Antibody to epha2

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of immunology, medicine and biotechnology. Claimed are versions of anti-EPHA2 antibodies. Claimed antibodies are bound with polypeptide, consisting of amino acids 426-534 in SEQ ID NO:8. Also described are hybridomes, which produce such antibodies, and pharmaceutical compositions and methods of application of said antibodies and compositions.

EFFECT: invention can be used in medicine.

74 cl, 14 dwg, 14 ex, 1 tbl

 

The technical field

The present invention relates to an antibody that has an inhibitory effect against malignant transformation of cells and/or growth of tumor cells. More specifically, the present invention relates to the antibody to EPHA2 and pharmaceutical composition containing the antibody.

Prior art

EPHA2 is a receptor tyrosinekinase c molecular mass of 130 kDa and one transmembrane domain (Molecular and Cellular Biology, 1990, vol. 10, p. 6316-6324). EPHA2 contains legal-binding domain and two fibronectin domain 3 type, located in the N-terminal extracellular region, and tyrosinekinase domain, and the domain of the α-motif sterility (SAM)located in the C-terminal intracellular region.

As EPHA2 ligands known proteins afrina, from Afrin-A1 to Afrin-A5, anchored to the plasma membrane through a GPI (Annual Review of Neuroscience, 1998, vol. 21, p. 309-345). The binding of ligand to EPHA2 leads to activation tyrosinekinase domain and phosphorylation of tyrosine residues present in the intracellular region EPHA2, which provides the transmission signal into the cell. Also described that EPHA2 associated with ligand, internalities into the cell by endocytosis and over time is cleaved by the proteasome (Molecular Cancer Research, 2002, vol. 1, p. 79-87).

It was the op who Sano, that high expression of EPHA2 clinically is manifested in many malignant tumors, in particular breast cancer, malignant tumor of the esophagus, malignant tumor of the prostate, malignant stomach tumor, non-small cell lung cancer, malignant tumor of the large intestine and the multiform glioblastoma (Cancer Research, 2001, vol. 61, p. 2301-2306; International Journal of Cancer, 2003, vol. 103, p. 657-663; The Prostate, 1999, vol. 41, p. 275-280; American Journal of Pathology, 2003, vol. 163, p. 2271-2276; Cancer Science, 2005, vol. 96, p. 42-47; Clinical Cancer Research, 2003, vol. 9, p. 613-618; Oncology Reports, 2004, vol. 11, p. 605-611; and Molecular Cancer Research, 2005, vol. 3, p. 541-551). Also described that: for malignant tumors of the esophagus in patients with expression of EPHA2 there is a tendency to metastasis of tumors with high frequency in the regional lymph nodes, to a large number of metastases in the lymph nodes and to a low degree of differentiation of the tumor, and to the low survival rate for five years (International Journal of Cancer, 2003, vol. 103, p. 657-663); in the case of non-small cell lung cancer patients with high expression of EPHA2 has a tendency to low relapse-free survival and susceptibility to relapse, in particular, metastases to the brain (Clinical Cancer Research, 2003, vol. 9, p. 613-618); and in the case of malignant tumors of the large intestine in patients with expression of EPHA2 there is a tendency to the appearance of METAS the Azov in the liver, the invasion of the tumor in the lymphatic vessels and metastasis in the lymph nodes, and many patients with late clinical stage malignant tumors are positive for the expression of EPHA2 (Oncology Reports, 2004, vol. 11, p. 605-611).

It also describes that with the introduction of EPHA2 gene in cells, non-malignant cells acquire a phenotype of malignant tumors, such as the ability to free growth of cells, the ability to form tubular structures in the matrix and the ability to grow cells in vivo (Cancer Research, 2001, vol. 61, p. 2301-2306), and malignant cells have increased invasiveness in the extracellular matrix (Biochemical and Biophysical Research Communications, 2004, vol. 320, p. 1096-1102; and Oncogene, 2004, vol. 23, p. 1448-1456). It also describes that: the invasiveness or free growth of malignant tumor cells and tumor growth in vivo inhibited by turning off the expression of EPHA2 using miRNAs (Oncogene, 2004, vol. 23, p. 1448-1456; and Cancer Research, 2002, vol. 62, p. 2840-2847); and the invasiveness of free growth and the ability of malignant cells to the formation of tubular structures inhibited by activation of EPHA2 using in the form of a fused protein to its ligand, ephrin-A1 and Fc-region of IgG human and thus causing degradation of EPHA2 by endocytosis (Cancer Research, 2001, vol. 61, p. 2301-2306; Molecular Cancer Research, 2005, vol. 3, p. 541-551; and Biochemical and Biophysical Research Communications, 2004, vol. 320, p. 1096-1102).

On the other hand, is written, that EPHA2 is expressed not only in malignant cells, but also within the tumor or in the surrounding blood vessels (Oncogene, 2000, vol. 19, p. 6043-6052). Described that mice signals EPHA2 involved in the angiogenesis induced by Eminem-A1 and, in particular, EPHA2, are expressed in vascular endothelial cells, required for the formation of vessel or survival of vascular endothelial cells (Journal of Cell Science, 2004, vol. 117, p. 2037-2049). Also described that the fused proteins of the extracellular region EPHA2 and Fc-region of human IgG inhibit angiogenesis in vivo and demonstrate antitumor effect (Oncogene, 2002, vol. 21, p. 7011-7026).

Monoclonal antibodies are suitable not only as a diagnostic medicines, but also as therapeutic drugs. Monoclonal antibodies are actively using, in particular, in the treatment of malignant tumors, and monoclonal antibodies to the receptor tyrosine kinases, such as HER2 and EGFR or extracellular regions CD20, is used in the treatment of malignant tumors and they demonstrate a superior effect (The New England Journal of Medicine, 2007, vol. 357, p. 39-51; Oncogene, 2007, vol. 26, p. 3661-3678; and Oncogene, 2007, vol. 26, p. 3603-3613). The mechanism of action of monoclonal antibodies used in the treatment of malignant tumors, includes the induction of apoptosis and inhibition of growth signals. In addition, it is believed that of all the ü also important is their effect, mediated through the immune response, such as ADCC or CDC. Indeed, it is described that the introduction of antibodies to HER2 (trastuzumab) or antibodies to CD20 (rituximab), they have a more weak antitumor activity in xenografts Nude mice (nude), which are defective in FcγR required for the induction of ADCC compared to bare mice without defect for FcγR (Nature Medicine, 2000, vol. 6, p. 443-446). Also described that antibodies to CD20 (rituximab), while antibodies have more weak antitumor activity in mice with deficiency of complement, which is caused by the introduction of Cobra venom, compared with mice without failure complement (Blood, 2004, vol. 103, p. 2738-2743).

For EPHA2 described that agonistic monoclonal antibody to EPHA2 activity induction of the phosphorylation of tyrosine residues EPHA2 and activity induction of degradation of EPHA2 as ligands inhibit the ability to the free growth of the cell line of breast cancer and the ability of cells to form tubular structures in the extracellular matrix (Cancer Research, 2002, vol. 62, p. 2840-2847). Also described that agonistic monoclonal antibody to EPHA2, which recognize the epitope on EPHA2 detected more frequently in malignant cells than in non-cancerous cells, and have activity induction of the phosphorylation of tyrosine residues EPHA2 and activity induction of degradation of EPHA2, reavley antitumor activity in vivo (Cancer Research, 2003, vol. 63, p. 7907-7912; and the application WO 03/094859). On the other hand, Kiewlich et al. described that their monoclonal antibody to EPHA2 had activity induction of the phosphorylation of tyrosine residues EPHA2 and activity induction of degradation of EPHA2, but had no antitumor activity in vivo (Neoplasia, 2006, vol. 8, p. 18-30).

In addition, in the application WO 2006/084226 described monoclonal antibody to EPHA2 LUCA19, SG5, LUCA40 and SPL1, obtained by immunization of mice with malignant cells and described that among these antibodies: LUCA19 and SG5 not affect the phosphorylation of tyrosine residues EPHA2; LUCA40 inhibits the growth of malignant cells in vitro; and LUCA19, SG5, and LUCA40 internalisers inside of malignant cells in the presence of antibodies to mouse antibodies, labeled toxin (caporino). The document also describes that LUCA40 and SPL1 show antitumor activity in vivo. However, it remains unclear whether these antibodies agonistic activity or not.

Despite these studies, still unknown is the epitope for the antibody to EPHA2, which exhibits antitumor activity in vivo. Has never been described that a certain amino acid sequence in the extracellular region EPHA2 suitable as the epitope for monoclonal antibody that targets for anticancer therapy.

Even antibodies to the same antigen differ in St. the named properties depending on differences in the epitopes or in their sequences. In addition, due to the differences in their properties, antibodies, when administered to humans, will cause different clinical response on the efficacy of drugs, frequency of therapeutic response, side effects, frequency of occurrence of resistance to drug etc.

Thus, the drug with the best clinical properties may also vary depending on the patient. In many cases, such properties are unknown before the actual introduction of the drug. Thus, it is necessary to develop a drug with a new mechanism of action. It is also imperative to develop antibody to EPHA2 with properties differing from the properties of traditional antibodies.

Description of the invention

The tasks to be solved by the invention of

The aim of the present invention to provide an antibody to EPHA2.

An additional objective of the present invention to provide pharmaceutical compositions and the like containing antibody to EPHA2, has a therapeutic effect on malignant tumor.

An additional objective of the present invention is to develop a method of obtaining antibodies.

To anitelea the aim of the present invention is to develop a method of inhibiting tumor growth, using antibodies, etc.

Means of solving problems

The authors of the present invention have conducted the necessary studies to achieve the goals and thus successfully received a new monoclonal antibody to EPHA2, which does not have an activity of inducing the phosphorylation of tyrosine residues EPHA2 and has ADCC activity and CDC activity against malignant cells expressing EPHA2. In addition, the authors of the present invention investigated the epitope for this antibody and the first discovered that the antibody, which binds to the region, including two domain fibronectin type 3 are presented in EPHA2, C-terminal domain, has excellent antitumor activity in vivo. The present invention is made on the basis of these discoveries.

Specifically, the present invention includes:

(1) the antibody recognizes an epitope that is recognized by the antibody produced by hybridomas SH348-1 (FERM BP-10836);

(2) the antibody according to (1), where the antibody has the following properties a)-d):

a) has no ability to phosphorylation of tyrosine residues EPHA2;

b) has ADCC activity against cells expressing EPHA2;

c) has CDC activity against cells expressing EPHA2; and

d) has antitumor activity in vivo;

(3) the antibody according to (1), where EN is Italo has the following properties (a)-(e):

a) has no ability to phosphorylation of tyrosine residues EPHA2;

b) demonstrates the effect of reducing the level of EPHA2 protein;

c) has ADCC activity against cells expressing EPHA2;

d) has CDC activity against cells expressing EPHA2; and

e) has antitumor activity in vivo;

(4) an antibody that is specific binds to the polypeptide consisting of the amino acid sequence corresponding to amino acids No. from 426 to 534 in SEQ ID NO: 8 in the list of sequences;

(5) an antibody that is specific binds to the polypeptide consisting of the amino acid sequence corresponding to amino acids No. from 426 to 534 in SEQ ID NO: 8 in the list of sequences and has the following properties a) to d):

a) has no ability to phosphorylation of tyrosine residues EPHA2;

b) has ADCC activity against cells expressing EPHA2;

c) has CDC activity against cells expressing EPHA2; and

d) has antitumor activity in vivo;

(6) an antibody that specifically binds to a peptide consisting of the amino acid sequence corresponding to amino acids No. from 426 to 534 in SEQ ID NO: 8 in the list of sequences and has the following properties a) to e):

a) has no way the spine to the phosphorylation of tyrosine residues EPHA2;

b) demonstrates the effect of reducing the level of EPHA2 protein;

c) has ADCC activity against cells expressing EPHA2;

d) has CDC activity against cells expressing EPHA2; and

e) has antitumor activity in vivo;

(7) the antibody according to any one of (1)to(6), where the antibody specifically binds to a peptide consisting of the amino acid sequence corresponding to amino acids No. 439 to 534 in SEQ ID NO: 8 in the list of sequences;

(8) the antibody according to any one of (1)to(7), where the antibody inhibits the phosphorylation of tyrosine residues EPHA2-induced EPHA2 ligand;

(9) the antibody according to any one of (1)to(7), where the antibody did not inhibit the binding of EPHA2 ligand with EPHA2, but inhibits the phosphorylation of tyrosine residues EPHA2-induced EPHA2 ligand;

(10) an antibody that is specific binds to the polypeptide consisting of the amino acid sequence corresponding to SEQ ID NO: 8 in the list of sequences, where the antibody has an amino acid sequence corresponding to SEQ ID No: 59, 61 and 63 in the list of sequences or amino acid sequence with deletion, substitution or insertion of one or several amino acids in the amino acid sequences as areas that define the complementarity variable regions of the heavy chain and the antibody is meet the amino acid sequence, corresponding to SEQ ID No: 65, 67 and 69 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequences, as areas that define the complementarity variable region of the light chain;

(11) an antibody that is specific binds to the polypeptide consisting of the amino acid sequence presented in SEQ ID NO: 8 in the list of sequences, characterized by the following 1) and 2):

1) with the peptide of the heavy chain containing the amino acid sequence represented by the General formula (I):

-FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4(I)

where FRH1denotes an arbitrary amino acid sequence containing from 18 to 30 amino acids; CDRH1indicates the amino acid sequence corresponding to SEQ ID NO: 59 in the list of sequences or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; FRH2denotes an arbitrary amino acid sequence consisting of 14 amino acids; CDRH2indicates the amino acid sequence corresponding to SEQ ID NO: 61 in the list of sequences or amino acid is a selected from a deletion, substitution or insertion of one or several amino acids in amino acid sequence; FRH3denotes an arbitrary amino acid sequence consisting of 32 amino acids; CDRH3indicates the amino acid sequence corresponding to SEQ ID NO: 63 in the list of sequences or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; and FRH4denotes an arbitrary amino acid sequence consisting of 11 amino acids, where these amino acids linked together by peptide bonds; and

2) having a light chain polypeptide containing the amino acid sequence represented by the General formula (II):

-FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4-(II)

where FRL1denotes an arbitrary amino acid sequence consisting of 23 amino acids; CDRL1indicates the amino acid sequence corresponding to SEQ ID NO: 65 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; FRL2denotes an arbitrary amino acid sequence consisting of 15 amino the slot; CDRL2indicates the amino acid sequence corresponding to SEQ ID NO: 67 in the list of sequences; FRL3denotes an arbitrary amino acid sequence consisting of 32 amino acids; CDRL3indicates the amino acid sequence corresponding to SEQ ID NO: 69 in the list of sequences or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; and FRL4denotes an arbitrary amino acid sequence consisting of 10 amino acids, where these amino acids linked together by peptide bonds;

(12) an antibody that recognizes the epitope recognized by the antibody produced by hybridomas SH357-1 (FERM BP-code 10837);

(13) the antibody according to (12), where the antibody has the following properties a) to d):

a) has no ability to phosphorylation of tyrosine residues EPHA2;

b) has ADCC activity against cells expressing EPHA2;

c) has CDC activity against cells expressing EPHA2; and

d) has antitumor activity in vivo;

(14) an antibody that specifically binds to a peptide consisting of the amino acid sequence corresponding to amino acids No. 426 to 534 in SEQID NO: 8 in the list of sequences and with the following properties, from a) to e):

a) has no ability to phosphorylation of tyrosine residues EPHA2;

b) demonstrates the effect of reducing the level of EPHA2 protein;

c) has ADCC activity against cells expressing EPHA2;

d) has CDC activity against cells expressing EPHA2; and

e) has antitumor activity in vivo;

(15) the antibody according to any one of (12)-(14), where the antibody specifically binds to a peptide consisting of the amino acid sequence corresponding to amino acids No. from 439 to 534 in SEQ ID NO: 8 in the list of sequences;

(16) the antibody according to any one of (12)-(15), where the antibody inhibits the phosphorylation of tyrosine residues EPHA2-induced EPHA2 ligand;

(17) the antibody according to any one of (12)-(15), where the antibody did not inhibit the binding of EPHA2 ligand with EPHA2, but inhibits the phosphorylation of tyrosine residues EPHA2-induced EPHA2 ligand;

(18) an antibody that specifically binds with a polypeptide consisting of the amino acid sequence represented in SEQ ID NO: 8 in the list of sequences, where the antibody has an amino acid sequence corresponding to SEQ ID No: 71, 73 and 75 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one or several amino acids in the amino acid posledovatelno is s, in the form of sections, determining the complementarity variable regions of the heavy chain and where the antibody has an amino acid sequence corresponding to SEQ ID No: 77, 79 and 81 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequences, in the form of sections, determining the complementarity variable region of the light chain;

(19) an antibody that is specific binds to the polypeptide consisting of the amino acid sequence corresponding to SEQ ID NO: 8 in the list of sequences, characterized by the following 1) and 2):

1) with the peptide of the heavy chain containing the amino acid sequence represented by the General formula (I):

-FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4(I)

where FRH1denotes an arbitrary amino acid sequence containing from 18 to 30 amino acids; CDRH1indicates the amino acid sequence corresponding to SEQ ID NO: 71 in the list of sequences or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; FRH2denotes an arbitrary amino acid sequence consisting of 14 aminoxy the lot; CDRH2indicates the amino acid sequence presented in SEQ ID NO: 73 in the list of sequences or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; FRH3denotes an arbitrary amino acid sequence consisting of 32 amino acids; CDRH3indicates the amino acid sequence presented in SEQ ID NO: 75 in the list of sequences or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; and FRH4denotes an arbitrary amino acid sequence consisting of 11 amino acids, where these amino acids linked together by peptide bonds; and

2) having a light chain polypeptide containing the amino acid sequence represented by the General formula (II):

-FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4-(II)

where FRL1denotes an arbitrary amino acid sequence consisting of 23 amino acids; CDRL1indicates the amino acid sequence corresponding to SEQ ID NO: 77 in the list of sequences or amino acid sequence with deletion, substitution or getting the oops of one or several amino acids in amino acid sequence; FRL2denotes an arbitrary amino acid sequence consisting of 15 amino acids; CDRL2indicates the amino acid sequence presented in SEQ ID NO: 79 in the list of sequences or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; FRL3denotes an arbitrary amino acid sequence consisting of 32 amino acids; CDRL3indicates the amino acid sequence presented in SEQ ID NO: 81 in the list of sequences or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; and FRL4denotes an arbitrary amino acid sequence consisting of 10 amino acids, where these amino acids linked together by peptide bonds;

(20) the antibody according to any one of (1)to(19), characterized in that the antibody is humanitariannet antibody;

(21) the antibody according to any one of (1)to(19), characterized in that the antibody is a human antibody;

(22) the antibody according to any one of (1)to(19), characterized in that the antibody is an IgG antibody;

(23) the antibody according to any one of (1)to(9) and (12)-(17), characterized by those who, what antibody is any selected from a Fab, F(ab')2, Fv, scFv, diately, linear antibodies and polyspecific antibodies;

(24) an antibody produced by hybridomas SH348-1 (FERM BP-10836);

(25) the antibody produced by hybridomas SH357-1 (FERM BP-code 10837);

(26) an antibody containing the following 1) and 2):

1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. from 1 to 119 in SEQ ID NO: 35 in the list of sequences or polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. from 1 to 119 in SEQ ID NO: 39 in the list of sequences; and

2) a light chain polypeptide containing an amino acid sequence corresponding to amino acids No. from 1 to 112 in SEQ ID NO: 37 in the list of sequences or polypeptide light chain containing the amino acid sequence corresponding to amino acids No. from 1 to 112 in SEQ ID NO: 41 in the list of sequences;

(27) an antibody containing the following 1) or 2):

1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. from 1 to 119 in SEQ ID NO: 35 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. from 1 to 112 in SEQ ID NO: 37 in the list of sequences; and

2) polypath is on the heavy chain, containing the amino acid sequence corresponding to amino acids No. from 1 to 119 in SEQ ID NO: 39 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. from 1 to 112 in SEQ ID NO: 41 in the list of sequences;

(28) an antibody containing the following 1) and 2):

1) polypeptide heavy chain containing the amino acid sequence corresponding to SEQ ID NO: 35 in the list of sequences or polypeptide heavy chain containing the amino acid sequence corresponding to SEQ ID NO: 39 in the list of sequences; and

2) a light chain polypeptide containing the amino acid sequence corresponding to SEQ ID NO: 37 in the list of sequences or polypeptide light chain containing the amino acid sequence corresponding to SEQ ID NO: 41 in the list of sequences;

(29) an antibody containing the following 1) or 2):

1) polypeptide heavy chain containing the amino acid sequence corresponding to SEQ ID NO: 35 in the list of sequences and a light chain polypeptide containing the amino acid sequence presented in SEQ ID NO: 37 in the list of sequences; and

2) polypeptide heavy chain containing the amino acid sequence corresponding to SEQ ID NO: 39 in the list of sequences and polypeptide light chain, containing the amino acid sequence corresponding to SEQ ID NO: 41 in the list of sequences;

(30) an antibody obtained by the humanization of the antibody according to any one of(24)-(29);

(31) an antibody containing the following 1) and 2):

1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. from 20 to 468 in SEQ ID NO: 107 in the list of sequences or polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. from 20 to 468 in SEQ ID NO: 115 in the list of sequences; and

2) a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. from 21 to 239 in SEQ ID NO: 91 in the list of sequences or polypeptide light chain containing the amino acid sequence corresponding to amino acids No. from 21 to 239 in SEQ ID NO: 99 in the list of sequences;

(32) an antibody containing the following 1) or 2):

1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. from 20 to 468 in SEQ ID NO: 107 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. from 21 to 239 in SEQ ID NO: 91 in the list of sequences; and

2) heavy polypeptide chain containing amino acid in sledovatelnot, corresponding to amino acids No. from 20 to 468 in SEQ ID NO: 115 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. from 21 to 239 in SEQ ID NO: 99 in the list of sequences;

(33) an antibody containing the following 1) and 2):

1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. from 20 to 468 in SEQ ID NO: 139 in the list of sequences or polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. from 20 to 468 in SEQ ID NO: 147 in the list of sequences; and

2) a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. from 21 to 239 in SEQ ID NO: 123 in the list of sequences or polypeptide light chain containing the amino acid sequence corresponding to amino acids No. from 21 to 239 in SEQ ID NO: 131 in the list of sequences;

(34) the antibody containing the following 1) or 2):

1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. from 20 to 468 in SEQ ID NO: 139 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. from 21 to 239 in SEQ ID NO: 123 in the list of sequences; and

2) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. from 20 to 468 in SEQ ID NO: 147 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. from 21 to 239 in SEQ ID NO: 131 in the list of sequences;

(35) A Fab, F(ab')2, Fv, scFv, ditelo, linear antibody or polyspecific antibody derived from the antibody according to any one of(24)-(34);

(36) any one polypeptide selected from the group consisting of the following 1)-20):

1) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 1 to 119 in SEQ ID NO: 35 in the list of sequences;

2) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 1 to 119 in SEQ ID NO: 39 in the list of sequences;

3) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 1 to 112 in SEQ ID NO: 37 in the list of sequences;

4) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 1 to 112 in SEQ ID NO: 41 in the list of sequences;

5) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 20 to 468 in SEQ ID NO: 107 in the list of sequences;

6) a polypeptide containing linakis is now the sequence corresponding to amino acids No. from 20 to 468 in SEQ ID NO: 115 in the list of sequences;

7) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 20 to 138 in SEQ ID NO: 107 in the list of sequences;

8) the polypeptide containing the amino acid sequence corresponding to amino acids No. from 20 to 138 in SEQ ID NO: 115 in the list of sequences;

9) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 21 to 239 in SEQ ID NO: 91 in the list of sequences;

10) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 21 to 239 in SEQ ID NO: 99 in the list of sequences;

11) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 21 to 134 in SEQ ID NO: 91 in the list of sequences;

12) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 21 to 134 in SEQ ID NO: 99 in the list of sequences;

13) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 20 to 468 in SEQ ID NO: 139 in the list of sequences;

14) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 20 to 468 in SEQ ID NO: 147 in the list of sequences;

15) polypep the ID, containing the amino acid sequence corresponding to amino acids No. from 20 to 138 in SEQ ID NO: 139 in the list of sequences;

16) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 20 to 138 in SEQ ID NO: 147 in the list of sequences;

17) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 21 to 239 in SEQ ID NO: 123 in the list of sequences;

18) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 21 to 239 in SEQ ID NO: 131 in the list of sequences;

19) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 21 to 134 in SEQ ID NO: 123 in the list of sequences; and

20) a polypeptide containing the amino acid sequence corresponding to amino acids No. from 21 to 134 in SEQ ID NO: 131 in the list of sequences;

(37) mouse hybridoma SH348-1 (FERM BP-10836);

(38) the mouse hybridoma SH357-1 (FERM BP-code 10837);

(39) a Pharmaceutical composition, characterized in that it contains at least one antibody selected from the antibodies by(1)-(35);

(40) a Pharmaceutical composition for the treatment of malignant tumors, characterized in that it contains at least one antibody selected from the antibodies by(1)-(35);

(41) the Method of Engibarov the tion of tumor growth in a mammal, where the method includes the introduction of any of the antibodies selected from the group consisting of antibodies(1)-(35), (39) and (40);

(42) a Method of inhibiting tumor growth by (41), characterized in that the tumor is a tumor expressing EPHA2;

(43) Polynucleotide that encodes the antibody or the polypeptide according to any one of(1)-(36);

(44) a host Cell transformed by polynucleotides (43); and

(45) a Method of obtaining antibodies using a host cell according to (44).

Advantages of the invention

In the present invention successfully obtained a new monoclonal antibody to EPHA2, which has no activity induction of the phosphorylation of tyrosine residues EPHA2 and has ADCC activity and CDC activity against malignant cells expressing EPHA2. In addition, we discovered that the antibody has excellent antitumor activity in vivo.

In addition, a pharmaceutical composition for the treatment of malignant tumors containing this antibody.

