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WO-2026093448-A1 - SINGLE-DOMAIN ANTIBODIES TARGETING THE HEPARIN-BINDING EGF-LIKE GROWTH FACTOR

WO2026093448A1WO 2026093448 A1WO2026093448 A1WO 2026093448A1WO-2026093448-A1

Abstract

The invention relates to a single domain antibody that binds Heparin-binding EGF-like growth factor (HB-EGF) for use in preventing, treating and/or diagnosing e.g. a brain disease, wherein the single domain antibody is in combination with a therapeutic compound and/or a diagnostic/imaging compound. The single domain antibody may in particular be a Variable domain of a Heavy chain only (VHH) antibody of camelid origin or an engineered single-domain antibody (sdAb) of other mammalian and be capable of transporting cargo such as peptides or other molecules into the brain across the blood-brain barrier (BBB) by receptor-mediated transcytosis (RMT). The brain disease may for example be Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, brain tumor, or Hunter syndrome.

Inventors

  • POMPE, Sara
  • QIU, Boning
  • XENAKI, Aikaterini-Titika
  • CAIAZZO, MASSIMILIANO
  • VAN BERGEN EN HENEGOUWEN, Paulus Martinus Petrus

Assignees

  • UNIVERSITEIT UTRECHT HOLDING B.V.

Dates

Publication Date
20260507
Application Date
20251030
Priority Date
20241030

Claims (20)

  1. CLAIMS
  2. 1. Single domain antibody that binds Heparin-binding EGF-like growth factor (HB-EGF) for use in imaging, treating and/or diagnosing a brain disease,
  3. wherein the single domain antibody is in combination with a therapeutic compound, imaging compound and/or a diagnostic compound.
  4. 2. Single domain antibody for use according to claim 1, wherein the brain disease is selected from a disease characterized by undesired protein aggregation, amyloidosis, Alzheimer’s Disease, Parkinson’s Disease, Huntington’s Disease, brain tumor, Hunter syndrome, Mucopolysaccharidosis type II (MPS II), encephalitis, headache, cluster headache, migraine, neurodevelopmental disorder, autism and epilepsy.
  5. 3. Single domain antibody for use according to any one of the previous claims, wherein the single domain antibody is a Variable domain of the Heavy chain of a Heavy-chain-only (VHH) antibody.
  6. 4. Single domain antibody for use according to claim 3, wherein the VHH antibody has the following structure with complementarity determining regions (CDR) and framework regions (FR):
  7. (N-terminus) FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 (C-terminus).
  8. 5. Single domain antibody for use according to claim 4, wherein the single domain antibody comprises:
  9. - CDR1 having an amino acid sequence of SEQ ID NO:30;
  10. - CDR2 having an amino acid sequence of SEQ ID NO:32;
  11. - CDR3 having an amino acid sequence of SEQ ID NO:34.
  12. 6. Single domain antibody for use according to claim 4, wherein the single domain antibody comprises:
  13. - CDR1 having an amino acid sequence of SEQ ID NO:44;
  14. - CDR2 having an amino acid sequence of SEQ ID NO:46;
  15. - CDR3 having an amino acid sequence of SEQ ID NO:48.
  16. 7. Single domain antibody for use according to any one of the previous claims wherein the single domain antibody comprises an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5, most preferably SEQ ID NO:3. 8. Single domain antibody for use according to any one of the previous claims, wherein the therapeutic compound, imaging compound and/or the diagnostic compound is conjugated to the single domain antibody.
  17. 9. Single domain antibody for use according to any one of the previous claims, wherein the therapeutic compound, imaging compound and/or a diagnostic compound is conjugated to the single domain antibody by means of
  18. - genetic fusion;
  19. - Cys-Maleimide conjugation;
  20. - NHS ester chemistry;

