AAV9 binding polypeptides

Abstract

The present invention provides antigen-binding polypeptides capable of binding to adeno-associated virus serotype 9 (AAV9), the polypeptides comprising single domain antibody (sdAb) variants having complementarity determining regions CDR1, CDR2 and CDR3 as disclosed herein. The antigen-binding polypeptides can be used as affinity ligands for affinity capture applications, such as affinity isolation.

Description

Technical Field

[0001] This invention relates to an antigen-binding polypeptide capable of binding adeno-associated virus (AAV) and a separation matrix comprising said antigen-binding polypeptide, and to its use in the context of affinity capture or affinity separation. Background Technology

[0002] Advanced therapeutic drugs (ATMPs) have emerged as a promising field for the treatment or prevention of serious diseases. This includes, for example, gene and cell therapies aimed at restoring or replacing defective genes, as well as other therapies based on recombinant nucleic acids.

[0003] A vector is typically required to deliver recombinant nucleic acids to recipient cells. Different vectors can be used depending on the type of therapy, the target cell type, and the nucleic acid to be delivered.

[0004] Adeno-associated virus (AAV) is a small, non-enveloped virus of the Parvoviridae family. It is naturally non-pathogenic and has low immunogenicity. AAV can be engineered to deliver DNA to target cells, and recombinant adeno-associated virus (rAAV) vectors have emerged as one of the most versatile and successful gene therapy delivery media.

[0005] Adeno-associated viruses (AAVs) possess a linear single-stranded DNA (ssDNA) genome contained within a capsid formed by capsid protein subunits. To date, AAV capsid serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 (AAVrh10), 11, and 12 have been identified. Different AAV serotypes exhibit different tropisms, i.e., specificity in infecting cell or tissue types. Most early AAV gene transfer studies used AAV serotype 2 (AAV2); however, later research and development have revealed that other serotypes may be more effective as therapeutic vectors. In recent years, AAV9 has attracted considerable interest. AAV9 is considered very promising for many gene therapies targeting the lungs, liver, heart, skeletal muscle, or CNS.

[0006] AAV vectors modified to contain the target gene can be produced in mammalian cells, such as human embryonic kidney cells. AAV vectors are harvested from cell cultures, clarified, and purified to remove cell debris, host cell DNA and proteins, and any other impurities. The purity, efficacy, and safety of clinical-grade vectors are critical, as impurities retained from the production stage can lead to adverse effects, reduced transduction efficiency, and even systemic immune or inflammatory responses. The vectors also need to retain viral activity throughout the purification process.

[0007] Typically, several filtration steps and several chromatographic steps are used to separate AAV particles from cell cultures. Ultracentrifugation can be effective, but it cannot be scaled up. Chromatographic techniques are used for adenovirus purification, but the results are often insufficient in terms of purity, yield, and capacity.

[0008] Affinity chromatography is a specific mode of chromatography in which an affinity ligand interacts with a target entity in a "lock-and-key" manner through biological affinity. Common examples of interactions that can be used in affinity chromatography include, for example, enzyme-substrate interactions, biotin-avidin interactions, and antibody-antigen interactions. The affinity ligand can specifically and reversibly bind to the desired target entity and is immobilized on the chromatographic carrier material. When contacted with a sample containing the target entity under binding conditions, the ligand selectively binds 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, the same material can be used for new affinity purification cycles. Affinity chromatography can potentially yield targets of very high purity.

[0009] For effective affinity capture processes, a high target binding capacity of the ligand is required. During affinity purification processes that involve many purification cycles, a deterioration in binding capacity is generally expected due to exposure to alkaline conditions during the aforementioned cleaning of the chromatographic material.

[0010] There is a need in this field to improve the purification methods for AAV-based (especially AAV9) viral vectors. Summary of the Invention

[0011] The objective of this invention is to overcome or at least partially mitigate the deficiencies of the prior art.

[0012] Therefore, the objective of this invention is to provide AAV affinity capture materials that allow for more efficient purification of AAV vectors.

[0013] In the first aspect, these and other targets are achieved by antigen-binding peptides capable of binding adeno-associated virus serotype 9 (AAV9), said peptides comprising single-domain antibody (sdAb) variants having complementarity-determining regions CDR1, CDR2 and CDR3 as defined herein.

[0014] CDR1 contains an amino acid sequence selected from the sequence group defined by SEQ ID NO: 1: X1X2X3SX5X6TMX9(SEQ ID NO: 1) Independently, X1 is R, L, or S, preferably R or L; X2 is T or R, preferably T; X3 is L or F, preferably L; X5 is D, N, or E, preferably D; X6 is Y, N, or F, preferably Y or F; and X9 is G or A, preferably G. For example, X6 can be F and X9 can be G or A.

[0015] CDR2 contains sequences selected from the sequence group defined by SEQ ID NO: 2: X1X2SWSGX7X8TX 10 (SEQ ID NO: 2) Independently, X1 is A, S, L, V, or I, preferably A; X2 is I or V, preferably I; X7 is A or S, preferably A; X8 is Y or F, preferably Y; and X 10 It is K, F, or Y. In the implementation scheme, X2 is I, X7 is A, X8 is Y and / or X 10 It's K.

[0016] CDR3 contains sequences selected from the sequence group defined by SEQ ID NO: 3: X1X2TX4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 (SEQ ID NO: 3) Independently, X1 is G or A, preferably G; X2 is P or S, preferably P; X4 is G or P, preferably G; X5 is L, T or P, preferably L or P; X6 is L or I, preferably L; X7 is S, T or A, preferably S or A; X8 is K, R, N or Q; X9 is K, H or R, preferably K or R; X 10 It is A, S, or T, preferably A or T; X 11 It is T, S, P, or A, preferably T; X 12 It is P, A, T, or S, preferably P; X 13 It is A, P, R, or G, preferably A or P; X 14 It is D, N, E, or Q, preferably D or E; X 15 It is Y or F, preferably Y; X 16 It is D, N, E, or Q, preferably D; and X 17 It is Y, F, or R, preferably Y or F.

[0017] On the other hand, this disclosure provides a multimeric polypeptide comprising at least two parts, each part comprising a single-domain antibody variant as defined herein.

[0018] On the other hand, a fusion protein is provided comprising at least one antigen-binding polypeptide or multimeric polypeptide as described herein and an additional polypeptide moiety.

[0019] In other respects, this disclosure provides isolated nucleic acids encoding the said antigen-binding polypeptide, multimeric polypeptide, or fusion protein, as well as expression vectors containing said nucleic acids and recombinant host cells containing said expression vectors.

[0020] In another aspect, a method for producing the antigen-binding polypeptide, multimeric polypeptide, or fusion protein is provided, the method comprising i) providing recombinant host cells; ii) culturing the host cells under conditions capable of expressing the antigen-binding polypeptide, multimeric polypeptide, or fusion protein; and iii) isolating the antigen-binding polypeptide, multimeric polypeptide, or fusion protein. Step iii) may include purifying the antigen-binding polypeptide, multimeric polypeptide, or fusion protein by chromatography, such as immobilized metal affinity chromatography (IMAC) or affinity chromatography.

[0021] In a further aspect, this disclosure provides the use of the antigen-binding polypeptide, polymer, or fusion protein as an affinity ligand for binding adeno-associated virus serotype 9 (AAV9). This use may, for example, be for the in vitro detection of AAV9 in a sample, or for the purification of AAV9 from a sample, optionally within the context of providing a method for a therapeutic composition comprising AAV9 as a viral vector.

[0022] In other respects, this disclosure provides separation matrices and separation methods.

[0023] The separation matrix comprises an antigen-binding polypeptide, polymeric polypeptide, or fusion protein coupled to a carrier material, thereby providing an affinity separation matrix using the antigen-binding polypeptide as an affinity ligand. The carrier material may be selected from particles, beads, fibers, fibrous membranes, filters, sheets, porous substrates, chips, plates, and wells. The carrier material may be a chromatographic matrix. For example, the chromatographic matrix may comprise a polysaccharide material, such as beads comprising agarose or its derivatives. Alternatively, the chromatographic matrix may comprise a polymeric fibrous matrix or membrane, such as a nonwoven fibrous matrix.

[0024] The separation method includes the following steps: (a) Provide the separation matrix of this disclosure, (b) Under conditions that allow the antigen-binding polypeptide to bind to AAV9 virus particles, the separation matrix is ​​brought into contact with a liquid sample containing an AAV9 virus particle carrier. (c) Optional washing of chromatographic materials. (d) Elution of bound AAV9 virus particles from the separation matrix, and optionally (e) Clean the separation matrix material with a cleaning liquid.

[0025] Step (e) typically involves cleaning the separation matrix with an alkaline cleaning liquid, wherein the cleaning liquid preferably contains 0.05-0.5 M NaOH. Attached Figure Description

[0026] These and other aspects of the invention will now be described in more detail with reference to the accompanying drawings, which illustrate embodiments of the invention, in which: Figure 1 This is a schematic diagram of the arrangement of the framework region and complementarity-determining region of a single-domain antibody.

[0027] Figure 2a -c is a schematic diagram of various structures of antigen-binding polypeptides according to this disclosure.

[0028] Figure 3a -c is a schematic diagram of a fusion protein containing an antigen-binding polypeptide, as disclosed herein, fused with another polypeptide moiety.

[0029] Figure 4a -b is a sensor plot showing the AAV9 binding capacity of two exemplary peptides.

[0030] Figure 5 This is a graph showing the basic stability of an exemplary antigen-binding polypeptide.

[0031] Figure 6 This is a graph showing the basic stability of an exemplary antigen-binding peptide and a commercially available affinity ligand.

[0032] Figure 7a -h is a sensor map showing the binding of exemplary antigen-binding peptides and commercially available affinity ligands to AAV9 and other AAV serotypes.

[0033] Figure 8a This is a graph showing the AAV9 binding response of antigen-binding peptides with or without fusion partner bodies (solid lines) to the binding cycle number on Biacore, with each cycle including 0.5 M NaOH exposure. The binding response to the second cycle (i.e., excluding the first cycle) is normalized. Figure 8b Showing the combined response from the same experiment, normalized to the response of the first cycle.

[0034] Figure 9aThese are PAGE gel images, confirming strong protein expression of the multimeric fusion protein and the individual antigen-binding polypeptide (lacking a fusion partner) according to embodiments of the present invention. Figure 9b This is a graph showing the AAV9 binding response of the multimeric fusion protein and the multimeric polypeptide lacking the fusion partner to the number of binding cycles on Biacore, with each cycle including 0.3 M NaOH exposure. The binding response to the second cycle (i.e., excluding the first cycle) is normalized.

[0035] Figure 10 The diagram shows the AAV9 binding response obtained for two fusion proteins according to embodiments of the present invention with increasing cycles including NaOH exposure.

[0036] Figure 11a is a chromatogram showing the elution peaks from a chromatographic column using an AAV9-binding polypeptide as an affinity ligand immobilized on the chromatographic matrix according to an embodiment of the present invention. Figure 11b is a partial magnified view of the elution peaks.

[0037] Figure 12 This is a photograph of an SDS-PAGE gel, showing the protein content of the eluent fraction from the chromatographic run of Example 8.

[0038] Figure 13 It is a graph showing the dynamic combined capacity, as evaluated in Example 8.

[0039] As illustrated in the accompanying drawings, 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.

[0040] definition As used herein, the terms “peptide” and “polypeptide” are used synonymously and refer to compounds formed from amino acid sequences, without limitation on size. “Protein” may be used to refer to larger compounds in this category. Amino acid sequences are written from left to right, from the amino (N) terminus to the carboxyl (C) terminus. According to standard nomenclature, amino acid residue sequences are represented by three-letter or single-letter codes as follows: 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). A "peptide" includes any oligopeptide, polypeptide, gene product, expression product, or protein. Peptides are composed of consecutive 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 through natural processes, such as post-translational processing, or through chemical modification techniques well known in the art.

[0041] In this paper, "single-chain polypeptide" refers to a polypeptide consisting of a single amino acid sequence, where the amino acid residues are linked by peptide bonds. Single-chain polypeptides may form other internal bonds, such as disulfide bonds, after folding.

[0042] The term "antigen-binding polypeptide" generally refers to a polypeptide having at least one binding region, and typically at least two, such as 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 they possess binding affinity for an antigen. However, some types of antigen-binding polypeptides that have been commonly developed and used as the basis for modifying antigen-binding polypeptides often have a backbone or framework structure that is generally conserved among antigen-binding polypeptides of the same type, but which bind different antigens. The binding regions of antigen-binding molecules can often be modified or evolved to bind specific antigens, while the backbone or framework structure remains substantially the same. Non-limiting examples of antigen-binding peptides include natural or engineered (i) antibodies, such as monoclonal antibodies; (ii) antibody fragments containing variable regions of the light and / or heavy chains having 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 having a bacterial backbone, such as immunoglobulin-binding bacterial proteins (e.g., *Goldobacter griseus*). Finegoldia magna Protein L and staphylococcal proteins A and G, or their domains, including wild-type and variants in which one or more antigen-binding regions have been modified.

[0043] The description of a "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 domains and light chain variable domains), single-domain antibodies (sdAbs), albumin-binding domains (ABDs), and polypeptides derived from domains of SpA (e.g., the B, C, or Z domains of SpA). 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 a disulfide bond). Preferably, the antigen-binding polypeptide of this disclosure may consist of a single polypeptide chain.

[0044] The term "single-domain antibody" refers to a variable domain that is a heavy chain variable domain (VH) of an antibody lacking a light chain. Therefore, a single-domain antibody completely lacks the antibody light chain, including the light chain variable domain (VL). Single-domain antibodies may also lack a structural feature essential for functional VH / VL interactions and present, for example, in the conventional VH of IgG1. Therefore, single-domain antibodies do not form part of a dimer structure containing 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 camels (e.g., dromedary camels, camels, llamas, and alpacas), also known as VHHs (heavy chain variable domains of heavy chain antibodies). Single-domain antibodies also include antibodies derived from cartilaginous fish (e.g., sharks), which are often referred to as VNARs (variable neoantigen receptors). 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 native 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.

[0045] Typically, terms such as "peptide," "protein," "antibody," "single-domain peptide," and "single-domain antibody" also include synthetic variants that are recombined and whose amino acid sequences can be modified relative to the amino acid sequences of their naturally occurring counterparts. The term "variant" may also be used when specifically referring to such modified variants. The terms "wild-type" or "wt" refer to the typical naturally occurring form. The single-domain antibody variants of this invention are synthetic, non-naturally occurring amino acid sequences.

[0046] Complementarity-determining regions (CDRs) are hypervariable regions of an antibody or a portion thereof that participate in binding to target epitopes. CDRs are typically 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, called CDR1, CDR2, and CDR3, located from the N-terminus of the polypeptide chain. The sequence length of the CDRs can vary, and the CDR1, CDR2, and especially CDR3 of the VH can have different lengths.

[0047] The antibody VH portion that does not form a CDR is called the framework region. There are typically four framework regions, referred to as framework regions (FW) 1–4, as counted from the N-terminus. The framework regions are responsible for the overall secondary and tertiary structure of the domain, and therefore for the localization and orientation of the CDR region. Framework regions can be referred to as the backbone structure. Although part of a variable domain, the variability of framework regions is lower than that of CDRs. However, they are generally tolerant of amino acid sequence changes that do not significantly alter the secondary or tertiary structure of the framework. Some portions or amino acid positions of the framework region can be conserved. The overall stability of the framework regions allows for a high degree of variation in CDRs.

[0048] 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," "selectively binding molecule," "selectively binding partner," "capture molecule," and "capture agent." An "affinity ligand" is 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, affinity ligands are polypeptides and may also be referred to as "affinity proteins." Affinity ligands may be immobilized to a solid carrier, such as a resin.

[0049] The term "target entity" in this document refers to an entity that forms a specific binding pair with a ligand and may also be referred to as an "analyte". Analytes or target entities of interest according to this disclosure are adeno-associated virus vectors, particularly AAV9 vectors.

[0050] As used herein, “affinity” in the context of peptides refers to the strength of the interaction between two molecules, at least one of which is a peptide. The interaction is selective, meaning there is a distinction between affinity-binding partner molecules and other molecules present. Binding affinity is also expressed as the dissociation equilibrium constant (KD).

[0051] As used herein, the term "binding capacity" refers to the ability of a ligand to bind a target molecule when it is immobilized on a surface. The binding capacity of a ligand can vary depending on the type of surface on which it is immobilized. For the purposes of this invention, binding capacity can be measured using surface plasmon resonance (SPR) techniques with instruments such as Biacore, as described in the embodiments herein. The binding capacity of an analyte at a given concentration is affected by the density of immobilized ligands on the surface. In the context of this invention, binding capacity is preferably determined at a ligand density in the range of at least about 3000 response units (RU), for example, 3000-4000 RU.

[0052] In the context of protein purification, the term "dynamic binding capacity" (abbreviated "DBC") for chromatographic columns refers to the binding capacity within a packed affinity column under operating conditions, specifically during sample loading. The DBC of a chromatographic resin is expressed as the amount of analyte bound to the resin under given flow conditions before significant breakthrough occurs in unbound analytes. 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 unbound analytes flow through the column. DBC can be determined on a breakthrough curve, for example, with a 10% loss of analyte. This is known as the QB10% value. The dynamic binding capacity of each resin is calculated at 10% breakthrough capacity, i.e., the amount of analyte sample loaded onto the column until the 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 known as the QB80% value.

[0053] As used herein, the term "basic stability" refers to the property of an antigen-binding peptide that involves its ability to withstand alkaline exposure without adverse effects on the structure and / or function of the antigen-binding 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 a thiol, N-hydroxysuccinimide (NHS), streptavidin-biotin, or another coupling that is inherently alkaline resistant. Typically, the first alkaline cleaning cycle can have a unique and 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.

[0054] In this context, an antigen-binding peptide is considered alkaline stable if, after 10 clean cycles, preferably 15 clean cycles, the fixed 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.

[0055] The term "solid support" herein refers to a non-aqueous matrix of a 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.

[0056] The term "surface" in this document refers to 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 individual fiber.

[0057] The term "separation matrix" is used herein to refer to a material containing a solid support with which one or more ligands are coupled. The ligands are capable of binding to a target entity (also referred to herein as an analyte), which is to be separated from its environment (e.g., a liquid sample), 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 is selected based on the intended use.

[0058] The separation matrix may further comprise compounds that couple ligands to a 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 whose ligands are affinity ligands.

[0059] The term "spacer" refers to a peptide or other chemical linker or element that extends the structure of a solid entity. Spacers, specifically amino acid spacers, attached to polypeptides can be provided at the N-terminus or C-terminus of the polypeptide. Spacers can link polypeptides to solid supports. Suitable spacers for coupling polypeptides to supports can generally be any spacer used in the art to link peptides, proteins, or other organic molecules to supports. Spacers can also be used to connect two polypeptides or polypeptide domains. Spacers connecting two polypeptide moieties can also be referred to as linkers.

[0060] 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. Therefore, a single-domain antibody can be considered as a monomeric polypeptide unit that can combine to form multimeric proteins, such as dimers, trimers, tetramers, pentamers, etc. The single-domain antibody units in a multimeric polymer may be identical in their amino acid sequences or may differ from each other. In addition to the single-domain antibody units described herein, the multimer may also contain one or more additional polypeptide units, such as fusion-coupled polypeptides or stable polypeptide units as described herein, which are not single-domain antibodies. Such additional polypeptide units may be present in the multimer in single or multiple copies.

