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WO1998035988A1 - Proteines - Google Patents

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Publication number
WO1998035988A1
WO1998035988A1 PCT/GB1998/000415 GB9800415W WO9835988A1 WO 1998035988 A1 WO1998035988 A1 WO 1998035988A1 GB 9800415 W GB9800415 W GB 9800415W WO 9835988 A1 WO9835988 A1 WO 9835988A1
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WIPO (PCT)
Prior art keywords
sequence
seq
pro
prodomain
modified
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PCT/GB1998/000415
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English (en)
Inventor
Michael Derek Edge
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Zeneca Limited
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Priority claimed from GBGB9703104.1A external-priority patent/GB9703104D0/en
Priority claimed from GBGB9722003.2A external-priority patent/GB9722003D0/en
Priority claimed from GBGB9722727.6A external-priority patent/GB9722727D0/en
Application filed by Zeneca Limited filed Critical Zeneca Limited
Priority to AU60006/98A priority Critical patent/AU6000698A/en
Publication of WO1998035988A1 publication Critical patent/WO1998035988A1/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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to modified carboxypeptidase B prodomains and their use in recombinant production of active carboxypeptidase B enzyme.
  • Many proteases are synthesised in vivo as pro-enzymes and the catalytically active protease is eventually cleaved from a larger precursor polypeptide.
  • the pro regions (also called “prodomain” or “pro sequence”) of pro-enzymes are usually N-terminal extensions of the mature proteins and in many cases it has been demonstrated that the pro regions are required for folding of their associated protease domains [reviewed by Baker, Shiau and Agard in Current Opinion in Cell Biology (1993) 5, 966-970].
  • CPB carboxypeptidase B
  • CPB carboxypeptidase B
  • the activation of porcine pancreatic CPB has been studied by V Villegas et al in Protein Science (1995), 4, 1792-1800. Activation of procarboxypeptidases has been reviewed by F X Aviles et al in Eur. J. Biochem. (1993), 211, 381-389.
  • proCPB is fused at its C terminus to the N-terminus of an antibody chain this allows removal of prodomain (e.g. by trypsin treatment) from the N-terminus ofthe fusion construct.
  • proCPB was attached to the C-terminus of an antibody chain then the problem arises of how to remove the prodomain from the "middle" ofthe construct.
  • the solution is to co-express the prodomain separately (in trans). Accordingly we have shown that by independently expressing the pro and protease domains of CPB. active human CPB (HCPB) is secreted from COS cells (see International Patent Application WO 96/2001 1).
  • the present invention is based on the discovery that modification ofthe prodomain sequence leads to improved recombinant expression yields of active carboxypeptidase B. According to one aspect ofthe present invention there is provided a modified prodomain of carboxypeptidase B which enhances recombinant expression thereof when co- expressed from a separate gene.
  • prodomain means an N-terminal sequence which is naturally responsible for assisting the folding of CPB into its active conformation before being subsequently removed to give active mature CPB.
  • the natural prodomain of pancreatic HCPB is set out in SEQ ID NO: 12 at positions 14-108.
  • the sequence of mature pancreatic HCPB is set out in SEQ ID NO: 12 at positions 109-415.
  • the prodomain attached to mature pancreatic HCPB is termed "proHCPB” and is set out in SEQ ID NO: 12 at positions 14-415.
  • modified prodomain in this context means that the prodomain is different from the naturally occurring prodomain for example by addition, deletion, substitution or insertion of amino acids.
  • An example of a suitable substitution is to change the Arg residue at the C-terminus ofthe prodomain of pancreatic HCPB (at position 108 in in SEQ ID NO: 12) into a hydrophobic amino acid such as leucine.
  • Suitable C-terminus additions are described herein for HCPB.
  • Suitable deletions include deletion of any of the last 3 amino acids ofthe natural prodomain of HCPB (positions 106-108 in SEQ ID NO: 12).
  • a skilled worker in the field will be able to select and test further modified prodomains based on the disclosures herein with reference in particular to the specific examples and proposed mechanism ofthe effect.
  • amino acid sequences are preferably of L-configuration.
  • the term "enhances recombinant expression” means that modifications tested by measuring expression levels of carboxypeptidase B in the presence of a modified prodomain compared with unmodified (i.e. natural) prodomain; enhanced levels of expression of at least 20 %, more preferably at least 50 % are within the scope ofthe invention.
  • Suitable expression systems for evaluation of enhanced expression include those set out in Reference Example 4 (for CPB) and Examples 14 & 19 (for "reversed polarity" mutants of CPB).
  • the prodomain is modified at its C-terminus.
  • the C- terminus amino acid ofthe prodomain after addition of the amino acid(s) is a hydrophobic amino acid.
  • the C-terminus ofthe added amino acids is a hydrophobic amino acid.
  • Preferred hydrophobic amino acids are leucine, isoleucine, valine, alanine or phenylalanine and especially leucine.
  • Preferred specific sequences are addition of any one of L, KDEL. KKAA or SDYQRL sequences and of these KKAA, SDYQRL or L are preferred, w ith L being especially preferred.
  • carboxypeptidase is human and especially human pancreatic CPB.
  • a preferred modified prodomain is a human pancreatic carboxypeptidase B prodomain with a leucine added at its C-terminus.
  • the carboxypeptidase enzyme may be fused to another protein such as for example an antibody chain.
  • Another suitable carboxypeptidase is plasma carboxypeptidase B (Eaton, D.L. et al., J.Biol. Chem.
  • CPB generally catalyses hydrolysis of basic amino acids from the C-terminus of polypeptides.
  • Carboxypeptidase mutants are also within the scope ofthe invention. Carboxypeptidase mutants include enzymes having altered substrate specificity.
  • International Patent Application WO 96/2001 published 4-Jul-96. we proposed a "reversed polarity" ADEPT system based on mutant human enzymes having the advantage of low immunogenicity compared with for example bacterial enzymes.
  • a particular host enzyme was human pancreatic CPB (see for example, Example 15 [D253K]human CPB & 16 [D253R]human CPB therein) and prodrugs therefor (see Examples 18 & 19 therein).
  • the host enzyme is mutated to give a change in mode of interaction between enzyme and prodrug in terms of recognition of substrate compared with the native host enzyme.
  • Preferred "reversed polarity" mutant carboxypeptidases are any one of [D253K]HCPB, [G251T,D253K]HCPB or [A248S,G251T,D253K]HCPB of which the latter is most preferred.
  • the carboxypeptidase enzyme may be in the form of a recombinant fusion protein with another peptide such as for example an antibody heavy or light chain.
  • the carboxypeptidase B is porcine.
  • porcine procarboxypeptidase B has been described in International patent application WO 95/14096, Eli Lilly. Without wishing to be bound by theoretical considerations the following text sets out our understanding ofthe mechanism behind the invention.
  • the C-terminal residue of the natural prodomain of pancreatic CPB is arginine. This is known to be removed readily by active CPB itself or by other enzymes for example carboxypeptidase H which is believed to be present in the Golgi apparatus (which along with the endoplasmic reticulum are the major organelles involved in intracellular trafficking of secreted proteins). This degradation is believed to reduce any interaction between the prodomain and mature enzyme.
  • CPB protein will be enhanced.
  • a C-terminal amino-acid (e.g. leucine) residue not cleaved by the active enzyme is believed to hinder degradation.
  • Another potential mechanism of enhanced expression is based on enhanced intracellular trafficking. Protein folding in eucaryotic cells takes place in the endoplasmic reticulum so modification ofthe prodomain with sequences (e.g. KKAA) which increase the concentration ofthe prodomain in intracellular compartments, especially the endoplasmic reticulum, are also believed to enhance expression yields of recombinantly expressed CPB.
  • sequences e.g. KKAA
  • a vector comprising a polynucleotide sequence capable of encoding a modified prodomain of the invention.
  • a host cell comprising a polynucleotide sequence, optionally in the form of a vector, capable of encoding a modified prodomain ofthe invention.
  • a method of recombinant carboxypeptidase B production which comprises simultaneously expressing in a host cell a carboxypeptidase B enzyme gene together with a separate gene encoding a modified prodomain ofthe invention and optionally at least partially purifying the recombinant carboxypeptidase B.
  • the carboxypeptidase B enzyme gene can be in the form of mature CPB enzyme or proCPB (with natural prodomain). In the case ofthe latter, without wishing to be bound by theoretical considerations, it is contemplated that separately expressed modified prodomain is able to interact with expressed CPB through participation in a dynamic equilibrium.
  • the host cell can be procaryotic or eucaryotic, preferabh eucaryotic.
  • an especially preferred host cell is in the form of a transgenic animal.
  • the separate genes may be on the same or a different genetic entity e.g. the same or different plasmids.
  • the carboxypeptidase B enzyme may be in the form of a recombinant fusion protein with another peptide such as for example an antibody heavy or light chain.
  • An especially preferred carboxypeptidase B fusion construct is described in Example 15 below.
  • Example 15 describes a gene encoding a humanised Fd heavy chain fragment of antibody 806.077 linked to enzyme [A248S.G251T,D253K]HCPB and its co-expression with a gene encoding a humanised light chain of 806.077 and a gene encoding the pro-L modified prodomain of human carboxypeptidase B to give the F(ab') 2 protein with a molecule of [A248S,G251T,D253K]HCPB at the C-terminus of each ofthe heavy chain fragments.
  • the constant and hinge regions of of the humanised Fd heavy chain fragment are derived from the human IgG3 antibody isotype.
