GB2421731A - Mutations in c-Src - Google Patents

Mutations in c-Src Download PDF

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GB2421731A
GB2421731A GB0526018A GB0526018A GB2421731A GB 2421731 A GB2421731 A GB 2421731A GB 0526018 A GB0526018 A GB 0526018A GB 0526018 A GB0526018 A GB 0526018A GB 2421731 A GB2421731 A GB 2421731A
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src
seq
positions
mutation
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John Craig Smith
Emma Donald
Rose Mccormack
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AstraZeneca AB
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism

Abstract

The present invention relates to detection of mutations in the kinase domain of c-Src in a tumour sample from a patient. Also claimed are mutant c-Src polynucleotides comprising mutations at position 829, 1032, 1270, 1342, 1359, 1372 or 1486, and mutant c-Src polypeptides comprising mutations at residues 277, 344, 424, 448, 458 or 496.

Description

METHOD
The present invention relates to detection of mutations in the kinase domain of c- Src in a tumour sample from a patient.
Protein kinases are crucial components of the signal transduction pathways that are central to cellular and biological functions. Inappropriate or uncontrolled activation, arising from over expression or mutation of many kinases has been shown to result in uncontrolled cell growth. pp60t tyrosine kinase (otherwise known as c-Src) is a non- receptor protein tyrosine kinase and was first identified as the cellular form 0f60VSrC tyrosine kinase (otherwise known as v-Src), the transforming gene product of the avian tumour virus Rous sarcoma virus. c- Src is a multifunctional protein involved in the regulation of a variety of processes, including proliferation, differentiation, survival, motility and angiogenesis.
The differential response of patients to chemotherapy treatments indicates that there is a need to find methods of predicting which treatment regimes best suit a particular patient. In addition, there is a need to identify methods that can predict a patients' response to a drug based on the results of a test for such factors that indicates whether the patient is likely to respond to treatment or to be resistant to treatment.
An increasing body of evidence suggests that patients' responses to numerous drugs may be related to a patients' genetic, genomic, proteomic and/or biochemical profile and that determination of the genetic factors that influence, for example, response to a particular drug could be used to provide a patient with a personalised treatment regime.
Such personalised treatment regimes offer the potential to maximise therapeutic benefit to the patient, whilst minimising, for example side effects that may be associated with alternative and less effective treatment regimes.
Studies have sought to identify mutations in c-Src which may influence response to a drug, however reports of mutations in c-Src in human tumours are extremely rare and when overexpressed in normal cells c-Src is non- or weakly oncogenic. The first reported activating c-Src mutation was a C-terminally truncated c-Src arising from a C-' T transition mutation at codon 531 which was detected in small subsets of colon and endometrial cancers (Irby, 1999). However, other studies have failed to detect such mutations in ovarian carcinomas, endometrial stromal sarcoma, rectal or colon cancer * *** S * * III * * . ** * S S S I I I * S IS * S S I S I S * . . S I S S 555 *** . S patients indicating that the codon 531 mutation is a very rare cause of c-Src activation in colorectal cancer (Nilbert, 2000; Laghi, 2001; Sugimura, 2000; Wang, 2000). Given the paucity of examples of mutational activation in human cancers, the involvement of c- Src in the etiology and progression of human cancers has been doubted for many years (Ischzawar, 2004, Tian, 2001).
The present invention is based on the discovery of mutations in the kinase domain of c-Src in tumour samples from patients; no such mutations were known beforehand.
In one aspect, the invention provides a method which comprises determining the presence of a mutation in the kinase domain of c-Src in a tumour sample from a patient wherein the mutation differs at one nucleic acid base position from SEQ ID NO: I and/or at one amino acid residue position from SEQ ID NO: 2. Preferably the method comprises determining the presence of a mutation in the kinase domain of c-Src in a tumour sample from a patient at any one of the following positions as defined in SEQ ID NO: 1: positions 744 to 1554 inclusive, and/or at any one of the following positions as defined in SEQ ID NO:2: positions 248 to 518 inclusive.
c-Src is known to share regions of homology with both the epidermal growth factor receptor (EGFR) and the oncogene abi, both of which are the targets of studies for anti- cancer therapeutic agents. Given the lack of mutations identified in c- Src despite the work of a number of groups it is surprising that we have identified mutations in c-Src within homologous regions occurring at or near to the same relative position as known mutations in EGFR and abl. Pharmacokinetic studies of anti-cancer therapeutic agents have identified a number of mutations in EGFR and abl which effect the likelihood and magnitude of patient response, for example see Lynch 2004, Paez 2004, La Rosee, 2002.
It is contemplated that the mutations in c-Src described herein will therefore affect the response of a patient to a c-Src drug.
All the mutations in c-Src described herein have been identified in the kinase domain (also known as the ATP-binding or catalytic domain). Without wishing to be bound by theoretical considerations it is considered that at least some such mutations are likely to affect the ATP binding and kinase activity of c-Src. To illustrate, Met 344, which we have found to be mutated in c-Src, is involved in hydrogen bonding to the quinazoline moiety of the AZ series of c-Src inhibitors and to PD173955 (PIe, 2004).
