POLYPEPTIDES AND USES THEREOF

Abstract

The invention relates to polypeptides, and in particular to polypeptides that are capable of inhibiting the activity or activation of the complement system. It also relates to nucleic acids that encode the polypeptides and to uses of the polypeptides.

The complement system helps or complements the ability of antibodies and phagocytic cells to clear pathogens from an organism. It forms part of the innate immune system. Down-regulation of complement activation has been demonstrated to be effective in treating several disease indications in animal models and in ex vivo studies. The present invention provides novel polypeptides that can be used for the treatment of diseases or disorders that relate to inappropriate activation of one or more of the complement pathways.

Claims

1. An isolated polypeptide comprising or consisting of: (a) the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; (b) a variant amino acid sequence having at least 60% sequence identity to (a); (c) an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to (a); or (d) an active fragment of (a), (b) or (c) that is at least 40, 42, 50, 60, 65, 70 or 75 amino acids in length.

2. The isolated polypeptide according to claim 1 consisting of: (a) the sequence set out in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; (b) a variant amino acid sequence having at least 60% sequence identity to (a); (c) an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to (a); or (d) an active fragment of (a), (b) or (c) that is at least 40, 42, 50, 60, 65, 70 or 75 amino acids in length.

3. An isolated polypeptide according to claim 1 or claim 2 comprising or consisting of: (e) the sequence set out in SEQ ID NO 12, 13, 14, 15, 16, 17, 18 or 19; or (f) an active variant amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to (e)

4. The isolated polypeptide according to any one of claims 1 to 3 comprising the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 12, 13, 14, 15, 16, 17, 18 or 19 with additional amino acids at one and/or both ends.

5. The isolated polypeptide according to any one of the preceding wherein the polypeptide consists of a fusion protein comprising a sequence according to (a), (b), (c) (d), (e) or (f) fused to one or more further polypeptides at the N- and/or C-terminal end.

6. The isolated polypeptide according to any one of the preceding claims wherein the polypeptide reduces the activity of the complement pathway or inhibits activation of the complement pathway.

7. The isolated polypeptide according to any one of the preceding claims wherein the polypeptide reduces the activity of or inhibits activation of the classical complement pathway, alternative complement pathway and/or the lectin mediated complement pathway.

8. The isolated polypeptide according to any one of the preceding claims wherein the polypeptide reduces the activity of or inhibits the activation of the classical complement pathway and/or lectin mediated complement pathway to a greater extent than it reduces the activity of or inhibits the activation of the alternative complement pathway.

9. The isolated polypeptide according to any one of the preceding claims wherein the polypeptide reduces complement activation in a suitable assay by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100%.

10. The isolated polypeptide according to any one of the preceding claims wherein the polypeptide reduces the total activity of the complement system in one or more specific tissues by more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80% more than 90%, more than 95%, more than 98%, more than 99% or the polypeptide may reduce the activity of the complement system in one or more specific tissues by 100%.

11. The isolated polypeptide according to any one of the preceding claims wherein the polypeptide binds to C5 and inhibits activation of C5, for example by a C5 convertase.

12. A polynucleotide encoding a polypeptide of the invention.

13. An expression vector, comprising a polynucleotide according to claim 12, for example a gene therapy vector.

14. A host cell comprising the polynucleotide according to claim 12 and/or the vector according to claim 13.

15. A composition comprising one or more isolated polypeptides according to any one of claims 1 to 11.

16. A pharmaceutical composition comprising one or more of (i) an isolated polypeptide according to any one of claims 1 to 11; (ii) a polynucleotide according to claim 12; (iii) a vector according to claim 13; and (iv) a host cell according to claim 14. and optionally the pharmaceutical composition may further comprise further ingredients, for example, one or more pharmaceutically acceptable excipient or carrier.

17. The pharmaceutical composition according to claim 16 further comprising one or more further active ingredients.

18. A composition comprising an isolated polypeptide according to any one of claims 1 to 11; a polynucleotide according to claim 12; a vector according to claim 13; a host cell according to claim 14; or a pharmaceutical composition according to claim 16 or claim 17, for use in medicine.

