Antibodies recognising epitope on cd43 and cea expressed on malignant cells and methods for using them

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is an antibody specifically binding to an epitope on CD43 and CEA and modified in a heavy and/or light chain constant region. There are disclosed polynucleotides, vector, host cell and methods for producing the antibody according to the invention, as well as pharmaceutical composition, kit and method of treating non-haemopoietic cancer.

EFFECT: this invention can find further application in therapy and diagnosing of CD43 or CEA mediated diseases.

35 cl, 15 tbl, 5 ex, 4 dwg

 

Cross-reference to related applications

This application claims the priority of provisional patent application U.S. No. 61/014716, filed December 18, 2007, which is fully incorporated into the present description by reference.

The technical field to which the invention relates

The present invention relates to antibodies, such as chimeric and humanized antibodies)that recognize carbohydrate-containing epitope on CD43 and carcinoembryonic the antigen (CEA)expressed on megamarketinc tumor or cancer cells. These antibodies are able to induce death (e.g. apoptosis) megamarketinc tumor or cancer cells in the absence of cytotoxin conjugation with, and immune effector functions. These antibodies can be used as diagnostic and therapeutic tools.

The level of technology

CD43 (also called sialophorin or leucovorine) represents strongly sililirovany molecule, expressed in large quantities in the majority of human leukocytes, including all T-cells and platelets with a molecular weight in the range from to 115000 135000. Expression of CD43 broken on T-cells of men suffering from syndrome Viskota-Aldrich, which is concatenated with the X-chromosome recessive immunodeficiency (Remold-O'donnell et al. (1987) Blood70(1):104-9; Remold-O Donnel et al. (1984) J. Exp. Med. 159:1705-23).

Functional studies showed that a monoclonal antibody against CD43 stimulates the proliferation of T-lymphocytes in the peripheral blood (Mentzer et al. (1987) J. Exp. Med. 1; 165 (5):1383-92; Park et al. (1991) Nature, 350:706-9) and activation of monocytes (Nong et al. (1989) J. Exp. Med. 1:170(1):259-67). Monoclonal antibody L11 against CD43 blocks the binding of T cells from lymph node and education of Meyerovich plaques. Antibody L11 inhibits the infiltration of T cells from the blood into the organized secondary lymphoid tissue (McEvoy et al. (1997) J. Exp. Med. 185:1493-8). Monoclonal antibody that recognizes a molecule CD43, causes apoptosis of hematopoietic progenitor cells (LDCs) bone marrow negative reaction to the marker line differentiation, which Express CD34 with a high degree of density (Bazil et al. (1996) Blood, 87(4):1271-81), and T-lymphoblastoid cells (Brown et al. (1996) J. Biol. Chem. 271:27686-95). Recently completed studies have shown that CD43 acts as a ligand for E-selectin on T cells (Matsumoto et al. (2005) J. Immunol. 175:8042-50; Fuhlbrigge et al. (2006) Blood 107:1421-6).

In addition, it was found that certain megamarketinc tumor cells, in particular cells of colorectal adenocarcinoma, also Express a molecule CD43 on the cell surface. Santamaria et al. (1996) Cancer Research 56:3526-9; Baeckstrom et al. (1995) J. Biol. Chem. 270:13688-92; Baeckstrom et al. (1997) J. Biol. Chem. 272:11503-; Sikut et al. (1997) Biochem. Biophy. Res. Commun. 238:612-6. It was found that glikana on CD43 expressed in a cancer cell line colon (COLO205), differ from glycans CD43 leukocytes (Baeckstrom et al. (1997) J. Biol. Chem. 272:11503-9). Although it has been suggested that overexpression of CD43 activates protein is a tumor suppressor p53 (Kadaja et al. (2004) Oncogene 23:2523-30) and, thus, suppresses the subpopulation of target genes of NF-Kappa-b, in part by inhibiting transcriptional activity of P65 (Laos et al. (2006) Int. J. Oncol. 28:695-704), still no direct evidence for the participation of CD43 in oncogenesis cancer of the colon. Standard antibody against CD43 has not found practical application as a therapeutic agent against megamarketinc tumor cells because of the strong binding of this antibody to either tumor cells, and immunocompetent T-cells. Therefore, there is a need to create antibodies that specifically associated with CD43 expressed on megamarketinc tumor or cancer cells, but not associated with a CD43 expressed on leukocytes or other hematopoietic cells. Such antibodies may be useful as therapeutic agents for the treatment of CD43-expressing cancer megamarketinccom origin.

CEA is usually expressed in different tissues of the glandular epithelium (such as VC is docno-intestinal tract, respiratory tract and urinary path)where the specified antigen localized on the apical cell surface (World, S. (1999) Semin. Cancer Biol. 9, 67-81). In tumors derived from these tissues, there is an increased level of expression of CEA, extending from the apical membrane region on the entire surface of the cells with the simultaneous secretion of protein in the blood (World, S. (1999) Semin. Cancer. Biol. 9, 67-81). Over-expression of CEA was observed in many types of cancer, including colorectal cancer, pancreatic cancer, lung cancer, gastric cancer, hepatocellular cancer, breast cancer and thyroid cancer. Therefore, CEA serves as a marker of tumor and immunological analyses, allowing to measure the high content of CEA in the blood of subjects with cancer, have long been used in clinical practice for the prevention and treatment of cancer (Gold P., et al. (1965) J. Expl. Med. 122:467-81; Chevinsky, A.H. (1991) Semin. Surg. Oncol. 7, 162-166; Shively, J.E. et al. (1985) Crit. Rev. Oncol. Hematol. 2, 355-399).

More importantly, CEA has become a potentially useful tumor-associated antigen for targeted therapy (Kuroki M., et al. (2002) Anticancer Res. 22:4255-64). Developed two basic strategies use of CEA as a target for immunotherapy of cancer. One method consists of directional effects on genes-suicide (gene synthase nitric oxide (iNOS)) (Kuroki M. et al., (2000) Anticancer Res. 20(6A):4067-71 or isotopes (R.W. Wilkinson et al., (2001) PNAS USA 98, 10256-60, Goldenberg, D.M. (1991) Am. J. Gastroenterol., 86:1392-1403, T. Olafsen et al., Protein Engineering, Design &Selection, 17, 21-27, 2004) to CEA-expressing tumor cells using antibodies against CEA. The method was also extended to the use of antibodies or fragments of antibodies conjugated to therapeutic agents such as drugs, toxins, radioactive isotopes, immunomodulators or cytokines. Another method is to use immunological cytolytic activity, in particular, antibody-dependent cretaceouspaleogene cytotoxicity (ADCC) or complementability cytotoxicity (CDC)to eliminate CEA-expressing tumor cells (Imakiire, T. et al., (2004) Int. J. Cancer. 108, 564-570). When implementing these methods often lead to the release of cytokines that cause systemic side effects.

Antibodies that recognize carbohydrate-containing epitope on CD43 and CEA expressed on megamarketinc cancer cells, as described in published patent application U.S. No. 2008/0171043 and PCT WO 07/146172. These antibodies can induce apoptosis megamarketinc cancer cells in the absence of cytotoxin conjugation with, and immune effector functions.

All references, publications and patent applications cited in the present invention, and fully incorporated into the present description by reference.

The invention

The present invention relates to antibodies, such as chimeric and humanized antibodies)that specifically bind to an epitope on CD43 and/or CEA expressed megamarketinc cancer cell, but not specifically associated with a CD43 expressed by a leukocyte or cell Jurkat, and can induce apoptosis megamarketinc cancer cell after binding to an epitope expressed on the surface megamarketinc cancer cell in the absence of cytotoxin conjugation with, and immune effector function, with the specified epitope contains a carbohydrate binding of antibodies to this epitope is inhibited by carbohydrate having the structure Leastructure Lea-lactose, the structure of LNDFH II or the structure of the LNT. In some embodiments of the invention the epitope bound to these antibodies, which are sensitive to fucose.

In some embodiments of the invention, these antibodies are chimeric or humanitarianism antibodies derived from the murine antibody m5F1 with the insertion, deletion or substitution of at least one of the amino acids in the hinge region constant region of the heavy chain.

Some embodiments of the invention relate to a selected antibody comprising a heavy chain and light chain, where (a) although ELA chain contains a variable region of the heavy chain, includes three areas that define the complementarity of amino acid sequence SEQ ID NO:1, and the constant region of the heavy chain of human IgG1, with the hinge region constant region of the heavy chain contains an insertion, a deletion or substitution of at least one amino acid; and (b) a light chain contains a variable region light chain comprising three areas that define the complementarity of amino acid sequence SEQ ID NO:2, and a constant region of light chain of the Kappa light-chain man or constant region of the light chain of the Kappa light-chain person, including insertions, deletions or replacing at least one amino acid. In some embodiments the invention, the constant region of the heavy chain contains the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:29.

In some embodiments of the invention from the side of the N-Terminus of amino acids C in the hinge region of human IgG1 inserted one, two, three, four, five, six, seven, eight, nine or ten amino acids, and the residue numbering corresponds to the numbering system of the EU. Cm. publication Burton, Mol. Immunol. 22:161-206, 1985. In some embodiments of the invention from the side of the N-Terminus of amino acids C inserted amino acid residues KSD.

In some embodiments of the invention antibodies include: (the), variable region of the heavy chain, comprising three CDR region of the amino acid sequence of SEQ ID NO:1, and the constant region of the heavy chain containing the amino acid sequence selected from the group consisting of SEQ ID nos:11-30; and (b) variable region light chain comprising three CDR region of the amino acid sequence of SEQ ID NO:2, and a constant region of the light chain containing the amino acid sequence selected from the group consisting of SEQ ID nos:10 and 31-37. In some embodiments of the invention, the specified antibody is a humanized antibody. In some embodiments of the invention, the specified antibody is a chimeric antibody. In some embodiments the invention, the variable region of the heavy chain comprises the amino acid sequence selected from the group consisting of SEQ ID NO:1, 3, and 87-91. In some embodiments the invention, the variable region of the light chain comprises the amino acid sequence selected from the group consisting of SEQ ID NO:2, 4, and 92-96. In some embodiments the invention, the variable region of the heavy chain of the antibody comprises the amino acid sequence of residues 20-137 SEQ ID NO:1 or SEQ ID NO:3 or the amino acid sequence of variable region of SEQ ID NO:1 or SEQ ID NO:3. In some embodiments of the invention variations is a recreational area of the light chain of the antibody comprises the amino acid sequence of residues 20-131 SEQ ID NO:2, amino acid sequence of variable region of SEQ ID NO:2, amino acid sequence of residues 21-132 SEQ ID NO:4 or the amino acid sequence of variable region of SEQ ID NO:4.

In some embodiments of the invention, the antibody of the present invention comprises a heavy chain and a light chain, heavy chain contains a variable region of a heavy chain including amino acid sequence of residues 20-137 SEQ ID NO:1 or the amino acid sequence of variable region of SEQ ID NO:1, and a constant region of a heavy chain including amino acid sequence SEQ ID NO:27 and a light chain contains a variable region light chain comprising the amino acid sequence of residues 20-131 SEQ ID NO:2 or the amino acid sequence of variable region of SEQ ID NO:2, and a constant region of light chain, comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments of the invention, the antibody of the present invention comprises a heavy chain and a light chain, heavy chain contains a variable region of a heavy chain including amino acid sequence of residues 20-137 SEQ ID NO:1 or the amino acid sequence of variable region of SEQ ID NO:1, and a constant region of a heavy chain including amino acid sequence is lnost SEQ ID NO:29, and light chain contains a variable region light chain comprising the amino acid sequence of residues 20-131 SEQ ID NO:2 or the amino acid sequence of variable region of SEQ ID NO:2, and a constant region light chain comprising the amino acid sequence of SEQ ID NO:34.

In some embodiments of the invention, the antibody of the present invention comprises a heavy chain and a light chain, heavy chain contains a variable region of a heavy chain including amino acid sequence of residues 20-137 SEQ ID NO:1 or the amino acid sequence of variable region of SEQ ID NO:1, and a constant region of a heavy chain including amino acid sequence SEQ ID NO:29 and a light chain contains a variable region light chain comprising the amino acid sequence of residues 20-131 SEQ ID NO:2 or the amino acid sequence of variable region of SEQ ID NO:2, and a constant region of light chain, comprising the amino acid sequence of SEQ ID NO:35.

The present invention relates also to antigennegative fragments of the antibodies disclosed in the present description of the invention.

The present invention relates also to pharmaceutical compositions comprising one or more antibodies of the present invention or their antigennegative fragments and headlights is asepticheski acceptable carrier.

The present invention relates to polynucleotides and vectors comprising the nucleic acid sequence encoding a heavy chain of the antibody of the present invention and/or light chain of the antibody of the present invention or its fragment. In some embodiments of the invention polynucleotides and vectors comprise a nucleic acid sequence encoding a heavy chain containing the variable region of the heavy chain comprising three CDR region of the amino acid sequence of SEQ ID NO:1, and a constant region of a heavy chain including amino acid sequence selected from the group consisting of SEQ ID nos:11-30. In some embodiments of the invention polynucleotides and vectors comprise a nucleic acid sequence encoding a light chain containing a variable region light chain comprising three CDR region of the amino acid sequence of SEQ ID NO:2, and a constant region light chain comprising the amino acid sequence selected from the group consisting of SEQ ID nos:10 and 31-37.

The present invention relates also to the cells of the host that contains polynucleotides and vectors disclosed in the present description of the invention.

The present invention further relates to methods of producing any of the antibodies or their antigenspecific fragments considered in the present description of the invention. These methods may include the stage of expression of one or more polynucleotides encoding antibodies (which can be expressed separately as a single heavy or light chain, or in a single vector can be expressed as the heavy chain and light chain) or antihistamie fragments in an appropriate cell host. In some embodiments of the invention expressed antibodies or their antigennegative fragments are separated and/or isolated. The present invention also relates to antibodies or antigennegative fragments obtained by the above methods.

The present invention relates to a method of treating cancer megamarketinccom of origin of the subject, which includes the introduction of a specified subject an effective amount of a composition containing one or more antibodies of the present invention that are associated with cancer cells in the body of the subject. In some embodiments of the invention cancer megamarketinccom origin is colorectal cancer, pancreatic cancer or gastric cancer. In some embodiments of the invention the antibody is conjugated to a cytotoxin.

The present invention relates to a method of slowing the development of cancer megamarketinccom origin (in particular, zamedleniyu and/or inhibition of cancer) in a subject, which includes the introduction of a specified subject an effective amount of a composition containing one or more antibodies of the present invention that are associated with cancer cells in the body of the subject. In some embodiments of the invention cancer megamarketinccom origin is colorectal cancer, pancreatic cancer or gastric cancer. In some embodiments of the invention the antibody is conjugated to a cytotoxin.

The present invention relates also to a method of treating cancer megamarketinccom of origin of the subject, which includes the introduction of a specified subject of a certain number of one or more antibodies of the present invention and a certain amount of another anticancer expense, one or more antibodies are associated with cancer cells in the body of the subject, allowing one or more antibodies together with anti-cancer provide effective treatment of cancer in a specified subject. In some embodiments of the invention cancer megamarketinccom origin is colorectal cancer, pancreatic cancer or gastric cancer. In some embodiments of the invention an anti-cancer agent is a chemotherapeutic agent.

Present and is gaining further relates to kits, includes a pharmaceutical composition comprising one or more antibodies of the present invention. In some embodiments of the invention, the kits will further include instructions for administration to a subject an effective amount of a pharmaceutical composition for the treatment of cancer megamarketinccom origin. In some embodiments of the invention, the kits include instructions for the introduction of a pharmaceutical composition together with other anti-cancer agent. In some embodiments of the invention, the antibody comprises (a) a variable region heavy chain comprising three CDR region of the amino acid sequence of SEQ ID NO:1, and a constant region of a heavy chain including amino acid sequence selected from the group consisting of SEQ ID nos:11-30; and (b) variable region light chain comprising three CDR region of the amino acid sequence of SEQ ID NO:2, and a constant region comprising the amino acid sequence selected from the group consisting of SEQ ID nos:10 and 31-37.

The present invention also relates to kits comprising a first pharmaceutical composition comprising the antibody or antigennegative fragment of the present invention, the second pharmaceutical composition comprising another anti-cancer agent, and instructions with the joint introduction to the subject of the first pharmaceutical composition and a second pharmaceutical composition for the treatment of cancer megamarketinccom origin.

It should be noted that some or all of the characteristics of various embodiments of the invention can be combined with the education of other embodiments of the present invention. Specialist in this field should be obvious the above and other objectives of the present invention.

Brief description of figures

Figure 1 shows the comparative analysis of amino acid sequences of the constant region of the heavy chain of IgG3 mouse (SEQ ID NO:138) and the constant region of the heavy chain of human IgG1 (SEQ ID NO:139). The hinge region underlined. As shown in this figure, the amino acid identity is 214/333 (64,3%), the similarity is 261/333 (78,4%) and gaps are 6/333 (1,8%).

Figure 2(a-E) shows the comparative analysis of amino acid sequences of the unmodified and modified constant regions of the heavy chain of human IgG1, and figure 2F shows the comparative analysis of amino acid sequences of the unmodified and modified constant regions of Kappa light-chain of human IgG1.

Figure 3 shows the binding of the antibody m5F1, c5F1v0, c5F1v15 and c5F1v16 with Colo205 cells according to the results of running cytometrical analysis using antibodies at different concentrations from 0.125 mg/ml to 4 μg/ml Background signals (MFI) for the control antibodies are: secondary antibody against m the Chi: 3; secondary antibody against human: 3; mouse IgG: 4; IgG-man: 5. All antibodies m5F1, c5F1v0, c5F1v15 and c5F1v16, significant binding to cells Colo205 compared to background signals.

