Compositions and methods for the therapy and diagnosis of breast cancer

专利摘要:
The present invention relates to compositions and methods for the treatment and diagnosis of cancer, such as breast cancer. The composition may comprise one or more breast tumor proteins, immunogenic portions thereof, or polynucleotides encoding such portions. Alternatively, the therapeutic composition may comprise antigen presenting cells expressing a breast tumor protein, or T cells specific for cells expressing such proteins. Such compositions can be used, for example, in the prevention and treatment of diseases (eg breast cancer). Also provided is a diagnostic method based on detecting a breast tumor protein, or an mRNA encoding such protein, in a sample.
公开号:KR20030008369A
申请号:KR1020027013944
申请日:2001-04-12
公开日:2003-01-25
发明作者:장위추；딜론데이빈씨；미참제니퍼엘；쉬장춘；하르락커수잔엘；헤플러윌리엄티
申请人:코릭사 코포레이션；
IPC主号:

专利说明:

Compositions and methods for treating and diagnosing breast cancer {Compositions and methods for the therapy and diagnosis of breast cancer}
[2] Breast cancer is becoming a significant health problem for women in the United States and around the world. Techniques for detecting and treating these diseases have advanced, but breast cancer is still the second largest cancer-related mortality rate among more than 180,000 women in the United States each year. For North American women, the survival rate for patients with breast cancer is now one in eight.
[3] No vaccines or other widely successful methods for the prevention or treatment of breast cancer are currently available. The management of the disease is currently only combined with an initial diagnosis (usually through the normal breast screening process) and active treatment that may include one or more of therapies, such as surgery, radiotherapy, chemotherapy and hormone therapy. Depends. The course of treatment for a particular breast cancer is often selected based on various prognostic parameters, including the analysis of specific tumor markers (Porter-Jordan and Lippman, Breast Cancer 8: 73-100 (1994)). However, the use of anchored markers often results in difficult judgments, and the high mortality observed in breast cancer patients indicates that techniques for the treatment, diagnosis, and prevention of these diseases need to be improved.
[4] Therefore, there is a need in the art for improved methods for the treatment and diagnosis of breast cancer. The present invention fulfills these needs and further provides other advantages in this regard.
[5] Summary of the Invention
[6] Briefly stated, the present invention provides compositions and methods for diagnosing and treating cancer, such as breast cancer. In one aspect, the invention provides a polypeptide comprising at least a portion of a breast tumor protein, or variant thereof. Because certain moieties and other variants are immunogenic, the ability of these variants to react with antigen-specific antisera is substantially not compromised. Within certain embodiments, the polypeptide comprises (a) the sequences set forth in SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489; (b) a variant of the sequence set forth in SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479,484, 486, and 489, and (c) a complementary sequence of (a) or (b) sequences It includes a sequence encoded by a polynucleotide sequence selected from among. In certain embodiments, the polypeptide of the invention comprises at least a schedule of tumor proteins comprising an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 176, 179, 181, 469-473, 475, 485, 487 and 488 and variants thereof Include the part.
[7] The present invention further provides a polynucleotide encoding a polypeptide as described above, or a portion thereof, wherein the portion encodes at least 15 amino acid residues of the breast tumor protein, an expression vector comprising such polynucleotide. And host cells transformed or transfected with the expression vector.
[8] Within other aspects, the present invention provides a pharmaceutical composition comprising a polypeptide or polynucleotide as mentioned above and a physiologically acceptable carrier.
[9] Within the relevant aspects of the invention, immunogenic compositions, or vaccines for prophylaxis or treatment, are provided. Such compositions comprise a polypeptide or polynucleotide as mentioned above and an immunostimulant.
[10] The invention further provides a pharmaceutical composition comprising (a) an antibody or antigen-binding fragment thereof that specifically binds to a breast tumor protein and (b) a physiologically acceptable carrier.
[11] In a further aspect, the present invention provides a pharmaceutical composition comprising (a) an antigen presenting cell expressing a polypeptide as mentioned above and (b) a pharmaceutically acceptable carrier or excipient. Antigen presenting cells include dendritic cells, macrophage, monocytes, fibroblasts and B cells.
[12] Within a related aspect, there is provided an immunogenic composition or vaccine comprising (a) an antigen presenting cell expressing a polypeptide as mentioned above and (b) an immunostimulant.
[13] The invention further provides, in one aspect, fusion proteins comprising one or more polypeptides as mentioned above, and polynucleotides encoding such fusion proteins. Fusion proteins exemplified in accordance with the present invention comprise a first amino acid moiety and a second amino acid moiety, said first amino acid moiety being nine or more contiguous from mammaglobin as set forth as amino acids 1-93 of SEQ ID NO: 493. Wherein the second amino acid portion comprises at least 9 contiguous amino acids from B726P as shown by SEQ ID NO: 475, SEQ ID NO: 469, or SEQ ID NO: 176, wherein the first amino acid portion is the amino terminus or carboxy of the second amino acid portion; It is connected to the end.
[14] A further aspect of the invention is that a first amino acid moiety may comprise IDELKECFLNQTDETLSNVE (amino acids 59-78 of SEQ ID NO: 493); TTNAIDELKECFLNQ (amino acids 55-69 of SEQ ID NO: 493); SQHCYAGSGCPLLENVISKTI (amino acids 13-33 of SEQ ID NO: 493); EYKELLQEFIDDNATTNAID (amino acids 41-60 of SEQ ID NO: 493); KLLMVLMLA (amino acids 2-10 of SEQ ID NO: 493); It provides a fusion protein is selected from the group consisting of N QEFIDD ATTNAI (amino acids 47-59 of SEQ ID NO: 493) and N LKECFL (amino acids 62-74 of SEQ ID NO: 493) QTDETL.
[15] Another aspect includes a combination of upstream and downstream open reading frames (ORFs) of B726P as shown in SEQ ID NO: 475; A fusion protein is provided comprising (2) an upstream ORF of B726P as shown in SEQ ID NO: 469 and at least 9 contiguous amino acids encoded by (3) a downstream ORF of B726P as shown in SEQ ID NO: 176. The fusion protein according to the invention may also comprise a second amino acid moiety comprising at least 9 contiguous amino acids from the amino acid sequence set forth as amino acids 1-129 of SEQ ID NO: 475. Also, examples of additional fusion proteins are SEQ ID NO: 493; Set forth herein as SEQ ID NO: 494 and SEQ ID NO: 495.
[16] While a fusion protein is provided in which the mamma globin amino acid portion is linked to the amino-terminus of the B726P amino acid portion, other fusion proteins are also provided in which the mamma globin amino acid portion is linked to the carboxy-terminus of the B726P amino acid portion. The linkage between the mamma globin amino acid portion and the B726P portion may be a covalent bond. In addition, amino acid extensions associated with or not associated with mammaglobin and / or B726P may be incorporated between amino- or carboxy-terminus to mammaglobin and / or B726P amino acid moieties.
[17] The present invention also provides isolated polynucleotides encoding all of the fusion proteins specifically described herein, as well as fusion proteins that can be achieved by experiments by one of ordinary skill in the art.
[18] Within a related aspect, there is provided a pharmaceutical composition comprising a fusion protein or a polynucleotide encoding such a fusion protein with a physiologically acceptable carrier.
[19] In other aspects, there is further provided a composition comprising, together with an immunosuppressive agent, a fusion protein or polynucleotide encoding such a fusion protein.
[20] Within a further aspect, the present invention provides a method of inhibiting cancer development in a patient, comprising administering to the patient a composition as mentioned above. The patient may develop breast cancer, in which case the method may be able to treat the disease, or prophylactically treat a patient deemed to be at risk of developing the disease.
[21] The present invention further provides, within other aspects, contacting a biological sample with a T cell that specifically reacts with a breast tumor protein, wherein the contacting step is a time sufficient to remove cells expressing the protein from the sample. And under conditions), a method for removing tumor cells from a particular biological sample.
[22] In a related aspect, a method of inhibiting cancer development in a patient is provided, including administering to the patient a biological sample treated as mentioned above.
[23] In other aspects, the T cells may comprise (i) a polypeptide as mentioned above; (ii) contacting at least one of the polynucleotides encoding such a polypeptide and / or (iii) one or more of the antigen-providing cells expressing said polypeptide with a time and condition sufficient to permit stimulation and / or expansion of the cell. Further provided are methods for stimulating and / or expanding T cells specific for breast tumor proteins, including. Also provided is an isolated T cell population comprising T cells prepared as mentioned above.
[24] Within a further aspect, the present invention provides a method of inhibiting cancer development in a patient, including administering to the patient an effective amount of a T cell population as mentioned above.
[25] The invention further comprises (a) a CD4 ⁺ and / or CD8 ⁺ T cell isolated from a particular patient ^; (i) a polypeptide comprising at least an immunogenic portion of a breast tumor protein; (ii) incubating with at least one of the polynucleotides encoding such a polypeptide and / or (iii) the antigen-providing cell expressing said polypeptide, and (b) thereby providing an effective amount of T cells proliferated to said patient. Inhibiting the occurrence of cancer in the patient by administering the method. Proliferated cells can be cloned prior to administration to a patient, but need not be.
[26] In a further aspect, the present invention provides a method of preparing a pharmaceutical composition comprising: (a) contacting a biological sample obtained from a patient with a binder that binds to a polypeptide as mentioned above; (b) detecting the amount of polypeptide binding to such a binder in the sample and (c) comparing the amount of the polypeptide with a predetermined cut-off value to determine whether cancer has developed in the patient. It provides a method of determining the onset of cancer in a patient, comprising the step of determining.
[27] The present invention also provides, within other aspects, a method of monitoring the progress of cancer in a patient. This method comprises the steps of (a) contacting a biological sample obtained from a patient at a first time point with a binder that binds to the polypeptide as mentioned above; (b) detecting the amount of polypeptide in the sample that binds to this binder; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a later time point and (d) detecting the amount of polypeptide detected in step (c) in step (b) Comparing the amount to the amount of the cancer, and monitoring the progress of the cancer from the patient.
[28] Within other aspects, the present invention further provides a method for preparing a breast tumor, comprising the steps of: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes with a polynucleotide encoding a breast tumor protein; (b) detecting the levels of polynucleotides, preferably mRNAs, which hybridize with such oligonucleotides in the sample, and (c) comparing the levels of polynucleotides that hybridize with the oligonucleotides to a predetermined cut-off value. Thereby providing a method of determining whether cancer develops in a patient, the method comprising determining whether cancer develops in the patient. In certain embodiments, for example, the amount of mRNA is detected through a polymerase chain reaction using a polynucleotide encoding a polypeptide as described above or one or more oligonucleotide primers that hybridize with the complementary sequence of such polynucleotides. do. In other embodiments, the amount of mRNA is detected using a hybridization technique using a polynucleotide encoding a polypeptide as described above or one or more oligonucleotide probes that hybridize with the complementary sequence of such polynucleotides.
[29] In a related aspect, (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes with a polynucleotide encoding a breast tumor protein; (b) detecting the amount of polynucleotide in the sample that hybridizes with such oligonucleotides; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a later time point and (d) detecting the amount of polynucleotide detected in step (c) in step (b) A method of monitoring cancer progression in a patient is provided, the method comprising comparing the amount of cancer to the amount of the cancer, and monitoring the progress of cancer in the patient.
[30] Within a further aspect, the present invention provides antibodies, such as monoclonal antibodies, and diagnostic kits comprising such antibodies that bind with a polypeptide as mentioned above. Also provided is a diagnostic kit comprising one or more oligonucleotide probes or primers as mentioned above.
[31] These and other aspects of the present invention will become apparent with reference to the following detailed description and the accompanying drawings. All references cited herein are hereby incorporated by reference in their entirety as if each had been individually inserted.
[1] The present invention generally relates to the treatment and diagnosis of cancer, for example breast cancer. More particularly, the present invention relates to polypeptides comprising at least a portion of breast tumor proteins, and polynucleotides encoding such polypeptides. Such polypeptides and polynucleotides can be used in compositions for the prevention and treatment of breast cancer, and compositions for the diagnosis and monitoring of breast cancer.
[32] 1 shows the results of a Northern blot of clone SYN18C6 (SEQ ID NO: 40).
[33] SEQ ID NO: 1 is the determined cDNA sequence of JBT2.
[34] SEQ ID NO: 2 is the determined cDNA sequence of JBT6.
[35] SEQ ID NO: 3 is the determined cDNA sequence of JBT7.
[36] SEQ ID NO: 4 is the determined cDNA sequence of JBT10.
[37] SEQ ID NO: 5 is the determined cDNA sequence of JBT13.
[38] SEQ ID NO: 6 is the determined cDNA sequence of JBT14.
[39] SEQ ID NO: 7 is the determined cDNA sequence of JBT15.
[40] SEQ ID NO: 8 is the determined cDNA sequence of JBT16.
[41] SEQ ID NO: 9 is the determined cDNA sequence of JBT17.
[42] SEQ ID NO: 10 is the determined cDNA sequence of JBT22.
[43] SEQ ID NO: 11 is the determined cDNA sequence of JBT25.
[44] SEQ ID NO: 12 is the determined cDNA sequence of JBT28.
[45] SEQ ID NO: 13 is the determined cDNA sequence of JBT32.
[46] SEQ ID NO: 14 is the determined cDNA sequence of JBT33.
[47] SEQ ID NO: 15 is the determined cDNA sequence of JBT34.
[48] SEQ ID NO: 16 is the determined cDNA sequence of JBT36.
[49] SEQ ID NO: 17 is the determined cDNA sequence of JBT37.
[50] SEQ ID NO: 18 is the determined cDNA sequence of JBT51.
[51] SEQ ID NO: 19 is the determined cDNA sequence of JBTT1.
[52] SEQ ID NO: 20 is the determined cDNA sequence of JBTT7.
[53] SEQ ID NO: 21 is the determined cDNA sequence of JBTT11.
[54] SEQ ID NO: 22 is the determined cDNA sequence of JBTT14.
[55] SEQ ID NO: 23 is the determined cDNA sequence of JBTT18.
[56] SEQ ID NO: 24 is the determined cDNA sequence of JBTT19.
[57] SEQ ID NO: 25 is the determined cDNA sequence of JBTT20.
[58] SEQ ID NO: 26 is the determined cDNA sequence of JBTT21.
[59] SEQ ID NO: 27 is the determined cDNA sequence of JBTT22.
[60] SEQ ID NO: 28 is the determined cDNA sequence of JBTT28.
[61] SEQ ID NO: 29 is the determined cDNA sequence of JBTT29.
[62] SEQ ID NO: 30 is the determined cDNA sequence of JBTT33.
[63] SEQ ID NO: 31 is the determined cDNA sequence of JBTT37.
[64] SEQ ID NO: 32 is the determined cDNA sequence of JBTT38.
[65] SEQ ID NO: 33 is the determined cDNA sequence of JBTT47.
[66] SEQ ID NO: 34 is the determined cDNA sequence of JBTT48.
[67] SEQ ID NO: 35 is the determined cDNA sequence of JBTT50.
[68] SEQ ID NO: 36 is the determined cDNA sequence of JBTT51.
[69] SEQ ID NO: 37 is the determined cDNA sequence of JBTT52.
[70] SEQ ID NO: 38 is the determined cDNA sequence of JBTT54.
[71] SEQ ID NO: 39 is the determined cDNA sequence of SYN17F4.
[72] SEQ ID NO: 40 is the determined cDNA sequence of SYN18C6 (also known as B709P).
[73] SEQ ID NO: 41 is the determined cDNA sequence of SYN19A2.
[74] SEQ ID NO: 42 is the determined cDNA sequence of SYN19C8.
[75] SEQ ID NO: 43 is the determined cDNA sequence of SYN20A12.
[76] SEQ ID NO: 44 is the determined cDNA sequence of SYN20G6.
[77] SEQ ID NO: 45 is the determined cDNA sequence of SYN20G6-2.
[78] SEQ ID NO: 46 is the determined cDNA sequence of SYN21B9.
[79] SEQ ID NO: 47 is the determined cDNA sequence of SYN21B9-2.
[80] SEQ ID NO: 48 is the determined cDNA sequence of SYN21C10.
[81] SEQ ID NO: 49 is the determined cDNA sequence of SYN21G10.
[82] SEQ ID NO: 50 is the determined cDNA sequence of SYN21G10-2.
[83] SEQ ID NO: 51 is the determined cDNA sequence of SYN21G11.
[84] SEQ ID NO: 52 is the determined cDNA sequence of SYN21G11-2.
[85] SEQ ID NO: 53 is the determined cDNA sequence of SYN21H8.
[86] SEQ ID NO: 54 is the determined cDNA sequence of SYN22A10.
[87] SEQ ID NO: 55 is the determined cDNA sequence of SYN22A10-2.
[88] SEQ ID NO: 56 is the determined cDNA sequence of SYN22A12.
[89] SEQ ID NO: 57 is the determined cDNA sequence of SYN22A2.
[90] SEQ ID NO: 58 is the determined cDNA sequence of SYN22B4.
[91] SEQ ID NO: 59 is the determined cDNA sequence of SYN22C2.
[92] SEQ ID NO: 60 is the determined cDNA sequence of SYN22E10.
[93] SEQ ID NO: 61 is the determined cDNA sequence of SYN22F2.
[94] SEQ ID NO: 62 is the putative amino acid sequence of SYN18C6 (also known as B709P).
[95] SEQ ID NO: 63 is the determined cDNA sequence of B723P.
[96] SEQ ID NO: 64 is the determined cDNA sequence of B724P.
[97] SEQ ID NO: 65 is the determined cDNA sequence of B770P.
[98] SEQ ID NO: 66 is the determined cDNA sequence of B716P.
[99] SEQ ID NO: 67 is the determined cDNA sequence of B725P.
[100] SEQ ID NO: 68 is the determined cDNA sequence of B717P.
[101] SEQ ID NO: 69 is the determined cDNA sequence of B771P.
[102] SEQ ID NO: 70 is the determined cDNA sequence of B722P.
[103] SEQ ID NO: 71 is the determined cDNA sequence of B726P.
[104] SEQ ID NO: 72 is the determined cDNA sequence of B727P.
[105] SEQ ID NO: 73 is the determined cDNA sequence of B728P.
[106] SEQ ID NOs: 74-87 are determined cDNA sequences of isolated clones that show homology with known sequences.
[107] SEQ ID NO: 88 is the determined cDNA sequence of 13053.
[108] SEQ ID NO: 89 is the determined cDNA sequence of 13057.
[109] SEQ ID NO: 90 is the determined cDNA sequence of 13059.
[110] SEQ ID NO: 91 is the determined cDNA sequence of 13065.
[111] SEQ ID NO: 92 is the determined cDNA sequence of 13067.
[112] SEQ ID NO: 93 is the determined cDNA sequence of 13068.
[113] SEQ ID NO: 94 is the determined cDNA sequence of 13071.
[114] SEQ ID NO: 95 is the determined cDNA sequence of 13072.
[115] SEQ ID NO: 96 is the determined cDNA sequence of 13073.
[116] SEQ ID NO: 97 is the determined cDNA sequence of 13075.
[117] SEQ ID NO: 98 is the determined cDNA sequence of 13078.
[118] SEQ ID NO: 99 is the determined cDNA sequence of 13079.
[119] SEQ ID NO: 100 is the determined cDNA sequence of 13081.
[120] SEQ ID NO: 101 is the determined cDNA sequence of 13082.
[121] SEQ ID NO: 102 is the determined cDNA sequence of 13092.
[122] SEQ ID NO: 103 is the determined cDNA sequence of 13097.
[123] SEQ ID NO: 104 is the determined cDNA sequence of 13101.
[124] SEQ ID NO: 105 is the determined cDNA sequence of 13102.
[125] SEQ ID NO: 106 is the determined cDNA sequence of 13119.
[126] SEQ ID NO: 107 is the determined cDNA sequence of 13131.
[127] SEQ ID NO: 108 is the determined cDNA sequence of 13133.
[128] SEQ ID NO: 109 is the determined cDNA sequence of 13135.
[129] SEQ ID NO: 110 is the determined cDNA sequence of 13139.
[130] SEQ ID NO: 111 is the determined cDNA sequence of 13140.
[131] SEQ ID NO: 112 is the determined cDNA sequence of 13146.
[132] SEQ ID NO: 113 is the determined cDNA sequence of 13147.
[133] SEQ ID NO: 114 is the determined cDNA sequence of 13148.
[134] SEQ ID NO: 115 is the determined cDNA sequence of 13149.
[135] SEQ ID NO: 116 is the determined cDNA sequence of 13151.
[136] SEQ ID NO: 117 is the determined cDNA sequence of 13051.
[137] SEQ ID NO: 118 is the determined cDNA sequence of 13052.
[138] SEQ ID NO: 119 is the determined cDNA sequence of 13055.
[139] SEQ ID NO: 120 is the determined cDNA sequence of 13058.
[140] SEQ ID NO: 121 is the determined cDNA sequence of 13062.
[141] SEQ ID NO: 122 is the determined cDNA sequence of 13064.
[142] SEQ ID NO: 123 is the determined cDNA sequence of 13080.
[143] SEQ ID NO: 124 is the determined cDNA sequence of 13093.
[144] SEQ ID NO: 125 is the determined cDNA sequence of 13094.
[145] SEQ ID NO: 126 is the determined cDNA sequence of 13095.
[146] SEQ ID NO: 127 is the determined cDNA sequence of 13096.
[147] SEQ ID NO: 128 is the determined cDNA sequence of 13099.
[148] SEQ ID NO: 129 is the determined cDNA sequence of 13100.
[149] SEQ ID NO: 130 is the determined cDNA sequence of 13103.
[150] SEQ ID NO: 131 is the determined cDNA sequence of 13106.
[151] SEQ ID NO: 132 is the determined cDNA sequence of 13107.
[152] SEQ ID NO: 133 is the determined cDNA sequence of 13108.
[153] SEQ ID NO: 134 is the determined cDNA sequence of 13121.
[154] SEQ ID NO: 135 is the determined cDNA sequence of 13126.
[155] SEQ ID NO: 136 is the determined cDNA sequence of 13129.
[156] SEQ ID NO: 137 is the determined cDNA sequence of 13130.
[157] SEQ ID NO: 138 is the determined cDNA sequence of 13134.
[158] SEQ ID NO: 139 is the determined cDNA sequence of 13141.
[159] SEQ ID NO: 140 is the determined cDNA sequence of 13142.
[160] SEQ ID NO: 141 is the determined cDNA sequence of 14376.
[161] SEQ ID NO: 142 is the determined cDNA sequence of 14377.
[162] SEQ ID NO: 143 is the determined cDNA sequence of 14383.
[163] SEQ ID NO: 144 is the determined cDNA sequence of 14384.
[164] SEQ ID NO: 145 is the determined cDNA sequence of 14387.
[165] SEQ ID NO: 146 is the determined cDNA sequence of 14392.
[166] SEQ ID NO: 147 is the determined cDNA sequence of 14394.
[167] SEQ ID NO: 148 is the determined cDNA sequence of 14398.
[168] SEQ ID NO: 149 is the determined cDNA sequence of 14401.
[169] SEQ ID NO: 150 is the determined cDNA sequence of 14402.
[170] SEQ ID NO: 151 is the determined cDNA sequence of 14405.
[171] SEQ ID NO: 152 is the determined cDNA sequence of 14409.
[172] SEQ ID NO: 153 is the determined cDNA sequence of 14412.
[173] SEQ ID NO: 154 is the determined cDNA sequence of 14414.
[174] SEQ ID NO: 155 is the determined cDNA sequence of 14415.
[175] SEQ ID NO: 156 is the determined cDNA sequence of 14416.
[176] SEQ ID NO: 157 is the determined cDNA sequence of 14419.
[177] SEQ ID NO: 158 is the determined cDNA sequence of 14426.
[178] SEQ ID NO: 159 is the determined cDNA sequence of 14427.
[179] SEQ ID NO: 160 is the determined cDNA sequence of 14375.
[180] SEQ ID NO: 161 is the determined cDNA sequence of 14378.
[181] SEQ ID NO: 162 is the determined cDNA sequence of 14379.
[182] SEQ ID NO: 163 is the determined cDNA sequence of 14380.
[183] SEQ ID NO: 164 is the determined cDNA sequence of 14381.
[184] SEQ ID NO: 165 is the determined cDNA sequence of 14382.
[185] SEQ ID NO: 166 is the determined cDNA sequence of 14388.
[186] SEQ ID NO: 167 is the determined cDNA sequence of 14399.
[187] SEQ ID NO: 168 is the determined cDNA sequence of 14406.
[188] SEQ ID NO: 169 is the determined cDNA sequence of 14407.
[189] SEQ ID NO: 170 is the determined cDNA sequence of 14408.
[190] SEQ ID NO: 171 is the determined cDNA sequence of 14417.
[191] SEQ ID NO: 172 is the determined cDNA sequence of 14418.
[192] SEQ ID NO: 173 is the determined cDNA sequence of 14423.
[193] SEQ ID NO: 174 is the determined cDNA sequence of 14424.
[194] SEQ ID NO: 175 is the determined cDNA sequence of B726P-20.
[195] SEQ ID NO: 176 is the putative amino acid sequence of B726P-20.
[196] SEQ ID NO: 177 is a PCR primer.
[197] SEQ ID NO: 178 is the determined cDNA sequence of B726P-74.
[198] SEQ ID NO: 179 is the putative amino acid sequence of B726P-74.
[199] SEQ ID NO: 180 is the determined cDNA sequence of B726P-79.
[200] SEQ ID NO: 181 is the putative amino acid sequence of B726P-79.
[201] SEQ ID NO: 182 is the determined cDNA sequence of 19439.1, showing homology with the mamma globin gene.
[202] SEQ ID NO: 183 is the determined cDNA sequence of 19407.1, showing homology with the human keratin gene.
[203] SEQ ID NO: 184 is the determined cDNA sequence of 19428.1, showing homology with the human chromosome 17 clone.
[204] SEQ ID NO: 185 is the determined cDNA sequence of B808P (19408), showing no actual homology with any known gene.
[205] SEQ ID NO: 186 is the determined cDNA sequence of 19460.1, which shows no actual homology with any known genes.
[206] SEQ ID NO: 187 is the determined cDNA sequence of 19419.1, showing homology with the Ig kappa light chain.
[207] SEQ ID NO: 188 is the determined cDNA sequence of 19411.1, showing homology with human alpha-1 collagen.
[208] SEQ ID NO: 189 is the determined cDNA sequence of 19420.1, showing homology with mus muscle proteinase-3.
[209] SEQ ID NO: 190 is the determined cDNA sequence of 19432.1, showing homology with the human high mobility group box.
[210] SEQ ID NO: 191 is the determined cDNA sequence of 19412.1, showing homology with the human plasminogen activator gene.
[211] SEQ ID NO: 192 is the determined cDNA sequence of 19415.1, showing homology with mitogen activated protein kinase.
[212] SEQ ID NO: 193 is the determined cDNA sequence of 19409.1, showing homology with the chondroitin sulfate proteoglycan protein.
[213] SEQ ID NO: 194 is the determined cDNA sequence of 19406.1, which exhibits no substantial homology to any known gene.
[214] SEQ ID NO: 195 is the determined cDNA sequence of 19421.1, showing homology with human fibronectin.
[215] SEQ ID NO: 196 is the determined cDNA sequence of 19426.1, showing homology with Retinoic acid receptor responder 3.
[216] SEQ ID NO: 197 is the determined cDNA sequence of 19425.1, showing homology with MyD88 mRNA.
[217] SEQ ID NO: 198 is the determined cDNA sequence of 19424.1, showing homology with the peptide transporter (TAP-1) mRNA.
[218] SEQ ID NO: 199 is the determined cDNA sequence of 19429.1, which shows no actual homology with any known gene.
[219] SEQ ID NO: 200 is the determined cDNA sequence of 19435.1, showing homology with human polymorphic epithelial mucin.
[220] SEQ ID NO: 201 is the determined cDNA sequence of B813P (19434.1), showing homology with human GATA-3 transcription factor.
[221] SEQ ID NO: 202 is the determined cDNA sequence of 19461.1, showing homology with the human AP-2 gene.
[222] SEQ ID NO: 203 is the determined cDNA sequence of 19450.1, showing homology with the DNA binding regulator.
[223] SEQ ID NO: 204 is the determined cDNA sequence of 19451.1, showing homology with the Na / H exchange regulatory cofactor.
[224] SEQ ID NO: 205 is the determined cDNA sequence of 19462.1, which shows no actual homology with any known genes.
[225] SEQ ID NO: 206 is the determined cDNA sequence of 19455.1, showing homology with human mRNA for histone HAS.Z.
[226] SEQ ID NO: 207 is the determined cDNA sequence of 19459.1, showing homology with PAC clone 179N16.
[227] SEQ ID NO: 208 is the determined cDNA sequence of 19464.1, which shows no actual homology with any known genes.
[228] SEQ ID NO: 209 is the determined cDNA sequence of 19414.1, showing homology with Lipophylline B.
[229] SEQ ID NO: 210 is the determined cDNA sequence of 19413.1, showing homology with chromosome 17 clone hRPK.209_J_20.
[230] SEQ ID NO: 211 is the determined cDNA sequence of 19416.1, showing no substantial homology with any known gene.
[231] SEQ ID NO: 212 is the determined cDNA sequence of 19437.1, showing homology with human clone 24976 mRNA.
[232] SEQ ID NO: 213 is the determined cDNA sequence of 19449.1, showing homology with mouse DNA for the PG-M core protein.
[233] SEQ ID NO: 214 is the determined cDNA sequence of 19446.1, which exhibits no substantial homology to any known gene.
[234] SEQ ID NO: 215 is the determined cDNA sequence of 19452.1, which shows no actual homology with any known genes.
[235] SEQ ID NO: 216 is the determined cDNA sequence of 19483.1, showing no substantial homology with any known gene.
[236] SEQ ID NO: 217 is the determined cDNA sequence of 19526.1, showing homology with human lipophylline C.
[237] SEQ ID NO: 218 is the determined cDNA sequence of 19484.1, showing homology with the secreted cement gland protein XAG-2.
[238] SEQ ID NO: 219 is the determined cDNA sequence of 19470.1, which shows no actual homology with any known genes.
[239] SEQ ID NO: 220 is the determined cDNA sequence of 19469.1, showing homology with the human HLA-DM gene.
[240] SEQ ID NO: 221 is the determined cDNA sequence of 19482.1, showing homology with the human pS2 protein gene.
[241] SEQ ID NO: 222 is the determined cDNA sequence of B805P (19468.1), which shows no actual homology with any known genes.
[242] SEQ ID NO: 223 is the determined cDNA sequence of 19467.1, showing homology with human thrombospondine mRNA.
[243] SEQ ID NO: 224 is the determined cDNA sequence of 19498.1, showing homology with the CDC2 gene involved in cell cycle regulation.
[244] SEQ ID NO: 225 is the determined cDNA sequence of 19506.1, showing homology with human cDNA for the TREB protein.
[245] SEQ ID NO: 226 is the determined cDNA sequence of B806P (19505.1), which shows no actual homology with any known genes.
[246] SEQ ID NO: 227 is the determined cDNA sequence of 19486.1, showing homology with Type I epidermal keratin.
[247] SEQ ID NO: 228 is the determined cDNA sequence of 19510.1, showing homology with the glucose transporter for the glycoprotein.
[248] SEQ ID NO: 229 is the determined cDNA sequence of 19512.1, showing homology with the human lysyl hydroxylase gene.
[249] SEQ ID NO: 230 is the determined cDNA sequence of 19511.1, showing homology with human palymotoyl-protein thioesterase.
[250] SEQ ID NO: 231 is the determined cDNA sequence of 19508.1, showing homology with human alpha enolase.
[251] SEQ ID NO: 232 is the determined cDNA sequence of B807P (19509.1), which shows no actual homology with any known genes.
[252] SEQ ID NO: 233 is the determined cDNA sequence of B809P (19520.1), showing homology with clone 102D24 on chromosome 11q13.31.
[253] SEQ ID NO: 234 is the determined cDNA sequence of 19507.1, showing homology with the prosome beta-subunit.
[254] SEQ ID NO: 235 is the determined cDNA sequence of 19525.1, showing homology with the human pro-urokinase precursor.
[255] SEQ ID NO: 236 is the determined cDNA sequence of 19513.1, showing no substantial homology with any known gene.
[256] SEQ ID NO: 237 is the determined cDNA sequence of 19517.1, showing homology with the human PAC 128M19 clone.
[257] SEQ ID NO: 238 is the determined cDNA sequence of 19564.1, showing homology with human cytochrome P450-IIB.
[258] SEQ ID NO: 239 is the determined cDNA sequence of 19553.1, showing homology with the human GABA-A receptor pi subunit.
[259] SEQ ID NO: 240 is the determined cDNA sequence of B811P (19575.1), which shows no actual homology with any known genes.
[260] SEQ ID NO: 241 is the determined cDNA sequence of B810P (19560.1), which shows no actual homology with any known genes.
[261] SEQ ID NO: 242 is the determined cDNA sequence of 19588.1, showing homology with the aortic carboxypeptidase-like protein.
[262] SEQ ID NO: 243 is the determined cDNA sequence of 19551.1, showing homology with the human BCL-1 gene.
[263] SEQ ID NO: 244 is the determined cDNA sequence of 19567.1, showing homology with human proteasome-related mRNA.
[264] SEQ ID NO: 245 is the determined cDNA sequence of B803P (19583.1), showing no actual homology with any known genes.
[265] SEQ ID NO: 246 is the determined cDNA sequence of B812P (19587.1), showing no actual homology with any known genes.
[266] SEQ ID NO: 247 is the determined cDNA sequence of B802P (19392.2), showing homology with human chromosome 17.
[267] SEQ ID NO: 248 is the determined cDNA sequence of 19393.2, showing homology with the human Niscein B2 chain.
[268] SEQ ID NO: 249 is the determined cDNA sequence of 19398.2, showing homology with human MHC class II DQ alpha mRNA.
[269] SEQ ID NO: 250 is the determined cDNA sequence of B804P (19399.2), showing homology with human Xp22 BAC GSHB-184P14.
[270] SEQ ID NO: 251 is the determined cDNA sequence of 19401.2, showing homology with the human ikB kinase-b gene.
[271] SEQ ID NO: 252 is the determined cDNA sequence of 20266, which shows no actual homology with any known genes.
[272] SEQ ID NO: 253 is the determined cDNA sequence of B826P (20270), showing no substantial homology with any known gene.
[273] SEQ ID NO: 254 is the determined cDNA sequence of 20274, which shows no actual homology with any known genes.
[274] SEQ ID NO: 255 is the determined cDNA sequence of 20276, which shows no actual homology with any known genes.
[275] SEQ ID NO: 256 is the determined cDNA sequence of 20277, which shows no actual homology with any known genes.
[276] SEQ ID NO: 257 is the determined cDNA sequence of B823P (20280), showing no substantial homology with any known gene.
[277] SEQ ID NO: 258 is the determined cDNA sequence of B821P (20281), which shows no actual homology with any known genes.
[278] SEQ ID NO: 259 is the determined cDNA sequence of B824P (20294), which shows no actual homology with any known genes.
[279] SEQ ID NO: 260 is the determined cDNA sequence of 20303, which shows no actual homology with any known genes.
[280] SEQ ID NO: 261 is the determined cDNA sequence of B820P (20310), which shows no actual homology with any known gene.
[281] SEQ ID NO: 262 is the determined cDNA sequence of B825P (20336), which shows no actual homology with any known genes.
[282] SEQ ID NO: 263 is the determined cDNA sequence of B827P (20341), which shows no actual homology with any known genes.
[283] SEQ ID NO: 264 is the determined cDNA sequence of 20941, which shows no actual homology with any known genes.
[284] SEQ ID NO: 265 is the determined cDNA sequence of 20954, which shows no actual homology with any known genes.
[285] SEQ ID NO: 266 is the determined cDNA sequence of 20961, showing no substantial homology with any known gene.
[286] SEQ ID NO: 267 is the determined cDNA sequence of 20965, which shows no actual homology with any known genes.
[287] SEQ ID NO: 268 is the determined cDNA sequence of 20975, which exhibits no substantial homology to any known gene.
[288] SEQ ID NO: 269 is the determined cDNA sequence of 20261, showing homology with human p120 catenin.
[289] SEQ ID NO: 270 is the determined cDNA sequence of B822P (20262), showing homology with human membrane glycoprotein 4F2.
[290] SEQ ID NO: 271 is the determined cDNA sequence of 20265, showing homology with human Na, K-ATPase alpha 1.
[291] SEQ ID NO: 272 is the determined cDNA sequence of 20267, showing homology with human heart HS 90, partial cds.
[292] SEQ ID NO: 273 is the determined cDNA sequence of 20268, showing homology with the human mRNA GPI-fixed protein p137.
[293] SEQ ID NO: 274 is the determined cDNA sequence of 20271, showing homology with the human cleavage stimulating factor 77 kDa subunit.
[294] SEQ ID NO: 275 is the determined cDNA sequence of 20272, showing homology with human p190-B.
[295] SEQ ID NO: 276 is the determined cDNA sequence of 20273, showing homology with human ribophorin.
[296] SEQ ID NO: 277 is the determined cDNA sequence of 20278, showing homology with human ornithine amino transferase.
[297] SEQ ID NO: 278 is the determined cDNA sequence of 20279, showing homology with human S-adenosylmethionine synthetase.
[298] SEQ ID NO: 279 is the determined cDNA sequence of 20293, showing homology with the human x inactivated transcript.
[299] SEQ ID NO: 280 is the determined cDNA sequence of 20300, showing homology with human cytochrome p450.
[300] SEQ ID NO: 281 is the determined cDNA sequence of 20305, showing homology with human extension factor-1 alpha.
[301] SEQ ID NO: 282 is the determined cDNA sequence of 20306, showing homology with the human epithelial ets protein.
[302] SEQ ID NO: 283 is the determined cDNA sequence of 20307, showing homology with the mRNA of a human signal transfer factor.
[303] SEQ ID NO: 284 is the determined cDNA sequence of 20313, showing homology with the human GABA-A receptor pi subunit mRNA.
[304] SEQ ID NO: 285 is the determined cDNA sequence of 20317, showing homology with human tyrosine phosphatase.
[305] SEQ ID NO: 286 is the determined cDNA sequence of 20318, showing homology with human cathepsin B proteinase.
[306] SEQ ID NO: 287 is the determined cDNA sequence of 20320, showing homology with human 2-phosphopyruvate-hydratase-alpha-enolase.
[307] SEQ ID NO: 288 is the determined cDNA sequence of 20321, showing homology with human E-cadherin.
[308] SEQ ID NO: 289 is the 2032 determined cDNA sequence, which shows homology with human hsp86.
[309] SEQ ID NO: 290 is the determined cDNA sequence of B828P (20326), showing homology with the human x inactivated transcript.
[310] SEQ ID NO: 291 is the determined cDNA sequence of 20333, showing homology with the human chromatin modulator, SMARCA5.
[311] SEQ ID NO: 292 is the determined cDNA sequence of 20335, showing homology with human sphingolipid activating factor protein 1.
[312] SEQ ID NO: 293 is the determined cDNA sequence of 20337, showing homology with human hepatocyte growth factor activation inhibitory factor type 2.
[313] SEQ ID NO: 294 is the determined cDNA sequence of 20338, showing homology with the human cell adhesion molecule CD44.
[314] SEQ ID NO: 295 is the determined cDNA sequence of 20340, showing homology with human nuclear factor (erythrocyte-derived) -like 1.
[315] SEQ ID NO: 296 is the determined cDNA sequence of 20938, showing homology with human vinculin mRNA.
[316] SEQ ID NO: 297 is the determined cDNA sequence of 20939, showing homology with the human elongation factor EF-1-alpha.
[317] SEQ ID NO: 298 is the determined cDNA sequence of 20940, showing homology with the human Nestin gene.
[318] SEQ ID NO: 299 is the determined cDNA sequence of 20942, showing homology with human pancreatic ribonucleases.
[319] SEQ ID NO: 300 is the determined cDNA sequence of 20943, showing homology with human transcobalamin I.
[320] SEQ ID NO: 301 is the determined cDNA sequence of 20944, showing homology with human beta-tubulin.
[321] SEQ ID NO: 302 is the determined cDNA sequence of 20946, showing homology with the human HS1 protein.
[322] SEQ ID NO: 303 is the determined cDNA sequence of 20947, showing homology with human cathepsin B.
[323] SEQ ID NO: 304 is the determined cDNA sequence of 20948, which shows homology with human testicular enhanced gene transcript.
[324] SEQ ID NO: 305 is the determined cDNA sequence of 20949, showing homology with the human elongation factor EF-1-alpha.
[325] SEQ ID NO: 306 is the determined cDNA sequence of 20950, showing homology with human ADP-ribosylation factor 3.
[326] SEQ ID NO: 307 is the determined cDNA sequence of 20951, showing homology with human IFP53 or WRS for tryptophanyl-tRNA synthetase.
[327] SEQ ID NO: 308 is the determined cDNA sequence of 20952, showing homology with human cyclin-dependent protein kinase.
[328] SEQ ID NO: 309 is the determined cDNA sequence of 20957, showing homology with human alpha-tubulin isoform 1.
[329] SEQ ID NO: 310 is the determined cDNA sequence of 20959, showing homology with human tyrosine phosphatase-61bp deletion.
[330] SEQ ID NO: 311 is the determined cDNA sequence of 20966, showing homology with human tyrosine phosphatase.
[331] SEQ ID NO: 312 is the determined cDNA sequence of B830P (20976), showing homology with human nuclear factor NF 45.
[332] SEQ ID NO: 313 is the determined cDNA sequence of B829P (20977), showing homology with the human delta-6 fatty acid desaturase.
[333] SEQ ID NO: 314 is the determined cDNA sequence of 20978, showing homology with human nuclear aconitase.
[334] SEQ ID NO: 315 is the determined cDNA sequence of clone 23176.
[335] SEQ ID NO: 316 is the determined cDNA sequence of clone 23140.
[336] SEQ ID NO: 317 is the determined cDNA sequence of clone 23166.
[337] SEQ ID NO: 318 is the determined cDNA sequence of clone 23167.
[338] SEQ ID NO: 319 is the determined cDNA sequence of clone 23177.
[339] SEQ ID NO: 320 is the determined cDNA sequence of clone 23217.
[340] SEQ ID NO: 321 is the determined cDNA sequence of clone 23169.
[341] SEQ ID NO: 322 is the determined cDNA sequence of clone 23160.
[342] SEQ ID NO: 323 is the determined cDNA sequence of clone 23182.
[343] SEQ ID NO: 324 is the determined cDNA sequence of clone 23232.
[344] SEQ ID NO: 325 is the determined cDNA sequence of clone 23203.
[345] SEQ ID NO: 326 is the determined cDNA sequence of clone 23198.
[346] SEQ ID NO: 327 is the determined cDNA sequence of clone 23224.
[347] SEQ ID NO: 328 is the determined cDNA sequence of clone 23142.
[348] SEQ ID NO: 329 is the determined cDNA sequence of clone 23138.
[349] SEQ ID NO: 330 is the determined cDNA sequence of clone 23147.
[350] SEQ ID NO: 331 is the determined cDNA sequence of clone 23148.
[351] SEQ ID NO: 332 is the determined cDNA sequence of clone 23149.
[352] SEQ ID NO: 333 is the determined cDNA sequence of clone 23172.
[353] SEQ ID NO: 334 is the determined cDNA sequence of clone 23158.
[354] SEQ ID NO: 335 is the determined cDNA sequence of clone 23156.
[355] SEQ ID NO: 336 is the determined cDNA sequence of clone 23221.
[356] SEQ ID NO: 337 is the determined cDNA sequence of clone 23223.
[357] SEQ ID NO: 338 is the determined cDNA sequence of clone 23155.
[358] SEQ ID NO: 339 is the determined cDNA sequence of clone 23225.
[359] SEQ ID NO: 340 is the determined cDNA sequence of clone 23226.
[360] SEQ ID NO: 341 is the determined cDNA sequence of clone 23228.
[361] SEQ ID NO: 342 is the determined cDNA sequence of clone 23229.
[362] SEQ ID NO: 343 is the determined cDNA sequence of clone 23231.
[363] SEQ ID NO: 344 is the determined cDNA sequence of clone 23154.
[364] SEQ ID NO: 345 is the determined cDNA sequence of clone 23157.
[365] SEQ ID NO: 346 is the determined cDNA sequence of clone 23153.
[366] SEQ ID NO: 347 is the determined cDNA sequence of clone 23159.
[367] SEQ ID NO: 348 is the determined cDNA sequence of clone 23152.
[368] SEQ ID NO: 349 is the determined cDNA sequence of clone 23161.
[369] SEQ ID NO: 350 is the determined cDNA sequence of clone 23162.
[370] SEQ ID NO: 351 is the determined cDNA sequence of clone 23163.
[371] SEQ ID NO: 352 is the determined cDNA sequence of clone 23164.
[372] SEQ ID NO: 353 is the determined cDNA sequence of clone 23165.
[373] SEQ ID NO: 354 is the determined cDNA sequence of clone 23151.
[374] SEQ ID NO: 355 is the determined cDNA sequence of clone 23150.
[375] SEQ ID NO: 356 is the determined cDNA sequence of clone 23168.
[376] SEQ ID NO: 357 is the determined cDNA sequence of clone 23146.
[377] SEQ ID NO: 358 is the determined cDNA sequence of clone 23170.
[378] SEQ ID NO: 359 is the determined cDNA sequence of clone 23171.
[379] SEQ ID NO: 360 is the determined cDNA sequence of clone 23145.
[380] SEQ ID NO: 361 is the determined cDNA sequence of clone 23174.
[381] SEQ ID NO: 362 is the determined cDNA sequence of clone 23175.
[382] SEQ ID NO: 363 is the determined cDNA sequence of clone 23144.
[383] SEQ ID NO: 364 is the determined cDNA sequence of clone 23178.
[384] SEQ ID NO: 365 is the determined cDNA sequence of clone 23179.
[385] SEQ ID NO: 366 is the determined cDNA sequence of clone 23180.
[386] SEQ ID NO: 367 is the determined cDNA sequence of clone 23181.
[387] SEQ ID NO: 368 is the determined cDNA sequence of clone 23143.
[388] SEQ ID NO: 369 is the determined cDNA sequence of clone 23183.
[389] SEQ ID NO: 370 is the determined cDNA sequence of clone 23184.
[390] SEQ ID NO: 371 is the determined cDNA sequence of clone 23185.
[391] SEQ ID NO: 372 is the determined cDNA sequence of clone 23186.
[392] SEQ ID NO: 373 is the determined cDNA sequence of clone 23187.
[393] SEQ ID NO: 374 is the determined cDNA sequence of clone 23190.
[394] SEQ ID NO: 375 is the determined cDNA sequence of clone 23189.
[395] SEQ ID NO: 376 is the determined cDNA sequence of clone 23202.
[396] SEQ ID NO: 378 is the determined cDNA sequence of clone 23191.
[397] SEQ ID NO: 379 is the determined cDNA sequence of clone 23188.
[398] SEQ ID NO: 380 is the determined cDNA sequence of clone 23194.
[399] SEQ ID NO: 381 is the determined cDNA sequence of clone 23196.
[400] SEQ ID NO: 382 is the determined cDNA sequence of clone 23195.
[401] SEQ ID NO: 383 is the determined cDNA sequence of clone 23193.
[402] SEQ ID NO: 384 is the determined cDNA sequence of clone 23199.
[403] SEQ ID NO: 385 is the determined cDNA sequence of clone 23200.
[404] SEQ ID NO: 386 is the determined cDNA sequence of clone 23192.
[405] SEQ ID NO: 387 is the determined cDNA sequence of clone 23201.
[406] SEQ ID NO: 388 is the determined cDNA sequence of clone 23141.
[407] SEQ ID NO: 389 is the determined cDNA sequence of clone 23139.
[408] SEQ ID NO: 390 is the determined cDNA sequence of clone 23204.
[409] SEQ ID NO: 391 is the determined cDNA sequence of clone 23205.
[410] SEQ ID NO: 392 is the determined cDNA sequence of clone 23206.
[411] SEQ ID NO: 393 is the determined cDNA sequence of clone 23207.
[412] SEQ ID NO: 394 is the determined cDNA sequence of clone 23208.
[413] SEQ ID NO: 395 is the determined cDNA sequence of clone 23209.
[414] SEQ ID NO: 396 is the determined cDNA sequence of clone 23210.
[415] SEQ ID NO: 397 is the determined cDNA sequence of clone 23211.
[416] SEQ ID NO: 398 is the determined cDNA sequence of clone 23212.
[417] SEQ ID NO: 399 is the determined cDNA sequence of clone 23214.
[418] SEQ ID NO: 400 is the determined cDNA sequence of clone 23215.
[419] SEQ ID NO: 401 is the determined cDNA sequence of clone 23216.
[420] SEQ ID NO: 402 is the determined cDNA sequence of clone 23137.
[421] SEQ ID NO: 403 is the determined cDNA sequence of clone 23218.
[422] SEQ ID NO: 404 is the determined cDNA sequence of clone 23220.
[423] SEQ ID NO: 405 is the determined cDNA sequence of clone 19462.
[424] SEQ ID NO: 406 is the determined cDNA sequence of clone 19430.
[425] SEQ ID NO: 407 is the determined cDNA sequence of clone 19407.
[426] SEQ ID NO: 408 is the determined cDNA sequence of clone 19448.
[427] SEQ ID NO: 409 is the determined cDNA sequence of clone 19447.
[428] SEQ ID NO: 410 is the determined cDNA sequence of clone 19426.
[429] SEQ ID NO: 411 is the determined cDNA sequence of clone 19441.
[430] SEQ ID NO: 412 is the determined cDNA sequence of clone 19454.
[431] SEQ ID NO: 413 is the determined cDNA sequence of clone 19463.
[432] SEQ ID NO: 414 is the determined cDNA sequence of clone 19419.
[433] SEQ ID NO: 415 is the determined cDNA sequence of clone 19434.
[434] SEQ ID NO: 416 is the determined extended cDNA sequence of B820P.
