WO2003025143A2 - Maxp1 - Google Patents
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- WO2003025143A2 WO2003025143A2 PCT/US2002/029643 US0229643W WO03025143A2 WO 2003025143 A2 WO2003025143 A2 WO 2003025143A2 US 0229643 W US0229643 W US 0229643W WO 03025143 A2 WO03025143 A2 WO 03025143A2
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- maxpl
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- nucleic acid
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- C07—ORGANIC CHEMISTRY
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- 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
Definitions
- This invention pertains to human Maxpl nucleic acids, vectors, host cells, polypeptides, compositions, monoclonal antibodies and cell lines therefor, and the use of human Maxpl in the diagnosis, prognosis and treatment of cancer, particularly lung cancer, colon cancer, and renal cancer.
- One popular way of detecting cancer early is to analyze the genetic makeup of an individual to detect the presence of or to measure expression levels of a marker gene(s) related to the cancer.
- a marker gene(s) related to the cancer For example, there are various diagnostic methods that analyze a certain gene or a pattern of genes to detect cancers of the breast, tongue, mouth, colon, rectum, cervix, prostate, testis, and skin.
- Ras proteins are guanine nucleotide-binding proteins that function by alternating between inactive GDP -bound and active GTP-bound forms. Ras activation is mediated by guanine nucleotide exchange factors that stimulate the release of bound GDP and its exchange for GTP. Activity of the Ras-GTP complex is then terminated by GTP hydrolysis, which is stimulated by the interaction of Ras-GTP with GTPase-activating proteins.
- Activated Ras is responsible for mediating multiple biological effects, including regulating the flow of mitogenic signals from cell-surface receptors to the internal cell signaling machinery. This process is achieved by various Ras effectors, which are stimulated downstream of Ras activation, either by binding directly to Ras or by associating with a molecule that has been activated by Ras. Thus, Ras stands at the forefront of a number of signaling pathways that are necessary for proper cell function. [0007] Recently, activated Ras has been determined to be involved with a variety of biological phenotypes associated with abnormal cell growth.
- activated Ras has been associated with a loss of contact inhibition (see, Huber et al., Oncogene, 3:245-256 (1988)), resistance to differentiation (see, e.g., Olson et al., Mol. Cell Biol, 7:2104-2111 (1987)), disruption of cytoskeletal architecture (Hall, Annu. Rev. Cell Biol, 10:31-54 (1994)), reduced requirement for growth factors (Andrejauskas et al., EMBO J., 8:2575- 2581 (1989)), invasiveness (see, e.g., Gelmann et al., Int. J.
- Ras also has been associated with cell growth and inhibition. Indeed, activated Ras may also induce senescence (Serrano et al., Cell, 88:593-602 (1997)), differentiation (Bar-Sagi et al., Cell, 42:841-848 (1985)), and apoptosis (see, e.g., Chen et al., Oncogene, 11 :1487-1498 (1995)). Thus, Ras can either drive processes that are normally associated with the acquisition of a transformed phenotype, or processes that promote growth arrest and death.
- the invention provides an isolated or purified nucleic acid molecule consisting essentially of (i) a nucleotide sequence encoding human Maxpl or a fragment thereof comprising at least 52 contiguous nucleotides, (ii) a nucleotide sequence encoding a variant human Maxpl, or (iii) a nucleotide sequence that is complementary to (i) or (ii), and related vectors, compositions and host cells.
- the invention also provides an isolated or purified polypeptide molecule consisting essentially of (i) an amino acid sequence encoding human Maxpl or a fragment thereof comprising at least 70 contiguous amino acids or (ii) an amino acid sequence encoding a variant human Maxpl or a fragment thereof comprising at least 70 contiguous amino acids, and related compositions, monoclonal antibodies (mAb), and mAb producing cell lines.
- isolated or purified polypeptide molecule consisting essentially of (i) an amino acid sequence encoding human Maxpl or a fragment thereof comprising at least 70 contiguous amino acids or (ii) an amino acid sequence encoding a variant human Maxpl or a fragment thereof comprising at least 70 contiguous amino acids, and related compositions, monoclonal antibodies (mAb), and mAb producing cell lines.
- the method comprises detecting a mutation in nucleic acid molecule comprising a nucleotide sequence encoding Maxpl, a decreased level of polypeptide molecule comprising an amino acid sequence encoding wild-type Maxpl, or a mutation in a polypeptide molecule comprising an amino acid sequence encoding Maxpl in a test sample obtained from the mammal.
- the detection of a mutation in the nucleic acid or polypeptide molecule encoding Maxpl, or the decreased level of wild- type Maxpl in the test sample is indicative of the cancer or a predisposition to the cancer in the mammal.
- the invention further provides a method of prognosticating a cancer in a mammal and a method of assessing the effectiveness of treatment of a cancer in a mammal.
- Maxpl is a marker for the cancer.
- These methods comprise measuring the level of Maxpl in a test sample obtained from the mammal. The level of Maxpl in the test sample is indicative of the prognosis or the effectiveness of treatment of the cancer in the mammal.
- the cancer is due to at least one mutation in a nucleic acid molecule comprising a nucleotide sequence encoding Maxpl, a decreased level of a polypeptide molecule comprising an amino acid sequence encoding wild-type Maxpl, or at least one mutation in a polypeptide molecule comprising an amino acid sequence encoding Maxpl.