Description

SINGLE-DOMAIN ANTIBODIES TARGETING THE HEPARIN-BINDING EGF-LIKE GROWTH FACTOR Technical field The present invention relates to the field of binding polypeptides, particularly for their use in the prevention, diagnosis, imaging and/or treatment of brain diseases such as brain tumors, Alzheimer’s Disease, Parkinson’s Disease, Huntington’s Disease, brain ischemia, metabolic diseases, neurodevelopmental disorders, or meningitis and encephalitis. Background of the invention The blood-brain barrier (BBB) represents a significant challenge for efficient drug delivery into the brain. The BBB maintains brain homeostasis via a tightly integrated multi-cellular structure and strictly selective transport mechanisms [1, 2], which usually keeps most molecules (such as pathogens) out of the brain. One strategy to selectively transport drugs across the BBB is to harness the receptor-mediated transcytosis (RMT) mechanism. RMT is a selective vesicular transport mechanism of brain endothelial cells, which facilitates the transport of large cargoes, such as peptides and proteins, across the BBB [3, 4], RMT is initiated by ligand-binding to its specific receptor expressed on the apical side of polarized brain endothelial cells, followed by the trafficking of the ligandreceptor complex in vesicles through the cell, and the release of the ligand at the basolateral side of the endothelial cell for its subsequent transport into the cerebrospinal fluid (CSF) [4], Hence, a therapeutic molecule conjugated to such a ligand can be delivered into the brain in a targeted manner. To date, several RMT-enabled receptors presented on the BBB have been explored for cerebral drug delivery, such as the transferrin receptor (TfR), insulin receptor (InsR), and low-density lipoprotein receptor (LDLR) [1, 3, 5, 6], However, these approaches share therapeutic limitations due to their binding and transport competition with endogenous ligands. This results in reduced drug delivery efficiency, or impediment of necessary cerebral nutrient transport, which leads to metabolic dysfunctions or cytotoxicity [7, 8], ProHB-EGF is the trans-membrane precursor of the heparin-binding EGF-like growth factor (HB-EGF). Apart from the juxtracrine effect, proHB-EGF undergoes ectodomain shedding to generate its mature version of the soluble HB-EGF (sHB-EGF), which binds to its downstream receptors (such as EGFR) to impose different physiological functions with regards to cell proliferation, survival, migration, and differentiation [9, 10], HB-EGF is constitutively expressed in the brain, like on endothelial cells, glial cells, and neurons [9], Cross-reacting material 197 (CRM197), a non-toxic derivative of the Diphtheria toxin (DT) [14], was found to be capable of binding proHB-EGF (also called DT receptor) and to carry large proteins (serving as proof-of-concept for drugs) across the BBB via RMT [13], While CRM 197 represented a promising BBB shuttle molecule, it was found to retain a certain level of toxicity, causing the inhibition of protein synthesis and endonucleolytic degradation of DNA [15-18], In addition, it was found that this approach shows a limited efficiency in people who have been vaccinated against Diphtheria. It is an objective of the present disclosure to overcome one or more of the above-identified or other problems in the art and/or to provide a new or improved strategy for the prevention, diagnosis and/or treatment of brain diseases, such as brain tumors, Alzheimer’s Disease, Parkinson’s Disease, Huntington’s Disease, Hunter syndrome, neurodevelopmental disorders or meningitis and encephalitis. Summary of the invention The present inventors present a novel approach utilizing the proHB-EGF-mediated BBB transcytosis. Previously, others have demonstrated the employment of single-domain antibodies derived from camelid heavy-chain-only antibodies, also known as the variable heavy chain of the heavy-chain-only antibody (VHH) or single-domain antibody (sdAb), for the transcytosis by the human polylmmunoglobulin receptor (plgR)[21], Being among the smallest antigen-binding units ever developed, sdAbs (-15 kDa) have gained particular attention over full-sized antibodies (-150 kDa) in the field of targeted drug delivery due to lower immunogenicity, better structural stability, and deeper tissue penetration [22, 23, 21], To date, several sdAbs have been generated that were able to transcytose the BBB by specifically targeting receptors such as TfR, a(2,3)-sialoglycoprotein receptor, insulin-like growth factor 1 receptor (IGF1R) and vascular cell adhesion molecule 1 (VCAM-1) [24, 25], The present inventors have identified multiple sdAbs, which selectively bind to proHB-EGF and sHB-EGF. These sdAbs were generated by immunization of llamas with the ectodomain of human proHB-EGF and sHB-EGF, followed by phase display selections. The inventors confirmed that the identified sdAbs can undergo RMT using in vitro transwell BBB models comprising of either primary h