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D13Tl~CTION OF CE:R~aIN MOLECULæS
This invention relates to assay systems for pyrogenic molecules which exert their effects through the transcriptional activation of genes.
At present, assay of such compounds may involve the treatment of specific mammalian cell lines with the test substance and the-subsequent assay of the cell line for synthesis of the gene product known to be produced in response to the test agent. The assay and quantitation of the newly synthesised gene product in such a system is then an indicator of the amount of active material in the test substance.
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Microbial contamination of parenterally administered therapeutic products commonly results in a pyrogenic reaction upon administration. Man is particularly sensitive to bacterial lipopolysaccharide (LPS) which, in even nanogram amounts, may induce fever. Pharmaceutical products which are intended for parenteral administration must therefore be screened for pyrogenic contaminants. The currently accepted methods for detecting pyrogens are the Limulus Amoebocyte Lysate (LAL) and the rabbit test. The LAL test is an in vitro test whilst the rabbit test directly measures the rise in body temperature in response to injection of the test substance. However, these tests are not definitive and at least one case of an E.coli ., . ~ . .
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derived recombinant product has been reported which was pyrogen negative in the above tests but which upon administration to man proved to be pyrogenic (Dinarello, et al., 1985).
The febrile response is mediated by the release from monocytes of cytokines, such as tumour necrosis factor and interleukin 1, which have a broad range of biological effects. A refinement of the pyrogen test is the direct measurement of cytokines released by monocytes in vitro using immuno methods. The synthesis and release of both TNF and interleukin-1 by monocytes following treatment with LPS are well characterised (Pinarello, 1984;
Phillip and Epstein, 1986) and a monocyte test for pyrogen has been developed based on these observations (Poole, et al., 1988).
' At least two distinct forms of interleukin 1 exist, termed IL-la and IL-l~ (March, et al., 1985). Of these, the IL-1~ gene is more responsive to LPS stimulation (Poole, et al., 1988).
Established monocytic cell lines are similarly responsive to LPS
although the sensitivity of such cell lines is reportedly not as great as that of fresh monocyte preparations (Poole, et al., 1988). Such detection methods, however, may be laborious and require the use of expensive reagents and equipment.
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We have now found that such potential problems may be reduced by .
the replacement of the known induced gene product with an easily quantifiable reporter gene within the mammalian or other cell 2~7~
line normally used for assay. Thus the reporter gene is induced in response to the test sample and then easily quantified as outlined in the description following. Additionally, such new cell lines can have the advantage of increased sensitivity since response is proportional to the copy number of the cloned promoter-reporter gene fusion and this copy number can be made to be more than one.
Thus, one aspect of the present invention provides a mammalian cell line having an inheritable genetic element comprising a TNF, IL-1~ or IL-l~ promoter and, under transcriptional control thereof, a coding region, wherein the promoter controls transcription of the coding region in response to the exposure of the said cell line to a pyrogen and the said coding region encodes a reporter polypeptide.
As uséd herein, the term ~'promoter" means a nucleotide sequence 5I to a TNF, IL-1~ or Ih-1~ coding region which controls transcription thereof; the promoter may comprise an enhancer system which may or may not be one normally associated with that promoter. The term "reporter polypeptide" is used to mean a polypeptide other than TNF, IL-1~ or IL-1~ and which polypeptide can be detected by means other than (i) immunological techniques and (ii) means requiring a second living cell.
2 0 ~ J 3 A second aspect of the invention provides an assay system comprising a cell line having an inheritable genetic element comprising a TNF, IL-1~ or IL-l~ promoter and, under transcriptional control thereof, a coding region, wherein the promoter controls transcription of the coding region in response to the exposure of the said cell line to a pyrogen and the said coding region encodes a reporter polypeptidé.
A third aspect of the invention provides a vector for transfecting a cell line, the vector comprising a TNF, IL-la or IL-1~ promoter and, under transcriptional control thereof, a coding region, wherein the promoter controls transcription of the coding region in response to the exposure of the said cell line to a pyrogen and the said coding region encodes a reporter polypeptide.
In a preferred embodiment of the invention, the cell line is derived from a human blood monocyte, such as the THP-l and U937 cell lines. Preferably, the promoter is human-derived.
