WO1995028489A1 - Compositions de detection de la dihydropyrimidine deshydrogenase (dpd) et procedes d'utilisation associes - Google Patents

Compositions de detection de la dihydropyrimidine deshydrogenase (dpd) et procedes d'utilisation associes Download PDF

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Publication number
WO1995028489A1
WO1995028489A1 PCT/US1995/004567 US9504567W WO9528489A1 WO 1995028489 A1 WO1995028489 A1 WO 1995028489A1 US 9504567 W US9504567 W US 9504567W WO 9528489 A1 WO9528489 A1 WO 9528489A1
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dpd
nucleic acid
seq
sequence
protein
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PCT/US1995/004567
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English (en)
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Robert B. Diasio
Zhihong Lu
Ruiwen Zhang
Martin Johnson
Xiaogang Cheng
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The Uab Research Foundation
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Priority to AU22894/95A priority Critical patent/AU2289495A/en
Publication of WO1995028489A1 publication Critical patent/WO1995028489A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01002—Formate dehydrogenase (1.2.1.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • a surprising aspect of the invention concerns the cDNA sequence of DPD from an individual with increased FUra sensitivity. Unlike the normal DPD cDNA obtained from individuals having normal DPD activity, the individual showing an increased sensitivity to FUra had an altered DPD gene.
  • immu ⁇ ologically effective amount an amount of a DPD protein or peptide composition that is capable of generating an immune response in the recipient animal. This includes both the generation of an antibody response (B-cell response), and/or the stimulation of a cytotoxic immune response (T-cell response).
  • B-cell response an antibody response
  • T-cell response cytotoxic immune response
  • the generation of such an immune response will have utility in both the production of useful bioreagents, e.g., cytotoxic T-lymphocytes (CTLs) and, more particularly, reactive antibodies, for use in diagnostic embodiments, and will also have utility in various prophylactic or therapeutic embodiments.
  • CTLs cytotoxic T-lymphocytes
  • kits may contain antigen or antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.
  • DNA segment refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding DPD refers to a DNA segment that contains DPD coding sequences yet is isolated away from, or purified free from, total genomic DNA of either bovine or human cells. Included within the term “DNA segment”, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Similarly, a DNA segment comprising an isolated or purified DPD gene refers to a
  • Sequences that are essentially the same as those set forth in SEQ ID N0:1 or SEQ ID N0:3 may also be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID N0:1 or SEQ ID N0:3 under relatively stringent conditions. Suitable relatively stringent hybridization conditions will be well known to those of skill in the art and are clearly set forth herein.
  • the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID N0:1 or SEQ ID N0:3. Nucleic acid sequences that are "complementary" are those that are capable of base- pairing according to the standard Watson-Crick complementarity rules.
  • intermediate lengths means any length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, etc , 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; 5,000-10,000 ranges, up to and including sequences of about 12,001, 12,002, 13,001, 13,002 and the like.
  • nucleic acid sequences disclosed herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 14 nucleotide long contiguous sequence of SEQ ID N0:1 or SEQ ID N0:3 will find particular utility.
  • Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of about 10, 15, 30, 50, or even of about 100 to about 200 nucleotides or so, identical or complementary to SEQ ID N0:1 or SEQ ID N0:3 are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. This would allow DPD structural or regulatory genes to be analyzed, both in eukar ⁇ otic and prokaryotic cells, including e.g., mammalian cells such as human and bovine cells, or various bacterial or other prokaryotic species. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment.
  • kits comprising DPD peptides or DPD-encoding nucleic acid segments comprise another aspect of the present invention.
  • Such kits will generally contain, in suitable container, a pharmaceutically acceptable formulation of DPD peptide or a DPD- encoding nucleic acid composition.
  • the kit may have a single container that contains the DPD composition or it may have distinct containers for the DPD composition and other reagents which may be included within such kits.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al., 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 ⁇ m) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made, as described (Couvreur et al., 1977; 1988).
