RU2311920C2 - Compositions and methods for treating and predicting pulmonary cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: illustrative compositions include one or several polypeptides of pulmonary tumor, their immunogenic parts, polynucleotides which code such polypeptides, an antigen-presenting cell that expresses such polypeptides, and T-cells being specific to cells that express such polypeptides. The compositions described could be applied, for example, during diagnostics, prophylaxis and/or treating diseases, especially pulmonary cancer.

EFFECT: increased specificity of diagnostics.

11 cl, 10 ex, 5 tbl

Description

Technical Field of the Invention

The present invention mainly relates to the treatment and diagnosis of a malignant tumor, such as lung cancer. The invention more specifically relates to polypeptides comprising at least a portion of a lung tumor protein, and to polynucleotides encoding such polypeptides. Such polypeptides and polynucleotides are used in pharmaceutical compositions, for example, in vaccines, and in other compositions for the diagnosis and treatment of lung cancer.

BACKGROUND OF THE INVENTION

Lung cancer is the leading cause of death from malignant tumors in the United States among both men and women, with an estimated 172,000 new cases reported in 1994. The ratio of survivors over five years is only 13% among all patients with lung cancer, regardless of the stage of the disease in the diagnosis. This contrasts with the ratio of survivors for five years, equal to 46%, among cases identified at the time when the disease was still localized. However, before the spread of the disease, only 16% of lung cancers are detected.

Early detection is difficult, because often the clinical symptoms are not noticeable until the disease reaches its expanded stage. Currently, chest x-rays, analysis of the type of cells contained in sputum, and the study of bronchial pathways using fiber optics are used to help diagnose. Treatment regimens are determined by the type and stage of the disease, and include surgery, radiation therapy and / or chemotherapy. Despite significant studies aimed at treating the disease, lung cancer remains intractable.

Accordingly, there remains a need in the art for improved vaccines, methods of treatment, and methods for diagnosing lung cancer.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to polynucleotide compositions selected from the group including:

(a) the sequences represented by SEQ ID NO: 217-390, 392, 394, 396, 398-420 422-424, 428-433 and 440-583;

(b) sequences complementary to the sequences of SEQ ID NO: 217-390, 392, 394, 396, 398-420, 422-424, 428-433 and 440-583;

(c) sequences consisting of at least 20 consecutive residues of the sequence represented by SEQ ID NO: 217-390, 392, 394, 396, 398-420 422-424, 428-433 and 440-583;

(d) sequences that hybridize to the sequence represented by SEQ ID NO: 217-390, 392, 394, 396, 398-420, 422-424, 428-433 and 440-583,

(e) sequences having at least 75% identity with the sequence of SEQ ID NO: 217-390, 392, 394, 396, 398-420, 422-424, 428-433 and 440-583;

(f) sequences having at least 90% identity with the sequence of SEQ ID NO: 217-390, 392, 394, 396, 398-420, 422-424, 428-433 and 440-583; and

(g) degenerate variants of the sequence represented by SEQ ID NO: 217-390, 392, 394, 396, 398-420, 422-424, 428-433 and 440-583.

In one preferred embodiment, the polynucleotide compositions of the invention are expressed in at least 20%, more preferably at least 30%, and most preferably in at least 50% of the tested lung tumor samples, at a concentration of at least about 2 times, more preferably at least about 5 times, and most preferably at least about 10 times higher than in normal tissues.

The present invention in another aspect relates to polypeptide compositions comprising an amino acid sequence that is encoded by the polynucleotide sequence described above.

In specific embodiments, the present invention relates to polypeptide compositions comprising an amino acid sequence selected from the group consisting of the sequences shown in SEQ ID NO: 391, 393, 395, 397, 421, 425-427, 434-439 and 584-587.

In certain preferred embodiments, the polypeptides and / or polynucleotides of the present invention are immunogenic, that is, they are capable of eliciting an immune response, in particular a humoral and / or cellular immune response, as described hereinafter.

The present invention also relates to fragments, variants and / or derivatives of the described polypeptide and / or polynucleotide sequences, where the fragments, variants and / or derivatives are preferably characterized by a level of biological activity of at least 50%, more preferably at least 70 %, and most preferably, at least 90% of the level of immunogenic activity of the polypeptide sequence set forth in SEQ ID NO: 391, 393, 395, 397, 421, 425-427, 434-439 and 584-587, or polynucleotide sequence STI, as set out in SEQ ID NO: 217-390, 392, 394, 396, 398-420 422-424, 428-433 and 440-583.

The present invention further relates to polynucleotides that encode the above polypeptide, to expression vectors encoding such half nucleotides, and to host cells transformed or transfected with such expression vectors.

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

In a related aspect of the present invention, pharmaceutical compositions, for example, vaccine compositions, are provided for prophylactic or therapeutic methods of use. Such compositions mainly include the immunogenic polypeptide or polynucleotide of the invention, and an immunostimulant such as an adjuvant.

The present invention further relates to pharmaceutical compositions which comprise (a) an antibody or antigen binding fragment thereof that specifically binds to a polypeptide of the present invention or a fragment thereof; and (b) a physiologically acceptable carrier.

In a further aspect, the present invention relates to pharmaceutical compositions comprising (a) antigen-presenting cells that express the above polypeptide and (b) a pharmaceutically acceptable carrier or excipient. Illustrative antigen-presenting cells include dendritic cells, macrophages, monocytes, fibroblasts, and B cells.

In related aspects, pharmaceutical compositions are provided that include: (a) an antigen presenting cell expressing the above polypeptide; and (b) an immunostimulant.

The present invention also, in other aspects, relates to fusion proteins that include at least one of the polypeptide described above, as well as polynucleotides encoding such fusion proteins, usually in the form of pharmaceutical compositions, for example, vaccine compositions comprising a physiologically acceptable carrier and / or immunostimulant. Fusion proteins may include multiple immunogenic polypeptides or parts or variants thereof, as described herein, or, in addition, may include one or more polypeptide segments that facilitate expression, purification and / or immunogenicity of the polypeptide (s).

In further aspects, the present invention relates to methods for stimulating an immune response in a patient, preferably a human T-cell response, comprising administering the pharmaceutical composition described herein. The patient may suffer from lung cancer, in which case the methods relate to treating the disease, or patients who are considered to be at risk of developing such a disease may receive prophylactic treatment.

In further aspects, the present invention relates to methods for inhibiting a patient from developing a malignant tumor, comprising administering to the patient the above pharmaceutical composition. The patient may suffer from lung cancer, in which case the methods relate to treating the disease, or patients who are considered to be at risk of developing such a disease may receive prophylactic treatment.

The present invention further relates, in other aspects, to methods for removing tumor cells from a biological sample, comprising contacting the biological sample with T cells that specifically react with a polypeptide of the present invention, wherein the step of the reaction is carried out under sufficient conditions and for sufficient time for the ability to remove from the sample cells expressing this protein.

In related aspects, methods are provided for inhibiting a patient from developing a malignant tumor, comprising administering to the patient a biological sample processed as described above.

In addition, in other aspects, methods are provided for stimulating and growing T cells specific for a polypeptide of the present invention, comprising contacting T cells with one or more of: (i) the polypeptides described above; (ii) polynucleotides encoding such a polypeptide; and / or (iii) antigen-presenting cells that express such a polypeptide; under sufficient conditions and for sufficient time to allow stimulation and growth of T cells. Isolated T-cell populations are also provided, including T cells obtained as described above.

In further aspects, the present invention relates to methods for inhibiting a patient from developing a malignant tumor, comprising administering to the patient an effective amount of a T cell population as described above.

The present invention further relates to methods for inhibiting a patient from developing a malignant tumor, comprising the steps of: (a) incubating CD4 ⁺ and / or CD8 ⁺ T cells isolated from a patient with one or more: (i) polypeptides, comprising at least an immunogenic portion of the polypeptide described herein; (ii) polynucleotides encoding such a polypeptide; and (iii) antigen-presenting cells that express such a polypeptide; and (b) administering to the patient an effective amount of proliferating T cells, and thereby inhibiting the development of a cancer in the patient. Proliferated cells may be cloned before administration to the patient, but this is not necessary.

In further aspects, the present invention relates to methods for determining a patient's presence or absence of a malignant tumor, preferably lung cancer, comprising: (a) reacting a biological sample obtained from the patient with a binding agent that binds the above polypeptide; (b) detecting in the sample an amount of a polypeptide that binds to a binding agent; and (c) linking the amount of the polypeptide to a predetermined cut-off level, and hence determining whether a patient has a malignant tumor. In preferred embodiments, the binding agent is an antibody, more preferably a monoclonal antibody.

The present invention also relates, in other aspects, to methods for monitoring cancer progression in a patient. Such methods include the steps of: (a) reacting a biological sample obtained from a patient at a first moment in time with a binding agent that binds the above polypeptide; (b) detecting in the sample an amount of a polypeptide that binds to a binding agent; (c) repeating steps (a) and (b) using a biological sample taken from a patient at a subsequent point in time; and (d) comparing the amount of the polypeptide detected in step (c) with the amount detected in step (b) and hence monitoring the progression of the cancer in the patient.

The present invention further relates, in other aspects, to methods for determining the presence or absence of a malignant tumor in a patient, comprising the steps of: (a) reacting a biological sample from a patient with an oligonucleotide that hybridizes with a polynucleotide that encodes a polypeptide of the present invention; (b) determining in the sample the level of a polynucleotide, preferably an mRNA that hybridizes with an oligonucleotide; and (c) comparing the level of the polynucleotide that hybridizes with the oligonucleotide with a predetermined cut-off level, and hence determining whether a patient has a malignant tumor. In specific embodiments, the amount of mRNA is detected by polymerase chain reaction, using, for example, at least one oligonucleotide primer that hybridizes to a polynucleotide encoding the above polypeptide, or to a sequence complementary to such a polynucleotide. In other implementations, the amount of mRNA is determined using a hybridization method using an oligonucleotide probe that hybridizes to a polynucleotide encoding the above polypeptide, or to a sequence complementary to such a polynucleotide.

In related aspects, methods are provided for monitoring the progression of a cancer in a patient, comprising the steps of: (a) reacting a biological sample from a patient with an oligonucleotide that hybridizes with a polynucleotide that encodes a polypeptide of the present invention; (b) determining in the sample the amount of polynucleotide that hybridizes with the oligonucleotide; (c) repeating steps (a) and (b) using a biological sample taken from a patient at a subsequent point in time; and (d) comparing the amount of polynucleotide detected in step (c) with the amount detected in step (b) and hence monitoring the progression of the cancer in the patient.

In further aspects, the present invention relates to antibodies, such as monoclonal antibodies that bind the polypeptide described above, as well as to diagnostic kits comprising such antibodies. Diagnostic kits are also provided, including one or more of the oligonucleotide probes or primers described above.

These and other aspects of the present invention will become apparent upon reference to the following detailed description. All references described herein are incorporated herein by reference in their entirety, as if each would be individually incorporated.

Sequence Identifiers

SEQ ID NO: 1 is the determined cDNA sequence for L363C1.cons.

SEQ ID NO: 2 is the determined cDNA sequence for L263C2.cons.

SEQ ID NO: 3 is the determined cDNA sequence for L263C2c.

SEQ ID NO: 4 is the determined cDNA sequence for L263C1.cons.

SEQ ID NO: 5 is the determined cDNA sequence for L263C1b.

SEQ ID NO: 6 is the determined cDNA sequence for L164C2.cons.

SEQ ID NO: 7 is the determined cDNA sequence for L164C1.cons.

SEQ ID NO: 8 is the determined cDNA sequence for L366C1a.

SEQ ID NO: 9 is the determined cDNA sequence for L260C1.cons.

SEQ ID NO: 10 is the determined cDNA sequence for L163C1c.

SEQ ID NO: 11 is the determined cDNA sequence for L163C1b.

SEQ ID NO: 12 is the determined cDNA sequence for L255C1.cons.

SEQ ID NO: 13 is the determined cDNA sequence for L255C1b.

SEQ ID NO: 14 is the determined cDNA sequence for L355C1.cons.

SEQ ID NO: 15 is the determined cDNA sequence for L366C1.cons.

SEQ ID NO: 16 is the determined cDNA sequence for L163C1a.

SEQ ID NO: 17 is the determined cDNA sequence for LT86-1.

SEQ ID NO: 18 is the determined cDNA sequence for LT86-2.

SEQ ID NO: 19 is the determined cDNA sequence for LT86-3.

SEQ ID NO: 20 is the determined cDNA sequence for LT86-4.

SEQ ID NO: 21 is the determined cDNA sequence for LT86-5.

SEQ ID NO: 22 is the determined cDNA sequence for LT86-6.

SEQ ID NO: 23 is the determined cDNA sequence for LT86-7.

SEQ ID NO: 24 is the determined cDNA sequence for LT86-8.

SEQ ID NO: 25 is the determined cDNA sequence for LT86-9.

SEQ ID NO: 26 is the determined cDNA sequence for LT86-10.

SEQ ID NO: 27 is the determined cDNA sequence for LT86-11.

SEQ ID NO: 28 is the determined cDNA sequence for LT86-12.

SEQ ID NO: 29 is the determined cDNA sequence for LT86-13.

SEQ ID NO: 30 is the determined cDNA sequence for LT86-14.

SEQ ID NO: 31 is the determined cDNA sequence for LT86-15.

SEQ ID NO: 32 is the predicted amino acid sequence for LT86-1.

SEQ ID NO: 33 is the predicted amino acid sequence for LT86-2.

SEQ ID NO: 34 is the predicted amino acid sequence for LT86-3.

SEQ ID NO: 35 is the predicted amino acid sequence for LT86-4.

SEQ ID NO: 36 is the predicted amino acid sequence for LT86-5.

SEQ ID NO: 37 is the predicted amino acid sequence for LT86-6.

SEQ ID NO: 38 is the predicted amino acid sequence for LT86-7.

SEQ ID NO: 39 is the predicted amino acid sequence for LT86-8.

SEQ ID NO: 40 is the predicted amino acid sequence for LT86-9.

SEQ ID NO: 41 is the predicted amino acid sequence for LT86-10.

SEQ ID NO: 42 is the predicted amino acid sequence for LT86-11.

SEQ ID NO: 43 is the predicted amino acid sequence for LT86-12.

SEQ ID NO: 44 is the predicted amino acid sequence for LT86-13.

SEQ ID NO: 45 is the predicted amino acid sequence for LT86-14.

SEQ ID NO: 46 is the predicted amino acid sequence for LT86-15.

SEQ ID NO: 47 is a (dT) ₁₂ AG primer.

SEQ ID NO: 48 is a primer.

SEQ ID NO: 49 is the determined 5'-cDNA sequence for L86S-3.

SEQ ID NO: 50 is the determined 5'-cDNA sequence for L86S-12.

SEQ ID NO: 51 is the determined 5'-cDNA sequence for L86S-16.

SEQ ID NO: 52 is the determined 5'-cDNA sequence for L86S-25.

SEQ ID NO: 53 is the determined 5'-cDNA sequence for L86S-36.

SEQ ID NO: 54 is the determined 5'-cDNA sequence for L86S-40.

SEQ ID NO: 55 is the determined 5'-cDNA sequence for L86S-46.

SEQ ID NO: 56 represents the predicted amino acid sequence for L86S-3.

SEQ ID NO: 57 is the predicted amino acid sequence for L86S-12.

SEQ ID NO: 58 is the predicted amino acid sequence for L86S-16.

SEQ ID NO: 59 is the predicted amino acid sequence for L86S-25.

SEQ ID NO: 60 is the predicted amino acid sequence for L86S-36.

SEQ ID NO: 61 is the predicted amino acid sequence for L86S-40.

SEQ ID NO: 62 is the predicted amino acid sequence for L86S-46.

SEQ ID NO: 63 is the determined 5'-cDNA sequence for L86S-30.

SEQ ID NO: 64 is the determined 5'-cDNA sequence for L86S-41.

SEQ ID NO: 65 is the predicted amino acid sequence for the 5'-end of LT86-9.

SEQ ID NO: 66 is the determined extended cDNA sequence for LT86-4.

SEQ ID NO: 67 is the predicted extended amino acid sequence for LT86-4.

SEQ ID NO: 68 is the determined 5'-cDNA sequence for LT86-20.

SEQ ID NO: 69 is the determined 3'-cDNA sequence for LT86-21.

SEQ ID NO: 70 is the determined 5'-cDNA sequence for LT86-22.

SEQ ID NO: 71 is the determined 5'-cDNA sequence for LT86-26.

SEQ ID NO: 72 is the determined 5'-cDNA sequence for LT86-27.

SEQ ID NO: 73 is the predicted amino acid sequence for LT86-20.

SEQ ID NO: 74 is the predicted amino acid sequence for LT86-21.

SEQ ID NO: 75 is the predicted amino acid sequence for LT86-22.

SEQ ID NO: 76 is the predicted amino acid sequence for LT86-26.

SEQ ID NO: 77 is the predicted amino acid sequence for LT86-27.

SEQ ID NO: 78 is the determined extended cDNA sequence for L86S-12.

SEQ ID NO: 79 is the determined extended cDNA sequence for L86S-36.

SEQ ID NO: 80 is the determined extended cDNA sequence for L86S-46.

SEQ ID NO: 81 is the predicted extended amino acid sequence for L86S-12.

SEQ ID NO: 82 is the predicted extended amino acid sequence for L86S-36.

SEQ ID NO: 83 is the predicted extended amino acid sequence for L86S-46.

SEQ ID NO: 84 is the determined 5'-cDNA sequence for L86S-6.

SEQ ID NO: 85 is the determined 5'-cDNA sequence for L86S-11.

SEQ ID NO: 86 is the determined 5'-cDNA sequence for L86S-14.

SEQ ID NO: 87 is the determined 5'-cDNA sequence for L86S-29.

SEQ ID NO: 88 is the determined 5'-cDNA sequence for L86S-34.

SEQ ID NO: 89 is the determined 5'-cDNA sequence for L86S-39.

SEQ ID NO: 90 is the determined 5'-cDNA sequence for L86S-47.

SEQ ID NO: 91 is the determined 5'-cDNA sequence for L86S-49.

SEQ ID NO: 92 is the determined 5'-cDNA sequence for L86S-51.

SEQ ID NO: 93 represents the predicted amino acid sequence for L86S-6.

SEQ ID NO: 94 is the predicted amino acid sequence for L86S-11.

SEQ ID NO: 95 is the predicted amino acid sequence for L86S-14.

SEQ ID NO: 96 is the predicted amino acid sequence for L86S-29.

SEQ ID NO: 97 is the predicted amino acid sequence for L86S-34.

SEQ ID NO: 98 is the predicted amino acid sequence for L86S-39.

SEQ ID NO: 99 is the predicted amino acid sequence for L86S-47.

SEQ ID NO: 100 is the predicted amino acid sequence for L86S-49.

SEQ ID NO: 101 is the predicted amino acid sequence for L86S-51.

SEQ ID NO: 102 is the determined DNA sequence for SLT-T1.

SEQ ID NO: 103 is the determined 5'-cDNA sequence for SLT-T2.

SEQ ID NO: 104 is the determined 5'-cDNA sequence for SLT-T3.

SEQ ID NO: 105 is the determined 5'-cDNA sequence for SLT-T5.

SEQ ID NO: 106 is the determined 5'-cDNA sequence for SLT-T7.

SEQ ID NO: 107 is the determined 5'-cDNA sequence for SLT-T9.

SEQ ID NO: 108 is the determined 5'-cDNA sequence for SLT-T10.

SEQ ID NO: 109 is the determined 5'-cDNA sequence for SLT-T11.

SEQ ID NO: 110 is the determined 5'-cDNA sequence for SLT-T12.

SEQ ID NO: 111 is the predicted amino acid sequence for SLT-T1.

SEQ ID NO: 112 is the predicted amino acid sequence for SLT-T2.

SEQ ID NO: 113 is the predicted amino acid sequence for SLT-T3.

SEQ ID NO: 114 is the predicted amino acid sequence for SLT-T10.

SEQ ID NO: 115 is the predicted amino acid sequence for SLT-T12.

SEQ ID NO: 116 is the determined 5'-cDNA sequence for SALT-T3.

SEQ ID NO: 117 is the determined 5'-cDNA sequence for SALT-T4.

SEQ ID NO: 118 is the determined 5'-cDNA sequence for SALT-T7.

SEQ ID NO: 119 is the determined 5'-cDNA sequence for SALT-T8.

SEQ ID NO: 120 is the determined 5'-cDNA sequence for SALT-T9.

SEQ ID NO: 121 is the predicted amino acid sequence for SALT-T3.

SEQ ID NO: 122 is the predicted amino acid sequence for SALT-T4.

SEQ ID NO: 123 is the predicted amino acid sequence for SALT-T7.

SEQ ID NO: 124 is the predicted amino acid sequence for SALT-T8.

SEQ ID NO: 125 represents the predicted amino acid sequence for SALT-T9.

SEQ ID NO: 126 is the determined cDNA sequence for PSLT-1.

SEQ ID NO: 127 is the determined cDNA sequence for PSLT-2.

SEQ ID NO: 128 is the determined cDNA sequence for PSLT-7.

SEQ ID NO: 129 is the determined cDNA sequence for PSLT-13.

SEQ ID NO: 130 is the determined cDNA sequence for PSLT-27.

SEQ ID NO: 131 is the determined cDNA sequence for PSLT-28.

SEQ ID NO: 132 is the determined cDNA sequence for PSLT-30.

SEQ ID NO: 133 is the determined cDNA sequence for PSLT-40.

SEQ ID NO: 134 is the determined cDNA sequence for PSLT-69.

SEQ ID NO: 135 is the determined cDNA sequence for PSLT-71.

SEQ ID NO: 136 is the determined cDNA sequence for PSLT-73.

SEQ ID NO: 137 is the determined cDNA sequence for PSLT-79.

SEQ ID NO: 138 is an established

cDNA sequence for PSLT-03.

SEQ ID NO: 139 is the determined cDNA sequence for PSLT-09.

SEQ ID NO: 140 is the determined cDNA sequence for PSLT-011.

SEQ ID NO: 141 is the determined cDNA sequence for PSLT-041.

SEQ ID NO: 142 is the determined cDNA sequence for PSLT-62.

SEQ ID NO: 143 is the determined cDNA sequence for PSLT-6.

SEQ ID NO: 144 is the determined cDNA sequence for PSLT-37.

SEQ ID NO: 145 is the determined cDNA sequence for PSLT-74.

SEQ ID NO: 146 is the determined cDNA sequence for PSLT-010.

SEQ ID NO: 147 is the determined cDNA sequence for PSLT-012.

SEQ ID NO: 148 is the determined cDNA sequence for PSLT-037.

SEQ ID NO: 149 is the determined 5'-cDNA sequence for SAL-3.

SEQ ID NO: 150 is the determined 5'-cDNA sequence for SAL-24.

SEQ ID NO: 151 is the determined 5'-cDNA sequence for SAL-25.

SEQ ID NO: 152 is the determined 5'-cDNA sequence for SAL-33.

SEQ ID NO: 153 is the determined 5'-cDNA sequence for SAL-50.

SEQ ID NO: 154 is the determined 5'-cDNA sequence for SAL-57.

SEQ ID NO: 155 is the determined 5'-cDNA sequence for SAL-66.

SEQ ID NO: 156 is the determined 5'-cDNA sequence for SAL-82.

SEQ ID NO: 157 is the determined 5'-cDNA sequence for SAL-99.

SEQ ID NO: 158 is the determined 5'-cDNA sequence for SAL-104.

SEQ ID NO: 159 is the determined 5'-cDNA sequence for SAL-109.

SEQ ID NO: 160 is the determined 5'-cDNA sequence for SAL-5.

SEQ ID NO: 161 is the determined 5'-cDNA sequence for SAL-8.

SEQ ID NO: 162 is the determined 5'-cDNA sequence for SAL-12.

SEQ ID NO: 163 is the determined 5'-cDNA sequence for SAL-14.

SEQ ID NO: 164 is the determined 5'-cDNA sequence for SAL-16.

SEQ ID NO: 165 is the determined 5'-cDNA sequence for SAL-23.

SEQ ID NO: 166 is the determined 5'-cDNA sequence for SAL-26.

SEQ ID NO: 167 is the determined 5'-cDNA sequence for SAL-29.

SEQ ID NO: 168 is the determined 5'-cDNA sequence for SAL-32.

SEQ ID NO: 169 is the determined 5'-cDNA sequence for SAL-39.

SEQ ID NO: 170 is the determined 5'-cDNA sequence for SAL-42.

SEQ ID NO: 171 is the determined 5'-cDNA sequence for SAL-43.

SEQ ID NO: 172 is the determined 5'-cDNA sequence for SAL-44.

SEQ ID NO: 173 is the determined 5'-cDNA sequence for SAL-48.

SEQ ID NO: 174 is the determined 5'-cDNA sequence for SAL-68.

SEQ ID NO: 175 is the determined 5'-cDNA sequence for SAL-72.

SEQ ID NO: 176 is the determined 5'-cDNA sequence for SAL-77.

SEQ ID NO: 177 is the determined 5'-cDNA sequence for SAL-86.

SEQ ID NO: 178 is the determined 5'-cDNA sequence for SAL-88.

SEQ ID NO: 179 is the determined 5'-cDNA sequence for SAL-93.

SEQ ID NO: 180 is the determined 5'-cDNA sequence for SAL-100.

SEQ ID NO: 181 is the determined 5'-cDNA sequence for SAL-105.

SEQ ID NO: 182 is the predicted amino acid sequence for SAL-3.

SEQ ID NO: 183 is the predicted amino acid sequence for SAL-24.

SEQ ID NO: 184 represents the first predicted amino acid sequence for SAL-25.

SEQ ID NO: 185 is the second predicted amino acid sequence for SAL-25.

SEQ ID NO: 186 is the predicted amino acid sequence for SAL-33.

SEQ ID NO: 187 is the first predicted amino acid sequence for SAL-50.

SEQ ID NO: 188 is the predicted amino acid sequence for SAL-57.

SEQ ID NO: 189 represents the first predicted amino acid sequence for SAL-66.

SEQ ID NO: 190 is the second predicted amino acid sequence for SAL-66.

SEQ ID NO: 191 is the predicted amino acid sequence for SAL-82.

SEQ ID NO: 192 is the predicted amino acid sequence for SAL-99.

SEQ ID NO: 193 is the predicted amino acid sequence for SAL-104.

SEQ ID NO: 194 is the predicted amino acid sequence for SAL-5.

SEQ ID NO: 195 is the predicted amino acid sequence for SAL-8.

SEQ ID NO: 196 is the predicted amino acid sequence for SAL-12.

SEQ ID NO: 197 is the predicted amino acid sequence for SAL-14.

SEQ ID NO: 198 is the predicted amino acid sequence for SAL-16.

SEQ ID NO: 199 is the predicted amino acid sequence for SAL-23.

SEQ ID NO: 200 is the predicted amino acid sequence for SAL-26.

SEQ ID NO: 201 is the predicted amino acid sequence for SAL-29.

SEQ ID NO: 202 is the predicted amino acid sequence for SAL-32.

SEQ ID NO: 203 is the predicted amino acid sequence for SAL-39.

SEQ ID NO: 204 is the predicted amino acid sequence for SAL-42.

SEQ ID NO: 205 represents the predicted amino acid sequence for SAL-43.

SEQ ID NO: 206 is the predicted amino acid sequence for SAL-44.

SEQ ID NO: 207 is the predicted amino acid sequence for SAL-48.

SEQ ID NO: 208 is the predicted amino acid sequence for SAL-68.

SEQ ID NO: 209 is the predicted amino acid sequence for SAL-72.

SEQ ID NO: 210 is the predicted amino acid sequence for SAL-77.

SEQ ID NO: 211 is the predicted amino acid sequence for SAL-86.

SEQ ID NO: 212 is the predicted amino acid sequence for SAL-88.

SEQ ID NO: 213 is the predicted amino acid sequence for SAL-93.

SEQ ID NO: 214 is the predicted amino acid sequence for SAL-100.

SEQ ID NO: 215 is the predicted amino acid sequence for SAL-105.

SEQ ID NO: 216 is the second predicted amino acid sequence for SAL-50.

SEQ ID NO: 217 is the determined cDNA sequence for SSLT-4.

SEQ ID NO: 218 is the determined cDNA sequence for SSLT-9.

SEQ ID NO: 219 is the determined cDNA sequence for SSLT-10.

SEQ ID NO: 220 is the determined cDNA sequence for SSLT-12.

SEQ ID NO: 221 is the determined cDNA sequence for SSLT-19.

SEQ ID NO: 222 is the determined cDNA sequence for SSLT-31.

SEQ ID NO: 223 is the determined cDNA sequence for SSLT-38.

SEQ ID NO: 224 is the determined cDNA sequence for LT4690-2.

SEQ ID NO: 225 is the determined cDNA sequence for LT4690-3.

SEQ ID NO: 226 is the determined cDNA sequence for LT4690-22.

SEQ ID NO: 227 is the determined cDNA sequence for LT4690-24.

SEQ ID NO: 228 is the determined cDNA sequence for LT4690-37.

SEQ ID NO: 229 is the determined cDNA sequence for LT4690-39.

SEQ ID NO: 230 is the determined cDNA sequence for LT4690-40.

SEQ ID NO: 231 is the determined cDNA sequence for LT4690-41.

SEQ ID NO: 232 is the determined cDNA sequence for LT4690-49.

SEQ ID NO: 233 is the determined 3'-cDNA sequence for LT4690-55.

SEQ ID NO: 234 is the determined 5'-cDNA sequence for LT4690-55.

SEQ ID NO: 235 is the determined cDNA sequence for LT4690-59.

SEQ ID NO: 236 is the determined cDNA sequence for LT4690-63.

SEQ ID NO: 237 is the determined cDNA sequence for LT4690-71.

SEQ ID NO: 238 is the determined cDNA sequence for 2LT-3.

SEQ ID NO: 239 is the determined cDNA sequence for 2LT-6.

SEQ ID NO: 240 is the determined cDNA sequence for 2LT-22.

SEQ ID NO: 241 is the determined cDNA sequence for 2LT-25.

SEQ ID NO: 242 is the determined cDNA sequence for 2LT-26.

SEQ ID NO: 243 is the determined cDNA sequence for 2LT-31.

SEQ ID NO: 244 is the determined cDNA sequence for 2LT-36.

SEQ ID NO: 245 is the determined cDNA sequence for 2LT-42.

SEQ ID NO: 246 is the determined cDNA sequence for 2LT-44.

SEQ ID NO: 247 is the determined cDNA sequence for 2LT-54.

SEQ ID NO: 248 is the determined cDNA sequence for 2LT-55.

SEQ ID NO: 249 is the determined cDNA sequence for 2LT-57.