19. A composition comprising an isolated polypeptide according to any one of claims 1 to 11; a polynucleotide according to claim 12; a vector according to claim 13; a host cell according to claim 14; or a pharmaceutical composition according to claim 16 or claim 17 for use in reducing activity of the complement pathway or inhibiting activation of the complement pathway.

20. A composition comprising an isolated polypeptide according to any one of claims 1 to 11 a polynucleotide according to claim 12; a vector according to claim 13; a host cell according to claim 14; or a pharmaceutical composition according to claim 16 or claim 17 for use in the prevention or treatment of a disease or disorder associated with increased activity in the complement pathway.

21. A composition comprising an isolated polypeptide according to any one of claims 1 to 11; a polynucleotide according to claim 12; a vector according to claim 13; a host cell according to claim 14; or a pharmaceutical composition according to claim 16 or claim 17, for use in the prophylactic or therapeutic treatment of a disease or a condition mediated by complement.

22. A composition comprising an isolated polypeptide according to any one of claims 1 to 11; a polynucleotide according to claim 12; a vector according to claim 13; a host cell according to claim 14; or a pharmaceutical composition according to claim 16 or claim 17, for use in the prophylactic or therapeutic treatment of acute rejection in organ transplantation; tissue damage resulting from deposition of autoantibodies and immune complexes, which may occur in autoimmune diseases such as systemic lupus erythematosus, myasthenia gravis and Goodpasture's syndrome; tissue injury in hyperacute xenograft rejection triggered by the direct binding of preformed host antibodies to the graft endothelium; ischemia and reperfusion injury occurring, for example, in stroke and myocardial infarction and after major surgery; anti-phospholipid syndrome and cold agglutinin disease; arthritis; neuromyelitis optica; thrombotic microangiopathies; Sjogren's Syndrome; psoriasis; bullous pemphigod and related skin disorders; cardiovascular pulmonary disease; or dense deposit disease.

23. A composition comprising an isolated polypeptide according to any one of claims 1 to 11; a polynucleotide according to claim 12; a vector according to claim 13; a host cell according to claim 14; or a pharmaceutical composition according to claim 16 or claim 17, for use in the prophylactic or therapeutic treatment of an inflammatory disease, ischemia, reperfusion injury, an autoimmune disease, an infection, an infection disease, transplant rejection, an ocular disease, a cancer, systemic lupus erythematosus, glomerulonephritis, rheumatoid arthritis, complications of cardiopulmonary bypass and hemodialysis, hyperacute rejection in organ transplantation, myocardial infarction, reperfusion injury, trauma, adult respiratory distress syndrome, thermal injury, asthma, anaphylactic shock, bowel inflammation, urticaria, angioedema, vasculitis, multiple sclerosis, myasthenia gravis, membranoproliferative glomerulonephritis, Sjogren's syndrome, renal disease, sepsis, paroxysmal nocturnal hemoglobinuria, psoriasis, transplant rejection, cancer, stroke, age-related macular degeneration, atypical haemolytic uremic syndrome, Crohn's disease and Alzheimer's disease, nerve disorders mediated by antibody mediated complement activation (e.g. myasthenia gravis, Guillain-Barre syndrome, Miller-Fisher syndrome, neuromyelitis optica) and anti-phospholipid syndrome.

24. Use of a polypeptide, polynucleotide or composition of the invention in an in vitro method.

25. Use of a polypeptide of the present invention or a polynucleotide of the present invention in a diagnostic assay to test the activation of the complement system.

26. A method of treating a disease or disorder in a subject associated with abnormal increased activity of the complement pathway wherein the method comprises administering to the subject an effective amount of a composition comprising an isolated polypeptide according to any one of claims 1 to 11; a polynucleotide according to claim 12; a vector according to claim 13; a host cell according to claim 14; or a pharmaceutical composition according to claim 16 or claim 17.

27. A method of providing a polypeptide according to any one of claims 1 to 11 comprising expressing the polypeptide in suitable cell.

