BG66184B1 - Means for extracorporeal plasmasorbtion of carcinoembrional antigen /cea/ - Google Patents

Abstract

The means is used in oncology. It represents means for extracorporeal plasmasorbtion of carcinoembrional antigen /CEA/, adhesion molecule CEACAM6 and polyamines, which comprises fixing of gram-negative bacteria or other bacteria with similar properties, whole bacterial bodies of Escherichia coli or other bacteria with similar adhesive properties on a bearing filtration column with a different fixation base with deployed area large enough to perform extracorporeal plasmasorbtion, the column acquiring the property to clean the plasma from the CEA molecules, the adhesion molecule CEACAM6 and polyamines, and is characterized by that on the column with high-affinity polymethacrylate bound albumen there are first adhered liposaccharides of gram-negative bacteria obtained from cell lysate, consecutively is loaded with a suspension of live gram-negative in concentration 109 live bacterial suspension Escherichia coli or other bacteria with similar properties.

Description

The invention relates to an extracorporeal sorption agent for carcinoembryonic antigen (CEA).

Field of expertise: Oncology, biological therapy of cancers.

BACKGROUND OF THE INVENTION

The role of CEA in the development of the disease process by assisting metastases has been established (Thompson et al. 1991; Ferroni et al. 2007; Ordofiez et al. 2000; Lantzis et al. 2002).

The following extracorporeal adsorption agents for carcinoembryonic antigen (CEA) are known:

1. Extracorporeal Plasma Immunoassay in Patients with Metastatic Colon Cancer with Formalin-Stabilized Immunoabsorbent Protein A, Contained in the Staphylococcus aureus Bacteria. The agent reduces the concentration of carcinoembryonic antigen and IgG immunoglobulins (Ray et al. 1982; Korec and Smith 1984).

2. Extracorporeal adsorption of CEA utilizes an avidin-agarose column loaded with humanized monoclonal antibodies (MoAb) that bind CEA for 2.5 h, administered to experimental rats (Wang et al. 2003).

The disadvantages of existing methods are:

1. The disadvantages of extracorporeal immunoadsorption with a filter column containing formalin-stabilized and immobilized protein A of Staphylococcus aureus are:

1.1. CEA is adsorbed from plasma but not that associated with CEA adhesion molecules on blood-forming elements.

1.2. The used columns of protein A immobilized with formalin also nonspecifically bind the immunoglobulin G molecule, which increases the risk of diminishing anti-infective immunity in patients with immune deficiency as most patients who have had cytostatic, radiation and / or radiation.

2. Column selective extracorporeal adsorption of CEA with monoclonal antibodies (MoAb) has the following disadvantages:

2.1. It selectively adheres only the CEA molecule from the liquid phase of the blood, and does not adhere to the CEA associated with the CEA adhesion molecules on neurophilic leukocytes, platelets and other blood-forming elements.

2.2. Hybridoma techniques for the production of MoAb are very expensive and difficult to access in large quantities, which requires the loading of CEA selective plasma adsorption columns.

3. Existing methods do not take advantage of the ability to selectively bind some key molecules of the CEA family, which would allow the blocking and / or elimination of those blood-forming elements that adhere to CEA or malignant cells expressing CEA (Zimmermann et al. 1999; Staubach et al. 2003; Ullrich et al. 2001; Kellum 2007).

3.1. Existing CEA adhering columns do not use the cross-reacting property of bacteria. Thus, the possibility of increasing the efficiency of the CEA adhering method on the basis of additional inclusion and loading of the column with bacterial bodies, respectively, is not used. using the selective adherence property of CEA by some bacteria such as Esherichia coli and Neisseria Meningitidis (Leusch et al. 1990).

3.2. The CEA adhering columns do not include or exploit the scientific fact that the bacterial bodies of Escherichia coli selectively bind CEA and CEACAM6 by producing high and sufficiently strong adhesion that is resistant even to detergent extraction (Berge et al. 2004) that CEACAM6 is an adhesion molecule for CEA and is also a receptor for the selective adhesion of Escherichia coli (Bamich et al. 2007).