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
  • MLVs generally have diameters of from 25 nm to 4 ⁇ m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • the following information may be utilized in generating liposomal formulations.
  • Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure.
  • the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.
  • Liposomes interact with cells via four different mechanisms: Endoc ⁇ tosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time. 8. DPD Compositions and FUra Treatment of Proliferative Cell Disorders
  • DPD eukaryotic expression system
  • baculovirus-based, glutamine synthase-based or dihydrofolate reductase-based systems could be employed.
  • piasmid vectors incorporating an origin of replication and an efficient eukaryotic promoter as exemplified by the eukaryotic vectors of the pCMV series, such as pCMV5, will be of most use.
  • a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal.
  • an immunogen comprising a polypeptide of the present invention
  • a wide range of animal species can be used for the production of antisera.
  • an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • FIG. 1A Cloning Strategy of Bovine Liver DPD cDNA.
  • a degenerate sense primer designated “Primer A”
  • an antisense primer designated “Primer B”
  • the primers correspond to the respective amino acid peptide sequences KAEASGA (SEQ ID NO: 10) and PHGMGER (SEQ ID N0:11) (denoted by boxes).
  • KAEASGA SEQ ID NO: 10
  • PHGMGER SEQ ID N0:11
  • FIG. 2 Alignment and Ligation of the Full-Length Bovine Liver DPD cDNA.
  • the full-length clone (4414 base pairs; SEQ ID N0:1) was generated by ligation of the three cDNA fragments (2360, 2076, and 237 base pairs). Each fragment was independently identified as a portion of the DPD clone by identification of specific peptide sequence derived from purified bovine liver DPD (as shown in boldface). Restriction sites common in only the overlapping regions (shown by hashed lines) were utilized to ligate the three fragments together.
  • FIG. 4 In vitro translation of bovine liver cDNA. RNA from in vitro transcription of full-length bovine liver cDNA was translated using a rabbit reticulocyte lysate system.
  • FIG. 10A In vitro transcription/translation of human DPD cDNA.
  • RNA from in vitro transcription of human lymphocyte DPD cDNA was translated in the presence of [ 35 S] methionine for labeling of the synthesized proteins;
  • lane 1 contains reaction products produced by human lymphocyte cDNA cloned from an individual with normal DPD enzyme activity and demonstrates a 108,000 dalton band.
  • Lane 2 contains reaction products produced using the cDNA cloned from the DPD-deficient patient and demonstrates a 40,000 dalton band corresponding to truncated DPD.
  • Lane 3 contains a lucif erase positive control demonstrating a 61,000 dalton band.
  • FIG. 10B In vitro transcription/translation of human DPD cDNA.
  • RNA from in vitro transcription of human lymphocyte DPD cDNA was translated in the presence of unlabeled amino acid for western blot analysis; lane 4 contains prestained molecular weight markers.
  • Lane 5 contains 0.2 ⁇ g purified DPD (Lu et al., 1992).
  • Lane 6 contains reaction products produced by human lymphocyte cDNA cloned from an individual with normal DPD enzyme activity and demonstrates a 108,000 dalton band corresponding to the band seen in lane 1.
  • Lane 7 contains reaction products produced by the cDNA cloned from the DPD deficient patient and demonstrates a 40,000 dalton band corresponding to the band seen in lane 2.
  • Lane 8 contains reaction products produced by luciferase positive control.
  • DPD Data base searches for amino acid sequences, similar to DPD, identified dihydroorotate dehydrogenase, thioredoxin reductase, and glutamate synthase with a partial amino acid sequence identity of 40, 37, and 38%, respectively. While these values are too low to support a common ancestry for these proteins (Doolittle, 1981), they do contain certain functional similarities to DPD.
  • Dihydroorotate dehydrogenase is a flavoprotein (using FAD as a cofactor) which catalyzes the fourth step in pyrimidine biosynthesis (Qui ⁇ n et al., 1991).
  • FAD FAD
  • both thioredoxin reductase and glutamate synthase use NADPH as a cofactor (Russel and Model, 1988; Oliver et al., 1987).
