Anti-tat226 antibodies and immunoconjugates

FIELD: medicine, pharmaceutics.

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

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

41 cl, 22 dwg, 3 tbl, 4 ex

 

This application claims the priority of provisional application U.S. No. 60/783746 filed March 17, 2006, the description of which is incorporated herein by reference in full.

The SCOPE of the INVENTION

The present invention relates to an anti-TAT226 antibodies and their immunoconjugates. The invention additionally relates to methods of using anti-TAT226 antibodies and their immunoconjugates.

The LEVEL of TECHNOLOGY

Antibodies that bind to the polypeptides expressed on the surface of cancer cells, has been effective in the ways of anti-cancer therapy. Such antibodies may function through a variety of mechanisms, including, for example, the activation of antibody-dependent cell-mediated cytotoxicity (ADCC), induction of antibody complementability cytotoxicity (CDC), increased release of cytokines and the induction of apoptosis. See, for example, Cardarelli et al. (2002) Cancer Immunol. Immunother. 51:15-24. For example, HERCEPTIN®and RITUXAN®(both from Genentech Inc., South San Francisco, California) are antibodies that have been successfully used to treat breast cancer and non-Hodgkin's lymphoma, respectively. HERCEPTIN®is a derived on the basis of recombinant DNA humanitariannet monoclonal antibody that selectively binds to the extracellular domain of proto the Cohen receptor 2 (HER2) and epidermal growth factor (human). The overexpression of HER2 protein is observed in 25-30% of primary breast cancers. RITUXAN®is a genetically engineered chimeric monoclonal antibody mouse/human directed against the CD20 antigen found on the surface of normal and malignant B-lymphocytes. Both of these antibodies receive recombinant method in CHO cells. Assume that HERCEPTIN®acts, at least partially, by inhibiting angiogenesis (Izumi et al. (2002) Nature 416:279-280) and, as suggested, RITUXAN®acts, at least in part, by inducing apoptosis (Cardarelli et al. (2002) Cancer Immunol. Immunother. 51:15-24).

Immunoconjugate, or conjugates of the antibody-drug"suitable for local delivery of cytotoxic agents in the treatment of cancer. See, for example, Syrigos et al. (1999) Anticancer Research 19:605-614; Niculescu-Duvaz et al. (1997) Adv. Drug Deliv. Rev. 26:151-172; U.S. patent No. 4975278. Immunoconjugate allow targeted delivery component of the drug in the tumor, while systemic injection of unconjugated cytotoxic agents may lead to undesirable levels of toxicity not only in tumor cells that you want to delete, but also in normal cells. Cm. Baldwin et al. (Mar. 15, 1986) Lancet p. 603-05; Thorpe (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological and Clinical Applications (A. Pinchera t al., eds.) p. 475-506. Immunoconjugate directed against polypeptides on the cell surface, was developed and continue to develop for the treatment of cancer. For an overview see, e.g., Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103.

Obviously, there is a growing need for diagnostic and/or therapeutic purposes in the media, which is directed against the polypeptides on the cell surface. The described invention meets this need and provides other benefits.

All cited in the present description references, including patent applications and publications cited as references in full.

The INVENTION

The invention relates to an anti-TAT226 antibodies and methods of use thereof.

One aspect relates to an antibody that binds to TAT226, where the antibody contains at least one, two, three, four, five or six HVR chosen from:

(1) HVR-H1 containing the amino acid sequence of SEQ ID NO:4;

(2) the HVR-H2 containing the amino acid sequence of SEQ ID NO:5;

(3) the HVR-H3 containing the amino acid sequence that corresponds to the consensus sequence SEQ ID NO:11;

(4) the HVR-L1 containing the amino acid sequence of SEQ ID NO:12;

(5) the HVR-L2, containing the amino acid sequence of SEQ ID NO:13; and

(6) the HVR-L3, containing aminokislotna the sequence, which corresponds to the consensus sequence SEQ ID NO:19.

In another aspect, the antibody that binds to TAT226, contains (a) HVR-H3 containing the amino acid sequence that corresponds to the consensus sequence SEQ ID NO:11, and (b)at least one, two, three, four or five HVR chosen from:

(1) HVR-H1 containing the amino acid sequence of SEQ ID NO:4;

(2) the HVR-H2 containing the amino acid sequence of SEQ ID NO:5;

(3) the HVR-L1 containing the amino acid sequence of SEQ ID NO:12;

(4) the HVR-L2, containing the amino acid sequence of SEQ ID NO:13; and

(5) the HVR-L3 containing the amino acid sequence that corresponds to the consensus sequence SEQ ID NO:19.

In one of the embodiments the antibody contains HVR-L3 containing the amino acid sequence that corresponds to the consensus sequence SEQ ID NO:19. In one of the embodiments, the antibody further comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:4, and HVR-H2 containing the amino acid sequence of SEQ ID NO:5. In one of the embodiments, the antibody further comprises HVR-L1, containing the amino acid sequence of SEQ NO:12, and HVR-L2 containing the amino acid sequence of SEQ ID NO:13.

In one of the embodiments the antibody sod is RIT HVR-H3, containing the amino acid sequence selected from SEQ ID nos:6-10. In one of the embodiments, the antibody further comprises HVR-L3 containing the amino acid sequence selected from SEQ ID nos:14-18. In one embodiment, the implementation of the HVR-H3 contains the amino acid sequence of SEQ ID NO:9 and HVR-L3 contains the amino acid sequence of SEQ ID NO:17. In one embodiment, the implementation of the HVR-H3 contains the amino acid sequence of SEQ ID NO:10 and HVR-L3 contains the amino acid sequence of SEQ ID NO:18. In one of the embodiments, the antibody further comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:4, and HVR-H2 containing the amino acid sequence of SEQ ID NO:5. In one of the embodiments, the antibody further comprises HVR-L1, containing the amino acid sequence of SEQ NO:12, and HVR-L2 containing the amino acid sequence of SEQ ID NO:13.

One aspect relates to an antibody that binds to TAT226, where the antibody contains at least one, two, three, four, five or six HVR chosen from:

(1) HVR-H1 containing the amino acid sequence of SEQ ID NO:1;

(2) the HVR-H2 containing the amino acid sequence of SEQ ID NO:2;

(3) the HVR-H3 containing the amino acid sequence of SEQ ID NO:3;

(4) the HVR-L1 containing the amino acid sequence of SEQ ID NO:12;

(5) the HVR-L2, containing the amino acid sequence of SEQ ID NO:13; and

(6) the HVR-L3 containing the amino acid sequence of SEQ ID NO:14.

In another aspect, the antibody that binds to TAT226, contains (a) HVR-H3 containing the amino acid sequence of SEQ ID NO:3, and (b)at least one, two, three, four or five HVR chosen from:

(1) HVR-H1 containing the amino acid sequence of SEQ ID NO:1;

(2) the HVR-H2 containing the amino acid sequence of SEQ ID NO:2;

(3) the HVR-L1 containing the amino acid sequence of SEQ ID NO:12;

(4) the HVR-L2, containing the amino acid sequence of SEQ ID NO:13; and

(5) the HVR-L3 containing the amino acid sequence of SEQ ID NO:14.

In one of the embodiments the antibody contains HVR-L3 containing the amino acid sequence of SEQ ID NO:14. In one of the embodiments, the antibody further comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:1, and HVR-H2 containing the amino acid sequence of SEQ ID NO:2. In one of the embodiments, the antibody further comprises HVR-L1, containing the amino acid sequence of SEQ NO:12, and HVR-L2 containing the amino acid sequence of SEQ ID NO:13.

In some embodiments, the implementation of any of the above antibodies further comprises at least one frame region selected from consensu the Noi frame region VH subgroup III consensus framework region VL subgroup I.

One aspect relates to an antibody that binds to TAT226, where the antibody contains a variable domain of a heavy chain having at least 90% sequence identity with the amino acid sequence selected from SEQ ID nos:21-25. In one of the embodiments, the antibody further comprises a variable domain light chain having at least 90% sequence identity with the amino acid sequence selected from SEQ ID nos:26-31. In one of the embodiments, the antibody contains a variable domain of a heavy chain having at least 90% sequence identity with the amino acid sequence SEQ ID NO:24. In one of the embodiments, the antibody further comprises a variable domain light chain having at least 90% sequence identity with the amino acid sequence SEQ ID NO:29. In one embodiment, the implementation of the variable domain of the heavy chain contains the amino acid sequence of SEQ ID NO:24 and the variable domain of the light chain contains the amino acid sequence of SEQ ID NO:29. In one of the embodiments, the antibody contains a variable domain of a heavy chain having at least 90% sequence identity with the amino acid sequence SEQ ID NO:25. In one of the VA is Ianto implementation of the antibody further comprises a variable domain light chain, having at least 90% sequence identity with the amino acid sequence SEQ ID NO:30. In one embodiment, the implementation of the variable domain of the heavy chain contains the amino acid sequence of SEQ ID NO:25 and a variable domain light chain contains the amino acid sequence of SEQ ID NO:30.

One aspect relates to an antibody that binds to TAT226, where the antibody contains a variable domain of a heavy chain having at least 90% sequence identity with the amino acid sequence SEQ ID NO:20. In one of the embodiments, the antibody further comprises a variable domain light chain having at least 90% sequence identity with the amino acid sequence SEQ ID NO:26. In one embodiment, the implementation of the variable domain of the heavy chain contains the amino acid sequence of SEQ ID NO:20 and the variable domain of the light chain contains the amino acid sequence of SEQ ID NO:26.

Some embodiments of concern to polynucleotide, codereuse any of the above antibodies. One of the embodiments relates to a vector containing polynucleotide. One of the embodiments refers to the cell host containing the vector. In one of the embodiments a host cell is PI is eroticheskoe. In one of the embodiments a host cell is a CHO cell. One of the embodiments relates to a method for obtaining anti-TAT226 antibodies, where the method comprises culturing the host cell under conditions suitable for expression of polynucleotide encoding the antibody, and isolating the antibody.

One aspect relates to an antibody that binds to TAT226 expressed on the cell surface. In one of the embodiments the antibody binds to an epitope within a region of TAT226 amino acids 21-115 SEQ ID NO:75. In one of the embodiments the cell is a cancer cell. In one of the embodiments the cancer cell is an ovarian cancer cell, a cell brain tumor or cell Wilms tumor.

In some embodiments, the implementation of any of the above antibodies is a monoclonal antibody. In one of the embodiments the antibody is an antibody fragment selected from a Fab fragment, Fab'-SH, Fv, scFv, or (Fab')2. In one of the embodiments the antibody is humanized. In one of the embodiments the antibody is human. In one of the embodiments the antibody binds to the same epitope as an antibody selected from YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6.

One aspect relates to a method of detecting the presence the of conduct TAT226 in the biological sample, where the method involves contacting a biological sample with any of the above antibodies under conditions that allow the antibody to contact TAT226, and detection is also established whether a complex between the antibody and TAT226. In one of the embodiments the biological sample contains cells of an ovarian tumor, the tumor cells of the brain or cells Wilms tumor.

One aspect relates to a method of diagnosing disorders of cell proliferation associated with increased expression of TAT226, where the method includes contacting the test cell with any of the above antibodies; determining the level of expression of TAT226 by detecting binding of an antibody to TAT226; and comparing the level of expression of TAT226 the test cell with the level of expression of TAT226 a control cell, where a higher level of expression of TAT226 the test cell compared to the control cell indicates the presence of disorders of cell proliferation associated with increased expression of TAT226. In one embodiment, the implementation of the test cell is a cell of the patient, which suggest the presence of disorders of cell proliferation. In one embodiment, the implementation of a violation of cell proliferation is selected from ovarian cancer, and Wilms tumor. In one of the embodiments the method comprises determining the level of expression of TAT226 on the surface of the test which has been created cell and comparing the level of expression of TAT226 on the surface of the test cell with the level of expression of TAT226 on the surface of the control cells.

The invention additionally relates to immunoconjugates and methods of use thereof.

In one aspect of immunoconjugate contains any of the above anti-TAT226 antibodies covalently attached to a cytotoxic agent. In one embodiment, the implementation of the cytotoxic agent selected from a toxin, a chemotherapeutic drug, an antibiotic, a radioactive isotope and nucleotidase enzyme.

One aspect relates to immunoconjugate having the formula Ab-(L-D)p, where:

(a) Ab represents any of the above anti-TAT226 antibodies

(b) L is a linker;

(c) D is a drug of the formula DEor DF

,

and where each R2and R6represents methyl, each R3and R4represents isopropyl, R7represents sec-butyl, each R8independently selected from CH3, O-CH3, OH, and H; R9represents H; R10represents aryl; Z represents-O - or-NH-; R11represents H, C1-C8alkyl or -(CH2)2-O-(CH2)2-O-(CH2)2-O-CH3; and R18represents-C(R8)2-C(R8)2-aryl; and

(d) p ranges from about 1 to 8.

In one of the embodiments the antibody (Ab) contains 1) the HVR-H3, containing the amino acid sequence that corresponds to the consensus sequence SEQ ID NO:11, and 2) at least one, two, three, four or five HVR chosen from:

(i) HVR-H1 containing the amino acid sequence of SEQ ID NO:4;

(ii) HVR-H2 containing the amino acid sequence of SEQ ID NO:5;

(iii) HVR-L1 containing the amino acid sequence of SEQ ID NO:12;

(iv) HVR-L2 containing the amino acid sequence of SEQ ID NO:13; and

(v) HVR-L3 containing the amino acid sequence that corresponds to the consensus sequence SEQ ID NO:19.

In one of the embodiments the antibody contains HVR-L3 containing the amino acid sequence that corresponds to the consensus sequence SEQ ID NO:19. In one of the embodiments the antibody contains HVR-H3 containing the amino acid sequence of SEQ ID NO:9, and HVR-L3, containing the amino acid sequence of SEQ ID NO:17. In one of the embodiments the antibody contains HVR-H3 containing the amino acid sequence of SEQ ID NO:10, and HVR-L3, containing the amino acid sequence of SEQ ID NO:18. In one of the embodiments, the antibody further comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:4, HVR-H2 containing the amino acid sequence of SEQ ID NO:5, the HVR-L1 containing aminokislotna the sequence of SEQ ID NO:12, and HVR-L2 containing the amino acid sequence of SEQ ID NO:13. In one of the embodiments, the antibody contains a variable region heavy chain having at least 90% sequence identity with the amino acid sequence selected from SEQ ID nos:21-25, and variable region light chain having at least 90% sequence identity with the amino acid sequence selected from SEQ ID nos:26-31. In one of the embodiments, the antibody contains a variable region heavy chain having at least 90% sequence identity with the amino acid sequence SEQ ID NO:24, and the variable region light chain having at least 90% sequence identity with the amino acid sequence SEQ ID NO:29. In one of the embodiments, the antibody contains a variable region heavy chain containing the amino acid sequence of SEQ ID NO:24, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:29. In one of the embodiments, the antibody contains a variable region heavy chain having at least 90% sequence identity with the amino acid sequence SEQ ID NO:25, and a variable region light chain having at least 90% identity p is coherence with the amino acid sequence SEQ ID NO:30. In one of the embodiments, the antibody contains a variable region heavy chain containing the amino acid sequence of SEQ ID NO:25, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:30.

The following implementation options for further relate to any of the above immunoconjugates. In one of the embodiments immunoconjugate has activity against destruction of cellsin vitroorin vivo. In one of the embodiments the linker is attached to the antibody through Tilney groups on the antibody. In one of the embodiments, the linker is cleaved by a protease. In one of the embodiments, the linker contains the dipeptide val-cit. In one of the embodiments, the linker contains a link p-aminobenzyl. In one embodiment, the implementation of the link p-aminobenzyl is located between the medicinal product and cleaved by a protease site linker. In one embodiment, the implementation of the link p-aminobenzyl is a p-aminobenzeneboronic (PAB). In one of the embodiments, the linker contains 6-maleimidomethyl. In one embodiment, the implementation of the 6-maleimidomethyl is located between the antibody and cleaved by a protease site linker. The above options exercise can be done individually, is whether in any combination with each other.

In one embodiment, the implementation of a drug selected from the MMAE and MMAF. In one of the embodiments immunoconjugate has the formula

,

where Ab is any of the above anti-TAT226 antibodies, S represents a sulfur atom, and p ranges from 2 to 5. In one of the embodiments immunoconjugate has the formula

,

where Ab is any of the above anti-TAT226 antibodies, S represents a sulfur atom, and p ranges from 2 to 5.

One aspect relates to pharmaceutical compositions containing any of the above immunoconjugates and a pharmaceutically acceptable carrier. One aspect relates to a method of treatment of disorders of cell proliferation, where the method includes the administration to an individual a pharmaceutical composition. In one embodiment, the implementation of a violation of cell proliferation is selected from ovarian cancer, cancer of uterine body, brain tumors and Wilms tumor. In one embodiment, the implementation of a violation of cell proliferation is associated with increased expression of TAT226 on the cell surface.

One aspect relates to a method of inhibiting proliferation of cells, where the method includes the impact on the cell any of the above immunoconjugates under conditions that allow immunoconjugate contact TAT226. In od the ohms of the embodiments the cell is a tumor cell. In one of the embodiments the tumor cell is a cell tumors of the ovaries, the cell tumors of body of uterus, cell brain tumor or cell Wilms tumor. In one of the embodiments the cell is a xenograft. In one embodiment, the exercise of influence exercised byin vitro. In one embodiment, the exercise of influence exercised byin vivo.

BRIEF DESCRIPTION of FIGURES

The figure 1 presents an alignment of TAT226 human, macaque-having ("cyno"), mice and rats. The remains enclosed in the frame, identical in these species. The remaining residues are different, at least two of the four species. The percentage of amino acid identity among the sequences TAT226 human, macaque-having, mice and rats are presented in the table below the alignment. The percentage identity was calculated using the program ClustalW.

The figure 2 presents the sequence H1, H2 and H3 hypervariable region (HVR) of the heavy chain of monoclonal anti-TAT226 antibodies, designated as YWO.32 and YWO.49 as described in example B. the provisions of the amino acids are numbered according to the numbering system according to Kabat as described below.

The figure 3 presents the sequence L1, L2 and L3 light chain HVR monoclonal anti-TAT226 antibodies, designated as YWO.32 and YWO.49 as described in example B. the provisions of the of minislot numbered according to the numbering system according to Kabat, as is described below.

The figure 4 shows the sequence of HVR-H3, HVR-L3 YWO.49 and YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 obtained by affinity maturation YWO.49 using soft randomized libraries based on HVR-H3, HVR-L3, as described in example B. Also presents consensus sequences of HVR-H3, HVR-L3.

In figures 5A and 5B presents illustrative acceptor sequence of the variable heavy (VH) consensus framework region of a person for use in practice of the present invention with sequence identifiers as follows:

- a consensus of the frame region "A" VH subgroup I of the person minus Kabat CDRs by (SEQ ID NO:32, 33, 34, 35);

- consensus framework region "B", "C" and "D" VH subgroup I of the person minus extended hypervariable regions (SEQ ID nos:36, 37, 34, 35; SEQ ID NO:36, 37, 38, 35; SEQ ID NO:36, 37, 39, 35);

- a consensus of the frame region "A" VH subgroup II of the person minus Kabat CDRs by (SEQ ID NO:40, 41, 42, 35);

- consensus framework region "B", "C" and "D" VH subgroup II of the person minus extended hypervariable regions (SEQ ID nos:43, 44, 42, 35; SEQ ID NO:43, 44, 45, 35; and SEQ ID NO:43, 44, 46 and 35);

- a consensus of the frame region "A" VH subgroup III of the person minus Kabat CDRs by (SEQ ID NO:47, 48, 49, 35);

- consensus framework region "B", "C" and "D" VH subgroup III of the person minus the extended hyperv riebeling areas (SEQ ID NO:50, 51, 49, 35; SEQ ID NO:50, 51, 52, 35; SEQ ID NO:50, 51, 53, 35);

- acceptor framework region "A" VH person minus Kabat CDRs by (SEQ ID NO:54, 48, 55, 35);

- acceptor framework region "B" and "C" VH person minus extended hypervariable regions (SEQ ID nos:50, 51, 55, 35; SEQ ID NO:50, 51, 56, 35);

- acceptor framework region 2 "A" VH person minus Kabat CDRs by (SEQ ID NO:54, 48, 57, 35);

- acceptor framework region 2 "B", "C" and "D" VH person minus extended hypervariable regions (SEQ ID nos:50, 51, 57, 35; SEQ ID NO:50, 51, 58, 35; SEQ ID NO:50, 51, 59, 35).

In figures 6A and 6B presents illustrative acceptor sequence of the variable light (VL) consensus framework region of a person for use in practice of the present invention with sequence identifiers as follows:

- a consensus of the frame region (κv1) subgroups I VL Kappa person: SEQ ID NO:60, 61, 62, 63;

- a consensus of the frame region (κv2) subgroup II VL Kappa person: SEQ ID NO:64, 65, 66, 63;

- a consensus of the frame region (κv3) subgroup III VL Kappa person: SEQ ID NO:67, 68, 69, 63;

- a consensus of the frame region (κv4) subgroup IV VL Kappa person: SEQ ID NO:70, 71, 72, 63.

The figure 7 presents the sequence of frame region light and heavy chains huMAb4D5-8. Numbers in Superscript indexes/bold indicate the position of amino acids according to Kabat.

On the figure 8 shows the sequence of frame region light and heavy chains huMAb4D5-8 with specified modifications. Numbers in Superscript indexes/bold indicate the position of amino acids according to Kabat.

The figure 9 presents the sequence of the variable region of the heavy chain (VH) YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. HVR underlined.

The figure 10 shows the sequence of the variable region of the light chain (VL) YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. VL-sequence gumanitarnogo monoclonal antibody 4D5-8 ("huMAb4D5-8") and "modified" huMAb4D5-8 is also represented in SEQ ID NO:31 and SEQ ID NO:26, respectively. YWO.32 and YWO.49 have the same VL sequence as "modified" VL huMAb4D5-8 (SEQ ID NO:26), which contains the following substitutions relative to SEQ ID NO:31: N30S, R66G and H91S. HVR underlined.

The figure 11 presents the alignment of sequences of the variable regions of the heavy chain YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. HVR are enclosed in boxes. The remains of HVR-H3 YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6, which differ from the corresponding residues HVR-H3 YWO.49, shaded.

The figure 12 presents the alignment of sequences of the variable region of the light chain YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. HVR are enclosed in boxes. The remains of the HVR-L3 YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6, which differ from the corresponding residues HVR-L3 YWO.49, shaded.

The figure 13 shows a graphical representation of the levels of expression of TAT226 person different who's tissues, as described in example A.

The figure 14 shows the graphical representation of the levels of expression of TAT226 person in normal ovary; normal fallopian tube; ovary cancer of clear, mucinous and serous subtypes of cystadenocarcinoma; metastatic cancer of ovary and other types of ovarian cancer, as described in example A.

Figure 15 presents the results of sorting cells with activation of fluorescence (FACS) OVCAR3 cells in the absence or presence of the indicated anti-TAT226 antibodies as described in example D.

The figure 16 shows the expression of mRNA and protein TAT226, as determined by analysis using 5'-nuclease (TaqMan®) and immunohistochemistry (IHC)performed on the OVCAR3 cells and panel samples of ovarian cancer, as described in example F.

Figure 17 presents the activity ofin vitrovarious conjugates antibody-drug (ADC) YWO.49.H2 and YWO.49.H6 in the analysis of the destruction of OVCAR3 cells as described in example H.

Figure 18 presents the activity ofin vitrodifferent ADC YWO.49.H2 and YWO.49.H6 in the analysis of cell killing using stable transfectants HCT116#9-4, as described in example H.

Figure 19 presents the activity ofin vivoADC YWO.49.H6 using xenografts in mice as described in example H.

Figure 20 presents the activity ofin vivoADC YWO.49.H6 using Xen the grafts in mice derived from tumors of patients-people, as described in example H.

DETAILED DESCRIPTION of embodiments of the INVENTION

The proposed selected antibodies that bind to TAT226. Additionally, the proposed immunoconjugate containing anti-TAT226 antibodies. Antibodies and immunoconjugates according to the invention are suitable, for example, for the diagnosis or treatment of disorders associated with altered expression of, for example, increased expression of TAT226. In some embodiments, the implementation of antibodies or immunoconjugate according to the invention is suitable for diagnosis or treatment of disorders of cell proliferation, such as tumor or cancer. In some embodiments, the implementation of antibodies or immunoconjugate according to the invention is suitable for detection TAT226, for example TAT226 expressed on the cell surface.

The proposed polynucleotide encoding an anti-TAT226 antibodies. The proposed vectors containing polynucleotide encoding an anti-TAT226 antibodies and proposed cell host containing such vectors. Also proposed compositions, including pharmaceutical compositions comprising any of one or more polynucleotides, anti-TAT226 antibodies or immunoconjugates according to the invention.

I. GENERAL METHODS

The methods and procedures described or cited here, as a rule, are well understood and applied in obsidiana the th practice using conventional methods specialists in this field, such as, for example, are widely used methods described in Sambrook et al.,Molecular Cloning: A Laboratory Manual3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Current Protocols in Molecular Biology(F. M. Ausubel et al. eds., (2003)); seriesMethods in Enzymology(Academic Press, Inc.):Pcr 2: A Practical Approach(M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane eds. (1988)Antibodies, A Laboratory ManualandAnimal Cell Culture(R. I. Freshney, ed. (1987));Oligonucleotide Synthesis(M. J. Gait, ed., 1984);Methods in Molecular Biology, Humana Press;Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture(R. I. Freshney), ed., 1987);Introduction to Cell and Tissue Culture(J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures(A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons;Handbook of Experimental Immunology(D. M. Weir and C. C. Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells(J. M. Miller & M. P. Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology(J. E. Coligan et al., eds., 1991);Short Protocols in Molecular Biology(Wiley and Sons, 1999);Immunobiology(C. A. Janeway and P. Travers, 1997);Antibodies(P. Finch, 1997);Antibodies: A Practical Approach(D. Catty., ed., IRL Press, 1988-1989);Monoclonal Antibodies: A Practical Approach(P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using Antibodies: A Laboratory Manual(E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999));The Antibodies(M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); andCancer: Principles and Practice of Oncology(V. T. DeVita et al., eds., J.B. Lippincott Company, 1993).

II. DEFINITIONS AND ABBREVIATIONS

Definitions A.

"Isolated" antibody is an antibody which we identify owano and isolated and/or extracted from a component of its natural environment. Contaminant components of its natural environment are materials that may obstruct the use of antibodies for the study, diagnosis, or treatment, and may include enzymes, hormones and other protein or non-protein solute. In some embodiments, the implementation of the antibody purified (1) to more than 95% of the mass. antibodies, as determined, for example, the Lowry method, and in some embodiments, the implementation of more than 99 wt. -%, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by, for example, sequencing machine with rotating cups, or (3) to homogeneity with SDS-PAGE in reducing or non conditions using, for example, Kumasi blue or silver staining. The selected antibody includes antibodyin situwithin recombinant cells, since in this case there is no at least one component of the natural environment antibodies. However, as a rule, the selected antibody receive through at least one stage of cleaning.

"Isolated" nucleic acid molecule is a nucleic acid molecule that is separated from at least one other nucleic acid molecule with which it is normally associated, for example, with the OEM natural environment. The selected nucleic acid molecule further includes a nucleic acid molecule contained in cells that normally Express the nucleic acid molecule, but the location of the nucleic acid molecules outside the chromosome or chromosome differs from its natural location on the chromosome.

"Purified" means that the molecule is present in the sample at a concentration of at least 95 wt. -%, or, at least 98% of the mass. the sample in which it is contained.

The term "essentially similar" or "essentially identical", as used herein, denotes a sufficiently high degree of similarity between two numeric values (for example, one value associated with the antibody according to the invention, and other value associated with a standard antibody/antibody for comparison) so that the specialist in this field can assume that the difference between the two values is negligible, or no biological and/or statistical significance within the context of the biological characteristic measured by using the specified values (e.g., Kd values). The difference between these two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20% and/or less than approximately 10% is a function from the standard value/value to compare.

The phrase "materially reduced" or "essentially different", as used herein, denotes a sufficiently high degree of difference between two numeric values (generally one value associated with the molecule, and another value that is associated with the standard molecule/molecule for comparison) so that the specialist in this field can assume that the difference between the two values is statistically significant in the context of the biological characteristic measured by using the specified values (e.g., Kd values). The difference between these two values is, for example, more than about 10%, more than about 20%, more than about 30%, more than about 40% and/or greater than about 50% as a function of the value for the standard molecule/molecule for comparison.

The term "vector"as used herein is intended to refer to nucleic acid molecules that can carry another nucleic acid to which it is associated. One type of vector is a "plasmid", which refers to the circular double-stranded DNA, which can be ligitamate additional segments of DNA. Another type of vector is a phage vector. Another type of vector is a viral vector, where additionally the s segments of DNA can be ligitamate in the viral genome. Certain vectors are capable of Autonomous replication in a cell host, in which they are administered (e.g., bacterial vectors having a bacterial replication origin and epilimnia vectors mammals). Other vectors (e.g., napisanie vectors mammals) can be integrated into the genome of a host cell upon introduction into the cell of the host, and thus, they are replicated together with the genome of the host. Moreover, certain vectors are capable of driving the expression of genes with which they are functionally linked. Such vectors are denoted herein as "recombinant expressing vectors" or simply "expressing vectors. As a rule, expressing the vectors used in the methods of recombinant DNA, are often in the form of plasmids. In the present description "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.

"Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substance that can be embedded into a polymer using DNA - Il is RNA polymerase, or using synthetic reactions. Polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogues. In the presence of modified nucleotide can be performed prior to Assembly of the polymer or after it. The nucleotide sequence can be interrupted dinucleotide components. Polynucleotide may contain the modification(s)obtained(s) after synthesis, such as conjugation with a label. Other types of modifications include, for example, "caps", substitution of one or more of the natural nucleotide analog, mezhnukleotidnyh modifications such as, for example, modification of uncharged bridges (for example, methylphosphonate, phosphotriesterase, potamididae, carbamates and the like) and charged bridges (for example, phosphorothioate, phosphorodithioate etc.), modification, containing suspended components, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine and the like), modification intercalating agents (e.g., acridine, psoralen and the like), modification, containing chelators (e.g., metals, radioactive metals, boron, metals, oxidizing agents, and the like), modification, containing alkylating agents, modification modified bridges (e.g., alpha anomeric nucleic acids, etc), as well as remodify the new forms of polynucleotide(s). Further, any hydroxyl group, usually present in sugars, can be replaced, for example, phosphonate groups, phosphate groups, protected by conventional protective groups or activated for more links with additional nucleotides, or they can be konjugierte with solid or semi-solid media. 5'- and 3'-terminal OH you can fosforilirovanii or replace amines or organic components of the blocking group of 1-20 carbon atoms. You can also get other derivatives of hydroxyl with conventional protective groups. Polynucleotides can also contain analogous forms of sugars ribose or deoxyribose, which are widely known in this field, including, for example, 2'-O-methyl, 2'-O-allyl, 2'-fluoro - or 2'-isidoros, carbocyclic analogues of sugars, α-anomeric sugars, epimeria sugars such as arabinose, xylose or lyxose, pyranose form of sugars, furanose form of sugars, sedoheptulose, acyclic analogs and basic nucleoside analogues, such as methylribose. One or more fosfolipidnyh relations can be replaced by alternative linking groups. These alternative linking groups include as non-limiting examples of variants of implementation, where the phosphate is replaced by P(O)S ("diatom"), P(S)S ("dayata"), "(O)NR2("amidate"), P(O)R, P(O)O', CO or CH2("formatitem"), in which each R or R' independently represents H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. You do not want all the links in polynucleotide was identical. The preceding description applies to all mentioned here polynucleotides, including RNA and DNA.

"Oligonucleotide", as used herein, generally refers to short, usually single-stranded, as a rule, synthetic polynucleotides that, typically, but not necessarily, consist of less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description for polynucleotides equally and fully applicable to oligonucleotides.

"Percent (%) amino acid sequence identity" with respect to the standard polypeptide sequence is defined as the percentage of amino acid residues in the sequence that are identical with amino acid residues in a standard polypeptide sequence, after aligning the sequences and, if necessary, the inclusion of passes to achieve the maximum percent identical to the t sequence, and not considering any conservative substitutions in the context of the identity sequence. Alignment for purposes of determining percent amino acid sequence identity can be obtained in various ways that are available to the person skilled in the art, for example, using publicly available software, such as software BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Specialists in this field can determine appropriate parameters for sequence alignment, including any algorithms needed to achieve maximal alignment over the entire full length of the compared sequences. However, for the purposes of this work the value of the % identity of amino acid sequence is obtained using a computer program to compare sequences ALIGN-2. A computer program for comparing sequences ALIGN-2 developed by Genentech, Inc., and the source code documentation for the user submitted to the U.S. Copyright Office, Washington DC, 20559, where it is registered under U.S. Copyright Registration no TXU510087. The program ALIGN-2 publicly available from Genentech, Inc., South San Francisco, California or may be compiled from the source program. The program ALIGN-2 should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All the parameters against which of the sequences are set by the program ALIGN-2 and do not change.

In situations where the use of ALIGN-2 for comparisons of amino acid sequence % identity of amino acid sequences for a given amino acid sequence A is compared with a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has a certain % amino acid sequence identity or include it in comparison with the given amino acid sequence B) is calculated as follows:

100 multiplied by the ratio X/Y,

where X represents the number of amino acid residues, marked as identical matches by the program for sequence alignment ALIGN-2 when aligning A and B of this program, and where Y represents the total number of amino acid residues in B. it Will be taken into account that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A compared to B will not equal the % amino acid sequence identity of B compared to A. Unless specifically stated otherwise, all values used here % identity of amino acid sequences obtained as described in the immediately preceding paragraph, use what Itanium computer program ALIGN-2.

The term "TAT226", as used herein, refers to any natural TAT226 received, unless specified otherwise, from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g. mice and rats). The term encompasses "full-size", reprezentirovanii TAT226, as well as any form of TAT226, which occurs when the processing in the cell. The term also includes natural options TAT226, for example splanirowannya variants or allelic variants. "Mature form" TAT226 is a form of TAT226 who has been subjected to processing, such as the form TAT226, which was subjected to N-terminal (for example, cleavage of the signal sequence) and/or C-terminal cleavage and/or modifications attach GPI-anchor. In one embodiment, the implementation of the "Mature form" TAT226 is expressed on the cell surface.

"Antibody" (Ab) and "immunoglobulin" (Ig) are glycoproteins with similar structural characteristics. While antibodies have binding specificity to a specific antigen, immunoglobulins include both antibodies and other antitelephone molecules, which, generally speaking, devoid of antigenic specificity. The polypeptides of the latter type, for example, are produced in low quantities lymphatic system and upgraded the s quantities myeloma.

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, polyspecific antibodies (for example, bespecifically antibodies provided that they possess the desired biological activity) and may also include certain antibody fragments (as described here in more detail). The antibody may be a chimeric, human, humanized and/or affinity-Mature.

The term "anti-TAT226 antibody" or "antibody that binds to TAT226" refers to an antibody that is capable of connecting to TAT226 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent upon exposure to TAT226. Preferably the degree of binding of anti-TAT226 antibodies not specific to TAT226 protein is less than about 10% of the binding of an antibody to TAT226 when measuring, for example, radioimmunoassay analysis (RIA). In some embodiments, the implementation of the antibody that binds to TAT226, has a dissociation constant (Kd) ≤ 1 ám ≤ 100 nm ≤ 10 nm, ≤ 1 nm or ≤ 0.1 nm. In some embodiments, the implementation of anti-TAT226 antibody binds to the epitope TAT226, which is what I am conservative to TAT226 of different types.

"Variable region" or "variable domain" antibody refers to aminocentesis the domains of the heavy or light chain antibodies. Variable domain of the heavy chain can be described as "VH". Variable domain light chain can be described as "VL". These domains usually are the most variable parts of the antibodies and contain antigennegative areas.

