Monoclonal antibodies against activated protein c

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of immunology and biotechnology. Claimed is monoclonal antibody or its functional fragment, where said antibody and fragment bind with activated protein C and inhibit anticoagulant activity, but do not bind and do not inhibit activation of inactivated protein C, where said antibody is obtained by immunisation of mammal by APC and screening of binding ability of said antibody with APC, but not with protein C. Also described is pharmaceutical composition for treating diseases associated with anticoagulation activity of APC, including said antibody in effective amount and pharmaceutically acceptable carrier. Claimed are: method of inhibiting anticoagulation activity of activated protein C in subject, method of inhibiting amidolytic activity of activated protein C in subject, method of treating subject, requiring blood coagulation; method of treating subject with haemophilia; method of modulating haemostasis in subject; as well as method of modulating thrombogenesis in subject, which include introduction of effective quality of said antibody to subject. In addition, described is method of treating subject with sepsis, including introduction of effective quality of said antibody and activated protein C.

EFFECT: invention makes it possible to obtain monoclonal antibody or its functional fragment, where said antibody and fragment bind with activated protein C and inhibit anticoagulation activity, but do not bind and do not inhibit activation of inactivated protein C.

17 cl, 11 dwg, 6 ex

 

The present invention claims priority of the provisional application U.S. No. 60/983092, filed October 26, 2007, the entire contents of which is hereby incorporated into this description by reference.

The technical FIELD TO WHICH the INVENTION RELATES

The present invention relates generally to the field of antibodies. More specifically, the present invention describes the identification and use of monoclonal antibodies and fragments of antibodies that selectively directed against activated protein C (APC).

Description of the prior art

Blood clotting is a process consisting of a complex interaction of various blood components, or factors, which results in the formation of a fibrin clot. In General, blood components involved in the cascade of reactions of coagulation are proenzymes or simagename - enzymatically inactive proteins, which become active form under the action of the activator. Regulation of blood coagulation is mainly carried out by enzymatic proteolytic inactivation of factors procoagulative Va and VIIIa, achieved with activated protein C (APC) (Esmon, 1989).

Protein C is a predecessor of the APC, a potent natural anticoagulant. The protein C is activated by thrombin in complex with thrombomodulin (TM). Activation of usalive the Xia endothelial receptor protein (EPCR). TM and EPCR can be deactivated with the help of a mediator of inflammation, such as tumor necrosis factor considered Esmon (1999). Also found that TM and EPCR reduced in some forms of septic shock, in particular when meningococcemia. Because EPCR and TM are expressed on the endothelium, it is impossible to determine directly how they function without removing the blood vessels.

APC acts as an anticoagulant by proteolytic cleavage and down-regulation procoagulation factors. APC also performs important functions as an antiapoptotic agent, anti-inflammatory molecules and cytoprotective agent. Disorders of coagulation, in which the regulation of the hemostatic status disrupted as a result of losing a key factor, as, for example, the absence of factor VIII in hemophilia, or trauma patients, in those cases where the wound process leads to a temporary loss of hemostasis, can be treated by removing the APC. However, such treatment may result in harmful unwanted effects associated with the removal of favorable functions of APC in addition to the removal of anticoagulant action. It is therefore desirable to have a drug that selectively directed to the anticoagulant activity of APC and at the same time leaves the other functions of the molecule intact.

The ENTITY FROM WHOM RETENE

Created and described herein a method of treating disorders of coagulation, including the use of monoclonal antibodies that recognize the activated protein C, but does not recognize non-activated protein C. In this regard, the present invention also provides monoclonal antibodies that selectively bind and/or inhibit the proteolytic active site of activated protein C. These antibodies may inhibit the anticoagulant activity of activated protein C, but cannot affect any activity non-activated protein C in certain embodiments. These antibodies can also support the cytoprotective effects of activated protein C in certain embodiments. Thus, the methods of the present invention also include treatment with the use of these monoclonal antibodies, in cases where it is desirable to selectively inhibit the anticoagulant activity of activated protein C.

Accordingly, some General aspects of the present invention involve monoclonal antibody, where the specified antibody binds and inhibits activated protein C, but not bound or not inhibits non-activated protein C. for Example, certain embodiments of the present invention before the look monoclonal antibody, where the specified antibody binds and inhibits anticoagulant activity of activated protein C, but not bound or not inhibits the activation of non-activated protein C. In certain embodiments the monoclonal antibody of the present invention is a HAPC1573. Antibodies of the present invention can bind and inhibit activated protein C and its anticoagulant activityin vivoand/orin vitro.

Several other monoclonal antibodies of the present invention, such as an antibody that inhibits the binding of activated or non-activated endothelial protein C receptor protein (EPCR) or phospholipids and inhibits the activation of non-activated protein C. In certain aspects, the specified antibody bound to the Gla domain of non-activated protein C mouse. These antibodies can be used in contextsin vitroorin vivo.

The antibody of the present invention may constitute, for example, the antibody is a mouse antibody of the present invention may constitute, for example, humanitariannet antibody. The antibody of the present invention may be included in a pharmaceutical composition, where the pharmaceutical composition also includes a pharmaceutically acceptable carrier. The antibody of the present izobreteny which can also be used in ways where the antibody is in contact with the cellin vitroorin vivo.

The present invention provides a method of inhibiting the anticoagulant activity of activated protein C in a subject, comprising introducing an effective amount of the antibody of the present invention a specified subject (e.g., a mammal such as man). In the specified method or any other method of the present invention, which includes the introduction of the antibodies of the present invention, the cytoprotective effects of activated protein C in certain embodiments may be described or may remain within normal values.

Also provides methods of inhibiting amylolyticus activity of activated protein C in a subject, comprising introducing an effective amount of the antibody of the present invention the specified entity.

The present invention also provides a method of treatment of a subject that needs blood coagulation, comprising introducing an effective amount of the antibody of the present invention a specified subject. The specified entity may support, for example, hemophilia or haemorrhage.

In the present description also provides a method of treatment of a subject having sepsis, which includes the introduction of an effective amount of the antibodies is as the present invention. These methods can also include the introduction of activated protein C.

Also provides methods of treatment of a subject having hemophilia, which include the introduction of an effective amount of the antibody of the present invention.

Antibodies of the present invention can also be used, for example, in methods of modulating hemostasis in a subject or modulation of thrombosis in a subject, comprising introducing an effective amount of the antibody of the present invention. These methods can also include the introduction of activated protein C.

Certain methods of the present invention involve a method of inhibiting activation of non-activated protein C, which includes an introduction to the subject an effective amount of a monoclonal antibody of the present invention. Antibodies used in the above methods, can also inhibit the binding of activated or non-activated endothelial protein C receptor protein (EPCR) or phospholipids.

According to the present invention, the subject may represent, for example, a mammal such as a mouse, rat, rabbit, dog, horse or man.

If not stated otherwise, any antibody described herein, may be a fragment of the antibody. For example, the antibody EXT is niteline can be defined as Fab', Fab, F(ab')2, a single domain antibody, Fv or scFv, which are well-known types of fragments of antibodies. Unless otherwise specified, the antibody of the present invention also includes the fragments.

The term "antibody" is used to refer to any antibody like molecule that has an antigen-binding site, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single-chain Fv), etc. described later. Techniques for production and application of various designs based on the antibodies and fragments of antibodies are well known in the art. Means for receiving and characteristics of the antibodies is also well known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated into the description by reference).

Another aspect of the invention involves a variable area, which includes changing the hypervariable regions or CDRs and framework regions or " FR. CDR are sequences within the variable regions, which generally produce antigenic specificity.

The invention also encompasses antibody fragments that include the sequence of the variable segment, sufficient to provide antigen binding. Antibody fragments include, without ogran the treatment Fab, Fab', F(ab')2, Fv, SFv, scFv (single-chain Fv), obtained either by proteolytic cleavage of intact antibodies, such as cleavage by papain or pepsin, or by using recombinant methods in which affect cDNA intact heavy and light chains to obtain fragments of the heavy and light chains, either individually or as part of the same polypeptide.