SEQ ID NO: 250 is the determined cDNA sequence for 2LT-58.

SEQ ID NO: 251 is the determined cDNA sequence for 2LT-59.

SEQ ID NO: 252 is the determined cDNA sequence for 2LT-62.

SEQ ID NO: 253 is the determined cDNA sequence for 2LT-63.

SEQ ID NO: 254 is the determined cDNA sequence for 2LT-65.

SEQ ID NO: 255 is the determined cDNA sequence for 2LT-66

SEQ ID NO: 256 is the determined cDNA sequence for 2LT-70.

SEQ ID NO: 257 is the determined cDNA sequence for 2LT-73.

SEQ ID NO: 258 is the determined cDNA sequence for 2LT-74.

SEQ ID NO: 259 is the determined cDNA sequence for 2LT-76.

SEQ ID NO: 260 is the determined cDNA sequence for 2LT-77.

SEQ ID NO: 261 is the determined cDNA sequence for 2LT-78.

SEQ ID NO: 262 is the determined cDNA sequence for 2LT-80.

SEQ ID NO: 263 is the determined cDNA sequence for 2LT-85.

SEQ ID NO: 264 is the determined cDNA sequence for 2LT-87.

SEQ ID NO: 265 is the determined cDNA sequence for 2LT-89.

SEQ ID NO: 266 is the determined cDNA sequence for 2LT-94.

SEQ ID NO: 267 is the determined cDNA sequence for 2LT-95.

SEQ ID NO: 268 is the determined cDNA sequence for 2LT-98.

SEQ ID NO: 269 is the determined cDNA sequence for 2LT-100.

SEQ ID NO: 270 is the determined cDNA sequence for 2LT-103.

SEQ ID NO: 271 is the determined cDNA sequence for 2LT-105.

SEQ ID NO: 272 is the determined cDNA sequence for 2LT-107.

SEQ ID NO: 273 is the determined cDNA sequence for 2LT-108.

SEQ ID NO: 274 is the determined cDNA sequence for 2LT-109.

SEQ ID NO: 275 is the determined cDNA sequence for 2LT-118.

SEQ ID NO: 276 is the determined cDNA sequence for 2LT-120.

SEQ ID NO: 277 is the determined cDNA sequence for 2LT-121.

SEQ ID NO: 278 is the determined cDNA sequence for 2LT-122.

SEQ ID NO: 279 is the determined cDNA sequence for 2LT-124.

SEQ ID NO: 280 is the determined cDNA sequence for 2LT-126.

SEQ ID NO: 281 is the determined cDNA sequence for 2LT-127.

SEQ ID NO: 282 is the determined cDNA sequence for 2LT-128.

SEQ ID NO: 283 is the determined cDNA sequence for 2LT-129.

SEQ ID NO: 284 is the determined cDNA sequence for 2LT-133.

SEQ ID NO: 285 is the determined cDNA sequence for 2LT-137

SEQ ID NO: 286 is the determined cDNA sequence for LT4690-71.

SEQ ID NO: 287 is the determined cDNA sequence for LT4690-82.

SEQ ID NO: 288 is the determined full-length cDNA sequence for SSLT-74.

SEQ ID NO: 289 is the determined cDNA sequence for SSLT-78.

SEQ ID NO: 290 is the determined cDNA sequence for SCC1-8.

SEQ ID NO: 291 is the determined cDNA sequence for SCC1-12.

SEQ ID NO: 292 is the determined cDNA sequence for SCC1-336.

SEQ ID NO: 293 is the determined cDNA sequence for SCC1-344.

SEQ ID NO: 294 is the determined cDNA sequence for SCC1-345.

SEQ ID NO: 295 is the determined cDNA sequence for SCC1-346.

SEQ ID NO: 296 is the determined cDNA sequence for SCC1-348.

SEQ ID NO: 297 is the determined cDNA sequence for SCC1-350.

SEQ ID NO: 298 is the determined cDNA sequence for SCC1-352.

SEQ ID NO: 299 is the determined cDNA sequence for SCC1-354.

SEQ ID NO: 300 is the determined cDNA sequence for SCC1-355.

SEQ ID NO: 301 is the determined cDNA sequence for SCC1-356.

SEQ ID NO: 302 is the determined cDNA sequence for SCC1-357.

SEQ ID NO: 303 is the determined cDNA sequence for SCC1-501.

SEQ ID NO: 304 is the determined cDNA sequence for SCC1-503.

SEQ ID NO: 305 is the determined cDNA sequence for SCC1-513.

SEQ ID NO: 306 is the determined cDNA sequence for SCC1-516.

SEQ ID NO: 307 is the determined cDNA sequence for SCC1-518.

SEQ ID NO: 308 is the determined cDNA sequence for SCC1-519.

SEQ ID NO: 309 is the determined cDNA sequence for SCC1-522.

SEQ ID NO: 310 is the determined cDNA sequence for SCC1-523.

SEQ ID NO: 311 is the determined cDNA sequence for SCC1-525.

SEQ ID NO: 312 is the determined cDNA sequence for SCC1-527.

SEQ ID NO: 313 is the determined cDNA sequence for SCC1-529.

SEQ ID NO: 314 is the determined cDNA sequence for SCC1-530.

SEQ ID NO: 315 is the determined cDNA sequence for SCC1-531.

SEQ ID NO: 316 is the determined cDNA sequence for SCC1-532.

SEQ ID NO: 317 is the determined cDNA sequence for SCC1-533.

SEQ ID NO: 318 is the determined cDNA sequence for SCC1-536.

SEQ ID NO: 319 is the determined cDNA sequence for SCC1-538.

SEQ ID NO: 320 is the determined cDNA sequence for SCC1-539.

SEQ ID NO: 321 is the determined cDNA sequence for SCC1-541.

SEQ ID NO: 322 is the determined cDNA sequence for SCC1-542.

SEQ ID NO: 323 is the determined cDNA sequence for SCC1-546.

SEQ ID NO: 324 is the determined cDNA sequence for SCC1-549.

SEQ ID NO: 325 is the determined cDNA sequence for SCC1-551.

SEQ ID NO: 326 is the determined cDNA sequence for SCC1-552.

SEQ ID NO: 327 is the determined cDNA sequence for SCC1-554.

SEQ ID NO: 328 is the determined cDNA sequence for SCC1-558.

SEQ ID NO: 329 is the determined cDNA sequence for SCC1-559.

SEQ ID NO: 330 is the determined cDNA sequence for SCC1-561.

SEQ ID NO: 331 is the determined cDNA sequence for SCC1-562.

SEQ ID NO: 332 is the determined cDNA sequence for SCC1-564.

SEQ ID NO: 333 is the determined cDNA sequence for SCC1-565.

SEQ ID NO: 334 is the determined cDNA sequence for SCC1-566.

SEQ ID NO: 335 is the determined cDNA sequence for SCC1-567.

SEQ ID NO: 336 is the determined cDNA sequence for SCC1-568.

SEQ ID NO: 337 is the determined cDNA sequence for SCC1-570.

SEQ ID NO: 338 is the determined cDNA sequence for SCC1-572.

SEQ ID NO: 339 is the determined cDNA sequence for SCC1-575.

SEQ ID NO: 340 is the determined cDNA sequence for SCC1-576.

SEQ ID NO: 341 is the determined cDNA sequence for SCC1-577.

SEQ ID NO: 342 is the determined cDNA sequence for SCC1-578.

SEQ ID NO: 343 is the determined cDNA sequence for SCC1-582.

SEQ ID NO: 344 is the determined cDNA sequence for SCC1-583.

SEQ ID NO: 345 is the determined cDNA sequence for SCC1-586.

SEQ ID NO: 346 is the determined cDNA sequence for SCC1-588.

SEQ ID NO: 347 is the determined cDNA sequence for SCC1-590.

SEQ ID NO: 348 is the determined cDNA sequence for SCC1-591.

SEQ ID NO: 349 is the determined cDNA sequence for SCC1-592.

SEQ ID NO: 350 is the determined cDNA sequence for SCC1-593.

SEQ ID NO: 351 is the determined cDNA sequence for SCC1-594.

SEQ ID NO: 352 is the determined cDNA sequence for SCC1-595.

SEQ ID NO: 353 is the determined cDNA sequence for SCC1-596.

SEQ ID NO: 354 is the determined cDNA sequence for SCC1-598.

SEQ ID NO: 355 is the determined cDNA sequence for SCC1-599.

SEQ ID NO: 356 is the determined cDNA sequence for SCC1-602.

SEQ ID NO: 357 is the determined cDNA sequence for SCC1-604.

SEQ ID NO: 358 is the determined cDNA sequence for SCC1-605.

SEQ ID NO: 359 is the determined cDNA sequence for SCC1-606.

SEQ ID NO: 360 is the determined cDNA sequence for SCC1-607.

SEQ ID NO: 361 is the determined cDNA sequence for SCC1-608.

SEQ ID NO: 362 is the determined cDNA sequence for SCC1-610.

SEQ ID NO: 363 is the determined cDNA sequence for clone DMS79T1.

SEQ ID NO: 364 is the determined cDNA sequence for clone DMS79T2.

SEQ ID NO: 365 is the determined cDNA sequence for clone DMS79T3.

SEQ ID NO: 366 is the determined cDNA sequence for clone DMS79T5.

SEQ ID NO: 367 is the determined cDNA sequence for clone DMS79T6.

SEQ ID NO: 368 is the determined cDNA sequence for clone DMS79T7.

SEQ ID NO: 369 is the determined cDNA sequence for clone DMS79T9.

SEQ ID NO: 370 is the determined cDNA sequence for clone DMS79T10.

SEQ ID NO: 371 is the determined cDNA sequence for clone DMS79T11.

SEQ ID NO: 372 is the determined cDNA sequence for clone 128T1.

SEQ ID NO: 373 is the determined cDNA sequence for clone 128T2.

SEQ ID NO: 374 is the determined cDNA sequence for clone 128T3.

SEQ ID NO: 375 is the determined cDNA sequence for clone 128T4.

SEQ ID NO: 376 is the determined cDNA sequence for clone 128T5.

SEQ ID NO: 377 is the determined cDNA sequence for clone 128T7.

SEQ ID NO: 378 is the determined cDNA sequence for clone 128T9.

SEQ ID NO: 379 is the determined cDNA sequence for clone 128T10.

SEQ ID NO: 380 is the determined cDNA sequence for clone 128T11.

SEQ ID NO: 381 is the determined cDNA sequence for clone 128T12.

SEQ ID NO: 382 is the determined cDNA sequence for clone NCIH69T3.

SEQ ID NO: 383 is the determined cDNA sequence for clone NCIH69T5.

SEQ ID NO: 384 is the determined cDNA sequence for clone NCIH69T6.

SEQ ID NO: 385 is the determined cDNA sequence for clone NCIH69T7.

SEQ ID NO: 386 is the determined cDNA sequence for clone NCIH69T9.

SEQ ID NO: 387 is the determined cDNA sequence for clone NCIH69T10.

SEQ ID NO: 388 is the determined cDNA sequence for clone NCIH69T11.

SEQ ID NO: 389 is the determined cDNA sequence for clone NCIH69T12.

SEQ ID NO: 390 is the full length cDNA sequence for 128T1.

SEQ ID NO: 391 is the amino acid sequence for 128T1.

SEQ ID NO: 392 is the full length cDNA sequence for 2LT-128.

SEQ ID NO: 393 is the amino acid sequence for 2LT-128.

SEQ ID NO: 394 is an extended cDNA sequence for clone SCC1-542.

SEQ ID NO: 395 is the amino acid sequence corresponding to SEQ ID NO: 394.

SEQ ID NO: 396 is an extended cDNA sequence for clone SCC1-593.

SEQ ID NO: 397 is the amino acid sequence corresponding to SEQ ID NO: 396.

SEQ ID NO: 398 is the determined cDNA sequence for 55508.1.

SEQ ID NO: 399 is the determined cDNA sequence for 55509.1.

SEQ ID NO: 400 is the determined cDNA sequence for 54243.1.

SEQ ID NO: 401 is the determined cDNA sequence for 54251.1.

SEQ ID NO: 402 is the determined cDNA sequence for 54252.1.

SEQ ID NO: 403 is the determined cDNA sequence for 54253.1.

SEQ ID NO: 404 is the determined cDNA sequence for 55518.1.

SEQ ID NO: 405 is the determined cDNA sequence for 54258.1.

SEQ ID NO: 406 is the determined cDNA sequence for 54575.1.

SEQ ID NO: 407 is the determined cDNA sequence for 54577.1.

SEQ ID NO: 408 is the determined cDNA sequence for 54584.1.

SEQ ID NO: 409 is the determined cDNA sequence for 55521.1.

SEQ ID NO: 410 is the determined cDNA sequence for 54589.1.

SEQ ID NO: 411 is the determined cDNA sequence for 54592.1.

SEQ ID NO: 412 is the determined cDNA sequence for 55134.1.

SEQ ID NO: 413 is the determined cDNA sequence for 55137.1.

SEQ ID NO: 414 is the determined cDNA sequence for 55140.1.

SEQ ID NO: 415 is the determined cDNA sequence for 55531.1.

SEQ ID NO: 416 is the determined cDNA sequence for 55532.1.

SEQ ID NO: 417 is the determined cDNA sequence for 54621.1.

SEQ ID NO: 418 is the determined cDNA sequence for 55548.1.

SEQ ID NO: 419 is the determined cDNA sequence for 54623.1.

SEQ ID NO: 420 is the determined cDNA sequence for L39.

SEQ ID NO: 421 is the predicted amino acid sequence for L39.

SEQ ID NO: 422 is the determined cDNA sequence for SCC2-29.

SEQ ID NO: 423 is the determined cDNA sequence for SCC2-36.

SEQ ID NO: 424 is the determined cDNA sequence for SCC2-60.

SEQ ID NO: 425 represents the predicted amino acid sequence for SCC2-29.

SEQ ID NO: 426 is the predicted amino acid sequence for SCC2-36.

SEQ ID NO: 427 is the predicted amino acid sequence for SCC2-60.

SEQ ID NO: 428 is an extended cDNA sequence for clone 20129, also referred to as 2LT-3 as set forth in SEQ ID NO: 238.

SEQ ID NO: 429 is an extended cDNA sequence for clone 20347, which is also referred to as 2LT-26 as set forth in SEQ ID NO: 242.

SEQ ID NO: 430 is an extended cDNA sequence for clone 21282, which is also referred to as 2LT-57 set in SEQ ID NO: 249.

SEQ ID NO: 431 is an extended cDNA sequence for clone 21283, which is also referred to as 2LT-58 set in SEQ ID NO: 250.

SEQ ID NO: 432 is an extended cDNA sequence for clone 21484, which is also referred to as 2LT-98 set forth in SEQ ID NO: 268.

SEQ ID NO: 433 is an extended cDNA sequence for clone 21871, which is also referred to as 2LT-124 set forth in SEQ ID NO: 279.

SEQ ID NO: 434 is the amino acid sequence encoded by SEQ ID NO: 428.

SEQ ID NO: 435 is the amino acid sequence encoded by SEQ ID NO: 429.

SEQ ID NO: 436 is the amino acid sequence encoded by SEQ ID NO: 430.

SEQ ID NO: 437 is the amino acid sequence encoded by SEQ ID NO: 431.

SEQ ID NO: 438 is the amino acid sequence encoded by SEQ ID NO: 432.

SEQ ID NO: 439 is the amino acid sequence encoded by SEQ ID NO: 433.

SEQ ID NO: 440 is the determined cDNA sequence for clone 19A4.

SEQ ID NO: 441 is the determined full-length cDNA sequence for clone 14F10.

SEQ ID NO: 442 is the determined 5'-cDNA sequence for clone 20E10.

SEQ ID NO: 443 represents the first identified cDNA sequence for clone 55153.

SEQ ID NO: 444 represents the second installed cDNA sequence for clone 55153.

SEQ ID NO: 445 represents the first identified cDNA sequence for clone 55154.

SEQ ID NO: 446 represents the second installed cDNA sequence for clone 55154.

SEQ ID NO: 447 is the determined cDNA sequence for clone 55155.

SEQ ID NO: 448 represents the first identified cDNA sequence for clone 55156.

SEQ ID NO: 449 represents the second installed cDNA sequence for clone 55156.

SEQ ID NO: 450 represents the first identified cDNA sequence for clone 55157.

SEQ ID NO: 451 represents the second installed cDNA sequence for clone 55157.

SEQ ID NO: 452 is the determined cDNA sequence for clone 55158.

SEQ ID NO: 453 is the determined cDNA sequence for clone 55159.

SEQ ID NO: 454 represents the first identified cDNA sequence for clone 55161.

SEQ ID NO: 455 represents the second installed cDNA sequence for clone 55161.

SEQ ID NO: 456 represents the first identified cDNA sequence for clone 55162.

SEQ ID NO: 457 represents the second installed cDNA sequence for clone 55162.

SEQ ID NO: 458 represents the first identified cDNA sequence for clone 55163.

SEQ ID NO: 459 represents the second installed cDNA sequence for clone 55163.

SEQ ID NO: 460 represents the first identified cDNA sequence for clone 55164.

SEQ ID NO: 461 represents the second installed cDNA sequence for clone 55164.

SEQ ID NO: 462 represents the first identified cDNA sequence for clone 55165.

SEQ ID NO: 463 represents the second installed cDNA sequence for clone 55165.

SEQ ID NO: 464 represents the first identified cDNA sequence for clone 55166.

SEQ ID NO: 465 represents the second installed cDNA sequence for clone 55156.

SEQ ID NO: 466 represents the first identified cDNA sequence for clone 55167.

SEQ ID NO: 467 represents the second installed cDNA sequence for clone 55167.

SEQ ID NO: 468 represents the first identified cDNA sequence for clone 55168.

SEQ ID NO: 469 represents the second installed cDNA sequence for clone 55168.

SEQ ID NO: 470 represents the first identified cDNA sequence for clone 55169.

SEQ ID NO: 471 represents the second installed cDNA sequence for clone 55169.

SEQ ID NO: 472 represents the first identified cDNA sequence for clone 55170.

SEQ ID NO: 473 represents the second installed cDNA sequence for clone 55170.

SEQ ID NO: 474 is the determined cDNA sequence for clone 55171.

SEQ ID NO: 475 is the determined cDNA sequence for clone 55172.

SEQ ID NO: 476 is the determined cDNA sequence for clone 55173.

SEQ ID NO: 477 represents the first identified cDNA sequence for clone 55174.

SEQ ID NO: 478 represents the second installed cDNA sequence for clone 55174.

SEQ ID NO: 479 is the determined cDNA sequence for clone 55175.

SEQ ID NO: 480 is the determined cDNA sequence for clone 55176.

SEQ ID NO: 481 is the determined cDNA sequence for contig 525.

SEQ ID NO: 482 is the determined cDNA sequence for contig 526.

SEQ ID NO: 483 is the determined cDNA sequence for contig 527.

SEQ ID NO: 484 is the determined cDNA sequence for contig 528.

SEQ ID NO: 485 is the determined cDNA sequence for contig 529.

SEQ ID NO: 486 is the determined cDNA sequence for contig 530.

SEQ ID NO: 487 is the determined cDNA sequence for contig 531.

SEQ ID NO: 488 is the determined cDNA sequence for contig 532.

SEQ ID NO: 489 is the determined cDNA sequence for contig 533.

SEQ ID NO: 490 is the determined cDNA sequence for contig 534.

SEQ ID NO: 491 is the determined cDNA sequence for contig 535.

SEQ ID NO: 492 is the determined cDNA sequence for contig 536.

SEQ ID NO: 493 is the determined cDNA sequence for contig 537.

SEQ ID NO: 494 is the determined cDNA sequence for contig 538.

SEQ ID NO: 495 is the determined cDNA sequence for contig 539.

SEQ ID NO: 496 is the determined cDNA sequence for contig 540.

SEQ ID NO: 497 is the determined cDNA sequence for contig 541.

SEQ ID NO: 498 is the determined cDNA sequence for contig 542.

SEQ ID NO: 499 is the determined cDNA sequence for contig 543.

SEQ ID NO: 500 is the determined cDNA sequence for contig 544.

SEQ ID NO: 501 is the determined cDNA sequence for contig 545.

SEQ ID NO: 502 is the determined cDNA sequence for contig 546.

SEQ ID NO: 503 is the determined cDNA sequence for contig 547.

SEQ ID NO: 504 is the determined cDNA sequence for contig 548.

SEQ ID NO: 505 is the determined cDNA sequence for contig 549.

SEQ ID NO: 506 is the determined cDNA sequence for contig 550.

SEQ ID NO: 507 is the determined cDNA sequence for contig 551.

SEQ ID NO: 508 is the determined cDNA sequence for contig 552.

SEQ ID NO: 509 is the determined cDNA sequence for contig 553.

SEQ ID NO: 510 is the determined cDNA sequence for contig 554.

SEQ ID NO: 511 is the determined cDNA sequence for contig 555.

SEQ ID NO: 512 is the determined cDNA sequence for clone 57207.

SEQ ID NO: 513 is the determined cDNA sequence for clone 57209.

SEQ ID NO: 514 is the determined cDNA sequence for clone 57210.

SEQ ID NO: 515 is the determined cDNA sequence for clone 57211.

SEQ ID NO: 516 is the determined cDNA sequence for clone 57212.

SEQ ID NO: 517 is the determined cDNA sequence for clone 57213.

SEQ ID NO: 518 is the determined cDNA sequence for clone 57215.

SEQ ID NO: 519 is the determined cDNA sequence for clone 57219.

SEQ ID NO: 520 is the determined cDNA sequence for clone 57221.

SEQ ID NO: 521 is the determined cDNA sequence for clone 57222.

SEQ ID NO: 522 is the determined cDNA sequence for clone 57223.

SEQ ID NO: 523 is the determined cDNA sequence for clone 57225.

SEQ ID NO: 524 is the determined cDNA sequence for clone 57227.

SEQ ID NO: 525 is the determined cDNA sequence for clone 57228.

SEQ ID NO: 526 is the determined cDNA sequence for clone 57229.

SEQ ID NO: 527 is the determined cDNA sequence for clone 57230.

SEQ ID NO: 528 is the determined cDNA sequence for clone 57231.

SEQ ID NO: 529 is the determined cDNA sequence for clone 57232.

SEQ ID NO: 530 is the determined cDNA sequence for clone 57233.

SEQ ID NO: 531 is the determined cDNA sequence for clone 57234.

SEQ ID NO: 532 is the determined cDNA sequence for clone 57235.

SEQ ID NO: 533 is the determined cDNA sequence for clone 57236.

SEQ ID NO: 534 is the determined cDNA sequence for clone 57237.

SEQ ID NO: 535 is the determined cDNA sequence for clone 57238.

SEQ ID NO: 536 is the determined cDNA sequence for clone 57239.

SEQ ID NO: 537 is the determined cDNA sequence for clone 57240.

SEQ ID-NO: 538 is the determined cDNA sequence for clone 57242.

SEQ ID NO: 539 is the determined cDNA sequence for clone 57243.

SEQ ID NO: 540 is the determined cDNA sequence for clone 57245.

SEQ ID NO: 541 is the determined cDNA sequence for clone 57248.

SEQ ID NO: 542 is the determined cDNA sequence for clone 57249.

SEQ ID NO: 543 is the determined cDNA sequence for clone 57250.

SEQ ID NO: 544 is the determined cDNA sequence for clone 57251.

SEQ ID NO: 545 is the determined cDNA sequence for clone 57253.

SEQ ID NO: 546 is the determined cDNA sequence for clone 57254.

SEQ ID NO: 547 is the determined cDNA sequence for clone 57255.

SEQ ID NO: 548 is the determined cDNA sequence for clone 57257.

SEQ ID NO: 549 is the determined cDNA sequence for clone 57258.

SEQ ID NO: 550 is the determined cDNA sequence for clone 57259.

SEQ ID NO: 551 is the determined cDNA sequence for clone 57261.

SEQ ID NO: 552 is the determined cDNA sequence for clone 57262.

SEQ ID NO: 553 is the determined cDNA sequence for clone 57263.

SEQ ID NO: 554 is the determined cDNA sequence for clone 57264.

SEQ ID NO: 555 is the determined cDNA sequence for clone 57265.

SEQ ID NO: 556 is the determined cDNA sequence for clone 57266.

SEQ ID NO: 557 is the determined cDNA sequence for clone 57267.

SEQ ID NO: 558 is the determined cDNA sequence for clone 57268.

SEQ ID NO: 559 is the determined cDNA sequence for clone 57269.

SEQ ID NO: 560 is the determined cDNA sequence for clone 57270.

SEQ ID NO: 561 is the determined cDNA sequence for clone 57271.

SEQ ID NO: 562 is the determined cDNA sequence for clone 57272.

SEQ ID NO: 563 is the determined cDNA sequence for clone 57274.

SEQ ID NO: 564 is the determined cDNA sequence for clone 57275.

SEQ ID NO: 565 is the determined cDNA sequence for clone 57277.

SEQ ID NO: 566 is the determined cDNA sequence for clone 57280.

SEQ ID NO: 567 is the determined cDNA sequence for clone 57281.

SEQ ID NO: 568 is the determined cDNA sequence for clone 57282.

SEQ ID NO: 569 is the determined cDNA sequence for clone 57283.

SEQ ID NO: 570 is the determined cDNA sequence for clone 57285.

SEQ ID NO: 571 is the determined cDNA sequence for clone 57287.

SEQ ID NO: 572 is the determined cDNA sequence for clone 57288.

SEQ ID NO: 573 is the determined cDNA sequence for clone 57289.

SEQ ID NO: 574 is the determined cDNA sequence for clone 57290.

SEQ ID NO: 575 is the determined cDNA sequence for clone 57292.

SEQ ID NO: 576 is the determined cDNA sequence for clone 57295.

SEQ ID NO: 577 is the determined cDNA sequence for clone 57296.

SEQ ID NO: 578 is the determined cDNA sequence for clone 57297.

SEQ ID NO: 579 is the determined cDNA sequence for clone 57299.

SEQ ID NO: 580 is the determined cDNA sequence for clone 57301.

SEQ ID NO: 581 is the determined cDNA sequence for clone 57302.

SEQ ID NO: 582 is the determined cDNA sequence for the beta chain of a tumor specific lung T-cell receptor.

SEQ ID NO: 583 is the determined cDNA sequence for the beta chain of a tumor specific T cell receptor.

SEQ ID NO: 584 is the amino acid sequence encoded by SEQ ID NO: 583.

SEQ ID NO: 585 is the amino acid sequence encoded by SEQ ID NO: 582.

SEQ ID NO: 586 is the amino acid sequence encoded by the 5 ′ end of 14F10.

SEQ ID-NO: 587 is the amino acid sequence of a T-cell epitope contained within SEQ ID NO: 586.

DETAILED DESCRIPTION OF THE INVENTION

The present invention mainly relates to compositions and their use in the treatment and diagnosis of a malignant tumor, in particular lung cancer. As described below, illustrative compositions of the present invention include, but are not limited to, polypeptides, in particular immunogenic polypeptides, polynucleotides encoding such polypeptides, antibodies and other binding agents, antigen-presenting cells (APCs) and immune system cells (e.g., T cells )

The execution of the present invention involves within the scope of the skills available in this field, with the exception of specifically indicated opposite cases, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA technologies, many of which are described below for purposes of illustration. Such methods are fully disclosed in the literature. See, for example, Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).

All publications, patents, and patent applications cited herein, above or below, are hereby incorporated by reference in their entireties.

When used in this specification and the appended claims, the singular include reference to the plural, except in cases where the meaning clearly indicates otherwise.

Polypeptide Compositions

As used herein, the term "polypeptide" is used in its generally accepted sense, i.e. as a sequence of amino acids. Polypeptides are not limited to the specific length of the product; thus, peptides, oligopeptides and proteins are included in the definition of a polypeptide, and such terms may be used interchangeably herein, unless otherwise specifically defined. This term also does not apply or excludes post-expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like, as well as other modifications known in the art, both naturally occurring and not found in nature. The polypeptide may be a whole protein or its subsequence. Particular polypeptides of interest in the context of this invention are amino acid subsequences including epitopes, i.e. antigenic determinants essentially responsible for the immunogenic properties of the polypeptide and capable of eliciting an immune response.

In particular, the illustrative peptides of the present invention include those encoded by the polynucleotide sequence shown in any of SEQ ID NOs: 217-390, 392, 394, 396, 398-420, 422-424, 428-433 and 440-583, or a sequence that hybridizes under moderately stringent conditions, or, alternatively, under highly stringent conditions with the polynucleotide sequence shown in any of SEQ ID NO: 217-390, 392, 394, 396, 398-420, 422-424, 428- 433 and 440-583. Other specific illustrative polypeptides of the invention include amino acid sequences as set forth in any of SEQ ID NOs: 391, 393, 395, 397, 421, 425-427, 434-439 and 584-587.

The polypeptides of the present invention are sometimes referred to herein as lung tumor proteins or lung tumor polypeptides, to indicate that their identification is based at least in part on an increase in their expression level in lung tumor samples. Thus, a “lung tumor polypeptide” or “lung tumor protein” mainly refers to a polypeptide sequence of the present invention, or a polynucleotide sequence encoding such a polypeptide that is expressed in a significant proportion of lung tumor samples, for example, preferably in more than 20% more preferably more than 30%, and most preferably more than 50% and higher of the tested lung tumor samples, at a level that is at least double, and preferably at least ive times the level of expression in normal tissues, as determined using a representative assay provided herein. The sequence of the lung tumor polypeptide of the invention, based on its increased expression level in tumor cells, is particularly useful as a diagnostic marker and also as a target for therapy, as further described below.

In certain preferred embodiments, the polypeptides of the invention are immunogenic, i.e. they detectably respond in an immunological assay (such as an ELISA or T cell stimulation assay) with the antisera and / or T cells of a patient with lung cancer. Screening for immunogenic activity may be performed using methods well known to one of skill in the art. For example, such screening can be performed using methods such as described by Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one illustrative example, the polypeptide can be immobilized on a solid basis and can be contacted with patient sera to bind serum antibodies with immobilized polypeptide. Unbound serum can then be removed and bound antibodies can be detected, for example, using ¹²⁵ I labeled protein A.

As is clear to a person skilled in the art, the immunogenic parts of the polypeptides described herein also relate to the present invention. As used herein, the term "immunogenic portion" represents a fragment of an immunogenic polypeptide of the invention, which itself is immunologically responsive (i.e., specifically binds) to B-cell or T-cell surface antigen receptors that recognize the polypeptide. Immunogenic parts can be mainly identified using well-known methods, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and the references cited here. Such methods include screening polypeptides for their ability to interact with antigen-specific antibodies, antisera and / or T cell lines and clones. As used herein, antisera and antibodies are “antigen specific” if they specifically bind to the antigen (ie, interact with a protein in an ELISA or other immunological assay, and do not react detectably with unrelated proteins). Such antisera and antibodies may be prepared as described herein and using well-known methods.