  • a preferred method of recombinant carboxypeptidase B production is one in which the eucaryotic host cell is mammalian and: i) the recombinant carboxypeptidase B is in the form of a humanised 806.077 F(ab , ) 2 -
  • the carboxypeptidase B enzyme gene is in the form of a gene encoding a humanised Fd heavy chain fragment of antibody 806.077 linked to enzyme
  • [A248S,G251T,D253K]HCPB iii) the separate gene encoding a modified prodomain encodes human pancreatic carboxypeptidase B prodomain with a leucine added at its C-terminus; and iv) a further gene is co-expressed which encodes a humanised light chain of antibody 806.077; and wherein the fusion protein is in the form of a F(ab') 2 with a molecule of [A248S,G251T,D253K]HCPB at a C-terminus of each of its heavy chain fragments.
  • Hybridoma 806.077 antibody was deposited at the European Collection of Animal Cell Cultures (ECACC), PHLS Centre for Applied Microbiology & Research, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom on 29th February 1996 under accession no. 96022936 in accordance with the Budapest Treaty. Humanisation of antibody 806.077 has been described in International Patent Application WO 97/42329, Zeneca Limited, published 13-Nov-97. Some expression systems involve transforming a host cell with a vector; such systems are well known such as for example in E. coli, yeast and mammalian hosts (see Methods in Enzymology 185, Academic Press 1990).
  • transgenic non-human mammals in which the gene of interest, preferably cut out from a vector and preferably in association with a mammary promoter to direct expressed protein into the animal's milk, is introduced into the pronucleus of a mammalian zygote (usually by micro injection into one ofthe two nuclei (usually the male nucleus) in the pronucleus) and thereafter implanted into a foster mother.
  • a mammalian zygote usually by micro injection into one ofthe two nuclei (usually the male nucleus) in the pronucleus
  • a proportion ofthe animals produced by the foster mother will carry and express the introduced gene which has integrated into a chromosome.
  • the integrated gene is passed on to offspring by conventional breeding thus allowing ready expansion of stock.
  • the protein of interest is simply harvested from the milk of female transgenic animals.
  • Transgenic plant technology is also contemplated such as for example described in the following publications: Swain W.F. (1991) TIBTECH 9: 107-109; Ma J.K.C. et_al (1994) Eur. J. Immunology 24: 131-138; Hiatt A. et al (1992) FEBS Letters 307:71-75; Hein M.B. et al (1991) Biotechnology Progress 7: 455-461; Duering K. (1990) Plant Molecular Biology jo: 281-294.
  • host genes can be inactivated or modified using standard procedures as outlined briefly below and as described for example in "Gene Targeting: A Practical Approach", IRL Press 1993.
  • the target gene or portion of it is preferably cloned into a vector with a selection marker (such as Neo) inserted into the gene to disrupt its function.
  • the vector is linearised then transformed (usually by electroporation) into embryonic stem (ES) cells (eg derived from a 129/Ola strain of mouse) and thereafter homologous recombination events take place in a proportion ofthe stem cells.
  • ES embryonic stem
  • the stem cells containing the gene disruption are expanded and injected into a blastocyst (such as for example from a C57BL/6J mouse) and implanted into a foster mother for development.
  • blastocyst such as for example from a C57BL/6J mouse
  • Chimaeric offspring can be identified by coat colour markers. Chimeras are bred to ascertain the contribution ofthe ES cells to the germ line by mating to mice with genetic markers which allow a distinction to be made between ES derived and host blastocyst derived gametes. Half of the ES cell derived gametes will carry the gene modification. Offspring are screened (eg by Southern blotting) to identify those with a gene disruption (about 50 % of progeny). These selected offspring will be heterozygous and therefore can be bred with another heterozygote and homozygous offspring selected thereafter (about 25 % of progeny).
  • Transgenic animals with a gene knockout can be crossed with transgenic animals produced by known techniques such as microinjection of DNA into pronuclei, sphaeroplast fusion (Jakobovits et al. (1993) Nature 362:255-258) or lipid mediated transfection (Lamb et al. (1993) Nature Genetics 5 22-29) of ES cells to yield transgenic animals with an endogenous gene knockout and foreign gene replacement.
  • ES cells containing a targeted gene disruption can be further modified by transforming with the target gene sequence containing a specific alteration, which is preferably cloned into a vector and linearised prior to transformation. Following homologous recombination the altered gene is introduced into the genome. These embryonic stem cells can subsequently be used to create transgenics as described above.
  • host cell includes any procaryotic or eucaryotic cell suitable for expression technology such as for example bacteria, yeasts, plant cells and non-human mammalian zygotes, oocytes, blastocysts, embryonic stem cells and any other suitable cells for transgenic technology. If the context so permits the term "host cell” also includes a transgenic plant or non-human mammal developed from transformed non-human mammalian zygotes. oocytes. blastocysts, embryonic stem cells, plant cells and any other suitable cells for transgenic technology.
  • conservative amino acid analogues of specific prodomain amino acid sequences are contemplated which substantially retain the properties of a prodomain of the invention but differ in sequence by one or more conservative amino acid substitutions, deletions or additions. It is known in the art that N-terminus additions can often be accommodated. A skilled worker in the field will be able to select and test conservative analogues using disclosures herein together with general knowledge.
  • the 3 dimensional structure of porcine CPB is known from Coll et al, 1991, The EMBO Journal, J O, 1-9.
  • a skilled worker can use the 3D structure to select potential non-critical regions for analogue production. For example, non-critical loop regions may be selected for analogue production.
  • nucleic acid variations in this specification nucleic acid variations (deletions, substitutions and additions) of specific nucleic acid sequences are contemplated which retain which the ability hybridise under stringent conditions to the specific sequence in question. However specifically listed nucleic acid sequences are preferred. Abbreviations used herein include:
  • FCS foetal calf serum
  • Fd heavy chain of Fab, Fab ' or F(ab') 2 optionally containing a hinge
  • HCPB human carboxypeptidase B preferably pancreatic
  • Figure 1 illustrates pancreatic HCPB cloning.
  • Figure 2 illustrates pancreatic HCPB sequencing.
  • Figure 3 illustrates vector pICI1266.
  • Figure 4 illustrates pICI1266 expression vector gene cloning.
  • DNA is recovered and purified by use of GENECLEANTM II kit (Stratech Scientific Ltd. or Bio 101 Inc.).
  • the kit contains: 1) 6M sodium iodide; 2) a concentrated solution of sodium chloride, Tris and EDTA for making a sodium chloride/ethanol/ water wash; 3)
  • Glassmilk (5- 10ml) is added, mixed well and left to stand for 10 min at ambient temperature.
  • the glassmilk is spun down and washed 3 times with NEW r WASH (0.5ml) from the kit.
  • the wash buffer is removed from the Glassmilk which is to dry in air.
  • the DNA is eluted by incubating the dried Glassmilk with water (5-10ml) at 55 C for 5-10 min.
  • the aqueous supernatant containing the eluted DNA is recovered by centrifugation.
  • 20 elution step can be repeated and supernatants pooled
  • Competent E. coli DH5a cells were obtained from Life Technologies Ltd (MAX efficiency DH5 ⁇ competent cells);
  • Serum free medium is OPTIMEMTM I Reduced Serum Medium, GibcoBRL Cat. No. 31985;
  • DNA preparation kit from BiolOl Inc. (cat. No 2070-400) or a similar product - the kit contains alkaline lysis solution to liberate plasmid DNA from bacterial cells and glassmilk in a spinfilter to adsorb liberated DNA which is then eluted with sterile water or 10 mM Tris- HC1, I mM EDTA, pH 7.5;
  • LIPOFECTINTM Reagent (GibcoBRL Cat. No. 18292-01 1) is a 1 : 1 (w/w) liposome formulation ofthe cationic lipid N-[l-(2,3-dioleyloxy)propyl]-n,n.n-trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE) in membrane filtered water. It binds spontaneously with DNA to form a lipid-DNA complex - see Feigner et al. in Proc. Natl. Acad. Sci. USA (1987) 84, 7431;
  • DOTMA l-(2,3-dioleyloxy)propyl]-n,n.n-trimethylammonium chloride
  • DOPE dioleoyl phosphatidylethanolamine
  • Oligonucleotide sequences were prepared in an Applied Biosystems DNA synthesiser from 5'dimethoxytrityl base-protected nucleoside-2-cyanoethyl-N,N'-di-isopropyl- phosphoramidites and protected nucleoside linked to controlled-pore glass supports on a 0.2 ⁇ mol scale, according to the protocols supplied by Applied Biosystems Inc.;
  • AMPLITAQTM available from Perkin-Elmer Cetus, is used as the source of thermostable DNA polymerase; T4 DNA ligase was obtained from New England Biolabs Inc.; and
  • Laemmli Loading Buffer is 0.125 M Tris-HCl pH 6.8, containing 2 % SDS. 2 % ⁇ -mercaptoethanol, 10 % glycerol and 0.1 % Bromophenol blue.
  • Buffer A is 200 mM Tris (hydroxymethyl)aminomethane hydrochloride (TRIS-HC1). 20 % sucrose, pH 8.0.
  • Elution Buffer is 100 mM sodium carbonate, 500 mM sodium chloride, pH 1 1.4.
  • Lysis Buffer is 50 mM Tris-HCl, 15 % sucrose, pH 8.0.
  • 1 OX Enzyme Buffer is 500 mM KCl, 100 mM Tris (pH8.3). 15 mM MgCl 2 and 0.1 % gelatin.
  • PCR Buffer is 10 mM Tris-HCl (pH 8.3), 50 mM KCL, 1.5 mM MgCk 0.125 mM each of dATP, dCTP, dGTP and dTTP.
  • Phosphate Buffer is 50 mM phosphate buffer, pH 6.5. All temperatures are in degrees centigrade.
  • the coding sequence for human pancreatic carboxypeptidase B was obtained from a human pancreatic cDNA library cloned in the ⁇ gtlO vector (Clontech. Human pancreas 5' STRETCH cDNA, HLl 163a) using PCR technology, and cloned into the plasmid vector pBluescript II KS+ (Stratagene).
