C3B INACTIVATING POLYPEPTIDE

Abstract

Polypeptides comprising a C3b binding region and a C3d inactivating region are disclosed, as well as nucleic acids and vectors encoding proteolytic such polypeptides. Also disclosed are cells and compositions comprising such polypeptides, and uses and methods using the same.

Claims

1. A polypeptide comprising a C3b binding region and a C3b inactivating region.

2. The polypeptide according to claim 1, wherein the C3b inactivating region is capable of proteolytic cleavage of C3b.

3. The polypeptide according to claim 1 or claim 2, wherein the C3b inactivating region is capable of cleaving C3 chain at positions 1303 and/or 1320.

4. The polypeptide according to any one of claims 1 to 3, wherein the C3b inactivating region comprises, or consists of, an amino acid sequence having at least 65% sequence identity to the amino acid sequence of SEQ ID NO:9.

5. The polypeptide according to any one of claims 1 to 4, wherein the C3b binding region binds to C3b in the region bound by a co-factor for Complement Factor I.

6. The polypeptide according to any one of claims 1 to 5, wherein the C3b binding region binds to C3b in the region bound by one of Complement Factor H, CR1, CD46, CD55 or C4-binding protein.

7. The polypeptide according to any one of claims 1 to 6, wherein the C3b binding region binds to C3b in the region bound by Complement Factor H, or the region bound by Complement Receptor 1 (CR1).

8. The polypeptide according to any one of claims 1 to 7, wherein the C3b binding region binds to C3b in the region bound by Complement Factor H complement control protein (CCP) domains 1-4, or the region bound by CR1 CCP domains 8-10 or 15-17.

9. The polypeptide according to any one of claims 1 to 8, wherein the C3b binding region comprises, or consists of, an amino acid sequence having at least 65% sequence identity to the amino acid sequence of SEQ ID NO:11, 13 or 14.

10. The polypeptide according to any one of claims 1 to 9, which is capable of diffusing across Bruch's membrane (BrM).

11. The polypeptide according to any one of claims 1 to 10, wherein the polypeptide is not glycosylated.

12. The polypeptide according to any one of claims 1 to 11, wherein the C3b inactivating region lacks an amino acid sequence conforming to the consensus sequence of SEQ ID NO:27.

13. The polypeptide according to any one of claims 1 to 12, wherein the polypeptide comprises a detection sequence, wherein the detection sequence comprises or consists of a cleavage site for a proteolytic enzyme, and wherein cleavage of the polypeptide with the proteolytic enzyme results in the production of a non-endogenous peptide.

14. The polypeptide according to any one of claims 1 to 13, comprising, or consisting of, an amino acid sequence having at least 65% sequence identity to the amino acid sequence of SEQ ID NO:32, 33, 34, 35, 36, 37, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 69, 70, 71, 72 or 73.

15. The polypeptide according to any one of claims 1 to 14, additionally comprising a secretory pathway sequence.

16. The polypeptide according to claim 15, wherein the secretory pathway sequence comprises one or more copies of an amino acid sequence conforming to the consensus sequence of SEQ ID NO:27, and wherein the polypeptide additionally comprises a cleavage site for removing the secretory pathway sequence.

17. The polypeptide according to claim 16, wherein the cleavage site for removing the secretory pathway sequence is a furin endoprotease cleavage site.

18. A nucleic acid encoding the polypeptide according to any one of claims 1 to 17.

19. A vector comprising the nucleic acid of claim 18.

20. A cell comprising the polypeptide according to any one of claims 1 to 17, the nucleic acid according to claim 18, or the vector according to claim 19.

21. A method for producing a polypeptide, comprising introducing into a cell a nucleic acid according to claim 18 or a vector according to claim 19, and culturing the cell under conditions suitable for expression of the polypeptide.

22. A cell which is obtained or obtainable by the method according to claim 21.

23. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 17, the nucleic acid according to claim 18, the vector according to claim 19, or the cell according to claim 20 or claim 22, and a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

24. The polypeptide according to any one of claims 1 to 16, the nucleic acid according to claim 17, the vector according to claim 18, the cell according to claim 19 or claim 21 or the pharmaceutical composition according to claim 22, for use in a method of treating or preventing a disease or condition.