Within the scope of the invention mAb also include sequences corresponding to the human antibodies, antibody animals and their combinations. The term "chimeric antibody" used in the description, includes antibodies that have variable plots obtained from the animal antibodies, such as antibodies rat or mouse, merged with another molecule, for example with a constant domains derived from human antibodies. One type of chimeric antibodies, "humanized antibody" has variable plots, modified through mutagenesis or CDR transfer) to match as possible known sequences of human variable regions. The CDR transfer includes the transfer of the CDRs of the antibodies with the desired specificity for FR human antibodies, with a significant portion of inhuman sequence replace the human sequence. Therefore, humanized antibodies are more suitable for testout (amino acid sequence) of the sequence of known human antibodies. By using humanization of murine monoclonal antibodies to reduce the severity of the response to human antimurine antibody, or HAMA. The invention further includes a fully human antibodies that would prevent, as far as possible, the answer to HAMA. Obtaining humanized antibodies are described in more detail later.

Used in the description of the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, agents that delay absorption, salts, preservatives, drugs, stabilizers medications, gels, binders, excipients, dezintegriruetsja agents, lubricants, sweeteners, flavorings, colorings, similar materials and combinations thereof, which shall be known to the person skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Unless any conventional carrier is incompatible with the active ingredient, provided its use in therapeutic or pharmaceutical compositions.

The term "contact", in relation to a cell, are used herein to describe the process by which the compound of the invention is delivered to the tap-hole of a target or is in direct contact with the cell-target.

The term "effective", as the term is used in the present description and/or claims (for example, "effective amount"), means sufficient to achieve the desired, expected or intended result.

The term "mainly" and its variants mean largely, but not necessarily all that obvious to a person skilled in the art, and in one non-limiting embodiment mainly refers to ranges within 10%, within 5%, within 1% or 0.5%

The terms "inhibiting," "reducing," or "prevention" or any variation of these terms, when used in the claims and/or in the description, include any measurable decrease or complete inhibition to achieve a desired result. For example, there may be a decrease in activity compared to the normal condition, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, or a decrease in the activity in any range derived from these values here.

In this application, the term "approximately" is used to indicate that a value includes fatal deviation of the error of the device or method used to determine the value, or deviation, which is among the subjects of the research. For example, "is roughly" may be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.

The term presented in the singular, if it is used in combination with the term "comprising" in the claims and/or description, can mean "one", but he also consistent with the value "one or more", "at least one" and "one or more than one."

The term "or" in the claims is used to mean "and/or", unless otherwise specified, the reference applies only to alternatives, or if the alternatives are mutually exclusive, although the disclosure of the invention supports a definition that refers to only alternatives and options "and/or".

Used in this description and in paragraph (paragraphs) of the claims, the words "comprising" (and any form of "consisting of", such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of “including”, such as "contains" and "contain" or "containing" (and any form of “containing”, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, not listed elements or stages of the method.

It is assumed that any embodiment discussed in the present description, may be susestudio in respect of any connection the method or composition of the invention, and Vice versa.

Other objects, features and advantages of the present invention become apparent from the subsequent detailed description. However, it should be understood that the detailed description and specific examples, which show the preferred embodiment of the invention, are given only for illustrative purpose, since various changes and modifications in the spirit and the invention will be obvious to those skilled in the art from this detailed description.

BRIEF DESCRIPTION of DRAWINGS

The following drawings form part of the present description and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG.1. A standard curve APC ELISA.

FIG.2. HAP1573 enhances the binding of APC to the endothelium.

FIG. 3. HAPC1573 promotes the internalization of APC in cell EA.

FIG.4. HAPC1573 changes amylolyticus APC activity towards the chromogenic substrate.

FIG.5. HAPCl573 blocks anticoagulant activity of APC in the analysis (coagulating activity of blood plasma.

FIG.6. HAPCl573 enhances cleavage of histone APC.

FIG.7A-B. E. the effect HAPC1573 on APC-cytoprotection against histones.

FIG.8A-C. MPC1609 and MAPC1591 inhibit the anticoagulant activity of APC. (FIG.8A) bEnd3 Cells were incubated with 100 nm FL-APC without or in the presence of 125 nm MPC1609 or MAPC1591 for 15 min on ice and were analyzed by flow cytometry. (FIG.8B) bEnd3 Cells were incubated with 100 nm protein C and 5 nm thrombin in the presence or without 100 nm MPC 1609 or MAPCl 591 for 15 min at 37°C and measured the activity of APC using a chromogenic substrate PCa. (FIG.8C) Time one-stage coagulation of blood plasma was measured with 200 ng/ml, without or in the presence of 5 μg/ml MPC1609 or MAPC1591. Tests the activation of protein s and coagulation was performed in duplicate, and all errors were within 5%.

FIG.9. MPC1609, but not MAPCl591 increased mortality in mice when introduced together with a sublethal dose of LPS. BL6 mice were administered an intravenous injection of 10 mg/kg LPS 10 mg/kg MPC1609 or MAPC1591 and recorded survival rates.

FIG.10A-D. body Temperature, concentrations in serum IL-6, urea and creatinine in mice after injection of LPS and MPC1609 or MAPCl 591. BL6 mice (4 mice in each group) were injected intravenously injection of saline, 10 mg/kg LPS or 10 mg/kg LPS 10 mg/kg MPC1609 or MAPC1591. (FIG.10A) the body Temperature of the mouse (FIG.10B), the concentration of serum IL-6 (FIG. 10C-D), urea and creatinine were measured after 3 hours or 18 hours after injection.

FIG.11A-C. MAPC1591 enhances APC-resplendissant. (FIG.11A) 100 mg/ml of histone H3 calf thymus (left panel) or H4 (right panel) were incubated in Opti-MEM with or without added 100 nm APC, without or in the presence of 200 nm MAPC1591 for 1 hour at 37°C. Then carried out the analysis of samples by electrophoresis in LTO-SDS page with Coomassie blue staining blue. (FIG.11B) Cells EA.hy926 were cultured with histones calf thymus (50 µg/ml) without or in the presence of APC (100 nm) and MAPC1591 (200 nm) for 1 hour at 37°C. the Number of dead cells was measured using flow cytometry on positive staining with PI (FL3). (FIG.11C) BL6 Mice were administered an intravenous injection of saline, 10 mg/kg LPS or 10 mg/kg LPS 10 mg/kg MAPC1591 or MPC1609. Plasma samples were obtained after 18 hours after injection and were analyzed by electrophoresis LTOs-SDS page and Western blotting using goat antibodies against histone H3.

DESCRIPTION of ILLUSTRATIVE EMBODIMENTS

The present invention relates to the disclosure of monoclonal antibodies that selectively bind to activated protein C, but not associated with neaktivirovannye protein, and specifically inhibit the anticoagulant activity of activated protein C. These and other aspects of the invention are described in detail below.

A. the Structure of antibodies

Antibodies constitute a large family of glycoproteins with in common with nocturnum features. The antibody consists of four polypeptide, which form a three-dimensional structure, which is similar to the letter Y. Typically, the antibody consists of two different polypeptide heavy chain and light chain. The antibody molecule consists of one or more Y-units, each unit of Y consists of two heavy chains and two light chains.

The antibody molecule typically consists of three functional domains: Fc, Fab and antigennegative plot. The domain Fc is located at the base of the Y. the “Shoulders” Y contain Fab domains. Antigennegative the site is located on the end of each “shoulder”. The area of the fulcrum shoulders Y represents the hinge area.

There are five different types of polypeptide heavy chains, called α, δ, ε, γ and μ. There are two different types of polypeptide light chains, denoted by κ and λ. The antibody typically is composed of only one type of heavy chain and only one type of light chain, although any light chain can connect with any heavy chain.

