Fusion proteins for affinity capture

Abstract

A fusion protein comprising at least one first polypeptide moiety and at least one second polypeptide moiety wherein the first polypeptide moiety is a single-chain polypeptide capable of binding to a target entity and the second polypeptide moiety is a stabilizing polypeptide and comprises a single-chain alpha-helix-containing domain, wherein the second polypeptide moiety has no binding affinity for the target entity. The presence of the second polypeptide moiety may improve at least one property of the fusion protein as compared to the property of the first polypeptide moiety alone, where the improved property is selected from the group consisting of: alkaline stability, recombinant protein expression, and coupling to a vector.

Description

Technical Field

[0001] This invention relates to fusion proteins capable of affinity interaction with target entities and methods of using them in vitro or industrial applications, such as for affinity separation. The invention further relates to adsorbent materials comprising said fusion proteins and 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, WO2016 / 079033A1, and WO2022 / 013272). Meanwhile, there is a significant need in the art for improved affinity ligands capable of binding to biological entities other than immunoglobulins. Summary of the Invention

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

[0009] Therefore, the objective of this invention is to provide improved affinity ligands that can be used for affinity capture of various target entities, such as for detection or separation purposes.

[0010] This, along with other targets, is achieved through fusion proteins, in which a polypeptide portion capable of binding to the target entity is fused with a second polypeptide portion.

[0011] Therefore, in one aspect, the present invention provides a fusion protein comprising at least one first polypeptide moiety and at least one second polypeptide moiety, wherein the first polypeptide moiety is a single-chain polypeptide capable of binding to a target entity, and the second polypeptide moiety comprises a single-chain containing an α-helical domain, wherein the second polypeptide moiety has no binding affinity to the target entity.

[0012] The presence of the second polypeptide moiety improves at least one property of the fusion protein compared to the properties of the first polypeptide moiety alone. The improved property may optionally be selected from: basic stability, recombinant protein expression, and vector conjugation. The second polypeptide moiety may be a stable polypeptide. The second moiety may be a basic stable polypeptide as defined herein.

[0013] The first polypeptide moiety may be a natural or synthetic antibody domain or antibody fragment or a modified variant thereof, such as a single-chain variable fragment or single-domain antibody (sdAb) or a variant thereof. Typically, the first polypeptide moiety alone has lower basic stability than the second polypeptide moiety alone.

[0014] The second polypeptide moiety may be an α-helical bundle domain, and optionally a protein domain derived from SpA. The second polypeptide moiety may have reduced affinity for the Fc and VH3 regions of trastuzumab.

[0015] The fusion protein may be provided as a multimeric fusion protein comprising at least two of the first polypeptide moieties and / or at least two of the second polypeptide moieties.

[0016] In another aspect, the present invention provides the use of a basic stable polypeptide comprising a single-chain α-helix domain to improve the in vitro basic stability of a single-chain polypeptide capable of binding a target entity. This use includes fusing a polypeptide comprising a single-chain α-helix domain to the C-terminus (optionally via a linker peptide) of a single-chain polypeptide capable of binding a target entity.

[0017] In another aspect, the present invention provides an adsorbent material comprising a fusion protein as described herein coupled to a carrier. The carrier may be selected from chips, plates, pores, sheets, fibers, particles, beads, fiber matrices, membranes, filters, and porous substrates. The carrier may be a chromatographic matrix.

[0018] In other respects, the present invention provides the use of the fusion protein or adsorbent material for detecting or separating target entities, such as from a sample containing target entities and other components.

[0019] In another aspect, a separation method is provided, comprising the steps of: providing an adsorbent material as described herein, wherein the fusion protein has a binding capacity for a target entity; contacting the adsorbent material with a liquid sample containing the target entity under conditions allowing the target entity to bind to a first polypeptide of the fusion protein; optionally washing the adsorbent material; eluting the target entity from the adsorbent material; and cleaning the adsorbent material with a cleaning liquid. The cleaning liquid is typically alkaline and may, for example, contain 0.05-0.5 M NaOH or KOH.

[0020] In other respects, this disclosure provides isolated nucleic acid sequences encoding the fusion protein, expression vectors containing the nucleic acid sequences, recombinant cells containing the expression vectors, and methods for producing the fusion protein.

[0021] Preferred aspects of this disclosure are described below in the detailed description, examples, list of embodiments, and in the dependent claims. It should be noted that the invention relates to all possible combinations of the features recited in the claims. Attached Figure Description

[0022] These and other aspects of the invention will now be described in more detail with reference to the accompanying drawings, in which: Figure 1A -B is a schematic diagram of a fusion protein according to an embodiment of the present invention.

[0023] Figure 2A -B is a schematic diagram of a multimeric fusion protein according to various embodiments of the present invention.

[0024] Figure 3A This is a graph showing the AAV9 binding response of the fusion protein (solid line) and the reference (dashed line) to the number of binding cycles on Biacore, with each cycle including 0.5 M NaOH exposure. The binding response is normalized for the second cycle (i.e., excluding the first cycle). Figure 3B The combined response from the same experiment is shown, normalized to the response of the first cycle. (Example 2A).

[0025] Figure 4A The images are PAGE gel images, confirming that the multimeric fusion protein with a stable peptide exhibits stronger protein expression compared to a reference lacking a stable peptide. Figure 4B This is a graph showing the AAV9 binding response of the multimeric fusion protein and the reference pair on Biacore for the number of binding cycles, each cycle including 0.3 M NaOH exposure. The binding response is normalized for the second cycle (i.e., excluding the first cycle). (Example 2B).

[0026] Figure 5A -C displays the options for those that can combine with AAV9 ( Figure 5A ), GFP ( Figure 5B ) or EGFR Figure 5C The fusion protein of [a specific protein] exhibited target binding responses to increasing cycles including NaOH exposure. The responses were normalized to the response of the first cycle. (Example 4A).

[0027] Figure 6A -C displays the options for those that can combine with AAV9 ( Figure 6A ), GFP ( Figure 6B ) or EGFR Figure 6C The fusion protein of [a specific protein] exhibited target binding responses to increasing cycles including NaOH exposure. The responses were normalized to the response of the first cycle. (Example 4B).

[0028] Figure 7A This is a bar chart showing the binding response of the fusion protein according to the invention to the target (GFP) and immunoglobulins (trastuzumab, Gammanorm) for increasing cycles including NaOH exposure. Figure 7B This shows the average values ​​for a group of fusion proteins that have variations in the amino acid sequence of a stable polypeptide. (Example 3).

[0029] Figure 8 This is a graph (Example 5) showing the target binding response (unnormalized) obtained for a fusion protein containing an IgG-binding polypeptide domain as the first polypeptide part with increasing cycles including NaOH exposure.

[0030] Figure 9A -C is a graph showing the target binding response (unnormalized) obtained for a fusion protein containing IgG-binding polypeptide domains (SpA domains C, D, E) as the first polypeptide part for increasing cycles including NaOH exposure (Example 6).

[0031] Figure 10A -B is a graph showing the target binding response (unnormalized) obtained for a fusion protein with scFv as the first polypeptide motif for increasing cycle numbers including NaOH exposure (Example 7).

[0032] Figure 11A It is a chromatogram showing the elution peaks from a chromatographic column using a fusion protein according to an embodiment of the present invention as an affinity ligand immobilized on a chromatographic matrix. Figure 11B This is a magnified view of a portion of the elution peak.

[0033] Figure 12 This is a photograph of an SDS-PAGE gel, showing the protein content of the eluent fraction from the chromatographic run of Example 8.

[0034] Figure 13 It is a graph showing the dynamic combined capacity, as evaluated in Example 8.

[0035] Figure 14 is a graph showing the binding capacity of sdAbs with different stable peptides, as evaluated in Example 9.

[0036] Figure 15This is a graph showing the binding capacity and basic stability of the purified and biotinylated construct on an SA-chip according to Example 10.

[0037] Figure 16 is a graph showing the binding capacity and basic stability of the construct of Example 11.

[0038] As illustrated in the accompanying drawings, some features and portions 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.

[0039] definition As used herein, the terms “peptide” and “polypeptide” are used synonymously and refer to compounds formed from amino acid sequences, without limitation on size. “Protein” may be used to refer to larger compounds in this category. Amino acid sequences are written from left to right, from the amino (N) terminus to the carboxyl (C) terminus. According to standard nomenclature, amino acid residue sequences are represented by three-letter or single-letter codes as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). A "peptide" includes any oligopeptide, polypeptide, gene product, expression product, or protein. Peptides are composed of consecutive amino acids and include naturally occurring or synthetic molecules. Furthermore, as used herein, the term "peptide" refers to amino acids linked together by peptide bonds or modified peptide bonds, such as isosteres, and may contain modified amino acids in addition to the 20 genetically encoded amino acids. Peptides can be modified through natural processes, such as post-translational processing, or through chemical modification techniques well known in the art.

[0040] 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.

[0041] The term "antigen-binding polypeptide" generally refers to a polypeptide having at least one binding region, such as at least two or three binding regions, that gives the polypeptide 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 typically 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 such 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 chain and / or heavy chain with complementarity-determining regions (CDRs), such as Fab (antigen-binding fragment), Fv (variable fragment), scFab (single-chain antigen-binding fragment), and scFv (single-chain variable fragment), (iii) single-domain antibodies, and (iv) peptides having a bacterial-derived backbone, such as immunoglobulin-binding bacterial proteins (e.g., Coptis chinensis protein L or Staphylococcus aureus protein A and protein G) or their domains, including wild-type and variants in which one or more antigen-binding regions have been engineered.

[0042] 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 domains and light chain variable domains), 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 L domain of protein). 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.

[0043] The term "single-domain antibody" refers to a variable domain that is a heavy chain variable domain (VH) of an antibody but lacks a light chain variable domain, either as part of or derived from the antibody. Therefore, a single-domain antibody completely lacks the antibody light chain, including the light chain variable domain (VL). Single-domain antibodies may also lack a structural feature essential for functional VH / VL interactions and present, for example, in the conventional VH of IgG1. Therefore, single-domain antibodies do not form part of a dimer structure containing the antibody light chain variable domain. Single-domain antibodies include antibodies that naturally lack the antibody light chain, such as sdAbs derived from camels (e.g., dromedary camels, camels, llamas, and alpacas) of IgG2 or IgG3, also known as VHHs (heavy chain variable domains of heavy chain antibodies). Single-domain antibodies that naturally lack the light chain also include so-called VNARs (variable neoantigen receptors), which are antibodies derived from cartilaginous fish (e.g., sharks). Alternatively, single-domain antibodies can be synthetic variants based on the amino acid sequence of the variable domain of the antibody heavy chain from species that do not naturally produce sdAbs (e.g., cows, rats, mice, or rabbits), and modified, for example, relative to the amino acids in the native VH / VL interaction of such antibodies to mimic native sdAbs. Such modifications may include substituting amino acids in the VH / VL interface region to increase hydrophilicity or solubility.

[0044] Typically, terms such as "peptide," "protein," "antibody," "single-domain peptide," and "single-domain antibody" also include synthetic variants, which 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] The term "target entity" in this paper refers to the entity that forms a specific binding pair with the ligand and may also be referred to as "analyte".

[0049] 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).

[0050] 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.

[0051] 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, i.e., during sample application. The DBC of a chromatographic resin is expressed as the amount of analyte bound to the resin under the provided flow conditions before significant breakthrough occurs of unbound analyte. DBC is determined by loading a sample containing a known concentration of analyte and monitoring the flow. The analyte will bind to the resin until a breakthrough point before unbound analyte flows 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 of analyte in the column effluent is 10% of the concentration of the analyte sample in the feed. If the dynamic binding capacity of each resin is calculated at 80% breakthrough capacity, this is known as the QB80% value.

[0052] As used herein, the term "basic stability" refers to a peptide property relating to its ability to withstand alkaline exposure without adverse effects on the structure and / or function of the peptide. For peptides capable of binding to a target entity, basic stability is determined based on affinity for the target entity after exposure to an alkaline reagent (typically NaOH). The basic stability of an antigen-binding peptide can be evaluated by immobilizing the 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, the peptide is immobilized on an SPR chip by covalent coupling with thiols, N-hydroxysuccinimide, streptavidin-biotin, or another coupling that is inherently alkaline-resistant, at a ligand density corresponding to at least 1000 RU, preferably at least about 3000 RU, and for a predetermined analyte concentration. Typically, the first alkaline cleaning cycle can have a unique and significant effect on binding capacity, for example, due to the removal of non-covalently bound 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 (i.e., the binding response normalized to the second cycle).

[0053] In this context, a peptide can be considered alkaline stable if, after 10 clean cycles, preferably 15 clean cycles, the antigen-binding peptide of the fusion protein retains at least 50% of its target binding capacity compared to its binding capacity after the first clean cycle (i.e., before the first alkaline exposure event, without considering the binding capacity of the first cycle). The clean cycle includes 600 seconds of exposure to at least 0.3 M NaOH, for example, at least 0.5 M NaOH.

[0054] Any polypeptide capable of binding antigens and immobilized on a carrier can be evaluated in this manner regarding its basic stability. For polypeptides that have no binding affinity to other entities (e.g., stable polypeptides according to some embodiments of the invention based on the SpA protein domain whose IgG binding affinity has been deprecated), it is envisioned that the basic stability of a stable polypeptide that has no binding affinity to another entity (e.g., IgG) can be evaluated by providing a single affinity binder capable of binding the stable polypeptide, immobilizing said single affinity binder on a carrier (e.g., an SPR chip or chromatographic matrix), and evaluating its interaction with the stable polypeptide as an analyte before and after exposure to NaOH (similar to that described elsewhere herein) (e.g., binding response or dynamic binding capacity QB10%).

