Conjugate antimelanoma antibody and a cytotoxic agent and a method of inhibiting melanoma

 

(57) Abstract:

Conjugate antimelanoma antibody and cytotoxic agent is a conjugate of a monoclonal antibody ZME-018 antigen GP 240 cancerous melanoma cells, and as a cytotoxic agent it contains toxins, or cytocidal money, or drugs, the method of inhibiting melanoma is the introduction to a mammal with melanoma conjugate parenterally in a dose of from 0.1 to 10 mg per 1 kg of body weight. 2 S. and 2 C.p. f-crystals, 13 Il, 1 table.

The branch of science to which the invention relates.

The present invention relates to the field immunoconjugate and more specifically to the use of immunoconjugate in the treatment of cancer. The invention relates also to the treatment of melanoma by conjugates of monoclonal antibodies (MAB) and cytotoxic components, such as gelonin, ribosomebinding protein, other cytotoxic components of plant origin or cytotoxic or cytostatic biological response modifiers.

The background to the invention.

The need for accurate orientation cancer therapy is critical for the reason that adequate tumor responses dependence is and tumor treatment has been the target of several researchers, using monoclonal antibodies as specialized carriers of therapeutic agents.

Cancer is one of the main causes of mortality and morbidity in the Western world. There are many types of cancer, each of which has its own characteristics. However, at least one feature is common to all forms of cancer. It is associated with defects in the regulation of cell growth.

Melanoma, the most dangerous kind of skin cancer, is a disease with active development of metastasis, affecting both sexes and almost certainly fatal within five years after diagnosis. It is proved that surgical removal of the identified malignant tumors can only be effective when there are initial signs of the disease. If the disease has developed, surgical intervention should be supplemented by other, more General treatments of the sick or destruction of malignant cells. The most commonly used additional treatments, such as radiation or chemotherapy, are not limited to tumor cells, and although they have proportionally more destructive effect of whole Express antigens or antigenic determinants, which in a very weak degree expressed or not expressed at all by normal cells. Some tumor cells Express antigens that are expressed by cells of the embryos, but not expressed by normal cells of an adult animal. These abnormally expressed antigens known as antigens associated with tumors. The peculiarity of these antigens is that in our time as a specific antigen can be expressed by more than one tumor, this antigen is normally expressed in all or most cells of certain tumors expressing it. Cell tumors may Express one or more associated with tumor antigens. These associated with tumor antigens can be expressed on the cell surface (antigen cell surface) can be secreted by tumor cell secreted antigens) or may remain inside the cell (intracellular antigen).

The presence of these associated with tumor antigens used for detection, diagnosis and localisation of the tumour. In some cases, the presence associated with tumor antigens on the tumor cells allows nalivat the body are proteins, that the immune system of the animal forms in response to alien antigens or antigenic determinants. Antibodies bind to specific antigens against which they are directed. For example, antibodies to other antigens of melanoma are used to ensure systematic intraperitoneal introduction to show the specific location of the tumor.

One method of targeting therapeutic agents to tumor cells and prevent their effects on normal cells became possible after the creation of the MAB, directed against antigens on the tumor cells, which is not found on normal cells. Monoclonal antibodies directed to specific antigens or antigenic determinants can be obtained in large quantities.

Antibodies can be labeled, so they can be used for localization and treatment of malignant tumors. Such labeled with radioactive isotopes monoclonal antibodies to surface antigens of tumor cells has been successfully used to represent the tumor from the patient through the outer scintography. (Deland, Lemin. Nucl. Med. 19(3) : 158-65 (Review) (1989) Tuhl. Heptagastroenterology 36(1) : 27-32 (Review), (1989)). Antibodies in complex with Lek the VA target tumor cells of a particular type, against which the antibody that is associated with the unique ability of antibodies to localize the site of the tumor after a systematic introduction. Antibodies can also be combined with toxins and act thus as a delivery system designed to target toxins directly on certain tumor cells.

Cytotoxic tools, often used in the conjugates of antibodies, fall generally into three main classes: toxins, radionuclides and chemotherapeutic agents. Conjugates of antibodies with each of these types of funds offer promising opportunities, but this creates certain problems (Frankel and others Aun. Rev. Med. 37 : 125 - 142 (1986), Reimann and others T. Clin. Invest 82(1) : 129-138 (1988)). Immunoconjugate, including plant toxins, have unique advantages compared with other types of conjugates of antibodies, because:

1. Dose immunotoxins required for antitumor effects in General, significantly lower than that required in the conjugates of antibodies with drugs.

2. Coupling of toxins with antibodies shows no negative impacts on sites of antibodies.

Gelonin is a glycoprotein (mainactivity plant toxins. Other members of this class ribosome-inactivating plant toxins are chain abrin, ricin and medecine. Gelonin as abrin and ricin, inhibits protein synthesis, damaging the subgroup of 60 S ribosome mammals. Although the A chain of ricin (MOUTH) has found wide application for use in immunotoxins, gelonin is more resistant to chemical and physical influence than the MOUTH (Basbieri and other Cancer Surv. 1 : 439-520 (1982)). In addition, he gelonin is not associated with the cells and therefore is non-toxic (except high concentrations) and secure in laboratory studies. Inactivation of ribosomes irreversible, it seems, that does not entail any side effects and comes with efficiency, suggesting that Galanin enzyme acts.

Many researchers in the past have said or suggested connection cytotoxic funds antibodies with the aim of obtaining "immunotoxins". Of particular interest is the immunotoxins of monoclonal antibodies, coupled with enzymatic active parts (chain A) toxins of bacterial or plant origin, such as ricin or abrin (Nevetta and others, Immunol. Rev. 62 : 75 - 92), Ross and others, European J. Biochem 104 (1980), Vitteta and others , Im Osada among the most active toxins inhibiting protein synthesis on the basis of weight of a protein. Gelonin 10 - 1000 times more active in the inhibition of protein synthesis compared to the A chain of ricin. The peptides of the type of ricin and abrin consist of two chains, of which the chain is A toxic component, and a chain B communicates with the cells. Unlike ricin and abrin gelonin consists of one chain, and because it has no chain B for communication with the cell, it itself is relatively nontoxic to healthy cells (Stirpe and other J. Biol. Chem. 255 : 6947 - 6953 (1980)). The mammalian cells clearly lacks the ability to link and/or the introduction of natural molecules gelonin. Conjugates gelonin with aimed at the tumor monoclonal antibody such as a monoclonal antibody ZME directed to the antigen, is expressed on some tumor cells such as melanoma cells, provide as a certain way of linking gelonin with the cage, and the way of implementation of complex gelonin-antibody. One of the advantages of using toxin gelonin compared to the use of toxins such as the A chain of ricin, is to reduce the toxicity to normal cells in comparison with the A chain of ricin. Gelonin in complex with monoclonal directed to the associated with tumor 2">

