for altered phosphorylation of
CK.alpha. through interaction with NS1. Considering that MVM
capsids become substrate for CKII.alpha. in the presence of NS1 it
can be expected that the interaction of NS1 is a key-element for
MVM induced oncolytic activities. This conclusion is supported by
the findings that cell lines expressing a dominant-negative mutant
form of CKII.alpha. become highly resistant for virus induced
cytopathic effects.
[0023] Effector proteins useful for the fusion polypeptide of the
invention comprise protein kinases, phosphatases, enzymes exerting
other post-translational modifications etc. which are able to
destabilize or stabilize the cytoskeleton or nuclear lamina
according to the desired effects with casein kinase (CKII.alpha.)
being preferred.
[0024] In a more preferred embodiment of the fusion polypeptide of
the invention, the binding site is derived from parvovirus NS1,
e.g. the Tropomyosin binding region of TnT, or the Tropomyosin
binding subunit of Troponin.
[0025] In an even more preferred embodiment of the fusion
polypeptide of the invention, the binding site for the cytoskeleton
component comprises the amino acid sequence from positions 235 to
379 of NS1 (included in SEQ ID NO: 12) (Astell et al., 1983, Nucl.
Acids Res. 11, 999-1018).
[0026] Particularly preferred is a fusion polypeptide of the
invention, wherein part (b) is a binding site for casein kinase II
(CKII.alpha.) comprising the amino acid sequence DLEPDEELED (SEQ ID
NO: 2).
[0027] To enhance the specificity of the fusion polypeptide of the
invention as a toxin, e.g., for neoplastically transformed cells,
it might be desirable to include regulatory features. In the
experiments shown in Examples 1 and 2, the NS1 interaction domain
with tropomyosin contains PKC phosphorylation sites, of which T363
upon mutagenesis to alanine obliterates binding to TM and in
consequence reduces the toxic potential of NS1. Likewise, the other
domain of NS1 interacting with CKII.alpha. is phosphorylated by
PKC.lamda. at two amino acids T435 and S473, which seem to be
crucial for NS1 to induce morphological alterations. NS1 mutants
that abolish interaction with CKII.alpha. are rather well tolerated
by the host cell in comparison to the wild type polypeptide. Since
members of the PKC family are often upregulated upon
transformation, it seems possible that phosphorylation-dependent
interaction with the appropriate cellular proteins is a feature for
the oncolytic activity of autonomous parvoviruses. Thus, the fusion
polypeptide of the invention could be attributed with this or
similar features in order to render the interaction-site cell type
specific, particularly for transformed cells. Such signals may
consist of specific phosphorylation sites for target kinases, but
also for acetylation-, methylation-, myristilation-, palmitylation-
or other signals for post-translational modifications. In addition,
the fusion polypeptide of the invention could contain signals,
which induce conformational changes in order to expose the
interaction sites upon a desired signal, analogous to the
activation cascade of protein kinase C, or it could contain
additional targeting and/or anchoring or secretion signal, such as
NLS, NES, transmembrane domains, etc. (FIG. 4).
[0028] Thus, in a further preferred embodiment of the fusion
polypeptide of the invention, modifications are present in parts
(a) and/or (b) inducing or enhancing morphological changes of the
host cell.
[0029] The fusion polypeptide of the invention may be used directly
or it can be supplied to the cells by intracellular expression and
subsequent secretion. Thus, the present invention also relates to a
nucleic acid sequence encoding a fusion polypeptide of the
invention as well as a recombinant vector containing said nucleic
acid sequence. Preferably, the recombinant vectors are plasmids,
cosmids, viruses, bacteriophages, cells, and other vectors usually
used in the field of genetic engineering. Vectors suitable for use
in the present invention include, but are not limited to the
CMV-based expression vector for expression in mammalian cells and
baculovirus-derived vectors for expression in insect cells.
