Coversin (OmCI) for the treatment of autoimmune blistering diseases: bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA)

Abstract

The present invention relates to methods of treating or preventing AIBD.

Claims

1. A method of treating an autoimmune blistering disease (AIBD) in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of an agent which is a protein comprising the sequence of amino acids 19 to 168 of the amino acid sequence of SEQ ID NO: 2 or a functional equivalent of this protein, wherein the functional equivalent is: a) 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; or b) a fragment of a protein, the 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 wherein the functional equivalent binds C5 to prevent the cleavage of complement C5 by convertase into complement C5a and complement C5b and binds to Leukotriene B4 (LTB4), wherein the AIBD is bullous pemphigoid or epidermolysis bullosa acquisita (EBA).

2. A method of treating an autoimmune blistering disease (AIBD) in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of an agent which is a nucleic acid molecule encoding a protein comprising the sequence of amino acids 19 to 168 of the amino acid sequence of SEQ ID NO: 2 or a functional equivalent of this protein, wherein the functional equivalent is: a) 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; or b) a fragment of a protein, the 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 wherein the functional equivalent binds C5 to prevent the cleavage of complement C5 by convertase into complement C5a and complement C5b and binds to Leukotriene B4 (LTB4), wherein the AIBD is bullous pemphigoid or epidermolysis bullosa acquisita (EBA).

3. The method of claim 1, wherein the agent is 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 convertase into complement C5a and complement C5b and/or binds to LTB4.

4. The method of claim 1, wherein the agent is a protein comprising or consisting of the sequence of amino acids 19 to 168 of SEQ ID NO: 2.

5. The method of claim 1, wherein the agent is a protein comprising the sequence of amino acids 19 to 168 of SEQ ID NO: 2, in which up to 10 amino acid substitutions, insertions or deletions have been made, and the protein binds C5 to prevent the cleavage of complement C5 by convertase into complement C5a and complement C5b and binds to LTB4.

6. The method of claim 1, wherein the agent is administered subcutaneously.

7. The method of claim 1, wherein the subject is a human.

8. The method of claim 1, wherein the agent is administered in a dose sufficient to bind as much available C5 and/or LTB4 as possible in the subject, or all available C5.

9. The method of claim 1, wherein the agent is administered in a dose 1.5 times the molar dose needed to bind all available C5 and/or LTB4 in the subject.

10. The method of claim 1, wherein the method comprises administering to the subject an initial loading dose of the agent and then administering maintenance doses thereof.

11. The method of claim 10, wherein there is an initial maintenance dose and one or more further maintenance doses.

12. The method of claim 1, wherein the method further comprises the administration of a second AIBD treatment.

13. The method of claim 12, wherein the second AIBD treatment is selected from systemic corticosteroid therapy, topical corticosteroid therapy, immunosuppressive therapy and immunosuppressive biological therapy.

14. The method of claim 13, wherein the corticosteroid is selected from prednisone and prednisolone, the immunosuppressive therapy is selected from methylprednisolone, mycophenolate, azathioprine, dapsone and cyclophosphamide and the immunosuppressive biological therapy is selected from rituximab and intravenous immunoglobulin G (IVIG).

15. The method of claim 1, wherein the AIBD is EBA.

16. The method of claim 1, 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 having at least 90% sequence identity to the sequence of amino acids 19 to 168 of SEQ ID NO: 2, and (b) a second sequence and said fusion protein binds C5 to prevent the cleavage of complement C5 by convertase into complement C5a and complement C5b and binds LTB4.

17. The method of claim 16, wherein said second sequence is a PAS sequence.

18. The method of claim 16, wherein said fusion protein comprises (a) a PAS sequence consisting of 30 copies of SEQ ID NO:15 and (b) (i) amino acids 19-168 of SEQ ID NO:2, wherein (a) is fused to the N terminus of (b).

