Method for generating active antibodies against resistance antigen, antibodies produced by same method and using them

FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology and medicine. What is presented is a method for generating an antibody and its functional fragments against a tumour antigen expressed on the tumour surface resistant to at least one anti-tumour compound by applying ground homogenate, and/or suspension, and/or cell lysate originated from the same tumour for immunisation. There are also disclosed using the method according to the invention for producing the monoclonal antibodies and their functional fragments, the monoclonal antibodies produced by the method, nucleic acids coding them, an expression vector, a host cell, and a method for preparing the antibody with using them, as well as hybridomes secreting these antibodies and their functional fragments for preparing a drug, the anti-tumour composition and using it as a drug.

EFFECT: invention can find further application in therapy of resistant tumours.

42 cl, 7 dwg, 4 ex, 6 tbl

 

The present invention relates to a new approach to create an exclusively monoclonal antibodies with therapeutic and/or diagnostic purposes. More specifically, the present invention is the creation of antibodies directed against the tumor antigen is absent on the surface of tumor cells amplified tumors and which may appear after conducting cancer treatment these native tumor antigen, which may possibly be involved in the resistance of these tumors to these antitumor treatments. In addition, this invention includes such antibodies, and the use of these antibodies as a drug for the prophylactic and/or therapeutic treatment of resistant tumors. Finally, the invention includes compositions containing such antibodies, which can be combined with anti-cancer agents, and their use for preventing and/or treating certain types of cancer.

The discovery of antibodies and more specifically the development of ways to generate antibodies has been a revolution in therapeutic treatment or diagnosis and, more specifically, related to the cancer field. Indeed, for the first time this new tool gave the opportunity to carry out targeted treatment due to the presence probability of inherent ability of the antibodies is. One of the main tasks of these last decades is to develop ways to create antibodies, regardless of whether they are in the first phase, polyclonal antibodies, monoclonal antibodies or even fragments of antibodies or similar structures.

Today, specialists in the field of technology has developed several technologies, with which you can create antibodies with therapeutic and/or diagnostic purposes.

The first antibodies are polyclonal antibodies, i.e. heterogeneous population of antibodies contained in the serum of immunized animals. For some animals (mice, rats, rabbits, goats, ...) are subjected to immunization by injection of a given natural or synthetic antigen, in combination with one or more adjuvants, such as beta-blockers and aluminum hydroxide, aimed at stimulating and strengthening the immune response. Then make the blood of animals and get the serum or anticigarette containing as antibodies, more or less effectively recognize several epitopes of a given antigen (polyclonal antibodies), and other antibodies of the animal. Purification of the obtained antibodies is performed, for example by affinity chromatography.

The first development was the creation of monoclonal antibodies to the e can be obtained from hybridomas according to the technique described by KOHLER and MIELSTEIN (1975). This technique allows to obtain specific monoclonal antibodies predetermining antigen consists in combining clone of lymphocytes with myeloma (myelomatous) cell with obtaining cells called hybridomas. This hybridoma characterized not only as immortalitya, but also constantly producing monoclonal and, therefore, monospecific antibodies.

Also described other methods based on the same principle, such as the method using trioma or another hybridoma technique described by KOZBOR and others (1983).

These methods, originally used to create mouse antibodies were developed, which resulted in the creation of humanized or even human chimeric antibodies. All these techniques are now well known to the person skilled in the technical field.

Human antibodies can be produced using the technology of genetically modified mice, the so-called "xanomeline" technology, which is described in patents US 5814318 and US 5939598.

In addition, a new modern ways of creating human monoclonal antibodies and, more specifically, fragments of antibodies, such as methods using banks or libraries of phages as described RIDDER and others (1995), or the so-called technology "phage di is play", based on the extraction of mRNA from folder (directory) of human cells derived from the peripheral blood, the construction of the Bank or cDNA libraries, comprising sequences of the variable regions, and on the insert cDNA in phages with the aim of producing variable regions of the antibodies in the form of fragments, preferably Fab-fragments.

Although these methods differ, they all have the same characteristic, i.e. immunization known antigen or volume of libraries or banks, the screening of these banks using the same known and specific antigen.

The present invention differs from these methods is the fact that known and a specific antigen is no longer used, and is used directly all the tumor or part of it. More specifically, the present invention relates to the direct application of the lysate, and/or suspension and/or crushed cell tumor homogenate. In the treatment of cancer observed for several years that a number of patients who were characterized by a full and satisfactory answer after the first therapeutic treatment, had a tendency to relapse. In a more specific case, it is known that tumors are able to modify your genotype and/or phenotype to be resistant to different types of PR is changing to them treatment.

This phenomenon takes place for these types of treatments like radiation therapy and chemotherapy and for the treatment of monoclonal antibodies.

Historically, the first chemotherapeutic compounds for the treatment of cancer have become the object of clinical trials in the 1940's. These were mainly agents with a short half-life, such as corticosteroids, antifolates or even vinylchloride. However, even if it was achieved full remission, it usually lasted for not more than one year, and systematically observed relapse associated with resistance to chemotherapeutic compound used for the treatment (Lehnert M., Eur. J. Cancer, 1996, 32A: 912-920).

In response to this phenomenon was proposed to carry out the so-called "combined" treatment using a variety of antitumor compounds. By simultaneous or successive use of different compounds were really received more good results In fact, in the case of the use of different anticancer compounds, each of which has cytotoxic activity, but different mechanisms of action, the tumor cell-resistant connection can still be sensitive to at least one of the other types of connections.

However, despite the use of R. the importance of antitumor compounds or agents, the resistance of tumors remains a major problem in the framework of chemotherapy. The use of combinations entail a delay of the phenomenon of sustainability, but does not lead to its disappearance. Resistance to chemotherapeutic means, as with the initial treatment, and appearing after a relapse after a favorable initial response, there is in almost all cases, so-called "curable" cancers.

As an example, mention may be made of patent application WO 2005/077385, WO 2004/026293 or even WO 2004/110497, which is well illustrated by the search for new methods of control phenomena sustainability, all of these claims are based on the principle of additional introduction generally, a new chemical molecule.

Demonstrated other mechanisms for the acquisition of resistance in tumors that are initially sensitive to treatment. According to the most widely held assumption implies that the phenomenon of resistance could be due to the accumulation of spontaneous and randomly occurring somatic mutations in the genotype of the tumor cells (Cancer Chemotherapy and Biotherapy: Principles and practice, Chabner & Lango editors, 1996, chapter 1).

Other known and described the phenomenon of resistance, called "MDR" (multi-drug resistance (multidrug resistance); also polyresistance; also resis intesti to many drugs), based on the ability of tumor cells to survive lethal concentrations of a large number of pharmacological, chemical or even structurally different cytotoxic compounds. Cells exhibiting "MDR", have the ability to reduce the accumulation inside the cells of cytotoxic compounds in the excretion of these compounds transport proteins. These proteins are called "carriers of drugs, providing multi-drug resistance (multi-drug transporters are membrane proteins that are able to display a wide range of toxic molecules out of the cell. These "carriers of drugs, providing multi-drug resistance" belong to the superfamily of ATP-binding cassette (ABC; ATP-binding cassette)transport proteins that use the energy of ATP hydrolysis for its activity. It is shown that this "MDR" responsible several mechanisms. The best known and most documented genome with the resistance associated with the mechanism of excretion (expulsion mechanism)dependent on ATP, is the MDR1 gene.

Concurrent with this phenomenon of resistance induced by chemotherapeutic treatment, also described induced resistance after treatment with monoclonal antibodies. This resistance may be related to the of ipom master, pharmacological considerations or it may be inherent in the tumor itself.

Before, during initial use of antibodies specific to mouse antibodies, or antibody-based test result received response against these mouse antibodies, the so-called HAMA (human antimachine or antibody-based test (human anti-mouse antibody)response. This form of resistance related to the owner, settled partially by the fact that murine antibodies have ceased to apply, and began to use human or humanized antibodies.

In different tests with the use of such human or humanized antibodies demonstrated new phenomena, in which the tumor may be resistant to immunological treatments. At the heart of these new mechanisms mainly lie mutations induced in the tumor, constitutive activation of the receptors and their subsequent cleavage (or leddingham) or even loss of expression of a target antigen.

As will be apparent hereinafter from Example 1, studies conducted by the applicant show, for example, that when triple therapy using antibodies against IGF-1R (receptor insulin-like growth factor type 1), EGFR (receptor epidermal growth factor) and Her/2neu it is possible to achieve a significant reduction in tumor growth A cells in Nude naked m the necks until complete disappearance of tumors in 90% of treated animals. Despite the fact that such multiple therapy is significantly more effective than monotherapy or bi-therapy, apparently tumors that still completely regressed gradually reappear, despite continuation of treatment, i.e. they become resistant to multiple treatment.

Based on this observation, the applicant for the first time offers a direct application of such resistant tumors to generate antibodies, preferably therapeutic and/or diagnostic monoclonal antibodies capable of recognizing antigens, the expression of which is induced on the surface of tumor cells of this tumor resistant to anticancer treatment, and which will not be expressed on the surface of tumor cells of native (untreated) tumor, these antigens may also be involved in the resistance of tumors to anticancer treatment.

Therefore, according to the first aspect of the present invention is the use of milled homogenate and/or suspension and/or cell lysate of tumor resistant to at least one anticancer compound, for immunization and establishment of in vitro antibody or one of its functional fragments, directed against the tumor antigen expressed on the surface of tumor glue is OK specified drug-resistant tumors this tumor antigen preferably is not expressed on the surface of tumor cells native tumor, which is resistant tumor, and the tumor antigen may be involved in the resistance specified tumors to anticancer connection.

The phrase "crushed homogenate or cell homogenate" understand the mixture of cells and/or cell fragments derived from tumors (xenotransplantation, orthotopic grafts, syngeneic grafts, surgical specimens from treated patients who had recurrence of the tumor, or samples of cell cultures), obtained by mechanical dissociation, for example, using ultrasonic mill type "Potter", mill type Ultraturax®, etc.

The term "suspension" or "cell suspension" means suspension of cells obtained after in vitro cultivation with or without processing and detached from their supporting environment by enzymatic solutions or non-enzymatic solutions for dissociation.

The term "lysate" or "cell lysate" understand the mixture of cells and/or cell fragments derived from tumors (xenotransplantation, orthotopic grafts, syngeneic grafts, surgical specimens from patients patients who who's had a relapse of the tumor, or samples of cell cultures), obtained through enzymatic degradation (enzymatic dissociation).

Under the expression "resistant tumor" understand the tumor, which does not meet or no longer responds to the applied(s) treatment(I). As described above, such resistance may occur or after treatment with chemotherapy, radiation therapy, hormonal therapy, or even in the specific use of antibodies, these treatments can be used individually or in combination. The origins of this ability to stability there may be different mechanisms, both known and not known at the present time. And finally, this resistance can result in the loss or reduction of the effect of the initially applied treatment.

Undoubtedly, known and described the phenomenon of "escapes"are a part of the phenomenon of sustainability, which aim to control with the use of this invention

Under native tumor (or the original tumor) in this description to understand the designation of the tumor, which is resistant tumor, this native tumor is not subjected to any anticancer treatment in contrast to resistant tumors.

Tumor antigen is understood herein as referring, in particular, to antigens, expre serwerem on the surface of tumor cells, regardless of whether they come from native tumor or from resistant tumors, and the tumor antigen is not expressed on the surface of healthy cells. In General these tumor antigens are natural macromolecules (which can be synthesized with which the specific antibody may be contacted. The tumor antigen can be, in particular, polypeptide, polysaccharide, carbohydrate, nucleic acid, lipid, hapten, or any other compound that is naturally present on the surface of tumor cells.

Finally, the expression "antibody" or "immunoglobulin" are used herein interchangeably in their broadest sense and include monoclonal antibodies (e.g., whole or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, polyspecific antibodies (for example, bispecific antibodies, provided that they exhibit the desired biological activity). Chimeric or humanized antibodies also fall under this designation.

More specifically, this molecule is a glycoprotein containing at least two heavy chains (H) and two light chains (L), connected together by a disulfide bridges. Each heavy chain comprises variable region (or domain) of the heavy chain (HCVR or VH) and a constant is blasti heavy chain. The constant region of the heavy chain consists of three domains CN, CH2 and CH3. Each light chain consists of a variable region light chain (LCVR or VL) and a constant region of the light chain. The constant region of the heavy chain contains the LC-domain VH - and VL-region can be subdivided into hypervariable sites, called CDR (complementarity determining areas; "complementary determining regions"), alternating with the more conservative areas, called frame regions (FR). Each VH and VL is composed of three CDRs and four FR, ranging from the N-terminal amino acids to the C-terminal amino acids in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant region of the antibodies may mediate the binding of immunoglobulin with tissues or host factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the system standard complement.

They may also include certain antibody fragments (expression will be described in more detail)showing the desired affinity and specificity of the source or type of immunoglobulin (IgG, IgE, IgM, IgA, etc).

As a rule, to obtain monoclonal antibodies or their functional fragments, especially of murine origin is to be placed, you can make reference to the techniques described, in particular, in the directory "Antibodies" (Hariow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp.726, 1988), or on methods of obtaining from hybridomas described by Kohler and Milstein (Nature, 256: 495-497, 1975).

Among antibodies, an attempt to create which is solved by the present invention, a preferred antibody, as defined above, and the so-called "active" antibodies, i.e. with antitumor activity, aimed at the tumor cells resistant to therapy.