In one preferred embodiment, the immunogenic portion of the polypeptide of the present invention is a portion that interacts with antibodies and / or T cells at a level substantially not less than that when interacting with a full-sized polypeptide (e.g., in an ELISA and / or interaction analysis T cells). Preferably, the level of immunogenic activity of the immunological part is at least about 50%, preferably at least about 70%, and most preferably more than about 90% of the immunogenicity of the full length peptide. In some cases, preferred immunogenic parts are identified having an immunogenic activity level higher than that of the corresponding full-sized polypeptide, for example, having more than about 100%, or 150%, or more immunogenic activity.

In other specific embodiments, exemplary immunogenic portions may include peptides in which the N-terminal leader sequence and / or transmembrane domain are deleted. Other illustrative immunogenic parts contain a small N-terminal and / or C-terminal deletion (for example, 1-30 amino acids, preferably 5-15 amino acids) with respect to the mature protein.

In another embodiment, the polypeptide composition of the invention may also include one or more polypeptides that immunologically interact with T cells and / or antibodies generated against a polypeptide of the invention, in particular a polypeptide having the amino acid sequence described herein, or an immunogenic fragment or variant thereof .

In another embodiment of the invention, polypeptides are provided that include one or more polypeptides capable of producing T cells and / or antibodies that immunologically interact with one or more of the polypeptides described herein, or one or more polypeptides encoded by contiguous nucleic acid sequences contained in polynucleotide sequences described herein, or immunogenic fragments or variants thereof, or one or more n sequences kleinovyh acids which hybridize with one or more of these sequences under conditions of moderate to high stringency.

The present invention in another aspect relates to polypeptide fragments comprising at least about 5, 10, 15, 20, 25, 50, or 100 consecutive amino acids, or more, including all intermediate sizes of the polypeptide compositions described herein, such as those shown in SEQ ID NO: 391, 393, 395, 397, 421, 425-427, 434-439 and 584-587, or those encoded by the polynucleotide sequence shown in the sequence of SEQ ID NO: 217-390, 392, 394, 396, 398-420, 422-424, 428-433 and 440-583.

In another aspect, the present invention relates to variants of the polypeptide compositions described herein. In general, the polypeptide variants encompassed by the present invention typically exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or more, of identity (as set forth below) along their length with respect to the polypeptide sequences shown here.

In one preferred embodiment, the polypeptide fragments and variants of the present invention immunologically interact with an antibody and / or T cell that reacts with the full length polypeptide specifically set forth herein.

In another preferred embodiment, the polypeptide fragments and variants of the present invention exhibit a level of biological activity of at least about 50%, preferably at least about 70%, and most preferably at least about 90%, or more, from the fact that the full-length polypeptide sequence specifically set forth herein exhibits.

As used herein, a polypeptide "variant" is a polypeptide that usually differs from the polypeptide specifically described herein by one or more substitutions, deletions, additions and / or inserts. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating their immunogenic activity, as described herein and / or using any of a variety of methods well known in the art.

For example, specific illustrative variants of the polypeptides of the invention include those in which one or more parts, such as an N-terminal leader sequence or transmembrane domain, are deleted. Other illustrative options include those in which a small portion is removed from the N- and / or C-terminus of the mature protein (e.g., 1-30 amino acids, preferably 5-15 amino acids).

In many cases, the option contains conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, so that a person skilled in the art of peptide chemistry will expect that the secondary structure and the hydropathic nature of the polypeptide will not change significantly. As described above, modifications can be made to the structure of the polynucleotides and polypeptides of the present invention, and yet a functional molecule can be obtained that encodes a variant or derivative polypeptide with the desired characteristics, for example, immunogenic characteristics. When you want to change the amino acid sequence of a polypeptide to create an equivalent, or even improved immunogenic variant or part of a polypeptide of the invention, one of ordinary skill in the art will typically change one or more codons of the DNA coding sequence of Table 1.

For example, specific amino acids can be replaced in the protein structure with other amino acids without noticeable loss of binding ability when interacting with structures, such as, for example, antigen-binding regions of antibodies or binding sites of substrate molecules. Since it is the ability to interact and the nature of the protein that determine the biological functional activity of the protein, specific amino acid substitutions can be made in the protein sequence and, of course, in the underlying coding DNA sequence, and despite this, a protein with similar properties can be obtained. Thus, it is contemplated that various changes can be made to the peptide sequences of the compositions described or to the corresponding DNA sequences that encode the peptides without a noticeable loss of their biological functionality or activity.

When making such changes, the hydropathic amino acid index can be considered. In this area, the significance of the hydropathic amino acid index in assigning a biological interaction function to a protein has been generally established (Kyte and Doolittle, 1982; incorporated herein by reference). It is believed that the relative hydropathic nature of the amino acid contributes to the secondary structure of the resulting protein, which, in turn, determines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid is assigned a hydropathic index based on hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: 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); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).

It is known in the art that certain amino acids can be replaced by other amino acids having a similar hydropathic index or count, and yet, this can lead to the appearance of a protein with similar biological activity, i.e. to obtain a biologically functionally equivalent protein. With the introduction of such changes, it is preferable to replace amino acids whose hydropathic indices are in the range of ± 2, particularly preferred are indices in the range of ± 1, and even more particularly preferred are indices in the range of ± 0.5. It has also been found in the art that the replacement of such amino acids can be effectively done based on hydrophilicity. US Pat. No. 4,554,101 (specifically incorporated by reference in its entirety) establishes that the highest local average hydrophilicity of a protein, determined by the hydrophilicity of its successive amino acids, correlates with the biological properties of the protein.

As described in detail in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+ 3.0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); 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). It was found that the amino acid can be replaced by another having a similar hydrophilicity value, and, nevertheless, can be obtained biologically equivalent, and, in particular, immunologically equivalent protein. With such changes, it is preferable to replace amino acids whose hydrophilicity values are within ± 2, particularly preferred are values in the range of ± 1, and even more preferred are values in the range of ± 0.5.

Therefore, as reflected above, amino acid substitutions are generally based on the relative similarity of substituents of the side groups of amino acids, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Typical substitutions that take into account the various above-mentioned characteristics are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

In addition, any polynucleotides can be further modified to increase in vivo stability. Possible modifications include, but are not limited to, the addition of flanking sequences from the 5 ′ and / or 3 ′ ends; the use of phosphorothioacid or 2'-O-methyl instead of phosphodiesterase bonds in the skeleton; and / or the inclusion of unconventional bases such as inosine, queosin and vibutosin, as well as acetyl, methyl, thio and other modified forms of adenine, cytidine, guanine, thymine and uridine.

Amino acid substitutions, in addition, can be carried out on the basis of the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative substitutions 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. A variant may also or alternatively comprise non-conservative substitutions. In a preferred embodiment, variant polypeptides are distinguished from the native sequence by substitution, deletion or addition of five amino acids or less. Variants can also (or alternatively) be modified, for example, by deletion or addition of amino acids that have minimal effect on immunogenicity, secondary structure, and the hydropathic nature of the polypeptide.

As noted above, polypeptides may include a signal (or leader) sequence at the N-terminus of a protein that directs protein transfer together with or after translation. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His) or to facilitate the binding of the polypeptide to a solid base. For example, the polypeptide may conjugate to the immunoglobulin Fc region.

When comparing polypeptide sequences, two sequences are said to be “identical” if the amino acid sequence in the two sequences matches when aligned to the maximum match, as described below. Comparisons between two sequences are usually performed by matching sequences in comparison windows to identify and compare areas of local sequence similarity. As used herein, a “comparison window” refers to a segment consisting of at least about 20 consecutive positions, usually from about 30 to about 75, from about 40 to about 50, in which the sequence can be compared with the comparison sequence with the same number successive positions after two sequences have been optimally aligned.

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

Alternatively, optimal sequence alignment for comparison may be performed using the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2: 482, by Alignment Identity Algorithm Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, through computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by examination.

One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described by Altschul et al. (1977) Nucl. Acids Res. 25: 3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215: 403-410, respectively. BLAST and BLAST 2.0 can, for example, be used with the parameters described herein to determine the percent identity of the polynucleotide and polypeptide sequences of the invention. BLAST assay software is publicly available through the National Center for Biotechnology Information. In the case of amino acid sequences, a score matrix can be used to calculate the cumulative score. The expansion of short sections of coincidence (words) in each direction stops when the cumulative alignment score decreases by the amount of X from its maximum value; when the cumulative account becomes equal to zero or lower due to the accumulation of one or more alignments of residues with a negative account; or when the end of each of the sequences is reached. The BLAST W, T, and X algorithm parameters determine the sensitivity and speed of alignment.

In one preferred approach, the “percent sequence identity” is determined by comparing two optimally aligned sequences with a comparison window including at least 20 positions, where part of the polypeptide sequence in the comparison window may include additions or deletions (ie, insertions) in 20 percent or less, typically 5 to 15 percent, or 10 to 12 percent, relative to comparison sequences (which do not include additions or deletions), for optimal alignment Vuh sequences. The percentage is calculated by determining the number of positions in which the identical amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison sequence (i.e., window size), and multiplying the results by 100 to obtain a percentage expression of sequence identity.

In other illustrative embodiments, the polypeptide may be a fusion polypeptide that includes the multiple polypeptides described herein, or which includes at least one polypeptide described herein and an unrelated sequence, such as a known tumor protein. A fusion partner may, for example, facilitate the provision of a T-helper epitope (immunological fusion partner), preferably a human-recognized T-helper epitope, or may promote protein expression (expression enhancer) with a higher yield than that of a native recombinant protein. Specific preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners can be selected so that they increase the solubility of the polypeptide or make it capable of being directed to the desired intracellular compartments. Other fusion partners include affinity tags that facilitate the purification of the polypeptide.

Fusion polypeptides can mainly be obtained using standard methods, including chemical conjugation. Preferably, the fusion polypeptide is expressed as a recombinant polypeptide, allowing for production at higher concentrations in relation to the non-fused polypeptide in the expression system. In summary, DNA sequences encoding the polypeptide components can be assembled separately and ligated into a suitable expression vector. The 3 ′ end of the DNA sequence encoding the first polypeptide component is ligated using or without a peptide linker, with the 5 ′ end of the DNA sequence encoding the second polypeptide component, so that the reading frames of the sequences are in phase. This allows translation into a single fusion polypeptide, which preserves the biological activity of both constituent polypeptides.

The peptide linker sequence can be used to separate the first and second polypeptide component at a distance that is essential to ensure that each polypeptide folds to form its secondary and tertiary structure. Such a peptide linker sequence is included in the fusion polypeptide using standard methods well known in the art. Suitable peptide linker sequences may be selected based on the following factors: (1) their ability to accept a flexible, stretched conformation; (2) their inability to form a secondary structure that can interact with functional epitopes on the first and second polypeptide; and (3) the absence of hydrophobic or charged residues that can interact with polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other neutral amino acids, such as Thr and Ala, can also be used in the linker sequence. Amino acid sequences that may conveniently be used as linkers include those described by Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83: 8258-8262, 1986; in US patent No. 4935233 and US patent No. 4751180. The linker sequence can be generally from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides possess non-essential N-terminal regions that can be used to separate functional domains and prevent steric hindrance.

Ligated DNA sequences are functionally linked to transcriptional or translational regulatory elements. The regulatory elements responsible for DNA expression are localized only in the 5'-direction from the DNA sequence encoding the first polypeptides. Similarly, the stop codons required to complete the translation and transcription termination signals are present only in the 3 ′ direction from the DNA sequence encoding the second polypeptide.

The fusion polypeptide may include the polypeptide described herein together with an unrelated immunogenic protein, such as an immunogenic protein, capable of enhancing the reverse response. Examples of such proteins include tetanus, tuberculosis and hepatitis pathogen proteins (see, for example, Stoute et al. New Engl. J. Med., 336: 86-91, 1997).

In one preferred embodiment, the immunological fusion partner is derived from Mycobacterium sp. As a Ra12 fragment derived from Mycobacterium tuberculosis. Ra12 compositions and methods for their use to enhance expression and / or immunogenicity of heterologous polynucleotide / polypeptide sequences are described in US Patent Application 60/158585, the disclosure of which is incorporated herein by reference in its entirety. Briefly, Ra12 refers to the polynucleotide region, which is a nucleic acid subsequence of Mycobacterium tuberculosis MTB32A. MTB32A is a 32 kDa serine protease encoded by the gene of virulent and non-virulent M. tuberculosis strains. The nucleotide sequence and amino acid sequence of MTB32A are described (for example, US Patent Application 60/158585; see also Skeiky et al., Infection, and Immun. (1999) 67: 3998-4007, incorporated herein by reference). C-terminal fragments of the MTB32A coding sequence are expressed in high concentrations and remain as soluble polypeptides during the purification procedure. Moreover, Ra12 can enhance the immunogenicity of heterologous immunogenic polypeptides with which it is fused. One of the preferred fusion polypeptides with Ra12 includes a 14 kDa C-terminal fragment corresponding to the amino acid residues MTB32A from 192 to 323. Other preferred Ra12 polynucleotides mainly include at least about 15 consecutive nucleotides, at least about 30 nucleotides of at least about 60 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides that encode a portion of the Ra12 polypeptide. Ra12 polynucleotides may include a native sequence (i.e., an endogenous sequence that encodes a Ra12 polypeptide or part thereof) or may include a variant of such a sequence. Variants of the Ra12 polynucleotide may contain one or more substitutions, additions, deletions and / or inserts, such that the biological activity of the encoded fusion polypeptide is substantially reduced relative to that of the fusion polypeptide comprising the native Ra12 polypeptide. Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity, and most preferably at least about 90% identity with respect to the polynucleotide sequence encoding the native Ra12 polypeptide or part thereof.

In other preferred embodiments, the immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). Preferably, the protein D derivative comprises approximately the first third of the protein (for example, the first N-terminal 100-110 amino acids), and the protein D derivative can be lipidated. In specific preferred embodiments, the first 109 residues of the lipoprotein D-based fusion partner are included at the N-terminus to produce a polypeptide with additional exogenous T-cell epitopes and to increase expression in E. coli (thus functioning as an expression enhancer). The lipid tail provides optimal antigen presentation to antigen-presenting cells. Other fusion partners include a non-structural protein from the influenza virus, NS1 (hemaglutinin). 81 N-terminal amino acids are typically used, although different fragments that include T-helper epitopes can be used.

In another embodiment, the immunological fusion partner is LYTA or a portion thereof (preferably a C-terminal portion). LYTA originates from Streptococcus pneumoniae, which synthesizes N-acetyl-L-alaninamidase, known as LYTA amidase (encoded by LytA gene; Gene 43: 265-292, 1986). LYTA is an autolysin that specifically destroys certain bonds in the peptidoglycan skeleton. The C-terminal domain of the LYTA protein is responsible for the affinity for choline or some choline analogues, such as DEAE. This property is used to develop C-LYTA expressing E. coli plasmids that can express fusion proteins. The purification of fusion proteins containing a C-LYTA fragment at the N-terminus is described (see Biotechnology 10: 795-798, 1992). In a preferred embodiment, a portion of the repeating LYTA may be included in the fusion polypeptide. A repeat part was found in the C-terminal region starting at residue 178. A particularly preferred repeat part includes residues 188-305.

Another exemplary embodiment includes fusion proteins and polynucleotides encoding them, wherein the fusion partner includes a targeted signal capable of directing the polypeptide to the endosomal / lysosomal compartment, as described in US Pat. No. 5,663,234. The immunogenic peptide of the invention will be more efficiently associated with fusion with this targeted signal with MHC molecules of class II and thus will provide enhanced in vivo stimulation of CD4 ⁺ T cells specific for this polypeptide.

The polypeptides of the invention are prepared using any of a variety of well-known synthetic and / or recombinant methods, the latter of which are further described below. Polypeptides, parts, and other variants, generally smaller than about 150 amino acids, can be generated by synthetic means using methods well known to those of ordinary skill in the art. In one illustrative example, such polypeptides are synthesized using any of the commercially available solid phase methods, such as the Merrifield solid phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J Am. Chem. Soc. 85: 2149-2146, 1963. Equipment for the automatic synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer / Applied BioSystems Division (Foster City, CA) and can be controlled according to the manufacturer's instructions.

In general, the polypeptide compositions (including fusion polypeptides) of the invention are isolated. An “isolated” polypeptide is one that is removed from its original environment. For example, a naturally occurring protein or polypeptide is isolated if it is separated from some or all of the substances coexisting in the natural system. Preferably, such polypeptides are also purified, for example, are at least about 90% pure, more preferably at least about 95% pure, and most preferably at least about 99% pure.

Polynucleotide Compositions

The present invention in other aspects relates to polynucleotide compositions. The terms “DNA” and “polynucleotide” are used interchangeably herein and refer to a DNA molecule that is isolated in the absence of all genomic DNA of a given species. “Isolated” as used herein means that the polynucleotide is substantially removed from other coding sequences and that the DNA molecule does not contain large amounts of unrelated coding DNA, such as large chromosome fragments or other functional genes or polypeptide coding regions. This, of course, applies to DNA that was originally isolated, and does not exclude genes or coding regions added to the segment later by human actions.

As will be appreciated by those skilled in the art, polynucleotide compositions of the present invention may include genomic sequences, extragenomic and plasmid encoded sequences, and smaller engineered gene segments that express, or can be adapted to express, proteins, polypeptides, peptides, and the like. Such segments can be isolated from natural sources or modified synthetically by human actions.

As is also clear to a person skilled in the art, the polynucleotides of the invention can be single-stranded (encoding or antisense) or double-stranded, and can be DNA molecules (genomic, cDNA, or synthetic) or RNA. RNA molecules may include Hn-RNA molecules that contain introns and correspond to a one-to-one DNA molecule, mRNA molecules that do not contain introns. Additional coding and non-coding sequences may optionally be provided in the polynucleotide of the present invention, and the polynucleotide may optionally be associated with other molecules and / or substrate materials.

Polynucleotides may include a native sequence (i.e., an endogenous sequence that encodes a polypeptide / protein of the invention or a part thereof) or may include a sequence that encodes a variant or derivative, preferably an immunogenic variant or derivative of such a sequence.

Therefore, in another aspect of the present invention, polynucleotide compositions are provided that include some or all of the polynucleotide sequences shown in any of SEQ ID NOs: 217-390, 392, 394, 396, 398-420, 422-424, 428-433 and 440- 583, sequences complementary to the polynucleotide sequence shown in any of SEQ ID NOs: 217-390, 392, 394, 396, 398-420, 422-424, 428-433 and 440-583, and degenerate variants of the polynucleotide sequence shown in any of SEQ ID NO: 217-390, 392, 394, 396, 398-420, 422-424, 428-433 and 440-583. In specific preferred embodiments, the polynucleotide sequences provided herein encode immunogenic polypeptides as described above.

In other related embodiments, the present invention relates to polynucleotide variants characterized by substantial identity with respect to the sequences described herein in SEQ ID NOs: 217-390, 392, 394, 396, 398-420, 422-424, 428-433 and 440- 583, for example, to those that include at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity compared to the polynucleotide sequence of this invention, using those described herein with individuals (e.g., BLAST-analysis using standard parameters, as described below). One skilled in the art will recognize that these values can be suitably altered to determine the corresponding identity of the proteins encoded by the two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

Typically, polynucleotide variants comprise one or more substitutions, additions, deletions and / or inserts, preferably such that the immunogenicity of the polypeptide encoded by the variant polynucleotide is not significantly reduced with respect to that of the polypeptide encoded by the polynucleotide sequence specifically described herein. The term "variants" should also be understood as encompassing homologous genes of foreign origin.

In additional implementations, the present invention relates to polynucleotide fragments containing different lengths of successive segments of a sequence identical to or complementary to one or more of the sequences described herein. For example, polynucleotides that include at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, or 1000, or more consecutively, belong to this invention. nucleotides of one or more of the sequences described here, as well as sections of the intermediate between the specified lengths. It is easy to understand that "intermediate length" in this context means any length between the given values, such as 16, 17, 18, 19, etc .; 21, 22, 23, etc .; 30, 31, 32, etc .; 50, 51, 52, 53, etc .; 100, 101, 102, 103, etc .; 150, 151, 152, 153, etc .; including all integers between 200-500; 500-1000 and the like.

In another embodiment of the invention, polynucleotide compositions are provided that are capable of hybridizing under moderate to severe conditions with the polynucleotide sequence or fragment thereof described herein, or a sequence complementary thereto. Hybridization methods are well known in the field of molecular biology. For purposes of illustration, suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include pre-washing in a solution of 5 × SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization at 50 ° C-60 ° C, 5 × SSC, overnight; followed by washing twice at 65 ° C for 20 minutes each time with 2 ×, 0.5 × and 0.2 × SSC containing 0.1% SDS. One skilled in the art will recognize that the stringency of hybridization can be easily controlled, for example, by changing the salt content of the hybridization solution and / or the temperature at which hybridization is performed. For example, in another embodiment, suitable highly stringent hybridization conditions include those described above, except that the hybridization temperature is raised, for example, to 60-65 ° C or 65-70 ° C.

In some preferred embodiments, the polynucleotides described above, for example polynucleotide variants, fragments, and hybridizing sequences, encode polypeptides that exhibit cross-immunoreactivity with the polypeptide sequence specifically set forth herein. In other preferred embodiments, such polynucleotides encode polypeptides that are characterized by an immunogenicity level of at least 50%, preferably at least 70%, and more preferably at least 90% of the immunogenicity level of the polypeptide sequence provided herein.

The polynucleotides of the present invention or fragments thereof, regardless of the length of the coding sequence as such, can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, so that the total length may vary markedly. Therefore, it is contemplated to use a nucleic acid fragment of almost any length, with a total length that should preferably be limited by the ease of preparation and use of the designated recombinant DNA protocol. For example, it is contemplated that exemplary polynucleotide segments with a total length of about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs and the like (including all intermediate lengths) can be used in many implementations of the present invention.

When comparing polynucleotide sequences, two sequences are said to be “identical” if the nucleotide sequence in the two sequences coincides when aligned to the maximum match, as described below. Comparisons between two sequences are usually performed by matching sequences in comparison windows to identify and compare areas of local sequence similarity. As used herein, a “comparison window” refers to a segment consisting of at least about 20 consecutive positions, usually from about 30 to about 75, from about 40 to about 50, in which the sequence can be compared with the comparison sequence with the same number successive positions after two sequences have been optimally aligned.

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

Alternatively, optimal sequence alignment for comparison may be performed using the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2: 482, by Alignment Identity Algorithm Needleman and Wunsch (1970) J Mol. Biol. 48: 443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, through computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by examination.

One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described by Altschul et al. (1977) Nucl. Acids Res. 25: 3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215: 403-410, respectively. BLAST and BLAST 2.0 can, for example, be used, with the parameters described here, to determine the percent identity of the polynucleotide sequences of the invention. BLAST assay software is publicly available through the National Center for Biotechnology Information. In one illustrative example, cumulative count values can be calculated using for the nucleotide sequences the parameters M (reward score for a pair of matched residues; always> 0) and N (penalty count for mismatched residues; always <0). The expansion of short sections of coincidence (words) in each direction stops when the cumulative alignment score decreases by the amount of X from its maximum value; when the cumulative account becomes equal to zero or lower due to the accumulation of one or more alignments of residues with a negative account; or when the end of each of the sequences is reached. The BLAST W, T, and X algorithm parameters determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) uses the default word length (W) of 11 and expectation (E) of 10 and alignment with the BLOSUM62 score matrix (see Henikoff and Henikoff (1989) Proc. Natl Acad. Sci. USA 89: 10915), (B) equal to 50, expectation (E) equal to 10, M = 5, N = -4, and a comparison of both chains.

Preferably, the “percent sequence identity” is determined by comparing two optimally aligned sequences with a comparison window including at least 20 positions, where a portion of the polypeptide sequence in the comparison window may include 20 percent additions or deletions (ie, insertions) or less, usually from 5 to 15 percent, or from 10 to 12 percent, relative to comparison sequences (which do not include additions or deletions), for optimal alignment of the two sequences stey. The percentage is calculated by determining the number of positions in which an identical nucleic acid base occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison sequence (i.e., window size), and multiplying the results by 100 to obtain a percentage expression of sequence identity.

It will be understood by one of ordinary skill in the art that due to the degeneracy of the genetic code, there are many nucleotide sequences encoding the polypeptide described herein. Some of these polynucleotides have minimal homology with respect to the nucleotide sequence of any native gene. Despite this, polynucleotides that vary due to the use of codons are specifically contemplated by the present invention. In addition, alleles of genes including the polynucleotide sequences described herein are included in the scope of the present invention. Alleles are endogenous genes that change as a result of one or more mutations, such as deletions, additions and / or substitutions of nucleotides. The resulting mRNA and protein may optionally have an altered structure or function. Alleles can be identified by standard methods (such as hybridization, amplification and / or comparison of sequences from databases).

Therefore, in another embodiment of the invention, a mutagenesis approach, such as site-specific mutagenesis, is used to produce immunogenic variants and / or derivatives of the polypeptides described herein. Using this approach, specific modifications in the polypeptide sequence can be obtained by mutagenesis of the main polynucleotides encoding it. Such methods provide direct approaches for preparing and testing sequence variants, for example, combining one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into a polynucleotide.

Site-specific mutagenesis allows the use of specific oligonucleotide sequences to obtain mutants that encode a DNA sequence with the desired mutation, as well as a sufficient number of nearby nucleotides, to ensure a primer sequence with sufficient size and sequence complexity to form a stable duplex on both sides of the intersected compound after deletion. Mutations can be used in the selected polynucleotide sequence to improve, modify, reduce modification or other methods of changing the properties of the polynucleotide itself and / or changing the properties, activity, structure, stability or primary sequence of the encoded polypeptide.

In certain embodiments of the present invention, the inventors provide for the mutagenesis of the described polynucleotide sequence to alter one or more properties of the encoded polypeptide, such as the immunogenicity of the polypeptide vaccine. Site-specific mutagenesis methods are well known in the art and are widely used to create variants of both polypeptides and polynucleotides. For example, site-specific mutagenesis is often used to modify a specific part of a DNA molecule. In such embodiments, a primer typically corresponding to about 14-25 nucleotides in length or the like is used with about 5-10 residues on both sides of the contact of the variable sequence.

In site-specific mutagenesis methods, a phage vector that is highly regarded by those skilled in the art that exists in both single and double chain forms is often used. Typical vectors suitable for site-specific mutagenesis include vectors such as phage M13. This phage is readily available commercially and its use is generally well known to those skilled in the art. Double-stranded plasmids are also regularly used for site-specific mutagenesis, which eliminates the step of transferring the gene of interest from the plasmid to the phage.

In general, site-specific mutagenesis in accordance with this was carried out on the obtained single-stranded vector or separated by melting of two chains of a double-stranded vector, including the DNA sequence encoding the desired peptide. A nucleotide primer carrying the desired mutant sequence is obtained mainly by synthesis. This primer is then annealed with a single chain vector, and subjected to DNA polymerizing enzymes, such as the Maple fragment of polymerase I from E. coli, in order to complete the synthesis of the mutation-carrying chain. Thus, a heteroduplex is formed, where one of the chains encodes the original non-mutant sequence, and the second chain carries the desired mutation. This heteroduplex vector was then used to transform the corresponding cells, such as E. coli cells, from which clones including recombinant vectors carrying the mutant sequence were selected.

Obtaining variants of the sequences of selected regions of DNA encoding a peptide using the site-specific mutagenesis method provides means for the production of potentially useful varieties and is not intended to limit any other ways in which variants of the sequences of peptides or DNA sequences encoding them can be obtained. . For example, to obtain sequence variants, recombinant vectors encoding the desired peptide sequences can be treated with mutagenic agents such as hydroxylamine. Specific details regarding these methods and protocols are found in the manuals of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each of which is included here for this purpose.

As used herein, the term “oligonucleotide-directed mutagenesis method” refers to matrix dependent processes and vector-mediated propagation resulting in an increase in the concentration of specific nucleic acid molecules relative to their initial concentration or an increase in the concentration of a detectable signal, such as amplification. As used herein, the term “oligonucleotide-directed mutagenesis method” refers to a process comprising a matrix dependent extension of a primer molecule. The term matrix dependent process refers to the synthesis of nucleic acid, RNA, or DNA molecules, where the sequence of the newly synthesized nucleic acid chain is dictated by well-known base pair complementarity rules (see, for example, Watson, 1987). Typically, vector-mediated procedures include introducing a nucleic acid fragment into a DNA or RNA vector, amplifying the clones of the vector, and isolating the amplified nucleic acid fragment. Examples of such procedures described by US Pat. No. 4,237,224 are hereby incorporated by reference in their entireties.

In another approach, recursive sequence recombination, as described in US Pat. No. 5,837,458, can be used to produce variants of the polypeptides of the present invention. In this approach, repeated cycles of recombination and screening or selection are carried out to "develop" individual polynucleotide variants of the invention having, for example, increased immunogenic activity.

In other implementations of the present invention, the polynucleotide sequences described herein can be used primarily as probes or primers for nucleic acid hybridization. Essentially, it is contemplated that nucleic acid regions will be particularly useful, including a region of a sequence consisting of a sequence of at least about 15 contiguous nucleotides that has the same or complementary to the sequence of 15 contiguous nucleotides described herein. Longer sections of identical or complementary sequences, such as approximately 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to full-length sequences, may also find application in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize to the sequence of interest will enable them to be used to detect the presence of complementary sequences in a given sample. However, other application possibilities can also be imagined, such as the use of sequence information to obtain mutant types of primers or primers for use in other genetic constructs.

Polynucleotide molecules having sequences consisting of regions of sequences consisting of segments, successive nucleotides of 10-14, 15-20, 30, 50, or even 100-200 nucleotides or so (including also intermediate lengths) that are identical or complementary to the polynucleotide sequences described herein are specifically considered as probes for use, for example, for southern and northern blots. This will allow you to analyze the product of the gene or its fragment in both different types of cells, as well as in various bacterial cells. The overall size of the fragment, as well as the size of the complementary segment (s), will depend largely on the intended use or application of a particular nucleic acid segment. Smaller fragments will mainly find application in hybridization implementations where the length of the adjacent complementary region can be varied within the range of about 15 to 100 nucleotides, but longer adjacent complementary regions can be used, corresponding to the length of the complementary sequences to be detected.

The use of probes with a length of approximately 15-25 nucleotides allows the formation of a duplex molecule, which is both stable and selective. Molecules having adjacent complementary sequences over lengths of more than 15 bases in length are generally preferred, at least in order to increase the stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. In general, it is preferable to construct nucleic acid molecules having regions complementary to the genes from 15-25 successive nucleotides or even longer if necessary.