[435] SEQ ID NO: 417 is the determined extended cDNA sequence of B821P.
[436] SEQ ID NO: 418 is the determined extended cDNA sequence of B822P.
[437] SEQ ID NO: 419 is the determined extended cDNA sequence of B823P.
[438] SEQ ID NO: 420 is the determined extended cDNA sequence of B824P.
[439] SEQ ID NO: 421 is the determined extended cDNA sequence of B825P.
[440] SEQ ID NO: 422 is the determined extended cDNA sequence of B826P.
[441] SEQ ID NO: 423 is the determined extended cDNA sequence of B827P.
[442] SEQ ID NO: 424 is the determined extended cDNA sequence of B828P.
[443] SEQ ID NO: 425 is the determined extended cDNA sequence of B829P.
[444] SEQ ID NO: 426 is the determined extended cDNA sequence of B830P.
[445] SEQ ID NO: 427 is the determined cDNA sequence of clone 266B4.
[446] SEQ ID NO: 428 is the determined cDNA sequence of clone 22892.
[447] SEQ ID NO: 429 is the determined cDNA sequence of clone 266G3.
[448] SEQ ID NO: 430 is the determined cDNA sequence of clone 22890.
[449] SEQ ID NO: 431 is the determined cDNA sequence of clone 264B4.
[450] SEQ ID NO: 432 is the determined cDNA sequence of clone 22883.
[451] SEQ ID NO: 433 is the determined cDNA sequence of clone 22882.
[452] SEQ ID NO: 434 is the determined cDNA sequence of clone 22880.
[453] SEQ ID NO: 435 is the determined cDNA sequence of clone 263G1.
[454] SEQ ID NO: 436 is the determined cDNA sequence of clone 263G6.
[455] SEQ ID NO: 437 is the determined cDNA sequence of clone 262B2.
[456] SEQ ID NO: 438 is the determined cDNA sequence of clone 262B6.
[457] SEQ ID NO: 439 is the determined cDNA sequence of clone 22869.
[458] SEQ ID NO: 440 is the determined cDNA sequence of clone 21374.
[459] SEQ ID NO: 441 is the determined cDNA sequence of clone 21362.
[460] SEQ ID NO: 442 is the determined cDNA sequence of clone 21349.
[461] SEQ ID NO: 443 is the determined cDNA sequence of clone 21309.
[462] SEQ ID NO: 444 is the determined cDNA sequence of clone 21097.
[463] SEQ ID NO: 445 is the determined cDNA sequence of clone 21096.
[464] SEQ ID NO: 446 is the determined cDNA sequence of clone 21094.
[465] SEQ ID NO: 447 is the determined cDNA sequence of clone 21093.
[466] SEQ ID NO: 448 is the determined cDNA sequence of clone 21091.
[467] SEQ ID NO: 449 is the determined cDNA sequence of clone 21089.
[468] SEQ ID NO: 450 is the determined cDNA sequence of clone 21087.
[469] SEQ ID NO: 451 is the determined cDNA sequence of clone 21085.
[470] SEQ ID NO: 452 is the determined cDNA sequence of clone 21084.
[471] SEQ ID NO: 453 is the first partial cDNA sequence of clone 2BT1-40.
[472] SEQ ID NO: 454 is the second partial cDNA sequence of clone 2BT1-40.
[473] SEQ ID NO: 455 is the determined cDNA sequence of clone 21063.
[474] SEQ ID NO: 456 is the determined cDNA sequence of clone 21062.
[475] SEQ ID NO: 457 is the determined cDNA sequence of clone 21060.
[476] SEQ ID NO: 458 is the determined cDNA sequence of clone 21053.
[477] SEQ ID NO: 459 is the determined cDNA sequence of clone 21050.
[478] SEQ ID NO: 460 is the determined cDNA sequence of clone 21036.
[479] SEQ ID NO: 461 is the determined cDNA sequence of clone 21037.
[480] SEQ ID NO: 462 is the determined cDNA sequence of clone 21048.
[481] SEQ ID NO: 463 is the conserved cDNA sequence of B726P (referred to as B726P-spliced_seq_B726P).
[482] SEQ ID NO: 464 is the determined cDNA sequence (referred to as 27490.seq_B726P) of the second splice form of B726P.
[483] SEQ ID NO: 465 is the determined cDNA sequence (referred to as 27068.seq_B726P) of the third splice form of B726P.
[484] SEQ ID NO: 466 is the determined cDNA sequence (referred to as 23113. seq_B726P) of the second splice form of B726P.
[485] SEQ ID NO: 467 is the determined cDNA sequence (referred to as 23103.seq_B726P) of the second splice form of B726P.
[486] SEQ ID NO: 468 is the determined cDNA sequence (referred to as 19310.seq_B726P) of the second splice form of B726P.
[487] SEQ ID NO: 469 is the putative amino acid sequence encoded by the upstream ORF of SEQ ID NO: 463.
[488] SEQ ID NO: 470 is the putative amino acid sequence encoded by SEQ ID NO: 464.
[489] SEQ ID NO: 471 is the putative amino acid sequence encoded by SEQ ID NO: 465.
[490] SEQ ID NO: 472 is the putative amino acid sequence encoded by SEQ ID NO: 466.
[491] SEQ ID NO: 473 is the putative amino acid sequence encoded by SEQ ID NO: 467.
[492] SEQ ID NO: 474 is the determined cDNA sequence for an alternative splice form of B726P.
[493] SEQ ID NO: 475 is the amino acid sequence encoded by SEQ ID NO: 474.
[494] SEQ ID NO: 476 is the isolated cDNA sequence of B720P.
[495] SEQ ID NO: 477 is the cDNA sequence of a known keratin gene.
[496] SEQ ID NO: 478 is the amino acid sequence encoded by SEQ ID NO: 477.
[497] SEQ ID NO: 479 is the determined cDNA sequence for clone 19465.
[498] SEQ ID NOs: 480 and 481 are PCR primers.
[499] SEQ ID NO: 482 is the cDNA sequence for the expressed downstream ORF of B726P.
[500] SEQ ID NO: 483 is the amino acid sequence for the expressed recombinant downstream ORF of B726P.
[501] SEQ ID NO: 484 is the determined full length cDNA sequence for B720P.
[502] SEQ ID NO: 485 is the amino acid sequence encoded by SEQ ID NO: 484.
[503] SEQ ID NO: 486 is a determined cDNA sequence of the truncated form of B729P (referred to as B720P-tr).
[504] SEQ ID NO: 487 is the amino acid sequence of B720P-tr.
[505] SEQ ID NO: 488 is the amino acid sequence of a naturally processed epitope of B726P recognized by B726P-specific CTL.
[506] SEQ ID NO: 489 is a DNA sequence expressing a B726P epitope set forth in SEQ ID NO: 488.
[507] SEQ ID NO: 490 is a mammagglobin fused to B726P combined upstream and downstream open reading frame (ORF), wherein the amino acid sequence of this B726P combined ORF is described herein as SEQ ID NO: 475 encoded by the DNA sequence of SEQ ID NO: 474. DNA sequence encoding the fusion protein.
[508] SEQ ID NO: 491 is a DNA sequence encoding a fusion protein in which a mammaglobin is fused to a B726P upstream ORF (the amino acid sequence of this B726P upstream ORF is described herein as SEQ ID NO: 469 encoded by the DNA sequence of SEQ ID NO: 463).
[509] SEQ ID NO: 492 is a DNA sequence encoding a fusion protein into which a mammaglobin is fused to a B726P downstream ORF (the amino acid sequence of this B726P downstream ORF is described herein as SEQ ID NO: 176 encoded by the DNA sequence of SEQ ID NO: 175).
[510] SEQ ID NO: 493 is an amino acid sequence encoded by a DNA sequence of SEQ ID NO: 490.
[511] SEQ ID NO: 494 is an amino acid sequence encoded by the DNA sequence of SEQ ID NO: 491.
[512] SEQ ID NO: 495 is an amino acid sequence encoded by the DNA sequence of SEQ ID NO: 492.
[513] As noted above, the present invention generally relates to compositions for the treatment and diagnosis of cancer, for example breast cancer, and methods of using such compositions in the treatment and diagnosis of the breast cancer. Specific examples of the compositions described herein include breast tumor polypeptides, polynucleotides encoding such polypeptides, binding agents, such as antibodies, antigen presenting cells (APCs) and / or immune system cells (eg T cells). The term “breast tumor protein” as used herein generally refers to a breast tumor at a level that is at least two times, preferably at least five times greater than the level of expression in normal tissue, as determined using the representative assays provided herein. Refers to a protein expressed in a cell. Certain breast tumor proteins are tumor proteins that react with the antisera of patients with breast cancer at detectable levels (immunoassays, such as in an ELISA or Western blot).
[514] Thus, and as described further below, the present invention relates to polynucleotides having the sequences set forth in SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489. Composition; A polypeptide composition having the amino acid sequence set forth in SEQ ID NOs: 176, 179, 181, 469-473, 475, 485, 487, and 488; Antibody compositions that can bind such polypeptides and numerous additional embodiments that utilize such compositions, for example, in the detection, diagnosis, and / or treatment of human breast cancer, are provided.
[515] Polynucleotide composition
[516] As used herein, the terms “DNA segment” and “polynucleotide” refer to DNA molecules isolated from whole genomic DNA of a particular species. Thus, a DNA segment encoding a polypeptide refers to a DNA segment containing one or more coding sequences that are substantially separated from, or purified from, the entire genomic DNA of the species from which the DNA segment is obtained. The terms "DNA segment" and "polynucleotide" include DNA segments and smaller fragments of such segments, and recombinant vectors such as plasmids, cosmids, phagemids, phages, viruses, and the like.
[517] As will be appreciated by those skilled in the art, the DNA segments of the present invention are genomic sequences, extra-genomic and plasmid-encoded sequences and more genetically engineered that can be expressed or adapted to express proteins, polypeptides, peptides, and the like. Small gene segments may be included. Such segments can be naturally isolated or can be manually modified synthetically.
[518] As used herein, “isolated” means that a large portion of the coding DNA, for example, a large chromosomal fragment or other functional gene, in which the polynucleotide has substantially no other coding sequence and the DNA segment is not involved, or It does not contain a polypeptide coding sequence. Of course, this refers to the DNA segment as originally isolated and does not manually exclude coding regions or genes that are later added to the segment.
[519] As will be appreciated by those skilled in the art, polynucleotides may be single stranded (coding or antisense) or double stranded, and may be DNA (genome, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules that contain introns and correspond to DNA molecules in a one-to-one manner, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences may be present in the polynucleotides of the invention, but need not necessarily exist, and the polynucleotides may be linked to other molecules and / or support materials, but need not necessarily be linked.
[520] The polynucleotides may comprise native sequences (ie, endogenous sequences encoding breast tumor proteins or portions thereof) or may include variants or biological or antigenic functional equivalents of such sequences. The polynucleotide variant may contain one or more substituents, adducts, deletions and / or inserts, as further described below, preferably wherein the immunogenicity of the encoded polypeptide is compared with the original tumor protein. Should not be degraded. The effect on the immunogenicity of the encoded polypeptide can generally be assessed as described herein. The term “variant” also encompasses homologous genes of heterologous origin.
[521] When comparing polynucleotide or polypeptide sequences, two sequences are considered "identical" if the nucleotide sequence or amino acid sequence in both sequences is identical when aligned for maximum correspondence, as described below. Comparison between two sequences is typically performed by comparing the sequences through a comparison window that identifies and compares local regions with sequence similarity. As used herein, a “comparison window” is about 20 or more, typically 30 to about 75, 40, that allows for optimal alignment of two sequences and then one sequence can be compared with a reference sequence of the same number of contiguous positions. To segments of about 50 contiguous positions.
[522] Optimal alignment of sequences for comparison uses omission parameters, using the Megalign program (Lasergene, WI, DNASTAR, Inc., WI) within the Lasergene Suite of Bioinformatics software. Can be done. Such programs include several alignment schemes described in the following references: Dayhoff, MO (1978) A model of evolutionary change in protein-Matrices for detecting distant relationships; Dayhoff, MO (ed.) Atlas of Proteins 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, DG and Sharp, PM (1989) CABIOS 5: 151-153; Myers, EW and Muller W. (1988) CABIOS 4: 11-17; Robinson, ED (1971) Comb. Theor 11 : 105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4 : 406-425; Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy-the Principles and Practice fo Numerical Taxonomy , Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ (1983) Proc. Natl. Acad., Sci. USA 80 : 726-730.
[523] On the other hand, optimal alignment of sequences for comparison is described in Smith and Waterman (1981) Add. APL. Math. 2: 482 local equality algorithm; See Needleman and Wunshc (1970) J. Mol. Biol. 48: 443; See Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444; a method for similarity; Transitioning these algorithms to computers [GAP, BESTFIT, BLAST, FASTA and TFASTA by Wisconsin Genetics Software Package, Genetics Computer Group (GCG) (575 Science Dr., Madison, Wis.)]; Or by inspection.
[524] One preferred example of a suitable algorithm for determining sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25: 3389-3402; and Altschul et al. (1990) J. Mol. Biol. 215: 403-410. BLAST and BLAST 2.0 can use the parameters described herein to determine, for example, sequence identity for the polynucleotides and polypeptides of the invention. Software for performing BLAST analysis is available from the National Center for Biotechnology Information. In one example, for nucleotide sequences, the cumulative score is calculated using parameters M (compensation score for matching residue pairs; always> 0) and N (penalty score for mismatching residues; always <0). Can be calculated For amino acid sequences, a scoring matrix can be used to calculate cumulative scores. The cumulative alignment score drops by an amount X from its maximum achievement; Due to accumulation of one or more negative-scoring residue alignments, the cumulative score becomes zero or less; Or when reaching the end of either sequence, the expansion of word hit in each direction is stopped. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) is omitted for word length (W) of 11, expected value of 10 (E) and BLOSUM62 scoring matrix (Henikoff and Henikoff (1989) P roc. Natl. Acad. Sci. USA 89: 10915] comparisons for alignment, 50 (B), expected value (E) of 10, M = 5, N = -4 and both chains.
[525] Preferably, “sequence identity” is determined by comparing two optimally aligned sequences through a comparison window of 20 or more positions, wherein a portion of the polynucleotide or polypeptide sequence within the comparison window is the optimal of the two sequences. It may comprise up to 20%, typically 5-15% or 10-12%, additions or deletions (ie gaps) compared to the reference sequence (not including adducts or deletions) for alignment. This percentage% determines the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences, yielding the number of matched positions; The number of matched positions thus calculated is divided by the total number of positions in the reference sequence (ie window size); The result is calculated by multiplying the result by 100 to calculate the sequence identity rate.
[526] Accordingly, the present invention is directed to using polynucleotide and polypeptide sequences that exhibit significant identity with the sequences described herein, for example, the methods described herein (e.g., BLAST analysis using standard parameters, as described below). Sequence identity at least 50%, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% compared to the polynucleotide or polypeptide sequences of the invention Polynucleotide and polypeptide sequences that are at least 97%, 98% or 99%. Those skilled in the art will recognize that by appropriately adjusting these values, one can determine the corresponding identity of the protein encoded by the two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, read frame position, and the like.
[527] In a further aspect, the present invention provides isolated polynucleotides and polypeptides comprising extensions of various lengths in succession of sequences that are identical or complementary to one or more sequences described herein. For example, at least about 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, or 1000 consecutive nucleotides of one or more sequences described herein, as well as all intermediate lengths therebetween. Polynucleotides comprising the nucleotides of are provided by the present invention. In this context “medium length” means any length between the recited values, eg, 16, 17, 18, 19, and the like; 21, 22, 23, and the like; 30, 31, 32, and the like; 50, 51, 52, 53, and the like; 100, 101, 102, 103, and the like; It will be readily appreciated that it means 150, 151, 152, 153, etc. (including any integer between 200-500, 500-1,000, etc.).
[528] The polynucleotides or fragments thereof of the present invention may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., regardless of the length of the coding sequence itself. As they can, their overall length can vary considerably. Accordingly, it is contemplated that nucleic acid fragments of almost any length can be used, with the total length being preferably limited depending on the ease of manufacture and the use in the intended recombinant DNA protocol. For example, DNA segments exemplified as having a total length of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs (including all intermediate lengths) It is considered useful for many implementations.
[529] In other embodiments, the invention relates to polynucleotides, or fragments or complements thereof, that are capable of hybridizing with the polynucleotide sequences provided herein under suitably stringent conditions. Hybridization techniques are well known in the field of molecular biology. For illustrative purposes, suitable stringent conditions suitable for testing the hybridization of polynucleotides and other polynucleotides of the present invention include pre-washing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA, pH 8.0; Hybridization overnight at 50-65 ° C., 5 × SSC; Washing twice at 65 ° C. for 20 minutes using 2 ×, 0.5 × and 0.2 × SSCs containing 0.1% SDS, respectively.
[530] Furthermore, one of ordinary skill in the art will recognize that due to the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides as described herein. Some of these polynucleotides may be combined with the nucleotide sequence of any native gene. Minimal homology. Nevertheless, polynucleotides that have been diversified due to differences in codon utilization are specifically contemplated by the present invention. In addition, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered due to deletion, addition and / or substitution of one or more mutations, eg, nucleotides. The mRNAs and proteins thus produced may, but need not, have a changed structure or function. Alleles can be identified using standard techniques (eg, hybridization, amplification, and / or database sequence comparison).
[531] Probes and Primers
[532] In other embodiments of the present invention, the polynucleotide sequences provided herein can advantageously be used as probes or primers for nucleic acid hybridization. As such, it is contemplated that a nucleic acid segment comprising a sequence region of about 15 or more nucleotides in length contiguous sequence having a sequence identical to or complementary to the 15 nucleotides in length contiguous sequences described herein will have particular use. . Longer contiguous identical or complementary sequences such as about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even full length sequences will be used in certain embodiments.
[533] Such nucleic acid probes have the ability to specifically hybridize with the sequence of interest such that the probe can be used to detect the presence or absence of complementary sequences in a given sample. However, other uses, such as the use of sequence information to prepare primers for use in the preparation of mutant species primers or other genetic constructs, are also contemplated.
[534] Sequence region consisting of 10-14, 15-20, 30, 50 or even 100-200 nucleotides (including intermediate lengths) in sequence, identical or complementary to the polynucleotide sequences described herein, Polynucleotide molecules having are particularly contemplated as probes for hybridization, for example for use in Southern and Northern blotting. This allows gene products or fragments thereof to be analyzed in various bacterial cells as well as in various cell types. The total size of the fragments as well as the size of the complementary extension (s) will ultimately depend on the desired use or application of the particular nucleic acid segment. Smaller fragments will generally be used in hybridization embodiments, where the length of contiguous complementarity regions may vary from about 15 to about 100 nucleotides, depending on the length of the complementary sequence to be detected, resulting in larger contiguous complementarity extensions. Can be used.
[535] By using hybridization probes of about 15 to 25 nucleotides in length, information regarding stable and selective double-stranded molecules is allowed. However, in order to improve the quality and level of specific hybrid molecules obtained by increasing the stability and selectivity of hybrids, molecules with complementary sequences that are continuous over extensions greater than 15 bases in length are generally preferred. It would generally be desirable to design nucleic acid molecules with 15-25 consecutive nucleotides, or optionally even longer, gene-complementary extensions of nucleotides.
[536] Hybridization probes can be selected from any portion of all sequences described herein. The sequence set forth in SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489, to be utilized as a probe or primer, or such sequence that is about 15 to 25 nucleotides in length It is a requirement to consider all contiguous parts of (including sequences of full length). The choice of probe and primer sequences can be determined by a variety of factors. For example, primers from the terminal side of the entire sequence may be used.
[537] Small polynucleotide segments or fragments can be readily prepared, for example, by direct synthesis of such fragments by chemical means, as is commonly practiced using automated oligonucleotide synthesizers. In addition, fragments can be applied to nucleic acid regeneration techniques, such as by applying the PCR ™ technique of US Pat. No. 4,683,202, which is incorporated herein by reference, methods of introducing selected sequences into recombinant production for recombinant production, and molecules. It can be obtained by other recombinant DNA techniques generally known to those skilled in the biology field.
[538] To determine the ability to selectively form complementary extensions and double-stranded molecules of the entire gene or gene fragment of interest, the nucleotide sequences of the invention can be used. Depending on the field of application contemplated, it is typically desired to use various hybridization conditions to achieve varying selectivity of the probe for the target sequence. For applications requiring high selectivity, relatively stringent conditions will have to be used to form the hybrids, for example, salts of from about 0.02 M to about 0.15 M salts at temperatures of about 50 ° C. to about 70 ° C. As indicated by the concentration, relatively low salt and / or high temperature conditions should be chosen. However, if there is a mismatch between the probe and the template or target chain, it will be particularly difficult to tolerate these optional conditions, which would be particularly suitable for separating related sequences.
[539] Of course, for some applications, for example, to prepare a mutant using a mutant primer chain hybridized with an underlying template, it is typically less likely to allow the formation of hetero-backbones. Strict (restricted) hybridization conditions will be required. Under such circumstances, it may be desirable to use a temperature of about 20 ° C. to about 55 ° C. and salt conditions of about 0.15 M to about 0.9 M. Thereby, the species hybridizing cross-hybrid can be easily identified as a signal that hybridizes positively compared to the control hybridization. In any case, it is readily recognized that the conditions can be made more stringent by adding increasing amounts of formamide which act to destabilize the hybrid double strand in the same manner as the temperature increase. Thus, the hybridization conditions can be easily manipulated, which would be one method of selection depending on the desired result.
[540] Polynucleotide Identification and Identification
[541] Polynucleotides can be identified, prepared and / or manipulated using a variety of well established techniques. For example, by screening a microarray of cDNA to determine tumor related expression (ie, expression that occurs more than twice as often in tumors as determined using representative assays provided herein), Polynucleotides can be identified as described in more detail below. Such screens can be performed according to manufacturer's instructions using, for example, Synteni microarrays (Palo Alto CA) and are essentially described in 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. Alternatively, polynucleotides can be amplified from cDNA prepared from cells expressing the proteins described herein, eg, breast tumor cells. Such polynucleotides can be amplified via polymerase chain reaction (PCR). For this approach, sequence-specific primers can be designed based on the sequences provided herein, which can be purchased or synthesized.
[542] The amplified portion of the polynucleotides of the present invention can be used to isolate full length genes from suitable libraries (eg, tumor breast cDNA libraries) using well known techniques. Within this technique, the library (cDNA or genome) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, the library is size-selected to include larger molecules. Randomly primed libraries may be suitable for identifying the 5 'and upstream regions of a gene. Genomic libraries are preferred for obtaining introns and extending the 5 'sequence.
[543] In the case of hybridization techniques, well-known sequences can be used to label partial sequences (eg, nick-detox or end-label at ³² P). Subsequently, bacterial or bacteriophage libraries are generally screened by hybridizing filters containing denatured bacterial colonies (or lawns containing phage plaques) with labeled probes as described above. Sambrook et al. , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989. Colonies or plaques that hybridize are screened and expanded and DNA is isolated for further analysis. cDNA clones can be analyzed to determine additional sequence amounts by PCR using primers from these partial sequences and primers from vectors. Restriction maps and partial sequences can be generated to identify one or more overlapping clones. The complete sequence can then be determined using standard techniques, which can include generating a series of deletion clones. The resulting overlapping sequences can then be assembled into a single continuous sequence. By linking suitable fragments using well known techniques, one can generate full length cDNA molecules.
[544] On the other hand, there are a number of amplification techniques for obtaining full length coding sequences from partial cDNA sequences. Within this technique, amplification is generally performed via PCR. Any commercially available kit can be used to perform the amplification step. For example, primers can be designed using software well known in the art. The primer is preferably 22-30 nucleotides in length, has a GC content of at least 50%, and anneals to the target sequence at a temperature of about 68-70 ° C. The regions thus amplified can be sequenced as mentioned above, and the overlapping sequences are assembled into contiguous sequences.
[545] One such amplification technique is inverse PCR, which uses restriction enzymes to generate specific fragments in known regions of genes. See Triglia et al., Nucl. Acids Res. 16 : 8186, 1988]. The fragments are then circularized by intramolecular linkage and used as templates for PCR with various primers derived from known regions. Within another approach, sequences adjacent to partial sequences can be searched by amplification with primers for linker sequences and primers specific for known regions. With this amplified sequence, two rounds of amplification are typically performed with the same linker primer and a second primer specific for the known region. A modification to this process is described in WO 96/38591, using two primers that initiate extension in opposite directions from known sequences. Another such technique is known as "a rapid amplification technique of the cDNA terminus" or RACE. The technique involves identifying sequences that are 5 'or 3' of a known sequence using internal and external primers that hybridize with the poly A region or vector sequence. Additional techniques include capture PCR (Lagerstrom et al., PCR Methods Applic. 1: 111-19, 1991] and walking PCR (Parker et al., Nucl. Acids. Res. 19: 3055-60, 1991. Other techniques using amplification can also be used to obtain full length cDNA sequences.
[546] In certain cases, it is possible to obtain a full length cDNA sequence by analyzing the sequences provided in the expressed sequence tag (EST) database, such as those available from GenBank. Investigations of overlapping ESTs can generally be performed using well known programs (eg, NCBI BLAST surveys), and these ESTs can be used to generate contiguous, full length sequences. Full length cDNA sequences can also be obtained by analyzing genomic fragments.
[547] Polynucleotide Expression in Host Cells
[548] In other embodiments of the invention, a polypeptide of the invention, or a polynucleotide or fragment thereof encoding a fusion protein or functional equivalent thereof, may be used in a recombinant DNA molecule to direct expression of the polypeptide in a suitable host cell. have. Due to the inherent degeneracy of the genetic code, it is possible to generate other DNA sequences that encode substantially identical or functionally equivalent amino acid sequences, which can be used to clone and express certain polypeptides. .
[549] As will be appreciated by those skilled in the art, in some cases it may be advantageous to generate polypeptide-encoding nucleotide sequences that possess non-natural codons. For example, selection of a preferred codon by a particular prokaryotic or eukaryotic host can increase the rate of protein expression, or have a longer half-life than the half-life of the transcript generated from the desired property, eg, a natural sequence. Recombinant RNA transcripts can be generated.
[550] Moreover, the polynucleotide sequences of the present invention are generally known in the art to vary polypeptide coding sequences for various reasons, including but not limited to modifications that modify the cloning, processing and / or expression of gene products. Engineering can be done using known methods. For example, nucleotide sequences can be engineered using DNA shuffling by PCR reassembly and random fragmentation of gene fragments and synthetic oligonucleotides. In addition, site-directed mutagenesis can be used to insert new restriction sites, change glycosylation patterns, change codon preferences, generate splice variants, or introduce mutations.
[551] In another aspect of the invention, a naturally modified or recombinant nucleic acid sequence can be linked to a heterologous sequence to encode a fusion protein. For example, to screen peptide libraries for polypeptide activity inhibitors, it may be useful to encode chimeric proteins that can be recognized by commercial antibodies. The fusion protein can also be engineered to contain a cleavage site located between the polypeptide-encoding sequence and the heterologous protein sequence so that the polypeptide can be cleaved and purified from the heterologous moiety.
[552] Sequences encoding the polypeptide of interest can be synthesized completely 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]. Alternatively, the protein itself may be produced using chemical methods of synthesizing the amino acid sequence of a polypeptide or a portion thereof. For example, peptide synthesis can be performed using various solid phase techniques, see Roberge, J. Y. et al. (1995) Science 269: 202-204, for example, an ABI 431A peptide synthesizer (Perkin Elmer, Palo Alto, Calif.) Can be used to achieve automated synthesis.
[553] The newly synthesized peptides can be prepared by formulated high performance liquid chromatography (Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, NY) or other comparable technology available in the art. It can be substantially purified by. The composition of the peptide thus synthesized can be confirmed by amino acid analysis or sequence analysis (eg, Edman degradation process). Additionally, the amino acid sequence of the polypeptide, or portions thereof, can be changed during direct synthesis and / or combined with sequences from other proteins or portions thereof using chemical methods to generate variant polypeptides.
[554] To express a polypeptide of interest, a nucleotide sequence encoding such polypeptide or a functional equivalent thereof can be inserted into a suitable expression vector, ie, a vector containing elements necessary for the transcription and translation of the inserted coding sequence. Methods well known to those skilled in the art can be used to construct expression vectors containing sequences encoding polypeptides of interest and containing appropriate transcriptional and translational regulatory elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination methods. Such techniques are described in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F.M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
[555] Various expression vectors / host systems can be utilized to contain and express polynucleotide sequences. These include bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; Yeast transformed with yeast expression vectors; Insect cell systems infected with virus expression vectors (eg, baculovirus); Viral expression vectors such as cauliflower mosaic virus (CaMV); Tobacco mosaic virus (TMV)] or bacterial expression vector (eg Ti or pBR322 plasmid); Or animal cell systems, including but not limited to.
[556] A “regulatory element” or “regulatory sequence” present in an expression vector is a non-translated region of that vector that interacts with the host cellular protein to perform transcription and translation--enhancer, promoter, 5 'and 3' non- Decryption realm. These factors can vary in strength and specificity. Depending on the vector system and host employed, any suitable number of transcriptional and translational elements may be used, including homeostatic and inducible promoters. For example, when cloning in a bacterial system, an inducible promoter such as PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) Or hybrid lacZ promoter of PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD) can be used. In mammalian cell systems, promoters from mammalian genes or mammalian viruses are generally preferred. If it is necessary to generate cell lines containing multiple copies of the sequence encoding a particular polypeptide, it may be advantageous to use SV40 or EBV-based vectors with appropriate selection markers.
[557] In bacterial systems, a number of expression vectors can be selected depending on the intended use for the expressed polypeptide. For example, if a large amount is needed to induce antibodies, a vector may be used that directs the expression of high levels of readily purified fusion proteins. These vectors contain multifunctional E. coli. E. coli cloning and expression vectors, for example BLUESCRIPT (Stratagene), wherein the sequence encoding the polypeptide of interest is the sequence for the amino terminal Met of beta-galactosidase and the subsequent seven residues. May be linked into the vector within the same frame to produce a hybrid protein; pIN vectors [Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509, etc., but is not limited thereto. Foreign polypeptides can also be expressed as fusion proteins with glutathione S-transferase (GST) using pGEX vectors (Promega, Madison, Wis.). In general, such fusion proteins are soluble and can be readily purified from fused cells by adsorption to glutathione-agarose beads and then eluting in the presence of free glutathione. Proteins made in such a system can be designed to contain heparin, thrombin, or factor XA protease cleavage sites to release the cloned polypeptide of interest from the GST residue at will.
[558] In yeast Saccharomyces cerevisiae, numerous vectors containing homeostatic or inducible promoters such as alpha factor, alcohol oxidase and PGH can be used. See Ausubel et al. (See above) and Grant et al. (1987) Methods Enzymol. 153: 516-544.
[559] When using plant expression vectors, expression of the sequence encoding the polypeptide can be driven by any number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with omega leader sequences from TMV. Takamatsu, N. (1987) EMBO J. 6: 307-311. Alternatively, plant promoters such as small subunits or heat shock promoters of RUBISCO can be used. See 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 constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are generally described in the numerous literature available. Hobbs, S. or Murry, L.E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y .; pp. 191-196.
[560] Insect systems may also be used to express a polypeptide of interest. For example, in one such system, Autographa california nuclear polyhedrosis virus (AcNPV) is used in Spodoptera frugiperda cells or Trichoplusia larvae. It is used as a vector for expressing a foreign gene. The sequence encoding the polypeptide is cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of this polyhedrin promoter. Successful insertion of this polypeptide-encoding sequence will result in inactivation of the polyhedrin gene and production of a recombinant virus lacking a coat protein. Then, using the recombinant virus, for example, S. a. It can infect S. frugiperda cells or Tricoflucia larva, where the polypeptide of interest can be expressed. Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. 91: 3224-3227.
[561] In mammalian host cells, a number of virus-utilized expression systems are generally available. For example, when adenoviruses are used as expression vectors, sequences encoding polypeptides of interest can be linked to adenovirus transcription / detoxification complexes consisting of late promoters and three-part leader sequences. Insertions in the non-essential El or E3 regions of the viral genome can be used to obtain viable viruses capable of expressing the polypeptide in infected host cells. See Logan, J. and Shenk, T. ( 1984) Proc. Natl. Acad. Sci. 81: 3655-3659. In addition, transcriptional enhancers, such as the Raus sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
[562] Specific initiation signals may be used to more efficiently translate sequences encoding polypeptides of interest. Such signals include ATG start codons and contiguous sequences. When inserting the sequence encoding the polypeptide, its start codon and upstream sequence into a suitable expression vector, no additional transcriptional or translational control signal may be required. However, if only the coding sequence or portion thereof is inserted, an exogenous translational control sequence comprising the ATG initiation codon should be provided. Furthermore, the start codon must be in the correct reading frame to ensure readout of the entire insert. Exogenous detoxification elements and initiation codons can be of various origins, both natural and synthetic. Expression efficiency is described by Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162 may be augmented by incorporating an appropriate enhancer into the particular cellular system used.
[563] It is also possible to select host cell strains which have the ability to regulate the expression of the inserted sequences or to process the expressed proteins in the desired manner. Modifications of such polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing that cleaves the “prepro” form of the protein may be used to facilitate correct insertion, folding and / or function. Different host cells, such as CHO, HeLa, MDCK, HEK293 and WI38, which have specific cellular mechanisms and characteristic mechanisms for this post-translational activity can be selected to ensure accurate modification and processing of foreign proteins. .
[564] In order to produce recombinant proteins in high yield for a long time, stable expression is generally preferred. For example, expression vectors that may contain viral replication origin and / or endogenous expression elements and selection markers on the same vector or on separate vectors may be used to transform cell lines stably expressing the polynucleotide of interest. Can be. After introduction of these vectors, cells can be grown in enrichment medium for 1 to 2 days and then they can be run in selection medium. The purpose of the selection marker is to confer resistance to the selection, and its presence allows the growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type.
[565] Numerous screening systems can be used to recover transformed cell lines. These include herpes simplex virus thymidine kinase, which can be used for tk.sup.- or aprt.sup.-cells, respectively. Wigler, M. et al. (1977) Cell 11: 223-32] and adenine phosphoribosyltransferases [Lowy, I. et al. (1990) Cell 22: 817-23, but are not limited to these. In addition, antimetabolic, antibiotic or herbicide tolerance can be used as a screening criterion; For example, dhfr confers resistance to methotrexate [Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77: 3567-70; Npt confers resistance to aminoglycosides, neomycin and G-418 [Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14 and als and pat [Murry supra] which confer resistance to chlorsulfuron and phosphinothricin acetyltransferase, respectively. Additional selection genes such as trpB, which allows cells to utilize indole instead of tryptophan, or hisD, which allows cells to utilize histinol instead of histidine, are described in Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85: 8047-51. Recently, the use of visible markers has gained popularity, and markers such as anthocyanins, beta-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin are not only widely used to identify transformants, It is widely used to quantify transient or stable protein expression levels that may contribute to specific vector systems. Rhodes, CA et al. (1995) Methods Mol. Biol. 55: 121-131.
[566] Although the presence / absence of marker gene expression suggests that the gene of interest exists, there is a need to confirm its presence and expression. For example, when inserting a sequence encoding a polypeptide into a marker gene sequence, recombinant cells containing the sequence can be identified as lacking marker gene function. Alternatively, the marker gene can be positioned in line with the polypeptide coding sequence under the control of a single promoter. Expression of marker genes in response to induction or selection usually directs the expression of the in-line genes.
[567] On the other hand, host cells containing and expressing the desired polynucleotide sequences can be identified by a variety of procedures known to those skilled in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques, which include membrane, solution, or chip utilized techniques for detecting and / or quantifying nucleic acids or proteins. It is not limited.
[568] Various protocols are known in the art for detecting and measuring expression of polynucleotide-encoded products using polyclonal or monoclonal antibodies specific for that product. Examples thereof include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site monoclonal used immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide may be preferred for some applications, but competitive binding assays may also be used. . These and other assays are described, inter alia, in 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).
[569] A wide range of labels and conjugation techniques are known to those skilled in the art and can be used in a variety of nucleic acid and amino acid assays. Means for generating labeled hybridization or PCR probes for detecting sequences associated with polynucleotides include PCR amplification using oligolabeling, nick translation, end-labeling or labeled nucleotides. Alternatively, the sequence, or portions thereof, can be cloned into a vector to generate an mRNA probe. Such vectors are known in the art and are commercially available and can be used to synthesize RNA probes in vitro by adding appropriate RNA polymerases such as T7, T3 or SP6 and labeled nucleotides. These procedures can be performed using various commercial kits. Suitable reporter molecules or labels that can be used include radionuclides, enzymes, phosphors, chemiluminescent or pigmenting agents, as well as substrates, cofactors, inhibitors, magnetic particles and the like.
[570] Host cells transformed with the polynucleotide sequence of interest can be cultured under conditions suitable for the expression and recovery of the protein of interest from the cell culture. Proteins produced by recombinant cells may be secreted intracellularly or contained within cells, depending on the sequence and / or vector used. As will be appreciated by those skilled in the art, expression vectors containing polynucleotides of the invention can be designed to contain signal sequences that direct the secretion of the encoded polypeptide through prokaryotic or eukaryotic cell membranes. Other recombinant construction techniques can be used to link a sequence encoding a polypeptide of interest to a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins. Such purification facilitation domains include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals; Protein A domains that allow purification on immobilized immunoglobulins and domains used in FLAGS extension / affinity purification systems (Immunex Corp., Seattle, Wash.) Are included, but are not limited thereto. A cleavable linker sequence between the purification domain and the encoded polypeptide may be incorporated, for example, a linker sequence specific for Factor XA or enterokinase (Invitrogen, San Diego, Calif.) To facilitate purification. This expression vector expresses a fusion protein containing the polypeptide of interest and the nucleic acid encoding the six histidine residues in front of the thioredoxin or enterokinase cleavage site. Such histidine residues are described in Porath, J. et al. (1992, Prot. Exp. Purif. 3: 263-281), while promoting purification on IMIAC (immobilized metal ion affinity chromatography), whereas the enterokinase cleavage site is the desired protein from the fusion protein. It provides a means to purify the polypeptide. A discussion of vectors containing fusion proteins is provided in Kroll, D.J. et al. (1993; DNA Cell Biol. 12: 441-453).
[571] In addition to recombinant production methods, the polypeptides and fragments thereof of the invention can be prepared by direct peptide synthesis using solid phase techniques. See Merrifield J. (1963) J. Am. Chem. Soc. 85: 2149-2154. Protein synthesis can be performed using manual or automated techniques. Automated synthesis is described, for example, in the following automated machines: Applied Biosystems 431A Peptide Synthesizer; Perkin Elmer]. Alternatively, the various fragments can be chemically separated and combined using chemical methods that produce molecules of full length.