Among antibodies, an attempt to create which is solved by the present invention, in a particular aspect of the invention, the preferred antibodies defined above, and those that are able also to the manifestation of antitumor activity against tumor cells with an aggressive phenotype, which naturally (before treatment) Express the antigen against which these antibodies are directed.

In the first embodiment of the use according to the invention is characterized by the fact that these drug-resistant tumor is obtained directly by biopsy and/or surgery from the patient, which passes or has passed therapeutic treatment using at least the specified antitumor compounds able to induce resistance, or resistance to which this tumor set.

In this embodied and adapted to each patient treatment can be considered in the sense of in response to immunization carried out using the whole-resistant tumor or portion thereof derived from real patient developed antibodies according to the invention. Thus, the patient may be offered the necessary therapy ex vivo.

In the second embodiment of the use according to the invention is characterized by the fact that these resistant tumors induce through transplantation of tumor lines and/or all human tumors or part of the animal and then by treatment of the animal by injection, usually by injection, at least one anticancer compound, stability to which it is desirable to induce or set.

More specifically, at least one specified antitumor compound selected from chemotherapeutic agents, such as chemical molecules, or even of therapeutic antibodies, or even agents of radiation therapy.

In General throughout this description the terms "anticancer agent" or "anti-cancer therapeutic agent" is intended to refer to a substance that, when administered to a patient that cures cancer or prevents the development of cancer in the patient.

As non-limiting examples of such agents may be mentioned the so-called "cytotoxic agents", such as "al yliluoma agents, antimetabolites, antitumor antibiotics, inhibitors of mitosis inhibitors functioning of chromatin, antiangiogenesis agents, antiestrogens, antiandrogens, or immunomodulators.

Such agents are referred to, for example, in the Handbook VIDAL 2006 edition page dedicated to compounds related to Oncology and Hematology, in the column "Cytotoxic means; these cytotoxic compounds referred to by reference into this document, referred to in this description as the preferred cytotoxic agents.

The term "alkylating agent" refers to any substance that can form a covalent bond or alkilirovanii any molecule, preferably a nucleic acid (e.g. DNA)in the cell. As examples of such alkylating agents may be mentioned nitrogen mustard analogues, such as mechlorethamine, chlorambucil, melphalan, hydrochloride, pipobroman, prednimustine, phosphate disodium or estramustine; oxazaphosphorine, such as cyclophosphamide, altretamine, trofosfamide, sulforophane or ifosfamide; aziridine or ethylenimine, such as thiotepa, triethylamine or altretamine; nitrosoanatabine, such as carmustine, streptozocin, fotemustine or lomustin, alkyl sulphonates such as busulfan, treosulfan or improsulfan; triazine, such as dacarbazine; what if even platinum complexes, such as cisplatin, oxaliplatin or carboplatin.

The term "antimetabolite" refers to substances that block the growth and/or metabolism of cells by exposure to certain types of activity, particularly on DNA synthesis. Examples of antimetabolites include methotrexate, 5-fluorouracil, floxuridine, 5-ftordezoksiuridin, capecitabine, cytarabine, fludarabine, citizenoriented, 6-mercaptopurine (6-MP), 6-tioguanin (6-TG), chloromethoxypropyl, 5-azacytidine, gemcitabine, cladribine, deoxycoformycin and pentostatin.

The term "antitumor antibiotic" refers to compounds that can inhibit the synthesis or inhibit synthesis of DNA, RNA and/or protein. Examples of such antitumor antibiotics include doxorubicin, daunorubicin, idarubitsin, valrubicin, mitoxantrone, dactinomycin, mithramycin, plicamycin, mitomycin C, bleomycin and procarbazine.

"Inhibitors of mitosis" interfere with the normal passage of the cell cycle and mitosis. In General, inhibitors of microtubules or "taxoid", such as paclitaxel and docetaxel, are able to inhibit mitosis. Also capable to inhibit the mitosis vinylchloride, such as vinblastine, vincristine, vindesine and vinorelbine.

The term "inhibitors of the function of chromatin or inhibitors of topoisomerases" refers to substances which inhibit normal is a great function modeling chromatin proteins, such as topoisomerase I and II. Examples of such inhibitors include, for topoisomerase I - camptothecin and its derivatives, such as irinotecan or topotecan, and topoisomerase II - etoposide phosphate of etoposide and teniposide.

The term "antiangiogenesis agents" refers to any drug, compound, substance or agent that inhibit the growth of blood vessels. Examples antiangiogenetic agents include, without any limitation, retuxin (razoxin), marimastat, batimastat, prinomastat, tonemaster, ilomastat, CGS-27023A, halofuginone, COL-3, neovastat, BMS-275291, thalidomide, CDC 501, DMXAA, L-651582, squalamine, endostatin, SU5416, SU6668, alpha-interferon, EMD121974, interleukin-12, IM862, angiostatin, vitaxin.

The term "antiestrogens" or "anti-estrogenic agents" refers to any substance that weakens, antagonisitic or inhibits the action of estrogen. Examples of such agents are tamoxifen, toremifene, raloxifene, droloxifene, idoxifene (lodoxyfene), anastrozole, letrozole, and exemestane.

The term "anti-androgens" or "antiandrogenna agents" refers to any substance that weakens, antagonisitic or inhibits the action of androgen. Examples of anti-androgens are flutamide, nilutamide, bikalutamid, spironolactone, ciproteron acetate, finasteride and cimetidin.

Immunomodulators are ve is esta, which stimulate the immune system. Examples of immunomodulators include interferon, interleukins, such as aldesleukin, OST-43, denileukin deflators or interleukin-2, tumor necrosis, such as tasonermin, or other types of immunomodulators, such as lentinan, sizofiran, roquinimex, pidotimod, pegademase, thymopentin, poly-I:C (polyinosine policitally acid) or levamisole in combination with 5-fluorouracil.

More detailed information specialist in the art can be found in the Handbook edited by the Association Francaise des Enseignants de Chimie Therapeutique called "traité de chimie therapeutique, Vol.6, Medicaments antitumoraux et perspectives dans le traitement des cancers, edition TEC &DOC, 2003".

As anticancer agents can also be referred to agents, radiation therapy, hormonal therapy, or therapeutic agents directed small molecules, such as inhibitors tyrosinekinase. And finally, according to a preferred embodiment the antibodies are part of antitumor agents that can be used according to the invention. More specifically, as a non-limiting example, one can mention the following antibodies: antibodies against EGFR, such as cetuximab (S or Erbitux), matuzumab, huR3, HuMax-EGFR or panicum; antibodies against VEGF (vascular endothelial growth factor), such as bevels the MAB (Avastin) or S; antibodies against IGF-1R, such as S, h7C10, hEM164, AVH-IGF-1R, Mao (monoclonal antibody (mAb also)) 39, N or 4G11, antibody against HER2, such as trastuzumab (Herceptin) or pertuzumab, antibodies against CD20 (leukocyte antigen 20), such as commericial (commentuximab), ibritumomab or tositumomab, antibody against CD33, such as gemtuzumab or lintuzumab; antibodies against CD22, such as epratuzumab, antibody against CD52, such as alemtuzumab; antibodies against Ersem (adhesion molecule epithelially cells), such as edrecolomab, Ch 17-1A or IGN-101; antibodies against STR or 16, such as Xactin; antibodies against DNA-Ag, such as131I Cotara TNT-1 (131I Kotar - agent 1 therapy of tumor necrosis); antibodies against MUC1 (mucin-1), such as pemtumomab or R1150; antibodies against MUC18, such as AVH-MA; antibodies against GD3, such as mitooma; antibodies against CEA (carcinoembryonic antigen), such as CeaVac or labetuzumab; antibodies against SA, such as OvaRex; antibodies against HLA (major histocompatibility complex)-DR, such as apolizumab; antibodies against CTLA4 (cytotoxic T-lymphocyte antigen 4), such as MDX-010; antibodies against PSMA (membrane antigen of the prostate), such as MDX-070,111In and90Y-J591,177Lu J591, J591-DM1; antibodies directed against the antigen Lewis (anti-Lewis Y antibodies), such as IGN311; antiangiogenesis antibodies, the same is how AS1405 and 90YmuBd; antibodies against Trail-R1 (TNF-related apoptosis-induced ligand receptor 1; receptor 1 related to the tumor necrosis factor apoptosis inducing ligand), such as TRAIL R1mAb or TRAIL R2mAb; or even any antibody directed against tyrosinekinase receptor other than those referred to above, or RON (recepteur d'origine nantais) receptor, estimates receptor, CXCR 2 (receptors chemokines type C-X-C), CXCR4, the receptor iminovogo type, etc. like therapies directed action using small chemical molecules, such as inhibitors tyrosinekinase.

Undoubtedly, this list is by no means limiting, and its purpose is simply the mention of the antibodies used or developed to date.

According to a specific embodiment of the invention assumes that the resistant tumor is resistant to various therapies or anticancer agents, the latter may be of various nature.

More specifically, the use according to the invention is characterized by the fact that the at least one anticancer compound consists of at least two, preferably at least three compounds of different nature and/or having different mechanisms of action, and/or directed to different proteins.

Under different mechanism of action see, for example, that protivoopuxolevye agents will affect different biological functions of cells, such as angiogenesis, DNA synthesis or even mitosis.

More specifically, directed action on different proteins refers to the case of anticancer agents are antibodies that can bind with proteins or receptors of different nature.

Obviously, you can consider either combining only antibodies with each other or only chemotherapeutic agents with each other, or a combination of both of these families of compounds with each other or with radiation therapy, hormonereleasing treatments or therapies directed action, using small chemical molecules, as described above.

Also preferably, though not necessarily to all of antitumor compounds would have been a different mechanism of action or they would operate at a different target.

According to the second aspect of the present invention is a method of creating an in vitro antibody or one of its functional fragments, directed against the tumor antigen expressed on the surface of a tumor resistant to at least one anticancer compound, and specified a tumor antigen preferably is not expressed on the surface of tumor cells of the corresponding native (untreated) tumor and the tumor antigen may be involved in ostoich the stability of the indicated tumor, resistant to anticancer treatment, the method comprising a stage consisting of immunization of animals directly crushed homogenate, and/or suspension and/or cell lysate from a specified drug-resistant tumors, and stage, consisting of a selection of antibodies that recognize the resistant tumor, not native tumor, which is resistant tumor.

According to a particular aspect of the method according to the invention described method of creating an in vitro antibody or one of its functional fragments, directed against the tumor antigen expressed on the surface of the resistant tumor, possibly involved in the resistance specified drug-resistant tumors, including immunization of animals directly crushed homogenate, and/or suspension and/or cell lysates from the indicated tumor resistant to at least one anticancer compound (antitumor compound, stability to which this tumor was installed or resistance to which it is desirable to induce)or after tolerization animals crushed homogenate, and/or suspension, and/or cell lysate from native tumors (tumors that are not subjected to any treatment)or without tolerization. The purpose of this tolerization carried out with the use of immunosuppressive and the enta type of cyclophosphamide, is the suppression of immune response against all surface antigens that are present to conduct cancer treatment(s)administered to the animal or person for the purpose of induction of resistance or in the treatment relating to people. Thus, the immune response after administration, especially through injections, preparations from resistant tumors will focus on surface structures induced by treatment and potentially involved in establishing the stability of the tumor. The effectiveness of tolerization evaluated on the basis of the subsequent disappearance of serum titer established after immunization of animals with tumor cells from native tumor.

Then produce a sampling spleens of mice immunized according to the aforementioned method, and produce the fusion of splenocytes with myeloma cells according to standard procedures known to a person skilled in the technical field.

Screening of hybridomas obtained after cell fusion, will be conducted through "differential immunohistochemistry on slides prepared in advance and bearing sections (untreated) native tumors or slices of resistant tumors (treated as one, and many anti-tumor agents). With the aim of producing the antibodies and tested for their antitumor activity will be selected, cloned and frozen only hybridoma producing antibodies that recognize the resistant tumor, but not native tumor. This whole method will be schematically depicted later in figure 2 and 3. It should be noted that this approach can also be offered for search and detection of intracellular molecules involved in the resistance.

As defined above, antibodies of the present invention preferably represent a specific, particularly murine, chimeric or humanized monoclonal antibodies which can be obtained according to standard methods, well known to the person skilled in the technical field.

In the General case to obtain monoclonal antibodies or their functional fragments, especially of murine origin, it is possible to refer to the techniques described, in particular, in the directory "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp.726, 1988) or on the method of obtaining from hybridomas described by Kohler and Milstein (Nature, 256: 495-497, 1975).

As the antibodies of the present invention also includes chimeric or humanized antibodies.

A chimeric antibody is intended to mean an antibody, which contains a natural variable region (light chain and heavy chain)derived from antibodies of a certain type, in Association with a constant area is s light chain and heavy chain antibody, heterologous specified specified type (mouse, horses, rabbits, dogs, cows, chicken etc).

Antibodies or their fragments, chimeric type according to the invention can be obtained by using techniques of genetic recombination. For example, the chimeric antibody can be obtained by cloning, recombinant DNA comprising a promoter and a sequence encoding the variable region is not human, especially murine, monoclonal antibodies according to the invention, and a sequence encoding a constant region of a human antibody. Chimeric antibody according to the invention encoded by this recombinant genome, will constitute, for example, human-mouse design, and the specificity of this antibody will be determined by the variable region derived from a murine DNA and its isotypes - constant region derived from human DNA. Regarding ways to obtain chimeric antibodies can be referenced, for example in the document Verhoeyn and others (BioEssays, 8: 74, 1988), Morrison and others (Proc. Natl. Acad Sci. USA 82: 6851-6855, 1984) or U.S. patent 4816567.

