Antibodies recognising carbohydrate containing epitope on cd-43 and cea expressed on cancer cells, and methods of using such

FIELD: medicine.

SUBSTANCE: invention represents versions of antibodies specifically binding to an extracellular domain of human CD43 or human CEA expressed by nonhematopoietic cancer cells. Said antibodies are characterised by amino acid sequences of either three heavy and light chain CDR, or amino acid sequences VH and VL. There are also presented an antibody conjugate with a cytotoxic agent; a host cell producing the antibody; a composition for nonhematopoietic cancer; a polynucleotide; an antibody expression vector.

EFFECT: versions of a method of treating nonhematopoietic cancer.

29 cl, 16 dwg, 6 tbl, 10 ex

 

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority based on provisional application for the grant of a U.S. patent with a serial number 60/811850, filed June 1, 2006, which is incorporated in this description by reference in full.

The technical FIELD TO WHICH the INVENTION RELATES

The present invention relates to novel monoclonal antibodies that recognize carbohydrate epitope on CD43 and embryonal tumor antigen (CEA)expressed on nonhematopoietic tumor or malignant cells. Such antibodies have the ability to induce cell death (e.g. apoptosis) such nonhematopoietic tumor or malignant cells in the absence of cytotoxic conjugation and immune effector function. Such monoclonal antibodies are applicable as diagnostic and therapeutic tools.

The LEVEL of TECHNOLOGY

CD43 (also called sialophorin or leucovorine), to a large extent sililirovany molecule, is expressed at high levels 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 in T-cells of men with the syndrome Wiskott-Aldrich associated with X-chromosome recessive immunodeficient the disorder (Remold-O'donnell et al. (1987) Blood 70(1): 104-9; Remold-O Donnel et al. (1984) J. Exp. Med. 159: 1705-23).

Functional studies showed that anti-CD43 monoclonal antibody 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 anti-CD43-antibody L11 blocks the binding of T-cells from lymph node and HEV in pierovich plaques. Antibody L11 inhibits the radiolabeled 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, induces apoptosis of hematopoietic progenitor cells (HPC) bone marrow negative marker lines that Express CD34 high density (Bazil et al. (1996) Blood, 87(4): 1272-81), and T-lymphoblastoid cells (Brown et al. (1996) J. Biol. Chem. 271: 27686-95). In addition, recent studies have shown that CD43 functions 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).

Interestingly, scientists also found that some nonhematopoietic tumor cells, in particular adenocarcinoma colorectal really 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: 11503-9; Sikut et al.(1997) Biochem. Biophy. Res. Commun. 238: 612-6. It was shown that glikana on CD43 expressed on the cell line carcinoma of the colon (COLO 205)differ from glycans on CD43 leukocytes (Baeckstrom et al. (1997) J. Biol. Chem. 272: 11503-9). Although it was assumed that the overexpression of CD43 causes activation of the protein of the tumor suppressor p53 (Kadaja et al. (2004) Oncogene 23: 2523-30) and suppresses the subset of target genes of NF-kappaB, partly due to the inhibition of the transcriptional activity of p65 (Laos et al. (2006) Int. J. Oncol. 28: 695-704), direct evidence showing the role of CD43 as a cause of cancer of the colon, is still missing. The conventional anti-CD43 antibodies as a therapeutic agent in the case of nonhematopoietic tumor cells is impossible in practice, due to its strong binding with tumor cells and immune T-cells. There remains a need in the creation of antibodies that are specific contact CD43 expressed on nonhematopoietic tumor or malignant cells, but not associated with a CD43 expressed on leukocytes or other cells of hematopoietic origin. Such antibodies can be used as therapeutic agents for treating nonhematopoietic malignant tumors expressing CD43.

CEA normal is expressed in various glandular epithelial tissues (n is an example, in the gastrointestinal tract, respiratory and urogenital tracts), where it is localized on the apical cell surface (World, S. (1999) Semin. Cancer Biol. 9, 67-81). In tumors derived from these tissues, there is a growing prevalence of expression of CEA from the apical membrane domain to the whole cell surface, along with the secretion of protein in the blood (World, S. (1999) Semin. Cancer Biol. 9, 67-81). Over-expression of CEA is observed in many types of malignant tumors, including cancer, colorectal cancer, pancreatic cancer, lung cancer, gastric cancer, hepatocellular carcinoma, breast cancer and thyroid cancer. Therefore, CEA used as a marker for tumors and immunological assays to measure the increased amount of CEA in the blood of patients with malignant tumor, long used in the clinic to predict and therapy of malignant tumors (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 became potentially applicable associated with tumor antigen for targeted therapy (Kuroki M, et al. (2002) Anticancer Res 22: 4255-64). There are two basic methods of using CEA as a target for immunotherapy of malignant tumors. One way is ecifica purposeful introduction of genes-suicide (gene synthase nitric oxide (iNOS)) (Kuroki M. et al., (2000) Anticancer Res. 20(6A): 4067-71) or isotopes (Wilkinson R. W. 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) in CEA-expressing tumor cells using anti-CEA antibodies. This method was also extended to the use of the antibody or antibody fragment conjugated to a therapeutic means, such as drugs, toxins, radionucleotides, immunomodulators or cytokines. Another way is to use immunological cytolytic activities, in particular through-dependent antibodies cellular cytotoxicity (ADCC) or dependent complement cytotoxicity (CDC), for elimination of tumor cells expressing CEA (Imakiire, T. et al., (2004) Int. J. Cancer: 108, 564-570). Such methods often cause the release of cytokines, leading to systemic side effects.

All communications, publications, and applications for patents mentioned in this specification, are included in the description in full by reference.

The INVENTION

In one aspect the present invention relates to novel antibodies that are specific contact epitope on CD43 and CEA expressed by nonhematopoietic cancer cell, but not associated with specific CD43 expressed by a leukocyte (e.g., peripheral T-cells) and the and Jurkat cell (lymphoblastoid leukemia cell). The epitope bound antibody contains carbohydrate. Such antibodies are able to induce cell death in these nonhematopoietic malignant cells in the absence of cytotoxic conjugation with antibodies and immune effector functions.

The invention relates to the antibody, and the antibody specific binds to the epitope on CD43 and/or CEA expressed by nonhematopoietic cancer cell, but not associated with specific CD43 expressed by a leukocyte or Jurkat cell, and is able to induce apoptosis nonhematopoietic cancer cells after binding to an epitope expressed on the cell surface nonhematopoietic malignant cells in the absence of cytotoxic conjugation and immune effector function, while the epitope contains carbohydrate, and the binding of an antibody to the epitope is inhibited by carbohydrate structure containing Leastructure Lea-lactose, the structure of LNDFH II or the structure of the LNT. In some embodiments, the epitope to which the antibody binds is sensitive to fucose.

Nonhematopoietic malignant cells include, without limitation, cancer cells colorectal and gastric cancer.

In some embodiments, the antibody described in this publication, is a monoclonal antibody. In some variants of the Ah antibody described in this publication is murine, human, humanized or chimeric antibody.

In some embodiments, the antibody described in this publication, when the binding epitope expressed on the cell surface of the nonhematopoietic cancer cells, reduces the number of malignant cells and/or inhibits the growth or proliferation of malignant cells. For example, reducing the number of cells or the inhibition of cell growth in the presence of the antibody is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 65%, about 75% or more compared with the number of cells or cell growth in the absence of antibodies.

In some embodiments, the antibody described in this publication, recognizes a conformational epitope on the extracellular domain of CD43 and CEA, which is expressed nonhematopoietic malignant cell, while the conformational epitope contains a structure with physical and chemical properties equivalent to the properties of the patterns formed by the Tripeptide N'-Trp-Pro-Ile-C'. In some embodiments, the antibody described in this publication, binds to the polypeptide containing the amino acid sequence of N'-Trp-Pro-Ile-C' N-end of the polypeptide.

In some embodiments, the antibody described in this audience is AI, competes with the antibody containing the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO: 1, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 2, for binding to the epitope present on the cell surface nonhematopoietic malignant cells.

In some embodiments, the antibody described in this publication that competes with the antibody containing the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO: 3, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 4, for binding to the epitope present on the cell surface nonhematopoietic malignant cells.

In some embodiments, the antibody described in this publication that competes with the antibody containing the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO: 5, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 6, for binding to the epitope expressed on the cell surface nonhematopoietic malignant cells.

In some embodiments, the antibody contains a variable region of the heavy chain with three CDRs of the amino acid sequence of SEQ ID NO: 1, and the variable region of the light CE and, containing the three CDRs of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibody contains a variable region heavy chain containing the amino acid sequence of SEQ ID NO: 1, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the antibody contains a variable region of the heavy chain with three CDRs of the amino acid sequence of SEQ ID NO: 3, and the variable region of the light chain with three CDRs of the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody contains a variable region heavy chain containing the amino acid sequence of SEQ ID NO: 3, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the antibody contains a variable region of the heavy chain with three CDRs of the amino acid sequence of SEQ ID NO: 5, and the variable region of the light chain with three CDRs of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody contains a variable region heavy chain containing the amino acid sequence of SEQ ID NO: 5, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the antibody is humanitariannet antibody, the content is ASEE variable region of the heavy chain, containing the amino acid sequence of SEQ ID NO: 7, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 8.

In another aspect, the present invention relates to polypeptides containing a heavy chain and/or light chain or a fragment of the antibodies described in this publication. The invention also relates to polypeptides derived from any of the antibodies described in this publication, and the specific polypeptides associated with epitope on CD43 and CEA expressed by nonhematopoietic cancer cell, but not associated with specific CD43 expressed by a leukocyte or Jurkat cell, and is able to induce apoptosis nonhematopoietic cancer cells after binding to CD43 expressed on nonhematopoietic cell surface of malignant cells in the absence of cytotoxic conjugation and immune effector function.

In another aspect, the present invention relates to polynucleotides, encoding any of the antibodies or polypeptides described in this publication. The invention also relates to vectors (such as expressing vectors)containing any of polynucleotides described in this publication. The invention also relates to cells of the host that contains any of polynucleotides or vectors described in D. the authorized publication.

In another aspect, the present invention relates to compositions containing any of the antibodies or polypeptides described in this publication. In some embodiments, the antibodies or polypeptides associated with the agent. In some embodiments, the agent is a therapeutic agent (e.g., radioactive residues, cytotoxins and chemotherapeutics). In some embodiments, the agent is a label (for example, enzymes, fluorescent molecules and Biotin).

The invention also relates to pharmaceutical compositions containing an effective amount of any of the antibodies or polypeptides described in this publication, or polynucleotides encoding antibodies or polypeptides and a pharmaceutically acceptable carrier. In some embodiments, the antibodies or polypeptides associated with therapeutic tool. In some embodiments, the composition is prepared for administration by intraperitoneal, intravenous, subcutaneous and intramuscular injections and other forms of administration, such as oral, mucosal, inhalation, sublingual, etc.

In some embodiments, the composition may contain more than one antibody according to the invention or one antibody according to the invention with one or more other anti-tumor antibodies or other anti-tumor agents.

In the other aspect of the present invention relates to methods of generating antibodies or polypeptide, described in this publication, which includes culturing the host cell or its progeny under conditions that allow production of the antibody or polypeptide, with a host cell contains expressing vector that encodes the antibody or polypeptide. In some embodiments, the method further includes cleaning the antibody or polypeptide.

In another aspect the invention relates to methods of generating any of the antibodies described in this publication, by the expression of one or more polynucleotides encoding the antibody (which can be expressed separately as a single light or heavy chain or both chains, and light and heavy, are expressed from a single vector) in a suitable cell, usually with subsequent extraction and/or selection of interest antibodies or polypeptides.

In another aspect, the present invention relates to methods of inducing apoptosis in nonhematopoietic malignant cell expressing the epitope on the cell surface, which includes the implementation of contact malignant cells with an antibody or a polypeptide described in this publication. In some embodiments, the malignant cell is in the body of the individual.

In another aspect, the present invention relates to FPIC is BAM treating nonhematopoietic malignant tumor in the subject, includes introduction to the subject an effective amount of a composition containing an antibody or a polypeptide described in this publication, the antibody or polypeptide is associated with malignant cells in the body of the subject. In some embodiments, the malignant tumor is a cancer of the rectum and colon, pancreas, stomach or lung.

In another aspect, the present invention relates to methods for treating nonhematopoietic malignant tumor in a subject, comprising the administration to a subject of a certain amount of the antibody or polypeptide described in this publication, and a number of other anti-cancer agents, the antibody or polypeptide is associated with malignant cells in the body of the subject and the antibody or polypeptide and an antitumor agent work together to provide effective treatment of malignant tumors in the subject.

In another aspect, the present invention relates to kits for treating nonhematopoietic malignant tumors of the subject containing the antibody or a polypeptide described in this publication. Such kits may further include instructions for administering the antibody or polypeptide to a subject for treatment of malignant tumors.

In another aspect of this is General the invention relates to methods of detecting or diagnosing nonhematopoietic malignant tumors, the identification of the individual with nonhematopoietic malignant tumor, to treat, or to monitor the progression of nonhematopoietic malignancies, including implementation of contact of the sample with the antibody or a polypeptide described in this publication; and recording the presence or absence or level of binding of the antibody or polypeptide to a cell in the sample. The presence of binding between the antibody and the cell in the sample indicates that the sample may contain malignant cells, and/or that of the subject having malignant tumor can be treated by the antibody described in this publication. The methods may additionally include a phase comparing the level of binding to a control.

In another aspect, the present invention relates to kits for the detection or diagnosis of nonhematopoietic malignant tumors, identification of the individual with nonhematopoietic malignant tumor for treatment or monitoring progression of nonhematopoietic malignant tumor containing antibody or a polypeptide described in this publication, and reagents for detecting binding of the antibody or polypeptide to a cell in the sample.

BRIEF DESCRIPTION of DRAWINGS

The figure 1 shows the results of identification of the protein target 5F1. Protein valuate with 5F1-immunoaffinity column of lysate COLO 205 (lanes 1 and 3) or lysate COLO 320 (lanes 2 and 4) were subjected to Western blot turns using commercial anti-CD43 antibodies AF2038 (lanes 1 and 2) or antibody 5F1 (lanes 3 and 4).

Figure 2A shows the results of flow-cytometrical analysis of the binding of the antibody 5F1 with three malignant cell lines: cells of colorectal cancer and colon (COLO 205 and DLD-1) and gastric cancer cells (NCI-N87).

Figure 2B shows the results of flow-cytometrical analysis of the binding of the antibody 5F1 with normal endothelial cells (HUVEC), normal (embryonic) cells of the lung (MRC-5), normal epithelial breast cells (MCF-10A), normal cells colorectal (CCD841-CoN), activated T-lymphocytes (activated within seven days) or normal mononuclear cells of peripheral blood (PBMC).

The figure 3 shows the average increase in the number of nucleosomes in the cytoplasm of COLO 205 cells after incubation in the presence of antibody 5F1 or 9E10 (anti-myc-antibody) or in control medium for 6, 24, and 48 hours.

The figure 4 shows the results of cell growth of COLO 205, measured in the analysis of WST-1 after incubation with the antibody 5F1, 9E10 or azide in vitro for 72 hours.

The figure 5 shows the results of cell growth, as measured in the analysis of WST-1. Cell carcinoma, rectal and colon cancer COLO 205 and the line of normal cells colorectal CCD841-CoN either not treated or incubated with 9E10, 5F1 (called "m133-5F1"), or with 0.5% azide.

The figure 6 shows the results of what you're MTT staining of COLO 205 cells after incubation with different concentrations 5F1 (0, 2, 4, 8, 16, 32, 64 µg/ml), 9E10 (64 µg/ml) or with 0.5% azide.

The figure 7 shows the effect in vivo (tumor size) antibody 5F1 (also called "m133-5F1") on tumor COLO 205 human SCID mice. Antibody 5F1 (500 μg/injection) or control antibody 9E10 (500 μg/injection) or PBS (untreated) were injected with in 0, 3, 5, 7, 10, 12, 14 and the 17th day.

The figure 8 shows the effect in vivo (tumor size) antibody 5F1 with chemical drugs 5FU/LV in the tumor COLO 205 human SCID mice. 5FU/LV were injected with intravenous through day four doses of 25 mg/kg one week after inoculation of COLO 205 cells. Antibody 5F1 were injected with intraperitoneally at a dose of 0, 6.25 mg/kg 12.5 mg/kg and 25 mg/kg twice a week for 3 weeks 7 days after implantation of the tumor.

Figure 9A shows the results of flow-cytometrical analysis of the binding of the chimeric antibody 5F1 with COLO 205 cell. Figure 9B shows the percentage positive for annexin V and PI cells after incubation of COLO 205 cells with control medium (untreated), sodium azide (0.5%), and murine antibody 5F1 (m5F1, 2-32 μg/ml) or chimeric antibody 5F1 (c5F1, 2-32 μg/ml).

The figure 10 shows the staining of COLO 205 cells and NCI-N87 against apoptotic cell death after incubation with different antibodies. COLO 205 cells and NCI-N87 cells were then incubated overnight with control 9E10 (30 µg/ml), m5F1 (10 μg/ml) or m5F1 (30 µg/ml).Then, the cells were dye YO-PRO-I (A) or annexin-V in combination with PI (B). The percentage of staining for each condition is shown in the histogram.

The figure 11 shows that m5F1 associated with recombinant CEA person (rhCEA), expressed in COLO 205 cells, but does not recognize rhCEA, expressed on cells COS-7. In figure 11A lysates of COLO 205 cells expressing flag-tagged CEA man was immunoprecipitated anti-Flag-M2 antibody, immunoprecipitated proteins were subjected to separation in SDS-page and then transferred onto the paper NC. Paper NC incubated with anti-Flag M2, m5F1, 51-41, 138-10 or CEA/Ab-3, as indicated. In figure 11B cell lysates of cells COS-7 expressing flag-tagged CEA man (+), or cells COS-7, not expressing CEA (-), was immunoprecipitated anti-Flag-M2 antibody, immunoprecipitated proteins were subjected to separation in SDS-page and then transferred onto the paper NC. Paper NC incubated with anti-Flag M2, m5F1 or CEA/Ab-3, as indicated.

The figure 12 shows that m5F1 associated with recombinant CD43 (rhCD43), expressed in COLO 205 cells, but does not recognize rhCD43, expressed on cells COS-7. In figure 12A soluble CD43 expressed by COLO 205 cells, purified using beads with protein A-separate, dispersed in SDS-page and transferred onto paper NC, paper NC were subjected to Western-blotting with the antibody m5F1, 51-14 or 138-10. In figure 12B the cell lysates of cells COS-7, transfected hCD43, hCD43/myc-His, or retransfusion the s cells were subjected to separation in SDS-page and transferred onto paper NC and paper NC were subjected to Western-blotting with anti-CD43 (MEM59) (left panel) or m5F1 (right panel).

The figure 13 shows that the antibody m5F1 recognizes fucose-dependent Glyco-epitope. rhCEA, downregulation of COLO 205 cells were treated with 0, 0.01, then of 0.03, 0.1 millidynes α-1→(2,3,4)-fucosidase. After processing the proteins were subjected to separation in SDS-page and then painted Kumasi blue (right panel) or subjected to Western-blotting with the antibody m5F1.

The figure 14 shows the structure of a Lewisa-lactose (Lea-lactose), Lewisb-lactose (Leb-lactose), Lewisx-lactose (Lex-lactose), lactose, Lewisy(Ley), sialyl-Lewisx(sialyl-Lex), Lewisa(Lea), lacto-N-tetraone (LNT) and lacto-N-divulgacion II (LNDFH II).