* I.. * * S *I* * * I IS S * S S
S S S S S IS
* I S S S U S * S S S * U S * I.. S 555 SI* * S Preferably the invention provides a method which comprises determining the presence of a mutation in the kinase domain of c-Src in a tumour sample from a patient at any one of the following positions as defined in SEQ ID NO:1: positions 744 to 1554 inclusive, and/or at any one of the following positions as defined in SEQ ID NO:2: positions 248 to 518 inclusive. Preferably the method comprises determining the presence of a mutation in the kinase domain of c-Src in a tumour sample from a patient wherein the mutation is selected from any one of the following at positions as defined in SEQ ID NO:1: position 829 is not G; position 1032 is not G; position 1270 is not G; position 1342 is not G; position 1359 is not C; or position 1372 is not C; or position 1486 is not G, andlor any one of the following at positions as defined in SEQ ID NO:2: position 277 is not glycine; position 344 is not methionine; position 424 is not glycine; position 448 is not valine; or position 458 is not leucine; or position 496 is not aspartic acid.
Preferably the method comprises determining the presence of a mutation in the kinase domain of c-Src in a tumour sample from a patient wherein the mutation is selected from any one of the following at positions as defined in SEQ ID NO:1: position 829 is A; position 1032 is A; position 1270 is A; position 1342 is A; position 1359 is A; or position 1372 is T; or position 1486 is A, andlor any one of the following at positions as defined in SEQ ID NO:2: position 277 is serine; position 344 is isoleucine; position 424 is serine; position 448 is methionine; or position 458 is phenylalanine; or position 496 is asparagine.
Another aspect of the invention provides a method as described hereinabove to assess the pharmacogenetics of a c-Src drug.
Preferably the method for detection of a nucleic acid mutation is selected from amplification refractory mutation system and restriction fragment length polymorphism.
Preferably the invention provides a primer or an oligonucleotide probe capable of detecting a mutation in the kinase domain of c-Src in a tumour sample from a patient at any one of the following positions as defined in SEQ ID NO: 1: positions 744 to 1554 inclusive. More preferably the invention provides a primer or an oligonucleotide probe capable of detecting a mutation in the kinase domain of c-Src in a tumour sample from a patient at any one of the following positions as defined in SEQ ID NO: 1: 829, 1032, 1270, 1342, 1359, 1372 or 1486.
Preferably the invention provides a mutant human c-Src polynucleotide comprising any one of the following nucleic acid bases at positions as defined in SEQ ID NO: 1: * I., * I * *t.
* S a.. * * S S S S * S p 55 * S S I I I * * * S S S S * S *s* a a.. s.. * * positions 744 to 1554 inclusive, or a fragment thereof, comprising at least 20 bases provided that the fragment comprises at least one of the mutant bases at positions 744 to 1554 inclusive. Preferably the invention provides a mutant human c-Src polynucleotide comprising any one of the following nucleic acid bases at positions as defined in SEQ ID NO: 1: position 829 is A; position 1032 is A; position 1270 is A; position 1342 is A; position 1359 is A; or position 1372 is T; or position 1486 is A, or a fragment thereof, comprising at least 20 bases provided that the fragment comprises at least one of the mutant bases at positions 829, 1032, 1270, 1342, 1359, 1372, 1486.
Preferably the invention provides a mutant human c-Src polypeptide comprising any one of the following amino acid residues at positions as defined in SEQ ID NO:2: positions 248 to 518 inclusive, or a fragment thereof, comprising at least 10 residues provided that the fragment comprises at least one of the mutant residues at positions 248 to 518 inclusive. Preferably the invention provides a mutant human c-Src polypeptide comprising any one of the following amino acid residues at positions as defined in SEQ ID NO:2: position 277 is serine; position 344 is isoleucine; position 424 is serine; position 448 is methionine; or position 458 is phenylalanine; or position 496 is asparagine, or a fragment thereof, comprising at least 10 residues provided that the fragment comprises at least one of the mutant residues at positions 277, 344, 424, 448, 458 or 496.
Another aspect the invention provides an antibody specific for the mutant human c- Src polypeptides as defined hereinbefore.
Another aspect of the invention provides a kit for detection of a mutation in c-Src comprising an antibody or a primer or oligonucleotide probe.
Preferably the tumour is selected from the group consisting of leukaemia, multiple myeloma, lymphoma, and also solid tumours, for example bile duct, bone, bladder, brain, CNS, glioblastoma, breast, colorectal, cervical, endometrial, gastric, head, neck, hepatic, lung, muscle, neuronal, oesophageal, ovarian, pancreatic, pleural membrane, peritoneal membrane, prostate, renal, skin, testicular, thyroid, uterine and vulval.