[572] Site specific mutagenesis
[573] Site specific mutagenesis is a technique useful for preparing polypeptides that are individual peptides or biologically functional equivalents through specific mutations of the base polynucleotides encoding the individual peptides or polypeptides that are biologically functional equivalents. The techniques well known to those skilled in the art further provide the ability to immediately prepare and test sequence variants by introducing one or more nucleotide sequence variants into the DNA, for example, incorporating one or more of the considerations described above. . Site specific mutagenesis not only produces mutants through the use of specific oligonucleotide sequences encoding the DNA sequence of the desired mutant, but also forms stable backbones on both sides of the deletion junctions that cross each other. It allows the production of a sufficient number of contiguous polynucleotides to provide primer sequences of sufficient size and sequence complexity. Mutants can be used in selected polynucleotide sequences to enhance, change, degrade, modify or change the properties of the polynucleotide itself and / or to alter the properties, activity, composition, stability or primary sequence of the encoded polypeptide. You can.
[574] In certain aspects of the invention, we contemplate mutagenesis of the polynucleotide sequences described herein to alter one or more properties of the encoded polypeptide, eg, the antigenicity of the polypeptide vaccine. Site specific mutagenesis techniques are well known in the art and are widely used to generate variants for both polypeptides and polynucleotides. For example, site specific mutagenesis is often used to alter specific portions of DNA molecules. In such embodiments, primers that typically comprise about 14 to about 25 nucleotides, and the like, can be used, varying from about 5 to about 10 residues on either side of the junction of the sequence.
[575] As will be appreciated by those skilled in the art, site specific mutagenesis techniques often utilize phage vectors that exist in both single and double strand forms. Typical vectors useful for site specific mutagenesis include vectors such as M13 phage. These phages are readily available and their use is generally well known to those skilled in the art. Double-stranded plasmids are also commonly used for site directed mutagenesis, wherein the step of transferring the gene of interest from the plasmid to phage is omitted.
[576] In general, site directed mutagenesis according to the present invention is carried out by first obtaining a single chain vector or by separately separating the two chains of a double stranded vector comprising a DNA sequence encoding within the sequence encoding the desired peptide. . Oligonucleotide primers carrying the desired mutated sequences are generally synthetically prepared. The primers are then annealed together with single chain vectors, to which DNA polymerases such as E. coli. E. coli polymerase I Klenow fragments are applied to complete the synthesis of the mutant-bearing chain. This results in a hetero-backbone in which one chain encodes the original unmuted sequence and the second chain carries the desired mutation. Subsequently, the hetero-double stranded vector can be used to provide appropriate cells, such as E. coli. E. coli cells are transformed and clones comprising the recombinant vector with the mutated sequence sequence are selected.
[577] By using site directed mutagenesis to prepare sequence variants of the peptide encoding DNA segments selected as above, it is possible to provide a means for generating a species that is beneficially useful, which is a sequence variant of the peptide and the DNA sequence encoding it. It should not be construed as limiting the existence of other ways to obtain. For example, a recombinant vector encoding the desired peptide sequence can be treated with a mutagen, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods and protocols can be found in Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982; Each of which is incorporated herein by reference.
[578] As used herein, the term “oligonucleotide directed mutagenesis process” refers to a template that increases the concentration of a specific nucleic acid molecule relative to the initial concentration or increases the concentration of a detectable signal, eg, amplification concentration. -Refers to dependent processes and vector-mediated proliferation processes. As used herein, the term “oligonucleotide directed mutagenesis process” refers to a process involving template-dependent extension of a primer molecule. The term template-dependent process refers to the process of nucleic acid synthesis of RNA or DNA molecules, in which the sequence of the newly synthesized nucleic acid chain is dictated by the well-known complementary base pairing law (see Watson, 1987). Typically, vector mediated methodologies include introducing nucleic acid fragments into DNA or RNA, clonal amplifying such vectors, and recovering such amplified nucleated fragments. Examples of such methodologies are shown in US Pat. No. 4,237,224, the entirety of which is incorporated herein by reference.
[579] Polynucleotide Amplification Technology
[580] Numerous template dependent processes can be used to amplify the target sequence of interest present in the sample. One of the best known amplification methods is the polymerase chain reaction (PCR ™) described in detail in US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, the entire contents of which are incorporated herein by reference. . Briefly, PCR ™ produces two primer sequences that are complementary to regions on opposite complementary chains of a target sequence. Excess deoxynucleoside triphosphate is added along with the DNA polymerase (eg Taq polymerase) to the reaction mixture. If the target sequence is present in the sample, the primer is bound to this target and the polymerase will allow the primer to be extended along the target sequence by adding the primer onto the nucleotide. By raising or lowering the temperature of the reaction mixture, such extended primers will dissociate from the target to form the reaction product, and excess primer will bind to the target and reaction product, repeating this process. Preferably, reverse transcription and PCR ™ amplification procedures can be performed to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.
[581] Another method of amplification is the ligase chain reaction (referred to as LCR) described in EP-A-320,308, the entirety of which is incorporated herein by reference. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair binds to the opposite complementary chain of the target, thereby contiguous. In the presence of ligase two probe pairs will link to form a single unit. By temperature cycles, as in PCR ™, bound and linked units dissociate from the target and act as “target sequences” for linking excess probe pairs. US Pat. No. 4,883,750, which is incorporated herein by reference in its entirety, describes an alternative amplification method similar to LCR for binding probe pairs to target sequences.
[582] The Qbeta replicator described in PCR international patent application PCT / US87 / 00880, which is incorporated herein by reference in its entirety, may also be used as another amplification method of the present invention. In this method, a replication sequence of RNA having a region complementary to that of a particular target is added to the sample in the presence of RNA polymerase. Such a polymerase will copy the replication sequence, which can then be detected.
[583] Isothermal amplification methods using restriction endonucleases and ligases to achieve amplification of target molecules containing nucleotide 5 ′-[α-thio] triphosphate in one chain of restriction sites. Walker et al. , 1992; The entirety of which is incorporated herein by reference, may also be useful for amplifying a nucleic acid in the present invention.
[584] Chain replacement amplification (SDA), which involves performing chain replacement and synthesis, ie, nick deciphering, is another method of performing isothermal amplification of nucleic acids. A similar method, called repair chain reaction (RCR), is another amplification method that may be useful in the present invention, in which only two of the four bases are annealed after annealing several probes across the region targeted for amplification. Carrying out the existing repair reaction. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used for SDA.
[585] Cyclic probe reactions (CPR) can also be used to detect sequences. In CPR, probes having 3 'and 5' sequences of non-target DNA and internal or "intermediate" sequences of target protein specific RNA are hybridized with DNA present in the sample. Upon hybridization, the product of the probe is identified as a unique product by treating the reaction with RNAse and generating a signal that is released after degradation. The original template is annealed to another cycling probe and the reaction is repeated. Thus, CPR involves amplifying a signal generated by hybridizing a probe to a target gene specific expressed nucleic acid.
[586] Other amplification methods described in British Patent Application No. 2 202 328 and PCT International Patent Application PCT / US89 / 01025, the entire contents of which are incorporated herein by reference, can be used in accordance with the present invention. In the former application, "modified" primers are used for PCR-like, template and enzyme dependent synthesis. The primers can be modified by labeling with capture residues (eg biotin) and / or detector factor residues (eg enzymes). In the latter application, excess labeled probe is added to the sample. In the presence of the target sequence, the probe is bound and catalytically cleaved. After cleavage, the target sequence is released intact and bound by excess probe. Cleavage of the labeled probe signals that the target sequence is present.
[587] Other nucleic acid amplification procedures include transcriptional used amplification systems (TAS), including nucleic acid sequence used amplification (NASBA) and 3SR (Kwoh et al., 1989; PCT International Publication WO 88/10315; The entirety of which is incorporated herein by reference. At NASBA, nucleic acids are prepared for amplification by standard phenol / chloroform extraction, thermal denaturation of samples, treatment with lysis buffer and minispin columns to separate DNA and RNA, or guanidinium chloride extraction of RNA. can do. These amplification techniques include annealing primers with sequences specific to the target sequence. After polymerization, DNA / RNA hybrids degrade with RNAse H, while double-stranded DNA molecules are again denatured. In either case, by adding a second target specific primer and then polymerizing it, single stranded DNA is made double stranded. This double-stranded DNA molecule is then multiplexed by polymerase such as T7 or SP6. In an isothermal cyclic reaction, RNA is reverse transcribed into DNA and once again transcribed with a polymerase such as T7 or SP6. The resulting product, whether truncated or complete, indicates the target specific sequence.
[588] EP 329,822, the entirety of which is incorporated herein by reference, discloses the periodic synthesis of single-stranded RNA ("ssRNA"), ssDNA and double-stranded DNA (dsDNA), which can be used according to the present invention. Nucleic acid amplification methods comprising the same are described. ssRNA is the first template for the first primer oligonucleotide, which is stretched by reverse transcriptase (RNA-dependent DNA polymerase). Subsequently, by the action of ribonuclease H (RNAse H, which is RNAse specific for RNA in the double strand with DNA or RNA), RNA is removed from the DNA: RNA double strand produced as above. The resulting ssDNA is the second template for the second primer, which, in its homology, comprises the sequence of the RNA polymerase promoter (exemplified as T7 RNA polymerase) which is 5 'to its template. The primers are then extended by DNA polymerase (illustrated as the large "cleno" fragment of E. coli DNA polymerase I), which has the same sequence as the original RNA sequence between the two primers. It is produced as a double stranded DNA ("dsDNA") molecule with an additional promoter sequence at its end. Such promoter sequences can be used by appropriate RNA polymerase to make numerous RNA copies of DNA. These copies can then be reintroduced in a cycle that results in extremely rapid amplification. By appropriately selecting an enzyme, the amplification can be performed isothermally without adding an enzyme to each cycle. Due to the circulating nature of the method, the starting sequence can be selected in the form of DNA or RNA.
[589] PCT International Publication No. WO 89/06700, which is incorporated herein by reference in its entirety, discloses the hybridization of a promoter / primer sequence with a target single-stranded DNA (“ssDNA”) followed by transcription of many RNA copies of that sequence. A nucleic acid sequence amplification scheme comprising is described. This scheme is not circular, i.e. no new template is produced from the resulting RNA transcript. Other amplification methods include "RACE" (Frohman, 1990) and "one-sided PCR" (Ohara, 1989), which are well known to those skilled in the art.
[590] Methods of using amplifying said di-oligonucleotides by linking two (or more) oligonucleotides in the presence of a nucleic acid sequence having the sequence of the resulting "di-oligonucleotide" [Wu and Dean, 1996; Incorporated herein by reference in its entirety, may also be used for amplification of the DNA sequences of the present invention.
[591] Biologically functional equivalents
[592] The internal structure of the polynucleotides and polypeptides of the present invention can be modified and changed, thereby obtaining a functional ingredient encoding a polypeptide having the desired characteristics. As mentioned above, it is often desirable to introduce one or more mutations into specific polynucleotide sequences. Under certain circumstances, the resulting encoded polypeptide sequence is altered by the mutation, and in other cases, the sequence of the polypeptide is not altered by one or more mutations in the polynucleotide encoding it.
[593] If one wants to change the amino acid sequence of the polypeptide to produce an equivalent or even improved second generation molecule, the amino acid changes can be achieved by changing one or more codons of the encoded DNA sequence, according to Table 1.
[594] For example, certain amino acids may be replaced with other amino acids in the protein structure without cognitively losing their ability to interact with certain structures, eg, antigen-binding regions or binding sites on the substrate molecule of the antibody. Can be. Because defining the biological activity of a protein is the nature and interactivity of such a protein, it is possible to obtain a protein with similar properties, although specific amino acid substitutions are made in the protein sequence and, of course, the base DNA coding sequence. have. Thus, it is contemplated by the inventors that various changes may be made in the peptide sequences of the compositions described herein, or corresponding DNA sequences encoding such peptides, without cognitively losing their biological utility or activity. do.
[595] Amino acid codons 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 Lys Ile I AUA AUC K AAA Aleucine Leu L UUA UUG CUA CUC 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 UCC UCG UCU Leo T ACA ACC ACG ACU Valine non-Thr V GUA GUC GUG GUU Tryptophan Trp Val W UGG Tyrosine Tyr Y UAC UAU
[596] In making these changes, one can consider the hydropathic index of amino acids. The importance of hydrotherapeutic amino acid indices in contributing interactive biological actions to proteins is well recognized in the art (Kyte and Doolittle, 1982, incorporated herein by reference). This is due to the relative hydrotherapeutic character of the amino acids due to the secondary structure of the resulting protein (which in turn defines the interaction of the protein with other molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.) It is tolerated as. Each amino acid is assigned a hydrotherapy index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are as follows: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cystine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9) and arginine (-4.5).
[597] It is known in the art that certain amino acids can be substituted with other amino acids having similar hydropathic indices or scores, thereby producing proteins with similar biological activity, ie, proteins that are biologically functional equivalents. In making such a change, substitution of amino acids whose hydropathic index is within ± 2 is preferred, substitutions of amino acids within ± 1 are particularly preferred, and substitutions of amino acids within ± 0.5 are more particularly preferred. It is also recognized in the art that substitutions of similar amino acids can be made effectively based on hydrophilicity. U.S. Patent No. 4,554,101, the entirety of which is incorporated herein by reference, states that the largest local mean hydrophobicity of a protein correlates with the biological properties of such protein, as dictated by the hydrophobicity of its adjacent amino acids. .
[598] As described in US Pat. No. 4,554,101, the following hydrophilicity values were assigned to amino acid residues: arginine (+3.0); Lysine (+3.0); Aspartate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+3.0); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-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). The amino acid can be substituted with another amino acid having a similar hydrophilicity value, thereby obtaining a biological equivalent, in particular an immunologically equivalent protein. In making such a change, substitution of amino acids with hydrophilicity values within ± 2 is preferred, substitutions of amino acids within ± 1 are particularly preferred, and substitutions of amino acids within ± 0.5 are more particularly preferred.
[599] Thus, as mentioned above, amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, and the like. Examples of substitutions in view of the various features described above are well known to those skilled in the art, including alignin and lysine; Glutamate and aspartate; Serine and threonine; Glutamine and asparagine and valine, leucine and isoleucine.
[600] In addition, any polynucleotide can be further modified to increase in vivo stability. Possible modifications include the addition of flanking sequences at the 5 'and / or 3' ends; Methods of using phosphorothioate or 2'O-methyl other than phosphodiesterase linkages in the backbone and / or incorporating non-traditional bases such as inosine, queocin and wibutocin as well as adenine Methods include, but are not limited to, acetyl-, methyl-, thio- and other modified forms of cytidine, guanine, thymine and uridine.
[601] In vivo polynucleotide delivery technology
[602] In a further aspect, genetic constructs comprising one or more polynucleotides of the invention are introduced into a cell in vivo. This can be accomplished using any of the well-known approaches or various approaches, some of which are summarized below for illustrative purposes.
[603] Adenovirus
[604] One preferred method for delivering one or more nucleic acid sequences in vivo involves using an adenovirus expression vector. An "adenovirus expression vector" comprises a construct containing (a) sufficient adenovirus sequences to support the packaging of the construct and (b) sufficient expression of the polynucleotide cloned therein in a sense or antisense orientation. Means that. Of course, in the context of antisense constructs, expression does not necessarily require the synthesis of gene products.
[605] The expression vector comprises adenovirus in a genetically engineered form. Using knowledge of the genetic system of adenoviruses, the fact that it is a 36 kb linear double stranded DNA virus, large pieces of adenovirus DNA can be replaced with foreign sequences of 7 kb or less. Grunhaus and Horwitz, 1992]. Unlike retroviruses, adenoviral infection of host cells results in no chromosomal integration, because adenovirus DNA can replicate in an episomal fashion without potential genotoxicity. In addition, adenoviruses are structurally stable and no genomic rearrangements have been detected after extended amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. To date, adenovirus infections are believed to be associated only with mild diseases such as acute respiratory disease in humans.
[606] Adenoviruses are particularly suitable for use as gene transfer vectors because their genomes are medium in size, easy to manipulate, have high titer, broad target cell range and high infectivity. Both ends of the viral genome contain 100 to 200 base pair reverse sequences (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of this genome contain different transcription units that are divided by viral DNA replication initiation. The El regions (E1A and E1B) encode proteins that are involved in the transcriptional regulation of the viral genome and several cellular genes. When the E2 regions (E2A and E2B) are expressed, proteins for viral DNA replication are synthesized. These proteins are involved in DNA replication, late gene expression and host cell shut-off [Renan, 1990]. The product of later genes, including most viral capsid proteins, is expressed only after the actual processing of a single primary transcript released by the major late promoter (MLP). These MLPs (located at 16.8 m.u.) are particularly efficient during the late phase of infection, and all mRNAs released from these promoters have a 5'-tripart leader (TPL) sequence, which makes them desirable mRNAs for translation.
[607] In the current system, the prepared adenovirus is generated from homologous recombination between a shuttle vector and a proviral vector. Since recombination is possible between two proviral vectors, wild-type adenoviruses can be generated in this way. Therefore, it is important to isolate a single clone of virus from individual plaques and to examine their genomic structure.
[608] The development and proliferation of current adenovirus vectors that are replication deficient depends on a unique helper cell line (named 293) that transforms from human embryonic kidney cells with A5d DNA fragments and constitutively expresses the E1 protein. Graham et. al., 1977. Since the E3 region can be removed from the adenovirus genome [Jones and Shenk, 1978], current adenovirus vectors carry foreign DNA in either E1, D3 or both regions with the help of 293 cells (Graham and Prevec, 1991]. Originally, adenovirus can package approximately 105% of the wild type genome while providing a capacity for about 2 extra kB of DNA (Ghosh-Choudhury et al., 1987). By combining with approximately 5.5 kB of DNA replaceable in the E1 or E3 region, the current adenovirus vector has a maximum capacity of 7.5 kB or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone, which is a source of cytotoxicity resulting from the vector. In addition, replication deficiency of E1-deleted viruses is incomplete. For example, viral gene expression leaks were observed using commercially available vectors with high multiplicity of infection (MOI) (Mulligan, 1993).
[609] Helper cell lines can be derived from human cells, such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. On the other hand, helper cells can be derived from other mammalian species cells that allow replication of human adenovirus. Such cells include, for example, Vero cells or other monkey embryonic mesenchymal or epithelial cells. As mentioned above, the presently preferred helper cell line is 293.
[610] Recently, improved methods of culturing 293 cells and propagating adenoviruses have been described in Racher et al. (1995). In one format, natural cell aggregates are grown by inoculating individual cells into a 1 liter siliconized spinner flask (Techne, Cambridge, UK) containing 100-200 ml of medium. After stirring at 40 rpm, cell viability is assessed with trypan blue. In another format, a Fibra-Cel microcarrier (Bibby Sterlin, Stone, UK) (5 g / l) is used as follows. Cell inoculum resuspended in 5 ml medium is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left to stand for 1 to 4 hours with occasional shaking. The medium is then replaced with 50 ml of fresh medium and shaking is started. For virus production, cells are grown to about 80% density, then the medium is replaced (to 25% final volume) and adenovirus is added at MOI 0.05. The culture is left in the stationary phase overnight, then the volume is increased to 100% and shaking is started for 72 more hours.
[611] The nature of adenovirus vectors other than the requirement that adenovirus vectors be replication deficient or at least conditionally defective should not be deemed critical to the successful implementation of the invention. The adenovirus may be any of 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is a preferred starting material for obtaining conditional replication-defective adenovirus vectors for use in the present invention, where adenovirus type 5 is known with significant amounts of biochemical and genetic information. It has been used for a long time in most constructions using adenovirus as a vector.
[612] As mentioned above, typical vectors according to the present invention are replication defective and will not have an adenovirus E1 region. Thus, it would be most convenient to introduce a polynucleotide encoding the gene of interest at the position from which the E1-coding sequence was removed. However, the position of insertion of the construct within the adenovirus sequence is not critical to the invention. Polynucleotides encoding genes of interest are described in place of the deleted E3 region in Karlsson et al. (1986), or may be inserted into an E3 substitution vector, or into an E4 region, where a helper cell line or helper virus compensates for E defects.
[613] Adenoviruses grow well, are easy to manipulate, and exhibit a broad host range in vitro and in vivo. Such viral groups can be obtained at high titers, for example, 10 ⁹ to 10 ¹¹ plaque forming units / ml, which are highly infectious. The life cycle of adenoviruses does not require integration into the host cell genome. Foreign genes delivered by adenovirus vectors are episomal and therefore have low cytotoxicity against host cells. No adverse reactions have been reported in the study of vaccination with wild-type adenovirus [Couch et al., 1963; Top et al., 1971, which demonstrates that they have therapeutic efficacy and are safe as gene transfer vectors in vivo.
[614] Adenovirus vectors express eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992] and vaccine development [Grunhaus and Horwitz, 1992; Graham and Prevec, 1992]. Recently, animal studies suggest that recombinant adenovirus can be used in gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al., 1990; Rich et al., 1993]. Studies in administering recombinant adenoviruses to different tissues include organ loading (Rosenfeld et al., 1991; Rosenfeld et al., 1992], intramuscular injection [Ragot et al., 1993], peripheral intravenous injection [Herz and Gerard, 1993] and stereotactic immunization into the brain [Le Gal La Salle et al. , 1993].
[615] 2. Retrovirus
[616] Retroviruses are a group of single stranded RNA viruses that characterize their ability to convert their RNA into double stranded DNA in infected cells by reverse transcription processes. The DNA thus generated is then stably integrated into the cellular chromosome as a provirus and directs the synthesis of viral proteins. This integration results in the viral gene sequence staying in the receptor cell and its progeny. The retroviral genome contains three genes, gag, pol and env, which encode capsid proteins, polymerase enzymes, and envelope components, respectively. The sequence found upstream from the gag gene contains a signal for packaging the genome into a virion. Two long terminal repeat (LTR) sequences are present at the 5 'and 3' ends of the viral genome. They contain potent promoter and enhancer sequences and are also required for integration into the host cell genome (Coffin, 1990).
[617] To construct retroviral vectors, nucleic acids encoding one or more oligonucleotides or polynucleotides of interest are inserted into the viral genome instead of a specific viral sequence to produce a replication defective virus. To generate virions, packaging cell lines containing the gag, pol and env genes but no LTR and packaging components are constructed (Mann et al., 1983). When a recombinant plasmid containing cDNA is introduced into the cell line together with retroviral LTR and packaging sequence (e.g., by calcium phosphate precipitation), the packaging sequence is such that the RNA transcript of the recombinant plasmid is a viral particle. Packaged into and then secreted into the culture medium. Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983. The medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors can infect a wide range of cell types. However, for integrated and stable expression, host cells must be divided (Paskind et al., 1975).
[618] New approaches designed to specifically target retroviral vectors have recently been developed, based on chemically modifying retroviruses by chemically adding lactose residues to the viral envelope. Due to this modification, hepatic cells could be specifically infected through the sialoglycoprotein receptor.
[619] Different approaches have been devised to target recombinant retroviruses, using biotinylated antibodies against retroviral envelope proteins and biotinylated antibodies against specific cellular receptors. The antibody was coupled through the biotin component by using streptavidin (Rux et al., 1989). Antibodies to major histocompatibility complex class I and class II antigens have been used to demonstrate infection of various human cells that make surface antigens difficult with ecotropic viruses in vitro (Rux et al., 1989). ].
[620] 3. Adeno-associated Virus
[621] AAV [Ridgeway, 1988; Hermonat and Muzycska, 1984 are paroviruses found as contaminants of adenovirus stocks. This is a ubiquitous virus that has never been associated with any disease (85% of Americans have their antibodies). It is also classified as a dependent virus because its replication is dependent on the presence of helper viruses, such as adenoviruses. Five serotypes were isolated, the best of which was AAV-2. AAV has single-stranded linear DNA encapsidated into capsid proteins VP1, VP2 and VP3 to form icosahedral virions with a diameter of 20 to 24 nm (Muzyczka and McLaughlin, 1988).
[622] AAV DNA is approximately 4.7 kilobases in length. It contains two open reading frames and is flanked by two LTRs. There are two main genes, rep and cap , in the AAV genome. The rep gene encodes a protein involved in viral replication, while the cap gene encodes the capsid protein VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeat sequences are the only essential cis component of AAV for chromosomal integration. Thus, AAV can be used as a vector where all viral coding sequences have been removed and replaced with a gene cassette for delivery. Three viral promoters have been identified, named p5, p19 and p40 depending on their map position. Transcription from p5 and p19 produces a rep protein and transcription from p40 produces a capsid protein (Hermonat and Muzyczka, 1984).
[623] There are several factors that encourage research into whether rAAV can be used as an expression vector. One is that surprisingly little is required to deliver these genes to integrate them into the host chromosome. It is necessary to have a 145-bp ITR, which is only 6% of the AAV genome. This allows the vector to assemble a 4.5 kb DNA insert. This carrying capacity may prevent AAV from delivering large genes, but it is well suited to delivering antisense constructs of the invention.
[624] AAV is also chosen as an excellent delivery vehicle due to its safety. There is a relatively complex protective mechanism; The wild-type adenovirus as well as the AAV gene must immobilize rAAV. Likewise, AAV is not pathogenic and therefore not associated with any disease. RAAV does not elicit an inflammatory response because removing the viral coding sequence minimizes the immune response to viral gene expression.
[625] 4. Other Viral Vectors as Expression Constructs
[626] Other viral vectors can be used as expression constructs in the present invention to deliver oligonucleotide or polynucleotide sequences to host cells. Vaccinia virus [Ridgeway, 1988; Coupar et al., 1988], vectors derived from viruses such as lentiviruses, polio viruses and herpes viruses can be used. They confer some interesting aspects on various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Coupar et al., 1988; Horwich et al., 1990.
[627] Recent recognition of defective hepatitis B virus has led to a new review of the structure-function relationship of different viral sequences. In vitro studies have shown that the virus can retain the ability to perform helper-dependent packaging and reverse transcription despite up to 80% of its genome being deleted (Horwich et al., 1990). This suggests that much of the genome can be replaced with foreign genetic material. Hepatic affinity and persistence (integration) are of particular interest in liver-directed gene transfer. Chang et al. (1991) introduced the chloramphenicol acetyltransferase (CAT) gene into the duke hepatitis B virus genome in place of the polymerase, surface and pre-surface coding sequences. Wild type virus was used to cotransfect into avian liver cancer cell lines. Culture medium containing high titre recombinant virus was used to infect primary tucking hepatocytes. At least 24 days after transfection, stable CAT gene expression was detected (Chang et al., 1991).
[628] 5. Non-viral vectors
[629] To express an oligonucleotide or polynucleotide sequence of the invention, the expression construct must be delivered intracellularly. Such delivery can be performed in vitro, such as in laboratory procedures for transforming cell lines, or in vivo or ex vivo, such as to treat a particular disease state. As mentioned above, one preferred mechanism of delivery is through viral infection that encapsulates the expression construct in infectious viral particles.
[630] Once the expression construct is delivered into the cell, the nucleic acid encoding the desired oligonucleotide or polynucleotide sequence can be located and expressed at different positions. In certain embodiments, the nucleic acid encoding the construct can be stably integrated into the genome of the cell. Such integration may be at specific positions and orientations via homologous recombination (gene substitution) or randomly integrated into non-specific positions (gene augmentation). In a further embodiment, the nucleic acid can be stably maintained in the cell as a separate episomal segment of DNA. Such nucleic acid segments or “episosomes” encode sufficient sequence to be maintained and replicated independently of or simultaneously with the host cell cycle. However, expression constructs are delivered to specific cells and the nucleic acid remaining in these cells depends on the type of expression construct used.
[631] In certain embodiments of the invention, an expression construct comprising one or more oligonucleotide or polynucleotide sequences may consist of simply exposed recombinant DNA or plasmid. The transfer of such constructs can be carried out by any of the methods mentioned above that can penetrate the cell membrane physically or chemically. It is particularly applicable to in vitro metastasis, but it can also be applied to in vivo applications. See Dubensky et al. (1984) describe the successful injection of polyomavirus DNA into the liver and spleen of adult and neonatal mice in the form of calcium phosphate precipitates, demonstrating active viral replication and acute infection. Benvenisty and Reshef (1986) demonstrated that direct intraperitoneal injection of calcium phosphate precipitated plasmids resulted in the expression of transfected genes. It is contemplated that DNA encoding a gene of interest can also be transferred in vivo and express gene products in a similar manner.
[632] Another aspect of the invention for transferring exposed DNA expression constructs into cells involves particle bombardment. This method relies on the ability to accelerate DNA-coated microposers at high speed so that they can penetrate the cell membrane and enter into these cells without killing the cells (Klein et al., 1987). Several devices have been developed to accelerate small particles. One such device relies on a high voltage discharge that provides the driving force by generating a current (Yang et al., 1990). The microcosoids used consist of biologically inert materials, for example tungsten or gold beads.
[633] Selected organs, including liver, skin and muscle tissues of rats and mice were shocked in vivo (Yang et al., 1990; Zelenin et al., 1991]. In order to remove any interfering tissue between the gun and the target organ, i.e. for ex vivo treatment, it is necessary to surgically expose the tissue or cells. Again, DNA encoding a particular gene can be delivered via this method, which can be incorporated by the present invention.
[634] Antisense oligonucleotides
[635] The end result of the genetic information flow is the synthesis of proteins. DNA is transcribed into messenger RNA by polymerase and translated on ribosomes to yield folded functional protein. Thus, along this pathway, there are several steps that can inhibit protein synthesis. The original DNA segment encoding the polypeptides described herein has two chains, like all mammalian DNA chains, a sense chain and an antisense chain, held together by hydrogen bonds. The messenger RNA encoding the polypeptide has the same nucleotide sequence as the sense DNA chain except that DNA thymidine is replaced with uridine. Thus, synthetic antisense nucleotide sequences will bind mRNA and inhibit the expression of the protein encoded by such mRNA.
[636] Thus, targeting antisense oligonucleotides to mRNA is one mechanism by which protein synthesis is stopped, which represents a powerful and targeted therapeutic approach. For example, the synthesis of polygalactauronase and muscarinic type 2 acetylcholine receptors is inhibited by antisense oligonucleotides indicated in their respective mRNA sequences (US Pat. Nos. 5,739,119 and 5,759,829; respectively). Incorporated herein by reference in its entirety. Additionally, nuclear protein cyclins, multiple drug resistance genes (MDG1), ICAM-1, E-selectin, STK-1, striped GABA _A receptors and human EGF were used to demonstrate antisense inhibition examples. Jaskulski et al. , 1988; Vasanthakumar and Ahmed, 1989; Peris et al., 1998; US Patent No. 5,801,154; US Patent No. 5,789,573; U.S. Patent 5,718,709 and U.S. Patent 5,610,288; Each full text is incorporated herein by reference]. Antisense constructs have also been reported that can be used to inhibit and treat various abnormal cell proliferations, such as cancer (US Pat. Nos. 5,747,470; 5,591,317 and 5,783,683; each of which is incorporated herein by reference in its entirety. Inserted).
[637] Thus, in exemplary embodiments, the present invention provides oligonucleotide sequences comprising all or part of a sequence capable of specifically binding to a polynucleotide sequence described herein or its complementary sequence. In one embodiment, the antisense oligonucleotides comprise DNA or derivatives thereof. In another embodiment, the oligonucleotides comprise RNA or derivatives thereof. In a third embodiment, the oligonucleotide is a modified DNA comprising a phosphorothioated modified backbone. In a fourth embodiment, the oligonucleotide sequence comprises a peptide nucleic acid or derivative thereof. In each case, preferred compositions comprise sequence regions that are complementary, more preferably substantially complementary, even more preferably completely complementary to one or more portions of the polynucleotides described herein.
[638] The selection of antisense compositions specific for a given gene sequence is based on analysis of selected target sequences (ie, in the illustrative example, rat and human sequences) and determinations on secondary structure, T _m , binding energy, relative stability. Antisense compositions are selected based on their relative ability to form dimers, hairpins or other secondary structures that may degrade or inhibit specific binding to target mRNAs in host cells.
[639] Highly preferred target regions for mRNA are sequences that are substantially complementary to the AUG translational initiation codon region or region near it, and the 5 'region of the mRNA. These secondary structure analysis and target site selection considerations are performed using v.4 of the OLIGO Primer Analysis Software (Rychlik, 1997) and the BLASTN 2.0.5 Algorithm Software (Altschul et al., 1997).
[640] The use of antisense delivery methods using short peptide vectors named MPG (27 residues) is also contemplated. MPG peptides contain a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of the SV40 T-antigen (Morris et al., 1997). It has been demonstrated that several molecules of MPG peptides can coat antisense oligonucleotides and deliver them into mammalian cells cultured in less than one hour with relatively high efficiency (90%). In addition, interaction with MPG significantly increases both the stability of oligonucleotides to nucleases and their ability to cross the plasma membrane (Morris et al., 1997).
[641] Ribozyme
[642] Although proteins have traditionally been used to catalyze nucleic acids, another class of macromolecules has been shown to be useful in these attempts. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site specific manner. Ribozymes have specific and catalytic domains that retain endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987; Forster and Symon, 1987]. For example, many ribozymes often accelerate only one of several phosphoesters in an oligonucleotide substrate while accelerating the phosphoester transfer reaction to high specificity (Cech et al., 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992]. This specificity stems from the requirement that the substrate be bound to the ribozyme's internal guide sequence (“IGS”) prior to chemical reactions through specific base pairing interactions.
[643] Ribozyme catalysis has been observed primarily as part of sequence specific cleavage / linking reactions involving nucleic acids. Joyce, 1989; Cech et al., 1981. For example, US Pat. No. 5,354,855, the entirety of which is incorporated herein by reference, discloses that a particular ribozyme has an endo sequence having greater sequence specificity than known ribonucleases and sequence specificity close to DNA restriction enzymes. It is reported that it can act as a nuclease. Thus, sequence specific ribozyme mediated inhibition of gene expression may be particularly suitable for therapeutic applications. Scanlon et al., 1991; Sarver et al., 1990. Recently, ribozymes have caused genetic changes in some cell lines to which they have been applied; Such altered genes have been reported to include genes of oncogene H- ras , c- fos and HIV. Most of this work involves modification of target mRNAs based on specific mutant codons cleaved by specific ribozymes.
[644] Six basic variants of native enzymatic RNA are currently known. Each can cleave other RNA molecules by catalyzing the hydrolysis of RNA phosphodiester bonds with trans under physiological conditions. In general, enzymatic nucleic acids act by first binding to target RNA. This binding is via the target binding portion of the enzymatic nucleic acid, which is kept in close contact with the enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid is first recognized and then bound to the target RNA via complementary base pairing, and once bound to the correct position, it acts enzymatically to cleave the target RNA. This strategic cleavage of the target RNA will destroy its ability to direct the synthesis of the encoded protein. After enzymatic nucleic acid is bound and its RNA target is cleaved off, it can be released from the RNA and examined for another target, followed by repeated binding and cleavage of the new target.
[645] The enzymatic properties of ribozymes are advantageous over many techniques, such as antisense techniques, where the nucleic acid molecule simply binds to the nucleic acid target and blocks its translation, which is the ribozyme necessary to affect the therapeutic treatment. This is because the concentration of is lower than that of the antisense oligonucleotide. This advantage reflects the ability of ribozymes to act enzymatically. Thus, a single ribozyme molecule can cleave many target RNA molecules. In addition, ribozymes are highly specific inhibitors, the inhibitory specificity of which depends not only on the base pairing mechanism of binding to the target RNA but also on the target RNA cleavage mechanism. Base-substituted or single mismatches near the cleavage site can completely eliminate the catalytic activity of ribozyme. Similar mismatches in antisense molecules do not interfere with their action (Woolf et al., 1992). Thus, the specificity of action of ribozymes is greater than antisense oligonucleotides that bind the same RNA site.
[646] Enzymatic nucleic acid molecules can be formed with hammerheads, hairpins, hepatitis δ virus, group I introns or RNAseP RNAs (associated with RNA guide sequences) or neurospora VS RNA motifs. Examples of hammerhead motifs are described in Rossi et al., 1992. Examples of hairpin motifs are described in Hampel et al., Eur. Pat. Appl. Publ. No. EP 0360257; Hampel and Tritz (1989), Hampel et al., (1990) and US Pat. No. 5,631,359, which is specifically incorporated herein by reference. examples of hepatitis δ viral motifs are described in Perrotta and Been (1992); Examples of RNAseP RNA motifs are described in Guerrier-Takada et al., (1983); Neurospora VS RNA ribozyme motifs are described in Saville and Collins, 1990; Saville and Collins, 1991; Collins and Olive, 1993; Examples of group I introns are described in US Pat. No. 4,987,071, which is specifically incorporated herein by reference. What is important for the enzymatic nucleic acid molecule of the present invention is a nucleotide sequence having a specific substrate binding site complementary to at least one target gene RNA region and also surrounding or surrounding the substrate binding site that confers RNA cleavage activity to the molecule. Have Thus, ribozyme constructs need not be limited to the specific motifs mentioned herein.
[647] In certain embodiments, it may be important to produce an enzymatic cleavage agent that exhibits high specificity for the desired target, eg, an RNA of one of the sequences described herein. It is desirable to target the enzymatic nucleic acid molecule to the highly homeostatic sequence region of the target mRNA. Such enzymatic nucleic acid molecules can be exogenously delivered to specific cells as needed. Alternatively, ribozymes can be expressed from DNA or RNA vectors delivered to specific cells.