The figure 15 shows the results of the analysis of inhibition of binding of adding oligosaccharides, creating competition binding m5F1, 138-10, and 51-41 with COLO 205 cells. Oligosaccharides (LNDFH II, LNT, sLe(x), Le(y), lactose, Le(x)-lactose, Le(b)-lactose, Le(a)-lactose or Le(a), each at a concentration of 1 mm) was added to different wells containing 2 x 105COLO 205 cells, followed by the addition of antibodies (138-10, 51-41 or m5F1) or without addition of antibodies as a control. Antibody binding to COLO 205 cells was measured in the analysis using flow cytometry. Inhibition of binding of the oligosaccharides for each antibody are shown in percentage of inhibition determined by the average value-free is tion, as shown in the figure.

DETAILED description of the INVENTION

Definitions

"Antibody" means an immunoglobulin molecule capable of specific contact with a target, such as carbohydrate, polynucleotide, lipid, polypeptide, etc. through at least one site of antigen recognition, located in the variable regions of immunoglobulin molecules. Used in this sense, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain (ScFv), mutants fused proteins containing part of the antibodies, and any other modified form of immunoglobulin molecules, which contains the site of antigen recognition. The antibody includes the antibody of any class such as IgG, IgA or IgM (or their subclasses), and the antibody does not necessarily relate to any particular class. Depending on the amino acid sequence of the constant domain of the heavy chains of the antibodies, the immunoglobulins can be attributed to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The constant domains of the heavy chain, which correspond to the different classes of immunoglobulins is in, called alpha, Delta, Epsilon, gamma and mu, respectively. The subunit structures and three-dimensional structure of immunoglobulins of different classes are well known.

It is assumed that the antibody according to the present invention further includes bispecific, polyspecific, single-chain, and chimeric and humanized molecules having affinity to the polypeptide attached to at least one CDR region of the antibody. Antibodies according to the present invention also include single-domain antibodies that are either variable domain heavy chain antibody, or a variable domain light chain antibodies. Holt et al., Trends Biotechnol. 21: 484-490, 2003. Methods of obtaining domain antibodies containing either variable domain heavy chain antibody, or a variable domain light chain antibodies containing three of the six naturally occurring complementarity determining regions of antibodies, also known in this field. See, for example, Muyldermans, Rev. MoI. Biotechnol. 74: 277-302, 2001.

In the present description, the term "monoclonal antibody" refers to an antibody obtained from an essentially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. M is nacionalnye antibodies are highly specific, directed against a single antigenic site. In addition, unlike drugs polyclonal antibodies, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The definition of "monoclonal" indicates the character of the antibody as being obtained from an essentially homogeneous population of antibodies, and it should not be construed as requiring receipt of antibodies specific way. For example, the monoclonal antibodies used according to the present invention can be obtained by a method based on a hybrid, first described by Kohler and Milstein, 1975, Nature 256: 495, or may be obtained by methods based on recombinant DNA, such as the method described in U.S. patent No. 4816567. Monoclonal antibodies can also be isolated from phage libraries generated using the methods described, for example, McCafferty et al., 1990, Nature 348: 552-554.

In the present description, the term "chimeric antibody" refers to an antibody having a variable region or part of the variable regions of the first type of organisms and the constant region of the second type of organisms. The intact chimeric antibody contains two copies of the chimeric light chain and two copies of the chimeric heavy chain. Obtaining chimeric antibodies are known in this region the STI (Cabilly et al. (1984), Proc. Natl. Acad. Sci. USA, 81: 3273-3277; Harlow and Lane (1988), Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory). Usually in such chimeric antibody variable region light and heavy chains simulates variable regions of antibodies derived from one species of mammals, whereas the constant part of the homologous sequences in antibodies derived from another species. One of the obvious advantages of such chimeric forms is that the variable regions, for example, can be easily obtained from the currently known sources using readily available hybrid or B-cells of the host body, not a person, in combination with a constant regions derived from, for example, preparations of human cells. While the advantage of the variable region is the ease of obtaining and specificity are not affected its source, constant region, if it is human, less likely to cause an immune response in humans by injection of antibodies than in the case when a constant region derived from a source other than human. However, the definition is not limited to such specific example.

An "isolated" antibody is an antibody that has been identified and separated and/or removed from a component of its natural environment.

In the present description, the term "essentially cleared the, refers to the substance that has been cleared (i.e. does not contain impurities)at least 50%, more preferably cleaned at least 90%, more preferably cleaned at least 95%, more preferably cleaned, at least 98%, more preferably cleaned at least 99%.

In the present description, the term "humanized" antibody refers to forms of antibodies in animals other than humans (e.g., mice)that are specific chimeric immunoglobulins, chains of immunoglobulins or fragments thereof (such as Fv, Fab, Fab', F(ab')2or other antigennegative subsequences of antibodies)which contain minimal sequence derived from the immunoglobulin of the animal other than human. Mainly, humanized antibodies are human immunoglobulins (recipient antibody)in which residues from a complementarity determining region (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 capacity. In some cases the remains of the frame Fv region (FR) of a human immunoglobulin replace the corresponding residues of the animal other than human. In addition, humanitariannet antibody may contain the substance remains, not found neither in the recipient antibody nor in the imported CDR sequences or frame sequences, but were introduced to further improve or optimize the efficiency of antibody. In General, humanitariannet antibody will contain essentially entirely, at least one, and typically two, variable domain, in which all or substantially all of the CDR field correspond to the CDR regions of an antibody of an animal other than human, and all or essentially all areas of FR are areas FR from the sequence of human immunoglobulin. Humanitariannet antibody optionally also will comprise at least a portion of the constant region or domain of immunoglobulin (Fc), typically a constant region of human immunoglobulin. Antibodies can be Fc-region, modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six)which are altered compared to the original antibody, which are also referred to as one or more CDRs "derived from" one or more CDRs of the source antibody.

In the present description, the term "human antibody" means an antibody that has an amino acid sequence corresponding to the amino acid sequence of the antibody from which organisme person, and/or obtained using any method of obtaining a human antibody, which is known in this field or described in this publication. This definition of human antibodies includes an antibody that contains at least one polypeptide heavy chains of a human or at least one polypeptide light chain of a human. One such example is an antibody containing light chain polypeptides mouse and heavy chains of a human. Human antibodies can be produced using various methods known in this field. In one embodiment, the human antibody is selected from ragovoy library, this phage library 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 the loci of human immunoglobulins in transgenic animals, such as mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This method is described, for example, in U.S. patents№ 5545807, 5545806, 5569825, 5625126, 5633425 and 5661016. Alternatively, a human antibody can be obtained by immortalization of B-lymphocytes person producing antibody directed against a target antigen (such B-lymphocytes can b the th extracted from the human body or may be immunized in vitro. See, for example, 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.

"Variable region" of an antibody refers to the variable region of the light chain of the antibody or variable region of the heavy chain of the antibody, either individually or in combination. Each of the variable regions of the heavy and light chain consists of four frame regions (FR), connected by three complementarity determining regions (CDR), also known as the hypervariable region. CDRs in each chain are held together in close proximity by the FR and together with the CDRs from the other chain, contribute to the education antigennegative plot antibodies. There are at least two methods of determining CDR: (1) the method based on the variability of the sequences of different species (e.g., 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)). Used in this sense, the CDR may refer to CDR, determined by any method or combination of both.

"Constant region" of an antibody refers to the constant region of the light chain of the antibody or the constant region of heavy chain antibodies, either individually or in combination. Konsta the percentage area antibodies usually provides structural stability and other biological functions, such as Association of chain antibody secretion, moving through the placenta and bind complement, but does not participate in binding to the antigen. Amino acid sequence and the corresponding sequence of the exons in the gene constant region will depend on the species from which it was obtained; however, changes in amino acid sequence from this species, leading to allotypes will be relatively limited in relation to a specific constant regions. The variable region of each chain is linked to a constant region of the binding polypeptide sequence. Linking sequence is encoded by a sequence of "J" in the gene for the light chain, and a combination of sequence "D" and series "J" in the gene for the heavy chain.

In the present description the term "dependent antibody-mediated cell cytotoxicity" and "ADCC" refer to mediated cell response, in which nonspecific cytotoxic cells that Express Fc receptors (FcR) (e.g., natural killer cells (NK), neutrophils, and macrophages) recognize bound antibody on the target cell and then cause lysis of the target cells. ADCC activity of interest molecules can be assessed using assays ADCC in vitro, such as the assays described in U.S. patent No. 5500362 or 5821337. Note the removable effector cells for such assays include mononuclear cells of peripheral blood (PBMC) and NK-cells. Alternative or additionally, ADCC activity of interest molecules can be assessed in vivo, e.g., in a animal model such as the model described in Clynes et al., PNAS (USA) 95: 652-656 (1998).

"Complement-dependent cytotoxicity" and "CDC" refers to the lysis of the target in the presence of complement. Path activation of complement is initiated by binding of the first component of the complement system (C1q) with a molecule (e.g. antibody) in complex with its antigen. To assess activation of the complement, you can analyze the CDC, for example, as described in Gazzano-three-bet et al., J. Immunol. Methods 202: 163 (1996).

The terms "polypeptide", "Oligopeptide", "polypeptide" and "protein" are used in the present description are interchangeable with respect to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it can be interrupted by structures other than amino acids. The terms also encompass a polymer of amino acids that has been modified naturally or by intervention; for example, by the formation of a disulfide bond, glycosylation, lipidization, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with the aiming component. Also included in the definition, for example, polypeptides containing one or bore the only amino acid analogues (including, for example, unnatural amino acids, etc), as well as other modifications known in this field. It should be understood that because the polypeptides according to the invention is based on the antibody polypeptides may occur in a separate circuit or associate circuit.

The terms "polynucleotide" or "nucleic acid"as used in this description interchangeably, refer to polymers of nucleotides of any length, and include DNA and RNA. The 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 contain modified nucleotides, such as methylated nucleotides and their analogues. If there is a modification in the structure of the nucleotide can be entered before or after Assembly of the polymer. The nucleotide sequence can be interrupted dinucleotide components. Polynucleotide can be further modified after polymerization, such as by conjugation with aiming component. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides a similar, mezhnukleotidnyh modifications such as, for example, modifications Natarajan the diversified bonds (for example, methylphosphonate, phosphotriester, phosphoamide, carbamates, etc) and charged linkages (e.g., phosphorothioate, phosphorodithioate etc), modification containing side residues, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine and so on), modification by intercalators (e.g., acridine, psoralen, etc.), modification, containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc), modification, containing alkylator, polynucleotide with modified linkages (e.g., alpha anomeric nucleic acids, etc), as well as unmodified forms of polynucleotide(s). In addition, any of the hydroxyl groups normally present in the sugars may be replaced, for example, phosphonate groups, phosphate groups, protected by standard protective groups or activated for more links with additional nucleotides, or may be conjugated to a solid substrate. 5'- and 3'-terminal OH group can be phosphorylated or substituted amines or organic kapinowski groups of 1-20 carbon atoms. Other hydroxyl can also be derivativea standard protective groups. Polynucleotides can also contain analogous forms of sugars ribose and deoxyribose, which are generally known in the Noi area, including, for example, 2'-O-methyl, 2'-O-allyl, 2'-fluoro - or 2'-isidoros, carbocyclic analogues of sugars, α-anomeric sugars, epimeria sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugar, sedoheptulose, acyclic analogs and deprived of reason nucleoside analogues, such as methylribose. One or more fosfolipidnyh ties can be replaced by alternative linking groups. These alternative linking groups include, without limitation, variants, in which the phosphate is replaced by P(O)S ("diatom"), P(S)S ("dayata"), (O)NR2("amidate"), P(O)R, P(O)OR', CO or CH2("formatitem"), where each R or R' independently denotes H or substituted or unsubstituted alkyl (1-20C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. Not all communication polynucleotide must be identical. The preceding description applies to all specified polynucleotides, including RNA and DNA.

In the present description, the term "vector" means a construct that is able to deliver and it is preferable to Express one or more genes of interest or sequences in the cell host. Examples of vectors include, without limitation, viral vectors expressing vectors on the basis of "naked" DNA or RNA, plasmid, cosignee lifehave vectors, DNA - or RNA-expressing vectors associated with cationic condensing agents, DNA - or RNA-expressing vectors encapsulated in liposomes, and some eukaryotic cells, such as cells-producers.

In the present description the term "sequence expression regulation" means a nucleic acid sequence that controls transcription of the nucleic acid. Sequence regulating the expression can be a promoter such as a constitutive or inducible promoter or enhancer. Sequence regulating the expression of the operatively linked to the transcribed sequence of the nucleic acid.

In the present description the term "effective dose" or "effective amount" of the drug, compound or pharmaceutical composition is an amount sufficient for obtaining beneficial or desired results. In the case of prophylactic use beneficial or desired results include results such as the elimination or reduction of risk, reduction of the severity or delay the appearance of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes manifested during the development of the disease. the case of therapeutic use of beneficial or desired clinical results include the results such as reducing one or more symptoms resulting from the disease, improving the quality of life of patients suffering from this disease, decreasing the dose of other medications required to treat the disease, enhancing the effect of other medicines, for example, through targeted delivery, slow the progression of the disease and/or life extension. In the case of cancer or tumor effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., to some degree, slow and preferably stop) infiltration of malignant cells into peripheral organs; inhibit (i.e., to some degree, slow and preferably stop) metastasis of tumors; to inhibit to some extent, tumor growth; and/or to some extent weaken one or more symptoms associated with the disorder. Effective dose can be entered for one or more injections. For the purposes of the present invention, an effective dose of the drug, compound or pharmaceutical composition is an amount sufficient to perform a prophylactic or therapeutic treatment, either directly or indirectly. As is clear from liricheskoy point of view, the effective dose of the drug, compound or pharmaceutical composition can be achieved or not achieved in conjunction with another drug, compound or pharmaceutical composition. Thus, the "effective dose" can be viewed in the context of the introduction of one or more therapeutic agents, and we can assume that one tool introduced in an effective amount if, together with one or more other means can be achieved or achieved the desired result.

In the present description the term "together with" refers to the introduction of a single treatment in addition to another treatment. As such, the term "together with" refers to the introduction of one treatment before, during or after administration to a subject other means of treatment.

In the present description, the term "treatment" means a method of obtaining beneficial or desired results, preferably including clinical results. For the purposes of this invention, beneficial or desired clinical results include, without limitation, one or more of the following results: reduction of proliferation (or destruction) of malignant cells, decreasing symptoms resulting from the disease, improve the quality of life of subjects suffering from the disease, reduction to the s other medicines, necessary for treatment of the disease, slow the progression of the disease and/or prolong the life of patients.

In the present description the term "delaying development of a disease" means a delay, braking, deceleration, containment, stabilization and/or delay the development of diseases such as malignant tumor). This delay may vary in duration depending on the history of disease and/or of the subject being treated. As is clear to a person skilled in the art, a sufficient or significant delay can really include prevention, in the case of which the subject does not develop the disease. For example, it may be delayed a malignant tumor in the last stage, such as the development of metastases.

"Individual" or "subject" is a mammal, more preferably human. Mammals include, without limitation, farm animals, sport animals, Pets (such as cats, dogs, horses), primates, mice and rats.

In the present description, the term "specific recognizes" or "specific binds" refers to a measurable and repeatable interactions, such as attracting and binding between the target and the antibody that is the defining characteristic of the presence of the target in the presence of heterogen the th population of molecules, including biological molecules. For example, an antibody that is specific or preferentially binds to the epitope is an antibody that binds to such an epitope with greater affinity, avidity, more readily, and/or longer than it binds to other epitopes of the target or epitopes that are not related to the target. It is also clear that when reading the above definition, for example, that the antibody (or component or epitope)that is specific or preferentially binds to the first target, you may contact or may not contact the specific or preferably with the second target. Essentially a "specific binding" or "preferred binding" does not necessarily require (although it may enable) exclusive binding. The antibody that is specific associated with the target may have a constant Association constituting at least about 103M-1or 104M-1sometimes about 105M-1or 106M-1in other cases, approximately 106M-1or 107M-1approximately 108M-1up to 109M-1or from about 1010M-1up to 1011M-1or higher. You can use various forms immunoassay for screening of antibodies specific immunological interacting with con the specific protein. For example, typically use solid-phase ELISA immunoassays for screening monoclonal antibodies immunologically specific interacting proteins. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, which describes the forms and conditions of immunoassays that can be used to determine specific immunoreactivity.

In the present description, the terms "malignant tumor", "tumor", "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth(proliferation). Examples of malignant tumors include, without limitation, cancer, including adenocarcinoma, lymphoma, blastoma, melanoma and sarcoma. More specific examples of such malignant tumors 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, Hodgkins and nahodkinskuju lymphoma, pancreatic cancer, glioblastoma, cervical cancer, glioma, ovarian cancer, liver cancer, such as carcinoma of the liver and hepatoma, bladder cancer, breast cancer, cancer colon, cancer of the rectum and colon cancer (colorectal cancer), carcinoma of the endometrium or uterine carcinoma of the salivary glands, a cancer on the EC, such as pochernkletocny carcinoma and Wilms tumor, basal cell carcinoma, melanoma, prostate cancer, thyroid cancer, cancer of the testis, cancer of the esophagus and various types of head and neck cancer.

In used in the present description and in the accompanying claims, the singular number include the plural forms unless the context clearly indicates otherwise. For example, reference to "an antibody" is a reference to antibodies in number from one to many, for example in a molar quantities, and includes cash equivalents, known to specialists in this field, and so on

It is clear that the aspects and variations of the invention described in this publication, include cases of "comprising" and/or "consisting essentially" of the aspects and options.

Antibodies and polypeptides that have been specifically associated with the carbohydrate epitope on CD43 and CEA expressed on nonhematopoietic malignant cells

The invention relates to isolated antibodies and polypeptides derived from antibodies that are specific contact epitope on CD43 and/or CEA expressed by nonhematopoietic cancer cells, but not associated with specific CD43 expressed by a leukocyte such as peripheral T-cell) or Jurkat cell. Antibodies and polypeptides according to the invention may additionally methodno or more of the following properties: (a) the binding of an antibody or polypeptide epitope is reduced, if a molecule containing an epitope treated α-1→(2,3,4)-fucosidase; (b) the binding of an antibody or polypeptide epitope is inhibited by carbohydrate structure containing Leastructure Lea-lactose, the structure of LNDFH II and/or structure of the LNT; (c) induce the death of the nonhematopoietic cancer cells (e.g., apoptosis) after binding with the epitope expressed on the cell surface of malignant cells in the absence of cytotoxic conjugation and immune effector function; (d) inhibit cell growth or proliferation nonhematopoietic cancer cells after binding to an epitope expressed on the cell surface of malignant cells; and (e) treat or prevent an individual nonhematopoietic malignant tumors expressing the epitope on the cell surface, such as cancer colorectal and gastric cancer.

In the present description, the term "inhibition" includes partial or total inhibition. For example, the binding of an antibody or polypeptide epitope on CD43 and CEA is suppressed by 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 IU the e is approximately 90% carbohydrate, containing the structure Leastructure Lea-lactose, the structure of LNDFH II or the structure of the LNT. The binding of an antibody to an epitope can be ingibirovalo through direct competition or other mechanisms.

Examples of nonhematopoietic malignant cells expressing the epitope include, without limitation, cells of cancer colorectal (such as COLO 205 and DLD-1) and gastric cancer cells (such as NCI-N87).

Antibodies and polypeptides according to the present invention can recognize the extracellular domain of CD43, which is present on nonhematopoietic malignant cells, but not in contact with the extracellular domain of CD43 leukocytes (e.g., peripheral T-cells), or the extracellular domain of CD43, which is expressed on Jurkat cells (lymphoblastoid leukemia cell). In some embodiments, new antibodies or polypeptides according to the invention are not associated with specific CD43 expressed by cells of hematopoietic origin.

Antibodies according to the invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, Fv, Fc, etc), chimeric antibodies, single-chain antibodies (ScFv), mutants fused proteins containing part of the antibodies, and any other modified structure of the molecules of the immunoglobulin, which contains a plot of RA is learning antigen with desired specificity. The antibody may be an antibody mouse, rat, camel, human, or have any other origin (including humanized antibodies).

