Modified serpins for the treatment of bradykinin-mediated disease

Abstract

The present invention relates to modified serpins for use in the treatment of bradykinin-mediated diseases. The modified serine protease inhibitors (serpins) have mutations in one or more of the P4, P3, P2, P1 and P1 residues of their reactive center loop, which mutations increase the serpin's inhibition of plasma kallikrein (PK) as compared to the corresponding unmodified serpin. The mutations in the modified serpins of the invention further ensure that serpins display substantially no inhibition of at least thrombin and activated protein C. A modified serpin of the invention further preferably shows increased inhibition of at least one of an active form of Factor XII (FXII) and plasmin as compared to the corresponding unmodified serpin, and, preferably, the serpin inhibits at least one of an active form of FXII and PK stronger than they are inhibited by C1 esterase inhibitor. Preferably the modified serpin is a modified 1-antitrypsin. The invention further pertains to nucleic acid molecule encoding the modified serpins of the invention, e.g. a gene therapy vector, and to pharmaceutical compositions comprising the modified serpins of the invention or such gene therapy vectors.

Claims

1.-15. (canceled)

16. A method of treating a bradykinin-mediated disease, wherein the method comprises administering to a subject in need thereof an effective amount of a modified serpin wherein the serpin has mutations in one or more of the P4, P3, P2, P1 and P1 residues of its reactive center loop (RCL), wherein the P1 residue is lysine or arginine, the P2 residue is not proline and wherein the P4 residue is serine, wherein the mutations increase the inhibition of plasma kallikrein (PK) as compared to the corresponding unmodified serpin, wherein the serpin more strongly inhibits PK than the serpin inhibits thrombin, wherein preferably, the serpin more strongly inhibits PK than the serpin inhibits activated protein C (APC) and wherein the serpin comprises an amino acid sequence that has at least 70% sequence identity to the sequence of a wild-type serpin selected from the group consisting of 1-antitrypsin (1AT) (SERPINA1), 1-antichymotrypsin (SERPINA3), C1-esterase inhibitor (SERPING1), 2-antiplasmin (SERPINF2), antithrombin (ATIII) (SERPINC1), heparin cofactor II (HCII) (SERPIND1), protein C inhibitor (PCI) (SERPINA5), Kallistatin (SERPINA4), Plasminogen activator inhibitor (SERPINE1), Protease nexin 1 (SERPINE2) and Protein Z-dependent protease inhibitor (SERPINA10).

17. The method of claim 16, wherein the serpin comprises an amino acid sequence that has at least 70% sequence identity to wild-type serpin 1 AT.

18. The method of claim 16, wherein the mutations increase the inhibition of at least one of an active form of factor XII (FXII) and plasmin as compared to the corresponding unmodified serpin, and wherein, preferably, the serpin inhibits at least one of an active form of FXII and PK as strong or stronger than they are inhibited by C1 esterase inhibitor.

19. The method of claim 16, wherein the minimal amount of the serpin that inhibits PK activity by at least 50%, is an amount that inhibits thrombin activity by no more than 15%.

20. The method of claim 19, wherein the minimal amount of the serpin that inhibits PK activity by at least 50%, is an amount that inhibits at least one of: a) plasmin by at least 15%; b) an active form of FXII by at least 15%; and, c) APC by no more than 15%.

21. The method of claim 16, wherein the serpin comprises an RCL wherein the residues P4-P1 of the RCL have an amino acid sequence selected from the group consisting of SMTR, SGQR, SVTR, SATR, SFNR, SWKK, SEAR, SVVK, SDYK, SVRK, SPRR, SMDR, SLGR, SKGR, SGNR, and SMHR, or wherein the residues P4-P1 of the RCL have the amino acid sequence SLLR and wherein the P1 residue is selected from the group consisting of: Q, F, H, R, K, C, L, Y, N, I, D, W, E, V, M, A, T and P, of which K, L, Y, I, D, E, A, T, M, R and V are preferred, and I and V are most preferred.

22. The method of claim 16, wherein the serpin further has a mutation whereby the P1 residue of its reactive center loop (RCL) is selected from the group consisting of: Q, F, H, R, K, C, L, Y, N, I, D, W, E, V, M, A, T and P, of which K, L, Y, I, D, E, A, T, M, R and V are preferred, and I and V are most preferred.

