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Definitions

• the present invention relates to recombinant factor H and variants and conjugates thereof and methods of their production, as well as uses and methods of treatment involving said materials.
• FH complement system regulatory glycoprotein, factor H
• AMD age-related macular degeneration
• aHUS atypical haemolytic uraemic syndrome
• DDD dense deposit disease
• US2007/0020647 discusses the expression of human CFH in a variety of eukaryotic and prokaryotic protein-overproduction vectors and in mammalian cell lines, but only explicitly exemplifies expression in the human lung carcinoma cell line A549. The quantities of recombinant protein obtained from this cell line are not disclosed, but based on precedent and in the absence of any evidence to the contrary the amounts are expected to be inadequate for therapeutic purposes.
• WO2007/038995 describes the use of human factor H to treat aHUS.
• EP1336618 describes using full length or fragments of porcine Factor H as a soluble complement regulator, for use as a therapeutic. It is suggested that porcine factor H could be purified from pig plasma or as exemplified in this patent, made recombinantly using Baculovirus.. However, no quantification of the amount of full length porcine factor H from a standard fermentation nor any functional data for the full length protein (rather than only fragments) is shown. However, there is no disclosure or teaching of how to express functional human Factor H.
• porcine Factor H naturally carries the risk of infection with cross-species zoonotic infections. Moreover, there is not complete DNA sequence or amino acid homology between human factor H and porcine factor H (62% homology Hegasy G.A. et al., Pig complement regulator factor H: molecular cloning and functional characterization. Immunogenetics. 2003 Oct;55(7):462-71 ). It is therefore very likely autoantibodies to porcine Factor H would be made, which would again limit therapeutic usage.
• WO 2008/135237 describes use of a therapeutic which combines a short consensus repeat (SCR) of Factor H with a pathogen recognition binding molecule e.g. an antibody. It specifically mentions use of fragments/peptide chains of less than 100 amino acids ( ⁇ 2 SCRs). It does not suggest use of a full length Factor H molecule with a pathogen recognition binding molecule. Also, its focus is for the use of treating infections or for cancer, not renal or opthalmological diseases.
• SCR short consensus repeat
• FH-replacement clinical therapy is achieved by means of infusing donated pooled plasma, of which FH is only one of many protein components. It is not possible clinically to routinely obtain plasma containing only the FH Y402 allotype (which is protective against AMD); when purified in bulk from pooled plasma, FH is heterogeneous in terms of both its heterotypic and glycoform variations and hence this material is ill-suited for therapy; antibody-affinity based purification methods generally yield only small amounts (a few mg at most) of material that can be enriched only for a single variant at a specific site of variation (e.g. for Y402) but will be heterogeneous with respect to other polymorphic sites (e.g. V62I). Any use of plasma-purified human proteins would in any case may carry unacceptable risks, of infection with both unknown viral and prion proteins, and of sensitisation to contaminating plasma components, when used on the repetitive basis proposed for AMD, aHUS and DDD therapies.
• the invention is based on work carried out by the present inventors towards providing high- yield production of versions of FH tailored for animal and human trials and therapeutic applications, which is based on the use of codon-optimised chemically synthesised genes that are transfected into, for example and preferably, Pichia pastoris followed by expression in a fermentor and purification using a sequence of chromatographic procedures.
• a process for making recombinant mammalian FH comprising the steps of: expressing in a chosen host organism a codon-optimised nucleic acid sequence which encodes said mammalian FH or variants thereof and which nucleic acid sequence has been codon optimised for expression in a chosen host organism and inserted into an appropriately designed vector; in order to obtain said mammalian FH or variants thereof.
• the codon-optimised nucleic acid sequence can initially be chemically synthesised rather than cloned and mutagenised in order to generate the necessary codon optimisation.
• the methods of the present invention may produce protein yields of at least 0.5 mg of recombinant FH (or its variants) per liter of culture medium, such as at least 1 mg, 5mg, 10 mg, 50 mg, 100 mg, 200 mg or 500 mg per litre of culture medium. It will therefore be appreciated that it is possible following the methods of the present invention, when using industrial-scale fermentors, to produce hundreds of milligrams or gramsoreven kilogram-quantities of recombinant FH and variants thereof, which was simply not possible using conventionally cloned recombinantly expressed FH.
• the above process may further comprise purifying said proteins from the cell and/or culture medium in which the cell is grown.
• Purification may typically involve the use of chromatographic methodologies, such as fast-protein liquid chromatographic or high-performance (pressure) liquid chromatographic techniques known in the art.