The reporter polypeptide is conveniently ~-galactosidase, chloramphenicol acetyl transferase, luciferase, phosphatase or a-galactosidase.
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Transcriptional and translational s-tart and stop codons ar.d downstream regulatory regions may be included as necessary and in known ways.
Preferred embodiments of the present invention will now be described by way of example and with reference to the accompanying drawings, in which:
Figure 1 shows the partial sequence of the IL-l~ gene (from Bensi et al, 1987), with the exons boxed and the positions at which probes 1 and 2 hybridize also indicated;
Figure 2 illustrates schematically the subcloning of the IL-l~
promoter into plasmid ml3mp8 to form plasmid mp8.1;
Figure 3 illustrates schematically the site-directed mutagenesis of plasmid mp8.1 to give plasmid mp8.1.1; and Figure 4 illustrates schematically the construction of plasmid pEK007.
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~ 6 EXAMPLE 1 : A CELL LINE FOR DETECTION OF INTERLEUKIN 1 SYNTHESIS
Outline The IL-l~ gene promoter is fused to the lacZ gene of E.coli which encodes ~-galactosidase and this cassette is inserted into a suitable cell line. Stimulation of this cell line by LPS or other pyrogen will result in the synthesis of ~-galactosidase which may then be detected in a simple colorimetric assay. Thus pyrogen may be measured indirectly, obviating the requirement for the relatively complex, time consuming and expensive immunological techniques used in the direct detection of TNF and IL-l~. The construction of such a cell line is given below.
The molecular biological techniques are generally those taught by Maniatis et al, 1982.
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Isolation of the IL-1~ Gene Promoter : :, A human leukocyte genomic DNA bank in the vector EMBL3 may be obtained from Clontech Laboratories Inc. (Palo Alto, California). The sequence of the 5' end of the IL-l~ gene is shown in Figure 1 (Bensi et al., 1987). Oligonucleotide probes which hybridize to the positions underlined in Figure 1 are constructed on an automatic DNA synthesiser using '': ~ ' ' ~
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~ 7 phosphoramidite chemistry and reagents supplied by the manufacturer (Applied Biosystems, Foster City, California).
These structures are as follows:
Probe 1 G A C C A C A T T A A A A T A C T G G A T T T T C C C A C G
Probe 2 C A G G T A C T T C T G C C A T G G C T G C T T C A G A C A C
These oligonucleotides are labelled with 32p and used to probe the gene bank by plaque hybridization as described by Maniatis et al., (1982)-Positive clones, those hybridizing to both primers, are furthercharacterized by restriction mapping and comparison with the published restriction data available via analysis of the DNA
sequence described previously (Bensi et al., 1987). In this way the sequence may be extended 5' to that reported by Bensi et al (1987) and the upstream sequence encompassing the promoter or regulatory regions isolated. This is most probably located in a region extending to 3kb 5' from the transcription start site.
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Next the insert or partial insert of the EMBL-3 clone which spans the desired sequence is subcloned into M13mp8 (Messing, 1983). This is carried out by insertion of a blunt-ended DNA
fragment into the BamHI site of M13mp8 which has been similarly blunt-ended as described previously (GB-A-2175590) as shown in Fig 2. A clone (designated mp8.1) is chosen in which exon 2 is adjacent to the SalI site of mp8.
Next, mp8.1 is manipulated to create a BqlII site in exon 2 which is compatible with the tailored ~-galactosidase gene from plasmid pMC1924 (Nielsen et al, 1983). This i8 most readily carried out by changing two nucleotides in exon 2 as shown in Fig 3 using the technique of oligonucleotide-directed mutagenesis (Zoller and Smith, 1983) to create mp8.1.1.
The lL-l~ promoter-N-terminus is now located on a SmaI-BqlII
fragmént, the SmaI site being derived from M13mp8. This fragment is then cloned into PvuII/BamHI digested pMC1924 (Nielsen et al, 1983) to give the plasmid pEX007 (Fig 4).
The E. coli ~-galactosidase gene has been tailored in pMC1924 such that insertion of the above fragment will result in an in-frame fusion of the IL~ N-terminus and ~-galactosidase. The fusion will have ~-galactosidase enzyme activity.
