JP2002516659A - Compounds for the treatment and diagnosis of lung cancer and methods of using them - Google Patents

Abstract

  (57) [Summary] Compounds and methods for treating lung cancer are presented. The compounds of the present invention contain a polypeptide that includes at least a portion of a lung tumor protein. Vaccines and pharmaceutical compositions for the immunotherapy of lung cancer comprising such polypeptides, or polynucleotides encoding such polypeptides, are also provided with the polynucleotides for preparing the polypeptides of the invention. You.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates generally to compositions and methods for treating lung cancer. The invention more specifically relates to nucleotide sequences that are preferentially expressed in lung tumor tissue, with the polypeptide encoded by such a nucleotide sequence. The nucleotide sequences and polypeptides of the invention can be used in vaccines and pharmaceutical compositions for treating lung cancer.

BACKGROUND OF THE INVENTION Lung cancer is the leading cause of cancer death in both men and women in the United States, with an estimated 172,000 new cases reported in 1994. The 5-year survival rate in all lung cancer patients is only 13%, regardless of the stage of the disease at the time of diagnosis. This is in contrast to the 5-year survival rate of 46% in the cases detected, but the disease is still localized. However, only 16% of lung cancers are found before the disease spreads.

[0003] Early detection is difficult. Because clinical symptoms are often not seen until the disease reaches an advanced stage. At present, diagnosis is aided by the use of chest x-rays, analysis of cell types contained in sputum, and bronchial fiber optic tests. Treatment regimens are determined by the type and stage of the cancer and include surgery, radiation therapy and / or chemotherapy. Despite many studies on the treatment of this disease,
Lung cancer remains difficult to treat.

[0004] Thus, there remains a need in the art for improved vaccines, treatment methods and diagnostic techniques for lung cancer.

SUMMARY OF THE INVENTION Briefly, the present invention provides compounds and methods for treating lung cancer. In a first aspect, there is provided an isolated polynucleotide encoding a lung tumor polypeptide, wherein such polynucleotide comprises a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 1 11, 19, 22 to 25, 2
7-31, 51, 53, 55, 63, 70, 72, 79, 80, 86, 87, 8
9, 90, 102 to 107, 109, 139, 143 to 149, 151 to 154
And (b) SEQ ID NOs: 1-11, 19
, 22-25, 27-31, 51, 53, 55, 63, 70, 72, 79, 80
, 86, 87, 89, 90, 102-107, 109, 139, 143-149
, 151-154 and 156-158; and a sequence complementary to (b) (a) or (b).

[0006] In a second aspect, there is provided an isolated polypeptide comprising at least an immunogenic portion of a lung tumor protein or a variant thereof. In certain embodiments, such polypeptides comprise an amino acid sequence encoded by a DNA sequence comprising a nucleotide sequence selected from the group consisting of: (a) SEQ ID NOs: 1
11, 19, 22 to 25, 27 to 31, 51, 53, 55, 63, 70, 72,
79, 80, 86, 87, 89, 90, 102 to 107, 109, 139, 14
The sequences listed in 3-149, 151-154 and 156-158;
b) SEQ ID NOS: 1 to 11, 19, 22 to 25, 27 to 31, 51, 53, 55, 6
3, 70, 72, 79, 80, 86, 87, 89, 90, 102 to 107, 10
9, 139, 143-149, 151-154 and 156-158; sequences complementary to; and (c) variants of the sequence of (a) or (b).

[0007] In a related aspect, an expression vector comprising a polynucleotide of the invention is provided along with a host cell transformed or transfected with such an expression vector. In a preferred embodiment, the host cell is
. E. coli, yeast and mammalian cells.

[0008] In another aspect, there is provided a first and second polypeptide of the present invention, or a fusion protein comprising the polypeptide of the present invention and a known lung tumor antigen.

[0009] The invention further provides a pharmaceutical composition comprising one or more of the above-described polypeptides, fusion proteins or fusion polynucleotides and a physiologically acceptable carrier, in combination with an immune response enhancer. And a vaccine comprising such a polypeptide, fusion protein or fusion polynucleotide.

[0010] In a related aspect, the invention provides a method for inhibiting the development of lung cancer in a patient, the method comprising: administering an effective amount of at least one of the above pharmaceutical compositions and / or vaccines to the patient. Administering.

[0011] In a still further aspect of the invention, there is provided a method for detecting lung cancer in a patient, the method comprising the steps of: (a) binding to a polypeptide disclosed herein; Contacting the resulting binding agent with a biological sample obtained from the patient; and (b) detecting a protein or polypeptide in the sample that binds to the binding agent. In a preferred embodiment, the binding agent is an antibody, most preferably a monoclonal antibody.

[0012] In a related aspect, there is provided a method for monitoring the progression of lung cancer in a patient, the method comprising the steps of: (a) selecting one of the polypeptides disclosed herein; Contacting a biological sample obtained from the patient with a binding agent capable of binding; (b) detecting an effective amount of a protein or polypeptide in the sample that binds the binding agent; (c) step (a). ) And (b); and comparing the amount of polypeptide detected in steps (b) and (c).

[0013] Within related aspects, the invention relates to antibodies, preferably monoclonal antibodies, that bind to the polypeptides of the invention, and diagnostic kits containing such antibodies, and to inhibit the development of lung cancer. Methods for using various antibodies are provided.

The present invention further provides a method for detecting lung cancer, comprising the steps of: (a) obtaining a biological sample from a patient; And contacting with a second oligonucleotide primer, wherein at least one of the oligonucleotide primers is specific for a polynucleotide encoding one of the polypeptides disclosed herein;
And (c) detecting in the sample a DNA sequence to be amplified in the presence of the first and second oligonucleotide primers. In a preferred embodiment, at least one of the oligonucleotide primers comprises SEQ ID NOS: 1-31, 49-
55, 63, 64, 66, 68-72, 78-80, 84-92, 102-11
0, 116-120 and at least about 10 contiguous nucleotides of a polynucleotide comprising a sequence selected from the group consisting of 126-181.

[0015] In a further aspect, the invention provides a method for detecting lung cancer in a patient, comprising the steps of: a) obtaining a biological sample from the patient: (b)
A) contacting the sample with an oligonucleotide probe specific for a polynucleotide encoding one of the polypeptides disclosed herein; and (c) DNA in the sample that hybridizes to the oligonucleotide probe.
Detecting the sequence. Preferably, the oligonucleotide probe comprises SEQ ID NOs: 1-31, 49-55, 63, 64, 66, 68-72, 78-80, 84-
At least about 15 contiguous nucleotides of a polynucleotide comprising a sequence selected from the group consisting of 92, 102-110, 116-120 and 126-181. In a related aspect, there is provided a diagnostic kit comprising the above oligonucleotide probe or primer.

[0016] In a still further aspect, there is provided a method for the treatment of lung cancer in a patient, the method comprising obtaining PBMC from the patient, converting the PBMC to a polypeptide of the invention (or a polypeptide encoding such a polypeptide). Nucleotides) and providing the incubated T cells; and administering the incubated T cells to a patient. In the present invention, further, a step of incubating the antigen-presenting cells with the polypeptide of the present invention (or a polynucleotide encoding such a polypeptide) to provide the incubated antigen-presenting cells, and the incubated antigen-presenting cells Administering to a patient a method for treating lung cancer. In certain embodiments, the antigen presenting cells are selected from the group consisting of dendritic cells and macrophages. Also provided is a composition for treating lung cancer comprising T cells or antigen presenting cells incubated with a polypeptide or polynucleotide of the invention. These and other aspects of the invention will be apparent with reference to the following detailed description. All references disclosed herein are incorporated by reference in their entirety as if each were individually incorporated.

(Sequence IDENTIFIERS) SEQ ID NO: 1 corresponds to L363C1. cDNA sequence determined for cons. SEQ ID NO: 2 is L263C2. cDNA sequence determined for cons. SEQ ID NO: 3 is the determined cDNA sequence for L263C2c. SEQ ID NO: 4 shows L263C1. cDNA sequence determined for cons. SEQ ID NO: 5 is the cDNA sequence determined for L263C1b. SEQ ID NO: 6 shows L164C2. cDNA sequence determined for cons. SEQ ID NO: 7 shows L164C1. cDNA sequence determined for cons. SEQ ID NO: 8 is the cDNA sequence determined for L366C1a. SEQ ID NO: 9 shows L260C1. cDNA sequence determined for cons. SEQ ID NO: 10 is the cDNA sequence determined for L163C1c. SEQ ID NO: 11 is the cDNA sequence determined for L163C1b. SEQ ID NO: 12 shows L255C1. cDNA sequence determined for cons. SEQ ID NO: 13 is the cDNA sequence determined for L255C1b. SEQ ID NO: 14 shows L355C1. cDNA sequence determined for cons. SEQ ID NO: 15 is L366C1. cDNA sequence determined for cons. SEQ ID NO: 16 is the determined cDNA sequence for L163C1a. SEQ ID NO: 17 is the cDNA sequence determined 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 cDNA sequence determined 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 5 'cDNA sequence determined for L86S-3. SEQ ID NO: 50 is the 5 'cDNA sequence determined for L86S-12. SEQ ID NO: 51 is the 5 'cDNA sequence determined for L86S-16. SEQ ID NO: 52 is the 5 'cDNA sequence determined for L86S-25. SEQ ID NO: 53 is the 5 'cDNA sequence determined for L86S-36. SEQ ID NO: 54 is the 5 'cDNA sequence determined for L86S-40. SEQ ID NO: 55 is the 5 'cDNA sequence determined for L86S-46. SEQ ID NO: 56 is 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 5 'cDNA sequence determined for L86S-30. SEQ ID NO: 64 is the 5 'cDNA sequence determined for L86S-41. SEQ ID NO: 65 is a predicted amino acid sequence derived from the 5 'end of LT86-9. SEQ ID NO: 66 is the determined cDNA sequence extension for LT86-4. SEQ ID NO: 67 is the deduced extended amino acid sequence for LT86-4. SEQ ID NO: 68 is the 5 'cDNA sequence determined for LT86-20. SEQ ID NO: 69 is the 3 'cDNA sequence determined for LT86-21. SEQ ID NO: 70 is the 5 'cDNA sequence determined for LT86-22. SEQ ID NO: 71 is the 5 'cDNA sequence determined for LT86-26. SEQ ID NO: 72 is the 5 'cDNA sequence determined 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 extended cDNA sequence determined for L86S-12. SEQ ID NO: 79 is the extended cDNA sequence determined for L86S-36. SEQ ID NO: 80 is the extended cDNA sequence determined 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 5 'cDNA sequence determined for L86S-6. SEQ ID NO: 85 is the 5 'cDNA sequence determined for L86S-11. SEQ ID NO: 86 is the 5 'cDNA sequence determined for L86S-14. SEQ ID NO: 87 is the 5 'cDNA sequence determined for L86S-29. SEQ ID NO: 88 is the 5 'cDNA sequence determined for L86S-34. SEQ ID NO: 89 is the 5 'cDNA sequence determined for L86S-39. SEQ ID NO: 90 is the 5 'cDNA sequence determined for L86S-47. SEQ ID NO: 91 is the 5 'cDNA sequence determined for L86S-49. SEQ ID NO: 92 is the 5 'cDNA sequence determined for L86S-51. SEQ ID NO: 93 is 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 DNA sequence determined for SLT-T1. SEQ ID NO: 103 is the 5 'DNA sequence determined for SLT-T2. SEQ ID NO: 104 is the 5 'DNA sequence determined for SLT-T3. SEQ ID NO: 105 is the 5 'DNA sequence determined for SLT-T5. SEQ ID NO: 106 is the 5 'DNA sequence determined for SLT-T7. SEQ ID NO: 107 is the 5 ′ DNA sequence determined for SLT-T9. SEQ ID NO: 108 is the 5 'DNA sequence determined for SLT-T10. SEQ ID NO: 109 is the 5 'DNA sequence determined for SLT-T11. SEQ ID NO: 110 is the 5 'DNA sequence determined 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 5 'cDNA sequence determined for SALT-T3. SEQ ID NO: 117 is the 5 'cDNA sequence determined for SALT-T4. SEQ ID NO: 118 is the 5 'cDNA sequence determined for SALT-T7. SEQ ID NO: 119 is the 5 'cDNA sequence determined for SALT-T8. SEQ ID NO: 120 is the 5 'cDNA sequence determined 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 is 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 cDNA sequence determined for PSLT-71. SEQ ID NO: 136 is the determined cDNA sequence for PSLT-73. SEQ ID NO: 137 is the cDNA sequence determined for PSLT-79. SEQ ID NO: 138 is the determined 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 cDNA sequence determined 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 5 'cDNA sequence determined for SAL-3. SEQ ID NO: 150 is the 5 'cDNA sequence determined for SAL-24. SEQ ID NO: 151 is the 5 'cDNA sequence determined for SAL-25. SEQ ID NO: 152 is the 5 'cDNA sequence determined for SAL-33. SEQ ID NO: 153 is the 5 'cDNA sequence determined for SAL-50. SEQ ID NO: 154 is the 5 'cDNA sequence determined for SAL-57. SEQ ID NO: 155 is the 5 'cDNA sequence determined for SAL-66. SEQ ID NO: 156 is the 5 'cDNA sequence determined for SAL-82. SEQ ID NO: 157 is the 5 ′ cDNA sequence determined for SAL-99. SEQ ID NO: 158 is the 5 'cDNA sequence determined for SAL-104. SEQ ID NO: 159 is the 5 'cDNA sequence determined for SAL-109. SEQ ID NO: 160 is the 5 'cDNA sequence determined for SAL-5. SEQ ID NO: 161 is the 5 'cDNA sequence determined for SAL-8. SEQ ID NO: 162 is the 5 'cDNA sequence determined for SAL-12. SEQ ID NO: 163 is the 5 'cDNA sequence determined for SAL-14. SEQ ID NO: 164 is the 5 'cDNA sequence determined for SAL-16. SEQ ID NO: 165 is the 5 'cDNA sequence determined for SAL-23. SEQ ID NO: 166 is the 5 'cDNA sequence determined for SAL-26. SEQ ID NO: 167 is the 5 'cDNA sequence determined for SAL-29. SEQ ID NO: 168 is the 5 'cDNA sequence determined for SAL-32. SEQ ID NO: 169 is the 5 'cDNA sequence determined for SAL-39. SEQ ID NO: 170 is the 5 ′ cDNA sequence determined for SAL-42. SEQ ID NO: 171 is the 5 'cDNA sequence determined for SAL-43. SEQ ID NO: 172 is the 5 'cDNA sequence determined for SAL-44. SEQ ID NO: 173 is the 5 'cDNA sequence determined for SAL-48. SEQ ID NO: 174 is the 5 'cDNA sequence determined for SAL-68. SEQ ID NO: 175 is the 5 'cDNA sequence determined for SAL-72. SEQ ID NO: 176 is the 5 'cDNA sequence determined for SAL-77. SEQ ID NO: 177 is the 5 'cDNA sequence determined for SAL-86. SEQ ID NO: 178 is the 5 'cDNA sequence determined for SAL-88. SEQ ID NO: 179 is the 5 'cDNA sequence determined for SAL-93. SEQ ID NO: 180 is the 5 'cDNA sequence determined for SAL-100. SEQ ID NO: 181 is the 5 'cDNA sequence determined 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 is the first deduced amino acid sequence for SAL-25. SEQ ID NO: 185 is the second deduced 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 deduced amino acid sequence for SAL-50. SEQ ID NO: 188 is the predicted amino acid sequence for SAL-57. SEQ ID NO: 189 is the first deduced 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 deduced 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 deduced 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 deduced 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 is 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 deduced 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 deduced amino acid sequence for SAL-50.