[0055] The term "solid support" herein refers to a non-aqueous matrix of solid-phase material. Suitable solid-phase materials include, but are not limited to, glass, silica (e.g., silica gel), polysaccharides (e.g., polysaccharide matrices) such as agarose and cellulose, and organic polymers such as polyacrylamide, methyl methacrylate, and polystyrene-divinylbenzene copolymers. The solid phase may be porous or non-porous and may be compressible or incompressible. For example, the solid phase may be a polymer matrix or agarose particles or beads. Preferred solid support materials will be physically and chemically resistant to the conditions used in the separation process, including pumping and cross-flow filtration, as well as the temperature, pH, and other properties of the liquid used.

[0056] The term "surface" in this document refers to all external surfaces of a solid structure. In the case of porous supports (e.g., beads used in, for example, chromatography), the term "surface" includes both the outer surface and the pore surface. In the case of fibrous membranes, the term "surface" includes the outer surface of each individual fiber.

[0057] The term "separation matrix" is used herein to refer to a material containing a solid support with which one or more ligands are coupled. The ligands are capable of binding to a target entity (also referred to herein as an analyte), which is to be separated from its environment (e.g., a liquid sample), and / or from other components present in the liquid sample. Separation matrices can be used in a variety of scenarios, including analytical assays and for purifying targets for analytical or preparative purposes. The type of support is selected based on the intended use.

[0058] The separation matrix may further comprise compounds that couple ligands to a support. The terms “spacer,” “extender,” and “surface extender” are used to describe such compounds, as further described herein. The term “resin” is sometimes used in the art for separation matrices. The terms “chromatographic material” and “chromatographic matrix” are used herein to denote a class of separation matrices. Affinity separation matrices are separation matrices whose ligands are affinity ligands.

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

[0060] 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 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.

[0061] 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 may 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.

[0062] 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. Detailed Implementation

[0063] The inventors have surprisingly discovered that the basic stability of otherwise base-sensitive affinity ligands can potentially be improved by fusing with peptide domains containing α-helical polypeptide domains, such as those corresponding to or based on SpA protein domains. Specifically, basic-stable variants of SpA domains B, C, and Z have been considered for this purpose. Such modified variants are described in WO2003080655A1 and WO2016079033A1, the patent disclosures of which are incorporated herein by reference in their entirety.

[0064] As demonstrated herein, the fusion partner, referred to as the second polypeptide moiety, improves the basic stability of the affinity ligand polypeptide, resulting in a fusion protein with higher basic stability than the individual affinity ligand (referred to herein as the first polypeptide moiety). The second polypeptide moiety can therefore be referred to as the stable polypeptide.

[0065] Another benefit of the fusion protein of the present invention is that the second polypeptide moiety can promote or increase the protein expression of the fusion protein in recombinant host cells, especially when the first polypeptide moiety alone is difficult to express.

[0066] Furthermore, the second polypeptide moiety has the potential to function as a purification tag, specifically for purification by affinity chromatography using ligands with affinity for the second polypeptide moiety, and / or as a tool for coupling the first polypeptide moiety to a carrier, potentially functioning as a spacer group, and / or providing a detection tool, for example by providing a binding site for a labeled binding partner. The second moiety can therefore also be more generally referred to as an accessory protein.

[0067] Several variants of the fusion coupler (second polypeptide moiety) have been found to result in the aforementioned effects, such as the polypeptides defined in SEQ ID NOs 7-9, 28-30, and 61-110. The effects have been demonstrated for several different types of first polypeptide moiety, such as native or synthetic antibody domains or antibody fragments or modified variants thereof; polypeptides comprising variable domains of immunoglobulin heavy chains, variable domains of heavy chains of single-domain antibodies (sdAbs), VHHs, VNARs, immunoglobulin single-chain variable fragments (scFvs), and synthetic variants of antibody heavy chain variable domains in which native VH / VL interaction sites have been modified to mimic naturally occurring sdAbs; polypeptides comprising β-sheet secondary structures; polypeptides comprising α-helical structures, such as α-helical bundle domains, such as polypeptides comprising or derived from staphylococcal protein A (SpA) or albumin-binding domains (ABDs); or polypeptides comprising a combination of β-sheets and α-helical bundles, such as protein G domain C2. Even though any stable second polypeptide moiety can be used with any first polypeptide moiety, the second polypeptide moiety can be selected based on the first polypeptide moiety to make a preferred combination, and / or selected according to the desired effect to be achieved (e.g., basic stability, protein expression, or vector conjugation). Preferably, the second polypeptide moiety may have a deprecated binding capacity to the Fc and VH regions.

[0068] Figure 1A The diagram schematically illustrates a fusion protein 100 comprising a first polypeptide portion 200 and a second polypeptide portion 301 connected via an optional peptide linker 204 in the N-terminal to C-terminal direction. In this figure, the second polypeptide portion 301 is located at the C-terminus of the first polypeptide portion 200. It is also contemplated that the second polypeptide portion may be located at the N-terminus of the first polypeptide portion.

[0069] Figure 1B This is an alternative description of a fusion protein according to an embodiment of the invention. Fusion protein 110 includes an additional N-terminal amino acid 201, such as a leader peptide, and a single-chain polypeptide 202 capable of binding to a target entity. A linker 204 is provided at the C-terminus of the single-chain polypeptide 202, followed by a stabilizing polypeptide 302, and finally an optional C-terminal amino acid sequence 203, which may be, for example, a purification tag and may include a linker connecting sequence 203 to the stabilizing polypeptide 302 (not shown in the figure).

[0070] The first polypeptide portion 200 may contain only, as shown in the example Figure 1B The single-chain polypeptide 202 described herein may be composed of or may comprise or be composed of the single-chain polypeptide 202 and additional amino acids (e.g., additional amino acid sequences 201 and / or linker 204). Similarly, Figure 1AThe second polypeptide portion 301 may contain or be composed of a stable polypeptide 202, or may contain or be composed of a stable polypeptide 202 and additional N- or C-terminal amino acids (e.g., linker 204 and / or C-terminal sequence 203).

[0071] The fusion protein disclosed herein can be a multimeric protein, meaning it can contain more than one affinity ligand or more than one stable polypeptide. Multimeric variants containing multiple copies of the affinity protein (first polypeptide moiety) may be advantageous because they offer increased binding capacity compared to fusion proteins containing only one copy. Multimeric fusion proteins containing multiple stable polypeptides offer improved stability, such as basic stability, compared to fusion proteins containing a single stable polypeptide.

[0072] Therefore, the fusion protein of this disclosure may optionally be provided in the form of a multimeric fusion protein (“multimer”) containing at least two of the first polypeptide moieties and / or at least two of the second polypeptide moieties, such as Figure 2A -B is an illustrative example. In Figure 2A In this embodiment, the multimeric fusion protein 310 comprises multiple units of a first polypeptide moiety 200 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 first polypeptide moiety 200 may be identical or may differ from each other in their amino acid sequences. Figure 2A Three polypeptide moieties 200 (“trimers”) are shown, but the fusion protein is envisioned to contain any suitable number of the first polypeptide moieties 200. For example, the fusion protein may contain at least two first polypeptide moieties, or at least 3, 4, 5, or 6 units of the first polypeptide moieties. The fusion protein may contain, for example, up to 10 first polypeptide moieties 200. The fusion protein may optionally contain N-terminal and / or C-terminal amino acids, as described elsewhere in the text.

[0073] In addition, the fusion protein may contain at least two, for example three, four, five or six units of the second polypeptide moiety, for example up to ten units. Figure 2BA fusion protein 320 containing one first polypeptide moiety 200 and three second polypeptide moiety 301 is described. The linker 204 is optional and may be the same or different for each occurrence. The second polypeptide moiety 301 may be the same or may differ in their amino acid sequences. In other embodiments, the fusion protein may contain two stable polypeptides and any suitable number of the first polypeptide moiety 200 as described above. For example, the fusion protein may contain a first polypeptide moiety side-attached to two stable polypeptides. When the fusion protein comprises multiple first polypeptide moietyes and multiple stable polypeptides, such as two or three each, it is envisioned that the different polypeptide moiety 200, 301 may be arranged in an alternating manner, instead of as... Figure 2A (for the first polypeptide portion 200) and Figure 2B (For stable polypeptide 301) polypeptides of the same kind are arranged sequentially. For example, a multimeric fusion protein containing two units of each polypeptide may have the following general structure: [first polypeptide moiety]-[second polypeptide moiety]-[first polypeptide moiety]-[second polypeptide moiety]. In embodiments where the fusion protein comprises at least two target-binding polypeptides (i.e., at least two first polypeptide moieties), the target-binding polypeptides may be the same or different, and if different, may target (be able to bind) different targets.

[0074] In instances where the fusion protein comprises at least two second polypeptide moieties, it is likely preferred that at least one is located at the C-terminus of the fusion protein. Optionally, the at least two second polypeptide moieties may be sequentially arranged at the C-terminus of the fusion protein.

[0075] At the N-terminus of a fusion protein (e.g., a multimeric fusion protein), a short amino acid sequence, such as a leader peptide or signal peptide, may be present. At the C-terminus of a fusion protein (e.g., a multimeric fusion protein), a tag or spacer or another short amino acid sequence may be optionally provided as needed.

[0076] In some embodiments, the second polypeptide moiety is not located at or near the N-terminus of the fusion protein.

[0077] For clarity, any references to the fusion proteins of the present invention herein also include their multimeric variants, unless otherwise specified.

[0078] The first polypeptide moiety of the fusion protein is capable of binding to a target entity and may be referred to as an affinity ligand or affinity binder for or against the target. The first polypeptide moiety may also be referred to as an antigen-binding polypeptide or protein, or a target-binding polypeptide or protein. Therefore, the fusion protein is capable of binding to the target entity through the interaction between the first polypeptide moiety and the target entity.

[0079] The target entity can be a biological entity, such as a biomolecule or a larger structure. The target entity can be a therapeutic molecule or entity intended for use in a drug or therapy. The target entity can be intended as a medium, such as a cell or gene therapy vector. For example, a biological entity can be an antibody or a portion thereof, such as a monoclonal antibody, antibody fragment, or antibody domain. As another example, a biological entity can be a protein, such as a recombinant protein. Alternatively, a biological 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, a 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 biological entities include exosomes and lipid nanoparticles. In yet another example, a biological entity can be a nucleic acid molecule, such as DNA or RNA, such as mRNA. The first polypeptide portion may have affinity for molecules exposed on the surface of a biological entity, such as viral capsid or envelope proteins, membrane receptors, or surface exposure markers.

[0080] Typically, antigen-binding peptides targeting desired targets can be identified or generated by any method known to those skilled in the art, including but not limited to immunizing animals (e.g., mice, rabbits, or camelids) with the target antigen, via phage display, ribosome display, cell surface display, or bacterial display, via computer simulation, and combinations thereof. When an antigen-binding peptide has been identified, the amino acid sequence, optionally modified, can be used to design fusion protein constructs together with a second peptide motif as disclosed herein. For example, methods for generating high-affinity antigen-binding single-domain antibodies are described, for example, in Schmitz et al, 2013, Structure 21 , 1214-1224 and Fleetwood et al., Cell. Mol. LifeSci. (2013) 70:1081-1093.

[0081] The first polypeptide moiety is typically a single-chain polypeptide and may form a polypeptide domain.

[0082] In some embodiments, the first polypeptide moiety may have a secondary structure comprising or predominantly containing β-chains, β-sheets, and / or β-sandwiches. However, a structure with a predominance of β-chains or β-sheets does not preclude the presence of other structural elements such as α-helices, loops, or disordered regions. Examples of protein domains characterized by a secondary structure of β-chains or β-sheets include antibody variable domains. Therefore, the first polypeptide moiety may comprise natural or synthetic variants of antibody heavy chain variable domains and / or antibody light chain variable domains, such as those selected from: heavy chain variable domains and variants thereof of heavy chain antibodies (VHH), VNARs and variants thereof, and immunoglobulin single-chain variable fragments (scFv). The first polypeptide may therefore be or be derived from a single-domain antibody (sdAb). Single-domain antibodies lack the light chain of a full-length antibody and also lack a heavy chain constant domain. When the first polypeptide moiety is antibody-based, it may lack the antibody heavy chain constant region.

[0083] In some embodiments, the first polypeptide moiety may have a secondary structure comprising one or more α-helices. Some polypeptides may have a mixed structure comprising at least one β-sheet and at least one α-helix; examples of such polypeptides include protein L protein domains and protein G protein domains.

[0084] In other embodiments, the first polypeptide moiety may be or include an α-helical bundle protein domain. Examples of α-helical bundle domains include the SpA protein domain and the albumin-binding domain (ABD). Specifically, the first polypeptide moiety may include a protein domain that is or is derived from SpA, such as a polypeptide containing domain A (SEQ ID NO: 1), domain B (SEQ ID NO: 2), domain C (SEQ ID NO: 3), domain D (SEQ ID NO: 4), or domain E (SEQ ID NO: 5). For example, the first polypeptide moiety may include an amino acid sequence selected from SEQ ID NO: 1-7 and an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 1-7.

[0085] The first polypeptide may or may not be modified to increase its basic stability. Therefore, the first polypeptide moiety itself is not necessarily basic stable.

[0086] The first polypeptide moiety, along with the fusion protein as a whole, has an affinity for the target entity. Therefore, the presence of the second polypeptide moiety should not interfere with the binding capacity of the first polypeptide to the target molecule.