[648] Small enzymatic nucleic acid motifs (such as hammerhead or hairpin structure motifs) can also be used for exogenous delivery. The simple structure of these molecules enhances the ability of enzymatic nucleic acids to invade the targeting regions of mRNA structures. Alternatively, catalytic RNA molecules can be expressed in cells from eukaryotic promoters (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. One skilled in the art recognizes that any ribozyme can be expressed in eukaryotic cells from a suitable DNA vector. The activity of such ribozymes can be enhanced by their release from primary transcripts by secondary ribozymes (both WO 93/23569 and WO 94/02595, both incorporated herein by reference; Ohkawa et al. al., 1992; Taira et al., 1991; and Ventura et al., 1993).
[649] Ribozyme can be added directly to the target cell or can be complexed with cationic lipids, lipid complexes, packaged in liposomes or delivered to the target cell by other means. Such RNA or RNA complexes can be administered topically to relevant tissue in vitro or in vivo via injection, aerosol inhalation, infusion pump or stent, without incorporating or incorporating them into the biopolymer.
[650] Ribozymes can be designed as described in International Publications WO 93/23569 and WO 94/02595, which are specifically incorporated herein by reference, and can be synthesized and tested in vitro and in vivo as described. Such ribozymes may be optimized for delivery. Although specific examples have been provided, those skilled in the art recognize that equivalent RNA targets in other species may be used as needed.
[651] Hammerhead or hairpin ribozymes can be analyzed separately by computer folding (Jaeger et al., 1989), which assesses whether the ribozyme sequence folds into a suitable secondary structure. Ribozymes that exhibit undesired intramolecular interactions between the binding arm and the catalytic core are excluded from consideration. The length of the various binding arms can be chosen to optimize the activity. In general, five or more bases on each cancer can bind to or interact with the target RNA.
[652] Ribozymes of hammerhead or hairpin motifs can be designed to anneal to various sites in an mRNA message, which can be chemically synthesized. Synthetic methods used are described in Usman et al. (1987) and Scaringe et al. (1990), following procedures for normal RNA synthesis, and conventional nucleic acid protection and coupling groups, such as dimethoxytrityl at the 5'-end and phosphoramidite at the 3'-end. Use The average step yield is typically> 98%. Hairpin ribozymes can be synthesized in two parts and annealed to reconstitute active ribozymes (Chowrira and Burke, 1992). Ribozymes are stable by modification with nuclease resistant groups such as 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-o-methyl, 2'-H. May be extended to enhance the activity of the bacteria (Usman and Cedergren, 1992). Ribozyme can be purified and resuspended in water by gel electrophoresis or high pressure liquid chromatography using conventional methods.
[653] Ribozyme activity may be modified to alter the length of ribozyme binding cancers or to prevent their degradation by serum ribonucleases (see, eg, WO 92/07065; Perrault et al., 1990; Pieken et al., 1991; Usman and Cedergren, 1992; International Publication WO 93/15187; WO 91/03162; European Publication No. 92110298; US Patent No. 5,334,711 and WO 94/13688; These describe various chemical modifications that can be made to sugar residues of enzymatic DNA molecules], modifications that enhance their efficacy in cells, and shorten RNA synthesis time and reduce chemical requirements. It can be optimized by chemically synthesizing ribozymes with the removal of the stem II base.
[654] International Publication WO 94/02595 to Sullivan et al. Describes a general method for delivering enzymatic RNA molecules. Ribozymes are known to those of skill in the art, including, but not limited to, encapsulation, electrophoresis, or incorporation into liposomes into other vehicles such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. It can be administered to cells by various methods. For some indications, ribozyme can be delivered ex vivo directly to the cell or tissue, with or without the vehicle described above. Alternatively, the RNA / vehicle combination can be directly inhaled, directly injected, or locally delivered by using a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (in tablet or pill form), topical, systemic, intraocular, intraperitoneal and / or intravesicular delivery. More details regarding ribozyme delivery and administration are provided in WO 94/02595 and WO 93/23569, each of which is specifically incorporated herein by reference.
[655] Another way to accumulate high concentrations of ribozyme in cells is to incorporate the ribozyme coding sequence into the DNA expression vector. Transcription of ribozyme sequences is driven from promoters for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II) or RNA polymerase III (pol III). Transcripts from the pol II or pol III promoters will be expressed at high levels in all cells; The level of a given pol II promoter in a given cell type depends on the type of gene regulatory sequence present (enhancer, silencer, etc.). Prokaryotic RNA polymerase promoters can also be used, such prokaryotic RNA polymerase enzymes must be expressed in suitable 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. 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 may be plasmid DNA vectors, viral DNA vectors (eg adenovirus or adeno-associated vectors) or viral RNA vectors (eg retroviral, semliki forest virus, sindbis virus). Vectors] can be incorporated into various vectors for introduction into mammalian cells, including but not limited to.
[656] Ribozyme can be used as a diagnostic tool for examining gene immobilization and mutations in diseased cells. They can also be used to assess the level of target RNA molecules. The close relationship between the ribozyme activity and the structure of the target RNA allows detection of mutations in all regions of the molecule that change the base pair formation of the target RNA and its three-dimensional structure. By using multiple ribozymes, it is possible to map nucleotide changes important to RNA structure and function, as well as in vitro and in cells and tissues. Ribozymes can be used to cleave target RNA to inhibit gene expression and define the role (essential) of the specialized gene product in disease progression. In this way, other genetic targets can be defined as important mediators of the disease. These studies have shown intermittent combination therapy (eg, multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or combinations of ribozymes and / or other chemical or biological molecules). By giving the possibility of treatment, the progress of the disease can be better treated. Other in vitro uses of ribozymes are well known in the art and include detecting the presence of mRNA associated with IL-5 related conditions. Such RNA is detected by determining the presence of cleavage products after treatment with ribozymes using standard methods.
[657] Peptide Nucleic Acids
[658] In certain embodiments, the use of peptide nucleic acids (PNAs) in practicing the methods of the present invention is contemplated. PNA is a DNA mimetic that has a nucleobase attached to the pseudopeptide backbone (Good and Nielsen, 1997). PNA can utilize a number of methods that have traditionally been used for RNA or DNA. Often PNA sequences are performed in specific techniques better than corresponding RNA or DNA sequences and have utility not inherent in RNA or DNA. A review of the preparation, features and methods of using PNAs is described in Corey (1997); Incorporated herein by reference. As such, in certain embodiments, PNA sequences complementary to one or more portions of an ACE mRNA sequence can be prepared, and such PNA compositions can be used to modulate, change, reduce or decrease the translation of ACE-specific mRNA, Such PNA compositions can alter the level of ACE activity in the host cell administered.
[659] PNA has a 2-aminoethyl-glycine linkage that replaces the normal phosphodiester backbone of DNA (Nielsen et al., 1991; Hanvery et al., 1992; Hyrup and Nielsen, 1996; Neilsen, 1996]. This chemistry has three important consequences: first, unlike DNA or phosphorothioate oligonucleotides, PNA is a neutral molecule; Second, PNA is achiral, requiring no development of stereoselective synthesis; Third, PNA synthesis has been used in other methods, including the modified Merrifield method (Christensen et al., 1995), but the standard Boc for solid phase peptide synthesis [Dueholm et al., 1994]. Or Fmoc (Thomson et al., 1995) protocol.
[660] PNA monomers or ready-made oligomers may be obtained from PerSeptive Biosystems; Framingham, MA; commercially available. PNA synthesis by the Boc or Fmoc protocol is a simple method using manual or automated protocols (Norton et al., 1995). Passive protocols allow the production of chemically modified PNAs or the simultaneous synthesis of closely related PNA families.
[661] As with peptide synthesis, the success of a particular PNA synthesis will depend on the nature of the selected sequence. For example, while in theory PNA can be incorporated into any combination of nucleotide bases, the presence of adjacent purines can result in the deletion of one or more residues in the product. In anticipation of this difficulty, it has been proposed that in the generation of PNAs with adjacent purines, the coupling of residues that would be added inefficiently must be repeated. The PNA should then be purified by reverse phase high pressure liquid chromatography (Norton et al., 1995) to provide product yields and purity similar to those observed during peptide synthesis.
[662] Modification of PNA for a given application can be accomplished by coupling amino acids during solid phase synthesis or by attaching compounds containing carboxylic acid groups to the exposed N-terminal amines. Alternatively, PNA can be modified after synthesis by coupling to lysine or cysteine introduced. Due to the ease of PNA modification, optimization for better solubility or optimization for specific functional requirements is facilitated. Once synthesized, the entities of PNA and their derivatives can be identified by mass spectrometry. Several studies have been made and used to modify PNA [Norton et al., 1995; Haaima et al., 1996; Stetsenko et al., 1996; Petersen et al., 1995; Ulman 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]. US Pat. No. 5,700,922 describes PNA-DNA-PNA chimeric molecules and their use in treating diseases susceptible to diagnosis, protein regulation and treatment in an organism.
[663] Unlike DNA and RNA, which contain negatively charged linkages, the PNA backbone is neutral. Despite these dramatic changes, PNA recognizes complementary DNA and RNA by Watson-Crick pairing (Egholm et al., 1993), thereby allowing Nielsen et al., 1991] confirmed initiation modeling. PNAs lack 3 'to 5' polarity and can be coupled in parallel or in series, preferably in series (Egholm et al., 1993).
[664] Hybridization of DNA oligonucleotides to DNA and RNA is destabilized due to the electrostatic repulsion between the negatively charged phosphate backbones of the complementary chains. In contrast, since there is no charge repulsion in the PNA-DNA or PNA-RNA double strand, the melting point (T _m ) is increased and the dependence of T _m on the concentration of monovalent or divalent cations is lowered. ; Nielsen et al., 1991]. Increasing the rate and affinity of hybridization is important because it is involved in the PNA's incredible ability to perform chain invasion of complementary sequences in relaxed double stranded DNA. In addition, efficient hybridization in reverse iterations suggests that PNA can efficiently recognize secondary structures in double-stranded DNA. Increased recognition is also achieved using PNA immobilized on the surface, Wang et al. Found that support-bound PNAs can be used to detect hybridization events (Wang et al., 1996). .
[665] Tight binding of PNA to complementary sequences can be expected to increase binding with similar (but not identical) sequences while decreasing PNA recognition sequence specificity. However, as with DNA hybridization, selective recognition can be achieved by balancing oligomer length and incubation temperature. Moreover, selective hybridization of PNA is encouraged by PNA-DNA hybridization, which is less resistant to base mismatch than DNA-DNA hybridization. For example, a single mismatch in a 16 pb PNA-DNA double strand can lower T _m by less than 15 ° C. (Egholm et al., 1993). Due to this high level of decay, several PNA-used strategies for analyzing point mutations have been developed (Wang et al., 1996; Carlsson et al., 1996; Thiede et al., 1996; Webb and Hurskainen, 1996; Perry-O'Keefe et al., 1996].
[666] High affinity binding offers clear advantages in molecular recognition and development of new applications for PNA. For example, 11-13 nucleotide PNAs inhibit the activity of telomerase, a ribonucleotide-protein that extends the telomer end using essential RNA templates, while similar DNA oligomers do not. Norton et al., 1996].
[667] Since neutral PNAs are more hydrophobic than similar DNA oligomers, it may be difficult to solubilize them at neutral pH, especially if the PNA content of the PNA is high or the PNA has the potential to form secondary structures. Their solubility can be enhanced by attaching one or more positive charges to the PNA terminus (Nielsen et al., 1991).
[668] The findings of Allfrey and his colleagues suggest that chain invasion will occur spontaneously in sequences within chromosomal DNA (Boffa et al., 1995; Boffa et al., 1996]. These studies targeted PNA to triple repeats of nucleotide CAGs, using this recognition to purify transcriptionally active DNA (Boffa et al., 1995) and inhibit transcription [Boffa et al., 1996]. . These results suggest that if PNA can be delivered intracellularly, it will have the potential to be a general sequence-specific regulator of gene expression. Studies and considerations regarding the use of PNAs as antisense and anti-gene agents include Nielsen et al., 1993b; Hanvey et al., 1992; and Good and Nielsen, 1997]. Koppelhus (1997) et al. Used PNA to inhibit HIV-1 reverse transcription, demonstrating that PNA can be used for antiviral treatment.
[669] Methods for characterizing the antisense binding properties of PNAs are discussed in Rose (1993) and Jensen et al. (1997). Rose uses capillary gel electrophoresis to determine if PNA binds to its complementary oligonucleotides, thereby measuring relative binding kinetics and stoichiometry. A similar type of measurement was made by Jensen et al. Using BIAcore ™ technology.
[670] Other applications of PNA include DNA chain invasion [Nielsen et al., 1991], antisense inhibition [Hanvey et al., 1992], mutation analysis [Orum et al., 1993], transcriptional enhancers [see : Mollegaard et al., 1994], nucleic acid purification [Orum et al., 1995], isolation of transcriptionally active genes [Boffa et al., 1995], blocking transcription factor binding [Vickers et al., 1995], genome cleavage [Velselkov et al., 1996], biosensor [Wang et al., 1996], in situ hybridization [Thisted et al., 1996] and alternative approaches to Southern blotting Use in Perry-O'Keefe, 1996.
[671] Polypeptide Composition
[672] In other aspects, the present invention provides a polypeptide composition. In general, the polypeptide of the invention will be an isolated polypeptide (or epitope, variant or active fragment thereof) derived from a mammalian species. Preferably, the polypeptide is encoded by a polynucleotide sequence described herein or a sequence that hybridizes with the polynucleotide sequence described herein under appropriate stringent conditions. Alternatively, the polypeptide can be defined as a polypeptide comprising a contiguous amino acid sequence from an amino acid sequence described herein, or a polypeptide comprising a complete amino acid sequence described herein.
[673] In the present invention, the polypeptide composition is also a polypeptide of the invention, in particular a polypeptide having the amino acid sequence set forth in SEQ ID NOs: 176, 179, 181, 469-473, 475, 485, 487 and 488, or an active fragment, variant thereof. Or at least one polypeptide that is immunologically reactive with an antibody produced to a biologically functional equivalent.
[674] Likewise, polypeptide compositions of the present invention comprise one or more contiguous nucleic acid sequences set forth in SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489, or active fragments, variants thereof. Or one or more polypeptides capable of inducing an antibody that is immunologically reactive with one or more polypeptides encoded by one or more nucleic acid sequences that hybridize with one or more of these sequences under moderately stringent or fairly stringent conditions. I understand. Particularly exemplified polypeptides include the amino acid sequences set forth in SEQ ID NOs: 176, 179, 181, 469-473, 475, 485, 487 and 488.
[675] As used herein, active fragments of a polypeptide include all or a portion of a polypeptide that is modified by conventional techniques, such as mutagenesis, addition, deletion or substitution, although such active fragments are described herein. Exhibit substantially the same structure, function, antigenicity, and the like as the polypeptide as described above.
[676] In certain exemplary embodiments, polypeptides of the invention will comprise at least immunogenic portions of breast tumor proteins or variants thereof as described herein. As recognized above, a "mammary tumor protein" is a protein expressed by breast tumor cells. Proteins, which are breast tumor proteins, also react at detectable levels with antisera from patients with breast cancer in immunoassays (eg, ELISA). Polypeptides as described herein can be any length. There may be additional sequences and / or duplex sequences derived from the original protein, which sequences may retain (but are not necessarily required) additional immunogenic or antigenic properties.
[677] As used herein, an “immunogenic portion” is a portion of a protein that is recognized (ie, specifically bound) by B-cell and / or T-cell surface antigen receptors. Such immunogenic moieties generally comprise at least 5, more preferably at least 10, even more preferably at least 20 amino acid residues of the breast tumor protein or variant thereof. Certain preferred immunogenic moieties include peptides that have deleted the N-terminal leader sequence and / or transmembrane domain. Other preferred immunogenic moieties may contain small N- and / or C-terminal deletions (eg, 1-30 amino acids, preferably 5-15 amino acids) relative to mature protein.
[678] Immunogenic moieties can generally be identified using well known techniques as summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening for the ability to react with antigen specific antibodies, antisera and / or T-cell lines or clones.Antisera and antibodies, as used herein, may be used when they specifically bind to an antigen. (Ie, when they react with proteins in an ELISA or other immunoassay and do not react at detectable levels with unrelated proteins). These antisera and antibodies use well known techniques and The immunogenic portion of the original breast tumor protein is substantially no less than a full length polypeptide. And part that reacts with the antiserum and / or T-cells (e.g., by ELISA and / or T-cell reactivity assay) These immunogenic moieties correspond to the reactivity of the full length polypeptide in the assay. Such screens may be reacted at similar or greater levels, such screens are generally described in methods well known to those skilled in the art, such as, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, a polypeptide can be immobilized on a solid support, which can be contacted with patient serum to allow antibodies in such serum to bind to the immobilized polypeptide. Unbound serum can be removed and then bound antibody can be detected using, for example, ¹²⁵ I-labeled Protein A.
[679] As mentioned above, the composition may comprise variants of the original breast tumor protein. A polypeptide “variant” as used herein is a polypeptide that differs from the original breast tumor protein in that it has one or more substitutions, deletions, additions and / or insertions that do not substantially reduce the immunogenicity of the polypeptide. . In other words, the ability of the variant to react with antigen specific antiserum may be enhanced or unchanged relative to the original protein, or may be lowered by less than 50%, preferably less than 20%, relative to the original protein. Such variants can generally be identified by modifying one of the polypeptide sequences and then evaluating the reactivity of the modified polypeptide with an antigen specific antibody or antiserum as described herein. Preferred variants include variants in which one or more moieties are removed, eg, an N-terminal leader sequence or transmembrane domain. Other preferred variants include those in which a small portion (eg, 1-30 amino acids, preferably 5-15 amino acids) is removed from the N- and / or C-terminus of the mature protein.
[680] Polypeptide variants encompassed by the present invention include about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 with a polypeptide as described herein. Variants that exhibit the same percentage (as determined above) of at least%, 97%, 98% or 99% are included.
[681] Preferably, variants contain conservative substitutions. "Conservative substitutions" are the replacement of one amino acid with another amino acid with similar properties, so that those skilled in peptide chemistry will expect that the secondary structure and hydrotherapy properties of such polypeptides will not substantially change. . Amino acid substitutions can generally be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphiphilic nature of the residue. For example, negatively charged amino acids include aspartic acid and glutamic acid; Positively charged amino acids include lysine and arginine; Amino acids having uncharged polar head groups and similar hydrophilic values include leucine, isoleucine and valine; Glycine and alanine; Asparagine and glutamine and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may exhibit 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. Variants may also have non-conservative changes. In a preferred embodiment, variant polypeptides differ from the original sequence by replacing, deleting or adding up to 5 amino acids. Variants can also be modified, for example, by deleting or adding amino acids with minimal impact on the immunogenicity, secondary structure and amphipathic properties of the polypeptide of interest.
[682] As mentioned above, a polypeptide may comprise a signal (or leader) sequence at the N-terminus of such a protein that directs the translation of the protein at the same time or after translation. The polypeptide may also be conjugated to a linker or other sequence (eg poly-His) to facilitate synthesis, purification or identification of such polypeptide, or to enhance binding of the polypeptide to a solid support. have. For example, the polypeptide can be conjugated to an immunoglobulin Fc region.
[683] Polypeptides can be prepared using a variety of well known techniques. Recombinant polypeptide encoded by a DNA sequence as mentioned above can be readily prepared from the DNA sequence using any of a variety of expression vectors well known to those skilled in the art. Expression can be achieved in any suitable host cell transformed or transfected with an expression vector containing a DNA molecule encoding a recombinant polypeptide. Suitable host cells include prokaryote, yeast and higher eukaryotic cells such as mammalian cells and plant cells. Preferably, the host cell used is E. coli. E. coli, yeast or mammalian cell lines such as COS or CHO. Supernatants from suitable host / vector systems that secrete recombinant polypeptides or proteins into the culture medium may first be concentrated using commercially available filters. After concentration, this concentrate can be applied to a suitable preparative matrix, for example an affinity matrix or an ion exchange resin. Finally, the recombinant polypeptide can be further purified using one or more reversed phase HPLC steps.
[684] Techniques well known to those skilled in the art can also be used to generate moieties and other variants having less than about 100 amino acids, typically less than about 50 amino acids, by synthetic methods. For example, such polypeptides can be synthesized using commercially available solid phase techniques, such as the Merrifield solid phase synthesis method, which sequentially adds amino acids to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85: 2149-2146, 1963. Automated synthesis equipment for polypeptides is available from suppliers [eg, Perkin Elmer / Applied BioSystems Division; Foster City, CA, which is commercially available and can operate according to manufacturer's instructions.
[685] Within certain embodiments, the polypeptide comprises a fusion protein comprising multiple polypeptides as described herein, or one or more polypeptides as described herein and unrelated sequences, eg, known tumor proteins. May be a fusion protein. The fusion protein helps to provide, for example, a T helper epitope (immunological fusion partner), preferably a T helper epitope recognized by human, or in higher yield than the original recombinant protein (expression enhancer). May help to express it. Certain preferred fusion partners are fusion partners that are both immunological and expression enhancing. Other fusion partners may be selected to increase the solubility of the protein of interest or to allow the protein to be targeted to the desired intracellular compartment. Further fusion partners also include an affinity tag that facilitates purification of the protein.
[686] Fusion proteins can generally be prepared using standard techniques, including chemical conjugation. Preferably, the fusion protein is expressed as a recombinant protein that allows for increased levels in the expression system as compared to the unfused protein. In brief, the DNA sequences encoding the polypeptide components can be assembled separately and linked to appropriate expression vectors. The 3 'end of the DNA sequence encoding one polypeptide component is linked to the 5' end of the DNA sequence encoding a second polypeptide component, with or without a peptide linker, so that the reading frame of these sequences Be on the same phase. This allows translation into a single fusion protein that retains the biological activity of both component polypeptides.
[687] Peptide linker sequences can be used to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide is folded into its secondary and tertiary structure. Such peptide linker sequences are incorporated into fusion proteins using standard techniques well known in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) their ability to adapt to flexible extended conformations; (2) their inability to adapt to secondary structures capable of reacting with functional epitopes on the first and second polypeptides, and (3) hydrophobic or charged moieties that may react with polypeptide functional epitopes. Lack of. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other closely neutral amino acids such as Thr and Ala may also be used in the linker sequence. Amino acid sequences that can be usefully used as linkers include those described in Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83: 8258-8262, 1986; US Pat. Nos. 4,935,233 and 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. If the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate functional domains and prevent steric interference, no linker sequence is required.
[688] The DNA sequence so linked is operably linked to a suitable transcriptional or translational regulatory element. Regulatory elements involved in the expression of DNA are located only 5 'of the DNA sequence encoding the first polypeptide. Similarly, stop codons and transcription termination signals required for terminal translation are present only at 3 'of the DNA sequence encoding the second polypeptide.
[689] Fusion proteins are also provided. Such proteins include polypeptides as described herein, along with unrelated immunogenic proteins. Preferably, the immunogenic protein may elicit a recall reaction. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins. See Stoute et al., New Engl. J. Med., 336: 86-91, 1997].
[690] Within a preferred embodiment, an immunological fusion partner is derived from protein D, a surface protein of Gram-negative bacteria Haemophilus influenza B (WO 91/18926). Preferably, the protein D derivative comprises approximately the first 1/3 of the protein (eg, the first N-terminal 100-110 amino acids) and can lipidize the protein D derivative. Within certain preferred embodiments, the first 109 residues of the lipoprotein D fusion partner are included on the N terminus to provide a polypeptide having additional exogenous T-cell epitopes. Increase the expression level in E. coli, thereby acting as an expression enhancer. The lipid tail ensures optimal labeling of antigens for antigen presenting cells. Other fusion partners include non-structural proteins (hemagglutinin) from influenza virus NS1. Different fragments comprising T-helper epitopes may be used, but typically, N-terminal 81 amino acids are used.
[691] In another embodiment, the immunological fusion partner is a protein known as LYTA, or a portion thereof (preferably C-terminal portion). LYTA is derived from Streptococcus pneumoniae , which synthesizes N-acetyl-L-alanine amidase known as amidase LYTA (encoded by LytA gene; Gene 43: 265-292, 1986). LYTA is an autolysin that specifically cleaves specific bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is involved in affinity for choline or some choline homologues such as DEAE. This property is useful for the expression of fusion proteins. This led to the development of E. coli C-LYTA expressing plasmids. Purification of hybrid proteins containing C-LYTA fragments at the amino terminus has been reported in the literature (Biotechnology 10: 795-798, 1992). Within a preferred embodiment, repeating sequence portions of LYTA can be incorporated into the fusion protein. The repeat sequence portion is found in the C-terminal region starting at residue 178. Particularly preferred repeat sequence portions incorporate residues 188-305.
[692] Generally, polypeptides (including fusion proteins) and polynucleotides as described herein are isolated. A "isolated" polypeptide or polynucleotide is one that is removed from its original environment. For example, natural proteins are separated when they are separated from all or some of the coexisting materials in the natural system. Preferably, such polypeptides have a purity of at least about 90%, more preferably at least about 95%, most preferably at least about 99%. Polynucleotides are considered isolated, for example, when they are cloned into a vector that is not part of the natural environment.
[693] In order to enhance the antigenic and / or immunogenicity of breast tumor proteins according to the invention, fusion proteins comprising the antigenic and / or immunogenic portions of two or more breast tumor proteins can be prepared. Exemplary breast tumor fusion proteins include DNA sequences encoding mamma globin, including (1) a combined B726P upstream and downstream ORF (SEQ ID NO: 490); It can be prepared via conventional recombinant DNA techniques by combining with DNA sequences encoding either (2) upstream B726P ORF (SEQ ID NO: 491) and / or (3) downstream B726P ORF (SEQ ID NO: 492). Ausubel , FM et al. "Short Protocols in Molecular" (4 ^th ed. 1999); The entirety of which is incorporated herein by reference). Exemplary fusion proteins are described herein as SEQ ID NO: 493 (mamma globulin-combined B726P ORF), SEQ ID NO: 494 (mamma globulin-upstream B726P ORF), and SEQ ID NO: 495 (mamma globulin-downstream B726P ORF). DNA sequences encoding mamma globin are described herein as nucleotides 1-279 of SEQ ID NOs: 490-492, and corresponding mamma globin amino acid sequences are described herein as amino acids 1-93 of amino acids 493-495. Patent 5,668,267; 5,922,836; 5,922,836; No. 5,855,889; 5,968,754 and 6,004,756; Its starch is incorporated herein by reference].
[694] In addition to fusion proteins exemplified by the fusion of full length mammaglobin coding regions with various B726P coding regions, the present invention further provides at least 9 contiguous amino acids from either or both of mamma globin and B726P. It provides a fusion protein comprising an immunogenic portion of. More preferably, the immunogenic portion is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more contiguous amino acids from either or both of mamma globin and / or B726P. Can be. Alternatively, the immunogenic portion may be at least 25, 30, 35, 40, 45, 50, 75, 100, 250, 500 or 1095 consecutive amino acids from any or both of mamma globin and / or B726P. And may comprise 20 to 1095 consecutive any integer amino acids from either or both of mammaglobin and / or B726P.
[695] Representative immunogenic portions of mamma globin are described in currently pending US patent application Ser. No. 60 / 136,528. Exemplary immunogenic moieties include the following mammaglobin peptide sequences: IDELKECFLNQTDETLSNVE (amino acids 59-78 of SEQ ID NO: 493); TTNAIDELKECFLNQ (amino acids 55-69 of SEQ ID NO: 493); SQHCYAGSGCPLLENVISKTI (amino acids 13-33 of SEQ ID NO: 493); EYKELLQEFIDDNATTNAID (amino acids 41-60 of SEQ ID NO: 493) and / or KLLMVLMLA (amino acids 2-10 of SEQ ID NO: 493). Other preferred epitopes include glycosylation sites of mamma globin. Such epitopes are particularly useful for the production of antibodies that specifically bind to glycosylated mamma globin. Two such sites are the N-linked glycosylation site Asparagine (Asp) -53 (QEFIDD N ATTNAI; amino acids 47-59 of SEQ ID NO: 493) and Asp-68 (LKECFL N QTDETL; amino acids 62-74 of SEQ ID NO: 493).
[696] The invention also contemplates that a wide range of immunogenic portions from the B726P combined amino acid sequence, the B726P upstream amino acid sequence, and / or the B726P downstream amino acid sequence can be used for the mammaglobin-B726P fusion protein. For example, particularly suitable mammaglobin-B726P fusion proteins include downstream B726P epitopes recognized by the B726P-specific CTL clones described in Example 4 herein, such epitopes are included in the N-terminus of the downstream region of B726P. (Eg, amino acids 1-129 of SEQ ID NO: 176), for example, by fusing mamma globin.
[697] It will be apparent to those skilled in the art that the exact amino acid sequence and primary sequence arrangement of the mammaglobin and / or B726P portion of the fusion protein may vary without departing from the scope of the present invention. For example, conservative amino acid substitutions in either or both of the mammaglobin or B726P moieties can be performed to achieve fusion proteins with improved properties such as, for example, increased protein stability and / or immunogenicity. Can be. In addition, the present invention allows the mammaglobin moiety to be fused to either the N-terminus or C-terminus of the B726P moiety to achieve a fusion protein with the desired antigenic and / or immunogenic properties.
[698] The fusion proteins according to the invention, as exemplified by the above examples as the mammaglobin-B726P fusion proteins described herein, will be useful as cancer vaccines, as reagents for antibody treatment and / or for various diagnostic assays. It is expected that these fusion proteins will have improved antigenic and / or immunogenic properties compared to mamma globin and / or B726P protein alone.
[699] Binding agent
[700] The present invention further provides agents, such as antibodies and antigen-binding fragments thereof, that specifically bind to breast tumor proteins. As used herein, an antibody or antigen-binding fragment thereof reacts at detectable levels with breast tumor proteins (eg, in an ELISA) and at detectable levels with proteins not related under similar conditions. If not, it is believed to be "specifically bound" to the breast tumor protein. As used herein, “binding” refers to a noncovalent association between two separate molecules such that a complex is formed. Binding capacity can be assessed, for example, by determining the binding constants for the formation of the complex. This binding constant is the concentration of the complex divided by the component product concentration. In general, when the binding constant for complex formation exceeds about 10 ³ L / mol, both compounds are considered to be "bonded" in the context of the present invention. Binding constants can be determined using methods well known in the art.
[701] Binding agents can be used to distinguish between patients who have cancer and those who do not, using the representative assays provided herein. In other words, an antibody or other binding agent that binds to a breast tumor protein will generate a signal indicative of the presence of cancer in about 20% or more of patients with cancer disease, and in about 90% or more of individuals who do not have cancer It will generate a negative signal indicating the absence of the disease. To determine whether a binding agent meets these requirements, biological samples from patients with cancer and non-cancer patients (as determined using standard clinical trials), such as blood, serum, saliva, urine and And / or tumor biopsies) can be assayed as described herein to determine the presence of a polypeptide that binds to the binding agent. It is clear that statistically significant numbers of diseased and non- diseased samples should be tested. While each binding agent must meet the above criteria, one skilled in the art will recognize that binding agents can be used in combination to improve sensitivity.
[702] Any agent that meets the above requirements can be a binding agent. For example, the binding agent may be a ribosome, RNA molecule or polypeptide with or without peptide component. In a preferred embodiment, the binding agent is an antibody or antigen binding fragment thereof. Such antibodies can be prepared by various techniques known to those skilled in the art (Harlow and Lane, Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, 1988). In general, antibodies are produced by cell culture techniques, including the production of monoclonal antibodies as described herein, or by transfecting an antibody gene into a suitable bacterial or mammalian cell host to allow production of recombinant antibodies. You can. In one technique, an immunogen comprising the polypeptide is initially injected into a wide variety of mammals (eg, mouse, rat, rabbit, sheep or goat). In this step, the polypeptide of the present invention can act as an immunogen without modification. On the other hand, particularly for relatively short polypeptides, an excellent immune response can be induced when the polypeptide is linked to a carrier protein such as bovine serum albumin or keyhole limpet hemocyanion. Such immunogens are injected into the animal host, preferably according to a predetermined schedule with one or more booster immunization processes, and blood is periodically withdrawn from the animal. Polyclonal antibodies specific for that polypeptide can then be purified from the antiserum by, for example, affinity chromatography using a polypeptide coupled to a suitable solid support.
[703] Monoclonal antibodies specific for the antigenic polypeptide of interest are described, for example, in Kohler and Milstein, Eur. J. Immunol. 6 : 511-519 (1976), and improvements therefor. Briefly stated, these methods include preparing immortal cell lines capable of producing antibodies with the desired specificity (ie, reactivity with the polypeptide of interest). Such cell lines can be generated, for example, from spleen cells obtained from animals immunized as mentioned above. Such spleen cells are then immortalized by fusing, for example, a myeloma cell fusion partner, preferably with a genotype common with the immunized animal. Various fusion techniques can be used. For example, splenocytes and myeloma cells may be combined together in a nonionic detergent for several minutes and then plated at low density on a selection medium that supports hybrid cell growth but not myeloma cell growth. Preferred screening techniques use HAT (hypoxanthine, aminopterin, thymidine) screening. After sufficient time, typically about 1 to 2 weeks, hybrid colonies are observed. Single colonies were selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas with high reactivity and specificity are preferred.
[704] Monoclonal antibodies can be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques, such as injecting hybridoma cell lines into the peritoneal cavity of a suitable vertebrate host such as a mouse, can be used to increase yield. The monoclonal antibodies can then be harvested from ascites or blood. Contaminants can be removed from the antibody by conventional techniques such as chromatography, gel filtration, precipitation and extraction. Polypeptides of the invention can be used in purification processes, for example, by affinity chromatography steps.
[705] Within certain embodiments, it may be desirable to use antigen binding fragments of antibodies. Such fragments include Fab fragments that can be prepared using standard techniques. Briefly, immunoglobulins can be purified from rabbit serum by affinity chromatography on a Protein A bead column (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain. Fab and Fc fragments can be obtained. These Fab and Fc fragments can be separated by affinity chromatography on a Protein A bead column.
[706] The monoclonal antibodies of the invention can be coupled to one or more therapeutic agents. Suitable therapeutic agents in this regard include radionuclides, differentiation inducing agents, drugs, toxins, and derivatives thereof. Preferred radionuclides include ⁹⁰ Y, ¹²³ I, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹¹ At and ²¹² Bi. Preferred medicaments include methotrexate, and pyrimidine and purine homologues. Preferred differentiated derivatives include phorbol esters and butyric acid. Preferred toxins include lysine, abrin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, shigella toxin and pokeweed antiviral protein.
[707] The therapeutic agent may be coupled (eg covalently bound) to a suitable monoclonal antibody either directly or indirectly (eg via a linker group). If each has a substituent that can react with each other, a direct reaction between the therapeutic agent and the antibody is possible. For example, either nucleophilic group, e.g., amino or sulfhydryl group, may be an alkyl group containing another good leaving group (e.g. halide), or a carbonyl containing group, e.g. It can react with anhydrides or acid halides.
[708] Alternatively, it may be desirable to couple the therapeutic agent and antibody through a linker group. The linker group can act as a spacer that spaces between the antibody and the therapeutic agent to avoid interfering binding capacity. The linker group may act to increase the coupling efficiency by increasing the chemical reactivity of the substituents on the agent or antibody. Due to the increased chemical reactivity, it may promote the use of agents, or functional groups on the agents, which was not possible.
[709] It will be apparent to those skilled in the art that various di- or multi-functional reagents that are homo- and hetero-functional (eg, those listed in the catalog of Pierce Chemical Co., Rockford, IL) can be used as linker groups. The coupling reaction can be carried out, for example, via an amino group, carboxyl group, sulfhydryl group or oxidized carbohydrate moiety. There are a number of references described about this method (eg, US Pat. No. 4,671,958 to Rodwell et al.).
[710] If the therapeutic agent is more potent when isolated from the antibody portion of the immunoconjugates of the invention, it may be desirable to use a cleavable linker group during or during internal migration into cells. Many different cleavable linker groups have been reported. Intracellular release mechanisms of agents from these linker groups include methods of cleaving by reducing disulfide bonds (eg, US Pat. No. 4,489,710 (Spitler)), methods of irradiating light labile bonds [eg, US Pat. No. 4,625,014 Senter et al., Methods for hydrolyzing derivatized amino acid side chains (eg, US Pat. No. 4,638,045 (Kohn et al.)), Serum complement mediated hydrolysis reactions [eg, US Patent 4,671,958 (Rodwell et al.) And acid catalyzed hydrolysis reactions (eg US Pat. No. 4,569,789 (Blattler et al.)).
[711] It may be desirable to couple one or more agents to the antibody. In one embodiment, multiple molecules of the agent are coupled to one antibody molecule. In another embodiment, more than one type of agent can be coupled to one antibody. Regardless of certain embodiments, immunoconjugates with one or more agents can be prepared by a variety of methods. For example, linkers can be used that couple one or more agents directly to the antibody molecule, or provide multiple sites for attachment. Alternatively, a carrier can be used.
[712] The carrier can retain the agent in a variety of ways, including direct covalent bonds or covalent bonds through linker groups. Suitable carriers include proteins, such as albumin (Kato et al.), Peptides and polysaccharides, such as aminodextran (Shi et al., US Pat. No. 4,699,784). al.)]. The carrier may also retain certain agents by noncovalent bonds or by encapsulation in liposome vesicles (see US Pat. Nos. 4,429,008 and 4,873,088). Carriers specific to radionuclide preparations include radiohalogenated small molecules and chelated compounds. For example, US Pat. No. 4,735,792 describes representative radiohalogenated small molecules and their synthesis. Radionuclide chelates may be formed from chelating compounds, including those containing nitrogen and sulfur atoms as donor atoms for binding metal or metal oxide radionuclides. For example, US Pat. No. 4,673,562 (Davison et al.) Describes representative chelating compounds and their synthesis.
[713] Various routes of administration for antibodies and immunoconjugates can be used. Typically, administration will be intravenously, intramuscularly, subcutaneously, or in incisional tumor phase. It is clear that the exact dose of antibody / immunoconjugate will vary depending on the antibody used, antigen density to the tumor, and the clearance rate of the antibody.
[714] T cell
[715] The immunotherapeutic composition may also include T cells specific for breast tumor proteins. Such cells can generally be prepared in vitro or ex vivo using standard procedures. For example, T cells are available from the supplier Nexell Therapeutics, Inc .; Commercial cell separation systems, such as the Isolex ™ system available from Irvine, CA, can be used to isolate from a patient's bone marrow, peripheral blood, or certain fractions of bone marrow or peripheral blood. See US Pat. No. 5,240,856; 5,215,926; 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243. Alternatively, T cells can be derived from related or unrelated human, non-human mammals, cell lines or cultures.
[716] T cells can be stimulated with breast tumor polypeptides, polynucleotides encoding breast tumor polypeptides, and / or antigen presenting cells (APCs) expressing such polypeptides. Such stimulation is carried out under conditions and for a time sufficient to generate T cells specific for the polypeptide. Preferably, the breast tumor polypeptide or polynucleotide is present in a delivery vehicle, eg, microspheres, to promote the development of specific T cells.