Brief description of drawings

Figure 1 is a drawing showing the results of a Western blot demonstrating the presence or absence of activity induction by antibody to EPHA2 phosphorylation of tyrosine residues EPHA2. Figa) is a figure showing the results obtained in the lack of the s cross-linking antibodies, where the upper part shows the results for antibody 4G10, and the lower part shows the results for antibody to EPHA2 (D7). On figv) presents a figure showing the results obtained in the presence of cross-linking antibodies, where in the upper part presents the results for antibody 4G10, and the lower part shows the results for antibody to EPHA2 (D7);

Figure 2 presents a picture showing the results of a Western blot showing the presence or absence of activity induction reduce EPHA2 protein by antibody to EPHA2. On figa) presents a figure showing the results obtained in the absence of cross-linking antibodies, where the upper part shows the results for antibody to EPHA2 (D7), and the lower part shows the results for antibody to β-actin. On figv) presents a figure showing the results obtained in the presence of cross-linking antibodies, where in the upper part presents the results for antibody to EPHA2 (D7), and the lower part shows the results for antibody to β-actin;

Figure 3 presents graphs showing the presence or absence of antibody to EPHA2 ADCC activity against different cell lines. On the chart "**" indicates significance P<0,01 and***represents significance at P<0,001. On figa) presents a graph on which the azan ADCC activity relative to cells MDA-MB-231. On figv) presents a graph showing the ADCC activity against A549 cells. On figs) presents a graph showing the ADCC activity relative to cells PC-3;

Figure 4 presents graphs showing the presence or absence of CDC activity of the antibody to EPHA2 in relation to different cells. On the chart "***" indicates significance P<0,001. On figa) presents a graph showing the activity of CDC SH348-1 compared to cells MDA-MB-231. On figv) presents a graph showing the activity of CDC SH348-1 against A549 cells. On figs) presents a graph showing the activity of CDC SH348-1 compared to cells PC-3. On fig.4D) presents a graph showing the activity of CDC SH357-1 compared to cells MDA-MB-231. On file) presents a graph showing the activity of CDC SH357-1 against A549 cells. On fig.4F) presents a graph showing the activity of CDC SH357-1 compared to cells PC-3;

On figa) presents the figure, which shows the calculation domain structure EPHA2 and regulations EPHA2-ECD, FNIII-NC, FNIII-N and FNIII-C EPHA2, which are peptides to determine the epitope. Figure ligand-BD denotes legal-binding domain, FN3 refers to the domain of fibronectin type 3, TM denotes the transmembrane region, Trk kinase indicates tyrosinekinase SAM domain and denotes SAM to the EN;

On figv) presents a graph showing the presence or absence of the binding activity of the antibody to EPHA2 with EPHA2-ECD, FNIII-NC, FNIII-N and FNIII-C;

On figa) presents a graph showing the antitumor activity of SH348-1 compared to cells MDA-MB-231, transplanted mice;

On figv) presents a graph showing the antitumor activity of SH357-1 compared to cells MDA-MB-231, transplanted mice. The graph trims errors indicate standard error (n=9);

On figa) presents a graph showing binding activity of SH348-1 polypeptide extracellular region EPHA2;

On figv) presents a graph showing binding activity of SH357-1 polypeptide extracellular region EPHA2;

Figs) is a chart showing binding activity of Ab96-1 polypeptide extracellular region EPHA2;

On Fig presents a graph showing the ligand-binding inhibitory activity of SH348-1 AND SH357-1 and Ab96-1;

Figure 9 presents a graph showing that the SH348-1 and SH357-1 have activity of inhibiting the phosphorylation of tyrosine residues EPHA2-dependent afrina-A1;

Figure 10 presents a graph which shows the diagram of the deletion mutants to identify EPHA2 epitope;

On figa) presents the figure, a description of the m shows the ability of SH348-1 to interact with deletion mutants EPHA2;

On figv) presents a picture showing the detection of deletion mutant EPHA2 in figa) on PVDF membrane;

On figs) presents the figure, which shows the ability of SH357-1 to interact with deletion mutants EPHA2;

On fig.11D) presents a picture showing the detection of deletion mutant EPHA2 in figs) on PVDF membrane;

On figa) presents a graph showing binding activity of hSH348-1-T1 polypeptide extracellular region EPHA2;;

On figv) presents a graph showing binding activity of hSH348-1-T3 polypeptide extracellular region EPHA2;;

On figs) presents a graph showing binding activity of hSH357-1-T1 polypeptide extracellular region EPHA2;

On fig.12D) presents a graph showing binding activity of hSH357-1-T3 polypeptide extracellular region EPHA2;

On figa) presents a graph showing the ability of competitive inhibition hSH348-1-T1 and hSH348-1-T3 with respect to antigen binding SH348-1;

On figv) presents a graph showing the competitive inhibitory activity hSH357-1-T1 and hSH357-1-T3 with respect to antigen binding SH357-1;

On figa) presents a graph showing the activity-dependent inhibition afrina-A1 phosphorylation of tyrosine residues EPHA2 what exploits hSH348-1-T1 or hSH348-1-T3; and

On figv) presents a graph showing the activity-dependent inhibition afrina-A1 phosphorylation of tyrosine residues EPHA2 through hSH357-1-T1 or hSH357-1-T3.

The best option of carrying out the invention

1. Definition

In the present description, the terms "cancer" and "tumor" are used in the same value.

In the present description, the term "gene" includes not only DNA, but also its mRNA, cDNA and its crnc. Thus, the term "EPHA2 gene" in the present invention refers to DNA, mRNA, cDNA and crnc EPHA2.

In the present description, the term "polynucleotide" used in the same meaning as nucleic acid, and it also includes DNA, RNA, probes, oligonucleotides and primers.

In the present description, the terms "polypeptide" and "protein" are used without distinction between them.

In the present description, the term "cell" includes cells of the specimen and cultured cells.

In the present description, the term "malignant transformation of cells" means cells exhibit abnormal growth, for example, lose their sensitivity to contact inhibition or show the free growth of cells. Cells showing such abnormal growth is designated as "malignant cells".

In the present description the protein having the equivalent functions on the reference to the activity of malignant transformation and/or activity of cell growth or similar EPHA2, also known as EPHA2.

In the present description, the term "phosphorylation of tyrosine residues" means that the tyrosine residues contained in the amino acid sequence of a peptide phosphorylate. Whether residues of tyrosine phosphorylated or not, you can determine, for example, based on the affinity of the peptide for the antibody to phosphotyrosine (for example, Anti-Phosphotyrosine, recombinant HRP conjugate 4G10 (Millipore (Upstate), #16-184)). The tyrosine residues can be defined as phosphorylated, if the peptide binds to the antibody.

In the present description, the term "ability to phosphorylation of tyrosine residues EPHA2" refers to the ability to phosphorylation of tyrosine residues in the amino acid sequence of EPHA2. Has the antibody or no ability to induce phosphorylation of tyrosine residues EPHA2 can be defined, for example, by incubating antibodies and EPHA2 and further research the presence or absence of affinity to EPHA2 antibody to phosphotyrosine.

In the present description, the expression "reduced level of expression of EPHA2 protein" means that the EPHA2 protein level is reduced. Possesses or no antibody effect of reducing levels of EPHA2 protein can be determined, for example, by incubating antibodies and EPHA2 and subsequent determination of the level of EPHA2.

In the present description, the term EPHA2 ligand" refers to a substance, which is able to perform the function of EPHA2 ligand. Specific examples thereof may include proteins afrina, from Afrin-A1 to Afrin-A5, anchored to the plasma membrane through a GPI (Annual Review of Neuroscience, 1998, vol. 21, p. 309-345).

In the present description, the term "cytotoxicity" refers to any pathological changes in cells and determines not only direct damage to cells, but also any structural or functional damage in cells, such as DNA cleavage, dimerization bases, chromosome break, damage to the mitotic apparatus and the reduction of various enzymatic functions.

In the present description, the term "cytotoxic activity" refers to activity that causes cytotoxicity.

In the present description ADCC is synonymous with antibody-dependent cellular cytotoxicity and refers to reactions in which the cells bearing the receptor Fcγ, joined by Fcγ receptors for the Fc regions of antibodies associated with surface antigens on target cells and destroy target cells. The ADCC activity also indicate antibody-dependent cytotoxic activity and belongs to the activity that causes the reaction. The ADCC activity can be measured by methods commonly used by experts in this field and can be measured, for example, according to the method, isonomy in the present description in paragraphs (3)-2 example 3.

In the present description, the term "CDC" is synonymous with complementability cytotoxicity. The CDC activity refers to activity that causes complementability cytotoxicity. The CDC activity can be measured by methods commonly used by experts in this field and can be measured, for example, according to the method described in the present description in paragraphs (3)-3 example 3.

In the present description, the expression "has antitumor activity in vivo" refers to the activity of inhibiting or reducing tumor growth in animals animals-media tumors. Whether antibody to EPHA2 has antitumor activity in vivo" or not can be determined by methods commonly used by experts in this field, and you can also define, for example, in the following way: the appropriate dose of the antibody to EPHA2 administered intraperitoneally administered to mice in the tumor cells (e.g. cells MDA-MB-231, transplanted subcutaneously naked mice) (e.g., BALB/cAJc1-nu/nu; received in CLEA Japan, Inc.) as a test substance, and depending on time, comparing changes in the size of tumors in Nude mice and control mice that were not injected antibody to EPHA2. Antibody to EPHA2, as a test substance can be defined as "having antitumor activity in vivo", when tumor volume at e is sperimentali mice is significantly less compared to a control.

As is known, each of the heavy and light chains of antibody molecules has three complementarity determining region (CDR). In the present description, region, defining a complementary antibodies, represented by areas CDRH1, CDRH2and CDRH3for areas that define the complementary heavy chain and CDRL1, CDRL2and CDRL3for areas that define the complementary light chain.

In the present description, the term "epitope" refers to an incomplete EPHA2 peptide having antigenic and/or immunogenic properties in vivo in animals, preferably mammals, more preferably in mice or humans. The epitope in the form of incomplete EPHA2 peptide possessing antigenic properties, can be determined by methods well known to specialists in this field, such as immunological analysis and can be defined, for example, in accordance with the following method, which receive various incomplete sequence EPHA2. To obtain partial sequences, we can use the methods of synthesis of oligopeptides, known in this area. For example, a number of successively shortened from the C - or N-Terminus of the polypeptide EPHA2 get the proper length, using the methods of recombination of genes is well known to specialists in this field. Then study the ability of the antibodies in topath in response to these polypeptides. Approximately determine the area of recognition and then synthesized short peptides. You can explore the reactivity of these peptides, thereby defining the epitope.

1. Description EPHA2

(1) Gene EPHA2

The nucleotide sequence of an EPHA2 gene and its amino acid sequence registered in GenBank as EPH receptor A2 (registration number No: NM_004431 and NP_004422, respectively). In addition, the nucleotide sequence of an open reading frame (ORF) in the EPHA2 gene described in SEQ ID NO: 1 in the list of sequences. Amino acid ORF sequence described in SEQ ID NO: 2 in the list of sequences.

In this regard, EPHA2 also includes proteins that consist of an amino acid sequence obtained by replacement, deletion or insertion of one or several amino acids in the amino acid sequence of EPHA2, and which possess biological activity equivalent to the enzyme.

(2) Site-specific expression of EPHA2 gene in malignant tumors.

Described that the EPHA2 gene is highly expressed in many malignant tumors, in particular breast cancer, malignant tumors of the esophagus, prostate cancer, malignant tumors of the stomach, non-small cell lung cancer, cancer of the colon and the multiform glioblastoma.

The company is and, you can measure the level of EPHA2 expression in each cell and/or each tissue, thereby determining the degree of malignant transformation and/or growth of malignant cells, which may be due to the overexpression of EPHA2 in the tested subjects.

In addition, the tool that inhibits the expression and/or activity of EPHA2 has activity of inhibiting malignant transformation of cells and/or growth of malignant cells associated with EPHA2.

Thus, the test substance are contacted with cells expressing EPHA2 and a tool that inhibits the expression and/or activity of EPHA2 can thus be selected for screening antitumor agent.

In this regard, miRNAs against EPHA2 inhibits the expression of EPHA2 and can be used as antitumor agents. miRNAs against EPHA2 can be obtained: construction on the basis of the nucleotide sequence of EPHA2 mRNA, RNA, containing a partial sequence of EPHA2 mRNA (sense RNA) and RNA containing a nucleotide sequence complementary to the nucleotide sequence of the RNA (antisense RNA); RNA synthesis by the method of chemical synthesis, essentially known in the field; and hybridization both derived RNA. Preferably, the sequence of one or more nucleotides, called the " sticky consistency should contact the 3'-end of each sense or antisense RNA, representing miRNAs. The length of the masking sequence, in particular, limit, because it protects the RNA from nucleases. You can use any sequence, preferably from 1 to 10 nucleotides, more preferably from 1 to 4 nucleotides, even more preferably 2 nucleotides.

2. Antibody to EPHA2

(1) Obtaining antigen

Examples of antigens to generate antibodies to EPHA2 the present invention may include a full-sized polypeptide EPHA2 and its incomplete polypeptides and, more specifically, may include a full-sized polypeptide EPHA2 and preferably, the extracellular region of the polypeptide EPHA2 (consisting of amino acid sequence corresponding to amino acids No. 1-534 in SEQ ID NO: 8 in the list of sequences), more preferably, a partial polypeptide extracellular region of EPHA2 polypeptide containing the amino acid sequence corresponding to amino acids No. 426-534 in SEQ ID NO: 8 in the list of sequences, even more preferably a partial polypeptide extracellular region of EPHA2 polypeptide containing the amino acid sequence corresponding to amino acids No. 439-534 in SEQ ID NO: 8 in the list of sequences, and derivatives obtained by adding an arbitrary amino acid p is coherence or media for these sequences. Additional examples thereof may include polypeptides consisting of the successive partial amino acid sequence of at least 6 amino acids and derivatives obtained by adding an arbitrary amino acid sequence or media to these sequences.

Thus, a full-size EPHA2 polypeptide or its partial polypeptide used as an antigen can be obtained, providing the expression of the EPHA2 gene or genes incomplete polypeptides in the cells of the host by means of genetic engineering.

EPHA2 to apply can be cleared directly from tumor tissues or tumor cells. In addition, a full-sized polypeptide EPHA2 or incomplete polypeptides can be synthesized in vitro or receive, ensuring the production of polypeptides in the cells of the host by means of genetic engineering.

In genetic engineering, genes encoding EPHA2 or incomplete polypeptides include, in particular, the vectors that can Express EPHA2 or incomplete polypeptides, and EPHA2 or incomplete polypeptides can then be synthesized in a solution containing enzymes, substrates and energy substances necessary for transcription and translation. The alternative, however, to obtain the desired protein cells are the owners of other prokaryotes or eukaryotes which you can transform and to ensure the expression of EPHA2 or incomplete polypeptides.

cDNA incomplete EPHA2 polypeptides can be obtained, for example, the so-called polymerase chain reaction (hereinafter referred to as "PCR"), a method in which PCR is performed using as matrices cDNA library expressing EPHA2 and specific primers for the amplification EPHA2 cDNA or DNA that encodes a partial polypeptide (see Saiki, R.K., et al. Science (1988) 239, p. 487-489).

Examples of polypeptide synthesis in vitro include as non-limiting examples of Rapid Translation System (RTS)produced by Roche Diagnotics Corp.

Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis. For transformation of these cells in host genes of interest, cell host transformed plasmid vectors containing replicon, i.e. plot the beginning of replication and a regulatory sequence derived from species compatible with the host. In addition, preferably, the vectors must have the sequence, which can provide a phenotypic trait (phenotype), selective for transformed cells.

Eukaryotic cell hosts include cells of vertebrates, insects, yeast, etc. Such as vertebrate cells often use cell lines COS monkey (Gluzman, Y., Cell (1981) 23, p. 175-182, ATCC CRL-1650), mouse fibroblasts NIH3T3 (ATCC No. CRL-1658) and cell line Chinese hamster ovary defective in digit politicalize (CHO cells, ATCC CCL-61) (Urlaub, G. and Chasin, L.A., Proc. Natl. Acad. Sci. USA (1980) 77, p. 4126-4220), although cells of vertebrates are not limited to.

Thus obtained transformants can be grown by standard methods, and in the culture they are able to intracellular or extracellular production of the polypeptide of interest.

The medium used for cultivation, you can choose accordingly depending on the chosen host cells from a variety of commonly used media. For Escherichia coli, for example, LB medium, optionally supplemented with antibiotic (eg, ampicillin) or you can use IPTG.

Recombinant protein, intracellular or extracellular produced by the transformants in a culture, you can select and clear the various ways of allocating, using the physical properties of the protein, chemical properties of a protein or the like

As an example one can cite, in particular, ways of handling traditional means to initiate the precipitation, ultrafiltration, various methods of liquid chromatography, such as chromatography on a molecular sieve (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography and high performance liquid chromatography (HPLC), dialysis, and combinations thereof.

In addition, the recombinant protein to be expressed, can be associated with residues of histidine, for effective cleaning by means of this interesting protein on the column for affinity chromatography with Nickel.

By combining these methods, the desired polypeptide can be obtained easily in large quantities, with high yield and high purity.

The antibody of the present invention can be obtained by immunization of animals with the antigen in accordance with the standard method, and receiving antibodies produced in vivo, with subsequent cleaning.

In addition, producing antibodies, the cells that produce antibody to EPHA2, subjected to fusion with myeloma cells according to a method known in this field (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497; and Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, N.Y. (1980)), thereby creating hybridoma, of which it is also possible to obtain monoclonal antibodies.

(2) Obtaining a monoclonal antibody to EPHA2

Examples of antibodies that are specific contact EPHA2 may include monoclonal antibodies that are specific contact EPHA2. A method of producing antibodies are described below.

To obtain monoclonal antibodies necessary to carry out the following process:

(a) the stage of purification of biopolymers used as antigens

(b) stage of immunization of animals with antigens by injection, then the selection of blood in animals, the study of the titer of anti-Christ. ate to determine the time of splenectomy and then getting producing antibodies cells

(c) the stage of obtaining myeloma cells (hereinafter referred to as "myeloma"),

(d) stage of implementation of cell fusion between producing antibodies cells and myeloma,

(e) stage select hybridoma group, which produces the desired antibody

(f) separation stage to separate cell clones (cloning),

(g) stage culturing hybridomas to obtain large quantities of monoclonal antibodies or raising animals transplanted with hybridomas,

(h) stage study of the biological activity and binding specificity of monoclonal antibodies thus obtained or the study of the properties as reagents for labeling, etc.

Further detail, in accordance with these stages will be described a method of obtaining monoclonal antibodies, although antibodies are not restricted in this way. For example, in addition to spleen cells and myeloma, you can use other producing antibodies cells.

(a) Purification of antigens

EPHA2 or incomplete polypeptides obtained as described above can be used as antigens.

In addition, you can also use as antigens partial peptides of the proteins of the present invention, which are synthesized chemically, using fractions of plasma membranes obtained from recombin ntih somatic cells, expressing EPHA2 or recombinant somatic cells expressing EPHA2, in accordance with the method, well known to specialists in this field.

(b) Obtaining producing antibodies cells

The antigens obtained in stage (a) is mixed with adjuvants well known to specialists in this field, for example, complete or incomplete adjuvant-blockers or other auxiliary means, such as potassium aluminum sulfate, and experimental animals subjected to immunization these immunogenum. The animals used in obtaining hybridoma ways that are known in this field can be used as experimental animals. In particular, for example, you can use mice, rats, goats, sheep, cows and horses. However, as animals, subject to immunization, use preferably mice or rats, from the viewpoint of easy availability of myeloma cells to be merged in, with a dedicated producing antibodies cells, etc.

In addition, the actually used line mice and rats are not specifically limited. For example, you can use mice such lines as A, AKR, BALB/c, BDP, BA, CE, C3H, 57BL, C57BR, C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB and 129 and rats such lines as Low, Lewis, Sprague-Dawley, ACI, BN and Fischer.

These mice and rats can be obtained, for example, from institutions in growing/on TASCAM experimental animals such as CLEA Japan, Inc. and Charles River Laboratories Japan, Inc.

Among these lines, particularly preferred as animals, subject to immunization are mouse strain BALB/c mice and rats line Low for compatibility reasons fusion with myeloma cells, as described below.

In addition, it is also preferable to use mice with disturbed biological mechanism of removal of autoantibodies, i.e. mice with autoimmune disease, given antigenic homology between humans and mice.

Preferably, in the period of immunization age these mice or rats ranged from 5 to 12 weeks, more preferably from 6 to 8 weeks.

For immunization of animals EPHA2 or recombinant antigens can be used the methods known in this field, described in detail in, for example, Weir, D.M., Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987), and Kabat, E.A. and Mayer, M.M., Experimental Immunochemistry, Charles C Thomas Publisher, Springfield, Illinois (1964).

Of these methods, immunization, specifically illustrated preferred in the present invention method, as described below.

Specifically, animals initially intradermally or administered intraperitoneally injected fractions of membrane proteins, which are used as antigens or cells that Express the antigen.

However, preferred is the combined use of both sponsorowane, to increase the efficiency of immunization. Efficacy can be increased, in particular through intradermal injection in the first immunization and intraperitoneal later immunizations or only in the last immunization.

The scheme is the introduction of antigens varies, depending on species, subject to immunization, their individual differences, etc. typically, the antigen is administered preferably from calculation 3-6 doses in the range of from 2 to 6 weeks, more preferably estimated 3-4 doses in the range of from 2 to 4 weeks.

In addition, doses of antigens vary depending on animal species, subject to immunization, their individual differences, etc. and, as a rule, the value of the order of 0.05-5 mg, preferably 0.1 to 0.5 mg.

Repeated immunization spend 1-6 weeks later, preferably 2-4 weeks later, more preferably 2-3 weeks later, from the above introduction of the antigen.

In this regard, the dose of antigens with repeated immunization vary depending on species, size, etc. and, as a rule, the value of the order of 0.05-5 mg, preferably 0.1-0.5 mg, more preferably 0.1-0.2 mg, for example, for mice.

1-10 days later, preferably 2-5 days later, more preferably 2-3 days later from re-immunization cells is lesenki or lymphocytes, contains producing antibodies cells in sterile conditions extracted from animals subject to immunization.

In this way measure the antibody titer, and animals that have significantly increased the antibody titer can be used as sources producing antibodies cells, thereby increasing the efficiency of the subsequent methods.

Examples of ways to measure the titer of antibodies, which are used in this document may include, but are not limited to, RIA and ELISA.

Measurement of the antibody titer of the present invention can be accomplished by methods as described, for example, in accordance with ELISA.

First, purified or partially purified antigens absorb on the surface of a solid phase, such as 96-well plates to ELISA. In addition, on the surface of the solid phase absorbed without antigen causing proteins that are not antigens, for example, bovine serum albumin (hereinafter referred to as "BSA"). The surface is washed and then subjected to interaction with serially diluted samples (e.g., mouse serum as the primary antibody, thus, antibodies in the sample bind to the antigens.

In addition, as secondary antibodies labeled add enzyme antibodies to murine antibodies, so that the secondary antibody binds to murine what ntially. After washing, they added substrates for the enzyme and, for example, measure the change in absorption caused by the development of painting, based on degradation of the substrate, counting, thus, the titer of antibodies.

Producing antibodies, the cells can be separated from those of the splenic cells or lymphocytes according to the methods known in this field (e.g., Kohler et al., Nature (1975) 256, p. 495; Kohler et al., Eur. J. Immunol. (1977) 6, p. 511; Milstein et al., Nature (1977), 266, p. 550; and Walsh, Nature, (1977) 266, p. 495).

For example, spleen cells, you can select a generic way, which includes the division of the sample of cells on a flat fragments, filtering them through a stainless steel sieve and then, based on this, the division producing antibodies cells by immersing in minimal supportive environments Needle (MEM)

(C) Obtaining myeloma cells (hereinafter referred to as "myeloma")

The myeloma cells used for cell fusion, are not specifically limited, and may be selected for use appropriately from cell lines known in this field. However, it is preferable to use lines defective in HGPRT (hypoxanthineguanine), for which efficient methods of selection, based facilities hybridoma selection of fused cells.

Their specific examples include: obtained from mice X63-Ag8 (X63), NS1-ANS/1 (NS1) P3X63-Ag8.U1 (P3U1), X63-Ag8.653 (X63.653), SP2/0-Ag14 (SP2/0), MPC11-45.6TG1.7 (45.6TG), FO, S149/5XXO and BU.1; received in rats 210.RSY3.Ag.1,2.3 (Y3); and obtained from the person U266AR (SKO-007), GM1500.GTG-A12 (GM1500), UC729-6, LICR-LON-HMy2 (HMy2) and 8226AR/NIP4-1 (NP41).

Such lines defective in HGPRT, can be obtained, for example, in the American type culture collection (ATCC).

These cell lines are subcultured in an appropriate environment, for example, Wednesday from 8-azaguanine [medium RPMI-1640 supplemented with glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter referred to as "ETS") and optionally supplemented with 8-azaguanine], modified according to the method of Claims environment, Dulbecco (hereinafter referred to as "IMDM"), or modified, Dulbecco Wednesday Needle (hereinafter referred to as "DMEM") for 3-4 days before the merge cells subcultured in a normal environment [for example, ASF104 medium (Ajinomoto Co., Inc.), containing 10% ETS]. Night get merge cells at 2×107or more.

(d) Merge cells

The merger between producing antibodies cells and myeloma cells can be performed appropriately in conditions that are not much lower rates of cell survival, well-known in this field (for example, Weir, D.M., Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987); and Kabat, E.A. and Mayer, M.M., Experimental Immunochemistry, Charles C Thomas Publisher, Springfield, Illinois (1964)).

For example, in such ways and the use of chemical method, which involves mixing producing antibodies cells and myeloma cells in a solution of highly concentrated polymer (e.g. polyethylene glycol) and the physical way in which use electrical stimulation.

Of these methods below specifically illustrated by the chemical method.

In particular, when the polymer in concentrated polymer solution using ethylene glycol producing antibodies cells and myeloma cells are mixed in a solution of polyethylene glycol having a molecular weight of from 1500 to 6000, preferably from 2000 to 4000, at a temperature of from 30°to 40 ° C, preferably from 35 to 38°C, for 1 to 10 minutes, preferably from 5 to 8 minutes.

(e) Selection of hybridoma group

The method for selection of hybridomas obtained by cell fusion is not specifically limited, and usually use the method of selection HAT (gipoksantin-aminopterin-thymidine) (Kohler et al., Nature (1975) 256, p. 495; Milstein et al., Nature (1977) 266, p. 550).

This method is effective for obtaining a hybrid, using line myeloma cells defective in HGPRT, which are not able to survive in aminopterine.

Specifically, no merged cells and hybridoma can be grown in the environment of the HAT, thereby causing the preservation and growth of the only hybrid that is resistant to aminopterin.

(f) Separation of individual cell clones (cloning)

In this way microplate sow feeding cells, such as lines of fibroblasts derived from embryos of rats or normal mouse spleen cells, thymus or ascitic cells.

On the other hand, hybridoma pre-diluted in medium to 0.2-0.5 cells/0,2 ml of This solution containing floating in it diluted hybridoma add in a concentration of 0.1 ml/well, and hybridoma can be continuously cultivated for about 2 weeks, with approximately 1/3 of the medium replaced with fresh, at regular intervals (for example, interval 3 day), thus growing the hybrid clones.

For holes with significant titers of antibodies cloning repeat 2 to 4 times, for example, by the method of limiting dilution and clones that have consistently see the titer of antibodies can be selected as a hybridoma lines producing monoclonal antibody to EPHA2.

Examples of the hybridoma lines, cloned thus, may include hybridoma SH348-1 and hybridoma SH357-1 Hybridoma SH348-1 and hybridoma SH357-1 deposited on 8 June 2007 at the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (address: Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan). Hybridoma SH348-1 designated as SH348-1 with registration number FERM BP-10836, and hybridoma SH357-1 designated as SH357-1 with registration number FERM BP-code 10837.

In the present description, the antibody produced by hybridomas SH348-1, designated as "SH348-1 and the antibody produced by hybridomas SH357-1, designated as "SH357-1".

(g) Obtaining monoclonal antibodies by culturing hybridomas

Selected so hybridoma you can cultivate, to efficiently retrieve, thus, monoclonal antibodies. Before cultivation preferably, hybridoma producing interest monoclonal antibody were subjected to screening.

For this screening, you can choose the methods are essentially known in this field.

Measurement of the antibody titer of the present invention can be realized, for example, by ELISA, as described in paragraph (b).

Hybridoma obtained by the method as described above, can be subjected to cryoconservation in liquid nitrogen or in a freezer at -80°C or below.

In addition, a fully cloned hybridoma you several times to re-plant in the HT environment (environment HAT except aminopterin) and then cultured in normal medium, which is then replaced.

Large-scale Kul is iferouane carried out by rotary cultivation, using large culture vessels or cultivation with constant stirring.

The supernatant obtained in this large-scale cultivation, can be cleaned by methods well known to specialists in this field, such as gel filtration to obtain monoclonal antibodies that are specific contacted with the protein of the present invention.

In addition, hybridoma can be administered intraperitoneally to be injected mice of the same line (for example, BALB/c mice) or mice Nu/Nu and grow with getting ascitic fluids containing large quantities of monoclonal antibodies of the present invention.

To obtain ascitic fluid in large quantities before intraperitoneally the introduction of pre - (from 3 to 7 days before administration) to introduce a mineral oil, such as 2,6,10,14-tetramethylpentane (piers).

For example, mice of the same line and hybridoma previously administered intraperitoneally injected IMMUNOSUPRESSIVE tool for inactivation of T-cells. After 20 days in medium without serum dissolves from 106up to 107cell hybridoma clone (0.5 ml) and the solution administered intraperitoneally injected to mice. As a rule, ascitic fluid of mice harvested when the accumulated ascitic fluid leads to an increase in the abdominal cavity.

In this way Paul is anxious monoclonal antibodies with a concentration of about 100 times greater, than in the culture solution.

Monoclonal antibodies obtained in this way, you can clear the methods described, for example, Weir, D.M.: Handbook of Experimental Immunology, Vol. I, II, III, Blackwell Scientific Publications, Oxford (1978).

Their specific examples include precipitation with ammonium sulfate, gel filtration, ion exchange chromatography and affinity chromatography.

For cleaning as suitable methods can also be used commercially available kits for purification of monoclonal antibodies (for example, MAbTrap GII Kit; production Pharmacia Inc.) etc.

