Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the present invention relates generally to compositions and methods for the treatment of lung cancer.
• the invention is more specifically related to nucleotide sequences that are preferentially expressed in lung tumor tissue, together with polypeptides encoded by such nucleotide sequences.
• the inventive nucleotide sequences and polypeptides may be used in vaccines and pharmaceutical compositions for the treatment of lung cancer.
• Lung cancer is the primary cause of cancer death among both men and women in the U.S., with an estimated 172,000 new cases being reported in 1994.
• the five-year survival rate among all lung cancer patients, regardless of the stage of disease at diagnosis, is only 13%. This contrasts with a five-year survival rate of 46% among cases detected while the disease is still localized. However, only 16% of lung cancers are discovered before the disease has spread.
• isolated polynucleotides encoding lung tumor polypeptides comprising a nucleotide sequence selected from the group consisting of: (a) sequences provided in SEQ ID NO: 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) sequences complementary to a sequence provided in SEQ ID NO: 1-11.
• isolated polypeptides comprise at least an immunogenic portion of a lung tumor protein or a variant thereof.
• such polypeptides comprise an amino acid sequence encoded by a DNA sequence comprising a nucleotide sequence selected from the- group consisting of (a) sequences recited in SEQ ID NO: 1-11, 19, 22-25, 27-31, 51.
• expression vectors comprising the inventive polynucleotides. together with host cells transformed or transfected with such expression vectors are provided.
• the host cells are selected from the group consisting of E. coli, yeast and mammalian cells.
• fusion proteins comprising a first and a second inventive polypeptide or. alternatively, an inventive polypeptide and a known lung tumor antigen, are provided.
• the present invention further provides pharmaceutical compositions comprising one or more of the above polypeptides, fusion proteins or polynucleotides and a physiologically acceptable carrier, together with vaccines comprising one or more such polypeptides.
• fusion proteins or polynucleotides in combination with an immune response enhancer comprising one or more of the above polypeptides, fusion proteins or polynucleotides and a physiologically acceptable carrier, together with vaccines comprising one or more such polypeptides.
• fusion proteins or polynucleotides in combination with an immune response enhancer.
• the present invention provides methods for inhibiting the development of lung cancer in a patient, comprising administering to a patient an effective amount of at least one of the above pharmaceutical compositions and/or vaccines.
• methods for detecting lung cancer in a patient comprising: (a) contacting a biological sample obtained from a patient with a binding agent that is capable of binding to a polypeptide disclosed herein; and (b) detecting in the sample a protein or polypeptide that binds to the binding agent.
• the binding agent is an antibody, most preferably a monoclonal antibody.
• methods for monitoring the progression of lung cancer in a patient comprising: (a) contacting a biological sample obtained from a patient with a binding agent that is capable of binding to one of the polypeptides disclosed herein; (b) determining in the sample an amount of a protein or polypeptide that binds to the binding agent; (c) repeating steps (a) and (b); and comparing the amounts of polypeptide detected in steps (b) and (c).
• the present invention provides antibodies, preferably monoclonal antibodies, that bind to the inventive polypeptides. as well as diagnostic kits comprising such antibodies, and methods of using such antibodies to inhibit the development of lung cancer.
• the present invention further provides methods for detecting lung cancer comprising: (a) obtaining a biological sample from a patient; (b) contacting the sample with a first and a second oligonucleotide primer in a polymerase chain reaction, at least one of the oligonucleotide primers being specific for a polynucleotide that encodes one of the polypeptides disclosed herein; and (c) detecting in the sample a DNA sequence that amplifies in the presence of the first and second oligonucleotide primers.
• At least one of the oligonucleotide primers comprises at least about 10 contiguous nucleotides of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 1- 31, 49-55, 63, 64, 66, 68-72, 78-80, 84-92, 102-110, 116-120 and 126-181.
• the present invention provides a method for detecting lung cancer in a patient comprising: (a) obtaining a biological sample from the patient; (b) contacting the sample with an oligonucleotide probe specific for a polynucleotide that encodes one of the polypeptides disclosed herein; and (c) detecting in the sample a DNA sequence that hybridizes to the oligonucleotide probe.
• the oligonucleotide probe comprises at least about 15 contiguous nucleotides of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 1-31, 49-55, 63, 64, 66, 68-72, 78-80, 84- 92, 102-110, 116-120 and 126-181.
• diagnostic kits comprising the above oligonucleotide probes or primers are provided.
• methods for the treatment of lung cancer in a patient comprising obtaining PBMC from the patient, incubating the PBMC with a polypeptide of the present invention (or a polynucleotide that encodes such a polypeptide) to provide incubated T cells and administering the incubated T cells to the patient.
• present invention additionally provides methods for the treatment of lung cancer that comprise incubating antigen presenting cells with a polypeptide of the present invention (or a polynucleotide that encodes such a polypeptide) to provide incubated antigen presenting cells and administering the incubated antigen presenting cells to the patient.
• the-antigen presenting cells are selected from the group consisting of dendritic cells and macrophages.
• Compositions for the treatment of lung cancer comprising T cells or antigen presenting cells that have been incubated with a polypeptide or polynucleotide of the present invention are also provided.
• SEQ ID NO: 1 is the determined cDNA sequence for L363Cl.cons
• SEQ ID NO: 2 is the determined cDNA sequence for L263C2.
• Cons SEQ ID NO: 3 is the determined cDNA sequence for L263C2c
• SEQ ID NO: 4 is the determined cDNA sequence for L263Cl.cons
• SEQ ID NO: 5 is the determined cDNA sequence for L263Clb
• SEQ ID NO: 6 is the determined cDNA sequence for L164C2.cons
• SEQ ID NO: 7 is the determined cDNA sequence for L164Cl.
• SEQ ID NO: 8 is the determined cDNA sequence for L366Cla
• SEQ ID NO: 9 is the determined cDNA sequence for L260Cl.cons
• SEQ ID NO: 10 is the determined cDNA sequence for L163Clc
• SEQ ID NO: 1 1 is the determined cDNA sequence for L163Clb
• SEQ ID NO: 12 is the determined cDNA sequence for L255Cl.
• cons SEQ ID NO: 13 is the determined cDNA sequence for L255Clb
• SEQ ID NO: 14 is the determined cDNA sequence for L355C1.
• cons SEQ ID NO: 15 is the determined cDNA sequence for L366Cl.