[0087] The ability of the first polypeptide moiety to bind to the target entity is provided by one or more binding regions. For example, the first polypeptide moiety may comprise a single-chain polypeptide having a complementarity-determining region 1 (CDR1), a complementarity-determining region 2 (CDR2), and a complementarity-determining region 3 (CDR3). CDR1, CDR2, and CDR3 may be CDRs of variable domains of natural or synthetic antibody heavy chains. CDRs may optionally be modified to provide desired binding characteristics.

[0088] For example, the first polypeptide moiety may have an sdAb-based amino acid sequence comprising a framework region (FWR) and a complementarity-determining region (CDR) provided in the N-terminal to C-terminal direction as follows: [FWR1]-[CDR1]-[FWR2]-[CDR2]-[FWR3]-[CDR3]-[FWR4] The framed areas can be collectively referred to as "frames".

[0089] Table 1 summarizes the Kabat amino acid numbering system described in this paper (Kabat et al., 1991). J. Immunol. FWR and CDR are defined in 147(5), 1709-1719. For the sdAb according to the embodiments of this disclosure, frame region 1 (FWR1) is formed by amino acids at positions 1-26. Then, 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.

[0090] Table 1 In cases where the first polypeptide portion comprises an sdAb variant, each frame region may have an amino acid sequence that is at least 70%, at least 75%, at least 80%, or at least 85% sequence identical to the corresponding frame region of SEQ ID NO: 127, 133, or 137. The SEQ ID NOs of the various frame regions of these sdAbs are shown in Table 1 above.

[0091] The frame regions of the sdAb are relatively conserved, and preferably, are minimally modified or unmodified relative to, for example, SEQ ID NO: 127, 133, 137, or 139. The conserved regions are defined herein by Kabat positions 1-5, 7-14, 25-26, 41-42, 46, 48-49, 59, 61-75, 77-78, 80-82a, 82c-94, and 103-113. Optionally, each of FWR1-3, and optionally FWR4, may have at least 80%, for example, at least 85%, for example, at least 90% sequence identity with the corresponding frame regions of SEQ ID NO: 127, 133, 137, or 139 at conserved amino acid positions (i.e., other positions are omitted for comparison). As an example, FWR1 contains 26 amino acids, of which 21 amino acids represent conserved positions as defined above. Replacing two of these amino acids results in 90.5% identity, while replacing three amino acids results in 85.7% identity.

[0092] The sdAb variant may have an amino acid sequence in which positions 1-5, 7-13, 25-26, 36-39, 41-42, 46, 48-49, 59, 61-75, 77-78, 80-82a, and 82c-94 of the Kabat sequence comprises up to 15, for example, up to 14, for example, up to 13, for example, 12, 11, or 10 amino acid residues substituted relative to at least one of SEQ ID NO: 127, 133, 137, or 139. 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 substitutions. In FWR2, positions 36-39, 41-42, 46, and 48-49 (14 residues) of the Kabat sequence may together have 1 or 2 amino acid substitutions. In FWR3, the Kabat positions 59, 61-75, 77-78, 80-82a, and 82c-94 (34 residues) can have up to 6 amino acid substitutions.

[0093] For example, the first polypeptide moiety may include an sdAb framework, which has - FWR1 selected from SEQ ID NO:156-177 (FWR1 variant); and / or - FWR2 selected from SEQ ID NO:178-202 (FWR2 variant); and / or - Selected from SEQ ID NO:203-230 (FWR3 variant) of FWR3.

[0094] Optionally, the sdAb variant FWR1 may have an E at Kabat position 1. Therefore, any sdAb amino acid sequence disclosed herein in which Kabat position 1 is not E (e.g., is Q) may have an E instead at Kabat position 1.

[0095] Optionally, one or more framework regions may be modified relative to the corresponding natural framework regions (e.g., the framework regions of SEQ ID NO:127 or SEQ ID NO:133) to provide improvements in the properties of the framework or the sdAb itself, such as improvements in basic stability.

[0096] For example, it has been found that certain amino acid positions in the frame region of the sdAb not only tolerate certain mutations of amino acid residues at said positions without impairing functionality, but can even lead to performance improvements in the resulting sdAb variants, particularly improvements in basic stability. Specifically, the following amino acid positions according to the Kabat numbering system have been identified as variable and potentially usable for improving the basic stability of sdAb variants: positions 6, 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b, and 89. Thus, in embodiments, the first polypeptide of the fusion protein of the present invention comprises a single-domain antibody variant comprising an amino acid sequence in which at least one of the Kabat amino acid positions 6, 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b, and 89 is modified relative to the corresponding amino acid of SEQ ID NO: 127 and wherein the sdAb frame has increased basic stability compared to SEQ ID NO: 127.

[0097] Stable modifications are outlined in Table 2. Therefore, the first polypeptide portion of the fusion protein of the present invention may optionally comprise a single-domain antibody variant comprising an amino acid sequence in which at least one, for example at least two, for example three, four, five, six, seven, eight, nine, or ten of the Kabat positions 19, 23, 24, 40, 43, 44, 45, 76, 79, and / or 89, and preferably at least eight residues selected from the stable amino acids indicated in Table 2.

[0098] Table 2: Exemplary Frame Stability Modifications Kabat location Stable amino acids Preferred stable amino acids 19 R, S, K, T R, T 23 T, S, V, A T 24 I, V, S 40 R, I, K, T R, I 43 R, K, Q, E, G R, K 44 E, Q, A, D, G 45 R, I, L R 76 N, Q 79 Y, W, F Y, W, F 89 V, I V, I In an implementation scheme, the first polypeptide portion may include an sdAb framework, which has - FWR1 selected from SEQ ID NO:158-177 (stable variants of FWR1); and / or - FWR2 selected from SEQ ID NO:178-183 and 185-199 (stable variants of FWR2); and / or - FWR3 selected from SEQ ID NO: 205-211, 216-217, 220-225, 227 and 229-230 (stable variants of FWR3).

[0099] A stable variant of FWR2 was specifically envisioned.

[0100] Despite the foregoing, the use of a second polypeptide moiety is envisioned as potentially very useful, wherein the first polypeptide moiety itself has relatively poor basic stability, or at least has basic stability inferior to that of the second polypeptide moiety itself. This could be, for example, when the first polypeptide moiety alone retains 75% or less, e.g., 50% or less, of its target binding capacity after exposure to 0.5 M NaOH for up to 20 hours, such as after 10 hours, or after up to 120 repeated NaOH exposure cycles, such as after 60 repeated NaOH exposure cycles, where one of the NaOH exposure cycles includes contact with 0.5 M NaOH for 10 minutes.

[0101] The second polypeptide moiety has the ability to stabilize or enhance the first polypeptide moiety in some respects, such as with regard to basic stability. Therefore, in cases where the first polypeptide moiety has undesirably low basic stability for its intended use, the fusion of the second polypeptide moiety with the first polypeptide moiety can provide a fusion protein with basic stability higher than that of the first polypeptide moiety alone. For this purpose, the second polypeptide moiety, when considered alone, has basic stability higher than that of the first polypeptide moiety alone.

[0102] Basic stability is a significant advantage for many applications where the fusion protein is immobilized on a solid support and subjected to basic conditions as part of its intended use. This is, for example, in affinity chromatography, where chromatographic materials are routinely cleaned with basic reagents (e.g., NaOH or KOH) between purification cycles. Increased basic stability of the affinity ligands means that the chromatographic material can be used for more purification cycles before the binding capacity becomes unacceptably low.

[0103] Basic stability is typically determined based on binding capacity after a certain degree of basic exposure (usually a certain number of basic treatment cycles). In this context, the terms "treatment," "exposure," and "cleaning" are used interchangeably. For example, "improved basic stability" could mean that the peptide can withstand a greater number of basic cleaning cycles, or the same number of cycles under more stringent basic conditions, without compromising target binding capacity. Alternatively, "improved basic stability" could mean that the peptide retains a higher percentage of binding capacity after an equal number of cleaning cycles under the same conditions. Basic stability may be particularly relevant for peptides intended to be immobilized on solid supports.

[0104] Alkaline treatment may include contacting or incubating with 0.01-1 M NaOH, such as 0.05-0.5 M, 0.1-0.5 M, or 0.3-0.5 M NaOH, for 5-15 minutes, such as 10 minutes (600 seconds) or a time period of about 10 minutes. Milder treatment conditions, such as 0.01-0.1 M, may be used, especially where the first polypeptide moiety itself is very sensitive to alkaline conditions.

[0105] The basic stability of the fusion protein may depend in part on the basic stability of the first polypeptide moiety itself. For example, in cases where the first polypeptide moiety alone has poor stability, the fusion protein of the present invention can provide small but valuable improvements, or relatively large improvements, to the point where a more acceptable level of stability is achieved. For the first polypeptide moiety itself having moderate or good basic stability, the fusion protein may provide relatively small improvements. However, in such cases, the improvement can still, for example, result in excellent basic stability.

[0106] It is worth noting that fusion proteins containing a first polypeptide moiety that is inherently less stable may not achieve the same level of basic stability as fusion proteins based on a first polypeptide moiety that is inherently more basic. Therefore, if very high basic stability is required, the basic stability of the first polypeptide moiety itself should not be too low.

[0107] For example, in cases where the fusion protein of the present invention is based on a first polypeptide moiety that is highly sensitive to alkaline conditions (e.g., retaining 50% of its target-binding capacity for very few cleaning cycles, such as only 1 or 2 cycles), a stabilizing polypeptide can be used to increase alkaline stability such that the fusion protein retains at least 50% binding capacity for twice the number of cycles, or for at least 2 cycles, such as at least 3, 4, 6, or 8 cycles, as long as this represents an increase. A greater number of cycles retaining at least 50% binding capacity indicates improved alkaline stability. It will be understood that each cycle includes target binding followed by a cleaning step. The cleaning step may be, for example, an incubation of approximately 5-15 minutes with 0.1-0.5M NaOH, such as approximately 5, 10, or 15 minutes. For example, the incubation time in contact with 0.5 M NaOH can be 5 ± 0.5 minutes, 6 ± 0.5 minutes, 7 ± 0.5 minutes, or 8 ± 0.5 minutes, 9 ± 0.5 minutes, 10 ± 0.5 minutes, 11 ± 0.5 minutes, 12 ± 0.5 minutes, 13 ± 0.5 minutes, 14 ± 0.5 minutes, or 15 ± 0.5 minutes. The incubation can be carried out, for example, at 22 ± 2 °C.

[0108] In some embodiments, the fusion protein of the present invention can retain a relatively high target binding capacity after repeated clean cycles including 600 seconds of exposure to at least 0.01 M, such as 0.1 M, 0.3 M, or 0.5 M NaOH. For example, compared to the target binding capacity after the first NaOH exposure cycle (i.e., excluding the binding capacity recorded for the first cycle from the comparison), the fusion protein can retain at least 50% target binding capacity after at least 10 repeated cycles of binding and clean NaOH (e.g., at least 0.1 M NaOH or at least 0.3 M NaOH). In embodiments, the fusion protein can retain at least 50% binding capacity after at least 15 cycles, such as 20 cycles.

[0109] The second polypeptide moiety contains an α-helical protein domain and may contain at least one α-helix, such as at least two α-helices, such as three, four, five, or six α-helices, or up to ten α-helices. Specifically, the second polypeptide moiety 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 aggregated and form the major portion of the protein domain, which may lack any other major disordered regions or β-structural regions. Examples of α-helical bundle domains include the SpA protein domain and the albumin-binding domain (ABD). Therefore, the second polypeptide moiety of this disclosure may contain sequences derived from SpA domain A (SEQ ID NO:1), domain B (SEQ ID NO:2), domain C (SEQ ID NO:3), domain D (SEQ ID NO:4), or domain E (SEQ ID NO:5). In this context, "amino acid sequence derived from the SpA protein domain" means an amino acid sequence having at least 70% identity with any of the following sequences: amino acid residues 9-28 of any of SEQ ID NO:1-3, residues 12-31 of SEQ ID NO:4, residues 7-26 of SEQ ID NO:5, residues 29-51 of any of SEQ ID NO:1-3, residues 30-54 of SEQ ID NO:4, and residues 27-49 of SEQ ID NO:5. Polypeptides derived from the SpA protein domain include SEQ ID NO:6-110. Specifically, the second polypeptide may comprise an α-helix bundle domain having an amino acid sequence selected from SEQ ID NO:1-110 and having at least 70% identity with any of the sequences, and, for example, a derivative thereof having at least 80%, at least 85%, at least 90%, or at least 95% identity with any of the sequences (and specifically with SEQ ID NO:8 or 64). In an embodiment, the second polypeptide portion may include an α-helical bundle domain having an amino acid sequence having at least 85% identity with amino acid residues 9-28 of SEQ ID NO:8 or amino acids 29-51 of SEQ ID NO:8.

[0110] The second polypeptide moiety may be a basic stable protein. For example, the second polypeptide moiety may contain an amino acid sequence derived from a natural protein but modified relative to the natural protein. Modifications may provide increased basic stability, reduced interaction with the natural binding partner, or both. For example, the second polypeptide moiety may contain a SpA-derived protein domain that has optionally been modified to improve its basic stability. Preferably, the second polypeptide moiety itself may have basic stability, measured as the number of base-cleaning cycles (or the ability to bind to affinity ligands, as described above for the non-binding second polypeptide moiety) with at least 50% retained target binding capacity, expressed as at least 10 cycles, and preferably at least 20 cycles, such as at least 30, 40, or 50 cycles.

[0111] Any modifications made relative to the native amino acid sequence containing α-helical protein domains, such as the SpA domain, should not disrupt the helical structure involved. The amino acid changes proposed for SpA-derived peptides in this paper take into account that certain amino acids readily form α-helical structures, and therefore, it is expected that such structures will be preserved.

