WO2006110478A2 - Mutations et polymorphismes du recepteur du facteur de croissance epidermique - Google Patents

Mutations et polymorphismes du recepteur du facteur de croissance epidermique Download PDF

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WO2006110478A2
WO2006110478A2 PCT/US2006/012878 US2006012878W WO2006110478A2 WO 2006110478 A2 WO2006110478 A2 WO 2006110478A2 US 2006012878 W US2006012878 W US 2006012878W WO 2006110478 A2 WO2006110478 A2 WO 2006110478A2
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egfr
cancer
seq
mutations
polypeptide
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PCT/US2006/012878
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WO2006110478A3 (fr
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Kenneth W. Culver
Jian Zhu
Stan Lilleberg
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Novartis Ag
Novartis Pharma Gmbh
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Publication of WO2006110478A3 publication Critical patent/WO2006110478A3/fr

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    • 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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • EGF epidermal growth factor
  • EGFR epidermal growth factor
  • EGFR receptor for EGF
  • EGFR-mediated cell growth is increased in a variety of solid tumours including non-small cell lung cancer, prostate cancer, breast cancer, gastric cancer, and tumours of the head and neck.
  • Salomon DS et ah Critical Reviews in Oncology/Haematology, 19:183-232 (1995).
  • excessive activation of EGFR on the cancer cell surface is now known to be associated with advanced, disease, the development of a metastatic phenotype and a poor prognosis in cancer patients.
  • the invention provides a method for diagnosing cancer in a subject and a method for choosing subjects for inclusion in a clinical trial for determining efficacy of an EGFR modulating agent; in both these methods the genotype and/or haplotype of a subject is interrogated at an EGFR gene locus. Also provided by the invention are kits for use in determining a treatment strategy for cancer.
  • the aromatase inhibitor FEMARA® is a treatment for advanced breast cancer in postmenopausal women. It blocks the use of oestrogen by certain types of breast cancer that require oestrogen to grow. Janicke F, Breast 13 Suppl 1: S 10-8 (December 2004); Mouridsen H et al, Oncologist 9(5):489-96 (2004).
  • ZOMET A® is a treatment for hypocalcaemia of malignancy (HCM)I and for the treatment of bone metastases across a broad range of tumour types. These tumours include multiple myeloma, prostrate cancer, breast cancer, lung cancer, renal cancer and other solid tumours. Rosen LS et al, Cancer 100(12):2613-21 (June 15, 2004).
  • Vatalanib (l-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a multi- VEGF receptor (VEGFR) inhibitor that may block the creation of new blood vessels to prevent tumour growth.
  • VEGFR VEGF receptor
  • This compound inhibits all known VEGF receptor tyrosine kinases, blocking angiogenesis and lymphangiogenesis. Drevs J et al, Cancer Res. 60:4819-4824 (2000); Wood JM et al, Cancer Res. 60:2178-2189 (2000).
  • Gimatecan is a novel oral inhibitor of topoisomerase I (topo I). Gimatecan blocks cell division in cells that divide rapidly, such as cancer cells, which activates apoptosis. Preclinical data indicate that gimatecan is not a substrate for multidrug resistance pumps, and that it increases the drug-target interaction. De Cesare M et al, Cancer Res. 61:7189-7195 (2001). Phase I clinical studies indicate that the dose-limiting toxicity of gimatecan is myelosuppression.
  • Patupilone is a microtubule stabilizer.
  • Altmann K-H Curr. Opin. Chem. Biol. 5:424- 431 (2001); Altmann K-H et ah, Biochim Biophys Acta. 470:M79-M91 (2000); O'Neill V et ah, 36th Annual Meeting of the American Society of Clinical Oncology; May 19-23, 2000; New La, LA, Abstract 829; Calvert PM et al. Proceedings of the 11th National Cancer Institute-European Organization for Research and Treatment of Cancer/American Association for Cancer Research Symposium on New Drugs in Cancer Therapy; November 7- 10, 2000; Amsterdam, The Netherlands, Abstract 575.
  • Midostaurin is an inhibitor of multiple signalling proteins. By targeting specific receptor tyrosine kinases and components of several signal transduction pathways, midostaurin impacts several targets involved in cell growth (e.g., KIT, PDGFR, PKC), leukaemic cell proliferation (e.g., FLT3), and angiogenesis (e.g.; VEGFR2).
  • KIT KIT
  • PDGFR PDGFR
  • PKC leukaemic cell proliferation
  • angiogenesis e.g.; VEGFR2
  • midostaurin showed broad antiproliferative activity against various tumour cell lines, including those that were resistant to several other chemotherapeutic agents.
  • locus means a location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature.
  • small molecule means a composition that has a molecular weight of less than about 5 kDa and more preferably less than about 2 kDa.
  • Small molecules can be, e.g., nucleic acids, peptides, polypeptides, glycopeptides, peptidomimetics, carbohydrates, lipids, lipopolysaccharides, combinations of these, or other organic or inorganic molecules.
  • SNP nucleic acid means a nucleic acid sequence, which comprises a nucleotide that is variable within an otherwise identical nucleotide sequence between individuals or groups of individuals, thus, existing as alleles. Such SNP nucleic acids are preferably from about 15 to about 500 nucleotides in length.
  • the SNP nucleic acids may be part of a chromosome, or they may be an exact copy of a part of a diiM- ⁇ osome, e.g., by amplification of such a part of a chromosome through PCR or through cloning.
  • the SNP nucleic acids are referred to hereafter simply as "SNPs".
  • the SNP probes according to the invention are oligonucleotides that are complementary to a SNP nucleic acid. In a particular embodiment, the SNP is in the EGFR gene.
  • EXAMPLE 1 Studies to determine EGFR mutations in colorectal cancer and lung cancer are summarized in EXAMPLE 1. Bioinformatics analyses of the EGFR mutations of the invention are further detailed in EXAMPLE 2.
  • SNPs Due to their prevalence and widespread nature, SNPs have the potential to be important tools for locating genes that are involved in human disease conditions. See e.g., Wang et al, Science 280: 1077-1082 (1998)).