• cons SEQ ID NO: 16 is the determined cDNA sequence for L163Cla
• SEQ ID NO: 17 is the determined cDNA sequence for LT86-1
• SEQ ID NO: 18 is the determined cDNA sequence for LT86-2
• 19 is the determined cDNA sequence for LT86-3
• 20 is the determined cDNA sequence for LT86-4
• 21 is the determined cDNA sequence for LT86-5
• 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
• 25 is the determined
• SEQ ID NO: 195 is the predicted amino acid sequence for SAL-8
• SEQ ID NO: 196 is the predicted amino acid sequence for SAL- 12
• SEQ ID NO: 197 is the predicted amino acid sequence for SAL- 14
• SEQ ID NO: 198 is the predicted amino acid sequence for SAL- 16
• SEQ ID NO: 199 is the predicted amino acid sequence for SAL-23
• SEQ ID NO: 200 is the predicted amino acid sequence for SAL-26
• SEQ ID NO: 201 is the predicted amino acid sequence for SAL-29
• SEQ ID NO: 202 is the predicted amino-acid sequence for SAL-32
• SEQ ID NO: 203 is the predicted amino acid sequence for SAL-39
• SEQ ID NO: 204 is the predicted amino acid sequence for SAL-42
• SEQ ID NO: 205 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 predicted amino acid sequence for SAL-48
• SEQ ID NO: 208 is the predicted amino acid sequence for SAL-68
• SEQ ID NO: 209 is the predicted amino acid sequence for SAL-72
• SEQ ID NO: 210 is the predicted amino acid sequence for SAL-77
• SEQ ID NO: 211 is the predicted amino acid sequence for SAL-86
• SEQ ID NO: 212 is the predicted amino acid sequence for SAL-88
• SEQ ID NO: 213 is the predicted amino acid sequence for SAL-93
• SEQ ID NO: 214 is the predicted amino acid sequence for SAL- 100
• SEQ ID NO: 215 is the predicted amino acid sequence for SAL- 105
• SEQ ID NO: 216 is a second predicted amino acid sequence for SAL-50
• the present invention is generally directed to compositions and methods for the therapy of lung cancer.
• the compositions described herein include polypeptides, fusion proteins and polynucleotides.
• molecules such as an antibody or fragment thereof that bind to the inventive polypeptides.
• binding agents such molecules are referred to herein as "binding agents.”
• the inventive polypeptides comprise at least a portion of a protein that is expressed at a greater level in human lung tumor tissue than in normal lung tissue.
• the level of RNA encoding the polypeptide is at least 2-fold higher in tumor tissue.
• Such polypeptides include, but are not limited to. polypeptides (and immunogenic portions thereof) encoded by the nucleotide sequences provided in SEQ ID NO: 1-16 and variants thereof.
• the inventive polypeptides comprise at least a portion of a immunogenic lung tumor protein, including but not limited to polypeptides wherein the lung tumor protein includes an amino acid sequence encoded by a polynucleotide including a sequence selected from the group consisting of (a) nucleotide sequences recited in SEQ ID NO: 17-31, 49-55, 63,64, 66, 68-72, 78-80 and 84-92, (b) the complements of said nucleotide sequences, and (c) variants of such sequences.
• the inventive polypeptides comprise at least a portion of a lung tumor protein, including polypeptides wherein the lung tumor protein includes an amino acid sequence encoded by a polynucleotide including a sequence selected from the group consisting of (a) nucleotide sequences recited in SEQ ID NO: 102-110, 116-120 and 126-181, (b) the complements of said nucleotide sequences, and (c) variants of such sequences.
• polypeptide encompasses amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
• a polypeptide comprising a portion of one of the above lung tumor proteins may consist entirely of the portion, or the portion may be present within a larger polypeptide that contains additional sequences.
• the additional sequences may be derived from the native protein or may be heterologous, and such sequences may (but need not) be immunoreactive and/or antigenic.
• polypeptides may be isolated from lung tumor tissue or prepared by synthetic or recombinant means.
• an "immunogenic portion" of a lung tumor protein is a portion that is capable of eliciting an immune response in a patient inflicted with lung cancer and as such binds to antibodies present within sera from a lung cancer patient.
• Such immunogenic portions generally comprise at least about 5 amino acid residues, more preferably at least about 10, and most preferably at least about 20 amino acid residues.
• Immunogenic portions of the proteins described herein may be identified in antibody binding assays. Such assays may generally be performed using any of a variety of means known to those of ordinary skill in the art, as described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988.
• a polypeptide may be immobilized on a solid support (as described below) and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, 125 I-labeled Protein A.
• a polypeptide may be used to generate monoclonal and polyclonal antibodies for use in detection of the polypeptide in blood or other fluids of lung cancer patients. Methods for preparing and identifying immunogenic portions of antigens of known sequence are well known in the art and include those summarized in Paul, Fundamental Immunology, 3 rd ed., Raven Press, 1993, pp. 243-247.
• polynucleotide(s), means a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and corresponding RNA molecules, including HnRNA and mRNA molecules, both sense and anti-sense strands, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides.
• An HnRNA molecule contains introns and corresponds to a DNA molecule in a generally one-to-one manner.
• An mRNA molecule corresponds to an HnRNA and DNA molecule from which the introns have been excised.
• a polynucleotide may consist of an entire gene, or any portion thereof.
• Operable anti-sense polynucleotides may comprise a fragment of the corresponding polynucleotide, and the definition of "polynucleotide” therefore includes all such operable anti-sense fragments.
• compositions and methods of the present invention also encompass variants of the above polypeptides and polynucleotides.
• a polypeptide "variant,” as used herein, is a polypeptide that differs from the recited polypeptide only in conservative substitutions and/or modifications, such that the antigenic properties of the polypeptide are retained.
• variant polypeptides differ from an identified sequence by substitution, deletion or addition of five amino acids or fewer.
• Such variants may generally be identified by modifying one of the above polypeptide sequences, and evaluating the antigenic properties of the modified polypeptide using, for example, the representative procedures described herein.
• Polypeptide variants preferably exhibit at least about 70%, more preferably at least about 90% and most preferably at least about 95% identity (determined as described below) to the identified polypeptides.
• a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
• the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gin, asn, ser. thr; (2) cys, ser, tyr, thr; (3) val. ile, leu, met, ala. phe; (4) lys, arg, his; and (5) phe, tyr, tip, his.
• Variants may also, or alternatively, contain other modifications, including the deletion or addition of amino acids that have minimal influence on the antigenic properties, secondary structure and hydropathic nature of the polypeptide.
• a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein.
• the polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.
• a polypeptide may be conjugated to an immunoglobulin Fc region.
