RECOMBINANT FACTOR H AND VARIANTS AND CONJUGATES THEREOF

Abstract

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 the materials.

Claims

1-20. (canceled)

21. A mammalian factor H (FH) polypeptide deletion variant encoded by a nucleic acid, wherein the nucleic acid comprises a nucleic acid sequence that is at least 85% identical to a nucleic acid deletion variant of SEQ ID NO: 2, wherein the polypeptide deletion variant comprises domains 1-4 and 19-20 of mammalian FH, and wherein the polypeptide deletion variant is capable of binding to C3b.

22. The polypeptide deletion variant of claim 21, wherein the nucleic acid comprises a nucleic acid sequence that is at least 90% identical to a nucleic acid deletion variant of SEQ ID NO: 2.

23. The polypeptide deletion variant of claim 21, wherein the nucleic acid comprises a nucleic acid sequence that is at least 95% identical to a nucleic acid deletion variant of SEQ ID NO: 2.

24. The polypeptide deletion variant thereof of claim 21, wherein the nucleic acid comprises a nucleic acid deletion variant of SEQ ID NO: 2.

25. The polypeptide deletion variant of claim 21, wherein the polypeptide deletion variant lacks domains 8-18 of FH.

26. The polypeptide deletion variant of claim 21, wherein the polypeptide deletion variant lacks domains 5-18 of FH.

27. The polypeptide deletion variant of claim 21, wherein the polypeptide deletion variant comprises any one or more of the following polymorphisms: a) a valine at the amino acid position corresponding to position 62 of the amino acid sequence encoded by Swiss-Prot P08603; b) a histidine at the amino acid position corresponding to position 402 of the amino acid sequence encoded by Swiss-Prot P08603; or c) a cysteine at the amino acid position corresponding to position 1210 of the amino acid sequence encoded by Swiss-Prot P08603.

28. The polypeptide deletion variant of claim 21, wherein the FH polypeptide deletion variant comprises any one or more of the following polymorphisms: a) an isoleucine at the amino acid position corresponding to position 62 of the amino acid sequence encoded by Swiss-Prot P08603; b) a tyrosine at the amino acid position corresponding to position 402 of the amino acid sequence encoded by Swiss-Prot P08603; or c) an arginine at the amino acid position corresponding to position 1210 of the amino acid sequence encoded by Swiss-Prot P08603.

29. The polypeptide deletion variant of claim 21, wherein the polypeptide deletion variant comprises one or more modified natural or non-naturally encoded variant amino acids at any of the amino acid positions corresponding to positions 511, 700, 784, 804, 864, 893, 1011, or 1077 of the amino acid sequence encoded by SEQ ID NO: 1.

30. A nucleic acid encoding a mammalian factor H (FH) polypeptide, wherein the nucleic acid comprises a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 2, wherein the polypeptide is capable of binding C3b.

31. The nucleic acid of claim 30, wherein the nucleic acid comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 2.

32. The nucleic acid of claim 30, wherein the nucleic acid comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 2.

33. The nucleic acid of claim 30, wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 2.

34. The nucleic acid of claim 30, wherein the nucleic acid sequence encodes a mammalian factor H (FH) polypeptide comprising any one or more of the following polymorphisms: a) a valine at the amino acid position corresponding to position 62 of the amino acid sequence encoded by Swiss-Prot P08603; b) a histidine at the amino acid position corresponding to position 402 of the amino acid sequence encoded by Swiss-Prot P08603; or c) a cysteine at the amino acid position corresponding to position 1210 of the amino acid sequence encoded by Swiss-Prot P08603.

35. The nucleic acid of claim 30, wherein the nucleic acid sequence encodes a mammalian factor H (FH) polypeptide comprising any one or more of the following polymorphisms: a) an isoleucine at the amino acid position corresponding to position 62 of the amino acid sequence encoded by Swiss-Prot P08603; b) a tyrosine at the amino acid position corresponding to position 402 of the amino acid sequence encoded by Swiss-Prot P08603; or c) an arginine at the amino acid position corresponding to position 1210 of the amino acid sequence encoded by Swiss-Prot P08603.

