PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Classification 6 ;
`C12N 15/54, 9/12, C12Q 1/48, 1/68,
`GOIN 33/50, C12N 15/11, CO7K 16/40,
`A61K 48/00 // CO7K 14/39
`
`(11) International Publication Number:
`
`WO 97/09433
`
`
`
`(43) International Publication Date:
`
`13 March 1997 (13.03.97)
`
`(21) International Application Number:
`
`PCT/GB96/02197
`
`(22) International Filing Date:
`
`6 September 1996 (06.09.96)
`
`(30) Priority Data:
`9518220.0
`
`6 September 1995 (06.09.95)
`
`GB
`
`(71) Applicant (for all designated States except US): MEDICAL
`RESEARCH COUNCIL [GB/GB}; 20 Park Crescent, Lon-
`don W1N 4AL (GB).
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): CARR, Antony, Michael
`[GB/GB]; MRC Cell Mutation Unit, University of Sussex,
`Falmer, Brighton BN1 9RR (GB).
`
`(74) Agents: WOODS, Geoffrey, Corlett et al.; JA. Kemp & Co.,
`14 South Square, Gray’s Inn, London WC1R 5LX (GB).
`
`(81) Designated States: AL, AM, AT, AU, AZ, BB, BG, BR, BY,
`CA, CH, CN,CU, CZ, DE, DK, EE, ES, FI, GB, GE, HU,
`IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT,
`LU, LV, MD, MG, MK, MN, MW,MX, NO,NZ,PL, PT,
`RO,RU,SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG,
`US, UZ, VN, ARIPOpatent (KE, LS, MW,SD, SZ, UG),
`Eurasian patent (AM, AZ, BY, Kae MD,RU,TI, TM),
`GR,IE, IT, LU, MC, NL,PT, srE), OAPI patent (BF, BI,
`European patent (AT, BE, CH,
`DE, DK, ES, FI, FR, GB,
`CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG).
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(54) Title: CELL-CYCLE CHECKPOINT GENES
`
`(57) Abstract
`
`This invention relates to a class of checkpoint genes and their polypeptide products which control progression through the cell cycle in
`eukaryotic cells. In particular this invention relates to Schizosaccharomyces pombe rad3 gene, to its human homologue (ATR) and to their
`encoded proteins. The invention further relates to assay methodsfor selecting compounds which modulate the activity of the polypeptide
`products of these checkpoint genes and the use of the selected compounds in anticancer therapy.
`
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`KELONIA EXHIBIT 1023
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`

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`SeWE
`
`
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`AM
`AT
`AU
`BB
`
`Armenia
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazi}
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cdte d’Ivoire
`Cameroon
`China
`Czechoslovakia
`Czech Republic
`Germany
`Denmark
`Estonia
`Spain
`Finland
`France
`Gabon
`
`GB
`GE
`GN
`GR
`HU
`
`IT
`
`Baas
`
`R58
`LK
`LR
`LT
`LU
`LV
`MC
`MD
`MG
`ML
`MN
`MR
`
`United Kingdom
`Georgia
`Guinea
`Greece
`Hungary
`Treland
`Ttaly
`Japan
`Kenya
`Kyrgystan
`Democratic People’s Republic
`of Korea
`Republic of Korea
`Kazakhstan
`Liechtenstein
`Sri Lanka
`Liberia
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`Mali
`Mongolia
`Mauritania
`
`MW
`MX
`NE
`NL
`
`NZ
`PL
`
`RO
`RU
`SD
`SE
`SG
`SI
`SK
`SN
`SZ
`TD
`TG
`TJ
`
`UA
`UG
`us
`UZ
`
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`
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`WO 97/09433
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`-l-
`
`Cell-cycle checkpoint genes
`
`Thepresentinventionrelates to a class of checkpoint genes which control progression through
`the cell cycle in eukaryotic cells.
`
`Background to the invention.