23. The method of claim 16, wherein the wild type serpin has been modified to reduce the susceptibility to oxidation.

24. The method of claim 16, wherein the bradykinin-mediated disease is selected from the group consisting of hereditary angioedema, idiopathic angioedema, rheumatoid arthritis, Crohn's disease, ulcerative colitis, lupus, Alzheimer's disease, septic shock, burn injury, brain ischemia/reperfusion injury, cerebral edema, diabetic retinopathy, diabetic nephropathy, macular edema, vasculitis, arterial or venous thrombosis, thrombosis associated with ventricular assist devices or stents, heparin-induced thrombocytopenia with thrombosis, thromboembolic disease, and coronary heart disease with unstable angina pectoris, edema, eye disease, gout, intestinal bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spine disease, post-operative ileus, aortic aneurysm, osteoarthritis, hereditary angioedema, pulmonary embolism, stroke, head trauma or peri-tumor brain edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosis nephritis, an autoimmune disease, an inflammatory disease, such as (chronic) auto-inflammatory urticaria, a cardiovascular disease, a neurological disease, a disease associated with protein misfolding, a disease associated with angiogenesis, hypertensive nephropathy and diabetic nephropathy, allergic and respiratory diseases (e.g. anaphylaxis, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, persistent, rhinitis, urticarial and histamine dependent angioedema), tissue injuries (e.g. burn or chemical injury) and poli-trauma (e.g. car crashed and battle injuries).

25. A gene therapy vector comprising a nucleotide sequence encoding a modified serpin as defined in claim 16, for use in the treatment of a bradykinin-mediated disease, wherein, preferably, the bradykinin-mediated disease is selected from the group consisting of hereditary angioedema, idiopathic angioedema, rheumatoid arthritis, Crohn's disease, ulcerative colitis, lupus, Alzheimer's disease, septic shock, burn injury, brain ischemia/reperfusion injury, cerebral edema, diabetic retinopathy, diabetic nephropathy, macular edema, vasculitis, arterial or venous thrombosis, thrombosis associated with ventricular assist devices or stents, heparin-induced thrombocytopenia with thrombosis, thromboembolic disease, and coronary heart disease with unstable angina pectoris, edema, eye disease, gout, intestinal bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spine disease, post-operative ileus, aortic aneurysm, osteoarthritis, hereditary angioedema, pulmonary embolism, stroke, head trauma or peri-tumor brain edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosis nephritis, an autoimmune disease, an inflammatory disease, such as (chronic) auto-inflammatory urticaria, a cardiovascular disease, a neurological disease, a disease associated with protein misfolding, a disease associated with angiogenesis, hypertensive nephropathy and diabetic nephropathy, allergic and respiratory diseases (e.g. anaphylaxis, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, persistent, rhinitis, urticarial and histamine dependent angioedema) tissue injuries (e.g. burn or chemical injury) and poli-trauma (e.g. car crashed and battle injuries).

26. A modified 1-antitrypsin having mutations in one or more of the P4, P3, P2, P1 and P1 residues of its reactive center loop (RCL), wherein the mutations increase the inhibition of plasma kallikrein (PK) as compared to the corresponding unmodified serpin, and wherein the serpin more strongly inhibits PK than the serpin inhibits either one of thrombin and APC.

27. The modified 1-antitrypsin of claim 26, wherein the 1-antitrypsin comprises an RCL wherein the residues P4-P1 of the RCL have an amino acid sequence selected from the group consisting of SMTR, SGQR, SVTR, SATR, SFNR, SWKK, SEAR, SVVK, SDYK, SVRK, SPRR, SMDR, SLGR, SKGR, SGNR, SMHR and SLLR, and wherein preferably, the P1 residue is selected from the group consisting of: Q, F, H, R, K, C, L, Y, N, I, D, W, E, V, M, A, T and P, of which K, L, Y, I, D, E, A, T, M, R and V are preferred, and I and V are most preferred, and wherein, optionally the 1-antitrypsin has been further modified to have a reduced susceptibility to oxidation.