Another aspect of the invention provides a method as described hereinbefore which may be used to predict the likelihood that a patient who is a candidate for treatment with a c-Src drug will respond to said treatment. Preferably the invention provides a method for determining the presence of a mutation in the kinase domain of c-Src in a tumour sample from a patient at the following position as defined in SEQ ID NO: 1; position 1032 is not G, **I * b * *SI * S S 1* 5 S * a S S I S * * I. e S 5 * * * * 5 S S S * S S ISS S 555 uS * S and/or at the following position as defined in SEQ ID NO:2: position 344 is not methionine, whereby to predict an increased likelihood of response to the c-Src drug.
More preferably the invention provides a method for determining the presence of a mutation in the kinase domain of c-Src in a tumour sample from a patient at the following position as defined in SEQ ID NO: I; position 1032 is A, and/or at the following position as defined in SEQ ID NO:2: position 344 is isoleucine, whereby to predict an increased likelihood of response to the c-Src drug.
Another aspect of the invention provides a method as described hereinbefore which may be used to predict the likelihood that a patient who is a candidate for treatment with a c-Src drug will respond to said treatment. Preferably the invention provides a method for determining the presence of a mutation in the kinase domain of c-Src in a tumour sample from a patient at the following position as defined in SEQ ID NO:1; position 1032 is not G, and/or at the following position as defined in SEQ ID NO:2: position 344 is not methionine, whereby to predict a decreased likelihood of response to the c-Src drug. More preferably the invention provides a method for determining the presence of a mutation in the kinase domain of c-Src in a tumour sample from a patient at the following position as defined in SEQ ID NO: 1; position 1032 is A, and/or at the following position as defined in SEQ ID NO:2: position 344 is isoleucine, whereby to predict a decreased likelihood of response to the c-Src drug.
According to another aspect of the invention, there is provided a method of treating a patient in need of treatment with a c-Src drug, comprising determining the presence of a mutation in the kinase domain of c-Src in a tumour sample from the patient according to the method as described hereinbefore and administering an effective amount of the drug.
According to another aspect of the invention, there is provided use of a c-Src drug in preparation of a medicament for treating a disease in a patient wherein the presence of a mutation in the kinase domain of c-Src in a tumour sample from the patient has been determined according to the method as described hereinbefore.
According to another aspect, the invention permits the prediction of sensitivity of tumours to anti-cancer therapeutic agents based on the sequence of a given cellular protein or encoding gene. Hence, the suitability of tumour patients for treatment with such therapeutic agents can be assessed by determining the gene or protein sequence in a tumour tissue sample.
4.4, 4 4 S I Is q * * * S I I * p * - I * S 4 * * I * I I s** II. I..Ia I,. * The present invention permits the improved selection of a patient, who is a candidate for treatment with an anti-cancer therapeutic agent, in order to predict an increased likelihood of response to the therapeutic agent.
Preferably the c-Src drug is an inhibitor of the Src family of nonreceptor tyrosine kinases. Said inhibitor may be specific or selective to the Src family of non-receptor tyrosine kinases, or may also inhibit other proteins, for example the oncogene abl. In a preferred embodiment the c-Src inhibitor is a c-Src kinase inhibitor.
In a more preferred embodiment the c-Src inhibitor may be selected from the quinazoline derivatives disclosed in International Patent Applications WO 01/94341, WO 02/16352, WO 02/30924, WO 02/30926, WO 02/34744, WO 02/085895, WO 02/092577, WO 02/092578, WO 02/092579 and the quinoline derivatives described in WO 03/008409, WO 03/047584 and WO 03/048 159 and the quinazoline derivatives described in co- pending International Application PCT/GBO3/04703 (arising from European Patent Application No. 02292736.2) and PCT/GBO3/290900. In an alternative embodiment the c- Src inhibitor may be selected from the 4-anilino-3-cyanoquinoline c-Src inhibitor known as SKI 606 (Cancer Research, 2003, 63, 375), or the compounds described in J Bone Mineral Research, 1999, 14 (Suppl. 1), S487, Molecular Cell, 1999, 3, 639-647, Journal Medicinal Chemistry, 1997, 40, 2296-2303, Journal Medicinal Chemistry, 1998, 41, 3276- 3292 and Bioorganic & Medicinal Chemistry Letters, 2002, 12, 1361 and 3153. In an alternative embodiment the c-Src inhibitor may be selected from the compounds described in, for example, International Patent Applications WO 96/10028, WO 97/0713 1, WO 97/08193, WO 97/16452, WO 97/28161, WO 97/32879 and WO 97/49706. In an alternative embodiment the c- Src inhibitor may be selected from compounds described in, for example, International Patent Application WO 03/0 13540 [particularly the compounds disclosed therein by way of Fonnulae Ito VIII and compounds based on Formulae VII and VIII but wherein the 2,6-dimethylphenyl group is replaced by a 2,6- dichlorophenyl or a 2- chloro-6-methylphenyl group]. In an alternative embodiment the c-Src inhibitor may be selected from the compounds described in J Med Chem 2004, 47, 87 1-888. Preferably the c-Src inhibitor is selected from AZDO53O, PD 173955, PD180970, Glivec (ST1571), CGP76030 or BMS-354825. More preferably the c-Src tyrosine kinase inhibitor is AZDO53O.
a q,, * * a a, * e * , a V a a * p, * , a p * * * a, S I S IS. I all 4!S S S We contemplate that c-Src drugs may be used as monotherapy or in combination with other drugs of the same or different classes. The present invention is also useful in adjuvant, or as a first-line, therapy.