  • An association between SNP's and/or mutations and a particular phenotype does not necessarily indicate or require that the SNP or mutation is causative of the phenotype. Instead, an association with a SNP may merely be due to genome proximity between a SNP and those genetic factors actually responsible for a given phenotype, such that the SNP and said genetic factors are closely linked. That is, a SNP may be in linkage disequilibrium ("LD") with the "true” functional variant.
  • LD linkage disequilibrium
  • a SNP may serve as a marker that has value by virtue of its proximity to a mutation or other DNA alteration (e.g. gene duplication) that causes a particular phenotype.
  • multiple polymorphic and/or mutant sites may be investigated by simultaneously amplifying multiple regions of the nucleic acid using sets of allele-specific primers as described in WO 89/10414.
  • the invention provides methods and compositions for haplotyping and/or genotyping the genetic polymorphisms (and possibly mutations) in an individual.
  • the terms "genotype” and “haplotype” mean the genotype or haplotype containing the nucleotide pair or nucleotide, respectively, that is present at one or more of the novel polymorphic (or mutant) sites described herein and may optionally also include the nucleotide pair or nucleotide present at one or more additional polymorphic (or mutant) sites in the gene.
  • the additional polymorphic (and mutant) sites may be currently known polymorphic/mutant sites or sites that are subsequently discovered.
  • the nucleic acid mixture is isolated from a biological sample taken from the individual, such as a blood sample, tumour or tissue sample.
  • tissue samples include whole blood, tumour or as part of any tissue type, semen, saliva, tears, urine, fecal material, sweat, buccal smears, skin and hair.
  • the nucleic acid mixture may be comprised of genomic DNA, mRNA, or cDNA and, in the latter two cases, the biological sample must be obtained from an organ in which the gene may be expressed.
  • mRNA or cDNA preparations would not be used to detect polymorphisms or mutations located in introns or in 5' and 3' nontranscribed regions.
  • first and second copies of the gene are labelled with different first and second fluorescent dyes, respectively, and an allele-specific oligonucleotide labelled with yet a third different fluorescent dye is used to assay the polymorphic/mutant sites, then detecting a combination of the first and third dyes would identify the polymorphism or mutation in the first gene copy, while detecting a combination of the second and third dyes would identify the polymorphism or mutation in the second gene copy.
  • the identity of a nucleotide (or nucleotide pair) at a polymorphic and/or mutant site may be determined by amplifying a target region containing the polymorphic and/or mutant sites directly from one or both copies of the gene, or fragments thereof, and sequencing the amplified regions by conventional methods. It will be readily appreciated by the skilled artisan that only one nucleotide will be detected at a polymorphic or mutant site in individuals who are homozygous at that site, while two different nucleotides will be detected if the individual is heterozygous for that site.
  • the polymorphism or mutation may be identified directly, known as positive-type identification, or by inference, referred to as negative-type identification.
  • a site may be positively determined to be either guanine or eviusi ⁇ e for all individuals homozygous at that site, or both guanine and cytosine, if the individual is heterozygous at that site.
  • the site may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanine/guanine).
  • the present invention provides a method for determining the frequency of a genotype or haplotype in a population.
  • the method comprises determining the genotype or the haplotype for a gene present in each member of the population, wherein the genotype ⁇ r haplotype comprises the nucleotide pair or nucleotide detected at one or more of the polymorphic sites in the gene and mutations identified in the region, and calculating the . frequency at which the genotype or haplotype is found in the population.
  • frequency data for genotypes and/or haplotypes found in a reference population are used in a method for identifying an association between a trait and a genotype or a haplotype.
  • the trait may be any detectable phenotype, including but not limited to cancer, susceptibility to a disease or response to a treatment.
  • the method involves obtaining data on the frequency of the genotypes or haplotypes of interest in a reference population and comparing the data to the frequency of the genotypes or haplotypes in a population exhibiting the trait.
  • Frequency data for one or both of the reference and trait populations may be obtained by genotyping or haplotyping each individual in the populations using one of the methods described above.
  • the haplotypes for the trait population may be determined directly or, alternatively, by the predictive genotype to haplotype approach described above.
  • the trait is susceptibility to a disease, severity of a disease, the staging of a disease or response to a drug.
  • Such methods have applicability in developing diagnostic tests and therapeutic treatments for all pharmacogenetic applications where there is the potential for an association between a genotype and a treatment outcome, including efficacy measurements, PD measurements, PK measurements and side effect measurements.
  • the frequency data for the reference and/or trait populations are obtained by accessing previously determined frequency data, which may be in written or electronic form.
  • the frequency data may be present in a database that is . accessible by a computer. Once the frequency data are obtained, the frequencies of the genotypes or haplotypes of interest in the reference and trait populations are compared.
  • a statistically significant difference between the observed and expected haplotype frequencies could be due to one or more factors including significant inbreeding in the population group, strong selective pressure on the gene, sampling bias, and/or errors in the genotyping process.
  • the assigning step involves performing the following analysis.
  • genotype or haplotype data is obtained on the clinical responses exhibited by a population of individuals who received the treatment, hereinafter the "clinical population". This clinical data may be obtained by analyzing the results of a clinical trial that has already been previously conducted and/or by designing and carrying out one or more new clinical trials.
  • the individuals included in the clinical population be graded for the existence of the medical condition of interest. This grading of potential patients could employ a standard physical exam or one or more lab tests. Alternatively, grading of patients could use genotyping or haplotyping for situations where there is a strong correlation between haplotype pair and disease susceptibility or severity.
  • the therapeutic treatment of interest is administered to each individual in the trial population, and each individual's response to the treatment is measured using one or more predetermined criteria. It is contemplated that in many cases, the trial population will exhibit a range of responses, and that the investigator may choose more than one responder groups (e.g., low, medium, high) made up by the various responses. In addition, the gene for each individual in the trial population is genotyped and/or haplotyped, which may be done before or after administering the treatment.
  • Correlations may also be analyzed using analysis of variation (ANOVA) techniques to determine how much of the variation in the clinical data is explained by different subsets of * the polymorphic and mutant sites in the gene.
  • ANOVA is used to test hypotheses about whether a response variable is caused by or correlates with one or more traits or variables that can be measured (Fisher & vanBelle, supra, Ch. 10).