Humanitariannet antibody is intended to mean an antibody which contains the CDR-plots derived from antibodies of human origin, and other parts of this antibody molecules derived from the same (or more than one) person is human antibodies. In addition, some of the remnants of the segments of the skeleton (skeleton) (referred to as FR) can be replaced with the goal of preserving the affinity of binding (Jones et al., Nature, 321: 522-525, 1986; Verhoeyen et al., Science, 239: 1534-1536, 1988, Riechmann et al., Nature, 332: 323-327, 1988).

Humanized antibodies according to the invention or their fragments can be obtained using methods known to the person skilled in the art (such as, for example, described in Singer et al., J. Immun. 150: 2844-2857, 1992, Mountain et al., Biotechnol Genet. Eng Rev., 10. 1-142, 1992; or (Bebbington et al., Bio/Technology 10: 169-175, 1992).

Specialist in the art are also aware of other methods of obtaining humanized antibodies, such as, for example, the method "CDR-cultivation" ("CDR Grafting"), described in PDL (Public Documentation License), which is the subject of invention patents EP 0451261, EP 0682040, EP 0939127, EP 0566647 or even US 5530101, US 6180370, US 5585089 and US 5693761. You can also mention the patents US 5639641 or even US 6054297, US 5886152 and US 5877293.

Functional fragment of the antibody according to the invention is intended in particular to refer to this fragment of the antibody, as the fragments Fv, scFv (single-chain Fv; sc). simple chain), Fab, F(ab')2, Fab', scFv-Fc, or diately or any part of, the duration (lifetime) of which would be increased by chemical modification, such as adding poly(alkylene)glycol such as polyethylene glycol (PEG), in the "Page is investing", or by incorporation in a complementary mechanism. The specified fragment typically contains at least one characteristic of the CDR of the antibody from which it comes, and are usually able to manifest at least partial activity of the antibody from which it comes.

Preferably, these functional segments will consist of a partial sequence or contain a partial sequence of the heavy or variable light chain of the antibody from which they originate, and the specified partial sequence essentially will properly retain the same binding specificity as the antibody from which it occurs, and sufficient affinity, preferably at least equivalent to 1/100, more preferably at least 1/10 of the affinity of the antibody from which it comes.

Such a functional fragment will comprise at least 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids from the sequence of the antibody from which it comes.

Preferably, these functional fragments will represent fragments of type Fv, scFv, Fab, F(ab')2F(ab'), scFv-Fc or diately, which usually have the same binding specificity as the antibody from which they originate. According to the present invention, fragments of antibodies image is meniu can be obtained from antibodies so, as described previously, through methods such as digestion by enzymes, such as pepsin or papain and/or by cleavage of disulfide bonds by chemical recovery. Different fragments of the antibodies of the present invention can be obtained using techniques of genetic recombination, a well-known specialist in this field of technology, or even by means of peptide synthesis, for example using an automatic peptide synthesizer, such as, for example, produced by Applied Biosystems, etc.

More preferably, the invention includes antibodies or their functional fragments according to the present invention, particularly a chimeric or humanized antibodies obtained by genetic recombination or by chemical synthesis.

According to a preferred embodiment of the method according to the invention specified conduct immunization by intraperitoneal and/or subcutaneous and/or intravenous, and/or vnutrikletochnoi injection. Because each method of immunization is equivalent to another, the selection of one of them relative to another made in accordance with the animals and the knowledge and experience of a specialist in this field of technology.

More specifically, according to the first embodiment of the resulting tumor is obtained directly by biopsie/or surgery from the patient, which passes or has passed therapeutic treatment using at least one anticancer compound capable to induce resistance, or resistance to which (the tumor) is installed.

According to the second embodiment of the resistant tumor induce through transplantation of tumor lines and/or all human tumors or part of the animal and then by treatment of the animal by injecting the injection of at least one anticancer compound, stability to which it is desirable to induce or set.

Whatever the above-described preferred embodiment, the method according to the invention is characterized by the fact that the active antibody or one of its functional fragments, is a monoclonal antibody.

More specifically, the specified monoclonal antibody or one of its functional fragments is an immunoglobulin selected from the group consisting of IgG, IgA, IgM, IgD or IgE.

Even more preferably, when the specified monoclonal antibody or one of its functional fragments is an IgG isotype gamma 1, gamma-2 or gamma-4.

However, it should be understood that according to the invention preferred immunoglobulins types of IgG1 and IgG2 because of their ability to induce factorie functions.

Usually, the person skilled in the art it will be obvious that the effector functions include, as a non-limiting example, the binding of C1q, CDC (complementability cytotoxicity), binding to the Fc receptor, ADCC (antibody-dependent cellular cytotoxicity and phagocytosis.

More specifically, the preferred effector functions according to the invention are ADCC and CDC.

In the General case, functional fragments, to which this invention relates, selected from the fragments Fv, Fab, (Fab')2, Fab', scFv, scFv-Fc and dietel or any part of, the duration of which will be increased, like Paglierani slices.

In more concrete, but in no way a non-limiting way, the method according to the invention includes at least the following stages:

i) direct immunization of animals crushed homogenate, and/or suspension and/or cell lysate derived from resistant tumors,

ii) the fusion of spleen cells of an animal immunized at stage i), with myeloma cells to obtain hybridomas and

iii) the selection by differential selection (differential selection hybridomas secreting antibodies that recognize specific antigens expressed on the surface of tumor cells resistant tumors, and the expression of which is induced in the FR is loopholing treatment.

Under "differential selection" is understood as any kind of selection, based on the ways in which induced antigens can be distinguished by the emergence of resistance to treatment of antigens present on the cells to establish this stability.

As a non-limiting example of a method of providing such "differential selection", you can mention the immunohistochemistry on frozen sections or included in paraffin samples or approaches to type "protein arrays or gene arrays".

More specifically, the use of so-called methods of "tissue array" is preferred. The method of "tissue array" is the building blocks of tissues, included in paraffin or frozen, new units, containing from tens to a few hundreds of cores (cores) of these tissues, so that after cutting these blocks "tissue array" had the possibility of making slides microscope containing from tens to a few hundreds of tissue sections.

According to a preferred embodiment of the method according to the invention further includes a front stage i)described above, the following stages:

a) selection of animals and transplantation animal tumor lines and/or all the so-called "native" of the tumor or part thereof,

b) the treatment part is quiet subjected to transplant animal at least one anticancer compound

c) removing the whole or parts of the so-called "native" tumors from untreated animals subjected to transplantation at the stage a),

d) removing the whole or parts of resistant tumors from animals treated at the stage b),

e) preparation of tools for the differential selection of antibodies from tumors, fully or partially extracted in stages C) and d), respectively, and

f) preparation of milled homogenate and/or a cell lysate of resistant tumors, fully or partially extracted in stage d).

It should be understood that in the present description the expression "native tumor" and "original tumor" are equivalent and will be used independently.

According to the embodiment of the invention specified tumor line and/or the so-called "native" tumor selected from the group of tumor cells (A) from the lungs, and in addition, but not limited to, colon, prostate, breast, ovarian, or any tumor resistance to treatment is installed.

In addition, the antitumor compound used according to the invention, selected from chemotherapeutic agents, agents, radiation therapy, chemical molecules or antibodies, and all of these different compounds defined above in the present description.

According to the preferred embodiment is subramania specified at least one anticancer compound is a monoclonal antibody, moreover, the specified monoclonal antibody, even more preferably selected from the group of antibodies or their functional fragments, directed against receptors of growth factors, molecules involved in angiogenesis, or even chemokines and integrins involved in cell migration phenomenon.

Under "growth receptors," you see any transmembrane protein that upon binding of ligand(s) or independent changes in the conformation of the ligand either Homo - or heterodimerization with other membrane proteins to mediate the proliferative response. Under "molecules involved in angiogenesis", understood as either a membrane receptor, which upon binding of ligand(s) or independent changes in the conformation of the ligand either Homo - or heterodimerization with other membrane proteins will cause the formation of blood vessels.

Under the "chemokines and integrins involved in cell migration phenomenon," see any soluble molecule, capable of activity of digestion of the extracellular matrix and/or chemoattractant activity.

According to another embodiment of the invention the method is characterized by the fact that the at least one anticancer compound is a combination of at least two, preferably at least three of antitumor compounds R is slicnoj nature, and/or characterized by different mechanisms of action, and/or directed to different proteins.

Preferably, the specified combination of antitumor compounds consists of a combination of monoclonal antibodies or their functional fragments.

Even more preferably, the specified combination of monoclonal antibodies or their functional fragments consists of a combination of an antibody selected from antibodies against IGF-1R, against EGFR, against Her/2neu, against VEGF, against VEGFR (VEGF receptor), against CXCR, against estimates against RON, iminovogo antibodies, etc.

According to the embodiment, as a non-limiting example, the method according to the invention is characterized by the fact that this combination is a combination of antibodies against IGF-1R antibodies against EGFR and antibodies against Her/2neu, with the above mentioned antibodies preferably are monochloramine antibodies S, 225 and h4D5, respectively.

Monoclonal antibody S is an antibody described in the patent application WO 03/059951 filed by the applicant on 20 January 2003, and the contents of which are incorporated in this description by reference.

Monoclonal antibody 225 is an antibody produced by hybridomas deposited in ATSS under reference number HB-8508.

Monoclonal antibody h4D5 is an existing prod the e Herceptin, for example in ICR (Institut Claudius Regaud).

According to another preferred embodiment of the method according to the invention is characterized by the fact that the specified stage iii) for selection of hybridomas secreting antibodies that recognize antigens of the so-called "native" tumor cells, is accomplished by differential screening between untreated cloth so-called "native" of the tumor and tissue resistant tumors and/or tumor, which is made resistant.

Under "differential screening refers to screening, by which induced antigens can be distinguished by the emergence of resistance to treatment of antigens present on the cells to establish this stability. Preferably, when the differential screening is conducted using the means obtained in stage e), as described above.

And finally, according to the last embodiment of the invention it is preferable that the method includes the additional step of tolerization before stage i).

Under the "tolerization" refers to the weakening of the immune response induced by immunosuppressive compounds such as cyclophosphamide. In practice, the specified stage of tolerization may consist of:

- introduction, especially by injection, the animals milled homogenate and/or suspension and/or cell lysate, receiving the data from the so-called native tumors, originating from stage (C), and

treatment of these animals by the immunosuppressive drug with the purpose of elimination of b-cells activated by injection at the above stage, and thereby inhibiting any possible response against specified the so-called native tumor.

Undoubtedly, any similar method or practical activities which can be obtained the same result, should be considered equivalent and included in the scope of the invention. Under the "immunosuppressant", understand any substance capable of Deplete (depleting) cell population of the immune system. As a non-limiting example may be mentioned cyclophosphamide.

In another aspect, the present invention relates to a method for creation and selection in vitro antibody or one of its functional fragments, capable to inhibit the resistance of tumors to anticancer compound, or the way of their formation and selection in vitro antibody or one of its functional fragments, directed against the tumor antigen expressed on the surface of a tumor resistant to at least one anticancer compound, and specified a tumor antigen involved in the resistance specified tumors to anticancer compound, method, characterized in that it includes:

a) a way to create in vitro is ntotila or one of its functional fragments according to the invention, moreover, the specified antibody directed against the indicated tumor antigen-specific expressed on the surface of the resistant tumors indicated a tumor antigen is not expressed on the cell surface of native tumor from which this resistant tumor occurs;

b) bringing obtained in stage a) antibodies in vitro or in vivo in contact with a tumor that is resistant to anticancer compound, and

c) the selection of the specified antibodies, if demonstrated inhibitory effect of this antibody on the resistance of tumors to anticancer connection.

According to another embodiment may be tested in vitro or in vivo to identify those involved whether the antigen whose expression induce antitumor treatment, the phenomenon of resistance or not. According to the preferred embodiment of this stage can be tested in vitro or in vivo antibody obtained in accordance with the invention, in resistant tumors and supervision, develops whether resistant tumor inhibiting the sustainability of the activity.

One other aspect of the present invention relates to a method for creation and selection in vitro antibody or one of its functional fragments, capable to exhibit antitumor activity, particularly by inhibiting the proliferation of tumor what's cells, expressing the antigen against which directed the specified antibody, characterized in that the method includes:

a) a way to create in vitro antibody or one of its functional fragments according to the invention, with the indicated antibody directed against the indicated tumor antigen-specific expressed on the surface of the resistant tumors indicated a tumor antigen is not expressroute on the surface of the native cells of the tumor from which the resistant tumor occurs;

b) bringing obtained in stage a) antibodies in vitro or in vivo in contact with tumor cells which Express the indicated tumor antigen, preferably with the specified resistant tumor used in stage a), or tumor with aggressive phenotype; and

c) the selection of the specified antibodies, if this tumor demonstrated antitumor effect, in particular the inhibition of cell proliferation of this tumor.

At the stage b) of the method described earlier, it should be understood that the tumor cells which Express the indicated tumor antigen, is not necessarily a tumor that is resistant to antitumor compound used to create the indicated antibodies. At the stage b) can be used any tumor, especially with the aggressive phenotype of tumor cells is toroi Express a tumor antigen, recognizes the specified antibody.

In addition, the invention obviously applies to the use of the above-described method for creating therapeutic and/or diagnostic monoclonal antibodies. The invention also encompasses partial use of such a method which can be developed by an expert in the field of technology to meet specific criteria or otherwise, with the aim of completely full of differences from the present description. According to a third aspect of the invention proposed monoclonal antibody or one of its functional fragments obtained(output) the result of applying the above method according to the invention.

