U.S. patent application number 10/638537 was filed with the patent office on 2004-06-10 for nucleic acid construct for expressing active substances which can be activated by proteases, and preparation and use.
Invention is credited to Heidtmann, Hans Heinrich, Mueller, Rolf, Sedlacek, Hans-Harald.
Application Number | 20040110682 10/638537 |
Document ID | / |
Family ID | 7817422 |
Filed Date | 2004-06-10 |
United States Patent
Application |
20040110682 |
Kind Code |
A1 |
Heidtmann, Hans Heinrich ;
et al. |
June 10, 2004 |
Nucleic acid construct for expressing active substances which can
be activated by proteases, and preparation and use
Abstract
The invention relates to a nucleic acid construct for expressing
an active substance which is activated by an enzyme which is
released from mammalian cells, which construct comprises the
following components: a) at least one promoter element, b) at least
one DNA sequence which encodes an active compound (protein B), c) a
least one DNA sequence which encodes an amino acid sequence (part
structure C) which can be cleaved specifically by an enzyme which
is released from a mammalian cell, and d) at least one DNA sequence
which encodes a peptide or protein (part structure D) which is
bound to the active compound (protein B) by way of the cleavable
amino acid sequence (part structure C) and inhibits the activity of
the active compound (protein B), and also to the use of the nucleic
acid construct for preparing a drug for treating diseases.
Inventors: |
Heidtmann, Hans Heinrich;
(Marburg, DE) ; Mueller, Rolf; (Marburg, DE)
; Sedlacek, Hans-Harald; (Marburg, DE) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
1666 K STREET,NW
SUITE 300
WASHINGTON
DC
20006
US
|
Family ID: |
7817422 |
Appl. No.: |
10/638537 |
Filed: |
August 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10638537 |
Aug 12, 2003 |
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09256237 |
Feb 24, 1999 |
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6670147 |
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09256237 |
Feb 24, 1999 |
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09008308 |
Jan 16, 1998 |
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6080575 |
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Current U.S.
Class: |
424/85.4 ;
514/13.3; 514/13.6; 514/14.1; 514/14.3; 514/14.4; 514/14.7;
514/20.3; 514/3.7; 514/44R |
Current CPC
Class: |
A61K 48/00 20130101;
C12Y 304/21006 20130101; C12N 9/6432 20130101; C12N 2830/008
20130101; C12N 15/63 20130101; C12N 15/85 20130101; C12N 2840/20
20130101; C12N 2830/007 20130101 |
Class at
Publication: |
514/012 ;
514/044 |
International
Class: |
A61K 048/00; A61K
038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 16, 1997 |
DE |
197 01 141.1 |
Claims
What is claimed is:
1. A nucleic acid construct for expressing an active substance
which is activated by an enzyme which is released from mammalian
cells, wherein said construct comprises the following nucleic acid
sequences in the following order: a) at least one promoter element
operably linked to; b) at least one nucleic acid sequence which
encodes an active compound, wherein said active compound is
endogenous to mammals, operably linked to; c) at least one nucleic
acid sequence which encodes an amino acid sequence cleavable
specifically by an enzyme which is released from a mammalian cell,
operably linked to; d) at least one DNA sequence which encodes a
polypeptide which is bound to said active compound by said
cleavable amino acid sequence and inhibits the activity of said
active compound, and wherein said nucleic acid component c) does
not naturally occur as operably linking said nucleic acid sequence
b) to said nucleic acid d).
2. A nucleic acid construct as claimed in claim 1, wherein said
enzyme is a protease.
3. A nucleic acid construct as claimed in claim 1, wherein said
enzyme is a prostate specific antigen, a plasminogen activator, a
cathepsin or a matrix metalloproteinase.
4. A nucleic acid construct as claimed in claim 1, wherein said
mammalian cells are tumor cells, leukemia cells, endothelial cells,
macrophages, lymphocytes, muscle cells, epithelial cells, glia
cells, synovial cells or virus-infected cells.
5. A nucleic acid construct as claimed in claim 1, wherein said
nucleic acid construct further comprises a nucleic acid sequence
operably linked to said construct of claim 1, wherein said nucleic
acid sequence encodes a ligand which binds said active compound to
a target structure.
6. A nucleic acid construct as claimed in claim 1, wherein said
nucleic acid sequences b) and d) of claim 1 encode parts of a
natural precursor of a protein active compound, wherein the nucleic
acid sequence encoding the cleavage sequence naturally occurring
between said nucleic acid sequences b) and d) has been replaced by
said nucleic acid sequence c), which does not naturally occur
between said nucleic acid sequences b) and d).
7. A nucleic acid construct as claimed in claim 1, wherein said
polypeptide encoded by said nucleic acid sequence d) is part of a
natural precursor of a protein active compound.
8. A nucleic acid construct as claimed in claim 1, wherein said
construct is operably inserted into a plasmid or a viral
vector.
9. A nucleic acid construct as claimed in claim 1, wherein said
nucleic acid sequence a) is a promoter sequence which can be
activated nonspecifically, cell-specifically, virus-specifically,
metabolically, cell cycle-specifically or by tetracycline.
10. A nucleic acid construct as claimed in claim 1, wherein said
nucleic acid sequence a) comprises at least two identical or two
different promoter sequences.
11. A nucleic acid construct as claimed claim 9, wherein said
nucleic acid sequence a) is activated in endothelial cells, in
cells adjoining activated endothelial cells, in muscle cells, in
leukemia cells, in tumor cells, in glia cells, in lymphocytes, in
macrophages or in synovial cells.
12. A nucleic acid construct as claimed in claim 1, wherein said
active compound activates or inhibits a biological activation
cascade or is an active component of this cascade, or activates or
inhibits the coagulation system, activates fibrinolysis, activates
the complement system or activates the kinin system, or is an
enzyme which converts the inactive precursor of a pharmacological
substance into the active substance, or which itself is a
pharmacologically active substance.
13. A nucleic acid construct as claimed in claim 12, wherein said
active compound is a coagulation factor which is selected from the
group consisting of thrombin, factor Va, factor VIIa, factor IXa,
factor Xa, TF coagulation-active fragments or factor XIIa; thrombin
which is mutated in the region of the Arg-Thr cleavage site (amino
acid position 327/328); a fibrinolytic protein which is selected
from urokinase, tPA or functional hybrids thereof; a complement
factor which is selected from CVF, C3b or functional cleavage
products thereof; an antithrombotic protein which is selected from
protein C, C-1S inhibitor, .alpha.1-antitrypsin, hirudin, AT-III,
TFPI, PAI-1, PAI-2 or PAI-3; a kallikrein; a cytostatic, cytotoxic
or inflammation-eliciting protein; an antiangiogenic protein; an
immunomodulatory protein; an antiinflammatory protein; a protein
which relieves damage to the nervous system; a protein which
inhibits or neutralizes the neurotoxic effect of TNF.alpha.; an
angiogenesis-stimulating protein; a hypotensive protein; an
antiviral protein; a cytokine; an interferon; a tumor necrosis
factor; oncostatin M or LIF; a cytokine receptor; the moiety of a
cytokine receptor which is external to the cell; a cytokine
antagonist; a growth factor; a growth factor receptor; the moiety
of a growth factor receptor which is external to the cell; a
chemokine; angiostatin; platelet factor 4; TIMP-1, TIMP-2 or
TIMP-3; a nitroreductase; a .beta.-glucuronidase; a
carboxypeptidase; a .beta.-lactamase; a cytosine deaminase; a
catalase; a peroxidase; a phosphatase; an oxidase; kallikrein or an
endothelial cell nitric oxide synthase.
14. A nucleic acid construct as claimed in claim 1, which further
comprises a nucleic acid sequence b') which encodes a ligand which
binds to a cell membrane receptor, a cell membrane antigen, a cell
membrane-located adhesion molecule, or to the extracellular matrix
or component thereof.
15. A nucleic acid construct as claimed in claim 14, wherein said
ligand is an antibody or an antibody fragment which binds
specifically to a cell membrane antigen or to an antigen on the
extracellular matrix, or is a polypeptide which binds to receptor
on the cell membrane wherein said polypeptide is a growth factor, a
cytokine, an interferon, a tumor necrosis factor, a chemokine, a
receptor-binding part sequence of these ligands, a peptide hormone,
angiotensin, kinin, folic acid, an adhesion molecule or the part
sequence of the adhesion molecule which binds to the corresponding
adhesion molecule or to the extracellular matrix, an extracellular
moiety of an Fc receptor, a glycoprotein of a virus, a part
sequence of the glycoprotein which binds to these cells, the
transmembrane domain of a receptor or of a viral glycoprotein, or a
glycophospholipid anchor.
16. A nucleic acid construct as claimed in claim 14, wherein said
ligand binds to activated or proliferating endothelial cells, to
tumor cells, to muscle cells, preferably smooth muscle cells, to
fibroblasts, to macrophages, to lymphocytes, to liver cells, to
kidney cells, to synovial cells, to inflammatory cells, to
virus-infected cells, to bronchial epithelial cells, to glia cells
or to leukemia cells.
17. A nucleic acid construct as claimed in claim 14, wherein the
construct comprises at least two identical or different nucleic
acid sequences b)c)d) or b')b)c)d), which nucleic acid sequences
are linked to each other by way of an internal ribosomal entry
site.
18. A process for preparing a nucleic acid construct according to
claim 1, which comprises operably linking said nucleic acid
sequences of claim 1.
19. A method for the treatment or prophylaxis of tumors, leukemias,
allergies, autoimmune diseases, infections, inflammations,
transplant rejection reactions, thromboses, blood vessel
occlusions, blood coagulation, blood circulation disturbances,
injuries to tissues, or damage to the nervous system, comprising
administering to a mammal an effective amount of a polypeptide
expressed by the nucleic acid construct of claim 1.
20. A method for preparing a recombinantly altered cell, comprising
transducing a suitable cell with said nucleic acid construct of
claim 1.
21. A method for preparing a polypeptide which is encoded by said
nucleic acid construct of claim 1, comprising transducing a
suitable cell with said construct, expressing said polypeptide in
said cell, and isolating said expressed polypeptide.
22. The method of claim 20, wherein said cell is an endothelial
cell, a lymphocyte, a macrophage, a glia cell, a fibroblast, a
liver cell, a kidney cell, a muscle cell, a cell of the bone or
cartilage tissue, a synovial cell, a peritoneal cell, a skin cell,
an epithelial cell, a leukemia cell or a tumor cell.
23. The method of claim 21, wherein said cell is an endothelial
cell, a lymphocyte, a macrophage, a glia cell, a fibroblast, a
liver cell, a kidney cell, a muscle cell, a cell of the bone or
cartilage tissue, a synovial cell, a peritoneal cell, a skin cell,
an epithelial cell, a leukemia cell or a tumor cell.
24. A cell transduced with said nucleic acid construct of claim
1.
25. A protein encoded by said nucleic acid construct of claim 1.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a nucleic acid construct
for expressing active substances which can be activated by
proteases and to its preparation and use.
[0002] Like inflamed areas, tumors are distinguished from the
surrounding normal tissue by a substantial increase in the
formation and secretion of proteases [Schmitt et al., Fibrinol. 6,
3 (1992), Cottam et al., Int. J. Oncol. 2, 861 (1993), Tryggvason
et al., Breast Cancer Res. And Treatm. 24, 209 (1993), Leto et al.,
Anticancer Res. 12, 235 (1992), Hart, Fibrinol. 6, 11 (1992),
Albini et al., J. Natl. Cancer Inst. 83, 735 (1991)]. Examples of
these proteases are plasminogen activators, cathepsins and matrix
metalloproteinases.
[0003] An essential function of these tumor proteases is to
dissolve the extracellular matrix to allow the tumor cells to
invade, and grow in an infiltrative manner in, normal tissue. At
the same time, these proteases protect the tumor from the defence
mechanisms of the body insofar as the active compounds which are
required for defence are cleaved, and thereby inactivated, by the
proteases which are formed by the tumor. Thus, for example,
antibodies, cytokines and growth factors, complement factors,
coagulation factors and mediators are inactivated by tumor
proteases.