- the method comprises administering to the mammal a composition comprising a carrier and (i) a nucleic acid molecule comprising and expressing a nucleotide sequence encoding wild-type Maxpl or a fragment thereof, (ii) a nucleic acid molecule comprising and expressing a nucleotide sequence encoding a variant Maxpl or a fragment thereof, (iii) a polypeptide molecule comprising an amino acid sequence encoding wild-type Maxpl or a fragment thereof, or (iv) a polypeptide molecule comprising an amino acid sequence encoding a variant Maxpl or a fragment thereof, wherein the composition is administered to the mammal in an amount sufficient to treat prophylactically or therapeutically the mammal for the cancer.
- Fig. 1 represents the nucleotide sequence (SEQ ID NO: 1) of human Maxpl cDNA.
- Fig. 2 represents the amino acid sequence (SEQ ID NO: 2) of the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1.
- the present invention provides an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding human Maxpl or a fragment thereof comprising at least 52 contiguous nucleotides.
- the isolated or purified nucleic acid molecule (i) encodes the amino acid sequence of SEQ ID NO:2 or a fragment thereof comprising at least 70 contiguous amino acids, (ii) consists essentially of the nucleotide sequence of SEQ ED NO:l or a fragment thereof comprising at least 52 contiguous nucleotides, (iii) hybridizes under highly stringent conditions to an isolated of purified nucleic acid molecule consisting essentially of the nucleotide sequence that is complementary to SEQ ID NO:l or a fragment thereof, or (iv) shares 85%> or more identity with SEQ ID NO: 1.
- nucleic acid molecule of the invention consists essentially of a nucleotide sequence encoding human Maxpl or a fragment thereof comprising at least 52 contiguous nucleotides, larger fragments of human Maxpl are also contemplated.
- nucleic acid molecule of the invention it is suitable for the isolated or purified nucleic acid molecule of the invention to consist essentially of a nucleotide sequence encoding human Maxpl or a fragment thereof comprising at least 75 contiguous nucleotides, at least 100 contiguous nucleotides, at least 125 contiguous nucleotides, at least 150 contiguous nucleotides, at least 175 contiguous nucleotides, or even at least 200 contiguous nucleotides.
- fragments of human Maxpl are also contemplated, such as fragments comprising at least 300 contiguous nucleotides, at least 400 contiguous nucleotides, at least 500 contiguous nucleotides, or even at least 600 contiguous nucleotides.
- any size fragment is contemplated as long as the fragment comprises contiguous nucleotides spanning 4.4% or more, 10% or more, or even 20% or more of the nucleic acid molecule consisting essentially of the nucleic acid molecule consisting essentially of a nucleotide sequence encoding human Maxpl.
- the present invention also provides an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding human Maxpl or a fragment thereof comprising at least 70 contiguous amino acids, either one of which is optionally glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated or converted into an acid addition salt and or optionally dimerized or polymerized.
- the isolated or purified polypeptide molecule (i) is encoded by the nucleotide sequence of SEQ ID NO:l or a fragment thereof comprising at least 210 contiguous nucleotides, (ii) consists essentially of the amino acid sequence of SEQ ID NO:2 or a fragment thereof comprising at least 70 contiguous amino acids, or (iii) shares 84% or more identity with SEQ ID NO:2.
- the isolated or purified polypeptide molecule of the present invention consists essentially of an amino acid sequence encoding human Maxpl or a fragment thereof comprising at least 70 contiguous amino acids, larger fragments of human Maxpl are also contemplated.
- the isolated or purified polypeptide molecule of the invention is suitable for consist essentially of an amino acid sequence encoding human Maxpl or a fragment thereof comprising at least 75 contiguous amino acids, at least 100 contiguous amino acids, at least 125 contiguous amino acids, at least 150 contiguous amino acids, at least 175 contiguous amino acids, or even at least 200 contiguous amino acids.
- Still larger fragments of human Maxpl are also contemplated, such as fragments comprising at least 225 contiguous amino acids, at least 250 contiguous amino acids, at least 275 contiguous amino acids, or even at least 300 contiguous amino acids.
- any size fragment is contemplated as long as the fragment comprises contiguous amino acids spanning 17.9%> or more, 25%> or more, or even 30% or more of the polypeptide molecule consisting essentially of an amino acid sequence encoding human Maxpl.
- isolated is meant the removal of a nucleic acid or polypeptide molecule from its natural environment.
- purified is meant that a given nucleic acid or polypeptide molecule, whether one that has been removed from nature or synthesized and/or amplified under laboratory conditions, has been increased in purity, wherein “purity” is a relative term, not “absolute purity”.
- nucleic acid molecule is intended to encompass a polymer of DNA or RNA, (i.e., a polynucleotide), which can be single-stranded or double- stranded and which can contain non-natural or altered nucleotides.
- a polypeptide molecule is intended to encompass a linear sequence of amino acids (i.e., a primary protein structure) but also can include secondary, tertiary, and quaternary protein structures, all of which can contain non-natural or altered amino acids.
- nucleic acid molecule will code for a "non-variant" human Maxpl .
- Such a nucleic acid molecule will code for a "variant” human Maxpl.
- the present invention provides an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding a variant human Maxpl or a fragment thereof comprising at least 52 contiguous nucleotides.