Previous studies describe a number of conjugate antibody toxin containing gelonin (Lambert and others, J. Biol. Chem. 260 : 12035-12038 (1985) Jhorpe and others , Eur. J. Biochem. 116 : 447-454 (1981) Lingh and others, J. Biol. Chem. 264 : 3089-95 (1989) Lcott, etc., J. Natl. Cancer Lnst. 79 : 1163-72 (1987) Tedoler etc., J. Immunol. 137(4) : 1387-91 (1986)). Recently Asavari and other Int. J. Cancer. 43 : 152 - 157 created helaney immunotoxin comprising an antibody 467 B, which binds to the cellular receptor for epidermal growth factor (EDF). This conjugate B 467 and gelonin has a high toxicity towards cells expressing the receptor for the EFF, but not toxic to cells lacking the receptor. Sivam and others (Cancer Research 47 : 3169-3173 (1987)) has created a conjugate antimelanoma antibodies 9.2.2.7 with Galanina and compared in vivo and in vitro cytotoxic effect with conjugate 9.2.2.7 with A chain abrin and ricin. These studies showed that conjugates with gelonida demonstrate in vitro a significant selective cytotoxic effect against the cells positive for the antigen. Experiments in vivo showed that cheloninae conjugates are not toxic at the dose of all antibodies, reaching 2 mg/mouse and that multiple introduction I. V. gelasinospora immunotoxin significantly slows the growth of subcutaneous human xenograft tumors in naked mice. P is yatelnosti and tumor localization with more significant therapeutic effect in vivo.

As the antibody with which is combined a drug, toxin or radioisotope label is associated only with tumor cells expressing a specific antigen, kill only tumor cells. In contrast, radiation therapy radiation from compounds labeled with radioactive isotopes, not limited to tumor cells that are being targeted exposure. So, for example, metabolic or enzymatic decomposition of antibodies may lead to the release of radioactive label and allow it to spread to other tissues such as kidney or bone marrow, leading to unacceptable radiation damage to these organs. Antibodies with radioactive labels are faced with challenges that limit or complicate their use as therapeutic agents.

A brief statement of the substance of the invention.

The present invention provides immunoconjugate with the antibody (designated here as ZME - 018), which recognizes the antigen GP 240 on cancer melanoma cells. One of the antibodies (225. 28S), reviewed by Wilson and others (Wilson and other Int. J. Cancer 28 : 293 (1981)) recognizes the antigen membrane of melanoma. This antigen is indicated here SIM the embodiment, the antibody is combined with a toxin, selected from the group consisting of gelonin, chain A of ricin and A chain abrina. In another embodiment, the antibody ZME can be combined with cytocidal means, such as adriamycin, or with modifier biological reactions, such as a lymphokine or cytokine. In another embodiment, the antibody may be labeled with a different label, such as label, radioactive isotope, chemoluminescence, fluorescers or enzyme label. Cytocidal immunoconjugate useful for the treatment and prevention of recurrence of tumors bearing associated with tumor GP 240 by introducing these cytokinin immunoconjugates in accordance with the individual characteristics in each individual case. Distinguishable marked immunoconjugate ZME useful for the diagnosis and localization of tumors by means known to specialists in this field. These labeled immunoconjugate also useful when analyzing for the presence of antigen GP 240 in biological samples and localizing tumors in vivo by means known to the specialists.

One of the purposes of the present invention is to offer a special compound that selectively binds to tumor cells and kills them. In particular the contact and kill tumor cells, expressing the antigen GP 240 as described above. Antibody ZME-018 was prepared "Fibretex" with the application of salt fractionation and chromatography on DEAE, and homogeneity was determined by SDS PAGE (Wilson and others, Int. J. Cancel. 28 : 293-300 (1981)). Another aspect of the present invention relates to a method of destruction of melanoma cells or other tumor cells expressing the antigen ZME (GP 240), through contact of the cells with cytochalasin effective amount of immunotoxin.

Another objective of the present invention is to propose such a composition, which would be toxic to tumor cells, but would cause minimal damage to healthy tissues.

Description of the drawings.

In Fig. 1 shows the wiring diagram and purification of complex ZME-gelonin.

In Fig. 2 shows the purification of ZME-gelonin method exclusion chromatography gel S-300.

In Fig. 3 shows the elution profile in the column Cyboron blue on sepharose after receipt of material with a high molecular weight after chromatography on S-300 and its elution with a linear salt gradient (0-300 mm NaCl). There are two protein peak: peak flow (fraction 14-20) and boundary peak elution prinjolata ZME-gelonin.

In Fig. 5 shows comparative data for analysis by ELISA ZME (white circles) and ZME-gelonin (black circles).

In Fig. 6 shows the cytotoxicity ZME-gelonin and free gelonin on log-phase cells AAB-527 after 72 hours of exposure.

In Fig. 7 shows the cytotoxicity ZME-gelonin and free gelonin on log-phase cells AAB-527.

In Fig. 8 shows the cytotoxicity ZME-gelonin positive for the antigen target cells melanoma (AAB-527) and negative for the antigen cells T-24 in culture.

In Fig. 9 shows the effect of free antibodies to the cytotoxicity ZME-gelonin.

In Fig. 10 shows the effect of IFN- , IFN - and TNF on cytotoxicity ZME-gelonin. Black circles show the response to dose only ZME-gelonin. White diamonds indicate the response to the dose ZME-gelonin and IFN - . White triangles show the response to a dose of ZME-gelonin in the presence of a specified number of TNF - . Black circles with a dashed line curve represent the response to a dose of ZME-gelonin in the presence of a fixed number of IFN - .

In Fig. 11 shows the impact of ZME-gelonin positive (A-375, black circles) and negative (CEM, white squares) on the antigen cells in the sample stem clench lines of stem cells, obtained from fresh samples for biopsy from 4 different patients.