• nucleotide “variant” is a sequence that differs from the recited nucleotide sequence in having one or more nucleotide deletions, substitutions or additions. Such modifications may be readily introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis as taught, for example, by Adelman et al. (DNA, 2:183. 1983). Nucleotide variants may be naturally occurring allelic variants, or non- naturally occurring variants. Variant nucleotide sequences preferably exhibit at least about 70%, more preferably at least about 80% and most preferably at least about 90% identity (determined as described below) to the recited sequence.
• the lung tumor antigens provided by the present invention include variants that are encoded by DNA sequences which are substantially homologous to one or more of the DNA sequences specifically recited herein.
• “Substantial homology,” as used herein, refers to DNA sequences that are capable of hybridizing under moderately stringent conditions. Suitable moderately stringent conditions include prewashing in a solution of 5X SSC, 0.5% SDS. 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-65°C, 5X SSC, overnight or, in the event of cross-species homology, at 45°C with 0.5X SSC; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS.
• Such hybridizing DNA sequences are also within the scope of this invention, as are nucleotide sequences that, due to code degeneracy, encode an immunogenic polypeptide that is encoded by a hybridizing DNA sequence.
• Two nucleotide or polypeptide sequences are said to be “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. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
• a “comparison window" as used herein refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Optimal alignment of sequences for comparison may be conducted using the
• the "percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e. gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• the percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e. the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
• the lung tumor polypeptides of the present invention may be isolated from lung tumor tissue using any of a variety of methods well known in the art.
• cDNA molecules encoding polypeptides preferentially expressed in lung tumor tissue may be cloned on the basis of the lung tumor- specific expression of the corresponding mRNAs, using differential display PCR. This technique compares the amplified products from RNA templates prepared from normal lung and lung tumor tissue.
• cDNA may be prepared by reverse transcription of RNA using a (dT) 12 AG primer.
• a band corresponding to an amplified product specific to the tumor RNA may be cut out from a silver stained gel and subcloned into a suitable vector.
• cDNA sequences that may be isolated using this procedure include those provided in SEQ ID NO: 1-16.
• cDNA molecules encoding immunogenic lung tumor polypeptides may be prepared by screening a cDNA expression library prepared from a lung tumor sample with sera from the same patient as the tumor sample, as described in detail in Example 2 below. Examples of cDNA sequences that may be isolated using this procedure include those provided in SEQ ID NO: 17-31.
• cDNA molecules encoding lung tumor polypeptides may be obtained by screening such a cDNA expression library with mouse anti- lung tumor serum as described below in Example 3. Examples of cDNA sequences that may thus be isolated are provided in SEQ ID NO: 49-55, 63, 64 and 126-148. cDNA sequences encoding lung tumor antigens may also be isolated by screening of lung tumor cDNA libraries prepared from SCID mice with mouse anti-tumor sera, as described below in Example 4. Examples of cDNA sequences that may be isolated using this technique are provided in SEQ ID NO: 149-181.
• a gene encoding a polypeptide described herein may, alternatively, be amplified from human genomic DNA, or from lung tumor cDNA, via polymerase chain reaction.
• sequence-specific primers may be designed based on the nucleotide sequences provided herein and may be purchased or synthesized.
• An amplified portion of a specific nucleotide sequence may then be used to isolate the full length gene from a human genomic DNA library or from a lung tumor cDNA library, using well known techniques, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1989).
• the polypeptide may be produced recombinantly by inserting the DNA sequence into an expression vector and expressing the polypeptide in an appropriate host.
• Any of a variety of expression vectors known to those of ordinary skill in the art may be employed to express recombinant polypeptides of this invention. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a polynucleotide that encodes the recombinant polypeptide.
• Suitable host cells include prokaryotes, yeast and higher eukaryotic cells.
• the host cells employed are E. coli, yeast or a mammalian cell line, such as COS 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 suitable host/vector systems which secrete the recombinant polypeptide may be first concentrated using a commercially available filter. The concentrate may then be applied to a suitable purification matrix, such as an affinity matrix or ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify the recombinant polypeptide.
• Such techniques may also be used to prepare polypeptides comprising portions or variants of the native polypeptides. Portions and other variants having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques well known to those of ordinary skill in the art.
• polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain (see, for example, Merrifield, J. Am. Chem. Soc. 55:2149-2146, 1963).
• Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/ Applied BioSystems Division (Foster City, CA), and may be operated according to the manufacturer's instructions.
• the polypeptides disclosed herein are prepared in an isolated, substantially pure form (i.e., the polypeptides are homogenous as determined by amino acid composition and primary sequence analysis).
• the polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.
• the substantially pure polypeptides are incorporated into pharmaceutical compositions or vaccines for use in one or more of the methods disclosed herein.
• the present invention provides fusion proteins comprising a first and a second inventive polypeptide or, alternatively, a polypeptide of the present invention and a known lung tumor antigen, together with variants of such fusion proteins.
• the fusion proteins of the present invention may (but need not) include a linker peptide between the first and second polypeptides.
• a DNA sequence encoding a fusion protein of the present invention is constructed using known recombinant DNA techniques to assemble separate DNA sequences encoding the first and second polypeptides into an appropriate expression vector.
• the 3' end of a DNA sequence encoding the first polypeptide is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide so that the reading frames of the sequences are in phase to permit mRNA translation of the two DNA sequences into a single fusion protein that retains the biological activity of both the first and the second polypeptides.
• a peptide linker sequence may be employed to separate the first and the second polypeptides by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures.
• Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art.
• Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides: and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
• Preferred peptide linker sequences contain Gly, Asn and Ser residues.
• linker sequence may be used in other near neutral amino acids, such as Thr and Ala.
• Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 53:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180.
• the linker sequence may be from 1 to about 50 amino acids in length. Peptide sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
• the ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements.
• the regulatory elements responsible for expression of DNA are located only 5' to the DNA sequence encoding the first polypeptides.
• stop codons require to end translation and transcription termination signals are only present 3' to the DNA sequence encoding the second polypeptide.
• Fusion proteins are also provided that comprise a polypeptide of the present invention together with an unrelated immunogenic protein.
• the immunogenic protein is capable of eliciting a recall response.
• examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al. New Engl. J. Med., 336:86- 91 (1997)).
• Polypeptides that comprise an immunogenic portion of a lung tumor protein may generally be used for therapy of lung cancer, wherein the polypeptide stimulates the patient's own immune response to lung tumor cells.
• the present invention thus provides methods for using one or more of the compounds described herein (which may be polypeptides. polynucleotides or fusion proteins) for immunotherapy of lung cancer in a patient.
• a "patient” refers to any warm-blooded animal, preferably a human.
• a patient may be afflicted with disease, or may be free of detectable disease.
• the compounds disclosed herein may be used to treat lung cancer or to inhibit the development of lung cancer.