Thus obtained monoclonal antibodies are highly antigenic specificity to EPHA2.

(h) Analysis of monoclonal antibodies

Isotypes and subclass of the monoclonal antibodies thus obtained can be determined as described below.

First, examples of methods of detection include a method of Ouchterlony, ELISA and RIA.

Way Ouchterlony usable, but requires such concentration methods for monoclonal antibodies with low concentration.

On the other hand, when using ELISA or RIA, the culture supernatant directly reacts with the antigen adsorbed on the solid phase and, in addition, as a secondary antibody can be used antibodies corresponding to different the immunoglobulin isotypes and subclasses, thereby determining the isotype or subclass of m is nacionalnih antibodies.

In addition, as a more suitable methods can also be used commercially available kits for detection (for example, Mouse Typer Kit; production of Bio-Rad Laboratories, Inc.) etc.

In addition, quantification of the protein can be determined according to the method Polina-Lowry (Folin-Lowry) and the calculation method using the absorption at 280 nm [1,4 (OD280) = 1 mg/ml immunoglobulin].

(3) Other antibodies

The antibody of the present invention includes a monoclonal antibody to EPHA2 and genetic recombinant antibodies artificially modified with the purpose of, for example, reduce xenoantigens actions on people, for example, chimeric antibodies, humanized antibodies and human antibodies. These antibodies can be obtained by known methods.

Examples of chimeric antibodies include antibody with variable and constant regions derived from species differing from each other, and specifically, includes a chimeric antibody containing the variable regions of murine origin and a constant region of a person connected together (see Proc. Natl. Acad. Sci. U.S.A., 81, 6851-6855, (1984)).

Examples gumanitarnogo antibodies can include antibody containing antibody man with complementarity determining regions (CDR)are replaced with the corresponding region of other species (see Nature (1986) 321, p. 522-525) and antibodies is, containing a human antibody with CDR sequences and some amino acid residues of the frame area, is replaced by the corresponding sequences of CDR and some amino acid residues from other species by "CDR grafting" (see WO 90/07861 and US 6972323).

Further examples of antibodies of the present invention may include a human antibody. The human antibody to EPHA2 indicates antibody only person with the gene sequence of an antibody derived from a human chromosome. The human antibody to EPHA2 can be obtained by methods using mice producing human antibody having a fragment of human chromosome that contains genes of the H and L chains of human antibodies (see, for example, Tomizuka, K. et al., Nature Genetics (1997) 16, p. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, p. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects, vol. 10, p. 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; and Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, p. 722-727).

To obtain such transgenic animals, specifically, genetic recombinant animals in which endogenous loci heavy and light chains of antibodies from non-human mammals destroyed, and instead they entered the loci heavy and light chains of human immunoglobulins by means of vectors based on a yeast artificial chromosome (YAC) or similar, you can get p is the means of obtaining knock-out and transgenic animals and breeding of these animals.

In addition, eukaryotic cells transformed by cDNA encoding both heavy and light chains of human antibodies, preferably vectors containing cDNA by means of genetic recombination, and the transformed cells producing genetic recombinant monoclonal human antibodies can also be cultivated, and thus, the antibodies from the culture supernatant.

To do this, as hosts can be used, for example, eukaryotic cells, preferably mammalian cells such as CHO cells, lymphocytes and myeloma.

In addition, known methods for producing human antibodies selected from libraries of human antibodies, exposed on the phage (see, for example, Wormstone, I.M. et al., Investigative Ophthalmology & Visual Science. (2002) 43 (7), p. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p. 189-203; and Siriwardena, D. et al., Ophthalmology (2002) 109 (3), p. 427-431).

For example, you can use the method of phage display, which includes the implementation of the expression of the variable regions of human antibodies in single-chain antibody (scFv) on the surface of phage and selection of phage that bind antigens (Nature Biotechnology(2005), 23, (9), p. 1105-1116).

In addition, you can also use another method of phage display, which includes the implementation of the expression of Fab-fragment (antigennegative fragment) antibody of man and selection of phages, binding antigens (WO 97/08320 and WO 01/05950).

The genes of the phage selected on the basis of binding to the antigen can be analyzed, determining thus, DNA sequences encoding the variable regions of human antibodies bind to the antigens.

When determining the DNA sequence of scFv or Fab, to bind to the antigens, it receives the sequence of CDR and you can get and enter the appropriate hosts expressing the vectors with these sequences, with the subsequent expression of genes with obtaining human antibodies (WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388, Annu. Rev. Immunol (1994) 12, p. 433-455, and Nature Biotechnology (2005) 23 (9), p. 1105-1116).

Genes of antibodies can temporarily select, and then enter the appropriate owners to generate antibodies. In this case, the appropriate hosts and expressing vectors can be combined for use.

In the case of use as hosts of eukaryotic cells, it is possible to use animal cells, plant cells and eukaryotic microorganisms.

Examples of the animal cells may include (1) mammalian cells, for example, cell lines COS monkey (Gluzman, Y., Cell (1981) 23, p. 175-182, ATCC CRL-1650), mouse fibroblasts NIH3T3 (ATCC No. CRL-1658) and lines defective in dihydropyrimidinase (Urlaub, G. and Chasin, L.A., Proc. Natl. Acad. Sci. USA (1980) 77, p. 4126-4220) cells Chinese hamster ovary (CHO cells, ACC CCL-61).

In addition, to use these owners also can be modified to Express the antibodies with a modified structure of chains of sugars and increased activity of ADCC (antibody-dependent cytotoxic activity or CDC activity. Examples of such hosts may include CHO cells, containing included genes that encode molecules, antibodies, and producing the antibody composition in which chains of sugars N-acetylglucosamine, is not associated with Fuksas, at their reducing ends, hold 20% or more chains of sugars connected N-glycoside complex type that binds Fc region of the antibody (see WO 02/31140).

When using prokaryotic cells their examples can include Escherichia coli and Bacillus subtilis.

The gene of interest antibodies injected into these cells by transformation, and the transformed cells are cultivated in vitro with antibodies.

The isotype of the antibodies of the present invention is not limited and examples include IgG (IgG1, IgG2, IgG3 or IgG4), IgM, IgA (IgA1 or IgA2), IgD, and IgE, and may preferably include IgG and IgM.

In addition, the antibody of the present invention may be a fragment of the antibody with antigennegative plot antibody or its modified form.

Examples of the antibody fragment include Fab, F(ab')2, Fv, single-chain Fv (scFv)containing heavy the light chain Fv, associated with a suitable linker, ditelo, linear antibody and polyspecific antibody, formed by fragments of antibodies.

Additionally, the antibody of the present invention may be a polyspecific antibody, having specificity for at least two different antigens.

Such a molecule usually connects two antigen (i.e., bespecifically antibody). "Polyspecific antibody" of the present invention refers to an antibody with specificity for many (e.g., three) antigens.

Polyspecific antibody is used as the antibody of the present invention, can be a full-sized antibody or a fragment of such an antibody (for example, F(ab')2 bespecifically antibody). Bespecifically antibody can be obtained by linking the heavy and light chains (pair HL) two antibodies or also can be obtained by merging hybridomas producing monoclonal antibodies are distinct from each other, obtaining especifismo antibodies produced by fused cell (Millstein et al., Nature (1983) 305, p. 537-539).

The antibody of the present invention may be a single-chain antibody (also referred to as scFv). Single-chain antibody receive by connecting the V regions of the heavy and light chain antibody polypeptide through a linker (Puckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenberg and Moore ed., Springer Verlag, New York, p. 269-315 (1994)); and Nature Biotechnology (2005), 23, p. 1126-1136).

Methods of obtaining single-chain antibodies are well known in the art (see for example, U.S. patent No. 4946778, 5260203, 5091513, and 5455030). For scFv, the V region heavy and light chain connected by a linker, which does not form a conjugate, preferably a polypeptide linker (Huston, J.S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988), 85, p. 5879-5883). V region heavy and light chain scFv can be obtained from the same antibody, or can be obtained from other antibodies.

For example, the peptide linker to connect the V regions use random single-stranded peptide from residues 12-19.

DNA encoding scFv get: amplificare, as matrix, a full-sized sequence or partial sequence (encoding the desired amino acid sequence) DNA that encodes a heavy chain or the V region of the heavy chain of the antibody and the DNA that encodes its light chain or the V region of the light chain, the method of PCR using pairs of primers designed for both ends; and subsequently optionally amplificare DNA encoding the peptide linker portion in combination with a pair of primers designed to communicate appropriately both ends of the linker sequence with sequences of heavy and light chains.

The AOC is e, after obtaining DNA encoding scFv can be obtained expressing the vectors containing the DNA and hosts transformed expressing vectors by standard methods. In addition, using the hosts, you can use a standard ways to obtain scFv.

For these fragments, antibodies, genes receive and Express in the same way as above, and you can ensure the production of fragments of antibodies by the host.

The antibody of the present invention may be a polyclonal antibody, which is a mixture of many EPHA2 antibodies to different amino acid sequences. One example of a polyclonal antibody can include a mixture of multiple antibodies with different CDR. Cultivate a mixture of cells producing antibodies that differ from each other, and as such polyclonal antibodies can be used purified from culture antibody (see WO 2004/061104).

As well as modified forms of antibodies can be used antibodies obtained by binding antibodies of the present invention with various molecules such as polyethylene glycol (PEG).

In addition, the antibody of the present invention may be a conjugate of these antibodies formed with other drugs (immunoconjugate). Examples of such antibodies may include the conjugates, obtained through the binding of these antibodies with radioactive substances or compounds having pharmacological activity (Nature Biotechnology (2005) 23, p. 1137-1146).

The resulting antibodies can be purified to a homogeneous state. In the separation and purification of antibodies, you can use any method of separation/purification, used for normal proteins.

Antibodies can be selected and cleared by selecting and combining, as appropriate, for example, the use of columns for chromatography, filter, ultrafiltration, salting out, dialysis, electrophoresis in polyacrylamide gel for the preparation and isoelectrofocusing (cleaning methods and study the properties of proteins: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); and antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), however, the method of separation/purification is not limited to them.

Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, and adsorption chromatography.

These methods chromatography can be performed using liquid chromatography, such as HPLC or FPLC.

Examples of columns used in affinity chromatography include column with protein A and protein G.

Examples of columns based on the column with protein A include Hyper D, POROS, Sepharose F.F. (Pharmacia Inc.)./p>

In addition, antibodies can also be clear on the basis of their affinity to antigens, using the antigen immobilized on the carrier.

3. Properties of antibodies of the present invention

The antibody of the present invention to EPHA2, obtained by the method has the following properties:

(1) one antibody according to the present invention has the following properties a) to e):

a) has no ability to phosphorylation of tyrosine residues EPHA2;

b) has ADCC activity against cells expressing EPHA2;

c) has CDC activity against cells expressing EPHA2;

d) has antitumor activity in vivo; and

e) specific binds to the polypeptide consisting of the amino acid sequence corresponding to amino acids No. 426-534 in SEQ ID NO: 8 in the list of sequences.

Examples of antibodies having similar properties may include any antibody selected from the group consisting of the following 1)-8):

1) SH348-1,

2) the antibody that recognizes the epitope recognized by the antibody produced by hybridomas SH348-1 (FERM BP-10836),

3) the antibody with the amino acid sequences represented by SEQ ID No: 59, 61 and 63 in the list of sequences as complementarity determining regions in the variable regions of the heavy chain and the amino acid is posledovatelnostei, corresponding to SEQ ID No: 65, 67 and 69 in the list of sequences as complementarity determining regions in the variable region of the light chain,

4) the antibody described by the following i) and ii):

i) a peptide having the heavy chain containing the amino acid sequence represented by the General formula (I):

-FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4(I)

where FRH1denotes an arbitrary amino acid sequence consisting of from 18 to 30 amino acids; CDRH1indicates the amino acid sequence corresponding to SEQ ID NO: 59 in the list of sequences; FRH2denotes an arbitrary amino acid sequence consisting of 14 amino acids; CDRH2indicates the amino acid sequence represented by SEQ ID NO: 61 in the list of sequences; FRH3denotes an arbitrary amino acid sequence consisting of 32 amino acids; CDRH3indicates the amino acid sequence represented by SEQ ID NO: 63 in the list of sequences; and FRH4denotes an arbitrary amino acid sequence consisting of 11 amino acids, where these amino acids linked together by peptide bonds; and

ii) having a light chain polypeptide containing and inoculate sequence, represented by the General formula (II):

-FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4-(II)

where FRL1denotes an arbitrary amino acid sequence consisting of 23 amino acids; CDRL1indicates the amino acid sequence represented by SEQ ID NO: 65 in the list of sequences; FRL2denotes an arbitrary amino acid sequence consisting of 15 amino acids; CDRL2indicates the amino acid sequence represented by SEQ ID NO: 67 in the list of sequences; FRL3denotes an arbitrary amino acid sequence consisting of 32 amino acids; CDRL3indicates the amino acid sequence represented by SEQ ID NO: 69 in the list of sequences; and FRL4denotes an arbitrary amino acid sequence consisting of 10 amino acids, where these amino acids linked together by peptide bonds.

5) SH357-1,

6) an antibody that recognizes the epitope recognized by the antibody produced by hybridomas SH357-1 (FERM BP-code 10837),

7) the antibody with the amino acid sequences represented by SEQ ID No: 71, 73 and 75 in the list of sequences as complementarity determining regions in the variable regions of the heavy chain and amino acids is diversified sequences corresponding to SEQ ID No: 77, 79 and 81 in the list of sequences as complementarity determining regions in the variable region of the light chain,

8) the antibody according to any one of (5)-(7), characterized by the following i) and ii):

i) a peptide having the heavy chain containing the amino acid sequence represented by the General formula (I):

-FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4(I)

where FRH1denotes an arbitrary amino acid sequence consisting of from 18 to 30 amino acid sequence; CDRH1indicates the amino acid sequence represented by SEQ ID NO: 71 in the list of sequences; FRH2denotes an arbitrary amino acid sequence consisting of 14 amino acids; CDRH2indicates the amino acid sequence represented by SEQ ID NO: 73 in the list of sequences; FRH3denotes an arbitrary amino acid sequence consisting of 32 amino acids; CDRH3indicates the amino acid sequence represented by SEQ ID NO: 75 in the list of sequences; and FRH4denotes an arbitrary amino acid sequence consisting of 11 amino acids, where these amino acids linked together by peptide bonds; and

ii) have its light chain polypeptide, containing the amino acid sequence represented by the General formula (II):

-FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4-(II)

where FRL1denotes an arbitrary amino acid sequence consisting of 23 amino acids; CDRL1indicates the amino acid sequence represented by SEQ ID NO: 77 in the list of sequences; FRL2denotes an arbitrary amino acid sequence consisting of 15 amino acids; CDRL2indicates the amino acid sequence represented by SEQ ID NO: 79 in the list of sequences; FRL3denotes an arbitrary amino acid sequence consisting of 32 amino acids; CDRL3indicates the amino acid sequence represented by SEQ ID NO: 81 in the list of sequences; and FRL4denotes an arbitrary amino acid sequence consisting of 10 amino acids, where these amino acids linked together by peptide bonds.

(2) another antibody of the present invention has the following properties a) to f):

a) has no ability to phosphorylation of tyrosine residues EPHA2;

b) demonstrates the effect of reducing the level of EPHA2 protein;

c) has ADCC activity against cells expressing EPHA2;

d) about lady CDC activity relative to cells, expressing EPHA2;

e) has antitumor activity in vivo; and

f) specific binds to the polypeptide consisting of the amino acid sequence corresponding to amino acids No. from 426 to 534 in SEQ ID NO: 8 in the list of sequences.

Examples of antibodies having such properties can include any antibody selected from the group consisting of the following 1)-4):

1) SH348-1,

2) the antibody that recognizes the epitope recognized by the antibody produced by hybridomas SH348-1 (FERM BP-10836),

3) the antibody with the amino acid sequences represented by SEQ ID No: 59, 61 and 63 in the list of sequences as complementarity determining regions in the variable regions of the heavy chain and the amino acid sequences represented by SEQ ID No: 65, 67 and 69 in the list of sequences as complementarity determining regions in the variable region of the light chain,

4) the antibody described by the following i) and ii):

i) a peptide having the heavy chain containing the amino acid sequence represented by the General formula (I):

-FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4(I)

where FRH1denotes an arbitrary amino acid sequence consisting of from 18 to 30 amino acids; CDRH1means aminokislotna the sequence, represented by SEQ ID NO: 59 in the list of sequences; FRH2denotes an arbitrary amino acid sequence consisting of 14 amino acids; CDRH2indicates the amino acid sequence represented by SEQ ID NO: 61 in the list of sequences; FRH3denotes an arbitrary amino acid sequence consisting of 32 amino acids; CDRH3indicates the amino acid sequence represented by SEQ ID NO: 63 in the list of sequences; and FRH4denotes an arbitrary amino acid sequence consisting of 11 amino acids, where these amino acids linked together by peptide bonds; and

ii) having a light chain polypeptide containing the amino acid sequence represented by the General formula (II):

-FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4-(II)

where FRL1denotes an arbitrary amino acid sequence consisting of 23 amino acids; CDRL1indicates the amino acid sequence represented by SEQ ID NO: 65 in the list of sequences; FRL2denotes an arbitrary amino acid sequence consisting of 15 amino acids; CDRL2indicates the amino acid sequence represented by SEQ ID NO: 67 in the list is posledovatelnostei; FRL3denotes an arbitrary amino acid sequence consisting of 32 amino acids; CDRL3indicates the amino acid sequence represented by SEQ ID NO: 69 in the list of sequences; and FRL4denotes an arbitrary amino acid sequence consisting of 10 amino acids, where these amino acids linked together by peptide bonds.

(3) another antibody of the present invention has the following properties a) to e):

a) has no ability to phosphorylation of tyrosine residues EPHA2;

b) demonstrates the effect of reducing the level of EPHA2 protein;

c) has ADCC activity;

d) has CDC activity;

e) has antitumor activity in vivo; and

f) specifically binds with a polypeptide containing the amino acid sequence corresponding to amino acids No. from 426 to 534 in SEQ ID NO: 8 in the list of sequences.

Examples of antibodies having similar properties, may include any one antibody selected from the group consisting of the following 1)-4):

1) SH357-1,

2) the antibody that recognizes the epitope recognized by the antibody produced by hybridomas SH357-1 (FERM BP-10836),

3) the antibody with the amino acid sequences represented by SEQ ID No: 71, 73 and 75 in the list of sequences as the e complementarity determining regions in the variable regions of the heavy chain and amino acid sequences presented in SEQ ID No: 77, 79, and 81 in the list of sequences as complementarity determining regions in the variable region of the light chain,

4) the antibody has the following properties i) and ii):

i) a peptide having the heavy chain containing the amino acid sequence represented by the General formula (I):

-FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4(I)

where FRH1denotes an arbitrary amino acid sequence consisting of from 18 to 30 amino acids; CDRH1indicates the amino acid sequence corresponding to SEQ ID NO: 71 in the list of sequences; FRH2denotes an arbitrary amino acid sequence consisting of 14 amino acids; CDRH2indicates the amino acid sequence represented by SEQ ID NO: 73 in the list of sequences; FRH3denotes an arbitrary amino acid sequence consisting of 32 amino acids; CDRH3indicates the amino acid sequence represented by SEQ ID NO: 75 in the list of sequences; and FRH4denotes an arbitrary amino acid sequence consisting of 11 amino acids, where these amino acids linked together by peptide bonds; and

ii) having a light chain polypeptide containing the amino acid placenta is the sequence, represented by the General formula (II):

-FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4-(II)

where FRL1denotes an arbitrary amino acid sequence consisting of 23 amino acids; CDRL1indicates the amino acid sequence represented by SEQ ID NO: 77 in the list of sequences; FRL2denotes an arbitrary amino acid sequence consisting of 15 amino acids; CDRL2indicates the amino acid sequence represented by SEQ ID NO: 79 in the list of sequences; FRL3denotes an arbitrary amino acid sequence consisting of 32 amino acids; CDRL3indicates the amino acid sequence represented by SEQ ID NO: 81 in the list of sequences; and FRL4denotes an arbitrary amino acid sequence consisting of 10 amino acids, where these amino acids linked together by peptide bonds.

4. The pharmaceutical preparation containing the antibody to EPHA2

Antibody to EPHA2 the present invention is suitable as pharmaceuticals, in particular, the pharmaceutical composition is intended for the treatment of malignant tumors or as antibodies for immunological diagnosis of such diseases.

Predpochtite the further examples of the types of malignant tumors may include, but not limited to, breast cancer, cancer of the esophagus, prostate cancer, malignant stomach tumor, non-small cell lung cancer, colon cancer and glioblastoma multiforme.

The present invention also relates to pharmaceutical compositions containing a therapeutically effective amount of the antibody to EPHA2 and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.

Preferably, pharmaceutically used tools, which are suitable for pharmaceutical compositions of the present invention must be non-toxic in the dose or type of concentration is for individuals who receive pharmaceutical composition.

The pharmaceutical composition of the present invention may contain a pharmaceutical agent for modifying, maintaining or preserving pH, osmotic pressure, viscosity, clarity, color, isotonicity, sterility, stability, rate of dissolution, the speed of a slow-release, absorption or permeability.

Examples of pharmaceutical substances may include, but are not limited to, the following: amino acids, such as glycine, alanine, glutamine, asparagine, arginine and lysine); antimicrobials; Antioch identy, such as ascorbic acid, sodium sulfate and sodium bisulfite; buffers such as phosphate, citrate and borate buffers, bicarbonate and a solution of Tris-HCl; fillers such as mannitol and glycine; chelating agents such as ethylenediaminetetraacetic acid (EDTA)); complexing agents, such as caffeine, polyvinyl pyrrolidine, β-cyclodextrin and hydroxypropyl-β-cyclodextrin; fillers such as glucose, mannose and dextrin; monosaccharides, disaccharides, glucose, mannose, and other hydrocarbons, such as dextrin; colorants; fragrances; thinners; emulsifiers; hydrophilic polymers such as polyvinyl pyrrolidine; low molecular weight polypeptides; soleobrazutaya counterions; antiseptics, such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, finitely alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and hydrogen peroxide; solvents, such as glycerol, propylene glycol and polyethylene glycol; sugar alcohols such as mannitol and sorbitol; suspendresume means; surfactants, such as PEG, arbitarily ester, Polysorbate, such as Polysorbate 20 and Polysorbate 80, Triton, tromethamine, lecithin, and cholesterol; a means of reinforcing stability, such as sucrose and sorbitol; a means of increasing elasticity, such as the sodium chloride, potassium chloride, mannitol and sorbitol; vehicles; diluents; excipients and/or pharmaceutical adjuvants.

The amount of these added pharmaceuticals preferably in quantities of from 0.01 to 100 times, particularly, from 0.1 to 10 times greater than the mass of the antibody to EPHA2.

In this context, the present invention also includes a pharmaceutical composition comprising immunoliposome, including antibody to EPHA2 in the liposome or antibody to EPHA2 associated with the liposome (U.S. patent No. 6214388).

The preferred composition of the pharmaceutical composition in the preparation can properly be determined by the person skilled in the art, depending on the disease, the route of administration, etc.

Excipients or carriers in the pharmaceutical composition may be liquid or solid. Suitable excipients or carriers may be a water for injection, saline solution, cerebrospinal fluid or other means normally used for parenteral administration.

As a carrier you can also use a neutral saline solution or saline solution containing serum albumin. The pharmaceutical composition may also contain buffer Tris (pH 7.0 to 8.5) or acetate buffer (pH 4,0-5,5), as well as sorbitol or other compounds. Pharmaceutical to the nosily of the present invention receive in dried or liquid form as a suitable medicinal product with the selected composition and the required purity.

Pharmaceutical composition comprising the antibody to EPHA2, also available in dried form, using a suitable excipient, such as sucrose.

The pharmaceutical composition of the present invention can be obtained for parenteral administration or also available for absorption in the gastrointestinal tract.

The composition and concentration of the drug can be installed depending on the method of introduction. If antibody to EPHA2 contained in the pharmaceutical compositions of the present invention, has a higher affinity to EPHA2, i.e. higher affinity (low Kd) to EPHA2 relative dissociation constants (Kd), the medicinal product containing the antibody may be effective for people in low dose. Based on these results it is also possible to determine the dose of the pharmaceutical composition of the present invention for a person.

The dose of antibody to EPHA2 for people, as a rule, may be approximately from 0.1 to 100 mg/kg once a period of from 1 to 180 days.

Examples of dosage forms of the pharmaceutical compositions of the present invention include injections, including drops, suppositories, nasal drugs, sublingual means, suction transdermal.

Introduction the pharmaceutical composition according to the present which the invention can inhibit tumor growth, expressing EPHA2.

Examples

Hereinafter the present invention will be described more specifically with reference to examples. However, it should be understood that this invention is not limited.

In the examples below, unless otherwise indicated, the methods related to genetic engineering carried out by the methods described in "Molecular Cloning" (Sambrook, J., Fritsch, E.F., and Maniatis, T., published by Cold Spring Harbor Laboratory Press, 1989), or by methods described in other experimental handbooks used by specialists in the field or in accordance with instructions included in commercially available reagents or used sets.

(Example 1) preparation of plasmid

1)-1 Receipt of vectors expressing EPHA2 person

1)-1-1 Obtaining vectors expressing full-sized human EPHA2

cDNA encoding human EPHA2, amplified by PCR reaction using cDNA synthesized from total RNA obtained from SK-OV-3 cells, as a matrix, and a set of primers:

Primer 1: 5'-ggggacaagtttgtacaaaaaagcaggcttcggggatcggaccgagagcgagaag-3'

(in the list of sequences sequence ID No. 3); and

Primer 2: 5'-ggggaccactttgtacaagaaagctgggtcctagatggggatccccacagtgttcacctggtcctt-3'

(in the list of sequences sequence ID No. 4).

The PCR product was incorporated into pDONR221 (Invitrogen Corp.) using BP Clonase (Invitrogen Corp.) for the teachings of the initial vector. The termination codon was removed from the EPHA2 gene in primary vector using site-specific mutagenesis GeneTailor (produced by Invitrogen Corp.) and the primer set:

Primer 3: 5'-ctgtggggatccccatcgacccagctttc-3' (in the list of sequences sequence ID No. 5); and

Primer 4: 5'-gatggggatccccacagtgttcacctggtc-3' (in the list of sequences sequence ID No. 6).

The recombination reaction between the obtained initial vector and pcDNA-DEST40 Gateway Vector (Invitrogen Corp.) was performed using LR Clonase (Invitrogen Corp.) to obtain pcDNA-DEST40-EPHA2 (this vector has a nucleotide sequence corresponding to SEQ ID NO: 7 in the list of sequences between attB1 and attB2 sequences). In addition, the sequence of the ORF region EPHA2 gene cloned in this vector, represented by nucleotides with No. from 33 to 2960 in SEQ ID NO: 7 in the list of sequences. In addition, EPHA2 amino acid sequence presented in SEQ ID NO: 8 in the list of sequences.

1)-1-2 Receiving expressing vector for extracellular region EPHA2

cDNA encoding the polypeptide extracellular region of human EPHA2 (consisting of amino acid sequence corresponding to amino acids No. from 1 to 534 in SEQ ID NO: 8 in the list of sequences; hereinafter, abbreviated to "EPHA2-ECD") is amplified by PCR reaction using the primer set:

Primer 5: 5'-aaaaagcttatggagctccaggcagcccgc-3' (in the list of sequences sequence ID No. 9); and

Primer 6: 5'-aaagggccctcagttgccagatccctccgg-3' (in the list of sequences sequence ID No. 10).

The resulting PCR product was digested HindIII and ApaI and cloned into the HindIII site/ApaI pcDNA3,1 (hereinafter, the resulting vector is reduced to "pcDNA3.1-EPHA2-ECD"; and in the description hereinafter and in the drawings, a recombinant protein expressed "pcDNA3.1-EPHA2-ECD" refers to as "rEPHA2-ECD").