DETAILED DESCRIPTION OF THE INVENTION As noted above, the present invention generally relates to compositions and methods for the treatment of lung cancer. The compositions described herein include polypeptides, fusion proteins and polynucleotides. The invention also includes molecules that bind to a polypeptide of the invention (eg, an antibody or fragment thereof). Such molecules are referred to herein as "binding agents."

In one embodiment, a polypeptide of the invention comprises at least a portion of a protein that is expressed at higher levels in lung tumor tissue than in normal human lung tissue. Preferably, the level of the RNA encoding the polypeptide is at least 2-fold higher in tumor tissue. Such polypeptides include, but are not limited to, the polypeptides encoded by the nucleotide sequences provided in SEQ ID NOs: 1-16 and variants thereof (and immunogenic portions thereof).

In a second embodiment, a polypeptide of the invention comprises at least a portion of an immunogenic lung oncoprotein, including but not limited to a polypeptide, wherein the lung oncoprotein comprises ( a) SEQ ID NOs: 17 to 31, 49 to 55, 63, 6
4, 66, 68-72, 78-80 and 84-92, (b) a complement of this nucleotide sequence, and (c) a sequence selected from the group consisting of variants of such a sequence. And amino acid sequences encoded by polynucleotides containing

[0021] In a third embodiment, a polypeptide of the invention comprises at least a portion of a lung oncoprotein comprising the polypeptide, wherein the lung oncoprotein comprises (a)
A) a nucleotide sequence set forth in SEQ ID NOs: 102-110, 116-120, and 126-181; (b) a complement of this nucleotide sequence; and (c) a sequence selected from the group consisting of variants of such sequences. And amino acid sequences encoded by polynucleotides containing

As used herein, the term “polypeptide” includes a chain of amino acids of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds. Thus, a polypeptide comprising a portion of one of the above lung tumor proteins may consist entirely of this portion, or this portion may be present within a larger polypeptide comprising additional sequences. The additional sequence may be derived from a native protein or may be heterologous, and such a sequence may be (
This need not be the case), but may be immunoreactive and / or antigenic. As detailed below, such polypeptides can be isolated from lung tumor tissue or prepared by synthetic or recombinant means.

As used herein, an “immunogenic portion” of a lung oncoprotein is capable of eliciting an immune response in a patient suffering from lung cancer, and as such is an antibody present in serum from a lung cancer patient Is the part that combines with Such immunogenic portions generally comprise at least about 5 amino acid residues, more preferably at least about 10 amino acid residues, and most preferably at least about 20 amino acid residues. The immunogenic portion of the proteins described herein can be identified in an antibody binding assay. Such assays are generally performed using any of a variety of means known to those of skill in the art (eg, Harlow and Lane, Antibody).
s: A Laboratory Manual, Cold Spring Ha
rbor Laboratory, Cold Spring Harbor, N
Y, 1988). For example, the polypeptide can be immobilized on a solid support (as described below) and contacted with the serum of a patient to allow binding of antibodies in the serum to the immobilized polypeptide.
Unbound serum is then removed and bound antibody can be detected using, for example, ¹²⁵ I-labeled protein A. Alternatively, the polypeptide can be used to produce monoclonal and polyclonal antibodies for use in detecting the polypeptide in the blood or other fluid of lung cancer patients. Methods for preparing and identifying immunogenic portions of an antigen of known sequence are well known in the art and are summarized in Paul, Fundamental Immunology, Third Edition, Raven Press, 1993, pp. 243-247. Including methods.

As used herein, the term “polynucleotide” means a single- or double-stranded polymer of deoxyribonucleotide bases or ribonucleotide bases and includes DNA molecules and corresponding RNA molecules. This includes HnR
It includes NA and mRNA molecules, both sense and antisense strands, and includes cDNA, genomic and recombinant DNA, and fully or partially synthetic polynucleotides. HnRNA molecules contain introns and generally correspond to DNA molecules in a one-to-one manner. mRNA molecules correspond to HnRNA and DNA molecules with introns removed. The polynucleotide may consist of the entire gene or any part thereof. An operable antisense polynucleotide can include a fragment of the corresponding polynucleotide, and thus the definition of "polynucleotide" includes all such operable antisense fragments.

The compositions and methods of the present invention also include variants of the above-described polypeptides and polynucleotides.

As used herein, a “variant” of a polypeptide is only with conservative substitutions and / or modifications that preserve the antigenic properties of the polypeptide.
A polypeptide different from this polypeptide. In a preferred embodiment, the modified polypeptide differs from the identified sequence by no more than 5 amino acid substitutions, deletions or additions. Such variants are generally obtained by altering one of the polypeptide sequences described above, and using, for example, the representative procedures described herein.
It can be identified by evaluating the antigenic properties of the modified polypeptide. The polypeptide variant preferably has at least about 70%, more preferably at least about 90%, and most preferably at least about 95% identity to the identified polypeptide (as described below). Determined).

As used herein, a “conservative substitution” is one in which an amino acid is replaced with another amino acid having similar properties, so that those skilled in peptide chemistry will have substantially altered Secondary structure and hydrophobicity index (h
hydrotropic) characteristics. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gln, as
n, ser, thr; (2) cys, ser, tyr, thr; (3) val,
ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

[0028] The variants may also (or) include other modifications, such as amino acids that have a minimal effect on the antigenic properties, secondary structure, and characteristics of the hydropathic index of the polypeptide. Deletion or addition. For example, the polypeptide can be linked to a signal (or leader) sequence at the N-terminus of the protein that directs translocation of the protein either translationally or post-translationally. The polypeptide may also be linked to a linker or other sequence to facilitate synthesis, purification or identification of the polypeptide (eg, poly-His) or to enhance binding of the polypeptide to a solid support. Can be done. For example, a polypeptide can be linked to an immunoglobulin Fc region.

A nucleotide “variant” is a sequence that differs from the recited nucleotide sequence in that it has one or more nucleotide deletions, substitutions or additions. Such modifications are made using standard mutagenesis techniques (eg, Adelman et al. (DNA, 2: 1).
83, 1983) as directed in oligonucleotide directed site-directed mutagenesis.
specific mutagenesis)). Nucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. The variant nucleotide sequence preferably has at least about 70%, more preferably at least about 80%, and most preferably at least about 90% identity (as described below) to the recited sequence. Determined).

Lung tumor antigens provided by the present invention include variants encoded by DNA sequences that are substantially homologous to one or more of the DNA sequences specifically recited herein. As used herein, “substantial homology” refers to a DNA sequence that is capable of hybridizing under moderately stringent conditions. Suitable moderately stringent conditions include: 5 × SSC
Pre-wash in a solution of 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization at 50 ° C. to 65 ° C., 5 × SSC overnight (or in the case of species cross homology, Using 0.5 × SSC at 45 ° C.); followed by 0.1% SD
20 minutes using each of 2 ×, 0.5 × and 0.2 × SSC containing S
2 washes at 5 ° C. Such hybridizing DNA sequences are also within the scope of the present invention. Because the nucleotide sequence (due to the degeneracy of the code)
It encodes the immunogenic polypeptide encoded by the hybridizing DNA sequence.

[0031] Two nucleotide or polypeptide sequences are "identical" if the sequence of nucleotides or amino acid residues in the two sequences is the same when aligned for maximum correspondence as described below. It is said that Comparisons between two sequences can typically be performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50 segments, where sequences are determined after the two sequences are optimally aligned. , Can be compared to a reference sequence at the same number of consecutive positions.

Optimal alignment of the sequences for comparison was performed using the default parameters and the bioinformatics software (DNASTAR, Inc., Madiso).
n, WI) of the Lasergene suite. This program integrates several alignment schemes described in the following references: Dayhoff, M .; O. (1978)
(A model of evolutionary change in p
proteins-Matrics for detecting distant
t relation,), Dayhoff, M .; O. (Edit) Atlas
of Protein Sequence and Structure, N
national Biomedical Research Fundati
on, Washington DC, Vol. 5, Supplement 3, pages 345-358; He.
in J. (1990) Unified Approach to Align
ment and Phylogenes 626-645 Methods
in Enzymology 183, Academic Press, I
nc. Higgins, D., San Diego, CA; G. FIG. And Shar
p, p. M. (1989) Fast and Sensitive multi
ple sequence alignments on a microco
mputer CABIOS 5: 151-153; W. And Muller W.S. (1988) Optimal alignments i
n linear space CABIOS 4: 11-17; Robins
on, E. D. (1971) Comb. Theor 11: 105; Santo
u, N. Nes, M .; (1987) The neighbor joining
method. A new method for reconstruct
ing phylogenetic trees Mol. Biol. Evol
. 4: 406-425; Snearth, P .; H. A. And Sokal, R .; R
. (1973) Numerical Taxonomy-the Princi
ples and Practice of Numerical Taxon
omy, Freeman Press, San Francisco, CA; W
ilbur, W.C. J. and Lipman, D.C. J. (1983) Rapid
similarity searches of nucleic acid
and protein data banks Proc. Natl. Ac
ad. Sci. ScL USA 80: 726-730.

