Stabilized polypeptides

Abstract

The present disclosure provides a polypeptide comprising an antibody heavy chain variable domain (VH) variant comprising a plurality of antigen-binding regions and a framework comprising framework region 1, framework region 2 and framework region 3 (FWR3) wherein the VH variant has an amino acid sequence satisfying at least eight of the following criteria i)-x): i) a residue at Kabat position 19 is R, S, K or T; ii) the residue at position 23 of Kabat is T, V, A or S; iii) the residue of Kabat position 24 is I, V or S; iv) the residue at position 40 of Kabat is selected from R, I, T or K; v) the residue at Kabat position 43 is R, K, Q, E or G; vi) the residue at Kabat position 44 is E, Q, A, D or G; vii) the residue at position 45 of Kabat is R, I or L; viii) the residue at position 76 of Kabat is N or Q; if) the residue of Kabat position 79 is Y, W or F; and x) the residue of Kabat position 89 is V or I. The VH variants have improved alkaline stability and are useful as scaffolds for affinity binding agents for in vitro applications, such as the detection, analysis or isolation of analytes based on affinity interactions.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480067293.7 (22) Application Date 2024.10.18 (30) Priority Data 23204844.7 2023.10.20 EP (85) PCT International Application Entering National Phase Date 2026.04.20 (86) PCT International Application Application Data PCT / EP2024 / 079593 2024.10.18 (87) PCT International Application Publication Data WO2025 / 083266 EN 2025.04.24 (71) Applicant: Siter Bioprocess Technology R&D Ltd. Address: Uppsala, Sweden (72) Inventors: A.L.M. Jungson, G. Millind, E. Merlin, P. Jaffer, M.C. Astraland (74) Patent Agency: China Patent Agency (Hong Kong) Ltd. 72001 Patent Attorneys: Huang Denggao, Zhang Hua (51) Int.Cl. C07K 16 / 18 (2006.01) B01D 15 / 38 (2006.01) C07K 1 / 22 (2006.01) C07K 16 / 08 (2026.01) C07K 16 / 28 (2006.01) G01N 30 / 00 (2006.01) (54) Invention Title: Stable Peptide (57) Abstract: This disclosure provides peptides comprising antibody heavy chain variable domain (VH) variants, said VH variants comprising multiple antigen- The binding region and the framework comprising frame region 1, frame region 2 and frame region 3 (FWR3), wherein the VH variant has an amino acid sequence satisfying at least eight of the following criteria i)-x): i) the residue at Kabat position 19 is R, S, K or T; ii) the residue at Kabat position 23 is T, V, A or S; iii) the residue at Kabat position 24 is I, V or S; iv) the residue at Kabat position 40 is selected from R, I, T or K; v) the residue at Kabat position 43 is R, K, Q, E or G; vi) the residue at Kabat position 44 is E, Q, A, D or G; vii) the residue at Kabat position 45 is R, I or L; viiii) the residue at Kabat position 76 is N or Q; ix) the residue at Kabat position 79 is Y, W or F; and x) the residue at Kabat position 89 is V or I. The VH variant possesses improved basic stability and can be used as a backbone for affinity binding agents for in vitro applications, such as the detection, analysis, or separation of analytes based on affinity interactions. Claims: 5 pages; Description: 68 pages; Sequence Listing (electronic publication); Figures: 14 pages; CN 122138977 A; 2026.06.02 CN 1 22 13 8977 A 1. A polypeptide comprising a heavy chain variable domain (VH) variant, such as a single-domain antibody (sdAb) variant, said variant comprising a plurality of antigen-binding regions and a frame comprising frame region 1 (FWR1), frame region 2 (FWR2), and frame region 3 (FWR3), wherein, compared with i) a natural camelid frame as defined in any one of SEQ ID NO: 152, SEQ ID NO: 155, or SEQ ID NO: 157, or ii) a synthetic frame as defined in any one of SEQ ID NO: 72 or SEQ ID NO: 73, said VH variant has a plurality of mutated amino acid sequences comprising, wherein at least eight of the following criteria i)-x) are satisfied: i) a residue at Kabat position 19 is R, S, K, or T; ii) a residue at Kabat position 23 is T, V, A, or S, preferably T; iii) a residue at Kabat position 24 is I, V, or S; iv) The residue at Kabat position 40 is selected from R, I, T or K, preferably R; v) The residue at Kabat position 43 is R, K, Q, E or G, preferably R or K; vi) The residue at Kabat position 44 is E, Q, A, D or G; vii) The residue at Kabat position 45 is R, I or L, preferably R; viii) The residue at Kabat position 76 is N or Q; ix) The residue at Kabat position 79 is Y, W or F; and x) The residue at Kabat position 89 is V or I. 2. The polypeptide of claim 1, wherein the VH variant has an amino acid sequence satisfying at least five of the following criteria i)-vi): i) the residue at Kabat position 24 is I, V, or S; ii) the residue at Kabat position 40 is selected from R, I, K, or T, preferably R; iii) the residue at Kabat position 45 is R, I, or L, preferably R; iv) the residue at Kabat position 76 is N or Q; v) the residue at Kabat position 79 is Y, W, or F; and vi) the residue at Kabat position 89 is V or I. 3. The polypeptide according to any one of the preceding claims, wherein the VH variant has a frame region 1 (FWR1) comprising an amino acid sequence according to the following sequence: X1X2QLX5X6SGGGX11VQX14GGSLX19LSCX23X24SG (SEQ ID NO: 1) wherein independently, X1 is Q, V, D, or E, preferably Q or E; X2 is V or D, preferably V; X5 is Q, V, or E, preferably Q; X6 is Q or E, preferably Q; X11 is S or L, preferably S; X14 is A, S, or T, preferably A; X19 is R, S, K, or T, preferably R or T; X23 is T, V, A, or S, preferably T; andX24 is I, V, or S, preferably I; Frame region 2 (FWR2) comprising an amino acid sequence according to the following sequence: WFRQX5PGX8X9X10EX12VA (SEQ ID NO: 2) Claims 1 / 5 page 2 CN 122138977 A Wherein independently, X5 is R, I, T, or K, preferably R; X8 is R, K, Q, E, or G, preferably K or R; X9 is E, Q, A, D, or G; X10 is R, I, or L, preferably R; and X12 is F or L, preferably F; and Frame region 3 (FWR3) comprising an amino acid sequence according to the following sequence: YX2X3X4VX6GRFTISRDNAKX18TX20X21LQMNX26LKPEDTAX34YYCAA (SEQ ID NO: 3) Wherein independently, X2 is A, T, N, Q, or S, preferably A; X3 is D or S, preferably D; X4 is S or A, preferably S; X6 is K or A, preferably K; X18 is N or Q, preferably N; X20 is V or A, preferably V; X21 is Y, F, or W; X26 is N, S, T, or Q, preferably S; and X34 is V or I. 4. The polypeptide according to any one of the preceding claims, wherein the VH variant comprises frame region 4 (FWR4) containing the amino acid sequence WGQGTQVTVSS (SEQ ID NO: 71). 5. The polypeptide according to any one of claims 1-3, wherein each of FWR1, FWR2, and FWR3 has at least 80%, for example, at least 85%, 90%, or 95% identity with the amino acid sequence of the corresponding frame region of SEQ ID NO: 80, wherein Kabat positions 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b, and 89, and optionally position 6, are omitted for the purpose of determining sequence identity. 6. The polypeptide according to any one of claims 2-5, wherein all criteria i)-x) of claim 1 and all criteria i)-vi) of claim 2 are satisfied. 7. The polypeptide according to any one of claims 1-6, wherein the amino acid sequence of the VH variant further satisfies any one, for example, more than one, for example, all of the following: the residue at Kabat position 6 is Q or E; the residue at Kabat position 14 is A, S, or T; the residue at Kabat position 47 is F or L, preferably F; the residue at Kabat position 60 is A, T, N, Q, or S, preferably A or S; and the residue at Kabat position 82b is N, S, T, or Q. 8. The polypeptide according to any one of the preceding claims, wherein frame region 2 (FWR2) has a sequence selected from SEQ ID NO.9. The polypeptide according to any one of the preceding claims, wherein frame region 2 (FWR2) has an amino acid sequence selected from SEQ ID NO: 23-24 and 26-41, for example selected from SEQ ID NO: 26, 32-39 and 41, for example selected from SEQ ID NO: 26, 38 and 41. Claims 2 / 5 Page 3 CN 122138977 A 10. The polypeptide according to any one of the preceding claims, which is an antigen-binding polypeptide. 11. The polypeptide according to any one of the preceding claims, which is a single-chain polypeptide. 12. The polypeptide according to any one of the preceding claims, wherein the VH variant is a variable domain of the heavy chain of a heavy chain antibody (VHH) variant. 13. The polypeptide according to any one of the preceding claims, wherein the VH variant has a frame corresponding to a frame of a VH variant selected from SEQ ID NO: 76, 78, 80-114, 118-121, 126-127, 130-135, 137, 139-151, 156, and 158. 14. The polypeptide according to any one of the preceding claims, wherein the VH variant does not contain any histidine (H) residues. 15. The polypeptide according to any one of the preceding claims, comprising an additional amino acid sequence optionally selected from: a leader peptide, a signal peptide, a purification tag, an affinity tag, a coupling peptide, a linker peptide, or a spacer peptide. 16. The polypeptide according to claim 15, wherein the additional amino acid sequence is located at the N-terminus or C-terminus of the polypeptide. 17. The polypeptide according to claim 15 or 16, wherein the additional amino acid sequence is a leader peptide or a signal peptide. 18. The polypeptide according to claim 15, wherein the additional amino acid sequence comprises a plurality of histidines, such as the (His)6 sequence. 19. A multimeric polypeptide comprising at least two moieties, each moiety being a VH variant as defined in any of the preceding claims, said moieties optionally linked by a peptide linker. 20. The multimeric polypeptide of claim 19, comprising three VH variants, wherein said VH variants are identical or have different CDRs. 21. A fusion protein comprising at least one polypeptide according to any one of claims 1-18 or a multimer according to claims 19-20 and at least one additional polypeptide moieties. 22. The fusion protein of claim 21, wherein said additional polypeptide moieties comprise an α-helical domain, such as an α-helical bundle domain. 23. The fusion protein of claim 22, wherein said additional polypeptide moieties are derived from a protein domain of staphylococcal protein A (SpA). 24.The fusion protein of claims 22-23, wherein the additional polypeptide moiety comprises a sequence having at least 80% identity, for example at least 85% or at least 90% identity, with respect to SEQ ID NO: 159 or SEQ ID NO: 160. 25. The fusion protein of any one of claims 21-24, wherein the additional polypeptide moiety is located at the C-terminus of the VH variant. 26. The fusion protein according to any one of claims 21-25, comprising the structure ([A-L1]m-[Z-L2]n)p, wherein A represents a polypeptide as defined in any one of claims 1-18, L1 may or may not be present for each occurrence, and when present represents a linker or spacer, Z represents an additional polypeptide portion according to any one of claims 22-24, L2 may or may not be present for each occurrence, and when present represents a linker or spacer, m represents an integer from 1 to 4, n represents an integer from 1 to 4, or when m ≥ 2, n represents 0 or an integer from 1 to 4, and p represents an integer from 1 to 4. 27. The fusion protein according to claim 26, wherein i) m is 2 or 3, n is 1 and p is 1, or ii) m is 1, n is 1 and p is 2 or 3. 28. The fusion protein of any one of claims 21-26, comprising a C-terminal tag containing a plurality of histidine residues, preferably at least six histidine residues. 29. The fusion protein of claim 28, wherein the fusion protein contains no histidine residues other than the tag. 30. An isolated nucleic acid encoding a polypeptide, polymer, or fusion protein of any one of claims 1-29. 31. An expression vector comprising the nucleic acid of claim 30. 32. A recombinant host cell for producing a polypeptide, polymer, or fusion protein of any one of claims 1-29, wherein the host cell comprises the expression vector of claim 31. 33. A method for producing a polypeptide, polymer, or fusion protein of any one of claims 1-29, comprising i. providing a recombinant host cell of claim 32; ii. culturing the host cell under conditions capable of expressing the polypeptide, polymer, or fusion protein; and iii. isolating the polypeptide, polymer, or fusion protein. 34. The method of claim 33, wherein step iii comprises purifying the polypeptide, polymer, or fusion protein by affinity chromatography. 35. The method of claim 34, wherein the affinity chromatography uses an affinity ligand that binds to the framework region of the VH variant. 36. The polypeptide according to any one of claims 1-18, the polymer according to claims 19-20, or according to...37. Use of the fusion protein of claims 21-29 as an affinity ligand for capturing target entities. 38. Use of the use of claim 37 for in vitro detection and / or purification of target entities. 39. An adsorbent material comprising a polypeptide of any one of claims 1-18, a polymer of claims 19-20, or a fusion protein of claims 21-29 coupled to a solid support. 30. The adsorbent material of claim 38, wherein the support material is selected from particles, beads, fibers, fibrous membranes, filters, sheets, porous substrates, chips, plates, and pores. 41. The adsorbent material of any one of claims 38-39, wherein the support material is a fibrous matrix, such as a nonwoven fibrous matrix. 42. The adsorbent material of any one of claims 38-39, wherein the support comprises agar or agarose or a derivative thereof, such as cross-linked agarose. 43. The adsorbent material of any one of claims 38-41, wherein the support is a chromatographic matrix. 44. Use of the adsorbent material of any one of claims 38-42 for separating the target entity from other components of a sample. 44. A separation method comprising the steps of: (a) providing an adsorbent material according to any one of claims 38-42, wherein the VH variant of the polypeptide has binding affinity for a target entity; (b) contacting the adsorbent material with a liquid sample containing the target entity, allowing the target entity to bind the polypeptide; (c) optionally washing the adsorbent material; (d) eluting the target entity from the adsorbent material; and (e) cleaning the adsorbent material with a cleaning liquid, such as an alkaline cleaning liquid. 45. The method of claim 44, wherein the cleaning liquid is alkaline and preferably contains 0.05-0.5 M NaOH, for example 0.1-0.5 M NaOH. 46. The method of any one of claims 44-45, wherein steps (a)-(e) are repeated at least 10 times. 47. The method according to any one of claims 44-46, wherein after 12 cycles of contact with an alkaline cleaning liquid, the polypeptide or fusion protein retains at least 50%, for example, at least 60%, for example, at least 80%, for example, at least 90%, of the initial target binding capacity. 48. The method according to any one of claims 44-46, wherein after 12 cycles of contact with an alkaline cleaning liquid, the polypeptide or fusion protein retains at least 50%, for example, at least 60%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 98%, of the target binding capacity in the second cycle. (Claims 5 / 5, Page 6, CN)122138977 A Stable Peptide Technology

[0001] This invention relates to peptides based on the variable domain (VH) of antibody heavy chains and their use as affinity ligand backbones, and to adsorbent materials comprising such peptides. The invention also relates to affinity separation methods. Background Art

[0002] Affinity-based binding events between molecules, where a ligand and a target entity interact in a “lock-and-key” manner, are used in many therapeutic and non-therapeutic applications. Non-therapeutic applications include, for example, the in vitro detection, analysis, and separation of targets or analytes present in a sample.

[0003] Affinity ligands are capable of selectively and reversibly binding to target entities. General examples of interactions are, for example, enzyme-substrate interactions, biotin-avidin interactions, antibody-antigen interactions, etc. In many applications, affinity ligands can be immobilized on a carrier and used to capture target entities, for example, for detection or purification purposes.

[0004] Affinity chromatography is a specific type of chromatography that utilizes the selectivity of the interaction between a ligand and a target entity. Affinity ligands are immobilized on a chromatographic support material (also known as a stationary phase). When contacted with a sample containing a target entity under binding conditions, the ligands selectively bind to the target entity, while other types of sample can be eluted. The captured target entity can then typically be eluted by changing buffer conditions, such as conductivity or salt concentration and / or pH. After subsequent cleaning and regeneration of the chromatographic material, it can be used for new affinity purification cycles. Affinity chromatography can potentially yield target entities of very high purity.

[0005] Affinity chromatography is commonly used to purify biomolecules, including biopharmaceuticals and, for example, monoclonal antibodies, antibody fragments, and recombinant proteins, as well as nucleic acids and viral particles (e.g., for vaccine production or gene therapy).

[0006] Preparative chromatography using carriers with affinity ligands immobilized thereon, and many analytical applications, require comprehensive attention to the definitive removal of contaminants from the carrier between capture / purification cycles. Such contaminants can be, for example, non-elutable molecules adsorbed onto the carrier or stationary phase, such as unwanted biomolecules or microorganisms, including, for example, proteins, carbohydrates, lipids, bacteria, and viruses. In affinity chromatography, removal of such contaminants from the support is typically performed after the initial elution of the desired product to regenerate the stationary phase before subsequent use. Such removal usually involves a procedure called in-situ cleaning (CIP), in which reagents capable of eluting contaminants from the stationary phase are used. One commonly used class of such reagents is an alkaline solution that flows through the stationary phase. Currently, the most widely used cleaning and disinfecting reagent is NaOH, and its concentration ranges from 0.05 M to, for example, 1 M, depending on the degree and nature of the contamination. This strategy involves exposing the stationary phase to solutions with pH values ​​of approximately 13 and higher. Such an alkaline environment is very harsh for many affinity chromatographic materials containing proteinaceous affinity ligands.The conditions are harsh, and therefore the capacity is reduced due to the high pH instability of the ligand pair involved.

[0007] Staphylococcal protein A (SpA)-based affinity reagents, which are the most widely used affinity media for separating immunoglobulins and their fragments, have been developed to be better resistant to alkaline conditions (see, for example, WO2003080655A1 and WO 2016 / 079033A1). Meanwhile, there is a great need in the art for improved affinity ligands capable of binding biomolecules other than immunoglobulins. Summary of the Invention

[0008] The objective of the present invention is to overcome or at least partially mitigate the deficiencies of the prior art. Specification 1 / 68 pages 7 CN 122138977 A

[0009] Therefore, the objective of the present invention is to provide a backbone for use in novel affinity ligands, which is alkaline stable.

[0010] These and other objectives are achieved by polypeptides comprising heavy chain variable domain (VH) variants in which at least one frame region, such as frame region 2 (FWR2), is modified to provide improved alkaline stability.

[0011] In a first aspect, the present invention provides a polypeptide comprising a heavy chain variable domain (VH) variant, said VH variant comprising a plurality of antigen-binding regions and a framework comprising framework region 1 (FWR1), framework region 2 (FWR2), and framework region 3 (FWR3), wherein said VH variant, compared with other framework sequences, such as existing frameworks, such as i) a natural camelid framework as defined in any one of SEQ ID NO: 157, SEQ ID NO: 155, or SEQ ID NO: 157, or ii) a synthetic framework as defined in any one of SEQ ID NO: 72 or SEQ ID NO: 73, has a plurality of mutated amino acid sequences, wherein at least eight of the following criteria i)-x) are satisfied: i) the residue at Kabat position 19 is R, S, K, or T; ii) the residue at Kabat position 23 is T, V, A, or S, preferably T; iii) the residue at Kabat position 24 is I, V, or S; iv) The residue at Kabat position 40 is selected from R, I, T, or K, preferably R; v) The residue at Kabat position 43 is R, K, Q, E, or G, preferably R or K; vi) The residue at Kabat position 44 is E, Q, A, D, or G; vii) The residue at Kabat position 45 is R, I, or L, preferably R; viii) The residue at Kabat position 76 is N or Q; ix) The residue at Kabat position 79 is Y, W, or F; and x) The residue at Kabat position 89 is V or I.

[0012] Optionally, at least nine of the criteria i) to ix) are satisfied, for example, all of them.

[0013] In a second aspect, a polypeptide comprising a heavy chain variable domain (VH) variant is provided, the VH variant comprising multiple antigen-binding regions and a framework comprising framework region 1 (FWR1), framework region 2 (FWR2), and framework region 3 (FWR3), wherein the VH variant, compared with other framework sequences, such as i) a natural camelid framework as defined in any one of SEQ ID NO: 152, SEQ ID NO: 155, or SEQ ID NO: 157, or ii) a synthetic framework as defined in any one of SEQ ID NO: 72 or SEQ ID NO: 73, has a sequence comprising multiple mutant amino acids, wherein at least five of the following criteria i)-vi) are satisfied: i) the residue at Kabat position 24 is I, V, or S; ii) the residue at Kabat position 40 is selected from R, I, K, or T, preferably R; iii) the residue at Kabat position 45 is R, I, or L, preferably R; iv) the residue at Kabat position 76 is N or Q; v) The residue at Kabat position 79 is Y, W, or F; and vi) the residue at Kabat position 89 is V or I.

[0014] Optionally, all six criteria i)-vi) may be satisfied.

[0015] The polypeptide of this disclosure may satisfy at least eight of the criteria i)-x) of the first aspect and at least five of the criteria i)-vi) of the second aspect.

[0016] In the following, all references to the polypeptide of this disclosure refer to both the first and second aspects unless otherwise provided.

[0017] Optionally, FWR1, FWR2, and FWR3 may each have at least 80% identity with the amino acid sequence of the corresponding frame region of SEQ ID NO: 80, wherein for the purpose of determining sequence identity, Kabat positions 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b, and 89, and optionally position 6, are omitted.

[0018] The VH variant may be a single-domain antibody (sdAb) variant.

[0019] Furthermore, the amino acid sequence of the VH variant may also satisfy any one, for example, more than one, for example, all of the following criteria: the residue at Kabat position 6 is Q or E; the residue at Kabat position 14 is A, S, or T; the residue at Kabat position 47 is F or L, preferably F; the residue at Kabat position 60 is A, T, N, Q, or S, preferably A or S; the residue at Kabat position 82b is N, S, T, or Q.

[0020] Specifically, the amino acid sequence of the VH variant may contain phenylalanine (F) or leucine at Kabat position 47.(L) residues. However, even in embodiments where the residue at Kabat position 47 is not limited to F or L, preferably it is not G, S, T, or Y.

[0021] In another aspect, the present invention provides a multimeric polypeptide comprising at least two parts, each part being a VH variant as defined herein, said parts optionally being linked by a peptide linker.

[0022] In yet another aspect, a fusion protein is provided comprising at least one polypeptide or multimer as defined herein and at least one additional polypeptide moiety.

[0023] In a further aspect, the present invention provides an adsorbent material comprising said polypeptide, multimer, or fusion protein coupled to a solid support. The solid support may be selected from particles, beads, fibers, fibrous membranes, filters, sheets, porous substrates, chips, plates, and pores. The support may be a chromatographic matrix.

[0024] In a further aspect, the present invention provides the use of said polypeptide, multimer, or fusion protein as an affinity ligand for capturing target entities, and the use of the aforementioned adsorbent material for separating target entities from other components of a sample. The target entity is an entity to which the VH variant has an affinity. A separation method is also provided, comprising the steps of: (a) providing an adsorbent material as described herein, wherein the VH variant of the peptide has a binding affinity for the target entity; (b) contacting the adsorbent material with a liquid sample containing the target entity, under conditions allowing the target entity to bind to the peptide; (c) optionally, washing the adsorbent material; (d) eluting the target entity from the adsorbent material; and (e) cleaning the adsorbent material with a cleaning liquid.

[0025] The cleaning liquid is typically alkaline and may contain 0.05–0.5 M NaOH. Steps (a)–I may be repeated at least 10 times.

[0026] Advantageously, the base-stable VH variant of the peptide may retain a high target binding capacity even after repeated or prolonged exposure to alkaline conditions.

[0027] These and other aspects of the invention will now be described in more detail with reference to the accompanying drawings, which show embodiments of the invention, in which: FIG1 is a schematic diagram of the arrangement of the frame region and complementarity-determining region of a single-domain antibody. Specification 3 / 68 pages 9 CN 122138977 A

[0028] FIG2a-c are schematic diagrams of various structures of polypeptides according to the present disclosure.

[0029] FIG3a-c are schematic diagrams of fusion proteins comprising at least one polypeptide as disclosed herein, fused with another polypeptide portion.

[0030] FIG4 is a graph showing the results of a basic stability study of the polypeptide described in Example 2A.

[0031] FIG5 is a graph showing the results of a basic stability study of the polypeptide described in Example 2B.

[0032] FIG6a-c show the results of a basic stability study of polypeptides capable of binding AAV9 (FIG. 6a), GFP (FIG. 6b), or EGFR (FIG. 6c), respectively.For increasing the number of cycles including NaOH exposure, the target binding response was obtained (Example 4A). The response was normalized to the response of the first cycle.

[0033] Figures 7a-c show the target binding response obtained for increasing the number of cycles including NaOH exposure for fusion proteins capable of binding AAV9 (Figure 7a), GFP (Figure 7b), or EGFR (Figure 7c), respectively (Example 4B). The response was normalized to the response of the first cycle.

[0034] Figure 8 is a graph showing the basic stability of the exemplary peptide (Example 5).

[0035] Figure 9 is a graph showing the basic stability of the exemplary peptide and a commercially available affinity ligand (Example 6).

[0036] Figure 10a is a chromatogram showing the elution peaks from a chromatographic column using the fusion protein according to an embodiment of the invention as an affinity ligand immobilized on a chromatographic matrix. Figure 10b is a partial magnified view of the elution peaks.

[0037] Figure 11 is a photograph of an SDS-PAGE gel showing the protein content of the eluent fraction from the chromatographic run of Example 7.

[0038] Figure 12 is a graph showing the dynamic binding capacity, as evaluated in Example 7.

[0039] Figure 13 is a graph showing the basic stability evaluated in Example 8.

[0040] Figure 14 is a graph showing the binding affinity and basic stability on the SA-chip of the purified and biotinylated construct according to Example 12.

[0041] As illustrated in the figures, some features may be exaggerated for illustrative purposes and are therefore provided to illustrate the general structure of embodiments of the invention. Throughout the text, the same reference numerals refer to the same elements.

[0042] Definitions as used herein: The terms “peptide” and “polypeptide” are used synonymously herein and refer to compounds formed from amino acid sequences, without limitation on size. “Protein” may be used to refer to larger compounds of this class. Amino acid sequences are written from left to right, from the amino (N) terminus to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are represented by three-letter or single-letter codes as shown below: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). "Peptide" includes any oligopeptide, polypeptide, gene product, expression product, or protein.Peptides are composed of a continuous sequence of amino acids and include naturally occurring or synthetic molecules. Furthermore, as used herein, the term "peptide" refers to amino acids linked together by peptide bonds or modified peptide bonds, such as isosteres, and may contain modified amino acids in addition to the 20 genetically encoded amino acids. Peptides can be modified by natural processes, such as post-translational processing, or by chemical modification techniques well known in the art.