The term "variable" refers to the fact that the sequence of certain portions of the variable domains differ considerably among antibodies and are used in the binding and specificity of each particular antibody against the specific antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called scopes that define complementarity, (CDRs) or hypervariable regions (HVR), variable domains, both light chain and heavy chain. More highly conserved part of the variable domains are called the frame regions (FR). Each of the variable domains of the natural heavy and light chain contains four FR-region, largely host configuration beta layer, connected by three CDRs, which form loops connecting structure of beta-layers and in some cases forming part thereof. CDRs in each chain are located is ogeny together in close proximity with regions FR and together with other CDR circuits are involved in the formation antigennegative site of antibodies (see Kabat et al.,Sequences of Protein of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding the antibody to the antigen, but they have different effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species on the basis of amino acid sequences of their constant domains can be attributed to one of two clearly distinct types, called Kappa (κ) and lambda (λ).

Depending on the amino acid sequence of the constant domain of their heavy chains of antibodies (immunoglobulins) can be attributed to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA1and IgA2. The constant domains of the heavy chain, which correspond to the different classes of immunoglobulins are called α, δ, ε, γ and µ, respectively. Structures of subunits and three-dimensional configurations of different classes of immunoglobulins are well known and described in, for example, Abbas et al.Cellular and Mol. Immunology. 4th ed. (2000). The antibody may be part of a larger fused molecule, formed by covalent or non-covalent binding of an antibody to one and the and several other proteins or peptides.

The terms "full-size antibody", "intact antibody" and "whole antibody" are used here interchangeably to refer to antibodies in its essentially intact form, not in the form of fragments of antibodies, as defined below. The terms, in particular, relate to the antibody heavy chains that contain an Fc-region.

"Fragments of the antibodies contain only a portion of an intact antibody, where the portion stores at least one and maximum most or all of the functions normally associated with this part when it is present in an intact antibody. In one of the embodiments the antibody fragment contains antigennegative the site of the intact antibody and thus retains the ability to bind to the antigen. In another embodiment, the antibody fragment, for example a fragment which contains the Fc-region, retains at least one biological functions normally associated with the Fc-region, when it is present in an intact antibody, such as FcRn binding, modulation of time half-life of antibodies, ADCC function and binding of complement. In one embodiment, the implementation of a fragment of the antibody is a monovalent antibody, which hasin vivothe half-life, is essentially the same as the intact antibody. For example, such an antibody fragment may contain ant is enswathe shoulder, associated with the Fc sequence, can givein vivothe stability of the fragment.

Cleavage of antibodies with papain produces two identical antigenspecific fragment, called "Fab-fragments, each with a single antigennegative site, and a residual "Fc-fragment, whose name reflects its ability to easily crystallize. Treatment with pepsin yields F(ab')2-the fragment that has two antihistamine areas, and he is still capable of forming cross-linkage with the antigen.

"Fv" is the minimum antibody fragment which contains a complete antigennegative plot. In one embodiment, the implementation of double-stranded variant Fv consists of a variable domain of the dimer of one heavy and one light chain with a strong non-covalent bond. In the single-stranded species Fv (scFV) variable domain of one heavy and one light chain can be covalently linked by a flexible peptide linker, so that the light and heavy chains can be connected in a "dimeric" structure analogous to the structure of double-stranded varieties Fv. In this configuration, the three CDRs of each variable domain interact with the formation antigennegative area on the surface of the dimer VH-VL. In aggregate, the six CDR give antigennegative specificity is nitely. However, even a single variable domain (or half of an Fv, containing only three CDRs specific antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

Fab-fragment contains the variable domains of the heavy and light chains and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab'fragments differ from Fab fragments by the addition of a few residues at the C-end of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody. Fab'-SH is the designation for Fab'in which the residue(s) cysteine constant domains bear(ut) free thiol group. Antibody fragments F(ab')2the source was given as pairs of Fab'-fragments, which are articulated cysteine each other. Also other known chemical cross-linking of fragments of antibodies.

"Single-chain Fv" or "scFv-fragments of the antibodies contain the VH and VL domains of antibodies, where these domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun inThe Pharmacology of Monoclonal Antibodiesvol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, p. 269-315 (1994).

The term "dim rye antibody" refers to small fragments of antibodies with two antihistamine sites where fragments contain the variable domain of the heavy chain (VH)connected to the variable domain of the light chain (VL) in the same polypeptide chain (VH-VL). When using a linker that is too short to provide the pairing between the two domains on the same chain, the domains are forced to pair with complementary domains of another chain and create two antigenspecific plot. Dimeric antibodies may be bivalent or bespecifically. Dimeric antibodies are more fully described in, for example, in EP 404097; WO 93/1161; Hudson et al. (2003) Nat. Med. 9:129-134; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). The trimeric antibodies and four-dimensional antibodies are also described in Hudson et al. (2003) Nat. Med. 9:129-134.

The term "monoclonal antibody"as used herein refers to an antibody obtained from a population essentially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, such as naturally occurring mutations that may be present in minor amounts. Thus, the definition of "monoclonal" indicates the character of the antibodies, because they are not a mixture of separate antibodies. In some embodiments, the implementation of such monoclonal antibody generally includes the antibody containing polypeptide posledovatel the face, which binds the target where the binding target polypeptide sequence is received by a process that includes the selection of one binds a target polypeptide sequence from a set of polypeptide sequences. For example, the selection process can be a selection of a particular clone from a variety of clones, such as the combination of hybridoma clones, phage clones or clones of recombinant DNA. It should be understood that the selected binding the target sequence can be further modified, for example, to increase the affinity against the target for the humanization of binding the target sequence, to improve its products in cell culture, to reduce its immunogenicityin vivoto create a polyspecific antibodies, etc. and that the antibody containing the modified binding the target sequence, is also a monoclonal antibody of the present invention. In contrast to the preparations of polyclonal antibodies, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in the preparation of monoclonal antibodies directed against the same antigen determinants. In addition to their specificity preparations of monoclonal antibodies have p what ekosistem is they usually do not contain impurities of other immunoglobulins.

The definition of "monoclonal" indicates the sign of antibodies that get essentially homogeneous population of antibodies, and does not imply that the antibody must be obtained to any particular method. For example, monoclonal antibodies intended for use in accordance with the present invention, can be obtained in a variety of ways, including, for example, a hybrid method (for example, Kohler et al., Nature, 256:495 (1975); Harlow et al.,Antibodies: A Laboratory Manual(Cold Spring Harbor Laboratory Press, 2nded. 1988); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N. Y., 1981)), by way of recombinant DNA (see, for example, U.S. patent No. 4816567), the technology of phage display (see, for example, Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol, 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol., 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods 284(1-2):119-132 (2004), and methods for the production of human antibodies or antibodies, such antibodies man, in animals that have part or all of the loci of the human immunoglobulin genes or coding sequences of human immunoglobulin (see, for example, WO98/24893; WO96/34096; WO96/33735; WO91/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. patent No. 5545807; 5545806; 5569825; 525126; 5633425; 5661016; Marks et al., Bio. Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

Monoclonal antibodies in the present description, in particular, include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a specific class or subclass of antibody, while the remainder of the chain(s) is(are) identical(s) or homologous(s) corresponding sequences in antibodies derived from another species or belonging to another class or subclass antibodies as well as fragments of such antibodies, provided that they possess the desired biological activity (patent U.S. No. 4816567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

"Humanized" forms of non-human (e.g. murine) antibodies are chimeric antibodies that contain minimal sequence derived from the immunoglobulin, which is not human. In one of the embodiments humanitariannet antibody is a human immunoglobulin (recipient antibody)in which residues of the hypervariable region of the recipient to replace the remnants of the ISU is rvariables area species non-human (donor antibody)such as mouse, rat, rabbit or non-human Primate, which has the desired specificity, affinity and/or activity. In some cases, remnants of the framework region (FR) of a human immunoglobulin replace the corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not present in the recipient antibody or in the donor antibody. These modifications can be done to further improve the properties of the antibodies. As a rule, humanitariannet antibody contains essentially all, of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to loops immunoglobulin, non-human, and all or substantially all of the FR represent the sequence of FR of a human immunoglobulin. Also humanized antibodies, not necessarily contain at least part of a constant region (Fc) of an immunoglobulin, typically a constant region of human immunoglobulin. For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Cm. the following cited here, review articles and links: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); arris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

"Human antibody" is an antibody that has an amino acid sequence that corresponds to the sequence of the antibody produced by the person, and/or received using any of the methods for producing human antibodies, as described in this paper. This definition of human antibodies, in particular, excludes humanitariannet antibody containing non-human antigennegative remains.

The term "hypervariable region", "HVR" or "HV" when used in this description refers to the regions of the variable domain of antibodies, which are hypervariable in sequence and/or form a structured loops. Typically, antibodies contain six hypervariable regions; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In natural antibodies H3 and L3 have the greatest diversity of the six hypervariable regions, and suggest that H3, in particular, plays a crucial role in giving fine specificity of the antibodies. Xu et al. (2000) Immunity 13:37-45; Johnson and Wu (2003) in Methods in Molecular Biology 248:1-25 (Lo, ed., Humana Press, Totowa, NJ). Indeed, existing in the nature of the antibodies of camelids, consisting only of heavy chains are functional and stable in the absence of light chain. Hamers-Castrman et al. (1993) Nature 363:446-448; Sheriff et al. (1996) Nature Struct. Biol. 3:733-736.

Use a range of definitions hypervariable region, and they are considered in this description. By Kabat, region complementarity determining (CDR), determined on the basis of the variability of the sequences and their use most often (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Instead, Chothia refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). Hypervariable region AbM are a compromise between Kabat CDRs by and structural hinges on Chothia, and their use in software for modeling antibodies Oxford Molecular's AbM. "Contact" hypervariable region determined on the basis of analysis of available crystal structures of the complexes. The remnants of each of these hypervariable regions are highlighted below.

LoopKabatAbMChothiaContact
L1L24-L34L24-L34L26-L32L30-L36
L2L50-L56 L50-L56L50-L52L46-L55
L3L89-L97L89-L97L91-L96L89-L96
H1H31-H35BH26-H35BH26-H32H30-H35B
Numbering according to Kabat
H1H31-H35H26-H35H26-H32H30-H35
Chothia numbering
H2H50-H65H50-H58H53-H55H47-H58
H3H95-H102H95-H102H96-H101H93-H101

Hypervariable region may contain "extended hypervariable region"as follows: 24-36 or 24-34 (L1), 46-6 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2)and 93-102, or 95-102 (H3) in the VH. The remains of the variable domain are numbered according to Kabat et al. above for each of these definitions.

"Framework" or "FR" residues are those residues of the variable domain, which differ from the residues of the hypervariable region, as defined in this specification.

The term "residue numbering variable domain, as in Kabat" or "numbering provisions of amino acids, as in Kabat" and its variation refers to the numbering system used for the variable domains of the heavy chain or variable light chain domains, from the collection of the data antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). When using this numbering system the actual linear amino acid sequence may contain fewer amino acids or more amino acids, which corresponds to the shortening FR or HVR variable domain or paste in them. For example, the variable domain of the heavy chain may contain a single insertion of amino acids (residue 52a according to Kabat) after residue 52 H2 and inserted residues (e.g. residues 82a, 82b and 82c, etc. according to Kabat) after residue 82 heavy chain FR. The residue numbering according to Kabat you can define for a given antibody by flattening the areas of homologisation antibodies with the "standard" are numbered according to Kabat sequence.

"Affinity-matured" antibody is an antibody with one or more changes in one or more of their HVR, which leads to increased affinity of antibodies against the antigen, compared to a parent antibody which does not have these(and) change(s). In one embodiment, the implementation of the affine-Mature antibody possesses nanomolar or even picomolar values of affinity against the target antigen. Affine-Mature antibodies produced using methods known in this field. In Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by the rearrangement of VH and VL domains. Random mutagenesis of residues in the HVR and/or frame residues described in Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169: 147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

"Blocking" antibody or antibody antagonist" is an antibody that inhibits or reduces biological activity of the antigen with which it is associated. Specific blocking antibodies or antibody antagonist essentially or completely inhibit the biological activity of the antigen.

"Antibody agonist", as used herein, is an antibody that mimicries, at least one of the functional activities of int representing the Republic of the polypeptide.

"Effector functions" antibodies refer to such kinds of biological activity which is inherent in the Fc-region (natural sequence Fc region or a variant amino acid sequence Fc region) of the antibody, and vary depending on the isotype of the antibody. Examples of effector functions of antibodies include C1q binding and complementability cytotoxicity; binding Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; negative regulation of cell surface receptors (e.g. B cell receptor); and activation of B-cells.

The terms "Fc receptor" or "FcR" describe a receptor that binds to the Fc region of antibodies. In some embodiments, the implementation of FcR is a natural FcR person. In some embodiments, the implementation of FcR is a receptor that binds to an IgG antibody (a gamma receptor) and includes receptors of the subclasses of the FcγRI, FcγRII and FcγRIII, including allelic variants and alternatively splanirowannya forms of these receptors. The FcγRII receptors include FcγRIIA (an"activating receptor") and FcγRIIB (an"inhibiting receptor"), which have similar amino acid sequences that differ primarily by their cytoplasmic domains. Activating receptor FcγRIIA in its cytoplasmic domain contains anoreceptive activating motif-based tyrosine (ITAM). Inhibiting receptor FcγRIIB in its cytoplasmic domain contains immunoreceptor inhibitory motif-based tyrosine (ITIM) (see Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcR reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). The term "FcR" in the present description include other FcR, including FcR, which will be identified in the future.

The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.24:249 (1994)) and the regulation of homeostasis of immunoglobulins. Known methods of measuring binding to FcRn (see, e.g., Ghetie 1997, Hinton 2004). You can analyze the binding to human FcRnin vivoand the half-life in serum polypeptides with high affinity binding to human FcRn, for example, in transgenic mice or transfected with the human cell lines expressing human FcRn, or primates, which enter the polypeptide variants of the Fc.

In WO00/42072 (Presta) described variants of the antibodies with increased or reduced binding to FcR. The contents of this patent publication, in particular, contained in this document as a reference. Cm. also Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

"Effector cells" are leukocytes which Express one or more what about the FcR and perform effector functions. In some embodiments, the implementation of the cells Express at least FcγRIII and perform effector(s) function(s) ADCC. Examples of human leukocytes which carry out ADCC include mononuclear cells of peripheral blood (PBMC), natural killer cells (NK), monocytes, cytotoxic T cells and neutrophils. Effector cells can be distinguished from native source, for example from the blood.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which the secretory Ig associated with Fc-receptors (FcR)present on certain cytotoxic cells (e.g., natural cells killer cells (NK), neutrophils and macrophages), specific causes for these cytotoxic effector cells to bind to the carrier antigen-cell target, and then they destroy the target cell by means of cytotoxins. The basic cell for the implementation of the ADCC, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is summarized in table 3 on page 464 in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To evaluate the activity of interest molecules against ADCC can analyze ADCCin vitrosuch as analysis, described in U.S. patent No. 5500362 or 5821337 or in U.S. patent No. 6737056, Presta. Suitable for that is their analyses of effector cells include mononuclear cells of peripheral blood (PBMC) and natural killer cells (NK). Alternative or additional activity of interest molecules against ADCC can be evaluatedin vivofor example, in animal models, such as described in Clynes et al. PNAS (USA) 95:652-656 (1998).

"Complementability cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement cascade begins with the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass)that are associated with them recognizable antigen. To assess activation of complement can be analyzed by the CDC, for example, as described in Gazzano-three-bet et al., J. Immunol. Methods 202:163 (1996).

Variants of polypeptides with altered amino acid sequences of the Fc-region and increased or decreased ability to bind C1q described in U.S. patent No. 6194551B1 and WO99/51642. The contents of these patent publications, particularly herein by reference. Cm. also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

The term "polypeptide containing an Fc region" refers to the polypeptide, such as an antibody or immunoadhesin, which contains the Fc-region. C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during purification of the polypeptide or by recombinant engineering of nucleic acid that encodes a polypeptide. Thus, the comp is position, containing polypeptide with Fc-region according to this invention may include polypeptides with K447 all of the remote K447, or a mixture of polypeptides with the K447 residue and without it.

"Acceptor framework region of a person" for the purposes of this work is a frame region containing the amino acid sequence of framework region VL or VH derived from a frame region of a human immunoglobulin or a consensus of the frame area. The acceptor framework region of a human, "originating from" framework region of human immunoglobulin or consensus framework region of a human, can contain the same as they, amino acid sequence, or it may contain pre-existing changes in amino acid sequence. In some embodiments, the implementation of a number of preexisting changes of amino acids is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. Where pre-existing amino acid changes are present in VH, it is preferable that these changes occurred only in three, two or one of the provisions 71H, 73H and 78H; for example, amino acid residues in these positions can represent 71A, 73T and/or 78A. In one of the embodiments of the acceptor frame region is here VL person identical in sequence with the sequence frame of the VL region of a human immunoglobulin or the consensus sequence of frame area.

"The consensus of the frame region of man" is the frame area, which represents the most frequently occurring amino acid residues at the selection sequence frame region VL or VH of a human immunoglobulin. Typically, selection of the sequences of the VL or VH immunoglobulin exercise of subgroups of sequences of the variable domain. Generally, the subgroup of sequences is a subgroup as in Kabat et al.,Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD (1991). In one of the embodiments for VL subgroup is a subgroup Kappa I, as in Kabat et al., above. In one of the embodiments for VH subgroup is a subgroup III, as in Kabat et al., above.

"The consensus frame region VH subgroup III contains a consensus sequence derived from the amino acid sequences of variable subgroup III heavy chain according to Kabat et al., above. In one embodiment, the implementation of the amino acid sequence of the consensus frame region VH subgroup III contains at least part or all of any of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:50)-H1-WVRQAPGKGLEWV (SEQ ID NO:51)-H2-RFTISADTSKNTAYLQMNSLRAEDTAVYYC (SEQ ID NO:59)-H3-WGQGTLVTVSS (SEQ ID NO:35).

"The consensus frame region VL subgroup I contains a consensus on sledovatelnot, derived from the amino acid sequences of variable subgroup I light chain Kappa by Kabat et al., above. In one embodiment, the implementation of the amino acid sequence of the consensus frame region VL subgroup I contains at least part or all of any of the following sequences: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:60)-L1-WYQQKPGKAPKLLIY (SEQ ID NO:61)-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:62)-L3-FGQGTKVEIK (SEQ ID NO:63).

"The binding affinity of"generally refers to the force of the total amount of non-covalent interactions between a single binding site of a molecule (e.g. antibody) and communicating with it by a partner (e.g., antigen). Unless otherwise specified, as used herein, "binding affinity of" refers to the self-affinity of binding, which reflects the interaction of 1:1 between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X with respect to its partner Y can usually presented via dissociation constants (Kd). The affinity can be measured by using common methods known in this field, including those described herein ways. Antibodies with low affinity, usually associated with antigen slowly and have a tendency to mild dissociation, whereas antibodies with high affinity, usually associated with antigen b is faster and tend to stay longer linked. Many ways to measure the affinity of binding is known in this field, any of which can be used for the purposes of the present invention. Specific illustrative embodiments of described below.

In one embodiment, the implementation of the "Kd" or "Kd value" according to this invention is measured by analyzing the binding of radioactively labelled antigen (RIA)performed with the Fab version of interest antibody and its antigen as described by the following analysis. The binding affinity of the solution to Fab in respect of the antigen is measured by balancing Fab minimum concentration (125I)-labeled antigen in the presence of a serial dilution of unlabeled antigen with subsequent removal of the associated antigen on the tablet coated with an antibody to Fab (Chen et al. (1999) J. Mol. Biol. 293:865-881). To determine the conditions of the analysis of tablets for micrometrology (Dynex) cover during the night of 5 μg/ml capture antibody anti-Fab (Cappel Labs) in 50 mm sodium carbonate (pH 9,6) and then blocked with 2% (wt./about.) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In readsorbing tablet (Nunc #269620) 100 PM or 26 PM [125I]-antigen are mixed with serial dilutions of interest Fab (for example, in accordance with the assessment and is Titel anti-VEGF, Fab-12, in Presta et al. (1997) Cancer Res. 57:4593-4599). Interest Fab then incubated over night; however, the incubation can be continued for a longer period (for example, 65 hours)to ensure achievement of equilibrium. After this mixture is transferred into a tablet for capture to incubate at room temperature (for example, within one hour). The solution is then removed and the plate washed eight times with 0.1% tween-20 in PBS. After the tablets are dried, add the scintillator (MicroScint-20; Packard) at the rate of 150 μl/well and take readings with tablets on the gamma counter Topcount (Packard) for ten minutes. The concentration of each Fab, which give less than 20% or equal to 20% of the maximum binding is chosen for use in the analysis of competitive binding.

According to another variant implementation of the Kd or Kd value is measured by use of methods of analysis based on surface plasmon resonance using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with CM5 chips with immobilized antigen at ~10 units resonance signal (RU). In summary, biosensor chips with carboxyethylgermanium dextran (CM5, BIAcore Inc.) activate the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. Antigen-fu is completed with 10 mm sodium acetate, pH of 4.8, to 5 μg/ml (~0.2 μm) before injection with flow rate of 5 μl/minute to achieve approximately 10 units of the resonance signal (RU) associated protein. After injection of the antigen 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements twofold serial dilution of Fab (0.78 nm to 500 nm) in PBS with 0.05% tween-20 (PBST) is injected at 25°C at a flow rate of approximately 25 μl/min Rate of Association (konand the rate of dissociation (koff) is calculated using a simple model linking one-to-one the Langmuir (software to calculate BIAcore, version 3.2) by simultaneous approximation sensogram Association and dissociation. The equilibrium dissociation constant (Kd) is calculated as the ratio of koff/kon. See, for example, Chen, Y. et al. (1999) J. Mol. Biol. 293:865-881. If the velocity of the Association exceed 106M-1with-1in the above analysis based on surface plasmon resonance, the velocity of the Association can be determined using the method of quenching the fluorescence, which measures the increase or decrease of the intensity of the emitted fluorescence (excitation = 295 nm; emission = 340 nm, a bandwidth of 16 nm) at 25°C 20 nm of the antibody to the antigen (Fab form) in PBS, pH of 7.2, in the presence of increasing concentrations of antigen in the measurement in spectromet is e, such as a spectrophotometer, equipped with a stopped stream (Aviv Instruments)or a spectrophotometer SLM-Aminco 8000 series (ThermoSpectronic) with a stirred cuvette.

"Rate of Association"or "speed link", or "kon"according to the invention can also be defined, as described above, using the BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ).

"Violation" is any condition or illness, which may have a beneficial effect of the treatment substance/molecule or method according to the invention. It includes chronic and acute disorders, including pathological conditions that make the mammal is predisposed to the disorder. Non-limiting examples of disorders to be treated according to this description, include cancerous conditions, such as tumors, such as carcinomas (epithelial tumors) and blastoma (embryonal tumors arising from tissues), and in some embodiments, the implementation of ovarian carcinoma, uterine (including endometrial cancer), brain tumor (e.g., astrocytomas and gliomas) and kidney cancer, including neuroblastoma (e.g., Wilms tumor).

The terms "impaired cell proliferation and proliferative violation" refers to disorders that are associated with some degree of pathological proliferation kletok.v one of the embodiments violation of cell proliferation is a cancer.

"Tumor", as used herein, refers to any neoplastic growth and proliferation of cells, or malignant or benign, and any pre-cancerous and cancerous cells and tissues. As used herein, the terms "cancer", "cancer", "violation of cell proliferation", "proliferative violation" and "tumor" are not mutually exclusive.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically differs unregulated cell growth/proliferation. Examples of cancer include, without limitation, carcinoma, lymphoma (e.g., Hodgkins and nahodkinskuju lymphoma), blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, liver cell cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, cancer of the colon and rectum, carcinoma of the endometrium or uterine carcinoma of the salivary glands, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, the hepatocarcinoma, leukemia and other lymphoprolif is exploring disorders and various types of head and neck cancer.

As used in the present description, the "treatment" (and variations such as "treat" or "treatment"refers to clinical intervention aimed at changing the natural course of the disease of the individual or cell being treated, and it can be done or for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, decrease the speed of disease progression, improvement or temporary relief of the disease state, and remission or improved prognosis. In some embodiments, the implementation of the antibodies according to the invention are used to delay development of a disease or impairment of, or slowing the progression of disease or impairment.

"Individual" is a vertebrate animal. In certain embodiments of the implementation of the vertebrate is a mammal. Mammals include as non-limiting examples of farm animals (such as cows), animals used in sports, Pets (such as cats, dogs and horses), primates, mice and rats. In certain embodiments of the implementation of the mammalian what the future is the man.

"Effective amount" refers to an amount effective, at dosages and within the required time periods to achieve the desired therapeutic or prophylactic result.

"Therapeutically effective amount" of a substance/molecule of the invention can vary due to factors such as the disease state, age, sex and weight of the individual and the ability of a substance/molecule to cause the desired response in the individual. Therapeutically effective amount includes an amount by which any toxic or detrimental effects of the substance/molecule were outweighed by therapeutically beneficial effects. "Prophylactically effective amount" refers to an amount effective, at dosages and within the required time periods to achieve the desired prophylactic result. Typically, but not necessarily as a prophylactic dose is used in subjects prior to disease or at its early stage, the prophylactically effective amount will be less than therapeutically effective amount.

The term "cytotoxic agent"as used in the present description, refers to a substance that inhibits the functioning of cells or stops it and/or cause the death or destruction of cells. It is assumed that the term includes adioactive isotopes (for example, At211I131I125, Y90That Re186That Re188Sm153Bi212, P32, Pb212and radioactive isotopes of Lu), chemotherapeutic agents (e.g., methotrexate, adriamicin, Vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating tools, enzymes and fragments thereof such as nucleotidase enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants, and the various antitumor or anticancer means, described below). Other cytotoxic tools described below. Comorienne means causes destruction of tumor cells.

Toxin is any substance capable of exerting a negative impact on the growth or proliferation of the cell.

"Chemotherapeutic agent" is a chemical compound suitable for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide CYTOXAN®; alkyl sulphonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodepa, carboquone, matureup and uredepa; etilene the us and methylmelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenemelamine; acetogenins (especially bullatacin, bullatacin); Delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; Betulinol acid; camptothecin (including the synthetic analogue topotecan (COSMEGEN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcystein, scopoletin and 9-aminocamptothecin); bryostatin; callistemon; CC-1065 (including its synthetic analogues of adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllin acid; teniposide; cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard gas analogues, such as chlorambucil, chlornaphazine, halophosphate, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novemberin, finestein, prednimustine, trofosfamide, uramustine; nitrosoanatabine, such as carmustine, chlorozotocin, fotemustine, lomustin, nimustine and ranimustine; antibiotics such as andinavia antibiotics (for example, calicheamicin, especially calicheamicin gamma and calicheamicin omega (see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994)); dynemicin, including dynemicin A, es is eromycin; as well as the chromophore neocarzinostatin and similar chromophores chromoproteins indinavir antibiotics, aclacinomycin, actinomycin, autralian, azaserine, bleomycin, actinomycin, carubicin, karminomitsin, calcination, chromomycin, dactinomycin, daunorubicin, distrubition, 6-diazo-5-oxo-L-norleucine, doxorubicin ADRIAMYCIN®(including morphosyntactical, cyanomethaemoglobin, 2-pyrrolidinecarbonyl and desoxidation), epirubicin, zorubicin, idarubitsin, marsellaise, mitomycin, such as mitomycin C, mycofenolate acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, colomycin, radiobeacon, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); analogs of folic acid, such as deeperin, methotrexate, peripherin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, timipre, tioguanin; pyrimidine analogues such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; means, depressing the function of the adrenal cortex, such as aminoglutethimide, mitotane, trilostane; compensator folic acid, is the aka as folinovaya acid; Eagleton; glycoside aldophosphamide; aminolevulinic acid; eniluracil; amsacrine; astranomical; bisantrene; edatrexate; defaming; demecolcine; diazinon; eflornithine; the acetate slipline; epothilone; etoposide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoid, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitrogen; pentostatin; penomet; pirarubicin; losoxantrone; 2-acylhydrazides; procarbazine; polysaccharide complex PSK®(JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trihlortrietilamin; trichothecenes (especially toxin T-2, verrucarin a, roridin a and unguided); urethane; vindesine (ELDISINE®, FIELDSIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; Galitsin; arabinoside ("Ara-C"); thiotepa; taxoid, such as paclitaxel (TAXOL®(Bristol-Myers Squibb Oncology, Princeton, NJ.), designed on the basis of the albumin preparation of nanoparticles of paclitaxel, without cremophor, ABRAXANE™ (American Pharmaceutical Partners, Schaumberg, Illinois) and docetaxel TEXTER®(Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR®); 6-tioguanin; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; adopted (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVINthe ), oxaliplatin; leucovorin; vinorelbine (NAVELBINE®); Novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; deformational (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

Also in this definition included antihormone funds, which act through regulation, decreasing, blocking or inhibiting effects of hormones that can stimulate the growth of cancer tumors, and they are often used in the form of systemic treatment or treatment of the whole body. They can be the hormones. Examples include antiestrogens and selective modulators of estrogen receptors (SERM), including, for example, tamoxifen (including tamoxifen NOLVADEX®), raloxifene EVISTA®droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene FARESTON®; antiprogesterone; negative regulators of estrogen receptors (ERD); means the, in operation, braking or stopping the functioning of the ovaries, such as releasing hormone agonists of luteinizing hormone (LHRH), such as LUPRON®and leuprolide acetate, ELIGARD®, goserelin acetate, buserelin acetate and triptorelin; other anti-androgens such as flutamide, nilutamide and bikalutamid; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutetimid, megestrol acetate MEGAS®, exemestane AROMASIN®, formestane, fadrozole, vorozole REVISOR®, letrozole FEMARA®and anastrozole ARIMIDEX®. In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS®or OSTAC®), etidronate DIDROCAL®, NE-58095, zoledronicaa acid/zoledronate ZOMETA®, alendronate FOSAMAX®, pamidronate AREDIA®tiludronate SKELID®or risedronate ACTONEL®; and troxacitabine (the analogue of the nucleoside cytosine-based 1,3-dioxolane); antisense oligonucleotides, particularly oligonucleotides that inhibit expression of genes in signaling pathways implicated in impaired cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and gene receptor epider the social growth factor (EGF-R); vaccines, such as vaccines CERATOP®vaccines and for gene therapy, for example a vaccine ALLOVECTIN®vaccine LEUVECTIN®and vaccine WAXED®; topoisomerase inhibitor 1 LURTOTECAN®; ABARELIX®rmRH; lapatinib ditosylate (low molecular weight dual inhibitor tyrosinekinase ErbB-2 and EGFR, also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any of the above.

"A means of inhibiting the growth" when used in this specification refers to a compound or composition which inhibits growth of the cell (such as cell expressing TAT226) orin vitroorin vivo. Thus, a means of inhibiting the growth can be a tool that significantly reduces the percentage of cells (such as cell expressing TAT226) in S-phase. Examples of means of inhibiting the growth include tools that block the passage of the cell cycle (at a different stage compared to S-phase), such as tools, which induce a stop in G1 and stop in M-phase. Classic blockers M-phases include the Vinca alkaloids (vincristine and vinblastine), taxanes and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. The means that cause a stop in G1, as well as a side result is to provide a stop in S-phase, for example tools, alkylating DNA, such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and Ara-C. further information can be found inThe Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs", Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs, both obtained from Tissa. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®Bristol-Myers Squibb). Paclitaxel and docetaxel stimulate the Assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization, resulting in the inhibition of mitosis in cells.

The term "intracellular metabolite" refers to the compound formed in a metabolic process or reaction inside a cell of the conjugate antibody-drug (ADC). Metabolic process or reaction may be enzymatic processes, such as proteolytic cleavage of the peptide linker of the ADC, or the hydrolysis of functional groups, such as hydrazone, ester or amide. Intracellular metabolites include as non-limiting examples of antibody and drug free cf is the rotary, which are intracellular cleavage after receipt, diffusion, absorption or transport into the cell.

The term "intracellular split" and "intracellular cleavage" refers to a metabolic process or reaction inside a cell to conjugate the antibody-drug (ADC), in which covalent bridge, i.e. the linker between the component drugs (D) and antibody (Ab) is destroyed, which leads to dissociation of the free drug and the antibody inside the cell. Split ADC components, therefore, are intracellular metabolites.

The term "bioavailability" refers to the system availability (i.e. levels of blood/plasma) given the number of drugs administered to the patient. Bioavailability is an absolute term that indicates the measurement time (speed) for the medicinal product and its total amount (extent)that achieves the overall flow of the entered dosage forms.

The term "cytotoxic activity" refers to the effect of the destruction of the cells, the cytotoxic effect or the effect of inhibiting the growth to conjugate the antibody-drug or intracellular metabolite conjugate antibody-drug. Cytotoxic activity can be expressed in the de values IC 50, which represents the concentration (molar or mass) per unit volume, which remains alive half cells.

"Alkyl" represents a hydrocarbon, C1-C18containing normal, secondary, tertiary or cyclic carbon atoms. Examples include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, isopropyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, isobutyl, -CH2CH(CH3)2), 2-butyl (s-Bu, sec-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, tert-butyl, -C(CH3)3), 1 pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), The 4th is Teal-2-pentyl (-CH(CH 3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3.

The term "C1-C8alkyl", as used herein, refers to an unbranched or branched, saturated or unsaturated hydrocarbon containing from 1 to 8 carbon atoms. Representative "C1-C8alkyl groups" include as non-limiting examples methyl, -ethyl, -n-propyl, n-butyl, -n-pentyl, n-hexyl, n-heptyl, -n-octyl, ' n ' nonyl and-n-decyl; while branched C1-C8alkali include as non-limiting examples,- isopropyl, -sec-butyl, -isobutyl, -tert-butyl-isopentyl, 2-methylbutyl, unsaturated C1-C8alkali include as non-limiting examples,- vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylene, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -PROPYNYL, -1-butynyl, -2-butynyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butynyl, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl is l, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-etylhexyl, 2,2-dimethylpentyl, 2,4-dimethylpentyl, 2.5-diethylhexyl, 3,5-dimethylhexane, 2,4-dimethylpentyl, 2-methylheptan, 3-methylheptan, n-heptyl, isoheptyl, n-octyl and isooctyl. C1-C8the alkyl group may be unsubstituted or substituted by one or more groups, including as non-limiting examples of-C1-C8alkyl, -O- (C1-C8alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)other', -C(O)N(R')2-NHC(O)R', -SO3R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2and-CN; where each R' is independently selected from H, -C1-C8of alkyl and aryl.

"Alkenyl" represents a C2-C18the hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one unsaturated site, i.e. a carbon-carbon doublesp2-communication. Examples include, without limitation: ethylene or vinyl (-CH=CH2), allyl (-CH2CH=CH2), cyclopentenyl (-C5H7) and 5-hexenyl (-CH2CH2CH2CH2CH=CH2).

"Quinil" represents a C2-C18the hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one unsaturated site, i.e. the carbon is low carbon, triplesp-communication. Examples include, without limitation: acetylenic (-C≡CH) and propargyl (-CH2C≡CH).

"Alkylene" refers to a saturated, branched or unbranched, or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two monovalent radical centers, which is obtained by removing two hydrogen atoms from the same or two different carbon atoms of the alkane source. Typical alkylene radicals include as non-limiting examples include methylene (-CH2-) 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2and so on

"C1-C10alkylene" is an unbranched, saturated hydrocarbon group of the formula -(CH2)1-10-. Examples of C1-C10alkylene include methylene, ethylene, propylene, butylene, pentile, hexylen, reptile, octiles, Nonlin and decalin.

"Albaniles" refers to an unsaturated, branched or unbranched, or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers, which is obtained by removing two hydrogen atoms from the same or two different carbon atoms of the original alkene. Typical alkenylamine radicals in luchot as non-limiting examples: 1,2-ethylene (-CH=CH-).

"Akinyan" refers to an unsaturated, branched or unbranched, or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers, which is obtained by removing two hydrogen atoms from the same or two different carbon atoms of the original alkyne. Typical akinleye radicals include as non-limiting examples: acetylene (-C≡C-), propargyl (-CH2C≡C-) and 4-pentenyl (-CH2CH2CH2C≡C-).

"Aryl" refers to a carbocyclic aromatic group. Examples of aryl groups include as non-limiting examples of phenyl, naphthyl and anthracene. Carbocyclic aromatic group or heterocyclic aromatic group may be unsubstituted or substituted by one or more groups, including as non-limiting examples of-C1-C8alkyl, -O-(C1-C8alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, - C(O)other', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), - N(R')2and-CN; where each R' is independently selected from H, -C1-C8of alkyl and aryl.