Carboxyl end of the polypeptide each heavy chain is called the constant (Fc) region. Aminocore polypeptide of each heavy and light chain is called variable (V) region. Inside chain variable regions are hypervariable sites, called plots, complementarity determining (CDR). Variable plots one heavy the ETUI and one light chain are connected with the formation of antigennegative plot. Each heavy chain and each light chain comprises three CDRs. Six CDR antigennegative plot identify amino acid residues that form the actually existing binding site of the antigen. The CDR variability causes a variety of recognized antigens.

B. Obtaining monoclonal antibodies of the present invention

The present invention relates to the preparation and application of molecules that are capable of specific binding with another molecule. In the present description the molecule referred to as capable of specific binding with another molecule if the specified binding depends on the respective structures of molecules. Known for the ability of antibodies to contact the immunogen is an example of a "specific binding". These interactions are the opposite of nonspecific binding, which involved classes of compounds regardless of their chemical structure (for example, the protein binding of the nitrocellulose, and so on) Most preferably, the antibody of the present invention will demonstrate "specific binding" such that the antibody is incapable or substantially incapable of binding of cognate heterologous molecules. Indeed, preferred monoclonal antibodies of the present image is the shadow demonstrate the ability to bind to activated protein C, but mostly not able to contact neaktivirovannye protein C. In other embodiments the monoclonal antibodies specifically inhibit only the anticoagulant activity of APC by binding and blocking the proteolytic active site of APC.

Thus, in one embodiment, these molecules include fragments such as F(ab'), F(ab')2), which are obtained, for example, by proteolytic cleavage mAb, or single-chain immunoglobulins obtained, for example, by recombinant methods. These derived antibodies are monovalent. In one embodiment, these fragments can be connected with each other or with other fragments of the antibodies or receptor ligands, with the formation of "chimeric" binding molecules. Such chimeric molecules largely may contain substituents capable of binding to different epitopes of the same molecule, or they may be capable of binding to the epitope of activated protein C epitope "non-activated protein C".

Monoclonal antibody can be easily obtained by well known methods such as the methods presented in example in U.S. patent 4196265, which is included in the description by reference. Typically the method includes the first immunosorbant right W the animal with a selected antigen (e.g., polypeptide or polynucleotide of the present invention), in a manner sufficient to provide an immune response. Rodents, such as mice and rats are preferred. The spleen cells of immunized animal are then combined with the cell immortalizing myeloma cell lines. If the immunized animal is a mouse, preferred myeloma cell line is a cell line NS-I myeloma mice.

Merged cells “spleen/myeloma” cultured in selective medium to separate merged cells “spleen/myeloma” from the parent cells. Merged cells isolated from the mixture Nikitich parent cells, for example, by adding agents that block the synthesis of nucleotidesde novoin the environment of tissue culture. Typical and preferred agents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block the synthesis ofde novopurines and pyrimidines, whereas azaserine blocks only purine synthesis. If you use aminopterin or methotrexate, Wednesday complement gipoksantin and thymidine as a source of nucleotides. If you use azaserine, Wednesday complement gipoksantin.

Specified cultivation provides a hybrid population from which select specific hybridoma. Usually, selecting the hybridomas is performed by culturing cells from the breeding of one clone in titration microplate, with further testing supernatants individual clones on the reactivity with the antigen polypeptides. Selected clones can then be propagated indefinitely to obtain monoclonal antibodies.

As a concrete example, in order to obtain a monoclonal antibody to mice injected intraperitoneally approximately 1-200 μg of antigen containing polypeptide. B-lymphocytes are stimulated to growth by the introduction of an antigen together with an adjuvant, for example with complete adjuvant's adjuvant (a non-specific stimulator of the immune response containing killedMycobacterium tuberculosis). At some point (for example, two weeks) after the first injection, mice repeatedly subjected to immunization by injection of the second dose of antigen mixed with incomplete adjuvant-blockers.

A few weeks after the second injection in mice are collecting blood from the tail vein and serum titrated using the method thus with isotope-labeled antigen. Preferably, the process of re-immunization and titration is repeated until reaching a suitable title. Allocate the spleen of the mouse with the highest titer and spleen lymphocytes obtained by homogenizing the spleen with a syringe. Usually the spleen of immunized animal contains approximately from 5×10 to 2×10 lymphocytes.

Mutant lymphocytic cells, known the nye as the myeloma cells, receive from laboratory animals, which induce the growth of these cells using a number of well known ways. The myeloma cells do not have "reutilization" pathway of biosynthesis of nucleotides. As the myeloma cells are tumor cells, they can grow indefinitely in tissue culture and are therefore called immortal. There are numerous cell lines myeloma cells of mice and rats, for example, myeloma cells of mouse NS-I.

The myeloma cells are combined under conditions conducive to merge with normal antibody-producing cells obtained from the spleen of a mouse or rat, which introduced the antigen/polypeptide. Conditions of the merger include, for example, the presence of polyethylene glycol. The resulting fused cells are hybridoma cells. Like the myeloma cells, the cells hybridoma can grow in culture indefinitely.

Hybridoma cells secrete from Nikitich myeloma cells by culturing in a selective medium, such as medium HAT (gipoksantin, aminopterin, thymidine). Naslite myeloma cells do not have the enzymes necessary for the synthesis of nucleotides by "reutilization" way, because they die in the presence of aminopterin, methotrexate or azaserine. Naslite lymphocytes also can't continue to grow in tissue culture. T is thus, only cells that have successfully merged (hybridoma cells), can grow in the selective medium.

Each of the surviving hybridoma cells producing a single antibody. This was followed by the screening of these cells for the production of specific antibodies immunoreactive against the antigen/polypeptide. By the serial dilution method produce separate hybridoma. Hybridoma serially diluted many times, after dilution leave for growth, the supernatant is tested for the presence of monoclonal antibodies. Clones producing the specified antibody, then cultivated in large quantities in order to obtain the antibody in an appropriate quantity.

Liaw et al. (2003) included in the present description in its entirety by reference, describes methods of obtaining specific mouse monoclonal antibodies against activated and non-activated protein C human.

If antibodies or fragments thereof are used for therapeutic purposes, it may be desirable to "humanize" them, in order to mitigate any immune response. These humanized antibodies can be studied in the context ofin vitroorin vivo. Humanized antibodies can be obtained, for example, by replacing the immunogenic part of the antibodies to the corresponding, but non-immunogenic portion (i.e., chimeric antibodies). Robinson et al., the patent application U.S. is under PCT PCT/US86/02269; Akira et al., European patent application 184187; Taniguchi, European patent application 171496; Morrison et al., European patent application 173494; Neuberger et al., International patent application under PCT WO 86/01533; Cabilly et al., European patent application 125023; Better et al. (1988); Liu et al. (1987); Liu et al. (1987); Sun et al. (1987); Nishimura et al. (1987); Wood et al. (1985); Shaw et al. (1988); which are incorporated in the description by reference. General reviews of "humanized" chimeric antibodies are provided by Morrison (1985) and Oi et al. (1986); these reviews are included in the description by reference).

Suitable "humanized" antibodies alternative can be obtained by replacing the CDR or CEA. Jones et al. (1986); Verhoeyan et al. (1988); Beidler et al. (1988); all these documents are included in the description by reference.

D. Pharmaceutical compositions

The pharmaceutical compositions of the present invention include an effective amount of one or more antibodies, therapeutic agents or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. Aqueous compositions of the present invention include an effective amount of the antibody, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The expression "pharmaceutically or pharmacologically acceptable" refer to molecular substances and compositions that are not in the home side, allergic or other adverse reaction with the introduction of the animal or the person appropriately.