[0004] In the past, the aim was, therefore, to inhibit the
infiltrative growth and metastatic growth of tumors, and
inactivation of the defence mechanisms of the body, by inhibiting
the tumor cell proteases [Hocman, Int. J. Biochem. 24, 1365 (1992),
Troll et al., JNCI 73, 1245 (1984), Ray et al., Eur. Respir. 7,
2062 (1994), Koop et al., Cancer Res. 54, 4791 (1994), Chiriri et
al., Int. J. Cancer 58, 460 (1994), Denhardt et al., 59, 329
(1993), Melchiori et al., Cancer Res. 52, 2353 (1992)]. However,
particularly for stoichiometric and pharmacokinetic reasons, little
success has previously been achieved in inhibiting tumor cell
proteases.
[0005] An attempt was therefore made to use the tumor cell
proteases to activate bacterial toxins such as Staphylococcus
aureus .alpha.-hemolysin [Panchal et.al., Nature Biotechn. 14, 852
(1996)]. For this, an amino acid sequence, i.e. XX-Arg-X, was
inserted into positions 129 to 132 of the .alpha.-hemolysin and in
this way inactive mutants were produced which are only cleaved, and
thereby activated, by tumor proteases such as cathepsin B.
[0006] Based on these results, proimmunolysins were proposed
[Panchal et al., Nature Biotechn. 14, 852 (1996)], which
proimmunolysins comprise an antibody which is coupled to a
Staphylococcus aureus .alpha.-hemolysin which can be activated by
tumor proteases or to a sea anemone equinatoxin II, with the
antibody determining the target cell specificity of the coupling
product.
[0007] However, the proposed concept suffers from the following
disadvantages in relation to its use in tumor therapy:
[0008] In the first place, the authors chose xenogeneic
nonendogenous lysins and/or toxins which are immunogenic for the
host organism (for example, patients) and as a result induce an
immune reaction in the host organism, which immune reaction
neutralizes and inactivates the antibody/toxin conjugate. In the
second place, it is known [Sedlacek et al., Antibodies as Carriers
of Cytotoxicity, Contrib. to Oncol. 43, Karger Verlag, Munich,
1992] that, due to their molecular size and to the Theological
conditions at the tumor, tumor-specific antibodies and immunotoxins
only accrue in very small quantities (0.01-0.001% of the given
antibody or immunotoxin/g of tumor) at the tumor and only penetrate
the tumor to an incomplete extent so that it is either not possible
to destroy all the tumor cells or only possible to destroy a small
portion of the cells of a tumor. Then again, the extent to which
tumor antigens, against which the antibody is directed, are
expressed usually differs between the individual tumor cells, and
the variable, antigen-negative tumor cells readily evade the attack
by the antibodies or the immunotoxins. In addition to this,
antigens which are secreted by the tumor cells neutralize the
antibodies at the periphery of the tumor (Sedlacek et al.,
Monoclonal Antibodies in Tumor Therapy, Contrib. to Oncol., Karger
Verlag, 1988).
[0009] Consequently, there is still a great need for a target
cell-specific therapy for tumors and inflammations.
SUMMARY OF THE INVENTION
[0010] An object of the present invention is therefore to provide
an active compound against tumors and inflammations, which active
compound does not exhibit said disadvantages. The present invention
therefore relates to a novel technique which uses the secretion of
enzymes in tumors or areas of inflammation to achieve the local
release of active compounds whose inactive precursors are expressed
in tumor cells, tumor-associated cells or inflammatory cells.
[0011] One part of the subject-matter of the present invention is
therefore a nucleic acid construct for expressing an active
substance which is activated by an enzyme which is released from
mammalian cells, which nucleic acid construct comprises the
following components:
[0012] a) at least one promoter element,
[0013] b) at least one DNA sequence which encodes an active
compound (protein B),
[0014] c) at least one DNA sequence which encodes an amino acid
sequence (part structure C) which can be cleaved specifically by an
enzyme which is released from a mammalian cell, and
[0015] d) at least one DNA sequence which encodes a peptide or
protein (part structure D) which is bound to the active compound
(protein B) by way of the cleavable amino acid sequence (part
structure C) and inhibits the activity of the active compound
(protein B).
[0016] Further objects of the instant invention are described as
follows:
[0017] 1. A nucleic acid construct for expressing an active
substance which is activated by an enzyme which is released from
mammalian cells, wherein the construct comprises the following
nucleic acid sequences in the following order:
[0018] a) at least one promoter element operably linked to;
[0019] b) at least one nucleic acid sequence which encodes an
active compound, wherein the active compound is endogenous to
mammals, operably linked to;
[0020] c) at least one nucleic acid sequence which encodes an amino
acid sequence cleavable specifically by an enzyme which is released
from a mammalian cell, operably linked to;
[0021] d) at least one DNA sequence which encodes a polypeptide
which is bound to the active compound by the cleavable amino acid
sequence and inhibits the activity of the active compound, and
wherein the nucleic acid component c) does not naturally occur as
operably linking the nucleic acid sequence b) to the nucleic acid
d).
[0022] 2. A nucleic acid construct as described in 1, wherein the
enzyme is a protease.
[0023] 3. A nucleic acid construct as described in 1, wherein the
enzyme is a prostate specific antigen, a plasminogen activator, a
cathepsin or a matrix metalloproteinase.
[0024] 4. A nucleic acid construct as described in 1, wherein the
mammalian cells are tumor cells, leukemia cells, endothelial cells,
macrophages, lymphocytes, muscle cells, epithelial cells, glia
cells, synovial cells or virus-infected cells.
[0025] 5. A nucleic acid construct as described in 1, wherein the
nucleic acid construct further comprises a nucleic acid sequence
operably linked to the construct of 1, wherein the nucleic acid
sequence encodes a ligand which binds the active compound to a
target structure.
[0026] 6. A nucleic acid construct as described in 1, wherein the
nucleic acid sequences b) and d) of 1 encode parts of a natural
precursor of a protein active compound, wherein the nucleic acid
sequence encoding the cleavage sequence naturally occurring between
the nucleic acid sequences b) and d) has been replaced by the
nucleic acid sequence c), which does not naturally occur between
the nucleic acid sequences b) and d).
[0027] 7. A nucleic acid construct as described in 1, wherein the
polypeptide encoded by the nucleic acid sequence d) is part of a
natural precursor of a protein active compound.
[0028] 8. A nucleic acid construct as described in 1, wherein the
construct is operably inserted into a plasmid or a viral
vector.
[0029] 9. A nucleic acid construct as described in 1, wherein the
nucleic acid sequence a) is a promoter sequence which can be
activated nonspecifically, cell-specifically, virus-specifically,
metabolically, cell cycle-specifically or by tetracycline.
[0030] 10. A nucleic acid construct as described in 1, wherein the
nucleic acid sequence a) comprises at least two identical or two
different promoter sequences.
[0031] 11. A nucleic acid construct as described 9, wherein the
nucleic acid sequence a) is activated in endothelial cells, in
cells adjoining activated endothelial cells, in muscle cells, in
leukemia cells, in tumor cells, in glia cells, in lymphocytes, in
macrophages or in synovial cells.
[0032] 12. A nucleic acid construct as described in 1, wherein the
active compound activates or inhibits a biological activation
cascade or is an active component of this cascade, or activates or
inhibits the coagulation system, activates fibrinolysis, activates
the complement system or activates the kinin system, or is an
enzyme which converts the inactive precursor of a pharmacological
substance into the active substance, or which itself is a
pharmacologically active substance.
[0033] 13. A nucleic acid construct as described in 12, wherein the
active compound is a coagulation factor which is selected from the
group consisting of thrombin, factor Va, factor VIIa, factor IXa,
factor Xa, TF coagulation-active fragments or factor XIIa; thrombin
which is mutated in the region of the Arg-Thr cleavage site (amino
acid position 327/328); a fibrinolytic protein which is selected
from urokinase, tPA or functional hybrids thereof; a complement
factor which is selected from CVF, C3b or functional cleavage
products thereof; an antithrombotic protein which is selected from
protein C, C-1S inhibitor, a1-antitrypsin, hirudin, AT-III, TFPI,
PAI-1, PAI-2 or PAI-3; a kallikrein; a cytostatic, cytotoxic or
inflammation-eliciting protein; an antiangiogenic protein; an
immunomodulatory protein; an antiinflammatory protein; a protein
which relieves damage to the nervous system; a protein which
inhibits or neutralizes the neurotoxic effect of TNF.alpha.; an
angiogenesis-stimulating protein; a hypotensive protein; an
antiviral protein; a cytokine; an interferon; a tumor necrosis
factor; oncostatin M or LIF; a cytokine receptor; the moiety of a
cytokine receptor which is external to the cell; a cytokine
antagonist; a growth factor; a growth factor receptor; the moiety
of a growth factor receptor which is external to the cell; a
chemokine; angiostatin; platelet factor 4; TIMP-1, TIMP-2 or
TIMP-3; a nitroreductase; a .beta.-glucuronidase; a
carboxypeptidase; a .beta.-lactamase; a cytosine deaminase; a
catalase; a peroxidase; a phosphatase; an oxidase; kallikrein or an
endothelial cell nitric oxide synthase.
[0034] 14. A nucleic acid construct as described in 1, which
further comprises a nucleic acid sequence b') which encodes a
ligand which binds to a cell membrane receptor, a cell membrane
antigen, a cell membrane-located adhesion molecule, or to the
extracellular matrix or component thereof.
[0035] 15. A nucleic acid construct as described in 14, wherein the
ligand is an antibody or an antibody fragment which binds
specifically to a cell membrane antigen or to an antigen on the
extracellular matrix, or is a polypeptide which binds to receptor
on the cell membrane wherein the polypeptide is a growth factor, a
cytokine, an interferon, a tumor necrosis factor, a chemokine, a
receptor-binding part sequence of these ligands, a peptide hormone,
angiotensin, kinin, folic acid, an adhesion molecule or the part
sequence of the adhesion molecule which binds to the corresponding
adhesion molecule or to the extracellular matrix, an extracellular
moiety of an Fc receptor, a glycoprotein of a virus, a part
sequence of the glycoprotein which binds to these cells, the
transmembrane domain of a receptor or of a viral glycoprotein, or a
glycophospholipid anchor.
[0036] 16. A nucleic acid construct as described in 14, wherein the
ligand binds to activated or proliferating endothelial cells, to
tumor cells, to muscle cells, preferably smooth muscle cells, to
fibroblasts, to macrophages, to lymphocytes, to liver cells, to
kidney cells, to synovial cells, to inflammatory cells, to
virus-infected cells, to bronchial epithelial cells, to glia cells
or to leukemia cells.
[0037] 17. A nucleic acid construct as described in 14, wherein the
construct comprises at least two identical or different nucleic
acid sequences b)c)d) or b')b)c)d), which nucleic acid sequences
are linked to each other by way of an internal ribosomal entry
site.
[0038] 18. A process for preparing a nucleic acid construct
according to 1, which comprises operably linking the nucleic acid
sequences of 1.
[0039] 19. A method for the treatment or prophylaxis of tumors,
leukemias, allergies, autoimmune diseases, infections,
inflammations, transplant rejection reactions, thromboses, blood
vessel occlusions, blood coagulation, blood circulation
disturbances, injuries to tissues, or damage to the nervous system,
comprising administering to a mammal an effective amount of a
polypeptide expressed by the nucleic acid construct of 1.
[0040] 20. A method for preparing a recombinantly altered cell,
comprising transducing a suitable cell with the nucleic acid
construct of 1.
[0041] 21. A method for preparing a polypeptide which is encoded by
the nucleic acid construct of 1, comprising transducing a suitable
cell with the construct, expressing the polypeptide in the cell,
and isolating the expressed polypeptide.
[0042] 22. The method of 20, wherein the cell is an endothelial
cell, a lymphocyte, a macrophage, a glia cell, a fibroblast, a
liver cell, a kidney cell, a muscle cell, a cell of the bone or
cartilage tissue, a synovial cell, a peritoneal cell, a skin cell,
an epithelial cell, a leukemia cell or a tumor cell.
[0043] 23. The method of 21, wherein the cell is an endothelial
cell, a lymphocyte, a macrophage, a glia cell, a fibroblast, a
liver cell, a kidney cell, a muscle cell, a cell of the bone or
cartilage tissue, a synovial cell, a peritoneal cell, a skin cell,
an epithelial cell, a leukemia cell or a tumor cell.
[0044] 24. A cell transduced with the nucleic acid construct of
1.
[0045] 25. A protein encoded by the nucleic acid construct of
1.
[0046] The term "endogenous to mammals" as used to describe the
active compound of the instant invention denotes a polypeptide that
is naturally expressed in mammals or a derivative thereof as
discussed herein.