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`to the growth and maintenance of eukaryotic
`Control of the cell cycle is fundamental
`organisms, from yeasts to mammals. Eukaryotic cells have evolved control pathways, termed
`"checkpoints" which ensurethat individual Steps of the cell cycle are completed before the
`next step occurs. In response to DNA damage,cell survivalis increased both by direct DNA
`repair mechanisms and by delaying progression through the cell cycle. Depending on the
`position of the cell within the cycle at the time of irradiation, DNA damage in mammalian
`cells can prevent (a) passage from G1 into S phase, (b) progression through S phase or(c)
`passage from G2 into mitosis. Such checkpoints are thoughtto preventdeleterious events such
`as replication of damaged DNAand the Segregation of fragmented chromosomes during
`mitosis (Hartwell and Kastan, 1994).
`
`The rad3 gene of Schizosaccharomyces pombeis required for the checkpoints that respond
`to DNA damageandreplication blocks. Rad3 is a memberofthe lipid kinase subclass of
`kinases which possess regions having sequence homologyto the lipid kinase domain ofthe
`p110 subunit of phosphatidylinositol-3 kinase (PI-3 kinase). This subclass also includes the
`ATM protein defective in ataxia-telangiectasia patients. Cells from ataxia telangiectasia
`patients (AT cells) have lost the delay to S phase following irradiation andaresaid to display
`‘radio resistant DNA synthesis (Painter and Young, 1989). AT cells irradiated in S phase
`accumulate in G2 with lethal damage, presumably as a consequence of attempting to replicate
`damaged DNA.
`ATcells irradiated during G2 display a different phenotype: they do not
`arrest mitosis after DNA damage, and progress through mitosis with damaged DNA (Beamish
`and Lavin, 1994). Mutations at the A-T locus, to which the ATM gene has been mapped,
`thus result in disruption of several checkpoints required for an appropriate response to
`ionising radiation. Other members ofthis lipid kinase subclass include: Tellp (Greenwellet
`al. 1985), a gene involved in maintaining proper telomere Jength in Saccharomyces
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`cerevisiae, Estip; Mecip and the product of the Drosophila meianogaster mei-41 checkpoint
`gene (Hari er al. 1995).
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`Disclosure of the invention.
`
`Wehave analyzed the S. pombe rad3 gene and found thatit has a full length amino acid
`sequence of 2386 aminoacids, not the 1070 amino acids described by Seaton et al. 1992.
`We have determinedthat this is the direct homologue of S. cerevisiae Esrip, and thatit
`shares the same overall structure as the ATM gene. The C-terminal region of the rad3
`protein contains a lipid kinase domain, which is required for Rad3 function. We have shown
`that Rad3 is capable of self association. We have also identified a protein kinase activity
`associated with Rad3.
`
`Further, we have found a human homologueto rad3. This gene, which we have named ATR
`(ataxia and rad related), displays significantly higher homology to rad3 than it does to the
`ATM gene.
`
`The human ATR cDNAsequenceis set out as Seq. ID No. 1. The aminoacid sequence of
`the ORF from nucleotides 80 and 8011 is set out as Seq. ID No. 2.
`
`The DNAsequenceof the open reading frame (ORF) of rad3 is shown as Seq. ID. No. 3.
`The 2386 aminoacid translation of the gene (nucleotides 585 to 7742 of Seq. ID No.3) is
`shown as Seq. ID. No.4.
`
`Accordingly, in a first aspect, the invention provides the ATR protein of Seq. ID. 2 and
`homologues thereof, polypeptide fragments thereof, as well as antibodies capable of binding
`the ATR protein or polypeptide fragments thereof. ATR proteins. homologues and fragments
`thereof are referred to below as polypeptides of the invention.
`
`In another aspect, the present invention provides a polynucleotide in substantially isolated
`form capable of hybridising selectively to Seq.ID No 1 or to the complement(i.e. opposite
`strand) thereof. Also provided are polynucleotides encoding polypeptides of the invention.
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`A
`Such polynucleotides will be referred to as a polynucleotide of the invention.
`polynucleotides of the invention includes DNA of Seq.ID Nos 1 and fragments thereof
`capable ofselectively hybridising to this gene.
`
`In a further aspect, the invention provides recombinant vectors carrying a polynucleotide of
`the invention, including expression vectors, and methodsof growing such vectors inasuitable
`host cell, for example under conditions in which expression of a protein or polypeptide
`encoded by a sequence ofthe invention occurs.