36. The nucleic acid of claim 30, wherein the nucleic acid sequence encodes a mammalian factor H (FH) polypeptide comprising one or more modified natural or non-naturally encoded variant amino acids at any of the amino acid positions corresponding to positions 511, 700, 784, 804, 864, 893, 1011, or 1077 of the amino acid sequence encoded by SEQ ID NO: 1.

Description

DETAILED DESCRIPTION

[0061] The present invention will now be further described by way of example and with reference to the figures which show:

[0062] FIGS. 1A-1E show 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.

[0063] FIG. 2 shows western-dot-blot results from non-codon optimised FH gene expression.

[0064] FIGS. 3A-3J shows production and characterisation of recombinant complement factor H:

[0065] FIG. 3A—Elution from an anion-exchange column (MonoQ) (A.sup.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 NaCl; conductivity on right-hand y-axis).

[0066] FIG. 3B—Fractions eluted from the MonoQ column (see FIG. 3A) were subjected to SDS-PAGE and protein bands were visualised with Coomassie blue. Lanes 1-8 (reducing conditions—i.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.

[0067] FIG. 3C—Two antibodies that recognise epitopes within the C-terminal CCP modules (domains) of FH, were used in western blots. Plasma FH (left lane) and recombinant rFH (middle lane) were detected with (i) MAb-SC47686_L20/3or Mab-Abnova-0167. MW=molecular weight markers—see right-hand side of gel (ii).

[0068] FIG. 3D—The abilities of FH (lanes 1-5) and rFH (lanes 7-11) to act as cofactors for factor I-catalysed cleavage of C3b to iC3b were assessed by visualising the 43-kDa and 68-kDa proteolytic fragments of the a′-chain using SDS-PAGE followed by Coomassie blue staining. Incubation times were 0 to 30 minutes, as indicated. Both versions of FH have similar activities in this semi-quantitative assay such that the a′-chain of C3b is completely processed within five minutes. MW =molecular weight markers of (from top) 250, 150, 100, 75, 50, 37, 25 and 20 kDa.

[0069] FIG. 3E—For comparison with FIG. 3D, the cofactor activity of soluble complement receptor type 1 (sCR1), at the same concentration was followed over the same time intervals. Note that (in agreement with literature) sCR1, but (from FIG. 3D) neither rFH nor plasma-purified FH, promoted the further degradation of the a′-chain to C3dg and a 30-kDa fragment. MW, as in FIG. 3D.

[0070] FIG. 3F—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 CMS (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. At similar concentrations (0.5 1AM), rFH is a more effective decay accelerator in this assay than plasma-purified FH. The control proteins, BSA and FH modules 19-20, have no effect on decay.

[0071] FIG. 3G—(i) and (ii)—Use of SPR to measure affinity of (i) rFH and (ii)plasma-purified FH for C3b coupled to aCM5 sensor chip (Biacore). Duplicate sensorgrams are shown for a concentration series (5.4 μM, 1.0 TIM, 0.5 JAM, 0.1 μM) flowed over 1540 response units of immobilised C3b.

[0072] (iii) and (iv)—Plots of response units versus (iii) rFH or (iv)plasma-purified FH concentrations for two different flow cells with either 1540 RUs (lower curve in each plot) or 3030 RUs (upper curve in each plot) of C3b. The dashed vertical line indicates the KD fitted in each case to both plots simultaneously, and yielding 1.4 μM for rFH and 2.9 μM for plasma-purified FH.

[0073] FIG. 3H—The candidate recombinant FH (peaks a and c correspond to double-charged and single-charged species, respectively) and an internal standard (IgG.sub.i; peaks b and d correspond to double-charged and single-charged species, respectively) were analysed on a MALDI-ToF mass spectrometer.