The present invention is advanced in the sense that at the moment no one antibody having such properties, moreover, obtained in this way.

Preferably, these functional fragments according to the present invention will be selected from the fragments Fv, scFv, Fab, (Fab')2, Fab', scFv-Fc or datel or any functional fragment, the duration of which would be increased by chemical modification, especially in the "Paglierani, or by incorporation into lysosome. Undoubtedly, this list in no way is limiting, or any other type of fragment, known by the CSOs skilled in the art, should be considered as part of the invention. As an illustrative example of the invention hereinafter described 5 antibodies, obtained by applying the method according to the invention. These antibodies, called as A, A, A, S and 3G7 may be murine, chimeric or humanitarianism.

According to the first embodiment of the present invention relates to a monoclonal antibody or one of its functional fragments, characterized by the fact that he/it contains:

- light chain contains the CDR portions of the sequences SEQ ID No 1, 2 and 3, or for which sequences have at least 80% identity after optimal alignment with sequences SEQ ID No 1, 2 and 3; and

- heavy chain contains the CDR portions of sequences SEQ ID nos 4, 5 and 6, and for which sequences have at least 80% identity after optimal alignment with sequences SEQ ID nos 4, 5 and 6.

In the present description, the terms polypeptide, polypeptide sequences, peptides and proteins associated with antibodies or their sequences are interchangeable.

It should be understood that the invention is not of antibodies in their natural form, i.e. they are not taken in their natural surroundings, and there is a possibility of their isolation and by purification from natural sources or also receive the Deposit them with the use of genetic recombination or by chemical synthesis, and so they can include non-natural amino acids, as will be described below.

CDR-plot(s) or CDRs are intended to denote the hypervariable areas of heavy and light chains of immunoglobulins, as defined by Kabat and others (Kabat et al., Sequences of proteins of immunological interest, 5th Ed, U.S. Department of Health and Human Services, NIH, 1991, and later editions). There are 3 CDRs of the heavy chain and the 3 CDRs of the light chain. The term CDR or CDRs used in this description to denote, depending on the case, one of these sites or more or even all of these sites, which contain most of the amino acid residues responsible for the affinity binding of the antibody to the antigen or epitope it recognizes.

"Percentage identity" between two sequences of nucleic acids or amino acids in the context of the present invention is intended to denote the percentage of nucleotides or identical amino acid residues between the two compared sequences, obtained after the best alignment (optimal alignment), despite the fact that this percentage is purely statistical and the differences between the two sequences randomly distributed throughout their length. Comparison of sequences of two nucleic acids or two amino acid sequences are traditionally performed with Aviva these sequences after aligning them in an optimal way, while this comparison may be performed by comparing segments or by using the "window of comparison". Optimal alignment of sequences for comparison can also be entered manually using algorithm local homology Smith and Waterman (1981) (Ad. App. Math 2: 482), with the application of the algorithm the local homology Neddleman et Wunsch (1970) J. Mol. Biol. 48:443), using the same search method of Pearson and Lipman (1988) (Proc. Natl. Acad. Sci. USA 85: 2444), with the use of computer software using these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the software package Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or even with the use of software packages for this comparison, the BLAST N or BLAST P).

The percent identity between two sequences of nucleic acids or amino acids determined by comparing these two sequences aligned in an optimal way, while comparing the sequence of nucleic acids or amino acids may contain additives or deletions relative to the reference sequence (reference sequence for optimal alignment between the two sequences. The percentage of identity is calculated, determining the number of identical positions where nucleotide or amino acid residue is identical in the two sequences, and p is Ozoda dividing this number of identical positions by the total number of positions in the comparison window, and generating a multiplication of the result by 100 to obtain the percentage identity between these two sequences.

For example, you can use the BLAST program "BLAST 2 sequences" (Tatusova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Environ. Lett 174: 247-250)available on the site http://www.ncbi.nlm.nih.gov/gorf/bl2.html with the parameters used by default (in particular for the parameters "a penalty to make deletions" ("gap open penalty"): 5, and the penalty for continued deletions" ("extension gap penalty": 2; the selected matrix is, for example, the matrix BLOSUM 62", proposed for the program), the percent identity between the two compared sequences is calculated directly by this program. You can also use other programs, such as software packages "ALIGN or Megalign" (DNASTAR).

Among amino acid sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity to the reference amino acid sequence, the preferred sequence having against the reference sequence, certain modifications, in particular a deletion, Supplement or replacement of at least one amino acid, shortening of, or insertion. In case of replacement of one or more adjacent or not adjacent amino acids preferred replacement, in which the substituted amino acids are replaced with "equivalent" amino acids. The expression "equivalent amino acids" this is the description of the proposed to denote any amino acid, where you can replace one amino acid basic structure, however, without significant changes of the biological activities of the corresponding antibodies, which will be determined later, especially in the examples.

These equivalent amino acids can be defined, relying either on their structural homology with amino acids, which they replace, or on the results of comparative tests of biological activity between different antibodies that can be done.

As a non-limiting example in the following Table 1 repeated opportunities of substitutions that can be carried out without much influence on the biological activity of the corresponding modified antibodies, reverse replacement naturally can be considered in the same terms.

Cys (C)
Table 1
The original balanceReplacement(s)
Ala (A)Val, Gly, Pro
Arg (R)Lis, His,
Asn (N)Gln
Asp (D)Glu
Ser
Gln (Q)Asn
Glu (G)Asp
Gly (G)Ala
His (H)Arg
Ile (1)Leu
Leu (L)Ile, Val, Met
Lys (K)Arg
Met (M)Leu
Phe (F)Tyr
Pro (P)Ala
Ser (S)Thr, Cys
Thr (T)Ser
Trp (W)Tyr
Tyr (Y)Phe, Trp
Val (V)Leu, Ala

Preferred is the above-described antibody,called A and contains a sequence of the heavy chain, corresponding to the sequence SEQ ID No 7, and a light chain containing the sequence SEQ ID No 8.

More specifically, the present invention also relates to petty hybridoma (A), are able to secrete antibody A that described above. In the present description independently use the same encoding when it comes to petty hybridoma or also about the antibody produced by the same hybridomas.

This mouse hybridoma is hybridoma deposited in the CNCM (Collection Nationale de Cultures de Microorganismes), Institut Pasteur, Pans (France) on may 31, 2006 under No. 1-3612. Among the six short CDR sequences of the third CDR of the heavy chain (CDRH3) is characterized by a high degree of variability (large variety of essentially due to mechanisms of genes, leading to it). It can be as short as 2 amino acids, whereas the largest known size is equal to 26. Functionally CDRH3 plays a distinct role in determining the specificity of antibodies (Segal et al., PNAS, 71: 4298-4302, 1974; Amit et al., Science, 233: 747-753, 1986; Chothia et al, J Mol Biol, 196: 901-917, 1987, Chothia et al., Nature, 342: 877-883, 1989; Caton et al., J. Immunol, 144: 1965-1968, 1990, Sharon et al, PNAS, 87: 4814-4817, 1990, Sharon et al., J. Immunol, 144: 4863-4869, 1990, Kabat et al., J. Immunol, 147:1709-1719, 1991).

It is known that only a small percentage of amino acids in the CDRs contributes to the structure of the binding site of this antibody, however, these balances must be maintained in all the ü specific three-dimensional conformation.

According to the second embodiment of the invention relates to a monoclonal antibody or one of its functional fragments, characterized in that it contains:

- light chain contains the CDR portions of the sequences SEQ ID No 9, 10 and 11, or for which sequences have at least 80% identity after optimal alignment with sequences SEQ ID No 9, 10 and 11; and

- heavy chain contains the CDR portions of sequences SEQ ID nos 12, 13 and 14, or for which sequences have at least 80% identity after optimal alignment with sequences SEQ ID nos 12, 13 and 14.

According to another embodiment of the specified antibody according to the invention, called A, contains a sequence of the heavy chain corresponding to the sequence SEQ ID No 15, and a light chain containing the sequence SEQ ID No 16.

According to another aspect of the claimed rat hybridoma (A), are able to secrete antibody, as defined above.

And finally, according to a preferred embodiment of the invention included mouse hybridoma A deposited in the CNCM (Collection Nationale de Cultures de Microorganismes), Institut Pasteur, Pans (France) on may 31, 2006 under the number I-3613.

According to a third embodiment of the invention relates to a monoclonal antibody or one of its functional fragments, obtained according to the lady of the invention and containing:

- light chain contains the CDR-sites with sequences SEQ ID No 17, 18 and 19, or for which sequences have at least 80% identity after optimal alignment with sequences SEQ No 17, 18 and 19, and

- heavy chain contains the CDR portions of sequences SEQ ID nos 20, 21 and 22, or for which sequences have at least 80% identity after optimal alignment with sequences SEQ ID nos 20, 21 and 22.

Preferably, the specified antibody according to the invention, called A, contains a sequence of the heavy chain that contains the sequence SEQ ID No 23, and a light chain containing the sequence SEQ ID No 24.

According to another aspect of the invention also includes a mouse hybridoma E able to secrete the antibody described above.

More specifically, the specified mouse hybridoma is hybridoma deposited in the CNCM (Collection Nationale de Cultures de Microorganismes), Institut Pasteur, Pans (France) on may 31, 2006 under the number I-3615.

According to a fourth embodiment of the invention relates to monochlorophenol the antibody or one of its functional fragments obtained as described above and containing:

- light chain contains the CDR portions of sequences SEQ ID nos 25, 26 and 27, or for which sequences have at least 80% identity after optimalnog the alignment with sequences SEQ ID No 25, 26 and 27; and

- heavy chain contains the CDR portions of the sequences SEQ ID No 28, 29 and 30, or for which sequences have at least 80% identity after optimal alignment with sequences SEQ ID No 28, 29 and 30.

More specifically, the specified antibody according to the invention, called S, contains a sequence of the heavy chain that contains the sequence SEQ ID No 31, and a light chain containing the sequence SEQ ID No 32.

According to another aspect of the invention also applies to petty hybridoma IS able to secrete the antibody described above.

This hybridoma preferably represents hybridoma deposited in the CNCM (Collection Nationale de Cultures de Microorganismes), Institut Pasteur, Pans (France) on may 31, 2006 under the number I-3614.

And finally, according to the fifth embodiment of the invention, the latter relates to a monoclonal antibody or one of its functional fragments, obtained by applying the method according to the present invention and containing:

- light chain contains the CDR portions of the sequences SEQ ID No 33, 34, or 35, or for which sequences have at least 80% identity after optimal alignment with sequences SEQ ID nos 33, 34 and 35; and

- heavy chain contains the CDR portions of the sequences SEQ ID No 36, 37 and 38, or for which posledovatelnostyu at least 80% identity after optimal alignment with sequences SEQ ID No 36, 37 and 38.

Preferably, the specified antibody, called 3G7, contains a sequence of the heavy chain that contains the sequence SEQ ID No 39, and a light chain containing the sequence SEQ ID No 40.

According to another aspect of the invention the mouse hybridoma 3G7, able to secrete the antibody described above.

This hybridoma preferably represents hybridoma deposited in 1A CNCM (Collection Nationale de Cultures de Microorganismes), Institut Pasteur, Pans (France) on may 31, 2006 under the number I-3616. For clarity, it is shown in Table 2 below, which shows the correspondence between the antibodies according to the invention and corresponding amino acid sequences of CDRs of these antibodies.

4
Table 2
AntibodyHeavy chainLight chainSEQ ID No
ACDR11
CDR22
CDR33
CDR1
CDR25
CDR36
ACDR19
CDR210
CDR311
CDR112
CDR213
CDR314
E
CDR117
CDR218
CDR319
CDR120
CDR221
CDR322
SCDR125
CDR226
CDR327
CDR128
CDR229
CDR330
3G7CDR133
CDR234
CDR335
CDR136
CDR237
CDR338

In Table 3, are given in the description below, in this respect, the compliance of the IU what do these same antibodies according to the invention and corresponding amino acid sequences of the heavy and light chains of these antibodies.

Table 3
AntibodyHeavy chainLight chainSEQ ID No
Aa7
a8
Aa15
a16
Ea23
a24
Sa31
a32
3G7a39
a40

And finally, the following Table 4 of this description summarizes the names of each of the antibodies according to the invention with a Deposit rooms at the CNCM.

Table 4
HybridomaDeposit No. in CNCM
A1-3612
A1-3613
E1-3614
S1-3615
3G71-3616

Undoubtedly, the entire set of properties or modifications described above for antibodies A, is applicable to other antibodies according to the invention, and more specifically to antibodies, identified as A, E, S and 3G7.

In the same way as described above for any of the antibodies obtained according to the method according to the invention, also found that the entire set of desired characteristics, properties or modifications should also be considered as applicable to the antibodies identified in this application.

More specifically, it is established that any functional fragment is selected from the FR is mentov Fv, scFv, Fab, (Fab')2, Fab', scFv-Fc and dietel or any part of, the duration of which would be increased, for example Paglierani fragments.

Preferably, the antibody of the present invention is preferably represented the antibody or one of its functional fragments, Sekretareva(th), one of the hydride, as described above, i.e. by hybridomas I-3612, 3613, 3614, 3615, or I-3616.

According to the next embodiment of the invention, the specified antibody is a chimeric antibody and further comprises a constant region of the light chain and heavy chain derived from heterologous antibodies to mice type.