The binding affinity of the polypeptide (including antibody) with 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 binding affinity of can be expressed in the form KDor dissociation constants, and high affinity binding corresponds to the reduced KD. One way to determine the affinity of antibody binding to CD43 or CEA is to measure the affinity of binding of monofunctional Fab fragments of antibodies. To get monofunctional Fab fragments of the antibody (e.g., IgG) can be split papain or to Express recombinante. The affinity of the Fab-fragments of antibodies can be determined using surface plasmon resonance measuring system of the surface plasmon resonance (SPR) BIAcore3000TM, BIAcore, INC, Piscaway NJ) and ELISA. Get the kinetic rate of Association (konand the rate of dissociation (koff) (usually measured at 25°C) and calculate values of the equilibrium dissociation constants (KD) in the form koff/kon.

Not what which variants of the antibodies and polypeptides according to the invention reduce the number of malignant cells and/or inhibit cell growth and proliferation of tumor or malignant cells, which have an epitope. Preferably there is a decrease in the number of cells or the inhibition of cell growth or proliferation, at least about 10%, about 20%, about 30%, about 40%, about 50%, about 65%, about 75% or more compared to a cell not treated with the antibody or polypeptides. Malignant cells include, without limitation, cancer, colorectal cancer, pancreatic cancer, lung cancer, cancer of the stomach.

In some embodiments, the antibodies and polypeptides according to the invention are able to induce cell death, e.g., through apoptosis after binding epitope located on the cell surface of the nonhematopoietic cancer cells. The term "induce cell death" in the present description means that the antibodies or polypeptides according to the present invention can directly interact with a molecule located on the cell surface, and only the binding/interaction is sufficient for the induction of cell death in cells without the help of other factors, such as the conjugation of cytotoxic or other immune effector functions, i.e., the complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis.

In the present description, the term "and optos" genes belong to a managed process of intracellular destruction of cells. Apoptosis differs from necrosis; it includes the destruction of the cytoskeleton, compression and condensation of the cytoplasm, the expression of phosphatidylserine on the outer surface of the cell membrane and bubble swelling, leading to the formation associated with cell membrane vesicles or apoptotic cells. The process is also called "programmed cell death". During apoptosis observed characteristic phenomena, such as curved cell surface, 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 for detecting cell death (such as apoptosis) include, without limitation, identification of morphology, DNA fragmentation, enzymatic activity and decay of polypeptides, etc. Cm. Siman et al., U.S. patent No. 6048703; Martin and Green (1995), Cell, 82: 349-52; Thomberry and Lazebnik (1998), Science, 281: 1312-6; Zou et al., U.S. patent 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(1): 23-32, these publications are included in this description by reference.

In some embodiments, the antibodies and polypeptides according to the invention recognize a conformational epitope expressed on neemat the poetic malignant cell, and this epitope contains a structure with physical and chemical properties equivalent to the properties of the patterns formed by the Tripeptide N'-Trp-Pro-Ile-C'. In the present description the term "epitope that contains the structure with physical and chemical properties equivalent to the properties of the patterns formed by peptide" means that both structures have similar physical and chemical properties in relation to the binding of the antibody so that the antibody, which is associated with one specific structure, can communicate with both. In some embodiments, the antibodies and polypeptides associated with the polypeptide containing the amino acid sequence of N'-Trp-Pro-Ile-C' N-end of the polypeptide.

In some embodiments, the antibodies and polypeptides according to the invention competes with the antibody 5F1, 138-10 or 51-41 for binding to the epitope expressed on the cell surface of malignant cells. In some embodiments, the antibodies or polypeptides according to the invention associated with epitope on CD43 or CEA is associated with at least one antibody 5F1, 138-10 and 51-41.

You can use competitive analysis to determine link whether two antibodies to the same epitope, through recognition of identical or sterically overlapping epitopes, or one antibody competition will ingibiruet the binding of another antibody to the antigen. Such assays are known in this field and are described in detail in the examples. Typically, the antigen or expressing the antigen cells immobilized to advance the tablet and measure the ability of unlabeled antibodies to block the binding of labeled antibodies. Usual labels for such competitive assays are radioactive label or an enzyme label.

In some embodiments, the antibody according to the invention is an antibody or antibody 5F1 obtained from the 5F1. Variable sequence of the heavy chain and light chain 5F1 shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The invention relates to the antibody or the polypeptide containing a fragment or antibody 5F1. In one embodiment, the fragment is a light chain of the antibody 5F1. In another embodiment, the fragment is a heavy chain antibody 5F1. In yet another embodiment, the fragment contains one or more variable regions from a light chain and/or heavy chain of the antibody 5F1. In yet another embodiment, the fragment contains one, two or three CDRs from a light chain and/or heavy chain of the antibody 5F1. In some embodiments, the antibody is humanitariannet antibody 5F1, such as h5F1 containing the variable region of the heavy chain shown in SEQ ID NO: 7, and the variable region of the light chain shown in SEQ ID NO: 8. In some embodiments, one or more CDRs derived from and is Titel 5F1, 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, at least one, at least two, at least three, at least four, at least five or at least six CDR 5F1.

In some embodiments, the antibody according to the invention is the antibody 138-10 or antibody derived from 138-10. Variable sequence of the heavy chain and light chain 138-10 shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The invention relates to the antibody or the polypeptide containing a fragment or antibody 138-10. In one embodiment, the fragment is a light chain antibodies 138-10. In another embodiment, the fragment is a heavy chain antibody 138-10. In yet another embodiment, the fragment contains one or more variable regions from a light chain and/or heavy chain antibodies 138-10. In yet another embodiment, the fragment contains one, two or three CDRs from a light chain and/or heavy chain of the anti-Christ. ate 138-10. In some embodiments, the antibody is humanitariannet antibody 138-10. In some embodiments, one or more CDRs derived from an antibody 138-10, 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 138-10.

In some embodiments, the antibody according to the invention is the antibody 51-41 or antibody derived from 51-41. Variable sequence of the heavy chain and light chain 51-41 shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The invention relates to the antibody or the polypeptide containing a fragment or antibody 51-41. In one embodiment, the fragment is a light chain antibodies 51-41. In another embodiment, the fragment is a heavy chain antibody 51-41. In yet another embodiment, the fragment contains one or more variable allstays light chain and/or heavy chain antibodies 51-41. In yet another embodiment, the fragment contains one, two or three CDRs from a light chain and/or heavy chain antibodies 51-41. In some embodiments, the antibody is humanitariannet antibody 51-41. In some embodiments, one or more CDRs derived from an antibody 51-41 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, at least one, at least two, at least three, at least four, at least five or at least six CDR 51-41.

In some embodiments, CDR represent the CDRs according to Kabat. In other embodiments, the CDR represent a CDR according to Chothia. In other embodiments, the CDR represents the combination of CDRs according to Kabat and Chothia (also called "combined CDR" or "advanced CDR"). In other words, in the case of any particular option that contains more than one CDR, CDR can be any, according to Kabat, Chothia and/or combined.

Methods for producing antibodies and the of peptides, originating from antibodies known in the field and described in this publication. Monoclonal antibodies according to the present invention can be obtained using well-developed methods. For example, monoclonal antibodies can be obtained using hybrid methods such as the method described in Kohler and Milstein (1975, Nature, 256: 495. In case-based hybridomas method, a mouse, hamster, or other appropriate animal host is usually subjected to immunization immunizing agent (for example, malignant cells expressing CD43 or CEA, CD43 or CEA (including extracellular domain and its fragments expressed by the malignant cell), which can be purified using antibodies described herein, or a polypeptide containing the amino acid sequence of N'-Trp-Pro-Ile-C' N-end of the polypeptide)to generate lymphocytes that produce or are capable of producing antibodies that will be specific to contact immunizing agent. Alternative lymphocytes may be immunized in vitro. Then lymphocytes merge with immortalizing a cell line using a suitable merge tool, such as polyethylene glycol, with the formation of cell hybridoma (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-1031). Termed lines to etoc are usually transformed mammalian cells, particularly myeloma cells of rodent, rabbit, bovine and human. Usually use a line of myeloma cells in rats or mice. Cell hybridoma can be grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or viability Nikitich immortalized cells. For example, if the original cells lacking the enzyme hypoxanthineguanine (HGPRT or HPRT), the culture medium for the hybridomas typically will contain gipoksantin, aminopterin and thymidine ("medium HAT"), and such substances prevent the growth of HGPRT-deficient cells.

Preferred termed the cell lines are cell line, which effectively merge, support stable high level expression of the antibodies selected producing antibodies cells and are sensitive to this environment, as the environment HAT. More preferred termed the cell lines are lines myeloma mice, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif, and the American type culture collection (ATSS), Manassas, Va. Described cell line of human myeloma and heteromyinae mouse-human to obtain monoclonal human antibodies (Kozbor, J. Immunol. (1984), 133: 3001; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp 51-63).

The cultural environment in which cultured cells hybridoma, can then be evaluated for the presence of monoclonal antibodies. Antibodies can be subjected to screening for specific binding to an epitope on CD-43 or CEA expressed by nonhematopoietic malignant tumor or neoplastic cells, and the absence of specific binding to CD43 expressed by white blood cells, Jurkat cells and/or other expressing CD43-cells of hematopoietic origin. For screening can be used to malignant cells or the extracellular domain (including its fragments containing the epitope. For example, screening can be used CEA-N-A2, downregulation of COLO 205 cells, described in example 10.

Cell line Jurkat line is lymphoblastoid leukemia cells was established from the peripheral blood of a 14-year-old boy Schneider et al. Schneider et al., Int. J. Cancer 19: 621-626, 1977. Different cell line Jurkat are commercially available, for example, from American type culture collection (for example, ATCC TIB-152, ATCC TIB-153, ATCC CRL-2678).

Preferably, the binding specificity of monoclonal antibodies produced by cells of hybridoma determine immunoprecipitate or using the analysis of binding in vitro, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such is the ways and assays known in the field. The binding affinity of the monoclonal antibody can be determined, for example, using analysis of Scatchard according to Munson and Pollard (1980), Anal. Biochem., 107: 220.

Identified antibodies can then be tested for their ability to induce cell death (e.g. apoptosis) and/or to inhibit cell growth or proliferation using methods known in this field and described in this publication.

After identifying the required cells hybridoma the clones may be subcloned by limiting ways of breeding and grown by standard methods (Goding, above). Suitable culture media for this purpose include, for example, modified by Dulbecco Wednesday Needle or medium RPMI-1640. Alternative cell hybridoma can be grown in vivo as ascites in a mammal.

Monoclonal antibodies can be generated by culturing cells hybridoma, and the antibodies secreted by the cells hybridoma, can then be selected or cleared. Antibodies may be isolated or purified from the culture medium or ascitic fluid by conventional means of purification of immunoglobulins, such as, for example, chromatography on protein A-sepharose, hydroxyapatite, gel electrophoresis, dialysis, or affinity chromatography.

Antibodies according to the invention can also be on the particular ways based on recombination of DNA, such as the methods described in U.S. patent No. 4816567 and 6331415, which are included in this description by reference, for example, DNA encoding the monoclonal antibodies according to the invention, can be easily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that are capable of specific contact with the genes encoding the heavy and light chains of murine antibodies). Cell hybridoma according to the invention serve as a preferred source of such DNA. After DNA extraction can be placed in expressing vectors, which are then transferout in cell host, such as cells, monkey COS cells Chinese hamster ovary (CHO) or myeloma cells, which in other circumstances not producing a protein of the immunoglobulin, to obtain the synthesis of monoclonal antibodies in the recombinant cell host. The DNA also may be modified, for example, by replacing the coding sequence for the constant domains of the heavy and light chain human instead of the homologous murine sequences (U.S. patent No. 4816567) or covalent binding sequence that encodes an immunoglobulin, all or part of a sequence that encodes nimmanahaeminda polypeptide. So nimmanahaeminda the polypeptide can replace the IC on the constant domains of the antibodies according to the invention or you can replace the variable domains of one antigennegative centre antibodies according to the invention, to create a chimeric bivalent antibody.

In some embodiments, the antibodies according to the present invention is expressed with two expressing vectors. First expressing the vector encodes a heavy chain antibodies (e.g., gumanitarnogo antibodies) and contains the first part encoding the 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, the first part encodes the variable region having the amino acid sequence represented in SEQ ID NO: 7. The second expressing the vector encodes the light chain of the antibody and contains a first portion encoding a variable region light chain antibody, and a second part encoding a constant region of light chain antibodies. In some embodiments, the first part encodes the variable region having the amino acid sequence represented in SEQ ID NO: 8.

Alternative antibodies (for example, humanitariannet antibody) according to the present invention is expressed with one expressing vector. One expressing vector and encodes a heavy chain and light chain of an antibody according to the present invention. In some embodiments, expressing the vector contains a polynucleotide sequence encoding a variable region of a heavy chain having the General amino acid sequence, presented in SEQ ID NO: 7, and the variable region of light chain having the amino acid sequence represented in SEQ ID NO: 8.

Usually expressing the vector has a sequence regulation of transcription and translation, which are derived from species compatible with the host-cell. In addition, the vector typically carries a specific gene (genes), which is able (able) to provide phenotypic selection of the transformed cells.

Know a wide variety of expression systems of recombinant host-vector for eukaryotic cells, and they can be used in the invention. For example, Saccharomyces cerevisiae, or common Baker's yeast is the most commonly used among eukaryotic microorganisms, although some other strains, such as Pichia pastoris. Line of cells derived from multicellular organisms, such as Sp2/0 or cells of the Chinese hamster ovary (CHO), which are available from ATCC, can also be used as hosts. Normal vector plasmids suitable for transforming eukaryotic cells are, for example, pSV2neo and pSV2gpt (ATCC), pSVL and pSVK3 (Pharmacia), pBPV-1/pML2d (International Biotechnology, Inc.).

Eukaryotic cells masters applicable in the present invention, preferred are cells hybridoma, myeloma, plasmacytoma or lymphoma. However, other power plant is eroticheskie cells-owners can be appropriately used, provided that the cells of the host mammal is able to recognize the transcription and translation of the DNA sequence for protein expression; processional leader peptide by cleavage of the leader sequence and to secrete proteins; and to provide for post-translational modification of proteins, for example, glycosylation.

Accordingly, the present invention relates to eukaryotic cells-the hosts, who transformed expressing recombinant vectors containing the structure of DNA described in this publication, and are able to Express the antibodies or polypeptides according to the present invention. Therefore, in some embodiments, the transformed cell host according to the invention contain at least one design DNA containing the DNA sequence of light and heavy chains described in this publication, and transcriptional and translational regulatory sequences, which are located relative to the DNA sequences encoding the light and heavy chain so as to control the expression of the antibodies or polypeptides.

Cell owners used in the invention can be transformed in various ways using standard transfection methods are well known in this field. To the standard SPO is obam transfection, which can be used include methods of electroporation, methods of fusion of protoplasts and precipitation of calcium phosphate. Such techniques are in General described in 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 two such vectors can be transferred into the cell host separately one after another or together (joint transfer or cotransfected).

The present invention also relates to a method of obtaining an antibody or polypeptide, which comprises culturing the host cell containing the expressing vector(s)encoding the antibodies or polypeptides, and removing the antibodies or polypeptides from the culture by methods well known specialist in this field.

In addition, the desired antibody can be obtained in the body of the transgenic animals. Suitable transgenic animal can be obtained by standard methods, which include microinjection into oocytes expressing suitable vectors, transfer eggs to pseudoriemannian females and the selection of the descendants of expressing the desired antibody.

The present invention also relates to chimeric antibodies that recognize specific epitope on CD43 and CEA expressed PLN is a quality cage. For example, variable and constant region of the chimeric antibody receive from different species. In some embodiments, the variable region and the heavy chain and light chain derived from a murine antibody described in this publication. In some embodiments, the variable region containing the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2. In some embodiments, the variable region containing the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4. In some embodiments, the variable region containing the amino acid sequence shown in SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, the constant region and the heavy chain and light chain derived from human antibodies.

Chimeric antibody according to the present invention can be obtained by methods well developed in this area. 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. 5677427, each of which is incorporated in this description by reference. In General, the chimeric antibody can be produced by obtaining cDNA encoding the variable regions of the heavy and light antibody chains, embedding expressing the cDNA in the vector, which when introduced into eukaryotic cells-owners expresses chimeric antibody according to the present invention. Preferably expressing the sector carries a complete constant sequence of the heavy or light chain, so any variable sequence of the heavy or light chain can be easily embedded in expressing vector.

The present invention relates to humanitarianlaw the antibody that recognizes specific epitope on CD43 and CEA expressed by nonhematopoietic malignant cell. Humanitariannet antibody is typically a human antibody in which residues from a CDR replaced by residues from a CDR of a species other than human, such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, the frame remains Fv antibodies person replace the corresponding residues derived from species other than human.

There are four main stages of humanization of monoclonal antibodies. Namely: (1) determination of the nucleotide and the calculation of the amino acid sequences of variable domains of light and heavy chains of the original antibody; (2) designing gumanitarnogo antibodies, i.e. deciding which frame region of the antibodies used in the process of humanization; (3) the implementation of techniques/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, the constant region can be constructed that is, so it was more similar to the constant region of a person in order to avoid the immune response in the case of the use of antibodies in clinical trials in the treatment of humans. See, for example, U.S. patent No. 5997867 and 5866692.

It is important that antibodies were humanitarian with retention of high affinity towards the antigen and other preferred biological properties. To achieve this goal humanized antibodies can be obtained by way of analysis of the source sequences and various conceptual humanized products using three-dimensional models of the source and humanized sequences. Three-dimensional models of immunoglobulins public and well-known experts in this field. There are computer programs that illustrate and demonstrate image possible three-dimensional conformational structures of selected for research immunoglobulin sequences. Viewing these images allows for an analysis of the likely role of the residues in the functioning selected for the study of immunoglobulin sequence, i.e., the analysis of residues that influence the ability selected for the study of immunoglobulin to bind its antigen. This way, FR residues can be selected and combined from the consensus and imported sequence is lnasty so, to obtain the desired property of the antibodies, such as increased affinity to the target antigen. In General, the CDR residues are directly and most heavily involved in influencing antigen binding. Humanized antibodies may also contain modifications in the hinge region, to improve one or more characteristics of the antibody.