Figure 4(a and b) shows a comparative analysis of the amino acid sequence of VH(a) and VL(b) antibodies h5F1M, h5F1A Va, h5F1A Vs h5F1M Va and h5F1M Vs.

Detailed description of the invention

Definitions

The term “antibody” means an immunoglobulin molecule that can specifically bind to a target, such as carbohydrate, polynucleotide, lipid, polypeptide, etc. using at least one site of recognition of antigen, located in the variable regions of immunoglobulin molecules. In used here the value of the specified term means not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain fragment (ScFv), mutants fused proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecules, which includes the site of antigen recognition. The antibody is an antibody of any class such as IgG, IgA or IgM (or subclass), but such an antibody does not necessarily refer to any particular class. Depending on the amino acid sequence of the constant region of the heavy chain the antibodies immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The constant region of the heavy chain, which correspond to the different classes of immunoglobulins are called, respectively, alpha, Delta, Epsilon, gamma and mu. Well-known subunit structures and three-dimensional configurations of different classes of immunoglobulins.

The antibody of the present invention further includes bespecifically, polyspecific, single-chain, and chimeric and humanized molecules having affinity for the polypeptide, reported at least one area defining complementarity, (the CDR) of the antibody. Antibodies of the present invention also include antibodies with one area that is or variable region of the heavy chain of the antibody or variable region of the light chain of the antibody. Holt et al., (2003), Trends Biotechnol. 21:484-490. In this area is well known methods for producing antibodies with a single field containing or variable region of the heavy chain of the antibody or variable region light chain antibodies, including three of the six natural regions defining complementarity, antibodies. See, for example, the publication Muyldermans, Rev. Mol. Biotechnol. 74:277-302, 2001.

In used here is Ter is in “monoclonal antibody” means an antibody from a population essentially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally mutations that may be present in minor amounts. Monoclonal antibodies are usually highly specific, targeted effect on one area of the determinants in the molecule of the antigen. In addition, unlike drugs based on polyclonal antibodies, which typically include different antibodies directed affecting different determinants (epitopes), each monoclonal antibody is directed affects one determinant of the antigen. The definition of “monoclonal” indicates the character of the antibody produced from essentially homologous population of antibodies, and does not limit the receipt of this antibody by any particular method. For example, monoclonal antibodies intended for use in accordance with the present invention, can be obtained by the hybrid method, first described in the publication Kohler and Milstein, (1975), Nature, 256:495, or by means of recombinant DNA, are described in U.S. patent No. 4816567. Monoclonal antibodies may also be isolated from phage libraries generated using the methods described, for example, McCafferty et al., (1990), Nature, 348:552-554.

In this sense, the term “chimeric antibody” means an antibody with the variable containing a series of the area or part of the variable regions of the first species and the constant region of the second kind. The intact chimeric antibody includes two copies of the chimeric light chain and two copies of the chimeric heavy chain. In this area known to produce chimeric antibodies (Cabilly et al. (1984), Proc. Natl. Acad. Sci. USA, 81:3273-3277; Harlow and Lane (1988), Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory). In these chimeric antibody variable region of both light and heavy chains usually mimics the variable regions of antibodies derived from one species of mammals, while the constant region homologous to the sequences in antibodies isolated from other species. In some embodiments of the invention in variable regions and/or constant region can be modified amino acids.

“Isolated” antibody is an antibody that has been identified and isolated from the natural environment.

In this sense, the term “essentially pure” means a substance that is clean (i.e. free from contaminants) at least 50%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%.

In used here is “humanized” antibodies mean forms of antibodies, non-human (e.g. murine), which are specific himem the mi immunoglobulins, chains of immunoglobulins or their fragments such as Fv, Fab, Fab', F(ab')2or other antigennegative sequence antibodies which contain minimal sequence selected from immunoglobulin other than human. In most cases, humanized antibodies are human immunoglobulins (recipient antibody)in which residues from a region that defines complementarity, (the CDR) of the recipient are replaced by residues from a CDR of a species other than human (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinity and antigennegative ability. In some cases, remnants of the framework region (FR) Fv-fragment of the human immunoglobulin are replaced by corresponding residues other than human. In addition, humanitariannet antibody can include residues that are absent in the recipient antibody or in the input sequences of the CDR or framework region, but included for further improvement and optimization of characteristics of the antibodies. As a rule, humanitariannet antibody includes essentially all variable regions, at least one, and usually two, in which all or substantially all of the CDR regions correspond to the specified regions of immunoglobulin other than human, and all or substantially all of the frame region is STI frame are areas of consensus sequence of human immunoglobulin. Humanitariannet antibody optimally also includes at least a portion of a constant region or domain (Fc) of an immunoglobulin, usually immunoglobulin. Antibodies can have an Fc region modified in accordance with the description given in the publication WO 99/58572. Other forms of humanized antibodies have one or more CDR regions (one, two, three, four, five, six), modified compared to the original antibody, which are also referred to as one or more CDR regions, “isolated from” one or more CDR regions of the original antibody.

In this sense, the term “human antibody” means an antibody containing the amino acid sequence corresponding to the amino acid sequence of the antibody produced by a human and/or received by any means of obtaining human antibodies known in the field or reviewed in the present description of the invention. This definition of human antibodies include antibodies comprising at least one polypeptide heavy chains of a human or at least one polypeptide light chain of a human. As one example, the antibody comprising a light chain polypeptides mouse and heavy chains of a human. Human antibodies can be obtained p the testing methods, known in this field. In one embodiment of the invention, the human antibody is selected from a library of phage that expresses human antibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., (1998), PNAS, (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., (1991), J. Mol. Biol., 222:581). Human antibodies can also be obtained by introducing loci of human immunoglobulin transgenic animals, e.g. mice, which have been partially or completely inactivated genes endogenous immunoglobulin. This method is described in U.S. patent№№ 5545807, 5545806, 5569825, 5625126, 5633425 and 5661016. Alternative human antibody can be obtained by immortalization of human lymphocytes producing the antibody against the target antigen (such b cells can be isolated from a subject or may be immunized in vitro. See, for example, the publication of Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., (1991), J. Immunol., 147(1):86-95; and U.S. patent No. 5750373.

The term “variable region” of an antibody means the variable region of the light chain of the antibody or variable region of the heavy chain of the antibody, considered separately or in combination. Variable regions of the heavy and light chains are composed of four frame regions (FR), connected by three areas, complementarity determining (CDR), also known as hypervariable the e plots. CDR-regions in each chain are held in close proximity to each other frame regions (FR) and together with the CDR regions from the other chain, contribute to the formation of antigennegative centre antibodies. There are at least two methods of determining the CDR-regions: (1) method based on the variability of the sequences in different species (Kabat et al., Sequences of Proteins of Immunological Interest, (5thed., 1991, National Institutes of Health, Bethesda, MD)); and (2) the method based on crystallographic studies of complexes of antigen-antibody (Al-lazikani et al., (1997) J. Molec. Biol. 273:927-948)). In used here the value of the CDR may mean CDR region, identified by any method or combination of both methods.

The term “constant region” of an antibody means constant region light chain antibody, or a constant region of heavy chain antibodies, considered separately or in combination. The constant region of antibodies usually provides resistance patterns and perform other biological functions, such as Assembly chain antibody secretion, transplacental mobility and binding of complement, but does not participate in binding to the antigen. Amino acid sequence and the sequence corresponding to exons in the genes const areas depend on the species from which they are selected; however, the changes of amino acid sequences leading to the education of allotypes, very limited to a specific constant regions of a certain type. The variable region of each chain is attached to the constant region by means of the linker polypeptide sequence. The linker sequence is denoted as a sequence of “J” in the gene for the light chain and how the combination of sequence “D” and series “J” in the gene for the heavy chain.

In this sense, the terms “antibody-dependent cretaceouspaleogene cytotoxicity” and “ADCC” refer cretaceouspaleogene reaction in which nonspecific cytotoxic cells that Express Fc receptors (FcR) (e.g., natural killer cells (NK-cells), neutrophils, and macrophages) recognize bound antibody on the target cell and cause lysis of such target cells. The ADCC activity of interest molecules can be assessed by using analysis of ADCC in vitro, similar to that described in U.S. patent No. 5500362 or 5821337. Effector cells suitable for performing such analyses include mononuclear cells (RVMS) and NK-cells in peripheral blood. Alternative or additionally, ADCC activity of interest molecules can be determined in vivo, for example, in animal models, similar to those described in the publication Clynes et al., 1998, PNAS (USA), 95:652-656.

The terms “complementability cytotoxins the ü” and “CDC” means the lysis of the target in the presence of complement. Path activation of complement is initiated by binding of the first component of complement (C1q) with a molecule (e.g. antibody), forming a complex with a cognate antigen. To assess activation of complement can be analyzed by the CDC, for example, similar to those described in the publication Gazzano-three-bet et al., J. Immunol. Methods, 202:163 (1996).

The terms “polypeptide”, “Oligopeptide”, “polypeptide” and “protein” are used interchangeably values in the present description of the invention and means a polymer of amino acids of any length. The polymer may be linear or branched, it may include modified amino acids and can be interrupted by acids other than the amino acids. In the definition of these terms is also a polymer of amino acids, modified natural or artificial means, for example, by the formation of a disulfide bond, glycosylation, limitirovaniya, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a component that is used as a label. In defining these terms also include, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc), as well as other modifications known in this field. It should be noted that because the polypeptides according to the present which the invention is based on antibodies, these polypeptides may exist as separate circuits or associated circuits.

The terms “polynucleotide” or “nucleic acid” are used interchangeably values in the present description of the invention means polymers of nucleotides of any length, and include DNA and RNA. These nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or their analogs, or any substrate that can be introduced into the polymer of DNA or RNA polymerase. Polynucleotide may include modified nucleotides, such as methylated nucleotides and their analogues. If modification of the structure of the nucleotide may be modified before or after Assembly of the polymer. The nucleotide sequence may be interrupted components, non-nucleotides. Polynucleotide can be further modified after polymerization, such as by conjugation with a component that is used as a label. Other types of modifications include, for example, “caps”, substitution of one or more natural nucleotide analog, mezhnukleotidnyh modification, for example, replacement with non-polarized connections (such as, for example, methylphosphonate, complex phosphocreatine, phosphoamide, carbamates, etc. and replace with polarized connections (such as, for example, tosporte the ATA, phosphorodithioate etc), replacement containing side chains, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine and so on), replace with intercalators (such as, for example, acridine, psoralen, and so on), replacing containing chelators (such as, for example, metals, radioactive metals, boron, oxidative metals, etc), replacing containing alkylator, replace with modified linkages (such as, e.g., alpha anomeric nucleic acids, etc), as well as unmodified forms of polynucleotides. In addition, any hydroxyl group, usually present in the sugars may be replaced, for example, phosphonate groups, phosphate groups, protected by standard protective groups activated with the purpose of obtaining additional links to additional nucleotides, or conjugated to a solid media. 5'- and 3'-terminal group, IT can be phosphorylated or substituted amines or organic kapinowski groups containing 1-20 carbon atoms. Other hydroxyl can also be replaced using standard protective groups. Polynucleotide may also contain analogues of sugars ribose or deoxyribose known in this field, which include, for example, 2'-O-methyl, 2'-O-allyl-, 2'-fluoro - or 2'-isidoros, carbocyclic analogues of sugars, α-ANO is Ernie sugar, epimeria sugars such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs and basic nucleoside analogues, such as methylribose. One or more complex fosfolipidnyh ties can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, cases in which the phosphate is replaced by P(O)S (”tiat”), R(S)S (”ditial”), (O)NR2(amidate”), P(O)R, P(O)OR', CO or CH2(formatall”), where R or R' independently denote H, substituted or unsubstituted alkyl (1-20 carbon atoms), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldi. Not all communication polynucleotide must be identical. The above description applies to all polynucleotides given in this description of the invention, including DNA and RNA.

In this sense, the term “vector” means a construct capable of delivering and preferably to Express one or more genes of interest or sequences in the cell host. Examples of vectors include, but are not limited to, viral vectors expressing vectors based deproteinizing DNA or RNA, plasmid, kosmidou or phage vectors expressing vectors based on DNA or RNA associated with cationic condensing agents, expressing vectors based on DNA or RNA encapsulated in liposomes, and certain eukaryotic cells, such as cells-producers.

In this sense, the term “sequence that regulates the expression” means a nucleic acid sequence that directs transcription of the nucleic acid. Sequence regulating expression may be a promoter such as a constitutive or inducible promoter or enhancer. Sequence regulating the expression of functionally associated with the transcribed sequence of the nucleic acid.

In this sense, the term “effective dose” or “effective amount” of the drug, compound or pharmaceutical composition refers to the amount sufficient to achieve a favorable or desired results. For prophylactic use favorable or desired outcomes mean eliminating or reducing risk, reducing the severity, or delay the onset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes arising in the process of development of the disease. Therapeutic application of favorable or desired outcomes of oz is achut weakening of one or more symptoms of the disease, improving the quality of life of subjects suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect achieved another drug, such as directional impact, slowing disease progression and/or longer life expectancy. In the case of cancer or tumor effective amount of a medicinal product can reduce the number of cancer cells; reduce the tumor volume; inhibition (that is, slowing to some extent, preferably stopping) infiltration of cancer cells into peripheral organs; inhibit (i.e., the slowing to some extent, preferably stopping) of metastasis of the tumor; inhibition, to some extent, of tumor growth and/or weakening to some extent one or more symptoms associated with this disorder. Effective dose can be administered in one or several stages. In accordance with the purposes of the present invention, an effective dose of the drug, compound or pharmaceutical composition is an amount sufficient for prophylactic or therapeutic treatment of direct or indirect methods. As should be understood in the context of clinical practice, effective doses of the medicinal product, the compounds or pharmaceutical compositions can be obtained or not obtained when used in conjunction with another drug, compound or pharmaceutical composition. Thus, the “effective dose” can be considered from the point of view of the introduction of one or more drugs, it is considered that one tool introduced in an effective amount, if joint application with one or more other means can be achieved or is the desired result.

In this sense, the term “in combination with” means the use of one modality in addition to the other modality. Thus, the term “in combination with” means the use of one modality before, during or after using another modality to needy entity.

In this sense, the term “treatment” means a method of achieving a favorable or desired results, including clinical preferably results. In accordance with the purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following results: reduction of proliferation (or destruct the tion) of cancer cells, relieving symptoms caused by this disease, improving the quality of life of subjects suffering from the disease, reducing the dose of other medications required to treat the condition, slowing disease progression and/or longer life expectancy of subjects.

In this sense, the term “slow disease” means a delay, hindrance, deceleration, braking, stability and/or delay the development of disease (such as cancer). The delay may have a different duration depending on the history of disease and/or condition of the subject to be treated. As should be known to the person skilled in the art, a sufficient or significant delay can, in fact, be tantamount to prevent development of the disease in the subject. For example, you can slow down the development of cancer at a later stage, in particular, to prevent the formation of metastases.

The term “subject” means a mammal, more preferably human. Mammals also include, but are not limited to, farm animals, sport animals, Pets (such as cats, dogs, horses), primates, mice and rats.

In this sense, the term “specifically recognizes” or “specifically binds” means measured is whether reproducible interaction, such as attraction or binding of the target antibody, which is an indicator of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically or preferentially binds to the epitope is an antibody that binds to a specified epitope with greater affinity, avidity, more easily and/or over a longer period than with other epitopes targets or epitopes of molecules that are not targets. This definition implies that, for example, the antibody (or epitope), which specifically or preferentially binds to the first target, may or may not specifically or preferentially to contact with the second target. Thus, the term “specific binding” or “preferred binding” is not necessarily (but may include) the exclusive binding. The antibody specifically binding to the target can have a constant Association equal to at least about 103M-1or 104M-1sometimes about 105M-1or 106M-1in other cases, approximately 106M-1or 107M-1approximately 108M-1- 109M-1approximately 1010M-1- 1011M-1or higher. For the selection of the antibodies is, having specific immunoreactivity against a specific protein can be used in various immunoassays. For example, for selection of monoclonal antibodies having specific immunoreactivity against a protein typically use solid-phase enzyme-linked immunosorbent assay (ELISA). See, for example, Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, to become familiar with the description of immunoassays and conditions, which can be used to determine specific immunoreactivity.

In this sense, the terms “cancer”, “tumor”, “cancer” and “malignant” define or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, including adenocarcinoma, lymphoma, blastoma, melanoma and sarcoma. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the gastrointestinal tract, Hodgkin's lymphoma and nahodkinskuju lymphoma, pancreatic cancer, glioblastoma, cervical cancer, glioma, ovarian cancer, liver cancer, such as carcinoma of the liver and hepatoma, bladder cancer, cancer of m is Nochnoi gland, cancer of the colon, colorectal cancer, carcinoma of the endometrium or uterine carcinoma of the salivary gland, kidney cancer such as renal cell carcinoma and Wilms tumor, basal cell cancer, melanoma, prostate cancer, thyroid cancer, testicular cancer, esophageal cancer and different types of cancer of the head and neck.

The value used in the present description of the invention and the attached claims, the singular number also include the plural, except in those cases where the context makes clear otherwise. For example, the term “antibody” in the singular means both one and many of antibodies, in particular, the molar amount, and includes its equivalents, known to specialists in this field, and so on.