1)-1-3 Obtaining expressing vectors truncated proteins EPHA2

To create vectors expressing region consisting of the amino acid sequence corresponding to amino acids No. from 315 up to 540 in SEQ ID NO: 8 in the list of sequences EPHA2 (hereinafter referred to as "FnIII-NC"), the region consisting of the amino acid sequence corresponding to amino acids No. from 315 to 430 thereof (hereinafter referred to as "FnIII-N") or a region consisting of the amino acid sequence corresponding to amino acids No. from 426 to 540 thereof (hereinafter referred to as "FnIII-C")was subjected to PCR reaction with pcDNA-DEST40-EPHA2 as matrices, using each set of primers:

A set of primers for amplification of FnIII-NC:

Primer 7: 5'-gcaggcttcatcgaaggtcgtgggcgggcacctcaggacccag-3' (in the list of sequences sequence ID No. 11); and

Primer 8: 5'-gtacaagaaagctgggtgctagccgccaatcaccgccaag-3' (list the sequence is sequence ID No. 12);

A set of primers for amplification of FnIII-N:

Primer 7, and

Primer 9: 5'-gtacaagaaagctgggtgctaggcagtacggaagctgcgg-3' (in the list of sequences sequence ID No. 13);

A set of primers for amplification of FnIII-C:

Primer 10: 5'-gcaggcttcatcgaaggtcgtgggagcttccgtactgccagtg-3' (in the list of sequences sequence ID No. 14); and

Primer 8.

To add sites attB1 and attB2 to both ends of the obtained PCR products were PCR reaction with each PCR product as the template, using a primer set:

Primer 11: 5'-ggggacaagtttgtacaaaaaagcaggcttcatcgaaggtcgtggg-3' (in the list of sequences sequence ID No. 15); and

Primer 12: 5'-ggggaccactttgtacaagaaagctgggt-3' (in the list of sequences sequence ID No. 16).

The PCR products obtained in this way were built into pDONR221 using BP Clonase to obtain the initial vectors. The reaction of recombination between each initial vector and the target vector obtained by cleavage sites NdeI and BamHI pET15b (Novagen) enzymes, zatuplenie” split plots and subsequent legirovaniem cassette reading frame C.1 Gateway Vector Conversion System (Invitrogen Corp.) in “blunt” plots was performed using LR Clonase order to obtain expressing vectors (hereinafter recombinant proteins expressed by FnIII-NC, FnIII-N - and FnIII-C-built-ex is desiroush vectors indicate how rFnIII-NC, rFnIII-N and rFnIII-C, respectively).

1)-2 Obtaining expressing vector extracellular region of the human EPHB2

cDNA encoding human EPHB2, was obtained by PCR reaction using cDNA synthesized on total RNA obtained from HCC70 cells as template and primer set:

Primer 13: 5'-ggggacaagtttgtacaaaaaagcaggcttcgccccgggaagcgcagcc-3' (in the list of sequences sequence ID No. 17); and

Primer 14:

5'-ggggaccactttgtacaagaaagctgggtcctaaacctccacagactgaatctggttcatctg-3' (in the list of sequences sequence ID No. 18).

The nucleotide sequence of cDNA EPHB2 person represented by SEQ ID NO: 19 in the list of sequences. Amino acid sequence thereof represented by SEQ ID NO: 20 in the list of sequences. The PCR reaction was performed using the primer set for amplifying the cDNA encoding the extracellular region of human EPHB2 (the region consisting of the amino acid sequence corresponding to amino acids No. from 1 to 542 in SEQ ID NO: 20 in the list of sequences) (hereinafter abbreviated to "EPHB2-ECD"):

Primer 15: 5'-aaaaagcttatggctctgcggaggctgggg-3' (in the list of sequences sequence ID No. 21); and

Primer 16: 5'-aaagatatctcatggcaacttctcctggat-3' (in the list of sequences sequence ID No. 22).

The resulting PCR product was digested HindIII and EcoRV and cloned into the HindIII site/EcoRV pcDNA3.1 (hereinafter the resulting vector is ukrashayut to "pcDNA3.1-EPHB2-ECD"; in the description hereinafter and in the drawings, a recombinant protein expressed "pcDNA3.1-EPHB2-ECD" refers to how rEPHB2-ECD).

1)-3 Obtaining expressing vector ERBB2 person

Carried out a PCR reaction with a collection of clones of man (produced by STRATAGENE, #C33830) as a matrix, using the primer set:

Primer 17: 5'-caccatggagctggcggccttg-3' (in the list of sequences sequence ID No. 23); and

Primer 18: 5'-tcccactggcacgtccagacc-3' (in the list of sequences sequence ID No. 24).

The resulting PCR product was built into the pENTR/D-TOPO (Invitrogen Corp.), using the pENTR Directional TOPO Cloning kit (Invitrogen Corp.) to obtain an initial vector. To repair mutations caused amino acid substitutions, the initial vector was digested EcoRI and among the obtained fragments fragment containing a sequence derived from pENTR/D-TOPO ligated with the second largest fragment (approximately 1,6 TPN) among fragments obtained by cleavage EcoRI Human clone collection (produced by STRATAGENE, #C14640). The recombination reaction between the obtained initial vector and pcDNA-DEST40 Gateway vector was carried out using LR Clonase order to obtain pcDNA-DEST40-ERBB2 (the vector has a nucleotide sequence represented by SEQ ID NO: 25 in the list of sequences between sequences attB1 and attB2).

(Example 2) Obtaining monoclonal antibodies

2)1 Getting antigen

For the expression of EPHA2-ECD, cells FreeStyle 293-F (produced by Invitrogen Corp.) was transfusional pcDNA3.1-EPHA2-ECD using 293fectin (Invitrogen Corp.) and were cultured at 37°C in 8% CO2within 5 days. After cultivation, the culture supernatant was collected by centrifugation and used as a source for purification rEPHA2-ECD. The obtained culture supernatant were dialyzed against 20 mm Tris-HCl, pH 7.5, using a dialysis tube with a marginal value of the molecular weight of 15,000, then filtered through a filter (0.45 mm, PES) and then was applied to a HiPrep 16/10 Q XL (manufactured by GE Healthcare Bio-Sciences Corp.), balanced 20 mm Tris-HCl, pH 7.5. Elution was performed at a linear concentration gradient of NaCl (20 mm Tris-HCl, pH 7.5, 0-1M NaCl). The aliquot buervenich fractions were subjected to separation by SDS-polyacrylamide gel electrophoresis (hereinafter abbreviated to "SDS-PAGE"). Then the gel was stained with Kumasi diamond blue (hereinafter abbreviated to "CBB-stained") to confirm the presence of the fractions containing rEPHA2-ECD. Next, the fractions containing rEPHA2-ECD, were combined and applied on a HiLoad 26/60 Superdex 200 PG (GE Healthcare Bio-Sciences Corp.), balanced PBS. After elution with PBS, aliquot buervenich fractions were subjected to separation in SDS-PAGE. Next, the gel was stained with CBB to confirm the presence of the fractions containing rEPHA2-ECD. The fractions containing rEPHA2-ECD, were combined and used and as antigen for immunization, and antigen for determination of the epitope. The protein concentration was measured using the reagent for studying protein BCA (PIERCE).

2)-2 Immunization

Used mice of BALB/cAnNCrlCrlj aged 4 to 6 weeks (Charles River Laboratories Japan, Inc.). At day 0, a mixture of 50 μg rEPHA2-ECD and complete adjuvant's adjuvant H37 Rv (produced by Wako Pure Chemical Industries, Ltd.) (1:1 in terms of volume ratio) was subcutaneously injected mouse back into the region. In addition, a mixture of 50 μg rEPHA2-ECD and TiterMax Gold Adjuvant (Sigma-Aldrich, Inc.) (1:1 in terms of volume ratio) was subcutaneously injected into the back of another individual. On 22 and 36 days, a mixture of 50 μg rEPHA2-ECD and incomplete adjuvant's adjuvant (produced by Wako Pure Chemical Industries, Ltd.) (1:1 in terms of volume ratio) was subcutaneously injected in the back region of each mouse. 53 day, 50 mcg rEPHA2-ECD was administered intraperitoneally administered to each mouse. At 56 days, provided the spleen of the mice used in obtaining hybridoma.

2)-3 Obtaining hybridoma

To obtain hybridomas were cell fusion between spleen cells and the cells of the mouse myeloma P3X63Ag8U.1 using PEG4000 (produced IBL (Measurement-Biological Laboratories, Co., Ltd.)). The culture supernatant obtained hybridomas used for screening hybridomas producing antibody to EPHA2.

2)-4 Screening antibodies

2)-4-1 Obtaining cells expressing the gene encoding the antigen

Cell line 293T were sown in Koli is este 5×10 4cells/cm2on the tablet, coated with collagen type I (IWAKI) and cultivated overnight at 37°C in 5% CO2in DMEM containing 10% ETS. The next day, 293T cells were transfusional pcDNA-DEST40-EPHA2 or pcDNA-DEST40-ERBB2, using as control Lipofectamine 2000 (Invitrogen Corp.) and additionally incubated over night at 37°C in 5% CO2. The next day transfetsirovannyh 293T cells were treated with trypsin, and then washed with DMEM containing 10% ETS, and then suspended in PBS containing 5% ETS. The cell suspension was used in ELISA cell-based and flow-cytometrical analysis.

2)-4-2 ELISA cell-based

The cell suspension obtained in paragraph (2)-4-1 centrifuged and the supernatant was removed. Then 293T cells expressing EPHA2 and 293T cells expressing ERBB2, separately suspended, by adding the supernatant of the culture hybridoma and incubated at 4°C for 1 hour. Cells in the wells washed twice PBS containing 5% ETS. Next, the cells are suspended, adding conjugated with peroxidase antibodies goat against mouse IgG (produced by Millipore (Chemicon), #AP181P)diluted 500 times in PBS containing 5% ETS, and incubated at 4°C for 1 hour. Cells in the wells washed twice PBS containing 5% ETS. Then, the solution for the manifestation of color OPD (o-phenylenediamine dihydrochlor is d (produced by Wako Pure Chemical Industries, Ltd.) and H2O2was dissolved to a concentration of 0.4 mg/ml and 0.6% (vol./vol.), accordingly, in a solution of OPD (0,05M trinitrate sodium, 0,1M sodium phosphate disubstituted 12-water, pH 4.5)), was added at the rate of 100 μl/well. Color reaction was performed with stirring and was stopped by adding 1M HCl at the rate of 100 μl/well. Cells were besieged by centrifugation and then the supernatant was excavated to a new 96-well flat-bottomed microplate. Measured the absorbance at 490 nm, using a spectrophotometer to read the tablets (ARVO, PerkinElmer). When selecting hybridomas producing antibodies that are specific contact EPHA2 presented on the surface of the cell membrane, positive production of antibody to EPHA2 were selected hybridoma, forming a supernatant of the culture, which exhibits a higher absorption for 293T cells expressing EPHA2, than to 293T cells expressing ERBB2 (control).

2)-4-3 Flow-cytometrics analysis

To exclude false-positive results in ELISA-based cells, antibodies produced by hybridomas defined as positive in the ELISA-based cells were additionally tested for their specificity of binding to EPHA2 by flow cytometry. The cell suspension obtained in paragraph 2)-4-1 centrifuged and supernatant was removed. The ZAT is separately suspended cells 293T, expressing EPHA2, and 293T cells expressing ERBB2, adding the hybridoma supernatant culture, and incubated at 4°C for 1 hour. Cells in the wells washed twice PBS containing 5% ETS. Next, the cells are suspended, adding, "Conjugate with fluorescein fraction of goat IgG to IgG mouse (Whole Molecule) (ICN Pharmaceuticals, Inc., #55493)diluted 1000 times PBS containing 5% ETS, and incubated at 4°C for 1 hour. Cells were twice washed in PBS containing 5% ETS, and then resuspendable in PBS containing 5% ETS, and additionally containing 2 μg/ml 7-aminoantipyrine D (produced by Invitrogen Corp. (Molecular Probes)), with subsequent analysis using flow cytometer (FC500, Beckman Coulter, Inc.). Data were analyzed using Flowjo (Tree Star, Inc.). Dead cells, positive for 7-aminoantipyrine D excluded, by setting the gate (window). Then displayed in a histogram, the intensity of FITC fluorescence in living cells. Hybridoma producing samples that the histogram of fluorescence intensity in 293T cells expressing EPHA2, provided a stronger fluorescence intensity than the histogram for 293T cells expressing ERBB2 presented as a control, was recognized as hybridomas producing antibody to EPHA2.

2)-5 hybrid Separation into individual clones

Hybridoma, producer the existing antibody to EPHA2, bred in the environment ClonaCell-HY Selection Medium D (StemCell Technologies, #03804) and were cultured, and educated clones were selected as individual clones. The selected clones were cultured separately and investigated their activity binding to EPHA2 using supernatant culture in the same way as in paragraph 2)-4-3, to identify hybridomas producing monoclonal antibody to EPHA2 (SH348-1 AND SH357-1, Ab57-1, Ab65-1, Ab96-1, Ab100-1, Ab105-1, Ab106-13, Ab136-1, Ab148-1, Ab151-4, Ab230-1, Ab373-1 and Ab382-1).

2)-6 Confirmation of binding activity of monoclonal antibodies for the line of malignant cells

Contact or no monoclonal antibodies obtained in paragraph 2)-5, with malignant cells highly expressing EPHA2, investigated by flow-cytometrical method in the same way as in paragraph 2)-4-3. Line of malignant cells human breast (MDA-MB-231), line malignant cells of the human lung (A549) and malignant prostate cells human (PC-3) was used instead of transfected 293T cells. The result was confirmed that all of the tested monoclonal antibodies bind to these malignant cell lines.

2)-7 determination of the isotype of the monoclonal antibody

The isotypes of the monoclonal antibodies was determined using a set of Mouse monoclonal isotyping (AbD Serotec). As a result, isotypes, not only is there a IgG1 (Ab57-1 and Ab230-1), IgG2a (SH348-1 AND SH357-1, Ab65-1, Ab96-1, Ab100-1, Ab136-1, Ab148-1 and Ab151-4) and IgG2b (Ab105-1, Ab106-13, Ab373-1 and Ab382-1).

2)-8 Obtaining monoclonal antibodies

Monoclonal antibodies were purified from ascitic fluid of mice, which transplanted hybridoma or from hybridoma supernatant of culture (hereinafter referred to as "the source for the purification of antibodies").

Mouse ascitic fluid was obtained as follows: first, mice of BALB/cAJcl-nu/nu aged 7 to 8 weeks (CLEA Japan, Inc.) subjected to the action of the Wharf (produced by Sigma-Aldrich, Inc.). Approximately 3 weeks later, administered intraperitoneally transplanted hybridoma, washed with saline, in the amount of 1×107cells/mouse. After 1-2 weeks, ascitic fluid accumulated in the abdominal cavity, collected, and then subjected to sterilization through to 0.22-μm filter and used as a source for purification of antibodies.

Hybridoma supernatant of the culture was obtained using CELLine (BD Biosciences). Hybridoma were cultured according to the manufacturer's instructions, except that used as the medium ClonaCell-HY Growth Medium E (StemCell Technologies, #03805). The collected supernatant was filtered through a 0,45 µm filter and used as a source for purification of antibodies.

Antibodies were purified using a column for affinity chromatography rPA50 containing recombinant protein A (about Sweden RepliGen Corp.), immobilized on Formyl-Cellulofine (Seikagaku Corp.) (hereinafter abbreviated to "Formyl-Cellulofine with protein A") or HiTrap MabSelect SuRe (GE Healthcare Bio-Sciences Corp.). For Formyl-Cellulofine with protein And a source for the purification of antibodies were diluted three times with binding buffer (3M NaCl, 1,5M glycine, pH 8.9) and was applied on the column, which was then washed with binding buffer, followed by elution of 0,1M citric acid, pH of 4.0. On the other hand, for HiTrap MabSelect SuRe, the source for the purification of antibodies was applied on the column, which was then washed with PBS, followed by elution of 2M arginine-HCl, pH of 4.0. Erwerbende antibody neutralized and then replaced on PBS buffer.

The antibody concentration was determined by elution of antibodies associated with POROS G 20 ám Column, PEEK, 4.6 mm×100 mm, 1.7 ml (Applied Biosystems) and measuring the absorbance of the eluate (OP 280 nm). In particular, the sample antibodies, diluted in PBS was applied to a POROS G 20 microns, balanced balanced buffer (30,6 mm sodium phosphate one-deputizing 12-water, 19,5 mm monopotassium phosphate potassium, 0,15M NaCl, pH 7.0). The column was washed equilibrating buffer and then suirable antibody associated with the column, additionally separated by only 0.1% (vol./about.) HCl, 0,15M NaCl). Measured peak area of the absorption of the eluate (OP 280 nm) and was calculated concentration by the following equation:

The concentration of the sample antibody (mg/ml) = (peak Area of sample antibody)/(peak area of control (IgG1 is the ne)) × Concentration control (mg/ml) × ratio of breeding.

In addition, the concentration of endotoxin contained in the obtained antibodies were measured using a set of Endospecy ES-50M (Seikagaku Corp., #020150) and set Endotoxin Standard CSE-L (Seikagaku Corp., #020055), and confirmed the presence of 1 EE/mg or lower. The resulting antibody was used in the subsequent experiments.

(Example 3) properties of SH348-1 and SH357-1

3)-1 Research activity induction of the phosphorylation of tyrosine residues EPHA2 activity and induction of reducing levels of EPHA2 protein in antibody to EPHA2.

3)-1-1 Receiving cell lysates stimulated by antibodies. MDA-MB-231 cells suspended in RPMI1640 containing 10% ETS, 50 units/ml penicillin and 50 µg/ml streptomycin (hereinafter abbreviated to "RPMI1640 containing 10% ETS (with antibiotics)") were sown in the amount of 6×105cells/well in 6-well plate and cultured overnight at 37°C in 5% CO2. The next day the medium was poured and added RPMI1640. Cells were additionally cultured overnight at 37°C in 5% CO2. The next day SH348-1 AND SH357-1, izotopicheskii control IgG2Amouse (in the description hereinafter and in the figures reduced to "mIgG2a"; produced R&D Systems, Inc., #MAB003) as izotopicheskogo control antibodies, recombinant Chimera Ephrin-A1/Fc mouse (in the description hereinafter and in the figures, reduced to "Ephrin-A1/Fc"; produced R&D Systems, Inc., #602-A1-200) as a soluble ligand of EPHA2 and recomb nanny human Fc IgG 1(in the description hereinafter and in the figures, abbreviated to "hG1Fc"; produced R&D Systems, Inc., #110-HG-100) as a control protein for soluble ligand, were separately diluted with RPMI1640 to the concentration shown in figure 1 or 2 (SH348-1 AND SH357-1 and mIgG2a: 10 μg/ml or 50 μg/ml figa and 50 µg/ml figa, Ephrin-A1/Fc and hG1Fc: 1 µg/ml figure 1 and 2). The resulting solution was added to the cells MDA-MB-231 after removal of medium, and incubated at 37°C in 5% CO2within the time specified in 5% CO2. Furthermore, in experiments in the presence of cross-linking antibodies, as cross-linking antibodies SH348-1 AND SH357-1 or mIgG2a and antibodies goats that are specific to the Fcγ fragment of mouse IgG (min X Hu, Bov, Hrs Sr Prot) (Jackson ImmunoResearch Laboratories, Inc., #115-005-071) every mixed with RPMI1640 to a concentration of 10 μg/ml or 50 μg/ml (pigv) and 50 µg/ml (pigv). The resulting solution after removal of the medium was added to cells MDA-MB-231, and incubated at 37°C in 5% CO2within a specified time. At the specified time removing the supernatant and literally cells, adding a buffer for lysis of 1×Cell (Cell Signaling Technology, Inc.), containing 1 mm PMSF (Sigma-Aldrich, Inc.), and was centrifuged at 15000 rpm./min. for 5 minutes. Supernatant cell lysates were used as samples in thus and Western-blotting. The concentration of proteins in the mod is SCE measured, using the reagent for studying protein BCA (PIERCE).

3)-1-2 Checking activity induction of the phosphorylation of tyrosine residues EPHA2

For the formation of immunoprecipitated EPHA2, first, 8 μg of clone D7 to Eck/EphA2 (in the description hereinafter and in the figures reduced to "the antibody to EPHA2 (D7)"; produced by Millipore (Upstate), #05-480) was added to 25 μl of the suspension with magnetic particles with protein G (NEW ENGLAND BioLabs, Inc.) on the sample and the mixture was mixed by inversion at 4°C within 2 hours.

Next, to the mixture was added ETS to a final concentration of 10% and the mixture was additionally mixed by inversion at 4°C for 30 minutes. The particles were washed three times in 1×buffer for lysis of cells, containing 1 mm PMSF. Then thereto was added 200 mg of supernatant cell lysate obtained under paragraph (3)-1-1, and the mixture was mixed by inversion overnight at 4°C. the next day, the particles were washed three times in 1×buffer for lysis of cells, containing 1 mm PMSF. Further, the buffer SDS-Sample (56,3 mm Tris-HCl, pH 6,8, 1,8% (wt./about.) SDS, 9% glycerol, 0,72M 2-mercaptoethanol, 0,045 mg/ml bromophenol blue) was added to the particles and the mixture was heated at 98°C for 5 minutes. Proteins, dissociative with particles were separated by SDS-PAGE.

For Western blotting, the proteins from the gels were transferred to membranes from polyvinylidenedifluoride (gave the e reduced to "PVDF-membrane"; with pore size 0.45 μm; produced by Millipore). After transfer, the PVDF membrane was blocked by shaking in blocking solution (one package of powder Block Ace (Dainippon Sumitomo Pharma Co., Ltd. (Snow Brand Milk Products Co., Ltd.)) was dissolved in 100 ml of ultrapure water, then added Tween 20 and sodium azide to a final concentration of 0.1% (vol./about.) and 0.02% (wt./vol.), respectively). First, to determine the resulting immunoprecipitate EPHA2, PVDF membrane, blocked in this way was immersed in a solution of antibody (D7) to EPHA2, diluted to 0.25 μg/ml blocking solution and was shaken at room temperature for 1 hour. PVDF-membrane was washed for 10 minutes three times with TBST (50 mm Tris-HCl, pH 8.0, 138 mm NaCl, 2.7 mm KCl, 0,1% (vol./about.) Tween 20). Next, the PVDF membrane was immersed in the solution associated with the HRP whole Ab sheep to mouse Ig (manufactured by GE Healthcare Bio-Sciences Corp.), diluted 3,000 times with TBST and were shaken at room temperature for 30 minutes. PVDF-membrane was further washed in TBST for 10 minutes three times. Next, on the film for chemiluminescence was detected signals using ECL Plus (GE Healthcare Bio-Sciences Corp.).

Further, removal of antibodies from these PVDF membrane, PVDF membrane was soaked with a solution to remove (50 mm Tris-HCl, pH of 6.8, 2% (wt./about.) SDS, 100 mm 2-mercaptoethanol) and were shaken at 55°C for 30 minutes. Next, the PVDF membrane was immersed in the process is to extinguish (TBST, containing 1% (vol./about.) H2O2and 0.1% (wt./about.) NaN3), then stirred at room temperature for 20 minutes and additionally washed in TBST for 10 minutes three times. To determine the phosphorylated state of tyrosine residues EPHA2, these PVDF membrane was blocked by shaking in blocking solution without sodium azide (one sachet of powder Block Ace was dissolved in 100 ml of ultrapure water, then added Tween 20 to a final concentration of 0.1% (vol./vol.)). Next, the PVDF membrane was immersed in a solution conjugated with HRP recombinant 4G10 to phosphotyrosine (figures reduced to "4G10 antibody"; produced by Millipore (Upstate), #16-184) diluted 10,000 times with the blocking solution without sodium azide, and were shaken at room temperature for 1 hour. PVDF membrane was washed in TBST for 10 minutes three times, and then further washed with H2O for 5 minutes three times. Signals were detected on the film for chemiluminescence using ECL Plus.

As a result, adding a soluble ligand, Ephrin-A1/Fc, the tyrosine residues of EPHA2 were phosphorylated within 10 minutes. In contrast, the effect of induction of the phosphorylation of tyrosine residues EPHA2 observed in ligand was not observed after addition of antibodies SH348-1 and SH357-1 at a concentration of 10 μg/ml or 50 µg/ml in all intervals of time (10 minutes, 30 minutes and 60 minutes) (figa). Similarly, the effect of induction of the phosphorylation of tyrosine residues EPHA2 observed in ligand were observed in the presence of antibodies SH348-1 and SH357-1 even in the presence of cross-linking antibodies (pigv).

3)-1-3 Checking activity induction reduce EPHA2 protein level

10 µg supernatant cell lysates obtained under paragraph (3)-1-1, were separated by SDS-PAGE. The proteins from the gel were transferred to PVDF membrane and subjected to Western-blotting using an antibody (D7) to EPHA2 and, as control, for example, level of protein, monoclonal antibody to β-actin, clone AC-15 (in the description hereinafter and in the figures, abbreviated to "an antibody to β-actin"; produced by Sigma-Aldrich, Inc., #A-5441). In particular, after transfer, the PVDF membrane was blocked by shaking in blocking solution and then cut with a razor, in the region of molecular weight of 70 kDa in two parts. PVDF-membranes containing proteins of 70 kDa or more, was immersed in a solution of antibody (D7) to EPHA2, diluted to 0.25 μg/ml blocking solution, while the PVDF membrane containing proteins of 70 kDa or less immersed in the solution of antibodies to β-actin (diluted 1000-fold with the blocking solution. Each PVDF-membrane was shaken at room temperature for 1 hour. Each PVDF-membrane was washed in TBST for 10 minutes three times. Next, each PVDF-membrane was immersed in the solution include the tion with HRP whole Ab sheep to mouse Ig, diluted 3000 times TBST and were shaken at room temperature for 30 minutes. Each PVDF-membrane was washed in TBST for 10 minutes three times. Then were detected signals using ECL Plus and NightOWL LB983 (Berthold Technologies GmBH & Co. KG). Next, quantify the intensity of the signal from bands using Gel-Pro Analyzer Version 4.5 for Windows (registered trademark; Media Cybernetics, Inc.).

By adding a soluble ligand, Ephrin-A1/Fc observed a significant decrease in the level of EPHA2 protein (figa and 2B). Adding antibodies SH348-1, both in the presence and in the absence of cross-linking antibodies, was observed a decrease in EPHA2 protein level, although weaker than the effect of the ligand (figa and 2B). On the other hand, for antibodies SH357-1 is almost not seen changes in the level of EPHA2 protein, regardless of the presence or absence of cross-linking antibodies (figa and 2B).

To analyze the EPHA2 protein level after 24 hours from the moment of addition of SH348-1, was calculated the average value of {the intensity of the signal strip EPHA2/intensity of the signal band of β-actin} of the three experimental values, adjusted for the sample with no added ligand/antibody. As a result, after 24 hours from the moment of addition of SH348-1 value was 70% in the absence of cross-linking antibodies and 69% in the presence of cross-svyazivalsa what about the antibodies, while after 24 hours from the moment of addition of mIgG2a is defined as 100%.

3)-2 activity ADCC

3)-2-1 Getting effector cells

Naked mice CAnN.Cg-Foxn1nu/CrlCrlj (Charles River Laboratories Japan, Inc.) under sterile conditions removed spleen. Extracted spleen homogenized two object glasses and was subjected to hemolysis using BD Pharm Lyse (BD Biosciences, #555899). The obtained spleen cells suspended in RPMI1640 without phenol red (Invitrogen Corp.), containing 10% fetal calf serum, Ultra-low IgG (Invitrogen Corp.) (hereinafter abbreviated to "medium for ADCC") and was passed through a cell sieve size of pores (40 μm; manufactured BD Biosciences). Then counted the number of living cells by excluding test Trifanova blue. Suspension of spleen cells was centrifuged and then deleted the environment. Cells resuspendable in the medium for ADCC to the density of living cells equal to 1.5×107cells/ml and used as effector cells.

3)-2-2 Obtaining target cells

Cells MDA-MB-231, A549 or PC-3 were treated with trypsin. Cells were washed in RPMI1640 containing 10% ETS, and then resuspendable in RPMI1640 containing 10% ETS. Each cell line (4×106cells) was mixed with chromium-51 (5550 BCF), were subjected to sterilization through a 0,22 µm filter and immediately were labeled at 37°C in 5% CO2 within 1 hour. Labeled cells were washed three times with medium for ADCC and resuspendable in the medium for ADCC to a concentration of 2×105cells/ml to obtain the target cells.