25. Use of the polypeptide according to any one of claims 1 to 17, the nucleic acid according to claim 18, the vector according to claim 19, the cell according to claim 20 or claim 22 or the pharmaceutical composition according to claim 23, in the manufacture of a medicament for treating or preventing a disease or condition.

26. A method of treating or preventing a disease or condition, comprising administering to a subject the polypeptide according to any one of claims 1 to 17, the nucleic acid according to claim 18, the vector according to claim 19, the cell according to claim 20 or claim 22 or the pharmaceutical composition according to claim 23.

27. A method of treating or preventing a disease or condition in a subject, comprising modifying at least one cell of the subject to express or comprise a nucleic acid according to claim 18, or a vector according to claim 19.

28. The polypeptide, nucleic acid, vector, cell, or pharmaceutical composition for use according to claim 24, the use according to claim 25, or the method according to claim 26 to or claim 27, wherein the disease or condition is a disease or condition in which C3b or a C3b-containing complex, an activity/response associated with C3b or a C3b-containing complex, or a product of an activity/response associated with C3b or a C3b-containing complex is pathologically implicated.

29. The polypeptide, nucleic acid, vector, cell, or pharmaceutical composition for use, the use, or the method according to any one of claims 24 to 28, wherein the disease or condition is age-related macular degeneration (AMD).

30. A kit of parts comprising a predetermined quantity of the polypeptide according to any one of claims 1 to 17, the nucleic acid according to claim 18, the vector according to claim 19, the cell according to claim 20 or claim 22 or the pharmaceutical composition according to claim 23.

31. A method of detecting a polypeptide in a sample, comprising: (i) contacting a sample suspected to contain a polypeptide according to claim 13 with a proteolytic enzyme specific for the proteolytic cleavage site of the detection sequence; and (ii) detecting the presence of the non-endogenous peptide.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0257] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.

[0258] FIG. 1. Schematic representations of C3b inactivating polypeptides comprising a Complement Factor H co-factor region. The C3b inactivating polypeptides comprise the C3b binding region of Complement Factor H (i.e. CCPs 1-4), a flexible linker comprising an engineered proteolytic cleavage site (shown by *) to generate a unique peptide designed to allow detection in biological samples by mass spectrometry, and the proteolytic domain of Complement Factor I. FIG. 1A represents the polypeptide comprising the N-glycans on the proteolytic domain of Complement Factor I. FIG. 1B represents the polypeptide wherein the proteolytic domain of Complement Factor I has been mutated to remove sites for N-glycosylation. FIG. 1C represents the polypeptide wherein the proteolytic domain of Complement Factor I has been mutated to remove sites for N-glycosylation, and the polypeptide comprises a surrogate glycosylation sequence at the N-terminus of the protein, and an endopeptidase cleavage site for removing the surrogate glycosylation sequence from the protein.

[0259] FIG. 2. Photograph showing the results of western blot of analysis of C3b breakdown. * indicates iC3b product of C3b breakdown. Chimera refers to the polypeptide represented schematically in FIG. 1A.

[0260] FIG. 3. Photograph showing of Instant Blue stained gel showing band shift following enzymatic deglycosylation of the polypeptide represented schematically in FIG. 1A. dChimera refers to the deglycosylated polypeptide.

[0261] FIG. 4. Photographs of (FIG. 4A) western blot detecting the presence of soluble complement proteins in whole human serum after diffusion through BrM, and (FIG. 4B) Instant Blue stained gel showing the same diffusion pattern was observed for pure complement proteins as that seen in whole human serum.

[0262] FIG. 5. Photographs of Instant Blue stained gel analysis of C3b breakdown in the presence of different purified proteins or diffusate from BrM diffusion assay. (A) showing the results of analysis of the ability of Complement Factor I to diffuse across the BrM and breakdown C3b. (B) showing deglycosylation of native Complement Factor I (dFI) and the resulting band shift pattern. (C) showing the results of analysis of the ability of degylcosylated Complement Factor I to diffuse across the BrM and breakdown C3b.

[0263] FIG. 6. Photograph of the results of a western blot detecting the presence of the deglycosylated chimeric FH-FI polypeptide (dFH-FI) represented schematically in FIG. 1C in the sample and diffusate chambers following analysis of ability to diffuse across the BrM.

