CA2236888C

CA2236888C - Medicament, in particular for modulation of the immune response in the control of viruses, tumors, bacteria and parasites

Info

Publication number: CA2236888C
Application number: CA002236888A
Authority: CA
Inventors: Joachim Robert Kalden; Martin Herrmann; Reinhard Voll; Wolf Maximilian Bertling; Klaus Von Der Mark; Otmar Zoller
Original assignee: Individual
Current assignee: Individual
Priority date: 1995-11-06
Filing date: 1996-11-04
Publication date: 2009-10-13
Anticipated expiration: 2016-11-04
Also published as: DE19541284C2; EP0859628B1; WO1997017084A1; ES2205065T3; EP0859628A1; EP1356818A2; EP0859628B9; DE19541284A1; JP2000500124A; CA2236888A1; EP1356818A3; ATE245995T1; DE59610633D1

Abstract

The invention relates to a medicament, in particular for modulation of the immune response in the control of viruses, tumors, bacteria and parasites, where an active compound selected from the following group is contained for influencing the phosphatidlyserine-dependent [sic] phagocytosis: annexin, annexin antibodies, annexin ligands or phosphatidlyserine [sic].

Description

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Kalden 1 - 1 - 11.04.96 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 Kalden 1 - 2 - 11_04.96 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 Kalden 1 - 3 - 11.04.96 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 Kalden 1 - 4 - 11.04.96 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 Kalden 1 - 5 - 11.04.96 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.

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 - 6a -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]

Kalden 1 - 7 - 11.04.96 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 Kalden 1 - 8 - 11.04.96 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.

Kalden 1 - 9 - 11.04.96 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.

Kalden 1 - 10 - 11.04.96 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 Kalden 1 - 11 - 11.04.96 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.

Claims

CLAIMS:

1. 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.

2. A pharmaceutical composition according to claim 1, wherein the annexin of type V is a constituent of a fusion protein.

3. A pharmaceutical composition according to claim 2, wherein the fusion protein is coupled to an RNA vector.

4. A pharmaceutical composition according to claim 3, wherein the RNA vector is derived from poliovirus.

5. 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 anti-annexin V antibody.

6. A use according to claim 5, where the annexin of type V is a constituent of a fusion protein.

7. A use according to claim 6, where the fusion protein is coupled to an RNA vector.

8. A use according to claim 7, wherein the RNA vector is derived from poliovirus.

9. A use according to any one of claims 5 to 8, wherein the active compound produces a blockade of phosphotidlyserine-dependent phagocytosis.

10. A use according to any one of claims 5 to 8, wherein the active compound produces a stimulation of phosphotidlyserine-dependent phagocytosis.

11. A use according to claim 10, wherein an immunostimulation is brought about by one or more of a blockade, a disguising, a masking and a removal of phosphotidlyserine localized on an extracellular membrane.

12. A use according to any one of claims 5 to 11, wherein the active compound is the modulator of the immune response.

13. A use according to any one of claims 5 to 11, wherein the active compound is an adjuvant in one or more of tumor therapy, tumor vaccines, virus therapy and treatment of malaria.

14. A use according to claim 13, wherein the virus therapy is for treatment of a retrovirus infection, a lentivirus infection or an HIV infection.

15. A use according to any one of claims 5 to 11, wherein the active compound is an adjuvant in malaria immunization.

16. A use according to any one of claims 5 to 11, for therapy of sickle cell anemia.