19. The method of claim 13, wherein the corticosteroid is prednisone.

20. The method of claim 13, wherein the corticosteroid is prednisolone.

21. The method of claim 1, wherein the AIBD is bullous pemphigoid.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1: Schematic diagram of classical and alternative pathways of complement activation. Enzymatic components, dark grey. Anaphylatoxins enclosed in starbursts.

(2) FIG. 2A: Primary sequence of Coversin. Signal sequence underlined. Cysteine residues in bold type. Nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) number indicated at right.

(3) FIG. 2B: Examples of Coversin variants

(4) FIG. 3: Clinical score of experimental EBA in vehicle versus mice treated with prophylactic Coversin demonstrate ameliorated disease in mice treated with all doses of Coversin as compared to the vehicle control group and the methylprednisolone group, experiment 1.

(5) FIG. 4: Clinical score of experimental EBA in vehicle versus mice treated with prophylactic Coversin demonstrate ameliorated disease in mice treated with all doses of Coversin as compared to the vehicle control group and the methylprednisolone group, experiment 2. In comparison to the first experiment, Coversin only had a significant effect compared to the vehicle control group at the 2.5 mg/kg dose. Notably, in this experiment, several mice in the control group did not develop a significant level of disease, which is likely the reason for this difference compared to the first experiment.

(6) FIG. 5: Clinical score of experimental EBA in vehicle versus mice treated with prophylactic Coversin demonstrate ameliorated disease in mice treated with all doses of Coversin as compared to the vehicle control group and the methylprednisolone group, combined results of experiments 1 and 2, Two-way ANOVA analysis reveals a statistically significant difference between the vehicle control and Coversin in the doses of 250 and 2500 μg/ml.

(7) FIG. 6A: Clinical score of experimental EBA in vehicle versus mice treated with prophylactic Coversin and prophylactic PAS-L-Coversin demonstrate ameliorated disease in mice treated with 2.5 mg/kg Coversin (Example 4, experiment 1).

(8) FIG. 6B: Clinical score of experimental EBA in vehicle versus mice treated with prophylactic Coversin and prophylactic PAS-L-Coversin demonstrate ameliorated disease in mice treated with 2.5 mg/kg Coversin (Example 4, experiment 2). Here the 10 mg/kg dose of PAS-L-Coversin and 2.5 mg/kg Coversin both show a statistically significant effect on ABSA.

(9) FIG. 6C: Clinical score of experimental EBA in vehicle versus mice treated with prophylactic Coversin and prophylactic PAS-L-Coversin. This shows the combined results of Example 4, experiments 1 and 2.

(10) FIG. 7: Effect of therapeutically administered Coversin on the course of pemphigoid disease-like skin inflammation in the passive EBA mouse model (Example 2, experiment 1). Coversin dose-dependently ameliorated skin inflammation. Data are presented as means+/−SEM; N=5 mice per group; Two-way ANOVA testing for statistical significance confirms significant differences between the vehicle control and Coversin in all doses used. The experiment was conducted exclusively in females.

(11) FIG. 8: Effect of therapeutically administered Coversin on the course of pemphigoid disease-like skin inflammation in the passive EBA mouse model (Example 2, experiment 2). Coversin dose-dependently ameliorated skin inflammation. Data are presented as means+/−SEM; N=5 mice per group; Two-way ANOVA testing for statistical significance confirms significant differences between the vehicle control and coversin in all doses used. The experiment was conducted exclusively in males.

(12) FIG. 9A shows C5a levels in blister fluid of bullous pemphigoid patients.

(13) FIG. 9B shows LTB4 levels in blister fluid of bullous pemphigoid patients.

(14) FIG. 10 shows the sequence of certain modified Coversin polypeptides with reduced or absent C5-binding activity but which retain LTB-4-binding ability

EXAMPLES

Example 1

Effect of Coversin in EBA Transfer Model with Coversin Administered Prophylactically

(15) Antibody transfer EBA was induced using a modified version of the protocol described by Sitaru et al., 2005 [43]. Five mice were tested in each treatment group. Briefly, mice were injected subcutaneously with 50 μg affinity-purified anti-Col7 IgG on days 0, 2, and 4. Mice were injected subcutaneously twice daily with three varying doses of Coversin starting four days before the first injection of anti-Col7 IgG (day −2). This application was continued throughout the experiment until its last day on day 11. In the control groups the mice received only the vehicle subcutaneously, or 20 mg/kg methylprednisolone once daily.