Preferably, the nucleic acid molecule of the invention is
operatively linked to the regulatory elements in the recombinant
vector of the invention that guarantee the transcription and
synthesis of an mRNA in prokaryotic and/or eukaryotic cells that
can be translated. The nucleotide sequence to be transcribed can be
operably linked to a promoter like a T7, metallothionein I,
polyhydrin, or CMV early promotor.
[0030] In a further embodiment, the present invention relates to
recombinant host cells transiently or stably containing the nucleic
acid sequences or vectors of the invention. A host cell is
understood to be an organism that is capable to take up in vitro
recombinant DNA and, if the case may be, to synthesize the fusion
polypeptides encoded by the nucleic acid molecules of the
invention. Preferably, these cells are prokaryotic or eukaryotic
cells, for example mammalian cells, bacterial cells, insect cells
or yeast cells.
[0031] The present invention also relates to an antibody that binds
specifically to a fusion polypeptide of the invention. The term
"antibody", preferably, relates to antibodies that consist
essentially of pooled monoclonal antibodies with different epitopic
specificities, as well as distinct monoclonal antibody
preparations. Monoclonal antibodies are made from an antigen
containing (fragments of) the polypeptides of the invention by
methods well known to those skilled in the art (see, e.g., Kohler
et al., Nature 256 (1975), 495). As used herein, the term
"antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include
intact molecules as well as antibody fragments (such as, for
example, Fab and F(ab') 2 fragments) which are capable of
specifically binding to protein. Fab and f(ab').sub.2 fragments
lack the Fc fragment of intact antibody, clear more rapidly from
the circulation, and may have less non-specific tissue binding than
an intact antibody. (Wahl et al., J. Nucl. Med. 24: 316-325
(1983)). Thus, these fragments are preferred, as well as the
products of a FAB or other immunoglobulin expression library.
Moreover, antibodies of the present invention include chimerical,
single chain, and humanized antibodies.
[0032] For certain purposes, e.g. diagnostic methods or for
assaying the half-life or clearance of the fusion polypeptide
within an organism, the antibody of the present invention can be
detectably labelled, for example, with a radioisotope, a
bioluminescent compound, a chemiluminescent compound, a fluorescent
compound, a metal chelate, or an enzyme.
[0033] The invention also relates to a transgenic non-human animal
such as transgenic mouse, rats, hamsters, dogs, monkeys, rabbits,
pigs, C. elegans and fish such as torpedo fish comprising a nucleic
acid molecule or vector of the invention, preferably wherein said
nucleic acid molecule or vector is stably integrated into the
genome of said non-human animal, preferably such that the presence
of said nucleic acid molecule or vector leads to the expression of
a fusion polypeptide of the invention. Said animal may have one or
several copies of the same or different nucleic acid molecules
encoding one or several forms of said fusion polypeptide. This
animal has numerous utilities, including as a research model for
development/progression of carcinomas and therefore, presents a
novel and valuable animal in the development of therapies,
treatment, etc. for carcinomas. Accordingly, in this instance, the
non-human mammal is preferably a laboratory animal such as a mouse
or rat. It might be also desirable to inactivate expression or
function of the fusion polypeptide at a certain stage of
development and/or life of the transgenic animal. This can be
achieved by using, for example, tissue specific, developmental
and/or cell regulated and/or inducible promoters which drive the
expression of, e.g., an antisense or ribozyme directed against the
RNA transcript encoding the fusion polypeptide. A suitable
inducible system is for example tetracycline-regulated gene
expression as described, e.g., by Gossen and Bujard (Proc. Natl.
Acad. Sci. 89 USA (1992), 5547-5551) and Gossen et al. (Trends
Biotech. 12 (1994), 58-62).
[0034] Methods for the production of a transgenic non-human animal
of the present invention, preferably transgenic mouse, are well
known to the person skilled in the art. Such methods, e.g.,
comprise the introduction of a nucleic acid sequence or vector of
the invention into a germ cell, an embryonic cell, stem cell or an
egg or a cell derived therefrom. Production of transgenic embryos
and screening of those can be performed, e.g., as described by A.