  • an aliquot ofthe cDNA library (5 ⁇ l at a titre of >10 ⁇ pfu/ml) was mixed with lOOpMols of two oligonucleotide primers.
  • BPT1 and BPB1 (SEQ ID NO: 1 and SEQ ID NO: 2), dNTPs to a final concentration of 200 ⁇ M, thermostable DNA polymerase reaction buffer, and 2.5U of thermostable DNA polymerase in a final volume of lOO ⁇ l.
  • the mixture was heated at 94° for 10 minutes prior to addition to the thermostable DNA enzyme, and the PCR incubation was carried out using 30 cycles of 94° for 1.5 minutes, 50° for 2 minutes, and 72° for 2 minutes, followed by a single incubation of 72° for 9.9 minutes at the end ofthe reaction.
  • the two oligonucleotide primers were designed to allow PCR extension from the 5' ofthe gene from BPT1 (SEQ ID NO: 1), between the start of the pre-sequence and the start ofthe pro-sequence, and PCR extension back from the 3' end of the gene from BPB1(SEQ ID NO: 2), as shown in Figure 1.
  • BPT1 and BPB1 are also designed to introduce unique restriction sites, SacI and Xhol respectively, into the PCR product.
  • pBluescript II KS+ double stranded DNA (Stratagene) was restriction digested with SacI enzyme, and the product dephosphorylation treated with calf intestinal alkaline phosphatase to remove 5'phosphoryl groups and reduce re-ligation and vector background following transformation.
  • the DNA product was purified from enzyme reaction contaminants using glass-milk, and then restriction digested with Xhol enzyme. DNA of the correct size (about 2850 base pairs) was purified. Aliquots of both restricted and purified DNA samples were checked for purity and concentration estimation using agarose gel electrophoresis compared with known standards.
  • ligation mixes were prepared to clone the HCPB gene into the vector, using a molar ratio of about 1 vector to 2.5 insert (1 pBluescript II KS+ to 2.5 HCPB PCR product), and a final DNA concentration of about 2.5 ng/ ⁇ l, in the presence of T4 DNA ligase, 1 mM ATP and enzyme buffer. Following the ligation reaction the DNA mixture was used to transform E.coli strain DH5 ⁇ . Cell aliquots were plated on L-agar nutrient media containing 100 ⁇ g/ml ampicillin as selection for plasmid vector, and incubated over-night at 37°. Colonies containing plasmids with inserts of interest were identified by hybridisation.
  • the filter was then placed on a filter paper (Whatman) soaked in lOxSSC and the denatured DNA cross-linked to the nitro-cellulose by ultra violet light treatment (Spectrolinker XL- 1500 UV crosslinker).
  • the filters were then allowed to air dry at room temperature, and then pre -hybridised at 60° for one hour in a solution of 6xSSC with gentle agitation (for example using a Techne HB-ID hybridizer). Pre-hybridization blocks non-specific DNA binding sites on the filters.
  • the DNA crosslinked to the nitro-cellulose filter was hybridised with a radio-labelled 3 p .
  • rjNA probe prepared from HCPB PCR product ofthe pancreatic cDNA library (see above).
  • DNA was labelled with 50 ⁇ Ci of 32 P-dCTP ( ⁇ 3000Ci/ mMol using T7 DNA polymerase in a total volume of 50 ⁇ l (Pharmacia T7 QUICKPRIMETM kit), and the reaction allowed to proceed for 15 minutes at 37°.
  • the labelled probe was then heated to 95° for 2 minutes, to denature the double stranded DNA, immediately added to 10ml of 6xSSC at 60°, and this solution used to replace the pre-hybridisation solution on the filters. Incubation with gentle agitation was continued for about 3 hours at 60°. After this time the hybridisation solution was drained off, and the filters washed twice at 60° in 2xSSC for 15 minutes each time. Filters were then gently blotted dry, covered with cling film
  • the selected isolates were checked by PCR for inserts ofthe correct size, using primers BPTl and BPBl, (SEQ ID NO: 1 and SEQ ID NO: 2), and for priming with an internal primer BPT2 (SEQ ID NO: 3) and BPBl .
  • BPT2 is designed to prime at the end of the pro-sequence, prior to the start ofthe mature gene and to introduce an Xbal restriction site.
  • PCR screening colonies of the selected isolates were picked and dispersed into 200 ⁇ l of distilled water and heated at 100° for 10 minutes in a sealed Eppendorf tube. The suspensions were then centrifuged for 10 minutes in a microfuge to pellet cell debris, and 1 ⁇ l ofthe supernatant used as the DNA template in PCR screening. Typically, 1 ⁇ l of supernatant was mixed with 20pMols of two oligonucleotide primers, BPTl and BPBl. or BPT2 and BPBl, dNTPs to a final concentration of 200 ⁇ M, thermostable DNA polymerase reaction buffer, and 0.5U of thermostable DNA polymerase in a final ⁇ olume of 20 ⁇ l.
  • the PCR incubation was carried out using 25 cycles of 94° for 1.5 minutes. 50° for 2 minutes, and 72° for 2 minutes, followed by a single incubation of 72° for 9.9 minutes at the end ofthe reaction.
  • the PCR products were analysed for DNA ofthe correct size (about 1250 base pairs from primers BPTl to BPBl, and about 900 base pairs from primers BPT2 to BPBl, see Figure 1) by agarose gel electrophoresis.
  • Ten ofthe twelve clones gave PCR DNA products ofthe correct size.
  • Six ofthe ten clones were then taken for plasmid DNA preparation (using Qiagen Maxi kits, from 100ml of over-night culture at 37° in L-broth with lOO ⁇ g/ml ampicillin).
  • plasmid DNA preparations were then sequenced over the region of PCR product insert using an USB Sequenase DNA sequencing kit, which incorporates bacteriophage T7 DNA polymerase.
  • Each clone was sequenced using eight separate oligonucleotide primers, known as 676, 336, 337, 679, 677. 1280, 1279 and 1281 (SEQ ID NOs: 3 to 10).
  • the positioning ofthe sequencing primers within the HCPB sequence is shown diagramatically in Figure 2, primers 336. 1279, 676, 1280, 677 and 1281 being 'forward', and 337 and 679 'backwards'.
  • SEQ ID NO: 1 1 The translation ofthe DNA sequence into its polypeptide sequence is shown in SEQ ID NO: 12.
  • the start ofthe mature protein sequence is amino acid residue 109.
  • Amino acid numbered 14 marks the start ofthe putative pro-enzyme sequence. Only part of the enzyme secretion leader sequence (pre-sequence) is present in the cloned PCR generated DNA.
  • the polypeptide sequence shows an aspartate residue at position 361, which when the whole sequence is aligned with other mammalian carboxypeptidase A and B sequences indicates a B type specificity (see amino acids numbered 255 by Catasus L, et al, Biochem J., 287, 299-303, 1992, and discussion).
  • the cysteine residue at position 243 in the cloned sequence is not observed in other published human pancreatic carboxypeptidase B sequences, as highlighted by Yamamoto et al, in the Journal of Biological Chemistry, v267, 2575-2581, 1992, where she shows a gap in her sequence following the position numbered 244.
  • pICI266 in a bacterial host MSD522 suitable for controlled expression, has been deposited on 11 October 1993 with the National Collection of Industrial and Marine Bacteria Limited (Aberdeen AB2 1RY, Scotland) and has the designation NCIMB 40589.
  • a plasmid map of pICI266 is shown in Figure 3.
  • the plasmid has genes for tetracycline resistance and induction (TetA and TetR), an AraB operator and promoter sequence for inserted gene expression, and an AraC gene for expression control.
  • the promoter sequence is followed by the PelB translation leader sequence which directs the polypeptide sequence following it to the periplasm.
  • the site of gene cloning has several unique restriction sites and is followed by a phage T4 transcription terminator sequence. The DNA sequence in this region and the features for gene cloning are shown diagramatically in Figure 4.
  • HCPB DNA For the cloning of the mature HCPB sequence into pICI266 it was decided to generate HCPB DNA by PCR, and to make some alterations to the codon usage at the start ofthe mature gene to introduce E.coli preferred codons. Also, to help with detection and purification ofthe expression construct a C-term peptide tag, known as (His)5_c-myc was added to the enzyme.
  • the tag consists of 6 histi dines, a tri-peptide linker (EPE) and a peptide sequence (EQKLISEEDL) from c-myc which is recognised by the antibody 9E10 (as published by Evan et al, Mol Cell Biol, 5, 129-136, 1985, and available from Cambridge Research Biochemicals and other antibody suppliers).
  • the C-terminus is completed by the addition of an Asparagine.
  • the 6 histidine residues should allow the purification ofthe expressed protein on a metal chelate column (for example Ni-NTA Agarose from Qiagen).
  • the PCR primers are used to introduce unique restriction sites at the 5' (Fspl) and 3' (EcoRI) ofthe gene to facilitate the introduction of the PCR product into the expression vector.
  • the sequence ofthe two primers, known as FSPTS1 and 6HIS9E10R1BS1 are shown in SEQ ID NOs: 13 and 14.
  • PCRs were set up using lOOpMols of primers FSPTS1 and 6HIS9E10R1BS1 in the presence of approximately 5ng of pICI1698 DNA, dNTPs to a final concentration of 200 ⁇ M, thermostable DNA polymerase reaction buffer, and 2.5U of thermostable DNA polymerase in a final volume of lOO ⁇ l.
  • the mixture was heated at 94° for 10 minutes prior to addition to the thermostable DNA enzyme, and the PCR incubation was carried out using 30 cycles of 94° for 1.5 minutes, 50° for 2 minutes, and 72° for 2 minutes, followed by a single incubation of 72° for 9.9 minutes at the end ofthe reaction.
