Stable peptides

Abstract

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

Description

Technical Field

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

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

[0003] Affinity ligands can selectively and reversibly bind to target entities. Common examples of interactions include, for instance, enzyme-substrate interactions, biotin-avidin interactions, and antibody-antigen interactions. In many applications, affinity ligands can be immobilized on vectors and used to capture target entities, for example, for detection or purification purposes.

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

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

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

[0007] Staphylococcal protein A (SpA)-based affinity reagents, which are the most widely used affinity media for separating immunoglobulins and their fragments, have been developed to better tolerate alkaline conditions (see, for example, WO2003080655A1 and WO2016 / 079033A1). Meanwhile, there is a great need in the art for improved affinity ligands capable of binding to biomolecules other than immunoglobulins. Summary of the Invention

[0008] The objective of this invention is to overcome or at least partially mitigate the deficiencies of the prior art.

[0009] Therefore, the objective of this invention is to provide a framework that can be used for novel affinity ligands, the framework being base-stable.

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

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

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

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

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

[0015] The polypeptides disclosed herein may satisfy at least eight of the criteria i)-x) of the first aspect and at least five of the criteria i)-vi) of the second aspect.

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

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

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

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

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

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

[0022] In another aspect, fusion proteins are provided that comprise at least one polypeptide or polymer as defined herein and at least one additional polypeptide moiety.

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

[0024] In a further aspect, the present invention provides the use of the aforementioned polypeptide, polymer, or fusion protein as an affinity ligand for capturing a target entity, and the use of the aforementioned adsorbent material for separating the target entity from other components of a sample. The target entity is an entity to which the VH variant has an affinity. A separation method is also provided, comprising the following steps: (a) Providing an adsorbent material as described herein, wherein the VH variant of said peptide has binding affinity for the target entity. (b) Under conditions that allow the target entity to bind to the polypeptide, the adsorbent material is brought into contact with a liquid sample containing the target entity. (c) Optionally, the adsorbent material is washed. (d) Elution of the target entity from the adsorbent material, and (e) Clean the adsorbent material with a cleaning liquid.

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

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

[0027] 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.

[0028] Figure 2a -c is a schematic diagram of various structures of the polypeptide according to this disclosure.

[0029] Figure 3a-c is a schematic diagram of a fusion protein comprising at least one polypeptide disclosed herein fused with another polypeptide moiety.

[0030] Figure 4 This is a graph showing the results of the alkaline stability study of the polypeptide described in Example 2A.

[0031] Figure 5 This is a graph showing the results of the alkaline stability study of the polypeptide described in Example 2B.

[0032] Figure 6a -c displays the options for those that can combine with AAV9 ( Figure 6a ), GFP ( Figure 6b ) or EGFR ( Figure 6c The peptide was subjected to target binding responses for increasing cycles including NaOH exposure (Example 4A). The responses were normalized to the responses of the first cycle.

[0033] Figure 7a -c displays the options for those that can combine with AAV9 ( Figure 7a ), GFP ( Figure 7b ) or EGFR ( Figure 7c The fusion protein of the protein, for an increasing number of cycles including NaOH exposure, obtained a target binding response (Example 4B). The response was normalized to the response of the first cycle.

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

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

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

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

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

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

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

[0041] 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.

[0042] 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 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.

[0043] 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.

[0044] 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 or staphylococcal protein A and protein G) or their domains, including wild-type and variants in which one or more antigen-binding regions have been modified.

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

[0046] 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, or is derived from such a domain. Therefore, a single-domain antibody completely lacks the antibody light chain, including the light chain variable domain (VL). Single-domain antibodies may also lack structural features 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). This article includes reference peptides having such a natural framework, such as the AAV9-binding sdAb of SEQ ID NO: 152 (vh342), the GFP-binding sdAb of SEQ ID NO: 155 (vh60), and the EGFR-binding sdAb of SEQ ID NO: 157 (vh166). Single-domain antibodies naturally lacking the light chain also include so-called VNARs (variable neoantigen receptors), which are antibodies derived from cartilaginous fish (e.g., sharks). Alternatively, single-domain antibodies can be synthetic variants based on the amino acid sequence of the variable domain of the antibody heavy chain from species that do not naturally produce sdAbs (e.g., bovine, rat, mouse, or rabbit), and modified, for example, relative to the amino acids in the natural VH / VL interaction of such antibodies to mimic sdAbs. Such modifications may include substituting amino acids in the VH / VL interface region to increase hydrophilicity or solubility.

[0047] 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, which may also be described as "natural" or "unmodified" when compared to modified variants. The single-domain antibody variants of this invention are synthetic, non-naturally occurring amino acid sequences.

[0048] Complementarity-determining regions (CDRs) are hypervariable regions of an antibody or a portion thereof that participate in binding to target epitopes. CDRs are 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.

[0049] 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.

[0050] As used herein, the term "ligand" is a molecule that has a known or unknown affinity for a given entity. The term "ligand" is used interchangeably herein with the terms "affinity ligand," "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.

[0051] 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.

[0052] 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).

[0053] 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, such as with instruments like Biacore, as described in the examples 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 3000 response units (RU), for example, 3000-4000 RU.

[0054] 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.

[0055] As used herein, the term "basic stability" refers to the property of an antigen-binding peptide that withstands alkaline exposure without adverse effects on the peptide's structure and / or function. 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, 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.

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

[0057] In this context, an antigen-binding peptide is considered alkaline stable if, after at least 12 clean cycles, such as after 15 clean cycles, preferably after 20 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, such as at least 0.5 M NaOH.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] As used herein, the terms "multimer" and "multimeric protein" refer to a protein containing at least two repeating units of the 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 the multimeric form may be identical in their amino acid sequences or may differ slightly 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 stable polypeptide units as described herein, which are not single-domain antibodies. Such additional polypeptide units may exist in the multimer in single or multiple copies.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] The terms "%identity" or "%sequence identity" as used throughout this disclosure may be calculated, for example, as follows. The query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al, Nucleic Acids Research, 22: 4673-4680 (1994)). Comparisons are made 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. When %sequence identity is determined, if an amino acid position is omitted, the sequence identity is determined based on the shorter sequence. For example, in the FWR2 sequence with a total of 14 amino acid residues, when Kabat positions 40, 43, 44, 45, and 47 are omitted from the sequence identity determination, these amino acids are removed from both the query and target statements, resulting in a sequence comparison on a 9-amino acid sequence instead. Detailed Implementation

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

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

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

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

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

[0073] Generally, single-domain antibodies are more readily expressed in prokaryotic and eukaryotic cells compared to conventional full-length antibodies and Fab fragments or single-chain fragments comprising both light and heavy chains. Despite having only three CDRs, their target-binding properties are often satisfactory, unlike larger antibodies and antibody fragments with six CDRs. However, the stability of natural single-domain antibodies, such as camel VHHs, under alkaline conditions is problematic, and in many cases, poor alkaline stability will prevent such VHHs from being used as affinity ligands in chromatographic applications.