Used in the description of the term "pharmaceutically acceptable carrier" includes any or all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, agents that delay absorption, salts, preservatives, drugs, stabilizers medications, gels, binders, excipients, dezintegriruetsja agents, lubricants, sweeteners, flavorings, colorings, similar materials and combinations thereof, which shall be known to the person skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329 included in the present description by reference). The application of such media and agents for pharmaceutical active substances is well known in the art. The use of any conventional medium or agent in the composition of therapeutic compositions is considered only in those cases where they are not incompatible with the active ingredient. Supplementary active ingredients can also be included in the composition. For the introduction of human drugs must meet the standards of sterility, progenote, General b the safety and purity according to the service requirements of the FDA for biological standards.

Biological material must be carefully dialserver to remove unwanted molecules with a low molecular weight and/or lyophilized for more easy cooking drugs in the desired media, when appropriate. Then active compounds typically include in compositions for parenteral administration, for example, include in the composition for injection, intravenous, intramuscular, subcutaneous, intranasal, vnutrenniy or even intraperitoneal route. Typically, such compositions can be prepared in the form of drugs for injection or in the form of liquid solutions or suspensions; can also be prepared in solid form, easy to prepare solutions or suspensions upon the addition of a liquid prior to injection of a; and the preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or suspensions; compositions containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for extemporanea preparation of sterile injectable solutions or dispersion systems. In all cases, the dosage form must be sterile and must be fluid, provided that there is the ability to easily introduce Lekarstvo the th form via syringe. Dosage form should be stable under conditions of manufacture and storage and must be protected from contaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free bases or pharmacologically acceptable salts can be prepared in water, respectively, mixed with a surfactant, for example, hydroxypropylcellulose. The dispersion system may also be prepared in glycerol, liquid polyethylene glycols and their mixtures and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

Antibodies of the present invention can be included in the composition in a free base form, in neutral form or in salt form. Pharmaceutically acceptable salts include acid salts of accession (formed with free amino groups of protein and salts, which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, almond acid, etc., Salts formed with the free carboxyl groups can also be derived from inorganic bases, such as, for example, hydroxides of sodium, potassium, ammonium, calcium or iron hydroxide, and so is x organic bases as Isopropylamine, trimethylamine, histidine, procaine, etc.,

The media can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol, and so on), suitable mixtures and vegetable oil. The proper fluidity can be maintained, for example, by applying a coating such as lecithin, by storing the required particle size in the case of disperse systems and through the use of surfactants. Protection from the action of microorganisms can be carried out using various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc., In many cases it is preferable to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be made using the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions prepared by combining the active compounds in the required amount in the appropriate solvent with various other ingredients listed above, as required, followed by sterilization by filtration. In General, the dispersed system is obtained by introducing again the ranks sterilized active ingredients into a sterile medium, which contains the basic dispersion medium and other necessary ingredients from gradients above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are drying in vacuum or freeze-thaw, which results in a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution. Also provides for the preparation of more, or highly concentrated solutions for direct injection in cases where the use of DMSO as solvent, resulting in a very rapid penetration with delivery of high concentrations of active agents in a small area.

After creating the solutions can be introduced in a way appropriate dosage of the composition and in an amount that is therapeutically effective. The compositions are easily entered into various dosage forms, such as the type of injectable solutions described above, but can also be used capsule with the release of medicines and so on

For parenteral administration in an aqueous solution, for example, the solution should be properly buffered if necessary and the liquid solvent must be given in advance in isotonic the mechanical condition sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intranasal and intra-abdominal injection. In this connection, sterile aqueous medium, which may be used is known to the specialist in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of liquid for subcutaneous infusion, or injected at the proposed location of the infusion (see, for example, "Remington''s Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Definitely going to be some variation in dosage depending on the condition of the subject who receives the treatment. Specialist, responsible for the introduction of drugs, in any case, will determine the appropriate dose for a specific subject.

In addition to the compounds in compositions for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solid forms for oral administration; liposomal composition; capsules with prolonged action; and any other form currently used, including creams.

In certain embodiments provided by the use of liposomes and/or nanoparticles for photomediarecovery forms and the introduction of antibodies and/or their analogues. The formation and use of liposomes in General known to the specialist in the art and are also described below.

Nanocapsules, basically, can capture compounds are stable and repeatable manner. To avoid side effects due to intracellular polymeric overloading, these ultramercial particles (approximately 0.1 μm) should be developed using polymers able to be destroyedin vivo. Biodegradable polyalkylacrylate nanoparticles that meet these requirements are provided for use in the present invention, and these particles are easily obtained.

Liposomes are formed from phospholipids that are dispersed in water, they spontaneously form a multilayer concentric bilayer vesicles (also called multilamellar vesicles, (MLVs)). MLV typically have a diameter from 25 nm to 4 μm. Sonication of MLV leads to the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200-500 Å, containing in the centre of the aqueous solution.

The following information can also be used in creating the liposomal compositions. Phospholipids can form various patterns in addition to liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low soo is wearing liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength and presence of divalent cations. Liposomes may exhibit low permeability to ionic and polar substances, but at higher temperatures they undergo a phase transition, which significantly alters their permeability. The phase transition involves changing tightly Packed, ordered structure, known as a gel-like state, loosely Packed, disorganized structure, known as the liquid state. This process occurs at a characteristic temperature of the phase transition and results in increased permeability to ions, sugars and drugs.

Liposomes interact with cells of four different mechanisms: endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by using nonspecific weak hydrophobic or electrostatic forces, or through specific interactions with components of the cell surface; fusion with the plasma cell membrane by inclusion of a lipid bilayer of the liposome into the plasma membrane, with simultaneous release of the contents of liposomes in the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular m is mpany, or, on the contrary, without any merge the contents of the liposomes. By varying the composition of the liposomes, it is possible to change the existing mechanism, while at the same time can work more than one mechanism.

therapeutic agent may include various types of media, depending on, whether he is introduced in solid, liquid or aerosol form, and whether it should be sterile for such routes of administration as injection. The composition according to the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, into the wound, intracranial, inside the joint, inside the prostate, vnutriplevralno intratrahealno, intranasal, intravitreal, vnutrivaginalno, intrarectal, locally, intratumorally, intramuscularly, subcutaneously, podklyuchenie, intravesicular, through the mucous membranes, intrapericardial, through the umbilical cord, orally, topically, locally, inhalation (e.g. aerosol inhalation), by injection, infusion, continuous infusion, localized perfusion, directly surrounding the target cells, through the catheter, through irrigation, creams, in lipid compositions (e.g., liposomes), or by using other methods or any combination of the methods mentioned above, as should be known to the person skilled in the art (see, for example, Remington's Pharmaceutical Scienes, 18th Ed. Mack Printing Company, 1990, included in the description by reference).

The actual size of the dosage of the composition of the present invention, introduce the animal to the patient, can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy the patient and the route of administration of the composition. The practitioner responsible for the introduction, in any case will be to determine the concentration of the active ingredient (ingredients) in the composition and the dose (the dose) that is designed for each specific subject.

In certain embodiments the pharmaceutical compositions can include, for example, at least about 0.1% of active compound. In other embodiments, the active compound may range from about 2% to about 75% by weight of one unit dosage, or, for example, from about 25% to about 60%, and any range derived from these values. In other non-limiting examples, a dose may also comprise from about 1 micrograms/kg body weight, about 5 microgram/kg / body weight, about 10 microgram/kg / body weight, about 50 microgram/kg / body weight, about 100 micrograms/kg of body weight, will bring the flax 200 micrograms/kg of body weight, approximately 350 micrograms/kg body weight, about 500 microgram/kg / body weight, about 1 mg/kg body weight, about 5 mg/kg body weight, about 10 mg/kg body weight, about 50 mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kg body weight, about 350 mg/kg body weight, about 500 mg/kg body weight to about 1000 mg/kg body weight or more per medication, and any range, derived from these values. In non-limiting examples of the range derived from these values, the dose may be entered in the range from about 5 mg/kg body weight to about 100 mg/kg body weight, from about 5 micrograms/kg body weight to about 500 mg/kg body weight, and so on, based on the values described above.

