Tumour-targeted monoclonal antibodies against fzd10 and their application

FIELD: medicine.

SUBSTANCE: invention relates to biotechnology and represents antibody or its fragment, which is able to bind with homologue 10 of protein Frizzled (FZD10), such as monoclonal mouse antibody, hybrid antibody, chimeric and humanised antibody. Also claimed are hybridoma clones, producing antibody, as well as method of treatment of prevention of FZD10-associated disease; method of diagnostics or prediction of FZD10-associated disease; method of visualisation in vivo FZD10 in a subject; pharmaceutical composition and set, containing antibody.

EFFECT: treatment with claimed antibodies makes it possible to improve clinical outcome in case of diseases associated with homologue to of protein Frizzled.

44 cl, 45 dwg, 8 ex

 

Cross-reference to related application

This application claims the priority of provisional patent application U.S. No. 60/815257, filed June 21, 2006, the Full contents of the above applications is hereby incorporated into this description by reference in full.

The technical field to which the invention relates

The present invention relates to an antibody or its fragment, which is capable of binding homolog 10 protein Frizzled (FZD10), such as monoclonal antibody mouse hybrid antibody and humanitariannet antibody. The present invention also relates to a method of treatment and/or prevention of diseases associated with FZD10; method of diagnosis or prognosis of diseases associated with FZD10; and the visualization method FZD10 the subject in vivo.

Prior art

Monoclonal antibodies against molecules that are specific for cancer, as proved suitable for the treatment of cancer (Harris, M. (2004). Lancet Oncol, 5, 292-302). In addition to the examples of the clinical use of humanized or hybrid antibody such as trastuzumab (Baselga, J. (2004). Oncology, 61, Supl 2 14-21), rituximab (Maloney, D.G., et al. (1997). Blood, 90, 2188-95) and bevacizumab (Ferrara, N., et al. (2004). Nat Rev Drug Discov, 3, 391-400) in breast cancer, malignant lymphoma and colon cancer, a number of monoclonal antibodies against other molecular mission is St is in the development and evaluated for their antitumor activity. These monoclonal antibodies are expected to give hope to patients with tumors that are not amenable to effective treatment. One of the other important advantages of these monoclonal antibodies is to achieve selective therapeutic effects on cancer cells without severe toxic effects, due to their specific interaction with cells expressing the target molecules (Crist, W.M., et al. (2001). J Clin Oncol, 19, 3091-102. Wunder, J.S., et al. (1998). J Bone Joint Surg Am, 80, 1020-33; Ferguson, W.S. and Goorin, A.M. (2001) Cancer Invest 19, 292-315).

In addition to soft tissue sarcoma, osteosarcoma, Ewing sarcoma and rhabdomyosarcoma sensitive to chemotherapy and these diseases may respond to treatment with chemotherapy. On the other hand, ureterolithotomy sarcoma resistant to chemotherapy and radiation, and having their patients are characterized by poor prognosis. For synovial sarcoma (SS) is effective surgical treatment of patients at an early stage, but for patients in advanced stage of effective therapeutic medication is not available. Therefore, the development of new therapeutic modalities, as expected, change the prognosis of patients in a better way.

Wide genomic analysis of gene expression in tumors provides useful information for the identification of new molecular targets for the development of new anticancer is karstenii tools and markers of tumors. In the previous study, the authors present invention have analyzed the expression profile of genes of a number of soft tissue sarcomas using genome wide analysis micrometrical cDNA consisting of 23040 genes, and demonstrated that the homologue 10 Frizzled (FZD10) (GenBank NO. access AB027464 (SEQ ID NO:1) and BAA84093 (SEQ ID NO:2)) specific and often positively regulated in SS (Nagayama, S., et al. (2002) Cancer Res 62, 5859-66; and WO2004/020668). Product FZD10 gene is a member of the Frizzled family and the assumed receptor that transmits signals WNT (Koike engineering Germany, J., et al. (1999). Biochem Biophys Res Commun, 262, 39-43). Additional analysis showed that FZD10-specific antigen is expressed in SS, and is not expressed or is expressed at almost undetectable levels in other normal organs, with the exception of the placenta, suggesting that drug agents targeting this molecule, should not cause or will cause a few side effects (Nagayama, S., et al. (2002). Cancer Res, 62, 5859-66). Experiments with mRNA showed that FZD10 largely involved in tumor growth SS (WO2006/013733). Moreover, the authors of the present invention have developed a polyclonal antibody rabbit against the extracellular domain FZD10 (FZD10-ECD) and found that this antibody has antitumor activity in mouse xenograft models SS (Nagayama, S., et al. (2005). Oncogene, 24, 6201-12; and WO2005/004912). In General, the treatment of the antibodies is AMI against FZD10 maybe expected to improve the clinical outcome of the SS.

Summary of the invention

In the present description below it is reported that established mouse monoclonal antibodies against FZD10 using the method of cell immunization for possible clinical application. The activity of these antibodies in relation to binding to tumor in vivo was assessed using imaging systems fluorescence in vivo fluorescence in the near infrared part of the spectrum in addition to the traditional method with radioisotopes. In the present description, the authors found the binding specificity of a monoclonal antibody against FZD10 both in vitro and in vivo, as well as the internalization of these antibodies in cells expressing FZD10, and found that in mice bearing xenograft SYO-1 processed once through the tail vein Mab against FZD10 labeled90Y dose of 100 µci, there was a significant antitumor effect.

Based on the data presented above, the authors of the present invention have come to the conclusion that the mouse monoclonal antibody against FZD10 have therapeutic potential in the treatment and diagnosis of SS and other tumors, sverkhekspressiya FZD10.

Therefore, in the first aspect of the present invention features an antibody or its fragment, which includes the V (variable) region H (heavy is th) chain, including region, complementarity determining (CDR)with the amino acid sequence represented in SEQ ID NO: 15, 17 and 19, or CDRs functionally equivalent, and V (variable) region L (light) chain comprising CDRs having the amino acid sequence represented in SEQ ID NO: 23, 25 and 27, CDR, functionally equivalent, which is capable of binding homolog 10 protein Frizzled (FZD10) or its partial peptide.

In one embodiment, the antibody or fragment is selected from the group consisting of mouse antibodies, hybrid antibodies, gumanitarnogo antibodies, fragments of antibodies and single-chain antibodies.

In a preferred embodiment, the antibody is an antibody of a mouse. Preferably, the antibody of the mouse includes an H chain having the amino acid sequence represented in SEQ ID NO: 57, and/or L chain having the amino acid sequence represented in SEQ ID NO: 59. For example, antibody mouse can be produced by a clone of hybridoma 92-13 (FERM BP-10628).

In an alternative preferred embodiment, the antibody is a hybrid antibody. Preferably, the hybrid antibody comprises the V region of the H chain having the amino acid sequence represented in SEQ ID NO: 13, for example, a hybrid antibody may include an H chain having the amino acid is th sequence, presented in SEQ ID NO: 46. Preferably, the hybrid antibody comprises the V region of L chain having the amino acid sequence represented in SEQ ID NO: 21, for example, a hybrid antibody may include an L chain having the amino acid sequence represented in SEQ ID NO: 48.

More preferably, the hybrid antibody comprises the V region of the H chain having the amino acid sequence represented in SEQ ID NO: 13, and the V region of L chain having the amino acid sequence represented in SEQ ID NO: 21. For example, a hybrid antibody comprises an H chain having the amino acid sequence represented in SEQ ID NO: 46, and an L chain having the amino acid sequence represented in SEQ ID NO: 48.

In one embodiment, a hybrid antibody further includes a C (constant) region of human antibodies.

In an alternative preferred embodiment, the antibody is humanitariannet antibody. In one embodiment, humanitariannet antibody further includes FR (frame) region of human antibodies and/or C-region of human antibodies.

In the second aspect of the present invention features an antibody or its fragment, which includes the V (variable) region H (heavy) chain, including the region complementarity determining (CDR), have a second amino acid sequence, presented in SEQ ID NO: 31, 33 and 35, or CDRs functionally equivalent, and V-region L (light) chain comprising CDRs having the amino acid sequence represented in SEQ ID NO: 39, 41 and 43, or CDRs functionally equivalent, which is capable of binding homolog 10 protein Frizzled (FZD10) or its partial peptide.

In one embodiment, the antibody or fragment is selected from the group consisting of mouse antibodies, hybrid antibodies, gumanitarnogo antibodies, fragments of antibodies and single-chain antibodies.

In a preferred embodiment, the antibody is an antibody of a mouse. Preferably, the antibody of the mouse includes an H chain having the amino acid sequence represented in SEQ ID NO: 61 and/or L chain having the amino acid sequence represented in SEQ ID NO: 63. For example, the antibody of the mouse can be produced by clone hybridoma 93-22 (FERM BP-10620).

In an alternative preferred embodiment, the antibody is a hybrid antibody. Preferably, the hybrid antibody comprises the V region of the H chain having the amino acid sequence represented in SEQ ID NO: 29, for example, a hybrid antibody comprises an H chain having the amino acid sequence represented in SEQ ID NO: 50. Preferably, the hybrid antibody comprises the V region of the L chain, having the General amino acid sequence, presented in SEQ ID NO: 37, for example, a hybrid antibody comprises an L chain having the amino acid sequence represented in SEQ ID NO: 52.

More preferably, the hybrid antibody comprises the V region of the H chain having the amino acid sequence represented in SEQ ID NO: 29, and the V region of L chain having the amino acid sequence represented in SEQ ID NO: 37. For example, a hybrid antibody comprises an H chain having the amino acid sequence represented in SEQ ID NO: 50, and the L chain having the amino acid sequence represented in SEQ ID NO: 52.

In one embodiment, a hybrid antibody further includes a C (constant) region of human antibodies.

In an alternative preferred embodiment, the antibody is humanitariannet antibody. In one embodiment, humanitariannet antibody further includes FR (frame) region of human antibodies and/or C-region of human antibodies.

In yet another alternative embodiment, the antibody or its fragment may be labeled with the radioisotope labels or fluorescent label. Such radioisotope label includes90yttrium (90Y)125iodine (125I) and111indium (111In.

In the third aspect of the present invention proposes a clone of hybridoma 92-13(FERM BP-10628), which produces monoclonal antibody 92-13 mouse.

In the fourth aspect of the present invention proposes a clone of hybridoma 93-22 (FERM BP-10620), which produces monoclonal antibody 93-22 mouse.

In the fifth aspect of the present invention proposes a method of treatment or prevention of disease, which is associated with 10 homologue protein Frizzled (FZD10) in a subject comprising administration to the subject an effective amount of the antibody or its fragment. In one embodiment, the disease, which is associated with FZD10 selected from synovial sarcoma (SS), colorectal cancer, gastric cancer, chronic myeloid leukemia (CML) and acute myeloid leukemia (AML).

In the sixth aspect of the present invention proposes a method for the diagnosis or prediction of disease that is associated with a 10 homologue protein Frizzled (FZD10), or predisposition to the development of the disease in the subject, including

(a) contacting the sample or a preparation from a subject with the antibody or fragment mentioned above;

(b) determination of FZD10 protein in the sample or the drug; and

(c) the decision suffers or not the subject's disease or at risk for its development, based on the relative content of FZD10 protein compared to control.

In one embodiment, the disease, which is associated with FZD10 selected from synovial sarcoma is (SS), colorectal cancer, gastric cancer, chronic myeloid leukemia (CML) and acute myeloid leukemia (AML).

In the seventh aspect of the present invention proposes a method of in vivo imaging of 10 homologue protein Frizzled (FZD10) in a subject comprising administration to the subject an effective amount of antibody or fragment, above.

In the eighth aspect of the present invention features a pharmaceutical composition for the treatment or prevention of diseases associated with the homologue 10 Frizzled (FZD10), containing the antibody or the fragment above, and a pharmaceutically acceptable carrier or excipient.

In the ninth aspect of the present invention features a kit for the diagnosis or prediction of disease-related homolog 10 Frizzled (FZD10), containing the antibody or the fragment above.

In the tenth aspect of the present invention features a pharmaceutical composition for in vivo imaging of 10 homologue protein Frizzled (FZD10), containing the antibody or the fragment above.

In the eleventh aspect of the present invention features the use of the antibody or fragment mentioned above, to obtain set for the diagnosis or prediction of disease-related homolog 10 Frizzled (FZD10).

In the twelfth aspect of the present invention features the use of antibodies or fragm the NTA, above, to obtain a composition for prevention or treatment of diseases associated with the homologue 10 Frizzled (FZD10).

The term "disease, which is associated with FZD10" (associated with FZD10 disease), refers to a disease that is associated with overexpression of FZD10 protein. Such diseases include, but are not limited to, synovial sarcoma (SS), colorectal cancer, gastric cancer, chronic myeloid leukemia (CML) and acute myeloid leukemia (AML).

The term "fragment" refers to any fragment of the antibody that can be obtained from the antibody against FZD10 protein, and contains some CDR. Such fragments include, but are not limited to, Fab fragment, F(ab')2the fragment, and Fv fragment.

The term "modified antibody" means any antibody that can occur from antibody against FZD10, and contains some CDR. Such a modified antibody includes, but is not limited to, PEG-modified antibody. The fragment of the antibody or a modified fragment can be easily recognized by a person skilled in the technical field and are produced using any method known in this technical field.

The term "subject" in the present description refers to a subject who suffers from a disease associated with FZD10, and also to the subject, are thought to have the disease, links the data with FZD10. The subject of the present invention can be animals, including mammals and birds. For example, mammals include humans, mice, rats, monkeys, rabbits and dogs.

Brief description of drawings

In Fig. 1a through 1f presents the characterization of the specificity of binding of two monoclonal antibodies against FZD10.

In Fig. 1a presents the analysis using flow cytometry four antibodies, 39-2 and 39-10 (disclosed in patent application WO2005/004912), 92-13 and 93-22, with five lines SS (SYO-1, YaFuSS, HS-SY-2, Fuji and 1973/99) and one line of colorectal cancer cells (LoVo). Solid lines show the fluorescence intensity, which is defined by each mAb; dashed lines depicts the fluorescence intensity of the cells, preincubating with non-immune mouse IgG as a negative control.

In Fig. 1b presents semi-quantitative RT-PCR FZD10 in the same lines of tumor cells, as used in Fig. 1a. Expression of β2-microglobulin (β2-MG) served as internal control.

In Fig. 1c presents the analysis using flow cytometry antibodies 92-13 (top panel) and 93-22 (bottom panel) against exogenous FZD10. Cell line colon cancer SNU-C5 was transfusional empty vector pCAGGS (left panel) or pCAGGS-FZD10-myc/His (right panel) and analyzed 48 hours after transfection. The solid line is the mi shows the intensity of fluorescence, determined by each mAb; dashed lines depicts the fluorescence intensity of the cells, preincubating with non-immune mouse IgG as a negative control.

In Fig. 1d presents the labeled binding125I 39-10, 39-2, 92-13 and 93-22 with normal human blood cells. Radioactively labeled Mab were each incubated with freshly isolated normal human blood cells from three subjects (A, B, and C) in the absence (empty bars) or presence (shaded bars) is identical to the unlabeled antibody.

In Fig. 1e presents binding activity of labeled125I Mab. A constant amount of radioactively labeled Mab were incubated with cell SYO-1 and increasing amounts of unlabeled antibody. Put the percentage of radioactivity associated with the cells against the number of unlabeled antibody. Shaded circle; 92-13, unfilled circle; 93-22.