  • correlations between individual response and genotype or haplotype content are created. Correlations may be produced in several ways. In one method, individuals are grouped by their genotype or haplotype (or haplotype pair) (also referred to as a polymorphism/mutation group), and then the averages and standard deviations of clinical responses exhibited by the members of each polymorphism/mutation group are calculated.
  • the identification of an association between a clinical response and a genotype or haplotype (or haplotype pair) for the gene may be the basis for designing a diagnostic method to determine those Individuals who will or will not respond to the treatment, or alternatively, will respond at a lower level and thus may require more treatment, i.e., a greater dose of a drug or suffer an adverse reaction.
  • the diagnostic method may take one of several forms: for example, a direct DNA test (i.e., genotyping or haplotyping one or more of the polymorphic/mutant sites in the gene), a serological test, or a physical exam measurement. The only requirement is that there be a good correlation between the diagnostic test results and the underlying genotype or haplotype. In a preferred embodiment, this diagnostic method uses the predictive genotyping/haplotyping method described above.
  • polymorphism and mutation data may be stored on the computer's hard drive or may, for example, be stored on a CD-ROM or on one or more other storage devices accessible by the computer.
  • the data may be stored on one or more databases in communication with the computer via a network.
  • the invention provides SNP and mutation probes, which are useful in classifying subjects according to their types of genetic variation.
  • the SNP and mutation probes according to the invention are oligonucleotides, which discriminate between SNPs or mutations and the wild-type sequence in conventional allelic discrimination assays.
  • the oligonucleotides according to this aspect of the invention are complementary to one allele of the SNP/mutant nucleic acid, but not to any other allele of the SNP/Mutant nucleic acid. Oligonucleotides according to this embodiment of the invention can discriminate between SNPs and mutations in various ways.
  • an oligonucleotide of appropriate length will hybridize to one SNP or mutation, but not to any other.
  • the oligonucleotide may be labelled using a radiolabel or a fluorescent mole ⁇ ular tag.
  • an oligonucleotide of appropriate length can be used as a primer for PCR, wherein the 3' terminal nucleotide is complementary to one allele containing a SNP or mutation, but not to any other allele.
  • the presence or absence of amplification by PCR determines the haplotype of the SNP or the specific mutation.
  • Genomic and cDNA fragments of the invention comprise at least one novel polymorphic site or mutation identified herein, have a length of at least 10 nucleotides, and may range up to the full length of the gene.
  • a fragment according to the present invention is between 100 and 3000 nucleotides in length, and more preferably between 200 and 2000 nucleotides in length, and most preferably between 500 and 1000 nucleotides in length.
  • kits of the Invention provides nucleic acid and polypeptide detection kits useful for haplotyping and/or genotyping the genes in an individual. Such kits are useful for classifying individuals for the purpose of classifying individuals. Specifically, the invention encompasses kits for detecting the presence of a polypeptide or nucleic acid corresponding to a marker of the invention in a biological sample, e.g., any tissue or bodily fluid including, but not limited to, serum, plasma, lymph, cystic fluid, urine, stool, cerebrospinal fluid, ascites fluid or blood, and including biopsy samples of body tissue.
  • a biological sample e.g., any tissue or bodily fluid including, but not limited to, serum, plasma, lymph, cystic fluid, urine, stool, cerebrospinal fluid, ascites fluid or blood, and including biopsy samples of body tissue.
  • the kit can comprise a labelled compound or agent capable of detecting a polypeptide or an mRNA encoding a polypeptide corresponding to a marker of the invention in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample, e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide.
  • Kits can also include instructions for interpreting the results obtained using the kit.
  • the invention provides a kit comprising at least two genotyping oligonucleotides packaged in separate containers.
  • the kit may also contain other components such as hybridization buffer (where the oligonucleotides are to be used as a probe) packaged in a separate container.
  • the kit may contain, packaged in separate containers, a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase, such as in the case of PCR.
  • such kit may further comprise a DNA sample collecting means.
  • the genotyping primer composition may comprise at least two sets of allele specific primer pairs.
  • the two genotyping oligonucleotides are packaged in separate containers.
  • the kit can comprise, e.g., (1) a first antibody, e.g., attached to a solid support, which binds to a polypeptide corresponding to a marker or the invention; and, optionally (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label.
  • a first antibody e.g., attached to a solid support
  • a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label.
  • the kit can comprise, e.g., (1) an oligonucleotide, e.g., a detectably-labelled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention; or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention.
  • the kit can also comprise, e.g., a buffering agent, a preservative or a protein- stabilizing agent.
  • the kit can further comprise components necessary for detecting the detectable-label, e.g., an enzyme or a substrate.
  • the kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample.
  • Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single pdokage, alo ⁇ g with instructions for interpreting the results of the assays performed using the kit.
  • a typical prehybridization, hybridization, and wash protocol is as follows: (1) prehybridization: incubate nitrocellulose filters containing the denatured target DNA for 3-4 hours at 55 0 C in 5xDenhardt's solution, 6xSSC (2OxSSC consists of 175 g NaCl, 88.2 g sodium citrate in 800 ml H 2 O adjusted to pH.
  • Another aspect of the invention includes vectors containing one or more nucleic acid sequences encoding a mutant or polymorphic polypeptide.
  • the nucleic acid containing all or a portion of the nucleotide sequence encoding the polypeptide is inserted into an appropriate cloning vector, or an expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as detailed below.
  • an expression vector i.e., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence
  • expression vectors useful in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • Such viral vectors permit infection of a subject and expression in that subject of a compound. Becker et al, Meth. Cell Biol. 43: 161 89 (1994). . .
  • the recombinant expression vectors of the invention comprise a nucleic acid encoding a mutant or polymorphic polypeptide in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression that is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to. mean that the nucleotide sequence of interest is linked to the regulatory sequences in a manner that allows for expression of the nucleotide sequence (e.g. , in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods In Enzymology (Academic Press, San Diego, Calif., 1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells ⁇ e.g., tissue specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides, encoded by nucleic acids as described herein (e.g., mutant polypeptides and mutant-derived fusion polypeptides, etc.).