Description

DESCRIPTION OF THE FIGURES

[0124] FIG. 1. Inhibition of plasma kallikrein-like activity in human plasma by various 1-Antitrypsin variants as indicated compared to inhibition by plasma-derived C1 esterase inhibitor. Plasma kallikrein-like activity activity is determined using the chromogenic substrate L2120.

[0125] FIG. 2. Inhibition of plasma kallikrein-like activity in mouse plasma by various 1-Antitrypsin variants as indicated. Plasma kallikrein-like activity activity is determined using the chromogenic substrate L2120.

[0126] FIG. 3. Inhibition of Factor XIIa activity by 1-Antitrypsin Pittsburgh variants wherein the P1 prime (P1) position was altered to every natural occurring amino acid as indicated.

[0127] FIG. 4. Inhibition of plasma kallikrein activity by 1-Antitrypsin Pittsburgh variants wherein the P1 prime (P1) position was altered to every natural occurring amino acid as indicated.

[0128] FIG. 5. Inhibition of plasmin activity by 1-Antitrypsin Pittsburgh variants wherein the P1 prime (P1) position was altered to every natural occurring amino acid as indicated.

[0129] FIG. 6. Inhibition of thrombin activity by 1-Antitrypsin Pittsburgh variants wherein the P1 prime (P1) position was altered to every natural occurring amino acid as indicated.

[0130] FIG. 7. Inhibition of activated protein C activity by 1-Antitrypsin Pittsburgh variants wherein the P1 prime (P1) position was altered to every natural occurring amino acid as indicated.

EXAMPLES

Example 1

[0131] Modified Serpin Construction

[0132] The SERPINA1 cDNA sequence was obtained from the NCBI database (NM_001127707.1). The sequence coding for the signal peptide was discarded and replaced by a sequence coding for a Tobacco Etch Virus cleavage site. At the 5 side an EcoRI digestion site was added and at the 3 side and NotI digestion was added after the STOP codon of SERPINA1. Hereafter the .sup.354AIPM.sup.358 sequence (amino acid numbering without the signal peptide) was replaced with SMTR sequence (Codon sequence was grafted from wild type F12 NCBI: NM_000505.3). The construct was obtained from IDT (Integrated DNA Technologies, Leuven, Belgium)) as a custom gene construct. The custom gene construct was propagated in top E. coli TOP10 and selected by ampicillin resistance. Obtained plasmid DNA was digested by EcoR1 and NotI. The resulting insert (1223 bp) was separated on and isolated from agarose gel and ligated into a modified pcDNA6 expression vector (pSM2) (De Maat et al, 2016 Clin Immunol November; 30; 138(5):1414-23)). pSM2 encodes for a N-terminal murine IgK secretion signal and a double STREP isolation tag where after the SerpinA1 construct is ligated.

[0133] For the construction of the wild type SERPINA1 or the other mutants the C-terminal part of the SERPINA1 sequence was replaced via BstelI-NotI digestion. The new constructs were ordered from IDT as gene block and ligated in to the digested SERPINA1-.sup.354SMTR.sup.358-pSM2 backbone.

Example 2

[0134] 1-Antitrypsin Expression

[0135] The SERPINA1-pSM2 constructs were transfected into HEK293 FreeStyle cells using 239Fectin as instructed by the manufacturer (ThermoFisher). After 4 days of protein production, the cells were spun down at 2000g for 5 minutes. Hereafter the supernatant was collected and stored at 20 C. until further use.

Example 3

[0136] Enzyme Inhibition by 1-Antitrypsin Variants

[0137] 10 l of supernatant containing the 1-Antitrypsin variants was incubated with 10 l of enzyme and 20 l of buffer (0.2% w/v bovine serum albumin in Hepes buffered saline: 10 mM HEPES, 150 mM NaCl, pH=7.4). After a 5 minute incubation the substrate was added and substrate conversion was monitored according to substrate specifications. Inhibition percentage was determined within the linear part of the substrate conversion, where substrate conversion without the presence of any l-AT variant was set as 0% inhibition.