Probes can be selected from any part of any of the sequences described here. All that is required is a review of the sequences provided herein or any extended portion of sequences of about 15-25 nucleotides or more in length, including the full-length sequence that you want to use as a probe or primer. The choice of probe or primer sequence may be determined by various factors. For example, there may be a need to apply primers to the end of the sequence.

Small polynucleotide regions or fragments can be easily obtained, for example, by direct synthesis of fragments by chemical methods, as is usually the case using an automatic oligonucleotide synthesizer. Fragments can also be obtained by applying a nucleic acid reproduction method, such as PCR ™ from US Pat. No. 4,683,202 (incorporated by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA methods known primarily to those skilled in the art. in the field of molecular biology.

The nucleotide sequences of the invention can be used because of their ability to selectively form duplex molecules with complementary regions of the whole gene or gene fragments of interest. Depending on the intended use, it is usually required to apply different hybridization conditions to achieve varying degrees of selectivity of the probe to the target sequence. For applications requiring high selectivity, it is usually required to apply relatively stringent conditions for the formation of hybrids, for example, it is necessary to choose conditions with a relatively low salt concentration and / or high temperature, such as those provided by a salt concentration of from about 0.02 M to 0.15 M at temperatures from about 50 ° C to 70 ° C. Such selective conditions hardly allow, if at all, a mismatch between the probe and the sample or the target chain, and are particularly suitable for isolating similar sequences.

Of course, for some applications, for example, where it is required to obtain mutants using the mutant primer chain hybridized to the underlying sample, less stringent (reduced stringency) hybridization conditions are usually required to allow heteroduplex formation. Under these circumstances, it may be necessary to apply salt conditions, such as from about 0.15 M to 0.9 M, with temperature fluctuations from about 20 ° C to 55 ° C. Cross-hybridizing species can thus be easily identified as positively hybridizing signals with respect to control hybridizations. In any case, in general, it is taken into account that the conditions can be made more stringent by adding increased amounts of formamide, which serves to destabilize the hybrid duplex in the same way as higher temperatures. Thus, hybridization conditions can be easily manipulated and, thus, in general, they can be the method of choice, depending on the desired results.

According to another embodiment of the present invention, polynucleotide compositions are provided comprising antisense oligonucleotides. Antisense oligonucleotides have been shown to be effective and targeted inhibitors of protein synthesis and therefore provide a therapeutic approach by which the disease can be treated by inhibiting the synthesis of proteins that contribute to the disease. The effectiveness of antisense oligonucleotides for inhibiting protein synthesis is well established. For example, the synthesis of polygalactorouonase and muscarin type 2 acetylcholine receptor is inhibited by antisense oligonucleotides directed to their respective mRNA sequences (US Pat. No. 5,739,119 and US Pat. No. 5,758,929). In addition, examples of antisense inhibition have been shown with the nuclear protein cyclin, the multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, the striatum GABA _A receptor, and human EGF (Jaskulski et al., Science. 1988. 1988 Jun 10; 240 (4858): 1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989; 1 (4): 225-32; Peris et al., Brain Res Mol Brain Res. 1998 Juh 15; 57 (2): 310-20; US patent 5801154; US patent 5789573; US patent 5718709 and US patent 5610288). Also, the antisense constructs described are inhibitory and can be used to treat a variety of cases of abnormal cell proliferation, for example, malignant tumors (US Pat. No. 5,747,470; US Pat. No. 5,591,317; US Pat. No. 5,783,683).

Therefore, in certain implementations, the present invention relates to oligonucleotide sequences that include all or parts of any sequence that is capable of specifically binding to the polynucleotide sequence described herein, or its complementary sequence. In one embodiment, antisense oligonucleotides include DNA or its derivatives. In another embodiment, oligonucleotides include RNA or its derivatives. In a third embodiment, the oligonucleotides are modified DNAs containing a phosphorothioacid modified backbone. In a fourth embodiment, the oligonucleotide sequences comprise peptide nucleic acids or derivatives thereof. In each case, preferred compositions comprise a region of a sequence that is complementary and, more preferably, highly complementary, and even more preferably, fully complementary to one or more portions of the polynucleotides described herein. The selection of antisense compositions specific to a given gene sequence is based on the analysis of the selected target sequence and determination of the secondary structure, T _m , binding energy and relative stability. Antisense compositions can be selected based on their relative inability to form dimers, hairpins, or other secondary structures that can weaken or inhibit binding to the target mRNA in the host cell. Highly preferred are those target mRNA regions that are located near or close to the AUG translation initiation codon, and those sequences that are highly complementary to the 5 ′ regions of mRNA. These analyzes of secondary structures and considerations for selecting target sites can be performed, for example, using version 4 of the OLIGO primer analysis software and / or the BLASTN 2.0.5 algorithmic software (Altschul et al., Nucleic Acids Res. 1997, 25 (17) : 3389-402).

It is also contemplated to use an antisense delivery method using a short peptide vector called MPG (27 residues). The MPG peptide contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain from the nuclear localization sequence of the SV40 T antigen (Morris et al., Nucleic Acids Res. 1997 Jul 15; 25 (14): 2730 -6). It has been shown that several molecules of the MPG peptide cover antisense oligonucleotides and can be delivered to cultured mammalian cells in less than 1 hour with relatively high efficiency (90%). Moreover, interaction with MPG greatly increases both the stability of the oligonucleotide to nuclease and the ability to cross the plasma membrane.

According to another embodiment of the present invention, the polynucleotide compositions described herein are used to design and produce ribozyme molecules to suppress the expression of the tumor polypeptides and proteins of the present study in tumor cells. Ribozymes are RNA protein complexes that cleave nucleic acids in a site-specific manner. Ribozymes contain specific catalytic domains that have endonuclease activity (Kim and Cech, Proc Natl Acad Sci USA. 1987 Dec; 84 (24): 8788-92; Forster and Symons, Cell. 1987 Apr 24; 49 (2): 211- twenty). For example, a large number of ribozymes accelerate highly specific phosphoester metabolism reactions, often cleaving only one of several phosphoesters in the oligonucleotide substrate (Cech et al., Cell. 1981 Dec; 27 (3 Pt 2): 487-96; Michel and Westhof, J. Mol. Biol. 1990 Dec 5; 216 (3): 585-610; Reinhold-Hurek and Shub, Nature. 1992 May 14; 357 (6374): 173-6). This specificity was explained by the requirement of substrate binding through the interaction of base pairs with the internal adapter sequence (“IGS”) of the preceding chemical reaction.

Currently, six variants of natural enzymatic RNA are known. Under physiological conditions, each can catalyze the hydrolysis of phosphodiester RNA bonds in the trans position (and thus can cleave other RNA molecules). In general, enzymatic nucleic acids act through primary binding to a target RNA. This binding occurs using a portion of the enzymatic nucleic acids that binds to the target, which is held in close proximity to the enzymatic part of the molecule, which cleaves the target RNA. Thus, enzymatic nucleic acid first recognizes and then binds substrate RNA by forming complementary pairs of nucleotides and, after binding to the correct site, enzymatically cleaves the substrate RNA. Rapid cleavage of such substrate RNA destroys their ability to direct the synthesis of the encoded protein. After the enzymatic nucleic acid has bound and cleaved the target RNA, it is freed from this RNA to search for another target and can re-bind and cleave new targets.

The enzymatic nature of the ribozyme has advantages when used in many technologies, such as the technology of antisense sequences (where the nucleic acid molecule simply binds to the target nucleic acid, blocking its translation), since the concentration of the ribozyme required to provide a therapeutic effect is lower than that antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act as an enzyme. So, one ribozyme molecule is able to cleave many molecules of the target RNA. In addition, ribozyme is a highly specific inhibitor in which the specificity of the inhibition depends not only on the mechanism of base binding to the target RNA, but also on the mechanism of cleavage of the target RNA. Single mismatches or substitutions of the base near the cleavage site can completely destroy the catalytic activity of the ribozyme. Similar discrepancies in the molecules of antisense sequences do not interfere with their action (Woolf et al., Proc. Natl. Acad. Sci. USA. 1992 Aug. 15; 89 (16): 7305-9). Thus, the specificity of the action of the ribozyme is higher than that of the oligonucleotide antisense sequence that binds the same RNA site.

The enzymatic nucleic acid molecule can be formed in a RNA of a hammer-like structure, hairpin, hepatitis δ virus, intron of group I or RNase P (in connection with the RNA guide sequence) or RNA motif Neurospora VS. Exemplary motifs of the hammer structure are described by Rossi et al., Nucleic Acids Res. 1992 Sep 11; 20 (17): 4559-65. Examples of hairpin motifs are described by Hampel et al. (publication of the application for the grant of European patent No. EP 0360257), Hampel and Tritz, Biochemistry 1989 Jun 13; 28 (12): 4929-33; Hampel et al. Nucleic Acids Res. 1990 Jan 25; 18 (2): 299-304 and US Pat. No. 5,631,359. An example of a hepatitis δ virus motif is described by Perrotta and Been, Biochemistry, 1992 Dec 1; 31 (47): 11843-52; an example of an RNase P motif is described by Guerrier-Takada et al., Cell. 1983 Dec; 35 (3 Pt 2): 849-57; The neurospore ribozyme RNA motif VS is described by Collins (Saville and Collins, Cell. 1990 May 18; 61 (4): 685-96; Saville and Collins, Proc. Natl. Acad. Sci. USA. 1991 Oct 1; 88 (19): 8826-30; Collins and Olive, Biochemistry. 1993 Mar. 23; 32 (11): 2795-9); and an example of a group I intron is described in US Pat. No. 4,987,071. All that matters in the enzymatic nucleic acid molecule of this invention is that it has a specific substrate-binding region that is complementary to one or more regions of the target gene RNA, and that it has a nucleotide sequence within or near this substrate-binding region, which gives the molecule activity in terms of RNA cleavage. Thus, ribozyme constructs should not be limited to the specific motives indicated here.

Ribozymes can be designed as described in Int. Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595 (each specifically incorporated herein by reference) and, as described, synthesized for in vitro and in vivo testing. Such ribozymes can also be optimized for delivery. Although specific examples are provided, it is clear to those skilled in the art that, if necessary, equivalent RNA targets of other species may be involved.

Ribozyme activity can be optimized by changing the length of the connecting shoulders of the ribozyme or by synthesizing ribozymes with modifications that prevent their degradation by serum ribonuclease (see, for example, Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; European patent Appl. Publ. No. 92110298.4; US patent 5334711; and Int. Pat. Appl. Publ. No. WO 94 / 13688, which describe various modifications that can be made with the sugar components of enzymatic RNA molecules), with modifications that increase their efficiency in cells and removal Stem II bases to shorten RNA synthesis periods and reduce chemical requirements.

Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describe basic methods for the delivery of enzymatic RNA molecules. Ribozymes can be sent to cells in a variety of ways known to those skilled in the art, including but not limited to, encapsulation in liposomes, iontophoresis (iontophoresis) or placement in other transporters such as hydrogels, cyclodextrins, biodegradable nanocapsules and bioadhesive microspheres. For some indications, ribozymes can be delivered directly to cells or tissues ex vivo with or without the above carriers. Alternatively, the RNA / vehicle combination can be delivered in situ by direct inhalation, by injection, or by use of a catheter, infusion pump or stent. Other delivery routes include, but are not limited to, intravascular, intramuscular, subcutaneous, or intraarticular injection, aerosol inhalation, oral route (tablets or pills), topical, systemic, ophthalmic, intraperitoneal, and / or intrathecal delivery. More detailed descriptions of the delivery and use of ribozyme are described in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl. Publ. No. WO 93/23569, each specifically incorporated herein by reference.

Other ways of accumulating high concentrations of ribozyme (s) in cells is to embed the ribozyme coding sequence into an expression vector. The transcription of the ribozyme sequence is driven by a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II) or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed in cells at a high level, levels for a given pol II promoter in this type of cell will depend on the nature of the gene regulatory sequences present (enhancers, silencers, etc.). Promoters of prokaryotic RNA polymerase can also be used, provided that prokaryotic RNA polymerase is expressed in the corresponding cells. Such transcription units can be introduced into a number of vectors for introduction into mammalian cells, including plasmid DNA vectors, virus DNA vectors (such as adenovirus and adeno-associated vectors) or virus RNA vectors (such as retroviral vectors, semliki forest and sindbis virus vectors), but not limited to them.

In another embodiment of the present invention, peptide nucleic acids (PNAs) are provided. PNAs mimic DNA in which nucleotides are attached to a pseudopeptide backbone (Good and Nielsen, Antisense Nucleic Acid Drug Dev. 1997 7 (4): 431-37). PNA can be used in a number of methods that traditionally use DNA or RNA. Often PNA sequences function better in methods than the corresponding RNA or DNA sequences, and find uses that are not characteristic of RNA or DNA. A review of PNA, including preparation methods, its characteristics and methods of application, is provided by Corey (Trends Biotechnol 1997 Jun; 15 (6): 224-9). Essentially, in some implementations, PNA sequences complementary to one or more parts of the ACE mRNA sequence can be obtained, and such PNA compositions can be used to regulate, alter, reduce or reduce translation of ACE-specific mRNAs and thus alter the level of ACE activity in a host cell to which such a PNA composition has been applied.

PNAs contain 2-aminoethyl-glycine bonds replacing the normal phosphodiester DNA skeleton (Nielsen. Et al., Science 1991 Dec 6; 254 (5037): 1497-500; Hanvey et al., Science. 1992 Nov 27; 258 (5087) : 1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996 Jan; 4 (1): 5-23). This chemistry has three important consequences: firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAs are neutral molecules, secondly, PNAs are achiral, which avoids the need to develop stereoselective synthesis, and thirdly, PNA synthesis uses standard Boc protocols or Fmoc for solid phase peptide synthesis, although other methods are used, including a modified Merrifield method.

PNA monomers or off-the-shelf oligomers are commercially available from PerSeptive Biosystems (Framingham, MA). PNA synthesis for each of the Boc or Fmoc protocols is carried out directly using manuals or automated protocols (Norton et al., Bioorg Med Chem. 1995 Apr; 3 (4): 437-45). The management protocol is used to obtain chemically modified PNAs or the simultaneous synthesis of families of closely related PNAs.

As with peptide synthesis, the success of a particular PNA synthesis depends on the properties of the selected sequence. For example, although in theory PNAs can include any combination of nucleotide bases, the presence of adjacent purines can lead to deletions of one or more residues in the product. In anticipation of such a difficulty, it was suggested that upon receipt of PNA with adjacent purines, the addition of residues that are likely to be added inefficiently should be repeated. This may follow the purification of PNA using high performance liquid chromatography of the described products and purification of products similar to that carried out in the synthesis of peptides.

PNA modifications for these applications can be carried out by addition of amino acids during solid-phase synthesis or by adding compounds that contain carboxylic acid groups on an unprotected N-terminal amine. Alternatively, PNAs can be modified after synthesis by attachment to the inserted lysine or cysteine. The simplicity with which PNAs can be modified facilitates optimization for improved solubility or for specific functional requirements. After synthesis, the identity of PNAs and their derivatives can be confirmed using mass spectrometry. In some studies, modifications of PNAs were obtained and used (e.g., Norton et al., Bioorg Med Chem. 1995 Apr; 3 (4): 437-45; Petersen et al., J Pept Sci. 1995 May-Jun; 1 (3 ): 175-83; Orum et al., Biotechniques. 1995 Sep; 19 (3): 472-80; Footer et al :, Biochemistry. 1996 Aug 20; 35 (33): 10673-9; Griffith et al., Nucleic Acids Res. 1995 Aug 11; 23 (15): 3003-8; Pardridge et al, Proc Natl Acad Sci USA. 1995 Jun 6; 92 (12): 5592-6; Boffa et al., Proc Natl Acad Sci USA . 1995 Mar 14; 92 (6): 1901-5; Gambacorti-Passerini et al., Blood. 1996 Aug 15; 88 (4): 1411-7; Armitage et al., Proc Natl Acad Sci USA. 1997 Nov 11 ; 94 (23): 12320-5; Seeger et al., Biotechniques. 1997 Sep; 23 (3): 512-7). US Pat. No. 5,700,922 discusses chimeric PNA-DNA-PNA molecules and their use in the diagnosis, modulation of body proteins, and treatment of treatment-sensitive conditions.

Methods for characterizing PNA binding properties of antisense sequences are discussed by Rose (Anal Chem. 1993 Dec 15; 65 (24): 3545-9) and Jensen et al. (Biochemistry. 1997 Apr 22; 36 (16): 5072-7). Rose uses capillary gel electrophoresis to determine the binding of PNA to their complementary oligonucleotide, measure kinetics of relative binding and stoichiometry. Simple measurement types were performed by Jensen et al. Using BIAcore ™ technology.

Other described applications of PNA that are obvious to those skilled in the art include use in DNA strand embedding, antisense inhibition, mutation analysis, transcription enhancers, nucleic acid purification, isolation of transcriptionally active genes, blocking transcription factor binding, genome cleavage, biosensors, hybridization in situ and the like.

Polynucleotide Identification, Characterization and Expression

Polynucleotide compositions of the present invention can be identified, prepared and / or manipulated using any of a variety of well-designed methods (generally see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY , 1989 and other similar references). For example, polynucleotides can be identified as described in more detail below by screening a cDNA microfield for tumor-associated expression (i.e., expression that is at least twice as large in a tumor as in normal tissue, as determined using the representative analysis described here). For example, such screenings can be performed using Affymetrix, Inc. microfield technology. (Santa Clara, CA) according to the manufacturer's instructions (and essentially as 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 with cDNA derived from cells, such as tumor cells expressing the proteins described herein.

Many matrix-dependent processes are available to amplify the target sequence of interest present in the sample. One of the best known amplification methods is polymerase chain reaction (PCR ™), which is described in detail in US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein by reference in its entirety. Briefly, two primer sequences are prepared for PCR ™ that are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates is added to the reaction mixture along with DNA polymerase (usually Taq polymerase). If the target sequence is present in the sample, the primers bind to the target, and the polymerase causes elongation of the primers along the target sequence by adding nucleotides. By increasing and decreasing the temperature of the reaction mixture, the elongated primers dissociate to form the reaction product, the excess primers bind to the target and the reaction product, and the process repeats. Reverse transcription and PCR ™ are preferably used to count the amount of amplified mRNA. Polymerase chain reaction methods are well known in the art.

A number of other matrix dependent processes, many of which are variants of the PCR ™ amplification method, are well known and available in the art. To demonstrate, some of these methods include a ligase chain reaction (referred to as LCR), as described, for example, in European Patent Application Publication No. 320308 and US Pat. No. 4,883,750; the Q-beta replicase described in PCT International Patent Application Publication No. PCT / US87 / 00880; chain offset amplification (SDA) and chain recovery reaction (RCR). In addition, other amplification methods are described in application for the grant of a patent of Great Britain No. 2202328 and in the publication of international patent application PCT No. PCT / US89 / 01025. Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) (PCT International Patent Application Publication No. WO 88/10315), including nucleic acid sequence-based amplification (NASBA) and European Patent Application Publication No. 3298 3SR describes a process nucleic acid amplification involving cyclically synthesized single-stranded RNA ("csRNA"), csDNA, double-stranded DNA (dsDNA). PCT Publication No. WO 89/06700 describes a nucleic acid sequence amplification scheme based on hybridization of a promoter / primer sequence to a single stranded target DNA (“csDNA”) following transcription of many RNA copies of a sequence. Other amplification methods, such as "RACE" (Frohman, 1990) and "one-way PCR" (Ohara, 1989) are also well known to those skilled in the art.

Using well-known methods, the amplified portion of the polynucleotide of the present invention can be used to isolate a full-sized gene from a suitable library (e.g., a tumor cDNA library). In this method, a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, the size of the library is selected so that it would include large molecules. Libraries obtained using random primers may be preferred for identifying 5 ′ regions and gene regions upstream of the start point of gene transcription. Genomic libraries are preferred for producing introns and extended 5'-sequences.

Using well-known methods, incomplete sequences may be labeled for hybridization methods (e.g., using nick translation or ³² P termination). Bacteria or bacteriophage libraries are then screened mainly by hybridizing filters containing denatured bacterial colonies (or a lawn containing phage plaques) with labeled probes (see Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratories, Cold Spring Harbor , NY, 1989). Hybridized colonies or plaques are selected and expanded and DNA is isolated for further analysis. cDNA clones can be analyzed to determine the number of additional sequences using, for example, PCR, using a primer from an incomplete sequence and a primer from a vector. There may be restriction charts and incomplete sequences obtained to determine one or more overlapping clones. The complete sequence can be determined using standard methods, which may include the generation of deletion clones. The resulting overlapping sequences can then be assembled into a single continuous sequence. A full-sized cDNA molecule can be obtained by ligation of suitable fragments using well-known methods.

Alternatively, amplification methods, such as those described above, may be useful for obtaining a full-length coding sequence from incomplete cDNA sequences. One such amplification method is inverted PCR (see Triglia et al., Nucl. Acids Res. 16: 8186, 1988), which uses restriction enzymes to produce a fragment in a known region of the gene. Then the fragment is cyclized by intramolecular ligation and used as a matrix for PCR with various primers obtained from a known region. Within an alternative approach, sequences adjacent to an incomplete sequence can be restored by amplification with a primer to a linker sequence and a primer specific to a known region. Amplified sequences are usually used in the second round of amplification with the same linker primer and a second primer specific to a known region. Variants of this procedure that use two primers from a known sequence that initiate expansion in opposite directions are described in WO 96/38591. Another such method is known as "rapid amplification of cDNA ends" or RACE. This method for identifying 5 'or 3' sequences of a known sequence involves the use of internal and external primers that hybridize to the polyA region or to the vector sequence. Additional approaches include breathtaking 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 approaches using amplification can also be used to obtain the full length cDNA sequence.

In certain cases, a full-length cDNA sequence can be obtained by analysis of the sequences provided in the database of expressed marker sequences (EST) such as are available in GenBank. Searches for overlapping ESTs can generally be performed using well-known programs (e.g., NCBI BLAST searches), and such ESTs can be used to generate a continuous full-length sequence. Full-length DNA sequences can be obtained by analysis of genomic fragments.

In other embodiments of the invention, polynucleotide sequences or fragments thereof that encode the polypeptides of the invention, or fusion proteins or their functional equivalents, can be used in recombinant DNA molecules to direct expression of the polypeptides in the respective host cells. Due to the degeneracy inherent in the genetic code, other DNA sequences encoding essentially the same or functionally equivalent amino acid sequences can be obtained, and these sequences can be used to clone and express these polypeptides.

As experts in this field understand, in some cases it may be useful to produce nucleotide sequences encoding polypeptides containing codons not found in natural conditions. For example, codons that are preferred for particular prokaryotic or eukaryotic hosts can be selected to increase the level of protein expression or to obtain a recombinant RNA transcript possessing the necessary properties, such as a longer half-life than the transcript that forms from a naturally occurring sequence.

Moreover, polynucleotide sequences of the present invention can be constructed using methods well known in the art in order to alter, but not limited to, modifications to the coding polypeptides that modify the cloning, processing and / or expression of the gene product. For example, DNA “shuffled” (mixed) by random fragmentation and again assembled using PCR gene fragments with synthetic oligonucleotides can be used to construct nucleotide sequences. In addition, site-specific mutagenesis can be used to insert new restriction sites, change glycosylation patterns, change codon preferences, obtain splicing or mutation options, and so on.

In another embodiment of the invention, natural, modified or recombinant nucleic acid sequences may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening polypeptide activity inhibitors in peptide libraries, it may be convenient to encode a chimeric protein that can be recognized by a commercially available antibody. A fusion protein can be designed so that it contains a cleavage site located between the sequence encoding the polypeptide and the heterologous protein sequence so that the polypeptide can be cleaved and purified from the heterologous part.

Sequences encoding the desired polypeptide can be synthesized in whole or in part using chemical methods well known in the art (see Caruthers, MH 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 can be prepared using chemical methods for synthesizing the amino acid sequence of a polypeptide or part thereof. For example, peptide synthesis can be carried out using various solid-state methods (Roberge, JY et al. (1995) Science 269: 202-204) and where automatic synthesis can be achieved, for example, using an ABI 431A peptide synthesizer (Perkin Elmer, Palo Alto , CA).

The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g. Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, NY) or other comparable method available in this area. The composition of the synthetic peptide can be confirmed by amino acid analysis or sequencing (e.g., Edman degradation reaction). Additionally, the amino acid sequence of the polypeptide or any part thereof during direct synthesis can be changed and / or, using chemical methods, combined with the sequence of another peptide or part thereof, to obtain variants of the polypeptide.

To obtain the desired polypeptide, nucleotide sequences encoding the polypeptide or functional equivalents can be inserted into the corresponding expression vector, that is, a vector containing the necessary elements for transcription and translation of the inserted coding sequences. Methods well known to those skilled in the art can be used to construct expression vectors containing sequences encoding polypeptides of interest and suitable transcriptional and translational control elements. These methods include in vitro recombinant DNA methods, synthesis methods, and in vivo genetic recombination. Such methods are described, for example, 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.

A variety of expression vector / host systems can be used to retain and express polynucleotide sequences. These include microorganisms, such as bacteria, transformed with recombinant bacteriophage, plasmid or cosmid expressing DNA vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (for example, plasmid Ti or pBR322); animal cell systems, but are not limited to.

The “control elements” or “regulatory sequences” that exist in expression vectors are untranslated regions of the vector — enhancers, promoters of 5′- and 3′-untranslated regions — that interact with host cell proteins to transcribe and translate. Such elements can change their strength and specificity. Depending on the vector and host systems used, several suitable transcriptional and translational elements may be used, including constitutive and inducible promoters. For example, for cloning in bacterial systems, inducible promoters such as the lacZ hybrid promoter of the PBLUESCRIPT phasmid (Stratagene, La Jolla, Calif.) Or the plasmid PSPORT1 (Gibco BRL, Gaithersburg, MD) or the like can be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. If necessary, to create a cell line containing multiple copies of a sequence encoding a polypeptide, it is preferable to use vectors based on SV40 or EBV with a suitable selective marker.

In bacterial systems, any of a variety of expression vectors can be selected, depending on the intended use of the expressed polypeptide. For example, when large quantities are needed, for example, to induce antibodies, vectors can be used that direct a high level of expression of fusion proteins that are easily purified. Such vectors include E. coli multifunctional cloning and expression vectors, such as BLUESCRIPT (Stratagene), in which a sequence encoding a polypeptide of interest can be ligated into a vector in a frame with sequences for the amino terminal Met and the subsequent 7 beta-galactosidase residues so that a hybrid is produced protein; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem; 264: 5503-5509) and the like, but are not limited to. PGEX vectors (Promega, Madison, Wis.) Can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be isolated from lysed cells by adsorption to glutathione-agarose granules after elution in the presence of free glutathione. Proteins obtained in such systems can be designed to include heparin, thrombin, or factor XA protease cleavage sites so that cloned polypeptides of interest, if necessary, can be freed from the GST portion.

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

In cases where plant expression vectors are used, expression of sequences encoding polypeptides may be from any of a variety of promoters. For example, viral promoters, such as 35S and 19S CaMV promoters, can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6: 307-311). Alternatively, plant promoters, such as the RUBISCO small subunit promoters or heat shock promoters (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). Such constructs can be introduced into plant cells using direct DNA transformation or pathogen-mediated transfection. Such methods are described in several widely available reviews (see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N. Y .; pp. 191-196).

Insect systems can also be used to express the polypeptides of interest. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for the expression of foreign genes in Spodoptera frugiperda cells or Trichoplusia larvae. Sequences encoding the polypeptide can be cloned into non-essential regions of the virus, such as the polyhedrosis gene and placed under the control of the polyhedrin promoter. Successful insertion of a sequence encoding a polypeptide leads to inactivation of the polyhedrosis gene and production of a recombinant virus lacking a membrane protein. Recombinant viruses can then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which polypeptides of interest can be expressed (Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. 91: 3224-3227).

For mammalian host cells, several virus-based expression systems are widely available. For example, in the case where an adenovirus is used as the expression vector, the sequences encoding the polypeptide of interest can be ligated into an adenovirus transcription / translation complex containing a late promoter and a triple leading sequence. To obtain viable viruses that will be capable of expressing polypeptides in host cells, insertion into the non-essential region of the E1 or E3 viral genome can be used (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81: 3655-3659). In addition, transcriptional enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to enhance expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficient translation of sequences encoding the polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where the sequences encoding the polypeptide, their initiation codon and the sequences located upstream of the transcription are inserted into a suitable expression vector, no additional transcription or translation control signals are needed. However, in cases where only a coding sequence or part thereof is inserted, exogenous translation control signals including the ATG initiator codon must be provided. Moreover, the initiator codon must be in the correct reading frame to ensure translation of the whole insert. Exogenous translational elements and initiator codons can be of various origins: both natural and synthetic. Expression efficiency can be enhanced by the inclusion of enhancers that are suitable for the particular cell system that is used, such as described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125 -162).

In addition, host cell strains can be selected for their ability to modulate the expression of the inserted sequence or process the expressed protein in the desired manner. Such modifications of the polypeptide include, but are not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipoylation and acylation. Post-translational processing, splitting the "prepro" forms of proteins, can also be used to facilitate the correction of insertion, folding and / or function. You can select various host cells, such as CHO, COS, HeLa, MDCK, HEK293 and WI38, which have a specific cell apparatus and characteristic mechanisms of post-translational activity, to ensure the correct modification and processing of a foreign protein.

For long-term, highly productive production of recombinant proteins, stable expression is generally preferred. For example, cell lines that stably express polynucleotides of interest can be transformed using expression vectors that may contain viral origin replication elements and / or endogenous expression elements and a selective marker gene on the same or on a separate vector. After the introduction of the vector, it is possible to allow the cells to grow for 1-2 days in an enriched medium before switching to a selective medium. The purpose of the selective marker is to provide resistance to selection and its presence makes it possible to grow and obtain cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be proliferated using a tissue culture method according to cell type.

Some of the selection systems can be used to obtain transformed cell lines. These include herpes simplex virus thymidine kinase genes (Wigler, M. et al. (1977) Cell 11: 223-32) and adenine phosphoribosyl transferases (Lowy, I. et al. (1990) Cell 22: 817-23) that can be used at tk.sup.- or aprt. sup.-cells, respectively, but not limited to. Also, as a basis for selection, resistance to antimetabolites, antibiotics or herbicides can be used; for example, dhfr, which gives resistance to metrotrexate (Wigler, M. et al. (1980) Poc. Natl. Acad. Sci. 77: 3567-70); npt, which gives resistance to aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150: 1-14) and als or pat, which give resistance to chlorosulfuron and phosphoinotricin acetyltransferase, respectively (Murry, supra). Additional selective genes are described, for example trpB, which allows cells to utilize indole instead of tryptophan or hisD, which allows cells to utilize histinol instead of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85: 8047-51). The use of visible markers with markers such as anthocyanin, beta-glucuronidase and its substrate GUS, luciferase and its substrate luciferin, which are widely used not only to identify transformants, but also to calculate the amount of short-term or stable expression of proteins related to a specific one, has gained popularity. vector system (Rhodes, CA et al. (1995) Methods Mol, Biol. 55: 121-131).