  • the occurrence of even minute amounts of immunocomplexes may be determined. As mentioned above, this may be achieved by subjecting the first immunocomplex to a second antibody having specificity for the first, or even a third antibody having specificity for the second. Where a second antibody alone is used, given that the control and test Fas samples will typically be of human origin, the second antibody will preferably be an antibody having specificity in general for human Fas. Where a third antibody is also used, the second antibody will still preferably be an antibody having specificity for human Fas, and the third antibody will then be an antibody having specificity in general for the second antibody. A second murine antibody and a third anti-mouse Ig antibody is a particular example.
  • the second or third antibody will have an associated label to allow detection.
  • this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
  • a urease, glucose oxidase or peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS/Tween ⁇ ).
  • nucleic acid segment that includes a contiguous sequence from within SEQ ID N0:1 or SEQ ID N0:3 may alternatively be described as preparing a nucleic acid fragment.
  • fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.
  • fragments may be obtained by application of nucleic acid reproduction technology, such as the PCRTM technology of U.S. Patent 4,683,202 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
  • the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase.
  • the test DNA or RNA
  • the test DNA is adsorbed or otherwise affixed to a selected matrix or surface.
  • This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions.
  • the selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+ C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.).
  • An epitopic core sequence is a relatively short stretch of amino acids that is "complementary" to, and therefore will bind, antigen binding sites on transferrin-bi ⁇ ding protein antibodies. Additionally or alternatively, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present disclosure, the term “complementary” refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the corresponding protein-directed antisera.
  • the size of the polypeptide antigen is not believed to be particulariy crucial, so long as it is at least large enough to carry the identified core sequence or sequences.
  • the smallest useful core sequence anticipated by the present disclosure would generally be on the order of about 8 to about 10 amino acids in length, with sequences on the order of 15 to 25 being more preferred. Thus, this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention.
  • the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.
  • the identification of epitopic core sequences is known to those of skill in the art, for example, as described in U.S.
  • the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal.
  • Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fiuorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard. 7.
  • Vaccines include enzymatically-, radiolabel-, or fiuorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard. 7.
  • Such vaccines are prepared as injectables. Either as liquid solutions or suspensions: solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified.
  • the active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired.
  • Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations. The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • nucleic acid sequences contemplated herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least an about 14-nucleotide long contiguous sequence that has the same sequence as, or is complementary to, an about 14- ⁇ ucleotide long contiguous DNA segment of SEQ ID N0:1 or SEQ ID N0:3 will find particular utility.
  • hybridization probe of about 14 nucleotides in length allows the formation of a duplex molecule that is both stable and selective.
  • Molecules having contiguous complementary sequences over stretches greater than 14 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained.
  • compositions disclosed herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1 % of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit.
  • the proteins were transferred from the gel to a nitrocellulose filter (Towbin et al., 1979).
  • the nitrocellulose filter was incubated overnight at 4°C with the polyclonal antibody (Ig G) purified by protein A column (diluted 1:2000) in a 120 mM borate-saline solution containing 1% (w/v) BSA, pH 8.5.
  • the nitrocellulose filter was washed with borate-saline containing 0.1 % Tween-20TM (w/v) and incubated with a secondary, alkaline phosphatase-labeled goat anti- rabbit antibody.
  • the amount of protein in the sample was determined using Bio-Rad protein determination reagent with BSA as a standard (Lowry et al., 1951). 17. Enzyme Purification
  • Protein activity 3 activity 3 mg nmollmin nmol/min/mg * ⁇ fold
  • Purified human liver DPD had an amber color (in buffer A) and showed the characteristic absorption spectrum of a reduced flavoprotei ⁇ .
  • the nature of the flavin cofactor in the enzyme molecule was shown by HPLC to be FAD and FMN. No conversion of FAD to FMN was detectable under these conditions. FAD and FMN were quantitated by a simultaneous fluorometric assay. As illustrated in Table 3, human DPD contains approximately 2 mol each of FAD ad FMN per mol of enzyme. 5.