[0138] Enzymes [0139] Human FXIIa (final concentration: 2 g/mL) [0140] Human FXIIa (final concentration: 0.7 g/mL) [0141] Plasma kallikrein (final concentration: 2 g/mL) [0142] Thrombin (final concentration: 2 U/mL) [0143] Plasmin (final concentration: 2 g/mL) [0144] Activated protein C (final concentration: 2 g/mL)

[0145] Substrates

TABLE-US-00004 Final Enzyme Substrate Concentration Method Wavelength FXIIa L2120 0.5 mM Absorption 405 nm PK L2120 0.5 mM Absorption 405 nm Thrombin I1140 2 mM Fluorescence Ext: 380 Em: 460 Plasmin I1390 0.5 mM Fluorescence Ext: 380 Em: 460 APC S2366 0.5 mM Absorption 405 nm

[0146] The following substrates where used: [0147] L2120 (Bachem, Cat#L-2120.0100), H-D-Pro-Phe-Arg-pNA-2 HCl; a chromogenic substrate for the determination of plasma kallikrein-like activity used as described in de Maat et al. (2016 Clin Immunol November; 30; 138(5):1414-23). [0148] I1140 (Bachem, Cat#I-1140.0100), Benzyloxycarbonyl-Gly-Gly-Arg-7-amido-4-methylcoumarin.HCl; can used as substrate in a direct fluorometric assay of urokinase and tissue-type plasminogen activator as well as for assaying trypsin and thrombin used as described in Chowdary et al. (2015, Br. J. Haematol., 168: 719). [0149] I1390 (Bachem, Cat#I-1390.0050), H-D-Val-Leu-Lys-7-amido-4-methylcoumarin acetate salt; a sensitive, highly specific fluorescent substrate for plasmin used as described in de Maat et al. (2016 Clin Immunol November; 30; 138(5):1414-23). [0150] S2366 (Chromogenix; Cat# S2366), pyroGlu-Pro-Arg-pNA-HCl; Chromogenic substrate for activated protein C and factor XIa as described in Hubbard et al. (1988, Thromb Haemost; 59, 464-467).

[0151] Results of the inhibition of the relevant proteases by the various 1AT variants are provide in Table 1.

TABLE-US-00005 TABLE 1 Overview of the percentage inhibition of the relevant proteases by the various 1-Antitrypsin variants of the invention. Plasma Amino acid residues in RCL positions FXIIa FXIIa Kallikrein Plasmin Thrombin APC 1-AT P8 and P4-P4 Aver- St Aver- St Aver- St Aver- St Aver- St Aver- St variants P8 P4 P3 P2 P1 P1 P2 P3 P4 age Dev age Dev age Dev age Dev age Dev age Dev WT M A I P M S I P P 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 M358R M A I P R S I P P 94.0 2.8 90.0 91.5 3.5 100.0 0.0 100.0 0.0 94.0 4.2 357AR358 M A I A R S I P P 62.5 10.6 49.0 98.0 0.0 99.0 1.4 97.5 0.7 66.5 17.7 SMTR M S M T R S I P P 88.5 9.2 85.0 98.5 0.7 94.0 1.4 10.5 0.7 75.0 18.4 SMTRox* V S M T R S I P P 92.5 3.5 89.2 4.8 98.7 0.8 94.5 1.4 10.3 1.5 76.5 8.4 SMTRox*- V S M T R V V G G NT 29.0 47.0 8.0 1.0 0.0 VVGG SMTRox*- V S M T R G V G G NT 9.0 39.0 20.0 3.0 3.0 GVGG SGQRox* V S G Q R S I P P 4.5 2.1 4.0 88.0 1.4 5.0 7.1 14.5 0.7 6.5 0.7 SVTRox* V S V T R S I P P 47.0 39.0 99.0 97.0 7.0 49.0 SATRox* V S A T R S I P P 17.0 15.0 94.0 0.0 3.0 10.0 SFNRox* V S F N R S I P P 5.0 3.0 94.0 3.0 7.0 5.0 SWKKox* V S W K K S I P P NT 5.0 94.0 47.0 0.0 0.0 SEARox* V S E A R S I P P NT 6.0 92.0 13.0 0.0 3.0 SLLRox* V S L L R S I P P NT 41.0 3.5 97.7 1.5 94.7 1.5 62.3 8.1 9.7 5.0 SLLRox*-V V S L L R V I P P NT 47.0 8.5 97.5 0.7 68.0 1.4 4.5 4.9 3.0 2.8 SLLRox*-I V S L L R I I P P NT 40.3 8.5 91.3 5.0 66.7 2.9 3.7 3.2 2.3 2.1 SVVKox* V S V V K S I P P 4.0 2.0 55.0 95.0 6.0 8.0 SDYKox* V S D Y K S I P P NT 7.0 50.0 7.0 0.0 1.0 SVRKox* V S V R K S I P P 2.0 0.0 91.0 97.0 1.0 4.0 SPRRox* V S P R R S I P P 1.0 2.0 97.0 19.0 0.0 1.0 SMDRox* V S M D R S I P P NT 4.0 87.0 20.0 0.0 0.0 SLGRox* V S L G R S I P P NT 17.3 2.9 95.5 0.6 5.3 2.4 9.5 2.1 1.0 1.2 SKGRox* V S K G R S I P P NT 22.0 97.0 37.0 7.0 0.0 SGNRox* V S G W R S I P P NT 5.2 1.3 71.6 1.9 1.0 2.2 0.0 0.0 1.0 1.0 SYARox* V S Y A R S I P P NT 20.0 97.0 0.0 62.0 7.0 SMHRox* V S M H R S I P P NT 18.3 4.0 96.3 0.6 73.0 6.1 10.0 1.0 34.0 5.6 SNSRox* V S N S R S I P P NT 20.0 97.0 0.0 62.0 7.0 *ox indicates that methionine (M) residues 351 (P8) is replaced with a serine (S) residues to prevent oxidative inactivation of 1AT