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`In an additional aspect, the invention provides kits comprising polynucleotides, polypeptides
`or antibodies of the invention and methods of using such kits in diagnosing the presence of
`absence of ATR andits homologues,or variants thereof, including deleterious ATR mutants.
`
`The invention further provides assay methods for screening candidate substancesfor use as
`compounds forinhibiting or activating ATR activity, or the activity of mutated forms of ATR
`which are deficient in checkpoint activity. The invention also provides assay methods for
`screening candidate substancesforuse as compoundsforinhibiting interactions between ATR
`and other compoundsthat interact with ATR, including ATR itself.
`
`In a related aspect, the invention also provides a polynucleotide sequence of Seq. ID No. 3
`in substantially isolated form, andthe protein of Seq. ID No.4 in substantially isolated form,
`and novel fragments and variants thereof.
`
`Detailed description of the invention.
`
`A. Polynucleotides.
`
`Polynucleotides of the invention may comprise DNA or RNA. They may also be
`polynucleotides which include within them synthetic or modified nucleotides. A numberof
`different types of modification to oligonucleotides are known in the art. These include
`methylphosphonate and phosphorothioate backbones,addition of acridine or polylysine chains
`at the 3° and/or 5’ ends of the molecule. Forthe purposesofthe present invention,it is to
`be understood that the polynucleotides described herein may be modified by any method
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`available in the art. Such modifications may be carried out in order to enhancethe in vivo
`activity or lifespan of polynucleotides of the invention.
`
`Polynucleotides of the invention capable ofselectively hybridizing to the DNA of Seq. ID
`No. 1 will be generally at least 70%, preferably at least 80 or 90% and more preferably at
`least 95% homologousto the corresponding DNAof Seq. ID No. 1 overa region ofatleast
`20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous
`
`nucleotides.
`It is to be understood that skilled persons may, using routine techniques, make nucleotide
`substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of
`the invention to reflect the codon usage of any particular host organism in which the
`polypeptides of the invention are to be expressed.
`
`Any combination of the above mentioned degrees of homology and minimum sizes may be
`used to define polynucleotides of the invention, with the more stringent combinations (i.e.
`higher homology over longer lengths) being preferred. Thus for example a polynucleotide
`whichis at least 80% homologousover 25, preferably 30 nucleotides forms oneaspectofthe
`invention, as does a polynucleotide whichis at least 90% homologous over 40 nucleotides.
`
`Polynucleotides of the invention may be used to produce a primer, e.g. a PCR primer, a
`primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label
`by conventional means using radioactive or non-radioactive labels, or the polynucleotides may
`be cloned into vectors.
`Such primers, probes and other fragments will be at least 15,
`preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also
`€ncompassed by the term polynucleotides of the invention as used herein.
`
`.
`
`Polynucleotides such as a DNApolynucleotide and primers according to the invention may
`be produced recombinantly, synthetically, or by any means available to those ofskill in the
`art. They mayalso be cloned by standard techniques,
`
`In general, primers will be produced by synthetic means, involving a step wise manufacture
`of the desired nucleic acid sequence one nucleotide ata time. Techniques for accomplishing
`this using automated techniques are readily available in the art.
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`Longer polynucleotides will generally be produced using recombinant means, for example
`using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair
`of primers(e.g. of about 15-30 nucleotides) to a region of the ATR gene whichit is desired
`to clone, bringing the primersinto contact with mRNA or cDNAobtained from a human cell
`(e.g. a dividing cell such as a peripheral blood leukocyte), performing a polymerase chain
`reaction under conditions which bring about amplification of the desired region, isolating the
`amplified fragment(e.g. by purifying the reaction mixture on an agarose gel) and recovering
`the amplified DNA. The primers may be designed to contain suitable restriction enzyme
`recognition sites so that the amplified DNA can be clonedinto a suitable cloning vector.
`
`Such techniques may be used to obtain all or part of the ATR sequence described herein.
`Genomic clones containing the ATR geneandits introns and promoter regions may also be
`obtained in an analogous manner,starting with genomic DNA from a human cell,e.g. a liver
`
`cell.