It should be noted that the objects and embodiments of the invention described in the present description of the invention, include objects and options defined by the terms “consisting of” and/or “consisting essentially of”.

Antibodies and polypeptides that are specifically associated with the carbohydrate epitope on CD43 and CEA expressed on megamarketinc cancer cells

The present invention relates to selected antibodies and polypeptides derived from antibodies that specifically bind to an epitope on CD43 and/or CEA expressed by the and megamarketinc cancer cells, but specifically not associated with a CD43 expressed by a leukocyte such as peripheral T-cell) or cell Jurkat.

Some embodiments of the invention relates to an antibody comprising the variable region of the heavy chain that contains one or more CDR regions of SEQ ID NO:1, and the constant region of the heavy chain of human IgG1. In some embodiments of the invention, the specified antibody comprises the variable region of the light chain containing one or more CDR regions of SEQ ID NO:2, and a constant region of the Kappa light-chain.

In some embodiments of the invention, one or more amino acid residues in the constant region of the heavy chain and/or constant region of the light chain of the antibody is modified (including insertion, deletion and substitution of amino acids). For example, amino acid residues may be modified as shown in the “Examples”section.

Some embodiments of the invention relates to the antibody comprises: (a) the variable region of the heavy chain that contains one or more CDR regions from the amino acid sequence of SEQ ID NO:1, and the constant region of the heavy chain containing the amino acid sequence selected from the group consisting of SEQ ID nos:11-30; and (b) variable region light chain containing one or more of CDR region is of the amino acid sequence of SEQ ID NO:2, and the constant region of the light chain containing the amino acid sequence selected from the group consisting of SEQ ID nos:10 and 31-37. In some embodiments of the invention, one or more CDR regions from the amino acid sequence of SEQ ID NO:1 are the three CDR regions from the amino acid sequence of SEQ ID NO:1. In some embodiments of the invention, one or more CDR regions from the amino acid sequence of SEQ ID NO:2 are the three CDR regions from the amino acid sequence of SEQ ID NO:2. In some embodiments the invention, the CDR1, CDR2 and CDR3 in the heavy chain comprise, respectively, amino acid sequences SYVMH (SEQ ID NO:168), YINPYNGGTQYNEKFKG (SEQ ID NO:169) and RTFPYYFDY (SEQ ID NO:170). In some embodiments the invention, the CDR1, CDR2 and CDR3 light chain comprise, respectively, amino acid sequences RSSQSILHSNGNTYLE (SEQ ID NO:171), KVSNRFS (SEQ ID NO:172) and FQGSHAPLT (SEQ ID NO:173). In some embodiments the invention, the variable region of the heavy chain comprises the amino acid sequence of variable region of SEQ ID NO:1 or 3. In some embodiments the invention, the variable region of the light chain comprises the amino acid sequence of variable region of SEQ ID NO:2 or 4.

In some embodiments of the invention, one or more of CDR region is, selected from the amino acid sequence of SEQ ID NO:1 and/or SEQ ID NO:2, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to at least one, at least two, at least three, at least four, at least five or at least six CDR regions of SEQ ID NO:1 and/or SEQ ID NO:2.

Antibodies and polypeptides of the present invention may further be characterized by one or more of the following characteristics: (a) the binding of an antibody or polypeptide epitope is reduced during processing of molecules, including the specified epitope of α-1→(2,3,4)-fucosidase; (b) the binding of an antibody or polypeptide epitope is inhibited by carbohydrate having the structure Leastructure Lea-lactose, the structure of LNDFH II and/or structure of the LNT; (C) the antibody or the polypeptide induces death megamarketinc cancer cells (apoptosis) after binding with the epitope expressed on the surface of okovoi cells, in the absence of cytotoxin conjugation with, and immune effector functions; (d) the antibody or the polypeptide inhibits the growth or proliferation megamarketinc cancer cell after binding to an epitope expressed on the surface of a cancer cell; and (e) the antibody or the polypeptide removes or prevents the subject has cancer megamarketinccom origin, expressing the epitope on the cell surface, such as colorectal cancer and gastric cancer.

In this sense, the term “inhibition” means the partial or complete inhibition. For example, a carbohydrate having the structure Leastructure Lea-lactose, the structure of LNDFH II and/or structure of the LNT, inhibits the binding of an antibody or polypeptide epitope on CD43 or CEA at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%. The binding of an antibody to an epitope can be ingibirovalo direct competition or other mechanisms.

Examples megamarketinc cancer cells expressing the indicated epitope include, but are not limited to, colorectal cancer cells (such as COLO205 and DLD-1), stomach cancer cells (such as NCI-N87) and cancer cells populadon the th cancer (such as SU.86.86, NO. ATS CRL-1837).

Antibodies and polypeptides of the present invention can recognize the extracellular domain of CD43 present at megamarketinc cancer cells, but not in contact with the extracellular domain of CD43 leukocyte (e.g., peripheral T-cells or the extracellular domain of CD43 expressed on the cell Jurkat (cell lymphoblastoid leukemia). In some embodiments of the invention new antibodies or polypeptides of the present invention is specifically not associated with a CD43 expressed by cells of hematopoietic origin.

In the scope of the present invention includes modifications of the antibodies or polypeptides of the present invention, including functionally equivalent antibodies without significant changes in the properties and variants with increased or decreased activity and/or affinity. For example, the amino acid sequence of the antibody may be metirovan order to obtain antibodies with the desired affinity binding to CD43 or CEA expressing cancer cell. Modification of polypeptides is a common practice in this field and does not require detailed description. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids, which do not cause the value is positive harmful changes in the functional activity, or the use of chemical analogues.

Insertions of amino acid sequences include the merger of the amino and/or carboxyl end of the length from one residue to polypeptides containing a hundred or more residues, as well as insertions of one or several amino acid residues within the sequence. Examples of terminal insertions include an antibody with an N-terminal nationalsim residue or the antibody fused with a tag epitope. Other options insertion of antibody molecules include the fusion to the N - or C-end of the antibody of an enzyme or a polypeptide which increases the half-life of antibodies in the blood line.

Options for replacement include removing at least one amino acid residue in the molecule antibodies and the introduction in its place of another residue. The sites of greatest interest for mutagenesis by the method of substitution, include hypervariable sites, but it is also possible substitutions in the framework region (FR). Conservative substitutions are shown in the table below under the heading “conservative substitutions”. In cases where such changes cause a change in biological activity, can be produced more significant replacement shown in the table below under the heading “illustrative replacement or described below with respect to classes of amino acids, and performed screening products

Table 1
Replacement of amino acids
The original balanceConservative substitutionsIllustrative replacement

Ala (A)ValVal; Leu; Ile
Arg (R)LysLys; Gln; Asn
Asn (N)GlnGln; His; Asp; Lys; Arg
Asp (D)GluGlu; Asn
Cys (C)SerSer; Ala
Gln (Q)AsnAsn; Glu
Glu (E)AspAsp; Gln
Gly (G)AlaAla
His (H)ArgAsn; Gln; Lys; Arg
Ile (I)Leu Leu; Val; Met; Ala; Phe; norleucine
Leu (L)IleNorleucine; Ile; Val; Met; Ala; Phe
Lys (K)ArgArg; Gln; Asn
Met (M)LeuLeu; Phe; Ile
Phe (F)TyrLeu; Val; Ile; Ala; Tyr
Pro (P)AlaAla
Ser (S)ThrThr
Thr (T)SerSer
Trp (W)TyrTyr; Phe
Tyr (Y)PheTrp; Phe; Thr; Ser
Val (V)LeuIle; Leu; Met; Phe; Ala; norleucine

Significant changes in the biological properties of the antibody are achieved by selecting substitutions that have significantly different effects on (a) the structure of the polypeptide backbone in place of the substitution, for example, forming a layer of natou or helical structure, (b) the charge or hydrophobicity of the molecule at the target site, or (C) the structure of the side chain. Natural residues are divided into groups on the basis of the General properties of the side chains:

(1) non-Polar: norleucine, Met, Ala, Val, Leu, Ile;

(2) Polar without charge: Cys, Ser, Thr, Asn, Gln;

(3) Acidic (negatively charged): Asp, Glu;

(4) Basic (positively charged): Lys, Arg;

(5) Residues that influence chain orientation: Gly, Pro; and

(6) Aromatic: Trp, Tyr, Phe, His.

Nonconservative substitutions produce, replacing a member of one of these classes with a member of another class.

Any cysteine residue not involved in maintaining the appropriate structure of antibodies, can also be substituted, generally with serine, to improve stability of the molecule oxidation and prevent aberrant cross-stitching. Conversely, the antibody can be entered one or more cysteine linkages to improve its stability, particularly when the antibody is an antibody fragment such as an Fv fragment.

Amino acid modifications can have a wide range from the replacement of one or more amino acids from the total change area, such as the variable region. Change variable regions can change the affinity of binding and/or specificity. In some embodiments of the invention in a CDR region may be made which were not more than one to five conservative substitutions of amino acids. In other embodiments of the invention in a CDR region may be made no more than one to three conservative substitutions of amino acids. In other embodiments the invention, the CDR is a CDR H3 and/or CDR L3.

In addition, modifications include glycosylated and deglycosylated polypeptides, and polypeptides with other posttranslational modifications, such as glycosylation of different sugars, acetylation and phosphorylation. Antibodies glycosylases in the conservative provisions of the constant regions (Hefferis and Lund (1997), Chem. Immunol. 65:111-128; Wright and Morrison, (1997), TibTECH 15:26-32). Oligosaccharide side chains of immunoglobulins affect the function of a protein (Boyd et al., (1996), Mol. Immunol. 32:1311-1318; Wittwe and Howard, (1990), Biochem. 29:4175-4180) and intramolecular interactions between parts of the glycoprotein, which may be altered conformation and three-dimensional surface of the glycoprotein (Hefferis and Lund, see above; Wyss and Wagner, (1996), Current Opin. Biotech. 7:409-416). Oligosaccharides can also be used for directed delivery of this glycoprotein to specific molecules on the basis of specific structures of recognition. It was found that glycosylation of antibodies affect antibody-dependent cretaceouspaleogene cytotoxicity (ADCC). In the scientific literature, in particular, it was noted that the cells of SNO with adjustable tetr what cyclin expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), glycosyltransferases that catalyze the formation of bisecting GlcNAc, are characterized by the best ADCC activity (Umana et al., (1999), Mature Biotech. 17:176-180).

Glycosylation of antibodies is typically N-linked or O-linked. N-linked glycosylation involves the joining of the carbohydrate portion to a side chain of an asparagine residue. Tripeptide sequence asparagine-X-serine, asparagine-X-threonine and asparagine-X-cysteine, where X is any amino acid except Proline, are sequences of recognition for enzymatic joining carbohydrate portion to the side chain of asparagine. Thus, the presence of such Tripeptide sequences in the polypeptide creates a potential site glycolythiourea. O-linked glycosylation involves the connection of one of the sugars N-atsetilgalaktozamin, galactose, or xylose to hydroxynicotinate, usually serine or threonine, although it may be used 5-hydroxyproline or 5-hydroxylysine.

The glycosylation sites are usually introduced into the antibody by modifying the amino acid sequence such that it contains one or more of the above Tripeptide sequences (for sites of N-linked glycosylation). Change can also be made by adding or replacing one Il is several residues of serine or threonine in the sequence of the original antibody (sites for O-linked glycosylation).

The glycosylation pattern of the antibodies can also be changed without changing the nucleotide sequence. Glycosylation largely depends on the host cell used for expression of the antibody. As the cell used for expression of recombinant glycoproteins, such as antibodies as potential drugs is rarely a native cell, you can expect changes in the glycosylation pattern of the antibodies (see, for example, the publication of the Hse et al., (1997), J. Biol. Chem. 272:9062-9070).

Antibodies of the present invention may include antibody fragments (such as Fab, Fab', F(ab')2, Fv, Fc, etc), chimeric antibodies, single-chain (ScFv), mutants fused proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecules, which includes the site of recognition of desired antigen specificity. Antibodies may be murine, rat, camel, human, or can have any other origin (including humanized antibodies).

Affinity binding polypeptide (including antibody) to CD43 or CEA may be less than about 500 nm, about 400 nm, about 300 nm, about 200 nm, about 100 nm, about 50 nm, about 10 nm, about 1 nm, about 500 PM, about 100 PM, or about 50 PM. As is well known in this field, the affinity binding can batterijen in the form of K Dor dissociation constants, and high affinity binding corresponds to a lower value of KD. One method of determining the affinity of binding of antibodies to CD43 or CEA is the measurement of the affinity of binding of monofunctional Fab fragments of antibodies. To obtain a monofunctional Fab fragments of the antibody (e.g., IgG) can be split papain or to Express the recombinant method. The affinity of the Fab fragment of the antibody may be determined by the method of surface plasmon resonance (measuring system of the surface plasmon resonance (SPR) BIAcore3000™, BIAcore, INC, Piscaway, NJ) and ELISA. Determine the speed of the kinetic Association (konand the rate of dissociation (koff) (measurement is usually carried out at 25°C) and calculate the values of equilibrium constants of dissociation (KDfrom the relations koff/kon.

In some embodiments the invention, the antibodies and polypeptides of the present invention reduce the number of cancer cells and/or inhibit the growth or proliferation of tumor or cancer cells that contain the epitope. The decrease in the number of cells or inhibition of growth or proliferation of the cells is preferably at least about 10%, about 20%, about 30%, about 40%, about 50%, about 65%, about 75% or more compared with cells not subjected to the hcpa is setiu antibodies or polypeptides. Cancer cells include, but are not limited to, colorectal cancer, pancreatic cancer, lung cancer and stomach cancer.

In some embodiments the invention, the antibodies and polypeptides of the present invention can induce cell death, e.g. apoptosis after binding with the specified epitope expressed on the surface megamarketinc cancer cells. The term “induce cell death” used here is meant that the antibodies or polypeptides of the present invention can directly interact with a molecule expressed on the cell surface, with one binding/interaction is sufficient to induce cell death without the help of other factors, such as conjugation with a cytotoxin or other immune effector functions, i.e complementability cytotoxicity (CDC), antibody-dependent cretaceouspaleogene cytotoxicity (ADCC) or phagocytosis.

In this sense, the term “apoptosis” means aimed at genes the process of intracellular destruction of cells. Apoptosis can be distinguished from necrosis; apoptosis involves the destruction of the cytoskeleton, compression and condensation of the cytoplasm, the expression of phosphatidylserine on the outer surface of the cell membrane and the allocation of Ozerkov, resulting in the formation of bubbles associated with the cellular membrane, or apoptotic cells. This process is also called “programmed cell death”. In the process of apoptosis observed typical phenomena as the curvature of the cell surfaces, condensation of nuclear chromatin, fragmentation of chromosomal DNA and loss of mitochondrial function. For detection of apoptosis can be used various known methods, such as staining of cells with annexin V, iodide of propecia, analysis of DNA fragmentation and YO-PRO-1 (Invitrogen).

Methods of detecting cell death (such as apoptosis) include, but are not limited to, identification of morphology, DNA fragmentation, enzymatic activity, cleavage of polypeptides, etc. Cm. publishingSiman et al., U.S. Pat. No. 6048703; Martin and Green (1995), Cell, 82:349-52; Thomberry and Lazebnik (1998), Science, 281:1312-6; Zou et al., U.S. Pat. No. 6291643; Scovassi and Poirier (1999), Mol. Cell Biochem., 199:125-37; Wyllie et al. (1980), Int. Rev. Cytol., 68:251-306; Belhocine et al. (2004), Technol. Cancer Res. Treat., 3(l):23-32, which is incorporated into this description by reference.

In some embodiments the invention, the antibodies and polypeptides of the present invention recognize the structural epitope expressed on megamarketinc the cancer cell, which has the structure, physical and chemical characteristics of which are the equivalents of the structure, about zovannoj the Tripeptide, N'-Trp-Pro-Ile-C'. In this sense, the term “epitope having the structure, physical and chemical characteristics which are equivalent to the structure formed by peptide” means that both structures have the same physical and chemical property related to the binding with the antibody, allowing the antibody that specifically binds to the same structure, will be contacted with both. In some embodiments the invention, the antibodies and polypeptides associated with the polypeptide comprising the amino acid sequence N'-Trp-Pro-Ile-C' at the N-Terminus of the polypeptide.

In some embodiments the invention, the antibodies and polypeptides of the present invention compete with the antibody m5F1 or h5F1 for binding to the epitope expressed on the surface of a cancer cell. In some embodiments the invention, the antibodies or polypeptides of the present invention associated with the epitope on CD43 or CEA is associated with at least one of the antibodies m5F1 or h5F1.

To determine whether the two are linked antibodies to the same epitope as the result of recognition is identical or sterically overlapping epitopes, or one antibody competitively inhibits the binding of another antibody to this antigen can be used competitively binding the f tests. Such assays are known in this field. Typically, the antigen or antigenantibody cells immobilized to advance the tablet and measure the ability of unlabeled antibodies to block the binding of labeled antibodies. In such competitive binding assays using conventional labels, such as radioactive labels or enzyme labels.

In some embodiments of the invention, the area determining complementarity is a CDR region of Kabat. In other embodiments of the invention, the area determining complementarity is the CDR-region Hatia. In other embodiments of the invention, the area determining complementarity, is a combination of CDR regions of Kabat, Hatia (also referred to as “United CDR region” or “extended CDR region”). In other words, in any embodiment of the invention, in which used several CDR regions, such regions can be CDR regions of Kabat, Hatia and/or combined CDR regions.