• the nucleic-acid sequence may be designed to encode a secretion-signal sequence of amino-acid residues fused to the N-terminus of FH so that FH is secreted into the media (whereupon said signal-sequence peptide is cleaved off) and thereby it is separated from intracellular P. pastoris proteins at the outset.
• crude material may, for example, be loaded onto an affinity chromatography column, such as a heparin-sepharose column equilibrated in phosphate-buffered saline (PBS), and eluted by application of a gradient, over multiple column volumes, to PBS substituted with high salt (e.g.1 M NaCI); in a further step, FH-containing fractions from the previous step may be loaded onto, for example, an ion- exchange resin-containing column, such as a GEHealthcare-supplied onoQ column that has been equilibrated in 20 mM glycine buffer (typically pH 9.5, 150 m NaCI), and then eluted with a gradient, over many column volumes, with the equilibration buffer at the same pH but substituted with high salt (e.g. 1 M NaCI).
• PBS phosphate-buffered saline
• the preferred choice of host organism is Pichia pastoris on the grounds that no re-folding of the expressed protein is required, the protein may be secreted into the media and therefore easily accessible, and specific glycoconjugates or non-natural amino acid residues may be incorporated into the recombinant product; but other prokaryotic (e.g. Escherichia co!i) and eukaryotic (e.g. Sacchyromyces cerevisiae) host organisms may also be envisaged.
• prokaryotic e.g. Escherichia co!i
• eukaryotic e.g. Sacchyromyces cerevisiae
• the mammalian FH referred to may be human FH or FH from another primate or other mammalian FH, such as that from mouse, rat, hamster, rabbit, dog, horse, cow, pig, sheep, camel, cat, guinea pig, or the like.
• the deoxyribonucleic nucleic acid (DNA) sequence may comprise unique restriction endonuclease sites at the 5' and 3' ends of the nucleic acid, to facilitate cloning into an appropriately restricted expression vector.
• Preferred restriction sites are Pstt, BamH ⁇ , Not ⁇ and Xbal, although others may easily be envisaged by the skilled addressee.
• the nucleic acid sequence encoding FH may relate to one of a number of wild-type sequences (known in the art as polymorphic variants) or may be a mutant sequence.
• the sequence may comprise one or more single-nucleotide polymorphisms known in the art.
• Preferred single-nucleotide polymorphisms that may be incorporated, individually or in combination, into the codon-optimised nucleic acid sequence could code for the following variations in the protein sequence: Ile62 (rather than Val), Tyr402 (rather than His), Glu936 (rather than Asp) and/or Arg1210 (rather than Cys) (all numbers refer to the sequence of the encoded protein prior to cleavage of the signal sequence (Swiss-Prot: P08603.4)).
• Such single-nucleotide polymorphisms and haplotypes have been reported to be associated with a lower-than-average risk of developing AMD (Hageman GS ef al.
• a common haplotype in the complement regulatory gene factor H predisposes individuals to age-related macular degeneration.
• mutant sequences may be designed to specifically alter the FH polypeptide sequence, for example to include one or more natural (encoded) or non-naturally encoded variant amino acids as described in more detail herein below.
• the conjugate refers to a molecule that consists of a polypeptide corresponding to FH or a variant of FH to which is covalently attached, normally via one or more amino-acid residue side-chains, to a chemical moiety or moieties intended to improve the biotherapeutic properties of said molecule.
• the attached moieties could include: natural polymers such as glycosaminoglycans and their derivatives or polysialic acids, dextran (-1 ,6 polyglucose), dextran (-1 ,4 polyglucose), hyaluronic acid, and chitosans; unnatural polymers such as any of a large family of linear or branched polyethylene glycols, polyether polyols, A/-(2-hydroxypropyl) methacrylamide copolymers, poly(vinylpyrrolidone), poly(ethyleneimine), or linear polyamidoamines; or pseudosynthetic polymers, such as poly(L-lysine), poly(glutamic acid), poly(malic acid) and poly(aspartamides) (see for example The dawning era of polymer therapeutics. Duncan R. Nature Reviews Drug Discovery 2003 2:347-360).
• natural polymers such as glycosaminoglycans and their derivatives or polysialic acids, dex
• the present invention is based on an initial chemical synthesis of the codon-optimised DNA molecules encoding said FH (and variants thereof), using gene design and synthesis techniques in the art (e.g.Gene composer: database software for protein construct design, codon engineering, and gene synthesis. Lorimer D, Raymond A, Walchli J, Mixon M, Barrow A, Wallace E, Grice R, Burgin A, Stewart L. BMC Biotechnol. 2009 9:36). In this manner, the codon-optimised nucleic acid is synthesised de novo prior to cloning into a suitable expression vector.