[0074] FIG. 3I—Dynamic light scattering was performed on rFH in PBS at a concentration of 1 mg/ml.

[0075] FIG. 3J—Sheep erythrocytes were incubated in physiological buffer, with 1.5 1AM FH modules 6-8 (negative control), 0.4 FLM plasma-purified FH or 0.4 mM rFH prior to exposure (for 20 minutes at 37 ° C.) to human serum that had been depleted of FH. The reaction was quenched and A412 was measured. The results shown were the average (plus or minus standard deviation) of four experiments.

[0076] FIG. 4A shows a schematic representation of human factor H (FH) showing certain SNP's and the eight N-linked glycans. FIG. 4B 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 pPICZα-B) are based on pPIC3.5K. Vector numbers 1-3 and 11 incorporate DNA for the human secretion signal peptide (hum. signal pept.) while vector numbers 7, 9 and 10incorporate 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. The encoded variants of FH (sequences in FIGS. 5A-5B) are indicated—the protective (prot.) and at-risk haplotypes are detailed in the text; “all-Q” and “one amber Q” or “two amber 0” refer to substitutions of Asn residues for Gln and one or two pPa residues (for example), respectively, as described in the text; “delta 10-15” indicates removal of FH domains 10-15 as described in the text; K/R indicates substitution of lysines and arginines with glutamines as described in the text.

[0077] FIGS. 5A and 5B are a summary of DNA sequences encoding (a) human and (b) mouse FH variants that have been inserted into vector numbers 1-11.

[0078] FIG. 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 Hf (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. For comparison (middle gel), rhFH (prior to purification) migrates as a fuzzy band until it is Endo Hf treated (right-hand gel). The sample of “delta10-15” rFH was eluted from an anion-exchange column and six peak fractions collected and run on SDS-PAGE under reducing (R) or (for four fractions) non-reducing (NR) conditions (right-hand gel), then stained with Coomassie blue.MW=molecular weight markers as indicated to left and right of the gels.

[0079] FIG. 7 is a schematic summary of a route to therapeutic versions of FH.

EXAMPLE 1

Attempted Expression of Non-Codon-Optimised DNA Encoding FH

[0080] 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. We next checked to see if protein expression by transformed cells could be detected under ideal expression conditions (as may be achieved in a one-litre fermentor in which oxygen and nutrient levels are maintained at near-optimal levels) and by using more sensitive detection methods (Western-dot-blot, see FIG. 2); notwithstanding these steps and even with the additional use of a larger-scale (three-litre) fermentation, no recombinant FH product could be detected.

[0081] In further attempts to find evidence for the expression of even small amounts of recombinant FH, a portion of the supernatant was concentrated (for Western-dot-blot) while the remainder was diluted (to reduce salt concentration) and loaded onto a HiTrap (GE Healthcare) heparin-affinity chromatography column at pH 6. A sample from a one-step elution (expected to wash all of the protein off in a small volume) with 1 M NaCl (in the equilibration buffer used for the HiTrap heparin column) was also assayed in a Western-dot-blot.

[0082] 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 FIG. 2).

[0083] Thus, we demonstrated that provision of multiple-milligram, let alone multiple-gram, quantities of recombinant FH from wt FH-encoding DNA, despite the use of a heterologous expression system that is known to be particularly suitable for extracellular proteins containing disulfides and that has been used for expression of shorter segments of FH, is far from a straightforward matter.

EXAMPLE 2

Development. Purification and Characterisation of Codon-Optimised Human Factor H

[0084] 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 Gene Optimizer® software.

[0085] 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 FIGS. 1A-1E).

[0086] The codon-optimised DNA sequence was synthesised by Geneart and then cloned into an Invitrogen-purchased P. pastoris-based expression vector, pPICZ alpha B-vector, which had been restricted using appropriate restriction enzymes.