In another alternative embodiment, antibodies can be subjected to screening and received recombinante using the technique of phage display. See, for example, U.S. patent No. 5565332, 5580717, 5733743 and 6265150; and Winter et al., Annu. Rev. Immunol. 12: 433-455 (1994). Alternative methods of phage display (McCafferty et al., Nature 348: 552-553 (1990)) can be used to produce human antibodies and fragments of antibodies in vitro from the repertoire of genes of variable (V) domains of immunoglobulins unimmunized donors. According to this technique, genes V-domain antibody clone in frame in the gene for either the primary or minor envelope protein of filamentous bacteriophage, such as M13 or fd, and are as functional fragments of the antibodies on the surface ragovoy particles. Because filamentous particle contains a single-stranded copy of DNA of phage genome, the selection based on the functional properties of antibodies, also leads to the selection of the gene encoding the antibody exhibiting such t the VA. Thus, the phage mimics some properties of B-cells. Phage display can be implemented in various forms; see the overview, for example, in Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3, 564-571 (1993). For phage display technique, you can use several sources sections V-genes. Clackson et al., Nature 352: 624-628 (1991)identified a variety of antibodies against oxazolone from a small random combinatorial library of V genes derived from the spleens of immunized mice. You can construct a repertoire of V genes neimmunizirovannah people, and antibodies to the matrix of a variety of antigens (including autoimmune antigens) can be isolated essentially according to the method described in Mark et al., J. Mol. Biol. 222: 581-597 (1991), or Griffith et al., EMBO J. 12: 725-734 (1993). When the natural immune response genes of antibodies with high speed accumulate mutations (somatic giperbolinovaya). Some of the changes will be to give a higher affinity, and during subsequent antigenic stimulation preferably replicated and are differentiated B-cells, representing high-affinity surface immunoglobulin. This natural process can be simulated using the method known as "shuffling circuits". Marks, et al., Bio/Technol. 10: 779-783 (1992)). In the specified way affinity "primary" human antibodies produced using phage display can be improved sequential replacement gene V-region heavy and light chain repertoires of naturally occurring variants (repertoires) genes V-domains, obtained from unimmunized donors. This method allows to obtain antibodies and antibody fragments with affinity in the PM-nm range. The method of obtaining very large repertoires of antibodies in phage (also known as "mother-of-all libraries" (library, which is the source of all antibodies) described in Waterhouse et al., Nucl. Acids Res. 21: 2265-2266 (1993). Shuffling of genes can also be used for obtaining human antibodies from antibodies rodents, the human antibody has similar affinity and specificity of the original antibody rodents. According to this method, also referred to as "epitope imprinting", gene V-domain of a heavy or light chain of the antibody rodents, obtained by the method based on phage display, replace the repertoire of genes V-domains of a person, creating chimeras rodent-human. Selection against the antigen leads to the separation of variable regions of a human, is capable of restoring a functional antigennegative plot, i.e. the epitope determines the choice of partner (performs imprinting). When the process is repeated to replace the remaining V-domain rodents, get a human antibody (see PCT publication no WO 93/06213, published April 1, 1993). Unlike traditional humanization of rodent antibodies by CDR grafting, this method provides a fully human anti the La, which have no frame of residues or residues of the CDRs derived from rodents. It is obvious that although the above discussion relates to humanized antibodies, the General principles discussed are applicable to fit the antibodies for use, for example, dogs, cats, primates, horses and cows.

In some embodiments, the antibody is a fully human antibody. Antibodies of animals other than humans that have been specifically bind the antigen, can be used to produce fully human antibodies, which binds to this antigen. For example, the person skilled in the art can use the method of permutation circuits, in which the heavy chain of the antibody of an animal other than humans Express in conjunction with the library for expression, expressing different light chains of human rights. The resulting hybrid antibody contains one light chain of a human and one heavy chain of an animal other than human, then subjected to screening for binding 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 subjected to screening for binding antigen. Methods such as the method, further described in the patent With The And 5565332. In addition, you can use antigen for vaccination of an animal that is transgenic for genes of human immunoglobulins. See, for example, U.S. patent 5661016.

The antibody can be bispecific antibody, monoclonal antibody, which has specificnosti binding against at least two different antigens, and can be obtained using the antibodies disclosed in the present description. Methods of obtaining bispecific antibodies known in the art (see, for example, Suresh et al., 1986, Methods in Enzymology 121: 210). Traditionally, the recombinant getting bispecific antibodies is based on the co-expression of two pairs of heavy chain-light chain immunoglobulins, with two heavy chains have different specificities (Millstein and Cuello, 1983, Nature 305, 537-539).

According to one method of obtaining bispecific antibody variable domains of antibodies with the desired specificnosti binding (antigennegative plots antibodies) merge sequences of the constant domains of immunoglobulin. The fusion preferably is performed with a constant domain of the heavy chain of immunoglobulin containing at least part of the hinge region, CH2 and CH3. It is preferred that the first constant region of the heavy chain (CH1)containing the site necessary for binding to the light chain present, at the ore, in one of the mergers. DNA encoding the fusion heavy chain immunoglobulin and optionally the light chain immunoglobulin is inserted into the individual expressing vectors and cotransfected in a suitable body of Hasan. This provides greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in those embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum outputs. However, you can embed the coding sequences for two or all three polypeptide chains in one expressing vector when the expression of at least two polypeptide chains in equal ratios results in high yields, or when the ratios are of no particular value.

In the same way bispecific antibodies are composed of a hybrid heavy chain immunoglobulin with a first binding specificity in one arm and hybrid pairs of heavy chain-light chain immunoglobulin (providing a second binding specificity) in the other shoulder. Such an asymmetric structure with a light chain immunoglobulin in only one half of bispecific molecules facilitates the separation of the desired bispecific connections from unwanted combinations of chains of immunoglobulin. This method is described in WO 94/04690, published March 3, 1994

Gets reconjugated antibodies containing two covalently linked antibodies are also included in the scope of the invention. Such antibodies are used to direct the immune system cells to unwanted cells (U.S. patent No. 4676980), and for the treatment of HIV infection (PCT publication WO 91/00360 and WO 92/200373, and EP 03089). Heteroconjugate antibodies can be obtained using any of the methods of formation of cross-links. Suitable cross-linking agents and methods well known in the field and described in U.S. patent No. 4676980.

Can also be derived single-chain Fv fragments, such as fragments, described in Iliades et al., 1997, FEBS Letters, 409: 437-441. Linking such single-stranded fragments using different linkers described in Kortt et al., 1997, Protein Engineering 10: 423-433. Various methods of recombinant production and processing of antibodies is well known in this field.

It is assumed that the present invention covers not only the monoclonal antibodies described above, but also any fragments thereof containing the active binding region of the antibodies, such as Fab fragments, F(ab')2, scFv, Fv and the like. Such fragments can be obtained from the monoclonal antibodies described herein, using methods that are well developed in this area (Rousseaux et al. (1986), in Methods Enzymol., 121: 663-69 Academic Press).

Methods of obtaining a fragment of the antibodies is well known in this field. For example, the antibody fragment can be obtained by enzymatic cleavage of antibodies with pepsin to obtain a fragment of 100 kDa, called F(ab')2. This fragment can be further split using restorative thiol agent and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds, with the receipt of monovalent Fab'-fragments of 50 kDa. Alternative enzymatic cleavage using papain immediately produces two monovalent Fab fragments and an Fc fragment. Such methods are described, for example, in U.S. patent No. 4036945 and 4331647 and listed in the references, patents are included in this description by reference. Also see 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 can be obtained by injection of DNA encoding Fab of the antibody, in expressing vector for prokaryote or expressing vector for eukaryote, and introducing the vector into a cell of a prokaryote or eukaryote to Express the Fab.

The invention covers the modifications of the antibodies or polypeptides described in this publication, including functionally equivalent antibodies which do not significantly affect their properties and variants which have enhanced or decreased activity and/or affinity. For example, amino acid sequentially the th antibody 5F1 or gumanitarnogo antibodies can be subjected to mutagenesis, to obtain an antibody with the desired binding affinity of with CD43 or CEA expressed by a malignant cell. Modification of polypeptides is a common practice in this field and needs no detailed description in this publication. 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 contribute significant adverse changes in the functional activity, or by the use of chemical analogues.

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

Variants with substitutions have at least one amino acid residue in the molecule antibodies, which are removed and in its place build a different balance. The plots present the determinant of the greatest interest to based on the replacement mutagenesis, include the hypervariable region, but also is expected to change FR. Conservative substitutions are shown in the table below under the heading "conservative substitutions". If such substitutions result in a change in biological activity, can be entered more substantial changes, indicated in the table below under the heading "examples of replacement or addition described below in the description of classes of amino acids, and the products are subjected to screening.

Amino acid replacement

The original balanceConservative substitutionsExamples of substitutions
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)LeuLeu; 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
ValV) LeuIle; Leu; Met; Phe; Ala; Norleucine

Significant modification of the biological properties of the antibody is carried out by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, in the form of a folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Naturally occurring groups 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) uncharged polar: Cys, Ser, Thr, Asn, Gln;

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

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

(5) residues that influence the orientation of the chains, Gly, Pro; and

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

Nonconservative replacement is performed by replacing the representative of one of the specified classes representative of another class.

Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve stability of the molecule to oxidation and to prevent the formation of abnormal cross-links. Conversely, cysteine bond (connection) can be added to the antibody to improve stability, especially when antitelomerase a fragment of the antibody, such as an Fv fragment.

The range of amino acid modifications can range from changing or modifying one or more amino acids to the complete reconstruction of the area, such as the variable region. Changes in variable regions can modify the affinity and/or specificity of binding. In some embodiments, the domain CDR carry no more than one to five conservative amino acid substitutions. In other embodiments, the domain CDR carry no more than one to three conservative amino acid substitutions. In other embodiments, the domain CDR is a CDRH3, and/or CDRL3.

Modifications also include glycosylated and deglycosylated polypeptides, and polypeptides with other posttranslational modifications, such as glycosylation of different sugars, acetylation and phosphorylation. Antibodies undergo glycosylation in conservative positions in the constant regions (Jefferis 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 the intramolecular interaction between the parts of glikoproteid, which can affect the conformation and presented three-dimensional surface glikoproteid (Hefferis and Lund, above; Wyss and Wagner, 1996, Current Opin. Biotech. 7: 409-416). Oligosacharide the s can also serve for to target this glycoprotein on some molecules on the basis of specific structures for recognition. It was also reported that glycosylation of antibodies affect dependent antibodies cell-mediated cytotoxicity (ADCC). In particular, it was reported that CHO cells controlled by the tetracycline expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase gene, catalyzes the formation of GlcNAc branching, have improved ADCC activity (Umana et al., 1999, Mature Biotech. 17: 176-180).

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the binding of the carbohydrate residue with a side chain of an asparagine residue. Tripeptide sequence asparagine-X-serine, asparagine-X-threonine and asparagine-X-cysteine, where X means any amino acid except Proline, are recognized sequences for enzymatic binding of the carbohydrate residue with a side chain of asparagine. Thus, the presence of any of these Tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the binding of one of the sugars N-atsetilgalaktozamin, galactose or xylose with hydroxyquinolinato, most commonly serine or threonine, although can also be used 5-hydroxyprop is h or 5-hydroxylysine.

Addition of glycosylation sites to the antibody is usually accompanied by a change in amino acid sequence, so that it contains one or more of the above described Tripeptide sequences (in the case of sites of N-linked glycosylation). Change can be realized by adding or replacing one or more residues of serine or threonine to the sequence of the original antibody (in the case of sites of O-linked glycosylation).

Picture glycosylation of antibodies can also be changed without changing the underlying nucleotide sequence. Glycosylation largely depends on the host cell used for expression of the antibody. As the cell type used for expression of recombinant glycoproteins, such as antibodies as potential therapeutic agents rarely presents native cell, one can expect the variability pattern of glycosylation of antibodies (see, for example, Hse et al., 1997, J. Biol. Chem. 272: 9062-9070).

In addition to selecting host cells, factors that affect glycosylation during recombinant generate antibodies include the mode of growth, the composition of the medium, the density of the culture, oxygen saturation, pH, purification and the like. Proposed different methods of altering the glycosylation pattern implemented in to kratom the body-master, including the introduction or overexpression of certain enzymes involved in the production of oligosaccharides (U.S. patent No. 5047335, 5510261 and 5278299). Glycosylation or some types of glycosylation can be enzymatically removed from glikoproteid, 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 so that it was defective in the processing of certain types of polysaccharides. These and similar methods well known in the field.

Other methods of modification include the application of the binding techniques known in this field, including, without limitation, enzymatic methods, oxidative substitution and chelation. Modification can be used, for example, to associate the labels in the case of immunoassay. Modified polypeptides receive, using the methods developed in this area, and they can be subjected to screening using standard assays known in this area, some of which are described below and in the examples.

The antibody or polypeptide according to the invention can be konjugierte (e.g., bind) with this agent as a therapeutic agent or label. Examples of therapeutic agents are pleased aktivnye residues, cytotoxins or chemotherapeutic molecule.

The antibody (or polypeptide) according to the present invention can be associated with a label such as a fluorescent molecule, a radioactive molecule, an enzyme or any others labels known in this field. Used in this sense, the term "label" refers to any molecule that can be registered. In some embodiments, the antibody can be marked by the introduction of radioactively labeled amino acids. In some embodiments, with the antibody can be bound residues of Biotin that can be detected using labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In some embodiments, a label may be imposed or associated with another reagent, which, in turn, communicates with interest by the antibody. For example, the label may be included or associated with the antibody, which, in turn, associated with specific interest antibody. In some embodiments, a label or marker can also be a therapeutic tool. In this area are known and can be used in a variety of ways of labeling polypeptides and glycoproteins. Some penetration the major classes of labels include, without limitation, enzymatic, fluorescent, chemiluminescent, and radioactive labels. Examples of labels for polypeptides include, without limitation, the following labels: radioisotopes or radionuclides (e.g.,3H,14C,15N35S90Y99Tc111In125I131I), fluorescent labels (e.g., isothiocyanate fluorescein (FITZ), rhodamine, lanthanide phosphors, phycoerythrin (PE)), enzymatic labels (such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, glucose-6-phosphatedehydrogenase, alcoholdehydrogenase, malatdegidrogenaza, penicillinase, luciferase), chemiluminescent labels, group biotinyl, pre-defined polypeptide epitopes recognized by a secondary reporter (e.g., pairwise sequence latinboy lightning, the binding sites for the second antibody, the binding domains of metals, epitope tags). In some embodiments, the label is associated spacer elements shoulders of various lengths to reduce potential steric hindrance.

The invention also relates to pharmaceutical compositions containing the antibodies or polypeptides described herein, and a pharmaceutically acceptable carrier or excipients. Pharmaceutically acceptable excipients are known in this field and represent the th relatively inert substances, to facilitate the introduction of pharmacologically effective substances. For example, excipient can give form or consistency or acts as a diluent. Suitable excipients include, without limitation, stabilizing agents, humectants and emulsifiers, salts for modifying the osmotic pressure, means for encapsulating, buffers and means to enhance penetration through the skin. Excipients, as well as preparations for parenteral and aparentally drug delivery specified in Remington, The Science and Practice of Pharmacy 20thEd. Mack Publishing (2000).

In some embodiments, the invention relates to compositions (described in this publication) for use in any of the ways described in this publication, or in the context of use as a medicinal product and/or application for the manufacture of a medicinal product.

Polynucleotides, vectors and cells-owners

The invention also relates to polynucleotide containing the nucleotide sequence encoding any of the monoclonal antibodies and polypeptides described in this publication. In some embodiments, the polypeptides contain a sequence of variable regions of the light chain and heavy chain.

In some embodiments, polynucleotide contain the nucleic acid sequence, coderush the th variable region of the heavy chain, specified in SEQ ID NO: 1 and/or the sequence of a nucleic acid encoding a variable region light chain found in SEQ ID NO: 2. In some embodiments, polynucleotide contain the nucleic acid sequence encoding the variable region of the heavy chain, containing one, two or three CDRs of sequence SEQ ID NO: 1 and/or the sequence of a nucleic acid encoding a variable region light chain containing one, two or three CDRs of sequence SEQ ID NO: 2. In some embodiments, polynucleotide contain the nucleic acid sequence specified in SEQ ID NO: 9, and/or a nucleic acid sequence specified in SEQ ID NO: 10.

In some embodiments, polynucleotide contain the nucleic acid sequence encoding the variable region of the heavy chain are indicated in SEQ ID NO: 3, and/or a nucleic acid sequence encoding a variable region light chain found in SEQ ID NO: 4. In some embodiments, polynucleotide contain the nucleic acid sequence encoding the variable region of the heavy chain, containing one, two or three CDRs of sequence SEQ ID NO: 3, and/or a nucleic acid sequence encoding the variable region of the light chain containing one, two or three CDRs of sequence SEQ ID NO: 4. In some embodiments, Pauline is cleotide contain the nucleic acid sequence, specified in SEQ ID NO: 11 and/or the nucleic acid sequence specified in SEQ ID NO: 12.

In some embodiments, polynucleotide contain the nucleic acid sequence encoding the variable region of the heavy chain are indicated in SEQ ID NO: 5, and/or a nucleic acid sequence encoding a variable region light chain found in SEQ ID NO: 6. In some embodiments, polynucleotide contain the nucleic acid sequence encoding the variable region of the heavy chain, containing one, two or three CDRs of sequence SEQ ID NO: 5, and/or a nucleic acid sequence encoding the variable region of the light chain containing one, two or three CDRs of sequence SEQ ID NO: 6. In some embodiments, polynucleotide contain the nucleic acid sequence specified in SEQ ID NO: 13, and/or a nucleic acid sequence specified in SEQ ID NO: 14.

In some embodiments, polynucleotide contain the nucleic acid sequence encoding the variable region of the heavy chain are indicated in SEQ ID NO: 7 and/or the sequence of a nucleic acid encoding a variable region light chain found in SEQ ID NO: 8. In some embodiments, polynucleotide contain the nucleic acid sequence specified in SEQ ID NO: 15, and/or sequence of nucleic KIS is the notes, specified in SEQ ID NO: 16.

Specialists in this field it is clear that as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptide described in this publication. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Thus, polynucleotide that vary due to differences in the use of codons that are specifically included in the present invention. In addition, alleles of genes containing the polynucleotide sequence presented in the present description, are included in the scope of the present invention. Alleles are endogenous genes that changed as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein can, but not necessarily, have a modified structure or function. Alleys can be identified using standard methods (such as hybridization, amplification and/or comparison of sequences in the database).

Polynucleotide according to the present invention can be produced using chemical synthesis methods recombination or PCR. The methods of chemical synthesis polynucleotides well known in this field and does not require the Ute detailed descriptions in this publication. The person skilled in the art can use the sequence proposed in the present invention, and a commercial DNA synthesizer to obtain the desired DNA sequence.

To obtain polynucleotides using the recombination polynucleotide containing the desired sequence may be embedded into a suitable vector and the vector in turn can be introduced into a suitable cell host for replication and amplification, as discussed later in the present description. Polynucleotide can be embedded in the cells are the owners of the methods known in this field. Cells transformed by the introduction of exogenous polynucleotide through direct uptake, endocytosis, transfection, F-mating or electroporation. After the introduction of exogenous polynucleotide can be stored in the cell in the form of reintegrating 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).

Alternatively, PCR allows the reproduction of DNA sequences. The PCR technique is well known in this field and are described in U.S. patent No. 4683195, 4800159, 4754065 and 4683202, as well as in PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, oston (1994).

The invention also relates to vectors (for example, cloning vectors expressing vectors, containing a nucleic acid sequence encoding any of the polypeptides (including antibodies)described in this publication. Suitable cloning vectors can be constructed according to standard methods, or can be selected from a large number of cloning vectors are available in this area. Although the choice of the cloning vector may vary depending on the host cell, which intend to use the applicable cloning vectors, in General, have the ability or infect other programs, can have a single target for a specific restriction enzyme and/or may carry genes token that can be used in selection of clones containing the vector. Suitable examples include plasmids and viruses bacteria, for example, 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 pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene and Invitrogen.

Expressing vectors, in General, are replicable polynucleotide constructs that contain polynucleotide according to the invention. Expressing the vector can replicate in the cells-Asaiah or AMISOM, either as an integral part of the chromosomal DNA. Suitable expressing vectors include, without limitation, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, Comedy and expressing vector (vectors)that are described in PCT publication WO 87/04462. Vector components generally may include, without limitation, one or more of the following: a signal sequence, the start of replication, one or more marker genes; bets the regulation of transcription (such as promoters, enhancers and terminators). For expression (i.e. broadcast) also typically require one or more elements of the regulation of translation, such as the binding sites of ribosomes, the sites of translation initiation and stop codons.

Vectors containing interest polynucleotide, can be introduced into the cell host any of a variety of suitable methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; the bombing of the microparticles; lipofection and infection (for example, in the case where the vector is an infectious agent such as a virus vaccine). The choice of the method of introducing vectors or polynucleotides will often depend on the particular host cell.

The invention also relates the I to the cells of the host, containing any of polynucleotides or vectors described in this publication. Any cell-hosts capable of sverkhekspressiya heterologous DNA can be used for isolation of genes encoding an antibody polypeptide or protein. Non-limiting examples of host cells of mammals include, without limitation, COS cells, HeLa and CHO. Cm. also PCT publication no WO 87/04462. Appropriate cell hosts other than mammalian cells include prokaryotes (such asE. coliorB. subtillis) and yeast (such asS. cerevisae,S.pombeorK. lactis).