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

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

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

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

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

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

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

[0081] Table 2: Variable positions that potentially enhance basic stability Furthermore, it has been found that variations at some other locations can be tolerated without necessarily improving basic stability in the same way as seen for the locations described in Table 2. Such potentially variable locations with lower impact include Kabat positions 1, 2, 5, 11, 61, 62, 64, and 78. In this context, "lower impact" refers to a stabilizing effect.

[0082] However, it is conceivable that even further amino acid positions could exist within the framework region, which could tolerate more than one type of amino acid without impairing functionality.

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

[0084] Currently, 15 amino acid residues have been identified as variable and potentially positively impacting peptide stability. For each position, a limited set of amino acids found to contribute to improved stability can be selected. The proposed amino acids for each of these positions are provided in Tables 3-5 below.

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

[0086] Table 3a Kabat location Stable amino acids Preferred amino acids 24 I, V, S 40 R, I, K, T R, I 45 R, I, L R 76 N, Q N 79 Y, W, F Y, W, F 89 V, I V, I The VH variants of this disclosure may have any one of the positions selected independently as per Table 3a, or any combination or subgroup of these positions. For example, the amino acid residue at Kabat position 24 may be selected from I, V, and S, and / or the amino acid residue at Kabat position 40 may be selected from R, I, K, and T. The amino acid residue at Kabat position 76 may be selected from N and Q.

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

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

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

[0090] In addition to the six positions indicated in Table 3a, Kabat positions 19, 23, 43, and 44 have been found to contribute to the stability of the VH variant when selected from amino acids as outlined in Table 3b. Table 3b Kabat location Stable amino acids Preferred amino acids 19 R, S, K, T R, T 23 T, S, V T 43 R, K, Q, E, G R, K 44 E, Q, A, D, G E Preferably, at least one residue at Kabat position 19 and position 23 is T. It has been found that having T at Kabat position 19 promotes basic stability, especially when the amino acid at Kabat position 23 is V (see Example 2). On the other hand, Kabat position 19 has been found to participate in the interaction between VH3 and protein A. Where maintaining this interaction capability is required, the amino acid residue at position 19 is advantageously R.

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

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

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

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

[0095] For the remaining variable and potentially stable positions in the VH framework region, one or more of these positions may have amino acids indicating independent selection as shown in Table 4: Table 4 Kabat location Stable amino acids Preferred amino acids 6 Q, E 14 A, S, T 47 F, L F 60 A, T, N, Q, S A, S 82b N, S, T, Q Preferably, the residue at Kabat position 47 is selected from F and L, and more preferably F. In embodiments where the residue at Kabat position 47 is not selected according to Table 4, it is preferably not G, S, T, or Y.

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

[0097] Kabat position 82b has been found to be involved in the interaction between VH3 and protein A. To maintain this interaction capability, the amino acid residues at 82b are advantageously selected from S and N.

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

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

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

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

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

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

[0104] The VH variant of this disclosure may have an amino acid sequence in which at least eight of the Kabat positions 19, 23, 24, 40, 43, 44, 45, 76, 79, and 89 are independently selected according to Tables 3a-b and 4, or at least five of the Kabat positions 24, 40, 45, 76, 79, and 89 are selected according to Table 3a. Furthermore, the VH variant may have an amino acid sequence in which, relative to SEQ ID NO: 80 or SEQ ID NO: 195, Kabat positions 1-5, 7-13, 25-26, 36-39, 41-42, 46, 48-49, 59, 61-75, 77-78, 80-82a, and 82c-94 may include up to 15, for example up to 14, for example up to 13, for example 12, 11, or 10 substituted amino acid residues. For example, in FWR1, positions 1-5, 7-13, and 25-26 (21 residues) may together have up to 4, for example, up to 3, amino acid residues different from the corresponding positions in SEQ ID NO: 80 or SEQ ID NO: 195. In FWR2, positions 36-39, 41-42, 46, and 48-49 (14 residues) in Kabat may together have 1 or 2 amino acid residues different from the corresponding positions in SEQ ID NO: 80. In FWR3, positions 59, 61-75, 77-78, 80-82a, and 82c-94 (34 residues) in Kabat may have up to 6 amino acid residues different from the corresponding positions in SEQ ID NO: 80.

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

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

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

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

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

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

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

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

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

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

[0115] In some embodiments, the VH variant may have a frame region 1 (FWR1) comprising an amino acid sequence according to the following sequence: X1X2QLX5X6SGGGX 11 VQX 14 GGSLX 19 LSCX 23 X 24 SG (SEQ ID NO: 1) Independently, X1 is Q, V, D or E, preferably Q or E; X2 is V or D, preferably V; X5 is Q, V, or E, preferably Q; X6 is either Q or E, preferably Q; X 11 It is S or L, preferably S; X 14 It is A, S, or T, with A being the preferred choice; X 19 It is R, S, K or T, preferably R or T; X 23 It is T, V, A, or S, preferably T; and X 24 It is I, V, or S, preferably I.

[0116] Alternatively or additionally, the VH variant may have a frame region 2 (FWR2) comprising an amino acid sequence according to the following sequence: WFRQX5PGX8X9X 10 EX 12 VA (SEQ ID NO: 2) Independently, X5 is R, I, T, or K, preferably R; X8 is R, K, Q, E or G, preferably K or R; X9 is E, Q, A, D, or G; X 10 It is R, I, or L, preferably R; and X 12 It is F or L, preferably F.

[0117] Alternatively or additionally, the VH variant may have a frame region 3 (FWR3) comprising an amino acid sequence according to the following sequence: YX2X3X4VX6GRFTISRDNAKX 18 TX 20 X 21 LQMNX 26 LKPEDTAX 34 YYCAA (SEQ ID NO: 3) Independently, X2 is A, T, N, Q, or S, preferably A; X3 is either D or S, preferably D; X4 is either S or A, preferably S; X6 is either K or A, preferably K; X 18 It is N or Q, preferably N; X 20 It is either V or A, preferably V; X 21 It is Y, W, or F, preferably Y or W; X 26 It is N, S, T, or Q, preferably S; and X 34 It is either V or I.

[0118] In the implementation scheme, X6 in SEQ ID NO: 3 is K, X 21 It is Y and X 26 It is S.

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

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

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

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

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

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

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

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

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

[0128] The exemplary arrangement of the polypeptides in this disclosure is as follows Figure 2a -c is an illustrative example. In Figure 2a In this study, peptide 200 comprises an N-terminal amino acid sequence 201, a VH 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.

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

[0130] For example, the polypeptide may contain two VH variants (“dimers”) or three VH variants (“trimers”). Figure 2c An exemplary trimer 230 is shown, which comprises three VH variants 202.

[0131] Optionally, the multimeric polypeptide may contain at least one additional amino acid sequence that does not represent the VH variant, such as a stable polypeptide as described elsewhere in this document in conjunction with fusion proteins.