[0061] Within a polymer, individual polypeptide units are linked by peptide bonds, typically through linker peptides. Therefore, multimeric proteins can be formed from a single polypeptide chain.

[0062] The term "fusion protein" refers to a protein formed by two or more separate proteins (fusion couplers), produced by recombinant protein expression as a single polypeptide containing two fusion couplers. Fusion couplers can be linked sequentially. The genes encoding the individual proteins are linked at the gene level. Fusion couplers do not typically exist naturally fused together. Fusion proteins may contain additional amino acid sequences between the fusion couplers, such as linkers.

[0063] As used herein, a "linker" refers to a peptide or other chemical bond that serves to connect otherwise independent functional or structural 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 an otherwise independent functional or structural domain, or between two ligands. A suitable linker for coupling two or more linked units can generally be any linker in the art used to connect peptides, proteins, or other organic molecules. A linker peptide can be a segment of amino acids, 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.

[0064] In this paper, the term "vector" is used to refer to viral particles, typically recombinant viral particles, designed to facilitate gene transfer to modify specific cell types or tissues. Viral particles may, for example, be modified to provide vectors for expressing therapeutic genes. Several viral types are currently under investigation for delivering genetic material (e.g., genes) into cells to provide transient or permanent transgenic expression. These include adenoviruses, retroviruses (gamma retroviruses and lentiviruses), poxviruses, adeno-associated viruses (AAVs), baculoviruses, and herpes simplex viruses. The term "capsid" refers to the outer shell of the viral particle.

[0065] Sequence identity can be assessed by any conventional method. However, to determine the degree of sequence identity between sequences, computer programs for pairwise or multiple sequence alignments can be used, such as EMBOSS Needle or EMBOSSstretcher for pairwise sequence alignment, and Clustal or MUSCLE for multiple sequence alignment, but any other suitable program can be used. Whether the alignment is pairwise or multiple, it must be performed globally (i.e., across the entire reference sequence) rather than locally. Sequence alignment and %identity calculation can be determined using, for example, the standard Clustal Omega parameters: matrix Gonnet, gap opening penalty of 6, and gap extension penalty of 1. The term "%identity" as used throughout this disclosure can be calculated, for example, as follows. Using the CLUSTAL W algorithm (Thompson et al, Nucleic Acids Research, 22: 4673-4680 (1994)), the query sequence is aligned with the target sequence. The comparison is performed on a 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. Detailed Implementation

[0066] The inventors have discovered that novel antigen-binding peptides capable of binding adeno-associated virus (AAV) serotype 9 (AAV9) can be used for affinity capture of AAV9-based vectors. The antigen-binding peptides are typically single-chain peptides. These peptides are based on single-domain antibodies (sdAbs) but may optionally contain additional amino acids, for example, located at their N-terminus or C-terminus. The antigen-binding peptides of this invention can be derived from camel-derived single-domain antibodies, also known as VHH (variable domain of the heavy chain of a heavy chain antibody).

[0067] Single-domain antibody variants lack the light chain of conventional antibodies such as IgG1, and also lack the heavy chain constant domain. Therefore, the single-domain antibodies of the antigen-binding peptides disclosed herein contain only the heavy chain variable domain, which includes three complementarity-determining regions (CDRs). Thus, the antigen-binding peptides do not contain antibody light chain variable regions or light chain CDRs.

[0068] Generally, single-domain antibodies are more readily expressed in prokaryotic and eukaryotic cells compared to full-length antibodies such as IgG and Fab fragments or single-chain fragments that include both light and heavy chains. Despite having only three CDRs, their target-binding properties are satisfactory compared to larger antibodies and antibody fragments with six CDRs.

[0069] The portions of an sdAb that do not form CDRs are called framework regions. There are typically four framework regions in an sdAb, referred to herein as framework regions (FW) 1-4, which are responsible for the overall secondary and tertiary structure of the sdAb. Framework regions form two β-sheets, which create a sandwich structure connected by cysteine ​​bridges. CDRs are typically presented as three surface rings, one CDR per ring.

[0070] Another advantage of the sdAb disclosed herein is that the CDR sequence can be longer than that of conventional antibodies. Therefore, the surface loop can extend further spatially from the sdAb body, resulting in a better ability to bind into the pocket of the target epitope. Notably, these relatively long CDR sequences do not lead to sdAb instability, as is the case with long CDRs that may exist in the antigen-binding backbone (Adnektin™) derived from the tenth type III domain of human fibronectin.

[0071] Typically, sdAb variants may contain the frame region and complementarity determination region in the following order from the N-terminus to the C-terminus: [FWR1]-[CDR1]-[FWR2]-[CDR2]-[FWR3]-[CDR3]-[FWR4]. Figure 1 The diagram illustrates the sequence of the framework region and complementarity-determining region (CDR) of the sdAb from the N-terminus to the C-terminus of the amino acid sequence. Furthermore, Table 1 summarizes the information regarding the Kabat amino acid numbering system (Kabat et al., 1991). J. Immunol. 147(5), 1709-1719), as defined herein, FWR and CDR. For the sdAb variant of this disclosure, frame region 1 (FWR1) is formed by amino acids at positions 1 to 26. Then, amino acids at positions 27 to 35d of Kabat form CDR1, with positions 35a-d of Kabat optional. Frame region 2 (FWR2) is formed by amino acids at positions 36 to 49 of Kabat. CDR2 is formed by amino acids at positions 50 to 58 of Kabat (52a optional). Frame region 3 (FWR3) is formed by amino acids at positions 59 to 94 of Kabat. CDR3 is formed by amino acids at positions 95 to 102 of Kabat (100a-j optional). Finally, frame region 4 (FWR4) is formed by amino acids at positions 103 to 113 of Kabat. It may be noted that the definitions of CDR and frame regions within this disclosure do not correspond exactly to the definitions of CDR and frame regions of Kabat et al.; however, the same amino acid numbering system is used. The frame area can be collectively referred to as "frame".

[0072] Table 1 Amino acid position (Kabat) 1-26 27-35d 36-49 50-58 59-94 95-102 103-113 area FWR1 CDR1 FWR2 CDR2 FWR3 CDR3 FWR4 Instance sequence SEQ ID NO:5 SEQ ID NO:6 SEQ ID NO:7 SEQ ID NO:8 The sdAb of the present invention may have a frame with a certain degree of sequence identity to the VHH frame of natural camels. For example, each frame region of the sdAb of the present invention may have a sequence identity ranging from 70 to 100% with the corresponding frame region of SEQ ID NO: 4 (represented by SEQ ID NO: 5 (FWR1), SEQ ID NO: 6 (FWR2), SEQ ID NO: 7 (FWR3), and SEQ ID NO: 8 (FWR4), respectively). Advantageously, at least one frame region of the sdAb of the present invention may contain an amino acid substitution relative to SEQ ID NO: 4 (i.e., one or more of SEQ ID NO: 5-8, preferably one or more of SEQ ID NO: 5-7, for example at least SEQ ID NO: 7), wherein the sdAb exhibits improved basic stability (e.g., assessed by exposure to at least 0.1 M NaOH) compared to SEQ ID NO: 4. Stable sdAb frames are described in more detail elsewhere herein.

[0073] Optionally, the frame regions of the sdAb of the present invention may have a certain degree of sequence identity with the synthetic frame regions of SEQ ID NO: 91 or SEQ ID NO: 92. The frame regions of SEQ ID NO: 92 are represented by SEQ ID NO: 151 (FWR1), SEQ ID NO: 154 (FWR2), SEQ ID NO: 157 (FWR3), and SEQ ID NO: 8 (FWR4), and the frame regions of SEQ ID NO: 91 are represented by SEQ ID NO: 149 (FWR1), SEQ ID NO: 152 (FWR2), SEQ ID NO: 155 (FWR3), and SEQ ID NO: 8 (FWR4). Alternatively, the frame regions of the sdAb of the present invention may have a certain degree of sequence identity with the frame regions of SEQ ID NO: 150 (FWR1), SEQ ID NO: 153 (FWR2), SEQ ID NO: 156 (FWR3), and SEQ ID NO: 8 (FWR4). Alternatively, the frame regions of the sdAb of the present invention may have a certain degree of sequence identity with the synthetic frame regions of any one of SEQ ID NO: 177-181. The frame regions of FW1 are defined by SEQ ID NO: 183-186, the frame regions of FW2 by SEQ ID NO: 187-189, and the frame regions of FW3 by SEQ ID NO: 190-192.

[0074] For example, frame regions 1, 2, and 3 of the sdAb of the present invention may each have at least 70% sequence identity with the reference frame region. FWR4 may have less than 70% sequence identity with the reference frame region. Optionally, FWR4 may also have at least 70% sequence identity with the reference frame region.

[0075] The frames of sdAb are relatively conserved, and preferably, are minimally modified or unmodified relative to, for example, SEQ ID NO:4, 91, or 92. These frames are defined herein by Kabat positions 1-5, 7-14, 25-26, 41-42, 46, 48-49, 59, 61-75, 77-78, 80-82a, 82c-94, and 103-113. Advantageously, each of FWR1-3, and optionally FWR4, may have at least 80%, for example, at least 85%, for example, at least 90% sequence identity with the corresponding frames of SEQ ID NO:4, 91, or 92 at conserved amino acid positions (i.e., other positions omitted from comparison). As an example, FWR1 contains 26 amino acids, of which 21 amino acids represent conserved positions as defined above. Substituting two of these amino acids results in 90.5% identity, while substituting three amino acids results in 85.7% identity. The sdAb variant may have an amino acid sequence in which 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 include up to 15, for example, up to 14, for example, up to 13, for example, 12, 11, or 10 amino acid residues that are substituted relative to at least one of SEQ ID NO: 4, 91, 92, 131, 171, and 172. For example, in FWR1, positions 1-5, 7-13, and 25-26 (21 residues) may together have up to four, for example, up to three, amino acid residues that are different from the corresponding positions in SEQ ID NO: 4, 91, 92, or 171. 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: 4, 91, or 92. 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: 4, 91, or 92.

[0076] The amino acid at position 1 of the Kabat position can be advantageously selected from Q, V, D, and E, and can be particularly Q or E. For example, any sdAb amino acid sequence disclosed herein in which position 1 of the Kabat position is not E (e.g., is Q) may instead have E at position 1 of the Kabat position.

[0077] The framework region of an sdAb typically has a secondary structure comprising β-chains and / or β-sheets. The framework region often forms a β-sandwich structure, which typically contains cysteine ​​bridges. CDRs are usually presented as three surface rings at one end of the structure, one CDR per ring.

[0078] The advantage of using sdAb as an affinity binding agent is that the CDR sequence can be more diverse in length than that of conventional antibodies. The surface loop formed by the CDR can therefore extend further spatially from the sdAb body, thus having a better ability to bind into the pocket of the target epitope, or the surface loop can be shorter, forming a flatter surface, which can promote binding to other epitopes.

[0079] The inventors have identified novel amino acid sequences that exhibit excellent AAV9 binding properties when used as a CDR within an sdAb framework. These binding properties surprisingly provide a good capacity for AAV9 binding when peptides containing sdAbs are immobilized on a vector. Furthermore, compared to other sdAbs that bind AAV, the sdAbs of this invention can distinguish between different AAV serotypes, allowing them to selectively bind AAV9, while other AAV sdAbs bind to several serotypes. The binding of the sdAbs of this invention is therefore more specific than that of the prior art.

[0080] Furthermore, the CDR sequences disclosed herein have been found to promote the overall basic stability of sdAbs. It is a well-known fact in the art that sdAbs (also known as “nanobodies”) have poor basic stability. This is also a common characteristic of previous AAV sdAb ligands, as described, for example, in patent application WO2020242988A2, which relates to affinity reagents containing ligands that bind AAV, stating in paragraph

[0005] that “currently, only three affinity resins are available for purifying AAV: POROS™ CaptureSelect™ AAV9, POROS™ CaptureSelect™ AAVX, and AVB Sepharose. These resins have two main drawbacks: they cannot be cleaned with sodium hydroxide and can only be reused for a few cycles. This increases resin consumption, thus leading to high resin costs for purification.” Therefore, it is surprising to find that the sdAbs of the present invention not only bind AAV selectively and with high capacity (good affinity), but they are also basic stable. Certain CDR sequences were identified as significantly increasing basic stability, especially when combined with modified sdAb frameworks, where the resulting sdAbs became even more stable. Furthermore, when sdAbs were fused with additional polypeptide portions of protein domains derived from Staphylococcus aureus protein A (SpA), the resulting fusion proteins exhibited even better properties.

[0081] Previous sdAbs capable of binding AAV9 exist in the prior art, for example, disclosed in CN116751284, but they are not specific for AAV9, and more importantly, they are not as basic as the sdAb of the present invention. CN116396381 discloses an sdAb that is allegedly capable of binding to multiple serotypes (including AAV9) and is acid-base resistant. However, when tested, the sdAb has extremely low affinity for AAV9. Five variants (sequences represented as SEQ ID NO: 7, 14, 21, 28, and 35, as described in claim 2 of CN116396381) were expressed and analyzed in the same manner as the AAV9-selective single-domain antibody of the present invention, and of these five, one (SEQ ID NO: 14) was not expressed, while the other four showed very low interactions (~100 Ru, instead of the 1000 Ru of the polypeptide described above for the present disclosure), the binding affinity being a similar “sticky / nonspecific” interaction, i.e., no affinity. Therefore, the affinity is too low, and the binding with AAV9 may be questionable. Furthermore, regarding the so-called "acid-base tolerance," for example, in Example 7 of CN116396381, a CBS buffer (the buffer is not further specified, only the pH is indicated) is presumably a sodium carbonate-sodium bicarbonate buffer with different pH values. It is well known that the type, ionic strength, and molar concentration of the buffer greatly affect the stability of the peptide. The buffer used in CN116396381 does not correspond to or resemble the NaOH buffer used for in-situ cleaning (CIP), and the basic stability of the protein cannot be determined. It should be noted that to achieve basic stability as defined in this disclosure, the peptide should be able to undergo CIP with at least 0.05 M, for example, 0.05-1 M NaOH or KOH. Therefore, no basic washing was performed, and basic stability was not demonstrated.

[0082] The CDR of an sdAb can have a variable number of amino acids. CDR1 can comprise a sequence of 9-13 amino acids. In the exemplary polypeptide shown herein, CDR1 is formed of 9 amino acids. However, it is envisioned that the amino acid sequence of CDR1 described herein could be supplemented with up to 4 additional amino acids, particularly at the C-terminus. In the Kabat numbering system, CDR1 can be formed from amino acid positions 27, 28, 29, 30, 31, 32, 33, 34, and 35 (9 amino acids) or positions 27-35 and up to 35d (and thus up to 13 amino acids).

[0083] CDR2 may comprise 10-13 amino acids. In the exemplary polypeptide shown herein, CDR2 is formed from 10 amino acids. In the Kabat numbering system, CDR2 may be composed of positions 50, 51, 52, 52a, 53, 54, 55, 56, 57, and 58.

[0084] CDR3 may comprise up to 18 amino acids. In the exemplary polypeptide shown herein, CDR3 is formed from 17 amino acids. In the Kabat numbering system, CDR3 may be formed from amino acid positions 95, 96, 97, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, 100g, 100h, 100i, 101, and 102.

[0085] More specifically, in the sdAb variant of the present invention, CDR1 may be constituted by the amino acid positions outlined in Table 2a below, according to the Kabat numbering system.

[0086] Table 2a: Location of CDR1 Location (Kabat) 27 28 29 30 31 32 33 34 35 35a 35b 35c 35d Position in SEQ ID NO: 1 1 2 3 4 5 6 7 8 9 - - - - Available amino acids according to the contents of this disclosure R, L, S T, R L, F S D, N, E Y, N, F T M A, G Preferred amino acids R T L S D Y, F T M G - - - - Therefore, in CDR1 / SEQ ID NO:1, for each position, the amino acid can be independently selected from the available and preferred amino acids indicated in Table 2a. The inventors have found that the amino acid sequence of CDR1 has a significant impact on the binding properties and stability of the antigen-binding peptide, and has the potential to improve one or both of these features. Overall, it was found that amino acid substitutions in CDR1 affect binding capacity and stability to a greater extent than amino acid substitutions in CDR3, which appears to be resistant to even greater degrees of variation.

[0087] Specifically, amino acids at Kabat positions 30, 33, and 34 (corresponding to positions 4, 7, and 8 of SEQ ID NO: 1) have been advantageously fixed as serine (S), threonine (T), and methionine (M), respectively. Meanwhile, other positions of CDR1 (e.g., Kabat position 32) have been found to allow for some variability, with acceptable or further improved binding capacities. At Kabat positions 27-29, an amino acid sequence RTL is considered particularly advantageous; however, an RTF is also envisioned.

[0088] Table 2b shows exemplary CDR1 sequences of the sdAb disclosed herein.

[0089] Table 2b: Exemplary CDR1 sequences CDR1 variant SEQ ID NO: RTFSDYTMG 9 LTLSDYTMG 10 RTLSNYTMG 11 RTLSEYTMG 12 RTLSDFTMG 13 RTLSDNTMG 14 RTLSDYTMA 15 RTLSDYTMG 16 RTLSDFTMA 17 In some implementations, CDR1 may be selected from SEQ ID NO:9-17, such as SEQ ID NO:13, 14, 16 and 17.

[0090] In the sdAb variant of the present invention, CDR2 can be formed from the amino acid positions outlined in Table 3a below, according to the Kabat numbering system.

[0091] Table 3a: CDR2 Location Location (Kabat) 50 51 52 52a 52b 52c 52d 53 54 55 56 57 58 Position in SEQ ID NO: 2 1 2 3 4 - - - 5 6 7 8 9 10 Available amino acids according to the contents of this disclosure A, S, L, I, V I, V S, T W S G A, S Y, F T H, K, Y, F Preferred amino acids A I S W - - - S G A Y T K, Y, F It has been found that certain amino acid substitutions in CDR2 affect the binding properties and stability of antigen-binding peptides, and have the potential to improve one or both of these characteristics. On the other hand, it has been found that amino acid substitutions at some positions do not improve binding capacity and / or stability, or even impair binding capacity and / or stability; therefore, positions 4, 5, 6, and 9 of CDR2 were selected, as indicated in Table 3a.

[0092] Therefore, in CDR2 / SEQ ID NO:2, for each position, the amino acid can be independently selected from the available and preferred amino acids indicated in Table 2a. For example, optionally, in SEQ ID NO:2, X2 can be I; X7 can be A; X8 can be Y; and / or X 10 It can be K. In some embodiments, in CDR2 / SEQ ID NO: 2, X2 is I and X8 is Y. In some embodiments, in CDR2 / SEQ ID NO: 2, i) X1 is A, or ii) X1 is S, L or I and X7 is A.

[0093] Table 3b shows exemplary CDR2 sequences of the sdAb variants disclosed herein.

