Compositions and methods of mammary gland cancer therapy and diagnostics

FIELD: medicine.

SUBSTANCE: invention claims compositions which can include one or several mammary gland tumour proteins, their immunogenic parts or polynucleotides encoding such parts. Alternatively the therapeutic composition can include antigen-presenting cell expressing mammary gland tumour protein, or T-cell specific to cells expressing such protein. These compositions can be applied in prevention and treatment of such diseases as mammary gland cancer. Invention also claims diagnostic methods based on determination of mammary gland tumour protein or mRNA encoding such protein in sample.

EFFECT: use of peptides obtained from protein expressed from mammary gland by tumour in diagnostics and therapy of mammary gland cancer.

37 cl, 6 ex, 1 dwg

 

TECHNICAL FIELD OF THE INVENTION.

This invention in General relates to the treatment and diagnosis of malignant tumors, such as breast cancer. More specifically the invention relates to polypeptides containing at least a portion of the protein of breast cancer, and to polynucleotides, encoding such polypeptides. Such polypeptides and polynucleotide can be used in compositions for the prevention and treatment of breast cancer and for the diagnosis and monitoring of such malignant tumors.

THE PREMISE OF THE INVENTION.

Breast cancer represents a major problem for the health of women and the United States and around the world. While advances have been made in detection and treatment, breast cancer remains the second leading cause associated with malignancy deaths among women, affecting every year, more than 180000 women in the United States. For women in North America the chance of getting breast cancer in a lifetime is one in eight.

There is currently no vaccine or other universal successful method of preventing or treating breast cancer. Therapy of the disease is currently based on a combination of early diagnosis through regular breast cancer screening and aggressive treatment, which may include one or more of the many methods of treatment, such as surgery, radiation therapy, chemotherapy and hormonal therapy. The course of treatment in the specific case of breast cancer is often chosen on the basis of a number of prognostic parameters, including analysis of specific tumor markers. See, for example, Porter-Jordan and Lippman, Breast Cancer 8: 73-100 (1994). However, the use of established markers often leads to the result that it is difficult to interpret, and the high mortality observed among patients with breast cancer, suggests that improvements are needed in the treatment, diagnosis and prevention of disease.

Therefore, in this area there is a need for improved methods of treatment and diagnosis of breast cancer. This invention satisfies these needs, and also provides other related advantages.

THE ESSENCE OF THE INVENTION.

Briefly, this invention relates to compositions and methods for diagnosis and therapy of malignant tumors, such as breast cancer. In one aspect this invention relates to polypeptides containing at least a portion of the protein of breast cancer or its variant. Certain parts and other options are immunogenic, so that the ability of the variant to react with antigen-anticorodal not substantially snijaetsa. In some embodiments, the polypeptide includes a sequence that encodes a polynucleotide sequence selected from the group consisting of: sequence listed SEQ ID NO: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486 and 489; (b) variants of a sequence provided in SEQ ID NO: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486 and 489; and (c) complements of a sequence (a) or (b). In specific embodiments, the polypeptides according to the invention contain at least a portion of the protein of the tumor, which comprises amino acid sequence selected from the group consisting of sequences listed in SEQ ID NO: 176, 179, 181, 469-473, 475, 485, 487 and 488, and their variants.

This invention also relates to polynucleotides that encode the polypeptide described above, or portions thereof (such as a portion encoding at least 15 amino acid residues of the protein of breast cancer), to expressing vectors containing such polynucleotide, and cell host transformed or transfitsirovannykh such expressing vectors.

In other aspects this invention relates to pharmaceutical compositions containing the polypeptide or polynucleotide that described above, and a physiologically acceptable carrier.

In a related aspect, the invention relates to immunogenic compositions or in which Kinam for prophylactic or therapeutic applications. Such compositions contain a polypeptide or polynucleotide that described above, and immunostimulant.

This invention also relates to pharmaceutical compositions that contain: (a) the antibody or antigennegative fragment that is specific contacts with the protein of breast cancer; and (b) a physiologically acceptable carrier.

The following aspects this invention relates to pharmaceutical compositions containing: (a) the antigen-presenting cell that expresses the polypeptide described above, and (b) a pharmaceutically acceptable carrier or excipient. Antigen-presenting cells include dendritic cells, macrophages, monocytes, fibroblasts and b cells.

In related aspects are presented immunogenic compositions or vaccines that contain: (a) the antigen-presenting cell that expresses the polypeptide described above, and (b) an immunostimulant.

In other aspects of this invention, furthermore, relates to hybrid proteins that contain at least one polypeptide as described above and polynucleotides coding for these hybrid proteins. Sample hybrid proteins according to the invention contain the first part of the amino acid and the second amino acid, where the first part of amino acids includes 9 or more continuously SL is blowing one after another amino acid mammaglobin, which depicts the amino acids 1-93 SEQ ID NO: 493; where the second part of the amino acids include 9 or more continuously consecutive amino acids of B726P, which are represented in SEQ ID NO: 475, SEQ ID NO: 469 or SEQ ID NO: 176; and where the first part of the amino acids associated with either aminocom.com or carboxyl end of the second part of the amino acids.

In addition, in the following embodiments, the present invention relates to hybrid proteins in which the specified first part of the amino acids selected from the group consisting of:

IDELKECFLNQTDETLSNVE (amino acids 59-78 SEQ ID NO: 493);

TTNAIDELKECFLNQ (amino acids 55-69 SEQ ID NO: 493);

SQHCYAGSGCPLLENVISKTI (amino acids 13 to 33 SEQ ID NO: 493);

EYKELLQEFIDDNATTNAID (amino acids 41-60 SEQ ID NO: 493);

KLLMVLMLA (amino acids 2-10 SEQ ID NO: 493);

QEFIDDNATTNAI (amino acids 47-59 SEQ ID NO: 493); and

LKECFLNQTDETL (amino acids 62-74 SEQ ID NO: 493).

In alternative embodiments presents a hybrid proteins in which the second part of the amino acids include 9 or more continuously consecutive amino acids encoded by (1) the joint open reading frame (ORF) of the "left" and "right" ORF, B726P specified in SEQ ID NO: 475; (2) "left" ORF B726P, which is shown in SEQ ID NO: 469; and (3) "right" ORF B726P specified in SEQ ID NO: 176. Hybrid proteins according to the invention can also contain the second part of the amino acids, which includes 9 or more continuously following one after the other the m amino acids of the amino acid sequence, the depicted amino acids 1-129 SEQ ID NO: 475. In addition, further examples of hybrid proteins is depicted here as SEQ ID NO: 493; SEQ ID NO: 494 SEQ ID NO: 495.

Presents a hybrid proteins in which a part of amino acids mammaglobin associated with aminocom.com part of amino acids B726P, along with the fact that presents other hybrid proteins in which a part of amino acids mammaglobin associated with carboxyl end of the amino acids B726P. The relationship between a part of the amino acids mammaglobin and part of B726P can be represented by a covalent bond. In addition, the area of amino acids either unrelated or related mammaglobin and/or B726P, may be included between or at the amino or carboxyl end part of the amino acids of mammaglobin and/or B726P.

This invention also relates to isolated polynucleotides that encode any of the hybrid proteins that are specifically described, as well as those of the hybrid proteins, which can be obtained by conventional experimentation specialist in this field.

Related aspects include pharmaceutical compositions containing hybrid protein or polynucleotide encoding a hybrid protein, in combination with a physiologically acceptable carrier.

In addition, other aspects of the presented composition, which contains a hybrid protein or polynucleotide encoding g is Britny protein, in combination with an immunostimulant.

The following aspects this invention relates to methods for inhibiting the development of malignant tumors in a patient, comprising the administration to a patient the composition specified above. The patient may be affected by breast cancer, and in this case the methods provide treatment for the disease, or the patient who assumes the risk of disease development may be carried out preventive treatment.

In other aspects of this invention, furthermore, relates to a method of removing tumor cells from a biological sample, comprising effecting contact a biological sample with T cells that react with specific proteins of breast cancer, with stage contacting performed under conditions and for a time sufficient to ensure the removal of cells expressing the protein from a sample.

In related aspects, the methods of inhibiting the development of malignant tumors in a patient, comprising the administration to a patient biological sample treated as described above.

In other aspects, in addition, presents methods of stimulation and/or expansion of T cells, specific for the protein of breast cancer, including the implementation of contact of T cells with one or several is likemy of the following factors: (i) a polypeptide, described above; (ii) polynucleotides, encoding such a polypeptide; and/or (iii) an antigen-presenting cell that expresses such a polypeptide; under conditions and for a time sufficient to provide stimulation and/or expansion of T cells. Also presents isolated populations of T cells, including T cells, obtained as described above.

The following aspects this invention relates to methods for inhibiting the development of malignant tumors in a patient, comprising the administration to a patient an effective amount of a population of T cells, as described above.

This invention also relates to methods of inhibiting the development of malignant tumors in a patient, comprising the following stages: (a) incubating CD4+and/or CD8+T cells isolated from a patient with one or more of the following factors: (i) a polypeptide containing at least immunogenic portion of the protein of breast cancer; (ii) polynucleotides, encoding such a polypeptide; and (iii) an antigen-presenting cell that expresses such a polypeptide; and (b) introduction to the patient an effective amount of proliferating T cells, and thereby inhibiting the development of malignant tumors in a patient. Proliferating cells prior to administration to the patient, but not about the sory, to clone.

The following aspects this invention relates to methods of determining the presence or absence of a malignant tumor in a patient, comprising: (a) effecting contact of a biological sample obtained from the patient with a binding agent that binds to a polypeptide listed above; (b) determining in the sample the amount of polypeptide that binds to the binding agent; and (C) comparing the amount of polypeptide with a pre-defined cut-off value, and determining on the basis of the presence or absence of a malignant tumor in a patient. In preferred embodiments, the binding agent is an antibody, preferably a monoclonal antibody. Malignant tumor is breast cancer.

In other aspects, the invention also relates to methods of monitoring the progression of a malignant tumor in a patient. Such methods include the following stages: (a) effecting contact of a biological sample obtained from the patient at a first point in time with a binding agent that binds a polypeptide listed above; (b) determining in the sample the amount of polypeptide that binds to the binding agent; (C) repeating steps (a) and (b) using a biological sample obtained from the human patient at a later point in time; and (d) comparing the amount of polypeptide defined in stage (C) with the amount determined at stage (b), and thereby monitoring the progression of a malignant tumor in a patient.

In other aspects of this invention, furthermore, relates to a method of determining the presence or absence of a malignant tumor in a patient, comprising the following stages: (a) effecting contact of a biological sample obtained from the patient with an oligonucleotide that hybridizes with polynucleotide, which encodes a protein of breast cancer; (b) determining in the sample the level of polynucleotide, preferably mRNA, which hybridizes with the oligonucleotide; and (C) comparing the level of polynucleotide that hybridizes with the oligonucleotide, with pre-defined cut-off value, and thereby determining the presence or absence of malignant tumors patient. In some embodiments, the amount of mRNA are determined by polymerase chain reaction, for example, using at least one oligonucleotide primer that hybridizes with polynucleotide coding for the polypeptide listed above, or the complement of such polynucleotide. In other embodiments, the amount of mRNA is determined with the method of the hybridization using oligonucleic tiny probe, which hybridizes with polynucleotide that encodes a polypeptide indicated above, or the complement of such polynucleotide.

In related aspects, the methods of monitoring the progression of a malignant tumor in a patient, comprising the following stages: (a) effecting contact of a biological sample obtained from the patient with an oligonucleotide that hybridizes with polynucleotide, which encodes a protein of breast cancer; (b) determining in the sample the number of polynucleotide that hybridizes with the oligonucleotide, (C) repeating steps (a) and (b) using a biological sample obtained from the patient at a later point in time; and (d) comparing the number of polynucleotide defined at the stage (C), with the number determined at stage (b), and thereby monitoring the progression of a malignant tumor in a patient.

The following aspects this invention relates to antibodies, such as monoclonal antibodies that bind to the polypeptide described above, and to diagnostic kits containing such antibodies. Also provides diagnostic kits containing one or more oligonucleotide probes or primers described above.

These and other aspects of the present invention will become Acevi is generated when referring to the following detailed description and drawing. All reported here references incorporated by reference in full, as they would have been included in a separate citation.

A BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE IDENTIFIERS.

The drawing shows the results of Northern blot clone SYN18C6 (SEQ ID NO: 40).

SEQ ID NO: 1 is a cDNA sequence JBT2.

SEQ ID NO: 2 is a sequence cDNA JBT6.

SEQ ID NO: 3 is a sequence cDNA JBT7.

SEQ ID NO: 4 is a sequence cDNA JBT10.

SEQ ID NO: 5 is a sequence cDNA JBT13.

SEQ ID NO: 6 is a sequence cDNA JBT14.

SEQ ID NO: 7 is a sequence cDNA JBT15.

SEQ ID NO: 8 is a sequence cDNA JBT16.

SEQ ID NO: 9 is a sequence cDNA JBT17.

SEQ ID NO: 10 is a sequence cDNA JBT22.

SEQ ID NO: 11 is a sequence cDNA JBT25.

SEQ ID NO: 12 is a sequence cDNA JBT28.

SEQ ID NO: 13 is a sequence cDNA JBT32.

SEQ ID NO: 14 is a sequence cDNA JBT33.

SEQ ID NO: 15 performance is made by a sequence cDNA JBT34.

SEQ ID NO: 16 is a sequence cDNA JBT36.

SEQ ID NO: 17 is a sequence cDNA JBT37.

SEQ ID NO: 18 is a sequence cDNA JBT51.

SEQ ID NO: 19 is a sequence cDNA JBTT1.

SEQ ID NO: 20 is a sequence cDNA JBTT7.

SEQ ID NO: 21 is a sequence cDNA JBTT11.

SEQ ID NO: 22 is a sequence cDNA JBTT14.

SEQ ID NO: 23 is a sequence cDNA JBTT18.

SEQ ID NO: 24 is a sequence cDNA JBTT19.

SEQ ID NO: 25 is a sequence cDNA JBTT20.

SEQ ID NO: 26 is a sequence cDNA JBTT21.

SEQ ID NO: 27 is a sequence cDNA JBTT22.

SEQ ID NO: 28 is a sequence cDNA JBTT28.

SEQ ID NO: 29 is a sequence cDNA JBTT29.

SEQ ID NO: 30 is a sequence cDNA JBTT33.

SEQ ID NO: 31 is a sequence cDNA JBTT37.

SEQ ID NO: 32 is a sequence cDNA JBTT38.

SEQ ID NO: 33 is a definite the th cDNA sequence JBTT47.

SEQ ID NO: 34 is a sequence cDNA JBTT48.

SEQ ID NO: 35 is a sequence cDNA JBTT50.

SEQ ID NO: 36 is a sequence cDNA JBTT51.

SEQ ID NO: 37 is a sequence cDNA JBTT52.

SEQ ID NO: 38 is a sequence cDNA JBTT54.

SEQ ID NO: 39 is a sequence cDNA SYN17F4.

SEQ ID NO: 40 is a sequence cDNA SYN18C6 (also known as VR).

SEQ ID NO: 41 is a sequence cDNA SYN19A2.

SEQ ID NO: 42 is a sequence cDNA SYN19C8.

SEQ ID NO: 43 is a sequence cDNA SYN20A12.

SEQ ID NO: 44 is a sequence cDNA SYN20G6.

SEQ ID NO: 45 is a sequence cDNA SYN20G6-2.

SEQ ID NO: 46 is a sequence cDNA SYN21B9.

SEQ ID NO: 47 is a sequence cDNA SYN21B9-2.

SEQ ID NO: 48 is a sequence cDNA SYN21C10.

SEQ ID NO: 49 is a sequence cDNA SYN21G10.

SEQ ID NO: 50 is a sequence cDNA SYN21G10-2.

SEQ ID NO: 51 is a sequence cDNA SYN21G11.

SEQ ID NO: 52 is a sequence cDNA SYN21G11-2.

SEQ ID NO: 53 is a sequence cDNA SYN21H8.

SEQ ID NO: 54 is a sequence cDNA SYN22A10.

SEQ ID NO: 55 is a sequence cDNA SYN22A10-2.

SEQ ID NO: 56 is a sequence cDNA SYN22A12.

SEQ ID NO: 57 is a sequence cDNA SYN22A2.

SEQ ID NO: 58 is a sequence cDNA SYN22B4.

SEQ ID NO: 59 is a sequence cDNA SYN22C2.

SEQ ID NO: 60 is a sequence cDNA SYN22E10.

SEQ ID NO: 61 is a sequence cDNA SYN22F2.

SEQ ID NO: 62 represents the estimated amino acid sequence SYN18C6 (also known as B709P).

SEQ ID NO: 63 is a sequence cDNA B723P.

SEQ ID NO: 64 is a sequence cDNA B724P.

SEQ ID NO: 65 is a sequence cDNA B770P.

SEQ ID NO: 66 is a sequence cDNA B716P.

SEQ ID NO: 67 is a sequence cDNA B72P.SEQ ID NO: 68 is a sequence cDNA B717P.

SEQ ID NO: 69 is a sequence cDNA B771P.

SEQ ID NO: 70 is a sequence cDNA B722P.

SEQ ID NO: 71 is a sequence cDNA B726P.

SEQ ID NO: 72 is a sequence cDNA B727P.

SEQ ID NO: 73 is a sequence cDNA B728P.

SEQ ID NO: 74-87 represent a certain sequence selected cDNA clones, which show homology with known sequences.

SEQ ID NO: 88 is a sequence cDNA 13053.

SEQ ID NO: 89 is a sequence cDNA 13057.

SEQ ID NO: 90 is a sequence cDNA 13059.

SEQ ID NO: 91 is a sequence cDNA 13065.

SEQ ID NO: 92 is a sequence cDNA 13067.

SEQ ID NO: 93 is a sequence cDNA 13068.

SEQ ID NO: 94 is a sequence cDNA 13071.

SEQ ID NO: 95 is a sequence cDNA 13072.

SEQ ID NO: 96 is a sequence cDNA 13073.

SEQ ID NO: 97 is a sequence cDNA 13075.

SEQ ID NO: 98 is a defined the th cDNA sequence 13078.

SEQ ID NO: 99 is a sequence cDNA 13079.

SEQ ID NO: 100 is a sequence cDNA 13081.

SEQ ID NO: 101 is a sequence cDNA 13082.

SEQ ID NO: 102 is a sequence cDNA 13092.

SEQ ID NO: 103 is a sequence cDNA 13097.

SEQ ID NO: 104 is a sequence cDNA 13101.

SEQ ID NO: 105 is a sequence cDNA 13102.

SEQ ID NO: 106 is a sequence cDNA 13119.

SEQ ID NO: 107 is a sequence cDNA 13131.

SEQ ID NO: 108 is a sequence cDNA 13133.

SEQ ID NO: 109 is a sequence cDNA 13135.

SEQ ID NO: 110 is a sequence cDNA 13139.

SEQ ID NO: 111 is a sequence cDNA 13140.

SEQ ID NO: 112 is a sequence cDNA 13146.

SEQ ID NO: 113 is a sequence cDNA 13147.

SEQ ID NO: 114 is a sequence cDNA 13148.

SEQ ID NO: 115 is a sequence cDNA 13149.

SEQ ID NO: 116 represents the definition of the complete cDNA sequence 13151.

SEQ ID NO: 117 is a sequence cDNA 13051.

SEQ ID NO: 118 is a sequence cDNA 13052.

SEQ ID NO: 119 is a sequence cDNA 13055.

SEQ ID NO: 120 is a sequence cDNA 13058.

SEQ ID NO: 121 is a sequence cDNA 13062.

SEQ ID NO: 122 is a sequence cDNA 13064

SEQ ID NO: 123 is a sequence cDNA 13080

SEQ ID NO: 124 is a sequence cDNA 13093.

SEQ ID NO: 125 is a sequence cDNA 13094.

SEQ ID NO: 126 is a sequence cDNA 13095.

SEQ ID NO: 127 is a sequence cDNA 13096.

SEQ ID NO: 128 is a sequence cDNA 13099.

SEQ ID NO: 129 is a sequence cDNA 13100

SEQ ID NO: 130 is a sequence cDNA 13103.

SEQ ID NO: 131 is a sequence cDNA 13106.

SEQ ID NO: 132 is a sequence cDNA 13107.

SEQ ID NO: 133 is a sequence cDNA 13108.

SEQ ID NO: 134 is a defined the th cDNA sequence 13121

SEQ ID NO: 135 is a sequence cDNA 13126.

SEQ ID NO: 136 is a sequence cDNA 13129.

SEQ ID NO: 137 is a sequence cDNA 13130.

SEQ ID NO: 138 is a sequence cDNA 13134.

SEQ ID NO: 139 is a sequence cDNA 13141.

SEQ ID NO: 140 is a sequence cDNA 13142.

SEQ ID NO: 141 is a sequence cDNA 14376.

SEQ ID NO: 142 is a sequence cDNA 14377.

SEQ ID NO: 143 is a sequence cDNA 14383.

SEQ ID NO: 144 is a sequence cDNA 14384.

SEQ ID NO: 145 is a sequence cDNA 14387.

SEQ ID NO: 146 is a sequence cDNA 14392.

SEQ ID NO: 147 is a sequence cDNA 14394.

SEQ ID NO: 148 is a sequence cDNA 14398.

SEQ ID NO: 149 is a sequence cDNA 14401.

SEQ ID NO: 150 is a sequence cDNA 14402.

SEQ ID NO: 151 is a sequence cDNA 14405.

SEQ ID NO: 152 is defined is nnow the cDNA sequence 14409.

SEQ ID NO: 153 is a sequence cDNA 14412.

SEQ ID NO: 154 is a sequence cDNA 14414.

SEQ ID NO: 155 is a sequence cDNA 14415.

SEQ ID NO: 156 is a sequence cDNA 14416.

SEQ ID NO: 157 is a sequence cDNA 14419.

SEQ ID NO: 158 is a sequence cDNA 14426.

SEQ ID NO: 159 is a sequence cDNA 14427.

SEQ ID NO: 160 is a sequence cDNA 14375.

SEQ ID NO: 161 is a sequence cDNA 14378 collection items.

SEQ ID NO: 162 is a sequence cDNA 14379.

SEQ ID NO: 163 is a sequence cDNA 14380.

SEQ ID NO: 164 is a sequence cDNA 14381.

SEQ ID NO: 165 is a sequence cDNA 14382.

SEQ ID NO: 166 is a sequence cDNA 14388.

SEQ ID NO: 167 is a sequence cDNA 14399.

SEQ ID NO: 168 is a sequence cDNA 14406.

SEQ ID NO: 169 is a sequence cDNA 14407.

SEQ ID NO: 170 is defined is nnow the cDNA sequence 14408.

SEQ ID NO: 171 is a sequence cDNA 14417.

SEQ ID NO: 172 is a sequence cDNA 14418.

SEQ ID NO: 173 is a sequence cDNA 14423.

SEQ ID NO: 174 is a sequence cDNA 14424.

SEQ ID NO: 175 represents a specific sequence of cDNA B726P-20.

SEQ ID NO: 176 is the amino acid sequence B726P-20.

SEQ ID NO: 177 is a PCR primer.

SEQ ID NO: 178 is a sequence cDNA B726P-74.

SEQ ID NO: 179 is designed amino acid sequence B726P-74.

SEQ ID NO: 180 is a sequence cDNA B726P-79.

SEQ ID NO: 181 is the amino acid sequence B726P-79.

SEQ ID NO: 182 represents a specific sequence of cDNA 19439.1 exhibiting homology with the genome of mammaglobin.

SEQ ID NO: 183 is a sequence cDNA 19407.1 exhibiting homology with the genome of human keratin.

SEQ ID NO: 184 is a sequence cDNA 19428.1 showing homology with clone chromosome 17 people.

SEQ ID NO: 185 is a sequence cDNA B808P (19408), does not exhibit significant homology with any known what Yong.

SEQ ID NO: 186 is a sequence cDNA 19460.1, does not exhibit significant homology with any known gene.

SEQ ID NO: 187 is a sequence cDNA 19419.1 showing homology with light chain Kappa Ig.

SEQ ID NO: 188 is a sequence cDNA 19411.1 showing homology with collagen alpha-1 person.

SEQ ID NO: 189 is a sequence cDNA 19420.1 showing homology with proteinase-3 mus musculus.

SEQ ID NO: 190 is a sequence cDNA 19432.1 showing homology with Boxing highly mobile group of people.

SEQ ID NO: 191 is a sequence cDNA 19412.1 exhibiting homology with the gene plasminogen activator person.

SEQ ID NO: 192 is a sequence cDNA 19415.1, showing homology to mitogen-activated protein kinase.

SEQ ID NO: 193 is a sequence cDNA 19409.1 showing homology with protein chondroitin sulfate-proteoglycan.

SEQ ID NO: 194 is a sequence cDNA 19406.1, does not exhibit significant homology with any known gene.

SEQ ID NO: 195 is is a sequence cDNA 19421.1 showing th is ologie with human fibronectin.

SEQ ID NO: 196 is a sequence cDNA 19426.1 showing homology with item 3, corresponding to the receptor for retinoic acid.

SEQ ID NO: 197 is a sequence cDNA 19425.1 showing homology with MyD88 mRNA.

SEQ ID NO: 198 is a sequence cDNA 19424.1 showing homology with an mRNA peptide Transporter (TAP-1)

SEQ ID NO: 199 is a sequence cDNA 19429.1, does not exhibit significant homology with any known gene.

SEQ ID NO: 200 is a sequence cDNA 19435.1 showing homology with polymorphic mucin epithelial person.

SEQ ID NO: 201 is a sequence cDNA B813P (19434.1), showing homology with transcription factor GATA-3 people.

SEQ ID NO: 202 is a sequence cDNA 19461.1 exhibiting homology with the gene of AP-2 people.

SEQ ID NO: 203 is a sequence cDNA 19450.1 exhibiting homology with the regulatory factor that binds DNA.

SEQ ID NO: 204 is a sequence cDNA 19451.1 exhibiting homology with the regulatory cofactor exchange of Na/h

SEQ ID NO: 205 is a sequence cDNA 19462.1, not shown C is achimas homology with any known gene.

SEQ ID NO: 206 represents a specific sequence of cDNA 19455.1 showing homology with an mRNA of histone HAS.Z person.

SEQ ID NO: 207 is a sequence cDNA 19459.1 showing homology with clone 179N16 RACES.

SEQ ID NO: 208 is a sequence cDNA 19464.1, does not exhibit significant homology with any known gene.

SEQ ID NO: 209 is a sequence cDNA 19414.1 showing homology with lipofilling .SEQ ID NO: 210 is a sequence cDNA 19413.1 showing homology with clone hRPK.209_J_20 chromosome 17.

SEQ ID NO: 211 is a sequence cDNA 19416.1, does not exhibit significant homology with any known gene.

SEQ ID NO: 212 is a sequence cDNA 19437.1 showing homology with an mRNA clone 24976 person.

SEQ ID NO: 213 is a sequence cDNA 19449.1 exhibiting homology with the DNA of mouse crustal protein PG-M.

SEQ ID NO: 214 is a sequence cDNA 19446.1, does not exhibit significant homology with any known gene.

SEQ ID NO: 215 is a sequence cDNA 19452.1, does not exhibit significant homology with any known gene.

SEQ ID NO: 216 depict is to place a sequence cDNA 19483.1, does not exhibit significant homology with any known gene.

SEQ ID NO: 217 is a sequence cDNA 19526.1 showing homology with lipofilling.

SEQ ID NO: 218 represents a specific sequence of cDNA 19484.1 showing homology with secretively protein XAG-2 cement gland.

SEQ ID NO: 219 represents a specific sequence of cDNA 19470.1, does not exhibit significant homology with any known gene.

SEQ ID NO: 220 is a sequence cDNA 19469.1 exhibiting homology with the gene HLA-DM of the person.

SEQ ID NO: 221 is a sequence cDNA 19482.1 exhibiting homology with the gene of the protein pS2 man.

SEQ ID NO: 222 is a sequence cDNA B805P (19468.1), does not exhibit significant homology with any known gene.

SEQ ID NO: 223 represents a specific sequence of cDNA 19467.1 showing homology with an mRNA of thrombospondin person.

SEQ ID NO: 224 is a sequence cDNA 19498.1, showing homology to CDC2 gene involved in the control of cell cycle.

SEQ ID NO: 225 is a sequence cDNA 19506.1 showing homology with human cDNA for protein TREB.

SEQ ID NO: 226 represents the definition is by the cDNA sequence B806P (19505.1), does not exhibit significant homology with any known gene.

SEQ ID NO: 227 represents a specific sequence of cDNA 19486.1 showing homology with epidermal keratin type I.

SEQ ID NO: 228 represents a specific sequence of cDNA 19510.1 showing homology with glucose Transporter glikoproteid.

SEQ ID NO: 229 is a sequence cDNA 19512.1 exhibiting homology with the genome of lysergically person.

SEQ ID NO: 230 represents a specific sequence of cDNA 19511.1 showing homology with Palmitoyl-proteinaceous person.

SEQ ID NO: 231 is a sequence cDNA 19508.1 showing homology with alpha enolate person.

SEQ ID NO: 232 represents a specific sequence of cDNA B807P (19509.1), does not exhibit significant homology with any known gene.

SEQ ID NO: 233 is a sequence cDNA B809P (19520.1), showing homology to clone 102D24 chromosome 11ql3.31.

SEQ ID NO: 234 represents a specific sequence of cDNA 19507.1 showing homology with beta-subunit of taproom.

SEQ ID NO: 235 is a sequence cDNA 19525.1 showing homology with prourokinase predecessor of the person.

SEQ ID NO: 236 ol dstanley a sequence cDNA 19513.1, does not exhibit significant homology with any known gene.

SEQ ID NO: 237 represents a specific sequence of cDNA 19517.1 showing homology with clone 128M19 of human RACES.

SEQ ID NO: 238 represents a specific sequence of cDNA 19564.1 showing homology with cytochrome P450-IIB person.

SEQ ID NO: 239 is a sequence cDNA 19553.1 exhibiting homology with the subunit of pi GABA receptor is A human.

SEQ ID NO: 240 represents a specific sequence of cDNA B811P (19575.1), does not exhibit significant homology with any known gene.

SEQ ID NO: 241 represents a specific sequence of cDNA B810P (19560.1), does not exhibit significant homology with any known gene.

SEQ ID NO: 242 represents a specific sequence of cDNA 19588.1 exhibiting homology with the protein, such as the aortic carboxypeptidase.

SEQ ID NO: 243 is a sequence cDNA 19551.1 exhibiting homology with the gene BCL-1 person.

SEQ ID NO: 244 is a sequence cDNA 19567.1 showing homology with related proteasome mRNA person.

SEQ ID NO: 245 is a sequence cDNA B803P (19583.1), does not exhibit significant homology with any known gene.

SEQ ID NO: 246 before the hat is a specific sequence of cDNA B812P (19587.1), does not exhibit significant homology with any known gene.

SEQ ID NO: 247 is a sequence cDNA B802P (19392.2), showing homology with chromosome 17 people.

SEQ ID NO: 248 represents a specific sequence of cDNA 19393.2 showing homology with circuit B2 of nicein person.

SEQ ID NO: 249 is a sequence cDNA 19398.2, mRNA class II DQ alpha MNS person.

SEQ ID NO: 250 is a sequence cDNA B804P (19399.2), showing homology with GSHB-184P14 YOU HR person.

SEQ ID NO: 251 represents a specific sequence of cDNA 19401.2 exhibiting homology with the gene kinase-b ikB person.

SEQ ID NO: 252 is a sequence cDNA 20266, does not exhibit significant homology with any known gene.

SEQ ID NO: 253 is a sequence cDNA B826P (20270), does not exhibit significant homology with any known gene.

SEQ ID NO: 254 is a sequence cDNA 20274, does not exhibit significant homology with any known gene.

SEQ ID NO: 255 is a sequence cDNA 20276, does not exhibit significant homology with any known gene.

SEQ ID NO: 256 represents a certain posledovatelno the ü cDNA 20277, does not exhibit significant homology with any known gene.

SEQ ID NO: 257 is a sequence cDNA B823P (20280), does not exhibit significant homology with any known gene.

SEQ ID NO: 258 is a sequence cDNA B821P (20281), does not exhibit significant homology with any known gene.

SEQ ID NO: 259 is a sequence cDNA B824P (20294), does not exhibit significant homology with any known gene.

SEQ ID NO: 260 represents a specific sequence of cDNA 20303, does not exhibit significant homology with any known gene.

SEQ ID NO: 261 represents a specific sequence of cDNA B820P (20310), does not exhibit significant homology with any known .SEQ ID NO: 262 represents a specific sequence of cDNA B825P (20336), does not exhibit significant homology with any known gene.

SEQ ID NO: 263 represents a specific sequence of cDNA B827P (20341), does not exhibit significant homology with any known gene.