In Fig. 13 shows the distribution of tissue antibody ZME and conjugate ZME-gelonin naked mice bearing human melanoma xenograft.

Detailed description of the invention.

Used herein, the term "monoclonal antibody" means a composition having a homogeneous population of antibodies. Do not assume the existence of limitations as a source of antibodies, and the way they are received.

Melanoma cells Express on the cell surface antigen 240 KD (GP 249). Produce antibodies to this antigen. Antibody ZME-018 (from "Fibretex") is a mouse monoclonal antibody IgG2recognizing glycoprotein 240 KD, present in most cells of human melanoma. Can be obtained monoclonal antibodies isotopes IgG1, IgG2aand IgG2bthat there are epitopes of the antigen 240 KD. This epitope 240 KD antigen ZME will be designated for the purposes of the present invention as the epitope ZME. Thus, all of the antibodies that recognize the epitope ZME, functionally equivalent.

These representative culture hybrid, whose cells secrete antur at 12301 Parklawn Drive, Rockville, PCs. Maryland 20852 ("ATCC").

These monoclonal antibodies can be obtained by methods known to experts in this field. Characteristics and procedure for obtaining cultures of these cells hybrid that creates these antibodies, are described in detail. (Wilson and other Int. J. Cancer 28 : 293 (1981), Jmai and others, Transplant broc. 12: 380-383 (1980)). In short we can say that hybridoma were obtained from myeloma cells of mice with Sp2/0-Ag-14 and splenocytes of mice immunized with cells of the myeloma line M21, as described by Imai and others ((1980) Transplant broc. 12: 380-383). Hybridoma, secreting monoclonal antibodies (mean) 225. 28 and 465. 12 subcloned and reproduced in vitro and in vivo. Both monoclonal antibodies belong to the subclass of Igand G2aand before using were purified from ascitic fluid of mice by absorption and elution from sepharose 4B protein A ("Pharmacia", Piscatway, PCs, new Jersey, USA).

Hybridoma forming antibodies that react with human melanoma cells but not with normal human cells, was characterized further. Antibodies formed by the cell line ZME and hybridomas, forming functionally equivalent antibody reacted with the antigen ZME on human melanoma cells. They reacted with 70-80% p is to shown in the table.

The term "Functionally equivalent" as used here is taken as an example, murine monoclonal antibody acting against human melanoma, means a monoclonal antibody that: (1) cross-blocked from the one shown in example a monoclonal antibody, (2) selectively binds to cells expressing the antigen ZME, such as cells of human melanoma, (3) has isotypes G or M, (4) binds to the antigen ZME identified by immunoprecipitating or sandwich immunoassay, and (5) when the clutch with gelonida dose inhibition of tissue culture is at least 50% against at least one of the cell lines AAV-527 or a 375 when using doses of 80-100 IU/ml

Antibody ZME coupled with gelonida using N-succinyl-3-/2-pyridylthio/propionate (people) or 2-aminosilane as the coupling reagent. The conjugates were tested on cells AAV-527 and 375 at exposure on tissue culture for 72 hours Conjugates of antibodies demonstrated acceptable antiproliferative effect (dose inhibiting tissue culture 50% from less than 10 IU/ml) against both strains of these cells.

For more details, characterizing antibodies, predestine, with paramount importance, are immunotoxins (conjugates of the antibody ZME and cytotoxic component or modifier biological reactions) and labeled (for example, radioactive isotopes, enzymes or phosphors) derivatives, in which the label is a means of identification of immune complexes, including labeled antibody.

Cytotoxic component of immunotoxin can be represented cytotoxic drug or an enzymatically active toxin of bacterial or plant origin (gelonin), or enzymatically active fragment of (a chain) of such a toxin. Enzymatically active toxins and fragments thereof and their preferred examples are gelonin, a chain of diphtheria, nesviazana active fragments of diphtheria toxin, a chain of exotoxin a (from Pseudomonas aeruginosa), a chain of ricin, a chain abrina, chain And modeccin, alfacell, proteins Aleuretes fosdii, proteins diantin, proteins Phytoiacca americana (RAR, RAR and PAP-S) inhibitor madica CHARANTIA, Curtin, krotin; inhibitor saponaria officinalis, mitogillin, restrictocin, vanomycin and inomycin. Most preferred is the compound with gelonida.

The biological reaction modifiers, which is, such as IL - 1, IL - 2, interferons (or / TNF, LT, TGF - and IL - 6, but are not limited to. These modifiers biological reactions have on the tumor cells varied impacts. Among the options the impact of the increase of killed tumor cells under direct influence, as well as the increase in the number of dead cells due to the processes which contribute to immune protection. Connection antibody ZME with these modifiers biological reactions contributes to the selective localization within cells and therefore improve antiproliferative actions while suppressing unwanted effects, leading to toxicity towards cells that are not targets.

Cytotoxic drugs that are useful for the present invention include adriamycin (and its derivatives), CIS-platinum complex (and its derivatives), bleomycin, methotrexa (and its derivatives), but are not limited to. These cytotoxic drugs are sometimes useful for clinical treatment of recurrent tumors and in particular melanoma, but their use is complicated by serious side effects and damage to the cells that are not goals. Antibody ZME can serve as a useful carrier of such drugs, obespechivayutsya shipping antibodies cytotoxic drugs to tumors will protect sensitive organs, such as the liver, kidneys and bone marrow, from the effects of chemotherapeutic agents. The use of medications, United antibody ZME as a delivery system, can reduce the dose of drugs, because all of the medicinal components are connected with antibodies concentrated inside the tumor.

Conjugates of monoclonal antibodies can be obtained by using different biofunctional protein coupling means. Examples of such reagents can serve SPD p. IT biofunctional derivatives imidocarb, such as dimethylacetamide, HCl, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis connection-azido, such as bis /n-azidobenzoyl /hexanediamine, derivatives of bis-page, such as bis-n-disoriented/-Ethylenediamine, diisocyanates such as toluene 2,6-di-isocyanate and bis-active fluorine compounds such as 1,5-di-fluorescent-2,4-dinitrobenzene.

When used for the destruction of human melanoma cells in vitro diagnostic and therapeutic conjugates typically will be added to the medium culture with a concentration of at least about 10 nm. Technology of preparation and route of administration is by ltural or medium perfusion.