In any case, the composition may include a variety of antioxidants to reduce the oxidation of one or more components.

In embodiments where the composition is provided in liquid form, the carrier can be a solvent or dispersion medium containing, without limitation water, ethanol, polyol (e.g. glycerol, propylene glycol, liquid polyethylene glycol, and so on), lipids (e.g., triglycerides, vegetable oils, liposomes), and combinations thereof. Appropriate fluidity can be maintained, for example, by applying a coating such as lecithin, by storing the desired particle size by using the dispersion in the medium, such as, for example, liquid polyol or lipids; using surfactants, such as, for example, hydroxypropylcellulose; or combinations thereof. In many cases it is preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

In other embodiments of the present invention can be applied eye drops, nasal solutions or sprays, aerosols or inhalers. These compositions typically develop compatible with the type of the target tissue. In a non-limiting example, nasal solutions are generally aqueous solutions, designed for introduction into the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects with nazalnam secret in order to maintain the normal operation of the ciliary apparatus. Thus, in preferred embodiments the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH from about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to the preservatives used the eye medicines medications or appropriate stabilizers medications, if necessary, can be included in the composition. For example, there are various commercial nasal preparations, and they include medications such as antibiotics or antihistamines.

In certain embodiments the antibody is prepared for insertion through such intake path, as oral ingestion. In these embodiments, the solid composition may include, for example, solutions, suspensions, emulsion, tablets, pills, capsules (e.g. hard or soft coated gelatin capsules), composition with delayed release, buccal compositions, lozenges, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions can be incorporated directly with the food of the diet. Preferred carriers for oral administration include inert diluents, digestible edible carriers or combinations thereof. In other aspects of the invention, the oral composition may be prepared in the form of a syrup or elixir. A syrup or elixir may include, for example, at least one active agent, sweetener, preservative, flavoring, dye, or combinations thereof.

In certain preferred embodiments of the oral composition may VK is ucati in one or more binders, fillers, dezintegriruetsja agents, lubricants, flavors, and combinations thereof. In certain embodiments the composition may include one or more of the following: a binder, such as, for example, tragacanth gum, acacia gum, corn starch, gelatin or combinations thereof; excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate or combinations thereof; desynchronosis agent, such as, for example, corn starch, potato starch, alginic acid, or combinations thereof; a lubricant, such as magnesium stearate; sweetening matter, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring, such as peppermint, Wintergreen oil, cherry flavoring, orange flavoring, and so on; or a combination of the above. If the standard dosage form is a capsule, it may contain, in addition to materials of the above type media, such as a lipid carrier. Various other materials may be provided as a coating or modification otherwise the physical form of a unit dosage. For example, tablets, pills or capsules can be coated with shellac, sugar or them about Jimi.

Additional compositions that are suitable for routes of administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually impregnated drug for introduction into the rectum, vagina or urethra. After the introduction of suppositories soften, melt or dissolve in the abdominal fluid. In General, suppositories, common carriers may include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In certain embodiments suppositories can be formed from mixtures containing, for example, the active ingredient in the range of from about 0.5% to about 10% and preferably from about 1% to about 2%.

The composition should be stable under conditions of manufacture and storage and must be protected from contaminating action of microorganisms, such as bacteria and fungi. It should be understood that the endotoxin contamination should be kept at the minimum safe level, for example less than 0.5 ng/mg protein.

In specific embodiments prolonged absorption of injectable compositions can be made using the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin, or a combination of them.

E. Sets

Any compositions described herein can be included in the set. Sets, thus, will include antibody and/or an additional agent of the present invention in a suitable container device. The authors of the present invention include other components that may be included in the set. Therapeutic kits of the present invention include in a suitable container device pharmaceutically acceptable composition antibodies in a pharmaceutically acceptable composition. A set can have one container device and/or can have a separate container device for each connection.

If the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution is particularly preferred. The antibody may also be included in the composition is introduced by syringe, and in this case, as such may be a syringe, pipette, and/or other similar device, from which the composition may be applied on the affected area of the body, injected animal and/or even applied to and/or mixed with other components of the set.

However, the components of the kit may be provided in the form of dry powders (powders). If the reagents and/or components are provided in VI is e dry powder, the powder can be dissolved by adding a suitable solvent. It is envisaged that the solvent may also be provided in another container device.

The container device typically includes at least one vial, test tube, flask, bottle, syringe and/or other container device, which is placed antibody/composition of the antibodies, preferably, respectively placed. The kits can also include a second container device, which comprises a sterile, pharmaceutically acceptable buffer and/or other diluent.

The kits of the present invention also include a device to hold the bubbles in a closed protective casing for commercial sale, such as, for example, injection and/or made of blow-molded plastic containers in which to store the desired bubbles.

Regardless of the number and/or type of containers, the kits of the invention also may include and/or be packaged together with an auxiliary tool for the injection/administration and/or placement of the resulting antibodies inside the body of the animal. Such an instrument may be a syringe, pipette, forceps and/or any such medical approved tool for drug delivery.

F. Examples

The following examples are included, that is to demonstrate the preferred embodiment of the invention. Specialists in the art should understand that the methods disclosed in the examples that follow represent techniques disclosed by the inventor for the proper functioning of the invention in practice, and thus can be considered as representing the preferred modes of carrying out the invention in practice. However, specialists in the art should be understood in the light of this disclosure that numerous changes may be made in the specific embodiments that are disclosed and received the same or similar result without violating the essence and inventions.

EXAMPLE 1. Methods of screening, identification and application of human MAb of the present invention

Materials. Human protein C, bovine thrombin was obtained as described (Esmon et al., 1993; included in the description in its entirety by reference).

Recombinant APC (Xigris) was obtained from Eli Lilly. Spectroxyme PCa were obtained from American Diagnostica. 1-Palmitoyl-2-oleoyl-phosphatidylcholine (PC), 1-Palmitoyl-2-oleoyl-phosphatidylserine (PS) and 1-Palmitoyl-2-oleoyl-phosphatidylethanolamine (PE) were obtained from Avanti Polar Lipids, Inc. Endothelial cells line EA.hy926 were maintained in DMEM (modified by way of Dulbecco Wednesday Needle), supplemented with 10% fetal calf serum, L-glutami the om and HAT (gipoksantin, aminopterin, thymidine). Labeled with APC fluorescein (FL-APC) were obtained using the kit for labeling of proteins Fluorescein-EX from Molecular Probes according to the manufacturer's instructions. In these examples, "protein C" without defining "activated" refers to to nonactivated protein C.

Getting mouse antibodies against a protein With human and APC monoclonal antibodies. Mouse monoclonal antibody (mAb) against human protein C or APC were obtained using standard techniques (Rezaie and Esmon, 1992).