- the nucleotide sequence encoding such a variant human Maxpl will have a deletion spanning nucleotides 396 to 1173 (i.e., will comprise nucleotides 1 to 395).
- one or more substitution(s) is present in the isolated or purified nucleic acid molecule encoding the variant human Maxpl, it is preferred that such a substitution(s) results in the substitution of an amino acid of the encoded variant human Maxpl with another amino acid of approximately equivalent mass, structure and charge.
- it is preferred that no insertions, deletions, substitutions and/or abnormal post-translational modifications are present in the polypeptide molecule.
- Such a polypeptide molecule will code for a non-variant human Maxpl .
- Such a polypeptide molecule will code for a variant human Maxpl .
- the present invention provides an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding a variant human Maxpl or a fragment thereof comprising at least 70 contiguous amino acids.
- the amino acid sequence encoding such a variant human Maxpl will have a deletion spanning amino acids 132 to 391 (i.e., will comprise amino acids 1 to 131).
- the variant human Maxpl will not differ functionally from the corresponding non-variant human Maxpl.
- any insertions, deletions, inversions and/or substitutions contained within the nucleic acid molecule comprising a nucleotide sequence encoding the variant human Maxpl will not (i) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the nucleotide sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
- the one or more substitution(s) result(s) in the substitution of an amino acid with another amino acid of approximately equivalent mass, structure and charge.
- nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to a nucleotide sequence encoding human Maxpl, a variant human Maxpl or a fragment of either of the foregoing comprising at least 52 contiguous nucleotides.
- such an isolated or purified nucleic acid molecule (i) is complementary to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2, (ii) is complementary to the nucleotide sequence of SEQ ID NO:l or a fragment thereof comprising at least 52 contiguous nucleotides, (iii) hybridizes under highly stringent conditions to an isolated or purified nucleic acid molecule consisting essentially of SEQ ID NO: 1 or a fragment thereof, or (iv) shares 85% or more identity with the nucleotide sequence that is complementary to SEQ ID NO:l.
- hybridizes to refers to the selective binding of a single-stranded nucleic acid probe to a single-stranded target DNA or RNA sequence of complementary sequence when the target sequence is present in a preparation of heterogeneous DNA and/or RNA.
- Stringent conditions are sequence-dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 20 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
- hybridization is preferably carried out using a standard hybridization buffer at a temperature ranging from about 50 °C to about 75 °C, even more preferably from about 60 °C to about 70 °C, and optimally from about 65 °C to about 68 °C.
- formamide can be included in the hybridization reaction, and the temperature of hybridization can be reduced to preferably from about 35 °C to about 45 °C, even more preferably from about 40 °C to about 45 °C, and optimally to about 42 °C.
- formamide is included in the hybridization reaction at a concentration of from about 30% to about 50%), preferably from about 35%> to about 85%), and optimally at about 40%>.
- the hybridized sequences are washed (if necessary to reduce non-specific binding) under relatively highly stringent conditions, as that term is understood by those skilled in the art.
- the hybridized sequences are washed one or more times using a solution comprising salt and detergent, preferably at a temperature of from about 50 °C to about 75 °C, even more preferably at from about 60 °C to about 70 °C, and optimally from about 65 °C to about 68 °C.
- a salt e.g., such as sodium chloride
- a detergent e.g., such as sodium dodecyl sulfate
- concentration of from about 0.01 % to about 1.0% is included in the wash solution at a concentration of from about 0.01 % to about 1.0%.
- hybridization conditions :
- washing conditions :
- “highly stringent conditions” preferably allow for up to 15%) mismatch, more preferably up to about 12%> mismatch, and most preferably up to 10% mismatch (such as 5%, 4%>, 3%, 2% or 1%>).
- “Moderately stringent conditions” preferably allow for up to about 40% mismatch, more preferably up to about 30% mismatch, and most preferably up to about 20% mismatch.
- “Low stringent conditions” preferably allow for up to about 60% mismatch, more preferably up to about 50%o mismatch, and most preferably up to about 40%) mismatch. With respect to the preceding ranges of mismatch, 1%> mismatch corresponds to one degree decrease in the melting temperature. It is generally appreciated that the stringent conditions can be manipulated by adjusting the concentration of formamide in the hybridization reaction. For example, conditions can be rendered more stringent by the addition of increasing amounts of formamide.
- nucleic acid and polypeptide molecules also can be characterized in terms of "percentage of sequence identity.”
- a given nucleic acid or polypeptide molecule as described above can be compared to a nucleic acid or polypeptide molecule encoding human Maxpl by optimally aligning the nucleotide or amino acid sequences over a comparison window, wherein the portion of the nucleotide or amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence, which does not comprise additions or deletions, for optimal alignment of the two sequences.
- the percentage of sequence identity is calculated by determining the number of positions at which the identical nucleotide or amino acid occurs in both sequences, i.e., the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
- Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms (e.g., GAP, BESTFIT, FAST A, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI; BlastN and BlastP available from the National Center for Biotechnology Information, Bethesda, MD; or ClustalW available from the European Bioinformatics Institute, Cambridgeshire, UK), or by inspection.