• the compounds are administered either prior to or following surgical removal of primary tumors and/or treatment by administration of radiotherapy and conventional chemotherapeutic drugs.
• the inventive polypeptide is generally present within a pharmaceutical composition or a vaccine.
• Pharmaceutical compositions may comprise one or more polypeptides, each of which may contain one or more of the above sequences (or variants thereof), and a physiologically acceptable carrier.
• the vaccines may comprise one or more such polypeptides and an immune response enhancer, such as an adjuvant, biodegradable microsphere (e.g., polylactic galactide) or a liposome (into which the polypeptide is incorporated).
• Pharmaceutical compositions and vaccines may also contain other epitopes of lung tumor antigens, either incorporated into a fusion protein as described above (/. e. , a single polypeptide that contains multiple epitopes) or present within a separate polypeptide.
• a pharmaceutical composition or vaccine may contain DNA encoding one or more of the above polypeptides and/or fusion proteins, such that the polypeptide is generated in situ.
• the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter).
• Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerri ⁇ ) that expresses an epitope of a lung cell antigen on its cell surface.
• the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retro virus, or adenovirus). which may involve the use of a non-pathogenic (defective), replication competent virus.
• a viral expression system e.g., vaccinia or other pox virus, retro virus, or adenovirus
• vaccinia or other pox virus, retro virus, or adenovirus may involve the use of a non-pathogenic (defective), replication competent virus.
• Suitable systems are disclosed, for example, in Fisher-Hoch et al, PNAS 5.5:317-321, 1989; Flexner et al., Ann. N Y. Acad. Sci. 569:86-103, 1989: Flexner et al., Vaccine 5:17-21, 1990; U.S. Patent Nos. 4,603,112, 4,769,330, and 5.017,487; WO 89/01973; U.S. Patent No.
• the DNA may also be "naked,” as described, for example, in published PCT application WO 90/11092, and Ulmer et al., Science 259:1745-1749, 1993, reviewed by Cohen, Science 259:1691-1692. 1993.
• the uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
• compositions and vaccines may be administered by injection (e.g., intracutaneous. intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Between 1 and 10 doses may be administered over a 3-24 week period. Preferably, 4 doses are administered, at an interval of 3 months, and booster administrations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients.
• a suitable dose is an amount of polypeptide or DNA that is effective to raise an immune response (cellular and/or humoral) against lung tumor cells in a treated patient.
• a suitable immune response is at least 10-50% above the basal (i.e., untreated) level.
• the amount of polypeptide present in a dose ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 ⁇ g.
• Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.01 mL to about 5 mL.
• the type of carrier will vary depending on the mode of administration.
• the carrier preferably comprises water, saline, alcohol, a lipid, a wax and/or a buffer.
• 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 may be employed.
• Biodegradable microspheres e.g., polylactic glycolide
• Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4.897,268 and 5,075,109. Any of a variety of immune response enhancers may be employed in the vaccines of this invention.
• an adjuvant may be included.
• Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a nonspecific stimulator of immune response, such as lipid A, Bordello pertussis or Mycobacterium tuberculosis.
• Such adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI), and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ).
• polynucleotides of the present invention may be formulated so as to permit entry into a cell of a mammal, preferably a human, and expression therein. Such formulations are particularly useful for therapeutic purposes.
• a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or other pox virus (e.g. avian pox virus). Techniques for incorporating DNA into such vectors are well known to those of skill in the art.
• a retroviral vector may additionally transfer or incorporate a targeting moiety, such as a gene that encodes for a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody, by methods know to those of ordinary skill in the art.
• a targeting moiety such as a gene that encodes for a ligand for a receptor on a specific target cell
• Polypeptides disclosed herein may also be employed in adoptive immunotherapy for the treatment of cancer.
• Adoptive immunotherapy may be broadly classified into either active or passive immunotherapy.
• active immunotherapy treatment relies on the in vivo stimulation of the endogenous host immune system to react against tumors with the administration of immune response-modifying agents (for example, tumor vaccines, bacterial adjuvants, and/or cytokines).
• immune response-modifying agents for example, tumor vaccines, bacterial adjuvants, and/or cytokines.
• effector cells include T lymphocytes (for example, CD8+ cytotoxic T-lymphocyte, CD4+ T-helper. tumor-infiltrating lymphocytes), killer cells (Natural Killer cells, lymphokine-activated killer cells), B cells, or antigen presenting cells (such as dendritic cells and macrophages) expressing the disclosed antigens.
• T lymphocytes for example, CD8+ cytotoxic T-lymphocyte, CD4+ T-helper. tumor-infiltrating lymphocytes
• killer cells Natural Killer cells, lymphokine-activated killer cells
• B cells or antigen presenting cells (such as dendritic cells and macrophages) expressing the disclosed antigens.
• antigen presenting cells such as dendritic cells and macrophages
• T-cells for passive immunotherapy.
• the predominant method of procuring adequate numbers of T-cells for adoptive immunotherapy is to grow immune T-cells in vitro.
• Culture conditions for expanding single antigen-specific T-cells to several billion in number with retention of antigen recognition in vivo are well known in the art.
• These in vitro culture conditions typically utilize intermittent stimulation with antigen, often in the presence of cytokines, such as IL-2, and non-dividing feeder cells.
• cytokines such as IL-2
• non-dividing feeder cells for non-dividing feeder cells.
• the immunoreactive polypeptides described herein may be used to rapidly expand antigen-specific T cell cultures in order to generate sufficient number of cells for immunotherapy.
• antigen-presenting cells such as dendritic, macrophage or B-cells
• antigen-presenting cells may be pulsed with immunoreactive polypeptides or transfected with a polynucleotide sequence(s), using standard techniques well known in the art.
• the cultured T-cells must be able to grow and distribute widely and to survive long term in vivo. Studies have demonstrated that cultured T-cells can be induced to grow in vivo and to survive long term in substantial numbers by repeated stimulation with antigen supplemented with IL-2 (see, for example, Cheever et al. Ibid).
• the polypeptides disclosed herein may also be employed to generate and/or isolate tumor-reactive T-cells, which can then be administered to the patient.
• antigen-specific T-cell lines may be generated by in vivo immunization with short peptides corresponding to immunogenic portions of the disclosed polypeptides.
• the resulting antigen specific CD8+ CTL clones may be isolated from the patient, expanded using standard tissue culture techniques, and returned to the patient.
• peptides corresponding to immunogenic portions of the polypeptides may be employed to generate tumor reactive T cell subsets by selective in vitro stimulation and expansion of autologous T cells to provide antigen-specific T cells which may be subsequently transferred to the patient as described, for example, by Chang et al. (Crit. Rev. Oncol. HematoL 22(3), 213, 1996).