  • pICI266 double stranded DNA prepared using standard DNA technology (Qiagen plasmid kits or similar), was restriction digested with Kpnl enzyme, being very careful to ensure complete digestion. The enzyme was then inactivated by heating at 65° for 10 minutes, and then cooling on ice. The 3' over-hang generated by the Kpnl was then enzymatically digested by the addition of T4 DNA polymerase as recommended by the supplier (New England BioLabs), in the presence of dNTPs and incubation at 16° for 15 minutes. The reaction was stopped by inactivating the enzyme by heating at 70° for 15 minutes.
  • the DNA product was purified from enzyme reaction contaminants using glass-milk, an aliquot checked for yield by agarose gel electrophoresis, and the remainder restriction digested with EcoRI enzyme. Again care was taken to ensure complete restriction digest. DNA ofthe correct size (about 5600 base pairs) was purified. Aliquots of both restricted and purified DNA samples were checked for purity and concentration estimation using agarose gel electrophoresis compared with known standards.
  • ligation mixes were prepared to clone the HCPB gene into the vector, using a molar ratio of about 1 vector to 2.5 insert (1 pICI266 to 2.5 HCPB PCR product), and a final DNA concentration of about 2.5ng/ ⁇ l, in the presence of T4 DNA ligase, 1 mM ATP and enzyme buffer, using conditions suitable for the ligation of blunt ended DNA (Fspl to T4 DNA polymerase treated Kpnl).
  • the selected isolates were checked by PCR for inserts ofthe correct size, using primers FSPTS1 and 6HIS9E10R1BS1, (SEQ ID NO: 13 and SEQ ID NO: 14). and for priming with an internal primer BPB2 (SEQ ID NO: 6) and FSPT1.
  • BPB2 is designed to prime within the mature gene and generate a fragment of about 430 base pairs.
  • PCR screening colonies ofthe selected isolates were picked and dispersed into 200 ⁇ l of distilled water and heated at 100° for 10 minutes in a sealed tube. The suspensions were then centrifuged for 10 minutes in a microfuge to pellet cell debris, and 1 ⁇ l ofthe supernatant used as the DNA template in PCR screening. Typically. 1 ⁇ l of supernatant was mixed with 20 pMols of two oligonucleotide primers, FSPT1 and
  • the PCR incubation was carried out using 25 cycles of 94° for 1.5 minutes. 50° for 2 minutes, and 72° for 2 minutes, followed by a single incubation of 72° for 9.9 minutes at the end ofthe reaction.
  • the PCR products were analysed for DNA of the correct size (about 1000 base pairs from primers FSPTS1 to 6HIS9E10R1BS1, and about 430 base pairs from primers FSPTS 1 to BPB2) by agarose gel electrophoresis.
  • the pICI1712 plasmid DNA was transformed into calcium chloride transformation competent E.coli expression strains. Included amongst these strains were a number which were incapable of growing with arabinose as the major carbon source, and were chromosome deleted for the arabinose (Ara) operon.
  • a preferred strain is known as MSD213 (strain MCI 000 of Casadaban et al. Journal of Molecular Biology, L38, 179-208, 1980), and has the partial genotype.
  • MSD525 strain MCI 061
  • MSD525 strain MCI 061
  • E.coli strains of similar genotype, suitable for controlled expression of genes from the AraB promoter in plasmid pICI266, may be obtained from The E.coli Genetic Stock Centre, Department of Biology, Yale University, CT, USA. Selection for transformation was on L-agar nutrient media containing lO ⁇ g/ml tetracycline, over night at 37°.
  • a single colony was used to inoculate 10 ml of L-broth nutrient media containing 10 ⁇ g/ml tetracycline in a 25 ml universal container, and incubated over night at 37° with shaking.
  • the samples were denatured by heating at 100° for 10 minutes, and then centrifuged to separate the viscous cell debris from the supernatant.
  • Expression samples usually 20 ⁇ l ofthe supernatant, typically were loaded onto 17 % SDS acrylamide gels for electrophoretic separation ofthe proteins.
  • Duplicate gels were generally prepared so that one could be stained for total protein (using Coomassie or similar stain and standard conditions), and the other could be processed to indicate specific products using Western analysis.
  • the washed membrane was then incubated with the primary antibody, monoclonal 9E10 mouse anti-c-myc peptide (see above), at a suitable dilution (typically 1 in 10,000 for ascites or 1 in 40 for hybridoma culture supernatant) in PBS containing 0.05 % TweenTM 20 and 0.5 % low fat milk powder, at room temperature with gentle agitation over night.
  • the membrane was then washed 3 times at room temperature with gentle agitation for at least 5 minutes each time in PBS containing 0.05 % TWEENTM 20.
  • the washed membrane was then incubated with the secondary antibody, horseradish peroxidase labelled anti-mouse IgG (typically raised in goat, such as A4416 from Sigma), at a suitable dilution (typically 1 in 10,000) in PBS containing 0.05 % TWEENTM 20 and 0.5 % low fat milk powder, at room temperature with gentle agitation for at least three hours.
  • the membrane was then washed 3 times at room temperature with gentle agitation for at least 10 minutes each time in PBS containing 0.05 % TWEENTM 20.
  • the membrane was then processed using a chemiluminescence Western detection kit (Amersham ECLTM) and exposed against film (Amersham HYPERFILM ECLTM) for 30 seconds in the first instance, and then for appropriate times to give a clear image ofthe expressed protein bands.
  • chemiluminescence Western detection kit Amersham ECLTM
  • Amersham HYPERFILM ECLTM exposed against film
  • Other methods of similar sensitivity for the detection of peroxidase labelled proteins on membranes may be used.
  • a system has been determined for the initial purification of the native and the different mutant enzymes via two routes.
  • the preferred route is described first.
  • Recombinant E.coli cell paste containing the recombinant enzyme was taken from storage at -70° and allowed to thaw.
  • the weight of cell paste was measured in grams and the paste resuspended with the addition of Buffer A to a volume equal to the initial weight ofthe cell paste.
  • the cell suspension was incubated at room temperature for 20 minutes with occasional gentle mixing before an equal volume of distilled water was added and thoroughly mixed in.
  • the cell suspension was again incubated at room temperature for 20 minutes with occasional gentle mixing.
  • the resulting crude osmotic shockate was clarified by centrifugation at 98000 x g for 90 minutes at 4° after which the supernatant was decanted off from the pelleted insoluble fraction.
  • Deoxyribonuclease 1 was added to the supernatant to a final concentration of 0.1 mg/ml The mixture was incubated at room temperature, with continuous shaking, until the vicosity was reduced enough for it to be loaded on to a Carboxypeptidase Inhibitor CNBr activated affinity column (CNBr activated SEPHAROSETM 4B from Pharmacia) and carboxypeptidase inhibitor from potato tuber (c-0279, Sigma).
  • the supernatant was adjusted to pH8.0 and loaded on to the affinity column, pre-equilibrated with 10 mM TRIS-HCL 500 mM sodium chloride, pH 8.0. After loading the supernatant the column was washed until the absorbance ofthe flow through was back to baseline before the bound material was eluted from the column by Elution Buffer.
  • the eluted fractions were frozen at -20° whilst those containing the recombinant carboxypeptidase were determined by Western blot analysis using an anti- c-myc tag antibody ( 9E10), followed by an anti-mouse -horse radish peroxidase conjugate (A-9044, Sigma) that gave a colour reaction with exposure to 4-chloronaphthol and hydrogen peroxide.
  • 9E10 anti- c-myc tag antibody
  • A-9044 anti-mouse -horse radish peroxidase conjugate
  • Fractions containing the recombinant carboxypeptidase B were pooled, concentrated and the pH adjusted to pH 7.5 before being snap-frozen and stored at -20°. Further purification ofthe pooled sample, utilising known methods such as ion exchange and gel permeation chromatography may performed if required.
  • the second route involves the total lysis ofthe E.coli cells as opposed to a periplasmic shock, as used in the preferred route.
  • Recombinant E.coli cell paste containing the recombinant enzyme was taken and resuspended in Lysis Buffer. Lysozyme was added to a concentration of 1 mg/ml and at the same time lithium dodecyl sulphate (LDS) was added (80 ⁇ l of a 25 % solution per 25ml of suspension). The suspension was incubated on ice for 30minutes with occasional shaking, followed by the addition deoxyribonuclease 1 to a concentration of 1 mg/ml and again the suspension was incubated on ice for 30 minutes with occasion shaking. The suspension was subsequently divided in to 200ml volumes and sonicated to complete the
  • Sonicated suspensions were centrifuged at 98,000x g for 90 minutes at 4° after which the supernatant was decanted off from the pelleted insoluble fraction.
  • the supernatant was adjusted to pH 8.0 and loaded on to the affinity column, pre-equilibrated with 10 mM TRIS-HC1, 500 mM sodium chloride, pH 8.0. After loading the supernatant the column 0 was washed until the absorbance ofthe flow through was back to baseline before the bound material was eluted from the column by Elution Buffer.
  • Coomassie stained nitrocellulose blot provided Coomassie stained bands at the correct molecular weight for the recombinant carboxypeptidase B's. These bands sequenced by an automated protein/peptide sequencer using the Edman degradation technique gave positive matches for the particular recombinant carboxypeptidase B being purified. 5
  • the preHCPB gene was digested for lh at 37° with EcoRI and HindlH in a lOO ⁇ l reaction containing 100 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 50 mM NaCl, 0.025 % TRITONTM X-100, and 25u each of HindlH and EcoRI (New England Biolabs).
  • the digested fragment was purified and cloned into pBluescript (Stratagene Cloning Systems).
  • pBluescript KS+ DNA (5 ⁇ g) was digested to completion with EcoRI and HindlH (25u each) in a lOO ⁇ l reaction as described above.
