CA2072626A1

CA2072626A1 - Drug containing cd14

Info

Publication number: CA2072626A1
Application number: CA002072626A
Authority: CA
Inventors: Christine Schuett; Rudolf Kunze
Original assignee: Individual
Current assignee: IMTOX PRIVATINSTITUT fur IMMUNBIOLOGISCHE FORSCHUNG GmbH
Priority date: 1990-09-14
Filing date: 1991-09-06
Publication date: 1992-03-15
Also published as: EP0500844A1; WO1992004908A1; JPH05502893A; DE59108104D1; EP0500844B1; ATE141510T1; DE4029227A1

Abstract

The new CD14-containing drug enables medical conditions to be treated in which the immune system plays a decisive role. The use of CD14 is proposed in the preparation of drugs for the therapy or prophylaxis of endotoxin-induced conditions (sepsis) or for the treatment of tumours or for the preservation of cell cultures or organ or tissue transplants.

Description

2072~

The present invention relates to a new medicamentcontaining CD14 which has been isolated from human or animal serum or other body liquids or from other bio-logical sources, and to the use of CD14 for preparing medicaments for the therapy or prophylaxis of diseases, and especially for the therapy of a sepsis. Moreover, the present invention relates to the use of CD14 as an endotoxin-neutralizing principle.

CD14 (Cluster of Differentiation) is a glycosylated protein occurring in the human organism. It character-izes the so-called monocytes which are present, inter alia, in the peripheral human blood. This molecule may be detected on the cell surface by means of modern immunodiagnostic methods and by the use of monoclonal antibodies. From the scientific literature it has further been known that CD14 is present not only on surfaces of monocytes and activated neutrophilic granulocytes, but that it can be determined in the same or in a slightly modified molecular form as soluble CD14 ~sCD14) in serum and in other body liquids. In addition 2072~2~

thereto, it has been known that in the case of certain diseases said molecule is present in the serum either at an elevated level or at a reduced level, which in all events are beyond the normal limits of tolerance.

The endotoxic shock constitutes a great clinical problem. The endotoxic shock may occur in the course of a sepsis and may be due to various causes. Monokines such as tumor necrosis factor (TNF-alpha), interleukin-1, interleukin-6 or PAF and free oxygen radicals play a key role in the pathophysiology of the traumata in the course of which sepsis and shock do occur.

Endotoxins are potent monocyte and endothelial cell activators. Due to subsequent further humoral and cellular activation processes, endothelial and tissue lesions will occur. After these excessive immuno-reactions, the local immune defense and also barrier are no longer in working order.

From A. Haziot et al., J. Immunol. 141 (lg88), pages 547 to 552, there has been known the CD14 sequence. In said publication it has further been stated that CD14 couples to the cell membrane through phosphatidyl inositol bonds. It has further been known from S. M. Goyert et al., Science, 239 (1988), pages 497 to 500, that CD14 comprises 5 sites of N-glycosylation.

Soluble CD14 (sCD14) has a molecular weight of about 55,000 Dalton. It may be recovered from human serum or urine (if a high CD1~ level is determined upon certain syndromes) using known methods. An appropriate preparation is given hereinbelow by way of an example.

2~7~62~

Material: Urin o~ a patient suffering from secondary amyloidosis 1. Ammonium sulfate precipitation (516 g/liter);
washing; lyophilization.

2. Affinity chromatography (using a monoclonal antibody against CD14, which in turn has bee~
coupled to CNBr-activated Sepharose 4B (5 mg of antibody/ml of gel);
Washing with PBS (Phosphate-Buffered Saline);
Elution of adsorbed proteins with 0.1 M
ethanolamine;
Take-up in 1 M phosphate buffer (pH 7.2), Titration with acetic acid to pH 7Ø

3. Repetition of the affinity chromatography (cf.
2).

4. Adsorption to porcine anti-human-Ig-antibody.

5. Dialysis against 10 mM ammonium hydrogen-carbonate solution.

6. Lyophilization.

There has further been described by Goyert et alO
(c~. above) the preparation of a c~NA which codes for the CD14. Thus, the preparation of CD14 and/or sCD14 is also feasible by genetic engineering.