[0112] The first polypeptide moiety and the second polypeptide moiety are different from each other. Therefore, in embodiments where the first polypeptide moiety includes an α-helical protein domain, such as an α-helical bundle domain, the amino acid sequence containing the α-helical domain is different. In such embodiments, the second polypeptide moiety has higher basic stability than the first polypeptide moiety.

[0113] Examples of SpA-derived basic stable polypeptides that can be particularly used as stable polypeptides of the present invention include the amino acid sequences of SEQ ID NO: 7-9, 28-30, and 61-110. Therefore, the second polypeptide moiety may comprise an amino acid sequence selected from SEQ ID NO: 7-9, 28-30, and 61-110, and derivatives thereof having at least 80% identity with any of said sequences, preferably having at least 85, 90, or 95% identity with any of said sequences, such as SEQ ID NO: 8 or 64. In an embodiment, the second polypeptide may comprise a basic stable α-helical bundle domain having an amino acid sequence having at least 85% identity with amino acid residues 9-28 of SEQ ID NO: 8 or amino acids 29-51 of SEQ ID NO: 8.

[0114] To avoid interfering with the affinity capture function of the first polypeptide moiety, the second polypeptide moiety should not have binding affinity for the target entity intended to bind to the first polypeptide moiety. Therefore, in this embodiment, the second polypeptide is unable to bind to the target entity.

[0115] Furthermore, to avoid unwanted interactions between the first and second polypeptide moieties, such as the formation of dimers or aggregates, the first polypeptide moieties preferably have no binding affinity for the second polypeptide moieties, and the second polypeptide moieties have no affinity for the first polypeptide moieties. Additionally, where the second polypeptide is not intended to link the fusion protein to another moieties (e.g., during purification or for coupling the fusion protein to a surface), it may be preferable that it has no binding affinity for another molecule, or at least no binding affinity for any polypeptide present during the production of the target protein, to avoid competitive interactions that could adversely affect the interaction between the first polypeptide moieties and their target antigen.

[0116] For example, it may be advantageous if the second polypeptide moiety has no binding affinity for immunoglobulins. Therefore, in cases where the second polypeptide is derived from the protein domain of the SpA of the naturally binding Fc and / or VH moiety of immunoglobulins, the amino acid sequence of the second polypeptide can, for example, disenchant its binding to Fc or VH3 or both. In embodiments, the second polypeptide moiety may therefore be unable to bind to the Fc or VH3 or both of immunoglobulins. This can also be referred to as the SpA-derived polypeptide being "Fc knocked out" and "VH knocked out," or, if both bindings to Fc and VH3 have been disenchanted, as "double knocked out." Disenchanting both bindings to Fc and VH3 has shown significant advantages in some applications because undesirable bindings are avoided.

[0117] However, it is envisioned that binding to IgG may be necessary in certain applications, and in such cases, the second peptide may be able to bind any desired IgG moiety, specifically the Fc and / or VH3 moiety. For example, the second peptide may bind the Fc moiety but not the VH3 of IgG. Alternatively, the second peptide may bind the VH3 but not the Fc moiety of IgG.

[0118] The second polypeptide moiety can be derived from the protein domain of SpA as described above, and through position X 33 X 40 and X 51 Mutations are introduced to modify the binding to VH3, as described in WO 2023 / 046886. Name X 33 X 40 and X 51 This refers to positions 33, 40, and 51 within the SpA domain represented by any one of SEQ ID NO: 1-3. The positions X of these polypeptides... 33 X 40 and X 51Certain mutations significantly reduce or eliminate the binding affinity for VH3. In such peptides, X 33 Selected from T, S, G, Q, A, E, H, R, P, D, K, and N; X 40 Selected from E, G, R, D, K, Q, N, H, and S, and X 51 Selected from L, V, S, I, R, and G; the condition is that when X 51 When it is L, then X 33 X 40 Selected from AD, HK, EG, ER, GR, AK, AR, PK, RR, and KK, and when X 51 When it is G, then X 33 X 40 It's TK.

[0119] Reduced VH affinity can be defined as the second polypeptide moiety having binding affinity to the Fc region of an immunoglobulin and a lower binding affinity to the VH3 region of trastuzumab compared to the binding affinity of SEQ ID NO: 6 (corresponding to SEQ ID NO: 59 of WO2023 / 046886) to the same VH3 region. WO2023 / 046886 describes a polypeptide with reduced VH affinity and is incorporated herein by reference in its entirety. Trastuzumab is a monoclonal antibody also known by the trade names Herceptin™ (Roche) and Trazimera™ (Pfizer).

[0120] The second polypeptide moiety of the fusion protein of the present invention may have an X selected from T, S, G, Q, A, E, H and R. 33 X selected from E, G, R, D, K, Q, H and S 40 And X selected from L, V, S, I, R and G 51 The condition is when X 51 When it is L, then X 33 X 40 Selected from AD, HK, EG, ER, GR, AK, AR, and RR, and when X 51 When it is G, then X 33 X 40 It's TK.

[0121] Alternatively or additionally, the second polypeptide moiety may be derived from the protein domain of SpA (as described above) and modified to at least reduce the affinity of a significant portion for the Fc region while retaining the affinity for the VH3 region. This may be done, for example, by providing selected amino acids at positions 9-11, 13-14, 17-18, 26, 28, and 29 of the SpA domain (e.g., represented by any one of SEQ ID NO: 1-3). International application PCT / EP23 / 056401 describes a polypeptide with reduced Fc affinity and is incorporated herein by reference in its entirety. Reduced Fc affinity can be defined as the second polypeptide having binding affinity for the VH3 region of the immunoglobulin and a lower binding affinity for the Fc region of trastuzumab compared to the binding affinity of SEQ ID NO: 8 for the same Fc region.

[0122] The second polypeptide moiety may comprise an amino acid sequence derived from the SpA protein domain, said amino acid sequence having reduced or eliminated binding to both VH3 and Fc. In such variants, the mutations described above for reducing VH3 affinity and Fc affinity, respectively, can be combined.

[0123] SEQ ID NO: 64 represents one of the preferred variants of the second polypeptide portion of this disclosure. However, the sequence can be modified, and a preferred second polypeptide portion can still be obtained. For example, the second polypeptide portion may comprise or may be an amino acid sequence derived from a protein domain of SpA, wherein... The amino acid residue at position 9 of SEQ ID NO:64 is selected from L, E, R, A, and Y, for example, it is A; The amino acid residue at position 10 of SEQ ID NO:64 is selected from L, E, R, A and Y, for example, Y; The amino acid residue at position 11 of SEQ ID NO:64 is selected from L, E, R, A, and Y, for example, R; The amino acid residue at position 13 of SEQ ID NO:64 is selected from L, E, R, A and Q, for example, L, R, A or Q; The amino acid residue at position 14 of SEQ ID NO:64 is selected from L, E, R, A, Q, and K, for example, it is A or R; The amino acid residue at position 17 of SEQ ID NO:64 is selected from L, E, R, and A, for example, A; The amino acid residue at position 18 of SEQ ID NO:64 is selected from L, E, R, and A, for example, R; The amino acid residue at position 28 of SEQ ID NO:64 is selected from L, E, R, and A, for example, R or A; The amino acid residue at position 29 of SEQ ID NO:64 is selected from L, E, R, and A, for example, R or A; The amino acid residue at position 33 of SEQ ID NO:64 is selected from T, S, G, Q, A, E, H and R, for example, S; The amino acid residue at position 40 of SEQ ID NO:64 is selected from E, G, R, D, K, Q, H, and S, for example, G; and The amino acid residue at position 51 of SEQ ID NO:64 is selected from L, V, S, I, R, and G, for example, V.

[0124] For example, the second polypeptide portion comprises an amino acid sequence according to the following: VDAKFDKEX9X 10 X 11 AX 13 X 14 EIX 17 X 18 LPNLTEEQRX 28 X 29 FIQX 33 LKDDPSX 40 SKAILAEAKKX 51 NDAQAPK (SEQ ID NO: 235) As described in this article, X9 and X are independently... 10 X 11 X 13 X 14 X 17 X 18 X 28 X 29 X 33 X 40 and X 51 Make a selection.

[0125] For clarity, in the variant, the expression "amino acid residue at position X" refers to the position in the second polypeptide portion corresponding to position X of SEQ ID NO: 64.

[0126] The amino acid residues proposed above for positions 9-11, 13-14, 17-18, 28-29, 33, 40 and 51 are expected to maintain the helical structure containing the α-helical polypeptide domain, and functional variants that provide stable polypeptides have been found.

[0127] In some variants, the amino acids at positions 9-11 are AYR. In other variants, positions 9-11 are independently selected from L, E, R, A, and Y, and positions 28, 29, 33, 40, and 41 are different from SEQ ID NO: 64, i.e., they are not A, A, S, G, and V, respectively. In some variants, positions 13-14 are selected from LA, RA, AR, and QA.

[0128] For example, the second polypeptide moiety may contain an amino acid sequence selected from SEQ ID NO:64-110.

[0129] In some embodiments, the second polypeptide portion may comprise an amino acid sequence based on a protein domain of SpA as defined above, wherein the amino acid at position 31 corresponding to any one of SEQ ID NO:8-10 is isoleucine.

[0130] In other embodiments, the second polypeptide moiety may comprise an amino acid sequence based on a protein domain of SpA as defined above, wherein the amino acid at position 8 corresponding to any one of SEQ ID NO: 8-10 is not isoleucine, or is not an amino acid selected from Ile, Leu, Val, Tyr, Phe, and Trp. In such embodiments, the first polypeptide moiety may be capable of binding IgG.

[0131] In the implementation scheme, neither the second polypeptide moiety nor the first polypeptide moiety has affinity for trastuzumab.

[0132] Specifically, the second polypeptide moiety may consist of the SpA-derived polypeptide described herein and optionally additional amino acids, such as a short N-terminal amino acid sequence and / or optionally a short C-terminal amino acid sequence.

[0133] More generally, the fusion proteins of this disclosure may contain additional amino acids as mentioned above (e.g., with respect to Figures 1 and 2) at the N-terminus, at the C-terminus, and / or between different polypeptide units or portions. Fusion proteins as defined herein may contain any suitable number of additional amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, for example, up to 20 additional amino acid residues, optionally in a continuous sequence. For example, the sequence of additional amino acids may consist of 1-12 amino acids, and several such sequences may be present in the fusion protein. The additional amino acids may be remnants from recombinant protein expression, leader peptides, signal peptides, purification 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 support, such as a solid support described in conjunction with aspects of an adsorbent material or separation matrix).

[0134] Fusion proteins or polymers as defined herein may contain at least one linker. For example, a linker may be present between each monomer within the polymer, and each linker may contain up to 15 or 30 amino acids, such as 1-5, 1-10, or 5-10 amino acids. In the presence of multiple linkers, the linkers may be identical or different. Those skilled in the art are familiar with different types of linkers with varying properties, such as flexible peptide linkers, rigid peptide linkers, and cleavable peptide linkers. Peptide linkers may be structured or unstructured. Generally, unstructured linkers are more flexible and less rigid than structured linkers. Linkers can be used, for example, to increase the stability of the fusion protein or to improve its folding. Linkers may also ensure a certain spatial distance between the polypeptide moieties to which they are linked, which may be advantageous for antigens to approach the binding surface of the antigen-binding polypeptide. The presence of linkers within a fusion protein generally does not significantly affect the target-binding capacity of its properly folded antigen-binding polypeptide moieties. Therefore, the fusion proteins or portions thereof disclosed herein may be directly linked to each other via peptide bonds between the C-terminus and N-terminus of the polypeptide.

[0135] The linker should preferably not destabilize the spatial conformation of the protein units 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 units. 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.

[0136] In some embodiments, the fusion protein disclosed above comprises one or more coupling elements selected from at least one cysteine ​​residue, multiple lysine residues, and multiple histidine residues, and combinations thereof. Such coupling elements can be provided anywhere in the fusion protein, but may optionally be located near the C- or N-terminus of the polypeptide or arranged at the C- or N-terminus of the polypeptide. The coupling element may, for example, be a single cysteine ​​residue at the C-terminus of the fusion protein. The coupling element may be directly linked to the C- or N-terminus, or may be linked via a linker comprising up to 15 amino acids, such as 1-5, 1-10, or 5-10 amino acids. Such an amino acid sequence should also preferably be sufficiently stable in an alkaline environment without impairing the properties of the fusion protein. For this purpose, it is advantageous if the sequence does not contain asparagine. It may also be additionally advantageous if the sequence does not contain glutamine. The advantage of having a C-terminal cysteine ​​is that the terminal 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 of the coupled protein.

[0137] The fusion protein of the present invention can be produced by recombinant protein production using genetically modified host cells, through conventional biotechnological methods.

[0138] Therefore, in a further aspect, this disclosure provides a polynucleotide encoding a fusion protein as described herein; an expression vector comprising said polynucleotide; and a host cell comprising said expression vector.

[0139] This disclosure also includes a method for producing a fusion protein as described herein, comprising culturing the host cell under conditions that allow expression of the fusion protein from its expression vector, and isolating the fusion protein.

[0140] 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 biotechnology for protein production, such as Chinese hamster ovary (CHO) cells.

[0141] After isolating the fusion protein, it is purified, for example, by conventional methods known in the art. For instance, if the fusion protein contains a histidine tag, the purification step may include immobilized metal affinity chromatography (IMAC). Other purification methods that may be used include affinity chromatography using ligands that bind to either the first or second polypeptide moiety. For example, affinity ligands that bind to the framework region of the first polypeptide may be used, or affinity chromatography may use SpA-based affinity ligands that bind to the VH region of the single-chain polypeptide of the first polypeptide moiety, such as VH3. Alternatively, the affinity ligand may bind to the second polypeptide moiety of the fusion protein, for example, an affinity tag included in the amino acid sequence of the second polypeptide moiety.