SEQ ID NO: 264 represents a specific sequence of cDNA 20941, does not exhibit significant homology with any known gene.

SEQ ID NO: 265 is a sequence cDNA 20954, does not exhibit significant homology sakim any known gene.

SEQ ID NO: 266 represents a specific sequence of cDNA 20961, does not exhibit significant homology with any known gene.

SEQ ID NO: 267 is a sequence cDNA 20965, does not exhibit significant homology with any known gene.

SEQ ID NO: 268 represents a specific sequence of cDNA 20975, does not exhibit significant homology with any known gene.

SEQ ID NO: 269 is a sequence cDNA 20261, showing homology with catenin-R120 person.

SEQ ID NO: 270 is a sequence cDNA B822P (20262), showing homology with membrane glikoproteinom 4F2 person.

SEQ ID NO: 271 represents a specific sequence of cDNA 20265 exhibiting homology with the Na,K-ATPase alpha 1 person.

SEQ ID NO: 272 represents a specific sequence of cDNA 20267, showing homology with HS 90 human heart, incomplete coding sequence.

SEQ ID NO: 273 is a sequence cDNA 20268, showing homology with an mRNA GPI-anchored protein R person.

SEQ ID NO: 274 represents a specific sequence of cDNA 20271, showing homology with subunit with Mm 77 KD factor human, stimulate lipolysis.

SEQ ID NO: 275 is particularly the specific sequence of cDNA 20272, showing homology with p190-B person.

SEQ ID NO: 276 represents a specific sequence of cDNA 20273, showing homology with ribavirina person.

SEQ ID NO: 277 is a sequence cDNA 20278, showing homology with initializationstring person.

SEQ ID NO: 278 represents a specific sequence of cDNA 20279, showing homology with S-adenosylmethionine person.

SEQ ID NO: 279 is a sequence cDNA 20293, showing homology with transcript X-inactivation person.

SEQ ID NO: 280 represents a specific sequence of cDNA 20300, showing homology with cytochrome p450 person.

SEQ ID NO: 281 is a sequence cDNA 20305, showing homology to elongation factor 1 alpha man.

SEQ ID NO: 282 represents a specific sequence of cDNA 20306, showing homology with epithelial protein ets person.

SEQ ID NO: 283 is a sequence cDNA 20307, showing homology to the mRNA of transducer signal person.

SEQ ID NO: 284 represents a specific sequence of cDNA 20313, showing homology with an mRNA subunit of pi GABA receptor is A human.

SEQ ID NO: 285 is a sequence to the NC 20317, showing homology with trointestinal person.

SEQ ID NO: 286 represents a specific sequence of cDNA 20318, showing homology with proteinase cathepsin In person.

SEQ ID NO: 287 represents a specific sequence of cDNA of 20,320 showing homology with 2 posterolateralis-alpha enolate person.

SEQ ID NO: 288 represents a specific sequence of cDNA 20321, showing homology with E-cadherins person.

SEQ ID NO: 289 is a sequence cDNA 20322, showing homology with hsp86 person.

SEQ ID NO: 290 represents a specific sequence of cDNA B828P (20326), showing homology with transcript X-inactivation person.

SEQ ID NO: 291 represents a specific sequence of cDNA 20333, showing homology with the regulator of chromatin person SMARCA5.

SEQ ID NO: 292 represents a specific sequence of cDNA 20335, showing homology with protein 1 activator of sphingolipids person.

SEQ ID NO: 293 is a sequence cDNA 20337, showing homology with inhibitor type 2 activator growth factor of human hepatocytes.

SEQ ID NO: 294 represents a specific sequence of cDNA 20338, showing homology with the cell adhesion molecule CD44 person.

SEQ ID N: 295 is a sequence cDNA 20340, showing homology with nuclear factor 1 human-like factor erythroid origin.

SEQ ID NO: 296 represents a specific sequence of cDNA 20938, showing homology with an mRNA vinculin person.

SEQ ID NO: 297 is a sequence cDNA 20939, showing homology to elongation factor EF-1 alpha human.

SEQ ID NO: 298 represents a specific sequence of cDNA 20940 exhibiting homology with the gene nestin person.

SEQ ID NO: 299 is a sequence cDNA 20942, showing homology with pancreatic ribonuclease person.

SEQ ID NO: 300 is a sequence cDNA 20943, showing homology with transcobalamin I man.

SEQ ID NO: 301 is a sequence cDNA 20944, showing homology with beta-tubulin person.

SEQ ID NO: 302 is a sequence cDNA 20946, showing homology with protein HS1 person.

SEQ ID NO: 303 represents a specific sequence of cDNA 20947, showing homology with cathepsin In person.

SEQ ID NO: 304 is a sequence cDNA 20948, showing homology with transcript reinforced testicular gene of the person.

SEQ ID NO: 305 represents ODA is divided by the cDNA sequence 20949, showing homology to elongation factor EF-1 alpha human.

SEQ ID NO: 306 represents a specific sequence of cDNA 20950, showing homology with factor 3 ADP-ribosylate person.

SEQ ID NO: 307 represents a specific sequence of cDNA 20951, showing homology with IFP53 or WRS person for tryptophanyl-tRNA synthetase.

SEQ ID NO: 308 is a sequence cDNA 20952, showing homology with dependent cycline protein kinase person.

SEQ ID NO: 309 represents a specific sequence of cDNA 20957, showing homology with isoform 1 of alpha-tubulin in person.

SEQ ID NO: 310 represents a specific sequence of cDNA 20959, showing homology with a deletion of length 61 P.N. tyrosinosis person.

SEQ ID NO: 311 represents a specific sequence of cDNA 20966, showing homology with trointestinal person.

SEQ ID NO: 312 represents a specific sequence of cDNA B830P (20976), showing homology to nuclear factor NF 45 .SEQ ID NO: 313 is a sequence cDNA B829P (20977), showing homology with Desaturate fatty acid Delta-6 persons.

SEQ ID NO: 314 is a sequence cDNA 20978, showing homology to nuclear aconitate is the ne.

SEQ ID NO: 315 is defined by the sequence of the cDNA clone 23176.

SEQ ID NO: 316 is defined by the sequence of the cDNA clone 23140.

SEQ ID NO: 317 is a certain sequence of cDNA clone 23166.

SEQ ID NO: 318 is defined by the sequence of the cDNA clone 23167.

SEQ ID NO: 319 is a certain sequence of cDNA clone 23177.

SEQ ID NO: 320 is defined by the sequence of the cDNA clone 23217.

SEQ ID NO: 321 is a certain sequence of cDNA clone 23169.

SEQ ID NO: 322 is defined by the sequence of the cDNA clone 23160.

SEQ ID NO: 323 is defined by the sequence of the cDNA clone 23182.

SEQ ID NO: 324 is defined by the sequence of the cDNA clone 23232.

SEQ ID NO: 325 is a certain sequence of cDNA clone 23203.

SEQ ID NO: 326 is defined by the sequence of the cDNA clone 23198.

SEQ ID NO: 327 is a certain sequence of cDNA clone 23224.

SEQ ID NO: 328 is defined by the sequence of the cDNA clone 23142.

SEQ ID NO: 329 is a certain sequence of cDNA clone 23138.

SEQ ID NO: 330 is defined by the sequence of the cDNA clone 23147.

SEQ ID NO: 331 is a certain sequence of cDNA clone 23148.

SEQ ID NO: 332 is defined by the sequence of the cDNA clone 23149.

SEQ ID NO: 333 op is delannoy the cDNA sequence of clone 23172.

SEQ ID NO: 334 is defined by the sequence of the cDNA clone 23158.

SEQ ID NO: 335 is a certain sequence of cDNA clone 23156.

SEQ ID NO: 336 is defined by the sequence of the cDNA clone 23221.

SEQ ID NO: 337 is a certain sequence of cDNA clone 23223.

SEQ ID NO: 338 is defined by the sequence of the cDNA clone 23155.

SEQ ID NO: 339 is a certain sequence of cDNA clone 23225.

SEQ ID NO: 340 is defined by the sequence of the cDNA clone 23226.

SEQ ID NO: 341 is defined by the sequence of the cDNA clone 23228.

SEQ ID NO: 342 is defined by the sequence of the cDNA clone 23229.

SEQ ID NO: 343 is a certain sequence of cDNA clone 23231.

SEQ ID NO: 344 is defined by the sequence of the cDNA clone 23154.

SEQ ID NO: 345 is a certain sequence of cDNA clone 23157.

SEQ ID NO: 346 is defined by the sequence of the cDNA clone 23153.

SEQ ID NO: 347 is a certain sequence of cDNA clone 23159.

SEQ ID NO: 348 is a certain sequence of cDNA clone 23152.

SEQ ID NO: 349 is a certain sequence of cDNA clone 23161.

SEQ ID NO: 350 is a certain sequence of cDNA clone 23162.

SEQ ID NO: 351 is a certain sequence of cDNA clone 23163.

SEQ ID NO: 352 op is delannoy the cDNA sequence of clone 23164.

SEQ ID NO: 353 is a certain sequence of cDNA clone 23165.

SEQ ID NO: 354 is defined by the sequence of the cDNA clone 23151.

SEQ ID NO: 355 is a certain sequence of cDNA clone 23150.

SEQ ID NO: 356 is defined by the sequence of the cDNA clone 23168.

SEQ ID NO: 357 is a certain sequence of cDNA clone 23146.

SEQ ID NO: 358 is a certain sequence of cDNA clone 23170.

SEQ ID NO: 359 is a certain sequence of cDNA clone 23171.

SEQ ID NO: 360 is a certain sequence of cDNA clone 23145.

SEQ ID NO: 361 is a certain sequence of cDNA clone 23174.

SEQ ID NO: 362 is defined by the sequence of the cDNA clone 23175.

SEQ ID NO: 363 is a certain sequence of cDNA clone 23144.

SEQ ID NO: 364 is a certain sequence of cDNA clone 23178.

SEQ ID NO: 365 is a certain sequence of cDNA clone 23179.

SEQ ID NO: 366 is a certain sequence of cDNA clone 23180.

SEQ ID NO: 367 is a certain sequence of cDNA clone 23181.

SEQ ID NO: 368 is a certain sequence of cDNA clone 23143.

SEQ ID NO: 369 is a certain sequence of cDNA clone 23183.

SEQ ID NO: 370 is a certain sequence of cDNA clone 23184.

SEQ ID NO: 371 op is delannoy the cDNA sequence of clone 23185.

SEQ ID NO: 372 is a certain sequence of cDNA clone 23186.

SEQ ID NO: 373 is a certain sequence of cDNA clone 23187.

SEQ ID NO: 374 is a certain sequence of cDNA clone 23190.

SEQ ID NO: 375 is a certain sequence of cDNA clone 23189.

SEQ ID NO: 376 is a certain sequence of cDNA clone 23202.

SEQ ID NO: 378 is a certain sequence of cDNA clone 23191.

SEQ ID NO: 379 is a certain sequence of cDNA clone 23188.

SEQ ID NO: 380 is defined by the sequence of the cDNA clone 23194.

SEQ ID NO: 381 is a certain sequence of cDNA clone 23196.

SEQ ID NO: 382 is a certain sequence of cDNA clone 23195.

SEQ ID NO: 383 is a certain sequence of cDNA clone 23193.

SEQ ID NO: 384 is defined by the sequence of the cDNA clone 23199.

SEQ ID NO: 385 is a certain sequence of cDNA clone 23200.

SEQ ID NO: 386 is a certain sequence of cDNA clone 23192.

SEQ ID NO: 387 is a certain sequence of cDNA clone 23201.

SEQ ID NO: 388 is a certain sequence of cDNA clone 23141.

SEQ ID NO: 389 is a certain sequence of cDNA clone 23139.

SEQ ID NO: 390 is a certain sequence of cDNA clone 23204.

SEQ ID NO: 391 op is delannoy the cDNA sequence of clone 23205.

SEQ ID NO: 392 is a certain sequence of cDNA clone 23206.

SEQ ID NO: 393 is a certain sequence of cDNA clone 23207.

SEQ ID NO: 394 is a certain sequence of cDNA clone 23208.

SEQ ID NO: 395 is a certain sequence of cDNA clone 23209.

SEQ ID NO: 396 is a certain sequence of cDNA clone 23210.

SEQ ID NO: 397 is a certain sequence of cDNA clone 23211.

SEQ ID NO: 398 is a certain sequence of cDNA clone 23212.

SEQ ID NO: 399 is a certain sequence of cDNA clone 23214.

SEQ ID NO: 400 is a certain sequence of cDNA clone 23215.

SEQ ID NO: 401 is a certain sequence of cDNA clone 23216.

SEQ ID NO: 402 is defined by the sequence of the cDNA clone 23137.

SEQ ID NO: 403 is a certain sequence of cDNA clone 23218.

SEQ ID NO: 404 is defined by the sequence of the cDNA clone 23220.

SEQ ID NO: 405 is defined by the sequence of the cDNA clone 19462.

SEQ ID NO: 406 is defined by the sequence of the cDNA clone 19430.

SEQ ID NO: 407 is defined by the sequence of the cDNA clone 19407.

SEQ ID NO: 408 is defined by the sequence of the cDNA clone 19448.

SEQ ID NO: 409 is a certain sequence of cDNA clone 19447.

SEQ ID NO: 410 is op is delannoy the cDNA sequence of clone 19426.

SEQ ID NO: 411 is a certain sequence of cDNA clone 19441.

SEQ ID NO: 412 is defined by the sequence of the cDNA clone 19454.

SEQ ID NO: 413 is defined by the sequence of the cDNA clone 19463.

SEQ ID NO: 414 is defined by the sequence of the cDNA clone 19419.

SEQ ID NO: 415 is a certain sequence of cDNA clone 19434.

SEQ ID NO: 416 is defined extended cDNA sequence VR.

SEQ ID NO: 417 is defined extended cDNA sequence VR.

SEQ ID NO: 418 is defined extended cDNA sequence VR.

SEQ ID NO: 419 is defined extended cDNA sequence VR.

SEQ ID NO: 420 is defined extended cDNA sequence VR.

SEQ ID NO: 421 is defined extended cDNA sequence VR.

SEQ ID NO: 422 is defined extended cDNA sequence VR.

SEQ ID NO: 423 is defined extended cDNA sequence VR.

SEQ ID NO: 424 is defined extended cDNA sequence VR.

SEQ ID NO: 425 is defined extended cDNA sequence VR.

SEQ ID NO: 426 is defined extended cDNA sequence VR.

SEQ ID NO: 427 is a certain sequence of cDNA clone W.

SEQ ID NO: 428 is about the particular sequence of the cDNA clone 22892.

SEQ ID NO: 429 is a certain sequence of cDNA clone 266G3.

SEQ ID NO: 430 is defined by the sequence of the cDNA clone 22890.

SEQ ID NO: 431 is a certain sequence of cDNA clone W.

SEQ ID NO: 432 is defined by the sequence of the cDNA clone 22883.

SEQ ID NO: 433 is a certain sequence of cDNA clone 22882.

SEQ ID NO: 434 is defined by the sequence of the cDNA clone 22880.

SEQ ID NO: 435 is a certain sequence of cDNA clone 263G1.

SEQ ID NO: 436 is a certain sequence of cDNA clone 263G6.

SEQ ID NO: 437 is a certain sequence of cDNA clone 262B2.

SEQ ID NO: 438 is a certain sequence of cDNA clone 262B6.

SEQ ID NO: 439 is a certain sequence of cDNA clone 22869.

SEQ ID NO: 440 is defined by the sequence of the cDNA clone 21374.

SEQ ID NO: 441 is a certain sequence of cDNA clone 21362.

SEQ ID NO: 442 is defined by the sequence of the cDNA clone 21349.

SEQ ID NO: 443 is a certain sequence of cDNA clone 21309.

SEQ ID NO: 444 is a certain sequence of cDNA clone 21097.

SEQ ID NO: 445 is a certain sequence of cDNA clone 21096.

SEQ ID NO: 446 is a certain sequence of cDNA clone 21094.

SEQ ID NO: 447 op is delannoy the cDNA sequence of clone 21093.

SEQ ID NO: 448 is a certain sequence of cDNA clone 21091.

SEQ ID NO: 449 is a certain sequence of cDNA clone 21089.

SEQ ID NO: 450 is a certain sequence of cDNA clone 21087.

SEQ ID NO: 451 is a certain sequence of cDNA clone 21085.

SEQ ID NO: 452 is defined by the sequence of the cDNA clone 21084.

SEQ ID NO: 453 is the first partial sequence of cDNA clone VT-40.

SEQ ID NO: 454 is the second partial sequence of cDNA clone VT-40.

SEQ ID NO: 455 is a certain sequence of cDNA clone 21063.

SEQ ID NO: 456 is a certain sequence of cDNA clone 21062.

SEQ ID NO: 457 is a certain sequence of cDNA clone 21060.

SEQ ID NO: 458 is a certain sequence of cDNA clone 21053.

SEQ ID NO: 459 is a certain sequence of cDNA clone 21050.

SEQ ID NO: 460 is a certain sequence of cDNA clone 21036.

SEQ ID NO: 461 is a certain sequence of cDNA clone 21037.

SEQ ID NO: 462 is a certain sequence of cDNA clone 21048.

SEQ ID NO: 463 is a consensus DNA sequence VR (denoted B726P-spliced_seq_B726P).

SEQ ID NO: 464 is a cDNA sequence of the second splanirovannaya form B726P (denoted 27490.seq_B726P).

SEQ ID NO: 465 is the tsya specific cDNA sequence of the third splanirovannaya form B726P (denoted 27068.seq_B726P).

SEQ ID NO: 466 is a cDNA sequence of the second splanirovannaya form B726P (denoted 23113.seq_B726P).

SEQ ID NO: 467 is a cDNA sequence of the second splanirovannaya form B726P (denoted 23103.seq_B726P).

SEQ ID NO: 468 is a cDNA sequence of the second splanirovannaya form B726P (denoted 19310.seq_B726P).

SEQ ID NO: 469 is calculated by the amino acid sequence encoded "left" ORF SEQ ID NO: 463.

SEQ ID NO: 470 is calculated by the amino acid sequence encoded by SEQ ID NO: 464.

SEQ ID NO: 471 is calculated by the amino acid sequence encoded by SEQ ID NO: 465.

SEQ ID NO: 472 is calculated by the amino acid sequence encoded by SEQ ID NO: 466.

SEQ ID NO: 473 is calculated by the amino acid sequence encoded by SEQ ID NO: 467.

SEQ ID NO: 474 is a certain sequence of cDNA alternative splanirovannaya form B726P.

SEQ ID NO: 475 is the amino acid sequence encoded by SEQ ID NO: 474.

SEQ ID NO: 476 is an isolated cDNA sequence B720P.

SEQ ID NO: 477 is the cDNA sequence of a known gene keratin.

SEQ ID NO: 478 is the amino acid sequence encoded by SEQ ID NO: 477.

SEQ ID NO: 479 is a certain sequence of cDNA clone 19465.

SEQ ID NO: 480 and 481 PR is astavliaut the PCR primers.

SEQ ID NO: 482 is a cDNA sequence expressed by the "right" ORF B726P.

SEQ ID NO: 483 is the amino acid sequence of expressed recombinant "right" ORF B726P.

SEQ ID NO: 484 is a certain sequence of full length cDNA for B720P.

SEQ ID NO: 485 is the amino acid sequence encoded by SEQ ID NO: 484.

SEQ ID NO: 486 is a cDNA sequence of a shortened form B720P denoted B720P-tr.

SEQ ID NO: 487 is the amino acid sequence B720P-tr.

SEQ ID NO: 488 is the amino acid sequence of naturally processioning epitope B726P, recognizable B726P-specific CTL.

SEQ ID NO: 489 is a DNA sequence that encodes the epitope B726P specified in SEQ ID NO: 488.

SEQ ID NO: 490 is a DNA sequence that encodes a hybrid protein in which mammaglobin merged with United open reading frame (ORF) of B726P "left" and "right" ORF (amino acid sequence United ORF B726P here presented in SEQ ID NO: 475, which is encoded by the DNA sequence SEQ ID NO: 474).

SEQ ID NO: 491 is a DNA sequence that encodes a hybrid protein in which mammaglobin merged with the "left" ORF B726P, (amino acid sequence "left" ORF B726P, here presented in SEQ ID NO: 469, which is encoded by the sequence DN is SEQ ID NO: 463).

SEQ ID NO: 492 is a DNA sequence that encodes a hybrid protein in which mammaglobin merged with the "right" ORF B726P, (amino acid sequence "right" ORF B726P, here presented in SEQ ID NO: 176, which is encoded by the DNA sequence SEQ ID NO: 175).

SEQ ID NO: 493 is the amino acid sequence encoded by the DNA sequence SEQ ID NO: 490.

SEQ ID NO: 494 is the amino acid sequence encoded by the DNA sequence SEQ ID NO: 491.

SEQ ID NO: 495 is the amino acid sequence encoded by the DNA sequence SEQ ID NO: 492.

DETAILED DESCRIPTION OF THE INVENTION.

As stated above, in General, the direction of the present invention are compositions and methods of using the compositions, for example, in the treatment and diagnosis of malignant tumors, such as breast cancer. Some are described here for illustrative compositions include polypeptides tumors of the breast, polynucleotide encoding such polypeptides, binding agents such as antibodies, antigen-presenting cells (APCS) and/or immune system cells (such as T-cells). Used herein, the term "protein of breast cancer", in General, refers to a protein that is expressed by tumor cells of the breast at a level that is at least twice, and preferably, Melsheimer, five times the level of expression in normal tissues, which are determined with the help presented here is a typical analysis. Some proteins of breast cancer are tumor proteins that react with anticorodal patient affected by breast cancer, so that it is registered (in the immunoassay, such as ELISA or Western blot).

Therefore, in accordance with the above and as described hereinafter, the present invention presents illustrative polynucleotide compositions having the sequence specified in SEQ ID NO: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486 and 489, illustrative polypeptide compositions having the amino acid sequence indicated in SEQ ID NO: 176, 179, 181, 469-473, 475, 485, 487 and 488, the compositions of antibodies which are able to bind to such polypeptides, and numerous additional applications of such compositions, for example, when the detection, diagnosis and/or therapy of breast cancer in humans.

POLYNUCLEOTIDE COMPOSITIONS.

In the sense used here, the term "DNA segment" or "polynucleotide" refers to a DNA molecule that was isolated from total genomic DNA of a particular species. Therefore, DNA encoding the polypeptide, refers to a segment of DNA that contains one or more coding sequences, the AOC is e, largely isolated or purified from total genomic DNA of the species from which the DNA segment. The term "DNA segment" or "polynucleotide" include DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, Comedy, family, phages, viruses and the like.

As will be clear to experts in this area, segments of DNA according to this invention can include genomic sequences, vaginalnye and encoded by plasmid sequences and smaller engineered gene segments that Express, or may be adapted for the expression of proteins, polypeptides, peptides and the like. Such segments can be distinguished in natural form or can be artificially modified synthetic method.

"Isolated" in the sense used here means that polynucleotide largely exempt from other coding sequences, and that the DNA segment does not contain large portions not related coding DNA, such as large chromosomal fragments or other functional genes or regions encoding polypeptides. Of course, it has to do with the segment of DNA that is initially allocated, and does not exclude genes or coding regions, artificially dobavlennye segment later.

As will be clear to experts in the field, polynucleotide can be single-stranded (coding or antimuslim) or Dantewada and may be DNA (genomic, cDNA or synthetic) or RNA. RNA molecules include molecules garns, which contain introns and are consistent with the DNA molecule of one-to-one, and the mRNA molecules that do not contain introns. In polynucleotide according to the invention can, but need not, be present additional coding or not coding sequences, and polynucleotide may, but need not necessarily, be associated with other molecules and/or materials-carriers.

Polynucleotide can contain native sequence (i.e. endogenous sequence that encodes a protein of breast cancer or a portion thereof) or may contain a variant, or a biological or antigenic functional equivalent of such a sequence. Options polynucleotide may contain one or more substitutions, accessions, deletions and/or insertions, which are described below, preferably such that the immunogenicity of the encoded polypeptide relative to the native protein of the tumor was reduced. The impact on the immunogenicity of the encoded polypeptide can usually be assessed as described here. The term "variants" also encompasses homologous gene is xenogenic nature. When comparing polynucleotide or polypeptide sequences say that two sequences are "identical"if the sequence of nucleotides or amino acids in the two sequences is the same when aligned in relation to the maximum correspondence as described below. Comparison between the two sequences are typically performed by comparing sequences over the window of comparison, to identify and compare local regions of sequence similarity. "Comparison window" in the sense used here refers to the segment length of at least about 20 continuously successive positions, usually from 30 to about 75, from about 40 to about 50, on which a sequence can be compared to a reference sequence of the same number continuously following each other regulations after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison can be done using the Megalign program in the set of computer programs in bioinformatics Lasergene (DNASTAR, Inc., Madison, WI)using the default settings. This program includes several equalization schemes, described in the following sources: Dayhoff, M. O. (1978) A model of evolutionary change in proteins " Matrices for detecting distan relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5: 151-153; Myers, E. W. and Muller W. (1988) CABIOS 4: 11-17; Robinson, E. D. (1971) Comb. Theor 11: 105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4: 406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80: 726-730.

Alternative optimal alignment of sequences for comparison can be carried out through the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2: 482, by the algorithm of alignment identity Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, search methods similarity Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, a computerized implementation of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the package of computer programs Wisconsin Genetics, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI) or view.

One preferred example of algorithms that are suitable for determining sequence identity and sequence similarity in percent, are algorithms BLAST and BLAST 2.0, which is described in Altschul et al. (1977) Nucl. Acids Res. 25: 3389-3402 and Altschul et al. (1990) J Mol. Biol. 215: 403-410, respectively. BLAST and BLAST 2.0 can use, for example, with the parameters described here, to determine the percent identity of the sequences of polynuclear is the Chida and polypeptides according to the invention. A computer program for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one illustrative example, the total number of points can be calculated using, for nucleotide sequences, the parameters M (reward points for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, you can use the matrix scoring to calculate the total number of points. The continuation of matching words in each direction stop in the case when: the total number of points aligning reduced by the amount of X in comparison with it reached a maximum value; the cumulative score goes to zero or below, due to the accumulation of one or more negatively evaluated residue alignments; or reaches the end of any sequence. The parameters W, T and X of the BLAST algorithm to determine the sensitivity and speed of the alignment. In the BLAST program (for nucleotide sequences), the default length of words (W), is equal to 11, and expectation (E) of 10, and the alignment matrix BLOSUM62 scoring (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: of 10,915) (B) of 50, expectation (E) of 10, M=5, N=-4 and a comparison of both strands.

Preferably, the percent identity p is sledovatelnot determine when comparing two optimally aligned sequences over the window of comparison, at least 20 positions, where the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e. gaps)that comprise 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, compared to the reference sequence (which does not include additions or deletions) for optimal alignment of two sequences. The percentage is calculated by determining the number of positions in which both sequences are identical to the nucleic acid bases or amino acid residues, to get the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e. window size), and multiplying the results by 100 to get the percent identity of the sequences.

Thus, the invention includes a polynucleotide or polypeptide sequences having substantial identity with the sequences described here, for example, sequences having at least 50% sequence identity, preferably at least, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity when compared to the polynucleotide reprivatize sequence according to this invention by methods described here (for example, BLAST analysis using standard parameters, as described below). The person skilled in the art will understand that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account the degeneracy of codons, the similarity of amino acids, the location of the reading frames and the like.

In additional embodiments, the present invention relates to isolated polynucleotides and polypeptides containing different along the length of the fragment sequence that is identical or complementary to one or more sequences described here. For example, the present invention presents polynucleotide, which contain at least about 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more continuously consecutive nucleotides of one or more sequences described here, as well as all intermediate lengths between them. It will be easy to understand that "intermediate lengths", in this context, means any length between the specified values, such as 16, 17, 18, 19, etc; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc; including all integers between 200-500; 500-1000, and the like.

Polynucleotide according to this invention or their f is Agency regardless of the length of the coding sequence, can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional sites of enzymes, multiple cloning sites, other coding segments, and the like, so that their overall length may vary significantly. Therefore, it is assumed that it is possible to use the fragment of the nucleic acid of almost any length, with the total length preferably is limited by the ease of production and use in the planned Protocol of recombinant DNA. For example, it is assumed that the illustrative segments of DNA with a total length of about 10,000, about 5,000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length, and the like (including all intermediate lengths) are suitable for many practical embodiments of the present invention.

In other embodiments, the direction of the present invention are polynucleotide that under conditions of moderate hardness can gibridizatsiya presented here polynucleotide sequence or its fragment, or she complementary sequence. Methods of hybridization are well known in the field of molecular biology. For purposes of illustration, suitable conditions of moderate stringency for testing the hybridization of polynucleotide according to the invention on the other is their polynucleotide include pre-washing in a solution of 5 X SSC, of 0.5% SDS, 1.0 mm EDTA (pH 8.0), hybridization at 50°C - 65°C, 5 X SSC, overnight; followed by washing at 65°C for 20 minutes twice each of the solutions of 2X, 0.5x and 0.2X SSC containing 0.1% SDS.

In addition, professionals in this field will be clear that as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptide described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Despite this, polynucleotide that vary as a result of differences in the use of codons that are specifically provided by this invention. In addition, the scope of this invention are alleles of the genes presented here contains polynucleotide sequence. Alleles are endogenous genes that changed as a result of one or more mutations, such as deletion, addition and/or substitution of nucleotides. The resulting mRNA and protein can, but not necessarily, have a modified structure or function. Alleles can be identified using standard methods (such as hybridization, amplification and/or comparison with sequences in the database).

PROBES AND PRIMERS.

In other embodiments of this invention present in the estuaries and the ü polynucleotide sequence can preferably be used as probes or primers for hybridization of nucleic acids. Essentially it is assumed that the segments of nucleic acids that contain a region of sequence which is a contiguous sequence of length of at least about 15 nucleotides, which has the same sequence or is complementary stated here a continuous sequence length of 15 nucleotides, will find the particular application. Longer identical or complementary to a contiguous sequence, for example, the sequence from about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to the full-length sequences will also be suitable in some embodiments.

The ability of such nucleic acid probes specific to gibridizatsiya sequence, which is of interest, provides the possibility of their use to detect the presence of complementary sequences in a given sample. However, it is also assumed other applications, such as using the information recorded in the sequence, to obtain mutant primers, or primers for use in accessing other genetic constructions.

Molecules polynucleotides having regions of sequence consisting of nucleotide sites of Contigo approximately 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or OK is lo (also including intermediate length), identical or complementary stated here polynucleotide sequence, in particular, are assumed as hybridization probes for use in, for example, southern and Northern-blotting. This will allow you to analyze the product of the gene or its fragment as in a variety of cell types and in different bacterial cells. The total size of the fragment, and the size of the complementary site(s) ultimately will depend on the intended use or application of a particular segment of nucleic acid. Smaller portions will mainly find application in embodiments, hybridization, while the length of the contiguous complementary region may be varied, for example, between about 15 and 100 nucleotides, but can be used over a long continuous complementary areas, according to the length complementary sequences that you want to find.

The use of probes for hybridization with a length of about 15-25 nucleotides allows for the formation of molecules of the duplex, which is both stable and selective. Although, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and level of specific hybrid molecules, as a rule, preferred molecules with continuous complementary the e sequence on sites longer than 15 bases. Mostly preferred is the design nucleic acid molecules having complementary genes plots ranging in length from 15 to 25 continuous consecutive nucleotides, or, if desired, even longer.

Probes for hybridization can be selected from any part of any of the claimed sequences here. All that is required is to analyze the sequence specified in SEQ ID NO: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486 and 489 or to any continuous portion of the sequence, a length of from about 15-25 nucleotides up to the full sequence length, inclusive, which is required for use as a probe or primer. The choice of sequence probe or primer can be caused by various factors. For example, desirable may be the use of primers from end to end a common sequence.

Small segments or fragments of polynucleotide easy to get, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated synthesizer oligonucleotides. The fragments can be obtained by applying the technology of reproduction of nucleic acids, such as PCR (PCRTM)set forth in U.S. patent 4683202 (incorporated herein by reference), by introducing selected posledovatelno is she in recombinant vectors for production of recombinant, or by other recombinant DNA technologies generally known to experts in the field of molecular biology.

Nucleotide sequence according to this invention can be used due to their ability to selectively form duplex molecules with complementary sections of the full gene or gene fragments of interest. Depending on the intended application, typically require use of a various conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. In the case of applications requiring high selectivity, as a rule, desirable to use a relatively stringent conditions to form the hybrids, for example, will be selected terms at a relatively low salt concentration and/or high temperatures, such as conditions, which provides a salt concentration from about 0.02 M to 0.15 M salt, at temperatures from about 50°With up to 70°C. Such selective conditions tolerate little, if any, allow erroneous base pairing between the probe and the matrix, or the target thread, and may be particularly suitable for the selection of related sequences.