`
`Although in general the techniques mentioned herein are well knownin the art, reference may
`be madein particular to Sambrookef al. (Molecular Cloning: A Laboratory Manual, 1989).
`
`Polynucieotides which are not 100% homologousto the sequences ofthe present invention
`but fall within the scope of the invention can be obtained in a number of ways.
`
`Other human allelic variants of the ATR sequence described herein may be obtained for
`example by probing genomic DNAlibraries made from a range of individuals, for example
`individuals from different populations.
`
`rats or rabbits), more
`In addition, other animal, particularily mammalian (e.g. mice,
`particularly primate, homologues of ATR may be obtained and such homologues and
`fragments thereof in general will be capable of selectively hybridizing to Seq. ID No. 1.
`Such sequences may be obtained by probing cDNAlibraries made from dividing cells or
`lissues or genomic DNAlibraries from other animal species, and probing suchlibraries with
`probes comprising all or part of Seq. ID. 1 under conditions of medium to high stringency
`(for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about
`60°C).
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`Allelic variants and species homologues may also be obtained using degenerate PCR which
`will use primers designed to target sequences within the variants and homologues encoding
`conserved amino acid sequences. Conserved sequences can be predicted from aligning the
`ATR amino acid sequence with that of rad3.
`The primers will contain one or more
`degenerate positions and will be used at stringency conditions lower than those used for
`cloning sequences with single sequence primers against known sequences.
`
`Alternatively, such polynucleotides may be obtained by site directed mutagenesis of the ATR
`sequencesorallelic variants thereof. This may be useful where for example silent codon
`changes are required to sequences to optimise codonpreferences for a particular host cell in
`which the polynucleotide sequencesare being expressed. Other sequence changes may be
`desired in order to introduce restriction enzyme recognition sites, or to alter the property or
`function of the polypeptides encoded by the polynucleotides. Further changes may be
`desirable to represent particular coding changes found in ATR which giverise to mutant ATR
`genes which havelost the checkpoint function. Probes based on such changes can be used
`as diagnostic probes to detect such ATR mutants.
`
`The invention further provides double stranded polynucleotides comprising a polynucleotide
`of the invention and its complement.
`
`Polynucleotides or primers of the invention may carry a revealing label. Suitable labels
`include radioisotopes such as *P or *S, enzymelabels, or other protein labels suchasbiotin.
`Such labels may be added to polynucleotides or primers of the invention and may bedetected
`using by techniques knownperse.
`
`Polynucieotides or primers of the invention or fragments thereof labelled or unlabelled may
`be used by a person skilled in the art in nucleic acid-based tests for detecting or sequencing
`ATR in the human or animalbody.
`
`Such tests for detecting generally comprise bringing a human or animal body sample
`containing DNA or RNAinto contact with a probe comprising a polynucleotide or primerof
`the invention under hybridizing conditions and detecting any duplex formed betweenthe probe
`and nucleic acid in the sample. Such detection may be achieved using techniques such as
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`PCRor by immobilizing the probe onasolid support, removing nucleic acid in the sample
`whichis not hybridized to the probe, and then detecting nucleic acid which has hybridized
`to the probe. Alternatively, the sample nucleic acid may be immobilized ona solid support,
`and the amountof probe bound to such a Support can be detected. Suitable assay methods
`of this any other formats can be foundin for example WO89/03891 and WO90/13667.
`
`Tests for sequencing ATR include bringing a human oranimal body sample containing target
`DNA or RNA into contact with a probe comprising a polynucleotide or primer of the
`invention under hybridizing conditions and determining the sequence by, for example the
`Sanger dideoxy chain termination method (see Sambrook efal.).
`
`in the presence of suitable reagents, the
`Such a method generally comprises elongating,
`primer by synthesis of a strand complementary to the target DNA or RNA and selectively
`terminating the elongation reaction at one or more of an A, C, G or T/U residue: allowing
`Strand elongation and termination reaction to occur; separating out according to size the
`elongated products to determinethe sequenceofthe nucleotides at which selective termination
`has occurred. Suitable reagents include a DNA polymerase enzyme, the deoxynucleotides
`dATP, dCTP, dGTP and dTTP,a buffer and ATP. Dideoxynucleotidesare usedforselective
`
`termination.