28. A method for providing a polypeptide according to any one of claims 1 to 11 comprising expressing the polypeptide in a Drosophila S2 cell.

29. A polypeptide, composition, pharmaceutical composition or method as described herein with reference to the figures and examples.

Description

[0092] There now follows by way of example only a detailed description of the present invention with reference to the accompanying drawings, in which;

[0093] FIG. 1shows an overview of the classical pathway, lectin pathway and alternative pathway of the complement system.

[0094] FIG. 2shows the amino acid sequence of RaCI. SEQ ID NO 1 is also referred to herein as RaCI, it has no signal sequence or 6 His tag. SEQ ID NOs 2, 3, 4, 5, 6, 7 and 8 show homologues of SEQ ID NO: 1 (RaCI) and all have no signal sequence or 6 His tag. SEQ ID NO: 9 shows SEQ ID NO: 1 (RaCI) with the signal sequence underlined at the N-terminal end. SEQ ID NO: 10 shows SEQ ID NO: 1 (RaCI) with the signal sequence underlined followed by a 6His tag. SEQ ID NO; 11 shows SEQ ID NO: 1 (RaCI) with a 6 His tag and no signal sequence.

[0095] FIG. 3 shows a CLUSTAL 2.1 multiple sequence alignment of SEQ ID NOs 1 to 8.

[0096] FIG. 4 shows a Percentage Identity Matrixcreated by Clustal 2.1 of SEQ ID NOs 1 to 8.

[0097] FIG. 5shows the results of complement inhibition assays using the peptide of SEQ ID NO: 1 (labelled RaCI) and SEQ ID NO: 11 (labelled His-RaCI) for each of the classical pathway, the lectin pathway and the alternative pathway. The His-Neg polypeptide is a supernatant of a non-related His-tagged protein used as a negative control. 17 g of purified Coversin (OmCI) is used as a positive control, this inhibits all the complement pathways as it inhibits the Terminal Pathway of the complement system. The activity of serum only is set to 100%. The values shown are the average of two repeats,

[0098] FIG. 6 shows effect of 6 His-RaCI on the Classical Pathway (CP), Lectin Pathway (LP) and Alternative Pathway (AP). The maximum and minimum values of each of the fitted curves were set to 100% and 0% activity respectively. Values are averages of three repeats and error bars indicate the standard error.

[0099] FIG. 7 shows complement inhibiting activity of RaCI and various of its homologues. Proteins corresponding to SEQ ID NOs 1-6 were tested. Some of the proteins were tested with the 6 His tag attached to the N-terminus as shown, e.g. His-Seq_ID2 some of the proteins were tested without the 6 His tag, e.g SEQ_ID2. It was found that the 6 His tag did not significantly affect activity. Supernatants of stable S2 cell lines were checked for inhibiting activity in a hemolysis assay using sensitised sheep red blood cells, following the protocol described by Giclas P C, (National Jewish Center for Immunology and Respiratory Medicine, Denver, Colo., Publication Name: Current Protocols in Immunology, Unit Number: Unit 13.1, DOI: 10.1002/0471142735.im1301s09)

[0100] Normal human serum was used to a final dilution of 1/80,

[0101] FIG. 8 shows the results of a pulldown assay of C5 by inhibitors of the present invention. Four inhibitors were covalently coupled to NHS-activated magnetic beads (Pierce Thermo Scientific). These all bound C5 from human serum as confirmed by western blot analysis with a poly-clonal anti-C5 antibody (CompTech, USA). C5 depleted serum indicated by () was used as a negative control. These inhibitors all work by binding C5 as evidenced by the fact that we can use inhibitors covalently coupled to beads to pull down human C5 from serum samples,

[0102] FIG. 9 shows a size exclusion elution profile and coomassie gel of the C5-OmCI-RmCI-Eculizumab complex. C5 was purified from human serum using His-tagged OmCI. Pure OmCI-C5 complexes were then mixed with threefold molar excess of Eculizumab (Ab) and RmCI. The insert gel shows the pooled fractions of the first peak. The second peak contains free antibody and RmCI. These inhibitors bind C5 in a different way to both the previous tick protein (OmCI/Coversin) or Eculizumab as evidenced by the fact we can form a quaternary complex of the four proteins,

