Coversin for Use in the Treatment of Rheumatic Diseases

Abstract

The present invention relates to methods of treating or preventing rheumatic disease.

Claims

1. A method of treating or preventing a rheumatic disease in a subject, which comprises administering to the subject a therapeutically or prophylactically effective amount of an agent which is a protein comprising amino acids 19 to 168 of the amino acid sequence in FIG. 2 (SEQ ID NO: 2) or a functional equivalent of this protein.

2. An agent which is a protein comprising amino acids 19 to 168 of the amino acid sequence in FIG. 2 (SEQ ID NO: 2) or a functional equivalent of this protein for use in a method of treating or preventing a rheumatic disease in a subject.

3. A method of treating or preventing a rheumatic disease in a subject, which comprises administering to the subject a therapeutically or prophylactically effective amount of an agent which is a nucleic acid molecule encoding a protein comprising amino acids 19 to 168 of the amino acid sequence in FIG. 2 (SEQ ID NO: 2) or a functional equivalent of this protein.

4. An agent which is a nucleic acid molecule encoding a protein comprising amino acids 19 to 168 of the amino acid sequence in FIG. 2 (SEQ ID NO: 2) or a functional equivalent of this protein for use in a method of treating or preventing a rheumatic disease in a subject.

5. The method of any one of claim 1 or 3 or the agent for use of any one of claim 2, or 4, wherein the agent is, or encodes, a protein comprising a sequence having at least 90% sequence identity to the sequence of amino acids 19 to 168 of SEQ ID NO: 2, and said protein binds C5 to prevent the cleavage of complement C5 by C5 convertase into complement C5a and complement C5b and binds to LTB4.

6. The method of any one of claim 1, 3 or 5 or the agent for use of any one of claim 2, 4 or 5, wherein the agent is, or encodes, a protein comprising a sequence having at least 95% sequence identity to the sequence of amino acids 19 to 168 of SEQ ID NO: 2, and said protein binds C5 to prevent the cleavage of complement C5 by C5 convertase into complement C5a and complement C5b and binds to LTB4.

7. The method of any one of claims 1, 3 or 5 to 6, or the agent for use of any one of claims 2, 4 or 5 to 6, wherein the agent is, or encodes, a protein comprising or consisting of the sequence of amino acids 19 to 168 of SEQ ID NO: 2.

8. The method of any one of claim 1 or 3, or the agent for use of any one of claim 2 or 4, wherein agent is, or encodes, a protein comprising the sequence of amino acids 19 to 168 of SEQ ID NO: 2, in which up to 50 amino acid substitutions, insertions or deletions have been made, and the protein binds C5 to prevent the cleavage of complement C5 by C5 convertase into complement C5a and complement C5b and binds to LTB4, wherein each of the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of the mature Coversin molecule as set out in SEQ ID NO: 4 is retained and at least five, ten or fifteen or each of the LTB4 binding residues and at least five, ten or fifteen or twenty or each of C5 binding residues set is retained or is subject to a conservative modification, wherein the LTB4 binding residues are Phe18, Tyr25, Arg36, Leu39, Gly41, Pro43, Leu52, Val54, Met56, Phe58, Thr67, Trp69, Phe71, Gln87, Arg89, His99, His101, Asp103, and Trp115 (numbering according to SEQ ID NO:4) and the C5 binding residues are Val26, Val28, Arg29, Ala44, Gly45, Gly61, Thr62, Ser97, His99, His101, Met 114, Met 116, Leu117, Asp118, Ala119, Gly120, Gly121, Leu122, Glu123, Val124, Glu125, Glu127, His146, Leu147 and Asp 149 (numbering according to SEQ ID NO:4).

9. The method or agent for use of claim 8 wherein up to 2, 3, 4, 5, 10, 15, 20 of the LTB4 and C5 binding residues are subject to a conservative modification.

10. The method or agent for use of claim 8 or 9 wherein at least five, ten or fifteen or each of the LTB4 binding residues and at least five, ten or fifteen or twenty or each of C5 binding residues is retained.