Although the presence / absence of expression of marker genes means that the gene of interest is also represented, its presence and expression may need confirmation. For example, if a sequence encoding a polypeptide is inserted into a marker gene sequence, recombinant cells containing sequences can be identified by the lack of marker gene function. Alternatively, marker genes may be located in tandem with a sequence encoding a polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually also means expression of the tandem gene.

Alternatively, host cells that contain and express the desired polynucleotide sequence can be identified by a variety of methods known to those skilled in the art. These methods include DNA-DNA or DNA-RNA hybridization and methods of protein bioassay or immunoassay, which include but are not limited to, for example, methods based on the use of membranes, solutions or chips for the detection and / or counting of nucleic acids or proteins.

A variety of protocols are known in the art using any polyclonal or monoclonal antibody specific for a product to detect and measure the expression of products encoded by polynucleotides. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescence-activated cell sorting (FACS). For some applications, a two-site immunological assay based on the use of monoclonal antibodies using monoclonal antibodies that react with two non-overlapping epitopes of a given polypeptide may be preferred, but a competitive binding assay can also be used. These and other assays are described, among other items, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.) And Maddox, D. E. et al. (l983, J. Exp. Med. 158: 1211-1216).

A wide variety of labeling and conjugation methods known to those skilled in the art can be used in various analyzes of amino acids or nucleic acids. Methods for producing labeled probes or PCR primers for detecting sequences associated with polynucleotides include oligonucleotide labeling, nick translation, terminal labeling or PCR amplification using labeled nucleotides. Alternatively, sequences or any part thereof can be cloned into a vector to obtain an mRNA probe. Such vectors, well known in the art, are commercially available and can be used to synthesize RNA probes in vivo by adding an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. Such procedures can be carried out using a variety of commercially available kits. Suitable reporter molecules or labels that can be used include radionuclides, enzymes, fluorescent, chemiluminescent or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles and the like.

Host cells transformed with the polynucleotide sequences of interest can be cultured under conditions suitable for expression and production of protein from cell culture. The protein produced by the recombinant cell may be secreted or intracellularly, depending on the sequence and / or vector used. As those skilled in the art will understand, expression vectors containing polynucleotides of the invention can be designed to contain signal sequences that direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane. Other recombinant constructs can be used to bind sequences encoding the polypeptides of interest to a nucleotide sequence encoding a polypeptide domain that facilitates the purification of soluble proteins. Such purification facilitating 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 the domain used in but not limited to FLAGS exclusion / affinity purification systems by them. The inclusion of cleavable linker sequences specific for factor XA or enterokinase (Invitrogen. San-Diego, Calif.) Between the purification domain and the encoded polypeptide can be used to facilitate purification. One of these expression vectors involves the expression of a fusion protein containing the polypeptide of interest and a nucleic acid encoding 6 histidine residues preceding the thioredoxin or enterokinase cleavage site. Histidine residues facilitate purification using IMIAC (metal affinity chromatography) as described by Porath, J. et al. (1992, Prot. Exp. Purif. 3: 263-281), while the enterokinase cleavage site provides methods for purifying the desired polypeptide from a fusion protein. A detailed discussion of vectors containing fusion proteins is provided by Kroll, D. J. et al. (1993; DNA Cell Biol. 12: 441-453).

In addition to methods for producing recombinants, the polypeptides of the invention or fragments thereof can be prepared by direct peptide synthesis using solid phase synthesis methods (Merrifield J. (1963) J. Am. Chem. Soc. 85: 2149-2154). Protein synthesis can be performed using manuals or automatic methods. Automatic synthesis can be performed, for example, using an Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Alternatively, various fragments can be synthesized separately and combined using chemical methods to synthesize a full-sized molecule.

Compositions of antibodies, fragments thereof and other binding agents

In another aspect, the present invention further relates to binding agents, such as antibodies or antigen binding fragments thereof, which are characterized by immunological binding to the tumor proteins described herein, parts, variants or derivatives thereof. An antibody or antigen-binding fragment thereof is said to “specifically bind” or “immunologically bind” and / or “immunologically reactive” to a polypeptide of the invention if it reacts at a detectable level (for example, by ELSIA analysis) with the polypeptide and does not detectably interact with unrelated polypeptides under the same conditions.

Immunological binding when used in this context usually refers to non-covalent interactions of the type that occurs between the immunoglobulin molecule and the antigen to which the immunoglobulin is specific. The strength or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K _d ) of the interaction, where a lower K _d represents greater affinity. The immunological binding properties of selected polypeptides can be calculated using methods well known in the art. One of these methods is associated with measuring the rates of formation and dissociation of antigen-binding site / antigen complexes, where these rates depend on the concentrations of the partners forming the complex, the affinity of interaction, and geometric parameters that equally affect the speed in both directions. So, both the “forward speed constant” (K _on ) and the “forward speed constant” (K _off ) can be determined by counting the concentration and current association and dissociation rates. The K _on / K _off ratio makes it possible to reduce all parameters not related to affinity, and is equivalent to the dissociation constant K _d . Mostly see Davies. et. al. (1990) Annual Rev. Biochem.

59: 439-473.

An “antigen binding site” or “binding part” of an antibody refers to parts of an immunoglobulin molecule that is involved in antigen binding. The antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three very different regions in the V regions of the heavy and light chains are referred to as “hypervariable regions” which are located between the more conservative marginal regions, known as “structural regions” or “FR”. Thus, the term "FR" refers to amino acid sequences that are found in immunoglobulins in nature between and near hypervariable regions. In an antibody molecule, three hypervariable regions of the light chain and three hypervariable regions of the heavy chain are located relative to each other in three-dimensional space so as to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the antigen to be bound, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity determining regions” or “CDRs”.

Binding agents may contribute to the ability to distinguish between patients with a malignant tumor, such as lung cancer, and without it, using the representative assays described herein. For example, antibodies or other binding agents that bind to a tumor protein preferably generate a signal indicating the presence of a malignant tumor in at least about 20% of patients with the disease, more preferably in 30% of patients. Alternatively or in addition, the antibody will generate a signal indicating the absence of disease in at least 90% of individuals without cancer. To determine whether a binding agent satisfies this condition, biological samples (e.g. blood, serum, sputum, urine and / or tumor biopsies) of patients with and without cancer (as determined using standard clinical tests) can be analyzed as described above for the presence of polypeptides that bind to binding agents. It is preferable to analyze a statistically significant number of samples with and without disease. Each binding agent must satisfy the criteria indicated above, however, it is clear to those skilled in the art that these binding agents can be used in combination to increase sensitivity.

Any agent that satisfies the above conditions may be a binding agent. For example, the binding agent may be a ribozyme, with or without a peptide component, an RNA molecule, or a polypeptide. In a preferred embodiment, the binding agent is an antibody or antigen binding fragment thereof. Antibodies can be obtained using any of a variety of methods known to specialists in this field. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced using cell culture methods, including the production of monoclonal antibodies, as described here, or by transfection of antibody genes in suitable bacterial or mammalian host cells in order to enable the production of recombinant antibodies. In one method, an immunogen containing a polypeptide is first introduced into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the polypeptides of the invention can serve as an immunogen without modification. Alternatively, especially for relatively short polypeptides, a primary immune response may be elicited if the polypeptide is bound to a carrier protein, such as bovine serum albumin or hemocyanin of a marine saucer. The immunogen is administered to the host animal, preferably according to a specific protocol, including one or more support immunizations with periodic blood sampling from the animal. Polypeptide-specific polyclonal antibodies can then be purified from such antiserum using, for example, affinity chromatography, using polypeptides bound to a suitable solid base.

Monoclonal antibodies specific for the antigenic peptide of interest can be obtained, for example, using the method of Kohler and Milstein, Eur. J. Immunol. 6: 511-519, 1976 and its improvements. Briefly, these methods include obtaining immortal cell lines capable of producing antibodies having the necessary specificity (for example, reacting with the polypeptide of interest). Such cell lines can be prepared, for example, from spleen cells obtained from animals immunized as described above. The spleen cells are then immortalized, for example, by fusion with a fusion partner from myeloma cells, preferably syngeneic with the immunized animal. Many merging methods can be applied. For example, spleen cells and myeloma cells can be combined for several minutes with a non-ionic detergent and then placed at a low density on a selective medium that supports the growth of hybrid but not myeloma cells. A preferred selection method utilizes HAT selection (hypoxanthine, aminopterin, thymidine). After sufficient time, usually about 1-2 weeks, hybrid colonies are observed. Single colonies are selected and the supernatant of their cultures is tested for binding activity against the polypeptide. Hybridomas with high reactivity and specificity are preferred.

Monoclonal antibodies can be isolated from supernatants of growing hybridoma colonies. In addition, various methods can be used to increase yield, such as introducing a hybridoma cell line into the abdominal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies can then be collected from ascites fluid or blood. Contaminants can be removed from antibodies by conventional methods, such as chromatography, gel filtration, precipitation and extraction. The polypeptides of the invention can be used in a purification process, for example, in an affinity chromatography step.

A number of molecules are known in the art that are useful in the treatment of molecules, including antigen binding sites that are capable of exhibiting immunological binding properties of an antibody molecule. The papain proteolytic enzyme preferably cleaves IgG molecules into several fragments, two of which ("F (ab)" fragments) contain a covalent heterodimer including an intact antigen-binding site. The pepsin enzyme is capable of cleaving IgG molecules to produce several fragments, including "F (ab ') ₂ " fragments that contain both antigen-binding sites. The "Fv" fragment can be obtained by the preferred proteolytic cleavage of the immunoglobulin IgM molecule and, in rare cases, IgG or IgA. However, Fv fragments are most often obtained using recombinant methods known in the art. The Fv fragment includes a non-covalently bound V _H :: V _L dimer including an antigen binding site that retains most of the antigen recognition and binding capabilities of a natural antibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69: 2659-2662; Hochman rt al. (1976) Biochem 15: 2706-2710 and Ehrlich et al. (1980) Biochem 19: 4091-4096.

A single chain Fv polypeptide ("sFv") is a covalently linked V _H :: V _L heterodimer that is expressed from a fusion gene including genes encoding V _H - and V _L - linked by a peptide-linking encoder. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85 (16): 5879-5883. A number of methods have been described for choosing chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains from the V region of an antibody into an sFv molecule, which will form a three-dimensional structure that is basically similar to the structure of the antigen-binding site. See, for example, US Pat. Nos. 5,091,513 and 5,132,405 issued to Huston et al .; and U.S. Patent No. 4,946,778 to Landner et al.

Each of the molecules described above includes a set of heavy chain and light chain CDRs, respectively located between the heavy chain and light chain FR set, which provide CDRS support and define spatial relationships of CDRs relative to each other. As used herein, the term “CDR kit” refers to three hypervariable regions of the heavy and light chain V regions. Starting from the N-terminus of the heavy or light chain, these regions are designated “CDR1”, “CDR2” and “CDR3”, respectively. Therefore, the antigen binding site containing a set of CDRs of each of the heavy and light chains of the V region includes six CDRs. A polypeptide containing a single CDR (eg, CDR1, CDR2, or CDR3) refers to a “molecular recognition unit”. Crystallographic analysis of a number of antigen-antibody complexes showed that the amino acid residues of the CDRs form a spatial contact with the associated antigen, where the largest antigen contact occurs with heavy chain CDR3. Thus, molecular recognition units are primarily responsible for the specificity of the V antigen-binding site.

As used herein, the term “FR kit” refers to four side amino acid sequences that frame the CDRs of the V-region heavy and light chain CDRs. Some FR residues may be in contact with the associated antigen, however, FR is mainly responsible for coagulation of the V region at the antigen binding site, in particular FR residues directly adjacent to the CDRS. Within the FR, some amino acid residues and some structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V regions collapse into a binding site, CDRs are exposed as protruding loop motifs that form an antigen-binding surface. In general, it is clear that there are conserved structural regions of FR that influence folding of the CDR loops into certain "canonical" structures, regardless of the exact amino acid sequence of the CDR. Moreover, it is known that some FR residues are involved in non-covalent cross-domain contacts that stabilize the interaction of the antibody heavy and light chains.

A number of molecules of “humanized” antibodies have been described that contain an antigen-binding site derived from non-human immunoglobulin, including chimeric antibodies containing rodent V regions and associated CDRs fused to human constant domains (Winter et al. (1991) Nature 349: 293- 299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA 86: 4220-4224; Shaw et al. (1987) J Immunol. 138: 4534-4538; and Brown et al. (1987). Cancer Res. 47: 3577-3583), rodent CDR grafted into supporting human FRs before fusion with the constant domain of the corresponding human antibody (Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (19 88) Science 239: 1534-1536; and Jones et al. (1986) Nature 321: 522-525) and rodent CDRs supported by recombinantly protected rodent FRs (European Patent Publication No. 519596, published December 23, 1992). These “humanized” molecules are designed to minimize the unwanted immune response to rodent anti-human antibody molecules, which limits the duration and effectiveness of therapeutic applications of such components in human recipients.

As used herein, the terms “protected FR” and “recombinantly protected FR” refer to selectively replacing FR residues, for example, rodent heavy and light chain V regions, with human FR residues, to preserve a xenogenetic molecule containing an antigen binding site that generally retains all the natural folding structure of the FR polypeptide. Protection methods are based on the understanding that the ligand binding characteristics of the antigen binding site are mainly determined by the structure and relative position of the heavy and light chain CDR sets within the antigen binding surface. Davies et al. (1990) Ann. Rev. Biochem. 59: 439-473. So, in a humanized antibody, the specificity of antigen binding can only be preserved where the CDR structures, their interaction with each other, and their interaction with the base of the V-domain domains are carefully preserved. By using protection methods, external (e.g., solvent accessible) FR residues that are readily recognized by the immune system are selectively replaced by human-specific residues to produce a hybrid molecule that contains either a weakly immunogenic or substantially non-immunogenic protected surface.

The protection procedure uses available sequence data for the variable domains of human antibodies collected by Kabat et al., In Sequences of Proteins of Immunological Interest, 4th ed., (US Dept. of Health and Human Services, US Government Printing Office, 1987), updates Kabat databases and other available US and foreign databases (both nucleic acid and protein). Solvent availability of V-region amino acids can be inferred from the known three-dimensional structure of fragments of human and mouse antibodies. There are two main steps in protecting a mouse antigen binding site. First, the FR variable domains of the antibody molecule of interest are compared with the corresponding FR sequence of human variable domains obtained from the sources identified above. The V regions with the highest homology are then compared residue by residue with the corresponding murine amino acids. Residues in murine FRs that differ from the human copy are replaced by residues present in the human part using recombinant methods known in the art. Residues are replaced in groups that are at least partially open (accessible to the solvent), and caution is required when replacing amino acid residues that can have a significant effect on the tertiary structure of V-domain domains, such as proline, glycine, and charged amino acids.

Thus, the resulting “protected” mouse antigen-binding sites are designed to retain the remains of murine CDRs, residues mainly adjacent to CDRs, residues identified as closed or mostly closed (inaccessible to the solvent), residues presumably involved in non-covalent ( for example, electrostatic or hydrophobic) contacts between the domains of the heavy and light chains, and the remnants of the conservative structural regions of the FR, which are supposed to affect the “canonical” tertiary CDR loop structures. These design criteria are used to obtain recombinant nucleotide sequences that combine the heavy and light chain CDRs of the mouse antigen binding site with human FRs and which can be used to transfect mammalian cells to express recombinant human antibodies that detect the antigenic specificity of the mouse antibody molecule.

In another embodiment of the present invention, monoclonal antibodies can be coupled to one or more therapeutic agents. Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins and their derivatives. Preferred radionuclides include ⁹⁰ Y, ¹²³ I, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹¹ At and ²¹² Bi. Preferred drugs include methotrexate and an analog of pyrimidine and purine. Preferred differentiation inducers include phorbol ester and butyric acid. Preferred toxins include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin and phytolakia antiviral protein.

Therapeutic agents may be attached (e.g., covalently linked) to a suitable monoclonal antibody, either directly or indirectly (e.g., via a linker group). A direct reaction between the agent and the antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a well-leaving group (e.g., a halide) on the other.

Alternatively, it may be necessary to attach the therapeutic agent to the antibody via a linker group. The linker group can function as a separator to distance the antibody from the agent in order to avoid interference with binding abilities. The linker group may also serve to enhance the chemical activity of the substituent on the agent or antibody and, thus, enhance the binding efficiency. An increase in chemical activity may also promote the use of agents or functional groups on agents, which is otherwise not possible.

It will be apparent to those skilled in the art that a variety of bifunctional and multifunctional reagents, or homo- or heterofunctional reagents (such as those described in the catalog of Pierce Chemical Co., Rockford, IL) can be used as linker groups. The compound can be carried out, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues. There are numerous references to where such methods are described, for example, US Pat. No. 4,671,958 to Rodwell et al.

In cases where the therapeutic agent is more effective if it is freed from the portion of the immunoconjugate of the present invention containing the antibody, it may be necessary to use a linker group that cleaves during internalization into the cell. A number of different fissile linker groups are described. Mechanisms for intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., US Pat. No. 4,489,710 issued to Spitler), irradiation of a photosensitive bond (e.g., US Pat. No. 4,625,014, issued by Senter et al.), Hydrolysis of derivatized amino acid side chains (e.g. US Pat. No. 4,638,045 to Kohn et al.), serum complement-mediated hydrolysis (e.g., US Pat. No. 4,671,958 to Rodwell et al.) and acid hydrolysis (e.g., US Pat. No. 4,569,789 to Blattler et al.).

It may be necessary to attach more than one agent to the antibody. In one embodiment, multiple agent molecules are attached to a single antibody molecule. In another embodiment, more than one type of agent may be coupled to one antibody. Regardless of the specific implementation, immunoconjugates with more than one agent can be obtained in many ways. For example, more than one agent can be attached directly to the antibody molecule, or linkers that provide multiple sites for attachment can be used. Alternatively, carriers may be used.

The carrier can carry agents in a variety of ways, including covalently linking each directly or via a linker group. Useful carriers include proteins such as albumin (e.g., US Pat. No. 4,507,234 to Kato et al.), Peptides and polysaccharides such as aminodextran (e.g., US Pat. No. 4,699,784 to Shih et al.). The carrier may also carry the agent by non-covalent binding or by encapsulation, as in a liposome vesicle (for example, US patent No. 4429008 and 4873088). Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds. For example, US Patent No. 4,735,792 describes typical radiohalogenated small molecules and their synthesis. A radionuclide chelate can be formed from chelating compounds that include a radionuclide that contains nitrogen and sulfur atoms as donor atoms to bind a metal or metal oxide. For example, US Patent No. 4,673,562 to Davison et al. Describes typical chelating compounds and their synthesis.

T Cell Compositions

The present invention, in another aspect, relates to T cells specific for the tumor polypeptide described herein, or a variant or derivative thereof. Such cells can usually be obtained in vitro or ex vivo using standard procedures. For example, T cells can be isolated from bone marrow, peripheral blood, or part of a patient’s bone marrow or peripheral blood using commercially available cell separation systems, such as the Isolex ^TM System, available from Nexell Therapeutics, Inc. (Irvine, CA; see also US patent No. 5240856; US patent No. 5215926; WO 89/06280; WO 91/16116 and WO 92/07243). Alternatively, T cells can be obtained from relatives or unrelated people, other mammals, cell lines or cultures.

T cells can be stimulated with a polypeptide, a polynucleotide encoding a polypeptide and / or an antigen presenting cell (APC) that expresses such a polypeptide. Such stimulation is carried out under conditions and such a time as to allow the generation of T cells that are specific for the polypeptide of interest. Preferably, the tumor polypeptide or polynucleotide of the invention is located in a delivery vehicle, such as a microsphere, to facilitate generation of specific T cells.

T cells are believed to be specific for the polypeptide of the present invention if the T cells specifically proliferate, secrete cytokines, or kill target cells coated with the polypeptide or expressing genes encoding the polypeptide. T cell specificity can be evaluated by any of a variety of standard methods. For example, in a chromium loss analysis or proliferation analysis, more than double the stimulation index during lysis and / or proliferation compared to negative controls, means T-cell specificity. Such assays can be carried out, for example, as described by Chen et al., Cancer Res. 54: 1065-1070, 1994. Alternatively, detection of T cell proliferation can be carried out by a variety of known methods. For example, T-cell proliferation can be detected by measuring an increased rate of DNA synthesis (for example, by pulse-labeling T-cell cultures with thymidine with tritium and measuring the amount of thymidine with tritium incorporated in DNA). Contact with a tumor polypeptide (100 ng / ml -100 μg / ml, preferably 200 ng / ml -25 μg / ml) for 3-7 days usually results in at least a twofold increase in T-cell proliferation. Such as described above, contact within 2-3 hours led to the activation of T cells, as measured using standard cytokine analysis, in which a twofold increase in the level of release of cytokines (e.g., TNF or IFN-γ) means the activation of T cells ( see Coligan et al., Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998)). T cells that have been activated in response to a tumor polypeptide, polynucleotide, or APC expressing polypeptide may be CD4 ⁺ and / or CD8 ⁺ . The number of T cells specific for the tumor polypeptide can be increased using standard methods. In a preferred embodiment, T cells are obtained from a patient and a related or unrelated donor and are administered to the patient after stimulation and increase in amount.

For therapeutic purposes, the number of CD4 ⁺ or CD8 ⁺ T cells that proliferate in response to a tumor polypeptide, polynucleotide, or APC can be increased either in vitro or in vivo. In vivo proliferation of such T cells can be accomplished in various ways. For example, T cells can be re-exposed to a tumor polypeptide or short peptide corresponding to the immunogenic portion of such a polypeptide, with or without the addition of T-cell growth factors, such as interleukin-2 and / or stimulatory cells that synthesize the tumor polypeptide. Alternatively, the number of one or more T cells that proliferate in the presence of a tumor polypeptide can be increased by cloning. Cell cloning methods are well known in the art and include limiting dilutions.

Pharmaceutical Compositions

In further embodiments, the present invention provides a preparation of one or more of the compositions of polynucleotides, polypeptides, T cells and / or antibodies described herein in pharmaceutically acceptable carriers for administration to a cell or animal, either alone or in combination with one or more other therapies.

Obviously, if required, the composition described herein can also be administered in combination with other agents, for example, with other proteins or polypeptides, or various pharmaceutically active agents. In fact, the number of other components that can also be included is, in essence, not limited, while additional agents will not cause significant adverse effects in contact with target cells or host tissues. Thus, the composition can be delivered with various other means, as required in a particular case. Such compositions may be purified from host cells or other biological sources, or, alternatively, may be chemically synthesized as described herein. Similarly, such compositions may further include substituted or modified RNA or DNA compositions.

Therefore, in another aspect of the present invention, pharmaceutical compositions are described comprising one or more of the compositions of polynucleotides, polypeptides, antibodies and / or T cells described herein in combination with a physiologically acceptable carrier. In some preferred embodiments, the pharmaceutical compositions of the invention include compositions of immunogenic polynucleotides and / or polypeptides of the invention for use in the use of prophylactic and therapeutic vaccines. Vaccine formulations are generally described, for example, by M. F. Powell and M. J. Newman, eds., "Vaccine Design (the subunit and adjuvant approach)," Ple - Press (NY, 1995). Typically, such compositions include one or more polynucleotide and / or polypeptide compositions of the present invention in combination with one or more immunostimulants.

Obviously, any of the pharmaceutical compositions described herein may contain pharmaceutically acceptable salts of the polynucleotides and polypeptides of the invention. Such salts can be obtained, for example, from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts) .

In another embodiment, exemplary immunogenic compositions of the present invention, for example, vaccine compositions, include DNA encoding one or more of the polypeptides described above, such that the polypeptide is generated in situ. As noted above, the polynucleotide can be introduced as part of any of the various delivery systems known in the usual practice of this field. Indeed, many gene delivery methods are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15: 143-198, 1998, and in the references cited here. Suitable polynucleotide expression systems, of course, contain the necessary regulatory DNA sequences for expression in the patient's body (such as suitable promoters and termination signals). Alternatively, bacterial delivery systems may include the introduction of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses or secrets the immunogenic portion of the polypeptide on its cell surface.

Therefore, in some embodiments, polynucleotides encoding the immunogenic polypeptides described herein are introduced into suitable mammalian host cells for expression using any of a number of virus-based systems. In one illustrative implementation, retroviruses provide a convenient and efficient platform for gene delivery systems. The selected nucleotide sequence encoding the polypeptide of the present invention can be introduced into a vector and packaged into retroviral particles using methods known in the art. Then, the recombinant virus can be isolated and delivered to the subject. A number of illustrative retroviral systems have been described (e.g., US Pat. No. 5,217,940; Miller and Rosman (1989) BioTechniques 7: 980-990; Miller, AD (1990) Human Gene Therapy 1: 5-14; Scarpa et al. (1991) Virology 180: 849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90: 8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3: 102-109 .

In addition, a number of illustrative adenovirus-based systems have been described. Unlike retroviruses that integrate into the host genome, adenoviruses persist outside the chromosome, thereby minimizing the risk of insertional mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57: 267-274; Bett et al. (1993) J. Virol. 67: 5911-5921; Mittereder et al. (1994) Human Gene Therapy 5: 717-729; Seth et al. (1994) J. Virol. 68: 933-940; Barr et al. (1994) Gene Therapy 1: 51-58; Berkner, KL (1988) BioTechniques 6: 616-629; and Rich et al. (1993) Human Gene Therapy 4: 461-476).

Also, various adeno-associated viral (AAV) vector systems have been developed for the delivery of polynucleotides. AAV vectors can be easily constructed using methods well known in the art. See, for example, US patents No. 5173414 and 5139941; International Publication No. WO 92/01070 and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8: 3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3: 533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158: 97-129; Kotin, R. M. (1994) Human Gene Therapy 5: 793-801; Shelling and Smith (1994) Gene Therapy 1: 165-169; and Zhou et al. (1994) J. Exp. Med. 179: 1867-1875.

Additional viral vectors that can be used to deliver polynucleotides encoding the polypeptides of the present invention by gene transfer include those from the family of poxviruses, such as vaccinia and avian poxvirus. For example, vaccinia virus recombinants expressing new molecules can be constructed as follows. DNA encoding the polypeptide must first be inserted into a suitable vector so that it is adjacent to the vaccinia virus promoter and flanking sequences of vaccinia virus DNA, such as a sequence encoding thymidine kinase (TK). This vector is then used to transfect cells that simultaneously infect cowpox. Homologous recombination is used to insert the vaccinia virus promoter together with the gene encoding the polypeptide of interest into the viral genome. The resulting recombinant TK.sup. (-) can be selected by culturing cells in the presence of 5-bromodeoxyuridine and capturing viral plaques that are resistant to it.

The vaccinia-virus-based infection / transfection system can conveniently be used to provide inducible transient expression or co-expression of one or more of the polypeptides described herein in host cells. In this particular system, cells are first infected in vitro with a vaccinia virus recombinant that encodes a T7 bacteriophage RNA polymerase. This polymerase shows exceptional specificity in that it only transcribes matrices carrying T7 promoters. After infection, the cells are transfected with the polynucleotide of interest or polynucleotides followed by the T7 promoter. A polymerase expressed in the cytoplasm from a recombinant vaccinia virus virus transcribes transfected DNA into RNA, which is then translated into the polypeptide via the host translation system. The method provides temporary cytoplasmic production in a high concentration of large quantities of RNA and its translation products. See, for example, Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87: 6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83: 8122-8126.

Alternatively, bird poxviruses, such as poultry and canary poxviruses, can also be used to deliver coding sequences of interest. Recombinant avian poxviruses expressing immunogens from mammalian pathogens are known to provide protective immunity when administered to non-avian species. The use of the Avipox vector in humans and other mammalian species is especially desirable, since members of the Avipox genus can be productively replicated only in bird species sensitive to them and therefore are not infectious with respect to mammalian cells. Methods for producing recombinant avian poxviruses are known in the art and involve genetic recombination as described above with respect to the production of vaccinia viruses. See, for example, WO 91/12882; WO 89/03429; and WO 92/03545.

Any of a number of alphavirus vectors can also be used to deliver polynucleotide compositions of the present invention, such as those described in US Pat. Nos. 5,843,723; 6015686; 6008035 and 6015694. Some vectors based on the Venezuelan horse encephalitis virus (VEE) virus can also be used, and illustrative examples thereof can be found in US Pat. Nos. 5,505,947 and 5,643,576.

Moreover, molecular conjugate vectors, such as chimeric adenoviral vectors described by Michael et al., J. Biol, can also be used to deliver the genes of the invention. Chem. (1993) 268: 6866-6869; and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89: 6099-6103.

Additional illustrative information on known virus-based delivery systems can be found, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86: 317-321, 1989; Flexner et al., Ann. N. Y. Acad. Sci 569: 86-103, 1989; Flexner et al., Vaccine 8: 17-21, 1990; U.S. Patent No. 4603112, 4769330 and 5017487; WO 89/01973; U.S. Patent No. 4,777,127; GB 2200651; 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; Guzrnan et al., Circulation 88: 2838-2848, 1993; and Guzman et al., Cir. Res. 73: 1202-1207, 1993.

In some implementations, the polynucleotide can integrate into the genome of the target cell. This integration can occur in specific localization and orientation through homologous recombination (gene replacement), or it can integrate in a random, non-specific site (gene increment). In further embodiments, the polynucleotide can be stably maintained in the cell as a separate episomal DNA segment. Such polynucleotide segments or “episomes” encode sequences sufficient to support and replicate independently or synchronously with the host cell cycle. The manner in which the expression construct is delivered to the cell, and where the polynucleotide resides in the cell, depends on the type of expression construct used.

In another embodiment of the invention, the polynucleotide is introduced / delivered as naked DNA, for example, as described by Ulmer et al., Science 259: 1745-1749, 1993 and in Cohen, Science 259: 1691-1692, 1993. Capturing naked DNA may amplified by coating the DNA with granules degrading in the biological medium, which are effectively transported into the cell.