Preferably, "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions. Here, the portion of the polynucleotide sequence in the comparison window is less than 20%, usually 5-15%, or 10-10% when compared to the reference sequence (without additions or deletions) for optimal alignment of the two sequences. It may include 12% additions or deletions (ie, gaps). This percentage determines the number of positions where the same nucleobase or amino acid residue is present in both sequences to calculate the number of matched positions; the number of matched positions is the total number of positions in the reference sequence. (Ie, window size) and multiplying the result obtained to calculate the percentage sequence identity by 100.

[0034] Lung tumor polypeptides of the invention, and polynucleotides encoding such polypeptides, can be isolated from lung tumor tissue using any of a variety of methods well known in the art. For example, a cDNA molecule encoding a polypeptide that is preferentially expressed in lung tumor tissue can be identified using differential display PCR.
It can be cloned based on lung tumor-specific expression of the corresponding mRNA. This technique compares amplification products from RNA templates prepared from normal lung tissue and lung tumor tissue. cDNA can be prepared by reverse transcription of RNA using (dT) ₁₂ AG primers. After amplification of cDNA using random primers, bands corresponding to amplification products specific for tumor RNA can be excised from the silver stained gel and subcloned into an appropriate vector. Examples of cDNA sequences that can be isolated using this procedure include the sequences given in SEQ ID NOs: 1-16.

A cDNA molecule encoding an immunogenic lung tumor polypeptide is described in Example 2 below.
As described in detail below, using a serum from the same patient as the tumor sample, by screening a cDNA expression library prepared from a lung tumor sample. Examples of cDNA sequences that can be isolated using this procedure include those provided in SEQ ID NOs: 17-31. Additional cDNA molecules encoding lung tumor polypeptides can be obtained by screening such a cDNA expression library with mouse anti-lung tumor serum, as described below in Example 3. Thus, examples of cDNA sequences that can be isolated are provided in SEQ ID NOs: 49-55, 63, 64 and 126-148.
A cDNA sequence encoding a lung tumor antigen was also isolated by screening a lung tumor cDNA library prepared from SCID mice using mouse anti-tumor serum, as described below in Example 4. Can be done. Examples of cDNA sequences that can be isolated using this technique are provided in SEQ ID NOs: 149-181.

The gene encoding the polypeptides described herein (or portions thereof) can be selectively amplified from human genomic cDNA or from lung tumor cDNA via the polymerase chain reaction. For this approach, sequence-specific primers can be designed based on the nucleotide sequence provided herein and purchased or synthesized. The amplified portion of the particular nucleotide sequence is then used and described in Sambrook et al., Molecular Cloth.
ning: A Laboratory Manual, Cold Spring
Harbor Laboratories, Cold Spring Har
Full-length genes can be isolated from human genomic DNA libraries or from lung tumor cDNA libraries using well-known techniques, such as those described in Bor, NY (1989).

[0037] Once the DNA sequence encoding the polypeptide is obtained, the polypeptide is produced recombinantly by inserting the DNA sequence into an expression vector and expressing the polypeptide in a suitable host. obtain. Any of a variety of expression vectors known to those of skill in the art can be used to express a recombinant polypeptide of the present invention. Expression can be achieved in any suitable host cell transformed or transfected with an expression vector containing a polynucleotide encoding the recombinant polypeptide. Suitable host cells include prokaryotic cells, yeast cells and higher eukaryotic cells. Preferably, the host cell used is E. coli. coli
, Yeast or mammalian cell lines (eg, COS cells or CHO cells). DNA sequences expressed in this manner may encode naturally occurring polypeptides, portions of naturally occurring polypeptides, or other variants thereof. Supernatants from a suitable host / vector system that secretes the recombinant polypeptide can be initially concentrated using commercially available filters. The concentrate can then be subjected to a suitable purification matrix, such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps are used, and the recombinant polypeptide can be further purified.

[0038] Such techniques can also be used to prepare polypeptides that contain parts or variants of a native polypeptide. Portions and other variants with less than about 100 amino acids, and generally less than about 50 amino acids, can be produced using techniques well known to those skilled in the art. For example, such polypeptides can be synthesized using any commercially available solid phase technique, such as the Merrifield solid phase synthesis method, in which amino acids are continuously added and amino acid chains are grown (eg, Merr.
iffield, J.M. Am. Chem. Soc. 85: 2149-2146,19
63). Equipment for automated synthesis of polypeptides is available from Perkin
Elmer / Applied BioSystems Division (Fo
(Ster City, CA) and can be operated according to the manufacturer's instructions.

In general, regardless of the method of preparation, the polypeptides disclosed herein are isolated and prepared in substantially pure form (ie, the polypeptides have an amino acid composition and 1 (Determined by subsequent sequence analysis). Preferably, the polypeptide is at least about 90% pure, more preferably, at least about 95% pure, and most preferably, at least about 99% pure. In certain preferred embodiments, a substantially pure polypeptide is a pharmaceutical composition or vaccine for use in one or more of the methods disclosed herein, as described in more detail below. Mixed.

In a related aspect, the invention provides a fusion protein comprising the polypeptides of the first and second inventions, or a fusion protein comprising the polypeptide of the invention and a known lung tumor antigen. Provided with a variant of the protein. The fusion proteins of the invention may, but need not, include a linker peptide between the first and second polypeptides.

The DNA sequence encoding the fusion protein of the present invention can be prepared using known recombinant DNA techniques by combining separate DNA sequences encoding the first polypeptide and the second polypeptide into an appropriate expression vector. Is constructed so that The 3 'end of the DNA sequence encoding the first polypeptide may be 5' of the DNA sequence encoding the second polypeptide, with or without a peptide linker.
'Connected to the end. As a result, the reading frame of this sequence is that the mRNA of the two DNA sequences is translated into a single fusion protein that retains the biological activity of both the first and second polypeptides. Is in phase to allow

A peptide linker sequence is used to ensure that each polypeptide folds into its secondary and tertiary structure, at a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structure. Can be separated. Such a peptide linker is incorporated into the fusion protein using standard techniques well known in the art. A suitable peptide linker sequence may be selected based on the following factors: (1) its ability to adopt a flexible, elongated conformation; (2) on the first and second polypeptides. Its ability to adopt a secondary structure that can interact with a functional epitope of
And (3) deletion of hydrophobic or charged residues capable of reacting with a functional epitope of the polypeptide. Preferred epitope linker sequences include Gly, Asn and Ser residues. Other, almost neutral amino acids (eg, Thr and Ala)
Can also be used for this linker sequence. Amino acid sequences that can be usefully used as linkers are described in Maratea et al., Gene 40: 39-46, 1985;
Murphy et al., Proc. Natl. Acad. Sci. USA 83:82
58-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,7;
51,180. This linker sequence has a length of 1 to about 5
It can be zero amino acids. A peptide sequence is not required if the first and second polypeptides have a non-essential N-terminal amino acid region that can be used for separation of functional domains and prevention of steric hindrance.

The ligated DNA sequence is operably linked to a suitable transcriptional or translational regulatory element. The regulatory element responsible for the expression of the DNA is located only 5 'to the DNA sequence encoding the first polypeptide. Similarly, the termination codon and transcription termination signal required to terminate translation are only present 3 'to the DNA sequence encoding the second polypeptide.

[0044] Fusion proteins comprising a polypeptide of the invention together with an irrelevant immunogenic protein are also provided. Preferably, the immunogenic protein comprises a recall (r
ecall) can elicit a response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (eg, Stout et al., New York).
Engl. J. Med. 336: 86-91 (1997)).

A polypeptide comprising an immunogenic portion of a lung tumor protein can generally be used for the treatment of lung cancer, where the polypeptide stimulates the patient's own immune response to lung tumor cells. Accordingly, the present invention provides a method of using one or more of the compounds described herein, which may be a polypeptide, polynucleotide, or fusion protein, for immunotherapy of lung cancer in a patient. provide. As used herein, "patient" refers to any warm-blooded animal, preferably a human. the patient,
The disease may be affected or may be free of detectable disease. Therefore,
The compounds disclosed herein can be used to treat lung cancer or to inhibit the development of lung cancer. In a preferred embodiment, the compound is administered either before or after surgical removal of the primary tumor and / or treatment with radiation therapy and administration of conventional chemotherapeutic drugs.

In these aspects, the polypeptides of the invention are generally present in a pharmaceutical composition or vaccine. Pharmaceutical compositions may include one or more polypeptides, each of which may include one or more of the above sequences (or variants thereof) and a physiologically acceptable carrier. Vaccines comprise one or more such polypeptides and an immune response enhancer (eg, an adjuvant, biodegradable microparticles (eg, polylactic galactide),
Or liposomes, which incorporate the polypeptide). Pharmaceutical compositions and vaccines may also include other epitopes of a lung tumor antigen, which may be incorporated into the fusion protein described above (ie, a single polypeptide comprising multiple epitopes), or may comprise another epitope. Either present in the polypeptide.

Alternatively, a pharmaceutical composition or vaccine may include DNA encoding one or more of the above polypeptides and / or fusion proteins, such that the polypeptide is produced in situ. In such pharmaceutical compositions and vaccines, the DNA can be in any of a variety of delivery systems known to those of skill in the art, including nucleic acid expression systems, bacterial expression systems, and viral expression systems. Suitable nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (eg, a suitable promoter). Bacterial delivery systems involve the administration of bacteria (eg, Bacillus-Calmette-Guerrin) that express an epitope of a lung cell antigen on their cell surface. In a preferred embodiment, the DNA can be introduced using a viral expression system (eg, vaccinia or other poxvirus, retrovirus, or adenovirus), wherein the viral expression system is non-pathogenic (defective). And the use of replication competent viruses. Suitable systems are, for example, Fischer-
Hoch et al., PNAS 86: 317-321, 1989; Flexner et al.,
Ann. N. Y. Acad. Sci. 569: 86-103, 1989; Fle
xner et al., Vaccine 8: 17-21, 1990; U.S. Pat. No. 4,60.
Nos. 3,112, 4,769,330 and 5,017,487;
WO 89/01973; U.S. Patent No. 4,777,127, GB 2,200,
No. 651; EP 0,345,242; WO 91/02805; Berkne
r, Biotechniques 6: 616-627, 1988; Rosen
Feld et al., Science 252: 431-434, 1991; Kolls.
Kass-Eisler et al., PNAS 91: 215-219, 1994;
PNAS 90: 11498-11502, 1993; Guzman et al., Cir.
circulation 88: 2838-2848, 1993; and Guzma
n et al., Cir. Res. 73: 1202-1207, 1993. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. This DNA is also disclosed, for example, in published PCT applications WO 90/11092, and Ulme.
r et al., Science 259: 1745-1749, 1993;
ohen, Science 259: 1691-1692, 1993. Uptake of naked DNA can be increased by coating the DNA on biodegradable beads, which are efficiently transported into cells.

The route and frequency of administration, as well as the dose, will vary from individual to individual and may correspond to those currently used in immunotherapy of other diseases. In general, pharmaceutical compositions and vaccines may be administered by injection (eg, intradermal, intramuscular, intravenous, or subcutaneous), nasally (eg, by inhalation), or orally. Doses between 1 and 10 may be administered over a period of 3 to 24 weeks. Preferably, at three-month intervals,
Four doses are administered and booster doses can then be made periodically thereafter. Alternative protocols may be appropriate for individual patients. A suitable dose is that amount of polypeptide or DNA that is effective to raise an immune response (cellular and / or humoral) against lung tumor cells in the treated patient. A suitable immune response is at least 10-50% above basal (ie, untreated) levels. Generally, the amount of polypeptide present in a dose (or produced in situ by DNA in a dose) will be from about 1 pg to about 100 mg / kg of host, typically about 10 mg / kg of host.
pg to about 1 mg, and preferably about 100 pg to about 1 μg. Suitable dose sizes will vary with the size of the patient, but will typically be about 0.01 m
It ranges from L to about 5 mL.

[0049] Any suitable carrier known to those skilled in the art can be used in the pharmaceutical compositions of the present invention, but the type of carrier will vary depending on the mode of administration. For parenteral administration (eg, subcutaneous injection), the carrier preferably includes water, saline, alcohol, lipids, waxes and / or buffers. For oral administration, any of the above carriers, or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and / or magnesium carbonate can be used. . Biodegradable microparticles, such as polylactic glycolide, can also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microparticles are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.