Cytotoxic radiopharmaceuticals for the diagnosis and treatment of tumor bearing the antigen ZME, such as melanoma, can be obtained by combining isotopes with high linear energy transfer with antibodies. The term "cytotoxic component" used here should include isotopes.

The labels that are used in the preparation of labeled variants of antibodies, include components that can be detected directly, such as fluorescene and radioactive labels, as well as components, such as enzymes, that must be included in the reaction or to be entered in order to be marked. Examples of such labels are 32P, 125I, 3H, 14Cfluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferia, 2,3-dihydropteridine, horseradish peroxidase, alkaline phosphatase, lysozyme dehydrogenase and glucose-6-phosphate. Antibodies may be associated with such marks by known methods. So, you may be using to connect the antibodies with the above fluorescent, chemiluminescent or enzyme labels coupling agents, such as aldehydes, carbodiimides, timelimit, imidate, su is used in various immunolocalization and immunoanalytical operations in order to detect the presence of the patient's tumor, expressing the antigen ZME, such as melanoma, or to determine the status of such a cancer in a patient, the diagnosis of which have already been delivered. When used to determine the status of cancer can be applied quantitative immunoassay. Such surveys are conducted periodically, and compare results to determine increases or decreases the tumor of the patient. Conventional methods of analysis that can be applied include direct and indirect analysis. Direct analysis involves the cultivation of a sample of tissue or cells of a patient with a labeled antibody. If included in the sample bearing the antigen ZME cells include cells of melanoma, the labeled antibody will bind to these cells. After washing the tissue or cells to remove unbound labeled antibody, the tissue sample is examined for presence of labeled immune complexes.

For use in diagnostic purposes, the antibodies will be offered in kit form. These kits will typically include: antibody in labeled form, in suitable packaging, reagents for growing and leaching and nutrient medium or separating reagents depending on the nature of the label. In addition, there may also be included means counter the Oia, the person who was diagnosed with the presence of tumor antigenic determinant ZME, will allow you to focus and concentrate cytotoxic agent in the place that you want to destroy tumor cells. Such targeting cytotoxic reagents allows to avoid or reduce undesirable toxic effects on other organs, tissues and cells.

When used for therapy in vivo immunotoxins are introduced to the patient in therapeutically effective amounts (i.e., quantities that allow to eliminate or reduce the tumor of the patient). Usually they will be introduced parenterally, preferably intravenously. The dose and regimen of medicines will usually depend on the nature of the cancer (primary or metastasis) and its population, the characteristics of the specific immunotoxin, for example, its therapeutic index, the patient, medical history. The number of immunotoxin entered the patient will typically be in the range from 0.1 to about 10 mg/kg weight of the patient.

For parenteral administration immunotoxins will be prepared in a form convenient for dosing and injection (solution, suspension, emulsion), in combination with a pharmaceutically acceptable parented fillers are water, saline, ringer's solution, dextrose and 5% serum albumin human. Anhydrous fillers such as fixed oils and ethyl oleate, may be used similarly. As carriers can be used liposomes. The filler may contain small amounts of additives such as substances that improve the chemical stability and isotonicity, such as buffers and preservatives. The immunotoxin will usually be prepared with fillers at a concentration of about 0.1 - 10 mg/ml

Toxin gelonin was purified from the seeds of gelonium Polygonum way Stirpe and others , in short, gelonin was extracted from sowing by homogenization in buffer saline solution (pH of 7.4). The supernatant was subjected to concentration after dialysis against 5 mm sodium phosphate (pH 6.5), then gelonin subjected to further cleaning ionoobmennoi chromatography; as shown in example 1. The purity of the toxin gelonin was determined by liquid chromatography high pressure (ghvd) and electrophoresis in dodecyl sulphate gel-polyacrilamide sodium (SDS-page). Toxin gelonin moved as a single band with an approximate molecular weight 29-30000 Dalton.

The effectiveness of the toxin gelonin measured fitiavana, as described in example 5, SPDP, it is connected with gelonida modified aminosilanes, as described in examples 3 and 6. Antibody associated with Galanina, was subjected to purification as described in example 7 by the method of column chromatography in a column of Sephadex G - 75.

Toxicity antibodies connected with gelonida was determined by the inhibition of protein synthesis, and through experiments in vitro and in vivo was determined by its antiproliferative effect.

The following examples provide a detailed description of the preparation, characterization and use immunotoxin monoclonal antibodies, which are the subject of the present invention. These examples in no way limit the scope of the present invention.

Example 1.

Cleaning gelonin

The seeds of Gelonium Polygonum were released from the shell and the core to grind in the homogenizer with eight volumes of 0.14 M NaCl containing 5 mm of sodium phosphate (pH 7,4). The homogenate was left overnight at 4oC under continuous stirring, cooled on ice and centrifuged at 35 thousand g for 20 min at a temperature of 0oC. the Supernatant was removed, was subjected to dialysis against 5 mm sodium phosphate (pH 6.5) and concentration by means of the filter 10 millionoC. Was obtained five fractions of 1 ml Fractions were measured on the spectrophotometer at a wavelength of 280 nm. Gelonin was elyuirovaniya in fractions of approximately 55-70 and account for the last peak gelonin. Faction 55-70 combined, subjected to dialysis against distilled water and the concentration by lyophilization. The purity and molecular weight of each preparation was examined using liquid chromatography high pressure using the method exclusion chromatography in column (TSK 3000 with a buffer of 50 mm sodium phosphate, pH 7.4 and by electrophoresis in 15% gel diecisiete-polyacrylamide sodium (SDS - page). Gelonin migrated in the form of a single band with an approximate molecular weight 29-30 thousand daltons.

Example 2

The determination of the activity of gelonin

Activity gelonin was determined in the course of experiments on the inhibition of the synthesis of cell-free protein. Experiments on the inhibition of the synthesis of cell-free protein was performed by sequentially adding to 50 µs lysate of reticulocyte rabbit subjected to thaw immediately before use, the following components: 0.5 ml of 0.2 M Tris HCl (pH 7.8) 7 8,9 ml of ethylene glycol and 0.25 ml of 1 M HCl, PE is ostoja of 0,375 M KCl; 10 mm Mg (CH3CO2)2, 15 mm glucose, 0.25 to 10 mm of the amino acids except leucine), 15 mm ATP, 1 mm Tris-HCl (pH 7,6), 10 ul of creatinine-phosphate-creatinine phosphokinase, 8 str14C leucine (Amersham 348 mCi mm), with the addition of 1.5 ul of solutions containing different concentrations of the mixture gelonin. The mixture was incubated for 60 min at 30oC. Implementation of 14C - leucine was observed in the aliquot of the mixture by precipitation of the synthesized protein on glass fiber filters, washing trichloroacetic acid and acetone and measurement of radioactivity counter beta-particles using Scintilla fluid Aquasol. For coupling with antibodies used gelonin with specific activity of not less than 4 of 109ate/mg a Unit of activity gelonin is the amount of protein gelonin causing 50% inhibition of the introduction of [14C] leucine into protein in a free sample of cells.

