Monoclonal antibody ico specific to the antigen mis1, antineoplastic

 

(57) Abstract:

The invention relates to immunology and medicine and can be used for immunotherapy of malignant tumors. Monoclonal antibody ICO specific to the antigen MIS1, belongs to IgG1 and has the constant Association 310-5M defined on the model cell culture human lung cancer OUT. On the basis of monoclonal antibodies ICO create an antitumor agent, which contains a monoclonal antibody ICO of 0.5% Polysorbate-80 2.0% and a phosphate-saline buffer else. The invention allows to selectively affect tumor cells by intravenous drug. 2 S. and 1 C.p. f-crystals.

The group of inventions relates to medicine and is a drug intended for immunotherapy of malignant tumors, the main active ingredient which is a monoclonal antibody (hereinafter MCAT), and the method of use of the medicinal product.

Known attempts to use antibodies directed to tumor antigens, such as the antitumor drug. This approach is based on the fact that when interacting with cancer cells, antibodies can OK is dependent on the specificity and the type of target cells. In the latter case, the development of mediated humoral and cellular cytotoxic reactions can lead to the death of tumor cells.

It is now known, MCAT BW835 interacting with polymorphic epithelial mucin - antigen associated with tumors of the breast, prostate, ovarian and lung (U.S. patent N 5695758). The claimed medicinal remedy known MCAT distinguishes the different specificity of the antigenic determinants.

Known monoclonal antiidiotypic antibody and a pharmaceutical composition based on it for therapeutic immunization against cancer of epithelial origin (application, publ. 21.01.97). Described below drug from known drug distinguish different specificity and the nature of antibodies and other therapeutic orientation effects.

A known method of destruction of tumor target cells using immunotoxin synthesized on the basis of MCAT directed to the antigen encoded by the gene MUC1 (application 98118574, publ. 20.07.2000). Claimed the drug differs from known immunotoxin that its active principle is the unconjugated ant is P> In Oncology, there are two potential medicinal product on the basis of MCAT. This is MabThera production company Genentech (USA), intended for the treatment of relapsed b-cell non-Hodgkin's lymphoma of low-grade and follicular b-cell non-Hodgkin lymphoma (Maloney et al. That "Blood" 1997a; 90(6): 2188-95) and Herceptin (produced by the same company) for the treatment of breast cancer in cases of overexpression of this gene antigen Her2/neu (Slamon et al., "In Proc. Am. Soc. Clin. Oncol.", 1998; 17: 98A). Drug from MabThera in contrast to the claimed medicinal product is not intended for the treatment of tumors of epithelial origin. From Herceptin claimed medicinal product features a different specific focus antibodies and other (presumably mediated) mechanism of therapeutic action.

Also known pharmaceutical composition for treating cancer containing the conjugate of a monoclonal antibody and is designed as a solution for injection or infusion (RF patent 2125889, publ. 10.02.99. Bull. N 4). The claimed medicinal product from known compositions are distinguished using unconjugated antibodies and other specific ispolzuemaya by introducing the patient an effective amount of antiidiotypic monoclonal antibodies (application 94046321, publ. 21.01.97). The inventive method of treatment known distinguishes different nature and specificity to be used as the beginning of the current antibodies.

Also known is a method of treatment of cancer by administration in the form of injection or infusion solution of the conjugate antibodies, acting on target cells (RF patent 2125889, publ. 10.02.99. Bull. N 4). The inventive method of treatment known features using unconjugated antibodies with different specificity of the used antibodies.

The claimed group of inventions aimed at solving common tasks - create a highly effective, safe biological product for immunotherapy of malignant tumors of epithelial origin and development of methods of treatment for these diseases.

Use in the pharmaceutical and clinical practice of the claimed MCAT, of the drug on the basis and method of its use allows to achieve several technical and therapeutic results:

- high selective effect on tumor cells;

- the absence of severe toxicity and side effects;

- the possibility of systemic effects on the body by intravenous n the s on object MCAT achieved by what is claimed MCAT, as well as known, interacts with the antigen-specific cells and tumors of epithelial origin. The feature of the claimed MCAT obtained according to SU 1698287, is that it is a mouse monoclonal antibody ICO received by vysokoochishchennogo membrane epithelial mucin MUC1 isolated from the membranes of the fat globules of milk, and represents the immunoglobulin class IgG1 constant Association 310-9defined on the model cell culture human lung cancer OUT, and is characterized by the following amino acid and nucleotide primary structure of the N-terminal regions of the heavy (H) and light (L) chains are:

Vh: DVQLQQSGPDLVKPS

Vl: DVLTQSPASLAVSLG

These common technical and therapeutic results for the group of inventions on object antitumor agent is achieved due to the fact that the claimed medicinal preparation as is known, contains MCAT and a pharmaceutically acceptable carrier. The feature of the claimed anti-cancer drugs is that its basis is MCAT ICO has a composition, wt.%:

Monoclonal antibody ECO - 0,5

Polysorbate-80 - 2,0

Phosphate the major technical and therapeutic results in the exercise of invention the object of the treatment is achieved by that the inventive method as is known, represents the immunotherapy of cancer. The feature of the proposed method lies in the fact that immunotherapy is carried out by systemic administration of medicinal products containing the mouse MCAT ICO. While the inventive method is used in the treatment of malignant tumors of epithelial origin by intravenous maintenance of the claimed medicinal preparation.