  • Calf-intestinal alkaline phosphatase (1 ⁇ l; New England Biolabs. lOu/ ⁇ l) was the added to the digested plasmid to remove 5' phosphate groups and incubation continued at 37° for a further 30 minutes. Phosphatase activity was destroyed by incubation at 70° for 10 minutes.
  • the EcoRI-Hindlll cut plasmid was purified from an agarose gel as described above.
  • the EcoRI-Hindlll digested preHCPB gene (50 ng) was ligated with the above cut plasmid DNA in 20 ⁇ l of a solution containing 30 mM Tris-Hcl (pH 7.8), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP. 50 ⁇ g/ml
  • GS-System(TM) system Celltech Biologies
  • WO 86/05807 and WO 89/10404 The procedure requires cloning the preHCPB gene into the Hindlll-EcoRI region of vector pEE12 [this vector is similar to pSV2.GS described in Bebbington et al. (1992) Bio/Technology 10, 169-175, with a number of restriction sites originally present in pSV2.GS removed by site-directed mutagenesis to provide unique sites in the multi-linker region].
  • plasmids ⁇ EE12 and pMF15 were digested with EcoRI and HindlH as described above.
  • the appropriate vector (from pEE12) and insert (from pMF15) from each digest were isloated from a 1 % agarose gel and ligated together and used to transform competent DH5 ⁇ cells.
  • the transformed cells were were plated onto L agar plus ampicillin (lOO ⁇ g/ml). Colonies were screened by PCR as described above, with oligos which prime within the CMV promoter (SEQ ID NO: 23) and in the HCPB gene (SEQ ID NO: 24). Clones producing a 1.365kb PCR product were used for large scale plasmid DNA preparation and the sequence ofthe insert confirmed by DNA sequence analysis.
  • the plasmid containing the preHCPB sequence in pEE12 was named pMF48.
  • a second eukaryotic expression plasmid, pEE12 containing the prepro sequence of preproHCPB was prepared as described above.
  • a gene for preproHCPB was prepared by PCR using as template pICI1689 and oligos SEQ ID NOS: 19 and 25 to give a 1270bp PCR product.
  • the gene was digested with EcoRI and HindlH and cloned initially into pBluescript KS+ to give pMFl 8.
  • oligos SEQ ID NOS: 25 and 26 were used in a PCR to isolate a gene for the prepro sequence from pMF18.
  • PCR was performed with a hot start procedure by first incubating the mix without thermostable DNA polymerase for 5 min at 94°. Thermostable DNA polymerase (2.5u) was then added and the PCR continued through the 25 cycles as described above.
  • the 356bp fragment was purified then digested with EcoRI and HindlH and cloned into pBluescript to give pMF66 and subsequently into pEE12 (screening by PCR with SEQ ID NOS 25 and 26) to give pMF67.
  • COS-7 cells For expression in eukaryotic cells, vectors containing genes capable of expressing HCPB (amino acid residues 109 to 415 of SEQ ID NO: 12) and the pro sequence (amino acid residues 14 to 108 of SEQ ID NO: 12) were cotransfected into COS-7 cells.
  • COS cells are an African green monkey kidney cell line, CV-1, transformed with an origin-defective SV40 virus and have been widely used for short-term transient expression of a variety of proteins because of their capacity to replicate circular plasmids containing an SV40 origin of replication to very high copy number.
  • COS-1 and COS-7 There are two widely available COS cell clones.
  • the cells were incubated at 37° in a CO 2 incubator for 20h.
  • the mix of plasmid DNA in serum-free medium (200 ⁇ l) was mixed gently with LIPOFECTINTM reagent (12 ⁇ l) and incubated at ambient temperature for 15min.
  • the cells were washed with serum-free medium (2ml).
  • Serum-free medium 600 ⁇ l was added to the DNA/LIPOFECTINTM and the mix overlaid onto the cells which were incubated at 37° for 6h in a CO 2 incubator.
  • the DNA containing medium was replaced with normal DMEM containing 10% FCS and the cells incubated as before for 72h.
  • This example describes the preparation of a plasmid for expression of [G251T.D253K]HCPB-His 6 -cMyc.
  • Plasmids pMF48 (10 ⁇ g; described in Reference Example 4) and pMC46.4.1 (10 ⁇ g; described in Example 14 of International Patent Application WO 97/07769 Zeneca Ltd, published 6 March, 1997) were digested separately to completion with Xmal (10 units; New England Biolabs) in a 100 ⁇ l reaction containing 10 mM Bis Tris-Propane-HCl (pH 7.0), 10 mM magnesium chloride, 1 mM DTT at 37°.
  • the cut plasmids were purified by electrophoresis on a 1 % agarose gel (Agarose type I, Sigma A-6013 followed by excision ofthe band from the gel and isolation ofthe DNA fragment.
  • the Xma cut fragments were further digested with EcoRI (140 units; New England Biolabs) in a 100 ⁇ l reaction containing 100 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 50 mM NaCl, 0.025 % TritonTM X-100 at 37° for 1 h.
  • the digested vector fragment from pMF48 (7833 bp) and insert fragment (271 bp) from pMC46.4.1 were isolated from a 1 % agarose gel as described above.
  • the pMF48 vector fragment and pMF46.4.1 insert fragment were ligated in 20 ⁇ l of a solution containing 30 mM Tris-HCl (pH 7.8), 10 mM MgCl 2 . 10 mM
  • This example describes the preparation of a vector containing a gene for the human IgG3 heavy chain constant and hinge region.
  • a gene containing the sequence shown in SEQ ID NO: 39 was prepared by PCR by a method similar to that described by Jayaraman et al. (1991) Proc. Natl. Acad. Sci USA 88, 4084-4088.
  • IgG3A and IgG3B The gene was made in two parts, known as IgG3A and IgG3B. These were cloned separately into the SacI and Xmal sites of pBluescript KS+ (Stratagene Cloning Systems) to give vectors IgG3A-pBSIIKS+ clone A7 and IgG3B-pBSIIKS+ clone B17 respectively.
  • IgG3A was made to extend past the PmaCI restriction site (CACGTG at positions 334-339 in SEQ ID NO: 39).
  • IgG3B was made such that the 5' end ofthe sequence was upstream ofthe PmaCI restriction site.
  • the intermediate IgG3A and IgG3B vectors were cut with Afllll and PmaCI.
  • the vector fragment (2823bp) from IgG3A-pBSIIKS+ clone A7, and insert fragment from IgG3B- pBSIIKS ⁇ clone B17 (666bp) were isolated by electrophoresis in a 1 % agarose gel and purified. The fragments were ligated and the ligation mix used to transform E. coli strain DH5 ⁇ . Clones containing the required gene were identified by digestion of isolated DNA with SacI and Xmal to give a 520bp fragment. The sequence ofthe insert was confirmed by DNA sequence analysis and clone number F3 was designated IgG3-pBSIIKS+.
  • a synthetic DNA sequence of SEQ ID NO: 49 was prepared using PCR by a method similar to that described by Jayaraman et al. (1991) Proc. Natl. Acad. Sci USA 88. 4084-4088 and cloned into pNG-VKss-HuCK-Neo (NCIMB deposit no. 40799, deposited 1 l-Apr-96 at National Collection of Industrial and Marine Bacteria Limited, 23 St. Machar Drive,
  • Reaction A used the synthetic oligonucleotide sequence primers SEQ ID NOS: 50 and 51 and reaction B the synthetic oligonucleotide sequence primers SEQ ID NOS: 52 and 53.
  • the products of these PCR reactions (A and B) were fragments of length 535 base pairs and 205 base pairs respectively. These reaction products were run on a 2 % agarose gel and separated from any background products. Bands ofthe expected size were excised from the gel and recovered. Mixtures of varying amounts ofthe products A and B were made and PCR reactions performed using the synthetic oligonucleotides SEQ ID NOS: 50 and 52.
  • the resulting product (ca.700 base pairs) was digested with the restriction enzymes SacII and Xhol and the cleavage products separated on a 2 % agarose gel. The band ofthe expected 310 base pairs size was excised from the gel and recovered. This fragment was then ligated into the vector pNG3-806.077HuVKl-HuVK-Neo vector (which had been previously cut with the restriction enzymes SacII/XhoI and subsequently isolated) and thus created HuVK4 DNA sequence (SEQ ID NO: 45) in the vector pNG3-Vkss-806.077HuVK4-HuCK-Neo.
  • Plasmid pMF 161 was prepared by PCR from pMF 18 as described for the unmodified prepro sequence, but using oligos SEQ ID NOS: 7 and 28. The 359bp fragment was cloned into pBluescript to give pMF141 and subsequently into pEE12 (Screening by PCR with SEQ ID NOS: 7 and 28) to give pMFl ⁇ l.
  • Example 5 Cell supernatants diluted 1 in 80 (250 ⁇ l) were analysed for HCPB activity against Hipp-Arg (5h assay) as described in Example 5. The result is shown in Example 5 and is expressed as the percentage conversion of substrate into product at 37°.
  • pro-KDEL sequence is the natural pro sequence with a C-terminal KDEL tetrapeptide residue.
  • the amino acid sequence of pro- KDEL is shown in SEQ ID NO: 29.
  • Plasmid pMF164 was prepared by PCR from pMF18 as described for the unmodified prepro sequence, but using oligos SEQ ID NOS: 7 and 30.
  • 365bp fragment was cloned into pBluescript to give pMF149 and subsequently into pEE12 (screening by PCR with SEQ ID NOS: 7 and 30) to give pMF164.
  • Cell supernatants diluted 1 in 80 (250 ⁇ l) were analysed for HCPB activity against
  • Example 5 Hipp-Arg (5h assay) as described in Example 5. The result is shown in Example 5 and is expressed as the percentage conversion of substrate into product at 37°.