[0087] The vector was transformed into E. coli in order to amplify the DNA, yielding several 10 s 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.

[0088] After filtration to remove cells, 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 NaCl. Fractions containing protein were pooled and the glycans were removed by incubating the sample with Endoglycosidase H-mannose binding protein fusion protein (Endo Hf, New England Biolabs) at 37° C. Protein was then applied to a Concanavalin A (GE Healthcare) column and then to mannose-binding-resin (New England Biolabs) to remove P. pastoris-derived glycans and the Endo H.sub.f. As an alternative to Endo H.sub.f, an exoglycosydase may be utilised so as to retain more of the glycans on the recombinant product, which might enhance solubility.

[0089] The sample was further purified on a self-poured Poros-Heparin chromatography column and eluted, over 20 column volumes, with a linear gradient from PBS to PBS plus 1 M NaCl. The final purification step involved anion exchange on a MonoQ column. The protein was eluted by a gradient, over 20 column volumes, from 20 mM glycine buffer (pH 9.5) to the same buffer supplemented with 1 M NaCl.

[0090] Exemplary results of such a purification, followed by extensive biophysical and functional characterisation and validation, are shown in FIGS. 3A-3J. The yield of protein from this procedure, that had not been optimised, was about 1.5-2.5 mg of protein from one litre.

EXAMPLE 3

Further Development of Human and Mouse FH Variants Using Codon-Optimised DNA; Elaboration to Enhance Therapeutic Efficacy.

[0091] In a first step, a set of 11 plasmid vectors (vector numbers 1 through 11) was designed by the inventors (FIGS. 4A-4B) 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.

[0092] In a second step, the 11 DNA inserts (see FIGS. 5A-5B 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 FIG. 7 aimed at amassing the information required to optimise a biotherapeutic product derived from FH.

[0093] In a third step, codon optimisation and gene synthesis to create construct numbers 1 through 11 (summarised in FIGS. 5A-5B) were carried out by Geneart (Regensburg, Germany) using their proprietary techniques and GeneOptimizer® software. Geneart were also contracted to incorporate the 11 constructs into inventor-supplied plasmids to generate vector numbers 1 through 11 (FIGS. 4A-4B).

[0094] In the production of recombinant human (rhFH) described in Example 2 we employed a pre-pro leader (signal) sequence to direct secretion of rhFH, thereby facilitating purification. In that work, 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 stel 3-gene product to perform Glu-Ala removal. Incomplete cleavage by stel3 sometimes resulted in potentially immunogenic N-terminal Glu-Ala pairs. To eliminate this possibility, codons encoding one or both of said Glu-Ala dipeptides were avoided during creation of vector number 1 and additionally construct 1 was designed to exploit the native secretion signal sequence of hFH and processing by yeast secretion-pathway enzymes. Hence, using vector number 1 the N-terminal expression artefact (NH.sub.2-Glu-Ala) that was included in our initial recombinant hFH is absent, and the presence of a previously present cloning artefact (Ala-Gly) is circumvented; in addition, using vector number 1, rhFH is in effect mutated to yield the protective haplotype (162, Y402) (creating IY-hFH).

[0095] 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. On the other hand, 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 Gln residues (FIGS. 5A-5B) (to create allQ-IY-hFH). Thus 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. Thus using 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 FIG. 6). We demonstrated that this material was glycan-free on the basis that no difference was observed in migration on SDS-PAGE before and after treatment with Endo Hf.