The diagnostic application

The present invention relates to a method of using the antibodies, polypeptides and polynucleotides according to the present invention for detection, diagnosis and monitoring of disease, disorder or condition associated with expression of epitopes (or increased, or decreased compared with the normal sample, and/or inappropriate expression, for example, in the presence of expression in tissues (tissues) and/or the cell (cells)in which the normal expression of the epitope is absent).

In some embodiments, the method includes the registration of the expression of the epitope in the sample obtained from the subject, which assumes the presence of a malignant tumor, such as cancer of the rectum and colon Ki is Ki, pancreas, stomach and lung. Preferably, the method of registering includes the implementation of contact of the sample with the antibody, polypeptide or polynucleotide according to the present invention and determining whether the level of binding of the level of binding in a control sample or sample for comparison. The method is also applicable to determine whether the antibodies or polypeptides described in this publication, the appropriate treatment for the patient.

Used in this sense, the term "sample" or "biological sample" refers to a whole organism or a subset of its tissues, cells or component parts (e.g. body fluids, including, without limitation, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). "Sample" or "biological sample"also refers to the homogenate, lysate or extract derived from a whole organism or a subgroup of its tissues, cells or component parts or fractions or parts, including without limitation, for example plasma, serum, cerebrospinal fluid, lymph, the external sections of the skin, respiratory, intestinal and genitourinary tracts, tears, saliva, milk, blood cells, tumors, of which Ghana. Most often, the sample isolated from the animal, but the term "sample" or "biological sample" can also refer to cells or tissue analyzed in vivo, i.e. without removing from the body of the animal. Usually "sample" or "biological sample" will contain cells from the animal, but the 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 levels associated with malignant tumor polynucleotides or polypeptides. "Sample" or "biological sample"also refers to such an environment as nutrient broth or gel, which was developed by the body, which contains cellular components such as proteins or nucleic acid molecule.

In one embodiment, the cells or cell lysate/tissue is subjected to contact with the antibody and define the binding between the antibody and the cell. When in the tested cells shows the binding activity compared with the control cell of the same tissue type, this may indicate that the test cell is carcinogenic. In some embodiments, the test cells derived from human tissue.

You can use various methods known in this field, to identify specific binding of the antibody-ant the gene. Examples of immunoassays which can be realized according to the invention include fluorescent polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), values of the inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Indicator residual or aiming the group may be associated with antibodies, they are chosen so that they satisfy the requirements of different applications of the method, which is often determined by the availability of equipment for analysis and compatible ways immunoassay. Suitable labels include, without limitation, radionuclides (e.g.,125I131I35S3H or32P), enzymes (e.g. alkaline phosphatase, horseradish peroxidase, luciferase or β-galactosidase), fluorescent residues or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP) or fluorescent residues (e.g., nanoparticles QdotTMsupplied by the Quantum Dot Corporation, Palo Alto, CA). The General methods used in the implementation of various immunoassays mentioned above, well-known experts in this field.

For diagnostic purposes the polypeptides, including antibodies, can be registered mark of the remnant, including, without limitation, radioisotopes, fluorescent labels, and various enzyme-substr the local label, known in this field. Methods of conjugating labels to the antibody known in this field.

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

Antibodies according to the present invention can use any known method of analysis, such as analysis of competitive binding, direct and indirect tests of sandwich type and analysis, based on thus. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc. 1987).

Antibodies and polypeptides can also be used for diagnostic tests in vivo, such as visualization in vivo. In General, the antibody or polypeptide been labelled with a radionuclide (such as111In99Tc14C,131I125I or3H), so that the interest of the cells or tissue can be localized using immunoscintigraphy.

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

Therapeutic use

A surprising and unexpected feature of the antibodies according to the present invention is related to their ability to induce the death of nonhematopoietic Slokas the state of the cell. Thus, the present invention relates to therapeutic uses of the antibodies and polypeptides according to the present invention for the treatment of malignant tumors, such as cancer, colorectal cancer, lung cancer, pancreatic cancer, stomach cancer, breast cancer, hepatocellular carcinoma, and thyroid cancer. You can treat any malignant tumor, such as cancer, colon cancer, colorectal cancer, lung cancer, breast cancer, brain tumor, malignant melanoma, pochernkletocny carcinoma, bladder cancer, lymphoma, T-cell lymphoma, multiple myeloma, gastric cancer, pancreatic cancer, cervical cancer, endometrial carcinoma, ovarian cancer, esophageal cancer, liver cancer, squamocellular carcinoma of the head and neck, skin cancer, carcinoma of the urinary tract, prostate cancer, horiokartsinoma, pharynx 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, thyroid cancer, Wilms tumor, provided that evil is acetona cell expresses the epitope recognized by antibodies described in this publication. The method further may include the stage of detecting binding between the antibody or a polypeptide described in this publication, and tumor or malignant cell of the individual being treated.

In General, an effective amount of a composition comprising the antibody or polypeptide, is administered to a subject in need of treatment, thereby inhibiting the growth of malignant cells and/or inducyruya the death of malignant cells. Preferably the composition is prepared with a pharmaceutically acceptable carrier.

In one embodiment, the composition is prepared for administration by intraperitoneal, intravenous, subcutaneous and intramuscular injections and other forms of administration, such as oral, mucosal, inhalation, sublingual, etc.

In another embodiment, the present invention also covers the introduction of the compositions containing the antibodies or polypeptides according to the present invention conjugated with other molecules, such as registered label or a therapeutic or cytotoxic funds. Tools may include, without limitation, radioisotopes, toxins, toxoid, inflammatory remedies, enzymes, antisense molecules, peptides, cytokines or chemotherapeutic agents. Methods of conjugation of antibodies with the same and molecules in General well-known experts in this field. See, for example, PCT publication WO 92/08495, WO 91/14438, WO 89/12624, U.S. patent No. 5314995 and EP 396387; descriptions of which are included in this description by reference in full.

In one embodiment, the composition comprises the antibody or polypeptide conjugated with a cytotoxic agent. Cytotoxic means may include any means that are harmful to cells. A preferred class of cytotoxic agents that can be conjugated with antibodies or fragments may include, without limitation, 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, and puromycin and their analogues or homologues.

The dose required for treatment depends on the choice of route of administration, the nature of the drug, the nature of the disease in the subject, height, weight, surface area, age and sex of the subject; the other input of medicines and opinions of the treating physician. Suitable doses are in the range of 0.01-1000,0 mg/kg

In General, you can use any of the following doses: dose comprising at least about 50 mg/kg body weight; at least about 10 mg/kg of body weight; for men the necks least approximately 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 microgram/kg of body weight or less. In case of multiple injections over several days or longer, depending on the condition, the treatment lasts as long as is desired suppression of disease symptoms. The approximate scheme of dispensing includes the introduction of a weekly dose of approximately 6 mg/kg of antibody. However, there may be other applicable dosing schedules depending on the pharmacokinetic pattern, which, in the opinion of the doctor, you want to achieve. Empirical factors, such as half-life, as a rule, will contribute to the dose determination. The success of this therapy is easily monitored by conventional means and tests.

Some subjects may require more than one dose. The frequency of injection can be determined and adjusted during therapy. For example, the frequency of injection may be determined or adjusted based on the type and stage of malignant tumors exposed Leche is tion, from enter whether the tool for preventive or therapeutic purposes, previous therapy, the patient's medical history and response to the agent and the decision of the attending physician. Usually the physician will enter therapeutic antibody (such as humanitariannet 5F1), until it reaches the appropriate dose to achieve the desired result. In some cases it may be appropriate preparations of antibodies continuous release. A variety of drugs and devices to achieve prolonged release known in this field.

In one embodiment, the dose of the antibodies or polypeptides can be determined empirically in subjects who received one or more injections. Subjects injected with increasing doses of the antibodies or polypeptides. To assess 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 measured by evaluating the mass or volume of the tumor, time to disease progression (TDP) and/or determine the extent of compliance to treatment (RR).

Introduction antibody or polypeptide by the method according to the present invention can be continuous or intermittent, depending, for example, on the physiological state of the recipient, what the purpose of therapeuti the definition or preventive, and other factors known in the art. Introduction antibody or polypeptide may be essentially continuous over a preselected period of time or may be in the form of a series separated in time doses.

Other drugs include a suitable form for delivery known in this field, including, without limitation, carriers, such as liposomes. See, for example, Mahato et al. (1997) Pharm. Res. 14: 853-859. Liposomal drugs include, without limitation, citavecchia, multi-layered vesicles and single-layer vesicles.

In another embodiment, the composition may contain one or more anticancer agents, one or more antibodies described in this publication or the antibody or polypeptide that 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 agents can be part of one of the drug (for example, in the mixture, as they are often called in this area), or they are different drugs, but they are administered simultaneously or sequentially, and they, in particular, applicable to the treatment of a wider range of patient populations.

Polynucleotide encoding any of the antibodies or polypeptides according to the present izaberete the s (for example, antibody 5F1 or humanitarian form), can also be used for delivery and expression of any of the antibodies or polypeptides according to the present invention in the desired cell. Obviously, expressing the vector can be used to control expression of the antibody or polypeptide. Expressing the vector, you can enter by any means known in this field, for example intraperitoneally, intravenously, intramuscularly, subcutaneously, intrathecally, intraventricular, oral, enterline, parenteral, intranasal, dermal, sublingual or inhalation. For example, the introduction of expressing vectors includes local and systemic injection, including injection, oral introduction, introduction to the use of "guns" for delivery of particles or introduction to the use of the catheter and the local introduction. The person skilled in the art known to the introduction of expressing vectors to obtain the expression of exogenous protein in vivo. See, for example, U.S. patent No. 6436908, 6413942 and 6376471.

You can also use targeted delivery of therapeutic compositions containing polynucleotide encoding any of the antibodies or polypeptides according to the present invention. Ways mediated by receptors DNA delivery are described, for example, Findeis et al., Trends Biotechnol. (1993) 11: 202; Chiou et al., Gene Therapeutics: Methods and Applications of Direct Gene Tranfer (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 in the range from about 100 ng to about 200 mg of DNA for local introduction according to the Protocol of gene therapy. In the gene therapy Protocol you can also use the concentration ranges 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 according to the present invention can be delivered using carriers for gene delivery. The carrier for gene delivery can be viral or non-viral origin (see 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 mammalian promoters and heterologous promoters. The expression of the coding sequence can be either constitutive or regulated.

Based on virus vectors for delivery of the desired polynucleotide and expression in the target cell are well known in this field. The examples are based on the virus carriers include, without limitation, recomb Nannie retroviruses, for example, described in PCT publications 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, patent GB No. 2200651 and the patent EP No. 0345242 based on the alpha viruses vectors, such as vectors on the basis of virus Sindbis virus, Semliki forest (ATCC VR-67; ATCC VR-1247), virus Ross river (ATCC VR-373; ATCC VR-1246) and the virus Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and vectors based on adeno-associated virus (AAV), for example, PCT publication WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655. You can also use the introduction of DNA associated with inaktivirovannye adenovirus, which is described in Curiel (1992), Hum. Gene Ther. 3: 147.

You can also use non-viral carriers for delivery and methods, including, without limitation, poly-condensed DNA linked or not linked with inaktivirovannye adenovirus (see, e.g., Curiel (1992), Hum. Gene Ther. 3: 147); associated with ligand DNA (see, for example, Wu (1989), J. Biol. Chem. 264: 16985); carriers for delivery to eukaryotic cells (see, for example, U.S. patent No. 5814482, PCT publication 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.

You can also use "naked" DNA. Examples of methods of introducing naked DNA is described in PCT publication WO 90/11092 and U.S. patent No. 5580859. Liposomes, which can act as carriers for DOS is where it is refuelled genes described in U.S. patent No. 5422120, PCT publication WO 95/13796, WO 94/23697, WO 91/14445 and the patent EP No. 0524968. Additional methods are described in Philip (1994), Mol. Cell Biol. 14: 2411 and in Woffendin (1994), Proc. Natl. Acad. Sci. 91: 1581.

A composition comprising the antibody according to the present invention, can be introduced sequentially or simultaneously with one or more other therapeutic means, such as chemotherapeutic agents (such as 5-FU, 5-FU/MTX, 5-FU/leucovorin, levamisole, irinotecan, oxaliplatin, capecitabine or uracil/tegafur), immunoadjuvant inhibiting growth funds, cytotoxic tools and cytokines, etc. the number of antibodies and therapeutic tools depend on the type of medication used, the pathological condition being treated, and the modes and routes of administration, but usually will be less than when applied separately.

After administration of the composition containing the antibody described in this publication, the effectiveness of the composition can be evaluated both in vitro and in vivo in a variety of ways, well known to the person skilled in the art. Various animal models are well known for testing the anticancer activity selected for study composition. Such models include xenotransplantation of human tumor Nude mice or mice scid/scid, or genetic model the tumors of mice, such as mouse, knocked out by p53. The nature of in vivo such animal models makes them, in particular, models that predict the answers to the sick people. Such models can be created by injecting the cells into the body of syngeneic mice using standard methods, for example, subcutaneous injection, injection into the tail vein, implantation of spleen, intraperitoneal implantation and implantation under the kidney capsule, etc.

Sets

The invention also relates to kits for use in the proposed methods. Kits according to the invention include one or more tanks containing a purified antibody or a polypeptide described herein, and instructions for use according to any one of the methods proposed in the invention described in this publication. In some embodiments, such instructions contain a description of the introduction of antibodies for treating nonhematopoietic malignant tumors, such as colorectal cancer and colon, according to any of the methods described in this publication. The kit can additionally contain a description of the choice of the individual suitable for treatment based on the identification of the individual that has the disease, and the identification stage of the disease or on the basis of the detection, whether expressed epitope on malignant cells is individuum.

In some embodiments, the kits for the detection of malignant cells in the sample containing the antibody or a polypeptide described in this publication, and reagents for detecting binding of the antibody or polypeptide to a cell in the sample.

Instructions relating to the use of antibodies or polypeptides for the treatment of malignant tumors usually contain information on doses, dosing schedules and routes of administration in the case of an alleged treatment. Containers may be a unit dose packages containing not divided into doses party (for example, packages containing the drug for multiple admission) or sub-unit doses. The instructions that came with the kits according to the invention, are typically written instructions on the label or the liner in the package (for example, paper sheet included in this kit), but also acceptable machine readable instructions (e.g., instructions on magnetic or optical storage disk).

On the label or the liner in the package indicated that the composition is used for treatment of malignant tumors described in this publication. Instructions can be proposed for the practical implementation of any of the methods described in this publication.

Kits according to the present invention are in suitable packaging. Suitable packing which includes, without limitation, vials, bottles, jars, flexible packaging (e.g., sealed Mylar bags or plastic bags), and the like. Also offers packaging for use in conjunction with a special device such as an inhaler, a device for nasal administration (e.g., spray gun) or a device for infusion, such as a mini-pump. The set may have a sterile access hole (for example, the container may be a bag of intravenous solution or vial having a stopper, which is punctured with a needle for subcutaneous injection). Capacity also may have a sterile access hole (for example, the container may be a bag of intravenous solution or vial having a stopper, which is punctured with a needle for subcutaneous injection). At least one active agent in the composition is an antibody described in this publication. The container may further comprise a second pharmaceutically active agent.

The kits optionally can contain additional active ingredients, such as buffers, and interpreted information. Typically, the kit contains a container and a label or an insert in the package located on the container or attached to the vessel.

EXAMPLES

The following examples are offered to illustrate, but not limited to the I invention.

Example 1: generation and characterization of monoclonal antibodies that are specific contact CD43 expressed on malignant cells

The creation of monoclonal antibodies

Cell line adenocarcinoma colorectal human COLO 205 (ATCC CCL-222) was purchased from Food Industry Research and Development Institute (CCRC 60054), Hsin-chu, Taiwan, and were grown in RPMI medium 1640 (GIBCO BRL) with 10% FBS (Hyclone), 100 units/ml penicillin and 100 µg/ml streptomycin (GIBCO BRL) at 37°C in OBLASTNOI atmosphere containing 5% CO2. Female mice Balb/c mice 8 weeks of age were immunized three times with 2×107COLO 205 cells in 500 ál of PBS or 10 micrograms partially purified protein in CFA every two weeks, and finally spent booster immunization using 2×106COLO 205 cells or 10 micrograms of partially purified protein in 200 µl PBS. Five days after the last booster immunization, the spleen cells were merged with cells of the myeloma X63. Hybridoma were subjected to selection using DMEM with addition of 10% FBS (Hyclone) and HAT (Hybri-Max®, Sigma H0262, at a final concentration of 100 μm gipoksantina, 0.4 µm of aminopterin, 16 μm thymidine). Was established three lines of cells hybridoma m5F1, m51-41, m138-10, which secrete monoclonal antibody 5F1, 51-41 and 138-10.

Identification and characterization of target antigen for monoclonal antibody 5F1

Membrane proteins from tissue malignant tumor direct the Oh and colon or COLO 205 cells were isolated using a buffer for extraction (50 mm Tris-HCl, a pH of 7.4, 150 mm NaCl, 1% Nonidet P-40)containing protease inhibitors (Complete tabs; Roche Molecular Biochemicals). Lysates membrane proteins first pre-osvetleni on a column volume of 1 ml containing IgG non-immune mouse, immobilized on sepharose with protein G (Amersham Pharmacia Biotech Inc., N.J., U.S.A.), and passing through the column part directly inflicted on a column volume of 1 ml containing 5F1 associated with protein G-separate. The column was washed and was suirable protein target 5F1. The purity of the selected protein visualized by staining with silver and also identified using Western blotting after separation in 8% SDS-PAG. Isolated protein used for immunization of mice with the aim of obtaining other 5F1-like antibodies, such as 138-10 or 51-41.

Experiments thus membrane proteins were incubated with 5F1 or anti-CD43-antibody (AF2038, R&D System, Inc.) followed by incubation with protein G-separate (Amersham Pharmacia Biotech Inc., N.J., U.S.A.). The precipitates were subjected to separation in 8% SDS-page and subjected to Western blot analysis (immunoassay).

Proteins were mixed with an equal volume of sample buffer (50 mm Tris-HCl, pH of 6.8, 100 mm DTT, 2% SDS, 0,1% bromophenol blue, 10% glycerol), were separated in 8% SDS-page and then transferred onto nitrocellulose membrane (Hybond-C Super, Amersham). Then, the nitrocellulose membrane was blocked by 5% skim milk in PBS and Incubus is listed with a 5F1 or anti-CD43-Mat (AF2038). Then the blot was treated with conjugated with horseradish peroxidase antibody goat against mouse immunoglobulin (Jackson ImmunoResearch Laboratories, West Grove, Pa.) and showed, using chemiluminescent reagents (ECL, Amersham, UK).

To test that detects whether 5F1 antigen CD43, used commercially available anti-CD43-Mat (AF2038, R&D system, Inc.) to confirm the identity of the affinity purified protein 5F1 from lysates COLO 205 by Western blot analysis. Lysates of cell line COLO 320, negative in relation linking 5F1, was used as control. These results (Fig. 1) showed that both antibodies anti-CD43 (AF2038) and 5F1 interacted with the protein captured on the 5F1-immunoaffinity column that clearly indicates that 5F1 really recognize CD43.

Monoclonal antibody 5F1 specific recognizes CD43 expressed on the cell surface nonhematopoietic malignant cells

2×105COLO 205 cells were sown in each well of 96-well plate with a v-shaped bottom, and incubated with different concentrations of antibody 5F1 in the range from 0.33 to 1 mcg/ml at 4°C for 1 hour. Cells are washed twice with 200 μl of FACS buffer (1×PBS+1% FBS), painted 100 μl of a solution of antibody goat against mouse IgG associated with PE (Southern Biotech.), at a concentration of 1 μg/ml in FACS buffer) and then incubated at 4°C for 30 minutes Cells TRIG the s were washed with FACS buffer and were analyzed by flow cytometry (BD LSR, BD Life Sciences).

