EXTRACORPOREAL DEVICES FOR METHODS FOR TREATING DISEASES ASSOCIATED WITH ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODIES

Abstract

The invention relates to a blood treatment device configured to remove anti-neutrophil cytoplasmic antibodies (ANCAs) from the blood or blood plasma of a person in need thereof in an extracorporeal blood circuit, wherein the device comprises a matrix, and wherein said matrix comprises a monomeric form of proteinase 3 (PR3). The invention further relates to an extra-corporeal blood circuit comprising a blood treatment device of the invention and to the blood treatment device for use as a medicament or to methods of treating a medical condition associated with ANCA.

Claims

1.-15. (canceled)

16. A blood treatment device configured to remove anti-neutrophil cytoplasmic antibodies (ANCAs) from the blood or blood plasma of a person in need thereof in an extracorporeal blood circuit, wherein the device comprises a matrix, and wherein said matrix comprises a monomeric form of proteinase 3 (PR3).

17. The blood treatment device according to claim 16, wherein the monomeric form of proteinase 3 (PR3) comprises at least one mutation at Ile221 and/or Trp222.

18. The blood treatment device according to claim 16, wherein the monomeric form of proteinase 3 (PR3) comprises at least mutations at Ile221 and Trp222.

19. The blood treatment device according to claim 16, wherein the monomeric form of proteinase 3 (PR3) comprises the Ile221Ala and/or Trp222Ala mutations.

20. The blood treatment device according to claim 16, wherein the monomeric form of proteinase 3 (PR3) comprises a mutation that reduces or abolishes protease activity.

21. The blood treatment device according to claim 16, wherein the matrix comprises a support to which the monomeric form of proteinase 3 (PR3) is bound.

22. The blood treatment device according to claim 16, wherein the blood treatment device is an adsorption cartridge and is perfused with whole blood.

23. The blood treatment device according to claim 16, wherein the blood treatment device is located in an extracorporeal blood circuit through which the blood of the patient passes and which is configured for transporting blood from the patient's vascular system to the blood treatment device at a defined flow rate and for returning the treated blood back to the patient.

24. The blood treatment device according to claim 23, wherein the extracorporeal blood circuit in which the blood treatment device is located further comprises a hemodialyzer which is located upstream or downstream of the blood treatment device.

25. An extracorporeal blood circuit comprising a blood treatment device according to claim 16, wherein the extracorporeal blood circuit comprises means for transporting blood or blood plasma from the patient's vascular system to the blood treatment device at a defined flow rate and means for returning the treated blood or blood plasma back to the patient.

26. The extracorporeal blood circuit according to claim 25, wherein the extracorporeal blood circuit further comprises a hemodialyzer for the hemodialysis of blood, wherein the hemodialyzer is located upstream or downstream of the blood treatment device.

27. The extracorporeal blood circuit according to claim 25, wherein said extracorporeal blood circuit further comprises a plasma dialyzer or centrifuge-based plasma separation system configured to separate blood plasma from blood, wherein the blood plasma is passed through the blood treatment device, wherein the blood treatment device is located downstream of the plasma outlet port of the plasma dialyzer.

28. A method of treating a medical condition associated with anti-neutrophil cytoplasmic antibodies (ANCA), said method comprising removing anti-neutrophil cytoplasmic antibodies (ANCAs) from the blood or blood plasma of a person in need thereof using the blood treatment device according to claim 16.

29. The method of treatment according to claim 28, wherein the medical condition associated with anti-neutrophil cytoplasmic antibodies (ANCAs) is an autoimmune disease associated with the presence of anti-PR3 autoantibodies.

30. The method of treatment according to claim 28, wherein the medical condition associated with anti-neutrophil cytoplasmic antibodies (ANCAs) is anti-neutrophil cytoplasmic autoantibody (ANCA)-associated autoimmune vasculitis (AAV).

31. The blood treatment device according to claim 20, wherein the mutation that reduces or abolishes protease activity is selected from one or more mutations in the group consisting of His71, Asp118 and/or Ser203.

32. The blood treatment device according to claim 31, wherein the mutation that reduces or abolishes protease activity is His71Glu, Asp118Ala and/or Ser203Ala.