[0264] FIG. 7. Photograph showing the results of western blot of analysis of C3b breakdown assay using the deglycosylated version of the chimeric FH-FI polypeptide represented schematically in FIG. 1A (dFH-FI).

[0265] FIG. 8. Schematic representation of the distinct complementone regions in the eye maintained By Bruch's membrane (BrM).

[0266] FIG. 9. Tables and graphs showing the binding affinities of glycosylated and deglycosylated chimeric FH-FI polypeptides (9A) and FH and FHL-1 (9B) for C3b, measured by Bio-Layer Interferometry.

[0267] FIG. 10. Schematic representations of C3b inactivating polypeptides comprising Complement Receptor 1 co-factor regions. The C3b inactivating polypeptides comprise the C3b binding region CCPs 8-10 or 15-17 of Complement Receptor 1, a motif for creating a mass-spectrometry-compatible detection peptide (shown by M), a flexible linker comprising a unique tryptic peptide designed to allow detection in biological samples by mass spectrometry (shown by *), and the proteolytic domain of Complement Factor I. FIG. 10A represents the polypeptide comprising CR1 CCPs 8-10 and N-glycans on the co-factor and proteolytic domains (CR1a-FI). FIG. 10B represents the polypeptide comprising CR1 CCPs 8-10 and wherein the co-factor and proteolytic domains have been mutated to remove sites for N-glycosylation (nCR1a-FI). FIG. 10C represents the polypeptide comprising CR1 CCPs 15-17 and N-glycans on the co-factor and proteolytic domains (CR1b-FI). FIG. 10D represents the polypeptide comprising CR1 CCPs 15-17 and wherein the co-factor and proteolytic domains have been mutated to remove sites for N-glycosylation (nCR1b-FI).

EXAMPLES

[0268] In the following Examples, the inventors describe the design of chimeric, C3b inactivating polypeptides comprising the C3b binding co-factor region of Complement Factor H and the C3b inactivating proteolytic region of Complement Factor I, and the ability of deglycosylated chimeric polypeptide to diffuse across Bruch's membrane (BrM) and breakdown C3b to iC3b.

Example 1: Generation of Chimeric C3b Inactivating Polypeptides Comprising a Complement Factor H Co-Factor Region

[0269] DNA inserts encoding the amino acid sequences shown in SEQ ID NOs:32, 33 and 34 were prepared by recombinant DNA techniques, and cloned into a vector to generate constructs for recombinant expression of chimeric proteins. The amino acid sequences and features thereof are shown below.

TABLE-US-00004 [00001] [00002] [00003] [00004] [00005] [00006] [00007] [00008]

[0270] Signal peptide; 6His tag; Tobacco Etch Virus (TEV) protease cleavage site; Human Complement Factor H co-factor region (UniProt: P08603 residues, 19-264); G4S-R-(G4S).sub.2 linker: Human Complement Factor I proteolytic domain (UniProt: P05156 residues 340-574).

TABLE-US-00005 [00009] [00010] [00011] [00012] [00013] [00014] [00015] [00016]

[0271] Signal peptide; 6His tag; Tobacco Etch Virus (TEV) protease cleavage site; Human Complement Factor H co-factor region (UniProt: P08603 residues, 19-264); G4S-R-(G4S).sub.2 linker; Human Complement Factor proteolytic domain (UniProt: P05156 residues 340-574) comprising substitutions N464Q, N494Q, and N536Q.

TABLE-US-00006 [00017] [00018] [00019] [00020] [00021] [00022] [00023] [00024]

[0272] Signal peptide; Human Complement Factor H co-factor region (UniProt: P08603 residues, 19-264); G4S-R-(G4S).sub.2 linker; Human Complement Factor I proteolytic domain (UniProt: P05156 residues 340-574) comprising substitutions N464Q, N494Q, and N536Q.

[0273] FIG. 1A shows a schematic representation of the protein FH-FI having the amino acid sequence SEQ ID NO:32 after treatment with TEV protease to remove the N-terminal 6His tag.

[0274] FIG. 1B shows a schematic representation of the protein FH-FI having the amino acid sequence SEQ ID NO:32 after treatment with TEV protease to remove the N-terminal 6His tag, and treatment with peptide:N-glycosidase (PNGase) to remove N-glycans (i.e. dChimera).