[0142] Alternatively, fusion proteins as disclosed herein can be produced by in vitro translation or by non-biological peptide synthesis using amino acids and / or amino acid derivatives having protected reactive side chains. Non-biological peptide synthesis may include: - Stepwise coupling of amino acids and / or amino acid derivatives to form peptides with protected reactive side chains, - Removal of protecting groups from the reactive side chains of peptides, and - Folding peptides in aqueous solutions.

[0143] Generally, the fusion protein of the present invention can be used to capture target entities to which the first polypeptide moiety has an affinity. However, preferably, the fusion protein itself is not intended to be used as a therapeutic compound, and preferably is not intended for in vivo use. Therefore, the fusion protein of the present invention can be used to capture target entities in vitro.

[0144] The capture can be used to detect target entities within a sample or to separate target entities from other components in the sample. The fusion protein can be used for analytical separation of the target entity, or it can be used for preparative purification of the target entity. The term "preparation" refers to the process of preparing a purified target entity, specifically in the production of products containing the target entity, such as pharmaceuticals. Alternatively, the separation, detection, and / or quantification of the fusion protein using this disclosure can be performed in the context of sensor applications.

[0145] Specifically, the fusion protein of the present invention can be used in applications involving the interaction between the first polypeptide moiety and its target entity, wherein at least one of the fusion protein and the target entity is coupled to a vector. When the fusion protein is coupled to or immobilized to a vector, the first polypeptide moiety can still bind to its target entity.

[0146] In the case of the fusion protein 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 fusion protein to the carrier can be provided by a stable polypeptide. For example, where the second polypeptide moiety is located at the C-terminus of the first polypeptide moiety, the second polypeptide moiety may have a C-terminal amino acid sequence suitable for coupling with the carrier. For example, a tag or spacer terminally marked with a cysteine ​​(C) can be provided for coupling via a thioether bond. Therefore, enhanced coupling can be achieved by a fusion protein comprising the second polypeptide moiety of this disclosure.

[0147] When the fusion protein is not coupled to a carrier, it can be used, for example, in detection assays where a target or analyte is present on a surface and in contact with the fusion protein present in solution. In such cases, a reporter entity capable of binding the second polypeptide moiety is envisioned for generating a detectable signal. The reporter entity can be, for example, a fluorescently labeled or radiolabeled entity, as known in the art. If the second polypeptide moiety is capable of binding IgG, the reporter entity can be IgG-based.

[0148] Because of the stabilizing effect of the second polypeptide moiety, the fusion protein is particularly suitable for applications where it can be exposed to alkaline conditions, such as treatment with NaOH. Such treatment is commonly used in the field of chromatography to remove impurities bound to the chromatographic matrix prior to the next purification cycle (in-situ cleaning, as described above). However, alkaline exposure or cleaning of the binding surface can also be used for other backgrounds.

[0149] The carrier may be a solid carrier and optionally a porous material.

[0150] The carrier may be or comprise a surface thereon on which the fusion protein is coupled. Examples of such carrier materials include conventional protein-binding carriers and surfaces, such as chips, plates, wells, and sheets. The carrier may optionally be provided in other forms, such as fibers, membranes, fiber matrices, filters, porous bulk materials, particles, or beads, such as gel beads used in chromatographic resins. Particles or beads may be porous or non-porous. Particles or beads may include magnetic beads. Carriers in bead or particle form may be used as packed beds or in suspension. Suspension forms include those known as expanded beds and pure suspensions, in which the particles or beads move freely. In the cases of bulk materials, packed beds, and expanded beds, the separation procedure typically follows conventional chromatography with a concentration gradient. In the case of pure suspensions, batch mode will be used.

[0151] The carrier can be prepared from any suitable material, as described in more detail below. As a non-limiting example, conventional affinity separation matrices are typically organic in nature and are based on exposing hydrophilic surfaces to the aqueous medium used, i.e., on their outer surfaces and, if present, on their inner surfaces, to polymers with hydroxyl (-OH), carboxyl (-COOH), formamide (-CONH2, possibly in N-substituted form), amino (-NH2, possibly in substituted form), oligomeric, or polyoxyethylene groups.

[0152] The fusion protein can be linked to the carrier using known coupling techniques, such as those utilizing thiol, amino, and / or carboxyl groups present in the fusion protein. Biepoxides, epichlorohydrins, CNBr, N-hydroxysuccinimide (NHS), etc., are well-known coupling agents. A spacer molecule can be introduced between the carrier and the fusion protein to improve the availability of the first polypeptide moiety and / or promote the chemical coupling of the fusion protein to the carrier. Depending on the properties of the fusion protein 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). Alternatively, the fusion protein can be linked to the carrier via non-covalent bonding, such as physical adsorption or biospecific adsorption.

[0153] The fusion protein can be coupled to the support 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 passes through terminal or proximal cysteine ​​residues on the fusion protein, the motility of the coupled fusion protein is enhanced, providing improved binding capacity and binding kinetics. In some embodiments, the fusion protein 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 electrophilic groups on the support, such as epoxide groups, halool groups, etc., resulting in thioether-bridged coupling.

[0154] 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.

[0155] 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.

[0156] Alternatively, the solid carrier according to the present invention comprises an inorganic carrier, such as silicon dioxide, zirconium oxide, etc.

[0157] In this embodiment, the fusion protein may be coupled to a carrier, which is a convection-based chromatographic matrix. Such a convection-based chromatographic matrix may comprise a porous polymer membrane, a filter, a fibrous matrix, or a porous bulk material. Examples of porous polymer membranes include Mustang. TM Membrane (Cytiva) and Sartobind TMMembrane (Sartorius). The fiber carrier can be based on electrospun polymer fibers or cellulose fibers, optionally nonwoven fibers. The fiber matrix can therefore be a fiber nonwoven 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.

[0158] The adsorbent materials or separation matrices described herein can be used for the same purposes mentioned above for the fusion protein.

[0159] Therefore, in one aspect, the present invention provides chromatographic materials comprising adsorbent materials or separation matrices as disclosed herein, and chromatographic columns or devices comprising such adsorbent materials or separation matrices.

[0160] On the other hand, the adsorbent material can be a sensor surface designed for use in sensors or other detection or quantification devices.

[0161] This invention provides a method for separating or isolating target entities, wherein an adsorbent material or separation matrix 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 made under conditions where the target entity can bind the first polypeptide moiety. 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 10 times, for example at least 20 times.

[0162] 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. The sample may have undergone one or more steps of filtration via tangential flow filtration (TFF).

[0163] 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 of the present invention can be used to separate the target entity from such impurities, and after the above process, the eluent containing the target entity has a reduced level of at least one of the said impurities.

[0164] Optionally, after separation based on affinity capture as described herein, the eluent containing the purified target entity may undergo one or more steps of filtration, concentration, dilution, or buffer exchange, or chromatographic steps not based on affinity chromatography, such as ion exchange chromatography, hydrophobic interaction chromatography, multimode chromatography, or size exclusion chromatography. Further chromatographic steps performed after the affinity separation steps of this disclosure may be referred to as purification steps.

[0165] 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.

[0166] The use of the verb "comprising" and its variations does not exclude the presence of elements or steps other than those specified. For example, a polypeptide "comprising" 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. The simple fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to produce benefits. Example

[0167] Example 1A: Preparation of glycerol stock solution from IDT oligonucleotides This example describes the generation of a glycerol reservoir for the production of the candidate peptide used in Example 4B.

[0168] The materials and equipment used are 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); 14ml Falcon™ round-bottom tubes (Corning™); PlasmidPrep Mini Spin kit (Cytiva™); Inforrs HT shaking incubator.

[0169] 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.

[0170] 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.

[0171] Example 1B: Preparation of glycerol stock solution from IDT plasmid This embodiment describes the generation of a glycerol reservoir for the production of candidate peptides used in Examples 2A, 2B, 4A, and 5-7.

[0172] The materials and equipment used are as follows: plasmid DNA of the candidate peptide to be produced; chemocompetent BL21(DE3) E. coli (prepared internally); KCM buffer (5X) 0.5M KCl, 0.15M CaCl2, 0.25M MgCl2; LB medium (Invitrogen™); carbenicillin (100 mg / ml); agar plates supplemented with carbenicillin (100 µg / ml) (PanReacAppliChem™) (BDBACTO™ agar); 14 ml Falcon™ Round-Bottom (Corning™); Inforrs HT shaking incubator.

[0173] 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.

[0174] Example 2A: AAV9-binding peptide fusion with Z – monomer construct A single-domain antibody capable of binding to AAV9 was fused with a stable peptide, and its effects on protein expression, antigen-binding capacity, and basic stability were investigated.

[0175] The peptides tested are identified in Table 3. Single-domain antibodies were compared with fusions of the same single-domain antibody and a stable peptide. The single-domain antibody (referred to as “sdAb”) was an AAV9-binding VHH variant (SEQ ID NO: 139). The stable peptide (referred to as “Z”) was a SpA domain Z variant (SEQ ID NO: 64) in which binding to the Fc and VH3 moieties of IgG was eliminated. Each construct had a C-terminal linker followed by a (His)6 tag.

[0176] Table 3 Candidate number Builder description Construct sequence 2A-1 [sdAb] SEQ ID NO:140 2A-2 [sdAb]-[Z] SEQ ID NO:142 Specifically, the following aspects were evaluated: (1) Affinity assessment of AAV9 interactions tested by high-concentration AAV9 injection; (2) Alkaline stability of AAV9 binding reduced by treatment with 0.5 M NaOH in increments of 0.5 M.

[0177] Materials and methods MabSelect PrismA™ purification of peptides In a 14 ml round-bottom tube, 5 µl of glycerol stock solution generated from peptides 2A-1 and 2A-2 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.

[0178] Protein expression medium prepared from Terrific Broth (TB) medium supplemented with 100 µg / ml carbenicillin and 2 mM MgCl2 was 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 hours, until the OD600 reached 1.0. Then, 50 µl of IPTG (1 M) was added to a final concentration of 1 mM, and the flasks were incubated in an Infortrère HT shaker at 27°C and 140 rpm for 18 hours. The culture was then transferred to Falcon tubes (50 ml). To produce crude variant lysates, Falcon tubes were incubated in a 48°C water bath for 2 hours, followed by centrifugation at 8000 g for 10 minutes to precipitate cell debris, and the supernatant was filtered through a 0.22 µm filter.

[0179] 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 for 5 CVs.

[0180] 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™).

[0181] Biacore analysis of AAV9 binding affinity To assess binding with AAV9, peptides 2A-1 and 2A-2, as described above, were immobilized on a Biacore™ S-Series CM5 chip using an amine conjugation kit (Cytiva™) and analyzed using a Biacore™ 8K+ instrument (Cytiva™). Samples containing AAV9 viral particles (vp) at a concentration of 2E12 vp / ml were used as analytes.

[0182] Using the standard method in Biacore software, peptides were immobilized by coupling in flow cell 2 (FC2) and activation / inactivation in flow cell 1 (FC1). Peptides were diluted in Biacore™ acetate buffer pH 5.0 (Cytiva™) at concentrations ranging from 17–30 µg / ml, depending on their molecular weight. Immobilization levels varied between different peptide variants corresponding to their molecular weights. In each run, the peptide was immobilized in FC2.

[0183] The Biacore™ method for binding assays: Run buffer: PBS-P+; Flow rate: 5 µl / min; Sample injection: 2400 s / 40 min on both FC1 and FC2; Dissociation time: 2400 s / 40 min; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle), the analyte (AAV9) was injected as follows: run buffer, AAV9 2E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

[0184] 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.

[0185] Biacore analysis of alkaline stability After evaluating AAV9 binding (see above), the same chip underwent repeated cycles of AAV9 binding (5E11 vp / ml) followed by NaOH (0.5 M) injection to assess alkaline stability.

[0186] 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.

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

[0188] result Bis-Tris PAGE analysis of expression levels showed that the expression levels of peptides 2A-1 (sdAb, unfused) and 2A-2 (sdAb with a C-terminal stable peptide) were comparable (data not shown). This sdAb has previously been found to express well in monomeric form.

[0189] The fixed levels on the CM5 chip were 2134 or 3560 RU, respectively. For the tested fusion protein 2A-2, the AAV9 binding capacity at the maximum viral concentration used (2E12 vp / ml) was approximately 10% higher than that of sdAb 2A-1 alone.

[0190] Figure 3A The AAV9 binding capacity is shown for incremental cycles including 0.5 M NaOH exposure (sample injection 2 above), normalized to the response after the first cycle (i.e., excluding the first cycle). The first cleaning cycle removes loosely bound peptides from the surface. As can be seen in the figure, fusion protein 2A-2, contained in a single-domain antibody variant fused to a stable peptide at its C-terminus, shows significantly improved basic stability compared to the single-domain antibody 2A-1 alone. This improvement is even more pronounced if the response after each cycle is normalized to the response of the first cycle before the first NaOH exposure, as shown in the figure. Figure 3B As shown in the image. Figure 3B As observed, the decrease in AAV9 binding capacity experienced between the first and second cycles was only 20% for the fusion protein 2A-2, compared to a 60% decrease in response for sdAb, which is not part of the fusion protein. It is hypothesized that the smaller decrease between the first and second binding cycles may be at least partially due to surface coupling of the fusion protein. Therefore, it is considered that the second polypeptide moiety provides a coupling mechanism more resistant to alkali exposure than coupling by sdAb itself.

[0191] Table 4 summarizes the results of AAV9 binding capacity and basic stability measurements.