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

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

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

[0135] The additional amino residues may be coupled to VH or multimers by chemical conjugation (using known organic chemical methods) or by any other means, such as expression as a fusion protein or by any other means (directly or through a linker, such as the peptide linker described above).

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

[0137] As those skilled in the art will understand, the construction of multimers, such as fusion proteins, typically involves the use of linkers between the monomeric moieties to be fused. Monomeric moieties can refer to single-domain peptides as described herein. Those skilled in the art will understand that different types of linkers exist 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 improve its folding. Linkers can also ensure a certain spatial distance between the VH variant and the fusion partner peptide, which can be advantageous for target proximity to the binding surface of the VH variant. The presence of linkers within a fusion protein generally does not significantly affect the target-binding capacity of correctly folded antigen-binding peptide monomers, such as single-domain antibodies.

[0138] Therefore, a polymer as defined herein may further include at least one linker. For example, a linker exists between each monomer within the polymer. In the presence of multiple linkers, the linkers may be the same or different. For each occurrence, the linker may be selected, for example, from flexible amino acid linkers, rigid amino acid linkers, and cleavable peptide linkers. Peptide linkers may be structured or unstructured. Generally, unstructured linkers are more flexible and less rigid than structured linkers. Alternatively, the linker may be a non-peptide linker. Thus, the polypeptides or subunits thereof 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 protein was excised. The number of additional amino acid residues may be, for example, 15 or fewer, such as 10 or fewer, or 5 or fewer.

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

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

[0141] In some embodiments, the polypeptide may be a fusion protein containing at least one VH variant as disclosed herein and 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 contain or be composed of the single-chain polypeptide 202 and other amino acids (e.g., other amino acid sequences 201 and / or linkers 204).

[0142] Specifically, the fusion protein of this disclosure may comprise one or more VH variants fused to an α-helical polypeptide (e.g., a protein domain). The α-helical polypeptide may contain at least one α-helix, 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 partner polypeptide may contain a sequence having at least 80% identity, such as at least 85% or at least 90% identity, with respect to SEQ ID NO: 159 or SEQ ID NO: 160.

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

[0144] Therefore, the fusion protein may optionally be provided as a multimeric fusion protein (“multimer”) containing at least two peptides comprising the VH variant 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 a plurality of polypeptide 200 units arranged sequentially from the N-terminus to the C-terminus, and these units may optionally be linked to each other by a linker sequence 204, which may be the same or different for each occurrence. The VH-based polypeptides 200 may be identical or different from each other in terms of their amino acid sequences. Figure 3b Three polypeptides 200 (“trimers”) are shown, but the fusion protein is envisioned to contain any suitable number of said polypeptides 200. For example, the fusion protein may contain at least 2 or at least 3, 4, 5 or 6 antigen-binding polypeptide units. The fusion protein may contain, for example, up to 10 antigen-binding polypeptides 200.

[0145] In addition, the fusion protein may contain at least two, for example three, four, five or six fusion partner polypeptide units, for example up to ten fusion partner polypeptide units. Figure 3c A fusion protein 320 containing a first antigen-binding polypeptide 200 and three polypeptide moieties 301 is described. The linker 204 is optional and may be the same or different for each occurrence. The polypeptide moieties 301 may be the same 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 stable polypeptides, or the fusion may contain an antigen-binding polypeptide comprising a VH variant, which has a different antigen-binding polypeptide attached to one side and a stable polypeptide attached to the other side. In cases where the fusion protein contains multiple antigen-binding polypeptides and multiple stable 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 polypeptide, such as... Figure 3b (for peptide 200) and Figure 3c (As described in Stable Peptide 301). For example, multimeric fusion proteins of each peptide containing two units may have the following general structure: [antigen-binding peptide]-[fusion partner peptide]-[antigen-binding peptide]-[fusion partner peptide].

[0146] More typically, the fusion protein according to this disclosure may have the following structure: ([A-L1)) m -[Z-L2] n ) p Where 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, may be at most 10, for example, at most 8.

[0147] 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.

[0148] For reference purposes, 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 3cIn the exemplary fusion protein, m=1, n=3, and p=1.

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

[0150] 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 group, or another short amino acid sequence, may be optionally provided as needed. For example, multiple histidine residues may be provided, as described elsewhere in the text.

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

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

[0153] The peptides of the present invention, which exhibit improved basic stability compared to peptides based on the native VHH framework, can be advantageously used to generate affinity binders for suitable targets by transplanting a CDR sequence targeting a desired target into a VH variant framework as disclosed herein. Known CDR1-3 sequences for a specific antigen can be identified or predicted from relevant literature, or new CDR sequences can be generated or identified by any method known to those skilled in the art, including but not limited to immunizing animals (e.g., camelids), phage display, ribosome display, cell surface display, bacterial display, computer simulation, and combinations thereof. When a satisfactory binding peptide has been identified, the amino acid sequence of the CDR can be selected and used to design stable antigen-binding peptides having a stable framework sequence as described herein. Exemplary methods for generating high-affinity antigen-binding VHHs with transplantable CDR sequences are described, for example, in Schmitz et al., 2013, Structure 21 , 1214-1224 and Fleetwood et al., Cell. Mol. Life Sci. (2013) 70:1081-1093.

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

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

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

[0157] This disclosure also includes a method for producing a polypeptide as described herein, comprising culturing the host cell under conditions that allow expression of the polypeptide, multimer, or fusion protein from its expression vector, and isolating the polypeptide, multimer, or fusion protein.

[0158] 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.

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

[0160] Alternatively, peptides 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.

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

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

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

[0164] When the peptide is coupled 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 separating target entities from other components. The coupling of the peptide to the carrier of the present invention can optionally be provided by a C-terminal amino acid sequence suitable for coupling with the carrier. For example, a cysteine-terminated tag or spacer can be provided for coupling via a thioether bond, as described in more detail below. When the peptide forms part of a fusion protein, the C-terminal fusion partner peptide may have such a C-terminal amino acid sequence suitable for coupling with the carrier described below.

[0165] When the 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 a polypeptide present in solution (capable of binding the target). In such cases, a reporter entity is envisioned for generating a detectable signal, wherein the reporter entity is capable of binding the polypeptide, or, in the case of a fusion protein, of binding the fusion partner. The reporter entity may be, for example, a fluorescently labeled or radiolabeled entity, as known in the art. If the polypeptide or, in its presence, the fusion partner polypeptide is capable of binding IgG, the reporter entity may be IgG-based.

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

[0167] The carrier may be a solid carrier and optionally a porous material.

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

[0169] The support may optionally be provided in other forms, such as fibers, membranes, fiber matrices, filters, porous bulk materials, particles, or beads, such as gel beads used in chromatographic resins. The particles or beads may be porous or non-porous. The particles or beads may include magnetic beads. Supports 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.