`
`Tests for detecting or sequencing ATR in the human or animal body may be used to
`determine ATR sequences within cells in individuals whohave, or are Suspected to have, an
`altered ATR gene sequence, for example within cancercells including leukaemic cells and
`solid tumours such as breast, ovary, lung, colon, pancreas,testes, liver, brain, muscle and
`‘bone tumours.
`
`In addition, the discovery of ATR will allow the role of this gene in hereditary diseases to
`be investigated,
`in a manner analogous to the ATM gene.
`In general,
`this will involve
`establishing the status of ATR (e.g using PCR sequence analysis) in cells derived from
`patients with diseases that may be connected with damage to replicating cells, e.g. familial
`Predisposition to cancer, chromosome breakage or instability phenotype or repair-damage
`sensitivity phenotype.
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`The probes of the invention may conveniently be packaged in the form ofa test kit in a
`Suitable container.
`In suchkits the probe may be bound to a solid support where the assay
`format for which thekit is designed requires such binding. The kit may also contain suitable
`reagents for treating the sample to be probed, hybridizing the probe to nucleic acid in the
`sample, control reagents, instructions, and the like.
`
`invention also provides polynucleotides encoding the polypeptides of the
`The present
`invention described below. Because such polynucleotides will be useful as sequences for
`recombinant production of polypeptides of the invention, it is not necessary for them to be
`selectively hybridizable to the sequence Seq. ID No. 1, although this will generally be
`desirable. Otherwise, such polynucleotides may be labelled, used, and made as described
`aboveif desired. Polypeptides of the invention are described below.
`
`Particularly preferred polynucleotides ofthe invention are those derived from the lipid kinase
`domain of ATR,its allelic variants and species homologues. The lipid kinase domain is
`represented by nucleotides 7054 to 8011 of Seq. ID. 1. Polynucleotides of the invention
`which comprise this domain are particularly preferred. The term "lipid kinase domain"refers
`to a domain which has homology to other known lipid kinases, in particular the p110 subunit
`of PI-3 kinase, as determined by sequence alignments.
`
`Other preferred polynucleotides of the invention those which comprise nucleotides encoding
`amino acids 181 to 302 of Seq. ID No. 2 (nucleotides 620 to 985 of Seq. ID No. 1), which
`is believed to be a leucine zipper region, a putative site of protein-protein interaction, and
`amino acids 1358 to 1366 (nucleotides 4151 to 4177), which is also conserved.
`
`In an additional aspect, polynucleotides of the invention include those of Seq. ID No. 3 and
`fragments thereof capable of selectively hybridizing to this sequence other than the fragment
`consisting of nucleotides 2482 to 6599 in which the following changes have been made:
`Deletion of residues 2499, 2501, 2507 & 2509; insertion of C between 5918/5919.
`
`Particularly preferred fragments include those comprising residues 6826 to 7334 (the lipid
`kinase domain) and theleucine zipper regions 1476 to 1625 and 2310to 2357. Additionally,
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`the fragment comprising the conserved region 3891 to 3917 is preferred. Such polypeptides
`
`and fragments may be made and used as described above.
`
`B. Polypeptides.
`
`Polypeptides of the invention include polypeptides in substantially isolated form which
`
`comprise the sequenceset out in Seq ID No.2.
`
`Polypeptides further include variants of such sequences, including naturally occurringallelic
`
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`variants and synthetic variants which are substantially homologousto said polypeptides.
`
`In
`
`this context, substantial homology is regarded as a sequence which has at least 70%, e.g.
`
`80% or 90% amino acid homology (identity) over 30 amino acids with the sequence of Seq.
`
`ID No.2 except for the lipid kinase domain and C-terminal portion (residues 2326 to 2644)
`
`wheresubstantial homologyis regardedas at least 80% homology, preferably 90% homology
`
`15
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`(identity) over 50 aminoacids.