[0050] Any of a variety of immune response enhancers can be used in the vaccines of the invention. For example, an adjuvant may be included. Most adjuvants are substances (eg, aluminum hydroxide or mineral oil) designed to protect the antigen from rapid metabolism and non-specific stimulators of the immune response (eg, lipid A, Bordella).
pertussis or Mycobacterium tuberculo
sis). Such adjuvants are, for example, incomplete Freund's adjuvant and complete Freund's adjuvant (Difco Laboratori)
es, Detroit, MI) and Merck Adjuvant 65 (Merck
and Company, Inc. , Rahway, NJ).

In certain embodiments, the polynucleotides of the present invention may be formulated to allow for entry into and expression of mammalian (preferably human) cells. Such formulations are particularly useful for therapeutic purposes. One of skill in the art understands that there are many ways to achieve expression of a polynucleotide in a target cell, and that any suitable method can be used. For example, the polynucleotide can be incorporated into a viral vector, such as, but not limited to, an adenovirus, an adeno-associated virus, a retrovirus, or a vaccinia or other poxvirus, such as an avian poxvirus. Techniques for incorporating DNA into such vectors are well known to those skilled in the art. Retroviral vectors can further transfer or incorporate a targeting moiety, such as a gene encoding a ligand for a receptor on a particular target cell, making the vector target-specific.
Targeting can also be achieved using antigens by methods known to those skilled in the art.

[0052] The polypeptides disclosed herein can also be used in adoptive immunotherapy for the treatment of cancer. Adoptive immunotherapy can be broadly classified as either active immunotherapy or passive immunotherapy. In active immunotherapy, treatment is with an immune response modifier (mo
Relying on the in vivo stimulation of the endogenous host immune system to respond to tumors by administration of differenting agents (eg, tumor vaccines, bacterial adjuvants, and / or cytokines).

In a passive immune response, treatment involves the delivery of a biological reagent (eg, effector cells or antibodies) with established tumor immunoreactivity. This reagent mediates, directly or indirectly, the antitumor effect and does not necessarily depend on the intact host immune system. Examples of effector cells are T lymphocytes (eg, CD + 8 cytotoxic T lymphocytes, CD4 + T helpers, tumor infiltrating lymphocytes), killer cells (natural killer cells, lymphokine-activated killer cells), B cells, or disclosed. Antigen presenting cells (eg, dendritic cells and macrophages) that express the antigen.
The polypeptides disclosed herein can also be used to generate antibodies or anti-idiotype antibodies (such as US Pat. No. 4,918,164) for passive immunotherapy.

A powerful way to obtain a sufficient number of T cells for adoptive immunotherapy is to expand immune T cells in vitro. Culture conditions for growing single antigen-specific T cells to billions while retaining antigen recognition in vivo are well known in the art. These in vitro culture conditions typically use intermittent stimulation with antigen (eg, IL-2) and non-dividing feeder cells, often in the presence of cytokines. As described above, the immunoreactive polypeptides described herein comprise:
It can be used to rapidly expand antigen-specific T cells in order to generate a sufficient number of cells for immunotherapy. In particular, antigen presenting cells (eg, dendritic cells, macrophage cells, or B cells) can be obtained using standard techniques well known in the art.
It can be pulsed with an immunoreactive polypeptide or transfected with a polynucleotide sequence. For a therapeutically effective cultured T cell, the cultured T cells must be able to proliferate in vivo and be widely and evenly distributed, and be able to survive for long periods of time. Studies have shown that cultured T cells can be induced to proliferate in vivo and survive in substantial numbers over long periods of time by repeated stimulation with an antigen supplemented with IL-2 (e.g., , Cheever et al., Ibid.).

The polypeptides disclosed herein also generate and / or produce tumor-reactive T cells.
Alternatively, the T cells can then be administered to a patient. In one technique, antigen-specific T cell lines can be generated by in vivo immunization with short peptides corresponding to the immunogenic portions of the disclosed polypeptides. The resulting antigen-specific CD8 + CTL clone can be isolated from the patient, expanded using standard tissue culture techniques, and returned to the patient.

Alternatively, a peptide corresponding to the immunogenic portion of the polypeptide can generate a subset of tumor-reactive T cells by selective in vitro stimulation and expansion of autologous T cells to provide antigen-specific T cells The antigen-specific T cells can then be used, for example, in Chang et al. (Crit. Rev. Oncol. Hem.
atol. , 22 (3), 213, 1996).

In another embodiment, syngeneic or autologous dendritic cells can be pulsed with a peptide corresponding to at least the immunogenic portion of a polypeptide disclosed herein. The resulting antigen-specific dendritic cells can either be transferred to the patient or used to stimulate T cells to provide antigen-specific T cells that can then be administered to the patient is there. The use of dendritic cells pulsed with peptides to generate antigen-specific T cells and the subsequent use of such antigen-specific T cells to eradicate tumors in a mouse model has been described by Cheever et al. Therapy
With Cultured T Cells: Principles Re
visited ”, Immunological Reviews, 157: 1
77, 1997).

In addition, vectors that express the disclosed polynucleotides can be introduced into stem cells from a patient and clonally propagated in vitro for autologous transplantation into the same patient.

In one embodiment, cells of the immune system (eg, T cells) are obtained from a commercially available cell separation system (eg, CellPro Incorporated's (Bothel)
1, WA) CEPRATE ^™ system) can be isolated from the peripheral blood of a patient (US Pat. Nos. 5,240,856; 5,215,926; WO 89 /
06280; WO 91/16116 and WO 92/07243). The separated cells can be stimulated with one or more immunoreactive polypeptides contained in a delivery vehicle (eg, microparticles) to provide antigen-specific T cells. The population of tumor antigen-specific T cells is then expanded using standard techniques, and the cells are administered back to the patient. The polypeptides and fusion proteins of the invention can also be used to generate binding agents (eg, antibodies, or fragments thereof) that can detect metastatic human lung tumors. The binding agents of the invention can generally be prepared using methods known to those of skill in the art, including the exemplary procedures described herein. A binding agent may use the representative assays described herein to distinguish between patients with lung cancer and those without lung cancer. In other words, antibodies or other binding agents raised against lung tumor proteins or appropriate portions thereof indicate the presence of primary or metastatic lung cancer in at least about 20% of patients affected by the disease. It produces a signal and in at least about 90% of individuals who are not primary or metastatic lung cancer, produces a negative signal indicating the absence of the disease. A suitable portion of such a lung tumor protein is substantially all of the patients with lung cancer (ie,
At least about 80%, and preferably at least about 90%), indicates the presence of primary or metastatic lung cancer, and in substantially all of the samples that are negative when tested with the full length protein. , A moiety capable of producing a binding agent indicating the absence of lung cancer. Representative assays described below (
For example, a two-antibody sandwich assay) can generally be used to assess the ability of a binding agent to detect metastatic lung tumors.

The ability of a polypeptide prepared as described herein to generate antibodies capable of detecting a primary human lung tumor or a metastatic human lung tumor is generally one that is directed to this polypeptide. Such antibodies can be raised (e.g., using the exemplary methods described herein) and evaluated in a patient by determining the ability of such antibodies to detect such tumors. . This determination can be made by assaying a biological sample from a patient with or without primary or metastatic lung cancer for the presence of a polypeptide that binds to the antibody produced. Such test assays can be performed, for example, using the representative procedures described below. Polypeptides that produce antibodies that can detect at least 20% of primary or metastatic lung tumors by such a procedure are useful in assays for detecting primary or metastatic lung tumors. it is conceivable that. Polypeptide-specific antibodies can be used alone or in combination to improve sensitivity.

A polypeptide capable of detecting a primary or metastatic human lung tumor can be used as a marker for diagnosing lung cancer or for monitoring disease progression in a patient. In one embodiment, lung cancer in a patient is determined by determining a biological sample obtained from the patient by a predetermined cut-off value (cut-o).
ff value) can be diagnosed by assessing for the level of one or more of the polypeptides described above. As used herein, suitable "biological samples" include blood, serum, urine, and / or pulmonary secretions.

[0062] The level of one or more of the above polypeptides can be assessed using any binding agent specific for the polypeptide. A “binding agent” is, in the context of the present invention, any agent (eg, compound or cell) that binds to a polypeptide as described above. As used herein, “bound” refers to two separate molecules, each of which may be free (ie, in solution) or may be present on the surface of a cell or solid support ) Between non-covalent bonds (eg, a “complex” is formed)
Say. Such a complex may be free or immobilized (either covalently or non-covalently) on a solid support. The ability to bind can generally be assessed by determining the binding constant for the formation of this complex. The binding constant is the value obtained when the concentration of the complex is divided by the product of the concentrations of the components. Generally, two compounds are said to "bind" in the context of the present invention if the binding constant for complex formation is greater than about 10 ³ L / mol. Binding constants can be determined using methods well known to those skilled in the art.

Any substance that satisfies the above requirements can be a binding agent. For example, the binding agent can be a ribosome with or without a peptide component, an RNA molecule, or a peptide. In a preferred embodiment, the binding partner is an antibody, or a fragment thereof. Such antibodies can be polyclonal or monoclonal. Further, the antibody can be a single chain antibody, a chimeric antibody, a CDR-grafted antibody, or a humanized antibody. Antibodies can be prepared by the methods described herein, and by other methods well known to those skilled in the art.

There are a variety of assay formats known to those of skill in the art that use a binding partner to detect a polypeptide marker in a sample. For example, Harlow
And Lane, Antibodies: A Laboratory Manu
al, Cold Spring Harbor Laboratory, 198
See FIG. In a preferred embodiment, the assay involves the use of a binding partner immobilized on a solid support to bind and remove polypeptide from the remaining sample. The binding polypeptide can then be detected using a second binding partner that includes a reporter group. Suitable second binding partners include antibodies that bind to the binding partner / polypeptide complex. Alternatively, a competition assay may be used in which the polypeptide is labeled with a reporter group to allow binding of the immobilized binding partner after incubation of the binding partner with the sample. The extent to which a component of a sample inhibits binding of a labeled polypeptide to a binding partner is indicative of the reactivity of the sample with the immobilized binding partner.

The solid support can be any material known to those skilled in the art to which an antigen can be attached to the solid support. For example, a solid support is a microtiter (microt)
iter) can be successfully tested on plates or nitrocellulose or other suitable membranes. Alternatively, the support is a glass, fiberglass, rubber or plastic material (eg, polystyrene or polyvinyl chloride)
Bead or disc. The support may also be a magnetic particle or a fiber optic sensor, for example, as disclosed in US Pat. No. 5,359,681. Binding agents can be immobilized on a solid support using various techniques known to those skilled in the art, which are described in detail in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to non-covalent association and covalent attachment, such as adsorption, which may be a direct bond between an antigen and a functional group on a support. Or may be linked by a cross-linking agent). Immobilization by adsorption to wells or membranes in microtiter plates is preferred. In such cases, adsorption can be achieved by contacting the binding agent with the solid support in a suitable buffer for a suitable length of time. Contact time varies with temperature, but is typically between about 1 hour and about 1 day. Generally, plastic microtiter plates (eg,
Contacting the wells of polystyrene or polyvinyl chloride) with an amount of binding agent in the range of about 10 ng to about 10 μg, and preferably in the range of about 100 ng to about 1 μg, to immobilize the appropriate amount of binding agent It is enough.

The covalent attachment of a binding agent to a solid support is generally accomplished using a bifunctional reagent that reacts with both the support and a functional group (eg, a hydroxyl or amino group). This can be achieved by first reacting the support above. For example, the binding agent can be covalently attached to the support with a suitable polymer coating using benzoquinone or by condensation of the amine and active hydrogen of the binding partner with the aldehyde group of the support. (For example, Pierce Immunotechnology Catalog)
d Handbook, 1991, A12-A13).

In certain embodiments, the assay is a two-antibody sandwich (two-ant)
idy sandwich) assay. The assay may be performed by first contacting the antibody with a sample on a solid support (typically a well of a microplate) so that the polypeptides in the sample can bind to the immobilized antibody. Is fixed. Unbound sample is then removed from the immobilized polypeptide-antibody complex, and a second antibody (including a reporter group) capable of binding to a different site on the polypeptide is added. . The amount of the second antibody that remains bound to the solid support is then determined using a method appropriate for the particular reporter group.