[0170] 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.

[0171] The 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 polypeptide to improve the utilization of the binding region and / or promote the chemical coupling of the polypeptide to the carrier. Depending on the properties of the polypeptide and the coupling conditions, the coupling can be random or multi-point coupling (e.g., via multiple lysine or histidine residues) or single-point coupling (e.g., via a single cysteine ​​residue). In embodiments, to improve coupling control, it may be preferred that the VH variant itself does not contain any histidine residues. However, in the same case, the polypeptide as a whole may contain a histidine tag, such as a (His)6 tag. Alternatively, the polypeptide can be linked to the carrier via non-covalent bonding, such as physical adsorption or biospecific adsorption.

[0172] The polypeptide can be coupled to the carrier via a thioether bond. 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 polypeptide, the motility of the coupled antigen-binding polypeptide is enhanced, providing improved binding capacity and binding kinetics. In some embodiments, the polypeptide is coupled via a C-terminal cysteine ​​residue provided on the protein as described above. This allows the cysteine ​​thiol group to be effectively coupled to an electrophilic group on the carrier, such as an epoxide group, a halool group, etc., resulting in thioether-bridged coupling.

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

[0174] Synthetic polymers that can be used as carrier materials include polyvinyl alcohol, polyhydroxyalkyl 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.

[0175] Alternatively, the solid carrier according to the present invention comprises an inorganic carrier, such as silicon dioxide, zirconium oxide, etc.

[0176] In this embodiment, the polypeptide may be coupled to a carrier, which is a convection-based chromatographic matrix. Such a convection-based chromatographic matrix may comprise a porous polymer membrane, a filter, a fibrous matrix, or a porous monolith. Examples of porous polymer membranes include Mustang. TM Membrane (Cytiva) and Sartobind TMThe 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.

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

[0178] 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.

[0179] 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.

[0180] This invention provides a method for separating or isolating target entities, wherein a separation matrix or adsorbent material as disclosed herein is used. In some embodiments, the method includes contacting a liquid sample containing the target entity with an adsorbent material as disclosed herein. Contact is carried out under conditions where the target entity can bind a VH variant of the peptide. The method may further include washing the adsorbent material with a washing liquid, eluting the target entity from the adsorbent material with an elution liquid, and optionally cleaning the adsorbent material with a cleaning liquid. The cleaning liquid may also be referred to as a clean-in-situ (CIP) liquid. The cleaning liquid is typically an alkaline solution, 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 5 times, for example at least 10 times, for example at least 20 times.

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

[0182] 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).

[0183] Prior to contact with the adsorbent material 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 adsorbent material can be used to separate the target entity from such impurities, and after the above process, the eluent containing the target entity has a reduced level of at least one of the impurities.

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

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

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

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

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

[0189] The materials and equipment used were as follows: kanamycin-resistant vector plasmids; G-blocks peptide candidates (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.

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

[0191] 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, the cells and ligation mixture were incubated on ice for 20 min, followed by incubation in KCM buffer 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 on ice for 20 min, followed by incubation in KCM buffer 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 then 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.

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

[0193] The materials and equipment used are as follows: plasmid DNA of the candidate peptide to be produced; chemically competent 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™) (BD BACTO™ agar); 14 ml Falcon™ Round-Bottom (Corning™); Infors HT shaking incubator.

[0194] 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.

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

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

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

[0198] Table 6 The following aspects were evaluated: (1) Affinity assessment of AAV9 interactions, which was tested by injecting high concentrations of AAV9. (2) Basic stability, which was tested by the reduction in AAV9 binding after treatment with 0.3 M NaOH in increments of 100%.

[0199] Materials and methods Generation of candidate peptides purified from IgG Sepharose In a 14 ml round-bottom tube, 5 µl of each variant of K12-017 glycerol stock solution as described in Example 1A was inoculated into 4 ml of LB medium supplemented with 50 µg / ml kanamycin and incubated overnight at 37°C with stirring at 160 rpm.

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

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

[0202] Following the manufacturer's instructions, use NanoDrop (Thermo Scientific) to measure the concentration of the purified IgG Sepharose sample. After purification, keep the sample in an Eppendorf tube in the refrigerator until all measurements have been performed, and then keep it in the freezer until Biacore analysis.

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

[0204] The materials and equipment used are as follows: Biacore™ S-Series CM5 sensor chip (Cytiva™); Biacore™ Amine Conjugation Kit (Cytiva™); Biacore™ Acetate Buffer pH 5.0 (Cytiva™); Biacore™ 8K+ Instrument (Cytiva™); AAV9 (prepared internally); and IgG Sepharose purified candidate peptides as described above.

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

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

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

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

[0209] All sensor maps 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.

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

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

[0212] All sensor maps 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.

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

[0214] Figure 4 The results of the alkaline stability studies are shown, with all responses normalized to the response of the first cycle (i.e., prior to the first NaOH exposure). VH98 is moderately stable under 0.3 M NaOH exposure, while VH100 and VH103 show slight improvements. VH105 confirms further improved stability, with a slower degradation rate than VH103. The results are summarized in Table 7. It can be concluded that both 19T and 23T are beneficial for stability. P40R also leads to improved stability.

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

[0216] The candidate VHH sequence is identified in Table 8. The CDR is identical across all candidates.

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

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

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

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

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

[0222] Using standard methods in Biacore software, candidate peptides were immobilized by coupling in flow cell 2 (FC2) and activation / inactivation in flow cell 1 (FC1). Candidate peptides were diluted in acetate buffer at a concentration of 25 µg / ml. Immobilization levels did vary slightly between different peptide variants (mean 4319 RU + SD 640 RU). Peptides were immobilized in FC2.

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

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

[0225] 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.

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

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

[0228] All sensor 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.

[0229] result All candidates were found to have similar and high AAV9 binding capacities (Biacore response values ​​ranged from approximately 12,000 to 15,000 RU; fixed levels varied slightly among the candidates), with vh97 exhibiting the highest capacity. It was concluded that variations in the testing framework had no significant impact on target binding capacity.

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

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

[0232] Compared to vh119, candidate vh118 exhibits better basic stability, and vh122 is more stable than vh123 (also omitted from the figure; its stability is similar to vh121), indicating that replacing E6Q can improve NaOH tolerance.

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

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

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

[0236] The following aspects were evaluated: (1) Affinity assessment of AAV9 interactions (tested by high-concentration AAV9 injection). (2) Basic stability (tested by the reduction of AAV9 binding after treatment with 0.5 M NaOH in increments of 100%.

[0237] Materials and methods Candidate peptides purified by immobilized metal affinity chromatography (IMAC) In a 14 ml round-bottom tube, 5 µl of each variant of BL21(DE3) glycerol stock solution as described in Example 1B was inoculated into 4 ml of LB supplemented with 100 µg / ml carbenicillin and incubated overnight at 37°C and 160 rpm.