4. The general disadvantage of the described extracorporeal column adsorption agents for CEA is that the methods described target the adsorption of CEA only from the liquid phase of the blood. CEA has been found to bind to adherent molecules that are found on blood-forming elements (neutrophil leukocytes, platelets), endothelial cells, and other

66184 Bl cells. When the vacancies of CEA adhesion molecules are occupied and the amount of CEA released from the cancer cells exceeds the capacity of the CEA adhesion molecule receptor, then the CEA and plasma levels begin to increase. It has been found that it is the blood-shaped elements such as neutrophilic leukocytes and platelets that participate in and assist the processes of metastasis, with the blood-forming elements adhering and transporting both CEA and malignant cells.

For this reason, it is important to find an approach, a means and a method to allow the extracorporeal adsorption of CEA from whole blood. There are no known methods for extracorporeal adsorption of CEA from whole blood, and known methods described in the literature have the disadvantage that they do not have the property of adhering simultaneously the free (plasma) and the bound, adherent to blood-forming elements of CEA. It is known that some of the blood-forming elements adhere to and transport not only CEA but also malignant cells that express CEA, thereby promoting metastasis.

SUMMARY OF THE INVENTION:

The disadvantages described in the extracorporeal sorption columns of carcinoembryonic antigen are avoided by the extracorporeal sorption column of the CEA, characterized in that it consists of a column of activated carbon particles pre-loaded with heat-killed whole bacterial bodies in one bacterial body. 10 ⁹ ) from: Escherichia coli, Neisseriae meningititis, Staphylococcus aureus.

An exemplary embodiment of the invention

The columnar system for extracorporeal sorption of CEA composed of activated carbon particles is loaded with heat treated at 60 ° C for 25 min whole bacterial bodies in one billionth suspension (10 ⁹ ) from: Escherichia coli, Neisseriae meningititis, Staphylococcus aureus. After standing in a refrigerator at 8 ° C for 30 min, the bacterial bodies in the column were fixed with formaldehyde 3% in phosphate buffer (pH 7.4) for 10 min at room temperature. The column was dried under sterile air for 10 min. Wash twice with sterile saline (pH 7.4) and dry again with sterile air for 10 minutes. The column thus treated acquires a new property of adsorption of the free CEA molecules in the liquid phase of the blood and of the CEA adhered to some blood-shaped elements. To check the efficiency of the column, a certain amount (50 ml) of heparinized whole blood is passed through it, in which the level of plasma CEA was determined before filtration and after passage through the column by a standard immunoassay or radioimmunoassay method. Prior to filtration, the CEA value by the immunoenzyme method was 50 µg / Ι. After passing through the column, the value is reported to be 10 microg / Ι. By indirect immunofluorescence method / antibodies against CEA from Abbott, additionally loaded with total human FITC-labeled immunoglobulin /, whole blood cell conglomerates, mainly platelets and leukocytes adhering to carcinogenic embryos, are visible on the column.

Field of application

The field of application is determined by the role and importance of CEA in carcinogenesis, metastasis of malignant cells, namely: in oncology, as an integral part of conventional or complex intensive biological therapy of cancers in the preoperative and postoperative period, to reduce the risk of metastasis, by time or after radiotherapy and chemotherapy, as well as for the prevention of relapses to reduce conditions and the risk of metastasis, which creates and favors increased levels of CEA.

Advantages of the invention

The advantages of the tool are:

1. The extracorporeal sorption column loaded with a bacterial suspension of the three bacterial species listed above has a qualitatively new property, expressed by binding of plasma-free CEA, as well as CEA adherent from blood-forming elements, other cells that have bound CEA or express CEA.

2. The tool is technically simpler and easier to implement, more affordable than

66184 Bl loading of columns with monoclonal antibodies (MoAlb) whose biotechnologies are incomparably more complicated, time-consuming and expensive.

3. The agent allows for use in secondary and tertiary prophylaxis, in the complex therapy of cancer patients in combination with intensive conventional therapy (chemo- and radiation), in complex biological therapy in cancer to reduce conditions and risk of metastasis. ] θ

Claims (1)

1. Carcinoembryonic antigen (CEA) extracorporeal sorption column comprising activated carbon particles, characterized in that it is preloaded with heat treated whole bacterial bodies in a one billion (10 ⁹ ) concentration of Escherichia coli, Neisseriae meningocitis, Stiss aureus, which are fixed with formaldehyde 3% in phosphate-20 phat buffer (pH 7.4), washed with sterile saline (pH 7.4) and dried with sterile air.