`
`Polypeptides also include other those encoding ATR homologuesfrom other species including
`
`animals such as mammals (e.g. mice, rats or rabbits), especially primates, and variants
`
`thereof as defined above.
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`
`Polypeptides of the invention also include fragments of the above mentioned full length
`
`polypeptides and variants thereof, including fragments of the sequenceset out in Seq. ID No.
`
`2.
`
`25
`
`Preferred fragments include those which include an epitope, especially an epitope. Suitable
`fragments will be at least about 5, e.g. 10, 12, 15 or 20 amino acids in size. Polypeptide
`
`fragments of the ATR protein and allelic and species variants thereof may contain one or
`
`more (e.g. 2, 3, 5, or 10) substimtions, deletions or insertions,
`
`including conserved
`
`substitutions.
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`Conserved substitutions may be made accordingto the following table indicates conservative
`
`substitutions, where amino acids on the same block in the second column and preferably in
`
`the same line in the third column maybesubstituted for each other:
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`Non-polar
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`GAP
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`
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`
`
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`Polar - uncharged
`
`CSTM
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`5
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`Variants of the polypeptides of the invention may also comprise polypeptides wherein one or
`
`more of the specified (i.e., naturally encoded) aminoacids is deleted or replaced or wherein
`
`one or more nonspecified amino acids are added: (1) without loss of the kinase activity
`
`specific to the polypeptides of the invention; or (2) with disablement of the kinase activity
`
`specific to the polypeptides of the invention; or (3) with disablementof the ability to interact
`
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`with members or regulators of the cell cycle checkpoint pathway.
`
`Epitopes may be determined either by techniques such as peptide scanning techniques as
`
`described by Geysen et al. Mol. Immunol., 23; 709-715 (1986).
`
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`Polypeptides of the invention may be in a substantially isolated form.
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`It will be understood
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`that the polypeptide may be mixed with carriers or diluents which will not interfere with the
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`intended purpose of the polypeptide and still be regarded as substantially isolated. A
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`polypeptide of the invention mayalso be in a substantially purified form, in whichcaseit will
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`generally comprise the polypeptide in a preparation in which more than 90%, e.g. 95%, 98%
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`or 99% of the polypeptide in the preparation is a polypeptide of the invention. Polypeptides
`of the invention may be modified for example by the addition of Histidine residues to assist
`their purification or by the addition of a signal sequence to promote their secretion from a
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`cell.
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`_A polypeptide of the invention may be labelled with a revealing label. The revealing label
`may be any suitable label which allowsthe polypeptide to be detected. Suitable labels include
`radioisotopes, ¢.g.
`'°I, enzymes, antibodies, polynucleotides and linkers
`such asbiotin.
`Labelled polypeptides of the invention may be used in diagnostic procedures such as
`immunoassays in order to determine the amountof a polypeptide ofthe invention in a sample.
`Polypeptides or labelled polypeptides of the invention may also be used in serological or cell
`mediated immune assays for the detection of immune reactivity to said polypeptides in
`animals and humans using standard protocols.
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`A polypeptide or labelled polypeptide of the invention or fragment thereof mayalso befixed
`to a solid phase, for example the surface of an immunoassay well or dipstick.
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`Such labelled and/or immobilized polypeptides may be packaged into kits in a suitable
`container along with suitable reagents, controls, instructions and the like.
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`Such polypeptides and kits may be used in methods of detection of antibodies to the ATR
`protein orits allelic or species variants by immunoassay.
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`Immunoassay methodsare well known in the art and will generally comprise:
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`(a)_providing a polypeptide comprising an epitope bindable by an antibody against
`said protein;
`incubating a biological sample with said polypeptide under conditions which
`allow for the formation of an antibody-antigen complex; and
`determining whether antibody-antigen complex comprising said polypeptide is
`formed.
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`(b)
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`(c)
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`Polypeptides of the invention may be may by synthetic means (e.g. as described by Geysen
`et al.) or recombinantly, as described below.
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`Particularly preferred polypeptides of the invention include those spanningor within the lipid
`kinase domain, namely from amino acids 2326 to 2644 of Seq.
`ID. 2. or sequences
`Substantially homologous thereto.