• syngeneic or autologous dendritic cells may be pulsed with peptides corresponding to at least an immunogenic portion of a polypeptide disclosed herein. The resulting antigen-specific dendritic cells may either be transferred into a patient, or employed to stimulate T cells to provide antigen-specific T cells which may, in turn, be administered to a patient.
• vectors expressing-the disclosed polynucleotides may be introduced into stem cells taken from the patient and clonally propagated in vitro for autologous transplant back into the same patient.
• cells of the immune system may be isolated from the peripheral blood of a patient, using a commercially available cell separation system, such as CellPro Incorporated's (Bothell, WA) CEPRATETM system (see U.S. Patent No. 5,240,856; U.S. Patent No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243).
• the separated cells are stimulated with one or more of the immunoreactive polypeptides contained within a delivery vehicle, such as a microsphere, to provide antigen-specific T cells.
• Binding agents of the present invention may generally be prepared using methods known to those of ordinary skill in the art, including the representative procedures described herein. Binding agents are capable of differentiating between patients with and without lung cancer, using the representative assays described herein.
• antibodies or other binding agents raised against a lung tumor protein, or a suitable portion thereof will generate a signal indicating the presence of primary or metastatic lung cancer in at least about 20%) of patients afflicted with the disease, and will generate a negative signal indicating the absence of the disease in at least about 90% of individuals without primary or metastatic lung cancer.
• Suitable portions of such lung tumor proteins are portions that are able to generate a binding agent that indicates the presence of primary or metastatic lung cancer in substantially all (i.e., at least about 80%, and preferably at least about 90%) of the patients for which lung cancer would be indicated using the full length protein, and that indicate the absence of lung cancer in substantially all of those samples that would be negative when tested with full length protein.
• the representative assays described below, such as the two-antibody sandwich assay may generally be employed for evaluating the ability of a binding agent to detect metastatic human lung tumors.
• the ability of a polypeptide prepared as described herein to generate antibodies capable of detecting primary or metastatic human lung tumors may generally be evaluated by raising one or more antibodies against the polypeptide (using, for example, a representative method described herein) and determining the ability of such antibodies to detect such tumors in patients. This determination may be made by assaying biological samples from patients with and without primary or metastatic lung cancer for the presence of a polypeptide that binds to the generated antibodies. Such test assays may be performed, for example, using a representative procedure described below. Polypeptides that generate antibodies capable of detecting at least 20% of primary or metastatic lung tumors by such procedures are considered to be useful in assays for detecting primary or metastatic human lung tumors. Polypeptide specific antibodies may be used alone or in combination to improve sensitivity.
• Polypeptides capable of detecting primary or metastatic human lung tumors may be used as markers for diagnosing lung cancer or for monitoring disease progression in patients.
• lung cancer in a patient may be diagnosed by evaluating a biological sample obtained from the patient for the level of one or more of the above polypeptides. relative to a predetermined cut-off value.
• suitable "biological samples” include blood, sera, urine and/or lung secretions.
• the level of one or more of the above polypeptides may be evaluated using any binding agent specific for the polypeptide(s).
• a "binding agent,” in the context of this invention, is any agent (such as a compound or a cell) that binds to a polypeptide as described above.
• binding refers to a noncovalent association between two separate molecules (each of which may be free (i.e., in solution) or present on the surface of a cell or a solid support), such that a "complex" is formed. Such a complex may be free or immobilized (either covalently or noncovalently) on a support material.
• the ability to bind may generally be evaluated by determining a binding constant for the formation of the complex.
• the binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations.
• two compounds are said to "bind” in the context of the present invention when the binding constant for complex formation exceeds about 10 3 L/mol.
• the binding constant may be determined using methods well known to those of ordinary skill in the art.
• a binding agent may be a ribosome with or without a peptide component, an RNA molecule or a peptide.
• the binding partner is an antibody, or a fragment thereof.
• Such antibodies may be polyclonal, or monoclonal.
• the antibodies may be single chain, chimeric, CDR-grafted or humanized.
• Antibodies may be prepared by the methods described herein and by other methods well known to those of skill in the art.
• the assay involves the use of binding partner immobilized on a solid support to bind to and remove the polypeptide from the remainder of the sample.
• the bound polypeptide may then be detected using a second binding partner that contains a reporter group.
• Suitable second binding partners include antibodies that bind to the binding partner/polypeptide complex.
• a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding partner after incubation of the binding partner with the sample.
• the extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding partner is indicative of the reactivity of the sample with the immobilized binding partner.
• the solid support may be any material known to those of ordinary skill in the art to which the antigen may be attached.
• the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.
• the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride.
• the support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Patent No. 5,359,681.
• the binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature.
• immobilization refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the antigen and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day.
• contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 ⁇ g, and preferably about 100 ng to about 1 ⁇ g, is sufficient to immobilize an adequate amount of binding agent.
• a plastic microtiter plate such as polystyrene or polyvinylchloride
• Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent.
• a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent.
• the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991. at A12-A13).
• the assay is a two-antibody sandwich assay.
• This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a second antibody (containing a reporter group) capable of binding to a different site on the polypeptide is added. The amount of second antibody that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.
• the immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody.
• the sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation.
• PBS phosphate-buffered saline
• an appropriate contact time i.e., incubation time is that period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual with lung cancer.
• the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide.
• a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide.
• the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.
• Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20TM.
• the second antibody which contains a reporter group, may then be added to the solid support.
• Preferred reporter groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.
• the conjugation of antibody to reporter group may be achieved using standard methods known to those of ordinary skill in the art.
• the second antibody is then incubated with the immobilized antibody- polypeptide complex for an amount of time sufficient to detect the bound polypeptide.
• An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound second antibody is then removed and bound second antibody is detected using the reporter group.
• the method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme).
• Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
• the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value.
• the cut-off value is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without lung cancer.
• a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for lung cancer.
• the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result.
• the cut-off value on the plot that is the closest to the upper left-hand corner is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive.
• the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate.
• a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for lung cancer.
• the assay is performed in a flow-through or strip test format, wherein the antibody is immobilized on a membrane, such as nitrocellulose.
• a membrane such as nitrocellulose.
• polypeptides within the sample bind to the immobilized antibody as the sample passes through the membrane.
• a second, labeled antibody then binds to the antibody- polypeptide complex as a solution containing the second antibody flows through the membrane.
• the detection of bound second antibody may then be performed as described above.
• the strip test format one end of the membrane to which antibody is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second antibody and to the area of immobilized antibody. Concentration of second antibody at the area of immobilized antibody indicates the presence of lung cancer.