[717] T cells, when these T cells specifically proliferate, secrete cytokines, or die from target cells that are coated with a breast tumor polypeptide or express genes encoding these polypeptides, the breast tumor polypeptide It is considered to be specific to. T cell specificity can be assessed using any of a variety of standard techniques. For example, within chromium release assays or proliferation assays, stimulation indices that increase fusion and / or proliferation more than twofold compared to negative controls indicate T cell specificity. Such assays are described, for example, in Chen et al., Cancer Res. 54: 1065-1070, 1994. On the other hand, detection of T cell proliferation can be carried out by various known techniques. For example, T cell proliferation may be determined by measuring the rate of DNA synthesis increase (eg, by pulse-labeling T cell culture with tritiated thymidine and then measuring the amount of tritiated thymidine incorporated into the DNA). Can be detected. Contact with the breast tumor polypeptide (100 ng / ml to 100 μg / ml, preferably 200 ng / ml to 25 μg / ml) for 3 to 7 days should result in a two-fold increase in the proliferation of T cells. As mentioned above, after 2-3 hours of contact, T cells should be activated using a standard cytokine assay where a 2-fold increase in cytokine release (eg, TNF or IFN-γ) indicates T cell activation. (See Coligan et al., Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998). T cells activated in response to a mammary tumor polypeptide, polynucleotide or polypeptide expressing APC may be CD4 ⁺ and / or CD8 ⁺ . Breast tumor protein specific T cells can be expanded using standard techniques. Within a preferred embodiment, these T cells are derived from the patient, associated donor or unrelated donor, stimulated and expanded and then administered to the patient.
[718] For therapeutic purposes, CD4 ⁺ or CD8 ⁺ T cells that proliferate in response to breast tumor polypeptide, polynucleotides or APCs can be expanded numerically in vitro or in vivo. Proliferation of such T cells in vitro can be accomplished in a variety of ways. T cells are exposed to or reexposed to breast tumor polypeptides with or without adding T cell growth factors, such as interleukin-2, and / or stimulator cells that synthesize breast tumor polypeptides. The short peptide corresponding to the immunogenic portion of the polypeptide can be exposed again. On the other hand, one or more T cells that proliferate in the presence of breast tumor proteins can be expanded numerically by cloning. Methods of cloning cells are well known in the art and include limiting dilution.
[719] Pharmaceutical composition
[720] In a further aspect, one or more polynucleotides, polypeptides, T cells described herein in a pharmaceutically acceptable solution for administration to the cell or animal alone or in combination with one or more other therapeutic modalities. And / or formulation of the antibody composition.
[721] If desired, nucleic acid segments, RNA, DNA or PNA compositions expressing a polypeptide as described herein may be administered in combination with other agents, such as other proteins or polypeptides or various pharmaceutically active agents. You should be aware. In fact, as long as the additional agent does not cause significant adverse effects upon contact with the target cell or host tissue, the other components that may be included are hardly limited. As such, the compositions can be delivered with various other agents as desired in certain cases. Such compositions may be purified from host cells or other biological sources, or may be chemically synthesized as described herein. Likewise, the composition may further comprise a substituted or derivatized RNA or DNA composition.
[722] Formulations of pharmaceutically acceptable excipients and carrier solutions are well known to those skilled in the art and employ particular compositions described herein in various therapeutic regimens, including oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation. Suitable dosages and treatment regimens for the development are under development.
[723] 1. Oral Delivery
[724] In certain applications, the pharmaceutical compositions described herein may be delivered to an animal via oral administration. As such, these compositions may be formulated with inert diluents or assimilable edible carriers, or they may be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into dietary foods. Can be.
[725] The active compound may even be incorporated with excipients and used in the form of ingestible tablets, intraballinal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Mathiowitz et al., 1997; Hwang et al., 1998; US Patent No. 5,641,515; 5,580,579 and 5,792,451; Each full text is specifically incorporated herein by reference. The tablets, troches, pills, capsules and the like may contain the following: binders such as tragacand gum, acacia gum, corn starch or gelatin; Excipients such as dicalcium phosphate; Disintegrants such as corn starch, potato starch, alginic acid; Lubricants, such as magnesium stearate, and sweetening agents, such as sucrose, lactose, and saccharin, or flavoring agents, such as peppermint, wintergreen extract anti-oil, and cherry flavor. When the dosage unit form is a capsule, in addition to the substances of the above type, it may contain a liquid carrier. Various other materials may be present as coatings or to change the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar or both. Elixir syrups may contain the active compound together with sucrose as a sweetening agent, methyl and propylparaben as preservatives, dyes and flavoring agents such as cherry or orange flavors. Of course, the materials used to prepare any dosage unit form must be pharmaceutically pure and substantially nontoxic in the amounts employed. In addition, the active compounds may be incorporated into sustained release formulations and formulations.
[726] Typically, these formulations may contain at least about 0.1% of the active compound, but the ratio of the active ingredient (s) may, of course, vary, conveniently about 1 or based on the weight or dose of the total formulation. From 2% to about 60 or 70%. Originally, the amount of active compound (s) in each therapeutically useful composition is that amount which can be obtained in such a way that a suitable dosage will be obtained within the desired unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product storage cycle, as well as other pharmacological considerations will be considered by those skilled in the art of preparing the pharmaceutical formulations, and therefore various dosages and treatments. A regime may be desired.
[727] For oral administration, the compositions of the present invention may be incorporated with one or more excipients in the form of mouthwashes, toothpastes, buccal tablets, oral sprays, or sublingual orally administered formulations. For example, mouthwashes can be prepared by incorporating the required amount of active ingredient in a suitable solvent, such as Dobell's Solution. Alternatively, it may be incorporated into an oral solution such as a solution comprising sodium borate, glycerin and potassium bicarbonate, dispersed in toothpaste, or in a therapeutically effective amount, such as water, binders, abrasives, It can be added to compositions that may include flavors, antifoams, and humectants. Alternatively, the composition may be in the form of a tablet or tablet that can be placed under the tongue or dissolved in the mouth.
[728] 2. Injection Delivery
[729] In certain circumstances, the pharmaceutical compositions described herein are disclosed in US Pat. No. 5,543,158; Parenteral, intravenous, intramuscular or even intraperitoneal delivery will be desirable, as described in US Pat. Nos. 5,641,515 and 5,399,363, each of which is specifically incorporated by reference herein. Solvents of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersants can also be prepared in glycerol, liquid polyethylene glycols, and mixtures and oils thereof. Under ordinary conditions of storage and use, these preparations contain a preservative to inhibit the growth of microorganisms.
[730] Pharmaceutical forms suitable for injection include sterile aqueous solvents or dispersants and sterile powders for improvisation of sterile injectable solutions or dispersants (US Pat. No. 5,466,468; the disclosure of which is specifically incorporated herein by reference in its entirety). Inserted). In all cases, the form must be sterile and must be fluid to the extent that it can be easily injected. It must be stable under the conditions of manufacture and storage and must inhibit the action of microorganisms (eg bacteria and fungi). The carrier may be, for example, a dispersion medium or solvent containing water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycols, etc.), suitable mixtures thereof and / or vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, in the case of dispersants, by maintaining the required particle size and by using surfactants. Inhibition of microbial action can be promoted by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable composition can be achieved by the use of agents which delay absorption in the composition, for example, aluminum monostearate and gelatin.
[731] For parenteral administration in the form of an aqueous solvent, for example, the solvent must be suitably buffered as necessary and the liquid diluent first made isotonic with sufficient saline or glucose. These particular aqueous solvents are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be employed will be known to those skilled in the art in view of this specification. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of bulk subcutaneous injection or injected at the proposed injection site. See Remington's Pharmaceutical Sciences 15th Edition, pages 1035-1038 and 1570. -1580]. Some dose change will necessarily occur depending on the condition of the subject being tested. In any case, the person involved in the administration will determine the appropriate dose for the individual subject. Moreover, when administered to humans, it must meet sterile, pyrogenic, and general safety and purity standards as required by the FDA for biological standards.
[732] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with the various other ingredients enumerated above, followed by filtered sterilization. Generally, dispersants are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solvents, the preferred method of preparation is the vacuum drying and lyophilization methods, whereby a powder of the active ingredient plus the additional desired ingredient is produced from its previously sterile-purified solvent.
[733] The compositions described herein may be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein), and salts or organic acids formed with inorganic acids, such as hydrochloric acid or phosphoric acid, such as acetic acid, oxalic acid, tartaric acid, Salts formed with delic acid and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium or iron oxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. have. When formulated, the solvent will be administered in a manner consistent with the dosage form and in a therapeutically effective amount. Such formulations are readily administered in a variety of dosage forms, such as injectable solutions, drug-release capsules, and the like.
[734] As used herein, "carrier" includes all solvents, dispersion media, vehicles, coatings, diluents, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as conventional media or agents are incompatible with the active ingredient, their use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
[735] “Pharmaceutically acceptable” refers to molecular entities and compositions that, when administered to a human, do not cause an allergic or similar undesirable reaction. The preparation of aqueous compositions containing proteins as active ingredients is well known in the art. Typically, such compositions are prepared as injectable liquid solutions or suspensions; Solid forms suitable for dissolving or suspending in liquids may also be prepared prior to injection. The formulation may also be emulsified.
[736] 3. intranasal delivery
[737] In certain embodiments, the pharmaceutical composition can be delivered by intranasal spray, inhalation, and / or other aerosol delivery vehicles. Methods for delivering genes, nucleic acids and peptide compositions directly to the lungs via nasal aerosol sprays are described, for example, in US Pat. Nos. 5,756,353 and 5,804,212, each of which is specifically incorporated herein by reference in their entirety. Reported. Similarly, intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds [US Pat. No. 5,725,871; It is well known in the pharmacy art to deliver a drug using its full text, specifically incorporated herein by reference. Likewise, methods for transmucosal drug delivery in the form of polytetrafluoroethylene support matrices are described in US Pat. No. 5,780,045, the entirety of which is specifically incorporated herein by reference.
[738] 4. Liposomal-, Nanocapsule- and Microparticle-mediated Delivery
[739] In certain embodiments, we contemplate using liposomes, nanocapsules, microparticles, microspheres, liquid particles, endoplasmic reticulum and the like to introduce the compositions of the invention into suitable host cells. In particular, the compositions of the present invention can be formulated to deliver encapsulated liquid particles, liposomes, endoplasmic reticulum, nanospheres or nanoparticles and the like.
[740] Such formulations may be desirable for introducing a pharmaceutically acceptable formulation of a nucleic acid or construct described herein. The formation and use of liposomes is generally known to those skilled in the art. See, for example, Couvreur et al., 1977; Couvreur, 1988; Lasic, 1988; This describes the use of liposomes and nanocapsules in targeted antibiotic therapy for intracellular bacterial infections and diseases. Recently, liposomes with improved serum stability and circulating half-life have been developed [Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987; US Patent No. 5,741,516; Each full text is specifically incorporated herein by reference. In addition, various methods of liposomes and liposome-like preparations as potent drug carriers have been reported. Takakura, 1998; Chandran et al., 1997; Margalit, 1995; US Patent No. 5,567,434; 5,552,157; 5,565,213; 5,565,213; 5,738,868 and 5,795,587; Each full text is specifically incorporated herein by reference.
[741] Liposomes have been used successfully with numerous cell types that are normally resistant to transfection by other processes, including T cell suspensions, primary hepatocyte cultures, and PC 12 cells (Renneisen et al., 1990; Muller). et al., 1990). In addition, liposomes lack the DNA length suppressor typical of virus-utilized delivery systems. Liposomes are genes, drugs (Heath and Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989; Fresta and Puglisi, 1996), radiotherapy (Pikul et al., 1987), enzymes (Imaizumi et al. , 1990a; Imaizumi et al., 1990b), viruses (Faller and Baltimore, 1984), transcription factors and homosteric effectors (Nicolauand Gersonde, 1979) have been effectively used to introduce into various cultured cell lines and animals. In addition, several successful clinical trials have been completed to examine the efficacy of liposome mediated drug delivery. Lopez-Berestein et al., 1985a; 1985b; Coune, 1988; Sculier et al., 1988]. In addition, some studies suggest that the use of liposomes is not associated with autoimmune responses, toxins, and gonad localization following systemic delivery (Mori and Fukatsu, 1992).
[742] Liposomes are formed from phospholipids dispersed in an aqueous medium, which spontaneously form multilayered concentric bilayer vesicles (also referred to as multilayer vesicles (MLV)). MLVs generally have a diameter of 25 nm to 4 μm. Sonication of the MLV results in the formation of small monolayer vesicles (SUVs) with a diameter in the range of 200 to 500 kPa, containing an aqueous solution.
[743] Liposomes possess something similar to cellular membranes and are contemplated for use in connection with the present invention as carriers for peptide compositions. They are widely suitable because both water soluble and lipid soluble materials can be captured in the aqueous space and in the bilayer itself, respectively. Drug bearing liposomes can also be used to site-specifically deliver an active agent by selectively modifying the liposome formulation.
[744] In addition to the teachings of Couvreur et al., 1977, the following information can be utilized to generate liposome formulations. Phospholipids, when dispersed in water, can form various structures other than liposomes, depending on the molar ratio of lipid to water. At low molar ratios, liposomes are the preferred structure. The physical characteristics of liposomes depend on the pH, ionic strength and presence of divalent cations. Liposomes may exhibit low permeability to ionic and polar materials, but at elevated temperatures, phase transitions occur that significantly change their permeability. This phase transition involves a change from a tightly packed and ordered structure known as the gel state to a loosely packed and less ordered structure known as the fluid state. It occurs at characteristic phase transition temperatures, thereby increasing the permeability to ions, sugars and drugs.
[745] In addition to temperature, exposure to proteins can change the permeability of liposomes. Certain soluble proteins, such as cytochrome c, bind to, modify, and penetrate the bilayer, causing changes in permeability. Cholesterol inhibits the penetration of such proteins by more tightly and visually packing the phospholipids. It is contemplated that the liposome formulations most useful for antibiotic and inhibitor delivery will contain cholesterol.
[746] The ability to capture solutes varies between different types of liposomes. For example, MLVs are reasonably efficient at capturing solutes, but SUVs are extremely inefficient. However, SUVs offer uniformity and reproducibility advantages in size distribution and are imparted by large monolayer vesicles (LUVs) with a large trade-off between size and collection efficiency. They are prepared by ether evaporation and are 3-4 times more efficient than MLV in solute capture.
[747] In addition to liposome characterization, an important determinant in capturing a compound is the physicochemical properties of the compound itself. When the polar compound is trapped in the aqueous space, the nonpolar compound is bound to the lipid bilayer of the endoplasmic reticulum. Polar compounds are released through permeation or when the bilayer is broken, but nonpolar compounds remain associated with the bilayer unless it is ruptured by temperature or exposure to lipoproteins. Both types exhibit maximum outflow rates at phase transition temperatures.
[748] Liposomes interact with cells through four different mechanisms: endocytosis by phagocytes of reticulocytes such as macrophages and neutrophils; Adsorption to the cell surface by nonspecific weak hydrophobic or electrostatic forces or by specific interaction with cell surface components; Fusion with the plasma cell membrane by inserting the liposome lipid bilayer into the plasma membrane while simultaneously releasing the liposome contents into the cytoplasm and transferring the liposome lipid to the intracellular membrane or subcellular membrane without any association with the liposome contents, or On the contrary. Determining if a mechanism works and determining whether more than one can work at the same time is often difficult.
[749] The fate and tendency of liposomes injected intravenously depend on their physical properties such as size, flow and surface charge. They may last from several hours to several days in the tissue and, depending on their composition, may have a half-life in blood of several minutes to several hours. Larger liposomes such as MLV and LUV are rapidly absorbed by phagocytes of the reticuloendothelial system, but the physiology of the circulatory system inhibits the release of this large species at most sites. They may be released only at locations where large openings or voids are present in the capillary endothelium, eg, the sinusoid of the liver or spleen. Therefore, these organs are preferential sites of absorption. SUVs, on the other hand, exhibit a broader tissue distribution, but are still highly blocked in the liver and spleen. In general, this in vivo behavior is limited to the efficient targeting of liposomes only to those organs and tissues that have access to their large size. These include blood, liver, spleen, bone marrow and lymphatic organs.
[750] Targeting is generally not limited only in the context of the present invention. However, where specific targeting is desired, methods available to it should be achieved. Antibodies can be used to bind to liposome surfaces, and antibodies and their drug contents can be directed to specific antigenic receptors located on specific cell type surfaces. Carbohydrate determinants (glycoproteins or glycolipid cell-surface components that play specific roles in cell-cell recognition, interaction, and adhesion) can also be used as recognition sites, which are ineffective in directing liposomes to specific cell types. Because there is. In most cases, intravenous injection of liposome preparations can be used, but other routes of administration are possible.
[751] On the other hand, the present invention provides pharmaceutically acceptable nanocapsule formulations of the compositions of the present invention. Nanocapsules can generally capture compounds in a stable and reproducible manner (Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998; Douglas et al., 1987. In order to avoid side effects caused by intracellular polymeric overload, for example ultra-fine particles (approximately 0.1 μm in size), they must be designed with polymers that can degrade in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention. Such particles are described in Couvreur et al., 1980; 1988; zur Muhlen et al., 1998; Zambaux et al., 1998; Pinto-Alphandry et al., 1995 and US Pat. No. 5,145,684; Each full text is specifically incorporated by reference herein, and can be readily prepared.
[752] Immunogenic Compositions
[753] In certain preferred embodiments of the invention, an immunogenic composition or vaccine is provided. Such immunogenic compositions generally comprise one or more pharmaceutical compositions as discussed above in combination with immunostimulating agents. The immunostimulatory agent may be any substance that enhances or enhances an immune response (antibody and / or cell mediated immune response) to an exogenous antigen. Examples of immunostimulating agents include the adjuvant, biodegradable microspheres (eg polylactic galactide) and liposomes [these compounds are incorporated into these liposomes; See, US Pat. No. 4,235,877 to Fullerton. Vaccine formulations are generally described, for example, in M.F. Powell and M.J. Newman, eds., "Vaccins Design (the subunit and adjuvant approach)", Plenum Press (NY, 1995). Pharmaceutical compositions and immunogenic compositions within the scope of the present invention may contain other compounds that may be biologically active or inactive. For example, one or more immunogenic sites of other tumor antigens may be present in the composition, incorporated into the fusion polypeptide, or as separate compounds.
[754] As the illustrated immunogenic compositions may contain DNA encoding one or more polypeptides as mentioned above, such polypeptides are produced in situ. As noted above, the DNA may be present in a variety of delivery systems known to those of skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Numerous gene delivery techniques are well known in the art and are described, for example, in Rolland, Crit. Rev. Therap. Drug Carrier Systems 15: 143-198, 1998 and references cited therein. Suitable nucleic acid expression systems contain DNA sequences necessary for expression in a patient, such as suitable promoters and termination signals. Bacterial delivery systems involve the administration of bacteria (eg Bacillus-Calmette-Guerrin ) that express or secrete an immunogenic portion of a polypeptide on its cell surface. In a preferred embodiment, the DNA is used using a viral expression system (eg vaccinia or other pox virus, retrovirus or adenovirus), which may include the use of a non-pathogenic (defective) replication competent virus. Can be introduced. Suitable systems are described, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86: 317-321, 1989; Flexner et al., Ann. NY Acad. Sci. 569: 86-103, 1989; Flexner et al., Vaccine 8: 17-21, 1990; U.S. Patents 4,603,112, 4,769,330 and 5,017,487; WO 89/01973; US Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; 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; and Guzman et al., Cir. Res. 73: 1202-1207, 1993. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. Such DNA is described, for example, in Ulmer et al., Science 259: 1745-1749, 1993 and as reviewed by Cohn, Science 259: 1691-1692, 1993, It may be "exposed". Uptake of exposed DNA can be increased by coating such DNA onto biodegradable beads that are efficiently transported into cells. It will be apparent that the immunogenic composition may comprise both polynucleotide components and polypeptide components. Such immunogenic compositions can provide an enhanced immune response.
[755] It will be apparent that the immunogenic compositions may contain pharmaceutically acceptable salts of the polynucleotides and polypeptides provided herein. Such salts include pharmaceutically acceptable ratios including organic bases (eg salts of primary, secondary and tertiary amines and basic amino acids) and inorganic salts such as sodium, potassium, lithium, ammonium, calcium and magnesium salts. It can be prepared from toxic bases.
[756] Although any suitable carrier known to those skilled in the art can be used in the compositions of the present invention, the type of carrier will depend upon the mode of administration. The compositions of the present invention may be formulated for suitable modes of administration, including, for example, topical, oral, intranasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, eg subcutaneous injection, the carrier preferably comprises water, saline, alcohol, fat, wax or buffer. For oral administration, solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose and magnesium carbonate or any of the above carriers can be used. Biodegradable microspheres such as polylactate polyglycolate can also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are described, for example, in US Pat. No. 4,897,268; 5,075,109; 5,075,109; 5,928,647; 5,928,647; 5,811,128; 5,811,128; 5,820,883; 5,820,883; 5,853,763; 5,814,344 and 5,942,252. Carriers comprising the microparticle-protein complexes described in US Pat. No. 5,928,647, which can induce a class I-limited cytotoxic T lymphocyte response in a host, can also be used.
[757] Such compositions may include buffers (eg neutral buffered saline or phosphate buffered saline), carbohydrates (eg glucose, mannose, sucrose or dextran), mannitol, proteins, polypeptides or amino acids such as glycine, oxidation Inhibitors, disinfectants, chelating agents, for example EDTA or glutathione, adjuvants (e.g. aluminum hydroxide), solutes, suspending agents, thickeners, which render the formulation isotonic, hypotonic or weak hypertonic with the blood of the receptor and And / or preservatives. Alternatively, the compositions of the present invention can be formulated as lyophilisates. Well known techniques can also be used to encapsulate a compound in liposomes.
[758] Various immunostimulants can be used in the immunogenic compositions of the present invention. For example, an adjuvant may be included. Most adjuvants are materials designed to prevent catabolism of antigens rapidly, such as aluminum hydroxide or mineral oil, and immune response stimulants such as lipid A, Bortadella pertussis or mycobacterium. Mycobacterium tuberculosis derived proteins. Suitable adjuvants include, for example, Freund's incomplete adjuvant and complete adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 from Merck and Company, Inc., Rahway, NJ; AS-2 from SmithKline Beecham, Philadelphia, PA; Aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; Calcium, iron or zinc salts; Insoluble suspending agents of acylated tyrosine; Acylated sugars; Polysaccharides derivatized cationic or anionic; Polyphosphazenes; Biodegradable microspheres; Commercially available as monophosphoryl lipid A and quill A. GM-CSF or cytokines such as interleukin-2, -7 or -12 may be used as the adjuvant.
[759] Within the immunogenic compositions provided herein, it is desirable to design the adjuvant composition primarily to induce an Th1 type immune response. High levels of Th1-type cytokines such as IFN-γ, TNFα, IL-2 and IL-12 tend to induce cell mediated immune responses to the administered antigen. In contrast, high levels of Th2-type cytokines (eg, IL-4, IL-5, IL-6, and IL-10) tend to elicit primarily humoral immune responses. After administering an immunogenic composition as provided herein, the patient will tolerate an immune response, including Th1- and Th2-type responses. Within a preferred embodiment where the response is mainly Th1-type, the level of Th1-type cytokines will increase much more than the level of Th2-type cytokines. The levels of these cytokines can be readily assessed using standard assays. For a review of cytokine families, see Mosmann and Coffman, Ann. Rev. Immunol. 7 : 145-173, 1989.
[760] Preferred adjuvants for use mainly in inducing Th1-type reactions are, for example, monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) and aluminum salts. There is a combination with. MPL adjuvant is available from the following sources (Corixa Corporation, Seattle, WA; see US Pat. Nos. 4,436,727, 4,877,611, 4,866,034, and 4,912,094). CpG-containing oligonucleotides, wherein the CpG dinucleotides are not methylated, also mainly induce Th1 responses. Such oligonucleotides are well known and are described, for example, in WO 96/02555 and WO 99/33488, US Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, in Sato et al., Science 273 : 352, 1996. Another preferred adjuvant is saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, MA), which may be used alone or in combination with other adjuvants. For example, an enhanced system may be a combination of monophosphoryl lipid A with a saponin derivative, for example a combination of 3D-MPL and QS21 as described in WO 94/00153, or as described in WO 96/33739. Likewise, QS21 includes less reactive developmental compositions that are quenched with cholesterol. Other preferred formulations include water-in-oil emulsions and tocopherols. Particularly effective adjuvant formulations comprising QS21, 3D-MPL and tocopherol in water-in-oil emulsions are described in WO 95/17210.
[761] Other preferred adjuvants include Montanide ISA 720 (Seppic, France), as described in pending US patent applications 08 / 853,826 and 09 / 074,720, which are incorporated herein by reference in their entirety. , SAF (Source: Chiron, California, United States), ISCOMS (Source: CSL), MF-59 (Source: Chiron), Adjuvant of the SBAS series (e.g. SBAS-2 or SBAS-4 (Source: SmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other aminoalkyl glucosaminide 4-phosphates (AGPs).
[762] Immunogenic compositions provided herein can be prepared using methods well known to produce combinations of antigens, immune response enhancers, and suitable carriers or excipients. The compositions described herein can be administered as part of sustained release formulations (ie, formulations that slowly release the compound after administration, eg, capsules, sponges, or gels (eg, consisting of polysaccharides)). Such formulations may generally be prepared using well known techniques (Coombers et al., Vaccine 14 : 1429-1438, 1996) and may be administered or desired by, for example, oral, rectal or subcutaneous infusion. Administration can be by infusion at the target site. Sustained release formulations may contain polypeptides, polynucleotides or antibodies, contained in a reservoir dispersed in a carrier matrix and / or surrounded by a rate controlling membrane.
[763] Carriers for use in such formulations are biologically suitable and can also be biodegradable; Preferably, the formulation provides a relatively constant level of active ingredient release. Such carriers include microparticles such as poly (lactide-co-glycolide), polyacrylates, latex, starch, cellulose, dextran and the like. Other delayed release carriers include supramolecular biovectors comprising a non-liquid hydrophilic core (eg, crosslinked polysaccharides or oligosaccharides), and optionally an outer layer comprising an amphiphilic compound such as a phospholipid [ See, US Patent Nos. 5,151,254 and PCT Publication Nos. WO 94/20078, WO 94/23701 and WO 96/06638. The amount of active compound contained in a sustained release formulation depends on the site of infusion, the rate of release and the expected duration, and the type of disease to be treated or prevented.
[764] Various delivery vehicles that facilitate the generation of antigen-specific immune responses that target tumor cells can be used in pharmaceutical compositions and immunogenic compositions. Delivery vehicles include antigen presenting cells (APCs) such as dendritic cells, macrophages, B cells, monocytes, and other cells that can be engineered to be efficient APCs. Genetically modify these cells to increase antigen-labeling ability, enhance activation and / or maintenance of T cell responses, have an anti-tumor effect itself and / or be immunologically compatible with the recipient (ie matched HLA) Hemisphere), but it is not necessary. APCs can generally be isolated from various biological fluids and organs, including tumor tissue and surrounding tumor tissue, which can be autologous, allogeneic, common genotypic or heterologous cells.
[765] Certain preferred embodiments of the invention use dendritic cells or their progeny as antigen presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392 : 245-251, 1998) and have been shown to be effective as physiological adjuvants for inducing prophylactic or therapeutic anti-tumor immunity. Timmerman and Levy, Ann. Rev. Med. 50 : 507-529, 1999]. In general, dendritic cells have their typical appearance (star-shaped in situ, which represents the remarkable cytoplasmic process (dendritic crystal) seen in vitro), the ability to absorb, process and label antigens with high efficiency, and the original T Identification can be based on the ability to activate cellular responses. Dendritic cells, of course, can be engineered to express specific cell-surface receptors or ligands not normally found in in vivo or ex vivo dendritic cells, and such modified dendritic cells are contemplated as the present invention. As a substitute for dendritic cells, secreted vesicle antigen-loaded dendritic cells (called exosomes) can be used in immunogenic compositions. See Zitvogel et al., Nature Med. 4 : 594-600, 1998].
[766] Dendritic cells and their progeny can be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, tissue surrounding tumor-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or other suitable tissues or fluids. For example, dendritic cells can be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and / or TNFα to monocyte cultures harvested from peripheral blood. Alternatively, a combination of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand, and / or other compound (s) that induces differentiation, maturation and proliferation of dendritic cells is added to the culture medium. By addition, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow can be differentiated into dendritic cells.
[767] Dendritic cells are commonly classified as "mature" cells and "mature" cells, which is a simple way to distinguish two widely characterized phenotypes. However, this nomenclature is not intended to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APCs with high antigen uptake and processing ability correlated with high expression of Fcγ receptors and mannose receptors. Mature phenotypes typically express low levels of these markers, but are cell surface molecules involved in T cell activation, such as class I and class II MHCs, adhesion molecules (eg CD54 and CD11) and costimulatory molecules ( Examples: CD40, CD80, CD86 and 4-1BB) are characterized by high levels of expression.
[768] APCs can generally be transfected with polynucleotides (or portions thereof or other variants thereof) encoding breast tumor proteins such that the breast tumor protein or immunogenic portion thereof is expressed on the cell surface. Such transfection can be performed ex vivo, and compositions comprising such transfected cells can be used for therapeutic purposes as described herein. Alternatively, transfection may be achieved in vivo by administering to the patient a gene transfer vehicle that targets dendritic cells or other antigen presenting cells. In vivo and ex vivo transfection of dendritic cells is generally carried out using methods known in the art as described, for example, in WO 97/24447, or by Mahvi et al., Immunology and Cell Biology. 75 : 456-460, 1997]. The antigenic load of dendritic cells is incubated with the dendritic cells or their progeny cells with a breast tumor polypeptide, DNA (exposed or contained in a plasmid vector) or RNA or antigen-expressing recombinant bacteria or virus (e.g., vaccinia). Nia, infectious epithelial, adenovirus or lentiviral vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner (eg, carrier molecule) that provides a T cell helper. Alternatively, dendritic cells can be pulsed individually or in the presence of a polypeptide as a non-conjugated immunological partner.
[769] Immunogenic compositions and pharmaceutical compositions may be provided in unit-dose or several-dose containers, eg, sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve the sterility of the formulation until used. In general, formulations may be stored as suspensions, solvents or emulsions in oily or aqueous vehicles. Alternatively, the immunogenic composition or pharmaceutical composition may be stored under lyophilized conditions requiring only the addition of a sterile liquid carrier immediately before use.
[770] Cancer treatment
[771] In a further aspect of the invention, the compositions described herein can be used for immunotherapy of cancer, such as breast cancer. Within such methods, pharmaceutical compositions and immunogenic compositions are typically administered to a patient. "Patient" as used herein refers to all warm-blooded animals, preferably humans. The patient may or may not have cancer. Thus, the pharmaceutical compositions and immunogenic compositions can be used to prevent the development of cancer or to treat patients with cancer. Cancer can be diagnosed using criteria generally accepted in the art, including the presence of malignant tumors. Pharmaceutical compositions and immunogenic compositions are administered before or after treatment by surgically removing the primary tumor and / or by administering radiation therapy or conventional chemotherapy drugs. Administration may be by any suitable method, including intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal, anal, vaginal, topical and oral administration.
[772] In certain embodiments, immunotherapy may be active immunotherapy wherein the treatment is an endogenous host immune system that responds to the tumor using administration of an immune response-modifying agent (eg, polypeptides and polynucleotides as provided herein). Depends on stimulation in vivo.
[773] In other embodiments, immunotherapy may be passive immunotherapy wherein the treatment exhibits an established tumor-immune responsiveness that may directly or indirectly mediate antitumor effects and does not necessarily depend solely on the original host immune system. Eg, effector cells or antibodies). Examples of effector cells include T cells, T lymphocytes (eg, CD8 ⁺ cytotoxic T-lymphocytes and CD4 ⁺ T-helper tumor-infiltrating lymphocytes), killer cells (natural killer cells and lymphokine-activation as mentioned above). Killer cells), B cells, and antigen presenting cells (eg, dendritic cells and macrophages) that express the polypeptides provided herein. Antibody receptors and T cell receptors specific for the polypeptides cited herein can be transferred into other vector or effector cells for cloning, expression, and proton immunotherapy. The polypeptides provided herein can also be used to generate antibodies or anti-genetic antibodies (such as those mentioned above and described in US Pat. No. 4,918,164) for passive immunotherapy.
[774] Effector cells can generally be obtained in an amount sufficient for quantum immunotherapy by growing in vitro as described herein. Culture conditions for expanding billions of single antigen specific effector cells while maintaining antigen recognition in vivo are well known in the art. Such in vitro culture conditions typically utilize intermittent stimulation with the antigen, often in the presence of cytokines (eg IL-2) and non-dividing feeder cells. As mentioned above, rapid expansion of antigen specific T cell cultures using immunoreactive polypeptides as provided herein results in a sufficient number of cells for immunotherapy. In particular, antigen presenting cells, such as dendritic cells, macrophages, monocytes, fibroblasts and / or B cells, can be pulsed with an immunoreactive polypeptide, or they can be prepared using one or more of the following techniques using standard techniques well known in the art. It can be transfected with a peptide. For example, antigen presenting cells can be transfected with polynucleotides having promoters suitable for increasing expression in recombinant viruses or other expression systems. Effector cells cultured for use in therapy must be widely grown and distributed and must survive long term in vivo. Studies have shown that by repeating stimulation with antigens supplemented with IL-2, it is possible to induce effector cells grown in vivo and cultured to a large extent for long-term survival. Cheever et al., Immunological Reviews 157 : 177, 1997].
[775] Alternatively, the polypeptides cited herein can be introduced into antigen presenting cells taken from a patient and cloned ex vivo in order to inject the implant back into the same patient. The transfected cells can be reintroduced to the patient by intravenous, intranasal, intraperitoneal or intratumoral administration, preferably in sterile form, using any means known in the art.
[776] The dosage as well as the route of administration and the frequency of administration of the therapeutic composition will vary from individual to individual, which can be readily established using standard techniques. In general, the pharmaceutical compositions and immunogenic compositions may be administered by injection (eg intradermal, intramuscular, intravenous or subcutaneous), intranasal (eg by aspiration) or orally. Preferably, 1 to 10 doses may be administered for 52 weeks. Preferably, six doses may be administered at monthly intervals, followed by periodic booster vaccination. Another protocol may be suitable for an individual patient. Suitable doses are those amounts of compounds capable of enhancing an anti-tumor immune response when administered as mentioned above, which are at least 10-50% of the baseline (ie untreated) level. This response can be monitored by measuring anti-tumor antibodies in the patient or by vaccine-dependent production of cell soluble effector cells capable of killing the patient's tumor cells in vitro. Such immunogenic compositions may also elicit an immune response that results in improved clinical outcomes (eg, more frequent differences in disease, complete, partial or longer survival) in treated patients compared to untreated patients. It should be possible. Generally, for pharmaceutical compositions and immunogenic compositions comprising one or more polypeptides, the amount of each polypeptide present in the dose ranges from about 25 μg to 5 mg per kg host. Suitable dosage sizes will vary depending on the size of the patient, but typically range from about 0.1 ml to about 5 ml.
[777] In general, suitable dosages and therapeutic regimens provide sufficient amount of active compound (s) to provide therapeutic and / or prophylactic benefits. This response can be monitored by establishing improved clinical outcomes (eg, more frequent differences in illness, complete, partial or longer survival) in treated patients compared to untreated patients. Increased pre-existing immune responses to breast tumor proteins generally correlate with improved clinical outcomes. This immune response can generally be assessed using standard proliferation, cytotoxicity or cytokine assays, which can be performed using samples obtained from patients before and after treatment.
[778] Cancer Detection and Diagnosis
[779] Generally, cancer is based on the presence of one or more breast tumor proteins and / or polynucleotides encoding such proteins in biological samples obtained from a patient (eg, blood, serum, saliva, urine and / or tumor biopsy). It can be detected in the patient. In other words, such proteins can be used as markers to indicate the presence or absence of cancer (eg, breast cancer). In addition, the protein may be useful for detecting other cancers. Binding agents provided herein generally allow for the detection of the level of antigen bound to the agent in a biological sample. Polynucleotide primers and probes can be used to detect the level of mRNA encoding tumor protein, which also indicates the presence or absence of cancer. In general, breast tumor proteins should be present at levels three or more times higher in tumor tissue than in normal tissue.
[780] There are a variety of assay formats known to those skilled in the art to use binding agents to detect polypeptide markers in certain samples (Harlow and Lane, Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, 1988). In general, whether a patient has cancer or is absent comprises (a) contacting a biological sample obtained from the patient with said binding agent; (b) in this sample, detecting the level of polypeptide bound to the binding agent; (c) can be determined by comparing the level of said polypeptide with a cut-off estimated value.
[781] In another embodiment, the assay comprises binding to the polypeptide using a binding agent immobilized on a solid support and removing it from the remaining sample. The bound polypeptide can then be detected using a detection reagent containing a reporter group and specifically bound to the binding agent / polypeptide complex. Such detection reagents can be, for example, binding agents or antibodies that specifically bind to polypeptides, or other agents that specifically bind to such binding agents, such as anti-immunoglobulins, protein G, protein A Or lectins. Alternatively, a competitive assay can be used that labels the reporter group on the polypeptide and allows the sample to be incubated with the binding agent and then bound to the immobilized binding agent. The degree to which the sample component inhibits the binding of the labeled polypeptide to the binding agent indicates the reactivity between the immobilized binding agent and the sample. Suitable polypeptides for use within such assays include full length breast tumor proteins and portions thereof to which binding agents are bound, as mentioned above.