The present invention is particularly suitable for use in predicting the response to the c-Src drug as described hereinbefore, in those patients or a patient population with a tumour which is dependent alone, or in part, on c-Src. Such tumours include, for example, non-solid tumours such as leukaemia, multiple myeloma or lymphoma, and also solid tumours, for example bile duct, bone, bladder, brainlCNS, glioblastoma, breast, colorectal, cervical, endometrial, gastric, head and neck, hepatic, lung, muscle, neuronal, oesophageal, ovarian, pancreatic, pleural/peritoneal membranes, prostate, renal, skin, testicular, thyroid, uterine and vulval tumours. In a more preferred embodiment the present invention is particularly suitable for use in predicting the response to one of the cSrc drugs as described hereinbefore in patients with colorectal or lung tumours.
In another aspect the invention provides a kit comprising an antibody specific for a mutant human c-Src polypeptide or a primer or oligonucleotide probe as described hereinbefore, for use in a method of predicting the responsiveness of a patient or patient population with a tumour as described hereinbefore.
In another aspect there is provided a method for determining the presence of a mutation in the kinase domain of c-Src in a sample from a patient with a somatic mutation, for example a germ line mutation such as osteoporosis.
The invention is illustrated by the following non-limiting examples. General molecular biology techniques are described in "Current Protocols in Molecular Biology Volume 1-3, edited by F M Asubel, R Brent and R E Kingston; published by John Wiley, 1998 and Sambrook, J. and Russell, D.W., Molecular Cloning: A Laboratory Manual, the Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
Example 1 - DNA extraction from slide section Volumes are given for extraction of one section. Region of tumour was identified by histopathology on one section and isolated from adjacent sections by scraping relevant area from slide into an eppendorf tube. The material from a 20 micron section was resuspended in 100 L 0.5% Tween-20 (Sigma Aldrich), heated to 90 C for 10 minutes q *q* , * I Ifs * I I II I * * i S S I I S Its, I 5 5 II * a * * SSI ,* I 115 ItS S S then cooled to 55 C. Proteinase K (2 jtL, lOmg!mL (Fluka)) was added to the suspension, the solution was mixed and incubated at 55 C for 3 hours with occasional mixing. Chelex- (100 tL, 5% in Tris EDTA (Sigma)) was added and the suspension was incubated at 99 C for 10 minutes. The extracted DNA was recovered by centrifugation at 10500 x g for 15 minutes, the solution below the wax layer which formed was transferred to a clean tube.
The solution was heated to 45 C before adding chloroform (100tL (Sigma)). The suspension was mixed before centrifugation at 10500 x g, DNA was then recovered from the upper aqueous layer by ethanol precipitation. The DNA pellet was rinsed in 70% ethanol, recovered by centrifugation, air dried and dissolved in water (50 pL).
Example 2- PCR amp4/lcation of the kinase domain Table I PCR primers used to amplify the kinase domain of c-Src (EMBL Accession number AL133293) Exon Length Tag Untagged Position in PCR conditions number sequence kLl 33293 Exonli 21 M13F SEQIDNO:09 W178-20198 Tm61 23 M13R SEQ IDNO:1O 20398-20420 Exon 12 22 M13F SEQ ID NO: 11 22489-22510 Touchdown 63 23 M13R SEQ IDNO:12 22682-22704 Exonl2 22 M13F SEQIDNO:13 22626-22647 Tm61 23 MI3R SEQIDNO:14 22789-22811 Exonl3 23 M13F SEQIDNO:15 23961-23983 Touchdown6l 22 M13R SEQ ID NO:16 4213-24234 Exon 14 20 M13F SEQ ID NO:17 24818-24837 Touchdown 63 23 MI3R SEQIDNO:18 5101-25123 ExonlS 21 M13F SEQIDNO:19 25131-25151 Touchdown63 21 MI3R SEQIDNO:20 25384-25404 Exon 16 22 M13F SEQ ID NO:21 25490-25511 Touchdown 61 MI3R SEQ ID NO:22 25708-25727 * a.. * * p sea * . S as S S S S S
S S I I S I IS
* . I S S I S * . I S I S S * I.. * *li SI. Tags:
M13F: SEQ ID NO:23 MI3R: SEQ ID NO:24 Reaction Conditions: Exons 11-16 of c-Src were amplified individually using the primers listed in table 1. PCR was performed on 5l of 1:5 and 1:10 dilutions of extracted genomic DNA in a total reaction volume of 50.tl. 3.75 units of Amplitaq gold DNA polymerase (Applied Biosystems) was used in each reaction with final concentrations of 2mM magnesium chloride, 400p.M dNTPs and 0.3tM of each primer. Cycle conditions varied from amplicon to amplicon.