  • mutant and polymorphic Polypeptide-Expressing Host Cells Another aspect of the invention pertains to mutant and polymorphic polypeptide-expressing host cells, which contain a nucleic acid encoding one or more mutant/polymorphic polypeptides of the invention.
  • the desired isogene may be introduced into a host cell in a vector such that the isogene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location.
  • the isogene is introduced into a cell in such a way that it recombines with the endogenous gene present in the cell.
  • Such recombination requires the • occurrence of a double recombination event, thereby resulting in the desired gene polymorphism or mutation.
  • Vectors for the introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector or vector construct may be used in the invention. Methods such as electroporation, particle bombardment, calcium phosphate co-precipitation and viral transduction for introducing DNA into cells are known in the art; therefore, the choice of method may lie with the competence and preference of the skilled practitioner.
  • the recombinant expression vectors of the invention can be designed for expression of mutant polypeptides in prokaryotic or eukaryotic cells.
  • mutant/polymorphic polypeptides can be expressed in bacterial ⁇ elU such as Escherichia coli (E. coli), insect cells (using baculovirus expression vectors), fungal cells, e.g., yeast, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods In Enzymology (Academic Press, San Diego, Calif., 1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • the SMP2 promoter is useful in the expression of polypeptides in smooth muscle cells (Qian et ah, Endocrinology 140(4): 1826 (1999)).
  • Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide.
  • Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant polypeptide; (ii) to increase the solubility of the recombinant polypeptide; and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, Gene 67: 31 40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, NJ.) that fuse glutathione S transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.
  • GST glutathione S transferase
  • suitable inducible non fusion E include glutathione S transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.
  • coli expression .vectors include pTrc (Amrann et al, Gene 69:301 315 (1988)) and pET 1 Id (Studier et al, Gene Expression Technology: Methods In En ⁇ ymology (Academic Press, San Diego, Calif, 1990) pp. 60-89.).
  • One strategy to maximize recombinant polypeptide expression in E. coli is to express the polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide. See, e.g., Gottesman, Gene Expression Technology: Methods In Enzymology (Academic Press, San Diego, Calif, 1990) 119 128.
  • the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al. , Molecular Cloning: A Laboratory Manual, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type ⁇ e.g., tissue specific regulatory elements are used to express the nucleic acid).
  • tissue specific regulatory elements are known in the art.
  • suitable tissue specific promoters include the albumin promoter (liver specific; Pinkert, et al., Genes Dev. 1 : 268 277 (1987)), lymphoid specific promoters (Calame & Eaton, Adv. Immunol. 43: 235 275 (1988)), in particular promoters of T cell receptors (Winoto & Baltimore, EMBO J.
  • promoters are also encompassed, e.g., the murine hox promoters (Kessel & Gruss, Science 249: 374 379 (1990)) and the ⁇ -fetoprotein promoter (Campes & Tilghman, Genes Dev. 3: 537 546 (1989)).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to a mutant polypeptide mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • mutant polypeptide can be exp'ressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • CHO Chinese hamster ovary cells
  • COS cells Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art recognized teelmiquea ?> >, ⁇ introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co precipitation, DEAE dextran mediated transfection, lipofection, or electroporation.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989), and other laboratory manuals.
  • a host cell that includes a compound of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) recombinant mutant/polymorphic polypeptide.
  • the method comprises culturing the host cell of invention (into, which a recombinant expression vector encoding mutant/polymorphic polypeptide has been introduced) in a suitable medium such that mutant polypeptide is produced.
  • the method further comprises the step of isolating mutant/polymorphic polypeptide from the medium or the host cell.
  • Purification of recombinant polypeptides is well known in the art and includes ion exchange purification techniques, or affinity purification techniques, for example with an antibody to the compound. Methods of creating antibodies to the compounds of the present invention are discussed below.
  • Transgenic Animals Recombinant organisms, i.e., transgenic animals, expressing a variant gene of the invention are prepared using standard procedures known in the art. Transgenic animals carrying the constructs of the invention can be made by several methods known to those having skill in the art. See, e.g., U.S. Pat. No. 5,610,053 and "The Introduction of Foreign Genes into Mice" and the cited references therein, in: Recombinant DNA, Eds. Watson JD, Gilman M, Witkowski J & Zolbr M (W.H, Freeman and Company, New York) pp. 254-272.
  • RNA isolation technique that does not select against the isolation of niRNA can be utilized for the purification of RNA from cells. See, e.g., Ausubel et al, Ed., Curt: Prot. MoI. Biol.
  • the isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, PCR analyses a ⁇ id probe arrays.
  • One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected.
  • the nucleic acid probe can be, e.g., a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to an mRNA or genomic DNA encoding a marker of the present invention.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed.
  • the probes are immobilized on a solid surface and the mRNA is contacted with the probes, for example, in an Affymetrix gene chip array (Affymetrix, Calif. USA).
  • amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice- versa) and contain a short region in between. In general, amplification primers are from about 10-30 nucleotides in length and flank a region from about 50-200 nucleotides in length.
  • RT-PCR Real-time quantitative PCR
  • various host animals may be immunized by injection with the polypeptide, or a portion thereof.
  • host animals may include, but are not limited to, rabbits, mice and rats.
  • Various adjuvants may be used to increase the immunological response, depending on the host species including, but not limited to, Freund's (complete and incomplete), mineral gels, such as aluminium hydroxide; surface active' substances, such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol; and potentially useful human adjuvants, such as bacille Camette-Guerin (BCG) and Corynebacterium parvum.
  • BCG Bacille Camette-Guerin
  • chimeric antibodies are a molecule in which different portions are derived from different animal species, such as those having a variable or hypervariable region derived form a murine mAb and a human immunoglobulin constant region.
  • Antibodies or antibody fragments can be used in methods, such as Western blots or immunofluorescence techniques, to detect the expressed proteins. In such uses, it is generally preferable to immobilize either the antibody or proteins on a solid support.
  • Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody.
  • Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros and magnetite.
  • a useful method for ease of detection, is the sandwich ELISA, of which a number of variations exist, all of which are intended to be used in the methods and assays of the present invention.
