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EP-4735111-A1 - SINGLE-DOMAIN ANTIBODY FOR INHIBITION OF NEUTROPHIL ELASTASE ACTIVITY

EP4735111A1EP 4735111 A1EP4735111 A1EP 4735111A1EP-4735111-A1

Abstract

The present invention relates to a binding agent capable of specifically binding to and inhibiting competitively a neutrophil elastase, which binding agent comprises an immunoglobulin single variable domain (ISVD), as well as related products, methods, and uses, such as in particular methods and uses aimed at the prevention, treatment, or diagnostics of inflammatory diseases.

Inventors

  • DUMOULIN, Mireille
  • MORALES YÀNEZ, Francisco
  • REDEGHIERI, Paola
  • VANBEVER, RITA
  • VANDERSTRAETEN, Jessica
  • LEAL, Teresinha
  • MOTTAIS, Angélique
  • MUYLDERMANS, SERGE
  • VINCKE, CÉCILE

Assignees

  • Université de Liège
  • Université catholique de Louvain
  • Vrije Universiteit Brussel

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. A binding agent capable of specifically binding to and inhibiting a neutrophil elastase (NE), wherein the binding agent comprises an immunoglobulin single variable domain (ISVD) and the binding agent competes with Elastase Inhibitor 3 (EI3) for binding to NE.
  2. 2. A binding agent according to claim 1, wherein the binding agent comprises an immunoglobulin single variable domain (ISVD) binding to at least one of the residues R36, A60, N61, P96, V97, S195, G218 and G219, preferably two or more of the residues R36, A60, N61, P96, V97, S195, G218 and G219, more preferably three or more of the residues R36, A60, N61,P96, V97, S195, G218 and G219, more preferably all of the residues R36, A60, N61, P96, V97, S195, G218 and G219 of the NE, such as to at least the residues R36, A60, N61, P96, V97 and S195 of the NE.
  3. 3. The binding agent according to claim 1 or 2, wherein the binding agent comprises an immunoglobulin single variable domain (ISVD) comprising a complementarity determining region 1 (CDR1) having the sequence set forth in SEQ ID NO: 1 (GRTISLYR), a CDR2 having the sequence set forth in SEQ ID NO:2 (INWSGDMT) and a CDR3 having the sequence set forth in SEQ ID NO:3 (TADPKLLPLADS SYGY) .
  4. 4. The binding agent according to claim 1 or 2, wherein the binding agent comprises an ISVD comprising a CDR1, CDR2 and CDR3, each as present in SEQ ID NO: 4 wherein the CDR1, CDR2 and CDR3 are annotated according to anyone of IMGT, Kabat, Chlotia, Martin or AHo numbering systems.
  5. 5. The binding agent according to claim 1 to 4, which comprises or consists of a VHH.
  6. 6. The binding agent according to any one of claims 1 to 5, wherein the ISVD comprises an amino acid sequence with at least 90% identity to the amino acid sequence set forth in SEQ ID NO:4 (QVQLQESGGGLVQAGGSLRLSCVVPGRTISLYRMGWFRQAPGKEREFVAGINWSGDMT DYVDSVKGRFTISRDNAKNTVYLEMNSLKPEDTAIYYCTADPKLLPLADSSYGYWGQGTQ VTVSS).
  7. 7. The binding agent according to any one of claims 1 to 6, wherein the ISVD comprises the amino acid sequence set forth in SEQ ID NO:4 (QVQLQESGGGLVQAGGSLRLSCVVPGRTISLYRMGWFRQAPGKEREFVAGINWSGDMT DYVDSVKGRFTISRDNAKNTVYLEMNSLKPEDTAIYYCTADPKLLPLADSSYGYWGQGTQ VTVSS).
  8. 8. The binding agent according to any one of claims 1 to 7, wherein the binding agent binds to at least a portion of the active site of the NE.
  9. 9. A nucleic acid molecule comprising a polynucleotide sequence encoding the binding agent according to any one of claims 1 to 8, or a vector comprising the nucleic acid molecule.
  10. 10. A cell or a virus comprising the nucleic acid molecule or vector according to claim 9, optionally wherein the cell is capable of expressing or expresses the binding agent or the virus is configured to induce expression of the binding agent in a recipient cell infected with the virus.
  11. 11. A pharmaceutical composition comprising the binding agent according to any one of claims 1 to 8, the nucleic acid molecule or vector according to claim 9, or the cell or virus according to claim 10, and a pharmaceutically acceptable carrier, diluent, and/or excipient, optionally wherein the pharmaceutical composition comprises a further inhibitor of the NE.
  12. 12. The pharmaceutical composition according to claim 11, wherein the pharmaceutical composition is suitable for nasal or pulmonary administration to a subject, optionally wherein the pharmaceutical composition is formulated as an inhalable dry powder, optionally wherein the pharmaceutical composition is configured for a nebulizer or a metered-dose inhaler.
  13. 13. Device comprising the pharmaceutical composition according to claim 11 or 12, wherein the device is a nebulizer or a metered-dose inhaler or a dry powder inhaler.
  14. 14. A kit such as a diagnostic kit comprising the binding agent according to any one of claims 1 to 8, the nucleic acid molecule or vector according to claim 9, or the cell or virus according to claim 10.
  15. 15. The binding agent according to any one of claims 1 to 8, the nucleic acid molecule or vector according to claim 9, the cell or virus according to claim 10, or the pharmaceutical composition according to claim 11, for use in medicine.
  16. 16. The binding agent according to any one of claims 1 to 8, the nucleic acid molecule or vector according to claim 9, the cell or virus according to claim 10, or the pharmaceutical composition according to claim 11, for use in the prevention or treatment of an inflammatory disease.
  17. 17. The binding agent, the nucleic acid molecule, the vector, the cell or virus, or the pharmaceutical composition for use according to claim 16, wherein the inflammatory disease is a pulmonary disease, preferably a disease selected from the group consisting of a muco-obstructive lung disease such as cystic fibrosis, chronic obstructive pulmonary disease (COPD), bronchiectasis, ciliary diskinesia and acute respiratory distress syndrome, inflammatory nose and sinus diseases, or the inflammatory disease is inflammatory bowel disease or the inflammatory disease is an inflammatory skin disease.
  18. 18. The binding agent according to any one of claims 1 to 8, the nucleic acid molecule or vector according to claim 9, the cell or virus according to claim 10, or the kit according to claim 14, for use in the diagnosis of an inflammatory disease in a subject.
  19. 19. A method for determining the level of active neutrophil elastase in a specimen, comprising contacting the specimen with the binding agent according to any one of claims 1 to 8, and detecting at least the neutrophil elastase bound to the binding agent.
  20. 20. A method for the diagnosis, prognosis and/or monitoring of an inflammatory disease, optionally any inflammatory disease as defined in claim 17, in a subject, comprising: - contacting a biological sample obtained from the subject with the binding agent according to any one of claims 1 to 8, and - determining the level of active neutrophil elastase in the sample by detecting at least the neutrophil elastase bound to the binding agent.