Preferably, the specified heterologous species is the human species.

Even more preferred that the chimeric antibody or one of its functional fragments according to the invention is characterized by the fact that the constant region of the light chain and heavy chain derived from human antibodies, represent the areas of Kappa and gamma-1, gamma-2 or gamma-4, respectively.

And finally, even more preferably, the specified antibody is humanitariannet antibody and contains a light chain and/or heavy chain, while the segments FR1-FR4 frame specified light chain and/or heavy chains come from segments FR1-FR4 frame, respectively, egcoa chain and/or heavy chains of human antibodies.

In a particular aspect the present invention relates to a chimeric antibody or one of its functional fragments according to the invention, characterized in that said antibody further comprises a constant region of the light chain and heavy chain derived from heterologous antibodies to mice species, especially humans, and it is preferable that the constant region of the light chain and heavy chain derived from human antibodies, represent the areas of Kappa and gamma-1, gamma-2 or gamma-4, respectively.

According to further aspect of the invention described selected nucleic acid, characterized in that it is chosen from the following nucleic acids:

a) nucleic acid, DNA or RNA) that encodes the antibody or one of its functional fragments according to the invention;

b) a nucleic acid complementary to the nucleic acid defined in a);

c) a nucleic acid consisting of at least 18 nucleotides capable of gibridizatsiya in conditions of severe austerity at least one of the CDRs of sequence SEQ ID No 41-46, 49-54, 57-62, 65 to 70, 73-78, or with a sequence having at least 80% identity after optimal alignment with sequences SEQ ID No 41-46, 49-54, 57-62, 65 to 70, 73-78; and

d) a nucleic acid consisting of at least 18 well is Leonidov, can gibridizatsiya in conditions of severe austerity at least one of the light chain of sequence SEQ ID No 48, 56, 64, 72 or 80 and/or one heavy chain sequence of SEQ ID No 47, 55, 63, 71 or 79, or a sequence having at least 80% identity after optimal alignment with sequences SEQ ID No 47, 48, 55, 56, 63, 64, 71, 72, 79 or 80. The terms "nucleic acid", "nucleic acid sequence" or "nukleinovokisly sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence", "nucleotide sequence", which will be equally used in the present description, consists in indicating the specific binding of nucleotides or modified or not, by which it is possible to define a fragment or a region of nucleic acid, which either include non-natural nucleotides, or not, and which may correspond to as double-stranded DNA and single-stranded DNA and the transcription products of the indicated DNA.

It also should be understood that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. under natural conditions. These are sequences that are isolated and/or purified, i.e. selected as samples, directly or indirectly, for example, by copying, while the environment is at least partially modified. The meaning is also in the notation in this description of the selected nucleic acid obtained by genetic recombination with host cells, for example, or obtained by chemical synthesis.

Nucleic acid sequence having a percentage identity of at least 80%, preferably 85%, 90%, 95% and 98%, after optimal alignment with the preferred sequence, are intended to refer to nucleic acid sequences with relatively the reference nucleic acid sequence some modifications, such as in particular a deletion, cut, paste, chimeric fusion and/or especially to change the type of point-like substitution). They are preferably sequences that encoded the same amino acid sequence as the reference sequence, and this is due to the degeneracy of the genetic code, or complementary sequences, which can be specific to gibridizatsiya with the preferred reference sequence under conditions of severe austerity, especially as hereinafter defined.

Hybridization under conditions of severe austerity means that the conditions of temperature and ionic strength selected in such a way that allow to maintain the hybridization between two complementary DNA fragments. As an illustration, a harsh austerity phase hybridization to determine polynucleotide fragments described above, preferably are as follows.

DNA-DNA or DNA-RNA hybridization is carried out in two stages: (1) prehybridization at 42°C for 3 hours in phosphate buffer (20 mm, pH 7.5)containing 5×SSC (1×SSC corresponds to a 0.15 M NaCl+0.015 G M solution of sodium citrate), 50% formamide, 7% of dodecylsulfate sodium (SDS), 10 × denhardt's solution, 5% doctranslate and 1% of the DNA from salmon ROE; (2) real hybridization for 20 hours at a temperature depending on the size of the probe (i.e.: 42°C for probe size >100 nucleotides), followed by two 20-minute washes at 20°C in 2×SSC+2% SDS, 20-minute wash at 20°C in 0.1×SSC+0.1% of SDS. The last washing is carried out in 0.1×SSC+0.1% of SDS for 30 minutes at 60°C for probe size >100 nucleotides. Conditions of hybridization of tough austerity described above for polynucleotide a certain size, can be adapted by a person skilled in the technical field for oligonucleotides larger or smaller in accordance with the manual Sambrook and others (1989, Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor).

For greater clarity, the correspondence between the antibodies according to the invention, more specifically the CDR sequences, as well as flexible circuits and their nucleotide pic what egovernance also summarized in Table 5, below.

53
Table 5
AntibodyHeavy chainLight chainSEQ ID No
ACDR141
CDR242
CDR343
CDR144
CDR245
CDR346
ACDR149
CDR250
CDR351
CDR152
CDR2
CDR354
E
CDR157
CDR258
CDR359
CDR160
CDR261
CDR362
SCDR165
CDR266
CDR367
CDR168
CDR269
CDR370
3G7CDR173
CDR274
CDR375
CDR176
CDR277
CDR378
Aa47
a48
Aa55
a56
Ea63
a64
Sa 71
a72
3G7a79
a80

The invention also relates to a vector containing a nucleic acid of the present invention.

The invention particularly relates to the cloning and/or expression vectors containing the nucleotide sequence according to the invention.

The vectors according to the invention preferably include elements that allow the expression and/or secretion of a nucleotide sequence in a particular host cell. Therefore, the vector should include a promoter, signal the initiation and termination of translation, as well as appropriate areas for the regulation of transcription. He has stably maintained in the host cell and may possibly have specific signals for specific secretion of the translated protein. These different elements are selected and optimized by a specialist in the art depending on the host cell. To this end a nucleotide sequence according to the invention can be inserted in sa is replicalouis vectors in the selected host or present an integrative vectors selected host.

Such vectors obtained by the methods currently used by an expert in the field of technology, and obtained a clone can be entered in a suitable host by standard methods, such as lipofection, electroporation, thermal shock or chemical methods.

Vectors according to the invention, for example, are the vectors of plasmid or viral origin. They are useful for transformation of host cells with the aim of cloning and expression of the nucleotide sequences according to the invention.

The invention also includes host cells transformed by the vector or containing the vector according to the invention.

A host cell may be selected from prokaryotic or eukaryotic systems, such as bacterial cells, but also cells of yeast or animal cells, in particular mammalian cells. You can also use insect cells or plant cells.

In addition, the invention relates to animals, except humans, containing the transformed cell according to the invention.

In another aspect the invention is a method of obtaining antibodies or one of its functional fragments according to the invention, characterized in that it comprises the following stages:

a) culturing the host cell according to the invention in the environment and in suitable culture conditions; and

<> b) the allocation thus obtained indicated antibodies or one of their functional fragments from culture medium or from these cultured cells.

Transformed cells according to the invention can be used in the methods of production of recombinant polypeptides according to the invention. The actual methods for producing the polypeptide according to the invention in recombinant form, characterized by the fact that they used the vector and/or a cell transformed by the vector according to the invention included in the present invention. Preferably, the cell transformed by the vector according to the invention is cultivated under conditions that allow to be expressed to the specified polypeptide, and the indicated recombinant peptide allocate.

As established, a host cell may be selected from prokaryotic or eukaryotic systems. In particular, we can identify nucleotide sequences according to the invention, which facilitates secretion in such prokaryotic or eukaryotic system. Therefore, the vector according to the invention, bearing the following sequence can be preferably used for producing recombinant proteins destined for secretion. Indeed, treatment of these interest recombinant proteins, it will be easier because they are more settled largely in the supernatant of the cell culture, than inside the cells of the host.

The polypeptides according to the invention can also be obtained by chemical synthesis. This method of obtaining is an objective of the invention. Specialist in the art is aware of methods of chemical synthesis, such as methods that use solid phase (see especially Steward et al, Solid phase peptides synthesis, Pierce Chem. Company, Rockford, 111, 2nd ed. (1984)), or methods that use partially solid phase condensation of fragments or synthesis in normal solution. The polypeptides obtained by chemical synthesis and which can include the corresponding unnatural amino acids are also included in the invention.

Antibodies, or one of their functional fragments, which can be obtained by the method according to the invention, are also included in the present invention. Bispecific or bifunctional antibodies form a second generation of monoclonal antibodies that have the same molecule combined two different variable regions (Hollinger and Bohlen, 1999, Cancer and metastasis rev. 18: 411-419). Their applicability has been demonstrated both in the field of diagnosis and therapy due to their ability to recruit new effector functions or targeted delivery to different molecules on the surface of tumor cells. These antibodies can be obtained by chemical methods (Glenne MJ et al., 1987, J. Immunol. 139, 2367-2375; Repp R. et al, 1995, J. Hemat. 377-382) or physical (Staerz U D. and Bevan MJ., 1986, PNAS 83, 1453-1457; MR Suresh. et al, 1986, Method Enzymol., 121: 210-228), but also and preferably using techniques of genetic engineering, which heterodimerization can be forced and which therefore facilitate the purification method of the desired antibody (Merchand et al., 1998, Nature Biotech., 16: 677-681).

These bispecific antibodies can be constructed in the form of whole IgG, as bispecific Fab'2in the form Fab'-PEG or datel or even bispecific scFv, as well as tetravalent bispecific antibodies or antibody with two binding sites for each target antigen (Park et al., 2000, Mol. Immunol., 37(18): 1123-30) or its fragments, as described above.

In addition to the economic benefits that the acquisition and introduction bispecific antibodies is less expensive than getting two specific antibodies, the use of such bispecific antibodies has the advantage of lowering the toxicity of treatment. Indeed, the use of bispecific antibodies can reduce the total number of circulating antibodies and, therefore, possible toxicity.

In the preferred embodiment of the invention bispecific antibody is a bivalent or tetravalent antibody.

In practice, the benefits from using terava entogo bispecific antibodies is it has a greater avidity compared to bivalent antibody due to the presence of two binding sites for each target.

Similarly, for selection of functional fragments of the antibodies described above, the specified second structure selected from the fragments Fv, Fab, (Fab')2, Fab', Fab'-PEG, scFv, scFv-Fc and dietel or any form, the duration of which would be increased.

In still another aspect the invention is an antibody or one of its functional fragments according to the invention as a drug, preferably humanitariannet antibody, which is defined previously. In the framework of the present description under antibody understand how the antibody obtained by the use of certain of the above method according to the invention and the antibody is selected from the identified and called antibodies A, A, A, S or 3G7.

The invention also relates to pharmaceutical compositions containing as active ingredient the compound constituting the antibody or one of its functional fragments according to the invention, preferably with added pharmaceutically acceptable excipients and/or the media.

More specifically, the invention encompasses a composition comprising as active ingredient the compound constituting the antibody, and is one of its functional fragments, as, for example, as described above or produced by hybridoma I-3612, 3613, 3614, 3615, or I-3616.

According to still one another embodiment of the present invention also relates to pharmaceutical compositions described above, optionally containing as a combination product for simultaneous, separate application or posted in time of application of the chemotherapeutic agent, the agent is radiation therapy or antibody. Under "concurrent use" means the introduction of the two compounds of the composition according to the invention is contained in one and the same pharmaceutical form.

Under the "single use," see the introduction to one and the same time both compounds the composition of the invention contained in separate pharmaceutical forms.

Under the "remoteness in time of the use of" understand the sequential introduction of the two compounds of the composition according to the invention, each of which is contained in a separate pharmaceutical form. Under chemotherapeutic agent understands any compound falling within the definition and the list shown above in the present description and which forms an integral part of the invention.

In a particular preferred embodiment of this composition as a combination product according to the invention is characterized by the fact that the cytotoxic who the Gent is linked by a chemical bond with the indicated antibody for simultaneous use.

In a particular preferred embodiment of the specified composition according to the invention is characterized by the fact that said cytotoxic/cytostatic agent is selected from agents that inhibit or stabilize the spindle, preferably vinorelbine and/or vinflunine.

In order to facilitate the formation of ties between the specified cytotoxic agent and the specified antibody according to the invention between the two link connections may particularly be entered spacer elements molecules, such as polyalkylene glycols such as polyethylene glycol, or even amino acids, or in another embodiment can be used active derivatives of these cytotoxic agents that have the functional group capable of interacting with the indicated antibody according to the invention. These combination techniques well known to the specialist in the art and will not be disclosed in the present description.

The invention in another aspect relates to a composition, characterized in that at least one of these antibodies, or one of their functional fragments represents a conjugate with a cell toxin and/or a radioactive element.

Preferably, the specified toxin is a toxin from enterobacteria, especially exotoxin a from Pseudomonas.

The conjugate of a toxin or radioactive e the ment of at least one antibody or one of its functional fragments according to the invention is intended to denote any means, with the help of which the specified toxin or specified radioactive element can be associated with at least one specified antibody, particularly through the formation of covalent bonds between the two compounds, with the introduction or without the introduction of a binding molecule. Another form of binding may consist in the application of chelate compounds with ion (ion chelator), providing non-covalent complexation, such as, for example, EDTA, DOTA, or even complex type99mTc.

Also preferably, the specified at least one antibody that is involved in the education of the specified conjugate according to the invention, was selected from its functional fragments, particularly fragments, separated from their Fc component, such as scFv fragments.