• site-directed mutagenesis techniques known in the art to carry out codon optimisation of the FH gene would be unfeasibly time-consuming, if not impossible due to the high risk of introducing additional mutational variations during the requisite repeated rounds of site- directed mutagenesis.
• site-directed mutagenesis may be used following cloning of the synthetic codon-optimised CFH, in order to accomplish one or a combination of site-specific mutations in the product
• Codon optimisation is carried out in order to enhance the expression levels of the mammalian FH and its variants in the desired host organism, such as P. pastoris.
• Said optimisation involves one or more of the following: adapting codon bias to match that of the chosen host organism; avoiding regions of high (>80%) or low ( ⁇ 30%) GC content; minimising any potential internal TATA boxes, chi- sites and ribosome-entry sites; minimising AT-rich or GC-rich stretches of sequence, avoiding repeat sequence and RNA secondary structures, minimising any (cryptic) splice-donor and/or splice-acceptor sites; and ensuring any desired restriction endonuclease sites are only found at the extreme 5' and 3' ends of the nucleic acid to facilitate cloning.
• the DNA sequence encoding mammalian FH is a CFH sequence which has been optimised for expression in the host, P. pastoris.
• a P. pastoris codon-optimised human CFH sequence (encoding for Y at position 402, I at position 62 and E at position 936) is compared to the wild-type cDNA sequence in Figure 1. It will be appreciated that this codon-optimised sequence may be varied in order to still further optimise the sequence for overproduction in P. pastoris.
• the sequence may be easily varied in order to allow for expression of various allotypes.
• certain nucleotide bases may be changed in order to specifically alter the amino-acid residue sequence of the FH protein.
• amino-acid residues may be replaced with, for example, alternative amino-acid residues that may be rare or non-naturally occurring amino-acid residues, so as to allow for the generation of recombinant FH proteins with one or even a combination of modifications leading to: altered glycosylation patterns; reduced immunogenicity; enhanced plasma half-life; and/or site-specific conjugation with moieties designed to improve pharmacokinetic and/or pharmacodynamic properties. It will be appreciated that all such modifications can be carried out whilst taking account of any codon optimisation considerations.
• the present invention provides a nucleic acid sequence capable of expressing a FH polypeptide or variant thereof, the nucleic acid sequence being codon optimised for expression in a host organism, such as P. pastoris.
• a mammalian FH polypeptide or variant thereof obtained from a nucleic acid sequence according to the present invention.
• the sequence is codon optimised for expression by P. pastoris, in which case the nucleic acid sequence may be the codon-optimised human sequence shown in Figure 1 or any of the sequences represented in Figure 5, or be substantially similar to them.
• substantially similar is understood that the sequence is greater than 70%, 75%, 80%, 85%, 90%, 95% or even 99% identical to the sequence shown in Figures 1 or 5.
• the present invention also relates to vectors which include a codon-optimised FH-encoding DNA sequence of the present invention, host cells which are genetically engineered with said recombinant vectors, and the production and purification of the encoded FH and FH-like polypeptides by recombinant techniques, and the conjugated products of said polypeptides.
• Recombinant constructs may be introduced into host cells using well-known techniques such as infection, transduction, transfection, transvection, electroporation and transformation.
• the vector may be, for example, a phage, plasmid, viral or retroviral vector.
• Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
• the polynucleotides of interest may be contained within a vector containing a selectable marker for propagation in a host.
• a plasmid vector is introduced in the form of a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
• vectors comprising c/s-acting control regions to the polynucleotide of interest.
• Appropriate frans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
• the vectors provide for specific expression and may be inducible and/or cell type-specific. Suitable vectors include those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
• Expression vectors useful in the present invention include chromosomal-, episomal- and virus- derived vectors, for example vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as cosmids and phagemids.
• the DNA insert should be operatively linked to an appropriate promoter.
• Known bacterial promoters suitable for use in the present invention include the E.
• coli lacl and lacZ promoters the T3 and 17 promoters, the gpt promoter, the phage lambda P R and P L promoters and the tac and trp promoter.
• Suitable eukaryotic promoters include the cytomegalovirus immediate early promoter, the herpes simplex virus thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral long terminal repeats (LTRs), such as those of the Rous sarcoma virus and metallothionein promoters, such as the mouse metallothionein-l promoter. Promoters specific to P.