[0096] Construct 3 exploits the amber codon to allow replacement of a potentially N-glycosylated Asn residues in/Y-hFH with an unnatural amino acid such as p-(propargoxy)phenylalanine (pPpa) (to create unN-IY-hFH) (see FIGS. 5A-5B). 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 F M, Pasut G. Drug Discov Today. 2005; 10:1451-8). Alternatives to 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. Gregoriadis G, Jain S, Papaioannou I, Laing P. Int J Pharm 2005 300:125-30). It will be understood that 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. We are able to exploit this desirable option thanks to our use of P. pastoris as our preferred expression system. Indeed, a very significant advantage of P. pastoris over a non-yeast eukaryotic expression system is the possibility of easily replacing one or possibly two relevant Asn residues with non-naturally encoded amino acid residues (this is possible with other eukaryotic expression systems but is less straightforward and would not be expected to produce protein in the required yields).

[0097] Thus by transfecting P. pastoris with vector number 3, along with a plasmid carrying the requisite tRNAs and aminoacyl tRNA transferase, we introduce the option of site-specific covalent modification with a chemically synthesised polymer that should mask the altered residue and eliminate a glycosylation site while potentially enhancing other biotherapeutic properties of the protein. It should be noted that these residues are not directly involved in binding to other proteins since they are normally N-glycosylated and they lie within modules of FH that we have previously shown not to be involved in C3b or GAG-binding. The system for incorporation of an unnatural amino acid used is the one developed by Schultz (Expanding the genetic repertoire of the methylotrophic meast Pichia pastoris. Young T S, Ahmad I, Brock A, Schultz P G. Biochemistry 2009 48:2643-2653) for incorporation of pPpa that is suitable for side-chain modification using “click” chemistry. This utilises an orthogonal tRNA/tRNA and aminoacyl-tRNA synthetase pair developed in E. coli using directed evolution. This allows, in the first place, the biological and biophysical properties of unN-IY-hFH to be compared to those of /Y-FH (after enzymatic deglycosylation) and allQ-IY-hFH. It will be understood that another unnatural amino acid could be incorporated instead of pPpa, which would provide alternative chemical routes to conjugation; for example, we could incorporate an unnatural amino acid with an azo-group or other reactive group. Many such possibilities are discussed in the above-cited paper by Young et al the contents of which are hereby incorporated in its entirety by reference. It will also be understood that other residues besides the Asn residues in N-glycosylation sites, for example the Ser or Thr residue that is found two residues after the Asn residue, could be replaced with unnatural amino acids.

[0098] Subsequently, click chemistry is utilised to PEGylate unN-IY-hFH creating our candidate therapeutic product, PEGylated-hFH (FIG. 7); for comparison, we non-specifically PEGylate Lys residues within allQ-IY-hFH (to create PEG.sup.x-hFH). The creation of these proteins is as follows. Azo- derivitised PEGs are available commercially and these react with the propargyl group of pPpa in a Cu(I)-catalysed azide-alkyne cycloaddition to give a high yield of the 1,2,3-triazole. It will be understood that it is possible to incorporate azo-amino acid residues instead of pPpa and then to use propargyl-PEG as a conjugate. It will also be understood that conjugations with other polymers would be equally feasible. In this way we create site-specifically PEGylated versions of FH. It is possible to explore different chain lengths, and the use of branched chains. For comparison with the products of site-specific conjugation, we use well-established protocols that randomly conjugate succinamide-ester activated PEGs to primary amines of the recombinant protein (see Peptide and protein PEGylation: a review of problems and solutions. Veronese F M. Biomaterials. 2001, 22:405-17 and references therein). Using homogenous preparations of activated PEGs at appropriate stochiometric ratios and by fractionating and characterising the products, one obtains well-defined positional isomers of mono/di-PEGylated protein. These operations are performed on IY-hFH creating PEG″-IY-hFH. Thus it is possible to compare the relative merits of site-specific and random PEGylation. It will be understood that a similar approach may readily be extended to polysialylation instead of PEGylation.