The present invention additionally includes the use of a composition according to the invention for the manufacture of drugs.

The present invention is also the use of the antibodies described above or even obtained according to the method according to the invention, also described above, for the preparation of medicaments intended for the prevention or treatment of cancer.

In addition, the present invention is the use of antibodies, which are described above or even obtained according to the method according to the invention, and as described above, for the manufacture of drugs intended for inhibiting the resistance of tumors to anticancer treatment in a patient within the framework of the prevention and treatment of cancer in this patient.

Preferably, the specified cancer is a cancer resistant type selected from cancer of the colon, prostate, breast, lung, testicular, or pancreas.

According to another embodiment of the invention also stated the use of the antibody or one of its functional fragments according to the invention for the manufacture of drugs intended for specific targeted delivery of biologically active compounds for drug-resistant tumors and/or tumors detected at a late stage. Labeled antibodies according to the invention or their functional fragments include, for example, so-called immunoconjugates antibodies, which may be, for example, conjugated with enzymes like peroxidase, alkaline phosphatase, α-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malatdegidrogenaza or glucose-6-phosphatedehydrogenase, or with such molecule as Biotin, digoxygenin or 5-bromosuccinimide. With antibodies or their functional fragments according to the invention can also be conjugated to fluorescent markers, and they specifically include fluorescein and its derivatives, fluorochrome, rhodamine and its derivatives, GFP (green fluorescent protein), dansyl, umbelliferone etc. Such conjugates with antibodies according to the invention and the and their functional fragments can be obtained by means well-known specialist in this field of technology. They (antibodies) can be associated with enzymes or fluorescent markers directly or through a spacer elements group or a linking group, such as polyallelic, such as glutaric aldehyde, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), or in the presence of the agents of the combination, such as periodic etc. Conjugates comprising markers fluoresceine type can be obtained in the reaction with isothiocyanates.

Other conjugates can also include chemiluminescent markers, such as luminal and dioxetane, bioluminescent markers, such as luciferase and luciferin, or even radioactive markers.

The biologically active compound is understood to refer in this description of any compounds capable of modulating, especially inhibiting the activity of the cells, in particular their growth, proliferation, gene transcription or translation.

In the present description pharmaceutically acceptable diluent is understood to refer to compounds or combinations of compounds included in the pharmaceutical composition, which does not cause adverse reactions and which, for example, can contribute to the introduction of the active compound(s)may increase the duration of its existence is its effectiveness in the body, can increase its solubility in solution or may even improve its safety. These pharmaceutically acceptable carriers well known and will be adapted by professionals in the art depending on the nature and method of implementation of the selected active connection(s).

Preferably, these compounds will be introduced systemically, in particular by intravenous, intramuscular, intradermal, intraperitoneal or subcutaneous route, or oral. More preferably, the composition comprising the antibody according to the invention will be entered repeatedly, with the passing time.

Methods of administration, dosage forms and optimal glenavy forms can be determined according to the criteria generally taken into consideration when establishing a treatment tailored to the patient, such as age or body weight of the patient, the severity of his/her status, tolerability and marked side effects.

Other characteristics and advantages of the invention will be apparent from the following description in the form of examples and graphic materials, including signatures, which are listed below.

And finally, according to the last aspect of the invention the application of the method according to the invention for the identification of new therapeutic and/or diagnostic intra - or extracellular targets, vowl is received in the phenomenon of resistance.

And finally, the identification of new therapeutic and/or diagnostic intra - or extracellular targets involved in the phenomenon of resistance, characterized by the fact that it represents the application of the above-described method according to the invention with the purpose of obtaining monoclonal antibodies and the subsequent identification of compounds, in particular protein, recognized by the specified monoclonal antibody.

Such identification connections, especially protein, can be carried out by any method known to a person skilled in the art such as, for example, immunoprecipitate and/or immunohisto cell lysate and protein identification using techniques Western blotting in combination with standard proteomic methods.

Caption graphic materials and examples, the following are intended to illustrate the invention without limiting in any way the volume.

CAPTION GRAPHIC MATERIALS

Figure 1: figure 1 illustrates the change in time of tumor volume after conducting triple therapy using h4D5, 225 and S and shows the emergence of resistance to these treatments.

Figure 2: figure 2 illustrates, in the first illustrative form chart illustrating an embodiment of the invention.

1. The sampling control tumors, divide by d the e parts.

2. Tolerization mice. Preparation of cells for immunization: lysate, cell suspensions, etc. Tolerization by/b (intraperitoneal) injection of cyclophosphamide or other topersuade agent.

3. Slides for IHC(immunohistochemical)-screening. Freezing or enable (paraffin) tumors: preparation of slides for tagging = negative control slides.

4. Sampling resistant tumors, the division into two parts.

5. Preparation of cells for immunization: lysate, cell suspensions, etc.

6. Freezing or enable (paraffin) tumors: preparation of slides for tagging = test slides.

7. The sampling control tumors.

8. Immunization of mice: preparation of cells from control tumors.

9. Control IHC-slides.

10. Tolerization by injection(s) immunosuppressive agent.

11. Samples of sera to control the lack of response to native cells.

12. Sampling resistant tumors.

13. Immunization tolerized mice: preparation of cells from resistant tumors.

14. Tested IHC-slides.

15. Samples of sera for IHC-tagging tumors in comparison with resistant tumors.

16. Breeding hybrid: no tagging on slides to control the different tumors, positive staining for resistant tumors (no tagging on stromal cells, staining of tumor cells).

Figure 3: figure 3 also illustrates, but this time in the form of a diagram, an embodiment of the method according to the invention.

Figure 4: figure 4 illustrates the profile of the antibodies according to the invention on slides type "tissue array", created from tumors, which are used for tolerization and immunization.

Figure 5: figure 5 illustrates the evaluation of antitumor activity of antibodies A and E in xenotransplanted colon NT.

6: figure 6 shows the corresponding sequence of the heavy and light chains of antibodies A, A, A, S and 3G7. The corresponding CDRs are underlined and in bold.

7: figure 7 illustrates the evaluation of antitumor activity of antibodies E in xenotransplanted pancreas Whrs.

Example 1. Demonstration of resistance of tumor cells A to triple therapy with antibodies against IGF-1R, EGFR and Her/2neu

Spent transplantation A-cells in mice. After injection of the tumor, the mice were subjected to treatment twice a week with a mixture containing 300 µg of each of the following antibodies, ie: h4D5 (Herceptin provided by ICR, Institut Claude Regaud, service pharmacie, 20-24 rue du Pont Saint-Pierre, 31052 Toulouse), 225 (ATCC no HB-8508) and S.

He later first week of treatment in some of the s mice can be observed regression of tumor growth until the complete disappearance of the tumor. Treatment support despite the complete or partial regression of the tumor.

Subsequently see the re-appearance of these tumors despite the continuity of treatment, which shows the formation of tumors that are resistant to triple therapy.

Figure 1 illustrates the example in which demonstrated tumor regression to 15 days and the re-emergence of tumors, since 15 days, which indicates that the development of resistance.

Example 2. Creating active monoclonal antibodies directed against the antigen involved in resistance to combination therapy with antibodies against IGF-1R, EGFR and Her/2neu

Spent transplantation A cells two batches of 10 mice. The first party receives fortnightly injections of PBS (phosphate buffered saline). The second part is treated with a combination of antibodies (C, 225 and h4D5)directed against receptors IGF-1R, EGFR and Her/2neu, respectively. This combination of antibodies causes tumor regression, leading to the disappearance of these tumors in 60% of treated animals. Despite such a radical effect of the tumor again occur in all animals.

In animals treated with PBS, A-tumor extract, when they reach 100-200 mm3.

Each of these tumors are divided into two parts: one freeze at low temperatures to create immunol tokimeki slides type "tissue array", another part is used to tolerization BALB/c mice. This tolerization is performed by intraperitoneal injection of crushed homogenate of cells from the tumor, followed by the introduction of cyclophosphamide and its goal is the complete eradication response against untreated "source" A-tumors. During this tumor recurrence also collect tumors, when they reach a volume of 100-200 mm3. Then they are divided into two equal parts. One part will be frozen at low temperatures to create immunohistochemical slides type "tissue array", as described above for the control of tumors using PBS. The second part will be cooked milled homogenate and used for immunization of mice tolerized in advance untreated A-tumors. For all of these all 3 mice received intraperitoneal injections of crushed homogenates of tumors that are resistant to triple therapy, were reinforced complete adjuvant's adjuvant for the first injection and incomplete adjuvant's adjuvant for subsequent injections.

Then perform the merge cells and the resulting merge hybrid conduct screening using immunohistochemical analysis on slides type "tissue array", prepared in advance.

Hybridoma, secreting ant the body, recognizing resistant fabric and does not recognize any of the tumor from the control of the party with PBS or native A cage, which was used to generate tumors in vivo, clone and freeze. Then antibodies are produced in ascitic fluid, cleaned, re-testing immunohistochemically, and then tested in vivo in cells expressing on the surface structure or the antigen recognized by antibodies (these cells were selected in advance using FACSCAN analysis of non-invasive panel of tumor lines available for each antibody).

Figure 4 shows the profile held antibodies.

The following Table 6 in this respect, the grouped cells are recognized by these antibodies (Detection antibody selected in (data) histology using a FACSCAN device, different cell lines).

Table 6
HybridomaCloneThe isotypeIn vivo model
AC7IgM kMCF7/Colo205
D10LnCap/HT29
AE11IgG1 kHepG2/H69/LnCap
F1
EA2IgG1 kHt29/BxPC3/H69
B11
EA8IgG1 kHt29/BxPC3/H69
B9
SE8IgM kColo205
E
3G7A7IgMDU145
B2

Example 3. Evaluation of antitumor activity of antibodies generated according to the invention (more specifically A and E) in the model of xenotransplanted rectum NC

5·106/sup> cells NT transplanted mice Swiss-Nude". Five days after transplantation of the tumor become measurable, and mice are divided into three parties of six mice with tumors of homogeneous type. Mice subjected to treatment with either PBS (negative control)or 0.5 mg of antibody A or E, three times a week (injection of the first dose for the presentation: 1 mg/mouse).

The control tumor volume exercise two times per week, taking into account that the volume of the tumor is usually calculated according to the following formula: (π/6)×(I)×(L)×(e), where I = measured width, L = measured length and e = measured thickness.

The results are illustrated in figure 5.

T-cells can be considered as cell aggressive phenotype. The results obtained demonstrate the possibility of using antibodies directed against the antigens of tumor cells derived from tumors treated with anti-tumor composition (see Example 2) and also sustainable, as antitumor compounds for the treatment of tumors with an aggressive phenotype.

The results obtained in this experiment, confirm the correctness of this original approach to generate antibodies and the concept of creation of antibodies for therapeutic purposes based on induced in mice sustainability. This method can be applied to any other combination Lakers the governmental funds, including chemotherapeutic drugs, either individually or in combination with each other or with antibodies or tyrosine kinase inhibitors or proteasome inhibitors, acting the same way.

Example 4. Evaluation of antitumor activity of antibodies E created according to the invention, in the model of xenotransplanted pancreas Whrs

10·106cells Whrs transplanted Nude "Nude" mice. Five days after transplantation of the tumor become measurable, and mice are divided into two parties of six mice with tumors of homogeneous type. Then mice subjected to treatment with either PBS (negative control), or antibody A (1 mg/dose)twice a week. The control tumor volume exercise twice a week.

The results are illustrated in Fig.7.

The results obtained in this experiment, justify the functional activity of antibodies A, as well as the concept of creating antibodies for therapeutic purposes based on induced in mice stability.

1. Method of creating an antibody or one of its functional fragments, directed against the tumor antigen expressed on the surface of a tumor resistant to at least one anticancer compound, characterized in that it comprises the following stages:
i) direct rodstvennuju immunization of animals crushed homogenate, and/or suspension and/or cell lysate derived from resistant tumors
ii) the fusion of spleen cells of an animal immunized at stage i), with myeloma cells to obtain hybridomas,
iii) selecting antibodies secreted by the hybridomas,
iv) the selection by differential selection (differential selection hybridomas secreting antibodies that recognize specific antigens expressed on the surface of tumor cells resistant tumors, and the expression of which is induced as the result of cancer treatment, where indicated resistant tumor obtained by a method selected from the following: (a) biopsy and/or surgery from the patient, which passes or has passed therapeutic treatment using said at least one anticancer compound capable to induce the resistance of the tumor, b) transplantation of tumor lines and/or all human tumors or part of the animal and then treatment of this animal by introducing at least one anticancer compound, stability to which it is desirable to induce, and
v) if necessary, obtain functional fragments of the selected antibodies.

2. The method according to claim 1, characterized in that the specified conduct immunization by intraperitoneal and/or subcutaneous, and/or nutrion the th, and/or vnutrikletochnoi injection.

3. The method according to claim 1, characterized in that said antibody or one of its functional fragments, directed(th) against the tumor antigen expressed on the surface of the specified drug-resistant tumors, is a monoclonal antibody.

4. The method according to claim 1, characterized in that said antibody or one of its functional fragments is an immunoglobulin selected from the group IgG, IgA, IgM, IgD or IgE.

5. The method according to claim 4, characterized in that said antibody or one of its functional fragments is an IgG isotype gamma 1, gamma-2 or gamma-4.

6. The method according to claim 1, characterized in that the said functional fragment is selected from the fragments Fv, Fab, (Fab')2, Fab', scFv (single-chain Fv), scFv-Fc and dietel or any part of, the duration of which would be increased, such as Paglierani fragments.