EP-A-O 330 191 describes a quick immunoselection-cloning method. Said method has been realized in cloning cell surface antigens of human lymphocytesO

21~262~

EP-A-O 330 191 mentions the use of CD molecules produced by genetic engineering as therapeutic and diagnostic tools for various purposes. It is also described there-in that actlve fragments of the CD molecules produced by genetic engineering could be employed for these pur-poses. Expressly emphasized is the use of recombinant CD molecules, but just not CD molecules recovered from natural sources.

It is the object of the present invention to provide a medicament for the therapy or prophylaxis of viral or bacterial infections, and especially of those related to sepsis and to the endothelial shock. The active principle of this medicament should also have a high endotoxin-binding capacity and, when formulated in an appropriate form, should be suitable for use in pre-ventive medicine, for example for preventing iatrogenic actions in the form of importing endotoxins during surgery or organ transplantation.

This object, surprisingly, is attained by a new medicament containing CD14 which has been recovered from serum or other body liquids of human or animal origin or from other biological sources. These may be, for examp-le, human fibroblast cell cultures. The preparation of synthetic peptides comprising the CD14 amino acid sequence or functionally relevant parts thereof is another possibility of providing CDl4, and especially of soluble CD14.

In laboratory experiments it was found that sCD14 beneficially affects the function of monocytes and neutrophilic granulocytes (phagocytosis). It is very likely that sCD14 is involved in the clearance of microbial toxins from the organism or blood.

_ 5 _ 2~72~

This observation was surprising. In prior art altogether there i5 not found any reference to such action of CD14. Corresponding properties have hitherto not been determined for other soluble CD molecules of leukocytes, e.g. the soluble interleukin-2 receptor or the soluble CD8 receptor molecule, and they are not very likely either.

Also surprising was the high binding capacity of CD14, and especially of soluble CD14 (sCD14), for endotoxins.

A preferred embodiment of the medicament according to the invention contains soluble CD14 (sCD14). This agent may also contain pharmacologically active partial sequences of CD14 or CD14 derivatives, optionally in addition to CD14. The term "pharmacologically active partial sequences of CD14 or CD14 derivatives" as used herein is understood to denote that structural constitu-ents are still present which are capable of reacting with a specific CD14-antibody. Thus, the CD14 pre-ferably has a molecular weight of from 45,000 to 55,000 Dalton. It is preferred to employ the glycosyl-ated CD14 derivatives among the variety of CD14 known or conceivable by the artisan.

~ n a further embodiment, the medicament according to the invention contains carrier-bonded CD14 and/or C14 derivatives. The medicament according to the invention is preferred to also contain CD~4 and/or CD14 derivati-ves coupled to immunoglobulins or immunoglobulin fractions. These contain, dependently on the method of preparation, no sugar moiety or a modified sugar moiety.

2~72~2~

The present invention further relates to th~ use of CD14 for the preparation of a medicament for the therapy or prophylaxis of diseases involving the cellular immune system.

According to the invention, CD14 may be used for the preparation of a medicament which neutralizes either endotoxins or the changes caused by endotoxins.

The present invention further relates to the use of CDl~ for the therapy or prophylaxis of viral or bacteri-al infections. The use according to the invention also includes medicaments for the therapy of multiple sclerosis and of other autoimmune and also infectious diseases, and especially those wherein a secondary immmune deficiency may occur. A secondary immune deficiency occurs, for example, as a result of radio- or chemotherapy. It may also be due to old age.

The present invention further relates to the use of CD14 for the preparation of a medicament for the therapy of burns.

The present invention further relates to the use of CD14 for the preparation of a medicament for the therapy of a sepsis and of an endotoxic shock.

The present invention further relates to the use of CD14 for the preparation of a medicament for the therapy or prophylaxis of diseases wherein a phagocytosis caused by monocytes and/or granulocytes is of crucial importan-ce for healing. The use according to the present invention preferably relates to diseases where micro-organisms and/or bacterial endotoxins are eliminated by the monocytes and/or granulocytes.