"Allen" represents an aryl group which has two covalent bonds and can be in ortho-, meta - or preconfiguration, as shown in the following the x structures:

in which the phenyl group may be unsubstituted or substituted groups, up to four, including as non-limiting examples of-C1-C8alkyl, -O-(C1-C8alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, - C(O)other', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), - N(R')2and-CN; where each R' is independently selected from H, -C1-C8of alkyl and aryl.

"Arylalkyl" refers to an acyclic alkyl, the radical, in which one of the hydrogen atoms associated with carbon atom, as a rule, limit, orsp3-carbon atom, substituted aryl radical. Typical arylalkyl groups include as non-limiting examples are benzyl, 2-Penilaian-1-yl, 2-Penilaian-1-yl, naphthylmethyl, 2-Nettleton-1-yl, 2-naphthalate-1-yl, naphthalenyl, 2-naphthenate-1-yl, etc. Arylalkyl group contains from 6 to 20 carbon atoms, for example an alkyl group, including albanello, alkenylphenol or alkenylphenol group, arylalkyl group consists of 1-6 carbon atoms, and aryl group consists of 5-14 carbon atoms.

"Heteroaromatic" refers to an acyclic alkyl, the radical, in which one of the hydrogen atoms associated with carbon atom, as a rule, limit, orsp3-carbon atom, is substituted by heteroaryl radical. Typical heteroallyl groups include as non-limiting examples of 2-benzimidazolylthio, 2-purolater etc. Heteroallyl group contains from 6 to 20 carbon atoms, for example an alkyl group, including albanello, alkenylphenol or alkenylphenol group, heteroallyl group consists of 1-6 carbon atoms, and a heteroaryl group consists of 5-14 carbon atoms, and from 1 to 3 heteroatoms selected from N, O, P and S. the Heteroaryl component heteroallyl group may be a monocycle containing from 3 to 7 ring elements (2-6 carbon atoms) or Bicycle containing from 7 to 10 elements of the ring (4-9 carbon atoms, and from 1 to 3 heteroatoms selected from N, O, P and S), for example, the system bicyclo[4.5], [5.5], [5.6] or [6.6].

"Substituted alkyl", "substituted aryl" and "substituted arylalkyl" mean respectively an alkyl, aryl and arylalkyl in which any one or more hydrogen atoms are independently replaced with a substituent. Typical substituents include as non-limiting examples of-X, -R, -O-, -OR, -SR, -S-, -NR2, -NR3, =NR, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, NC(=O)R, -C(=O)R, -C(=O)NR2, -SO3-, -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NR, -S(=O)R, -OP(=O)(OR)2, -P(=O)(OR)2, -PO-3, -PO3H2, -C(=O)R, -C(=O)X, -C(=SR, -CO2R, -CO2-, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NR2, -C(=S)NR2, -C(=NR)NR2where each X independently represents a halogen: F, Cl, Br or I; and each R independently represents-H, C2-C18alkyl, C6-C20aryl, C3-C14a heterocycle, a protective group or component prodrugs. Alkylene, alkenylamine and alkenylamine group, as described above, it is possible to substitute a similar manner.

"Heteroaryl" and "heterocycle" refers to a ring system in which one or more ring atoms are heteroatoms, for example nitrogen, oxygen and sulfur. Heterocyclic radical contains from 1 to 20 carbon atoms, and from 1 to 3 heteroatoms selected from N, O, P and S. the Heterocycle may be a monocycle containing from 3 to 7 ring elements (2-6 carbon atoms, and from 1 to 3 heteroatoms selected from N, O, P and S) or Bicycle containing from 7 to 10 elements of the ring (4-9 carbon atoms, and from 1 to 3 heteroatoms selected from N, O, P and S), for example, the system bicyclo[4.5], [5.5], [5.6] or [6.6].

The heterocycles described in Paquette, Leo A., "Principles of Modern Heterocyclic Chemistry" (W.A. Benjamin, New York, 1968), particularly chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), in particular volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocycles include, as non-limiting examples of PI is ideal, dihydropyridin, tetrahydropyranyl (piperidyl), thiazolyl, tetrahydrothiophene, tetrahydrothiophene with oxidized sulphur, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, tianeptine, indolyl, indolinyl, chinoline, ethenolysis, benzimidazolyl, piperidinyl, 4-piperidinyl, pyrrolidinyl, 2-pyrrolidinyl, pyrrolyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydropyranyl, tetrahydroisoquinoline, decahydroquinoline, octahydronaphthalene, azocines, triazinyl, 6H-1,2,5-thiadiazine, 2H,6H-1,5,2-detainer, thienyl, thianthrene, pyranyl, isobenzofuranyl, bromanil, xantener, femoxetine, 2H-pyrrolyl, isothiazolin, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazole, purinol, 4H-hemolysins, phthalazine, naphthyridine, honokalani, hintline, cinnoline, pteridine, 4aH-carbazolyl, carbazolyl, β-carbolines, phenanthridines, acridines, pyrimidinyl, phenanthrolines, phenazines, phenothiazines, furutani, phenoxazines, isopropanol, bromanil, imidazolidinyl, imidazolyl, pyrazolidine, pyrazoline, piperazinil, indolinyl, isoindolyl, hinokitiol, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazole, oxindoles, benzoxazolyl and satanail.

As non-limiting examples, linked to the s with carbon heterocycles are linked in position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5 or 6 pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5 or 6 pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, position 2, 4 or 5 oxazole, imidazole or thiazole, position 3, 4 or 5 isoxazol, pyrazole or isothiazole, position 2 or 3 of aziridine, position 2, 3 or 4 azetidine, position 2, 3, 4, 5, 6, 7 or 8 of a quinoline or position 1, 3, 4, 5, 6, 7 or 8 of isoquinoline. More specifically associated with carbon heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl or 5-thiazolyl.

As non-limiting examples related nitrogen heterocycles are connected to position 1 of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of the research and position 9 carbazole or β-carboline. More specifically related nitrogen heterocycles include 1-ezyrider, 1-azetidin, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl and 1-piperidinyl.

The C 3-C8a heterocycle" refers to an aromatic or non-aromatic C3-C8carbocycle, in which from one to four carbon atoms in the ring is independently replaced by a heteroatom from the group consisting of O, S and N. Representative examples of C3-C8heterocycles include, without limitation, benzofuranyl, benzothiophene, indolyl, benzimidazolyl, coumarinyl, ethenolysis, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, chinoline, pyrimidinyl, pyridinyl, pyridinyl, pyrazinyl, pyridazinyl, isothiazolin, isoxazolyl and tetrazolyl. C3-C8the heterocycle may be unsubstituted or substituted groups, up to seven, including as non-limiting examples of-C1-C8alkyl, -O-(C1-C8alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)other', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2and-CN; where each R' is independently selected from H, -C1-C8of alkyl and aryl.

"C3-C8heterocycle" refers to defined above, C3-C8heterocyclic group, where one of the hydrogen atoms of the heterocyclic group substituted communication. C3-C8heterocycle may be unsubstituted or substituted groups, up to six, including as non-limiting examples of-C1-C alkyl, -O-(C1-C8alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)other', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), - N(R')2and-CN; where each R' is independently selected from H, -C1-C8of alkyl and aryl.

"Carbocycle" means a saturated or unsaturated ring containing from 3 to 7 carbon atoms in the form of a monocycle or from 7 to 12 carbon atoms in the form of Bicycle. Monocyclic carbocycle contain from 3 to 6 atoms in the ring, more specifically, 5 or 6 atoms in the ring. Bicyclic carbocycle contain from 7 to 12 atoms in the ring, for example, arranged in the form of bicyclo[4,5], [5,5], [5,6] or [6,6], or 9 or 10 atoms in the ring, arranged in the form of bicyclo [5,6] or [6,6]. Examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl and cyclooctyl.

"C3-C8carbocycle" is a 3-, 4-, 5-, 6-, 7 - or 8-membered saturated or unsaturated non-aromatic carbocyclic ring. Representative C3-C8carbocycle include as non-limiting examples cyclopropyl-cyclobutyl-cyclopentyl, cyclopentadienyl, cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cyclo is heptyl, -1,3-cycloheptadiene, -1,3,5-cycloheptatriene, -cyclooctyl and-cyclooctadiene. C3-C8the carbocyclic group may be unsubstituted or substituted by one or more groups that include as non-limiting examples of-C1-C8alkyl, -O-(-C1-C8alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, - C(O)other', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2and-CN; where each R' is independently selected from H, -C1-C8of alkyl and aryl.

"C3-C8carbocycle" refers to defined above, C3-C8carbocyclic group, where one of the hydrogen atoms of the carbocyclic group substituted connection.

"Linker" refers to a chemical component that contains covalent bond or a chain of atoms that covalently attach the antibody to a component of a medicinal product. In various embodiments, the implementation of linkers include a divalent radical such as alkerdeel, areldil, heteroaryl, components such as: -(CR2)nO(CR2)n-, repeating units of alkyloxy (for example, polietilene, PEG, polymethylenes), alkylamino (for example, polyethylenimine, Jeffamine™); and esters and amides of dibasic acids, including succinate, succinamide, diglycolate, malonate and caproamide.

The term "chiral" otnositsa molecules, which possess the property of impossibility of combining with its reflection, while the term "achiral" refers to molecules that can be combined with its reflection.

The term "stereoisomers" refers to compounds which have identical chemical structure but differ in the arrangement of atoms or groups in space.

"Diastereoisomer" refers to a stereoisomer with two or more centers of chirality and those molecules that are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral characteristics, and reaction capabilities. A mixture of diastereoisomers can be divided analytical methods high resolution, such as electrophoresis and chromatography.

"Enantiomers" refers to two stereoisomers of compounds that are incompatible mirror images of each other.

Used here stereochemical definitions and conventions generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they have the ability breatplate plane-polarized light. In describing an optically active compound, the prefixes D and L, orRandSuse to denote the absolute configuration of the molecules in the area of chiral(s) centre(s). The prefixes d and l or (+) and (-), used to designate the sign of rotation of plane-polarized light by the compound, where (-) or l means that the connection is levogyrate. Connection with the prefix (+) or d is Pervouralsk. For a given chemical structure of these stereoisomers are identical except that they are mirror images of each other. A specific stereoisomer may also be described as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. The mixture of enantiomers of 50:50 is designated as a racemic mixture or a racemate, which can be formed where there is no stereoselectivity or stereospecificity chemical reaction or process. The terms "racemic mixture" and "racemate" refers to an equimolar mixture of two forms of the enantiomers, which is devoid of optical activity.

"Leaving group" refers to a functional group that can substitute another functional group. Specific leaving groups are well known in this field, and examples include, without limitation halide (e.g. chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluensulfonyl (tosyl), Tr is pharmacysulfacet (triplet) and triftormetilfullerenov.

B. Abbreviations

COMPONENTS of the LINKER:

MC = 6-maleimidomethyl

Val-Cit or vc" = valine-citrulline (illustrative dipeptide in cleaved by the protease linker)

The citrulline = 2-amino-5-breedopedia acid

PAB = p-aminobenzeneboronic (an example of "self sacrifice" component linker)

Me-Val-Cit = N-methyl-valine-citrulline (where the linker peptide bond has been modified to prevent its cleavage by cathepsin B)

MC(PEG)6-OH = maleimidopropionamide (can be attached to zisteinom antibodies)

CYTOTOXIC DRUGS:

MMAE = monomethylaniline E (MW 718)

MMAF = option auristatin E (MMAE) with a phenylalanine at the C-end medicines (MW 731.5)

MMAF-DMAEA = MMAF with DMAEA (diethylaminoethylamine) in amide bond with a C-terminal phenylalanine (MW 801.5)

MMAF-TEG = MMAF with tetraethylene glycol, tarifitsirovannyim phenylalanine

MMAF-NtBu = N-tert-butyl, attached in the form of amide to the C-end of MMAF

Additional abbreviations are as follows: AE is auristatin E, Boc represents N-(tert-butoxycarbonyl), cit is citrulline, dap is dalapon, DCC is a 1,3-dicyclohexylcarbodiimide, DCM represents dichloromethane, DEA is diethylamin, DEAD is diethylazodicarboxylate, DEPC before the hat is diethylphosphoramidite, DIAD is diisopropylsalicylic, DIEA represents N,N-diisopropylethylamine, dil is daisosasen, DMA is a dimethylacetamide, DMAP is 4-dimethylaminopyridine, DME is a dimethyl ether of ethylene glycol (or 1,2-dimethoxyethane), DMF represents N,N-dimethylformamide, DMSO is a sulfoxide, doe is draftin, dov is a N,N-dimethylamine, DTNB is a 5,5'-dithiobis(2-nitrobenzoic acid), DTPA is diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is a 2-ethoxy-1-etoxycarbonyl-1,2-dihydroquinoline, ES-MS is a mass spectrometry with elektrorazpredelenie, EtOAc is an acid ethyl ester, Fmoc represents N-(9-fluorenylmethoxycarbonyl), gly represents glycine, HATU is aO-(7-asobancaria-1-yl)-N,N,N',N'-tetramethyluronium-hexaphosphate, HOBt represents 1-hydroxybenzotriazole, HPLC is a liquid chromatography high pressure, ile represents isoleucine, lys is a lysine, MeCN (CH3CN) is acetonitrile, MeOH is a methanol, Mtr is a 4-Anis diphenylmethyl (or 4-methoxytrityl), nor is a (1S,2R)-(+)-norephedrine, PBS is a phosphate buffer saline solution (pH 7,4), PEG represents a polyethylene glycol, Ph represents phenyl, Pnp is a p-nitrophenyl, MC represents a 6-maleimidomethyl, phe represents L-phenylalanine, PyBrop represents bromo-Tris-pyrrolidinone-hexaphosphate, SEC represents exclusion chromatography, Su represents succinimide, TFA represents triperoxonane acid, TLC is a thin-layer chromatography, UV is the ultraviolet light, and val is a valine.

III. COMPOSITIONS AND METHODS for THEIR preparation

Presents antibodies that bind to TAT226. Presents immunoconjugate containing anti-TAT226 antibodies. Antibodies and immunoconjugates according to the invention are suitable, for example, for the diagnosis or treatment of disorders associated with altered expression of, for example, increased expression of TAT226. In some embodiments, the implementation of the antibody or immunoconjugate according to the invention is suitable for diagnosis or treatment of disorders of cell proliferation, such as cancer.

A. Anti-TAT226 antibodies

TAT226 ("tumor-associated antigenic target No. 226") is a protein which is processed and expressed on the surface of certain cell types,including tumor cells. In particular, it was previously reported that human TAT226 sverkhekspressiya in certain types of tumors, including tumors of the ovaries, uterus, endometrium, kidney, lung, pancreas, adrenal glands and liver cells. See, for example, the publication of patent applications, US no US 2003/0148408 A1, US 2004/0229277 A1 and US 2003/0100712 A1 (where TAT226 designated as "PRO9917"); and U.S. patent No. 6710170 B2 (SEQ ID NO:215). Other components and descriptions in the databases associated with TAT226, are as follows: Depository No. NCBI AY358628_1 (where TAT226 person designated as "PSCA Hlog"); Depository No. NCBI AAQ88991.1 and "gi" No. 37182378 (where TAT226 person designated as "PSCA Hlog"); RIKEN cDNA 2700050C12; US 2003/0096961 A1 (SEQ ID NO:16); US 2003/0129192 A1 (SEQ ID NO:215); US 2003/0206918 A1 (example 5; SEQ ID NO:215); US 2003/0232056 A1 (SEQ ID NO:215); US 2004/0044179 A1 (SEQ ID NO:16); US 2004/0044180 A1 (SEQ ID NO:16); US 2005/0238649 A1 (where TAT226 person designated as "PSCA Hlog"); WO 2003/025148 (SEQ ID NO:292); WO 2003/105758 (SEQ ID NO:14) and EP 1347046 (SEQ ID NO:2640).

Full-TAT226 subjected to processing in the cell with the formation of the Mature form of the protein, which is expressed on the cell surface. For example, a full-sized human TAT226 as shown in SEQ ID NO:75, contains the expected sequence of the signal peptide from amino acids 1-20 or 1-22, which are believed to be cleaved from the protein. C-end amino acid 116-141, as suggested, is cleaved from the protein and component of the GPI is attached to the amino acid 115 Belkacem guess the Mature form of human TAT226 of amino acids 21-115 or 23-115 SEQ ID NO:75 is anchored on the cell surface using the GPI. TAT226 monkeys and rodents (see, for example, SEQ ID NO:76-78) highly similar to human TAT226 and, thus, assumed to be cleaved and modified in equivalent positions of amino acids. Cm. figure 1. The resulting Mature forms TAT226 humans, monkeys and rodents from amino acids 21-115 or 23-115 (as shown in figure 1) is 100%identical.

Other characteristics of human TAT226 include the proposed site of N-glycosylation at amino acid position 45, which is confirmed experimentally, and the estimated domain Ly6/u-PAR from amino acids 94-107 SEQ ID NO:75. TAT226 person has approximately 32% amino acid homology with stem cell antigen of the prostate (PSCA), specific for cancer of the prostate tumor antigen, which is expressed on the cell surface via a GPI-anchor. Cm. Reiter et al. (1998) Proc. Natl. Acad. Sci. USA 95:1735-1740. PSCA sverkhekspressiya in over 80% of prostate cancers. Id. Like TAT226, it contains estimated domain Ly6/u-PAR, which is involved in cellular functions such as signal transduction and cell adhesion. Id.

In one aspect the invention relates to antibodies that bind to TAT226. In some embodiments, the implementation of performance is aulani antibodies which are associated with Mature form of TAT226. In one of these embodiments Mature form TAT226 has the amino acid sequence of amino acids 21-115 or 23-115 SEQ ID NO:75. In some embodiments, the implementation of TAT226 antibody to bind with Mature form of TAT226, expressed on the cell surface. In some embodiments, the implementation of an antibody that binds to a Mature form of TAT226, expressed on the cell surface and inhibits cell growth. In some embodiments, the implementation of anti-TAT226 antibody binds to a Mature form of TAT226, expressed on the cell surface and inhibits cell proliferation. In some embodiments, the implementation of anti-TAT226 antibody binds to a Mature form of TAT226, expressed on a cell surface and causing cell death. In some embodiments, the implementation of anti-TAT226 antibody binds to a Mature form of TAT226, expressed on the surface of cancer cells. In some embodiments, the implementation of anti-TAT226 antibody binds to a Mature form of TAT226, which sverkhekspressiya on the surface of cancer cells compared to normal cells derived from the same cloth.

In one aspect of the anti-TAT226 antibody is a monoclonal antibody. In one aspect of the anti-TAT226 antibody is an antibody fragment, e.g. the fragment Fab, Fab'-SH, Fv, scFv, or (Fab')2. In one aspect of the anti-TAT226 antibody is a chimeric, humanized or human antibody. In one aspect of any of these anti-TAT226 antibody is purified.

Illustrative monoclonal antibodies obtained from ragovoy library presented here, as described in example B. the Antigen used for screening the library was a polypeptide having the sequence of amino acids 1-115 SEQ ID NO:75, which corresponds to the shape of TAT226, devoid of amino acids that are C-terminal to the intended site of attachment of GPI. Antibodies obtained from screening libraries indicated YWO.32 and YWO.49. YWO.49 was subjected to affinity maturation with education YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. Sequence alignment of the variable domains of the heavy and light chains YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 shown in figures 11 and 12, respectively.

In one aspect presents monoclonal antibodies that compete with YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 or YWO.49.H6 for binding to TAT226. Also presents monoclonal antibodies that bind to the same epitope as YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 or YWO.49.H6.

In one aspect of the invention presents polynucleotide encoding an anti-TAT226 antibody. In some embodiments, the implementation representation is Lena vectors, containing polynucleotide encoding an anti-TAT226 antibodies. In some embodiments, the implementation of the presented cell host containing such vectors. In another aspect of the invention presents a composition containing an anti-TAT226 antibodies or polynucleotide encoding an anti-TAT226 antibodies. In some embodiments, the implementation of the composition according to the invention is a pharmaceutical composition for the treatment of disorders of cell proliferation, such as violations listed here.

Detailed description of illustrative anti-TAT226 antibodies is as follows:

1. Specific embodiments of anti-TAT226 antibodies

In one aspect the invention relates to an antibody that contains at least one, two, three, four, five or six HVR selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:4; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5; (c) HVR-H3 containing the amino acid sequence selected from SEQ ID NO:3 and 6-11; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO:12; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO:13; and (f) HVR-L3 containing the amino acid sequence selected from SEQ ID NO:14-19.

In one aspect the invention relates to an anti-TAT226 antibody that contains at least one, m is Nisha least two or all three sequences VH HVR selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:4; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5; and (c) HVR-H3 containing the amino acid sequence selected from SEQ ID NO:3 and 6-11. In one aspect the invention relates to an anti-TAT226 antibody containing HVR-H1 containing the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:4. In one aspect the invention relates to an anti-TAT226 antibody containing HVR-H2 containing the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5. In one aspect the invention relates to an anti-TAT226 antibody containing HVR-H3 containing the amino acid sequence selected from SEQ ID NO:3 and 6-11. In one aspect the invention relates to an anti-TAT226 antibody containing HVR-H3 containing the amino acid sequence of SEQ ID NO:9 or 10.

In one aspect the invention relates to an anti-TAT226 antibody containing HVR-H3 containing the amino acid sequence selected from SEQ ID NO:3 and 6-11, and HVR-H1 containing the amino acid sequence selected from SEQ ID NO:1 and SEQ ID NO:4. In one aspect the invention relates to an anti-TAT226 antibody containing HVR-H3 containing the amino acid sequence selected from SEQ ID NO:6-11, and HVR-H1 containing SEQ ID NO:4. In one aspect of the invention regarding the priority for anti-TAT226 antibody, containing HVR-H3 containing SEQ ID NO:9 or 10, and HVR-H1 containing SEQ ID NO:4. In one aspect the invention relates to an anti-TAT226 antibody containing HVR-H3 containing SEQ ID NO:3, HVR-H1 containing SEQ ID NO:1.

In one aspect the invention relates to an anti-TAT226 antibody containing HVR-H3 containing the amino acid sequence selected from SEQ ID NO:3 and 6-11, and HVR-H2 containing the amino acid sequence selected from SEQ ID NO:2 and SEQ ID NO:5. In one aspect the invention relates to an anti-TAT226 antibody containing HVR-H3 containing the amino acid sequence selected from SEQ ID NO:6-11, and HVR-H2 containing SEQ ID NO:5. In one aspect the invention relates to an anti-TAT226 antibody containing HVR-H3 containing SEQ ID NO:9 or 10, and HVR-H2 containing SEQ ID NO:5. In one aspect the invention relates to an anti-TAT226 antibody containing HVR-H3 containing SEQ ID NO:3, and HVR-H2 containing SEQ ID NO:2.

In one aspect the invention relates to an anti-TAT226 antibody containing HVR-H1 containing the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:4; HVR-H2 containing the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5; and HVR-H3 containing the amino acid sequence selected from SEQ ID NO:3 and 6-11. In one embodiment, the implementation of HVR-H1 contains the amino acid sequence of SEQ ID NO:4; HVR-H2 contains the amino acid sequence of SEQ ID NO:5; and HVR-H3 content is t amino acid sequence, selected from SEQ ID NO:6-11. In one embodiment, the implementation of HVR-H1 contains the amino acid sequence of SEQ ID NO:4; HVR-H2 contains the amino acid sequence of SEQ ID NO:5; and HVR-H3 contains SEQ ID NO:9 or 10. In one embodiment, the implementation of HVR-H1 contains the amino acid sequence of SEQ ID NO:1; HVR-H2 contains the amino acid sequence of SEQ ID NO:2; and HVR-H3 contains the amino acid sequence of SEQ ID NO:3.

In one aspect the invention relates to an anti-TAT226 antibody that contains at least one, at least two, or all three VL sequence HVR selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO:12; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO:13; and (c) HVR-H3 containing the amino acid sequence selected from SEQ ID NO:14-19. In one aspect the invention relates to an anti-TAT226 antibody containing HVR-L3 containing the amino acid sequence selected from SEQ ID NO:14-19. In one aspect the invention relates to an anti-TAT226 antibody containing HVR-L3 containing the amino acid sequence of SEQ ID NO:17 or 18.

In one aspect the invention relates to an anti-TAT226 antibody containing (a) HVR-H3 containing the amino acid sequence selected from SEQ ID NO:3 and 6-11, and (b) HVR-L3 containing the amino acid sequence selected from SEQ ID NO:14-19. In one the m aspect of the invention relates to an anti-TAT226 antibody, containing (a) HVR-H3 containing the amino acid sequence of SEQ ID NO:3, and (b) HVR-L3 containing the amino acid sequence of SEQ ID NO:14. In some embodiments, the implementation of the antibody to TAT226 further comprises (a) HVR-H1 containing SEQ ID NO:1, and HVR-H2 containing SEQ ID NO:2.

In one aspect the invention relates to an anti-TAT226 antibody containing (a) HVR-H3 containing the amino acid sequence selected from SEQ ID NO:6-11, and (b) HVR-L3 containing the amino acid sequence selected from SEQ ID NO:14-19. In some embodiments, the implementation of the HVR-H3 contains the amino acid sequence of SEQ ID NO:9, and HVR-L3 contains the amino acid sequence of SEQ ID NO:17. In some embodiments, the implementation of the HVR-H3 contains the amino acid sequence of SEQ ID NO:10, and HVR-L3 contains the amino acid sequence of SEQ ID NO:18. In some embodiments, the implementation of the antibody to TAT226 further comprises HVR-H1 containing SEQ ID NO:4, and HVR-H2 containing SEQ ID NO:5.

In one aspect the invention relates to an anti-TAT226 antibody containing (a) HVR-H1 containing the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:4; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5; (c) HVR-H3 containing the amino acid sequence selected from SEQ ID NO:3 and 6-11; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO:12; (e) HVR-L2 containing AMI is kislotno sequence of SEQ ID NO:13; and (f) HVR-L3 containing the amino acid sequence selected from SEQ ID NO:14-19.

In one aspect the invention relates to an anti-TAT226 antibody containing (a) HVR-H1 containing the amino acid sequence of SEQ ID NO:1; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO:2; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO:3; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO:12; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO:13; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO:14.

In one aspect the invention relates to an anti-TAT226 antibody containing (a) HVR-H1 containing the amino acid sequence of SEQ ID NO:4; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO:5; (c) HVR-H3 containing the amino acid sequence selected from SEQ ID NO:6-11; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO:12; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO:13; and (f) HVR-L3 containing the amino acid sequence selected from SEQ ID NO:14-19.

In one aspect the invention relates to an anti-TAT226 antibody containing (a) HVR-H1 containing the amino acid sequence of SEQ ID NO:4; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO:5; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO:9; (d) HVR-L1 containing amino acid pic is egovernance SEQ ID NO:12; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO:13; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO:17.

In one aspect the invention relates to an anti-TAT226 antibody containing (a) HVR-H1 containing the amino acid sequence of SEQ ID NO:4; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO:5; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO:10; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO:12; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO:13; and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO:18.

In some embodiments, the implementation of anti-TAT226 antibody is subjected to affinity maturation to obtain the required affinity of binding with the target. In some embodiments, the implementation of any of one or more amino acids of the antibody replace the following provisions of the HVR (numbering according to Kabat): H98, H99, H100, H100B, L90, L92, L93, L96 and L97. For example, in some embodiments, the exercise of any one or more of the following substitutions can be obtained in any combination:

in HVR-H3 (SEQ ID NO:6): V98I; S99T; R100L or I; G100bA, S, or P;

in HVR-L3 (SEQ ID NO:14): Q90R, K, H or N; Y92V; T93F, N, G or A; P96F; T97I or A.

All possible combinations of the above substitutions are covered by the consensus sequences SEQ ID NO:11 (HVR-H3) and SEQ ID NO:19 (HVR-L3).

Anti-TAT226 antibody may contain lupuloides the frame sequence of the variable region domain, provided that the antibody retains the ability to bind to TAT226. For example, in some embodiments, the implementation of anti-TAT226 antibodies according to the invention contain a consensus sequence of a frame region of the heavy chain subgroup III person. In one of the embodiments of these antibodies consensus sequence frame region of the heavy chain contains the substitution of(I) at position 71, 73 and/or 78. In one of the embodiments of these antibodies, position 71 is a, A, position 73 is a T and/or position 78 is an A. In one embodiment, the implementation of these antibodies contain the frame sequence field variable domain of the heavy chain huMAb4D5-8, for example SEQ ID NO:50, 51, 59, 35 (FR1, 2, 3, 4, respectively). huMAb4D5-8 has a commercial name HERCEPTIN®, Genentech, Inc., South San Francisco, CA, USA; also discussed in U.S. patent No. 6407213 & 5821337, and Lee et al., J. Mol. Biol. (2004), 340(5):1073-93. In one of these embodiments, these antibodies also include a consensus sequence of a frame region of the light chain κI person. In one of these embodiments, these antibodies contain the frame sequence field variable domain of the light chain of huMAb4D5-8.

In one embodiment, the implementation of anti-TAT226 antibody contains a variable domain of the heavy chain, containing th is sequence frame region and the hypervariable region, where the frame sequence field contains a sequence FR1-FR4, selected from the sequences shown in figures 5A and 5B; HVR-H1 contains the amino acid sequence of SEQ ID NO:4; HVR-H2 contains the amino acid sequence of SEQ ID NO:5; and HVR-H3 contains the amino acid sequence selected from SEQ ID NO:6-11. In one of the embodiments of these antibodies HVR-H3 contains the amino acid sequence of SEQ ID NO:9 or 10. In one embodiment, the implementation of anti-TAT226 antibody contains a variable domain light chain containing a sequence of frame region and the hypervariable region, where the frame sequence field contains a sequence FR1-FR4, selected from the sequences shown in figures 6A and 6B; HVR-L1 contains the amino acid sequence of SEQ ID NO:12; HVR-L2 contains the amino acid sequence of SEQ ID NO:13; and HVR-L3 contains the amino acid sequence selected from SEQ ID NO:14-19. In one of the embodiments of these antibodies HVR-L3 contains the amino acid sequence of SEQ ID NO:17 or 18.

In one embodiment, the implementation of anti-TAT226 antibody contains a variable domain of a heavy chain containing a sequence of frame region and the hypervariable region, where the frame sequence field contains a sequence FR-FR4 SEQ ID NO:50, 51, 59 and 35, as shown in figure 7; HVR-H1 contains the amino acid sequence of SEQ ID NO:4; HVR-H2 contains the amino acid sequence of SEQ ID NO:5; and HVR-H3 contains the amino acid sequence selected from SEQ ID NO:6-11. In one of the embodiments of these antibodies HVR-H3 contains the amino acid sequence of SEQ ID NO:9 or 10. In one embodiment, the implementation of anti-TAT226 antibody contains a variable domain light chain containing a sequence of frame region and the hypervariable region, where the frame sequence field contains a sequence FR1-FR4 SEQ ID NO:60, 61, 62 and 63, as shown in figures 6A and 6B; HVR-L1 contains the amino acid sequence of SEQ ID NO:12; HVR-L2 contains the amino acid sequence of SEQ ID NO:13; and HVR-L3 contains the amino acid sequence selected from SEQ ID NO:14-19. In one of the embodiments of these antibodies HVR-L3 contains the amino acid sequence of SEQ ID NO:17 or 18.

In one embodiment, the implementation of anti-TAT226 antibody contains a variable domain of a heavy chain containing a sequence of frame region and the hypervariable region, where the frame sequence field contains a sequence FR1-FR4 SEQ ID NO:50, 51, 53 and 35, as shown in figure 8; HVR-H1 contains the amino acid sequence of SEQ ID NO:4; HVR-H2 contains linakis is now the sequence of SEQ ID NO:5; and HVR-H3 contains the amino acid sequence selected from SEQ ID NO:6-11. In one of the embodiments of these antibodies HVR-H3 contains the amino acid sequence of SEQ ID NO:9 or 10. In one embodiment, the implementation of anti-TAT226 antibody contains a variable domain light chain containing a sequence of frame region and the hypervariable region, where the frame sequence field contains a sequence FR1-FR4 SEQ ID NO:60, 61, 62 and 74, as shown in figure 8; HVR-L1 contains the amino acid sequence of SEQ ID NO:12; HVR-L2 contains the amino acid sequence of SEQ ID NO:13; and HVR-L3 contains the amino acid sequence selected from SEQ ID NO:14-19. In one of the embodiments of these antibodies HVR-L3 contains the amino acid sequence of SEQ ID NO:17 or 18.

In some embodiments implementing the invention relates to an anti-TAT226 antibody containing the variable domain of the heavy chain containing the amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence selected from SEQ ID nos:20-25. In some embodiments, the implementation of the amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity contains substitutions, insertions of alidelete relative to a reference sequence, but the antibody containing the amino acid sequence retains the ability to bind to TAT226. In some embodiments, the implementation of a total of from 1 to 10 amino acids are substituted, inserted or deleted in a sequence selected from SEQ ID nos:20-25. In some embodiments, the implementation of substitution insertions or deletions occur in regions outside of the HVR (i.e., in FR). In some embodiments, the implementation of anti-TAT226 antibody contains a variable domain of a heavy chain containing the amino acid sequence selected from SEQ ID nos:20-25.

In some embodiments implementing the invention relates to an anti-TAT226 antibody containing the variable domain light chain gumanitarnogo antibody 4D5 (huMAb4D5-8) (HERCEPTIN®, Genentech, Inc., South San Francisco, CA, USA) (also discussed in U.S. patent No. 6407213 and Lee et al. J. Mol. Biol. (2004), 340(5):1073-93)as shown in SEQ ID NO:31 below.

1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr CysArg Ala Ser Gln Asp ValAsnThr Ala Val AlaTrp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile TyrSer Ala Ser Phe Leu Tyr SerGly Val Pro Ser Arg Phe Ser Gly SerArgSer Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr CysGln GlnHisTyr Thr Thr Pro Pro ThrPhe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 108 (SEQ ID NO:31) (HVR residues are underlined).

In some embodiments, the implementation sequence of the variable domain of the light chain of huMAb4D5-8 modified in one or more position is at 30, 66 and 91 (Asn, Arg and His, respectively, as described above in bold/italics). In one embodiment, the implementation of the modified sequence of huMAb4D5-8 contains a Ser at position 30, Gly at position 66 and/or Ser at position 91. Accordingly, in one of the embodiments the antibody according to the invention contains a variable domain of the light chain containing the sequence represented by SEQ ID NO:26 below:

1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr CysArg Ala Ser Gln Asp ValSerThr Ala Val AlaTrp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile TyrSer Ala Ser Phe Leu Tyr SerGly Val Pro Ser Arg Phe Ser Gly SerGlySer Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr CysGln GlnSerTyr Thr Thr Pro Pro ThrPhe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 108 (SEQ ID NO:26) (HVR residues are underlined).

Substituted remains relatively huMAb4D5-8 described above in bold/italic.

In one aspect the invention relates to an anti-TAT226 antibody containing the variable domain of the light chain containing the amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence selected from SEQ ID nos:26-31. In some embodiments, the implementation of the amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity contains substitutions, to whom Alenia or deletions relative to a reference sequence, but the antibody containing the amino acid sequence retains the ability to bind to TAT226. In some embodiments, the implementation of a total of from 1 to 10 amino acids are substituted, inserted or deleted in a sequence selected from SEQ ID nos:26-31. In some embodiments, the implementation of substitutions, insertions, or deletions occur in regions outside of the HVR (i.e., in FR). In some embodiments, the implementation of anti-TAT226 antibody contains a variable domain of the light chain containing the amino acid sequence selected from SEQ ID nos:26-31.

In one aspect the invention relates to an anti-TAT226 antibody containing (a) a variable domain of a heavy chain containing the amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence selected from SEQ ID nos:20-25; and (b) the variable domain of the light chain containing the amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence selected from SEQ ID nos:26-31. In some embodiments, the implementation of the amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity contains substitutions, additions or deletions consider is Ino check sequence, but the antibody containing the amino acid sequence retains the ability to bind to TAT226. In some embodiments, the implementation of a total of from 1 to 10 amino acids are substituted, inserted or deleted in the control sequence. In some embodiments, the implementation of substitutions, insertions, or deletions occur in regions outside of the HVR (i.e., in FR). In some embodiments, the implementation of anti-TAT226 antibody contains a variable domain of a heavy chain containing the amino acid sequence selected from SEQ ID nos:20-25, and the variable domain of the light chain containing the amino acid sequence selected from SEQ ID nos:26-31.