In Fig. 1f presents the analysis using flow cytometry of autoblok and cross-block. Alexa-488-labeled 92-13 (top panel) and 93-22 (lower panel) were incubated with cell SYO-1 (i) PBS, or in the presence of 100 µg (ii) unlabeled 92-13 and (iii) unlabeled 93-22. On the hatched histogram shows the intensity of fluorescence was determined using each of the Alexa-488-labeled Mab; dashed lines represent the fluorescence intensity of the cells, and kopirovannym with PBS as a negative control.

In Fig. 2 shows immunohistochemical analysis in SS and normal frozen sections of human tissue without antibodies (a, d, g, j and m), 92-13 (b, e, h, k and n) and 93-22 (c, f, i, l and o). (a-c), synovial sarcoma; (d-f)kidney; (g-i), liver, (j-l)heart; (m-o), the brain. Original magnification: ×100.

In Fig. 3 presents the biological distribution111In-labeled and125I-labeled antibodies. 10 CVR (a),111In-labeled 92-13, (b),125I-labeled 92-13, (c),111In-labeled 93-22 and (d),125I-labeled 93-22 was injected intravenously naked BALB/c mice bearing tumor SYO-1. The organs and the tumor is cut out after 1 hour (open bar), 24 hours (hatched bars) and 48 hours (shaded bar) and the measured radioactivity. The data presented are representative data of two independent experiments.

In Fig. 4a presents the visualization of the in vivo fluorescence of mice bearing tumor SYO-1, after the introduction of the Alexa 647-labeled 92-13 or 93-22. Fluorescently labeled Mab was administered at a dose of 20 μg per mouse intraperitoneally. All fluorescent images were obtained with a 60-second exposure time, f/stop = 2) before inserting, immediately after injection (0 hour), after 24, 48 and 96 hours. Arrows indicate the location of the tumor. S.C. tumor localized dorsally much farther to 92-13 (top panel) and trunk for 93-22 (bottom panel). Fluorescent signal from Alexa647 was pseudo is krashen in accordance with a color bar, shown on the right. If 93-22 (bottom panel) head arrows indicate the injection.

In Fig. 4b and 4c presents illustrative visualization of the removed organs and tumors from mice shown in Fig. 4a, 4b; 92-13, and 4c; 93-22. i, the tumor SYO-1; ii, liver; iii, spleen; iv, kidney; v, pancreas; vi, the colon.

In Fig. 5a presents the visualization of in vivo fluorescence mice bearing LoVo tumor, after the introduction of Alexa647-labeled 92-13 or 93-22. Fluorescently labeled Mab was injected as in Fig. 4. All fluorescent images were obtained with a 60-second exposure time, f/stop = 2) immediately after injection (0 hour), after 48, 72, 96 and 120 hours (h). Arrow indicates the location of the tumor. S.c. the tumor is localized in the right forearm for 92-13 (top panel)and 93-22 (bottom panel).

In Fig. 5b and 5c presents illustrative visualization of the removed organs and tumors of mice shown in Fig. 5a, 5b; 92-13, and 5c; 93-22. i LoVo tumor; ii, liver; iii, spleen; iv, kidney; v, pancreas; vi, the colon.

In Fig. 6 presents the internalization 92-13 and 93-22, measured by confocal microscopy. Cells were treated with PBS (a, d and g), 50 μg/ml 92-13 (b, e and h) or 93-22 (c, f, i) for 3 hours at 37oC, 5% CO2. Antibodies associated with the cell surface, was desirerable in the acidic environment of 0.1 M glycinol the m buffer (pH 2,5). The cells were fixed, was permeability.avi and then blocked with 3% BSA. Intracellular antibodies were determined using goat artemisinin IgG-Alexa488, and the nucleus was stained with DAPI. (a - c), SYO-1; (d - f), YaFuSS; (g - i) LoVo.

In Fig. 7 presents the effect of90Y-labeled 92-13 on tumor growth. When the tumors were grafted in (0,4-2,7cm3), the mice were injected once into the tail vein of 100 µci90Y-labeled 92-13.

In Fig. 8 presents induced hybrid as 92-13 and 93-22, ADCC specifically in relation to do overexpress FZD10 cells SYO-1. 1 µg/ml hybrid antibodies 93-22 (ch93-22) or hybrid antibodies 92-13 (ch92-13) was added in different ratio effector:target. As effector cells used PBMC from different people donors; (a), (c) ADCC hybrid 92-13 against cells SYO-1 with five healthy donor PBMC. (b), (d) ADCC hybrid 93-22 against LoVo cells with two healthy donor PBMC. Quantitative assessment of cytotoxicity by LDH activity described in (Nagayama, S., et al. Oncogene, 24, 6201-12).

Detailed description and preferred embodiments of the present invention

Homolog 10 Frizzled (FZD10) is a member of the Frizzled family, which is a receptor, a conductive signal from Wnt. As described in the present invention below, the authors have successfully created mouse monoclonal antibodies and hybrid antibodies against FZD10 protein, which may be the ü suitable for use in medicine.

Mouse monoclonal antibodies specific for FZD10 (92-13 and 93-22 Mab)generated by immunization of mice with cells, transfitsirovannykh FZD10. As shown by analysis using flow cytometry (FACS), Mab, as 92-13 and 93-22, have specific binding activity against FZD10 in cell line SS, the cells SYO-1 cells and COS7, transfitsirovannykh FZD10. To confirm the specific binding activity of these antibodies in vivo, the authors present invention was injected fluorescently labeled Mab intraperitoneally or intravenously to mice carrying xenografts SS, and found through the use of fluorescence imaging in vivo and radioactivity that these Mab are associated with tumors expressing FZD10, and not with any other normal tissues of mice. Subsequent immunohistochemical analysis with Mab confirmed the absence or barely detectable level of FZD10 protein in normal human organs except the placenta. Moreover, it is interesting that the authors of the present invention have found, using confocal laser scanning microscopy that Mab internalized in cell lines SS, SYO-1, but not in a negative against FZD10 cell line, LoVo. It was unexpectedly found that in mice bearing xenograft SYO-1, with a single introduction into the tail vein of90Y-labeled Mab (92-13) against FZD10 in a dose of 100 µci was observed su is the natural enemy anti-tumor effect. In summary, the authors concluded that these specific Mab against FZD10 can be used as a new diagnostic marker or for the treatment of SS with minimal risk of adverse reactions or lack of it.

Often it is very difficult to produce antibodies against proteins with seven transmembrane domains due to the complicated structure of these proteins. In the previous study, the authors present invention showed that FZD10 forms homologoues (Nagayama, S., et al. (2005). Oncogene, 24, 6201-12). After many failed attempts to establish a monoclonal antibody against FZD10, which can learn the natural shape FZD10, through the use of full or partial recombinant protein FZD10 the authors present invention finally used the immunization by injection of living cells COS-7, do overexpress FZD10, pads of Balb/c mice and successfully got two hybridoma producing antibodies against FZD10, which had the ability to know the natural form of FZD10 in living cells, as shown by FACS analysis. Because these antibodies do not define FZD10 protein in Western-blotting, the authors of the present invention suggested that these Mab recognize the tertiary structure of FZD10.

To examine the distribution in vivo Mab 92-13 and 93-22 the authors of the present invention used two methods; one based on radioisotope IU the odes using 125I and111In-labeled antibodies, and the other was based on fluorescent imaging using antibodies labeled in the near infrared part of the spectrum (Alexa647). Fluorescent in the near infrared part of the spectrum, mainly indocyanine dye, now widely used for imaging in vivo for diagnostic purposes, as the light of this wavelength penetrates living tissue quite effectively (Chen, X., et al. (2004). Cancer Res, 64, 8009-14). The results obtained using the two approaches were very consistent with each other and pointed to the fact that 92-13 and 93-22 contact with tumor cells SYO-1, but not with other normal tissues. To confirm whether these antibodies may be used for clinical application, the authors present invention has further examined the binding activity of antibodies against normal blood cells. Binding activity125I-labeled 92-13 and 93-22 against normal human blood cells was not detectable in all three individual donors (Fig. 1d). These results are consistent with those obtained by FACS analysis using mononuclear cells of peripheral blood (data not shown), suggesting the clinical applicability of these two antibodies with a small chance of side effects in patients with SS, thanks to the high-affinity binding is the affinity molecule FZD10. Moreover, in vitro experiments using confocal microscopy revealed that specic binding of Mab 92-13 and 93-22 with FZD10 cell surface induces the internalization of the antibodies (Fig. 6). As described previously (Stein, R., et al. (2001). Criti Rev Oncol Hematol, 39, 173-80; Stein, R., et al. (2005). Clin Cancer Res, 11, 2727-34)if labeled Mab internalized after binding125I-labeled antibody is metabolized in lysosomes and diffuses from tumor target cells, whereas111In-labeled antibody remains in the lysosomes. As can be seen in Fig. 3, the radioactivity111In-labeled antibody and125I-labeled antibody in tumors were significantly different (Fig. 3, a and b, c and d). These data suggest that Mab 92-13 (and 93-22) can specifically internalities in SS cells through FZD10 protein.

When antibodies are used to treat cancer, the following three mechanisms are believed to induce antitumor activity; (i) if the molecule-target involved in the stimulation of growth, neutralizing antibodies to block the transmission of growth signals and then to suppress the growth of tumor cells; (ii) the second possibility is the effector activity in terms of induction of antibody-dependent cellular cytotoxicity (ADCC) or dependent complement cytotoxicity (CDC). (iii) the Third case are radioactive isotopes or antitumor Lek is RSTO, which konjugierte with antibodies and efficiently delivered to tumor target cells. Although the authors of the present invention have previously shown that the molecule-target FZD10 involved in tumor growth SS, neither Mab 92-13, no 93-22 showed no neutralizing effect in vitro when added to cell culture media (data not shown) or in vivo when administered to mice, the carriers of tumour (data not shown).

Conjugated with antibodies radioactive isotope or an anti-cancer drug, such as zevalin (antibody against CD20 conjugated with90yttrium) and mylotarg (antibody against CD33 conjugated to calicheamicin), as proven, are highly effective in terms of providing cytotoxicity antibody (Wiseman, G.A. and Witzig, T.E. (2005). Cancer Biother Radiopharm, 20, 185-8; van der Velden, VH, et al. (2001). Blood, 97, 3197-204; Carter, P. (2001). Nat Rev Cancer, 1, 118-29). Mylotarg exerts its antitumor activity by release of anticancer drugs, calicheamicin, in cancer cells after internalization (van der Velden, VH, et al. (2001). Blood, 97, 3197-204). In the examples for therapeutic experiments established conjugate90yttrium-DTPA-92-13 and investigated its antitumor activity. In the model of xenograft mouse tumor was rapidly decreased after treatment90yttrium-DTPA-92-13 (Fig. 7). It is observed that tumor, including tumor larger volume (> 1 cm 3), did not manifest refraction to 34 days after injection, and was not observed strong toxicity. Since antibodies 92-13 and 93-22 against FZD10, clearly, effectively internalizations in antigen-positive cells, as shown in Fig. 6, the conjugation of anticancer drugs with both Mab, 92-13 and 93-22, is also expected to show strong anti-cancer effect on the cells of SS. Relative to the effector activity, as hybrid 92-13 and 93-22, induced specific ADCC against FZD10-do overexpress cells SYO-1 (Fig. 8, a and c), but not against FZD10-negative LoVo cells (Fig. 8, b and d). Especially hybrid 92-13 showed higher induction of cytotoxicity compared to the hybrid 93-22, however, their activity depended on donor effector cells, possibly due to polymorphism of the Fc receptor. In conclusion, the authors present invention successfully received monoclonal antibodies which can specifically bind with FZD10 for FZD10-do overexpress tumor cells in vitro and in vivo. In summary, the authors of the present invention believe that the monoclonal antibody against FZD10 have great potential in developing new drug therapies for the treatment of SS and other tumors that hyperexpression FZD10.

1. Obtaining antibodies

Antibodies that can be used in the present invention, specific n is focused against FZD10 protein, obtained by disease associated with FZD10. Used in the present description, the term "antibody" means an antibody molecule as a whole, and of its fragments, such as Fab fragments, F(ab')2fragments and Fv fragments, which can be contacted with the protein or its peptide fragments as antigens. The antibody may be either a polyclonal antibody or a monoclonal antibody. It may also be humanized or hybrid antibody or single-chain Fv (scFv) antibody. Antibodies (polyclonal antibodies and monoclonal antibodies) for use in the present invention can be obtained, for example, using the following method.

(1) Monoclonal antibody

The source receives the antigen for production of antibodies suitable for the present invention. FZD10 protein or its partial peptide can be used as an immunogenic protein. Alternatively, a cell expressing the FZD10 protein or its partial peptide, can also be used as an immunogen. Amino acid sequence of FZD10 protein used as immunogen in the present invention, and the cDNA sequence encoding the protein is in the public domain in GenBank No. access BAA84093 (SEQ ID NO: 1) and AB027464 (SEQ ID NO: 2), respectively. FZD10 protein or its partial peptide for use in the project as an immunogen can be obtained synthetically in accordance with the procedure known in the art, such as a method of solid-phase peptide synthesis, using available information about the amino acid sequence. Peptide fragments FZD10 protein include, but are not limited to, a peptide containing residues 1-225 of the amino acid sequence represented in SEQ ID NO: 1, which corresponds to the N-terminal extracellular domain of the protein FZD10 (FZD10-ECD).

Protein or its partial peptide, or expressing their cell can be obtained by using the sequence information of cDNA that encodes a FZD10 protein or its partial peptide, in accordance with known procedure recombination of genes. Obtaining a protein or peptide fragment, as well as expressing their cells in accordance with such procedure genetic recombination is illustrated below.

Recombinant vector for production of the protein can be obtained by blending the above sequence of cDNA with a suitable vector. The transformant can be obtained by introducing a recombinant vector for production of the protein in the host, so that the protein-target FZD10 or its partial peptide can be expressed.

As a vector using the phage or plasmid that is able to autonomously replicate in the host. Examples of plasmid DNA include pCAGGS, pET28, pGEX4T, pUC118, pUC119, pUC1, pUC19 and other plasmid DNA originating from Escherichia coli; pUB110, pTP5 and other plasmid DNA derived from Bacillus subtilis; and YEp13, YEp24, YCp50 and other plasmid DNA derived from yeast. Examples of DNA phages include the phage lambda, such as λgt11 and λZAP. In addition, can be used in the vectors of animal viruses such as retrovirus vector, and the vector of cowpox and can also be used in the vectors of insect viruses such as baculovirus vector.

DNA encoding FZD10 protein or its partial peptide (referred to in the present description hereinafter referred to as DNA FZD10), inserted into a vector, for example, by using the following method. In this method, the purified DNA is digested with the appropriate restriction enzymes and inserted into the restriction site of the enzyme or in the multiple cloning site of a suitable DNA vector for ligation into the vector.

In addition to the promoter and DNA FZD10 any enhancers and other CIS-elements, splicing signals, the signal adding poly-A, a selective marker, the site of ribosome binding (RBS), and other items can be legirovanyh in the recombinant vector for production of the protein for use in mammalian cells, if desired.

For ligation of DNA fragment with a fragment of the vector can be used a known DNA ligase. The DNA fragment and the vector fragment is annealed and are ligated thereby obtaining a recombinant vector for production of the protein.