  • sandwich assay is intended to encompass all variations on the basic two-site technique. Immunofluorescence and EIA techniques are both very well- established in the art. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules may also be employed. It will be readily apparent to the skilled artisan how t ⁇ vmy tUc procedure to suit the required use.
  • Whole genome monitoring of protein i.e., the "proteome” can be carried out by constructing a microarray in which binding sites comprise immobilized, preferably monoclonal, antibodies specific to a plurality of protein species encoded by the cell genome.
  • binding sites comprise immobilized, preferably monoclonal, antibodies specific to a plurality of protein species encoded by the cell genome.
  • antibodies are present for a substantial fraction of the encoded proteins, or at least for those proteins relevant to testing or confirming a biological network model of interest.
  • methods for making monoclonal antibodies are well-known. See, e.g., Harlow & Lane, Antibodies: A Laboratory Manual” (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988)).
  • monoclonal antibodies are raised against synthetic peptide fragments designed based on genomic sequence of the cell. With such an antibody array, proteins from the cell are contacted to the array and their binding is measured with assays known in the art.
  • MS-based analysis methodology is useful for analysis of isolated target polypeptide as well as analysis of target polypeptide in a biological sample.
  • MS formats for use in analyzing a target polypeptide include ionization (I) techniques, such as, but not limited to, matrix assisted laser desorption (MALDI), continuous or pulsed electrospray ionization (ESI) and related methods, such as ionspray or thermospray, and massive cluster impact (MCI).
  • I ionization
  • MALDI matrix assisted laser desorption
  • ESI electrospray ionization
  • MCI massive cluster impact
  • Such ion sources can be matched with detection formats, including linear or non-linear reflectron time of flight (TOF), single or multiple quadrupole, single or multiple magnetic sector Fourier transform ion cyclotron resonance (FTICR), ion trap and combinations thereof such as ion-trap/TOF.
  • TOF linear or non-linear reflectron time of flight
  • FTICR magnetic sector Fourier transform ion cyclotron resonance
  • ion trap and combinations thereof such as ion-trap/TOF.
  • numerous matrix/wavelength combinations ⁇ e.g., matrix assisted laser desorption (MALDI)) or solvent combinations ⁇ e.g., ESI) can be employed.
  • MALDI matrix assisted laser desorption
  • ESI solvent combinations
  • a solvent is selected that minimizes the risk that the target polypeptide will be decomposed by the energy introduced for the vaporization process.
  • a reduced risk of target polypeptide decomposition can be achieved, e.g., by embedding the sample in a matrix.
  • a suitable matrix can be an organic compound such as a sugar, e.g., a pentose or hexose, or a polysaccharide such as cellulose. Such compounds are decomposed thermolytically into CO 2 and H 2 O such that no residues are formed that can lead to chemical reactions.
  • the matrix also can be an inorganic compound, such as nitrate of ammonium, which is decomposed essentially without leaving any residue.
  • an inorganic compound such as nitrate of ammonium, which is decomposed essentially without leaving any residue.
  • Use of these and other solvents is known to those of skill in the art. See, e.g., U.S. Pat. No. 5,062,935.
  • Electrospray MS has been described by Fenn et al, J. Phys. Chem. 88: 4451-4459 (1984); and PCT Application No. WO 90/14148; and current applications are summarized in review articles. See Smith et al., Anal Chem. 62: 882-89 (1990); and Ardrey, Spectroscopy 4: 10-18 (1992).
  • Matrix Assisted Laser Desorption The level of the target protein in a biological sample, e.g., body fluid or tissue sample, may be measured by means of mass spectrometric (MS) methods including, but not limited to, those techniques known in the art as matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI- TOF-MS) and surfaces enhanced for laser desorption/ionization, time-of-flight mass spectrometry (SELDI-TOF-MS) as further detailed below.
  • MS mass spectrometric
  • Methods for performing MALDI arc v/ellrknown to those of skill in the art. See, e.g., Juhasz et al, Analysis, Anal. Chem.
  • MALDI-TOF-MS has been described by Hillenkamp et al, Biological Mass Spectrometry, Burlingame & McCloskey, eds. (Elsevier Science PubL, Amsterdam, 1990) pp. 49-60. [161] A variety of techniques for marker detection using mass spectroscopy can be used. See Bordeaux Mass Spectrometry Conference Report, Hillenkamp, Ed., pp.
  • MS techniques allow the successful volatilization of high molecular weight biopolymers, without fragmentation, and have enabled a wide variety of biological macromolecules to be analyzed by mass spectrometry.
  • SMDI Surfaces Enhanced for Laser Desorption/Ionization
  • Other techniques are used which employ new MS probe element compositions with surfaces that allow the probe element to actively participate in the capture and docking of specific analytes, described as Affinity Mass Spectrometry (AMS). See SELDI patents U.S. Pat. Nos. 5,719,060; 5,894,063; 6,020,208; 6,027,942; 6,124,137; and U.S. Patent application No. U.S. 2003/0003465.
  • SEAC probe elements have been designed with Surfaces Enhanced for Affinity Capture (SEAC). See Hutchens & Yip, Rapid Commiin. Mass Spectrom. 7: 576-580 (1993).
  • SEAC probe elements have been used successfully to retrieve and tether different classes of biopolymers, particularly proteins, by exploiting what is known about protein surface structures and biospecific molecular recognition.
  • the immobilized affinity capture devices on the MS probe element surface, i.e., SEAC determines the location and affinity (specificity) of the analyte for the probe surface, therefore the subsequent analytical MS process is efficient.
  • SELDI Surfaces Enhanced for Neat Desorption
  • the probe element surfaces i.e., sample presenting means
  • SEAC Energy Absorbing Molecules
  • the probe element surfaces, i.e., sample presenting means w- designed to contain chemically defined and/or biologically defined affinity capture devices to facilitate either the specific or non-specific attachment or adsorption (so-called docking or tethering) of analytes to the probe surface, by a variety of mechanisms (mostly non-covalent).