3)-2-3 Analysis release51Cr

Target cells (2×105cells/ml) were excavated at 50 μl/well in 96-well microplate with a U-shaped bottom. Thereto was added 50 μl of SH348-1 AND SH357-1 or izotopicheskogo control antibodies (mIgG2a), diluted medium for ADCC to 2.5 µg/ml (in terms of the final concentration after adding effector cells) and incubated at 4°C for 1 hour. Was added 100 μl of effector cells (1.5 x 107cells/ml) and incubated over night at 37°C in 5% CO2. The next day, the supernatant was collected in a Luma-tablet (produced by PerkinElmer). The dose emitted gamma rays were measured using a count of gamma radiation. The degree of cell lysis associated with the ADCC activity was calculated by the following equation:

The degree of cell lysis (%) = (A-B)/(C-B)×100

A: the number of pulses in the wells with samples

B: mean (n=3) number of pulses in spontaneous release (wells, which were not added to the antibody/effector cell). Instead of antibodies and effector cells were added to 50 μl and 100 μl, respectively, of the medium for ADCC. Other procedures were performed in the same way as in the wells with samples.

C: average (n=3) number of pulses premaxilla release (wells, containing target cells, dissolved in the detergent). Instead antibody was added 50 μl of medium for ADCC and instead of effector cells were added to 100 μl of medium for ADCC, containing 2% (vol./about.) Triton-X100. Other procedures were performed in the same way as in the wells with samples.

Figure 3 shows the average value of three experiments, where the standard error is the standard deviation, and the P value was calculated by student's t test. As a result, antibodies SH348-1 shows the activity of cell lysis by 8.2%, 9.1% and 4,7% compared to cells MDA-MB-231 (figa), A549 cells (pigv) and cells PC-3 (figs), respectively. For antibodies SH357-1 shows the activity of cell lysis of 8.8%, 13.0% and 9,0% compared to cells MDA-MB-231 (figa), A549 cells (pigv) and cells PC-3 (figs), respectively. These results showed that both antibodies have ADCC activity relative to cells MDA-MB-231, A549 cells and cells PC-3.

3)-3 activity CDC

Cells MDA-MB-231, A549 or PC-3, suspended in RPMI1640 containing 10% ETS (with antibiotics) were sown at the rate of 5000 cells/well on 96-well microplate, and were cultured overnight at 37°C in 5% CO2. The next day, they added SH348-1 AND SH357-1 or izotopicheskie control antibody (mIgG2a), diluted with RPMI1640 containing 10% ETS (with antibiotics), 25 µg/ml (in terms of the final concentration after dopaminecontaining of the complement system) and incubated at 4°C for 1 hour. There was added diluted RPMI1640 up to 30% component of the system of rabbit complement (CEDARLANE, #CL3051) calculated final concentration of 5%, then incubated at 37°C in 5% CO2within 1 hour and additionally left standing at room temperature for 30 minutes. To measure cell viability reagent for analysis of cell viability CellTiter-Glo Luminescent (produced by Promega Corp.) added in a quantity equal to the amount of the culture solution and the mixture was stirred at room temperature for 10 minutes. Then measured the amount of emitted light using a spectrophotometer to read the tablets. Cell viability was calculated by the following equation:

Cell viability (%) = (a-b)/(c-b)×100

a: the amount of light emitted from the wells with samples

b: mean (n=8) the amount of emitted light in the background (wells, which are not added to the cell/antibody). Instead of seeded cells was added RPMI1640 containing 10% ETS (with antibiotics), in number equal to the number of cell suspension and instead antibody was added RPMI1640 containing 10% ETS (with antibiotics), in the amount equal to the dilution of antibodies. Other procedures were performed in the same way as in the wells with samples.

c: average (n=3) the amount of emitted light from the holes in which no added antibody. Instead antibody is added RPMI1640, containing 10% ETS (with antibiotics), in number equal to the number of breeding antibodies. Other procedures were performed in the same way as in the wells with samples.

Figure 4 shows the average value of three experiments, where the standard error is the standard deviation, and the P value was calculated by student's criterion. As a result, the antibody SH348-1 induced 44%, 31%, and 41% decrease in cell viability of MDA-MB-231 (figa), A549 cells (pigv) and cells PC-3 (figs), respectively, in the presence of a component of the complement system. Antibody SH357-1 also induced 65%, 60% and 65% decrease in cell viability of MDA-MB-231 (fig.4D), A549 cells (fige) and cells PC-3 (fig.4F), respectively, in the presence of a component of the complement system. These results showed that both antibodies have CDC activity relative to cells MDA-MB-231, A549 cells and cells PC-3.

3)-4 determination of the epitope

3)-4-1 Getting shorter polypeptide EPHA2 (rFnIII-NC, rFnIII-N and rFnIII-C)

Escherichia coli BL21 and E. coli Origami (DE3) (Novagen) separately transformed expressing the plasmid obtained in paragraph (1)-1-3, and were cultivated in LB medium, with the addition of 50 μg/ml of ampicillin (Sigma-Aldrich, Inc.). The expression of truncated polypeptides induced EPHA2, using a system of self-induction for BL21 (Novagen) and the addition of 0.5 mm IPTG for Origami (DE3). bacterially cells were collected by centrifugation at 6000 rpm./minutes within 20 minutes, and then suspended in buffer for homogenization (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% (vol./about.) Triton-X100, 10% (vol./about.) glycerol) and then on ice subjected to the action of ultrasound. The supernatant was collected by centrifugation at 14000 rpm./minutes within 15 minutes and was applied to a 0.5 ml Ni-NTA (produced by Invitrogen Corp.). Ni-NTA were washed in washing buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 50 mm imidazole, 10% (vol./about.) glycerol), followed by elution buffer for elution (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 400 mm imidazole, 10% (vol./about.) glycerine). Erwerbende samples were additionally purified by column chromatography gel filtration (Superdex 75 10/300; manufactured by GE Healthcare Bio-Sciences Corp.), using PBS as the solvent. The protein concentration of the obtained recombinant proteins was measured using Protein Assay (Bio-Rad Laboratories, Inc).

3)-4-2 Obtaining the extracellular region of the polypeptide EPHB2 (rEPHB2-ECD)

To Express EPHB2-ECD, cells FreeStyle 293-F was transfusional pcDNA3.1-EphA2-ECD using 293fectin, and cultivated at 37°C in 8% CO2within 72 hours. After cultivation, the culture solution was collected by centrifugation and used as a source for purification rEPHB2-ED. The obtained culture supernatant were dialyzed against 20 mm Tris-HCl, pH 7.5, using a dialysis tube with a marginal value of molecular weight 1500, then filtered through a filter (0.45 µm, PES), and then was applied to a HiPrep 16/10 Q XL, balanced 20 mm Tris-HCl, pH 7.5. The elution was performed at a linear concentration gradient of NaCl (20 mm Tris-HCl, pH 7.5, 0-1M NaCl). The aliquot buervenich fractions were separated in SDS-PAGE. Next, the gel was stained with CBB to confirm the presence of the fractions containing rEPHB2-ECD. Next, the fractions containing rEPHB2-ECD, were combined and applied on a HiLoad 26/60 Superdex 200 PG, balanced PBS. After elution PBS, aliquot of elution fractions were separated in SDS-PAGE. Next, the gel was stained with CBB to confirm the presence of the fractions containing rEPHB2-ECD. The fractions containing rEPHB2-ECD, were combined and used as antigen to determine the epitope. The protein concentration was measured using the reagent for protein analysis BCA.

3)-4-3 Determination of binding sites on the antigen by ELISA

rEPHA2-ECD, rFnIII-NC, rFnIII-N, rFnIII-C or control protein rEPHB2-ECD was diluted with PBS to 1 μg/ml, was then excavated 100 μl/well to immunoplate (Nunc, #442404) and incubated over night at 4°C, thereby absorbing protein on the tablet. The next day, the solution from the wells was removed and a solution of Block Ace (one sachet of powder Block Ace was dissolved in 100 ml of ultrapure water), diluted 4 times in PBS, was excavated at 200 μl/well, and incubated at room temperature for 1 hour. The solution from the wells was removed and then added 50 μl/well SH348-1 AND SH357-1, or from the typical control antibodies (mIgG2a), diluted to 5 μg/ml of the buffer for dilution (PBS, 0.05% of (about./about.) Tween 20). Tablet incubated at room temperature for 1 hour. Next, remove the solution from the wells and the wells washed twice with buffer for cultivation. Was added at 50 µl/well antibody goat to mouse IgG, kongugirovanne with horseradish peroxidase, diluted 3000 times the buffer for dilution and incubated at room temperature for 1 hour. The solution from the wells was removed, and the wells washed twice with buffer for cultivation. Then there was a color reaction, with stirring, adding the solution to the degree of color development OPD at the rate of 100 μl/well. After staining, the color reaction was stopped by adding 1M HCl at the rate of 100 μl/well. Measured the absorbance at 490 nm, using a spectrophotometer to read the tablets.

On figa shows the calculation domain structure EPHA2 (NCBI CDD version 2,11, CBS TMHMM Server V. 2,0) and the location of the EPHA2-ECD, FnIII-NC, FnIII-N and FnIII-C EPHA2. Ligand-BD denotes legal-binding domain, FN3 refers to the domain of fibronectin type 3, TM denotes the transmembrane region, Trk kinase indicates tyrosinekinase domain, and SAM denotes the SAM domain.

Received recombinant proteins of the extracellular region EPHA2 (EPHA2-ECD), a region that contains two domain fibronectin type 3 (FnIII-NC), the region containing the N-terminal domain of fibronectin type 3 (FnIII-N), and the region containing the C-terminal domain fibranet is on type 3 (FnIII-C), and investigated their binding activity in respect of SH348-1 and SH357-1. As a result, antibodies SH348-1 and SH357-1 showed a binding activity against rEPHA2-ECD, rFnIII-NC and rFnIII-C (figure 5B). Thus, for antibodies SH348-1 and SH357-1 shows the binding region of amino acids from 426 to 534, containing the C-terminal domain of fibronectin type 3 (amino acid sequence corresponding to amino acids No. 426-534 in SEQ ID NO: 8 in the list of sequences).

(Example 4) anti-tumor effect in vivo

Cells MDA-MB-231 were separated from the culture flask by treatment with trypsin and then suspended in RPMI1640 containing 10% ETS (with antibiotics). After centrifugation supernatant was removed. Cells were twice washed with the same medium described above, and then suspended in BD Matrigel Basement Membrane Matrix (BD Biosciences) and subcutaneously transplanted at a concentration of 5×106cells/mouse into the back of 6-week-old mice BALB/cAJcl-nu/nu ((CLEA Japan, Inc.). If the day of transplantation is designated as day 0, SH348-1 and SH357-1 was administered intraperitoneally administered at a dose of 500 μg/mouse on 9, 16, 23 and 30 day. In the same volume (500 μl), and the amount of antibody administered intraperitoneally injected PBS as a control. Tumor volume was measured on 9, 13, 16, 20, 23, 28, 30, 34 and 37 days to study the antitumor effect attributed to the introduction of antibodies. As a result, the growth of tumors and significantly inhibited in groups, which was introduced SH348-1 and SH357-1, compared with the group which was administered PBS (tumor volume, compared with the group which was administered PBS at 37 days, for SH348-1 and SH357-1 values of P for both was P<0,001; P values were calculated by student's criterion). In addition, the degree of inhibition of tumor growth (= 100-(mean volume of tumors in the group, which was administered antibody)/(average value of the volumes of the tumors in the group, which was administered PBS)×100) 37 day amounted to 89.5% for SH348-1 and 84,1% for SH357-1. Their intense anti-tumor effects observed in vivo (figa and 6B).

14 monoclonal antibody to EPHA2, are investigated for their antitumor effects exerted on mice transplanted with cells MDA-MB-231, the efficiency was shown only for antibodies SH348-1 and SH357-1, communicating with the FnIII-C (table 1).

Table 1
ANTIBODYThe ACTIVITY of BINDING TO the ANTIGENThe INHIBITORY EFFECT ON TUMOR GROWTH IN VIVO
rEPHA2-ECDrFnIII-NCrFnIII-NrFnIII-C
SH348-1 +-++
SH357-1++-++
Ab 57-1+++--
AB 65-1+----

AB 96-1+----
Ab 100-1+----
AB 105-1+----
AB 106-13+- ---
Ab 136-1+----
AB 148-1+----
Ab 151-4+----
Ab 230-1+++--
Ab 373-1+----
Ab 382-1+----

These results showed that the SH348-1 and SH357-1 are antibodies that recognize a previously undescribed epitope (amino acid sequence corresponding to amino acid is with No. 426-534 in SEQ ID NO: 8 in the list of sequences) and antineoplastic effect. In addition, it was shown that the area where contact SH348-1 and SH357-1, serves as a promising target for anticancer monoclonal antibodies aimed at EPHA2.

(Example 5) Identification of antibody genes SH348-1 and SH357-1

To determine N-terminal amino acid sequences of the heavy and light chain antibody SH348-1 and SH357-1 mouse to human EPHA2, an aliquot of solution containing SH348-1 and SH357-1, treated in paragraph 2)-8 were separated in SDS-PAGE. Proteins separated thus in the gel, was transferred from the gel onto PVDF-membrane (size of pores of 0.45 μm; produced by Invitrogen Corp.). PVDF-membrane was washed in washing buffer (25 mm NaCl, buffered with 10 mm sodium borate, pH 8.0), and then were stained by immersion in the dye solution (50% methanol, 20% acetic acid, 0.05% of Kumasi brilliant blue) for 5 minutes, and then decolorized 90% methanol. The area of the bands corresponding to the heavy and light chains (heavy chain: the band with lower mobility, light chain: the band with higher mobility), visualized on a PVDF-membrane, cut out and attempted to identify the corresponding N-terminal amino acid sequence of the automatic method of Edman (see Edman, P., et al. (1967) Eur. J. Biochem. 1, 80), using a Procise (zaregistrirovannogo brand) cLC Protein Sequencer Model 492cLC (Applied Biosystems). For the heavy chain SH348-1 failed to identify amino acid sequence by the method. Thus, the deleted N-terminal pyroglutamic acid using Pyroglutamate-aminopeptidase Pfu (produced by TAKARA BIO INC.), and then performed the same procedure as described above, to identify the amino acid sequence starting at the second amino acid from the N-end.

As a result, the amino acid sequence (starting with the second amino acid from the N-end) bands corresponding to the heavy chain SH348-1 was a

I-Q-L-V-Q-S-G-P (SEQ ID NO: 26 in the list of sequences).

N-terminal amino acid sequence of the band, which corresponds to the light chain of SH348-1 was a

D-V-L-M-T-Q-S-P-L-S-L (SEQ ID NO: 27 in the list of sequences).

N-terminal amino acid sequence of the bands corresponding to the heavy chain SH357-1 was a

Q-I-Q-L-V-Q-S-G-P (SEQ ID NO: 28 in the list of sequences).

N-terminal amino acid sequence of the band corresponding to the light chain of SH357-1 was a

D-V-L-M-T-Q-T-P-L-S-L-P-V-S-L-G-D-Q-A (SEQ ID NO: 29 in the list of sequences).

These amino acid sequences were compared with amino acid sequences of the antibodies in the database, obtained Kabat et al. (see Kabat, E.A. et al., (1991) in Seguences of Proteins of Immunological Interest, Vol. I and II, U.S. Department of Halth and Human Services). As a result, the subtype of the heavy chain SH348-1 (γ2a chain) were mixed, and the subtype of the light chain represented light chain Kappa II. In addition, the subtype of the heavy chain SH357-1 (γ2a chain) identified as mixed, and the light chain subtype was identified as a light chain Kappa II.

Thus, the synthesized following oligonucleotide primers that hybridized respectively with 5'-terminal region of the antibody gene, which encodes a region related to the subtypes of the mouse and its 3'-terminal region that contains the stop codon (see Kabat et al., ibid; Matti Kartinen et al. (1988) 25, 859-865; and Heinrich, G. et al. (1984) J. Exp. Med. 159, p. 417-435):

5'-cagatccagttggtgcagtctggacct-3' (DB3F1: list of sequences of sequence ID No. 30)

5'-aagatatctcatttacccggagtccgggagaa-3' (MIG2AEVR1: in the list of sequences sequence ID No. 31)

5'-aagaattcatgaagttgcctgttagg-3' (MK19EIF1: in the list of sequences sequence ID No. 32)

5'-aagatatcttaacactcattcctgttgaagct-3' (KEVR1: in the list of sequences sequence ID No. 33)

To clone the cDNA encoding the heavy and light chains SH348-1 and SH357-1, received mRNA from hybridomas that produce SH348-1 and SH357-1, using the cleaning kit mRNA Quick Prep (GE Healthcare Bio-Sciences Corp., #27-9254-01). Each mRNA, thus obtained, amplified cDNA encoding a heavy or light chain of each antibody using a set of PCR RNA TaKaRa One Step (AMV) (produced TAKAR BIO INC., #RR024A) and a set of primers for the synthesis of the heavy chain (combination DB3F1 and MIG2AEVR1) or a set of primers for the synthesis of the light chain (combination MK19EIF1 and KEVR1). Such cDNA, amplificatoare by PCR, cloned using the kit to clone the Zero Blunt TOPO PCR (Invitrogen Corp.). Was determined for each of the cloned nucleotide sequences of the light and heavy chains, using the analyzer gene sequences ("ABI PRISM 3700 DN Analyzer; Applied Biosystems" or "Applied Biosystems 3730xl Analyzer; Applied Biosystems"). For sequencing reactions used the GeneAmp 9700 (Applied Biosystems).

Installed the nucleotide sequence of the cDNA that encodes a heavy chain SH348-1 corresponds to SEQ ID NO: 34 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 35. The nucleotide sequence of cDNA encoding the light chain of SH348-1 corresponds to SEQ ID NO: 36 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 37 in the list of sequences. The nucleotide sequence of the cDNA that encodes a heavy chain SH357-1 corresponds to SEQ ID NO: 38 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 39. The nucleotide sequence of the cDNA that encodes a light chain SH357-1 corresponds to SEQ ID NO: 40 in the list of sequences, and its amino acid sequence corresponds SQ ID NO: 41. The sequence corresponding to nucleotides with No. from 1 to 27 and from 1327 to 1350 in SEQ ID NO: 34, the sequence corresponding to nucleotides with No. from 637 to 660 in SEQ ID NO: 36, sequence corresponding to nucleotides with No. from 1 to 27 and from 1327 to 1350 in SEQ ID NO: 38, and the sequence corresponding to nucleotides with No. from 637 to 660 in SEQ ID NO: 40 represent sequences derived from the primers.

In addition, amino acid sequences of these heavy and light chains were analyzed, comparing with the amino acid sequences of the antibodies in the database, obtained Kabat et al. (see Kabat, E.A., et al. (1991) in "Sequence of Proteins of Immunological Interest, Vol. I and II; U.S. Department of Health and Human Services). The result showed that the heavy chain SH348-1 has the amino acid sequence corresponding to the amino acids of the variable region with No. from 1 to 119 in SEQ ID NO: 35 in the list of sequences and has an amino acid sequence corresponding to the amino acids of the constant region with No. from 120 to 449, ibid. In addition, it was shown that light chain SH348-1 has the amino acid sequence corresponding to the amino acids of the variable region with No. from 1 to 112 in SEQ ID NO: 37 in the list of sequences and has an amino acid sequence corresponding to the amino acids of the constant region with No. from 113 to 219, ibid.

Showed that the heavy chain SH357-1 shall meet the amino acid sequence, the corresponding amino acids of the variable region with No. from 1 to 119 in SEQ ID NO: 39 in the list of sequences and has an amino acid sequence corresponding to the amino acids of the constant region with No. from 120 to 449, ibid. In addition, it was shown that light chain SH357-1 has the amino acid sequence corresponding to the amino acids of the variable region with No. from 1 to 112 in SEQ ID NO: 41 in the list of sequences and has an amino acid sequence corresponding to the amino acids of the constant region with No. from 113 to 219, ibid.

The nucleotide sequence encoding the variable region of the heavy chain SH348-1 corresponds to SEQ ID NO: 42 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 43. The nucleotide sequence encoding the constant region of the heavy chain SH348-1 corresponds to SEQ ID NO: 44, and its amino acid sequence corresponds to SEQ ID NO: 45. The nucleotide sequence encoding the variable region of the light chain of SH348-1 corresponds to SEQ ID NO: 46 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 47. The nucleotide sequence encoding the constant region of the light chain of SH348-1 corresponds to SEQ ID NO: 48, and its amino acid sequence corresponds to SEQ ID NO: 49. Sequence, with the matter of the nucleotides with No. from 1 to 27 in SEQ ID NO: 42, the sequence corresponding to nucleotides with No. from 970 to 993 in SEQ ID NO: 44, and the sequence corresponding to nucleotides with No. 301 to 324 in SEQ ID NO: 48, represent sequences derived from the primers.

In addition, the nucleotide sequence encoding the variable region of the heavy chain SH357-1 corresponds to SEQ ID NO: 50 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 51. The nucleotide sequence encoding the constant region of the heavy chain SH357-1 corresponds to SEQ ID NO: 52, and its amino acid sequence corresponds to SEQ ID NO: 53. The nucleotide sequence encoding the variable region of the light chain SH357-1 corresponds to SEQ ID NO: 54 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 55. The nucleotide sequence encoding the constant region of the light chain SH357-1 corresponds to SEQ ID NO: 56, and its amino acid sequence corresponds to SEQ ID NO: 57. The sequence corresponding to nucleotides with No. from 1 to 27 in SEQ ID NO: 50, the sequence corresponding to nucleotides with No. from 970 to 993 in SEQ ID NO: 52, and the sequence corresponding to nucleotides with No. from 301 to 324 in SEQ ID NO: 56 represent sequences derived from the primers.

In addition, the provisions and consequences of the successive CDRs in each of the amino acid sequences of variable regions of heavy and light chains was analyzed and was determined by comparing the homology with the amino acid sequences of the antibodies in the database, received Kabat et al. (see Kabat, E.A., et al. (1991) ibid). In accordance with this document, even different antibodies have a frame region, which have almost equal amino acid in length and they see the presence of similarity of amino acid sequences in variable regions, if they are in the same subtype. On the other hand, CDR represent a specific sequence, flanked these frame areas. Thus, the amino acid sequences of the heavy and light chains were analyzed by comparison with the amino acid sequences of the heavy and light chains of the same subtype. As a result determined that the CDRs in the heavy chain SH348-1 has the amino acid sequence corresponding to amino acids No. from 26 to 35 in SEQ ID NO: 35 in the list of sequences (CDRH1), amino acids with No. from 50 to 66 ibid (CDRH2) and amino acids with No. from 99 to 108 ibid (CDRH3). Has determined that the CDRs in the light chain of SH348-1 has the amino acid sequence corresponding to amino acids No. from 24 to 39 in SEQ ID NO: 37 in the list of sequences (CDRL1), amino acids with No. from 55 to 61 ibid (CDRL2) and amino acids with No. from 94 to 102 ibid (CDRL3). Has determined that the CDRs in the heavy chain SH357-1 has the amino acid sequence corresponding to amino acids No. from 26 to 35 in SEQ I NO: 39 in the list of sequences (CDRH 1), amino acids with No. from 50 to 66 ibid (CDRH2) and amino acids with No. from 99 to 108 ibid (CDRH3). Has determined that the CDRs in the light chain SH357-1 has the amino acid sequence corresponding to amino acids No. from 24 to 39 in SEQ ID NO: 41 in the list of sequences (CDRL1), amino acids with No. from 55 to 61 ibid (CDRL2) and amino acids with No. from 94 to 102 ibid (CDRL3).

The nucleotide sequence encoding CDRH1SH348-1 corresponds to SEQ ID NO: 58 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 59. The nucleotide sequence encoding CDRH2SH348-1 corresponds to SEQ ID NO: 60, and its amino acid sequence corresponds to SEQ ID NO: 61. The nucleotide sequence encoding CDRH3SH348-1 corresponds to SEQ ID NO: 62, and its amino acid sequence corresponds to SEQ ID NO: 63. The nucleotide sequence encoding a CDRL1SH348-1 corresponds to SEQ ID NO: 64, and its amino acid sequence corresponds to SEQ ID NO: 65. The nucleotide sequence encoding a CDRL2SH348-1 corresponds to SEQ ID NO: 66, and its amino acid sequence corresponds to SEQ ID NO: 67. The nucleotide sequence encoding a CDRL3SH348-1 corresponds to SEQ ID NO: 68, and its amino acid sequence corresponds to SEQ ID NO: 69. In addition, nucleotide n is the sequence, coding CDRH1SH357-1 corresponds to SEQ ID NO: 70, and its amino acid sequence corresponds to SEQ ID NO: 71. The nucleotide sequence encoding CDRH2SH357-1 corresponds to SEQ ID NO: 72, and its amino acid sequence corresponds to SEQ ID NO: 73. The nucleotide sequence encoding CDRH3SH357-1 corresponds to SEQ ID NO: 74, and its amino acid sequence corresponds to SEQ ID NO: 75. The nucleotide sequence encoding a CDRL1SH357-1 corresponds to SEQ ID NO: 76, and its amino acid sequence corresponds to SEQ ID NO: 77. The nucleotide sequence encoding a CDRL2SH357-1 corresponds to SEQ ID NO: 78, and its amino acid sequence corresponds to SEQ ID NO: 79. The nucleotide sequence encoding a CDRL3SH357-1 corresponds to SEQ ID NO: 80, and its amino acid sequence corresponds to SEQ ID NO: 81.

(Example 6) the binding Activity of the antibody to EPHA2 with extracellular region EPHA2

The solution of the polypeptide extracellular region EPHA2 (produced R&D Systems, Inc., #3035-A2-100) or bovine serum albumin as a control (in the description hereinafter and in the figures, abbreviated to "BSA"), diluted with PBS to 1 μg/ml was applied at the rate of 100 μl/well to immunoplate (Nunc, #442404) and incubated over night at 4°C, thereby absorbing protein on the tablet.

For CL, the blowing day solution from the wells was removed, and the solution of Block Ace (one sachet of powder Block Ace (Dainippon Sumitomo Pharma Co., Ltd. (Show Brand Milk Products Co., Ltd.)) was dissolved in 100 ml of ultrapure water), diluted in PBS 4 times was applied at the rate of 200 µl/well and incubated at room temperature for 1 hour. The wells were washed twice with a buffer for dilution (PBS, 0.05% of (about./about.) Tween 20). Further, separately diluted PBS SH348-1 AND SH357-1 and Ab96-1 to a concentration of 1.25×10-4μg/ml, 1,25×10-3μg/ml, 1,25×10-2μg/ml, 1,25×10-1µg/ml of 1.25 ág/ml 12.5 ág/ml 125 ág/ml and the resulting solution (containing 0.05% (vol./about.) (final concentration) Tween 20) was added at the rate of 100 μl/well.

Tablet incubated at room temperature for 1 hour. Then, the solution from the wells was removed, and the wells washed twice with buffer for cultivation. Antibody goat to mouse IgG conjugated to peroxidase (produced by Millipore (Chemicon), #AP181P)diluted 1000 times with a buffer for dilution, was added at the rate of 100 μl/well and incubated at room temperature for 1 hour. The solution from the wells was removed, and the wells washed twice with buffer for cultivation. Next, the color reaction was performed with stirring, adding the solution to the degree of color development OPD at the rate of 100 μl/well. After staining, the color reaction was stopped by adding 1M HCl at the rate of 100 µl/well. Measured the absorbance at 490 nm using spectrophotometers reading tablets (Fig.7).

On figa) shows the results for the SH348-1, pigv) shows the results for SH357-1 and figs) shows the results for Ab96-1. On each graph, the absorption is shown as mean ± standard deviation (n=3). Stronger absorption indicates a stronger binding activity. As shown in the graphs, all antibodies SH348-1 AND SH357-1 and Ab96-1, does not exhibit affinity for BSA, demonstrate that they are specific in contact with the extracellular region of EPHA2.