[0099] With regard to comparisons of the various products—e.g. hFH, FY-hFH, allQ-IY-hFH, unN-IY-hFH, PEG-hFH and PEG.sup.x-hFH—we explore their C3b- and GAG-binding properties and their bioactivities. Thus, pure and authenticated samples are tested for the following: (i) Ability to act as a cofactor for factor I-catalysed cleavage of C3b (see FIG. 3D) (Enzymic assay of C3b receptor on intact cells and solubilised cells. Sim E, Sim R B. Biochem 1 1983, 210: 567-76); (ii) Ability to promote acceleration of decay of C3bBb assembled on a surface plasmon resonance (SPR) sensor chip (Decay-accelerating factor must bind both components of the complement alternative pathway C3 convertase to mediate efficient decay. Harris C L, Pettigrew D M, Lea S M, Morgan B P. J Immunol. 2007 178:352-9)(see FIG. 3F); (iii) Affinity for C3b immobilised on a sensor chip as measured by SPR (A new map of glycosaminoglycan and C3b-binding sites on factor H. Schmidt C Q, Herbert A P, Kavanagh D, Gandy C, Fenton C J, Blaum B S, Lyon M, Uhrin D, Barlow P N. J Immunol. 2008 181:2610-9)(see FIG. 3G; (iv) Affinity for GAGs as measured by heparin-affinity chromatography or gel-mobility shift assay (Disease-associated sequence variations congregate in a polyanion recognition patch on human factor H revealed in three-dimensional structure. Herbert A P, Uhrin D, Lyon M, Pangburn M K, Barlow P N. J Biol Chem. 2006 281:16512-20); (v) Ability to protect sheep erythrocytes from complement-mediated haemolysis by FH-depleted human sera (available from Complement Technology)—a standard biological assay for human FH (Critical role of the C-terminal domains of factor H in regulating complement activation at cell surfaces. Ferreira V P, Herbert A P, Hocking H G, Barlow P N, Pangburn M K. J Immunol. 2006 177:6308-16)(see FIG. 3J); (vi) Ability to protect human cells from complement-mediated injury (Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells. Barilla-LaBarca M K, Liszewski M K, Lambris J D, Hourcade D, Atkinson J P. J Immunol. 2002 168:6298-304; Inhibiting complement activation on cells at the step of C3 cleavage. Liszewski M K, Fang C J, Atkinson J P. Vaccine. 2008, 26 Suppl 8:122-7.sub.—

[0100] In construct number 4 two amber codons have been incorporated and the protein product is suitable for site-specific placement of a pair of conjugates. With this construct it will be possible to explore the feasibility of introducing a second PEGylation site although it is expected that there may be a decrease in yield that generally accompanies each unnatural amino acid-residue incorporation. In this example, we have chosen conjugation sites on adjacent modules (modules 12 and 13) in the middle of the protein. Not only could these sites by PEGylated without compromising binding sites lying elsewhere in the FH molecule, they could be used for attachment of fluorescent probes resulting in fluorescent versions of human FH with potential applications in fluorescent microscopy and histology as well as diagnostics. Alternatively these sites could be used for conjugation with paramagnetic moieties that can be exploited in electron paramagnetic resonance spectroscopy to provide distance measurements between probes and, by inference, structural information that will help to generate hypotheses and the design of protein engineering approaches aimed at optimising FH efficacy.

[0101] 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.sub.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.

[0102] 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. In the current example (vector 6) modules 10-15 are deleted (for result, see FIG. 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). It is also facile to replace any of these deleted domains with homologous or non-homologous domains from other proteins. 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. In this example, nine basic amino acid residues have been replaced with Gln (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 C Q, Herbert A P, Mertens H D, Guariento M, Soares D C, Uhrin D, Rowe A J, Svergun D I, Barlow P N. J Mol Biol. 2009 Epub. Oct 14.) which seems unlikely to have evolved by chance and may have an as yet unrecognised binding role in the biological mechanism of action of FH. Thus we exploit our protein production strategy both to make therapeutic proteins and to make versions of FH for assay that shed light on structure-function relationships and hence on engineering of designer versions of FH with superior therapeutic efficacy.