• the concentration of second antibody at that site generates a pattern, such as a line, that can be read visually.
• a pattern such as a line
• the amount of antibody immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above.
• the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 ⁇ g, and more preferably from about 50 ng to about 500 ng.
• Such tests can typically be performed with a very small amount of biological sample.
• numerous other assay protocols exist that are suitable for use with the antigens or antibodies of the present invention. The above descriptions are intended to be exemplary only.
• the above polypeptides may be used as markers for the progression of lung cancer.
• assays as described above for the diagnosis of lung cancer may be performed over time, and the change in the level of reactive polypeptide(s) evaluated.
• the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed.
• lung cancer is progressing in those patients in whom the level of polypeptide detected by the binding agent increases over time.
• lung cancer is not progressing when the level of reactive polypeptide either remains constant or decreases with time.
• Antibodies for use in the above methods may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
• an immunogen comprising the antigenic polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep and goats).
• the polypeptides of this invention may serve as the immunogen without modification.
• a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin.
• the immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically.
• Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
• Monoclonal antibodies specific for the antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. (5:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed.
• the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated 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 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.
• Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies.
• various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse.
• Monoclonal antibodies may then be harvested from the ascites fluid or the blood.
• Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction.
• the polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.
• Monoclonal antibodies of the present invention may also be used as therapeutic reagents, to diminish or eliminate lung tumors.
• the antibodies may be used on their own (for instance, to inhibit metastases) or coupled to one or more therapeutic agents.
• Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof.
• Preferred radionuclides include 90 Y, I, ,25 I, 131 I, 186 Re, I88 Re, 211 At, and 212 Bi.
• Preferred drugs include methotrexate, and pyrimidine and purine analogs.
• Preferred differentiation inducers include phorbol esters and butyric acid.
• Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.
• a therapeutic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody either directly or indirectly (e.g., via a linker group).
• a direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other.
• a nucleophilic group such as an amino or sulfhydryl group
• on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide. or with an alkyl group containing a good leaving group (e.g., a halide) on the other.
• a linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities.
• a linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible.
• a linker group which is cleavable during or upon intemalization into a cell.
• a number of different cleavable linker groups have been described.
• the mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Patent No. 4,489,710, to Spitler), by irradiation of a photolabile bond (e.g., U.S. Patent No. 4,625,014, to Senter et al.), by hydrolysis of derivatized amino acid side chains (e.g., U.S.
• Patent No. 4.638,045 to Kohn et al.
• serum complement-mediated hydrolysis e.g., U.S. Patent No. 4.671,958, to Rodwell et al.
• acid-catalyzed hydrolysis e.g., U.S. Patent No. 4,569,789, to Blattler et al.
• immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers which provide multiple sites for attachment can be used. Alternatively, a carrier can be used.
• a carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group.
• Suitable carriers include proteins such as albumins (e.g., U.S. Patent No. 4,507,234, to Kato et al.), peptides and polysaccharides such as aminodextran (e.g., U.S. Patent No. 4,699,784, to Shih et al.).
• a carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Patent Nos. 4,429.008 and 4,873,088).
• Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds.
• U.S. Patent No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis.
• a radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide.
• U.S. Patent No. 4,673,562 to Davison et al. discloses representative chelating compounds and their synthesis.
• a variety of routes of administration for the antibodies and immunoconjugates may be used. Typically, administration will be intravenous, intramuscular, subcutaneous or in the bed of a resected tumor. It will be evident that the precise dose of the antibody/immunoconjugate will vary depending upon the antibody used, the antigen density on the tumor, and the rate of clearance of the antibody.
• Diagnostic reagents of the present invention may also comprise DNA sequences encoding one or more of the above polypeptides, or one or more portions thereof.
• at least two oligonucleotide primers may be employed in a polymerase chain reaction (PCR) based assay to amplify lung tumor-specific cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for a polynucleotide encoding a lung tumor protein of the present invention.
• PCR polymerase chain reaction
• the presence of the amplified cDNA is then detected using techniques well known in the art, such as gel electrophoresis.
• oligonucleotide probes specific for a polynucleotide encoding a lung tumor protein of the present invention may be used in a hybridization assay to detect the presence of an inventive polypeptide in a biological sample.
• oligonucleotide primer/probe specific for a polynucleotide means an oligonucleotide sequence that has at least about 60%, preferably at least about 75% and more preferably at least about 90%, identity to the polynucleotide in question.
• Oligonucleotide primers and/or probes which may be usefully employed in the inventive diagnostic methods preferably have at least about 10-40 nucleotides.
• the oligonucleotide primers comprise at least about 10 contiguous nucleotides of a polynucleotide having a partial sequence selected from SEQ ID NO: 1-31, 49-55, 63, 64. 66, 68-72, 78-80, 84-92, 102-110, 1 16-120 and 126-181.
• oligonucleotide probes for use in the inventive diagnostic methods comprise at least about 15 contiguous oligonucleotides of a polynucleotide having a partial sequence provided in SEQ ID NO: 1-31, 49-55, 63, 64, 66, 68-72, 78-80, 84-92, 102-110, 116-120 and 126-181.
• Techniques for both PCR based assays and hybridization assays are well known in the art (see, for example, Mullis et al. Ibid; Ehrlich, Ibid). Primers or probes may thus be used to detect lung tumor- specific sequences in biological samples, including blood, semen, lung tissue and/or lung tumor tissue.
• This example illustrates the preparation of cDNA molecules encoding lung tumor-specific polypeptides using a differential display screen.
• Tissue samples were prepared from breast tumor and normal tissue of a patient with lung cancer that was confirmed by pathology after removal of samples from the patient.
• Normal RNA and tumor RNA was extracted from the samples and mRNA was isolated and converted into cDNA using a (dT) I2 AG (SEQ ID NO: 47) anchored 3' primer.
• Differential display PCR was then executed using a randomly chosen primer (SEQ ID NO: 48).
• Amplification conditions were standard buffer containing 1.5 mM MgC , 20 pmol of primer, 500 pmol dNTP and 1 unit of Taq DNA polymerase (Perkin-Elmer, Branchburg, NJ).
• This example illustrates the isolation of cDNA sequences encoding lung tumor antigens by expression screening of lung tumor samples with autologous patient sera.
• a human lung tumor directional cDNA expression library was constructed employing the Lambda ZAP Express expression system (Stratagene, La Jolla, CA). Total RNA for the library was taken from a late SCID mouse passaged human squamous epithelial lung carcinoma and poly A+ RNA was isolated using the Message Maker kit (Gibco BRL, Gaithersburg, MD). The resulting library was screened using E.