The owner for use in transformation is not specifically limited as long as he allows to be expressed in it FZD10 protein or its partial peptide. Examples of the host include bacteria, for example E. coli and Bacillus; yeast such as Saccharomyces cerevisiae; animal cells such as COS cells, cells of the Chinese hamster ovary (CHO) cells and insects.

For example, when the host uses a bacterium, a recombinant vector for production of the protein should preferably be capable of Autonomous replication in bacteria-host and enable the promoter site of the ribosomal binding, DNA FZD10 and the sequence termination of transcription. The recombinant vector may further include a gene for the regulation of the promoter. The example of Escherichia coli include Escherichia coli BRL, and example Bacillus is Bacillus subtilis. In the present description can be used any promoter that can be expressed in the host, such as Escherichia coli.

The recombinant vector may be introduced into the bacterium-host using any of the methods known in the art. Such methods include, for example, a method using calcium ions and electroporation. When the owner uses the yeast cell, animal cell or insect cell, the transformant can be obtained in accordance with a method known in the village is authorized technical field and then FZD10 protein or its partial peptide can be obtained from the owner (transformant).

FZD10 protein or its partial peptide for use as an immunogen in the present invention can be obtained from the culture created above transformant. "Culture" refers to any culture supernatant, cultured cells, cultured microorganisms and their homogenates. The transformant is cultivated in culture medium using the traditional method of cultivation of the host.

Culture media for cultivation of transformant obtained using Escherichia coli, yeast or other microorganisms as hosts, can be either a natural medium or a synthetic medium, so long as it contains a carbon source, nitrogen source, inorganic salts and other components used by the microorganism, and allows transformant effectively to grow.

The transformant is usually cultivated by shaking culture or aeration of the culture with stirring under aerobic conditions at 25°C to 37°C for 3 to 6 hours. During cultivation, the pH is maintained at about neutral by bringing, for example, inorganic or organic acid and alkaline solution. In the process of cultivation to the environment can be added antibiotics, t is such as ampicillin or tetracycline, in accordance with the selective marker is included in a recombinant expression vector, if necessary.

After cultivation, when FZD10 protein or its partial peptide is produced in the microorganism or cell, the protein or its partial peptide is extracted by homogenization of the microorganism or cell. When FZD10 protein or its partial peptide is secreted from the microorganism or cell culture medium is used as it is, or remains of a microorganism or cells are removed from culture medium, for example by centrifugation. After that, FZD10 protein or its partial peptide can be isolated from the culture and purified by using conventional biochemical method for isolation and purification of proteins, such as precipitation with ammonium sulfate, gel chromatography, ion exchange chromatography and affinity chromatography, either individually or in combination.

The FZD10 protein or its partial peptide can be confirmed by, for example, by polyacrylamide gel electrophoresis with SDS.

Then the FZD10 protein or its partial peptide or the transformant is dissolved in a buffer that will receive the immunogen. When necessary, there may be added adjuvant for effective immunization. Such adjuvants include, for example, a commercially available full beta-blockers and incomplete adjus the NT's adjuvant. Any of these adjuvants may be used individually or in combination.

Thus prepared immunogen is administered to a mammal such as a rabbit, rat or mouse. The immunization is carried out mainly through intravenous, subcutaneous or intraperitoneal administration. Spacing the immunizations are not limited specifically, and mammal immunities from one to 3 times at intervals ranging from several days to weeks. The cells producing the antibodies are harvested from 1 to 7 days after the last immunization. Examples of cells producing antibodies include cells, spleen cells, lymph node cells and peripheral blood.

To obtain hybridoma cell producing antibodies, and the myeloma cell is drained. As the myeloma cells for fusion with the cell producing antibodies, can be used normally available established cell line. Preferably, the used cell line would have a selectivity of medicines, and of such a nature that it could not survive in HAT selective medium (containing gipoksantin, aminopterin and thymidine) in nakitai form and could only survive when merged with cell producing antibodies. Possible myeloma cells include, for example, lines are murine myeloma cells, such as P3X63-Ag.8.U1 (P3U1) and NS-I.

Then jamnow the cage and the cage, producing antibodies, poured. To merge these cells were mixed, preferably in the cells producing antibodies to the myeloma cells was 5:1 in culture medium for animal cells, which does not contain serum, such as DMEM and RPMI-1640, and was merged in the presence of an agent that stimulates cell fusion, such as polyethylene glycol (PEG). The cell fusion can be carried out using commercially available devices to merge cells using electroporation.

Then hybridoma selected from the cells after the above processing to merge. For example, the suspension of cells is appropriately diluted, for example, medium RPMI-1640 containing fetal calf serum, and then dropped off on the tablet for micrometrology. To each well add selective medium, and the cells were cultured with appropriate replacement of the selective environment. As a result, in the form of hybridoma can be derived cells that grow approximately 30 days after the start of culturing in a selective medium.

This is followed by screening the culture supernatant of the growing hybridoma for the presence of antibodies, which interacts with the FZD10 protein or its partial peptide. Screening hybridoma can be carried out in accordance with conventional procedure, for example, using tverdova the aqueous enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). Merged cell clone by serial dilution to create hybridoma, which produces interesting monoclonal antibody.

Monoclonal antibody can be collected from the generated hybridoma, for example, using the conventional method of culturing cells or by the production of ascites. If necessary, the antibody can be purified in the above-described method of collection of antibodies in accordance with the known method, such as precipitation with ammonium sulfate, ion exchange chromatography, gel filtration, affinity chromatography, or a combination thereof.

Type of globulin monoclonal antibodies useful in the present invention is not specifically limited, as long as they can specifically bind with FZD10 protein, and they can be any of IgG, IgM, IgA, IgE and IgD. Among them, preferred are IgG and IgM.

In the present invention murine monoclonal antibodies 93-22 and 92-13 successfully created and are preferably used. Clone hybridoma 93-22 producing monoclonal antibody mouse 93-22, was put on the international storage Shuichi Nakatsuru in IPOD International Patent Organism Depository of the National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-Ken, 305-8566 Japan) on June 14, 2006 under registration number FERM BP-10620. Also, the clone of hybridoma 92-3, producing monoclonal antibody mouse 92-13, was put on the international storage Shuichi Nakatsuru in IPOD International Patent Organism Depository of the National Institute of AIST June 28, 2006 under registration number FERM BP-10628. Monoclonal antibody is produced by hybridomas, may preferably be used in the present invention.

In the present invention can also be used monoclonal antibody recombinant type, which can be obtained by cloning the gene of the antibody from hybridoma, integration of the gene of the antibody in an appropriate vector, introducing the vector into the host, production of antibodies by the host in accordance with generally accepted ways recombination of genes (see, for example, Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-75).

More specifically, mRNA encoding the variable (V) region of mouse monoclonal antibodies against FZD10, separated from hybridoma producing antibodies (e.g., such as described above). The selection of mRNA is carried out by obtaining total RNA using a known method such as a method of ultracentrifugation in guanidine (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-9) and AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-9), and then get the desired mRNA from total RNA using a kit for purification of mRNA (Pharmacia) or the like. Alternatively, mRNA can also be derived directly using the set QuickPrep mRNA Purificatin (Pharmacia).

Then from the mRNA using reverse transcriptase synthesizes cDNA for the V region of the antibody. The cDNA synthesis can be performed by using a commercially available kit, such as Gene RacerTM(Invitrogen). cDNA can be synthesized or amplified by the method of 5'RACE (Frohman, M.A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-32) with a set of 5'-Ampli FINDER RACE (Clontech) in combination with PCR method.

Amino acid sequence of H-chain and L-chain monoclonal antibodies 92-13 mouse are represented in SEQ ID NO: 57 and 59, respectively (encoded by the nucleotide sequence as shown in SEQ ID NO: 58, and 60, respectively). Amino acid sequence of H-chain and L-chain monoclonal antibodies 93-22 mouse are represented in SEQ ID NO: 61 and 63, respectively (encoded by the nucleotide sequence as shown in SEQ ID NO: 62 and 64, respectively). Based on sequence information, using the conventional method can be created primers used for amplification of the H-chain or L-chain of interest monoclonal antibodies.

The desired DNA fragment is isolated and purified from the obtained PCR product and then are ligated with a DNA vector to obtain a recombinant vector. The recombinant vector is introduced into the host, such as E. coli, and selected colony, containing the Yu desired recombinant vector. The nucleotide sequence of DNA of interest in the recombinant vector is confirmed by using, for example, automatic sequencing machine.

After obtaining the DNA encoding the V region of the antibody against FZD10, DNA integrate into the expression vector containing DNA encoding the constant (C) region of the antibody.

To obtain antibody against FZD10 used in the present invention, the antibody gene is integrated into the expression vector so that the gene of the antibody could be expressed under the control elements controlling the expression (e.g., enhancer, promoter). A host cell transformed expression vector for the expression of antibodies.

When expression of the gene of the antibody DNA encoding the heavy (H) chain and DNA encoding a light (L) chain antibodies, can be integrated into separate expression vectors, and then a host cell transformed together obtained recombinant expression vectors. Alternatively, DNA encoding the H-chain and DNA encoding the L-chain antibodies, can be integrated together into a single expression vector, and then a host cell transformed the obtained recombinant expression vector (for example, in patent WO 94/11523).

Gene antibodies can be expressed using known methods. For expression in the cell mlekovita the x can be operatively linked traditionally used the promoter, gene antibody expression for the expected and the signal poly(A) (localized below the 3'-end of the gene of the antibody). For example, as a suitable system promoter/enhancer can be used in the system of immediate early promoter/enhancer of cytomegalovirus cheloveka.

Other systems the promoter/enhancer, for example, derived from viruses (e.g., retrovirus, virus polyoma, adenovirus and virus 40 monkeys (SV40) and originating from mammalian cells (for example, factor 1-alpha elongation person (HEF1 alpha)), can also be used for expression of the antibodies in the present invention.

When using the promoter/enhancer of SV40, the gene expression can easily be done using the method of Mulligan et al. (Nature (1979) 277, 108-14). When using the promoter/enhancer HEF1 alpha gene expression can easily be carried out by the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322).

For expression in E. coli can be functionally linked traditionally used the promoter, signal sequence for secretion of specific antibodies and antibody gene. As a promoter can be used lacZ promoter or araB promoter. When using the lacZ promoter, the gene expression can be performed using the method of Ward et al. (Nature (1098) 341, 544-6; FASBE J. (1992) 6, 2422-7), while, when using the araB promoter, expression of the gene can realize is taken using the method of Better et al. (Science (1988) 240, 1041-3).

In relation to the signal sequence for secretion of the antibody when the antibody of interest is intended for secretion in periplasmatic space of E. coli, can be used to signal the pelB sequence (Lei, S.P. et al., J. Bacteriol. (1987) 169, 4379-83). Antibody Sekretareva in periplasmatic space, isolated and then re-stack so that the antibody has taken the appropriate configuration.

Can be used origin of replication derived from viruses (such as SV40, a virus polyoma, adenovirus, papilloma virus cows (BPV), or the like. In order to increase the number of gene copies in the system of the host cell, the expression vector may additionally contain a gene selective marker such as a gene aminoglycosidetherapy (APH), gene timedancing (TK), gene xanthine-guaninephosphoribosyltransferase (Ecogpt) gene digidrofolatreduktazy (dhfr).

To obtain antibodies used in the present invention may be used in any expression system, including eukaryotic and prokaryotic cell system. Eukaryotic cell includes recognized animal cell lines (e.g., mammals, insects, moulds and fungi, yeast). Prokaryotic cell includes bacterial cells such as E. coli cells. Preferably, h is Oba antibody used in the present invention, expressed in a cell of a mammal, such as a CHO cell, COS, myeloma, BHK, Vero, and HeLa.

Then the transformed cell host cultivated in vitro or in vivo to receive the necessary antibodies. Culturing the host cell can be carried out using any known method. Cultural environment that can be used in the present description, can be a DMEM, MEM, RPMI 1640, or IMDM. The culture medium may contain an additive serum such as fetal calf serum (FCS).

Upon receipt of recombinant antibodies, in addition to the above host cells, as the owner can also be used transgenic animal. For example, the antibody gene is inserted into a predetermined site of the gene encoding the protein, which tend to be produced in the milk of the animal (e.g., beta-casein) to obtain a hybrid gene. The DNA fragment containing the hybrid gene with the introduced gene antibody, is administered to the animal embryo, which is not a person, and the embryo then bring in the female animal. The female animal, which carries the embryo carries a transgenic animal, not a person. The antibody of interest is secreted into the milk of the transgenic animal, not a person, or his progeny. In order increased the I milk containing antibody transgenic animals may be entered in the appropriate hormone (Ebert, K.M. et al., Bio/Technology (1994) 12, 699-702).

The antibody expressed and produced as described above can be isolated from cells or body of an animal host and purified. Isolation and purification of antibodies used in the present invention, can be carried out on the affinity column. Can also be used in other ways, traditionally used for isolation and purification of antibodies; thus, the method is not particularly limited. For example, for isolation and purification of the antibody of interest can be used by various chromatographic methods, filtration, ultrafiltration, salting out and dialysis, singly or in combination (Antibodies A Laboratory Manual. Ed. Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).

(2) Hybrid antibody and humanitariannet antibody

In the present invention can be used artificially modified recombinant antibody, including hybrid antibody and humanitariannet antibody. These modified antibodies can be obtained by any known method. For example, there can be used methods developed for "hybrid antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81: 6851-5; Neuberger et al., 1984, Nature, 312: 604-8; Takeda et al., 1985, Nature, 314: 452-4). Hybrid antibody is a molecule, inwhich different parts come from different species of animals, such as a molecule, having a variable region derived from mAb mouse, and a constant region of human immunoglobulin, e.g., "humanized antibodies".

Hybrid antibody of the present invention can be obtained by ligating the DNA encoding the V region of the antibody, DNA encoding the C region of human antibodies integration of the product of ligation into the expression vector, and introducing the obtained recombinant expression vector into the host for hybrid antibodies.

Humanitariannet antibody referred to as "antibody man with the restored form, in which region complementarity determining (CDR)of an antibody of a mammal, not a person (e.g., a mouse), connected with those of human antibodies. The usual method of genetic recombination to obtain such gumanitarnogo antibodies are also known (for example, in patents EP 125023; WO 96/02576).

Specifically, it creates a DNA sequence in which the CDRs of the antibodies of the mouse with the subsequent ligation through the frame region (FR), and is synthesized using the PCR method with the use of multiple oligonucleotides as primers, which are created with areas overlapping end region CDR and FR. The resulting DNA are ligated with DNA encoding the C region of human antibodies, and the product of the League of the Finance integrate in the expression vector. The obtained recombinant expression vector introduced into the host, thereby obtaining humanitariannet antibody (for example, patent WO 96/02576).

FR, legirovannye through CDR are selected so that the CDR was able to form functional antigennegative website. If necessary, amino acid(s) in FR V region of the antibody can be substituted so that the CDR of human antibodies with the restored form could form the corresponding antigennegative site (Sato, K. et al., Cancer Res. (1993) 53, 851-6).

Hybrid antibody consists of V-regions, derived from the antibody of a mammal, not a person, and C-regions, derived from human antibodies. Humanitariannet antibody comprises a CDR derived from an antibody of a mammal, not a person, and FR and C-regions, derived from human antibodies. Humanitariannet antibody may be suitable for clinical use, because the antigenicity of the antibody against the human body is reduced.