• pastoris include alcohol oxidase 1(AOX1), AOX2 (both methanol inducible), CUP1 (copper inducible), GAP (glycerol inducuble, constitutively active on various carbon sources), FLD1 (formaldehyde dehydrogenase gene)http://faculty.kgi.edu/cregg/PP strains.htm : pblhisix, PEX8 (moderate promoter)http://faculty.kgi.edu/cregg/PP strains.htm - pblhisix, YPT1 (moderate promoter, constitutively active on various carbon sources)http://faculty.kgi.edu/cregg PP strains.htm - pblhisix, DAS1 (dihydroxyacetone svnthase)http://faculty.kgi.edu/cregg/PP strains.htm - pblhisix, ADH1
• the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome-binding site for translation.
• the coding portion of the mature transcripts expressed by the constructs will include a translation-initiating AUG at the beginning and a termination codon appropriately positioned at the end of the nucleic acid sequence to be translated. It is facile, using synthetic genes, to optimise all of these features of the insert to maximise gene- expression levels and recombinant-protein yields.
• the expression vectors will preferably include at least one selectable marker.
• markers include e.g. dihydrofolate reductase or neomycin or zeocin resistance for eukaryotic cell culture ande.g. tetracycline or ampicillin-resistance genes for culturing in E. coli and other bacteria.
• Representative examples of appropriate hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells like P.
• yeast pastoris Kluyveromyceslactis and Sacchyromyces cerevisiae
• insect cells such as Drosophila meianogastor S2 and Spodoptera frugiperda 9 cells
• animal cells such as Chinese hamster ovary, COS and Bowes melanoma cells
• plant cells Appropriate culture media and conditions for the above-described host cells are known in the art.
• the host organism is the methylotropic yeast P. pastoris. Strains of P.
• Vectors preferred for use in bacteria include pA2, pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
• preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, p SG and pSVL available from Pharmacia.
• Vectors preferred for use in P. pastoris include pPIC9K, pHIL-D2, pHIL-S1 , pPIC3.5K, pGAPZ, pGAPZalpha, pPICZalpha-A, pPICZalpha-B, pPICZalpha-C, pPICZalpha-E, pPICZalpha-E/Uni, pPIC3.5, pPIC9, pPICZ-A, pPICZ-B, pPICZ-C, pPICZ-E from Invitrogen.
• Other suitable vectors will be readily apparent to the skilled artisan.
• introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods.
• calcium phosphate transfection DEAE-dextran mediated transfection
• cationic lipid-mediated transfection electroporation, transduction, infection or other methods.
• electroporation transduction, infection or other methods.
• introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods.
• Such methods are described in many standard laboratory manuals, such as Davis LGG et al., Basic Methods in Molecular Biology, (2 nd Ed., McGraw-Hill, 1995).
• Enhancers are c/s-acting elements of DNA, usually from about 10 to about 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
• enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early-promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
• secretion signals may be incorporated into the expressed polypeptide.
• the signals may be endogenous to the polypeptide or they may be heterologous. Examples of such sequences that may be used in P. pastoris include the native human or mouse (or other mammalian) FH-secretion signals and the yeast alpha-mating factor.
• the polypeptide of interest may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions.
• a region of additional amino-acid residues, particularly charged amino-acid residues may be added to the N terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage.
• peptide moieties may be fused to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. Additions of peptide moieties to polypeptides in order to engender secretion or excretion, to improve stability and to facilitate purification, amongst others, are familiar and routine techniques in the art.
• the FH protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion-exchange or cation-exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, reverse-phase chromatography, size-exclusion chromatography and lectin chromatography. Most preferably, heparin- affinity is followed by ion-exchange chromatography.
• amino-acid residue sequence of aFH polypeptide may be selectively varied without having a significantly detrimental effect on the structural integrity or functional properties of the protein. If such differences in sequence are contemplated, it should be remembered that there are regions of the protein that are critical to its biological activity. There will also be residues that are critical to the folding of the protein or for stabilisation of its folded structure. Some residues serve as glycosylation sites, recognised by enzymes that covalently attach glycans to, for example, Asn side-chains. In general, it may be possible to safely replace residues that contribute directly or indirectly to structure or function by other residues that are chemically similar (this is known as a conservative substitution). In the cases of amino-acid residues that contribute neither to structural integrity nor to functional sites, it may be possible to safely replace such a residue with an amino-acid residue of a different chemical nature (a non-conservative replacement).
• the invention further includes variations of the FH polypeptide which variants show substantially FH-like biological activity.