[0047] The term "does not naturally occur" as used to describe the
linking nucleic acid component c) denotes that the described
component c) of the instant invention is not found in nature as
operably linking components b) and d).
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 is a diagrammatic representation of a novel nucleic
acid construct comprising components a), b), c) and d);
[0049] FIG. 2 is a diagrammatic representation of a novel nucleic
acid construct which has been enlarged by adding component b');
and
[0050] FIG. 3 is diagrammatic representation of a nucleic acid
construct for PSA-activatable factor X.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0051] In their simplest form, the individual components can be
arranged, for example, as shown in FIG. 1. In this case, expression
of a protein BCD, encoded by components b), c) and d), is induced
by activating the promoter sequence [component a)]. The amino acid
sequence C of the expression product is then cleaved by cellular
enzymes, e.g. proteases, as a result of which protein B, which
constitutes the active compound, is released. Within the meaning of
the present invention, proteases or enzymes are to be understood as
being one or more proteases or enzymes.
[0052] In another embodiment, said enzyme is a protease, in
particular a plasminogen activator, a cathepsin or a matrix
metalloproteinase. Said mammalian cells are preferably tumor cells,
leukemia cellsi endothelial cells, macrophages, lymphocytes, muscle
cells, epithelial cells, glia cells, synovial cells or
virus-infected cells.
[0053] Enzymes are preferably released, in an organism, by tumors
and tumor cells and also by cells which are involved in an
inflammatory process [Barrett et al., Mammalian Proteases, Academic
Press, London 1980; Sedlacek and Moroy, Immune Reactions, Springer
Verlag, 1995)].
[0054] According to the present invention, component c) is
consequently selected such that the expressed protein, e.g. BCD, is
preferably cleaved, in its part structure C, by proteases which are
formed in tumors or secreted by tumor cells or inflammatory cells.
Examples of these proteases are plasminogen activators, such as
plasminogen activator of the urokinase type or tissue plasminogen
activator; cathepsins, such as cathepsin B, cathepsin D, cathepsin
L, cathepsin E or cathepsin H, or their precursors (procathepsins);
matrix metalloproteinases (MMP), such as collagenases, for example
of groups I, II, III, IV or V; stromelysin 1, stromelysin 2 or
stromelysin 3; metrilysins; gelatinases, such as gelatinase A (MMP
2), and progelatinase B (MMP 9) and progelatinase A [Pappot et al.,
Lung Cancer 12, 1 (1995), Schmitt et al., Fibronolysis 614, 3
(1992), Monsky et al., Cancer Biol. 4, 251 (1993), Rochefort et
al., Medicine/Sciences 7, 30 (1991), Kao et al., 46, 1349 (1986),
Fridman et al., Cancer Res. 55, 2548 (1995), Ray et al., Eur.
Respir. J. 7, 2062 (1994), Cottam et al., Int. J. Oncol. 2, 861
(1993), Tryggvason et al., Breast Cancer Res. and Treatm. 24, 209
(1993)]; tumor cell surface proteases [surface-expressed
proteases=seprase; Monsky et al., Cancer Res. 54, 5702 (1994)];
elastase [Kao et al., Cancer Res. 46, 1355 (1986)];
prostate-specific antigen [Lundwall, Biochem. Biophys. Res. Commun.
161, 1151 (1989), Riegman et al., Biochem. Biophys. Res. Commun.
159, 95 (1989)] or pancreatic trypsinogens [Miszuk-Jamska et al.,
FEBS Lett. 294, 175 (1991)].
[0055] In accordance with another embodiment of the present
invention, the nucleotide sequence for component b) can be extended
by the addition of a component b'). This component b') encodes a
ligand (part structure B') which can bind the active compound to a
target structure. Component b') is, for example, arranged as shown
in FIG. 2. Expression of the nucleic acid construct corresponding
to FIG. 2 results in a protein, i.e. B'BCD, which binds to a target
structure by way of the ligand (part structure B'). The part
structure C is then cleaved by an enzyme, for example, cellular
proteases, thereby releasing the active compound, i.e. protein
B'B.
[0056] In a particular embodiment, said protein B and the part
structure D are parts of the natural precursors of protein active
compounds, with the natural cleavage sequence, which connects the
part structures B and D, having been replaced by the part structure
C; in particular, said part structure D is the part structure of a
natural precursor of a protein active compound.
[0057] The novel nucleic acid constructs are preferably composed of
DNA. The term "nucleic acid constructs" is understood to mean
artificial nucleic acid structures which can be transcribed in the
target cells. They are preferably inserted into a vector, with
plasmid vectors or viral vectors being particularly preferred.
[0058] Depending on the choice of the promoter element [component
a)], the novel nucleic acid constructs express a structural gene
[components b)+c)+d) or b')+b)+c)+d)] either nonspecifically,
cell-specifically, virus-specifically, under particular metabolic
conditions, cell cycle-specifically or in the presence of
tetracycline. At least two identical or different promoter elements
can also be combined together for the purpose of modifying the
expression of the structural gene depending on the choice of these
promoter elements. Component a) is preferably activated in
endothelial cells, in cells adjoining activated endothelial cells,
in muscle cells, in leukemia cells, in tumor cells, in glia cells,
in lymphocytes, in macrophages and/or in synovial cells.
[0059] The part structure B (protein B) of the protein encoded by
the novel structural gene constitutes the actual novel active
compound which is released or activated by cleavage of the part
structure C and thereby converted from the inhibited state, e.g. as
protein BCD or as protein B'BCD, into the active state, e.g. as
protein B or as protein B'B.
[0060] According to the invention, this active compound can be an
enzyme which activates or inhibits a biological activation cascade
and/or is an active component of this cascade. Examples of
biological activation cascades of this nature are the coagulation
system, which can be activated or inhibited, fibrinolysis, which is
preferably activated, the complement system, which is likewise
preferably activated, or the kinin system, which is also preferably
activated. The active compound can also be an enzyme which converts
the inactive precursor of a pharmacological substance into the
active substance or which itself is a pharmacologically active
substance. Particular preference is given to an active compound
(protein B) which is a coagulation factor which is selected from
thrombin, factor Va, factor VIIa, factor IXa, factor Xa, TF
coagulation-active fragments or factor XIIa; thrombin which is
mutated in the region of the Arg-Thr cleavage site (amino acid
position 327/328); a fibrinolytic protein which is selected from
urokinase, tPA or functional hybrids thereof; a complement factor
which is selected from CVF, C3b or functional cleavage products
thereof; an antithrombotic protein which is selected from protein
C, C-1S inhibitor, .alpha.1-antitrypsin, hirudin, AT-III, TFPI,
PAI-1, PAI-2 or PAI-3; a kallikrein; a cytostatic, cytotoxic or
inflammation-eliciting protein; an antiangiogenic protein; an
immunomodulatory protein; an antiinflammatory protein; a protein
which relieves damage to the nervous system; a protein which
inhibits or neutralizes the neurotoxic effect of TNF.alpha.; an
angiogenesis-stimulating protein; a hypotensive protein; an
antiviral protein; a cytokine; an interferon; a tumor necrosis
factor; oncostatin M or LIF; a cytokine receptor; the moiety of a
cytokine receptor which is external to the cell; a cytokine
antagonist; a growth factor; a growth factor receptor; the moiety
of a growth factor receptor which is external to the cell; a
chemokine; angiostatin; platelet factor 4; TIMP 1, TIMP 2 or TIMP
3; a nitroreductase; a .beta.-glucuronidase; a carboxypeptidase; a
.beta.-lactamase; a cytosine deaminase; a catalase; a peroxidase; a
phosphatase; an oxidase; kallikrein or an endothelial cell nitric
oxide synthase.
[0061] The part structure B' of the protein encoded by the novel
structural gene constitutes the novel ligand for binding the active
compound (protein B) to a target structure. A preferred target
structure is the surface of cells, preferably a cell membrane
receptor, a cell membrane antigen, a cell membrane-located adhesion
molecule, or the extracellular matrix, for example of endothelial
cells, in particular of activated or proliferating endothelial
cells, tumor cells, muscle cells, in particular smooth muscle
cells, fibroblasts, macrophages, lymphocytes, liver cells, kidney
cells, synovial cells, inflammatory cells, virus-infected cells,
bronchial epithelial cells, glia cells, leukemia cells or cells of
other tissues and organs. A particularly preferred target structure
is the surface of activated and/or proliferating endothelial
cells.
[0062] Another preferred target structure is constituted by
components of the extracellular matrix, for example collagens
[Prockop et al., Annu. Rev. Biochem. 64, 403 (1995), Wetzels et
al., Am. J. Pathol. 139, 451 (1991)]; ficolin [Ichijo et al., J.
Biol. Chem. 268, 14505 (1993)]; sialoprotein [Bellahcene et al.,
Cancer Res. 54, 2823 (1994)]; laminin [von der Mark et al.,
Biochem. Biophys. Acta 823, 147 (1985); Hunt. Expl. Cell Biol. 57,
165 (1989)]; proteoglycans [Schmidtchen et al., Biomed.
Chromatography 7, 48 (1993)] or tenascin [Oyama et al., Cancer Res.
51, 4876 (1991); Herlyn et al., Cancer Res. 51, 4853 (1991)].
[0063] The novel ligand (part structure B') can, for example, be an
antibody or an antibody fragment, such as the epitope-binding
moiety of an antibody, Fab, Fv, single-chain Fv or Fc, which binds
specifically to a cell membrane antigen or to an antigen on the
extracellular matrix, or another peptide or protein which binds to
a receptor on the relevant cell membrane. These include, for
example, growth factors, cytokines, interferons, tumor necrosis
factor, chemokines, their receptor-binding part sequences, peptide
hormones, angiotensin, kinin or folic acid. The ligand can also be
an adhesion molecule or its adhesion sequence which binds to a
corresponding molecule on the cell membrane or on the extracellular
matrix, or the target cell-binding moiety, an extracellular moiety
of an Fc receptor, a target cell-binding glycoprotein of a virus
which has a tropism for selected cells, or a part sequence of the
glycoprotein which binds to these cells, or a peptide by whose help
the active compound is anchored in the cell membrane of the cell
which is expressing it. Examples of these anchoring peptides are
the transmembrane domains of receptors or virus proteins or
glycophospholipid anchors.
[0064] Component d) encodes a peptide (part structure D) which is
bonded to protein B or protein B'B by way of part structure C and
thereby inhibits the activity of protein B. Component d) can be any
arbitrary nucleic acid sequence. Preferably, however, it is
composed of nucleic acid sequences which encode endogenous peptides
or proteins, in order to avoid or decrease the danger of an immune
reaction. In another preferred embodiment, components b) and d) of
the novel structural gene encode endogenous proteins or
peptides.
[0065] A considerable number of protein active compounds occur in
nature in the form of inactive precursors (protein BSD). A
precursor of this type is activated by enzymes cleaving this
precursor into a part structure which constitutes the actual
protein active compound (protein B) and into an inactive part
structure (part structure D). This precursor is cleaved at least
one defined amino acid sequence, i.e. the so-called cleavage
sequence (part structure S).
[0066] It is a particular part of the subject-matter of this
invention that this cleavage sequence (part structure S) which
occurs naturally in precursors of protein active compounds is
replaced by the part structure C. This replacement is effected by
the sequence encoding the part structure S being replaced by
component c), encoding part structure C, in the nucleic acid
sequence which encodes the natural precursor (protein BSD). After
components a) and, where appropriate, b') have been added on, a
novel nucleic acid construct is produced which comprises, for
example, components a)b')b)c)d) or a)b)c)d), the part structure C
of whose expression product, i.e. protein B'BCD or BCD,
respectively, is cleaved by proteases which are formed in tumors or
secreted by tumor cells or inflammatory cells, such that the active
compound, i.e. protein B'B or B, can be formed.
[0067] In another embodiment, the novel construct comprises at
least two identical or different components b)c)d) and/or
b')b)c)d), which components are linked to each other by way of a
so-called internal ribosomal entry site (IRES).
[0068] Having been inserted into a nonviral vector or viral vector,
the novel nucleic acid construct is generally, for the prophylaxis
and/or therapy of disorders, administered locally or injected into
the blood circulation. These disorders particularly include tumor
diseases and inflammations. Such inflammations can be triggered,
for example, by physicochemical damage, by an infection or by an
immune reaction against endogenous or foreign tissue.