- the isolated of purified nucleic acid molecule consists essentially of a nucleotide sequence which shares 85%» or more identity with SEQ ID NO:l
- the isolated or purified polypeptide molecule consists essentially of an amino acid sequence which shares 84%> or more identity with SEQ ID NO:2. It will be understood, however, that the percentage of sequence identity may vary slightly when using the different computerized programs since they implement different algorithms. The invention is intended to cover such variations but will generally share the percentage of sequence identities above using at least one computerized program and its respective algorithm.
- the present invention also provides a vector comprising an above-described isolated or purified nucleic acid molecule.
- a nucleic acid molecule as described above can be cloned into any suitable vector and can be used to transform or transfect any suitable host.
- the selection of vectors and methods to construct them are commonly known to persons of ordinary skill in the art and are described in general technical references (see, in general, "Recombinant DNA Part D,” Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987)).
- Constructs of vectors which are circular or linear, can be prepared to contain an entire nucleic acid molecule as described above or a portion thereof ligated to a replication system functional in a prokaryotic or eukaryotic host cell.
- Replication systems can be derived from ColEl, 2 m ⁇ plasmid, ⁇ , SV40, bovine papilloma virus, and the like.
- the construct can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like.
- Suitable vectors include those designed for propagation and expansion or for expression or both.
- a preferred cloning vector is selected from the group consisting of the pUC series, the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clonetech, Palo Alto, CA).
- Bacteriophage vectors such as ⁇ GTIO, ⁇ GTl l, ⁇ ZapII (Stratagene), ⁇ EMBL4, and ⁇ NM1149, also can be used.
- plant expression vectors examples include pBHOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clonetech, Palo Alto, CA).
- animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clonetech).
- An expression vector can comprise a native or nonnative regulatory sequence operably linked to an isolated or purified nucleic acid molecule as described above. If more than one nucleic acid sequence is included in the nucleic acid molecule, each sequence can be operably linked to its own regulatory sequence.
- the "regulatory sequence” is typically a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the nucleic acid to which it is operably linked.
- the regulatory sequence can, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter.
- Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus, Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus and other retroviruses, and Herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art.
- the promoters useful as regulatory sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions.
- inducible promoters are known to those of ordinary skill in the art and can be used in the context of the invention, when desired.
- the selection of promoters e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the skill in the art.
- the combining of a nucleic acid molecule as described above with a promoter is also within the skill in the art.
- operably linked can be defined when a nucleic acid molecule and the regulatory sequence are covalently linked in such a way as to place the expression of the nucleic acid coding sequence under the influence or control of the regulatory sequence.
- a regulatory sequence would be operably linked to a nucleic acid molecule if the regulatory sequence were capable of effecting transcription of that nucleic acid molecule such that the resulting transcript is translated into the desired protein or polypeptide.
- the present invention provides a cell (i.e., a host cell) comprising an isolated or purified nucleic acid molecule or a vector as described above.
- a host cell i.e., a host cell
- examples of host cells include, but are not limited to, a human cell, a human cell line, E. coli, B. subtilis, P. aerugenosa, S. cerevisiae, and N crassa.
- E. coli in particular E. coli TB-1, TG-2, DH5 ⁇ , XL-Blue MRF' (Stratagene), SA2821 and Y1090 are preferred hosts.
- Any appropriate expression vector e.g., as described in Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevior, ⁇ Y, (1985)
- suitable host can be employed for production of recombinant polypeptides.
- Expression hosts include, but are not limited to, bacterial species within the genera Escherichia, Bacillus, Pseudomonas, Salmonella, mammalian or insect host cell systems including baculovirus systems (e.g., as described by Luckow et al., Bio/Technology, 6, 47 (1988)), and established cell lines such as the COS-7, C127, 3T3, CHO, HeLa, BHK cell line, and the like.
- polypeptide molecules of the invention can be modified, for instance, by glycosylation, amidation, carboxylation, or phosphorylation, or by the creation of acid addition salts, amides, esters, in particular C-terminal esters, and N-acyl derivatives of the polypeptide molecules of the invention.
- polypeptide molecules also can be dimerized or polymerized. Moreover, the polypeptide molecules can be modified to create polypeptide derivatives by forming covalent or noncovalent complexes with other moieties in accordance with methods known in the art. Covalently-bound complexes can be prepared by linking the chemical moieties to functional groups on the side chains of amino acids comprising the polypeptides, or at the N- or C- terminus.
- hybridomas are known in the art (see, e.g., Roitt I., Immunology, 4 th Ed., Mosby, NY (1996)).
- the present invention also provides a monoclonal antibody produced by the hybridoma cell line.
- monoclonal antibodies are typically employed for diagnostic applications as they are described herein.
- Maxpl exhibits all the basic biological and biochemical characteristics of a Ras effector, i.e., a protein which directly mediates the biological effects of Ras. However, unlike most previously described Ras effectors, Maxpl serves to inhibit cell growth. This growth inhibition is enhanced in the presence of an activated form of Ras, and reduced in the presence of a dominant negative form of Ras. Thus, Ras appears to regulate the growth inhibitory properties of Maxpl. Maxpl serves to connect Ras to numerous signal transduction pathways, including the CREB pathway. Maxpl is, however, a relatively poor activator of NFkB and it is possible that activation of CREB in the absence of NFkB activation promotes apoptotic cell death (Saeki et al, Biochem J.
- Maxpl expression is lost or severely reduced in many lung, breast and colon tumors and tumor cell lines.