33. The blood treatment device according to claim 21, wherein the support comprises or consists of a material selected from the group consisting of hollow fiber membrane, flat sheet membrane, fiber mat, resin, non-woven and open porous foams

34. The blood treatment device according to claim 33, wherein the support comprises or consists of polyurethane (PU) foam.

35. The blood treatment device according to claim 23, wherein the extracorporeal blood circuit in which the blood treatment device is located further comprises a plasma dialyzer or centrifuge-based plasma separation system which allows for the separation of a plasma fraction from the blood, and wherein the blood treatment device is located downstream of the plasma outlet port of the plasma dialyzer.

36. The blood treatment device according to claim 25, wherein the blood treatment device is a hemodialyzer for the hemodialysis of blood, and wherein the hemodialyzer comprises a monomeric form of proteinase 3 (PR3) and is configured to immobilize anti-neutrophil cytoplasmic antibodies (ANCAs).

37. The blood treatment device according to claim 25, wherein the blood treatment device is a plasma dialyzer configured to separate blood plasma from blood, and wherein the plasma dialyzer comprises a monomeric form of proteinase 3 (PR3) and is configured to immobilize anti-neutrophil cytoplasmic antibodies (ANCAs).

38. The blood treatment device according to claim 25, wherein the autoimmune disease associated with the presence of anti-PR3 autoantibodies is an anti-neutrophil cytoplasmic antibody (ANCA) vasculitides, pauci-immune crescentic glomerulonephritis or eosinophilic granulomatosis with polyangiitis.

39. The blood treatment device according to claim 38, wherein the anti-neutrophil cytoplasmic antibody (ANCA) vasculitides is a granulomatosis with polyangiitis or microscopic polyangiitis.

Description

FIGURES

[0167] The invention is further described by the following figures. There are intended to represent a more detailed illustration of a number of preferred non-limiting embodiments or aspects of the invention without limiting the scope of the invention described herein.

[0168] FIG. 1: Comparison of wtPR3 and monomeric PR3 properties by size exclusion chromatography. (A) shows a size exclusion chromatogram (S200) with the elution profile of wtPR3. The protein does not elute in a discrete peak but rather as a collection of large aggregates with approximate sizes ranging from monomeric (ca. 26 kD) to over 600 kD. (B) shows the analogous chromatogram of a monomeric Trp222Ala PR3 variant showing a more uniform elution primarily at the predicted retention time of a monomeric species.

[0169] FIG. 2: Comparison of wtPR3 and monomeric PR3 properties by SPR and ELISA. (A) shows a raw SPR sensorgram of the binding interaction of a concentration series of soluble monomeric Trp222Ala PR3 variant to immobilized CD177. The affinity of the interaction was measured to be 5.7×10-8M. (B) shows an ELISA assay using two different anti-PR3 antibodies (anti-PR3 40 and anti-PR3 81) purified from hybridoma supernatants. The antibodies each recognize distinct, non-overlapping epitopes on PR3. wtPR3 (left bar) and the monomeric Trp222Ala PR3 variant (right bar) are equally well recognized by the antibodies.

[0170] FIG. 3: Schematic representation of an extracorporeal treatment circuit comprising a blood treatment device. The device can be a cartridge or filter comprising a membrane, resin or non-woven based support to which a ligand having affinity for a target protein has been bound. The circuit can be operated in hemoperfusion mode. In cases where the blood treatment device is a hollow fiber membrane filter device the treatment mode can be hemodialysis, hemodiafiltration, hemofiltration or hemoperfusion of the filter with closed dialysate/filtrate ports.

[0171] FIG. 4: Schematic representation of an extracorporeal treatment circuit comprising a blood treatment device. The device can be an adsorption cartridge comprising a resin or non-woven or a filter comprising a membrane, to which a ligand having affinity for a target protein has been bound, respectively. The blood treatment device can be located upstream of a hemodialyzer (pre-dialyzer setting, FIG. 4A) or downstream of a hemodialyzer (post-dialyzer setting, FIG. 4B). The non-functionalized hemodialyzer in the circuit can be operated in different treatment modes depending on the medical need, including hemodialysis, hemodiafiltration or hemofiltration mode.