`Fragments as defined above from this region are
`particularly preferred. The polypeptides and fragments thereof may contain amino acid
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`alterations as defined above, including substitutions at one or more of positions 2475, 2480
`and 2494, which correspond to the positions of the rad3 substitutions described in the
`examples below. Preferred substitutions include D2475A, N2480K and D2494E.
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`Polypeptides of the invention may be usedin in vitro or in vivo cell culture systems to study
`the role of ATR as a checkpoint gene. For example, truncated or modified (e.g. modified
`in the lipid kinase domain) ATRs may be introduced into a cell to disrupt the normal
`checkpoint functions which occur in the cell.
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`The polypeptides of the invention may be introducedinto the cell by in situ expression of the
`polypeptide from a recombinant expression vector (see below). The expression vector
`optionally carries an inducible promoter to control the expression of the polypeptide.
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`The use of mammalian host cells is expected to provide for such post-translational
`modifications (e.g., myristolation, glycosylation, truncation, lapidation and tyrosine, serine
`or threonine phosphorylation) as may be needed to confer optimal biological activity on
`recombinant expression products of the invention.
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`Suchcell culture systems in which polypeptide of the invention are expressed may be used
`in assay systems to identify candidate substances which interfere or enhance checkpoint
`functions in the cell (see below).
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`In an additional aspect, polypeptides of the invention include the protein of Seq. ID No. 4
`_ and fragments thereof from the region other than the fragment consisting of amino acids 713
`to 1778. Particularly preferred fragments include those comprising residues 2082 to 2386
`(the lipid kinase domain) and the leucine zipper regions 298 to 347 and 576 to 591.
`Additionally, the fragment comprising the conserved region 1103 to 1111 is preferred. Such
`polypeptides and fragments may be made and used as described above.
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`The invention also provides polypeptides substantially homologousto the protein of Seq. ID
`No. 4, and fragments thereof. In this context, substantial homology is regarded as a sequence
`which has at least 70%, e.g. 80% or 90% amino acid homology (identity) over 30 amino
`acids with the sequence of Seq. ID No. 4 except for the lipid kinase domain and C-terminal
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`portion (residues 2082 to 2386) where substantial homology is regarded as at least 80%,
`preferably at least 90% homology (identity) over 50 amino acids.
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`C.. Vectors.
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`Polynucieotides of the invention can be incorporated into a recombinant replicable vector.
`The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a
`further embodiment,
`the invention provides a method of making polynucleotides of the
`invention by introducing a polynucleotide ofthe invention into a replicable vector, introducing
`the vector into a compatible hostcell, and growing the host cell under conditions which bring
`aboutreplication of the vector. The vector may be recovered from the host cell. Suitable
`host cells are described below in connection with expression vectors.
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`D. Expression Vectors.
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`Preferably, a polynucleotide of the invention in a vector is operably linked to a control
`sequence which is capable of providing for the expression of the coding sequence by the host
`cell, i.e. the vector is an expression vector.
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`The term "operably linked" refers to a juxtaposition wherein the components described are
`in a relationship permitting them to function in their intended manner. A control sequence
`“operably linked" to a coding sequenceis ligated in such a way that expression of the coding
`sequence is achieved under condition compatible with the control sequences.
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`Such vectors may be transformed into a suitable host cell as described above to provide for
`expression of a polypeptide of the invention. Thus, ina further aspectthe invention provides
`a process for preparing polypeptides according to the invention which comprises cultivating
`a host cell transformed or transfected with an expression vector as described above under
`conditions to provide for expression by the vector of a coding sequence encoding the
`polypeptides, and recovering the expressed polypeptides.
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`The vectors may be for example, plasmid, virus or phage vectors provided with anorigin of
`replication, optionally a promoter for the expression ofthe said polynucleotide and optionally
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`a regulator of the promoter. The vectors may contain one or moreselectable marker genes,
`for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin
`resistance gene for a mammalian vector. Vectors may be usedin vitro, for example for the
`production of RNA orused to transfect or transformahostcell. The vector may also be
`adapted to be used in vivo, for example in a method of gene therapy.
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`A further embodimentofthe invention provides hostcells transformedor