2~7~

The medicament according to the invention may be used for inhibiting the release of mediators (e.g. o~
tumor necrosis factor, interleukin-l, complement) induced by endotoxins and of the increased production of oxygen radicals as observed, for example, in ischemic cardiac and organ failure. CD14, and more particularly soluble CD14, can be used for preparing a ~edicament for the treatment of tumors by means of lymphokin-activated killer cells produced ln vitro (LAK therapy). In this therapy, lymphocytes and monocytes are isolated ~rom peripheral blood of cancer patients and are cultured in cell cultures over an extended period of time. These cells, after the augmentation and activation thereof, are returned into the blood circulation of the donor where they now will attack cancer cells. This operation involves the problem o~ that the cells produced outside the human body are to be obtained free from endotoxin.
Here, CD14 acts as a preventive against endotoxin.

In a similar manner, CD14, and more particularly soluble CD14, is employed as an additive to materials such as culture media, sera, plastics materials as used in clinical and biological laboratories for initial cultivation and maintenance of cell cultures.

By using CD14, the endotoxin is kind of neutral-izedj so that also cells can be grown which are only cultivatable in an endotoxin-free environment.

Moreover, CD14, and more particularly soluble CD14, is suitable for a use as additive in organ-flushing solutions (e.g. for dialysis), perfusion solutions or other solutions for storing organ or tissue transplants.

2~72~26 The application of medicaments produc~d via genetic engineering gives rise to problems, since it must be warranted that the medicaments thus produced are endotoxin-free. The use of CD14, and especially of soluble CD14, as an additive to solutions or application forms of prepared recombinant medicaments eliminates the need of removing the endotoxins in a troublesome and expensive procedure from medicaments produced via genetic engineering.

Claims

C L A I M S :

1. A medicament containing CD14 which has been recovered from serum or other body liquids of human or animal origin or from other biological sources.

2. The medicament according to claim 1, characterized in that it contains soluble CD14 (sCD14) and/or pharma-cologically active fragments of CD14.

3. The medicament according to any of claims 1 and/or 2, characterized in that it contains glycosylated CD14.

4. The medicament according to at least one of claims 1 to 3, characterized in that it contains carrier-bonded CD14 and/or CD14 derivatives.

5. The medicament according to at least one of claims 1 to 4, characterized in that it contains CD14 and/or CD14 derivatives coupled to immunoglobulins or immuno-globulin fractions.

6. The use of CD14 as neutralizing agent for endo-toxins.

7. The use of CD14 for the preparation of a medicament for the therapy or prophylaxis of viral or bacterial infections, for inhibiting the release of mediators such as tumor necrosis factor (TNF), inter-leukins, complement, induced by endotoxins, for the therapy of burns and for the neutralization of endo-toxins and/or for the therapy of diseases caused by endotoxins.

8. The use of CD14 for the preparation of a medicament for the therapy or prophylaxis of the increased production of oxygen radicals as observed, for example, in ischemic cardiac and organ failure.

9. The use of CD14 for the preparation of a medicament for the therapy of a sepsis and of an endo-toxic shock.

10. The use of CD14 for the preparation of a medicament for the therapy or prophylaxis of diseases wherein a phagocytosis caused by monocytes and/or granulocytes is of crucial importance for healing.

11. The use according to claim 10, wherein micro-organisms and/or bacterial endotoxins are eliminated by the monocytes and/or granulocytes.

12. The use of CD14 for the preparation of a medicament for the therapy of tumors by means of lymphokin-activated killer cells produced in vitro (LAK
therapy).

13. The use of CD14 as an additive to materials such as culture media, additives, sera, plastics materials as used in clinical and biological laboratories for initial cultivation and maintenance of cell cultures.

14. The use of CD14 as an additive to materials such as additive in organ-flushing solutions, perfusion solutions or other solutions for storing organ or tissue transplants.

15. The use of CD14 as an additive to materials such as solutions or other application forms of medicaments prepared by recombinant techniques.

16. The use according to at least one of claims 6 to 15, characterized in that pharmacologically active fragments of CD14 are used as the CD14.