[0094] Table 3b: Exemplary CDR2 amino acid sequences CDR2 variant SEQ ID NO AVSWSGSFTY 21 AISWSGSFTY 22 SISWSGAYTY 23 LISWSGAYTY 24 IISWSGAYTY 25 AISWSGAYTY 26 AVSWSGAYTY 27 AVSWSGAYTK 28 AISWSGAYTK 29 AISWSGAYTH 30 AISWSGAYTF 31 Therefore, in some implementations, CDR2 may be selected from SEQ ID NO: 21-31, for example from SEQ ID NO: 26, 28, 29 and 31.

[0095] In the sdAb variants of this disclosure, CDR3 can be constructed from the amino acid positions outlined in Table 4a below, according to the Kabat numbering system. The sdAbs of the present invention are resistant to a relatively high degree of variability in the CDR3 amino acid sequence without compromising binding capacity or basic stability. However, it has been found that certain amino acids are particularly advantageous for certain positions for the purpose of improving binding capacity or basic stability. Therefore, for CDR3, amino acids (available and preferred, respectively) are selected as indicated in Table 4a.

[0096] Table 4a: CDR3 Location Therefore, in CDR3 / SEQ ID NO:3, for each position, the amino acid can be independently selected from the available and preferred amino acids indicated in Table 4a. For example, optionally, in SEQ ID NO:3, X2 is P; X7 is S; X8 is K, N, or R, such as K or R; X9 is K; X 10 It is A or T; X11 It is T; X 13 It is A; and / or X 17 It's Y.

[0097] Optionally, in SEQ ID NO: 3, at least one of X8 and X9 can be K.

[0098] Optionally, in SEQ ID NO: 3, at least one of X5 and X6 can be L, and preferably both X5 and X6 are L.

[0099] Optionally, in SEQ ID NO: 3, X 13 It is A, and preferably X. 10 It is A.

[0100] Optionally, in SEQ ID NO: 3, X 14 It is D, and X 16 It is D.

[0101] Table 4b shows exemplary CDR3 sequences of the sdAb disclosed herein.

[0102] Table 4b: Exemplary CDR3 sequences CDR3 sequence SEQ ID NO CDR3 sequence SEQ ID NO GPTGPLSRRSSPPDYDY 34 GPTGLLSRKSTPADYDY 54 GSTGPLTRRSTPPDYDY 35 GPTGLLSKKATPADYDY 55 APTGLLSKKATPADYDY 36 GPTGLLSKKTTPADYDY 56 GSTGLLSKKATPADYDY 37 GPTGLLSKKAAPADYDY 57 GPTGLLSKKSTPADYDY 38 GPTGLLSKKATSADYDY 58 GPTGPLSKKATPADYDY 39 GPTGLLSKKATTADYDY 59 GPTGTLSKKATPADYDY 40 GPTGLLSKKATPRDYDY 60 GPTGLISKKATPADYDY 41 GPTGLLSKKATPANYDY 61 GPTGLLAKKATPADYDY 42 GPTGLLSKKATPAEYDY 62 GPTGLLSNKATPADYDY 43 GPTGLLSKKATPAQYDY 63 GPTGLLSQKATPADYDY 44 GPTGLLSKKATPADFDY 64 GPTGLLSRKATPADYDY 45 GPTGLLSKKATPADYNY 65 GPTGLLSKRATPADYDY 46 GPTGLLSKKATPADYEY 66 GPTGLLSKHATPADYDY 47 GPTGLLSKKATPADYQY 67 GPTGLLSNRATPADYDY 48 GPTGLLSKKATPADYDF 68 GPTGLLSQRATPADYDY 49 GPTGLLSKKATPADYDR 69 GPTGLLSRRATPADYDY 50 GPTGLLSRKTTPADYQY 70 GPTGLLSNHATPADYDY 51 GPTGLLSKKATPPDYDY 71 GPTGLLSQHATPADYDY 52 GPTGPLSKKTTPADYDY 72 GPTGLLSRHATPADYDY 53 GPTGLLSQKTTPADYDY 173 Therefore, in some implementations, CDR3 may be selected from SEQ ID NO: 34-72 and 173, for example selected from SEQ ID NO: 36, 37, 39, 40, 42-46, 48-50, 55, 56, 62, 64, 68, 70-72 and 173.

[0103] Exemplary CDR sequence combinations are listed in Table 5. This table also relates to exemplary sdAb variant sequences having said CDR combinations. All listed peptides show good or excellent binding to AAV9, as demonstrated in the examples.

[0104] Table 5 For example, any combination of CDRs may be present in the sdAb variant of this disclosure, wherein CDR1 is selected from SEQ ID NO:9-17, for example from SEQ ID NO:13, 14, 16 and 17; CDR2 is selected from SEQ ID NO:21-31, for example from SEQ ID NO:26, 28, 29 and 31; and CDR3 is selected from SEQ ID NO:34-72, for example from SEQ ID NO:36, 37, 39, 40, 42-46, 48-50, 55, 56, 62, 64, 68, 70-72 and 173.

[0105] Further variants that share SEQ ID NO:92 with CDR1 and CDR2 and differ only slightly in CDR3 are represented by SEQ ID NO: 106, 107, 109-124, 126-130 and 132-139.

[0106] Therefore, for example, the sdAb variant may comprise a) an amino acid sequence selected from SEQ ID NO: 75-93, 95-98, 101, 102, 105-108, 109-124, 126-146, 174, for example selected from SEQ ID NO: 92, 97, 109, 116, 131, 140-146 and 174; or b) an amino acid sequence having at least 90%, for example at least 95%, identity with the sequence as defined in a), provided that the CDR is as defined for SEQ ID NO: 1-3. Optionally, sdAb variants according to option b) having less than 100% identity with any of the said sequences may differ in terms of the amino acid residues of the CDR (still within the definition of SEQ ID NO: 1-3) and / or the framework amino acid residues at Kabat positions 6, 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b and / or 89.

[0107] As used in this disclosure, the terms “capable of binding AAV9,” “AAV9 binding,” “capable of interacting with AAV9,” and “interacting with AAV9” refer to events or properties of the peptide that can be detected, for example, by ELISA or by surface plasmon resonance (SPR) technology.

[0108] AAV9 interactions can be tested experimentally in which the peptide to be tested is immobilized on the sensor chip of a surface plasmon resonance (SPR) instrument, and a liquid sample containing AAV9 (the target or analyte) passes through the chip. Alternatively, the target (here, AAV9) can be immobilized on a surface, and the peptide to be tested is contained in a liquid sample that passes through the surface. The results obtained through such experiments can then be interpreted by a technician to at least establish a qualitative measure of the binding affinity of the peptide to AAV9.

[0109] AAV9-binding peptides can "selectively" bind to AAV9, meaning they bind to AAV9 but not to other AAV serotypes, or bind to other AAV serotypes to a much lower degree. The term "selectivity" for a binding peptide's target, sometimes called "specificity," means that the binding peptide will bind to the target with high affinity but typically not to other antigens. Selective or specific binding peptides / single-domain antibodies will not cross-react with targets other than the intended antigen, or will cross-react with targets other than the intended antigen to a low degree. Therefore, "specific" binding means that the binding protein binds to its target (i.e., AAV9) in a way that distinguishes it from binding to non-target molecules; more specifically, the binding peptide binds to its target (AAV9) with a greater binding affinity than it binds to other molecules. That is, the binding peptide does not bind to other non-target molecules, or binds to them to a lesser degree, or binds to such molecules with a lower affinity than it binds to AAV9. In the case of this invention, the binding peptide binds to AAV9 but not to other serotypes (or binds to very low levels). For example, the interaction with other AAV serotypes can be less than 20% of the AAV9 interaction response, such as less than 10% or less than 5%, as measured on a Biacore instrument, as detailed in the examples below. Other serotypes refer to other different serotypes, such as AAV2, AAV5, and AAV8. As shown in Example 5B below, the peptide of this invention selectively binds to AAV9 and has no affinity or very low affinity for other serotypes. It should be noted that closely related sequence variants, such as AAV-PHP.eB, are not considered different serotypes in this sense and are therefore not included in the "other serotypes" that do not bind.

[0110] Compared to known sdAb-based AAV9 affinity ligands, the AAV9-binding peptides disclosed herein exhibit improved AAV9 binding properties. Specifically, the improved binding property is the binding capacity. Therefore, the AAV9-binding peptides of the present invention can have improved AAV9 binding capacity.

[0111] Furthermore, the inventors have discovered that, in addition to exhibiting an initial high binding capacity for AAV9, the peptide retains a high AAV9 binding capacity even after basic treatment. This improved basic stability is a significant advantage in the context of affinity chromatography, as chromatographic materials are typically cleaned with basic reagents (e.g., NaOH or KOH) between purification cycles. Increased basic stability of the affinity ligand means that the chromatographic material can be used for more purification cycles before the binding capacity becomes unacceptably low.

[0112] Basic stability is typically determined based on the binding capacity after a certain number of basic treatment cycles. In this context, the terms "treatment," "exposure," and "cleaning" are used interchangeably. For example, "improved basic stability" may mean that the peptide can withstand more basic cleaning cycles, or the same number of cycles but with more stringent basic conditions, without compromising binding capacity. Alternatively, "improved basic stability" may mean that after an equal number of cleaning cycles under the same conditions, the peptide retains a higher percentage of its initial binding capacity. Basic stability can be particularly relevant for peptides intended to be immobilized on a solid support.

[0113] Alkaline treatment may include contact or incubation with 0.05-1 M NaOH or 0.1-0.5 M NaOH, such as 0.3-0.5 M NaOH, for 5-15 minutes, such as 10 minutes (600 seconds) or a period of about 10 minutes.

[0114] Preferably, the AAV9 peptide of the present invention retains a high AAV9 binding capacity after repeated cleaning cycles including 600 s exposure to 0.3 or 0.5 M NaOH. For example, compared to the AAV9 binding capacity after the first NaOH exposure cycle (i.e., excluding the binding capacity recorded for the first cycle from the comparison), the AAV9 binding peptide disclosed herein retains at least 50% of its AAV9 binding capacity after at least 8 repeated binding cycles followed by cleaning with 0.5 M NaOH.

[0115] In an embodiment, the AAV9-binding peptide retains at least 50% binding capacity after at least 10 cycles of alkaline exposure, for example, after at least 12 cycles. More preferably, the AAV9-binding peptide retains at least 50% binding capacity after at least 20, 22, or 24 cycles of exposure to 0.5 M NaOH. A higher number of cycles retaining at least 50% binding capacity indicates improved alkaline stability. Furthermore, a higher binding capacity retained after the same number of cycles of exposure to a cleaning liquid (e.g., at least 0.1 M NaOH, such as 0.3 M NaOH or 0.5 M NaOH) indicates improved alkaline stability. In an embodiment, after 10 cycles of contact with a cleaning liquid, the peptide retains at least 50%, for example, at least 60%, at least 70%, at least 80%, or at least 90% of its initial target-binding capacity, and / or after 20 cycles of contact with a cleaning liquid, the peptide retains at least 50%, at least 60%, for example, at least 70%, or at least 80% of its initial target-binding capacity. In the implementation, after 10 cycles of contact with an alkaline cleaning liquid, the peptide retains at least 60%, at least 70%, at least 80%, at least 90%, or for example at least 95% of the target entity binding capacity of the second cycle, and / or after 20 cycles of contact with an alkaline cleaning liquid, the peptide retains at least 60%, at least 70%, at least 80%, at least 90%, or for example at least 95% of the target entity binding capacity of the second cycle. The cycles of exposure to the alkaline cleaning liquid can be chromatographic cycles as described herein, or alkaline stability assessment cycles as described elsewhere in the text.

[0116] It will be understood that each binding cycle is followed by a cleaning step. The cleaning step may be, for example, an incubation of approximately 5-15 minutes in contact with 0.1-0.5 M NaOH, such as approximately 5, 10, or 15 minutes. For example, the incubation time in contact with 0.5 M NaOH may be 5 ± 0.5 minutes, 6 ± 0.5 minutes, 7 ± 0.5 minutes, or 8 ± 0.5 minutes, 9 ± 0.5 minutes, 10 ± 0.5 minutes, 11 ± 0.5 minutes, 13 ± 0.5 minutes, 14 ± 0.5 minutes, or 15 ± 0.5 minutes. The incubation may be carried out, for example, at 22 ± 2 °C.

[0117] The polypeptides of the present invention may have a CDR that promotes improved basic stability.

[0118] Furthermore, the peptides disclosed herein may possess a stable framework, meaning that at least one framework region is modified relative to the corresponding framework region of SEQ ID NO: 4 in a manner that promotes increased basic stability of the peptide. Therefore, the peptide as a whole may possess improved basic stability.

[0119] For example, certain amino acid positions in the frame region of sdAbs have been found where amino acid residues not only tolerate certain mutations without impairing functionality, but can even lead to performance improvements in the resulting sdAb variants, particularly improvements in basic stability. Specifically, 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 sdAb variants: positions 6, 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b, and 89. Therefore, the antigen-binding polypeptide may comprise a single-domain antibody variant comprising an amino acid sequence in which at least one of amino acid positions 6, 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b, and 89 is modified relative to the corresponding amino acid of SEQ ID NO: 4 and wherein the sdAb frame has increased basic stability compared to SEQ ID NO: 4.

[0120] Particularly advantageous modifications are outlined in Table 6. Thus, the antigen-binding polypeptide may optionally comprise a single-domain antibody variant comprising an amino acid sequence in which at least one, for example two, for example three, four, five, six, seven, eight, nine, or ten residues at Kabat positions 19, 23, 24, 40, 43, 44, 45, 76, 79, and / or 89 are selected from the stable amino acids indicated in Table 6. For example, at least eight, for example at least nine, of these positions may have correspondingly selected amino acids.

[0121] Table 6: Exemplary Stable Frame Modifications Kabat location Stable amino acids Preferred stable amino acids 19 R, S, K, T R, T 23 T, S, V T 24 I, V, S 40 R, I, K, T R, I 43 R, K, Q, E, G R, K 44 E, Q, A, D, G 45 R, I, L R 76 N, Q 79 Y, W, F Y, W, F 89 V, I V, I Figure 2a -c schematically illustrates an exemplary structure of the antigen-binding polypeptide of this disclosure. Figure 2a In this study, antigen-binding polypeptide 200 comprises an N-terminal amino acid sequence 201, an sdAb variant 202, and a C-terminal amino acid sequence 203. Notably, both the N-terminal amino acid sequence 201 and the C-terminal sequence 203 are optional.

[0122] The antigen-binding polypeptide may optionally be used in the following ways: Figure 2b The multimeric antigen-binding polypeptide (“multimer”) illustrated in section -c is provided in the form of a schematic representation. Figure 2bIn this embodiment, the multimeric polypeptide 220 comprises a plurality of single-domain antibody 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 2-6 amino acids, such as 4 amino acids. The C-terminal sequence 203 is optional and may contain a purification tag, optionally including a linker. The sdAb variant units 202 may be identical in their amino acid sequences or may differ from each other. For example, the polypeptide may comprise two sdAb variants (“dimers”) or three sdAb variants (“trimers”). Figure 2c An exemplary trimer 230 is shown, which contains three sdAb variants 202.

[0123] Optionally, the multimeric antigen-binding polypeptide may contain at least one additional amino acid sequence that does not represent an sdAb variant, such as a fusion partner polypeptide as described elsewhere in this document for fusion proteins.

[0124] Multimeric variants of the peptides of the present invention may be advantageous because they provide increased binding capacity and / or improved stability compared to peptides containing a single copy of the sdAb variant.

[0125] As indicated above, the antigen-binding polypeptide or polymer may optionally contain additional amino acids located at its N and / or C terminus, in addition to the sdAb variant, for example... Figure 2a As described in -d. Such a polypeptide or polymer should be understood as having one or more additional amino acid residues at the very beginning and / or end of the polypeptide chain, i.e., at the N and / or C-terminus of the polypeptide or polymer. Thus, an antigen-binding polypeptide as defined herein 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, optionally in a continuous sequence. For example, the sequence of additional amino acids may consist of 1-12 amino acids, and an antigen-binding polypeptide may contain several such sequences. The additional amino acids may be remnants from recombinant protein expression, leader peptides, signal peptides (e.g., PelB or OmpA), purification 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. Additional amino acid residues can, 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 in relation to the separation matrix). 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.

[0126] Furthermore, when the antigen-binding peptide is provided as a polymer comprising multiple single-domain antibody units, the additional amino acid residues may not be repeated for each occurrence of the single-domain antibody. For example, when the antigen-binding peptide is provided as a polymer, the additional amino acid may appear only at the N-terminus or C-terminus of the polymer.

[0127] The additional amino residues may be chemically conjugated (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 a peptide linker as described above), to the sdAb or multimer.

[0128] In some embodiments, the polypeptides and / or polymers disclosed above further include one or more tag or coupling elements at the C-terminus or N-terminus, selected from cysteine ​​residues, multiple lysine residues, and multiple histidine residues, and combinations thereof. Exemplary tag or coupling peptides include (His)6, (His)6-Cys, and AEAAAAKHHHHHHC (SEQ ID NO:159).

[0129] The coupling element can be, for example, a single cysteine ​​residue at the C-terminus. The coupling element can be directly attached to the C or N-terminus, or it / they can be linked via a linker containing up to 15 amino acids, such as 1-5, 1-10, or 5-10 amino acids. The segment should preferably be sufficiently stable even in alkaline environments without impairing the protein's properties. For this purpose, it is advantageous if the segment does not contain asparagine. It may also be advantageous if the segment does not contain glutamine. The advantage of having a C-terminal cysteine ​​residue is that terminal coupling of the protein can be achieved through the reaction of the cysteine ​​thiol group with an electrophilic group on the carrier. This provides excellent motility for the coupled protein.

[0130] As those skilled in the art will understand, the construction of polymers (e.g., as fusion proteins) typically involves the use of linkers between monomeric moieties to be fused. A monomeric moieties can refer to single-domain peptides as described herein. Those skilled in the art are familiar with different types of linkers with varying 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 to improve its folding. Linkers can also ensure a certain spatial distance between two sdAb variants or between an sdAb variant and a fusion partner peptide, in either case which may facilitate target access to the binding surface of the sdAb variant. The presence of linkers within the fusion protein generally does not significantly affect the target-binding capacity of a properly folded antigen-binding peptide monomer, such as a single-domain antibody.

[0131] Therefore, a polymer as defined herein may further include at least one linker. For example, the linker is present between each monomer within the polymer. The linker may be selected, for example, from flexible amino acid linkers, rigid amino acid linkers, and cleavable amino acid 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 polypeptide or its subunits disclosed herein may be directly linked to each other via peptide bonds between the C-terminus and N-terminus of the polypeptide. Alternatively, two or more monomers (in other words, monomer units or portions) within the polymer may be linked by elements comprising oligomer or polymer species, 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 include 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 the polymer was cut. The number of additional amino acid residues may be, for example, 15 or fewer, such as 10 or fewer, or 5 or fewer.

[0132] Antigen-binding polypeptides can be provided in the form of fusion proteins. As indicated above, it may contain multiple sdAb variants produced as a single fusion protein, optionally with additional amino acids, including linkers as described above.