• Variants might include conservative substitutions (for example, substituting one hydrophilic residue for another, or one hydrophobic residue for another), but would be unlikely to include replacements of strongly hydrophilic residues for strongly hydrophobic ones (or vice versa).
• Variants might include conservative substitutions within N-glycosylation sites that result in loss of such sites.
• Variants may also include deletions of one or more of the 20 protein domains within the FH molecule.
• deletion of one or a combination of domains [such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or Udomains] between and including domains 8 and 18 would be unlikely to have a detrimental effect on the functionally critical individual binding sites located in domains 1-4, 6-7 (or 6-8) and 19-20.
• Variants could also include deletions of one or a combination of domains from the region of FH between and including domains 5-18 since this would preserve C3b-binding sites (1-4, and 19-20) and one (in 19-20) of two cell surface-recognition sites within FH (see e.g. A new map of glycosaminoglycan and C3b-binding sites on factor H.
• Variants might also include hybrids, in which, for example one or more deleted domains from the domains 8-18, or 5-18, regions of FH are replaced with one or more similar domains derived from other proteins, for example from complement receptor type I or type II; alternatively they might be replaced by one or more dissimilar domains derived from a wide range of other proteins such as proteins of the extracellular matrix or the clotting or complement cascades.
• Non-conservative substitutions could include substitutions with both naturally encoded amino-acid residues and a non-naturally encoded (unnatural) amino-acid residue.
• the unnatural amino-acid residue could be one that serves as a site-specific attachment sites for conjugation with chemical moieties (such as polyethylene glycols (PEGs) and other polymers), or with biochemical groups (such as glycans) that enhance the therapeutic efficacy of FH.
• chemical moieties such as polyethylene glycols (PEGs) and other polymers
• biochemical groups such as glycans
• Proteolysis results in a loss of the target protein thus lowering yield and also makes purification more difficult. Proteolysis may be reduced by recognition of proteolytic sites via computational prediction or empirical means and conservative substitutions therein. Possible modifications of particular relevance to mammalian FH include mutating one or more
• Asn residues to Gin residues in order to minimise glycosylation of the FH protein.
• one or even two Asn residues of the FH protein may be replaced by any of a large number of unnatural amino-acid residues, such as p-(propargoxy)-phenylalamine (pPpa) residues (Expanding the genetic repertoire of the methylotrophic yeast Pichia pastoris. Young TS, Ahmad I, Brock A, Schultz PG. Biochemistry 2009 48:2643-53).
• Such an unnatural residue could be further modified by PEGylation or sialylation techniques known in the art.
• the present invention provides a recombinantly expressed variant of mammalian, especially human, FH obtained from a codon-optimised nucleic acid wherein the variation comprises one or more amino-acid residue substitutions designed to modulate one or more biological properties of said FH variant as compared to a native FH.
• amino acid substitution(s) do not relate to polymorphic changes to the FH protein, as known in the art. Said substitution(s) may result in modulation of, for example, immunogenicity and/or a physiological property of said FH variant as compared to a native FH.
• Exemplar modifications include substituting one or more Asn residues for another amino acid residue, such as Gin, or a non-naturally occurring amino acid residue, such as pPpa, in order to vary the glycosylation state of said FH variant and/or allow further modification of said FH variant using chemistry known in the art in order to allow the variant to be specifically modified at said substituted sites by a molecule such as PEG or polysialyl chains.
• the target FH-like polypeptide may be: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code; or (ii) one in which one or more of the amino acid residues is conjugated with another molecule or includes a substituent group; or (iii) one in which the mature polypeptide may be covalently linked to another compound or compounds, such as a compound to increase the half-life of the polypeptide (for example, PEG or polysialic acid); or (iv) one in which additional amino acid residues are fused to the mature polypeptide, such as an IgG Fc fusion-region peptide or leader or secretion-signal sequence or a sequence which is employed for purification of the mature polypeptide or a pro-protein sequence; or (v) one with an altered (compared to the native glycoforms of FH) pattern of attached glycans
• polypeptides/variants may be used therapeutically to treat or prevent age-related macular degeneration (AMD) or to prevent or slow the progression of this disease, in genetically susceptible individuals and facilitate treatment in those with AMD, as well as in the treatment/prevention of two life-threatening kidney conditions known as atypical haemolytic uraemic syndrome (aHUS) and dense deposit disease (DDD).