[0043] "Single-chain polypeptide" herein refers to a polypeptide formed from a single amino acid sequence, wherein the amino acid residues are linked by peptide bonds. Single-chain polypeptides may form additional internal bonds, such as disulfide bonds, upon folding.

[0044] The expression "antigen-binding polypeptide" generally refers to a polypeptide having at least one binding region, and typically at least two, for example three, binding regions, such that the polypeptide has a binding affinity for a molecule (called an antigen or "target"). Antigen-binding polypeptides can have any protein structure, as long as there is a binding affinity for the antigen. However, some types of antigen-binding peptides that have been commonly developed and used as the basis for modifying antigen-binding peptides typically have a backbone or framework structure that is generally conserved among antigen-binding peptides of the same type, but which bind different antigens. The binding region of an antigen-binding molecule can often be modified or evolved to bind a specific antigen, while the backbone or framework structure remains substantially the same. Non-limiting examples of antigen-binding peptides include natural or modified (i) antibodies, such as monoclonal antibodies, (ii) antibody fragments containing variable regions of the light chain and / or heavy chain with complementarity-determining regions (CDRs), such as Fab (antigen-binding fragment), Fv (variable fragment), scFab (single-chain antigen-binding fragment), and scFv (single-chain variable fragment), (iii) single-domain antibodies, and (iv) peptides with a bacterial-derived backbone, such as immunoglobulin-binding bacterial proteins (e.g., Finegoldia magna protein L or Staphylococcus aureus proteins A and G) or their domains, including wild-type and variants in which one or more of the antigen-binding regions have been modified.

[0045] The expression "single-domain polypeptide" or "single-domain amino acid sequence" refers to a polypeptide that forms a single protein domain and does not contain any other single protein domains. Examples of single-domain polypeptides include antibody fragments (e.g., heavy chain variable domain (VH) and light chain variable domain (VL)), single-domain antibodies (sdAbs), albumin-binding domains (ABDs), and polypeptides derived from bacterial protein domains (e.g., A, B, C, D, E, or Z domains of SpA or protein domain L). Single-domain antibodies are particularly considered. Single-domain polypeptides typically consist of a single-chain polypeptide. A single-domain polypeptide is itself a monomer, but it can form part of a multimer, as described elsewhere in this text. A single-domain polypeptide can be linked to another polypeptide (e.g., via a peptide bond).(or via disulfide bonds). Preferably, in the context of this disclosure, the antigen-binding polypeptide may consist of a single polypeptide chain.

[0046] The term "single-domain antibody" refers to such a variable domain that is or is derived from the heavy chain variable domain (VH) of an antibody lacking a light chain. Thus, a single-domain antibody completely lacks the antibody light chain, including the light chain variable domain (VL). A single-domain antibody may also lack such a structural feature that is essential for functional VH / VL interactions and is present, for example, in the conventional VH of IgG1. Thus, a single-domain antibody does not form part of a dimer structure having the antibody light chain variable domain. Single-domain antibodies include antibodies that are naturally lacking the antibody light chain, such as sdAbs derived from IgG2 or IgG3 of cameloidea (e.g., dromedary camel, camel, llama, and alpaca), which are also referred to as VHHs (heavy chain variable domains of heavy chain antibodies). This article includes reference peptides with such a natural framework, such as the AAV9-binding sdAb of SEQ ID NO: 152 (vh342), the GFP-binding sdAb of SEQ ID NO: 155 (vh60), and the EGFR-binding sdAb of SEQ ID NO: 157 (vh166). Single-domain antibodies that naturally lack the light chain also include so-called VNARs (variable neoantigen receptors), which are antibodies derived from cartilaginous fish (e.g., sharks). Alternatively, single-domain antibodies can be synthetic variants based on the amino acid sequence of the variable domain of the antibody heavy chain from species that do not naturally produce sdAbs (e.g., bovine, rat, mouse, or rabbit), and modified, for example, relative to the amino acids in the natural VH / VL interaction of such antibodies to mimic sdAbs. Such modifications may include substituting amino acids in the VH / VL interface region to increase hydrophilicity or solubility.

[0047] Generally, terms such as “peptide,” “protein,” “antibody,” “single-domain peptide,” “single-domain antibody,” etc., also include synthetic variants that are recombined and whose amino acid sequences may be modified relative to the amino acid sequences of naturally occurring counterparts. The term “variant” may also be used where specifically referring to such modified variants. The terms “wild-type” or “wt” refer to the typical naturally occurring form, which may also be referred to as “natural” or “unmodified” when compared to modified variants. The single-domain antibody variants of the present invention are synthetic, non-naturally occurring amino acid sequences.

[0048] Complementarity-determining regions (CDRs) are hypervariable regions of an antibody or a portion thereof that participate in binding to target epitopes. CDRs are generally defined by their individual amino acid sequences, although in antibodies, CDRs together form a three-dimensional binding site for the target antigen. The variable domain (VH) of the antibody heavy chain has three CDRs, referred to as CDR1, CDR2, and CDR3, as directed from the N-terminus of the polypeptide chain. CDR Specification 5 / 68The sequence length of page 11 CN 122138977 A can be varied, and the CDR1, CDR2, and especially CDR3 of VH can have different lengths.

[0049] The antibody VH portion that does not form CDRs is called the frame region. There are usually four frame regions, and they are referred to as frame regions (FW) 1-4, as calculated from the N-terminus. The frame regions are responsible for the overall secondary and tertiary structure of the domains, and therefore for the localization and orientation of the CDR regions. The frame regions may be referred to as the backbone structure. Although as part of a variable domain, the variability of the frame regions is lower than that of the CDRs. However, amino acid sequence changes that do not significantly alter the secondary or tertiary structure of the frame are generally tolerable. Some portions or amino acid positions of the frame regions may be conserved. The overall stability of the frame regions allows for a high degree of variation in the CDRs.

[0050] As used herein, the term “ligand” is a molecule that has a known or unknown affinity for a given entity. The term “ligand” is used interchangeably herein with the terms “affinity ligand,” “selective binding molecule,” “selective binding partner,” “capture molecule,” and “capture agent.” "Affinity ligand" refers to a portion or molecule that binds reversibly and selectively, or preferably with high affinity, to a target entity through a specific interaction with the binding site of a component. In the context of this invention, the affinity ligand is a polypeptide and may also be referred to as an "affinity protein". Affinity ligands may be immobilized to a solid carrier, such as a resin.

[0051] The term "target entity" herein refers to an entity that forms a specific binding partner with the ligand and may also be referred to as an "analyte". An analyte or target entity of interest according to this disclosure is an adeno-associated virus vector, particularly an AAV9 vector.

[0052] As used herein, "affinity" in the context of a polypeptide refers to the strength of an interaction between two molecules, where at least one molecule is a polypeptide. The interaction is selective, i.e., there is a distinction between the affinity-binding partner and other molecules present. Binding affinity is also expressed as a dissociation equilibrium constant (KD).

[0053] As used herein, the term "binding capacity" refers to the ability of a ligand to bind a target molecule when the ligand is immobilized on a surface. The binding capacity of a ligand can vary depending on the type of surface on which the ligand is immobilized. For the purposes of this invention, the binding capacity can be measured using surface plasmon resonance (SPR) technology with instruments such as Biacore, as described in the embodiments herein. The binding capacity for a given concentration of analyte is affected by the density of immobilized ligands on the surface. In the context of this invention, the binding capacity is preferably determined at a ligand density in the range of at least 3000 response units (RU), for example, 3000–4000 RU.

[0054] In the context of protein purification, the term “dynamic binding capacity” (abbreviated “DBC”) for a chromatographic column is used when operating under certain conditions.The dynamic binding capacity (DBC) of a chromatographic resin is the amount of analyte bound to the resin under given flow conditions before significant breakthrough occurs in the unbound analyte. DBC is determined by loading a sample containing a known concentration of analyte and monitoring the flow. The analyte will bind to the resin until a breakthrough point occurs before the unbound analyte flows through the column. DBC can be determined on a breakthrough curve, for example, with a 10% analyte loss. This is called the QB10% value. The dynamic binding capacity of each resin is calculated at 10% breakthrough capacity, i.e., the amount of analyte loaded onto the column until the analyte concentration in the column effluent is 10% of the analyte concentration in the feed. If the dynamic binding capacity of each resin is calculated at 80% breakthrough capacity, this is called the QB80% value.

[0055] As used herein, the term "basic stability" refers to the property of an antigen-binding peptide that is able to withstand alkaline exposure without adverse effects on the structure and / or function of the peptide. For peptides capable of binding to a target entity, basic stability is determined based on the affinity for the target entity after exposure to NaOH. The basic stability of an antigen-binding peptide can be evaluated by immobilizing the antigen-binding peptide on a carrier and measuring the target binding capacity before and after one or more cycles of exposure to NaOH using the methods described in the examples below. Specifically, it can be evaluated on an SPR chip at a ligand density corresponding to at least 1000 RU and preferably at least 3000 RU and for a predetermined analyte concentration, wherein the antigen-binding peptide is immobilized on the SPR chip by covalent coupling with thiols, N-hydroxysuccinimide, streptavidin-biotin, or another coupling that is inherently alkali-resistant. Typically, the first alkaline clean cycle can have a uniquely significant impact on binding capacity, for example, due to the removal of non-covalently bound antigen-binding peptides from the carrier surface. Therefore, binding capacity is sometimes expressed as the binding capacity normalized to that measured after the first alkaline exposure cycle.

[0056] For example, “improved alkaline stability” can mean that the antigen-binding peptide can tolerate a higher number of alkaline clean cycles, or the same number of cycles but more stringent alkaline conditions, without compromising target binding capacity. Alternatively, “improved alkaline stability” can mean that the peptide retains a higher percentage of binding capacity after an equal number of clean cycles using the same conditions. Alkaline stability can be particularly important for peptides intended to be immobilized on a solid carrier. Alkaline treatment may include treatment with 0.05–1 M NaOH, such as 0.1–0.5 M or 0.3–0.5 M.NaOH contact or incubation for 5–15 minutes, for example, 10 minutes (600 seconds) or a period of about 10 minutes. The treatment can be performed, for example, at 22 ± 2 °C. Antigen-binding peptides exhibiting improved alkaline stability as defined above and below may be referred to as “alkaline stable” or “base-stable”.

[0057] In this context, an antigen-binding peptide is considered base-stable if, after at least 12 clean cycles, for example, after 15 clean cycles, preferably after 20 clean cycles, the immobilized antigen-binding peptide retains at least 50% of its target binding capacity compared to its binding capacity after the first clean cycle (i.e., regardless of the binding capacity of the first cycle before the first alkaline exposure event), wherein the clean cycle comprises 600 seconds of exposure to at least 0.3 M NaOH, for example, at least 0.5 M NaOH.

[0058] The term “solid carrier” herein refers to a non-aqueous matrix of solid-phase material. Suitable solid-phase materials include, but are not limited to, glass, silica (e.g., silica gel), polysaccharides (e.g., polysaccharide matrices) such as agarose and cellulose, and organic polymers such as polyacrylamide, methyl methacrylate, and polystyrene-divinylbenzene copolymers. The solid phase may be porous or non-porous and may be compressible or incompressible. For example, the solid phase may be a polymer matrix or agarose particles or beads. Preferred solid support materials will be physically and chemically resistant to the conditions used in the purification process, including pumping and cross-flow filtration, as well as the temperature, pH, and other aspects of the liquid used.

[0059] The term “surface” herein means all external surfaces of a solid structure. In the case of porous supports (e.g., beads used in, for example, chromatography), the term “surface” includes both the outer surface and the pore surface. In the case of fibrous membranes, the term “surface” includes the outer surface of each fiber.

[0060] The term “separation matrix” herein is used to refer to a material containing a solid support with which one or more ligands have been coupled. Ligands are capable of binding to target entities (also referred to herein as analytes) that are to be separated from their environment (e.g., liquid samples) and / or from other components present in the liquid sample. Separation matrices can be used in a variety of scenarios, including analytical assays and for purifying targets for analytical or preparative purposes. The type of support can be selected according to the intended use.

[0061] Separation matrices may further comprise compounds that couple ligands to the support. The terms “spacer,” “extender,” and “surface extender” are used to describe such compounds, as further described herein. The term “resin” is sometimes used in the art for separation matrices. The terms “chromatographic material” and “chromatographic matrix” are used herein to denote a class of separation matrices. Affinity separation matrices are separation matrices in which the ligand is an affinity ligand.

[0062] The term “spacer” refers to a peptide or other chemical bond or element that extends the structure of the entity. A spacer linked to a polypeptideA spacer, specifically an amino acid spacer, may be provided at the N-terminus or C-terminus of a polypeptide. The spacer may link the polypeptide to a solid carrier. A suitable spacer for coupling a polypeptide to a carrier may generally be any spacer used in the art to link peptides, proteins, or other organic molecules to a carrier. A spacer may also be used to link two polypeptides or polypeptide domains. A spacer linking two polypeptide moieties may also be referred to as a linker.

[0063] As used herein, the terms “multimer” and “multimeric protein” refer to a protein comprising at least two repeating units of a single-domain antibody disclosed herein. Thus, a single-domain antibody may be considered as a monomeric polypeptide unit that can be combined to form multimeric proteins, such as dimers, trimers, tetramers, pentamers, etc. Multimeric single-domain antibody units may be identical in their amino acid sequences or may be slightly different from each other. In addition to the said single-domain antibody units, a multimer may also contain one or more other polypeptide units, such as stable polypeptide units as described herein, which are not single-domain antibodies. Such additional polypeptide units may exist in the polymer as a single copy or multiple copies.

[0064] Within the polymer, the individual polypeptide units may be linked by peptide bonds, typically by a linker peptide. Thus, the multimeric protein may be formed from a single polypeptide chain.

[0065] The term "fusion protein" refers to a protein formed from two or more separate proteins (fusion couplers) produced by recombinant protein expression as a single polypeptide containing two fusion couplers. The fusion couplers may be linked sequentially. The genes encoding the individual proteins are linked at the gene level. Fusion couplers typically do not naturally exist fused together. Fusion proteins may contain additional amino acid sequences, such as linkers, between the fusion couplers.

[0066] As used herein, "linker" refers to a peptide or other chemical link that serves to connect otherwise independent functional domains. A linker may be located between two polypeptide units, monomers, or domains, for example, between a ligand (e.g., sdAb) and another polypeptide component containing otherwise independent functional or structural domains, or between two ligands. A suitable linker for coupling two or more linked units may generally be any linker in the art used to connect peptides, proteins, or other organic molecules. The linker peptide can be a single amino acid, preferably ranging from 1 to 20 amino acids, for example 2 to 15, such as 2 to 8, 2 to 4, or 4 to 12 amino acids.

[0067] The terms "%identity" or "%sequence identity" as used throughout this disclosure can be calculated, for example, using the CLUSTAL W algorithm (Thompson et al., Nucleic Acids Research, 22: 4673-4680).(1994)) The query sequence is compared with the target sequence. The comparison is made on the window corresponding to the shortest aligned sequence. The shortest aligned sequence may be the target sequence in some cases. In other cases, the query sequence may constitute the shortest aligned sequence. The amino acid residues at each position are compared, and the percentage of positions in the query sequence that have the same consistency as the target sequence is reported as % identity. When % sequence identity is determined, if an amino acid position is omitted, the sequence identity is determined based on the shorter sequence. For example, in the FWR2 sequence with a total of 14 amino acid residues, when the Kabat positions 40, 43, 44, 45, and 47 are omitted from the sequence identity determination, these amino acids are removed from both the query and target statements, so that the sequence is compared on a 9-amino acid sequence instead. Detailed Description

[0068] Antigen-binding peptides have gained attention for in vitro applications, such as affinity ligands in chromatographic applications. Even if it is possible to modify ligands to have high affinity and specificity for a target, many ligands have defects, such as low basic stability, and therefore cannot withstand the harsh conditions of regenerated chromatographic columns, i.e., when performing in-situ cleaning (CIP). Therefore, the purpose of this disclosure is to enhance the basic stability and other properties of antigen-binding peptides.

[0069] The inventors have discovered that heavy chain variable domain (VH) backbones can be stabilized to make them particularly usable as affinity binders for in vitro applications, such as the detection, analysis, or separation of analytes based on affinity interactions. Thus, the backbone can be used to present a CDR with affinity for the analyte to be captured, for example, for detection or separation purposes. Specifically, the backbone can be used to provide affinity ligands for affinity capture, such as affinity chromatography. Thus, the peptide can be an antigen-binding peptide. However, it is conceivable that a stable VH backbone (referred to herein as a “framework”) may also be useful in cases where a functional CDR is not required.

[0070] This disclosure provides scaffold modifications to improve the basic stability of peptides, meaning that antigen-binding functionality is less affected by exposure to basic conditions compared to unmodified counterparts, such as naturally occurring single-domain antibody sdAbs. Specification 8 / 68 pages 14 CN 122138977 A The use of sdAbs for affinity capture applications has gained attention due to their ease of expression and good affinity. However, sdAbs are widely known to lack basic stability, which is a significant drawback when it comes to affinity chromatography. Improved basic stability is a great benefit in the context of affinity chromatography, as chromatographic materials are routinely cleaned with basic reagents (typically NaOH) between purification cycles. Increased basic stability of affinity ligands means that chromatographic materials can be used for more purification cycles before the binding capacity becomes unacceptably low.

[0071] The peptides of this disclosure contain heavy chain variable domain (VH) variants, such as single-domain antibody variants, for example...VHH variants, or synthetic antibody fragments, such as single-chain variable fragments (scFv). The peptides of this disclosure can therefore be single-chain peptides.

[0072] Single-domain antibodies, and the VH variants of this disclosure, lack the light chain of a full-length antibody and also lack the heavy chain constant domain. Therefore, the VH variants of the peptides disclosed herein contain only the heavy chain variable domain, which includes three complementarity-determining regions (CDRs). Therefore, the VH variants of this disclosure do not contain antibody light chain variable regions or light chain CDRs. However, in embodiments of the peptides of this disclosure that include single-chain variable fragments, it includes antibody light chain variable domains in addition to the VH domain.

[0073] In general, single-domain antibodies are more readily expressed in prokaryotic and eukaryotic cells compared to conventional full-length antibodies, and Fab fragments or single-chain fragments that include both light and heavy chain portions. Although they have only three CDRs, their target-binding properties may be satisfactory, unlike the six CDRs of larger antibodies and antibody fragments. However, the stability of natural single-domain antibodies, such as camel VHH, under alkaline conditions is problematic, and in many cases, poor alkaline stability will prevent such VHH from being used as affinity ligands for chromatographic applications.

[0074] The portion of the heavy chain variable domain (VH) that does not form a CDR is called a framework. The VH framework is formed by four regions, referred to herein as framework regions (FWRs) 1–4, which are responsible for the overall secondary and tertiary protein structure. The framework regions of the VH form a β-sandwich structure, which typically contains cysteine ​​bridges. CDRs are typically presented at one end as three surface loops, one CDR per loop.

[0075] An advantage of using sdAbs as affinity binders is that the CDR sequences of sdAbs can be more diverse in length than those of conventional antibodies. For example, the surface loops formed by sdAb CDRs may extend further spatially from the sdAb body, thus having a better ability to bind into the pocket of the target epitope, or the surface loops may be shorter, forming a flatter surface, which can facilitate binding to other epitopes.

[0076] Therefore, the polypeptides of this disclosure comprise variants of the heavy chain variable region (VH) of antibodies, and more specifically, variants of single-domain antibodies (sdAbs) (also referred to as VHH or VH). Single-domain antibodies lack a light chain and are naturally found, for example, in camels. The polypeptides of this invention can therefore be synthetic VHs, initially derived from sequences of, for example, camels (or other species), in which various modifications have been introduced. Cameloid VH sequences (also referred to as nanobodies when expressed in bacterial hosts) exhibit a high degree of framework conservation – in fact even above the VH domain of full-length antibodies (Mitchell & Colwell, Proteins. 2018; 86:697-706, see, for example, Figures 2 and 3). Thus, the natural cameloid framework,For example, the frame region of vh166 / SEQ ID NO:157 (lamb) can be considered as the “original” starting sequence into which modifications (mutations) are introduced. Alternatively, vh98 / SEQ ID NO:73, representing a synthetic sequence obtained by the inventors by introducing certain modifications into a natural camelid sequence, can be used as a starting point for introducing further mutations, because even though the basic stability of vh98 is significantly improved relative to the natural camelid sequence, it can be further improved for use in the context of affinity chromatography. Therefore, further modifications are proposed, which have been found to further increase basic stability. Therefore, the modified “starting” frame region can be based on a frame region from a natural camelid sequence, such as EGFR-binding VHH (vh166, SEQ ID NO: 157), AAV9-binding VHH (vh342, SEQ ID NO: 152), or GFP-binding VHH (vh60, SEQ ID NO: 155), or on a synthetic frame, such as the frame region of vh97 / vh98 (SEQ ID NO: 72 / SEQ ID NO: 73). The problem addressed by these modifications is the very poor basicity stability of the natural camelid VHH domain, as illustrated in Example 4B and Figure 7c below, where the EGFR-binding VHH (vh166) with a frame region obtained from a llama shows almost complete loss of binding activity after only two cycles of basic exposure.

[0077] Figure 1 schematically illustrates the sequence of the frame region and complementarity-determining region (CDR) of the VH (e.g., sdAb) from the N-terminus to the C-terminus of the amino acid sequence. Furthermore, Table 1 summarizes the FWR and CDR as defined herein with respect to the Kabat amino acid numbering system (Kabat et al., 1991, J. Immunol. 147(5), 1709–1719), where an example sequence of FWR is derived from vh97. For the VH variant of this disclosure, frame region 1 (FWR1) is formed by amino acids at positions 1–26. Next, amino acids at Kabat positions 27–35d form CDR1, with Kabat positions 35a–d being optional. Frame region 2 (FWR2) is formed by amino acids at Kabat positions 36–49. CDR2 is formed by amino acids at Kabat positions 50–58 (52a being optional). Frame region 3 (FWR3) is formed by amino acids at Kabat positions 59–94. CDR3 is formed from amino acids at positions 95-102 of the Kabat structure (100a-j are optional). Finally, framework region 4 (FWR4) is formed from amino acids at positions 103-113 of the Kabat structure. It can be noted that within this disclosure...The definitions of the CDR and frame region do not correspond exactly to the definitions of the CDR and frame region by Kabat et al.; however, the same amino acid numbering system is used.

[0078] Table 1 The inventors have identified that at some amino acid positions in the frame region, not only are certain mutations of the amino acid residues at said positions tolerated without impairing functionality, but can even lead to improved performance of the VH variant, particularly improved basic stability. On the other hand, there also appear to be amino acid positions that are less tolerant of mutations. Therefore, the frame region, which does not include the hypervariable CDR region, contains regions or positions that allow some variability, as well as positions that are more conserved.

[0079] Among the various frame regions, FWR2 has been identified as having the most significant effect on basic stability. Therefore, the VH variant of this disclosure may have frame region 2 as described herein, and specifically, amino acids at Kabat positions 40, 43, 44, 45 and / or 47 as described herein.

[0080] Considering FWR1, FWR2, and FWR3, the following amino acid positions according to the Kabat numbering system have been identified as variable and potentially usable for improving the basic stability of the VH variant.

[0081] Table 2: Variable positions that potentially enhance basic stability (10 / 68 pages, 16 CN 122138977 A) Furthermore, it has been found that variations at some other positions can be tolerated without necessarily improving basic stability in the same way as seen with respect to the positions described in Table 2. Such potentially variable positions with lower impact include Kabat positions 1, 2, 5, 11, 61, 62, 64, and 78. In this context, “lower impact” refers to a stabilizing effect.

[0082] However, it is envisioned that even further amino acid positions may exist in the frame region that can tolerate more than one type of amino acid without impairing functionality.

[0083] In summary, the frame regions FWR1–FWR3 as described in this disclosure may contain 79 amino acids. As mentioned above, at least 23 positions in FWR1-3 may allow more than one amino acid. With this in mind, the VH variant amino acid sequences contained in this disclosure represent at most about 30% sequence variability in frame regions 1-3 (FWR1-3) relative to any of the individual VH sequences. Therefore, for example, this disclosure includes stable VH variant sequences whose frame regions have at least 70% of the same amino acids as SEQ ID NO:80; and sequences whose frame regions have more than 70% sequence identity with SEQ ID NO:80, for example, at least 75%, for example, at least 80%, for example, at least 85% sequence identity.

[0084] Currently identified as variable amino acid residues at 15 positions that have a potentially positive impact on peptide stability.For each position, the amino acid can be selected from a limited set of amino acids that have been found to contribute to improved stability. The envisioned amino acids for each of these positions are provided in Tables 3-5 below.

[0085] Of these 15 variable positions, the amino acid positions (Kabat numbers) described in Table 3a have been identified as particularly important.

[0086] Table 3a Kabat Position Stable Amino Acids Preferred Amino Acids 24 I, V, S 40 R, I, K, TR, I 45 R, I, LR 76 N, QN 79 Y, W, FY, W, F 89 V, IV, I The VH variant of this disclosure may have any position independently selected according to Table 3a, or any combination or subgroup of these positions. For example, the amino acid residues at Kabat position 24 may be selected from I, V, and S, and / or the amino acid residues at Kabat position 40 may be selected from R, I, K, and T. The amino acid residue at position 76 of Kabat may be selected from N and Q.

[0087] Although some single-point mutations have been found to significantly improve basic stability, it is thought that amino acid residues at multiple positions interact to provide a stable VH. For this reason, it is ideal to select more than one amino acid residue according to Table 3. For example, at least four or at least five of the six positions may therefore be selected. For example, positions 24 or 40 may have amino acids that fall outside the groups indicated in Table 3, still resulting in a stable VH variant, such as the sdAb variant having alanine (A) at position 24 of Kabat or proline (P) at position 40 of Kabat, as shown in Examples 2A and 2B. However, if the residue at position 24 of Kabat is A, it may be preferred that the residue at position 40 of Kabat is not A, and vice versa (so that the residues at positions 24 and 40 of Kabat are not both A). In some embodiments, it may be preferred that the residue at position 40 of Kabat is not A.

[0088] In some embodiments, the residue at position 76 of Kabat is not T. Alternatively or additionally, in the embodiment, the residue at Kabat position 89 is not E.

[0089] Furthermore, it is assumed that amino acids at some positions may have a stronger interaction or effect on stability together with one or more amino acids at some other positions, which may be due, for example, to the spatial position and orientation that allow the individual amino acids in VH to interact. For example, data suggest that the combination of Y, F, or W at Kabat position 79 with V or I at Kabat position 89 may be particularly advantageous. It is envisioned that appropriate selection of amino acid residues at positions 79 and 89 may provide a stabilizing effect such that less preferred amino acids, such as 24A and / or 40P, can be tolerated at other positions, such as position 24 (FWR1) and / or position 40 (FWR2).