[0172] FIG. 5: Schematic representation of an extracorporeal treatment circuit comprising a blood treatment device. The device is perfused with blood plasma. In the embodiment shown, a plasma separation filter is used to separate blood plasma from whole blood. The plasma filter generates a plasma fraction comprising the target protein by means of pore sizes ranging from 0.03 μm and 2 μm. The plasma is perfused through the blood treatment device which comprises a matrix based on a non-woven, resin or membrane support to which a ligand having an affinity to a target protein has been bound.

[0173] FIG. 6: Schematic representation of the covalent coupling of a target protein to an epoxy-activated or an amino support. The support can be a resin, a membrane, including hollow fiber membranes, flat sheet membranes or fiber mats, or a non-woven. (A) shows the direct coupling of the protein via amino groups of the protein to the support (Example 6). (B) shows the covalent immobilization of enzymes is based on the use of amino resins. Amino resins can be pre-activated with glutaraldehyde and then used in for covalent immobilization of enzymes. Reaction of an aldehyde group with an amino group of the target proteins is fast and forms a Schiff base (imine), resulting in a stable multipoint covalent binding between enzyme and carrier. The imine double bonds can be further reduced with borohydrides.

EXAMPLES

[0174] The invention is further described by the following examples. These are intended to present support for the workability of a number of preferred non-limiting embodiments or aspects of the invention without limiting the scope of the invention described herein.

Example 1: Production of Monomer PR3 Protein Variants

[0175] Monomeric PR3 protein variants were created essentially as described in Jerke et al (2017, Scientific Reports 7:43328). The proteins were produced from plasmid pTT5 as C-terminal fusions with a human Ig1 Fc, secreted from 293_6E EBNA cells (NRC, Canada) via an N-terminal Ig1 secretion signal peptide and purified from the culture supernatant by passage over immobilized Protein A. Protein A eluted material was immediately neutralized with 1 M HEPES pH 7.5 and Fc removed by addition of TEV protease and incubation overnight at 4° C. The flowthrough of a subsequent Protein A trap column was concentrated and passed over a Superdex 200 size exclusion in 20 mM HEPES, 150 mM NaCl, pH 7.5 for CD177 and in the same buffer plus 0.02% LM for PR3 variants. PR3 variants were activated by enterokinase removal of an N-terminal FLAG peptide and passage over anti-M2 agarose.

Example 2: Comparison of wtPR3 and Monomeric PR3 Properties by Size Exclusion Chromatography

[0176] PR3 proteins obtained as described above were assessed using size exclusion chromatography for molecular weight. PR3 proteins were diluted in suitable buffer (500 μl @ 1.2 mg/ml, 20 mM HEPES, 150 mM NaCl, 0.02% lauryl-maltoside, pH 7.4), passed through a 0.45 μm filter and subsequently applied to a Superdex S200 gel filtration column at a flow rate of 0.5 ml/min, at 4 degrees C. and applied to a gel filtration column. High molecular weight aggregates of wtPR3 eluted at approx. 8.8 mL elution volume (FIG. 1A), whereas the Trp222Ala PR3 variant eluted primarily at approx. 16.5 mL elution volume, consistent with a monomeric form of the protein (FIG. 1B). Similar results are obtained for Ile221Ala, Ile221Ala+Trp222Ala, Ile221Ala+Ser203Ala, and Ile221Ala+Trp222Ala+Ser203Ala. Further PR3 mutants are being assessed.

Example 3: Comparison of wtPR3 and Monomeric PR3 Properties by SPR

[0177] Experiments were performed on a ProteOn XPR36 instrument (BioRad) with proteins immobilized to GLH sensor chips (BioRad) using standard amine chemistry. The binding interaction of a concentration series of soluble monomeric Trp222Ala PR3 variant with immobilized CD177 was assessed (FIG. 2A). The affinity of the interaction was measured to be 5.7×10-8M, indicating that the PR3 variant shows no loss in biding to its natural protein target CD177.

Example 4: Comparison of wtPR3 and Monomeric PR3 Properties by ELISA

[0178] PR3 ELISA was used to quantitatively assess PR3 amounts. Briefly, anti-PR3 capture antibodies (anti-PR3 40 and anti-PR3 81) were coated, blocked and incubated with PR3 samples of WT or monomeric variants. After washing, biotinylated anti-PR3 detection mab was added. A streptavidin-HRPO conjugate and OPD substrate were used to visualize binding. The absorbance was determined at 405 nm in a plate reader. FIG. 2B shows that wtPR3 (left bar) and the monomeric Trp222Ala PR3 variant (right bar) are equally well recognized by the antibodies.