[0069] The present invention furthermore relates, therefore, to the
use of a novel nucleic acid construct for preparing a drug for
local or systemic administration for the prophylaxis and/or therapy
of tumors, leukemias, allergies, autoimmune diseases, infections,
inflammations, transplant rejection reactions, thromboses, blood
vessel occlusions, blood coagulation and blood circulation
disturbances, and injuries to tissues and/or damage to the nervous
system.
[0070] The choice of the components of the novel nucleic acid
construct depends on the disease which is to be treated by
administering the nucleic acid construct and can be made as
follows:
[0071] Promoter Sequences [component a)]:
[0072] According to the present invention, particular preference is
given, on the one hand, to promoter sequences [component a)] which
are promoters and activator sequences which can be activated in an
unrestricted manner, such as the promoter of RNA polymerase III,
the promoter of RNA polymerase II, etc., the CMV promoter and CMV
enhancer, or the SV40 promoter, and, on the other hand, to viral
promoter and activator sequences, such as HBV, HCV, HSV, HPV, EBV,
HTLV or HIV.
[0073] For example, in the case of the HIV promoter, the entire LTR
sequence, including the TAR sequence [positions .ltoreq.-453 to
.gtoreq.-80, Rosen et al., Cell 41, 813 (1985)] can be used as a
virus-specific promoter.
[0074] Metabolically activatable promoter and enhancer sequences,
such as the hypoxia-inducible enhancer, promoters which can be
activated in a cell cycle-specific manner, such as the promoters of
the cdc25C gene, the cyclin A gene, the cdc2 gene, the Bmyb gene,
the DHFR gene or the E2F-1 gene, or tetracyline-activatable
promoters, such as the tetracycline operator in combination with an
appropriate repressor, are also particularly preferred as component
a).
[0075] According to the present invention, nucleotide sequences
which, after binding transcription factors, activate the
transcription of a structural gene which adjoins them at the 3' end
are also to be used as promoter sequences.
[0076] In addition, promoters which can be activated in a
cell-specific manner are particularly preferred as component a).
These promoters preferably include promoters or activator sequences
composed of promoters or enhancers from those genes which
preferably encode proteins in selected cells. For example,
promoters for the following proteins are preferably to be used in
the following cells:
[0077] Promoter and activator sequences which are activated in
endothelial cells, such as brain-specific, endothelial glucose-I
transporter, endoglin, VEGF receptor 2 (flt-1), VEGF receptor 2
(flk-1, KDR), tiel-1 or tiel-2, B61 receptor (Eck receptor), B61,
endothelin, especially endothelin B and endothelin 1, endothelin
receptors, in particular the endothelin B receptor, mannose
6-phosphate receptors, von Willebrand factor, IL-1.alpha.,
IL-1.beta., IL-1 receptor, vascular cell adhesion molecule (VCAM 1)
or synthetic activator sequences.
[0078] As an alternative to natural, endothelial cell-specific
promoters, use can also be made of synthetic activator sequences
which are composed of oligomerized binding sites for transcription
factors which are preferentially or selectively active in
endothelial cells. An example is transcription factor GATA 2, whose
binding site in the endothelin 1 gene is 5'-TTATCT-3' [Lee et al.,
Biol. Chem. 266, 16188 (1991), Dormann et al., J. Biol. Chem. 267,
1279 (1992) and Wilson et al., Mol. Cell. Biol. 10, 4854
(1990)].
[0079] Promoters or activator sequences which are activated in
cells in the vicinity of activated endothelial cells, in particular
in smooth muscle cells, are present, for example, in the VEGF gene.
The gene-regulatory sequences for the VEGF gene are the 5'-flanking
region, the 3'-flanking region, the c-Src gene or the v-Src
gene.
[0080] Steroid hormone receptors and their promoter elements, in
particular the mouse mammary tumor virus promoter, or promoter
elements of the gene encoding tropomyosin, .alpha.-actin,
.alpha.-myosin, the receptor for PDGF, the receptor for FGF, MRF-4,
phosphofructokinase A, phosphoglycerate mutase, troponin C,
myogens, receptors for endothelin A, desmin or separate
"artificial" promoters, are also suitable. Promoter elements to
which the factors of the helix-loop-helix (HLH) family (MyoD, Myf
5, myogens and MRF4 [review in Olson and Klein, Genes Dev. 8, 1
(1994)]) can bind, as muscle-specific transcription factors, are
likewise suitable. The muscle-specific transcription factors also
include the zinc finger protein GATA-4 (Arceci et al., Mol. Cell
Biol. 13, 2235 (1993), Ip et al., Mol. Cell Biol. 14, 7517 (1994)]
and the groups of the MEF transcription factors [Yu et al., Gene
Dev. 6, 1783 (1992)].
[0081] The HLH proteins, and also GATA 4, exhibit a similar
muscle-specific transcription not only with promoters from
muscle-specific genes but also in a heterologous context, that is
with "artificial" promoters. Examples of such artificial promoters
are multiple copies of the (DNA) binding site for muscle-specific
HLH proteins, such as the E box (myo D), e.g. 4.times.
AGCAGGTGTTGGGAGGC, [Weintraub et al., PNAS 87, 5623 (1990)] or
multiple copies of the DNA binding site for GATA 4 of the
.alpha.-myosin heavy chain gene, e.g.
5'-GGCCGATGGGCAGATAGAGGGGGCCGATGGGCAGATAGAGG3' [Molkentin et al.,
Mol. Cell Biol. 14, 4947 (1994)].
[0082] Examples of promoters and activator sequences which are
activated in leukemia cells are promoters for c-myc, HSP-70,
bcl-1/cyclin D-1, bcl-2, IL-6, IL-10, TNF.alpha., TNF.beta.,
HOX-11, BCR-Ab1, E2A-PBX-1 or PML-RATA.
[0083] Examples of promoters or activator sequences which are
activated in tumor cells are promoter or activator sequences which
interact with the transcription factors which are formed, or are
active, in tumor cells. These preferred promoter or activator
sequences include gene-regulatory sequences or elements from genes
which encode proteins which are formed, in particular, in cancer
cells or sarcoma cells. Thus, for example, the promoter of the
N-CAM protein is used in the case of small-cell bronchial
carcinomas, the promoter of the hepatitis growth factor receptor or
of L-plastin is used in the case of ovarian carcinomas, and the
promoter of L-plastin or of polymorphic epithelial mucin (PEM) is
used in the cas of pancreatic carcinomas.
[0084] Promoters and activator sequences which are activated in
glia cells are, in particular, the gene-regulatory sequences or
elements from genes which encode, for example, the following
proteins: the Schwann cell-specific protein periaxin, glutamine
synthetase, glia cell-specific protein (glial fibrillary acid
protein=GFAP), the glia cell protein SlOOb, IL-6 (CNTF), 5-HT
receptors, TNF.alpha., IL-10, insulin-like growth factor receptor I
and II or VEGF. The gene-regulatory sequences for the VEGF gene
have already been listed above.
[0085] Examples of promoters and activator sequences which are
activated in lymphocytes and/or macrophages are the promoter and
activator sequences of the gene encoding cytokines, cytokine
receptors and adhesion molecules, and receptors for the Fc fragment
of antibodies. Examples of these are: IL-1 receptor, IL-1.alpha.,
IL-1.beta., IL-2, IL-2 receptor, IL-3, IL-3 receptor (.alpha.
subunit), IL-3 receptor (.beta. subunit), IL-4, IL-4 receptor,
IL-5, IL-6, IL-6 receptor, interferon regulatory factor 1 (IRF-1),
(the promoter of IRF-1 is activated to the same extent by IL-6 as
by IFN.gamma. or IFN.beta.), IFN.gamma.-responsive promoter, IL-7,
IL-8, IL-10, IL-11, IFN.gamma., GM-CSF, GM-CSF receptor (.alpha.
chain), IL-13, LIF, macrophage colony stimulating factor (M-CSF)
receptor, type I and II macrophage scavenger receptors, MAC-1
(leukocyte function antigen), LFA-1.alpha. (leukocyte function
antigen) or p150,95 (leukocyte function antigen).
[0086] Examples of promoter and activator sequences which are
activated in synovial cells are the promoter sequences for matrix
metalloproteinases (MMP), for example for: MMP-1 (interstitial
collagenase), or MMP-3 (stromelysin/transin). These also include
the promoter sequences for tissue inhibitors of metalloproteinases
(TIMP), for example TIMP-1, TIMP-2 and TIMP-3.
[0087] According to the present invention, several of the promoter
sequences which have been listed by way of example can be combined
with each other in order to achieve the highest possible target
cell specificity in the expression of the novel nucleic acid
construct. Two identical promoters can also be combined. Several
promoter sequences can be combined, for example, using chimeric
promoters or hybrid promoters. A chimeric promoter is the
combination of an upstream activator sequence, which can be
activated cell-specifically, metabolically or virus-specifically,
with a downstream promoter module which binds the transcription
factors of the CDF and CHF families or the E2F and CHF families and
can thereby inhibit activation of the upstream activator sequence
in the GO and Gi phases of the cell cycle (Lucibello et al., EMBO
J. 14, 132 (1994)].
[0088] In the case of hybrid promoters, the TATA box of a promoter
is, for example, mutated, with this mutation being compensated for
by a corresponding mutation in the gene for a TATA-binding protein,
and this TATA-binding protein being under the control of another
promoter.
[0089] Nucleic Acid Sequence Component b')1. Which Encodes a Ligand
(Part Structure B'):
[0090] According to the present invention, the ligand is a
substance which binds a membrane antigen to a receptor or to an
adhesion molecule on the target cell or which is integrated in the
cell membrane and/or binds to the extracellular matrix. Reviews of
the important cytokines and growth factors and their receptors,
adhesion molecules and extracellular matrix proteins are provided
by Ayad et al., The Extracellular Matrix, Academic Press 1994;
Callard et al., The Cytokine, Academic Press 1994; Pigott et al.,
The Adhesion Molecule, Academic Press 1994, and Barclay et al., The
Leucocyte Antigen, Academic Press 1994.
[0091] Examples of substances which bind to receptors are growth
factors, such as VEGF, PDGF, EGF, TGFA, TGF.beta., KGP, SDGF, FGF,
IGF, HGF, NGF, BDNF, neurotrophins, BMF, bombesin, M-CSF,
thrombopoietin, erythropoietin, SCF, SDGF, oncostatin, PDEGF or
endothelin-1, cytokines, such as IL-1, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,
interferons .alpha., .beta. and .gamma., tumor necrosis factors
TNF.alpha. and TNF.beta., chemokines, such as RANTES, MCAF,
MIP-1.alpha. or MIP-1.beta., NAP or .beta.-thromboglobulin, peptide
hormones, such as SRH, SIH or STH, MRH or MSH, PRH, PIH or
prolactin, LH-RH, FSH-RH, LH/ICSH or FSH, TRH or TSH, CRH or ACTH,
angiotensin, kinins, homologs or analogs thereof, or vitamins, such
as folic acid.
[0092] According to the present invention, the ligand can also be
an adhesion molecule, a part of an adhesion molecule or an analog
of an adhesion molecule which binds to a corresponding adhesion
molecule which is located in the cell membrane or to another
specific binding structure for an adhesion molecule on the target
cell or in the extracellular matrix.
[0093] Examples of such adhesion molecules which are capable of
functioning as ligands are Lewis X (for GMP-140), S Lewis X (for
ELAM-1), LFA-1 (for ICAM-1 and ICAM-2), MAC-1 (for ICAM-1), VLA-4
(for VCAM-1), PECAM (for PECAM), vitronectin (for the vitronectin
receptor), GMP-140 (for Lewis X), S Lewis X (for ELAM-1), ICAM-1,
ICAM-2 (for LFA-1 and MAC-1), VCAM-1 (for VLA-4), fibronectin (for
VLA-4), laminin (for VLA-6), laminin (for VLA-1, VLA-2 and VLA-3),
fibrinogen (for GPIIb-IIIa), B7 (for CD28), CD28 (for B7), CD40
(for CD40L) or CD40L (for CD40).
[0094] According to the present invention, the ligand can also be
the extracellular moiety of an Fc receptor [Dougherty et al.,
Transfusion Science 17, 121 (1996)]. Furthermore, the ligand can
also be an antibody molecule or the epitope-binding moiety of an
antibody molecule. The murine monoclonal antibodies should
preferably be employed in humanized form. The humanization is
effected in the manner described by Winter et al. Nature 349, 293
(1991) and Hoogenbooms et al. Rev. Tr. Transfus. Hemobiol. 36, 19
(1993).