[0090] In addition to the six positions indicated in Table 3a, it has been found that Kabat positions 19, 23, 43, and 44, when selected from the amino acids outlined in Table 3b, contribute to the stability of the VH variant: Table 3b Preferred stable amino acids for Kabat positions 19 R, S, K, TR, T 23 T, S, VT 43 R, K, Q, E, GR, K 44 E, Q, A, D, GE It is likely preferred that at least one residue in Kabat positions 19 and 23 is T. It has been found that having T at Kabat position 19 promotes basic stability, especially when the amino acid at Kabat position 23 is V (see Example 2). On the other hand, it has been found that Kabat position 19 is involved in the interaction between VH3 and protein A. Where it is necessary to maintain such interaction ability, the amino acid residue at position 19 is advantageously R.

[0091] Tables 3a-b above define a total of ten positions. Ideally, at least eight of these ten positions have amino acids selected from the stable amino acids according to Tables 3a-b. Preferably, at least nine, for example all positions, are selected as outlined in Tables 3a-b.

[0092] When only eight positions have amino acids selected according to Tables 3a-b, it is possible that only four of positions 24, 40, 45, 76, 79, and 89 are as defined in Table 3a. For example, such a polypeptide may have 24A and 40P.

[0093] In embodiments, at least five of positions 24, 40, 45, 76, 79, and 89 are as defined in Table 3a, and optionally, any one of positions 19, 23, 43, and 44 is selected according to Table 3b.

[0094] In some embodiments, the residue at Kabat position 43 is R, K, Q, or G.

[0095] For the remaining variable and potentially stable positions in the VH framework region, one or more of these positions may have amino acids selected independently as indicated in Table 4: Table 4 Specification 12 / 68 pages 18 CN 122138977 A Kabat position stable amino acid preferred amino acid 6 Q, E 14 A, S, T 47 F, LF 60 A, T, N, Q, SA, S 82b N, S, T, Q Preferably, the residues at Kabat position 47 are selected from F and L, and more preferably F. In embodiments where the residues at Kabat position 47 are not selected according to Table 4, it is preferably not G, S, T, or Y.

[0096] Position 60 can tolerate some variability while maintaining acceptable basic stability. However, preferably, the amino residues at position 60 may be selected from A, T, N, and S.

[0097] Kabat position 82b has been found to participate in the interaction between VH3 and protein A. Where it is necessary to maintain such interaction capability, the amino acid residues at 82b are advantageously selected from S and N.

[0098] Optionally, more than one of the Kabat positions 6, 14, 47, 60, and 82b may have an amino acid selected according to Table 4. For example, at least two, at least three, at least four, or all five of these positions may have a correspondingly selected stable amino acid.

[0099] Considering all the Kabat positions 6, 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b, and 89, it may be preferred that the VH variant according to this disclosure have at least 13 positions selected from amino acid residues according to Tables 3a-b and 4.

[0100] As indicated above, 15 positions in the frame region have been identified as sensitive to modifications that improve stability. Regarding the remaining portions of the frame region, amino acids at some positions may be replaced with another amino acid, but in general, these portions of the frame region are highly conserved and may preferably remain largely constant. These portions are defined herein by Kabat positions 1–5, 7–13, 25–26, 36–39, 41–42, 46, 48–49, 59, 61–75, 77–78, 80–82a, 82c–94, and 103–113. Optionally, position 6 may be included in this group. The VH variants of the present invention can therefore have a relatively high degree of sequence identity at these amino acid positions, which represent portions of the framework region with lower variability.

[0101] Therefore, in the VH variant of this disclosure, FWR1, FWR2, and FWR3 each have at least 80%, for example at least 85%, at least 88%, or at least 90% identity with the amino acid sequence of the corresponding frame region of SEQ ID NO: 80, wherein Kabat positions 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b, and 89, and optionally Kabat position 6, are omitted from the comparison for the purpose of determining sequence identity. As an example, FWR1 contains 26 amino acids, and after omitting positions 6, 14, 19, 23, and 24, sequence identity is determined based on the remaining 21 amino acids, which represent more conserved positions. Substituting two of these amino acids relative to FWR1 of SEQ ID NO: 80 results in 90.5% identity, while substituting three amino acids relative to FWR1 of SEQ ID NO: 80 results in 85.7% identity. FWR1 of SEQ ID NO: 80 is represented by SEQ ID NO: 8.

[0102] Considering frame regions 1-3 as a whole, FWR1-3 together may have at least 90% sequence identity with the corresponding amino acid of any other VH in this disclosure. As an example, FWR1-3 may have at least 90%, for example at least 92%, for example at least 94%, for example at least 96% sequence identity with the corresponding amino acid (FWR1-3) of SEQ ID NO: 80, wherein in order to determine the sequenceFor the purpose of identity, Kabat positions 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b, and 89, and optionally position 6, are omitted.

[0103] Despite the foregoing, it has been found that the amino acid at position 1 can advantageously be selected from Q, V, D, and E, and can be particularly E. For example, any VH variant amino acid sequence disclosed herein in which Kabat position 1 is not E (e.g., is Q) may instead have E at Kabat position 1. At Kabat position 2, V or D can be used, but in some embodiments V may be preferred. Thus, the amino acids at positions 1 and 2 can optionally be QV or EV. Specification 13 / 68 pages 19 CN 122138977 A

[0104] The VH variant of this disclosure may have an amino acid sequence in which at least eight of the Kabat positions 19, 23, 24, 40, 43, 44, 45, 76, 79 and 89 are independently selected according to Tables 3a-b and 4, or at least five of the Kabat positions 24, 40, 45, 76, 79 and 89 are selected according to Table 3a. Furthermore, the VH variant may have an amino acid sequence in which, relative to SEQ ID NO: 80 or SEQ ID NO: 195, Kabat positions 1-5, 7-13, 25-26, 36-39, 41-42, 46, 48-49, 59, 61-75, 77-78, 80-82a, and 82c-94 may comprise up to 15, for example, up to 14, for example, up to 13, for example, 12, 11, or 10 substituted amino acid residues. For example, in FWR1, positions 1-5, 7-13, and 25-26 (21 residues) may together have up to 4, for example, up to 3, amino acid residues different from those at the corresponding positions in SEQ ID NO: 80 or SEQ ID NO: 195. In FWR2, Kabat positions 36-39, 41-42, 46, and 48-49 (14 residues) may together have one or two amino acid residues different from the corresponding positions in SEQ ID NO: 80. In FWR3, Kabat positions 59, 61-75, 77-78, 80-82a, and 82c-94 (34 residues) may have up to six amino acid residues different from the corresponding positions in SEQ ID NO: 80.

[0105] It may be particularly advantageous to retain some amino acids in portions of the framework region with lower variability as defined herein. For example, VH variants may have an amino acid sequence having one or more of the following amino acid residues: - 15G, 17S, 59Y, 64K, 65G, 66R, 68T, 70S, 81Q and / or 82aN, all of which are involved in the interaction between VH and protein A - 22C, 92C - 36W, 103W - 38R.

[0106] The framework region of a VH domain or sdAb typically has a secondary structure comprising a β-sheet, which is stabilized by intramolecular interactions between side chains of amino acids located at different parts of the primary protein structure (amino acid sequence). Certain amino acid residues have been identified as interacting with one or more other amino acid residues in the VH variant. For example, residues at position 76 and also at position 44 have been found to interact with several other residues. A large number of interactions are considered to provide high stability.

[0107] Furthermore, due to the central position of FWR2 within the three-dimensional structure of the VH variant, FWR2 is considered particularly important for the stability of the VH variant compared to FWR1, FWR3, and FWR4.

[0108] Therefore, a large number of mutations that contribute to peptide stability are disclosed herein, some of which may be more important than others. The peptide may be an sdAb, but stability also exists for scFv, as shown in Example 12 of the Examples section below. Therefore, it is assumed that the mutations increase the stability of the peptide, regardless of the peptide in which the framework is present. Therefore, starting with any polypeptide having at least three CDRs and four surrounding frame regions, the following mutations can be introduced to increase the stability of the polypeptide.

[0109] Therefore, in some embodiments, the present invention provides a polypeptide comprising a heavy chain variable domain (VH) variant, said VH variant comprising a plurality of antigen-binding regions and a framework comprising a framework region 1 (FWR1), a framework region 2 (FWR2), and a framework region 3 (FWR3), said VH variant having an amino acid sequence satisfying at least eight of the following criteria i)-x): i) The residue at Kabat position 19 is R, S, K, or T; ii) The residue at Kabat position 23 is T, V, A, or S, preferably T; iii) The residue at Kabat position 24 is I, V, or S; iv) The residue at Kabat position 40 is selected from R, I, T, or K, preferably R; v) The residue at Kabat position 43 is R, K, Q, E, or G, preferably R or K; vi) The residue at Kabat position 44 is E, Q, A, D, or G; vii) The residue at Kabat position 45 is R, I, or L, preferably R; Specification 14 / 68 pages 20 CN 122138977 A viii) The residue at Kabat position 76 is N or Q; ix) The residue at Kabat position 79 is Y, W or F; and x) The residue at Kabat position 89 is V or I.

[0110] Optionally, at least nine of the criteria i)-ix) are satisfied, for example, all of them.

[0111] In a further embodiment, a polypeptide comprising a heavy chain variable domain (VH) variant is provided, the VH variant comprising a plurality of antigen-binding regions and comprising frame region 1 (FWR1), frame region 2 (FWR2) and frame region 3.The framework of (FWR3), wherein the VH variant has an amino acid sequence that satisfies at least five of the following criteria i)-vi): i) the residue at Kabat position 24 is I, V, or S; ii) the residue at Kabat position 40 is selected from R, I, K, or T, preferably R; iii) the residue at Kabat position 45 is R, I, or L, preferably R; iv) the residue at Kabat position 76 is N or Q; v) the residue at Kabat position 79 is Y, W, or F; and vi) the residue at Kabat position 89 is V or I.

[0112] Optionally, all six criteria i)-vi) may be satisfied.

[0113] The polypeptide of this disclosure may satisfy at least eight of the criteria i)-x) of the first embodiment and simultaneously satisfy at least five of the criteria i)-vi) of the second embodiment.

[0114] In some embodiments, the criteria defined in the embodiments above refer to mutations compared to i) a natural camelid framework as defined in any of SEQ ID NO: 157, SEQ ID NO: 155, or SEQ ID NO: 157, or ii) a synthetic framework as defined in any of SEQ ID NO: 72 or SEQ ID NO: 73. The mutation may also be based on sdAb or other natural or synthetic frameworks that require stabilization and contain at least three CDRs and four surrounding framework regions (e.g., scFv).

[0115] In some embodiments, the VH variant may have a frame region 1 (FWR1) comprising an amino acid sequence according to the following sequence: X1X2QLX5X6SGGGX11VQX14GGSLX19LSCX23X24SG (SEQ ID NO: 1) wherein independently, X1 is Q, V, D or E, preferably Q or E; X2 is V or D, preferably V; X5 is Q, V or E, preferably Q; X6 is Q or E, preferably Q; X11 is S or L, preferably S; X14 is A, S or T, preferably A; X19 is R, S, K or T, preferably R or T; X23 is T, V, A or S, preferably T; and X24 is I, V or S, preferably I.

[0116] Alternatively or additionally, the VH variant may have a frame region 2 (FWR2) comprising an amino acid sequence according to the following sequence: WFRQX5PGX8X9X10EX12VA (SEQ ID NO: 2) wherein independently, X5 is R, I, T, or K, preferably R; X8 is R, K, Q, E, or G, preferably K or R; X9 is E, Q, A, D, or G; X10 is R, I, or L, preferably R; and X12 is F or L, preferably F.

[0117] Alternatively or additionally, the VH variant may have a frame region 3 (FWR3) comprising an amino acid sequence according to the following sequence: YX2X3X4VX6GRFTISRDNAKX18TX20X21LQMNX26LKPEDTAX34YYCAA (SEQ ID NO: 3) wherein independently, X2 is A, T, N, Q or S, preferably A; X3 is D or S, preferably D; X4 is S or A, preferably S; X6 is K or A, preferably K; X18 is N or Q, preferably N; X20 is V or A, preferably V; X21 is Y, W or F, preferably Y or W; X26 is N, S, T or Q, preferably S; and X34 is V or I.

[0118] In an embodiment, in SEQ ID NO: 3, X6 is K, X21 is Y and X26 is S.

[0119] Frame region 4 (FWR4) typically does not form part of the core structure of the VH, but rather forms a peripheral tail structure at one end of the polypeptide, resembling a linker or spacer. Compared to FWR1-3, the amino acid sequence of FWR4 may have a less significant impact on the stability of the VH and / or target binding affinity, and therefore may have lower potential for improving basic stability. VH variants of this disclosure may have an FRW4 amino acid sequence corresponding to, for example, the native FWR4 of camel VHH. Optionally, the VH variant may comprise frame region 4 (FWR4) which comprises or consists of the following amino acid sequence: WGQGTQVTVSS (SEQ ID NO: 71) or an amino acid sequence having at least 70%, for example, at least 75%, for example, at least 80% identity with SEQ ID NO: 4, provided that the amino acid residue at position 1 is W.

[0120] However, it is conceivable that VH may lack the conventional FWR4, or may have a partially or completely different structure instead of VH FWR4, for example, a peptide linker as disclosed elsewhere herein.

[0121] Referring to the position in the Kabat numbering system and the relative position in the corresponding SEQ ID NO: 1-3, Tables 5a-c provide exemplary amino acids for each complete frame region FWR1-3.

[0122] Table 5a: Examples of frame region 1 Table 5b: Examples of frame region 2 Specification 16 / 68 pages 22 CN 122138977 A Table 5c: Examples of frame region 3 The VH variant of the present invention may have an FWR2 amino acid sequence having at least 85%, for example at least 90%, sequence identity with Kabat position amino acids 36-49 (FWR2) of SEQ ID NO: 80, wherein Kabat positions 40, 43, 44, 45 and 47 are omitted for the purpose of determining sequence identity. Alternatively, FWR2 may have an amino acid sequence selected from SEQ ID NO: 23-24 and 26-41.The sequence has an amino acid sequence with at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 85% identity, including Kabat positions 40, 43, 44, 45, and 47 in the comparison. Furthermore, in this case, at least eight of the ten positions identified in Tables 3a-b may have stable amino acids indicated in Tables 3a-b, or at least five of the six positions in Table 3a may have correspondingly selected stable amino acids. Preferably, the residues at Kabat positions 40, 43, 44, 45, and 47 are selected as outlined in Tables 3a-b and Table 4. FWR2 may have an amino acid sequence selected from SEQ ID NO: 23-24 and 26-41, for example, selected from SEQ ID NO: 26, 32-39, and 41, or selected from SEQ ID NO: 26, 38, and 41.

[0123] Alternatively or additionally, the VH variant of the present invention may have an FWR1 amino acid sequence having at least 85%, for example, at least 90% sequence identity with the Kabat positions 1-26 (FWR1) of SEQ ID NO: 80, wherein Kabat positions 6, 14, 19, 23, and 24 are omitted for the purpose of determining sequence identity. For example, the FWR1 may have an amino acid sequence having at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 85% identity with the amino acid sequences selected from SEQ ID NO: 4-22, 190, and 194, including Kabat positions 6, 14, 19, 23, and 24 in the comparison. Furthermore, in this case, at least eight of the ten positions identified in Tables 3a-b may have correspondingly selected stable amino acids, or at least five of the six positions in Table 3a may have correspondingly selected stable amino acids. Preferably, the residues at Kabat positions 6, 14, 19, 23, and 24 are selected as outlined in Tables 3a-b and Table 4. FWR1 may optionally have an amino acid sequence selected from SEQ ID NO: 4-22, 190, and 194.

[0124] Alternatively or additionally, the VH variant of the present invention may have an FWR3 amino acid sequence having at least 85%, for example, at least 90% sequence identity with the Kabat positions 59-94 (FWR3) of SEQ ID NO: 80, wherein Kabat positions 60, 76, 79, 82b, and 89 are omitted for the purpose of determining sequence identity. For example, FWR3 may have an amino acid sequence having at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 85% identity with sequences selected from SEQ ID NO: 45-49, 54-55, 58-63, 65, 67-70, and 191, including Kabat positions 60, 76, 79, 82b, and 89 in the comparison. Furthermore, in thisIn this case, at least eight of the ten positions identified in Tables 3a-b may have the corresponding selected stable amino acids, or at least five of the Kabat positions in Table 3a may have the corresponding selected stable amino acids. Preferably, the residues at Kabat positions 60, 76, 79, 82b, and 89 are selected as outlined in Tables 3a-b and Table 4. FWR3 may optionally have an amino acid sequence selected from SEQ ID NO: 45-49, 54-55, 58-63, 65, 67-70, and 191.

[0125] The framework of the VH variant of the present invention as a whole may have at least 90% sequence identity with the framework of SEQ ID NO: 80, wherein Kabat positions 6, 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b, and 89 are omitted for the purpose of determining sequence identity. For example, the frame region of the VH of the present invention may have at least 92%, for example at least 94%, for example at least 97% sequence identity with the corresponding frame region of SEQ ID NO:80, wherein, for the purpose of establishing sequence identity, positions 6, 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b and 89 are omitted.

[0126] In an embodiment, the frame of the VH variant of the present invention may have a frame amino acid sequence corresponding to the frame of the VH variant selected from SEQ ID NO:72-78, 80-114, 118-121, 126-127, 130-135, 137, 139-151, 156, 158, 192 and 193. For example, a VH variant may have a frame corresponding to a frame of a VH selected from SEQ ID NO: 76, 78, 80-114, 118-121, 126-127, 130-135, 137, 139-151, 156, and 158.

[0127] The VH variants disclosed herein may form a portion of a polypeptide containing additional amino acids besides the amino acid sequence of VH. Such a polypeptide is capable of binding an antigen via a CDR and may be referred to as an antigen-binding polypeptide. The main portion of an antigen-binding polypeptide may consist of one or more VH variants as disclosed herein.

[0128] Exemplary arrangements of the polypeptides of this disclosure are schematically illustrated in Figures 2a-c. In Figure 2a, polypeptide 200 comprises an N-terminal amino acid sequence 201, a VH variant 202, and a C-terminal amino acid sequence 203. It is noteworthy that both the N-terminal amino acid sequence 201 and the C-terminal sequence 203 are optional.

[0129] The polypeptides of this disclosure may optionally be multimeric polypeptides (“multimers”) schematically illustrated in Figures 2b-c.Provided in the form of. In Figure 2b, the multimeric polypeptide 220 comprises a plurality of VH variants 202 as disclosed herein, which may optionally be linked by a linker sequence 204. The N-terminal amino acid sequence 201' is optional and may be, for example, a leader peptide of 1-6, for example, 4 amino acids. The C-terminal sequence 203 is optional and may contain a purification tag. The VH variant units 202 may be identical in their amino acid sequences or may be slightly different from each other. Optionally, the VH variants may have different CDRs and may even have affinity for different targets. It is also possible that the multimeric VH variants have different framework sequences, for example, concerning amino acid residues capable of interacting with IgG (VH3) binding proteins, such that at least one VH variant of the multimeric may be able to perform such an interaction, while at least one other VH variant of the multimeric may not be able to perform such an interaction.

[0130] For example, the polypeptide may comprise two VH variants (“dimer”) or three VH variants (“trimer”). Figure 2c shows an exemplary trimer 230 comprising three VH variants 202.

[0131] Optionally, the multimeric polypeptide may contain at least one additional amino acid sequence that does not represent a VH variant, for example, a stable polypeptide as described elsewhere herein regarding binding fusion proteins.

[0132] Multimeric variants of the polypeptides of the present invention may be advantageous because they can provide increased binding capacity and / or improved stability compared to polypeptides comprising a single copy of a VH variant.

[0133] In addition to the VH variant, the polypeptide may contain additional amino acids or at least one additional amino acid sequence, typically located at the C-terminus and / or N-terminus, or as a linker between the various VH variants of the multimeric or fusion protein. The polypeptide may contain any suitable number of additional amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, for example, up to 20 additional amino acid residues. The additional amino acids may be remnants from recombinant protein expression, leader peptides, signal peptides, purification tags, affinity tags, peptides intended for conjugation to a vector, etc. The additional amino acids may be spacers or linkers as defined elsewhere herein, or have the ability to function as spacers or linkers. The additional amino acid residues may individually or collectively improve the production, purification, in vitro stabilization, or conjugation of the peptide to a target substrate (e.g., a solid carrier, such as the solid carrier described herein). Such additional N-terminal or C-terminal amino acid residues generally do not affect the functional properties of the antigen-binding peptide or single-domain antibody itself, such as binding affinity.

[0134] When the peptides of the present invention are provided as polymers comprising multiple VH variants or as fusion proteins as described below, the additional amino acid residues may, for example, not be repeated for each occurrence of a VH variant. For example, in antigen-binding...When the polypeptide is provided in the form of a polymer, such additional amino acids may appear only at the N-terminus or C-terminus of the polymer.

[0135] The additional amino residues may be coupled to the VH or polymer by chemical conjugation (using known organic chemical methods) or by any other means, such as expression as a fusion protein or by any other means (directly or through a linker, such as the peptide linker described above on page 18 / 68 of the specification, CN 122138977 A).

[0136] In some embodiments, the polypeptide and / or polymer disclosed above further comprises one or more coupling elements selected from at least one cysteine ​​residue, a plurality of lysine residues and a plurality of histidine residues, such as the (His)6 sequence, and combinations thereof. Such coupling elements may be provided anywhere in the polypeptide, but may optionally be near or arranged at the C-terminus or N-terminus of the polypeptide. The coupling element may be, for example, a single cysteine ​​residue at the C-terminus of the polypeptide. The coupling element can be directly attached to the C or N terminus, or it can be attached via a linker containing up to 15 amino acids, such as 1-5, 1-10, or 5-10 amino acids. Such a segment should preferably be sufficiently stable even in alkaline environments without impairing the properties of the protein. For this purpose, it is more advantageous if the segment does not contain asparagine. It may also be advantageous if the segment does not contain glutamine. Having a C-terminal cysteine ​​is advantageous because the endpoint coupling of the protein can be achieved by the reaction of the cysteine ​​thiol group with an electrophilic group on the support. This provides excellent motility of the coupled protein.

[0137] As those skilled in the art will understand, the construction of polymers, such as fusion proteins, typically involves the use of linkers between the monomeric moieties to be fused. A monomeric moieties can refer to a single-domain polypeptide as described herein. Those skilled in the art will understand different kinds of linkers with different properties, such as flexible peptide linkers, rigid peptide linkers, and cleavable peptide linkers. Linkers can be used, for example, to increase the stability of the fusion protein or improve its folding. The linker also ensures a certain spatial distance between the VH variant and the fusion partner peptide, which can be advantageous for the target to be close to the binding surface of the VH variant. The presence of a linker within the fusion protein generally does not significantly affect the target binding capacity of the correctly folded antigen-binding peptide monomer, such as a single-domain antibody.

[0138] Therefore, a multimer as defined herein may further include at least one linker. For example, the linker exists between each monomer within the multimer. In the case of multiple linkers, the linkers may be the same or different. For each occurrence, the linker may be selected, for example, from flexible amino acid linkers, rigid amino acid linkers, and cleavable peptide linkers. Peptide linkers may be structured or unstructured. Generally, unstructured linkers are more flexible and less rigid than structured linkers. Alternatively, the linker may be a non-peptide linker. Thus, the peptides or subunits thereof disclosed herein may be connected to each other via peptide bonds between the C-terminus and N-terminus of the peptide.Direct linkage. Alternatively, two or more monomers (in other words, monomer units or portions) within a polymer can be linked by elements comprising oligomers or polymers, for example, elements comprising up to 15 or 30 amino acids, such as 1-5, 1-10, or 5-10 amino acids. In an embodiment, the linker comprises up to 15 amino acid residues. The properties of such a linker should preferably not destabilize the spatial conformation of the protein unit (i.e., the monomer within the polymer). This can be achieved, for example, by avoiding the presence of proline in the linker. Furthermore, the linker should preferably be sufficiently stable in an alkaline environment without impairing the properties of the protein unit. For this purpose, it is advantageous if the linker does not contain asparagine. It may also be advantageous if the linker does not contain glutamine. The polymer may further comprise additional amino acid residues at its N-terminus as described above, for example, residues derived from the cloning process or constituting a signal sequence from which a cleavage has occurred. The number of additional amino acid residues may be, for example, 15 or fewer, such as 10 or fewer, or 5 or fewer.

[0139] The antigen-binding polypeptide may be in the form of a fusion protein. As indicated above, it may contain multiple VH variants generated as a single fusion protein, said fusion protein optionally having additional amino acids as described above, including linkers.

[0140] In some embodiments, the antigen-binding polypeptide may be a fusion protein containing at least one VH variant as disclosed herein and at least one additional polypeptide moiety (referred to as a fusion partner polypeptide). The fusion partner polypeptide may be a polypeptide that modifies or adds at least one functional or desired property. For example, the fusion partner polypeptide may be another antigen-binding polypeptide targeting the same antigen as the VH variant of the present invention, or may target a different antigen. It is contemplated that said other antigen-binding polypeptide need not be a VH variant as disclosed herein, but may be an antigen-binding polypeptide based on a different backbone, for example, a single-domain polypeptide as defined above. For example, the fusion partner polypeptide may be a stable polypeptide, i.e., a polypeptide that further improves the stability of the antigen-binding polypeptide, such as a polypeptide with basic stability. As another example, the fusion partner polypeptide may be a polypeptide that improves the expression of an antigen-binding polypeptide in recombinant cells, such as Escherichia coli, as described on page 25 of this specification (page 19 / 68). As another example, fusion partner peptides can provide the opportunity to purify antigen-binding peptides by affinity chromatography. As yet another example, fusion partner peptides can improve the coupling of said peptides to solid carriers.

[0141] In some embodiments, said peptides may be fusion proteins containing at least one VH variant as disclosed herein and a fusion partner peptide. Figure 3a schematically illustrates a fusion protein 300 comprising a first polypeptide moiety 200 and a second polypeptide moiety 301 in the N-terminal to C-terminal direction, which are linked by an optional peptide linker 204. In this figure, the second polypeptide moiety301 is located at the C-terminus of the first polypeptide portion 200. It is also contemplated that the second polypeptide portion may be located at the N-terminus of the first polypeptide portion. The first polypeptide portion 200 may comprise only or consist of the single-chain polypeptide 202 illustrated in FIG. 2a, or may comprise the single-chain polypeptide 202 and additional amino acids (e.g., additional amino acid sequences 201 and / or linkers 204) or consist of them.