Cycle conditions: Tm 61 - 95 C for 10 minutes followed by 35 cycles of 95 C for 1 minute, 61 C for 1 minute and 72 C for 1 minute. A final extension step of 72 C for 10 minutes was used.
Touchdown 61 - 95 C for 10 minutes followed by 13 cycles of 94 C for 20 seconds, 61 C for 1 minute (dropping 0.5 C/cycle), 72 C for 1 minute. Standard cycling conditions were then carried out for a further 29 cycles at an annealing temperature of 54 C.
Touchdown 63 - 95 C for 10 minutes followed by 13 cycles of 95 C for 30 seconds, 63 C for 30 seconds (dropping 0.5 C/cycle), 72 C for 1 minute. Standard cycling conditions were then carried out for a further 19 cycles at an annealing temperature of 58 C.
PCR products (10 p.1) were treated with ExoSAP-IT (usb) (1 p.!, 1:2 dilution) to remove unincorporated oligonucleotides and nucleotides.
Example 3 - DNA sequencing Sequencing reactions were performed using ABI Big Dye Terminator chemistry (at a 1:16 dilution) and then run on an ABI 3730 sequencer as described in the Applied Biosystems manual. Sequence chromatograms were analysed using Mutation Surveyor software.
* *** * * * 0*S * I S IS I I S I a I * I I II * I I * I S * I I I I * I 5 I III 115 * S Example 4- Selection ofpatients for treatment Determination of the sequence of c-Src in a tumour sample from a patient can be used to select patients for treatment with a c-Src drug, for example AZDO53O, either as monotherapy or in combination therapy.
References Irby, R., (1999), Nat Genet, 21, 187-190.
Ischzawar, (2004), Cancer Cell, 6, 209-2 14.
Laghi, L., (2001), British Journal of Cancer, 84, 196-198.
La Rosee., (2002), Cancer Research, 62, 7149-7 153.
Lynch et al.,(2004) New England Journal of Medicine, 350(21) p2129.
Nilbert, M., (2000), Cancer Genet Cytogenet, 121, 94-95.
Paez et al. (2004) Science, 304, 1497-1500.
Ple et al., (2004) J. Med. Chem, 47, 87 1-887.
Sugimura (2000) Jpn J Cancer Res., 91(4), 395-8.
Tian, Biochem (2001), 40, 70847091.
Wang, N., (2000), Cancer Letters, 150, 201-204.
S 5Sf * S * * S S IS a a * * a I S S * S I IS I I S 5 5 8 S * * * I I I S U 555 5 III 585 S S
SEQUENCE LISTING
<110> AstraZeneca UK Limited <120> Method <130> CCH 101646 <160> 24 <170> Patentln version 3.2 <210> 1 <211> 1611 <212> DNA <213> Homo Sapiens <400> 1 atgggtagca acaagagcaa gcccaaggat gccagccagc ggcgccgcag cctggagccc 60 gccgagaacg tgcacggcgc tggcgggggc gctttccccg cctcgcagac ccccagcaag 120 ccagcctcgg ccgacggcca ccgcggcccc agcgcggcct tcgcccccgc ggccgccgag 180 cccaagctgt tcggaggctt caactcctcg gacaccgtca cctccccgca gagggcgggc 240 ccgctggccg gtggagtgac cacctttgtg gccctctatg actatgagtc taggacggag 300 acagacctgt ccttcaagaa aggcgagcgg ctccagattg tcaacaacac agagggagac 360 tggtggctgg cccactcgct cagcacagga cagacaggct acatccccag caactacgtg 420 gcgccctccg actccatcca ggctgaggag tgtatttt gcaa5atcac cagacgggag 480 tcagagcggt tactgctcaa tgcagagaac ccgagaggga ccttcctcgt gcgagaaagt 540 gagaccacga aaggtgccta ctgcctctca gtgtctgact tcgacaacgc caagggcctc 600 aacgtgaagc actacaagat ccgcaagctg gacagcggcg gcttctacat cacctcccgc 660 acccagttca acagcctgca gcagctggtg gcctactact ccaaacacgc cgatggcctg 720 tgccaccgcc tcaccaccgt