• LT86-1 - LT86-15 Fifteen clones were isolated, referred to hereinafter as LT86-1 - LT86-15.
• the isolated cDNA sequences for LT86-1 - LT86-8 and LT86-10 - LT86-15 are provided in SEQ ID NO: 17-24 and 26-31, respectively, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 32-39 and 41-46, respectively.
• the determined cDNA sequence for LT86-9 is provided in SEQ ID NO: 25, with the corresponding predicted amino acid sequences from the 3' and 5' ends being provided in SEQ ID NO: 40 and 65, respectively. These sequences were compared to those in the gene bank as described above.
• Clones LT86-3, LT86-6 - LT86-9, LT86-11 - LT86-13 and LT86-15 were found to show some homology to previously identified expressed sequence tags (ESTs), with clones LT86-6, LT86-8, LT86-11. LT86-12 and LT86- 15 appearing to be similar or identical to each other.
• Clone LT86-3 was found to show some homology with a human transcription repressor.
• Clones LT86-6, 8, 9, 11, 12 and 15 were found to show some homology to a yeast RNA Pol II transcription regulation mediator.
• Clone LT86-13 was found to show some homology with a C.
• Clone LT86-9 appears to contain two inserts, with the 5 * sequence showing homology to the previously identified antisense sequence of interferon alpha-induced P27, and the 3' sequence being similar to LT86-6.
• Clone LT86-14 (SEQ ID NO: 30) was found to show some homology to the trithorax gene and has an "RGD" cell attachment sequence and a beta-Lactamase A site which functions in hydrolysis of penicillin.
• Clones LT86-1, LT86-2, LT86-4, LT86-5 and LT86-10 (SEQ ID NOS: 17, 18, 20, 21 and 26, respectively) were found to show homology to previously identified genes.
• LT86-20, LT86-21, LT86-22, LT86-26 and LT86-27 The determined 5' cDNA sequences for LT86-20, LT86-22, LT86-26 and LT86-27 are provided in SEQ ID NO: 68 and 70-72, respectively, with the determined 3' cDNA sequences for LT86-21 being provided in SEQ ID NO: 69.
• the corresponding predicted amino acid sequences for LT86-20, LT86-21, LT86- 22, LT86-26 and LT86-27 are provided in SEQ ID NO: 73-77, respectively.
• LT86-22 and LT86-27 were found to be highly similar to each other. Comparison of these sequences to those in the gene bank as described above, revealed no significant homologies to LT86-22 and LT86-27.
• LT86-20, LT86-21 and LT86-26 were found to show homology to previously identified genes.
• TUMOR ANTIGENS This example illustrates the isolation of cDNA sequences encoding lung tumor antigens by screening of lung tumor cDNA libraries with mouse anti-tumor sera.
• a directional cDNA lung tumor expression library was prepared as described above in Example 2.
• Sera was obtained from SCID mice containing late passaged human squamous cell and adenocarcinoma tumors. These sera were pooled and injected into normal mice to produce anti-lung tumor serum. Approximately 200,000 PFUs were screened from the unamplified library using this antiserum. Using a goat anti-mouse IgG-A-M (H+L) alkaline phosphatase second antibody developed with NBT/BCIP (BRL Labs.), approximately 40 positive plaques were identified.
• Phage was purified and phagemid excised for 9 clones with inserts in a pBK-CMV vector for expression in prokaryotic or eukaryotic cells.
• the determined cDNA sequences for 7 of the isolated clones (hereinafter referred to as L86S-3, L86S-12, L86S-16, L86S-25, L86S-36, L86S-40 and L86S-46) are provided in SEQ ID NO: 49-55, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 56-62, respectively.
• the 5' cDNA sequences for the remaining 2 clones (hereinafter referred to as L86S-30 and L86S-41) are provided in SEQ ID NO: 63 and 64.
• L86S-36 and L86S-46 were subsequently determined to represent the same gene. Comparison of these sequences with those in the public database as described above, revealed no significant homologies to clones L86S-30, L86S-36 and L86S-46 (SEQ ID NO: 63, 53 and 55, respectively). L86S-16 (SEQ ID NO: 51) was found to show some homology to an EST previously identified in fetal lung and germ cell tumor. The remaining clones were found to show at least some degree of homology to previously identified human genes.
• L86S-47 L86S-49 and L86S-51 (SEQ ID NO: 84-92, respectively).
• the corresponding predicted amino acid sequences are provided in SEQ ID NO: 93-101. respectively.
• L86S-30, L86S-39 and L86S-47 were found to be similar to each other. Comparison of these sequences with those in the gene bank as described above, revealed no significant homologies to L86S-14.
• L86S-29 was found to show some homology to a previously identified EST.
• L86S-39, L86S-47, L86S-49 and L86S-51 were found to show some homology to previously identified genes.
• RNA for the library was isolated from two primary squamous lung tumors and poly A+ RNA was isolated using an oligo dT column.
• Antiserum was developed in normal mice using a pool of sera from three SCID mice implanted with human squamous lung carcinomas. Approximately 700,000 PFUs were screened from the unamplified library with E. coli absorbed mouse anti-SCID tumor serum. Positive plaques were identified as described above. Phage was purified and phagemid excised for 180 clones with inserts in a pBK-CMV vector for expression in prokaryotic or eukaryotic cells.
• the determined cDNA sequences for 23 of the isolated clones are provided in SEQ ID NO: 126-148. Comparison of these sequences with those in the public database as described above revealed no significant homologies to the sequences of SEQ ID NO: 139 and 143-148. The sequences of SEQ ID NO: 126-138 and 140-142 were found to show homology previously identified human polynucleotide sequences.
• This example illustrates the isolation of cDNA sequences encoding lung tumor antigens by screening of lung tumor cDNA libraries prepared from SCID mice with mouse anti-tumor sera.
• a directional cDNA lung tumor expression library was prepared using a Stratagene kit with a Lambda Zap Express vector.
• Total RNA for the library was taken from a late passaged lung adenocarcinoma grown in SCID mice.
• Poly A+ RNA was isolated using a Message Maker Kit (Gibco BRL).
• Sera was obtained from two SCID mice implanted with lung adenocarcinomas. These sera were pooled and injected into normal mice to produce anti-lung tumor serum. Approximately 700,000 PFUs were screened from the unamplified library with E. e /t-absorbed mouse anti-SCID tumor serum.
• SEQ ID NO: 149-181 The determined 5' cDNA sequences for 33 of the isolated clones are provided in SEQ ID NO: 149-181.