In yet another embodiment, the composition of the present invention can be delivered by particle bombardment approaches, many of which have been described. In one illustrative example, gas-directed particle acceleration can be achieved using devices such as those manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc. (Madison, WI), some examples thereof are described in US patent No. 5846796; 6010478, 5865796; 5,584,807; and EP Patent No. 0500799. This approach involves a needleless delivery method, where a dried powdery preparation of microscopic particles, such as polynucleotide or polypeptide particles, is accelerated to a high speed by a jet of helium gas generated by a hand-held device spraying particles into a target tissue of interest.

In a related embodiment, other devices and methods that can be used for gas-directed needleless injection of the compositions of the present invention include those provided by Bioject, Inc. (Portland, OR), some examples thereof are described in US patent 4790824; 5,064,413; 5,312,335; 5,383,851; 5,399,163; 5520639 and 5993412.

In another embodiment, the pharmaceutical compositions described herein include one or more immunostimulants in addition to the composition of the immunogenic polynucleotides, polypeptides, antibodies, T cells, or APCs of the present invention. An immunostimulant refers essentially to any substance that enhances or promotes an immune response (mediated by antibodies and / or cells) to a foreign antigen. One of the preferred types of immunostimulants includes an adjuvant. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and an immune response stimulator such as lipid A, proteins derived from Bortadella pertussis or Mycobacterium tuberculosis. Some adjuvants are commercially available, for example, Freund's incomplete and complete adjuvant (Difco Laboratories, Detroit, MI); adjuvant Merck 65 (Merck and Company, Inc ,, Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; insoluble suspension of acylated tyrosine; acylated sugars; polysaccharides modified with cations or anions; polyphosphazenes; microspheres degrading in the biological environment; monophosphoryl lipid A and Quil A. Cytokines, such as GM-CSF, interleukin-2, -7, -12, and other similar growth factors, can also be used as adjuvants.

In some embodiments of the invention, an adjuvant composition that induces an immune response predominantly of the Th1 type is preferred. High concentrations of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2, and IL-12) are prone to induce cell-mediated immune responses to the introduced antigen. High concentrations of Th2-type cytokines (e.g., IL-4, IL-5, IL-6, and IL-10), on the contrary, are prone to inducing humoral immune responses. After application of the vaccine described herein, an immune response is maintained in a patient including Th1 and Th2 type responses. In a preferred embodiment in which the response is predominantly Th1-type, the concentration of Th1-type cytokines increases more than the concentration of Th2-type cytokines. Concentrations of these cytokines can be easily estimated using standard assay methods. For a review of cytokine families see Mosmann and Coffman, Ann. Rev. Immunol. 7: 145-173, 1989.

Some preferred adjuvants in order to elicit a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, with an aluminum salt. MPL® adjuvants are available from Corixa Corporation (Seattle, WA; see, for example, US Pat. No. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is not methylated) also primarily induce a Th1-type response. Such oligonucleotides are well known and described, for example, in WO 96/02555, WO 99/33488 and US patent No. 6008200 and 5856462. Immunostimulatory DNA sequences are also described, for example, Sato et al, Science 273: 352, 1996. Another preferred adjuvant includes saponin, such as Quil A, or its derivatives, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA); escin; digitonin; or saponins of Gypsophila or Chenopodium quinoa. Other preferred formulations include more than one saponin in a combination of adjuvants of the present invention, for example, in a combination of at least two of the following group, including QS21, QS7, Quil A, β-escin or digitonin.

Alternatively, saponin preparations can be combined with vaccine carriers consisting of chitosan or other polycationic polymers, polyactides and polyactide-co-glycalides, particles, a poly-N-acetylglucosamine polymer matrix, particles consisting of polysaccharides or chemically modified polysaccharides, liposomes and parts lipid-based particles consisting of glycerol monoesters, etc. Saponins can also be formulated in the presence of cholesterol together to form particulate structures such as liposomes or ISCOM. Moreover, saponins can also be formulated in the presence of a polyoxyethylene ester or ether, which is either a particle-free solution or a suspension, or a particulate structure, such as weak-lamellar liposomes or ISCOM. Saponins may also be formulated with fillers such as Carbopol® to increase viscosity, or the compositions may be prepared as a dry powder with a powder filler such as lactose.

In one preferred embodiment, the adjuvant system comprises a combination of monophosphoryl lipid A and a saponin derivative, such as a combination of QS21 and 3D-MPL® adjuvant as described in WO 94/00153, or a less reactogenic composition where QS21 is quenched with cholesterol as described in WO 96 / 33739. Other preferred compositions include an oil in water emulsion and tocopherol. Another particularly preferred adjuvant preparation using QS21, 3D-MPL adjuvant and tocopherol oil-in-water emulsion is described in WO 95/17210.

Another enhanced adjuvant system includes a combination of a CpG-containing oligonucleotide and a saponin derivative, in particular, a combination of CpG and QS21 is described in WO 00/09159. Preferably, the preparation further includes an oil in water emulsion and tocopherol.

Additional illustrative adjuvants for use in the pharmaceutical compositions of the invention include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), a series of SBAS adjuvants (e.g. SBAS- 2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Enhanzyn®) (Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other aminoalkylglucosaminide-4-phosphates (AGP), such as those described in pending U.S. patent applications serial numbers 08/853826 and 09/074720, the descriptions of which are incorporated herein by reference in their entirety, and adjuvants for again polyoxyethylene ethers, such as those described in WO 99 / 52549A1.

Other preferred adjuvants include adjuvant molecules of the general formula:

(I): BUT (CH ₂ CH ₂ O) _n -AR,

where n is 1-50, A is a bond or —C (O) -, R is C _1-50 alkyl or phenyl C _1-50 alkyl.

One of the implementations of the present invention consists of a vaccine preparation comprising a polyoxyethylene ether of the general formula (I), where n is between 1 and 50, preferably in the range of 4-24, most preferably is 9; component R is C _1-50 , preferably C ₄ -C ₂₀ alkyl, and most preferably C ₁₂ alkyl, and A is a bond. The concentration of polyoxyethylene ethers is in the range of 0.1-20%, preferably 0.1-10%, and most preferably in the range of 0.1-1%. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-stearyl ether, polyoxyethylene-8-stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether and polyoxyethylene -23-lauryl ether. Polyoxyethylene ethers, such as polyoxyethylene lauryl ether, are described in the Merck catalog (12th edition: article 7717). These adjuvant molecules are described in WO 99/52549.

The polyoxyethylene ether according to the above general formula (I), if desired, can be combined with another adjuvant. For example, a preferred combination of adjuvants is preferably a combination of CpG, as described in pending UK patent application GB 9820956.2.

In another embodiment of the invention, the immunogenic composition described herein is delivered to the host via antigen presenting cells (APCs) such as dendritic cells, macrophages, B cells, monocytes and other cells that can be selected as effective APCs. Such cells may optionally be genetically modified to increase the ability to present antigen, to improve activation and / or maintenance of the T-cell response, so that they have an antitumor effect on their own and / or are immunologically compatible with the recipient (i.e., coincident haplotype HLA). APCs can mainly be isolated from any various biological fluids and organs, including tumor and near-tumor tissues, and can be autologous, allogeneic, syngeneic or xenogenic.

In some preferred embodiments of the present invention, dendritic cells or their precursors, such as antigen presenting cells, are used. Dendritic cells are potent APCs (Banchereau and Steinman, Nature 392: 245-251, 1998) and are shown to be effective as physiological adjuvants for the induction of prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Ann. Rev. Med 50: 507-529, 1999). In general, dendritic cells can be identified based on their typical shape (stellate in situ, with noticeable cytoplasmic processes (dendrites) visible in vitro), their ability to capture, process and present with high efficiency, and their ability to activate naive T- responses cells. Of course, dendritic cells expressing specific cell surface receptors or ligands that are not normally found on dendritic cells in vivo or ex vivo can be constructed, and dendritic cells thus modified are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles of antigen-loaded dendritic cells (called exosomes) can be used with the vaccine (see Zitvogel et al., Nature Med. 4: 594-600, 1998).

Dendritic cells and precursors can be obtained from peripheral blood, bone marrow, near-tumor tumor infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood, or from any other tissue or fluid. 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 collected from peripheral blood. Alternatively, CD34-positive cells collected from peripheral blood, umbilical cord blood or bone marrow can differentiate into dendritic cells by adding a combination of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and / or the other (s) compound (s) that induce the differentiation, maturation and proliferation of dendritic cells.

Dendritic cells are usually divided into "immature" and "mature" cells, which allows a simple way to separate two well-characterized phenotypes. However, this nomenclature should not be construed as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high ability to capture and process antigen, which correlates with high levels of expression of the Fcγ receptor and mannose receptor. The mature phenotype is usually characterized as lower expression of these markers, but higher expression of cell surface molecules responsible for T-cell activation, such as MHC classes I and II, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40 , CD80, CD86 and 4-1BB).

Typically, APCs can be transfected with a polynucleotide of the invention (or a part thereof, or another variant thereof) such that the polypeptide encoded by it or its immunogenic part is expressed on the cell surface. Such transfection can occur ex vivo, and a pharmaceutical composition comprising such transfected cells can then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle targeted at dendritic or other antigen presenting cells can be administered to a patient, resulting in transfection that occurs in vivo. Transfection of dendritic cells in vivo and ex vivo, for example, can generally be carried out using any methods known in the art, such as those described in WO 97/24447, or as the gene gun approach described by Mahvi et al., Immunology and Cell Biology 75: 456-460, 1997. The loading of dendritic cells with antigen can be achieved by incubating dendritic or progenitor cells with a tumor polypeptide, DNA (naked or as part of a plasmid vector) or RNA; or antigen expressing recombinant bacteria or viruses (e.g. vectors based on vaccinia virus, poultry poxvirus, adenovirus or lentivirus). Before loading, the polypeptide can be covalently conjugated to an immunogenic partner that provides T cell assistance (e.g., to a carrier molecule). Alternatively, a dendritic cell may be stimulated with an unconjugated immunological partner, alone or in the presence of a polypeptide.

Although any suitable carrier known to those of ordinary skill in the art can be used in the pharmaceutical compositions of the invention, the type of carrier will usually vary depending on the route of administration. The compositions of the present invention can be formulated for any suitable route of administration, including, for example, topical, oral, nasal, mucosal, intravenous, intracranial, intraperitoneal, subcutaneous and intramuscular administration.

Carriers for use in such pharmaceutical compositions are biocompatible and may also be capable of degradation under biological conditions. In some embodiments, the preparation preferably provides a relatively constant release level of the active ingredient. In other implementations, however, a higher release rate may be required immediately after administration. The preparation of such compositions is well known at the usual level in this field and is carried out using known methods. Illustrative carriers useful in this regard include microparticles of poly (lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative sustained release vehicles include supramolecular bio-vectors, which include a non-liquid hydrophilic core (eg, cross-linked polysaccharides or oligosaccharides) and, optionally, an outer layer comprising an amphiphilic compound such as a phospholipid (see, for example, US Pat. No. 5,151,254 and PCT applications WO 94/20078, WO 94/23701 and WO 96/06638). The amount of active ingredient contained within a sustained release preparation depends on the implantation site, rate and expected duration of release, and the nature of the condition to be treated or prevented.

In another illustrative embodiment, biodegradable microspheres (e.g., polylactate-polyglycolate) are used as carriers for the compositions of this invention. Suitable biodegradable microspheres are described, for example, in US Pat. Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5814344, 5407609 and 5942252. Carrier systems based on modified core proteins of hepatitis B virus, such as those described in WO 99/40934 and the references cited here, can also be used in many applications. Another exemplary carrier / delivery system utilizes a carrier comprising particle and protein complexes, such as those described in US Pat. No. 5,928,647, which are capable of inducing class I-restricted cytotoxic T-lymphocyte responses in the host.

The pharmaceutical compositions of the invention also often include one or more buffers (e.g., neutral saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextran), mannitol, proteins, polypeptides or amino acids, such as glycine , antioxidants, bacteriostatics, chelating agents such as EDTA or glutathione, adjuvants (e.g. aluminum hydroxide), solutions leading to an isotonic, hypotonic or slightly hypertonic state with respect to the recipient's blood, su spelling agents, thickeners and / or preservatives. Alternatively, the compositions of the present invention can be formulated as a lyophilisate.

The pharmaceutical compositions described herein may be presented in containers containing one or more doses, such as sealed ampoules or vials. Such containers are usually closed in such a way as to preserve the sterility and stability of the drug before testing. In general, preparations can be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, the pharmaceutical compositions can be stored under freeze-drying conditions, which only requires the addition of a sterile liquid carrier immediately before the study.

The development of suitable dosage and treatment regimens for the application of the specific pharmaceutical compositions described herein among a variety of treatment regimens, including, for example, oral, parenteral, intravenous, intranasal and intramuscular administration and formulation, is well known in the art, and some of these regimens are briefly discussed below in mainly for the purpose of illustration.

In some applications, the pharmaceutical composition described herein can be delivered by oral administration. In such a case, these compositions may be formulated in an inert solvent or in an assimilable edible carrier or may be placed inside soft or hard shell gelatine capsules, or they may be compressed into tablets, or they may be included in diet foods.

The active compounds can even be combined with carriers and can be used in the form of digestible tablets, sucking tablets, lozenges, capsules, elixirs, suspensions, syrups, cachets and the like (see, for example, Mathiowitz et al., Nature 1997 Mar 27; 386 (6623): 410-4; Hwang et al., Crit Rev Ther Drug Carrier Syst 1998; 15 (3): 243-84; U.S. Patent 5,641,515; U.S. Patent 5,580,579 and U.S. Patent 5,792,451). Tablets, lozenges, pills, capsules, and the like, may also contain any kind of additional components, for example, a binding agent such as tragacanth gum, gum arabic, corn starch or gelatin; fillers, such as dibasic calcium phosphate; disintegrating agents such as corn starch, potato starch, alginic acid and the like can be added; a lubricant such as magnesium stearate; and a sweetener, such as sucrose, lactose, or saccharin, or flavors, such as peppermint, buttercup, or cherry flavors. When the dosage unit form is a capsule, it may contain a liquid carrier in addition to the materials of the above type. Various other materials may be presented in the form of coatings or for otherwise modifying the physical state of the dosage unit. For example, tablets, pills or capsules may be coated with shellac, sugar, or both. Of course, any material used to produce any form of dosage unit must be pharmaceutically pure and not show significant toxicity in the amounts used. In addition, the active compounds may be included in sustained release formulations and compositions.

Typically, these compositions contain at least about 0.1% or more active ingredient, although the percentage of active ingredient (s) may, of course, vary and may suitably be from about 1 or 2% to about 60%. or 70% or more, by weight or volume of the total composition. Naturally, the amount of active (s) component (s) in each therapeutically applicable composition can be prepared in such a way that a suitable dosage can be obtained in each unit dose administered. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations will be considered by a person skilled in the art of the preparation of such pharmaceutical preparations, and in essence, various dosages and treatment regimens may be required.

For oral administration, the compositions of the present invention can alternatively be combined with one or more excipients in the form of a rinse, toothpaste, sucking candy, mouth spray or orally administered under the tongue of the composition. Alternatively, the active ingredient may be introduced into an oral solution, such as containing sodium borate, glycerin and potassium bicarbonate, or may be dispersed in toothpaste, or may be added in a therapeutically effective amount to the composition, which may include water, carriers, abrasives, flavors, foaming agents and moisturizers. Alternatively, tablets or solution forms may be formed from the compositions, which may be placed under the tongue or otherwise dissolved in the mouth.

In some circumstances, it is required to deliver the pharmaceutical compositions described herein parenterally, intravenously, intramuscularly, or even intraperitoneally. Such approaches are well known to those skilled in the art, some of which are further described, for example, in US Pat. No. 5,543,158; U.S. Pat. Dispersions can also be obtained in glycerol, liquid polyethylene glycols and their mixtures, as well as in oils. Under normal conditions of storage and use, these preparations usually contain a preservative to prevent the growth of microorganisms.

Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the urgent preparation of sterile injectable solutions or dispersions (for example, see US Pat. No. 5,466,468). In all cases, the form must be sterile and must be fluid to the extent that easy use of the syringe is possible. It must be stable under the conditions of production and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyhydric alcohol (e.g. glycerin, propylene glycol and liquid polyethylene glycol and the like), suitable mixtures thereof and / or vegetable oils. Proper fluidity can be maintained, for example, by applying a coating, such as lecithin, by maintaining the required particle size in the case of dispersion and / or by using surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars and sodium chloride. Prolonged absorption of the injected compositions can be carried out by the use of compositions of agents delaying absorption, for example, aluminum monostearates and gelatin.

In one embodiment for parenteral administration in an aqueous solution, the solution must be suitably buffered if necessary, and the liquid solvent is first rendered isotonic with a sufficient amount of saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, a sterile aqueous medium that can be used is known to those skilled in the art in light of the present description. For example, one dosage may be dissolved in 1 ml of isotonic solution of NaCl and may be either added to 1000 ml gipodermoklizisa liquid (hypodermoclysis) or injected at the proposed infusion section (see., E.g., "Remington's Pharmaceutical Sciences", 15 ^th edition, p. 1035-1038 and 1570-1580). Some variations in dosage necessarily occur depending on the condition of the patient being treated. Moreover, preparations for administration to humans are, of course, preferably characterized by sterility, pyrogenicity, and meet the general safety and purity standards of the FDA Office of Biologics standards.

In another embodiment of the invention, the compositions described herein may be formulated in neutral or salt form. Illustrative pharmaceutically acceptable salts include acid addition salts (formed by the free amino groups of a protein), and they are formed by inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, almond, and the like. Salts formed by free carboxy groups can also be derived from inorganic bases, such as, for example, hydroxides of sodium, potassium, ammonium, calcium or iron, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. After preparation, the solutions are administered in a manner compatible with the dosed preparation and in such an amount as therapeutically effective.

These carriers may further include any and all of solvents, dispersion media, carriers, coatings, solvents, antibacterial and antifungal agents, isotonic agents and absorption delay agents, buffers, carrier solutions, suspensions, colloids and the like. The use of such media and agents with respect to pharmaceutically active substances is well known in the art. Unless a conventional medium or agent is found to be incompatible with the active ingredient, their use in a pharmaceutical composition is contemplated. Additional active ingredients may also be included in the composition. The phrase "pharmaceutically acceptable" refers to molecular formulations and compositions that do not lead to allergic and similar adverse reactions when administered to humans.

In some embodiments, the pharmaceutical compositions may be delivered via intranasal sprays, inhalation and / or other aerosol delivery vehicles. Methods for delivering genes, nucleic acids, and peptide compositions for direct delivery to the lungs via nasal aerosol sprays are described, for example, in US Pat. No. 5,756,353 and US Pat. Controlled Release 1998 Mar 2; 52 (1 -2): 81-7) and lysophosphatidylglycerol compounds (US Pat. No. 5,725,871) are also well known in the field of pharmacology. Similarly, exemplary mucosal drug delivery in the form of a polytetrafluoroethylene base matrix is described in US Pat. No. 5,780,045.

In some embodiments, liposomes, nanocapsules, microparticles, lipid particles, vesicles and the like are used to introduce the compositions of the present invention into suitable host cells / organisms. In particular, the compositions of the present invention can be formulated for delivery encapsulated in a lipid particle, liposome, vesicle, nanosphere or nanoparticle, or the like. Alternatively, the compositions of the present invention may be covalently or non-covalently linked to the surface of such carrier vesicles.

The preparation and use of liposomes and liposome-like preparations as potential drug carriers is generally known to those skilled in the art (see, for example, Lasic, Trends Biotechnol 1998 Jul; 16 (7): 307-21; Takakura, Nippon Rinsho 1998 Mar; 56 (3): 691-5; Chandran et al., Indian J Exp Biol. 1997 Aug; 35 (8): 801-9; Margalit, Crit Rev Ther Drug Carrier Syst. 1995; 12 (2-3): 233- 61; US patent 5567434; US patent 555257; US patent 5565213; US patent 5738868 and US patent 5795587, each of which is specifically incorporated herein by reference in its entirety).

Liposomes have been successfully used with a number of cell types that are usually difficult to transfect with other methods, including suspensions, T cells, primary hepatocyte cultures and PC 12 cells (Renneisen et al., J. Biol. Chem. 1990 Sep 25; 265 (27) : 16337-42; Muller et al., DNA Cell Biol. 1990 Apr; 9 (3): 221-9). In addition, liposomes are not characterized by DNA length difficulties that are typical of virus-based delivery systems. Liposomes were effectively used to introduce genes, various drugs, radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric effectors, and the like into a variety of cultured cell lines and in different animals. Moreover, it appears that the use of liposomes is not associated with autoimmune reactions or unacceptable toxicity after systemic administration.

In some embodiments, liposomes are formed from phosphoslipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLV).

Alternatively, in other embodiments, the invention relates to pharmaceutically acceptable nanocapsule formulations of the compositions of the present invention. Nanocapsules can typically capture compounds in a stable and reproducible manner (see, for example, Quintanar-Guerrero et al., Drug. Dev. Ind. Pharm. 1998 Dec; 24 (12): 1113-28). To avoid side effects due to intracellular polymer overload, such ultra-thin particles (about 0.1 microns in size) can be constructed using polymers capable of degrading in vivo. Such particles can be obtained as described, for example, Couvreur et al., Crit. Rev. Ther. Drug. Carrier Syst. 1988; 5 (1): 1-20; zur Muhlen et al., Eur. J. Pharm. Biopharm. 1998 Mar; 45 (2): 149 -55; Zambaux et al., J. Controlled Release. 1998 Jan 2; 50 (1-3): 31-40; and U.S. Patent 5,145,684.

Methods of treating malignant tumors

In further aspects of the present invention, the pharmaceutical compositions described herein can be used to treat malignant tumors, in particular for immunotherapy of lung cancer. With such methods, the described pharmaceutical compositions are administered to a patient, usually a warm-blooded animal, preferably a human. The patient may or may not suffer from a malignant tumor. Accordingly, the above pharmaceutical compositions can be used to prevent the development of a malignant tumor or to treat a patient suffering from a malignant tumor. Pharmaceutical compositions and vaccines can be administered either before or after surgical removal of the primary tumors and / or treatment, such as radiotherapy or conventional chemotherapy drugs. As discussed above, the administration of pharmaceutical compositions can be carried out by any suitable method, including administration by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal, anal, vaginal, local and oral routes.

In some embodiments, the immunotherapy may be an active immunotherapy, in which the treatment is based on stimulating the in vivo antitumor response of the endogenous immune system of the host by introducing modulating the immune response agents (such as the polypeptides and polynucleotides described herein).

In other implementations, immunotherapy may be a passive immunotherapy, in which the treatment includes the delivery of funds with established antitumor immunoreactivity (such as effector cells or antibodies), which can directly or indirectly mediate the antitumor effect and does not necessarily depend on the intact immune system of the host. Examples of effector cells include the T cells discussed above, T lymphocytes (such as CD8 ⁺ cytotoxic T lymphocytes and CD4 ⁺ T helper, tumor infiltrating lymphocytes), killer cells (such as natural killers and lymphokine-activated killer cells ), B cells and antigen presenting cells (such as dendritic cells and macrophages) expressing the polypeptide described herein. T cell receptors and immunoglobulin receptors specific for the polypeptides shown here can be cloned, expressed and transfected into other vectors and effector cells for adaptive immunotherapy. The polypeptides described herein can also be used to produce antibodies and anti-idiotypic antibodies (as described above and in US Pat. No. 4,918,164) for passive immunotherapy.

Effector cells, as a rule, can be obtained in sufficient quantities for immunotherapy by growing in vitro, as described here. Cultivation conditions are well known in the art for the expansion of a single antigen-specific effector cell up to several billion in number, while maintaining antigen recognition in vivo. Under such in vitro culture conditions, intermittent antigen stimulation is usually used, often in the presence of cytokines (such as IL-2) and non-dividing nurse cells. As noted above, the immunoreactive polypeptides described herein can be used to rapidly expand antigen-specific cultures of T cells, to obtain a sufficient number of cells for immunotherapy. In particular, antigen-presenting cells such as dendritic cells, macrophages, monocytes, fibroblasts and / or B cells can be exposed to immunoreactive polypeptides or transfected with one or more polynucleotides using standard methods well known in the art. For example, antigen-presenting cells can be transfected with a polynucleotide having a promoter suitable for increasing expression in a recombinant virus or other expression system. Cultured effector cells for use in treatment should be able to grow and spread widely, and survive in vivo for a long time. Studies have shown that in vivo growth and long-term survival of cultured effector cells in significant amounts can be induced by repeated stimulation with antigen supplemented with IL-2 (see, for example, Cheever et al., Immunological Reviews 157: 177, 1997).

Alternatively, a vector expressing the polypeptides shown herein may be introduced into antigen-presenting cells taken from a patient and subjected to ex vivo clonal expansion for reverse transplantation to the same patient. Transfected cells can be re-introduced to the patient using any means known in the art, preferably in sterile form, by intravenous, intracavitary, intraperitoneal or intratumoral administration.

The routes and frequency of administration of the therapeutic compositions described herein, as well as the dosage, vary from subject to subject, and can be readily established using standard methods. In general, pharmaceutical compositions may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration), or orally. Preferably, from 1 to 10 doses may be administered over a period of 52 weeks. Preferably, 6 doses are administered at 1 month intervals, and thereafter, maintenance vaccinations may be given. Alternative protocols may be applied to specific patients. A suitable dose is an amount of a compound which, when administered as described above, is capable of eliciting an antitumor immune response that is at least 10-50% higher than the basal (i.e., untreated) one. This response can be monitored by measuring the amount of antitumor antibody in the patient or by vaccine-dependent production of cytolytic effector cells capable of killing patient tumor cells in vitro. Such vaccines are also capable of eliciting an immune response that leads to an improved clinical outcome (for example, more frequent remissions, complete or partial, or prolonged disease-free survival) in vaccinated patients compared to unvaccinated patients. In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in dosage ranges from about 25 μg to 5 mg per kg of host weight. Suitable dosage volumes vary depending on the size of the patient, but usually vary from about 0.1 ml to 5 ml.

In general, a suitable dosage and treatment regimen provides the active compound (s) in an amount sufficient to provide therapeutic and / or prophylactic benefits. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or long, disease-free survival) in treated patients compared to untreated patients. An increase in preexisting immune responses to tumor protein tends to correlate with improved clinical outcome. Such immune responses can generally be assessed using standard analyzes of proliferation, cytotoxicity, or cytokines, which can be performed using samples obtained from the patient before and after treatment.

Malignant tumor detection and diagnostic compositions, methods and kits

In general, a malignant tumor can be detected in a patient based on the presence of one or more lung tumor proteins and / or polynucleotides encoding such proteins in a biological sample (e.g., blood, serum, sputum, urine and / or tumor biopsy) obtained from the patient. In other words, such proteins can be used as markers to indicate the presence or absence of a malignant tumor, such as lung cancer. In addition, such proteins can be used to detect other malignant tumors. The binding agents described herein typically allow detection of the level of antigen that binds to the agent in a biological sample. Polynucleotide primers and probes can be used to detect the level of mRNA encoding a tumor protein, which is also an indicator of the presence or absence of a malignant tumor. In general, the sequence of a lung tumor should be present at a concentration of at least three times that of the tumor tissue than normal tissue.

There are a diverse number of assay formats known to those of ordinary skill in the art for using a binding agent in the detection of polypeptide markers in a sample. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of a malignant tumor in a patient can be determined (a) by the interaction of a biological sample obtained from the patient with a binding agent; (b) detecting in the sample an amount of a polypeptide that binds to a binding agent; and (c) comparing the level of the polypeptide with a predetermined cutoff level.

In a preferred embodiment, the assay involves the use of a solid-state immobilized binding agent to bind the polypeptide to it and remove the polypeptide from the remainder of the sample. The bound polypeptide can then be detected using a detection reagent that contains a reporter group and specifically binds to a binding agent / polypeptide complex. Such detection reagents may include, for example, a binding agent that specifically binds to a polypeptide or antibody, or another agent that specifically binds to a binding agent, such as anti-immunoglobulin, protein G, protein A or lectin. Alternatively, a competitive assay may be used in which the polypeptide is labeled with a reporter group and is bound to bind to the immobilized binding agent after incubation of the binding agent with the sample. The degree to which the components of the sample inhibit the binding of the labeled polypeptide to the binding agent is an indicator of the reactivity of the sample with respect to the immobilized binding agent. Suitable polypeptides for use in such assay methods include full-size tumor proteins and their polypeptide portions to which a binding agent binds, as described above.

The solid base may be of any material known to those skilled in the art to which the tumor protein may bind. For example, the solid base may be a microtiter plate test well or a nitrocellulose or other suitable membrane. Alternatively, the base may be a granule or disk, such as glass, fiberglass, latex, or a plastic material, such as polystyrene or polyvinyl chloride. The base can also be a magnetic particle or an optical fiber sensor, such as those described, for example, in US Pat. No. 5,359,681. The binding agent can be immobilized on a solid basis using a variety of methods known to those skilled in the art that are fully described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to non-covalent association, such as adsorption, and covalent binding (which may be a direct bond between the agent and the functional groups of the base, or may be a bond through a cross-linking agent). Immobilization by absorption onto a well of a microtiter plate or onto a membrane is preferred. In such cases, adsorption can be achieved by reacting the binding agent with a solid base in a suitable buffer for a suitable period of time. Contact time varies with temperature, but usually ranges from about 1 hour to 1 day. In general, the interaction of a well of a plastic microtiter plate (such as polystyrene or polyvinyl chloride) with an amount of a binding agent ranging from about 10 ng to 10 μg, and preferably from about 100 ng to about 1 μg, is sufficient to immobilize an adequate amount of binding agent agent.

The covalent binding of a binding agent to a solid base can generally be achieved initially by reacting the base with a bifunctional reagent that will react with both the base and the functional group of the binding agent, such as hydroxyl or amino. For example, a binding agent can be covalently linked to bases having an appropriate polymer coating, using benzoquinone or by condensing the aldehyde group of the base with an amine and active hydrogen on a binding partner (see, for example, Pierce Immunotechnology Catalog and Handbook, 1991, at A12- A13).

In some implementations, the analysis is a sandwich analysis with two antibodies. This assay can be performed by initially reacting an antibody that is immobilized on a solid basis, usually in a well of a microtiter plate, with a sample so that the polypeptides in the sample are able to bind to the immobilized antibody. The unbound portion of the sample is then removed from the immobilized polypeptide-antibody complexes, and a detection reagent (preferably a secondary antibody capable of binding to another portion of the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound on a solid basis is then determined using a method suitable for a particular reporter group.

