Note: Descriptions are shown in the official language in which they were submitted.
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Medicament, in particular for modulation of the immune
response in the control of viruses, tumors, bacteria
and parasites
The invention relates to a medicament, in particular
for modulation of the immune response in the control of
viruses, tumors, bacteria and parasites. The invention
further relates to the use of an active compound for
the production of a medicament.
Phosphatidylserine is a negatively charged phospholipid
which is found on the inside of the cytoplasmic
membrane in all cells. Occasionally, however, a
phosphatidylserine molecule can jump through the
membrane and thus reach the outside of the cytoplasmic
membrane. In living healthy cells, phosphatidylserine
which has reached the outside is immediately
transported back to the inside of the cytoplasmic
membrane enzymatically. In old and in Plasmodium
falciparum-infected erythrocytes, in sickle cells,
post-inflammatory granulocytes and in apoptotic cells,
the phosphatidylserine, however, remains on the
outside. From a certain phosphatidylserine density, the
cells bind through the "phosphatidylserine receptor" to
phagocytes. If the phosphatidylserine density increases
further and in the course of this reaches a certain
threshold value, the cells are extremely rapidly
phagocytozed (engulfment phagocytosis). In this
process, neither release of the cell contents nor
activation of the immune system occurs. For this
reason, this phagocytosis pathway is called
"noninflammatory".
In the elimination of cells which have grown old, e.g.
old erythrocytes and apoptotic cells, such as post-
inflammatory granulocytes, a specific immunosuppression
is quite useful and desirable, since in these cases an
inflammatory phagocytosis could even lead to autoimmune
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phenomena. The noninflammatory phagocytosis of
Plasmodium falciparum-infected erythrocytes, however,
is responsible, inter alia, for the extremely poor
immune response and difficult immunization against
malaria. No measure described to date or prophylaxis
against malaria considers the circumstance in which
Plasmodium falciparum-infected erythrocytes are
phagocytozed by means of the phosphatidylserine-
dependent engulfment phagocytosis pathway. -
Medicaments which affect this phagocytosis pathway are
presently unknown.
The behavior is similar in virus infections. Viruses
which are taken up in phagocytes by means of
phosphatidylserine-dependent phagocytosis of apoptotic
cells can thus escape immunosurveillance. Thus, for
example, the uptake of HIV in monocytes, which takes
place without triggering of the "respiratory burst", is
responsible for the penetration of the HIV, which is
early and unnoticed by the immune system, into the
long-lived monocyte pool. This infection of the
monocytes/macrophages, which is presently not
understood, is held causally responsible for the
persistence of HIV and thus for the formation of the
AIDS syndrome. Although the route of infection of
monocytes/macrophages with HIV is presently still not
clearly identified in molecular terms, an involvement
of phosphatidylserine and phosphatidylserine receptor
is probable because of the noninflammatory
phagocytosis. It was thus possible to show, for
example, that retrovirus genomes from apoptotic debris
in cells can be taken up and these cells thus infected.
Since HIV can survive for a very long time in the
monocytes and, possibly even years after infection, is
spontaneously released, the human immune system cannot
completely eliminate the HIV from the body. Since the
HIV damages the immune system somewhat on each release
by destroying the CD 4-positive T cells, full-blown
AIDS can thus be formed in the course of usually
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several years. Similar problems also exist in the
elimination of other viruses persisting or replicating
in phagocytes. Other retroviruses and particularly the
subgroup of the lentiviruses can especially be
mentioned here. Some of these viruses- (EIAV, meadi
[sic] visna virus, CAEV) persist in the phagocytes of
hoofed animals and lead to autoimmune diseases there.
No -oreviouslv described measure or prophvlaxis aaainst
HIV infection or infection with other viruses surviving
in phagocytes considers the circumstance in which
apoptotic cells can be phagocytozed via the
phosphatidylserine-dependent pathway. - Medicaments
which block this phagocytosis pathway are presently
unknown.
The situation is different in patients with sickle cell
anemia. Owing to the continuous and extremely rapid
phagocytosis of autologous, genetically modified
erythrocytes, anemia occurs in the patients which,
untreated, can lead to death in severe cases. Here, the
fact is less prominent that the phosphatidylserine-
mediated phagocytosis proceeds in a noninflammatory
manner, rather than the fact that it extremely rapidly
and efficiently- eliminates phosphatidylserine-bearing
cells. - Since at present there are no medicaments
which block this phagocytosis pathway, sickle cell
anemia is presently treated with repeated blood
transfusions.