L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford
University Press. The DNA of the embryonal membranes of embryos can
be analyzed using, e.g., Southern blots with an appropriate
probe.
[0035] Due to the intrinsic cytotoxicity of the fusion polypeptide
of the invention targeted at transformed cells, it can be used as
an oncotoxin. The fusion polypeptide is able to target effector
proteins (modifying cellular enzymes) to distinct targets within
the cell in order to destroy or at least inhibit proliferation.
Designed in the Examples to specifically kill cancer cells,
however, the fusion polypeptide of the invention has a wider
spectrum of action. Particularly, besides transformed cells, other
aberrant cell populations (e.g. HIV infected cells) can be targeted
and destroyed in a similar way, considering the specificity can be
granted or at least enhanced compared to the healthy population.
The fusion polypeptide of the invention has several advantages. As
a genetic element, the specificity can be reached by targeted
genetics and gene expression. Moreover, as exemplified with the
parvovirus NS1 protein, binding sites and location within the cell
can be further subject for regulation in order to achieve efficient
cell killing in the desired environment. Last but not least, it is
also possible to apply the fusion polypeptide of the invention
directly as a compound attributed with the appropriate features.
Treatment can be given to cell cultures or disease bearing
organisms.
[0036] Thus, the present invention also relates to a pharmaceutical
composition comprising a fusion polypeptide, nucleic acid sequence
or recombinant vector of the invention and a pharmaceutically
acceptable excipient, diluent or carrier. Examples of suitable
pharmaceutical carriers etc. are well known in the art and include
phosphate buffered saline solutions, water, emulsions, such as
oil/water emulsions, various types of wetting agents, sterile
solutions etc. Such carriers can be formulated by conventional
methods and can be administered to the subject at a suitable dose.
Administration of the suitable compositions may be effected by
different ways, e.g. by intravenous, intraperetoneal, subcutaneous,
intramuscular, topical or intradermal administration. The route of
administration, of course, depends on the nature of the disease,
its localisation and the kind of compound contained in the
pharmaceutical composition. The dosage regimen will be determined
by the attending physician and other clinical factors. As is well
known in the medical arts, dosages for any one patient depends on
many factors, including the patient's size, body surface area, age,
sex, the particular compound to be administered, time and route of
administration, the kind and stage of a disease, general health and
other drugs being administered concurrently.
[0037] The delivery of the nucleic acid sequences of the invention
can be achieved by direct application or, preferably, by using a
recombinant expression vector such as a chimeric virus containing
these compounds or a colloidal dispersion system. Direct
application to the target site can be performed, e.g., by ballistic
delivery, as a colloidal dispersion system or by catheter to a site
in artery. The colloidal dispersion systems which can be used for
delivery of the above nucleic acid sequences include macromolecule
complexes, nanocapsules, microspheres, beads and lipid-based
systems including oil-in-water emulsions (mixed), micelles,
liposomes and lipoplexes, The preferred colloidal system is a
liposome. The composition of the liposome is usually a combination
of phospholipids and steroids, especially cholesterol. The skilled
person is in a position to select such liposomes that are suitable
for the delivery of the desired nucleic acid sequence.
Organ-specific or cell-specific liposomes can be used in order to
achieve delivery only to the desired tissue. The targeting of
liposomes can be carried out by the person skilled in the art by
applying commonly known methods. This targeting includes passive
targeting (utilizing the natural tendency of the liposomes to
distribute to cells of the RES in organs which contain sinusoidal
capillaries) or active targeting (for example by coupling the
liposome to a specific ligand, e.g., an antibody, a receptor,
sugar, glycolipid, protein etc., by well known methods). In the
present invention monoclonal antibodies are preferably used to
target liposomes to specific tissues, e.g., tumors, via specific
cell-surface ligands.