Example 5: Preparation of a Matrix Comprising an Epoxy-Functionalized Resin

[0179] First, the resin is equilibrated. The resin is washed with immobilization buffer and filtered. A resin/buffer ratio of 1/1 (w/v) is preferable. The immobilization buffer is chosen to be compatible with PR3. The process is repeated 2-4 times. The PR3 solution is prepared by dissolving the protein in immobilization buffer. For example, 100-200 mg PR3 can be loaded per gram of wet resin. Protein concentration can be determined by using standard protein content assays. The PR3 is dissolved in a sufficient amount of buffer to obtain a ratio resin/buffer of 1:4 (w/v). This ratio can be optimized depending on the PR protein used (range can vary from 1:1-1:4). Immobilization begins with the transfer of the immobilization buffer containing the PR3 protein into the immobilization vessel. The epoxy-functionalized resin, for example the Purolite® Lifetech™ resin described herein, is then added. The slurry is gently mixed at 70-80 rpm for 18 h and afterwards left without mixing for another 20 h. Magnetic stirring during protein immobilization should be avoided as this can damage the beads. Immobilization can be performed at temperatures of 20° C.-30° C., depending on the protein stability. Immobilizations should not be performed at high temperatures as this can cause degradation of the epoxy rings (hydrolysis) and facilitate microbial growth. Finally, the liquid phase is filtered off and collected. The protein content in the liquid is determined and the immobilization yield calculated. The resin is then washed with washing buffer. The process is repeated 2-4 times under gentle stirring or in column wash. An additional washing step using a 0.5 M NaCl containing buffer for complete desorption of non-covalently bound proteins can be performed. Excess water is removed by filtration. The immobilized PR3 protein can then be characterized in terms of moisture content and specific binding activity.

Example 6: Preparation of a Matrix Comprising an Epoxy-Functionalized Resin

[0180] First, the resin is equilibrated. The resin is washed with immobilization buffer and filtered. A resin/buffer ratio of 1:1 (w/v) is preferable. The immobilization buffer is chosen to be compatible with the PR3 protein. In a second step 2% glutaraldehyde buffer is prepared starting from a solution of 25% (w/v) glutaraldehyde. A 2% glutaraldehyde (v/v) solution is prepared using the immobilization buffer. In a third step, the amino resin is activated by adding the 2% glutaraldehyde buffer prepared in step 2 to the resin. The optimal volume of 2% glutaraldehyde buffer should be in the range of resin/buffer ratio of 1:4 (w/v). The slurry is left to mix for 60 min at 20° C.-25° C. The beads are then filtered and washed with immobilization buffer using a resin/buffer ratio of 1:4 (w/v). It should be avoided to store pre-activated resin for a period longer than 48 h. Beads are then ready for the immobilization step. In a fourth step the PR3 protein solution is prepared. To that end, the protein is dissolved in immobilization buffer. For example, between 1 mg and 100 mg PR3 protein can be loaded per gram of wet resin. The protein concentration can be determined by using standard protein content assays.

[0181] The protein is dissolved in buffer to obtain a ratio resin/buffer of 1:4 (w/v). Optimization of this ratio can be pursued in further trials (range can vary from 1:1-1:4). In a fifth step, the protein is immobilized. The immobilization buffer is transferred into the immobilization vessel and the pre-activated amino resin (e.g. from Purolite®, Lifetech™) as prepared in step 3 is added. The slurry is gently mixed for 18 h at 70-80 rpm. Magnetic stirring should be avoided during immobilization as this can damage the beads. The immobilization can be performed at 20° C.-30° C. accordingly to PR3 protein stability. The immobilization should not be performed at high temperatures since this might cause side reactions of the aldehyde groups on the resin formed during step 3. Finally, the liquid phase is filtered off and collected. The protein content in the liquid is determined and the immobilization yield calculated. The resin is then washed with washing buffer. The process is repeated 2-4 times under gentle stirring or in column wash. An additional washing step using a 0.5 M NaCl containing buffer for complete desorption of non-covalently bound proteins can be performed. Excess water is removed by filtration. The immobilized PR3 protein can then be characterized in terms of moisture content and specific binding activity.