A problem similar to that in sickle cell anemia also
occurs in the storage of erythrocytes for transfusion.
Even during storage under blood bank conditions, an
increasing number of erythrocytes express
phosphatidylserine on their surface. After the
transfusion, this leads to these erythrocytes very
rapidly being cleared by phagocytes and thus becoming
ineffective. - Since there are presently no medicaments
or additives to conserved blood which prevent this
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phagocytosis, the storage of erythrocytes is strictly
limited in terms of time.
In the preparation of tumor vaccines, the tumor cells
returned to the bodies of the patients or experimental
animals are irradiated in order to prevent a further
spread of the tumor. Since under these circumstances
apoptosis is induced in the tumor cells and these are
then eliminated in a noninflammatory manner via the
phosphatidylserine-dependent engulfment phagocytosis
pathway, a relatively weak immune response usually
occurs to the respective tumor vaccines. - Since at
present no substances are known which block the
phosphatidylserine-dependent phagocytosis pathway, at
present classical immunization routes and adjuvants are
used in order to increase the immune response to tumor
cells.
According to the prior art, annexins are additionally
known. These form a highly conserved family of cellular
proteins which are found in higher plants, through
invertebrates, fish and birds up to the mammals
(nomenclature: Crumpton MJ and Dedman JR, 1990:
"Protein terminology tangle"; Nature 345: 212) . These
cytoplasmic membrane-associated proteins either have a
relatively low (32 kD to 38 kD) or else a relatively
high (about 67 kD) molecular weight. They are
additionally distinguished by an affinity to Ca++ and
phospholipids. A highly conserved core structure
consists of four or eight repetitions of 70 amino acids
each, which in each case contain an endonexin region
having 17 amino acids which are involved in the Ca++
binding. No significant similarities are found in the
amino-terminal regions of the different annexins, which
has led to the speculation that this domain is
responsible for the different functions of the
annexins. Although a multiplicity of functions have
been postulated for annexins, such as, for example, the
inhibition of phospholipase A2 and blood clotting, as
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well as a possible involvement in signal transduction,
cell growth and differentiation, it was previously not
possible to establish any clear biological role for the
annexins (Morgan MO and Femandez MP, 1991: -Annexins
and signal transduction" in Bailey JM (ed): "Advances
in Prostaglandine [sic], leukotrien [sic], Lipoxin and
PAF research"; New York: Plenum Press: 107-122).
The X-ray structural analysis of the annexins of type V
from humans, chickens and rats showed that annexin V is
a channel-forming integral membrane protein (Huber R,
Romisch J, Paques E, 1990: "The crystal and molecular
structure of human Annexin V, an anticoagulant protein
that binds to calcium and membranes"; The EMBO Journal
9: 3867-3874). The protein is found in the cytoplasm
and nucleus. Although it is only partially integrated
into the cytoplasmic membrane in the inside, it was
possible to detect a voltage-dependent calcium channel
activity. Additionally, annexin V is also found on the
surface of chondrocytes, where it is involved in
cartilage calcification (Pfaffle M, Ruggiero F, Hofmann
H, Fernadez MP, Selmin J, Yamada Y, Garrone R and von
der Mark K, 1988: "Biosynthesis, secretion and
extracellular locafization [sic] of anchorin CII, a
collagen-binding protein of the calpactin family"; The
EMBO Journal 7/8, 2335-2342).
The medicaments and procedures known according to the
prior art, in particular the adjuvants employed today,
stimulate the i.mmune system nonspecifically. To date,
no agent is yet described which prevents the engulfment
phagocytosis of phosphatidylserine-bearing cells and
thus leads to a specific immunostimulation. The
disadvantages which occur in noninflammatory clearing
of vaccines and viruses are particularly to be
emphasized. On the one hand, this leads to the
ineffective breakdown of vaccines, on the other hand to
virus persistence.