A specific example of a hybrid antibody or gumanitarnogo antibodies used in the present invention is an antibody in which CDR occur from mouse monoclonal antibodies 92-13, or an antibody in which CDR occur from mouse monoclonal antibodies 93-22. A method of obtaining such hybrid antibodies and humanized antibodies are described below.

For the klonirovania DNA comprising a nucleotide sequence encoding a V region of mouse monoclonal antibody against FZD10, from hybridomas may be isolated mRNA using reverse transcriptase inhibitor can be synthesized each cDNA in the V-L - and H-chains, as described above. During the synthesis of cDNA can be used oligo-dT primer or other suitable primer, which hybridizes C-area L - or H-chain. For example, can be used, but are not limited to, CH1 (IgG2a) primer having the nucleotide sequence as shown in SEQ ID NO: 3, V-N-chain, and CL1 (Kappa) primer having the nucleotide sequence as shown in SEQ ID NO: 4, for the V region of the L-chain.

Amplification of cDNA as L-and H-chains, can be carried out by PCR (polymerase chain reaction) using a commercially available kit (for example, set GeneRacerTMfrom Invitrogen) or using the known method, including the 5'-RACE (Frohman, M.A. et al., Proc. Natl. Acad. Sci. USA 85, 8998-9002, 1988; Belyavsky, A. et al., Nucleic Acids Res., 17, 2919-32, 1989).

Specific primers for DNA amplification of the V-regions of mouse monoclonal antibodies 92-13 include primers having the nucleotide sequences shown in SEQ ID NO: 5 and 6 for the V region of H chain, and the primers having the nucleotide sequences shown in SEQ ID NO: 7 and 8 for the V region of the L-chain. When using the years of these primers can be amplified DNA encoding a V region of H chain having the amino acid sequence as shown in SEQ ID NO: 13, and DNA encoding the V region of the L-chain having the amino acid sequence as shown in SEQ ID NO: 21. Specific primers for DNA amplification of the V-regions of mouse monoclonal antibodies 93-22 include primers having the nucleotide sequences shown in SEQ ID NO: 53 and 54 for the V region of H chain, and the primers having the nucleotide sequences shown in SEQ ID NO: 55 and 56 for the V region of the L-chain. When using these primers can be amplified DNA encoding the V region of H chain having the amino acid sequence as shown in SEQ ID NO: 29, and DNA encoding the V region of the L-chain having the amino acid sequence as shown in SEQ ID NO: 37.

Then amplificatoare products are subjected to agarose gel electrophoresis in accordance with conventional methods, and the desired DNA fragments are cut out, isolated and are ligated with a DNA vector.

The obtained DNA and the DNA vector can be ligitamate using a known set for ligation to create a recombinant vector. DNA vector can be obtained using a known method: J. Sambrook, et al., "Molecular Cloning", Cold Spring Harbor Laboratory Press, 1989. DNA vector hydrolyzing enzyme(Tami) restriction analysis and nucleotide consequently the efficiency of the desired DNA can be determined by a known method or by using an automatic sequencing machine.

After cloning the DNA fragments encoding the V region of the L - and H-chains of the monoclonal antibodies of the mouse (in the present description further L - or H-chain antibodies can sometimes be referred to as "L - or H-chain mouse to mouse antibodies and L - or H-chain of human to human antibodies, DNA encoding the V region of a mouse, and a DNA encoding the constant region of human antibodies are ligated and Express obtaining hybrid antibodies.

The standard method of obtaining a hybrid antibody includes ligation leader sequence of the mouse and the sequence V-region present in the cloned cDNA sequence, encoding the C region of human antibodies already present in the expression vector for mammalian cells. Alternatively, a leader sequence of the mouse and the sequence V-region present in the cloned cDNA are ligated to the sequence encoding the C region of human antibodies with subsequent legirovaniem in the expression vector for mammalian cells.

The polypeptide comprising the C region of human antibodies can be any H - or L-chain C-region of antibodies, including, for example, C gamma 1, C gamma 2, C gamma 3 or C gamma 4 for N-chains of a human or C lambda or C Kappa L-chain.

For hybrid antibodies first construct the two expression the vector; this expression vector containing DNA encoding the V region of the L-chain of the mouse and the C-region L-chain man, under control element controlling the expression such as a promoter/enhancer, and the expression vector containing DNA encoding the V region of H chain of the mouse and the C region of the H chain of a human, under control element controlling the expression such as a promoter/enhancer. The cells are then-owners, such as mammalian cells (such as COS cell), jointly transformed with these expression vectors, and transformed cells are cultivated in vitro or in vivo for hybrid antibodies: see, for example, patent WO91/16928.

Alternatively, a leader sequence of the mouse, which is present in the cloned cDNA, and DNA encoding the V region of the L-mouse chain and the C region of the L-chain of a person, and a leader sequence of the mouse and the DNA encoding the V region of H chain of the mouse and the C region of the H chain of a human, is administered in a single expression vector (see, for example, patent WO94/11523), and the specified vector is used for transformation of the host cell; then the transformed host is cultivated in vivo or in vitro to produce the desired hybrid antibodies.

The vector for the expression of the H chain hybrid antibodies can be obtained by introducing a cDNA comprising a nucleotide sequence encoding a V region of Ncepi mouse (in the present description hereinafter referred to as "cDNA V-N-chain"), into a suitable expression vector containing the genomic DNA comprising the nucleotide sequence encoding the C region of the H-chain of human antibodies (in the present description hereinafter referred to as "genomic DNA C-N-chain"), or cDNA, encoding the specified region (in the present description hereinafter referred to as "cDNA C-N-chain"). C-region N-chain includes, for example, region C gamma 1, C gamma 2, C gamma 3 or C gamma 4.

Expression vectors having the genomic DNA encoding the C region of the H chain, particularly those that encode region C gamma 1 include, for example, HEF-PMh-g gamma 1 (patent WO92/19759) and DHER - INCREMENT E-RVh-PM1-f (patent WO92/19759). Alternatively, the library of the constant regions of a human can be obtained using cDNA from human PBMC (mononuclear peripheral blood cells), as described previously (Liu, A.Y. et al., Proc. Natl. Acad. Sci. USA, Vol.84, 3439-43, 1987; Reff, M.E. et al., Blood, Vol.83, No.2, 435-45, 1994).

When cDNA encoding V region of H chain of the mouse, is inserted into these expression vectors, the published cDNA may be inserted a suitable nucleotide sequence using PCR. For example, PCR can be carried out using a PCR primer that is created so that this cDNA has at its 5'-end of recognizable sequence for a suitable restriction enzyme and the consensus sequence To the aka immediately before the initiation codon so, to improve the efficiency of transcription and PCR primer that is created so that this cDNA has at its 3'-end of recognizable sequence for a suitable restriction enzyme and the donor site for the proper splicing splicing products of primary transcription of genomic DNA with obtaining mRNA for the introduction of these suitable nucleotide sequences in the expression vector.

The thus created cDNA encoding V region of H chain of the mouse treated with the appropriate(s) enzyme(Tami) restriction, then place it in the specified expression vector to create a hybrid expression vector H-chain containing genomic DNA encoding the C region (region C gamma 1) N-chain.

The thus created cDNA encoding V region of H chain of the mouse treated with the appropriate(s) enzyme(Tami) restriction, are ligated with cDNA that encodes a C-region C gamma 1 N-chain, and insert in the expression vector, such as pQCXIH (Clontech) to create an expression vector containing cDNA encoding a hybrid N-chain.

The vector for the expression of L-chain hybrid antibodies can be obtained by ligating the cDNA encoding V region of the L-chain of mouse genomic DNA or cDNA coding for the C region of the L-chain of human antibodies, and the introduction into a suitable expression vector. C-area L-chain includes, e.g. the measures the Kappa chain and lambda chain.

When creating an expression vector containing cDNA encoding V region of the L-chain of the mouse, the expression vector by PCR can be entered in the appropriate nucleotide sequence, such as a recognizable sequence or a consensus Kozak sequence.

The complete nucleotide sequence of cDNA encoding the C region of the L chain lambda person, can be synthesized using a DNA synthesizer and constructed using the PCR method. It is known that the C-region of the L chain lambda person contains at least 4 different isotype, and each isotype can be used to construct expression vector.

Constructed cDNA encoding the C region of the L chain lambda man, and the above constructed cDNA encoding V region of the L-chain of a mouse, can be legirovanyh between suitable sites of enzymes and inserted into the expression vector, such as pQCXIH (Clontech)to construct the expression vector containing cDNA encoding the L chain lambda hybrid antibodies.

DNA encoding the C region of the L chain Kappa person intended for ligating DNA encoding the V region of the L-chain of a mouse, can be constructed, for example, from HEF-PM1k-gk containing genomic DNA (see WO92/19759). Alternatively, using cDNA from PBMC (Monon learnig peripheral blood cells) may be obtained library constant region of a human, as described previously (Liu, A.Y. et al., Proc. Natl. Acad. Sci. USA, Vol.84, 3439-43, 1987; Reff, M.E. et al., Blood, Vol.83, No.2, 435-45, 1994).

Recognizable suitable enzymes sequences can be introduced by PCR at the 5'- and 3'-ends of the DNA encoding the C region of the L chain Kappa, and DNA encoding the V region of the L-chain of mouse, constructed above, and the DNA encoding the C region of the L chain Kappa, may be Legerova with each of them and inserted in the expression vector, such as pQCXIH (Clontech)to create an expression vector containing cDNA encoding the L chain Kappa hybrid antibodies.

To obtain gumanitarnogo antibody in which the CDR of mouse monoclonal antibodies connected with human antibody, preferably having high homology between FR mouse monoclonal antibodies and FR human antibodies. Accordingly produces a mapping between V regions of H and L-chains of mouse monoclonal antibodies against FZD10 and V-regions of all known antibodies, the structure of which was elucidated using Protein Data Bank. In addition, they are compared simultaneously with the sub-human antibodies (HSG: Human subgroup), which are classified according to Kabat et al. on the basis of length FR of the antibody amino acid homology and the like: Kabat, E.A. et al., US Dep, Health and Human Services, US Government Printing Offices, 1991.

The first stage of creating a DNA that encodes a V-region gumanitarnogo antibodies, is to select the V-region is t human antibodies as the basis for design. For example, to get gumanitarnogo antibodies can be used FR V-region of human antibodies with the homology of more than 80% with FR V region antibody mouse.

In humanitariannet the antibody C-region and framework (FR) region V-region of the specified antibodies originate from a person, and region complementarity determining (CDR) V-region, come from the mouse. Polypeptide comprising V region gumanitarnogo antibodies can be obtained by a method called grafting CDR, using the PCR method, subject to the availability of a DNA fragment of human antibodies as a matrix. The term "CDR grafting" refers to a method in which get a DNA fragment that encodes a CDR of a mouse, and replace them with CDR human antibodies as a matrix.

If the DNA fragment of the human antibodies to be used as a matrix, not available, can be synthesized on a DNA synthesizer nucleotide sequence available in the database, and using the PCR method can be derived DNA V-region gumanitarnogo antibodies. In addition, when the database contains only the amino acid sequence of the full nucleotide sequence can be deduced from amino acid sequences on the basis of knowledge about the use of codons in antibodies, as reported Kabat, E.A. et al. US Dep. Health and Human Services, US Government Printing Offices, 1991. Nucleate the percent sequence synthesized on a DNA synthesizer, and DNA V-region gumanitarnogo antibodies can be obtained by PCR and introduced into a suitable host, with subsequent expression to obtain the desired polypeptide.

General procedures CDR grafting using the PCR method described below for the case when there is a DNA fragment of human antibodies as a matrix.

Primarily synthesize DNA fragments corresponding interest CDR. CDR 1 to 3 are synthesized based on the nucleotide sequences of the previously cloned V-regions of H and L-chains of a mouse. For example, when humanitariannet antibody are derived from mouse monoclonal antibodies 92-13, the CDR sequence of the V region of H chain can be an amino acid sequence represented in SEQ ID NO: 15 (VH CDR1), 17 (VH CDR2) and 19 (VH CDR3); and a CDR sequence of the V region of the L-chain can be an amino acid sequence represented in SEQ ID NO: 23 (VL CDR1), 25 (VL CDR2) and 27 (VL CDR3). When humanitariannet antibody are derived from mouse monoclonal antibodies 93-22, the CDR sequence of the V region of H chain can be an amino acid sequence represented in SEQ ID NO: 31 (VH CDR1), 33 (VH CDR2) and 35 (VH CDR3); and a CDR sequence of the V region of the L-chain can be an amino acid sequence represented in SEQ ID NO: 39 (VL CDR1), 41 (VL CDR2) and 43 (VL CDR3).

DNA V-area and H-chain gumanitarnogo antibodies can be Legerova with the DNA of any C-N-chain of human antibodies for example, region C gamma 1 N-chain man. As mentioned above, DNA V-N chain may be treated with a suitable restriction enzyme and Legerova with DNA encoding the C region of the H chain of a human, the expression of which is controlled by the controlling element such as an enhancer/promoter system for obtaining expression vector containing the DNA of the humanized V region of H chain and the C region of the H chain of a human.

DNA V-region L-chain gumanitarnogo antibodies can be Legerova with the DNA of any C-region L-chains of human antibodies, for example, region C lambda L-chain man. DNA V region of the L chain may be treated with a suitable restriction enzyme and Legerova with DNA encoding the C region of the L-chain lambda man, whose expression is controlled by regulatory element such as an enhancer/promoter system for obtaining expression vector containing the DNA of the humanized V region of the L-chain and the C region of the L-chain lambda man.

DNA encoding the V region of H chain gumanitarnogo antibodies and C-N-chain and DNA encoding humanitarian V region of the L-chain and the C region of the L chain may be introduced into a single expression vector, such as disclosed in the patent WO94/11523, and the specified vector can be used to transform a host cell, the transformed host mo which should be cultivated in vivo or in vitro with the desired gumanitarnogo antibodies.

For hybrid or gumanitarnogo antibodies should get the two above-mentioned expression vector. So, in the case of the hybrid antibody construct expression vector comprising DNA encoding the V region of H chain of the mouse and the C region of the H chain of human-driven controlling element in the expression, such as an enhancer/promoter, and expression vector comprising DNA encoding the V region of the L-mouse chain and the C region of the L-chain of human-driven controlling element in the expression. If gumanitarnogo antibody construct expression vector comprising DNA encoding humanitarian V region of H chain and the C region of the H-chain of human-driven controlling element in the expression, and expression vector comprising DNA encoding humanitarian V region of the L-chain and the C region of the L-chain of human-driven controlling element in the expression.

After that, the cell host, such as a cell of a mammal (for example, a COS cell), jointly transform these expression vectors, and the resulting transformed cell can be cultivated in vitro or in vivo with getting a hybrid or gumanitarnogo antibodies (see, for example, patent WO91/16928).

Alternatively, DNA encoding the V and C-N-chain and DNA encoding the V and C-region L-chain can be legirovanyh in a single victory transformed into a suitable cell host with obtaining antibodies. Thus, when the expression of the hybrid antibody DNA encoding a leader sequence of the mouse in the cloned cDNA, V region of H chain of the mouse and the C region of the H chain of a human, as well as DNA encoding a leader sequence of the mouse V region of the L-chain of the mouse and the C-region L-chains may be introduced into a single expression vector, such as, for example, disclosed in patent WO94/11523. In the case of expression gumanitarnogo antibodies DNA encoding humanitarian V region of H chain and the C region of the H chain of a human, and DNA encoding humanitarian V region of the L-chain and the C region of the L chain may be introduced into a single expression vector, such as, for example, disclosed in patent WO94/11523. This vector is used for transformation of the host cell, and the transformed cell is cultivated in vivo or in vitro to obtain a hybrid or gumanitarnogo antibodies.