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

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

[0240] Following the manufacturer's instructions, use NanoDrop (Thermo Scientific) to measure the concentration of the HisTrap™ FF purified sample. After purification, keep the sample in an Eppendorf tube in the refrigerator until all measurements are performed, and then keep it in the freezer until Biacore analysis.

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

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

[0243] Immobilization was performed using standard methods in Biacore software, by coupling AAV9-binding peptide variants in flow cell 2 (FC2) and activating / inactivating them in flow cell 1 (FC1). The AAV9-binding peptide variants were diluted at a concentration of 25 µg / ml in acetate buffer at pH 5.0. Immobilization levels varied slightly between the different peptide variants (mean 3763 RU ± SD500 RU).

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

[0245] 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, AAV9 2E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

[0246] 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.

[0247] Biacore analysis of alkaline stability After evaluating AAV9 binding (see above), the same chip underwent 40 cycles of AAV9 binding (5E11 vp / ml) followed by NaOH (0.5 M) injection.

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

[0249] 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.

[0250] result The response levels of the candidate peptides to the AAV9 interaction were normalized to the response of SEQ ID NO: 80 and are summarized in Tables 9a-c. In this regard, it should be noted that vh118 (SEQ ID NO: 80) contains a preferred frame with a near-optimal combination of mutations for the CDRs currently tested, compared to the native or synthetic starting frame. Therefore, most other variants will exhibit lower basic stability compared to SEQ ID NO: 80. The variants described below all exhibit stability compared to the virtually non-existent stability of the native frame and the moderate stability of the synthetic starting frame. Therefore, the mutations introduced in these variants are still stable compared to the initial frame, but less so than the most preferred mutations. A comparison between SEQ ID NO: 80 and the least preferred mutations is included in Example 8 below.

[0251] Alkaline stability was recorded as the number of cycles in which at least 50% of the binding capacity was retained. The first cycle was excluded, and the binding capacity value was 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 10-minute contact time. Results for each candidate are provided in Tables 9a-c.

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

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

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

[0255] Table 9a: FWR1 variants Table 9b: FWR2 variants Table 9c: FWR3 variants Example 4A: Basic stability of natural and modified sdAb frameworks This embodiment compares single-domain antibodies with a natural camel sdAb framework with sdAbs with a framework modified to enhance basic stability. The following aspects were evaluated: (1) Affinity assessment of target molecule interactions (tested by injecting target molecules at various concentrations). (2) Basic stability (tested by the reduction in target binding after treatment with 0.1 M or 0.3 M NaOH in increments of 0.1 M or 0.3 M).

[0256] The single-domain antibodies are VHH variants targeting AAV9, green fluorescent protein (GFP), or epidermal growth factor receptor (EGFR), and are based on natural camel VHH frames or modified frames to enhance the basic stability of the VHH variants themselves. The natural camel frames correspond to Schmitz et al., 2013, Structure 21 The frame sequence of VHH “EgA1” or “9G8” described in 1214-1224, or corresponding to Fleetwood et al. Cell. Mol. Life Science. The GFP-binding VHH “S-Nb3” framework sequence described in (2013) 70:1081-1093 is used, except that the first two amino acids are replaced by VD for the latter. The CDR region is the same for candidates that bind the same target.

[0257] Each sdAb was expressed as a fusion protein fused to a C-terminal SpA domain Z variant (SEQ ID NO: 160), in which binding to the Fc and VH3 moieties of IgG was deactivated. All constructs had a C-terminal linker (GS) followed by a His6 tag.

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

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

[0260] 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 filter the supernatant through a 0.22 µm filter.

[0261] Samples were purified using a HisTrap™ FF 1 mL column (Cytiva). The clarified lysate was loaded onto a column 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. Protein was 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) pH 7.4 using a gravity flow column pre-packed with Sephadex™ G-25 resin (Cytiva).

[0262] Following the manufacturer's instructions, use NanoDrop (Thermo Scientific) to measure the concentration of purified and buffer-exchanged samples. After purification, keep the samples in Eppendorf tubes in the refrigerator until all measurements are performed, and then keep them in the freezer until Biacore analysis.

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

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

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

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

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

[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 Following the binding assay (see above), the same CM5 chip with the immobilized peptide underwent repeated cycles of binding the corresponding analyte followed by NaOH injection to assess alkaline stability (0.3 M for AAV9-binding peptides and 0.1 M for GFP and EGFR-binding peptides, respectively).

[0270] Biacore method for alkaline stability (per cycle): Run buffer: PBS-P+; Flow rate: 10 µl / min; Sample injection 1 (AAV9 5E11 vp / ml or GFP 50 nM or EGFR 75 nM): 300 s in both flow cells; Dissociation time 1: 30 s; Sample injection 2 (0.1 M or 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 target response.

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

[0273] Table 11a: AAV9 binding candidates Table 11b: EGFR-binding candidates Table 11c: GFP binding candidates Target binding capacity (late-report point binding) measured at the highest analyte concentration showed little or no difference in target binding capacity between candidates with different framework sequences. However, the difference in basic stability was significant. Figure 6a -c indicates the presence of AAV9-binding peptides ( Figure 6a ), GFP-binding peptide ( Figure 6b ) and EGFR-binding peptide ( Figure 6c The binding capacity was normalized to the response of the first cycle, with increasing cycle numbers including NaOH exposure (sample injection 2 above). As can be seen in these figures, VHH affinity ligands based on stable VHH frameworks (4A-3, 4A-5, 4A-8) exhibit improved basic stability compared to a reference VHH with a natural camelid VHH framework. In fact, after the first cycle, unstable candidates 4A-1 and 4A-2 almost completely lost their target binding response.

[0274] Example 4B: Basic stability of various sdAb frameworks This example compares single-domain antibodies with natural camel-derived or synthetic sdAb frameworks with sdAbs having frameworks modified to enhance basic stability. The following aspects were evaluated: (1) Affinity assessment of target molecule interactions (tested by high-concentration injection of target molecules).

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

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

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

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

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

[0280] 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.

[0281] Samples were purified using gravity flow on 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 buffer was exchanged for phosphate-buffered saline (Medicago) at pH 7.4 using a gravity flow column (PD-10 (Cytiva)).

[0282] Following the manufacturer's instructions, use NanoDrop (Thermo Scientific) to measure the concentration of the purified IgG Sepharose sample. After purification, keep the sample in an Eppendorf tube in the refrigerator until all measurements are performed, and then keep it in the freezer until Biacore analysis.

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

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

[0285] Biacore™ method for AAV9 binding analysis: 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, 1.25E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

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

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

[0288] 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.

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

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

[0291] Biacore method for GFP basic stability (per cycle): Run buffer: PBS-P+; Flow rate: 10 µl / min; Blocking solution injection: (Fc 2 µM from mAb) 90 s in both flow cells; Block dissociation time: 0 s; Sample injection 1 (trastuzumab-GFP 2 µM): 300 s in both flow cells; Dissociation time 1: 30 s; Sample injection 2 (0.3 M NaOH): 600 s in both flow cells; Dissociation time 2: 0 s; Regeneration: 10 mM glycine-HCl pH1.5, 30 µl / min, 2 x 30 s.