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Since, in sickle cell anemia, even young
erythrocytes bear phosphatidylserine on their surface, they
are removed by the endogenous phagocytes. This contributes
disadvantageously to the anemia of the patients.
Even for the storage of blood and erythrocytes, no
medicaments or additives are known which prevent the
breakdown of the donor erythrocytes by the recipient
phagocytes after transfusion. This presently leads to a
relatively short shelf life of conserved blood and
erythrocyte concentrates and to a marked loss of activity in
preserves stored for a longer time.
The object of the present invention is to
eliminate the disadvantages according to the prior art. In
particular, a medicament or the use of an active compound
which brings about an increase in the immunity to viruses,
tumors, bacteria and parasites will be specified.
According to one aspect of the present invention,
there is provided a pharmaceutical composition for
influencing phosphotidylserine-dependent phagocytosis in
modulation of an immune response in control of a virus, a
tumor, a bacteria, a genetic disorder or a parasite,
comprising an active compound selected from: annexin of type
V and anti-annexin V antibody, and a pharmaceutically
acceptable carrier therefor.
According to another aspect of the present
invention, there is provided a use of an active compound in
production of a medicament for influencing
phosphotidylserine-dependent phagocytosis in modulation of
an immune response in control of a virus, a tumor, a
bacteria, a genetic disorder or a parasite, wherein the
active compound is selected from: annexin of type V and
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anti-annexin V antibody. _
According to the invention, an active compound
selected from the following group is contained in a
medicament for influencing the phosphatidylserine-dependent
phagocytosis, in particular for modulation of the immune
response in the control of viruses, tumors, bacteria and
parasites: annexin, annexin antibodies, annexin ligands,
phosatidylserine [sic] or phophodiesterase [sic].
According to the invention, the use of an active
compound for the production of a medicament, in particular
for modulation of the immune response in the control of
viruses, tumors, bacteria and parasites, is additionally
proposed, the active compound for influencing the
phosphatidylserine-dependent [sic]
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phagocytosis being selected from the following group:
annexin, annexin antibodies, annexin ligands,
phosatidylserine [sic] or phophodiesderase [sic].
By means of the addition of one of the active compounds
according to the invention, the annexin level is
modified and/or the distribution of annexins is
modified. In addition, the sites of action or active
components of the phospatidylserine [sic] can thus be
modified.
By means of annexin, preferably by means of annexin V,
the phopstidlyserine-dependent [sic] phagocytosis, in
particular the engulfment phagocytosis, can be
modulated or inhibited. Additionally, for example, by
removal or a blockade of annexins, in particular of
annexin V, the engulfment phagocytosis can be modulated
or stimulated.
Particular importance is to be ascribed to use in the
human or veterinary medicine field, where, in many
established forms of therapy, but also in experimental
forms of therapy, immunomodulation is desirable. Thus
in the treatment of oncoses and of virus infections,
immunostimulation is often desirable, whereas in
disorders of the rheumatic type and in autoimmune
disorders immunosuppression is more desirable.
An important field of use for the blockade of
noninflammatory phosphatidylserine-dependent engulfment
phagocytosis follows from the specific `N adjuvant
action" resulting therefrom. Phosphatidylserine-bearing
cells are phagocytozed by means of an inflammatory
immunostimulatory alternative pathway after the
blockade of the noninflammatory phosphatidylserine-
dependent engulfment phagocytosis pathway, which is
accompanied by a massively increased immune response.
For this "adjuvant action", all kinds of areas of use
result, e.g. in human medicine. On the one hand, the
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immunogenicity of tumor vaccines can be increased
thereby, if these consist of irradiated and thus for
the most part apoptotic tumor cells. It is furthermore
possible to achieve an immune response to those tumor
cells which are radioactively irradiated in situ for
therapeutic reasons. In this case, a tumor-specific
immune response would increase the therapeutic success
in the elimination of the residual tumor mass. In
parallel with a cytostatic therapy with apoptosis-
inducing agents, such as, for example, cisplatin and
hydroxyurea, a similar effect can also lead to a
massive tumor-specific immunostimulation. In the
treatment of virus infections as well, e.g. of those
viruses which persist in phagocytes, the blocking of
the phosphatidylserine-dependent engulfment
phagocytosis pathway leads to a specific
immunostimulation. The treatment of infections with
lentiviruses and HIV must be regarded as a particularly
important example in this connection. A
phosphatidylserine-dependent penetration of the viruses
"unnoticed" by the cell leads to virus persistence in
the long-lived monocyte/macrophage pool. Virus
persistence leads to death in by far the most infected
people after a longer or shorter latency period.