[0095] Recombinant antibody fragments are either prepared directly
from existing hybridomas or are isolated from libraries of murine
or human antibody fragments [Winter et al., Annu. Rev. Immunol. 12,
433 (1994)] using the phage-display technique [Smith, Science 228,
1315 (1985)]. The antibody fragments are then employed directly, at
the genetic level, for further manipulations, e.g. for fusion with
other proteins.
[0096] In order to prepare recombinant antibody fragments from
hybridomas, the genetic information which encodes the
antigen-binding domains (VH and VL) of the antibodies is obtained
by isolating the mRNA, reverse-transcribing the RNA into cDNA and
then amplifying the cDNA by means of the polymerase chain reaction
[Saiki et al., Science 230, 1350 (1985)] and using oligonucleotides
which are complementary to the 5' and 3' ends of the variable
fragments (Orlandi et al., 1989). The VH and VL fragments are then
cloned into bacterial expression vectors, for example in the form
of Fv fragments [Skerra & Pluckthun, Science 240, 1038 (1988)],
single-chain Fv fragments (scFv) [Bird et al., Science 242, 423
(1988), Huston et al., PNAS-USA 85, 5879 (1988)] or as Fab
fragments [Better et al., Science 240, 1041 (1988)].
[0097] The phage-display technique can also be used to isolate new
antibody fragments directly from antibody libraries (immune
libraries or naive libraries) of murine or human origin. In the
phage-display of antibody fragments, the antigen-binding domains
are cloned, as protein fusions with the coat protein g3P of
filamentous bacteriophages, either into the phage genome
[McCafferty et al., Nature 348, 552 (1990)] or into phagemid
vectors [Breitling et al., Gene 104, 147 (1991)] in the form of
scFv fragments [McCafferty et al., Nature 348, 552 (1990)] or as
Fab fragments [Hoogenboom et al., Nucl. Acid Res. 19, 4133 (1991),
Barbas et al., PNAS-USA 88, 7978 (1991)]. Antigen-binding phages
are selected on antigen-loaded plastic vessels (panning) [Marks et
al., J. Mol. Biol. 222, 581 (1991)], on antigen-conjugated,
paramagnetic beads [Hawkins et al., J. Mol. Biol. 226, 889 (1992)]
or by binding to cell surfaces [Marks et al., Bio/Technol. 11, 1145
(1993)].
[0098] Immune libraries are prepared by subjecting the variable
antibody fragments from the B lymphocytes of immunized animals
[Sastry et al., PNAS-USA 86, 5728 (1989), Ward et al., Nature 341,
544 (1989), Clackson et al., Nature 352, 624 (1991)] or patients
[Mullinax et al., PNAS-USA, 87, 8095 (1990), Barbas et al.,
PNAS-USA, 88, 7978 (1991)] to PCR amplification. For this, use is
made of combinations of oligonucleotides which are specific for
murine [Orlandi et al., PNAS-USA, 86, 3833 (1989), Sastry et al.,
PNAS-USA, 86, 5728 (1989)] or human immunoglobulin genes [Larrick
et al., BBRC 160, 1250 (1989)] or for the human immunoglobulin gene
families [Marks et al., Eur. J. Immunol. 21, 985 (1991)].
[0099] Naive libraries can be prepared, for example, using
nonimmunized donors as the source of the immunoglobulin genes
[Marks et al., J. Mol. Biol. 222, 581 (1991)]. Alternatively,
immunoglobulin germ line genes can be used to prepare semisynthetic
antibody repertoires, with the complementarity-determining region 3
of the variable fragments being amplified by PCR using degenerate
primers [Hoogenboom & Winter, J. Mol. biol. 227, 381 (1992),
Barbas et al., PNAS-USA, 89, 4457 (1992), Nissim et al., EMBO J.
13, 692 (1994), Griffiths et al., EMBO J. 13, 3245 (1994)]. As
compared with immune libraries, these so-called single-pot
libraries have the advantage that antibody fragments against a
large number of antigens can be isolated from one single library
[Nissim et al., EMBO J, 13, 692 (1994)].
[0100] The phage-display technique can be used to increase the
affinity of antibody fragments still further, with new libraries
being prepared from already existing antibody fragments by random
[Hawkins et al., J. Mol. Biol. 226, 889 (1992), Gram et al.,
PNAS-USA, 89, 3576 (1992)], codon-based [Glaser et al., J. Immunol.
149, 3903 (1992)] or site-directed mutagenesis [Balint &
Larrick, Gene 137, 109 (1993)], by shuffling the chains of
individual domains with those of fragments from naive repertoires
[Marks et al., Bio/Technol 10, 779 (1992)] or by using bacterial
mutator strains [Low et al., J. Mol. Biol. 26, 359 (1996)], and
antibody fragments having improved properties being isolated by
reselecting under stringent conditions [Hawkins et al., J. Mol.
Biol. 226, 889 (1992)]. In addition, murine antibody fragments can
be humanized by a stepwise replacement of one of the variable
domains with a human repertoire and then selecting with the
original antigen (guided selection) [Jespers et al., Bio/Technol,
12, 889 (1994)]. Alternatively, murine antibodies are humanized by
specifically replacing the hypervariable regions of human
antibodies with the corresponding regions of the original murine
antibody [Jones et al., Nature 321, 522 (1987)].
[0101] According to the present invention, the ligand can also be
the nucleotide sequence encoding a coat protein, or a part of a
coat protein, of viruses which specifically bind to selected cells
by way of their coat protein.
[0102] The ligand can also be a peptide, with whose help the active
compound (protein B) is anchored in the cell membrane of the
expressing cells. These anchoring peptides include the
transmembrane domains of cell membrane-located receptors or of
virus proteins, such as the transmembrane sequence of human
macrophage colony-stimulating factor [DNA position .ltoreq.1485 to
.gtoreq.1554; Cosman et al., Behring Inst. Mitt. 83, 15 (1988)] or
the DNA sequence for the signal and transmembrane regions of human
respiratory syncytial virus (RSV) glycoprotein G [amino acids 1 to
63 or their part sequences, amino acids 38 to 63; Vijaya et al.,
Mol. Cell Biol. 8, 1709 (1988); Lichtenstein et al., J. Gen. Virol.
77, 109 (1996)] or the DNA sequence for the signal and
transmembrane region of influenza virus neuraminidase [amino acids
7 to 35 or the part sequence of amino acids 7 to 27, Brown et al.,
J. Virol. 62; 3824 (1988)].
[0103] However, the nucleotide sequence for a glycophospholipid
anchor [review of glycophospholipid-anchored membrane proteins in
Ferguson et al., (Arn. Rev. Biochem. 57, 285 (1988))] can also be
inserted for the purpose of anchoring the active compound in the
cell membrane of the transduced cells which form the active
compound. Glycophospholipid anchors have been described, for
example, for CEA [DNA position <893 to >1079; Berling et al.,
Cancer Res. 50 6534 (1990)], for N-CAM [Cunningham et al., Science
236, 799 (1987)] and for other membrane proteins such as Thy-1
[Clissold, Biochem. J. 281, 129 (1992)] or CD16 [Selvaray et al.,
Nature 333, 565 (1988)].
[0104] The choice of the ligand depends, first and foremost, on the
target cell which is to be transduced with the nucleic acid
construct. Ligands for activated endothelial cells are examples of
this. Within the meaning of the invention, these ligands include
antibodies or antibody fragments which are directed against
membrane structures of endothelial cells, as have been described,
for example, by Burrows et al. Pharmac. Ther. 64, 155 (1994),
Hughes et al., Cancer Res. 49, 6214 (1989) and Maruyama et al.,
PNAS-USA 87, 5744 (1990). In particular, these antibodies include
antibodies against actin, angiotensin II receptors, antibodies
against receptors for growth factors such as VEGF, FGF, PDGF or
EGF, and antibodies against adhesion molecules, for example against
the vitronectin receptor or ICAM 3.
[0105] The ligands furthermore include all active compounds which
bind to membrane structures or membrane receptors on endothelial
cells. Examples of these are IL-1 or growth factors, or their
fragments or part sequences thereof, which bind to receptors which
are expressed in endothelial cells, for example PDGF, bFGF, VEGF or
TGF.beta. [Pusztain et al., J. Pathol. 169, 191 (1993)].
[0106] The ligands furthermore include adhesion molecules which
bind to activated and/or proliferating endothelial cells. Adhesion
molecules of this nature, such as Slex, LFA-1, MAC-1, LECAM-1,
VLA-4 or vitronectin, have already been described [Augustin-Voss et
al., J. Cell Biol. 119, 483 (1992), Pauli et al., Cancer Metast.
Rev. 9, 175 (1990), Honn et al., Cancer Metast. Rev. 11, 353
(1992), Pigott et al., The Adhesion Molecule, Academic Press
(1994)].
[0107] The ligands within the meaning of this invention also
include, in particular, glycoproteins from the coats of viruses
which have a tropism for endothelial cells. Examples of these
viruses are filoviruses, such as Marburg virus with its coat
proteins GP (glycoprotein) and sGP (second glycoprotein) or Ebola
virus, in each case with its coat proteins GP and sG,
cytomegalovirus, particularly with its gB protein, herpes simplex
virus type I, HIV-1 virus, measles virus, Hantaan virus,
alphaviruses, such as Semliki forest virus, epidemic hemorrhagic
fever virus, polio virus or enteroviruses, such as ECHO 9, ECHO 12
and Coxsackie B3.
[0108] Antibodies or antibody fragments which are directed against
membrane structures of muscle cells, in particular of smooth muscle
cells, are examples of ligands for muscle cells. Examples of
antibodies of this nature are antibody 10F3, antibodies against
actin, antibodies against angiotensin II receptors, antibodies
against receptors for growth factors or antibodies, for example,
against EGF receptors, against PDGF receptors or against FGF
receptors, or antibodies against endothelin A receptors.
[0109] The ligands furthermore include nucleotide sequences for
active substances which bind to membrane structures or membrane
receptors on muscle cells [Pusztai et al., J. Pathol. 169, 191
(1993), Harris, Curr. opin. Biotechnol. 2, 260 (1991)]. Examples of
these ligands are growth factors, or their fragments or part
sequences thereof, which bind to receptors which are expressed in
smooth muscle cells, for example PDGF, EGF, TGF.beta., TGF.alpha.,
FGF or endothelin A.
[0110] The ligands also include glycoproteins from the coats of
those viruses which have a tropism for muscle cells. An example of
these viruses is cytomegalovirus [Speir et al., Science 265, 391
(1994)].
[0111] Examples of ligands for activated macrophages and/or
activated lymphocytes are, in addition, nucleotide sequences which
encode substances which bind specifically to the surface of immune
cells. These substances include antibodies or antibody fragments
which are directed against membrane structures of immune cells, as
have been described, for example, by Powelson et al., Biotech. Adv.
11, 725 (1993) and Barclay et al., The Leucocyte Antigen, Academic
Press (1994). The ligands also include monoclonal or polyclonal
antibodies or antibody fragments which bind, by their
antigen-binding variable moiety, to Fc.gamma., Fc.epsilon. or
Fc.mu. receptors of immune cells [Rojanasakul et al., Pharm. Res.
11, 1731 (1994)]. They furthermore include the Fc fragment of human
monoclonal or polyclonal immunoglobulin.
[0112] The ligands furthermore include all substances which bind to
membrane receptors on the surface of immune cells. These substances
include cytokines, such as IL-1, IL-2, IL-3, IL-4, IL-6, IL-10,
TNF.alpha., GM-CSF and M-CSF, and also growth factors, such as EGF,
TGF, FGF, IGF or PDGF, or their fragments or part sequences
thereof, which bind to receptors which are expressed in immune
cells [Callard et al., The Cytokine, Academic Press (1994)]. The
ligands also include adhesion molecules and other ligands which
bind to cell membrane structures on macrophages, and in spleen,
liver, lung and other tissues [Pigott et al., The Adhesion
Molecule, Academic Press (1994), Perales et al., Eur. J. Biochem.
226, 255 (1994)].
[0113] The ligands within the meaning of this invention also
include glycoproteins from the coats of those viruses which have a
tropism for lymphocytes and/or macrophages. Examples of these
macrophage-infecting viruses are HIV-1, in particular those strains
having mutations in the V3 region of gp120 which result in
increased binding to macrophages, HIV-2, Hantaviruses, for example
Punmalavirus, cytomegalovirus, respiratory syncytial virus, herpes
simplex virus or filoviruses.