More specifically, as soon as the antibody is immobilized on the basis of, as described above, the remaining binding sites of the protein are usually blocked. Some suitable blocking agents are known to those skilled in the art, such as bovine serum albumin or Tween 20 ™ (Sigma Chemical Co., St. Louis, MO). The immobilized antibody is then incubated with the sample and the polypeptide is allowed to bind to the antibody. The sample may be diluted with a suitable solvent before incubation, such as phosphate buffered saline (PBS). In general, a suitable contact time (i.e., incubation time) is a period of time that is sufficient to detect the presence of a polypeptide in a sample obtained from a subject with lung cancer. Preferably, the contact time is sufficient to achieve a binding level that is at least 95% of the time to reach equilibrium between the bound and unbound polypeptide. It will be apparent to those of ordinary skill in the art that the time required to reach equilibrium can be easily determined by analyzing the level of binding that occurs over a period of time. At room temperature, an incubation time of approximately 30 minutes is usually sufficient.

An unbound sample can then be removed by washing the solid base with a suitable buffer, such as PBS containing 0.1% Tween 20 ™. A secondary antibody that contains a reporter group can then be added to the solid base. Preferred reporter groups contain those groups described above.

Then, the detection reagent is incubated with the antibody-polypeptide complex immobilized for a period of time sufficient to detect the bound polypeptide. A suitable length of time can usually be determined by analyzing the level of binding that occurs over a period of time. The unbound detection reagent is then removed and the associated detection reagent is detected using a reporter group. A method involving detection of a reporter group depends on the nature of the reporter group. For radioactive groups, scintillation or autoradiography counting methods are generally suitable. Spectroscopic methods can be used to detect tags, luminescent groups and fluorescent groups. Biotin can be detected using avidin bound to various reporter groups (usually a radioactive or fluorescent group or enzyme). Enzymatic reporter groups, as a rule, can be detected by adding a substrate (mainly over a specific period of time), followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of a malignant tumor, such as lung cancer, a signal detected from a reporter group that remains connected to a solid base is typically compared with a signal that corresponds to a predetermined cutoff level. In one preferred embodiment, the cutoff level for cancer detection is the average of the signal obtained by incubating the immobilized antibody with samples from patients without a malignant tumor. In general, a sample whose signal exceeds the predefined cutoff level by three standard deviations is considered positive in the sense of a malignant tumor. In an alternative preferred embodiment, the cutoff level is determined using the Receiver Operator Curve according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cutoff level can be determined from a graph of pairs of truly positive relationships (i.e., sensitivity) and false positives (100% specificity) that correspond to each possible cutoff level for the result of the diagnostic test. The clipping value on the graph that is closest to the upper left corner (i.e., the value covering the largest area) is the most accurate clipping level, and a sample generating a signal above the clipping level determined by this method can be considered positive. Alternatively, the cutoff level can be shifted to the left according to the schedule to minimize the false positive ratio or to the right to minimize the false negative ratio. In general, a sample generating a signal above a cutoff level determined by this method is considered positive in the sense of a malignant tumor.

In a related embodiment, an analysis is performed in the form of a flow test or strip test where the binding agent is immobilized on a membrane such as nitrocellulose. In a flow test, the polypeptides of the sample bind to the immobilized binding agent while the sample passes through the membrane. Then, the second, labeled agent binds to the binding agent-polypeptide complex while the solution containing the second binding agent flows through the membrane. The detection of the bound second binding agent can then be carried out as described above. In a strip test format, one end of the membrane to which the binding agent is bound is immersed in a solution containing a sample. The sample migrates along the membrane through the region containing the second binding agent and to the location of the immobilized binding agent. The concentration of the second binding agent in the region of the immobilized antibody indicates the presence of a malignant tumor. Typically, the concentration of the second binding agent in this region leads to the formation of a pattern, such as a line, that can be visually determined. The absence of such a pattern indicates a negative result. In general, the amount of binding agent immobilized on the membrane is chosen so that a visually distinguishable pattern is formed when the biological sample contains a concentration of the polypeptide sufficient to form a positive signal in the sandwich analysis with two antibodies in the format discussed above. Preferred binding agents for use in such an assay are antibodies and antigen binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane varies from about 25 ng to 1 μg, and more preferably from about 50 ng to 500 ng. Such tests can usually be carried out with a very small amount of biological sample.

Of course, there are many other assay protocols that are suitable for use with the tumor proteins and binding agents of the present invention. The above descriptions are intended to be representative only. For example, those skilled in the art will recognize that the above protocols can be easily modified to use tumor polypeptides in detecting antibodies that bind to such polypeptides in a biological sample. Detection of such antibodies specific for tumor proteins may correlate with the presence of a malignant tumor.

A malignant tumor can also, or alternatively, be detected based on the presence of T cells that specifically react with the tumor protein in the biological sample. In specific methods, a biological sample comprising CD4 ⁺ or CD8 ⁺ T cells isolated from a patient’s body is incubated with a tumor polypeptide, a polynucleotide encoding such a polypeptide and / or APC expressing at least an immunogenic portion of such a polypeptide, and detect the presence or absence of T-cell activation. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells can be isolated from a patient’s body by routine methods (such as peripheral blood lymphocyte density gradient Ficoll / Hypaque centrifugation). T cells can be incubated in vitro for 2-9 days (usually 4 days) at 37 ° C with a polypeptide (for example, 5 -25 μg / ml). It may be necessary to incubate another aliquot of the T cell sample in the absence of a tumor polypeptide, which will serve as a control. Activation of CD4 ⁺ T cells is preferably detected by evaluating T cell proliferation. Activation of CD8 ⁺ T cells is preferably detected by assessing cytolytic activity. The level of proliferation, which is at least two times higher, and / or the level of cytolytic activity, which is at least 20% higher than those in healthy patients, indicates the presence of a malignant tumor in the patient.

As noted above, a malignant tumor can also, or alternatively, be detected based on the level of mRNA encoding the tumor protein in a biological sample. For example, at least two oligonucleotide primers can be used in a polymerase chain reaction (PCR) analysis to amplify a portion of a tumor cDNA isolated from a biological sample, where at least one of the oligonucleotide primers is specific for a polynucleotide encoding a tumor protein ( i.e. hybridizes with it). The amplified cDNA is then separated and detected using methods well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize with a tumor protein encoding polynucleotide to detect the presence of a tumor protein encoding polynucleotide in a biological sample can be used in a hybridization assay.

To allow hybridization under assay conditions, oligonucleotide primers and probes should include an oligonucleotide sequence having at least about 60% identity, preferably at least about 75% identity, and more preferably at least about 90% identity over relative to the portion of the polynucleotide encoding the tumor protein of the invention, which is at least 10 nucleotides, and preferably at least 20 nucleotides in length. Preferably, the oligonucleotide primers and / or probes hybridize to a polynucleotide encoding the polypeptide described herein under the moderately stringent conditions defined above. Oligonucleotide primers and / or probes that may suitably be used in the diagnostic methods described herein are preferably 10-40 nucleotides in length. In a preferred embodiment, the oligonucleotide primers comprise at least 10 consecutive nucleotides, more preferably at least 15 consecutive nucleotides from the DNA molecule described herein. Methods of all types of PCR-based analysis and hybridization analysis are well known in the art (see, for example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263, 1987; Erlich ed., PCR Technology , Stockton Press, NY, 1989).

One of the preferred analysis methods involves RT-PCR, in which PCR is used in combination with reverse transcription. Typically, RNA is extracted from a biological sample, such as tissue biopsy, and reverse transcribed to obtain cDNA molecules. Due to PCR amplification using a specific primer, a cDNA molecule is generated that can be detached and visualized, for example, using gel electrophoresis. Amplification can be carried out on biological samples taken from a test patient and from a subject not affected by a malignant tumor. The amplification reaction can be carried out at various dilutions of cDNA, covering two orders of magnitude. A twofold or greater increase in expression at different dilutions of a sample from a test patient compared to the same dilutions of a sample from a subject without a malignant tumor is usually considered positive.

In another embodiment, the compositions described herein can be used as markers for cancer progression. In this embodiment, the above analysis methods for the diagnosis of malignant tumors can be performed over time, and the change in the concentration of the reactive polypeptide (s) or polynucleotide (s) is evaluated. For example, tests can be done every 24-72 hours for a period of 6 months to 1 year, and then run them as needed. In general, a malignant tumor progresses in those patients in whom the concentration of the detectable polypeptide or polynucleotide increases. Conversely, a malignant tumor does not progress when the concentration of the reactive polypeptide or polynucleotide remains constant or decreases over time.

Some in vivo diagnostic tests can be performed directly on the tumor. One such assay involves the interaction of tumor cells with a binding agent. The bound binding agent can then be detected directly or indirectly through a reporter group. Such binding agents can also be used in histological applications. Alternatively, polynucleotide probes may be used in such applications.

As noted above, many tumor markers can be analyzed in this sample to improve sensitivity. It is also understood that binding agents specific for the different proteins described herein can be combined in one analysis method. In addition, different primers or probes can be used simultaneously. The choice of tumor protein markers may be based on routine experiments to determine the combination that leads to optimal sensitivity. In addition or alternatively, assays for the tumor proteins described herein may be combined with assays for other known tumor antigens.

The present invention further relates to kits for use in any of the above diagnostic methods. Such kits typically include two or more components necessary for performing a diagnostic analysis. Components may be compounds, reagents, containers and / or equipment. For example, one of the containers in the kit may contain monoclonal antibodies or a fragment thereof that specifically binds a tumor protein. Such antibodies or fragments can be supplied bound to the base material, as described above. One or more additional components may include elements such as reagents or buffers to be used in the analysis. Such kits can also, or alternatively, include a detection reagent, as described above, which contains a reporter group suitable for direct or indirect detection of antibody binding.

Alternatively, a kit can be designed to detect the level of mRNA encoding a tumor protein in a biological sample. Such kits mainly include at least one oligonucleotide probe or primer, as described above, which hybridizes with a polynucleotide encoding a tumor protein. Such an oligonucleotide, for example, can be used in a PCR or hybridization assay. Additional components that may be present within such kits include a second oligonucleotide and / or diagnostic reagent, or a container for detecting a polynucleotide encoding a tumor protein.

The following examples are offered for purposes of illustration and without purpose of limitation.

Example 1

Obtaining lung tumor specific cDNA sequences using RT-PCR differential display

This example illustrates the preparation of cDNA molecules encoding polypeptides specific for a lung tumor using screening by a differential display method.

Tissue samples were obtained from the tumor and normal lung tissue of a patient with lung cancer, which was confirmed by a pathology study after taking samples from the patient. Normal RNA and tumor RNA were extracted from the samples, and mRNA was isolated and converted to cDNA using an anchored (dT) ₁₂ AG (SEQ ID NO: 47) 3 ′ primer. Then, PCR differential display was performed using a randomly selected primer (SEQ ID NO: 48). The amplification conditions were a standard buffer containing 1.5 mM MgCl ₂ , 20 pmol primer, 500 pmol dNTP and 1 Taq DNA polymerase unit (Perkin-Elmer, Branchburg, NJ). Forty cycles of amplification were carried out using denaturation at 94 ° C for 30 seconds, annealing at 42 ° C for 1 minute, and elongation at 72 ° C for 30 seconds. Bands which, according to repeated observations, were specific for the tumor RNA fingerprint pattern, were cut out from a silver stained gel, subcloned into the pGEM-T vector (Promega, Madison, WI) and the sequence was determined. Selected 3'-sequences are presented in SEQ ID NO: 1-16.

When comparing these sequences with those of available databases using the BLASTN program, no significant homology was revealed with the sequences shown in SEQ ID NO: 1-11. According to the best information of the inventors, it was not previously shown that any of the selected DNA sequences was expressed at a high level in the tissue of a human lung tumor, in contrast to normal lung tissue.

Example 2

The use of patient sera to identify DNA sequences encoding lung tumor antigens

This example illustrates the isolation of cDNA sequences encoding lung tumor antigens by expression screening of lung tumor samples with autologous patient sera.

A targeted expression cDNA library of human lung tumors was constructed using the expression system based on ZAP Express lambda phage (Stratagene, La Jolla, CA). Total library RNA was taken from late human squamous epithelial carcinoma, which was inoculated with SCID mice, and polyA + RNA was isolated using the Message Maker kit (Gibco BRL, Gaithersburg, MD). The resulting library was screened using absorbed E. coli autologous patient serum as described by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989), with a secondary antibody, which is a goat anti-human IgG-AM (H + L) antibody conjugated to alkaline phosphatase, with the development of a reaction with NBT / BCIP (Gibco BRL). Purified positive plaques expressing immunoreactive antigen. Phagemids were removed from plaques and the nucleotide sequences of the clones were determined.

Fifteen clones were isolated, hereinafter referred to as LT86-1-LT86-15. Isolated cDNA sequences for LT86-1-LT86-8 and LT86-10-LT86-15 are presented in SEQ ID NOs: 17-24 and 26-31, respectively, with the corresponding predicted amino acid sequences presented in SEQ ID NO: 32-39 and 41-46, respectively.

The determined cDNA sequence for LT86-9 is presented in SEQ ID NO: 25, with the corresponding predicted amino acid sequences from the 3 ′ and 5 ′ ends shown in SEQ ID NO: 40 and 65, respectively. These sequences were compared with those of the gene bank, as described above. It was found that clones LT86-3, LT86-6-LT86-9, LT86-11-LT86-13 and LT86-15 (SEQ ID NO: 19, 22-25, 27-29 and 31, respectively) show some homology according to with respect to previously identified Expression Sequence Labels (EST), the clones LT86-6, LT86-8, LT86 -11, LT86-12 and LT86-15 being similar or identical to each other. Clone LT86-3 was found to exhibit some homology with respect to the human transcription repressor. It was found that clones LT86-6, 8, 9, 11, 12 and 15 show some homology with respect to the mediator of transcriptional regulation of yeast RNA-Pol II. Clone LT86-13 was found to exhibit some homology with respect to C. elegans leucine aminopeptidase. It turned out that clone LT86-9 contains two inserts with a 5'-sequence, showing homology with respect to the previously identified antisense sequence induced by interferon-alpha P27, and with a 3'-sequence similar to LT86-6. It was found that clone LT86-14 (SEQ ID NO: 30) shows some homology with respect to the tritorax gene and has a binding sequence with RGD cells, as well as a beta-lactamase A site that functions during the hydrolysis of penicillin. Clones LT86-1, LT86-2, LT86-4, LT86-5, and LT86-10 (SEQ ID NOs: 17, 18, 20, 21, and 26, respectively) were found to exhibit homology to the previously identified genes. The subsequently defined extended cDNA sequence for LT86-4 is presented in SEQ ID NO: 66, with the corresponding predicted amino acid sequence presented in SEQ ID NO: 67.

Subsequent studies led to the selection of five additional clones, designated as LT86-20, LT86-21, LT86-22, LT86-26 and LT86-27. Certain 5'-cDNA sequences for LT86-20, LT86-22, LT86-26 and LT86-27 are presented in SEQ ID NOs: 68 and 70-72, respectively, with certain 3'-cDNA sequences for LT86-21, which are represented in SEQ ID NO: 69. The corresponding predicted amino acid sequences for LT86-20, LT86-21, LT86-22, LT86-26 and LT86-27 are presented in SEQ ID NO: 73-77, respectively. It was found that LT86-22 and LT86-27 are very similar to each other. Comparison of these sequences with those of the gene bank, as described above, did not reveal significant homology with respect to LT86-22 and LT86-27. It was found that LT86-20, LT86 -21 and LT86-26 exhibit homology with respect to previously identified genes.

In further studies, an cDNA expression library was prepared using mRNA from a small cell lung carcinoma cell line in a ZAP Express vector (Stratagene) based on lambda phage and screened, as described above, by two pooled sera of small cell lung carcinoma patients. The pooled sera were absorbed with an E. coli lysate and human PBMC lysate was added to the serum to block antibodies to proteins found in normal tissue. Seventy-three clones were isolated. The determined cDNA sequences for these clones are presented in SEQ ID NO: 290-362. Found that the sequence of SEQ ID NO: 289-292, 294, 296-297, 300, 302, 303, 305, 307-315, 317-320, 322-325, 327-332, 334, 335, 338-341, 343-352, 354-358, 360, and 362 exhibit some homology with respect to the previously isolated genes. It was found that the sequences of SEQ ID NO: 293, 295, 298, 299, 301, 304, 306, 316, 321, 326, 333, 336, 337, 342, 353, 359, and 361 exhibit some homology with respect to the previously identified EST .

Example 3

The use of murine antisera to identify DNA sequences encoding lung tumor antigens

This example illustrates the isolation of cDNA sequences encoding lung tumor antigens by screening lung tumor cDNA libraries with mouse antitumor antisera.

A targeted expression cDNA library of lung tumors was obtained as described above in Example 2. Sera were obtained from SCID mice containing inoculated squamous tumor and human adenocarcinoma. Serum data were pooled and administered to normal mice to produce antiserum against lung tumor. From an unamplified library, approximately 200,000 PFUs were screened using this antiserum. Using a goat secondary antibody against mouse IgG-A-M (H + L) with alkaline phosphatase, approximately 40 positive plaques were identified by developing a reaction with NBT / BCIP (BRL Labs.). The phage were purified and phagemid was excised for 9 clones with insertions into the pBK-CMV vector for expression in prokaryotic or eukaryotic cells.

The determined cDNA sequences for 7 of the selected clones (hereinafter referred to as L86S-3, L86S-12, L86S -16, L86S-25, L86S-36, L86S-40 and L86S-46) are presented in SEQ ID NO: 49 -55, with the corresponding predicted amino acid sequences shown in SEQ ID NO: 56-62, respectively. The 5'-cDNA sequences for the remaining 2 clones (hereinafter referred to as L86S-30 and L86S-41) are presented in SEQ ID NO: 63 and 64. Subsequently, it was determined that L86S-36 and L86S-46 are the same gene. Comparison of these sequences with those in the available database, as described above, did not reveal significant homology for clones L86S-30, L86S-36 and L86S-46 (SEQ ID NO: 63, 53 and 55, respectively). L86S-16 (SEQ ID NO: 51) was found to exhibit some homology with respect to the EST previously identified in a fetal lung tumor and a germ line tumor. The remaining clones were found to exhibit at least some degree of homology to previously identified human genes. The subsequently determined extended cDNA sequences for L86S-12, L86S-36 and L86S-46 are presented in SEQ ID NO: 78-80, respectively, with the corresponding predicted amino acid sequences presented in SEQ ID NO: 81-83.

Subsequent studies led to the determination of 5'-cDNA sequences for an additional nine clones designated as L86S-6, L86S-11, L86S-14, L86S-29, L86S-34, L86S-39, L86S-47, L86S-49 and L86S -51 (SEQ ID NO: 84-92, respectively). The corresponding predicted amino acid sequences are presented in SEQ ID NO: 93-101, respectively. Found that L86S-30, L86S-39 and L86S-47 are similar to each other. Comparison of these sequences with those of the gene bank, as described above, did not reveal significant homology with respect to L86S-14. L86S -29 was found to exhibit some homology with respect to the previously identified EST. It was found that L86S-6, L86S-11, L86S-34, L86S-39, L86S-47, L86S-49 and L86S-51 exhibit some homology with respect to the previously identified genes.

In further studies, a targeted cDNA library was constructed using a Stratagene kit with a ZAP Express lambda phage vector. Total RNA for the library was taken from two primary squamous lung tumors, and polyA + RNA was isolated using an oligo-dT column. Antiserum was generated in normal mice using pooled sera from 3 SCID mice with implanted squamous cell carcinomas of the human lung. Approximately 700,000 PFUs were screened from an unamplified library by absorbed E. coli antitumor mouse serum with SCID. Positive plaques were identified as described above. The phage were purified and phagemid was cut for 180 clones with insertions into the pBK-CMV vector for expression in prokaryotic or eukaryotic cells.

The determined cDNA sequences for 23 of the selected clones are presented in SEQ ID NO: 126-148. Comparison of these sequences with those in an accessible database, as described above, did not reveal significant homology with respect to sequences SEQ ID NO: 139 and 143-148. The sequences of SEQ ID NOs: 126-138 and 140-142 were found to exhibit homology with respect to previously identified human polynucleotide sequences.

Example 4

The use of murine antisera for screening lung tumor libraries obtained from SCID mice

This example illustrates the isolation of cDNA sequences encoding lung tumor antigens by screening for lung tumor cDNA libraries obtained from SCID mice using murine antitumor sera.

Directional expression cDNA library of lung tumors was obtained using a Stratagene kit with a vector based on the phage lambda ZAP Express. Total RNA for the library was taken from late inoculated squamous adenocarcinoma of the lung growing in a mouse with SCID. PolyA + RNA was isolated using the Message Maker kit (Gibco BRL). Serum was obtained from two SCID mice implanted with lung adenocarcinomas. Serum data were pooled and administered to normal mice to produce antiserum against lung tumor. Approximately 700,000 PFUs were screened from an unamplified library by absorbed E. coli antitumor serum from SCID mice. Using goat anti-mouse IgG-A-M (H + L) secondary antibody with alkaline phosphatase, positive plaques were identified when developing a reaction with NBT / BCIP (Gibco BRL). The phage were purified and phagemid was excised for 100 clones inserted into the pBK-CMV vector for expression in prokaryotic or eukaryotic cells.

The determined 5 'cDNA sequences for 33 of the isolated clones are presented in SEQ ID NO: 149-181. The corresponding predicted amino acid sequences for SEQ ID NO: 149, 150, 152-154, 156-158 and 160-181 are presented in SEQ ID NO: 182, 183, 186, 188-193 and 194-215, respectively. The clone with SEQ ID NO: 151 (designated as SAL-25) was found to contain two open reading frames (ORFs). The predicted amino acid sequences encoded by the ORF data are described in SEQ ID NO: 184 and 185. The clone with SEQ ID NO: 153 (designated as SAL-50) was found to contain two open reading frames encoding the predicted amino acid sequences with SEQ ID NO: 187 and 216. Similarly, it was found that the clone with SEQ ID NO: 155 (designated as SAL -66) contained two open reading frames encoding the predicted amino acid sequences with SEQ ID NO: 189 and 190. Comparison of these sequences with those in an accessible database data did not reveal significant homology with respect to clones with SEQ ID NO: 151, 153 and 154. The sequences of SEQ ID NO: 149, 152, 156, 157 and 158 were found to exhibit some homology with respect to the previously isolated Expression Sequence Labels (EST). It was found that the sequences of SEQ ID NO: 150, 155 and 159 -181 exhibit homology with respect to sequences previously identified in humans.

Using the above procedures, two targeted cDNA libraries (designated LT46-90 and LT86-21) were obtained from two late inoculated squamous lung carcinomas grown in SCID mice and screened with sera obtained from SCID mice that were implanted with squamous lung carcinomas person. Certain cDNA sequences for isolated clones are presented in SEQ ID NO: 217-237 and 286-289. SEQ ID NO: 286 was found to be the longest sequence in LT4690-71 (SEQ ID NO: 237). Comparison of these sequences with those in the available database did not reveal a known homology for the sequences of SEQ ID NO: 219, 220, 225, 226, 287 and 288. It was found that the sequences of SEQ ID NO: 218, 221, 222 and 224 exhibit some homology in relation to previously identified sequences with unknown function. The sequence of SEQ ID NO: 236 was found to exhibit homology with respect to the sequence of known mRNA. The sequences of SEQ ID NO: 217, 223, 227-237, 286 and 289 showed some homology with respect to known human DNA and / or RNA sequences.

In further studies using the methods described above, one of the cDNA libraries described above (LT86-21) was screened by absorbed E. coli mouse serum against a tumor with SCID. This serum was obtained from normal mice immunized with 3 pooled sera from SCID mice with implanted human lung squamous cell carcinomas. The determined cDNA sequences for the isolated clones are presented in SEQ ID NO: 238-285. Comparison of these sequences with those in the available databases did not reveal significant homology for clones of SEQ ID NO: 253, 260, 277 and 285. It was found that the sequences of SEQ ID NO: 249, 250, 256, 266, 276 and 282 show some homology in relation to previously allocated labels of expressed sequences (EST). The sequences of SEQ ID NO: 238-248, 251, 252, 254, 255, 257-259, 261-263, 265, 267-275, 278-281, 283 and 284 were found to exhibit some homology with respect to the previously identified DNA sequences or human RNA.

The expression levels of some of the isolated antigens in lung tumor tissues compared to expression levels in normal tissues were determined using microfields technology. The results of these studies are shown below in table 2 together with the results of the analysis of the data bank for these sequences.

table 2 Clone SEQ ID NO Description LT + F / N SCC + M / N Squa / n Adeno / n 2LT-3 238 Unknown (KIAA0712) 2.2 3.8 3.3 - 2LT-6 239 Lactate-DG B 2,3 3.8 4.1 - 2LT-22 240 Fumarate hydratase - 3.0 - - 2LT-26 242 Cg1-39 - - 12.8 - 2LT-31 243 ADH7 - - 8.4 2.2 2LT-36 244 ADH7 - 2,4 2.0 - 2LT-42 245 HMG-CoA synthase 2.2 2.6 2.2 - 2LT-54 247 (Mus) Ninein - 2.1 - - 2LT-55 248 Ubiquitin 2.2 - 2,5 2.0 2LT-57 249 New 2.1 2.9 2,4 - 2LT-58 250 New 2,3 4.0 2.9 - 2LT-59 251 Unknown KIAA0784 2,4 3.0 2,3 2.0 2LT-60 252 Nuc Pore Cmplx- Ass. TPR protein - - - 2.1 2LT-70 256 Unknown KIAA0871 - 2,5 2.2 2.1 2LT-73 257 Mus, polyadenylate binding - 2.0 - - 2LT-76 259 Trans Golgi p230 2.1 - 2.6 - 2LT-85 263 Ribosomal Protein (LS29) - - - 2.1 2LT-89 265 Unknown PAC212G6 - 2.0 - - 2LT-98 268 Protein, ass. with diff. melanomas 9 - - - 2.2

2LT-100 269 Mus Collagen Alpha VI - - - 2.1 2LT-105 271 Antigen NY-CO-7 - 3.2 - - 2LT-108 273 Unknown RG363M04 - 3,1 - - 2LT-124 279 Galectin-9 (secreted) 2,3 2.7 2.0 - 2LT-126 280 L1 element L1.33 p40 2.5 - 3,1 - 2LT-128 282 New (Kappa B -ras 2) 2,3+ - 20,4 2.5 2LT-133 284 Spectrin Alpha II - 2,3 - - LT + F / N = Lung tumor plus fetal tissue relative to normal tissues
SCC + M / N = Small cell lung carcinoma plus metastatic normal tissue
Squa / N = Squamous cell tumor of the lung in relation to normal tissues
Aden / N = Adenocarcinoma with respect to normal tissues

Studies on the determination of the full-length sequence of the 2LT-128 antigen (SEQ ID NO: 282) resulted in the isolation of the full-size cDNA sequence shown in SEQ ID NO: 392. This amino acid sequence encoded by this full-size cDNA sequence is presented in SEQ ID NO: 393. This antigen characterized by a 20-fold increase in expression in squamous cell carcinoma and a 2.5-fold increase in expression in lung adenocarcinoma. This gene has been described as a potential ras oncogene (Fenwick et al. Science, 287: 869-873, 2000).

Extended sequence information was obtained for clones 2LT-3 (SEQ ID NO: 238), 2LT-26 (SEQ ID NO: 242), 2LT-57 (SEQ ID NO: 249), 2LT-58 (SEQ ID NO: 250 ), 2LT-98 (SEQ ID NO: 268) and 2LT-124 (SEQ ID NO: 279). Extended cDNA sequences for these clones are shown in SEQ ID NO: 428-433, respectively, and encode the polypeptide sequences shown in SEQ ID NO: 434-439, respectively.

Example 5

Determination of tissue specificity of lung tumor polypeptides

Using gene-specific primers, mRNA expression levels for representative lung tumor polypeptides in various normal and tumor tissues were evaluated by RT-PCR.

Briefly, total RNA was extracted from various normal and tumor tissues using Trizol reagent. The first strand was synthesized using 2 μg of total RNA with SuperScript II reverse transcriptase (BRL Life Technologies) at 42 ° C for one hour. Then the cDNA was amplified by PCR with gene-specific primers. To be sure of the semi-quantitative nature of RT-PCR, β-actin was used as an internal control in each of the evaluated tissues. 1 μl of a diluted 1:30 cDNA solution was used to start the linear region of amplification of the β-actin matrix, and this was sensitive enough to reflect differences in the initial number of copies. Using these conditions, the level of β-actin was determined for the reverse transcription reaction from each tissue. DNA contamination was minimized by treatment with DNase and by confirming the negative result of PCR using the first cDNA strand, which was obtained without the addition of reverse transcriptase.

MRNA expression levels were evaluated in five different types of tumor tissue (squamous cell lung tumor from 3 patients, lung adenocarcinoma, prostate tumor, colon tumor and lung tumor) in various normal tissues, including four patients lung, prostate gland, brain, kidney , liver, ovary, skeletal muscle, skin, small intestine, myocardium, retina and testicles. It was found that L86S-46 was expressed at a high level in a squamous cell tumor of the lung, a tumor of the colon and a tumor of the prostate, and could not be detected in other evaluated tissues. L86S-5 was found to be expressed in lung tumor samples and in 2 of 4 normal lung samples, but not in other normal or tumor tissues tested. L86S-16 was found to be expressed in all tissues except normal liver and normal stomach. Using real-time PCR, it was found that L86S-46 was overexpressed in the squamous tissue of the lungs and normal tissue of the tonsils, and its expression in all other tissues evaluated was low or not detected.

Example 6

Isolation of DNA sequences encoding lung tumor antigens

DNA sequences encoding antigens potentially involved in the formation of a squamous cell tumor of the lungs were isolated as follows.

A targeted expression cDNA library of lung tumors was constructed using a ZAP Express phage lambda expression system (Stratagene, La Jolla, CA). Total RNA for the library was taken from two pooled squamous epithelial tumors of a human lung, and polyA + RNA was isolated using oligo dT cellulose (Gibco BRL, Gaithersburg, MD). Phagemide was randomly extracted and the cDNA sequences of the isolated clones were determined.

The specific cDNA sequence for clone SLT-Tl is presented in SEQ ID NO: 102, with specific 5'-cDNA sequences for clones SLT-T2, SLT-T3, SLT-T5, SLT-T7, SLT-T9, S-LT-T10 , SLT-T11 and SLT-T12 presented in SEQ ID NO: 103-110, respectively. The corresponding predicted amino acid sequences for SLT-T1, SLT-T2, SLT-T3, SLT-T10 and SLT-T12 are presented in SEQ ID NO: 111-115, respectively. Comparison of the sequences SLT-T2, SLT-T3, SLT-T5, SLT-T7, SLT-T9 and SLT-T11 with those in the available databases, as described above, did not reveal significant homology. The sequences SLT-T10 and SLT-T12 were found to exhibit some homology with respect to sequences previously identified in humans.