gtgccccacg tccaagccgc agactcaggg cctggccaag 780 gatgcctggg agatccctc ggagtcgctg cggctggagg tcaagctggg ccagggctgc 840 tttggcgagg tgtggatggg gacctggaac ggtaccacca 5ggtggccat caaaaccctg 900 aagcctggca cgatgtctcc agaggccttc ctgcaggagg cccaggtcat gaagaagctg 960 aggcatgaga agctggtgca gttgtatgct gtggtttcag aggagcccat ttacatcgtc 1020 acggagtaca tgagcaaggg gagtttgctg gactttctca agggggagac aggcaagtac 1080 ctgcggctgc ctcagctggt ggacatggct gctcagatcg cctcaggcat ggcgtacgtg 1140 gagcggatga actactcca ccgggacctt cgtgcagcca acatcctggt gggagagaac 1200 ctggtgtca aagtggcgga ctttgggctg gctcggctca ttgaaacaa tgagtacacg 1260 gcgcggcaag gtgccaaatt ccccatcaag tggacggctc cagaagctgc cctctatggc 1320 cgcttcacca tcaagtcgga cgtgtg5tcc ttcgggatcc tgctgactga gctcaccaca 1380 aagggacggg tgccctaccc tggtatggtg aaccgcgagg tgctggacca ggtggagcgg 1440 ggctaccgga tgccctgccc gccggagtgt cccgagtccc tgcacgacct catgtgccag 1500 tgctggcgga aggagcctga ggagcggccc accttcgagt acctgcaggc cttcctggag 1560 S *i. . S 115 * . I IS * S * * S S * * . * I IS S S * a S * I * I I I S S a.. * a.. *5 S gactacttca cgtccaccga gccccagtac cagcccgggg agaacctcta g 1611 <210> 2 <211> 536 <212> PRT <213> Homo Sapiens <400> 2 Met Gly Ser Asn Lys Ser Lys Pro Lys Asp Ala Ser Gln Arg Arg Arg 1 5 10 15 Ser Leu Glu Pro Ala Glu Asn Val His Gly Ala Gly Gly Gly Ala Phe 25 30 Pro Ala Ser Gln Thr Pro Ser Lys Pro Ala Ser Ala Asp Gly His Arg 40 45 Gly Pro Ser Ala Ala Phe Ala Pro Ala Ala Ala Glu Pro Lys Leu Phe 55 60 Gly Gly Phe Asn Ser Ser Asp Thr Val Thr Ser Pro Gin Arg Ala Gly 70 75 80 Pro Leu Ala Gly Gly Val Thr Thr Phe Val Ala Leu Tyr Asp Tyr Glu 90 95 Ser Arg Thr Glu Thr Asp Leu Ser Phe Lys Lys Gly Glu Arg Leu Gln 105 110 Ile Val Asn Asn Thr Glu Gly Asp Trp Trp Leu Ala His Ser Leu Ser 120 125 Thr Gly Gin Thr Gly Tyr Ile Pro Ser Asn Tyr Val Ala Pro Ser Asp 135 140 Ser Ile Gln Ala Glu Glu Trp Tyr Phe Gly Lys Ile Thr Arg Arg Glu 150 155 160 Ser Glu Arg Leu Leu Leu Asn Ala Glu Asn Pro Arg Gly Thr Phe Leu 170 175 Val Arg Glu Ser Glu Thr Thr Lys Gly Ala Tyr Cys Leu Ser Val Ser 185 190 Asp Phe Asp Asn Ala Lys Gly Leu Asn Val Lys His Tyr Lys Ile Arg 200 205 Lys Leu Asp Ser Gly Gly Phe Tyr Ile Thr Ser Arg Thr Gln Phe Asn 210 215 220 Ser Leu Gin Gin Leu Val Ala Tyr Tyr Ser Lys His Ala Asp Gly Leu 225 230 235 240 0 0 o.o -3 t' (.3 0' 1J - 0 0 I-' CD I- 01 N I- U) CD QD< CD Cl) I-' OCD N C C < 010 U) U C U'N cr C CD 10 01cr Ii C -< U) -3 Z -3 -3 JCI) 0 < ( -3 Cl) M' t-' CD < U1CD N U) U) -.JI-' < F-* CD wIn U) CD H- N N N ON 0 C OU) U) N U) N O I-' C 11) C) 0 0 -3 C) U) 0 0 0 MI:-' I-'CD - < N N 3 1- -< f-' U1- I-' F-' N I-' -J'-< F-' N InC U) 10 0 CD 01C N U) ID l)J U1'-< CD C 0 -< UlU) U) 10 0 010 Z 0 0:r,D-3 0 0 1J< 0 -3 t' t'J F-' OF-' CD F-' F-' F-' MU N F-' F- ' .U) F-' CD 0,'< CD U OU rt U) ON U) 10 U C OF-' U) C N OU) C C) Do i-I U) F-I -3 -3 1,J -3 0 M3 F-' < U) N CD F-' F-' F-' 0 l)) U) F-' U 0 N) CD I-' F-' U) . U U) U) 010 rt CD U) U) U1F-' 0 CD N N InC U) N -< 0 U1N -3 C) < t' t' U) 1.J 0 0 < ti'o 1" 0 " -3 N -< -Jl)) CD CD N F-' IDCD F-' F--' F- U) i-'U N I-' F-' CD U) OF-' C C 10 U) OC U) C F--' OCD IC) U U) N 0 U) -3 0 1'(n t' -3 0 t M< 0 -3 CD N N U) U1CD F-' N -lCD F-' CD IOU) F-- ' N Di C tO 0 U InC N U 0 10 U1N U) N U C U1F-' -< 0 F-' 0 -3 0 0 0 0 1).