• the corresponding predicted amino acid sequences for SEQ ID NO: 149, 150, 152-154, 156-158 and 160-181 are provided in SEQ ID NO: 182, 183, 186, 188-193 and 194-215, respectively.
• SAL-25 The clone of SEQ ID NO: 151 (referred to as SAL-25) was found to contain two open reading frames (ORFs).
• ORFs open reading frames
• the predicted amino acid sequences encoded by these ORFs are provided in SEQ ID NO: 184 and 185.
• SAL-50 The clone of SEQ ID NO: 153 (referred to as SAL-50) was found to contain two open reading frames encoding the predicted amino acid sequences of SEQ ID NO: 187 and 216.
• SAL-66 the clone of SEQ ID NO: 155 (referred to as SAL-66) was found to contain two open reading frames encoding the predicted amino acid sequences of SEQ ID NO: 189 and 190.
• Comparison of the isolated sequences with those in the public database revealed no significant homologies to the sequences of SEQ ID NO: 151, 153 and 154.
• the sequences of SEQ ID NO: 149, 152, 156, 157 and 158 were found to show some homology to previously isolated expressed sequence tags (ESTs).
• ESTs previously isolated expressed sequence tags
• RNA expression levels for representative lung tumor polypeptides were examined in a variety of normal and tumor tissues using RT-PCR. Briefly, total RNA was extracted from a variety of normal and tumor tissues using Trizol reagent. First strand synthesis was carried out using 2 ⁇ g of total RNA with Superscript II reverse transcriptase (BRL Life Technologies) at 42 °C for one hour. The cDNA was then amplified by PCR with gene-specific primers. To ensure the semi- quantitative nature of the RT-PCR, ⁇ -actin was used as an internal control for each of the tissues examined.
• mRNA Expression levels were examined in five different types of tumor tissue (lung squamous tumor from 3 patients, lung adenocarcinoma, prostate tumor colon tumor and breast tumor), and different normal tissues, including lung from four patients, prostate, brain, kidney, liver, ovary, skeletal muscle, skin, small intestine, myocardium, retina and testes.
• L86S-46 was found to be expressed at high levels in lung squamous tumor, colon tumor and prostate tumor, and was undetectable in the other tissues examined.
• L86S-5 was found to be expressed in the lung tumor samples and in 2 out of 4 normal lung samples, but not in the other normal or tumor tissues tested.
• L86S-16 was found to be expressed in all tissues except normal liver and normal stomach.
• L86S-46 was found to be over- expressed in lung squamous tissue and normal tonsil, with expression being low or undetectable in all other tissues examined.
• Example 6 ISOLATION OF DNA SEQUENCES ENCODING LUNG TUMOR ANTIGENS
• DNA sequences encoding antigens potentially involved in squamous cell lung tumor formation were isolated as follows.
• a lung tumor directional cDNA expression library was constructed employing the Lambda ZAP Express expression system (Stratagene, La Jolla, CA). Total RNA for the library was taken from a pool of two human squamous epithelial lung carcinomas and poly A+ RNA was isolated using oligo-dT cellulose (Gibco BRL, Gaithersburg, MD). Phagemid were rescued at random and the cDNA sequences of isolated clones were determined.
• the determined cDNA sequence for the clone SLT-T 1 is provided in SEQ ID NO: 102, with the determined 5' cDNA sequences for the clones SLT-T2, SLT-T3, SLT-T5, SLT-T7, SLT-T9, SLT-T10, SLT-T11 and SLT-T12 being provided in SEQ ID NO: 103- 1 10, respectively.
• the corresponding predicted amino acid sequence for SLT-T1, SLT-T2, SLT-T3, SLT-T10 and SLT-T12 are provided in SEQ ID NO: 111-115, respectively.
• SLT-T2 Comparison of the sequences for SLT-T2, SLT-T3, SLT-T5, SLT-T7, SLT-T9 and SLT-T11 with those in the public databases as described above, revealed no significant homologies.
• the sequences for SLT-T10 and SLT-T 12 were found to show some homology to sequences previously identified in humans.
• the sequence of SLT-T 1 was determined to show some homology to a PAC clone of unknown protein function.
• the cDNA sequence of SLT-T1 (SEQ ID NO: 102) was found to contain a mutator (MUTT) domain.
• SLT-T1 may thus be of use in the treatment, by gene therapy, of lung cancers caused by, or associated with, a disruption in DNA repair.
• DNA sequences encoding antigens potentially involved in adenocarcinoma lung tumor formation were isolated as follows.
• a human lung tumor directional cDNA expression library was constructed employing the Lambda ZAP Express expression system (Stratagene, La Jolla, CA).
• Total RNA for the library was taken from a late SCID mouse passaged human adenocarcinoma and poly A+ RNA was isolated using the Message Maker kit (Gibco BRL, Gaithersburg, MD). Phagemid were rescued at random and the cDNA sequences of isolated clones were determined.
• SALT-T3, SALT-T4, SALT-T7, SALT-T8, and SALT-T9 The determined 5' cDNA sequences for five isolated clones (referred to as SALT-T3, SALT-T4, SALT-T7, SALT-T8, and SALT-T9) are provided in SEQ ID NO: 116- 120, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 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 the human homologue of the mouse H beta 58 gene.
• SALT-T7 was found to have 97% identity to human 3- mercaptopyruvate sulfurtransferase and SALT-T8 was found to show homology to human interferon-inducible protein 1-8U.
• SALT-T9 shows approximately 90% identity to human mucin MUC 5B.
• Polypeptides may be synthesized on a Perkin Elmer/ Applied Biosystems Division 43 OA peptide synthesizer using FMOC chemistry with HPTU (O-Benzotriazole- N,N,N',N'-tetramethyluronium hexafluorophosphate) activation.
• HPTU O-Benzotriazole- N,N,N',N'-tetramethyluronium hexafluorophosphate
• a Gly-Cys-Gly sequence may be attached to the amino terminus of the peptide to provide a method of conjugation, binding to an immobilized surface, or labeling of the peptide.
• Cleavage of the peptides from the solid support may be carried out using the following cleavage mixture: trifluoroacetic acid:ethanedithiol:thioanisole:water:phenol (40: 1 :2:2:3).
• the peptides may be precipitated in cold methyl-t-butyl-ether.
• the peptide pellets may then be dissolved in water containing 0.1% trifluoroacetic acid (TFA) and lyophilized prior to purification by C18 reverse phase HPLC.
• TFA trifluoroacetic acid
• a gradient of 0%-60% acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA) may be used to elute the peptides.
• the peptides may be characterized using electrospray or other types of mass spectrometry and by amino acid analysis.

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