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

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

[0294] 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.

[0295] result All fusion proteins showed satisfactory binding to their respective targets.

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

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

[0298] Example 5: AAV9-binding peptides with different frameworks and CDR variants This embodiment further investigated two different sets of CDR sequences (both targeting AAV9) within different stable sdAb frameworks. The candidate peptides tested were expressed as fusion proteins, with each sdAb having SEQ ID NO: 160 fused to its C-terminus via a linker and possessing a C-terminal (His) 6-tag. sdAb sequence variants are provided in Table 14. For all candidates, FWR4 was identical (SEQ ID NO: 71).

[0299] 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 ...

[0300] Table 14 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 1B was inoculated into 4 ml of LB medium supplemented with 100 µg / ml carbenicillin in a 14 ml round-bottom tube and grown overnight at 37°C and 200 rpm with stirring. Protein expression medium (TB medium supplemented with 100 µg / ml carbenicillin and 2 mM MgCl2) was prepared and added to 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 shaking incubator at 37°C and 140 rpm for approximately 3.5 hours until the OD600 reached 1.0. Next, 50 µl of IPTG (1 M) was added to a final concentration of 1 mM, and the 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.

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

[0302] 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™).

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

[0304] The materials and equipment used 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 peptide generation as described above.

[0305] 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. The peptide was immobilized in FC2 in each run.

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

[0307] All sensor maps 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.

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

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

[0310] All sensor 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.

[0311] result Using the maximum AAV9 concentration (2E12 vp / ml), the response level of the candidate peptides to AAV9 interaction was normalized to the response of vh118 and is summarized in Table 15, with basic stability recorded as the number of cycles retaining at least 50% of the binding capacity. Figure 8 Results of the alkaline stability assessment are provided, with binding capacity plotted against cycle number. Binding response values ​​are normalized against the binding capacity recorded in 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 10-minute contact time.

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

[0313] Table 15 Example 6: 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: 160 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 16. The FWR sequence was identical for all candidates.

[0314] Table 16 Materials and methods As described in Example 5, IMAC-purified candidate peptides are generated.

[0315] 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 2X molar excess of biotin.

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

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

[0318] The materials and equipment used 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™).

[0319] 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.

[0320] 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.

[0321] 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.

[0322] 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 with AAV9 (5E11 vp / ml), otherwise a run of PBS-P+ buffer was used for sample injection to minimize analyte consumption.

[0323] Biacore method for alkaline stability (per cycle): Run buffer: PBS-P+; flow rate: 10 µl / min; Sample injection 1 (AAV9 5E11 vp / ml or run buffer PBS-P+, every second cycle to reduce analyte consumption): 300 s 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.

[0324] 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.

[0325] result Table 17 shows the fixation levels obtained for the test 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:160 and therefore had a higher molecular weight than the sdAb itself. Relative fixation levels are fixation levels divided by molecular weight.

[0326] Table 17 *Existing technology Despite being fixed at a lower molar level than the existing technology reference CaptureSelect™ biotinylate-AAV9 conjugate, the AAV9-binding peptides of the present invention exhibited significantly higher AAV9 binding capacity. Furthermore, the candidates of the present invention demonstrated very high basic stability. In fact, for one candidate, vh377, the AAV9 response never decreased below 50% during 88 cycles.

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

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

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

[0330] Protein expression medium (TB medium supplemented with 100 µg / ml carbenicillin and 2 mM MgCl2) was prepared and added to pre-filled 500 ml baffled glass shake flasks (50 ml / flask). Approximately 500 µl of the previous overnight culture was inoculated into each flask to obtain an initial OD600 of 0.05. The flasks were incubated in an Infortrère HT shaker at 37°C and 140 rpm for approximately 3.5 h until the OD600 reached 1.0. Then, 50 µl of IPTG (1 M) was added to a final concentration of 1 mM, and the flasks were incubated in an Infortrère HT shaker at 27°C and 140 rpm for 18 h. 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 h, followed by centrifugation at 8000 g for 10 min to precipitate cell debris, and the supernatant was filtered through a 0.22 µm filter.

[0331] Clarified lysates of each sample were loaded onto a 1 ml HiTrap MabSelect™ PrismA column (Cytiva™) equilibrated in phosphate-buffered saline (PBS) 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.

[0332] 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™).

[0333] The fusion protein was immobilized on epoxy-activated chromatography resin (highly cross-linked agarose beads) using cysteine ​​coupling. 0.2 ml of resin was packed into a Tricorn 5 / 20 column (Cytiva). Using an ÄKTA pure 25 system (Cytiva), 50 ml of tangentially filtered 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.

[0334] 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.

[0335] 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 entire stability study consisted of 40 cycles, each involving contacting the chromatographic material with 0.5 M NaOH for 30 minutes. Every 10th cycle, a solution containing pure AAV9 particles was added, with a residence time of 15 seconds. PBS buffer was used for all other cycles.

[0336] result Figure 10a The chromatograms obtained from the purification of AAV9 TFF material using candidate vh118 as an affinity ligand are shown. Elution peaks are indicated by rectangles. Figure 10b Only the area of ​​the elution peak is displayed. Figure 11 Photograph of the gel is shown. VP1, VP2, and VP3 represent AAV9 virosomal proteins 1, 2, and 3, respectively.

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

[0338] The results of the alkaline stability assessment showed that Figure 12 In the figure, the binding capacity was plotted against the binding capacity normalized to the level before the first NaOH exposure. After 40 cycles of exposure to 0.5 M NaOH, corresponding to a cumulative exposure time of 20 hours, the target binding capacity decreased by less than 20%.

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

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

[0341] The VH sequence is expressed, with SEQ ID NO:160 serving as a C-terminal fusion via a peptide linker. The total molecular weight is 21 kDa.

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

[0343] B: sdAb targeting GFP As in Example A above, this example uses a different target than AAV9 to study the basic stability of the framework, wherein a GFP-binding VH is generated, one with multiple variations in the framework region, wherein the mutation is selected from less preferred mutations, one has a natural framework, and one has a framework with preferred modifications.

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

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

[0346] Table 19 The following aspects were evaluated: (1) Affinity assessment of AAV9 interactions, which was tested by injecting high concentrations of AAV9. (2) Basic stability, which was tested by the reduction in AAV9 binding after treatment with 0.1 M NaOH in increments of 0.1 M.

[0347] C: sdAb for EGFR As in Example B above, this example uses a different target than AAV9 to study the basic stability of the framework, wherein EGFR-binding VH is generated, one with multiple variations in the framework region, wherein the mutation is selected from less preferred mutations, one has a natural framework, and one has a framework with preferred modifications.