11. The method or agent for use of any of claims 8 to 10 wherein each of the LTB4 binding residues and each of C5 binding residues is retained or is subject to a conservative modification.

12. The method or agent for use of any of claims 8 to 11 wherein each of the LTB4 binding residues and each of C5 binding residues is retained or is subject to a conservative modification, wherein up to 2, 3, 4, 5, 10, 15, 20 of the C5 and/or LTB4 binding residues are subject to a conservative modification.

13. The method or agent for use of any of claims 8 to 12, wherein each of the LTB4 binding residues and each of the C5 binding residues is retained.

14. The method of any one of claim 1 or 3, or the agent for use of any one of claim 2 or 4, wherein the agent is, or encodes, a fragment of the protein as defined in any of the preceding claims, and the protein binds C5 to prevent the cleavage of complement C5 by C5 convertase into complement C5a and complement C5b and binds to LTB4.

15. The method or the agent for use of any preceding claim, wherein the agent is administered subcutaneously or intrasynovially, preferably subcutaneously.

16. The method or the agent for use of any preceding claim, wherein the subject is a human.

17. The method or the agent for use of any preceding claim, wherein the rheumatic disease is selected from ankylosing spondylitis, relapsing polychondritis, systemic lupus erythematosus, rheumatoid arthritis, gout, inflammatory arthritis, pseudogout, juvenile arthritis, Sjögren syndrome, scleroderma, polymyositis, dermatomyositis, Behçet's disease and psoriatic arthritis.

18. The method or the agent for use of any preceding claim, wherein the rheumatic disease is RA, optionally wherein the RA is RF and/or anti-CCP positive RA.

19. The method or the agent for use of claim 18, wherein the RA is accompanied by vasculitis.

20. The method or agent for use of any preceding claim, wherein there the method comprises administering to the subject an initial ablating regimen of the agent and then administering maintenance doses of the agent, optionally wherein there is an initial maintenance dose and one or more further maintenance doses.

21. The method or the agent for use of any preceding claim, wherein the method further comprises the administration of a second rheumatic disease treatment.

22. The method or the agent for use of claim 21, wherein the second rheumatic disease treatment is selected from a DMARD, an anti-inflammatory agent (e.g. an NSAID, or a glucocorticoid) and an analgesic.

23. The method or the agent for use of claim 22 wherein: (a) the DMARD is selected from cyclosporine, cyclophosphamide, hydroxychloroquine, gold salts, methotrexate, leflunomide, mycophenolate, sulfasalazine, etanercept, certolizumab pegol, golimumab, infliximab, and adalimumab, anakinra, rituximab and abatacept, preferably selected from etanercept, certolizumab pegol, golimumab, infliximab, and adalimumab, anakinra, rituximab and abatacept, (b) the anti-inflammatory agent is an NSAID, or a glucocorticoid, and/or (c) the analgesic is selected from paracetamol, compound analgesics and an opiod analgesic.

24. The method or the agent for use of any preceding claim wherein the functional equivalent of the protein comprising amino acids 19 to 168 of SEQ ID NO:2 is a fusion protein comprising (a) a sequence as defined in any of claims 6 to 14, and (b) a second sequence and said fusion protein binds C5 to prevent the cleavage of complement C5 by C5 convertase into complement C5a and complement C5b and binds LTB4.

25. The method or agent for use of claim 24 wherein said second sequence is a PAS sequence.

26. The method or agent for use of claim 23 or 24, wherein said fusion protein comprises multiple copies of one of ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 15); AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 16); APSSPSPSAPSSPSPASPSS (SEQ ID NO: 17), SAPSSPSPSAPSSPSPASPS (SEQ ID NO: 18), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 19), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 20) and ASAAAPAAASAAASAPSAAA (SEQ ID NO: 21), preferably 20-30 or 30 copies of one of SEQ ID NOs 15-21.

27. The method or agent for use of any of claims 24 to 26, wherein said fusion protein comprises (a) a PAS sequence consisting of 30 copies of SEQ ID NO:15 and (b) amino acids 19-168 of SEQ ID NO:2, wherein (a) is fused to the N terminus of (b).