Of course, for some applications, for example, when chelation is) to obtain mutants, using a thread mutant primer, hybridization with the main matrix is typically required less stringent (low stringency) conditions are hybridization in order to provide opportunity for education of heteroduplex. Under such circumstances it may be desirable to use the conditions at a concentration of salt as from about 0.15 M to about 0.9 M salt, at temperatures in the range from about 20°With up to approximately 55°C. this cross-hybridization variants can be easily defined as a positive hybridization signals relative to hybridization in control. In any case, is usually taken into account that the conditions can be made more rigid by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be easily manipulated, and therefore, as a rule, there is a method of choice depending on the desired results.

IDENTIFICATION AND CHARACTERIZATION OF POLYNUCLEOTIDES.

Polynucleotide can identify, receive and/or be manipulated using any of a variety of well-developed methods. For example, polynucleotide can be identified, as described in more detail below,by screening cDNA chips in relation to the expression, associated with the tumor (for example, the expression of which at least two times higher in tumor than in normal tissue based on the definitions provided in this paper, a typical analysis). Such screening assays can, for example, be accomplished using the chips Synteni (Palo Alto, CA) according to manufacturer's instructions (and essentially as described by Schena et al., Proc. Natl. Acad. Sci. USA 93: 10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA 94: 2150-2155, 1997). Alternative polynucleotide you can amplify from cDNA derived from cells expressing the proteins described herein, such as tumor cells of the breast. Such polynucleotide possible to amplify by polymerase chain reaction (PCR). Such an approach specific to the sequence of the primers can be designed based on the sequences presented here, and can be purchased or synthesized.

Amplified part of polynucleotide according to this invention can be used to highlight the full gene from a suitable library (e.g., cDNA library of breast cancer) using well known methods. In such methods, conduct screening of libraries (cDNA or genomic) using one or more polynucleotide probes or primers suitable for am the skill levels. Preferably filter size that the library consisted of larger molecules. Preferred libraries can be obtained by using random primers, in order to identify the 5'-regions and the left regions of genes. Genomic libraries preferred to obtain introns and extending 5'-sequences.

For the method for hybridization of a partial sequence can be marked (e.g., nick-translation, or by tagging all32P), using well known methods. Then, as a rule, conduct screening of libraries in bacteria or bacteriophages by hybridizing filters containing denatured bacterial colonies (or lawns containing phage plaques)with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). Hybridization of colonies or plaques are selected and are increasing, and DNA allocate for further analysis. The cDNA clones can be analyzed to determine the number of additional sequences, for example by PCR, using the primer of the partial sequence and the primer vector. You can create maps of restriction and partial sequence, to identify one or more overlapping clones. Then you can determine the complete sequence using the mill is Artie ways which may include the creation of a series of deletion clones. Then the resulting overlapping sequences can be assembled into a single contiguous sequence. Molecule cDNA is full length, you can create legirovaniem suitable fragments, using well known methods.

Alternative there are numerous ways amplification to obtain the full coding sequence of the partial cDNA sequence. In such methods, the amplification is typically performed by PCR. To perform stage of amplification you can use any of a variety of commercially available kits. Primers can be designed using, for example, computer programs well known in this field. The primers preferably have a length in the 22-30 nucleotides, have a GC content of at least 50%, and hybridize with the target sequence at temperatures about 68°With up to 72°C. Amplificatory region can be sequenced, as described above, and overlapping sequences to collect in a continuous sequence.

One way to do this amplification is the inverse PCR (see Triglia et al., Nucl. Acids Res. 16: 8186, 1988), in which use restriction enzymes to create a fragment in the known region of a gene. Then f is agent transfer in annular form intramolecular legirovaniem and used as template for PCR with divergent primers, obtained from the known area. An alternative approach sequence adjacent to the partial sequence can be found by amplification with primer to the linker sequence and a primer specific to the known region. Amplificatoare sequence is usually subjected to a second round of amplification with the same linker primer and a second primer specific to a known area. A variant of this procedure, which uses two primers, which initinput elongation in opposite directions from a known sequence, described in WO 96/38591. Another such method is known as "rapid amplification of cDNA ends" or RACE. This method involves the use of an internal primer and the external primer, which hybridizes with the poly-a region or a vector sequence, to identify sequences that are 5'- and 3'-side of the known sequence. Additional methods include PCR with carbon capture (Lagerstrom et al., PCR Methods Applic. 1: 111-19, 1991) and PCR-"walk" (Parker et al., Nucl. Acids. Res. 19: 3055-60, 1991). You can also use other methods with the use of amplification to get the full cDNA sequence.

In some cases, you can get the full cDNA sequence of the pic is edstam sequence analysis represented in the database marker expressed sequences (EST), such as those available from GenBank. The search for overlapping EST usually can be performed using well-known programs (e.g., search NCBI BLAST). Such EST can be used to create a continuous full-size sequence. The DNA sequence of a full length can also be obtained through analysis of genomic fragments.

EXPRESSION OF POLYNUCLEOTIDES IN THE CELLS OF THE HOST.

In other embodiments of the invention, polynucleotide sequences or fragments thereof that encode the polypeptides according to the invention, or hybrid proteins or their functional equivalents, can be used in recombinant DNA molecules to direct expression of the polypeptide in the appropriate cell hosts. Due to inherent genetic code degeneracy is possible to obtain other DNA sequences that encode essentially the same, or functionally equivalent amino acid sequence, and these sequences can be used to clone and Express the polypeptide.

As will be clear to experts in this field, preferred in some cases, you may be obtaining the nucleotide sequences encoding polypep the water, having codons are not of natural origin. For example, you can choose the codons preferred a particular prokaryotic or eukaryotic host, in order to increase the rate of expression of the protein, or to obtain recombinant RNA transcripts having desirable characteristics, such as a longer half-life, than transcripts, created on the basis of the sequence of natural origin.

In addition, the polynucleotide sequence according to this invention can be obtained genetically engineered by using methods generally known in this field to change the sequence encoding the polypeptide for a variety of reasons, including, but not limited to, changes which modify the cloning, processing and/or expression of the gene product. For example, to construct a nucleotide sequence, it is possible to use changes in DNA by obtaining random fragments and re-Assembly PCR of gene fragments and synthetic oligonucleotides. In addition, you can use site-specific mutagenesis to incorporate the new restriction sites, alter the nature of glycosylation, change the preferred use of codons, to obtain the variants of splicing, or to introduce mutations, and so on.

In another variant of the invention, natural, modified, or recombinant nucleic acid sequences can be ligitamate with a heterologous sequence to encode a hybrid protein. For example, for screening libraries of peptides in relation to inhibitors of polypeptide activity, it may be useful to encode a chimeric protein, which can be well-known commercially available antibody. A hybrid protein also can be constructed so that it contains the cleavage site, localized between sequence that encodes a polypeptide, and the sequence of the heterologous protein, so that the polypeptide can be split and cleaned heterologous component.

Sequence encoding the desired polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternative protein itself may be produced using chemical methods to synthesize the amino acid sequence of the polypeptide or part thereof. For example, peptide synthesis can be performed using various techniques of solid-phase synthesis (Roberge, J. Y. Et al. (1995) Science 269: 202-204) and it is possible to achieve automated synthesis using, for example, a peptide synthesizer ABI 431A (Perkin Elmer, Palo Alto, CA).

Novocin tiropanis peptide can be substantially clear preparative high performance liquid chromatography (for example, Creighton, T. (1983) Proteins, Structure and Molecular Principles, WH Freeman and Co., New York, N.Y.) or other compatible methods available in this area. The structure of synthetic peptides can confirm amino acid analysis or sequencing (e.g., by way of degradation of Admino). In addition, the amino acid sequence of the polypeptide or any portion thereof can be changed during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to obtain a variant of the polypeptide.

In order to Express the desired polypeptide, the nucleotide sequence encoding the polypeptide, or functional equivalents, can be embedded into a suitable expression vector, i.e. a vector which contains the necessary elements for the transcription and translation of the built-in coding sequence. Methods which are well known to specialists in this field can be used to construct expressing vectors containing sequences encoding interest polypeptide, and appropriate regulatory elements for transcription and translation. These methods include recombinant DNA technology in vitro, methods of synthesis and genetic recombination in vivo. Such methods are described in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y. and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

A variety expressing vectors/system owners can use to put in them and the expression of polynucleotide sequences. These include, but are not limited to these, microorganisms such as bacteria transformed by expressing DNA vectors based on recombinant bacteriophage, plasmid or cosmid; yeast transformed with yeast expression vectors; insect cells infected with virus expressing vectors (e.g. baculovirus); plant cell transformed by expressing viral vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressing vectors (e.g. plasmids Ti or pBR322); or animal cells.

"Control elements" or "regulatory sequences"that are present in expressing the vector represent untranslated regions in the vector - enhancers, promoters, 5'- and 3'-noncoding regions, which interact with proteins of the host cell to carry out transcription and translation. Such elements can vary in intensity and specificity. Depending on the vector system and host, you can use Liu is th number of bets transcription and translation, including constitutive and inducible promoters. For example, when cloning in bacterial systems, it is possible to use inducible promoters such as the hybrid lacZ promoter family PBLUESCRIPT (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD) and the like. In mammalian cells is generally preferable promoters of mammalian genes or viruses to mammals. If you want to create a cell line that contains multiple copies of a sequence that encodes a polypeptide, mainly you can use vectors based on SV40 or EBV, with appropriate breeding markers.

In bacterial systems, it is possible to choose a number expressing vectors depending on the intended use of the expressed polypeptide. For example, in the case when you need large quantities, for example, for the induction of antibodies, it is possible to use vectors that provide a high level of expression of a fused protein, which is easy to clean. Such vectors include, but are not limited to, multifunctional cloning and expressing the E. coli vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding interest polypeptide, can be Legerova into the vector in frame with sequences for aminoanisole Met and the subsequent 7 residues of beta-Gal is chaidez, in order to produce a hybrid protein; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264: 5503-5509); and the like. You can also use the pGEX vectors (Promega, Madison, Wis.), to Express foreign polypeptides as well as proteins, hybrid with glutathione-S-transferase (GST). In General, these hybrid proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins obtained in such systems can be designed so that they included the sites of cleavage by proteases heparin, thrombin or factor XA, so that interest cloned polypeptide, if desired, could be freed from the fragment GST.

In the yeast Saccharomyces cerevisiae can be used a number of vectors containing constitutive or inducible promoters such as the promoters of alpha-factor, alcoholiday and PGH. For an overview, see Ausubel et al. (above) and Grant et al. (1987) Methods Enzymol. 153: 516-544.

In those cases, use when expressing vectors of plants, the expression of sequences encoding polypeptides, can be controlled by any number of promoters. For example, viral promoters such as the 35S promoter and CaMV 19S, can be used alone or in combination with leader sequence omega TMV (Takamatsu, N. (1987) EMO J. 6: 307-311. Alternatively, you can use plant promoters such as the promoter of the small subunit of RUBISCO or heat shock (Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie, R. et al. (1984) Science 224: 838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17: 85-105). These designs can be entered into plant cells by direct DNA transformation or transfection mediated by pathogens. Such methods are described in a number of widely available reviews (see, for example, Hobbs, S. or Murry, L. E in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; p. 191-196).

The system of insects can also be used to Express the interest of the polypeptide. For example, in one such system is used in the nuclear polyhedrosis virus of Autographa californica NPV (AcNPV) as the vector for expression of foreign genes in cells of Spodoptera frugiperda or Trichoplusia larvae. Sequence encoding the polypeptide can be cloned into unimportant for living near a virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful embedding sequence that encodes a polypeptide, makes polyhedrin gene inactive and gives a recombinant virus that does not have a protein shell. Then recombinant viruses can be used, for example, to infect cells of S. frugiperda or Trichoplusia larvae in which interest polypeptide can expr sirovatka (Engelhard E.K. et al.(1994) Proc. Natl. Acad. Sci. 91: 3224-3227).

In the cells of the host mammal usually are expressing several systems based viruses. For example, in cases where as expressing vector using adenoviruses, sequences encoding interest polypeptide, can be ligitamate in adenovirus transcription/translation complex comprising the late promoter and consists of three parts leader sequence. You can use the insertion in a nonessential region of the viral genome E1, or E3, in order to obtain a viable virus which is capable to Express the interest of the polypeptide in infected cells of the host (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81: 3655-3659). In addition, you can use the transcription enhancers, such as enhancer of rous sarcoma virus (RSV), to enhance expression in the cells of the host mammal.

You can also use specific initiation signals, in order to achieve more efficient translation of sequences encoding interest polypeptide. Such signals include the initiating ATG codon and adjacent sequences. In cases where sequences encoding the polypeptide, their initiation codon and "left" sequence is inserted into the corresponding expressing vector, can the t absent the need for additional signals in the regulation of transcription or translation. However, in cases when only embed the coding sequence or part of it, you must provide exogenous signals, regulation of translation, including the initiating codon ATG. Furthermore, the initiation codon must be in the correct reading frame to ensure the broadcast of the full insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for specific cellular systems, such as enhancers described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162).

In addition, the cell line host, you can choose according to their ability to modulate the expression of the built-in sequences or processional expressed protein in the desired manner. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidization and acylation. In order to ensure proper integration, packaging, and/or operation can also be used post-translational processing which cleaves a "form preprally". To ensure the correct modification and processing of the foreign protein, you can choose different cells-owners, that is their as SNO, HeLa, MDCK, HEK293, and WI38, which have specific cellular apparatus and characteristic mechanisms for such post-translational activities.

For long-term high-yield production of recombinant proteins, as a rule, preferred stable expression. For example, cell lines which stably Express interest polynucleotide, can be transformed using expressing vectors that may contain early viral replication and/or endogenous elements of expression and breeding marker gene in the same or separate vectors. After the introduction of the vector, cells may be able to grow for 1-2 days in an enriched media before transferring them to the selective environment. The purpose of breeding token is giving resistance to selection, and its presence provides an opportunity to grow and extract of cells which successfully Express the introduced sequences. Resistant clones of stably transformed cells can be propagated using the methods of cultivation of tissues suitable for the cell type.

To obtain a transformed cell lines can be used any number of breeding systems. These systems include, but are not limited to, genes timedancing of herpes simplex virus (Wigler, M. et al. (1977) Cel 11: 223-32) and adrinfo.standortstr (Lowy, I. et al. (1990) Cell 22: 817-23)that can be used in cells tk.sup.- or aprt.sup.-, respectively. Also as the basis for selection, you can use resistance to antimetabolites, antibiotics or herbicides; for example, dhfr, which gives resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77: 3567-70); npt, which gives resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14); and als or pat, which give resistance to chlorsulfuron and phosphonomethylglycine, respectively (Murry, supra). Describes the advanced breeding genes, for example, trpB, which allows cells the ability to use indole instead of tryptophan, or hisD, which allows cells to use gastinel instead of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85: 8047-51). Recently gained popularity, the use of visible markers, such markers as anthocyanins, beta-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, however, they are widely used not only to identify transformants, but also to measure the amount of temporary or stable protein expression attributable to a specific vector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55: 121-131).

Although the presence/absence of expression of the marker gene indicates that the gene of interest also p is outstay, may require confirmation of its presence and expression. For example, if the sequence encoding the polypeptide has a sequence of the marker gene, recombinant cells containing sequences can be identified by the absence of functioning of the marker gene. Alternative marker gene can be placed in tandem with a sequence encoding the polypeptide, under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of tandem gene.

Alternative cell-hosts that contain and Express a desired polynucleotide sequence can be identified by a variety of procedures known to specialists in this field. These procedures include, but are not limited to, hybridization DNA-DNA or DNA-RNA and methods of bioanalysis, or immunoassay of protein, which include technology-based membranes, solutions or chips for detection and/or quantification of nucleic acid or protein.

In this area there are many protocols for detecting and measuring the expression products encoded by polynucleotide, using either polyclonal or monoclonal antibodies specific product. Examples include solid-phase IMM is kofermentnuu assay (ELISA), the radioimmunoassay (RIA) and fluorescence-activated sorting of cells (FACS). For some applications it may be preferred immunoassay based on monoclonal antibodies with two antiterminator, using monoclonal antibodies reactive to two non-overlapping with the epitope on the polypeptide, but you can also use the analysis of competitive binding. These and other assays are described, along with other publications, Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158: 1211-1216).

A wide variety of labels and methods of conjugation are known to specialists in this field and can be used in various analyses of nucleic acids and amino acids. Methods of obtaining labeled probes for hybridization and PCR for detecting sequences related to polynucleotides include tagging oligonucleotides, nick translation, end-labeling or PCR amplification using a labeled nucleotide. In the alternative case, sequence, or any part thereof, can be cloned into a vector to obtain an mRNA probe. Such vectors are known in the field and available for purchase, and can be used to synthesize RNA probes in vitro by adding the appropriate RNA polymerase such as T7, T3 or SP6, and the state of the nucleotide. These procedures can be performed using a variety of commercially available kits. Suitable reporter molecules or labels, which can be used include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like. Cell host transformed interest polynucleotide sequence can be cultivated under conditions suitable for the expression and extraction of the protein from cell culture. A protein produced by a recombinant cell may secretariats or is inside the cells, depending on the sequence and/or used in the vector. As will be clear to experts in the field, expressing the vectors containing polynucleotide according to the invention, can be constructed so that they contain signal sequences that control the secretion of the encoded polypeptide through the membrane prokaryotic or eukaryotic cells. You can use other recombinant constructions to join sequences encoding interest polypeptide with a nucleotide sequence that encodes a polypeptide domain which will facilitate purification of soluble proteins. These easy-to-clean domenicucci, but not limited to, peptides, chelating metals, such as modules histidine-tryptophan, which provide the purification on immobilized metals, protein domains And that provide purification on immobilized immunoglobulin, and the domain used in the system FLAGS extension/affinity purification (Immunex Corp., Seattle, Wash.). The inclusion of degradable linker sequences, such as sequences that are specific for factor XA or enterokinase (Invitrogen, San Diego, Calif.) between domain intended for cleaning, and encoded a polypeptide, can be used to facilitate purification. One of these expressing vector provides for expression of the fused protein containing of interest polypeptide, and a nucleic acid encoding 6 histidine residues preceding thioredoxin, or the site of cleavage by enterokinase. Residues of histidine provide treatment on IMIAC (affinity chromatography using immobilized metal ions)as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3: 263-281), while the site of cleavage by enterokinase provides a means for purification of the desired polypeptide of the fused protein. Discussion of vectors that contain hybrid proteins is given in Kroll, D. J. et al. (1993; DNA Cell Biol. 12: 441-453).

In addition to the methods of obtaining of recombinant polypeptides according to the invention or their f is Agency can be obtained by direct peptide synthesis, using the technique of solid-phase synthesis (Merrifield J. (1963) J. Am. Chem. Soc. 85: 2149-2154). Protein synthesis can be performed manually or automated methods. Automated synthesis, for example, can be performed using a peptide synthesizer Applied Biosystems 431A (Perkin Elmer). Alternatively, separate chemical methods to synthesize the various fragments and combine them, using chemical methods to obtain the full length molecule.

SITE-SPECIFIC MUTAGENESIS.

Site-specific mutagenesis is a method suitable for individual peptides, or biologically functional equivalent proteins through specific mutagenesis of the main polynucleotides that encode them. The method is well known to specialists in this field, in addition, provides an easily accessible opportunity to get and test sequence variants, for example, whereas one or more of the above considerations, by introducing one or more nucleotide changes in the sequence of the DNA. Site-specific mutagenesis allows to obtain mutants through the use of specific oligonucleotide sequences that encode the DNA sequence with a desired mutation, as well as a sufficient number of neighboring nucleotides to get consequently the efficiency of the primer is of sufficient size and complexity of sequences, to form a stable duplex on both sides of the overlapped connection with the deletion. Mutations can be used in the selected nucleotide sequence, in order to improve, change, reduce, modify, or otherwise change the properties of polynucleotide and/or change the properties, activity, structure, stability or the primary sequence of the encoded polypeptide.

In some embodiments of the present invention, the authors suggest the use of mutagenesis of the claimed polynucleotide sequences, in order to change one or more properties of the encoded polypeptide, such as a polypeptide antigenicity of the vaccine. Methods site-specific mutagenesis is well known in this field and are widely used to create variants as polypeptides and polynucleotides. For example, site-specific mutagenesis is often used to change a specific part of the DNA molecule. In such scenarios, use primer, usually containing from about 14 to 25 nucleotides or so in length, and about 5 to 10 residues on both sides of the connection variable sequence.

As will be clear to experts in the field, in the way of site-specific mutagenesis is often used phage vector that exists as of nonthavej, and in double form. Normal vectors, suitable for site-specific mutagenesis include vectors, as the M13 phage. This phage is easily available for purchase and its use is generally well known to specialists in this field. Denitive plasmids are also commonly used in site-specific mutagenesis, at which there is no phase transfer of the gene of interest from a plasmid to the phage.

In General, site-specific mutagenesis according to this invention perform, receiving a first single-stranded vector or melting to separate the two strands of double vector, which contains in its sequence a DNA sequence which encodes the desired peptide. Oligonucleotide primer bearing the desired mutated sequence, usually are synthetically. Then the specified primer annealed to single-stranded vector, and subjected to exposure to DNA-polymerizes enzymes, such as a piece maple polymerase I of E. coli, in order to complete the synthesis of the thread carrying the mutation. Thus, form heteroduplex, in which one strand encodes the source not the mutant sequence and the second strand carries the desired mutation. Then the specified heteroduplexes vector used to transform appropriate cells, such as E. coli cells, and selected clones that contain recombinant ve the Torah, bearing sequence with a mutant nucleotides arrangement.

Obtaining sequence variants of the selected segments of DNA encoding the peptide, using site-specific mutagenesis provides a means of obtaining potentially useful options and does not imply limitations, since there are other ways through which you can obtain the sequences of the peptides and their coding DNA sequences. For example, recombinant vectors encoding the desired peptide sequence can be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details related to the above mentioned methods and protocols are guidelines Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each of which with this purpose, incorporated herein by reference.

In the sense used here, the term "procedure site-specific mutagenesis using oligonucleotides" refers to processes that depend on the matrix, and reproduction-mediated vector, resulting in an increase in the concentration of molecules of a specific nucleic acid relative to its initial concentration, or an increase in the concentration recorded signal, such as amplification. Used here, the meaning is implied, the term "procedure site-specific mutagenesis using oligonucleotides" refers to a method that includes dependent matrix elongation of the primer molecules. The term "method-dependent matrix" refers to the synthesis of nucleic acid molecules of RNA or DNA in which the sequence of the newly synthesized strands of nucleic acid is dictated by the well-known rules of complementary mating grounds (see, for example, Watson, 1987). Usually methods-mediated vectors include the introduction of the fragment of the nucleic acid in a DNA or RNA vector, clonal amplification of the vector and extracting the amplified fragment of the nucleic acid. Examples of such methods are given in U.S. patent No. 4237224, specifically incorporated herein by reference in full.

METHODS AMPLIFICATION OF POLYNUCLEOTIDES.

There are a number dependent on matrix methods in order to amplify interest to a target sequence in the sample. One of the most known methods of amplification is the polymerase chain reaction (PCR), which is described in detail in U.S. patent No. 4683195, 4683202 and 4800159, each of which is incorporated herein by reference in full. Briefly, PCR prepare two primernye sequences that are complementary to regions on opposite the complementary strands of a target sequence. To the reaction mixture an excess of deoxynucleotidase together with a DNA polymerase (such as Taq polymerase). If the sequence of the target present in the sample, the primers will contact the target, and the polymerase will cause elongation of the primers along the target sequence by joining nucleotides. When raising and lowering the temperature of the reaction mixture, the extended primers will be separated from the target, forming reaction products, excess primers will bind with the target and with the reaction product and the process will repeat. Preferably, you can perform reverse transcription and PCR amplification in order to measure the amount of amplified mRNA. Methods polymerase chain reaction well known in this field.

Another method for amplification is the ligase chain reaction (called LCR), described in the published application for the grant of European patent No. 320308 (specifically incorporated herein by reference in full). When LCR prepare two pairs of complementary probes, and in the presence of a target sequence, each pair will communicate with the opposite complementary strands of the target, so that they are adjacent to each other. In the presence of ligase two pairs of probes are in contact, forming a single unit. Under cyclic change of temperature is tours, as with PCR, legirovannye associated units are separated from the target and then serve as "sequences are targets for ligating an excess of pairs of probes. In U.S. patent No. 4883750, incorporated herein by reference in full, describes an alternative method of amplification similar to LCR, to associate pairs of probes with the target sequence.

You can also use Q beta replicase described in published international application PCT patent application No. PCT/US87/00880, incorporated herein by reference in full, as another method of amplification according to this invention. In the specified way replicative RNA sequence that has a region complementary to the target area, add to the sample in the presence of RNA polymerase. Polymerase will copy the replicative sequence, which can then be detected.

The way isothermally amplification with using the restriction enzyme and ligase to achieve the amplification of target molecules that contain nucleotide-5'-[α-thio]triphosphates in one strand of a restriction site (Walker et al., 1992, work incorporated herein by reference in full), may also be suitable for nucleic acid amplification according to this invention.

Amplification preemptive chain (SDA) is another way the imp is in isothermally nucleic acid amplification, which includes multiple rounds of displacement of the chain and synthesis, i.e. by nick-translation. A similar method, called chain reaction repair (RCR), is another way of amplification, which may be suitable for this invention, and it includes several annealing of the probes along the length of the area selected as the target for amplification, followed by reaction of reparation, in which there are only two of the four bases. The other two bases can be added in the form of a biotinylated derivatives to facilitate registration. A similar approach is used in SDA.

The sequence can also be detected using a cyclic reaction with the probe (CPR). When CPR probe having 3'and 5'-DNA sequence, which is not a target, and internal or "average" RNA sequence-specific protein target hybridized with DNA, which is present in the sample. After hybridization, the reaction mixture is treated with RNase H, and the products of the probe identifies as characteristic products while generating signal, which is released after cleavage. The original matrix is annealed with other cyclic probe and the reaction repeated. Thus, CPR involves amplification of the signal generated by hybridization of a probe specific for the gene target expressed by nukleinovokisly.

According to this invention it is still possible to use other methods of amplification, as described in the application for the grant of a UK patent No. 2202328 and in published international PCT application No. PCT/US89/01025, each of which is incorporated herein by reference in full. In the first application of the "modified" the primers used in the course of such PCR-dependent matrix and enzyme synthesis. The primers can be modified by tagging fragment of capture (e.g., Biotin) and/or detector fragment (e.g., enzyme). In the last application to the sample add an excess of labeled probes. In the presence of a target sequence, the probe is bound and catalytically cleaved. After cleavage sequence of a target is released in intact form to contact the excess probe. Cleavage of the labeled probe gives a signal about the presence of the target sequence.

Other methods of nucleic acid amplification include amplification system based on transcription (TAS) (Kwoh et al., 1989; published international PCT application No. WO 88/10315, incorporated herein by reference and full), including amplification-based nucleic acid sequence (first NASBA) and 3SR. In the first NASBA nucleic acid amplification can be obtained by standard extraction with phenol/chloroform, thermal denaturation of which were acquired, processing lytic buffer and using centrifuge miniology for DNA and RNA, or RNA extraction with guanidinium. These methods of amplification include annealing primer that has a sequence that is specific to the target sequence. After polymerization of the hybrid DNA/RNA cleaved by RNase H, while denitive DNA molecules again denatured by heat. In each case, single-stranded DNA is made double by adding the second is specific to the target primer, followed by polymerization. Then denitive DNA molecules repeatedly transcribing polymerase such as T7 or SP6. When isothermally cyclic reaction RNA back transcribers in DNA and again transcribing polymerase such as T7 or SP6. The resulting products, shortened or in full, indicate specific for the target sequences.

In the published application for the European patent No. 329822, incorporated herein by reference in full, disclosed a method of nucleic acid amplification, comprising a cyclical synthesis of single-stranded RNA (ORNK), andnc and double DNA (dndn), which can be used according to this invention. onrc is the first matrix to the first oligonucleotide primer, which longerbut arr is based transcriptase (RNA-dependent DNA polymerase). Then the RNA is removed from the resulting duplex DNA:RNA, acting by ribonuclease H (Mcasa N, Unkasa, it is specific to the RNA in the duplex DNA or RNA). The resulting andnc is the second matrix for the second primer, which also contains the sequence of the promoter of RNA polymerase (an example of which is the RNA polymerase T7) at the 5'end of the sequencing primer with homology to the matrix. Then the specified primer lengthen DNA polymerase (an example of which is the large fragment of "maple" DNA polymerase I, E. coli), resulting in Dunaeva the DNA molecule ("dndrc")having a sequence identical to the sequence of the original RNA between the primers, and optionally having at one end a sequence of the promoter. This promoter sequence can be used the appropriate RNA polymerase to make many RNA copies of the DNA. Then these copies are again injected into the cycle that leads to very rapid amplification. With the right choice of enzymes such amplification can be performed isothermal without added enzymes in each cycle. Due to the cyclic nature of this process, the starting sequence can be selected in the form of either DNA or RNA.

In published international application PCT patent application No. WO 89/06700, VK is uchennai here by reference in full, the described scheme amplification, nucleic acid sequence-based hybridization sequence of a promoter/primer to single-stranded DNA ("ndnc"target followed by transcription of many RNA copies of the sequence. This scheme is not cyclical; that is, the new matrix is not produced on the basis of the resulting RNA transcripts. Other methods of amplification include "RACE" (Frohman, 1990) and "one-sided PCR" (Ohara, 1989), which are well known to specialists in this field.

Methods based on legirovanii two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "diagonality", thereby leading to amplification of diagonalised (the work of Wu and Dean, 1996, incorporated herein by reference in full), can also be used for amplification of DNA sequences according to the invention.

BIOLOGICALLY FUNCTIONAL EQUIVALENTS.

In the structure of polynucleotides and polypeptides according to this invention it is possible to make modifications and changes, and to get another functional molecule that encodes a polypeptide with desirable characteristics. As indicated above, it is often desirable to introduce one or more mutations in a specific sequence polynucleotide. In some the cases the resulting encoded sequence of the polypeptide is modified such mutation or in some cases, when one or more mutations encoding polynucleotide sequence of the polypeptide is not changed.

In the case when you want to change the amino acid sequence of a polypeptide to create an equivalent, or even superior molecule of the second generation, changes of amino acids can be achieved by changing one or more codons coding DNA sequence in accordance with table 1.

For example, in the structure of the protein, some amino acids can be replaced by other amino acids without significant loss of binding capacity for interaction with such structures as, for example, antigennegative regions of antibodies or binding sites on substrate molecules. Because this biological functional activity of the protein is determined by the ability to interact and nature of a protein in a protein sequence can make some substitutions of amino acid sequence, and, of course, her main coding DNA sequence, and nevertheless obtain a protein with like properties. Therefore, the authors of the present invention believe that it is possible to make various changes in the peptide sequences described structure or corresponding DNA sequences which encode these peptides without appreciable loss of their biological effectiveness or activity.

Table 1.

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Amino acid Codons

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Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic acid Asp D GAC GAU

Glutamic acid Glu E GAA GAG

Phenylalanine Phe F UUC UUU

Glycine Gly G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine Ile I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUC CUA CUG CUU

Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCA CCC CCG CCU

Glutamine Gln Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Valine Val V GUC GUA GUG GUU

Tryptophan Trp W UGG

Tyrosine Tyr Y UAC UAU

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When making such substitutions can be considered hydropathicity index of amino acids. Is hydropathical index of amino acids to make protein biological function of interaction, as a rule, considered in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is believed that the relative hydropathicity the nature of the amino acid contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens and the like. For each amino acid is defined hydropathicity index on the basis of its characteristics, hydrophobicity and charge (Kyte and Doolittle, 1982). These values are equal to: isoleucine (+4,5); valine (+4,2); leucine (is+3.8); phenylalanine (a+2.8); Cys is Ein/cystine (+2,5); methionine (+1,9); alanine (+1,8); glycine (-0,4); threonine (a-0.7); serine (of-0.8); tryptophan (of-0.9); tyrosine (-0,3); Proline (of-1.6); histidine (-3,2); glutamate (for 3,5); glutamine (for 3,5); aspartate (for 3,5); asparagine (for 3,5); lysine (-3,9); and arginine (-4,5).

In this area it is known that certain amino acids can be substituted by other amino acids having similar hydropathicity index or score and still get the protein with similar biological activity, i.e. still get the protein biologically functionally equivalent. When making such changes, the preferred replacement amino acids, hydropathicity index which is within ±2, especially preferred replacement within ±1, and even more preferred replacement within ±0.5 in. In this area it is also clear that the substitution of like amino acids can be efficiently implemented on the basis of hydrophilicity. In U.S. patent 4554101 (specifically incorporated herein by reference in full) found that the highest average local hydrophilicity of a protein, which is caused by the hydrophilic nature of the adjacent amino acids, correlates with a biological property of the protein.