[0103] The subset of 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- (i.e. amber) versions of human FH (i.e. as in the human versions, one or two of the N-glycosylation sites of mFH are re-engineered as sites of site-specific conjugation) (allQ-mFH and unN-mFH). PEGylated (or polysialylated proteins) are constructed as described for hFH. 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 Gln.

[0104] To evaluate clinical potential of the protein products of vectors 1-11, we begin with the products of vectors 7-10 and test these in (i) the FH-knockout mouse (FH.sup.−/−) that has uncontrolled plasma C3 activation and develops DDD (Uncontrolled C3 activation causes membranoproliferative glomerulonephritis in mice deficient in complement factor H. Pickering M C, Cook H T, Warren J, Bygrave A E, Moss J, Walport M J, Botto M. Nat Genet 2002 31:424-8) and retinal abnormalities (Complement factor H deficiency in aged mice causes retinal abnormalities and visual dysfunction. Coffey P J, Gias C, McDermott C J, Lundh P, Pickering M C, Sethi C, Bird A, Fitzke F W, Maass A, Chen L L, Holder G E, Luthert P J, Salt T E, Moss S E, Greenwood J. Proc Natl Acad Sci USA. 2007 104:16651-6), and (ii) the FH transgenic mouse (CFH-/-delta16-20 (in which, effectively, the truncated FH consisting of modules 1-15 replaces full-length FH) that develops aHUS (Spontaneous hemolytic uremic syndrome triggered by complement factor H lacking surface recognition domains. Pickering M C, de Jorge E G, Martinez-Barricarte R, Recalde S, Garcia-Layana A, Rose K L, Moss J, Walport M J, Cook H T, de Córdoba S R, Botto M. J Exp Med 2007 204:1249-56.). We select the best candidate(s) based on a range of considerations including yield of protein, bioassays and standard toxicology studies. For example, allQ-mFH, PEG-mFH and/or PEG.sup.x-mFH (likely to have low immunogenicity) will be injected i.v./i.p. into the FH.sup.−/−− mouse. Levels of complement components C3, factor B and naturally expressed mouse FH (as well as the recombinant mFH) are measured by ELISA to titrate optimal doses of mFH needed to achieve maximal complement regulation in the serum and to assess mFH half-lives. With the dosing schedule optimised we evaluate the efficacy of mFH against DDD and retinal abnormalities. Survival, renal function (urinary albumin, serum urea) and retinal abnormalities (behavioural and electrophysiological studies) of the FH.sup.−/− mice over a period of eight months (kidney)/24 months (retina) will be assessed and compared to untreated FH.sup.−/− mice. Histological studies (light microscopy, immunofluoresence and fluorescent and electron microscopy) are used to assess differences in glomerular and retinal pathology in the two groups. Any generation of antibodies against mFH in these FH-deficient mice is assessed by ELISA-based assays. The utility of our product(s) in aHUS is determined in analogous experiments in the CFH.sup.−/− delta 16-20 mouse.

[0105] We are continuing to improve the yields of hFH by further DNA manipulation and optimisation of fermentation technology, aiming to achieve production levels in the region of grams of protein per 10-litre fermentation. In the literature on P. pastoris, expression levels of 100-500 mg or more protein per litre have been reported. Numerous strategies available for the improvement of yield include: further enhancements of DNA sequence to decrease RNA secondary structure; elimination of potential proteolytic sites where possible; wider screening and selection for high copy-number transformants arising from multiple integration events; choice of culture conditions e.g. agitation, oxygen supply, pH, temperature, and addition of reagents (e.g. EDTA, amine salts, casamino acids) to minimize proteolysis; timing and rates of glycerol/methanol feeds (reviewed in for example Expression of recombinant proteins in Pichia pastoris. Li P, Anumanthan A, Gao X G, Ilangovan K, Suzara V V, Duzgune N, Renugopalakrishnan V.Appl Biochem Biotechnol. 2007 142:105-24).