[0133] In some embodiments, the antigen-binding polypeptide can be a fusion protein containing at least one sdAb variant as disclosed herein and an additional polypeptide motif (also known as a fusion partner polypeptide). Figure 3a The diagram schematically illustrates a fusion protein 300 comprising a first polypeptide moiety 200 and a second polypeptide moiety 301 along an N-terminal to C-terminal direction, linked by an optional peptide linker 204. In this figure, the second polypeptide moiety 301 is located at the C-terminus of the first polypeptide moiety 200. It is also contemplated that the second polypeptide moiety may be located at the N-terminus of the first polypeptide moiety. The first polypeptide moiety 200 may contain only... Figure 2a The single-chain polypeptide 202 described herein may be composed of or may comprise or be composed of a single-chain polypeptide 202 and additional amino acids (e.g., additional amino acid sequences 201 and / or linkers 204). The second polypeptide portion 301 may be referred to as a fusion partner polypeptide.

[0134] Fusion coupler peptide 301 can be a peptide that improves or adds at least one functional or desirable property. For example, the fusion coupler peptide can be a stable peptide that further improves the stability of the antigen-binding peptide, such as basic stability. As another example, the fusion coupler peptide can be a peptide that improves the expression of the antigen-binding peptide in recombinant cells, such as *E. coli*. As yet another example, the fusion coupler peptide can provide the opportunity to purify the antigen-binding peptide by affinity chromatography.

[0135] The fusion protein of this disclosure may specifically comprise one or more sdAb variants fused to an α-helical polypeptide (e.g., a protein domain). The α-helical polypeptide may contain at least one α-helix, such as at least two α-helices, such as three, four, five, or six α-helices, such as 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 coupler polypeptide may contain an amino acid sequence that has at least 80% identity with SEQ ID NO: 161 or 162, such as at least 85% or at least 90% identity.

[0136] Furthermore, the fusion protein of this disclosure can be a multimeric protein, meaning it can contain more than one sdAb or more than one fusion partner polypeptide. Multimeric variants containing multiple copies of the sdAb may be advantageous because they provide increased binding capacity compared to fusion proteins containing only one copy. Multimeric fusion proteins containing multiple fusion partner polypeptides can provide improved stability.

[0137] Therefore, the fusion protein may optionally be provided as a multimeric fusion protein (“multimer”) containing at least two antigen-binding peptides and / or at least two fusion partner peptides, such as Figure 3b The -c option is used for illustration. Figure 3b In this embodiment, the multimeric fusion protein 310 comprises multiple units of antigen-binding polypeptide 200 arranged sequentially from the N-terminus to the C-terminus, and these units are optionally linked to each other by a linker sequence 204, which may be the same or different for each occurrence. The antigen-binding polypeptides 200 may be identical in their amino acid sequences or may be different from each other. Figure 3bThe diagram shows three antigen-binding polypeptides 200 (“trimers”), but it is envisioned that the fusion protein may contain any suitable number of the antigen-binding polypeptides 200. For example, the fusion protein may contain at least two, three, four, five, or six antigen-binding polypeptide units. The fusion protein may contain, for example, up to ten antigen-binding polypeptides 200.

[0138] In addition, the fusion protein may contain at least two, for example three, four, five or six units of the fusion partner polypeptide, for example up to ten units. Figure 3c A fusion protein 320 is described, comprising a first antigen-binding polypeptide 200 and three polypeptide moieties 301. The linker 204 is optional and may be the same or different for each occurrence. The polypeptide moieties 301 may be identical or different in 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 fusion partner polypeptides. In cases where the fusion protein comprises multiple antigen-binding polypeptides and multiple fusion partner polypeptides (e.g., two or three each), it is envisioned that the different polypeptide moieties 200, 301 may be arranged in an alternating manner, rather than sequentially arranged with the same type of polypeptides, such as... Figure 3b (for antigen-binding polypeptide 200) and Figure 3c (For fusion partner polypeptide 301) as described. For example, a multimeric fusion protein of each polypeptide containing two units may have the following general structure: [antigen-binding polypeptide]-[fusion partner polypeptide]-[antigen-binding polypeptide]-[fusion partner polypeptide].

[0139] More typically, the fusion protein according to this disclosure may have the following structure: ([A-L1)) m -[Z-L2] n ) p Wherein A represents the polypeptide as described herein; Z represents another polypeptide motif (fusion partner polypeptide) as described herein; L1 may or may not be present for each occurrence, and when present, represents a linker or spacer group; L2 may or may not be present 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 motifs A and Z in the fusion protein, can be at most 10, for example, at most 8.

[0140] In some implementations, n=1, m=1, and p=1. In other implementations, m is 2 or 3, n is 1, and p is 1. In still other implementations, m is 1, n is 1, and p is 2 or 3. For reference purposes, in Figure 3a In the exemplary fusion protein, m=1, n=1, and p=1. Figure 3b In the exemplary fusion protein, m=3, n=1, and p=1. Figure 3c In the exemplary fusion protein, m=1, n=3, and p=1. In the candidate construct "6B-3" of Example 6B (see Table 17), m=1, n=1, and p=3. In instances where the fusion protein comprises at least two fusion partner polypeptides, at least one is preferably 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.

[0141] At the N-terminus of a 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 a fusion protein (e.g., a multimeric fusion protein), a tag or spacer or another short amino acid sequence may optionally be provided as needed.

[0142] In the implementation scheme, the fusion partner polypeptide is not located at or near the N-terminus of the fusion protein.

[0143] For clarity, any reference in this document to antigen-binding peptides of this disclosure also includes their multimeric variants and fusion proteins, unless otherwise indicated.

[0144] The antigen-binding polypeptide of the present invention can be produced by recombinant protein production using genetically modified host cells, through conventional biotechnological methods.

[0145] Therefore, in a further aspect, this disclosure provides isolated nucleic acids encoding antigen-binding polypeptides as described herein; expression vectors containing said nucleic acids; and host cells containing said expression vectors.

[0146] This disclosure also includes a method for producing an antigen-binding polypeptide as described herein, comprising culturing the host cell under conditions that allow expression of the antigen-binding polypeptide from its expression vector, and isolating the antigen-binding polypeptide.

[0147] The host cell can be a prokaryotic cell, such as bacteria, or a eukaryotic cell. An exemplary prokaryotic host cell is *Escherichia coli*. Suitable eukaryotic cells can be yeast cells, such as *Saccharomyces cerevisiae* and *Pichia pastoris*, or animal cells, such as insect cells, or mammalian cells commonly used in the biotechnology field for protein production, such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells.

[0148] After isolating the antigen-binding peptide from the host cell (which can be done by conventional methods such as harvesting, clarification, and / or filtration), the antigen-binding peptide can be purified, for example, by conventional methods known in the art. For example, in cases where the antigen-binding peptide contains a histidine tag, the purification steps may include immobilized metal affinity chromatography (IMAC). Other purification methods that can be used include affinity chromatography. For example, affinity ligands that bind to the framework region of the sdAb variant can be used, or affinity chromatography can use affinity ligands based on the SpA domain, wherein the affinity ligand binds to the VH region of the antigen-binding peptide, such as VH3. Therefore, the sdAb variants of this disclosure may be capable of VH3-SpA domain or variants thereof interacting.

[0149] Alternatively, in cases where the antigen-binding polypeptide forms part of a fusion protein, the affinity ligand can bind to the fusion partner, such as an affinity tag included in the amino acid sequence of the fusion partner polypeptide.

[0150] Alternatively, antigen-binding peptides as disclosed herein can be produced 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 peptides with protected reactive side chains, - Removal of protecting groups from reactive side chains, and - Folding peptides in aqueous solutions.

[0151] Generally, the antigen-binding peptides of the present invention can be used to capture target entities to which the antigen-binding peptides have an affinity. However, preferably, the antigen-binding peptides themselves are not intended to be used as therapeutic compounds, and preferably are not intended for in vivo use. Therefore, the antigen-binding peptides of the present invention can be used to capture target entities in vitro.

[0152] The antigen-binding peptide can be used to detect target entities within a sample or to separate target entities from other components in the sample. The antigen-binding peptide can be used for analytical separation of the target entity, or it can be used for preparative purification of the target entity. 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 the antigen-binding peptide using this disclosure can be performed in the context of sensor applications.

[0153] An in vitro method for detecting AAV9 viral particles in a sample may include: providing a sample containing AAV9 viral particles; contacting the sample with an antigen-binding polypeptide, polymer, or fusion protein as described herein, under conditions that allow sdAb variants to bind to the AAV9 viral particles, wherein the binding event generates a detectable signal; and detecting the detectable signal. Suitable methods for generating a detectable signal in response to a binding event are known in the art.

[0154] Specifically, the antigen-binding peptide of the present invention can be used in applications involving interactions between the antigen-binding peptide and its target entity, wherein the antigen-binding peptide or target entity is coupled to a carrier. When the antigen-binding peptide is coupled to or immobilized to a carrier, it remains able to bind to its target entity.

[0155] In the case of conjugation of the antigen-binding peptide to a carrier, this can be referred to as an adsorbent material or affinity trapping 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. Separation matrices as described herein are adsorbent materials designed for the separation of target entities from other components. The conjugation of the antigen-binding peptide to the carrier can optionally be provided by a C-terminal amino acid sequence suitable for conjugation to the carrier. For example, a cysteine-terminated tag or spacer can be provided for conjugation via a thioether bond, as described in more detail below. In the case where the antigen-binding peptide forms part of a fusion protein, the C-terminal fusion partner peptide may have such a C-terminal amino acid sequence suitable for conjugation to the carrier described below.

[0156] When the antigen-binding polypeptide is not coupled to a carrier, it can be used, for example, in detection assays where a target or analyte is present on a surface and in contact with the antigen-binding polypeptide present in solution. In such cases, a reporter entity is envisioned for generating a detectable signal, wherein the reporter entity is capable of binding the antigen-binding polypeptide, or, in the case of a fusion protein, of binding a fusion coupler. The reporter entity can be, for example, a fluorescently labeled or radiolabeled entity, as known in the art. If the antigen-binding polypeptide or fusion protein is capable of binding IgG, the reporter entity can be IgG-based.

[0157] The antigen-binding 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 bound impurities from 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.

[0158] The carrier may be a solid carrier and optionally a porous material.

[0159] The carrier may be or comprise a surface thereon on which the antigen-binding polypeptide is coupled. Examples of such carrier materials include conventional protein-binding carriers and surfaces, such as chips, plates, wells, and sheets. 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. Particles or beads may be porous or non-porous. 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 cases of bulk materials, packed beds, and expanded beds, the separation procedure typically follows conventional chromatography with a concentration gradient. In the case of pure suspensions, batch mode will be used.

[0160] The carrier can be prepared from any suitable material, as outlined in more detail below. As a non-limiting example, conventional affinity separation matrices are typically organic in nature and based on exposing hydrophilic surfaces to the aqueous medium used, i.e., on their outer surfaces and, if present, also on their inner surfaces, polymers containing hydroxyl (-OH), carboxyl (-COOH), formamide (-CONH2, possibly in N-substituted form), amino (-NH2, possibly in substituted form), oligomeric, or polyoxyethylene oxy groups.

[0161] The antigen-binding polypeptide can be linked to the carrier using known coupling techniques, such as those utilizing thiol, amino, and / or carboxyl groups present in the antigen-binding polypeptide. Biepoxides, epichlorohydrins, CNBr, N-hydroxysuccinimide (NHS), etc., are well-known coupling agents. A spacer molecule can be introduced between the carrier and the antigen-binding polypeptide, which improves the availability of the first polypeptide moiety and / or promotes the chemical coupling of the antigen-binding polypeptide to the carrier. Depending on the properties of the antigen-binding 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 increase coupling control, it may be preferred that the sdAb variant itself does not contain any histidine residues. However, in such cases, the antigen-binding polypeptide as a whole may contain a histidine tag, such as a (His)6 tag. Alternatively, the antigen-binding polypeptide can be linked to the carrier via non-covalent bonding, such as physical adsorption or biospecific adsorption.

[0162] The antigen-binding polypeptide can be coupled to the carrier via a thioether bond. Methods for performing 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 antigen-binding polypeptide, the motility of the coupled antigen-binding polypeptide is enhanced, providing improved binding capacity and binding kinetics. In some embodiments, the antigen-binding 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 carrier, such as an epoxide group, a halool group, etc., resulting in thioether-bridged coupling.

[0163] Regarding the specific materials, the support may comprise polymeric materials. Polymeric materials include natural or synthetic polymers and combinations thereof. For example, the support may comprise a polyhydroxy polymer, such as a polysaccharide. Examples of polysaccharides include, for example, dextran, starch, cellulose, pullulan, agar, agarose, etc., including derivatives thereof. The advantageous characteristics of these polysaccharides can be further enhanced by covalent cross-linking, making them particularly suitable for enzyme immobilization using various derivatization methods (utilizing chemical modification of the polymer's hydroxyl moiety), wherein the hydroxyl functionalities can be partially or completely derivatized. These derivatives and methods for derivatizing polysaccharides are well known to those skilled in the art. For example, agarose derivatives include, for example, agarose-protein A / G / L, agarose-biotin, agarose-carboxyl, agarose-amine, agarose-sulfonate, and agarose-maltose. 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 processes. The carrier may comprise agar or agarose, such as cross-linked agarose. Such a carrier used in this invention can be readily prepared by standard methods, such as reverse-phase suspension gelation (SHjerten: 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). This makes the matrix more suitable for high flow rates.

[0164] Synthetic polymers that can be used as carrier materials include polyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamide, polymethacrylamide, etc. In the case of hydrophobic polymers (e.g., matrices based on divinyl and monovinyl-substituted benzenes), the matrix surface can be hydrophilized to expose hydrophilic groups, as defined above, to the surrounding aqueous liquid. Such polymers are readily produced using standard methods. Alternatively, commercially available products such as SOURCE™ (Cytiva™) can be used.

[0165] Alternatively, the solid carrier according to the present invention comprises an inorganic carrier, such as silicon dioxide, zirconium oxide, etc.

[0166] In this embodiment, the antigen-binding 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 bulk material. Examples of porous polymer membranes include Mustang. TM Membrane (Cytiva) and Sartobind TM The membrane (Sartorius) is used. The fiber carrier can be based on electrospun polymer fibers or cellulose fibers, optionally nonwoven fibers. The fiber matrix can therefore be a nonwoven fiber matrix. The fibers can have a cross-sectional diameter of 10-1000 nm, for example 200-800 nm, 200-400 nm, or 300-400 nm. Such fiber carriers are seen in the HiTrap Fibro™ device (Cytiva™). Alternative fiber carriers are disclosed, for example, in WO2019 / 137869 and WO2018 / 011600.

[0167] The adsorbent materials or separation matrices described herein can be used for the same purposes mentioned above for the antigen-binding peptides.

[0168] Therefore, in one aspect, the present invention provides chromatographic materials comprising separation matrices as disclosed herein, and chromatographic columns or devices comprising such separation matrices.

[0169] On the other hand, the adsorbent material or separation matrix can be a sensor surface designed for use in sensors or other detection or quantification devices.

[0170] This invention provides a method for separating or isolating a target entity, wherein a separation matrix as disclosed herein is used. In some embodiments, the method includes contacting a liquid sample containing the target entity with a separation matrix as disclosed herein. Contact is made under conditions where the target entity can bind to the first polypeptide moiety. The method may further include washing the separation matrix with a washing liquid, eluting the target entity from the separation matrix with an elution liquid, and optionally cleaning the separation matrix 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, such as NaOH or KOH containing at least 0.05 M, for example 0.05-1 M, for example 0.05-0.5 M, for example at least 0.1 M, for example 0.1-0.5 M, for example 0.3 M or 0.5 M. The contact (incubation) time may be at least 10 minutes. The binding, elution and cleaning steps may advantageously be repeated at least 10 times, for example at least 20 times.

[0171] Technicians will understand that the liquid sample to be purified can be any sample containing the target entity to be purified or isolated from the environment. The sample may be obtained from cell cultures, such as clarified cell culture harvests, and may have undergone one or more routine 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 harvests. In some cases, the sample may have already undergone one or more filtration steps via tangential flow filtration (TFF).

[0172] Prior to contact with the separation matrix of the present invention, the liquid sample contains a target entity and at least one impurity, such as a host cell protein (HCP) or host cell nucleic acid. The separation matrix of the present invention 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 the said impurities.

[0173] 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.

[0174] Although the invention has been described herein with reference to exemplary embodiments, those skilled in the art will understand that the invention is not limited to these embodiments. As will be appreciated by those skilled in the art, the function of any polypeptide depends on its tertiary structure. Therefore, it is possible to make minor changes to the amino acid sequence of a polypeptide without affecting its function. Thus, this disclosure includes modified variants of antigen-binding polypeptides. Such modified variants at least retain their affinity for the target AAV vector (specifically AAV9).

[0175] The use of the verb "comprising" and its variations does not exclude the presence of elements or steps other than those specified. For example, a polypeptide "comprising" complementarity-determining region 1 (CDR1) may also have further CDRs, such as CDR2 and CDR3, and may have other regions, such as FWRs, and optionally other structures or sequences. The article "a" or "an" preceding an element does not exclude the presence of multiple such elements. The fact that certain measures are recited in mutually different dependent claims does not in itself indicate that a combination of these measures cannot be used to produce benefits. Example

[0176] Example 1: Production of polypeptides This example describes the generation of a glycerol reservoir for the production of candidate peptides used in Examples 3-6 and 8.

[0177] The materials and equipment used are as follows: plasmid DNA of the candidate peptide to be produced; chemocompetent BL21(DE3) E. 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) (PanReacAppliChem™) (BDBACTO™ agar); 14 ml Falcon™ Round-Bottom (Corning™); Inforrs HT shaking incubator.

[0178] The desired amino acid sequences of the candidate peptides were reverse-translated into DNA and optimized to remove rare codons. Plasmid DNA was ordered from expression vectors containing the T5 promoter, OmpA signal peptide, ampicillin resistance, and pUC replication origin. The plasmid DNA was transformed into chemocompetent BL21(DE3) *E. 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 into 4 mL of LB medium supplemented with 100 µg / ml carbenicillin in 14 mL round-bottom tubes and grown overnight at 37°C with stirring at 200 rpm. Glyceryl stock solutions for each variant were prepared using 900 µl of the overnight culture and 500 µl of 50% glycerol and stored at -80°C until further use.

[0179] Example 2: AAV9 binding of sdAb variants with CDR2 / CDR3 variations The ability of candidate peptides to bind AAV9 was investigated using a Biacore™ instrument (Cytiva, Sweden) via surface plasmon resonance analysis. Each candidate peptide contained an sdAb with CDR2 and CDR3 amino acid sequence variations as outlined in Table 7. All candidates shared the same CDR1 amino acid sequence RTLSDYTMG (SEQ ID NO: 16).

[0180] Table 7 Materials and methods Generation of purified candidate peptides Candidate peptides were ordered from GenScript Biotech for protein expression and purification. A construct with the pelB signal peptide and C-terminal tag (AEAAAKHHHHHHC, SEQ ID NO: 161) was expressed in *E. coli*. Protein purification was performed using immobilized metal affinity chromatography (IMAC). The purified candidate peptides were transported to Cytiva, Sweden.

[0181] Biotinylation of purified candidate peptides The purified candidate peptide was biotinylated at the C-terminal cysteine ​​residue using EZ-Link™ maleimide-PEG2-biotin, No-Weigh™ form (ThermoFisherScientific), with a 5X molar excess of biotin. Following biotinylation, the buffer was replaced with PBS using a PD MidiTrap™ G-25 column (Cytiva).