  • the amino acid composition of carboxymethylated DPD was determined and the results are presented in Table 4. These data represent the mean of four separate DPD preparations.
  • the amino acid compositions of rat and pig liver DPDs were identical to the human sequence.
  • Table 6 summarizes the kinetic studies of purified human liver DPD, with comparison to rat and pig liver enzymes.
  • enzyme kinetic studies revealed apparent K m values for uracil, thymine, and FUra of 4.9, 4.8 and 3.3 ⁇ M, with corresponding V ma ⁇ values of 0.6, 0.7, and 0.9 ⁇ mol/min/mg protein, respectively.
  • substrate inhibition was observed for all substrates examined in the study.
  • sequence of the purified, CNBr-generated fragment was determined by the Protein Analysis and Peptide Synthesis Core Facility in the Comprehensive Cancer Center at University of Alabama at Birmingham using an Applied Biosystems Model 470 protein sequencer with an on-line 120A PTH analyzer. Analysis of two separate samples generated identical sequence data.
  • Bovine liver was obtained directly from a local slaughter house and snap frozen in dry ice/methanol.
  • Total RNA was isolated by the method of Ausebel (Ausebel et al., 1987). Purification of poly(A) + RNA was performed using an Oligotex-dT mRNA kit (Qiagen) according to the manufacturers instructions.
  • VLSKDVADIESILALN amino acids from the amino-termi ⁇ ai end of purified bovine liver DPD were determined.
  • a degenerate oligonucleotide, primer D sense: 5'-AARGAYGTIGCIGATATCGA-3' was designed to the portion of the N- terminal amino acid sequence KDVADIE (SEQ ID N0:6). Sequence data obtained from the 2360 base pair fragment (FIG.
  • the specific antisense primer (5'-GTCGTGTGCTTGATGTCATC-3') (SEQ ID N0:17) was used for first-strand cDNA synthesis followed by PCRTM amplification with the specific antisense primer (5'-GCTTCTCGCAATTAAAGCAG-3') (SEQ ID N0:18).
  • the sense primer 5'-GTCGTGTGCTTGATGTCATC-3' was used for first-strand cDNA synthesis followed by PCRTM amplification with the specific antisense primer (5'-GCTTCTCGCAATTAAAGCAG-3') (SEQ ID N0:18).
  • Total and poly(A) + RNA were prepared from bovine liver by the methods described above for cDNA synthesis. Radiolabeled probe (specific activity - 1 x 10 1 1 cpm/ ⁇ g) was prepared with a Pharmacia Oligolabeliing Kit using full-length 4414 base pair bovine liver cDNA as the template.
  • Total RNA (30 ⁇ g) and poly(A) + RNA (1 ⁇ g) were resolved by electrophoresis in a 1.5% agarose-formaldehyde denaturing gel and transferred to a NYTRANTM nylon membrane (Schleicher & Schuell). The filters were UV cross-linked, prehybridized for 30 min and then hybridized for two hours at 60° C in 10 ml QuickHybTM solution (Stratagene). The filters were washed under stringent conditions following the manufacturer's instructions.
  • the full-length 4414-bp bovine liver cDNA was constructed in the pCRII® vector downstream from the SP6 RNA polymerase promoter.
  • In vitro transcription and translation was conducted with the TNTTM SP6 coupled reticulocyte lysate system (Promega) using [J5S]methionine for labeling of the synthesized proteins.
  • the translated products were resolved by SDS-PAGE in an 8% polyacrylamide gel (Lu et al., 1993). The gels were vacuum-dried at 65° C and exposed to autoradiography film for 6 hr.
  • Fusion protein was eluted with buffer A containing 10 mM maltose and the eluent concentrated in an Amicon Ce ⁇ triprepTM 30 concentrator.
  • the maltose binding protein was cleaved from the expressed DPD using factor Xa according to manufacturer's instructions.