- the human gene has been mapped to lq32.1-2, a site which has been described as the location of an unknown tumor suppressor in kidney tumors (Steiner et al. Cancer Res, 56:50044-5046 (1996)).
- Maxpl appears to be a Ras effector which is also a tumor suppressor. Accordingly, the invention provides a method of diagnosing a mammal with a cancer or a predisposition to a cancer.
- the method comprises detecting either (i) a mutation in a nucleic acid molecule comprising a nucleotide sequence encoding Maxpl, (ii) a decreased level of a polypeptide molecule comprising an amino acid sequence encoding wild-type Maxpl, or (iii) a mutation in a polypeptide molecule comprising an amino acid sequence encoding Maxpl in a test sample obtained from the mammal.
- the detection of (i), (ii), or (iii) in the test sample is indicative of the cancer or a predisposition to the cancer in the mammal.
- the nucleic acid molecule comprising the nucleotide sequence encoding Maxpl comprises SEQ ID NO:l and the polypeptide molecule comprising the amino acid encoding Maxpl comprises SEQ ID NO:2.
- Down-regulation can be due to promoter methylation.
- the test sample used in conjunction with the invention can be any of those typically used in the art and will vary depending on the condition of the mammal (i.e., whether or not a cancer has developed in the mammal).
- the test sample can be saliva, tissue or blood.
- the tissue is metastatic (e.g., cancerous) and is obtained by means of a biopsy.
- tissue can include bone marrow, lymph nodes, skin, and any organ that may develop cancerous cells.
- the test sample is one which is least invasive to the mammal, such as a saliva or blood sample.
- assays are contemplated for use in analyzing a given test sample of the present invention.
- the term "assay” can be defined as any quantitative or qualitative analysis of a nucleic acid or polypeptide molecule that is known in the art. A variety of these assays are contemplated for use in the invention, many of which are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2" Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, (1989). Microarrays, such as those described in U.S. Patent Nos. 6,197,506 and 6,040,138, also can be used to detect and quantify Maxpl .
- assay used will depend on whether a nucleic acid or polypeptide molecule is being assayed for and whether the detection or quantification of the nucleic acid or polypeptide molecule is sought.
- various assays can be used to detect or to measure the level of Maxpl in a given test sample. For example, when only the detection of Maxpl or the identification of a mutation in Maxpl is necessary to diagnose effectively the cancer or a predisposition to the cancer, assays including PCR and microarray analysis can be used. In certain embodiments it may be necessary to detect the quantity of Maxpl present.
- Maxpl comprises DNA
- hybridization techniques can include, for example, Southern hybridization (i.e., a Southern blot), in situ hybridization and microarray analysis.
- Southern hybridization i.e., a Southern blot
- Northern hybridization i.e., a Northern blot
- in situ hybridization i.e., a Northern blot
- a nucleotide sequence that specifically binds to or associates with a nucleic acid molecule comprising a nucleotide sequence encoding Maxpl, whether DNA or RNA can be attached to a label for determining hybridization.
- appropriate labels are known in the art, including fluorescent, radioactive, and enzymatic labels as well as ligands, such as avidin/biotin, which are capable of being detected.
- a fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, is used instead of a radioactive or other environmentally undesirable label.
- enzyme tags colorimetric indicator substrates are known which can be employed to provide a detection means visible to the human eye or spectrophotometrically to identify specific hybridization with complementary Maxpl nucleic acid-containing samples.
- nucleic acid molecule comprising a nucleotide sequence encoding Maxpl
- the nucleic acid used as a template for amplification is isolated from cells contained in the test sample, according to standard methodologies (see, e.g., Sambrook et al., (1989), supra).
- the nucleic acid can be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it can be desirable to convert the RNA to cDNA.
- pairs of primers that selectively hybridize to nucleic acids corresponding to Maxpl are contacted with the nucleic acid under conditions that permit selective hybridization. Once hybridized, the nucleic acid-primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles,” are conducted until a sufficient amount of amplification product is produced.
- RNA polymerase chain reaction PCR
- RT-PCR reverse transcriptase PCR
- Alternative methods for reverse transcription utilize thermostable DNA polymerases and are described in WO 90/07641, for example.
- LCR ligase chain reaction
- isothermal amplification in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha-thio]-triphosphates in one strand (Walker et al., Proc. Natl Acad. Sci.
- strand displacement amplification SDA
- RCR repair chain reaction
- a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA, which is present in a sample.
- the reaction Upon hybridization, the reaction is treated with RNase H, and the products of the probe are identified as distinctive products, which are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated. A number of other amplification processes are contemplated; however, the invention is not limited as to which method is used. [0050] Following amplification of Maxpl, it can be desirable to separate the amplification product from the template and the excess primer for the purpose of determining whether specific amplification has occurred. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al. (1989), supra. [0051] Alternatively, chromatographic techniques can be employed to effect separation.
- Amplification products must be visualized in order to confirm amplification of the Maxpl sequence.
- One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
- the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
- visualization is achieved indirectly.
- a labeled, nucleic acid probe is brought into contact with the amplified Maxpl sequence.
- the probe preferably is conjugated to a chromophore but may be radiolabeled.
- the probe is conjugated to a binding partner, such as an antibody or biotin, where the other member of the binding pair carries a detectable moiety (i.e., a label).