In some embodiments, the implementation of anti-TAT226 antibody contains a variable region heavy chain containing the amino acid sequence of SEQ ID NO:20, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:26. In some embodiments, the implementation of anti-TAT226 antibody contains a variable region heavy chain containing the amino acid sequence of SEQ ID NO:21, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:26. In some embodiments, the implementation of anti-TAT226 antibody contains a variable region heavy chain containing the amino acid sequence of SEQ ID NO:22, and the variable region of leglize, containing the amino acid sequence of SEQ ID NO:27. In some embodiments, the implementation of anti-TAT226 antibody contains a variable region heavy chain containing the amino acid sequence of SEQ ID NO:23, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:28. In some embodiments, the implementation of anti-TAT226 antibody contains a variable region heavy chain containing the amino acid sequence of SEQ ID NO:24, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:29. In some embodiments, the implementation of anti-TAT226 antibody contains a variable region heavy chain containing the amino acid sequence of SEQ ID NO:25, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:30.

In one aspect the invention relates to an anti-TAT226 antibody containing (a) one, two or three VH HVR selected from VH HVR shown in figures 2 and 4, and/or (b) one, two or three VL HVR selected from VL HVR shown in figures 3 and 4. In one aspect the invention relates to an anti-TAT226 antibody containing the variable domain of the heavy chain selected from the variable domains, are shown in figures 9 and 11, and the variable domain of the light chain selected from the variable domains, are shown in figures 10 and 12.

Fragment antibodies

The present invention relates to fragments of antibodies. Antibody fragments can be obtained by conventional methods, such as enzymatic cleavage or recombinant methods. In certain circumstances there are advantages to using fragments of antibodies instead of whole antibodies. The smaller size of the fragments provides quick clearance and may lead to improved access to solid tumors. For an overview of some fragments of antibodies, see Hudson et al. (2003) Nat. Med. 9:129-134.

To obtain fragments of antibodies have been developed in different ways. Traditionally, these fragments were obtained by proteolytic cleavage of intact antibodies (see, e.g., Morimoto et al.Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be obtained directly by recombinant host cells. All Fab, Fv and ScFv fragments of antibodies can be Express inE. coliand they can secretariats fromE. coliproviding a way to easily obtain large amounts of these fragments. Antibody fragments can be isolated from phage libraries of antibodies discussed above. Alternative fragments, Fab'-SH, you can select directly from theE. coliand chemically combine with the formation of fragments F(ab')2(Carter et al., Bio/Technology 10:163-167 (1992)). In accordance with other approaches the house fragments F(ab') 2you can select directly from recombinant cell culture-owners. The fragment Fab and F(ab')2with increased half-life ofin vivocontaining residues of the epitope that binds the receptor of salvation described in U.S. patent No. 5869046. Other methods of obtaining fragments of antibodies will be apparent to the skilled technician. In some embodiments, the implementation of the antibody is a single-chain Fv fragment (scFv). Cm. WO 93/16185, U.S. patent No. 5571894 and 5587458. Fv and sFv are the only types with the intact active centers, which are devoid of constant regions; thus, they may be able to reduce nonspecific binding when usingin vivo. Merged sFv proteins can be constructed with getting effector protein, fused or N-end or C-end of the sFv. SeeAntibody Engineering, ed. Borrebaeck, above. Also the fragment of the antibody may be a "linear antibody", e.g., as described in U.S. patent No. 5641870. Such linear antibodies can be monospecificity or especificada.

3. Humanized antibodies

The present invention relates to humanized antibodies. Various methods of humanizing antibodies, non-human, known in this area. For example, humanitariannet the antibody may possess one or more amanakis what now remains, built it from a source that is not human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization, mainly, can be carried out in accordance with the method of Winter and colleagues (Jones et al.(1986) Nature 321:522-525; Riechmann et al.(1988) Nature 332:323-327; Verhoeyen et al.(1988) Science 239:1534-1536), by replacing the corresponding sequences of human antibodies with sequences of the hypervariable regions. Thus, such "humanized" antibodies are chimeric antibodies (U.S. patent No. 4816567), where essentially less intact variable domain of the human substituted by the corresponding sequence type, non-human. In practice, humanized antibodies, as a rule, are human antibodies in which some hypervariable residues region and possibly some FR residues substituted by residues from analogous sites of antibodies rodents.

The choice of the variable domains of a man, as light and heavy chains, for use when obtaining humanized antibodies is very important to reduce antigenicity. In accordance with the so-called method of "best match" sequence variabeln the th domain antibodies rodent analyze relative to a library of known sequences of the variable domains of a human. Then the sequence of the human being, which is the most similar to the sequence of the rodent, is considered acceptable as the frame area for gumanitarnogo antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). In another method uses a particular framework region derived from a consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same frame area can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

Moreover, as a rule, requires that antibodies were humanitarianism with retention of high affinity towards the antigen and other positive biological properties. To achieve this objective, according to one method, humanized antibodies produced using the process of analysis of the parental sequences and various alleged humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are widely available and well-known experts in this field. Available are computer programs which illustrate and display probable three-dimensional conformational structures of selected who's suspected of sequences of immunoglobulins. The study of these displayed data allows an analysis of the possible role of the residues in the functioning of the anticipated sequences of immunoglobulins, i.e., the analysis of residues that influence the ability of the proposed immunoglobulin to bind its antigen. This way, FR residues can be selected and combined from the recipient and import sequences so that to achieve the desired properties of the antibodies, such as increased affinity for the antigen(s)target(s). Typically, the immediate and most significant effect on the binding of antigen have remnants of the hypervariable region.

4. Human antibodies

Anti-TAT226 antibodies man according to the invention can be obtained by combining sequence(s) of the variable domain of an Fv clone selected(s) from a library of phage display derived from the person with the known(s) sequence(s) of the constant domain of a human, as described above. Alternative human monoclonal anti-TAT226 antibodies according to the invention can be obtained hybridoma method. Cell line the human myeloma and heteromyinae mouse-human to obtain human monoclonal antibodies are described, e.g., Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, p.51-63 (Marcel Dekker, Inc., New York, 1987); and oerner et al., J. Immunol., 147:86 (1991).

Currently you can obtain transgenic animals (e.g. mice)that when immunization capable of producing a full repertoire of human antibodies in the absence of endogenous production of immunoglobulins. For example, it has been described that the homozygous deletion of the gene region of heavy chain antibodies (JH) in chimeric and bearing mutations in the germ line of mice results in complete inhibition of endogenous production of antibodies. The transfer of immunoglobulin genes germline of the person so carrying mutations in the germline of mice with antigenic stimulation will lead to the production of antibodies person. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993).

For obtaining human antibodies from non-human antibodies, such as antibodies rodents, where the human antibody has similar appendectomy and specificnosti in relation to the original, does not belong to a human antibody, it is also possible to use a permutation of genes. According to this method, also referred to as "epitope imprinting", the variable region or a heavy or light chain not owned by the person of the fragment of the antibody, which is produced by means of phage display as described in this work, replace the repertoire of genes V-domain h the rights, obtaining a population of chimeric molecules scFv or Fab, consisting of not belonging to/owned by the man chains. Selection against the antigen leads to the secretion of chimeric molecules scFv or Fab, consisting of not belonging to/owned by the man chains, where the chains of man is restored antigennegative area disturbed by deleting the matching does not belong to the human chain in the primary clone phage display, i.e. the epitope determines (detects) the choice of the partner chain man. Human antibodies were obtained when repeating the process of replacing the remaining not owned by the person chain (see PCT WO 93/06213, published April 1, 1993). Unlike traditional humanizing non-human antibodies by CDR grafting, this method leads to the production of fully human antibodies, which do not contain residues of FR or CDR is not associated with human origin.

5. Bespecifically antibodies

Bespecifically antibodies are monoclonal antibodies that have specificnosti binding in respect of at least two different antigens. In some embodiments, the implementation bespecifically antibodies are human or humanized antibodies. In some embodiments, the implementation of one of specificdate binding the apply is to TAT226 and the other refers to any other antigen. In some embodiments, the implementation bespecifically antibodies can bind to two different epitopes TAT226. Bespecifically antibodies can also be used for targeted delivery of cytotoxic agents to cells which Express TAT226. These antibodies possess a part to associate with TAT226 and part of which is associated with a cytotoxic agent, such as, for example, saporin, anti-interferon-α, Vinca alkaloid, A-chain of ricin, methotrexate or hapten with a radioactive isotope. Bespecifically antibodies can be obtained as full-length antibodies or fragments of antibodies (e.g., bespecifically antibody F(ab')2).

Methods of obtaining bespecifically antibodies known in the field. Traditionally, the recombinant getting bespecifically antibodies based on simultaneous expression of two pairs of heavy chain-light chain immunoglobulins, where the two heavy chains have different specificnosti (Milstein and Cuello, Nature, 305:537 (1983)). Due to the random Assembly of the heavy and light chains of immunoglobulins such hybridoma (quadroma) produce a mixture of potentially 10 different antibody molecules, of which only one has the correct bespecifically structure. Purification of the correct molecule, which is usually carried out by stages affinity chromatography, is quite difficult, and the exit of the product is low. Similar methods are described in WO 93/08829 and in Traunecker et al., EMBO J., 10:3655 (1991).

In accordance with another approach, conduct the merge variable domains of antibodies with the desired specificnosti binding (the binding sites of the antibody-antigen) with the sequences of the constant domains of immunoglobulins. Merger, for example, is carried out with a constant domain of the heavy chain of immunoglobulin containing at least part of the hinge regions CH2 and CH3. In some embodiments, the first constant region of the heavy chain (CH1)containing the site necessary for binding to the light chain, is present in at least one of the components to merge. DNA molecules encoding designed to merge the components of the heavy chains of immunoglobulins and, if required, the light chain immunoglobulin is inserted into the individual expressing vectors and implement joint transfection of them fit of the host body. This integration provides significant flexibility in the regulation of the mutual ratios of the three polypeptide fragments in the variants of implementation, where unequal ratios of the three polypeptide chains used in the construction provide the optimum yield. However, you can embed the coding sequences for two or all three polypeptide chains in one EC is preserue vector, if the expression of at least two polypeptide chains in equal ratios results in high product yield or if the ratios are of no particular value.

In one embodiment, the implementation of this approach bespecifically antibodies comprise the heavy chain hybrid immunoglobulin with a first binding specificity in one part and a pair of heavy chain-light chain hybrid immunoglobulin (having a second binding specificity) in the other part. Found that this asymmetric structure facilitates the separation of the desired especifismo compounds and undesirable combinations of chains of immunoglobulins, as the presence of light chain immunoglobulin in only one half of bespecifically molecules provides an easy way of separation. This method is described in WO 94/04690. For a more detailed description of obtaining bespecifically antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another method, you can construct a contact area between a pair of antibody molecules to maximize the percentage of heterodimers, which is recovered from recombinant cell culture. The contact area contains at least part of the CH3-domain constant domain of antibodies. In this way one or more small amino acid side chains from the field to the of ntact first molecule antibodies replace larger side chains (for example, tyrosine or tryptophan). In the contact region of the second molecule antibodies create compensatory "cavities" of identical or similar with the major(s) side(s) chain(s) size by replacing large amino acid side chains are chains of smaller sizes (e.g., alanine or threonine). This replacement provides a mechanism to enhance the yield of heterodimer compared to other unwanted end-products such as homodimers.

Bespecifically antibodies include cross stitched antibodies or heteroconjugate" antibodies. For example, one of the antibodies in heteroconjugate you can connect with Avidya, and the other with Biotin. Such antibodies, for example, proposed to target immune system cells against unwanted cells (U.S. patent No. 4676980) and for the treatment of HIV infection (WO 91/00360, WO 92/00373 and EP 03089). Heteroconjugate antibodies can be obtained using any suitable methods cross-linkage. Suitable substances for cross-linkage are well known in this field and are described in U.S. patent No. 4676980 together with several ways of cross-linkage.

Methods of obtaining bespecifically antibodies, fragments of antibodies are also described in the literature. For example, bespecifically antibodies can be obtained by using the formation of chemical bonds. In Brennan et al., Science, 229:81 (1985) openspool, in which intact antibodies proteoliticeski split to obtain F(ab')2-fragments. These fragments regenerate in the presence of arsenite sodium, a substance that forms complexes with developed, in order to stabilize neighboring dithioles and prevent the formation of intermolecular disulfide. Then, the resulting Fab'-fragments are converted into derivatives of dinitrobenzoate (TNB). Then one of the derivatives of Fab'-TNB re-turn in the Fab'-thiol by restoring mercaptoethylamine and mixed with equimolar amounts of the other derived Fab'-TNB obtaining especifismo antibodies. Received bespecifically antibodies can be used as a means for the selective immobilization of enzymes.

Recent advances have simplified the direct selection ofE. colifragments, Fab'-SH, which can chemically combine with the formation of bespecifically antibodies. In Shalaby et al., J. Exp. Med., 175:217-225 (1992) describe how to get a fully humanized molecule F(ab')2especifismo antibodies. Carried secretion fromE. colieach Fab'fragment individually and they were subjected to direct chemical bindingin vitrowith the formation of especifismo antibodies. Bespecifically antibody thus obtained, possessed the ability to bind to cells, sverkhekspressiya receptor HER2, normal T-cell is mi man and run the lytic activity of cytotoxic lymphocytes against targets tumor human breast.

Also describes the various methods of obtaining and allocating fragments bespecifically antibodies directly from recombinant cell culture. For example, the received bespecifically antibodies using "latinovich lightning", Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The peptides "latinboy lightning" from proteins Fos and Jun were connected with the Fab'portions of two different antibodies by gene fusion. Homodimeric antibodies were restored in the hinge region to form monomers and then re-oxidized with the formation of heterodimeric antibodies. This method can also be used to obtain homodimeric antibodies. The way dimeric antibodies described in Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), provides an alternative mechanism for obtaining bespecifically fragments of antibodies. The fragments contain the variable domain of the heavy chain (VH)connected to the variable domain of the light chain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the domains VH and VL of one fragment are forced to pair with complementary domains VL and VH of another fragment, thereby forming two antigen is vyzyvayushih plot. Also described another method of obtaining fragments bespecifically antibodies using dimers of single-chain Fv (sFv). Cm. Gruber et al., J. Immunol., 152:5368 (1994).

Also provides antibodies with more than two valencies. For example, you can get thespecification antibodies, Tutt et al. J. Immunol. 147:60 (1991).

6. Multivalent antibodies

Multivalent antibody can be faster than bivalent antibody, internalizacao (and/or metabolizirovannom) a cell expressing the antigen, which bind antibodies. Antibodies of the present invention may be a polyvalent antibodies (which are different from IgM) with three antihistamine sites or more (for example, tetravalent antibodies), which can easily be obtained by recombinant expression of a nucleic acid that encodes a polypeptide chain of the antibody. Multivalent antibody may contain dimerization domain, and three or more antigenspecific plot. In some embodiments, the implementation dimerization domain contains (or consists of) Fc-region or a hinge region. In this case, the antibody contains a Fc region, and three or more antigenspecific plot from N-Terminus to the Fc-region. In some embodiments, the implementation of polyvalent antibody contains (or consists of) three to approximately the seven antigenspecific areas. In one of these embodiments polyvalent antibody contains (or consists of) four antigenspecific plot. Polyvalent antibody contains at least one polypeptide chain (for example, two polypeptide, where the polypeptide(s) chain(s) contains(at) two or more variable domains. For example, the polypeptide(s) chain(s) may contain VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n represents 0 or 1. For example, the polypeptide(s) chain(s) may contain: chain VH-CH1-flexible linker-VH-CH1-Fc region; or chain VH-CH1-VH-CH1-Fc region. In this work, the multivalent antibody can optionally contain at least two (e.g., four) of the polypeptide of variable domains of the light chain. In this paper polyvalent antibody, for example, may contain from about two to about eight polypeptides variable domain of the light chain. Polypeptides variable domain of the light chain provided in this work contain a variable domain light chain and, optionally, further contain a CL domain.

7. Single-domain antibodies

In some Islands Ianto implementation of the antibody according to the invention is a single domain antibody. Single-domain antibody is a single polypeptide chain, containing the entire variable domain heavy chain antibody or a part or the entire variable domain of the light chain of the antibody or a part of it. In some embodiments, the implementation of the single domain antibody is a single domain antibody man (Domantis, Inc., Waltham, MA; see, for example, U.S. patent No. 6248516 B1). In one embodiment, the implementation of single-domain antibody consists of the entire variable domain of the heavy chain of the antibody or its parts.

8. Variants of antibodies

In some embodiments, the implementation provided by(s) modification(s) the amino acid sequences described in this paper antibodies. For example, it may be desirable to improve the affinity of binding and/or other biological properties of the antibody. Variants of the amino acid sequences of antibodies can be obtained by making appropriate substitutions in the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions of residues, and/or insertion of residues, and/or substitutions of residues of the amino acid sequences of the antibodies. To obtain the final design can be performed by any combination of deletions, insertions and substitutions, provided that the final construct possesses the desired properties is AMI. Amino acid modifications can be made in the considered amino acid sequence of the antibody at the time of receiving the order.

Suitable method of identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called "alanine scanning by mutagenesis"as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this paper reveal a residue or group of residues of target proteins (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine)to affect the interaction of amino acids with antigen. Those provisions of amino acids, which are the functional sensitivity to the substitutions then adjust through the introduction of additional or other options in the areas of replacement or instead of them. Thus, despite the fact that the plot for a change in the amino acid sequence is predetermined, there is no need in the immediate certainty of the nature of the mutation. For example, analysis of the action of mutations in a given area, the codon-target or area target spend alanine scanning or random mutagenesis and expressed immunoglobulins conduct screening for the presence of desired activity.

Insert amino acid sequences include intended for fusion to the N - and/or C-end of the sequence, having a length in the range from one residue to polypeptides containing a hundred or more residues, as well as to insert into the sequence of one or more amino acid residues. Examples of terminal insertions include an antibody with an N-terminal residue of methionine. Other options insertions in the antibody molecule include the fusion to the N - or C-Terminus of an antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the half-life of antibodies in the serum.

In some embodiments, the implementation of the antibody according to the invention to change to increase or decrease the degree of glycosylation of antibodies. Glycosylation of polypeptides typically represents or N-glycosylation or O-glycosylation. N-glycosylation refers to the attachment of a carbohydrate group to the side chain of an asparagine residue. Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except Proline, are sequences that are recognized for enzymatic joining of carbohydrate groups to the side chain of asparagine. Thus, the presence of any of these Tripeptide sequences in the polypeptide POPs the AET potential glycosylation site. O-glycosylation refers to the attachment of one of the sugars, which represents the N-atsetilgalaktozamin, galactose, or xylose to hydroxynicotinate, as a rule, to serine or threonine, although you can also use 5-hydroxyproline or 5-hydroxylysine.

Adding or removing sites of glycosylation of antibodies conveniently be done by changing the amino acid sequence so that was the creation or deletion of one or more of the above Tripeptide sequences (in the case of sites of N-glycosylation). Also, the change can be made by adding or deleting one or more residues of serine or threonine, or by substitution of one or more residues of serine or threonine in the sequence of the original antibody (in the case of sites of O-glycosylation).

In that case, if antibodies contain the Fc-region, you can change attached to it is a carbohydrate. For example, antibodies with a Mature carbohydrate structure that lacks fucose attached to the Fc region of the antibodies described in the patent application U.S. No. US 2003/0157108 (Presta, L.). Cm. also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies containing separating N-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc region of the antibodies discussed in WO 2003/011878, Jean-Mairet et al., and U.S. patent No. 6602684, Umana et al. Antibodies, on ageusia, at least one galactose residue in the oligosaccharide attached to the Fc region of the antibodies discussed in WO 1997/30087, Patel et al. Cm. also WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.) relative to antibodies with altered carbohydrate attached to an Fc region of antibodies. Cm. also US 2005/0123546 (Umana et al.) relatively antigenspecific molecules with modified glycosylation.

In some embodiments, the implementation variant glycosylation contains a Fc region, where the carbohydrate structure attached to the Fc region lacks fucose. Such variants have enhanced function in relation to the ADCC. Optional Fc-region further comprises one or more amino acid substitutions, which further improves ADCC, for example, substitutions in positions 298, 333, and/or 334 of the Fc region (residue numbering Eu). Examples of publications related to "deforsirovannym" or "devoid of fucose" antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Examples of cell lines producing deoksigenirovanii antibodies include cells Lec13 CHO, have violated fokusirovanie protein (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); patent application U.S. No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., in particular in example 1), and knockout cell lines, such as knockout CHO cells by gene alpha-1,6-fucosyltransferase,FUT8(Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004)).

Another type of variant is the variant with the substitution of amino acids. Such variants have at least one amino acid residue in the antibody molecule, which is replaced by another residue. Areas of interest for carrying out mutagenesis with replacement, include the hypervariable sites, but also is expected to change FR. Conservative substitutions are shown in table 1 under the heading of "preferred substitutions". If the result of such substitutions is the desired change in biological activity, then you can make more substantial changes, indicated in table 1 as "illustrative replacement", or as described hereinafter relative to the classes of amino acids, and conduct screening products.

Table 1
The original balanceIllustrative replacementPreferred replacement
Ala (A)Val; Leu; IleVal
Arg (R)Lys; Gln; AsnLys
Asn (N)Gln; His; Asp, Lys; ArgGln
Asp (D)Glu; AsnGlu
Cys (C)Ser; AlaSer
Gln (Q)Asn; GluAsn
Glu (E)Asp; GlnAsp
Gly (G)AlaAla
His (H)Asn; Gln; Lys; ArgArg
Ile (I)Leu; Val; Met; Ala;
Phe; norleucine
Leu
Leu (L)Norleucine; Ile; Val;
Met; Ala; Phe
Ile
Lys (K)Arg; Gln; AsnArg
Met (M)Leu; Phe; IleLeu
Phe (F)Trp; Leu; Val; Ile; Ala; TyrTyr
Pro (P)Ala Ala
Ser (S)ThrThr
Thr (T)Val; SerSer
Trp (W)Tyr; PheTyr
Tyr (Y)Trp; Phe; Thr; SerPhe
Val (V)Ile; Leu; Met; Phe;
Ala; norleucine
Leu

Significant modification of the biological properties of the antibodies is done by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of replacement, such as conformation in the form of a layer or a spiral, (b) the charge or hydrophobicity of the molecule at the site of the target or (c) the size of the side chain. Amino acids can be grouped according to the similarity of the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., p.73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala (A)Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M;)

(2) uncharged polar: Gly (G), Ser (S)Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q);

(3) acidic: Asp (D)Glu (E);

(4) basic: Lys (K), Arg (R), His(H).

Alternative naturally occurring residues may be divided into groups based on common properties of the side chains:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

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

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will lead to the replacement of a member of one of these classes to a member from another class. Such substituted residues can also be entered in sections a conservative substitution, or in the remaining (non-conservative) areas.

One of the variant with the substitution includes replacement of one or more residues of the hypervariable region of the parent antibody (e.g., gumanitarnogo antibodies or human antibodies). Generally, the obtained(s) option(s)selected(s) for further development, has(have) modified (improved biological properties relative to the parent antibody from which it(they) is obtained(s). A convenient way of obtaining such variants with substitutions includes affinity maturation using phage display. In summary, in several sections of hypervariable region (for example, in 6-7 sites) make mutations to obtain all possible amino acid substitutions at each site. The antibodies thus obtained produce the particles of filamentous phages as antibodies, fused, at least with the part about protein is rocki phage (for example, product of the gene III M13), Packed in each particle. Then conduct the screening produced in phage variants on their biological activity (e.g. binding affinity of). In order to identify areas hypervariable region that are candidates for modification, it is possible to carry out scanning mutagenesis (for example, alanine scanning mutagenesis to identify residues of the hypervariable region, largely involved in the binding to the antigen. Alternative or in addition it may be useful to analyze the crystal structure of the complex antigen-antibody to identify potential areas of contact between the antibody and the antigen. Such forming the contact residues and neighboring residues are candidates for replacement in accordance with well-known in this field of ways, including the methods developed in this work. Upon receipt of such options panel of variants is subjected to screening using methods known in this field, including the methods described in this paper, and for the further development it is possible to select antibodies with improved properties through one or more suitable methods of analysis.

The nucleic acid molecules encoding amino acid sequence variants of antibodies, receive a variety of ways known in this is blasty. These methods include as non-limiting examples of selection from a natural source (in the case of naturally occurring amino acid sequence) or the receipt by oligonucleotide-mediated (or site-specific) mutagenesis, mutagenesis using PCR and cassette mutagenesis previously obtained variant or variant antibodies.

It may be desirable to make one or more modifications of amino acids in the Fc-region of an antibody according to the invention, thereby obtaining a variant Fc region. The variant Fc region may contain a sequence of Fc-region of a person (e.g., Fc region of IgG1, IgG2, IgG3, or IgG4 human), containing the amino acid modification (e.g. a substitution) at one or more positions of the amino acids, including the provisions of the hinge cysteine.

In accordance with this description and ideas in this area suggest that in some embodiments, the implementation of the antibody according to the invention may contain one or more modifications compared to the same antibody wild-type, for example, in the Fc-region. These antibodies can, however, save essentially the same characteristics required for therapeutic use compared with their analogous to wild-type. For example, assume that in the Fc-region can be performed ODA is divided modifications which can lead to change (either improvement or deterioration) binding to C1q and/or complementability cytotoxicity (CDC), for example as described in WO 99/51642. Cm. also Duncan & Winter Nature 322:738-40 (1988); U.S. patent No. 5648260; U.S. patent No. 5624821; and WO 94/29351 relative to other examples of variants of the Fc region. In WO 00/42072 (Presta) and WO 2004/056312 (Lowman) described variants of the antibodies with improved or worsened by binding to FcR. The contents of these patent publications, particularly herein by reference. Cm. also Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001). Antibodies with increased half-life and enhanced binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), described in US 2005/0014934 A1 (Hinton et al.). These antibodies contain the Fc region with one or more substitutions that improve the binding of the Fc region with FcRn. Variants of polypeptides with altered amino acid sequences of the Fc-region and increased or reduced ability to bind to C1q described in U.S. patent No. 6194551 B1, WO 99/51642. The contents of these patent publications, particularly herein by reference. Cm. also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

In one aspect the invention relates to antibodies containing modifications in the contact area Fc-polypeptides, with whom containing a series Fc-region, where modifications to facilitate heterodimerization and/or contribute to it. These modifications include the introduction of a protruding part of the first Fc polypeptide and the cavity of the second Fc polypeptide, where the protruding portion is located in the cavity so as to facilitate the formation of the complex from the first and second Fc polypeptides. Methods for producing antibodies with such modifications known in this field, for example as described in U.S. patent No. 5731168.

9. Derived antibodies

Antibodies of the present invention can be further modified to contain additional non-protein groups, which are known in this area and easily accessible. Preferably groups which are suitable for obtaining derivatives of antibodies, are water-soluble polymers. Non-limiting examples of water-soluble polymers include without limitation polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, a copolymer of ethylene/maleic anhydride, polyaminoamide (or homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, homopolymers of probabilistically, copolymers of polypropyleneoxide/ethylene oxide, polyoxyethylene poly the crystals (for example, glycerine), polyvinyl alcohol and mixtures thereof. Polyethylenepolypropylene may have advantages in production due to its stability in water. The polymer may have any molecular weight, and may be branched or unbranched. The number attached to the antibodies of the polymers can vary, and if you attach more than one polymer, they may represent the same or different molecules. Typically, the number and/or type of polymers used to obtain the derivatives can be determined on the basis of factors that include as non-limiting examples of specific properties or functions of antibodies, which needs to be improved, the fact will be whether to use a derived antibodies in the treatment of certain conditions, etc.

In another embodiment, presents the conjugates of the antibody and the protein component, which can selectively be heated by exposure to radiation. In one embodiment, the implementation of the non-protein component is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. 102:11600-11605 (2005)). Irradiation may have any wavelength and includes, as non-limiting examples of wavelengths that do not harm normal cells, but which heat the nonprotein component to a temperature at which the peg is but cells, located in close proximity to conjugate the antibody is a non-protein component.

B. Some methods of obtaining antibodies

1. Some methods based on hybrid

Monoclonal anti-TAT226 antibodies according to the invention can be obtained using the hybridoma method first described in Kohler et al., Nature, 256:495 (1975), or they can be obtained by means of recombinant DNA (U.S. patent No. 4816567).

In the hybridoma method, a mouse or other suitable animal host, such as a hamster, subjected to immunization to obtain lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. The induction of anti-TAT226 antibodies, as a rule, carried out in animals, multiple subcutaneous (sc) or intraperitoneal (ip) injections TAT226 and adjuvant. TAT226 can be obtained using methods well known in the field, some of which are further described here. For example, TAT226 can be obtained by recombinant means. In one of the embodiments of animals subjected to immunization derived TAT226, which contains the extracellular part of TAT226, fused with the Fc-part of the heavy chain of the immunoglobulin. In one of the embodiments of animals subjected to immunization fused protein TAT226-IgG1. In one of the embodiments of animals is Monserrat immunogenic derivative TAT226 in solution with monophosphoryl-lipid A (MPL)/dicenomicon trehalose (TDM) (Ribi Immunochem. Research, Inc., Hamilton, MT) and the solution is injected intradermally in many areas. Two weeks later the animals repeated immunization. From seven to fourteen days later the animals are taken blood and serum will be analyzed by the titer of anti-TAT226. Animals repeated immunization up until the titer stabilizes.

Alternative lymphocytes can givein vitro. Then produce a fusion of lymphocytes with myeloma cells using a suitable substance which causes the merge, such as polyethylene glycol, to obtain a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, p.59-103 (Academic Press, 1986)).

The thus obtained hybridoma cells were seeded in a suitable culture medium and grown in it, for example in an environment that contains one or more substances that inhibit the growth or survival is not affected by the merger of the parent myeloma cells. For example, if the parental myeloma cells lacking the enzyme hypoxanthineguanine (HGPRT or HPRT), the culture medium for the hybridomas typically will include gipoksantin, aminopterin and thymidine (Wednesday HAT), substances that prevent the growth of cells with a defect HGPRT.

In some embodiments, the implementation of the myeloma cells are cells that are effectively merging maintain a stable antibody production at a high level electrovanne producing antibodies cells and are sensitive to the environment, such as the environment HAT. Illustrative myeloma cells include as non-limiting examples of murine myeloma lines, such as lines originating from tumors of mice MOPC-21 and MPC-11, available at the Salk Institute Cell Distribution Center, San Diego, California USA, cells and SP-2 or X63-Ag8-653, available at the American Type Culture Collection, Rockville, Maryland USA. Cell line the human myeloma and heteromyinae mouse-man is also described for obtaining human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, p.51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma is grown cells, analyze on developing monoclonal antibodies that bind to TAT226. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or analysis of bindingin vitrosuch as radioimmune assay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of monoclonal antibodies, for example, can be determined by analysis of Scatchard by Munson et al., Anal. Biochem., 107:220 (1980).

After the identification of those hybridoma cells that produce antibodies of the desired specificity, affinity and/or activity, the clones can be subclinical by means of serial dilutions and grown using standard who methods (Goding, Monoclonal Antibodies: Principles and Practice, p.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, medium (D-MEM or RPMI-1640. In addition, the hybridoma cells can be grownin vivoas ascitic tumors in the animal. Monoclonal antibodies secreted by the subclones, accordingly isolated from the culture medium, ascites fluid, or serum by conventional methods of purification of immunoglobulins, such as, for example, protein A-sepharose, chromatography with hydroxyapatite, gel electrophoresis, dialysis, or affinity chromatography.

2. Some methods of screening libraries

Anti-TAT226 antibodies according to the invention can be obtained by using combinatorial libraries to screen for antibodies with the desired activity or activities. For example, many methods known in this field to obtain libraries of phage display and screening such libraries for antibodies with the desired binding characteristics. Such methods are broadly described in Hoogenboom et al. (2001) in Methods in Molecular Biology 178:1-37 (O'brien et al., ed., Human Press, Totowa, NJ), and in some embodiments, the implementation in Lee et al. (2004) J. Mol. Biol. 340:1073-1093.

Essentially clones of synthetic antibodies are selected by screening phage libraries containing the phage, which exposed various fragments of variable about the Asti (Fv) antibody fused with the protein shell of the phage. Such phage library selected by affinity chromatography with respect to the desired antigen. Clones expressing Fv-fragments, the ability to communicate with the desired antigen, adsorbed on the antigen and, thus, separated from unbound clones in the library. Bound peroxidase clones then elute with antigen, and can further be enriched by additional cycles of adsorption of the antigen/elution with him. Any of the anti-TAT226 antibodies according to the invention can be obtained by designing a suitable screening method using antigen for selection of interest phage clone then get a clone of the full-size anti-TAT226 antibodies using the Fv sequences from the interest of phage clone and sequence a suitable constant region (Fc), described by Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

In some embodiments, the implementation antigennegative domain antibodies formed by two variable (V) regions of approximately 110 amino acids, in one area of the light (VL) and heavy (VH) chains, which both give three hypervariable loops (HVR) or region complementarity determining (CDR). Variable domains can functionally be exposed on the phage, or in the form of tnotebook the s Fv fragments (scFv), in which VH and VL are covalently bound through a short, flexible peptide, or as Fab fragments, in which each of them fused with the constant domain, and they interact ecovalence, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). As used in this study, phage clones encoding scFv, phage clones encoding Fab, collectively referred to as "Fv phage-clones" or "Fv-clones".

The gene repertoires of VH and VL can be individually cloned using polymerase chain reaction (PCR) and randomly recombine in phage libraries, which you can then search antigenspecific clones, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Libraries from immunized sources give high-affinity antibodies to the immunogen without the need to create a hybrid. Alternative naive repertoire can be cloned to create a single source of human antibodies to a wide range of nauto and proteins without any immunization, as described in Griffiths et al., EMBO J 12:725-734 (1993). In conclusion, naive libraries can also be obtained synthetically by cloning nepristoinyi segments V-genes from stem cells and using primers for PCR containing a random sequence to encode vysokopribylnyj CDR3 regions and for the implementation of perestroikain vitro as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

In some embodiments, the implementation of the filamentous phage is used to display fragments of antibodies by fusion with the minor protein shell pIII. Antibody fragments can be exhibited in the form of single-chain Fv fragments, in which VH and VL domains are linked by a single polypeptide chain flexible polypeptide spacer, for example, as described in Marks et al., J. Mol. Biol., 222:581-597 (1991), or as Fab fragments, in which one chain is subjected to fusion with pIII and other secreted in periplasm bacterial host cell, where the Assembly structure of the Fab-protein shell, which begins to be exhibited on the surface of phage by the replacement of some membrane proteins of wild-type, for example, as described in Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (1991).

Typically, nucleic acids encoding fragments of antibody genes, derived from immune cells isolated from human or animal. If you want a library enriched clones against TAT226, the subject is subjected to immunization through TAT226 to obtain a humoral immune response, and spleen cells and/or circulating B cells other peripheral blood lymphocytes (PBL) allocate to create the library. In a preferred embodiment, a library of gene fragments of human antibodies enriched clones against TAT226, which are square-the induction of humoral immune response against TAT226 in transgenic mice functional set of genes of human immunoglobulins (and lacking a functional system the production of endogenous antibodies) such that immunization by TAT226 leads to the production of B-cells of the human anti-TAT226 antibodies. Obtaining transgenic mice that produce human antibodies, as described below.

Additional enrichment of populations of activated cells against TAT226 can be obtained by using a suitable screening method for the selection of B-cells expressing specific to TAT226 membrane-bound antibody, for example, by separating cells by affinity chromatography on the basis of TAT226 or adsorption of cells to labeled with fluorochrome TAT226 followed fluorescence-activated cell sorting (FACS).