As described in detail in U.S. patent 4554101 for amino acid residues has the following hydrophilicity values: arginine (+3,0); lysine (+3,0); aspartate (+3,0 ±1); glutamate (+3,0 ±1); serine (+0,3); asparagine (+0,); glutamine (at+0.2); glycine (0); threonine (-0,4); Proline (-0,5 ±1); alanine (-0,5); a histidine at (- 0.5); cysteine (-1,0); methionine (-1,3); valine (-1,5); leucine (-1,8); isoleucine (-1,8); tyrosine (-2,3); phenylalanine (-2,5); tryptophan (-3,4). It is clear that the amino acid can be replaced with another having a similar hydrophilicity value and still obtain a biological equivalent, and, in particular, immunologically equivalent protein. When such changes are preferred replacement amino acids, hydrophilicity values are within ±2, especially preferred replacement within ±1, and even more preferred replacement within ±0.5 in.

Therefore, as noted above, the replacement amino acids are usually based on the relative similarity of the substituents of the side chains of amino acids, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Examples of substitutions, which takes into account the above characteristics are well known to specialists in this area and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

In addition, polynucleotide can be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the accession of flanking sequences at the 5' and/or 3'ends; the use of fosforo iatneh or 2'-O-methyl, not fosfolipidnyh linkages in the main chain; and/or the inclusion of nontraditional bases such as inosine, quitin and wybutosine, as well as acetyl-, methyl-, thio - and other modified forms of adenine, cytidine, guanine, thymine and uridine.

METHODS OF DELIVERY OF POLYNUCLEOTIDES IN VIVO.

In additional embodiments, the cells injected in vivo genetic constructs containing one or more polynucleotides according to the invention. This can be achieved using any of a variety of well-known approaches, some of which are shown below for illustrative purposes.

1. ADENOVIRUS.

One of their preferred methods of delivery in vivo one or more sequences of nucleic acids involves the use of adenovirus expressing vector. The term "adenovirus expressing vector" means that it includes structures that contain sequences of adenovirus, sufficient to (a) ensure packaging design and (b) to Express polynucleotide, which was cloned in sense or antisense orientation. Of course in the context of antisense construction, expression does not require was synthesized gene product.

Expressing the vector contains a genetically engineered form of the adenovirus. Knowledge of the genetic organization of adenovirus is a, linear donateware DNA virus size 36 TPN allows substitution of large areas adenoviral DNA alien sequences of length up to 7 TPN (Grunhaus and Horwitz, 1992). Unlike retrovirus adenovirus infection of host cells does not result in integration into the chromosome, because adenoviral DNA can be replicated in the form of Epsom without the possible genotoxicity. Moreover, adenoviruses are structurally stable, and after a large amplification is not detected rearrangement of the genome. Adenovirus can infect virtually all epithelial cells, regardless of the stage of the cell cycle. To date adenoviral infection detected only in connection with moderate disease, such as acute respiratory disease in humans.

Adenovirus, in particular, suitable for use as a vector for gene transfer due to mid-sized genome, ease of manipulation, high titer, wide range of target cells and high infectivity. Both ends of the viral genome contain inverted repeats with a length of 100-200 BP (ITR), which are CIS-elements necessary for replication of viral DNA and packaging. Early (E) and late (L) regions of the genome contain different transcription units, which are separated by the beginning of the replication of viral DNA. Area E1 (E1 and IV) encodes proteins, responsible for regulation of transcription of the viral genome and several cellular genes. The result of the expression of area E2 (EA and EB) is a protein synthesis replication of viral DNA. These proteins are involved in DNA replication, the expression of late genes and termination of the functioning of the host cell (Renan, 1990). The products of the late genes, including most viral capsid proteins, are expressed only after significant processing of a single primary transcript, which is formed under the main control of the late promoter (MLP). MLP (localized in position 16,8% genetic map) is particularly efficient during the late phase of infection, and all of mRNA produced from this promoter possess a 5'-leader sequence consisting of three parts (TPL), which makes them preferred for translation of mRNA.

In this system, recombinant adenovirus give the result of homologous recombination between the Shuttle vector and proviral vector. Due to the possible recombination between the two proviral vectors in the result of this process may be wild-type adenovirus. Therefore, it is important to allocate a separate clone of the virus from individual plaques and examine the structure of its genome.

The creation and reproduction of data adenoviral vectors that are deficient in replication depend on the unique the first line helper cells, named 293, which was transformed from cells embryonic kidney human Ad5 DNA fragments and which constitutively expresses proteins E1 (Graham et al., 1977). As the area E3 is not essential for adenovirus genome (Jones and Shenk, 1978) currently available adenoviral vectors using 293 cells carry foreign DNA in either the E1, D3, or both regions (Graham and Prevec, 1991). In nature, the adenovirus can package approximately 105% of the genome of wild-type (Ghosh-Choudhury et al., 1987), providing accommodation for about 2 TPN additional DNA. Together with approximately 5.5 TPN DNA, which is replaced in the E1 and E3 regions, the maximum capacity of this adenoviral vector is about 7.5 TPN, or approximately 15% of the total length of the vector. More than 80% of the viral genome of the adenovirus is stored in the skeleton of the vector and is the source of cytotoxicity associated with the vector. Moreover, the deficit in the replication of the virus with a deletion of the E1 incomplete. For example, observed a "trickle down" the expression of viral genes when using the currently available vectors at high multiplicity infection (MOI) (Mulligan, 1993).

Line helper cells can be obtained from human cells, such as cells of embryonic human kidney, muscle cells, hematopoietic cells or other mesenchymal or epithelial cells of the human embryo. Alternative helper cells can p is to be obtained from cells of other mammalian species, which are permissive for human adenovirus. Such cells include, for example, Vero cells, or other mesenchymal or epithelial cells of the embryo monkeys. As noted above, in the present preferred line helper cell line is 293.

Recently Racher et al. (1995) stated about improved methods of culturing 293 cells and propagation of adenoviruses. In one form, the natural aggregates of cells grown by insulinopenia individual cells in a rotating siliconized vials with a volume of 1 liter (Techne, Cambridge, UK)containing 100-200 ml of medium. After shaking at 40 rpm and the cell viability was determined Trifanova blue. In another form using microneedle Fibra-Cel (Bibby Sterlin, Stone, UK) (5 g/l) as follows. The inoculate cells, resuspending in 5 ml of medium, is added to the medium (50 ml) in an Erlenmeyer flask of 250 ml and left in a stationary state at a rare shaking for 1 to 4 hours. Then Wednesday replace 50 ml of fresh medium and begin to shake. For the production of viruses to cells provide the opportunity to grow to confluence of approximately 80%, after this point the environment replace (up to 25% of the final volume) and add adenoviruses at MOI of 0.05. Culture left in a stationary state during the night, after which the volume is increased to 100%, and start shaking in ECENA the next 72 hours.

In addition to the requirement that an adenoviral vector was replication defective, or at least conditionally defective, it is assumed that the nature of the adenovirus vector is not decisive for the successful practical application of the invention. The adenovirus may be any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication defective adenovirus vector for use in this invention, since the adenovirus type 5 is a human adenovirus about which we know a huge number of biochemical and genetic information, and its historically used for most structures, using adenovirus as a vector.

As stated above the normal vector according to the invention has a defect in replication and will not have the adenoviral area E1. Thus, the most appropriate will be the introduction of polynucleotide encoding gene of interest in the position from which the deleted sequence encoding E1. However, the position of insertion in the design within the adenovirus sequences is not critical to the invention. A polypeptide that encodes a gene of interest, can also be embedded instead of doing the one area E3 in the vectors with substitutions E3, as described by Karlsson et al. (1986), or in the area E4, in cases when the line helper cells or helper virus will complementery the E4 defect.

Adenovirus is easy to grow and circulation and detects a wide host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g. 109-1011plaque-forming units per ml, and they are highly infectious. The life cycle of adenovirus does not require integration into the genome of the host cell. Foreign genes delivered by adenoviral vectors are epitanime, therefore, have low genotoxicity in relation to the cell master. No reported side effects in the study of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al., 1971), indicating that their safety and possible therapeutic applications as vectors for gene transfer in vivo.

Adenoviral vectors used for the expression of eukaryotic genes (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies have shown that recombinant adenovirus can be used in gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al., 1990; Rich et al., 1993). Research on the introduction of recombinant adenovirus to different tissues include instillation into the trachea (Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injects the Yu (Ragot et al., 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

2. RETROVIRUSES.

Retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA into Dunaeva DNA in infected cells by a process of reverse transcription (Coffin, 1990). Then, the resulting DNA is stably integrated into the cellular chromosomes in the form of provirus and directs the synthesis of viral proteins. The result of the integration is the preservation of sequences of viral genes into the recipient cell and its progeny. The genome of the retrovirus contains three genes, gag, pol and env, which encode proteins of the capsid, the enzyme polymerase and shell components, respectively. Sequence detected "left" gag gene contains a signal for packaging of the genome into virions. On the 5'- and 3'-ends of the viral genome contains two sequences of the long terminal repeats (LTR). These sequences contain sequences of a strong promoter and enhancer, and also necessary for integration into the genome of the host cell (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding one or more oligonucleotide or polynucleotide sequences of interest, f is awayt in the viral genome in place of some viral sequences, to get a virus that is replication defective. For the production of virions design line packing of cells containing the genes gag, pol and env, but without the LTR and packaging components (Mann et al., 1983). In that case, when a recombinant plasmid containing a cDNA, together with the retroviral LTR and sequences packing, enter into the specified cell line (for example, by precipitation of calcium phosphate), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The medium containing recombinant retroviruses, then collected, optionally concentrated and used for gene transfer. Retroviral vectors are able to infect a wide variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).

Recently developed a new approach, designed to provide specific delivery to target retroviral vectors based on chemical modification of retrovirus chemical accession residues of lactose to the viral membrane. This modification may allow for specific infection of hepatocytes by receptor sialoglycoproteins.

Was developed another approach to the delivery to mi is Yeni recombinant retroviruses, when used biotinylated antibodies against the envelope protein of the retrovirus and against specific receptor cells. Antibodies were bound by bilinovich components using streptavidin (Roux et al., 1989). Using antibodies against antigens of class I and class II major histocompatibility complex, the authors have demonstrated the infection of a variety of human cells that bore these surface antigens, ectropium virus in vitro (Roux et al., 1989).

3. ADENO-ASSOCIATED VIRUSES.

AAV (Ridgeway, 1988; Hermonat and Muzycska, 1984) is provirus detected as pollution adenovirus strains. This is a widespread virus (antibodies have 85% of the human population in the USA), which was not associated with any disease. The virus is also classified as dependecies because its replication depends on the presence of helper virus, such as adenovirus. Selected five serotypes, of which the best characterized AAV-2. AAV has a single-stranded linear DNA, which is enclosed in a capsid proteins VP1, VP2 and VP3, forming the virion in the form of an icosahedron from 20 to 24 nm in diameter (Muzyczka and McLaughlin, 1988).

DNA AAV has a length of about 4.7 thousand bases. It contains two open reading frames and flanked by two ITRS. In the AAV genome contains two major gene: rep and cap. Gene rep encodes proteins responsible for p is plicatio viruses, while cap encodes a protein capsid VP1-3. Each ITR forms a hairpin structure having the form So These terminal repeats are the only important CIS-AAV components for integration into the chromosome. Therefore, AAV can be used as a vector in the deletion of all viral coding sequences and their replacement by a cassette of genes for delivery. Identified three viral promoter and named P5, P19 and R40 in accordance with their position on the map. The result of transcription from the P5 and P19 is the production of rep proteins, and transcription with R40 leads to the production of proteins capsid (Hermonat and Muzyczka, 1984).

There are several factors that lead researchers to explore the possibility of using rAAV as expressing vector. One of them is that the requirements for delivery of gene and integrating into a chromosome of the host surprisingly little. You must have the ITR with a length of 145 BP, which make up only 6% of the genome of AAV. This gives the location in the vector in order to compose the insert DNA with a length of 4.5 so-called Although it is possible this capacity to migrate does not allow delivery by AAV large genes, it is quite suitable for the delivery of antisense constructions according to this invention.

AAV is also a good choice among carriers for delivery due to its security. There is relative is a recreational complex mechanism of "salvation": not only the wild-type adenovirus, but also genes required for AAV mobilization of rAAV. Moreover, AAV is not pathogenic and is not associated with any disease. Deletion of the coding sequences of the virus minimizes the immune response on the expression of viral genes, and therefore, rAAV does not cause inflammatory response.

4. OTHER VIRAL VECTORS AS EXPRESSING STRUCTURES.

In this invention as expressing structures for the delivery of oligonucleotide or polynucleotide sequences in a cell host, you can use other viral vectors. You can use vectors derived from virus such as vaccinia virus (Ridgeway, 1988; Coupar et al., 1988), lentiviruses, poliovirus and herpes viruses. These viruses have several attractive for various mammalian cell features (Friedmann, 1989; Ridgeway, 1988; Coupar et al., 1988; Horwich et al., 1990).

In connection with the recent detection of defective hepatitis b viruses obtained a new view about the relationship between structure and function of different viral sequences. In vitro studies showed that the virus may be capable of dependent helper packaging and reverse transcription, despite the deletion of up to 80% of its genome (Horwich et al., 1990). This suggests that large parts of the genome can be replaced with alien genetic material. Hepatotropic the persistence (integration) were particularly attractive properties for targeted gene transfer to the liver. Chang et al. (1991) have introduced gene chloramphenicolchloramphenicol (CAT) gene of hepatitis b virus In ducks instead of the sequences coding for the polymerase, coating and pre-coating. The virus was co-transfusional with wild-type virus in cell line hepatoma birds. Culture medium containing high titers of recombinant virus was used to infect primary duck hepatocytes. Was revealed stable expression of the gene SAT for at least 24 days after transfection (Chang et al., 1991).

5. NON-VIRAL VECTORS.

In order to realize the expression of oligonucleotide or polynucleotide sequences according to the invention, expressing the construct must be delivered into the cell. Such delivery can be performed in vitro, as in laboratory procedures for transformation of cell lines, or in vivo or ex vivo, as in the treatment of certain pathological conditions. As described above, one preferred mechanism for delivery is via viral infection, which expresses the design is encapsulated in an infectious viral particle.

After expressing design delivered into the cell the nucleic acid encoding the desired oligonucleotide or polynucleotide sequences may possess the change and expressed in different places. In some embodiments, the nucleic acid encoding the construct can be stably integrated into the host cell genome. This integration may be in a specific position and orientation by means of homologous recombination (gene replacement) or can be integrated at random is not specific position (adding genes). In the following embodiments, the nucleic acid may be stably maintained in the cell as a separate episunago segment of DNA. Such nucleic acid segments or "episome" encode sequence, sufficient to preserve and replicate independently of the cell cycle of the host or synchronously with it. As delivered expressing construct into the cell, and where in the cell is maintained nucleic acid depends on the type of expressing design.

In some embodiments of the invention expressing construct that contains one or more oligonucleotide or polynucleotide sequences may simply consist of naked recombinant DNA or plasmids. Transfer design can be accomplished in any of the above methods that are physically or chemically make the cell membrane permeable. In particular, this applies to migrate in vitro, but can also be used for application in vivo. Dubensky et al. (1984) successfully injecion is whether DNA polyomavirus in the form of precipitates calcium phosphate in the liver and spleen of adult and newborn mice, which in this was manifested the active viral replication and acute infection. Benvenisty and Reshef (1986) also demonstrated that direct intraperitoneal injection precipitating calcium phosphate plasmids results in expression of transfected genes. It is assumed that DNA encoding a gene of interest, in a similar manner may also be transliterowany in vivo and can Express the gene product.

Another variant of the invention for transferring expressing constructs on the basis of "naked" DNA into cells may involve particle bombardment. This method is due to the possibility to accelerate covered DNA microarray to high speeds, allowing them to pass through cell membranes and enter cells without killing them (Klein et al., 1987). Developed several devices to accelerate small particles. One of such devices is based on the high-voltage discharge to generate electric current, which in turn provides the driving force (Yang et al., 1990). Used microarray consisted of biologically inert substances, such as balls of tungsten or gold.

Selected organs of rats and mice, including liver, skin, and muscle tissue, bombarded in vivo (Yang et al., 1990; Zelenin et al., 1991). When this may be necessary surgical effect on the tissue or cells to eliminate any is for intermediate tissue between the firing unit and the target organ, i.e. treatment ex vivo. And in this case, DNA encoding a specific gene can be delivered by this method and thus to be included in this invention.

ANTISENSE OLIGONUCLEOTIDES.

The end result of the flow of genetic information is protein synthesis. DNA is transcribed by polymerase into messenger RNA and translated on ribosomes, giving Packed functional protein. Thus, along the way there are several steps that can be Engibarov protein synthesis. The native DNA segment that encodes a polypeptide described herein, and all such chains of mammalian DNA has two strands: sense chain and antisense chain held together by hydrogen bonds. Messenger RNA encoding a polypeptide that has the same nucleotide sequence as the sense strand of DNA, except that the thymidine DNA replaced by uridine. Thus, synthetic antisense nucleotide sequence will contact the mRNA and inhibit expression of the protein encoded by this mRNA.

Targeted delivery of antisense oligonucleotides to the target mRNA, thus, is one of the mechanisms shutdown of protein synthesis, and thus represents a powerful and targeted therapeutic approach. For example, the syntheses polygalacturonase and scarimbolo acetylcholine receptor type 2 inhibited antimyeloma oligonucleotide, directed to their corresponding mRNA sequences (U.S. patent 5739119 and U.S. patent 5759829, each specifically incorporated herein by reference in full). In addition, examples of antisense inhibition is shown for nuclear protein cyclina, gene, multidrug resistance (MDG1), ICAM-1, E-selectin, STK-1, the receptor for GABAAstriped body and human EGF (Jaskulski et al., 1988; Vasanthakumar and Ahmed, 1989; Peris et al., 1998; U.S. patent 5801154; U.S. patent 5789573; U.S. patent 5718709 and U.S. patent 5610288, each publication is specifically incorporated herein by reference in full). Also described antisense constructs that inhibit and can be used for the treatment of various abnormalities of cell proliferation, such as malignant tumors (U.S. patent 5747470; U.S. patent 5591317 and U.S. patent 5783683, each specifically incorporated herein by reference in full).

Thus, in illustrative embodiments, the invention relates to oligonucleotide sequences that contain all or part of any sequence that is capable of specific contact described here polynucleotide sequence or she complementary sequence. In one embodiment, the antisense oligonucleotides comprise DNA or its derivatives. In another embodiment, the oligonucleotides contain RNA or her proizvodi the E. In the third embodiment, the oligonucleotides are modified DNA containing the modified frame with phosphorothioate links. In the fourth embodiment, oligonucleotide sequences contain peptidoglycan acids or their derivatives. In each case, the preferred structure contains a region of sequence that is complementary, and more preferably substantially complementary, and more preferably completely complementary to one or more parts of the declared here polynucleotides.

The selection of antisense compounds specific to the gene sequence, based on the analysis of the selected target sequence (i.e. in these illustrative examples, the sequences of rat and human) and the determination of the secondary structure, Tmthe binding energy, relative stability, and the antisense compounds were chosen on the basis of their relative inability to form dimers, hairpins or other secondary structures that would reduce or prevent specific binding to the mRNA target in the cell host.

Highly preferred regions of mRNA are areas, which are located at the codon of translation initiation AUG or close to it, and those sequences that are substantially complementary the s 5'-regions of mRNA. These analyses of the secondary structure and the analyses associated with the site selection of the target was performed using version 4 of the computer program for the analysis of OLIGO primers (Rychlik, 1997) and computer program algorithm BLASTN 2.0.5 (Altschul et al., 1997).

Also provides for the application of the method of delivery of antisense sequences using short peptide vector, called MPG (27 residues). The MPG peptide contains a hydrophobic domain derived from the sequence of the fusion of the HIV gp41, and a hydrophilic domain from the nuclear localization sequence of the T-antigen of SV40 (Morris et al., 1997). It is shown that several molecules of peptide MPG cover the antisense oligonucleotides can be delivered into cultured mammalian cells in less than 1 hour with relatively high efficiency (90%). In addition, interaction with MPG strongly increases as the stability of the oligonucleotide to nucleases, and the ability to cross the plasma membrane (Morris et al., 1997).

RIBOZYMES.

Although catalysis of nucleic acids traditionally used proteins, appeared another class of macromolecules that are suitable for this approach. Ribozymes are RNA-protein complexes that break down nucleic acids site-specific manner. Ribozymes have specific catalytic domains that possess their endonuclease and what activity (Kim and Cech, 1987; Gerlach et al., 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate reactions transfer Postavarul with a high degree of specificity, often splitting only one of several Postavarul in an oligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity is due to the fact that a necessary condition is the binding of a substrate prior to chemical reaction with the internal guide sequence ("IGS") of the ribozyme through specific interactions mating grounds.

First ribozymes catalysis was discovered as part of a specific sequence of reactions cleavage/ligation involving nucleic acids (Joyce, 1989; Cech et al., 1981). For example, in U.S. patent No. 5354855 (specifically incorporated herein by reference) reported that some of the ribozymes can act as an endonuclease with greater specificity in relation to sequences than the known specificity of ribonuclease approaching specificity of the enzymes DNA. Thus, specific sequences mediated by ribozymes in the inhibition of gene expression can be extremely useful for therapeutic applications (Scanlon et al., 1991; Sarver et al., 1990). Recently reported that ribozymes caused genetic changes in some lines of the tile is to, for which they were used; the modified genes include oncogenes H-ras, c-fos and genes of HIV. Most of this work included modification of mRNA targets based on specific mutant codon, which splits specific ribozyme.

Currently, there are six major types of enzymatic RNA of natural origin. Each species can catalyze the hydrolysis fosfolipidnyh relations RNA in trans-position (and, thus, can cleave other RNA molecules) under physiological conditions. In General, enzymatic nucleic acids are first through binding to the RNA target. Such binding occurs through part of the enzymatic nucleic acid binding target, which has a near part of the enzyme molecule, which acts, splitting RNA target. Thus, the enzymatic nucleic acid first recognizes and then binds to the RNA target by means of the complementary mating grounds, and after contact with the correct site, acts enzymatically, slitting RNA target. Splitting in a strategically important part of such RNA target would be to violate its ability to direct synthesis of the encoded protein. After enzymatic nucleic acid will be contacted and will split its RNA target, it is released from such RNA, to find another Misha is ü, and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme possesses advantages in comparison with many ways such as technology antisense sequences (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation), as the concentration of ribozyme necessary to have a therapeutic effect, is lower than the concentration of antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of RNA targets. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition, caused not only by the mechanism of coupling substrates in the binding of the RNA target, but also the mechanism of cleavage of the RNA target. The only erroneous base pairing or replacing grounds near the site of cleavage can completely eliminate the catalytic activity of the ribozyme. Similar erroneous base pairing in the antisense molecules do not prevent action (Woolf et al., 1992). Thus, the specificity of action of the ribozyme is higher than the specificity of antisense oligonucleotide binding to the same site RNA.

Molecule enzymatic nucleic acid which acid can be formed in the motif of the head of the hammer, hairpin, hepatitis δ, group I intron or RNA RNase P (in connection with the guide RNA molecule or RNA in Neurospora VS. Examples of motifs as head of the hammer is described by Rossi et al. (1992). Examples of motifs in the form of studs described by Hampel et al. (published application for the grant of European patent No. EP 0360257), Hampel and Tritz (1989), Hampel et al. (1990) and U.S. patent 5631359 (specifically incorporated herein by reference). An example of the motif of hepatitis δ described by Perrotta and Been (1992); an example of a motif Mozyr described by Guerrier-Takada et al. (1983); motif ribozyme RNA in Neurospora VS described by Collins (Saville and Collins, 1990; Saville and Collins, 1991; Collins and Olive, 1993); and an example of a group I intron is described in U.S. patent 4987071 (specifically incorporated herein by reference). The only thing that is important in the molecule, the enzymatic nucleic acid according to this invention is that it has a specific binding site of the substrate, which is complementary to one or more regions of the RNA of the target genes, and that she has the nucleotide sequence in substrate binding site or around it, which give the molecule the activity in the cleavage of RNA. Thus, ribozyme design do not need the restriction listed here are specific motives.

In some embodiments, it may be important to obtain the enzymatic splitting of funds that exhibit a high degree of specificity in respect of RNA Proc. of the required target such as the one stated here sequences. Molecule enzymatic nucleic acid is preferably targeted to a highly conserved region of the sequence of the mRNA target. Such molecules enzymatic nucleic acids can be delivered exogenously to a specific cell. Alternative ribozymes can be Express with DNA or RNA vectors that are delivered to specific cells. Small motifs enzymatic nucleic acids (for example, the structure of the head of the hammer or studs) can also be used for exogenous delivery. The simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade selected as target areas in the structure of the mRNA. Alternative catalytic RNA molecules can be Express within cells from eukaryotic promoters (e.g., Scanlon et al., 1991; Kashani-Sabet et al., 1992; Dropulic et al., 1992; Weerasinghe et al., 1991; Ojwang et al., 1992; Chen et al., 1992; Sarver et al., 1990). Specialists in this field understand that any ribozyme can be Express in eukaryotic cells with a suitable DNA vector. The activity of these ribozymes can be improved by their release from the primary transcript by a second ribozyme (published international application patent No. WO 93/23569, and published international application patent No. WO 94/0259, however, both applications incorporated by reference; Ohkawa et al., 1992; Taira et al., 1991; Ventura et al., 1993).

Ribozymes can be added directly to the target cells, or to deliver in a complex with cationic lipids, lipid complexes, Packed in liposomes, or other way. RNA or RNA complexes can be entered locally in the relevant tissues ex vivo, or in vivo through injection, aerosol inhalation, pump for infusion or stent, with the inclusion or without inclusion in biopolymers. Ribozymes can be constructed as described in published international application patent No. WO 93/23569 and published international application patent No. WO 94/02595 (each of which is specifically incorporated herein by reference) and synthesized for testing in vitro and in vivo, as described. Such ribozymes can also optimize for delivery. Despite the fact that provided specific examples of specialists in this field will understand that if necessary, you can use the equivalent RNA target other species.

Laying of ribozymes in the form of a hammer head or stud can be separately analyzed using the computer (Jaeger et al., 1989), to determine whether the sequence of the ribozyme in the appropriate secondary structure. Ribozymes with unsuccessful intramolecular interactions between the connecting shoulders and was pushing the optical core is excluded from consideration. You can choose different lengths connecting the shoulder to optimize activity. Typically, at least 5 or so bases of each arm is able to contact or interact in other ways with the RNA target.

Ribozymes with the motif of the head of the hammer or studs can be designed to anneal to various sites of mRNA-matrix, and can be synthesized by chemical means. In the used method of synthesis adhere to the procedures of synthesis of normal RNA, as described in Usman et al. (1987) and in Scaringe et al. (1990), and use the normal groups for protection and binding of nucleic acids, such as dimethoxytrityl at the 5'-end and phosphoramidite on the 3'-end. Normal speed linking usually gives output >98%. Ribozymes having the form of a stud, can be synthesized in two parts, and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992). Ribozymes can largely be modified to enhance stability by modification resistant to nucleases groups, for example, 2'-amino, 2'-C-allyl, 2'-fluorine, 2'-o-stands, 2'-H (for a review see for example Usman and Cedergren, 1992). Ribozymes can be cleaned by gel-electrophoresis, using conventional methods, or liquid chromatography high pressure and resuspending in the water.

The activity of the ribozyme can be optimizare the performance by changing the length of the connecting arms of the ribozyme or chemically synthesizing ribozymes with modifications, that prevent their degradation by serum ribonuclease (see, for example, published international application for patent No. WO 92/07065; Perrault et al. 1990; Pieken et al., 1991; Usman and Cedergren, 1992; published international application for patent No. WO 93/15187; published international application for patent No. WO 91/03162; published an application for the grant of European patent No. 92110298.4; U.S. patent 5334711; and published international application for patent No. WO 94/13688, which describe various chemical modifications that can be made with respect to the components of the molecules of the enzyme RNA, presents sugars), modifications which enhance their efficacy in cells, and with the exception of the bases of the barrel II to reduce the time of RNA synthesis and reduce chemical requirements.

Sullivan et al. (published international application for a patent WO 94/02595) describes conventional methods of delivery of molecules of enzyme RNA. Ribozymes can be entered into cells by various methods known to those familiar with this area, including, but not limited to, encapsulation in liposomes, by iontophoresis, or by incorporation into other media, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. Some indications ribozymes can directly before the presentation to cells or tissues ex vivo with the above carriers or without them. Alternative combination of RNA/media can locally deliver direct inhalation, direct injection or by use of a catheter, a pump for infusion or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous injection or injection into the joint, aerosol inhalation, orally (in the form of tablets or pills), local, systemic, ocular, intraperitoneal and/or intrathecal delivery. A more detailed description of the delivery of ribozymes and the introduction given in the published international application patent No. WO 94/02595 and published international application patent No. WO 93/23569, each of which is specifically incorporated herein by reference.

Another way to accumulate high concentrations of ribozyme(s) within cells is the inclusion of a sequence encoding a ribozyme, in expressing the DNA vector. Transcription of sequences of the ribozyme is controlled by a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from promoter pol II or pol III will be expressed at high levels in all cells; the levels of a given promoter pol II in a given cell type will depend on the nature of the audience next regulatory gene sequences (enhancers, silencers, etc). You can also use the ü promoters prokaryotic RNA polymerase, provided that the prokaryotic enzyme RNA polymerase is expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gao and Huang, 1993; Lieber et al., 1993; Zhou et al., 1990). Ribozymes expressed from such promoters can function in mammalian cells (e.g., Kashani-Saber et al., 1992; Ojwang et al., 1992; Chen et al., 1992 Yu et al., 1993; L Huillier et al., 1992; Lisziewicz et al., 1993). Such transcription units can be incorporated into different vectors for introduction into mammalian cells, including, but not limited to, vectors based on the plasmid DNA, vectors based on viral DNA (such as adenovirus or adeno-associated vectors) or vectors on the basis of viral RNA (such as vectors based on retroviruses, virus, Semliki forest virus, Sindbis).

Ribozymes can be used as diagnostic tools to examine genetic drift and mutations within diseased cells. Ribozymes can also be used to assess levels of RNA molecules target. A close relationship between the activity of the ribozyme and the structure of the RNA target allows to identify mutations in any region of the molecule, which alters the base pairing and three-dimensional structure of the RNA target. Using multiple ribozymes can be mapped changes of nucleotides that are important for structure and function of RNA in vitro and in cells and tissues. The splitting of P Is To target the ribozymes can be used to inhibit gene expression and define the role (essentially) of specific gene products in the progression of the disease. Thus, it is possible to install other genetic targets as important mediators of the disease. These studies will lead to improved treatment for the disease by providing the possibility of combination therapies (e.g., multiple ribozymes targeting different genes, ribozymes, associated with known small molecules inhibitors, or intermittent treatment with combinations of ribozymes and/or combinations of other chemical or biological molecules). Other applications of ribozymes in vitro are well known in this field and include detection of the presence of mRNAs associated with a condition associated with IL-5. Such RNA is detected, determining the presence of cleavage products after processing the ribozyme using the standard method.

PEPTIDOGLYCAN ACID.

In some embodiments, the authors of the invention involve the use of peptidomimetic acids (NCP) in the practice of the methods of the invention. NCP is an imitation of DNA, in which the bases of the nucleotides associated with pseudopeptides skeleton (Good and Nielsen, 1997). NCP can be used in a number of ways in which traditionally used RNA or DNA. Often the sequence NCP are more effective ways than the corresponding sequences of RNA or DNA, and have the application, which is not characteristic of RNA or DNA. A survey on the NCP, including methods of production, characteristics and applications, see Corey (1997) and incorporated herein by reference. Essentially in some embodiments can be obtained sequence NCP, which are complementary to one or more parts of a sequence, mRNA ACE, and such compounds NCP can be used to regulate, change, reduce or reduce broadcast ACE-specific mRNA, and thereby alter the activity of ACE in the cell host, in which such compounds NCP were introduced.

NCP have 2-aminomethylpyridine connection instead of the usual phosphodieterase backbone of DNA (Nielsen et al., 1991; Hanvey et al., 1992; Hyrup and Nielsen, 1996; Nielsen, 1996). This chemical structure has three important consequences: firstly, in contrast to DNA or phosphorothioate oligonucleotides, NCP are neutral molecules; secondly, the NCP are the organization of the achiral molecules that cancels the need to develop a stereoselective synthesis; and third, during the synthesis of the NCP uses the standard Boc Protocol (Dueholm et al., 1994) or Fmoc-Protocol (Thomson et al., 1995) for solid-phase synthesis of peptides, although I've used other methods, including a modified method of Merrifield (Christensen et al., 1995).