The results of antibody binding, the obtained flow cytometry, are shown in table 1 below. Monoclonal antibody, such as 5F1 (isotype: IgG3) or 138-10 (IgM isotype) or 51-41 (IgM isotype) recognizes surface CD43 expressed on the cytoplasmic membranes of malignant cells rectal and colon cancer COLO 205 and malignant cells of the stomach NCI-N87, but nepereversheny T cells or Jurkat cells (line lymphoblastoid leukemia cells; ATCC TIB-152). Cm. table 1 below. Data flow cytometry (Fig. 2A and Fig. 2B) also show that the 5F1 is associated with other types of malignant cells, such as malignant cells rectum and colon (DLD-1) and malignant cells of the stomach (NCI-N87), but not associated with normal endothelial cells (HUVEC), normal cells of the lung (MRC-5), normal epithelial breast cells (MCF-10A), normal cells colorectal (CCD841-CoN), activated T-lymphocytes (activated within seven days) or normal mononuclear cells of peripheral blood (PBMC).

Table 1 shows antigennegative properties anti-CD43 antibodies, such as 5F1, 138-10 or 51-41 in relation to malignant cells, colorectal, peripheral T cells and Jurkat cells (line lymphoblastoid leukemia cells).

% binding/AtPeripheral T-cellJurkat (leukemia cells)COLO 205NCI-N87 (malignant cells of the stomach)5F1<10%<10%>80%>80%138-10<10%<10%>80%>80%51-41<10%<10%>80%>80%Anti-CD162>80%>80%<10%not testedMEM-59 (anti-CD43; Biovendor, Candler, NC)>80%not tested>70%not testedAt control isotype<10%<10%<10% <10%

To detect expression of the protein target 5F1 in COLO 205 cells and tissues cancer colorectal person used a standard immunohistochemical method. Briefly, the cells or tissue were fixed for immunostaining 1 µg/ml 5F1, incubated with labeled Biotin antibody against mouse IgG and then incubated with a complex of avidin-Biotin-peroxidase (Vector Laboratories, Inc. Burlingame, CA, U.S.A.) and was painted by the Chromogen tetrachloride 3,3'-diaminobenzidine. Immunohistochemical study showed that 52.5 per cent of the tissues of patients with cancer colorectal (31/59) were positively stained 5F1.

Example 2. Apoptotic activity of the antibodies that are specific contact CD43 expressed by malignant cells

Detection of apoptosis induced 5F1, in COLO 205 cells by ELISA

To assess the type of cell death induced 5F1, malignant cells colorectal grew up in cups for cultivation, and incubated in the presence or in the absence of a 5F1. Level mineclearance (apoptotic) DNA fragmentation was determined using an indirect antibody capture and registration complexes histone-DNA, associated with cytoplasmic mononucleosomes and oligonucleosome using the kit to detect cell death ELISAPLUS(Roche, is in the directory 1774425). The ELISA was performed according to the manufacturer's instructions. Briefly, 1×104COLO 205 cells were sown in each well of 96-well plates and incubated with 5F1 or 9E10 (anti-myc-antibody) at a concentration of 10 μg/ml or with the control environment. After 6-, 24 - or 48-hour incubation at 37°C cells were washed and incubated with 200 ál lyse buffer for 30 minutes After deposition nuclei (200×g, 10 min) 20 μl of the supernatant (cytoplasmic fraction)containing fragmented DNA, was transferred to a tablet for micrometrology, coated with streptavidin, which were incubated with biotinylated monoclonal antihistone antibody. The amount of fragmented DNA nucleosomes associated with antihistone antibody was evaluated using conjugated to peroxidase monoclonal anti-DNA antibodies using ABTS (6-diammonium salt of 2,2-asindi[3-ethylbenzthiazolinesulfonic]) as a substrate. Finally, we determined the optical density at 405 nm after incubation with peroxidase substrate within 10-20 minutes, using a reading device for microplate (Molecular Devices, SPECTRA max M2). Values for wells containing lyse buffer and only one substrate, subtracted as background values. Data were analyzed in the form of specific enrichment of mono - and oligonucleosomal released into the cytoplasm, on the basis of the research Institute of the obtained values, using the following formula:

honey sample (dying/dead cells)
Enrichment factor (E.F.) =________________________________________
honey control environment (cells without treatment)

m Ed. = optical density [10-3]

The data shown in figure 3, indicate that 5F1 induced enrichment of nucleosomes in the cytoplasm of COLO 205 cells after 24-hour incubation. The number of registered fragmented DNA in processed 5F1 the cytoplasm was increased more than 4 times compared with the control environment. This enrichment was not observed in cells treated with either control antibody 9E10 (anti-myc-antibody), or only the environment, suggesting that the antibody 5F1 causes apoptosis in malignant cells.

Detection of apoptosis induced 5F1, 138-10 and 51-41 in COLO 205 cells, using staining with annexin V and PI

Annexin V stains phospholipids, which are on the outer side of the cytoplasmic membrane at the early stage of apoptosis. Thus, the staining of annexin V, which analyzed using FACS analysis shows that h is of the cells undergo apoptosis. of 1.2×105COLO 205 cells were sown in each well of 96-hole tablet and then the cells were added different concentrations 5F1 (2-16 μg/ml)diluted in medium or fresh medium (untreated control). To test whether cross-linking agent (CL) in order 5F1 induced apoptosis in tumor cells, 20 μg/ml of rabbit antibodies against mouse IgG (Jackson ImmunoResearch, No. in catalogue 315-005-045) was added to the set of samples for comparison. After 6-hour incubation at 37°C the cells were stained with 0.25 μl conjugated with FITZ annexin V (Strong Biotech Corporation) in 100 μl of buffer for the binding of annexin V at room temperature for 15 minutes and Then the cells were stained iodide of propecia (PI, dye DNA) and were analyzed by flow cytometry.

Data of flow cytometry showed that the treatment 5F1 in the absence of cross-linking agent within 6 hours at a concentration of 4 μg/ml or higher had caused the emergence of a significant number of malignant cells that have undergone apoptosis, suggesting that the antibody 5F1 separately can effectively induce apoptosis in COLO 205 cells.

Also tested the induction of apoptosis in COLO 205 cells under the influence of other anti-C43-antibodies, 138-10 and 51-41. Table 2 below shows that 5F1, 138-10 or 51-41 induced apoptosis in malignant cells of the rectum and colon. Cell the COLO 205 cells were then incubated with 32 micrograms/ml of each antibody for 6 hours and stained with annexin-V, then PI and subsequent FACS analysis.

Table 2
Induction of apoptosis in COLO 205 cells by antibody 5F1, 138-10 and 51-41
5F1138-1051-41The control Antibody (negative control)
% apoptosis (annexin-V+)>40%>30%>30%<15%

The above results show that such treatment with antibodies specific against malignant tumors expressing CD43, can induce apoptosis in malignant cell.

Detection of apoptosis induced m5F1 in cells NCI-87, using colouring YO-PRO-1 or staining with annexin V and PI

4×105cells NCI-N87 (line of carcinoma cells of human stomach) or 5×105COLO 205 cells were sown in 12-well plate for culturing (Nunc No. in catalogue 150628). After culturing overnight, the medium was replaced and added to the antibody at the concentrations indicated in the figure 10, or azide, and incubated for 6 hours. Then cells were treated with trypsin and collected for staining YO-RO-1 (Invitrogen, No. in the directory Y3603) or double staining with annexin-V-FITZ and PI (Strong Biotech, kits for detection of apoptosis, No. in catalogue AVK250).

As shown in Fig. 10A and 10B, the antibody m5F1 induced apoptosis in cells NCI-87 and COLO 205, which was measured by staining with YO-PRO-1 (A) and double staining with annexin-V-FITZ and PI (B). The data obtained show that m5F1 can also induce apoptosis in carcinoma cells of the stomach.

Example 3. Inhibition of growth of malignant cells by antibody 5F1

Processing separately 5F1 inhibits the growth of malignant cells

Malignant cells rectum and colon (COLO 205) and normal endothelial cells (HUVEC) were sown, and incubated with a 5F1 or control antibody 9E10. As negative controls included untreated cells. Cell viability was assessed using MTT assays and WST-1 to determine the proliferative activity, as described in this publication.

Analysis of WST-1 based on the splitting of the salt of tetrazole WST-1 to formazan mitochondrial dehydrogenase cells. Get formazan can be quantified using a spectrophotometer by measuring the optical density at 450 nm. The proliferation of viable cells leads to an increase in total enzyme activity; and the reduction of the enzymatic activity indicates the inhibition to tochnogo growth. Thus, the analysis of WST-1 was used to assess the viability of tumor cells after treatment 5F1. Briefly, 4×103COLO 205 cells in 100 µl of culture medium were sown in 5 repetitions for each treatment in 96-well tablet for cultivation. Then added 10 μg/ml 5F1, control antibody 9E10 (anti-myc antibodies) or fresh medium (untreated control). Treatment with 0.5% sodium azide (NaN3also included in the analysis as control of cytotoxicity. After three days incubation at 37°C, each well was added 20 μl of the reagent WST-1 (Roche, No. in catalogue 1664807) and the mixture is incubated at 37°C for 30 minutes was Measured by optical density at 450 nm, which reflects the survival of the treated cells. The results of the WST-1 analysis of COLO 205 cells is shown in figure 4. Evidence suggests that the growth of COLO 205 cells was significantly Engibarov (Fig. 4), whereas in HUVEC (data not shown) processing 5F1 had no effect. Survival percentage decreased to values less than 50%, in the treated antibody 5F1 malignant cells colorectal compared with cells treated with antibody 9E10 used to control isotype. In the positive control group treated with 0.5% sodium azide, observed survival constituting 19%.

In another experiment, cells (2×103/sup> ) were sown in each well of 96-well plate to cultivation, and incubated with 10 micrograms/ml of monoclonal antibody 5F1 or control antibody 9E10 (antibody against c-myc). Untreated cells were used as negative controls; and cells treated with 0.5% sodium azide, were used as positive controls. After two - or three-day incubation period at 37°C, each well was added 10 μl of the reagent WST-1 cells and additionally incubated for 30 minutes Then was carried out by WST-1 analysis of cell viability, as described above. The results, shown in figure 5, indicate that the growth of carcinoma cells colorectal, such as COLO 205, was largely Engibarov antibody 5F1, but the antibody had no effect on the line of normal cells colorectal (such as CCD841-CoN).

MTT is another way based on tetrazole way of measuring the viability and proliferation of cells. Yellow tetrazolium MTT (bromide 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium) restored metabolically active cells, in part through the action of dehydrogenase enzymes, with the formation of the restored equivalents such as NADH and NADPH. Resultant intracellular purple formazan can be solubilisation and quantified SPECT is fotometricheskie ways. Cells (5×103) were sown in each well of 96-well plate to cultivation, and incubated with a monoclonal antibody 5F1 (concentration range 0-64 μg/ml) or control antibody 9E10 (64 µg/ml) against c-myc. Untreated cells were used as negative controls; and cells treated with 0.5% sodium azide, were used as positive controls. After 72-hour incubation period at 37°C, each well was added 10 μl of MTT reagent and cells additionally incubated for 2-4 hours before until a purple precipitate became visible. Added 100 μl of detergent (DMSO). Recorded optical density of samples at 570 nm. Data MTT analysis showed that 5F1 inhibited the proliferation of COLO 205 cells dependent on the dose of the image (from 0 to 64 µg/ml) and the ED50 (effective dose for 50% inhibition) was 8 μg/ml (Fig. 6). As also shown in Fig. 6, found a marked inhibition of cell growth when using 64 µg/ml 5F1, whereas the control antibody 9E10 had no such influence in the same concentration.

Evaluation of antitumor effects 5F1 in vivo

Antitumor effects 5F1 in vivo was analyzed in a murine model of xenotransplantation tumors. 5×106COLO 205 cells were implanted subcutaneously in the posterior lateral region of SCID mice at day 0. A week after inoculate the cells in one experiment, mice were treated with 500 micrograms 5F1 or PBS by intraperitoneal injection. In another experiment, four groups of mice (6 mice in each group), bearing developed tumors, intravenous processed through day four doses of 25 mg/kg of 5-fluorouracil plus leucovorin (5 FU/LV) and different doses 5F1 by intraperitoneal injection twice a week or without 5F1.

In one experiment implantation of tumors in SCID mice was performed by subcutaneous injection of malignant cells rectal and colon cancer COLO 205 1×107cells in the mouse at day 0 and then were treated by intraperitoneal injection of a monoclonal antibody 5F1 (500 μg per dose) or a control monoclonal antibody 9E10 (antibody against c-myc) or PBS in 0, 3, 5, 7, 10, 12, 14 and the 17th day. In each group in experiment used fifteen mice. Tumor size was measured from 5 day to 24 days, getting the work width, width and length (W×W×L) and expressed in mm3. As shown in figure 7, a monoclonal antibody 5F1 effectively inhibited tumor growth compared with control antibody 9E10 and PBS (without processing).

To identify the combined effects 5F1 and chemotherapeutic agents, such 5FU/LV, mice intravenously every other day were injected with 4 doses of 25 mg/kg 5FU/LV and was intraperitoneally injected with or not were injected with different amounts of antibody 5F1 twice a week for 3 weeks 7 days after implantation of the tumor. Growth is the tumor was estimated based on the measurements twice a week and tumor volume (mm 3) with a caliper and the tumor size was calculated using the formula: π/6×larger diameter×(smaller diameter)2(Kievit, E., Cancer Research, 60: 6649-55). As shown in Fig. 8, a combination of treatments antibody 5F1 and 5FU/LV significantly inhibited tumor growth colorectal person compared to treatment with a chemotherapeutic agent separately (figure 8).

Example 4. Three new anti-CD43 antibodies, 5F1, 138-10 and 51-41, recognize a similar epitope expressed on malignant cells

To determine the binding properties of three new anti-CD43 antibodies (5F1, 138-10 and 51-41) used FACS analysis. COLO 205 cells (100000) painted 1 micrograms/ml biotinylated 5F1 at 4°C for 1 hour in the presence of different amounts of nebutiniausias antibodies 5Fl, 138-10 and 51-41. After washes, cells were additionally stained with streptavidin-FITZ in the same conditions for 30 minutes the Cells were washed and analyzed in FACS. The data shown in table 3 below represent the average fluorescence intensity from one typical experiment. Both antibodies 51-41 and 138-10 able to compete with biotinylated 5F1 for binding to COLO 205 cells, suggesting that all three antibodies bind similar binding sites expressed on the surface of COLO 205 cells.

Table 3
5F1, 138-10 and 51-41 recognize similar epitopes expressed on the cells of COLO 205
The concentration of the competing antibodies (micrograms/ml)The average fluorescence intensity in the presence of unlabeled 5F1, 51-41 or 138-10
5F151-41138-10
2009,7875,66199,7
66,760,28165,62297,03
22,221,67325,23395,53
7,4129,04606,20640,42
2,46733,36783,81702,79
0,821004789,23724,15
0,2731027901,36683,08
0,09825869,61717,19
0,03888860,13704,33

Example 5. Determination of epitope antibody 5F1

To further determine the structure of the epitope recognized by monoclonal antibody 5F1 specified monoclonal antibody used to test his abilities to the specific interaction with the different polypeptide sequences. 96-well plates to micrometrology covered antibody 5F1 50 ál per well at a concentration of 10 μg/ml in buffer for covering containing 0.1m NaHCO3(pH 8,6), overnight at 4°C. After washing, the plates were blocked by incubation with blocking buffer containing 0.1m NaHCO3(the pH to 8.6), 5 mg/ml BSA, 0.02% of NaN3(150 µl/well), at least for one hour at 4°C. Then the plates were incubated with fused proteins containing different fragments of the polypeptides, in different concentrations for one hour at room temperature. After washing TBS containing 0.5% tween related fused with protein polypeptides were suirable 0,2M buffer glycine-HCl (pH 2,2)containing 1 mg/ml BSA, and neutralized 1M Tris-HCl (pH 9,1). Then determined the amino acid sequence elwira the data merged with protein polypeptides. The polypeptide that binds to the antibody 5F1 contain Tripeptide sequence Trp-Pro-Ile (WPI) from N-Terminus to the C-end. This Tripeptide amino acid sequence is not present in the amino acid sequence of CD43. Pallant et al., Proc. Natl. Acad. Sci. USA 86: 1328-32, 1989; Shelley et al., Proc. Natl. Acad. Sci. USA 86: 2819-23, 1989.

For additional confirmation tripeptides epitope that binds an antibody 5F1, was carried out by ELISA "sandwich". 96-well plates to micrometrology covered antibody (5F1 or control antibody 9E10) at 50 μl per well at a concentration of 1 μg/ml overnight at 4°C. the plates were blocked by incubation with 0.25% BSA in PBS (150 μl/well) for 1 hour at 37°C. Then the plates were incubated with fused proteins containing different fragments of the polypeptides, fused to a protein carrier, for 2 hours at room temperature. After washing 4 times with PBS containing 0.05% tween-20, plates were incubated with specific respect to protein-media antibody at a concentration of 2 µg/ml for 1.5 hours at room temperature. After incubation tablets 4 times washed with PBST. Then to each well was added 50 μl diluted 1:3000 antibodies goat, against antibodies specific to the protein-carrier conjugated with HRP, and the plates were incubated for 1 hour at 37°C. the Enzymatic reaction was performed by adding to the of strata enzyme HRP, to determine the reactivity of the specified polypeptide relative to the two antibodies (5Fl and 9E10). Table 4 below shows the data analysis ELISA for the case when the tablet was immobilized 5F1 or 9E10. "+" means that the polypeptide binds to a monoclonal antibody; and "-" means that the polypeptide is not bound to a monoclonal antibody. Monoclonal antibody 5F1 recognizes Tripeptide epitope structure of WPI. As Tripeptide sequence WPI is not present in CD43, our data indicate that the 5F1 recognizes a conformational epitope that includes a structure having a physical and/or chemical parameters, similar or equivalent to the structure formed by the Tripeptide WPI.

Table 4
The structure of the epitopes recognized by 5F1
The name proteinAmino acid sequence5F19E10

Example 6. Cloner is of variable regions of light and heavy chains 5F1, 138-10 and 51-41 and humanization of antibodies

cDNA of the variable regions of light and heavy chains 5F1 amplified in PCR and synthesized cDNA was subcloned into pCRII (Invitrogen) for sequence detection. Nucleotide sequences were obtained and analyzed from several independent clones. Selected identical to the cDNA sequence of independent clones representing the V-region of the light or heavy chain of each antibody. Table 5 below shows the translated amino acid sequence and nucleotide sequence encoding a V-region light and heavy chains 5F1, 138-10, 51-41 and gumanitarnogo 5F1 (h5F1Vc).

Table 5. Amino acid sequences of the variable regions of the antibodies and nucleic acid sequence encoding the variable region of antibodies (CDRs are underlined)

Amino acid sequence (SEQ ID NO: 1) and nucleotide sequence (SEQ ID NO: 9) heavy chain 5F1

Amino acid sequence (SEQ ID NO: 2) and nucleotide sequence (SEQ ID NO: 10) light chain 5F1

Amino acid sequence (SEQ ID NO: 3) and nucleotide sequence (SEQ ID NO: 11) heavy chain 138-10

The amino acid the percentage sequence (SEQ ID NO: 4) and nucleotide sequence (SEQ ID NO: 12) light chain 138-10

Amino acid sequence (SEQ ID NO: 5) and nucleotide sequence (SEQ ID NO: 13) heavy chain 51-41

Amino acid sequence (SEQ ID NO: 6) and nucleotide sequence (SEQ ID NO: 14) light chain 51-41

Amino acid sequence (SEQ ID NO: 7) and nucleotide sequence (SEQ ID NO: 15) heavy chain h5F1Vc

Amino acid sequence (SEQ ID NO: 8) and nucleotide sequence (SEQ ID NO: 16) light chain h5F1Vc

Example 7. Fabrication and characterization of chimeric antibody 5F1

Designing and obtaining chimeric antibody 5F1 [c5F1)

To construct vectors for expression of a chimeric antibody V-region light chain 5F1 was subcloned into the plasmid pVk. pVk contains a CMV promoter and a constant region of light chain of a human. Sequence and biological information on the constant region of the light chain of a human can be found in Hieter, P.A., et al. (1980), Cloned human and mouse kappa immunoglobulin constant and J region genes conserve homology in functional segments. Cell, 22 (1 Pt 1): p. 197-207.