For hybrid or gumanitarnogo antibody against FZD10 protein of the present invention can be used in any expression system. For example, eukaryotic cells include animal cells, such as stable lines of mammalian cells, fungi cells and yeast cells; prokaryotic cells include bacterial cells such as Escherichia coli. Preferably, hybrid or humanitariannet antibody to the present is the invention Express in the cell of a mammal, such as cell COS or CHO.

Can be used any traditional promoters suitable for expression in mammalian cells. For example, preferably using the immediate early promoter of human cytomegalovirus (HCMV). In addition, the promoters for gene expression in mammalian cells may include viral promoters such as the promoter of retrovirus, virus polyoma, adenovirus and simian virus (SV) 40, and the promoters from mammalian cells, such as the promoter factor-1-alpha elongation of polypeptide chain man (HEF-1 alpha). For example, the SV40 promoter can easily be used according to the method of Mulligan et al. (Nature, 277, 108-14, 1979); the method of Mizushima, S. et al. (Nucleic Acids Research, 18, 5322, 1990) can be easily used with the promoter HEF-1 alpha.

The origin of replication includes originy replication of the SV40 virus, polyoma, adenovirus and human papilloma virus in cattle (BPV). In addition, the expression vector can include the gene phosphotransferase (APH(3') II or I (neo), timedancing (TK), xanthine-guaninephosphoribosyltransferase E. coli (Ecogpt) or digidrofolatreduktazy (DHFR) as a marker for selection to increase the number of copies of the gene in the system host cell.

Hybrid or humanitariannet antibody of interest, which is obtained thus by culturing transformant transformed DNA, coderush the th hybrid or humanitariannet antibody can be isolated from cells and then purified.

Isolation and purification of hybrid or gumanitarnogo antibodies of interest can be carried out using a column of protein A-agarose, but can also be produced using any of the methods used for isolation and purification of the protein, and thus are not limiting. For example, for isolation and purification of the hybrid or gumanitarnogo antibodies can be optionally selected or used in conjunction chromatography, ultrafiltration, salting out or dialysis.

After separation of the hybrid antibodies or gumanitarnogo antibody concentration of purified antibodies can be determined using ELISA.

Definition antigennegative activity or other activity types hybrid antibodies or gumanitarnogo antibodies, including binding activity against normal cells, can be produced using any known method (Antibodies A Laboratory Manual, Ed. Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).

As for the method of determining antigennegative activity of the antibody can be used methods such as ELISA (enzyme-linked immunosorbent assay), ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay analysis) or fluorescence analysis.

(3) a fragment of the antibody and the modified ant the body

The antibody used in the present invention may be a fragment of an antibody or a modified antibody, until it binds FZD10 protein and inhibits its activity. For example, the antibody fragment include Fab, F(ab')2, Fv or single-chain Fv (scFv), consisting of an Fv fragment N-chain or fragment Fv L-chain, linked by a suitable linker. Specifically, these fragments of antibodies can be obtained by cleavage of the antibody to an enzyme (e.g., papain, pepsin) to fragments of antibodies or by constructing a gene encoding the antibody fragment and insertion of the gene in the expression vector, and introducing the obtained recombinant expression vector in a suitable cell host, expressing thus the fragment antibodies (see, for example, Co, M. S., et al., J. Immunol. (1994), 152, 2968-76; Better, M. &Horwitz, A.H., Methods in Enzymology (1989), 178, 476-96, Academic Press, Inc.; Pluckthun, A. &Skerra, A., Methods in Enzymology (1989) 178, 497-515, Academic Press, Inc.; Lamoyi, E., Methods in Enzymology (1989) 121, 652-63; Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-9; and Bird, R. E. et al., Trends Biotechnol. (1991) 9, 132-7). Alternatively, it may be constructed expression libraries Fab (Huse et al., 1989, Science, 246: 1275-81) to allow quick and easy identification of monoclonal Fab fragments with the desired specificity.

scFv can be obtained by legirovaniem V-region of the H-chain V-region L-chain via a linker, n is edocfile peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-83). V region of H chain and the V region of the L-chain scFv can occur from any of the described in the present description of the antibodies. A peptide linker linking the V-region, can be any single-stranded peptide, for example, consisting of 12 to 19 amino acid residues.

As modified antibodies can also be used, for example, antibody against FZD10 or its fragment, conjugated to any molecule (e.g., polyethylene glycol). Such modified antibodies are also covered by the term "antibody" of the present invention. The modified antibodies can be obtained by chemical modification of the antibody. Methods of chemical modification, are suitable for this purpose have already been installed in this area of technology.

2. Therapeutic use

Below are methods and pharmaceutical compositions for the treatment and/or prevention associated with FZD10 diseases using the antibodies of the present invention. The result of treatment is at least a positive effect on the treated subject, which in the case of tumors includes, but is not limited to, the remission of tumors, symptomatic relief of tumors and control of metastatic spread of tumors.

Specifically, the method of treatment and/or prevention associated with FZD10 for the of Alemania the subject of the present invention includes an introduction to the needy in the subject the antibody or fragment, above.

The term "subject" in the present description refers to a person who is suffering associated with FZD10 disease, as well as to the subject, presumably having associated with FZD10 disease. The subject of the present invention may be an animal, including mammals and birds. For example, mammals can include humans, mice, rats, monkeys, rabbits and dogs.

The term "associated with FZD10 disease" in the present description refers to a disease associated with excessive expression of FZD10 protein. Specifically associated with FZD10 diseases include, but are not limited to, synovial sarcoma (SS), colorectal cancer, gastric cancer, chronic myeloid leukemia (CML) and acute myeloid leukemia (AML).

Described in the present description, the antibody or its fragment may specifically bind to a protein FZD10, so with the introduction of the antibody or its fragment to the subject it is associated with FZD10 protein from the subject, and FZD10 protein activity can be suppressed. Alternatively, when the antibody or its fragment can be conjugated to a therapeutic part and introduced the subject, it is delivered in the region, which expresses FZD10 protein (i.e. suffering region) of the subject, and therapeutic part can be selectively delivered to the affected area and work on it. Such therapeutic part of the mod is no represent any known drug or agent, which will be developed in the future, have a curative effect on associated with FZD10 disease and includes, without limitation, radioisotope label and chemotherapeutic agent. Radioisotope label, which can be used as a therapeutic agent, may be selected depending on many factors, including energy β-radiation and the efficiency of its emission, the presence or absence of γ-radiation, its energy and the efficiency of emission, the physical half-life and procedures prokachivanija. Usually can be used radioisotope label yttrium (such as90Y) and iodine (such as125I and131I). The chemotherapeutic agent may be any known agent or the agent that will be created in the future, for the treatment associated with FZD10 disease and include, but not be limited to, methotrexate, Taxol, mercaptopurine, tioguanin, cisplatin, carboplatin, mitomycin, bleomycin, doxorubicin, idarubitsin, daunorubicin, dactinomycin, vinblastine, vincristine, vinorelbine, paclitaxel and docetaxel. Described in the present description, the antibody or its fragment may be selectively contact the FZD10 protein and not to contact with normal cells, causing side effects, which is caused by the antibody or its fragment or a radioisotope and is and chemotherapeutic agent can be effectively neutralized, and therefore, therapeutic efficacy can be high.

The antibody or fragment described in the present description, can be administered to the subject in an effective dose to treat or prevent associated with FZD10 disease. Effective dose refers to an amount of the antibody or its fragment, which is sufficient to provide beneficial effects in exposed treatment of the subject. The compositions and methods of administration that can be used in cases where the pharmaceutical composition comprises the antibody of the present invention, is described below.

Pharmaceutical compositions for use according to the present invention can be prepared in the traditional way using one or more pharmaceutically acceptable carriers or excipients.

Antibodies or fragments thereof may be formulated for parenteral administration (i.e. intravenous or intramuscular), by injection, for example bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage forms, such as ampoules or containers with multiple doses, with added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous carriers, and can sod is neigh for education to form agents, such as suspendida, stabilizing and/or dispersing agents. Alternatively, the antibody may be in the form of liofilizirovannogo powder for connection with a suitable vehicle, e.g. sterile, free of pyrogens water, before use.

Toxicity and therapeutic efficacy of the antibody or fragment or therapeutic portion attached to them, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The ratio of doses that cause toxic and therapeutic effects is a therapeutic index and can be expressed as the ratio LD/ED.

Preferred antibodies or therapeutic part exhibiting high therapeutic indices. In case of using the antibodies or portions exhibiting toxic side effects, it is necessary to provide a delivery system that directs such antibodies or part of a section of damaged tissue to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained in the analysis of cell cultures and in animal studies, you can use the formation ranges of dosages for use in humans. The dose of such antibodies preferably is in the range of concentrations in the plasma of the circulating blood, including ED50 with low or no toxicity. The dosage may vary within this range depending on the shape of the dose, the route of administration and the type and quantity of conjugated therapeutic part. For any of the antibodies used in the method of the invention, the effective dose can be established initially on the basis of tests in cell cultures. The dose can be determined in animal models to provide the range of concentrations in the plasma of the circulating blood, including IC50 (i.e. the concentration of test antibody, which provides premaxillae inhibition of symptoms)as defined in cell culture. This information can be used for more accurate determination of the dose that is suitable for a person. Levels in plasma may be measured, for example, by high performance liquid chromatography.

Depending on the condition and age of the subject and/or route of administration specialist in the art can select a suitable dose of the pharmaceutical composition of the present invention. For example, the pharmaceutical composition of the present invention is administered in such an amount that the antibody of the present invention is introduced is objektu in the amount of from about 3 to about 15 μg per kg of body weight of the subject, and preferably from about 10 to about 15 μg per kg of body weight of the subject. The interval between the introduction and the time of its introduction can be selected in accordance with the condition and age of the subject, route of administration and response to the pharmaceutical composition. For example, the pharmaceutical composition can enter the subject of from one to 5 times, preferably 1 time a day for 5 to 10 days.

The pharmaceutical composition can be entered systemically or topically. Preferably its introduction way directed delivery for delivery of the active component in the area of corruption.

In some cases, the implementation of the methods and compositions of the present invention is used for treatment or prevention associated with FZD10 disease together with a chemotherapeutic agent or combination of chemotherapeutic agents, including, but not limited to these, methotrexate, Taxol, mercaptopurine, tioguanin, cisplatin, carboplatin, mitomycin, bleomycin, doxorubicin, idarubitsin, daunorubicin, dactinomycin, vinblastine, vincristine, vinorelbine, paclitaxel and docetaxel.

As for therapy, it can be used by any Protocol therapy depending on the type being treated is associated with FZD10 disease. For example, but not limitation, can be used x-ray irradiation. For exposure of tissues can also be used emitting ha is mA-radiation radioisotopes, such as radioactive isotopes of radium, cobalt and other elements.

In another embodiment, the chemotherapy or radiotherapy is used for at least one hour, five hours, 12 hours, day, week, month or more, preferably several months (e.g., up to three months) after using the methods and compositions containing the antibody of the present invention. Chemotherapy or radiotherapy used to, simultaneously with or after treatment using the methods and compositions of the present invention, can be used with any known in the art method.

3. Diagnostic and prognostic use

Antibody against FZD10 protein or its fragments can also be used as diagnostic and prognostic agents, as described in the present description. Such diagnostic methods can be used to detect the presence or absence of associated with FZD10 disease and the risk of developing such disease. A method for diagnosing and/or predicting the present invention associated with FZD10 disease includes immunologically detecting or identifying in the sample FZD10 protein, resulting from disease using the antibody or its fragment according to the present invention. Specifically, a method for diagnosing and/or before the punishment associated with FZD10 disease or predisposition of a subject to this disease according to the present invention includes:

(a) introducing the contact of the sample from the subject with an antibody against FZD10 protein or its fragment;

(b) identification of FZD10 protein in the sample; and

(c) deciding, suffer if the subject against the disease or risk of disease, on the basis of the relative excess FZD10 protein compared to control.

A method for diagnosing and/or predicting the present invention can be implemented on the basis of any of the procedures, as they represent the analysis using antibodies, i.e. immunological analysis. Thus, FZD10 protein can be determined using the antibody or its fragment according to the present invention as the antibodies used for analysis. For example, FZD10 protein may be detected using immunohistochemical staining, immunoassays such as enzyme-linked immunosorbent assay (ELISA and ELISA), immunofluorescent analysis, radioimmunological assay (RIA) or Western blotting.

The sample to be tested by the method of diagnosis and/or prediction associated with FZD10 diseases according to the present invention has no particular restrictions as long as he is a biological sample that may contain FZD10 protein, resulting associated with FZD10 disease. Examples of the sample include an extract of cells or organ and the tissue section, and the blood, with which the crank, the plasma supernatant of cultured lymphocytes, urine, cerebrospinal fluid, saliva, sweat and ascites. Excess FZD10 protein detected in these samples, as tumor tissue, a biopsy of the tumor and the tissue metastasis, using the antibody or its fragment according to the present invention may be particularly suitable as indicator associated with FZD10 disease.

For example, antibodies or fragments thereof described in the present description, can be used for quantitative or qualitative detection of FZD10 protein. The antibodies (or fragments thereof) of the present invention, furthermore, can be used for histology, for example, immunofluorescence or immunoelectron microscopy, for the detection of FZD10 protein in situ. The in situ detection may be accomplished by selection of histological sample from the subject, such as prisoners in the paraffin-embedded tissue sections (such as samples obtained during surgical operations), and application of labeled antibodies of the present invention. The antibody (or fragment) is preferably applied by coating the sample labeled antibody (or fragment). Specialists in the art should be quite clear that when using the present invention to achieve such in situ detection of any of a variety of histological methods (such as staining procedure) m which can be modified.

Immunological analyses FZD10 protein usually include incubation explored sample from the subject, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells incubated in cell culture, in the presence of the antibodies of the present invention, labeled detectable label, and detecting bound antibody by any of a number of ways, well known to specialists in this field of technology.

The sample can be introduced into contact with and immobilized on a solid phase substrate or carrier such as nitrocellulose, or other solid substrate, which is capable of immobilizative cells, cell particles or soluble proteins. The substrate can then be washed with suitable buffers and then process labeled detectable label, antibody against FZD10. Solid-phase substrate can then be rinsed with buffer a second time to remove unbound antibody. Then you can determine with traditional means, the amount of bound label on the solid substrate.

The term "solid-phase substrate or carrier" means any substrate capable of binding the antigen or antibody. Specialists in the art should be aware of many suitable carriers for binding of antibodies or antigens, or specialists must be able to identify such media p is the traditional experimentation.

Binding activity of this lot of antibody against FZD10 can be determined using well known methods. Experts in the art should be able to define work and optimal conditions of analysis for each definition using traditional experimentation.

The interaction between the antibody (or fragment) of the present invention and FZD10 protein, easily obtained in the sample from the area affected by associated with FZD10 disease, can be detected directly by prokachivanija antibodies of the present invention or indirect by prokachivanija secondary antibodies. The last procedure of indirect detection, such as a sandwich analysis or competitive ELISA, preferably used in the method according to the present invention with improved sensitivity.