Screening specific mAb to protein C or APC. Monoclonal antibody to human protein C mAb 1575 and 1580 (HPCl575 and HPCl580) was obtained by screening the blocking ability of mAb when linking FL-APC cells EA.hy926 using FACS analysis. Briefly, cells EA.hy926 cells were then incubated with 50 nm FL-protein C and 100 nm of different monoclonal antibodies against protein C in HBSS buffer (balanced salt Hanks solution) containing 0.5% BSA, 3 mm CaCl2and 0.6 mm MgCl2within 30 min on ice and performed the FACS analysis. Antibody against human APC, mAb1573 (HAPC1573) was obtained by screening of binding properties of mAb with APC, but not with protein C, using ELISA method. Briefly, 96-well plate MaxiSorp (NUNC) was applied 5 μg/ml of various mAb in the buffer 15 mm Na2CO3, 35 mm NaHCO3pH of 9.6 was left overnight at 4°C. the Tablet was washed with a solution of TTBS (TBS containing the s 0,05% Tween-20), containing 1 mm CaC12(TTBS-calcium buffer), blocked with 0.1% gelatin in TBS (20 mm Tris-HCl, 150 mm NaCl, pH 7.5) for 1 hour, again washed TTBS-calcium buffer, incubated with 100 ng/ml of protein C or APC in TTBS-calcium buffer for 1 hour. After washing the TTBS-calcium buffer tablet incubated with 2 μg/ml biotinylated HPC1580 for 1 hour, again washed TTBS-calcium buffer, incubated with 1 μg/ml conjugate streptavidin-alkaline phosphatase in TTBS-calcium buffer for one hour. The absorbance at the end point at 405 nm was read using a microplate reader production Vmax after the final washing TTBS-calcium buffer and add the liquid to the substrate p-nitrophenyl phosphate (Sigma).

Method ELISA for determination of APC in plasma. The method was modified based on a previously described ELISA method for screening mAb against APC. Briefly, the tablet was applied 5 μg/ml HAPC1573, blocked TBS containing 1X casein (Vector Lab), and again washed TTBS-calcium buffer. Contributed recombinant APC in concentrations of 0-8 ng/ml in TTBS containing 10 mm benzamidine, 1 mm EDAT and 0.25 X casein buffer (buffer for breeding), or in a dilution of human plasma 1:4 and incubated the samples in tablet within one hour. After washing the TTBS-calcium buffer tablet incubated with 1 μg/ml biotinylated HPCl575 in TTBS containing 10 mm benzamide is a, 5 mm CaCl2and 0.25 X casein buffer for one hour. After washing the TTBS-calcium buffer tablet incubated with 0.5 μg/ml of streptavidin-HRP in TTBS containing 10 mm of benzamidine, 5 mm CaCl2and 0.25 X casein buffer for one hour, again washed TTBS-calcium buffer and staining was performed with the substrate Ultra-TMB (Pierce). OD450 was determined after addition of 0.5 M H2SO4to stop the enzymatic reaction with horseradish peroxidase.

The effect of mAb on the binding of FL-APC on the endothelium. Cells EA.hy926 cells were then incubated with 50 nm FL-APC in HBSS buffer containing 0.5% BSA, 3 mm CaCl2and 0.6 mm MgCl2without and in the presence of HAPCl573 or HPCl575 in various concentrations for 30 min on ice and performed the FACS analysis.

The effect of mAb on the anticoagulant activity of APC in the analysis of the coagulating activity of blood plasma. The effect of mAb on the anticoagulant activity of APC in plasma were determined using a modified one-stage analysis of coagulation of blood plasma with factor Xa using coagulometer ST4 (Diagnostica Stago). In the standard analysis in normal human pulirovaniya plasma was added to a set number of X-CP, factor X-activating enzyme from the venom of vipers Russell, to obtain a 30-second clotting time in a mixture of phospholipid vesicles (final concentration 10 μg/ml of 40% PE, 20% PS and 40% PC, masses/about) and CaCl22. Before adding CaCl2added APC (final concentration 200 ng/ml) or HAPC 1573 (final concentration 20 μg/ml).

The effect of mAb on amylolyticus APC activity towards the chromogenic substrate. Amylolyticus activity 50 μl of 10 nm APC in HBSS buffer (HBSS containing 0.1% bovine serum albumin, 3 mm CaCl2, 0.6 mm MgCl2) without or in the presence of 66.7 nm HPC1555 or HAPC1573 was determined by adding 50 μl of 0-2 mm Spectrozyme PCa, serially diluted in 50 mm HEPES buffer, 100 mm NaCl, pH 7.5.

Analysis of cytotoxicity of histone. Cells EA.hy926 cells were then incubated with histone calf thymus H3 or H4 (Roche) without and in the presence of 100 nm APC and 200 nm HAPC1573 in the medium Opti-MEM (Invitrogen) for 1 hour at 37°C and then for 5 min at room temperature after addition of 10 μg/ml of iodide of propecia (PI). Cells were washed and separated using EDTA/PBS and were analyzed by flow cytometry for PI-positive staining.

EXAMPLE 2.The results of the screening, identification and application of human MAb of the present invention

HAPC1573 enhances the binding of APC to the endothelium. To check whether HAPC 1575 any effect on APC binding to the endothelium, the inventors incubated the cells of the EA.hy926 with FL-APC without and in the presence of HAPC1573 or HPC1575 and measured the binding of FL-APC on ledah using the method of flow cytometry. The histogram data analysis flow cytometry showed that HAPC1573 increased the binding of FL-APC on endothelial cells, whereas HPC1575 inhibited the binding of FL-APC cells (FIG.2). HAPC1573 promotes the internalization of APC on the endothelium. FL-APC could be internalized into cells EA.hy926 due to the interaction of GIa-domain APC and EPCR on the cells, and this internalization could be blocked by either EPCR-blocking Ab (JRK1494) or a domain GIa-blocking Ab (HPCl575) (FIG.3). HAPCl573 could contribute to the internalization of the FL-APC in cells, and this effect could be completely blocked EPCR-blocking Ab (FIG.3).

HAPC 1573 changes amylolyticus APC activity towards the chromogenic substrate. Because HAPC1573 recognize APC on the tablet in the ELISA method and the endothelial cell, the inventors were interested in whether HAPC1573 affect amylolyticus APC activity towards the chromogenic substrate. Synthetic peptide substrates are typically small molecules with a molecular weight of several hundred daltons, most antibodies against serine proteases in plasma have a minor effect on the enzymatic activity of these small substrates. However HAPC1573 dramatically altered kinetic parameters APC for its chromogenic substrate Spectrozyme PCa (FIG.4). The km value for APC Spectrozyme PCa pillar is about 15 nm in the presence of HAPC1573, compared to 270 nm without Ab or in the presence of HPC1555. The value of kcat for APC Spectrozyme PCa was 18 in the presence of HAPC1573, compared to 67 without Ab or in the presence of HPC1555. This pronounced change in the activity of APC in relation to a small peptide substrate in the presence of HAPC1573 shows that this mAb recognizes an epitope located near the active site of APC, and the interaction between Ab and antigen dramatically increased the affinity of APC for a small peptide substrate, but reduced the rate of dissociation of the product from the catalytic site of APC.

HAPC1573 blocks anticoagulant activity of APC in plasma. In FIG.5 shows that HAPC1573 almost completely reduces the prolonged action of APC in the analysis of one-stage coagulation of blood plasma initiated by factor XA, suggesting that the interaction HAPC1573 and APC inhibits the APC cleavage of factor Va.

Influence HAPC1573 on the APC cleavage of extracellular histones.Recently, the inventors have found that APC can cleave extracellular histones and to protect the endothelium from the cytotoxicity of histone manuscripts prepared for publication). Because HAPCl573 changed amylolyticus APC activity by chromogenic substrate and blocked the anticoagulant activity of APC in plasma, the inventors were interested in whether mAb to change APC-cleavage of unclutch what's histones H3 and H4 and to influence cytoprotection APC on the endothelium against the cytotoxicity of histones H3 and H4. Surprisingly, HAPCl573 not inhibited, and really increased the APC cleavage of histone H3 and H4 (FIG.6). According, HAPC1573 not inhibited, but few have strengthened the cytoprotective effects of APC on endothelial against histone H3 and H4 (FIG. 7A-B).

EXAMPLE 3. Discussion of screening, identification and application of human MAb of the present invention

The protein C is activated by thrombin complex and thrombomodulin on the surface of the endothelium. In contrast to the very short time half-life of active thrombinin vivoaverage of a few seconds, the human APC has a half-life of about 20 minutes after its formation in the circulation (Berg, et al., 2003). Therefore, it is possible in practice to measure the level of APC in plasma in order to study its regulation in various pathological situations.