[0348] Three candidate VH sequences were tested: one with an unmodified frame (vh166), one with a frame containing amino acid substitutions preferred over the unmodified frame (vh167), and one with a combination of less preferred mutations (vh541). The amino acid substitutions (Kabat numbers) and their corresponding full-length sdAb sequences for each candidate VHH are illustrated in Table 20. The CDR sequence was identical for all candidates.

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

[0350] Table 20 The following aspects were evaluated: (1) Affinity assessment of AAV9 interactions, which was tested by injecting high concentrations of AAV9. (2) Basic stability, which was tested by the reduction in AAV9 binding after treatment with 0.1 M NaOH in increments of 0.1 M.

[0351] Materials and methods Candidate peptides were generated and purified on an IMAC (as described in Example 5 above) or using PrismA (as described in Example 7 above). Biacore analysis of the binding affinity for AAV9 and basic stability analysis were also performed as described in the examples above.

[0352] result For AAV-binding VHH candidates, both vh540 and vh118 showed good AAV binding, while vh342 showed slightly lower binding (data not shown). When assessing basic stability (up to 10% stability at cycle 1), vh342 with the native framework showed zero stability at 0.3 M NaOH, meaning it is not basic stable. Candidates including several less preferred point mutations showed basic stability for 12 cycles at 0.3 M NaOH. Therefore, even though vh540 is less stable than vh118, it shows good basic stability, especially compared to the native framework used in vh342 (which showed no stability at all). This suggests that combinations of less preferred mutations will also result in stable frameworks compared to the native framework.

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

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

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

[0356] Example 9: AAV9 binding peptides in different frameworks and two groups of CDRs This embodiment further investigated five additional different sdAb frames as well as the same sdAb CDR sequences. A frame was also tested with two different sets of CDR sequences. All sdAbs had the same CDR1 and CDR2 sequences, while vh516 had a different CDR3 than vh511-vh515. Vh511-515 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.

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

[0358] The following aspects were evaluated: (1) Affinity assessment of AAV9 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).

[0359] Table 21 Table 22 Materials and methods Generation of candidate peptides purified by immobilized metal affinity chromatography (IMAC) For each variant, 5 µl of the glycerol stock solution as described in Example 1 was inoculated into 4 ml of LB medium 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 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.

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

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

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

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

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

[0365] The materials and equipment used 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™).

[0366] Using standard methods in Biacore™ software, the biotinylated AAV9-binding peptide variants were immobilized by coupling them in flow cell 2 (FC2) and then activating / inactivating them in flow cell 1 (FC1). The biotinylated AAV9-binding peptide variants were diluted in PBS at a concentration of 100 µg / ml.

[0367] 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.

[0368] 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.

[0369] 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 with AAV9 (5E11 vp / ml), otherwise a run of PBS-P+ buffer was used for sample injection to minimize analyte consumption.

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

[0371] 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.

[0372] 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, recording basic stability as the number of cycles in which at least 50% of the binding capacity was retained. Binding response values ​​were normalized to the binding capacity recorded for the second cycle (i.e., excluding the first cycle). In each cycle, 0.3 M NaOH was injected at a rate of 10 µl / min for a 10-minute contact time.

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

[0374] Table 23 Example 10: 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.

[0375] Materials and methods Four peptides were tested: vh118 (SEQ ID NO: 80) and vh498 (SEQ ID NO: 195), with vh498 differing from vh118 in that the first position is replaced by E instead of Q. Similarly, vh324 (SEQ ID NO: 187) and vh459 (SEQ ID NO: 219) were tested, with vh458 having the first position replaced by E instead of Q. Constructs were generated and tested, purified on PrismA, and conjugated to a CM5 chip.

[0376] Generation of candidate peptides purified by MabSelect PrismA™ For each candidate, 5 µl of the glycerol stock solution as described in Example 1 above was inoculated into 4 ml of LB medium supplemented with 100 µg / ml carbenicillin in a 14 ml round-bottom tube and grown overnight at 37°C and 200 rpm with stirring. Protein expression medium (TB medium supplemented with 100 µg / ml carbenicillin and 2 mM MgCl2) was prepared and added to 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 shaking incubator at 37°C and 140 rpm for approximately 3.5 hours until the OD600 reached 1.0. Next, 50 µl of IPTG (1 M) was added to a final concentration of 1 mM, and the 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.

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

[0378] 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™).

[0379] 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.

[0380] 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.

[0381] 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.

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

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

[0384] All sensor maps 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.

[0385] 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).

[0386] 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.

[0387] 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.

[0388] result Even for peptides with E as the first amino acid in the framework, the stability of the peptides 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 24 below. Vh118 with E substitution at position 1 is referred to as vh118 (Q1E). vh324 is named accordingly.

[0389] Table 24 Example 11: Measurement of basic stability for AAV9, EGFR and GFP relative to ZVH3 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 increased the basic stability of the peptide. The data obtained from this example will be compared with the results for AAV9-binding sdAb in the previous examples.

[0390] 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.

[0391] Biacore analysis of alkaline stability The materials and equipment used are 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 candidate peptides as described above.

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

[0393] 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.

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

[0395] Table 25 For the GFP, EGFR, and AAV9 binding molecules, basic stability was further evaluated, comparing target binding with ZVH3 binding. The same constructs as in Example 8 above were used. For GFP and EGFR, the NaOH concentration was 0.1 M, and for AAV9, it was 0.5 M. Results are provided in Table 26 below.

[0396] Table 26 Candidates SEQ ID NO: target NaOH stability (retaining at least 50% of target binding in exposure cycles) NaOH stability (retaining at least 50% of the ZVH3 binding exposure cycle count) vh60 155 GFP 2 1 vh113 156 GFP 16 17 vh539 217 GFP 1 NA vh166 157 EGFR 1 1 vh167 158 EGFR 6 9 vh541 218 EGFR 1 NA vh342 152 AAV9 2 3 vh118 80 AAV9 22 26 vh540 216 AAV9 6 NA Example 12: Basic stability of the scFv framework scFv, a fusion of two variable domains (VH and VL) with a flexible linker, is a suitable framework for target interaction but lacks some biologically relevant properties. Therefore, the framework mutations of this disclosure were used to assess the potential for stabilizing the scFv framework. A publicly available scFv fragment targeting human serum albumin (HSA) was used as the starting point.

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

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

[0399] The first Biacore experiment was performed on an SA-chip with biotinylation affinity to compare ScFvHSA with and without peptide moiety fusion. The binding assay used HAS concentrations of 0, 10, 50, 100, 500, and 1000 nM, and the alkaline stability assay used 1000 nM HAS with CIP conditions for 10 minutes and 0.1 M NaOH. The alkaline stability was improved by peptide moiety fusion according to SEQ ID NO: 160 compared to the native formulation, and the stability can be further improved by framework mutations, such as... Figure 14 As shown in the image.