Methods of making antibodies and polypeptides derived from antibodies are well known in this field and described in the present description of the invention. The resulting antibodies can be tested for the presence of specific binding to an epitope on CD43 or CEA expressed megamarketinccom cancer Il the tumor cells, and the lack of specific binding to CD43-expressing the leukocyte cells of Jurkat and/or other CD43-expressing cells of hematopoietic origin. Testing can be used by cancer cells or the extracellular domain (including fragments)containing a specified epitope.

The cell line of Jurkat is a cell lymphoblastoid leukemia and was isolated from peripheral blood of a 14-year-old boy Schneider and others (Schneider et al., Int. J. Cancer 19:621-626, 1977). Different cell lines of Jurkat can be purchased commercially, for example, from American type culture collection (for example, ADS TIB-152, ATCC TIB-153, ATCC CRL-2678).

The binding specificity of the resulting antibodies can be identified by thus or analysis of binding in vitro, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such methods and assays well known in this field. The affinity of binding of the monoclonal antibody can, for example, be determined using the analysis of Scatchard described in the publication of Munson and Pollard (1980), Anal. Biochem., 107-220.

Identified antibodies can be further tested for their ability to induce the death of cells (e.g., apoptosis) and/or inhibit the growth or proliferation of the cells using methods known in this field and Russ is utrennih in the present description of the invention.

Antibodies of the present invention can be also obtained by the methods of recombinant DNA, are described in U.S. patent No. 4816567 and 6331415, which are included in the present description of the invention in the form of links. For example, DNA encoding the monoclonal antibodies of the present invention, can be easily isolated and sequenced by standard methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of murine antibodies). Cell hybridoma of the present invention serve as a preferred source of such DNA. The selected DNA can be introduced in expressing vectors, which are then transferout in cell host, such as COS cells monkeys, cells of the Chinese hamster ovary (Cho or myeloma cells that otherwise do not produce the protein of the immunoglobulin, for the synthesis of monoclonal antibodies in the recombinant cell host. DNA can also be modified, for example, by introducing the coding sequence for the constant regions of the heavy and light chains of the person instead of the homologous murine sequences (U.S. patent No. 4816567) or by covalent joining the coding sequence of the immunoglobulin whole or part of the coding sequence for a polypeptide, which is immunoglobulin the om. Such a polypeptide, non-immunoglobulin can be entered instead of the constant regions of the antibodies of the present invention or instead of the variable regions of one antigennegative centre antibodies of the present invention to create a chimeric bivalent antibody.

In some embodiments the invention, the antibodies of the present invention Express two expressing vectors. First expressing the vector encodes a heavy chain antibodies (e.g., gumanitarnogo antibodies) and includes a first portion encoding a variable region of the heavy chain antibody, and a second part encoding a constant region of the heavy chain of the antibody. In some embodiments of the invention the first part encodes a heavy chain containing the variable region of the heavy chain comprising one or more CDR regions from the amino acid sequence of SEQ ID NO:1, and a constant region of a heavy chain including amino acid sequence selected from the group consisting of SEQ ID nos:11-30. In some embodiments of the invention, one or more CDR regions from the amino acid sequence of SEQ ID NO:1 are the three CDR regions from the amino acid sequence of SEQ ID NO:1. The second expressing the vector encodes the light chain of the antibody and includes the first hour of the ü, encoding the variable region of the light chain of the antibody, and the second part encoding a constant region of light chain antibodies. In some embodiments of the invention the first part encodes the light chain containing the variable region light chain comprising one or more CDR regions from the amino acid sequence of SEQ ID NO:2, and a constant region light chain comprising the amino acid sequence selected from the group consisting of SEQ ID nos:10 and 31-37. In some embodiments of the invention, one or more CDR regions from the amino acid sequence of SEQ ID NO:2 are the three CDR regions from the amino acid sequence of SEQ ID NO:2.

Alternative antibodies (for example, humanitariannet antibody) of the present invention Express from a single expressing vector. A unit expressing the vector encodes both heavy chain and light chain of the antibody of the present invention. In some embodiments of the invention expressing the vector includes a polynucleotide sequence encoding a heavy chain containing the variable region of the heavy chain comprising one or more CDR regions from the amino acid sequence of SEQ ID NO:1, and a constant region of a heavy chain including amino acid sequence, select the Yu group, consisting of SEQ ID nos:11-30, and variable region light chain comprising one or more CDR regions from the amino acid sequence of SEQ ID NO:2, and a constant region light chain comprising the amino acid sequence selected from the group consisting of SEQ ID nos:10 and 31-37. In some embodiments of the invention, one or more CDR regions from the amino acid sequence of SEQ ID NO:1 are the three CDR regions from the amino acid sequence of SEQ ID NO:1. In some embodiments of the invention, one or more CDR regions from the amino acid sequence of SEQ ID NO:2 are the three CDR regions from the amino acid sequence of SEQ ID NO:2.

Expressing the vector typically contains sequences that regulate transcription and translation, which are extracted from species compatible with the host-cell. In addition, the vector typically carries out specific genes capable of producing a selection on phenotype in transformed cells.

There are a number of recombinant expressing systems host-vector for eukaryotic cells, which can be used in the present invention. For example, most often for eukaryotic microorganisms using Saccharomyces cerevisiae, or common Baker's yeast, although there are a number of other strains, such as Pichia pastoris. As a researcher who, as owners can be also used cell line, isolated from multicellular organisms, such as Sp2/0 or cells of the Chinese hamster ovary (Cho), which can be purchased from ATS. A typical plasmid vectors suitable for transformation of eukaryotic cells are, for example, pSV2neo and pSV2gpt (ATCC), pSVL and pSVK3 (Pharmacia) and pBPV-1/pML2d (International Biotechnology, Inc.).

Eukaryotic cell hosts suitable for use in the present invention, preferred are cells hybridoma, myeloma cells, cells plasmacytoma or lymphoma. However, there may be used other eukaryotic cells-owners, provided that such cells are the owners of mammals are able to identify the transcription and translation of the DNA sequence for the expression of proteins, processional leader peptide by cleavage of the leader sequence and secretion of proteins and to perform post-translational modification of proteins, for example, glycosylation.

Thus, the present invention relates to eukaryotic cells-the hosts, who transformed expressing recombinant vectors, including designs based on the DNA described in the present description of the invention, and are able to Express the antibodies or polypeptides of the present invention. In some embodiments the invention, the transformed cells and the masters of the present invention include at least one design on the basis of DNA containing the DNA sequence of light and heavy chains and sequences regulating transcription and translation, which are located relative to the DNA sequences encoding the light and heavy chain, with the ability to control expression of the antibodies or polypeptides.

Cell owners used in the present invention, can be transformed by different standard methods of transfection are well known in this field. Standard transfection methods that can be used include methods of electroporation, fusion of protoplasts and precipitation of calcium phosphate. Such methods are described in publications F. Toneguzzo et al. (1986), Mol. Cell. Biol., 6:703-706; G. Chu et al., Nucleic Acid Res. (1987), 15:1311-1325; D. Rice et al., Proc. Natl. Acad. Sci. USA (1979), 79:7862-7865; and V. Oi et al., Proc. Natl. Acad. Sci. USA (1983), 80:825-829.

In the case of two expressing vectors expressing both vectors can be transliterowany in cell host each other separately or together (coperto or cotransfected).

The present invention relates also to a method of producing an antibody or polypeptide, which comprises culturing the host cell containing expressing vectors encoding the antibodies or polypeptides, and the selection of the antibodies or polypeptides from the culture by methods well known to the person skilled in the art. In some vari is ntah the invention, the antibodies may be isolated or purified by standard methods of purification of immunoglobulins, such as, for example, purification of protein a-separate, chromatography on hydroxyapatite, gel electrophoresis, dialysis, or affinity chromatography.

In addition, the desired antibody can be produced in the body transgenic animal. Acceptable transgenic animal can be obtained by standard methods, which include microinjection in egg relevant expressing vectors, introduction of eggs to pseudoriemannian females and selection of progeny expressing the desired antibody.

The present invention relates to chimeric antibodies, which specifically recognize the specified epitope on CD43 and CEA expressing cancer cell. For example, variable and constant region of the chimeric body belong to different species. In some embodiments the invention, the variable region of the heavy chain and light chain, derived from mouse antibodies described in the present description of the invention. In some embodiments the invention, the variable regions include amino acid sequences of the variable regions of SEQ ID NO:1 and SEQ ID NO:2 or residues 20-137 SEQ ID NO:1 and residues 20-131 SEQ ID NO:2. In some embodiments the invention, the constant region of the heavy chain and light chain derived from a human antibody.

Chimeric antibody truly the invention can be obtained by, well known in this field. See, for example, U.S. patent No. 6808901, U.S. patent No. 6652852, U.S. patent No. 6329508, U.S. patent No. 6120767 and U.S. patent No. 5677423, which are included in the present description of the invention in the form of links. Typically, a chimeric antibody can be produced by obtaining cDNA encoding the variable regions of the heavy and light chains of the antibodies, the introduction of expressing the cDNA in the vector, which after transfer in eukaryotic cells-owners expresses a chimeric antibody of the present invention. Expressing the vector preferably carries a functionally complete sequence of the constant region of the heavy or light chain that allows you to easily enter in expressing vector any sequence variable regions of the heavy or light chain.

The present invention relates to humanitarianlaw the antibody that specifically recognizes the specified epitope on CD43 and CEA expressed megamarketinc cancer cell. Humanitariannet antibody typically is a human antibody in which residues from a CDR regions replaced by residues from a CDR regions of a species other than human, such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, remnants of the Fv-fragment frame region of a human antibody is replaced by a suitable and residues other than human.

Monoclonal antibody humanitarium, performing four General stages. These stages include: (1) determination of the nucleotide and proposed amino acid sequence of the original variable regions of light and heavy chains; (2) constructing gumanitarnogo antibodies, i.e. deciding which frame region of the antibodies will be used in the humanization process; (3) carrying out the actual methods of humanization and (4) transfection and expression of gumanitarnogo antibodies. See, for example, U.S. patents№ 4816567, 5807715, 5866692, 6331415, 5530101, 5693761, 5693762, 5585089, 6180370 and 6548640. For example, a constant region may have more to remind the constant region of a person in order to avoid the immune response, if the antibody used in clinical studies for the treatment of people. See, for example, U.S. patent No. 5997867 and 5866692.

It is important that antibodies were humanitarian with retention of high affinity for the antigen and other favorable biological properties. To achieve this chain of humanized antibody can be obtained by analysis of the source sequences and various conceptual humanized products using three-dimensional models of the source and humanized sequences. Specialists in this about the region familiar to the public three-dimensional model of an antibody. There are computer programs that illustrate and reproduce probable three-dimensional conformational structures of selected sequences of the candidate immunoglobulin. The study received images allows an analysis of the possible role of the residues in the functioning sequence of the candidate immunoglobulin, that is, the analysis of residues that influence the ability of the candidate immunoglobulin to communicate with its antigen. Thus can be selected residues frame region and the joint of consensus and input sequence to achieve the desired characteristics of the antibodies, such as increased affinity for the target antigen. Typically, the CDR residues are directly and significantly affect the binding to the antigen. Humanized antibodies may also contain modifications in the hinge region, to improve one or more characteristics of the antibody.

In accordance with another alternative antibodies can be studied and developed the recombinant method of display on the phage. See, for example, U.S. patent No. 5565332, 5580717, 5733743 and 6265150 and publication Winter et al., Annu. Rev. Immunol. 12:433-455 (1994). An alternative method is displayed on the phage (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and fragments of antibodies in vitro from a set of genes is areabelow (V) region of the immunoglobulin, obtained from unimmunized donors. In accordance with the method of the genes of the V region of the antibody clone in frame read in gene major or minor envelope protein of filamentous bacteriophage, such as M13 or fd, and display it in the form of the functional fragments of the antibodies on the surface ragovoy particles. Because filamentous particle contains a copy of the single-stranded DNA genome of phage selection on the basis of the functional properties of antibodies also leads to the selection of the gene encoding the antibody, with the specified properties. Thus, the phage mimics some properties of b-cells. The display on the phage can be made in different formats; see, for example, the publication of Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3, 564-571 (1993). To display on the phage can be used several sources of segments of the gene V-region. Horn and others (Clackson et al., Nature 352:624-628 (1991)) have identified a variety of antibodies against oxazolone from a small random combinatorial library of V genes-region derived from the spleens of immunized mice. Can be created in a range of gene V-region, obtained from unimmunized human donors, and antibodies to a diverse set of antigens (including autoimmune antigens) can be isolated by the methods described in the publication by Mark et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993). In the case of the natural the public immune response antibody genes accumulate mutations with high frequency (somatic spermatazoa). Some of the changes reported higher affinity, and b cells displaying surface immunoglobulin with high affinity, preferably replicate and differentiate during subsequent antigenic stimulation. The above natural process can be simulated using a technique known as “moving the chains”. Marks et al., Bio/Technol. 10:779-783 (1992). When performing this method, the affinity of primary human antibodies obtained by the method of display on the phage, can be improved by sequential replacement gene V-region heavy and light chains of natural variants (sets) gene V-region, obtained from unimmunized donors. The method allows to obtain antibodies and antibody fragments with an affinity in the range of PM-nm. The strategy of obtaining a very large spectrum of antibodies on phage (also known as “mother's library”) described in the publication Waterhouse et al., Nucl. Acids Res. 21:2265-2266 (1993). Permutation method genes can also be used for obtaining human antibodies from antibodies rodents, the human antibody has an affinity and specificity similar to the original rodent antibody. In accordance with the specified method, which is also known as “epitope imprinting”, gene V-region heavy or light chain of the antibody rodents,obtained by the method of display on the phage, is replaced by the genes of the V-region of a person, resulting in a rodent-human Chimera. Selection on antigen allows you to isolate the variable regions of a human, is capable of restoring a functional antigennegative centre, i.e. the epitope controls (captures) the choice of a partner. By repeating this process with the aim of replacing the remaining V-region of the rodent get a human antibody (see PCT publication no WO 93/06213, published April 1, 1993). Unlike traditional humanization of rodent antibodies by CDR grafting specified method allows to obtain fully human antibodies that do not contain residues of the framework region and CDR rodents. It is obvious that, although the above description relates to a humanized antibody, the General principles applicable to the creation of antibodies, for example, for dogs, cats, primates, horses and cows.

In some embodiments of the invention, the specified antibody is a fully human antibody. Antibodies, non-human that specifically bind to the antigen, can be used to produce fully human antibodies that bind with the antigen. For example, a qualified technician can use the method to replace the chain, which heavy chain antibodies, on the personal from the human, coexpression library expressing different light chains of human rights. The resulting hybrid antibody contains one light chain of a human and one heavy chain, non-human, then examined in relation to binding to the antigen. Light chains, which are involved in binding to the antigen, then coexpression with a library of heavy chains of human antibodies. Derived human antibodies again examined for binding to the antigen. Such methods are described in U.S. patent No. 5565332. In addition, the antigen can be used to vaccinate an animal that is transgenic for genes of human immunoglobulin. See, for example, U.S. patent No. 5661016.

The specified antibody can be bespecifically antibody, namely a monoclonal antibody which has binding specificity against at least two different antigens and can be obtained using the antibodies of the present invention. Methods of obtaining bespecifically antibodies known in the art (see, for example, Suresh et al., (1986), Methods in Enzymology 121:210). Recombinant methods of producing bespecifically antibodies traditionally based on the co-expression of two pairs of heavy chain-light chain immunoglobulin with two heavy chains, having a different specificity (Millstein and Cuello, (1983)Nature 305, 537-539).

The fit is accordance with one method of obtaining bespecifically antibody variable regions of the antibody with the desired binding specificity (sites combining the antigen-antibody) merge sequences of the constant region of the immunoglobulin. The fusion preferably is produced from a constant region of a heavy chain immunoglobulin comprising at least part of the hinge region, CH2 and CH3 regions. It is desirable that the first constant region of the heavy chain (SN)containing the site necessary for binding to the light chain was present in at least one of the mergers. DNA encoding the fusion heavy chain immunoglobulin and, if desired, the light chain immunoglobulin is injected into different expressing vectors and cotransferred acceptable in the body of the host. This method provides more flexibility in the regulation of the mutual proportions of the three polypeptide fragments in those embodiments of the invention, when different ratios of the three polypeptide chains used in the construction, to produce the optimum output. However, you can enter the coding sequences for two or all three polypeptide chains in one expressing the vector when the expression of at least two polypeptide chains in equal ratios allows to obtain a high output, or when the ratio does not matter.

In accordance with one method bespecifically antibodies are composed of a hybrid heavy chain of immunoglobulin, in which the first binding specificity is in the same domain, and hybrid pair of heavy chains became the first chain of immunoglobulin (providing a second binding specificity) is in a different domain. Such an asymmetric structure in the presence of light chain immunoglobulin in only one half of bespecifically molecules facilitates the separation of the desired especifismo connections from unwanted combinations of chains of immunoglobulin. The method is described in PCT publication no WO 94/04690, published March 3, 1994

Heteroconjugate antibodies containing two covalently linked antibodies are also included in the scope of the present invention. Such antibodies can be used for directed delivery of immune system cells to unwanted cells (U.S. patent No. 4676980) and for the treatment of HIV infection (PCT publication no WO 91/00360 and WO 92/200373; and European patent No. 03089). Heteroconjugate antibodies can be obtained by standard methods of cross-stitching. Acceptable cross-linking agents and methods well known in the field and described in U.S. patent No. 4676980.