Existing methods of measuring APC-based capture enzyme, which is used antibody exciting APC, and then measure the activity using chromogenic substrates. Since all antibodies used in these analyses recognize not only the APC, but also his proferment, protein C, and since the concentration of protein C is approximately 1000 times higher than the concentration of APC in normal circulation, measuring APC using these methods are not clinically significant. Fast and reliable SP is a way to measure APC demand for diagnostics, and for a treat. The results presented above demonstrate that mAb, HAPC1573 recognizes APC, but not the protein, and demonstrates the creation of a convenient ELISA method for measuring the level of APC in plasmain vivo. Typically, the measurement in this way a single sample of blood plasma containing 1 ng/ml APC is less than 4 hours, compared to 19 hours or even weeks when measured using assays based capture enzyme (Gruber and Griffen, 1992; Liaw et al., 2003).

Recent studies have shown that anticoagulant activity of APC is optional for its cytoprotective function, but splitting activity of APC relative PARl may be required for its antiapoptotic actions (Mosnier et al., 2004). However, the cytoprotective effects of APC has been shown not only on endothelial cells, which Express EPCR, but also on other cells, such as neurons and keratinocytes, which does not Express EPCR on their cell surface (Guo et al., 2004; Berg et al., 2003), which indicates the possibility of existence of other mechanisms in addition to PARl-mediated signaling system APC. HAPC1573 changed splitting activity of APC toward chromogenic peptide substrate and also blocked the anticoagulant activity of APC in the analysis of the coagulating activity of blood plasma, suggesting that this mAb recognizes an epitope of the poison with the active center of the APC and changes its catalytic activity upon binding of the antigen-antibody.

On the other hand, HAPC1573 not inhibited, but actually increased the APC cleavage of extracellular histones and increased cytoprotective activity of APC on the endothelium against histones, which indicates that the anticoagulant activity of APC cleavage of activated factors V and VIII is not required for its cytoprotective activity in the cleavage of extracellular histones. Cleavage of extracellular histone-independent anticoagulant activity may be one of the molecular mechanisms of APC-regulation of inflammation and cytoprotective.

For example, HAPC1573 can be applied in the treatment of patients with hemophilia A. APC cleaves factor VIIIa and factor Va and, thus, negatively regulates blood clotting. In patients with hemophilia And the levels of factor VIII are low, and the inactivation of factor Va by using the APC is probably the main route of regulation of hemostasis and thrombosis in these patients. Recent clinical reports have shown that the Leiden mutation, in which the factor V is resistant to APC-cleavage was favorable for patients with hemophilia A in relation to the symptom of bleeding (van't Zant el. ah, 1997). Blocking anticoagulant activity of APC against factor Vain vivousing mAb, such as HAPC1573, may be an alternative approach in the treatment of hemophilia a, especially in those patients, who who have high levels of inhibitors of factor VIII, provided that substitution therapy of factor VIII had a special effect.

Other possible clinical application of HAPC1573 is used in the treatment of trauma patients with impaired hemostasis, possibly profuse bleeding, and surgical intervention lay for the recovery of the haemostatic status. Treatment HAPC1573 can selectively restore procoagulant state without deleting cytoprotective or anti-inflammatory activity of activated protein C.

Other possible clinical application of HAPC1573 is its combination with APC in the treatment of sepsis. Currently, APC is the only FDA approved drug for the treatment of severe sepsis. Its side effects, such as bleeding in patients due to the anticoagulant activity of APC. HAPC1573 blocked anticoagulant activity of APC and at the same time preserved and even strengthened cytoprotective effects of APC. This complex mAb-APC may be the best medicine, than used separately APC, whereas its side effects bleeding.

EXAMPLE 4. Methods of screening and use of mouse monoclonal antibodies of the present invention

Materials and methods. Recombinant mouse protein C, APC, rat mAb MPC1609 and MAPC1591 received in the laboratory ACC is accordance with standard techniques. Labeled with APC fluorescein (FL-APC) was obtained with the use of the kit for labeling of proteins Fluorescein-EX from Molecular Probes according to the manufacturer's instructions.

Research on animals. Male mice breed BL6 aged 6-12 weeks were used in this study in accordance with the Protocol approved by the Committee on institutional animal care and use medical and research Foundation of Oklahoma.

Cell culture. The bEnd3 cell line of endothelial cells of the brain of a mouse) were maintained in DMEM (modified by way of Dulbecco Wednesday Needle), supplemented with 10% fetal calf serum and L-glutamine. Cells EA.hy926 (line human vascular endothelial cells) were cultured in DMEM, supplemented with 10% fetal bovine serum, L - glutamine and HAT (gipoksantin, aminopterin, thymidine).

The binding of FL-APC and activation of protein C in the endothelium. The bEnd3 cells were incubated with 100 nm FL-APC in HBSS buffer containing 0.5% BSA, 3 mm CaCl2and 0.6 mm MgCl2without or in the presence of 125 nm MPC1609 or MAPC1591 for 15 min on ice and were analyzed by FACS method.

The effect of mAb on the activation of protein C on endothelial cells.The bEnd3 cells in 24-hole tablet was washed once with HBSS buffer (HBSS containing 0.1% bovine serum albumin, 3 mm CaCl2, 0.6 mm MgC12and preincubated is whether within 5 min with HBSS buffer, containing 0.1 μm of protein C and 0.1 μm MPC1609 or MAPC1591. The activation reaction was initiated by adding 5 nm bovine thrombin in a total volume of 0.2 ml Reaction was performed for 15 min at 37°C and stopped by adding 50 μl of bovine anti-thrombin III (1.67 mg/ml). Fifty μl of supernatant was transferred into a 96-well microplate and determined the rate of activation of protein C by using a tablet reader Vmax at 405 nm with 50 μl of 0.4 mm substrate Spectrozyme PCa buffer 100 mm NaCl, 50 mm HEPES-NaOH, pH 7.5.

Analysis of the anticoagulant activity of APC. The effect of mAb on the anticoagulant activity of APC in plasma was determined using a modified analysis of one-stage coagulation of blood plasma with factor Xa using coagulometer ST4 (Diagnostica Stago). In this analysis in plasma (50% mouse plasma and 50% normal human polerowanie plasma) was added to a set number of X-CP, factor X-activating enzyme from the venom of vipers Russell to get a 30-second clotting time in a mixture of phospholipid vesicles (final concentration 10 μg/ml of 40% phosphatidylethanolamine, 20% phosphatidylserine and 40% phosphatidylcholine, wt./about.) and CaCl2(6,35 nm) in buffer 20 mm Tris-HCl, 150 mm NaCl (pH 7.5) containing 0.1% BSA. The coagulation was initiated by addition of CaCl2. APC (final concentration 200 ng/ml) and MPC1609 (final concentration 5 μg/ml) or MAPCl591 (final concentration 5 µg/m is) was made before adding CaCl 2.

Analysis of cytotoxicity of histone. Cells EA.hy926 cells were then incubated with 50 μg/ml histone calf thymus (Sigma) without or in the presence of 100 nm APC and 200 nm MAPCl 591 in the medium Opti-MEM (Invitrogen) for 1 hour at 37°C and then for 5 min at room temperature after addition of 10 μg/ml of iodide of propecia (PI). Cells were washed and separated using 0,526 mm EDTA in PBS and were analyzed by flow cytometry for PI-positive staining.

Analysis of IL-6, urea and creatinine. In the serum of mice was determined by the concentration of urea and creatinine using a biochemical analyzer Vitros 250 (Ortho-Clinical Diagnostics). The level of serum IL-6 was measured using the Quantikine kit for carrying out colorimetric sandwich variant of the method of ELISA (R&D Systems).