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

[0401] result Compared to other constructs, expression and purification showed an approximately 2-fold increase in yield for the stable third construct, scFvHSAStab1-Z. Experiments on the SA-chip demonstrated improved stability for scFvHSA-Z, and even further improved stability for scFvHSAStab1-Z, indicating that the stable VHH framework further improves stability. Basic stability results from the SA-chip experiments showed… Figure 14 The diagram shows a standardized plot of the alkaline stability experiment. For the second Biacore experiment, a CM5-chip with NHS coupling was used, and it was shown that the construct with the polypeptide motif according to SEQ ID NO: 160 had good coupling efficiency compared to natural scFvHSA alone (data not shown).

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

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

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

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

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

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

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

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

[0410] 9. The polypeptide according to any one of the foregoing items is an antigen-binding polypeptide.

[0411] 10. The polypeptide according to any one of the foregoing items is a single-chain polypeptide.

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

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

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

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

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

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

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

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

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

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

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

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

[0424] 23. The polypeptide according to any one of the preceding items, wherein FWR1 has at least 85%, for example at least 90%, sequence identity with amino acids 1-26 (FWR1) at the Kabat position of SEQ ID NO: 80, wherein Kabat positions 6, 14, 19, 23 and 24 are omitted for the purpose of determining sequence identity.

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

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

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

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

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

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

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

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

[0433] 32. The polypeptide according to any one of the preceding items, wherein said VH variant has a frame region 1 (FWR1) comprising an amino acid sequence according to the following sequence: X1X2QLX5X6SGGGX 11 VQX 14 GGSLX 19 LSCX 23 X 24 SG (SEQ ID NO: 1) Independently, X1 is Q, V, D or E, preferably Q or E; X2 is V or D, preferably V; X5 is Q, V, or E, preferably Q; X6 is either Q or E, preferably Q; X 11 It is S or L, preferably S; X 14 It is A, S, or T, with A being the preferred choice; X 19 It is R, S, K or T, preferably R or T; X 23 It is T, V, A, or S, preferably T; and X 24 It is I, V, or S, preferably I.

[0434] 33. The polypeptide according to any one of the preceding items, wherein the VH variant has a frame region 2 (FWR2) comprising an amino acid sequence according to the following sequence: WFRQX5PGX8X9X 10 EX 12 VA (SEQ ID NO: 2) Independently, X5 is R, I, T, or K, preferably R; X8 is R, K, Q, E or G, preferably K or R; X9 is E, Q, A, D, or G; X 10 It is R, I, or L, preferably R; and X 12 It is F or L, preferably F.

[0435] 34. The polypeptide according to any one of the preceding items, wherein said VH variant has a frame region 3 (FWR3) comprising an amino acid sequence according to the following sequence: YX2X3X4VX6GRFTISRDNAKX 18 TX 20 X 21 LQMNX 26 LKPEDTAX 34 YYCAA (SEQ ID NO: 3) Independently, X2 is A, T, N, Q, or S, preferably A; X3 is either D or S, preferably D; X4 is either S or A, preferably S; X6 is either K or A, preferably K; X18 It is N or Q, preferably N; X 20 It is either V or A, preferably V; X 21 It is Y, W, or F, preferably Y or W; X 26 It is N, S, T, or Q, preferably S; and X 34 It is either V or I.

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

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

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

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

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

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

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

[0443] 42. The polypeptide according to item 39, wherein the additional amino acid sequence comprises a plurality of histidines, such as the (His)6 sequence.

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

[0445] 44. The multimeric polypeptide according to item 43, which contains three VH variants.

[0446] 45. The multimeric polypeptide according to item 43 or 44, wherein the VH variant is identical.

[0447] 46. ​​The multimeric polypeptide according to any one of items 43-45, wherein the VH variant has a different CDR.

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

[0449] 48 . According to the fusion protein described in Item 47, the additional polypeptide portion of which contains an α-helical domain.

[0450] 49. The fusion protein according to item 48, wherein the additional polypeptide portion comprises an α-helical bundle domain.

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

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

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

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

[0455] 54. The fusion protein according to any one of items 47-52, comprising the following structure ([A-L1)) m -[Z-L2] n) p in A represents a polypeptide as defined in any of items 1-42. 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 48-51. 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.

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

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

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

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

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

[0461] 60. An expression vector comprising the nucleic acid as described in item 59.

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

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

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

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

[0466] 65. The recombinant host cell according to item 63, wherein the eukaryotic cell is an insect cell.

[0467] 66. The recombinant host cell according to item 63, wherein the eukaryotic cell is an animal cell, such as a mammalian cell, such as a Chinese hamster ovary (CHO) cell or a human embryonic kidney (HEK) cell.

[0468] 67. A method for producing a polypeptide, polymer, or fusion protein according to any one of items 1-58, comprising: i. Provide recombinant host cells according to any one of items 61-66; ii. Culture the host cells under conditions that enable the expression of the polypeptide, polymer, or fusion protein; and iii. Isolate the polypeptide, polymer, or fusion protein.

[0469] 68. The method according to any one of items 62-67, wherein step iii comprises purifying the polypeptide, polymer, or fusion protein by affinity chromatography.

[0470] 69. The method of claim 68, wherein the affinity chromatography uses an affinity ligand that binds to the frame region of the VH variant.

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

[0472] 71. For use as described in item 70, for in vitro detection and / or purification of the target entity.

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

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

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

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

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

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

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

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

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

[0482] 81. The adsorbent material according to item 80, wherein the carrier material is a chromatographic matrix selected from fiber matrices, membranes, filters and bulk materials.

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

[0484] 83. For the purpose described in item 82, for detecting the target entity within a sample.

[0485] 84. As described in item 82, for separating the target entity from other components of a sample.

[0486] 85. For analytical separation of target entities, as described in item 84.

[0487] 86. For the preparative purification of target entities, as described in item 82.

[0488] 87. A separation method comprising the following steps: (a) Providing an adsorbent material according to any one of items 72-81, wherein the VH variant of said polypeptide has binding affinity for the target entity. (b) Under conditions that allow the target entity to bind to the polypeptide, the adsorbent material is brought into contact with a liquid sample containing the target entity. (c) Optionally, the adsorbent material is washed. (d) Elution of the target entity from the adsorbent material, and (e) Clean the adsorbent material with a cleaning liquid.

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

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

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

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

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

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

[0495] Table 18: 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) WO2003080655A1 WO2016079033A1 S Hjerten, Biochim Biophys Acta 79(2), 393-398 (1964) US6602990 US7396467 WO2019137869A1 WO2018011600A1 Schmitz et al, Structure 21, 1214-1224 (2013) Fleetwood et al., Cell. Mol. Life Sci. 70:1081-1093 (2013) Mitchell&Colwell, Proteins 86, 697-706 (2018) Adams R. Et al, MABS VOL. 8, NO. 7, 1336–1346 (2016) WO2023174900A1

Claims

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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