Alternative use of spleen cells and/or B cells or other PBL from an unimmunized donor provides a better representation of the possible repertoire of antibodies, and also allows the creation of a library of antibodies using any kind of animal (human or non-human), in which TAT226 is not antigenic. For libraries, including the construction of antibody genesin vitrostem cells isolated from the subject to obtain nucleic acids encoding nepristoinye segments of antibody genes. Interest IMM is nnye cells can be obtained from many species of animals, species such as human, mouse, rat, hares, rabbit, dog, cat, pig, bovine, equine and poultry etc.

Nucleic acid encoding the variable segments of the gene of the antibody (including VH - and VL segments), isolated from the interest of cells and amplified. In the case of libraries rebuilt VH and VL genes to the desired DNA can be obtained by selection of the genomic DNA or mRNA from lymphocytes with subsequent polymerase chain reaction (PCR) with primers complementary to the 5'- and 3'-ends rebuilt VH and VL genes, as described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86:3833-3837 (1989), thus creating a varied repertoire of V genes for expression. V-genes can amplify from cDNA and genomic DNA with a reverse primer at the 5'end of the exon encoding the Mature V-domain, and direct primers located within the J-segment, as described in Orlandi et al. (1989) and Ward et al., Nature, 341:544-546 (1989). However, for amplification with cDNA reverse primers can also be placed in the leader exon, as described in Jones et al., Biotechnol., 9:88-89 (1991), and direct the primers within the constant region as described in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86:5728-5732 (1989). To maximize complementarities primers can include degeneracy, as described in Orlandi et al. (1989) or Sastry et al. (1989). In some embodiments, the implementation of a variety of libraries to maximize the use of what Finance primers for PCR, aimed at every family of V-gene in order to amplify all available VH - and VL-configuration present in the sample nucleic acid from immune cells, for example, as described in the method according to Marks et al., J. Mol. Biol., 222:581-597 (1991), or as described in the method according Orum et al., Nucleic Acids Res., 21:4491-4498 (1993). For cloning of amplified DNA in expressing vectors can be entered rare restriction sites in primer for PCR in the form of a label on one end, as described in Orlandi et al. (1989), or more amplification by PCR with labeled primers as described in Clackson et al., Nature, 352:624-628 (1991).

The repertoire of synthetically reconstructed V-genes can be obtainedin vitrobased on segments V-genes. Most segments VH-human genes cloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet., 3:88-94 (1993); these cloned segments (including all the major conformation of the H1 - and H2-loop) can be used to obtain different repertoires of VH genes with primers for PCR, coding H3-loops of different sequences and lengths, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH-playlists can also be obtained in a variety of sequences that is concentrated in long-H3-loop the same length, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89:4457-4461 (1992). Segments Vκ and Vλ human cloned and behold generovany (reported in Williams and Winter, Eur. J. Immunol., 23:1456-1461 (1993)), and can be used to obtain synthetic repertoires light chain. The repertoire of synthetic V-gene-based range VH - and VL-bends and L3 and H3-length encode antibodies with considerable structural diversity. After amplification of the DNA encoding the V-gene segments V-genes in the germ line can be rebuiltin vitroaccording to the ways in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

The repertoires of fragments of antibodies can be designed by combining together repertoires of VH and VL genes in several ways. Each repertoire can be obtained in different vectors, and the vectors recombinein vitrofor example, as described in Hogrefe et al., Gene, 128:119-126 (1993), orin vivoconcomitant infection, for example, by loxP system described in Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993). In the way that recombinationin vivouse the double-stranded nature of Fab fragments to overcome the limitations of the size of the library, which limit the efficiency of transformationE. coli. "Naive" VH - and VL-clone repertoires separately, one in formigny and another in the phage vector. Two libraries are then combined by ragovoy infections contain fahmida bacteria so that each cell contains a different combination and the size of the library is limited only by the number of cells present (approximately 102 clones). Both vectors contain the signals of recombinationin vivoso that VH - and VL-genes recombine into a single replicon together and packaged into phage virions. These huge libraries provide large number of different antibodies with good affinity (Kd-1approximately 10-8M).

Alternative repertoires can be cloned sequentially into the same vector, for example, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991), or to collect together using PCR and then cloned, for example, as described in Clackson et al., Nature, 352:624-628 (1991). Assembly using PCR can also be used to join DNA VH and VL DNA encoding a flexible peptide spacer, for the formation of a repertoire of single-chain Fv (scFv). In another method of Assembly using PCR in the cell" is used for combining VH and VL genes in lymphocytes using PCR and then cloned repertoires connected genes, as described in Embleton et al., Nucl. Acids Res., 20:3831-3837 (1992).

Antibodies formed naive libraries (or natural or synthetic), may have moderate affinity (Kd-1approximately 106up to 107M-1), but affinity maturation can also mimic thein vitrodesign and re-selection based on secondary libraries, as described in Winter et al. (1994) above. For example, the mutation may accidentally enterin vito using inaccurate polymerase (reported in Leung et al., Technique, 1:11-15 (1989)) in the method according to Hawkins et al., J. Mol. Biol., 226:889-896 (1992) or in the method according to Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992). In addition, affinity maturation can be performed random mutagenesis of one or more CDRs, for example, using PCR with primers carrying a random sequence, covering the interest CDR, the selected individual Fv-clones and screened for clones with higher affinity. In WO 9607754 (published on 14 March 1996) described a method for inducing mutagenesis in a complementarity determining region light chain immunoglobulin to obtain a library of light chain genes. Another effective method is a recombination of VH or VL domains selected using phage display, with repertoires of naturally occurring variants of the V-domain, obtained from unimmunized donors, and screening for higher affinity in a few cycles adjustment circuit, as described in Marks et al., Biotechnol., 10:779-783 (1992). This method allows to obtain antibodies and antibody fragments with affiniscape approximately 10-9M or less.

Screening libraries can be implemented in various ways known in this field. For example, TAT226 can be used to coat the wells of adsorption tablets, to Express on the cell of the host, attached to the adsorption tablets, or used in cell sorting, or konjugierte with Biotin for capture streptavidin coated beads, or used in any other way for panning libraries phage display.

Samples ragovoy library contact with immobilized TAT226 under conditions suitable to bind at least a portion of the phage particles with the adsorbent. Usually conditions, including pH, ionic strength, temperature and the like, are chosen to mimic physiological conditions. Phages associated with the solid phase, washed and then elute the acid, for example, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991), or alkali, for example, as described in Marks et al., J. Mol. Biol., 222:581-597 (1991), or competition with antigen TAT226, for example, in the way similar to the way competition with antigen in Clackson et al., Nature, 352:624-628 (1991). Phages may be enriched in the 20-1000 times in one cycle of selection. Moreover, the enriched phage can be grown in bacterial culture and be subjected to further cycles of selection.

The effectiveness of selection depends on many factors, including the kinetics of dissociation during washing, and on whether the multiple fragments of antibodies on a single phage simultaneously to contact with the antigen. Antibodies with fast kinetics of dissociation and low affiniscape binding) can be kept in the line is the use of short cleaning, polyvalent phage display and high-density coating antigen solid phase. High density not only stabilizes the phage through multivalent interactions, but promotes the re-linking dissociative phage. Selection of antibodies with slow dissociation kinetics (and good affiniscape binding) can facilitate the use of long-term cleaning and monovalent phage display as described in Bass et al., Proteins, 8:309-314 (1990) and in WO 92/09690, and low density coating antigen, as described in Marks et al., Biotechnol., 10:779-783 (1992).

Possible breeding TAT226 between fagbemi antibodies of different affinely, even affinely, which slightly differ. However, random mutagenesis breeding antibodies (for example, in the implementation of some methods of affinity maturation), will likely lead to the formation of many mutants, most of which are linked to the antigen, and the few with a higher affinity. Given the limited number of TAT226 few phages with high affinity can be eliminated. To save all mutants with higher affinity phages can be incubated with excess biotinylated TAT226, but with biotinylated TAT226 in concentrations lower polyarnosti than the planned value of molar affinity constants for TAT226. Phages with high quality, the second affinity binding can then capture on streptavidin coated paramagnetic beads. This "equilibrium capture" enables discriminates antibodies according to their affinity of binding with sensitivity, which allows you to isolate mutant clones with affinity, up to two times higher, from a large excess of phages with lower affinity. Used washing conditions phages associated with the solid phase, can also be manipulated to separate on the basis of the kinetics of dissociation.

Clones of anti-TAT226 can be discriminates activity. In some embodiments implementing the invention relates to an anti-TAT226 antibodies that bind to living cells, which Express TAT226 in natural conditions. In one of the embodiments the invention relates to an anti-TAT226 antibodies that block the binding between the ligand TAT226 and TAT226, but do not block the binding between the ligand TAT226 and the second protein. Fv clones corresponding to such anti-TAT226 antibodies, can be discriminates (1) selection of clones of anti-TAT226 of ragovoy library, as described above, and optional amplification of the selected population of phage clones by growing population in a suitable bacterial host; (2) the selection of TAT226 and a second protein against which respectively require blocking and non-blocking activity; (3) adsorption of phage clones anti-TAT226 on immobilized TAT226; (4) used is eating an excess of the second protein elution any undesired clones that that recognize determinants of binding TAT226 that overlap with the determinants of binding of the second protein or coincide with them; and (5) elution of the clones which remain adsorbed in the course of stage (4). Optional clones with the desired properties of the blocking/non-blocking can further enrich the repetition here of the procedures for the selection of one or more times.

DNA encoding obtained from a hybrid monoclonal antibodies or Fv clones of phage display technique according to the invention, is easily isolated and is sequenced using conventional methods (e.g., using oligonucleotide primers designed for specific amplification of interest regions encoding the heavy and light chain from hybridoma or ragovoy matrix DNA). After DNA extraction can be embedded in expressing vectors, which are then transferout in cell host, such as cellsE. colicells , COS monkey cells Chinese hamster ovary (CHO) or myeloma cells that do not otherwise produce protein-immunoglobulin, for the synthesis of the required monoclonal antibodies in the recombinant cell host. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256 (1993) and Pluckthun, Immunol. Revs, 130:151 (1992).

DNA to yuushuu Fv-clones according to the invention, can be combined with known DNA sequences encoding the constant region of the heavy chain and/or light chain (e.g., the appropriate DNA sequence may be obtained from Kabat et al., above), to obtain clones encoding the heavy and/or light chain of the full or partial length. Will be taken into account that you can use for this purpose a constant region of any isotype, including the constant region of IgG, IgM, IgA, IgD and IgE, and that such constant region can be obtained from either human or animal. As used in the present description, Fv-clone originating from DNA variable domain of one species of animal (such as man) and then merged with the DNA of a constant region of another species of animal, with the formation of the sequence(s), its(their) "hybrid"full size heavy chain and/or light chain include in the definition of "chimeric" and "hybrid" antibodies. In some embodiments, the implementation of the Fv-clone originating from variable human DNA, are merged with the DNA of a constant region of a human with the formation of the sequence(s), its(their) heavy and/or light chain of the full or partial length.

DNA encoding anti-TAT226 antibodies originating from hybridoma according to the invention can also be modified, for example, by replacing the sequence encoding the intercept is ntie domains of the heavy and light chains of a human, instead of the homologous murine sequences derived from clone hybridoma (for example, as in the method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). DNA encoding the antibody or fragment originating from hybridoma or Fv-clone, can be further modified by covalent joining sequence that encodes an immunoglobulin, all or part of a sequence that encodes a polypeptide that is not an immunoglobulin. Thus, you receive a "chimeric" or "hybrid" antibodies that have the binding specificity of the antibodies according to the invention, originating from Fv-clone or clone hybridoma.

3. The vectors, cells of the host and recombinant methods

For recombinant obtain antibodies according to the invention encoding its nucleic acid is isolated and inserted into can replicate the vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody, easy to produce and is sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). Many vectors. The choice of vector depends in part on the used host cell. Typically, cells are the owners have or prokaryotic, or eukaryotic (usually from the milk of itausa) origin. Take into account that for this purpose you can use the constant region of any isotype, including the constant region of IgG, IgM, IgA, IgD and IgE, and that such constant region can be obtained from either human or animal.

a) Obtaining antibodies using prokaryotic host cells:

(1) Construction of vectors

Polynucleotide sequences encoding polypeptide components of the antibodies according to the invention, can be obtained using conventional recombinant methods. The desired polynucleotide sequence can be extracted and sequenced from producing the antibody of cells, such as cell hybrid. Alternative polynucleotide can be synthesized using nucleotide synthesizer or methods based on PCR. After receiving the sequence encoding the polypeptides are inserted into the recombinant vector capable of replication and expression of heterologous polynucleotides in prokaryotic hosts. Many vectors are available and known in this field can be used for the purpose of the present invention. The selection of the appropriate vector will depend largely on the size of nucleic acids which are inserted into the vector and the particular host cell which is transformed with the vector. Each vector contains various components in C is depending on its function (amplification or expression of heterologous polynucleotide or, and more) and its compatibility with a particular cell of the host in which it is located. Vector components generally include, as non-limiting examples: the origin (beginning) of replication, a marker gene for selection, the promoter, the binding site with the ribosome (RBS), signal sequence, the insertion of a heterologous nucleic acid and the sequence termination of transcription.

Generally, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host-cell, used in relation to these hosts. Vector, as a rule, is the site of replication and a marker sequence that can ensure the selection of transformed cells by phenotype. For example,E. coliusually transformed using pBR322, a plasmid derived fromE. coli. pBR322 contains genes encoding resistance to ampicillin (Amp) and tetracycline (Tet) and, thus, provides an easy way of identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or can be modified so that they contain the promoters that can be used in the microbial organism for expression of endogenous proteins. Examples of derivatives of pBR322, used for the expression of Conques is to maintain antibody, described in Carter et al., U.S. patent No. 5648237.

In addition, phage vectors containing replicon and control sequences that are compatible with the microorganism host, can be used as transformation vectors in relation to these hosts. For example, bacteriophage such as λGEM.TM.-11, can be used to obtain a recombinant vector that can be used to transform susceptible host cells such asE. coliLE392.

Expressing the vector according to the invention may contain two or more pairs of the promoter-cistron that encode each of the polypeptide components. The promoter is a noncoding regulatory sequence above (5') cistron, which modulates its expression. Prokaryotic promoters, as a rule, belong to two classes, inducible and constitutive. The inducible promoter is a promoter, which stimulates increased levels of transcription of cistron under its control, in response to changes in culture conditions, such as the presence or absence of nutrients or temperature changes.

It is well known a large number of promoters that are recognized by a variety of potential host cells. The selected promoter can be functional in order to associate with castronno DNA encoding the light or heavy chain, highlighting the promoter from the source DNA by cleavage with restriction enzymes and inserting the selected promoter sequence in the vector according to the invention. As a natural promoter sequence and many heterologous promoters may be used to control the amplification and/or expression of target genes. In some embodiments, the implementation of the use of heterologous promoters, as they typically provide a higher level of transcription and higher output expressed by the target gene in comparison with the natural promoter of the target polypeptide.

Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the promoter system β-galactosi and lactose promoter system tryptophan (trp) and hybrid promoters such as the tac promoter or the trc. However, also other suitable promoters that are functional in bacteria (such as other known bacterial or phage promoters). Their nucleotide sequences are published, thus allowing qualified functionally ligitamate them with cisternae coding target light and heavy chains (Siebenlist et al. (1980) Cell 20:269) using linkers or adapters to provide any necessary phase is mi restriction.

In one aspect of the invention, each cistron in the recombinant vector contains a component of the secretory signal sequence, which directs the movement of expressed polypeptides across the membrane. Typically, the signal sequence may be a component of the vector, or it may be part of the coding target polypeptide DNA that is inserted into a vector. The signal sequence selected, for the purposes of this invention, should be recognized by the host-cell and processionals (i.e. to split the signal peptidases) in it. For prokaryotic host cells that do not recognize and do not ProcessInput signal sequence characteristic of heterologous polypeptides, the signal sequence is substituted prokaryotic signal sequence selected, for example, from the group consisting of the leader sequence of alkaline phosphatase, penitsillinazy, Ipp or thermostable enterotoxin II (STII), LamB, PhoE, PelB, OmpA and MBP. In one of the embodiments of the invention the signal sequence used for both Castronovo expression systems represent the STII signal sequences or their variants.

In another aspect of the production of the immunoglobulins according to the invention can occur in the cytoplasm of a cell-hosay is a and, thus, does not require a secretory signal sequences in each cistron. In this case, the light and heavy chains of immunoglobulins are expressed, are minimized and going with the formation of a functional immunoglobulin in the cytoplasm. Some strains hosts (for example, strains ofE. colitrxB-) create conditions in the cytoplasm, which is favorable for the formation of a disulfide bond, thus ensuring the correct folding and Assembly of expressed protein subunits, Proba and Pluckthun, Gene, 159:203 (1995).

Antibodies according to the invention can also be produced by using expression systems, in which the quantitative ratio of the expressed polypeptide components can be modulated to achieve the maximum yield of secreted and correctly assembled antibodies according to the invention. Such modulation is carried out, at least partially simultaneous modulation efficiencies broadcast polypeptide components.

One way of modulating the efficiency of translation is described in Simmons et al., U.S. patent No. 5840523. It uses the options field of translation initiation (TIR) cistron. For this TIR you can get a number of options amino acid sequence or nucleic acid sequence with a range of efficiencies broadcast, so the way giving a convenient means to adjust this factor to the desired level of expression of a particular circuit. Options TIR can be obtained by using common methods of mutagenesis, which lead to the replacement of codons that can change the amino acid sequence. In some embodiments, the implementation of substitutions in the nucleotide sequence are silent. Changes in TIR may include, for example, change the number or location of sequences Shine-Dalgarno, together with changes in the signal sequence. One way of obtaining mutant signal sequences consists in obtaining Bank codons" in the beginning of the coding sequence, which does not change the amino acid sequence of the signal sequence (i.e. substitutions are silent). This can be done by changing the third nucleotide position of each codon; in addition, some amino acids such as leucine, serine and arginine, have several the first and second positions, which may complicate the receiving Bank. This method of mutagenesis is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.

In one embodiment, the implementation of a set of vectors to be obtained with a range of efficiencies TIR for each cistron in these vectors. This limited set provides a comparison of expression levels of each circuit, and the output of the desired product antibodies at different combinations of the efficiencies of TIR. The effectiveness of TIR is possible to quantify the level of expression of a reporter gene, as described in Simmons et al., U.S. patent No. 5840523. Based on a comparison of the effectiveness of broadcast select desired specific TIR for combination in constructions expressing vectors according to the invention.

Prokaryotic cells are the owners that are suitable for expression of the antibodies of the invention include archaebacteria and eubacteria, such as gram-negative or gram-positive organisms. Examples of suitable bacteria includeEscherichia(for example,E. coli),Bacilli(for example,B. subtilis), enterobacteria, the speciesPseudomonas(for example,P. aeruginosa),Salmonellatyphimurium,Serratiamarcescans,Klebsiella,Proteus,Shigella,Rhizobia,VitreoscillaorParacoccus. In one of the embodiments use the gram-negative cells. In one of the embodiments using cellsE. colias hosts for constructions according to the invention. Examples of strains ofE. coliinclude strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), p.1190-1219; depositing in ATCC No. 27325) and its derivatives, including strain 33D3 having genotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41 kanR (U.S. patent No. 5639635). Also suitable for other strains and derivatives thereof, such asE. coli294 (ATCC 31446),E. coliB,E. coliλ 1776 (ATCC 31537), andE. coliRV308 (ATCC 31608). These examples are illustrative, and not limiting. The methods of constructing derivatives of any of the above bacteria, possessing certain genotypes, known in this field and are described, for example, Bass et al., Proteins, 8:309-314 (1990). As a rule, you must select the appropriate bacteria, whereas replenishement replicon cells bacteria. For example, the speciesE. coli,SerratiaorSalmonellacan appropriately be used as a master, if for replication uses well known plasmids such as pBR322, pBR325, pACYC177, or pKN410. Typically, a host cell must secrete minimal amounts of proteolytic enzymes, and optionally in the cell culture can contribute additional protease inhibitors.

(2) the Production of antibodies

Cell owners transform the above expressing vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Transformation means introducing DNA into a prokaryotic host such a way that DNA is replicated or as an extrachromosomal element, or as a chromosomal integrant. Depending on the host cell transformation exercise is the use of common methods, suitable for such cells. The processing of calcium using calcium chloride as a rule, used for bacterial cells, which have significant barriers in the form of the cell wall. In another method transformation using polyethylene glycol/DMSO. Another method is electroporation.

Prokaryotic cells used for the production of polypeptides according to the invention are grown in environments known in the field and suitable for culturing a selected host cells. Examples of suitable media include environment Luria (LB) plus the necessary nutrients. In some embodiments, the implementation of the environment also contain a substance for making selection based constructs expressing vector to provide selective growth of prokaryotic cells containing expressing vector. For example, ampicillin is added to the media for the growth of cells expressing the gene of resistance to ampicillin.

Any necessary additives in addition to the sources of carbon, nitrogen and inorganic phosphate can also be included in relevant concentrations, making them individually or in a mixture with other additives or environment, such as a complex nitrogen source. Optional culture medium may contain one or more reducing the medium is in, selected from the group consisting of glutathione, cysteine, applied, thioglycolate, dithioerythritol and dithiothreitol.

Prokaryotic cells are the owners of cultivated under suitable temperatures. In some embodiments, the implementation for the cultivation ofE. colithe temperature for growing the range from approximately 20°C to about 39°C, from about 25°C to about 37°C, or about 30°C. the pH of the medium may be any pH ranging from about 5 to about 9, generally depending on the host body. In some embodiments, the implementation forE. colipH is from about 6.8 to about or approximately 7,4 7,0.

If expressing the vector according to the invention using inducible promoter, expression of the protein induce under conditions suitable for activation of the promoter. In one aspect of the invention using the PhoA promoter for regulating transcription of the polypeptide. Thus, the transformed cell hosts are cultivated in the medium for induction, containing a limited amount of phosphate. In some embodiments, the implementation of a medium containing a limited amount of phosphate is CRAP (see, for example, Simmons et al., J. Immunol. Methods (2002), 263:133-147). You can use many other inductors in accordance with used the th design vector, as is well known in this field.

In one embodiment, the implementation of the expressed polypeptides of the present invention are secreted into periplasm host cells, and distinguish them from it. The selection of protein typically involves the destruction of the microorganism, usually through methods such as osmotic shock, sonication or lysis. After the destruction of the cells remains of cells or whole cells can be removed by centrifugation or filtration. Proteins can be further clear, for example, by affinity chromatography on a resin. Alternative proteins can be transferred into culture medium and to select from them. Cells can be removed from the culture and the culture supernatant filtered and concentrated for further purification of proteins produced. Expressed polypeptides can then be isolated and identified using conventional methods such as polyacrylamide gel electrophoresis (PAGE) and analyzed using Western blotting.

In one aspect of the invention the production of antibodies is carried out in large quantities by fermentation process. Various large-scale fermentation procedure under cultivation with water available for the production of recombinant proteins. Large-scale methods of fermentation include at least a capacity of 1000 liters and in some the older versions of the implementation capacity of about 1,000 to 100,000 liters. In these fermenters use paddle stirrers for the distribution of oxygen and nutrients, especially glucose (the preferred source of carbon/energy). Small-scale fermentation means, as a rule, the fermentation in the fermenter, the volumetric capacity of which does not exceed approximately 100 liters and can vary from about 1 liter to about 100 liters.

In the fermentation process the induction of expression of the protein, typically starting after the cells grow under suitable conditions to the desired density, for example OD550approximately 180-220, at this stage the cells are in early stationary phase. You can use a variety of inductors in the design vector, as is well known in this field and described above. Cells can be grown for more than short periods before induction. Cells typically induce approximately 12-50 hours, although you can use a longer or shorter induction time.

To improve the yield and quality of the polypeptides according to the invention can be modified in different fermentation conditions. For example, to improve the proper Assembly and folding of secreted polypeptides antibodies in a joint transformation of prokaryotic host cells can be used further the nutrient vectors, sverkhekspressiya protein chaperones, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and/or DsbG) or FkpA (peptideprophet-CIS,TRANS-isomerase with chaperone activity). It is shown that protein chaperones promote the proper folding and solubility of heterologous proteins produced by bacterial cells masters, Chen et al. (1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. patent No. 6083715; Georgiou et al., U.S. patent No. 6027888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol. Environ. 39:199-210.

For minimizing proteolysis of expressed heterologous proteins (in particular proteins, which are sensitive to proteolysis) can be used for the present invention, certain strains hosts with impaired proteolytic enzymes. For example, strains of host cells can be modified to obtain genetic(a) mutation(s) in genes encoding known bacterial proteases such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI and combinations thereof. Some strains ofE. colidisturbed proteases are available and are described, for example, in Joly et al. (1998), above; Georgiou et al., U.S. patent No. 5264365; Georgiou et al., U.S. patent No. 5508192; Hara et al., Microbial Drug Resistance, 2:pp.63-72 (1996).

In one embodiment, the implementation of the strains ofE. coliwith impaired proteolytic enzymes and transformed plasmids, surgex residuosity one or more proteins, chaperones, used as host cells in the expression system according to the invention.

(3) Purification of antibodies

In one of the embodiments obtained in this study protein antibodies optionally purified to obtain preparations that are essentially homogeneous for further analyses and applications. You can use the conventional methods of protein purification known in this field. The following methods are of appropriate cleaning methods as example: fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, HPLC with reversed phase chromatography on silica or cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, precipitation with ammonium sulfate and gel filtration using, for example, Sephadex G-75.

In one aspect, the protein A immobilized on a solid phase, for use immunoaffinity cleaning products antibodies according to the invention. Protein a is a protein of the cell wall mass of 41 kDa fromStaphylococcusaureasthat binds with high affinity to Fc region of antibodies, Lindmark et al. (1983) J. Immunol. Meth. 62:1-13. Solid phase on which the immobilized protein a may be a column containing glass or silica surface, or a glass column with a controlled pore size, or column based on kremna the Oh of the acid. In some embodiments, the implementation of the column cover with a reagent, such as glycerin, to possibly prevent nonspecific adsorption of impurities.

As the first stage of purification of the product obtained from the cell culture, as described above, is placed on a solid phase with immobilized protein A to ensure specific binding interest antibodies with protein A. the Solid phase can then be washed to remove impurities, nonspecific contacting the solid phase. In conclusion, interest antibody is removed from the solid phase by elution.

b) Obtaining antibodies using eukaryotic host cells:

Vector for use in eukaryotic cells is the owner, as a rule, contains, as non-limiting examples of one or more of the following: a signal sequence, the origin (beginning) of replication, one or more marker genes, an enhancer element, a promoter and a sequence of termination of transcription.

(1) The signal sequence

Vector for use in eukaryotic cells-the owner may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-end interest of the Mature protein or polypeptide. Wybran what I heterologous signal sequence may be a sequence, which is recognized by the host-cell and processed (i.e. cleaved signal peptidases) in it. For expression in mammalian cell available signal sequence mammals, as well as viral secretory leader sequence, for example, gD-signal herpes simplex virus. The DNA for such field-predecessor are ligated in reading frame with the coding antibody DNA.

(2) the origin (beginning) replication

Generally, the origin (beginning) replication is not necessary for expressing vectors mammals. For example, the SV40 origin may typically be used only because it contains the early promoter.

(3) a Component of a gene, providing a selection

Expressing and cloning vectors may contain a gene that provides the selection, also known breeding marker. Typical genes selection, encode proteins that (a) give resistance to antibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate, or tetracycline, (b) complementary auxotrophic failure in appropriate circumstances, or (c) provide vital nutrients that are not available in complex environments.

In one example of the selection process used medication to stop the Ki growth of the host cell. Those cells that are successfully transformed with a heterologous gene produce a protein which imparts resistance to the drug, and thus survive in the conditions of selection. Examples of such dominant selection includes drugs neomycin, mycophenolate acid and hygromycin.

Another example of suitable breeding markers for mammalian cells are a marker such as DHFR, thymidine kinase, metallothionein-I and-II, preferably genes metallothionein primates, adenoidectomies, ornithindecarboxilase, etc. that make it possible to identify cells that are competent in respect of the absorption of the nucleic acid that encodes the antibody.

For example, in some embodiments, the implementation of cells transformed by providing a selection of DHFR gene, first identified in the cultivation of all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. In some embodiments, the implementation using DHFR wild type corresponding to the host-cell is a cell line of Chinese hamster ovary (CHO) with impaired DHFR activity (e.g., ATCC CRL-9096).

Alternative cell owners (particularly wild-type hosts that contain endogenous DHFR)transformed or co-transformed sequence of the DNA, encoding the antibody, protein DHFR wild-type and the other of breeding marker such as aminoglycoside-3'-phosphotransferase (APH)can be selected by cell growth in medium containing ensuring the selection of material for breeding marker such as aminoglycoside antibiotic, such as kanamycin, neomycin, or G418. Cm. U.S. patent No. 4965199.

(4) The promoter

Expressing and cloning vectors usually contain a promoter that is recognized by the body is the master and which is functionally linked to a nucleic acid that encodes an polypeptide (e.g. antibody). Promoter sequences are known for eukaryotes. For example, virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases above the site of transcription initiation. Another sequence found 70 to 80 bases above from the start of transcription of many genes is an area CNCAAT, where N can be any nucleotide. At the 3'end of most eukaryotic genes is the sequence AATAAA, which may be a signal to add a poly-A tail at the 3'end of the coding sequence. In some embodiments, the implementation of any or all of these sequences are properly inserted into E. kriticheskie expressing vectors.

Transcription vectors in the cells of the host mammal control, for example, promoters derived from the genomes of viruses such as virus polyoma, rinderpest virus of birds, adenovirus (such as adenovirus 2), the virus bovine papilloma virus sarcoma birds, cytomegalovirus, a retrovirus, hepatitis B virus and the monkey virus 40 (SV40), from heterologous mammalian promoters, e.g. the actin promoter or promoter of the immunoglobulin from the promoters of heat shock, provided such promoters are compatible with the systems of the host cell.

Early and late promoters of SV40 virus are conveniently obtained as SV40 restriction fragment which also contains the viral origin (beginning) of the SV40 replication. Pretani the promoter of the human cytomegalovirus is conveniently obtained as a HindIII restriction fragment E. the System for the expression of DNA in mammalian hosts using virus bovine papilloma as a vector described in U.S. patent No. 4419446. Modification of this system is described in U.S. patent No. 4601978. Cm. also Reyes et al., Nature 297:598-601 (1982) on the expression of cDNA β-interferon in human cells of mice under the control of the promoter timedancing from herpes simplex virus. Alternative long terminal repeat of rous sarcoma virus can be used as a promoter.

(5) The enhancer element

Transcription Of D Is K, encoding the antibody according to this invention, in higher eukaryotes is often increased by inserting enhancer sequence into the vector. Currently known many enhancer sequences from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). As a rule, however, use an enhancer from a eukaryotic virus cells. Examples include the SV40 enhancer on the late side from the origin of replication (BP 100-270), the enhancer early promoter of cytomegalovirus enhancer of virus polyoma on the late side from the origin of replication and adenovirus enhancers. Cm. also Yaniv, Nature 297:17-18 (1982) relative to the enhancer elements for activation of eukaryotic promoters. The enhancer can be embedded into a vector in the 5'- or 3'-position with respect to a sequence that encodes a polypeptide antibodies, but, as a rule, it is located at the 5'segment from the promoter.

(6) The termination of transcription

Expressing the vectors used in eukaryotic cells-the owners may also contain sequences necessary for the termination of transcription and stabilization of the mRNA. Such sequences are usually located in the 5'- and sometimes in the 3'untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as paladin the isolation of the fragments in the untranslated portion of the mRNA, encoding the antibody. One of the suitable components termination of transcription is an area polyadenylation growth hormone bull. Cm. WO94/11026 and described in this work expressing vector.

(7) Selection and transformation of host cells

In the present work appropriate cell hosts for cloning or expression of the DNA in the vectors include cells of higher eukaryotes, as described in the present work, including cells of vertebrate hosts. Propagation of vertebrate cells in culture (tissue culture) has become a common procedure. Examples of suitable cell lines of mammalian hosts represent a line of monkey kidney CV1 transformed by SV40 (COS-7, ATCC CRL 1651); a line of embryonic human kidney (293 or 293 cells, subcloned to grow in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); the cells of the kidneys of the newborn hamster (BHK, ATCC CCL 10); the cells of the Chinese hamster ovary/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); the Sertoli cells of mice (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); kidney cells monkeys (CV1 ATCC CCL 70); kidney cells of the African green monkey (VERO-76, ATCC CRL-1587); carcinoma cells human cervical (HELA, ATCC CCL 2); cells, dog kidney (MDCK, ATCC CCL 34); liver cells of rats Buffalo (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); the cells of the human liver (Hep G2, HB 8065); tumor of the mammary gland of mice (MMT 060562, ATCC CCL51);TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982)); the cells, MRC 5; FS4 cells and human hepatoma (Hep G2).

Cell owners transform the above expressing or cloning vectors for the production of antibodies and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

(8) Culturing host cells

Cell owners used to generate antibodies according to this invention, can be grown in many environments. For culturing host cells suitable commercially available medium such as ham's F10 (Sigma), minimal medium (MEM, Sigma), RPMI-1640 (Sigma) and Wednesday Needle in the modification of Dulbecco (DMEM, Sigma). Moreover, as culture media for the host cells can be used any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. patent No. 4767704, 4657866, 4927762, 4560655 or 5122469; WO 90/03430; WO 87/00195; or U.S. patent Re. 30985. In any of these environments can be added if necessary with hormones and/or other growth factors (such as insulin, transferrin, or a growth factor of the epidermis), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as Lekarstvo the e tool gentamicin™), trace elements (defined as inorganic compounds usually present at final concentrations of the order of the micromol), and glucose or an equivalent energy source. Any other additives can also be made in appropriate concentrations, which are known to experts in this field. Culturing conditions, such as temperature, pH, etc. represent the conditions used for the host cell selected for expression, and will be visible to an ordinary specialist.

(9) Purification of antibodies

Using recombinant methods, the antibody can be produced intracellularly or directly to secrete into the environment. If the antibody is produced intracellularly, as a first stage debris in the form of particles or cells of the owners, or lysed fragments can be removed, for example, by centrifugation or ultrafiltration. If the antibody is secreted into the medium, supernatants from such expression systems can be first concentrated using a commercially available filter for concentrating the protein, for example, a kit for ultrafiltration Amicon or Millipore Pellicon. You can make a protease inhibitor such as PMSF, on any of the above stages for the inhibition of proteolysis and you can make antibiotics to prevent the growth of casual contaminant is.

The composition of the antibodies obtained from cells can be purified using, for example, chromatography with hydroxyapatite, gel electrophoresis, dialysis, and affinity chromatography, where the affinity chromatography is a suitable method of purification. The suitability of protein A as an affinity ligand depends on the species and isotype of any Fc-domain of an immunoglobulin, which is the antibody. Protein A can be used for purification of antibodies, which are based on the heavy chain γ1, γ2, or γ4 human (Lindmark et al., J. Immunol. Methods 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for ø3 person (Guss et al., EMBO J. 5:1567-1575 (1986)). Matrix, which is attached with the affinity ligand may be an agarose is, however, available and other matrices. Mechanically stable matrices such as glass with controlled pore size or poly(Stradivari)benzene, provide higher flow rates and shorter processing time than those that can be achieved with agarose. If the antibody contains a CH3 domain, suitable for cleaning resin Bakerbond ABX™ (J. T. Baker, Phillipsburg, NJ). Also, depending on the antibody to be the selection that is available other methods of protein purification such as fractionation on an ion-exchange column, ethanol precipitation, HPLC with reversed phase chromatography on silica, chromatography on sepharose™ g is a Parin, chromatography on an anion or cation exchange resin (such as a column with poliasparaginovaya acid), chromatofocusing, SDS-PAGE and precipitation with ammonium sulfate.

After any(s) advance(s) phase(s) of purification of a mixture containing an antibody and impurities, can be subjected to additional purification, for example, by hydrophobic interaction chromatography with a low pH using a buffer for elution with a pH value of approximately between 2.5 to 4.5, which is preferably performed at low salt concentrations (e.g., approximately 0-0,25 M salt).

Generally, various methods for producing antibodies for use in research, testing and clinical use are well known in this field, consistent with the above-described method and/or assumed appropriate specialist in this field for a specific interest antibodies.