V-region heavy chain 5F1 was subcloned into the plasmid pVg1. Plasmid pVg1 has the CMV promoter and contains the constant region of the heavy chain of human IgG1. Serial is inost and biological information on the constant region of the heavy chain IgG1 person can be found in Ellison J. W., B.J. Berson and Hood LE (1981), The Nucleotide sequence of a human immunoglobulin C gamma 1 gene., Nucleic acids Res.10: 4071.

Plasmids expressing the light and heavy chains, then cotranslational cells Cos-7. Nadeshiko containing c5F1, collected and analyzed in relation to inducing apoptosis functions c5F1.

Functional testing c5F1

Adosados containing c5Fl, then tested with respect to its binding to COLO 205 cells using surface staining, and in relation to its functions in the analysis of apoptosis using annexin V as described above. For analysis of binding used 0,58 micrograms/ml c5F1. For analysis of apoptosis used 2~32 micrograms/ml m5F1, c5F1 and 9E10 (control anti-myc-antibody), and incubation continued for 16 hours. Adosados containing c5F1 connects COLO 205 cells and induces apoptosis in COLO 205 cells, like its mouse counterpart, suggesting that the cloned cDNA fragments indeed encode V-region 5F1 (Fig. 9A and Fig. 9B).

Example 8. Immunohistochemical study of malignant tissue colorectal using m5F1

Staining of tissue antibody m5F1

The expression of the target m5F1 investigated using immunohistochemistry on paraffin tissue samples of the primary tumor of patients with cancer colorectal (n=59). Tissue matrix filled in paraf the n tissue cancer colorectal received from SuperBioChips Laboratories (matrix of human tissue, No. in catalogue CD1). Used standard methods of staining for immunohistochemistry according to the manufacturer's instructions (kit VECTASTAIN Elite ABC, Vector Laboratories). All sections were heated at 58°C for 1 hour, the paraffin was removed, 5 times processing xylene, and consistently rehydratable in ethanol solutions of increasing concentrations. After blocking with normal serum (VECTASTAIN, PK6102) for 1 hour, sections were incubated with m5F1 at a concentration of 1 μg/ml for 1 hour at room temperature and then with a second biotinylated antibody against mouse Ig (VECTASTAIN, PK6102). Then the sections were incubated with a complex of streptavidin-Biotin (VECTASTAIN, PK6102). The slide was treated with a solution of diaminobenzidine. Finally cover contrasting dyed with hematoxylin, dehydrational, prosvetlili and made in 50% glycerol in PBS.

Assessment of the relative scales

The expression of antigen on each tissue section was evaluated by two independent researchers, and the degree of staining 5F1 was assessed using semi-quantitative empirical system: -, negative result; +-, weak staining; +, moderate staining; ++, strong staining.

Results

All slices staining m5F1 was primarily membrane. Only 31 out of 59 samples of tumors (52,5%) stained positively against the target of 5F1, 27 (45.8%) of the shallow high levels of expression. 19 samples (32,2%) had negative staining in respect of the expression of a target m5F1. Total colourings of all tested images cancer colorectal shown in table 6 below. The obtained data show that the antibody m5F1 can be used to diagnose cancer of the rectum and colon.

20 + +
Table 6
The frequency of expression of the target 5F1 in malignant tumors of colorectal man
the pattern of malignant tumors colorectal/colour m5F1
1 + +2 + +3 + +4 -5 + +6 +-7 + +8 -9 + +10 + +
11+ +12 -13+ +14 + -15 -16 -17 + +18 + +19 + -
21 +22 + -23 +24 -25 + +26 -27 -28 -29 + +30 + +
31 +-32 -33 -34 + +35 + +36 -37 -38+ +39 + +40 + +
41 +42 + +43 -44 -45 -46 -47 + -48 + +49 -50 +
51+ +52 + +53+-54 + +55 + +56 + +57 + - 58 + -59 -60 carbon atoms
++: 27/59 (45,76%)+: 4/59 (6,78%)+-: 9/59 (15,25%)-: 19/59 (32,20%)

Example 9. m5F1 associated with recombinant CEA person (rhCEA) and CD43 person (rhCD43), downregulation of COLO 205 cells, but does not recognize rhCEA or rhCD43, expressed by cells COS-7

Get sample protein

Immunoprecipitate against recombinant Flag-tagged CEA in COLO 205 cells and COS:cDNA encoding the full-size protein CEA (35~702 amino acids)was cloned in the plasmid pFlag-CMV-1. The constructed plasmid DNA was introduced into cells COLO 205 (to construct a stable cell line) by electroporation or COS cells (for experiments on temporal expression) using lipofectamine 2000 (Invitrogen, No. in catalogue 11668-019). Cells expressing the antigen, collected and literally in lyse buffer (50 mm Tris-HCl, pH 8.5, 150 mm NaCl, 1% NP40)containing protease inhibitors (Roche, No. in catalogue 11836145001). Adosados cell lysates were incubated with associated with anti-Flag (M2, Stratagene, No. in catalogue 200472) beads containing protein G-sepharose (GE Healthcare, no directory 17-0618-02) for 2 cha is s at 4°C. G protein-sepharose balls then three times washed lytic buffer. G protein-sepharose balls containing products thus used as samples for SDS-page and Western blot.

Purification of soluble recombinant protein in the case of Cr1-labeled CD43 expressed in COLO 205 cells:cDNA encoding the extracellular domain of CD43 protein, cloned in a modified plasmid pcDNA3, which contains an N-terminal tag Flag and C-terminal tag Cr1. The constructed plasmid DNA was injected in COLO 205 cells by electroporation to obtain a stable cell line. Soluble recombinant hCD4320-253expressed by COLO 205 cells, contains an N-terminal tag 3×Flag and C-terminal tag Cr1. Soluble protein was purified using beads containing protein A-sepharose (GE Healthcare, no directory 17-1279-02). After the glycine elution buffer and dialysis against PBS, the protein samples were stored at -20°C for later use.

Lysates of whole cells to obtain CD43, temporarily expressed in COS cells:The construct containing full-CD43 (pcDNA3.1myc-His), was introduced into COS cells using lipofectamine 2000 (Invitrogen, No. in catalogue 11668-019). Cells were literally RIPA buffer (50 mm Tris-HCl, pH 7,4, 150 mm NaCl, 1 mm EDTA, and 0.25% SDS, 1% NP-40)containing protease inhibitors, and centrifuged at is 21,900 g at 4°C for 10 minutes to obrat supernatant. After a quantitative assessment of Bio-Rad enough protein lysates were applied to SDS-page for Western blot analysis.

Western blot analysis

After adding sample buffer, the protein samples were boiled at 95°C, were applied to SDS-PAG-minigel and then transferred to NC paper (GE Healthcare, Hybond-ECL, No. in catalogue RPN303D). After blocking the membranes with 5% skim milk in TBS was added to the first antibody. The binding of the first antibody was detected using conjugated with HRP second antibody (NEN Life Science, HRP-antibody goat against mouse IgG, No. in catalogue NEF822, or Southern Biotech, HRP-antibody goat against mouse Ig, No. in catalogue 1010-05) and was treated with reagents for detection for Western blotting ECL (GE Healthcare, no directory RPN2106).

Results

In the experiment shown in Fig. 11A, cell lysates of COLO 205 cells expressing rhCEA, immunoprecipitation anti-Flag-antibody and immunoprecipitated proteins were separated in SDS-page and transferred to NC paper. The NC paper was subjected to blotting, using different antibodies: anti-Flag, m5F1, 50-14, 51-41, 138-10, 186-14, 280-6 or anti-CEA-antibody (CEA/Ab-3; the name of the clone COL-1 from NeoMarker, no catalog, MS-613-P1ABX). The data presented in Fig. 11A, showed that m5F1, 51-41 and 138-10 recognize rhCEA, downregulation of COLO 205 cells.

In the experiment shown in Fig. 11B, the cell lysates of cells COS-7 expressing rhCEA, and who was imunoprecipitation anti-Flag-antibody and immunoprecipitated proteins were subjected to separation in SDS-page and transferred to NC paper. The NC paper was subjected to blotting, using different antibodies: anti-Flag, m5F1 or anti-CEA (CEA/Ab-3). The data presented in Fig. 11B, showed that m5F1 is not associated with rhCEA, expressed by cells COS-7.

In the experiment shown in Fig. 12A, soluble proteins, downregulation of COLO 205 cells were purified using protein A-sepharose balls were separated in a SDS gel and transferred to NC paper. The NC paper was subjected to blotting, using different antibodies: m5F1, 51-41 or 138-10. The data presented in Fig. 12A, showed that m5F1, 51-41 and 138-10 recognize rhCD43, downregulation of COLO 205 cells.

In the experiment shown in Fig. 12B, soluble proteins expressed in COLO 205 cells, was purified using protein A-sepharose, were separated in SDS-page and transferred to NC paper. The NC paper was subjected to blotting using anti-CD43 (MEM59) (left panel) or m5F1 (right panel). The data presented in Fig. 12B, showed that m5F1 didn't recognize rhCD43, expressed by cells COS-7. The obtained data indicate that the epitope recognized m5F1 has a post-translational modification specific to certain cell types.

Example 10. The epitope recognized m5F1, 51-41 and 138-10, contains a Lewis structurea(Leadepends on fucose

Processing glycosidases

Recombinant CEA person (rhCEA) resulted in the expression of CEA protein is a ne in COLO 205 cells. cDNA encoding rhCEA (CEA-N-A2), which is a fusion containing amino acids 35-145 (N-domain) and amino acids 324-415 (A2 domain) CEA, cloned in a modified plasmid pcDNA3, which contains the Flag-tag. The position of the amino acid residues CEA based on the provisions of the amino acids in the pre-protein. The constructed plasmid DNA was introduced into cells COLO 205 to obtain a stable cell line by electroporation. Recombinant CEA protein was purified from the supernatant of cell culture, stable cell lines expressing recombinant CEA, using anti-Flag-antibody.

For each reaction, approximately 1.8 μg of recombinant protein (rhCEA) were incubated with different amounts (0, of 0.01, 0.03 or 0.1 to Millie.) solution of α-1→(2,3,4)-fucosidase Xanthomonas sp. (Sigma, No. in catalogue F1924) at 37°C for 20 hours. After processing, the protein samples were applied to SDS-page for coloring Kumasi blue (Fig. 13, left panel) or to identify Western blotting (Fig. 13, right panel) using m5F1.

As shown in Fig. 13, the linking m5F1 with rhCEA decreased in the case where the antigen was treated with α-1→(2,3,4)-fucosidase, which suggests that m5F1 recognizes sensitive to fucose picapica.

Competitive analysis of oligosaccharides

To further test picapica recognized m5F1, conducted competitive anal is SHL, using different oligosaccharides. Oligosaccharides were purchased (Lewisafrom Sigma, No. in catalogue 03499, Lewisb-lactose from Sigma, No. in catalogue L7033, Lewisx-lactose from Sigma, No. in catalogue L7777, Lewisyfrom Sigma, No. in catalogue L7784, serial-Lewisxfrom Sigma, No. in catalogue S1782, lacto-N-tetraaza from Sigma, No. in catalogue L6770, lacto-N-divulgacion II from Sigma, No. in catalogue L6645, Lewisafrom Calbiochem, No. in catalogue 434626, and β-lactose from Sigma, No. in catalogue L3750) and dissolved in PBS. The structures of these oligosaccharides is shown in Fig. 14. Oligosaccharides (final concentration 1 mm) was added to different wells containing 2 x 105COLO 205 cells, with the subsequent addition of the indicated antibodies (m5F1, 51-41 or 138-10; 0.25 μg/ml each). After incubation for one hour at 4°C the supernatant was discarded and added a second antibody (Southern Biotech, RPE-antibody goat against mouse IgG, No. in catalogue 1032-09, or Southern Biotech, RPE-antibody goat against mouse IgM, No. in catalogue 1022-09) and recorded signals binding to cells, using flow-cytometrics analysis.

As shown in Fig. 15, LNDFH II, Lea-lactose and Leainhibited at different levels of binding antibodies m5F1, 51-41 and 138-10 with COLO 205 cells; and LNT also inhibited the binding of the antibody 51-41 and 138-10, but significantly inhibited the binding m5F1 with COLO 205 cells. The results suggests that the epitope recognizable by the specified antibodies may contain Leaor similar structure and is sensitive to fucose. In addition, the antibody m5F1 largely depends on fucose than antibody 51-41 and 138-10.

Although the invention is described above in detail as an illustration and example for a clearer understanding, the description and examples should not be construed as limiting the scope of invention.

Links

Laos, S., Baeckstrom D, and Hansson, G.C. (2006) Inhibition of NF-kappaB activation and chemokine expression by the leukocyte glycoprotein, CD43, in colon cancer cells. Int. J. Oncol. 28(3): 695-704.

Fuhlbrigge, R.C., King, S.L., Sackstein, R., and Kupper TS. (2006) CD43 is a ligand for E-selectin on CLA+ human T cells. Blood. 15; 107(4): 1421-6.

Matsumoto, M., Atarashi, K., Umemoto, E., Furukawa, Y., Shigeta, A., Miyasaka, M., and Hirata, T. (2005) CD43 functions as a ligand for E-Selectin on activated T cells. J. Immunol. 15; 175(12): 8042-50.

Pimenidou, A., Madden, L.A., Topping, K.P., Smith, K.A., Monson, J.R., and Greenman, J. (2004) Novel CD43 specific phage antibodies react with early stage colorectal tumours. Oncol. Rep. 11(2): 327-31.

Kadaja, L., Laos, S., and Maimets, T. (2004) Overexpression of leukocyte marker CD43 causes activation of the tumor suppressor proteins p53 and ARF. Oncogene. 19; 23(37): 2523-2530.

Fernandez-Rodriguez, 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.

Cermak, L. Simova, S., Pintzas, A., Horejsi, V., and Andera, L. (2002) Molecular mechanisms involved in CD43-mediated apoptosis of TF-1 cells. Roles of reduced Daxx expression, and adhesion molecules. J Biol Chem. 8;277(10):7955-61.

Carlow, D.A., Corbel, S.Y., and Ziltener, H.J. (2001) Absence of CD43 fails to alter T cell development and responsiveness. J Immunol. 166(1): 256-61.

Nieto, M., Rodriguez-Fernandez, J.L., Navarro, F., Sancho, D., Frade, J.M., Melldo, M., Martinez-A C, Cabanas, C, and Sanchez-Madrid, F. (1999) Signaling through CD43 dosage natural killer cell activation, chemokine release, and PYK-2 activation. Blood. 94(8):2767-77.

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(1): 52-8.

Lopez, S., Seveau, S., Lesavre, P., Robinson, M.K., and Halbwachs-Mecarelli, L. (1998) CD43 (sialophorin, leukosialin) shedding is an initial event during neutrophil migration, which could be closely related to the spreading of adherent cells. Cell Adhes. Commun. 5(2): 151-60.

Stockton, V.M., Cheng, G., Manjunath, N., Ardman, B., and von Andrian, Uriah Heep show; (1998) Negative regulation of T cell homing by CD43. Immunity. 8(3): 373-81.

McEvoy, L.M., Jutila, M.A., Tsao, P.S., Cooke, J.P., and Butcher, E.G. (1997) Anti-CD43 inhibits monocyte-endothelial adhesion in inflammation and atherogenesis. Blood. 90(9): 3587-94.

Baeckstrom, D. (1997) Post-translational fate of a mucin-like leukocyte sialoglycoprotein (CD43) aberrantly expressed in a colon carcinoma cell line. J Biol Chem. 272(17): 11503-9.

McEvoy, L.M., Sun, H., Frelinger, J.G., and Butcher, E.C. (1997) Anti-CD43 inhibition of T cell homing. J Exp Med. 185(8): 1493-8.

Brown, T.J., Shuford, W.W., Wang, W.C., Nadler, S.G., Bailey, T.S., Marquardt, H., and Mittler, R.S. (1996) Characterization of a CD43/leukosialin-mediated pathway for inducing apoptosis in human T-lymphoblastoid cells. J Biol Chem. 271(44): 27686-95.

Santamaria, M., Lopez-Beltran, A., M., Pena, J., and Molina, I.J. (1996) Specific monoclonal antibodies against leukocyte-restricted cell surface molecule CD43 react with nonhematopoietic tumor cells. Cancer Res. 56(15): 526-9.

Bazil, V., Brandt, J., Chen, S., Roeding, M., Luens, K., Tsukamoto, A., and Hoffman, R. (1996) A monoclonal antibody recognizing CD43 (leukosialin) initiates apoptosis of human hematopoietic progenitor cells but not stem cells. Blood. 87(4): 272-81.

Manjunath, N., Correa, M., Ardman, M., and Ardman, B. (1995) Negative regulation of T-cell adhesion and activation by CD43. Nature. 377(6549): 535-8.

Nong, Y.H., Remold-O'donnell, E., LeBie, T.W., and Remold, H.G. (1989) A monoclonal antibody to sialophorin (CD43) dosage homotypic adhesion and activation of human monocytes. J Exp Med. 170(l): 259-67.

Mentzer, S.J., Remold-O'donnell, E., Crimmins, M.A., Bierer, B.E., Rosen, F.S., and Burakoff, S.J. (1987) Sialophorin, a surface sialoglycoprotein defective in the Wiskott-Aldrich syndrome, is involved in human T lymphocyte proliferation. J Exp Med. 165(5): 1383-92.

Pallant, A., Eskenazi, A., Mattei, MG., Fournier, R.E.K., Carlsson, S.R., Fukuda, M., and Frelinger, J.G. (1989) Characterization of cDNA encoding human leukosialin and localization of the leukosialin gene to chromosome 16. Proc. Natl. Acad. Sci. USA 86:1328-32.

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 characterization of sialophorin (CD43), the lymphocyte surface sialoglycoprotein defective in Wiskott-Aldrich syndrome. Proc. Natl. Acad. Sci. USA 86: 2819-23.

1. The selected antibody that is specific binds to the extracellular domain of CD43 person or CEA person expressing nonhematopoietic malignant human cells, where the antibody contains a variable region light chain comprising three CDRs of the amino acid sequence of SEQ ID NO:1, and the variable region of the light chain with three CDRs of the amino acid sequence of SEQ ID NO:2.

2. The antibody according to claim 1, where the antibody contains a sequence of variable regions of the heavy chain of the amino acid sequence of SEQ ID NO:1 and the sequence of the variable region of the light chain of the amino acid sequence of SEQ ID NO:2.

3. The selected antibody that is specific binds to the extracellular domain of CD43 person or that the person expressed by nonhematopoietic malignant human cells, where the antibody contains a variable region of the heavy chain with three CDRs of the amino acid sequence of SEQ ID NO:3, and the variable region of the light chain with three CDRs of the amino acid sequence of SEQ ID NO:4.

4. The antibody according to claim 3, where the antibody contains a sequence of variable regions of the heavy chain of the amino acid sequence of SEQ ID NO:3 and the sequence of the variable region of the light chain of the amino acid sequence of SEQ ID NO:4.

5. The selected antibody that is specific binds to the extracellular domain of CD43 person or CEA person expressing nonhematopoietic malignant human cells, where the antibody contains a variable region of the heavy chain with three CDRs of the amino acid sequence of SEQ ID NO:5, and the variable region of the light chain with three CDRs of the amino acid sequence of SEQ ID NO:6.