Examples of labels for use in the present description are as follows. In immunoassay analysis can be used peroxidase (POD), alkaline phosphatase, β-galactosidase, urease, catalase, glucose oxidase, lactate dehydrogenase, amylase and complexes of avidin-Biotin. In immunofluorescence analysis, you can use the fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), substituted rhodamine isothiocyanate, dichlorotriazines isothiocyanate and Alexa488. In radioimmunological analysis can what about the use of tritium, iodine (such as125I and131I) and indium (such as111In). In immunosenescence analysis you can use the test NADH-FMN2luciferase system luminal-peroxide-POD, esters of acridine and dioxetane connection.

The label can be attached to the antibody using conventional procedures. For example, the label can be attached to the antibody using glutaraldehyde way maleimide way pyridyldithio way or periodate way in immunoassay analysis and method of chloramine-T or method Bolton-hunter in radioimmunological analysis.

The analysis can be carried out in accordance with known procedure (Ausubel, F.M. et al. Eds., Short Protocols in Molecular Biology, Chapter 11 "Immunology" John Wiley & Sons, Inc. 1995).

For example, when the antibody of the present invention directly mark above the label, the sample is introduced into contact with the labeled antibody with the formation of the complex between the protein and FZD10 antibody. Then separated from unbound labeled antibody, and the FZD10 protein level in the sample can be determined on the basis of the amount of bound labeled antibody or the amount of unbound labeled antibodies.

When using a labeled secondary antibody to the antibody of the present invention allow to interact with the sample in the first reaction, and the resulting complex give browseimages who participate with labeled secondary antibody in the second reaction. The first reaction and the second reaction may be performed in reverse order, simultaneously, or after a certain period of time between them. The first and second reaction give the complex [FZD10 protein]-[antibody according to the invention]-[labeled secondary antibody] or complex [antibody according to the invention]-[FZD10 protein]-[labeled secondary antibody]. Unbound labeled secondary antibody is then separated, and the FZD10 protein level in the sample can be determined on the basis of the amount of bound labeled secondary antibody or the amount of unbound labeled secondary antibodies.

According to another variant implementation of the antibody of the present invention have been labelled with a radioisotope or fluorescent label, and the labeled antibody is administered parenteral subject. Thus using a non-invasive method can be quickly determined the localization of the primary tumor and the associated metastases tumors associated with FZD10 disease. This method of diagnosis is known as the visualization of a tumor in vivo, and specialist in the art can easily understand its procedures. Labeled antibody can be entered to the subject systemically or topically, preferably by parenteral, such as intravenous injection, intramuscular injection, intraperitoneal injection or subcutaneous injection.

Antibodies of the present invention specific EOI is modestou with FZD10 protein, as mentioned above, and can because of this be used in sets for the diagnosis and/or prediction associated with FZD10 disease.

A kit for the diagnosis and/or prediction of the present invention includes an antibody or fragment described above. By determining the FZD10 protein in the sample from the subject with suspicion associated with FZD10 disease with the use of the kit for diagnosing and/or predicting the present invention can quickly and easily identify suffer if the subject associated with FZD10 disease. Kits for diagnosis and/or prognosis of diseases by the use of such immunological reactions are widely known, and expert in the art can readily select appropriate components other than antibodies. Kits for diagnosing and/or predicting the present invention can be used in any way until they are methods of immunological analysis.

EXAMPLES

The present invention will be further illustrated by the following non-limiting examples.

Cell lines and tissue samples used in the following examples were obtained as described below. Specifically, cell line, originating from the synovial sarcoma (HS-SY-2, YaFuSS, 1973/99, Fuji and SYO-1), cancers of the colon (LoVo, SNU-C4 and SNU-C5), cells HEK93 and COS7 were grown as monolayers in appropriate media with addition of 10% fetal calf serum and 1% solution of the antibiotic/antifungal agent and maintained at 37°C in an atmosphere of air, containing 5% CO2. Samples of primary synovial sarcoma (SS) were obtained after informed consent, immediately frozen in liquid nitrogen immediately after resection and stored at -80°C.

Example 1

Obtaining a monoclonal antibody against FZD10

(1) preparation of monoclonal antibodies using cell immobilization

Mouse monoclonal antibodies against FZD10 (Mab) was obtained by immunization with a four-week female mice of Balb/c pads 2×107cells COS-7, transfected with 2×107pCAGGS/neo-FZD10-myc/His (Medical and Biological Laboratories, Nagoya, Japan). Construction of pCAGGS/neo-FZD10-myc/His was described previously (Nagayama, S., et al. (2005). Oncogene, 24, 6201-12), and it expresses a complete sequence, encoding cDNA and FZD10 Myc and tag epitope His at the C-end. Mice were immunized complete adjuvant's adjuvant (Mitsubishi Depending Iatron, Inc., Tokyo, Japan) one day prior to immunization with cells. From immunized mice received cells of the spleen, which was merged with cells of the myeloma line. Hybridoma was subclinically and tested using cell ELISA for the ability to secrete immunoglobulin that binds to the extracellular domain FZD10 (amino acid residues 1-225 FZD10). For cell ELISA cells COS-7 expressing FZD10-myc/His (full cDNA sequence that encodes a FZD10 and Myc and His epitope tag at its C-end), were sown in 96-well plates. ZAT is m in the plate was added 50 μl of culture supernatants, obtained from hybridomas, and incubated for 30 minutes at room temperature. After washing, cells were added to goat IgG-POD against mouse (Medical and Biological Laboratories, Nagoya, Japan) at a dilution of 1:10000, incubated for 30 minutes at room temperature. Bound antibodies were identified by OD450-620nm. Positive clones were additionally analyzed for specific binding activity. These clones include clones 39-2 and 39-10 (disclosed in the patent WO2005/004912 designated as 5F2)and 92-13 and 93-22. All Mab were of the IgG2a isotype determined using the IsoStrip kit for ittipiboon monoclonal mouse antibodies (Roche). Mab was subjected to affinity purification on protein G-sepharose for additional features.

Hybridoma clone 93-22 producing monoclonal antibody mouse 93-22, was put on the international storage Shuichi Nakatsuru in IPOD International Patent Organism Depository of the National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-Ken, 305-8566 Japan) on June 14, 2006 with registration number FERM BP-10620. Also hybridoma clone 92-13 producing monoclonal antibody mouse 92-13, was put on the international storage Shuichi Nakatsuru in IPOD International Patent Organism Depository of the National Institute of AIST, June 28, 2006 with registration number FERM BP-10628.

(2) Prokachivanie antibodies with radionuclides

Labeled125I Mab was obtained by way of chloramine T (Arano, Y., et al. (1999). Cancer Res, 59, 128-34). BK/2 µl of Na 125I was added to 10 μg of Mab in 100 ál of 0.3 M sodium phosphate buffer. Then add one μg of chloramine T in 3 ál of 0.3 M sodium phosphate buffer, incubated for 5 min at room temperature. Labeled antibody was purified using column 6 Biospin (Bio-Rad).

For prokachivanija Mab111In 1 mg of Mab in 100 μl of 50 mm borate buffer (pH 8.5) conjugatively with isothiocyanateconjugated acid (SCN-BZ-DTPA; Macrocyclics) in dimethylformamide in a molar ratio of 1:3. After incubation at 37°C for 20 hours conjugates Mab was purified using column 6 Biospin. 40 ál111In incubated in 60 μl of 0.25 M acetic acid buffer (pH 5.5) and included 10 μg/ml of conjugates of Mab-DTPA for one hour at room temperature. Labeled antibody was purified using column 6 Biospin.

To obtain 92-13, conjugated with90Y, 92-13 conjugatively with DTPA on lysine residues. DTPA-92-13 was labelled with yttrium to a specific activity of 100 µci/mg, and the immunoreactivity90Y-DTPA-92-13 was approximately 70%.

(3) Synthesis of Mab labeled with Alexa647

Prokachivanie Mab Alexa-Fluoro647 produced according to the manufacturer's instructions for using the kit prokachivanija Alexa647 monoclonal antibodies (Molecular Probes, Eugene, Oregon). Reactive dye Alexa647 contains Succinimidyl ester part, which interacts with the primary amines of proteins, and polucen the e conjugates Mab-dye was purified via column with the exception of size.

Example 2

Binding activity of monoclonal antibody against FZD10

The authors of the present invention used two methods to estimate the binding affinity of the monoclonal antibodies of the mouse; running cytometrics analysis with fluorescent dyes and measurement of radioactivity using125I.

(1) Flowing cytometrics (FACS) analysis

To study the affinity of binding to cell four antibodies, 39-2 and 39-10 (disclosed in patent application WO2005/004912), 92-13 and 93-22 applicants have conducted experiments with flow cytometry (FACS). For flow cytometrical analysis with indirect fluorescence suspension of 5×106cells were incubated with 10 μg/ml Mab or IgG non-immune mouse (Beckman Coulter) for 30 min at 4°C. After washing, PBS was added to 2 μg of fluorescent IgG goat against mouse (Alexa Fluor 488, Molecular Probes, Eugene, Oregon), and the cell suspension was incubated for 30 min at 4°C for analysis using a FACScan (Becton Dickinson, Franklin Lakes, NJ). For direct immunofluorescence analysis cells were incubated with 2 µg Alexa488-Mab in the presence or in the absence of excess (100 µg) unlabeled Mab for 30 min at 4°C and were analyzed using FACScan.

To confirm the expression of FZD10 in cell lines, the authors conducted RT-PCR. For experiments RT-PCR from cell lines was extracted total RNA using TRIzol reagent (nvitrogen, Carlsbad, CA, USA), and 3-µl aliquot of each total RNA was subjected to reverse transcription. Amplification by PCR was performed using cDNA as a matrix with the following primers: 5'-TATCGGGCTCTTCTCTGTGC-3' (SEQ ID NO: 9) and 5'-GACTGGGCAGGGATCTCATA-3' (SEQ ID NO: 10) for FZD10 and 5'-TTAGCTGTGCTCGCGCTACT-3' (SEQ ID NO: 11) and 5'-TCACATGGTTCACACGGCAG-3' (SEQ ID NO: 12) for β2-microglobulin (β2MG), internal control.

As shown in Fig. 1a, all four Mab, 39-2, 39-10, 92-13 and 93-22 contact with four expressing FZD10 SS cell lines, SYO-1, YafuSS, HS-SY-2, and Fuji, dependent on dose FZD10, but not in contact with the two cell lines, 1973/99 and LoVo, in which the transcript FZD10 was not identified. In table 1, below, shows the correlation between average relative fluorescence intensity (MFI) of these Mab and the expression of FZD10, shown in Fig. 1b. In addition, the applicants, in particular, showed that Mab 92-13 and 93-22 contact the SNU-C5, transfitsirovannykh construct FZD10-myc/His, whereas binding was not detected when using cells SNU-C5, transfected with the empty vector (Fig. 1c), suggesting specific binding of these Mab 92-13 and 93-22 against FZD10 protein.

Table 1
Binding of Mab against FZD10 with SS lines human cell SYO-1, YaFuSS, HS-SY-II, Fuji, 1973/99 and cell line cancer of docnoj intestine of man, LoVo
SYO-1YaFussHS-SY-IIFuji1973/99LoVo
39-217,6the 11.69,47,15,51,1
39-1018,411,89,96,9a 4.91,0
92-13the 4.73,03,01,30,91,0
93-223,32,72,41,11,01,1
MFI FZD10 measure flow cytometry as described above

(2) Binding activity with respect to the normal blood cells

To confirm that these antibodies can be used draconically goals, the authors present invention additionally tested binding activity of antibodies against normal blood cells. To assess nonspecific binding activity of Mab against normal blood cells labeled125I Mab were incubated with 100 µl of fresh blood of a healthy donor. After incubation for one hour at room temperature were measured radioactivity cellular precipitate, as described above.

Binding activity of labeled125I Mab 92-13 and 93-22 in respect of normal blood cells were not detected in all three individual donors, whereas the activity of Mab 39-2 and 39-10 were detected in all three individual donors (Fig. 1d). These results are consistent with the results of FACS analysis using the mononuclear cells of peripheral blood (data not shown), suggesting clinically useful only antibodies 92-13 and 93-22 with a low probability of adverse effects on patients with SS due to the very specific binding affinity to a molecule FZD10. Therefore, applicants have focused only on the antibody 92-13 and 93-22 for further analysis.

(3) Additional tests

In addition, the checked binding was performed using labeled125I Mab (see example 1 (2)) to estimate binding affinity towards molecules FZD10 on the cell surface. Disradioactive analysis obtained in example 1 (2) of 0.5 BCF (to 0.001 μg antibody) labeled with 125I Mab was added to 100 ál of cell suspension with different quantities of identical unlabeled Mab. After incubation for one hour at room temperature, the cell suspension was centrifuged at 800×g. The supernatant was removed, and measured the radioactivity of the cell precipitate.

The results showed a higher binding affinity antibodies 92-13 compared with the antibody 93-22; under the same conditions with cells contacted approximately 33% 92-13 and approximately 9% of the antibodies 93-22 (Fig. 1e). The amount of bound antibodies was decreased by adding unlabeled antibody dependent dose-dependent manner.

The authors then conducted a competitive binding analysis of Mab 92-13 and 93-22 using flow cytometry. Linking cells of both labeled with Alexa488 antibodies completely blocked by a large quantity of unlabeled antibody (Fig. 1f, ii and iii) adding to each of them, suggesting that Mab 92-13 and 93-22 probably learn very similar or the same epitope FZD10. These results suggest that these Mab can specifically learn FZD10 expressed on the surface of SS cells.

Example 3

Immunohistochemistry

To assess the binding specificity 92-13 and 93-22 with human tissues, the authors conducted immunohistochemical analysis using frozen tissue sections. Tissue with the drive frozen normal organs of an adult (BioChain, Hayward, California) were fixed in 4% paraformaldehyde at 4°C for 15 min, and incubated with 5 μg/ml Mab for one hour at room temperature. Then added mouse ENVISION Polymer Reagent (DAKO) and visualized by peroxidase substrate (3,3'-diaminobenzidine tetrahydrochloride).

The results are shown in Fig. 2. In Fig. 2 shows immunohistochemical analysis in SS and normal frozen slices of tissue of a healthy person without antibodies (a, d, g, j and m), with 92-13 (b, e, h, k and n) and 93-22 (c, f, i, l and o). (a-c), synovial sarcoma; (d-f), kidney (g-i), liver, (j-l)heart; (m-o), the brain. As expected, the authors observed a strong immune response against FZD10 in sample SS (Fig. 2, a, b and c) and the placenta (data not shown), but did not find it in the normal kidney, heart, brain and liver (Fig. 2, d-o), which corresponds to the experimental results of Northern blot and RT-PCR (Nagayama, S., et al. (2005). Oncogene, 24, 6201-6212).

Example 4

The biodistribution Mab against FZD10 in xenograft models in mice Balb/c

Distribution 92-13 and 93-22 model in vivo was analyzed in BALB/c mice using two independent methods, radionuclide imaging and fluorescent imaging.

