Note: Descriptions are shown in the official language in which they were submitted.
CA 02276564 1999-06-29
CENTEON PHARMA GMBH 199812007 - MA 1177 - C17
Modified C1 esterase inhibitor for blocking the infectiousness of HIV
The invention relates to a modified C1 esterase inhibitor which can be
employed for blocking the infectiousness of human immunodeficiency virus
(HIV) in vivo or in vitro.
It is known that the removal of HIV from biological fluids, but especially
from blood, blood plasma or blood serum, is an important prerequisite for
their risk-free use for all sorts of medical purposes. Numerous processes
have therefore also already been proposed using which removal of HIV
from biological fluids should be achieved. Thus a process has been
proposed in International Patent Application WO 97/07674 using which
HIV can be removed from biological fluids or inactivated by treating them
with certain ethylenimine oligomers. It is important in this case that other
constituents of the blood, in particular the cellular constituents, especially
the erythrocytes, are not damaged by a treatment of this type and the
removal of HIV can be carried out in a simple manner and short time in
order that sufficiently large amounts of purified blood can be obtained in an
economically justifiable process.
It has also already been proposed to remove HIV from biological fluids
adsorptively by means of filtration through a material impregnated with the
C1 inhibitor.
The C1 inhibitor, also called C1 esterase inhibitor, is a protein present in
the blood and is the main inhibitor of the classical pathway of the
complement system and of the contact system. The C1 inhibitor can inhibit
the activated form of factor XII and of kallikrein (Schapira M. et al., 1985,
Complement 2: 111; Davis A.E., 1988, Ann Rev Immunol 6: 595; Sim R.B.
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et al., 1979, FEBS Lett 97: 111; De Agostini A. et al., 1984, J Clin Invest
73: 1542; Pixley R.A. et al., 1985, J Biol Chem 260: 1723; Schapira M, et
al., 1982, J Clin Invest 69: 462; Van der Graaf F. et al., 1983, J Clin Invest
71: 149; Harpel P.C. et al., 1975, J clin Invest 55: 593). The C1 inhibitor
thus regulates the activities of two plasma cascades, namely the
complement system and the contact system, by which biologically active
peptides are produced. The C1 inhibitor is therefore also an important
regulator of the inflammatory system. The C1 inhibitor moreover inhibits
the activated factor XI (Meijers J.C.M. et al., 1988, Biochemistry 27: 959;
Wuillemin W.A. et al., 1995, Blood 85: 1517). It follows from this that the
C1 inhibitor can be considered as a coagulation inhibitor. The tissue
plasminogen activator and plasmin are also inhibited to a certain extent by
the C1 inhibitor, although that is not its main function (Harpel P.C. et al.,
1975, J Clin Invest 55: 149; Booth N.A. et al., 1987, Blood 69: 1600).
The C1 inhibitor is obtained to a considerable extent from plasma by
purification and utilized for clinical applications, in particular in the
treatment of hereditary angioedema, a disorder which is caused by a
genetically related tack of the C1 inhibitor. Moreover, it has already been
described that by administration of the C1 inhibitor in systemic
inflammations [International Patent Application WO 92/22320 (Genentech
Inc.)], in severe burns, pancreatitis, bone marrow transplants, cytokine
therapy and during use in a extracorporeal blood circulations [DE-A-4 227
762 (Behringwerke AG)] good therapeutic results were achieved.
The complete genomic and the cDNA which codes for the C1 inhibitor has
already been cloned (Bock S.C. et al., 1986, Biochemistry 25: 4292; Carter
P.E. et al., 1988, Eur J Biochem 173: 163). Various variants of the
recombinant C1 inhibitor with amino acid mutations in the P1 and the P3
and/or P5 positions of the reactive center and variants which were isolated
from patients with a hereditary angioedema have already been prepared
recombinantly (Eldering E. et al., 1988, J Biol Chem 263: 11776; Eldering
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E. et al., 1993, J Biol Chem 267: 7013; Eldering E. et al., 1993, J Clin
Invest 91: 1035; US-Patent 5,622,930; Davis A.E. et al., 1992, Nature
Genetics 1: 354; Eldering E. et al., 1995, J Biol Chem 270: 2579; Verpy et
al., 1995, J Clin Invest 95: 350).