  • Generation of the pMal-c2/bovine DPD construct resulted in the incorporation of a short segment of polylinker from the pCRII® vector (CTGGAATTCGGCTT) (SEQ ID N0:21) to the 5' end of bovine DPD cDNA.
  • the deduced amino acid sequence was examined, utilizing the MacVector 4.1 Sequence Analysis software. Several protein motifs were identified in the translated sequence. These include a GDP/GTP binding site at position 1060, a 4-Fe/4-S binding site at position 1010 and a cAMP phosphorylation site at position 782 (Gilman, 1987; Otaka and Ooi, 1989).
  • bovine liver DPD cDNA was used as the probe in Northern analysis to determine the size and number of messages in both total and poly(A) + RNA from bovine liver (FIG. 3). With both types of RNA, a single band was observed with a size of about 4400 nucleotides. These results suggest that the complete cDNA has been isolated and that there is only a single gene transcript encoding bovine liver DPD. 5. In Vitro Transcription and Translation of Bovine Liver cDNA In vitro transcription and translation were used to verify that the cloned cDNA translated a protein equivalent in size to bovine liver DPD. This procedure was performed prior to bacterial expression of the cDNA to confirm that the open reading frame contained no errant stop codons.
  • the full-length cDNA was cloned, sequenced, and expressed in a bacterial cell line. Comparison to other sequences in the GenBank database verified that this is a unique sequence. The conclusion that the cDNA clone contained the entire coding region of bovine liver DPD is based on the following observations:
  • the open-reading frame codes for a protein consisting of 1025 amino acids (molecular mass 111, 688 daltons) corresponding to that of purified enzyme (the active form of the enzyme is a homodimer, made up of two 108 kDa subunits (Lu et al., 1993);
  • the iron sulfur binding site is consistent with data obtained from purified DPD (Shiotani and Weber, 1981; Lu et ai., 1992).
  • Biological activity of DPD expressed using the prokaryotic pMAL vector was evaluated for both DPD/pMAL constructs and for vector controls. While only the pMAL construct containing the bovine DPD cDNA generated immunoreactive enzyme, neither sample contained significant DPD activity. This suggests the possibility that expression of the fusion protein could alter the folding of DPD and generate inactive enzyme.
  • An expressio ⁇ system such as baculovirus, which provides post-translational processing, might be more appropriate to generate enzymatically active DPD.
  • bovine liver DPD (together with the completely translated amino acid sequence) should permit further studies including: elucidation of tertiary structure; binding with known cofactors; and specific interactions with inactivators of this enzyme. Furthermore, the availability of the bovine liver DPD cDNA should allow the isolation of the full-length cDNA from other species including human. This in turn should provide insight into the molecular basis of the altered DPD activity observed with the inherited (pharmacogenetic) disorder (Diasio et ai., 1988; Lu ef al., 1993).
  • the human lymphocyte ⁇ gt10 cDNA library was obtained from Clontech (Palo Alto, CA). Histopaque was purchased from the Sigma Chemical Co. (St. Louis, MO). The TA cloning kit was purchased from Invitrogen (San Diego, CA). Restriction enzymes and DNA-modifying enzymes were from New England Biolabs (Beverly, MA). The random primer labeling kit and microspin columns were from Pharmacia LKB Biotechnology Inc (Piscataway, NJ). The TNT coupled in vitro transcription and translation system, piasmid and lambda purification kits were purchased from Promega (Madison, Wl). Specific oligonucleotides were synthesized by Midland Certified Reagent Co.
  • RNAzol® B Biotecx, Houston, TX
  • the nucleotide sequence and deduced amino acid sequence of full length human lymphocyte DPD cDNA are shown in FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D.
  • the ATG start codon (shown in bold face) has the canonical flanking sequence for a translational start site with the customary GCC at position -3 to -1, and the standard G at position 4 (Kozak, 1991).
  • the complete human lymphocyte DPD cDNA sequence is 4356 base pairs long, contains a 48 nucleotide 5'-nontra ⁇ slated region, and an open-reading frame of 3075 bases encoding a 1025 amino acid protein.
  • a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal.