  • SEPAR Photolabile Attachment and Release
  • the probe element surfaces i.e., sample presenting means
  • the analyte e.g., protein
  • the chemical specificities determining the type and number of the photolabile molecule attachment points between the SEPAR sample presenting means (i.e., probe element surface) and the analyte may involve any one or more of a number of different residues or chemical structures in the analyte (e.g., His, Lys, Arg, Tyr, Phe and Cys residues in the case of proteins and peptides).
  • a polypeptide of interest also can be modified to facilitate conjugation to a solid support.
  • a chemical or physical moiety can be incorporate into the polypeptide at an appropriate position.
  • a polypeptide of interest can be modified by adding an appropriate functional group to the carboxyl terminus or amino terminus of the polypeptide, or to an amino acid in the peptide, (e.g., to a reactive side chain, or to the peptide backbone.
  • a naturally-occurring amino acid normally present in the polypeptide also can contain a functional group suitable for conjugating the polypeptide to the solid support.
  • a cysteine residue present in the polypeptide can be used to conjugate the polypeptide to a support containing a sulfhydryl group through a disulfide linkage, e.g., a support having cysteine residues attached thereto.
  • thiol-reactive functionalities include, e.g., haloacetyls, such as iodoacetyl; diazoketones; epoxy ketones, alpha- and beta-unsaturated carbonyls, such as alpha-enones and beta-enones; and other reactive Michael acceptors, such as maleimide; acid halides; benzyl halides; and the like. See Greene & Wuts, Protective Groups in Organic Synthesis, 2 nd Edition (John Wiley & Sons, 1991).
  • the thiol groups can be blocked with a photocleavable protecting group, which then can be selectively cleaved, e.g., by photolithography, to provide portions of a surface activated for immobilization of a polypeptide of interest.
  • Photocleavable protecting groups are known in the art (see, e.g., published International PCT Application No. WO 92/10092; and McCray et ah, Ann. Rev. Biophys. Biophys. Chem. 18: 239-270 (1989)) and can be selectively de-blocked by irradiation of selected areas of the surface using, e.g., a photolithography mask.
  • Linkers A polypeptide of interest can be attached directly to a support via a linker. Any linkers known to those of skill in the art to be suitable for linking peptides or amino acids to supports, either directly or via a spacer, may be used. For example, the polypeptide can be conjugated to a support, such as a bead, through means of a variable spacer.
  • Linkers include, Rink amide linkers (see, e.g., Rink, Tetrahedron Lett. 28: 3787 (1976)); trityl chloride linkers (see, e.g., Leznoff, Ace Cheifi. Res.
  • linkers see, e.g., Bodansky et al, Peptide Synthesis, 2 nd Edition (Academic Press, New York, 1976)
  • trityl linkers are- known. See, e.g., U.S. Pat. Nos. 5,410,068 and 5,612,474.
  • Amino trityl linkers are also known. See, e.g., U.S. Pat. No. 5,198,531.
  • Other linkers include those that can be incorporated into fusion proteins and expressed in a host cell. Such linkers may be selected amino acids, enzyme substrates or any suitable peptide.
  • the linker may be made, e.g., by appropriate selection of primers when isolating the nucleic acid. Alternatively, they may be added by post-translational modification of the protein of interest.
  • Linkers that are suitable for chemically linking peptides to supports include disulfide bonds, thioether bonds, hindered disulfide bonds and covalent bonds between free reactive groups, such as amine and thiol groups.
  • linker that is cleavable under MS conditions, such as a silyl linkage or photocleavable linkage, can be combined with a linker, such as an avidin biotin linkage, that is not cleaved under these conditions, but may be cleaved under other conditions.
  • Acid-labile linkers are particularly useful chemically cleavable linkers for mass spectrometry, especially for MALDI-TOF, because the acid labile bond is cleaved during conditioning of the target polypeptide upon addition of a 3 -HPA matrix solution.
  • the acid labile bond can be introduced as a separate linker group, e.g., an acid labile trityl group, or can be incorporated in a synthetic linker by introducing one or more silyl bridges using diisopropylysilyl, thereby forming a diisopropylysilyl linkage between the polypeptide and the solid support.
  • the diisopropylysilyl linkage can be cleaved using mildly acidic conditions, such as 1.5% trifluoroacetic acid (TFA) or 3 -HP A/1 % TFA MALDI-TOF matrix solution.
  • TFA trifluoroacetic acid
  • Methods for the preparation of diisopropylysilyl linkages and analogues thereof are well-known in the art. See, e.g., Saha r a/., J Org. Chem. 58: 7827-7831 (1993).
  • the pin tool has a functional group attached to each pin tip, or a solid support, e.g., functionalized beads or paramagnetic beads are attached to each pin
  • the polypeptides in a well can be captured (1 pmol capacity).
  • the pins can be kept in motion (vertical, 1-2 mm travel) to increase the efficiency of the capture.
  • a reaction such as an in vitro transcription is being performed in the wells
  • movement of the pins can increase efficiency of the reaction.
  • Further immobilization can result by applying an electrical field to the pin tool.
  • the polypeptides are attracted to the anode or the cathode, depending on their net charge.
  • the pin tool (with or without voltage) can be modified to have conjugated thereto a reagent specific for the polypeptide of interest, such that only the polypeptides of interest are bound by the pins.
  • the pins can have nickel ions attached, such that only polypeptides containing a polyhistidine sequence are bound.
  • the pins can have antibodies specific for a target polypeptide attached thereto, or to beads that, in turn, are attached to the pins, such that only the target polypeptides, which contain the epitope recognized by the antibody, are bound by the pins.
  • Pin tools can be useful for immobilizing polypeptides of interest in spatially addressable manner on an array. Such spatially addressable or pre-addressable arrays are useful in a variety of processes, including, for example, quality control and amino acid sequencing diagnostics.
  • the pin tools described in the U.S. Application Nos. 08/786,988 and 08/787,639 and International PCT Application No. WO 98/20166 are serial and parallel .
  • dispensing tools that can be employed to generate multi-element arrays of polypeptides on a surface of the solid support.
  • the array surface can be flat, with beads or. geometrically altered to include wells, which can contain beads.
  • MS geometries can be adapted for accommodating a pin tool apparatus.