[0038] Preferred recombinant vectors useful for gene therapy are
viral vectors, e.g. adenovirus, herpes virus, vaccinia, Measles
virus, Parvovirus, or an RNA virus such as a retrovirus. Even more
preferably, the retroviral vector is a derivative of a murine or
avian retrovirus. Examples of such retroviral vectors which can be
used in the present invention are: Moloney murine leukemia virus
(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary
tumor virus (MuMTV) and Rous sarcoma virus (RSV). Most preferably,
a non-human primate retroviral vector is employed, such as the
gibbon ape leukemia virus (GaLV), providing a broader host range
compared to murine vectors. Since recombinant retroviruses are
defective, assistance is required in order to produce infectious
particles. Such assistance can be provided, e.g., by using helper
cell lines that contain plasmids encoding all of the structural
genes of the retrovirus under the control of regulatory sequences
within the LTR. Suitable helper cell lines are well known to those
skilled in the art. Said vectors can additionally contain a gene
encoding a selectable marker so that the transduced cells can be
identified. Moreover, the retroviral vectors can be modified in
such a way that they become target specific. This can be achieved,
e.g., by inserting a polynucleotide encoding a sugar, a glycolipid,
or a protein, preferably an antibody. Those skilled in the art know
additional methods for generating target specific vectors. Further
suitable vectors and methods for in vitro- or in vivo-gene therapy
are described in the literature and are known to the persons
skilled in the art; see, e.g., WO 94/29469 or WO 97/00957.
[0039] In order to achieve expression only in the target organ,
e.g., a tumor to be treated, the nucleic acid sequences of the
present invention can be linked to a tissue specific promoter and
used for gene therapy. Such promoters are well known to those
skilled in the art (see e.g. Zimmermann et al., (1994) Neuron 12,
11-24; Vidal et al.; (1990) EMBO J. 9, 833-840; Mayford et al.,
(1995), Cell 81, 891-904; Pinkert et al., (1987) Genes & Dev.
1, 268-76). For the treatment of a colon cancer, the use of a
wnt-pathway specific promoter (Korinek et al., 1997, Science 275,
1784-1787) is preferred, for the HIV treatment use of a
tat-responsive element. In addition, depending on the particular
needs alternative available or newly designed promoter/enhancer
elements can be used to drive expression of the nucleic acid
sequence of the invention.
[0040] The present invention also relates to the use of the above
compounds of the invention for the preparation of a pharmaceutical
composition for the treatment of a disease associated with the
presence of an aberrant cell population. Preferred diseases are
cancer and AIDS. Other diseases could include the genetic diseases
altering the cellular structure such as the
Wiscott-Aldrich-Syndrome, Cystis Fibrosis, or chronic viral
diseases such as Heptatitis B and Hepatitis C.
[0041] Finally, the present invention also relates to the targeting
and mediating of an effector protein, e.g., CKII.alpha. (activity)
by a protein like the NS1 protein or submolecular parts thereof to
candidate cellular proteins such as tropomyosin, tubulin and
gelsolin.
[0042] The following Examples illustrate the invention.
EXAMPLE 1
[0043] Generation of Fusion Polypeptide Constructs for Testing
Putative Oncotoxins for their Effects on Mammalian Cells
[0044] Given the supposition that NS1 works as a scaffold protein,
connecting the catalytic subunit of casein kinase II (CKII.alpha.)
to tropomyosin, artificial peptides were designed harboring the
tropomyosin binding region of MVM NS1 and connecting either
CKII.alpha. (or variants thereof) or just a known CKII.alpha.