[0182] Biacore analysis of AAV9 binding affinity To evaluate binding to AAV9, candidate peptides were immobilized on a Biacore™ S-series SA chip. AAV9 (2E12 viral particles (vp) / ml) was used as the analyte.

[0183] The materials and equipment used are as follows: Biacore™ S-series SA sensor chip, Biacore™ 8K+ instrument (all from Cytiva, Sweden); AAV9 (prepared in-house); and candidate peptides as described above.

[0184] Immobilization was performed using standard methods in Biacore™ software, by coupling the biotinylated peptide 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. The obtained immobilization level was 5503 + / - 324 RU.

[0185] In each run, the peptide is immobilized in FC2. Multiple sensor chips are used until all candidates are tested.

[0186] Biacore™ method for combined 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 2.5E11 vp / ml, AAV9 1E12 vp / ml, AAV9 2E12 vp / ml.

[0187] All sensor maps are generated by subtracting the reference, and the response values ​​are generated by the difference between the baseline before injection and the signal immediately before the end of injection.

[0188] result Table 8 below reports the fixed levels and maximum binding responses recorded for each analyte concentration. All candidates showed binding to AAV9, and specifically, vh29, vh30, vh33, and vh35-41 demonstrated excellent binding capacity, with even higher binding capacity for the two highest AAV9 concentrations tested.

[0189] Table 8 Candidates Fixed level (RU) Response (RU) 2.5E11 vp / ml Response (RU) 1E12 vp / ml Response (RU) 2E12 vp / ml vh28 5096 4214 7347 7680 vh29 5373 5421 9279 9505 vh30 4879 5542 9102 9306 vh31 5310 4829 7887 8191 vh32 5751 4651 7153 7394 vh33 5274 5327 9096 9295 vh34 4734 4602 7461 7715 vh35 6078 6892 11028 11353 vh36 5787 6829 10743 11052 vh37 5384 6633 10067 10385 vh38 5804 6195 9963 10322 vh39 5452 6702 10551 10954 vh40 5846 7189 10876 11170 vh41 5268 6832 10954 11281 Figure 4a-b is a representative sensor plot, showing the VH36 concentration for each AAV9 concentration: 2E12 vp / ml (upper curve), 1E12 v / ml (middle curve), and 2.5E11 vp / ml (lower curve). Figure 4a ) and vh41 ( Figure 4b The binding capacity of vh28-vh35 and vh37-40 produces curves of similar shape.

[0190] Example 3A: Binding and stability of further candidate peptides with CDR site modification This embodiment further investigated the effect of single-point amino acid modifications in the CDR region of candidate peptides on basic stability and target binding. The candidate peptides tested were expressed as fusion proteins, with each sdAb having SEQ ID NO:161 fused to its C-terminus via a linker. All candidates had an N-terminal OmpA signal peptide and a C-terminal (His)6 tag. The sdAb sequences are provided in Tables 9a-c. The sdAb of SEQ ID NO:92 was used as a reference, into which selected single-point mutations were introduced. As indicated in Tables 9a-c below, each candidate differed from SEQ ID NO:92 at one amino acid position in CDR1, CDR2, or CDR3.

[0191] The following aspects were evaluated: (1) Affinity assessment of AAV9 interactions (tested by injecting high concentrations of AAV9). (2) Basic stability (tested by the reduction in AAV9 binding after treatment with 0.5 M NaOH in increments of 10 ...

[0192] Materials and methods 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 pre-filled 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 incubated in an Infortr HT shaker 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 flask 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 generate 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.

[0193] Samples were purified using a HiTrap MabSelect PrismA™ column (Cytiva). For each sample, the clarified lysate was 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. Proteins were eluted using a stepwise gradient of 50 mM sodium acetate at pH 3.5, with a 5 CV elution time.

[0194] Following the manufacturer's instructions, the concentration of the 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. Protein mass was determined by liquid chromatography-mass spectrometry using a BioRessolve column (Waters™) on an ACQUITY Rda detector (Waters™).

[0195] Biacore analysis of AAV9 binding affinity To evaluate binding to AAV9, the candidate peptide was immobilized on a Biacore™ S-Series CM5 chip. AAV9 (2E12 viral particles (vp) / ml) was used as the analyte.

[0196] The materials and equipment used are 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 in-house); and candidate peptides as described above.

[0197] Immobilization was performed using the standard method in Biacore™ software by coupling 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 Biacore™ acetate buffer at pH 5.0. The obtained immobilization level was 3484 + / - 197 RU.

[0198] In each run, the peptide is immobilized in FC2. Multiple sensor chips are used until all candidates are tested.

[0199] 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.

[0200] All sensor maps are generated by subtracting the reference, and the response values ​​are generated by the difference between the baseline before injection and the signal immediately before the end of injection.

[0201] Biacore analysis of alkaline stability The alkaline stability of the candidate peptides was assessed by immobilizing them on a Biacore™ chip. After evaluating AAV9 binding (see above), the same chip underwent repeated cycles of AAV9 binding (5E11 vp / ml) followed by NaOH (0.5 M).

[0202] The Biacore method for basic 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.

[0203] All sensor maps are generated by subtracting the reference, and the response values ​​are generated by the difference between the baseline before injection and the signal immediately before the end of injection.

[0204] result The response levels of the AAV9 interaction of the candidate peptides were normalized to the response of candidate vh118 (with the sdAb sequence of SEQ ID NO: 92) using the maximum AAV9 concentration (2E12 vp / ml) and are summarized in Tables 9a-c.

[0205] Basic stability was recorded as the number of cycles in which at least 50% of the binding capacity was retained. The first cycle is excluded, and the binding capacity values ​​are 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 was injected at a rate of 10 µl / min for a contact time of 10 minutes. Results for each candidate are provided in Tables 9A-C.

[0206] Satisfactory AAV9 binding capacities were achieved for many of the point mutations studied. A binding capacity of at least 80% for vh118 was considered satisfactory. Examination of basic stability revealed that some point mutations in the CDR region (see SEQ ID NO: 13, 14, 37, 39, 42-44, 56) were particularly advantageous. In this respect, maintaining at least 50% of the binding capacity after 10 cycles was considered acceptable; maintaining at least 50% of the binding capacity after 15 cycles was preferred.

[0207] Particularly preferred are those variants that exhibit at least 100% normalized binding capacity and maintain at least 50% binding capacity for 20 cycles or more, such as at least 24 cycles.

[0208] On the other hand, unclaimed variants, such as vh194 with Y at position 3 of CDR1 and vh206 with R at position 4 of CDR2, show poor or extremely poor binding and basic stability.

[0209] Table 9a: Tested CDR1 variants sdAb CDR1 amino acid sequence CDR1 SEQ ID NO: sdAb SEQ ID NO: Standardized AAV9 binding capacity (%) NaOH stability (retaining at least 50% of the bound amount after exposure cycles) vh118 RTLSDYTMG 16 92 100.0 24 vh193 LTLSDYTMG 10 93 89.1 17 vh194 RTYSDYTMG 18 94 37.9 5 vh197 RTLSNYTMG 11 95 104.0 15 vh198 RTLSEYTMG 12 96 102.5 15 vh199 RTLSDFTMG 13 97 115.1 ≥40** vh200 RTLSDNTMG 14 98 85.5 24 vh202 RTLSDYAMG 19 99 16.4 1 vh203 RTLSDYTIG 20 100 72.3 8 vh204 RTLSDYTMA 15 101 110.0 18 ** Retains 62% binding capacity after 40 cycles Table 9b: Tested CDR2 variants sdAb CDR2 sequence CDR2 SEQ ID NO: sdAb SEQ ID NO: Standardized AAV9 binding capacity (%) NaOH stability (exposed cycle number to maintain at least 50% binding) vh118 AISWSGAYTK 29 92 100.0 24 vh210 AISWSGAYTF 31 105 111.1 22 vh205 AITWSGAYTK 169 102 78.6 8 vh206 AISRSGAYTK 32 103 0.4 N / A vh208 AISWSAAYTK 33 104 51.8 6 Table 9c: Tested CDR3 variants sdAb CDR3 amino acid sequence CDR3 SEQ ID NO sdAb SEQ ID NO Standardized AAV9 binding capacity (%) NaOH stability (exposed cycle number to maintain at least 50% binding) vh118 GPTGLLSKKATPADYDY 55 92 100.0 24 vh211 APTGLLSKKATPADYDY 36 106 103.1 22 vh212 GSTGLLSKKATPADYDY 37 107 112.8 28 vh213 GPSGLLSKKATPADYDY 73 108 40.5 3 vh214 GPTGPLSKKATPADYDY 39 109 110.8 38 vh215 GPTGTLSKKATPADYDY 40 110 109.0 23 vh216 GPTGLISKKATPADYDY 41 111 114.3 16 vh217 GPTGLLAKKATPADYDY 42 112 116.4 25 vh218 GPTGLLSNKATPADYDY 43 113 107.1 27 vh219 GPTGLLSQKATPADYDY 44 114 107.3 29 vh220 GPTGLLSKHATPADYDY 47 115 103.1 16 vh221 GPTGLLSKKTTPADYDY 56 116 101.6 ≥40* vh222 GPTGLLSKKAAPADYDY 57 117 112.0 18 vh223 GPTGLLSKKATSADYDY 58 118 110.0 15 vh224 GPTGLLSKKATTADYDY 59 119 108.0 17 vh225 GPTGLLSKKATPRDYDY 60 120 107.9 18 vh226 GPTGLLSKKATPANYDY 61 121 109.5 15 vh227 GPTGLLSKKATPAEYDY 62 122 105.0 24 vh228 GPTGLLSKKATPAQYDY 63 123 106.5 18 vh229 GPTGLLSKKATPADFDY 64 124 102.7 22 vh230 GPTGLLSKKATPADRDY 74 125 31.1 3 vh231 GPTGLLSKKATPADYNY 65 126 103.9 18 vh232 GPTGLLSKKATPADYEY 66 127 108.6 18 vh233 GPTGLLSKKATPADYQY 67 128 111.7 17 vh234 GPTGLLSKKATPADYDF 68 129 106.9 23 vh235 GPTGLLSKKATPADYDR 69 130 106.8 15 * Retains 51% binding capacity after 40 cycles Example 3B: Binding and stability of another candidate peptide with CDR3 site modification This embodiment further investigated the effect of amino acid modifications in the CDR3 region of the candidate peptides on basic stability and target binding. The candidate peptides tested were expressed as fusion proteins, wherein each sdAb had SEQ ID NO: 161 fused to its C-terminus via a linker. All candidates had an N-terminal OmpA signal peptide and a C-terminal (His)6 tag. The sequences of the tested sdAbs are provided in Table 9d. The sdAb of SEQ ID NO: 92 (vh118) was used as a reference, to which point mutations were introduced at amino acid positions 100b and / or 100c.

[0210] The following aspects were evaluated: (1) Affinity assessment of AAV9 interactions (tested by injecting high concentrations of AAV9). (2) Basic stability (tested by the reduction in AAV9 binding after treatment with 0.5 M NaOH in increments of 10 ...

[0211] Materials and methods Design, generate candidate peptides, and evaluate their binding affinity and basic stability on a Biacore instrument, as described in Example 3A.

[0212] result The obtained fixed level is 3394 + / - 183 RU.

[0213] The response levels of AAV9 interactions of candidate peptides were normalized to those of candidate vh118 using the maximum AAV9 concentration (2E12 vp / ml) and are summarized in Table 9d. Satisfactory AAV9 binding capacities were found for many of the point mutations studied.

[0214] Basic stability was recorded as the number of cycles in which at least 50% of the binding capacity was retained. The first cycle is excluded, and the binding capacity values ​​are normalized to the binding capacity recorded for the second cycle. In each cycle, 0.5 M NaOH was injected at 10 µl / min for a contact time of 10 minutes. Results for each candidate are provided in Table 9d.

[0215] It is noted that candidate peptides with R instead of K at position 9 of CDR3 performed even better than vh118.

[0216] Table 9d: Other CDR3 variants sdAb CDR3 amino acid sequence CDR3 SEQ ID NO sdAb SEQ ID NO Standardized AAV9 binding capacity (%) NaOH stability (exposed cycle number to maintain at least 50% binding) vh118 GPTGLLSKKATPADYDY 55 92 100.0 22 vh333 GPTGLLSRKATPADYDY 45 132 116.7 20 vh334 GPTGLLSKRATPADYDY 46 133 117.3 32 vh335 GPTGLLSNRATPADYDY 48 134 114.9 28 vh336 GPTGLLSQRATPADYDY 49 135 106.5 33 vh337 GPTGLLSRRATPADYDY 50 136 120.6 27 vh338 GPTGLLSNHATPADYDY 51 137 104.4 14 vh339 GPTGLLSQHATPADYDY 52 138 104.2 18 vh340 GPTGLLSRHATPADYDY 53 139 98.6 13 Example 4: AAV9 binding peptides exhibiting variations in CDR1, CDR2, CDR3, and different frameworks. This embodiment further investigated the CDR sequences of different groups within different sdAb frameworks. The candidate peptides tested were expressed as fusion proteins, wherein each sdAb had SEQ ID NO: 161 fused to its C-terminus via a linker and possessed a C-terminal (His) 6-tag. sdAb sequence variants are provided in Tables 10a-b.

[0217] The following aspects were evaluated: (1) Affinity assessment of AAV9 interactions (tested by injecting high concentrations of AAV9). (2) Basic stability (tested by the reduction in AAV9 binding after treatment with 0.5 M NaOH in increments of 10 ...

[0218] Table 10a Table 10b 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 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 pre-filled 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 shaker 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 generate 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.

[0219] The clarified lysate was loaded onto a HisTrap™ FF 1 mL column (Cytiva™) equilibrated with 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 replaced with phosphate-buffered saline (Medicago) at pH 7.4 using a gravity flow column pre-packed with Sephadex™ G-25 resin (Cytiva).

[0220] 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. Protein mass was determined by liquid chromatography-mass spectrometry using a BioRessolve column (Waters™) on an ACQUITY Rda detector (Waters™).

[0221] Biacore analysis of AAV9 binding affinity To evaluate binding to AAV9, the AAV9-binding peptide was immobilized on a Biacore™ S-Series CM5 chip. AAV9 (2E12 viral particles (vp) / ml) was used as the analyte.

[0222] The materials and equipment used are 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 in-house); and candidate peptides as described above.

[0223] Immobilization was performed using the standard method in Biacore™ software by coupling 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 at pH 5.0. The obtained immobilization level was 3001 + / - 159 RU.

[0224] In each run, the peptide is immobilized in FC2.

[0225] 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.

[0226] All sensor maps are generated by subtracting the reference, and the response values ​​are generated by the difference between the baseline before injection and the signal immediately before the end of injection.

[0227] Biacore analysis of alkaline stability The alkaline stability was evaluated on the Biacore™ chip prepared above. After evaluating AAV9 binding (see above), the sample was then subjected to repeated cycles of AAV9 binding (5E11 vp / ml) followed by NaOH (0.5 M).

[0228] 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.

[0229] All sensor maps are generated by subtracting the reference, and the response values ​​are generated by the difference between the baseline before injection and the signal immediately before the end of injection.

[0230] result 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 11. Basic stability was recorded as the number of cycles in which at least 50% of the binding capacity was retained. Figure 5 Results of the alkaline stability assessment are provided, with binding capacity plotted against cycle number. Binding response values ​​are normalized to the binding capacity recorded for the second cycle (i.e., excluding the first cycle). In each cycle, 0.5 M NaOH was injected at a rate of 10 µl / min for a contact time of 10 minutes.

[0231] Candidates vh374, vh359, and vh428, which share the same CDR group but differ in their framework regions, were found to exhibit even higher basic stability than vh118. A comparison of vh118 with vh428, which shares the same framework region, concludes that the CDR of vh428 / vh374 / vh359 positively contributes to basic stability.

[0232] Table 11 Standardized AAV9 binding capacity (%) NaOH stability (exposed cycle number to maintain at least 50% binding) vh118 100.0 22 vh374 102.2 30 vh359 86.4 34 vh428 107.6 40 Example 5A: Comparative Study of AAV9 Binding and Stability In this embodiment, seven candidate peptides were tested and compared with a commercially available affinity ligand (CaptureSelect™ biotinylate-AAV9 conjugate, ThermoFischer Scientific). The candidate peptides were expressed as fusion proteins, each sdAb having SEQ ID NO: 161 fused to its C-terminus via a peptide linker (AA), and all constructs having a C-terminal tag (HHHHHHC). sdAb sequence variants are provided in Table 12.

[0233] Table 12 Materials and methods As described in Example 4, IMAC-purified candidate peptides are generated.

[0234] Biotinylation of purified candidate peptides Using EZ-Link™ maleimide-PEG2-biotin, in the No-Weigh™ form (ThermoFisherScientific), the purified candidate peptide was biotinylated at the C-terminal cysteine ​​residue with a 2X molar excess of biotin.

[0235] SEC purification of biotinylated candidate peptides The protein was further purified by size exclusion chromatography (SEC) to separate it from 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 eluted as a sharp, symmetrical peak. The purest fractions (e.g., those analyzed by SDS-PAGE) were pooled for further analysis.

[0236] Biacore analysis of AAV9 binding affinity To evaluate binding to AAV9, an AAV9-binding peptide candidate and a reference CaptureSelect™ biotinylate anti-AAV9 conjugate (Thermo Scientific™) were immobilized on a Biacore™ S-series SA chip. AAV9 (2E12 viral particles (vp) / ml) was used as the analyte.

[0237] The materials and equipment used are as follows: Biacore™ S-series SA sensor chip, Biacore™ 8K+ instrument (all from Cytiva, Sweden); AAV9 (prepared in-house); candidate peptides as described above; CaptureSelect™ biotinylate-AAV9 conjugate (Thermo Scientific™).

[0238] Using standard methods in Biacore™ software, immobilization was performed by coupling a biotinylated AAV9-binding peptide variant and the CaptureSelect™ biotinylated anti-AAV9 conjugate (Thermo Scientific™) in flow cell 2 (FC2) and then 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™ biotinylated anti-AAV9 conjugate (Thermo Scientific™) was diluted in PBS at a concentration of 10 µg / ml.

[0239] The Biacore™ method for binding assays: 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.

[0240] All sensor maps are generated by subtracting the reference, and the response values ​​are generated by the difference between the baseline before injection and the signal immediately before the end of injection.

[0241] Biacore analysis of alkaline stability Basic stability was assessed on the same Biacore™ chip used for the binding assays 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 AAV9 (5E11 vp / ml), otherwise the PBS-P+ buffer was used for sample injection to minimize analyte consumption.

[0242] 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 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 88 times to track the stability of the AAV9 response.

[0243] All sensor maps are generated by subtracting the reference, and the response values ​​are generated by the difference between the baseline before injection and the signal immediately before the end of injection.

[0244] result Table 13 shows the fixation levels obtained for the tested peptides, as well as the AAV9 binding capacity and NaOH stability using the maximum AAV9 concentration. The candidate peptides were expressed as a fusion with SEQ ID NO: 161, and therefore had a higher molecular weight than the sdAb itself. Relative fixation levels are fixation levels divided by molecular weight.

[0245] Table 13 Despite being fixed at a lower molar level than the existing technology reference CaptureSelect™ biotinylate-AAV9 conjugate, the tested AAV9-binding peptides of the present invention exhibited significantly higher AAV9 binding capacity. Furthermore, the candidates of the present invention demonstrate very high basic stability. In fact, for one candidate, vh377, the AAV9 response never dropped below 50% during 88 cycles.