  • an immunogenic composition in accordance with the present invention
  • a wide range of animal species can be used for the production of antisera.
  • the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization.
  • a variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and i ⁇ traperitoneal).
  • the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved.
  • the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
  • NS-1 myeloma cell line also termed P3- NS-1-Ag4-1
  • Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
  • Krzyzek "Genetic transformation of maize cells by electroporation of cells pretreated with pectin degrading enzymes," U.S. Patent 5,384,253, 01/24/95. Kuby, "Immunology” 2nd Edition. W. H. Freeman & Company, New York, 1994.
  • Neote et al. Blood, 84:44, 1994.
  • Neote et al. Cell, 72:415-425, 1993.
  • AAG CAC ACG ACT CTT GGT GAG CGA GGA GCT CTC CGA GAA GCA ATG AGA 301 10 Lys His Thr Thr Leu Gly Glu Arg Gly Ala Leu Arg Glu Ala Met Arg en 65 70 75
  • ATC CCT CAG TTC CGG CTG CCA TAT 781 Val Gly Gly He Ser Thr Ser Glu He Pro Gin Phe Arg Leu Pro Tyr
  • GGC ACA GCC ATC AGA CCT ATT GCT TTG AGA GCT GTG ACC ACC ATT GCT 2413 Gly Thr Ala He Arg Pro lie Ala Leu Arg Ala Val Thr Thr He Ala 770 775 780
  • Ala Lys Met lie Phe Ser Asp Asn Pro Leu Gly Leu Thr Cys Gly Met 115 120 125
  • Gly Arg lie Val Ala Met Gin Phe Val Arg Thr Glu Gin Asp Glu Thr 405 410 415
  • Val Thr Glu Leu Lys Ala Asp Phe Pro Asp Asn lie Val He Ala Ser 625 630 635 640 He Met Cys Ser Tyr Asn Arg Asn Asp Trp Met Glu Leu Ser Arg Lys 645 650 655
  • GCT GGA GAC ACT GCC TTT GAC TGT GCA ACA TCT GCT CTA CGT TGT GGA 1113 Ala Gly Asp Thr Ala Phe Asp Cys Ala Thr Ser Ala Leu Arg Cys Gly 1365 1370 1375 1380
  • GCT GTC CCT GAG GAG ATG GAA CTT GCT AAG GAA GAA AAG TGT GAA TTT 1209 J 10 Ala Val Pro Glu Glu Met Glu Leu Ala Lys Glu Glu Lys Cys Glu Phe 1400 1405 1410
  • He Lys Ser Phe lie Thr Ser He Ala Asn Lys Asn Tyr Tyr Gly Ala 100 105 110 Ala Lys Met lie Phe Ser Asp Asn Pro Leu Gly Leu Thr Cys Gly Met 115 120 125
  • Ala lie Arg Pro He Ala Leu Arg Ala Val Thr Ser He Ala Arg Ala 770 775 780

Abstract

Procédés et compositions destinés à être utilisés pour détecter et quantifier la dihydropyrimidine (DPD) utile par exemple pour optimiser les doses de 5-fluoro-uracil données à des patients atteints de cancer. On décrit plus particulièrement des anticorps, y compris des anticorps monoclonaux, dirigés contre la forme humaine de DPD; des séquences d'ADN extraites de DPD d'origine bovine et humaine; des dispositifs immunologiques et de biologie moléculaire permettant de détecter la DPD; et des procédés de détermination de stratégies de traitement efficaces contre le cancer fondés sur les informations obtenues au sujet des taux de DPD. On décrit également une caractérisation moléculaire d'une lésion génétique induisant une carence en DPD chez l'homme et des procédés de diagnostic permettant d'effectuer le tri génétique de cette mutation pour les patients soumis à un traitement au 5-fluoro-uracil.