binding site (FIG. 1). PCR-derived fragments composed of a
tropomyosin binding site (TM.sub.B) derived from the parvovirus
MVMp NS1 protein (amino acids 235 to 279), the stabilizer
polypeptide EGFP derived from pEGFP (Becton Dickinson, Heidelberg),
and either a casein kinase II.alpha. binding site (derived from
CKII.beta.: DLEPDEELED) or the functional casein kinase 11
catalytic subunit CKII.alpha. (NCB1 L15618) isolated from the mouse
fibroblast cell line A9. (CKII.sub.B) were cloned directly into
pCR3.1 (Invitrogen, Karlsruhe), due to 3'adenosinetriphosphate
overhangs generated by Taq-polymerase (see Annex 1). EGFP, an in
eukaryotic cells tolerated protein, serves as a spacer and to
stabilize the fusion polypeptide. The plasmid constructs were then
transfected in eucaryotic cells A9, HEK 293 and the effector
proteins were expressed under the control of the cytomegalo virus
early promoter (PCMV). In addition, the constructs contain a
neomycin-resistance gene under the control of SV40
promoter/enhancer (P.sub.SV40/ori), which allows for selection of
transfected cells by their achieved resistance towards the drug
G418. PCR fragments harboring the desired properties are directly
ligated into the linearized pCR3.1 vectors, which contain 3'
terminal T-overhangs according to the manufacturer's suggestions
(Invitrogen, Karlsruhe). These expression plasmids allow to
determine toxicity of an appropriate gene by colony formation
inhibition assays using G418 sensitive cell lines.
[0045] The following effector constructs were generated and
analyzed for their impact on colony formation inhibition:
TM.sub.B-CKII.alpha. (the catalytic subunit of casein kinase II
(CKII.alpha.) linked to a tropomyosin binding site (derived from
parvovirus MVM NS1 protein) spaced by GFP)) and TM.sub.B-CKII.sub.B
(an adaptor construct harboring the binding sites for tropomyosin
as well as a casein kinase II (CKII.alpha.) binding site). The two
binding sites are fused to the enhanced green fluorescent protein
(EGFP). As negative controls, the following pseudo-effector
constructs were generated: GFP-CKII.alpha. (casein kinase II.alpha.
linked to GFP without a tropomyosin binding site), TM.sub.B-E81A
(tropomyosin binding site linked through GFP to a catalytic
inactive casein kinase II.alpha.), TM.sub.B-GFP (tropomyosin
binding site of NS1 linked to GFP without CKII.alpha. or
CKII.sub.B).
EXAMPLE 2
[0046] Toxicity Assays
[0047] Colony formation inhibition assays were performed with the
constructs described in Example 1. A9 or HEK293 (2.times.10.sup.5
cells per 25 cm.sup.2) were transfected with 10 .mu.g plasmid DNA
using 25 .mu.l lipofectamin in DMEM without serum according to the
manufacturer's conditions (Invitrogen). After 5 hr incubation
transfection medium was replaced with DMEM containing 10% FBS and
cells were grown for additional 48 h in absence of G418 before
subdividing into 150 cm.sup.2 plates where transfected cells were
selected for by addition of 400 .mu.g/ml G418 (SIGMA, Taufkirchen).
Growing colonies were fixed and stained according to McCoy after
two to three weeks growth under selective pressure. Two
representative experiments are shown in FIG. 2a and FIG. 2b. While
expression of the two effector proteins (TM.sub.B=CKII.alpha. (FIG.
2a) and TM.sub.B=CKII.sub.B (FIG. 2b) allowed only few colonies to
be generated in A9 cells in comparison to the control peptides,
almost similar transfectants were generated in a low passage HEK293
cell lines, reflecting the selective toxicity of the fusion
polypeptide of the invention.
[0048] Thus, in the presence of the designed toxin (e.g.
TM.sub.B-GFP-CKII.alpha. or TM.sub.B-GFP-CKII.sub.B), hardly any
colonies could be obtained after transfection of the MVM
susceptible fibroblast cell line A9, while the transfections of the
plasmids expressing the control peptides (peptides that do not
connect CKII.alpha.) generated >2000 colonies under G418
selection. It should be mentioned that all transfections delivered
green fluorescent cells 2 days post transfection, suggesting that
the proteins were indeed expressed during a certain period.
Interestingly, with TM.sub.B-GFP-CKII.sub.B significantly less
colonies were obtained than with GFP-NS1.sub.wt (data not shown),
suggesting that in absence of additional regulatory elements
present within the NS1 coding sequence, the newly designed toxin is
more effective than the original viral protein. In contrast to the
susceptible A9 cells, transfection of the effector constructs
TM.sub.B-CKII.alpha. or TM.sub.B-GFP-CKII.sub.B produced almost the
same amounts of colonies in HEK293 cells, demonstrating that the
newly designed toxins exert cell type specificity.