C. Immunoconjugate

The invention also relates to immunoconjugates (interchangeably referred to as "the conjugates of the antibody-drug" or "ADC"), containing any anti-TAT226 antibodies of the invention conjugated to one or more cytotoxic means, such as a chemotherapeutic agent, a drug, a means of inhibiting the growth, toxin (e.g., f is mentative active toxin of bacterial, fungal, plant or animal origin, or fragments), or a radioactive isotope (i.e radioconjugates).

Immunoconjugate can be used in the treatment of cancer for the local delivery of cytotoxic agents, i.e. drugs that destroy or inhibit the growth or proliferation of tumor cells (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; U.S. patent No. 4975278). Immunoconjugate provide targeted delivery of the component drugs to the tumor and its intracellular accumulation therein, if the system introduction unconjugated drugs can lead to unacceptable levels of toxicity to normal cells, as well as when you wish to eliminate tumor cells (Baldwin et al. (1986) Lancet (Mar. 15, 1986) p.603-05; Thorpe (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies'84: Biological And Clinical Applications (A. Pinchera et al., eds) p.475-506). It was reported that both polyclonal antibodies and monoclonal antibodies suitable for these methods (Rowland et al. (1986) Cancer Immunol. Immunother., 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al. (1986), above). Toxins used in the conjugates of the antibody-toxin include bacterial toxins such as diphtheria toxin, plant toxins such as is itzin, small molecule toxins, such as geldanamycin (Mandler et al. (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), maytansinoid (EP 1391213; Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623) and calicheamicin (Lode et al. (1998) Cancer Res. 58:2928; Hinman et al. (1993) Cancer Res. 53:3336-3342). Toxins can cause cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding or inhibition of topoisomerase. Some cytotoxic drugs have a tendency to inaktivirovanie or become less active during conjugation with major antibodies or protein ligands of receptors.

Of ZEVALIN®(ibritumomab tiuxetan, Biogen/Idec) is a conjugate of the antibody-radioisotope consisting of murine monoclonal antibodies IgG1 Kappa directed against the CD20 antigen presented on the surface of normal and malignant B-lymphocytes, and radioisotope111In or90Y related timesaving chelating linker (Wiseman et al. (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al. (2002) Blood 99(12):4336-42; Witzig et al. (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al. (2002) J. Clin. Oncol. 20(15):3262-69). Although the ZEVALIN has activity against B-cell non-Hodgkin lymphoma (NHL), its introduction causes severe and prolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab of autogamic is h, Wyeth Pharmaceuticals), conjugate antibodies with drug consisting of antibodies to huCD33 associated with calicheamicin, has received approval from 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future(2000) 25(7):686; U.S. patents№ 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001). Cantuzumab mertansine (Immunogen, Inc.), conjugate antibody with a drug consisting of the huC242 antibody linked via a disulfide linker SPP to the component of the medicinal product on the basis of maytansinoid, DM1, is undergoing phase II studies for the treatment of cancers in which to Express CanAg, such as colon cancer, pancreas, stomach, etc. MLN-2704 (Millennium Pharm., BZL Biologies, Immunogen, Inc.), conjugate antibody with a drug consisting of monoclonal antibodies against specific to prostate membrane antigen (PSMA)associated with a component of a medicinal product on the basis of maytansinoid, DM1, is under development for the potential treatment of prostate tumors. Auristatin peptides, auristatin E (AE) and monomethylmercury (MMAE), synthetic analogs of dolastatin, conjugatively with chimeric monoclonal antibodies cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies) (Doronina et al. (2003) Nature Biotechnology 21(7):778-784), and they are the state therapeutic development.

In some embodiments, the implementation immunoconjugate contains an anti-TAT226 antibody and chemotherapeutic agent or other toxin. Chemotherapeutic agents suitable for receiving immunoconjugates described in this paper (e.g., above). You can also use the enzymatically active toxins and fragments thereof, and they are described in this work.

In some embodiments, the implementation immunoconjugate contains an anti-TAT226 antibody and one or more low molecular weight toxins, including as non-limiting examples of low molecular weight drugs, such as calicheamicin, maytansinoid, dolastatin, auristatin, trichothecin and CC1065, and the derivatives of these drugs, which have cytotoxic activity. Examples of such immunoconjugates further detail below.

1. Illustrative immunoconjugate

Immunoconjugate (or conjugate antibody-drug" ("ADC")) according to the invention can have the formula I, below, where an anti-TAT226 antibody conjugated (i.e. covalently attached) with one or more components of the medicinal product (D) via a suitable linker (L).

Accordingly, an anti-TAT226 antibody can be konjugierte with drug either directly or through whether the Kera. In formula I, p is the average number of components of the drug to the antibody, which may vary, for example, from approximately 1 to approximately 20 components of the drug to the antibody, and in some embodiments, the implementation from 1 to about 8 components of the drug to the antibody.

(a) Illustrative linkers

The linker may consist of one or more components of the linker. Illustrative components of linkers include 6-maleimidomethyl ("MC"), maleimidomethyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe), p-aminobenzeneboronic ("PAB"), N-Succinimidyl-4-(2-pyridylthio)pentanoate ("SPP"), N-Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC) and N-Succinimidyl-(4-iodates)aminobenzoate ("fairs are forthcoming-Siab"). The various components of linkers known in this area, some of which are described below.

The linker can be a "biodegradable linker"facilitating release of the drug in the cell. For example, you can use unstable to acids linker (for example, hydrazone), sensitive to proteases (e.g., sensitive peptidase) linker, photolabile linker, dimethyl linker or dyslipidaemias linker (Chari et al. Cancer Research 52:127-131 (1992); U.S. patent No. 5208020).

In some variations the tah implementation component of the linker may contain "tensile link", which binds the antibody with another component of the linker or with a component of a medicinal product. Illustrative tensile links below (where the wavy line indicates the sites of covalent attachment to an antibody):

In some embodiments, the implementation component of the linker may contain amino acid level. In one of these embodiments the amino acid level provides cleavage of linker-protease, thus facilitating the release of drugs from immunoconjugate when exposed to intracellular proteases, such as lysosomal enzymes. See, for example, Doronina et al. (2003) Nat. Biotechnol. 21:778-784. Illustrative amino acid units include as non-limiting examples of dipeptide, Tripeptide, tetrapeptide and Pentapeptide. Illustrative dipeptides include valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys) or N-methyl-valine-citrulline (Me-val-cit). Illustrative tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid link may contain amino acid residues that exist in nature, as well as minor amino acids and do not exist in nature, similar amino acids such as citrulline. Amino acid chains can konstruirovanie and optimize their selectivity with respect to the enzymatic cleavage by a specific enzyme for example associated with tumor protease, cathepsin B, C and D or protease plasmin.

In some embodiments, the implementation component of the linker may contain "spacer elements" link that binds to the antibody component of the medicinal product, either directly or through a tensile element and/or amino acid level. Spacer elements link may be a "self sacrifice" or "negritude." "Negritude a" spacer elements link can be a link, in which part of the spacer elements parts or all of them remain associated with the component medicines enzymatic (e.g., proteolytic) the splitting of the ADC. Examples desertusa a spacer elements links include as non-limiting examples of glycine spacer elements link and glycine-glycine spacer elements link. Also provided by other combinations of peptide spacers, subject-specific with respect to the sequence of enzymatic cleavage. For example, enzymatic cleavage ADC containing a glycine-glycine spacer elements link associated with tumor cells by protease may lead to the release of glycine-glycine component of a medicinal product from the residue ADC. In one of these embodiments, the glycine-glycine component of the medicinal product the ZAT is subjected to a separate hydrolysis step in cancer cells, thus arseplay glycine-glycine spacer elements link from the component drugs.

"Self sacrifice" link spacer elements provide release component of a medicinal product without a separate hydrolysis step. In some embodiments, the implementation of the spacer elements link the linker contains a link p-aminobenzyl. In one of these embodiments p-aminobenzoyl alcohol attached to the amino acid chain via amide linkages between benzyl alcohol and the cytotoxic agent is formed carbamate, methylcarbamate or carbonate. See, for example, Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103. In one embodiment, the implementation of the spacer elements element is a p-aminobenzeneboronic (PAB). In some embodiments, the implementation fenelonov part of the link p-aminobenzyl substituted Qm, where Q represents-C1-C8alkyl, -O-(C1-C8alkyl), -halogen, -nitro or-cyano and m is an integer in the range 0-4. Examples the victim of a spacer elements units additionally include as non-limiting examples of aromatic compounds that are electronically similar to the p-aminobenzyl alcohol (see, for example, US 2005/0256030 A1), such as derivatives of 2-aminoimidazole-5-methanol (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho - or para-aminobenzoate. You can use pastry, subjected to cyclization by the hydrolysis of amide linkages, such as substituted and unsubstituted amides of 4-aminobutyric acid (Rodrigues et al., Chemistry Biology, 1995, 2, 223); appropriately substituted cyclic system bicyclo[2.2.1] bicyclo[2.2.2] (Storm, et al., J. Amer. Chem. Soc., 1972, 94, 5815); and amides of 2-aminophenylamino acid (Amsberry et al., J. Org. Chem., 1990, 55, 5867). Removal of aminecontaining medicines, which are substituted in the α-position of glycine (Kingsbury et al., J. Med. Chem., 1984, 27, 1447), also represents examples victim of a spacer suitable for the ADC.

In one embodiment, the implementation of the spacer elements chain is a branched link bis(hydroxymethyl)styrene (BHMS), as presented below, which can be used to activate and release of multiple drugs.

where Q represents-C1-C8alkyl, -O-(C1-C8alkyl), -halogen, -nitro or-cyano; m is an integer in the range 0-4; n is 0 or 1; and p ranges from 1 to about 20.

The linker may contain any one or more of the above components of the linker. In some embodiments, the implementation of the linker is the same as presented in parentheses in the following formula II ADC:

where A represents restivus is e link and a is an integer from 0 to 1; W represents the amino acid level and w is an integer from 0 to 12; Y is a spacer elements link and y represents 0, 1 or 2; and Ab, D, and p are defined as above for formula I. Illustrative embodiments of such linkers are described in US 2005-0238649 A1, which is expressly given here as a reference.

Illustrative components of the linker and their combinations are described below in the context of the ADC of formula II:

Val-Cit or VC

Components of linkers, including tensile, spacer elements and amino acid units can be synthesized by methods known in this field, such as the methods described in US 2005-0238649 A1.

b) Illustrative components of the medicinal product

(1) Maytansine and maytansinoids

In some embodiments, the implementation immunoconjugate contains the antibody of the invention conjugated to one or more molecules of maytansinoids. Maytansinoid are inhibitors of mitosis, which act by inhibiting tubulin polymerization. First mitanin was isolated from the East African shrubMaytenus serrata(U.S. patent No. 3896111). Then it was discovered that certain microbes also produce maytansinoid, such as maytas the Nol and esters maytansine C-3 (U.S. patent No. 4151042). Synthetic maytansines and its derivatives and analogs are described, for example, in U.S. patents№ 4137230; 4248870; 4256746; 4260608; 4265814; 4294757; 4307016; 4308268; 4308269; 4309428; 4313946; 4315929; 4317821; 4322348; 4331598; 4361650; 4364866; 4424219; 4450254; 4362663 and 4371533.

Components of the medicinal product on the basis of maytansinoids are attractive components of medicines in the conjugates of antibodies with drug due to the fact that they: (i) on available for fermentation or chemical modification upon receipt of derivative products of fermentation, (ii) suitable for obtaining derivatives with functional groups suitable for conjugation through ridiculing of linkers with antibodies, (iii) stable in plasma, and (iv) effective against many lines of tumor cells.

Connections on the basis of maytansine suitable for use as a component of a medicinal product on the basis of maytansinoids, well known in this field, and they can be isolated from natural sources according to known methods or obtained by using the methods of genetic engineering (see Yu et al. (2002) PNAS 99:7968-7973). Maytansines and analogues maytansine can also be obtained synthetically according to known methods.

Illustrative components of a medicinal product on the basis of maytansinoids include components possessing the e modified aromatic ring, such as: C-19-dechloro (U.S. patent No. 4256746) (obtained by reduction with lithium hydride-aluminum ansamitocins P2); C-20-hydroxy (or C-20-demethyl) +/-C-19-dechloro (U.S. patent No. 4361650 and 4307016) (obtained by demethylation usingStreptomycesorActinomycesor the removal of chlorine using LAH); and C-20-dimethoxy, C-20-acyloxy (-OCOR), +/-dechloro (U.S. patent No. 4294757) (obtained by acylation using acylchlorides), and components with modifications in other provisions.

Illustrative components of a medicinal product on the basis of maytansinoids also include components with modifications, such as: C-9-SH (U.S. patent No. 4424219) (obtained by the interaction maytansine with H2S or P2S5); C-14-alkoxymethyl(dimetoxy/CH2OR)(US 4331598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. patent No. 4450254) (derived fromNocardia); C-15-hydroxy/acyloxy (US 4364866) (obtained by the transformation maytansine withStreptomyces); C-15-methoxy (U.S. patent No. 4313946 and 4315929) (isolated fromTrewianudlflora); C-18-N-demethyl (U.S. patent No. 4362663 and 4322348) (obtained by demethylation maytansine withStreptomyces); and 4,5-deoxy (US 4371533) (obtained by reduction of trichloride titanium/LAH maytansine).

Illustrative embodiments of the components of the medicinal product on the basis of maytansinoids including the Ute: DM1, DM3 and DM4, having the structures:

where the wavy line indicates the covalent joining of the sulfur atom of the medicinal product to the linker (L) conjugate antibody-drug. It was reported about HERCEPTIN®(trastuzumab), linked via SMCC with DM1 (WO 2005/037992; US 2005/0276812 A1).

Other illustrative conjugates antibody-drug based maytansinoids have the following structures and abbreviations (where Ab represents the antibody and p is from 1 to about 8):

Illustrative conjugates antibody-drug where DM1 is connected through a linker BMPEO with Tilney group of antibodies, have the structure and reduction:

where Ab is an antibody; n represents 0, 1 or 2; and p represents 1, 2, 3, or 4.

Immunoconjugate containing maytansinoid, methods for their preparation and their therapeutic use are described, for example, in U.S. patent No. 5208020, 5416064, US 2005/0276812 A1 and European patent EP 0425235 B1 as described herein expressly by reference. In Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugate containing maitan inoid, referred to as DM1 associated with the monoclonal antibody C242 directed against colorectal cancer. Found that the conjugate is vysokorychlostnym in respect of cultured cells of large intestine cancer, and he showed antitumor activity in the analysis of tumor growthin vivo. In Chari et al. Cancer Research 52:127-131 (1992) described immunoconjugate in which maytansinoid conjugatively via a disulfide linker to the antibody mouse A7 binding to an antigen on the cell lines of the cancer of the large intestine of man, or with another monoclonal antibody mouse TA.1 that binds to the oncogene HER-2/neu. The cytotoxicity of the conjugate TA.1-maytansinoid testedin vitroon the line of breast cancer cells human SK-BR-3, which expresses 3×105surface antigens HER-2 on the cell. Conjugate with the medicinal product has reached the degree of cytotoxicity similar to the free drug on the basis of maytansinoids, which can be increased by increasing the number of molecules maytansinoid molecule antibodies. For conjugate A7-maytansinoid on mice shows low systemic cytotoxicity.

Conjugates of the antibody-maytansinoid get chemically linking an antibody to a molecule maytansinoid without a significant reduction in biological activity or antibodies, and molecules maytansinoid. See, for example, U.S. patent No. 5208020 (described herein expressly by reference). 3-4 molecules maytansinoid conjugate molecule antibodies are effective in enhancing the cytotoxicity against target cells without negative impacts on the feature or the solubility of the antibody, although even one molecule of the toxin to the antibody, as you might expect, enhances cytotoxicity compared using unconjugated antibodies. Maytansinoid well known in this field, and they can be synthesized by known methods or to isolate from natural sources. Suitable maytansinoid described, for example, in U.S. patent No. 5208020 and in other patents and non-patent publications referenced above in this description. Preferred maytansinoid are maytansines and analogues maytansine modified aromatic ring or other provisions of molecules maytansine, such as various esters maytansine.

In this area there are many linking groups for preparation of conjugates of the antibody-maytansinoid, including, for example, the groups described in U.S. patent No. 5208020 or in the European patent 0425235 B1, Chari et al. Cancer Research 52:127-131 (1992), and US 2005/016993 A1 described here explicitly as the e links. Conjugates of the antibody-maytansinoid containing linker component SMCC, can be obtained as described in US 2005/0276812 A1 "Antibody-drug conjugates and Methods". Linkers include disulfide groups, thioester group unstable to acids group, photolabile group unstable to the action of peptidases group or unstable to the action of esterases group, as described in the above patents. Additional linkers are described and shown as examples in this work.

Conjugates of the antibody and maytansinoid can be obtained using a variety of bifunctional substances to attach proteins, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylpiperidin-HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis-(p-azidobenzoyl)hexanediamine), derivatives of bis -, page (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). In some embodiments, the implementation of the substance to attach represents N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) or the N-Succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to ensure the formation of disulfide bonds.

The linker can be attached to the molecule maytansinoid in different positions depending on the type of communication. For example, the ester bond can be formed by reaction with a hydroxyl group using conventional methods of joining. The reaction may proceed in position C-3, having a hydroxyl group in position C-14, modified with hydroxymethyl, C-15, modified with hydroxyl group in position C-20 having a hydroxyl group. In one embodiment, the implementation of the bond is formed at the position C-3 maytansine or similar maytansine.

(2) Auristatin and dolastatin

In some embodiments, the implementation immunoconjugate contains the antibody of the invention conjugated to dolastatins or peptide analog and derivative of dolastatin, such as auristatin (U.S. patent No. 5635483; 5780588). It is shown that dolastatin and auristatin violate the dynamics of microtubules, the hydrolysis of GTP and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anti-cancer (U.S. patent No. 5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). A component of a medicinal product on the basis of dolastatin or auristatin can be attached to the antibody via N-Terminus (amino) or C-Terminus (carboxyl) peptide component Lek is stannage funds (WO 02/088172).

Illustrative embodiments of using auristatin include components of the medicinal product based on the associated N-end of monomethylaniline DE and DF, described in Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, presented March 28, 2004, the description of which is clearly shown by reference in full.

The peptide component of a medicinal product can be selected from the formulas DEand DFbelow:

,

where the wavy line for DEand DFindicates the site of covalent joining the antibody or the antibody-linker component and independently in each state:

R2selected from H and alkyl, C1-C8;

R3selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl, C1-C8of alkyl(C3-C8carbocycle), C3-C8heterocycle and C1-C8of alkyl(C3-C8a heterocycle);

R4selected from H, C1-C8of alkyl, carbocycle C3-C8, aryl, C1-C8alkylaryl, C1-C8of alkyl(C3-C8carbocycle), C3-C8heterocycle and C1-C8of alkyl(C3-C8a heterocycle);

R5selected from H and methyl;

or R4and R5jointly about who will formed carbocyclic ring and have the formula -(CR aRb)n-where Raand Rbindependently selected from H, C1-C8the alkyl and C3-C8carbocycle and n is selected from 2, 3, 4, 5 and 6;

R6selected from H and C1-C8of alkyl;

R7selected from H, C1-C8of alkyl, C3-C8carbocycle, aryl, C1-C8alkylaryl, C1-C8of alkyl(C3-C8carbocycle), C3-C8heterocycle and C1-C8of alkyl(C3-C8a heterocycle);

each R8independently selected from H, OH, C1-C8of alkyl, C3-C8carbocycle and O-(C1-C8alkyl);

R9selected from H and C1-C8of alkyl;

R10selected from aryl or C3-C8heterocycle;

Z represents O, S, NH or NR12where R12represents a C1-C8alkyl;

R11selected from H, C1-C20of alkyl, aryl, C3-C8heterocycle, -(R13O)m-R14or -(R13O)m-CH(R15)2;

m is an integer in the range 1-1000;

R13represents a C2-C8alkyl;

R14represents H or C1-C8alkyl;

each occurrence of R15independently represents H, COOH, -(CH2)n-N(R16)2, -(CH2)n-SO3H or -(CH2)n-SO3-C1-C8alkyl;

the each occurrence of R 16independently represents H, C1-C8alkyl or -(CH2)n-COOH;

R18selected from-C(R8)2-C(R8)2-aryl, -C(R8)2-C(R8)2-(C3-C8a heterocycle), and-C(R8)2-C(R8)2-(C3-C8carbocycle); and

n is an integer in the range 0-6.

In one embodiment, the implementation of R3, R4and R7independently represent an isopropyl or sec-butyl and R5represents-H or methyl. In the illustrative embodiment, each R3and R4represents isopropyl, R5represents-H, and R7represents a sec-butyl.

In another embodiment, each R2and R6represents methyl and R9is a-H.

In another embodiment, each occurrence of R8represents-OCH3.

In the illustrative embodiment, each R3and R4represents isopropyl; each R2and R6represents methyl, R5represents-H, R7represents sec-butyl, each occurrence of R8represents-OCH3and R9is a-H.

In one of the embodiments Z represents-O - or-NH-.

In one embodiment, assests is of R 10represents aryl.

In the illustrative embodiment, R10represents-phenyl.

In the illustrative embodiment, where Z represents-O-, R11represents-H, methyl or tert-butyl.

In one of the embodiments, where Z represents-NH, R11represents a-CH(R15)2where R15represents -(CH2)n-N(R16)2and R16represents a C1-C8alkyl or -(CH2)n-COOH.

In another embodiment, where Z represents-NH, R11represents a-CH(R15)2where R15represents -(CH2)n-SO3H.

Illustrative variant implementation using auristatin formula DErepresents MMAE, where the wavy line indicates the covalent joining the linker (L) conjugate antibody-drug:

Illustrative variant implementation using auristatin formula DFrepresents MMAF, where the wavy line indicates the covalent joining the linker (L) conjugate antibody-drug (see US 2005/0238649 and Doronina et al. (2006) Bioconjugate Chem. 17:114-124):

Other components of the drug include shadowdiamonds MMAF, where the wavy line indicates the covalent joining the linker (L) conjugate antibody-drug:

In one aspect of the hydrophilic group, including as non-limiting examples of esters of triethylene glycol (TEG), as shown above, can be attached to a component of a medicinal product in R11. Without going into specific theoretical details, the hydrophilic group promotes the internalization and the absence of aggregation of the component drugs.

Illustrative embodiments of using the ADC of formula I containing auristatin/dolastatin or their derivative, described in US 2005-0238649 A1 and Doronina et al. (2006) Bioconjugate Chem. 17:114-124, which explicitly herein by reference. In illustrative embodiments, the implementation of the ADC of formula I containing MMAE or MMAF and various components of the linker, have the following structures and abbreviations (where Ab is an antibody; p is from 1 to about 8, "Val-Cit" is a dipeptide valine-citrulline; and "S" is a sulfur atom):

Illustrative embodiments of using the ADC of formula I containing MMAF and various components of the linker optionally include AbMC-PAB-MMAF and Ab-PAB-MMAF. It is interesting to note that immunoconjugate containing MMAF, attached to the antibody using a linker that is not proteoliticeski split, as shown, have activity comparable to immunoconjugate containing MMAF, attached to the antibody using proteoliticeski biodegradable linker. See, Doronina et al. (2006) Bioconjugate Chem. 17:114-124. In such cases, it is believed that the release of drug occurs when the degradation of the antibody in the cell. Id.

Typically, the components of the medicinal product on the basis of peptides can be obtained by formation of a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be obtained, for example, according to the method of liquid-phase synthesis (see E. Schroder and K. Lubke. "The Peptides", volume 1, p.76-136, 1965, Academic Press)that is well known in the field of peptide chemistry. Components of the medicinal product on the basis of auristatin/dolastatin can be obtained according to methods: US 2005-0238649 A1; U.S. patent No. 5635483; U.S. patent No. 5780588; Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; Pettit G.R. et al. Synthesis, 1996, 719-725; Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat. Biotechnol. 21(7):778-784.

In particular, the components of the formula DFmedicines on the basis of auristatin/dolastatin, such as MMAF and its derivatives, can be obtained with the use of what Finance methods, described in US 2005-0238649 A1 and Doronina et al. (2006) Bioconjugate Chem. 17:114-124. The components of the formula DEmedicines on the basis of auristatin/dolastatin, such as MMAE and its derivatives, can be obtained using the methods described in Doronina et al. (2003) Nat. Biotech. 21:778-784. The components of the drug-linker-MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF and MC-vc-PAB-MMAE can appropriately to synthesize conventional ways, for example as described in Doronina et al. (2003) Nat. Biotech. 21:778-784 and the publication of patent application no US 2005/0238649 A1, and then konjugierte with interest the antibody.

(3) Calicheamicin

In other embodiments, implementation immunoconjugate contains the antibody of the invention conjugated to one or more molecules calicheamicin. Antibiotics family calicheamicin able to create double-stranded DNA breaks in subpicomolar concentrations. To obtain conjugates family calicheamicin see U.S. patent 5712374, 5714586, 5739116, 5767285, 5770701, 5770710, 5773001, 5877296 (all issued by American Cyanamid Company). Structural analogues calicheamicin that can be used include as non-limiting examples γ1Iα2Iα3IN-acetyl-γ1I, PSAG and θI1(Hinman et al. Cancer Research 53:3336-3342 (1993), Lode et al. Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents issued to American Cyanamid). Another item is tibooburra drug which can be konjugierte antibody is QFA, which is antifolate. As calicheamicin and QFA have intracellular action, and they are not easily pass through the plasma membrane. Thus, the cell capture these substances is mediated by antibody internalization significantly enhances their cytotoxic effects.

c) Other cytotoxic funds

Other antitumor agents that can be konjugierte with antibodies according to the invention include BCNU, streptozocin, vincristine and 5-fluorouracil, the family of funds, known under the collective name of complex LL-E33288 described in U.S. patent No. 5053394, 5770710 and espiramicina (U.S. patent No. 5877296).

Enzymatically active toxins and fragments thereof that can be used include the A-chain of diphtheria toxin, nesviazana active fragments of diphtheria toxin A-chain, exotoxin a (fromPseudomonasaeruginosa), A-chain of ricin A-chain abrina, A-chain medecine, alpha sarcin, proteinsAleuritesfordiiproteins of diantin, proteinsPhytolacaamericana(PAPI, PAPII, and PAP-S), inhibitor ofMomordicacharantiaCurtin, krotin, the inhibitor ofSapaonariaofficinalis, gelonin, mitogillin, restrictocin, vanomycin, inomycin and tricothecene. See, for example, WO 93/21232, published October 28, 1993.

The present invention also relates to immunoconjugate, formed by the antibody and the connection with nucleotidase activity (e.g., a ribonuclease or a DNA endonuclease such as desoksiribonukleaza; Ncasa).

In some embodiments, the implementation immunoconjugate may contain highly radioactive atom. To obtain radioactive conjugated antibodies are available various radioactive isotopes. Examples include At211I131I125, Y90That Re186That Re188Sm153Bi212, P32, Pb212and radioactive isotopes of Lu. In the case of the use of the conjugate for detection, it may contain a radioactive atom for scintigraphic studies, for example Tc99mor I123or a spin label for image acquisition of nuclear magnetic resonance (NMR) (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Radioactive or other labels can be embedded in the conjugate by known methods. For example, the peptide can biologically synthesize or can be synthesized by chemical amino acid synthesis using suitable amino acid precursors containing, for example, fluorine-19 instead of hydrogen. Labels such as Tc99mor I123That Re186That Re188and In111you can join through the STATCOM cysteine in the peptide. Yttrium-90 can be attached via a lysine residue. The way IODOPHENOL (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80:49-57) can be used for injection of iodine-123. In "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) described other ways.

In some embodiments, the implementation immunoconjugate may contain anti-TAT226 antibody of the invention conjugated with a prodrug activating enzyme which converts a prodrug (e.g., peptide chemotherapeutic agent, see WO 81/01145) in the active drug, such as an anti-cancer drug. Such immunoconjugate suitable antibody-dependent enzyme-mediated proletarienne therapy (ADEPT). Enzymes that can be konjugierte with anti-TAT226 antibody according to the invention include as non-limiting examples of alkaline phosphatase, which is suitable for converting phosphate-containing prodrugs into free drugs; Ukrainian, which is suitable for turning sulfidogenic prodrugs into free drugs; sitoindosides which is suitable for converting non-toxic 5-fertilizin in anti-cancer drug, 5-fluorouracil; proteases, such as proteaseSerratiathermolysin, subtilisin, carboxypeptidase and cathepsins (such as cathepsins B and L), which is odni to turn petitcodiac prodrugs into free drugs; D-alanismorissette, which is suitable for converting prodrugs that contain D-amino acid substituents; carbohydrate-splitting enzymes, such as β-galactosidase and neuraminidase, which are suitable for converting glycosylated prodrugs into free drugs; β-lactamase, which is suitable for making medicines, modified with β-lactams, into free drugs; and penicillinases, such as penicillin-V-amidase and penicillin-G-amidase, which is suitable for making medicines, modified by the nitrogen atoms of the amino group using phenoxyacetyl or phenylacetyl groups respectively, into free drugs. Enzymes can covalently bind with the anti-TAT226 antibodies according to the invention using methods of recombinant DNA, are well known in this field. See, for example, Neuberger et al., Nature 312:604-608 (1984).

d) Loading drug

Load the medicinal product is a p, the average number of components of the drug to the antibody in a molecule of formula I. Load medicinal product may vary from 1 to 20 components of the medicinal product (D) antibody. The ADC of formula I include the collection of antibodies conjugated with medicinal components is CSO funds in the range from 1 to 20. The average number of components of the drug to the antibody in preparations from ADC conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA and HPLC. You can also determine the quantitative distribution of the ADC in terms of p. In some instances, separation, purification, and characterization of homogeneous ADC, where p is a certain value from the ADC with the other loads of the medicinal product, can be achieved by methods such as HPLC with reversed phase or electrophoresis.

For some conjugates of the antibody-drug, p may be restricted by the number of lots attach to the antibody. For example, where the connection is done according to the thiol of cysteine, as in illustrative embodiments, the implementation of the above, the antibody may contain only one or a few tylnej groups of cysteine or may contain only one or more active enough tylnej groups, which can be attached to the linker. In some embodiments, the higher the load of the medicinal product, such as p>5, can cause aggregation, the insolubility, toxicity or loss of the ability to penetrate into cells for certain conjugates of the antibody-drug. In some embodiments, the implementation of load medicines for ADC is about the invention ranges from 1 to about 8, from about 2 to about 6, or from about 3 to about 5. Indeed, it is shown that for some ADC optimal ratio of components of the drug to the antibody may be less than 8 and may vary from about 2 to about 5. Cm. US 2005-0238649 A1.

In some embodiments, the implementation of a smaller number of components of the medicinal product compared to theoretical maximum kongugiruut with the antibody during the conjugation reaction. The antibody may contain, for example, lysine residues that do not interact with the intermediate connection of a medicinal product with a linker or linker reagent, as discussed below. Typically, the antibody does not contain a lot of free and active tylnej groups of cysteine, which can bind to a component of the medicinal product; in practice, most tylnej cysteine residues in the antibody exists in the form disulfide bridges. In some embodiments, implement to get active tylnej groups of cysteine antibody can be restored restoring means, such as dithiothreitol (DTT) or tricarbonylchromium (TCEP) in a partially or fully reducing conditions. In some embodiments, the implementation of the antibody is exposed to denaturing conditions to identify AK is active nucleophilic groups, such as lysine or cysteine.

The load ratio (drug/antibody) of the ADC can be controlled in various ways, for example through: (i) restrictions molar excess of intermediate compounds of the medicinal product with the linker or linker reagent relative to the antibody, (ii) the time limit or the reaction temperature conjugation and (iii) partial or limiting recovery conditions for modification of the thiol of cysteine.

It should be understood that if the intermediate connection of a medicinal product with a linker or linker reagent reacts more than one nucleophilic group with subsequent interaction with the reagent component of the medicinal product, the resulting product is a mixture of compounds of the ADC with the distribution of one or more components of the medicinal product, attached to the antibody. The average number of drugs per antibody can be calculated from the mixture using dual ELISA for antibodies that are specific for antibodies specific for the drug. Individual molecules of the ADC can be identified in the mixture by mass spectroscopy and divide by HPLC, for example, chromatography based on hydrophobic interactions (see, for example, K. J. Hamblett et al. "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 anibody-drug conjugate", Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of AACR, Volume 45, March 2004; Alley, S.C. et al. "Controlling the location of drug attachment in antibody-drug conjugates", Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of AACR, Volume 45, March 2004). In some embodiments, the implementation of homogeneous ADC with a single load value can be extracted from the conjugation mixture by electrophoresis or chromatography.

e) Some methods of obtaining immunoconjugates

The ADC of formula I can be obtained in several ways using the reactions of organic chemistry, conditions, and reagents known to specialists in this area, including: (1) the interaction of a nucleophilic group of an antibody with a bivalent linker reagent with the formation of Ab-L, via a covalent bond with the subsequent interaction with a component of a medicinal product D; and (2) the interaction between the nucleophilic group of a component of a medicinal product with a bivalent linker reagent with the formation of D-L, via a covalent bond with the subsequent interaction with the nucleophilic group of an antibody. Illustrative methods of obtaining the ADC of formula I through the last method described in US 2005-0238649 A1, which is clearly shown here as a reference.

Nucleophilic groups on the antibodies include as non-limiting examples: (i) N-terminal amino group, (ii) amino side CE and, for example lysine, (iii) thiol group on the side chain, for example cysteine, and (iv) a hydroxyl group or amino sugars, if the antibody is glycosylated. Amino, thiol and hydroxyl groups are nucleophiles and can interact with the formation of covalent bonds with electrophilic groups on the components of the linker, where the linker reagents include: (i) active esters such as NHS esters, esters, HOBt, trechaleidae derivatives and acid halides; (ii) alkyl and benzylchloride, such as haloacetic; (iii) aldehyde, ketone, carboxyl and maleinimide group. Some antibodies have recovered megamachine disulphide, i.e. cysteine bridges. Antibodies can be obtained in active form for conjugation with linker reagents processing regenerating reagent such as DTT (dithiothreitol) or tricarbonylchromium (TCEP), so that the antibody is fully or partially restored. Each cysteine bridge, thus, forms a theoretically two active thiol of the nucleophile. Additional nucleophilic groups can be introduced in antibodies by modifying lysine residues, for example of the interaction of the lysine residues with 2-aminothiophenol (reagent trout), which leads to the transformation of the amine in the thiol. Active thiol group which you can enter into the antibody by introducing one, two, three, four or more cysteine residues (for example, obtaining variants of antibodies that contain one or more non-natural amino acid cysteine residues).

Conjugates of antibodies with drugs according to the invention can also be obtained by the interaction between the electrophilic group on the antibody, such as an aldehyde or ketone carbonyl group and the nucleophilic group on the linker reagent or drug. Suitable nucleophilic groups on the linker reagent include as non-limiting examples of the hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazinecarboxamide and originated. In one of the embodiments the antibody is modified to introduce electrophilic components that can interact with nucleophilic substituents on the linker reagent or drug. In another embodiment, sugar glycosylated antibodies can oxidize, for example, oxidizing reagents based periodate with the formation of aldehyde or ketone groups that can react with the amino group of the linker reagents or components of the drug. The resulting groups iminovogo Schiff bases can form a stable bond, or they can be recovered, for example, using borohydride reagents with about what adowanie stable amine linkages. In one embodiment, the implementation of the interaction of the carbohydrate portion of a glycosylated antibody with either galactosialidosis or metaperiodate sodium can lead to the formation of carbonyl (aldehyde and ketone) groups in the antibody that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, antibodies that contain N-terminal residues of serine or threonine can interact with metaperiodate sodium, leading to the formation of aldehyde in place of the first amino acid (Geoghegan & Stroh (1992) Bioconjugate Chem. 3:138-146; US 5362852). This aldehyde can react with a component of a medicinal product or a nucleophile linker.

Nucleophilic groups on the component drugs include as non-limiting examples: amino, thiol, hydroxyl, hydrazide, Aksinya, hydrazine powered, thiosemicarbazone, hydrazinecarboxamide and arylhydrazines group capable of reacting with the formation of covalent bonds with electrophilic groups on the components of the linker, where the linker reagents include: (i) active esters such as NHS esters, esters, HOBt, trechaleidae derivatives and acid halides; (ii) alkyl and benzylchloride, such as haloacetic; (iii) aldehyde, ketone, carboxyl and m is Leinonen group.