[0114] Examples of lymphocyte-infecting viruses are varicella
zoster virus (VZV), since VZV infects T cells in particular,
herpesvirus 6 (HHV 6), since HHV 6 likewise infects T cells in
particular, rabies virus, since rabies virus coat protein binds to
TH2 cells in particular, HIV-1, since glycoprotein gp120 binds
preferably to the CD4 molecule of T cells, HTLV-II, since HTLV-II
infects B cells in particular, HTLV-I, since HTLV-I infects T cells
in particular, influenza C viruses, since influenza C viruses bind
to N-acetyl-9-.beta.-acetylneuraminic acid (Neu 5,9 Ac), which
preferentially occurs on B lymphocytes and to a lesser extent, or
not at all, on T lymphocytes, by way of the hemagglutinin-esterase
fusion (HEF) protein, influenza C viruses having a mutation in
nucleotide position 872, which encodes position 284 of the amino
acid sequence of the HEF, for example with the threonine being
replaced with isoleucine, since the surface protein HEF which
possesses this mutation has a markedly stronger affinity for the
N-acetyl-9-O-acetylneuraminic acid receptor than does the wild-type
virus, cleavage products of the influenza C virus HEF which contain
the structure for binding to N-acetyl-9-.beta.-acetylneuraminic
acid. This binding structure is defined by the catalytic triad
serine 71, histidine 368 or 369 and aspartic acid 261, Epstein-Barr
virus, since EBV infects B cells in particular, herpes simplex
virus 2, since HSV-2 infects T cells in particular, or measles
virus.
[0115] Examples of ligands for synovial cells and inflammatory
cells which are to be mentioned are nucleic acid sequences which
encode antibodies or antibody fragments which bind, by their
variable domains, to membrane structures of synovial cells or
inflammatory cells. Examples of these membrane structures are
vimentin [Miettinen et al., Am. J. Pathol. 117, 18 (1984)],
fibronectin [Wojciak et al., Clin. Exp. Immunol. 93, 108 (1993)] or
Fc receptors. These ligands also include antibodies or antibody
fragments which bind to the Fc receptor by their constant domains
[Rojanasakul et al., Pharm. Res. 11, 1731 (1994)].
[0116] These ligands furthermore include all active compounds which
bind to membrane structures or membrane receptors on synovial
cells. Examples of these are cytokines or growth factors, or their
fragments or part sequences thereof, which bind to receptors which
are expressed by synovial cells, for example IL-1-RA, TNF.alpha.,
IL-4, IL-6, IL-10, IGF or TGF.beta. [Callard et al., The Cytokine,
Academic Press (1994)].
[0117] Examples of ligands for virus-infected cells which are to be
mentioned are nucleic acid constructs which encode antibodies or
antibody fragments which are directed against the viral antigens
which are located on the cell membrane of virus-infected cells.
Antibodies of this nature are directed, for example, against
antigens of HBV, HCV, HSV, HPV, HIV, EBV or HTLV.
[0118] Examples of ligands for liver cells and other tissue cells
are all substances which bind to membrane structures or membrane
receptors on the surface of liver cells. Examples of these are
growth factors, such as cytokines, EGF, TGF, FGF or PDGF, or their
fragments or part sequences thereof, which bind to receptors which
are expressed in cells of this nature.
[0119] These ligands furthermore include ligands which bind to cell
membrane structures which are selective for particular tissues.
Examples are:
1 Ligand Tissue cells Transferrin receptor Transferrin Liver, other
tissue cells Insulin receptor Insulin Liver, other tissue cells
Fc.gamma. receptors Immunoglobulin G Reticuloendothelial system,
other tissue cells
[0120] These ligands and membrane structures are reviewed in
Perales et al., Eur. J. Biochem. 226, 255 (1994).
[0121] The ligands particularly include glycoproteins from the
coats of viruses which have a tropism for selected cells, such as
for bronchial epithelial cells (respiratory syncytial virus), liver
cells (hepatitis C virus), filoviruses, Marburg virus by way of the
asialoglycoprotein receptor of liver cells, hepatitis B virus, with
liver cells preferably binding to the preS2 and presi domains of
HBV by way of the asialoglycoprotein receptor, hepatitis D virus,
liver-sinusoiddl cells, and heptatis B virus, with HBV being bound
by way of fibronectin.
[0122] Examples of ligands for glia cells are nucleic acid
sequences which encode antibodies or antibody fragments which are
directed against membrane structures of glia cells, as have been
reported, for example, by Mirsky et al. [Cell and Tissue Res. 240,
723 (1985)], Coakham et al. [Prog. Exp. Tumor Res. 29, 57 (1985)]
and McKeever et al. [Neurobiol. 6, 119 (1991)]. These membrane
structures furthermore include neural adhesion molecules such as
N-CAM, in particular its polypeptide chain C [Nybroe et al., J.
Cell Biol. 101, 2310 (1985)]. These ligands furthermore include all
active compounds which bind to membrane structures or membrane
receptors on glia cells. Examples of these active compounds are
insulin and insulin-like growth factor, and those fragments of
these growth factors which bind to the relevant membrane
receptors.
[0123] The ligands within the meaning of the invention additionally
include nucleic acid sequences which encode glycoproteins of the
coats of those viruses which have a tropism for glia cells.
[0124] These viruses include, for example, HIV-1 subtype JRF1 or
herpes simplex virus I.
[0125] Examples of ligands for leukemia cells include nucleic acid
constructs which encode antibodies or antibody fragments which are
directed against membrane structures of leukemia cells. A large
number of monoclonal antibodies of this nature have already been
described for diagnostic and therapeutic procedures [Kristensen,
Danish Medical Bulletin 41, 52 (1994); Schranz, Therapia Hungarica
38, 3 (1990); Drexler et al., Leuk. Res. 10, 279 (1986); Naeim,
Dis. Markers 7, 1 (1989); Stickney et al., Curr. Opin. Oncol. 4,
847 (1992); Drexler et al., Blut 57, 327 (1988); Freedman et al.,
Cancer Invest. 9, 69 (1991)]. Depending on the type of leukemia,
monoclonal antibodies, or their antigen-binding antibody fragments,
of the following specificity are, for example, suitable as
ligands:
[0126] AML cells having the membrane antigens CD13, CD14, CD15,
CD33, CAMAL and sialosyl-Le; B-CLL cells having the membrane
antigens CD5, CD1c and CD23, and also idiotypes and isotypes of the
membrane immunoglobulins; T-CLL cells having the membrane antigens
CD33, M38, IL-2 receptors and T cell receptors; and ALL cells
having the membrane antigens CALLA and CD19, and also non-Hodgkin's
lymphoma.
[0127] The ligands furthermore include all active compounds which
bind to membrane structures or membrane receptors of leukemia
cells. Examples of these are growth factors, or their fragments or
part sequences thereof, which bind to receptors which are expressed
in leukemia cells.
[0128] Growth factors of this nature have already been described
[reviews in Cross et al., Cell 64, 271 (1991); Aulitzky et al.,
Drugs 48, 667 (1994); Moore, Clin. Cancer Res. 1, 3 (1995); Van
Kooten et al., Leuk. Lymph. 12, 27 (1993)]. For example, they
include IFN.alpha., in the case of non-Hodgkin's lymphomas, IL-2,
particularly in the case of T cell leukemias, FGF in the case of T
cell, monocytic, myeloid, erythrocytic and megakaryoblastic
leukemias, TGF.beta. in the case of leukemias, or retinoids, e.g.
retinoic acid, in the case of acute promyelocytic leukemia.
[0129] Examples of ligands for tumor cells include nucleic acid
sequences which encode antibodies, and fragments of these
antibodies, which are directed against membrane structures on tumor
cells. Antibodies of this nature have been reviewed, for example,
by Sedlacek et al., Contrib. to Oncol. 32, Karger Verlag, Munich
(1988) and Contrib. to Oncol. 43, Karger Verlag, Munich (1992).
[0130] Other examples are antibodies against sialyl Lewis, peptides
on tumors which are recognized by T cells, proteins expressed by
oncogenes, gangliosides such as GD3, GD2, GM2, 9-O-acetyl-GD3 and
fucosyl-GM1, blood group antigens and their precursors, antigens on
polymorphic epithelial mucine or antigens on heat shock
proteins.
[0131] Nucleic Acid Sequence [Component b)] Which Ecodes An Active
Compound (Protein B):
[0132] The active compound (protein B) according to the present
invention can be a substance which, for example, intervenes in a
biological activation cascade and/or is an active component of this
cascade. These substances include active compounds which activate
the coagulation cascade, for example thrombin [MacGillivray et al.,
Ann. N.Y. Acad. Sci. 485, 73 (1986)], thrombin which is mutated in
the region of the Arg-Thr cleavage site (amino acid position
327/328), factor Va [Cripe et al., Biochem. 31, 3777 (1992), Jenny
et al., PNAS-USA 84, 4846 (1987)], factor VIIa [O'Hara et al.,
PNAS-USA 84, 5158 (1987)], factor IXa [Yoshitake et al., Biochem.
24, 3736 (1985)], factor Xa [Messier et al., Gene 99, 291 (1991)]
or tissue factor and coagulation-active fragments thereof
[Morrissey et al., Cell 50, 29 (1987); Scarpati et al., Biochem.
26, 5234 (1987); Spicer et al., PNAS-USA 84, 5148 (1987);
Rehemtulla et al., Thromb. Heamost. 65, 521 (1991)] or which
inhibit the coagulation cascade or which activate fibrinolysis, for
example the plasminogen activator inhibitors PAI-1, PAI-2 and
PAI-3, hirudin, protein C, serine proteinase inhibitors, such as
C-1S inhibitor, .alpha.1-antitrypsin or antithrombin III, tissue
factor pathway inhibitor (TFPI), plasminogen activators such as
urokinase, tissue plasminogen activator (tPA), or hybrids thereof,
or which activate the complement cascade, for example cobra venom
factor (CVF) or part sequences of CVF which correspond functionally
to human complement factor C3b, i.e. which are able to bind to
complement factor B and which, after having been cleaved by factor
D, constitute a C3 convertase (the DNA sequence for CVF and its
part sequences were described by Fritzinger et al., Proc. Natl.
Acad. Sci. USA 91, 12775 (1994)), human complement factor C3b (the
DNA sequence for C3 and its part sequences were published by De
Bruijn et al., Proc. Natl. Acad. Sci. USA 82, 708 (1985), cleavage
products of human complement factor C3 which resemble CVF
functionally and structurally (such cleavage products have been
described by O'Keefe et al., J. Biol. Chem. 263, 12690 (1988) or
which activate the kinin system, the complement system and/or the
coagulation system, for example activated Hagemann factor (F XIIa)
[Shibuya et al., Biochem, Biophys. Acta 1206, 63 (1994), Que et
al., Biochem. 25, 1525 (1986), Tripodi et al., Nucl. Acid Res. 14,
3146 (1986)] or kallikrein [Chen et al., Biochem. J. 307, 481
(1995), Fukushima et al., Biochem. 24, 8037 (1985)].
[0133] The active compound (protein B) can also be a cytostatic,
cytotoxic or inflammation-eliciting protein, such as perforin,
granzyme, cytokines, such as IL-1, IL-2, TL-4, IL-12, IL-3, IL-5,
human leukemia inhibitory factor (LIF), IL-7, IL-11, IL-13, GM-CSF,
G-CSFb or M-CSF, interferons, such as IFN.alpha., IFN.beta. or
IFN.gamma., TNF, such as TNF.alpha. or TNF.beta., oncostatin M,
sphingomyelinase [Jarvis et al., PNAS USA 91, 73 (1994)], magainin
and magainin derivatives [Cruciani et al., PNAS USA 88, 3792
(1991)]; Jacob et al., Ciba Found. symp. 186, 197 (1994);
Peck-Miller et al., Cancer Chemother. Pharmac. 32, 109 (1993)] or
chemokines, such as RANTES (MCP-2), monocyte chemotactic and
activating factor (MCAF), IL-8, macrophage inflammatory protein 1
(MIP-1.alpha. or MIP-1.beta.) or neutrophil activating protein 2
(NAP-2).
[0134] The active compound (protein B) can also be an
antiangiogenic protein, such as angiostatin, interferons, such as
IFN.alpha., IFN.beta. or IFN.gamma., platelet factor 4, IL-12,
TIMP-1, TIMP-2 or TIMP-3.