The monomers NCP or ready oligomers are commercially available from PerSeptive Biosystems (Framingham, MA). The NCP synthesis by Boc - or Fmoc-Protocol is m are the direct synthesis using protocols manual or automated synthesis (Norton et al., 1995). Protocol synthesis manual is suitable for the production of chemically modified NCP or simultaneous synthesis of families of closely related NCP.

As in the case of peptide synthesis the success of the synthesis of specific NCP will depend on the properties of the selected sequence. For example, although theoretically NCP may include any combination of nucleotide bases, the presence nearby of purines can lead to deletions of one or more residues in the product. In anticipation of the mentioned difficulties, it is assumed that when receiving NCP nearby purines should be repeated binding residues, which are likely to join inefficient. Then should follow the cleaning NCP reversed-phase high-performance liquid chromatography (Norton et al., 1995), providing the outputs and the purity of the product, similar to those observed during the synthesis of peptides.

Modification NCP for this application can be accomplished by linking amino acids during solid-phase synthesis or coupling components which contain a carboxylic acid group, with an open N-terminal amine. Alternative NCP can be modified after synthesis by binding with the introduced lysine or cysteine. The ease with which you can modify the NCP, facilitates optimization for better solubility or for specific is unctionally requirements. After NCP synthesized, the identity of the NCP and their derivatives can confirm mass spectrometry. Several studies have been produced and used modifications NCP (Norton et al., 1995; Haaima et al., 1996; Stetsenko et al., 1996; Petersen et al., 1995; Ulmann et al., 1996; Koch et al., 1995; Orum et al., 1995; Footer et al., 1996; Griffith et al., 1995; Kremsky et al., 1996; Pardridge et al., 1995; Boffa et al., 1995; Landsdorp et al., 1996; Gambacorti-Passerini et al., 1996; Armitage et al., 1997; Seeger et al., 1997; Ruskowski et al., 1997). In U.S. patent No. 5700922 discussed chimeric molecules PNK-DNA-NCP and their application in diagnostics, modulation of protein in organisms and treatment of conditions amenable to treatment.

Unlike DNA and RNA, which contain negatively charged communication frame NCP neutral. Despite this significant change, the NCP recognize complementary DNA and RNA by forming pairs of Watson-Crick (Egholm et al., 1993), confirming the initial modeling Nielsen et al. (1991). NCP does not have polarity from 3' to 5'-end and can be reached either parallel or antiparallel manner with antiparallel is the preferred method (Egholm et al., 1993).

Hybridization of DNA oligonucleotides with DNA and RNA destabilized by electrostatic repulsion between the negatively charged phosphate cores complementary threads. In contrast, the absence of repulsion of charges in the duplexes PNK-DNA or PNK-RNA increases tempera is ur melting point (T mand reduces the dependence of Tmfrom the concentration of mono - and divalent cations (Nielsen et al., 1991). Increased speed and affinity hybridization are important because they are responsible for the amazing ability of the NCP to implement the invasion of complementary sequences in relacionado Dunaeva DNA. In addition, the effective hybridization in the inverted repeats suggests that the NCP can effectively learn the secondary structure in the double-DNA. The best learning takes place in the case of NCP, immobilized on surfaces, and Wang et al. showed that are associated with the substrate, the NCP can be used to detect hybridization events (Wang et al., 1996).

It can be expected that the strong binding of the NCP with complementary sequences will also increase the binding with similar (but not identical) sequences, reducing the specificity of the NCP in the recognition sequence. However, as in the case of DNA hybridization, selective recognition can be achieved by choosing the length of the oligomer and the incubation temperature. In addition, selective hybridization NCP contributes to the fact that PNK-DNA hybridization is less tolerant to erroneous base pairing than DNA-DNA hybridization. For example, a single erroneous mating within duplex PNK-DNA length 16 P.N. which may reduce T mon the value up to the 150(Egholm et al., 1993). This high level of recognition allowed to develop strategies based on the NCP for analysis of point mutations (Wang et al., 1996; Carlsson et al., 1996; Thiede et al., 1996; Webb and Hurskainen, 1996; Perry-O'keefe et al., 1996).

High-affinity binding provides clear advantages for molecular recognition and development of new applications NCP. For example, the NCP from 11-13 nucleotides inhibits the activity of telomerase, ribonucleoprotein, which lengthens the telomere ends, using the basic RNA-matrix, while similar DNA oligomers do not (Norton et al., 1996).

Neutral NCP more hydrophobic than similar DNA oligomers, and this can lead to difficulties of their dissolution at neutral pH, especially if the NCP has a high content of purine, or if they have the ability to form secondary structure. The solubility of the NCP can be increased by adding one or more positive charges to the ends of the NCP (Nielsen et al., 1991).

Data Allfrey and co-authors suggest that the invasion of the thread in the sequence in chromosomal DNA occurs spontaneously (Boffa et al., 1995; Boffa et al., 1996). In these studies, the NCP zeroing in on a target representing the repeated triplets of nucleotides CAG, and used this recognition to clear transcription is active DNA (Boffa et al., 1995) and to inhibit transcription (Boffa et al., 1996). The result suggests that if the NCP can be delivered into cells, they will be able to perform the role of the major-specific sequence regulators of gene expression. Studies and surveys relating to the application of NCP as antisense and antigenic agents include the work of Nielsen et al. (1993b), Hanvey et al., (1992) and Good and Nielsen (1997). Koppelhus et al. (1997) used the NCP in order to inhibit the reverse transcription of HIV-1, showing that the NCP can be used for antiviral therapy.

Methods to characterize the binding properties of the NCP in relation to the antisense sequences are discussed in Rose (1993) and Jensen et al. (1997). Rose used capillary gel electrophoresis in order to determine the binding NCP them with complementary oligonucleotide, measuring the relative binding kinetics and stoichiometry. Similar measurements were made Jensen et al. using BIAcore technologyTM.

Other applications NCP include applying for invasion strands of DNA (Nielsen et al., 1991), antisense inhibition (Hanvey et al., 1992), mutational analysis (Orum et al., 1993), enhancers of transcription (Mollegaard et al., 1994), purification of nucleic acids (Orum et al., 1995), isolation of transcriptionally active genes (Boffa et al., 1995), the blocking factor binding transcripts and (Vickers et al., 1995), cleavage of the genome (Veselkov et al., 1996), biosensors (Wang et al., 1996) in situ hybridization (Thisted et al., 1996) and as an alternative southern-blotting (Perry-O'keefe, 1996).

POLYPEPTIDE COMPOSITION.

In other aspects this invention relates to polypeptide compositions. In General, the polypeptide according to the invention is an isolated polypeptide or epitope variant, or active fragment)derived from mammalian species. Preferably the polypeptide is encoded stated here polynucleotide sequence or a sequence, which in conditions of moderate hardness hybridized stated here polynucleotide sequence. Alternative polypeptide can be defined as a polypeptide that contains a sequence of continuously consecutive amino acids from those expressed amino acid sequence, or polypeptide, which contains the full stated here the amino acid sequence.

The present invention also assumes that the polypeptide composition comprises one or more polypeptides that are immunologically reactive with respect to antibodies generated against a polypeptide according to the invention, in particular a polypeptide having the amino acid sequence represented in SEQ ID NO: 176, 179, 181, 469-473, 475, 485, 488 87 and, or its active fragments or variants, or biologically functional equivalents.

Similarly assumes that the polypeptide composition according to the invention contains one or more polypeptides, which can cause the appearance of antibodies that react immunologically with one or more polypeptides encoded by one or more contiguous sequences of nucleic acids that are part of SEQ ID NO: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486 and 489; or their active fragments or variants, or one or more sequences of nucleic acids, which in conditions of moderate to high hardness hybridize with one or more specified the sequences. In particular, given for illustration polypeptides contain amino acid sequence indicated in SEQ ID NO: 176, 179, 181, 469-473, 475, 485, 487 and 488.

In the sense used here, the active fragment of the polypeptide includes the whole or part of a polypeptide which is modified by conventional methods, for example, by mutagenesis or accession, deletion or replacement, but an active fragment substantially shows the same structure, function, antigenicity, etc. as described here polypeptide.

In some illustrative embodiments, poly is atidy according to the invention will contain, at least immunogenic portion of the protein of breast cancer or its variant, which is described here. As indicated above the protein of breast cancer" is a protein that is expressed by tumor cells of the breast. Proteins, which are proteins of the tumor in the breast, when the immunoassay (ELISA) is also responsive to the detected level with anticorodal from a patient with breast cancer. Described here polypeptides can be of any length. There may be additional sequences derived from the native protein, and/or a heterologous sequence, and such sequences may (but need not) be more immunogenic or antigenic properties.

"Immunogenic portion" in the sense used here is part of a protein that recognizes (i.e. specific links) surface receptor antigens In cells and/or T-cells. Such immunogenic parts usually contain at least 5 amino acid residues, more preferably at least 10, even more preferably at least 20 amino acid residues of the protein of breast cancer or its variants. Some preferred immunogenic parts include peptides in which was demeterova N-terminal leader sequence and/or transmembranal. Other preferred immunogenic portions may contain a small N - and/or C-terminal deletions (e.g., 1-30 amino acids, preferably 5-15 amino acids)relative to the Mature protein.

Immunogenic part, can generally be identified using well known methods, such as methods, are summarized in Paul, Fundamental Immunology, 3rded., 243-247 (Raven Press, 1993) and cited in this work references. Such methods include screening polypeptides for their ability to react with antigen-specific antibodies, anticorodal and/or lines or clones of T cells. In the sense used here, anticavity and antibodies are antigen-specific"if they are specific contact with antigen (i.e., they react with the protein in an ELISA or other immunoassay and do not respond to the detected level with unrelated proteins). Such antisera and antibodies can be obtained, as described here and using well known methods. Immunogenic part of a native protein of the tumor in the breast is the portion that reacts with such anticorodal and/or T-cells at a level that is not significantly lower than the reactivity of the full length polypeptide (e.g., in an ELISA and/or analysis of the reactivity of T cells). Such immunogenic portions may react in such analyses at a level similar to or higher than reactive is here polypeptide is full length. Typically, such screening can be performed using methods well known to specialists in this field, such as the methods described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, the polypeptide can be mobilitat on a solid medium and to carry out his contact with the patient's serum to ensure the binding of the antibody in the serum to the immobilized polypeptide. Then not bound peroxidase serum can be removed and bound peroxidase antibodies to detect using, for example,125I-labeled protein A.

As indicated above, the composition may contain a variant of a native protein of breast cancer. In the sense used here "variant" polypeptide is a polypeptide that differs from a native protein of breast cancer by one or more substitutions, deletions, connections and/or insertion, so that the immunogenicity of the polypeptide is not significantly reduced. In other words, the ability of the variant to react with antigen-specific anticorodal compared to the native protein can be increased or remain unchanged, or may be reduced less than 50%, and preferably less than 20% relative to the native protein. Typically, these variants can be identified by modifying one of the above polypeptide sequences and estimates the Wai reactivity of the modified polypeptide with antigen-specific antibodies or antisera, as described here. Preferred variants include variants in which one or more parts, such as the N-terminal leader sequence or transmembrane domain, have been removed. Other preferred variants include variants in which a small portion (e.g., 1-30 amino acids, preferably 5-15 amino acids) were removed from the N - and/or C-end of the Mature protein.

Variants of polypeptides covered by this invention include variants exhibiting at least, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity (defined as above) with those expressed polypeptides.

Preferably the variant contains a conservative replacement. "Conservative substitution" is a substitution, wherein the amino acid substituted by another amino acid that has similar properties, so that the expert in the field of peptide chemistry would expect the secondary structure and hydropathicity nature of the polypeptide is not substantially changed. Typically, replacement of amino acids can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipatic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include Liz is n and arginine; and amino acids with uncharged groups of the polar heads having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids, which can give conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Option may also or alternatively contain nonconservative changes. In a preferred embodiment, variants of the polypeptides differ from the native sequence by substitution, deletion or accession, five amino acids or less. Variants may also (or alternatively) modified, for example, by deletion or by joining amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathicity nature of the polypeptide.

As indicated above polypeptides may contain a signal (or leader) sequence at the N-end of the protein, which cotranslational or excision directs transfer of the protein. The polypeptide can also be konjugierte with a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (for example, poly-His), or to enhance binding of the polypeptide to a solid carrier. For example, the polypeptide can con wherewith with the Fc region of immunoglobulin.

The polypeptides can be obtained using any of a variety of well known methods. Recombinant polypeptides encoded by DNA sequences that are described above, can be easily obtained from DNA sequences using any of a number expressing vectors, known to specialists in this field. Expression can be achieved in any suitable cell host, which has been transformed or transliterowany expressing a vector containing a DNA molecule that encodes a recombinant polypeptide. Appropriate cell hosts include prokaryotes, yeast, and cells of higher eukaryotes, such as mammalian cells and plant cells. Preferably used cells hosts are E. coli, yeast or line of mammalian cells such as COS or CHO. Nadeshiko suitable systems owner/vector, which secrete recombinant protein or polypeptide into the culture medium can first be concentrated using a commercially available filter. After concentration, the concentrate can be applied to a suitable matrix for purification, such as affinity matrix, or ion exchange resin. Finally one or several stages of reversed-phase HPLC can be used to further purify the recombinant polypeptide.

Parts and other options with less than approximately you, and typically less than approximately 50 amino acids, can also be obtained by synthetic means, using techniques well known to specialists in this field. For example, such polypeptides can be synthesized using any commercially available solid-phase methods, such as solid-phase synthesis method of Merrifield, in which amino acids are sequentially added to a growing amino acid chain. See, Merrifield, J. Am. Chem. Soc. 85: 2149-2146, 1963. Equipment for automated synthesis of polypeptides is available for purchase from a vendor such as Perkin Elmer/Applied BioSystems Division (Foster City, CA), and you can work with it according to the manufacturers ' instructions.

In some specific embodiments, the polypeptide may be a hybrid protein that contains a composite polypeptides that are described herein or which contains at least one polypeptide, which is described here, and are unrelated sequence, such as the known protein tumor. Partner with the merger may, for example, to participate in providing T helper epitopes (an immunological partner in the hybrid), preferably T helper epitopes recognized by humans, or may contribute to the expression of the protein (enhancer of the expression) with a higher yield than the native recombinant protein. Some preferred the additional partners in the merger are as immunological, and amplifying the expression of the partners in the merger. Other partners in the merge, you can choose to increase the solubility of the protein or to provide for a targeted delivery of necessary protein in intracellular compartments. Still one of the partners in the merger include affinity tags that facilitate purification of the protein.

Hybrid proteins, as a rule, can be obtained using standard methods, including chemical binding. Preferably, the hybrid protein is expressed in the form of a recombinant protein, providing in expressing the system of production of increased levels of relatively hybrid protein. Briefly, the DNA sequence encoding polypeptide components can be packaged separately, or legirovanyh in an appropriate expression vector. the 3'End DNA sequence that encodes one component of the polypeptide, are ligated using a peptide linker with or without the 5'-end DNA sequence that encodes a second component of the polypeptide, so that the reading frames of the sequences were in phase. This provides the ability to broadcast a single hybrid protein that retains the biological activity of both components of the polypeptides.

You can use the sequence of peptide linker to separate the first and second components is of polypeptide distance sufficient to ensure the laying of each polypeptide secondary and tertiary structures. The following sequence of peptide linker include hybrid protein, using standard methods, well known in this field. The appropriate sequence of peptide linkers can be selected on the basis of the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the functional epitopes of the polypeptide. The preferred sequence of peptide linker contain residues Gly, Asn and Ser. In the linker sequence can also be used other close to neutral amino acids, such as Thr and Ala. Amino acid sequences that can be successfully used as linkers include sequence shown in Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83: 8258-8262, 1986; U.S. patent No. 4935233 and U.S. patent No. 4751180. Usually the linker sequence may have a length of from 1 to about 50 amino acids. Linker sequences are not required when the first and second polypeptides are not significant N-concave the areas of amino acids, which can be used to separate the functional domains and prevent steric interference.

Legirovannye DNA sequence is functionally linked to suitable transcriptional or translational regulatory elements. Regulatory elements responsible for expression of the DNA is localized only to the 5'-side of the DNA sequence that encodes a first polypeptide. Similarly, stop codons required to end translation and signals termination of transcription are present only with the 3'side relative to the DNA sequence that encodes a second polypeptide.

Also included are hybrid proteins. Such proteins contain a polypeptide, which is described here, together with an unrelated immunogenic protein. Preferably immunogenic protein can cause secondary immune response. Examples of such proteins include proteins tetanus, tuberculosis and hepatitis (see, for example, Stoute et al. New Engl. J. Med., 336: 86-91, 1997).

In preferred embodiments, the immunological partner to merge derived from protein D, a surface protein of the gram-negative bacteria Haemophilus influenzae B (WO 91/18926). Preferably derived protein D has approximately the first third of the protein (for example, the first N-terminal 100-110 amino acids), and the derived protein D can be associated with a lipid. In some predpochtite is selected embodiments, the first 109 residues partner in the merger, obtained from lipoprotein D, placed at the N-end to provide the polypeptide with additional exogenous T-cell epitopes and to increase the level of expression in E. coli (thus, the partner functions as power of expression). The lipid tail ensures optimal antigen presentation antigen-presenting cells. Other partners for mergers include non-structural proteins of influenza virus, NS1 (hemagglutinin). Usually use 81 N-terminal amino acids, although you can use various fragments, which include T-helper epitopes.

In another embodiment, the immunological partner in the merger is a protein known as LYTA, or part (preferably C-terminal part). LYTA is obtained from Streptococcus pneumoniae, which synthesizes N-acetyl-L-alaninemia known as amidase LYTA (encoded LytA gene; Gene 43: 265-292, 1986). LYTA is autolysins that specific causes degradation of some bonds in the main chain of the peptidoglycan. The C-terminal domain of the protein LYTA responsible for the affinity to the choline or some analogues of choline, such as DEAE. This property is used for developing expressing plasmids C-LYTA E. coli, suitable for expression of the hybrid proteins. Describes the purification of hybrid proteins containing the fragment of C-LYTA on aminocore (see Biotechnology 10: 795-798, 1992). In preferably the variant in the hybrid protein can be included part of the repeat LYTA. Part of the repetition found in the C-terminal region, starting with residue 178. Especially preferred part of the repeat contains the remains of 188-305.

In General, the polypeptides described herein (including hybrid proteins) or polynucleotide are isolated. An "isolated" polypeptide or polynucleotide is a polypeptide or polynucleotide that was removed from its original environment. For example, the protein of natural origin is isolated if it is separated from some or all together present in the natural system materials. Preferably, the purity of such polypeptides is at least about 90%, more preferably at least about 95%, and most preferably the purity is at least about 99%. Polynucleotide is considered isolated if, for example, clone in the vector, which is not part of the natural environment.

In order to increase the antigenicity and/or immunogenicity of the protein of breast cancer according to this invention can be obtained hybrid protein containing antigenic and/or immunogenic portions of two or more proteins of breast cancer. Examples of hybrid proteins of breast cancer you can get conventional recombinant DNA, combining the DNA sequence encoding mammaglobin, with what sledovatelnot DNA coding either (1) a United "left" and "right" ORF B726P (SEQ ID NO: 490); (2) "left" ORF B726P (SEQ ID NO: 491); and/or (3) "right" ORF B726P (SEQ ID NO: 492). See, for example, Ausubel, F.M. et al. "Short Protocols in Molecular Biology" (4nded. 1999); incorporated herein by reference in full). Examples of hybrid proteins are listed here in SEQ ID NO: 493 (mammaglobin - United ORF B726P), SEQ ID NO: 494 (mammaglobin - left ORF B726P) and SEQ ID NO: 495 (mammaglobin "right" ORF B726P). The DNA sequence encoding mammaglobin, pictured here nucleotides 1-279 SEQ ID NO: 490-492, and the corresponding amino acid sequence of mammaglobin pictured here amino acids 1-93 SEQ ID NO: 493-495. See also U.S. patent No. 5668267; U.S. patent No. 5922836; U.S. patent No. 5855889; U.S. patent No. 5968754; and U.S. patent No. 6004756, each of these U.S. patents is incorporated herein by reference in full.

In addition to the above as examples of hybrid proteins obtained by merging region encoding mammaglobin full length, with different coding regions of the B726P this invention also relates to hybrid proteins containing the immunogenic part of 9 or more continuously consecutive amino acids of any of one of the two or both proteins mammaglobin and B726P. Preferred immunogenic portions may be a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more continuously following each other and is of inoculat or one of the two, or both proteins mammaglobin and/or B726P. Alternative immunogenic portion may constitute at least, 25, 30, 35, 40, 45, 50, 75, 100, 250, 500, or 1095 continuously consecutive amino acids of any of one of the two or both proteins mammaglobin and/or B726P, or can also include any integer of amino acids 20 to 1095 continuously consecutive amino acids of any of one of the two or both proteins mammaglobin and/or B726P.

Typical immunogenic part of mammaglobin described in submitted to the joint consideration of the application for the grant of a U.S. patent 60/136528. Examples of immunogenic parts include the following peptide sequence mammaglobin:IDELKECFLNQTDETLSNVE (amino acids 59-78 SEQ ID NO: 493); TTNAIDELKECFLNQ (amino acids 55-69 SEQ ID NO: 493); SQHCYAGSGCPLLENVISKTI (amino acids 13 to 33 SEQ ID NO: 493); EYKELLQEFIDDNATTNAID (amino acids 41-60 SEQ ID NO: 493), and/or KLLMVLMLA (amino acids 2-10 SEQ ID NO: 493). Other preferred epitopes include the site of glycosylation of mammaglobin. Such epitopes, in particular, suitable to generate antibodies that are specific contact glycosylated mammaglobin. Two such customers represent N-linked glycosylation sites asparagine (Asp)-53 (QEFIDDNATTNAI; amino acids 47-59 SEQ ID NO: 493), and Asp-68 (LKECFLNQTDETL; amino acids 62-74 SEQ ID NO: 493).

The present invention also assumes that gibr is-breaking the proteins mammaglobin-VR can find application in a wide variety of immunogenic parts of the joint, "left" and/or "right" amino acid sequence VR. For example, particularly suitable hybrid protein mammaglobin-B726P can be obtained by merging mammaglobin with the "right" epitope B726P, recognizable B726P-specific CTL clones described herein in example 4, and the epitope is part of the N-end of the "right" area B726P (i.e. amino acids 1-129 SEQ ID NO: 176).

Specialists in this field will understand that the exact amino acid sequence and primary location of the sequence of parts of mammaglobin and/or B726P hybrid proteins can be varied without going beyond the scope of this invention. For example, you can make conservative amino acid substitutions in one or two, or in both parts - mammaglobin or B726P, for example, to ensure that the hybrid proteins had improved properties such as increased stability of the protein and/or immunogenicity. In addition, in this invention it is assumed that part of mammaglobin can be drained or N-end or From the end part of the B726P, to ensure that the hybrid proteins possessed the required antigenic and/or immunogenic properties.

Hybrid proteins according to this invention, an example of which is given hybrid proteins mammaglobin-B726P described here in this example, will find use as vaccines FR is in malignant tumors, reagents for therapeutics based on antibodies and/or in various diagnostic assays. It is assumed that these hybrid proteins will have improved antigenic and/or immunogenic properties compared to only either mammaglobin and/or protein B726P.

BINDING AGENTS.

This invention also relates to such agents, such as antibodies and their antigennegative fragments that are specific contacted with the protein of breast cancer. In the sense used here say that the antibody, or antigennegative slice-specific contacts with the protein of breast cancer, if it reacts at a detectable level(for example, when ELISA) protein of breast cancer, but does not respond to the recorded level with unrelated proteins under similar conditions. In the sense used here, "binding" refers to non-covalent Association between two separate molecules to form the complex. The ability to communicate can be estimated, for example, determining the binding constant for the complex formation. The binding constant is a value obtained by dividing the concentration of the complex to the product of the concentrations of the components. In General, in the context of this invention, they say that two connections are "tied" in the case when the con is Tanta binding for the formation of the complex is greater than about 10 3l/mol. The binding constant can be determined using methods well known in the field.

Binding agents also allow you to distinguish between patients who do not have a malignant tumor and having a malignant tumor, such as breast cancer, using provided here are representative analyses. In other words, antibodies and other binding agents that bind to a protein of breast cancer, will generate a signal indicating the presence of a malignant tumor, at least about 20% of patients with the disease, and will generate a negative signal, showing the absence of disease, at least 90% of subjects having a malignant tumor. To determine whether the binding agent this requirement, biological samples (e.g. blood, serum, sputum, urine and/or biopsy of the tumor) from patients with or without malignancy (which is determined using standard clinical tests), can be analyzed as described here, the presence of polypeptides that bind with a binding agent. It will be obvious that should be analyzed statistically significant number of samples in the presence of the disease and in its absence. Each binding is th agent must meet the above criteria; however, experts in this field will understand that linking agents can be used in combination to improve sensitivity.

Any agent which satisfies the above requirements, can be a binding agent. For example, the binding agent may be a ribosome, with or without peptide component, an RNA molecule or polypeptide. In a preferred embodiment, the binding agent is an antibody or antigennegative fragment. Antibodies can be obtained by any of many methods known to experts in this field. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In General, antibodies can be obtained through the techniques of cell culture, including the creation of monoclonal antibodies, as described here, or by transfection of antibody genes in a suitable cell host bacteria or mammals in order to ensure the production of recombinant antibodies. In the same way immunogen containing the polypeptide is first injected any of a wide range of mammals (e.g. mice, rats, rabbits, sheep or goats). At this stage, the polypeptides according to the invention can serve as an immunogen without modification. Alternative especially for relatively short polypeptides greater immune response can be called in if the polyp is Ted protein-bound carrier, such as bovine serum albumin or hemocyanin marine saucer "keyhole". The immunogen is injected animal host, preferably in a predefined mode, including one or more booster immunizations, and the animals periodically take blood. Then polyclonal antibodies specific for the polypeptide, can be cleared from a serum, for example, affinity chromatography using the polypeptide associated with a suitable solid carrier.

Monoclonal antibodies specific for interest antigenic polypeptide can be, for example, be obtained using the method of Kohler and Milstein, Eur. J. Immunol. 6: 511-519, 1976, and its modifications. Briefly, these methods involve the preparation of immortalized cell lines capable of producing antibodies having the desired specificity (i.e., the reactivity with respect to interest the polypeptide). Such cell lines can, for example, be obtained from spleen cells derived from animals immunized as described above. Then cells spleen immortalized, for example, the fusion hybrid ground - myeloma cells, preferably with a partner who is syngeneic with respect to the immunized animal. You can use a variety of technologies merge. For example, cells of the spleen and the notches of myeloma can be mixed with a nonionic detergent for a few minutes and then place in a low density on the selective medium, which supports the growth of hybrid cells, but not myeloma cells. In the preferred method of breeding using HAT-selection (gipoksantin, aminopterin, thymidine). After a sufficient period of time, usually about 1 to 2 weeks, see colonies of hybrids. Collect a separate colony and nadeshiko their cultures are tested against binding capacity, which is directed against the polypeptide. Preferred hybridoma with high reactivity and specificity.

Monoclonal antibodies can be distinguished from nadeshiko growing colonies of hybridoma. In addition, you can use various methods to increase yields, such as injection line cells hybridoma into the peritoneal cavity of a suitable host is a vertebrate, such as a mouse. Then, monoclonal antibodies can be collected from ascitic fluid or blood. Contamination can be removed from the antibodies by conventional methods such as chromatography, gel filtration, precipitation and extraction. The polypeptides according to the invention can be used in the cleaning process, for example at the stage affinity chromatography.

In some embodiments it may be preferable to use antigenspecific fragments of antibodies. Such fragments include Fab fragments, which can be obtained using standard methods. Briefly, immunoglobu the ins can be cleaned from rabbit serum by affinity chromatography on a column of beads with protein A (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and split papain, receiving fragments Fab and Fc. Fragments Fab and Fc can be divided by affinity chromatography on columns with balls containing protein A.

Monoclonal antibodies according to this invention can be associated with one or more therapeutic agents. Suitable in this respect means include radionuclides, inducers of differentiation, drugs, toxins and their derivatives. Preferred radionuclides include90Y123I125I131I186Re,188Re,211At and212Bi. Preferred drugs include methotrexate and analogues of pyrimidine and purine. Preferred inducers of differentiation include complex forblue esters and butyric acids. Preferred toxins include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin and virus protein of lacunosa.

A therapeutic agent may be associated (e.g., covalently linked) with the appropriate monoclonal antibody either directly or indirectly (e.g. via a linker group). Direct reaction between the agent and the antibody is possible in the case when each of them has a Deputy, capable of reacting with another Deputy. For example, a nucleophilic group, such as amino or sulfhydryl group, one reagent may be capable of reacting with a group containing a carbonyl, such as the anhydride or gelegenheid, or alkyl group containing an easily removable group (e.g., halide) on the other. An alternative may be desirable binding of therapeutic agent and the antibody via a linker group. The linker group can function as a spacer to position the antibody at a distance from the funds, so as not to interfere with the binding capacity. The linker group can also serve to increase the chemical reactivity of substituents on the tool or the antibody, and thus to increase the efficiency of binding. The increase in chemical reactivity can also facilitate the use of means or functional groups of drugs, which otherwise is not possible.

Specialists in this field will be obvious that as the linker group can be used a number of bifunctional or polyfunctional reagents, both Homo-and hetero-functional (such as reagents described in the catalog of the Pierce Chemical Co., Rockford, IL). Binding can, for example, implement, through amino groups, carboxyl groups, sulfhydryl groups or residues oxidized carbohydrate. There are numerous references describing such a method, for example, PA is UNT USA No. 4671958, Rodwell et al.

In those cases where therapeutic agent is more effective when it contains no part immunoconjugates according to this invention, represented by antibody desirable may be the use of the linker group that is cleaved during or after internalization into the cell. Described a number of different biodegradable linker groups. Mechanisms of intracellular release of funds from these linker groups include cleavage by reduction disulfide bond (e.g., U.S. patent No. 4489710, Spitler), irradiation photolabile communication (for example, U.S. patent No. 4625014, Senter et al.), the hydrolysis of the side chains derivatizing amino acids (for example, U.S. patent No. 4638045, Kohn et al.), hydrolysis mediated by serum complement (for example, U.S. patent No. 4671958, Rodwell et al.), and hydrolysis catalyzed by acid (e.g., U.S. patent No. 4569789, Blattler et al.).

Desirable may be linking more than one tool with the antibody. In one embodiment, numerous molecules means associated with one molecule of antibody. In another embodiment, one antibody may be associated with more than one media type. Regardless of the specific variant immunoconjugate with more than one drug can be obtained in various ways. For example, more than one tool can be associated directly with moleculo the antibody or you can use linkers, which provide multiple sites for binding. Alternative you can use the media.

The media can be agents of different ways, including covalent binding, either directly or through a linker group. Suitable carriers include proteins such as albumin (e.g., U.S. patent No. 4507234, Kato et al.), peptides and polysaccharides, such as amylodextrin (for example, U.S. patent No. 4699784, Shih et al.). The carrier can also be an agent through non-covalent binding or encapsulating, such as encapsulation in liposomal carrier (for example, U.S. patent No. 4429008 and 4873088). Media-specific radionuclide agents include small radiohalogenated molecules and chelating compounds. For example, in U.S. patent No. 4735792 described representative radiohalogenated small molecules and their synthesis. Chelating radionuclides agent can be formed from a chelating compounds, which include compounds containing nitrogen atoms and sulfur as donor atoms to bind the radionuclide, presents metal, or metal oxide. For example, in patent No. 4673562 Davison et al., the described examples of chelating compounds and their synthesis.

You can use a variety of routes of administration of antibodies and immunoconjugates. Usually the introduction is intravenous, intramuscular, subcutaneous or lodges in the tumor after resection. It will be obvious that the exact dose of the antibody/immunoconjugate will vary depending on the antibodies used, the density of antigens on the tumor and the rate of clearance of the antibody.

T-CELLS.

Immunotherapy compositions may also or alternatively contain T cells that are specific for the protein of breast cancer. These cells usually can be obtained in vitro or ex vivo, using standard procedures. For example, T cells can be isolated from bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood of the patient, using a commercially available system, cell division, such as the Isolex systemTMavailable from Nexell Therapeutics, Inc. (Irvine, CA; see U.S. patent No. 5240856; U.S. patent No. 5215926; WO 89/06280; WO 91/16116 and WO 92/07243). Alternative T-cells can be obtained from people - relatives and non-relatives, other mammals, lines or cell cultures.

T cells can be stimulated by the polypeptide of breast cancer, polynucleotides coding for the peptide of breast cancer, and/or antigen-presenting cell (APC)that expresses such a polypeptide. Such stimulation is performed under conditions and for a time sufficient to allow for formation of a T-cell specific polypeptide. Preferably the polypeptide tumor Moloch the th cancer or polynucleotide is the carrier for delivery, such as a microsphere, to facilitate the formation of specific T cells.