28. The method or agent for use of any of claims 24 to 27, wherein said fusion protein comprises the sequence of SEQ ID NO:22.

29. The method or agent for use of any one of claims 1 to 28, wherein the protein or fusion protein binds C5 to prevent the cleavage of complement C5 by C5 convertase into complement C5a and complement C5b and binds LTB4.

Description

BRIEF DESCRIPTION OF FIGURES

[0170] FIG. 1: Schematic diagram of classical and alternative pathways of complement activation. Anaphylatoxins are enclosed in starbursts.

[0171] FIG. 2A: Primary sequence of Coversin. Signal sequence underlined. Cysteine residues in bold type. Nucleotide and amino acid number indicated at right. The nucleotide sequence is SEQ ID NO: 1 and the amino acid sequence is SEQ ID NO: 2.

[0172] FIG. 2B: Examples of Coversin variants.

[0173] FIG. 3: Clinical score and paw width of experimental RA in vehicle versus mice treated with prophylactic Coversin, PAS-Coversin or Zileuton.

[0174] FIG. 4: Clinical score of experimental RA in vehicle versus mice treated with PAS-Coversin demonstrates ameliorated disease in mice treated with PAS-Coversin compared to the vehicle control group, Zileuton, and PAS-L Coversin, which does not bind C5.

[0175] FIG. 5: PAS-Coversin sequence.

EXAMPLES

Example 1—Effect of Coversin in K/B×N Serum Transfer Model (Prophylactic)

[0176] The K/B×N serum-transfer arthritis model was used to test whether development of arthritis could be prevented by Coversin. This model is a well-known murine model in which the immunological mechanisms occurring in rheumatoid arthritis (RA) are induced by transferring serum from arthritic transgenic K/B×N mice to naive mice. In the absence of treatment arthritic symptoms occur a few days later. This model is recognised to be highly relevant for RA, especially for the preclinical screening of new therapeutic targets for RA and perhaps other forms of inflammatory arthritis [44].

[0177] Experimental RA was induced using the protocol described by [45]. Five mice were tested in each treatment group.

[0178] On day 0 C57BL/6 mice were assessed for clinical score and paw thickness using standard protocols [45]. All mice received an injection of K/B×N serum on day 0 and on day 2. Each treatment was administered every day from day 0 to day 14. On days where serum was also injected (day 0 and day 2) drug was administered immediately after the serum injection. Clinical score and paw thickness were measured every other day.

[0179] Treatment groups were as follows:

[0180] Group 1: PBS (vehicle, subcutaneous, q24h)

[0181] Group 2: Treatment (Coversin 5 mg/kg subcutaneous, q12h)

[0182] Group 3: Treatment (Zileuton 50 mg/kg orally once daily)

[0183] Group 4: Treatment (PAS-Coversin 20 mg/kg subcutaneous, once daily)

[0184] The results of Experiment 1 are shown in FIG. 3. It can be seen that Coversin (q12h) & PAS-Coversin (q24h) completely inhibit the development of arthritis in this model. Zileuton (5-LOX inhibitor) partially ameliorates the development of arthritis in this model.

[0185] Coversin and PAS-Coversin reduced the development of arthritis in this model to a greater degree than N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea (Zileuton), which is a 5-lipoxygenase inhibition (5-LOX) oral inhibitor (an important enzyme of the arachidonic acid cascade and is involved in the formation of bioactive leukotrienes (LTs)).

[0186] The difference between PAS-Coversin and Coversin is that the PAS-Coversin is “PASylated”. This describes the genetic fusion of a protein with conformationally disordered polypeptide sequences composed of the amino acids Pro, Ala, and/or Ser. This is a technology developed by XL Protein (http://xl-protein.com/) and provides a simple way to attach a solvated random chain with large hydrodynamic volume to the protein to which it is fused. The polypeptide sequence adopts a random coil structure resulting in a large increase in apparent molecular weight and a reduced rate of clearance by kidney filtration. The sequence of PAS-Coversin used in this experiment is shown in FIG. 5 and SEQ ID NO:22.