The C1 inhibitor belongs to the large family of serine proteinase inhibitors
which are also called serpines (Travis J. et al., 1983, Ann Rev Biochem 52:
655; Carrel R.W. et al., 1985, Trends Bioch Sci 10: 20). On SDS
polyacrylamide gels, the C1 inhibitor exhibits a molecular weight of
approximately 105 KD. Its plasma concentration is approximately 270 mg/l
(Schapira M et al., 1.985, Complement 2: 111; Nuijens JH et al., 1989, J
Clin Invest 84: 443). The C1 inhibitor is a protein whose plasma level can
increase up to two-fold in uncomplicated infections and other
inflammations (Kalter ES et al., 1985, J Infect Dis 151: 1019). The
increased formation of the C1 inhibitor in inflammations probably serves to
protect the body against the harmful effects of the intravascular activation
of the complement system and of the contact system during the acute
reactions.
The serpines react as inhibitors by formation of bimolecular complexes
with the proteinase to be inhibited. In these complexes, the active center of
the proteinase is bound by the active center of the serpine and thus
inactive (Travis J. et al., 1983, Ann Rev Biochem 52: 655). The serpines
react specifically with certain proteinases, this specificity being determined
by the amino acid sequence of the reactive center.
The present invention starts from the observation that the infectiousness of
HIV can be blocked by the administration of a C1 inhibitor.
As is known, the first step in an HIV infection consists in helper T
lymphocytes (TH cells) of the immune system, which carry CD4 receptors
on their surface, entering into an interaction with the surface proteins of
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HIV. Antibodies against CD4 block the HIV infection of TH cells in vitro.
Even with an excess of free CD4 protein, the infection rate decreases in
vitro. Both treatments admittedly block the infection, but do not destroy the
virus. Similarly, the direct interaction between the surface proteins of HIV
and the CD4 receptors of the TH cells can be suppressed according to the
invention by binding of HIV to the C1 inhibitor.
The human C1 inhibitor, however, not only has a great affinity for the coat
proteins of HIV, but also a great affinity for the surface proteins of
lymphocytes. By means of the C1 inhibitor, a bridge-like connection can be
produced between the HIV and the TH cell in the manner of a spacer.
However, the infectiousness of HIV surprisingly still cannot be decreased
by this. This can be achieved, however, if a modified C1 esterase inhibitor
is employed which on the one hand has a high binding affinity for the coat
proteins of HIV, but on the other hand does not bind to human cell
membranes.
Modified C1 esterase inhibitors are available from different sources. Thus
the C1 esterase inhibitors obtained from the blood of cattle or other
mammals show no affinity for human cell membranes, in particular not for
the CD4 receptors of the human TH cells. Their affinity for the surface
proteins of HIV (gp120 and gp41), however, can be compared with that of
the human C1 esterase inhibitor. An animal C1 esterase inhibitor is
therefore able to bind to the surface proteins of HIV, but prevents any
contact of HIV with the human CD4 receptors, since the C1 inhibitor does
not adhere to the surfaces of the TH cells.
There is, however, also the possibility of preparing modified variants of the
human C1 inhibitor by a genetic engineering route. The exact amino acid
sequence of the human C1 inhibitor and the DNA sequence which codes
for this human C1 inhibitor have been described by Bock et al.,
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Biochemistry 25: 4292-4301, 1986 and by Davis et al., PNAS 83: 3161-
3165, 1986. Biologically active variants of the C1 esterase inhibitor are
known from the International Patent Application WO 91/06650. These
variants can be prepared by recombinant expression processes, the DNA
coding for the C1 esterase inhibitor having specific deletions, insertions or
substitutions of nucleotides.
Fundamentally, all non-human C1 esterase inhibitors which do not bind to
lymphocytes can be employed for blocking the infectiousness of HIV.
These are either animal C1 esterase inhibitors isolated from natural
material or animal C1 esterase inhibitors prepared recombinantly.
Moreover, a recombinantly modified, human C1 esterase inhibitor whose
binding ability to the CD4 receptors of the T-helper cells is abolished can
block the infectiousness of HIV. For this, modifications of the amino acid
sequence of the C1 inhibitor causing the binding to the CD4 receptor are
necessary, which can be carried out by recombinant methods known per
se and are described in particular in the International Patent Application
WO 92/22320.
The abovementioned non-human C1 esterase inhibitors and recombinantly
modified, human C1 esterase inhibitors are expediently administered
parenterally and in an amount sufficient for the therapeutic action. In
general, they are administered to the patient in a physiological saline
solution, a Ringer's solution or another excipient suitable for injection
purposes.