[782] The solid support can be any substance known to those skilled in the art to which the tumor protein can be attached. For example, such a solid support can be a test well or nitrocellulose or other suitable membrane in a microtiter plate. Alternatively, the support may be a bead or disc, for example glass, fiber glass, latex or plastic material, for example polystyrene or polyvinylchloride. The support may also be a fiber optical sensor or magnetic particles, for example as described in US Pat. No. 5,359,681. Binding agents can be immobilized on a solid support using a variety of techniques known to those skilled in the art, described in detail in patents and scientific journals. In the context of the present invention, the term "immobilization" refers to both non-covalent bonds such as adsorption and covalent attachment, which may be direct linkages between the agent and the functional group on a support or linkages by crosslinkers. . It is preferable to fix by adsorption to the well or membrane in a microtiter plate. In this case, adsorption can be achieved by contacting the binding agent in a suitable buffer with the solid support for a suitable time. Contact time varies with temperature, but is typically about 1 hour to 1 day. Generally, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with a binding agent in the range of about 10 ng to about 10 μg, preferably about 100 ng to 1 μg, is used to immobilize the appropriate amount of binding agent. Suffice.
[783] Covalent attachment of a binding agent to a solid support can generally be accomplished by first reacting with the support a bifunctional reagent that will react with both the support on the binding agent and a functional group, such as a hydroxyl or amino group. For example, the binding agent can be covalently attached to a support having a suitable polymer coating, either by using benzoquinone or by condensing aldehyde groups on the support with active hydrogens and amines on the binding partner. Pierce Immunotechnology Catalog and Handbook (1991) at A12-A13].
[784] In certain embodiments, the assay is a two-antibody sandwich assay. Such assays can be performed by first contacting an antibody immobilized on a well of a solid support, typically a microtiter plate, with a biological sample to allow the polypeptide in such sample to bind with the immobilized antibody. The unbound sample is then removed from this immobilized polypeptide-antibody complex and the detection reagent containing the reporter group (preferably, a second antibody capable of binding different sites on the polypeptide) Add. The amount of detection reagent bound to the solid support is then determined using methods appropriate to the specific reporter group.
[785] More specifically, once the antibody is immobilized on a support as mentioned above, the remaining protein binding site on this support is typically blocked. All suitable blockers such as bovine serum albumin or Tween 20 ^™ (Sigma Chemical Co., St. Louis, Mo.) are known to those skilled in the art. The immobilized antibody is then incubated with the sample and the polypeptide is allowed to bind with this antibody. Prior to incubation, the sample may be diluted with a suitable diluent, for example phosphate buffered saline (PBS). In general, a suitable contact time (ie, incubation time) is a time sufficient to detect the presence of the polypeptide in a sample obtained from an individual with breast cancer. Preferred contact time is a time sufficient to achieve at least about 95% of the binding level resulting in equilibrium between the bound polypeptide and the unbound polypeptide. Those skilled in the art will appreciate that the time required to achieve equilibrium can be readily determined by assaying the level of binding that occurs over time. At room temperature, incubation for about 30 minutes is usually sufficient.
[786] Unbound samples can be removed by washing the solid support with a suitable buffer such as PBS containing 0.1% Tween 20 ^™ . Subsequently, a second antibody containing a reporter group can be added to the solid support. Preferred reporter groups include the groups mentioned above.
[787] The detection reagent is then incubated with the immobilized antibody-polypeptide complex for a time sufficient to detect the bound polypeptide. The appropriate time can generally be determined by assaying the level of binding that occurs over time. The unbound detection reagent is then removed and the bound detection reagent is detected using a reporter group. The method used to detect the reporter group depends on the type of reporter group. In the case of radioactive groups, scintillation coefficients or self-spinning methods are generally suitable. Spectroscopy can be used to detect dyes, luminescent groups, and fluorescent groups. Biotin coupled with different reporter groups (typically radioactive or fluorescent groups or enzymes) can be detected using avidin. Enzyme reporter groups generally can add a substrate (typically for a certain time) and then detect this reaction product by spectroscopy or other analysis.
[788] To determine the presence or absence of cancer (eg, breast cancer), signals detected from a group of reporters that remain bound to a solid support are generally compared to signals corresponding to cut-off predetermined values. In one preferred embodiment, the cut-off value for detecting cancer is the signal average obtained when the immobilized antibody is incubated with a sample from a patient who does not have cancer. In general, a sample that generates a signal that is 3 standard deviations above the cut-off predetermined value may be considered positive for cancer. In another preferred embodiment, receiver operator curves according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine , Little Brown and Co., 1985, p. 106-7. Curve) to determine the cut-off value. In brief, in this embodiment, the cut-off value is plotted by plotting a true positive rate (ie, sensitivity) and false positive rate (100% -specificity) pairs corresponding to each possible cut-off value for the diagnostic test results. You can decide. The cut-off value (ie, the value covering the largest area) on the plot closest to the upstream left corner is the most accurate cut-off value, and the sample producing a signal higher than the cut-off value determined by this method is positive. Can be considered. On the other hand, this cut-off value may move to the left along the plot to minimize the false positive rate, or to the right to minimize the false negative rate. In general, samples that generate signals higher than the cut-off value determined by this method are considered positive for cancer.
[789] In a related embodiment, the assay is performed in a flow-through or strip test format in which the binding agent is immobilized on a membrane such as nitrocellulose. In this cocurrent test, the polypeptide in the sample is combined with an immobilized binding agent as the sample passes into the membrane. The second labeled binding agent is then combined with the binding agent-polypeptide complex as the solution containing the second binding agent flows into the membrane. The detection of the bound second binding agent can then be carried out as mentioned above. In the strip test format, one end of the membrane to which the binding agent is bound is impregnated into the sample containing solution. This sample travels along the membrane and into the immobilized binding agent region through the region containing the second binding agent. Concentration of the second binding agent in the immobilized antibody region indicates the presence of cancer. Typically, the concentration of the second binding agent at this site creates a specific pattern, such as a line that is visually readable. The absence of this pattern indicates a negative result. In general, the amount of binding agent immobilized on the membrane is visually discernible if, in the format discussed above, the biological sample contains polypeptide levels that may be sufficient to generate a positive signal in a two-antibody sandwich assay. Choose to create a pattern. Preferred binding agents for use in such assays are antibodies and antigen binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 μg, more preferably from about 50 ng to about 1 μg. Such testing can typically be performed using extremely small amounts of biological samples.
[790] Of course, there are many other assay protocols suitable for use with the tumor proteins or binding agents of the invention. The above description is only an example. For example, it will be apparent to those skilled in the art that the protocol can be readily modified to detect the antibody binding to the breast tumor polypeptide in a biological sample. Detection of such breast tumor protein specific antibodies may be correlated with the presence of cancer.
[791] Cancer can also be detected based on the presence of T cells that specifically react with breast tumor proteins in a biological sample. Within certain methods, a biological sample containing CD4 ⁺ and / or CD8 ⁺ T cells isolated from a patient expresses a breast tumor polypeptide, a polynucleotide encoding such polypeptide and / or at least an immunogenic portion of said polypeptide. Incubated with APC to detect whether or not specific activation of T cells is present. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells can be isolated from patients by conventional techniques (eg, peripheral blood lymphocytes by Ficoll / Hypaque density gradient centrifugation). T cells can be incubated in vitro with polypeptides (eg, 5-25 μg / ml) at 37 ° C. for 2-9 days (typically 4 days). It may be desirable to incubate another aliquot of T cell sample in the absence of a breast tumor polypeptide, which acts as a control. For CD4 ⁺ T cells, activation is preferably detected by assessing the proliferation of T cells. In the case of CD8 ⁺ T cells, activation is preferably detected by assessing cytolytic activity. More than two times higher proliferation levels and more than 20% greater cytolytic activity than patients without disease indicate that the patient has cancer.
[792] As mentioned above, cancer may also be detected based on the level of mRNA encoding a breast tumor protein in a biological sample. For example, two or more oligonucleotide primers can be used in an assay using polymerase chain reaction (PCR) to amplify a portion of breast tumor cDNA derived from a biological sample, wherein the one or more oligonucleotide primers Specific for (ie, hybridized to) polynucleotides encoding breast tumor proteins. The cDNA thus amplified is then isolated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to polynucleotides encoding breast tumor proteins can be used in hybridization assays to detect the presence of polynucleotides encoding breast tumor proteins in a biological sample.
[793] In order to perform hybridization under assay conditions, the oligonucleotide primers and probes are at least about 60% identical to a portion of a polynucleotide encoding a breast tumor protein that is at least 10, preferably at least 20 nucleotides in length. And, preferably, at least about 75%, more preferably at least about 90% oligonucleotide sequence. Preferably, the oligonucleotide primers and / or probes are hybridized with polynucleotides encoding the polypeptides described herein under suitable stringent conditions as mentioned above. Oligonucleotide primers and / or probes that can be usefully employed in the diagnostic methods described herein are preferably 10 to 40 or more nucleotides in length. In a preferred embodiment, the oligonucleotide primers comprise at least 10 contiguous nucleotides of a DNA molecule having the sequences set forth in SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486 and 489, Preferably at least 15 consecutive nucleotides. Techniques for both PCR using assays and hybridization assays are well known in the art. See Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263, 1987; Erlich et al., PCR Technology, Stockton Press, NY, 1989].
[794] One preferred assay uses RT-PCR, where PCR is applied in conjunction with reverse transcription. Typically, RNA is extracted from a biological sample, such as biopsy tissue, and reverse transcribed to produce cDNA molecules. PCR amplification using one or more specific primers results in cDNA molecules, which can be isolated and visualized using, for example, gel electrophoresis. Amplification can be performed on biological samples taken from test patients and biological samples taken from individuals who do not have cancer. The amplification reaction can be performed on several dilutions of cDNA extending to second order size. More than two-fold increase in expression in several dilutions of the test patient sample is typically considered positive compared to the same dilution of the sample from an individual who does not have cancer.
[795] In another embodiment, the compositions described herein can be used as markers of cancer progression. In this embodiment, the assay as described above is performed over time for the diagnosis of cancer and the level change of the reactive polypeptide (s) or polynucleotide (s) is evaluated. For example, the assay may be performed every 24 to 72 hours over 6 months to 1 year and then as needed. In general, cancer is progressing in patients whose detected polypeptide or polynucleotide levels increase with time. In contrast, when reactive polypeptide or polynucleotide levels are constant or decreased over time, cancer is not progressing.
[796] Certain in vivo diagnostic assays can be performed directly on tumors. One such assay involves contacting tumor cells with a binding agent. The bound binding agent can then be detected directly or indirectly through the reporter group. Such binding agents can also be used in histological applications. Alternatively, polynucleotide probes can be used within these applications.
[797] As mentioned above, to improve sensitivity, multiple breast tumor protein markers can be assayed in a given sample. Binding agents specific for the different proteins provided herein can be combined within a single assay. In addition, multiple primers or probes may be used simultaneously. The selection of tumor protein markers can be based on routine experimentation to determine the combination that yields optimal sensitivity. In addition, assays for tumor proteins provided herein can be combined with assays for other known tumor antigens.
[798] Diagnostic Kit
[799] The invention further provides a kit for use in said diagnostic method. Such kits typically include two or more components necessary to perform a diagnostic assay. The components may be compounds, reagents, containers and / or equipment. For example, one container in a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a breast tumor protein. Such antibodies or fragments may be provided attached to a support material, as mentioned above. One or more additional containers may contain elements to be used in the assay, such as reagents or buffers. Such kits may also contain a reagent for detection as mentioned above, containing a reporter group suitable for detecting antibody binding directly or indirectly.
[800] Alternatively, the kit can be designed to detect the level of mRNA encoding a breast tumor protein in a biological sample. Such kits generally comprise one or more oligonucleotide probes or primers as mentioned above that hybridize with the polynucleotides encoding the breast tumor protein. Such oligonucleotides can be used, for example, in PCR or hybridization assays. Additional components that may be present in the kit include second oligonucleotides and / or diagnostic reagents or containers that facilitate the detection of polynucleotides encoding breast tumor proteins.
[801] The following examples are illustrated and are not so limited.
[802] Example 1
[803] Isolation and Identification of Breast Tumor Polypeptides
[804] This example describes the isolation of breast tumor polypeptide from a breast tumor cDNA library.
[805] A cDNA subtraction library containing cDNA from a breast tumor from which normal breast cDNA was subtracted is constructed as follows. Total RNA is extracted from primary tissue using Trizol reagent [Gibco BRL Life Technologies, Gaithersburg, MD] as described by the manufacturer. Purify polyA + RNA using an oligo (dT) cellulose column according to standard protocols. Next Kit [Clontech PCR-Selet cDNA Subtraction Kit; Clontech, Palo Alto, CA] was used to synthesize first chain cDNA. Driver DNA consists of cDNA from two normal breast tissues, and tester cDNA is from three primary breast tumors. Double-stranded cDNAs for both tester and driver are synthesized and digested with a combination of endonucleases (MluI, MscI, PvuII, SalI and StuI) that recognize 6 base pair DNA. These modifications resulted in a significant increase in mean DNA size compared to cDNAs generated according to the Clontech (Palo, Alto, CA) protocol. The digested tester cDNA as described above is connected to two different adapters and the subtraction is performed according to the Clontech protocol. The cDNA deducted as described above is subjected to two rounds of PCR amplification according to the manufacturer's protocol. The resulting PCR product was acloned into TA cloning vector pCRII (Invitrogen, San Diego, Calif.) And electroporated by ElectroMax. Transform into E. coli DH10B cells [Gibco BRL Life, Technologies]. DNA was isolated from independent clones and sequenced using the following automated sequence analyzer model 373A (Perkin Elmer / Applied Biosystems Division (Foster City, Calif.)).
[806] 63 unique cDNA clones were found in the subtracted breast tumor specific cDNA library. One chain (5 'or 3') cDNA sequence determined for the clone is provided in SEQ ID NOs: 1-61, 72 and 73, respectively. Comparing the cDNA sequences with known sequences in the gene bank using EMBL and the Gene Bank Database (Release 97), SEQ ID NOs: 14, 21, 22, 27, 29, 30, 32, 38, 44, 45, 53, No substantial homology with the sequences provided in 72 and 73. The sequences 1, 3, 16, 17, 34, 48, 57, 60 and 61 were found to represent known human genes. SEQ ID NOs: 2, 4, 23, 39 and 50 were found to show some similarities with previously identified non-human genes. Remaining 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) Has been shown to exhibit at least some homology with the previously identified expressed sequence tag (EST).
[807] To determine the mRNA expression levels of the isolated cDNA clones, cDNA clones from the above mentioned breast deductions are randomly taken and colony PCR amplified. Microarray techniques [Synteni, Palo Alto, CA] are used to determine their mRNA expression levels in breast tumors, normal breasts and various other normal tissues. Briefly, PCR amplification products are arrayed on slides in an array format, each product occupying a unique position in the array. mRNA is extracted from the tissue sample to be tested, reverse transcribed, and a fluorescently labeled cDNA probe is generated. The microarray is probed with the labeled cDNA probe, the slide is scanned and the fluorescence intensity is measured. Data were analyzed using Synteni provided GEMTOOLS software. Of the 17 cDNA clones tested, clones of SEQ ID NOs: 40, 46, 59, and 73 were overexpressed in breast tumors and all normal tissues tested (breast, PBMC, colon, fetal tissue, salivary glands, bone marrow, lungs, pancreas, colon, Low levels in spinal cord, adrenal gland, kidney, pancreas, liver, stomach, skeletal muscle, heart, small intestine, skin, brain and human mammalian epithelial cells). Clones of SEQ ID NOs: 41 and 48 were overexpressed in breast tumors and found to be expressed at low levels in all other tissues tested except bone marrow. The clone of SEQ ID NO: 42 was overexpressed in breast tumors and found to be expressed at low levels in all other tissues tested except bone marrow and spinal cord. The clone of SEQ ID NO: 43 was overexpressed in breast tumors and found to be expressed at low levels in the spinal cord, heart and all other tissues tested except the small intestine. The clone of SEQ ID NO: 51 was overexpressed in breast tumors and found to be expressed at low levels in all other tissues tested except the large intestine. The clone of SEQ ID NO: 54 was overexpressed in breast tumors and found to be expressed at low levels in PBMC, all other tissues tested except the stomach and small intestine. The clone of SEQ ID NO 56 was overexpressed in breast tumors and found to be expressed at low levels in all other tissues tested except colon and small intestine, human breast epithelial cells and SCID mouse-passaged breast tumors. The clone of SEQ ID NO: 60 was overexpressed in breast tumors and found to be expressed at low levels in the spinal cord and all other tissues tested except the heart. The clone of SEQ ID NO: 61 was overexpressed in breast tumors and found to be expressed at low levels in all other tissues tested except the small intestine. The clone of SEQ ID NO: 72 was overexpressed in breast tumors and found to be expressed at low levels in colon and all other tissues tested except salivary glands.
[808] Northern blot analysis of clone SYN18C6 (SEQ ID NO: 40) is shown in FIG. 1. The expected protein sequence encoded by SYN18C6 is provided in SEQ ID NO: 62.
[809] Additional cDNA clones that are overexpressed in breast tumor tissue are isolated from the breast cDNA subtraction library as follows. A breast deduction library is prepared as described above by PCR-utilized deduction using a pool of breast tumor cDNA as a tester and a pool of cDNA from normal breast cDNA or other normal tissue as a driver. CDNA clones from breast deductions are randomly taken and colony PCR amplified and microarray techniques as mentioned above are used to determine their mRNA expression levels in breast tumors, normal breasts and various other normal tissues. 24 unique cDNA clones were overexpressed in breast tumors and all normal tissues tested (breast, brain, liver, pancreas, lung, salivary glands, stomach, colon, kidney, bone marrow, skeletal muscle, PBMC, heart, small intestine, adrenal gland, spinal cord, Large intestine and skin). The cDNA sequences determined for these clones are shown in SEQ ID NOs: 63-87. Comparing SEQ ID NOS: 74-87 with sequences in the gene bank as mentioned above showed homology with previously identified human genes. No substantial homology was found for the sequences of SEQ ID NOs: 63-73.
[810] Three DNA isotypes for the clone B726P (partial sequence provided in SEQ ID NO: 71) were isolated as follows. Radioactive probes are synthesized from B726P by cleaving B726P DNA from the pT7Blue vector (Novagen) by BamHI / XbaI restriction digestion and using the resulting DNA as a template for single chain PCR in the presence of [α-32P] dCTP. The sequence of primers used for such PCR is provided in SEQ ID NO: 177. The resulting radioactive probe is used to probe random primed cDNA libraries and directional cDNA libraries made using RNA isolated from breast tumors. 85 clones were identified, excised, purified and sequenced. Of these 85 clones, three were found to each contain a practical open reading frame. The determined cDNA sequence of isotype B726P-20 is provided in SEQ ID NO: 175 and the corresponding expected amino acid sequence is shown in SEQ ID NO: 176. The determined cDNA sequence of isotype B726P-74 is provided in SEQ ID NO: 178 and the corresponding expected amino acid sequence is shown in SEQ ID NO: 179. The determined cDNA sequence of isotype B726P-79 is provided in SEQ ID NO: 180, and the corresponding expected amino acid sequence is shown in SEQ ID NO: 181.
[811] As part of obtaining a full length B726P clone using standard techniques, five additional clones representing additional 5 'sequences of B726P were isolated. These clones appear to be alternative splice forms of the same gene. The determined cDNAs of these clones are shown in SEQ ID NOs: 464-468, and the expected amino acid sequences encoded by SEQ ID NOs: 464-467 are shown in SEQ ID NOs: 470-473, respectively. Standard computer techniques were used to generate a 3,681 bp consensus DNA sequence (SEQ ID NO: 463) containing two large open reading frames. The downstream ORF encodes the amino acid sequence of SEQ ID NO: 181. The expected amino acid sequence encoded by the upstream ORF is shown in SEQ ID NO: 469. Subsequent studies resulted in the isolation of additional splice forms of B726P with 184 bp inserts relative to the other forms. This 184 bp insert causes a frameshift that brings the downstream and upstream ORFs together into a single ORF of length 1002aa. The determined cDNA sequence of this alternative splice form is set forth in SEQ ID NO: 474 and the corresponding amino acid sequence is provided in SEQ ID NO: 475.
[812] Using the cDNA subtraction library technique mentioned above, individual clones that are overexpressed in breast tumor tissue are further isolated. In particular, a cDNA subtraction library containing cDNA from breast tumors subtracted with five other normal human tissue cDNAs (brain, liver, PBMC, pancreas and normal breast) was utilized for the screening. From the original deductions, 177 clones were selected for further characterization by DNA sequencing and microarray analysis. Microarray analysis demonstrated that the sequences in SEQ ID NOs: 182-251 and 479 were overexpressed twice in human breast tumor tissues compared to normal human tissues. For 19 clones 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 No homology was found, except for some previously identified expressed sequence tags (EST). The remaining clones are genes previously identified, specifically SEQ ID NOs: 181-184, 187-193, 195-198, 200-204, 206, 207, 209, 210, 212, 213, 217, 218, 220, 221, 223 Some homology with -225, 227-231, 233-235, 237-239, 242-244 and 247-251.
[813] One clone of the cDNA clone isolated by PCR subtraction as mentioned above, found to be overexpressed in breast tumor tissue by microarray, was found to be identical to the known keratin gene (SEQ ID NO: 176; referred to as B720P). The full length cDNA sequence of the known keratin gene is provided in SEQ ID NO: 477 and the corresponding amino acid sequence is shown in SEQ ID NO: 478. Primers were generated based on the sequence of SEQ ID NO: 477 and used to clone full length cDNA from mRNA obtained from total RNA exhibiting high expression of B720P in real time PCR analysis. The product was then cloned and sequenced. The full length cDNA sequence determined for B720P is provided in SEQ ID NO: 484 and the corresponding amino acid sequence is shown in SEQ ID NO: 485.
[814] In further studies, truncated forms of B720P (referred to as B720P-tr) have been identified in breast carcinoma. This antigen was cloned from mRNA derived from whole breast tumor RNA showing high expression of B720P-tr in real time PCR analysis. cDNA pools are generated using mRNA, which is then used as a template to amplify the cDNA corresponding to B720P-tr by PCR. The cDNA sequence determined for B720P-tr is provided in SEQ ID NO: 486. B720P-tr has an ORF of 708 base pairs that encodes a 236 amino acid protein (SEQ ID NO: 487). The size of the transcript was confirmed by Northern analysis.
[815] Of the 70 clones showing overexpression in breast tumor tissues, 15 showed particularly good expression levels in breast tumors compared to normal human tissues. The following 11 clones do not exhibit substantial homology with any known genes. Clone 19463.1 (SEQ ID NO: 185) was overexpressed in the majority of breast tumors and SCID breast tumors tested (see Example 2); In addition, overexpression has been found in the majority of normal breast tissues. Clone 19483.1 (SEQ ID NO: 216) showed overexpression in several breast tumors, but no overexpression in any normal tissue tested. Clone 19470.1 (SEQ ID NO: 219) was slightly overexpressed in some breast tissues. Clone 19468.1 (SEQ ID NO: 222) appeared to be slightly overexpressed in the majority of breast tumors tested. Clone 19505.1 (SEQ ID NO: 226) was slightly overexpressed in 50% of breast tumors and in SCID tumor tissue, with some overexpression in normal breasts. Clone 1509.1 (SEQ ID NO: 232) was rarely overexpressed in breast tumors, but showed a certain degree of overexpression in metastatic breast tumor tissues and no significant level of overexpression in normal tissues. Clone 19513.1 (SEQ ID NO: 236) showed slightly overexpression in only a few breast tumors, but no significant overexpression in normal tissues. Clone 19575.1 (SEQ ID NO: 240) showed low levels of overexpression in some breast tumors and normal breasts. Clone 19560.1 (SEQ ID NO: 241) was overexpressed in 50% of breast tumors tested and in some normal breast tissues. Clone 19583.1 (SEQ ID NO: 245) was slightly overexpressed in some breast tumors and extremely low levels of overexpression were found in normal tissues. Clone 19587.1 (SEQ ID NO: 246) showed low levels of overexpression in some breast tumors and no significant level of overexpression in normal tissues.
[816] Clone 19520.1 (SEQ ID NO: 233), showing homology with 102D24 on chromosome 11q13.31, was overexpressed in breast tumors and SCID tumors. Clone 19517.1 (SEQ ID NO: 237), which showed homology with the human PAC 128M19 clone, was shown to be slightly overexpressed in the majority of breast tumors tested. Clone 19392.2 (SEQ ID NO: 247), showing homology with human chromosome 17, was overexpressed in 50% of breast tumors tested. Clone 19399.2 (SEQ ID NO: 250), showing homology with human Xp22 BAC GSHB-184P14, was shown to be slightly overexpressed in a limited number of breast tumors tested.
[817] In subsequent studies, 64 individual clones were isolated from a subtracted cDNA library containing cDNA from a breast tumor pool subtracted with cDNA from five normal tissues (brain, liver, PBMC, pancreas and normal breast). The subtracted cDNA library was prepared as mentioned above with the following modifications. A combination of five six base cutters (MluI, MscI, PvuII, SalI and StuI) is used to degrade cDNA instead of RsaI. This increased the average insert size from 300 bp to 600 bp. 64 isolated clones are colonized PCR amplified and their mRNA expression levels in breast tumor tissue, normal breast and various other normal tissues are examined using microarray techniques as mentioned above. The determined cDNA sequences of 11 clones that have been shown to be overexpressed in breast tumor tissue are provided in SEQ ID NOs: 405-415. Comparison of these sequences with published databases as summarized above showed homology between SEQ ID NOs: 408, 411, 413 and 414 and previously isolated ESTs. The sequences 405-407, 409, 410, 412, and 415 were found to show some homology with previously identified sequences.
[818] In a further study, deduction from cDNA from metastatic breast tumors deducted from cDNA pools from five normal tissues (breast, brain, lung, pancreas and PBMC) using Clontech's PCR-deduction protocol as mentioned above Prepare cDNA library. The determined cDNA sequences of 90 clones isolated from the library are provided in SEQ ID NOs: 316-404. Comparing these sequences with published databases as summarized above did not show any substantial homology with SEQ ID NO: 366. The sequences 321-325, 343, 354, 368, 369, 377, 382, 385, 389, 395, 397 and 400 were found to show some homology with existing isolated ESTs. The remaining sequences were found to show homology with previously identified gene sequences.
[819] In a further study, from cDNA from breast tumors subtracted with cDNA pools from six normal tissues (liver, brain, stomach, small intestine, kidney and heart) using Clontech's PCR-subtraction protocol as mentioned above, A subtracted cDNA library (referred to as 2BT) is prepared. For cDNA clones isolated from this deduction, DNA microarray analysis as described above is performed and the resulting data is subjected to four modified GEMTOOLS analyzes. The first analysis compared 28 breast tumors with 28 non-breast normal tissues and used an average overexpression of at least 2.1-fold as the screening cut-off The second analysis used 29 ratios of 6 metastatic breast tumors. Compared to breast normal tissues Average overexpression of 2.5-fold or more was used as cut-off The third and fourth assays included two initial SCID mouse-passages Tumors and two late SCID mouse-passaged tumors were compared, with average overexpression of at least 2.0-fold in early or late passaged tumors as cut-off, and microarray data for 2BT clones. The determined cDNA sequences of the 13 clones identified in this visual analysis are provided in SEQ ID NOs: 427-439. The determined cDNA sequences of the 22 clones identified using the modified GEMTOOLS analysis were sequenced. 440-462, and SEQ ID NOs: 453 and 454 show two partial, non-overlapping sequences of the same clone.
[820] Analysis of the clone sequences of SEQ ID NOs: 436 and 437 (referred to as 263G6 and 262B2) with published databases as mentioned above revealed no substantial homology with previously identified sequences. SEQ ID NOs: 427, 429, 431, 435, 438, 441, 443, 444, 445, 446, 450, 453 and 454 (266B4, 266G3, 264B4, 263G1, 262B6, 2BT2-34, 2BT1-77, 2BT1-62, 2BT1 The sequences of -60, 61, 2BT1-59, 2BT1-52 and 2BT1-40, respectively, show some homology with the previously isolated expressed sequence tags (EST). SEQ ID NOs: 428, 430, 432, 433, 434, 439, 440, 442, 447, 448, 449, 451, 452 and 455-462 (clone 22892, 22890, 22883, 22882, 22880, 22869, 21374, 21349, 21093, 21091, 21089, 21085, 21084, 21063, 21062, 21060, 21053, 21050, 21036, 21037 and 21048, respectively), show some homology with gene sequences previously identified in humans.
[821] Example 2
[822] Isolation and Characterization of Breast Tumor Polypeptides Obtained by PCR-Used Subtraction Using SCID-Passitated Tumor RNA
[823] Human breast tumor antigens were obtained by PCR-utilized subtraction using SCID mouse passaged breast tumors as follows. Human breast tumors are implanted in SCID mice, as described in US Patent Application No. 08 / 556,659 (filed 11/13/95), US Patent No. 5,986,170, and upon the first or sixth series of passages Collect it. Genes that have been found to be differentially expressed between early and late passage SCID tumors are phase specific and therefore useful for therapeutic and diagnostic applications. Total RNA was prepared from snap frozen SCID passaged human breast tumors from both first and sixth passages.
[824] PCR utilization subtraction is performed essentially as mentioned above. In the first subtraction (referred to as T9), RNA from the first passaged tumor is subtracted from the sixth passaged tumor RNA to identify more aggressive, late passage specific antigens. Of the 64 clones isolated and sequenced from these subtractions, 30 clones showed no real homology, which was later 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, with the exception of some previously identified expressed sequence tags (EST). The cDNA sequences determined for these clones are shown in SEQ ID NOs: 88-116, respectively. The isolated cDNA sequences of SEQ ID NOs: 117-140 showed homology with known genes.
[825] In the second PCR use subtraction, RNA from the sixth passaged tumor is subtracted from the first passaged tumor RNA to identify downregulated antigens throughout multiple passages. Of the 36 clones isolated and sequenced, 19 clones showed no real homology, which was later 14376, 14377, 14383, 14384, 14387, 14392, 14394, 14398, 14401, 14402, 14405, 14409, 14412 , 14414-14416, 14419, 14426 and 14427, with the exception of some previously identified expressed sequence tags (EST). The cDNA sequences determined for these clones are shown in SEQ ID NOs: 141-159, respectively. The isolated cDNA sequences of SEQ ID NOs: 160-174 showed homology with existing known genes.
[826] Human breast tumor antigens were further analyzed through PCR using subtraction using the first and sixth passage SCID tumor RNA. 63 clones were found to be differentially expressed at intervals of two or more times, as determined by microarray analysis, ie higher levels in early passaged tumors compared to late passaged tumors. Seventeen of these clones showed no substantial homology with any known genes, but were found to show some homology with previously identified expressed sequence tags (EST), which were later identified as 20266, 20270, 20274, 20276, 20277, 20280, 20281, 20294, 20303, 20310, 20336, 20341, 20941, 20954, 20961, 20965 and 20975 (SEQ ID NOs 252-268, respectively). The remaining clones were found to show some homology with known genes and were identified in the Brief Description and Sequence Identification sections of this figure, later on 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. The cDNA sequences determined for these clones are provided in SEQ ID NOs: 269-314, respectively.
[827] To further analyze based on the results obtained using microarray analysis, clones 20310, 20281, 20262, 20280, 20303, 20336, 20270, 20341, 20326 and 20977 (B820P, B821P, B822P, B823P, B824P, B825P). , B826P, B827P, B828P and B829P, respectively). Specifically, microarray data analysis indicates that these clones are overexpressed 2-3 times more in breast tumor RNA compared to normal tissues tested. Subsequent studies determine the complete insertion sequences for clones B820P, B821P, B822P, B823P, B824P, B825P, B826P, B827P, B828P and B829P. These extended cDNA sequences are shown in SEQ ID NOs: 416-426, respectively.
[828] Example 3
[829] Synthesis of Polypeptides
[830] Using FMOC chemistry with HPTU (O-Benzotriazole-N, N, N ', N'-tetramethyluronium hexafluorophosphate) activation, the polystyrene was synthesized on a synthesizer [Perkin Elmer / Applied Biosystems Division 430A Peptide Synthesizer]. Peptides can be synthesized. The Gly-Cys-Gly sequence can be attached to the amino terminus of the peptide to provide a method for binding or labeling the conjugated, immobilized surface of the peptide. Cleavage of the peptide from the solid support can be accomplished using the following cleavage mixture: trifluoroacetic acid: ethanedithiol: thianizol: water: phenol (40: 1: 2: 2: 3). After cleavage for 2 hours, the peptide can be precipitated in cold methyl-t-butyl-ether. The peptide pellet can then be dissolved in water containing 0.1% trifluoroacetic acid (TFA), lyophilized and purified by C18 reversed phase HPLC. The peptide is eluted using a gradient of 0-60% acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA). After lyophilization of the pure fractions, the peptides can be characterized by electrospray or other types of mass spectroscopy and amino acid analysis.
[831] Example 4
[832] Induction of Breast Antigen-Specific CTL Responses in Human Blood
[833] This example illustrates the ability of breast specific antigen B726P to induce a cytotoxic T lymphocyte (CTL) response in peripheral blood lymphocytes from normal humans.
[834] Autologous dendritic cells (DCs) are differentiated from monocyte cultures derived from PBMCs of normal donors by growing for 5 days in RPMI medium containing 10% human serum, 30 ng / ml GM-CSF and 30 ng / ml IL-4. . After incubation for 5 days, DCs were transformed into M.O.I. adenoviruses expressing recombinant B726P (downstream ORF; SEQ ID NO: 181). Infect with 2.5 overnight and mature for 8 hours by adding 2 micrograms / ml CD40 ligand. CD8 positive cells are enriched by depletion of CD4 and CD14-positive cells. Priming cultures are initiated with cytokines IL-6 and IL-12 in individual wells of several 96-well plates. These cultures are restimulated in the presence of IL-2 using autologous fibroblasts treated with IFN-gamma and transduced with B726P and CD80. After stimulation three times, IFN-gamma elipot assay, using IFN-gamma treated autologous fibroblasts, transduced to express B726P or irrelevant control antigen as antigen presenting cells (APC), see Lalvani et al. ., J. Exp. Med. 186: 859-865, 1997] to assess the presence of B726P specific CTL activity. Of the approximately 96 lines, one line (referred to as 6-2B) was identified that specifically recognizes B726P-transduced APC but does not specifically recognize control antigen-transduced APC. Such microcultures are cloned using standard protocols. B726P specific CTLs are identified by ElliSpot assay and expanded for further analysis. These CTL clones demonstrated the recognition of B726P expressing fibroblasts but no control antigen MART-1 using a chromium-51 release assay. Furthermore, using a panel of allogeneic fibroblasts transduced with B726P in an antibody blocking assay, HLA restriction elements for these B726P specific CTLs were identified as HLA-B * 1501.
[835] To more precisely define the location of epitopes recognized by the B726P specific CTL clone, a deletion construct (B726Pdelta3 ') containing only the N-terminal half of B726P was constructed into the pBIB retroviral expression plasmid (aa 1-129). ). The plasmid and other plasmids containing B726P are transfected into COS-7 cells, alone or in combination with plasmids expressing HLA-B * 1501. Approximately 48 hours after transfection, B726P specific CTL clones (1-9B) are added at approximately 10e4 cells per well. The next day the wells are harvested and the amount of IFN-gamma released is measured by ELISA. CTLs responded to the gastric background (EGFP) against COS-7 cells transfected with both B726P and HLA-B * 1501. There was no background reaction for COS-7 cells transfected with B726P or HLA-B * 1501 alone. Importantly, higher responses were observed using COS-7 cells transfected with HLA-B * 1501 and B726delta3 ′. This result indicates that the epitope is located in the N-terminal region (a.a. 1-129) of B726P. The region was examined and HLA-B * 1501 peptide binding motifs were described in J. Immunol. 1999, 162: 7277-84, and amino acid sequences corresponding thereto were identified and synthesized. These peptides were pulsed overnight on autologous B-LCL at 10 μg / ml. The next day, the cells were washed and the ability of the cells to stimulate B726P specific CTL clone 1-9B was assayed by IFN-gamma ELISPOT assay. Of the 11 peptides tested, only one peptide with amino acid sequence SLTKRASQY (a.a. 76-84; SEQ ID NO: 488) could be recognized by CTL. This result confirms the peptide as a naturally-treated epitope recognized by the B726P specific CTL clone.
[836] Example 5
[837] Preparation and Characterization of Antibodies to Breast Tumor Polypeptides
[838] Polyclonal antibodies against breast tumor antigen B726P were prepared as follows.
[839] this. The downstream ORF of B726P (SEQ ID NO: 181) expressed in E. coli recombinant expression system is grown overnight in LB broth with the appropriate antibiotic at 37 ° C. in a shake incubator. The next morning, 10 ml of the culture was added overnight to 500 ml to 2 × YT plus appropriate antibiotics in a 2 L baffle Erlenmeyer flask. When the light density (at 560 nm) of the culture reaches 0.4 to 0.6, the cells are induced with IPTG (1 mM). 4 hours after induction with IPTG, cells are harvested by centrifugation. The cells are then washed with phosphate buffered saline and centrifuged again. The supernatant is decanted and the cells frozen for further use or immediate treatment. 20 ml of lysis buffer is added to the cell pellet and vortex. this. To disrupt the E. coli cells, the mixture is carried out at a pressure of 16,000 psi into the French press. The cells are then centrifuged again and the supernatant and pellets are checked by SDS-PAGE to fractionate recombinant proteins. For proteins localized in cell pellets, the pellets are resuspended in 10 mM Tris pH 8.0, 1% CHAPS, the inclusion body pellets are washed and then centrifuged again. This process was repeated two more times. Inclusion body pellets thus washed are solubilized with 8M urea or 6M guanidine HCL (containing 10 mM Tris pH 8.0 plus 10 mM imidazole). This solubilized protein is added to 5 ml of nickel-chelate resin (Qiagen) and incubated for 45 minutes to 1 hour at room temperature with continuous shaking. After incubation, the resin and protein mixtures are poured into a disposable column and the cocurrent is collected. The column is then washed with 10-20 column volumes of solubilization buffer. The antigen is then eluted from the column using 8M urea, 10 mM Tris pH 8.0 and 300 mM imidazole and collected in 3 ml fractions. SDS-PAGE gels are performed to determine the fractions collected for further purification.
[840] As a final purification step, a strong anion exchange resin, such as HiPrepQ (Biorad), is equilibrated with appropriate buffer and the fractions collected from above are loaded into the column. The antigen is eluted off the column with increasing salt gradient. Fractions are collected as the column is run and another SDS-PAGE gel is run to determine the fractions collected in the pool from the column. The fractions thus collected are dialyzed against 10 mM Tris pH 8.0. It is then filtered into a 0.22 micron filter and placed in vials and the antigens frozen until needed for immunization.
[841] 400 micrograms of B726P antigen are combined with 100 micrograms of muramyldipeptide (MDP). Every four weeks the rabbit is boosted to 100 micrograms mixed with equal volume of incomplete Freund's adjuvant (IFA). Seven days after each boost, the animals are bled. Serum is produced by incubating the blood at 4 ° C. for 12-24 hours followed by centrifugation.
[842] 96-well plates are coated with B726P antigen by incubating with 50 microliters (typically 1 microgram) of recombinant protein for 20 hours at 4 ° C. 250 microliters of BSA blocking buffer is added to the wells and incubated for 2 hours at room temperature. The plates are washed six times with PBS / 0.01% Tween. Rabbit serum is diluted in PBS. 50 microliters of diluted serum is added to each well and incubated for 30 minutes at room temperature. After washing the plates as mentioned above, 500 microliters of Gott anti-rabbit horseradish peroxidase (HRP) is added at a 1: 10000 dilution and incubated for 30 minutes at room temperature. The plate is washed again as mentioned above, and 100 microliters of TMB microwell peroxidase substrate is added to each well. After incubation for 15 min in the dark at room temperature, the colorimetric reaction is stopped using 100 microliters of 1N H ₂ SO ₄ and immediately read at 450 nm. Polyclonal antibodies showed immunoreactivity to B726P.
[843] Example 6
[844] Protein Expression of Breast Tumor Antigens
[845] The downstream ORF of B726P (SEQ ID NO: 181), along with the C-terminal 6X His tag, is expressed in insect cells using a baculovirus expression system as follows.
[846] CDNA for the full length downstream ORF of B726P is PCR amplified using the primers of SEQ ID NOs: 480 and 481. The PCR product with the expected size is recovered from the agarose gel, restriction digested with EcoRI and HindII, and linked to the transition plasmid pFastBac1, which is digested with the same restriction enzyme. The sequence of the insert is confirmed by DNA sequencing. Using recombinant transfer plasmid pFBB726P, recombinant backmid DNA and viruses are made using the Bac-To-Bac Baculovirus Expression System (BRL Life Technologies, Gaithersburg, MD). High five cells are infected with recombinant virus BVB726P to produce proteins. The cDNA and amino acid sequences of the expressed B726P recombinant protein are provided in SEQ ID NOs: 482 and 483, respectively.
[847] From the foregoing, while specific aspects of the invention have been described herein for purposes of illustration, it will be appreciated that various modifications may occur without departing from the spirit and scope of the invention.