[0246] The results of the alkaline stability assessment also showed that Figure 6 During the basic stability analysis, the Biacore run stopped between cycles 28 and 30, so the data point at cycle 30 was not measured. The run continued, and the next binding data was measured at cycle 32.

[0247] Example 5B: Comparative Study of Target Selectivity In this embodiment, binding selectivity to AAV9 was tested for the seven candidate peptides of Example 5A and commercially available AAV9 binding affinity ligands. The candidate peptides were expressed as fusion proteins, wherein each sdAb has SEQ ID NO: 161 fused to its C-terminus via a peptide linker (AA), and all constructs have a C-terminal tag (HHHHHHC).

[0248] Materials and methods The candidate peptide was purified by IMAC and produced in Example 5A.

[0249] Biotinylation of purified candidate peptides Using EZ-Link™ maleimide-PEG2-biotin, in the No-Weigh™ form (ThermoFisherScientific), the purified candidate peptide was biotinylated at the C-terminal cysteine ​​residue with a 2X molar excess of biotin.

[0250] Dialysis of biotinylated candidate peptides Proteins were separated from excess biotin by dialysis. Dialysis of biotinylated protein samples was performed using the Slide-A-Lyzer™ G2 dialysis cartridge 3 mL 10 kDa MWCO (Thermo Scientific™) protocol, with PBS as the dialysis buffer. The concentration of the dialysis samples was measured using NanoDrop (Thermo Scientific) according to the manufacturer's instructions, and the samples were then kept in a freezer until further analysis.

[0251] Biacore analysis of binding selectivity for different AAV serotypes To evaluate binding to different AAV serotypes, candidate peptides and CaptureSelect™ biotinylate-AAV9 conjugates (Thermo Scientific™) were immobilized on Biacore™ S-series SA chips. Tangential flow filtration (TFF) AAV materials were used as analytes for serotypes AAV2, AAV5, AAV8, and AAV9.

[0252] The materials and equipment used are as follows: Biacore™ S-series SA sensor chips, Biacore™ 8K+ instruments (all from Cytiva, Sweden); TFF materials for AAV2, AAV5, AAV8, and AAV9 (approximately 1E12 vp / ml for all AAV serotypes) (prepared in-house); candidate peptides as described above; CaptureSelect™ biotinylate-AAV9 conjugates (Thermo Scientific™).

[0253] Immobilization was performed using the standard method in Biacore™ software by coupling a biotinylated AAV9-binding peptide variant and a CaptureSelect™ biotinylated anti-AAV9 conjugate in flow cell 2 (FC2) and then activating / inactivating them 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™ biotinylated anti-AAV9 conjugate was diluted in PBS at a concentration of 10 µg / ml. Immobilization was performed to obtain similar molar amounts of AAV9-binding peptide, thus varying the immobilization level depending on the molecular weight.

[0254] The Biacore™ method for binding assays: Run buffer: PBS-P+; Flow rate: 10 µl / min; Sample injection: 500 s in both flow cells (FC1 and FC2); Dissociation time: 500 s; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle), the analyte is injected as follows: run buffer, AAV2 TFF, AAV5 TFF, AAV8 TFF, AAV9 TFF.

[0255] All sensor maps are generated by subtracting the reference, and the response values ​​are generated by the difference between the baseline before injection and the signal immediately before the end of injection.

[0256] result The obtained fixed levels are shown in Table 14. The candidate peptide was expressed as a fusion with SEQ ID NO: 161, and therefore had a higher molecular weight than the sdAb itself.

[0257] Table 14 Molecular weight (Da) Fixed level (RU) Relatively fixed level (RU / Da) vh118 21385 1207 0.09 vh97 21215 1841 0.06 vh376 21284 1258 0.06 vh324 21386 1139 0.05 vh377 21373 1497 0.07 vh378 21478 1312 0.06 vh379 21161 1915 0.09 CaptureSelect™ Biotin-AAV9 Conjugate 13000 983 0.08 Figure 7a -h is a sensor plot showing the binding response of TFF samples to different AAV serotypes: AAV9 (dashed line), AAV8 (dotted line), AAV2 (solid line), AAV5 (solid line). The run buffer response is also shown as a solid line. As clearly seen in these plots, candidate vh97 ( Figure 7a vh118 Figure 7b ), vh324 ( Figure 7c ), vh376 ( Figure 7d ), vh377 ( Figure 7e ), vh378 ( Figure 7f ) and vh379 ( Figure 7gIt is selective for AAV9 but does not bind to AAV2, AAV5, or AAV8 samples. In contrast, the reference affinity ligand also shows high binding to both AAV9 and AAV8 samples. Figure 7h ).

[0258] Example 6A: AAV9 binding peptide with or without a C-terminal fusion partner A single-domain antibody capable of binding to AAV9 was fused with a stable peptide, and the effects on protein expression, antigen-binding capacity, and basic stability were investigated.

[0259] The peptides tested are identified in Table 15. Single-domain antibodies (sdAbs) were compared with fusions of the same single-domain antibody and a stable peptide. The single-domain antibody (referred to as "sdAb") was an AAV9-binding VHH variant (SEQ ID NO: 92). The stable peptide (referred to as "Z") was a SpA domain Z variant (SEQ ID NO: 161) in which binding to the Fc and VH3 moieties of IgG was eliminated. Each construct had a C-terminal linker attachment (His)6 tag.

[0260] Table 15 Candidate number Builder description Construct sequence 6A-1 [sdAb] SEQ ID NO:147 6A-2 [sdAb]-[Z] SEQ ID NO:165 Specifically, the following aspects were evaluated: (1) Affinity assessment of AAV9 interactions (tested by injecting high concentrations of AAV9). (2) Basic stability (tested by the reduction in AAV9 binding after treatment with 0.5 M NaOH in increments of 10 ...

[0261] Materials and methods Production of purified peptides using MabSelect PrismA™ In a 14 ml round-bottom tube, 5 µl of glycerol stock solution generated for candidates 6A-1 and 6A-2 as described in Example 1 was inoculated into 4 ml of LB medium supplemented with 100 µg / ml carbenicillin and incubated overnight at 37°C and 160 rpm.

[0262] Prepare protein expression medium (TB medium supplemented with 100 µg / ml carbenicillin and 2 mM MgCl2) and add it to pre-filled 500 ml baffled glass shake flasks (50 ml / flask). Inoculate each flask with approximately 500 µl of the previous overnight culture to obtain an initial OD600 of 0.05. Incubate the flasks in an Inforrs HT shake incubator at 37°C and 140 rpm for approximately 3.5 h until the OD600 reaches 1.0. Then, add 50 µl of IPTG (1 M) to a final concentration of 1 mM and incubate the flasks in an Inforrs HT shake incubator at 27°C and 140 rpm for 18 h, after which 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 h, followed by centrifugation at 8000 g for 10 min to precipitate cell debris and filtering the supernatant through a 0.22 µm filter.

[0263] For each sample, the clarified lysate was loaded onto a 1 mL HiTrap MabSelect™ PrismA column (Cytiva™) 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. Proteins were eluted using a stepwise gradient of 50 mM sodium acetate at pH 3.5, with a 5 CV eluent.

[0264] Following the manufacturer's instructions, the concentration of the purified sample was measured using NanoDrop (ThermoFisher 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. Protein mass was determined by liquid chromatography-mass spectrometry using a BioRessolve column (Waters™) on an ACQUITY Rda detector (Waters™).

[0265] Biacore analysis of AAV9 binding affinity To evaluate binding with AAV9, candidates were immobilized on a Biacore™ S-Series CM5 chip using an amine conjugation kit (Cytiva™) and analyzed using a Biacore™ 8K+ instrument (Cytiva™). Samples containing AAV9 viral particles (vp) at a concentration of 2E12 vp / ml were used as analytes.

[0266] Using the standard method in Biacore software, peptides were immobilized by coupling in flow cell 2 (FC2) and activation / inactivation in flow cell 1 (FC1). Peptides were diluted in Biacore™ acetate buffer pH 5.0 (Cytiva™) at concentrations ranging from 17–30 µg / ml, depending on their molecular weight. Immobilization levels varied between different peptide variants corresponding to their molecular weights. In each run, the peptide was immobilized in FC2.

[0267] The Biacore™ method for binding assays: 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, AAV9 2E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

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

[0269] 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 (0.5 M) injection to assess alkaline stability.

[0270] 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.

[0271] All sensor maps are generated by subtracting the reference, and the response values ​​are generated by the difference between the baseline before injection and the signal immediately before the end of injection.

[0272] result Expression level analysis by Bis-Tris PAGE showed that the expression levels of peptides 6A-1 (sdAb, unfused) and 6A-2 (sdAb and C-terminal stable peptide) were comparable (data not shown).

[0273] The fixed levels on the CM5 chip were 2134 or 3560 RU, respectively. For the tested fusion protein 6A-2, the AAV9 binding capacity at the maximum viral concentration used (2E12 vp / ml) was approximately 10% higher than that of sdAb 6A-1.

[0274] Figure 8A illustrates the AAV9 binding capacity for incremental cycles including 0.5 M NaOH exposure (sample injection 2 above), normalized to the response after the first cycle (i.e., excluding the first cycle). As can be seen in this figure, the fusion protein 6A-2, which contains a single-domain antibody variant fused to a stable peptide at the C-terminus, exhibits improved basic stability compared to the standalone single-domain antibody 6A-1. The improvement is even more significant if the response after each cycle is normalized to the response of the first cycle prior to the first NaOH exposure, as shown in Figure 8A. Figure 6 As shown in B. (For example, in...) Figure 6 As seen in B, the decrease in AAV9 binding capacity experienced by the fusion protein between the first and second cycles was only 20%, compared to a 60% decrease in response to sdAb (which is not part of the fusion protein).

[0275] Table 16 summarizes the results of AAV9 binding capacity and basic stability measurements.

[0276] Table 16 Example 6B: AAV9-binding multimeric fusion protein Regarding protein expression, antigen-binding capacity, and basic stability, we studied multimeric constructs fused with single-domain antibodies that can bind to AAV9 and stable peptides.

[0277] The peptides tested are summarized in Table 17 below. The single-domain antibody (sdAb) trimer is compared with trimer fusion proteins containing one or three stable peptides, respectively. The single-domain antibody (referred to as “sdAb”) is an AAV9-binding VHH variant (SEQ ID NO: 92). The stable peptide (referred to as “Z”) is a SpA domain Z variant (SEQ ID NO: 161) in which binding to the Fc and VH3 moieties of IgG has been eliminated. Each construct has a C-terminal linker followed by a (His)6-Cys tag.

[0278] Table 17 Candidate number describe Construct sequence 6B-1 [sdAb]-[sdAb]-[sdAb] SEQ ID NO:166 6B-2 [sdAb]-[sdAb]-[sdAb]-[Z] SEQ ID NO:167 6B-3 [sdAb]-[Z]-[sdAb]-[Z]-[sdAb]-[Z] SEQ ID NO:168 The following aspects were evaluated: (1) Protein expression analysis by PAGE. (2) Affinity assessment of AAV9 interaction (tested by high-concentration AAV9 injection). (3) Basic stability (tested by the reduction of AAV9 binding after incremental treatment with 0.3 M NaOH).

[0279] Materials and methods Production of purified and biotinylated peptides using MabSelect PrismA™ The candidate polypeptide was generated and purified according to the method described in Example 6A.

[0280] Following the manufacturer's instructions, the concentration of the MabSelectPrismA™ purified sample was measured using NanoDrop (Thermo Scientific). After purification, the sample was reduced by adding 10 µl of 0.5 MDTT dissolved in sterile water to 490 µl of sample to achieve a final DTT concentration of 10 mM. The sample was incubated at room temperature for 1 hour, followed by exchange to PBS using Amersham NAP-5 column (Cytiva™) buffer, yielding 1 ml of eluent. EZ-Link™ maleimide-PEG2-biotin, No weight™ form (Thermo Scientific) was dissolved in 190 µl of PBS, and 5 µl of dissolved biotin was added to each 1 ml eluent to achieve a final biotin excess of 2–5 moles. The sample was incubated on ice for 2 hours, followed by dialyzing using the Slide-A-Lyzer™ G2 dialysis cartridge (Thermo Scientific). Following the manufacturer's instructions, the concentration of the dialysis sample was measured using NanoDrop (Thermo Scientific) and kept in an Eppendorf tube in a refrigerator until all measurements were completed, then kept in a freezer until further analysis. Protein mass was determined by liquid chromatography-mass spectrometry using a BioRessolve column (Waters™) on an ACQUITY Rda detector (Waters™).

[0281] Bis-Tris PAGE analysis To evaluate protein expression levels, Bis-Tris PAGE was performed on the filtered supernatant after heat treatment. The materials and equipment used were as follows: NuPAGE LDS sample buffer (x4), pre-stained Page Ruler, 4-12% Bis-Tris mini-gel, Invitrogen NP0321BOX, mini gel bath (Invitrogen), 20x MES buffer (Invitrogen), and QuickBlue dye.

[0282] Mix 15 µl of the filtered supernatant after heat treatment with 5 µl of LDS sample buffer. Heat the sample mixture to 70 °C for 10 min and centrifuge gently to collect the condensate from the cap. For each expression construct, load 15 µl of the sample mixture onto a gel. Load 5 µl of PageRuler as a marker. Run the gel at 200 V for 35 min and stain with QuickBlue dye for 2 h. Decolorize the gel overnight in deionized water.

[0283] Binding affinity analysis of AAV9 To evaluate binding with AAV9, the candidate was immobilized on a Biacore™ S-Series SA chip (Cytiva™) and analyzed using a Biacore™ 8K+ instrument (Cytiva™). Samples containing AAV9 viral particles (vp) at a concentration of 2E12 vp / ml were used as analytes.

[0284] The peptides were immobilized using the standard SA conjugation method in Biacore software by conjugating them in flow cell 2 (FC2) and activating / inactivating them in flow cell 1 (FC1). The peptides were diluted in PBS-P+ (Cytiva™) at a concentration of approximately 25 µg / ml. The immobilization levels varied slightly between different peptide variants, corresponding to their molecular weights. In each run, the peptides were immobilized in FC2.

[0285] The Biacore™ method for binding assays: 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, AAV9 2E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

[0286] All sensor maps are generated by subtracting the reference, and the response values ​​are generated by the difference between the baseline before injection and the signal immediately before the end of injection.

[0287] Alkali stability analysis 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.

[0288] 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 40 times to track the stability of the AAV9 response value.

[0289] All sensor maps are generated by subtracting the reference, and the response values ​​are generated by the difference between the baseline before injection and the signal immediately before the end of injection.

[0290] result Expression level analysis by Bis-Tris PAGE (Figure 9A) showed that multimeric fusion proteins containing stable peptides can improve protein expression.

[0291] The fixed level range on the SA chip was 3000-3500 RU. For the tested fusion proteins 6B-2 and 6B-3, the AAV9 binding capacity at the maximum viral concentration used was higher than that of the sdAb trimer without a stable peptide.

[0292] Figure 9B illustrates the AAV9 binding capacity for incremental cycles including 0.3 M NaOH exposure (sample injection 2 above), normalized to the response after the first cycle (i.e., excluding the first cycle). The first cycle removes loosely bound peptides from the surface. As can be seen in this figure, both fusion proteins 6B-2 and 6B-3 show a significant improvement in basic stability compared to sdAb trimer 6B-1. Normalizing the response to the first cycle, it was found that 6B-3 experienced a larger decrease in response between the first and second cycles than 6B-1 or 6B-2, but remained more stable than 6B-1 for subsequent cycles. 6B-1 lost more binding capacity than fusion proteins 6B-2 and 6B-3 during the experiment.

[0293] Table 18 summarizes the results of AAV9 binding capacity and basic stability measurements.

[0294] Table 18 Example 7: AAV9-binding peptide with C-terminal fusion partner This embodiment compares single-domain antibodies with different frame sequences, one of which has a frame modified to enhance basic stability. The following aspects were evaluated: (1) Affinity assessment of AAV9 interaction (tested by high-concentration AAV9 injection). (2) Basic stability (tested by the reduction in target binding after incremental treatment with 0.5 M NaOH).

[0295] The single-domain antibody is a VHH variant of AAV9. The CDR region is the same, but the candidate differs in the amino acid sequence of the frame region.

[0296] Each VHH was expressed as a fusion protein fused with a Z variant of the SpA domain at the C-terminus (SEQ ID NO: 162), which is capable of binding immunoglobulin Fc but not VH3.

[0297] The tested peptide candidates (fusion proteins) are summarized in Table 19.

[0298] Table 19 Candidate number VHH variant VHH Frame Type VHH sequence Full-length protein sequence 7-1 vh97 Synthetic, partially stable SEQ ID NO:91 SEQ ID NO:163 7-2 vh118 Synthetic, stable SEQ ID NO:92 SEQ ID NO:164 Materials and methods Preparation of glycerol reservoir from IDT oligonucleotides The materials and equipment used were as follows: kanamycin-resistant vector plasmids; G-block 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 (New England 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™); supplemented with kanamycin (50 mg / ml). Agar plates (µg / ml); 14 ml Falcon™ round-bottom tubes (Corning™); PlasmidPrep Mini Spin kit (Cytiva™); Inforrs HT shaking incubator.

[0299] The vector plasmid and the G-block 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 electrophoresis according to the manufacturer's protocol.

[0300] Restriction enzyme-digested G-blocks were ligated into gel-purified plasmids using T4 DNA ligase at room temperature for 2 h, and then transformed into Top10 *E. coli* cells. In short, cells and the ligation mixture were incubated in KCM buffer on ice for 20 min, followed by incubation at room temperature for 10 min. Cells were then transferred to SOC medium and incubated at 37°C for 1 h, followed by inoculation onto agar plates containing 50 µl / ml kanamycin and incubation overnight at 37°C. Single colonies were inoculated into 4 mL of LB medium supplemented with 50 µg / ml kanamycin in 14 mL round-bottom tubes and grown overnight at 37°C and 200 rpm. Plasmids were prepared according to the manufacturer's protocol, and the purified plasmids were transformed into KCM competent *E. coli* K12-017 or KCM competent *E. coli* BL21(DE3) cells. Cells and plasmids were incubated in KCM buffer on ice for 20 min, followed by incubation at room temperature for 10 min. Cells were then transferred to SOC medium and incubated at 37°C for 1 h, followed by inoculation onto agar plates containing 50 µg / ml kanamycin and incubation overnight at 37°C. Single colonies were inoculated into 4 mL of LB medium supplemented with 50 µg / ml kanamycin in 14 mL round-bottom tubes and grown overnight at 37°C and 200 rpm. Glycerol stock solutions for each variant were prepared using 900 µl of the overnight culture and 500 µl of 50% glycerol and stored at -80°C.

[0301] Protein expression and purification For each candidate, 5 µl of the BL21(DE3) glycerol stock solution as described above 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.