(Example 7) Effect of antibody to EPHA2 binding ligand

Polypeptide extracellular region EPHA2 (produced R&D Systems, Inc., #3035-A2-100), immobilized on immunoplate receive the same manner as described in example 6. The wells of immunoplate twice washed with buffer for cultivation. Next was added SH348-1 AND SH357-1, Ab96-1 or izotopicheskii control IgG2Amouse (in the description hereinafter and in the figures, abbreviated to "mIgG2a"; produced R&D Systems, Inc., #MAB003) as izotopicheskogo control antibodies, diluted with buffer diluted to 10 μg/ml or 50 μg/ml, 100 μl/well. Tablet incubated at room temperature for 1 hour. Next was added a recombinant Chimera Ephrin-A1/Fc mouse (in the description hereinafter and in the figures, abbreviated to "Ephrin-A1/Fc"; produced R&D Systems, Inc., #602-A1-200) as a soluble ligand or recombinant IgG Fc1person (in the description hereinafter and in the figures, with Krasnogo to "hG 1Fc"; produced R&D Systems, Inc., #110-HG-100) as a negative control protein to soluble ligand to a final concentration of 1 μg/ml and incubated at room temperature for 1 hour.

Further according to the method described in example 6, a solution from the wells was removed, and the wells were washed with buffer for cultivation. Added to them are conjugated with peroxidase antibodies goat affinity purity (AffiniPure)specific to Fcγ fragment of human IgG (Jackson ImmunoResearch Laboratories, Inc., #109-035-098). Color reaction was performed using the solution for the manifestation of color OPD. Measured the absorbance at 490 nm, using a spectrophotometer to read the tablets (Fig).

On Fig shows the absorption as mean ± standard deviation (n=3). Antibody Ab96-1 even at a concentration of 10 μg/ml strongly inhibited the binding of the ligand of EPHA2 ephrin-A1 with EPHA2. In contrast, antibodies SH348-1 and SH357-1, even added in a concentration of 50 μg/ml (five concentrations Ab96-1), is not inhibited binding of Ephrin-A1/Fc with EPHA2. These results showed that antibodies SH348-1 and SH357-1 does not inhibit the binding of Ephrin-A1/Fc with EPHA2.

(Example 8) Check the activity of inhibition of antibody to EPHA2 relative to the dependent from ephrin-A1 phosphorylation tyrosine residues EPHA2

8)-1 Obtain cell lysates

Cells MDA-MB-231, suspended in RPMI1640 containing 10% ETS, 50 units/ml penicilline 50 µg/ml streptomycin (hereinafter, abbreviated to "RPMI1640 containing 10% ETS (with antibiotics)") were sown at the rate 2.5×105cells/well on 12-well plate and cultured overnight at 37°C in 5% CO2. Then the medium from the wells was removed and the newly added RPMI1640. Cells were additionally cultured overnight at 37°C in 5% CO2. Then the medium from the wells was removed and only RPMI1640 or RPMI1640 containing antibody (mIgG2a, SH348-1 and SH357-1) at a concentration of 10 μg/ml or 50 μg/ml was added to each well and pre-incubated at 37°C in 5% CO2within 1 hour.

In wells only added RPMI1640, pre proinsurance thus added 1/50 volume Ephrin-A1/Fc or hG1Fc (final concentration 1 μg/ml) or RPMI1640 in the same volume (figure 9, presents (-)). In addition, wells with added mIgG2a, SH348-1 and SH357-1, was added 1/50 volume Ephrin-A1/Fc (final concentration 1 μg/ml).

Die additionally incubated at 37°C in 5% CO2within 15 minutes. After removal of the supernatant, for lysis of cells was added 1×buffer for lysis of the cells (Cell Signaling Technology, Inc.) containing 1 mm PMSF (Sigma-Aldrich, Inc.) and a cocktail of protease inhibitors (Sigma-Aldrich, Inc., #P8340) (hereinafter, abbreviated to PPCLB). The lysate was centrifuged at 15000 rpm./minutes within 5 minutes, and the resulting supernatant used as samples for immunoplate is Itachi. The protein concentration in the samples was measured using the reagent for protein analysis ICA (produced by PIERCE).

8)-2 Detection of phosphorylated forms of EPHA2 by thus

Added 25 μl of a suspension of magnetic particles with protein G (NEW ENGLAND BioLabs, Inc.) on the sample, and 4 µg of antibody to EphA2 (Santa Cruz Biotechnology, Inc., #sc-924), and the mixture turned for mixing at 4°C for 2 hours. Next to it was added by the ETS to a final concentration of 10% and the mixture is additionally turned for mixing at 4°C for 30 minutes. Next, the particles were washed PPCLB. Next to it was added 200 mg of supernatant cell lysate obtained in paragraph 8)-1 and the mixture turned to mix overnight at 4°C.

The next day the particles three times washed PPCLB. Next, the particles were added to the sample buffer SDS (56,3 mm Tris-HCl, pH 6,8, 1,8% (wt./about.) SDS, 9% glycerol, 0,72M 2-mercaptoethanol, 0,045 mg/ml bromophenol blue), and the mixture was let warm up for at 98°C for 5 minutes. Proteins, dissociative with particles were separated in SDS-PAGE.

Proteins transferred from the gel onto PVDF-membrane (size of pores of 0.45 μm; produced by Millipore). PVDF-membrane was blocked by shaking in blocking solution without sodium azide (one sachet of powder Block Ace was dissolved in 100 ml of ultrapure water, then added Tween 20 at a final concentration of 0.1% (vol./vol.)).

To determine phospho who pilirovanny form tyrosine residues EPHA2, PVDF-membrane was immersed in a solution of Anti-Phosphotyrosine, recombinant HRP-conjugate 4G10 (Upstate, #16-184)diluted 10,000 times with the blocking solution without sodium azide and left for reaction at room temperature for 1 hour. PVDF-membrane was washed three times for 10 minutes with TBST (50 mm Tris-HCl, pH 8.0, 138 mm NaCl, 2.7 mm KCl, 0,1% (vol./about.) Tween 20) and then further washed with H2O three times for 5 minutes. On the film for chemiluminescence was detected signals using ECL Plus (GE Healthcare Bio-Sciences Corp.).

Further, for detection immunoprecipitating EPHA2 that is present on this PVDF-membrane, a PVDF-membrane after detection of phosphorylated forms of tyrosine residues EPHA2 was immersed in the solution to remove (50 mm Tris-HCl, pH of 6.8, 2% (wt./about.) SDS, 100 mm 2-mercaptoethanol) and were shaken at 55°C for 30 minutes. Then, the PVDF-membrane was immersed in the solution for damping (TBST containing 1% (vol./about.) H2O2and 0.1% (wt./about.) NaN3) and was shaken at room temperature for 20 minutes to remove antibodies to PVDF-membrane. PVDF-membrane was washed in TBST three times for 10 minutes and blocked in blocking solution (one sachet of powder Block Ace was dissolved in 100 ml of ultrapure water, then added Tween 20 and sodium azide to a final concentration of 0.1% (vol./about.) and 0.02% (wt./vol.), respectively). Next, the PVDF-membrane was immersed in Astor antibody to EphA2 (Santa Cruz Biotechnology, Inc., #sc-924)diluted 4000 times in blocking solution and left to react at room temperature for 1 hour. PVDF-membrane was washed in TBST three times for 10 minutes. Next, the PVDF-membrane was immersed in the solution associated with the HRP antibodies to rabbit IgG (Cell Signaling Technology, Inc., #7074)diluted in TBST 10,000 times and left for reaction at room temperature for 30 minutes. On the PVDF-membrane was washed in TBST three times for 10 minutes. Next on the film for chemiluminescence was detected signals using ECL Plus.

Soluble ligand Ephrin-A1/Fc, inducing phosphorylation of tyrosine residues EPHA2, not inhibited ezotericheskim control mIgG2a antibody, but inhibited in a dose-dependent manner, adding antibodies SH348-1 and SH357-1 (Fig.9).

These results showed that antibodies SH348-1 and SH357-1 does not inhibit the binding of the ligand afrina-A1 with EPHA2, but inhibit induced by ligand phosphorylation of tyrosine residues EPHA2.

(Example 9) identification of the epitope for antibody to EPHA2

Received deletion of EPHA2 mutants containing the area indicated on figure 10, and determined the binding site with the SH348-1 and SH357-1.

9)-1 Obtain deletion mutants EPHA2

For the expression of deletion mutants of EPHA2 in the form of protein with GST-Tag and His-Tag added to the N-end and S-Tag added to the C-end, synthesized the following primers, and gene fragments, amplificatoare way below cloned in pET-49b (+) (Novagen):

5'-attaggatccgagcttccgtactgccagtgtc-3' (primer 1 sequence ID No. 82)

5'-attaggatccgccccccaaggtgaggct-3' (primer N2: sequence ID No. 83)

5'-attaggatccggtcacttaccgcaagaagggaga-3' (primer N3: sequence ID No. 84)

5'-attaggatccggtccaggtgcaggcactgacg-3' (primer N4: sequence ID No. 85)

5'-aattaagcttgccgccaatcaccgccaagtt-3' (primer C1: sequence ID No. 86)

5'-aattaagcttgttgccagatccctccggggac-3' (primer C2: sequence ID No. 87)

5'-aattaagcttcaggtaggtggtgtctggg-3' (primer C3: sequence ID No. 88)

5'-aattaagcttctcgtacttccacactcggc-3' (primer C4: sequence ID No. 89)

Each segment amplified by PCR reaction using pcDNA-DEST40-EPHA2 as a matrix and a set of primers: the primers N1 and C1 for the site consisting of the amino acid sequence corresponding to amino acids No. 426-540 in SEQ ID NO: 8 in the list of sequences (hereinafter, referred to as "m1"); primers N1 and C2 for the site consisting of the amino acid sequence corresponding to amino acids No. 426-534 ibid (hereinafter, referred to as "m2"); primers N1 and C3 for the site consisting of the amino acid sequence corresponding to amino acids No. 426-504 ibid (hereinafter, referred to as "m3"); primers N1 and C4 for the site consisting of the amino acid sequence corresponding to amino is the slot with No. 426-470 ibid (hereinafter, referred to as "m4"); primers N2 and C2 for the site consisting of the amino acid sequence corresponding to amino acids No. 439-534 ibid (hereinafter, referred to as "m5"); primers N3 and C2 for the site consisting of the amino acid sequence corresponding to amino acids No. 471-534 ibid (hereinafter, referred to as the "m6"); primers N4 and C2 for the site consisting of the amino acid sequence corresponding to amino acids No. 505-534 ibid (hereinafter, referred to as the "m7"); primers N2 and C3 for the site consisting of the amino acid sequence corresponding to amino acids No. 439-504 the same (hereinafter, referred to as "m8"); and primers N2 and C4 for the site consisting of the amino acid sequence corresponding to amino acids No. 439-470 ibid (hereinafter, referred to as "m9"). Obtained PCR products were digested BamHI and HindIII and subcloned into the BamHI site/HindIII pET-49b (+).

9)-2 Expression of deletion mutants EPHA2

Escherichia coli BL21 (DE3) transformed plasmid DNA constructed in paragraph 9)-1, or, as negative control, pET-49b (+). The obtained transformants were cultured in LB medium, supplemented with 30 µg/ml kanamycin (Invitrogen Corp.). Expression of deletion mutants induced using a system of self-induction (produced by Novagen). Bacterial cells were collected through the om centrifugation and washed with PBS. Then the bacterial cells were subjected to lysis solution 2% SDS containing 1 mm PMSF and a cocktail of protease inhibitors (Sigma-Aldrich, Inc., #P8340). The supernatant was collected by centrifugation and used in the identification of the epitope.

The level of protein expression (containing obtained with GST-Tag, His-Tag and S-Tag pET-49b (+) and linker elements for connecting them; in the description hereinafter and in the figures referred to as "Vec"), expressed in the bacterial lysate of Escherichia coli cells, transformed with pET-49b (+) was estimated by the method described hereinafter. A series of dilutions of the bacterial lysate of cells expressing Vec, and a series of dilutions of the marker proteins with 6x His (QIAGEN) were separately dissolved in sample buffer with SDS and warmed up at 98°C for 5 minutes. Next, proteins were separated in SDS-PAGE and the proteins in the gel were transferred to PVDF-membrane. PVDF-membrane was blocked in TBST containing 5% BSA. Next, the PVDF-membrane was immersed in a solution of Penta-His, HRP conjugate (QIAGEN), diluted 1000 times in TBST containing 5% BSA, and reaction was performed at room temperature for 1 hour. This PVDF-membrane was washed in TBST three times for 10 minutes. Next were detected signals using ECL Plus. Signal intensity was compared between a series of dilutions of the bacterial lysate of cells expressing Vec, and a series of dilutions of the marker proteins with 6x His, and the protein concentration Vec, Paul is obtained from the lysate of bacterial cells, expressing Vec, was assessed by the level of protein in the lane containing marker proteins with 6x His. Deletion mutants of EPHA2 with a number showing the reactivity equivalent to 20 ng Vec Western-blotting using monoclonal antibodies S-Tag (Novagen) was used in the subsequent experiment on the identification of the epitope.

9)-3) identification of the epitope

Cell lysates containing deletion mutants of EPHA2 obtained in paragraph 9)-2 was dissolved in sample buffer with SDS and warmed up at 98°C for 5 minutes. The samples obtained were separated in SDS-PAGE and the proteins in the gel were transferred to PVDF membrane. After transfer, PVDF-membrane was blocked by shaking in blocking solution (one sachet of powder Block Ace was dissolved in 100 ml of ultrapure water, then added Tween 20 and sodium azide in a final concentration of 0.1% (vol./about.) and 0.02% (wt./vol.), respectively). Then the PVDF membrane was left over night at 4°C in blocking solution containing 2 mg/ml of SH348-1 and SH357-1, for the reaction. These PVDF-membranes were washed in TBST three times for 10 minutes and was further subjected to reaction at room temperature for 30 minutes in a solution associated with HRP whole Ab sheep to mouse Ig, diluted 5000 times TBST. Subsequently, the PVDF membrane was washed in TBST three times for 10 minutes. Next on the film for chemiluminescence was detected signal is s, using ECL Plus.

Then, the PVDF membrane was kept in the solution for removal (50 mm Tris-HCl, pH of 6.8, 2% (wt./about.) SDS, 100 mm 2-mercaptoethanol), then were shaken at 55°C for 30 minutes, and then washed with TBST three times for 10 minutes. These PVDF-membrane was blocked in blocking solution and then washed with TBST three times for 10 minutes. PVDF-membrane was kept in the solution of monoclonal antibodies S-Tag, diluted 10,000 times in TBST, and left for reaction at room temperature for 30 minutes. The PVDF membrane was washed in TBST three times for 10 minutes. Then, the PVDF membrane was kept in the solution associated with the HRP whole Ab sheep to mouse Ig, diluted 5000 times TBST, and left for reaction at room temperature for 30 minutes. Next, the PVDF membrane was washed in TBST three times for 10 minutes. Then, on the film for chemiluminescence was detected signals using ECL Plus.

As a result, both antibodies SH348-1 (figa) and SH357-1 (figs) was active binding only with m1, m2 and m5. In addition, in this way, deletion mutants of EPHA2 were present in almost equal amounts on each of the PVDF-membranes (SH348-1: figv, SH357-1: fig.11D). These results showed that antibodies SH348-1 and SH357-1 are associated with a plot consisting of the amino acid sequence corresponding to amino acids No. 439-534 in SEQ ID NO: 8 in the list of amino acid sequences is posledovatelnosti EPHA2.

(Example 10) Design gumanitarnogo antibodies

10)-1 Designing gumanitarnogo antibodies SH348-1

10)-1-1 Molecular modeling variable regions of SH348-1.

Molecular modeling of the variable regions of SH348-1 was performed by homology modeling (Methods in Enzymology, 203, 121-153, (1991)). Primary sequence (available spatial structure obtained from x-ray crystal structure) variable regions of human immunoglobulins registered in the database of proteins (Nuc. Acid Res. 35, D301-D303 (2007)) was compared with the variable regions of SH348-1, defined in example 5. As a result, 2JEL and 1A4J have chosen, respectively, as the sequence with the highest homology sequence of the variable regions of the light and heavy chain SH348-1. The spatial structure of framework regions were obtained on the basis of "wireframe", obtained by combining the coordinates 2JEL and 1A4J corresponding light and heavy chains SH348-1. For CDR SH348-1, CDRL1, CDRL2, CDRL3, CDRH1and CDRH2consistent with the clusters 16A, 7A, 9A, 10A and 10A, respectively, according to the classification of Thornton et al. (J. Mol. Biol., 263, 800-815, (1996)). CDRH3classified in e (9) D according to the rules of H3 (FEBS letters 399, 1-8 (1996)). Subsequently, a typical conformation of each CDR included in the wireframe model.

In C the conclusion is, to obtain probable, from the point of view of energy, the molecular model of the variable regions of SH348-1, conducted energy calculation to exclude unfavorable atomic interactions. These methods were performed using the commercially available program Prime for prediction of protein spatial structure and program coordinate search MacroModel (Schrödinger, LLC).

10)-1-2 Designing amino acid sequence gumanitarnogo SH348-1

Humanized antibodies SH348-1 designed according to the “transplant CDR” (Proc. Natl. Acad. Sci. 86 USA 1002 9-10033 (1989)). Acceptor antibodies were selected on the basis of homology of the amino acids in the framework regions. Sequence frame areas SH348-1 compared with all frame areas of a person registered in the Kabat database (Nuc. Acid Res. 29, 205-206 (2001))that includes the amino acid sequence of the antibody. Antibody GSD2B5B10'CL was chosen as an acceptor due to the fact that it is 72%, the maximum sequence homology between their frame areas. Amino acid residues frame areas in GSD2B5B10'CL was aligned with the corresponding amino acid residues in the SH348-1 to identify provisions between them, which used a different amino acid. The position of these residues were analyzed using a model of the spatial structure of SH348-1, constructed in the same the way. Then, selected amino acid residues of the donor, subject to transplant to the acceptor, in accordance with the criteria provided by Queen et al. (Proc. Natl. Acad. Sci. 86 USA 1002 9-10033 (1989)). Humanized sequence SH348-1 was determined by transferring some selected residues of the donor to the acceptor antibody GSD2B5B10'CL. As a result, humanized sequence of two types of light chains and two types of heavy chains were obtained as described below. Further, the variable and constant region and CDR classified based on their database of amino acid sequences of the antibodies obtained Kabat and other

10-1-3) Humanization light chain SH348-1

10-1-3-1) Type hSH-348-T1L light chain:

Humanitarian light chain SH348-1 was designed by replacing amino acids with No. 2 (valine), 3 (leucine), 14 (serine), 15 (leucine), 17 (aspartic acid), 18 (glutamine), 50 (lysine), 79 (arginine), 88 (leucine), 105 (glycine), 109 (leucine) and 114 (alanine) of the variable region of the light chain SH348-1, are shown in SEQ ID NO: 37 list of sequences for isoleucine, valine, threonine, Proline, glutamic acid, Proline, glutamine, lysine, valine, glutamine, valine and threonine, respectively, was designated as "type a light chain hSH348-T1L".

The nucleotide sequence encoding the type of light chain hSH348-1-T1L corresponds to SEQ ID NO: 90 in the list of sequences, and the amino acid which corresponds to the selected SEQ ID NO: 91. The nucleotide sequence corresponding to nucleotides with No. 1-60 in SEQ ID NO: 90 is a signal sequence for secretion. The nucleotide sequence corresponding to nucleotides with No. 61-402, there is a variable region. The nucleotide sequence corresponding to nucleotides with No. 403-717, there is a constant region. The nucleotide sequence corresponding to nucleotides with No. 130-177, there is a CDRL1. The nucleotide sequence corresponding to nucleotides with No. 223-243, there is a CDRL2. The nucleotide sequence corresponding to nucleotides with No. 340-363, there is a CDRL3.

Amino acid sequence corresponding to amino acids No. 1-20 in SEQ ID NO: 91 list sequence is a signal sequence for secretion. Amino acid sequence corresponding to amino acids No. 21-134, there is a variable region. Amino acid sequence corresponding to amino acids No. 135-239, there is a constant region. Amino acid sequence corresponding to amino acids No. 44-59, there is a CDRL1. Amino acid sequence corresponding to and is inoculated with No. 75-81, there is a CDRL2. Amino acid sequence corresponding to amino acids No. 114-121, there is a CDRL3.

In addition, the nucleotide sequence encoding a CDRL1light chain type hSH348-1-T1L, corresponds to SEQ ID NO: 92 in the list of sequences, its amino acid sequence corresponds to SEQ ID NO: 93, the nucleotide sequence of CDRL2corresponds to SEQ ID NO: 94 in the list of sequences, its amino acid sequence corresponds to SEQ ID NO: 95, the nucleotide sequence of CDRL3corresponds to SEQ ID NO: 96 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 97.

10-1-3-2) Type hSH348-T3L light chain:

Humanitarian light chain SH348-1 was designed by replacing amino acids with No. 14 (serine), 15 (leucine), 17 (aspartic acid), 18 (glutamine), 50 (lysine), 79 (arginine), 88 (leucine), 105 (glycine), 109 (leucine) and 114 (alanine) of the variable region of the light chain SH348-1, are shown in SEQ ID NO: 37 list of sequences for threonine, Proline, glutamic acid, Proline, glutamine, lysine, valine, glutamine, valine and threonine, respectively, was designated as "type hSH348-1-T3L light chain".

The nucleotide sequence encoding the type hSH348-1-T3L light chain corresponds to SEQ ID NO: 98 in the list of sequences, is its amino acid sequence corresponds to SEQ ID NO: 99. The nucleotide sequence corresponding to nucleotides with No. 1-60 in SEQ ID NO: 98 is a signal sequence for secretion. The nucleotide sequence corresponding to nucleotides with No. 61-402, there is a variable region. The nucleotide sequence corresponding to nucleotides with No. 403-717, there is a constant region. The nucleotide sequence corresponding to nucleotides with No. 130-177, there is a CDRL1. The nucleotide sequence corresponding to nucleotides with No. 223-243, there is a CDRL2. The nucleotide sequence corresponding to nucleotides with No. 340-363, there is a CDRL3.

Amino acid sequence corresponding to amino acids No. 1-20 in SEQ ID NO: 99 list sequence is a signal sequence for secretion. Amino acid sequence corresponding to amino acids No. 21-134, there is a variable region. Amino acid sequence corresponding to amino acids No. 135-239, there is a constant region. Amino acid sequence corresponding to amino acids No. 44-59, there is a CDRL1. Amino acid sequence corresponding to and is inoculated with No. 75-81, there is a CDRL2. Amino acid sequence corresponding to amino acids No. 114-121, there is a CDRL3.

In addition, the nucleotide sequence encoding a CDRL1light chain type hSH348-1-T3L, corresponds to SEQ ID NO: 100 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 101, the nucleotide sequence of CDRL2corresponds to SEQ ID NO: 102 in the list of sequences, its amino acid sequence corresponds to SEQ ID NO: 103, the nucleotide sequence of CDRL3corresponds to SEQ ID NO: 104 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 105.

10-1-4) Humanization of the heavy chain SH348-1

10-1-4-1) Type hSH348-1-T1H heavy chain:

Humanitarian heavy chain SH348-1 was designed by replacing amino acids with No. 2 (isoleucine), 9 (Proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 38 (lysine), 43 (lysine), 46 (lysine), 68 (phenylalanine), 69 (alanine), 70 (phenylalanine), 71 (serine), 72 (leucine), 73 (glutamic acid), 76 (alanine), 80 (phenylalanine), 82 (glutamine), 83 (isoleucine), 84 (asparagine), 85 (asparagine), 87 (lysine), 88 (asparagine), 93 (threonine), 95 (phenylalanine), 114 (threonine) and 115 (leucine) variable regions of the heavy chain SH348-1 is presented in SEQ ID NO: 43 list of sequences on the'alene, alanine, valine, serine,serine, valine, arginine, glutamine, glutamic acid, valine, threonine, isoleucine, threonine, alanine, aspartic acid, threonine, tyrosine, glutamic acid, leucine, serine, serine, arginine, serine, valine, tyrosine, leucine and valine, respectively, and designated as "type hSH348-1-T1H heavy chain.

The nucleotide sequence encoding the type hSH348-1-T1H heavy chain corresponds to SEQ ID NO: 106 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 107. The nucleotide sequence corresponding to nucleotides with No. 1-57 in SEQ ID NO: 106 is a signal sequence for secretion. The nucleotide sequence corresponding to nucleotides with No. 58-414, there is a variable region. The nucleotide sequence corresponding to nucleotides with No. 415-1404, there is a constant region. The nucleotide sequence corresponding to nucleotides with No. 148-162, there is a CDRH1. The nucleotide sequence corresponding to nucleotides with No. 205-255, there is a CDRH2. The nucleotide sequence corresponding to nucleotides with No. 352-381, there is a CDRL3.

Amino acid sequence corresponding to amino acids No. 1-19 in SEQ ID NO: 107 list p is sledovatelnot is a signal sequence for secretion. Amino acid sequence corresponding to amino acids No. 20-138, there is a variable region. Amino acid sequence corresponding to amino acids No. 139-468, there is a constant region. Amino acid sequence corresponding to amino acids No. 50-54, there is a CDRH1. Amino acid sequence corresponding to amino acids No. 69-85, there is a CDRH2. Amino acid sequence corresponding to amino acids No. 118-127, there is a CDRH3.

In addition, the nucleotide sequence encoding CDRH1the heavy chaintype hSH348-1-T1H, corresponds to SEQ ID NO: 108 in the list of sequences, the amino acid sequence thereof corresponds to SEQ ID NO: 109, the nucleotide sequence of CDRH2corresponds to SEQ ID NO: 110 in the list of sequences, its amino acid sequence corresponds to SEQ ID NO: 111, the nucleotide sequence of CDRH3corresponds to SEQ ID NO: 112 in the list of sequences and amino acid sequence corresponds to SEQ ID NO: 113.

10-1-4-2) Type hSH348-1-T3H heavy chain:

Humanitarian heavy chain SH348-1 was designed by replacing amino acids with No. 9 (Proline), 11 (leucine), 16 (glutamine KIS the PTA), 17 (threonine), 20 (isoleucine), 38 (lysine), 43 (lysine), 73 (glutamic acid), 76 (alanine), 80 (phenylalanine), 82 (glutamine), 83 (isoleucine), 84 (asparagine), 85 (asparagine), 87 (lysine), 88 (asparagine), 93 (threonine), 95 (phenylalanine), 114 (threonine) and 115 (leucine) variable domains of the heavy chain SH348-1 is presented in SEQ ID NO: 43 list of sequences of alanine, valine, serine, serine, valine, arginine, glutamine, aspartic acid, threonine, tyrosine, glutamic acid, leucine, serine, serine, arginine, serine, valine, tyrosine, leucine and valine, respectively, and designated as "type hSH348-1-T3H heavy chain.

The nucleotide sequence encoding the type hSH348-1-T3H heavy chain corresponds to SEQ ID NO: 114 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 115. The nucleotide sequence corresponding to nucleotides with No. 1-57 in SEQ ID NO: 114 is a signal sequence for secretion. The nucleotide sequence corresponding to nucleotides with No. 58-414, there is a variable region. The nucleotide sequence corresponding to nucleotides with No. 415-1404, there is a constant region. The nucleotide sequence corresponding to nucleotides with No. 148-162, there is a CDRH1. The nucleotide sequence corresponding to nucleotides No. 205-255, there is a CDRH2. The nucleotide sequence corresponding to nucleotides with No. 352-381, there is a CDRL3.

Amino acid sequence corresponding to amino acids No. 1-19 in SEQ ID NO: 115 list sequence is a signal sequence for secretion. Amino acid sequence corresponding to amino acids No. 20-138, there is a variable region. Amino acid sequence corresponding to amino acids No. 139-468, there is a constant region. Amino acid sequence corresponding to amino acids No. 50-54, there is a CDRH1. Amino acid sequence corresponding to amino acids No. 69-85, there is a CDRH2. Amino acid sequence corresponding to amino acids No. 118-127, there is a CDRH3.

In addition, the nucleotide sequence encoding CDRH1heavy chain type hSH348-1-T3H, corresponds to SEQ ID NO: 116 in the list of sequences, its amino acid sequence corresponds to SEQ ID NO: 117, the nucleotide sequence of CDRH2corresponds to SEQ ID NO: 118 in the list of sequences, its amino acid sequence corresponds to SEQ ID NO: 119, the nucleotide is th sequence CDRH 3corresponds to SEQ ID NO: 120 in the list of sequences and amino acid sequence corresponds to SEQ ID NO: 121.