权利要求:
Claims (72)
[1" claim-type="Currently amended] (a) SEQ ID NOs: 2, 4-15, 18-33, 35-47, 49-56, 58, 59, 63-73, 88-116, 141-159, 175, 178, 180, 185, 186, 194, 199, 205, 208, 211, 214-216, 219, 222, 226, 232, 236, 240, 241, 245, 246, 252-268, 321-325, 343, 354, 367-369, 377, 382, 385, 389, 395, 397, 400, 408, 411, 413, 414, 416, 417, 419-423, 426, 427, 429, 431, 435-438, 441, 443-446, 450, 453, 454, The sequences set forth in 463-468, 474, 479, 484, 486, and 489;
(b) SEQ ID NOs: 2, 4-15, 18-33, 35-47, 49-56, 58, 59, 63-73, 88-116, 141-159, 175, 178, under appropriate stringent conditions 180, 185, 186, 194, 199, 205, 208, 211, 214-216, 219, 222, 226, 232, 236, 240, 241, 245, 246, 252-268, 321-325, 343, 354, 367-369, 377, 382, 385, 389, 395, 397, 400, 408, 411, 413, 414, 416, 417, 419-423, 426, 427, 429, 431, 435-438, 441, 443- Sequences that hybridize with the sequences set forth in any one of 446, 450, 453, 454, 463-468, 474, 479, 484, 486, and 489; and
(c) An isolated polypeptide comprising at least an immunogenic portion of a breast tumor protein comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of the complementary sequences of (a) or (b).
[2" claim-type="Currently amended] The compound of claim 1, SEQ ID NOs: 2, 4-15, 18-33, 35-47, 49-56, 58, 59, 63-73, 88-116, 141-159, 175, 178, 180, 185, 186 , 194, 199, 205, 208, 211, 214-216, 219, 222, 226, 232, 236, 240, 241, 245, 246, 252-268, 321-325, 343, 354, 367-369, 377 , 382, 385, 389, 395, 397, 400, 408, 411, 413, 414, 416, 417, 419-423, 426, 427, 429, 431, 435-438, 441, 443-446, 450, 453 An isolated polypeptide comprising the polynucleotide sequence set forth in any one of 454, 463-468, 474, 479, 484, 486 and 489, or an amino acid sequence encoded by the complementarity sequence of any one of these polynucleotide sequences.
[3" claim-type="Currently amended] An isolated polypeptide comprising the sequence set forth in any one of SEQ ID NOs: 176, 179, 181, 469-473, 475, 485, 487 and 488.
[4" claim-type="Currently amended] SEQ ID NOs: 2, 4-15, 18-33, 35-47, 49-56, 58, 59, 63-73, 88-116, 141-159, 175, 178, 180, 185, 186, 194, 199, 205 , 208, 211, 214-216, 219, 222, 226, 232, 236, 240, 241, 245, 246, 252-268, 321-325, 343, 354, 367-369, 377, 382, 385, 389 , 395, 397, 400, 408, 411, 413, 414, 416, 417, 419-423, 426, 427, 429, 431, 435-438, 441, 443-446, 450, 453, 454, 463-468 An isolated polynucleotide encoding at least 15 amino acid residues of a breast tumor protein comprising a polynucleotide comprising a sequence set forth in any one of 474, 479, 484 and 486 or an amino acid sequence encoded by a complementary sequence of such a sequence ; Or a variant thereof that differs in one or more substitutions, deletions, additions and / or insertions such that the ability of the variant to react with antigen specific antiserum is not substantially reduced.
[5" claim-type="Currently amended] SEQ ID NOs: 2, 4-15, 18-33, 35-47, 49-56, 58, 59, 63-73, 88-116, 141-159, 175, 178, 180, 185, 186, 194, 199, 205 , 208, 211, 214-216, 219, 222, 226, 232, 236, 240, 241, 245, 246, 252-268, 321-325, 343, 354, 367-369, 377, 382, 385, 389 , 395, 397, 400, 408, 411, 413, 414, 416, 417, 419-423, 426, 427, 429, 431, 435-438, 441, 443-446, 450, 453, 454, 463-468 , Polynucleotides comprising a sequence set forth in any one of 474, 479, 484, 486 and 489 or an isolated polynucleotide encoding a breast tumor protein comprising an amino acid sequence encoded by a complementary sequence of such sequences, or variants thereof .
[6" claim-type="Currently amended] SEQ ID NOs: 2, 4-15, 18-33, 35-47, 49-56, 58, 59, 63-73, 88-116, 141-159, 175, 178, 180, 185, 186, 194, 199, 205 , 208, 211, 214-216, 219, 222, 226, 232, 236, 240, 241, 245, 246, 252-268, 321-325, 343, 354, 367-369, 377, 382, 385, 389 , 395, 397, 400, 408, 411, 413, 414, 416, 417, 419-423, 426, 427, 429, 431, 435-438, 441, 443-446, 450, 453, 454, 463-468 An isolated polynucleotide comprising the sequence set forth in any one of 474, 479, 484, 486 and 489.
[7" claim-type="Currently amended] SEQ ID NO: 2, 4-15, 18-33, 35-47, 49-56, 58, 59, 63-73, 88-116, 141-159, 175, 178, 180, 185, 186, 194 under moderate stringent conditions , 199, 205, 208, 211, 214-216, 219, 222, 226, 232, 236, 240, 241, 245, 246, 252-268, 321-325, 343, 354, 367-369, 377, 382 , 385, 389, 395, 397, 400, 408, 411, 413, 414, 416, 417, 419-423, 426, 427, 429, 431, 435-438, 441, 443-446, 450, 453, 454 An isolated polynucleotide comprising a sequence that hybridizes with the sequence set forth in any one of 463-468, 474, 479, 484, 486 and 489.
[8" claim-type="Currently amended] An isolated polynucleotide complementary to the polynucleotide according to any one of claims 4 to 7.
[9" claim-type="Currently amended] An expression vector comprising a polynucleotide according to any one of claims 4 to 8.
[10" claim-type="Currently amended] A host cell transformed or transfected with an expression vector according to claim 9.
[11" claim-type="Currently amended] SEQ ID NOs: 2, 4-15, 18-33, 35-47, 49-56, 58, 59, 63-73, 88-116, 141-159, 175, 178, 180, 185, 186, 194, 199, 205 , 208, 211, 214-216, 219, 222, 226, 232, 236, 240, 241, 245, 246, 252-268, 321-325, 343, 354, 367-369, 377, 382, 385, 389 , 395, 397, 400, 408, 411, 413, 414, 416, 417, 419-423, 426, 427, 429, 431, 435-438, 441, 443-446, 450, 453, 454, 463-468 , An isolated antibody that specifically binds to a breast tumor protein comprising a polynucleotide sequence set forth in any one of 474, 479, 484, 486, and 489, or an amino acid sequence encoded by the complementary sequence of such a polynucleotide sequence, or Antigen binding fragments thereof.
[12" claim-type="Currently amended] A fusion protein comprising at least one polypeptide according to claim 1.
[13" claim-type="Currently amended] The fusion protein of claim 12, comprising an expression enhancer that increases expression of such fusion protein in a host cell transfected with a polynucleotide encoding the fusion protein.
[14" claim-type="Currently amended] The fusion protein of claim 12, comprising a T helper epitope that is not present in the polypeptide of claim 1.
[15" claim-type="Currently amended] The fusion protein of claim 12, comprising an affinity tag.
[16" claim-type="Currently amended] The first amino acid portion comprises at least 9 contiguous amino acids from mammaglobin as set forth as amino acids 1-93 of SEQ ID NO: 493, including a first amino acid portion and a second amino acid portion; The second amino acid portion comprises at least 9 contiguous amino acids from B726P as set forth as SEQ ID NO: 475, 469 or 176; A fusion protein, wherein said first amino acid moiety is linked to an amino or carboxy end of a second amino acid moiety.
[17" claim-type="Currently amended] The method of claim 16, wherein the first amino acid portion is an IDELKECFLNQTDETLSNVE (amino acids 59-78 of SEQ ID NO: 493); TTNAIDELKECFLNQ (amino acids 55-69 of SEQ ID NO: 493); SQHCYAGSGCPLLENVISKTI (amino acids 13-33 of SEQ ID NO: 493); EYKELLQEFIDDNATTNAID (amino acids 41-60 of SEQ ID NO: 493); KLLMVLMLA (amino acids 2-10 of SEQ ID NO: 493); Fusion protein is selected from the group consisting of N QEFIDD ATTNAI (amino acids 47-59 of SEQ ID NO: 493) and N LKECFL (amino acids 62-74 of SEQ ID NO: 493) QTDETL.
[18" claim-type="Currently amended] The fusion protein of claim 16, wherein the second amino acid portion comprises at least 9 contiguous amino acids encoded by the combined upstream and downstream open reading frame (ORF) of B726P as set forth in SEQ ID NO: 475.
[19" claim-type="Currently amended] The fusion protein of claim 16, wherein the second amino acid portion comprises nine or more contiguous amino acids encoded by an upstream ORF of B726P as set forth in SEQ ID NO: 469.
[20" claim-type="Currently amended] The fusion protein of claim 16, wherein the second amino acid portion comprises at least 9 contiguous amino acids encoded by a downstream ORF of B726P as set forth in SEQ ID NO: 176.
[21" claim-type="Currently amended] The fusion protein of claim 16, wherein the second amino acid portion comprises at least 9 contiguous amino acids from the amino acid sequence set forth as amino acids 1-129 of SEQ ID NO: 475.
[22" claim-type="Currently amended] The fusion protein of claim 16, as set forth in SEQ ID NO: 493.
[23" claim-type="Currently amended] The fusion protein of claim 16, as set forth in SEQ ID NO: 494.
[24" claim-type="Currently amended] The fusion protein of claim 16, as set forth in SEQ ID NO: 495.
[25" claim-type="Currently amended] An isolated polynucleotide encoding a fusion protein according to claim 12.
[26" claim-type="Currently amended] The fusion protein of claim 16, wherein the first amino acid portion is linked to the N-terminus of the second amino acid portion.
[27" claim-type="Currently amended] The fusion protein of claim 16, wherein the first amino acid portion is linked to the C-terminus of the second amino acid portion.
[28" claim-type="Currently amended] An isolated polynucleotide encoding a fusion protein according to claim 12.
[29" claim-type="Currently amended] (a) a polypeptide according to claim 1;
(b) a polynucleotide according to claim 4;
(c) the antibody according to claim 11;
(d) a fusion protein according to claim 12 or 16 and
(e) A pharmaceutical composition comprising at least one component selected from the group consisting of polynucleotides according to claim 28 and a physiologically acceptable carrier.
[30" claim-type="Currently amended] (a) a polypeptide according to claim 1;
(b) a polynucleotide according to claim 4;
(c) the antibody according to claim 11;
(d) a fusion protein according to claim 12 or 16 and
(e) An immunogenic composition comprising at least one component selected from the group consisting of polynucleotides according to claim 28 and an immunostimulant.
[31" claim-type="Currently amended] The immunogenic composition of claim 30 wherein the immunostimulatory agent is an adjuvant.
[32" claim-type="Currently amended] 31. The immunogenic composition of claim 30, wherein the immunostimulatory agent primarily induces a type I response.
[33" claim-type="Currently amended] A method of inhibiting cancer development in a patient, comprising administering to the patient an effective amount of the pharmaceutical composition according to claim 29.
[34" claim-type="Currently amended] A method of inhibiting cancer development in a subject, comprising administering to the patient an effective amount of an immunogenic composition according to claim 30.
[35" claim-type="Currently amended] A pharmaceutical composition comprising an antigen presenting cell expressing a polypeptide according to claim 1 together with a pharmaceutically acceptable carrier or excipient.
[36" claim-type="Currently amended] The pharmaceutical composition of claim 29, wherein the antigen presenting cells are dendritic cells or macrophages.
[37" claim-type="Currently amended] (a) the sequences set forth in SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489-492;
(b) sequences that hybridize with the sequence set forth in any one of SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489-492 under appropriate stringent conditions, and
(c) expressing a polypeptide comprising at least an immunogenic portion of a breast tumor protein comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of the complementary sequences of (a) or (b), or a variant thereof An immunogenic composition comprising an antigen-providing cell, together with an immunostimulant.
[38" claim-type="Currently amended] 38. The immunogenic composition of claim 37 wherein the immunostimulant is an adjuvant.
[39" claim-type="Currently amended] 38. The immunogenic composition of claim 37 wherein the immunostimulatory agent mainly induces a type I response.
[40" claim-type="Currently amended] The immunogenic composition of claim 37 wherein the antigen presenting cells are dendritic cells.
[41" claim-type="Currently amended] (a) the sequences set forth in SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489-492;
(b) sequences that hybridize with the sequence set forth in any one of SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489-492 under appropriate stringent conditions, and
(c) the sequence (a) or (b) encoded by the polynucleotide set forth in any one of SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486 and 489-492 By administering to a patient an effective amount of an antigen-providing cell expressing a polypeptide comprising at least an immunogenic portion of a breast tumor protein comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of the complementary sequences of A method of inhibiting the development of cancer in a subject, including inhibiting the development of cancer in the subject.
[42" claim-type="Currently amended] The method of claim 41, wherein the antigen presenting cells are dendritic cells.
[43" claim-type="Currently amended] 43. The method of any one of claims 33, 34 and 41, wherein the cancer is breast cancer.
[44" claim-type="Currently amended] (i) the polynucleotide set forth in any one of SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486 and 489-492 and
(ii) a T-cell that specifically reacts with a breast tumor protein comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of complementary polynucleotides of the polynucleotide, and a cell expressing the antigen. Contacting the biological sample under a time and condition sufficient to remove it from the tumor sample.
[45" claim-type="Currently amended] 45. The method of claim 44, wherein the biological sample is blood or fractions thereof.
[46" claim-type="Currently amended] 45. A method of inhibiting the occurrence of cancer in a patient, comprising administering to the patient a biological sample treated according to the method of claim 44.
[47" claim-type="Currently amended] (a) (i) the sequences set forth in SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486 and 489-492;
(ii) a sequence which hybridizes with the sequence set forth in any one of SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486 and 489-492 under appropriate stringent conditions and
(iii) a polypeptide comprising at least an immunogenic portion of a breast tumor protein comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of (i) or (ii) a complementary sequence of a sequence, or a variant thereof;
(b) a polynucleotide encoding the polypeptide of (a) and
(c) contacting the at least one component selected from the group consisting of antigen-presenting cells expressing the polypeptide of (a) with the T cells under a time and condition sufficient to stimulate and / or expand the T cells, A method of stimulating and / or expanding T cells specific for said breast tumor protein.
[48" claim-type="Currently amended] An isolated T cell population comprising T cells prepared according to the method of claim 47.
[49" claim-type="Currently amended] 50. A method of inhibiting the occurrence of cancer in a patient, comprising administering to the patient an effective amount of the T cell population according to claim 49.
[50" claim-type="Currently amended] (a) (i) (1) the sequences set forth in SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489-492;
(2) a sequence that hybridizes with the sequence set forth in any one of SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489-492 under appropriate stringent conditions, and
(3) a polypeptide comprising at least an immunogenic portion of a breast tumor protein comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of the complementary sequences of (1) or (2);
(ii) a polynucleotide encoding the polypeptide of (i) and
(iii) incubating at least one component selected from the group consisting of antigen-presenting cells expressing the polypeptide of (i) with CD4 ⁺ and / or CD8 ⁺ T cells isolated from the patient to propagate T cells; and
(b) inhibiting the development of cancer in the patient by administering to the patient an effective amount of such proliferated T cells.
[51" claim-type="Currently amended] (a) (i) (1) the sequences set forth in SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489-492;
(2) a sequence that hybridizes with the sequence set forth in any one of SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489-492 under appropriate stringent conditions, and
(3) a polypeptide comprising at least an immunogenic portion of a breast tumor protein comprising an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of the complementary sequences of (1) or (2);
(ii) a polynucleotide encoding the polypeptide of (i) and
(iii) incubating at least one component selected from the group consisting of antigen-presenting cells expressing the polypeptide of (i) with CD4 ⁺ and / or CD8 ⁺ T cells isolated from the patient to propagate the T cells;
(b) cloning one or more cells so proliferated to provide cloned T cells; and
(c) inhibiting the development of cancer in the patient by administering to the patient an effective amount of such cloned T cells.
[52" claim-type="Currently amended] (a) encoded by the polynucleotide sequence set forth in any one of SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489-492 or the complementary sequence of such a polynucleotide sequence Contacting a binding agent that binds to a breast tumor protein comprising an amino acid sequence, wherein the binding agent is in contact with a biological sample obtained from a particular patient;
(b) detecting the amount of polypeptide binding to the binding agent in the sample, and
(c) comparing the amount of such polypeptide to a cut-off predetermined value and determining therefrom cancer from the patient, thereby determining whether cancer develops in the patient. Way.
[53" claim-type="Currently amended] The method of claim 52, wherein the binding agent is an antibody.
[54" claim-type="Currently amended] The method of claim 53, wherein the antibody is a monoclonal antibody.
[55" claim-type="Currently amended] The method of claim 52, wherein the cancer is breast cancer.
[56" claim-type="Currently amended] (a) encoded by the polynucleotide sequence set forth in any one of SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489-492 or the complementary sequence of such a polynucleotide sequence Contacting a binding agent that binds to a breast tumor protein comprising the amino acid sequence thus prepared, at a first time point with a biological sample obtained from a particular patient;
(b) detecting the amount of polypeptide binding to the binding agent in the sample;
(c) repeating steps (a) and (b) using biological samples obtained from the patient at subsequent time points and
(d) comparing the amount of polypeptide detected in step (c) with the amount detected in step (b) and therefrom monitoring the progress of cancer in the patient. How to monitor.
[57" claim-type="Currently amended] The method of claim 56, wherein the binding agent is an antibody.
[58" claim-type="Currently amended] The method of claim 57, wherein the antibody is a monoclonal antibody.
[59" claim-type="Currently amended] The method of claim 56, wherein the cancer is breast cancer.
[60" claim-type="Currently amended] (a) encoded by the polynucleotide sequence set forth in any one of SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489-492 or the complementary sequence of such a polynucleotide sequence Contacting an oligonucleotide that hybridizes with a polynucleotide encoding a breast tumor protein comprising an amino acid sequence, to a biological sample obtained from a particular patient;
(b) detecting the amount of polynucleotide hybridizing with the oligonucleotide in the sample, and
(c) comparing the amount of polynucleotide hybridizing with such oligonucleotides to a cut-off estimated value and determining therefrom cancer from the patient, thereby determining whether cancer has occurred in the patient How to.
[61" claim-type="Currently amended] 61. The method of claim 60, wherein the amount of polynucleotide hybridizing with the oligonucleotide is determined using polymerase chain reaction.
[62" claim-type="Currently amended] 61. The method of claim 60, wherein the amount of polynucleotide hybridizing with the oligonucleotide is determined using a hybridization assay.
[63" claim-type="Currently amended] (a) encoded by the polynucleotide sequence set forth in any one of SEQ ID NOs: 1-175, 178, 180, 182-468, 474, 476, 477, 479, 484, 486, and 489-492 or the complementary sequence of such a polynucleotide sequence Contacting an oligonucleotide that hybridizes with a polynucleotide encoding a breast tumor protein comprising the amino acid sequence, to a biological sample obtained from a particular patient;
(b) detecting the amount of polynucleotide in the sample that hybridizes with the oligonucleotide;
(c) repeating steps (a) and (b) using biological samples obtained from the patient at subsequent time points and
(d) comparing the amount of polynucleotides detected in step (c) with the amount detected in step (b) and therefrom monitoring the progress of cancer in the patient. How to monitor.
[64" claim-type="Currently amended] 64. The method of claim 63, wherein the amount of polynucleotide hybridizing with the oligonucleotide is determined using polymerase chain reaction.
[65" claim-type="Currently amended] The method of claim 63, wherein the amount of polynucleotide hybridizing with the oligonucleotide is determined using a hybridization assay.
[66" claim-type="Currently amended] (a) at least one antibody according to claim 11 and
(b) A diagnostic kit comprising a reagent for detection comprising a reporter group.
[67" claim-type="Currently amended] The kit of claim 66 wherein the antibody is immobilized on a solid support.
[68" claim-type="Currently amended] 67. The kit of claim 66, wherein the reagent for detection comprises anti-immunoglobulin, protein G, protein A or lectin.
[69" claim-type="Currently amended] 67. The kit of claim 66, wherein the reporter group is selected from the group consisting of radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and dye particles.
[70" claim-type="Currently amended] Under appropriate stringent conditions, SEQ ID NOs: 2, 4-15, 18-33, 35-47, 49-56, 58, 59, 63-73, 88-116, 141-159, 175, 178, 180, 185, 186, 194, 199, 205, 208, 211, 214-216, 219, 222, 226, 232, 236, 240, 241, 245, 246, 252-268, 321-325, 343, 354, 367-369, 377, 382, 385, 389, 395, 397, 400, 408, 411, 413, 414, 416, 417, 419-423, 426, 427, 429, 431, 435-438, 441, 443-446, 450, 453, Polynucleotides encoding a breast tumor protein comprising a polynucleotide sequence set forth in any one of 454, 463-468, 474, 479, 484, 486, and 489-492 or an amino acid sequence encoded by the complementary sequence of such polynucleotide sequence Oligonucleotides comprising 10 to 40 consecutive nucleotides that hybridize with.
[71" claim-type="Currently amended] The method of claim 70, SEQ ID NO: 2, 4-15, 18-33, 35-47, 49-56, 58, 59, 63-73, 88-116, 141-159, 175, 178, 180, 185, 186 , 194, 199, 205, 208, 211, 214-216, 219, 222, 226, 232, 236, 240, 241, 245, 246, 252-268, 321-325, 343, 354, 367-369, 377 , 382, 385, 389, 395, 397, 400, 408, 411, 413, 414, 416, 417, 419-423, 426, 427, 429, 431, 435-438, 441, 443-446, 450, 453 Oligonucleotides comprising 10 to 40 consecutive nucleotides as set forth in any one of 454, 463-468, 474, 479, 484, 486 and 489-492.
[72" claim-type="Currently amended] (a) an oligonucleotide according to claim 71 and
(b) A diagnostic kit comprising a diagnostic reagent for use in a polymerase chain reaction or hybridization assay.