[0275] A further FH-FI construct was designed encoding the sequence shown in SEQ ID NO:34, additionally comprising an N-terminal surrogate glycosylation sequence and furin endoprotease cleavage site immediately upstream of the Complement Factor H co-factor region. A schematic representation of the FH-FI protein encoded by this construct is shown in FIG. 1C. Advantageously, the polypeptide shown schematically in FIG. 1C will be secreted in aglycosyl form by cells having endogenous expression of furin endoprotease. The construct encoding this protein is therefore useful for generating cells capable of producing the non-glycosylated polypeptide in vivo, e.g. at a desired location.

Example 2: C3b Breakdown to iC3b by Chimeric C3b Inactivating Polypeptides

[0276] The ability of the chimeric C3b inactivating FH-FI polypeptides to breakdown C3b was investigated in vitro, as described in Clark et al J. Immunol (2014) 193, 4962-4970. Briefly, reactions were conducted in a total volume of 20 l. Purified C3b, Factor I and Factor H; purified C3b and chimeric FH-FI polypeptide; or purified C3b and cell culture media control were mixed together in PBS and incubated at 37 C. for 15 min. Reactions were stopped by the addition of 5 l 5SDS reducing sample buffer and boiling at 100 C. for 10 min.

[0277] C3b and the iC3b 68 kDa iC3b product were subsequently detected by western blotting. Briefly, samples were run on pre-cast 4-12% NuPAGE Bis Tris SDS gels (Thermo Fisher Scientific, Altrincham, UK) for 60 minutes at 200 V in order to ensure the resolution of any closely migrating bands, and gels were then transferred onto nitrocellulose membrane at 80 mA for 1.5 hours using semi-dry transfer apparatus in transfer buffer (25 mM Tris, 192 mM glycine, 10% (v/v) Methanol). Membranes were blocked in PBS, 10% (w/v) milk, 0.2% (w/v) BSA for 16 hours at 4 C. before the addition of anti-C3b antibody clone 755 (Cambridge Biosciences, Cambridge, UK; catalogue no. 2072). at 0.5 g/ml, in PBS, 0.2% (v/v) Tween-20 (PBS-T) for 2 hours at room temperature. Membranes were washed 230 min in PBS-T before the addition of a 1:2000 dilution of HRP-conjugated secondary antibody for 2 hours at room temperature. Membranes were washed 230 min in PBS-T before the addition of SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, catalogue no. 34080) for 3 min at room temperature. Reactive bands were detected by exposing Super RX-N X-ray film (FujiFilm, catalogue no. PPB5080) to the treated membrane for 5 min at room temperature, and developed on an automated X-ray film developer.

[0278] FIG. 2 shows the results of analysis of C3b breakdown by the chimeric FH-FI polypeptide represented schematically in FIG. 1A. FH-FI was found to be able to breakdown C3b to iC3b, as designated by the detection of the iC3b 68 kDa band (*).

Example 3: Deglycosylation of Chimeric C3b Inactivating Polypeptides

[0279] 3.1 Deglycosylation of Chimeric C3b Inactivating Polypeptide

[0280] The chimeric FH-FI polypeptide represented schematically in FIG. 1A was deglycosylated to remove N-linked glycans as follows.

[0281] Remove-iT PNGase F (New England Biolabs, catalogue no. P0706S), which is tagged with a chitin-binding domain, was used to deglycosylate (by removing N-glycans) purified chimeric polypeptide under non-denaturing conditions. 2 l of GlycoBuffer 2 (10) was added to 20 g protein, in a total volume of 18 l. After gentle mixing by aspiration, 5 l of PNGase F was added and carefully mixed by aspiration. Reactions were left in a water bath at 37 C. for 24 hours. For the subsequent removal of PNGase F, 50 l of magnetic chitin beads (New England Biolabs, catalogue no. E8036S) were washed in PBS and pelleted using a magnetic eppendorf holder. Harvested beads were applied to the deglycosylation reaction and incubated at room temperature for 10 min. Magnetic chitin beads and associated PNGase F were pelleted using the magnetic stand and the supernatant containing the deglycosylated protein collected. Deglycosylated proteins were analysed by gel electrophoresis. Pre-cast 4-12% NuPAGE Bis Tris SDS gels (Thermo Fisher Scientific, Altrincham, UK) were run for 60 minutes at 200V in order to ensure the resolution of any closely migrating bands, and gels were then stained with Instant Blue stain (Expedeon, Harston, UK) for 60 minutes at room temperature.