Example 3.

Modification gelonin aminothiophenol

Concentration gelonin in physiological solution with phosphate buffer was brought to microconcentrators Centricon 10 to approximately 2 mg/ml of It was added the hydrochloride of triethanolamine (TEA/HCl) pH 8,8, ethylenediaminetetraacetic acid (EDTA) at a final concentration of E. then the sample was incubated for 90 min at 4oC in a stream of nitrogen gas.

Excess aminosilane removed by gel filtration column of Sephadex G - 25/1 24 cm/ pre-equilibrated with 5 mm bis-Tris-acetate buffer, pH 5.8, containing 5 mm NaCl and 1 mm EDTA. Fractions were analyzed for protein on the blade using the method of Bradford binding dye. For this purpose, in each cell was added 40 μl of sample, 100 ul of saline phosphate buffer and 40 ul of dye concentrate. The optical density at 600 nm was determined on an automatic reading device "Dynatex microelisa". Gelonin blueraven in the empty volume (about fractions 14-20). These fractions were merged together and focused on microconcentrators Centricon 10.

Example 4.

Preparation and identification of monoclonal antibodies to the antigen ZME melanoma

Hybridoma, secreting antibodies

The female mouse BAL B/c (Nursery SCRF La JOLLA, PCs California) 8 weeks of age administered intraperitoneally introduced 107cells M21 human melanoma, adding to them two weeks later 5 10 cells M21. The male mouse NZB/B (Nursery SCRF) 50 weeks of age were injected first with 5 106cells BW5 melanoma, adding that five injections of 5 to 106cells BW5, M51, who was lekali spleen, the scalpel was divided splenocytes with the purpose of obtaining a cell suspension. Suspension of spleen cells for 10 min were processed 0,17 J H4Cl 0.01 M Tris, pH 7.2, for the purpose lizirovania red blood cells. Then these splenocytes were merged with cells of the SP2/OAg 14, as described by Gefter, etc./ (1977) Somat. Cell. Genet 3:231 /with the following minor modifications : 5 107the spleen cells and 107cells SP2/OAg 14 was hybridisable with 0.3 ml of 30% (V/V) of polyethylene glycol 1000 (PEG) in the MEME. After incubation with PEG cells were washed and grown over night at a concentration of 2 to 106cells/ml in D-MEM. The next day, cells were diluted in suspension in 40 ml of medium NAT and pipette made in 400 cells/0.1 ml/cell/blade (Costar N 3596, Cambridge, pieces Massachusetts). At weekly intervals was added drop by drop approx. 25 ml of medium NAT. After 2-3 weeks in D-MEM with 10% FBS were grown to hybridoma selected for further studies. Hybridoma spread in tissue culture and were grown in the peritoneal cavity of a mouse BALB/c mice, which first introduced 0.5 ml of Piers/Pfalz & Bauer, Stamford, PCs Connecticut/, spent culture medium and acritically liquid were used as source of antibodies.

Clones of hybridoma were grown in vitro in the CE is

Then identified hybridoma forming antibodies that react with cells of the myeloma person, but not with healthy human cells.

As shown in the table, they do not react with most healthy tissues tested. Antibodies formed by the cell line ME, and functionally equivalent antibodies, hybridomas formed by, reacts with antigens ME on human melanoma cells, such as the M21.

Example 5

Modification of monoclonal antibody ME-018 by people

N - succinimide 3-/2-pyridylthio/propionate/ (people) in dimethylformamide was prepared as the main solution of 3 mg/ml in dry dimethylformamide. Since the crystal people are susceptible to hydrolysis, the actual concentration of the chemically active element, forming cross-links were determined by the spectrophotometric method of analysis of the optical density at 260 nm in the two-beam electrophotometer. The concentration of the basics people was calculated by the following formula

< / BR>
One milligram monoclonal antibody ME in 1.0 ml of saline phosphate buffer (PBS) was introduced into the glass tube. The main solution people was slowly added into the tube when the AP is Noah temperature, mixing during the incubation period every 5 minutes

Excess unreacted people were removed from the sample by using exclusion chromatography on a column of Sephadex C-25 (1 x 24 cm) pre-equilibrated with 100 mm buffer of sodium phosphate pH 7.0, containing 0.5 mm EDTA (buffer A). Fractions (0.5 ml) were collected and analyzed for protein content using the method of Bradford binding dye (Bradford, Anal. Biochem. 72: 248-254 (1976)). The optical density (600 nm) was determined in 96-cell microplate using an automatic reader Bio-Tex microplate". Antibodies were suirable in the void volume (fractions 14-20), these fractions are merged together and kept at 4oC. Protein was concentrated on microconcentrators "Centricon-30". The sediment in Centricon" washed 100 mm buffer of sodium phosphate pH 7.0, containing EDTA (0.5 mm). Antibodies were concentrated to a final volume of approximately 0.5 to 0.75 ml.

Example 6

The connection of modified people monoclonal antibody ZME-018 with modified imitational Galanina

One milligram of purified gelonin (2 mg/ml in PBS), prepared as described in example 1 was modified by imitational as described in example 3. Monoselenide, modified as shown in example 3. This proportion corresponds to a fivefold molar excess gelonin in comparison with antibodies. The pH of the mixture was brought to 7.0 by adding 0.05 M buffer Thea/HCl pH 8.0, after which the mixture is incubated for 20 h at 4oC in nitrogen atmosphere. Iodoacetamide (0.1 M) was added to a final concentration of 2 mm in order to block any remaining free sulfydryl group, then another hour of continued incubation at a temperature of approximately 25oC. the Reaction mixture was kept at 4oC prior to purification by gel filtration.