Single-chain Fv fragments can also be obtained by the method described in the publication Iliades et al., 1997, FEBS Letters, 409:437-441. Linking such single-stranded fragments with different linkers described in the publication Kortt et al., 1997, Protein Engineering 10:423-433. In this area is well known for different methods of recombinant receipt and manipulation of antibodies.

It should be noted that the scope of the present invention includes not only the above monoclone the performance communications antibodies but any fragments of these antibodies containing the active binding region of these antibodies, including Fab, F(ab')2, scFv, Fv fragments and the like. Such fragments can be obtained from the monoclonal antibodies of the present invention using methods well known in the field (Rousseaux et al. (1986), in Methods Enzynol., 121:663-69, Academic Press).

Methods of obtaining fragment antibodies are well known in this field. For example, the antibody fragment can be obtained by enzymatic cleavage of antibodies with pepsin to form fragment length 100 kDa, denoted F(ab')2. The specified fragment can be further cleaved thiol reducing agent, optionally with the use of a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds, with the formation of monovalent Fab'fragments with a length of 50 kDa. Alternative as a result of enzymatic cleavage by papain immediately formed two monovalent Fab fragments and an Fc fragment. These methods are described, for example, in U.S. patent No. 4036945 and 4331647 and contained in the links that are included in the present description of the invention in the form of links. Cm. publications Nisonoff et al. (1960), Arch. Biochem. Biophys. 89:230; Porter (1959), Biochem. J. 73:119, Edelman et al., in Methods in Enzymology, vol.1, page 422 (Academic Press, 1967).

Alternative Fab-fragment can be on what is learned through the introduction of DNA coding Fab-fragment antibodies in expressing vector for prokaryotic cells or expressing vector for eukaryotic cells, and the introduction of the specified vector in a prokaryotic or eukaryotic cell for the expression of Fab-fragment.

In addition to selecting host cells, factors that affect the glycosylation of the recombinant process of obtaining antibodies include the mode of cultivation, the medium composition, density culture, oxygen saturation, pH, treatment methods, and the like. Have been proposed various methods for modifying the glycosylation pattern achieved in a particular organism, the host, including the introduction and overexpression of certain enzymes involved in the production of oligosaccharides (U.S. patent No. 5047335, 5510261 and 5278299). Glycosylation or certain types of glycosylation can be enzymatically removed from the glycoprotein, for example, using endoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. In addition, recombinant a host cell can be genetically engineered defective when processing certain types of polysaccharides. The above and such methods are well known in this field.

In some embodiments the invention, the antibodies of the present invention can be modified using the methodology is binding, known in this field, which include, but are not limited to, enzymatic methods, oxidative substitution and helatoobrazovateli. Modifications can be used, for example, to attach labels for immunoassay. Modified polypeptides, obtained by the methods used in this field can be investigated using standard assays known in this area, some of which are described below and in the “Examples”section.

The antibody or polypeptide of the present invention can be anywhereman (e.g., connected) with this agent as a therapeutic agent and a label. Examples of therapeutic agents include radioactive parts, cytotoxins or chemotherapeutic molecule.

The antibody (or polypeptide) of the present invention may be associated with a label such as a fluorescent molecule, a radioactive molecule, an enzyme or any others labels known in this field. In this sense, the term “label” refers to any molecule that can be detected. In a particular embodiment of the invention, the antibody can be observed by introducing amino acid labeled with a radioactive isotope. In a particular embodiment of the invention the antibody can be attached Biotin that can be detected by labeled avidin is (for example, the streptavidin containing a fluorescent marker or possessing enzymatic activity that can be detected by optical or colorimetric methods). In certain embodiments of the invention, a label may be inserted or attached to another reagent, which in turn communicates with interest by the antibody. For example, the label may be inserted or attached to an antibody, which specifically binds to interest the antibody. In certain embodiments of the invention a label or marker can also be therapeutic agents. In this area known various methods of labeling polypeptides and glycoproteins, which can be used in the present invention. Certain General classes of labels include, but are not limited to, enzymatic, fluorescent, chemiluminescent, and radioactive labels. Examples of labels for polypeptides include, but are not limited to, radioactive isotopes or radionuclides (e.g.,3H,14C,15N35S90Y99Tc111In125I131I), fluorescent labels (e.g., isothiocyanate fluorescein (FITC), rhodamine, lanthanide phosphorus, phycoerythrin (D)), enzymatic labels (such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose is xidase, glucose-6-phosphate dehydrogenase, alcoholdehydrogenase, malatdegidrogenaza, penicillinase, luciferase), chemiluminescent substances, biotinylate groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., pairwise sequence latinovich zippers, binding sites for secondary antibodies, metallovedeniye domains, tag epitopes). In certain embodiments of the invention the label is attached by means of spacers of various lengths to reduce potential steric inconsistencies.

The present invention relates also to pharmaceutical compositions comprising the antibodies or polypeptides of the present invention and a pharmaceutically acceptable carrier or excipients. In this area known pharmaceutically acceptable excipients, which are relatively inert substances that facilitate the introduction of pharmacologically effective substances. For example, excipient can tell the shape or texture, or to act as a diluent. Acceptable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for modifying the osmotic concentration of a solution, encapsulating agents, buffers, and substances that enhance penetration through the skin. Excipients, as well as preparations for the pair is Teroldego and apparentering administration of drugs presented in Remington, The Science and Practice of Pharmacy, 20th Ed. Mack Publishing (2000).

Some embodiments of the invention relate to compositions (discussed in the present description of the invention, which are intended for use in any methods of the present invention, both as drugs and for obtaining medicines.

Polynucleotides, vectors and cells-owners

The present invention relates also to polynucleotides comprising a nucleotide sequence encoding any of the monoclonal antibodies and polypeptides of the present invention. In some embodiments of the invention polypeptides include a sequence of variable regions of the light chain and/or heavy chain.

In some embodiments of the invention polynucleotide include a nucleic acid sequence encoding a heavy chain containing the variable region of the heavy chain comprising one or more CDR regions from the amino acid sequence of SEQ ID NO:1, and a constant region of a heavy chain including amino acid sequence selected from the group consisting of SEQ ID nos:11-30, and/or a nucleic acid sequence encoding a light chain containing a variable region light chain comprising one or more CDR regions from the amino acid the sequence SEQ ID NO:2, and the constant region light chain comprising the amino acid sequence selected from the group consisting of SEQ ID nos:10 and 31-37. In some embodiments of the invention polynucleotide contain the nucleic acid sequence encoding a heavy chain containing the variable region of the heavy chain comprising three CDR region of the amino acid sequence of SEQ ID NO:1, and a constant region of a heavy chain including amino acid sequence selected from the group consisting of SEQ ID nos:11-30, and/or a nucleic acid sequence encoding a light chain containing a variable region light chain comprising three CDR region of the amino acid sequence of SEQ ID NO:2, and a constant region comprising the amino acid sequence selected from the group consisting of SEQ ID nos:10 and 31-37.

Professionals in this field should be aware that due to the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptide of the present invention. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Thus, polynucleotide, which differ due to differences in the use of the codon are included in the scope of the present invention. In addition to t the th, alleles of genes containing the polynucleotide sequence of the present invention, also included in the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The obtained mRNA and protein may, although not necessarily, have a modified structure or function. Alleles can be identified by standard methods (such as hybridization, amplification and/or comparison of sequences in the database).

Polynucleotide of the present invention can be obtained by chemical synthesis, recombinant DNA or polymerase chain reaction (PCR). Methods of chemical synthesis of polynucleotides well known in this field and do not require detailed consideration in the present description of the invention. The person skilled in the art can use the sequence according to the present invention and industrial DNA synthesizer to obtain the desired DNA sequence.

To obtain polynucleotides methods of recombinant DNA, polynucleotide comprising the desired sequence can be inserted at the appropriate vector, which in turn can be introduced in a suitable cell host for replication and amplification, as will be hereinafter rasm is treno in the present description of the invention. Polynucleotide can be introduced into host cells by any means known in this field. Cells transformed by the introduction of exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating or electroporation. After the introduction of exogenous polynucleotide can be in the cell in the form of non-embedded vector (such as plasmid) or integrated into the genome of the host cell. Amplificatory thus polynucleotide can be isolated from the host cell by methods well known in the field. See, for example, Sambrook et al. (1989).

Alternative DNA sequence can be reproduced by using polymerase chain reaction (PCR). Methods of performing PCR are well known in this field and are described in U.S. patent No. 4683195, 4800159, 4754065 and 4683202, as well as in the publication PCR: The Polymerase Chain Reaction, Mullis et al., eds., Birkauswer Press, Boston (1994).

The present invention also relates to vectors, such as cloning vectors expressing vectors), including the sequence of a nucleic acid encoding any of the polypeptides (including antibodies) of the present invention. Acceptable cloning vectors can be generated by standard methods, or can be selected from a large number of cloning vectors are available in this area. Despite the fact that wybran the th cloning vector may vary depending on allegedly used host cell, suitable cloning vectors usually able to autoreplacement, can have a single target for a particular restriction endonuclease and/or may carry genes for marker, which can be used in the selection of clones containing the specified vector. Acceptable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and Shuttle vectors such as pSA3 and rat. These and many other cloning vectors can be purchased from commercial suppliers, such as BioRad, Stratagene, and Invitrogen.

Expressing the vectors generally are replicable polynucleotide constructs that contain polynucleotide of the present invention. Expressing the vector can replicate in the cells of the host in the form of apicom or an integral part of the chromosomal DNA. Acceptable expressing vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, Comedy and expressing vectors, described in PCT publication no WO 87/04462. Vectors can include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an acceptable elements that control transcription (such as the promoters, the enhancers and the terminator). For expression (i.e. broadcast) are also usually required one or more elements to control the transmission, such as the binding sites of ribosomes, the sites of translation initiation and termination codons.

Vectors containing interest polynucleotide, can be introduced into the cell host by any acceptable means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances, microprojection bombing, lipofection and infection (for example, when the vector is an infectious agent, such as vaccinia virus). The choice of the method of introducing vectors or polynucleotides often depends on the characteristics of the host cell.

The present invention relates also to the cells of the host, including any polynucleotide or vectors of the present invention. Any cell-hosts capable of sverkhekspressiya heterologous DNA can be used to identify genes encoding an antibody polypeptide or protein. Non-limiting examples of host cells of mammals include, but are not limited to, COS cells, HeLa and Cho. Cm. also PCT publication no WO 87/04462. Acceptable cell owners other than mammalian cells, include procaryotic the Kie cells (such as E. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe or K. lactis).

The diagnostic application

The present invention relates to a method of using the antibodies, polypeptides and polynucleotides of the present invention for the detection, diagnosis and monitoring of diseases, disorders or conditions associated with expression of the indicated epitope (elevated or reduced level compared to a normal sample and/or pathological expression, for example, the presence of expression in tissues and/or cells in which usually there is no expression of this epitope).

In some embodiments of the invention the method includes detecting expression of the epitope in a sample obtained from the subject suspected of having cancer, such as colorectal cancer, pancreatic cancer, stomach cancer and lung cancer. This method of detection preferably includes contacting the sample with the antibody, polypeptide or polynucleotide of the present invention and detecting the difference between the binding from the same level in the control or comparative sample. This method can also be used to determine the suitability of the antibodies or polypeptides according to the present invention for treatment of a subject.

In this sense, the term “sample is or “biological sample” refers to the entire body or individual tissues, cells or components (e.g., biological fluids, which include, but are not limited to, blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluids and semen). The term “sample” or “biological sample” additionally means homogenate, lysate or extract derived from the whole body or individual tissues, cells or component, its fractions or parts, which include, but are not limited to, plasma, serum, cerebrospinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. Most often, the sample obtained from the animal, but the term “sample” or “biological sample” can also refer to cells or tissues, analyzed in vivo, i.e. without removal of the animal. Usually, if the “sample” or “biological sample” will contain cells from the animal, then this term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva or urine, which can be used to measure associated with cancer levels polynucleotides or polypeptides. The term “sample” or “biological sample” to omnitele means Wednesday, such as nutrient broth or gel, which was a reproduction of the organism containing the cell components such as proteins or nucleic acid molecules.

In one embodiment of the invention, the cells or cell/tissue lysates are contacted with the antibody and determine the binding of an antibody to a cell. If the analyzed cells exhibit a binding activity comparable with that of a control cell tissue of the same type, we can conclude that the analyzed cell is cancerous. In some embodiments of the invention the analyzed cells derived from human tissue.

For detection of specific binding of the antibody-antigen can be used various techniques known in this field. Typical immunoassays that can be performed in accordance with the present invention include fluorescent polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), immunonephelometric analysis of inhibition (NIA), enzyme-linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). The indicator portion or labeled group can be attached to the antibodies and selected in accordance with the needs of different applications of this method, which is often determined by the availability of equipment for analysis and% is urami, compatible with immunoassay. Acceptable labels include, but are not limited to, radioactive isotopes (e.g.,125I131I35S3H or32P), enzymes (e.g. alkaline phosphatase, horseradish peroxidase, luciferase or β-galactosidase), fluorescent part or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP) or fluorescent part (for example, nanoparticles Qdot™, supplied by the Corporation, Quantum Dot Corporation, Palo Alto, CA). Specialists in this area known common methods of performing various of the above immunoassays.

In accordance with the purposes of diagnosis polypeptide, including antibodies, can be mechen creation part, which includes, but is not limited to, radioactive isotopes, fluorescent labels, and different label enzyme substrate, known in the field. In this area known methods of conjugation label with the antibody.

In some embodiments of the invention, the polypeptides, including antibodies of the present invention, do not require labeling, and their presence can be detected using a labeled antibody which binds to the antibodies of the present invention.

Antibodies of the present invention can be used to run any well-known assays, such as competitive binding assays, sandwich assays PR is ICDO and indirect linking and immunoprecipitation analyses. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc. 1987).

Antibodies and polypeptides can also be used to run diagnostic tests in vivo, such as visualization in vivo. The antibody or polypeptide typically mark a radioactive isotope (such as111In99Tc14C,131I125I or3H)to represent the interest of the cells or tissue can be localized by using immunoscintigraphy.

The antibody can also be used as a coloring reagent in the case of pathology using methods that are well known in this field.

Therapeutic use

Antibodies of the present invention is able to induce the death of megamarketinc cancer cells. Thus, the present invention relates to therapeutic uses of the antibodies and polypeptides of the present invention for the treatment and/or delay the development of cancer, such as colorectal cancer, lung cancer, pancreatic cancer, stomach cancer, breast cancer, hepatocellular cancer and thyroid cancer. Antibodies of the present invention it is possible to treat any cancer, such as cancer of the colon, colorectal cancer, lung cancer, breast cancer, brain tumor, malignant melanoma, kidney cancer, bladder cancer, lymphoma, T-cell lymphoma, multiple whom I myeloma, stomach cancer, pancreatic cancer, cervical cancer, endometrial carcinoma, ovarian cancer, esophageal cancer, liver cancer, squamous cell head and neck cancer, skin cancer, cancer of the urinary tract, prostate cancer, horiokartsinoma, throat cancer, larynx cancer, hyperplasia of the stroma of the ovary, androblastoma, endometrial hyperplasia, endometriosis, embryoma, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, angioplasty, retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma, medulloblastoma, ganglioneuroblastoma, glioma, rhabdomyosarcoma, hamartoblastoma, osteogenic sarcoma, leiomyosarcoma sarcoma of the thyroid gland and Wilms tumor and if the cancer cell expresses the epitope recognized by the antibodies of the present invention. This method may optionally include the phase detecting binding of the antibody or polypeptide according to the present invention with a tumor or cancer cell from the subject in need of treatment.

To a subject in need of treatment is usually administered an effective amount of a composition comprising the antibody or polypeptide that allows you to inhibit cancer cell growth and/or induce the death of cancer cells. The composition is preferably obtained using pharmaceutically acceptable carrier.

In one embodiment of the invention developed the first composition is intended for administration in the form of intraperitoneal, intravenous, subcutaneous and intramuscular injection and other routes of administration such as oral administration, the introduction through the mucous membrane, the introduction of inhalation, sublingual introduction etc.

Another variant of implementation of the present invention also includes the introduction of compositions containing the antibodies or polypeptides of the present invention conjugated with other molecules, such as detected label, a therapeutic or cytotoxic agents. These agents can include, but are not limited to, radioactive isotopes, toxins, toxoid, inflammatory agents, enzymes, antisense molecules, peptides, cytokines or chemotherapeutic agents. Specialists in this area known methods of conjugation of antibodies with such molecules. See, for example, PCT publication WO 92/08495, WO 91/14438, WO 89/12624, U.S. patent No. 5314995 and European patent No. 396387, which are fully included in the present description by reference.

In one embodiment of the invention the composition comprises the antibody or polypeptide conjugated with a cytotoxic agent. Cytotoxic agents can include any agents that are detrimental to cells. A preferred class of cytotoxic agents that can be conjugated with antibodies or their fragments, can in luceti, not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and their analogues or homologues.