[0192] Table 4 sample AAV9 capacity (2E12 vp / ml) (RU) Maintain at least 50% binding cycle number (5E11 vp / ml, 100% = AAV9 response of the first cycle). Maintain at least 50% binding cycle number (5E11 vp / ml, 100% = AAV9 response in the second cycle). 2A-1 11294 1 10 2A-2 12421 14 19 Example 2B: AAV9-binding peptide fusion with Z – multimeric construct Regarding protein expression, antigen-binding capacity, and basic stability, we studied multimeric constructs of single-domain antibodies fused with stable peptides that can bind AAV9.

[0193] The peptides tested are summarized in Table 5 below. The single-domain antibody (sdAb) trimers were compared with trimer fusion proteins containing one or three stable peptides, respectively. The single-domain antibody (referred to as “sdAb”) was an AAV9-binding VHH variant (SEQ ID NO: 139). The stable peptide (referred to as “Z”) was a SpA domain Z variant (SEQ ID NO: 64) in which binding to the Fc and VH3 moieties of IgG was eliminated. Each construct had a C-terminal linker followed by a (His)6-Cys tag.

[0194] Table 5 Candidate number Builder description Construct sequence 2B-1 [sdAb]-[sdAb]-[sdAb] SEQ ID NO:143 2B-2 [sdAb]-[sdAb]-[sdAb]-[Z] SEQ ID NO:144 2B-3 [sdAb]-[Z]-[sdAb]-[Z]-[sdAb]-[Z] SEQ ID NO:145 The following aspects were evaluated: (1) Protein expression analysis by PAGE. (2) Affinity assessment of AAV9 interaction (tested by high concentration of AAV9 injection). (3) Basic stability (tested by the reduction of AAV9 binding after incremental treatment with 0.3 M NaOH).

[0195] Materials and methods MabSelect PrismA™ purification and biotinylated peptide generation The candidate peptide was generated and purified according to the method described in Example 2A.

[0196] Following the manufacturer's instructions, the concentration of the MabSelectPrismA™ purified sample was measured using NanoDrop (Thermo Scientific). After purification, the sample was reduced by adding 10 µl of 0.5 M DTT dissolved in sterile water to 490 µl of sample to achieve a final DTT concentration of 10 mM. The sample was incubated at room temperature for 1 hour, followed by buffer exchange to PBS using an Amersham NAP-5 column (Cytiva™) to produce 1 ml of eluent. EZ-Link™ maleimide-PEG2-biotin, No weight™ form (Thermo Scientific) was dissolved in 190 µl of PBS, and 5 µl of dissolved biotin was added to each 1 ml of eluent to achieve a final biotin excess of 2–5 moles. The sample was incubated on ice for 2 hours, followed by dialyzing using a Slide-A-Lyzer™ G2 dialysis cartridge (Thermo Scientific). Following the manufacturer's instructions, the concentration of the dialyzed sample was measured using NanoDrop (Thermo Scientific) and kept in an Eppendorf tube in a refrigerator until all measurements were completed, then kept in a freezer until further analysis. Protein mass was determined by liquid chromatography-mass spectrometry using a BioRessolve column (Waters™) on an ACQUITY RDa detector (Waters™).

[0197] Bis-Tris PAGE analysis To evaluate protein expression levels, Bis-Tris PAGE was performed on the filtered supernatant after heat treatment. The materials and equipment used were as follows: NuPAGE LDS sample buffer (x4); PageRuler pre-stained gel; 4-12% Bis-Tris mini-gel; Invitrogen NP0321BOX; mini-gel tray (Invitrogen); 20x MES buffer (Invitrogen); QuickBlue dye.

[0198] Mix 15 µl of filtered supernatant from the heat-treated gel with 5 µl of LDS sample buffer. Heat the sample mixture to 70 °C for 10 min and gently centrifuge to collect the condensate from the cap. For each expressed construct, load 15 µl of the sample mixture onto the gel. Load 5 µl of PageRuler as a marker. Run the gel at 200 V for 35 min and stain with QuickBlue dye for 2 h. Decolorize the gel overnight in deionized water.

[0199] Binding affinity analysis for AAV9 To evaluate binding with AAV9, peptides 2B-1, 2B-2, and 2B-3, as described above, were immobilized on a Biacore™ S-series SA chip (Cytiva™) and analyzed using a Biacore™ 8K+ instrument (Cytiva™). Samples containing AAV9 viral particles (vp) at a concentration of 2E12vp / ml were used as analytes.

[0200] The peptides were immobilized using the standard SA conjugation method in Biacore software by conjugating them in flow cell 2 (FC2) and activating / inactivating them in flow cell 1 (FC1). The peptides were diluted in PBS-P+ (Cytiva™) at a concentration of approximately 25 µg / ml. Immobilization levels varied slightly between different peptide variants corresponding to their molecular weights. In each run, the peptides were immobilized in FC2.

[0201] The Biacore™ method for binding assays: Run buffer: PBS-P+; Flow rate: 5 µl / min; Sample injection: 2400 s / 40 min on both FC1 and FC2; Dissociation time: 2400 s / 40 min; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle), the analyte (AAV9) was injected as follows: run buffer, AAV9 2E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

[0202] 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.

[0203] 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.

[0204] Biacore™ method for alkaline stability (per cycle): Run buffer: PBS-P+; Flow rate: 10 µl / min; Sample injection 1 (AAV9 5E11 vp / ml): 300 s in both flow cells; Dissociation time 1: 30 s; Sample injection 2 (0.3 M NaOH): 600 s in both flow cells; Dissociation time 2: 0 s; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. This cycle was repeated 40 times to track the stability of the AAV9 response value.

[0205] 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.

[0206] result via Bis-Tris PAGE ( Figure 4A Analysis of expression levels showed that multimeric fusion proteins containing stable peptides can improve protein expression.

[0207] The fixed level range on the SA chip was 3000-3500 RU. For the tested fusion proteins 2B-2 and 2B-3, the AAV9 binding capacity at the maximum viral concentration used was higher than that of the sdAb trimer without a stable peptide.

[0208] Figure 4BThe AAV9 binding capacity is shown for incremental cycles including 0.3 M NaOH exposure (sample injection 2 above), normalized to the response after the first cycle (i.e., excluding the first cycle). As can be seen in the figure, both fusion proteins 2B-2 and 2B-3 show significant improvements in basic stability compared to sdAb trimer 2B-1. By normalizing the response to the response of the first cycle, it was found that 2B-3 experienced a larger decrease in response between the first and second cycles than 2B-1 or 2B-2, but remained more stable than 2B-1 for subsequent cycles. 2B-1 lost more binding capacity than fusion proteins 2B-2 and 2B-3 during the test. Table 6 summarizes the results of the AAV9 binding capacity and basic stability measurements.

[0209] Table 6 sample AAV9 capacity (2E12 vp / ml, RU) Maintain at least 50% binding cycle number (5E11 vp / ml, 100% = AAV9 response of the first cycle) Maintain at least 50% binding cycle number (5E11 vp / ml, 100% = AAV9 response in the second cycle) 2B-1 13410 12 20 2B-2 14328 21 30 2B-3 14991 11 28 Example 3: GFP-binding peptides with different Z variants In this embodiment, 47 fusion proteins with the same GFP-binding VHH (SEQ ID NO: 120) and different stable peptides were evaluated. The stable peptides were SpA domain Z variants with different amino acid groups at positions 9, 10, 11, 13, 14, 17, 18, 28, 29, 33, 40, and 51, as summarized in Table 7. The stable peptides were fused to the C-terminus of the GFP-binding peptide via a peptide linker (QAS). The fusion proteins had a C-terminal (His)6 tag linked to the stable peptide via a GS linker.

[0210] Table 7: Details of the stabilizing peptides used for fusion proteins Evaluate the following aspects: (1) Affinity assessment of GFP target molecule interactions (by injecting assays with varying target molecule concentrations).

[0211] (2) Basic stability (assessed by the decrease in GFP binding after increasing numbers of NaOH (0.3 M) treatments).

[0212] (3) IgG interaction as tested by infusion of Gammanorm (Octapharma™) and trastuzumab (prepared internally).

[0213] Materials and methods plasmid preparation The materials and equipment used were as follows: plasmid pAM1050; G-blocks fusion protein candidate (IDT™); restriction enzymes HindIII and BamHI (NEB™); Antarcic phosphatase (NEB™); GFX™ PCR DNA and gel strip purification kit (Cytiva™); T4 DNA ligase (Thermo Fisher™); Top10 E. coli cells (One Shot™ TOP10 chemocompetent E. coli, ThermoFisher™); SOC medium (Invitrogen™); agar plates, 100 µl carbenicillin / ml.

[0214] Plasmid pAM1050 and G-blocks containing the DNA sequences of all candidates were digested with restriction enzymes HindIII and BamHI. The plasmids were dephosphorylated using antarctic phosphatase and then purified by gel electrophoresis according to the manufacturer's protocol.

[0215] 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 KCM competent Top10 *E. coli* cells. In short, the cells and ligation mixture were incubated on ice for 30 min, followed by incubation in KCM buffer at room temperature for 10 min. The cells were then transferred to SOC medium and incubated at 37°C for 1 h, followed by seeding onto agar plates containing 100 µl carbenicillin / ml and incubating overnight at 37°C.

[0216] protein expression The materials and equipment used were as follows: E. coli colonies transformed with the candidate fusion protein; LB medium, Terrific Broth (TB) medium, carbenicillin, IPTG 1 M, 125 ml and 250 ml glass shaker flasks with baffles, Infors HT shaking incubator; water bath; centrifuge (Beckman Coulter™).

[0217] Single colonies were selected from agar plates and inoculated overnight at 37°C in 3 ml of LB medium containing 100 µl carbenicillin / ml (stirring at 150 rpm). The next day, 250 µl of the overnight culture was transferred to 25 ml of TB medium containing 100 µl carbenicillin / ml and grown at 37°C with shaking at 150 rpm until the OD600 reached 0.6–0.9. IPTG was then added to a final concentration of 1 mM, and the flask was incubated overnight at 27°C with shaking at 150 rpm. The next day, the culture pellet (3000 g, 5 min) was resuspended in 25 ml of PBS. The cell suspension was heated to 48°C for 2 h, then centrifuged at 8000 g for 10 min. The supernatant was transferred to new tubes and stored at -20°C until purification.

[0218] Protein purification The materials and equipment used are as follows: ÄKTA Pure P150 system (Cytiva™); HiTrap MabSelectPrismA column (Cytiva™); NanoDrop One (Thermo Fisher™); NuPAGE™ 4-12%, Bis-Tris miniature protein gel (Thermo Fisher™); ACQUITY RDa detector (Waters™); BioRessolve column (Waters™).

[0219] The supernatant from protein expression was filtered (0.45 µm) and loaded onto a HiTrap MabSelect PrismA column using an ÄKTA Pure P150 system. Samples were washed with PBS and 50 mM sodium acetate (pH 6), followed by elution with 50 mM sodium acetate at pH 3.5. Protein concentration was determined using NanoDrop One, and purity was assessed on NuPAGE™ SDS-PAGE gels. Protein mass was determined by liquid chromatography-mass spectrometry using a BioRessolve column on an ACQUITY RDa detector system.

[0220] Biacore combined analysis The materials and equipment used are as follows: Biacore™ 8K+ instrument (Cytiva™); CM5 sensor chip (Cytiva™); Biacore™ amine conjugation kit (Cytiva™); fusion protein candidate as described above; green fluorescent protein (GFP, Merck™); Gammanorm (Octapharma™); trastuzumab (produced and purified from an internal production cell line (CHO-K1 background)).

[0221] All purified candidates were diluted to a concentration of 25 µg / ml in 10 mM sodium acetate buffer at pH 5. Immobilization was performed on the CM5 sensor chip using the standard immobilization method in the Biacore™ Amine Coupling Kit and Biacore software. The candidates were immobilized in flow cell 2 (Fc2), and flow cell 1 (Fc1) was activated / deactivated and used as a reference surface.

[0222] The following studies investigated the interactions between the candidate antibodies and trastuzumab, gammanorm, and GFP, respectively: One injection of 1µM Gammanorm and one injection of 1µM trastuzumab, contact time 240s, dissociation time 240s, flow rate 10µl / min.

[0223] Three concentrations of GFP were studied: 10 nM, 25 nM, and 100 nM. Association time was 600 s, dissociation time was 600 s, and flow rate was 10 µl / min.

[0224] After each cycle, the surface was regenerated with two injections (30 s, 30 µl / min) of 10 mM glycine-HCl pH 1.5. All generated sensor maps were subtracted from the reference, and the final response was provided as the signal difference between the baseline before injection and the end of sample injection.

[0225] Biacore alkaline stability analysis Following the binding analysis (see above), the same CM5 chip with the immobilized peptide underwent repeated cycles of binding GFP followed by injection of 0.3M NaOH to assess alkaline stability.

[0226] Methods for alkaline stability (per cycle): Start with PBS-P+ (only before the first sample injection).

[0227] Sample injection 1: 50 nM GFP in cycles 1 and 2. Then, for each subsequent cycle, GFP is injected alternately with PBSP+ (without analyte).

[0228] Association time 300 s, dissociation time 30 s, 10 µl / min.

[0229] Sample injection: 2: 0.3M NaOH. Contact time: 600s, dissociation time: 0s.

[0230] Regeneration: Glycine at pH 1.5 was injected twice consecutively at a rate of 30 µl / min for 30 seconds.

[0231] The method was repeated for a total of 40 cycles. All generated sensor maps were subtracted from the reference, and the final response was provided as the signal difference between the baseline before injection and the end of sample injection.

[0232] result All candidates were successfully cloned and transformed into Top10 E. coli, expressed, and purified with the expected protein quantities (data not shown). The fixed levels on the chip ranged from 3000 to 4500 RU.