[0142] Specifically, the fusion protein of this disclosure may comprise one or more VH variants fused to an α-helical polypeptide (e.g., a protein domain). The α-helical polypeptide may contain at least one α-helix, for example, at least two α-helices, such as three, four, five, or six α-helices, for example, up to ten α-helices. In embodiments, it may be an α-helical bundle domain, such as a triple-helical bundle domain. In an α-helical bundle domain, the α-helices are spatially clustered together and form the main portion of the protein domain, which may lack other prominent unstructured or β-structured regions. Examples of α-helical bundle domains include the protein domain and albumin-binding domain (ABD) of Staphylococcus aureus protein A (SpA). For example, the fusion partner polypeptide of the fusion protein of this disclosure may comprise an amino acid sequence derived from domain A, domain B, domain C, domain D, or domain E of SpA. For example, the fusion partner polypeptide may comprise a sequence having at least 80%, for example, at least 85%, or at least 90% identity with SEQ ID NO: 159 or SEQ ID NO: 160.

[0143] Furthermore, the fusion protein of this disclosure may be a multimeric protein, meaning that it may comprise more than one VH or more than one fusion partner. Multimeric variants containing multiple copies of VH may be advantageous because they provide increased binding capacity relative to fusion proteins containing only one copy. Multimeric fusion proteins containing multiple fusion partner polypeptides provide improved stability relative to fusion proteins containing a single stable polypeptide.

[0144] Therefore, the fusion protein may optionally be provided as a multimeric fusion protein (“multimer”) containing at least two peptides comprising a VH variant and / or at least two fusion partner peptides, as schematically illustrated in Figures 3b-c. In Figure 3b, the multimeric fusion protein 310 comprises a plurality of peptide 200 units arranged sequentially from the N-terminus to the C-terminus, and these units may optionally be linked to each other by a linker sequence 204, which may be the same or different for each occurrence. The VH-based peptides 200 may be the same or different from each other in terms of their amino acid sequences. Figure 3b shows three peptides 200 (“trimer”), but it is contemplated that the fusion protein may contain any suitable number of the peptides 200. For example, the fusion protein may contain at least 2 or at least 3, 4, 5 or 6 antigen-binding peptide units. The fusion protein may contain, for example, up to 10 antigen-binding peptide units.Peptide 200.

[0145] Furthermore, the fusion protein may contain at least two, for example, three, four, five, or six fusion partner polypeptide units, for example, up to ten fusion partner polypeptide units. Figure 3c illustrates a fusion protein 320 containing a first antigen-binding polypeptide 200 and three polypeptide moieties 301. The connector 204 is optional and may be the same or different for each occurrence. The polypeptide moieties 301 may be the same or different in terms of their amino acid sequences. In other embodiments, the fusion protein may contain two fusion partner polypeptides and any suitable number of antigen-binding polypeptides 200 as described above. For example, the fusion protein may contain one antigen-binding polypeptide flanked by two stable polypeptides, or the fusion may contain an antigen-binding polypeptide comprising a VH variant, which has a different antigen-binding polypeptide attached to one side and a stable polypeptide attached to the other side. In cases where a fusion protein comprises multiple antigen-binding peptides and multiple stable peptides (e.g., two or three of each), it is envisioned that the different peptide motifs 200 and 301 can be arranged in an alternating manner, rather than sequentially arranged with peptides of the same type, as described in Figure 3b (for peptide 200) and Figure 3c (for stable peptide 301) on page 26 of the specification, CN 122138977 A. For example, a multimeric fusion protein containing two units of each peptide can have the following general structure: [antigen-binding peptide] - [fusion partner peptide] - [antigen-binding peptide] - [fusion partner peptide].

[0146] More generally, the fusion protein according to this disclosure may have the following structure: ([A-L1]m-[Z-L2]n)p where A represents a polypeptide as described herein, Z represents an additional polypeptide moiety (fusion partner polypeptide) as described herein; L1 may be present or absent for each occurrence, and when present, represents a linker or spacer group; L2 may be present or absent for each occurrence, and when present, represents a linker or spacer group; m represents an integer from 1 to 4; n represents an integer from 1 to 4, or when m ≥ 2, n represents 0 or an integer from 1 to 4; and p represents an integer from 1 to 4. Preferably, (m+n)*p, i.e., the total number of polypeptide moiety A and Z in the fusion protein, may be at most 10, for example, at most 8.

[0147] In some embodiments, n=1, m=1, and p=1. In other embodiments, m is 2 or 3, n is 1, and p is 1. In still other embodiments, m is 1, n is 1, and p is 2 or 3.

[0148] For reference purposes, in the exemplary fusion protein of FIG. 3a, m=1, n=1, and p=1. In the exemplary fusion protein of FIG. 3b, m=3, n=1, and p=1. In the exemplary fusion protein of FIG. 3c, m=1, n=3, and p=1.

[0149] In instances where the fusion protein comprises at least two fusion partner polypeptides, it may be preferred that at least one is located at the C-terminus of the fusion protein. Optionally, at least two fusion partner polypeptides may be sequentially arranged at the C-terminus of the fusion protein.

[0150] At the N-terminus of the fusion protein (e.g., a multimeric fusion protein), a short amino acid sequence, such as a leader peptide or signal peptide, may be present. At the C-terminus of the fusion protein (e.g., a multimeric fusion protein), a tag or spacer group, or another short amino acid sequence, may be optionally provided as needed. For example, multiple histidine residues may be provided, as described elsewhere in the text.

[0151] In embodiments, the fusion partner polypeptide is not located at or near the N-terminus of the fusion protein.

[0152] For clarity, any reference herein to polypeptides or fusion proteins of this disclosure also includes their multimeric variants, unless otherwise indicated.

[0153] The polypeptides of the present invention, which have improved basic stability compared to polypeptides based on the natural VHH framework, can be advantageously used to generate affinity binders for suitable targets by transplanting a CDR sequence for a desired target into a VH variant framework as disclosed herein. Known CDR1-3 sequences for a specific antigen can be identified or predicted from relevant literature, or new CDR sequences can be generated or identified by any method known to those skilled in the art, including but not limited to immunizing animals (e.g., camelids), phage display, ribosome display, cell surface display, bacterial display, computer simulation, and combinations thereof. When a satisfactory binding polypeptide has been identified, the amino acid sequence of the CDR can be selected and used to design stable antigen-binding polypeptides having a stable framework sequence as described herein. Exemplary methods for generating high-affinity antigen-binding VHHs with CDR sequences suitable for transplantation are described, for example, in Schmitz et al., 2013, Structure21, 1214-1224 and Fleetwood et al., Cell. Mol. Life Sci. (2013) 70:1081-1093.

[0154] For example, the target entity may be an antibody or a portion thereof, such as a monoclonal antibody, antibody fragment, or antibody domain. As another example, the target entity may be a protein, such as a recombinant protein. Alternatively, the target entity may be a viral particle or viral vector, such as adenovirus, retrovirus (γ-retrovirus and lentivirus), poxvirus, adeno-associated virus (AAV), baculovirus, or herpes simplex virus. For example, the target entity may be adeno-associated virus (AAV), such as AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 (AAVrh10), 11, or 12. Other examples of target entities include exosomes and lipid nanoparticles.In other instances, the target entity may be a nucleic acid molecule, such as DNA or RNA, such as mRNA. A CDR that provides affinity for such antigens may be provided, as described above. Specification 21 / 68 pages 27 CN 122138977 A

[0155] The polypeptides of this disclosure, as well as the polymers and fusion proteins mentioned herein, can be produced by conventional biotechnological methods through recombinant protein production using genetically modified host cells.

[0156] Therefore, in a further aspect, this disclosure provides isolated nucleic acids encoding polypeptides, polymers, or fusion proteins as described herein; an expression vector containing said nucleic acid; and a host cell containing said expression vector.

[0157] This disclosure also includes a method for producing polypeptides as described herein, comprising culturing said host cells under conditions allowing expression of said polypeptide, polymer, or fusion protein from its expression vector, and isolating said polypeptide, polymer, or fusion protein.

[0158] The host cell may be a prokaryotic cell, such as bacteria, or a eukaryotic cell. An exemplary prokaryotic host cell is *Escherichia coli*. Suitable eukaryotic cells may be yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris, or animal cells, such as insect cells, or mammalian cells commonly used in the field of biotechnology for protein production, such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells.

[0159] After isolating the polypeptide from the host cell (which may be done by conventional methods such as harvesting, clarification, and / or filtration), the polypeptide may be purified, for example, by conventional methods known in the art. For example, if the polypeptide contains a histidine tag, the purification step may include immobilized metal affinity chromatography (IMAC). Other purification methods that may be used include affinity chromatography. For example, affinity ligands that bind to the framework region of the VH variant may be used, or affinity chromatography may use affinity ligands based on the SpA domain, wherein the affinity ligand binds to the VH region of the polypeptide, such as VH3. Alternatively, when the polypeptide forms part of a fusion protein, the affinity ligand may bind to a fusion partner, such as an affinity tag included in the amino acid sequence of the fusion partner polypeptide.

[0160] Alternatively, the polypeptides disclosed herein can be generated by in vitro translation or by non-biological peptide synthesis using amino acids and / or amino acid derivatives having protected reactive side chains. Non-biological peptide synthesis may include: - stepwise coupling of amino acids and / or amino acid derivatives to form a polypeptide having protected reactive side chains, - removal of protecting groups from the reactive side chains, and - folding the polypeptide in an aqueous solution.

[0161] In general, the polypeptides of the present invention can be used to capture target entities to which the polypeptide has an affinity. However, preferably, the polypeptides themselves are not intended to be used as therapeutic compounds, and preferably are not intended for in vivo use. Therefore, the polypeptides of the present invention can be used to capture target entities in vitro.

[0162] The capture can be used to detect target entities within a sample or to separate target entities from other components in the sample. Thus, the antigen-binding peptide can be used for analytical separation of target entities, or it can be used for preparative purification of target entities. The term "preparation" refers to the process of preparing a purified target entity, specifically in the production of products containing the target entity, such as pharmaceuticals or therapeutic compositions. Alternatively, the separation, detection, and / or quantification of peptides using the present disclosure can be performed in the context of sensor applications.

[0163] Specifically, the peptides of the present invention can be used in applications involving interactions between VH variants and their target entities, wherein the peptide and target entity are coupled to a carrier. When the peptide is coupled or immobilized to a carrier, the VH variant remains able to bind its target entity.

[0164] In the case of the peptide being coupled to a carrier, this can be referred to as an adsorbent material or affinity capture material. Adsorbent materials can be used in a variety of industrial or laboratory applications, including the separation, purification, detection, and / or quantification of target entities. The separation matrix described herein is an adsorbent material designed for separating target entities from other components. The conjugation of the polypeptide of the present invention to the carrier may optionally be provided by a C-terminal amino acid sequence suitable for conjugation to the carrier. For example, a cysteine-terminated tag or spacer may be provided for conjugation via a thioether bond, as described in more detail below. In the case of the polypeptide forming a fusion protein, as described in section 22 / 68 of CN 122138977 A, the C-terminal fusion partner polypeptide may have such a C-terminal amino acid sequence suitable for conjugation to the carrier described below.

[0165] In the absence of conjugation to the carrier, the polypeptide may be used, for example, in a detection assay where a target or analyte is present on a surface and in contact with a polypeptide present in solution (capable of binding the target). In such a case, a reporter entity is envisioned for generating a detectable signal, wherein the reporter entity is capable of binding the polypeptide, or, in the case of a fusion protein, of binding the fusion partner. The reporter entity may be, for example, a fluorescently labeled or radiolabeled entity, as known in the art. If the polypeptide or, in the presence, the fusion partner polypeptide is capable of binding IgG, the reporter entity may be based on IgG.

[0166] The polypeptide can be used in applications where it can be exposed to alkaline conditions, such as treatment with NaOH. Such treatment is commonly used in the field of chromatography to remove impurities bound to the chromatographic matrix prior to the next purification cycle (in-situ cleaning, as described above). However, alkaline exposure or cleaning of the binding surface can also be used for other backgrounds.

[0167] The support can be a solid support and optionally a porous material.

[0168] The support can be or comprise a surface on which the polypeptide is coupled. Examples of such support materials include conventional protein-binding supports and surfaces, such as chips, plates, wells, and sheets.

[0169] The carrier may optionally be provided in other forms, such as fibers, membranes, fiber matrices, filters, porous bulk materials, particles, or beads, such as gel beads used in chromatographic resins. The particles or beads may be porous or non-porous. The particles or beads may include magnetic beads. Carriers in bead or particle form may be used as packed beds or in suspension. Suspension forms include those known as expanded beds and pure suspensions, in which the particles or beads move freely. In the case of bulk, packed, and expanded beds, the separation procedure typically follows conventional chromatography with a concentration gradient. In the case of pure suspensions, a batch mode will be used.

[0170] The carrier may be prepared from any suitable material, as outlined in more detail below. As a non-limiting example, conventional affinity separation matrices typically have organic properties and are based on exposing hydrophilic surfaces to the aqueous medium used, i.e., on their outer surfaces and, if present, on their inner surfaces, polymers of hydroxyl (-OH), carboxyl (-COOH), formamide (-CONH2, possibly in N-substituted form), amino (-NH2, possibly in substituted form), oligomers, or polyoxymethylene groups.

[0171] The polypeptide can be linked to the carrier using, for example, the thiol, amino, and / or carboxyl groups present in the antigen-binding polypeptide via known coupling techniques. Biepoxides, epichlorohydrins, CNBr, N-hydroxysuccinimide (NHS), etc., are well-known coupling agents. A spacer molecule can be introduced between the carrier and the polypeptide, which improves the utilization of the binding region and / or promotes the chemical coupling of the polypeptide to the carrier. Depending on the properties of the polypeptide and the coupling conditions, the coupling can be random or multi-point coupling (e.g., via multiple lysine or histidine residues) or single-point coupling (e.g., via a single cysteine ​​residue). In embodiments, to improve coupling control, it may be preferred that the VH variant itself does not contain any histidine residues. However, in the same case, the polypeptide as a whole may contain a histidine tag, such as a (His)6 tag. Alternatively, the polypeptide can be linked to the carrier via non-covalent bonding, such as physical adsorption or biospecific adsorption.

[0172] The polypeptide can be coupled to the carrier via a thioether bond. The methods used to perform such coupling are well known in the art and readily performed by those skilled in the art using standard techniques and equipment. Thioether bonds are flexible and stable and are generally suitable for affinity chromatography. Specifically, when the thioether bond is via a terminal or proximal cysteine ​​residue on the polypeptide, the motility of the coupled antigen-binding polypeptide is enhanced, providing improved binding capacity and binding kinetics. In some embodiments, the polypeptide is coupled via a C-terminal cysteine ​​residue provided on the protein as described above. This allows the cysteine ​​thiol group to be effectively coupled to an electrophilic group on the support, such as an epoxide group, a halool group, etc., resulting in thioether-bridged coupling.

[0173] Regarding the specific materials, the carrier may comprise polymeric materials. Polymeric materials include natural or synthetic polymers and combinations thereof. For example, the carrier may comprise polyhydroxy polymers, such as polysaccharides. Examples of polysaccharides include, for example, dextran, starch, cellulose, pullulan, agar, agarose, etc., including their derivatives. Polysaccharides are inherently hydrophilic, have a low degree of nonspecific interactions, provide a high content of reactive (activatable) hydroxyl groups, and are generally stable to alkaline cleaning solutions used in biological treatments. The carrier may comprise agar or agarose, such as cross-linked agarose. Such carriers used in this invention can be readily prepared by standard methods, such as reverse-phase suspension gels (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964)). Alternatively, the base matrix is ​​a commercially available product, such as cross-linked agarose beads sold under the name SEPHAROSE™ FF (Cytiva™). In embodiments particularly advantageous for large-scale separations, the carrier has been modified to increase its rigidity using methods described in US6602990 or US7396467 (incorporated herein by reference in its entirety), thus making the matrix more suitable for high flow rates.

[0174] Synthetic polymers that can be used as carrier materials include polyvinyl alcohol, polyhydroxyalkyl acrylate, polyhydroxyalkyl methacrylate, polyacrylamide, polymethacrylamide, etc. In the case of hydrophobic polymers (e.g., matrices based on divinyl and monovinyl-substituted benzene), the matrix surface can be hydrophilized to expose hydrophilic groups as defined above to the surrounding aqueous liquid. Such polymers are readily produced according to standard methods. As an alternative, commercially available products such as SOURCE™ (Cytiva™) can be used.

[0175] Alternatively, the solid carrier according to the invention comprises a carrier of inorganic properties, such as silica, zirconium oxide, etc.

[0176] In an embodiment, the polypeptide may be coupled to a carrier, which is a convection-based chromatographic matrix. Such a convection-based chromatographic matrix may comprise a porous polymer membrane, a filter, a fibrous matrix, or a porous monolith. Examples of porous polymer membranes include Mustang™ membranes (Cytiva) and Sartobind™ membranes (Sartorius). The fibrous carrier may be based on electrospun polymer fibers or cellulose fibers, optionally nonwoven fibers. The fibrous matrix can therefore be a nonwoven fibrous matrix. The fibers may have a cross-sectional diameter of 10–1000 nm, for example 200–800 nm, 200–400 nm, or 300–400 nm. Such fibrous carriers are seen in HiTrap.Fibro™ device (Cytiva™). Alternative fiber carriers are disclosed, for example, WO2019 / 137869 and WO2018 / 011600.

[0177] Adsorbent materials or separation matrices as described herein can be used for the same purposes mentioned above for the peptides.

[0178] Thus, in one aspect, the present invention provides chromatographic materials comprising separation matrices as disclosed herein, and chromatographic columns or devices comprising such separation matrices.

[0179] In another aspect, the adsorbent material or separation matrix may be a sensor surface designed for use in sensors or other detection or quantification devices.

[0180] The present invention provides a method for separating or isolating target entities, wherein separation matrices or adsorbent materials as disclosed herein are used. In some embodiments, the method includes contacting a liquid sample containing a target entity with an adsorbent material as disclosed herein. Contact is made under conditions where the target entity can bind a VH variant of the peptide. The method may further include washing the adsorbent material with a washing liquid, eluting the target entity from the adsorbent material with an elution liquid, and optionally cleaning the adsorbent material with a cleaning liquid. The cleaning liquid may also be referred to as a clean-in-situ (CIP) liquid. The cleaning liquid is typically an alkaline solution, for example containing at least 0.05 M, such as 0.05–1 M, such as 0.05–0.5 M, such as at least 0.1 M, such as 0.1–0.5 M, such as 0.3 M or 0.5 M NaOH or KOH. The contact (incubation) time may be at least 10 minutes. The binding, elution and cleaning steps may advantageously be repeated at least 5 times, such as at least 10 times, such as at least 20 times.

[0181] As shown herein, the peptides of this disclosure have improved alkaline stability and are therefore more resistant to alkaline conditions, such as those used during the CIP step, and retain a high degree of binding capacity to the target entity even after repeated cycles of conventional CIP treatment (using, for example, 0.1–0.5 M NaOH). For example, after at least 12 cycles of contact with an alkaline liquid, such as after at least 15 cycles, or even after 20 cycles, the peptide may still retain at least 50%, such as at least 60%, such as at least 80%, such as at least 90% of the initial target binding capacity. Alternatively, after 12 or 15 cycles of contact with an alkaline liquid, or after 20 cycles, the peptide may retain at least 50%, such as at least 60%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 98% of the target entity binding capacity in the second cycle.

[0182] Those skilled in the art will understand that the liquid sample to be purified may be a target contained in the environment from which purification or separation is performed.Any sample of the target entity. The sample may be obtained from cell cultures, such as clarified cell culture harvest, and may have undergone one or more conventional steps of filtration, concentration, dilution, and / or buffer exchange, and optionally one or more initial chromatographic steps, specifically steps not based on affinity chromatography, such as ion exchange chromatography, hydrophobic interaction chromatography, multimode chromatography, or size exclusion chromatography. For example, the sample may be clarified and filtered cell culture harvest. In some cases, the sample may have undergone one or more filtration steps by tangential flow filtration (TFF).

[0183] Before contact with the adsorbent material of the present invention, the liquid sample contains the target entity and at least one impurity, such as host cell protein (HCP) or host cell nucleic acid. The adsorbent material can be used to separate the target entity from such impurities, and after the above process, the eluent containing the target entity has a reduced level of at least one of said impurities.

[0184] Optionally, after separation based on affinity capture as described herein, the eluent containing the purified target entity may undergo one or more steps of filtration, concentration, dilution, or buffer exchange, or chromatographic steps not based on affinity chromatography, such as ion exchange chromatography, hydrophobic interaction chromatography, multimode chromatography, or size exclusion chromatography. Further chromatographic steps performed after affinity separation according to this disclosure may be referred to as purification steps.

[0185] Although the invention has been described herein with respect to exemplary embodiments, those skilled in the art will understand that the invention is not limited to these embodiments. Any reference marks enclosed in parentheses in the claims should not be construed as limiting the claims.

[0186] The use of the verb “comprising” and variations thereof does not exclude the presence of elements or steps other than those specified. For example, a polypeptide “comprising” frame regions FWR1, FWR2, and FWR3 may also have further frame regions, such as FWR4, and may have other regions, such as CDRs, and optionally other structures or sequences. The article “a” or “an” preceding an element does not exclude the presence of multiple such elements.

[0187] The fact that certain measures are described in mutually different dependent claims does not in itself indicate that a combination of these measures cannot be used to produce benefits. Examples

[0188] Example 1A: Preparation of Glycerol Stock Solution from IDT Oligonucleotides This example describes the production of a glycerol stock solution for the production of candidate peptides used in Examples 2A-B and 4B.

[0189] The materials and equipment used are as follows: a vector plasmid with kanamycin resistance; G-blocks peptide candidate (IDT™); restriction enzymes KpnI-HF and HindIII-HF (NEB™); Antarcic phosphatase (NEB™); GFX™ PCR DNA and gel strip purification kit (Cytiva™); T4 DNA ligase (NewEngland Biolabs; Top 10 E. coli cells (ThermoFisher™); Chemocompetent BL21(DE3) E. coli cells (in-house preparation); Chemocompetent K12-017 E. coli cells (in-house preparation); SOC medium (Invitrogen™); Kanamycin stock solution 50 mg / ml (PanReac AppliChem™); KCM buffer (5X) 0.5M KCl, 0.15M CaCl2, 0.25M MgCl2; Luria-Bertani (LB) medium (Invitrogen™); Agar plates supplemented with kanamycin (50 µg / ml); 14 ml Falcon™ round-bottom tubes (Corning™); PlasmidPrep Mini Spin kit (Cytiva™); Inforrs HT shaking incubator. Instructions for Use, Pages 25 / 68, No. 31, CN 122138977 A

[0190] The vector plasmid and G-blocks containing the DNA sequences of all candidates were digested with restriction enzymes KpnI-HF and HindIII-HF. The plasmid was dephosphorylated using antarctic phosphatase and purified by gel according to the manufacturer's protocol.

[0191] The restriction enzyme-digested G-blocks were ligated to the gel-purified plasmid using T4 DNA ligase at room temperature for 2 h and transformed into Top10 E. coli cells. Briefly, the cells and ligation mixture were incubated on ice for 20 min and incubated in KCM buffer at room temperature for 10 min. The cells were then transferred to SOC medium and incubated at 37°C for 1 h, then seeded on agar plates containing 50 µl / ml kanamycin and incubated overnight at 37°C. Inoculate 4 mL of LB medium supplemented with 50 µg / mL kanamycin using a single colony in a 14 mL round-bottom tube and incubate overnight at 37 °C and 200 rpm. Prepare plasmids according to the manufacturer's protocol and transform the purified plasmids into KCM competent cells K12-017 or KCM competent cells BL21(DE3) E. coli. Incubate cells and plasmids on ice for 20 min and incubate in KCM buffer at room temperature for 10 min. Then transfer cells to SOC medium and incubate at 37 °C for 1 h, then inoculate onto an agar plate containing 50 µl / mL kanamycin and incubate overnight at 37 °C. Inoculate 4 mL of LB medium supplemented with 50 µg / mL kanamycin using a single colony in a 14 mL round-bottom tube and incubate overnight at 37 °C and 200 rpm. Prepare glycerol stock solutions for each variant using 900 µl of overnight culture and 500 µl of 50% glycerol and store at -80 °C.

[0192] Example 1B: Preparation of Glycerol Stock Solution from IDT Plasmid This example describes the preparation of a glycerol stock solution for the production of candidate peptides used in Examples 3, 4A, and 5-7.

[0193] The materials and equipment used are as follows: plasmid DNA of the candidate peptide to be produced; chemocompetent BL21(DE3) Escherichia coli (prepared internally); KCM buffer (5X) 0.5M KCl, 0.15M CaCl2, 0.25M MgCl2; LB medium (Invitrogen™); carbenicillin (100 mg / ml); agar plates supplemented with carbenicillin (100 µg / ml) (PanReac AppliChem™) (BD BACTO™ agar); 14 ml Falcon™ Round-Bottom (Corning™); Infors HT shaking incubator.

[0194] The desired amino acid sequence of the candidate peptide was reverse-translated into DNA and optimized to remove rare codons. Based on an expression vector containing a T5 promoter, OmpA signal peptide, ampicillin resistance, and pUC replication origin, plasmid DNA was ordered as a construct. The plasmid DNA was used to transform into chemically competent BL21(DE3) Escherichia coli, plated on agar plates supplemented with 100 µg / ml carbenicillin, and grown overnight at 37°C. For each candidate, a single colony was inoculated in 4 mL of LB medium supplemented with 100 µg / ml carbenicillin in a 14 mL round-bottom tube and grown overnight at 37°C with stirring at 200 rpm. Glycerol stock solutions for each variant were prepared with 900 µl of overnight culture and 500 µl of 50% glycerol and stored at -80°C until further use.

[0195] Example 2A: Basic stability of sdAb variants with frame variations This example investigated the basic stability of AAV9-binding VHH with synthetic FWR3 and further variations in frame regions 1 (FWR1) and 2 (FWR2). The effects of mutations in the VHH framework were investigated to identify amino acid positions that enable high basic stability and maintain AAV9 binding.

[0196] Candidate VHH sequences are described in Table 6. Amino acid substitutions (Kabat numbers) are described relative to SEQ ID NO:73, which is a synthetic VHH with a modified framework compared to the native VHH framework sequence and has been found to be resistant to repeated cleaning with 0.1 M NaOH (data not shown). The CDR sequence is identical for all candidates.