Compounds according to the invention explicitly include as non-limiting examples of the ADC obtained using the following cross-linking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, fairs are forthcoming-Siab, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-fairs are forthcoming-Siab, sulfo-SMCC, sulfo-SMPB, and SVSB (Succinimidyl-(4-vinylsulfonic)benzoate)which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A); see pages 467-498, 2003-2004 Applications Handbook and Catalog.

Immunoconjugate containing the antibody and cytotoxic agent, can also be obtained using a variety of bifunctional substances to attach a protein, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylpiperidin-HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis-(p-azidobenzoyl)hexanediamine), derivatives of bis -, page (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). For example, rezinovy immunotoxin can be obtained as described in Vitetta et al., Science, 238:1098 (1987). Labelled with carbon-14 1-isothiocyanatobenzene-3-methylvalerate minopensea acid (MX-DTPA) is an illustrative chelating agent for conjugation of radionucleotide with the antibody. Cm. WO94/11026.

Alternative protein containing the antibody and cytotoxic agent, can be obtained, for example, by recombinant methods or peptide synthesis. The recombinant DNA molecule may contain a region encoding or antibody-based test and the cytotoxic portion of the conjugate, or adjacent to each other, or separated by a region that encodes a linker peptide which does not violate the required properties of the conjugate.

In another embodiment, the antibody can be konjugierte with the "receptor" (such as streptavidin) for utilization in the pre - "the transformation of the tumor in the target, where the conjugate of the antibody-receptor is administered to the patient, followed by removal of unbound conjugate from the blood flow with the use of tools that allow for clearance, and then the introduction of a "ligand" (e.g., avidin), which anywhereman with a cytotoxic agent (e.g., radioactively labeled nucleotide).

D. Pharmaceutical compositions

In one aspect of the invention additionally relates to pharmaceutical compositions containing at least one anti-TAT226 antibody according to the invention and/or at least one immunoconjugate. In some embodiments, the implementation of the pharmaceutical composition comprises: 1) an anti-TAT226 antibody and/or its immunoconjugate and (2) pharmaceutically acceptable but Italy. In some embodiments, the implementation of the pharmaceutical composition comprises: 1) an anti-TAT226 antibody and/or its immunoconjugate and optional 2)at least one additional therapeutic agent. Additional therapeutic agents include as non-limiting examples of the tools described below in section E.2.

Pharmaceutical compositions containing the antibody or immunoconjugate according to the invention, receive for storage by mixing the antibody or immunoconjugate possessing the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of aqueous solutions or lyophilized or otherwise dehydrated compositions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the used doses and concentrations, and they include buffers such as phosphate, citrate, his-tag and on the basis of other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as chloride of octadecyltrimethoxysilane; chloride hexadecane; benzalkonium chloride, chloride benzathine); phenol, butyl or benzyl alcohol; alkylarene (such as methyl or propyl paraben; catechol; resorcinol; cyclohex anal; 3-pentanol; and m-cresol); low molecular weight (approximately less than 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; soleobrazutaya counterions such as sodium; metal complexes (e.g., complexes of Zn protein); and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Pharmaceutical compositions for use in the introduction ofin vivoas a rule, are sterile. It is easy to reach by filtration through sterile filtration membranes.

The active ingredients can also be incorporated into the microcapsules obtained, for example, ways koatservatsii or by interfacial polymerization, for example, microcapsules based hydroxymethylcellulose or gelatin and microcapsules on the basis of polymethylmethacrylate, respectively, in colloidal systems for delivery of a drug (for example, liposomes, albumen the microspheres, microemulsions, nanoparticles and nanocapsules) or in macroe is Oliseh. Such methods are described in Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

You can get drugs with a slow release. Examples of suitable drugs with a slow release include semi-permeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate according to the invention, where the matrices are in the form with a certain form of products, e.g. films, or microcapsules. Examples of matrices for sustained release include polyesters, hydrogels (for example, poly-2-hydroxyethylmethacrylate or polyvinyl alcohol), polylactide (U.S. patent No. 3773919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, degradiruem the ethylene vinyl acetate, degradiruete copolymers of lactic acid and glycolic acid, such as the LUPRON DEPOT™ (injectable microspheres composed of a copolymer of lactic acid and glycolic acid and acetate leuprolide), and poly-D-(-)-3-hydroxybutiric acid. While polymers such as based on vinyl acetate and lactic acid-glycolic acid capable of releasing molecules within 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies or immunoconjugate remain in the body for a long time, they can denaturing or egregia the step under the action of humidity at 37°C, which leads to the loss of biological activity and possible changes in immunogenicity. It is possible to develop rational strategies for stabilization depending on the mechanism involved. For example, if you discovered that the mechanism of aggregation is the formation of intermolecular S-S linkages through the conversion of thiol to disulfide, stabilization can be achieved by modifying sulfhydryl residues, lyophilisation from acidic solutions, controlling the water content, using appropriate additives, and developing specific composition based on a polymer matrix.

E. Methods of using anti-TAT226 antibodies and immunoconjugates

1. Diagnostic methods and methods of detection

In one aspect of the anti-TAT226 antibodies and immunoconjugates according to the invention is suitable for detecting the presence of TAT226 in a biological sample. The term "detection"as used herein, refers to qualitative or quantitative detection. In some embodiments, the implementation of the biological sample contains a cell or tissue, such as tissue, are listed in figure 13. In some embodiments, the implementation of such fabrics include normal and/or cancerous tissues that Express TAT226 at higher levels relative to other tissues, such as tissue of the ovaries, kidneys, brain, endomet the Oia, adrenal, bone, lung, skin and soft tissues.

In one aspect the invention relates to a method of detecting the presence of TAT226 in a biological sample. In some embodiments, the implementation of the method involves contacting a biological sample with an anti-TAT226 antibody under conditions ensuring the binding of anti-TAT226 antibodies with TAT226 and the detection of whether there has been the formation of a complex between the anti-TAT226 antibody and TAT226.

In one aspect the invention relates to a method for the diagnosis of disorders associated with increased expression of TAT226. In some embodiments, the implementation of the method comprises contacting the test cells with an anti-TAT226 antibody; determining the level of expression (or quantitatively, or qualitatively) TAT226 the test cell by detecting binding of an anti-TAT226 antibodies with TAT226; and comparing the level of expression of TAT226 the test cell with the level of expression of TAT226 control cell (e.g., a normal cell of the same tissue origin as the test cell or a cell that expresses TAT226 at levels comparable to the levels in normal cells), where a higher level of expression of TAT226 the test cell compared to the control cell indicates the presence of violations, associated with increased expression of TAT226. In some embodiments, the implementation of the test cell floor is given of the individual, which suggest the presence of disorders associated with increased expression of TAT226. In some embodiments, the implementation of the violation constitutes a violation of cell proliferation, such as cancer or a tumor.

Illustrative of disorders of cell proliferation, which can be diagnosed using an antibody of the invention include cancerous conditions, such as tumors, such as carcinomas (epithelial tumors) and blastoma (tumor arising from embryonic tissues) and in some embodiments, the implementation of ovarian carcinoma, uterine (including endometrial cancer and kidney cancer, including neuroblastoma (e.g., Wilms tumor). To cancer of the ovary, in particular, is a heterogeneous group of malignant tumors that arise from the ovary. Approximately 90% of malignant tumors of the ovary have an epithelial origin; the rest are tumors of germ cell and stromal tumors. Epithelial tumors of the ovary are classified by the following histological subtypes: serous adenocarcinoma (approximately 50% of epithelial tumors of the ovary); endometrioid adenocarcinoma (~20%); mucinous adenocarcinoma (~10%); clear cell carcinomas (~5-10%); tumor Brenner (transitional cell) (relatively rare). The prognosis of ovarian cancer, which is Stoica sixth place of the most common cancers in women, is usually unfavorable, where the levels of the five-year survival rates are in the range of 5-30%. For reviews on cancer of the ovary, see Fox et al. (2002) "Pathology of epithelial ovarian cancer"Ovarian Cancer ch. 9 (Jacobs et al., eds., Oxford University Press, New York); Morin et al. (2001) "Ovarian Cancer", in Encyclopedic Reference of Cancer, p.654-656 (Schwab, ed., Springer-Verlag, New York). The present invention provides methods for diagnosing or treating any of the above subtypes of epithelial tumors of the ovary and, in particular, subtype serous adenocarcinoma.

In some embodiments, the implementation of a method of diagnosis or detection, such as the above methods includes the detection of the binding of anti-TAT226 antibodies with TAT226, expressed on the cell surface or in a membrane preparation obtained from a cell expressing TAT226 on its surface. In some embodiments, the implementation of the method involves contacting cells with an anti-TAT226 antibody under conditions ensuring the binding of anti-TAT226 antibodies with TAT226 and the detection of whether there has been the formation of a complex between the anti-TAT226 antibody and TAT226 on the cell surface. Illustrative analysis for detection of the binding of anti-TAT226 antibodies with TAT226, expressed on the cell surface, is an analysis of the "FACS", such as the analysis described in example D below.

You can use some other methods of detecting the binding of anti-TAT226 antibodies TA226. Such methods include as non-limiting examples of analyses of binding antigen, which is well known in this area, such as Western blotting, types of radioimmune analysis, ELISA (enzyme-linked immunosorbant assay), "bivalent" immunoassays, analyses by thus, fluorescent immunoassays, immunoassays with protein A and immunohistochemistry (IHC).

In some embodiments, the implementation of anti-TAT226 antibodies are labeled. Labels include as non-limiting examples of labels or components that detect directly (such as fluorescent, chromophore, electron-dense, chemiluminescent, and radioactive labels), as well as components, such as enzymes or ligands that are detected indirectly, for example, by enzymatic reaction or molecular interaction. Illustrative labels include as non-limiting examples of radioisotopes32P,14C,125I3H and131I, fluorophore, such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, such as Firefly luciferase and bacterial luciferase (U.S. patent No. 4737456), luciferin, 2,3-dihydropteridine, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, l is Siim, saridakis, such as glucose oxidase, galactosidase, and glucose-6-phosphatedehydrogenase, heterocyclic oxidases such as uricase and the xanthine oxidase-related enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microbiocides, Biotin/avidin, spin labels, the labels on the basis of bacteriophages, stable free radicals, etc.

In some embodiments, the implementation of anti-TAT226 antibody immobilized on an insoluble matrix. Immobilization leads to the separation of anti-TAT226 antibodies from any TAT226, which remains free in solution. Immobilization conventional method is carried out or transfer to an insoluble form an anti-TAT226 antibody before the assay procedure, as by adsorption to a water-insoluble matrix or surface (Bennich et al., US 3720760), or covalent binding (e.g., using cross-linking by glutaraldehyde), or transfer to an insoluble form an anti-TAT226 antibody after formation of a complex between the anti-TAT226 antibody and TAT226, such as immunoprecipitate.

Any of the above embodiments diagnosis or detection can be performed using immunoconjugate according to the invention instead of the anti-TAT226 antibodies, or in addition to it.

2. Therapeutically the ways

The antibody or immunoconjugate according to the invention can be applied, for example, in therapeutic waysin vitro, ex vivo andin vivo. In one aspect the invention relates to methods for inhibiting cell growth or proliferation, orin vivoorin vitrowhere the method includes the impact on the cell with an anti-TAT226 antibodies or immunoconjugate the conditions for binding immunoconjugate with TAT226. "Inhibition of cell growth or proliferation" means the slowing down of cell growth or proliferation, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% and includes the induction of cell death. In some embodiments, the implementation of the cell is a tumor cell. In some embodiments, the implementation of the cell is a cell tumors of the ovaries, the cell tumors of body of uterus, cell brain tumor or cell tumors of the kidney. In some embodiments, the implementation of the cell represents a xenograft, for example, as shown as an example in this work.

In one aspect the antibody or immunoconjugate according to the invention is used for the treatment or prevention of disorders of cell proliferation. In some embodiments, the implementation of a violation of cell proliferation is associated with increased expression and/or activity TAT226. For example, in some embodiments, the implementation on Uchenie cell proliferation is associated with increased expression of TAT226 on the cell surface. In some embodiments, the implementation of the impaired proliferation of cells is a tumor or cancer. Examples of disorders of cell proliferation, which are subject to treatment with an antibody or immunoconjugate according to the invention include as non-limiting examples of cancer conditions, such as tumors, such as carcinomas (epithelial tumors) and blastoma (tumor arising from embryonic tissues) and in some embodiments, the implementation of endometrial cancer; brain tumors (e.g., astrocytomas, including advanced stages of gliomas, also referred to as glioblastoma multiforme); and ovarian cancer; cancer of the uterine body, including kidney cancer, including neuroblastoma (e.g., Wilms tumor).

In one aspect the invention relates to methods for treating disorders of cell proliferation, which include introduction to the individual an effective amount of an anti-TAT226 antibodies or immunoconjugate. In some embodiments, the implementation of the method of treatment of disorders of cell proliferation includes an introduction to the individual an effective amount of a pharmaceutical composition containing an anti-TAT226 antibody, and optionally at least one additional therapeutic agent, such as the one below means. In some embodiments, the implementation of the method of treatment of disorders of cell proliferation involved in the t introduction to the individual an effective amount of the pharmaceutical composition, containing: 1) immunoconjugate containing anti-TAT226 antibody and cytotoxic agent; and optionally 2)at least one additional therapeutic agent, such as below the means.

In one aspect, at least some of the antibodies or immunoconjugates according to the invention can be contacted with TAT226 from non-human species. Thus, antibodies or immunoconjugate according to the invention can be applied to associate with TAT226, for example, in a cell culture containing TAT226, in the body or in the body of other mammals, containing TAT226, which antibody or immunoconjugate according to the invention cross-reacts (e.g. chimpanzee, baboon, igranka,Cynomolgusand rhesus, pig or mouse). In one embodiment, the implementation of anti-TAT226 antibody or immunoconjugate can be used for inhibiting the activity TAT226 by contacting the antibody or immunoconjugate with TAT226 so, what happens inhibition activity TAT226. In one embodiment, the implementation of TAT226 is a human TAT226.

In one embodiment, the implementation of anti-TAT226 antibody or immunoconjugate can be used in the methods of linking TAT226 for a subject suffering from a disorder associated with increased expression and/or activity TAT226, where the method includes introduction to the subject of antic the La or immunoconjugate thus, what happens linking TAT226 the subject. In one embodiment, the implementation of TAT226 is a TAT226 person and the subject is human. Alternative a subject can be a mammal expressing TAT226 is associated with anti-TAT226 antibody. In addition, the subject may be a mammal, which introduced TAT226 (for example, with the introduction of TAT226 or expression of the transgene encoding TAT226).

Anti-TAT226 antibody or immunoconjugate you can enter to man for therapeutic purposes. Moreover, an anti-TAT226 antibody can enter non-human mammal expressing TAT226, which cross-reacts with the antibody (e.g., a Primate, pig, rat or mouse)for veterinary purposes or using it as an animal model for human disease. Regarding the above, these model animals can be useful to assess therapeutic efficacy of antibodies or immunoconjugates according to the invention (e.g., testing of dosages and duration of the course introduction).

The antibody or immunoconjugate according to the invention can be used in the treatment or separately, or in combination with other compositions. For example, the antibody or immunoconjugate according to the invention it is possible to introduce at least one additional therapeutic among the CTB and/or adjuvant. In some embodiments, the implementation of the additional therapeutic agent is a cytotoxic agent, chemotherapeutic agent or a means of inhibiting growth. In one of these embodiments chemotherapeutic agent is a tool or combination of tools that are used in the treatment of ovarian cancer, such as a platinum compound (e.g., cisplatin or carboplatin); taxon (e.g., paclitaxel or docetaxel); topotecan; anthracyclin (for example, doxorubicin (ADRIAMYCIN®or liposomal doxorubicin (DOXIL®)); gemcitabine; cyclophosphamide; vinorelbine (NAVELBINE®); hexamethylmelamine; ifosfamide and etoposide. In other such embodiments, the implementation of the chemotherapeutic agent is a tool or combination of tools that are used in the treatment of cancer of the uterus or endometrium, such as cisplatin, carboplatin, doxorubicin, paclitaxel, methotrexate, fluorouracil, and medroxyprogesterone. In other such embodiments, the implementation of the chemotherapeutic agent is a tool or combination of tools that are used in the treatment of brain tumors, such as nitrosoanatabine (for example, carmustine or lomustin); cytotoxic agent (e.g., irinotecan or temozolamide); antiangiogenic agent (for example, thalidomide, TNP-470, robotically factor 4, interferon, endostatin); a means of differentiation (e.g., retinoids, phenylbutyrate, phenylacetate and antineoplaston); antiinvasive tool (for example, inhibitors of matrix metalloprotease, such as marimastat); the modulator signal (for example, tamoxifen, bryostatin and O-6-benzoquinon); a topoisomerase inhibitor (e.g., irinotecan or topotecan); and inhibitors of growth factors (e.g., a tyrosine kinase inhibitor). In other such embodiments, the implementation of the chemotherapeutic agent is a tool or combination of tools that are used in the treatment of kidney cancer (e.g., Wilms tumor), such as vincristine, actinomycin D, adriamycin, doxorubicin, cyclophosphamide, ifosfamide, etoposide, and carboplatin. In some embodiments, the implementation of the antibody according to the invention can be combined with anti-inflammatory and/or antiseptic.

Such aforementioned combination therapy include combined administration (where two or more therapeutic agents are included in the same composition or different compositions) and a separate introduction, in which the introduction of antibodies or immunoconjugate according to the invention can be implemented before the introduction of the additional therapeutic agent and/or adjuvant, simultaneously and/or after it. Antibodies or immunoconjugate according to the invention can changepriority in combination with radiotherapy.

The antibody according to the invention (and any additional therapeutic agent or adjuvant) is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intra-lungs and intranasal, and, if required for local treatment, intranidus introduction. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antibody or immunoconjugate appropriately injected pulse infusion, particularly with declining doses of the antibody or immunoconjugate. The dose can be accomplished in any suitable way, for example, injections such as intravenous or subcutaneous injection, partly depending on whether the introduction of short-term or chronic.

If the target link is in the brain, in certain embodiments the invention provides that the antibody or immunoconjugate passes through the blood-brain barrier. There are several known in the field of methods for transport of molecules across the blood-brain barrier, including as non-limiting examples of physical methods, methods with the use of lipids, methods using stem cells and methods of using receptors and channels.

Physical methods of transport and is tetela or immunoconjugate through the blood-brain barrier include, as non-limiting examples a full crawl of the blood-brain barrier or creating holes in the blood-brain barrier. Workarounds include as non-limiting examples of direct injection into the brain (see, for example, Papanastassiou et al., Gene Therapy 9:398-406 (2002)), interstitial infusion/convection delivery (see, for example, Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994)) and the implantation of delivery vehicles in the brain (see, for example, Gill et al., Nature Med. 9:589-595 (2003); and Gliadel Wafers™, Guildford Pharmaceutical). How to create holes in the barrier include as non-limiting examples of ultrasound (see, for example, published U.S. patent No. 2002/0038086), osmotic pressure (for example, when a hypertonic mannitol (Neuwelt, E. A., Memorandum of the Blood-Brain Barrier and its Manipulation, Vols 1 & 2, Plenum Press, N. Y. (1989)), the permeability of, for example, bradykinin or permeabilisation A-7 (see, for example, U.S. patent No. 5112596, 5268164, 5506206 and 5686416) and transfection of neurons that surround the blood-brain barrier, vectors containing genes encoding the antibody (see, for example, published U.S. patent No. 2003/0083299).

Methods with the use of lipids for transport antibodies or immunoconjugate through the blood-brain barrier include, as non-limiting examples of encapsulation of antibodies or immunoconjugate in liposomes that are associated with antielastase fragments that bind to receptors on the vascular endothelium of the blood-brain barrier (see, for example, publication of a patent bid, medium, small the USA No. 20020025313), and the conclusion of the antibodies or immunoconjugates in particles of low-density lipoprotein (see, for example, the publication of the patent application U.S. No. 20040204354) or apolipoprotein E (see, for example, the publication of the patent application U.S. No. 20040131692).

Methods with the use of stem cells for the transport of antibodies or immunoconjugate through the blood-brain barrier include genetic engineering of neural progenitor cells (NPCs) for expression of interest antibodies or immunoconjugates and then implantation of stem cells in the brain being treated subject. Cm. Behrstock et al. (2005) Gene Ther. 15 Dec. 2005 anticipatory interactive publication (it is reported that NPC, genetically engineered for the expression of the neurotrophic factor GDNF, reduced the severity of symptoms in Parkinson's disease, if they are implanted in the brain in models in rodents and primates).

Methods with the use of receptors and channels for transport of antibodies or immunoconjugate through the blood-brain barrier include, as non-limiting examples, the use of glucocorticoid blockers to enhance the permeability of the blood-brain barrier (see, for example, the publication of patent applications U.S. No. 2002/0065259, 2003/0162695 and 2005/0124533); activation of potassium channels (see, for example, the publication of the patent application U.S. No. 2005/0089473); inhib is of the ABC-carriers of drugs (see, for example, the publication of the patent application U.S. No. 2003/0073713); floor antibodies or immunoconjugates by transferrin and modulating the activity of one or more transferrin receptors (see, for example, the publication of the patent application U.S. No. 2003/0129186); and cationization antibodies or immunoconjugates (see, for example, U.S. patent No. 5004697).

The composition of an antibody or immunoconjugate according to the invention are dosed and administered in accordance with good medical practice. Factors for consideration in this context include the specific violation is subject to treatment, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the violation, the section of the delivery means, the method of administration, the scheme of administration and other factors known to practitioners. The antibody or immunoconjugate not necessarily be in a composition with one or more tools, currently used for the prevention or treatment considered violations. The effective amount of such other funds depends on the amount of antibody or immunoconjugate present in the composition, the type of disorder or treatment, and other factors discussed above. They usually are used in the same dosages and routes of administration as described in this paper, or from about 1 to 99% from those given in the th work dosages, or in any dosage and in any way that is empirically/clinically determined as appropriate.

For the prevention or treatment of disease, the appropriate dosage of antibody and immunoconjugate according to the invention (when used as monotherapy or in combination with one or more other additional therapeutic means, such as chemotherapeutic agents) will depend on the type subject to treatment of the disease, the type of antibody and immunoconjugate, the severity and course of the disease, from enter whether the antibody and immunoconjugate with preventive or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody and immunoconjugate and discretion of the attending physician. Antibody and immunoconjugate suitably administered to the patient at one time or during the course of treatment. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg to 10 mg/kg) antibodies and immunoconjugates may be possible initial dosage for administration to the patient, or, for example, in the form of one or more separate injections or a continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the above factors. With repeated introductions in several the ducks or more depending on condition treatment continued until until the desired attenuation of the symptoms of the disease. One illustrative dosages of antibodies and immunoconjugates is in the range from about 0.05 mg/kg to about 10 mg/kg Thus, the patient can enter a single or multiple doses of approximately 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof). Such doses can be entered periodically, for example every week or every three weeks (e.g. such that the patient receives from about two to about twenty, e.g. about six doses of the antibody and immunoconjugate). You can enter a higher initial loading dose followed by one or more lower doses. Illustrative dispensing includes the introduction of an initial loading dose of approximately 4 mg/kg followed by a supporting dose for weeks at a level of approximately 2 mg/kg of antibody. However, you can use other dosing regimens. The progress of this treatment is easy to monitor by conventional methods and analysis.

3. Analyses

Anti-TAT226 antibodies and immunoconjugates according to the invention can be characterized for their physical/chemical properties and/or biological activities by various assays known in this field.

a) actively Analyses the t

One aspect relates to assays for identifying anti-TAT226 antibodies or their immunoconjugates possessing biological activity. Biological activity may include, for example, the ability to inhibit cell growth or proliferation (e.g., activity in the "cell killing") or the ability to induce cell death, including programmed cell death (apoptosis). Also provides antibodies or immunoconjugate having such biological activityin vivoand/orin vitro.

In some embodiments, the implementation of anti-TAT226 antibody or immunoconjugate tested for its ability to inhibit cell growth or proliferationin vitro. Analysis of inhibition of cell growth or proliferation are well known in this field. In certain assays cell proliferation, illustrated is described here analyses the destruction of the cells to measure cell viability. One of these analyses is a luminescent analysis of the viability of the cells CellTiter-Glo™, which is commercially available from Promega (Madison, WI). In this analysis, we determine the number of living cells in culture based on the determination of the quantity of ATP present, which is an indicator of metabolically active cells. Cm. Crouch et al. (1993) J. Immunol. Meth. 160:81-88, U.S. patent No. 6602677. The analysis of usestat in 96 - or 384-well format, making it suitable for automated high-throughput screening (HTS). Cm. Cree et al. (1995) Anticancer Drugs 6:398-404. The assay procedure involves adding a single reagent (reagent CellTiter-Glo®) directly to cultured cells. This leads to the lysis of cells and the formation of a fluorescent signal produced by the luciferase reaction. Luminescent signal proportional to the amount of ATP present, which is directly proportional to the number of living cells present in the culture. Data can be registered by luminometer or device for obtaining images using a CCD camera. The output luminescence is expressed as relative light units (RLU).

Another analysis of cell proliferation is an analysis of the "MTT", colorimetric analysis, which measures the oxidation of bromide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in formazan using mitochondrial reductase. Like the analysis of CellTiter-Glo™, this analysis indicates the number of metabolically active cells present in the cell culture. See, for example, Mosmann (1983) J. Immunol. Meth. 65:55 to 63 and Zhang et al. (2005) Cancer Res. 65:3877-3882.

In one aspect of the anti-TAT226 antibody is tested for its ability to induce cell deathin vitro. Analysis of the induction of cell death is well known in this field. In some embodiments done by the means of such tests measure the for example, the loss of integrity of the membranes, as indicated by the uptake of iodide propecia (PI), Trypanosoma blue (see Moore et al. (1995) Cytotechnology, 17:1-11) or 7AAD. Illustrative analysis of PI absorption cells cultured in the medium Needle in the modification of Dulbecco (D-MEM):medium ham's F-12 (50:50) supplemented with 10% V / V heat inactivated FBS (Hyclone) and 2 mm L-glutamine. Thus, the analysis carried out in the absence of complement and immune effector cells. Cells seeded on the basis of density of 3×106a Cup of 100×20 mm Cup and allow them to attach overnight. The medium is removed and replaced with clean fresh medium or medium containing various concentrations of the antibody or immunoconjugate. Cells are incubated for 3 days. After processing, the monolayer was washed with PBS and separated by treatment with trypsin. Cells are then centrifuged at 1200 rpm for 5 minutes at 4°C, the precipitate resuspended in 3 ml of cold buffer for the binding of Ca2+(10 mm Hepes, pH of 7.4, 140 mm NaCl, 2.5 mm CaCl2) and divided into aliquots in 12×75 mm test tubes equipped with 35-mm filter (1 ml per tube, 3 tubes per group processing) to remove clumps of cells. In a test tube then add PI (10 μg/ml). Samples analyzed using the flow cytometer FACSCAN™ and software FACSCONVERT™ CellQuest (Becton Dickinson). Thus, identify antibodies or immunoconjugate, to the that induce statistically significant levels of cell death, as determined by PI uptake.

In one aspect of the anti-TAT226 antibody or immunoconjugate tested for its ability to induce apoptosis (programmed cell death)in vitro. Illustrative analysis of antibodies or immunoconjugates, which induce apoptosis, represents the analysis of the binding of annexin. Illustrative analysis of binding of annexin cells were cultured and plated on cups, as discussed in the previous paragraph. The medium is removed and replaced with clean fresh medium or medium containing from 0.001 to 10 μg/ml antibody or immunoconjugate. After incubation for three days, the monolayer was washed with PBS and separated by treatment with trypsin. Cells are then centrifuged, resuspended in the buffer for the binding of Ca2+and divided into aliquots in test tubes, as discussed in the previous paragraph. In a test tube then add labeled annexin (e.g., annexin-V-FITC) (1 μg/ml). Samples analyzed using the flow cytometer FACSCAN™ and software FACSCONVERT™ CellQuest (BD Biosciences). Thus, identify antibodies or immunoconjugate that induce statistically significant levels of binding of annexin relative to control. Another illustrative analysis of antibodies or immunoconjugates, which induce apoptosis, is a colorimetric ELISA kistanova the DNA for detection mineclearance degradation of genomic DNA. Such analysis can be performed using, for example, set the Cell Death Detection ELISA (Roche, Palo Alto, CA).

Cells for use in any of the above testsin vitroinclude cells or cell lines that naturally Express TAT226 or which is designed for the expression of TAT226. Such cells include tumor cells that sverkhekspressiya TAT226 relative to normal cells, which originate from the same cloth. Such cells include cell lines (including tumor cell line), which Express TAT226, and cell line, which normally do not Express TAT226, but which transliterowany nucleic acid that encodes a TAT226. Illustrative cell line presented here for use in any of the above testsin vitroinclude the cell line of ovarian cancer man OVCAR3, which expresses TAT226, and the cancer cell line human colon HCT116, transtitional nucleic acid that encodes a TAT226.

In one aspect of the anti-TAT226 antibody or immunoconjugate tested for its ability to inhibit cell growth or proliferationin vivo. In some embodiments, the implementation of anti-TAT226 antibody or immunoconjugate tested for its ability to inhibit tumor growthin vivo. For such testing can be used to fashion the performance communications systems in vivosuch as the model using xenograft. In the illustrative system using xenograft tumor human cells enter a suitable non-human animal with a weakened immune system, for example Nude naked mouse. The antibody or immunoconjugate according to the invention is administered to an animal. Measure the ability of antibodies or immunoconjugate to inhibit or slow down tumor growth. In certain embodiments the above-described system using xenograft tumor cells are tumor cells of the patient. Such models using xenograft commercially available from Oncotest GmbH (Frieberg, Germany). In some embodiments, the implementation of the tumor cells are tumor cells lines of human cells, such as cells OVCAR3, as presented here. In some embodiments, the implementation of the tumor cells enter a suitable non-human animal with a weakened immune system subcutaneous injection or transplantation in a suitable site, such as the accumulation of fatty breast tissue.

b) Analysis of binding and other tests

In one aspect of the anti-TAT226 antibody test in his antigennegative activity. For example, in some embodiments, the implementation of the ant-Christ.-TAT226 antibody is tested for its ability to bind with TAT226, expressed on the cell surface. For this test, you can use the FACS analysis, as described in example D.

In one aspect, you can use competitive analysis to identify a monoclonal antibody which competes with YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 or YWO.49.H6 for binding to TAT226. In some embodiments, the implementation of such a competing antibody binds to the same epitope (e.g., linear or conformational epitope)that is bound YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 or YWO.49.H6. Illustrative competitive analyses include as non-limiting examples of conventional analyses, such as analyses presented in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed illustrative ways of mapping the epitope to which antibody binds presented in Morris (1996) "an epitope Mapping Protocols", Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ). They say that the two antibodies bind to the same epitope if each blocks the binding of the other by 50% or more.

In the illustrative competitive analysis of immobilized TAT226 incubated in a solution containing a first labeled antibody that binds to TAT226 (for example, YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 or YWO.49.H6)and a second unlabeled antibody that is tested by its ability to compete with the first antibodies the om for binding to TAT226. The second antibody may be present in the hybridoma supernatant. As a control, immobilized TAT226 incubated in the solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that ensure the binding of the first antibody with TAT226, remove excess unbound antibody and measuring the amount of label associated with immobilized TAT226. If the amount of label associated with immobilized TAT226 significantly reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to TAT226. In some embodiments, the implementation of the immobilized TAT226 is present on the cell surface or in a membrane preparation obtained from a cell expressing TAT226 on its surface.

In one aspect of the purified anti-TAT226 antibodies can be further characterized in a number of analyses, including as non-limiting examples of N-terminal sequencing, amino acids analysis, exclusion liquid chromatography high pressure (HPLC) in adenocarinoma conditions, mass spectrometry, ion exchange chromatography and papain cleavage.

In one of the embodiments the invention relates to a modified antibody, which has some, but not all effector the mi functions, which make it a desirable candidate for many applications in which it is important the half-life of antibodiesin vivohowever , certain effector functions (such as the binding of complement and ADCC) are unnecessary or harmful. In specific embodiments, the implementation of the measure activity Fc antibodies to make sure that these are only the required properties. Analysis of cytotoxicityin vitroand/orin vivocan be performed to confirm the reduction/attenuation of activities in relation to CDC and/or ADCC. For example, it is possible to conduct analyses of the binding of Fc-receptor (FcR) to make sure that the antibody not binding to FcγR (thus, the probable lack of activity against ADCC), but retained the ability to bind to FcRn. The basic cell for the implementation of the ADCC, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is summarized in table 3 on page 464 in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Example of analysis ofin vitroto evaluate the activity of interest molecules against ADCC described in U.S. patent No. 5500362 or 5821337. Suitable for such analyses effector cells include mononuclear cells of peripheral blood (PBMC) and natural killer cells (NK). Alternative or additional activity predstavlyaetes molecules against ADCC can be evaluated in vivofor example, in animal models, such as described in Clynes et al. PNAS (USA) 95:652-656 (1998). You can also carry out analyses of the binding of C1q to confirm that the antibody is unable to communicate with C1q and, thus, lost its activity against CDC. To assess activation of complement can analyze the CDC, for example, as described in Gazzano-three-bet et al., J. Immunol. Methods 202:163 (1996). The FcRn binding and defining clearance/time-life isin vivoyou can also be accomplished using methods known in this field.

F. Products

In another embodiment, the invention relates to a product containing substances suitable for the treatment, prevention and/or diagnosis of the above violations. The product includes a container and a label or an insert in the packaging, on the container or in connection with it. Suitable containers include, for example, bottles, vials, syringes, etc. Containers can be formed from various materials, such as glass or plastic. The container contains a composition which by itself or when combined with another composition is effective for the treatment, prevention and/or diagnosis of the condition and may have a hole for sterile access (for example, the container may be a package for intravenous solution or vial having a stopper, permeable to the allow for subcutaneous injection). At least one active agent in the composition is an antibody or immunoconjugate according to the invention. On the label or an insert in the packaging indicates that the composition is used to treat the selected state. Moreover, the product may contain (a) a first container containing a composition, where the composition comprises the antibody and immunoconjugate according to the invention; and (b) a second container containing a composition, where the composition contains additional cytotoxic or other therapeutic agent. The product in this embodiment of the invention may further comprise an insert in the packaging, which indicates that the composition can be used to treat a specific condition. Alternative or additionally, the product may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-saline buffer, ringer's solution and dextrose. In addition, it can optionally contain other necessary commercial and consumer point of view the materials, including other buffers, diluents, filters, needles and syringes.

IV. EXAMPLES

Examples of methods and compositions according to the invention. It should be understood that it is possible to apply various other vari the details of implementation based on the above General description.

A. Analysis of gene expression of TAT226

Gene expression of TAT226 man were analysed using a private database containing information on gene expression (spatial structures®, Gene Logic Inc., Gaithersburg, MD). Graphical analysis database spatial structures®was performed using a viewer profile microarray. Figure 13 is a graphical representation of the gene expression of TAT226 in various tissues, which are listed on the left. The scale in the upper part of the graph indicates the levels of gene expression based on the intensity of the hybridization signal. Points are located both above and below the line corresponding to each of the listed tissues. Points located above the line represent gene expression in normal tissue, and points below the line represent gene expression in the tumor or diseased tissue. In the figure 13 presents the trend toward increased gene expression of TAT226 in the tumor or diseased tissue relative to normal options. In particular, for TAT226 shown significant overexpression in the tumor and diseased ovary relative to normal ovary and in Wilms tumor relative to normal kidney. Other tissues, which shows overexpression in the tumor or diseased tissue relative to normal tissue includes tissue of the endometrium, adrenal glands,bones, lung, skin and soft tissue. In addition, TAT226 largely is expressed in normal brain tissue (such as the olfactory brain, hippocampus and basal ganglia) and in the tumor or diseased tissue of the brain, such as gliomas.