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同族专利:

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公开号 | 公开日

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-04-17|Priority to US09/551,621

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2000-04-17|Priority to US09/551,621

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2000-06-08|Priority to US09/590,751

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2000-06-08|Priority to US09/590,751

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2000-06-22|Priority to US09/604,287

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2000-06-22|Priority to US09/604,287

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2000-07-20|Priority to US09/620,405

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2000-07-20|Priority to US09/620,405

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2001-04-12|Application filed by 코릭사 코포레이션

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2003-01-25|Publication of KR20030008369A

优先权:

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申请号 | 申请日 | 专利标题

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US09/551,621|2000-04-17|

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US09/551,621|US6844325B2|1998-12-28|2000-04-17|Compositions for the treatment and diagnosis of breast cancer and methods for their use|

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US09/590,751|US6756477B1|1998-12-28|2000-06-08|Compositions and methods for the therapy and diagnosis of breast cancer|

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US09/590,751|2000-06-08|

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US09/604,287|2000-06-22|

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US09/604,287|US6586572B2|1998-12-28|2000-06-22|Compositions and methods for the therapy and diagnosis of breast cancer|

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US09/620,405|2000-07-20|

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US09/620,405|US6528054B1|1998-12-28|2000-07-20|Compositions and methods for the therapy and diagnosis of breast cancer|