7. The method according to claim 1, where the specified drug-resistant tumor obtained by transplantation of tumor lines and/or all human tumors or part of the animal and then treatment of this animal by introducing at least one anticancer compound, stability to which it is desirable to induce, characterized in that it further includes a front stage i) after the ith stage:
a) selection and transplantation of animal tumor lines and/or all of the original tumor or its parts,
b) treatment of these subjected to transplant animal at least one anticancer compound,
c) removing the whole or parts of the original tumor, originating from untreated animals subjected to transplantation at the stage a),
d) removing the whole or parts of resistant tumors originating from animals treated at the stage b),
e) preparation of tools for the differential selection of antibodies from tumors, fully or partially extracted in stages C) and d), respectively, and
f) preparation of milled homogenate and/or a cell lysate of resistant tumors, fully or partially extracted in stage d).

8. The method according to claim 7, characterized by the fact that this tumor line and/or the original tumor is selected from the group consisting of cancer cells of the lung, colon, prostate, breast, ovarian, or any tumor resistance to treatment is installed.

9. The method according to claim 1, characterized in that the at least one anticancer compound selected from chemotherapeutic agents, agents, radiation therapy, hormonotherapy agents, chemical molecules as tyrosine kinase inhibitors or antibodies or where indicated the data anticancer treatment is a radiotherapy.

10. The method according to claim 1, characterized in that the at least one anticancer compound preferably is a monoclonal antibody.

11. The method according to claim 1, characterized in that said antibody is a monoclonal antibody, and the fact that it is selected from the group of antibodies or their functional fragments, directed against receptors of growth factors, molecules involved in angiogenesis, or even chemokines and integrins involved in cell migration phenomenon.

12. The method according to claim 1, characterized in that the at least one anticancer compound is a combination of at least two, preferably at least three of antitumor compounds of different nature and/or having different mechanisms of action, and/or directed to different proteins.

13. The method according to item 12, characterized in that the specified combination of anticancer compounds is a combination of monoclonal antibodies or their functional fragments.

14. The method according to item 13, characterized in that the specified combination of monoclonal antibodies or their functional fragments is a combination of an antibody selected from antibodies against IGF-1R (receptor insulin-like growth factor type 1)against EGFR (receptor epiderm is a high growth factor), against Her/2neu, against VEGF (vascular endothelial growth factor), against VEGFR (VEGF receptor), against CXCR (receptors chemokines type C-X-C), against estimates against RON (recepteur d'origine nantais).

15. The method according to 14, characterized in that the specified combination is a combination of antibodies against IGF-1R antibodies against EGFR and antibodies against Her/2neu.

16. The method according to item 15, characterized in that the specified combination is a combination of monoclonal antibodies S, 225 and h4D5.

17. The method according to claim 1, characterized in that the specified stage iv) for selection of hybridomas secreting antibodies that do not recognize native antigens of tumor cells, is accomplished by differential screening of antibodies secreted by the hybridomas, between the native tumor and resistant tumor.

18. The method according to 17, characterized in that the differential screening is carried out by using a product obtained in stage e) of the method according to claim 7.

19. The method according to any one of claims 1 to 18, characterized in that it includes the additional step of weakening the immune response induced by immunosuppressive compounds before stage i).

20. The method according to claim 19, characterized in that the said stage of tolerization consists of:
- introduction to the animals milled homogenate and/or suspension, and/is the cellular lysate, derived from the so-called "native" tumor, originating from stage (C), and
treatment of these animals with immunosuppressant to remove b cells, activated in the introduction, held at the previous stage, thereby inhibiting any possible response against specified the so-called native tumor.

21. The way of generating and screening antibody or one of its functional fragments, capable to inhibit the resistance of tumors to anticancer compound and/or directed against a tumor antigen expressed on the surface of a tumor resistant to at least one anticancer compound, and specified a tumor antigen involved in the resistance specified tumor to the specified antitumor compound, and the method is characterized by the fact that it includes:
a) method of creating an antibody or one of its functional fragments according to any one of claims 1 to 18, with the indicated antibody directed against the indicated tumor antigen-specific expressed on the surface of the resistant tumors indicated a tumor antigen is not expressed on the cell surface of native tumor from which this resistant tumor occurs;
b) bringing obtained in stage a) of the antibody into contact with a tumor that is resistant to the anti-Christ. wholesome connection; and
(C) the selection of the specified antibodies, if demonstrated inhibitory effect of this antibody on the resistance of tumors to anticancer connection.

22. Application of the method according to any one of claims 1 to 21 for the creation of therapeutic monoclonal antibodies.

23. Monoclonal antibody directed against a tumor antigen expressed on the surface of a tumor resistant to at least one anticancer compound, or one of its functional fragments, obtained by applying the method according to item 15.

24. Monoclonal antibody according to item 23, characterized in that the said functional fragment is selected from the fragments Fv, Fab, (Fab')2, Fab', scFv, scFv-Fc and dietel or any part of, the duration of which would be increased, such as Paglierani fragments.

25. Monoclonal antibody or one of its functional fragments according to item 23, characterized in that selected from the group of antibodies consisting of:
(a) antibodies, which contains:
- light chain contains the CDR-areas (hypervariable sites) sequences SEQ ID No 1, 2 and 3; and
- heavy chain contains the CDR portions of sequences SEQ ID nos 4, 5 and 6;
(b) antibodies, which contains:
- light chain contains the CDR portions of the sequences SEQ ID No 9, 10 and 11; and
- heavy chain contains the CDR-sections of th is of Donatella SEQ ID No 12, 13 and 14;
(C) antibody, which contains:
- light chain contains the CDR portions of the sequences SEQ ID No 17, 18 and 19; and
a heavy chain contains the CDR portions of sequences SEQ ID nos 20, 21 and 22;
(d) antibodies, which contains:
- light chain contains the CDR portions of sequences SEQ ID nos 25, 26 and 27; and
- heavy chain contains the CDR portions of the sequences SEQ ID No 28, 29 and 30;
(d) antibodies, which contains:
- light chain contains the CDR portions of sequences SEQ ID nos 33, 34 and 35; and
- heavy chain contains the CDR portions of the sequences SEQ ID No 36, 37 and 38.

26. The antibody or one of its functional fragments according A.25, characterized in that said antibody is a chimeric antibody and further comprises a constant region of the light chain and heavy chain derived from heterologous antibodies to mice type.

27. The antibody or one of its functional fragments according p, characterized in that said antibody is a chimeric antibody and a specified heterologous species is the human species.

28. The selected nucleic acid, characterized in that it is chosen from the following nucleic acids:
a) nucleic acid, DNA or RNA) that encodes the antibody or one of its functional fragments according A.25;
b) a nucleic acid complementary nekleenov the th acid, defined in a).

29. Expressing a vector containing a nucleic acid according p.

30. Isolated a host cell containing a vector according to clause 29, to produce recombinant antibodies.

31. A method of producing an antibody or one of its functional fragments according A.25, characterized in that it comprises the following stages:
a) culturing cells according to item 30 in the environment and in suitable culture conditions; and
b) the allocation thus obtained indicated antibodies or one of their functional fragments from culture medium or from these cultured cells.

32. Antitumor composition containing as active ingredient an effective amount of the compound constituting the antibody or one of its functional fragments according A.25.

33. The composition according to p, characterized in that it further comprises as a combination product for simultaneous, separate applications or remoteness in time of application of the chemotherapeutic agent, the agent is radiation therapy or antibody.

34. The use of a composition according p as medicine.

35. The use of a composition according to clause 34 for the manufacture of medicaments intended for the prevention or treatment of cancer.

36. Use p, characterized in that the specified cancer represents the t of a cancer resistant type, selected from cancer of the colon, prostate, breast, lung, ovarian or pancreatic cancer.

37. The use of antibodies or one of its functional fragments according A.25 for the manufacture of drugs intended for specific targeted delivery of biologically active compounds for drug-resistant tumors and/or tumors detected at a late stage.

38. Mouse hybridoma able to secrete antibody on A.25 deposited in the CNCM (Collection Nationale de Cultures de Microorganismes), Institut Pasteur, Paris (France) under the number I-3612.

39. Mouse hybridoma able to secrete antibody on A.25 deposited in the CNCM (Collection Nationale de Cultures de Microorganismes), Institut Pasteur, Paris (France) under the number I-3613.

40. Mouse hybridoma able to secrete antibody on A.25 deposited in the CNCM (Collection Nationale de Cultures de Microorganismes), Institut Pasteur, Paris (France) under the number I-3614.

41. Mouse hybridoma able to secrete antibody on A.25 deposited in the CNCM (Collection Nationale de Cultures de Microorganismes), Institut Pasteur, Paris (France) under the number I-3615.

42. Mouse hybridoma able to secrete antibody on A.25 deposited in the CNCM (Collection Nationale de Cultures de Microorganismes), Institut Pasteur, Paris (France) under the number I-3616.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: chimeric monoclonal antibody is described, which specifically connects to human erythropoietin, characterised by the following criteria: a) Kd=2.4×10-9 M, isoelectric point in the range pH 7.5-8.0; b) sequence of the heavy chain SEQ ID NO:12; c) sequence of the light chain SEQ ID NO:14. A mouse hybridome strain is proposed, which is a producent of a monoclonal antibody to human erythropoietin, deposited in the Russian Academy of Agricultural Sciences, Specialised Collection of Cell Cultures of Farm and Game Animals under the No.84. Also a mouse anticlonal antibody is described, which specifically connects to human erythropoietin, produced by the specified hybridome and characterised by the following criteria: a) Kd=0.95×10-9 M, molecular weight = 160 kD, isopoint in the range pH 6.8-7.1; b) sequence of variable area of light chain SEQ ID NO:1; c) sequence of variable area of heavy chain SEQ ID NO:2; d) sequence of areas that define antibody complementarity: CDRH-1 - SEQ ID NO:5, CDRH-2 - SEQ ID NO:6, CDRH-3 - SEQ ID NO:7, CDRL-1 - SEQ ID NO:8, CDRL-2 - SEQ ID NO:9, CDRL-3 - SEQ ID NO:10.

EFFECT: invention makes it possible to expand arsenal of mouse antibodies against human erythropoietin.

3 cl, 3 dwg, 5 ex, 2 tbl

FIELD: biotechnologies.

SUBSTANCE: two antibodies against IL-21 of a human being are presented. The first antibody includes a variable region of a heavy chain, which includes SEQ ID NO: 31, 33 and 35, and a variable region of a light chain, which includes SEQ ID NO: 39, 41 and 43. The second antibody includes a variable region of heavy chain, which includes SEQ ID NO: 47, 49 and 51, and variable region of light chain, which includes SEQ ID NO: 55, 57 and 59. Besides, the invention describes hybridomes producing the first and the second antibodies against IL-21 of a human being and deposited in the collection of cultures "American Type Culture Collection" and have numbers "ATCC Patent Deposit Designation PTA-8790" and "ATCC Patent Deposit Designation PTA-8786" respectively.

EFFECT: invention allows obtaining antibodies to IL-21 of a human being.

48 cl, 4 dwg, 16 tbl, 23 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: this invention relates to biotechnology and immunology. One proposes: JAM-A protein antibody or functional fragment thereof, hybridoma secreting such antibody, nucleic acid, expression vector and host cell as well as a method for the antibody and composition production. One considers application of the JAM-A protein antibody or functional fragment thereof.

EFFECT: invention usage ensures creation of new JAM-A protein antibodies which may be further applied in treatment or prevention of diseases related to proliferation of tumour cells extracting JAM-A protein.

34 cl, 31 dwg, 5 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: invention presents cultivated hybrid cell strains of Mus. musculus animals Sp2/0-BC/rhPC-4F10, Sp2/0-BC/rhPC-1C6, Sp2/0-BC/rhPC-3H6, producers of monoclonal antibodies specific to human protein C. The strains are deposited in the Russian National Collection of Industrial Microorganisms of Federal Unitary Enterprise State Research Institute 'Genetics', No. (VKPM H-111), (VKPM H-112), respectively. The antibodies belong to hPROC-specific murine immunoglobulin G possessing cross-responsiveness, are selectively bound with human protein C and form a stable complex.

EFFECT: antibodies under the invention may be used for purposes of pharmaceutical and biomedical analytical studies, particularly for quantitative detection of the human recombinant factor C.

1 dwg, 4 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, namely to infectious diseases, and can be used for treating whooping cough and/or for Bordetella infection protection. Polypeptide under the invention represents a fragment of adenylate cyclase Bordetella containing CD11b/CD18 interaction domain from an amino acid sequence extended from position 1166 to position 1281 SEQ ID NO:1. Said invention also concerns the specific fragments of adenylate cyclase Bordetella containing CD11b/CD18 interaction domain, and to their application, particularly for targeting of molecules of interest to CD11b expressing cells.

EFFECT: use of the inventions allows extending the range of products for treatment and prevention of the Bordetella infection.

26 cl, 9 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: in invention described is hybridoma, producing monoclonal antibodies, which bind with protein 161P2F10B in a specific way. Invention describes versions of monoclonal antibodies, one of which includes amino acid sequence of heavy and light chain of antibody, produced by said hybridoma, and the other includes amono acid sequence of variable part of heavy and light chain of antibody, produced by said hybridoma, or amino acid sequence Fab, F(ab')2, Fv or Sfv of antibody fragment, which binds with protein 161P2F10B, 161P2F10B demonstrates tissue-specific expression in healthy, mature tissue and is abnormally expressed in case of oncologic diseases.