More specifically, once the antibody is immobilized on the support as described above,
The remaining protein binding sites on this support are typically blocked. Bovine serum albumin or Tween20 ^™ (Sigma Chemical C
o, St. (Louis, MO) Any suitable blocking agent is known to those skilled in the art. 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 diluent (eg, phosphate buffered saline (PBS)) prior to incubation.
Generally, a suitable contact time (ie, incubation time) is a length of time sufficient to detect the presence of the polypeptide in a sample obtained from an individual with lung cancer. Preferably, this contact time is sufficient to achieve a binding level that reaches at least about 95% of the equilibrium between the bound and unbound polypeptides. One skilled in the art will understand that the time required to reach equilibrium can be readily determined by assaying the level of binding that occurs over time. At room temperature, an incubation time of about 30 minutes is generally sufficient.

Unbound sample can then be removed by washing the solid support with a suitable buffer (eg, PBS containing 0.1% Tween 20 ^™ ). A second antibody, which contains a reporter group, can then be added to the solid support. Preferred reporter groups include enzymes (eg, horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups, and biotin. Binding of the antibody to the reporter group can be achieved using standard methods known to those skilled in the art.

[0070] The second antibody is then incubated with the immobilized antibody-polypeptide complex for a period of time sufficient to detect the bound polypeptide. The appropriate length of time can generally be determined by assaying the level of binding that occurs over time. The unbound second antibody is then removed, and the bound second antibody is detected using a reporter group. The method used to detect the reporter group depends on the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally suitable. Spectroscopy can be used to detect dyes, luminescent and fluorescent groups. Biotin can be detected using avidin, which is conjugated to a different reporter group (generally a radioactive or fluorescent group or an enzyme). The reporter group of the enzyme can generally be detected by addition of the substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction product.

To determine whether lung cancer is present or absent, the signal detected from the reporter group that remains attached to the solid support generally corresponds to a predetermined cut-off value. Compare with signal. In one preferred embodiment, the cut-off value is the average signal obtained when the immobilized antibody is incubated with a sample from a patient without lung cancer. Generally, a sample that produces a signal that is three standard deviations above a predetermined cutoff value is considered positive for lung cancer. In an alternative preferred embodiment, the cut-off value is determined by Sackett et al., Clinical E
ideology: A Basic Science for Clin
ical Medicine, Little Brown and Co. , 1
985, 106-7, receiver operator curve (Receiver).
(overoperator Curve). Briefly, in this embodiment, the cutoff value is a percentage of true positives (ie, susceptibility) and a percentage of false positives (100%), each corresponding to a possible cutoff value for the diagnostic test result. Specificity) pair plot. The cutoff value on this plot that is closest to the upper left hand corner (ie, the value surrounding the largest area) is the most accurate cutoff value, and signals that are higher than the cutoff value determined by this method The resulting sample can be considered positive. Alternatively, the cutoff value may be moved left along the plot to minimize the percentage of false positives, or may be moved right to minimize the percentage of false negatives. Generally, samples that produce a signal that is higher than the cutoff value determined by this method are considered positive for lung cancer.

[0072] In a related embodiment, the assay is flow-th
rough or strip test format, where the antibody is immobilized on a membrane (eg, nitrocellulose). In a flow-through test, the polypeptide in the sample binds to the immobilized antibody as the sample passes through the membrane. The second labeled antibody then binds to the antibody-polypeptide complex as the solution containing the second antibody flows through the membrane. Detection of the bound second antibody may then be performed as described above. In a strip test format, one end of the membrane to which the antibody binds is immersed in a solution containing the sample. The sample travels along the membrane through the area containing the second antibody and to the area of the immobilized antibody. The concentration of the second antibody in the region of the immobilized antibody indicates the presence of lung cancer. Typically, the concentration of the second antibody at the site produces a pattern (eg, a line) that can be seen visually. The absence of such a pattern indicates a negative result. Generally, the amount of antibody immobilized on the membrane is determined if the biological sample contains, in the format discussed above, a level of polypeptide that is sufficient to produce a positive signal in a two-antibody sandwich assay. , Are selected to produce a visually separable pattern. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 μg, and more preferably, from about 50 ng to about 500 ng. Such tests are
Typically, it can be performed with very small biological samples.

It will be appreciated that there are numerous other assay protocols that are suitable for use with the antigens or antibodies of the present invention. The above description is intended by way of example only.

[0074] In another embodiment, the above-described polypeptides can be used as markers for the progression of lung cancer. In this embodiment, assays as described above for diagnosing lung cancer may be performed over time and assess changes in the level of reactive polypeptide. For example, this assay may be used for a period of 6 months to 1 year.
It can be performed every 4 to 72 hours, and then as needed. Generally, lung cancer is progressing in patients whose levels of this polypeptide detected by the binding agent increase over time. In contrast, lung cancer has not progressed if the level of the reactive polypeptide either remains constant or decreases over time.

[0075] Antibodies for use in the above methods can be prepared by any of a variety of techniques known to those of skill in the art. For example, Harlow and Lane, Antibodi
es: A Laboratory Manual, Cold Spring H
See arbor Laboratory, 1988. In one such technique, an immunogen comprising an antigenic polypeptide can be first injected into any of a wide variety of mammals, such as mice, rats, rabbits, sheep and goats. In this step, the polypeptide of the present invention can be supplied as an immunogen without modification. Alternatively, particularly for relatively short polypeptides, a superior immune response can be induced when the polypeptide is linked to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin.
Preferably, the immunogen is injected into the animal's host and the animal is bled periodically according to a predetermined schedule incorporating one or more booster immunizations. A polyclonal antibody specific for the polypeptide is then purified from such antisera, for example, by affinity chromatography using the polypeptide bound to a suitable solid support.

[0076] Monoclonal antibodies specific for the antigenic polypeptide of interest include, for example, Koh
ler and Milstein, Eur. J. Immunol. 6: 511-5
19, 1976 and modifications thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies with the desired specificity (ie, reactivity with the polypeptide of interest). Such cell lines can be produced, for example, from spleen cells obtained from an immunized animal as described above. The splenocytes are then immortalized, for example, by fusion with a myeloma cell fusion partner, preferably with a partner that is a common gene with the immunized animal. Various fusion techniques may be used. For example, the splenocytes and myeloma cells can be combined with a non-ionic detergent for a few minutes and then seeded at low density on a selective medium that supports the growth of hybrid cells but not myeloma cells. . A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1-2 weeks, colonies of hybrids are observed. A single colony is selected and tested for binding activity to the polypeptide. Hybridomas with high reactivity and specificity are preferred.

[0077] Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, various techniques, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host (eg, mouse), can be used to increase yield. The monoclonal antibody can then be collected from ascites or blood. Contaminants can be removed from the antibody by conventional techniques such as chromatography, gel filtration, precipitation, and extraction. The polypeptides of the present invention can be used, for example, in a purification process in an affinity chromatography step.

The monoclonal antibodies of the invention also reduce or eliminate lung tumors
Can be used as therapeutic reagents. The antibodies themselves (eg, metastases)
To inhibit the enzyme) or in combination with one or more therapeutic agents
. In this regard, suitable drugs include radionuclides, inducers of differentiation, drugs
Substances, toxins, and derivatives thereof. Preferred radionuclides include ⁹⁰ Y,^{one two Three}I,¹²⁵I,¹³¹I,¹⁸⁶Re,¹⁸⁸Re,²¹¹At, and²¹²Bi is raised
I can do it. Preferred drugs include methotrexate and pyrimidine ana
Log and purine analogs. Preferred inducer of differentiation
Include phorbol esters and butyric acid. A preferred toxin is Rishi
, Abrin, diphtheria toxin, cholera toxin, gelonin, P
pseudomonas exotoxin, Shigella toxin, and pokeweed
C. Antiviral proteins.

Therapeutic agents can be attached (eg, covalently) to the appropriate monoclonal antibody either directly or indirectly (eg, via a linker group). A direct reaction between the agent and the antibody is possible if each has a substituent capable of reacting with the other. For example, a nucleophilic group (amino or sulfhydryl group) on one hand has the ability to react with an alkyl group containing a carbonyl-containing group (eg, anhydride or halogen acid) or a good leaving group (eg, halogen) on the other. Can have.

Alternatively, it may be desirable to couple the therapeutic agent to the antibody via a linker group. The linker group can function as a spacer to separate the antibody from the reagent to avoid interfering with the binding ability. The linker group can also act to increase the chemical reactivity of the drug or antibody substituent and increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of a drug, or a functional group of a drug, or otherwise not possible.

A variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (eg, those described in the catalog of Pierce Chemical Co., Rockford, IL) can be used as linker groups. That will be apparent to those skilled in the art. Binding may be affected, for example, by an amino group, a carboxyl group, a sulfhydryl group, or an oxidized carbohydrate residue. There are numerous references describing such methodologies (eg, US Pat. No. 4,671,958 to Rodwell et al.).

If the therapeutic agent without the antibody portion of the immunoconjugate of the invention is more potent, it is desirable to use a linker group that can be cleaved during or upon internalization into the cell. Can be done. Many different cleavable linker groups have been described. Mechanisms for the intracellular release of drugs from these linker groups include disulfide bond reduction (eg, US Pat. No. 4,489,727 to Spitler).
No. 10), irradiation of a photosensitive bond (see, for example, US Pat.
625,014), hydrolysis of derivatized amino acid side chains (eg, Koh
U.S. Pat. No. 4,638,045 to N. et al., serum complement mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958 to Rodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. Patent 4,569,789
).

It is desirable to couple one or more agents to the antibody. In one embodiment, multiple molecules of the agent are conjugated to one antibody molecule. In another embodiment,
One or more types of agents may be conjugated to one antibody. Regardless of the particular embodiment, immunoconjugates with one or more agents can be prepared in various ways. For example, one or more agents can be directly attached to an antibody molecule, or a linker, which provides multiple sites for attachment that can be used. Alternatively, a carrier can be used.

The carrier can carry the agent in a variety of ways, including covalently, either directly or via a linker group. Suitable carriers include proteins such as albumin (eg, US Pat. No. 4,507,23 to Kato et al.).
No. 4), peptides and polysaccharides such as aminodextran (eg,
U.S. Pat. No. 4,699,784 to Shih et al.). Carriers may also carry the drug by non-covalent attachments or by encapsulation such as in liposome vesicles (eg, US Pat. Nos. 4,429,008 and 4,429).
873,088). Carriers specific for radionuclide drugs include small radioactive halogen molecules and chelating compounds. For example, U.S. Pat.
, 792 discloses representative radiohalogenated small molecules and their synthesis. Radionuclide chelates can be formed from metal or metal chelates, including metal oxides, compounds containing nitrogen and sulfur atoms as donor atoms for binding the radionuclide. For example, U.S. Pat. No. 4,677 to Davison et al.
No. 3,562 discloses representative chelates and their synthesis.

Various routes of administration for antibodies and immunoconjugates can be used. Typically, administration will be intravenous, intramuscular, subcutaneous or in the bed of a resected tumor. Obviously, the exact dose of antibody / immunoconjugate will vary depending on the antibody used, the density of the antigen on the tumor, and the rate of clearance of the antibody.

The diagnostic reagents of the present invention may also include DNA sequences encoding one or more of the above polypeptides, or one or more portions thereof. For example, at least two oligonucleotide primers can be used in a polymerase chain reaction (PCR) based on an assay to amplify lung tumor-specific cDNA from a biological sample. Here, at least one oligonucleotide primer is specific for the polynucleotide encoding the lung tumor protein of the present invention. Then
The presence of the amplified cDNA is detected using techniques well known in the art, such as gel electrophoresis. Similarly, an oligonucleotide probe specific for a polynucleotide encoding a lung tumor protein of the invention can be used in biological samples to
It can be used in hybridization assays to detect the presence of the invented polypeptide.