EXAMPLE 3
[0049] Generation of Semi-Synthetic Toxins by Chimeric PCR
[0050] Fusion constructs are generated by consecutive PCR reactions
using overlapping primer pairs. In a first reaction the individual
PCR-elements generated: TM.sub.B(GFP): Lefthand primer A
5'-GATATCCCATGGGGAAAACTAACTTTTTAAAAGAAGGCGA-3' (SEQ ID NO: 3) with
righthand primer B: 5'-TCCTCGCCCTTGCTCACCATATGGCAACTTAACATAGGT-3'
(SEQ ID NO: 4) using pdBMVp (Kestler et al, 1999) as a template.
(TM.sub.B)-GFP.CKII.sub.B: C:
5'-ACTATGTTAAAGTTTGCCATATGGTGAGCAAGGGCGAGGA-3' (SEQ ID NO: 5) with
D: 5'-GCGGCCGCTCTAGATTAATCTTCCAATTCTTCATCGGGTTCCAAATCCCTCC
GATGCTTGTACAGCTCGTCCATGCCGAG-3' (SEQ ID NO: 6) using pEGFP (Becton
Dickinson) as a template. GFP-(CKII.alpha.): E:
5'-CCCGGGATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGG-3' (SEQ ID NO: 7) and
F: 5'-TCCTCGCCCTTGCTCACCATCTGCTGAGCGCCAGCGGCAGG-3' (SEQ ID NO: 8)
using pEGFP as a template. (TM.sub.B)-GFP-(CKII.alpha.): Primer A
and F using pEGFP as a template (GFP)-CKII.alpha.(wt or E81A): G:
5'-CTGCCGCTGGCGCTCAGCAGATGGTGAGCAAGGGCGAGGA-3' (SEQ ID NO: 9) and
H: 5'-GCGGCCGCTTACTGCTGAGCGCCAGCGGCAGCTGGTACGG-3'' (SEQ ID NO: 10)
using pCR2.1:mCKII.alpha. or pCR2.1:CKII-E81A, respectively (Nuesch
unpublished) as templates. (TM.sub.B)-GFP: primer C and I:
5'-ACGGTCTCGATGAGCGACCGGCGCTCAGTTGG-3' (SEQ ID NO: 11) with pEGFP
as a template.
[0051] In a second PCR two individual elements were combined to a
fusion-protein and amplified with N- and C-terminal primers: [0052]
TM.sub.B-GFP; TM.sub.B-(GFP) with (TM.sub.B)-GFP using primer A and
I. [0053] TM.sub.B-CKII.sub.B: TM.sub.B-(GFP) with
(TM.sub.B)-GFP-CKII.sub.B using primers A and D. [0054]
GFP-CKII.alpha.: GFP-(CKII.alpha.) with (GFP)--CKII.alpha.(wt)
using primers E and H. [0055] (TM.sub.B)-GFP-(CKII.alpha.):
TM.sub.B-(GFP) with GFP-(CKII.alpha.) using primers A and F.
[0056] In a third PCR the remaining triple fusion constructs were
generated: [0057] TM.sub.B-CKII.alpha.(wt):
TM.sub.B-GFP-(CKII.alpha.) with (GFP)--CKII.alpha.(wt) using
primers A and H. [0058] TM.sub.B-CKII.alpha.(E81A):
TM.sub.B-GFP-(CKII.alpha.) with (GFP)--CKII.alpha.(E81A) using
primers A and H.
[0059] The final PCT+R constructs [TM.sub.B-GFP,
TM.sub.B-CKII.sub.B, GFP-CKII.alpha., TM.sub.B-CKII.alpha.(wt), AND
TM.sub.B-CKII.alpha.(E81A)] were directly ligated into linearized
pCR3.1 according to the manufacture's conditions (Invitrogen) and
tested for their properties by sequencing (Microsynth GmbH, Balgach
Switzerland).