- a nucleic acid of partial sequence can be used to quantify the expression of a structurally related gene or the full-length genomic or cDNA clone from which it is derived.
- various assays i.e., immunobinding assays
- Maxpl or an antibody able to recognize antibodies that are specific for Maxpl (i.e., an anti- idiotypic antibody), can be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies can be prepared and employed to detect Maxpl or an anti-idiotypic antibody thereof.
- the steps of various useful immunodetection assays have been described in Nakamura et al., Handbook of Experimental Immunology (4 th Ed.), Wol.
- Enzyme Immunoassays Heterogenous and Homogenous Systems, Chapter 27 (1987) and include Western hybridization (i.e., Western blots), immunoaffmity purification, immunoaffinity detection, enzyme-linked immunosorbent assay (e.g., an ELISA), and radioimmunoassay.
- Western hybridization i.e., Western blots
- immunoaffmity purification i.e., immunoaffmity purification
- immunoaffinity detection e.g., an ELISA
- radioimmunoassay e.g., an ELISA
- a microarray also can be used to detect or measure the levels of Maxpl .
- the immunobinding assays involve obtaining a test sample suspected of containing a polypeptide molecule comprising an amino acid sequence encoding Maxpl or an antibody corresponding to human Maxpl, and contacting the test sample with an antibody in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes.
- a mammal can be diagnosed with a cancer or a predisposition to a cancer by either detecting or quantifying the levels of a polypeptide molecule comprising an amino acid sequence encoding Maxpl, an antibody that recognizes Maxpl, or an antibody that recognizes an antibody that is specific for Maxpl.
- any suitable antibody can be used in conjunction with the present invention.
- the antibody is specific for Maxpl, however, the antibody can recognize other antibodies (i.e., an anti-idiotypic antibody) present in a test sample that bind to Maxpl .
- the antibody can be specific for any region (i.e., epitope) within Maxpl.
- the epitope will be located in a Ras association domain, such as a C-terminal portion of a Ras association domain.
- the epitope can comprise a region spanning amino acids 311-327 of the wild-type human Maxpl protein.
- the antibody can be a polyclonal or a monoclonal antibody and can be identified using methods well known in the art.
- the immunobinding assays for use in the present invention include methods for detecting or quantifying the amount of Maxpl in a test sample, which methods require the detection or quantitation of any immune complexes formed during the binding process.
- a test sample suspected of containing a polypeptide molecule comprising an amino acid sequence encoding Maxpl or an antibody that is specific for Maxpl would be obtained from a mammal and subsequently contacted with an antibody. The detection or the quantification of the amount of immune complexes formed under the specific conditions is then performed.
- the first added component that becomes bound within the primary immune complexes can be detected by means of a second binding ligand that has binding affinity for the first antibody.
- the second binding ligand is, itself, often an antibody, which can be termed a "secondary" antibody.
- the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes.
- the secondary immune complexes are then washed to remove any non- specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
- Further methods include the detection of primary immune complexes by a two-step approach.
- a second binding ligand such as an antibody, that has binding affinity for the first antibody is used to form secondary immune complexes, as described above.
- the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
- the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed.
- diagnostic tests can be used in conjunction with the diagnostic tests described herein to enhance further the accuracy of diagnosing a cancer or a predisposition to a cancer in a mammal.
- a monoclonal antibody which is known to be specific for a cancer can be used in conjunction with the methods of the invention, or the detection of other genetic abnormalities known to be associated with cancer or a predisposition to a cancer can be employed.
- the present invention also provides a method of prognosticating a cancer in a mammal, wherein Maxpl is a marker for the cancer, which method comprises measuring the level of Maxpl in a test sample obtained from the mammal, wherein the level of Maxpl in the test sample is indicative of the prognosis of the cancer in the mammal.
- the level of Maxpl in the test sample can be measured by comparing the level of Maxpl in another test sample obtained from the mammal over time in accordance with the methods described above.
- a decrease in Maxpl levels from one sample to the next is indicative of growth and/or metastasis of the cancer (i.e., a negative prognosis), whereas an increase or no change in Maxpl levels from one sample to the next is indicative of halted growth or even reduction of the cancer (i.e., a positive prognosis).
- the invention also provides a method of assessing the effectiveness of treatment of a cancer in a mammal, wherein Maxpl is a marker for the cancer, which method comprises measuring the level of Maxpl in a test sample obtained from the mammal, wherein the level of Maxpl in the test sample is indicative of the effectiveness of the treatment of the cancer in the mammal.
- the level of Maxpl in the test sample can be measured by comparing the level of Maxpl in the test sample to the level of Maxpl in another test sample obtained from the mammal over time in accordance with the methods described above. A decrease or no change in Maxpl levels from one sample to the next is indicative of the treatment being ineffective, whereas an increase in Maxpl levels from one sample to the next is indicative of the treatment being effective.
- the term "decreased level” can be defined as detecting Maxpl in a test sample obtained from a mammal at a level below that which is considered normal. For example, the level of Maxpl in a test sample is decreased when the copy number of the gene encoding the Maxpl, the mRNA encoding Maxpl, or a polypeptide molecule comprising an amino acid sequence encoding Maxpl is detected at a level below that which is considered normal. Conversely, the term “increased level” can be defined as detecting Maxpl in a test sample obtained from a mammal at a level above that which is considered normal.