It was determined that the sequence of SLT-T1 shows some homology with respect to the PAC clone with unknown protein function. The SLT-T1 cDNA sequence (SEQ ID NO: 102) was found to contain a mutator domain (MUTT). Such domains are known to function in the removal of damaged guanine from DNA, which may result in a permutation of A instead of G (see, for example, el-Deiry, WS, 1997, Curr. Opin. Oncol. 9: 79-87; Okamoto, K . et al., 1996 Int. J. Cancer 65: 437-41; Wu, C. et al., 1995 Biochem. Biophys. Res. Commun. 214: 1239-45; Porter, DW et al., 1996 Chem. Res. Toxicol. 9: 1375-81). Thus, SLT-T1 can be used in the treatment through gene therapy of lung cancers caused or associated with impaired DNA repair.

In further studies, DNA sequences encoding antigens potentially involved in the formation of lung adenocarcinoma were isolated as follows. A targeted expression cDNA library of human lung tumors was constructed using the expression system based on ZAP Express lambda phage (Stratagene, La Jolla, CA). Total RNA for the library was taken from late human adenocarcinoma, which was inoculated with SCID mice, and polyA + RNA was isolated using the Message Maker kit (Gibco BRL, Gaithersburg, MD). Phagemide was randomly extracted and the cDNA sequences of the isolated clones were determined.

The defined 5'-cDNA sequences for the five selected clones (designated as SALT-T3, SALT-T4, SAL-T7, SALT-T8 and SALT-T9) are presented in SEQ ID NO: 116-120, with the corresponding predicted amino acid sequences represented by in SEQ ID NO: 121-125. SALT-T3 was found to be 98% identical to the previously identified human transducine-like TLE2 enhancer protein. SALT-T4 turned out to be the human homologue of the mouse H-beta 58 gene. SALT-T7 was found to have 97% identity with respect to human-mercaptopropyl sulfate transferase, and SALT-T8 was found to be homologous to the human interferon-induced protein 1-8U . SALT-T9 exhibits approximately 90% identity with respect to the human mucin MUC 5B.

The cDNA sequences encoding antigens potentially involved in the formation of small cell lung carcinoma were identified as follows. Expression cDNA libraries were constructed based on mRNA from small cell lung carcinoma cell lines NCIH69, NCIH128, and DMS79 (all available from the American Type Culture Collection, Manassas, VA) using a ZAP Express phage lambda expression system (Stratagene, La Jolla, CA). Phagemide was randomly extracted and the cDNA sequences of 27 isolated clones were determined. Comparison of certain cDNA sequences did not reveal significant homology with respect to the sequences of SEQ ID NO: 372 and 373. The sequences of SEQ ID NO: 364, 369, 377, 379 and 386 showed some homology with respect to the previously isolated EST. The sequences of the remaining 20 clones showed some homology with respect to the previously identified genes. The cDNA sequences of these clones are presented in SEQ ID NO: 363, 365-368, 370, 371, 374-376, 378, 380-385 and 387-389, where SEQ ID NO: 363, 366-368, 370, 375, 376 , 378, 380-382, 384, and 385 are full length sequences.

Comparison of the cDNA sequence of SEQ ID NO: 372 indicates that this clone (designated as 128T1) is a new member of the family of putative transmembrane proteins that penetrate the membrane seven times. More specifically, using the PSORT computer algorithm, this protein was predicted to be type IIIA transmembrane protein seven times permeating the membrane. A Genomic clone was identified in the Genbank database that contains the predicted 58 N-terminal amino acids lost in the amino acid sequence encoded by SEQ ID NO: 372. The determined full-length cDNA sequence for clone 128T1 is presented in SEQ ID NO: 390, with the corresponding predicted amino acid sequence, presented in SEQ ID NO: 391.

The expression levels of some of the isolated antigens in lung tumor tissues compared to expression levels in normal tissues were determined using microfields technology. The results of these studies are shown below in table 3 together with the results of the analysis of the data bank for these sequences.

Table 3 Clone SEQ ID NO Description LT + F / N SCC + M / N Squa / n Adeno / n DMS79-T1 363 STAT-Hindu inhib. cytokine - 2.0 - - DMS79-T6 367 Related to neural cell death - 2.2 - - DMS79-T9 369 New - 2.2 - - DMS79-T10 370 Ubiquitin Carrier Protein - 3.9 2.2 - DMS79-T11 371 HPV16E1 binding protein - 2.1 - - 128-T9 378 Elongation factor 1 alpha - 2.7 - - 128-T11 380 Malate dehydrogenase - 2,3 2.0 - 128-T12 381 Apuric / Apyrim. endonuclease - 5,4 - - NCIH69-T3 382 Sm-like CaSm protein - - 2,4 - NCIH69-T6 384 BTF3a Transcription Factor - 2.5 - - LT + F / N = Lung tumor plus fetal tissue relative to normal tissues
SCC + M / N = Small cell lung carcinoma plus metastatic normal tissue
Squa / N = Squamous cell tumor of the lung in relation to normal tissues
Aden / N = Adenocarcinoma with respect to normal tissues

Example 7

The synthesis of polypeptides

The polypeptides can be synthesized on a Perkin Elmer / Applied Biosystems Division 430A peptide synthesizer using FMOC chemistry with HPTU activation (O-benzotriazole-N, N, N ', N'-tetramethyluronium hexafluorophosphate). The Gly-Cys-Gly sequence can be linked at the N-terminus to provide a method for conjugation, binding to an immobilized surface, or labeling of a peptide. The peptide can be cleaved from the solid base using the following cleavage mixture: trifluoroacetic acid: ethanedithiol: thioanisole: water: phenol (40: 1: 2: 2: 3). After cleavage for 2 hours, the peptides can precipitate with cold methyl tert-butyl ether. Precipitated peptides can then be dissolved in water containing 0.1% trifluoroacetic acid (TFA) and lyophilized prior to purification by reverse phase C18 HPLC. A gradient of 0% -60% acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA) can be used to elute the peptides. After lyophilization of the pure fractions, the peptides can be characterized using electrospray or other types of mass spectrometry and by amino acid analysis.

Example 8

Isolation and characterization of DNA sequences encoding lung tumor antigens by expression cloning of T cells

Lung tumor antigens can also be identified by expression cloning of T cells. One of the sources of tumor-specific T cells is their isolation from surgical tumors of patients.

Non-small cell lung carcinoma was crushed and subjected to enzymatic treatment for several hours to release tumor cells and infiltrating lymphocytes (tumor infiltrating T-lymphocytes, or TIL). Cells were washed with HBSS buffer and passed through a continuous Ficoll gradient (100% / 75% / HBSS) to separate tumor cells and lymphocytes from non-viable cells. Two strips were taken from the interface; the upper band at the 75% / HBSS interface predominantly contained tumor cells, while the lower band at the 100% / 75% / HBSS interface contained most of the lymphocytes. TIL was propagated in culture or in 24-well plates with culture medium supplemented with 10 ng / ml IL-7 and 100 units / ml IL-2, or, alternatively, in 24-well plates that were previously coated with anti-CD3 monoclonal antibody OKT3. The resulting TIL cultures were analyzed by FACS to confirm that a large percentage of CD8 ⁺ T cells (> 90% of the selected population) with only a small percentage of CD4 ⁺ cells.

In addition, non-small cell lung carcinoma cells were propagated in culture using standard methods for deriving a tumor cell line (designated as LT391-06), and later it was confirmed by immunohistochemical analysis that it was a lung carcinoma cell line. This tumor cell line was transduced with a retroviral vector for expression of human CD80, and characterized by FACS analysis to confirm the high expression level of CD80, MHC class I molecules and MHC class II.

The ability of TIL lines to specifically recognize an autologous lung tumor was demonstrated by analysis of cytokine release (IFN-γ and TNF-α), as well as by analysis of ⁵¹ Cr release. Briefly, TIL cells from 21-day-old cultures were cultured together with autologous and allogeneic tumor cells, EBV-immortalized LCL, or Daudi and K562 control cell lines, and the culture supernatant was monitored by ELISA for cytokines. TILs specifically recognized an autologous tumor, but not an allogeneic tumor. In addition, EBV-immortalized LCL or control cell lines were not recognized, which means that TIL lines are tumor-specific and potentially recognize tumor antigens represented by autologous MHC molecules.

The characterized tumor-specific TIL lines were propagated to appropriate amounts for expression cloning of T cells using soluble anti-CD3 antibody in a culture with irradiated EBV-transformed LCL and PBL nurse cells in the presence of 20 units / ml IL 2. Clones from the expanded TIL lines were generated with standard limited breeding methods. More specifically, TIL cells were plated at 0.5 cells / well in a 96-well U-bottom plate and stimulated with autologous tumor cells with transduced CD-80, EBV-transformed LCL and PBL nurse cells in the presence of 50 u / ml IL -2. The specificity of these clones for an autologous tumor was confirmed by bioassays of ⁵¹ Cr microcytotoxicity and IFN-γ.

In antibody blocking experiments, it was demonstrated that these CTL clones were HLA-B / C restricted. A representative CTL clone was tested on an allogeneic lung carcinoma panel and it recognized an autologous tumor and squamous cell carcinoma of the lung (936T). Since the only common MHC class I for these tumors was HLA-Cw1203, this meant that it was the restriction element used by CTL. This result was confirmed by CTL recognition of a number of allogeneic lung carcinomas transduced with a retroviral vector encoding HLA-Cw1203.

PolyA mRNA was obtained from a lung tumor cell line designated LT391-06 using Message Maker (Life Technologies; Rockville, MD). Subsequent steps, including cDNA synthesis, were performed according to the Life Technologies cloning guide (SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning). Modifications to the protocol were carried out as follows. EcoRI adapters (New England Biolabs; Beverly, MA) were used at the stage of adding the adapter. Size-fractionated cDNAs ligated the HisMax A, B, C expression vector system (Invitrogen; Carlsbad, CA) to optimize protein expression in all three coding frames. The library plasmids were then aliquoted at approximately 100 CFU / well in a 96-well liquid amplification unit overnight. A reserve of these cultures in glycerol was obtained and the combined plasmid was obtained by an automated robot and pooled plasmid was (Qiagen; Valencia, CA). The concentration of plasmid DNA in each well of library plates was determined to be approximately 150 ng / μl. The initial characterization of the cDNA expression library was carried out by randomly determining the sequence of 24 primary transformants, and the resulting sequence was subjected to BLAST searches in available databases. The determined cDNA sequences are presented in SEQ ID NO: 443-480, with the BLAST search results shown in Table 4.

Table 4 Clone SEQ ID NO: GenBank Event Number Description 55163 458, 459   New Arrival at GenBank 55158 452   New Arrival at GenBank Clone SEQ ID NO: GenBank Event Number Description Homology with respect to known sequences with unknown function 55153 443, 444 7018516 H. sapiens mRNA; cDNA DKFZp434M035 55154 445, 446 6437562 H. sapiens, chrome. 22q11, clone PAC p393 55157 450, 451 2887408 H. sapiens KIAA0417 mRNA 55165 462, 463 3970871 H. sapiens HRIHFB2122 mRNA Homology with respect to known sequences with known function 55155 447 7677405 H. sapiens F-Box Protein (FBS) 55156 448, 449 3929584 Pseudogen H. sapiens EEN 55161 454, 455 4503350 DNA (cytosine-5 -) - methyltransferase 1 (DNMT1) H. sapiens 55162 456, 457 31220 ERK1 mRNA for serine-threonine protein kinase 55164 460, 461 6677666 RNA-binding protein (autoantigenic) H. sapiens (RALY) 55166 464, 465 3249540 The p40 subunit of H. sapiens ribonuclease protein (RPP40) 55167 466, 467 7657497 Kidney Tumor Antigen (RAGE) H. sapiens 55168 468, 469 2873376 exportin t H. sapiens mRNA 55169 470, 471 3135472 mRNA of a ^2nd protein similar to Cre binding protein, H. sapiens 55171 474 4759151 Spermine synthase H. sapiens (SMS) 55173 476 6688148 Part of the mice of NICE-3 H. sapiens protein 55174 477, 478 531394 PC4 human transcription coactivator

Clone SEQ ID NO: GenBank Event Number Description 55175 479 6563201 Delta subunit of H. sapiens translation initiation factor eIF-2b 55176 480 29860 Gene hCENP-B, autogenous centromere B (CENP-B) Ribosomal protein homology 55159 453 337494 mRNA of the large subunit of the ribosomal protein L7a (surf 3) 55170 472, 473 4506648 H.sapiens ribosomal protein mRNA m3 55172 475 388031 H. sapiens ribosomal protein L11

For T-cell screening, approximately 80 ng of the library plasmid DNA and 80 ng of the HLA-Cw1203 plasmid DNA were mixed with the Fugene lipid preparation according to the manufacturer's instructions, and transfected with the COS-7 cell mixture in two parallels. After incubation at 37 ° C for 48 hours, the transfection mixture was selected and 10,000 CTL LT391-06 was added to each well in fresh medium containing human serum.

The ability of T cells to recognize antigen in the library was evaluated by the release of cytokines after 6 hours (TNF-alpha, WEHI bioanalysis) or after 24 hours (IFN-gamma, ELISA). About 2.0 × 10 ⁵ clones (in pools of 100 plasmids) were screened using this system in COS-7 cells. Three plasmid pools (identified as 14F10, 19A4 and 20E10) were identified that were recognized by CTL LT391-06. Transfection of COS-7 cell plasmids with these pools resulted in CTL LT391-06 production of both IFN-gamma and TNF-alpha at concentrations significantly higher than background. The 14F10, 19A4, and 20E10 pools were “split” into several hundred individual plasmid DNAs and were tested again. The sequence of 24 new clones isolated from the pool 14F10 presented in SEQ ID NO: 481-511.

One plasmid (3D9) from the 14F10 pool, one plasmid from the 20E10 pool and 5 plasmids (2A6, 2E11, 2F12, 3F4, 3H8) from the 19A4 pool were able to reproduce recognition by T cells. Sequencing of these plasmids led to the identification of a cDNA insert of 7.8 thousand n.p. (designated as clone 14F10), 2.2 kbp cDNA inserts (designated as l9A4; SEQ ID NO: 440) and a clone designated as 20E10. The full-length cDNA sequence for 14F10 is presented in SEQ ID NO: 441. Clone 14F10 did not contain the first two nucleotides "G" detected from the 5'-end of 19A4, and the 3'-terminal 24 n.p. 19A4 differed from the corresponding region of 14F10 (nucleotides 2145-2165). Moreover, an additional 3837 bp were isolated for clone 14F10 3'-terminal sequence. The 5 ′ end cDNA sequence (337 bp) of clone 20E10 is shown in SEQ ID NO: 442. 20E10 contains an additional 3 nucleotides (compared to 19A4) at the 5 ′ end. The additional sequence from the 5'-end of clone 20E10 contains “ATG” and therefore appears to contain a translation start portion of a new open reading frame. When analyzing the BLAST search in GenBank databases, these sequences were identified as having significant homology for the truncated human cystine / glutamic acid transporter gene. However, unlike the published sequence, clones 14F10 and l9A4 contained a unique 5'-end, consisting of 181 nucleotides. This new sequence replaced the published 5'-region and resulted in a shift in the described initiating methionine (start codon) and the appearance of two additional amino acid residues compared to the described transporter protein. Therefore, the translated product of clones 14F10 and 19A4 is different from the cystine / glutamic acid transporter protein. Moreover, recognition by T cells of other lung tumors demonstrates that this antigen is also expressed in other tumors.

The epitope and amino acid sequence encoded in clones 19A4 and 14F10, which reproduces T cell recognition of anti-LT391-06 cells, were mapped as follows. Cos-7 cells transfected 80 ng / well of HLA-Cw1203 along with titratable amounts of cDNA encoding clone 19A4, a potential open reading frame located at the unique 5'-end of 19A4, or an open reading frame of the cystine / glutamic acid transporter gene (Cys-Glu ) were cloned into a eukaryotic expression vector, and tested for stimulation of anti-LT391-06-T cells in a TNF assay. As a positive control, Cos-7 cells were co-transfected with HLA-Cwl203 and the above positive plasmid clone 19A4. The Cys-Glu transporter expressing construct was isolated by PCR using 5'- and 3'-primers specific for the known ORF transporter, with 19A4 as template. In addition, each 5'-primer contained a Kozak translation initiation site and a start methionine for translation of the polypeptide. CTLs against LT391-06 did not recognize transfectants expressing the Cys-Glu transporter construct, but transfectants expressing 19A4 5'-ORF from 19A4 were recognized.

In subsequent experiments, Cos-7 cells were co-transfected with 80 ng / well HLA-Cw1203 together with titratable amounts of transpositional mutants F10 and C12 DNA, respectively, and tested for stimulation of anti-LT391-06-T cells in a TNF assay. As a positive control, Cos-7 cells were co-transfected with HLA-Cwl203 and clones with 5'-ORF 19A4. Transposition mutants F10 and C12 were obtained by transposon-mediated mutation of clone 14F10 and screening of the insertion sites by sequence analysis. The transposon of the F10 mutant was inserted at approximately 304 n.p. from the 5'-end region of cloning EcoRI cDNA 14F10. This mutation did not cancel the translation of the T-cell epitope. On the contrary, it was found that transposon C12, which was inserted at approximately 116 n.p. from the 5'-end region of the cloning of EcoRI cDNA 14F10, interrupted the translation of the T-cell epitope. Thus, the 14F10 epitope maps between these two transposon insertion sites. The amino acid sequence of the region between the transposon insertion sites C12 and F10 is shown in SEQ ID NO: 586.

A series of 11 overlapping 16-membered and 15-membered peptides was obtained for the region represented in SEQ ID NO: 586, and they were tested for stimulation of anti-LT391-06 cells, which was determined by the release of cytokines in IFN-γ TNF assays. The only peptide provided in SEQ ID NO: 587 (corresponding to residues 5-20 of SEQ ID NO: 586) stimulated the release of the cytokine. These studies demonstrate that the restricted HLA-Cw1203 antigen is contained in SEQ ID NO: 587.

Example 9

Isolation and characterization of DNA sequences encoding lung tumor antigens by subtracting PCR

This example describes the isolation and characterization of cDNA clones of an expression library obtained by subtracting PCR from the human lung tumor cell line LT391-06 described above.

The tester polyA-mRNA was obtained from the LT391-06 cell line as described above. The driver polyA-mRNA was isolated from the T-lymphocytic cell line of human acute T-cell leukemia (Jurkat), which is not of pulmonary origin and is not recognized by LT391-06-reactive T-cells. Subtraction was performed according to the Clontech method (Palo Alto, CA) with the following changes: 1) a second restriction digestion of cDNA was performed using combined enzymes (MscI, PvuII, StuI and DraI). This was a complement and difference from cleavage with the single restriction enzyme RsaI recommended by Clontech. Each restriction digestion kit was processed as a separate library to ensure that the final mixed library contained overlapping fragments. Thus, the epitope recognized by T cells should be represented on a fragment in the library and should not be destroyed due to the presence of a single restriction enzyme site. 2) The ratio of guide and test cDNA was increased in the hybridization step to increase the subtraction hardness. To analyze the effectiveness of subtraction in dilutions of subtracted as well as unaccustomed PCR samples, actin was amplified by PCR. In the second stage of the PCR, primers were used that were different from those commonly used. Three nested PCR primers were designed to contain a cleavable EcoRI site (not involved during cloning), which was in one of three frames. Thus, the result of the second amplification with these primers was products that could be ligated directly into the eukaryotic expressing plasmid pcDNA4His / Max-Topo (Invitrogen). This led to the formation of PCR subtracted and amplified fragments presented in a frame somewhere in the library. Due to the subtraction mechanism, only 50% of the fragments were in the correct orientation. The complexity and redundancy of the library was characterized by determining the sequence of 96 randomly selected clones from the finished pooled expression library obtained by subtracting PCR, designated as LT391-06PCR. Sequence data (SEQ ID NO: 512-581) was analyzed by comparison with sequences in freely available databases (Table 5).

Table 5 Clone SEQ ID NO Stock Number at GenBank Description 57235 532 New Arrival at GenBank 57255 547 New Arrival at GenBank 57264 554 New Arrival at GenBank Homology with respect to known sequences with unknown function 57215 518 5689540 mRNA for KIAA1102 H. sapiens protein 57223 522 2341006 Human Xq13 3'-end PAC92E23 57227 524 7022540 cDNA FLJ10480 fis H. sapiens, clone NT2RP2000126 57238 535 6807795 H. sapiens mRNA; cDNA DKFZp761 G02121 57239 536 5757546 Clone H. sapiens DJ0823F17 57243 539 7023805 cDNA FLJ11259 fis H. sapiens, clone PLACE1009045 57245 540 4884472 H. sapiens mRNA; cDNA DKFZp586O2223 57267 557 6808218 H. sapiens mRNA; cDNA DKFZp434O1519 57268 558 10040400 Sequence 12 of Patent WO9954460 57270 560 7959775 H. sapiens PRO1489 mRNA 57271 561 4500158 H. sapiens mRNA; cDNA DKFZp586B0918 Clone SEQ ID NO Stock Number at GenBank Description 57281 567 6560920 Clone H. sapiens RP11-501O7 57283 569 285962 Human mRNA for the KIAA0108 gene 57285 570 7019813 H. sapiens cDNA FLJ20002 fis, clone ADKA01577 Homology with respect to known sequences with known function 57207 512 517176 H. sapiens YAP65 mRNA 57210 514 6841233 H. sapiens HSPC292 mRNA 57211 515 2606093 mRNA of Cyr61 protein (CYR61) H. sapiens 57212 516 339648 human thioredoxin mRNA (TXN) 57219 519 4504616 Protein 3, binding to insulin-like growth factor (IGFBP3) H. sapiens 57221 520 7274241 New retinal pigment epithelial cell protein (NORPEG) H. sapiens 57222 521 189564 Human plasminogen activator inhibitor-1 gene 57228 525 4757755 Annexin A2 (ANXA2) H. sapiens 57230 527 180800 Type IV alpha-1 collagen human gene, exon 52 57232 529 6729061 Clone RPC11-98D12 of 7q31 H. sapiens 57233 530 338391 Spermidine / Spermine-N1-Acetyltransferase 57234 531 7305302 NCK Associated Protein 1 (NCKAP1) H. sapiens 57236 533 4929722 Protein CGI-127 H. sapiens

Clone SEQ ID NO Stock Number at GenBank Description 57242 538 4503558 Epithelial membrane protein 1 (EMP1) H. sapiens 57248 541 183585. Human pregnancy-specific beta-glycoprotein c 57250 543 4759283 C-terminal esterase of ubiquitin L1 (UCHL1) H. sapiens 57251 544 1236321 The human gene for gamma-2-chain laminin (LAMC2) 57253 545 213831 Isoform 2 of lysyl hydroxylase (PLOD2) H. sapiens 57254 546 536897 follicatin-related human protein precursor mRNA 57257 548 339656 Human endothelial cell thrombomodulin 57258 549 190467 human prion protein (PrP) mRNA 57261 551 338031 Human Serglicin Gene 57262 552 178430 Human Alphoid DNA (Alphoid Repeat Sequence) 57265 555 4502562 Calpain H. sapiens, Large L2 Polypeptide (CAPN2) 57266 556 398163 mRNA for protein 3 binding to insulin-like growth factor, H. sapiens 57269 559 7262375 Carboxyl esterase 2 (intestinal, hepatic) (CES2) 57272 562 467560 mRNA for cystindioxygenase type 1, H sapiens 57274 563 482664 Annexin A3 (ANXA3) H. sapiens

Clone SEQ ID NO Stock Number at GenBank Description 57275 564 2281904 Bruton tyrosine kinase (BTK), alpha-D-galactosidase A (GLA) H. sapiens 57277 565 4557498 C-terminal binding protein 2 (CTBP2) H. sapiens 57282 568 189245 Human NAD (P) H-Menadione Oxidoreductase mRNA 57287 571 28525 Human mRNA for Alzheimer's Disease Amyloid A4 Precursor 57288 572 4757755 Annexin A2 (ANXA2) H. sapiens 57289 573 5729841 glyoxalase I mRNA (GLO1) H. sapiens 57290 574 6103642 F-box protein FBX3 H. sapiens mRNA 57295 576 182513 human ferritin L chain mRNA 57299 579 37137 Human mRNA for thrombospondin 57301 580 179682 Beta chain of C4b binding protein (clone A12) 57302 581 6042205 Membrane metalloendopeptidase (neutral endopeptidase, enkephalinase, CALLA, CD10) (MME) H. sapiens 57213 517 2665791 Caveolin-2 mRNA H. sapiens 57259 550 2665791 Caveolin-2 mRNA H. sapiens 57225 523 179765 Human Calcicline Gene 57229 526 179765 Human Calcicline Gene 57237 534 186962 Human B2 Laminin Gene

Clone SEQ ID NO Stock Number at GenBank Description 57249 542 186962 Human B2 Laminin Gene 57231 528 4972626 Caveolin 1 gene (CAV1) H. sapiens 57296 577 4972626 Caveolin 1 gene (CAV1) H. sapiens 57297 578 4972626 Caveolin 1 gene (CAV1) H. sapiens 57240 537 266237 Protein 3 Binding Insulin-Like Growth Factor 57292 575 184522 The gene for human protein 3 that binds insulin-like growth factor 57263 553 4504618 Protein 7, binding to insulin-like growth factor (IGFBP7) H. sapiens 57280 566 4504618 Protein 7, binding to insulin-like growth factor (IGFBP7) H. sapiens Homology for ribosomal proteins 57209 513 337504 Human ribosomal protein S24 mRNA

Example 10

Isolation and characterization of T-cell receptors from a clone of T-cells specific for lung tumor antigen

This example describes the cloning and sequencing of the alpha and beta chains of a T cell receptor (TCR) from a clone of CD8-T cells specific for the antigen expressed by the LT391-06 lung tumor cell line. T cells have a limited lifespan. Cloning of TCR chains and subsequent transfer will essentially lead to the spread of various T cell specificities. Cloning of tumor antigen TCR chains allows specificity to be transferred to T cells isolated from patients who have MHC restriction TCR alleles. Such T cells can then undergo expansion and are used in adaptive transfer methods to introduce tumor antigen specificity to patients having tumors that express the antigen (see, for example, Clay et al. J Immunol. 163: 507 (1999)) .

Clones of cytotoxic T lymphocytes (CTLs) specific for the LT391-06 lung tumor cell line were obtained. Total mRNA from 2 × 10 ⁶ cells from 15 such clones was isolated using Trizol reagent and cDNA was synthesized using Ready-to-Go kits (Pharmacia). To determine the Va and Vb sequences in these clones, a panel of primers specific for the Va and Vb subtypes was synthesized and used in RT-PCR reactions with cDNA obtained for each of the clones. The RT-PCR reactions showed that each of their clones expressed the usual Vb sequence, which corresponded to the Vb13 subfamily. Using cDNA obtained from each of the clones (designated as 1105), the expressed Va sequence was determined as Va22. To clone full-length TCR alpha and beta chains from clone 1105, primers were designed to overlap TCR nucleotides encoding the initiator and terminator. Standard 35-cycle RT-PCR reactions were performed using cDNA synthesized from CTL clone and primers with PWO (BMB) as thermostable polymerase. The resulting specific bands (approximately 850 bp for the alpha chain and approximately 950 bp for the beta chain) were ligated into the blunt-ended PCR vector (Invitrogen) and transformed into E. coli. E. coli transformed with plasmids containing full-length alpha and beta chains was identified and large scale preparations of the corresponding plasmids were obtained. The sequence of plasmids containing full-length TCR alpha and beta chains was determined. The determined cDNA sequences for the alpha and beta chains are shown in SEQ ID NO: 583 and 582, respectively, with the corresponding amino acid sequences shown in SEQ ID NO: 584 and 585, respectively.

From the following it is understood that although specific embodiments of the invention are described here for illustrative purposes, various modifications may be made without departing from the spirit and scope of the invention. Therefore, the present invention is not limited by anything other than the appended claims.

Claims (13)

1. An isolated polynucleotide that is used to diagnose or treat lung cancer, containing a sequence selected from the group consisting of (a) the sequences shown in SEQ ID NO: 440, 441 and 442; (b) sequences complementary to the sequences presented in SEQ ID NO: 440, 441 and 442; (c) sequences that hybridize to the sequence set forth in SEQ ID NO: 440, 441, and 442 under moderately stringent conditions; (d) sequences having at least 90% identity with respect to the sequence of SEQ ID NO: 440, 441 and 442; and (e) degenerate variants of the sequence shown in SEQ ID NO: 440, 441 and 442. 2. An isolated polypeptide that is used to diagnose or treat lung cancer, containing an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 586 and 587; (b) sequences encoded by the polynucleotide according to claim 1; and (c) sequences having at least 90% identity with respect to the sequence encoded by the polynucleotide according to claim 1. 3. An expression vector containing the polynucleotide according to claim 1, operably linked by an expression control sequence. 4. An isolated antibody or antigen binding fragment thereof that specifically binds to the polypeptide of claim 2. 5. A method for determining the presence of a malignant tumor in a patient, comprising the steps of (a) receiving a biological sample from a patient; (b) contacting the biological sample with a binding agent that binds the polypeptide according to claim 2; (c) determining in the sample the amount of polypeptide that binds to a binding agent; and (d) comparing the amount of a given polypeptide with a predetermined cut-off level and thereby determining the presence of a malignant tumor in a patient. 6. The oligonucleotide used for the diagnosis of lung cancer, which hybridizes with the sequence presented in SEQ ID NO: 440, 441 and 442, under moderately stringent conditions. 7. A method of stimulating and / or expanding T cells specific for a tumor protein, comprising contacting the T cells with at least one component selected from the group consisting of (a) the polypeptides according to claim 2; (b) polynucleotides according to claim 1; and (c) antigen-presenting cells that express the polypeptide according to claim 2, under conditions and for a time sufficient to effect stimulation and / or expansion of T cells. 8. An isolated population of T cells containing T cells obtained by the method according to claim 7. 9. A method for determining the presence of a malignant tumor in a patient, comprising the steps of (a) receiving a biological sample from a patient; (b) contacting the biological sample with the oligonucleotide according to claim 6; (c) determining in the sample the amount of polynucleotide that hybridizes with the oligonucleotide; and (d) comparing the amount of polynucleotide that hybridizes with a given oligonucleotide with a predetermined cutoff level, and thereby determining the presence of a malignant tumor in a patient. 10. A diagnostic kit containing at least one oligonucleotide according to claim 6. 11. A diagnostic kit containing at least one antibody according to claim 4, and a detection reagent, where the detection reagent contains a reporter group. Priority on points and signs: 09/20/2000 according to claims 1-11 with respect to the characteristic of SEQ ID NO: 440. 11/01/2000 according to claims 1-11 with respect to the characteristics of SEQ ID NO: 441 and SEQ ID NO: 442.