[0233] Figure 7A -B shows the Biacore binding response (RU) of pAM1050 to trastuzumab (1µM), gammanorm (1µM), and GFP (10, 25, and 100 nM, respectively). Figure 7A ), and the average value of all constructs ( Figure 7B All fusion proteins showed affinity for GFP, and for all candidates, interaction with IgG was near zero. For the reference, in the same experimental setup (fixed level: 3807 RU), a fusion protein containing the same GFP-binding VHH fusion with the SpA domain derivative (SEQ ID NO:9) (with maintained Fc interaction but no VH3 interaction) provided a trastuzumab (1 µM) response of 9533 RU and a Gammanorm (1 µM) response of 8707 RU. Therefore, the ability of the stable peptides to interact with both the Fc and VH3 portions was negligible for the candidates tested.

[0234] Basic stability varied slightly among the candidates tested. Table 8 reports the GFP response (unnormalized) for cycles 1, 2, and 20 for basic stability assessment.

[0235] Table 8: Response to GFP injection (50 nM) after repeated cycles of exposure to 0.3 M NaOH. Data indicate that several substitutions at the tested positions are tolerated and result in high basic stability of the fusion protein. After 20 cycles, all tested candidates retained more than 50% of the binding response recorded in the second cycle. pAM1178-80, containing different amino acids at positions 13 and 14, performed particularly well. The results for pAM1034 and pAM1037 suggest that R at positions 28 or 29 may be advantageous.

[0236] Example 4A: Fusion of a single-domain antibody with Z, without IgG interaction. Different single-domain antibodies were fused to stable peptides via peptide linkers at the C-terminus, and the following aspects were evaluated: (1) Affinity assessment of target molecule interactions, which was tested by injecting target molecules at various concentrations. (2) Basic stability, which was 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.

[0237] Single-domain antibodies are VHH variants targeting AAV9, green fluorescent protein (GFP), or epidermal growth factor receptor (EGFR), and are based on natural camelid VHH frames or modified frames to enhance the basic stability of the VHH variants themselves. The natural camelid frames used 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 Sci. 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.

[0238] The stable peptide is a SpA domain Z variant (“Z0127”) (SEQ ID NO:64) in which the binding to the Fc and VH3 portions of IgG has been eliminated.

[0239] The tested peptide candidates (fusion proteins) are summarized in Table 9.

[0240] Table 9 Candidate number VHH variant target VHH Frame Type Stable peptides Full-length protein sequence 4A-1 vh342 AAV9 Natural (EgA1) Z0127 SEQ ID NO:134 4A-2 vh343 AAV9 Natural (9G8) Z0127 SEQ ID NO:136 4A-3 vh118 AAV9 stable Z0127 SEQ ID NO:142 4A-4 vh166 EGFR Natural (9G8) Z0127 SEQ ID NO:131 4A-5 vh167 EGFR stable Z0127 SEQ ID NO:132 4A-6 vh25 GFP Modified natural (S-Nb3) Z0127 SEQ ID NO:112 4A-7 vh60 GFP Natural (EgA1) Z0127 SEQ ID NO:116 4A-8 vh113 GFP stable Z0127 SEQ ID NO:123 Materials and methods Generation of candidate peptides purified by immobilized metal affinity chromatography (IMAC) For each candidate, 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 incubated overnight at 37°C and 160 rpm.

[0241] 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 that, 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.

[0242] 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 at pH 7.5–8 using a linear gradient (0–100%) at 10 CV. Prior to further analysis, the eluted protein buffer was exchanged for phosphate-buffered saline (Medicago) at pH 7.4 using a gravity flow column pre-packed with Sephadex™ G-25 resin (Cytiva).

[0243] 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.

[0244] 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 the Biacore™ Amine Conjugation Kit (Cytiva) and analyzed using a Biacore™ 8K+ instrument (Cytiva). The analytes used were AAV9 (2E12 vp / mL), GFP (1 g / L), and EGFR (0.2 g / L).

[0245] 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.

[0246] In each run, the peptide was immobilized in FC2. Multiple sensor chips were used until all candidates were tested.

[0247] The 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, 2E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

[0248] Biacore™ method for GFP or EGFR binding assays: Run buffer: PBS-P+; Flow rate: 10 µl / min; Sample injection: 600 s / 10 min on both FC1 and FC2; Dissociation time: 600 s / 10 min; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle) immobilized with the GFP-binding peptide, the analyte (GFP) was injected as follows: run buffer, 10 nM, 25 nM, and 100 nM. For each channel (cycle) immobilized with the EGFR-binding peptide, the analyte (EGFR) was injected as follows: run buffer, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM.

[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] 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).

[0251] 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.

[0252] 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.

[0253] result All fusion proteins showed satisfactory binding to their respective targets.

[0254] Tables 10a-c summarize the results of the binding capacity and basic stability measurements.

[0255] Table 10a: AAV9 binding candidates Table 10b: EGFR-binding candidates Table 10c: GFP binding candidates Figure 5A -C indicates that AAV9-binding fusion protein ( Figure 5A ), GFP-binding fusion protein ( Figure 5B ) and EGFR-binding fusion protein ( Figure 5C The binding capacity was calculated by normalizing the response to the first cycle for incremental cycles including NaOH exposure (sample injection 2 above). As can be seen in these figures, fusion proteins (4A-3, 4A-5, 4A-8) with VHH affinity ligands based on a stable VHH framework exhibited improved basic stability compared to their corresponding fusion proteins containing VHHs with a native camelid VHH framework.

[0256] Example 4B: Fusion of a single-domain antibody with Z Different single-domain antibodies were fused at their C-terminus to a stable polypeptide in the form of a SpA domain Z variant (“Z0091”) (SEQ ID NO:9) capable of binding immunoglobulin Fc but not VH3. The following aspects were evaluated: (1) affinity assessment of target-molecule interactions as tested by high-concentration injection of the target molecule; (2) alkaline stability as tested by reduction in target binding after treatment with 0.1 M, 0.3, or 0.5 M NaOH for several delivery cycles.

[0257] Single-domain antibodies are VHH variants targeting AAV9, green fluorescent protein (GFP), or epidermal growth factor receptor (EGFR), and are based on either a native camelid VHH framework or a modified framework to enhance the basic stability of the VHH variant itself. The native camelid framework corresponds to the framework sequences of VHH “EgA1” or “9G8” described in Schmitz et al., respectively. For candidates binding the same target, the CDR region is identical.

[0258] The peptide candidates (fusion proteins) tested are listed in Table 11.

[0259] Table 11 Candidate number VHH variant target VHH Frame Type Stable peptides Full-length protein sequence 4B-1 vh97 AAV9 Synthetic, partially stable Z0091 SEQ ID NO:138 4B-2 vh118 AAV9 Synthetic, stable Z0091 SEQ ID NO:141 4B-3 vh166 EGFR Natural (9G8) Z0091 SEQ ID NO:129 4B-4 vh167 EGFR Synthetic, stable Z0091 SEQ ID NO:130 4B-5 vh60 GFP Natural (EgA1) Z0091 SEQ ID NO:115 4B-6 vh113 GFP Synthetic, stable Z0091 SEQ ID NO:122 Materials and methods Generation of candidate peptides purified from IgG Sepharose For each candidate, 5 µl of the 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.

[0260] 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.

[0261] Samples were purified using gravity flow on a PD-10 column (Cytiva™) packed with IgG Sepharose 6FF chromatographic 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 using 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)).

[0262] Following the manufacturer's instructions, the concentration of the IgG Sepharose 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™).

[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 (in-house prepared), trastuzumab-GFP (in-house prepared), and EGFR (SinoBiological, 10001-H08H), with Fc from the mAb (in-house prepared).

[0264] 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 ± 463 RU. Peptides were immobilized in FC2.

[0265] 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.

[0266] The Biacore™ method for GFP binding assays: Run buffer: PBS-P+; Flow rate: 10 µl / min; Blocking 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 (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.

[0267] 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).

[0268] 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.

[0269] 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).

[0270] 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.

[0271] Biacore method for GFP basic stability (per cycle): Run buffer: PBS-P+; Flow rate: 10 µl / min; Blocking injection: (Fc 2 µM from mAb) 90 s in both flow cells; Dissociation time blocking: 0 s; Sample injection 1 (trastuzumab-GFP 2 µM): 300 s in both flow cells; Dissociation time 1: 30 s; Sample injection 2 (0.3 M NaOH): 600 s in both flow cells; Dissociation time 2: 0 s; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s.

[0272] 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.

[0273] These loops are repeated 30 times to track the stability of the target response values.

[0274] 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.

[0275] result All fusion proteins showed satisfactory binding to their respective targets.

[0276] Tables 12a-c summarize the results of the binding capacity and basic stability measurements.

[0277] Table 12a: AAV9 binding candidates Table 12b: EGFR-binding candidates Table 12c: GFP binding candidates Figures 6a-c show the binding capacity obtained for the AAV9-binding fusion protein (Figure 6a), the GFP-binding fusion protein (Figure 6b), and the EGFR-binding fusion protein (Figure 6c), normalized to the first cycle (i.e., including the first cycle), for incremental cycles including NaOH exposure (sample injection 2). As can be seen in these figures, fusion proteins (4B-2, 4B-4, 4B-6) with VHH affinity ligands based on a stable VHH framework, relative to the corresponding fusion proteins containing a natural camelid VHH framework (4B-3, 4B-5) or a synthetic but not optimally stable framework (4B-1), show improved basic stability.

[0278] Example 5: IgG-binding fusion protein IgG-binding peptides (representing the first peptide moiety) were expressed and evaluated, either alone or fused with a C-terminal stable peptide. The IgG-binding peptide was protein L-domain B3 and the C-terminal fusion partner was a SpA-derived peptide (Z0127) (SEQ ID NO: 64), wherein binding to the Fc and VH3 moieties of IgG was deactivated. The following constructs were tested: Table 13 protein Stable peptides Full-length protein sequence pL1 - SEQ ID NO:146 pL1-Z0127 Z0127 SEQ ID NO:147 The effect of stable fusion couplers on basic stability was assessed by measuring the binding after repeated exposure to NaOH 0.3 M.

[0279] Materials and methods Generation of candidate peptides purified by immobilized metal affinity chromatography (IMAC) For each variant, 1 µl of the glycerol stock solution as described in Example 1B was inoculated into 3 ml of LB medium supplemented with 100 µg / ml carbenicillin in a 14 ml round-bottom tube and incubated overnight at 37°C and 180 rpm.

[0280] Prepare protein expression medium (TB medium supplemented with 100 µg / ml carbenicillin and 2 mM MgCl2) and add it to pre-filled 100 ml baffled glass shake flasks (25 ml / flask). Inoculate each flask with 250 µl of the previous overnight culture to obtain an initial OD600 of approximately 0.05. Incubate the flasks in an Inforrs HT shake incubator at 37°C and 150 rpm for approximately 3.5 h until the OD600 reaches 1.0. Then, add 25 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 150 rpm for 18 h, after which transfer the culture to 50 ml polypropylene centrifuge tubes. To generate crude variant lysates, incubate the centrifuge tubes in a 48°C water bath for 2 h, followed by centrifugation at 8000 g for 10 min to precipitate cell debris and filtering the supernatant through a 0.22 µm filter.

[0281] The sample was purified using a HisTrap™ FF 1 mL column (Cytiva). The clarified lysate was loaded onto a column equilibrated with 50 mM sodium phosphate at pH 7.5 and 500 mM NaCl, allowing a residence time of approximately 2 minutes. The column was then washed with 15 column volumes (CV) of 50 mM sodium phosphate at pH 7.5 and 500 mM NaCl. Protein was eluted using a linear gradient (0–100%) of 0.5 M imidazole at pH 7.5–8 over a range of 10 CVs. Prior to further analysis, the eluted protein buffer was exchanged for phosphate-buffered saline (Medicago) at pH 7.4 using a gravity flow column pre-packed with Sephadex™ G-25 resin (Cytiva).

[0282] Following the manufacturer's instructions, use NanoDrop (Thermo Scientific) to measure the concentration of the purified and buffer-exchanged sample. After purification, keep the sample in a freezer until Biacore analysis.

[0283] Biacore binding and alkaline stability The binding interaction of protein L with IgG was assessed by measuring binding to adalimumab (prepared internally) on the Biacore 8K system (Cytiva). The proteins to be tested were amine-conjugated to the S-series CM 5 sensor chip using the Biacore Amine Conjugation Kit (Cytiva). Each protein was diluted to 10 µg / ml in NaAc pH 4.5 and immobilized in FC2 using a standard immobilization protocol. FC1 was activated and inactivated and used as a reference for all runs.

[0284] The alkaline stability was assessed by performing 20 cycles of the following sequential steps: (1) Adalimumab 200 nM was bound at 10 µl / min for 180 s, followed by dissociation for 100 s.

[0285] (2) Wash with 0.3 M NaOH at 10 µl / min for 600 s.

[0286] (3) Regenerate with glycine at pH 1.5 at 30 µl / min for 30 s.

[0287] Use the report point "Late Combination" to record the combination and plot against the cycle number.

[0288] result Table 14 shows the fixed levels obtained for each protein.

[0289] Table 14 protein Molecular weight (g / mol) Fixed level (RU) Standardized fixed level (g / (mol*RU)) pL1-Z0127 15816 1795 8.8 pL1 9347 1133 8.3 When considering molecular weight, fixation results in similar protein levels on the surface, although slightly higher for fusion proteins.

[0290] Figure 8 The figure shows the binding of adalimumab to the surface after 20 cycles of 0.3M NaOH exposure. As can be seen in the figure, the fusion protein (triangle) initially produces a slightly higher binding signal than the individual pL1 (dot) due to the higher fixation level, and degrades at a slower rate after repeated alkali exposure.