[0135] The active compound (protein B) can also be an enzyme which
is able to convert an inactive precursor of a pharmacological
active substance, for example a cytostatic agent, into the active
substance itself. Examples of such active compounds are bacterial
nitroreductase, bacterial .beta.-glucuronidase, plant
.beta.-glucuronidase derived from Secale cereale, human
.beta.-glucuronidase, human carboxypeptidase (CB), e.g. mast cell
CB-A or pancreas CB-B, or bacterial carboxypeptidase, bacterial
.beta.-lactamase, bacterial cytosine deaminase, human catalase or
peroxidase, phosphatase, in particular human alkaline phosphatase
or human acid prostate phosphatase, type 5 acid phosphatase,
oxidase, in particular human lysyl oxidase or human acid
D-aminooxidase, peroxidase, in particular human glutathione
peroxidase, human eosinophilic peroxidase or human thyroid
peroxidase.
[0136] The active compound (protein B) can also be a protein which
affects the immune system, for example a protein having an
antiallergic effect, such as IFN.beta., IFN.gamma., IL-10, soluble
IL-4 receptors, IL-12 or TGF.beta., or a protein which can prevent
the rejection of transplanted organs, such as IL-10, TGF.beta.,
soluble IL-1 receptors, soluble IL-2 receptors, IL-2 receptor
antagonists or soluble IL-6 receptors, or a protein for the therapy
of antibody-mediated autoimmune diseases, for example TGF.beta.,
IFN.alpha., IFN.beta., IFN.gamma., IL-12, soluble IL-4 receptors or
soluble IL-6 receptors, or a protein for the therapy of
cell-mediated autoimmune diseases, for example IL-6, IL-9, IL-10,
IL-13, TNF.alpha., IL-4 or TNF.beta., or a protein for the therapy
of arthritis. According to the present invention, structural genes
can also be selected whose expressed protein directly or indirectly
inhibits inflammation, for example in a joint, and/or promotes the
reconstitution of extracellular matrix (cartilage and connective
tissue) in the joint. These expressed proteins include, for
example, IL-1 receptor antagonists (IL-1-RA), since IL-1-RA
inhibits the binding of IL-1.alpha. and IL-1.beta., soluble IL-1
receptor, since soluble IL-1 receptor binds and inactivates IL-1,
IL-6, since IL-6 increases secretion of TIMP and superoxides and
decreases secretion of IL-1 and TNFa by synovial cells and
chondrocytes, soluble TNF receptor, since soluble TNF receptor
binds and activates TNF, IL-4, since IL-4 inhibits the formation
and secretion of IL-1, TNF.alpha. and MMP, IL-10, since IL-10
inhibits the formation and secretion of IL-1, TNF.alpha. and MMP
and increases the secretion of TIMP, insulin-like growth factor
(IGF-1), since IGF-1 stimulates the synthesis of extracellular
matrix, TGF.beta., especially TGF.beta.1 and TGF.beta.2, since
TGF.beta. stimulates the synthesis of extracellular matrix
superoxide dismutase, or TIMP (tissue inhibitors of
metalloproteinases), especially TIMP-1, TIMP-2 or TIMP-3.
[0137] The active compound (protein B) can also be a protein for
relieving damage to the nervous system, for example a growth
factor, such as FGF, nerve growth factor (NGF), brain-derived
neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4
(NT-4) or ciliary neurotrophic factor (CNTF), or a cytokine, or a
cytokine inhibitor, which is able to inhibit or neutralize the
neurotoxic effect of TNF.alpha., for example TGF.beta., soluble TNF
receptors, IL-10, since IL-10 inhibits the formation of IFN.gamma.,
TNF.alpha., IL-2 and IL-4, soluble IL1 receptors, such as IL-1
receptor I or IL-1 receptor II, since soluble IL-1 receptors
neutralize the activity of IL-1, IL-1 receptor antagonist or
soluble IL-6 receptors.
[0138] The active compound (protein B) can also be a protein which
stimulates angiogenesis, for example VEGF or FGF.
[0139] The active compound (protein B) can furthermore be a protein
which lowers blood pressure, for example kallikrein or endothelial
cell nitric oxide synthase.
[0140] The active compound (protein B) can also be a protein for
the therapy of chronic infectious diseases, for example a protein
which exhibits cytostatic or cytotoxic effects, or an enzyme which
cleaves a precursor of an antiviral or cytotoxic substance into the
active substance, or a cytotoxin having an antiviral effect or a
growth factor having an antiviral effect. Examples are IFN.alpha.,
IFN.beta., IFN.gamma., TNF.beta., TNF.alpha., IL-1 or
TGF.beta..
[0141] The present invention furthermore relates to a nucleic acid
construct in which two identical or two different DNA sequences,
which encode identical or different active compounds (protein B)
[component b) and b")] are combined.
[0142] In order to ensure that both DNA sequences are expressed,
the cDNA of an internal ribosome entry site (IRES) is preferably
intercalated, as a regulatory element, between the two structures.
An internal ribosome entry site makes it possible to express two
DNA sequences which are linked to each other by way of an IRES.
IRESs of this nature have been described, for example, by Montford
and Smith TIG 11, 179 (1995); Kaufman et al., Nucl. Acids Res. 19,
4485 (1991); Morgan et al., Nucl. Acids Res. 20, 1293 (1992); Dirks
et al., Gene 128, 247 (1993); Pelletier and Sonenberg, Nature 334,
320 (1988) and Sugitomo et al., BioTechn. 12, -694 (1994). Thus,
for example, the cDNA for the polio virus IRES sequence (positions
.ltoreq.140 to .gtoreq.630 of the 5' UTR [Pelletier and Sonenberg,
Nature 334, 320 (1988)] can be used to link the DNA of component c)
to the DNA of component d).
[0143] Nucleic Acid Sequences [Component c)] Which Encode The
Protease-Cleavable Part Structure C:
[0144] According to the present invention, part stucture C
comprises an amino acid sequence which is cleaved by proteases
which are formed in tumors or by tumor cells or inflammatory cells.
The nucleic acid sequence for this part structure C is inserted,
for example, into the nucleic acid sequence of the naturally
occurring precursor (protein BSD, where S is the naturally
occurring cleavage sequence) of the relevant active compound
(protein B) in place of the cleavage sequence S such that this
recombinant nucleic acid expresses protein BCD or B'BCD.
[0145] The nucleic acid sequence encoding part structure C is
chosen depending on the protease which is predominantly secreted in
the tumor or in the inflammation.
[0146] The following part structures C may, for example, be
employed for the following enzymes [Barrett et al., Mammalian
Proteases, Academic Press, London (1980), Panchal et al., Nature
Biotechnol. 14, 852 (1996); Pigott et al., Ayad et al., The
extracellular Matrix, Academic press (1994); Yoshida et al., Int.
J. Cancer 63, 863 (1995), Petersen et al., J. Biol. Chem. 265, 6104
(1990); Cramer et al., J. Urology 156, 526 (1995); Forsgen et al.,
FEBS Lett. 213, 254 (1987) Zhang et al. Chin. Chem. 41, 1567,
(1995)]:
2 Part structure C cleavage Enzyme A6 A5 A4 A3 A2 A1 A-1 (A-2)
Plasminogen Cys Pro Gly Arg Val (Ile) (Val) activator Gln Gly Arg
Gly Gly Arg Pro Arg Phe Lys Gly Lys Arg Prostrate- Pro Arg Phe Lys
Ile (Ile) (Val) specific Arg Pro Tyr antigen Arg Arg Phe Phe Leu
(Ile) (His) (Val) Tyr Ile Val Ser Phe Ser Ile Gln Tyr Ile Val Gly
Ser Gln Gln Leu Leu Ile Val Gly Ile Ser Ser Gln Tyr Ile Val
Cathepsins Pro Arg Phe Lys Ile Ile (Val) Tyr Lys Ser Arg Met (Ile)
Lys Met Arg Arg (Ile) Ile Arg Arg Arg (Ile) Arg Ala Arg Leu (Ile)
Gln Ala Arg Phe (Ile) Lys Leu Arg Leu (Ile) Lys Arg Val (Ile) Lys
Phe Arg Stromelysins Gly Gly Gly Ala Gln (Leu) Gln Leu Gly Val Met
(Gln) Ala Ala Ala Ser Leu (Lys) Val Ala Val Ser Ala (Lys) Leu Ala
Ala Asn Leu (Arg) Collagenase I Gly Pro Gln Gly Ile (Ala) Gly Pro
Gln Gly Leu (Leu) II Gly Pro Gln Gly Leu (Ala) III Gly Ile Ala Gly
Ile (Thr) VIII Gly Leu Pro Gly Ile (Gly) Gly Phe Pro Gly Ile (Gly)
XI Gly Pro Ala Gly Ile (Ser) Gly Pro Ala Gly Ile (Ala) Plasminogen
Ser Gly Thr Glu Ile (Val) The amino acid positions (A1-A6 and A-1
and A-2) were defined in accordance with Schechter and Bergr,
Biochem. Biophys. Res. Comm. 27, 157 (1967).
[0147] Nucleic Acid Sequences [Component d)] Which Encode Part
Structure D:
[0148] According to the present invention, the nucleic acid
sequence [component d)] encodes a peptide (part structure D) which
binds to the active compound (part structure B) by way of the part
structure C and inactivates this active compound by means of this
binding.
[0149] Preferably, those nucleic acid sequences are used for part
structure D which encode part structure D in the naturally
occurring precursors (protein BSD), with part structure S being the
natural cleavage sequence in protein BSD.
[0150] The structures of the naturally occurring precursors of
active compounds (protein B) have already been reviewed, for
example by Bartett et al., Mammalian Proteases, Academic Press,
London (1980) in the case of coagulation factors, complement
factors and kallikrein, by Callard et al., The Cytokine Facts Book,
Academic Press (1994) in the case of interleukins, chemokines and
growth factors, and by Denhardt et al., Pharmac. Ther. 59, 329
(1993) in the case of tissue inhibitors of metalloproteinases
(TIMPs).
[0151] When selecting active compounds which do not have any
naturally occurring precursors, and in the case of xenogeneic
active compounds, use should be made of nucleic acid sequences, as
component d), which encode any peptide, preferably, however, of
nucleic acid sequences which encode those part structures D which
naturally occur in the precursors of human active compounds.
[0152] In order to facilitate secretion of the protein BCD, or
B'BCD, which is expressed by the novel nucleic acid sequence, the
homologous signal sequence which may be present in the DNA sequence
of component b) can be replaced with a heterologous signal sequence
which improves extracellular secretion. Thus, for example, the
signal sequence for immunoglobulin [DNA positions .ltoreq.63 to
.gtoreq.107; Riechmann et al., Nature 332, 323 (1988)] or the
signal sequence for CEA [DNA positions .ltoreq.33 to .gtoreq.134,
Schrewe et al., Mol. Cell Biol. 10, 2738 (1990); Berling et al.,
Cancer Res. 50, 5634 (1990)] or the signal sequence of human
respiratory syncytial virus glycoproteins [cDNA of amino acids
.ltoreq.38 to .gtoreq.50 or 48 to 65; Lichtenstein et al., J. Gen.
Virol. 77, 109 (1996)] can be inserted.
[0153] In addition, in order to augment translation, the nucleotide
sequence GCCACC or GCCGCC [Kozak, J. Cell Biol. 108, 299 (1989)]
can be inserted at the 3' end of the promoter sequence and directly
at the 5' end of the start signal (ATG) of the signal sequence.
[0154] Preparation of the Novel Nucleic Acid Constructs
[0155] The novel nucleic acid constructs which have been described
are prepared by linking the individual components to each other
using standard molecular biological methods.
[0156] Applications:
[0157] The novel nucleic acid construct is particularly well suited
for treating diseases which are accompanied by an increased local
formation of proteases, such as tumor diseases, leukemias,
allergies, autoimmune diseases, infections, inflammations,
transplant rejection reactions, thromboses and blood vessel
occlusions and other disturbances of blood clotting and of blood
circulation, and tissue injuries, including injuries to the central
nervous system and damage to the nervous system. Administration is
effected locally (e.g. onto the skin), nasally, orally,
gastrointestinally, intrabronchially, intravesically,
intravaginally, into the uterus, sub-cutaneously, intramuscularly,
periarticularly, intraarticularly, into the cerebrospinal fluid,
into the brain tissue, into the spinal medulla, into wounds,
intraperitoneally or intrapleurally, or systemically, e.g.
intravenously, intraarterially, intraportally or into the
heart.