(1) Radionuclide imaging in vivo

The in vivo experiments was carried out in vivarium in accordance with the approved rules. Mice BALB/cA Jcl-nu (females aged 7 weeks) were injected with subcutaneous (s/C) tumor cells SYO- (5×10 6cells) in 0.1 ml PBS on the sides. To study bearsdley mice with fully developed tumors were injected 10 kBq (0.5-1 ág) labeled with125I Mab and 10 kBq (0.5-1 ág) labeled with111In Mab via the tail vein. After 1, 24, 48 hours, animals were killed, and measured the mass and radioactivity of the tissues. The distribution for all samples was expressed as % of injected dose/g of tissue. For optical imaging of bearsdley except mice with tumor SYO-1 used mice bearing LoVo tumor. Tumor cells LoVo (1×107cells) were injected with p/C mice BALB/cA Jcl-nu, as described above. After full development of tumors, mice were subjected to imaging research.

The results in Fig. 3a show that the radioactivity111In-92-13, associated with blood, fell from 35% of the injected dose per gram (%ID/g) one hour after injection up to 12% after 48 hours. Radioactivity111In-92-13-related liver, kidney, intestine, spleen, pancreas, lungs, heart, stomach and muscle, remained without significant changes or decreased in the process of observation (Fig. 3a). Radioactivity111In-92-13, associated with a tumor, and accumulated in the course of the experiment with 2%ID/g after one hour after injection to 11%ID/g at 48 hours. On the other hand, in Fig. 3b shows that the radioactivity of the labeled125I 92-13 associated with the tumor, the e increased significantly, although the radioactivity associated with the blood, decreased from 25% after one hour up to 7% after 48 hours and the radioactivity associated with other normal organs, remained constant. Perhaps labeled125I antibodies are degraded inside the cells after internalization. Labeled111In 93-22 also accumulated in the tumor SYO-1 at 48 hours after injection (Fig. 3c), and labeled125I 93-22 showed poor accumulation (Fig. 3d), suggesting internalization, as 92-13.

(2) Fluorescence imaging in vivo

Fluorescent imaging in vivo was performed using the imaging system 100 series IVISTM(Xenogen, Alameda, CA). To obtain fluorescence Alexa647-Mab in vivo used optimized cy5.5 filter. Mice native tumor SYO-1 were injected with 20 μg Alexa647 labeled Mab intraperitoneally and were subjected to fluorescence imaging through different periods of time. Mice were fed food that did not contain alfalfa for four days before injection of Mab to reduce background fluorescence. When getting images of mice were anestesiologi 2% isoflurane (Abbott Laboratories) and placed in the IVIS system. Mice were killed four days after injection of Mab, removed the tumor and major organs and obtained fluorescent image.

As shown in Fig. 4a, significant fluorescence was detected in the localization of the whole 24 hours after injection. Associated with tumor fluorescence was observed for both Mab, 92-13 and 93-22; the signal has reached its maximum level after approximately 48 hours after injection, and can be detected through 96 hours after injection. The authors present invention squashed these mice after 120 hours after injection and measured the fluorescence intensity in tumor and critical normal organs (liver, spleen, kidney, pancreas, colon) (Fig. 4b and 4c). Very strong fluorescent signal was observed in the extracted tumor, whereas in normal organs fluorescent signal was not detected. To confirm the binding specificity of the authors of the present invention was created xenografts using negative for antigen cell lines LoVo naked mice and were injected with Alexa647 labeled Mab conducting fluorescence imaging analysis. Mice-media LoVo fluorescence was not detected neither in the localization of the tumor (Fig. 5a), nor in the extracted tumor or other organs (Fig. 5b and 5c). These results show that Mab also can specifically bind with expressing FZD10 tumor cells in vivo.

Example 5

The internalization of Mab against FZD10 in antigen-positive cells

To study molecular behavior of these Mab after binding to cell what poverhnosti tracked their localization using visualization system in vitro.

The cells were placed in 8-hole closed glass slides (Nalge Nunc International, Naperville, IL) at a density of 5×104cells per well. Cells were incubated with Mab for three hours at 37°C in an air chamber containing 5% CO2. Mab associated with the cell surface was removed with acid removes buffer (0.1 M glycine, 500 mm NaCl, pH 2,5) at 4°C for 10 min and neutralization of 500 mm Tris (pH 7.5). Cells then were fixed with 3.7% formaldehyde for 15 min at room temperature and was permeability.avi exposure with 0.2% Triton X-100 for 10 min, followed by blocking with 3% bovine serum albumin for one hour at room temperature. To identify Mab, internalizing in the cell samples were incubated with Alexa488 labeled goat IgG against mouse (dilution 1:700) for one hour at room temperature. Glass made c DAPI (Vectashield, Vector Laboratories, Burlingame, CA) and analyzed using confocal optics Leica TCS SP1.

As shown in Fig. 6, according to the confocal microscopic visualization produced using labeled with Alexa488 goat IgG against mouse, both Mab 92-13 and 93-22 effectively included in the cytosol of cells SYO-1 cells and YaFuSS 3 hours after incubation with Mab cells (Fig. 6, a-f). On the other hand, the fluorescence signals of these Mab was barely detected in LoVo cells, not expressing FZD10 (Fig. 6, g-i)that while the displays, the specific binding of Mab with FZD10 cell surface induced internalization of antibodies.

Example 6

Specific cytotoxicity of Mab

92-13 and 93-22 had no effect on tumor cell growth when added directly to cultured cells (data not shown). For therapeutic studies of tumor SYO-1 were grown in mice BALB/cA Jcl-nu way similar to that described in example 4. The diameter of the tumors were measured using calipers, and the volume of the tumors was determined using the following formula: 0,5 × (larger diameter) × (smaller diameter)2as described previously (Nagayama, S., et al. (2005). Oncogene, 24, 6201-12). When tumor volume reached more than 0.4-2.8 cm3mice Balb/c-nude-bearing subcutaneous tumor SYO-1, randomly distributed in the processing group, and they did intravenous injection of 100 µci labeled90Y Mab or control Mab into the tail vein. Mice were weighed and recorded diameters of the tumor.

In Fig. 7 shows that the volume of the tumor was significantly decreased immediately after treatment, almost to trace values in one week all mice. When administered to mice 50 µci90Y-DTPA-92-13 tumor with a volume of >1 cm3recovered two weeks after treatment, although the size of the tumors immediately after treatment was significantly decreased. Mice were observed temporary reduction in weight (10-15%), but they withdrawles what if for one week, and there was no obvious signs of toxicity (data not shown).

Example 7

The hybrid antibodies

Hybrid antibodies ch92-13 and ch93-22, corresponding to the antibody 92-13 and 93-22 mouse, was created by porting the sequence of the variable regions of each antibody mouse constant region of IgG1person under the control of the CMV promoter. From hybridoma clones 92-13 and 93-22 were extracted total RNA. Of total RNA was synthesized cDNA using the GeneRacer kitTM(Invitrogen). Sequences of the variable regions of monoclonal antibodies amplified using the direct primer (GeneRacerTM5'Primer) and reverse primer; CH1 (IgG2a); 5'-AATTTTCTTGTCCACCTTGGTG-3' (SEQ ID NO: 3) for the heavy chain and CL1 (Kappa); 5'-CTAACACTCATTCCTGTTGAAGCTCT-3' (SEQ ID NO: 4) for the light chain. The PCR products sequenced, and identified sequence encoding the variable region m92-13 and m93-22.

In the identified amino acid sequences of the variable regions of the H-chain and the variable L-chain Ig:

92-13, the variable region of the H-chain:

MKCSWVIFFLMAVVTGVNSEVQLQQSGAELVKPGASVKLSCTASGFNINDTYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGARGSRFAYWGQGTLVTVSA (SEQ ID NO: 13)encoded by nucleotide sequence SEQ ID NO: 14, and

92-13, the variable region of the L-chain:

MSVPTQVLGLLLLWLTDARCDIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWGTPYTFGGGTKL(SEQ ID NO: 21), encoded by the nucleotide sequence of SEQ ID NO: 22; and

93-22, the variable region of the H-chain:

MGWSRIFLFLLSITAGVHCQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARGGNYGWFAYWGQGTLVTVSAGS (SEQ ID NO: 29)encoded by nucleotide sequence SEQ ID NO: 30, and

93-22, the variable region of the L-chain:

METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELYTFGGGTKLGS (SEQ ID NO: 37)encoded by nucleotide sequence SEQ ID NO: 38. Underline the marked signal sequence.

Identified the following sequence of CDR (region that defines complementarity) antibodies:

92-13, INDTYMH (SEQ ID NO: 15) as VH CDR1, RIDPANGNTKYD (SEQ ID NO: 17) as VH CDR2, and GSRFAY (SEQ ID NO: 19) as VH CDR3, RASENIYSNLA (SEQ ID NO: 23) as VL CDR1, VATNLAD (SEQ ID NO: 25) as VL CDR2, and QHFWGTPY (SEQ ID NO: 27) as VL CDR3; and

93-22, SSWMN (SEQ ID NO: 31) as VH CDR1, RIYPGDGDTNYN (SEQ ID NO: 33) as VH CDR2, and GGNYGWFAY (SEQ ID NO: 35) as VH CDR3, RASKSVSTSGYSYMH (SEQ ID NO: 39) as VL CDR1, LASNLES (SEQ ID NO: 41) as VL CDR2, and QHSRELY (SEQ ID NO: 43) as VL CDR3.

In addition, the following were identified amino acid sequences of H chain and L-chain monoclonal antibody mouse 92-13, and 93-22 39-10:

92-13, N-chain: SEQ ID NO: 58 (encoded by nucleotide sequence SEQ ID NO: 57);

92-13, L-chain: SEQ ID NO: 60 (encoded by nucleotide sequence SEQ ID NO: 59);

93-22, N-chain: SEQ ID NO: 62 (encoded by nucleotide is the selected SEQ ID NO: 61);

93-22, L-chain: SEQ ID NO: 64 (encoded by nucleotide sequence SEQ ID NO: 63);

39-10, N-chain: SEQ ID NO: 66 (encoded by nucleotide sequence SEQ ID NO: 65);

39-10, L-chain: SEQ ID NO: 68 (encoded by nucleotide sequence SEQ ID NO: 67).

In accordance with a certain sequence were designed specific primers for the variable region m92-13: 5'-AATAGCGGCCGCACCATGAAATGCAGCTGGGTTATCTT-3' (SEQ ID NO: 5) and 5'-AATAGCTAGCTGCAGAGACAGTGACCAGAGTCC-3' (SEQ ID NO: 6) for the heavy chain and the 5'-AATAGCGGCCGCACCATGAGTGTGCCCACTCAGG-3' (SEQ ID NO: 7) and 5'-TTCCAGCTTGGTCCCCCC-3' (SEQ ID NO: 8) for the light chain. In addition, were designed specific primers for the variable region m93-22, 5'-AATAGCGGCCGCACCATGGGATGGAGCCGGATCTTT-3' (SEQ ID NO: 53) and 5'-AATAGGATCCTGCAGAGACAGTGACCAGAGTCCCTT-3' (SEQ ID NO: 54) for the heavy chain and the 5'-AATAGCGGCCGCACCATGGAGACAGACACACTCCT-3' (SEQ ID NO: 55) and 5'-AATAGGATCCCAGCTTGGTCCCCCCTCCGAACGT-3' (SEQ ID NO: 56) for the light chain. To construct the expression vector for the hybrid antibody was obtained two cassette vector. The DNA fragment encoding IgG1 (CH1-CH3) man, was inserted in pQCXIH (Clontech) (pQCXCHIH), and the DNA fragment encoding Igκ (CL1) man, was inserted into pQCXIP (pQCXCLIP). To obtain DNA fragments encoding human IgG1 or human Igκ, was obtained library constant region of a person using cDNA from PBMC (mononuclear cells in peripheral blood) of a person using a previously described method (Liu, A.Y. et al., Proc. Natl. Acad. Sci. USA, Vol.84, 3439-43, 1987; Reff, M.E. et al., Blood, Vol.83, No.2, 435-45, 1994). DNA encoding the variable region of the heavy chain and light chain m92-13 and m93-22, amplified by PCR, sequenced and subcloned into pQCXCHIH and pQCXCLIP, respectively, using the sites NotI and BamHI. These vectors together was transfusional in CHO cells. Transfetsirovannyh cells were cultured in medium F-12 containing 500 μg/ml of hygromycin and 10 μg/ml puromycin. When the cells were grown to a state close to confluently, the medium was replaced with serum-free medium (CHO-S-SFM II; GIBCO), and hybrid antibody was purified from the supernatant using affinity column of protein A (GE Amersham) and sequenced. The sequence of the heavy chain hybrid antibodies ch92-13 includes SEQ ID NO: 46, encoded by the nucleotide sequence of SEQ ID NO: 45; and the sequence of the light chain of the hybrid antibodies ch92-13 includes SEQ ID NO: 48, encoded by the nucleotide sequence of SEQ ID NO: 47. The sequence of the heavy chain hybrid antibodies ch93-22 includes SEQ ID NO: 49, encoded by the nucleotide sequence of SEQ ID NO: 50; and the sequence of the light chain of the hybrid antibodies ch93-22 includes SEQ ID NO: 52, encoded by the nucleotide sequence of SEQ ID NO: 51.

Example 8

Binding activity of hybrid antibodies

Antibody-dependent cellular cytotoxic (ADCC) activity induced hybrid 92-13 and 93-22, was determined by what aktivnosti LDH, as described previously (Nagayama, S., et al. (2005). Oncogene, 24, 6201-6212). Fresh effector cells were isolated from heparinized peripheral blood of a healthy donor using Ficoll-Plaque (Amersham Bioscience). Effector cells (E) and target cells (T) (each in an amount of 5×103/well) was co-incubated for 6 h at 37°C in four Parallels in different ratios (E:T with hybrid 92-13, hybrid 93-22 or non-immune IgG human in 0.1 ml containing no phenol red RPMI medium 1640 with the addition of 5% FBS in 96-well pad. LDH allocated in the culture supernatant was determined by absorption at 490 nm. The percentage of specific cytotoxicity was calculated in accordance with the manufacturer's instructions.

As for effector activity, both hybrid 92-13 and 93-22 induced ADCC specific sverkhekspressiya FZD10 cells SYO-1 (Fig. 8, a and c), but not against FZD10-negative LoVo cells (Fig. 8, b and d). Specifically, hybrid 92-13 caused a large induction of cytotoxicity compared to the hybrid 93-22; however, their activity depended on donor effector cells, possibly due to polymorphism of the Fc receptor.

Each of the applications and patents mentioned in this document, and each document cited or referenced in the aforementioned applications and patents included in the present description by reference. Bol is e, all references cited in this text documents, and all documents cited or referenced in the documents cited in this text, is included, therefore, in the present description by reference.

Various modifications and variations of the described methods and systems of the invention and within the scope and spirit of the invention should be obvious to a person skilled in the art. Although the invention has been described in connection with specific preferred variant implementation, it should be understood that the invention as it is claimed, should not unreasonably restrict such specific choices of implementation and that within the scope of the invention it can be made a variety of modifications and additions. Indeed, various modifications of the described methods for carrying out the invention that are obvious to experts in the field of molecular biology or related fields, are considered as included in the scope of the claims. Moreover, various combinations of the following characteristic features of the dependent claims can be made with the characteristics of the independent claims without going beyond the scope of the present invention.