EFFECT: antibodies can be used as anti-cancer medication and in composition of pharmaceutical composition for treating cancer, in particular, kidney cancer, reducing tumour growth, as well as for detection of protein 161P2F10B in biological sample.

11 cl, 212 dwg, 8 tbl, 20 ex

FIELD: medicine.

SUBSTANCE: essence of invention includes contact of liquid sample, taken from mammal organism, with one or several monoclonal antibodies to Lawsonia intracellularis antigen, secreted by cell lines of ECACC hybridomes, which have registration numbers. Invention also includes diagnostic test-kit, containing antibodies specific for Lawsonia intracellularis.

EFFECT: increased specificity of antigen detection.

11 cl, 5 ex, 5 dwg

FIELD: medicine.

SUBSTANCE: invention aims at preparation of new strain of hybrid cells Mus. Musculus 6F3 - a producer of monoclonal antibody (MCA) to hemagglutinin protein of high-pathogen avian influenza virus A/duck/Novosibirsk/56/05. Strain 6F3 is prepared by fusing murine myeloma cells Sp2/0 with murine spleen cells BALB/c, immunised with a purified and inactivated preparation of avian influenza virus A/H5N1 (strain A/duck/Novosibirsk/56/05). Hybridoma produced MCA belong to IgA class. Strain 6F3 is deposited in the Collection of cell culture of Ivanovsky State Research Institution of Virology of the Russian Academy of Medical Sciences, No. 8/2/3. Using hybridoma allows producing specific monoclonal antibodies to hemagglutinin protein of avian influenza virus A/H5N1.

EFFECT: possibility to use antibodies to studying the antigenic structure of hemagglutinin for differential diagnostics of avian influenza virus A/H5 serotype.

1 dwg, 6 ex

FIELD: agriculture.

SUBSTANCE: invention relates to the field of genetic engineering and cloning. Transgenic cow is disclosed, having low activity of prion protein as a result of one or several genetically designed mutations. Specified transgenic cows are also genetically modified for expression of xenoantibodies.

EFFECT: as a result of their resistance to prion diseases, such as trembling disease of cattle, such cows are a safe source of human antibodies for use in pharmaceutics and a safe source of agricultural products.

12 cl, 112 dwg, 13 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: invention refers to antibody specifically getting bound with PRO87299 version. In addition, the antibody according to the invention has ability to block interaction HVEM and PRO87299 and to function as PRO87299 agonist. The antibody of agonist nature is produced by hybridoma Btig5F5.1 or Btig3B1.9. For the antibody, there is established amino acid sequence given in the description. The invention discloses the methods of using the antibodies to stimulate or reduction of immune response in immune-associated diseases connected, to relieve lymphoma, and inflammatory disease in requiring mammal, to detect polypeptide PRO87299 in a sample and to manage rejection of grafted cells.

EFFECT: antibody is an immunomodulator that allows applying therapeutically identical medicinal agents both to intensify and reduce immune response.

16 cl, 34 dwg, 7 tbl, 20 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biology and chemistry and deals with isolated nucleic acid, coding fluorescent protein with biosensor properties, expression cassettes, providing expression of said fluorescent protein, cells, producing said protein, and peculiarly fluorescent protein with biosensor properties. Obtained fluorescent protein has amino acid sequence, given in SEQ ID NO:4, and intended for changing NAD+/NADH ratio inside cells by increasing signal with displacement of NAD+/NADH ratio towards decrease of NADH concentration.

EFFECT: claimed invention makes it possible to carry out analysis of processes in cell in real time mode.

4 cl, 6 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Described is molecule of chimeric nucleic acid of porcine circovirus (PCV2Gen-1Rep), which includes molecule of nucleic acid, coding porcine circovirus of type II (PCV2), which contains sequence of nucleic acid, coding protein Rep of porcine circovirus of type 1 (PCV1). Chimeric molecule of nucleic acid is constructed by replacement of gene Rep ORF1 PCV2 with gene Rep ORF1 PCV1. Invention also includes biologically functional plasmid or viral vector, which contain unique molecules of chimeric nucleic acids, suitable host cells, transformed by plasmid or vector, infectious chimeric porcine circoviruses, which produce suitable host cells, method of obtaining immunogenic polypeptide product with application of novel chimera, viral vaccines, protecting pig against viral infection or syndrome of postweaning multisystem wasting syndrome (PMWS), caused by PCV2, methods of protecting pigs against viral infection or postweaning multisystem wasting syndrome (PMWS), caused by PCV2, methods of obtaining unique chimera PCV2Gen-1Rep and the like. Invention can be applied in veterinary.

EFFECT: invention additionally includes novel method of increasing level of replication and PCV2 titre in cell culture.

21 cl, 2 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Claimed is separated chimeric polynucleotide for amplification of production of heterologous protein of interest, which contains polynucleotide sequence of promoter SigA or SigH, functionally connected with polynucleotide, coding protein YmaH, with chimeric polynucleotide connecting sequence, which by, at least, 90% is identical to SEQ ID NO: 1, 2, 3 or 13. Also described are: expression vector, containing claimed nucleotide structure, and host cell Bacillus for production of heterologous protein of interest, which contains said vector. Claimed is method of obtaining modified Bacillus cell, including transformation of host cell of Bacillus-producent of heterologous protein of interest with said vector; and growing said modified cell in optimal conditions. Described is method of obtaining protein of interest in modified Bacillus cell, where method includes cultivation of said host cell; and growing said modified Bacillus cell in optimal conditions. Also described is method of amplification of expression of heterologous protein from Bacillus of interest includes obtaining said modified Bacillus cell; growing modified Bacillus cell in optimal conditions; and expression of said protein of interest in modified Bacillus cell, where expression of said heterologous protein of interest in modified Bacillus cell is amplified in comparison with expression of said protein of interest in said parent Bacillus host-cell.

EFFECT: invention makes it possible to increase output of target protein due to superexpression of protein YmaH.

30 cl, 4 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed is binding protein for binding one or more targets, which contains four polypeptide chains forming four functional antigen-binding sites. Four polypeptide chains contain VD1-(X1)n-VD2-C-(X2)n. VD1 stands for first variable domain of heavy chain, VD2 stands for second variable domain of heavy chain, C stands for CH1 domain, X1 stands for polypeptide linker, on condition that it is not constant domain, and X2 stands for Fc-region, and n equals 0 or 1. Two polypeptide chains contain VD1-(X1)n-VD2-C. VD1 stands for first variable domain of light chain, VD2 stands for second variable domain of light chain, C stands for CL domain, X1 stands for linker, on condition that it is not constant domain; and n equals 0 or 1. Conjugate of binding protein with visualising detecting cytotoxic or therapeutic agent is described. Disclosed are: nucleic acids (NA), coding polypeptide chains, as well as expressing vectors, vectors for replication, host cells which contain them, and method of obtaining antibody applying cells. Described is pharmaceutical composition for treatment or preventing target-associated disease or disorder based on binding protein. Method of treatment by introduction of binding protein is described.

EFFECT: application of invention provides new format (DVD-Ig) of antigen-binding molecules, which in the same dosage possess higher activity with respect to target than respective full-size antibodies, which can be applied in medicine for prevention and treatment of various diseases.

45 cl, 27 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a recombinant cell of Ralstonia eutropha, designed to obtain 2-hydroxyisobutyric acid. The cell is transformed by a plasmid with the sequence SEQ ID NO: 2.

EFFECT: cell bearing said plasmid produces 2-hydroxyisobutyric acid in concentration of up to 0,72 mmol/kg.

4 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to immunology. Described are antibodies against VEGF, one on which contains complementary regions with amino acid sequences SEQ ID NO:1, 2, 3, 4, 6 and 7, another contains complementary regions with amino acid sequences SEQ ID NO:1, 2, 3, 5, 6 and 7, disclosed in description. Also described are polynucleotides, coding said antibodies; espression vectors, containing said polynucleotides, and host cells, intended for obtaining antibodies in accordance with the claimed invention. Claimed is method of obtaining antibodies against VEGF, which includes expression of vector in host cell and separation of antibody. Disclosed is method of obtaining immunocongugate of antibody against VEGF, which includes conjugation of antibody with drug or cytotoxic agent. Described is method of VEGF identification, which includes identification of complex VEGF-antibody against VEGF in biological sample. In addition, described are compositions for treatment of VEGF-associated disease, one of which contains efficient quantity of antibody against VEGF, and another - efficient quantity of polynucleotide, coding said antibody. Also disclosed are methods of: 1) treating tumour, cancer or VEGF-associated cell proliferative disease; 2) inhibition if angiogenesis in subject and 3) inhibition of vascular permeability; consisting in introduction to subject of efficient quantity of antibody against VEGF in accordance with claimed invention.

EFFECT: invention makes it possible to obtain antibodies against VEGF and apply them for treatment of VEGF-associated diseases.

41 cl, 16 dwg, 2 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly a method for specific collection of DNA molecules (DNA aptamers) with high affinity for a recombinant protein target. Said method involves synthesis of a single polypeptide chain of a recombinant protein containing a fragment of glutathione S-transferase, a protein target, a peptide sequence split by the B. Anthracis lethal factor, a peptide which is biotinylated in vitro under the action of an E.coli biotin-ligase enzyme, binding the obtained recombinant polypeptide with an oligonucleotide library and immobilising the protein on paramagnetic particles bearing glutathione, washing the paramagnetic particles with the immobilised polypeptide from unbound oligonucleotides in a liquid stream, splitting the protein target with the bound DNA aptamers from the surface of paramagnetic particles with the B. anthracis lethal factor, separating and amplifying the DNA sequence with affinity to the recombinant protein target in a polymerase chain reaction and obtaining a set of single-chain DNA aptamers that are specific to the protein target.

EFFECT: invention provides efficient production of DNA aptamers with high affinity for recombinant protein targets.

4 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers immunology and medicine. What is presented is an antibody for the recovery of the central nervous system, comprising an antigen-binding site that specifically binds to human Nogo A polypeptide or human NiG described by (SEQ ID NO: 2 and 3, respectively, presented in the description), wherein the antigen-binding site comprises: CDR-H1-6A3 (SEQ ID NO:8), CDR-H2-6A3 (SEQ ID NO:9) and CDR-H3-6A3 (SEQ ID NO:10); and CDR-L1-6A3 (SEQ ID NO:11), CDR-L2-6A3 (SEQ ID NO:12) and CDR-L3-6A3 (SEQ ID NO:13). There are also described a polynucleotide coding the above antibody; an expression vector comprising the above polynucleotide; and a host cell specified in bacterium, yeast or mammalian cell line comprising myeloma, hybridoma, or immortalised B-cell for producing the antibody according to the present invention. A pharmaceutical composition for the CNS recovery comprising an effective amount of the above antibody mixed with at least one acceptable carrier or solvent is also described. Using the polynucleotide, the expression vector or the host cell for the above pharmaceutical composition is also described. The invention enables producing the human Nogo A or NiG antibody effective in treating CNS injuries.

EFFECT: what is presented is a method for producing the above antibody involving the polynucleotide or vector expression in the host cell.

16 cl, 11 dwg, 9 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry and discloses a polypeptide having antimicrobial activity, which includes an amino acid sequence having at least 70% identity with an amino acid sequence corresponding to positions 1-21 of SEQ ID NO:2. The invention also relates to structures of nucleic acids, vectors and host cells which include a polynucleotide which encodes the polypeptide according to the invention, as well as a method of producing such a polypeptide and use of the polypeptide to destroy microbe cells.

EFFECT: invention widens the range of antimicrobial polypeptides.

18 cl, 2 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biotechnology, in particular to novel peptide analogue of insulin-like growth factor-1 (IGF-1), which contains amino acid substitution of methionine in position 59 on Asn, Leu, Nle, Ile, Arg, A6c, Glu, Trp or Tyr, as well as other additional substitutions, inserts and deletions. Said peptide or its pharmaceutically acceptable salt is used in composition of pharmaceutical composition for treatment of IGF-1-mediated diseases, as well as in method of treating dwarfism.

EFFECT: invention makes it possible to obtain IGF-1 analogue-agonist, possessing higher biological activity with respect to native IGF-1.

17 cl, 2 tbl

FIELD: medicine.

SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed is binding protein for binding one or more targets, which contains four polypeptide chains forming four functional antigen-binding sites. Four polypeptide chains contain VD1-(X1)n-VD2-C-(X2)n. VD1 stands for first variable domain of heavy chain, VD2 stands for second variable domain of heavy chain, C stands for CH1 domain, X1 stands for polypeptide linker, on condition that it is not constant domain, and X2 stands for Fc-region, and n equals 0 or 1. Two polypeptide chains contain VD1-(X1)n-VD2-C. VD1 stands for first variable domain of light chain, VD2 stands for second variable domain of light chain, C stands for CL domain, X1 stands for linker, on condition that it is not constant domain; and n equals 0 or 1. Conjugate of binding protein with visualising detecting cytotoxic or therapeutic agent is described. Disclosed are: nucleic acids (NA), coding polypeptide chains, as well as expressing vectors, vectors for replication, host cells which contain them, and method of obtaining antibody applying cells. Described is pharmaceutical composition for treatment or preventing target-associated disease or disorder based on binding protein. Method of treatment by introduction of binding protein is described.

EFFECT: application of invention provides new format (DVD-Ig) of antigen-binding molecules, which in the same dosage possess higher activity with respect to target than respective full-size antibodies, which can be applied in medicine for prevention and treatment of various diseases.

45 cl, 27 tbl, 5 ex

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