Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of co-culturing cells
Reexamination Certificate
1999-03-22
2001-07-17
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of co-culturing cells
C435S325000, C435S366000, C435S372000, C424S093700, C424S093710
Reexamination Certificate
active
06261839
ABSTRACT:
The present invention relates to an ex vivo method for the induction of a NK cell-mediated immune response and a method of treating diseases caused by viruses, bacteria and fungi and tumor diseases.
Etherlipids such as 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, edelfosine) belong to the group of alkyl-lysophospholipids. Alkyl-lysophospholipids are synthetic analogs of the naturally occuring 2-lyso phosphatidylcholine. It has been known that alkyl-lysophospholipids are cytotoxic and selectively kill neoplastic cells. It has been known that at cytotoxic concentrations of alkyl-lysophospholipids tumor cell growth is inhibited in vitro and in vivo by direct destruction of tumor cells.
Although the mechanism of cytotoxicity induced by etherlipids is unknown it is believed that the plasma membrane is a main target of the cytotoxic activity of alkyl-lysophospholipids, e.g. of ET-18-OCH3. Thus, for example, it has been shown that by alkyl-lysophospholipid derivatives the membrane permeability, membrane fluidity, lipid composition of the membrane and its cholesterol content are affected. Furthermore, ET-18-OCH3 has been employed as a purging agent in preclinical models of autologous hematopoietic stem cell transplantation and in clinical phase I/II studies in the ex vivo purging.
However, it has been observed that e.g. some leukemic cell lines, for example K562, are relatively resistant against ET-18-OCH3-mediated cytotoxic effects (1). Therefore, a purging treatment could be associated with a higher risk for a new outbreak of the leukemic disease due to the reinfusion of highly resistant leukemic cells into the patient. To avoid this drawback and to increase the cytotoxic effects of ET-18-OCH3 other methods of treatment have been suggested in addition, for example a hyperthermal treatment to be conducted in vitro ((2)-(4)). Thus, the publication (2) discloses the treatment of HL-60, K562, and KG-1 leukemic cells from the bone marrow of leukemic patients with 50 &mgr;g/ml ET-18-OCH3 and a subsequent heat shock treatment at 42° C. for one hour. This achieves a direct cytotoxic effect on the tumor cells.
From (3) there has been known the treatment of BG-1 ovarian carcinoma cells with 1, 2, and 4 &mgr;M of ET-18-OCH3, respectively, (1 &mgr;m is equivalent to about 0.5 &mgr;g/ml) for a period of 1-12 days and a subsequent heat shock treatment at 42° C. or 44° C., respectively. This method kills the tumor cells by direct cytotoxic effects.
From (4) there has been known the treatment of BG1 cells with ET-18-OCH3 at a concentration of 2 and 8 &mgr;M, respectively, followed by a heat shock treatment at 42° C. for a period of 0-16 hours and at 44° C. for a period of 0-33 hours. This is said to be effective in achieving a direct enhanced cytotoxic effect on the tumor cells.
From (5) it has been known that upon non-toxic treatment of K562 cells with ET-18-OCH3 a significant increase in sensitivity of K562 cells for the lysis by interleukin-2-stimulated NK cells occurs. Moreover, this reference reports that similar effects may be achieved by a non-lethal heat shock treatment. However, the combination of the two methods of treatment and the imperative observance of a certain order in the treatment to achieve a stimulation of the cytolytic activity of NK cells for tumor cells or a synergistic effect is not suggested therein.
It is an object of the present invention to provide a method which is useful ex vivo for the induction of a NK cell-mediated immune response. Said method will be furthermore designed to render the cell suspension thus treated reinfusable into the patient for treatment of the tumor disease.
According to the invention this object has been solved by the method according to claim
1
of the appended claims. Preferred embodiments of the method arise from the dependent claims as well as the following specification and the examples.
The method according to the invention is effective in inducing an immune response mediated by natural killer cells. For this purpose a physiological cell suspension containing at least tumor cells or animal or human cells infected by viruses, bacteria and/or fungi, shortly referred to as target cells, and natural killer cells is treated by the following process steps in the order mentioned to increase the sensitivity of the target cells for lysis by NK cells:
a) Heat treating the cells contained in the suspension at a temperature of 38° C. to 43° C. for a period of at least one hour;
b) lowering the temperature to physiological cell temperature (about 37° C.) to give the cells contained in the suspension a recovery period of at least one hour;
c) addition of a compound increasing the portion of membrane-bound Hsp70 of the target cells in a concentration which is sublethal for the cells and allowing the compound to be effective for at least 30 minutes;
d) recovery period of at least 1 hour at 37° C.
Thus, according to the invention a method is provided for increasing the lytic activity of NK cells for tumor cells, in particular for leukemic cells, lymphoma cells, and cells derived from metastases of solid tumors. Futhermore, by the method according to the invention also cells may be effectively lysed which are infected by viruses, bacteria, and/or fungi. Also cells containing antigenic portions of these foreign organisms or tumor cells may be lysed by the method of the invention while the lysis is mediated by an increase in sensitivity of the target cells to an attack by NK cells.
The method of the invention achieving an increase in sensitivity of the target cells for lysis by natural killer cells is characterized in that a physiological cell suspension containing at least the target cells and natural killer cells under strict observance of a specific order is subjected to a combination of first a heat treatment, followed by a recovery phase after which a treatment with a sublethal concentration of a compound is performed which increases the portion of membrane-bound Hsp70 of the target cells. According to the invention, an alkyl-lysophospholipid is used as the compound increasing the portion of membrane-bound Hsp70 of the target cells. This is followed by another recovery phase. Using the method of the invention, a marked increase in sensitivity of the target cells for lysis by NK cells is achieved.
The NK cells may be isolated from the patient to be treated or a healthy donor by suitable methods. Preferably, however, the NK cells are present together with other peripheral mononucleated blood cells, for example in the form of buffy coat cells.
Buffy coat cells are obtained from the patient via the vein and are for example added with heparin to avoid aggregation of the cells. The whole blood added with heparin is collected in a sterile container (mostly a plastic bag) and is then centrifuged to achieve an enrichment of blood cells (=PBMC, peripheral mononucleated cells, e.g. lymphocytes, erythrocytes, granulocytes etc.). A major portion of the serum (⅔) is removed in sterile manner and stored for the patient who for example has to undergo surgery and may need his own serum. The lymphocyte concentrate remains in the container (plastic bag). In the case of healthy subjects a buffy coat consists of white and red blood cells (lymphocytes, erythrocytes etc.). In the case of a tumor patient the buffy coat consists not only of blood cells but also contains tumor cells (in the case of leukemia: leukemic cells=blasts; in the case of solid tumors e.g. metastases in the form of single cells).
The whole blood or the buffy coat cells containing peripheral mononucleated blood cells are employed in the form of a physiological cell suspension, preferably added with heparin. Heparin functions to inhibit the aggregation of cells.
There has been provided completely unexpected evidence as demonstrated herein using K562 leukemia cells as an example which exhibit a strong resistance against treatment with a compound increasing the portion of membrane-bound Hsp70 in the target cell, such as an alkyl-lysophospholipid, that the target cells
Botzler Claus
Multhoff Gabriele
Fish & Richardson P.C.
GSF-Forschungszentrum fur Umwelt und Gesundheit GmbH
Lankford , Jr. Leon B.
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