Sequence CWU 1
1
12 1 4 PRT Artificial Sequence Synthetic construct 1 Glu Gly Phe
Pro 1 2 10 PRT Artificial Sequence Synthetic Construct 2 Asp Leu
Glu Pro Asp Glu Glu Leu Glu Asp 1 5 10 3 40 DNA Artificial Sequence
Synthetic Construct 3 gatatcccat ggggaaaact aactttttaa aagaaggcga
40 4 39 DNA Artificial Sequence Synthetic Construct 4 tcctcgccct
tgctcaccat atggcaactt aacataggt 39 5 40 DNA Artificial Sequence
Synthetic Construct 5 actatgttaa agtttgccat atggtgagca agggcgagga
40 6 78 DNA Artificial Sequence Synthetic Construct 6 gcggccgctc
tagattaatc ttccaattct tcatcgggtt ccaaatccct ccgatgcttg 60
tacagctcgt ccatgccg 78 7 40 DNA Artificial Sequence Synthetic
Construct 7 cccgggatgg tgagcaaggg cgaggagctg ttcaccgggg 40 8 41 DNA
Artificial Sequence Synthetic Construct 8 tcctcgccct tgctcaccat
ctgctgagcg ccagcggcag g 41 9 40 DNA Artificial Sequence Synthetic
Construct 9 ctgccgctgg cgctcagcag atggtgagca agggcgagga 40 10 40
DNA Artificial Sequence Synthetic Construct 10 gcggccgctt
actgctgagc gccagcggca gctggtacgg 40 11 32 DNA Artificial Sequence
Synthetic Construct 11 acggtctcga tgagcgaccg gcgctcagtt gg 32 12
721 PRT Minute Virus of MIce (MVM) 12 Met Ile Ser Gly Ser Gly Ser
Leu Asn Gln Gly Ala Lys Arg Lys Trp 1 5 10 15 Ala Trp Phe Lys Val
Tyr Lys Gln Leu Leu Lys Ser Val Thr Tyr Leu 20 25 30 Phe Phe His
Ser Val Ser Arg Asp Ala Gln Lys Glu Ser Asn Gln Leu 35 40 45 Thr
Met Ala Gly Asn Ala Tyr Ser Asp Glu Val Leu Gly Ala Thr Asn 50 55
60 Trp Leu Lys Glu Lys Ser Asn Gln Glu Val Phe Ser Phe Val Phe Lys
65 70 75 80 Asn Glu Asn Val Gln Leu Asn Gly Lys Asp Ile Gly Trp Asn
Ser Tyr 85 90 95 Lys Lys Glu Leu Gln Glu Asp Glu Leu Lys Ser Leu
Gln Arg Gly Ala 100 105 110 Glu Thr Thr Trp Asp Gln Ser Glu Asp Met
Glu Trp Glu Thr Thr Val 115 120 125 Asp Glu Met Thr Lys Lys Gln Val
Phe Ile Phe Asp Ser Leu Val Lys 130 135 140 Lys Cys Leu Phe Glu Val
Leu Asn Thr Lys Asn Ile Phe Pro Gly Asp 145 150 155 160 Val Asn Trp
Phe Val Gln His Glu Trp Gly Lys Asp Gln Gly Trp His 165 170 175 Cys
His Val Leu Ile Gly Gly Lys Asp Phe Ser Gln Ala Gln Gly Lys 180 185
190 Trp Trp Arg Arg Gln Leu Asn Val Tyr Trp Ser Arg Trp Leu Val Thr
195 200 205 Ala Cys Asn Val Gln Leu Thr Pro Ala Glu Arg Ile Lys Leu
Arg Glu 210 215 220 Ile Ala Glu Asp Asn Glu Trp Val Thr Leu Leu Thr
Tyr Lys His Lys 225 230 235 240 Gln Thr Lys Lys Asp Tyr Thr Lys Cys