[0187] Because of the higher molecule weight of the PAS-Cov, 20 mg/kg PAS-Cov corresponds to 5 mg/kg Coversin, so in this experiment equivalent doses of the active Coversin agents are administered each day, but Coversin is administered twice as frequently (12 hourly). PAS-Coversin was as effective as Coversin in this experiment but with less frequent administration. Thus, there may be advantages in using longer half-life versions of Coversin, such as PAS Coversin therapeutically.

Example 2 Effect of Coversin in K/B×N Serum Transfer Model (Treatment)

[0188] The K/B×N serum-transfer arthritis (STA) model was used to test whether established arthritic disease could be ameliorated by Coversin.

[0189] Experimental RA was induced using the protocol described by [45]. Five mice were tested in each treatment group.

[0190] On day 0 C57BL/6 mice were assessed for clinical score and paw thickness using standard protocols [45]. All mice received an injection of K/B×N serum on day 0 and on day 2. The treatment was administered every day from day 4 to day 14. On days where serum was also injected (day 0 and day 2) drug was administered immediately after the serum injection. Clinical score and paw thickness were measured every other day.

[0191] Treatment groups were as follows:

[0192] Group 1: PBS (vehicle)

[0193] Group 2: Treatment (PAS-L Coversin 20 mg/kg subcutaneous once daily)

[0194] Group 3: Treatment (Zileuton 50 mg/kg orally once daily)

[0195] Group 4: Treatment (PAS-Coversin 20 mg/kg subcutaneous once daily)

[0196] The results of the clinical scores from the second experiment are shown in FIG. 4. It can be seen mice developed arthritis following serum injection. In the vehicle and Zileuton groups maximum clinical score was seen at day 6 of the experiment. Following administration of PAS-L-Coversin (q24h) and PAS-Coversin (q24h), the clinical score decreased for the PAS-Coversin and PAS-L-Coversin groups. For the PAS-L-Coversin group the clinical score was significantly lower than the vehicle and Zileuton treated groups.

[0197] The difference between PAS-Coversin and PAS-L-Coversin is that in the L-Coversin molecule the Coversin sequence has been mutated such that it binds LTB4 but does not bind C5 (referred to as “L-Coversin”). The sequence of the L-Coversin sequence is a variant of the mature Coversin sequence (SEQ ID NO: 4) in which the following residues have been modified: Ala44 to Asn, Met116 to Gln, Leu117 to Ser, Gly121 to Ala, Leu122 to Asp, Glu123 to Ala and Asp149 to Gly, (referred to as variant 2, sequence is dsesdctgse pvdafqafse gkeayvlvrs tdpkardclk gepNgekqdn tlpvmmtfkn gtdwastdwt ftldgakvta tlgnitqnre vvydsqshhc hvdkvekevp dyemwQSdag ADAveveccr qkleelasgr nqmyphlkGc (SEQ ID NO:23), where the changes relative to the native Coversin sequence of SEQ ID NO:4 are in capitals).

[0198] The PAS-L-Coversin was not as effective as the PAS-Coversin in ameliorating RA but was more effective than Zileuton. This suggests that the dual inhibitory activity of Coversin (C5 and LTB4 inhibition) provides improved therapeutic benefit in this model. It had been expected that combined inhibition of C5 activation and LTB4 would be only as effective in the therapeutic model as LTB4 inhibition alone. However unexpectedly combined inhibition of C5 and LTB4 by Coversin, as shown by the improved effected of Coversin compared to L-Coversin, proved much more effective in the therapeutic model than LTB4 inhibition alone.