[0103] FIG. 10 shows Sequence alignment of the three inhibitors for which we have the structure. The pattern of disulphide bonds is indicated by the yellow lines and numbering above the alignment. The extent of secondary structure (as defined by DSSP) in the highest resolution structure (DaCI) is indicated below in blue. The full structure of three of these inhibitors that represent the level of sequence diversity seen in the family (less than 50% sequence identity within the folded core of the protein that is required for function),

[0104] FIG. 11 shows cartoon representations of the DaCI structure (coloured Blue to Red from N-terminus to C-terminus) with the disulphides shown as sticks. The views are related by a 90 degree rotation,

[0105] FIG. 12 shows overlay of the ribbon representations of the three inhibitors for which we have structure DaCI (coloured blue to red), RaCI (black) and RmCI (grey). The root mean square deviation between the backbone atoms for the structures is 1 2. We have the full structures of each of these three inhibitors in complex with C5 to define exactly how the inhibitors interact with C5. This reveals that the only portion of the molecule needed for function are the 42 residues that lie between the first and last CYS in addition to revealing precisely which residues within this region are directly in contact with C5

[0106] FIG. 13 shows complement inhibition by deletion mutants of RmCI. Mutants and wildtype RmCI were expressed in E. coli Shuffle T7 cells and cell lysates were tested for inhibitory activity in a haemolysis assay. Empty vector serves as a negative control. Values are means of two experimental replicates.

[0107] FIG. 14 shows a sequence alignment based on structures of the inhibitors bound to C5. Residues coloured green are buried in formation of the complex with C5. Those shown in green italics forms specific salt bridges with residues in C5.

[0108] FIG. 15 shows Cross-species alignment of complement C5. Human C5 is the top sequence in each block and residues highlighted in blue are those that are buried in formation of the complex with DaCI/RaCI/RmCI. Highlighting in lower sequences shows which of the contact residues are conserved in other species. (We know that these inhibitors have full activity against human and guinea pig sera (top and bottom sequences in alignment) partial activity against pig sera (4th sequence from top) and no activity against rat and mouse (3rd and 4th sequences up from bottom.

[0109] FIG. 16 shows cross-species activity of DaCI/RaCI/RmCI/HmCI tested in a classical pathway haemolysis assay. Final serum concentrations in the assay are 1/80 (human) and 1/640 (guinea pig). Values are averages of three repeats and error bars indicate the standard error, except for the mouse assay in which values are averages of two repeats. Values are normalised to PBS (100% activity) and empty wells (0%).

[0110] FIG. 17 shows picture of complex between DaCI (coloured blue at N-terminus to red at C-terminus) and complement C5 (wheat). The present inventors have developed a competition assay which reveals that these inhibitors do not act by stopping interaction with the C5 convertase (as has been the assumed mechanism for earlier C5 inhibitors) and our structure suggests the mechanism of inhibition is more likely to be that the inhibitors lock C5 into a conformation which is incompatible with activation. In terms of therapy this means that presence in the patient of inhibited C5 will further act to prevent activation of any un-inhibited C5 present by competing for binding to the activating enzyme,

[0111] FIG. 18 shows Competition assay with C5-ligand complexes. Pure C5 (CompTech, USA) was mixed with two-fold molar excess ligand, and complexes were purified by a size exclusion chromatography step. Purified complexes were mixed with diluted serum in a Wieslab-based Classical pathway ELISA. Antibody is a Fab fragment based on the Eculizumab drug and Neg control is a tick protein not targeting Complement. Values are means of three repeats and error bars indicate the standard error.