T-cells are specific polypeptide of breast cancer, if T cells specific proliferate, secrete cytokines or kill target cells coated with the polypeptide or expressing a gene encoding the polypeptide. The specificity of T cells can be assessed using any of a variety of standard ways. For example, analysis of the release of chromium or analysis of cell proliferation, increased stimulation index in the lysis and/or proliferation by more than two times in comparison with the negative control indicates the specificity of T cells. Such analyses, for example, can be performed as described in Chen et al., Cancer Res. 54: 1065-1070, 1994. Alternative registration proliferation of T cells can be accomplished by several known methods. For example, T-cell proliferation can be registered by measuring the increased rate of DNA synthesis (e.g., a pulse phase cultures of T cells labeled with tritium thymidine and measuring the amount of tritium-labeled thymidine incorporated into DNA). Contact with the polypeptide of breast cancer (100 ng/ml - 100 μg/ml, preferably 200 ng/ml - 25 μg/ml) for 3-7 days should result to result, at least a twofold increase in the proliferation of T-cells. As described above, contact the CT for 2-3 hours should result in activation of T cells, what is measured using standard analysis of cytokines, with a two-fold increase in release of cytokines (e.g., TNF or IFN-γ) is a factor of activated T cells (see Coligan et al., Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1988)). T cells that were activated in response to the polypeptide of breast cancer, polynucleotide or expressing the polypeptide APC may be CD4+and/or CD8+. Specific to the protein of breast cancer T-cells can be propagated using standard methods. In preferred embodiments, T cells receive from the patient, donor family member or donor, not a relative, and administered to the patient after stimulation and reproduction.

For therapeutic purposes, CD4+or CD8+T cells that proliferate in response to the polypeptide of breast cancer, polynucleotide or APK, you can increase either in vitro or in vivo. The proliferation of these T cells in vitro can be accomplished in a number of ways. For example, T cells can be re-exposed to the polypeptide of breast cancer, or a short peptide corresponding to immunogenic portion of such a polypeptide, with or without added growth factors T cells, such as interleukin-2, and/or stimulating cells that synthesize the polypeptide tumor mo is full of cancer. Alternative, one or more T cells that proliferate in the presence of the protein of breast cancer, can be propagated by cloning. Methods cloning of cells are well known in this field and include limiting dilution.

PHARMACEUTICAL COMPOSITIONS.

In additional embodiments, the invention relates to the preparation of one or more stated here compositions of polynucleotide, polypeptide, T-cells and/or antibodies, in the form of a pharmaceutically acceptable solutions for administration to a cell or animal, either individually or in combination with one or more other therapies.

Also it will be clear that if desired, you can enter the composition of a segment of nucleic acid, RNA, DNA, or NCP, which Express stated here polypeptide, in combination with other agents, such as other proteins or polypeptides or various pharmaceutically active funds. In fact, there is virtually no limit to other components that can also be included, provided that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. Thus, in some cases, when required, the composition can be delivered along with various other agents. Such compositions can the sight of the notice from host cells or other biological sources, or alternatively, chemically synthesized, as described here. Also, such compositions may also contain substituted or derivationally RNA or DNA composition.

Technology of preparation of pharmaceutically acceptable fillers and solutions media well known to specialists in this area, as the development of suitable dosing schedules and treatment in the application described here, the specific compositions with different treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and methods of cooking.

1. ORAL DELIVERY.

In some applications, a stated here pharmaceutical compositions can be delivered via oral administration to an animal. As such, these compositions can be prepared in a composition with an inert diluent or with an assimilable edible carrier, or they can be enclosed in a gelatin capsule with a hard or soft shell, or they may be compressed into tablets, or they can directly be mixed with food.

Active components can even be mixed with excipients and used in the form taken inside tablets, buccal tablets, pastilles, capsules, elixirs, suspensions, syrups, wafers and the like (Mathiowitz et al., 1997; Hwang et al, 1998; U.S. patent 5641515; U.S. patent 5580579 and U.S. patent 5792451, each of which is specifically incorporated herein by reference in full). Tablets, lozenges, pills, capsules and the like may also contain the following: binder as tragacanth gum, Arabia gum, corn starch or gelatin; excipients such as calcium phosphate disubstituted; dezintegriruetsja tool, such as corn starch, potato starch, alginic acid and the like; a moving substance, such as magnesium stearate; and can be added sweetener, such as sucrose, lactose or saccharin, or corrigentov, such as corrective means of peppermint, oil of Grushenka or cherry. In the case when the dosage form is a capsule, it may contain in addition to the materials mentioned above, the carrier liquid. May contain various other materials as coatings or to otherwise modify the physical form of the dosage forms. For example, tablets, pills or capsules can be coated with shellac or sugar or both of these materials. A syrup or elixir may contain the active compound, sucrose as a sweetener, methyl - and propylparaben as preservatives, a dye and a corrective tool, such as Corrigan the cherry or orange. Of course, any material used in preparing any dosage form should be pharmaceutically pure and substantially non-toxic in the quantities used. In addition, the active compounds may be included in the preparation and composition of long-term release.

Typically, these compositions may contain at least 0.1% of active compound or more, although the percentage of active ingredient(s), of course, you can easily vary from about 1 or 2% to about 60% or 70% or more by weight or volume of the total composition. Naturally the number of active compound(s) in each therapeutically useful compositions can be prepared so that was received appropriate doses in any given a standard dose of a compound. A specialist in the field of preparing such pharmaceutical compositions will include factors such as solubility, bioavailability, time biological half-life, route of administration, the shelf life of the product, as well as other pharmacological considerations, and as such may require different dosing regimens and treatment.

For oral administration the compositions according to this invention an alternative can be mixed with one or more fillers in the form of a liquid for rinsing the mouth, cf is DSTV for brushing your teeth, buccal tablets, oral spray or sublingual oral input composition. For example, liquid mouth rinse can be prepared with the inclusion of the active ingredient in the required amount in an appropriate solvent, such as a solution of sodium borate (solution Dobelle). Alternative active ingredient can be included in the oral solution, such as a solution containing sodium borate, glycerin and potassium bicarbonate, or dispersing tool for brushing your teeth, or add in a therapeutically effective amount of the composition, which may include water, binders, abrasives, correcting means, the blowing means and moisturizers. Alternative compositions can be formed into tablets or solution that can be placed under the tongue or otherwise dissolve in the mouth.

2. INJECTABLE DELIVERY.

In some cases it will be desirable shipping stated here pharmaceutical compositions parenterally, intravenously, intramuscularly, or even intraperitoneally as described in U.S. patent 5543158; in U.S. patent 5641515 and U.S. patent 5399363 (each specifically incorporated herein by reference in full). Solutions of the active compounds as free base or pharmaceutically acceptable salts can be prepared in water suitably mixed the with surface-active substance, such as hydroxypropylcellulose. It is also possible to prepare dispersions in glycerol, liquid polyethylene glycols and their mixtures and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for prepared immediately prior to use of the preparation of sterile injectable solutions or dispersions (U.S. patent 5466468, specifically incorporated herein by reference in full). In all cases the form must be sterile and must be fluid to the extent to easily pass through the syringe. It must be stable under conditions of manufacture and storage and must be protected from contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, high molecular weight alcohol (e.g. glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures and/or vegetable oil. The proper fluidity can, for example, to support, applying a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by applying the surface is chestno-active substances. Prevention of the action of microorganisms can contribute to various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, the preferred is the incorporation of tools that isotonicity, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use in the compositions of agents that decrease the absorption of, for example, aluminum monostearate and gelatin.

For parenteral administration, for example, in aqueous solution, if necessary, must be properly buffered and the liquid diluent first brought to isotonic sufficient quantity of a solution of salt or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous medium, which can be used in the light of this proposal, is well known to specialists in this field. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of fluid for injection into the subcutaneous tissue or injected at the proposed location of the infusion (see, for example, "Remington''s Pharmaceutical Sciences" 15ththEdition, pages 1035-1038 and 1570-1580). Some of arrowana dosage will necessarily occur depending on the condition of the subject, undergoing treatment. The man responsible for the introduction, in any case will determine the appropriate dose for a specific subject. In addition, for the introduction of human drugs must meet the standards of sterility, progenote and General safety and purity, as required by the FDA standards for biological products.

Sterile injectable solutions are prepared by combining the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, which are necessary, and then sterilized by filtration. In General, dispersions are prepared, including a variety of sterile active ingredients into a sterile filler, which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are the means of vacuum drying and freeze-drying, which give a powder of the active ingredient plus any additional desired ingredient from their previously sterile-filtered solution.

Stated here compositions can be prepared in a neutral form or a salt form. Pharmaceutically acceptable salts include the acid additive is Oli (formed with free amino groups of the protein), and these salts are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, almond, and the like. You can also get the salts formed with free carboxyl groups, of inorganic bases, such as, for example, hydroxides of sodium, potassium, ammonium, calcium or iron, and such organic bases as Isopropylamine, trimethylamine, histidine, procaine and the like. After preparation, the solutions will introduce a way compatible with the measured composition, and in such a quantity that is therapeutically effective. The composition is easily administered in various dosage forms such as injectable solutions, capsules, releasing the drug, and the like.

In the sense used here, the term "carrier" includes any and all solvents, dispersion media, fillers, coatings, diluents, antibacterial and antifungal agents, means providing isotonicity and lowering the suction, buffers, media solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in this field. The use of any conventional medium or agent is provided in those who appenticeship compositions except for those restrictions, when they are not compatible with the active ingredient. The composition may also include additional active ingredients.

The phrase "pharmaceutically acceptable" refers to molecular particles and compositions, which do not produce an allergic or similar adverse reactions when administered to man. The preparation of an aqueous composition which contains as active ingredient a protein, is well known in this field. Typically such compositions are prepared in the form of injectable compositions either in the form of aqueous solutions or suspensions; also it is possible to prepare solid forms suitable for solution or suspension in liquid prior to injection. The preparation also can be emulsified.

3. NASAL DELIVERY.

In some embodiments, the pharmaceutical compositions can be delivered by intranasal sprays, inhalation, and/or other media for aerosol delivery. Methods of gene delivery, nucleic acid and peptide compositions directly to the lungs via nasal aerosol sprays are described, for example, in U.S. patent 5756353 and U.S. patent 5804212 (each specifically incorporated by reference in full). Similarly, in the pharmaceutical field is also well known for the delivery of drugs using intranasal resin with micro what astitsy (Takenaga et al., 1998) and compounds of lysophosphatidylserine (U.S. patent 5725871, specifically incorporated herein by reference in full). Similarly, the delivery of drugs through the mucosa in the form of a PTFE matrix media is described in U.S. patent 5780045 (specifically incorporated herein by reference in full).

4. SHIPPING, MEDIATED BY LIPOSOMES, NANOCAPSULES AND MICROPARTICLES.

In some embodiments, the authors of the invention include the use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like for the introduction of the compositions according to the invention in a suitable cell host. In particular, compositions according to this invention can be prepared for delivery either encapsulated in a lipid particle, a liposome, vesicles, nano scale, or nanoparticles, or the like.

Such compositions may be preferred for the introduction stated here pharmaceutically acceptable compositions of nucleic acids or structures. Mainly the formation and use of liposomes is known to specialists in this field (see, for example, Couvreur et al., 1977; Couvreur, 1988; Lasic, 1998, which describe the use of liposomes and nanocapsules in the targeted antibiotic therapy of intracellular bacterial infections and diseases). Recently develo the Tana liposomes with enhanced stability in serum and circulation half-life (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987; U.S. patent 5741516, specifically incorporated herein by reference in full). In addition, the reviews the different ways concerning preparations of liposomes and similar to liposomes particles as potential drug delivery vehicles (Takakura, 1998; Chandran et al., 1997; Margalit, 1995; U.S. patent 5567434; U.S. patent 5552157; U.S. patent 5565213; U.S. patent 5738868 and U.S. patent 5795587 (each specifically incorporated herein by reference in full).

Liposomes have been used for a number of types of cells that are normally resistant to transfection by other means, including suspension of T-cells, primary cultures of hepatocytes and cells of RS 12 (Renneisen et al., 1990; Muller et al., 1990). In addition, liposomes do not have restrictions on the length of DNA that is usually for delivery systems based viruses. Liposomes effectively used to introduce genes, drugs (Heath and Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989; Fresta and Puglisi, 1996), radiotherapy funds (Pikul et al., 1987), enzymes (Imaizumi et al., 1990a; Imaizumi et al., 1990b), viruses (Faller and Baltimore, 1984), transcription factors and allosteric effectors (Nicolau and Gersonde, 1979) in different lines of cultured cells and animals. In addition, completed several successful clinical trials evaluating the efficacy mediated by liposomes drug delivery (Lopez-Berestein et al., 1985 a; 1985b; Coune, 1988; Sculier et al., 1988). To the ome, several studies indicate that the use of liposomes is not associated with autoimmune responses, toxicity or localization in the gonads after systemic delivery (Mori and Fukatsu, 1992).

Liposomes formed from phospholipids that are dispersed in an aqueous medium and spontaneously form a multilayer concentric bubbles from belayneh structures (also called multilayer vesicles (MLV). MLV typically have diameters of from 25 nm to 4 μm. Processing MLV ultrasound leads to the formation of small single-layer vesicles (SUVs) with diameters in the range from 200 to 500 Åcontaining aqueous solution in the kernel.

Liposomes have similarities with cellular membranes and are planned for use in connection with this invention as carriers for peptide compositions. They are widely applicable, as it can capture both water-soluble and lipitorhistory substances, i.e. in a water-filled spaces in the bilayer, respectively. It is likely that liposomes bearing drug, even can be used for site-specific delivery of active agents through selective modification of the composition of liposomes.

In addition to the guidelines Couvreur et al. (1977; 1988) to create compositions of liposomes you can use the following information. Phospholipids can form a number of structures other than lipase is, in the case when they are dispersed in water, depending on the molar ratio of lipid to water. At low ratios, the preferred structures are liposomes. The physical characteristics of liposomes depend on pH, ionic strength and presence divalent cations. Liposomes can exhibit low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition, which significantly alters their permeability. The phase transition involves the transformation of tightly Packed, ordered structure, known as a gel-like state, loosely Packed less ordered structure, known as the liquid state. This occurs when the characteristic temperature of the phase transition and results in increased permeability to ions, sugars and medicines.

In addition to temperature, the permeability of liposomes may change when exposed proteins. Some soluble proteins, such as cytochrome C, contact, deform and penetrate into the bilayer, thereby causing changes in permeability. Cholesterol inhibits the specified penetration of proteins, presumably due to more dense packing of phospholipids. It is assumed that most suitable for the delivery of antibiotics and inhibitors liposomal education will contain cholesterol.

the Ability to pick different solute various different types of liposomes. For example, MLV moderately effective in the capture of dissolved substances, and SUV's extremely inefficient. However, SUV's provide advantage in homogeneity and reproducibility in the distribution by size, and a compromise between size and catching ability to provide large single-layer vesicles (LUV). They are obtained by evaporating the ether, and they are three to four times more effective in capturing than MLV.

In addition to the characteristics of liposomes important determining factor in the capture compounds are physico-chemical properties of the compounds. Polar compounds are captured in the space containing water and non-polar compounds are associated with the lipid Balaam bubble. Polar compounds are released, penetrate through the bilayer, or in the case when the bilayer is broken, and nonpolar compounds remain in the bilayer until then, until it is destroyed by heat or impact of lipoproteins. Both compounds show maximum speed output when the temperature of the phase transition.

Liposomes interact with cells via four different mechanisms: endocytosis faguoqitirute cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic forces, or e is khreschatikska forces, or by specific interactions with components of the cell surface; fusion with the plasma membrane of cells by injection of a lipid bilayer of the liposome into the plasma membrane, with simultaneous release of the contents of liposomes in the cytoplasm; and by transfer of lipids liposomes in the cellular or subcellular membranes, or Vice versa, without any content linking liposomes. It is often difficult to determine which mechanism operates, and at the same time can operate more than one mechanism.

The fate and location of intravenously injected liposomes depend on their physical properties such as size, fluidity and surface charge. They can persist in the tissues within hours or days, depending on their composition, and the half-life in blood is in the range from min to several hours. Large liposomes, such as MLV and LUV quickly captured faguoqitirute cells of the reticuloendothelial system, but the physiology of the circulation system limits the existence of such large species of particles in most places. They can only exist in places where there are large holes or pores in the endothelium of capillaries, such as sinusoids of the liver or spleen. Thus, these bodies are the predominant places of capture. On the other hand, SUV show Bo is its wide tissue distribution, but still at a high level are retained in the liver and spleen. In General, this behavior in vivo limits the possibility of targeted delivery of liposomes to target only the specified organs and tissues available for liposomes large size. Organs and tissues include blood, liver, spleen, bone marrow and lymphoid organs.

Targeted delivery to the target, in General, is not a limitation for the present invention. However, if you should require specific delivery to target, there are ways to do that. You can use antibodies to bind them to the surface of liposomes and direct the antibody and the content presented drug to specific antigenic receptors, localized on the surface of specific cell types. Carbohydrate determinants (glycoprotein or glycolipid components of the cell surface that play a role in intercellular recognition, interaction and adhesion) can also be used as recognition sites, as they have the potential to direct liposomes to a specific cell type. Mainly, it is assumed that can be used intravenous drugs liposomes, but also other possible ways of introduction.

Alternative the invention relates to pharmaceutically acceptable compositions of nanok the purs-based compositions according to this invention. Usually nanocapsules can capture compounds are stable and reproducible way (Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998; Douglas et al., 1987). To avoid side effects due to intracellular overload polymers, such ultra-fine particles (sized around 0.1 ám) should be designed using polymers able to be degraded in vivo. For use in this invention are available biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements. Such particles can be easily obtained as described (Couvreur et al., 1980; 1988; zur Muhlen et al., 1998; Zambaux et al.,1998; Pinto-Alphandry et al., 1995, and U.S. patent 5145684, specifically incorporated herein by reference in full).

IMMUNOGENIC COMPOSITIONS.

In some embodiments of the present invention provided immunogenic composition or vaccine. Immunogenic compositions typically will include one or more pharmaceutical compositions, such as discussed above, in combination with an immunostimulant. Immune stimulator can be any substance that increases or enhances the immune response mediated by antibodies and/or cells) to an exogenous antigen. Examples of Immunostimulants include adjuvants, biodegradable microspheres (e.g., polylacticacid) and liposomes (which include the connection; the show is, for example, Fullerton, U.S. patent No. 4235877). Getting vaccines mainly described, for example, in M.F. Powell and M.J. Newman, eds., "Vaccine Design (the subunit and adjuvant approach)," Plenum Press (NY, 1995). Pharmaceutical compositions and immunogenic compositions within this invention may also contain other compounds, which may be biologically active or inactive. For example, the composition may contain one or more pieces of other tumor antigens, either incorporated in a hybrid polypeptide, either in the form of a separate connection.

Illustrative immunogenic compositions may contain DNA encoding one or more polypeptides as described above, so that the polypeptide produced in situ. As described above, the DNA may be present in any of a variety of delivery systems known to specialists in this field, including systems for the expression of nucleic acids, bacteria and virus expressing system. In this area there are many methods of gene delivery, such as the methods described Rolland, Crit. Rev. Therap. Drug Carrier Systems 15: 143-198, 1998) and cited in this work references. Suitable expression system, a nucleic acid contain necessary for expression in the patient's DNA sequence (such as a suitable promoter and signal termination). Bacterial delivery systems involve the introduction of bacteria (takoyaki Bacillus-Calmette-Guerrin), which expresses the immunogenic portion of the polypeptide on its surface or secretes such an epitope. In a preferred embodiment, DNA can be entered using virus expressing the system (e.g., vaccinia virus or other poxvirus, retrovirus, or adenovirus), which may include the use of non-pathogenic (defective), competent for replication of the virus. Suitable systems are described, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86: 317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569: 86-103, 1989; Flexner et al., Vaccine 8: 17-21, 1990; U.S. patent No. 4603112, 4769330 and 5017487; WO 89/01973; U.S. patent No. 4777127; GB 2200651; EP 0345242; WO 91/02805; Berkner, Biotechniques 6: 616-627, 1988; Rosenfeld et al., Science 252: 431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91: 215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90: 11498-11502, 1993; Guzman et al. Circulation 88: 2838-2848, 1993; Guzman et al., Cir. Res. 73: 1202-1207, 1993. The methods include DNA expressing such systems are well known to specialists in this field. DNA can also be "naked," as described, for example, in Ulmer et al., Science 259: 1745-1749, 1993, and in the review of Cohen, Science 259: 1691-1692, 1993. The capture of "naked" DNA can be increased by coating the DNA biodegradable beads, which are efficiently transported into the cells. It will be obvious that the immunogenic composition may contain polynucleotide and polypeptide component. Such immunogenic compositions can provide enhanced immune response.

It will be obvious that the immunogenic composition may contain pharmaceutically acceptable salt provided here polynucleotides and polypeptides. Such salts can be obtained from pharmaceutically acceptable non-toxic bases, including organic bases (for example, salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (for example, salts of sodium, potassium, lithium, ammonium, calcium and magnesium).

Despite the fact that in the compositions according to the invention can use any suitable carrier known to specialists in this field, the type of carrier will vary depending on the method of introduction. It is possible to prepare compositions according to this invention for any suitable route of administration, including, for example, local, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably contains water, saline, alcohol, a fat, a wax or a buffer. For oral administration, you can use any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, saccharin sodium, talc, cellulose, glucose, sucrose, and magnesium carbonate. As carriers for the pharmaceutical compositions according to this invention can also be used biodegradable microspheres (e.g., polylactate-polyglycolide). Suitable bid gradirei microspheres described, for example, in U.S. patent No. 4897268; 5075109; 5928647; 5811128; 5820883; 5853763; 5814344 and 5942252. You can also use the media containing the complexes of particles with proteins, are described in U.S. patent 5928647, which is able to induce in the host responses of cytotoxic T lymphocytes restricted class I.

Such compositions may also contain buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g. glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostatic means, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), dissolved substances, which render the composition isotonic, gipotonichnaya or weakly hypertonic with respect to the recipient's blood, suspendresume agents, thickening agents and/or preservatives. Alternate compositions of this invention can be prepared in the form of a lyophilisate. Connections can also be encapsulated in liposomes, using well known methods.

In immunogenic compositions according to this invention can use any of a variety of Immunostimulants. For example, you can include adjuvant. Most adjuvants contain a substance designed to protect the antigen from rapid is th catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid And proteins derived from Bortadella pertussis or Mycobacterium tuberculosis. Suitable adjuvants are available for purchase, such as incomplete adjuvant's adjuvant and complete adjuvant's adjuvant (Difco Laboratories, Detroit, MI); Merck adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts such as gel aluminum hydroxide (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cation or anion derivateservlet polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl-lipid a and the derived saponin "quil A". As adjuvants can also be used cytokines, such as GM-CSF or interleukin-2, -7, or-12.

In this immunogenic compositions of the adjuvant composition is preferably designed to induce an immune response predominantly Th1-type. High levels of cytokines Th1-type (e.g., IFN-γ, TNFα, IL-2 and IL-12) favor the induction mediated by cells of the immune response to the administered antigen. In contrast, high levels of cytokines of the Th2 type (for example, IL-4, IL-5, IL-6 and IL-10) favor the induction of humoral immune responses. After application as provided herein, immunogenic compositions from PAC is enta will be supported immune response, which includes the responses of Th1 - and Th2-type. In a preferred embodiment, in which the response is primarily a Th1 response-type, the level of cytokines by Th1-type will increase to a greater extent than the level of Th2 cytokines. The levels of these cytokines can be easily estimated by standard tests. For an overview of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7: 145-173, 1989.

Preferred adjuvants used in order to cause a predominantly Th1 response-type include, for example, a combination of monophosphoryl-lipid A, preferably 3-de-O-acylated monophosphoryl-lipid A (3D-MPL)together with an aluminum salt. Adjuvants MPL available for purchase from Corixa Corporation (Seattle, WA; see U.S. patent No. 4436727, 4877611, 4866034 and 4912094). Oligonucleotides containing CpG (in which the CpG dinucleotide is not methylated), also predominantly induce a Th1 response. Such oligonucleotides are well known and described, for example, in WO 96/02555, WO 99/33488 and U.S. patent No. 6008200 and 5856462. Also described immunostimulatory DNA sequences, for example, Sato et al., Science 273: 352, 1996. Another preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, MA), which can be used separately or in combination with other adjuvants. For example, enhanced system involves the combination monophosphoryl-lipid a and the derived saponin, such as is ombinatio QS21 and 3D-MPL, as described in WO 94/00153, or a less reactogenic composition where the QS21 quenched with cholesterol, as described in WO 96/33739. Other preferred compositions contain an emulsion of the type oil-in-water and tocopherol. A particularly effective composition adjuvant comprising QS21, 3D-MPL and tocopherol in the emulsion of the type oil-in-water, is described in WO 95/17210.

Other preferred adjuvants include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOM (CSL), MF-59 (Chiron), a series of adjuvants SBAS (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other 4-phosphates of aminoalkylphosphonic (AGP), such as described in pending applications for the grant U.S. patent No. 08/853826 and 09/074720, materials which are incorporated herein by reference in full.

Any provided here immunogenic composition can be prepared using known methods, which are combination of antigen amplifier of the immune response and a suitable carrier or excipient. Described herein compositions can be entered as part of the composition of long-term releases (i.e. such compositions as capsule, sponge or gel (consisting of, e.g., polysaccharides), which provides slow release of the compound after administration). Such compositions typically can sentence is o be catching using well known techniques (see, for example, Coombes et al., Vaccine 14: 1429-1438, 1996) and enter, for example, oral, rectal or subcutaneous implantation, or by implantation into the required target. The slow release composition may include a polypeptide, polynucleotide or antibody dispersed in the matrix of the carrier and/or enclosed in a container surrounded by a membrane that controls the speed.

The media used in such compositions are biocompatible, and may also be biodegradable; preferably the composition provides a relatively constant release rate of the active component. Such media include microparticles of a copolymer of poly(lactide-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other slow release carriers include molecular Biofactory that contain non-liquid hydrophilic core (e.g., cross-cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see, for example, U.S. patent No. 5151254 and PCT application WO 94/20078, WO 94/23701 and WO 96/06638). The number of active compounds contained in the composition of extended release depends on the location of implantation, the rate and expected duration of release and the nature of the condition to be treated or p is ecotricity.

In the pharmaceutical compositions and immunogenic compositions you can use any of a variety of media for delivery to facilitate the production of antigen-specific immune response that targets which are tumor cells. Carriers for delivery include antigen-presenting cells (APCS)such as dendritic cells, macrophages, b cells, monocytes and other cells, which can be constructed so that they are effective APK. Such cells may, but need not, genetically modified to increase the capacity for presenting the antigen, to improve activation and/or save the response of T cells to the cells themselves possessed antitumor effect and/or were immunologically compatible with the recipient (i.e. the same HLA haplotype). Usually APK you can select from any of a variety of biological fluids or organs, including tumor or perioperative tissue, and they can be autologous, allogeneic, syngeneic or xenogeneic cells.

In some preferred embodiments of the present invention as antigen-presenting cells using dendritic cells or their precursors. Dendritic cells are highly effective agriculture (Banchereau and Steinman, Nature 392: 245-251, 1998), and it was shown that they are effective as a physiological what about adjuvant, calling prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50: 507-529, 1999). In General, dendritic cells can be identified based on their typical shape (a star in situ with marked cytoplasmic processes (dendrites), visible in vitro), their ability with high efficiency to capture, processional and presentation of antigens, and their ability to activate native T-cell responses. Of course, dendritic cells can be constructed so that they are expressed in a specific cell surface receptors or ligands that are not normally detected in dendritic cells in vivo or ex vivo, and such modified dendritic cells provided by this invention. Alternatively, dendritic cells in the immunogenic compositions can be used secreted vesicles loaded with antigen dendritic cells (called ectosomal) (see Zitvogel et al., Nature Med. 4: 594-600, 1998).

Dendritic cells and the precursors can be obtained from peripheral blood, bone marrow, cells, infiltra tumor cells, infiltra perioperative tissue, lymph nodes, spleen, skin, umbilical cord blood, or any other suitable tissue or fluid. For example, the differentiation of dendritic cells can be performed ex vivo by adding com is Inacio such cytokines, as GM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested from peripheral blood. Alternative CD34-positive cells collected from peripheral blood, cord blood or bone marrow can be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3, and/or other(s) connection(s), which induces differentiation, maturation and proliferation of dendritic cells.

Dendritic cells conveniently be divided into "immature" and "Mature" cells, which provides a simple way of recognition of two well-characterized phenotypes. However, you cannot assume that this terminology eliminates all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for capturing and processing of antigen, which correlates with high expression of the receptor Fcγ and the mannose receptor. The Mature phenotype is typically characterized by a lower expression of these markers, but a high expression of surface molecules of the cells responsible for the activation of T cells, such as molecules MHC class I and II, adhesion molecules (e.g., CD54 and CD11) and co-stimulating molecules (such as CD40, CD80, CD86 and 4-1BB).

Usually APK can be transliterate polynucleotide, encoding a protein of breast cancer (or part of it or others is another option) to the polypeptide of breast cancer or immunogenic part expressionlist on the cell surface. Such transfection may take place ex vivo, and composition containing such transfetsirovannyh cells can then be used for therapeutic purposes, as described here. Alternative patient, you can enter the carrier for delivery of genes whose target is dendritic or other antigen-presenting cell, resulting in obtaining the transfection, which occurs in vivo. Transfection of dendritic cells in vivo and ex vivo, as a rule, you can, for example, be performed using any method known in this field, such as the methods described in WO 97/24447, or the approach of gene gun"as described by Mahvi et al., Immunology and Cell Biology 75: 456-460, 1997. Antigen loading of dendritic cells may be achieved incubare dendritic cells or precursor cells with a polypeptide of breast cancer, DNA (naked or within a plasmid vector) or RNA; or with expressing the antigen recombinant bacterium or viruses (e.g., vectors based on vaccinia virus, poxvirus birds, adenovirus or lentivirus). Before loading, the peptide can be covalently konjugierte with an immunological partner who provides assistance T cells (for example, a molecule of media). An alternative to dendritic cell can and pulse impact unconjugated immunological partner, alone or in the presence of the polypeptide.

Immunogenic compositions and pharmaceutical compositions can be presented in the tanks containing the standard dose or multiple doses, such as sealed ampoules or vials. Such tanks are preferably hermetically sealed to preserve sterility of the composition until use. In General, the composition can be stored in the form of suspensions, solutions or emulsions in oily or aqueous fillers. Alternative immunogenic or pharmaceutical composition can be stored in dried by freezing condition requiring only the addition of sterile liquid carrier immediately prior to use.

THERAPY OF MALIGNANT TUMORS.

The following aspects of the invention described herein, the composition can be used for immunotherapy of malignant tumors, such as breast cancer. In this way the patient is usually administered pharmaceutical compositions and immunogenic compositions. In the sense used here, a "patient" refers to any warm-blooded animal, preferably a human. The patient may be affected or not affected by a malignant tumor. Accordingly, the above pharmaceutical compositions and immunogenic compositions can be used to prevent the development of malignant the first tumor or to treat the patient, affected by a malignant tumor. Cancer can be diagnosed using the criteria generally adopted in this area, including the presence of a malignant tumor. Pharmaceutical compositions and immunogenic compositions can be entered either before or after surgical removal of primary tumors and/or treatment, such as radiation therapy or introduction traditional chemotherapeutic drugs. The introduction can be accomplished in any suitable way, including intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal, anal, vaginal, local or oral routes. In some embodiments, immunotherapy may be an active immunotherapy, in which treatment is based on stimulation of the in vivo response of the endogenous immune system of the host against the tumor by administering agents that modify the immune response (such as presented here polypeptides and polynucleotide).

In other embodiments, immunotherapy may be a passive immunotherapy, in which treatment involves the delivery of agents with established immunoreactivity in relation to the tumor (such as effector cells or antibodies)that can directly or indirectly mediate antitumor activity and not necessarily C is hanging from the intact immune system of the host. Examples of effector cells include T-cells, which were discussed above, T lymphocytes (such as cytotoxic T-lymphocytes CD8+and ingeltrude tumor T-helper lymphocytes CD4+), killer cells (such as natural killer cells and activated by lymphokines killer cells), b cells and antigen-presenting cells (such as dendritic cells and macrophages)expressing the polypeptide. Receptors of T cells and receptors of antibodies specific for these polypeptides, it is possible to clone, Express and move into other vectors or effector cells for adoptive immunotherapy. The polypeptides can also be used to generate antibodies or antiidiotypic antibodies (as described above and in U.S. patent No. 4918164) for passive immunotherapy.