Example 7

Purification of complexes gelonin-monoclonal antibodies A

Gelonin not included in the conjugate, and products with low molecular weight was removed from the reaction mixtures of example 6 by gel filtration on a column of Sephadex S-300 (1.6 x 31 cm), previously equilibrated FBC.

Before loading the column Sephadex reaction mixture from example 6 was concentrated on the hub "Centricon 30" to approximately 1 ml Column was washed FBS. Were selected fractions of 1 ml and aliquots of 50 ul analyzed for protein by the method of Bradford binding dye (Bradford, Anal Biochem, 72: 248 (1976)).

As shown me how unrelated gelonin blueraven about faction 45 - 65.

To remove unbound ZME-018 high molecular weight peak (fraction 28-40) column S-300 was applied to the column affinity chromatography of Sepharose blue CL-6B (1 x 24 cm) pre-equilibrated with 10 mm phosphate buffer (pH of 7.2) containing 0.1 M NaCl. After loading the sample the column was washed with 30 ml buffer until complete elution of the unbound antibodies. The column was suirable with a linear salt gradient from 0.1 m to 2 M NaCl in 10 m M phosphate buffer pH of 7.2. The protein content in buervenich fractions was determined by the method of Bradford binding dye.

In Fig. 2 shows the elution profile in the column of S-300 and it is shown that Galanin can be separated from the conjugate gelonin-antibody and unbound antibodies, which together suiryudan in the first peak (about fractions 28-40). This picture elution confirmed by electrophoresis 50 ul aliquot of on 5-20% gradient nevosstanovlenie SDS polyacrylamide gels, as shown in Fig. 4. The coupling mixture was placed on track 3. Shows bands corresponding to free gelonin (lane 2), free antibodies (lane 1) and one molecule gelonin associated with the antibody molecule and two molecules connected in the molecule antibodies. The peak in the void volume of the column S-300, connecting free Antel, related gelonida by affinity chromatography on a column of Sepharose blue CL - 6B (1 x 24 cm) pre-equilibrated with 10 mm phosphate buffer, pH of 7.2, containing 0.1 M NaCl. After loading the sample eluate S-300 column was washed with 30 ml of the same buffer to completely eluted unbound antibody.

Antibodies associated with Galanina and detained in the column was suirable with a linear salt gradient of 0.1-2 NaCl in 10 mm phosphate buffer, pH 7,2. The complex of antibody-gelonin was suirable approximately 0.7 M NaCl as shown in Fig. 3, which shows the profile of elution in column Sepharose blue. The protein content in buervenich fractions was determined by the method of Bradford binding dye. Containing protein fractions were merged together, and the picture elution was confirmed by electrophoresis on a 5-20% gradient nevosstanovlenie polyacrylamide gel. Picture electrophoresis complex ZME-gelonin shown in Fig. 4. Peak flow (fractions 14-20) contains only free antibody (Fig. 4, lane 5), while the fraction of 50-80, erwerbende high in salt, contain conjugate ZME-gelonin, free from unrelated gelonin or antibodies (Fig. 4, lane 6). The final product contains antibody ZME-related 1, 2, and 3 molecules heII reticulocyte rabbit in vitro, described in example 2, was used to assess the activity of Galanin in a very pure complex gelonin-antibody ZME. One unit of activity in these experiments was defined as the amount of protein required for 50% inhibition of protein synthesis compared with the untreated control sample. Conducting these experiments gelonin and conjugate ME-halogen respectively 2 108u/mg and 8.2 105u/mg. Exceptionally clean gelonin-antibody ME active in the sample lysate of reticulocyte. Breeding in the ratio of 1:1000 initial sample resulted in approximately 50% inhibition of protein synthesis, i.e. 50% reduction implementation14C-leucine into protein. Thus, the activity of the original drug was 1000 u/ml

Example 8

Methods of obtaining a cell culture

Negative for the antigen ZME cell carcinoma of the human urinary bladder (T-24), carcinoma of the neck of a man or positive to the antigen of tumor cells metalicheskoy human melanoma AM or AAV-527 cultivated using minimal additions to the basic medium (MEM) with 10% deactivated by heat treatment of embryonic bovine serum plus 100 mm non-terrestrial amino acids, 2 mm L-glutamine, 1 mm sodium salts of the I impurities Mycoplasma.

A. Experience in cell proliferation

Cell lines were maintained in culture in complete medium at 37oC in an incubator with 5% CO2- humidified air. For experiments with combinations of TNF, immunotoxins IFN and IFN culture were washed, separated by versinon and re-suspended in complete medium at a density of 25 103cells/ml. Aliquots two hundred ml were distributed in 96-cell to microplasmas, then allow adherence of the cells. This led to a rare population of cells. After 24 h the medium was replaced with medium containing different concentrations of immunotoxin, toxins, TNF, IFNg or IFNa. Cells were incubated for 72 h and then perform analysis on the relative cell proliferation by crystal violet staining.

B. Staining with crystal violet.

Cells are washed three times FBS containing associated calcium and magnesium, and stained with 20% (V/V) methanol containing 0.5% (V/V) crystal violet. Bound dye was suirable 150 ul of citrate buffer of Sorenson /0.1 M sodium citrate, pH 4.2 to 50% /about/about ethanol/ for 2 h at room temperature. The optical density was measured at 600 nm using a reader blade "Bio-Tex". Relative cast new

Tumor samples for biopsies were obtained from patients with melanoma during clinically prescribed procedures biopsy. Aseptically prepared suspension of tumor cells (Leibowitz and others, Int. J. Cell Cloning 1 : 478 - 485 (1983)). Along with this experienced lines of melanoma cells AR and leukemia CEM from the American type culture collection (Rockville, PCs Maryland). Determine the impact of IU-gelonin suspension of fresh melanoma cells and cell lines was determined in samples of colony cells from human tumors using standard procedures lamellar distribution of tumor cells in semi-solid medium (agarose) in the presence of complete medium containing 10% embryonic calf serum, each of 0.5 ml of LPS to the culture contained 100,000 cells fresh tumor and 10000 cells from cell lines (Burger and others , Science 197 : 461 - 463 (1977), salmon, etc., N. Engl. J. Med. 298 : 1321 - 1327 (1987), salmon and other J. clin Oncol. : 1346 - 1350 (1989)). Conjugate ZME-gelonin, prepared as described above were tested by adding to the plate with culture soon after the application of tumor cells. ZME-gelonin added to triplicate plate at each of the three concentrations from 0.025 to 250 ng/ml In addition to the untreated control plates in parallel, cells were incubated for an average period of 10 days at a temperature of 37oC and 50% CO2on the air in the incubator with humidity, with the definition of colony formation by staining viability (shoemaker and others, Cancer Res. 45 : 2145 - 2153 (1985)) and by using an automated device image analysis, optimized for counting colonies (salmon and other, Int. J. Cell. Cloning 2 : 142 - 160 (1984)). The percentage survival of cultures treated with ZME-018 compared with both untreated control samples were determined in the same experiments. After that graphically curves were constructed response to dose.