  • aspects of the biological activity state, or mixed aspects can be measured in order to obtain drug and pathway responses.
  • the activities of proteins relevant to the characterization of cell function can be measured; and embodiments of this invention can be based on such measurements.
  • Activity measurements can be performed by any functional, biochemical or physical means appropriate to the particular activity being characterized. Where the activity involves a chemical transformation, the cellular protein can be contacted with natural substrates, and the rate of transformation measured. Where the activity involves association in multimeric units, e.g., association of an activated DNA binding complex with DNA, the .. amount of associated protein or secondary consequences of the association, such as amounts of mRNA transcribed, can be measured.
  • DHPLC analysis (Lilleberg SL, Curr. Opin. DrugDiscov. Devel. 6(2): 237-52 (March 2003) was conducted on test samples derived from tissues of nine human cancers and non- small-cell lung cancer (NSCLC) to identify EGFR mutations associated with these diseases.
  • NSCLC non- small-cell lung cancer
  • the EGFR genomic reference sequence was NT 079592.
  • Four isoforms of human EGFR are reported in the LocusLink database.
  • the mRNA and peptide reference sequences are NM_005228 and NP_005219 respectively.
  • Analysis of EGFR mutations in human lung cancer samples revealed the EGFR mutations, included, e.g., G719S; L858R; E746_A750del; L747_E749del; and A750P.
  • These EGFR mutations identified in NSCLC were different from the EGFR mutations identified in this invention.
  • Bioinformatics analysis of selected EGFR mutations identified in this invention is detailed below in EXAMPLE 2.
  • BIOINFORMATICS ANALYSIS OF THE EGFR MUTATIONS OF THE INVENTION [179] The EGFR missense mutations identified in human cancers (see TABLE 3 and TABLE 5) were analyzed using computational analysis tools to determine the effects of these mutations on EGFR function.
  • Allele 1 Allele 2: Ref SNP frequency frequency
  • T629T Synonymous T: 0.685 A: 0.315 rs2072454
  • the positions of the EGFR missense mutations identified in TABLE 3 which appear in the Pfam model of protein kinase domain are highlighted in bold underlined text. As shown in TABLE 5, twenty-eight (28) mutations identified in TABLE 3 are located in the protein kinase domain region and six (6) others are located in the receptor L domain. As shown in TABLE 7, alignment of the human wild- type EGFR sequence with the Pfam model of protein kinase domain indicates G729, K745, G779 and R932 are highly conserved, while G735, 1740 and L760 are moderately conserved. Mutations that change amino 'acid residues at conserved positions may potentially alter the protein function.
  • FIG. 1 is a schematic drawing of the three-dimensional structure of wild-type EGFR protein kinase domain rendered in Cn3d, a structure visualizing software provided by NCBL Locations of EGFR missense mutation are highlighted by arrows.
  • Position L760 is located in the middle of alpha- helix protein secondary structure. Proline can potentially break the helix structure. Change of leucine to proline at 760 (L760P) can have dramatic effect on the local secondary structure (See also infra Example 2, Section E).
  • EGFR missense mutations on known and potential protein phosphorylation sites of EGFR was analyzed.
  • Known EGFR phosphorylation sites include Thr678, Thr693 5 Ser695, Tyr869, Serl070, SerlO71, TyrlO92, Tyrl llO, Tyrl l72, and Tyrl 197 which are summarized below in TABLE 9.
  • the known EGFR phosphorylation sites are highlighted in bold text.
  • EGFR phosphorylation sites were identified by computational analysis using the NetPhos computational analysis tool. NetPhos produces neural network predictions for serine, threonine and tyrosine phosphorylation sites in eukaryotic proteins. Blom et al., J. MoI. Biol. 294(5): 1351-1362 (1999). Potential EGFR phosphorylation sites predicted by NetPhos are summarized below in TABLE 9. The known EGFR phosphorylation sites are highlighted in bold text.
  • Y285 is close to E282, T693, also a known threonine phosphorylation site, is mutated in NSCLC, T725 is mutated in ovarian cancer, T751 is mutated in prostate cancer, S752 is next to T751 and close to other mutation sites K754 and N756, Y764 is close to L760, T892 is adjacent to Y891.
  • mutations at the positions E282, T693, T725, T751, K754, N756, L760, and Y891 may influence the phosphorylation patterns of nearby sites.
  • Clustal W is a general purpose multiple sequence alignment program for DNA or proteins. It produces biologically meaningful multiple sequence alignments of divergent sequences. It calculates the best match for the selected sequences, and lines them up so that the identities, similarities and differences can be seen. Evolutionary relationships can be seen via viewing Cladograms or Phylograms. . [191] For every position with a mutation reported, the mutated residues were inspected for their occurrence in organisms other than human. It is hypothesized that if the mutated residue is present in the wild-type sequence of another species in the corresponding position, the amino acid change may not have any adverse effect on the protein function. The results of ClustalW analysis are summarized below in TABLE 12. In position 756 of human EGFR, a mutation of asparagine to serine was found present in fruit fly, a non-human species.
  • H618 Y "Q" is present in chimp and zebrafish; "F” is present in fruit fly
  • T725P "R” is present in fruit fly
  • I732T "V” is present in zebrafish and fruit fly
  • R748G “L” is present in fruit fly
  • N756S "S" is present in fruit fly
  • L760P "M” is present in zebrafish
  • N771D "H” is present in zebrafish and fruit fly
  • V774M "L” is present in fruit fly
  • G779S "A” is present in fruit fly
  • T725P This mutation eliminates a strand, changes the secondary structure of 3 amino acids
  • V738G This mutation changes the secondary structure of 1 amino acid
  • R748I This mutation extends a helix by 1 amino acid
  • T751A This mutation extends the upstream helix by 1 amino acid
  • N756S This mutation extends the downstream helix by 1 amino acid
  • mutations at positions E282, T693, T725, T751, K754, N756, L760, and Y891 may alter the phosphorylation patterns of the mutated and nearby sites.
  • T751 is predicted as a potential threonine phosphorylation site that is next a potential serine phosphorylation site at 752.