The dose required for treatment depends on the method of administration, the nature of the drug, the nature of the disease entity, height, body weight, surface area, age and sex of the subject; the other input of medicines and the decision of the attending physician. Acceptable doses are in the range of 0.01-1000,0 mg/kg

Can be used any of the following doses: a dose equal to at least about 50 mg/kg body weight; at least about 10 mg/kg body weight; at least about 3 mg/kg body weight; at least about 1 mg/kg body weight; at least about 750 μg/kg body weight; at least about 500 μg/kg body weight; at least about 250 μg/kg body weight; at least about 100 μg/kg body weight; at least about 50 μg/kg body weight; at least about 10 μg/kg body weight; at least about 1 μg/kg of body weight or less. Can be re-introducing several days or bol is e extended period of time depending on the condition of the subject to achieve the desired suppression of disease symptoms. Illustrative scheme is the introduction includes the introduction of a weekly dose of approximately 6 mg/kg of antibody. However, there may be used other schemes depending on the pharmacokinetic effects, which wants to reach the attending physician. Empirical evidence, such as half-life, usually help to determine the dose. The result of this treatment can be easily controlled using standard methods and analysis.

Some subjects may require the introduction of several doses. The frequency of injection can be determined and adjusted during treatment. For example, the frequency of injection can be determined or adjusted depending on the type and stage of cancer, subject to the treatment, prophylactic or therapeutic injection prior to treatment, the clinical history of the subject, the response to the utility and the decision of the attending physician. The doctor usually continues the introduction of therapeutic antibodies, such as chimeric antibody 5F1) up until the appropriate dose will not achieve the desired result. In some cases, there may be used an antibody preparations with prolonged action. In this area known different drugs and devices to achieve prolonged action.

In one embodiment of the invention the dose of the antibodies and polypeptides can be determined empirically for the subjects, which was made by one or more injections. Subjects injected with increasing doses of the antibodies or polypeptides. To evaluate the effectiveness of the antibodies or polypeptides can be controlled markers of disease symptoms, such as CD43 or CEA. Efficacy in vivo can also be determined by measuring tumor weight or volume, time of disease progression (TDP) and/or the reaction rate (RR).

Introduction antibody or polypeptide by the method according to the present invention can be continuous or intermittent, for example, depending on the physiological state of the recipient, therapeutic or prophylactic administration and other factors known to skilled doctors. Introduction antibody or polypeptide may be essentially continuous over a preselected period of time or may be periodic intervals between doses.

Other drugs are an acceptable form of delivery known in this field, which include, but are not limited to, devices such as liposomes. See, for example, the publication Mahato et al. (1997) Pharm. Res. 14:853-859. Liposomal drugs include, but are not limited to, citavecchia, multi-layered vesicles and single-layer vesicles.

In another embodiment of the invention the composition may include one or more anti-cancer tools, one or more anti who ate of the present invention or the antibody or polypeptide, which binds to a different antigen. Such a composition may contain at least one, at least two, at least three, at least four, at least five different antibodies. Antibodies and other anti-cancer remedies can be in the same drug (e.g., in a mixture, as these drugs are often spotted in this area) or in a separate preparations, administered simultaneously or sequentially, being especially useful in the treatment of a wider range of subjects.

Polynucleotide encoding any of the antibodies or polypeptides of the present invention, can also be used for delivery and expression of any of the antibodies or polypeptides of the present invention in the desired cell. Obviously, expressing the vector can be used to direct expression of the antibody or polypeptide. Expressing the vector can be introduced by any method known in this field, for example, intraperitoneally, intravenously, intramuscularly, subcutaneously, intrathecally, intraventricular, oral, enterline, parenteral, nasal, dermal, sublingually or by inhalation. For example, the introduction of expressing vectors includes local or systemic injection, including injection, oral administration, introduction by the apparatus for shooting particles or cat is tera and local introduction. The specialist in this area should be well-known methods of introducing expressing vectors for the expression of exogenous protein in vivo. See, for example, U.S. patent No. 6436908, 6413942 and 6376471.

Can also be produced by targeted delivery of therapeutic compositions comprising polynucleotide encoding any of the antibodies or polypeptides of the present invention. Mediated by receptor methods of DNA delivery are described, for example, in the following publicationsFindeis et al.,Trends Biotechnol. (1993) 11:202; Chiou et al.,Gene Therapeutics: Methods And Applications Of Direct Gene Transfer(J. A. Wolff, ed.) (1994); Wu et al.,J. Biol. Chem.(1988) 263:621; Wu et al.,J. Biol. Chem.(1994) 269:542; Zenke et al. (1990),Proc. Natl. Acad. Sci. USA, 87:3655; Wu et al. (1991),J. Biol. Chem. 266:338. Therapeutic compositions containing polynucleotide, enter the amount from about 100 ng to about 200 mg of DNA for local administration in accordance with the methods of gene therapy. In the process of gene therapy can be used in concentrations ranging from about 500 ng to about 50 mg, from about 1 μg to about 2 mg, about 5 μg to about 500 μg and about 20 μg to about 100 μg of DNA.

Therapeutic polynucleotide and polypeptides of the present invention can be delivered using vectors for gene delivery. Vector for gene delivery can be viral or non-viral origin (see publications Jolly (1994), Cancer Gene Therapy 1:51; Kimura (1994), Human Gene Therapy 5:845 Connelly (1985), Human Gene Therapy 1:185; and Kaplitt (1994), Nature Genetics 6:148). The expression of such coding sequences can be induced using endogenous or heterologous mammalian promoters. The expression of the coding sequence may be constitutive or regulated.

In this area are well known vectors based on viruses, intended for delivery to and expression of the necessary polynucleotide in the desired cell. Typical vectors based on viruses include, but are not limited to, vectors based on recombinant retroviruses described, for example, in PCT publications no WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. patent No. 5219740, 4777127; UK patent No. 2200651 and European patent No. 0345242; vectors based alphavirus, for example, vectors based virus Sindbis, fever virus Semliki forest (ATSS VR-67; ATCC VR-1247), virus Ross river (ATCC VR-373; ATCC VR-1246) and virus Venezuelan equine encephalitis (ATSS VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and vectors based on adeno-associated virus (AAV), described, for example, in PCT publications no WO 94/12649, WO 93/03769, WO 93/19191, WO 94/28938, WO 95/11984 and WO 95/00655. You can also enter DNA related to killed adenovirus, in accordance with the description given in the publication Curiel (1992), Hum. Gene Ther. 3:147.

Can also be used non-viral vectors for delivery and how they applied what I which include, but are not limited to, poly-condensed DNA linked or not linked to killed adenovirus (see, for example, the publication Curiel (1992), Hum. Gene. Ther. 3:147); DNA associated with ligand (see, for example, Wu (1989), J. Biol. Chem. 264:16985); vectors for delivery to eukaryotic cells (see, for example, U.S. patent No. 5814482, PCT publication no WO 95/07994, WO 96/17072, WO 95/30763 and WO 97/42338) and neutralizing the charge of the nucleic acid or fusion with cell membranes.

Can also be used deproteinizirovanny DNA. Typical methods of introducing deproteinizing DNA described in PCT publication no WO 90/11092 and U.S. patent No. 5580859. Liposomes, which can serve as carriers for gene delivery, as described in U.S. patent No. 5422120, PCT publications No. WO 95/13796, WO 94/23697, WO 91/14445 and European patent No. 0524968. Additional methods are described in publications Philip (1994), Mol. Cell Biol. 14:2411 and Woffendin (1994), PNAS 91:1581.

In addition, the present invention relates to a method of treating cancer in a subject which comprises: (a) the introduction of a specified subject an effective amount of a composition comprising the antibody of the present invention; and (b) conduct additional anti-cancer therapy. In some embodiments of the invention additional therapy includes surgery, radiation therapy, hormonal therapy, genethera the Oia, therapy with other antibodies and chemotherapy. Composition comprising the antibody, can be administered simultaneously with carrying out additional therapy (e.g., simultaneous administration) and/or sequentially (e.g., sequential administration). For example, a composition comprising the antibody can be entered separately from additional therapy with a time interval of no more than about 15 minutes, for example, about 10, 5 or 1 minute. Alternative composition comprising the antibody can be entered separately from additional therapy with a time interval of more than about 15 minutes, for example, about 20, 30, 40, 50 or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month or more.

Composition comprising the antibody of the present invention, can be entered sequentially or simultaneously with one or more other drugs, such as chemotherapeutic agents (such as 5-FU, 5-FU/MTX, 5-FU/leucovorin, levamisole, irinotecan, oxaliplatin, capecitabine or uracil/tegafur), immunoadjuvant, growth inhibitors, cytotoxic agents, cytokines, etc. the Amount of injected antibodies and drugs depends on the type of drugs, pathological condition to be treated, schemes and techniques, but usually less than the number entered in separate using each of the CSOs medicines.

After administration of the composition containing the antibody of the present invention, a variety of methods well known to the person skilled in the art, to determine the effectiveness of the composition both in vitro and in vivo. Well-known different animal models used to study anti-cancer activity of the composition candidate. Such methods include the transplantation of human tumors Nude naked mice or mice scid/scid, or genetic models of tumor mice, such as mice that lack the gene p53. The nature of these animal models allows them to predict in vivo reactions of people. Such models can be created by introducing cells syngenetic mice by standard methods, such as subcutaneous injection, injection into the tail vein of implantation in the spleen, intraperitoneal implantation, implantation under the kidney capsule, etc.

Sets

The present invention also relates to kits intended for use in implementing methods of the present invention. The kits of the present invention include one or more containers containing the purified antibody or polypeptide of the present invention and instructions for use in accordance with the methods of the present invention described in the present description of the invention. In some variations the tah the invention, these instructions comprise a description of the introduction of antibodies for the treatment and/or delay the development of cancer megamarketinccom origin, such as colorectal cancer, in accordance with the methods of the present invention. The specified set may additionally include a description of the choice of the subject suitable for treatment, taking into account the presence and stage of disease or the expression of epitopes on cancer cells of a given entity.

In some embodiments the invention, kits for detecting a cancer cell in a sample includes an antibody or polypeptide of the present invention and reagents required for detecting binding of the indicated antibody or polypeptide to a cell in the sample.

Instructions relating to the use of the antibodies or polypeptides to treat or slow the development of cancer, usually include information about the dose scheme introduction and method of administration for the intended treatment. The containers may contain a single dose of bulk drug (for example, packaging of drugs for multiple reception) or the dose is less than standard doses. The instructions that came with the kits of the present invention, usually printed on the label or the liner in the package (for example, in a set of nested sheet of paper), but also valid machine-readable instructions (e.g. instructions on magnetic or optical disk).

Label or an insert in the packaging contains information about what this song is the treatment for cancer, considered in the present description of the invention. Can be provided with instructions for performing any of the methods of the present invention.

The kits of the present invention are in acceptable packaging. Acceptable packaging includes, but is not limited to, vials, bottles, cans, flexible packaging (e.g., sealed mirovye or plastic bags), and the like. In the scope of the present invention also includes packaging intended for use in conjunction with a special device, such as an inhaler, a device for insertion into the nose (e.g., spray gun) or a device for infusion, such as a mini-pump. The set may provide an opportunity for sterile access (for example, the container may be a bag of intravenous solution or bottle with stopper, permeable to hypodermic needles). The container also may provide an opportunity for sterile access (for example, the container may be a bag of intravenous solution or bottle with stopper, permeable to hypodermic needles). At least one active agent in the composition is an antibody of the present invention. The specified container may further include a second pharmaceutically active agent.

The kits can optionally include additional components, is such as buffers and necessary information. The specified set typically includes a container and a label or the liner in the package or container, or attached to the container.

EXAMPLES

The following examples serve to illustrate the invention and do not limit its scope.

Example 1. Cloning of the variable regions of light and heavy chains of the antibody 5F1

As indicated in the application for U.S. patent No. 11/811303 filed 06/07/07 (publication U.S. No. 2008/0171043), cDNA of the variable regions of light and heavy chains of the antibody 5F1 amplified using PCR and synthesized cDNA was subcloned into pCRII (Invitrogen) for sequence determination. Nucleotide sequences were obtained from several independent clones and subjected to analysis. Identical to the cDNA sequence of independent clones were selected to represent the V-region of the light or heavy chain of each antibody. The following table 2 shows the translated amino acid sequence and nucleotide sequence encoding a V-region light and heavy chains of the murine antibody 5F1 (m5F1) and gumanitarnogo antibodies 5F1Vc (h5F1Vc).

Table 2. Amino acid sequences of the variable regions of the antibodies and nucleic acid sequence encoding the variable region of the antibody (CDR-region are underlined; the signal peptide is posledovatelnosti italics)

Amino acid sequence of heavy chain antibodies m5F1 (SEQ ID NO:1) and nucleotide sequence (SEQ ID NO:5)

Amino acid sequence of the light chain of the antibody m5F1 (SEQ ID NO:2) and nucleotide sequence (SEQ ID NO:6)

Amino acid sequence of heavy chain antibodies h5F1Vc (SEQ ID NO:3) and nucleotide sequence (SEQ ID NO:7)

Amino acid sequence of the light chain h5F1Vc (SEQ ID NO:4) and nucleotide sequence (SEQ ID NO:8)

Example 2. A modified version of the variants of the chimeric antibody 5F1

The isotype mouse antibody 5F1 is IgG3 mouse. To resolve the problem associated with the response to human antibodies against mouse (HAMA), and provide more efficient Fc-dependent functions in the human body was created chimeric form of the antibody 5F1 (c5F1) (c5F1-v0; heavy chain: SEQ ID NO:1 (VH), NO:9 (CH); light chain: SEQ ID NO:2 (VL), NO:10 (CL), see table 2 and figure 2) by combining the variable (V) region of the mouse antibody 5F1 with a constant region IgG1 person. Compare also the amino acid sequence of the constant region of the heavy chain, which includes SN-sphere is, the hinge region, CH2 and CH3-region of human IgG1 and IgG3 mouse. As a result of comparison of the sequences, it was found that SN-region and the hinge region are characterized by the greatest difference between IgG3 mouse IgG1 and human (figure 1). The value used in the present description of the invention when comparing sequences, “*” means that the residues in that column are identical in all sequences compared, “:” indicates that occurred conservative substitutions, and “.” means that was semi-conservative substitutions. To obtain antibodies c5F1 with inducing apoptosis activity, the equivalent figure murine antibody 5F1, was made several modifications in SN-area and/or hinge region of the heavy chain of the antibody c5F1 (table 3; the numbering of the residues in table 3 produced according to the EU numbering system described in the publication Burton, Mol. Immunol. 22:161-206, 1985), and several modifications in the light chain of the antibody c5F1 (table 4). In some cases, the modified heavy chain was expressed together with a modified near-end of the light chain (table 5). Cm. also Fig. 2 to familiarize yourself with the amino acid sequences of the heavy chain and light chain.

Table 3
Modification hard the chain v0[H] on the basis of a constant region of human IgG1

Table 4
Modifications to the constant region of the light chain v0[L] on the basis of Kappa-chain IgG1 human
Table 5
Chimeric antibodies comprising a combination of modified constant regions of the heavy and/or light chain

Example 3. Introduction changes in the constant region of the heavy and light chains of the chimeric antibody 5F1

In order to facilitate the production and purification of antibodies for expression of a chimeric antibody 5F1 (c5F1) used the vector pcDNA5-FRT-hIgG1 (created in the company AbGenomics)containing the constant region of the heavy chain and Kappa light-chain of human IgG1. Variable region genes for the heavy chain and light chain antibodies m5F1 amplified separately using PCRs using primer pairs, respectively m5F1HC-XbaI f/m5F1HC-XbaI r and m5F1LC-XbaI f/m5F1LC-XbaI r (table 6, primers A3/A7 and A8/A9). The PCR products were digested XbaI and sequentially inserted into the vector pcDNA5-FRT-hIgG1. The fully-assembled c5F1-expressing plasmid c5F1/pcDNA5-FRT-hIgG1, containing the gene for the heavy chain gene and the light chain c5F1, used for the expression of unmodified antibody c5F1. The same plasmid was used as a matrix for modifications to the antibody c5F1.

Constructs with deletion (v15) and replacing S (v16) in the position of the residue 220 (EU numbering) were created as a result of executing siteprovides mutagenesis using PCR using primers (table 6)introducing mutations in genes c5F1/pcDNA5-FRT-hIgG1, set for siteprovides QuickChange mutagenesis Multi (Stratagene, catalog No. 200531-5) according to the manufacturer's instructions. Oligonucleotide M1 (5'-CAGAGCCCAAATCTGACAAAACTCACAC-3' (SEQ ID NO:47)was used for the deletion of Cys at position residue 220 (v15), and oligonucleotide M2 (5'-CAGAGCCCAAATCTTCTGACAAAACTCACAC-3' (SEQ ID NO:48)) used for the replacement of Ser at position residue 220 (v16). In order to avoid possible random mutations introduced when performing PCR in the process siteprovides mutagenesis, DNA fragments containing modified, cut out by using AgeI (TN-region) and XmaI (CH3 region) and re-cloned in the original vector c5F1/pcDNA5-FRT-hIgG1 to replace the original unmodified areas.

Alternative for making other modifications you shall alkali PCR with overlapping (table 3-6). Two polymerase chain reactions were performed to generate two fragments of the products of DNA containing the desired mutations and the overlying sequence consisting of at least 20 nucleotides. Two of the PCR product was then mixed, denaturiruet and subjected to re-hybridization. Another PCR with two external primers (from the previous two PCR) used to amplify collected, reprezentirovannoe DNA fragment. For example, for v1 used a pair of primers A4/M23 and M24/A3 (table 6) with the aim of creating the first two fragments using PCR. The two fragments obtained by performing PCR, then mixed, re-hybridisierung and used an external primer (A3 and A4) to create a full-length PCR product. Finally, the DNA fragments containing modified, re-cloned in the original vector c5F1/pcDNA5-FRT-hIgG1. The fragment containing the modification SN-scope, re-cloned in the XbaI sites (at the beginning of the V-region heavy chain) and AgeI (TN-region). The fragment containing the modified hinge region, re-cloned sites AgeI (TN-region) and XmaI (CH3-region). To obtain C-terminal modification of the light chain PCR products were cloned sites AvrII (at the end of the V-region light chain) and BamHI (below the coding sequence for the light chain) to replace the original remotefilename the different sequences.