10)-2 Design gumanitarnogo antibodies SH357-1

10)-2-1 Molecular modeling variable regions SH357-1

2JEL and 1A4J was chosen as the sequence with the highest homology sequence of the variable regions of the light and heavy chain SH357-1, respectively, in the same way as in paragraph 10)-1-1. CDRL1, CDRL2, CDRL3, CDRH1and CDRH2consistent with the clusters 16A, 7A, 9A, 10A and 10A, respectively. CDRH3classified in e(9)D.

10)-2-2 Designing amino acid sequences for gumanitarnogo SH357-1

As acceptor selected antibody GSD2B5B10'CL, in the same way as in paragraph 10)-1-2, and determined the sequence gumanitarnogo SH357-1. As a result, humanized sequence of two types of light chains and two types of heavy chains were obtained, as shown next.

10-2-3) Humanization light chain SH357-1

10-2-3-1) Type hSH357-1-T1L light chain:

Humanitariannet light chain SH357-1, designed by replacing amino acids with No. 2 (valine), 3 (leucine), 7 (threonine), 14 (serine), 15 (leucine), 17 (aspartic acid), 18 (glutamine), 50 (lysine), 88 (leucine), 105 (glycine), 109 (leucine) and 114 (alanine) light chain SH357-1, is presented in SEQ ID NO: 41 JV is ska sequences for isoleucine, valine, serine, threonine, Proline, glutamic acid, Proline, glutamine, valine, glutamine, valine and threonine, respectively, was designated as "type hSH357-1-T1L light chain".

The nucleotide sequence encoding the type hSH357-1-T1L light chain corresponds to SEQ ID NO: 122 list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 123. The nucleotide sequence corresponding to nucleotides with No. 1-60 in SEQ ID NO: 122, is a signal sequence for secretion. The nucleotide sequence corresponding to nucleotides with No. 61-402, there is a variable region. The nucleotide sequence corresponding to nucleotides with No. 403-717, there is a constant region. The nucleotide sequence corresponding to nucleotides with No. 130-177, there is a CDRL1. The nucleotide sequence corresponding to nucleotides with No. 223-243, there is a CDRL2. The nucleotide sequence corresponding to nucleotides with No. 340-363, there is a CDRL3.

Amino acid sequence corresponding to amino acids No. 1-20 in SEQ ID NO: 123 list sequence is a signal sequence for secretion. Amino acid sequence, sootvetstvuyuschayatrebovaniyam with No. 21-134, there is a variable region. Amino acid sequence corresponding to amino acids No. 135-239, there is a constant region. Amino acid sequence corresponding to amino acids No. 44-59, there is a CDRL1. Amino acid sequence corresponding to amino acids No. 75-81, there is a CDRL2. Amino acid sequence corresponding to amino acids No. 114-121, there is a CDRL3.

In addition, the nucleotide sequence encoding a CDRL1light chain type hSH357-1-T1L, corresponds to SEQ ID NO: 124 in the list of sequences, its amino acid sequence corresponds to SEQ ID NO: 125, the nucleotide sequence of CDRL2corresponds to SEQ ID NO: 126 in the list of sequences, its amino acid sequence corresponds to SEQ ID NO: 127, the nucleotide sequence of CDRL3corresponds to SEQ ID NO: 128 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 129.

10-2-3-2) Type hSH357-1-T3L light chain:

Humanitarian light chain SH357-1 designed by replacing amino acids with No. 7 (threonine), 14 (serine), 15 (leucine), 17 (aspartic acid), 18 (glutamine), 50 (lysine), 88 (leucine), 105 (glycine), 109 (leucine) and 114 (alanine) is egcoa chain SH357-1, presented in SEQ ID NO: 41, serine, threonine, Proline, glutamic acid, Proline, glutamine, valine, glutamine, valine and threonine, respectively, was designated as "type hSH357-1-T3L light chain".

The nucleotide sequence encoding the type hSH357-1-T3L light chain corresponds to SEQ ID NO: 130 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 131. The nucleotide sequence corresponding to nucleotides with No. 1-60 in SEQ ID NO: 130, is a signal sequence for secretion. The nucleotide sequence corresponding to nucleotides with No. 61-402, there is a variable region. The nucleotide sequence corresponding to nucleotides with No. 403-717, there is a constant region. The nucleotide sequence corresponding to nucleotides with No. 130-177, there is a CDRL1. The nucleotide sequence corresponding to nucleotides with No. 223-243, there is a CDRL2. The nucleotide sequence corresponding to nucleotides with No. 340-363, there is a CDRL3.

Amino acid sequence corresponding to amino acids No. 1-20 in SEQ ID NO: 131 list sequence is a signal sequence for secretion. Amino acid sequence, matched with the appropriate amino acids with No. 21-134, there is a variable region. Amino acid sequence corresponding to amino acids No. 135-239, there is a constant region. Amino acid sequence corresponding to amino acids No. 44-59, there is a CDRL1. Amino acid sequence corresponding to amino acids No. 75-81, there is a CDRL2. Amino acid sequence corresponding to amino acids No. 114-121, there is a CDRL3.

In addition, the nucleotide sequence encoding a CDRL1light chain type hSH357-1-T3L, corresponds to SEQ ID NO: 132 at the list of sequences, its amino acid sequence corresponds to SEQ ID NO: 133, the nucleotide sequence of CDRL2corresponds to SEQ ID NO: 134 in the list of sequences, its amino acid sequence corresponds to SEQ ID NO: 135, the nucleotide sequence of CDRL3corresponds to SEQ ID NO: 136 list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 137.

10-2-4) Humanization of the heavy chain SH357-1

10-2-4-1) Type hSH357-1-T1H heavy chain:

Humanitarian heavy chain SH357-1 was designed by replacing amino acids with No. 2 (isoleucine), 9 (Proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 38 lysine), 43 (lysine), 46 (lysine), 68 (phenylalanine), 69 (alanine), 70 (phenylalanine), 71 (serine), 72 (leucine), 73 (glutamic acid), 76 (alanine), 82 (glutamine), 83 (isoleucine), 85 (asparagine), 87 (lysine), 88 (asparagine), 93 (serine), 95 (phenylalanine), 114 (threonine) and 115 (leucine) variable regions of the heavy chain SH357-1, presented in SEQ ID NO: 51 list of sequences, valine, alanine, valine, alanine, serine, valine, arginine, glutamine, glutamic acid, valine, threonine, isoleucine, threonine, alanine, aspartic acid, threonine, glutamic acid, leucine, serine, arginine, serine, valine, tyrosine, leucine and valine, respectively, and designated as "type hSH357-1-T1H heavy chain.

The nucleotide sequence encoding the type hSH357-1-T1H heavy chain corresponds to SEQ ID NO: 138 in the list of sequences, and its amino acid sequence corresponds to SEQ ID NO: 139. The nucleotide sequence corresponding to nucleotides with No. 1-57 in SEQ ID NO: 138, is a signal sequence for secretion. The nucleotide sequence corresponding to nucleotides with No. 58-414, there is a variable region. The nucleotide sequence corresponding to nucleotides with No. 415-1404, there is a constant region. The nucleotide sequence corresponding to nucleotides with No. 145-162, there is a CDRH1. ucleotide sequence, corresponding to nucleotides with No. 205-255, there is a CDRH2. The nucleotide sequence corresponding to nucleotides with No. 352-381, there is a CDRL3.

Amino acid sequence corresponding to amino acids No. 1-19 in SEQ ID NO: 139 list sequence is a signal sequence for secretion. Amino acid sequence corresponding to amino acids No. 20-138, there is a variable region. Amino acid sequence corresponding to amino acids No. 139-468, there is a constant region. Amino acid sequence corresponding to amino acids No. 49-54, there is a CDRH1. Amino acid sequence corresponding to amino acids No. 69-85, there is a CDRH2. Amino acid sequence corresponding to amino acids No. 118-127, there is a CDRH3.

In addition, the nucleotide sequence encoding CDRH1heavy chain type hSH357-1-T1H corresponds to SEQ ID NO: 140 in the list of sequences, the amino acid sequence thereof corresponds to SEQ ID NO: 141, nucleotide sequence CDRH2corresponds to SEQ ID NO: 142 in the list of sequences, the amino acid sequence is W hat corresponds to SEQ ID NO: 143, the nucleotide sequence of CDRH3corresponds to SEQ ID NO: 144 in the list of sequences, and amino acid sequence thereof corresponds to SEQ ID NO: 145.

10-2-4-2) Type hSH357-1-T3H heavy chain:

Humanitarian heavy chain SH357-1 was designed by replacing amino acids with No. 9 (Proline), 11 (leucine), 16 (glutamic acid), 17 (threonine), 20 (isoleucine), 38 (lysine), 43 (lysine), 73 (glutamic acid), 76 (alanine), 82 (glutamine), 83 (isoleucine), 85 (asparagine), 87 (lysine), 88 (asparagine), 93 (serine), 95 (phenylalanine), 114 (threonine) and 115 (leucine) variable regions of the heavy chain SH357-1, presents in SEQ ID NO: 51 by alanine, valine, alanine, serine, valine, arginine, glutamine, aspartic acid, threonine, glutamic acid, leucine, serine, arginine, serine, valine, tyrosine, leucine and valine, respectively, and designated as "type hSH357-1-T3H heavy chain.

The nucleotide sequence encoding the type hSH357-1-T3H heavy chain corresponds to SEQ ID NO: 146 in the list of sequences, and amino acid sequence thereof corresponds to SEQ ID NO: 147. The nucleotide sequence corresponding to nucleotides with No. 1-57 in SEQ ID NO: 146 is a signal sequence for secretion. The nucleotide sequence corresponding to nucleotides with No. 58-414 there is a variable region. Nucleotide follow etelnost, corresponding to nucleotides with No. 415-1404, there is a constant region. The nucleotide sequence corresponding to nucleotides with No. 145-162, there is a CDRH1. The nucleotide sequence corresponding to nucleotides with No. 205-255, there is a CDRH2. The nucleotide sequence corresponding to nucleotides with No. 352-381, there is a CDRL3.

Amino acid sequence corresponding to amino acids No. 1-19 in SEQ ID NO: 147 list sequence is a signal sequence for secretion. Amino acid sequence corresponding to amino acids No. 20-138, there is a variable region. Amino acid sequence corresponding to amino acids No. 139-468, there is a constant region. Amino acid sequence corresponding to amino acids No. 49-54, there is a CDRH1. Amino acid sequence corresponding to amino acids No. 69-85, there is a CDRH2. Amino acid sequence corresponding to amino acids No. 118-127, there is a CDRH3.

In addition, the nucleotide sequence encoding CDRH1the heavy chain of typehSH357-1-T3H, corresponds to SEQ ID N: 148 in the list of sequences, amino acid sequence thereof corresponds to SEQ ID NO: 149, the nucleotide sequence of CDRH2corresponds to SEQ ID NO: 150 in the list of sequences, the amino acid sequence thereof corresponds to SEQ ID NO: 151, the nucleotide sequence of CDRH3corresponds to SEQ ID NO: 152 in the list of sequences, and amino acid sequence thereof corresponds to SEQ ID NO: 153.

(Example 11) Receiving gumanitarnogo antibody to EPHA2

For measuring the activity of gumanitarnogo SH348-1 and gumanitarnogo SH357-1, a plasmid with the heavy and light chains gumanitarnogo antibody to EPHA2, obtained in example 10, was designed, as shown next.

11)-1 Designing expressing vectors for gumanitarnogo antibody to EPHA2

11)-1-1 Receipt of universal expressing vector for light chain gumanitarnogo antibodies (pEF6/KCL)

Synthesized the gene encoding the signal sequence of the light chain of human antibodies, and constant region of light chain (k chain) Ig person described in SEQ ID NO: 154 in the list of sequences (Invitrogen Corp.; service on the synthesis of artificial genes) and were digested with restriction enzymes NheI and PmeI. Cleaved DNA fragment was embedded in the site NheI/PmeI vector pEF6/V5-HisB (Invitrogen Corp.) to construct a generic expressing vecto is for light chain gumanitarnogo antibodies (pEF6/KCL).

11)-1-2 Getting universal expressing vector for the heavy chain gumanitarnogo antibodies (pEF1/FCCU-1)

Synthesized the gene encoding the signal sequence of the heavy chain of human antibodies and a constant region of human IgG1, is described in SEQ ID NO: 155 in the list of sequences (Invitrogen Corp.; service on the synthesis of artificial genes) and were digested with restriction enzymes NheI and PmeI. Cleaved DNA fragment was embedded in the site NheI/PmeI vector pEF1/myc-HisB (Invitrogen Corp.) to construct a generic expressing vector for the H chain gumanitarnogo antibodies (pEF1/FCCU-1).

11)-1-3 Expressing vectors for types hSH348-1-T1L and hSH348-1-T3L light chain

Synthesized each of the DNA containing the gene encoding the variable region of light chain type hSH348-1-T1L or hSH348-1-T3L presented in SEQ ID NO: 156 and 157 list of sequences, merged with the secretion signal (Invitrogen Corp., the service of the synthesis of artificial genes) and were digested with restriction enzymes NdeI and BsiWI. The resulting DNA fragments were separately embedded in parts of the universal vector for the expression of light chain gumanitarnogo antibodies (pEF6/KCL), previously cleaved with restriction enzymes NdeI and BsiWI, thereby creating expressing vectors for light chain type hSH348-1-T1L and hSH348-1-T3L. Received expressing vectors were designated as "pEF6/KCL/hSH348-1-T1L and pEF6/KCL/hSH348-1-T3L", respectively.

11)-1-4 Designing expressing vectors for hSH348-1-T1H and hSH348-1-T3H types of heavy chains

Synthesized each of the DNA containing the gene encoding the variable region hSH348-1-T1H or hSH348-1-T3H types of heavy chain, represented in SEQ ID NO: 158 and 159, respectively, of the list of sequences (Invitrogen Corp., the service of the synthesis of artificial genes) and were digested with restriction enzyme BlpI. The resulting DNA fragments were separately embedded in parts of the universal vector for the expression of H chain gumanitarnogo antibodies (pEF1/FCCU-1)previously cleaved with restriction enzyme BlpI, thereby creating expressing vectors for hSH348-1-T1H and hSH348-1-T3H types of heavy chains. Received expressing vectors were designated as "pEF1/FCCU/hSH348-1-T1H" and "pEF1/FCCU/hSH348-1-T3H", respectively.

11)-1-5 Designing expressing vectors for hSH357-1-T1L and hSH357-1-T3L types of light chain

Synthesized each of the DNA containing the gene encoding the variable region hSH357-1-T1L or hSH357-1-T3L types of light chain, represented in SEQ ID NO: 160 or 161 of the list of sequences, respectively, fused to the secretion signal (Invitrogen Corp., the service of the synthesis of artificial genes) and were digested with restriction enzymes NdeI and BsiWI. The resulting DNA fragments were separately embedded in parts of the universal vector for the expression of L chain humaniterian the th antibody (pEF6/KCL), pre-split by enzymes NdeI and BsiWI, thereby creating expressing vectors for light chain type hSH357-1-T1L and hSH357-1-T3L. Received expressing vectors were designated as "pEF6/KCL/hSH357-1-T1L and pEF6/KCL/hSH357-1-T3L", respectively.

11)-1-6 Designing expressing vectors for hSH357-1-T1H and hSH357-1-T3H types of heavy chains

Synthesized each of the DNA containing the gene encoding the variable region hSH357-1-T1H or hSH357-1-T3H types of heavy chain, represented in SEQ ID NO: 162 or 163 list of sequences, respectively (Invitrogen Corp., the service of the synthesis of artificial genes) and were digested with restriction enzyme BlpI. The resulting DNA fragments were separately embedded in parts of the universal vector for the expression of H chain gumanitarnogo antibodies (pEF1/FCCU-1)previously cleaved with restriction enzyme BlpI, thereby creating expressing vectors for hSH357-1-T1H and hSH357-1-T3H types of heavy chains. Received expressing vectors were designated as "pEF1/FCCU/hSH357-1-T1H" and "pEF1/FCCU/hSH357-1-T3H", respectively.

11)-2 Obtaining gumanitarnogo antibodies

of 1.2×109cell line 293 FreeStyle in the logarithmic phase of growth were sown in 1.2 l of fresh medium for the expression of FreeStyle 293 (Invitrogen Corp.) and cultivated with shaking (125 rpm./minutes) in an incubator at 37°C in 8% CO2. 12 mg polyethylenimine (Polyscience #24765) was dissolved in 40 ml Opti-Pro SFM (Invitrogen Corp.) and left at room temperature for 5 minutes. A plasmid for the expression of H chain (0.6 mg) and a plasmid for the expression of L chain (1.8 mg), obtained using the kit PureLink HiPure Plasmid (Invitrogen Corp.), suspended in 40 ml of medium Opti-Pro SFM (Invitrogen Corp.). Thus, 40 ml expressing plasmids mixed with OptiPro SFM was added to 40 ml of polyethylenimine mixed with OptiPro SFM, left at room temperature for 5 minutes and was further left at room temperature for 5 minutes. Next, 80 ml of a mixed solution of polyethylenimine expressing plasmids and OptiPro SFM was added to the cell suspension 293 FreeStyle and continued cultivation with shaking. After culturing for 7 days at 37°C in 8% CO2, collected the supernatant of the culture.

11)-3 Cleanup gumanitarnogo antibodies

The culture supernatant obtained in paragraph 11)-2, filtered through a Disposable filter Capsule (Advantec MFS Inc., #CCS-045-E1H) and then purified by column affinity chromatography with protein A. the culture Supernatant was applied to a column MabSelect SuRe HiTrap 1 ml (GE Healthcare Bio-sciences Corp.), balanced PBS and washed with PBS. Later on it was applied a solution of 2M arginine (pH 4.0)is to collect, thereby fractions containing the antibody. the pH was brought to 7 and the fractions containing the antibody were applied to the column for desalting HiPrep (26/10, 50 ml) (GE Healthcare Bio-sciences Corp.), pre-equilibrated PBS. After replacing the PBS and, the fractions containing the antibody was passed through the 0.2-μm filter to obtain purified samples.

The concentration of antibody was determined, elwira antibodies associated with column POROS G 20 microns, PEEK, 4.6 mm×100 mm, 1.7 ml (Applied Biosystems) and measuring the eluate absorbance (O.D. 280 nm), with subsequent comparison of the peak area with the control (human IgG1).

Humanitariannet antibody SH348-1 obtained by the combination between the pEF6/KCL/hSH348-1-T1L and pEF1/FCCU/hSH348-1-T1H, designated as "hSH348-1-T1; and humanitariannet antibody SH348-1 obtained by the combination between the pEF6/KCL/hSH348-1-T3L and pEF1/FCCU/hSH348-1-T3H, designated as "hSH348-1-T3".

In addition, humanitariannet antibody SH357-1, obtained by the combination between the pEF6/KCL/hSH357-1-T1L and pEF1/FCCU/hSH357-1-T1H, designated as "hSH357-1-T1; and humanitariannet antibody SH357-1, obtained by the combination between the pEF6/KCL/hSH357-1-T3L and pEF1/FCCU/hSH357-1-T3H, designated as "hSH357-1-T3".

(Example 12) Confirmation of binding activity gumanitarnogo antibody to EPHA2 antigen

The ability of antibodies hSH348-1-T1, hSH348-1-T3, hSH357-1-T1 and hSH357-1-T3 for binding to the antigen was confirmed by the method described in example 6, except that the secondary antibody used conjugated with peroxidase antibodies goat affinity purity (AffiniPure)specific to Fcγ fragment of human IgG (Jackson ImmunoResearch Laboratories, Inc., #109-035-098).

The graphs figa)-12D), the uptake of screening is but as mean±standard deviation (n=3). As a result, for all of humanized antibodies hSH348-1-T1, hSH348-1-T3, hSH357-1-T1 and hSH357-1-T3 to EPHA2 confirmed the presence of binding activity to the extracellular region of EPHA2.

(Example 13) Measurement of competitive inhibitory activity against the binding of SH348-1 and SH357-1 with EPHA2

Competitive inhibitory activity hSH348-1-T1 and hSH348-1-T3 relative to the binding of SH348-1 with EPHA2 and competitive inhibitory activity hSH357-1-T1 and hSH357-1-T3 relative to the binding of SH357-1 with EPHA2 was measured by the method described below.

Monoclonal antibodies SH348-1 and SH357-1 mouse was biotinilated individually using the set for biotinidase EZ-Link Sulfo-NHS-LC (manufactured by Thermo Fisher Scientific K.K., #21435), according to the Protocol included in a set (hereinafter biotinylated SH348-1 and SH357-1 was identified as "bSH348-1" and "bSH357-1, respectively). Concentration bSH348-1, bSH357-1 and unlabeled antibody (SH348-1 AND SH357-1, hSH348-1-T1, hSH348-1-T3, hSH357-1-T1, hSH357-1-T3 and Ab96-1)used in the experiment on competitive inhibition, measured using the reagent for protein analysis ICA (produced by PIERCE).

Polypeptide extracellular region EPHA2 (produced R&D Systems, Inc., #3035-A2-100) was diluted with PBS to 0.5 μg/ml, and then was applied at 100 μl/well to immunoplate (Nunc, #442404), and incubated overnight at 4°C for absorption thereby of protein per tablet. The next day, the wells once washed with buffer for cultivation (BS, of 0.05% (vol./about.) Tween 20). Then, the solution of Block Ace (one sachet of powder Block Ace was dissolved in 100 ml of ultrapure water), diluted in PBS 4 times, was applied at 200 μl/well and incubated at room temperature for 4 hours. The solution from the wells was removed. Then the mixed solutions of biotinylated antibodies (5 μg/ml) and various concentrations (0 μg/ml, 1 μg/ml, 5 μg/ml, 25 μg/ml, 50 μg/ml and 125 μg/ml) of unlabeled antibody (solvent: PBS containing 0.05% (vol./about.) (final concentration) Tween 20) separately applied at 100 μl/well and incubated at room temperature for 1 hour. The wells were washed twice with a buffer for dilution (PBS, 0.05% of (about./about.) Tween 20). Then, conjugate with streptavidin horseradish peroxidase (GE Healthcare Bio-Sciences Corp., #RPN1231V)diluted 500 times with a buffer for dilution, was added at 100 μl/well and incubated at room temperature for 1 hour. The solution from the wells was removed, and the wells were washed twice with buffer for cultivation. Then color reaction was performed with stirring, adding the solution to the degree of color development OPD 100 μl/well. After development of color staining reaction was stopped by adding 1M HCl 100 μl/well. Measured the absorbance at 490 nm, using a spectrophotometer to read the tablets.

As a result, the absorption in the wells, which was added only bSH348-1 or bSH357-1, was 0,780±0,016 and 0,978±0,007 (mean±St is hartsoe deviation (n=3)), respectively.

The graphs figa) and 13B), the absorption is shown as mean±standard deviation (n=3). Linking SH348-1 and SH357-1 with EPHA2 not inhibited Ab96-1, wherein, on the basis of the epitope. On the other hand, showed that binding of SH348-1 with EPHA2 inhibited by the antibody SH348-1 or humanitarianism antibodies hSH348-1-T1 and hSH348-1-T3 (figa). Similarly, showed that binding of SH357-1 with EPHA2 is subject to inhibition by the antibody SH357-1 or humanitarianism antibodies hSH357-1-T1 and hSH357-1-T3 (pigv).

(Example 14) Inhibitory effect gumanitarnogo antibody to EPHA2 dependent on ephrin-A1 phosphorylation of tyrosine residues EPHA2

The ability gumanitarnogo antibody to EPHA2 inhibition of the phosphorylation of tyrosine residues EPHA2, depending on ephrin-A1, investigated by the method described in example 8. As a result, all antibodies hSH348-1-T1, hSH348-1-T3, hSH357-1-T1 and hSH357-1-T3 showed the preservation of the activity of inhibiting the phosphorylation of tyrosine residues EPHA2, induced Ephrin-A1/Fc (Fig).

Industrial applicability

Antibody to EPHA2 the present invention has antitumor activity. Pharmaceutical composition comprising the antibody to EPHA2, can be used as anti-cancer tool.

1. Anti-ERA antibody, which binds to specific polypeptid the Ohm, consisting of the amino acid sequence corresponding to amino acids No. 426-534 in SEQ ID NO: 8 in the list of sequences,
in which complementarity determining region in the variable regions of the heavy and light chain antibody selected from the group consisting of:
1) CDRH1consists of SEQ ID No: 59; CDRH2consists of SEQ ID No: 61; CDRH3consists of SEQ ID No: 63; CDRL1consists of SEQ ID No: 65; CDRL2consists of SEQ ID No: 67; and CDRL3consists of SEQ ID No: 69;
2) CDRH1consists of SEQ ID No: 71; CDRH2consists of SEQ ID No: 73; CDRH3consists of SEQ ID No: 75; CDRL1consists of SEQ ID No: 77; CDRL2consists of SEQ ID No: 79; and CDRL3consists of SEQ ID No: 81;
3) CDRH1consists of SEQ ID No: 109; CDRH2consists of SEQ ID No: 111; CDRH3consists of SEQ ID No: 113; CDRL1consists of SEQ ID No: 93; CDRL2consists of SEQ ID No: 95; and CDRL3consists of SEQ ID No: 97; and
4) CDRH1consists of SEQ ID No: 141; CDRH2consists of SEQ ID No: 143; CDRH3consists of SEQ ID No: 145; CDRL1consists of SEQ ID No: 125; CDRL2consists of SEQ ID No: 127; and CDRL3consists of SEQ ID No: 129.

2. The antibody according to claim 1, where the antibody has the following properties a)-C):
a) has no ability to phosphorylation of tyrosine residues ERNA;
b) has ADCC activity at a concentration of 2.5 µg/ml against cells expressing ERA; and
c) has CDC activity at a concentration of 25 µg/mlpo towards cells, expressing IRNA.

3. The antibody according to claim 1, where the antibody can be obtained through the humanization of antibodies containing the following 1) and 2):
1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 1-119 in SEQ ID NO: 35 in the list of sequences or polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 1-119 in SEQ ID NO: 39 in the list of sequences; and
2) a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 1-112 in SEQ ID NO: 37 in the list of sequences, or a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 1-112 in SEQ ID NO: 41 in the list of sequences.

4. The antibody according to claim 3, where the antibody can be obtained through the humanization of antibodies containing the following 1) and 2):
1) polypeptide heavy chain containing the amino acid sequence represented in SEQ ID NO: 35 in the list of sequences or polypeptide heavy chain containing the amino acid sequence represented in SEQ ID NO: 39 in the list of sequences; and
2) a light chain polypeptide containing the amino acid sequence represented in SEQ ID NO: 37 in the list of sequences or polypeptide light chain, stereoselectivity sequence, presented in SEQ ID NO: 41 in the list of sequences.

5. The antibody according to claim 1, where the antibody has antitumor activity in vivo.

6. The antibody according to claim 1, where the antibody has the following properties a) and b):
a) demonstrates the effect of reducing the level of protein ERA; and
b) has antitumor activity in vivo.

7. Anti-ERA antibody according to claim 1, where the antibody is specific binds to the polypeptide consisting of the amino acid sequence corresponding to amino acids No. 439-534 in SEQ ID NO: 8 in the list of sequences.

8. The antibody according to claim 7, where the antibody has antitumor activity in vivo.

9. The antibody according to claim 7, where the antibody has the following properties a) and b):
a) demonstrates the effect of reducing the level of protein ERA; and
b) has antitumor activity in vivo.

10. The antibody according to claim 1, with the following properties a) and b):
a) does not demonstrate the effect of reducing the level of protein ERA; and
b) has antitumor activity in vivo.

11. The antibody according to any one of claims 1 to 6, where the antibody inhibits the phosphorylation of tyrosine residues ERNA induced ligand URNA.

12. The antibody according to any one of claims 1 to 6, where the antibody did not inhibit the binding of ligand ERNA with ERNA, but inhibits the phosphorylation of tyrosine residues ERNA induced ligand URNA.

13. The antibody according to any one of FG1-10, characterized in that the antibody is a humanized antibody.

14. The antibody according to any one of claims 1 to 10, characterized in that the antibody is a human antibody.

15. The antibody according to any one of claims 1 to 10, characterized in that the antibody is an antibody of the IgG class.