- the level of Maxpl in a test sample is increased when the copy number of the gene encoding the Maxpl, the mRNA encoding Maxpl, or a polypeptide molecule comprising an amino acid sequence encoding Maxpl is detected at a level above that which is considered normal.
- "Normal levels" pertain to an already determined range of Maxpl established from cancer- free mammals of the same species and are generally accepted and recognized in the art.
- Maxpl mediates a signaling pathway directed by Ras which leads to growth inhibition.
- a mutation in Maxpl which disrupts its normal function or a decreased level of Maxpl would, in effect, shut down the growth inhibitory effects of Ras, leaving the pro-transformation effector pathways intact.
- the present invention further provides a method of treating a mammal prophylactically or therapeutically for a cancer by administering to the mammal a composition comprising a carrier and (a) a nucleic acid molecule comprising and expressing a nucleotide sequence encoding wild-type Maxpl or a fragment thereof, (b) a nucleic acid molecule comprising and expressing a nucleotide sequence encoding a variant Maxpl or a fragment thereof, (c) a polypeptide molecule comprising an amino acid sequence encoding wild-type Maxpl or a fragment thereof, or (d) a polypeptide molecule comprising an amino acid sequence encoding a variant Maxpl or a fragment thereof, wherein the composition is administered to the mammal in an amount sufficient to treat prophylactically or therapeutically the mammal for the cancer.
- the cancer typically is due to (i) at least one mutation in a nucleic acid molecule comprising a nucleotide sequence encoding Maxpl, (ii) a decreased level of a polypeptide molecule comprising an amino acid sequence encoding wild-type Maxpl, or (iii) at least one mutation in a polypeptide molecule comprising an amino acid sequence encoding Maxpl.
- a mammal can be diagnosed with, or predisposed to, any cancer utilizing the methods of the invention.
- the method involving prognosticating a mammal for a cancer, assessing the effectiveness of treatment of a cancer, and treating a mammal prophylactically or therapeutically for a cancer can be utilized with any cancer.
- the cancer is lung cancer, including primary lung tumors, colon cancer, or renal cancer.
- the cancer can be metastatic.
- cancers contemplated in the invention include: anal cancer; bile duct cancer; bladder cancer; bone cancer; brain and spinal chord cancers; breast cancer; cervical cancer; lymphoma; endometrial cancer; esophageal cancer; gallbladder cancer; gastrointestinal cancer; laryngeal cancer; leukemia; liver cancer; multiple myeloma; neuroblastoma; ovarian cancer; pancreatic cancer; prostatic cancer; retinoblastoma; skin cancer (e.g., melanoma and non-melanoma); stomach cancer; testicular cancer; thymus cancer; thyroid cancer; as well as other carcinomas and sarcomas.
- anal cancer bile duct cancer; bladder cancer; bone cancer; brain and spinal chord cancers; breast cancer; cervical cancer; lymphoma; endometrial cancer; esophageal cancer; gallbladder cancer; gastrointestinal cancer; laryngeal cancer; leukemia; liver cancer; multiple myeloma;
- the present invention also provides a composition
- a composition comprising a carrier and either (i) an above-described isolated or purified nucleic acid molecule or a fragment thereof comprising at least 52 nucleotides, (ii) an above-described vector, or (iii) an above-described polypeptide molecule or a fragment thereof comprising at least 70 amino acids.
- the composition comprises a carrier and a nucleic acid molecule comprising a nucleotide sequence encoding a wild-type or variant Maxpl or a polypeptide molecule comprising an amino acid sequence encoding a wild-type or variant Maxpl.
- nucleic acid molecule encoding a wild-type or variant Maxpl in a recombinant vector is used, as described above.
- the polypeptide molecule comprises a deletion spanning amino acids 132 to 391 and that the nucleic acid molecule comprises a deletion spanning nucleotides 396 to 1173.
- Such a nucleic acid molecule will code for a variant Maxpl which comprises a deletion spanning amino acids 132 to 391.
- the composition can further comprise, or can be conjugated to, a targeting moiety.
- the targeting moiety is an antibody or an antigenically reactive fragment thereof.
- the antibody or antigenically reactive fragment thereof can be specific for cancer cells expressing Maxpl, and thus increase the affinity of the composition for the cancer cells.
- the targeting moiety can be a reporter group, including, but not limited to a radiolabel, a fluorescent label, an enzyme (e.g., that catalyzes a colorimetric or fluorometric reaction), a substrate, a solid matrix, or a carrier (e.g., biotin or avidin).
- an antigenically reactive fragment of the antibody e.g., Fab, Fc, etc.
- an antigenically reactive fragment of the antibody can be obtained from the antibodies produced as described above, by methods which include digestion with enzymes, such as pepsin or papain, and/or cleavage of disulfide bonds by chemical reduction.
- the composition can comprise more than one active ingredient, such as comprising more than one type of molecule of Maxpl.
- the composition can comprise another pharmaceutically active agent or drug.
- other anticancer compounds can be used in conjunction with the composition of the present invention and include, but are not limited to, all of the known anticancer compounds approved for marketing in the United States and those that will become approved in the future. See, for example, Table 1 and Table 2 of Boyd, Current Therapy in Oncology, Section 1. Introduction to Cancer Therapy (J.E. Niederhuber, ed.), Chapter 2, by B.C. Decker, Inc., Philadelphia, 1993, pp. 11-22.