Val Leu Phe Gly Asn Met Ile 245 250 255 Ala Tyr Tyr Phe Leu Thr Lys
Lys Lys Ile Ser Thr Ser Pro Pro Arg 260 265 270 Asp Gly Gly Tyr Phe
Leu Ser Ser Asp Ser Gly Trp Lys Thr Asn Phe 275 280 285 Leu Lys Glu
Gly Glu Arg His Leu Val Ser Lys Leu Tyr Thr Asp Asp 290 295 300 Met
Arg Pro Glu Thr Val Glu Thr Thr Val Thr Thr Ala Gln Glu Thr 305 310
315 320 Lys Arg Gly Arg Ile Gln Thr Lys Lys Glu Val Ser Ile Lys Thr
Thr 325 330 335 Leu Lys Glu Leu Val His Lys Arg Val Thr Ser Pro Glu
Asp Trp Met 340 345 350 Met Met Gln Pro Asp Ser Tyr Ile Glu Met Met
Ala Gln Pro Gly Gly 355 360 365 Glu Asn Leu Leu Lys Asn Thr Leu Glu
Ile Cys Thr Leu Thr Leu Ala 370 375 380 Arg Thr Lys Thr Ala Phe Asp
Leu Ile Leu Glu Lys Ala Glu Thr Ser 385 390 395 400 Lys Leu Thr Asn
Phe Ser Leu Pro Asp Thr Arg Thr Cys Arg Ile Phe 405 410 415 Ala Phe
His Gly Trp Asn Tyr Val Lys Val Cys His Ala Ile Cys Cys 420 425 430
Val Leu Asn Arg Gln Gly Gly Lys Arg Asn Thr Val Leu Phe His Gly 435
440 445 Pro Ala Ser Thr Gly Lys Ser Ile Ile Ala Gln Ala Ile Ala Gln
Ala 450 455 460 Val Gly Asn Val Gly Cys Tyr Asn Ala Ala Asn Val Asn
Phe Pro Phe 465 470 475 480 Asn Asp Cys Thr Asn Lys Asn Leu Ile Trp
Val Glu Glu Ala Gly Asn 485 490 495 Phe Gly Gln Gln Val Asn Gln Phe
Lys Ala Ile Cys Ser Gly Gln Thr 500 505 510 Ile Arg Ile Asp Gln Lys
Gly Lys Gly Ser Lys Gln Ile Glu Pro Thr 515 520 525 Pro Val Ile Met
Thr Thr Asn Glu Asn Ile Thr Val Val Arg Ile Gly 530 535 540 Cys Glu
Glu Arg Pro Glu His Thr Gln Pro Ile Arg Asp Arg Met Leu 545 550 555
560 Asn Ile His Leu Thr His Thr Leu Pro Gly Asp Phe Gly Leu Val Asp
565 570 575 Lys Asn Glu Trp Pro Met Ile Cys Ala Trp Leu Val Lys Asn
Gly Tyr 580 585 590 Gln Ser Thr Met Ala Ser Tyr Cys Ala Lys Trp Gly
Lys Val Pro Asp 595 600 605 Trp Ser Glu Asn Trp Ala Glu Pro Lys Val
Pro Thr Pro Ile Asn Leu 610 615 620 Leu Gly Ser Ala Arg Ser Pro Phe
Thr Thr Pro Lys Ser Thr Pro Leu 625 630 635 640 Ser Gln Asn Tyr Ala
Leu Thr Pro Leu Ala Ser Asp Leu Glu Asp Leu 645 650 655 Ala Leu Glu
Pro Trp Ser Thr Pro Asn Thr Pro Val Ala Gly Thr Ala 660 665 670 Glu
Thr Gln Asn Thr Gly Glu Ala Gly Ser Lys Ala Cys Gln Asp Gly 675 680
685 Gln Leu Ser Pro Thr Trp Ser Glu Ile Glu Glu Asp Leu Arg Ala Cys
690 695 700 Phe Gly Ala Glu Pro Leu Lys Lys Asp Phe Ser Glu Pro Leu
Asn Leu 705 710 715 720 Asp
* * * * *