[0112] Materials and Methods

[0113] Sequence and expression of polypeptides. Sequences of SEQ ID NO 1 to 11: were expressed from pExpres2-2 vectors in Drosophila S2 cells (ExpreS2ion Biotechnologies, Denmark). SEQ ID NO: 9 and SEQ ID NO: 10 are shown with the signal sequences underlined. SEQ ID NOs 1 to 8, 11 and 12 are shown without signal sequences. The signal sequences are cleaved during expression in Drosophila S2 cells to provide the peptides shown in SEQ ID NOs 1-8, 11 and 12. The signal sequences for the homologues SEQ ID NO 2 to 8 may be the same or similar to the signal sequence show underlined in SEQ ID NO: 9.

TABLE-US-00002 SEQIDNOs:9 (MNAMLVLFIASALFISEHNTEEVKTTPIPNHQCVNATCERKLDALGNAV ITKCPQGCLCVVRGASNIVPANGTCFQLATTKPPMAPGDNKDNKEEESN) and SEQIDNO:10 (MKLCILLAVVAFVGLSLGHHHHHHAGEEVKTTPIPNHQCVNATCERKLD ALGNAVITKCPQGCLCVVRGASNIVPANGTCFQLATTKPPMAPGDNKDNK EEESN)
contain signal peptides for secretion (underlined), as predicted by SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/). The proteins were transiently expressed for 72 hours according to the manufacturer's protocol (ExpreS2ion Biotechnologies, Denmark). Spent medium was cleared by centrifugation and tested for anti-complement activity using complement inhibition assays.

[0114] Purification of His6-RaCI. RaCI fused to an N-terminal His6-tag (His6-RaCI) was expressed from a pExpres2-2 vector in Drosophila S2 cells (ExpreS2ion Biotechnologies, Denmark). The generation of a stable cell line and expression of His6-RaCI were done according to the manufacturer's protocol (ExpreS2ion Biotechnologies, Denmark). Cell cultures were cleared by centrifugation and His6-RaCI was purified from the supernatant using a complete His-Tag Purification column (Roche), followed by a gel filtration step.

[0115] Complement inhibition assay for His-6-RaCI. Complement inhibiting activity of a dilution series of His6-RaCI was determined using the Complement System Screen WIESLAB (Euro Diagnostica, Sweden), according to the manufacturer's protocol with the following modifications. Normal human serum was used in all conditions. To test for complement inhibition 2 l of purified His6-RaCI to 100 l of diluted serum before the incubation step. The effect of His6-RaCI on each of the three pathways was tested with the different buffers and ELISA strips provided with the kit. The dilution series were used to calculate the IC50 values of His6-RaCI for each of the pathways.

[0116] The results of this assay are shown in FIG. 4. The effect of His6-RaCI on the Classical Pathway (CP), Lectin Pathway (LP) and Alternative Pathway (AP). The maximum and minimum values of each of the fitted curves were set to 100% and 0% activity respectively. Values are averages of three repeats and error bars indicate the standard error.

[0117] Complement inhibition assays for spent medium. Complement inhibiting activity of spent medium was determined using the Complement System Screen WIESLAB (Euro Diagnostica, Sweden), according to the manufacturer's protocol with the following modifications. Normal human serum was used in all conditions. To test for complement inhibition 5 l of spent medium was added to 100 l of diluted serum before the incubation step. The effect of SEQ ID NO: 1 on each of the three pathways was tested with the different buffers and ELISA strips provided with a Wieslab kit. The Wieslab kit is an ELISA kit having three different coatings on ELISA plates to activate each of the three pathways. Inhibitor is added to serum to before it is added to an ELISA well. % inhibition is proportional to amount of inhibitor added but should be up to 100%. The assay is performed as described in Seelen et al. Journal of Immunological Methods Volume 296, Issues 1-2, January 2005, Pages 187-198.

RESULTS

[0118] The results of the complement inhibition assays for spent medium are shown in FIG. 3 the effect of SEQ ID NO: 1 on the Classical Pathway, Lectin Pathway and Alternative Pathway is shown. His-Neg is a supernatant of a non-related His-tagged protein used as a negative control. 17 ug of purified Coversin (OmCI) is used as a positive control, it inhibits all pathways since it inhibits the Terminal Pathway of the complement system. The activity of serum only is set to 100%. Values are averages of two repeats.