Example 9

Mapping the coupling strength of the antibody ZME associated and not associated with Galanina, cells order.

Determined the ability associated and not associated with gelonida antibody ZME connection with a cell-purposes. Communication ZME-gelasinospora immunotoxin with positive cells (cells AAB-527) and negative (cells T-24) antigen was determined by the method of ELISA samples.

In each cell on microplate added 50 thousand cells-targets (cells AAB-527) or cells that are not targets (T-24). The cells were dried on the plates overnight at 37 ° oC. thereafter, the cells three times washed in three changes of phosphate buffer and during the night were air-dried. After the e application of the cells of the blade washed with wash buffer (9,68 g Tris, to 64.8 g of sodium chloride, 16 ml tween-20, 800 mg thimerasol 8 l double distillation). Samples antibodies were diluted in wash buffer containing 1% embryonic bovine serum (weight/about) (diluting buffer). In cells was added 50 μl of various concentrations from 0.02 to 50 ug/ml or with related or unrelated antibodies ME. After incubation for one hour at 4oC the supernatant was removed, and cells washed twice with wash buffer.

In each cell was added 50 μl of horseradish peroxidase linked goat anti-mouse Ig G, obtained from Bio-Rad and diluted in the ratio 1 : 1000 (V/V) (HPGAM) in razvodami buffer. Plates were incubated for 1 h at 4oC, and the cells washed twice with wash buffer. After incubation of the plates with 50 ml of substrate solution (80 mm citrate phosphate), pH 5.0 (1 mm 2.2 Azino bis/3-ethyl-Benz-thiazoline-6-sulfonic) (ABTS) demoniaca salt (Sigma chemical) and 4 ul of 30% hydrogen peroxide in the dark for 30 min at room temperature in each cell was added 25 ul chetyrehkamernoe sulfuric acid. The optical density at 492 nm was determined on the counter "Alice".

As shown in Fig. 5, as a natural ZME and conjugate ZME-gelonin well contact cell-chains of th the th antibody. This improvement was not associated with a modification of antibodies by people, as modified people ZME behaves similarly to natural ZME. The improvement is not associated with the communication cells-targets with janinejay part of the molecule, since pre-treatment of cells-targets natural gelonida not affect the relationship antibodies and immunotoxins.

In experiments "ALICE" was not determined relationship with negative antigen cells T-24 no ZME or ZME-gelonin.

Example 10

Cytotoxicity gelonin and complex gelonin-antibody ZME

Study the cytotoxicity of the conjugate ZME-gelonin conducted on positive by antigen cells after prolonged (72 h) exposure to the immunotoxin or natural gelonin. As shown in Fig. 6, when a positive for the antigen cells AAB-527 were exposed to 0.1/nm ZME-gelonin, there has been a loss of 50% of the cells. When cells were subjected to natural gelonin, it took concentration of 100 nm gelonin to reduce the number of cells to 50% of their numbers in the untreated control sample.

Cells-chain processed after this, different concentrations of ZME-gelonin or only gelonin on a unit basis, as the op is and, whereas to achieve the same effect required 1 107u/ml of free gelonin.

The effect ZME-gelonin was determined in the negative by antigen cells T-24 culture in the log phase. As shown in Fig. 8 only gelonin provides 50% cytotoxicity cells AAB-527 at a concentration of 100 u/ml, which is close to that found for cells AAB-527.*ZME-helanie provides 50% cytotoxicity cells-targets T-24 at a concentration of 10 μg/ml, However, the immunotoxin ZME-gelonin has no cytotoxicity against non-targets cells T-24, even at the highest tested concentrations.

In order to demonstrate that the cytotoxicity ZME-gelonin mediated through surface antigen ZME, fixed-dose and ZME-gelonin, reaching 80% cytotoxicity was added to log-phase cells-targets melanoma cells in culture in the presence of free antibody ZME or not related to this situation antibodies (15AB, an antibody that does not bind to melanoma cells). As shown in Fig. 9, the presence of increasing amounts of antibody ZME suppresses the cytotoxicity of the conjugate ZME-gelonin, while antibodies 15AB have no effect. Thus, the cytotoxicity of the conjugate ZME-gelonin separowanie cytotoxicity ME-gelonin by IFN, IFN and TNF

In order to demonstrate the impact on the cytotoxicity of immunotoxins processing different modifiers biologic response log-phase melanoma cells were treated for 24 h fixed-dose IFN/ (200 u/ ml), IFN/(20000 u/ml) orrTNF/(20000 u/ ml). Previously, it was determined that these doses have minimal (approximately 20%) cytotoxic effect on these cells. After that cells within 72 h were treated with different doses of ZME-gelonin. As shown in Fig. 10, processingrIFN leads to a twofold increase in the sensitivity to the immunotoxin ZME-gelonin. However, pre-treatment andrIFN and TNF in both cases leads to a twofold increase in the sensitivity to the immunotoxin. Adding fixed dose rIFN, or rIFNrTNF positive for the antigen to the cells leads to increased cytotoxicity of toxin ZME-gelonin. ProcessingrTNF provides the greatest increase in the cytotoxicity of immunotoxins, which is followed by processingrIFN andrIFN.

A significant increase in cytotoxic effects ZME-gelonin was observed in the pre-treatmentrIFN andrTNF, but notrIFN . While on the e antigens of melanoma, such as P-97, they have little effect on the antigen with a large molecular weight (GP 240) recognized ZME (Murray and others, Proc, Ain. Assoc. Cancer. Res. 27:313 (1986), Greiner and others, Cancer Res. 44:3208-3214 (1984), Greiner and other Cancer. Res. 46:4984-4990 (1986), Gregorini etc., J. Jmmunol 133:1649-1655 (1984), Imai and others, J. Jmmunol 127:505-509, Murray and others, J. Biol. Res. Mod. 7: 152-161 (1988)). Therefore, associated with the use of TNF and IFN mechanism of increased activity of the ZME-gelonin unclear, but may involve changes in the rate of adoption of antibodies, changes in the cellular action of immunotoxin or modulation of any of several enzymes mediated inteferons.