Example 4

[0152] Inhibition in Human and in Mouse Plasma

[0153] 1-Antitrypsin variants as indicated in FIGS. 1 and 2 were tested for their ability to inhibit plasma kallikrein-like activity as determined with the chromogenic substrate L2120 in respectively human and mouse plasma. In human plasma the inhibition by 1-Antitrypsin variants was also compared with that by plasma-derived C1 esterase inhibitor (Alpha Diagnostics; C1E15-N).

[0154] 50 l of human plasma (3.2% Citrate) was mixed with 20 l 1-Antitrypsin variant (20 g/mL final conc.) or C1 esterase inhibitor (40 g/mL final conc.), 20 l L2120 chromogenic PK substrate (0.5 mM final conc.) and 10 l Kaolin (18,75 g/mL final conc.). Inhibition of plasma kallikrein-like activity was determined as described in Example 3. Results are shown in FIG. 1.

[0155] 20 l of mouse plasma (3.2% Citrate) was mixed with 20 l 1-Antitrypsin variant (100 g/mL final conc.), 10 l L2120 chromogenic PK substrate (0.5 mM final conc.) and 5 l Kaolin (135 g/mL final conc.). Inhibition of plasma kallikrein-like activity was determined as described in Example 3. Results are shown in FIG. 2.

Example 5

[0156] Enzyme Inhibition by 1-Antitrypsin P1 Prime-Variants

[0157] The amino acid of the P1 prime (P1) position was altered to every natural occurring amino acid. As a template sequence we used the 1-Antitrypsin-Pittsburgh sequence having the P4-P3-P2-P1/P1-P2-P3-P4 sequence: AIPR/XIPP for the library, wherein X marks the P1 position in which the amino acid was varied.

[0158] 1-Antitrypsin variants were produced as described above in Example 2, except that supernatant was harvested after 5 days. Empty production vector was used as a negative control (No A1AT). Production of the 1-Antitrypsin variants was quantified by coomassie blue and supernatants were diluted in pSM2 media to equalize the concentrations of the different 1AT variants. Enzyme inhibition by the 1-Antitrypsin P1-variants was tested as described in Example 3 above.

[0159] Results are shown in FIG. 3 for the inhibition of Factor XIIa, FIG. 4 for the inhibition of plasma kallikrein, FIG. 5 for the inhibition of plasmin, FIG. 6 for the inhibition of thrombin and FIG. 7 for the inhibition of activated protein C. Results indicate that A, M, R and T are preferred residues at the P1 position.