PCT/US1995/004567 1994-04-13 1995-04-13 Compositions de detection de la dihydropyrimidine deshydrogenase (dpd) et procedes d'utilisation associes WO1995028489A1 (fr)

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US22735794A 1994-07-19 1994-07-19

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US6787306B1 (en) * 1996-03-20 2004-09-07 The United States Of America As Represented By The Department Of Health And Human Services Methods and compositions for detecting dihydropyrimidine dehydrogenase splicing mutations
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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996008568A3 (fr) * 1994-09-12 1996-05-17 Us Health Clonage et expression d'adn complementaire codant pour la dihydropyrimidine deshydrogenase humaine
US5856454A (en) * 1994-09-12 1999-01-05 The United States Of America As Represented By The Department Of Health And Human Services CDNA for human and pig dihydropyrimidine dehydrogenase
WO1996008568A2 (fr) * 1994-09-12 1996-03-21 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Clonage et expression d'adn complementaire codant pour la dihydropyrimidine deshydrogenase humaine
EP2009106A3 (fr) * 1994-09-12 2009-03-18 THE UNITED STATES OF AMERICA, as represented by the Secretary of the Department of Health and Human Services Clonage et expression de l'ADNc pour la dihydropyrimidine déshydrogénase humaine
US7402387B2 (en) 1996-03-20 2008-07-22 The United States Of America As Represented By The Department Of Health And Human Services Methods and compositions for detecting dihydropyrimidine dehydrogenase splicing mutations
US6787306B1 (en) * 1996-03-20 2004-09-07 The United States Of America As Represented By The Department Of Health And Human Services Methods and compositions for detecting dihydropyrimidine dehydrogenase splicing mutations
FR2767528A1 (fr) * 1997-08-22 1999-02-26 Hoffmann La Roche Methode et materiaux immunologiques pour le dosage de la dihydropyrimidine deshydrogenase
US6232448B1 (en) * 1997-08-22 2001-05-15 Roche Diagnostics Corporation Immunological materials and methods for detecting dihydropyrimidine dehydrogenase
US6730488B2 (en) * 1997-08-22 2004-05-04 Roche Diagnostics Corporation Immunological materials and methods for detecting dihydropyrimidine dehydrogenase
WO2001036686A2 (fr) * 1999-11-15 2001-05-25 University Of Southern California Prediction de reponse therapeutique d'apres le polymorphisme genomique
WO2001036686A3 (fr) * 1999-11-15 2002-03-07 Univ Southern California Prediction de reponse therapeutique d'apres le polymorphisme genomique
US7049059B2 (en) 2000-12-01 2006-05-23 Response Genetics, Inc. Method of determining a chemotherapeutic regimen based on ERCC1 and TS expression
US7132238B2 (en) 2000-12-01 2006-11-07 Response Genetics, Inc. Method of determining a chemotherapeutic regimen based on ERCC1 expression
US7560543B2 (en) 2000-12-01 2009-07-14 Response Genetics, Inc. Method of determining a chemotherapeutic regimen based on ERCC1 and TS expression
US7732144B2 (en) 2000-12-01 2010-06-08 Response Genetics, Inc. Method of determining a chemotherapeutic regimen based on ERCC1 and TS expression
US8026062B2 (en) 2000-12-01 2011-09-27 Response Genetics, Inc. Method of determining a chemotherapeutic regimen by assaying gene expression in primary tumors
US8586311B2 (en) 2000-12-01 2013-11-19 Response Genetics, Inc. Method of determining a chemotherapeutic regimen based on ERCC1 and TS expression
US6956111B2 (en) 2001-03-02 2005-10-18 Response Genetics, Inc. Method of determining dihydropyrimidine dehydrogenase gene expression
US7005278B2 (en) 2001-03-02 2006-02-28 Danenberg Kathleen D Method of determining dihydropyrimidine dehydrogenase gene expression
US6905821B2 (en) 2001-03-02 2005-06-14 Response Genetics, Inc. Method of determining Dihydropyrimidine dehydrogenase gene expression
US7138507B2 (en) 2001-06-14 2006-11-21 Response Genetics, Inc. Method of determining a chemotherapeutic regimen based on glutathione-s transferase pi expression

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