Database spatial structures®also used to analyze gene expression of TAT226 person in normal ovary; normal fallopian tube; ovarian cancer subtypes clear, mucinous and serous cystadenocarcinoma; metastatic cancer of ovary and other types of ovarian cancer. The results are presented graphically in figure 14, where the specific tissue types are indicated below the graph. The scale on the y-axis of the graph indicates the levels of gene expression based on the intensity of the hybridization signal. For serous cystadenocarcinoma and metastatic cancer of the ovary showed significant overexpression of TAT226 relative to normal ovary. For clear and mucinous subtypes shows the expression comparable to normal ovary. For normal fallopian tubes also shows significant expression of TAT226. It is noteworthy that the serous subtype of ovarian cancer histologically substantially similar to the epithelium of the fallopian tubes, and both ovaries and fallopian tubes originate from the same embryonic tissue. Cm. Fox et al. (2002) "Pathology of epithelial ovarian cancer"Ovarian Cancer ch. 9 (Jacobs et al, eds., Oxford University Press, New York).

B. Receiving anti-TAT226 antibodies

The TAT226 antibody was obtained by screening libraries of phage display using a recombinant fused protein "TAT226-His containing amino acids 1-115 SEQ ID NO:75 and C-terminal polyhistidine tag. Library of phage display was a synthetic library (Fab')2obtained using Fab'-zip-phage. Cm. Lee et al. (2004) J. Immunol. Methods 284:119-132. The library contained a library of heavy chain HVR in the frame region of the variable region of the heavy chain huMAb4D5-8 (see figures 5A and 5B, the second acceptor "B", SEQ ID NO:50, 51, 57, 35) and a fixed variable region light chain huMAb4D5-8 as shown in SEQ ID NO:26. Clones selected using phage display were subjected to screening against TAT226-His using phage ELISA (see, for example, Sidhu et al. (2004) J. Mol. Biol. 338:299-310). Clones YWO.32 and YWO.49 selected for further analysis.

To improve the affinity of YWO.49 library of phage display were obtained on the basis YWO.49 where HVR-H3, HVR-L3 were used as targets for soft randomization, in which selected amino acid residues at the indicated HVR kept constant, while the other was subjected to mutagenesis. The selected clones were subjected to screening using a phage ELISA. Affine Mature antibodies labeled YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6, were selected for further analysis. Nucleotide sequence that is tianye sequence and the sequence of the encoded polypeptide regions VH and VL YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 was determined, as shown in figures 9 and 10. Sequences of the heavy and light chain HVR YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 shown in figures 2-4. Consensus sequences of HVR-H3, HVR-L3, obtained from YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6, also presented in figure 4. YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 "transformed" in the form of full-size IgG inculcation of fragments, Fab', the corresponding constant region using recombinant methods. The following experiments were carried out using the transformed antibodies.

C. Characterization of the affinity of binding with recombinant antigen

The binding affinity of YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 with the recombinant antigen was determined by measurement using surface plasmon resonance using the BIACORE system®3000 (Biacore, Inc., Piscataway, NJ). In summary, biosensor chips with carboxyethylgermanium dextran (CM5, BIAcore Inc.) activated hydrochloride N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Anti-TAT226 antibody was diluted with 10 mm sodium acetate, pH of 4.8, to 5 μg/ml before injection with flow rate of 5 μl/minute to achieve approximately 500 units of the resonance signal (RU) associated antibodies. Next, 1 M ethanolamine injec is believed to block unreacted groups. For kinetics measurements twofold serial dilution TAT226-His (from 0.7 nm to 500 nm) were injected with PBS with 0.05% tween-20 (PBST) at 25°C at a flow rate of approximately 25 μl/min Rate of Association (konand the rate of dissociation (koff) was calculated using a simple model linking one-to-one the Langmuir (software BIAevaluation version 3.2). The equilibrium dissociation constant (Kd) was calculated as the ratio of koff/kon. The results of this experiment are presented in table 2 below.

Table 2
Clonekon/105koff/10-4Kd (nm)
YWO.490,0743,4947,16
YWO.49.B70,27<0,05<0,18
YWO.49.C91,53<0,05<0,03
YWO.49.H20,220,050,23
YWO.49.H6to 1.860,090,05

D. characterization of the binding of TAT226 antibodies on the cell surface

Investigated the ability of anti-TAT226 antibodies to contact TAT226, expressed on the surface of OVCAR3, cell line of ovarian cancer man. Carried out fluorescence-activated cell sorting (FACS) in OVCAR3 cells in the absence and in the presence of YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 or YWO.49.H6. In brief, detached cells were incubated with 5 µg/ml of primary antibodies for 1 hour on ice, washed and incubated with secondary antibody (IgG against human conjugated with phycoerythrin) for 30 minutes on ice. FACS was performed using the flow cytometer FACScan™ (BD Biosciences, San Jose, CA).

The results of FACS analysis for YWO.49, YWO.49.H2 and YWO.H6 presented on figure 15. The peaks on the left side of each graph represent the "background" binding, i.e. binding only with secondary antibody. The peaks on the right side of each graph represents the binding with the specified anti-TAT226 antibody. YWO.49.B7 not associated significantly with OVCAR3 cells according to FACS, although it is associated with a recombinant antigen with a Kd in the range of Kd found for YWO.49 and other affinity-Mature antibodies (see results of BIACORE analysis®tables is 2 above). Linking YWO.49.C9 was comparable to that found for YWO.49.H2 and YWO.H6.

E. Characterization of the affinity of binding to the antigen on the cell surface

The binding affinity of YWO.49.H2 and YWO.49.H6 with TAT226, expressed on the surface of OVCAR3 cells, was investigated using competitive analysis. In summary, labeled (iodirovannoye) YWO.49.H2 or YWO.49.H6 allowed to contact the OVCAR3 cells in the presence of unlabeled antibody. The binding affinity of the antibodies was determined in accordance with the method of analysis Scatchard, originally described in Munson et al., Anal. Biochem. 107:220 (1980). The results of this experiment are presented in table 3 below.

Table 3
CloneKd (nm)
YWO.49.H20,348
YWO.49.H60,404

Kd YWO.49.H2 and YWO.49.H6 above for TAT226, expressed on the surface of OVCAR3 cells, compared with recombinant TAT226-His (cf. Kd for YWO.49.H2 and YWO.49.H6 in tables 2 and 3), indicating that YWO.49.H2 and YWO.49.H6 associated with slightly higher affinity to recombinant TAT226-His than with TAT226, expressed on the surface of OVCAR3 cells.

F. Expression of mRNA and proteins is TAT226

The expression of mRNA TAT226 in OVCAR3 cells and in the Swatch panel of ovarian cancer were analyzed using analysis of the 5'-nuclease (TaqMan®) and quantitative real-time PCR. Samples of ovarian cancer, marked "HF####on figure 16, was a frozen tissue sections. RNA was extracted from tissue sections, amplified using a set of Ambion''s Message Amp II (Ambion, Austin, TX) and subjected to reverse transcription into cDNA. RNA was isolated from OVCAR3 cells and subjected to reverse transcription into cDNA. cDNA TAT226 amplified using real-time PCR in the presence of nedostrahovanija reporter probe, specific product amplification. The threshold cycle or Ct (the cycle in which the signal received by the cleavage of a reporter probe, exceeded the background) was determined and used to calculate the initial levels of mRNA TAT226. The mRNA level of TAT226 in the Swatch panel of ovarian cancer was expressed relative to the mRNA level of TAT226 in OVCAR3 cells, as shown in the bar chart in figure 16.

The expression of protein TAT226 in OVCAR3 cells and the panel above samples of ovarian cancer were analyzed using immunohistochemistry (IHC), as indicated below. The tissue sections (frozen or prisoners in paraffin samples of ovarian cancer were fixed for 5 minutes in acetone/ethanol. Sections were washed in PBS, blocked with Avidya, bioti is ω (Vector Laboratories, Inc., Burlingame, CA) for 10 minutes each, and again washed in PBS. The slices are then blocked with 10% serum for 20 minutes and subjected to blotting to remove excess whey. Primary antibody (YWO.49.H2 or YWO.49.H6) was then added to the sections at a concentration of 10 μg/ml for 1 hour. The slices were then washed in PBS. Biotinylated secondary antibody against the person was added to the samples for 30 minutes and then the sections were washed in PBS. The sections were then treated with reagents Vector ABC (Vector Laboratories, Inc., Burlingame, CA) for 30 minutes and then washed in PBS. The samples were then treated with diaminobenzidine (Pierce) for 5 minutes and washed in PBS. The samples are then contrasted with hematoxylin Mayer, covered with cover glass and visualized. IHC was performed on OVCAR3 cells using the same Protocol except that the cells first besieged, froze and then turned them into slices. The slices were then processed according to the above Protocol.

The results are qualitatively presented in figure 16, where the expression levels are ranked as "-", "±" or "+". As a rule, in General, there was a correlation between the level of expression of mRNA and expression of TAT226 protein TAT226 on the surface of OVCAR3 cells. Experiments with IHC also confirmed that the antibodies recognize TAT226 on the cell surface. Histological analysis of each cell in the panel cleto is of ovarian cancer is also presented in figure 16 with the abbreviation "adenoca", meaning "adenocarcinoma".

G. Receiving ADC anti-TAT226

ADC anti-TAT226 was obtained by conjugation YWO.49.H2 and YWO.49.H6 with the following components of the drug-linker: MC-vc-PAB-MMAE, MC-vc-PAB-MMAF and MC-MMAF, which are presented above in section III.C.1.b.2. Before conjugation of antibodies partially recovered using TCEP using conventional methods according to the method described in WO 2004/010957 A2. Partially restored antibodies conjugatively with the above mentioned components of the drug-linker using conventional methods according to the method described in Doronina et al. (2003) Nat. Biotechnol. 21:778-784 and US 2005/0238649 A1. In brief, partially restored antibodies combined with components of the drug-linker to provide conjugation components with cysteine residues. Stopped reactions of conjugation and cleansed the ADC. The load of the medicinal product (the average number of components of the drug to the antibody) for each ADC was determined by HPLC as follows:

ADCLoad drugs
YWO.49.H2-MC-vc-PAB-MMAEthe 3.8
YWO.49.H2-MC-vc-PAB-MMAF4,7
YWO.49.H2-MC-MMAFa 4.9
YWO.49.H6-MC-vc-PAB-MMAE4,4
YWO.49.H6-MC-vc-PAB-MMAF4,4
YWO.49.H6-MC-MMAF4,1

H. Analysis of cell killing

Conjugates of the antibody-drug (ADC) were tested for ability to inhibit proliferation of cells expressing TAT226, in the following analyses of cell killingin vitroandin vivo:

1. Analysis of the destruction of OVCAR3 cells in vitro

ADC YWO.49.H2 and YWO.49.H6 tested for ability to inhibit the proliferation of OVCAR3 cells. OVCAR3 cells were planted in 96-well plates in RPMI with 20% FBS. OVCAR3 cells at a density of 3000 cells per well were incubated with different concentrations of the ADC, as shown in figure 17. Anti-MUC16/CA125 antibody conjugated with MC-vc-PAB-MMAE, was used as positive control. MUC16/CA125 is a known antigen of ovarian cancer. See, for example, Yin et al. (2001) J. Biol. Chem. 276:27371-27375. Anti-IL-8 antibody conjugated with MC-vc-PAB-MMAE was used as a negative control. After 5 days of incubation, cell viability was measured using fluorescent analysis of cell viability CellTiter-Glo™ (Promega, Madison, WI) according to the instructions of p is aizvaditas. The scale on the y-axis of figure 17 indicates the relative light unit or "RLU" when luciferase luminescence, which is a measure of the viability of the cells.

Figure 17 shows that similarly to the positive control YWO.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF possessed significant activity against destruction of cells, in particular at a concentration of calculation and approximately 0.01 and 0.1 ág/ml YWO.49.H2-MC-vc-PAB-MMAE and YWO.49.H6-MC-vc-PAB-MMAE also had activity against destruction of cells, but to a lesser extent than the activity of YWO.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF. IC50for YWO.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF was approximately 0,005 nm, and the IC50for YWO.49.H2-MC-vc-PAB-MMAE and YWO.49.H6-MC-vc-PAB-MMAE was approximately 0.2 nm. IC50for free MMAE was approximately 0.1 nm. YWO.49.H2-MC-MMAF and YWO.49.H6-MC-MMAF had no significant activity against the destruction of the cells in this analysis. For this particular system of analysis it should be noted that at high concentrations of the ADC, including the ADC as a negative control, cell viability decreased significantly due to the overall high concentration of MMAE and MMAF.

2. Analysis of cell killing in vitro using transfected HCT116 cells

ADC YWO.49.H2 and YWO.49.H6 tested for ability to inhibit the proliferation of HCT116 cells that line the colon cancer cells, which article is stable was transfusional nucleic acid, coding TAT226 person. Nitrostilbene HCT116 cells at the rate of about 5-6 times less sensitive to the free (unconjugated) MMAE than OVCAR3 cells.

Briefly, HCT116 cells were transfusional as follows. Nucleic acid encoding associated with the epitope of human TAT226, designed in expressing vector mammals pcDNA3.1 (Invitrogen, Carlsbad, CA). Epitope tag consisted of amino acids 1-53 of glycoprotein D of herpes simplex virus type 1 (label "gD"), which was replaced with a signal sequence from amino acids 1-22 N-end human TAT226. The recombinant vector was transfusional in HCT116 cells using Lipofectamine200 (Invitrogen) according to the manufacturer's Protocol. Transfetsirovannyh HCT116 cells were cultured in medium McCoy's 5a with 10% FBS and 0.4 mg/ml G418. Cells were stained using antibodies anti-gD and sorted by FACS for the selection of individual clones expressing recombinant protein gD:human TAT226. One of the clones, designated HCT116#9-4, were selected for further analysis.

For analysis of cell killing cells HCT116#9-4 were sown in 96-well plates. Cells HCT116#9-4 density of 1000 cells per well were incubated with different concentrations of the ADC, as shown in figure 18. Anti-gp120 antibody conjugated with MC-vc-PAB-MMAE, was used as a negative is about control. After 3 days incubation, cell viability was measured using fluorescent analysis of cell viability CellTiter-Glo™ (Promega, Madison, WI) according to manufacturer's instructions.

The figure 18 shows that YWO.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF possessed significant activity against destruction of cells, in particular, at about 0.01 μg/ml and up to the highest of the tested concentrations. IC50for YWO.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF was about 0.05 nm, and the IC50for free MMAE was ~ 0.9 nm. YWO.49.H2-MC-vc-PAB-MMAE and YWO.49.H6-MC-vc-PAB-MMAE had no significant activity against the destruction of the cells relative to negative control in this particular analysis. The difference between the activity in respect of destruction of cells for YWO.49.H2-MC-vc-PAB-MMAE and YWO.49.H6-MC-vc-PAB-MMAE in this analysis compared to the analysis of the destruction of OVCAR3 cells (above) can be attributed to various factors, such as the density of cells and/or differences in sensitivity to the drug.

It should be noted that for some other tested cell lines that normally Express mRNA or protein TAT226, ADC YWO.49.H2 and YWO.49.H6 had no significant activity against destruction of cells. This can be attributed to various factors, such as effects that are specific to cell types, the level is her expression of TAT226 on the cell surface and/or differences in sensitivity to the drug.

3. Analysis of in vivo using xenograft HCT116#9-4

Modelin vivousing xenograft used for testing unconjugated and conjugated YWO.49.H6 by the ability to inhibit the proliferation of expressing TAT226 tumor cellsin vivo. The formation of tumors induced in Nude naked mice "nu-nu" subcutaneous injection of approximately 5×106cells HCT116#9-4 in dorsal groin mice. Tumors were given the opportunity to grow as long as they did not reach the average tumor volume of 200 mm3. This time point was designated as "0 days". As shown in figure 19, mice received intravenous injections at the rate of 3 mg/kg of these unconjugated antibody or ADC at 0, 7 and 16 days. Unconjugated and conjugated antibodies to agglutinin ambrosia (Ab) served as negative controls. The mean tumor volume was measured at 3, 7, 10, 16 and 21 days. As shown in figure 19, YWO.49.H6-MC-vc-PAB-MMAF possessed significant activity against the destruction of tumor cells in the measurement of the average tumor volume compared with anti agglutinins ambrosia Ab-MC-vc-PAB-MMAF in this particular model with the use of xenograft. YWO.49.H6-MC-vc-PAB-MMAE had no significant activity against the destruction of tumor cells relative to anti-agglutin is in the ragweed Ab-MC-vc-PAB-MMAE in this model using xenograft. However, this model using xenograft may not reflect the activity in respect of destruction of cells YWO.49.H6-MC-vc-PAB-MMAE, observablein vitrodue to many factors, for example due to differences in sensitivity to the drug or levels of expression of TAT226 on the cell surface in the microenvironment of xenograft tumors.

4. Other models using xenograft

Other models using xenograft can be used for testing unconjugated and conjugated anti-TAT226 antibodies for the ability to inhibit the proliferation of expressing TAT226 tumor cellsin vivo. For example, models using xenograft for tumors of the ovary and the brain can be obtained from publicly available sources, such as Oncotest GmbH (Frieberg, Germany) and Southern Research Institute (Birmingham, AL). Model Oncotest, in particular, developed by growing tumors patients in naked mice with weakened immune systems. The xenografts, which Express mRNA and/or protein TAT226, can be used to demonstrate activity against destruction of cells anti-TAT226 antibodiesin vivo.

Conjugated YWO.49.H6 tested in the model Oncotest OVXF1023 by the ability to inhibit the proliferation of tumor cells of the ovaryin vivo. Model Oncotest OVXF1023 comes from metastatic the laboratory differentiated papillary serous adenoma carcinoma ovary, stage M1. Mice OVXF1023 processed ADC, shown on figure 20. In figure 20 YWO.49.H6 designated as "H6"; antibody to agglutinin ambrosia (control) are indicated as "RW"; and the linker-MC-vc-PAB - abbreviated designated as "vc". ADC was implemented on the day and in the concentrations indicated in the figure 20. The results presented in figure 20 indicates that YWO.49.H6-MC-vc-PAB-MMAF and YWO.49.H6-MC-MMAF significantly reduce tumor volume relative to other ADC.

The above experiment was repeated using OVXF1023 in similar conditions, but with changes in dosage and in the control of the ADC. In the second experiment, mice were treated with a higher dose (5 mg/kg) YWO.49.H6-MC-vc-PAB-MMAE and lower doses (5 mg/kg) YWO.49.H6-MC-vc-PAB-MMAF and YWO.49.H6-MC-MMAF. The results showed that the ADC H6 (and, in particular, YWO.49.H6-MC-vc-PAB-MMAE) reduced tumor volume relative to the corresponding control ADC (anti-gp120-MC-vc-PAB-MMAE from 6.4 mg/kg; anti-gp120-MC-vc-PAB-MMAF from 7.2 mg/kg; and anti-gp120-MC-MMAF from 5.4 mg/kg), although the difference in effectiveness between the ADC H6 and corresponding ADC control was not statistically significant for some data values (data not shown). The difference between these results and the results obtained in the first experiment with OVXF1023, can be attributed to differences in dosages ADC H6 and the identification of the fact that ADC control anti-gp120 possessed unexpected AK is ewnetu in reducing tumor volume.

In addition, the ADC H6 also tested in another model Oncotest, OVXF899, which comes from a moderately differentiated papillary serous ovarian carcinoma (primary tumor). ADC H6 did not reduce tumor volume in this model (data not shown). However, this particular model Oncotest had a low expression of TAT226 that may be responsible for the observed results.

I. MAb

The obtained antibodies designed with cysteine or "MAb", in which the selected balance YWO.49.H6 substituted by cysteine to provide an additional site for conjugation with a component of the linker-drug. In particular, the substitution A118C (EU numbering) obtained in the heavy chain YWO.49.H6, or substitution V205C (numbering according to Kabat) obtained in the light chain YWO.49.H6. Received MAb A118C then conjugatively with MC-MMAF and received MAb V205C then conjugatively or MC-MMAF, or MC-vc-PAB-MMAE. All MAb was able to contact the OVCAR3 cells according to FACS analysis (data not shown).

Although the above invention is described in detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. Descriptions of all patents and publications, cited herein, explicitly cast as reference in full.

1. Monoclonal antibody that binds to THAT, where the antibody contains
(1) HVR-H1 containing the amino acid sequence of SEQ ID NO:4;
(2) the HVR-H2 containing the amino acid sequence of SEQ ID NO:5;
(3) the HVR-H3 containing the amino acid sequence that corresponds to the consensus sequence SEQ ID NO:11;
(4) the HVR-L1 containing the amino acid sequence of SEQ ID NO:12;
(5) the HVR-L2, containing the amino acid sequence of SEQ ID NO:13; and
(6) the HVR-L3 containing the amino acid sequence that corresponds to the consensus sequence SEQ ID NO:19.

2. The antibody according to claim 1, where the antibody contains HVR-H3 containing aminokislotna the sequence, selected from SEQ ID NO:6-10, and HVR-L3, containing the amino acid sequence selected from SEQ ID nos:14-18.

3. The antibody according to claim 2, where the HVR-H3 contains the amino acid sequence of SEQ ID NO:9, and HVR-L3 contains the amino acid sequence of SEQ ID NO:17.

4. The antibody according to claim 2, where the HVR-H3 contains the amino acid sequence of SEQ ID NO:10, and HVR-L3 contains the amino acid sequence of SEQ ID NO:18.

5. The antibody according to claim 1, additionally containing at least one frame region selected from the consensus frame region VH subgroup III consensus framework region VL subgroup I.

6. Monoclonal antibody that binds to THAT, where the antibody contains a variable domain of a heavy chain having at least 95% sequence identity with the amino acid sequence selected from SEQ ID nos:21-25 and variable domain light chain having at least 95% sequence identity with the amino acid sequence selected from SEQ ID nos:26-31, and where the specified antibody that binds to TAT, has a dissociation constant (KD)≤10 nm.

7. The antibody according to claim 6, where the antibody contains a variable domain of a heavy chain having at least 95% sequence identity with the amino acid sequence SEQ ID NO:24, and the variable domain of light chain, oblad the store, at least 95% sequence identity with the amino acid sequence SEQ ID NO:29.

8. The antibody according to claim 7, where the variable domain of the heavy chain contains the amino acid sequence of SEQ ID NO:24, and the variable domain of the light chain contains the amino acid sequence of SEQ ID NO:29.

9. The antibody according to claim 6, where the antibody contains a variable domain of a heavy chain having at least 95% sequence identity with the amino acid sequence SEQ ID NO:25, and the variable domain light chain having at least 95% sequence identity with the amino acid sequence SEQ ID NO:30.

10. The antibody according to claim 9, where the variable domain of the heavy chain contains the amino acid sequence of SEQ ID NO:25, and the variable domain of the light chain contains the amino acid sequence of SEQ ID NO:30.

11. The antibody according to claim 1 or 6, where the antibody is an antibody fragment selected from a Fab fragment, Fab'-SH, Fv, scFv, or (Fab')2.

12. The antibody according to claim 1 or 6, where the antibody is humanized.

13. The antibody according to claim 1 or 6, where the antibody is human.

14. Immunoconjugate containing the antibody, which binds to TET according to claim 1 or 6, covalently attached to a cytotoxic agent.

15. Immunoconjugate on 14, where the cytotoxic agent is selected from the toxin, chemotherapeutic the ski means, antibiotic, a radioactive isotope and nucleotidase enzyme.

16. Immunoconjugate having the formula Ab-(L-D)p, where:
(a) Ab is an antibody that binds to TAT, and which contains
(i) HVR-H1 containing the amino acid sequence of SEQ ID NO:4;
(ii) HVR-H2 containing the amino acid sequence of SEQ ID NO:5;
(iii) HVR-H3 containing the amino acid sequence that corresponds to the consensus sequence SEQ ID NO:11;
(iv) HVR-L1 containing the amino acid sequence of SEQ ID NO:12;
(v) HVR-L2 containing the amino acid sequence of SEQ ID NO:13; and
(vi) HVR-L3 containing the amino acid sequence that corresponds to the consensus sequence SEQ ID NO:19;
(b) L is a linker;
(C) D is a drug of the formula DEor DF

,
and where each R2and R6represents methyl, each R3and R4represents isopropyl, R7represents sec-butyl, each R8independently selected from CH3, O-CH3HE and H; R9represents H; R10represents aryl; Z represents-O - or-NH-; R11represents H, C1-C8alkyl or -(CH2)2-O-(CH2)2-O-(CH2) 2-O-CH3; and R18represents-C(R8)2-C(R8)2-aryl; and
(d) p ranges from about 1 to 8.

17. Immunoconjugate on clause 16, where the antibody contains HVR-H3 containing the amino acid sequence selected from SEQ ID NO:6-10, and HVR-L3, containing the amino acid sequence selected from SEQ ID nos:14-18.

18. Immunoconjugate on 17, where the HVR-H3 contains the amino acid sequence of SEQ ID NO:9, and HVR-L3 contains the amino acid sequence of SEQ ID NO:17.

19. Immunoconjugate on 17, where the HVR-H3 contains the amino acid sequence of SEQ ID NO:10, and HVR-L3 contains the amino acid sequence of SEQ ID NO:18.

20. Immunoconjugate having the formula Ab-(L-D)p, where:
(a) Ab is an antibody that binds to TAT and which contains the variable region of the heavy chain having at least 95% sequence identity with the amino acid sequence selected from SEQ ID nos:21-25, and variable region light chain having at least 95% sequence identity with the amino acid sequence selected from SEQ ID nos:26-31;
(b) L is a linker;
(c) D is a drug of the formula DEor DF

,
and where each R2and R6represents the t a methyl, each R3and R4represents isopropyl, R7represents sec-butyl, each R8independently selected from CH3, O-CH3HE and H; R9represents H; R10represents aryl; Z represents-O - or-NH-; R11represents H, C1-C8alkyl or -(CH2)2-O-(CH2)2-O-(CH2)2-O-CH3; and R18represents-C(R8)2-C(R8)2-aryl; and
(d) p ranges from about 1 to 8
and where the specified antibody that binds to TAT, has a dissociation constant (KD)≤10 nm.

21. Immunoconjugate according to claim 20, where the antibody contains a variable region heavy chain having at least 95% sequence identity with the amino acid sequence SEQ ID NO:24, and the variable region light chain having at least 95% sequence identity with the amino acid sequence SEQ ID NO:29.

22. Immunoconjugate on item 21, where the antibody contains a variable region heavy chain containing the amino acid sequence of SEQ ID NO:24, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:29.

23. Immunoconjugate according to claim 20, where the antibody contains a variable region heavy chain having at least 95% ID is ntichnosti sequence with amino acid sequence SEQ ID NO:25, and variable region light chain having at least 95% sequence identity with the amino acid sequence SEQ ID NO:30.

24. Immunoconjugate according to item 23, where the antibody contains a variable region heavy chain containing the amino acid sequence of SEQ ID NO:25, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:30.

25. Immunoconjugate in article 16, or 20, where the linker is attached to the antibody through Tilney groups on the antibody.

26. Immunoconjugate in article 16, or 20, where the drug is selected from MMAE and MMAF.

27. Immunoconjugate on p, where the drug is MMAE.

28. Immunoconjugate on p, where the drug is a MMAF.

29. Immunoconjugate on p, where the linker is cleaved by a protease.

30. Immunoconjugate on clause 29, where the linker contains the dipeptide val-cit.

31. Immunoconjugate on clause 29, where the linker contains a link p-aminobenzyl.

32. Immunoconjugate on clause 29, where the linker contains 6-maleimidomethyl.

33. Immunoconjugate on item 21, where immunoconjugate has the formula
,
where S represents a sulfur atom, and p ranges from 2 to 5.

34. Immunoconjugate on p, where Ab is a monoclonal antibody according to claim 3 or 8.

35. Immunoconjugate on p, where Ab represents sobaypanhellenic antibody according to claim 4 or 10.

36. Immunoconjugate on p where immunoconjugate has the formula
,
where S represents a sulfur atom, and p ranges from 2 to 5.

37. Immunoconjugate on p, where Ab is a monoclonal antibody according to claim 3 or 8.

38. Immunoconjugate on p, where Ab is a monoclonal antibody according to claim 4 or 10.

39. Pharmaceutical composition comprising an effective amount immunoconjugate on any of p-38, for use in the treatment of disorders of cell proliferation.

40. The pharmaceutical composition according to § 39, where the violation of cell proliferation is selected from ovarian cancer, cancer of uterine body, brain tumors and Wilms tumor.

41. The pharmaceutical composition according to § 39, where immunoconjugate is immunoconjugate in article 16, or 20.



 

Same patents:

FIELD: medicine.

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

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

227 cl, 25 dwg, 16 tbl, 14 ex

FIELD: medicine.

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

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

43 cl, 20 dwg, 10 ex

FIELD: medicine, pharmaceutics.

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

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

10 cl, 6 dwg, 8 ex

FIELD: chemistry.

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

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

24 cl, 5 dwg, 2 tbl

FIELD: chemistry.

SUBSTANCE: method involves conducting a conjugation reaction by mixing ciprofloxacin and carbodiimide in ratio of 1:1 for 5 minutes at room temperature, adding the reaction mixture obtained as a result of conjugation to a solution of a human alpha-fetoprotein fragment followed by incubation of the obtained solution for 20-30 minutes while stirring and monitoring pH. By-products obtained from adding the reaction mixture to the solution of the human alpha-fetoprotein fragment are removed via dialysis against a phosphate-salt buffer at pH 8.0.

EFFECT: invention widens the field of using methods of producing conjugates of human alpha-fetoprotein with ciprofloxacin.

1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: chimeric polypeptide contains a first domain which is a first intracellular loop (i1-loop) of chemokine receptor, and a second domain coupled with the first domain where the second domain is a cell-penetrating and membrane-bound natural or artificial hydrophobic residue. The produced polypeptide is used in treating a disease or a condition characterised by pathological activity of chemokine receptor.

EFFECT: invention enables producing chimeric polypeptide which is selectively reacting with related chemokine receptor.

13 cl, 33 dwg, 3 tbl, 15 ex

FIELD: medicine.

SUBSTANCE: invention refers to producing fused proteins. The fused construct consists of an amino acid sequence of glycosyl phosphatidylinositol anchored tissue inhibitor of metalloproteinase.

EFFECT: cictrisation prevention during skin injures treatment when using the fused construct.

10 cl, 33 dwg, 18 ex

FIELD: medicine.

SUBSTANCE: protein is capable to inhibit thrombin activity specifically. Besides, what is offered is a nucleic acid coding said chimeric protein, a vector containing this nucleic acid and a host cell carrying the vector. The present invention also refers to a pharmaceutical composition containing the recombinant chimeric protein of neutrophils and girugen inhibition factor. An effect of the given composition consists in thrombocyte aggregation inhibition or peripheral leukocyte activation inhibition.

EFFECT: composition can be used for treating a cardio-cerebrovascular disease or preventing a cerebral ischemic injury or a cerebral hematoma.

13 cl, 11 dwg, 16 tbl, 17 ex

FIELD: medicine.

SUBSTANCE: offered are versions of antibodies each of which is specifically bound with IGF-IR, inhibits its activity and is its antagonist, not exhibiting substantially IGF-IR agonist activity. Each of the antibodies is characterised at least by the presence of a variable area of a heavy and easy chain. There are described: antibody conjugates with cytotoxic agents, and also versions of a pharmaceutical composition for cancer diagnosing and therapy, methods of cancer treatment and diagnosing, a cancer diagnosing reagent - on the basis of the antibodies. There are disclosed: a method of producing the antibodies; nucleic acid (NA) coding the antibody; an expression vector containing NA. Offered is a hybridoma EM 164 producing the antibody under the invention, deposited in ATCC, No. PTA-4457, and also the use of the antibody for IGF-IR linkage. The use of the invention presents the antibodies which allow inhibiting MCF cell growth approximately in 12 times that is higher approximately in 5 times than hen using the antibody IR3, and can be used for cancer diagnosing and treatment expressing higher levels of receptor IGF-I, such as breast cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, synovial carcinoma and pancreatic cancer.

EFFECT: more efficient diagnosing and treatment of said cancers.

58 cl, 28 dwg, 10 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: offered is a fused protein containing from an amino-terminus to carboxyl terminus: binding domain polypeptide which is bound with a biological target molecule; human one-cysteine "link" region IgGl peptide; immunoglobulin heavy chain CH2 constant region polypeptide and immunoglobulin heavy chain CH3 constant region polypeptide. The fused protein represents mixed monomers and dimers, has ADCC, CDC or both cytotoxicities. Also, described are a pharmaceutical composition and a method of treating a B-cell disorder with using the fused protein.

EFFECT: use of the invention can find the further application in medicine in treating B-cell disorders.

26 cl, 26 dwg, 9 ex

FIELD: medicine.

SUBSTANCE: there are presented versions of antibodies specific to claudin 18A2 produced by immunisation by a related amino acid sequence or a nucleic acid or a host cell expressing said peptide. The antibodies possess an ability to mediate elimination of cancer cells expressing claudin 18A2. There are disclosed versions of antibody-producing hybridomas. What is described is a conjugate or a pharmaceutical composition on the basis of antibodies or conjugates for elimination and/or inhibition of a cancer cell expressing claudin 18A2. There are disclosed versions of the method for growth inhibition and/or elimination of the cancer cell, as well as for treating or preventing a disease or a disorder involving cancer cells expressing claudin 18A2 with using the antibodies, conjugate and pharmaceutical composition under the invention.

EFFECT: use of the invention can find further application in medicine for treating cancer cells expressing claudin 18A2.

39 cl, 33 dwg, 5 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of medicine and deals with bispecific binding agents for modulating biological activity. Essence of invention includes bispecific binding agent, which is cable of directed action on cancer cells by means of binding domain with high affinity with respect to first marker of cell surface, which does not induce significant biological effect, and binding domain with low affinity, which specifically binds with second marker of cell surface, producing significant and required biological effect. Also claimed are pharmaceutical compositions for treatment of malignant tumour, their application for production of medication and sets for diagnostics and visualisation of tumour.

EFFECT: intensification of antitumor action.

37 cl, 7 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: invention refers to immunology and biotechnology. What is offered is a TAT 10772 antibody which is characterised by the presence of six CDR. What is described is a method for identifying the antibody which binds TAT 10772 polypeptide. What is disclosed is using the antibody according to claim 1 for TAT 10772 expressing cell growth inhibition, and for therapeutic treatment of a tumour in a mammal where the tumour expresses TAT 10772. There are described: a presence-absence test of TAT 10772 protein in a sample which is supposed to contain it, and a method of diagnostic detection of a TAT 10772 expressing tumour, based on using the antibody according to claim 1.

EFFECT: presented inventions can find further application in therapy and diagnostics of TAT 10772 expressing tumours.

31 cl, 37 dwg, 11 tbl, 18 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, and represents an antibody able to bind a tumour-associated antigen TAT 10772 polypeptide. Besides, there are presented antibody conjugate with a drug, pharmaceutical composition, method of cell proliferation inhibition, method of treating the TAT 10772 expressing tumours , analysis for tumour cell detection, as well as kit for treating the tumours.

EFFECT: invention can be used in treating the tumours expressing the tumour associated antigen TAT 10772 polypeptide.

41 cl, 45 dwg, 11 tbl, 18 ex

FIELD: medicine.

SUBSTANCE: invention concerns anti-PSMA antibodies which contain variable sites of heavy and light chains; the antibody does not contain fucosyl residues. Amino acid sequences of said chains are presented in the formula. Antibodies under the invention are a monoclonal antibody, a humanised or chimeric antibody, a human antibody. Also, there is described a method of inhibition of PSMA+ cell growth, such as tumour cells by contact of said cells with the anti-PSMA antibodies.

EFFECT: antibodies show higher, antibody-dependent prostate cancer cell cytotoxicity as compared with a fucosyl antibody form.

32 cl, 3 tbl, 6 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

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: 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: 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: 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, pharmaceutics.

SUBSTANCE: invention relates to benzamide compounds of formula (IA) where R2, R3 and R5 each is independently selected from hydrogen or metal or their pharmaceutically acceptable salts. Compounds of formula (IA) are inhibitors of histone deacetilase.

EFFECT: elaboration of method of obtaining formula (IA) compounds, pharmaceutical composition, which contains them, and their application for preparation of medication for application as anti-proliferative means for prevention or treatment of tumours or other proliferative conditions, which are sensitive to inhibition of histone deacetilase.

17 cl, 2 tbl, 11 ex

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