EP-4739780-A1 - ENGINEERED AAV CAPSID PROTEINS

Abstract

The present disclosure relates to an engineered capsid protein comprising an AAV capsid protein and an antibody fragment, wherein the antibody fragment comprises a bovine ultralong CDR-H3, or a portion thereof. The present disclosure also relates to an engineered capsid protein comprising an AAV capsid protein and an antibody fragment, wherein the antibody fragment comprises a knob domain of an ultralong CDR-H3, or a portion thereof. The disclosure further relates to a capsid comprising an engineered capsid protein, and to recombinant AAVs comprising said engineered capsid protein or capsid, and their use in therapy. The present disclosure also extends to methods of preparing said engineered capsid proteins, capsids and AAVs.

Inventors

• KAS, Onur Yusuf
• KRAMER, Tal
• LAWSON, ALASTAIR DAVID GRIFFITHS
• MACPHERSON, ALEXANDER
• SCHULZE, Monika-Sarah Elisabeth Dorothea
• XU, Meiyu

Assignees

• UCB Biopharma SRL

Dates

Publication Date

20260513

Application Date

20240702

Claims (20)

1. 1. An engineered capsid protein comprising an AAV capsid protein and an antibody fragment, wherein the antibody fragment comprises a bovine ultralong CDR-H3, or a portion thereof.
2. 2. An engineered capsid protein comprising an AAV capsid protein and an antibody fragment, optionally according to claim 1, wherein the antibody fragment comprises a knob domain of an ultralong CDR-H3, or a portion thereof.
3. 3. The engineered capsid protein according to claim 2, wherein the antibody fragment does not comprise a stalk of an ultralong CDR-H3.
4. 4. The engineered capsid protein according to any one of the preceding claims, wherein the antibody fragment binds an antigen.
5. 5. The engineered capsid protein according to any one of the preceding claims, wherein the antibody fragment is 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length or more, 50 amino acids in length or more, 55 amino acids in length or more, or 60 amino acids in length or more.
6. 6. The engineered capsid protein according to any one of the preceding claims, wherein the antibody fragment is up to 69 amino acids in length.
7. 7. The engineered capsid protein according to any one of the preceding claims, wherein the antibody fragment is between 5 and 55, or between 15 and 50, or between 20 and 45 or between 25 and 40 amino acids in length.
8. 8. The capsid protein according to any one of the preceding claims, wherein the antibody fragment comprises a (Zi) Xi C X2 motif at its N-terminal extremity, wherein: Zi is present or absent, and when Zi is present, Zi represents 1 amino acid or 2, 3, 4, or 5 independently selected amino acids; Xi is any amino acid residue, preferably selected from the list consisting of Serine, Threonine, Asparagine, Alanine, Glycine, Proline, Histidine, Lysine, Valine, Arginine, Isoleucine, Leucine, Phenylalanine and Aspartic acid; and, C is cysteine; and, X2 is an amino acid selected from the list consisting of Proline, Arginine, Histidine, Lysine, Glycine and Serine.
9. 9. The engineered capsid protein according to any one of the preceding claims, wherein the antibody fragment comprises a sequence which is a variant of a naturally occurring sequence.
10. 10. The engineered capsid protein according to any one of the preceding claims, wherein the antibody fragment further comprises at least one bridging moiety between two amino acids, optionally wherein the bridging moiety is a disulphide bond.
11. 11. The engineered capsid protein according to any one of the preceding claims, wherein the antibody fragment is fully bovine.
12. 12. The engineered capsid protein according to any one of claims 1 to 10, wherein the antibody fragment is chimeric.
13. 13. The engineered capsid protein according to any one of the preceding claims, wherein the AAV capsid protein comprises a naturally occurring, or a variant or an artificial AAV sequence or a combination thereof.
14. 14. The engineered capsid protein according to any one of the preceding claims, wherein the AAV capsid protein comprises a sequence of an AAV selected from the group consisting of AAV1, AAV2, AAV true type (AAV-TT), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, or AAV13, or a combination thereof.
15. 15. The engineered capsid protein according to any one of the preceding claims, wherein the antibody fragment is inserted within the AAV capsid protein, optionally via a linker.
16. 16. The engineered capsid protein according to claim 15, wherein the antibody fragment is inserted within the AAV capsid protein via one linker, wherein optionally the linker is genetically fused to the antibody fragment, optionally at its C-terminal end.
17. 17. The engineered capsid protein according to claim 15, wherein the antibody fragment is inserted within the AAV capsid protein, via at least two linkers.
18. 18. The engineered capsid protein according to claim 17, wherein at least one linker is fused, optionally genetically, to the N-terminal end of the antibody fragment, and at least one linker is fused, optionally genetically, to the C-terminal end of the antibody fragment.
19. 19. The engineered capsid protein according to any one of the preceding claims, wherein the AAV capsid protein is a VP1, a VP2, or a VP3.
20. 20. The engineered capsid protein according to any one of the preceding claims, wherein the antibody fragment is inserted within the common VP3 region of the AAV capsid protein, optionally, within the GH loop of the common VP3 region.