• AMD age-related macular degeneration
• aHUS atypical haemolytic uraemic syndrome
• DDD dense deposit disease
• the recombinantly expressed mammalian FH of the present invention could have beneficial effects in the treatment or prevention of numerous other diseases or pathologies in which inadequate complement regulation contributes to aetiology or symptoms, for exampleAlzheimer's disease, ischemia, pre-eclampsia, early pregnancy loss, sepsis, multiple sclerosis, system lupus erythematosus and transplant rejection. See for example, Ischaemia-Reperfusion Injury: (Shah KG et al, J Surg Res. 2010 163 1:1 10-1 17; Yang J et al, Ann Surg. 2009 249 2:310-317; Zhang F et al, Regul Pept.
• the recombinant FH polypeptides and variants in accordance with the present invention may also find application in research and also in kits and the like.
• Figure 1 shows DNA sequence (Swiss-Prot: P08603.4) of native human FH; Sequence of P. pastoris codon-optimised human FH of the present invention; and an alignment of the wild-type (cDNA- derived) and codon-optimised FH gene sequences.
• Figure 2 shows western-dot-blot results from non-codon optimised FH gene expression.
• Figure 3 shows production and characterisation of recombinant complement factor H: A - Elution from an anion-exchange column ( onoQ) (A 280 in milli-absorbance units on left-hand y- axis) with a salt gradient (20 mM glycine buffer, pH 9.5, 0.12 - 1 M NaCI; conductivity on right-hand y- axis).
• onoQ anion-exchange column
• Lanes 1 -8 (reducing conditions-/. e. no disulfides present) correspond to elution volumes 23-30 .. No significant "clipping" of the polypeptide chain is evident.
• Lane 9 contains molecular weight markers (MW) as indicated on the right-hand side. Lanes 3', 4' and 5' correspond to lanes 3, 4 and 5 but were run under non-reducing conditions; the faster migration of bands in lanes 3', 4', and 5' (compared to lanes 3, 4 and 5) is typical for proteins that contain disulfide bonds.
• F -Surface plasmon resonance was used to monitor formation of the C3bBb (convertase) complex as factor D and factor B were flowed together over C3b that was amine-coupled to a CM5 (Biacore) sensor chip.
• the subsequent decline in response reflects decay of the complex as Bb is released from the chip surface.
• the rate of decay is accelerated by initiating (in this case 210 s into the natural decay process) a flow of reference FH or rFH.
• rFH is a more effective decay accelerator in this assay than plasma-purified FH.
• I - Dynamic light scattering was performed on rFH in PBS at a concentration of 1 mg/ml.
• FIG. 4(a) shows a schematic representation of human factor H (FH) showing certain SNP's and the eight N-linked glycans.
• FIG. 4(b) shows schematic representations of vector (plasmid) maps designed such that various FH molecules and variants can be prepared in accordance with the present invention. All except vector 4 (based on pPICZa-B) are based on pPIC3.5K.
• I I incorporate DNA for the human secretion signal peptide (hum. signal pept.) while vector numbers 7, 9 and lOincorporate the mouse equivalent.
• the other four vectors incorporate DNA for the yeast alpha- factor peptide with (vector number 4) or without (vectors 5, 6 and 8) EA dipeptides.
• Figure 5 is a summary of DNA sequences encoding (a) human and (b) mouse FH variants that have been inserted into vector numbers 1-11.
• Figure 6 illustrates the expression of two recombinant variants of FH.
• the sample of "all-Q" mutant of rhFH (left-hand gel) migrates as a single band during SDS-PAGE under reducing (R) and non-reducing (NR) conditions (stained by Coomassie blue).
• Endo H f (77 kDa) treatment causes no change in migration rate. This is consistent with the "all-Q" mutant having no N-glycosylation sites and being glycan-free.
• rhFH prior to purification migrates as a fuzzy band until it is Endo Hf treated (right-hand gel).
• Figure 7 is a schematic summary of a route to therapeutic versions of FH.
• Example 1 Attempted expression of non-codon-optimised DNA encoding FH Human FH-encoding DNA was amplified from cDNA, and inserted into the yeast expression vector pPICZalphaB, and KM71 H P. pastoris cells were duly transformed. Cell colonies grew on high antibiotic-containing plates, consistent with the presence of multiple copies of the gene in the transformed cells. We failed, however, to detect (on SDS-PAGE, stained with Coomassie Blue) any evidence of FH expression in mini-scale cultures. Nor was any detectable recombinant FH produced in shaker-flask cultures.