[0282] FIG. 3 shows that deglycosylation of the chimeric FH-FI polypeptide represented schematically in FIG. 1A causes a band shift. The increased band migration indicates of loss of glycans and consequent reduction in hydro-dynamic radius.

Example 4: Diffusion of Chimeric C3b Inactivating Polypeptides Across Bruch's Membrane

[0283] 4.1 Complement Factors I and H are Unable to Diffuse Across Bruch's Membrane

[0284] The ability of different complement proteins to diffuse across Bruch's membrane (BrM) was analysed.

[0285] Passive diffusion of soluble proteins through enriched macula BrM was performed as described Clark et al J. Immunol (2014) 193, 4962-4970. Briefly, the macular region of enriched BrM was isolated from donor eyes as described in McHarg et al., J Vis Exp (2015) 1-7 and mounted in an Ussing chamber (Harvard Apparatus, Hamden, USA); the eye tissue that the BrM was removed from did not show macroscopic evidence of AMD. Once mounted, the 5 mm diameter macular area was the only barrier between two identical compartments. Both sides of BrM were washed with 2 ml of PBS for 5 minutes at room temperature. For the experiment using whole human serum, human serum (Sigma-Aldrich, Poole, UK, catalogue no. H4522) was diluted 1:1 with PBS and 2 ml was added to the Ussing compartment designated the sample chamber. For the experiment using purified complement, the purified proteins were added to the sample chamber in PBS at 100 g/ml. After 1 minute if no leaks are detected into the second compartment (which would indicate a compromise in membrane integrity) 2 ml PBS alone was added to the second compartment of the Ussing chamber, designated the diffusate chamber, and the left at room temperature for 24 hours with gentle stirring in each compartment to avoid generating gradients of diffusing proteins. Samples from each chamber were subsequently analysed by gel electrophoresis, and either stained with Instant Blue stain (Expedeon, Harston, UK) for 60 minutes at room temperature or subjected to western blotting as described above. The following antibodies were used in western blotting experiments: anti-FI clone 271203 (R&D Systems, catalogue no. MAB3307), anti-FD clone 255706 (R&D Systems, catalogue no. MAB1824); and anti-FB clone 313011 (R&D Systems, catalogue no. MAB2739), anti-FH clone OX23, (ABcam catalogue no. ab17928), anti-C3b clone 755 (Cambridge Biosciences catalogue no. 2072), and polyclonal anti-FHL-1 antibody described in Clark et al J. Immunol (2014) 193, 4962-4970.

[0286] The results of the experiments are shown in FIGS. 4A (whole human serum) and 4B (purified complement proteins). Complement Factors H, B, I and C3b were found not to be able to diffuse through BrM, whereas Factor D and FHL-1 are able to diffuse across BrM.

[0287] Inability of Complement Factor I to diffuse across the BrM and breakdown C3b was confirmed in a C3b breakdown assay performed essentially as described in Example 2 above. Reactions were conducted in a total volume of 20 l, with 2 g purified C3b and 0.1 g FHL-1 mixed together in PBS, and 0.04 g either purified FI, or a 10 l sample taken from the diffusate chamber of a diffusion experiment in which diffusion of purified FI across the BrM was investigated.

[0288] The results are shown in FIG. 5A. It was possible to observe the breakdown of the -chain of C3b in the presence of FHL-1 and FI. Addition of FHL-1 and C3b to a sample from the diffusate chamber demonstrated a lack of C3b -chain breakdown, i.e. in an absence of FI. Assay validity was confirmed by the addition of purified FI directly to the same sample and the subsequent degradation of the C3b -chain into its constituent 43 kDa and 68 kDa iC3b breakdown products.

[0289] Complement Factor I was demonstrated not to be present in diffusate, as evidenced by the lack of iC3b products in the Diffusate+, C3b+, FHL-1+, supplemented FI- lane (i.e. lane 8 of the gel of FIG. 5A). This experiment also shows that FHL-1 alone is not capable of breaking down C3b (lanes 7 and 8 of the gel of FIG. 5A).