6. The antibody according to claim 5, where the antibody contains a sequence of variable regions of the heavy chain of the amino acid sequence of SEQ ID NO:5, and the sequence of the variable region of the light chain of the amino acid sequence of SEQ ID NO: 6.

7. The antibody according to any one of claims 1, 3 and 5, where the antibody is humanitariannet antibody.

8. Antelope any one of claims 1, 3 and 5, where the antibody is a chimeric antibody.

9. The antibody according to any one of claims 1, 3 and 5, where the antibody is a human antibody.

10. The selected antibody that is specific binds to the extracellular domain of CD43 person or CEA person expressing nonhematopoietic malignant human cells, where the antibody contains a sequence of variable regions of the heavy chain of the amino acid sequence of SEQ ID NO: 7, and the sequence of the variable region of the light chain of the amino acid sequence of SEQ ID NO:8.

11. Conjugate for treating nonhematopoietic malignant tumor containing monoclonal antibody conjugated to a cytotoxin, where the antibody comprises:
variable region of the heavy chain with three CDRs of the amino acid sequence of SEQ ID NO:1, and the variable region of the light chain with three CDRs of the amino acid sequence of SEQ ID NO:2;
variable region of the heavy chain with three CDRs of the amino acid sequence of SEQ ID NO:3, and the variable region of the light chain with three CDRs of the amino acid sequence of SEQ ID NO:4, or
variable region of the heavy chain with three CDRs of the amino acid sequence of SEQ ID NO:5, and the variable region of the light chain with three CDRs of the amino acid p is coherence SEQ ID NO:6.

12. The conjugate according to claim 11, where the cytotoxin is a radioactive residue or chemotherapeutic agent.

13. The conjugate according to claim 11, where the cytotoxin is selected from the group consisting of paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetina, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin.

14. A host cell which produces the antibody according to any one of claims 1 to 6 and 10.

15. Composition for treating nonhematopoietic malignant tumor containing an effective amount of the antibody according to any one of claims 1 to 6 and 10, or a conjugate according to any one of § § 11 to 13 and a pharmaceutically acceptable carrier.

16. Polynucleotide for expression of the antibody containing the sequence of the nucleic acid encoding the antibody according to any one of claims 1 to 6 and 10.

17. The vector for expression of the antibody containing the sequence of the nucleic acid encoding the antibody according to any one of claims 1 to 6 and 10.

18. A host cell for expression of the antibody containing the vector by 17.

19. A method of treating nonhematopoietic malignant tumor in an individual with malignancy, including the introduction of the individual is effectivnogo amount of the composition, containing the antibody according to any one of claims 1 to 6 and 10, or a conjugate according to any one of § § 11-13.

20. The method according to claim 19, in which nonhematopoietic malignant tumor is a cancer colorectal person, pancreas or stomach of the person.

21. A method of treating nonhematopoietic malignant tumor in an individual comprising the administration to an individual of a certain number of antibodies according to any one of claims 1 to 6 and 10, or a conjugate according to any one of § § 11-13 and a number of other anti-cancer agents, with antibody and anti-cancer agent in combination provide effective treatment of malignant tumors in the individual.

22. The method according to item 21, in which nonhematopoietic malignant tumor is a cancer colorectal, pancreas or stomach of the person.

23. The method according to item 21, in which the anticancer agent is a chemotherapeutic agent.

24. Kit for treating nonhematopoietic malignant tumor with a container, comprising an effective dose of a pharmaceutical composition comprising the antibody according to any one of claims 1 to 6 and 10, or a conjugate according to any one of § § 11-13.

25. Set point 24 additionally comprising instructions for the implementation of an effective amount of the pharmaceutical composition to an individual for treating nonhematopoietic of Sloka is the natural enemy of the tumor.

26. Set point 24, where nonhematopoietic malignant tumor is a cancer colorectal person, pancreas or stomach.

27. Set point 24, further comprising another anti-cancer agent for use in combination with one or more specified monoclonal antibodies.

28. The method of producing the antibody according to any one of claims 1 to 6 and 10, comprising culturing the host cell under conditions that allow production of antibodies, where a host cell that contains one or more expressing vectors, where expressing a vector containing the polynucleotide sequence encoding the antibody, or expressing the vectors contain polynucleotide encoding light and heavy chain antibodies.

29. The method according to p, further comprising purification of antibodies produced by the host-cell.



 

Same patents:

FIELD: medicine.

SUBSTANCE: what is presented is an antibody characterised by the fact that it binds the human p40 IL-12 subunit and comprises six CDR regions (CDR1, CDR2, CDR3 from the light chain and CDR1, CDR2, CDR3 from heavy chain). There are described: an antibody construct, an antibody conjugate, versions of coding nucleic acid, versions of vectors (for expression and replication), versions of a host cell, a method for producing the antibody, versions of compositions, versions of a method of treating an individual having unfavourable IL-12 activity, a method of reducing IL-12 activity in a human.

EFFECT: use of the invention can find application in medicine for prevention and treatment of acute and chronic diseases associated with activity of the human p40 subunit.

115 cl, 24 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: there are offered: there are offered: an agonist antibody or its functionally active fragment which specifically bind to an epitope containing the first domain Lin 12 Notch3 and activate the signal pathway Notch3. What is described is a labelled antibody and a humanised antibody of their basis. There are offered: a coding nucleic acid, a cell for producing the antibody containing said NA, as well as an expression vector. What is described is a method for producing the antibody by cell cultivation. What is disclosed is applying the antibody for preparing a drug for treating a Notch3-associated disease or disorder. What is described is applying the antibody for diagnosing the Notch3-associated disease or disorder.

EFFECT: use of the invention can find application in medicine in treating and diagnosing the CADASIL diseases.

26 cl, 11 dwg, 6 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: invention refers to biochemistry and represents versions of antibodies binding human anaplastic lymphoma kinase (ALK) protein. There are also presented: versions of radiolabelled or toxin antibodies; a DNA sequence coding the antibody, an expression vector containing the DNA sequence; a host cell transformed by the vector, a method for producing the antibody by cell cultivation, as well as using the antibody for preparing a drug for treating cancer or tumours.

EFFECT: invention may be effectively used for treating tumour or cancer.

32 cl, 5 dwg, 2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: there are disclosed new completely human VAP-1 binding antibodies containing three polypeptides CDR of heavy and light chains, and their VAP-1-binding fragments. There are also presented nucleic acids coding VAP-1 antibodies or their fragments, expression vectors and host cells including these nucleic acids for recombinant expression of VAP-1 antibodies. There is described a method for producing a completely human antibody by transformation of the proper host by the expression vector and cultivation with the antibodies to be collected and purified. There are also disclosed pharmaceutical compositions containing said antibodies, and their therapeutic application - for treating VAP-1-mediated inflammatory diseases in the patients.

EFFECT: method improvement.

21 cl, 20 dwg, 4 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: cellobiohydrolase polypeptide is selected from a group consisting of: a polypeptide containing an amino acid sequence having at least 95% identity to SEQ ID NO:6 and having cellobiohydrolase activity and an fragment a), having cellobiohydrolase activity. Such polypeptides can be obtained using a recombinant technique using suitable polynucleotides, expression vectors and host cells.

EFFECT: invention can be used in production of fermentable sugars and bioethanol from lignocellulose material via enzymatic conversion.

32 cl,15 dwg, 31 tbl, 32 ex

FIELD: chemistry.

SUBSTANCE: fused proteins contain an endoglucanase nucleus amino acid sequence having at least 95% identity to SEQ ID NO:2, fused with an amino acid sequence containing a linker and a cellulose-binding domain (CBD), having at least 95% identity to SEQ ID NO:15. Such fused proteins can be obtained via a recombinant technique using suitable polynucleotides, expression vectors and host cells.

EFFECT: invention provides cellulase, having low activity with respect to restaining, and can be used to treat cellulose material; disclosed fused proteins and enzyme preparations based thereon can be used to prepare detergent compositions or for improving quality of animal feed.

26 cl, 8 dwg, 10 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: invention is an isolated nucleic acid comprising a canine RNA polymerase I regulatory sequence and containing (i) at least 250 or at least 350 or at least 450 adjoining nucleotides or an entire nucleotide sequence, which is in form of SEQ ID NO:26, (ii) a nucleotide which is at least 80% identical to said nucleotide sequence (i) or includes a complementary or reverse complementary (i) or (ii) sequence. The nucleotide sequence (i) or (ii) is operably linked to cDNA which encodes influenza virus vRNA, and when produced in MDCK cells, is capable of directing expression of said influenza virus vRNA. The present invention also describes expression vectors and cells containing such nucleic acids, as well as methods of using such nucleic acids to obtain influenza viruses, including infectious influenza viruses.

EFFECT: canine plasmid rescue system pol I enables to obtain recombinant influenza viruses in a canine cell culture with high titre.

25 cl, 16 dwg, 7 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: there are offered: recovered polynucleotides and polypeptides for binding human EGFR as a part of an antibody, a vector and a host cell for antibody expression, a method for producing an anti-EGFR antibody and an antibody fragment, the anti-EGFR antibody and the antibody fragment. There are discussed a composition containing the anti-EGFR antibody or its fragment, as well as applying the antibody and its fragment for treating EGFR-associated disorders. Besides, there are described versions of glycosylation of the anti-EGFR antibody or its fragment.

EFFECT: invention can find further application in therapy of various EGFR-mediated diseases.

30 cl, 6 ex, 32 dwg, 39 tbl

FIELD: medicine.

SUBSTANCE: what is disclosed is a DNA plasmid for delivery and expression of an antigen, an epitope, an immunogen, a peptide or a polypeptide under the interest. The DNA plasmid contains a cartridge of kanamycin resistance (KanaR) gene wherein the promoter KanaR is modified. The DNA plasmid shows high expression and stability. There are also described a composition containing such DNA plasmid, and methods for immune system stimulation in an animal and immune response induction in an animal with the use of the composition containing the DNA plasmid.

EFFECT: invention is applicable for producing effective DNA vaccines.

18 cl, 21 dwg, 4 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: compound contains IL-15 indirectly bound by covalent links with a polypeptide containing a sushi domain of an extra-cellular region of the alpha IL-15R subunit.

EFFECT: invention allows inducing and stimulating activation and proliferation of said cells, and effectively treating the conditions and diseases which requires higher activity of IL-15.

44 cl, 42 dwg, 4 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: what is presented is an antibody characterised by the fact that it binds the human p40 IL-12 subunit and comprises six CDR regions (CDR1, CDR2, CDR3 from the light chain and CDR1, CDR2, CDR3 from heavy chain). There are described: an antibody construct, an antibody conjugate, versions of coding nucleic acid, versions of vectors (for expression and replication), versions of a host cell, a method for producing the antibody, versions of compositions, versions of a method of treating an individual having unfavourable IL-12 activity, a method of reducing IL-12 activity in a human.

EFFECT: use of the invention can find application in medicine for prevention and treatment of acute and chronic diseases associated with activity of the human p40 subunit.

115 cl, 24 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: recombinant plasmid DNA pTB323 under the invention coding the hybrid polypeptide glutathione-8-transferase (GST) and a shorter version of the protein MPT64 (rΔMPT64), has an average molecular weight 3.6 MDa, size 5574 base pairs, consists of: a) EcoRI-BamHI-fragment of the vector plasmid pGEX-2T of size 4938 base pairs containing the β-lactamase gene inducing tac-promotor, the internal gene Iaclg coding the lactose operone repressor protein, a glutathione-5-transferase gene fragment from S. japonicum with a multiple sites of gene cloning (MSC) in 3'-terminal part of this gene and a nucleotide sequence coding a thrombine proteolysis site and found in front of the MSC; b) EcoRI-BamHI-fragment of 636 base pairs containing a truncated gene MPT64 flanked by EcoRI and BamHI restriction endonuclease sites and prepared by amplification of the gene-related fragment with genome DNA M. tuberculosis; c) a genetic marker - β-lactamase gene determining resistance of pTB323 plasmid transformed cells E. coli to the antibiotic ampicillin; d) unique restriction sites: BamHI - 930/934, EcoRI ~ 1566/1570. The recombinant bacterial strain Escherichia coli BL21/pTB323 - producer of hybrid polypeptide GST-ΔMPT64 with the properties of the mycobacterial antigen ΔMPT64 is deposited in the Collection of Microorganisms of Federal State Research Institution State Science Centre Vector, No. B-1028. The recombinant polypeptide GST-ΔMPT64 produced by the recombinant strain under the invention contains as a carrier protein the N-terminal polypeptide fragment glutathione-S-transferase S.j. (226 amino acid residues, 26.31 kDa) and has a complete amino acid sequence (431 amino acid residues, 48.76 kDa) presented in the description.

EFFECT: using the invention enables developing the high-purity polypeptide in the preparation amounts with the preserved immunogenic properties and provided separation of the target protein from the amino acid sequence of the carrier protein for studying of the immunogenic properties of the target protein.

3 cl, 4 dwg, 4 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of: a SSMA monoclonal antibody and its antigen-binding fragment which are bound with a mark or a cytotoxic agent. There are described versions of a pharmaceutical composition and a diagnostic kit based on such antibodies. There are disclosed methods for identification in vitro of tumour cells, as well as for diagnostic identification of tumour cells based on such antibodies. What is described is an isolated polynucleotide for producing monoclonal antibodies.

EFFECT: using the invention provides the antibodies which can bind SSMA in its native form on the surface of tumour cells, are bound with LNCAP, but are not bound with cells with lost SSMA expression that can find further application in prostate cancer therapy.

15 cl, 21 dwg, 3 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: disclosed is an isolated thymic stromal lymphopoietin protein (TSLP) or antigenic fragment thereof for inducing immune response, as well as a nucleic acid molecule coding it.

EFFECT: invention can find further use in therapy of atopic diseases.

7 cl, 8 dwg, 2 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a method of producing recombinant spider-web proteins of orb-weaving spiders in yeast cells, fused proteins containing spider-web recombinant protein sequences of orb-weaving spiders, recombinant DNA encoding fused proteins, yeast host cells and expression vectors used to realise the method, as well as producer strains of recombinant proteins of orb-weaving spiders. The method involves constructing an expression vector containing a DNA sequence encoding recombinant spider-web proteins of orb-weaving proteins, fused with a sequence which encodes ubiquitin or an ubiquitin-lie protein SUMO of yeast Saccharomyces cerevisiae, which occupies the N-terminal position in the fused protein relative the recombinant spider-web protein, transformation of yeast cells with the obtained expression vector and expression of orb-weaving spider web protein in the transformed cells.

EFFECT: method increases production of recombinant spider-web protein during accumulation thereof in yeast cells in a water-soluble fraction in form of a protein which does not contain a hybrid component.

24 cl, 6 dwg, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to biochemistry and represents versions of monoclonal anti-TAT226 antibodies, their immunoconjugates and pharmaceutical compositions.

EFFECT: antibodies by invention can be efficiently applied for inhibition of tumour cell proliferation.

41 cl, 22 dwg, 3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies specific to CD22 epitope located from amino acid 22 to amino acid 240 CD22. There are disclosed: a coding polynucleotide, an expression vector, a based host cell and a method of producing an antibody with the use of the cell. There are described versions of a method of CD22 detection on the basis of the antibodies. There are disclosed versions of the CD22 immunoconjugate and based pharmaceutical compositions for treating disturbed B-cell proliferation, and also versions of a method of treating with the use of the pharmaceutical composition. There is disclosed a method of B-cell proliferation inhibition on a basis the immunoconjugate. There are described versions of an engineered cystein-substituted antibody specific to CD22 with one or more free cysteines of thiol reactance within the range 0.6 to 1.0. There are disclosed versions of the "antibody-drug" conjugate, the immunoconjugate and pharmaceutical formulaitons for treating disturbed B-cell proliferation. There are also described a method for protein CD22 detection in a sample on the basis of the immunoconjugate, a method for B-cell detection and a method of treating a malignant tumour on the basis of the "antibody-drug" conjugate. There are disclosed: a product for treating disturbed B-cell proliferation on the basis of the pharmaceutical formulation and a method of producing the "antibody-drug" conjugate.

EFFECT: use of the invention provides new specific CD22 antibodies and the based drugs of acceptable therapeutic efficacy with lower toxicity that can find application in therapy of tumours.

227 cl, 25 dwg, 16 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: by recombinant method obtained is fused protein, which contains natural molecule of human erythropoetine with cysteine residue near its C-end and Fc fragment of humal IgG, containing hinge region, N-end of said Fc fragment is connected to said C-end of said erythropoetine molecule, and said Fc fragment is natural, excluding mutation, consisting in substitution of cysteine residue in said hinge region, located the nearest of all to said erythropoetine molecule, with non-cysteine residue, which resulted in the fact that first cysteine residue of said hinge region, located the nearest of all to said N-end, is separated, by, at least, 12 or 17 amino acids from said cysteine residue of said erythropoetine molecule. Obtained peptide is used for stimulation of erythropoesis in mammal.

EFFECT: invention makes it possible to obtain fused protein, which possesses erythropoetine activity, has prolonged time of half-life in vivo in comparison with native human erythropoetine.

43 cl, 20 dwg, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to optimised fused protein for blocking BLyS or APRIL, which contains extracellular region of N-end of truncated TACI (transmembrane activator and CAML-partner) and Fc sequence IgG. TACI segment of fused protein contains sequence of amino-end region of extracellular region, starting with 13-th amino acid residue, complete sequence of stem area from TACI and is obtained from native sequence of TACI between 12-th and 120-th amino acids. Segment Fc of immunoglobulin IgG of fused protein contains hinge region, CH2 region and CH3 region, TACI segment and Fc segment are fused either directly or through linker sequence. In addition, claimed is DNA sequence which codes fused protein, expression vector, host-cell, pharmaceutical composition, containing fused protein, and application of fused protein for blocking BLyS or APRIL. Obtained fused protein does not degrade in process of expression, possesses high biological activity and high level of expression.

EFFECT: fused protein in accordance with claimed invention can be used in treatment of diseases, associated with abnormal immunologic functions and in treatment of diseases caused by abnormal proliferation of B-lymphocytes.

10 cl, 6 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Disclosed is a cell wall-destroying fused protein containing feruloyl esterase and xylanase, which do not contain a C-terminal hydrocarbon-binding molecule (CBM). If necessary, said fused protein contains CBM from a third enzyme, wherein the enzymes and CBM are recombinant proteins corresponding to native fungal proteins. Also disclosed is use of said fused protein to realise methods of destroying plant cell walls when producing compounds of interest from plants or plant by-products. The invention also discloses nucleic acid which codes said fused protein, an expression vector transformed by said nucleic acid and a host cell containing said vector. Described is a method of producing said fused proteins, involving culturing host cells, extraction and, if necessary, cleaning the fused proteins produced by said host cells in the culture. Disclosed is a method of producing desired compounds from plant products, comprising the following steps: 1) enzymatic treatment of plant products with fused proteins or transformed fungus cells, 2) if necessary, treating plant products with steam combined with the action of fused proteins, 3) if necessary, biotransformation of compounds extracted from cell walls during said enzymatic treatment, 4) extraction and, if necessary, cleaning the desired compounds.

EFFECT: invention increases the effect of destroying plant cell walls.

24 cl, 5 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: there are offered versions of: a SSMA monoclonal antibody and its antigen-binding fragment which are bound with a mark or a cytotoxic agent. There are described versions of a pharmaceutical composition and a diagnostic kit based on such antibodies. There are disclosed methods for identification in vitro of tumour cells, as well as for diagnostic identification of tumour cells based on such antibodies. What is described is an isolated polynucleotide for producing monoclonal antibodies.

EFFECT: using the invention provides the antibodies which can bind SSMA in its native form on the surface of tumour cells, are bound with LNCAP, but are not bound with cells with lost SSMA expression that can find further application in prostate cancer therapy.

15 cl, 21 dwg, 3 tbl, 18 ex

Up!