[0197] The VHH sequence with SEQ ID NO:159 as the C-terminal fusion was expressed via a peptide linker (AA), and all constructs had a C-terminal tag (HHHHHHC). The total molecular weight was 21 kDa.

[0198] Table 6 Specification 26 / 68 pages 32 CN122138977 A The following aspects were evaluated: (1) Affinity assessment of AAV9 interaction, which was tested by injecting high concentrations of AAV9. (2) Basic stability, which was tested by the reduction of AAV9 binding after treatment with 0.3 M NaOH in increments of 100 times.

[0199] Materials and Methods Generation of candidate peptides purified from IgG Sepharose 5 µl of each variant of the generated strain as described in Example 1A was inoculated into 4 ml of LB medium supplemented with 50 µg / ml kanamycin in 14 ml round-bottom tubes and incubated overnight at 37°C with stirring at 160 rpm.

[0200] Protein expression medium prepared from Terrific Broth (TB) medium supplemented with 50 µg / ml kanamycin and 2 mM MgCl2 (200 mM) was added to a 100 ml baffled glass shaker flask (20 ml / flask). Each vial was inoculated with approximately 100 µl of the previous overnight culture to obtain an initial OD600 of 0.05. The vials were incubated in an Inforrs HT shaking incubator at 37 °C and 140 rpm for approximately 3 hours until the OD600 reached 1.0. Then, 20 µl of IPTG (1 M) was added to a final concentration of 1 mM, and the vials were incubated in an Inforrs HT shaking incubator at 27 °C and 140 rpm for 18 hours, after which the culture was transferred to Falcon tubes (50 mL). To generate coarse variant lysates, the samples were sonicated on ice (40% amplitude, 1.5 s total on time, 1 s on / off pulse). Cell debris was precipitated by centrifugation at 12000 g for 10 min, and the supernatant was filtered through a 0.22 µm filter.

[0201] Samples were purified using gravity-flow IgG Sepharose 6FF chromatographic resin (Cytiva™) packed inside an empty PD-10 column (Cytiva™). The clarified lysate was loaded onto a column equilibrated in 50 mM Tris, 150 mM NaCl, and 0.05% Tween 20 (TST buffer). The column was washed with 10 column volumes (CV) of TST buffer, followed by 2 CV of mM NH4Ac at pH 5. Protein was eluted with 2.5 mL of 0.5 M HAc. Prior to further analysis, the eluted protein buffer was exchanged for phosphate-buffered saline (Medicago) at pH 7.4 using a gravity-flow PD-10 (column, Cytiva) column.

[0202] IgG Sepharose was measured using NanoDrop (Thermo Scientific) according to the manufacturer's instructions.The concentration of the purified sample. After purification, the sample was kept in an Eppendorf tube in a refrigerator until all measurements had been performed, and then kept in a freezer until Biacore analysis.

[0203] Biacore analysis of binding affinity to AAV9 To evaluate binding to AAV9, the candidate peptide was immobilized on a Biacore™ S-Series CM5 chip. AAV9 (2E12 vp / ml) was used as the analyte.

[0204] The materials and equipment used were as follows: Biacore™ S-Series CM5 sensor chip (Cytiva™); Biacore™ Amine Conjugation Kit (Cytiva™); Biacore™ Acetate Buffer pH 5.0 (Cytiva™); Biacore™ 8K+ Instrument (Cytiva™); AAV9 (prepared internally); IgG Sepharose purified candidate peptide as described above. Instruction manual, pages 27 / 68, 33 CN 122138977 A

[0205] Using the standard method in Biacore software, candidate peptides were immobilized by coupling in flow cell 2 (FC2) and activation / inactivation in flow cell 1 (FC1). Candidate peptides were diluted in acetate buffer at a concentration of 35 µg / ml. Immobilization levels varied slightly between different peptide variants (average 5200 ± SD 436 RU).

[0206] In each run, the peptides were immobilized in FC2. Multiple sensor chips were used until all candidates were tested.

[0207] Biacore™ method used for binding analysis: Run buffer: PBS-P+; Flow rate: 5 µl / min; Sample injection: 2400 s / 40 min in both flow cells (FC1 and FC2); Dissociation time: 2400 s / 40 min; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s.

[0208] For each channel (cycle), the analyte (AAV9) was injected as follows: run buffer, AAV9 6E10 vp / ml, 1.25E11 vp / ml, 2.5E11 vp / ml, 5E11 vp / ml, 1E12 vp / ml, 2E12 vp / ml.

[0209] All sensor maps were generated after subtracting the reference, and the response values ​​were generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0210] Biacore analysis of alkaline stability: After evaluating AAV9 binding (see above), the same chip underwent repeated cycles of AAV9 binding (5E11 vp / ml) followed by NaOH (0.3 M) injection to assess alkaline stability.

[0211] Biacore method for alkaline stability (per cycle): Run buffer: PBS-P+; flow rate: 10 µl / min; Sample injection 1 (AAV9 5E11 vp / ml): 300 s in both flow cells; dissociation time 1: 30 s; Sample injection 2 (0.3 M NaOH): 600 s in both flow cells; dissociation time 2: 0 s; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. This cycle was repeated 25 times to track the stability of the AAV9 response value.

[0212] All sensor maps were generated with reference subtraction, and the response value was generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0213] The results, combined with affinity analysis, showed that candidates vh98, vh100, vh103, and vh105 had similar relative binding responses to AAV9 and never reached saturation even at the highest analyte concentrations.

[0214] Figure 4 shows the results of the alkaline stability study, with all responses normalized to the response of the first cycle (i.e., before the first NaOH exposure). vh98 was moderately stable under 0.3 M NaOH exposure, while vh100 and vh103 showed slight improvements. vh105 confirmed further improved stability, with a slower degradation rate than vh103. The results are summarized in Table 7. It can be concluded that both 19T and 23T are beneficial for stability. P40R also leads to improved stability.

[0215] Table 7 Example 2B: Basic stability of sdAb variants with further frame variations This example investigated the basic stability of AAV9-binding VHH variants with the same FWR3 (SEQ ID NO:45) and further variations in frame regions 1 (FWR1) and 2 (FWR2). The effects of mutations in the VHH frame were investigated to identify amino acid positions that could retain high basic stability and AAV9 binding.

[0216] Candidate VHH sequences are identified in Table 8. The CDR is the same in all candidates. Specification 28 / 68 pages 34 CN 122138977 A

[0217] Table 8 Expression via peptide linker with sdAb sequence having SEQ ID NO:159 as C-terminal fusion, and all constructs have a C-terminal tag (HHHHHHC). The total molecular weight is 21 kDa.

[0218] The following aspects were evaluated: (1) Affinity assessment of AAV9 interaction, which was tested by injecting high concentrations of AAV9. (2) Basic stability, which was tested by the reduction of AAV9 binding after treatment with 0.5 M NaOH in increments of 100%.

[0219] Materials and Methods Generation of candidate peptides purified from IgG SepharoseAs described in Example 2A, candidate peptides were generated and purified, except that, in order to generate crude lysates, Falcon tubes were incubated in a 48°C water bath for 2 h, followed by centrifugation at 8000 g for 10 min to precipitate cell debris, instead of sonication on ice.

[0220] Biacore Affinity Assay To assess binding to AAV9, candidate peptides were immobilized on a Biacore™ S-Series CM5 chip. AAV9 (2E12 vp / ml) was used as the analyte.

[0221] The materials and equipment used were as follows: Biacore™ S-Series CM5 sensor chip (Cytiva™); Biacore™ Amine Conjugation Kit (Cytiva™); Biacore™ Acetate Buffer pH 5.0 (Cytiva™); Biacore™ 8K+ Instrument (Cytiva™); AAV9 (prepared internally); IgG Sepharose purified candidate peptides as described above.

[0222] The candidate peptide was immobilized using the standard method in Biacore software by conjugating it in flow cell 2 (FC2) and activating / inactivating it in flow cell 1 (FC1). The candidate peptide was diluted in acetate buffer at a concentration of 25 µg / ml. The immobilization level did vary slightly between different peptide variants (mean 4319 RU + SD 640 RU). The peptide was immobilized in FC2.

[0223] Biacore™ method for binding analysis: Run buffer: PBS-P+; Flow rate: 5 µl / min; Sample injection: 2400 s / 40 min for both flow cells (FC1 and FC2); Dissociation time: 2400 s / 40 min; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s.

[0224] For each channel (cycle), the analyte (AAV9) was injected as follows: run buffer, AAV9 1.25E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

[0225] All sensor maps were generated after subtracting the reference, and the response values ​​were generated as the difference between the baseline before sample injection and the signal immediately before the end of injection.

[0226] Biacore analysis of alkaline stability: After evaluating AAV9 binding (see above), the same chip underwent repeated cycles of binding AAV9 (3E11 vp / ml) followed by injection of NaOH (0.5 M) to assess alkaline stability.

[0227] Biacore method for alkaline stability (per cycle): run buffer: PBS-P+; flow rate: 10 µl / min; sample injection 1 (AAV9)3E11 vp / ml): 300 s in both flow cells; dissociation time 1: 30 s; Sample injection 2 (0.5 M NaOH): 600 s in both flow cells; dissociation time 2: 0 s; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. This cycle was repeated 30 times to track the stability of the AAV9 response value.

[0228] All sensor plots were generated with reference subtracted, and the response value was generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0229] The results showed that all candidates had similar and high AAV9 binding capacity (Biacore response values ​​ranged from approximately 12000 to 15000 RU; the fixed level varied slightly among different candidates), with vh97 showing the highest capacity. It was concluded that changes in the test framework had no significant effect on the target binding capacity.

[0230] Alkali stability assessment studies confirmed that candidates with one or more FWR1 or FWR2 variations as described in Table 8 were more resistant to alkalinity than vh97. Figure 5 shows the AAV9 response for incremental cycles including alkaline exposure (0.5 M NaOH), normalized to the response of the second cycle (i.e., excluding the first cycle).

[0231] As can be seen in Figure 5, vh118 and vh120 have very similar NaOH resistance, and vh122 and vh124 (both omitted from the figure) perform very similarly, at the same level as vh119. This means that A24I and A24V have a considerable effect on NaOH resistance. Candidates with R at position 40 perform particularly well.

[0232] Candidate vh118 has better alkaline stability than vh119, and vh122 is more stable than vh123 (also omitted from the figure; stability is similar to vh121), indicating that substitution of E6Q improves NaOH tolerance.

[0233] Regarding NaOH resistance, candidates with position 44 E performed better than the corresponding mutants with position 44 A (vh118 vs vh122; vh119 vs vh123; vh120 vs vh124; vh121 vs vh125). This indicates that position 44 E is more favorable for basic stability than A.

[0234] This example shows that even though previously tested FW mutation combinations, such as vh98, exhibit greatly enhanced basic stability compared to the native framework, other substitutions, such as vh118, can result in even more stable peptides. However, it is concluded that variants lacking all the optimal mutations are still considered to be basic stable and meet the goal of obtaining basic stable antigen-binding peptides of this disclosure.

[0235] Example 3: Binding and stability of candidate peptides with FW point modificationsThe effects of point mutations in the AAV9-binding VHH framework were investigated to identify amino acid positions that enable high basic stability and AAV9 binding. Based on SEQ ID NO:80, a class of AAV9-binding peptides with different point mutations were generated, as described in Tables 9a-c below.

[0236] The following aspects were evaluated: (1) Affinity assessment of AAV9 interaction (tested by high concentration injection of AAV9). (2) Basic stability (tested by reduction of AAV9 binding after treatment with 0.5 M NaOH in increments of 30 / 68 pages of the specification 36 CN 122138977 A).

[0237] Materials and Methods Immobilized metal affinity chromatography (IMAC) purified candidate peptides were generated in 14 ml round-bottom tubes. 5 µl of each variant generated as described in Example 1B was inoculated into 4 ml of LB supplemented with 100 µg / ml carbenicillin and incubated overnight at 37°C and 160 rpm.

[0238] Protein expression medium (TB medium supplemented with 100 µg / ml carbenicillin and 2 mM MgCl2) was prepared and added to pre-filled 500 ml baffled glass shake flasks (50 ml / flask). Approximately 500 µl of the previous overnight culture was inoculated into each flask to obtain an initial OD600 of 0.05. The flasks were incubated in an Infortr HT shaker incubator at 37°C and 140 rpm for approximately 3.5 hours until the OD600 reached 1.0. Then, 50 µl of IPTG (1 M) was added to a final concentration of 1 mM, and the flasks were incubated in an Infortr HT shaker incubator at 27°C and 140 rpm for 18 hours. The culture was then transferred to Falcon tubes (50 mL). To generate crude variant lysates, Falcon tubes were incubated in a 48°C water bath for 2 hours, followed by centrifugation at 8000 g for 10 min to precipitate cell debris and filtration of the supernatant using a 0.22 µm filter.

[0239] The clarified lysates were loaded onto a HisTrap™ FF 1 mL column (Cytiva™) equilibrated in 50 mM sodium phosphate pH 7.5 and 500 mM NaCl, allowing a residence time of approximately 2 minutes. The column was then washed with 15 column volumes (CV) of 50 mM sodium phosphate pH 7.5 and 500 mM NaCl. Proteins were eluted using 0.5 M imidazole pH 7.5–8 via a linear gradient of 10 CV (0–100%). Prior to further analysis, the eluted protein buffer was replaced with phosphate-buffered saline (Medicago) pH 7.4 using a gravity flow column pre-packed with Sephadex™ G-25 resin (Cytiva).

[0240] Following the manufacturer's instructions, the concentration of the HisTrap™ FF purified sample was measured using NanoDrop (Thermo Scientific). After purification, the sample was kept in an Eppendorf tube in a refrigerator until all measurements were performed, and then kept in a freezer until Biacore analysis.

[0241] Biacore analysis of binding affinity to AAV9 To assess binding to AAV9, candidate peptides were immobilized on a Biacore™ S-Series CM5 chip.

[0242] The materials and equipment used were as follows: Biacore™ S-Series CM5 sensor chip, Biacore™ amine conjugation kit, Biacore™ acetate buffer pH 5.0, Biacore™ 8K+ instrument (all from Cytiva™); AAV9 (in-house prepared); candidate peptides purified by HisTrap™ FF.

[0243] Immobilization was performed using the standard method in the Biacore software by conjugating the AAV9-binding peptide variant in flow cell 2 (FC2) and activating / inactivating it in flow cell 1 (FC1). AAV9-binding peptide variants were diluted at a concentration of 25 µg / ml in acetate buffer pH 5.0. Immobilization levels varied slightly between different peptide variants (mean 3763 RU ± SD 500 RU).

[0244] In each run, the peptide was immobilized in FC2. Multiple sensor chips were used until all candidates were tested.

[0245] Biacore™ method used for binding analysis: Run buffer: PBS-P+; Flow rate: 5 µl / min; Sample injection: 2400 s / 40 min in both flow cells (FC1 and FC2); Dissociation time: 2400 s / 40 min; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle), the analyte (AAV9) was injected as follows: run buffer, AAV9 2E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

[0246] All sensor maps were generated after subtracting the reference, and the response values ​​were generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0247] Biacore analysis of alkaline stability: After evaluating AAV9 binding (see above), the same chip underwent 40 repeated cycles of AAV9 binding (5E11 vp / ml) followed by NaOH (0.5 M). Specification 31 / 68 pages 37 CN 122138977 A

[0248] Biacore method for alkaline stability (per cycle): Run buffer: PBS-P+; Flow rate: 10 µl / min; Sample injection 1(AAV9 5E11 vp / ml): 300 s in both flow cells; dissociation time 1: 30 s; Sample injection 2 (0.5 M NaOH): 600 s in both flow cells; dissociation time 2: 0 s; regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. This cycle was repeated 40 times to track the stability of the AAV9 response values.

[0249] All sensor maps were generated with reference subtracted, and response values ​​were generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0250] The results normalize the response levels of the candidate peptides to the AAV9 interaction to the response of SEQ ID NO: 80 and are summarized in Tables 9a-c. In this respect, it should be noted that vh118 (SEQ ID NO: 80) contains a preferred frame with a near-optimal combination of mutations for the CDRs currently tested, compared to natural or synthetic starting frames. Therefore, most other variants will show lower basic stability compared to SEQ ID NO: 80. The variants below all show stability compared to the practically non-existent stability of the natural framework and the moderate stability of the synthetic starting framework. Therefore, the mutations introduced in these variants are still stable compared to the initial framework, but not as stable as the most preferred mutation. A comparison between SEQ ID NO: 80 and the least preferred mutation is included in Example 8 below.

[0251] Basic stability is recorded as the number of cycles in which at least 50% of the binding capacity is retained. The first cycle is excluded and the binding capacity value is normalized to the binding capacity of the second cycle (i.e., the binding after the first exposure to NaOH). In each cycle, 0.5 M NaOH is injected at 10 µl / min for a 10-minute contact time. Results for each candidate are provided in Tables 9a-c.

[0252] Satisfactory basic stability was found to be achieved for many of the point mutations studied, particularly in FWR1 and FWR2. At least 70% of the initial AAV9 binding capacity of SEQ ID NO: 80 is considered satisfactory. Regarding basic stability, it is preferred to maintain at least 50% binding capacity after 12 cycles; it is more preferred to retain at least 50% binding capacity after 15 cycles. Particularly preferred are those variants that maintain at least 50% binding capacity for 20 or more cycles of basic exposure, such as at least 24 cycles.

[0253] For example, it has been found that at position 40 of FWR2, all tested amino acids (R, T, I, K, A) result in a very stable peptide. Furthermore, at position 45, L and I provide good results in both target binding and basic stability. At position 47 of Kabat, amino acids F and L result in a very stable candidate. It has also been found that at FWR3...Point mutations at Kabat positions 76, 79, and 89 have a significant impact on basic stability. At position 76, N and Q were identified as favorable. At position 79, amino acids Y, F, and W provide excellent basic stability, and A is acceptable. At Kabat position 89, V, I, and L result in a very stable polypeptide.

[0254] In FWR 1, 24I / S / V provides excellent basic stability.

[0255] Table 9a: FWR1 variant specification 32 / 68 pages 38 CN 122138977 A Table 9b: FWR2 variant Table 9c: FWR3 variant specification 33 / 68 pages 39 CN 122138977 A Example 4A: Basic stability of natural and modified sdAb frameworks This example compares a single-domain antibody with a natural camel sdAb framework with an sdAb having a framework modified to enhance basic stability. The following aspects were evaluated: (1) Affinity assessment of target molecule interactions (tested by injecting target molecules at various concentrations). (2) Basic stability (tested by the reduction in target binding after treatment with 0.1 M or 0.3 M NaOH in increments of 0.1 M or 0.3 M).

[0256] The single-domain antibodies were VHH variants targeting AAV9, green fluorescent protein (GFP) or epidermal growth factor receptor (EGFR), and were based on the natural camel VHH framework or a modified framework to enhance the basic stability of the VHH variants themselves. The natural camelid frameworks correspond to the VHH “EgA1” or “9G8” framework sequences described in Schmitz et al., 2013, Structure21, 1214–1224, or to the GFP-binding VHH “S-Nb3” framework sequence described in Fleetwood et al., Cell. Mol. Life Sci. (2013) 70:1081–1093, except that the first two amino acids are replaced by VD in the latter. The CDR region is the same for candidates that bind to the same target. Specification 34 / 68 pages 40 CN 122138977 A

[0257] Each sdAb is expressed as a fusion protein fused to the C-terminus with the SpA domain Z variant (SEQ ID NO:160), in which binding to the Fc and VH3 portions of IgG has been deprecated. All constructs have a C-terminal linker (GS) followed by a His6 tag.

[0258] The tested peptide candidates (fusion proteins) are summarized in Table 10.

[0259] Table 10 Materials and Methods Generation of candidate peptides purified by immobilized metal affinity chromatography (IMAC) For each candidate, 5 µl of BL21(DE3) glycerol as described in Example 1B was added to a 14 ml round-bottom tube.The stock solution was inoculated in 4 ml of LB medium supplemented with 100 µg / ml carbenicillin and incubated overnight at 37°C and 160 rpm.

[0260] Protein expression medium (TB medium supplemented with 100 µg / ml carbenicillin and 2 mM MgCl2) was prepared and added to 500 ml baffled glass shake flasks (50 ml / flask). Approximately 500 µl of the previous overnight culture was inoculated into each flask to obtain an initial OD600 of 0.05. The flasks were incubated in an Inforrs HT shaker incubator at 37°C and 140 rpm for approximately 3.5 hours until the OD600 reached 1.0. Then, 50 µl of IPTG (1 M) was added to a final concentration of 1 mM, and the flasks were incubated in an Inforrs HT shaker incubator at 27°C and 140 rpm for 18 hours, after which the culture was transferred to Falcon tubes (50 mL). To generate crude variant lysates, Falcon tubes were incubated in a 48°C water bath for 2 hours, followed by centrifugation at 8000 g for 10 minutes to precipitate cell debris, and the supernatant was filtered through a 0.22 µm filter.

[0261] Samples were purified using a HisTrap™ FF 1 mL column (Cytiva). The clarified lysates were loaded onto a column equilibrated with 50 mM sodium phosphate at pH 7.5 and 500 mM NaCl, allowing a residence time of approximately 2 minutes. The column was then washed with 15 column volumes (CV) of 50 mM sodium phosphate at pH 7.5 and 500 mM NaCl. Proteins were eluted with 0.5 M imidazole at pH 7.5–8 using a linear gradient (0–100%) of 10 CV. Prior to further analysis, the eluted protein buffer was exchanged to phosphate-buffered saline (Medicago) pH 7.4 using a gravity flow column pre-filled with Sephadex™ G-25 resin (Cytiva).

[0262] The concentration of the purified and buffer-exchanged sample was measured using NanoDrop (Thermo Scientific) according to the manufacturer's instructions. After purification, the sample was kept in Eppendorf tubes in a refrigerator until all measurements were performed, and then kept in a freezer until Biacore analysis.

[0263] Biacore analysis for binding affinity to target molecules To assess binding to target molecules (AAV9, GFP, or EGFR), candidate peptides were immobilized on a Biacore™ S-Series CM5 chip (Cytiva) using an amine conjugation kit (Cytiva) and analyzed using a Biacore™ 8K+ instrument (Cytiva). The analyte used was AAV9 (2E13).vp / mL), GFP (1 g / L), and EGFR (0.2 g / L).

[0264] The peptide variants were immobilized using standard methods in Biacore software by conjugating them in flow cell 2 (FC2) and activating / inactivating them in flow cell 1 (FC1). The peptide variants were diluted at a concentration of 25 µg / mL in Biacore™ acetate buffer pH 5.0 (Cytiva™). The immobilization levels on the CM5 chip ranged from approximately 1000 to 3300 RU, but were typically around 3000 RU.

[0265] In each run, the peptides were immobilized in FC2. Multiple sensor chips were used until all candidates were tested.

[0266] Biacore™ method for AAV9 binding analysis: Run buffer: PBS-P+; Flow rate: 5 µl / min; Sample injection: 2400 s / 40 min in both flow cells (FC1 and FC2); Dissociation time: 2400 s / 40 min; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle), the analyte (AAV9) was injected as follows: run buffer, 2E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

[0267] Biacore™ method for GFP or EGFR binding assays: Run buffer: PBS-P+; Flow rate: 10 µl / min; Sample injection: 600 s / 10 min in both flow cells (FC1 and FC2); Dissociation time: 600 s / 10 min; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle) immobilized with the GFP-binding peptide, the analyte (GFP) was injected as follows: run buffer, 10 nM, 25 nM, and 100 nM. For each channel (cycle) immobilized with the EGFR-binding peptide, the analyte (EGFR) was injected as follows: run buffer, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM.

[0268] All sensor maps were generated after subtracting the reference, and the response values ​​were generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0269] Biacore analysis of basic stability was performed on the same CM5 chip with immobilized peptides after binding analysis (see above), involving repeated cycles of binding the corresponding analyte followed by injection of NaOH to assess basic stability (0.3 M for AAV9 binding peptides and 0.1 M for GFP and EGFR binding peptides, respectively).

[0270] Biacore method for alkaline stability (per cycle): Run buffer: PBS-P+; flow rate: 10 µl / min; Sample injection 1 (AAV9 5E11 vp / ml or GFP 50 nM or EGFR 75 nM): 300 s for both flow cells; dissociation time 1: 30 s; Sample injection 2 (0.1 M or 0.3 M NaOH): 600 s for both flow cells; dissociation time 2: 0 s; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. This cycle was repeated 40 times to track the stability of the target response values.

[0271] All sensor maps were generated with reference subtraction, and the response values ​​were generated as the difference between the baseline before injection and the signal immediately following the end of injection on pages 36 / 68 of the specification, CN 122138977 A.

[0272] All fusion proteins showed satisfactory binding to their respective targets, and Tables 11a-c summarize the results of binding capacity and basic stability measurements.

[0273] Table 11a: AAV9 binding candidates Table 11b: EGFR binding candidates Table 11c: GFP binding candidates Target binding capacity (late reporter point binding) measured at the highest test analyte concentrations showed little or no difference in target binding capacity among candidates with different frame sequences. However, the differences in basic stability were significant. Figures 6a-c show the binding capacity obtained for increasing cycle numbers including NaOH exposure (sample injection 2 above), normalized to the first cycle, for the AAV9-binding peptide (Figure 6a), GFP-binding peptide (Figure 6b), and EGFR-binding peptide (Figure 6c). As can be seen in these figures, VHH affinity ligands (4A-3, 4A-5, 4A-8) based on stable VHH frameworks show improved basic stability compared to reference VHHs with natural camel VHH frameworks. In fact, after the first cycle, unstable candidates 4A-1 and 4A-2 almost lost all target binding response.

[0274] Example 4B: Basic Stability of Various sdAb Frameworks This example compares single-domain antibodies with natural or synthetic sdAb frameworks with sdAbs with frameworks modified to enhance basic stability. The following aspects were evaluated: Specification 37 / 68 pages 43 CN 122138977 A (1) Affinity assessment of target molecule interactions (tested by high concentration injection of target molecules).

[0275] (2) Basic stability (tested by reduction in target binding after treatment with 0.1, 0.3, or 0.5 M NaOH in increments of 100%.

[0276] Single-domain antibodies are VHH variants targeting AAV9, green fluorescent protein (GFP), or epidermal growth factor receptor (EGFR), and are based on natural camel VHH frames, synthetic frames, or synthetically stable frames modified to enhance the basic stability of the VHH variants themselves. Natural camel frames correspond to the frame sequences of VHH “EgA1” or “9G8” described in Schmitz et al., respectively. For candidates binding the same target, the CDR is identical.