Plasmids containing or not containing the modification, then transfusional cells Flp-In-CHO (Invitrogen, catalog No. R758-07), using lipofectamine 2000 (Invitrogen, catalog No. 11668-019). Culture medium containing unmodified or modified antibodies c5F1, was collected and the antibody was purified by protein A. the Purified antibody was investigated in relation to binding and inducing apoptosis activity in COLO205 cells.

The analysis of binding

Purified antibodies m5F1, c5F1-v0, c5F1-v15 and c5F1-v16 in the concentration 0.125-4 mg/ml was added to the cells COLO205 in the amount of 1.5×105and incubated for 30 minutes at 4°C, washed twice with saline phosphate buffer (PBS)containing 2% FBS and 0.05% NaN3and incubated with 1 μg/ml of the respective secondary antibodies (R-PE-conjugated F(ab')2-a fragment of an antibody goat against IgG(H+L) mouse, Southern Biotech, catalog No. 1032-09; or R-PE-conjugated antibody goat IgG against human, Southern Biotech, catalog No. 2040-09) at 4°C for 30 minutes. At the end of the dyeing, the samples were twice washed in PBS containing 2% FBS and 0.05% NaN3and analyzed using a flow cytometer. All flow cytometrical analyses were performed in a flow cytometer BD-LSR (Becton Dickinson), using the software Cell Quest.

Analysis of apoptosis

Cells COLO205 in the amount of 1.5×105sown in the wells of 96-Luna the different tablets. Aliquots of purified antibodies m5F1, c5F1-v0, c5F1-v15, c5F1-v16 and control antibodies at a concentration of 2-32 μg/ml was obtained in culture medium immediately before use and added to each well. The sample treated m9E10 and h16C11A, was used as isotype control. The treated cells were kept in a thermostat at 37°C for 6 hours prior to analysis of apoptosis by the method of cell sorting with excitation fluorescence (FACS). When performing analysis of apoptosis was measured by staining with annexin V using the kit for detection of apoptosis, including annexin-V-FITC, (Strong Biotech, catalog No. AVK250), in accordance with the manufacturer's instructions. The treated cells were collected and resuspendable in binding buffer for annexin V, containing annexin V-FITC at room temperature. Cells were incubated in the dark for 15 minutes and then washed twice with 200 ál binding buffer for annexin V Before performing FACS analysis was added to 0.25 μg/ml of iodide of propecia (PI). All flow cytometrical analyses were performed in a flow cytometer BD-LSR (Becton Dickinson), using the software Cell Quest. Cells positive for annexin V and/or cells positive for PI are considered to be apoptotic cells.

Table 6br/> The primers used to introduce mutations into a gene antibodies c5F1

Result

Data binding and induction of apoptosis for variants of the antibody 5F1, the resulting execution flow cytometrical analysis, shown in figure 3 and the following table 7. Antibodies c5F1-v0, c5F1-v15 and c5F1-v16 contact with COLO205 cells and induce apoptosis in COLO205 cells as well as murine antibody m5F1. Antibodies c5F1-v15 and c5F1-v16 contact with COLO205 cells to a lesser extent compared with the antibody c5F1. With regard to induction of apoptosis, the effects observed in cells treated with antibody c5F1-v0, was not as effective as when using antibodies m5F1. However, when using forms with a modified hinge region (c5F1-v15 and c5F1-v16) inducing apoptosis activity was restored. Antibodies c5F1-v15 and c5F1-v16 induced apoptosis in COLO205 cells almost as effectively as the antibody m5F1, while h is about the activity of antibody binding c5F1-v15 and c5F1-v16 with COLO205 cells, apparently, was lower than that of antibodies c5F1-v0. Antibodies isotype control E (control Ig isotype mouse) and h16C11A (isotype control human IgG) at a concentration of 32 μg/ml did not induce apoptosis in COLO205 cells.

Table 7
Analysis of apoptosis induced by antibody 5F1 in COLO205 cells after six hours
(ág/ml)2481632
m5F13553769293
c5F1 v033466878
c5F1 v1564829396
c5F1 v1658789296
m9E0 23
h16C11A25
(% of cells positive for annexin V and/or PI)

Example 4. Humanization of an antibody 5F1

Was also created humanitarianly variant antibody 5F1 (figure 4) and put in expressing plasmids with modifications constant regions (see examples 2 and 3).

The area determining complementarity, (CDR) was used to create the variable regions gumanitarnogo antibody 5F1 (h5F1M), in which the CDR region of the variable region of mouse antibody 5F1 introduced in the framework region of the variable region of human IgG1 (acceptor antibody) by recombinant DNA. To determine the most suitable acceptor antibody for mouse antibody 5F1, the sequence of the variable region of mouse antibody 5F1 analyzed together with the database of immunoglobulins created in the company AbGenomics. Murine antibody M (Man Sung With et al., J. Immunol. 148(4):1149-1154 (15 February 1992) is best matched murine antibody 5F1. The human antibody Eu Man Sung With et al., J. Immunol. 148(4):1149-1154 (February 15, 1992) was selected as the acceptor antibody. Nucleotide sequences were designed and synthesized with the ability to create gumanitarnogo variant antibody 5F1 with three CDR regions of the murine antibody 5F1 entered in the frame region of the variable regions of the antibodies of the Eu.

To create each gene V-region antibody h5F1M were synthesized four pairs of oligonucleotides of length 55-70 grounds, which then formed the overlapping area, consisting of at least 18 nucleotides (table 8, for the heavy chain: N1-N8, light circuit: L1-L8). The Assembly and amplification of all genes of the V-region was performed in four steps: 1) four pairs of complementary oligonucleotides (for heavy chain: H1/H2, H3/H4, H5/H6 and H7/H8; light chain: L1/L2, L3/L4, L5/L6 and L7/L8) was hybridisable and region with the 3'-end gaps were filled fragment maple when performing separate reactions with the formation of four fragments of double-stranded DNA (dzanc); 2) received four fragments dzanc mixed pairs, denaturiruet, re hybridisable and the 3'end gap was filled as a result of executing two separate reactions with the formation of two fragments dzanc; 3) two of the received fragment dzanc mixed, denaturiruet, re hybridisable and the 3'end gap was filled with education fully esmersoy dzanc; and 4) is then performed PCR with two external primers (for heavy chain: a10 and A11, for light chain: A12 and A13 (table 8)containing the XbaI site, for amplification of the collected fragments of the VL and VH.

XbaI-containing fragments, VH and VL are then introduced into the vector pcDNA5-FRT-hIgG1 sites NheI and AvrII, respectively, for the heavy chain and light chain. Plasmid h5F1M/pcDNA5-FRT-hIgG1, expressing fully assembled antibody h5F1M and containing the gene for the heavy chain and light chain antibodies h5F1M, used for the expression of unmodified antibody h5F1M. The same plasmid was used as a matrix for modifications to the antibody h5F1M (figure 4).

Modification of antibodies h5F1M

Polymerase chain reaction with overlapping and siteprovides mutagenesis by PCR was used for modification of the variable regions of the antibodies h5F1M (figure 4) using the primers listed in tables 8 and 9. Unmodified or modified variable regions of antibodies h5F1 were introduced in the constant region (unmodified or modified) human IgG, as shown in examples 2 and 3. Expressing plasmid was then transfusional cells SNO. Supernatant collected and the antibodies were purified by protein A. the Purified antibodies were tested in regard to the characteristics of binding and induction of apoptosis of COLO205 cells.

Table 8
The list of primers used to create variants of humanized antibody 5F1

Table 9
Primers for modifying antibodies h5F1M

Example 5. The study of variants of the chimeric antibody 5F1

Antibody binding to cells Colo205

Purified antibodies m5F1, c5F1-v0, c5F1-v17, c5F1-v24 and c5F1-v25 concentration of 1 μg/ml was added to the cells Colo205 in the amount of 2×105, were incubated for 30 minutes at 4°C, washed twice PBS containing 1% FBS, and incubated with 1 μg/ml of the respective secondary antibodies (R-PE-conjugated F(ab')2-a fragment of an antibody goat against IgG(H+L) mouse, Southern Biotech, catalog No. 1032-09; or R-PE-conjugated antibody goat IgG against human othern Biotech, catalog No. 2040-09) at 4°C for 30 minutes. At the end of the dyeing, the samples were twice washed in PBS containing 1% FBS and 0.05% NaN3and analyzed using a flow cytometer. All flow cytometrical analyses were performed in a flow cytometer BD-LSR (Becton Dickinson), using the software Cell Quest. The data in table 10 show that all tested variants of the antibody 5F1 can contact Colo205 cells.

Table 10
Binding to cells Colo205
AntibodiesThe mean fluorescence intensity (MFI)
mIgG37
m5F1800
hIgG16
c5F1v02760
c5F1v172303
c5F1v243134
c5F1v253174

Analysis of apoptosis

Cells Colo205 in the amount of 1.5×105sown in the wells of 96-well plates. Aliquots of purified antibodies m5F1, c5F1, c5F1-v17, c5F1-v24, c5F1-v25 and control antibodies at stake is entrale 8-32 µg/ml was obtained in culture medium immediately before use and added to each well. The treated cells were kept in a thermostat at 37°C for 6 hours prior to analysis of apoptosis by the method of cell sorting with excitation fluorescence (FACS). When performing analysis of apoptosis was measured by staining with annexin V using the kit for detection of apoptosis, including annexin-V-FITC (Strong Biotech, catalog No. AVK250) according to the manufacturer's instructions. The treated cells were collected and resuspendable in binding buffer for annexin V, containing annexin V-FITC at room temperature. Cells were incubated in the dark for 15 minutes and then washed twice with 200 ál binding buffer for annexin V Before performing FACS analysis was added to 0.25 μg/ml of iodide of propecia (PI). All flow cytometrical analyses were performed in a flow cytometer BD-LSR (Becton Dickinson), using the software Cell Quest. Cells positive for annexin V and/or cells positive for PI are considered to be apoptotic cells. The data in table 11 show that all tested variants of the antibody 5F1 can induce apoptosis in Colo205 cells.

54
Table 11(a,b)
The induction of apoptosis cells Colo205
(a) Experiment 1
8 mcg/ml16 mcg/ml32 µg/ml
m5F1889292
c5F1v0346070
c5F1v24335262
c5F1v25264350
mIgG117
hIgG118
(% of cells positive for annexin V and/or PI)
(b) Experiment 2
8 mcg/ml16 mcg/ml32 µg/ml
m5F1899496
c5F1v06369
c5F1v17515660

mIgG126
hIgG127
(% of cells positive for annexin V and/or PI)

The study xenografts

The Colo205 cells (5×105implanted subcutaneously in the lateral region of the hind limb of 6-7-week-old SCID mice in the 0-th day. Mice intraperitoneally was injectively antibodies in the amount of 30 mg/kg in the 0-th day after inoculation of tumor cells and produced the re-introduction on the 4th, 7th, 11th, 14th and 18th day. In each experimental group was six mice. Tumor growth was determined by measuring tumor volume (mm3) with a caliper twice a week, and the size of the tumor was calculated by the formula: π/6 × larger diameter × (smaller diameter)2(Kievit, E., Cancer Research, 60:6649-55). Mice were killed on day 21, the tumor was removed and measured the mass of the tumor. The results are shown in table 12, the certificate shall indicate the antitumor effect of all tested antibodies compared with the introduction of PBS.

Table 12
The study xenografts
Tumor volume (mm3)The tumor weight (g)
PBS521,695±129,0060,3228±0,0707
c5F1v17 (30 mg/kg × 6 times)169,698±68,798*0,0925±0,0360*
c5F1v24 (30 mg/kg × 6 times)44,108±37,382*0,0170±0,0154*
c5F1v25 (30 mg/kg × 6 times)111,093±56,051*0,0682±0,0320*
*P<0,01 compared with the introduction of PBS on day 21 (t-student test).

The synergistic action of antibody 5F1 together with oxaliplatin in relation to induction of apoptosis cells Colo205

Cells Colo205 in the amount of 1.4×105sown in the wells of 96-well plates. Aliquots oxaliplatin, restored in 5% glucose solution was obtained immediately before use and added to each well at a final concentration of 1 and 10 μg/ml, alone or together with an aliquot of purified antibodies c5F1-v17, c5F1-v24, c5F1-v2 and the control antibody at final concentrations equal to 10 and 30 μg/ml Treated cells was kept in a thermostat at 37°C for 24 hours prior to analysis of apoptosis by FACS method. When performing analysis of apoptosis was measured by staining with annexin V using the kit for detection of apoptosis, including annexin-V-FITC (Strong Biotech, catalog No. AVK250) according to the manufacturer's instructions. The treated cells were collected and resuspendable in binding buffer for annexin V, containing annexin V-FITC at room temperature. Cells were incubated in the dark for 15 minutes and washed twice with 200 ál binding buffer for annexin V Before performing FACS analysis was added 0.5 µg of iodide of propecia (PI). All flow cytometrical analyses were performed in a flow cytometer BD-LSR (Becton Dickinson), using the software Cell Quest. Cells positive for annexin V and/or cells positive for PI are considered to be apoptotic cells. The data in table 13 indicate a synergistic effect of all tested antibody 5F1 together with oxaliplatin in relation to the induction of apoptosis of cancer cells Colo205.

Table 13
The effect of antibody 5F1 together with oxaliplatin
% apoptosis* Oxaliplatin 0Oxaliplatin 1 mg/mlOxaliplatin 10 mg/ml
Antibody 0026
HIg 30 mcg/ml142
c5F1v17 10 mg/ml273046
c5F1v17 30 mcg/ml495562
c5F1v24 10 mg/ml193042
c5F1v24 30 mcg/ml314954
c5F1v25 10 mg/ml203553
c5F1v25 30 mcg/ml445463
* Less background signal

The binding of an antibody m5F1 with SU.86.86 cells of pancreas glands and the induction of apoptosis of these cells

The purified antibody m5F1 and control antibodies at a concentration of 1 μg/ml was added to the SU.86.86 cells at 2×105and incubated for 1 hour at 4°C, washed twice PBS containing 1% FBS, and then incubated with 1 μg/ml of the respective secondary antibodies (R-PE-conjugated F(ab')2-a fragment of an antibody goat against IgG(H+L) mouse, Southern Biotech, catalog No. 1032-09) at 4°C for 1 hour. At the end of the dyeing, the samples were twice washed in PBS containing 1% FBS, and analyzed using a flow cytometer. All flow cytometrical analyses were performed in a flow cytometer BD-LSR (Becton Dickinson), using the software Cell Quest.

Table 14
The binding of an antibody 5F1 with SU.86.86 cells
AntibodiesMFI
only the secondary antibody6
m5F1131

The SU.86.86 cells at 2×105sown in wells of 12-well plates. Aliquots of purified antibodies m5F1 concentration 2-32 μg/ml was obtained in culture medium immediately before use and added to each well. Control antibody at a concentration of 32 μg/ml was used to measure the background signal. The treated cells were kept in a thermostat at 37°C for 6 hours prior to analysis by FACS method. When performing analysis of apoptosis was measured by staining with annexin V using the kit for detection of apoptosis, including annexin-V-FITC (Strong Biotech, catalog No. AVK250) according to the manufacturer's instructions. The treated cells were collected and resuspendable in binding buffer for annexin V, containing annexin V-FITC at room temperature. Cells were incubated in the dark for 15 minutes and washed twice with 200 ál binding buffer for annexin V Before performing FACS analysis was added to 0.25 μg/ml of iodide of propecia (PI). All flow cytometrical analyses were performed in a flow cytometer BD-LSR (Becton Dickinson), using the software Cell Quest. Cells positive for annexin V and/or cells positive for PI are considered to be apoptotic cells.

Table 15
Induction of apoptosis SU.86.86 antibody m5F1
02 mcg/ml4 mcg/ml8 mcg/ml16 mcg/ml32 µg/ml
mIgG1 no datano datano datano datano data36
m5F1366072788991
(% of cells positive for annexin V and/or PI)

The data shown in tables 14 and 15 show that the antibody m5F1 can contact SU.86.86 cell line cancer of the pancreas, while the binding of antibody m5F1 causes apoptosis SU.86.86 cells.

Experiments on binding were performed using antibodies c5F1.v15, c5F1.v16 and c5F1.v24. These antibodies were characterized by significant binding to SU.86.86 cells. Analysis of apoptosis was performed in respect of antibodies c5F1.v15. Data show that the antibody at a concentration of 8 μg/ml and 32 μg/ml induces apoptosis SU.86.86 cells only in the presence of cross-linker mouse IgG against human, which is a specific Fcγ fragment (Jackson ImmunoResearch 209-005-098).

Links

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Fernandez-Rodrigue, J., Andersson, C.X., Laos, S., Baeckstrom, D., Sikut, A., Sikut, R., and Hansson, G.C. (2002) The leukocyte antigen CD43 is expressed in different cell lines of nonhematopoietic origin. Tumour Biol. 23(4):193-201.

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Sikut, R., Andersson, C.X., Sikut, A., Fernandez-Rodriguez, J., Karlsson, N.G., and Hansson, G.C. (1999) Detection of CD43 (leukosialin) in colon adenoma and adenocarcinoma by novel monoclonal antibodies against its intracellular domain. Int. J. Cancer. 82(l):52-8.