As used herein, the term “polynucleotide-specific oligonucleotide primer / probe” refers to at least about 60%, preferably at least about 75%, of the polynucleotide of interest. And more preferably oligonucleotide sequences having at least about 90% identity. Oligonucleotide primers and / or probes that can be usefully used in the diagnostic methods of the present invention preferably have at least about 10-40 nucleotides. In a preferred embodiment, the oligonucleotide primers are
31, 49-55, 63, 64, 66, 68-72, 78-80, 84-92,
At least about 10 contiguous nucleotides of a polynucleotide having a partial sequence selected from 102-110, 116-120, and 126-181. Preferably, the oligonucleotide probe for use in the diagnostic method of the present invention comprises SEQ ID NOS: 1-31, 49-55, 63, 64, 66, 68-72, 7
8-80, 84-92, 102-110, 116-120 and 126-181
And at least 15 contiguous oligonucleotides of a polynucleotide having a partial sequence provided by: Techniques for both PCR-based and hybridization assays are well known in the art (eg, Mu
Ilis et al., supra; see Ehrlich, supra). Thus, primers or probes can be used to detect lung tumor-specific sequences in biological samples, including blood, semen, lung tissue and / or lung tumor tissue.

The following examples are offered by way of illustration, not by way of limitation.

Examples Example 1 Preparation of Lung Tumor-Specific cDNA Sequences Using Differential Display RT-PCR This example uses differential display screening,
The preparation of a cDNA molecule encoding a lung tumor-specific polypeptide is described.

A tissue sample was prepared from breast and normal tissue of a patient suffering from lung cancer, which was confirmed by pathology after removal of the sample from the patient. Normal RNA
And tumor RNA was extracted from this sample and mRNA was isolated and converted to cDNA using (dT) ₁₂ AG (SEQ ID NO: 47) immobilized 3 ′ primer. Then, a differential display PCR was performed using a randomly selected primer (SEQ ID NO: 48). Amplification conditions were 1.5 mM
MgCl ₂ , 20 pmol of primer, 500 pmol of dNTP and 1 unit of Taq DNA polymerase (Perkin-Elmer, Branchb
rug, NJ). 40 cycles of amplification at 94 ° C
For 30 seconds, annealing at 42 ° C. for 1 minute, and extension at 72 ° C. for 30 seconds. Bands that were repeatedly observed to be specific for the tumor RNA fingerprint pattern were excised from the silver stained gel and p
Subcloned into GEM-T vector (Promega, Madison, WI) and sequenced. The isolated 3 'sequences are provided in SEQ ID NOs: 1-16.

Comparison of these sequences with those in public databases using the BLASTN program did not reveal significant homology to the sequences provided in SEQ ID NOs: 1-11. To the knowledge of the inventors, any of the isolated DNA sequences
Previously, it had not been shown to be expressed at higher levels in human lung tumor tissue than in normal lung tissue.

Example 2 Use of Patient Sera to Identify DNA Sequences Encoding Lung Tumor Antigen This example demonstrates the use of patient autologous serum to screen lung tumor samples for expression of lung tumor antigens. 2 illustrates the isolation of a cDNA sequence to be expressed.

A human lung tumor-directed cDNA expression library was constructed using the λZAP Express
It was constructed using an expression system (Stratagene, La Jolla, CA). Total RNA for the library was replaced with late S cells passaged into human squamous cell lung cancer.
Obtained from CID mice and use the Message Maker kit (Gibco
BRL, Gaithersburg, MD) was used to isolate poly A + RNA. The obtained library was used in Sambrook et al. (Molecular C
loning: A Laboratory Manual, Cold Spri
ng Harbor Laboratories, Cold Spring H
arbor, NY, 1989). E. coli-absorbed autologous pat absorbed by E. coli
Screening was performed using a secondary antibody (Gibco BRL), which is a goat anti-human IgG-AM (H + L) conjugated to alkaline phosphatase developed using NBT / BCIP. Positive plaques expressing immunoreactive antigen were purified. Rescue plaque-derived phagemid,
The nucleotide sequence of the clone was determined.

Fifteen clones were isolated and are hereinafter referred to as LT86-1 to LT86-15. LT
The cDNA sequences isolated for 86-1 to LT86-8 and LT86-10 to LT86-15 are provided in SEQ ID NOS: 17 to 24 and 26 to 31, respectively.
The corresponding deduced amino acid sequences are set forth in SEQ ID NOs: 32-39 and 41-46, respectively.
To provide. The determined cDNA sequence for LT86-9 is provided in SEQ ID NO: 25, and the corresponding deduced amino acid sequences from the 3 'and 5' ends are provided in SEQ ID NOs: 40 and 65, respectively. These sequences were replaced with the ge described above.
The sequence was compared with that of ne bank. Clone LT86-3, LT86-6 to LT
86-9, LT86-11 to LT86-13, and LT86-15 (SEQ ID NOs: 19, 22 to 25, 27 to 29 and 31, respectively) have some relative to previously identified expressed sequence tags (ESTs). And the clone LT86-6
, LT86-8, LT86-11, LT86-12 and LT86-15 were found to be similar or identical to each other. Clone LT86-3
Was found to show some homology with the human transcription repressor. Clone LT86-6, 8, 9, 11, 12 and 15 are yeast RNA Pol II
It has been found to show some homology to the transcription regulatory mediator. Clone LT86-13 is C.I. elegans leucine aminopeptidase was found to show some homology. Clone LT86-9 has two inserts, with a 5 'sequence showing homology to the previously identified antisense sequence of interferon alpha-derived P27 and a 3' sequence similar to LT86-6. It seems to include. Clone LT86-14 (SEQ ID NO: 30) shows some homology to the trithorax gene and has a "RGD" cell adhesion sequence and a β-lactamase A site that functions in penicillin hydrolysis. Was found. Clones LT86-1, LT86-2,
LT86-4, LT86-5 and LT86-10 (SEQ ID NO: 17,
18, 20, 21 and 26) were found to show homology to previously identified genes. Subsequently, the extended cDNA sequence determined for LT86-4 is provided in SEQ ID NO: 66, and the corresponding deduced amino acid sequence is provided in SEQ ID NO: 67.

[0095] Subsequent studies indicate that five additional clones (LT86-20, LT86-
21, LT86-22, LT86-26 and LT86-27). LT86-20, LT86-22, LT86-26 and LT86-
The determined 5 'cDNA sequences for SEQ ID NO: 27 are shown in SEQ ID NOs: 68 and 7, respectively.
0-72 and the 3 'cDNA sequence determined for LT86-21 is provided in SEQ ID NO: 69. LT86-20, LT86-21, LT86-22, LT
The corresponding deduced amino acid sequences for 86-26 and LT86-27 are provided in SEQ ID NOs: 73-77, respectively. LT86-22 and LT86-27 are:
It has been found to have a high similarity to each other. The gene ban described above
Comparison of these sequences with k sequences did not show significant homology to LT86-22 and LT86-27. LT86-20, LT86-21 and L
T86-26 was found to show homology to previously identified genes.

Example 3 Use of Mouse Antiserum to Identify DNA Sequence Encoding Lung Tumor Antigen This example demonstrates the use of mouse antitumor serum to screen a lung tumor cDNA library 2 illustrates the isolation of the encoding cDNA sequence.

A directional cDNA lung tumor expression library was prepared as described in Example 2 above. Serum was obtained from SCID mice containing late passaged human squamous lung cells and adenocarcinoma tumors. These sera were pooled and injected into normal mice to produce anti-lung tumor sera. This antiserum was used to screen about 200,000 PFU from the unamplified library. About 40 positive plaques were identified using a goat anti-mouse IgG-AM (H + L) alkaline phosphatase secondary antibody (BRL Labs.) Developed using NBT / BCIP. Purifying the phage and for expression in prokaryotic or eukaryotic cells,
Phagemids were picked for nine clones with inserts in the pBK-CMV vector.

The seven isolated clones (hereinafter L86S-3, L86S-12, L86S
-16, L86S-25, L86S-36, L86S-40 and L86S-4
6) is provided in SEQ ID NOs: 49-55, and the corresponding deduced amino acid sequences are provided in SEQ ID NOs: 56-62, respectively. Remaining 2
5 clones (hereinafter referred to as L86S-30 and L86S-41)
'The cDNA sequence is provided in SEQ ID NOs: 63 and 64. Then, L86S-3
6 and L86S-46 were determined to represent the same gene. Comparison of these sequences with public database sequences described above was performed using clone L86S-
30, L86S-36 and L86S-46 (SEQ ID NOs: 63, 53 and 55, respectively) showed no significant homology. L86S-16 (SEQ ID NO: 5
1) was found to show some homology to ESTs previously identified in fetal lung and germ cell tumors. The remaining clones were found to show at least some homology to previously identified human genes. Subsequently, the extended cDNA sequences determined for L86S-12, L86S-36 and L86S-46 are provided in SEQ ID NOs: 78-80, respectively, and the corresponding deduced amino acid sequences are provided in SEQ ID NOs: 81-83.

[0099] Subsequent studies have shown that nine additional clones (L86S-6, L86S-1)
1, L86S-14, L86S-29, L86S-34, L86S-39, L8
The determination of the 5 'cDNA sequence for 6S-47, L86S-49 and L86S-51 (respectively SEQ ID NOs: 84-92) was guided. The corresponding deduced amino acid sequences are provided in SEQ ID NOs: 93-101, respectively. L86S-30, L8
6S-39 and L86S-47 were found to be similar to each other. A comparison of the gene bank sequences described above with these sequences is based on L86S-14.
Did not show significant homology to L86S-29 is a previously identified E
It was found to show some homology to ST. L86S-6, L86S-1
1, L86S-34, L86S-39, L86S-47, L86S-49 and L86S-51 were found to show some homology to previously identified genes.

In a further study, a directional cDNA library was constructed using λZap Exp
Constructed using the Stratagene kit with the less vector. Total RNA for the library was isolated from two primary squamous lung tumors and poly A + RNA was isolated using an oligo dT column. Antisera were generated in normal mice using serum pools from three SCID mice transplanted with human squamous cell lung cancer. Approximately 700,000 PFU was obtained from E.I. Screened from unamplified library with mouse anti-SCID tumor serum absorbed in E. coli. Positive plaques were identified as described above. The phage is purified and the phagemid is transformed into pBK-
180 clones with inserts in the CMV vector were picked.

The cDNA sequences determined for the 23 isolated clones were compared to SEQ ID NOs: 126-126.
148. Comparison of these sequences with the public database sequences described above showed no significant homology to the sequences of SEQ ID NOs: 139 and 143-148. The sequences of SEQ ID NOs: 126-138 and 140-142 were found to show homology to previously identified human polynucleotide sequences.

Example 4 Use of Mouse Antiserum to Screen a Lung Tumor Library Prepared from SCID Mice This example demonstrates a lung tumor cDNA library prepared from SCID mice using mouse antitumor serum Screening for cD encoding a lung tumor antigen
2 illustrates the isolation of the NA sequence.

A directional cDNA lung tumor expression library was prepared using the Stratagene kit with the λZap Express vector. Total RNA for the library was obtained from late passage lung adenocarcinoma grown in SCID mice. The polyA + RNA was converted to a Message Maker Kit (Gibco BR).
L). Serum was obtained from two SCID mice implanted with lung adenocarcinoma. These sera were pooled and injected into normal mice to produce anti-lung tumor serum. Approximately 700,000 PFU was obtained from E.I. Screening was performed from an unamplified library using mouse anti-SCID tumor serum absorbed by E. coli. Positive plaques were isolated from goat anti-mouse IgG-A- developed using NBT / BCIP.
M (H + L) alkaline phosphatase secondary antibody (Gibco BRL) was used for identification. Phages were purified and phagemids were picked for 100 clones with inserts in the pBK-CMV vector for expression in prokaryotic or eukaryotic cells.

The 5 ′ cDNA sequences determined for the 33 isolated clones are provided in SEQ ID NOs: 149-181. SEQ ID NOs: 149, 150, 152-154, 156
The corresponding deduced amino acid sequences for -158 and 160-181 are provided in SEQ ID NOs: 182, 183, 186, 188-193, and 194-215, respectively. The clone of SEQ ID NO: 151 (designated SAL-25) was found to contain two open reading frames (ORFs). These ORFs
Are provided in SEQ ID NOs: 184 and 185. The clone of SEQ ID NO: 153 (designated SAL-50) has the sequence of SEQ ID NO: 18.
7 and 216 were found to contain two open reading frames encoding the predicted amino acid sequences. Similarly, the clone of SEQ ID NO: 155 (SAL-
No. 66) was found to contain two open reading frames encoding the predicted amino acid sequences of SEQ ID NOs: 189 and 190. Comparisons of publicly available database sequences with these isolated sequences are shown in SEQ ID NOs: 151,153.
And 154 sequences showed no significant homology. SEQ ID NOs: 149, 1
The sequences of 52, 156, 157 and 158 correspond to the previously isolated expressed sequence tags (
EST) was found to show some homology. SEQ ID NO: 150,
The sequences 155 and 159-181 were found to show homology to previously identified sequences in humans.