[0290] The inventors next investigated whether the glycosylation status of Complement Factor I is important for the ability to diffuse across the BrM. Deglycosylated Complement Factor I (designated dFI in FIG. 5B) was prepared by treatment of Complement Factor I with Remove-iT PNGase F as described in Example 3.1 above. Deglycosylation is associated with a band shift; the heavy chain band is most glycosylated and therefore shows the greatest movement (FIG. 5B).

[0291] The ability of deglycosylated Complement Factor I to diffuse across the BrM and breakdown C3b was analysed in a C3b breakdown assay performed essentially as described in Example 2 above. Reactions were conducted in a total volume of 20 l, with 2 g purified C3b and 0.1 g FHL-1 mixed together in PBS, and a 10 l sample taken from the diffusate chamber of a diffusion experiment in which diffusion of dFI across the BrM was investigated.

[0292] The results are shown in FIG. 5C, Deglycosylated Complement Factor I was demonstrated to be present in diffusate, and was able to breakdown C3b as evidenced by the presence of iC3b products in the Diffusate+, C3b+, FHL-1+ lane (i.e. lane 7 of the gel of FIG. 5C).

[0293] 4.2 Deglycosylated Chimeric FH-FI C3b Inactivating Polypeptide Diffuses Across Bruch's Membrane

[0294] Deglycosylated chimeric FH-FI polypeptide prepared as described in Example 3 was analysed for its ability to diffuse across BrM in an assay as described in Example 4.1 above.

[0295] The results of the experiment are shown in FIG. 6. The deglycosylated chimeric FH-FI polypeptide (designated dFH-FI in FIG. 6) was shown to be able to diffuse across BrM.

Example 5: Deglycosylated Chimeric FH-FI C3b-Inactivating Polypeptide Retains C3b Breakdown Activity

[0296] Deglycosylated chimeric FH-FI polypeptide prepared as described in Example 3 was analysed for its ability to breakdown C3b to iC3b in an assay as described in Example 2 above.

[0297] The results of the experiment are shown in FIG. 7. The deglycosylated chimeric FH-FI polypeptide (designated dFH-FI in FIG. 7) was shown to be able to breakdown C3b to iC3b.

Example 6: Non-Glycosylated Chimeric FH-FI Polypeptide is Assessed for the Ability to Diffuse Across Bruch's Membrane, and Retain C3b Breakdown Activity

[0298] Non-glycosylated chimeric FH-FI polypeptide, e.g. as represented schematically in FIG. 1B, is analysed for its ability to diffuse across BrM in an assay as described in Example 4.1 above.

[0299] Non-glycosylated chimeric FH-FI polypeptide, e.g. as represented schematically in FIG. 1B, is analysed for its ability to breakdown C3b to iC3b in an assay as described in Example 2 above.

Example 7: Glycosylated and Deglycosylated Chimeric FH-FI Polypeptides Demonstrate Binding Affinity for C3b

[0300] The binding affinities of glycosylated chimeric FH-FI polypeptide (e.g. as shown schematically in FIG. 1A) and deglycosylated chimeric FH-FI polypeptide (prepared as described in Example 3) for C3b were assessed.

[0301] Affinity measurements were calculated using Bio-Layer Interferometry. The natural complement regulators and C3b-binding polypeptides FH and FHL-1 were included as positive controls.

[0302] The results are shown in FIGS. 9A and 9B. Both the glycosylated and deglycosylated forms of chimeric FH-FI showed binding affinity for C3b (FIG. 9A). Glycosylated chimeric FH-FI demonstrates the strongest binding to C3b at KD 5.21e.sup.9 M. Deglycosylated chimeric FH-FI binds less strongly at KD 4.76e.sup.8 M. Both chimeric FH-FI polypeptides bind C3b more strongly than either FH (KD 5.83e.sup.7 M) or FHL-1 (KD 1.17e.sup.6 M), shown in FIG. 9B.

Example 8: Generation of Chimeric C3b Inactivating Polypeptides Comprising Complement Receptor 1 Co-Factor Regions

[0303] DNA inserts encoding the amino acid sequences shown in SEQ ID NOs:50, 51, 52 and 53 were designed, and are produced by recombinant DNA techniques, and cloned into a vector to generate constructs for recombinant expression of chimeric proteins. The amino acid sequences and features thereof are shown below.