[0158] In general, the administered composition comprises, where
appropriate in addition to the customary additives and auxiliary
substances, either the novel nucleic acid construct or a cell which
is able to express the novel nucleic acid construct. The
administered composition can be administered for the prophylaxis or
therapy of a disease, as already described in detail above.
[0159] For administration purposes, an effective amount is
determined by the skilled artisan considering variables well known
in the art such as the nature of the applicable disease or
condition, the nature of the patient, mammal or cells being treated
and the method of administration.
[0160] Moreover, in addition to the methods of administration
discussed above, the present invention contemplates the
administration of the novel nucleic acid construct to a mammal by
ex vivo gene transfer of the cells of the subject mammal in a
clinical setting. Such techniques are well known to those of skill
in the art. In addition, the present invention contemplates
introduction of the novel nucleic acid construct into cells in vivo
[Rosenberg et al., Science 242:1575-1578 (1988) and Wolff et al.,
PMAS 86:9011-9014 (1989)]. In this regard, the routes of delivery
include systemic administration and administration in situ.
Well-known techniques include systemic administration with cationic
liposomes, and administration in situ with viral vectors. Any one
of the gene delivery methodologies described in the existing art is
suitable for the introduction of novel nucleic acid construct into
a target cell.
[0161] Said cell is prepared, for example, by transforming or
transfecting cells with the novel nucleic acid construct using
methods known to the skilled person.
[0162] Examples of suitable cells are endothelial cells,
lymphocytes, macrophages, glia cells, fibroblasts, liver cells,
kidney cells, muscle cells, cells of the bone or cartilage tissue,
synovial cells, peritoneal cells, skin cells, epithelial cells,
leukemia cells and/or tumor cells.
[0163] The novel cells are also suitable for preparing the protein
which is encoded by the novel nucleic acid construct and which can
be used directly as a drug.
[0164] The present invention furthermore relates, therefore, to the
use of the novel nucleic acid construct for preparing a
recombinantly altered cell, with the nucleic acid construct being
introduced into the cells, to the use of the novel nucleic acid
construct for preparing a protein which is encoded by the nucleic
acid construct, with the nucleic acid construct being caused to
express in a suitable cell and the protein which is formed being
isolated, and to a cell which harbors the novel nucleic acid
construct. The above-described cells are the preferred cells.
[0165] The following selection can, for example, be made from the
above-mentioned examples of promoter sequences and structural genes
(for the protein BCD or B'BCD) depending on the nature and site of
the disease and on the target cell to be transduced:
[0166] Therapy of Tumors:
[0167] Promoters [component a)]: endothelial cell-specific and cell
cycle-specific or cell-nonspecific or muscle cell-specific and cell
cycle-specific or tumor cell-specific (solid tumors, leukemias)
[0168] Ligands for the following target cells [component b')]:
proliferating endothelial cells or stroma cells and muscle cells
adjacent to the endothelial cell or tumor cells or leukemia
cells.
[0169] Structural genes [component b)c)d)]: for
coagulation-inducing factors, for complement factors, for
angiogenesis inhibitors, for cytostatic and cytotoxic proteins, for
inducers of inflammations or for enzymes for activating precursors
of cytostatic agents, for example for enzymes which cleave inactive
precursor substances (prodrugs) thereby forming active cytostatic
agents (drugs).
[0170] Therapy of Autoimmune Diseases and Inflammations:
[0171] Promoters [component a)]: endothelial cell-specific and cell
cycle-specific, or macrophage-specific and/or lymphocyte-specific
and/or cell cycle-specific or synovial cell-specific and/or cell
cycle-specific.
[0172] Ligands for the following target cells [component b')]:
proliferating endothelial cells, macrophages and/or lymphocytes or
synovial cells.
[0173] Structural genes [component b)c)d)]: for the therapy of
antibody-mediated autoimmune diseases, for inhibitors of cell
proliferation, cytostatic or cytotoxic proteins, enzymes for
activating precursors of cytostatic agents or for the therapy of
arthritis.
[0174] Therapy of Damage to the Nervous System:
[0175] Promoters [component a)]: glia cell-specific, endothelial
cell-specific and cell cycle-specific or nonspecific and cell
cycle-specific.
[0176] Ligands for the following target cells [component b')]: glia
cells or proliferating endothelial cells
[0177] Structural genes [component b)c)d)]: for neuronal growth
factors, for example for cytokines and cytokine inhibitors which
inhibit or neutralize the neurotoxic effect of TNF.alpha..
[0178] Therapy of disturbances of the blood coagulation system and
the blood circulation system:
[0179] Promoters [component a)]: cell-nonspecific, cell-nonspecific
and cell cycle-specific or specific for endothelial cells, smooth
muscle cells or macrophages, or specific for endothelial cells,
smooth muscle cells or macrophages and cell cycle-specific.
[0180] Ligands for the following target cells [component b')]:
endothelial cells, proliferating endothelial cells or somatic cells
in the vicinity of endothelial cells and smooth muscle cells or
macrophages.
[0181] Structural genes [component b)c)d)]: for the inhibition of
coagulation or for the promotion of fibrinolysis, for angiogenesis
factors, for hypotensive peptides, for an antiproliferative,
cytostatic or cytotoxic protein or for an enzyme for cleaving is
precursors of cytostatic agents, thereby forming cytostatic agents,
for inhibition of the proliferation of smooth muscle cells
following injury to the endothelial layer or for blood plasma
proteins, such as C1 inactivator, serum cholinesterase or
.alpha.1-antitrypsin.
[0182] Therapy of Chronic Infectious Diseases:
[0183] Promoters [component a)]: virus-specific, cell-specific or
virus-specific or cell-specific and cell cycle-specific.
[0184] Ligands for the following target cells [component b')]:
liver cells, lymphocyte and/or macrophage, epithelial cell or
endothelial cell.
[0185] Structural genes [components b) c) d)]: for a protein which
exhibits cytostatic or cytotoxic effects, an enzyme which cleaves a
precursor of an antiviral or cytotoxic substance thereby forming
the active substance, or for antiviral proteins such as antivirally
active cytokines and growth factors.
[0186] The invention is explained in more detail with the aid of
the following examples and figures without restricting it
thereto:
EXAMPLES
1. Preparation of a Nucleic Acid Construct Encoding
Prostate-Specific Antigen (PSA)-Activatable FX
[0187] This deals with the preparation of a therapeutic agent for
treating prostate carcinoma metastases. Despite the surgical
removal of a prostate which has become carcinomatous, metastases of
the prostate carcinoma frequently arise which are currently still
largely untreatable and which lead to the death of the patient.
Such prostate carcinoma metastases induce angiogenesis.
Furthermore, prostate carcinoma metastases secrete a
tissue-specific enzyme, i.e. prostate-specific antigen (PSA). In
accordance with the invention, a nucleic acid construct is prepared
which, having been introduced into proliferating endothelial cells,
leads to a modified FX coagulation factor being expressed. The
modification comprises replacing, in the gene for the natural FX,
the nucleotide sequence for the natural cleavage site, whose
cleavage results in coagulation-active FXa, with a nucleotide
sequence encoding a PSA-specific cleavage site. As a result, the
PSA which is secreted by prostate carcinoma metastases is able to
specifically activate the modified FX which is secreted by
proliferating endothelial cells in the vicinity of the metastases
and thereby to initiate the coagulation which leads to the blood
supply to the metastasis being interrupted and consequently to
necrosis of the metastasis.
[0188] The nucleic acid construct for the PSA-activatable FX is
prepared in accordance with a scheme which is depicted in FIG.
3.
[0189] The DNA sequences of the individual components are joined
together, in the 5' to 3' direction, as follows:
[0190] Component a), which contains the promoter sequence of the
cdc25C gene [nucleic acids: -290 to +121; Lucibello et al., EMBO J.
14, 132 (1995); Zwicker et al., Nucl. Acids Res. 23, 3822 (1995);
EMBO J. 14, 4514 (1995)], the sequence GCCACC (Kozak, J. Cell Biol.
108, 229 (1989)) and the cDNA for the immunoglobulin signal peptide
[nucleotide sequence .ltoreq.63 to .gtoreq.107; Riechmann et al.,
Nature 332, 323 (1988)], is fused to component b)c)d), which
contains the cDNA for human FX (nucleotide sequence 1 to
.gtoreq.1468) [Messier et al., Gene 99, 291 (1991)] in which amino
acid 194 has been mutated from Arg to Tyr.
[0191] The individual components of the construct are linked by way
of suitable restriction sites which are introduced at the termini
of the different elements by way of PCR amplification. The linking
is effected using enzymes which are specific for the restriction
sites and which are known to the skilled person, and DNA ligases.
These enzymes can be obtained commercially.
[0192] The nucleotide construct which has been prepared in this way
is cloned into pUC 18/19 or Bluescript-derived plasmid vectors.
2. Expression in Human Embryonic Kidney Cells
[0193] Proliferating human embryonic kidney cells [HEK 293; Racchi
et al., J. Biol. Chem. 268, 5735 (1993)] which are being maintained
in culture are transfected with the above-described plasmid using
the method known to the skilled person [Graham and van der Eb,
Virol. 52, 456 (1973)].
[0194] The mutated factor X is purified from the supernatant from
approx. 10.sup.7 transfected HEK 293 cells [Watzke et al., J. Clin.
Invest. 88, 1685 (1991)] and assayed in a coagulation test for
factor X with and without the addition of PSA. Purified PSA is
obtained from Chemicon (Temecula, Calif., USA).
[0195] In this test, the coagulation defect in human FX-deficient
plasma is counterbalanced by functionally active FXa.
[0196] Nonmutated (wild-type) FX (which is activated by Russel's
viper venom) is employed as a positive control. In addition to the
test mixture lacking PSA, a mock preparation from the supernatant
from untransfected HEK 293 cells is used as a negative control.
[0197] The coagulation activity of the mutated FX is measured by
recalcification time (Seitz R et al., Int. J. Cancer 53:514-520,
1993). 100 .mu.l of FX-deficient plasma (Behringwerke, Marburg) are
incubated, at 37.degree. C. for 120 sec, with 100 .mu.l of the FX
preparation from the cell supernatant. The FX preparation contains
PSA as activator. No PSA is added in the case of the negative
control. FX (wild-type) and Russel's viper venom (RVV) are employed
as the positive control. The coagulation reaction is augmented by
adding 100 .mu.l of 0.02 M CaCl2 and determined in a
coagulometer.
[0198] The following results are obtained:
[0199] The negative controls without any activation of coagulation
give a coagulation time of approx. 200 sec. By contrast,
significantly shorter coagulation times, of 50 sec, are achieved
when activated FX (mutated FX and PSA or wild-type FX and RVV) is
used.
[0200] It can be concluded from this that the transduced HEK 293
cells express mutated FX which, in the added presence of PSA,
counterbalances the coagulation defect of FX-deficient plasma.
3. Expression in Human Endothelial Cells
[0201] Human umbilical cord endothelial cells which are being
maintained in culture are transfected with the above-described
plasmid using the method known to the skilled person (Lucibello et
al., EMBO J. 14, 132 (1995).
[0202] In order to check cell cycle specificity, endothelial cells
are synchronized in G0/G1 by withdrawing methionine over a period
of 48 hours. After staining with Hoechst 33258 (Hoechst AG,
Frankfurt), the DNA content of the cells is determined in a
fluorescent-activated cell sorter (Lucibello et al., EMBO J. 14,
132 (1995).
[0203] The expression of the nucleic acid construct is assayed in
the supernatant from the endothelial cells in analogy with the
investigation carried out on the HEK 293 cells.
[0204] The following results are obtained:
[0205] The protein which is expressed by the transfected
endothelial cells counterbalances the coagulation defect of
FX-deficient plasma, in contrast to mock preparations from the
supernatant from untransfected endothelial cells.
[0206] A markedly higher concentration of mutated FX can be
detected in the supernatant from proliferating, transduced
endothelial cells (DNA>2S) as compared with the supernatant from
endothelial cells which have been synchronized in G0/G1
(DNA=2S).
[0207] Consequently, the above-described nucleic acid construct
leads to the gene for the mutated FX being expressed in a cell
cycle-dependent manner in endothelial cells, and this mutated FX
can be activated by PSA such that it brings about coagulation in
FX-deficient plasma.
[0208] Federal Republic of Germany priority application, DE
19701141.1, filed Jan. 16, 1997, including the specification,
drawings, claims and abstract, is hereby incorporated by reference.
Sequence CWU 1
1
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