Example 5 Determining Tissue Specificity of Lung Tumor Polypeptides The gene specific primers were used to determine the mRN of a representative lung tumor polypeptide.
The expression level of A was tested in various normal and tumor tissues using RT-PCR.

Briefly, total RNA was extracted from various normal and tumor tissues using Trizol reagent. First strand synthesis was performed with 2 μg of total RNA together with SuperScript II reverse transcriptase (BRL Life Technologies).
For 1 hour at 42 ° C. The cDNA was then amplified by PCR using gene-specific primers. To ensure the semi-quantitative nature of PT-PCR, β-actin was used as an internal control for each tissue tested. Using 1 μl of the 1:30 dilution of the cDNA allowed linear range amplification of the β-actin template and was sensitive enough to reflect differences in starting copy number. Using these conditions,
β-actin levels were determined for each reverse transcription reaction from each tissue. DNA contamination was minimized by DNase treatment and by confirming negative PCR results when using first strand cDNA prepared without the addition of reverse transcriptase.

The mRNA expression levels were measured for 5 different types of tumor tissue (lung squamous tumor, lung adenoma, prostate tumor, colon tumor and breast tumor from 3 patients), as well as different normal tissues (4 patients). (Including lung, prostate, brain, kidney, liver, ovary, skeletal muscle, skin, small intestine, myocardium, retina and testis) of interest. L86S-46 was found to be expressed at high levels in lung squamous, colon and prostate tumors and was not detected in other tissues tested. L8
6S-5 was found to be expressed in lung tumor samples and two of the four 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, L86S
-46 was found to be overexpressed in lung squamous epithelium and normal tonsil, whereas low or undetectable expression was observed in all other tissues tested.

Example 6 Isolation of DNA Sequences Encoding Lung Tumor Antigens DNA sequences encoding antigens potentially involved in squamous cell lung tumorigenesis were
Isolated as follows.

A lung tumor-directed cDNA expression library was constructed using the λZAP Express expression system (Stratagene, La Jolla, CA). Total RNA for the library was obtained from two pools of human squamous cell lung cancer, and poly A + RNA was obtained from oligo dT cellulose (Gibco BRL, Gaithe).
rsburg, MD). The phagemid was rescued randomly and the cDNA sequence of the isolated clone was determined.

The determined cDNA sequence for clone SLT-T1 is provided in SEQ ID NO: 102, and clones SLT-T2, SLT-T3, SLT-T5, SLT-T7, S
The 5 'cDNA sequences determined for LT-T9, SLT-T10, SLT-T11 and SLT-T12 are provided in SEQ ID NOs: 103-110, respectively. SL
T-T1, SLT-T2, SLT-T3, SLT-T10 and SLT-T12
Are provided in SEQ ID NOs: 111-115, respectively. Public database sequences described above and SLT-T2, SLT-T
3, Comparison with the sequence for SLT-T5, SLT-T7, SLT-T9 and SLT-T11 did not show significant homology. SLT-T10 and SLT
The sequence for -T12 was found to show some homology to previously identified sequences in humans.

The sequence of SLT-T1 was determined to show some homology to PAC clones of unknown protein function. The cDNA sequence of SLT-T1 (SEQ ID NO: 102) was found to contain a mutagenesis (MUTT) domain. Such domains are known to function in the removal of damaged guanine from DNA that can cause an A to G transversion (eg, elDeiry, W.
. S. , 1997 Curr. Opin. Oncol. 9: 79-87; Oka
moto, k. Et al. 1996 Int. J. Cancer 65: 437-41
Wu, C .; Et al. 1995 Biochem. Biophys. Res. Com
mun. 214: 1239-45; Porter, D .; W. Et al. 1996 Ch
em. Res. Toxicol. 9: 1375-81). Therefore,
SLT-T1 may be useful for the treatment (by gene therapy) of lung cancer caused by or involved in DNA repair disruption.

In further studies, DNA sequences encoding antigens were strongly involved in adenoma lung tumorigenesis, isolated as follows. A human lung tumor-directed cDNA expression library was prepared using the λZAP Express expression system (Stratagene, La Jo
11a, CA). Total RNA for the library was obtained from late SCID mouse passage human adenomas and poly A + RNA was
Isolated using a Maker kit (Gibco BRL, Gaithersburg, MD). The phagemid was rescued randomly and the cDNA sequence of the isolated clone was determined.

The five isolated clones (SALT-T3, SALT-T4, SALT-T
7, designated SALT-T8 and SALT-T9) are provided in SEQ ID NOs: 116-120 and the corresponding deduced amino acid sequences are provided in SEQ ID NOs: 121-125. SALT-T3 was found to show 98% identity to the previously identified human transducin-like enhancer protein TLE2. SALT-T4 appears to be a human homolog of the mouse Hβ58 gene. SALT-T7 was found to have 97% identity to human 3-mercaptopyruvate transferase, and SALT-T8 was found to be a human interferon-inducible protein 1-8.
It was found to show homology to U. SALT-T9 is a human mucin M
Shows about 90% identity to UC 5B.

Example 7 Synthesis of Polypeptides Polypeptides were converted to FMUC with HPTU (O-benzotriazole N, N, N ′, N′-tetramethyluronium hexafluorophosphate) activation. Using the action, Perkin Elmer / Applied Biosystem
s can be synthesized on a Division 430A peptide synthesizer. Gly
The -Cys-Gly sequence may be attached to the amino terminus of the peptide to provide a method of attachment, attachment to an immobilized surface, or labeling of the peptide. Cleavage of the peptide from the solid support can be performed using the following cleavage mixture: trifluoroacetic acid: ethanedithiol: thioanisole: water: phenol (40: 1: 2: 2).
: 3). After cleavage for 2 hours, the peptide can be precipitated in cold methyl-t-butyl-ether. The peptide pellet was then washed with 0.1% trifluoroacetic acid (TFA).
) And lyophilized prior to purification by C18 reverse phase HPLC. The peptide can be eluted using a gradient of 0% to 60% acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA).
Following lyophilization of the pure fractions, the peptide can be characterized using electrospray or other types of mass spectrometers and by amino acid analysis.

From the foregoing, while certain embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Is understood.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/395 A61P 11/00 4C085 35/00 4C087 A61P 11/00 C07K 14/47 4H045 35/00 16 / 18 C07K 14/47 19/00 16/18 C12N 1/19 19/00 1/21 C12N 1/19 C12P 21/08 1/21 C12Q 1/68 A 5/10 G01N 33/53 D C12P 21/08 33 / 574 A C12Q 1/68 33/577 B G01N 33/53 (C12N 1/21 33/574 C12R 1:19) 33/577 C12N 15/00 ZNAA // (C12N 1/21 A61K 37/02 C12R 1: 19) C12N 5/00 B (31) Priority claim number 09 / 040,828 (32) Priority date March 18, 1998 (March 18, 1998) (33) Priority claim country United States (US) ( 31) Priority claim number 09 / 040,8 1 (32) Priority date March 18, 1998 (March 18, 1998) (33) Priority claim country United States (US) (31) Priority claim number 09 / 122,192 (32) Priority date 1998 (23) Priority claim country United States (US) (31) Priority claim number 09 / 122,191 (32) Priority date July 23, 1998 (1998. 7.23) (33) Priority claim country United States (US) (31) Priority claim number 09 / 219,245 (32) Priority date December 22, 1998 (1998.22.22) (33) Priority Claimed State United States (US) (81) Designated State EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, U Z, VN, YU, ZW (72) Inventor Fuldakis, Tony N. United States Washington 99232-0232, Seattle, P. Oh. Box 99232 (72) Inventor Mohamas, Laodeau, USA Washington 98118, Seattle, South Morgan 4205 F-term (reference) 4B024 AA01 AA12 BA36 CA04 CA09 DA02 DA06 DA12 GA11 HA01 HA03 HA12 HA15 4B063 QA01 QA18 QQ02 QQ52 QR32 QR55 QR62QS14 QX02 QX07 4B064 AG01 AG27 CA10 CA19 CA20 CC24 CE07 CE08 CE10 DA01 DA14 4B065 AA26X AA90X AA91X AA91Y AA93Y AB01 AC14 BA02 CA24 CA45 CA46 4C084 AA02 AA07 BA01 BA20 BA22 CA53 ZB092 ABAA AB AA AB AA AB AA AA AA AA A BA A BA41 CA41 DA76 DA86 EA28 EA31 EA51 FA33 FA74 GA23 GA26

Claims (60)