Because immunotoxin gelonin-ZME kills only cells that contain on their surface antigen ZME, this immunotoxin is an effective way to identify and destroy cells containing the antigen ZME associated with the tumor, while minimizing or preventing damage to healthy cells, not associated with a tumor.

Example 12

The impact of immunotoxin ZME-gelonin on a sample of colonies of cells of the human tumor

Activity ZME-gelonin was also evaluated using a sample of colonies of cells of human tumors in connection with cells obtained by biopsy from four pazienti cells, described in example 8C. Different doses of immunotoxin ZME-gelonin was added to the cell lines, positive (A-375 melanoma) and negative (CEM) on the antigen. Survival of colonies was determined after 72 h after the addition. As shown in Fig. 11, the dose immunotoxin in the range from 0.25 to 2500 ng/ml contributed to the suppression of colony cell lines, positive to the antigen (black circles). ZME-018 and free gelonin separately or together, were not cytotoxic. Not impacted on the line, negative for the antigen (CEM) even at the highest concentration tested immunotoxin (light squares).

The impact of ZME-gelonin on 4 different fresh biopsy sample is shown in Fig. 12. At the highest tested dose immunotoxin (250 ng/ml) on two samples detected a reduction in the survival of melanoma cells forming a colony on 80 - 90%. One patient demonstrated at this dose suppression of cell growth by 50%, while one patient showed no cytotoxicity immunoconjugate. The third sample was noted only moderate (25%) reduction in colony. In the fourth sample at the highest dose of immunotoxin was marked by increased growth. In addition, increased growth was observed on the same sample at low the mi lines, adding unrelated ZME-018 and free gelonin when tested doses was not cytotoxic.

While samples from colonies of cells are not infallible, approximately 75% of clinically active anticancer agents positive in this test system. Means inactive in the colonies, so far turned out to be inactive and clinical conditions. Therefore, the activity of the conjugate ZME-gelonin has a 75% chance to demonstrate positive clinical value.

Example 13

The distribution of antibody ZME in fabric

Distribution in tissue labeled125I antibodies ME compared with immunotoxin relevant (ZME-gelonin) and immunotoxin, irrelevant (15A8-gelonin). Each antibody or conjugate antibody was injected intravenously into the tail vein of five Nude mice bearing xenograft human melanoma. Each animal received 10 mg of total protein labeled with 0.5 MS 125I in a total volume of 100 µl of physiological solution with phosphate buffer.

As shown in Fig. 13 not relevant conjugate 15A8-gelonin not localized specifically to tumor tissues (ratio T/B is 0.5). In contrast, as ZME, torenia T/B, respectively, of 2.0 and 1.5). Statistically significant differences in the absorption of 125I tumors after injection ZME or ZME-gelonin was not observed. The person skilled in the art can easily come to the conclusion that the present invention is well suited to achieve the objectives and obtain the above results and benefits, as well as inherent. Compounds, methods, procedures and techniques described herein are representative of preferred embodiments and are shown as examples, but should not define the limits of the scope of the present invention. Specialists in this field can imagine the changes that are within creatures, as well as the scope of the invention defined below by the claims.

1. Conjugate antimelanoma antibody and a cytotoxic agent, wherein as antimelanoma antibodies it contains monoclonal antibodies Z ME-018 antigen GP 240 cancerous melanoma cells, and as a cytotoxic agent it contains toxins, or cytocidal funds, or cytostatics.

2. Conjugate under item 1, characterized in that it further using labeled antibodies.

3. Conjugate under item 1, characterized in that th Method of inhibiting melanoma by injecting the mammal with melanoma conjugate, containing gelonin and antimelanoma antibody, characterized in that use conjugate conjugate under item 1 with a parenteral dose of from 0.1 to 10 mg per 1 kg of body weight.

 

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FIELD: medicine, oncology, immunology, tumor biology.

SUBSTANCE: invention relates, in particular, to methods for enhancing cytotoxicity based on applying anti-CD38-immune toxins. Method involves carrying out the treatment of patient with pathophysiological state taken among the group including myelomas and leukosis and involves the following stages: a) administration to the indicated patient the pharmacologically effective dose of retinoid that enhances expression of antigen CD38; and b) administration to the indicated patient the pharmacologically effective dose of immune toxin acting against effectively expressing antigen CD38. Method provides enhancing the cytotoxicity with respect to above said diseases in their resistance to anti-tumor medicinal agents.

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SUBSTANCE: method involves applying effective doses of epidermis growth factor receptors antagonists being active ingredient in drug for inhibiting various types of resistant human tumors. The epidermis growth factor receptor antagonists are combined with other chemotherapeutic agents like Cisplatin, Irinotecan or ionizing radiation.

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FIELD: medicine.

SUBSTANCE: invention concerns immunology area. Versions of the artificial fused protein consisting of an antibody (or its fragment) and cytokine, fused through a link peptide are offered. The antibody or its fragment is chosen from an antibody 225, 425, KS 1/4, 14.18, anti-CDx-antibody where x has the whole value 1-25. Each of versions of the fused protein has lowered quantity T-epitopes, at least, in the component of the fused protein presented by an antibody, and as consequence, possesses the lowered adjuvanticity, in comparison with an initial molecule. Identification of T-lymphocyte epitopes is performed by the automated calculation of sizes for the binding centres of class II MHC molecules with the subsequent experimental test of the obtained versions of protein for presence of the lowered adjuvanticity. The automated way of T-epitopes calculation is based on use of the Bjom's function modified in such manner that contribution of Van-der-vaals repulsion and lipophilic interaction in pairs between all lipophilic atoms of the chosen segments of the fused protein and a binding groove of a MHC P molecule is taken into account. Also a way of protein construction on the basis of the modified function Bjom's function with the subsequent experimental test of the received versions for presence of the lowered adjuvanticity is revealed, and also application of the fused protein for preparation of a pharmaceutical composition for tumour treatment is in addition considered.

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EFFECT: application of the invention provides low-immunogenicity antibody.

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