-3 X t' 0 MC) 0 C) MF--' N N U.F- F-' U I-' F-' C< CD CD F-' I-' (O< F-' OC U) 0 10 N O< CD U) < ON U) ow C U) X 010 0 0 0 o' 0 X t' t'C$) -3 0 F--i 0 MF-I 0 U] 0 F-' F--' F-' F--' N N) I-' F-' F-' CD CD CD -< F-' F-' U 0 F-' N 0 InC C C C IC) Ull)) -< U) rr C U1N N C CD CD Oil)) 0 -3 0,l)-C) 0 t' t' t 1 t' 0 0 N IO< U) CD < I-'CD U) F-' N -< 1')F--' F-' F-' N W N 0 OW F-' CD U) OC U U) IC) U) OU) U) U) < 0 ON 0 0 0 t' -3 -3 0 x 1,JF-4 1-3 0 < I)Q -3 0 0) F--' N -lCD F-' IDF-' -< CD F--' U) F-'F-' F-' N CD CD C 0 InC N N CD U) U1CD N C I-' U1 N C IC) N - -3 0 0 Di i-3 i-I 0 o' I: (/) < 0 I:-' F-' F--' U) O F-' N N CD U)U) N CD 0) U) OCD U) F-' II C C 0 ON CD 0 10 C OF-' 0 N F- F-' OC F--' C U) IflCi) 0' Cl) 0 t-' .o-t-' -i 0 t' Cl) t' M'-3 0) 0 MCD N CD I-' -< < F-' CD U) F-' ml-' CD CD CD < N CD N U1N IC) N U U) Oil)) CD C F-' C U1U C N rt U) U1'0 N 0 -3 Ui'0 t' 0 0) t-' - o:r t-' Lit-' 0 I:-' 0 Mt' 0 0 l-'N CD U) F- CD 1''-< F-' F-' N CD U1CD F-' -< N CD -)CD F-' N 00 C F- ' N OU) CD IC) C OC C U) 0 cr OC U O -3 0 -3.0 0 U) 1)Q t' 0 0 0) M.-3 F- ' U to I-'- F-' N ID N I-' F-' F--' CD U) U) Li F-' -< I-' F-' N 01 U C N 01(J) C IC) 0 0 InC C rr F-' 0 InC U) -< < 10 O1N 0 0 U) ,D-0) -3 0-r' 0" 0 Lit-' 3 -3 t 0 -. - N (I) N l)) I)) 0) OU) U) U) N MCD U U CD F-' * 0 CD 0 010 I-' F-' N Ii OU U 0 CD 0 OCN N C U * .. * * * Sn *. S
V *S I * b *II * I ** * I I V (I, I * * II * * S I I V I S *S* I *IV III S * Gin Tyr Gin Pro Giy Glu Asn Leu 530 535 <2i0> 3 <211> 50 <2i2> DNA <2i3> Homo Sapiens :400> 3 tcgctgcggc tggaggtcaa gctgggccag ggctgctttg gcgaggtgtg 50 <2i0> 4 <21i> 50 <2i2> DNA <2i3> Homo Sapiens <400> 4 catttacatc gtcacggat acatgagcaa ggggagttt ctggactttc 50 <210> 5 <2ii> 50 <2i2> DNA <213> Homo Sapiens <400> 5 gacaatgagt acacggcgcg gcaaggtgcc aaattcccca tcaagtggac 50 <2i0> 6 <2ii> 50 <2i2> DNA <2i3> Homo Sapiens <400> 6 ggccgcttca ccatcaagtc ggacgtgtgg tccttcggga tcctgctgac 50 <2i0> 7 <211> 50 <212> DNA <2i3> Homo Sapiens <400> 7 gtcggacgtg tggtccttcg ggatcctgct gactgactc accacaaagg 50 <2i0> 8 <2ii> 50 <212> DNA <213> Homo Sapiens <400> 8 tccttcggga tcctgctgac tgagctcacc acaaagggac gggtgcccta 50 <210> 9 <211> 21 <212> DNA <213> Homo Sapiens <400> 9 cgtccaagcc gcagactcag g 21 <210> 10 * II. S * e * I IS I 5 * * 5 I I 4 * , S II L) 5 * * S * $ I 5 4* II. I *g * * <211> 23 <212> DNA <213> Homo Sapiens <400> 10 ggagctccag atttacaaca ggg 23 <210> 11 <211> 22 <212> DNA <213> Homo Sapiens <400> 11 atccctgtgt gtaggagtt g 22 <210> 12 <211> 23 <212> DNA <213> Homo Sapiens <400> 12 tgtaaatggg ctcctctaa acc 23 <210> 13 <211> 22 <212> DNA <213> Honia Sapiens <400> 13 gaggcccagg tcatgaaaa gc 22 <210> 14 <211> 23 <212> DNA <213> Homo Sapiens <400> 14 ctagacatga gaggaaatga ggc 23 <210> 15 <211> 23 <212> DNA <213> Homo Sapiens <400> 15 ggatgggttt tgggaatcac tgc 23 <210> 16 <211> 22 <212> DNA <213> Homo Sapiens <400> 16 gtgtggaggc aatcaaggca gg 22 <210> 17 <211> 20 <212> DNA <213> Homo Sapiens <400> 17 tgggagggca tgggtg9cac 20 a i*a a *
I I I II
I I I I I * I * * a Io * * * III 4 I.e II.