[Claims] 1. An isolated polynucleotide comprising: (a) SEQ ID NOs: 1-11, 19, 22-25, 27-31, 51, 53, 55
, 63, 70, 72, 79, 80, 86, 87, 89, 90, 102-107,
109, 139, 143-149, 151-154 and 156-158; (b) SEQ ID NOs: 1-11, 19, 22-25, 27-31, 51, 53, 55
, 63, 70, 72, 79, 80, 86, 87, 89, 90, 102-107,
109, 139, 143-149, 151-154 and the complement of the sequence provided in 156-158; and (c) a variant of the sequence of (a) and (b), a nucleotide selected from the group consisting of: A polynucleotide comprising a sequence. 2. An isolated polypeptide comprising an immunogenic portion of a lung tumor protein or a variant thereof, wherein the protein is encoded by the polynucleotide of claim 1. A polypeptide comprising an amino acid sequence. 3. The isolated polypeptide of claim 2, wherein said polypeptide is a sequence selected from the group of sequences listed in SEQ ID NOs: 182, 184-193 and 216. A polypeptide. 4. A polynucleotide comprising a nucleotide sequence encoding the polypeptide of claim 3. An expression vector comprising the polynucleotide according to claim 1 or 4. 6. A host cell transformed with the expression vector according to claim 5. 7. The host cell according to claim 6, wherein the host cell is E. coli. c
a host cell selected from the group consisting of oli, yeast, and a mammalian cell line. 8. A pharmaceutical composition comprising the polypeptide of claim 2 and a physiologically acceptable carrier. 9. A vaccine comprising the polypeptide of claim 2 and an immune response enhancer. 10. The vaccine according to claim 9, wherein said immune response enhancer is an adjuvant. 11. A vaccine comprising the polynucleotide of claim 1 or 4 and an immune response enhancer. 12. The vaccine of claim 11, wherein said immune response enhancer is an adjuvant. 13. A pharmaceutical composition for the treatment of lung cancer comprising a polypeptide and a physiologically acceptable carrier, wherein said polypeptide is an immunogen of an immunogenic part of a lung protein or a variant thereof. Wherein the protein comprises:
The following: (a) SEQ ID NOs: 12 to 18, 20, 21, 26, 49, 50, 52, 54, 6
4, 66, 68, 69, 71, 78, 84, 85, 88, 91, 92, 116-
120, 126-138, 140-142, 150, 155 and 159-18
(B) SEQ ID NOS: 12 to 18, 20, 21, 26, 49, 50, 52, 54, 6
4, 66, 68, 69, 71, 78, 84, 85, 88, 91, 92, 116-
120, 126-138, 140-142, 150, 155 and 159-18
And (c) a variant of the sequence of (a) and (b), a pharmaceutical composition comprising an amino acid sequence encoded by a polynucleotide comprising a sequence selected from the group consisting of: object. 14. A vaccine for the treatment of lung cancer comprising a polypeptide and an immune response enhancer, wherein said polypeptide comprises an immunogenic portion of a lung protein or a variant thereof, wherein said immunogenic portion comprises The proteins are as follows: (a) SEQ ID NOs: 12-18, 20, 21, 26, 49, 50, 52, 54, 6
4, 66, 68, 69, 71, 78, 84, 85, 88, 91, 92, 116-
120, 126-138, 140-142, 150, 155 and 159-18
Sequences listed in 1: (b) SEQ ID NOs: 12-18, 20, 21, 26, 49, 50, 52, 54, 6
4, 66, 68, 69, 71, 78, 84, 85, 88, 91, 92, 116-
120, 126-138, 140-142, 150, 155 and 159-18
And (c) a variant of the sequence of (a) and (b). A vaccine comprising an amino acid sequence encoded by a polynucleotide comprising a sequence selected from the group consisting of: 15. A vaccine for the treatment of lung cancer comprising a polynucleotide and an immune response enhancer, said polynucleotide comprising: (a) SEQ ID NOS: 12-18, 20, 21, 26, 49, 50; 52, 54, 6
4, 66, 68, 69, 71, 78, 84, 85, 88, 91, 92, 116-
120, 126-138, 140-142, 150, 155 and 159-18
(B) SEQ ID NOS: 12 to 18, 20, 21, 26, 49, 50, 52, 54, 6
4, 66, 68, 69, 71, 78, 84, 85, 88, 91, 92, 116-
120, 126-138, 140-142, 150, 155 and 159-18
A vaccine complementary to the sequence of SEQ ID NO: 1; and (c) a variant of the sequence of (a) and (b). 16. A method of inhibiting the development of lung cancer in a patient, comprising the step of administering to the patient an effective amount of the pharmaceutical composition of claim 8 or 13. 17. A method for inhibiting the development of lung cancer in a patient, comprising administering to said patient an effective amount of said vaccine according to any one of claims 9, 11, 14 or 15. Including, methods. 18. A fusion protein comprising at least one polypeptide according to claim 2. 19. A fusion protein comprising at least two polypeptides according to claim 2. 20. A fusion protein comprising the polypeptide of claim 2 and a known lung cancer tumor antigen. 21. A pharmaceutical composition comprising a fusion protein according to any one of claims 18 to 20 and a physiologically acceptable carrier. 22. A vaccine comprising the fusion protein of any one of claims 18 to 20 and an immune response enhancer. 23. The vaccine of claim 22, wherein said immune response enhancer is an adjuvant. 24. A method for inhibiting the development of lung cancer in a patient, comprising the step of administering to the patient an effective amount of the pharmaceutical composition of claim 21. 25. A method for inhibiting the development of lung cancer in a patient, comprising the step of administering to said patient an effective amount of the vaccine of claim 22. 26. A method for inhibiting the development of lung cancer in a patient, wherein the polynucleotide is administered to the patient under conditions such that the polynucleotide enters the patient's cells and the polynucleotide is expressed therein. Wherein the polynucleotide comprises the following: (a) a sequence provided in SEQ ID NO: 102; (b) a sequence complementary to the sequence in SEQ ID NO: 102; and (c) a variant of the sequence in SEQ ID NO: 102. A method having a sequence selected from the group consisting of: 27. A method for detecting lung cancer in a patient, comprising: (a) contacting a binding agent capable of binding to a polypeptide with a biological sample obtained from the patient. Wherein the polypeptide comprises an immunogenic portion of a lung tumor protein or a variant thereof, wherein the protein comprises SEQ ID NOs: 1-31, 49-55, 63, 64, 66, 68-72. , 78-80, 84
-92, 102-110, 116-120 and 126-181, the amino acid sequence encoded by a polynucleotide comprising a nucleotide sequence selected from the group consisting of the complement of the sequence and variants thereof. And b) detecting in the sample a polypeptide that binds to the binding agent, thereby detecting lung cancer in the patient. 28. The binding agent according to claim 27, wherein the binding agent is a monoclonal antibody.
The method described in. 29. The binding agent according to claim 28, wherein the binding agent is a polyclonal antibody.
The method described in. 30. A method for monitoring the progression of lung cancer in a patient, comprising: (a) contacting a biological sample obtained from the patient with a binding agent capable of binding to the polypeptide. Wherein the polypeptide comprises an immunogenic portion of a lung tumor protein or a variant thereof, wherein the protein comprises SEQ ID NOs: 1-31, 49-55, 63, 64, 66, 68- 72, 78-80, 84-
92, 102-110, 116-120 and 126-181, including the amino acid sequence encoded by a polynucleotide comprising a nucleotide sequence selected from the group consisting of the complement of the sequence and variants thereof. (B) determining the amount of polypeptide that binds to the binding agent in the sample; (c) repeating steps (a) and (b); and (d) steps (b) and (c). B) comparing the amount of the polypeptide detected in the step (a), and monitoring the progress of lung cancer in the patient. 31. A monoclonal antibody that binds to a polypeptide comprising an immunogenic portion of a lung tumor protein or an immunogenic portion of a variant thereof, wherein the protein comprises: (a) SEQ ID NOS: 1-11 , 19,22-25,27-31,51,53,55
, 63, 70, 72, 79, 80, 86, 87, 89, 90, 102-107,
109, 139, 143-149, 151-154 and 156-158; (b) SEQ ID NOS: 1-11, 19, 22-25, 27-31, 51, 53, 55
, 63, 70, 72, 79, 80, 86, 87, 89, 90, 102-107,
109, 139, 143-149, 151-154 and 156-158; a complement of the nucleotide sequence listed; and (c) a variant of the sequence of (a) and (b). A monoclonal antibody comprising an amino acid sequence encoded by a polynucleotide comprising the sequence. 32. A method for inhibiting the development of lung cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of the monoclonal antibody of claim 31. 33. The monoclonal antibody is conjugated to a therapeutic agent.
33. The method according to claim 32. 34. A method for detecting lung cancer in a patient, comprising: (a) obtaining a biological sample from the patient; (b) at least two oligonucleotide primers in the polymerase chain reaction with the sample. Wherein at least one of said oligonucleotides is specific for a polynucleotide encoding a polypeptide comprising an immunogenic portion of a lung tumor protein or an immunogenic portion of a variant thereof, The protein has SEQ ID NOs: 1-31, 49-55, 63, 64, 66, 68-72.
, 78-80, 84-92, 102-110, 116-120 and 126-1
81. a sequence comprising an amino acid sequence encoded by a polynucleotide comprising a nucleotide sequence selected from the group consisting of the sequences listed under No. 81, complements of the sequences and variants thereof; and Detecting in the sample a DNA sequence that amplifies in the presence, thereby detecting lung cancer. 35. The method of claim 34, wherein the at least one oligonucleotide primer comprises SEQ ID NOs: 1-31, 49-55, 63.
, 64, 66, 68-72, 78-80, 84-92, 102-110, 116
A method comprising at least about 10 contiguous nucleotides of a polynucleotide comprising a sequence selected from -120 and 126-181. 36. A diagnostic kit, comprising: (a) one or more of the monoclonal antibodies of claim 31; and (b) a detection reagent. 37. The kit of claim 36, wherein said monoclonal antibody is immobilized on a solid support. 38. The kit of claim 37, wherein said solid support comprises nitrocellulose, latex, or a plastic member. 39. The kit of claim 36, wherein the detection reagent comprises a group of reporters bound to a binding agent. 40. The kit of claim 39, wherein said binding agent is selected from the group consisting of anti-immunoglobulin, protein G, protein A and lectin. 41. The kit of claim 39, wherein said reporter group is selected from the group consisting of radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and dye particles. 42. A diagnostic kit comprising at least two oligonucleotide primers, wherein said at least one oligonucleotide primer encodes a polypeptide comprising an immunogenic portion of a lung tumor protein or a variant thereof. SEQ ID NO: 1 is specific for the polynucleotide
~ 31, 49-55, 63, 64, 66, 68-72, 78-80, 84-92
, 102-110, 116-120 and 126-181, the amino acid sequence encoded by a polynucleotide comprising a nucleotide sequence selected from the group consisting of the complement of the sequence and variants thereof. kit. 43. The diagnostic kit of claim 42, wherein at least one of said oligonucleotide primers comprises SEQ ID NOS: 1-31, 49-55.
, 63, 64, 66, 68-72, 78-80, 84-92, 102-110,
A kit comprising at least about 10 contiguous nucleotides of a polynucleotide having a nucleotide sequence selected from the group consisting of 116-120 and 126-181, the complement of the sequence, and variants thereof. 44. A method for detecting lung cancer in a patient, comprising: (a) obtaining a biological sample from the patient; (b) an immunogenic portion of the biological sample and a lung tumor protein. Or contacting an oligonucleotide probe that is specific for a polynucleotide encoding a polypeptide comprising the immunogenic portion of a variant thereof, wherein the protein comprises:
SEQ ID NOs: 1-31, 49-55, 63, 64, 66, 68-72, 78-80,
Amino acids encoded by a polynucleotide comprising a nucleotide sequence selected from the group consisting of the sequences listed in 84-92, 102-110, 116-120 and 126-181, complements of the nucleotide sequences and variants thereof And (c) detecting in the sample a DNA sequence that hybridizes to the oligonucleotide probe, thereby detecting lung cancer in the patient. 45. The method of claim 44, wherein said oligonucleotide probe is selected from the group consisting of SEQ ID NOs: 1-31, 49-55, 63, 64, 66, 6
8-72, 78-80, 84-92, 102-110, 116-120 and 1
26. A method comprising at least about 15 contiguous nucleotides of a polynucleotide having a nucleotide sequence selected from the group consisting of the sequences listed in 26-181, the complement of said nucleotide sequence, and variants thereof. 46. A diagnostic kit comprising an oligonucleotide probe that is specific for a polynucleotide that encodes a polypeptide comprising an immunogenic portion of a lung tumor protein or an immunogenic portion of a variant thereof, wherein the protein comprises: SEQ ID NOs: 1-31, 49-55, 63, 64, 66, 68-72, 78-80, 84-9
2, the sequences listed in 102-110, 116-120 and 126-181,
A kit comprising an amino acid sequence encoded by a polynucleotide comprising a nucleotide sequence selected from the group consisting of the complement of the sequence as well as variants thereof. 47. The diagnostic kit according to claim 46, wherein said oligonucleotide probe is selected from the group consisting of SEQ ID NOs: 1-31, 49-55, 63, 64, 66.
, 68-72, 78-80, 84-92, and 102-110, at least about 15 of the polynucleotide having a nucleotide sequence selected from the group consisting of the complements of the sequences and variants thereof. A diagnostic kit comprising: 48. A method for treating lung cancer in a patient, comprising: (a) obtaining peripheral blood cells from the patient; (b) the method of claim 2, wherein the T cells are expanded. Incubating the cells in the presence of at least one of the described polypeptides; and (c) administering the expanded T cells to the patient. 49. A method for treating lung cancer in said patient, comprising: (a) obtaining peripheral blood cells from said patient; (b) so that the T cells proliferate. Incubating the cells in the presence of at least one of the polypeptides of claim 1; and (c) administering the expanded T cells to the patient. 50. The method of any one of claims 48 and 49, wherein the step of incubating the T cells is repeated one or more times. 51. The method according to any one of claims 48 and 49, wherein step (a) further comprises the step of separating T cells from said peripheral blood cells, and step (b). The method wherein the cells incubated with the T cells are the T cells. 52. The method according to any one of claims 48 and 49, wherein step (a) further comprises removing CD4 + cells or CD8 from said peripheral blood cells.
+ Separating the cells, and wherein the cells grown in step (b) are CD4 +
The method, which is a T cell or a CD8 + T cell. 53. The method according to any one of claims 48 and 49, wherein step (b) further comprises cloning one or more T cells grown in the presence of said polypeptide. A method comprising the steps of: 54. A composition for the treatment of lung cancer in a patient, comprising T cells grown in the presence of the polypeptide of claim 2 in combination with a pharmaceutically acceptable carrier. object. 55. A composition for the treatment of lung cancer in a patient, comprising T cells grown in the presence of the polypeptide of claim 1 in combination with a pharmaceutically acceptable carrier. object. 56. A method for treating lung cancer in a patient, comprising: (a) incubating an antigen presenting cell in the presence of at least one polypeptide of claim 2; and (b) Administering the incubated antigen presenting cells to the patient. 57. A method for treating lung cancer in a patient, comprising: (a) incubating an antigen presenting cell in the presence of at least one polynucleotide of claim 1; and (b) Administering the incubated antigen presenting cells to the patient. 58. The method of claim 54 or 55, wherein said antigen presenting cells are selected from the group consisting of dendritic cells and macrophage cells. 59. A composition for the treatment of lung cancer in a patient, comprising an antigen presenting cell incubated in the presence of the polypeptide of claim 2 in combination with a pharmaceutically acceptable carrier. Composition. 60. A composition for the treatment of lung cancer in a patient, comprising an antigen presenting cell incubated in the presence of the polypeptide of claim 1 in combination with a pharmaceutically acceptable carrier. Composition.