Annexins, preferably annexin V, are suitable for the
treatment of HIV-infected people, since inflammatory
phagocytozed apoptotic material in the phagocytes
triggers a"respiratory burst" and thus would lead to
the destruction of the virus genomes.
Furthermore, owing to the blocking of the
phosphatidylserine-dependent engulfment phagocytosis,
undesirable cell losses can be avoided in vivo and in
vitro. This is of great importance both in the storage
of erythrocyte-containing conserved blood and as a
medicament for patients with sickle cell anemia.
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Examples:
1. Use of annexins, preferably annexin V, as
adjuvants for tumor vaccines
For the production of tumor vaccines from tumor
cells isolated from patients, these are
radioactively irradiated before reinjection into
the patient in order to prevent growth. During the
apoptosis induced thereby, phosphatidylserine is
exposed on the surface of the tumor cells, which
leads to a weak immunogenicity of the tumor
vaccine. Directly before the injection, the
irradiated tumor cells are incubated ex corpore
with annexins, preferably annexin V, in order to
block the phosphatidylserine-dependent engulfment
phagocytosis in the patient. Additionally, an
annexin, preferably annexin V, bolus is placed in
the injection site in order further to increase
the action locally.
2. Use of annexins, preferably annexin V, as an
immunostimulant in chemo- and radiation therapy
On therapeutic radioactive tumor irradiation and
also on treatment with cytostatics, apoptosis is
induced in corpore in a multiplicity of tumor
cells. In order to prevent a noninflammatory
clearance of the dead cells and to increase the
weak immune response associated therewith,
annexins, preferably annexin V, are injected into
the tumor before or immediately after radiation
therapy or chemotherapy. The clearance of the dead
tumor cells thereby takes place via an
inflammatory phagocytosis pathway and thus leads
to an increased immune response to the residual
tumor.
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3. Storage of whole blood and erythrocyte
preparations
Annexins, preferably annexin V, are added to whole
blood or erythrocyte concentrates in'order to slow
the breakdown of the phosphatidylserine-bearing
erythrocytes after the transfusion and thus to
increase the efficiency of the transfusion. The
annexins, preferably annexin V, can in this case
be added either directly after taking blood or
else alternatively even before the transfusion.
4. Use of annexins, preferably annexin V, in patients
with sickle cell anemia
4a. Classical solution with annexin, preferably
annexin V, infusions
In order to prevent the phagocytosis of the sickle
cells, which in this syndrome contributes
decisively to the anemia, annexins, preferably an
annexin V solution, are administered
intraveneously to very severely anemic patients.
4.b Use of annexins, preferably annexin V, in the
transient gene therapy approach with vectors
derived from RNA virus
In this approach, a fusion protein from annexins,
preferably annexin V, with a leader peptide is
expressed in blood cells, e.g. monocytes, with the
aid of a transient RNA vector system (e.g. a
system derived from the poliovirus). In this
manner, this transient in situ production as an
infusion blocks the phagocytosis of the sickle
cells over a relatively long period of time. Since
RNA-dependent expression systems neither integrate
into the genomic DNA of the host cells nor spread
vertically, the expression of the annexins is only
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transient, which minimizes the risks of the
triggering of an autoimmune pathology.
5. In the treatment of infections with viruses as
well, e.g. of those which persist in phagocytes,
the blocking of the phosphatidylserine-dependent
engulfment phagocytosis pathway leads to a
specific immunostimulation. The treatment of
infection with lentiviruses and HIV must be
regarded as a particularly important example in
this connection. A penetration of the viruses
"unnoticed" by the cell leads to the virus
persistence in the long-lived monocyte/macrophage
pool and to death in by far the most infected
people after a longer or shorter latency period.
Annexins, preferably annexin V, are suitable for
the treatment of HIV, since inflammatory
phagocytozed apoptotic material triggers a
"respiratory burst" in the phagocytes and thus
leads to the destruction of the virus genomes.