• Detection was attempted using a standard Western-blotting technique with both a commercial polyclonal anti-FH antibody and secondary antibody coupled to horseradish peroxidase. With the exception of the positive controls (consisting of the primary anti-FH antibody, the secondary antibody, and human plasma-derived FH purchased from Complement Technology, Texas) no positive signal was detectable (see Figure 2).
• Codon optimisation aimed at human FH expression in P. pastoris was carried out by consultation between the inventors and Geneart (Regensburg, Germany) using their proprietary techniques and GeneOptimizer® software.
• the nucleic acid sequence of a codon-optimised form of human FH, for expression in P. pastoris is significantly different (it has 76% sequence identity) to the native DNA sequence (see Figure 1 ).
• the codon-optimised DNA sequence was synthesised by Geneart and then cloned into an
• the vector was transformed into E. coli in order to amplify the DNA, yielding several 10s of pg of plasmid DNA. This was purified, linearised (to enhance homologous recombination) and then transformed (using electroporation) into P. pastoris strain, KM71 H. Selection of P. pastoris clones containing the expression plasmid was achieved by streaking transformed yeast onto rich-media plates containing a range of concentrations of an antibiotic marker. Colonies that grew on high antibiotic- containing plates were screened for protein expression.
• the supernatant from the fermentor was diluted one-in-five with distilled water and applied to a self-poured XK-Heparin column (Heparin FastFlow resin - from GE Healthcare). Elution was accomplished with a linear gradient, over six column volumes, from 20 mM potassium phosphate buffer (pH 6.0) to the same buffer substituted with 1 M NaCI. Fractions containing protein were pooled and the glycans were removed by incubating the sample with Endoglycosidase H-mannose binding protein fusion protein (Endo H f , New England Biolabs) at 37°C.
• Endoglycosidase H-mannose binding protein fusion protein Endoglycosidase H-mannose binding protein fusion protein
• Protein was then applied to a Concanavalin A (GE Healthcare) column and then to mannose-binding- resin (New England Biolabs) to remove P. pasfor/s-derived glycans and the Endo H f .
• Concanavalin A GE Healthcare
• mannose-binding- resin New England Biolabs
• an exoglycosydase may be utilised so as to retain more of the glycans on the recombinant product, which might enhance solubility.
• the sample was further purified on a self-poured Poros-Heparin chromatography column and eiuted, over 20 column volumes, with a linear gradient from PBS to PBS plus 1 M NaCI.
• the final purification step involved anion exchange on a MonoQ column.
• the protein was eiuted by a gradient, over 20 column volumes, from 20 mM glycine buffer (pH 9.5) to the same buffer supplemented with 1 M NaCI.
• Example 3 Further development of human and mouse FH variants using codon-optimised DNA; elaboration to enhance therapeutic efficacy
• a set of 1 1 plasmid vectors (vector numbers 1 through 1 1 ) was designed by the inventors ( Figure 4) in order to further exemplify the utility and versatility of expression of a synthetic codon-optimised gene in P. pastoris.
• This set of vectors was designed so as to allow "cutting and pasting" of DNA encoding FH between vectors so as to maximise the number of secretion pathways that could be easily explored for each of the targeted FH variants.
• the aim was to produce mouse FH in addition to human FH, since mouse FH is needed for trials in mice.
• the 11 DNA inserts (see Figure 5 for sequence information) intended for codon optimisation were designed by the inventors based on (i) the desired amino acid residue sequences, (ii) the requirement for suitable endonuclease restriction sites, (iii) the incorporation of appropriate secretion signal sequences (peptides) at the N termini of the target proteins to promote secretion into the growth media, (iv) pursuit of the strategies summarised in Figure 7aimed at amassing the information required to optimise a biotherapeutic product derived from FH.
• codon optimisation and gene synthesis to create construct numbers 1 through 1 1 were carried out by Geneart (Regensburg, Germany) using their proprietary techniques and GeneOptimizer® software. Geneart were also contracted to incorporate the 1 1 constructs into inventor-supplied plasmids to generate vector numbers 1 through 11 ( Figure 4).
• rhFH recombinant human
• rhFH recombinant human
• Example 2 we employed a pre-pro leader (signal) sequence to direct secretion of rhFH, thereby facilitating purification.
• the pro-region was separated from the target sequence by an endopeptidase (kex2 protease)-cleavage site followed by two Glu-Ala dipeptides introduced to enhance cleavage-site accessibility.
• Native sequence generation relied upon kex2 protease to remove the pro-region, followed by dipeptidyl aminopeptidase action of the ste13-gene product to perform Glu-Ala removal. Incomplete cleavage by ste13 sometimes resulted in potentially immunogenic N-terminal Glu-Ala pairs.