1. The antibody or its fragment, which includes the V (variable) region H (heavy) chain, including the surrounding area, determining complementarity (CDRs)having the amino acid sequence represented in SEQ ID NO: 15, 17 and 19 and the V-region L (light) chain comprising CDRs having the amino acid sequence represented in SEQ ID NO: 23, 25 and 27, and which are capable of binding homolog 10 protein Frizzled (FZD10) or its partial peptide.

2. The antibody or fragment according to claim 1, where the antibody is selected from the group consisting of mouse antibodies, hybrid antibodies, gumanitarnogo antibodies, fragments of antibodies and single-chain antibodies.

3. The antibody or fragment according to claim 1, where the antibody is an antibody of a mouse.

4. The antibody or fragment according to claim 3, where the antibody is a mouse includes a H-chain having the amino acid sequence represented in SEQ ID NO: 57, and/or L-chain having the amino acid sequence represented in SEQ ID NO: 59.

5. The antibody or fragment according to claim 3, where the antibody mouse is produced by a clone of hybridoma 92-13 (FERM BP-10628).

6. The antibody or fragment according to claim 1, where the antibody is a hybrid antibody.

7. The antibody or fragment of claim 6, where the hybrid antibody comprises the V region of H chain having the amino acid sequence represented in SEQ ID NO: 13.

8. The antibody or fragment according to claim 6 or 7, where the hybrid antibody comprises an H-chain having the amino acid sequence represented by the SEQ ID NO: 46.

9. The antibody or fragment of claim 6, where the hybrid antibody comprises the V region of the L-chain having the amino acid sequence represented in SEQ ID NO: 21.

10. The antibody or fragment according to claim 6 or 9, where the hybrid antibody comprises an L-chain having the amino acid sequence represented in SEQ ID NO: 48.

11. The antibody or fragment of claim 6, where the hybrid antibody comprises the V region of H chain having the amino acid sequence represented in SEQ ID NO: 13, and V-region L-chain having the amino acid sequence represented in SEQ ID NO: 21.

12. The antibody or fragment according to claim 6 or 11, where the hybrid antibody comprises an H-chain having the amino acid sequence represented in SEQ ID NO: 46, and L-chain having the amino acid sequence represented in SEQ ID NO: 48.

13. The antibody or fragment of claim 6, where the hybrid antibody further includes a C (constant) region of human antibodies.

14. The antibody or fragment according to claim 1, where the antibody is humanitariannet antibody.

15. The antibody or fragment according to 14, where humanitariannet antibody further includes FR (frame) region of human antibodies and/or C region of human antibodies.

16. The antibody or its fragment, which includes the V (variable) region H (heavy) chain, including the area that defines complementarist (CDR), having the amino acid sequence represented in SEQ ID NO: 31, 33 and 35, and V-region L (light) chain comprising CDRs having the amino acid sequence represented in SEQ ID NO: 39, 41 and 43, and which are capable of binding homolog 10 protein Frizzled (FZD10) or its partial peptide.

17. The antibody or fragment according to item 16, where the antibody is selected from the group consisting of mouse antibodies, hybrid antibodies, gumanitarnogo antibodies, fragments of antibodies and single-chain antibodies.

18. The antibody or fragment according to item 16, where the antibody is an antibody of a mouse.

19. The antibody or fragment according p, where antibody mouse includes a H-chain having the amino acid sequence represented in SEQ ID NO: 61 and/or L-chain having the amino acid sequence represented in SEQ ID NO: 63.

20. The antibody or fragment according p, where antibody mouse is produced by a clone of hybridoma 93-22 (FERM BP-10620).

21. The antibody or fragment according to item 16, where the antibody is a hybrid antibody.

22. The antibody or fragment according to item 21, where the hybrid antibody comprises the V region of H chain having the amino acid sequence represented in SEQ ID NO: 29.

23. The antibody or fragment according to item 21 or 22, where the hybrid antibody comprises an H-chain having the amino acid sequence represented in SEQ ID NO: 50.

24. Antibody Il is his fragment on item 21, where hybrid antibody comprises the V region of the L-chain having the amino acid sequence represented in SEQ ID NO: 37.

25. The antibody or fragment according to item 21 or 24, where the hybrid antibody comprises an L-chain having the amino acid sequence represented in SEQ ID NO: 52.

26. The antibody or fragment according to item 21, where the hybrid antibody comprises the V region of H chain having the amino acid sequence represented in SEQ ID NO: 29, and the V region of the L-chain having the amino acid sequence represented in SEQ ID NO: 37.

27. The antibody or fragment according to item 21 or 26, where the hybrid antibody comprises an H-chain having the amino acid sequence represented in SEQ ID NO: 50, and L-chain having the amino acid sequence represented in SEQ ID NO: 52.

28. The antibody or fragment according to item 21, where the hybrid antibody further includes a C (constant) region of human antibodies.

29. The antibody or fragment according to item 16, where the antibody is humanitariannet antibody.

30. The antibody or its fragment by clause 29, where humanitariannet antibody further includes FR (frame) region of human antibodies and/or C region of human antibodies.

31. Chimeric or humanitariannet antibody or its fragment, in which the complementarity determining region (CDR) derived from mouse monoclonalantibody 92-13 (FERM BP-10628).

32. The antibody or fragment according p representing a chimeric antibody that contains:
(i) the V region of H chain having the amino acid sequence represented in SEQ ID NO: 13, and/or V region of the L-chain having the amino acid sequence represented in SEQ ID NO: 21; or
(ii) the H chain having the amino acid sequence represented in SEQ ID NO: 46, and/or L-chain having the amino acid sequence represented in SEQ ID NO: 48.

33. The antibody or fragment according to claim 1 or 31, which contains a radioisotope label or fluorescent label.

34. The antibody or fragment according p, where the radioisotope label is selected from the90yttrium (90Y)125iodine (125I) and111India (111In.

35. Clone hybridoma 92-13 (FERM BP-10628), which produces monoclonal antibody 92-13 mouse.

36. Clone hybridoma 93-22 (FERM BP-10620), which produces monoclonal antibody 93-22 mouse.

37. Method for the treatment or prevention of disease, which is associated with 10 homologue protein Frizzled (FZD10) in a subject comprising administration to the subject an effective amount of the antibody or fragment according to any one of claims 1 to 34.

38. The method according to clause 37, where the disease, which is associated with FZD10 selected from synovial sarcoma (SS), colorectal cancer, gastric cancer, chronic myeloid leukemia (CML) and acute myeloid leukemia (AML).

39. SPO is about the diagnosis or prediction of disease which is associated with 10 homologue protein Frizzled (FZD10), or predisposition to the development of the disease in the subject, including
(a) contacting the sample or a preparation from a subject with the antibody or fragment according to any one of claims 1 to 34;
(b) identification of FZD10 protein in the sample or the drug; and
(c) deciding, suffer if the subject's disease or a subject at risk of developing the disease, based on the relative content of FZD10 protein compared to control.

40. The method according to § 39, where the disease, which is associated with FZD10 selected from synovial sarcoma (SS), colorectal cancer, gastric cancer, chronic myeloid leukemia (CML) and acute myeloid leukemia (AML).

41. How to visualize in vivo homolog 10 protein Frizzled (FZD10) in a subject comprising administration to the subject an effective amount of the antibody or fragment according p or 34.

42. Pharmaceutical composition for treatment or prevention of diseases associated with the homologue 10 Frizzled (FZD10), containing the antibody or fragment according to any one of claims 1 to 34 and a pharmaceutically acceptable carrier or excipient.

43. A kit for the diagnosis or prediction of disease-related homolog 10 Frizzled (FZD10), containing the antibody or fragment according to any one of claims 1 to 34 and labeled secondary antibody.

44. Pharmaceutical composition for in vivo imaging homolog 10 Frizzled protein (FZD10), starasiantattoo or fragment according p or 34 and a pharmaceutically acceptable carrier or excipient.



 

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6 cl, 21 dwg, 1 tbl

FIELD: medicine, oncology, biochemistry.

SUBSTANCE: invention relates to fused proteins, namely to the multifunctional fused protein cytokine-antibody. This fused protein involves immunoglobulin region and cytokine fused protein of the formula IL-12-X or X-IL-12 wherein interleukin-12 (IL-12) represents the first cytokine and X represents the second cytokine taken among the group comprising IL-2, IL-4 and GM-CSF bound covalently either by amino-end or carboxyl-end to subunit p35 or p40 of interleukin-12 (IL-12) in its heterodimeric or a single-chain form. Indicated fused cytokine protein is fused by either its amino-end or carboxyl-end with indicated region of immunoglobulin. Multifunctional fused protein cytokine-antibody shows an anticancer activity.

EFFECT: valuable medicinal properties of protein complexes.

13 cl, 40 dwg, 18 ex

The invention relates to the field of immunobiotechnology and may find application in medicine

FIELD: medicine, oncology, biochemistry.

SUBSTANCE: invention relates to fused proteins, namely to the multifunctional fused protein cytokine-antibody. This fused protein involves immunoglobulin region and cytokine fused protein of the formula IL-12-X or X-IL-12 wherein interleukin-12 (IL-12) represents the first cytokine and X represents the second cytokine taken among the group comprising IL-2, IL-4 and GM-CSF bound covalently either by amino-end or carboxyl-end to subunit p35 or p40 of interleukin-12 (IL-12) in its heterodimeric or a single-chain form. Indicated fused cytokine protein is fused by either its amino-end or carboxyl-end with indicated region of immunoglobulin. Multifunctional fused protein cytokine-antibody shows an anticancer activity.

EFFECT: valuable medicinal properties of protein complexes.

13 cl, 40 dwg, 18 ex

FIELD: biotechnology, peptides.

SUBSTANCE: invention relates to a method for preparing antibodies raised to human leukocyte differentiation factor (HLDF) or to HLDF fragment (31-38) representing peptide of the following structure: Arg-Arg-Trp-His-Arg-Leu-Glu-Lys possessing with antigenic and nucleic acids-hydrolyzing properties, and for diagnostic aims also. Antibodies are prepared from rabbit plasma blood immunized with three injections of antigens wherein synthetic HLDF factor or conjugate is used as antigens. Diagnosis of anaplastic state of human cells is carried out by using solutions of antibodies to HLDF factor or HLDF fragment (31-38) in the concentration 0.0013 mg/ml as biological markers. Invention provides carrying out the differential diagnosis of tumors and normal organs and effective detecting initial stages in cell differentiation disturbances.

EFFECT: improved preparing method of antibody, improved method for diagnosis.

6 cl, 21 dwg, 1 tbl

FIELD: oncology and biotechnology.

SUBSTANCE: invention concerns conjugates used for treatment of malignant tumor. Conjugate includes staphylococcal or streptococcal wild-type superantigen or modified superantigen and antibody constituent. Bacterial superantigen is modified to reduce serum reactivity with preserved its antigenic activity. Amino acid sequence of superantigen incorporates A-E regions determining binding to TCR and MHC molecules class II. Invention is directed to preparing antitumor drug and also to preparing pharmaceutical composition.

EFFECT: use of the conjugate according to invention activate immune system and, therefore, resistance of mammalian against malignant tumor.

67 cl, 11 dwg, 1 tbl, 11 ex

FIELD: immunology, antibodies.

SUBSTANCE: invention elates to human monoclonal antibodies to MN and antibody fragments to MN that are targeted to repeat sequence GEEDLP within proteoglycan domain. Binding with a desired epitope is confirmed by competitive immunoenzyme analysis method ELISA wherein ELISA signal is attenuated in combined incubation with peptide comprising this repeat sequence (PGEEDLPGEEDLP). Binding inhibition can be confirmed by the Biacore study also wherein binding required antibodies with immobilized MN or proteoglycan peptides can be inhibited by peptide repeat sequence. In addition to binding with human peptide repeat sequence anti-MN can inhibit adhesion of CGL-1 cells to plastic plates covered by MN. Human antibodies anti-MN can be used in treatment of cancer diseases or for diagnosis of cancer diseases wherein the level of MN is increased.

EFFECT: valuable medicinal properties of antibodies.

11 cl, 8 dwg, 2 tbl, 13 ex

FIELD: medicine.

SUBSTANCE: versions of the bond intended for linkage with the external domain B (ED-B) of a fibronectin are offered. The bond includes an antigen-binding fragment of one-chained antibody L19 and a cysteinum-containing linker for hanging of a radioactive label. Versions of a pharmaceutical composition for diagnostics and treatment of angiogenic diseases on the basis of the specified bond are opened. Application of bond for linkage with radioactive bond is described. The method of reception of bond in eucariotic cells is opened, including in Pichia pastoris and a set for reception is radioactive labelled agents on the basis of bond.

EFFECT: high-avid bond accumulation in solid tumours.

23 cl, 4 dwg, 5 tbl, 15 ex

FIELD: chemistry.

SUBSTANCE: proposed is a recombinant single-strand trispecific antibody for treating tumours which express CEA. The said antibody consists of a series of three antibody fragments: anti-CEA-scFv, anti-CD3-scFv and VH CD28-antibody, linked by two intermediate linkers (intermediate linker Fc and intermediate linker HSA). If necessary, a c-myc-mark or (His)6-mark can be added at the C-end. Described is DNA, which codes the antibody, expression vector based on it and E.coli cell, containing the vector.

EFFECT: use of the invention is more beneficial in clinical use compared to bispecific antibodies and known trispecific antibodies, makes easier clearing and expression of an antibody, which can further be used in treating CEA-mediated tumours.

10 cl, 21 dwg, 11 ex

FIELD: medicine.

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

EFFECT: invention use allows obtaining the fused proteins with the lowered adjuvanticity and, basically, keeping identical biological activity in comparison with a parent molecule; it can be used in treatment of tumours.

4 cl, 6 dwg, 22 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: there is offered application of humanised fused protein for making a medicine used for stimulation of immune response and stabilisation of disease progressing in patients with GD2-positive tumours. The antibody contains antibody H14.18 caught with surface glycosphingolipid GD2 of human cells, and cytokine IL2. There is disclosed method of increase in ADCC and lysis activity of natural killers in cancer patients by introduction of the fused protein. The invention can be applied in GD2-overexpression cancer therapy.

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

2 cl, 8 dwg, 1 tbl, 2 ex

FIELD: pharmacology.

SUBSTANCE: present invention refers to immunology and biotechnology. There is offered recovered human antibody to RG1 polypeptide. There are described versions of antibodies, including one-chain antibody, and immunoconjugate based on said antibodies. There are disclosed methods of selective cell destruction, cell inhibition, treatment of disease state, detection of disease state, detection of RG1, monitoring of clinical course of prostate cancer, prediction in a person with using antibodies and immunoconjugate.

EFFECT: application of the invention provides new antibodies to RG1 polypeptide that can find application in treating tumours with RG1 overexpression.

16 cl, 4 dwg, 1 tbl, 13 ex

FIELD: medicine.

SUBSTANCE: invention relates to biotechnology and represents antibody or its fragment, which is able to bind with homologue 10 of protein Frizzled (FZD10), such as monoclonal mouse antibody, hybrid antibody, chimeric and humanised antibody. Also claimed are hybridoma clones, producing antibody, as well as method of treatment of prevention of FZD10-associated disease; method of diagnostics or prediction of FZD10-associated disease; method of visualisation in vivo FZD10 in a subject; pharmaceutical composition and set, containing antibody.

EFFECT: treatment with claimed antibodies makes it possible to improve clinical outcome in case of diseases associated with homologue to of protein Frizzled.

44 cl, 45 dwg, 8 ex

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