  • Amino acid alterations at positions 751 (T751I & T751A), 754 (K754R & K754E) and 756 (N756S) can alter the phosphorylation pattern and consequently the biological activity of the EGFR polypeptide. Mutations G729R, K745R, G779S and R932G are identified at highly conserved positions.
  • L760P appears to have most impact on the protein structure.
  • L760 is located in the middle of a alpha-helix of about ten amino acids. Mutation of leucine to proline at 760 can break the alpha-helix as indicated by the secondary structure analysis.
  • Other mutations at positions located within the protein kinase domain e.g., I732T, G735R, K754R, T751I and N756S etc., can also impact EGFR function by altering its structure.
  • mutations at positions E282, T693, T725, T751, K754, N756, L760, and Y891 may alter the phosphorylation patterns of the mutated and nearby sites.
  • T751 is predicted as a potential threonine phosphorylation site that is next a potential serine phosphorylation site at 752.
  • Amino acid alterations at positions 751 (T751I & T751A), 754 (K754R & K754E) and 756 (N756S) can alter the phosphorylation pattern and consequently the biological activity of the EGFR polypeptide. Mutations G729R, K745R, G779S and R932G are identified at highly conserved positions.
  • Q217R, G725R, M971R, N771D, H618Y, R748G, R748I, R932G, D587N, E709V, E758G, K754E and K757E changes the number of electric charges of EGFR.
  • An agent that modulates EGFR biological activity is administered to a patient with cancer, e.g., glioblastoma, breast cancer, cholangioma, non- small-cell lung cancer (NSCLC), melanoma, ovarian cancer, prostate cancer, colon cancer and myeloma, when the patient has a SNP/mutation pattern that correlates with the disease.
  • cancer e.g., glioblastoma, breast cancer, cholangioma, non- small-cell lung cancer (NSCLC), melanoma, ovarian cancer, prostate cancer, colon cancer and myeloma
  • SNPs and mutations are selected from the group consisting the EGFR mutations and polymorphisms summarized in TABLE 1.
  • the EGFR antagonist is AEE788, which inhibits multiple receptor tyrosine kinases including EGFR, HER2, and VEGFR, to stimulate tumour cell growth and angiogenesis.
  • AEE788 showed high target specificity and demonstrated antiproliferative effects against tumour cell lines and in animal models of cancer.
  • AEE788 also exhibited direct antiangiogenic activity.
  • AEE788 is currently in phase I clinical development.
  • the EGFR antagonist is gefitinib (Iressa®), which has been approved by the Food and Drug Administration (FDA) as a single agent for the treatment of non-small cell lung cancer (NSCLC) that has progressed after, or failed to respond to two other types of chemotherapy (drugs used to kill cancer cells).
  • Iressa® belongs to a group of anticancer drugs called epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR- TKI). Iressa® is given by mouth as a single tablet of 250 mg with or without food.
  • the EGFR antagonist is erlotinib (Tarceva®; OSI-774).
  • Erlotinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor used in molecular targeted therapy. Erlotinib is used to treat non-small cell lung cancer. It is also being studied in many other cancers including breast cancer. Erlotinib is a pill taken by mouth each day as directed with a large glass of water, at least one hour before or two hours after a meal.
  • EGFR-targeting agents include PKI166 (Fabbro D et al, Pharmacol Ther. 93(2- 3):79-98 (February-March 2002); Traxler P et al., Med. Res. Rev. 21(6):499-512 (November 2001)), C-225, ZD1839.

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Abstract

L'invention concerne généralement l'épreuve analytique de prélèvements de tissus in vitro et, en particulier, des aspects de polymorphismes et mutations génétiques du récepteur du facteur de croissance épidermique('epidermal growth factor receptor' ou EGFR). L'invention porte sur de nouvelles mutations du EGFR et de nouveaux polymorphismes à nucléotide unique ('single nucleotide polymorphisms' ou SNP), qui sont utilisés dans le diagnostic et le traitement de sujets qui en ont besoin. En conséquence, l'invention se rapporte, dans ses divers aspects, à des polynucléotides codant les mutations de l'EGFR de l'invention, à des vecteurs d'expression codant les polypeptides à EGFR mutant et à des organismes qui expriment les polynucléotides à EGFR mutant et polymorphes et/ou les polypeptides à EGFR mutant/polymorphe de l'invention. Les divers aspects de l'invention concernent en outre des procédés diagnostiques/théranostiques et des trousses qui font appel aux mutations et polymorphismes de l'EGFR de l'invention pour identifier les individus qui sont prédisposés à la maladie ou pour classer les individus eu égard à la réponse pharmacodynamique, aux effets secondaires ou à la dose optimale de médicament.
PCT/US2006/012878 2005-04-11 2006-04-07 Mutations et polymorphismes du recepteur du facteur de croissance epidermique WO2006110478A2 (fr)

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US8017321B2 (en) 2004-01-23 2011-09-13 The Regents Of The University Of Colorado, A Body Corporate Gefitinib sensitivity-related gene expression and products and methods related thereto
US9434994B2 (en) 2004-05-27 2016-09-06 The Regents Of The University Of Colorado, A Body Corporate Methods for prediction of clinical outcome to epidermal growth factor receptor inhibitors by non-small cell lung cancer patients
WO2008088885A2 (fr) * 2007-01-18 2008-07-24 Creighton University Mutations de récepteur de facteur de croissance épidermique et procédés d'utilisation
WO2008088885A3 (fr) * 2007-01-18 2008-10-02 Univ Creighton Mutations de récepteur de facteur de croissance épidermique et procédés d'utilisation
US7887805B2 (en) 2007-03-01 2011-02-15 Symphogen A/S Recombinant anti-epidermal growth factor receptor antibody compositions
US8414896B2 (en) 2007-03-01 2013-04-09 Symphogen A/S Recombinant anti-epidermal growth factor receptor antibody compositions
US9340601B2 (en) 2007-03-01 2016-05-17 The Board Of Trustees Of The Leland Stanford Junior University Splice variants of the EGF receptor
US8663640B2 (en) 2008-08-29 2014-03-04 Symphogen A/S Methods using recombinant anti-epidermal growth factor receptor antibody compositions

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