<210> 18 <211> 23 <212> DNA <213> Homo Sapiens <400> 18 taagcaccat gaggaatgac agc 23 <210> 19 <211> 21 <212> DNA <213> Homo Sapiens <400> 19 gagcggtcat gacaggaggt C 21 <210> 20 <211> 21 <212> DNA <213> Homo Sapiens <400> 20 gaaacagcc cagcaggagt c 21 <210> 21 <211> 22 <212> DNA <213> Homo sapiens <400> 21 tatgtcact cccaacctgt cc 22 <210> 22 <211> 20 <212> DNA <213> Homo Sapiens <400> 22 gctcctcagg ctccttccgc 20 <210> 23 <211> 20 <212> DNA <213> Homo Sapiens <400> 23 actgtaaaac gacggccagt 20 <210> 24 <211> 20 <212> DNA <213> Homo Sapiens <400> 24 accaggaaac agctatgacc 20

Claims (10)

  1. I.. * * I III * . . I. * I * * a a * * . a II I * * S I * * I * * I * S *
    S a.. * *.. a. . * * Claims 1. A method which comprises determining the presence of a mutation in the kinase domain of c-Src in a tumour sample from a patient wherein the mutation differs at one base position from SEQ ID NO: 1 andlor at one amino acid position from SEQ ID NO: 2.
  2. 2. A method according to claim I wherein the mutation is selected from any one of the following at positions as defined in SEQ ID NO: 1: position 829 is not G; position 1032 is not G; position 1270 is not G; position 1342 is not G; position 1359 is not C; position 1372 is not C; or position 1486 is not G, andlor any one of the following at positions as defined in SEQ ID NO:2: position 277 is not glycine; position 344 is not methionine; position 424 is not glycine; position 448 is not valine; position 458 is not leucine; or position 496 is not aspartic acid.
  3. 3. A method according to claim 2 wherein the mutation is selected from any one of the following at positions as defined in SEQ ID NO: 1: position 829 is A; position 1032 is A; position 1270 is A; position 1342 is A; position 1359 is A; position 1372 is T; or p *p* * * p isp * p * ** * * . p 5 I * p * * ** I p * * * S S S * 5. S S S I.. I 555 *** S position 1486 is A, andlor any one of the following at positions as defined in SEQ ID NO:2: position 277 is serine; position 344 is isoleucine; position 424 is serine; position 448 is methionine; position 458 is phenylalanine; or position 496 is asparagine.
  4. 4. Use of a method according to any of the preceding claims to assess the pharmacogenetics of a c-Src drug.
  5. 5. A method according to any one of the preceding claims wherein the method for detection of a nucleic acid mutation is selected from amplification refractory mutation system and restriction fragment length polymorphism.
  6. 6. A primer or an oligonucleotide probe capable of detecting a mutation in the kinase domain of c-Src in a tumour sample from a patient at any one of the following positions as defined in SEQ ID NO: 1: 829, 1032, 1270, 1342, 1359, 1372 or 1486.
  7. 7. A mutant human c-Src polynucleotide comprising any one of the following nucleic acid bases at positions as defined in SEQ ID NO: 1: position 829 is A; position 1032 is A; position 1270 is A; position 1342 is A; position 1359 is A; position 1372 is T; or position 1486 is A, or a fragment thereof comprising at least 20 bases provided that the fragment comprises at least one of the mutant bases at positions 829, 1032, 1270, 1342, 1359, 1372 or 1486.
  8. S 555 * S S 550 * . S *s S S 5 * S S I S S * S SI S 5 5 5 S S I S * * S S * S S.. 4 *i* S. . S * 8. A mutant human c-Src polypeptide comprising any one of the following amino acid residues at positions as defined in SEQ ID NO:2: position 277 is serine; position 344 is isoleucine; position 424 is serine; position 448 is methionine; position 458 is phenylalanine; or position 496 is asparagine, or a fragment thereof, comprising at least 10 residues provided that the fragment comprises at least one of the mutant residues at positions 277, 344, 424, 448, 458 or 496.
  9. 9. An antibody specific for a mutant human c-Src polypeptide as defined in claim 8.
  10. 10. A kit for detection of a mutation in c-Src comprising an antibody of claim 9 or a primer or oligonucleotide probe of claim 6.
GB0526018A 2004-12-28 2005-12-22 Mutations in c-Src Withdrawn GB2421731A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040261142A1 (en) * 1999-11-24 2004-12-23 Yeatman Timothy J. Mutated Src oncogene composition and methods

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040261142A1 (en) * 1999-11-24 2004-12-23 Yeatman Timothy J. Mutated Src oncogene composition and methods

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. Biol. Chem., Vol.270, 1995, Bjorge, J. D. et al., "Characterization of two activated...", pp. 24222-24228 *
P. N. A. S., Vol.83, 1986, Levy, J. B. et al., "Activation of the transforming potential...", pp.4228-4232 *

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