TABLE-US-00007 [00025] [00026] [00027] [00028] [00029] [00030] [00031] [00032]

[0304] Signal peptide; 6His tag; Tobacco Etch Virus (TEV) protease cleavage site; Human Complement Receptor 1 co-factor region CCPs 8-10 (UniProt: P17927 residues 491 to 684); Motif for creating a mass-spectrometry compatible detection peptide; G4S-R-(G4S).sub.2 linker; Human Complement Factor I proteolytic domain (UniProt: P05156 residues 340-574).

TABLE-US-00008 [00033] [00034] [00035] [00036] [00037] [00038] [00039] [00040]

[0305] Signal peptide; 6His tag; Tobacco Etch Virus (TEV) protease cleavage site; Human Complement Receptor 1 co-factor region CCPs 15-17 (UniProt: P17927 residues 941 to 1134); Motif for creating a mass-spectrometry compatible detection peptide; G4S-R-(G4S).sub.2 linker; Human Complement Factor I proteolytic domain (UniProt: P05156 residues 340-574).

TABLE-US-00009 [00041] [00042] [00043] [00044] [00045] [00046] [00047] [00048]

[0306] Signal peptide; 6His tag; Tobacco Etch Virus (TEV) protease cleavage site; Human Complement Receptor 1 co-factor region CCPs 8-10 (UniProt: P17927 residues 491 to 684) comprising substitutions N509Q and N578Q; Motif for creating a mass-spectrometry-compatible detection peptide; G4S-R-(G4S).sub.2 linker; Human Complement Factor I proteolytic domain UniProt: P05156 residues 340-574) comprising substitutions N464Q, N494Q, and N536Q.

TABLE-US-00010 [00049] [00050] [00051] [00052] [00053] [00054] [00055] [00056]

[0307] Signal peptide; 6His tag; Tobacco Etch Virus (TEV) protease cleavage site; Human Complement Receptor 1 co-factor region CCPs 15-17 (UniProt: P17927 residues 941 to 1134) comprising substitutions N959Q and N1028Q; Motif for creating a mass-spectrometry-compatible detection peptide; G4S-R-(G4S).sub.2 linker; Human Complement Factor I proteolytic domain UniProt: P05156 residues 340-574) comprising substitutions N464Q, N494Q, and N536Q.

[0308] FIGS. 10A-10D shows a schematic representations of the proteins of SEQ ID NOs:50 to 53 having after treatment with TEV protease to remove the N-terminal 6His tag.

Example 9: Chimeric CR1a-FI and CR1b-FI Polypeptides are Assessed for their Ability to Diffuse Across Bruch's Membrane, and Retain C3b Breakdown Activity

[0309] The chimeric CR1a-FI and CR1b-FI polypeptides represented schematically in FIGS. 10A and 10C are analysed for their ability to breakdown C3b to iC3b in an assay as described in Example 2 above.

[0310] The chimeric CR1a-FI and CR1b-FI polypeptides represented schematically in FIGS. 10A and 10C are analysed for their ability to diffuse across BrM in an assay as described in Example 4.1 above.

[0311] Deglycosylated versions of the chimeric CR1a-FI and CR1b-FI polypeptides represented schematically in FIGS. 10A and 10C are prepared by treatment with Remove-iT PNGase F as described in Example 3.1 above. Schematics of deglycosylated chimeric CR1a-FI and CR1b-FI polypeptides are shown in FIGS. 10B and 10D.

[0312] The deglycosylated versions of chimeric CR1a-FI and CR1b-FI polypeptides are analysed for their ability to diffuse across BrM in an assay as described in Example 4.1 above, and for their ability to breakdown C3b to iC3b in an assay as described in Example 2 above.

Example 10: Non-Glycosylated Diffusion of Chimeric C3b Inactivating Polypeptides Across Bruch's Membrane

[0313] Non-glycosylated chimeric nCR1a-FI and nCR1b-FI polypeptides, e.g. as represented schematically in FIGS. 10B and 10D, are analysed for their ability to diffuse across BrM in an assay as described in Example 4.1 above.

[0314] The non-glycosylated chimeric nCR1a-FI and nCR1b-FI polypeptides, e.g. as represented schematically in FIGS. 10C and 10D, are analysed for their ability to breakdown C3b to iC3b in an assay as described in Example 2 above.