[0302] Prepare protein expression medium (TB medium supplemented with 50 µg / ml kanamycin and 2 mM MgCl2) and add it to pre-filled 100 ml baffled glass shake flasks (20 ml / flask). Inoculate each flask with approximately 100 µl of the previous overnight culture to obtain an initial OD600 of 0.05. Incubate the flasks in an Inforrs HT shake incubator at 37°C and 140 rpm for approximately 3 hours until the OD600 reaches 1.0. Then, add 20 µl of IPTG (1 M) to a final concentration of 1 mM and incubate the flasks in an Inforrs HT shake incubator at 27°C and 140 rpm for 18 hours, after which 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 through a 0.22 µm filter.

[0303] Samples were purified using gravity flow in a PD-10 column (Cytiva™) internally packed with IgG Sepharose 6FF chromatography resin (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 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 was exchanged for phosphate-buffered saline (Medicago) at pH 7.4 using a gravity flow column (PD-10 (Cytiva)).

[0304] Following the manufacturer's instructions, the concentration of the purified IgG Sepharose 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. Protein mass was determined by liquid chromatography-mass spectrometry using a BioRessolve column (Waters™) on an ACQUITY Rda detector (Waters™).

[0305] Biacore analysis of AAV9 affinity To evaluate binding with AAV9, candidate peptides were immobilized on a Biacore™ S-Series CM5 chip using an amine conjugation kit and analyzed using a Biacore™ 8K+ instrument (all from Cytiva). The analyte used was AAV9 (prepared in-house).

[0306] The peptide variant was immobilized using standard methods in Biacore software by coupling it in flow cell 2 (FC2) and activating / inactivating it 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™), and the average immobilization level was 4319 RU + / - 640 RU. The peptide was immobilized in FC2.

[0307] The Biacore™ method for binding assays: 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.

[0308] All sensor maps were generated by subtracting the reference, and the response values ​​were generated by the difference between the baseline before sample injection and the signal immediately before the end of injection.

[0309] Biacore analysis of alkaline stability Following the binding analysis (see above), the same CM5 chip with the immobilized peptide underwent repeated cycles of binding AAV9 followed by NaOH injection to assess alkaline stability (0.5 M).

[0310] Biacore method (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 target response.

[0311] All sensor maps are generated by subtracting the reference, and the response values ​​are generated by the difference between the baseline before injection and the signal immediately before the end of injection.

[0312] result Both fusion proteins showed satisfactory binding to AAV9.

[0313] Table 20 summarizes the results of the binding capacity and basic stability measurements.

[0314] Table 20 Figure 10 The figure shows the binding capacity obtained for incremental cycles including NaOH exposure (sample injection 2), normalized to the first cycle (i.e., including the first cycle). As can be seen in the figure, the AAV9-binding peptide based on the stable VHH framework (7-2) shows improved basic stability compared to the corresponding candidate (7-1) with the same degree of stabilization modification of the VHH framework.

[0315] Example 8: Functional Evaluation – Affinity Chromatography This embodiment uses basic experiments to confirm the functionality of the AAV9 binding peptide as an affinity ligand immobilized on the separation matrix.

[0316] Materials and methods AAV9 binding candidate vh118 (SEQ ID NO:92) was generated and purified as a fusion protein with SEQ ID NO:161, as described in Example 6A.

[0317] The fusion protein (SEQ ID NO:170) was immobilized on epoxy-activated chromatography resin (highly cross-linked agarose beads) using cysteine ​​coupling. 0.2 ml of the resin was packed into a Tricorn 5 / 20 column (Cytiva). Using an ÄKTApure 25 system (Cytiva), 50 ml of tangential flow filtration (TFF) AAV9 material (in-house prepared) was loaded onto the column with a 4-minute residence time and eluted with 100 mM citrate buffer at pH 2.5.

[0318] The collected eluent fractions were analyzed on Coomassie SDS-PAGE gels. 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. 5 µl of sample was loaded and the gel was run at 200 V for 35 min, then incubated overnight in Coomassie solution. The gel was then destained with water until satisfactory staining was achieved. The gels were photographed using an ImageQuant 800.

[0319] To assess the basic stability of the immobilized fusion protein, the chromatographic resin was cleaned in situ (CIP) with 10 mM glycine-HCl at pH 1.5, followed by a basic stability study involving exposure to 0.5 M NaOH. The complete stability study consisted of 40 cycles, each involving contacting the chromatographic material with 0.5 M NaOH for 30 minutes. Every tenth cycle, a solution containing pure AAV9 particles was added, with a residence time of 15 seconds. PBS buffer was used for all other cycles.

[0320] result Figure 11a shows the chromatogram obtained from the purification of AAV9 TFF material using the candidate antigen-binding peptide as the affinity ligand. Elution peaks are indicated by rectangles. Figure 11b shows only the elution peak regions. Figure 12 The gel image is shown. VP1, VP2, and VP3 represent AAV9 virosomal proteins 1, 2, and 3, respectively.

[0321] The results showed that the candidate of the present invention could successfully purify AAV9 virus particles, with an estimated dynamic binding capacity (QB10%) of about 3E+14 virus particles / ml resin, and an estimated recovery rate of 70-80% for loaded virus particles.

[0322] The results of the alkaline stability assessment showed that Figure 13 The diagram shows the binding capacity normalized to the level prior to the first NaOH exposure. After 40 cycles of exposure to 0.5 M NaOH (corresponding to a cumulative exposure time of 20 hours), the AAV9 binding capacity decreased by less than 20%.

[0323] Example 9: AAV9 binding peptides using different frameworks and two sets of CDRs This embodiment further investigated five additional different sdAb frames with the same sdAb CDR sequences. A frame was also tested using two sets of different CDR sequences. All sdAbs had the same CDR1 and CDR2 sequences, while vh516 had a different CDR3 than vh511-vh515. Vh511-515 had the same CDR as vh324 but different frames. Vh324 had frame variant 1, while vh511-515 had five new frame arrangements, as shown in the table below.

[0324] The candidate peptides tested were expressed as fusion proteins, wherein each sdAb has SEQ ID NO: 161 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.

[0325] The following aspects were evaluated: (1) Affinity assessment of AAV9 interactions (tested by injecting high concentrations of AAV9). (2) Basic stability (tested by the reduction in AAV9 binding after treatment with 0.3 M NaOH in increments of 1000 times).

[0326] Table 21 Table 22 Materials and methods As described in Example 5, candidate peptides purified by IMAC were generated, and Biacore analysis of binding affinity and stability was performed accordingly.

[0327] result Using the maximum AAV9 concentration (2E12 vp / ml), the response levels of the AAV9 interaction of the candidate peptides were normalized to the response of vh377 and are summarized in Table 23. Basic stability was recorded as the number of cycles in which at least 50% of the binding capacity was retained. 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.

[0328] Candidates vh511-515, which have the same group of CDRs but different framework regions, were found to have even higher basic stability than vh377. Comparison of vh516 and vh515, which have the same framework region, concluded that different CDRs contribute equally to basic stability.

[0329] Table 23 Standardized AAV9 binding capacity (%) NaOH stability (exposed cycle number to maintain at least 50% binding) vh377 100.0 76 vh511 119 46 vh512 111 56 vh513 118 60 vh514 113 69 vh515 122 46 vh516 119 46 Example 10: Measurement of ligand basic stability By evaluating the ZVH3 hexamer (MabSelect) that targets AAV9 (5e11 vp / ml) or interacts with the ligand backbone, we were able to determine its effectiveness. TM The basic stability of the peptide was measured using a VH3 ligand (50 nM). Alternating binding with AAV9 or ZVH3, 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 increase the basic stability of the peptide. The data obtained from this example were compared with results for a previously tested AAV9-bound sdAb (shown in patent application EP23204844.7).

[0330] Materials and methods As described in WO2023174900A1, a ZVH3 hexamer is provided, the 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.

[0331] Biacore analysis of alkaline stability The materials and equipment used were as follows: Biacore™ S-Series CM5 sensor chip, Biacore™ amine coupling kit, Biacore™ acetate buffer pH 5.0, Biacore™ 8K+ instrument (all from Cytiva, Sweden); 0.5M NaOH; ZVH3-ligand; and the candidate peptide as described above. Basic stability was assessed on the Biacore™ chip prepared above. The peptide underwent 40 cycles of binding to AAV9 (5e11 vp / ml) or ZVH3 hexamer (50 nM) followed by 10 minutes of exposure to NaOH (0.5 M). The NaOH stability of the peptide was assessed by comparing the number of cycles at which the peptide lost 50% of its initial binding to AAV9 or ZVH3 hexamer.

[0332] All sensor maps are generated by subtracting the reference, and the response values ​​are generated by the difference between the baseline before injection and the signal immediately before the end of injection.

[0333] result The results of the assessment of the basic stability of the peptides are summarized in Table 24 below. For AAV9-binding peptides vh260-268, the binding of peptides CDR AAV9 was compared with the binding of AAV9 ZVH3 backbone. It was found that the basic stability also increased for this target. The unstable peptides vh267 and vh268 were unstable for both binding methods.

[0334] Table 24 SEQ ID NO NaOH stability cycling with 50% bound AAV9 NaOH with 50% ZVH3 binding stability cycling vh260 SEQ ID NO: 195 22 23 vh261 SEQ ID NO: 196 22 21 vh262 SEQ ID NO: 197 21 21 vh263 SEQ ID NO: 198 22 19 vh264 SEQ ID NO: 199 22 22 vh265 SEQ ID NO: 200 21 22 vh266 SEQ ID NO: 201 25 24 vh267 SEQ ID NO: 202 1 2 vh268 SEQ ID NO: 203 1 2 Example 11: The first position of the frame is replaced by E from Q. In this experiment, we investigated whether replacing Q with E at the first position of the frame (FW1) would affect the stability of the peptide.

[0335] Materials and methods Four peptides were tested: vh118 (SEQ ID NO: 92) and vh498 (SEQ ID NO: 171), with vh498 differing from vh118 in that the first position was replaced by E instead of Q. Similarly, vh324 (SEQ ID NO: 131) and vh458 (SEQ ID NO: 172) were tested, with vh458 having the first position replaced by E instead of Q. The constructs were generated and tested as described in Example 3A above, purified on PrismA, and conjugated to a CM5 chip.

[0336] result Even for peptides with E as the first amino acid in the framework, the stability of the peptide is slightly lower, but it has been confirmed that using E at the first position of FW1 also produces highly stable peptides, as outlined in Table 25 below. Vh118 with E substitution at position 1 is referred to as vh118 (Q1E). vh324 is named accordingly.

[0337] Table 25 SEQ ID NO: NaOH stability (exposed cycle number of cycles maintaining at least 80% binding) vh118 92 8 vh118 (Q1E) 171 7 Vh324 131 29 Vh324 (Q1E) 172 15 List of Implementation Plans 1. An antigen-binding polypeptide capable of binding adeno-associated virus serotype 9 (AAV9), said polypeptide comprising a single-domain antibody (sdAb) variant having complementarity-determining regions CDR1, CDR2, and CDR3, wherein CDR1 contains an amino acid sequence selected from the sequence group defined by SEQ ID NO: 1: X1X2X3SX5X6TMX9(SEQ ID NO: 1) Independently, X1 is R, L, or S, preferably R or L; X2 is either T or R, preferably T; X3 is L or F, preferably L; X5 is D, N, or E, preferably D; X6 is Y, N, or F, preferably Y or F; X9 is either G or A, preferably G; CDR2 contains sequences selected from the sequence group defined by SEQ ID NO: 2: X1X2SWSGX7X8TX 10 (SEQ ID NO: 2) Independently, X1 is A, S, L, V or I, preferably A; X2 is I or V, preferably I; X7 is either A or S, preferably A; X8 is either Y or F, preferably Y; X 10 Is it K, F, or Y? as well as CDR3 contains sequences selected from the sequence group defined by SEQ ID NO: 3: X1X2TX4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 (SEQ ID NO: 3) Independently, X1 is G or A, preferably G; X2 is either P or S, preferably P; X4 is G or P, preferably G; X5 is L, T, or P, preferably L or P; X6 is L or I, preferably L; X7 is S, T, or A, preferably S or A; X8 is K, R, N, or Q; X9 is K, H, or R, preferably K or R; X 10 It is A, S, or T, preferably A or T; X 11 It is T, S, P or A, preferably T; X 12 It is P, A, T or S, preferably P; X 13 It is A, P, R, or G, preferably A or P; X 14 It is D, N, E or Q, preferably D or E; X 15 It is either Y or F, preferably Y; X 16 It is D, N, E, or Q, preferably D; and X 17 It is Y, F, or R, preferably Y or F.

[0338] 2. The antigen-binding polypeptide described in Project 1 is a single-chain antigen-binding polypeptide.

[0339] 3. The antigen-binding polypeptide according to item 1 or 2, wherein the polypeptide lacks the antibody heavy chain constant domain.

[0340] 4. The antigen-binding polypeptide according to any one of the preceding items, wherein the sdAb variant is a variable domain of the heavy chain of a heavy chain antibody (VHH) variant.

[0341] 5. The antigen-binding polypeptide according to any one of the preceding items, wherein the polypeptide does not contain an antibody light chain or its domains, such as variable fragments (VLs) of the light chain.

[0342] 6. The antigen-binding polypeptide according to any one of the preceding items, wherein the sdAb variant comprises four frame regions FWR1, FWR2, FWR3 and FWR4.

[0343] 7. The antigen-binding polypeptide according to any one of the preceding items, wherein the sdAb variant comprises a framework region and a complementarity-determining region from the N-terminus to the C-terminus in the following order: [FWR1]-[CDR1]-[FWR2]-[CDR2]-[FWR3]-[CDR3]-[FWR4].

[0344] 8. The antigen-binding polypeptide according to item 7, wherein at least one of the framework regions comprises at least one amino acid substitution relative to SEQ ID NO: 4, and wherein the antigen-binding polypeptide exhibits improved basic stability compared to SEQ ID NO: 4.

[0345] 9. The antigen-binding polypeptide according to item 7 or 8, wherein FWR1, FWR2 and FWR3 each have an amino acid sequence that is at least 70% identical to the corresponding frame region of the sdAb selected from SEQ ID NO: 91 and 92.

[0346] 10. The antigen-binding polypeptide according to any one of the preceding items, wherein the sdAb variant comprises an amino acid sequence wherein at least one of the Kabat amino acid positions 6, 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b and 89 is modified relative to the corresponding amino acid of SEQ ID NO: 4.

[0347] 11. The antigen-binding polypeptide according to any one of the preceding items, wherein the sdAb variant is capable of VH3 interaction with the Staphylococcus protein A domain or a variant thereof.

[0348] 12. The antigen-binding polypeptide according to any one of the preceding items, wherein X1 in SEQ ID NO: 1 is R.

[0349] 13. The antigen-binding polypeptide according to any one of the preceding items, wherein X2 in SEQ ID NO: 1 is T.

[0350] 14. The antigen-binding polypeptide according to any one of the preceding items, wherein X3 in SEQ ID NO: 1 is L.

[0351] 15. The antigen-binding polypeptide according to any one of the preceding items, wherein X5 in SEQ ID NO: 1 is D.

[0352] 16. The antigen-binding polypeptide according to any one of the preceding items, wherein X6 in SEQ ID NO: 1 is Y or F.

[0353] 17. The antigen-binding polypeptide according to any one of the preceding items, wherein X9 in SEQ ID NO: 1 is G.

[0354] 18. The antigen-binding polypeptide according to any one of the preceding items, wherein in SEQ ID NO: 1 X1 is R, X2 is T and X3 is L or F.

[0355] 19. The antigen-binding polypeptide according to any one of the preceding items, wherein CDR1 comprises a sequence selected from SEQ ID NO: 9-17, for example selected from SEQ ID NO: 13, 14, 16 and 17.

[0356] 20. The antigen-binding polypeptide according to any one of the foregoing items, wherein i) In SEQ ID NO: 2, X1 is A, or ii) In SEQ ID NO: 2, X1 is S, L, or I, and in SEQ ID NO: 3, X 10 It is A.

[0357] 21. The antigen-binding polypeptide according to any one of the preceding items, wherein X2 in SEQ ID NO: 2 is I.

[0358] 22. The antigen-binding polypeptide according to any one of the preceding items, wherein X7 in SEQ ID NO: 2 is A.

[0359] 23. The antigen-binding polypeptide according to any one of the preceding items, wherein X8 in SEQ ID NO: 2 is Y.

[0360] 24. The antigen-binding polypeptide according to any one of the preceding items, wherein X in SEQ ID NO: 2 10 It's K.

[0361] 25. The antigen-binding polypeptide according to any one of the preceding items, wherein in SEQ ID NO: 2, X2 is I and X8 is Y.

[0362] 26. The antigen-binding polypeptide according to any one of the preceding items, wherein CDR2 comprises a sequence selected from SEQ ID NO: 21-31, for example selected from SEQ ID NO: 26, 28, 29 and 31.

[0363] 27. The antigen-binding polypeptide according to any one of the preceding items, wherein at least one of X8 and X9 in SEQ ID NO: 3 is K.

[0364] 28. The antigen-binding polypeptide according to any one of the preceding items, wherein X2 in SEQ ID NO: 3 is P.

[0365] 29. The antigen-binding polypeptide according to any one of the preceding items, wherein in SEQ ID NO: 3, at least one of X5 and X6 is L, and preferably both X5 and X6 are L.

[0366] 30. The antigen-binding polypeptide according to any one of the preceding items, wherein X7 in SEQ ID NO: 3 is S.

[0367] 31. The antigen-binding polypeptide according to any one of the preceding items, wherein X8 in SEQ ID NO: 3 is K, N or R, for example K or R.

[0368] 32. The antigen-binding polypeptide according to any one of the preceding items, wherein X9 in SEQ ID NO: 3 is K.

[0369] 33. The antigen-binding polypeptide according to any one of the preceding items, wherein X in SEQ ID NO: 3 10 It is either A or T.

[0370] 34. The antigen-binding polypeptide according to any one of the preceding items, wherein X in SEQ ID NO: 3 11 It's T.

[0371] 35. The antigen-binding polypeptide according to any one of the preceding items, wherein X in SEQ ID NO: 3 13 It is A, and preferably X. 10 It is A.

[0372] 36. The antigen-binding polypeptide according to any one of the preceding items, wherein X in SEQ ID NO: 3 14 It is D and X16 It is D.

[0373] 37. The antigen-binding polypeptide according to any one of the preceding items, wherein X in SEQ ID NO: 3 17 It's Y.

[0374] 38. The antigen-binding polypeptide according to any one of the preceding items, wherein CDR3 comprises a sequence selected from SEQ ID NO: 34-72, for example selected from SEQ ID NO: 36, 37, 39, 40, 42-46, 48-50, 55, 56, 62, 64, 68 and 70-72.

[0375] 39. The antigen-binding polypeptide according to any one of the preceding items, wherein CDR1 is selected from SEQ ID NO: 9-17, for example from SEQ ID NO: 13, 14, 16 and 17; CDR2 is selected from SEQ ID NO: 21-31, for example from SEQ ID NO: 26, 28, 29 and 31; and CDR3 is selected from SEQ ID NO: 34-72, for example from SEQ ID NO: 36, 37, 39, 40, 42-46, 48-50, 55, 56, 62, 64, 68 and 70-72.

[0376] 40. The antigen-binding polypeptide according to any one of items 1-7, wherein the sdAb variant has: a) An amino acid sequence selected from SEQ ID NO: 75-93, 95-98, 101, 102, 105-107, 109-124, and 126-146, for example, an amino acid sequence selected from SEQ ID NO: 92, 97, 109, 116, 131, and 140-146; or b) Has at least 90% identity with the sequence defined in a), for example, at least 95% identity with the amino acid sequence, provided that the CDR is as defined in Item 1.