Effector cells can usually be obtained in sufficient quantities for adoptive immunotherapy cultivation in vitro, as described here. The cultivation conditions for reproduction of individual antigen-specific effector cells in an amount up to several billion, with retention of antigen recognition in vivo are well known in this field. In such culture conditions in vitro usually use periodic stimulation by antigen, often in the presence of cytokines (still is how IL-2) and non-dividing feeder cells. As indicated above, is presented here immunoreactive polypeptides can be used to quickly reproduce the culture of antigen-specific T-cells in order to obtain a sufficient number of cells for immunotherapy. In particular, antigen-presenting cells such as dendritic cells, macrophages, monocytes, fibroblasts and/or b-cells, pulse can be exposed to immunoreactive polypeptides, or transliterate one or more polynucleotide using standard methods, well known in this field. For example, antigen-presenting cells can be transliterate polynucleotides with the promoter, suitable to increase expression in a recombinant virus or other expressing the system. Cultured effector cells for use in therapy must have the ability to grow and to spread widely and to survive for long periods of time in vivo. Studies have shown that cultured effector cells can be induced to grow in vivo and survival over a long period of time in large quantities by repeated stimulation with antigen by adding IL-2 (see, for example, Cheever et al., Immunological Reviews 157: 177, 1997).

Alternative vector expressing this polypeptide can be introduced into the antigen-presenting cells taken from the patient, and clonal propagated ex vivo for transplantation back to the same patient. Transfetsirovannyh cells can re-enter the patient using any of the methods known in this field, preferably in sterile form by intravenous, intracavitary, intraperitoneal or intratumoral injection.

Route and frequency of introduction described here therapeutic compositions and dosage will vary from person to person, and can be easily installed using standard methods. In General, pharmaceutical compositions and immunogenic compositions can be introduced through the injection (e.g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Preferably you can enter from 1 to 10 doses during the 52-week period. Preferably introduced 6 doses at intervals of 1 month and after that periodically can be a booster vaccination. For individual patients may be appropriate alternative protocols. A suitable dose is an amount of compound that, when introduced, as described above, is capable of stimulating an antitumor immune response and, at least 10-50% above the basic (i.e. untreated) level. Such response can be monitored by measuring tumor antibodies in a patient, Ileana basis dependent on vaccine production of cytolytic effector cells, able to kill the tumor cells of the patient in vitro. Such immunogenic compositions must also possess the ability to induce an immune response, which leads to improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer survival without signs of disease) in patients who had treatment, compared with patients for whom treatment was not carried out. In General, for pharmaceutical compositions and immunogenic compositions containing one or more polypeptides, the amount of each polypeptide is in the range of doses from about 25 μg to 5 mg per kg of body weight of the host. Appropriate values of doses will vary with a change in the weight of the patient, but will typically be in the range from about 0.1 ml to about 5 ml

In General, an appropriate dosage and treatment regimen provides the active compound(s) in a quantity sufficient to bring therapeutic and/or prophylactic benefit. This response can be observed when establishing improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer survival without signs of disease) in patients subjected to treatment, compared with patients who were not treated. Strengthening of preexisting immune responses to a protein of the tumor in the breast is usually corralero is with improved clinical outcome. Such immune responses are usually estimated using standard assays of proliferation, cytotoxicity or cytokine that can be performed using samples obtained from the patient before and after treatment.

DETECTION AND DIAGNOSIS OF MALIGNANT TUMORS.

In General, cancer patients can be detected based on the presence of one or more proteins of breast cancer and/or polynucleotides encoding such proteins in a biological sample (e.g. blood, serum, sputum, urine and/or biopsies of tumors), obtained from the patient. In other words, such proteins can be used as markers to indicate the presence or absence of malignant tumors, such as breast cancer. In addition, such proteins can be used to identify other types of malignant tumors. Presented here linking agents usually help to identify the level of antigen that is associated with the agent in the biological sample. Polynucleotide primers and probes can be used to determine the level of mRNA encoding the tumor protein, which is also an indicator of the presence or absence of a malignant tumor. In General, the sequence of a tumor of the breast should be at a level that is at least three times higher in tumors of the eve of the fabric, than in normal tissue.

There are many varieties of analysis, known to experts in the field, using a binding agent to detect polypeptide markers in a sample. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In General, the presence or absence of a malignant tumor in a patient can be detected by: (a) contact a biological sample obtained from a patient with a binding agent; (b) determining in the sample a level of polypeptide that binds to the binding agent; and (C) comparing the level of polypeptide with a pre-defined cut-off cut-off value.

In a preferred embodiment, the analysis includes the use of binding agent immobilized on a solid medium in order to bind and remove the polypeptide from the rest of the sample. Then associated polypeptide can be detected using a reagent for detection, which contains a reporter group, and specific binds to the complex-binding agent/polypeptide. Such reagents for detection may include, for example, a binding agent that is specific binds to the polypeptide or antibody or other agent that is associated with specific binding agent, such as anti-immunoglobulin, protein G, protein a or a lectin. Viola is rnative you can use competitive analysis, wherein the polypeptide have been labelled with a reporter group and give the opportunity to contact the immobilized binding agent after incubation of the binding agent with the sample. The degree to which connection of the sample inhibit the binding of the labeled polypeptide with a binding agent, is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include proteins of the tumor in the breast is full length, and parts thereof, which binds to the binding agent, as described above.

A solid carrier can be any material known to specialists in this field, which can bind the protein of the tumor. For example, a solid carrier can be experienced well in the tablet to micrometrology or nitrocellulose or other suitable membrane. Alternative media can be a bead or disc, such as a carrier made of glass, fiberglass, latex or a plastic, such as polystyrene or polyvinyl chloride. The carrier may also be a magnetic particle or a fiber-optical sensor, such as sensors, are described, for example, in U.S. patent No. 5359681. The binding agent may be immobilized on a solid medium using a variety of methods known to experts in this field, which is described in detail in the patent and scientific literature In the context of this invention, the term "immobilization" refers to both noncovalent Association, such as adsorption, and covalent binding (which may represent the formation of a direct communication between the agent and the functional groups of the carrier, or may be linking with a cross-linking agent). The preferred immobilization by adsorption in the hole of the tablet for micrometrology or on the membrane. In such cases, adsorption may be achieved by providing the contact linking agent in a suitable buffer with a solid carrier for the appropriate amount of time. The contact time varies with temperature, but is typically between about 1 hour and 1 day. In General, the contact hole plastic tablet for micrometrology (such as polystyrene or polyvinylchloride) with an amount of binding agent in the range from about 10 ng to about 10 μg, and preferably from about 100 ng to 1 μg, is sufficient to mobilitat an adequate amount of binding agent.

Covalent binding of a binding agent with a solid carrier, as a rule, can be done first by reaction of the carrier with a bifunctional reagent that will react with both the carrier and the functional group such as hydroxyl or amino group in the binding agent. For example, the binding agent can covalently bind with the media is, having an appropriate polymer coating using benzoquinone or by condensation of the aldehyde group of media with an amine and an active hydrogen binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).

In some embodiments, the analysis is the analysis with the two antibodies type "sandwich". The specified analysis can be performed, providing a first contact of the antibody that has been immobilized on a solid carrier, usually in the hole of the tablet for micrometrology, sample, so as to enable the polypeptides in the sample contacted with the immobilized antibody. Then the unbound sample is removed from the immobilized complexes of the polypeptide-antibody and add detection reagent (preferably a second antibody that is able to contact the other site of the polypeptide)that contains a reporter group. Then determine the number of the detecting agent, which remains associated with the solid media using the appropriate method for a specific reporter groups.

More specifically, after the antibody immobilized on the media, as described above, the remaining protein binding sites on the carrier usually block any suitable blocking agent known to specialists in this field, such as bovine serum albumin or tween-20TM(Sigma Chemical Co., St Louis, MO). Then the immobilized antibody is incubated with the sample and allow the polypeptide to contact with the antibody. The sample before incubation can be diluted with a suitable diluent such as phosphate buffered saline (PBS). In General, an appropriate contact time (i.e., the incubation time) is a period of time sufficient to detect the presence of the polypeptide in the sample obtained from the individual with breast cancer. Preferably the contact time is sufficient to achieve a level of binding that is at least approximately 95% of the level achieved at equilibrium between bound and unbound polypeptide. Specialists in this field will understand that the time required to reach equilibrium, which can be determined by analyzing the level of binding that occurs over a period of time. At room temperature is usually sufficient incubation time of about 30 minutes.

Then unbound sample can be removed by washing the solid media appropriate buffer, such as PBS containing 0.1% Tween-20TM. Then to solid media, you can add a second antibody that contains a reporter group. Preferred reporter groups include the groups listed above.

Then de is entirely reagent is incubated with the immobilized complex of the antibody-polypeptide over time, sufficient to detect the bound peptide. The relevant period of time can usually be determined by analyzing the level of binding that occurs within a certain period of time. Then unbound detection reagent was removed and the bound detection reagent determined using reporter group. Used to detect reporter group method depends on the nature of the reporter group. For radioactive groups usually suitable ways scintillation counting or autoradiography. Spectroscopic methods can be used for detection of dyes, luminescent groups and fluorescent groups. Biotin can be detected using avidin associated with a different reporter group (usually radioactive or fluorescent group or an enzyme). Enzyme reporter groups can usually be detected by adding a substrate (usually within a certain period of time), followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of malignant tumors, such as breast cancer, the signal detected from the reporter group that remains bound to the solid carrier, usually compared to a signal corresponding to a predefined cut-off (cut-off) value. One before Occitania variant cut-off value for the detection of malignant tumors is the average value of the signal obtained in the case when the immobilized antibody is incubated with samples from patients without malignancy. In General, a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive in relation to malignant tumors. In an alternative preferred embodiment, the cut-off value is determined using the operating characteristic curve according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value can be determined on the basis of the schedule of pairs of true positive assessments (i.e. sensitivity) and false-positive assessments (100%-specificity)that correspond to each possible cut-off value for the diagnostic test. Cut-off value on the chart that is closest to the top left corner (i.e. the value that covers the largest area)is the most accurate cut-off (cut-off) value, and a sample generating a signal that is higher than the cut-off value determined by this method can be considered positive. Alternative cut-off value can be shifted to the left along the graph to minimize false positive assessment, or right, in order to minimize false negative is the preliminary assessment. In General, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive in relation to malignant tumors.

In a related embodiment, the analysis is carried out in a test in flow mode or test strip strips where the binding agent is immobilized on the membrane, such as nitrocellulose. In running the test polypeptides in the sample bound to the immobilized binding agent as the sample passes through the membrane. Then the second labeled binding agent binds to the complex, the binding agent is a polypeptide as a solution containing the second binding agent flows through the membrane. Then you can spend the detection of the second binding agent, as described above. In the embodiment, test strip strip one end of the membrane, which is associated binding agent, immersed in a solution containing the sample. The sample migrates along the membrane through a region containing the second binding agent, and to the field of immobilized binding agent. The concentration of the second binding agent in the area of immobilized antibody indicates the presence of a malignant tumor. Typically, the concentration of the second binding agent in this place creates an image such as a line that can be observed visually. The absence of such a figure is NCA indicates a negative result. In General, the amount of binding agent immobilized on the membrane are chosen so that there were visible figure in the case, when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the composite analysis with the two antibodies discussed above in the form. Preferred binding agents for use in such assays are antibodies and antigen-binding fragments. Preferably the amount of antibody immobilized on the membrane is in the range of from about 25 ng to 1 μg, and more preferably from about 50 ng to 500 ng. These tests can usually be performed with very small amounts of biological sample.

Of course, there are many other protocols that are suitable for use with tumor proteins or binding agents according to this invention. The above descriptions are intended only as examples. For example, for professionals in this field will be obvious that the above protocols can be easily modified for use with the polypeptides of breast cancer, to identify antibodies that bind to such polypeptides in a biological sample. The detection of these antibodies specific for the protein of breast cancer, can short airavat with the presence of malignant tumors.

A malignant tumor also or alternatively, be detected based on the presence of T cells that react with specific proteins of breast cancer in a biological sample. In some methods, the biological sample containing CD4+and/or CD8+T-cells isolated from a patient is incubated with a polypeptide of breast cancer, polynucleotides, encoding such a polypeptide and/or an APC that expresses at least immunogenic portion of such a polypeptide, and identifying the presence or absence of specific activation of T cells. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells can be distinguished from the body of the patient by conventional means (such as by centrifugation of peripheral blood lymphocytes in density gradient Picola/Vipaka). T-cells can be incubated with the polypeptide (for example, 5-25 μg/ml) in vitro for 2-9 days (typically 4 days) at 370C. May require incubation of another aliquot of the sample T-cells in the absence of the polypeptide of breast cancer, which serves as a control. For T-cells CD4+the activation is preferably determined by assessing the proliferation of T-cells. For t cells, CD8+the activation is preferably determined by the assessment of cytolytic activity. Ur the level of proliferation, which is at least two times higher, and/or the level of cytolytic activity that is at least 20% higher than in patients without signs of disease, indicates the presence of a malignant tumor in a patient.

As indicated above, a malignant tumor can also, or alternatively, be detected based on the level of mRNA that encodes a protein of breast cancer in the biological sample. For example, you can use at least two oligonucleotide primers in the analysis based on polymerase chain reaction (PCR)to amplify part of the cDNA of breast cancer obtained from a biological sample, with at least one of the oligonucleotide primers is specific for polynucleotide (i.e. hybridizes with him), codereuse protein of breast cancer. Then amplified cDNA was separated and determined using methods well known in the field, such as gel electrophoresis. Similarly, oligonucleotide probes that are specific hybridize with polynucleotides, encoding a protein of breast cancer, can be used in hybridization analysis to detect the presence of polynucleotide encoding the tumor protein in a biological sample.

To allow hybridization under conditions of analysis of oligonucleotide p is aimery and probes must contain oligonucleotide sequence, which, at least about 60%, preferably at least about 75% and more preferably at least about 90% identical to part of polynucleotide encoding a protein of breast cancer, which has a length of at least 10 nucleotides, and preferably at least 20 nucleotides. Preferably the oligonucleotide primers and/or probes hybridize with polynucleotides coding for this polypeptide, under conditions of medium hardness, which is defined above. Oligonucleotide primers and/or probes that can be used in this diagnostic methods, preferably have a length of at least 10-40 nucleotides. In a preferred embodiment, the oligonucleotide primers contain at least 10 continuous consecutive nucleotides, more preferably at least 15 continuous consecutive nucleotides of the DNA molecule having the sequence specified in SEQ ID NO: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486 and 489. Methods of carrying out tests based on PCR and hybridization assays are well known in the art (see, for example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263, 1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989).

In one preferred analysis using RT-PCR, where PCR is used together with education is Noah transcription. Typically, RNA is extracted from a biological sample, such as tissue biopsy, and back transcribers to obtain cDNA molecules. PCR amplification using at least one specific primer provides a cDNA molecule, which can be separated and visualized, for example, using gel electrophoresis. Amplification can be performed on biological samples taken from the test patient and the individual who is not affected by a malignant tumor. Reaction amplification can be performed at multiple dilutions of cDNA, spanning two orders of magnitude values. A twofold or greater increase in expression in several dilutions of the sample of the patient being tested compared to the same dilutions non-cancerous sample is usually considered positive.

In another embodiment, compositions described herein can be used as markers of progression of malignant tumors. In this embodiment, assays as described above for the diagnosis of malignant tumors, can be completed within a certain period of time and to evaluate the change in the level of reactive polypeptide(Dov) or polynucleotide(Dov). For example, you can analyze every 24 to 72 hours over a period of time from 6 months to 1 year and after that to perform as needed. In General, cancer is what I am progressing in those patients in which the detected level of the polypeptide or polynucleotide increases over time. In contrast, a malignant tumor is not progressing when the level of reactive polypeptide or polynucleotide either remains constant or decreases with time.

Some diagnostic tests in vivo can be performed directly on the tumor. One such analysis includes the implementation of contact of tumor cells with a binding agent. Associated binding agent can then be detected directly or indirectly by means of a reporter group. Such binding agents can also be used for histological applications. An alternative for such applications can be used polynucleotide probes.

As described above, to improve sensitivity, the sample can be analyzed various protein markers of breast cancer. It will be obvious that the binding agents specific for different proteins presented here can be combined in a single analysis. In addition, you agreed to use multiple primers or probes. Choice of protein tumor marker may be based on routine experiments to determine combinations that results in optimal sensitivity. Additional or alternative analysis is s presented here proteins of the tumor can be combined with analyses of other known tumor antigens.

DIAGNOSTIC KITS.

This invention also relates to kits for use in any of the above diagnostic method. These kits usually contain two or more components necessary to perform diagnostic analysis. Components may be compounds, reagents, containers and/or equipment. For example, one container with the kit may contain a monoclonal antibody or fragment that is specific contacts with the protein of breast cancer. Such antibodies or fragments can be provided associated with material media, as described above. One or more additional containers may contain elements, such as reagents or buffers for use in the analysis. Such kits may also or alternatively contain the detecting reagent, as described above, which contains a reporter group suitable for direct or indirect detection of antibody binding sites.

An alternative set may be designed to determine the level of mRNA that encodes a protein of breast cancer in the biological sample. These kits usually contain at least one oligonucleotide probe or primer, as described above, which hybridizes with polynucleotide, encoding a protein of breast cancer. This oligonucleotide can be, for example, be used in PCR or hybridization analysis. Additional components that may be present within such kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of polynucleotide encoding a protein of breast cancer. The following examples are offered to illustrate but not to limit.

EXAMPLE 1.

Isolation AND CHARACTERIZATION of POLYPEPTIDE

TUMORS OF THE MAMMARY GLAND.

This example describes the selection of polypeptides of breast cancer from a cDNA library of breast cancer.

Subtracted cDNA library containing cDNA from breast tumors subtracted using the cDNA of the normal mammary gland, were designed as follows. Total RNA was extracted from primary tissues, using reagent Tryzol (Gibco BRL Life Technologies, Gaithersburg, MD)as described by the manufacturer. The poly+-RNA was purified using a column of oligo-dT-cellulose according to standard protocols. First strand cDNA was synthesized using primer supplied in the kit for cDNA subtraction with selective PCR Clontech (Clontech, Palo Alto, CA). Your DNA consisted of cDNA from two normal breast tissue, with the test DNA was from three primary breast tumors. Dunaeva cDNA was synthesized as in the case of the test, and in case your DNA and rasmala and a combination of endonucleases (MluI, MscI, PvuII, SalI and StuI), which recognize six base pairs of DNA. This modification significantly increased the average size of the cDNA in comparison with cDNA generated according to the Protocol of Clontech (Palo Alto, CA). Split testing cDNA ligated with two different adapters and subtraction were performed according to the Clontech Protocol. Subtracted cDNA was subjected to two rounds of PCR amplification, following the manufacturer's Protocol. The resulting PCR products were subcloned into the cloning vector THE pCRII (Invitrogen, San Diego, CA) and by electroporation was used to transform cells of E. coli ElectroMax DH10B (Gibco BRL Life Technologies). From independent clones were isolated DNA and sequenced using automated sequencing machine model A Perkin Elmer/Applied Biosystems Division (Foster City, CA).

In specific deducted for breast tumor cDNA library found sixty-three separate cDNA clone. Certain sequence of one strand cDNA (5' or 3') is presented in SEQ ID NO: 1-61, 72 and 73, respectively. Comparison of these cDNA sequences with known sequences in the gene Bank using databases EMBL and GenBank (release 97) did not reveal significant homology with the sequences shown in SEQ ID NO: 14, 21, 22, 27, 29, 30, 32, 38, 44, 45, 53, 72 and 73. Discovered that the sequence of SEQ ID NO: 1, 3, 16, 17, 34, 48, 57, 60 and 61 represent the known human genes. Found that pic is egovernance SEQ ID NO: 2, 4, 23, 39, and 50 show some similarity with previously identified genes are not humans, and other organisms. Discovered that the rest of the clones (SEQ ID nos: 5-13, 15, 18-20, 24-26, 28, 31, 33, 35-37, 40-43, 46, 47, 49, 51, 52, 54-56, 58 and 59) show at least some degree of homology with previously identified marker expressed sequences (EST).

To determine the levels of expression of mRNA isolated cDNA clones, randomly selected cDNA clones after the above-described subtraction in the case of the mammary gland and carried out PCR amplification of the colonies. Levels of mRNA expression of these clones in breast tumor, normal breast and various other normal tissues were determined using microarrays (Synteni, Palo Alto, CA). Briefly, the products of PCR amplification were placed on a glass slide in the form of a matrix, with each product took the only position in the matrix. mRNA was extracted from the tissue sample for testing, back transcribable and received fluorescently labeled cDNA probes. Chips were probed with labeled cDNA probes, the slides scanned and measured the fluorescence intensity. Data were analyzed using the computer program GEMTOOLS provided Synteni. Discovered that of the seventeen tested clone cDNA clone cDNA, shown is SEQ ID NO: 40, 46, 59 and 73, sverkhekspressiya in tumors of the breast, and at low levels expressibility all tested normal tissues (breast, PBMC, colon, fetal tissue, salivary gland, bone marrow, lung, large intestine, spinal cord, adrenal gland, kidney, liver, stomach, skeletal muscle, heart, small intestine, skin, brain and epithelial cells of the human mammary gland). Found that the clones of SEQ ID NO: 41 and 48 were sverkhekspressiya in tumors of the breast and expressibility at low levels in all other tested tissues except bone marrow. Found that the clone of SEQ ID NO: 42 was sverkhekspressiya in tumors of the breast and expressively at low levels in all other tested tissues except bone marrow and spinal cord. Found that the clone of SEQ ID NO: 43 was sverkhekspressiya in tumors of the breast and expressively at low levels in all other tested tissues, except the spinal cord, heart and small intestine. Found that the clone of SEQ ID NO: 51 was sverkhekspressiya in tumors of the breast and expressively at low levels in all other tested tissues, with the exception of the large intestine. Found that the clone of SEQ ID NO: 54 was sverkhekspressiya in tumors of the breast and expressively on low the levels in all other tested tissues, except PBMC, stomach and small intestine. Found that the clone of SEQ ID NO: 56 was sverkhekspressiya in tumors of the breast and expressively at low levels in all other tested tissues, with the exception of the colon and small intestine, the epithelial cells of the human mammary gland and mammary gland tumors, passaged in SCID mice. Found that the clone of SEQ ID NO: 60 sverkhekspressiya in tumors of the breast and expressively at low levels in all other tested tissues, except the spinal cord and heart. Found that the clone of SEQ ID NO: 61 was sverkhekspressiya in tumors of the breast and expressively at low levels in all other tested tissues, with the exception of the small intestine. Found that the clone of SEQ ID NO: 72 was sverkhekspressiya in tumors of the breast and expressively at low levels in all other tested tissues, with the exception of the colon and salivary glands.

The results of Northern blot analysis of clone SYN18C6 (SEQ ID NO: 40) is shown on the drawing. The calculated sequence of the protein encoded SYN18C6 presented in SEQ ID NO: 62.

Additional cDNA clones, which sverkhekspressiya in the tumor tissue of the breast, was isolated from a subtracted cDNA libraries breast cancer, as follows. Subtracted libraries breast cancer received as described you the e by PCR-based subtraction using cDNA pools of breast cancer as a test DNA or cDNA pools of normal mammary gland, or pools of cDNA from other normal tissues as your DNA. The cDNA clones from breast cancer after subtraction were selected randomly and carried out PCR amplification of the colonies, and determined the levels of expression of their mRNA in breast tumor, normal breast and various other normal tissues using microarrays described above. Found that twenty-four separate cDNA clone sverkhekspressiya in breast tumor and expressed at low levels in all tested normal tissues (breast, brain, liver, pancreas, lung, salivary gland, stomach, colon, kidney, bone marrow, skeletal muscle, PBMC, heart, small intestine, adrenal gland, spinal cord, large intestine and skin). Certain sequences of these cDNA clones represented in SEQ ID NO: 63-87. Comparison of the sequences of SEQ ID NO: 74-87 with sequences in the gene Bank as described above revealed homology with previously identified human genes. Revealed no significant homology with sequences SEQ ID NO: 63-73.

Three isoforms of DNA to clone B726P (incomplete sequence shown in SEQ ID NO: 71) were isolated as follows. Synthesized radioactive probe on the basis of B726P cutting DNA B726P from the vector pT7Blue (Novagen) by splitting what strictatime BamHI/XbaI and using the resulting DNA as matrix in the single-stranded PCR in the presence of [α -32P] dCTP. The sequence used primers for this PCR is presented in SEQ ID NO: 177. The resulting radioactive probe used to study the cDNA library obtained with directional primer, and cDNA library, obtained with a random primer created using RNA isolated from breast tumors. Eighty-five clones were identified, cut out, purified and sequenced. Found that, of these 85 three clones contain significant open reading frame. A specific sequence of cDNA isoforms B726P-20 represented in SEQ ID NO: 175, while the corresponding calculated amino acid sequence presented in SEQ ID NO: 176. A specific sequence of cDNA isoforms B726P-74 represented in SEQ ID NO: 178, and the corresponding calculated amino acid sequence presented in SEQ ID NO: 179. A specific sequence of cDNA isoforms B726P-79 presented in SEQ ID NO: 180 and the corresponding calculated amino acid sequence presented in SEQ ID NO: 181.

Attempt to get a full-sized clone of B726P using standard methods led to the isolation of five additional clones that represent additional 5'sequences B726P. These clones, apparently, are forms of alternative splicing and one that is of the same gene. Certain sequences of these cDNA clones are shown in SEQ ID NO: 464-468, and designed amino acid sequence encoded by SEQ ID NO: 464-467, respectively, are presented in SEQ ID NO: 470-473. Using standard software techniques, created a consensus DNA sequence with a length 3681 BP (SEQ ID NO: 463), which contains two large open reading frames. "Right ORF encodes the amino acid sequence of SEQ ID NO: 181. The calculated amino acid sequence encoded "left" ORF, provided in SEQ ID NO: 469. Subsequent studies led to the isolation of additional forms of splicing B726P, which has an insert size of 184 BP compared with other forms. The specified insert size of 184 BP causes a shift in the reading frame, which brings together the "left" and "right" ORF in one ORF, which has a length 1002 a/K. Specific cDNA sequence of the specified alternative splicing forms shown in SEQ ID NO: 474, and the corresponding amino acid sequence presented in SEQ ID NO: 475.

Further isolation of individual clones, which sverkhekspressiya in the tumor tissue of the mammary gland was performed using a technology library subtracted cDNA described above. In particular, in the specified screening used the library subtracted cDNA containing cDNA from tumor Molo is Noah gland, deducted using cDNA from five other normal tissues (brain, liver, PBMC, pancreatic cancer and normal mammary gland). Based on the initial subtracting a selected one hundred and seventy-seven clones for further characterization by DNA sequencing and analysis on microarrays. Analysis on microarrays showed that the sequence of SEQ ID NO: 182-251 and 479 expressibility in tumors of the breast of a person at a level two or more times the expression level in normal human tissues. Revealed no significant homology to nineteen of these clones, including SEQ ID NO: 185, 186, 194, 199, 205, 208, 211, 214-216, 219, 222, 226, 232, 236, 240, 241, 245, 246 and 479, except for certain previously identified marker expressed sequences (EST). The rest of the clones showed some homology to previously identified genes, in particular, SEQ ID NO: 181-184, 187-193, 195-198, 200-204, 206, 207, 209, 210, 212, 213, 217, 218, 220, 221, 223-225, 227-231, 233-235, 237-239, 242-244 and 247-251.

Discovered that one of the cDNA clones, isolated by subtraction-based PCR as described above (SEQ ID NO: 476; denoted B720P), which, as shown in the analysis on micrometric, sverkhekspressiya in tumors of the breast, identical to known gene keratin. The cDNA sequence of the full length known keratin gene represented in SEQ ID NO: 477, as appropriate to estoya amino acid sequence presented in SEQ ID NO: 478. On the basis of sequence SEQ ID NO: 477 created the primers used to clone the full length cDNA based on the mRNA, which was obtained from total RNA, giving a high level of expression B720P in the analysis of PCR in real-time. Products are then cloned and sequenced. A specific sequence of cDNA B720P full length represented in SEQ ID NO: 484, and the corresponding amino acid sequence presented in SEQ ID NO: 485.

In subsequent analyses identified a shortened form B720P (named B720P-tr) in carcinomas of the breast. The specified antigen cloned on the basis of mRNA obtained from total RNA tumor of the breast, which gave a high level of expression B720P-tr PCR analysis real-time. the mRNA used in order to create a pool of cDNA, which is then used as template for amplification by PCR of cDNA corresponding to B720P-tr. A specific sequence of cDNA B720P-tr are presented in SEQ ID NO: 486. B720P-tr has ORF size 708 nucleotides, which encodes a protein of 236 amino acids (SEQ ID NO: 487). The size of the transcript was confirmed by Northern blot analysis.

Seventy clones showing overexpression in tissues of breast tumors, fifteen showed particularly high levels of expression in breast tumors. who in normal human tissues. The following eleven clones did not show any significant homology with any known genes. Clone 19463.1 (SEQ ID NO: 185) sverkhekspressiya in most of the tested breast tumors and in breast tumors SCID (see example 2); in addition, overexpression was detected in most normal tissues of the breast. Clone 19483.1 (SEQ ID NO: 216) sverkhekspressiya in several breast tumors and not sverkhekspressiya in any of the tested normal tissues. Found that the clone 19470.1 (SEQ ID NO: 219) slightly sverkhekspressiya in some breast tumors. Found that the clone 19468.1 (SEQ ID NO: 222) slightly sverkhekspressiya in most of the tested breast tumors. Found that the clone 19505.1 (SEQ ID NO: 226) slightly sverkhekspressiya in 50% of breast tumors and in tissues of SCID tumors, with some degree of overexpression was detected in normal mammary gland. Found that the clone 1509.1 (SEQ ID NO: 232) sverkhekspressiya in a very few breast tumors, with some degree of overexpression was observed in tissues of metastatic tumors in the mammary gland, and also not found significant overexpression in normal tissues. Showed that clone 19513.1 (SEQ ID NO: 236) was slightly sverkhekspressiya in a few tumors of the s breast, in normal tissues did not reveal significant levels of overexpression. Clone 19575.1 (SEQ ID NO: 240) gave low level overexpression in some breast tumors, and normal mammary gland. Clone 19560.1 (SEQ ID NO: 241) sverkhekspressiya in 50% of tested breast tumors, as well as in some normal tissues of the breast. Clone 19583.1 (SEQ ID NO: 245) was slightly sverkhekspressiya in some breast tumors, normal tissues revealed very low levels of overexpression. Clone 19587.1 (SEQ ID NO: 246) showed low levels of overexpression in some breast tumors and did not give significant overexpression in normal tissues.

Found that the clone 19520.1 (SEQ ID NO: 233), showing homology to clone 102D24 chromosome 11q13.31, sverkhekspressiya in breast tumors and in tumors in SCID. Found that the clone 19517.1 (SEQ ID NO: 237), showing homology to clone M PAC man, slightly sverkhekspressiya in most of the tested breast tumors. Showed that clone 19392.2 (SEQ ID NO: 247), showing homology with chromosome 17 man, sverkhekspressiya in 50% of tested breast tumors. Showed that clone 19399.2 (SEQ ID NO: 250), showing homology with GSHB-184P14 YOU Her man, slightly sverkhekspressiya in a limited number of test is skilled breast tumors.

In subsequent studies have identified 64 individual clones from the subtracted cDNA library containing cDNA from a pool of breast tumors subtracted using cDNA from five normal tissues (brain, liver, PBMC, pancreatic cancer and normal mammary gland). Subtracted cDNA library was prepared as described above with the following modifications. To split cDNA instead of RsaI used a combination of five cutting six-nucleotide sites of enzymes (MluI, MscI, PvuII, SalI and StuI). The result was an increase in the average size of the insert from 300 BP to 600 BP 64 Selected clones were subjected to PCR amplification colonies and evaluated the levels of expression of their mRNA in the tumor tissue of the breast, normal breast and various other normal tissues using the technology of microarrays, as described above. Certain sequences of 11 cDNA clones that were found, were sverkhekspressiya in the tumor tissue of the breast, is presented in SEQ ID NO: 405-415. Comparison of these sequences with sequences published databases, as described above, revealed homology of the sequences SEQ ID NO: 408, 411, 413, and 414 and the previously selected EST. Found that the sequence of SEQ ID NO: 405-407, 409, 410, 412, and 415 showed some homology to previously identified sequences.

the following studies subtracted cDNA library were obtained on the basis of cDNA from metastatic tumors of the breast, deducted using pooled cDNA from five normal tissues (breast, brain, lung, pancreas and PBMC)using the Protocol of PCR-subtraction Clontech described above. Certain sequences of cDNA 90 clones isolated from the library, presented in SEQ ID NO: 316-404. Comparison of these sequences with sequences in the published databases, as described above, revealed no significant homology with the sequence SEQ ID NO: 366. Discovered that the sequence of SEQ ID NO: 321-325, 343, 354, 368, 369, 377, 382, 385, 389, 395, 397 and 400 showed some homology to previously allocated EST. Found that other sequences show homology with previously identified gene sequences.