The invention consists in the following.

The proposed method of therapeutic treatment based on the use of murine monoclonal antibodies to MUC1 mucin.

Normal mucin MUC1 is synthesized by cells of the glandular epithelium and is localized in the apical membrane of the cells. In cells of malignant neoplasms has increased synthesis of mucin MUC1 that is associated with the increase in the number of copies of the gene MUC1. In addition, tumor cells have a high content of MUC1 in the cytoplasm and across the cell membrane. Due to the redistribution of antigen and increase its expression in cells of the mucin in malignant tumors may be an effective target for cytotoxic antibodies. At the same time, antigens, mannai system, that reduces the likelihood of side reactions during the use of MCAT this specificity for therapeutic purposes.

According to immunohistochemical analysis MCAT ICO interacts with cells of normal epithelium of the breast, lung, salivary glands, skin glands, glands of the tongue, esophagus, stomach, intestine, epithelium of the urogenital system (kidneys, ureters, bladder, uterus, fallopian tubes), the epithelium of the pancreas, prostate gland, epithelium of the gallbladder and biliary excretory ducts of the liver.

MCAT ICO interacts with cells of malignant tumors of epithelial origin in the breast, lung, ovary, stomach, colon, salivary glands, the organs of the urinary system organs Oro-faringealni zone.

The antibody does not interact with blood cells, connective tissue, blood vessels and muscle fibers, nerve tissue, and cells of malignant tumors of non-epithelial origin.

In normal epithelium, MCAT ICO interacts with the apical membrane of the cells. In tumour cells (depending on the type of neoplasm) is observed okra is="ptx2">

The above data confirm these potential benefits of a medicinal product on the basis of MCAT ICO as high selective effect on tumor cells of epithelial origin, and no toxic and side effects.

The feature of the claimed MCAT also lies in the fact that it represents the immunoglobulin class IgG1 with unique specificity of antigenic determinants, with the constant Association 310-9defined on the model cell culture human lung cancer OUT.

The isotype of the immunoglobulin MCAT ICO was determined by the method of double immunodiffusion agar on Ouchterlony using antisera to the following isotypes of immunoglobulins: IgG1, IgG2a, IgG2b, IgG3IgM. Line precipitation revealed in the result of the interaction of antibodies with anticorodal to the isotype of the immunoglobulin IgG1. With antisera to other isotypes formation of precipitates was not detected. Thus, MCAT ICO has isotype IgG1.

The affinity of the antibodies was determined by a standard method (D. Rodbard, 1973) on the cell line OUT, the results of which MCAT ICO are characterized by relatively high Data characterize the primary structure of MCAT ICO obtained by analysis of N-terminal amino acid sequences of N - and L-chain antibodies, as well as sequencing of the genetic products (cloned cDNA materials chains) from ICO hybridoma cells.

Drug for immunotherapy of malignant tumors of epithelial origin is a sterile solution of immunoglobulin in 0,015 M phosphate-buffered saline, pH 7.5, containing 25 mg MCAT ICO at a concentration of 5 mg/ml and 2% Polysorbate-80. It is a colorless, slightly opalescent liquid with no odor. Applied in the form of sterile 0.025% solution, which should be prepared immediately before administration. The solvent was 0.9% sodium chloride solution.

The inventive method of immunotherapy of malignant tumors can be used to treat colon cancer and other malignant tumors of epithelial origin. The treatment consists of 2-fold or 4-fold intravenous drug in a dose of from 5 to 500 mg/kg with an interval between infusions 7 days. The duration of treatment, the dosage is determined depending on the severity of the disease, localization swollen is inoe antibody ICO, specific to the antigen MIS1, characterized in that represents the immunoglobulin class IgGI constant Association 310-9M defined on the model cell culture human lung cancer OUT, and is characterized by the following amino acid structure of the N-terminal regions of the heavy (H) and light (L) chains are:

Vh: D V Q L Q Q S G P D L V K P S

VI: D V L T Q S P A S L A V S L G

2. Antitumor agent comprising the monoclonal antibody and a pharmaceutically acceptable carrier, characterized in that it contains mouse monoclonal antibody ICO under item 1 and has a composition, wt.%:

Monoclonal antibody ECO - 0,5%

Polysorbate-80 - 2,0%

Phosphate-saline buffer - Rest

3. Antitumor agent under item 2, characterized in that it is a solution for intravenous injection.

 

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FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

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