[0277] Each sdAb was expressed as a fusion protein fused to the C-terminus of the SpA domain Z variant (SEQ ID NO: 159).

[0278] The tested peptide candidates (fusion proteins) are summarized in Table 12.

[0279] Table 12 Materials and Methods For each candidate, 5 µl of BL21(DE3) glycerol stock solution as described in Example 1A was inoculated into 4 ml of LB medium supplemented with 50 µg / ml kanamycin in a 14 ml round-bottom tube and incubated overnight at 37°C and 160 rpm.

[0280] Protein expression medium (TB medium supplemented with 50 µg / ml kanamycin and 2 mM MgCl2) was prepared and added to 100 ml baffled glass shake flasks (20 ml / flask). Approximately 100 µl of the previous overnight culture was inoculated into each flask to obtain an initial OD600 of 0.05. The flasks were incubated in an Infortr HT shaking incubator at 37°C and 140 rpm for approximately 3 hours until the OD600 reached 1.0. Next, 20 µl of IPTG (1 M) was added to a final concentration of 1 mM and the vial was incubated in an Inforrs HT shaking incubator at 27°C and 140 rpm for 18 hours. The culture was then transferred to Falcon tubes (50 mL). To produce crude variant lysates, the Falcon tubes were incubated in a 48°C water bath for 2 hours, followed by centrifugation at 8000 g for 10 minutes to precipitate cell debris and filtration of the supernatant using a 0.22 µm filter.

[0281] The sample was purified using gravity flow in a PD-10 column (Cytiva™) packed with IgG Sepharose 6FF chromatography resin (Cytiva™). The clarified lysates were loaded onto a column equilibrated in 50 mM Tris, 150 mM NaCl, and 0.05% Tween 20 (TST buffer). The column was washed with 10 column volumes (CV) of TST buffer, followed by 2 CV of 5 mM NH4Ac at pH 5. Protein was eluted with 2.5 ml of 0.5 M Hac. Prior to further analysis, the eluted protein buffer was replaced with phosphate-buffered saline (Medicago) at pH 7.4 using a gravity flow column PD-10 (Cytiva).

[0282] The concentration of the purified IgG Sepharose sample was measured using NanoDrop (Thermo Scientific) according to the manufacturer's instructions. After purification, the sample was kept in an Eppendorf tube in a refrigerator until all measurements were performed, and then kept in a freezer until Biacore analysis.

[0283] Biacore analysis instructions for binding affinity to target molecules, pages 38 / 68, CN 122138977 A. To evaluate binding to target molecules (AAV9, GFP, or EGFR), candidate peptides were immobilized on a Biacore™ S-Series CM5 chip (Cytiva) using an amine conjugation kit (Cytiva) and analyzed using a Biacore™ 8K+ instrument (Cytiva). The analytes used were AAV9 (in-house prepared), trastuzumab-GFP (in-house prepared), and EGFR (SinoBiological, 10001-H08H), Fc from mAb (in-house prepared).

[0284] Using standard methods in Biacore software, peptide variants were immobilized by conjugating them in flow cell 2 (FC2) and activating / inactivating them in flow cell 1 (FC1). AAV9 binding candidates were diluted at a concentration of 25 µg / ml in Biacore™ acetate buffer pH 5.0 (Cytiva™), with an average immobilization level of 4319 RU ± SD 640 RU. GFP binding peptide variants were diluted at a concentration of 25 µg / ml in Biacore™ acetate buffer pH 4.5 (Cytiva™), with an average immobilization level of 3803 RU ± SD 302 RU. EGFR binding peptide variants were diluted at a concentration of 25 µg / ml in Biacore™ acetate buffer pH 4.5 (Cytiva™), with an average immobilization level of 3719 RU ± SD 463 RU. Peptides were immobilized in FC2.

[0285] Biacore™ method for AAV9 binding assay: Run buffer: PBS-P+; Flow rate: 5 µl / min; Sample injection: 2400 s / 40 min on both FC1 and FC2; Dissociation time: 2400 s / 40 min; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle), the analyte (AAV9) was injected as follows: run buffer, 1.25 E11 vp / ml, 5 E11 vp / ml, 2 E12 vp / ml.

[0286] Biacore™ method for GFP binding assay: Run buffer: PBS-P+; Flow rate: 10 µl / min; BlockingLiquid injection: 90 s for both FC1 and FC2; Sample injection: 600 s for both FC1 and FC2; Dissociation time: 1800 s; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle), the blocking buffer (Fc) was injected at 2 µM. For each channel (cycle), the analyte (trastuzumab-GFP) was injected as follows: run buffer, 1, 10, 100, 1000 nM.

[0287] Biacore™ method for EGFR binding analysis: Run buffer: PBS-P+; Flow rate: 10 µl / min; Sample injection: 600 s for both FC1 and FC2; Dissociation time: 1800 s; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle), the analyte (EGFR) was injected as follows: run buffer, 0.1–200 nM (in a 1:1 dilution sequence).

[0288] All sensor maps were generated with reference subtraction, and response values ​​were generated as the difference between the baseline before sample injection and the signal immediately preceding the end of injection.

[0289] Biacore analysis of basic stability was performed after binding analysis (see above), with the same CM5 chip containing the immobilized peptide undergoing repeated cycles of binding the corresponding analyte followed by injection of NaOH to assess basic stability (0.1 / 0.3 / 0.5 M).

[0290] Biacore method for AAV9 basic stability (per cycle): Run buffer: PBS-P+; Flow rate: 10 µl / min; Sample injection 1 (AAV9 3E11 vp / ml): 300 s in both flow cells; Dissociation time 1: 30 s; Sample injection 2 (0.5 M NaOH): 600 s in both flow cells; Dissociation time 2: 0 s; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s.

[0291] Biacore method for GFP basic stability (per cycle): Run buffer: PBS-P+; Flow rate: 10 µl / min; Blocking solution injection: (Fc 2 µM from mAb) 90 s in both flow cells; Block dissociation time: 0 s; Sample injection 1 (trastuzumab-GFP 2 µM): 300 s in both flow cells; Dissociation time 1: 30 s; Sample injection 2 (0.3 M NaOH): 600 s in both flow cells; Dissociation time 2: 0 s; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s.

[0292] Biacore method for EGFR basic stability (per cycle): Run buffer: PBS-P+; Flow rate: 10 µl / min; Sample injection 1 (EGFR 0.1 M): 300 s in both flow cells; Dissociation time 1: 30 s; Sample injection 2 (0.1 M NaOH): 600 s in both flow cells; Dissociation time 2: 0 s; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s.

[0293] These cycles were repeated 30 times to track the stability of the target response values.

[0294] All sensor maps were generated with reference subtraction, and the response values ​​were generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0295] The results showed that all fusion proteins exhibited satisfactory binding to their respective targets.

[0296] Tables 13a-c summarize the results of the binding capacity and basic stability measurements.

[0297] Table 13a: AAV9 binding candidates Table 13b: EGFR binding candidates Table 13c: GFP binding candidates Figures 7a-c show the binding capacity obtained for AAV9-binding fusion proteins (Figure 7a), GFP-binding fusion proteins (Figure 7b), and EGFR-binding fusion proteins (Figure 7c), normalized to the first cycle (i.e., including the first cycle), for incremental cycles including NaOH exposure (sample injection 2). As can be seen in these figures, fusion proteins (4B-2, 4B-4, 4B-6) containing VHHs with natural camel VHH frameworks (4B-3, 4B-5) or synthetic but not optimally stable frameworks (4B-1), where VHH affinity ligands are based on stable VHH frameworks, show improved basic stability.

[0298] Example 5: AAV9-binding peptides with different frames and CDR variants This example further investigated two different sets of CDR sequences (both targeting AAV9) in different stable sdAb frames. The candidate peptides tested were expressed as fusion proteins, wherein each sdAb has SEQ ID NO: 160 fused to its C-terminus via a linker and has a C-terminal (His) 6-tag. sdAb sequence variants are provided in Table 14. For all candidates, the specification 40 / 68 pages 46 CN 122138977 A FWR4 is the same (SEQ ID NO: 71).

[0299] The following aspects were evaluated: (1) Affinity assessment of AAV9 interaction (tested by high concentration injection of AAV9). (2) Basic stability (tested by the reduction of AAV9 binding after treatment with 0.5 M NaOH in increments of 100%.

[0300] Table 14Materials and Methods Generation of candidate peptides purified by immobilized metal affinity chromatography (IMAC): For each variant, 5 µl of the glycerol stock solution as described in Example 1B was inoculated into 4 ml of LB medium supplemented with 100 µg / ml carbenicillin in a 14 ml round-bottom tube and grown overnight at 37°C and 200 rpm with stirring. Protein expression medium (TB medium supplemented with 100 µg / ml carbenicillin and 2 mM MgCl2) was prepared and added to 500 ml baffled glass shake flasks (50 ml / flask, 7 flasks in total). Approximately 500 µl of the previous overnight culture was inoculated into each flask to obtain an initial OD600 of 0.05. The flasks were incubated in an Infortr HT shaking incubator at 37°C and 140 rpm for approximately 3.5 hours until the OD600 reached 1.0. Next, 50 µl of IPTG (1 M) was added to a final concentration of 1 mM, and the vial was incubated in an Inforrs HT shaking incubator at 27°C and 140 rpm for 18 hours. The culture was then transferred to Falcon tubes (50 mL). To produce crude variant lysates, the Falcon tubes were incubated in a 48°C water bath for 2 hours, followed by centrifugation at 8000 g for 10 minutes to precipitate cell debris and filtration of the supernatant using a 0.45 µm filter.

[0301] The clarified lysates were loaded onto a HisTrap™ FF 1 mL column (Cytiva™) equilibrated in 50 mM sodium phosphate pH 7.5 and 500 mM NaCl, allowing a residence time of approximately 2 minutes. The column was then washed with 15 column volumes (CV) of 50 mM sodium phosphate pH 7.5 and 500 mM NaCl. Proteins were eluted with 0.5 M imidazole at pH 7.5–8 using a linear gradient of 10 CV (0–100%). Prior to further analysis, the protein buffer eluted (as per instructions page 41 / 68, 47 CN 122138977 A) was exchanged for phosphate-buffered saline (Medicago) at pH 7.4 using a gravity flow column pre-packed with Sephadex™ G-25 resin (Cytiva).

[0302] The concentration of the HisTrap™ FF purified samples was measured using a NanoDrop (Thermo Scientific) according to the manufacturer's instructions. After purification, the samples were kept in Eppendorf tubes in a refrigerator until all measurements were performed, and then kept in a freezer until Biacore analysis. Protein mass was determined by liquid chromatography-mass spectrometry using a BioRessolve column (Waters™) on an ACQUITY Rda detector (Waters™).

[0303] Biacore analysis of AAV9 binding affinity.To evaluate binding to AAV9, the AAV9-binding peptide was immobilized onto a Biacore™ S-Series CM5 chip. AAV9 (2E12 viral particles (vp) / ml) was used as the analyte.

[0304] The materials and equipment used were as follows: Biacore™ S-Series CM5 sensor chip, Biacore™ amine conjugation kit, Biacore™ acetate buffer pH 5.0, Biacore™ 8K+ instrument (all from Cytiva, Sweden); AAV9 (in-house prepared); candidate peptide generation as described above.

[0305] Immobilization was performed using standard methods in the Biacore™ software by conjugating the AAV9-binding peptide variant in flow cell 2 (FC2) and activating / inactivating it in flow cell 1 (FC1). The AAV9-binding peptide variant was diluted at a concentration of 25 µg / ml in acetate buffer pH 5.0. The immobilization level obtained was 3001 + / - 159 RU. In each run, the peptide was immobilized in FC2.

[0306] Run buffer: PBS-P+; flow rate: 5 µl / min; sample injection: 2400 s / 40 min in both flow cells (FC1 and FC2); dissociation time: 2400 s / 40 min; regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle), the analyte (AAV9) was injected as follows: run buffer, AAV9 2E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

[0307] All sensor maps were generated after subtracting the reference, and the response values ​​were generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0308] Basic stability was assessed using Biacore on the Biacore™ chip prepared above. After evaluating AAV9 binding (see above), the sample underwent repeated cycles of AAV9 binding (5E11 vp / ml) followed by NaOH (0.5 M).

[0309] Biacore method for alkaline stability (per cycle): Run buffer: PBS-P+; flow rate: 10 µl / min; Sample injection 1 (AAV9 5E11 vp / ml): 300 s in both flow cells; dissociation time 1: 30 s; Sample injection 2 (0.5 M NaOH): 600 s in both flow cells; dissociation time 2: 0 s; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. This cycle was repeated 40 times to track the stability of the AAV9 response value.

[0310] All sensor plots were generated after subtracting the reference, and response values ​​were generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0311] The results were obtained using the maximum AAV9 concentration (2E12 vp / ml), the response level of the AAV9 interaction of the candidate peptides was normalized to the response of vh118, and is summarized in Table 15 as the cycle number for which at least 50% of the binding capacity was retained for basic stability. Figure 8 provides the results of the basic stability assessment, plotting the binding capacity against the cycle number. The binding response values ​​were normalized against the binding capacity recorded for the second cycle (i.e., excluding the first cycle). In each cycle, 0.5 M NaOH was injected at 10 µl / min for a contact time of 10 minutes.

[0312] Candidates vh374, vh359, and vh428, which have the same group of CDRs but different framework regions, were found to have higher basic stability than vh118.

[0313] Table 15 Specification 42 / 68 pages 48 CN 122138977 A Example 6: Comparative Study of AAV9 Binding and Stability In this example, seven candidate peptides were tested and compared with commercially available affinity ligands (CaptureSelect™ biotinylate-AAV9 conjugate, ThermoFischer Scientific). The candidate peptides were expressed as fusion proteins, wherein each sdAb has SEQ ID NO: 160 fused to its C-terminus via a peptide linker (AA), and all constructs have a C-terminal tag (HHHHHHC). sdAb sequence variants are provided in Table 16. The FWR sequence is identical for all candidates.

[0314] Table 16 Materials and Methods As described in Example 5, IMAC-purified candidate peptides were produced.

[0315] Biotinylation of the purified candidate peptide was performed using EZ-Link™ maleimide-PEG2-biotin, No-Weigh™ form (ThermoFisher Scientific), with a 2X molar excess of biotin at the C-terminal cysteine ​​residue.

[0316] SEC purification of the biotinylated candidate peptide was further performed by size exclusion chromatography (SEC) to separate excess biotin, non-biotinylated protein dimers, and other impurities. SEC was performed using a Superdex™ 75 Increase 10 / 300 GL column (Cytiva) at a flow rate of 0.75 mL / min in phosphate-buffered saline (Medicago) pH 7.4. The target protein was eluted as a sharp, symmetrical peak. The purest fractions (e.g., analyzed by SDS-PAGE) were combined and used for further analysis.

[0317] Biacore analysis of AAV9 binding affinity: To evaluate binding to AAV9, an AAV9 binding peptide candidate and a reference CaptureSelect™ biotinylate-AAV9 conjugate (Thermo Scientific™) were immobilized onto a Biacore™ S-Series SA chip. AAV9 (2E12 viral particles (vp) / ml) was used as the analyte.

[0318] The materials and equipment used were as follows: Biacore™ S-Series SA sensor chip, Biacore™ 8K+ instrument (all from Cytiva, Sweden); AAV9 (in-house prepared); candidate peptide as described above; CaptureSelect™ biotinylate-AAV9 conjugate (Thermo Scientific™).

[0319] Immobilization was performed using the standard method in Biacore™ software by conjugating a biotinylated AAV9-binding peptide variant and the CaptureSelect™ biotinylate-AAV9 conjugate (Thermo Scientific™) in flow cell 2 (FC2) and activating / inactivating in flow cell 1 (FC1). The biotinylated AAV9-binding peptide variant was diluted in PBS at a concentration of 100 µg / ml, and the CaptureSelect™ biotinylate-AAV9 conjugate (Thermo Scientific™) was diluted in PBS at a concentration of 10 µg / ml.

[0320] Biacore™ method for binding analysis: Run buffer: PBS-P+; Flow rate: 5 µl / min; Sample injection: 2400 s / 40 min in both flow cells (FC1 and FC2); Dissociation time: 2400 s / 40 min; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle), the analyte (AAV9) was injected as follows: run buffer, 2E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

[0321] All sensor maps were generated after subtracting the reference, and response values ​​were generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0322] Biacore analysis of alkaline stability was performed on the same Biacore™ chip used for the binding analysis described above. After evaluating AAV9 binding (see above), the sample underwent 88 repeated cycles of exposure to NaOH (0.3 M). Each second cycle included binding with AAV9 (5E11 vp / ml), otherwise run buffer PBS-P+ was used for sample injection to reduce analyte consumption.

[0323] Biacore method for alkaline stability (per cycle): Run buffer: PBS-P+; flow rate: 10 µl / min; Sample injection 1 (AAV9 5E11 vp / ml or run buffer PBS-P+, every second cycle to reduce analyte consumption): 300 s on both flow cells; dissociation time 1: 30 s; Sample injection 2 (0.3 M NaOH): 600 s on both flow cells; dissociation time 2: 0 s; regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. This cycle was repeated 88 times to track the stability of the AAV9 response value.

[0324] All sensor plots were generated with reference subtraction, and the response value was generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0325] Results Table 17 shows the fixed levels obtained for the test peptides, as well as the AAV9 binding capacity and NaOH stability using the maximum AAV9 concentration. The candidate peptides are expressed as a fusion with SEQ ID NO:160, and therefore have a higher molecular weight than the sdAb itself. The relative fixation level is the fixation level divided by the molecular weight.

[0326] Table 17 Specification 44 / 68 pages 50 CN 122138977 A *Prior art has found that the AAV9 binding peptides of the present invention tested have significantly higher AAV9 binding capacity despite being fixed at a lower molar level than the prior art reference CaptureSelect™ biotin-AAV9 conjugate. In addition, the basic stability of the candidates of the present invention is very high. In fact, for one candidate vh377, the AAV9 response never dropped below 50% during 88 cycles.

[0327] The results of the basic stability assessment are also shown in Figure 9. During the basic stability analysis, the Biacore run stopped between cycles 28 and 30, so the data point for cycle 30 was not measured. The run continued, and the next binding data was measured in cycle 32.

[0328] Example 7: Functional Evaluation – Affinity Chromatography The AAV9-binding sdAb variant (SEQ ID NO: 80) generated as a fusion protein (SEQ ID NO: 161) was immobilized onto a support to form an affinity separation matrix and tested in a chromatographic experiment.

[0329] Materials and Methods 5 µl of glycerol stock solution as described in Example 1B was inoculated into 4 ml of LB medium supplemented with 100 µg / ml carbenicillin in a 14 ml round-bottom tube and incubated overnight at 37°C and 160 rpm.

[0330] Protein expression medium (TB medium supplemented with 100 µg / ml carbenicillin and 2 mM MgCl2) was prepared and added to a pre-filled 500 ml baffled glass shake flask (50(ml / vial). Each vise was inoculated with approximately 500 µl of the previous overnight culture to obtain an initial OD600 of 0.05. The vises were incubated in an Inforrs HT shaking incubator at 37°C and 140 rpm for approximately 3.5 h until the OD600 reached 1.0. Then, 50 µl of IPTG (1 M) was added to a final concentration of 1 mM, and the vises were incubated in an Inforrs HT shaking incubator at 27°C and 140 rpm for 18 h. The culture was then transferred to Falcon tubes (50 ml). To generate coarse variant lysates, the Falcon tubes were incubated in a 48°C water bath for 2 h, followed by centrifugation at 8000 g for 10 min to precipitate cell debris and filtration of the supernatant using a 0.22 µm filter.

[0331] Clarified lysates of each sample were loaded onto a 1 ml HiTrap MabSelect™ PrismA column (Cytiva™) equilibrated in phosphate-buffered saline (PBS) pH 7.4, allowing a residence time of approximately 2.4 minutes. The column was then washed with 5 column volumes (CV) of PBS pH 7.4, followed by 5 CV of 50 mM sodium acetate pH 6. Proteins were eluted using a stepwise gradient of 50 mM sodium acetate pH 3.5 at 5 CV.

[0332] The concentration of purified samples was measured using NanoDrop (ThermoFisher Scientific) according to the manufacturer's instructions. After purification, samples were kept in Eppendorf tubes in a refrigerator until all measurements were performed, and then kept in a freezer until Biacore analysis. Protein mass was determined by liquid chromatography-mass spectrometry using a BioRessolve column (Waters™) on an ACQUITY Rda detector (Waters™).

[0333] The fusion protein was immobilized on epoxy-activated chromatographic resin (highly cross-linked agarose beads) using cysteine ​​coupling. 0.2 ml of resin was packed into a Tricorn 5 / 20 column (Cytiva). Using an ÄKTA pure 25 system (Cytiva), 50 ml of tangentially flow filtered AAV9 material (in-house prepared) was loaded onto the column with a residence time of 4 min and eluted with 100 mM citrate buffer at pH 2.5.

[0334] The collected eluent fractions were analyzed on a Coomassie SDS-PAGE gel. Low pH eluted samples were neutralized with a 1:1 volume of Tris buffer (400 mM). 15 µl of sample and 5 µl of sample buffer + DTT were heated to 70 °C for 10 min. Load 5 μl of sample and run the gel at 200V for 35 minutes, then soak in Coomassie solution overnight. Then rinse with water.The gel was destained until satisfactory staining was achieved. The gel was photographed using an ImageQuant 800.

[0335] To evaluate the basic stability of the immobilized fusion protein, the chromatographic resin was cleaned in situ (CIP) with 10 mM glycine-HCl pH 1.5, followed by a basic stability study involving exposure to 0.5 M NaOH. The entire stability study consisted of 40 cycles, each of which involved contacting the chromatographic material with 0.5 M NaOH for 30 minutes. Every 10th cycle, a solution containing pure AAV9 particles was added, with a residence time of 15 seconds. PBS buffer was used for all other cycles.

[0336] Results Figure 10a shows the chromatogram obtained for purifying the AAV9 TFF material using candidate vh118 as an affinity ligand. Elution peaks are indicated by rectangles. Figure 10b shows only the regions of the elution peaks. Figure 11 shows a photograph of the gel. VP1, VP2, and VP3 represent AAV9 virosomal proteins 1, 2, and 3, respectively.

[0337] The results showed that the candidate could successfully purify AAV9 virus particles, with an estimated dynamic binding capacity (QB10%) of approximately 3E+14 virus particles / ml resin, and an estimated recovery rate of 70-80% for loaded virus particles.

[0338] The results of the basic stability assessment are shown in Figure 12, which plots the binding capacity normalized to the binding capacity before the first NaOH exposure. After 40 cycles of exposure to 0.5 M NaOH, corresponding to a cumulative exposure time of 20 hours, the target binding capacity decreased by less than 20%.

[0339] Example 8: Basic stability of natural and differently modified sdAb frameworks A: sdAbs for AAV This example investigated the basic stability of AAV9-binding VH with multiple variations in the framework region, wherein the mutations were selected from less preferred modifications to show that the combination of multiple less preferred modifications also resulted in increased basic stability compared to the unmodified (natural) framework. In this example, the basic stability of the natural framework, the framework with preferred modifications, and the framework with proposed but not preferred modifications were compared.

[0340] For the modified frame, amino acid substitutions were performed at positions 19, 23, 24, 40, 43, 44, 45, 76, 79, and 89 (Kabat numbers), one selected from a combination of preferred substitutions, and one selected from a combination of proposed but not preferred substitutions. Thus, three candidate VH sequences were tested: one with an unmodified frame (vh342), one with a frame having amino acid substitutions preferred compared to the unmodified frame (vh118), and one with a combination of possible but less preferred mutations (vh540). The amino acid substitutions (Kabat numbers) for each candidate VHH and their corresponding full-length sdAb sequences are illustrated in Table 18. For all candidates, the CDR sequence is identical (as in vh118).

[0341] The VH sequence was expressed, wherein SEQ ID NO:160 was used as a C-terminal fusion via a peptide linker. The total molecular weight was 21 kDa. Specification 46 / 68 pages 52 CN 122138977 A

[0342] Table 18 The following aspects were evaluated: (1) Affinity assessment of AAV9 interaction, which was tested by high-concentration injection of AAV9. (2) Basic stability, which was tested by the reduction of AAV9 binding after treatment with 0.3 M NaOH in increments of 100%.

[0343] B: sdAb against GFP As in Example A above, this example used a different target than AAV9 to study the basic stability of the frame, wherein GFP-binding VH was generated, one with multiple variations in the frame region, wherein the mutation was selected from less preferred mutations, one with a native frame, and one with a preferred modified frame.

[0344] Three candidate VH sequences were tested: one with an unmodified framework (vh 60), one with a framework having amino acid substitutions preferred compared to the unmodified framework (vh113), and one with a combination of less preferred mutations (vh540). The amino acid substitutions (Kabat numbers) of each candidate VHH and their corresponding full-length sdAb sequences are illustrated in Table 19. For all candidates, the CDR sequence was identical.

[0345] The VH sequence, wherein SEQ ID NO:160 was expressed as a C-terminal fusion via a peptide linker. The total molecular weight was 21 kDa.

[0346] Table 19 evaluates the following aspects: (1) Affinity assessment of AAV9 interaction, which was tested by high-concentration AAV9 injection. (2) Basic stability, which was tested by the reduction in AAV9 binding after treatment with 0.1 M NaOH in increments of 100%.

[0347] C: sdAb targeting EGFR As described in Example B above, this example uses a different target than AAV9 to study the basic stability of the framework, wherein EGFR-binding VHs are generated, one with multiple variations in the framework region, wherein the mutations are selected from less preferred mutations, one with the native framework, and one with the preferred modified framework.

[0348] Three candidate VH sequences were tested, one with an unmodified framework (vh166), one with a framework with amino acid substitutions preferred compared to the unmodified framework (vh167), and one combination with less preferred mutations (vh541). The amino acid substitutions (Kabat numbers) of each candidate VHH and their corresponding full-length sdAb sequences are described in Table 20. For all candidates, the CDR sequence is the same.

[0349] The VH sequence, wherein SEQ ID NO:160 is expressed as a C-terminal fusion via a peptide linker. The total molecular weight is 21 kDa.