In the following studies subtracted cDNA library (named 2BT) were obtained on the basis of cDNA from breast tumors subtracted using pooled cDNA six normal tissues (liver, brain, stomach, small intestine, kidney and heart) with the application of the Protocol PCR subtraction Clontech described above. The cDNA clones, selected after this subtraction, were subjected to DNA analysis on micrometric, as described above, and the resulting data were subjected to four modified analyses Gemtools. In the first analysis compared the 28 breast tumors from 28 normal TKA is s, not related to the breast. As cut-off values for the selection used, at least to 2.1 times the average overexpression. In the second analysis compared the 6 metastatic breast tumors from 29 normal tissues not related to breast cancer. As cut-off values used, at least 2.5 times the average overexpression. In the third and fourth analyses compared the 2 tumors early passages of SCID mice with two tumors late passages in SCID mice. As cut-off values for the selection used a 2.0-fold or higher secondary overexpression in tumors early or late passages. In addition, conducted a visual analysis of data from microarrays for clones 2BT. Certain sequences of 13 cDNA clones identified in the visual analysis presented in SEQ ID NO: 427-439. Certain sequences of cDNA 22 clones identified using a modified analysis Gemtools presented in SEQ ID NO: 440-462, where SEQ ID NO: 453, and 454 are two incomplete overlapping sequences of the same clone.

Comparison of the sequences of the clones of SEQ ID NO: 436 and 437 (named 263G6 and 262B2) with sequences in the published databases, as described above, revealed no significant homology with previously identified by what sledovatelnot. Sequence SEQ ID NO: 427, 429, 431, 435, 438, 441, 443, 444, 445, 446, 450, 453 and 454 (named 266B4, 266G3, 264B4, 263G1, 262B6, 2BT2-34, 2BT1-77, 2BT1-62, 2BT1-60,61, 2BT1-59, 2BT1-52 and 2BT1-40, respectively) showed some homology to previously selected marker expressing sequences (EST). Sequence SEQ ID NO: 428, 430, 432, 433, 434, 439, 440, 442, 447, 448, 449, 451, 452 and 455-462 (called clones 22892, 22890, 22883, 22882, 22880, 22869, 21374, 21349, 21093, 21091, 21089, 21085, 21084, 21063, 21062, 21060, 21053, 21050, 21036, 21037 and 21048, respectively) showed some homology with sequences of genes previously identified in humans.

EXAMPLE 2.

ISOLATION AND CHARACTERIZATION OF POLYPEPTIDES OF BREAST CANCER, OBTAINED BY PCR-BASED SUBTRACTION USING RNA TUMORS PASSAGED IN SCID MICE.

The tumor antigens on human breast were obtained by PCR-based subtraction using RNA tumor of the breast, passereau SCID mice, as follows. Tumor human breast implanted SCID mice and collected at the first or sixth serial passage, as described in the application for the grant of a patent registration No. 08/556659 filed 11/13/95, U.S. patent No. 5986170. Genes that were identified as differentially expressed in tumors of early and late passages of SCID can be specific stage, and therefore suitable for use in tera is AI and diagnosis. Total RNA was obtained from frozen breast tumor human passaged in SCID mice of both the first and sixth passages.

PCR-based subtraction was performed basically as described above. When you first subtract (called T9) RNA from the tumor first passage read from RNA tumor sixth passage to identify more aggressive antigens specific to late passages. 64 clones isolated and sequenced in the case of a specified subtraction, revealed no significant homology to 30 of these clones, hereinafter referred to: 13053, 13057, 13059, 13065, 13067, 13068, 13071-13073, 13075, 13078, 13079, 13081, 13082, 13092, 13097, 13101, 13102, 13131, 13133, 13119, 13135, 13139, 13140, 13146-13149 and 13151, except for certain previously identified marker expressing sequences (EST). Certain sequences of cDNA for these clones are provided in SEQ ID NO: 88-116, respectively. Selected cDNA sequence SEQ ID NO: 117-140 showed homology with known genes. In the second PCR-based subtraction RNA tumor sixth passage read from RNA tumor first passage to identify the antigens are suppressed in multiple passages. Of the 36 selected and sequenced clones revealed no significant homology to nineteen of these clones, hereinafter referred to: 14376, 14377, 14383, 14384, 14387, 14392, 14394, 14398, 1441, 14402, 14405, 14409, 14412, 14414-14416, 14419, and 14426 14427, except for certain previously identified marker expressing sequences (EST). Certain sequences of cDNA for these clones are provided in SEQ ID NO: 141-159, respectively. Detected that the selected cDNA sequence SEQ ID NO: 160-174 showed homology to previously known genes.

Further analyses of tumor antigens on human breast by PCR-based subtraction using RNA tumor SCID first and sixth passages. Found that sixty-three clone differentially expressed with a twofold or greater difference, as determined by analyses on micrometric, i.e. a higher expression in the tumor early passage compared with tumor late passage, or Vice versa. Seventeen of these clones did not show significant homology with any known genes, although it was revealed some degree of homology with previously identified marker expressing sequences (EST), and these clones is further marked by 20266, 20270, 20274, 20276, 20277, 20280, 20281, 20294, 20303, 20310, 20336, 20341, 20941, 20954, 20961, 20965 and 20975 (SEQ ID NO: 252-268, respectively). Discovered that the rest of the clones have some degree of homology with known genes identified in the brief description of the drawings and section identificat the Directors sequences and these clones is further marked by 20261, 20262, 20265, 20267, 20268, 20271, 20272, 20273, 20278, 20279, 20293, 20300, 20305, 20306, 20307, 20313, 20317, 20318, 20320, 20321, 20322, 20326, 20333, 20335, 20337, 20338, 20340, 20938, 20939, 20940, 20942, 20943, 20944, 20946, 20947, 20948, 20949, 20950, 20951, 20952, 20957, 20959, 20966, 20976, 20977 and 20978. Certain sequences of these cDNA clones represented in SEQ ID NO: 269-314, respectively.

Clones 20310, 20281, 20262, 20280, 20303, 20336, 20270, 20341, 20326 and 20977 (also called B820P, B821P, B822P, B823P, B824P, B825P, B826P, B827P, B828P and B829P, respectively) were selected for further analysis based on the results obtained in the analysis on micrometric. In particular, the analysis of the data obtained at micrometric, showed an overexpression of at least two-to threefold, these clones in RNA tumor of the breast in comparison with the tested normal tissues. Subsequent studies led to the identification of the full sequence of the inserts of clones B820P, B821P, B822P, B823P, B824P, B825P, B826P, B827P, B828P and B829P. These extra long cDNA sequence presented in SEQ ID NO: 416-426, respectively.

EXAMPLE 3.

SYNTHESIS OF POLYPEPTIDES.

Peptides can be synthesized on a peptide synthesizer such as Perkin Elmer/Applied Biosystems Division 430A, using FMOC-chemistry with activation HPTU (hexaphosphate 0-benzotriazole-N,N,N',N'-tetramethylurea). With aminocom.com peptide can bind the sequence Gly-Cys-Gly, to provide a method of conjugation, with Azania with immobilized surface or staining of peptide. Cleavage of the peptides from the solid media can be performed using the following mixture for removal: triperoxonane acid: acondition: thioanisole: water: phenol(40:1:2:2:3). After removal within 2 hours of the peptides can be precipitated with cold methyl tert-butylation. Then precipitation of the peptides can be dissolved in water containing 0.1% triperoxonane acid (TFU), and freeze-dried before purification by reversed-phase HPLC on a C18 column. In order eluted peptides, you can use a gradient of 0%-60% acetonitrile (containing 0.1% TFU) in water (containing 0.1% TFU). After lyophilization of pure fractions of peptides can be characterized using mass spectrometry with elektrorazpredelenie or other types of mass spectrometry and amino acids analysis.

EXAMPLE 4.

CALL-SPECIFIC ANTIGENS BREAST CTL RESPONSES IN HUMAN BLOOD.

This example illustrates the ability specific to breast cancer antigen B726P to provoke a response of cytotoxic T lymphocytes (CTL) in peripheral blood lymphocytes of healthy people.

Autologous dendritic cells (DC) were obtained by differentiation of the cultures of monocytes obtained from PBMC of healthy donors, when grown for five days in RPMI medium containing 10% human serum, 30 ng/ml GM-CSF and 30 ng/ml IL-4. After culturing for five days DK and who had ratified during the night, adenovirus, expressing recombinant B726P ("right" ORF; SEQ ID NO: 181)at M.O.I. of 2.5 and caused maturation by addition of 2 micrograms/ml CD40 ligand within 8 hours. Spent the enrichment of CD8-positive cells by depletion on CD4 and CD14-positive cells. Bromirovannye culture initiated in separate wells of several 96-well plates cytokines IL-6 and IL-12. These cultures re-stimulated in the presence of IL-2 using autologous fibroblasts treated with IFN-gamma and transduced B726P and CD80. After three cycles of stimulation was assessed by the presence of specific in relation to B726P CTL activity in the analysis of the IFN-gamma Elispot (Lalvani et al., J Exp. Med. 186: 859-865, 1997), using treated IFN-gamma autologous fibroblasts transduced to Express either B726P, or irrelevant control antigen as the antigen-presenting cells (APCS). Approximately 96 lines identified one line (called 6-2B), which seems to be specific learned transduced B726P APK, but did not recognize APK transduced control antigen. The resulting algae cloned, using standard protocols. CTL specific in relation to VR identified in the analysis of Elispot and were propagated for further analysis. Using the analysis of the release of chromium-51, showed that the CTL clones recognize fibroblasts Express the dominant B726P, but not the control antigen MART-1. In addition, using a panel of allogeneic fibroblasts transduced B726P, in the analysis of blocking antibodies identified element HLA-restriction for these specific in relation to WR CTL as HLA-B*1501.

In order to more accurately determine the position of the epitope, recognized by CTL clones specific in relation to VR, designed deletion construct containing only the N-terminal half of B726P (B726Pdelta3') (a/K 1-129), expressing retroviral plasmid pBIB. The resulting plasmid, as well as other plasmids containing B726P, transfusional cells COS-7 either alone or in combination with plasmid expressing HLA-B* 1501. Approximately 48 hours after transfection were added to the CTL clone, it is specific to the B726P (1-9B), in an amount of about 104cells per well. The contents of the wells were collected the next day and by ELISA measured the amount of released IFN-gamma. The CTL response in cells COS-7, which were transliterowany and B726P and HLA-B*1501, was above background (EGFP). There was no response above background on the cells COS-7, which were transliterowany only B726P or only HLA-B*1501. Importantly, a higher response was observed in the case of cells COS-7, which were transliterowany HLA-B*1501 and B726Pdelta3'. This result was confirmed that the epitope was probably localized in the N-terminal region (a/the 1-129) B726P. Investigated the specified area and identified and synthesized amino acid sequence that corresponded to the connecting peptide motif of HLA-B*1501 (J. Immunol. 1999, 162: 7277-84). These peptides pulse was added at a concentration of 10 μg/ml to autologous B-LCL during the night. The next day cells were washed and the ability of cells to stimulate specific to B726P CTL clone 1-9B were evaluated in the analysis of the IFN-gamma ELISPOT. Of the eleven tested peptides only one peptide having the amino acid sequence SLTKRASQY (a/K 76-84; SEQ ID NO: 488), could be well-known CTL. The result identifies the peptide as the epitope of natural origin, recognizable data is specific to B726P CTL clone.

EXAMPLE 5.

FABRICATION AND CHARACTERIZATION OF ANTIBODIES AGAINST POLYPEPTIDES OF BREAST CANCER.

Polyclonal antibodies against the antigen B726P breast tumors were obtained as follows.

"Right" ORF B726P (SEQ ID NO: 181), expressed in recombinant expressing the system of E. coli, was obtained by growing the culture overnight in LB broth with the appropriate antibiotics at 370C in an incubator with shaking. The next morning, 10 ml of overnight culture was added to 500 ml of 2x YT plus the appropriate antibiotics in the Erlenmeyer flask with a discharge volume of 2 L. When the optical tightly the TB (at 560 nm) of the culture reached 0.4 to 0.6, cells induced IPTG (1 mm). Four hours after induction with IPTG, the cells were collected by centrifugation. Then cells were washed in phosphate-saline buffer and again centrifuged. Adosados was decanted and cells were either frozen for future use or immediately processed. To precipitation cells were added twenty ml of lyse buffer and shook. To destroy E. coli cells, the resulting mixture is then chased through a French press at a pressure of 16,000 psi (110316142 PA). The cells are then centrifuged and adosados and the sediment was examined in SDS-page in relation to the distribution of the recombinant protein. In the case of proteins that were localized in the sediment cells, sediment resuspendable in 10 mm Tris, pH 8.0, 1% CHAPS and sediment Taurus enable washed and centrifuged. This procedure was repeated two more times. The washed precipitate Taurus inclusion was solubilizers 8 M urea or 6 M guanidine-HCl containing 10 mm Tris, pH 8.0, plus 10 mm imidazole. The solubilized protein was added to 5 ml of the Nickel-chelate resin (Qiagen) and incubated for 45 min to 1 hour at room temperature and continuous stirring. After incubation the resin and the protein mixture was passed through a disposable column and collected the passing fluid. Then the column was washed with 10-20 column volumes of buffer to solubilize. Then the antigen suirou and with speakers using 8 M urea, 10 mm Tris, pH 8.0, 300 mm imidazole, and collected fractions of 3 ml were Dispersed in SDS-page-gel to determine which faction to join for further purification.

As the final stage of purification of a strong anion exchange resin, such as HiPrepQ (Biorad), was balanced by a corresponding buffer and the column was applied combined fractions mentioned above. The antigen was suirable column with an increasing gradient of salt. Fractions as output from the column was collected and dispersed in different SDS-page-gel to determine which fractions from the column to combine. Combined fractions were dialyzed against 10 mm Tris, pH 8.0. Then the protein to hand out bottles after filtration through a filter with a pore diameter of 0.22 micron and the antigens were frozen before they will need for immunization.

Four hundred micrograms of antigen B726P was mixed with 100 micrograms of muramyldipeptide (MDP). Every four weeks the rabbits were subjected to booster immunization with 100 micrograms, mixed with an equal volume of incomplete adjuvant's adjuvant (IFA). Seven days after each booster immunization, the animal took the blood. Serum was obtained by incubating the blood at 4°With in 12-24 hours, followed by centrifugation.

Defenestration tablets were coated with antigen VR, incubare 50 Microlitre (typically 1 microgram) re ominotago protein at 4 °C for 20 hours. 250 Microlitres blocking buffer with BSA was added to each well and incubated at room temperature for 2 hours. Tablets 6 times washed with PBS/0.01% tween. The serum of rabbits was diluted in PBS. Fifty microlitres of diluted sera was added to each well and incubated at room temperature for 30 minutes, the Tablets were washed as described above before being added 50 microliters labeled with horseradish peroxidase (HRP) anti-rabbit antibodies goat in a dilution of 1:10000, and incubated at room temperature for 30 minutes the Tablets again washed as described above and to each well was added 100 microliters peroxidase substrate TMB Microwell. After incubation for 15 min in the dark at room temperature colorimetric reaction was stopped by adding 100 microliters 1N H2SO4and immediately recorded at 450 nm. Polyclonal antibodies showed immunoreactivity against B726P.

EXAMPLE 6.

PROTEIN EXPRESSION OF TUMOR ANTIGENS IN BREAST CANCER.

"Right" ORF VR (SEQ ID NO: 181) together with a C-terminal 6X His-tag expressed in insect cells using the baculovirus expression system, as follows.

cDNA "right" ORF B726P full length PCR-amplified using primers SEQ ID NO: 480 and 481. The PCR product of the expected size retrieve the Cali from agarose gel, were digested with restriction enzymes EcoRI and HindII and ligated into the plasmid to transfer pFastBac1, which was digested with the same enzymes. The sequence of the insert was confirmed by DNA sequencing. The recombinant plasmid to transfer pFBB726P used to obtain recombinant DNA backside and the virus using a baculovirus expression system Bac-To-Bac (BRL Life Technologies, Gaithersburg, MD). The High Five cells infected with recombinant virus BVB726P to get protein. The cDNA sequence and amino acids expressed recombinant protein VR presented in SEQ ID NO: 482, and 483, respectively.

Based on the foregoing, it will be obvious that although here for purposes of illustration, described specific variants of the invention, it is possible to make various modifications without departing from the essence and without leaving the scope of the invention.

1. An isolated polypeptide containing at least a portion of the protein of breast cancer or its variant, the polypeptide includes an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of

(a) the sequence given in SEQ ID NO: 474, 482 or 489;

(b) sequences that hybridize to the sequence given in SEQ ID NO: 474, 482, or 489, under conditions of moderate stringency; and

(c) is posledovatelnostei, complementary sequences (a) or (b).

2. An isolated polypeptide according to claim 1, where the polypeptide includes an amino acid sequence that is encoded by a polynucleotide sequence given in SEQ ID NO: 474, 482 or 489 or she complementary sequence.

3. Isolated polynucleotide encoding the polypeptide according to claim 1.

4. Isolated polynucleotide, encoding a protein of breast cancer, or its variant, while the tumor protein comprises the amino acid sequence that is encoded by polynucleotide containing the sequence specified in SEQ ID NO: 474, 482 or 489 or she complementary sequence.

5. Isolated polynucleotide containing the sequence specified in SEQ ID NO: 474, 482, or 489.

6. Isolated polynucleotide containing a sequence that hybridizes with the sequence given in SEQ ID NO: 474, 482, or 489, under conditions of moderate hardness.

7. Isolated polynucleotide, complementary polynucleotide on any of PP-6.

8. Expressing the vector containing polynucleotide on any of PP-7.

9. The vector of claim 8 for the transformation or transfection of a host cell.

10. An isolated antibody or antigennegative fragment that is specific contacts with the protein of breast cancer, the soda is containing amino acid sequence, which is encoded by a polynucleotide sequence given in SEQ ID NO: 474, 482 or 489 or she complementary sequence.

11. A hybrid protein containing the first part of the amino acids and the second part of amino acids, where this first part of the amino acids include 9 or more continuously consecutive amino acids from mammaglobin, which are represented by amino acids 59-78, 55-69, 13 to 33, 41-60, 2-10, 47-59, 62-74 SEQ ID NO: 493; where this second part of the amino acids include 9 or more continuously consecutive amino acids of VR, which are presented in the amino acid sequence of SEQ ID NO:

475 encoded by SEQ ID NO: 474, including T-helper epitope from the amino acid sequence represented SLTKRASQY;

and where this first part of the amino acids is due to the amino end or carboxyl end of the specified second part of the amino acids.

12. A hybrid protein according to claim 11, where the specified hybrid protein includes the amino acid sequence found in any of SEQ ID NO: 493,494 and 495.

13. A hybrid protein according to item 12, where this first part of the amino acid bonded to the amino end of the specified second part of the amino acids.

14. A hybrid protein according to item 12, where this first part of the amino acid is linked to the carboxyl end of the specified second part of the amino acids.

15. Isolated the polynucleotide, encoding a hybrid protein according to claim 11 or 12.

16. Method of removing tumor cells from the blood or fractions of blood, including the implementation of the specified contact blood or blood fraction of T-cells that react with specific proteins of breast cancer, while the tumor protein contains an amino acid sequence that is encoded by a polynucleotide sequence given in SEQ ID NO: 474, 482 or 489-492, her or complementary sequence, where the stage of contacting performed under conditions and for a time sufficient to remove from a sample of cells expressing the antigen.

17. Composition for use in the suppression of breast cancer in a patient, comprising the biological sample treated according to the method according to item 16.

18. The method of stimulation and/or expansion of T cells specific for the protein of breast cancer, including the implementation of ex vivo contact of T cells with at least one component selected from the group consisting of

(a) a polypeptide containing the amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of

(i) the sequence given in SEQ ID NO: 474, 482 or 489-492;

(ii) sequences that hybridize with the settlement of what ecovitality, specified in SEQ ID NO: 474, 482 or 489-492, under conditions of moderate stringency;and (iii) sequences complementary to the sequences of (i) or (ii);

(b) polynucleotides encoding the polypeptide (a); and

(c) antigen-presenting cells that Express a polypeptide (a);

under conditions and for a time sufficient to allow for stimulation and/or expansion

T-cells.

19. The method according to p, further comprising cloning at least one enlarged cells with maintenance of cloned T cells.

20. The isolated population of T cells containing T cells obtained according to the method according to p or 19.

21. A composition comprising antigen presenting cell that expresses a polypeptide containing at least immunogenic portion of the protein of breast cancer, or its variant, where the tumor protein contains an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of

(i) the sequence given in SEQ ID NO: 474, 482

or 489-492;

(ii) sequences that hybridize to the sequence given in SEQ ID NO: 474, 482 or 489-492, under conditions of moderate stringency; and

(iii) sequences complementary to the sequences of (i) or is (ii).

22. Composition for use in the suppression of breast cancer in a patient, comprising a population of T cells according to claim 20.

23. The method of determining the presence or absence of breast cancer in a patient, comprising the following stages:

(a) contacting a biological sample obtained from the patient with an antibody which binds to the protein of breast cancer, when the tumor protein contains an amino acid sequence that is encoded by a polynucleotide sequence given in SEQ ID NO: 474, 482 or 489-492, or she complementary sequence;

(b) determining in the sample the amount of polypeptide that binds to the binding agent; and

(c) comparing the amount of polypeptide with a pre-defined cut-off (cut-off) value, and determining based on the presence or absence of cancer in a patient.

24. The method according to item 23, where the antibody is a monoclonal antibody.

25. Method of monitoring the progression of breast cancer in a patient, comprising the following stages:

(a) contacting a biological sample obtained from the patient at a first point in time, with the antibody, which binds to the protein of breast cancer, when the tumor protein contains the amino acid sequence, the cat heaven is encoded by a polynucleotide sequence, specified in SEQ ID NO: 474, 482 or 489-492, or she complementary sequence;

(b) determining in the sample the amount of polypeptide that binds to the binding agent;

(c) repeating steps (a) and (B) using a biological sample obtained from the patient to the next point in time; and

(d) comparing the amount of polypeptide defined in stage (C), with the number determined at stage (B), and monitoring the progression of breast cancer patients.

26. The method according A.25, where the antibody is a monoclonal antibody.

27. The method of determining the presence or absence of breast cancer in a patient, comprising the following stages:

(a) contacting a biological sample obtained from the patient with an oligonucleotide that hybridizes in conditions of moderate hardness with polynucleotide, which encodes a protein of breast cancer where the tumor protein contains an amino acid sequence that is encoded by a polynucleotide sequence given in SEQ ID NO: 474, 482 or 489-492, or she complementary sequence;

(b) determining in the sample the number of polynucleotide that hybridizes with the oligonucleotide; and

(c) comparing the number of polynucleotide that hybridizes what the oligonucleotide, with pre-defined cut-off (cut-off) value, and based on this determination of the presence or absence of breast cancer in a patient.

28. The method according to item 27, where the number of polynucleotide that hybridizes with the oligonucleotide, determined using polymerase chain reaction.

29. The method according to item 27, where the number of polynucleotide that hybridizes with the oligonucleotide, determine, using hybridization analysis.

30. Method of monitoring the progression of breast cancer in a patient, comprising the following stages:

(a) contacting a biological sample obtained from the patient with an oligonucleotide that hybridizes in conditions of moderate hardness with polynucleotide, which encodes a protein of breast cancer, when the tumor protein contains an amino acid sequence that is encoded by a polynucleotide sequence given in SEQ ID NO: 474, 482 or 489-492, or she complementary sequence;

(b) determining in the sample the number of polynucleotide that hybridizes with the oligonucleotide;

(c) repeating steps (a) and (b) using a biological sample obtained from the patient to the next point in time; and

(d) comparing the number of polynucleotide defined at the stage (C), with the quantity of the particular stage (S), and on the basis of this monitoring the progression of cancer in a patient.

31. The method according to item 30, where the number of polynucleotide that hybridizes with the oligonucleotide, determined using polymerase chain reaction.

32. The method according to item 30, where the number of polynucleotide that hybridizes with the oligonucleotide, determine, using hybridization analysis.

33. Diagnostic kit containing:

(a) one or more antibodies of claim 10; and

(b) a reagent for detecting that contains a reporter group.

34. Set p, where the antibody is immobilized on a solid medium.

35. Set p, where the reagent for detection additionally includes anti-immunoglobulin, protein G, protein a or a lectin.

36. Set p, where the reporter group is selected from the group consisting of radioisotopes, fluorescent groups, luminescent groups, enzymes, Biotin and dye particles.

37. Diagnostic kit containing

(a) oligonucleotide containing 10 to 40 contiguous nucleotides, under conditions of moderate hardness hybridized with polynucleotide, which encodes a protein of breast cancer, when the tumor protein contains an amino acid sequence that is encoded by a polynucleotide sequence given in SEQ ID NO: 474, 482 or 489-492, or she complementary sequence; and

(b) a diagnostic reagent for use in the polymerase chain reaction or hybridization analysis.

Priority signs:

SEQ ID NO:474 and 475 dated 17.04.2000; SEQ ID NO: 482, and 489 from 22.06.2000 and SEQ ID NO: 490-495 from 20.07.2000.



 

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2 cl, 3 dwg, 6 ex

FIELD: gene engineering.

SUBSTANCE: the present innovation deals with the ways for obtaining transgenic poultry due to introducing retroviral vectors into blastodermal cells through the fissure in the shell of nonhatching egg from the side of its blunt end. With the help of insulin syringe one should introduce gene constructions for the depth of about 2-3 cm near a germinal disk. The innovation enables to simplify the procedure of introducing gene constructions into target cells at maintaining general efficiency of transgenesis that leads to the decrease of embryonic lethality.

EFFECT: higher efficiency.

FIELD: biotechnology, virology, medicine.

SUBSTANCE: invention relates to attenuated virus derived from modified Ankara vaccina virus. Said virus are not able for reproduction by replication in human cell lines. Also disclosed are application of virus or recombinant variants thereof as drug or vaccine, as well as method for inducing of immune response in patients with defected immunity, in patients having immunity to vaccine virus, or in patient during antiviral therapy.

EFFECT: variant of Ankara vaccina virus effective in medicine and veterinary.

86 cl, 15 dwg, 1 tbl, 2 ex

FIELD: biotechnology, medicine, in particular viral disease treatment.

SUBSTANCE: invention relates to recessive dividing retroviral vector useful in inhibition of wild-type retrovirus replication. Vector contains retroviral long terminal repeat sequences; retroviral packing signal; nucleotide sequence encoding (expressing) genetic antiviral agent; and optionally the second nucleotide sequence. Disclosed are method for production of said vector and reproduction thereof. Further isolated and purified nucleic acid (NA) molecule providing of selective advantage in regard to viral generation packing into virions is disclosed. Uses of retroviral vector in particular for specific antibody production are described.

EFFECT: new genetic antiviral agents generating prolonged and stable immunological responses in regard, for example, to AIDS and cancer viruses.

97 cl, 11 ex

FIELD: biotechnology, medicine, in particular viral disease treatment.

SUBSTANCE: invention relates to recessive dividing retroviral vector useful in inhibition of wild-type retrovirus replication. Vector contains retroviral long terminal repeat sequences; retroviral packing signal; nucleotide sequence encoding (expressing) genetic antiviral agent; and optionally the second nucleotide sequence. Disclosed are method for production of said vector and reproduction thereof. Further isolated and purified nucleic acid (NA) molecule providing of selective advantage in regard to viral generation packing into virions is disclosed. Uses of retroviral vector in particular for specific antibody production are described.

EFFECT: new genetic antiviral agents generating prolonged and stable immunological responses in regard, for example, to AIDS and cancer viruses.

97 cl, 11 ex

FIELD: genetic engineering, proteins, medicine, pharmacy.

SUBSTANCE: invention relates to a method for preparing a fused protein representing immunoglobulin Fc-fragment and interferon-alpha and can be used in treatment of hepatitis. Method involves construction of a fused protein comprising immunoglobulin Fc-fragment prepared from Ig G1 or Ig G3 in direction from N-end to C-end and the end protein comprising at least one interferon-alpha. Fc-fragment and the end protein are joined directly or by a polypeptide bridge. The fused protein is used for preparing a pharmaceutical composition used in treatment of liver diseases and in a method for targeting interferon-alpha into liver tissues. Invention provides preparing the fused protein eliciting with biological activity of interferon-alpha providing its concentrating in liver and showing enhanced solubility, prolonged half-time life in serum blood and enhanced binding with specific receptors.

EFFECT: improved targeting method, valuable biological properties of fused protein.

10 cl, 5 dwg, 9 ex

FIELD: genetic engineering, proteins, medicine, pharmacy.

SUBSTANCE: invention relates to a method for preparing a fused protein representing immunoglobulin Fc-fragment and interferon-alpha and can be used in treatment of hepatitis. Method involves construction of a fused protein comprising immunoglobulin Fc-fragment prepared from Ig G1 or Ig G3 in direction from N-end to C-end and the end protein comprising at least one interferon-alpha. Fc-fragment and the end protein are joined directly or by a polypeptide bridge. The fused protein is used for preparing a pharmaceutical composition used in treatment of liver diseases and in a method for targeting interferon-alpha into liver tissues. Invention provides preparing the fused protein eliciting with biological activity of interferon-alpha providing its concentrating in liver and showing enhanced solubility, prolonged half-time life in serum blood and enhanced binding with specific receptors.

EFFECT: improved targeting method, valuable biological properties of fused protein.

10 cl, 5 dwg, 9 ex

FIELD: gene engineering, in particular apoptosis inducing gene delivery vectors useful for cancer, hyperplasia, metaplasia and displasia diagnosis and treatment.

SUBSTANCE: recombinant adenovirus apoptin-containing vectors are obtained by cotransfection into 911 helper cell line of p.Amb-VP3 adaptor plasmids (in case of VP3 protein expression) or pMAb-VP2 plasmids (in case of VP2 protein expression) and JM17 DNA. p.Amb-VP3 plasmids carry apoptin gene in 5'-3'-orientation, expressing under control of adenoviral main late promoter. Plasmid JM17 DNA contains complete adenoviral DNA excepted E1 and E2 regions. pMAb-°VP2 plasmids carry apoptin gene with two point mutation in limits of coding region. Cotransfections are carried out by calcium phosphate method. Recombinant adenoviral DNA is formed by homologous recombination between homologous viral sequences representing in p.Amb-VP3 (or pMAb-VP2) plasmid and in adenoviral DNA from plasmid JM17 DNA. Cell infection of various human tumors with gene delivery vectors causes to tumor cell apoptosis induction and sufficiently reduced normal, diploid, non-transformed or non-pernicious cell apoptosis.

EFFECT: new gene delivery vector capable to induce cell apoptosis.

8cl, 7 dwg

FIELD: gene engineering, in particular apoptosis inducing gene delivery vectors useful for cancer, hyperplasia, metaplasia and displasia diagnosis and treatment.

SUBSTANCE: recombinant adenovirus apoptin-containing vectors are obtained by cotransfection into 911 helper cell line of p.Amb-VP3 adaptor plasmids (in case of VP3 protein expression) or pMAb-VP2 plasmids (in case of VP2 protein expression) and JM17 DNA. p.Amb-VP3 plasmids carry apoptin gene in 5'-3'-orientation, expressing under control of adenoviral main late promoter. Plasmid JM17 DNA contains complete adenoviral DNA excepted E1 and E2 regions. pMAb-°VP2 plasmids carry apoptin gene with two point mutation in limits of coding region. Cotransfections are carried out by calcium phosphate method. Recombinant adenoviral DNA is formed by homologous recombination between homologous viral sequences representing in p.Amb-VP3 (or pMAb-VP2) plasmid and in adenoviral DNA from plasmid JM17 DNA. Cell infection of various human tumors with gene delivery vectors causes to tumor cell apoptosis induction and sufficiently reduced normal, diploid, non-transformed or non-pernicious cell apoptosis.

EFFECT: new gene delivery vector capable to induce cell apoptosis.

8cl, 7 dwg

FIELD: medicine; pharmacology.

SUBSTANCE: immunogenic hybrid polypeptide includes mimetic peptide of V-cellular epitope of apolypoprotein B-100 in which C-end of mimetic peptide is merged with N-end of T-helper epitope. Amino acid sequences of polypeptide variants are presented in description. Described is method of specified polypeptide production providing application of host cell transformed with recombinant express vector including gene coding specified polypeptide. Besides, invention concerns vaccine composition including specified immunogenic hybrid polypeptide for obesity prevention or treatment, recombinant express vector and host cell.

EFFECT: excellent anti-obesity activity without induction of immune response or severe by-effects.

15 cl, 25 dwg, 4 tbl, 15 ex

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