  • pro-KKAA sequence is the natural pro sequence with a C-terminal KKAA tetrapeptide residue.
  • amino acid sequence of pro- KKAA is shown in SEQ ID NO: 31.
  • Plasmid pMF165 was prepared by PCR from pMF18 as described for the unmodified prepro sequence, but using oligos SEQ ID NOS: 7 and 32. The 365bp fragment was cloned into pBluescript to give pMF145 and subsequently into pEE12 (Screening by PCR with SEQ ID NOS: 7 and 32) to give pMF165. Cell supernatants diluted 1 in 80 (250 ⁇ l) were analysed for HCPB activity against
  • Example 5 Hipp-Arg (5h assay) as described in Example 5. The result is shown in Example 5 and is expressed as the percentage conversion of substrate into product at 37°.
  • pro-SDYQRL sequence is the natural pro sequence with a C-terminal SDYQRL hexapeptide residue.
  • the amino acid sequence of pro-SDYQRL is shown in SEQ ID NO: 33.
  • Plasmid pMF166 was prepared by PCR from pMF18 as described for the unmodified prepro sequence, but using oligos SEQ ID NOS: 7 and 34.
  • the 371bp fragment was cloned into pBluescript to give pMF148 and subsequently into pEE12 (Screening by PCR with SEQ ID NOS: 7 and 34) to give pMF166.
  • Cell supernatants diluted 1 in 80 (250 ⁇ l) were analysed for HCPB activity against
  • Example 5 Hipp-Arg (5h assay) as described in Example 5. The result is shown in Example 5 and is expressed as the percentage conversion of substrate into product at 37°.
  • Example 5 Enzymic activity of recombinant HCPB against Hipp-Arg.
  • Purified human CPB produced as described in Reference Example 3, or COS cell supernatant (from Reference Example 4 and Examples 1 to 4) was assayed for its ability to convert hippuryl-L-arginine (Hipp-Arg; Sigma) to hippuric acid using a HPLC assay.
  • the reaction mixture contained either purified human CPB, or COS cell supernatant diluted 1 in 8 or 1 in 80, and 0.5 mM Hipp-Arg in 0.025 M Tris-HCL, pH 7.5 (250 ⁇ l total volume). Samples were incubated for 5 hr at 37°. The reactions were terminated by the addition of 250 ⁇ l of 40 % methanol, 60 % Phosphate Buffer, 0.2 % trifluoroacetic acid and the amount of hippuric acid generated was quantified by HPLC.
  • HPLC analysis was carried out using a Hewlett Packard 1090 Series 11 (with diode array) HPLC system. Samples (50 ⁇ l) were injected onto a HICHROM Hi-RPBTM column (25 cm) and separated using a mobile phase of 20 % methanol, 80 % Phosphate Buffer at a flow rate of lml/min. The amount of product (hippuric acid, detected at 230nm) produced was determined from calibration curves generated with known amounts of hippuric acid (Sigma-H6375). The results are expressed as the percentage conversion of substrate into product in 5 hr at 37°.
  • Plasmid pMFl ⁇ l was prepared by PCR from pMF18 as described for the unmodified prepro sequence, but using oligos SEQ ID NOS: 7 and 28. The 359 bp fragment was cloned into pBluescript to give pMF141 and subsequently into pEE12 (Screening by PCR with SEQ ID NOS: 7 and 28) to give pMFl ⁇ l .
  • Example 9 Cell supernatants diluted 1 in 80 (250 ⁇ l) were analysed for HCPB activity against Hipp-Arg (5 h assay) as described in Example 9. The result is shown in Example 9 and is expressed as the percentage conversion of substrate into product at 37°.
  • Example 7
  • Example 9 Cell supernatants diluted 1 in 80 (250 ⁇ l) were analysed for HCPB activity against Hipp-Arg (5 h assay) as described in Example 9. The result is shown in Example 9 and is expressed as the percentage conversion of substrate into product at 37°.
  • pro-SDYQRL sequence is the natural pro sequence with a C-terminal SDYQRL hexapeptide residue.
  • amino acid sequence of pro-SDYQRL is shown in SEQ ID NO: 33.
  • Plasmid pMFl ⁇ was prepared by PCR from pMF18 as described for the unmodified prepro sequence, but using oligos SEQ ID NOS: 7 and 34. The 371 bp fragment was cloned into pBluescript to give pMF148 and subsequently into pEE12 (screening by PCR with SEQ ID NOS: 7 and 34) to give pMFl ⁇ . Cell supernatants diluted 1 in 80 (250 ⁇ l) were analysed for HCPB activity against Hipp-Arg (5 h assay) as described in Example 9. The result is shown in Example 9 and is expressed as the percentage conversion of substrate into product at 37°.
  • Example 9 Enzymic activity and antigen binding activity of recombinant murine A5B7 F(ab') 2 - (HCPB) 2 fusion protein
  • COS cell supernatant (from Reference Example 13 of International Patent Application WO 97/07769, Zeneca Ltd, published 6 March, 1997) and Examples 6 to 8) was assayed for its ability to convert hippuryl-L-arginine (Hipp-Arg; Sigma) to hippuric acid using a HPLC assay.
  • the reaction mixture contained either purified human CPB. or COS cell supernatant diluted 1 in 8 or 1 in 80, and 0.5 mM Hipp-Arg in 0.025 M Tris-HCL. pH 7.5 (250 ⁇ l total volume). Samples were incubated for 5 h at 37°. The reactions were terminated by the addition of 250 ⁇ l of 40 % methanol, 60 % Phosphate Buffer. 0.2 % trifluoroacetic acid and the amount of hippuric acid generated was quantified by HPLC. HPLC analysis was carried out using a suitable HPLC system (Hewlett Packard 1090 Series 11 with diode array).
  • COS cell supernatant were also assayed for antigen binding activity in a CEA ELISA assay performed essentially as described in Reference Example 13 of International Patent
  • Example 14 Cell supernatants diluted 1 in 10 (250 ⁇ l) were analysed for enzyme activity against Hipp-Glu (5 h assay) as described in Example 14. The result is shown in Example 14 and is expressed as the percentage conversion of substrate into product (hippuric acid) at 37°.
  • Example 11
  • pro-KDEL sequence is the natural pro sequence with a C-terminal KDEL tetrapeptide residue.
  • amino acid sequence of pro- KDEL is shown in SEQ ID NO: 29.
  • Plasmid pMF164 was prepared by PCR from pMF18 as described for the unmodified prepro sequence, but using oligos SEQ ID NOS: 7 and 30. The 365bp fragment was cloned into pBluescript to give pMF149 and subsequently into pEE12 (screening by PCR with SEQ ID NOS: 7 and 30) to give pMF164.
  • Example 14 Cell supernatants diluted 1 in 10 (250 ⁇ l) were analysed for enzyme activity against Hipp-Glu (5h assay) as described in Example 14. The result is shown in Example 14 and is expressed as the percentage conversion of substrate into product (hippuric acid) at 37°.
  • pro-KKAA sequence is the natural pro sequence with a C-terminal KKAA tetrapeptide residue.
  • the amino acid sequence of pro- KKAA is shown in SEQ ID NO: 31
  • Plasmid pMF165 was prepared by PCR from pMF18 as described for the unmodified prepro sequence, but using oligos SEQ ID NOS: 7 and 32. The 365bp fragment was cloned into pBluescript to give pMF145 and subsequently into pEE12 (Screening by PCR with SEQ ID NOS: 7 and 32) to give pMF165.
  • Example 14 Cell supernatants diluted 1 in 10 (250 ⁇ l) were analysed for enzyme activity against Hipp-Glu (5 h assay) as described in Example 14. The result is shown in Example 14 and is expressed as the percentage conversion of substrate into product (hippuric acid) at 37°.
  • Example 13
  • pro-SDYQRL sequence is the natural pro sequence with a C-terminal SDYQRL hexapeptide residue.
  • amino acid sequence of pro-SDYQRL is shown in SEQ ID NO: 33.
  • Plasmid pMFl ⁇ was prepared by PCR from pMF18 as described for the unmodified prepro sequence, but using oligos SEQ ID NOS: 7 and 34. The 371bp fragment was cloned into pBluescript to give pMF148 and subsequently into pEE12 (screening by PCR with SEQ ID NOS: 7 and 34) to give pMFl ⁇ .
  • Example 14 Cell supernatants diluted 1 in 10 (250 ⁇ l) were analysed for enzyme activity against Hipp-Glu (5 h assay) as described in Example 14. The result is shown in Example 14 and is expressed as the percentage conversion of substrate into product (hippuric acid) at 37°.
  • the reaction mixture contained COS cell supernatant diluted 1 in 10 in 0.025 M Tris-HCL, pH 7.5 and 0.5 mM Hipp-Glu in 0.025 M Tris-HCL, pH 7.5 (total volume 250 ⁇ l). Samples were incubated for 5 h at 37°. The reactions were terminated by the addition of 250 ⁇ l of 30 % methanol, 70 % Phosphate Buffer, 0.2 % trifluoroacetic acid and the amount of hippuric acid generated was quantified by HPLC.
  • HPLC analysis was carried out using a Hewlett Packard 1090 Series 11 (with diode array) HPLC system. Samples (50 ⁇ l) were injected onto a reverse phase, base deactivated, octyl/octadecylsilane column (HICHROM Hi-RPBTM) (25 cm) and separated using a mobile phase of 15 % methanol, 85 % Phosphate Buffer at a flow rate of lml/min. The amount of product (hippuric acid, detected at 230nm) produced was determined from calibration curves generated with known amounts of hippuric acid (Sigma-H6375). The results are expressed as the percentage conversion of substrate into product in 5 h at 37°.