- these other anticancer compounds include doxorubicin, bleomycin, vincristine, vinblastine, VP- 16, VW-26, cisplatin, carboplatin, procarbazine, and taxol for solid tumors in general; alkylating agents, such as BCNU, CCNU, methyl-CCNU and DTIC, for brain or kidney cancers; and antimetabolites, such as 5-FU and methotrexate, for colon cancer.
- the carrier can be any suitable carrier.
- the carrier is a pharmaceutically acceptable carrier.
- the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with Maxpl , and by the route of administration. It will be appreciated by one of skill in the art that, in addition to the above-described composition, the compositions of the present inventive methods can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.
- inclusion complexes such as cyclodextrin inclusion complexes, or liposomes.
- the pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the Maxpl and one which has no detrimental side effects or toxicity under the conditions of use.
- composition of the present invention The choice of carrier will be determined in part by the particular molecule of Maxpl involved, as well as by the particular method used to administer the composition. Accordingly, there are a variety of suitable formulations of the composition of the present invention.
- suitable formulations of the composition of the present invention The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intramuscular, interperitoneal, rectal, and vaginal administration are exemplary and are in no way limiting.
- a composition of the invention to a mammal, in particular a human
- suitable methods of administering a composition of the invention to a mammal are available, and, although more than one route can be used to administer a particular compound, a particular route can provide a more immediate and more effective reaction than another route. Accordingly, the herein-described methods are exemplary and are in no way limiting.
- the dose administered to a mammal, in particular a human should be sufficient to treat prophylactically or therapeutically the cancer in the mammal.
- dosage will depend upon a variety of factors including the strength of the particular composition employed, as well as the age, species, condition, and body weight of the mammal.
- the size of the dose will also be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular composition and the desired physiological effect.
- Suitable doses and dosage regimens can be determined by conventional range-finding techniques known to those of ordinary skill in the art. Generally, a composition is initially administered in smaller dosages, which are less than the optimum dose of the composition. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.
- the present inventive method will typically involve the administration of about 0.1-100 mg of one or more of the compositions described above per kg body weight.
- NIH 3T3 cells (ATCC, Rockville, MD) were grown in 10% calf serum in DMEM (BRL-Life technologies, Gaithersburg, MD). These cells were then transfected using the calcium phosphate technique (Clark, G.J., Methods in Enzymology, vol. 255, 395- 412 (1995)) with 200 ng of pZIPHA vector or pZIPHAmaxpl (Fiordalisi, J.J. et al., Methods Enzymol 332: 3-36 (2001)) in 60 mm plates.
- Maxpl mediates a Ras-dependent apoptosis. These results indicate that Maxpl is a novel Ras effector that is involved in a signaling pathway initiated by Ras, which leads to programmed cell death.
- Maxpl Maxpl and its use in diagnosing small cell lung cancer.
- Maxpl peptide i.e., amino acids 311-327
- the resulting polyclonal antibody was specific only for
- Maxpl and not specific for related proteins such as RASSFl and RASSF2.
- the resulting antibody is able to recognize human, mouse and rat Maxpl proteins.
- the polyclonal antibody described above was used to detect Maxpl expression levels in test samples known to comprise cancerous cells. Specifically, five randomly selected sections (i.e., pathology slides) of small cell lung carcinomas were incorporated in an immunohistochemical analysis for Maxpl expression. In four of the five sections,
- Maxpl expression was not present in test samples known to comprise cancerous cells. These results indicate that a polyclonal antibody of the invention can be used to diagnose a cancer in a mammal by detecting low levels or lack of expression of Maxpl.
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Abstract
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AU2002339946A AU2002339946A1 (en) | 2001-09-19 | 2002-09-18 | Maxp1 |
US10/489,906 US20040198970A1 (en) | 2002-09-18 | 2002-09-18 | Maxp1 |
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US32327401P | 2001-09-19 | 2001-09-19 | |
US60/323,274 | 2001-09-19 |
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US8951745B2 (en) | 2003-10-10 | 2015-02-10 | Deutsches Krebsforschungszentrum | Compositions for diagnosis and therapy of diseases associated with aberrant expression of futrins (R-Spondins) and/or Wnt |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6485910B1 (en) * | 1998-02-09 | 2002-11-26 | Incyte Genomics, Inc. | Ras association domain containing protein |
US20040048249A1 (en) * | 2000-01-21 | 2004-03-11 | Tang Y. Tom | Novel nucleic acids and secreted polypeptides |
-
2002
- 2002-09-18 WO PCT/US2002/029643 patent/WO2003025143A2/fr not_active Application Discontinuation
- 2002-09-18 AU AU2002339946A patent/AU2002339946A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6485910B1 (en) * | 1998-02-09 | 2002-11-26 | Incyte Genomics, Inc. | Ras association domain containing protein |
US20040048249A1 (en) * | 2000-01-21 | 2004-03-11 | Tang Y. Tom | Novel nucleic acids and secreted polypeptides |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US8951745B2 (en) | 2003-10-10 | 2015-02-10 | Deutsches Krebsforschungszentrum | Compositions for diagnosis and therapy of diseases associated with aberrant expression of futrins (R-Spondins) and/or Wnt |
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