Description

Single-domain antibody for inhibition of neutrophil elastase activity FIELD The invention is broadly situated in the medical field and particularly relates to agents, more specifically binding agents, and pharmaceutical formulations suitable for inhibition of neutrophil elastase (NE) activity or for the detection/quantification of active NE. BACKGROUND Neutrophil elastase (NE) is a protease that plays an important role in the immune response and host defence mechanisms in both physiological and disease-associated conditions. NE is stored in azurophilic granules of polymorphonuclear nucleophiles and can be released during neutrophil degranulation and neutrophil extracellular traps release (NETosis). Under physiological conditions, the amount of extracellular NE is tightly regulated, mainly by the antiproteases AAT (alphal- antitrypsin) and Elafin. However, in a number of pathological conditions, this balance is displaced towards exceeding extracellular NE levels, which are associated with progressive destruction of tissues and loss of their associated functions and impaired immune system regulation. Multiple types of inhibitors, including protein-based inhibitors and small molecule inhibitors, are known to inhibit NE activity. Regarding protein-based inhibitors, an inhaled version of AAT, an endogenous glycoprotein inhibitor of neutrophil elastase, has been tested for the treatment of respiratory pathologies. However, the effectiveness of inhaled AAT in reducing inflammatory parameters and improving pulmonary function is significantly limited. While it does not decrease the frequency of infectious exacerbations, it mitigates their severity. The need for administering high doses of inhaled AAT reflects the inadequate potency of the protein (Amin and Ratjen, Expert Opin Emerg Drugs 2014, vol. 19(1), 143-55). This inefficiency may be attributed to the rapid degradation of AAT in pulmonary tissue, as the glycoprotein is highly unstable in biological fluids due to its susceptibility to proteolysis and oxidation (Suter et al., Eur Respir J. 1991, vol. 4(1), 40-9). Moreover, the utilization of nebulizers for pulmonary administration to accommodate the high AAT doses necessitates extended periods of manipulation and inhalation, further adding to the therapy burden for patients. Additional drawbacks associated with AAT are its elevated cost and limited production capacity, as it is derived from human plasma through purification processes. Furthermore, small molecule inhibitors of NE have been studied extensively. However, only few of these molecules, such as Sivelestat, have resulted in an approved drug in the market for a limited number of conditions, partially due to significant organ toxicity. Challenges in the development of small molecules include inefficient efficacy in inhibiting elastase, a lack of specificity towards elastase, and inadequate metabolic stability in vivo. Therefore, there is a compelling demand from patients with significant unmet clinical needs for the development of new, potent agents that effectively inhibit NE. SUMMARY The present invention is at least in part based on the identification of immunoglobulin single variable domain (ISVD) binding agents which bind to and efficiently inhibit neutrophil elastase (NE). In particular, as evidenced in the experimental section of this specification the NbE201 ISVD, which illustrates an embodiment of the present invention, is a competitive inhibitor of NE and demonstrates a high affinity, a high specificity and a high inhibitory capacity of NE. Furthermore, the ISVD displays a high stability and long halftime in the lung, especially when PEGylated. This opens an avenue for diagnostic, prognostic, and therapeutic applications with the ISVDs disclosed herein. In view of these advantages, an aspect of the invention provides a binding agent capable of specifically binding to and inhibiting a neutrophil elastase (NE), wherein the binding agent comprises an immunoglobulin single variable domain (ISVD) and the binding agent competes with Elastase Inhibitor 3 (EI3) for binding to NE. Another aspect of the invention provides a binding agent capable of specifically binding to and inhibiting a neutrophil elastase (NE), wherein the binding agent comprises an immunoglobulin single variable domain (ISVD) binding to at least one of the residues R36, A60, N61,P96, V97, S195, G218 and G219 of the NE, preferably two or more of the residues R36, A60, N61,P96, V97, S195, G218 and G219, more preferably three or more of the residues R36, A60, N61, P96, V97, S195, G218 and G219, more preferably all of the residues R36, A60, N61, P96, V97, S195, G218 and G219 of the NE, wherein the residues are annotated according to the chymotrypsinogen scheme. In certain embodiments, the ISVD binds to at least the residues R36, A60, N61,P96, V97 and S195 of the NE. Another aspect of the invention provides a binding agent capable of specifically binding to and inhibiting a neutrophil ela