  • This Example describes the preparation of a gene encoding a humanised Fd heavy chain fragment of antibody 806.077 linked to enzyme [A248S,G251T,D253K]HCPB and its co-expression with a gene encoding a humanised light chain of 806.077 and a gene encoding the pro-L modified prodomain of human carboxypeptidase B to give the F(ab * ) 2 protein with a molecule of [A248S,G251T,D253K]HCPB at the C-terminus of each ofthe heavy chain fragments.
  • the constant and hinge regions of of the humanised Fd heavy chain fragment are derived from the human IgG3 antibody isotype.
  • the expressed protein is also referred to as antibody-enzyme fusion protein.
  • a gene encoding humanised 806.077 Fd linked to [A248S,G251T,D253K]HCPB was generated by PCR from pZEN1921 (this plasmid is also named pMC60.3 and is described in 5 Example 37 of International Patent Application WO 97/07769, Zeneca Ltd, published 6 March, 1997).
  • a first PCR was set up with template pZEN1921 (2 ng) and oligonucleofides SEQ ID NO: 35 and SEQ ID NO: 36 (100 pM of each) in PCR Buffer (100 ⁇ l). The reaction was incubated at 94 for 5 min then thermostable DNA polymerase (2.5 u, 0.5 ml) was added and the mixture overlaid with mineral oil (100 ⁇ l) and the reaction mixture incubated at 94
  • the PCR product of 536 base pairs was isolated by electrophoresis on a 1 % agarose (Agarose type I. Sigma A-6013) gel followed by excision ofthe band from the gel and isolation of the DNA fragment.
  • the PCR product of 1434 base pairs was isolated by electrophoresis on a 1 % agarose gel, purified and digested with Nhel (20 u) and Xbal (80 u) (New England Biolabs Inc.,) in a 25 total volume of 100 ⁇ l containing 10 mM Tris HCl (pH7.9), 50 mM NaCl, 10 mM MgCl 2 , 1 mM DTT and BSA (100 ⁇ g/ml) for 4 h at 37°. The resulting fragment was again isolated by electrophoresis on a 1 % agarose gel and purified.
  • vector pNG4-VHss- 806.077huVHl-HuIgG2CHl ' (10 ⁇ g; Example 1 1) was cut with Nhel and Xbal then calf intestinal alkaline phosphatase (1 ⁇ l; New England Biolabs, lOu/ ⁇ l) was added to the digested
  • the Nhel- Xbal cut plasmid was purified from an agarose gel.
  • the Nhel-Xbal digested PCR product from above (about 500 ng) was ligated with the above cut plasmid DNA (about 200 ng) in 20 ⁇ l of a solution containing 50 mM Tris-Hcl (pH 7.8), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 50 ⁇ g/ml BSA and 400u T4 DNA ligase at 25° for 4h.
  • a 1 ⁇ l aliquot ofthe reaction was used to transform 20 ⁇ l of competent E. coli DH5 ⁇ cells. Transformed cells were plated onto L-agar plus 100 ⁇ g/ml ampicillin. Potential clones containing the gene for humanised 806.077 Fd-[A248S,G251T,D253K]HCPB were identified by PCR. Each clone was subjected to PCR as described above with oligonucleofides SEQ ID NOS: 40 and 41. A sample (10 ⁇ l) ofthe PCR reaction was analysed by electrophoresis on a 1 % agarose gel. Clones containing the required gene were identified by the presence of a 512 base pairs PCR product.
  • Clones producing the 512 base pairs band were used for DNA minipreps.
  • the DNA samples were checked by digestion with HindlH and Xbal for the presence of 3751 base pairs and 1862 base pairs fragments.
  • Clones containing these fragments on digestion of the DNA with HindlH and Xbal were used for large scale plasmid DNA preparation and the sequence of the insert confirmed by DNA sequence analysis.
  • the sequence of the expected insert is shown in SEQ ID NO: 42 Ofthe clones examined above, 2 contained the expected sequence but with a single base mutation.
  • Clone 54 (also designated pMF195) had an T base at position 605 in SEQ ID NO: 42 in place ofthe A base, whereas clone 68 (also designated pMF198) had a C base at position 1825 instead of the expected T base.
  • the sequence shown in SEQ ID NO: 42 was prepared from pMF195 and and pMF198 by digesting both (10 ⁇ g of each) with Xmal (lOu) and Xbal (100 u) (New England Biolabs) in buffer (100 ⁇ l) containing 20 mM Tris acetate (pH 7.9) 50 mM potassium acetate, 10 mM Mg acetate, 1 mM DTT and BSA (100 ⁇ g/ml).
  • the 215 base pairs fragment from pMF195 and the vector fragment from pMF198 were isolated from a 1 % agarose gel and ligated together as described previously.
  • the ligation mix was used to transform competent DH5 ⁇ cells.
  • the transformed cells were plated onto L agar plus ampicillin and resulting colonies screened by digestion of the DNA with Xmal and Xbal for the presence of 5400 base pairs and 215 base pairs fragments. Positive clones were used for large scale plasmid DNA preparation and the sequence ofthe insert confirmed by DNA sequence analysis.
  • the plasmid containing the 806.077 Fd-[A248S,G251T,D253K]HCPB gene from clone number 102 was named pMF213.
  • the Hindlll-Xbal fragment from pMF213 was cloned into pEE ⁇ [this is a derivative of pEE ⁇ .hCMV - Stephens and Cockett (1989) Nucleic Acids Research 17, 7110 - in which a HindlH site upstream ofthe hCMV promoter has been converted to a BglH site] in DH5 ⁇ (screened by PCR with oligonucleofides SEQ ID NOS: 43 and 44 for a 2228 base pairs insert) to give pMF221.
  • WO 89/01036, WO 86/05807 and WO 89/10404 The procedure requires cloning the humanised antibody light chain gene into the HindlH-Xmal region of vector pEE14.
  • This vector is described by Bebbington in METHODS: A Companion to methods in Enzymolog ⁇ (1991) 2. 136-145.
  • plasmids pEE14 and pNG3-VKss- 806.077HuVK4-HuCK-Neo (Reference Example 7) were digested with HinHI and Xmal as described above.
  • the appropriate vector (from pEE14) and insert (732 base pairs from pNG3- VKss-806.077HuVK4-HuCK-Neo) from each digest were isolated from a 1 % agarose gel and ligated together and used to transform competent DH5 ⁇ cells.
  • the transformed cells v ⁇ ere were plated onto L agar plus ampicillin (100 ⁇ g/ml). Colonies were screened by restriction analysis of isloated DNA for the presence of a 732 base pairs fragment on digestion of the DNA with HindlH and Xmal. Clones producing a 732 base pairs restriction fragment were used for large scale plasmid DNA preparation and the sequence of the insert confirmed DNA sequence analysis.
  • the plasmid containing the humanised light chain sequence of SEQ ID NO: 45 in pEE14 was named pEE14-806.077HuVK4-HuCK.
  • pMF221 (10 ⁇ g) was cut with BglH (20 u) and Sail (40 U) in buffer (100 ⁇ l) containing 10 mM Tris-HCl (pH 7.9), 150 mM NaCl. 10 mM MgCl 2 , 1 mM DTT and BSA (lOO ⁇ g/ml) and the 4560 base pairs fragment isolated by agarose gel electrophoresis and purified.
  • pEE14-806.077HuVK4-HuCK was cut with BamHI (40 u) and Sail (40 u) and the 9.95 kb vector fragment isolated and ligated to the BglH-Sall fragment from pMF221 and cloned into DH5 ⁇ .
  • Colonies were screened by PCR with 2 sets of oligonucleofides (SEQ ID NOS: 40 and 41. and SEQ ID NOS: 46 and 47). Clones giving PCR products of 185 base pairs and 525 base pairs respectively were characterised by DNA sequencing. A clone with the correct sequence was named pMF228 - light chain/Fd-mutant HCPB co-expression vector in DH5 ⁇ .
  • the humanised Fd-mutant HCPB sequence is shown in SEQ ID NO: 48.
  • Residues 1 to 19 are the signal sequence
  • residues 20 to 242 are the humanised variable and IgG3 CHI region
  • residues 5 243 to 306 are the IgG3 hinge region
  • residues 307 to 613 are the mutant HCPB sequence with the changes at residues 248, 251 and 253 from the human HCPB sequence.
  • the changes in the HCPB sequence occur in SEQ ID NO: 48 at postions 554 (Ser), 557 (Thr) and 559 (Lys) respectively.
  • Western blot analysis was performed as described as follows. Aliquots (20 ⁇ l) of each supernatant sample were mixed with an equal volume of sample buffer (62.5 mM Tris, pH 6.8, 1 % SDS, 10 % sucrose and 0.05 % bromophenol blue) with and without reductant. The samples were incubated at 65° for 10 minutes before electrophoresis on a 5 8-18 % acrylamide gradient gel (EXCELTM gel system from Pharmacia Biotechnology Products) in a MULTIPHORTM II apparatus (LKB Rescue AB) according to the manufacturer's instructions.
  • sample buffer 62.5 mM Tris, pH 6.8, 1 % SDS, 10 % sucrose and 0.05 % bromophenol blue
  • the separated proteins were transfered to a membrane (HYBONDTM C-Super,Amersham International) using a NOVABLOTTM apparatus (LKB Stammer AB) according to protocols provided by the manufacturer. 0 After blotting, the membrane was air dried. The presence of antibody fragments was detected by the use of an anti-human kappa antibody (Sigma A7164, goat anti -human Kappa light chain peroxidase conjugate) used at 1 :2500 dilution. The presence of human antibody fragments was visualised using a chemiluminescence system (ECLTM detection system, Amersham International).
  • ECLTM detection system chemiluminescence system
  • Example 15 The procedure described in Example 15 was repeated but with pMFl ⁇ l, containing the gene for a pro-L modified prepro sequence, was replaced by plasmid pMF164 containing a gene for a pro-KDEL modified prepro sequence.