[0377] 41. The antigen-binding polypeptide according to any one of the preceding items, wherein the sdAb variant does not contain any histidine (H) residues.

[0378] 42. The antigen-binding polypeptide according to any one of the preceding items, comprising at least one additional amino acid sequence, optionally selected from leader peptides, signal peptides, purified tags, coupled peptides, spacer peptides and adaptor peptides, each additional amino acid sequence comprising up to 20 amino acids.

[0379] 43. The antigen-binding polypeptide according to item 42, wherein the additional amino acid sequence is located at the N-terminus or C-terminus of the sdAb variant.

[0380] 44. The antigen-binding polypeptide according to item 43, wherein the additional amino acid sequence is a PelB or OmpA signal peptide.

[0381] 45. The antigen-binding polypeptide according to any one of items 42-44, wherein the additional amino acid sequence comprises a plurality of histidines, such as the (His)6 sequence and optionally a C-terminal cysteine.

[0382] 46. ​​The antigen-binding polypeptide according to item 45, wherein the additional amino acid sequence comprises a C-terminal tag, for example selected from HHHHHH, HHHHHHC and AEAAAAKHHHHHHC (SEQ ID NO: 159).

[0383] 47. A multimeric polypeptide comprising at least two moieties, each moiety comprising a single-domain antibody variant as defined in any of the preceding projects, said moieties optionally being linked by a peptide linker.

[0384] 48. A fusion protein comprising at least one antigen-binding polypeptide according to any one of items 1-46 or a multimeric polypeptide according to item 47 and an additional polypeptide moiety.

[0385] 49. The fusion protein according to item 48, wherein the additional polypeptide portion comprises an α-helical polypeptide domain.

[0386] 50. The fusion protein according to item 49, wherein the additional polypeptide portion comprises an α-helical bundle domain.

[0387] 51. The fusion protein according to item 50, wherein the additional polypeptide portion is or is derived from a protein domain of staphylococcal protein A (SpA).

[0388] 52. The fusion protein according to item 50 or 51, wherein the additional polypeptide portion comprises an amino acid sequence having at least 80% identity, for example at least 85% or at least 90% identity with SEQ ID NO: 161 or 162.

[0389] 53. The fusion protein according to any one of items 48-52, wherein the additional polypeptide portion is located at the C-terminus of the antigen-binding polypeptide.

[0390] 54. The fusion protein according to any one of items 48-52, comprising two or more copies of the additional polypeptide moiety, optionally separated by a linker peptide.

[0391] 55. The fusion protein according to item 48 comprises the following structure: ([A-L1)) m -[Z-L2] n ) p in A represents an antigen-binding polypeptide as defined in any of items 1-46. 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 items 49-52. 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.

[0392] 56. The fusion protein according to item 55, wherein m is 2 or 3, n is 1 and p is 1.

[0393] 57. The fusion protein according to item 55, wherein m is 1, n is 1 and p is 2 or 3.

[0394] 58. The fusion protein according to any one of items 55-57, comprising a C-terminal tag containing a plurality of histidine residues, preferably at least six histidine residues, optionally wherein the fusion protein contains no histidine residues other than the tag.

[0395] 59. An isolated nucleic acid encoding an antigen-binding polypeptide according to any one of items 1-46, a multimeric polypeptide according to item 47, or a fusion protein according to any one of items 48-58.

[0396] 60. An expression vector comprising the nucleic acid as described in item 59.

[0397] 61. A recombinant host cell for producing an antigen-binding polypeptide according to any one of items 1-46, or a multimeric polypeptide according to item 47, or a fusion protein according to any one of items 48-58, comprising an expression vector according to item 60.

[0398] 62. A method for producing an antigen-binding polypeptide according to any one of items 1-46, or a multimeric polypeptide according to item 47, or a fusion protein according to any one of items 48-58, comprising: i. Provide recombinant host cells as described in item 61; ii. Culture the host cells under conditions that enable the expression of the antigen-binding polypeptide, multimeric polypeptide, or fusion protein; and iii. Isolate the antigen-binding polypeptide, multimeric polypeptide, or fusion protein.

[0399] 63. The method according to item 62, wherein the cell is a prokaryotic cell, such as an Escherichia coli cell.

[0400] 64. The method according to item 62, wherein the cell is a eukaryotic cell.

[0401] 65. The method according to item 64, wherein the eukaryotic cell is a yeast cell, such as a Saccharomyces cerevisiae or Pichia pastoris cell.

[0402] 66. The method according to item 64, 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.

[0403] 67. The method according to any one of items 62-66, wherein step iii comprises purifying the antigen-binding polypeptide, multimeric polypeptide, or fusion protein by affinity chromatography.

[0404] 68. The method of claim 67, wherein the affinity chromatography uses an affinity ligand that binds to the frame region of a single-domain antibody variant.

[0405] 69. Use of the antigen-binding polypeptide according to any one of items 1-46, or the multimeric polypeptide according to item 47, or the fusion protein according to any one of items 48-58 as an affinity ligand for binding adeno-associated virus serotype 9 (AAV9).

[0406] 70. For use as described in item 69, for in vitro detection and / or purification of AAV9.

[0407] 71. The method for preparing a pharmaceutical composition comprising an AAV9 viral vector, according to the use described in item 69.

[0408] 72. A separation matrix comprising an antigen-binding polypeptide according to any one of items 1-46, a multimeric polypeptide according to item 47, or a fusion protein according to any one of items 48-58 as an affinity ligand coupled to a carrier material.

[0409] 73 . According to the separation matrix described in Project 72, the carrier material is a surface.

[0410] 74. The separation matrix according to Item 72, wherein the carrier material is selected from particles, beads, fibers, fiber membranes, filters, sheets, porous substrates, chips, plates, and pores.

[0411] 75. The separation matrix according to item 74, wherein the support material is a chromatographic matrix.

[0412] 76. The separation matrix according to item 75, wherein the matrix comprises a polysaccharide-based material, such as agarose and its derivatives, or cellulose and its derivatives.

[0413] 77. The separation matrix according to item 76, wherein the matrix comprises cross-linked agarose.

[0414] 78. The separation matrix according to item 75, wherein the carrier material is a chromatographic matrix selected from fibrous matrices, membranes, filters and porous substrates.

[0415] 79. The separation matrix according to item 78, wherein the carrier material is a nonwoven fiber matrix.

[0416] 80. An in vitro method for detecting AAV9 viral particles present in a sample, comprising (a) providing a sample containing AAV9 viral particles; (b) contacting the sample with an antigen-binding polypeptide according to any one of items 1-46, a polymer according to item 47, or a fusion protein according to any one of items 48-58, under conditions allowing sdAb variants to bind to the AAV9 viral particles, wherein the binding event generates a detectable signal; and (c) detecting the detectable signal.

[0417] 81. A method for chromatographic separation of AAV9 virus particles, comprising the following steps: (a) Provide a separation matrix according to any one of items 72-79, (b) Under conditions that allow the antigen-binding polypeptide to bind to AAV9 virus particles, the separation matrix is ​​brought into contact with a liquid sample containing the AAV9 virus particles. (c) Optionally wash the separation matrix. (d) Eluting bound AAV9 virus particles from the separation matrix, and (e) Clean the separation matrix material with a cleaning liquid.

[0418] 82. The method according to item 81, wherein step (e) comprises cleaning the separation matrix with an alkaline cleaning liquid, wherein the cleaning liquid preferably contains 0.05-0.5 M NaOH.

[0419] 83. The method according to item 81 or 82, wherein steps (a)-(e) are repeated at least 10 times, for example at least 20 times.

[0420] 84. The method according to any one of items 81-83, wherein after 10 cycles of contact with a cleaning liquid, the polypeptide retains at least 50%, for example at least 60%, at least 70%, at least 80%, or at least 90% of its initial target entity binding capacity.

[0421] 85. The method according to any one of items 81-84, wherein after 20 cycles of contact with a cleaning liquid, the polypeptide retains at least 50%, at least 60%, for example at least 70% or at least 80% of its initial target entity binding capacity.

[0422] 86. The method according to any one of items 81-83, wherein after 10 cycles of contact with an alkaline cleaning liquid, the polypeptide retains at least 60%, at least 70%, at least 80%, at least 90%, or for example at least 95% of the target entity binding capacity of the second cycle.

[0423] 87. The method according to item 86, wherein after 20 cycles of contact with an alkaline cleaning liquid, the polypeptide retains at least 60%, at least 70%, at least 80%, at least 90%, or for example at least 95% of the target entity binding capacity of the second cycle.

[0424] Table 21: Exemplary amino acid sequences References Thompson et al, Nucleic Acids Research , 22: 4673-4680 (1994) Kabat et al., J. Immunol.147(5), 1709-1719 (1991) The Heart, Biochim Biophys Acta 79(2), 393-398 (1964) US6602990 US7396467 WO2019137869A1 WO2018011600A1 CN116396381A CN116751284 WO2020242988A2 WO2023174900A1.

Claims

1. An antigen-binding polypeptide capable of binding adeno-associated virus serotype 9 (AAV9), said polypeptide comprising a single-domain antibody (sdAb) variant having complementarity-determining regions CDR1, CDR2, and CDR3, wherein CDR1 contains an amino acid sequence selected from the sequence group defined by SEQ ID NO: 1: X1X2X3SX5X6TMX9 (SEQ ID NO: 1) Independently, X1 is R, L, or S, preferably R or L; X2 is either T or R, preferably T; X3 is L or F, preferably L; X5 is D, N, or E, preferably D; X6 is Y, N, or F, preferably Y or F; X9 is either G or A, preferably G; CDR2 contains sequences selected from the sequence group defined by SEQ ID NO: 2: X1X2SWSGX7X8TX 10 (SEQ ID NO: 2) Independently, X1 is A, S, L, V or I, preferably A; X2 is I or V, preferably I; X7 is either A or S, preferably A; X8 is either Y or F, preferably Y; X 10 Is it K, F, or Y? as well as CDR3 contains sequences selected from the sequence group defined by SEQ ID NO: 3: X1X2TX4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 (SEQ ID NO: 3) Independently, X1 is G or A, preferably G; X2 is either P or S, preferably P; X4 is G or P, preferably G; X5 is L, T, or P, preferably L or P; X6 is L or I, preferably L; X7 is S, T, or A, preferably S or A; X8 is K, R, N, or Q; X9 is K, H, or R, preferably K or R; X 10 It is A, S, or T, preferably A or T; X 11 It is T, S, P or A, preferably T; X 12 It is P, A, T or S, preferably P; X 13 It is A, P, R, or G, preferably A or P; X 14 It is D, N, E or Q, preferably D or E; X 15 It is either Y or F, preferably Y; X 16 It is D, N, E, or Q, preferably D; and X 17 It is Y, F, or R, preferably Y or F.

2. The antigen-binding polypeptide according to claim 2, wherein the antigen-binding polypeptide selectively binds AAV9.

3. The antigen-binding polypeptide according to claim 1 or 2, wherein the sdAb variant is a variable domain of the heavy chain of a heavy chain antibody (VHH) variant.

4. The antigen-binding polypeptide according to any one of claims 1-3, wherein the polypeptide is basic and stable.

5. The antigen-binding polypeptide according to any one of the preceding claims, wherein the sdAb variant comprises a framework region (FWR) and a complementarity-determining region (CDR) from the N-terminus to the C-terminus in the following order: [FWR1]-[CDR1]-[FWR2]-[CDR2]-[FWR3]-[CDR3]-[FWR4].

6. The antigen-binding polypeptide of claim 5, wherein at least one of the framework regions comprises at least one amino acid substitution relative to SEQ ID NO: 4, and wherein the antigen-binding polypeptide exhibits improved basic stability compared to SEQ ID NO:

4.

7. The antigen-binding polypeptide according to claim 5 or 6, wherein FWR1, FWR2 and FWR3 each have an amino acid sequence that is at least 70% identical to the corresponding frame region of the sdAb selected from SEQ ID NO: 91 and 92.

8. The antigen-binding polypeptide according to any one of the preceding claims, wherein the sdAb variant comprises an amino acid sequence in which at least one of the Kabat amino acid positions 6, 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b and 89 is modified relative to the corresponding amino acid of SEQ ID NO:

4.

9. The antigen-binding polypeptide according to any one of the preceding claims, wherein in SEQ ID NO: 1, X1 is R, X2 is T, and X3 is L.

10. The antigen-binding polypeptide according to any one of the preceding claims, wherein in SEQ ID NO: 1, X6 is F and X9 is G or A.

11. The antigen-binding polypeptide according to any one of the preceding claims, wherein in SEQ ID NO: 2, X2 is I.

12. The antigen-binding polypeptide according to any one of the preceding claims, wherein in SEQ ID NO: 2, X7 is A, X8 is Y, and / or X 10 It's K.

13. The antigen-binding polypeptide according to any one of the preceding claims, wherein in SEQ ID NO: 3, X5 is L or P, and / or X 10 It's T.

14. The antigen-binding polypeptide according to claim 1 or 2, wherein CDR1 is selected from SEQ ID NO: 9-17, for example from SEQ ID NO: 13, 14, 16 and 17; and / or CDR2 is selected from SEQ ID NO: 21-31, for example from SEQ ID NO: 26, 28, 29 and 31; and / or CDR3 is selected from SEQ ID NO: 34-72 and 173, for example from SEQ ID NO: 36, 37, 39, 40, 42-46, 48-50, 55, 56, 62, 64, 68, 70-72 and 173.

15. The antigen-binding polypeptide of claim 8, wherein each FWR is selected from SEQ ID NO: 149-151 (FWR1), SEQ ID NO: 152-154 (FWR2), SEQ ID NO: 155-157 (FWR3) and SEQ ID NO: 8 (FWR4) and sequences having 95% or higher, such as 96%, 97%, 98%, 99%, identity with them.

16. The antigen-binding polypeptide according to any one of the preceding claims, wherein the sdAb variant has a) An amino acid sequence selected from SEQ ID NO: 75-93, 95-98, 101, 102, 105-107, 109-124, 126-146, and 173, for example, an amino acid sequence selected from SEQ ID NO: 92, 97, 109, 116, 131, 140-146, and 173; or b) Has at least 90% identity with the sequence defined in a), for example, at least 95% identity with the amino acid sequence, provided that the CDR is as defined in claim 1.

17. The antigen-binding polypeptide according to any one of the preceding claims, wherein the antigen-binding polypeptide comprises a sequence selected from SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 97, SEQ ID NO: 131, SEQ ID NO: 173 and SEQ ID NO: 177-182 and sequences having 95% or higher, such as 96%, 97%, 98%, 99%, identity with them.

18. The antigen-binding polypeptide according to any one of the preceding claims, wherein the antigen-binding polypeptide comprises at least one additional amino acid sequence, optionally selected from leader peptides, signal peptides, purification tags, coupling peptides, spacer peptides, and adaptor peptides, each additional amino acid sequence comprising up to 20 amino acids.

19. The antigen-binding polypeptide of claim 18, wherein the additional amino acid sequence comprises a C-terminal tag, for example selected from HHHHHH, HHHHHHC and AEAAAAKHHHHHHC (SEQ ID NO: 159).

20. A multimeric polypeptide comprising at least two moieties, each moiety comprising a single-domain antibody variant as defined in any of the preceding claims, said moieties optionally being linked by peptide linkers.

21. A fusion protein comprising at least one antigen-binding polypeptide according to any one of claims 1-19 or a multimeric polypeptide according to claim 20 and additional polypeptide portions.

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

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

24. The fusion protein according to claims 21-23, wherein the additional polypeptide portion comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 161 or 162, for example at least 85% or at least 90% identity.

25. The fusion protein according to claim 21 or 22, comprising the following structure: ([A-L1] m -[Z-L2] n ) p in A represents an antigen-binding polypeptide as defined in any one of claims 1-19. 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 moiety, optionally as defined in 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.

26. An isolated nucleic acid encoding an antigen-binding polypeptide according to any one of claims 1-19, a multimeric polypeptide according to claim 20, or a fusion protein according to any one of claims 21-25.

27. An expression vector comprising the nucleic acid according to claim 26.

28. A recombinant host cell for producing an antigen-binding polypeptide according to any one of claims 1-19, a multimeric polypeptide according to claim 20, or a fusion protein according to any one of claims 21-25, comprising the expression vector according to claim 27.

29. A method for producing an antigen-binding polypeptide according to any one of claims 1-19, or a multimeric polypeptide according to claim 20, or a fusion protein according to any one of claims 21-25, comprising: i. Providing the recombinant host cell according to claim 28; ii. Culture the host cells under conditions that enable the expression of the antigen-binding polypeptide, multimeric polypeptide, or fusion protein; and iii. Isolate the antigen-binding polypeptide, multimeric polypeptide, or fusion protein.

30. Use as an affinity ligand for binding adeno-associated virus serotype 9 (AAV9) by any of claims 1-19, the multimeric polypeptide of claim 20, or the fusion protein of any of claims 21-25.

31. The use according to claim 30, for purifying AAV9 virus particles.

32. The use according to claim 30, for in vitro detection of AAV9.

33. A separation matrix comprising an antigen-binding polypeptide according to any one of claims 1-19, a multimeric polypeptide according to claim 20, or a fusion protein according to any one of claims 21-25, coupled to a carrier material.

34. The separation matrix according to claim 33, wherein the carrier material is selected from fibrous membranes, particles or beads, fibers, filters, sheets, porous substrates, chips, plates, and pores.

35. The separation matrix according to claim 33 or 34, wherein the support material is a chromatographic matrix.

36. The separation matrix according to claim 35, wherein the carrier material is a chromatographic matrix selected from fibrous matrices, membranes, filters, and porous substrates.

37. The separation matrix according to claim 36, wherein the carrier material is a nonwoven fiber matrix.

38. The separation matrix of claim 35, wherein the carrier material comprises beads, the beads comprising agarose or a derivative thereof.

39. A method for chromatographic separation of AAV9 virus particles, comprising the following steps: (a) Providing a separation matrix according to any one of claims 33-38, (b) The separation matrix is ​​brought into contact with a liquid sample containing the AAV9 virus particles, provided that the antigen-binding polypeptide is allowed to bind to the AAV9 virus particles. (c) Optionally wash the separation matrix, and (d) Eluting bound AAV9 virus particles from the separation matrix.

40. The method of claim 39, further comprising: (e) Clean the separation matrix material with a cleaning liquid.

41. The method of claim 40, wherein step (e) comprises cleaning the separation matrix with an alkaline cleaning liquid, wherein the cleaning liquid preferably contains 0.05-0.5 M NaOH.

42. The method according to any one of claims 40-41, wherein steps (a)-(e) are repeated at least 10 times, for example at least 20 times.

43. The method according to any one of claims 40-42, wherein after 10 cycles of contact with a cleaning liquid, the polypeptide retains at least 50%, for example at least 60%, at least 70%, at least 80%, or at least 90% of its initial target entity binding capacity.

44. The method according to any one of claims 40-42, wherein after 10 cycles of contact with an alkaline cleaning liquid, the polypeptide retains at least 60%, at least 70%, at least 80%, at least 90%, for example at least 95% of the target entity binding capacity of the second cycle.

Images