• Pichia pastoris normally introduces high mannose-type N-glycans at Asn-Xaa-Thr/Ser sequons resulting in heterogenous, potentially immunogenic, products. These glycans lack terminal sialic acids and are probably susceptible to rapid clearance via hepatic asialoglycoprotein receptors.
• glycosylation may assist folding and stability of the recombinant protein and in the original study we removed P. pastoris N-glycans from rhFH enzymatically after expression and before purification or after the first purification step.
• Construct number 2 was designed so that Asn residues at N- glycosylation sites are replaced with Gin residues ( Figure 5) (to create allQ-IY-hFH).
• vector number 2 allows assessment of the consequences of producing FH lacking eight normally occupied (out of nine potential) N-glycosylation sequons by mutating the relevant Asn residues to Gin residues.
• vector number 2 we produced, secreted (relying on the human-FH secretion signal sequence) and purified allQ-IY-hFH corresponding to the protective haplotype but with no N- glycosylation sites (see Figure 6).
• this material was glycan-free on the basis that no difference was observed in migration on SDS-PAGE before and after treatment with Endo H f .
• Construct 3 exploits the amber codon to allow replacement of a potentially N-glycosylated Asn residues in ⁇ -hFH with an unnatural amino acid such as p-(propargoxy)phenylalanine (pPpa) (to create unN-IY-hFH) (see Figure 5).
• pPpa p-(propargoxy)phenylalanine
• Low long-term immunogenicity and enhanced half-life are essential properties in biotherapeutics suitable for supplementation of human FH function in patients.
• Attachment of poly( ethylene) glycols (PEGs) is a proven strategy in this respect (see e.g. PEGylation, successful approach to drug delivery. Veronese FM, Pasut G. Drug Discov Today. 2005; 10:1451 -8).
• PEGylation include conjugation with biodegradable polysialic acid chains that may have advantages over PEGs where high and repeated doses are involved (see e.g. Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids.
• numerous other polymers could be conjugated to hFH to improve its biotherapeutic potential. Randomly placed PEGylation or polysialylation for example, on primary amines is straightforward but frequently results in a heterogenous product and steric interference with binding regions on the protein. Far more desirable is site-specific modification.
• Vectors 4 and 5 incorporate DNA encoding the yeast alpha-factor secretion signal peptide since it is potentially advantageous to explore secretion pathways other then the pathway that deals with the natural human FH secretion signal peptide.
• Vector 4 incorporates the codons for NH 2 -Glu-Ala, while vector 5 does not, thereby providing opportunities to examine the role of the Glu-Ala spacer in terms of efficiency of proteolytic processing of the secretion signal peptide.
• Vector 6 (utilising the alpha-factor/no-EA strategy) incorporates a construct encoding an example of a FH deletion.
• This term refers to versions of FH that are missing one or more central domains (or modules) within the region that connects together the two main C3b and GAG-binding sites proximal to the N and C termini. Such deletions represent an opportunity to create more compact version of hFH for research and therapeutic applications.
• modules 10-15 are deleted (for result, see Figure 6). It will be appreciated that given the modularity of the FH structure it is possible to delete any number or combinations of modules (or to truncate FH at either end to create FH truncations).
• Vector 11 has been designed for production of an example of a FH mutant that can readily be produced in useful amounts using our strategy.
• nine basic amino acid residues have been replaced with Gin (neutral) residues.
• the basic amino acids selected in this case form a striking electropositive patch on module 13 of human FH (The central portion of factor H (modules 10-15) is compact and contains a structurally deviant CCP module.
• Schmidt CQ Herbert AP, Mertens HD, Guariento M, Soares DC, Uhrin D, Rowe AJ, Svergun Dl, Barlow PN. J Mol Biol. 2009 Epub.
• vectors numbered 7 through 10 were designed for production of mouse FH (mFH) in P. pastoris using codon-optimised DNA. These protein products assist in the assessment of FH as a biotherapeutic in mouse-based models of disease.
• the natural mFH secretion signal sequence is exploited in vectors 7, 9 and 10 while vector 8 contains DNA for the yeast alpha-factor secretion signal (no Glu-Ala).
• Construct 7 encodes wild-type mFH and constructs 8 and 9 encode the mouse equivalents of the allQ- and unN- (/.e.amber) versions of human FH (i.e.
• Construct 10 encodes a two-amber-codon version of mFH in which the remaining glycosylation sites (except those in modules 1-4 and 19-20) have been substituted, Asn to Gin.

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