(16) To generate antibodies directed to murine type VII collagen (“anti-COL7”), New Zealand White rabbits were immunized with 200 μg of a protein mixture containing three different recombinant proteins (“Col7A, B, and C”) derived from the non-collagenous 1 (NC1) domain of collagen VII together with incomplete Freund's adjuvant. IgGs were isolated from the serum of immunized rabbits by use of protein G, and afterwards IgGs were affinity-purified with his-Col7 to specifically obtain rabbit anti-Col7 IgG. Antibodies were quality checked by determining the titer and the potency in the cryosection assay.

(17) Starting on day 4, the extent of skin lesions was scored in each individual mouse every other day. Skin areas exhibiting erythema, blisters, erosions, crusts, or alopecia were categorized as “affected” or “unaffected” by a trained observer [43]. The percentage of the total body surface affected by skin lesions (ABSA) was calculated.

(18) Two independent experiments were conducted.

(19) The results of the first experiment are shown in FIG. 4. It can be seen that the percentage ABSA in mice treated with Coversin at 25 μg/kg was reduced, and that treatment with Coversin at Coversin 2500 μg/kg, and Coversin 250 μg/kg was further reduced. There was a 63% difference between vehicle and the 0.25 mg/kg dose. The P value for this difference by two tailed t-test is 0.0023. The mean ABSA scores for vehicle and 0.25 mg/kg dose at day 10 were 6.23+1.1 and 2.65+0.4. The values after the means are SDs.

(20) Coversin reduced the percentage ABSA to a greater degree than previously tested molecules. For example, in previous similar experiments N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea (Zileuton), which is a 5-lipoxygenase inhibition (5-LO) oral inhibitor (an important enzyme of the arachidonic acid cascade and is involved in the formation of bioactive leukotrienes (LTs)) was administrated to C57Bl/6J 8-weeks-old female mice in a preventive dosing scheme. In this earlier experiment, mice were distributed into two groups and received 0.6-0.8 mg/mouse/day Zileuton or vehicle control supplemented into their drinking water. Experimental EBA was induced and evaluated [44]. The % ABSA was reduced, and it was concluded that “starting from day 7 of the experiment and until its end-point, mice treated with Zileuton exhibited a significantly reduced disease severity as compared to control group (clinical score day 7; control group: X.sup.−=9.2±0.7; Zileuton treated group: X.sup.−=5.9±0.5% of affected surface body area)”.

(21) In a similar earlier experiment in which 20 mg/kg/day of Methylprednisolone was administered in an antibody transfer model of EBA, 7.5±0.1% of the body surface area were affected by skin lesions in control mice at the end of the experiment, but this disease severity was significantly reduced to 4.7±0.4% in the Methylprednisolone treated mice [45].

Example 2

Effect of Coversin in EBA Transfer Model

(22) The study included the following experimental groups, with 5 mice in each experimental group: 1. Vehicle (PBS) control group 2. 125 μg/kg Coversin 2× daily s.c. 3. 2500 μg/kg Coversin 2× daily s.c.

(23) Antibody transfer EBA-like skin inflammation was induced, as described in Sezin et al [46]. Briefly, C57Bl/6J WT mice were injected subcutaneously (s.c.) with 50 μg affinity-purified anti-Col7 IgG on days 0, 2, and 4.

(24) The treatment of mice with Coversin started on Day 5 of the experiment (Day 0=day of first administration of anti-COL7 antibody). Afterwards, mice were injected 12 hourly s.c. in the scapular region with Coversin or vehicle control until the end of the experiment.