EXAMPLE 5. The results of the screening and application of murine monoclonal antibodies of the present invention

MPC1609 was directed against protein C and APC and could inhibit the binding of protein C and APC on the endothelium (FIG.8A and data not shown). MAPC1591 was directed against the APC, but not against protein C, and could enhance the binding of APC to the endothelium (FIG.8A and data not shown). Activation of protein C on the endothelium was sharply decreased in the presence of MPC1609 (FIG.8B). MAPC1591 partly reduced activation of protein C, probably as a result of strengthening their APC binding to the cells (FIG.8B). Both antibodies MPC1609 and MPC1591 could completely inhibit the anticoagulant activity of APC in the analysis of the coagulating activity of blood plasma (FIG.8C). Based on these studies,in vitrothe inventors came to the conclusion that MPC1609 inhibited the binding of protein C and APC on the endothelium or phospholipid by masking Gla domain of protein C or APC, which is responsible for binding of protein C or APC on the endothelium or phospholipid. MAPC1591 recognize APC, but not protein C by interacting with an epitope located near the active site of APC, and this interaction is inhibited anticoagulant activity of APC, probably by preventing the APC cleavage of factor Va.

For studying the molecular mechanism for the protective effect of APC in LPS-induced septic shock, the authors of the invention was injected into mice, the injection of a sublethal dose of LPS with MPC1609 or MAPC1591, and they found that mice that were injected LPS and MPC1609, died within 48 hours, mice that were injected LPS and MAPC1591, survived (FIG.9). 18 hours after infusion in mice which were injected LPS and MPC1609 observed symptoms of severe septic shock, including low body temperature, high content of neutrophils and low lymphocytes and platelets in the peripheral blood (FIG.10A and data not shown). Serum level of IL-6 was very high in these mice (FIG.10B), but did not observe a significant difference in the mRNA levels of IL-6 in heart, lung, liver, spleen, thymus and peripheral blood data we who she is and mice, receiving the injection of LPS and MAPC1591 (data not shown), suggesting that the synthesis of IL-6de novocan be nonprincipal cause sustained elevated levels of IL-6 in septic shock in mice. Serum levels of urea and creatinine in mice that received LPS and MPC1609 were higher than other mice (FIG.10C-D), indicating acute renal failure in these mice, which may contribute to the low ground clearance of IL-6. Interestingly, the levels of urea and creatinine in mice who received the injection of LPS and MAPC1591 were even lower than these levels in mice, which was introduced only LPS (FIG.10C), suggesting that the complex MAPC1591 and APC may be more effective than APC to protect kidneysin vivoexperiment with LPS.

Extracellular histones were detected on activated neutrophils and macrophages and apoptotic cells (Brinkmann et al., 2004; It et al., 2004), and they were cytotoxic to mammalian cells (Abakushin et al., 1999; Currie et al., 1997; Kleine et al., 1997). APC could split the histone H3 and histone H4, and MAPC1591 not inhibited, but actually increased the APC cleavage of these histones (FIG.10A). APC could reduce the cytotoxicity of histones in relation to the endothelium, and MAPC1591 could reinforce specified cytoprotective activity of APC (FIG.11B). Extracellular histone actually found in septic mouse serum after a call is placed injections of LPS and MPC1609, but was not detected in mice that were injected with the injection of LPS or LPS and MAPC1591 (FIG.11C), indicating a correlation ofin vivobetween the lack of activation of protein C by the presence of extracellular histones in circulation and lethality of mice with septic shock. Taking into account the data receivedin vitroand the results obtainedin vivothe inventors were able to separate the cytoprotective activity of APC from its anticoagulant activity by using MAPC1591, and splitting cytotoxic histone APC regardless of its anticoagulant activity provides a new molecular mechanism of action of APC, preventing the mice to the development of LPS-induced septic shock.

EXAMPLE 6. Discussion screening and application of murine monoclonal antibodies of the present invention

The path of the exchange protein C plays an important role in regulating blood clotting and inflammation (Esmon, 2006). It is shown that human APC significantly reduces mortality in severe sepsis, and APC was approved as the first drug for the treatment of severe sepsis (Bernard et al., 2001). However, the molecular mechanism of the protective effects of APC in sepsis is still poorly understood. The mutagenesis study showed that the anticoagulant activity of APC was apparently not essential for the antiapoptotic actions of APC on endothelial the entrances (Mosnier et al., 2004), and anti-inflammatory and antiapoptotic effects of the signaling system APC were mediated by protease-activated receptor 1 (PAR-1) in endothelial cells (Reiwald et al., 2002). However, mouse, deficient in PAR-1, had a similar phenotype with the control wild-type mice during the experiment with LPS, suggesting that PAR-1 may not be a major participant for APC in the regulation of inflammation and cytoprotectivein vivo(Pawlinski et al., 2004; Camerer 2006). Considering the Central role of APC in the regulation of physiological functionsin vivothe inventors have developed two mAb against mouse protein C and murine APC and used these two mAb to explore poorly understood mechanisms of the protective effect of APC in LPS-induced septic shock in mice.

All compositions and/or methods disclosed in the description and included in the claims, can be made and implemented without special experiments in the light of the present disclosure. Although the compositions and methods of the present invention is described on the example of the preferred embodiments, specialists in the art should be obvious that there may be changes in the composition and/or methods and in the steps or in the sequence of stages of the method described herein without violating the concept, sushnost the and scope of the invention. In more detail, it is apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while can be obtained the same or similar results. It is implied that all such similar substitutes and modifications obvious to a person skilled in the art, are within the nature, scope and concept of the invention as defined by the attached claims.

Links

Links mentioned throughout this application, including the following references, in cases where they provide the typical methodological or other details in addition to the details set forth herein, are specifically incorporated herein by reference.

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1. Monoclonal antibody or functional fragment, where the indicated antibody and the fragment associated with activated protein C and inhibit anticoagulant activity, but do not connect and do not inhibit the activation of non-activated protein C, where the aforementioned antibody is produced by immunizing a mammal APC and screening of binding properties of the decree is spent antibodies with APC, but not with protein C.

2. Monoclonal antibody under item 1, where the specified binding to activated protein C occurs in the active site of activated protein C and inhibits cytoprotective effects of activated protein C.

3. Monoclonal antibody under item 1, where the aforementioned inhibition of activated or non-activated protein C occurs in vivo.

4. Monoclonal antibody under item 1, where the aforementioned inhibition of activated or non-activated protein C occurs in vitro.

5. Monoclonal antibody under item 1, where the antibody is an antibody of a mouse.

6. Monoclonal antibody under item 1, where the antibody is an antibody of human rights.

7. Monoclonal antibody under item 1, where the fragment of the antibody is further defined as Fab', Fab, F(ab')2, single domain antibody, Fv or scFv.

8. Pharmaceutical composition for treatment of diseases associated with anticoagulant activity of APC, including antibody under item 1 in an effective amount and a pharmaceutically acceptable carrier.

9. The method of inhibition of the anticoagulant activity of activated protein C in a subject, comprising introducing an effective amount of the monoclonal antibody under item 1 the specified entity.

10.The method according to p. 9, where the cytoprotective effects of activated protein C is not reduced under the influence of the specified monoclone inogo antibodies.

11. Method of inhibiting amylolyticus activity of activated protein C in a subject, comprising introducing an effective amount of the antibody under item 1 the specified entity.

12. The method of treatment of a subject that needs blood coagulation, including the introduction of a specified subject an effective amount of the antibody under item 1.

13. The method of treatment of a subject with sepsis, including the introduction of an effective amount of the antibody under item 1, and activated protein C.

14. The method of treatment of a subject with hemophilia, including the introduction of an effective amount of the antibody under item 1.

15. Method of modulating hemostasis in a subject, comprising introducing an effective amount of the antibody under item 1.

16. The method according to p. 15, where the subject is a trauma patient.

17. Method of modulating blood clot formation in a subject, comprising introducing an effective amount of antibodies in p. 1.



 

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