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Shelley, C.S., Remold-O'donnell, E., Davis III, A.E., Bruns, G.A.P., Rosen, F.S., Carroll, M.C., and Whitehead, A.S. (1989) Molecular characterizationof sialophorin (CD43), the lymphocyte surface sialoglycoprotein defective in Wiskott-Aldrich syndrome. Proc. Natl. Acad. Sci. USA 86:2819-23.

1. The selected antibody that specifically binds to an epitope on CD43 and/or carcinoembryonic the antigen (CEA)expressed megamarketinc cancer cell, comprising a heavy chain and light chain, in which:
(a) a heavy chain contains a variable region heavy chain comprising three areas that define complementarity, (CDRs) from the amino acid sequence of SEQ ID NO:1, and a constant region of a heavy chain including amino acid sequence selected from the group consisting of SEQ ID nos:11-30; and
(b) a light chain contains a variable region light chain comprising three areas that define complementarity, (CDRs) from the amino acid sequence of SEQ ID NO:2, and a constant region light chain comprising the amino acid sequence selected from SEQ ID nos:10 and 31-37.

2. The antibody according to claim 1, which is a humanized antibody.

3. The antibody according to claim 1, which is a chimeric antibody.

4. The antibody according to claim 1, in which the constant region of the heavy chain comprises the amino acid sequence of SEQ ID NO:27.

5. The antibody according to claim 4, in which the constant region of the light chain comprises the amino acid sequence of SEQ ID NO:10.

6. The antibody according to claim 5, in which the variable region of the heavy chain comprises the amino acid is an explicit sequence of residues 20-137 of the sequence SEQ ID NO:1 and a constant region of the heavy chain comprises the amino acid sequence of SEQ ID NO:27, variable region light chain comprises the amino acid sequence of residues 20-131 of the sequence SEQ ID NO:2 and a constant region of the light chain comprises the amino acid sequence of SEQ ID NO:10.

7. The antibody according to claim 1, in which the constant region of the heavy chain comprises the amino acid sequence of SEQ ID NO:29.

8. The antibody according to claim 7, in which the constant region of the light chain comprises the amino acid sequence of SEQ ID NO:34.

9. The antibody of claim 8, in which the variable region of the heavy chain comprises the amino acid sequence of residues 20-137 of the sequence SEQ ID NO:1 and a constant region of the heavy chain comprises the amino acid sequence of SEQ ID NO:29, the variable region of the light chain comprises the amino acid sequence of residues 20-131 of the sequence SEQ ID NO:2 and a constant region of the light chain comprises the amino acid sequence of SEQ ID NO:34.

10. The antibody according to claim 7, in which the constant region of the light chain comprises the amino acid sequence of SEQ ID NO:35.

11. The antibody of claim 10, in which the variable region of the heavy chain comprises the amino acid sequence of residues 20-137 of the sequence SEQ ID NO:1 and a constant region of the heavy chain comprises the amino acid sequence of SEQ ID NO:29, the variable region of the light chain consists of amino acid after outermost residues 20-131 of the sequence SEQ ID NO:2 and a constant region of the light chain comprises the amino acid sequence of SEQ ID NO:35.

12. The antibody according to any one of claims 1 to 11, which specifically binds to an epitope on CD43 and carcinoembryonic antigen (CEA)expressed megamarketinc cancer cell selected from the group consisting of colorectal cancer cells, cancer cells of the pancreas or stomach cancer cells.

13. The antibody according to any one of claims 1 to 11, which is conjugated with a cytotoxic agent.

14. The antibody according to item 13, where the cytotoxic agent is a radioactive isotope or a toxin.

15. Polynucleotide for the expression of heavy chain antibodies, encoding a heavy chain of the antibody according to any one of claims 1 to 14.

16. Polynucleotide for the expression of the light chain of the antibody encoding light chain of the antibody according to any one of claims 1 to 14.

17. Polynucleotide for the expression of heavy and light chains of the antibody encoding heavy and light chains of the antibody according to any one of claims 1 to 14.

18. The vector for the expression of heavy and/or light chains of the antibodies, including the sequence of a nucleic acid encoding a heavy and/or light chain of the antibody according to any one of claims 1 to 14.

19. A host cell for expression of the antibody, comprising (a) one vector on p, where the vector encodes the light and heavy chain antibody, or (b) two vectors in p, where the first vector encodes a heavy chain of the antibody and the second vector encodes the light chain of the antibody.

20. A method of obtaining antibodies, of which the first includes culturing the host cell according to claim 19, producing the antibody encoded by nucleic acid and isolating the antibodies produced by the cell host.

21. A method of producing antibodies, which includes the expression in the cell-owner:
(a) polynucleotide comprising a nucleic acid sequence encoding a heavy chain containing the variable region of the heavy chain comprising three CDR region of the amino acid sequence of SEQ ID NO:1, and a constant region of a heavy chain including amino acid sequence selected from the group consisting of SEQ ID nos:11-30; and
(b) polynucleotide comprising a nucleic acid sequence encoding a light chain containing a variable region light chain comprising three CDR region of the amino acid sequence of SEQ ID NO:2, and a constant region light chain comprising the amino acid sequence selected from the group consisting of SEQ ID nos:10 and 31-37;
thus polynucleotide encoding heavy chain and light chain, coexpression in this cell the owner.

22. The method according to claim 20 or 21, in which the antibody is an antibody isolated from cell culture.

23. Pharmaceutical composition for treating cancer megamarketinccom origin, comprising the antibody according to any one of claims 1 to 14 in an effective amount and a pharmaceutically acceptable carrier.

24. Set the La cancer treatment megamarketinccom origin, includes a pharmaceutical composition comprising the antibody according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

25. Set point 24 which further includes instructions introduction to the subject an effective amount of a pharmaceutical composition for the treatment of cancer megamarketinccom origin.

26. Set point 24 which further includes instructions for administration to a subject a pharmaceutical composition together with other anti-cancer treatment for cancer megamarketinccom origin.

27. Set point 24 where the cancer megamarketinccom origin is colorectal cancer, pancreatic cancer or cancer of the stomach.

28. The kit for cancer treatment megamarketinccom origin, comprising a first pharmaceutical composition comprising the antibody according to any one of paragraphs. 1-14 and a pharmaceutically acceptable carrier, and a second pharmaceutical composition comprising another anti-cancer agent.

29. Set p, further comprising instructions introduction the subject of the first pharmaceutical composition and a second pharmaceutical composition for the joint treatment of cancer megamarketinccom origin.

30. Set p where the cancer megamarketinccom origin is colorectal cancer, pancreatic cancer or cancer of the stomach.

31. Method l the treatment of cancer megamarketinccom of origin of the subject, a cancer patient, which includes the introduction of a specified subject an effective amount of a composition comprising the antibody according to any one of claims 1 to 14.

32. The method according to p, in which the cancer megamarketinccom origin is colorectal cancer, pancreatic cancer or cancer of the stomach.

33. A method of treating cancer megamarketinccom of origin of the subject, which includes the introduction of a specified subject a certain amount of the antibody according to any one of claims 1 to 14 and a certain amount of another anticancer expense, the specified antibody together with the specified anti-cancer provide effective treatment of cancer in the subject.

34. The method according to p. 33, in which the anticancer agent is a chemotherapeutic agent.

35. The method according to p, in which the cancer megamarketinccom origin is colorectal cancer, pancreatic cancer or gastric cancer.



 

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FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of immunology. Claimed is a version of Fc polypeptide of human IgG with substitutions 2591 and 308F, where numeration of positions is given in accordance with EU Kabat index. Described is a version of the said polypeptide, including one or several substitutions of the following: 428L, 434S, 307Q, 319L, 250I in addition to the said ones. Disclosed are: a nucleic acid, coding the said versions, a host cell for production of the said versions of polypeptide, which contains the coding nucleic acid, a method of obtaining the said versions of polypeptide, including application of the cell expressing the said polypeptide and containing the nucleic acid, which codes the said polypeptide.

EFFECT: application of the invention provides polypeptide, demonstrating higher affinity with human FcRn, which can be applied in therapy of different diseases.

11 cl, 32 dwg, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a recovered human integrin α5β1 monoclonal antibody. The antibody is characterised by the fact that it contains 6 CDR, 3 CDR from a light chain and 3 CDR from a heavy chain. A nucleic acid (NA) coding the antibody according to the invention, an expression vector containing a NA molecule, a host cell containing the vector, and a method for preparing the antibody on the basis of the cell are described. There are disclosed: a composition and a method for growth inhibition of the tumour cells expressing human integrin α5β1 on the basis of the antibody. What is described is a version of the method for growth inhibition of the tumour cells expressing human integrin α5β1 using the composition.

EFFECT: invention provides the new antibodies with high (approximately nm, as measured by FACS) binding affinity for human integrin α5β1 that can find application in medicine in therapy of the tumours mediated by integrin α5β1 expression.

13 cl, 36 dwg, 3 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to biotechnology. There are presented versions of the recombinant bacterial strain Escherichia coli that are succinic acid producers and contain a gene coding pyruvate carboxylase. The bacterial strain Escherichia coli SGM1.0 [pPYC] possesses ackA, pta, poxB, ldhA, adhE inactivated genes. The bacterial strain Escherichia coli SGM1.0 [pPYC] possesses ackA, pta, poxB, ldhA, adhE and icIR inactivated genes. The bacterial strain Escherichia coli SGM2.0 [pPYC] possesses ackA, pta, poxB, ldhA, adhE inactivated genes and enhanced expression of aceE, aceF and lpdA genes. The bacterial strain Escherichia coli SGM2.1 [pPYC] possesses ackA, pta, poxB, ldhA, adhE, icIR inactivated genes and enhanced expression of aceE, aceF and lpdA genes. The bacterial strain Escherichia coli SGM3.0 [pPYC] possesses ackA, pta, poxB, ldhA, adhE, pflB inactivated genes and enhanced expression of aceE, aceF and lpdA genes. The bacterial strain Escherichia coli SGM3.1 [pPYC] possesses ackA, pta, poxB, ldhA, adhE, pflB, iclR inactivated genes and enhanced expression of aceE, aceF and lpdA genes. What is also presented is a method for preparing succinic acid with using the above strains.

EFFECT: group of inventions provides higher succinic acid yield.

17 cl, 2 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention concerns biotechnology and nanotechnology. The method includes transforming archaeal cells with a recombinant plasmid, growing cells, selecting flagella and modifying the surface of the flagella. The plasmid structure contains recombinant genes for synthesis of flagellins A1 and A2 which form flagella, wherein the sequence of flagellin A1 or flagellin A2 or sequences of flagellin A and flagellin A2 contain at least one peptide insert for selective binding of metal ions or nanoparticles. The point of the peptide insert in flagellin A1 is defined in the region between first and second glycosylation sites located between position 86 and position 96 of SEQ ID NO:2, and the point of the peptide insert in flagellin A2 is defined in the region between first and second glycosylation sites located between position 82 and position 92 of SEQ ID NO:3, where the length of the peptide insert is 5 to 60 amino acids. The method includes selecting archaeal flagella containing peptide inserts for non-covalent bonding with metal ions, performing fragmentation of flagella into fragments and modifying the surface of flagella by binding peptide inserts with metal ions and oxidising metals, washing, drying and packing the obtained nano-structured material.

EFFECT: method enables to obtain a coating for forming active surfaces on flexible and solid substrates or capsules using archaeal flagella, which enable non-covalent bonding of a wide range of substances such as metal ions, metal nanoparticles, semiconductors and other ligands.

6 cl, 11 dwg, 1 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: invention refers to genetic engineering and can be used for methane-producing cell permeability control. What is prepared is a polypeptide able to permeate into a methane-producing cell and to increase its permeability, characterised by an amino acid sequence SEQ ID NO:117, 118 or 119 or being at least 90% identical to the above sequence, or at least 15 sequential amino acids of the above sequence. What is also prepared is a polynucleotide coding the above polypeptide cloning and expressing vectors used for producing host cells producing the polypeptide or used for the vector replication. The polypeptide can contain a fluorescent tag on an N-terminal amino acid residue.

EFFECT: invention enables providing higher methane-producing cell permeability.

18 cl, 35 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to a plant, having high resistance to an AHAS-inhibiting herbicide, which includes at least one Shiloh-8 IMI nucleic acid, parts thereof, a plant cell and seeds. Described is a nucleic acid which encodes a polypeptide which increases herbicide resistance of a plant. Disclosed are an expression cassette and a plant transformation vector which include said nucleic acid. Described are methods of controlling weeds growing near a plant having high resistance to an AHAS-inhibiting herbicide. Disclosed is a method of producing a plant having high resistance to an AHAS-inhibiting herbicide, as well as a method of increasing AHAS activity in a plant. Described is a method of selecting a cell transformed by a vector containing IMI nucleic acid. Also disclosed is a method of increasing resistance to an AHAS-inhibiting herbicide and a weed control method which includes treatment with an AHAS-inhibiting herbicide.

EFFECT: invention enables to obtain a plant which is resistant to an AHAS-inhibiting herbicide, which provides effective control of weeds growing near said plant.

57 cl, 3 dwg, 5 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to methods for preparing next generation drug preparations and biologically active additives in bioreactors on the basis of transgenic producing mammals. The method for creating transgenic animals producing a protein with stable and high expression in milk, involves producing transgenic mammals with using a vector containing a reporter gene coding a target protein that is a goat beta-casein gene promoter, a bovine growth factor terminator and effective two-fold transcription terminators. The terminators surround an expression cartridge and possess an ability to break genome transcripts effectively in a mammalian genome by the effective protection of transgene expression in the mammalian genome against further repression. The effective two-fold terminators represent any mammalian genome site fulfilling the following conditions: 3'-sites of the two simultaneously expressing and opposite genes containing a site of the second-to-last exon, the last intervening sequence, the last exon and a polyadenylation signal, a space of two polyadenylation signals at different DNA strands is no more than 100 base pairs.

EFFECT: method can be used for creating the transgenic animals with high and stable target protein production in milk for medical and research purposes.

4 dwg, 4 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to the field of biotechnology. Synthetic 5'UTR regions are applied to enhance the transgene expression, with the 5'UTR regions being located between a promoter and a sequence, presenting an interest in an expression vector. The claimed invention also claims vectors, which contain the 5'UTR regions, and a method of enhancing the transgene expression in their application.

EFFECT: claimed invention provides the synthetic 5'UTR regions, which contain the first polynucleotide fragment in the form of the second intron of gene of calcium ATphase of the sarcoplasmic/endoplasmic reticulum and the second polynucleotide fragment, represented by a part of the 5' untranslated region (5'UTR) of casein gene.

25 cl, 17 dwg, 2 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biochemistry, in particular to clostridial neurotoxins with a modified persistence. Claimed is a polypeptide, containing HC-domain, the first and, at least, one additional LC-domain with amino acid sequences, at least, 90% identical to the respective sequences of a neurotoxic component of botulotoxin of a serotype A, B, C1, D, E, F or G. Also claimed are nucleic acid, an expression vector and a host cell, intended for the expression of the said polypeptide. Also claimed are a method of obtaining and application of the said polypeptide, including as a component of a pharmaceutical composition, for treatment of a condition, associated with hyperactive cholinergic innervations of a muscle or a exocrine gland, and for cosmetic procedures, associated with wrinkles.

EFFECT: invention makes it possible to controllably vary a period of activity of clostridial neurotoxins.

12 cl, 1 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a recombinant strain of Escherichia coli bacteria - a producer of biologically active flagellin. The described strain is obtained by transformation of an E. coli BL21[DE3] cell culture with recombinant plasmid DNA pET151FliC, which is obtained based on a pET151FliC vector in which was embedded a fliC gene which codes biologically active flagellin, having a nucleotide sequence represented in Seq ID No 3. The strain is deposited in the Russian National Collection of Industrial Microorganisms (RCIM) of the Research Institute for Genetics and Selection of Industrial Microorganisms under No B-11369.

EFFECT: present solution has higher production capacity with respect to recombinant flagellin, which is an effective adjuvant.

1 dwg, 2 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: carrier is proposed for targeted delivery of nucleic acids to cells expressing the receptor CXCR4, which consists of a sequence-ligand to the receptor CXCR4 with the amino acid sequence KPVSLSYRSPSRFFESH, the linker part of two molecules of ε-aminohexanoic acid linking the sequence-ligand to the sequence for compaction of nucleic acids, the sequence providing compaction of nucleic acids and the complex output from endosomes CHRRRRRRHC.

EFFECT: invention can be used for targeted delivery of genetic structures into cells with the receptor CXCR4 on the surfaces, such as malignant tumour and stem cells, in order to correct genetic defects, influence on processes of implementation of the genetic information and prevention of diseases.

3 cl, 7 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a recovered human integrin α5β1 monoclonal antibody. The antibody is characterised by the fact that it contains 6 CDR, 3 CDR from a light chain and 3 CDR from a heavy chain. A nucleic acid (NA) coding the antibody according to the invention, an expression vector containing a NA molecule, a host cell containing the vector, and a method for preparing the antibody on the basis of the cell are described. There are disclosed: a composition and a method for growth inhibition of the tumour cells expressing human integrin α5β1 on the basis of the antibody. What is described is a version of the method for growth inhibition of the tumour cells expressing human integrin α5β1 using the composition.

EFFECT: invention provides the new antibodies with high (approximately nm, as measured by FACS) binding affinity for human integrin α5β1 that can find application in medicine in therapy of the tumours mediated by integrin α5β1 expression.

13 cl, 36 dwg, 3 tbl, 11 ex

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