16. The antibody according to any one of claims 1 to 10, characterized in that the antibody is any selected from a Fab, F(ab')2, Fv, scFv, diately, linear antibodies and polyspecific antibodies.

17. The antibody according to claim 1, where the antibody is produced by hybridoma SH348-1 (FERM BP-10836) or hybridomas SH357-1 (PERM BP-code 10837).

18. The antibody that is specific binds to the polypeptide consisting of the amino acid sequence corresponding to SEQ ID NO: 8 in the list of sequences, where the antibody has an amino acid sequence corresponding to SEQ ID No: 59, and 61 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequences and amino acid sequence corresponding to SEQ ID No: 63 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one amino acid in the amino acid sequence as the complementarity determining regions in the variable regions of the heavy chain and has and is inoculate sequence, corresponding to SEQ ID No: 65 and 67 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one amino acid in the amino acid sequences and amino acid sequence corresponding to SEQ ID No: 69 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one amino acid in the amino acid sequence as the complementarity determining regions in the variable region of the light chain.

19. The antibody according p, where the antibody has an amino acid sequence corresponding to SEQ ID NO: 109 as CDRH1, 111 as CDRH2and 113 as CDRH3, and has an amino acid sequence corresponding to SEQ ID NO: 93 as CDRL1, 95 as CDRL2, 97 as CDRL3in the list of sequences.

20. The antibody according to any one of p or 19, characterized in that the antibody is a humanized antibody.

21. The antibody according to any one of p or 19, characterized in that the antibody is a human antibody.

22. The antibody according to any one of p or 19, characterized in that the antibody is an antibody of the IgG class.

23. The antibody that is specific binds to the polypeptide consisting of the amino acid sequence presented in SEQ ID NO: 8 in the list of the placenta is of valnontey, characterized by the following 1) and 2):
1) is a peptide of the heavy chain containing the amino acid sequence represented by the General formula (I):

where FRH1indicates the frame section consisting of from 18 to 30 amino acids; CDRH1indicates the amino acid sequence corresponding to SEQ ID NO: 59 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; FRH2indicates the frame section consisting of 14 amino acids; CDRH2indicates the amino acid sequence corresponding to SEQ ID NO: 61 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; FRH3indicates the frame section consisting of 32 amino acids; CDRH3indicates the amino acid sequence corresponding to SEQ ID NO: 63 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one amino acid in the amino acid sequence; and FRH4indicates the frame section consisting of 11 amino acids, where these amino acids linked together by peptide bonds; and
2) has polypeptide light chain, containing the amino acid sequence represented by the General formula (II):

where FRL1denotes a frame section, consisting of 23 amino acids; CDRL1indicates the amino acid sequence corresponding to SEQ ID NO: 65 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; FRL2indicates the frame section consisting of 15 amino acids; CDRL2indicates the amino acid sequence corresponding to SEQ ID NO: 67 in the list of sequences; FRL3indicates the frame section consisting of 32 amino acids; CDRL3indicates the amino acid sequence corresponding to SEQ ID NO: 69 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one amino acid in the amino acid sequence; and FRL4indicates the frame section consisting of 10 amino acids, where these amino acids linked together by peptide bonds.

24. The antibody according to item 23, wherein the antibody is a humanized antibody.

25. The antibody according to item 23, wherein the antibody is a human antibody.

26. The antibody according to item 23, wherein the antibodies is about is an antibody of the IgG class.

27. The antibody that is specific binds to the polypeptide consisting of the amino acid sequence corresponding to SEQ ID NO: 8 in the list of sequences, where the antibody has an amino acid sequence corresponding to SEQ ID No: 71 and 73 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequences and amino acid sequence corresponding to SEQ ID No: 75 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one amino acid in the amino acid sequence as the complementarity determining regions in the variable regions of the heavy chain has the amino acid sequence, corresponding to SEQ ID No: 77 and 79 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequences and amino acid sequence corresponding to SEQ ID No: 81 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one amino acid in the amino acid sequence as the complementarity determining regions in the variable region of the light chain.

28. Anti is eaten by item 27, where the antibody has an amino acid sequence corresponding to SEQ ID NO: 141 as CDRH1, 143 as CDRH2and 145 as CDRH3and has the amino acid sequence represented by SEQ ID NO: 125 as CDRL1, 127 as CDRL2, 129 as CDRL3in the list of sequences.

29. The antibody according to any one of p or 28, characterized in that the antibody is a humanized antibody.

30. The antibody according to any one of p or 28, characterized in that the antibody is a human antibody.

31. The antibody according to any one of p or 28, characterized in that the antibody is an antibody of the IgG class.

32. The antibody that is specific binds to the polypeptide consisting of the amino acid sequence presented in SEQ ID NO: 8 in the list of sequences, characterized by the following 1) and 2):
1) is a peptide of the heavy chain containing the amino acid sequence represented by the General formula (I):

where FRH1indicates the frame section consisting of from 18 to 30 amino acids; CDRH1indicates the amino acid sequence corresponding to SEQ ID NO: 71 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one or several amino acids in the amino acid posledovatel the activity; FRH2indicates the frame section consisting of 14 amino acids; CDRH2indicates the amino acid sequence corresponding to SEQ ID NO: 73 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; FRH3indicates the frame section consisting of 32 amino acids; CDRH3indicates the amino acid sequence corresponding to SEQ ID NO: 75 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one amino acid in the amino acid sequence; and FRH4indicates the frame section consisting of 11 amino acids, where these amino acids linked together by peptide bonds; and
2) has a light chain polypeptide containing the amino acid sequence represented by the General formula (II):

where FRL1denotes a frame section, consisting of 23 amino acids; CDRL1indicates the amino acid sequence corresponding to SEQ ID NO: 77 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; FRL2indicates the frame section, the status is of ADI of 15 amino acids; CDRL2indicates the amino acid sequence corresponding to SEQ ID NO: 79 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one or several amino acids in amino acid sequence; FRL3indicates the frame section consisting of 32 amino acids; CDRL3indicates the amino acid sequence corresponding to SEQ ID NO: 81 in the list of sequences, or amino acid sequence with deletion, substitution or insertion of one amino acid in the amino acid sequence; and FRL4indicates the frame section consisting of 10 amino acids, where these amino acids linked together by peptide bonds.

33. The antibody according p, wherein the antibody is a humanized antibody.

34. The antibody according p, wherein the antibody is a human antibody.

35. The antibody according p, wherein the antibody is an antibody of the IgG class.

36. Anti-ERA antibody consisting of the following 1) and 2):
1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 1-119 in SEQ ID NO: 35 in the list of sequences or polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 1-119 SEQ ID NO: 39 in the list of sequences; and
2) a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 1-112 in SEQ ID NO: 37 in the list of sequences, or a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 1-112 in SEQ ID NO: 41 in the list of sequences,
where the antibody has the following properties a)-C):
a) has no ability to phosphorylation of tyrosine residues ERNA;
a) has ADCC activity against cells expressing ERA; and
(C) has CDC activity against cells expressing ERA.

37. The antibody according p, consisting of the following 1) and 2);
1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 1-119 in SEQ ID NO: 35 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 1-112 in SEQ ID NO: 37 in the list of sequences; and
2) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 1-119 in SEQ ID NO: 39 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 1-112 in SEQ ID NO: 41 in the list of sequences.

38. The antibody according p, consisting of the following is 1) and 2):
1) polypeptide heavy chain containing the amino acid sequence corresponding to SEQ ID NO: 35 in the list of sequences or polypeptide heavy chain containing the amino acid sequence corresponding to SEQ ID NO: 39 in the list of sequences; and
2) a light chain polypeptide containing the amino acid sequence corresponding to SEQ ID NO: 37 in the list of sequences, or a light chain polypeptide containing the amino acid sequence corresponding to SEQ ID NO: 41 in the list of sequences.

39. The antibody according p, consisting of the following 1) or 2):
1) polypeptide heavy chain containing the amino acid sequence corresponding to SEQ ID NO: 35 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to SEQ ID NO: 37 in the list of sequences; and
2) polypeptide heavy chain containing the amino acid sequence corresponding to SEQ ID NO: 39 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to SEQ ID NO: 41 in the list of sequences.

40. Anti-ERA antibody obtained through the humanization of the antibody according to any one of p or 36-39, where the antibody has the following properties a)-C):
a) has no ability to tyrosine phosphorylation is o residues ERNA;
a) has ADCC activity against cells expressing ERA; and
(C) has CDC activity against cells expressing ERA.

41. Anti-ERA antibody consisting of the following 1) and 2):
1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 20-468 in SEQ ID NO: 107 in the list of sequences or polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 20-468 in SEQ ID NO: 115 in the list of sequences; and
2) a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-239 in SEQ ID NO: 91 in the list of sequences, or a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-239 in SEQ ID NO: 99 in the list of sequences,
where the antibody has the following properties a)-C):
a) has no ability to phosphorylation of tyrosine residues ERNA;
a) has ADCC activity against cells expressing ERA; and
(C) has CDC activity against cells expressing ERA.

42. The antibody according to paragraph 41, consisting of the following 1) or 2):
1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 20-468 in SEQ ID NO: 107 in the list is sledovatelnot, and the light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-239 in SEQ ID NO: 91 in the list of sequences; and
2) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 20-468 in SEQ ID NO: 115 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-239 in SEQ ID NO: 99 in the list of sequences.

43. Anti-ERA antibody consisting of the following 1) and 2):
1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 20-468 in SEQ ID NO: 139 in the list of sequences or polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 20-468 in SEQ ID NO: 147 in the list of sequences; and
2) a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-239 in SEQ ID NO: 123 in the list of sequences, or a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-239 in SEQ ID NO: 131 in the list of sequences,
where the antibody has the following properties a)-C):
a) has no ability to phosphorylation of tyrosine residues ERNA;
a) has ADCC activity to otnosheniy to the cells, expressing ERNA; and
(C) has CDC activity against cells expressing ERA.

44. The antibody according to item 43, consisting of the following 1) or 2):
1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 20-468 in SEQ ID NO: 139 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-239 in SEQ ID NO: 123 in the list of sequences; and
2) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 20-468 in SEQ ID NO: 147 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-239 in SEQ ID NO: 131 in the list of sequences.

45. Anti-ERA Fab, which can be derived from any antibody according to any one of p, 36-39 or 41-44.

46. Anti-ERNA F(ab')2, which can be derived from any antibody according to any one of p, 36-39 or 41-44.

47. Anti-ERNA Fv, which can be derived from any antibody according to any one of p, 36-39 or 41-44.

48. Anti-ERNA scFv, which can be derived from any antibody according to any one of p, 36-39 or 41-44.

49. Anti-ERNA diatel, which can be derived from any antibody according to any one of p, 36-39 or 41-44.

50. Anti-ERNA linear antibody, which can b shall be derived from any antibody according to any one of p, 36-39 or 41-44.

51. Anti-ERNA polyspecific antibody, which can be derived from any antibody according to any one of p, 36-39 or 41-44.

52. The polypeptide for use in obtaining anti-ERNA antibodies selected from the group consisting of the following 1)-20):
1) a polypeptide containing the amino acid sequence corresponding to amino acids No. 1-119 in SEQ ID NO: 35 in the list of sequences;
2) a polypeptide containing the amino acid sequence corresponding to amino acids No. 1-119 in SEQ ID NO: 39 in the list of sequences;
3) a polypeptide containing the amino acid sequence corresponding to amino acids No. 1-112 in SEQ ID NO: 37 in the list of sequences;
4) a polypeptide containing the amino acid sequence corresponding to amino acids No. 1-112 in SEQ ID NO: 41 in the list of sequences;
5) a polypeptide containing the amino acid sequence corresponding to amino acids No. 20-468 in SEQ ID NO: 107 in the list of sequences;
6) a polypeptide containing the amino acid sequence corresponding to amino acids No. 20-468 in SEQ ID NO: 115 in the list of sequences;
7) a polypeptide containing the amino acid sequence corresponding to amino acids No. 20-138 in SEQ ID NO: 107 in the list of sequences;
8) the polypeptide containing the amino acid series is here, corresponding to amino acids No. 20-138 in SEQ ID NO: 115 in the list of sequences;
9) a polypeptide containing the amino acid sequence corresponding to amino acids No. 21-239 in SEQ ID NO: 91 in the list of sequences;
10) a polypeptide containing the amino acid sequence corresponding to amino acids No. 21-239 in SEQ ID NO: 99 in the list of sequences;
11) a polypeptide containing the amino acid sequence corresponding to amino acids No. 21-134 in SEQ ID NO: 91 in the list of sequences;
12) a polypeptide containing the amino acid sequence corresponding to amino acids No. 21-134 in SEQ ID NO: 99 in the list of sequences;
13) a polypeptide containing the amino acid sequence corresponding to amino acids No. 20-468 in SEQ ID NO: 139 in the list of sequences;
14) a polypeptide containing the amino acid sequence corresponding to amino acids No. 20-468 in SEQ ID NO: 147 in the list of sequences;
15) a polypeptide containing the amino acid sequence corresponding to amino acids No. 20-138 in SEQ ID NO: 139 in the list of sequences;
16) a polypeptide containing the amino acid sequence corresponding to amino acids No. 20-138 in SEQ ID NO: 147 in the list of sequences;
17) a polypeptide containing the amino acid sequence, with the expansion of its amino acids with No. 21-239 in SEQ ID NO: 123 in the list of sequences;
18) a polypeptide containing the amino acid sequence corresponding to amino acids No. 21-239 in SEQ ID NO: 131 in the list of sequences;
19) a polypeptide containing the amino acid sequence corresponding to amino acids No. 21-134 in SEQ ID NO: 123 in the list of sequences; and
20) a polypeptide containing the amino acid sequence corresponding to amino acids No. 21-134 in SEQ ID NO: 131 in the list of sequences.

53. Hybridoma SH348-1 mouse (FERM BP-10836) for producing anti-ERNA antibodies.

54. Hybridoma SH357-1 mouse (FERM BP-code 10837) for producing anti-ERNA antibodies.

55. Pharmaceutical composition for treatment of cancer, characterized in that it contains at least one antibody selected from the antibodies according to claims 1-10, 17, 18, 19, 23, 27, 28, 32, 36-39 or 41-44.

56. Pharmaceutical composition for treatment of cancer, characterized in that it contains at least one antibody according p.

57. The use of antibodies according to claims 1-10, 17, 18, 19, 23, 27, 28, 32, 36-39 or 41-44 to obtain drugs for inhibiting tumor growth in a mammal.

58. The application of § 57, wherein the tumor is a tumor expressing IRNA.

59. The use of antibodies for p to obtain drugs for inhibiting tumor growth in a mammal.

60. P is the applicatio on p, characterized in that the tumor is a tumor expressing IRNA.

61. Method of inhibiting growth of a tumor in a mammal, including the introduction of any of the antibodies selected from the group consisting of the antibody according to claims 1-10, 17, 18, 19, 23, 27, 28, 32, 36-39 or 41-44, in an effective amount.

62. The method according to p, wherein the tumor is a tumor that contains URNA.

63. Method of inhibiting growth of a tumor in a mammal, including the introduction of any of the antibodies selected from the group consisting of antibodies p, in an effective amount.

64. The method according to p, wherein the tumor is a tumor that contains URNA.

65. Polynucleotide encoding anti-ERA antibody or polypeptide for use in obtaining anti-ERA antibody according to any one of claims 1 to-10, 17, 18, 19, 23, 27, 28, 32, 36-39, 41-44 or 52.

66. A host cell for producing anti-ERNA antibodies, transformed polynucleotides on p.

67. The use of host cell according p for producing antibodies.

68. Polynucleotide encoding anti-ERA antibody on p.

69. A host cell for producing anti-ERNA antibodies, transformed polynucleotides on p.

70. The use of host cell according p for producing antibodies.

71. The antibody according to claim 1, consisting of the following 1) and 2):
1) heavy polypeptide chain containing amino acid serial is lnost, corresponding to amino acids No. 20-138 in SEQ ID NO: 107 in the list of sequences or polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 20-138 in SEQ ID NO: 115 in the list of sequences; and
2) a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-134 in SEQ ID NO: 91 in the list of sequences, or a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-134 in SEQ ID NO: 99 in the list of sequences.

72. The antibody according to claim 1, consisting of the following 1) and 2):
1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 20-138 in SEQ ID NO: 107 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-134 in SEQ ID NO: 91 in the list of sequences; and
2) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 20-138 in SEQ ID NO: 115 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-134 in SEQ ID NO: 99 in the list of sequences.

73. The antibody according to claim 1, consisting of the following 1) and 2):
1) heavy polypeptide chain, containing the second amino acid sequence, corresponding to amino acids No. 20-138 in SEQ ID NO: 139 in the list of sequences or polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 20-138 in SEQ ID NO: 147 in the list of sequences; and
2) a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-134 in SEQ ID NO: 123 in the list of sequences, or a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-134 in SEQ ID NO: 131 in the list of sequences.

74. The antibody according to claim 1, consisting of the following 1) and 2):
1) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 20-138 in SEQ ID NO: 139 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-134 in SEQ ID NO: 123 in the list of sequences; and
2) polypeptide heavy chain containing the amino acid sequence corresponding to amino acids No. 20-138 in SEQ ID NO: 147 in the list of sequences and a light chain polypeptide containing the amino acid sequence corresponding to amino acids No. 21-134 in SEQ ID NO: 131 in the list of sequences.



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: invention relates to an agent for treatment of ischemic lesions of tissues, which is a mixture with the ratio of 1.5-3 of two cultures of mesenchymal stem cells, one of which is modified by the genetic structure based on the viral vector which provides hyperproduction of vascular endothelial growth factor, and the other is modified by the genetic structure based on the viral vector which provides hyperproduction of angiopoietin, and a method of treatment of ischemic lesions of tissues by puncture of ischemic tissue, and can be used in medicine.

EFFECT: invention enables to achieve effective vascularisation and repair of ischemic tissue.

4 cl, 4 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: inventions relate to the field of immunology. Claimed are a single-chain antibody, specific to a carcinoembryonic antigen, a chimeric mononuclear T-cell receptor, a vector, a host cell and a method of diagnostics or treatment of diseases, characterised by the presence of antigens, capable of binding with the chimeric receptor. Described is a genetic construction, coding chimeric monomolecular T-cell receptors, in which an effector fragment of the T-cell receptor is combined with an antigen-recognising part, which represents variable fragments of two different antibodies to the carcinoembryonic antigen (CEA).

EFFECT: claimed inventions can be used in T-cell cancer therapy.

7 cl, 4 dwg, 3 ex, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to chemical-pharmaceutical industry and represents a preparation for involving a mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow, which is introduced into the blood vessel or muscle and which contains any of components: (a) protein HMGB1; (b) HMGB1 protein-secreting cell; (c) a vector, into which HMGB1 protein-coding DNA is inserted; (d) protein HMGB2; (e) HMGB2 protein-secreting cell; (f) a vector, into which HMGB2 protein-coding DNA is inserted; (g) protein HMGB3; (h) HMGB3 protein-secreting cell; and (i) a vector, into which HMGB3 protein-coding DNA is inserted.

EFFECT: elaboration of the preparation for involving the mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow.

3 cl, 6 ex, 1 tbl, 14 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Method includes introduction of RNA molecule into a bird egg. Introduced RNA molecule contains double-stranded region and results in reduction of the level of molecule of RNA and/or protein, included into determination of sex in birds, in the egg. Invention can be used in poultry breeding.

EFFECT: claimed is method of changing sex characteristics in birds.

7 cl, 3 dwg, 6 tbl, 2 ex

FIELD: food industry.

SUBSTANCE: invention refers to the field of biotechnology and food industry. Presented is a barley plant that yields grain and is homozygotic in at least two loci for the genetic variations having been bred, representing: a) allele wherein most of or all the genes coding B-hordein in Locus Hor2 are removed, and b) mutant allele in the barley Locus Lys3 so that the grain contains neither B-, nor C- hordeins, the said genetic variations present in Lines Riso 56 and Riso 1508 barley accordingly; absence of B-hordeins is to be revealed by absence of amplified DNA using primers: 5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3', 3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3', while absence of C-hordeins is to be revealed by absence of the 70 kDa strip during study of the grain alcohol-soluble extract by means of SDS-PAGE. Additionally presented are: barley grain cropped from the said plant; B- and C-hordein-free products produced from the said grain such as flour, malt and beer. Additionally described are methods for production of food products barley (flour, whole-grain flour, starch, malt) and beverages using grain cropped from the barley plant having the above characteristics. Proposed is a method for identification of barley grain suitable for production of a malt-based food product and/or beverage suitable for consumption by a person suffering from gluten-sensitive enteropathy which method includes: a) production of one or more materials: i) sample of a plant capable to yield the said grain, ii) grain, iii) malt produced from the grain, and/or iv) extract of the said grain; b) analysis of Stage a) material for presence of at least one hordein and/or at least one hordein-coding gene with selection of grain having the gene pattern of the above plant.

EFFECT: invention allows to manufacture B- and C-hordein-free malt-based food products or beverages.

27 cl, 14 dwg, 10 tbl, 10 ex

FIELD: medicine, pharmaceutics.

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

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11 cl, 32 dwg, 14 ex

FIELD: chemistry.

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

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

4 cl, 6 dwg, 4 ex

FIELD: chemistry.

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

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21 cl, 2 dwg, 6 ex

Fused rage proteins // 2513695

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biochemistry. Claimed is fused protein for treating diseases, mediated by advanced glycation end products (AGE), consisting of a fragment of a version of human receptor of advanced glycation end products (RAGE), which has two point mutations H217R and R221H, and a fragment of constant domain of human immunoglobulin IgG4, joined with linker if necessary. In addition, considered are: nucleic acid and recombinant host cell for obtaining fused protein, as well as pharmaceutical composition for treatment of AGE-mediated diseases, which contain fused protein.

EFFECT: invention ensures lower aggregation of fused protein.

13 cl, 19 dwg, 3 ex, 9 tbl

FIELD: chemistry.

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

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

17 cl, 2 tbl

FIELD: medicine.

SUBSTANCE: inventions refer to biotechnology and concern a recombinant plasmid DNA pQE30/Derf2L and a bacterial strain Escherichia coli M15/pQE30/Derf2L. The characterised recombinant plasmid DNA codes protein Derf2L of a mite Dermatophagoides farinae and consists of BamHI/HindIII - a DNA fragment of a plasmid pQE30 and a DNA sequence coding BamHI/HindIII fragment including gene Derf2L Dermatophagoides farinae. The characterised bacterial strain Escherichia coli M15/pQE30/Derf2L, is produced by transformation of the cells Escherichia coli M15 by the recombinant plasmid DNA pQE30/Derf2L.

EFFECT: presented inventions can be applicable to develop a dust mite diagnosticum, as well as for conducting specific immune therapy.

2 cl, 3 dwg, 1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Method includes introduction of RNA molecule into a bird egg. Introduced RNA molecule contains double-stranded region and results in reduction of the level of molecule of RNA and/or protein, included into determination of sex in birds, in the egg. Invention can be used in poultry breeding.

EFFECT: claimed is method of changing sex characteristics in birds.

7 cl, 3 dwg, 6 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns an isolated polypeptide, a pharmaceutical composition containing such polypeptide, as well as a method of treating cancer. The presented polypeptide contains an amino acid sequence corresponding to SEQ. ID. NO: 2 or SEQ ID NO: 4. There are also characterised fragments or versions of the presented polypeptide.

EFFECT: group of inventions may be used to stimulate the immune system in treating malignant diseases and for diagnosing loss of immunologic activity.

7 cl, 7 dwg, 1 tbl, 6 ex

FIELD: chemistry.

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

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

4 cl, 6 dwg, 4 ex

Fused rage proteins // 2513695

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biochemistry. Claimed is fused protein for treating diseases, mediated by advanced glycation end products (AGE), consisting of a fragment of a version of human receptor of advanced glycation end products (RAGE), which has two point mutations H217R and R221H, and a fragment of constant domain of human immunoglobulin IgG4, joined with linker if necessary. In addition, considered are: nucleic acid and recombinant host cell for obtaining fused protein, as well as pharmaceutical composition for treatment of AGE-mediated diseases, which contain fused protein.

EFFECT: invention ensures lower aggregation of fused protein.

13 cl, 19 dwg, 3 ex, 9 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to recombinant fused protein dimers intended to inhibit or suppress immune response in a mammal, which bind human CD80 or human CD86 or the extracellular domain of any thereof, and has higher capacity for suppressing immune response than a dimer of the fused protein LEA29Y-Ig. Also disclosed are nucleic acids which code said dimers, expression vectors containing said nucleic acids, as well as recombinant host cells containing said nucleic acids and/or said vectors. Disclosed are pharmaceutical compositions for inhibiting or suppressing immune response in a mammal, which contain said fused protein dimers, as well as use of said dimers to produce drugs for inhibiting or suppressing immune response in a mammal, treating diseases or disorders of the immune system or treating organ or tissue transplant rejection in a mammal. Methods of producing said fused protein dimers are also disclosed.

EFFECT: invention provides effective inhibition or suppression of immune response in a mammal.

9 cl, 15 dwg, 11 tbl, 12 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents a method for obtaining recombinant DNAse I of a human or its mutein, as well as their conjugates with polyethylene glycol, using a bacterium belonging to Escherichia class, transformed with expression plasmid, containing a promoter functioning in a bacterial cell, DNA fragment coding a hexahistidine cluster, a fragment coding enterokinase recognition sequence amalgamated in frame with human DNAse I or its functionally active mutein containing replacements of asparagine with cysteine, transcription termination section, vector pET28a(+) fragment containing initiation section of replication of bacteriophage fl, sequence coding aminoglycoside-3'-phosphotransferase, area of beginning of plasmid pBR322 replication, gene RNA-organising protein Rop, sequence coding lactose operon repressor.

EFFECT: invention allows obtaining recombinant human DNAse I or its mutein with high yield.

18 cl, 7 dwg, 1 tbl, 12 ex

FIELD: chemistry.

SUBSTANCE: isolated peptide having cytotoxic T lymphocyte (CTL) inducing capacity in the presence of an antigen-presenting cell bearing HLA-A*2402, is used to obtain antigen-presenting cells and therefore CTL. The obtained CTL are used for targeted action against CDCA1-expressing cancer cells.

EFFECT: invention provides an effective vaccine for inducing anti-tumour immunity in a subject.

16 cl, 4 dwg, 1 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant hybrid inhibitor of angiogenesis represents a protein shown in dwg. 1. This protein includes amino acid sequence of plasminogen of a human being from amino acid 82 to 341 and sequence Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys, which are covalently connected to each other. An inhibitor production method involves expression of its gene in cells of E. coli producer strain, which are transfected with recombinant plasmid DNA pBSRK13 with a physical map presented in dwg. 2, which has the size of 4155 pairs of bases. This plasmid includes a gene coding the recombinant hybrid inhibitor of angiogenesis, as well as a gene of signal peptide OmpA, lac-operator, a gene of stability to kanamycin, replicative origin pUC ori and a gene coding the lac-operator under control of promoter T7. A target protein is extracted from periplasmatic area of bacterial cells by affine and gel-filtration chromatography. The invention can also be used in medicine for creation of new medicinal agents with antiangiogenic therapeutical effect.

EFFECT: invention allows producing a new protein having antiangiogenic activity and increased selectivity of action in relation of tumoral endothelium.

2 cl, 3 dwg, 1 tbl, 5 ex

Vns-met-histones // 2498997

FIELD: biotechnologies.

SUBSTANCE: nucleic acid molecule codes a polypeptide consisting of two residues of methionine as the first and the second N-end amino-acid residues connected through a peptide link to a mature eucariotic histone. Polypeptide is obtained by cultivation of a host cell transformed by an expression vector including the above molecule of nucleic acid. Polypeptide is used as part of pharmaceutical composition for therapy of cancer, bacterial, virus or fusarium infections. Besides, polypeptide is used as part of composition for diagnostics of a patient in relation to response to pharmaceutical composition containing the above polypeptide, or in relation to curability using it.

EFFECT: invention allows improving efficiency of recombinant expression and simplifying determination of the above polypeptide in presence of endogenic histones at preservation of biologic activity of mature eucariotic histone.

17 cl, 3 dwg, 6 tbl, 7 ex

FIELD: chemistry.

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

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

1 dwg, 2 tbl, 3 ex

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