REFERENCES

[0199] [1] Hoover et al, 1984, Proc. Nat. Acad. Sci. U.S.A. 81, 2191-2193 [0200] [2] Harrison and Murphy, 1995, J. Biol. Chem. 270, 17273-17276 [0201] [3] Ford-Hutchinson, 1990, Crit. Rev. Immunol. 10, 1-12 [0202] [4] Showell et al., 1995, J. Pharm. Exp. Ther. 273, 176-184 [0203] [5] Klaas et al, 2005 J. Exp. Med. 201, 1281-1292 [0204] [6] Del Prete et al, 2007 Blood, 109, 626-631 [0205] [7] Miyahara et al, 2006 Allergol Int. 55, 91-7 [0206] [8] Taube et al, 2006 J. Immunol. 176, 3157-3164 [0207] [9] Yamaoka et al, 1989 J. Immunol. 143, 1996-2000 [0208] [10] Yokomizo et al, 1997 Nature 387, 620-624 [0209] [11] Yokomizo et al, 2000 J. Exp. Med. 192, 421-432 [0210] [12] Tager and Luster, 2003 Prostaglandins Leukot. Essent. Fatty Acids 69, 123-134 [0211] [13] Yokomizo et al., 2001, J. Biol. Chem. 276, 12454-12459 [0212] [14] Kim, N. D. and Luster, A. D. (2007) The Scientific World Journal 7, 1307-1328. [0213] [15] Kim et al., 2006. J. Exp. Med. 203, 829-835 [0214] [16] Noiri et al., 2000 Proc Nat Acad Sci USA 97, 823-828 [0215] [17] Lundeen et al., 2006. J. Immunol. 177, 3439-3447 [0216] [18] Shao et al, 2006. J. Immunol. 176, 6254-6261 [0217] [19] Chen et al., 2006. J. Exp. Med. 203, 837-842 [0218] [20] Sebaldt et al., 1990 Proc Natl Acad Sd. U.S.A. 8, 6974-6978 [0219] [21] Curry et al., 2005 Journal of the American Animal Hospital Association 41, 298-309 [0220] [22] Dube et al., 1998. Zileuton: the first leukotriene inhibitor for use in the management of chronic asthma. In: Drazen J M, Dahlen S, Lee T H, eds. Five-lipoxygenase Products in Asthma. New York, N.Y.: Marcel Dekkar, Inc [0221] [23] Sharma and Mohammed, 2006 Immunopharmacology 14, 10-16 [0222] [24] GBD 2015 Disease and Injury Incidence and Prevalence, Collaborators. Lancet. 388 (10053): 1545-1602. (2016) [0223] [25] GBD 2013 Mortality and Causes of Death, Collaborators (17 Dec. 2014). Lancet. 385 (9963): 117-71. [0224] [26] WO2004/106369 [0225] [27] Jore, M. M. et al, Nature Structural & Molecular Biology 2016 volume 23, pages 378-386 [0226] [28] Pincus et al Rheumatology 2008; 47:345-349 [0227] [29] Oliver J et al Arthritis Res Ther. 2009; 11(3): 223. [0228] [30] Roversi, P et al Journal of Biological Chemistry 2013, 288(26) 18789-18802 [0229] [31] Guo, R. F. and P. A. Ward, Annu Rev Immunol, 2005, 23: p. 821-52 [0230] [32] Ricklin D & Lambris J, Nature Biotechnology, 25: 1265-1275 (2007) [0231] [33] Nishimura, J et al., New Engl J. Med., 30; 7: 632-639 (2014) [0232] [34] Breustedt D. A., Schonfeld D. L., Skerra A. (2006) Comparative ligand-binding analysis of ten human lipocalins. Biochim Biophys Acta 1764(2):161-173. [0233] [35] Terpe K, Appl Microbiol Biotechnol, 60: 523-33, 2003 [0234] [36] Schlapschy M, et al Protein Eng Des Sel. 2013 August; 26(8):489-501 [0235] [37] Kuhn et al Bioconjugate Chem., 2016, 27 (10), pp 2359-2371 [0236] [38] Sambrook et al (2000) [0237] [39] Fernandez & Hoeffler (1998) [0238] [40] Ausubel et al. (1991) [0239] [41] https://resources.rndsystems.com/pdfs/datasheets/kge006b.pdf [0240] [42] Remington's Pharmaceutical Sciences; Mack Pub. Co., N.J. 1991 [0241] [43] Patel et al Ther. Deliv. (2014) 5(3), 337-365 [0242] [44] Christensen, A. et al. Front Immunol. 2016; 7: 213. [0243] [45] Kim et al JEM 2006; 203(4) 829-835