Description

ENGINEERED AAV CAPSID PROTEINS Field of the Invention The present disclosure relates to an engineered capsid protein comprising an AAV capsid protein and an antibody fragment, wherein the antibody fragment comprises a bovine ultralong CDR-H3, or a portion thereof. The present disclosure also relates to an engineered capsid protein comprising an AAV capsid protein and an antibody fragment, wherein the antibody fragment comprises a knob domain of an ultralong CDR-H3, or a portion thereof. The disclosure further relates to a capsid comprising an engineered capsid protein, and to recombinant AAVs comprising said engineered capsid protein or capsid, and their use in therapy. The present disclosure also extends to methods of preparing said engineered capsid proteins, capsids and AAVs. Background Viral vector-mediated gene delivery has shown great promise for gene therapy applications, including approved treatments for inherited blindness, spinal muscular atrophy, and rare diseases. Among the viral vectors that are suitable for use in gene therapy, adeno-associated virus (AAV) has a number of advantageous features for clinical applications as compared to other gene therapy vectors such as adenovirus or lentivirus. AAV is non-pathogenic and is smaller in size than lentivirus, resulting in higher diffusion rates in tissues and greater potential for transducing large areas of tissue upon local injection. In addition, the AAV genome has minimal requirements for replication and packaging, enabling much of AAV to be replaced by a gene of interest and its regulatory elements. Genes delivered by AAV do not integrate into the host chromosome, which mitigates risk of undesired effects, and the genes nonetheless remain stable over long periods in host cells (Li & Samulski, Nature Reviews Genetics 21 (4): 255-272 (2020)). Finally, many naturally-occurring AAVs and AAV serotypes have been identified in nature, which may differ in their tissue tropism and in their transduction efficiency (Srivastava, Current Opinion in Virology 21 : 75-80 (2016)). This variety is an advantage in gene therapy, as certain AAVs may be preferred to transduce specific cells or tissues. The advantage of diversity has been further exploited by protein engineering to create artificial AAVs. The identity of the AAV is determined by the sequences of proteins that make up the protein shell, or capsid, of AAV. Among different variants, most sequence diversity is found in variable or hypervariable regions (VRs or HVRs) of the capsid proteins. By modifying amino acids in the variable regions or in other parts of the capsid proteins, it has been shown that it was possible to create AAVs with new or improved properties, such as improved transduction efficiency or altered tissue tropism. One approach to capsid engineering is a random mutagenesis, in which entirely new capsids with random mutations are created and screened for desired properties. In another approach, targeted changes are made to a capsid by substituting amino acid residues from the capsid of a first AAV serotype with corresponding residues from the capsid of a second AAV serotype and/or modifying specific amino acid residues in areas of the capsid that interact with binding partners on target cells. Alternative approaches also include engineering a capsid by inserting heterologous binding moieties, such as peptides, for example to try to enhance recognition of a binding partner on a target cell, thereby improving the ability of AAV to bind and transduce said target cell. For example, in one study, a 14-mer peptide (LI 4) derived from the laminin fragment Pl was inserted into the common VP3 region of AAV2, as L14 is a target of several cellular integrin receptors and can serve as a viral receptor (Girod et al., Nature Medicine 5: 1052-1056 (1999)). In other studies, insertions of peptides into capsid proteins increased infectivity of rAAVs for retinal cells (e.g. US Patent Application No. US2020121746), cardiac tissues (e.g. PCT Publication No. WO20205889 Al), hepatic tissues (e.g. PCT Publication No. WO2020193799), muscle cells (e.g. PCT Publication No. WO19207132 Al), and GPL anchored blood-brain barrier ligands (e.g. US Patent Application No. US2020325456). Peptides have also been inserted into capsid proteins to facilitate evasion of neutralizing antibodies (e.g. US Patent No. US10745447). In another example, a designed ankyrin repeat protein (DARPin), specific for the target HER2///CZ/, a receptor tyrosine kinase overexpressed on human cancer cells, was inserted into the N-terminus of VP2 capsid proteins in AAV2, and the resulting rAAV showed increased transduction efficiency of cells expressing Her2, as well as targeting of Her2-expressing tumors (Munch et al., Mol Therapy 21 (1): 109-118 (2013)). In another example, VHH antibodies selective for cell surface markers (CD38, ARTC2.2, or CD38) were inserted into a VP1 capsid protein to confer binding and transduce cells expressing the cell-s