[0350] Table 20 Specification page 47 / 68 53 CN122138977 A The following aspects were evaluated: (1) Affinity assessment of AAV9 interaction, which was tested by injecting high concentrations of AAV9. (2) Basic stability, which was tested by the reduction of AAV9 binding after treatment with 0.1 M NaOH in increments of 10 ... Candidates including several less preferred point mutations showed basic stability at 0.3 M NaOH for 12 cycles. Therefore, even though less stable than vh118, vh540 showed good basic stability, especially compared to the natural framework used in vh342 (which showed no stability at all). This suggests that combinations of less preferred mutations will also result in stable frameworks compared to the natural framework.

[0353] For GFP-binding VHH candidates, all three showed slightly lower target binding. vh60 with the natural framework showed stability at 0.1 M NaOH for 2 cycles, while vh539 with the non-preferred substitution framework showed basic stability at 0.1 M NaOH for 29 cycles (compared to over 30 cycles for vh113), a significant increase. Therefore, it can be concluded that the framework also greatly stabilizes GFP-binding CDR.

[0354] For EGFR, the modified framework showed good target binding, while the natural framework showed slightly lower binding. vh166 with the native framework showed no stability at 0.1M NaOH, while vh541 with the framework containing the non-preferred substitution showed basic stability at 0.1M NaOH for 3 cycles compared to vh167 with the preferred mutation for 12 cycles. Even though this is lower stability compared to other targets, it is still a huge increase compared to the native framework. However, this also shows that if CDR will affect stability, and that a more stable CDR when the framework is modified will also produce a more stable sdAb.

[0355] The results are shown in Figure 13, where Figure 13A shows the results for GFP, Figure 13B shows the results for EGFR, and Figure 13C shows the results for AAV9. Considering the remaining binding capacity of less than 8% for GFP as a target, it can be found thatFor vh60, it was reached after one cycle; for vh539, after 25 cycles; and for vh113, after 35 cycles. For EGFR as the target, vh166 was reached after only one cycle; vh541 after 3 cycles; and vh16 after 12 cycles. Finally, for AAV9 as the target, vh342 was reached after one cycle; vh540 after 13 cycles; and vh118 after more than 40 cycles.

[0356] Example 9: AAV9 binding peptides in different frames and two sets of CDRs This example further investigated five additional different sdAb frames and the same sdAb CDR sequences. One frame was also tested with two different sets of CDR sequences. All sdAbs had the same CDR1 and CDR2 sequences, while vh516 had a different CDR3 than vh511–vh515. Vh511-515 has the same CDR as vh324, but with a different frame. Vh324 has frame variant 1, while vh511-515 has five new frame arrangements, as shown in the table below. Specification 48 / 68 pages 54 CN 122138977 A

[0357] The candidate peptides tested were expressed as fusion proteins, wherein each sdAb has SEQ ID NO: 160 fused to its C-terminus via a linker and has a C-terminal (His) 6-tag. sdAb sequence variants are provided in Tables 21 and 22.

[0358] The following aspects were evaluated: (1) Affinity assessment of AAV9 interaction (tested by high concentration injection of AAV9). (2) Basic stability (tested by the reduction of AAV9 binding after treatment with 0.3 M NaOH in increments of 100%.

[0359] Table 21 Table 22 Materials and Methods Generation of candidate peptides purified by immobilized metal affinity chromatography (IMAC) For each variant, 5 µl of the glycerol stock solution as described in Example 1 was inoculated into 4 ml of LB medium containing 100 µg / ml carbenicillin in a 14 ml round-bottom tube and grown overnight at 37°C and 200 rpm with stirring. Protein expression medium (TB medium supplemented with 100 µg / ml carbenicillin and 2 mM MgCl2) was prepared and added to 500 ml baffled glass shake flasks (50 ml / flask, 7 flasks in total). Approximately 500 µl of the previous overnight culture was inoculated into each flask to obtain an initial OD600 of 0.05. The flasks were incubated in an Inforrs HT shaking incubator at 37°C and 140 rpm for approximately 3.5 hours until the OD600 reached 1.0. Then, add 50 µl of IPTG (1 M) to a final concentration of 1.mM, and the vials were incubated in an Inforrs HT shaking incubator at 27°C and 140 rpm for 18 hours, after which the culture was transferred to Falcon tubes (50 mL). To produce crude variant lysates, the Falcon tubes were incubated in a 48°C water bath for 2 hours, followed by centrifugation at 8000 g for 10 minutes to precipitate cell debris and filtration of the supernatant through a 0.45 µm filter.

[0360] The clarified lysates were loaded onto a HisTrap™ FF 1 mL column (Cytiva™) equilibrated in 50 mM sodium phosphate pH 7.5 and 500 mM NaCl, allowing a residence time of approximately 2 minutes. The column was then washed with 15 column volumes (CV) of 50 mM sodium phosphate pH 7.5 and 500 mM NaCl. Proteins were eluted with 0.5 M imidazole pH 7.5–8 via a linear gradient of 10 CV (0–100%). Prior to further analysis, the eluted protein buffer was exchanged for phosphate-buffered saline (Medicago) pH 7.4 using a gravity flow column pre-filled with Sephadex™ G-25 resin (Cytiva).

[0361] The concentration of HisTrap™ FF purified samples was measured using NanoDrop (Thermo Scientific) according to the manufacturer's instructions. After purification, the samples were kept in Eppendorf tubes in a refrigerator until all measurements were performed, and then kept in a freezer until Biacore analysis. Protein mass was determined by liquid chromatography-mass spectrometry using a BioRessolve column (Waters™) on an ACQUITY Rda detector (Waters™).

[0362] Biotinylation of purified candidate peptides The purified candidate peptides were biotinylated at the C-terminal cysteine ​​using EZ-Link™ maleimide-PEG2-biotin, No-Weigh™ form (ThermoFisher Scientific) with a 2X molar excess of biotin.

[0363] The biotinylated candidate peptide was further purified by size exclusion chromatography (SEC) to separate excess biotin, un-biotinylated protein dimers, and other impurities. SEC was performed in phosphate-buffered saline (Medicago) at pH 7.4 using a Superdex™ 75 Increase 10 / 300 GL column (Cytiva) at a flow rate of 0.75 mL / min. The target protein eluted as a sharp, symmetrical peak. The purest fractions (e.g., analyzed by SDS-PAGE) were combined and used for further analysis.

[0364] Biacore analysis of the binding affinity for AAV9.To evaluate binding to AAV9, AAV9-binding peptide candidates were immobilized on a Biacore™ S-Series SA chip. AAV9 (2E12 viral particles (vp) / ml) was used as the analyte.

[0365] The materials and equipment used were as follows: Biacore™ S-Series SA sensor chip, Biacore™ 8K+ instrument (all from Cytiva, Sweden); AAV9 (prepared internally); candidate peptides as described above; CaptureSelect™ biotinylated anti-AAV9 conjugate (Thermo Scientific™).

[0366] Immobilization was performed using standard methods in Biacore™ software by conjugating the biotinylated AAV9-binding peptide variant in flow cell 2 (FC2) and activating / inactivating it in flow cell 1 (FC1). The biotinylated AAV9-binding peptide variant was diluted in PBS at a concentration of 100 µg / ml.

[0367] Biacore™ method for binding analysis: Run buffer: PBS-P+; Flow rate: 5 µl / min; Sample injection: 2400 s / 40 min in both flow cells (FC1 and FC2); Dissociation time: 2400 s / 40 min; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle), the analyte (AAV9) was injected as follows: run buffer, 2E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

[0368] All sensor maps were generated with reference subtraction, and response values ​​were generated as the difference between the baseline before injection and the signal immediately following the end of injection on pages 50 / 68 of the specification, 56 CN 122138977 A.

[0369] The Biacore analysis of alkaline stability was performed on the same Biacore™ chip used for the binding analysis described above. After evaluating AAV9 binding (see above), the sample underwent 88 repeated cycles of exposure to NaOH (0.3 M). Each second cycle included binding with AAV9 (5E11 vp / ml), otherwise run buffer PBS-P+ was used for sample injection to reduce analyte consumption.

[0370] Biacore method for alkaline stability (per cycle): Run buffer: PBS-P+; Flow rate: 10 µl / min; Sample injection 1 (AAV9 5E11 vp / ml or run buffer, PBS-P+ for each second cycle to reduce analyte consumption): 300 s in both flow cells; Dissociation time 1: 30 s; Sample injection 2 (0.3 M NaOH): 600 s in both flow cells; Dissociation time 2: 0 ss; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. This cycle was repeated 88 times to track the stability of the AAV9 response values.

[0371] All sensor maps were generated with reference subtracted, and response values ​​were generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0372] The results were obtained using the maximum AAV9 concentration (2E12 vp / ml), and the response level of the AAV9 interaction of the candidate peptides was normalized to the response of vh377 and summarized in Table 23 as the number of cycles in which at least 50% of the binding capacity was retained for basic stability. The binding response values ​​were normalized for the binding capacity recorded for the second cycle (i.e., excluding the first cycle). In each cycle, 0.3 M NaOH was injected at 10 µl / min for a contact time of 10 min.

[0373] Candidates vh511-515, which have the same set of CDRs but different framework regions, were found to have higher basic stability than vh377. Comparison of vh516 and vh515, which have the same frame region, concludes that different CDRs contribute equally positively to basic stability.

[0374] Table 23 Example 10: First position of frame replaced by E In this experiment, it was investigated whether replacing Q with E at the first position of frame (FW1) would affect the stability of the peptide.

[0375] Materials and Methods Four peptides, vh118 (SEQ ID NO: 80) and vh498 (SEQ ID NO: 195), were tested, with vh498 differing from vh118 in that the first position was replaced by E. Similarly, vh324 (SEQ ID NO: 187) and vh459 (SEQ ID NO: 219) were tested, with vh458 having the first position replaced by E. Constructs were generated and tested, purified on PrismA, and coupled to CM5 chips.

[0376] Generation of candidate peptides purified by MabSelect PrismA™ For each candidate, 5 µl of the glycerol stock solution as described in Example 1 above was inoculated into 4 ml of LB medium supplemented with 100 µg / ml carbenicillin in a 14 ml round-bottom tube and grown overnight at 37°C and 200 rpm with stirring. Protein expression medium (TB medium supplemented with 100 µg / ml carbenicillin and 2 mM MgCl2) was prepared and added to 500 ml baffled glass shake flasks (50 ml / flask, 43 flasks in total). Approximately 500 µl of the previous overnight culture was inoculated into each flask to obtain an initial OD600 of 0.05. The flasks were placed in an Inforrs HT shaking incubator at 37°C and 140°C.Incubate for approximately 3.5 hours with shaking at rpm until the OD600 reaches 1.0. Then, add 50 µl of IPTG (1 M) to a final concentration of 1 mM and incubate the vial in an Inforrs HT shaking incubator at 27°C and 140 rpm for 18 hours. Afterward, transfer the culture to Falcon tubes (50 ml). To generate crude variant lysates, incubate the Falcon tubes in a 48°C water bath for 2 hours, followed by centrifugation at 8000 g for 10 minutes to precipitate cell debris and filtering the supernatant using a 0.45 µm filter.

[0377] Samples were purified using a HiTrap MabSelect PrismA™ column (Cytiva). Clarified lysates of each sample were loaded onto a 1 mL column equilibrated in phosphate-buffered saline (PBS) at pH 7.4, allowing a residence time of approximately 2.4 minutes. The column was then washed with 5 column volumes (CV) of PBS at pH 7.4, followed by 5 CV of 50 mM sodium acetate at pH 6. Protein was eluted with a stepwise gradient of 50 mM sodium acetate at pH 3.5 for 5 CV.

[0378] The concentration of the purified sample was measured using NanoDrop (Thermo Scientific) according to the manufacturer's instructions. After purification, the sample was kept in Eppendorf tubes in a refrigerator until all measurements were performed, and then kept in a freezer until Biacore analysis. Protein mass was determined by liquid chromatography-mass spectrometry using a BioRessolve column (Waters™) on an ACQUITY Rda detector (Waters™).

[0379] Biacore analysis of binding affinity for AAV9: To assess binding to AAV9, candidate peptides were immobilized on a Biacore™ S-Series CM5 chip. AAV9 (2E12 viral particles (vp) / ml) was used as the analyte.

[0380] The materials and equipment used were as follows: Biacore™ S-series CM5 sensor chip, Biacore™ amine conjugation kit, Biacore™ acetate buffer pH 5.0, Biacore™ 8K+ instrument (all from Cytiva, Sweden); AAV9 (prepared internally); candidate peptides as described above.

[0381] The AAV9-binding peptide variants were immobilized using standard methods in the Biacore™ software by conjugating them in flow cell 2 (FC2) and activating / inactivating them in flow cell 1 (FC1). The AAV9-binding peptide variants were diluted at a concentration of 25 µg / ml in Biacore™ acetate buffer pH 5.0. The immobilization level obtained was 3484 + / - 197 RU.

[0382] In each run, the peptide was immobilized in FC2. Multiple sensor chips were used until all candidates were tested.

[0383] Run buffer: PBS-P+; flow rate: 5 µl / min; sample injection: 2400 s / 40 min in both flow cells (FC1 and FC2); dissociation time: 2400 s / 40 min; regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle), the analyte (AAV9) was injected as follows: run buffer, 2E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

[0384] All sensor maps were generated after subtracting the reference, and the response values ​​were generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0385] Biacore analysis of basic stability was performed by immobilizing the candidate peptides on the Biacore™ chip to assess basic stability. After evaluating AAV9 binding (see above), the same chip underwent repeated cycles of AAV9 binding (5E11 vp / ml) followed by NaOH (0.5 M).

[0386] Biacore method for alkaline stability (per cycle): Run buffer: PBS-P+; flow rate: 10 µl / min; Sample injection 1 (AAV9 5E11 vp / ml): 300 s in both flow cells; dissociation time 1: 30 s; Sample injection 2 (0.5 M NaOH): 600 s in both flow cells; dissociation time 2: 0 s; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. This cycle was repeated 40 times to track the stability of the AAV9 response value.

[0387] All sensor maps were generated after subtracting the reference, and response values ​​were generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0388] The results showed that even for peptides with E as the first amino acid in the framework, the stability of the peptides was slightly lower, but it was confirmed that using E at the first position of FW1 also produced highly stable peptides, as outlined in Table 24 below. Vh118 with E replaced at position 1 is referred to as vh118 (Q1E). vh324 was named accordingly.

[0389] Table 24 Example 11: Basic stability measured relative to ZVH3 for AAV9, EGFR and GFP by evaluating the target binding to AAV9 (5e11 vp / ml) or the binding to the ligand backbone of the ZVH3 hexamer (MabSelectTMVH3 ligand).The basic stability of the peptide was measured at 50 nM. Alternating with AAV9 or ZVH3 binding, the peptide was exposed to NaOH (0.5 M) for 10 minutes for a total of 40 cycles. This was to demonstrate that both target binding to the CDR and the framework increased the basic stability of the peptide. Data obtained from this embodiment will be compared with results for AAV9-binding sdAb in previous embodiments.

[0390] Materials and methods as described in WO 20 231 7 4 90 0 A1, provide a ZVH3 hexamer having the sequence according to SEQ ID NO: 177 of WO2023174900A1, which is a hexamer of a monomer as defined in SEQ ID NO: 15 of WO2023174900A1, the patent publication of which is incorporated herein by reference.

[0391] The materials and equipment used for the Biacore analysis of alkaline stability were as follows: Biacore™ S-Series CM5 sensor chip, Biacore™ amine conjugation kit, Biacore™ acetate buffer pH 5.0, Biacore™ 8K+ instrument (all from Cytiva, Sweden); 0.5 M NaOH; ZVH3-ligand; candidate peptides as described above.

[0392] Alkaline stability was evaluated on the Biacore™ chip prepared above. The peptide underwent 40 repeated cycles of binding to AAV9 (5e11vp / ml) or ZVH3 hexamer (50 nM) followed by 10 minutes of exposure to NaOH (0.5 M). The NaOH stability of the peptide was evaluated by comparing the number of cycles when the peptide had lost 50% of its initial binding to AAV9 or ZVH3 hexamer.

[0393] All sensor maps were generated after subtracting the reference, and the response values ​​were generated as the difference between the baseline before injection and the signal immediately before the end of injection.

[0394] The results of the assessment of the basic stability of the peptides are summarized in Table 25 below. For AAV9-binding peptides vh260-268, peptide CDR AAV9 binding was compared with AAV9 ZVH3 backbone binding. It was found that basic stability also increased for this target. The unstable peptides vh267 and vh268 were unstable for both bindings.

[0395] Table 25 Specification 53 / 68 pages 59 CN 122138977 A For GFP, EGFR and AAV9-binding molecules, basic stability was further evaluated, with target binding compared with ZVH3 binding. The same construct as in Example 8 above was used. For GFP and EGFR, the NaOH concentration was 0.1M, and for AAV9 it was 0.5M. The results are provided in Table 26 below.

[0396] Table 26 Candidate SEQ ID NO: TargetNaOH stability (exposed cycles retaining at least 50% of target binding) NaOH stability (exposed cycles retaining at least 50% of ZVH3 binding) vh60 155 GFP 2 1 vh113 156 GFP 16 17 vh539 217 GFP 1 NA vh166 157 EGFR 1 1 vh167 158 EGFR 6 9 vh541 218 EGFR 1 NA vh342 152 AAV9 2 3 vh118 80 AAV9 22 26 vh540 216 AAV9 6 NA Example 12: Basic stability of the scFv backbone The scFv, a fusion of two variable domains (VH and VL) with a flexible linker, is a suitable backbone for target interaction, but lacks some biological process-related properties. Therefore, the possibility of stabilizing the scFv backbone was assessed using the framework mutations of this disclosure. Publicly available scFv fragments targeting human serum albumin (HSA) were used as starting points.

[0397] Materials and Methods Three constructs were designed for initial evaluation of expression, coupling, binding, and basic stability: i) a first construct using native HSA scFv; ii) a second construct modified to a fusion protein of native HSA scFv (scFvHSA) and the polypeptide moiety according to SEQ ID NO: 160 (scFvHSA-Z); and iii) a third construct modified to a stable HSA scFv (vh118 framework) and the polypeptide moiety according to SEQ ID NO: 160 (scFvHSAStab1-Z), i.e., the VH portion of the scFv backbone was stabilized by introducing mutations to preferred positions. Natural HSA scFv is as defined in Adams R. Et al (MABS VOL. 8, NO. 7, 1336–1346 (2016)), and the modified construct is designed by fusing with a polypeptide moiety according to SEQ ID NO: 160, and in one construct, the framework is modified according to the framework of the vh118 variant described herein.

[0398] As described in the previous examples, the construct was expressed in E. coli and purified on IMAC or PrismA.

[0399] The first Biacore experiment was performed on an SA-chip with biotinylation affinity to compare scFvHSA with and without fusion with the polypeptide moiety, wherein the binding experiment used HAS concentrations of 0, 10, 50, 100, 500 and 1000 nM, and the alkaline stability assay used 1000 nM HAS and CIP conditions for 10 min and 0.1 M NaOH.The improved basic stability of the peptide moiety fused to SEQ ID NO: 160 compared to the native form, and the stability can be further improved by framework mutation, as shown in Figure 14. Specification 54 / 68 pages 60 CN 122138977 A

[0400] Similar experiments were performed on an NHS-conjugated CM5-chip. Biacore evaluation of the binding of purified affinity ligands (i) and (ii) was performed on the CM5-chip using HAS concentrations of 0, 50, 100, 500, and 1000 nM. Biacore evaluation of basic stability was performed using 1000 nM HAS and CIP conditions for 10 min and 0.1 M NaOH. This example also shows improved basic stability by fusion of the peptide moiety according to SEQ ID NO: 160 compared to the native form, and further improvement by framework mutation (data not shown).

[0401] Results showed that for the stable third construct scFvHSAStab1-Z, expression and purification yields increased by approximately 2-fold compared to other constructs. Experiments on the SA-chip showed improved stability for ScFvHSA-Z, and even further improved stability for scFvHSAStab1-Z, indicating that a stable VHH framework further improves stability. The basic stability results from the SA-chip experiments are shown in Figure 14, which presents a normalized plot of the basic stability experiments. For the second Biacore experiment, a CM5-chip with NHS coupling was used, and it was shown that the construct with the peptide motif according to SEQ ID NO: 160 had good coupling efficiency compared to natural scFvHSA alone (data not shown).

[0402] List of Specific Embodiments 1. A polypeptide comprising a heavy chain variable domain (VH) variant, such as a single-domain antibody (sdAb) variant, said variant comprising a plurality of antigen-binding regions and a frame comprising frame region 1 (FWR1), frame region 2 (FWR2) and frame region 3 (FWR3), said VH variant having at least eight, at least nine, or, for example, all of the following amino acid sequences satisfying criterions i)-x): i) The residue at Kabat position 19 is R, S, K or T; ii) The residue at Kabat position 23 is T, V, A or S, preferably T; iii) The residue at Kabat position 24 is I, V or S; iv) The residue at Kabat position 40 is selected from R, I, T or K, preferably R; v) The residue at Kabat position 43 is R, K, Q, E or G, preferably R or K; vi) The residue at Kabat position 44 is E, Q, A, D or G; vii) The residue at position 45 of Kabat is R, I, or L, preferably R; viii) The residue at position 76 of Kabat is N or Q; ix)The residue at Kabat position 79 is Y, W, or F; and the residue at Kabat position 89 is V or I.

[0403] 2. The polypeptide according to item 1, wherein FWR1, FWR2, and FWR3 each have at least 80% identity with the amino acid sequence of the corresponding frame region of SEQ ID NO:80, wherein for the purpose of determining sequence identity, Kabat positions 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b, and 89, and optionally Kabat position 6, are omitted.

[0404] 3. A polypeptide comprising a VH variant, such as a single-domain antibody (sdAb) variant, said variant comprising a plurality of antigen-binding regions and a framework comprising a frame region 1 (FWR1), a frame region 2 (FWR2), and a frame region 3 (FWR3), wherein said VH variant has an amino acid sequence satisfying at least five of the following criteria i)-vi): i) the residue at Kabat position 24 is I, V, or S; ii) the residue at Kabat position 40 is selected from R, I, K, or T, preferably R; iii) the residue at Kabat position 45 is R, I, or L, preferably R; iv) the residue at Kabat position 76 is N or Q; v) the residue at Kabat position 79 is Y, W, or F; vi) the residue at Kabat position 89 is V or I; and wherein FWR1, FWR2, and FWR3 are each associated with SEQ ID NO: The amino acid sequence of the corresponding frame region of 80 has at least 80% identity, wherein, for the purpose of determining sequence identity, Kabat positions 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b and 89 and optionally position 6 are omitted.

[0405] 4. The polypeptide according to item 1 or 2, which also satisfies at least five of the criteria i)-vi) according to item 3.

[0406] 5. The polypeptide according to item 4, wherein all criteria i)-x) of item 1 and all criteria i)-vi) of item 3 are satisfied.

[0407] 6. The polypeptide according to any one of the preceding items, wherein the frame regions FWR1-3 as a whole have at least 90% sequence identity with the frame region of SEQ ID NO: 80, wherein Kabat positions 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b and 89 and optionally position 6 are omitted for the purpose of determining sequence identity.

[0408] 7. The polypeptide according to any one of items 1-6, wherein Kabat position 6 is omitted for the purpose of determining sequence identity.

[0409] 8.The polypeptide according to any one of items 1-6, wherein Kabat position 6 is not omitted for the purpose of determining sequence identity.

[0410] 9. The polypeptide according to any one of the preceding items, wherein it is an antigen-binding polypeptide.

[0411] 10. The polypeptide according to any one of the preceding items, wherein it is a single-chain polypeptide.

[0412] 11. The polypeptide according to any one of the preceding items, wherein the polypeptide lacks the antibody heavy chain constant domain.

[0413] 12. The polypeptide according to any one of the preceding items, wherein the VH variant is a variable domain of the heavy chain of a heavy chain antibody (VHH) variant.

[0414] 13. The polypeptide according to any one of the preceding items, wherein the amino acid sequence of the VH variant further comprises any one, for example, more than one, for example, all of the following: the residue at Kabat position 6 is Q or E; the residue at Kabat position 14 is A, S or T; the residue at Kabat position 47 is F or L, preferably F; the residue at Kabat position 60 is A, T, N, Q or S, preferably A or S; the residue at Kabat position 82b is N, S, T or Q.

[0415] 14. The polypeptide according to any one of the preceding items, wherein the residue at Kabat position 47 is F or L.

[0416] 15. The polypeptide according to any one of the preceding items, wherein the residue at Kabat position 47 is not G, S, T or Y.

[0417] 16. The polypeptide according to any one of the preceding items, wherein the residues at Kabat positions 24 and 40 are not both A.

[0418] 17. The polypeptide according to any one of the preceding items, wherein the residue at Kabat position 40 is not A.

[0419] 18. The polypeptide according to any one of the preceding items, wherein the residue at Kabat position 76 is not T.

[0420] 19. The polypeptide according to any one of the preceding items, wherein the residue at Kabat position 76 is N or Q.

[0421] 20. The polypeptide according to any one of the preceding items, wherein the residue at Kabat position 89 is not E.

[0422] 21. The polypeptide according to any one of the preceding items, wherein the residue at Kabat position 79 is Y, W or F, preferably Y or F, and the residue at Kabat position 89 is V or I.

[0423] 22. The polypeptide according to any one of the preceding items, wherein FWR2 has at least 85%, for example at least 88%, sequence identity with amino acids 36-49 (FWR2) at Kabat position of SEQ ID NO: 80, wherein Kabat positions 40, 43, 44, 45 and 47 are omitted for the purpose of determining sequence identity.

[0424] 23. The polypeptide according to any one of the preceding items, wherein FWR1 has at least 85%, for example at least 88% sequence identity with amino acids 36-49 (FWR2) at Kabat position of SEQ ID NO: 80, wherein Kabat positions 40, 43, 44, 45 and 47 are omitted.The Kabat positions of SEQ ID NO: 80, page 56 / 68, 62 CN 122138977 A, amino acids 1-26 (FWR1) have at least 85%, for example, at least 90% sequence identity, wherein Kabat positions 6, 14, 19, 23 and 24 are omitted for the purpose of determining sequence identity.

[0425] 24. The polypeptide according to any one of the preceding items, wherein the frame has at least 90% sequence identity with the frame of SEQ ID NO: 80, wherein Kabat positions 6, 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b and 89 are omitted for the purpose of determining sequence identity.

[0426] 25. The polypeptide according to any one of the preceding items, wherein frame region 1 (FWR1) has an amino acid sequence having at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 85% identity with the sequences selected from SEQ ID NO: 4-22 and 190.

[0427] 26. The polypeptide according to item 25, wherein frame region 1 (FWR1) has an amino acid sequence selected from SEQ ID NO: 4-22 and 190.