  • the pro-KDEL sequence is the natural pro sequence with a C-terminal KDEL tetrapeptide residue.
  • the amino acid sequence of pro- KDEL is shown in SEQ ID NO: 29. Plasmid pMF165 is described in Example 2.
  • Example 19 Cell supernatants diluted 1 in 10 (250 ⁇ l) were analysed for enzyme activity against Hipp-Glu (5 h assay) as described in Example 19. The result is shown in Example 19 and is expressed as the percentage conversion of substrate into product (hippuric acid) at 37°.
  • Example 15 The procedure described in Example 15 was repeated but with pMFl ⁇ l. containing the gene for a pro-L modified prepro sequence, was replaced by plasmid pMF165 containing a gene for a pro-KKAA modified prepro sequence.
  • the pro-KKAA sequence is the natural pro sequence with a C-terminal KKAA tetrapeptide residue.
  • the amino acid sequence of pro- KKAA is shown in SEQ ID NO: 31.
  • Plasmid pMF165 is described in Example 3.
  • Example 19 Cell supernatants diluted 1 in 10 (250 ⁇ l) were analysed for enzyme activity against Hipp-Glu (5 h assay) as described in Example 19. The result is shown in Example 19 and is expressed as the percentage conversion of substrate into product (hippuric acid) at 37°.
  • Example 18
  • Example 15 The procedure described in Example 15 was repeated but with pMFl ⁇ l, containing the gene for a pro-L modified prepro sequence, was replaced by plasmid pMFl ⁇ containing a gene for a pro- SDYQRL modified prepro sequence.
  • the pro- SDYQRL sequence is the natural pro sequence with a C-terminal KKAA tetrapeptide residue.
  • the amino acid sequence of pro- SDYQRL is shown in SEQ ID NO: 33.
  • Plasmid pMFl ⁇ is described in Example 4.
  • Cell supernatants diluted 1 in 10 (250 ⁇ l) were analysed for enzyme activity against Hipp-Glu (5 h assay) as described in Example 19. The result is shown in Example 19 and is expressed as the percentage conversion of substrate into product (hippuric acid) at 37°.
  • COS cell supernatant (from Examples 15 to 18) was assayed for its ability to convert hippuryl-L-glutamic acid (Hipp-Glu; described in Reference Example 1 of International Patent Application WO 97/07769, Zeneca Ltd, published 6 March, 1997) to hippuric acid using a HPLC assay.
  • Hipp-Glu hippuryl-L-glutamic acid
  • As a control sample the procedure described in Example 15 was repeated but with the plasmid pMFl ⁇ l (containing the gene for a pro-L modified prepro sequence) replaced by pMF67 (containing the unmodified prepro sequence) as described in Reference Example 4.
  • the reaction mixture (250 ⁇ l) contained COS cell supernatant diluted 1 in 5 in 0.025 M Tris-HCL, pH 7.5 (125 ⁇ l), and 0.5 mM Hipp-Glu in 0.025 M Tris-HCL, pH 7.5. Samples were incubated for 5 h at 37°. The reactions were terminated by the addition of 250 ⁇ l of 30 % methanol, 70 % Phosphate Buffer , 0.2 % trifluoroacetic acid and the amount of hippuric acid generated was quantified by HPLC.
  • HPLC analysis was carried out using a Hewlett Packard 1090 Series 11 (with diode array) HPLC system. Samples (50 ⁇ l) were injected onto a HICHROM Hi-RPBTM column (25 cm) and separated using a mobile phase of 15 % methanol, 85 % Phosphate Buffer at a flow rate of 1 ml/min. The amount of product (hippuric acid, detected at 230 nm) produced was determined from calibration curves generated with known amounts of hippuric acid (Sigma-H6375). The results are expressed as the percentage conversion of substrate into product in 5 h at 37°.
  • COS cell supernatant were also assayed for antigen binding activity in a CEA ELISA assay performed essentially as described in Reference Example 13 of International Patent Application WO 97/07769, Zeneca Ltd, published 6 March, 1997, but with 250 ng/well used instead of 50 ng/well of CEA.
  • the results are expressed as concentrations of fusion protein present in the supernatant. The amount of of 806.077 F(ab') 2 -
  • ⁇ [A248S,G251T,D253K]HCPB ⁇ 2 fusion protein present in the COS cell supernatants was determined from calibration curves generated with known amounts of of 806.077 F(ab') 2 - ⁇ [A248S,G251T,D253K]HCPB ⁇ 2 fusion protein.
  • the data demonstrate that co-expression of of 806.077 F(ab') 2 - ⁇ [A248S.G251T,D253K]HCPB ⁇ 2 fusion protein in the presence of pro sequences with additional C-terminal amino-acid residues is enhanced compared with expression in the presence of an unmodified pro sequence.
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO: 5:
  • MOLECULE TYPE protein (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
  • GGAATTCAGT GTGAGGTGCA GCTGCAGCAG AGCGGTCCAG GTCTCGTACG GCCTAGCCAG
  • CTACAC-TCCT CAGGACTCTA CTCCCTCAGC AGCGTGGTGA CCGTGCCCTC CAGCAGCTTG
  • ATCTCTCGCA GTGTTATCGG AACCACATTT GAGGGACGCG CTATTTACCT CCTGAAGGTT 1080

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Abstract

L'invention se situe dans le domaine de la production par recombinaison de carbopeptidase B. Elle concerne un prodomaine modifié de carboxypeptidase B qui améliore l'expression par recombinaison de cette dernière lorsqu'elle est co-exprimée à partir d'un gène séparé. Les prodomaines modifiés séparés préférés comportent au niveau de leur C-terminal des acides aminés ajoutés, plus particulièrement une des séquences suivantes: L, KDEL, KKAA ou SDYQRL. La carboxypeptidase est de préférence une carboxypeptidase B pancréatique humaine. Cette invention porte également sur des séquences polynucléotidiques correspondantes, des vecteurs, des cellules hôtes et des procédés de production par recombinaison de carboxypeptidase B.
PCT/GB1998/000415 1997-02-14 1998-02-10 Proteines WO1998035988A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000037615A1 (fr) * 1998-12-21 2000-06-29 Eli Lilly And Company Synthese par recombinaison de beta-lipotropine et d'autres peptides
WO2001023588A1 (fr) * 1999-09-29 2001-04-05 Lexicon Genetics Incorporated Carboxypeptidases humaines et polynucleotides codant ces dernieres
WO2002100431A1 (fr) * 2001-06-08 2002-12-19 The Forth Military Medical University Kit pharmaceutique comprenant une proteine de fusion carboxypeptidase humaine dirigee contre un anticorps a chaine simple de proteine plasmatique seminale et promedicament
WO2002000860A3 (fr) * 2000-06-26 2003-04-03 Sugen Inc Nouvelles proteases
WO2002008396A3 (fr) * 2000-07-21 2003-04-10 Incyte Genomics Inc Proteases
EP1632575A1 (fr) * 1999-09-29 2006-03-08 Lexicon Genetics Incorporated Carboxypeptidases humaines et polynucléotides les codant
US7291587B2 (en) 2001-11-09 2007-11-06 Novo Nordisk Healthcare A/G Pharmaceutical composition comprising factor VII polypeptides and TAFI polypeptides

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0585570A1 (fr) * 1992-08-05 1994-03-09 BEHRINGWERKE Aktiengesellschaft Greffon d'anticorps anti-granulocytes, sa préparation et usage
WO1996020011A1 (fr) * 1994-12-23 1996-07-04 Zeneca Limited Composes chimiques
WO1997042329A1 (fr) * 1996-05-04 1997-11-13 Zeneca Limited Anticorps monoclonal anti-cea, conjugues contenant cet anticorps et leur utilisation therapeutique dans un systeme adept

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0585570A1 (fr) * 1992-08-05 1994-03-09 BEHRINGWERKE Aktiengesellschaft Greffon d'anticorps anti-granulocytes, sa préparation et usage
WO1996020011A1 (fr) * 1994-12-23 1996-07-04 Zeneca Limited Composes chimiques
WO1997042329A1 (fr) * 1996-05-04 1997-11-13 Zeneca Limited Anticorps monoclonal anti-cea, conjugues contenant cet anticorps et leur utilisation therapeutique dans un systeme adept

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000037615A1 (fr) * 1998-12-21 2000-06-29 Eli Lilly And Company Synthese par recombinaison de beta-lipotropine et d'autres peptides
WO2001023588A1 (fr) * 1999-09-29 2001-04-05 Lexicon Genetics Incorporated Carboxypeptidases humaines et polynucleotides codant ces dernieres
US6441153B1 (en) 1999-09-29 2002-08-27 Lexicon Genetics Incorporated Human carboxypeptidases and polynucleotides encoding the same
US6780640B2 (en) 1999-09-29 2004-08-24 Lexicon Genetics Incorporated Human carboxypeptidases and polynucleotides encoding the same
EP1632575A1 (fr) * 1999-09-29 2006-03-08 Lexicon Genetics Incorporated Carboxypeptidases humaines et polynucléotides les codant
WO2002000860A3 (fr) * 2000-06-26 2003-04-03 Sugen Inc Nouvelles proteases
WO2002008396A3 (fr) * 2000-07-21 2003-04-10 Incyte Genomics Inc Proteases
WO2002100431A1 (fr) * 2001-06-08 2002-12-19 The Forth Military Medical University Kit pharmaceutique comprenant une proteine de fusion carboxypeptidase humaine dirigee contre un anticorps a chaine simple de proteine plasmatique seminale et promedicament
US7291587B2 (en) 2001-11-09 2007-11-06 Novo Nordisk Healthcare A/G Pharmaceutical composition comprising factor VII polypeptides and TAFI polypeptides

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