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Transfection of Cell Line The recombinant pMC1924 derivative containing the interleukin-1~
promoter, pEK007, is next transferred into a suitable cell line such as THP-l (Fenton, et al., 1987) as described by Gorman ~1985). Stable integrant clones are obtained via co-transfection of pSVNeo, encoding resistance to the aminoglycoside G418 (Southern and Berg, 1982). Co-integrants of pSVNeo and pER007 are screened for by resistance to G418 and subsequent Southern blotting of G418 resistance clones for co-insertion of the ~-galactosidase gene using the ~-galactosidase gene as a probe, as described by Maniatis et al., (1982).
Induction of ~-Galactosidase SYnthesis The transgenic THP-1 cell line is next induced with LPS as described in Fenton et al. (1987). ~-galactosidase may be assayed as described by Gorman (1985) and Nielsen (1983).
Analysis of LPS-stimulated clones shows that ~-galactosidase is produced and the amount produced corresponds with the degree of stimulation. It is probable that cell lines with different copy numbers of integrated pER007 will exhibit slightly different responses, with the degree of responsiveness and thus sensitivity being proportional to the number of integrated copies of the plasmid. Following these analyses, a suitable sub-line is chosen as the basis for the pyrogen assay and its response to a range of pyrogens in addition to LPS assessed.
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Additional Expression Siqnals Some mammalian and higher eukaryotic genes have additional expression regulatory elements within the gene itself or in the 3' non-coding region. There may well be such additional elements within the interleukin 1~ gene. Thus expression of the hybrid interleukin 1~ alactosidase fusion polypeptide may require such additional signals. If therefore LPS failed to stimulate ~-galactosidase synthesis, selected regions such as the 3' non-coding region of the interleukin 1~ gene may be attached to the fusion and the effect of these assessed.
A typical assay kit using a cell line of the invention may be supplied as a microtitre plate containing a sample of the cell line and the substrate for ~-galactosidase (X-gal) in each well along with cell culture medium and other essential factors. The plate may be supplied frozen and would be thawed prior to use and addition of the test sample to the culture dish well.
Incubation at 37C in a suitable incubator will proceed for a period of time sufficient for the pyrogen contained with the test sample to stimulate ~-galactosidase expression and ~
sufficient chromophore to be generated to facilitate its ~-measurement in a plate-reading spectrophotometer. The signal generated may finally be compared to that generated by known amounts of pyrogen in a parallel standard assay and thus the amount of pyrogen in the test sample assessed. -.'~
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FURTHER EXAMPLES
Alternative Promoters and Reporter Genes The effector cytokines for the pyrogenic response are believed to be TNF, IL-l~ and IL-la; however additional factors may be involved. Thus, TNF and IL-la gene promoters are alternative promoters in this system. Similarly there are other simple reporter genes such as chloramphenicol acetyl transferase (CAT), horseradish peroxidase, luciferase and phosphatase which may be substituted for ~-galactosidase to facilitate detection of cytokine synthesis. Gorman (1985), section 7.1, details an assay for CAT, whilst Van Zonneveld et al (1988) detail an assay for luciferase.
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REFERENCES
., Bensi, G., et al., (1987) Gene, 52; 95-101.
Dinarello, C.A. (1984) Rev.Infect.Dis. 6; 51-95~
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Dinarello, C.A., et al., (1985) J.Clin.Microbiol, 20; 323-329.
Fen~on, M.J., et al., (1987) J.Immunol, 138; 3972--3979.
Gorman, C. (1985) in "DNA Cloning Vol II~. Glover, D.M. (Ed) 143-189 IRL Press Oxford.
Maniatis, T., et al., (1982) Molecular Cloninq : A laboratory manual. Cold Sprinq Harbor Publications, N.Y. U.S.A.
March, C.J. et al., (1985) Nature. 315; 641-647.
Messing, J. (1983) Methods in Enz~mol. 100, 20-78.
Nielsen, D.A., et al., (1983) Proc. Natl. Acad. Sci (USA). 80;
5198-5202.
Phillip, R. and Epstein, L. (1986) Nature. 323, 86-89.
Poole, 5., et al., (1988) Lancet. I, 130. ;
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Van Zonneveld, A-J., Curriden, S.A. and Loskutoff, D.J. (1988) Proc. Natl. Acad. Sci. USA. 85; 5525-5529.
Zoller, M.J. and Smith M. (1983) Methods in Enzymol. 100; 468-500.
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