[0428] 27. The polypeptide according to any one of the preceding items, wherein frame region 2 (FWR2) has an amino acid sequence having at least 70%, for example at least 75%, for example at least 80%, for example at least 85% identity with sequences selected from SEQ ID NO: 23-24 and 26-41.

[0429] 28. The polypeptide according to item 27, wherein frame region 2 (FWR2) has an amino acid sequence selected from SEQ ID NO: 23-24 and 26-41, for example selected from SEQ ID NO: 26, 32-39 and 41, for example selected from SEQ ID NO: 26, 38 and 41.

[0430] 29. The polypeptide according to any one of the preceding items, wherein the frame region 3 (FWR3) has an amino acid sequence having at least 70%, for example at least 75%, for example at least 80%, or for example at least 85% identity with the sequence selected from SEQ ID NO: 45-49, 54-55, 58-63, 65, 67-70, and 191.

[0431] 30. The polypeptide according to item 29, wherein the frame region 3 (FWR3) has an amino acid sequence selected from SEQ ID NO: 45-49, 54-55, 58-63, 65, 67-70, and 191.

[0432] 31. The polypeptide according to any one of the preceding items, wherein the frame region as a whole has at least 92%, for example at least 94%, or for example at least 97% sequence identity with the frame region of SEQ ID NO: 80, wherein, in order to determine sequence identity...Objective: Kabat positions 6, 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b, and 89 are omitted.

[0433] 32. The polypeptide according to any one of the preceding items, wherein the VH variant has a frame region 1 (FWR1) comprising an amino acid sequence according to the following sequence: X1X2QLX5X6SGGGX11VQX14GGSLX19LSCX23X24SG (SEQ ID NO: 1) wherein independently, X1 is Q, V, D or E, preferably Q or E; X2 is V or D, preferably V; X5 is Q, V or E, preferably Q; X6 is Q or E, preferably Q; X11 is S or L, preferably S; X14 is A, S or T, preferably A; X19 is R, S, K or T, preferably R or T; X23 is T, V, A or S, preferably T; and X24 is I, V or S, preferably I.

[0434] 33. The polypeptide according to any one of the preceding items, wherein the VH variant has a frame region 2 (FWR2) comprising an amino acid sequence according to the following sequence: Specification 57 / 68 pages 63 CN 122138977 A WFRQX5PGX8X9X10EX12VA (SEQ ID NO: 2) wherein independently, X5 is R, I, T or K, preferably R; X8 is R, K, Q, E or G, preferably K or R; X9 is E, Q, A, D or G; X10 is R, I or L, preferably R; and X12 is F or L, preferably F.

[0435] 34. The polypeptide according to any one of the preceding items, wherein the VH variant has a frame region 3 (FWR3) comprising an amino acid sequence according to the following sequence: YX2X3X4VX6GRFTISRDNAKX18TX20X21LQMNX26LKPEDTAX34YYCAA (SEQ ID NO: 3) wherein independently, X2 is A, T, N, Q or S, preferably A; X3 is D or S, preferably D; X4 is S or A, preferably S; X6 is K or A, preferably K; X18 is N or Q, preferably N; X20 is V or A, preferably V; X21 is Y, W or F, preferably Y or W; X26 is N, S, T or Q, preferably S; and X34 is V or I.

[0436] 35. The polypeptide according to any one of the preceding items, wherein the VH variant comprises a frame region 4 (FWR4), the FWR4 comprising the amino acid sequence WGQGTQVTVSS (SEQ ID NO: 71) or an amino acid sequence having at least 70%, for example at least 75%, for example at least 80% identity with SEQ ID NO: 71, provided that the amino acid residue at position 1 is W.

[0437] 36.The polypeptide according to any one of the preceding items, wherein the VH variant has a frame corresponding to a frame of a VH variant selected from SEQ ID NO: 72-78, 80-114, 118-121, 126-127, 130-135, 137, 139-151, 156 and 158.

[0438] 37. The polypeptide according to item 36, wherein the VH variant has a frame corresponding to a frame of a VH selected from SEQ ID NO: 76, 78, 80-114, 118-121, 126-127, 130-135, 137, 139-151, 156 and 158.

[0439] 38. The polypeptide according to any one of the preceding items, wherein the VH variant does not contain any histidine (H) residues.

[0440] 39. The polypeptide according to any one of the preceding items, comprising an additional amino acid sequence optionally selected from: a leader peptide, a signal peptide, a purification tag, an affinity tag, a coupling peptide, a linker peptide, and a spacer peptide.

[0441] 40. The polypeptide according to item 39, wherein the additional amino acid sequence is located at the N-terminus or C-terminus of the polypeptide.

[0442] 41. The polypeptide according to item 39 or 40, wherein the additional amino acid sequence is a leader peptide or a signal peptide.

[0443] 42. The polypeptide according to item 39, wherein the additional amino acid sequence comprises a plurality of histidines, such as a (His)6 sequence. Specification 58 / 68 pages 64 CN 122138977 A

[0444] 43. A multimeric polypeptide comprising at least two moieties, each moieties being a VH variant as defined in any one of items 1-38, said moieties optionally linked by a peptide linker.

[0445] 44. The multimeric polypeptide of claim 43, comprising three VH variants.

[0446] 45. The multimeric polypeptide of claim 43 or 44, wherein the VH variants are identical.

[0447] 46. The multimeric polypeptide of any one of claims 43-45, wherein the VH variants have different CDRs.

[0448] 47. A fusion protein comprising at least one polypeptide of any one of the preceding claims and at least one additional polypeptide moiety.

[0449] 48. The fusion protein of claim 47, wherein the additional polypeptide moiety comprises an α-helix domain.

[0450] 49. The fusion protein of claim 48, wherein the additional polypeptide moiety comprises an α-helix bundle domain.

[0451] 50. The fusion protein of claim 49, wherein the additional polypeptide moiety is derived from a protein domain of staphylococcal protein A (SpA).

[0452] 51.The fusion protein according to item 49 or 50, wherein the additional polypeptide moiety comprises a sequence having at least 80% identity, for example at least 85% or at least 90% identity with SEQ ID NO: 159 or SEQ ID NO: 160.

[0453] 52. The fusion protein according to any one of items 47-51, wherein the additional polypeptide moiety is located at the C-terminus of the VH variant.

[0454] 53. The fusion protein according to any one of items 47-52, comprising two or more copies of the additional polypeptide moiety.

[0455] 54. A fusion protein according to any one of items 47-52, comprising the following structure ([A-L1]m-[Z-L2]n)p, wherein A represents a polypeptide as defined in any one of items 1-42, L1 may be present or absent for each occurrence and, when present, represents a linker or spacer, Z represents an additional polypeptide portion according to any one of items 48-51, L2 may be present or absent for each occurrence and, when present, represents a linker or spacer, m represents an integer from 1 to 4, n represents an integer from 1 to 4, or when m ≥ 2, n represents 0 or an integer from 1 to 4, and p represents an integer from 1 to 4.

[0456] 55. A fusion protein according to item 54, wherein m is 2 or 3, n is 1, and p is 1.

[0457] 56. A fusion protein according to item 54, wherein m is 1, n is 1, and p is 2 or 3.

[0458] 57. A fusion protein according to any one of items 47-56, comprising a C-terminal tag containing a plurality of histidine residues, preferably at least six histidine residues.

[0459] 58. A fusion protein according to item 57, wherein the fusion protein does not contain any histidine residues other than the tag.

[0460] 59. An isolated nucleic acid encoding a polypeptide, polymer, or fusion protein according to any one of items 1-58.

[0461] 60. An expression vector comprising the nucleic acid according to item 59. Specification 59 / 68 pages 65 CN 122138977 A

[0462] 61. A recombinant host cell for producing a polypeptide, polymer, or fusion protein according to any one of items 1-58, wherein the host cell comprises the expression vector according to item 60.

[0463] 62. A recombinant host cell according to item 61, wherein the cell is a prokaryotic cell, such as an Escherichia coli cell.

[0464] 63. The recombinant host cell according to item 61, wherein the cell is a eukaryotic cell.

[0465] 64. The recombinant host cell according to item 63, wherein the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae or Pichia pastoris.

[0466] 65.66. The recombinant host cell according to item 63, wherein the eukaryotic cell is an insect cell.

[0467] 67. The recombinant host cell according to item 63, wherein the eukaryotic cell is an animal cell, such as a mammalian cell, such as a Chinese hamster ovary (CHO) cell or a human embryonic kidney (HEK) cell.

[0468] 68. A method for producing a polypeptide, polymer, or fusion protein according to any one of items 1-58, comprising: i. providing a recombinant host cell according to any one of items 61-66; ii. culturing the host cell under conditions capable of expressing the polypeptide, polymer, or fusion protein; and iii. isolating the polypeptide, polymer, or fusion protein.

[0469] 69. The method according to any one of items 62-67, wherein step iii comprises purifying the polypeptide, polymer, or fusion protein by affinity chromatography.

[0470] 60. The method according to item 68, wherein the affinity chromatography uses an affinity ligand that binds to the frame region of the VH variant.

[0471] 70. Use as an affinity ligand for capturing a target entity by a polypeptide according to any one of items 1-42, a polymer according to any one of items 43-46, or a fusion protein according to any one of items 47-58.

[0472] 71. Use according to item 70 for in vitro detection and / or purification of the target entity.

[0473] 72. An adsorbent material comprising a polypeptide according to any one of items 1-42, a polymer according to any one of items 43-46, or a fusion protein according to any one of items 47-58 coupled to a solid support.

[0474] 73. The adsorbent material according to item 72, wherein the support material is a surface.

[0475] 74. The adsorbent material according to item 72 or 73, wherein the support is selected from particles, beads, fibers, fibrous membranes, filters, sheets, porous substrates, chips, plates, and pores.

[0476] 75. The adsorbent material according to any one of items 72-74, wherein the support comprises a polymer material.

[0477] 76. The adsorbent material according to item 75, wherein the support comprises a polysaccharide-based material, such as cellulose or agarose and derivatives thereof.

[0478] 77. The adsorbent material according to item 76, wherein the support comprises agar or agarose or derivatives thereof, such as cross-linked agarose.

[0479] 78. The adsorbent material according to any one of items 72-76, wherein the support material is a fibrous material, such as a material comprising nanofibers.

[0480] 79. The adsorbent material according to item 78, wherein the support material is a fibrous matrix, such as a nonwoven fibrous matrix.

[0481] 80.The adsorbent material according to any one of items 72-79, wherein the support is a chromatographic matrix.

[0482] 81. The adsorbent material according to item 80, wherein the support material is a chromatographic matrix selected from fiber matrices, membranes, filters, and bulk materials. Specification 60 / 68 pages 66 CN 122138977 A

[0483] 82. Use of the adsorbent material according to any one of items 72-81 for binding a target entity.

[0484] 83. Use according to item 82 for detecting the target entity in a sample.

[0485] 84. Use according to item 82 for separating the target entity from other components of a sample.

[0486] 85. Use according to item 84 for analytical separation of a target entity.

[0487] 86. Use according to item 82 for preparative purification of a target entity.

[0488] 87. A separation method comprising the steps of: (a) providing an adsorbent material according to any one of items 72-81, wherein the VH variant of the polypeptide has binding affinity for a target entity; (b) contacting the adsorbent material with a liquid sample containing the target entity, under conditions allowing the target entity to bind the polypeptide; (c) optionally washing the adsorbent material; (d) eluting the target entity from the adsorbent material; and (e) cleaning the adsorbent material with a cleaning liquid.

[0489] 88. The method according to item 87, wherein the cleaning liquid is alkaline and preferably contains 0.05-0.5 M NaOH, for example 0.1-0.5 M NaOH.

[0490] 89. The method according to item 87 or 88, wherein steps (a)-(e) are repeated at least 10 times, for example at least 20 times.

[0491] 90. The method according to any one of items 87-89, wherein after 12 cycles of contact with an alkaline cleaning liquid, the polypeptide or fusion protein retains at least 50%, for example at least 60%, for example at least 80%, for example at least 90%, of its initial target binding capacity.

[0492] 91. The method according to item 90, wherein after 15 cycles of contact with an alkaline cleaning liquid, the polypeptide or fusion protein retains at least 50%, for example at least 60%, for example at least 80%, for example at least 90%, of its initial target binding capacity.

[0493] 92. The method according to any one of items 87-89, wherein after 12 cycles of contact with an alkaline cleaning liquid, the polypeptide or fusion protein retains at least 50%, for example at least 60%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 98%, of its target binding capacity in the second cycle.

[0494] 93.According to the method of Project 92, after 15 cycles of contact with an alkaline cleaning liquid, the polypeptide or fusion protein retains at least 50%, for example at least 60%, for example at least 80%, for example at least 90%, for example at least 95%, of the target entity binding capacity of the second cycle.

[0495] Table 18: Exemplary amino acid sequences Specification 61 / 68 pages 67 CN 122138977 A Specification 62 / 68 pages 68 CN 122138977 A Specification 63 / 68 pages 69 CN 122138977 A Specification 64 / 68 pages 70 CN 122138977 A Specification 65 / 68 pages 71 CN 122138977 A Specification 66 / 68 pages 72 CN 122138977 A Specification 67 / 68 pages 73 CN 122138977 A References Thompson et al., Nucleic Acids Research, 22: 4673-4680 (1994) Kabat et al., J. Immunol. 147(5), 1709-1719 (1991) WO2003080655A1 WO2016079033A1 S Hjerten,Biochim Biophys Acta79(2), 393-398 (1964) US6602990 US7396467 WO2019137869A1 WO2018011600A1 Schmitz et al. al,Structure21, 1214‑1224 (2013) Fleetwood et al.,Cell. Mol. Life Sci.70:1081‑1093 (2013) Mitchell&Colwell, Proteins 86, 697‑706 (2018) Adams R. Et al, MABS VOL. 8, NO. 7, 1336–1346 (2016) WO2023174900A1 Instruction Manual 68 / 68 pages 74 CN 122138977 A Figure 1 Figure 2a Figure 2b Figure 2c Instruction Manual Drawings 1 / 14 pages 75 CN 122138977 A Figure 3a Figure 3b Figure 3c Instruction Manual Drawings 2 / 14 pages 76 CN 122138977 A Figure 4 Figure 5 Instruction Manual Drawings 3 / 14 pages 77 CN 122138977 A Figure 6a Figure 6b DescriptionFigure 4 / 14, page 78, CN 122138977 A, Figure 6c, Figure 7a; Figure 5 / 14, page 79, CN 122138977 A, Figure 7b, Figure 7c; Figure 6 / 14, page 80, CN 122138977 A, Figure 8; Figure 7 / 14, page 81, CN 122138977 A, Figure 9; Figure 8 / 14, page 82, CN 122138977 A, Figure 10a, Figure 10b; Figure 9 / 14, page 83, CN 122138977 A, Figure 11, Figure 12; Figure 10 / 14, page 84, CN 122138977 A, Figure 13A; Figure 11 / 14, page 85, CN 122138977 A, Figure 13B; Figure 12 / 14, page 86, CN 122138977 A, Figure 13C. Figure 14, Appendix 14, Page 14, CN 122138977 A (Instruction Manual)

Claims

1. A polypeptide comprising a heavy chain variable domain (VH) variant, such as a single-domain antibody (sdAb) variant, said variant comprising a plurality of antigen-binding regions and a frame comprising frame region 1 (FWR1), frame region 2 (FWR2), and frame region 3 (FWR3), wherein, compared with i) a natural camelid frame as defined in any one of SEQ ID NO: 152, SEQ ID NO: 155, or SEQ ID NO: 157, or ii) a synthetic frame as defined in any one of SEQ ID NO: 72 or SEQ ID NO: 73, said VH variant has a plurality of mutated amino acid sequences comprising, wherein at least eight of the following criteria i)-x) are satisfied: i) The residue at position 19 of Kabat is R, S, K, or T; ii) The residue at position 23 of Kabat is T, V, A or S, preferably T; iii) The residue at position 24 of Kabat is I, V, or S; iv) The residue at position 40 of Kabat is selected from R, I, T or K, preferably R; v) The residue at position 43 of Kabat is R, K, Q, E or G, preferably R or K; vi) The residue at position 44 of Kabat is E, Q, A, D, or G; vii) The residue at position 45 of Kabat is R, I, or L, preferably R; viii) The residue at position 76 of Kabat is N or Q; ix) The residue at position 79 of Kabat is Y, W, or F; and x) The residue at position 89 of Kabat is V or I.

2. The polypeptide of claim 1, wherein the VH variant has an amino acid sequence satisfying at least five of the following criteria i)-vi): i) The residue at position 24 of Kabat is I, V, or S; ii) The residue at position 40 of Kabat is selected from R, I, K or T, preferably R; iii) The residue at position 45 of Kabat is R, I, or L, preferably R; iv) The residue at position 76 of Kabat is N or Q; v) The residue at position 79 of Kabat is Y, W, or F; and vi) The residue at position 89 of Kabat is V or I.

3. The polypeptide according to any one of the preceding claims, wherein the VH variant has a frame region 1 (FWR1) comprising an amino acid sequence according to the following sequence: X1X2QLX5X6SGGGX 11 VQX 14 GGSLX 19 LSCX 23 X 24 SG (SEQ ID NO: 1) Independently, X1 is Q, V, D or E, preferably Q or E; X2 is V or D, preferably V; X5 is Q, V, or E, preferably Q; X6 is either Q or E, preferably Q; X 11 It is S or L, preferably S; X 14 It is A, S, or T, with A being the preferred choice; X 19 It is R, S, K or T, preferably R or T; X 23 It is T, V, A, or S, preferably T; and X 24 It is I, V, or S, preferably I; Frame region 2 (FWR2) contains an amino acid sequence based on the following sequence: WFRQX5PGX8X9X 10 FROM 12 VA (SEQ ID NO: 2) Independently, X5 is R, I, T, or K, preferably R; X8 is R, K, Q, E or G, preferably K or R; X9 is E, Q, A, D, or G; X 10 It is R, I, or L, preferably R; and X 12 It is F or L, preferably F; and Frame region 3 (FWR3) contains an amino acid sequence based on the following sequence: YX2X3X4VX6GRFTISRDNAKX 18 TX 20 X 21 LQMNX 26 LKPEDTAX 34 YYCAA (SEQ ID NO: 3) Independently, X2 is A, T, N, Q, or S, preferably A; X3 is either D or S, preferably D; X4 is either S or A, preferably S; X6 is either K or A, preferably K; X 18 It is N or Q, preferably N; X 20 It is either V or A, preferably V; X 21 Is it Y, F, or W? X 26 It is N, S, T, or Q, preferably S; and X 34 It is either V or I.

4. The polypeptide according to any one of the preceding claims, wherein the VH variant comprises a frame region 4 (FWR4) containing the amino acid sequence WGQGTQVTVSS (SEQ ID NO: 71).

5. The polypeptide according to any one of claims 1-3, wherein each of FWR1, FWR2 and FWR3 has at least 80%, for example at least 85%, 90% or 95% identity with the amino acid sequence of the corresponding frame region of SEQ ID NO:80, wherein Kabat positions 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b and 89 and optionally position 6 are omitted for the purpose of determining sequence identity.

6. The polypeptide according to any one of claims 2-5, wherein all criteria i)-x) of claim 1 and all criteria i)-vi) of claim 2 are satisfied.

7. The polypeptide according to any one of claims 1-6, wherein the amino acid sequence of said VH variant further satisfies any one, for example, more than one, for example, all of the following: The residue at position 6 of the Kabat is either Q or E; The residue at position 14 of Kabat is A, S, or T; The residue at position 47 of Kabat is F or L, preferably F; The residue at position 60 of Kabat is A, T, N, Q, or S, preferably A or S; The residue at position 82b of Kabat is N, S, T, or Q.

8. The polypeptide according to any one of the preceding claims, wherein frame region 2 (FWR2) has an amino acid sequence that is at least 70%, for example at least 75%, for example at least 80%, for example at least 85% identical to the sequence selected from SEQ ID NO: 23-24 and 26-41.

9. The polypeptide according to any one of the preceding claims, wherein the frame region 2 (FWR2) has an amino acid sequence selected from SEQ ID NO: 23-24 and 26-41, for example selected from SEQ ID NO: 26, 32-39 and 41, for example selected from SEQ ID NO: 26, 38 and 41.

10. The polypeptide according to any one of the preceding claims, wherein it is an antigen-binding polypeptide.

11. The polypeptide according to any one of the preceding claims, wherein it is a single-chain polypeptide.

12. The polypeptide according to any one of the preceding claims, wherein the VH variant is a variable domain of the heavy chain of a heavy chain antibody (VHH) variant.

13. The polypeptide according to any one of the preceding claims, wherein the VH variant has a frame corresponding to a frame selected from the VH variants of SEQ ID NO: 76, 78, 80-114, 118-121, 126-127, 130-135, 137, 139-151, 156 and 158.

14. The polypeptide according to any one of the preceding claims, wherein the VH variant does not contain any histidine (H) residues.

15. The polypeptide according to any one of the preceding claims, comprising an additional amino acid sequence optionally selected from: a leader peptide, a signal peptide, a purification tag, an affinity tag, a coupling peptide, a linker peptide, and a spacer peptide.

16. The polypeptide of claim 15, wherein the additional amino acid sequence is located at the N-terminus or C-terminus of the polypeptide.

17. The polypeptide according to claim 15 or 16, wherein the additional amino acid sequence is a leader peptide or a signal peptide.

18. The polypeptide of claim 15, wherein the additional amino acid sequence comprises a plurality of histidines, such as the (His)6 sequence.

19. A multimeric polypeptide comprising at least two moieties, each moiety being a VH variant as defined in any of the preceding claims, said moieties optionally being linked by a peptide linker.

20. The multimeric polypeptide of claim 19, comprising three VH variants, wherein the VH variants are identical or have different CDRs.

21. A fusion protein comprising at least one polypeptide according to any one of claims 1-18 or a polymer according to claims 19-20 and at least one additional polypeptide moiety.

22. The fusion protein of claim 21, wherein the additional polypeptide portion comprises an α-helical domain, such as an α-helical bundle domain.

23. The fusion protein of claim 22, wherein the additional polypeptide portion is derived from a protein domain of staphylococcal protein A (SpA).

24. The fusion protein according to claims 22-23, wherein the additional polypeptide moiety comprises a sequence having at least 80% identity with SEQ ID NO: 159 or SEQ ID NO: 160, for example at least 85% or at least 90% identity.

25. The fusion protein according to any one of claims 21-24, wherein the additional polypeptide moiety is located at the C-terminus of the VH variant.

26. The fusion protein according to any one of claims 21-25, comprising the following structure ([A-L1] m -[Z-L2] n ) p in A represents a polypeptide as defined in any one of claims 1-18. L1 may or may not exist for each occurrence, and when it exists, it indicates a joint or spacer base. Z represents an additional polypeptide portion according to any one of claims 22-24. L2 may or may not exist for each occurrence, and when it exists, it indicates a joint or spacer base. m represents an integer from 1 to 4. n represents an integer from 1 to 4, or when m ≥ 2, n represents 0 or an integer from 1 to 4. as well as p represents an integer from 1 to 4.

27. The fusion protein of claim 26, wherein i) m is 2 or 3, n is 1 and p is 1, or ii) m is 1, n is 1 and p is 2 or 3.

28. The fusion protein according to any one of claims 21-26, comprising a C-terminal tag, said tag comprising a plurality of histidine residues, preferably at least 6 histidine residues.

29. The fusion protein of claim 28, wherein the fusion protein contains no histidine residues other than the tag.

30. An isolated nucleic acid encoding a polypeptide, polymer, or fusion protein according to any one of claims 1-29.

31. An expression vector comprising the nucleic acid according to claim 30.

32. A recombinant host cell for producing a polypeptide, polymer, or fusion protein according to any one of claims 1-29, said host cell comprising the expression vector according to claim 31.

33. A method for producing a polypeptide, polymer, or fusion protein according to any one of claims 1-29, comprising: i. Providing the recombinant host cell according to claim 32; ii. Culture the host cells under conditions that enable the expression of the polypeptide, multimer, or fusion protein; and iii. Isolate the polypeptide, polymer, or fusion protein.

34. The method of claim 33, wherein step iii comprises purifying the polypeptide, polymer, or fusion protein by affinity chromatography.

35. The method of claim 34, wherein the affinity chromatography uses an affinity ligand that binds to the frame region of the VH variant.

36. Use of the polypeptide according to any one of claims 1-18, the polymer according to claims 19-20, or the fusion protein according to claims 21-29 as an affinity ligand for capturing a target entity.

37. The use according to claim 37, for in vitro detection and / or purification of target entities.

38. An adsorbent material comprising a polypeptide according to any one of claims 1-18, a polymer according to claims 19-20, or a fusion protein according to claims 21-29 coupled to a solid support.

39. The adsorbent material according to claim 38, wherein the carrier material is selected from particles, beads, fibers, fiber membranes, filters, sheets, porous materials, chips, plates, and pores.

40. The adsorbent material according to any one of claims 38-39, wherein the carrier material is a fiber matrix, such as a nonwoven fiber matrix.

41. The adsorbent material according to any one of claims 38-39, wherein the carrier comprises agar or agarose or a derivative thereof, such as cross-linked agarose.

42. The adsorbent material according to any one of claims 38-41, wherein the support is a chromatographic matrix.

43. Use of the adsorbent material according to any one of claims 38-42 for separating the target entity from other components of the sample.

44. A separation method, comprising the following steps: (a) Providing an adsorbent material according to any one of claims 38-42, wherein the VH variant of said polypeptide has binding affinity to the target entity. (b) Contacting the adsorbent material with a liquid sample containing the target entity, provided that the target entity is allowed to bind to the polypeptide. (c) Optionally, the adsorbent material is washed. (d) Elution of the target entity from the adsorbent material, and (e) Clean the adsorbent material with a cleaning liquid, such as an alkaline cleaning liquid.

45. The method of claim 44, wherein the cleaning liquid is alkaline and preferably contains 0.05-0.5 M NaOH, for example 0.1-0.5 M NaOH.

46. ​​The method according to any one of claims 44-45, wherein steps (a)-(e) are repeated at least 10 times.

47. The method according to any one of claims 44-46, wherein after 12 cycles of contact with an alkaline cleaning liquid, the polypeptide or fusion protein retains at least 50%, for example at least 60%, for example at least 80%, for example at least 90% of the initial target entity binding capacity.

48. The method according to any one of claims 44-46, wherein after 12 cycles of contact with an alkaline cleaning liquid, the polypeptide or fusion protein retains at least 50%, for example at least 60%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 98% of the target entity binding capacity of the second cycle.

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