(25) Disease severity was scored. To score disease severity, skin areas exhibiting erythema, blisters, erosions, crusts, or alopecia were categorized as “affected” by a single trained examiner blinded to treatment. The percentage of the absolute body surface affected by skin lesions (ABSA) was calculated. To score the disease, the mice were anesthetized every other day with a mixture of ketamine/xylazine administered i.p. starting day 4 of the experiment.

(26) Two independent experiments were conducted according to this protocol.

(27) Test Item Preparation

(28) 18 mg of Coversin were reconstituted with 0.6 ml of sterile water to achieve a concentration of 30 mg/ml, aliquoted, and stored at −20° C. until final dilution.

(29) Prior to the beginning of the experiment mean weight of the mice was calculated and 2.5 mg/kg and 0.125 mg/kg of the drug were prepared freshly to a final volume of 100 μl in PBS pH 7.2. The drug was prepared freshly, twice daily, 30 minutes before the final injection into the mice. Thereafter, based on the mean weight of the mice the drug was adjusted with PBS pH 7.2 to a concentration appropriate to provide the required mg/kg dose in a final volume of 100 μl. Drug was prepared freshly twice daily, 30 minutes before the final application into the animals. As a vehicle control 100 μl of PBS pH 7.2 was injected s.c into the mice.

(30) Statistical Analysis

(31) ABSA between treatment groups was evaluated by two-way ANOVA conducted using GraphPad Prism 7.

(32) Results

(33) Clinical Signs and Efficacy of Treatment

(34) In Experiment 1, Coversin dose-dependently ameliorated skin inflammation measured by absolute body surface area affected (ABSA). 2.5 mg/kg Coversin was effective in reducing ABSA (FIG. 6). The ABSA in the negative control (vehicle) group was about 7% in experiment 1, which is a typical value achieved in this model. All mice in the negative control group showed a similar inflammatory response.

(35) In Experiment 2, 2.5 mg/kg Coversin significantly (P<0.01) ameliorated skin inflammation measured by (ABSA) (FIG. 7). In this experiment, the ABSA in the negative control (vehicle) group peaked at a mean of about 4%, which is a little bit lower than the value mostly achieved in this model.

(36) There were no deaths during treatment of mice in the two experiments. Mice were terminated on day 12 after initiation of Col7 IgG passive transfer.

Example 3 C5 and LTB4 in Blister Fluid of Patients

(37) The levels of C5a and LTB4 in blister fluid from 4 bullous pemphigoid patients was tested. The results of each of the 4 patients are shown in FIGS. 9A (C5a) and 9B (LTB4). Blister fluid was aspirated with a syringe from blisters of four BP patients admitted to an inpatient clinic with acute BP. Samples were immediately frozen on liquid nitrogen. LTB4 and C5a levels were measured in the fluid using ELISA kits from R&D Systems.

Example 4

Comparison of Effect of Dual Action and Single Action Coversin

(38) In a further experiment, the effect of PAS-L-Coversin was assessed. Mice were treated with 0.1 mg/kg, 10 mg/kg, 1 mg/kg PAS-L-Coversin, or 2.5 mg/kg Coversin, with the experiment being carried out as described above. PAS-L-Coversin has a PAS sequence fused to the N-terminus of the Coversin sequence, which 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). Because of the higher molecule weight of the PAS-L-Cov, 10 mg/kg PAS-L-Cov corresponds to 2.5 mg/kg Coversin. In the first experiment (FIG. 6A) the 10 mg/kg dose but not 1 mg/kg or 0.1 mg/kg dose of PAS-L-Coversin reduced ABSA compared to control. In the second experiment (FIG. 6B), the 10 mg/kg dose and the 1 mg/kg but not the 0.1 mg/kg dose of PAS-L-Coversin reduced ABSA compared to control. The 10 mg/kg dose PAS-L-Coversin was not as effective as the molar equivalent 2.5 mg/kg dose of Coversin (although the 10 mg/kg dose of PAS-L-Coversin shows a statistically significant effect in the second experiment). This suggests that the dual inhibitory activity of Coversin (C5 and LTB4 inhibition) provides improved therapeutic benefit in this model.

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