[0291] Example 6: IgG-binding fusion protein This embodiment aims to express and evaluate IgG-binding peptides (each representing the first peptide moiety) alone or fused with a C-terminal stable peptide. The IgG-binding peptides are SpA domains A, B, C, D, and E, and the C-terminal fusion coupler is a SpA-derived peptide (Z0127, SEQ ID NO: 64) in which binding to the Fc and VH3 moieties of IgG has been eliminated. The effect of the stable fusion coupler was evaluated for: (1) soluble expression yield from E. coli cultures, and (2) basic stability assessed by measuring binding after repeated exposure to 0.3 M NaOH.

[0292] Materials and methods All experiments were conducted as detailed in Example 5.

[0293] For the basic stability test, each test protein was immobilized in a separate flow cell in the Biacore instrument, as described in Example 5, and its binding and basic stability were evaluated.

[0294] result It was found that fusion with a stable peptide improved the expression levels of Z0204 (domain E) and Z0205 (domain D). Similar expression levels were observed for Z0208 (domain C) and the corresponding fusions Z0208-Z0127. No expressed proteins were obtained for Z0206 (domain A) and Z0207 (domain B) individually, possibly due to issues with the expression vector. Because no reference was available, the fusion proteins Z0206-Z127 and Z0207-Z127 were not included in the alkaline stability assay.

[0295] Table 15 identifies the evaluated proteins.

[0296] Table 15 Table 16 shows the fixation levels obtained for each protein. When molecular weight is taken into account, fixation results in similar protein levels on the surface.

[0297] Table 16 protein Molecular weight (g / mol) Expression yield (mg / L) Fixed level (RU) Standardized fixed level (g / (mol*RU)) Z0204-Z127 14199 59 1336 10.6 Z0204 8522 15 655 13.0 Z0205-Z127 14911 61 1445 10.3 Z0205 9235 25 781 11.8 Z0208-Z127 14628 29 1609 9.1 Z0208 8952 26 987 9.0 Figure 9A -C shows the binding of adalimumab to each protein after 20 cycles, including 10 minutes of exposure to 0.3 M NaOH. In these figures, the SpA domain is referred to using the format "ZGE020X". Although fixed at similar or slightly lower levels, all fusion proteins consistently produced a higher binding response than their respective references, as in the case of domain C. Furthermore, for domain D ( Figure 9B ) and structural domain C ( Figure 9C Compared to their respective references (SpA domains without stable fusion partners), the fusion proteins experienced a less significant reduction in response between the first and second cycles. Overall, for the SpA domains tested, fusion with the stable peptide contributed to improved basic stability of the immobilized protein, especially within the first 15 cycles.

[0298] Example 7: HSA-binding fusion protein Single-chain variable fragments (scFvs) representing the first polypeptide moiety were expressed and evaluated, either alone or in combination with C-terminal stable polypeptides. scFvs exhibited affinity for human serum albumin (HSA), and the C-terminal fusion partner was a SpA-derived polypeptide (Z0127, SEQ ID NO:64), in which binding to the Fc and VH3 moieties of IgG was deprecated. Both constructs were expressed with a C-terminal (His)6 tag. The following constructs were tested: Table 17 protein Stable peptides Full-length protein sequence scFvHSA - SEQ ID NO:153 scFvHSA-Z127 Z0127 SEQ ID NO:154 The effect of stable fusion couples on basic stability was assessed by measuring binding after repeated exposure to 0.3 M NaOH.

[0299] Materials and methods All methods were performed as detailed in Example 5. In short, scFv was expressed with or without fusion with a stable peptide and immobilized in a flow cell for Biacore analysis. Binding and alkaline stability were assessed after 20 cycles. Two different NaOH concentrations, 0.01 M and 0.05 M, were used to facilitate comparison between constructs. Thus, each protein was immobilized on two surfaces, each using the same conditions.

[0300] result Table 18 shows the fixation levels obtained for each protein. Fixation resulted in similar levels when molecular weight (MW) was taken into account, although slightly lower for fusion proteins.

[0301] Table 18 Figure 10A-B shows results from alkaline stability assessments at 0.05 M NaOH and 0.01 M NaOH, respectively. The proteins exhibited the same initial binding signal, although the fusion protein was immobilized at slightly lower molar fixation levels. For the fusion protein, the decrease in response between the first and second cycles was less than that of the scFv reference, and at higher NaOH concentrations (0.05 M, ... Figure 10A Furthermore, the binding signal of the fusion protein decreased at a slower rate than that of the scFv alone. In 0.01 M NaOH ( Figure 10B At this level, the binding signal decreased and stabilized for both constructs, but the level was even lower for the construct without the stabilizing peptide. In summary, fusion with the stabilizing peptide improved the basic stability of anti-HSA scFv.

[0302] Example 8: Functional Evaluation This embodiment demonstrates, through basic experiments, the functionality of the peptide immobilized on the support in forming an affinity separation matrix. The peptide is a fusion of an AAV9-binding sdAb variant and a stable peptide (SEQ ID NO: 64).

[0303] Materials and methods The fusion protein (SEQ ID NO:155) was generated and purified according to the method described in Example 2A.

[0304] 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.

[0305] 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.

[0306] 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.

[0307] result Figure 11A The chromatograms obtained for purifying AAV9 TFF material using candidate vh118 as an affinity ligand are shown. Elution peaks are indicated by rectangles. Figure 11B Only the area of ​​the elution peak is displayed. Figure 12 Photograph of the gel is shown. VP1, VP2, and VP3 represent AAV9 virosomal proteins 1, 2, and 3, respectively.

[0308] The results showed that the existing 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.

[0309] The results of the alkaline stability assessment showed that Figure 13 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%.

[0310] Example 9: Evaluation of alternative combinations of stable second polypeptide moiety and target-binding first polypeptide moiety This embodiment demonstrates the efficiency of using several variants of the stable peptides Z127 (SEQ ID NO: 64), Z464 (SEQ ID NO: 103), and Z472 (SEQ ID NO: 110) in combination with different target-binding peptides. The test peptides were fusion proteins of different AAV9-binding sdAb variants vh503, vh498, vh459, and vh504 with the stable peptides Z464 and Z472.

[0311] Materials and methods The fusion proteins vh503-Z127 (SEQ ID NO:261), vh498-Z127 (SEQ ID NO:262), vh459-Z127 (SEQ ID NO:263), vh504-Z127 (SEQ ID NO:264), vh503-Z464 (SEQ ID NO:265), vh498-Z464 (SEQ ID NO:266), vh459-Z464 (SEQ ID NO:267), vh504-Z464 (SEQ ID NO:268), vh503-Z472 (SEQ ID NO:269), vh498-Z472 (SEQ ID NO:270), vh459-Z472 (SEQ ID NO:271), and vh504-Z472 (SEQ ID NO:272) were generated and purified according to the method described in Example 2A.

[0312] Biacore analysis of AAV9 binding affinity To assess binding with AAV9, the peptides described above were immobilized on a Biacore™ S-Series CM5 chip using an amine conjugation kit (Cytiva™) and analyzed using a Biacore™ 8K+ instrument (Cytiva™). Samples containing AAV9 viral particles (vp) at a concentration of 2E12 vp / ml were used as analytes.

[0313] Using the standard method in Biacore software, peptides were immobilized by coupling in flow cell 2 (FC2) and activation / inactivation in flow cell 1 (FC1). Peptides were diluted in Biacore™ acetate buffer pH 5.0 (Cytiva™) at concentrations ranging from 17–30 µg / ml, depending on their molecular weight. Immobilization levels varied between different peptide variants corresponding to their molecular weights. In each run, the peptide was immobilized in FC2.

[0314] The Biacore™ method for binding assays: Run buffer: PBS-P+; Flow rate: 5 µl / min; Sample injection: 2400 s / 40 min on both FC1 and FC2; Dissociation time: 2400 s / 40 min; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle), the analyte (AAV9) was injected as follows: run buffer, AAV9 2E11 vp / ml, 5E11 vp / ml, 2E12 vp / ml.

[0315] 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.

[0316] Biacore analysis of alkaline stability After evaluating AAV9 binding (see above), the same chip underwent repeated cycles of AAV9 binding (5E11 vp / ml) followed by NaOH (0.5 M) injection to assess alkaline stability.

[0317] 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.

[0318] 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.

[0319] result All constructs showed good binding capacity for AAV9 (data not shown). Regarding basic stability, the sdAbs showed small variations but generally similar basic stability for all three stable peptides (fusion couplers). Results for each sdAb are shown in... Figures 14A-14D Among them, 14A shows the results of vh503 with different fusion partners Z127, Z472 and Z472, 14B shows the results of vh498, 14C shows the results of vh459, and 14D shows the results of vh504.

[0320] Example 10: 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.

[0321] 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: 64 (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: 64 (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. (MABS VOL.8, NO. 7, 1336–1346 (2016)), and the modified constructs were designed by fusing with the polypeptide moiety according to SEQ ID NO: 64, and in one construct, the framework was modified according to the framework of the vh118 variant described herein.

[0322] As described in the previous examples, the construct was expressed in E. coli and purified on IMAC or PrismA.

[0323] The first Biacore experiment was performed on an SA-chip with biotinylation affinity to compare ScFvHSA with and without peptide moiety fusion. Binding was measured using HAS concentrations of 0, 10, 50, 100, 500, and 1000 nM, and alkaline stability was tested using 1000 nM HAS under CIP conditions for 10 min and 0.1 M NaOH. Alkaline stability was improved by peptide moiety fusion according to SEQ ID NO: 64 compared to native HAS, and stability can be further improved through framework mutations, such as... Figure 15 As shown in the image.

[0324] Similar experiments were performed on a CM5-chip with NHS conjugation. Biacore evaluation of the binding of purified affinity ligands (i) and (ii) was performed on the CM5-chip using HAS concentrations of 0, 50, 100, 500, and 1000 nM. Biacore evaluation of basic stability was performed using 1000 nM HAS 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: 64, and further improved by framework mutation (data not shown).

[0325] 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 showed improved stability for scFvHSA-Z, and even more for scFvHSAStab1-Z, indicating that the stable VHH framework further improves stability. The basic stability results from the SA-chip experiments are shown in Figure 14, which presents a normalized plot of the basic stability experiments. For the second Biacore experiment, a CM5-chip with NHS conjugation was used, and it was shown that the construct with the peptide motif according to SEQ ID NO: 64 had good conjugation efficiency compared to native scFvHSA alone (data not shown).

[0326] Example 11: Basic stability of the triple helix domain as the first polypeptide moiety Materials and methods In this embodiment, the stability of the triple helix domain as the first polypeptide moiety is evaluated using a stable polypeptide (second polypeptide portion) of the present disclosure. A first construct Z-0799-His (SEQ ID NO: 273) of a his-tagged triple helix Z0799 and a second construct Z0799-Z127-His (SEQ ID NO: 273) of a his-tagged triple helix Z0799 were generated, expressed, and purified according to the method described in Example 10 above.

[0327] Biacore analysis of binding affinity for polyclonal IgA To assess binding with polyhIgA, the peptides described above were immobilized on a Biacore™ S-Series CM5 chip using an amine coupling kit (Cytiva™) and analyzed using a Biacore™ 8K+ instrument (Cytiva™). Samples containing polyhIgA were used as analytes.

[0328] Using the standard method in Biacore software, peptides were immobilized by coupling in flow cell 2 (FC2) and activation / inactivation in flow cell 1 (FC1). Peptides were diluted in Biacore™ acetate buffer pH 5.0 (Cytiva™) at concentrations ranging from 17–30 µg / ml, depending on their molecular weight. Immobilization levels varied between different peptide variants corresponding to their molecular weights. In each run, the peptide was immobilized in FC2.

[0329] The Biacore™ method for binding analysis: Run buffer: PBS-P+; Flow rate: 10 µl / min; Sample injection: 300 s on both FC1 and FC2; Dissociation time: 300 s; Regeneration: 10 mM glycine-HCl pH 1.5, 30 µl / min, 2 x 30 s. For each channel (cycle), the analyte (polyhIgA) is injected as follows: run buffer, polyhIgA 0, 10, 50, 250, 1000 nM.

[0330] 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.

[0331] Biacore analysis of alkaline stability After evaluating the binding of polyhIgA (see above), the same chip underwent repeated cycles of binding polyhIgA (1000 nM) followed by injection of NaOH (0.5 M) to assess alkaline stability.

[0332] Biacore method for alkaline stability (per cycle): Run buffer: PBS-P+; flow rate: 10 µl / min; Sample injection 1 (polyhIgA (1000 nM)): 300 s in both flow cells; dissociation time 1: 300 s; Sample injection 2 (0.5 M NaOH): 10 min 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 polyhIgA response.

[0333] 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.

[0334] result The results showed that the construct containing the stable second peptide fusion moiety (SEQ ID NO: 274) exhibited better binding properties compared to the construct lacking the moiety (SEQ ID NO: 273). Binding sensor maps of the constructs were shown... Figure 16A middle.

[0335] Regarding basic stability, the results showed that the construct without the stable second peptide moiety retained 40% of its binding capacity after 7 cycles, while the construct containing the second peptide fusion moiety retained 40% of its initial binding capacity after 12 cycles. Figure 16B As shown in the figure. Therefore, stability is improved by fusing the triple helix polypeptide Z0799 with the stabilizing polypeptide Z127.

[0336] List of Implementation Plans 1. A fusion protein comprising at least one first polypeptide moiety and at least one second polypeptide moiety, wherein The first polypeptide portion is a single-chain polypeptide capable of binding to the target entity, and The second polypeptide moiety comprises a single-chain containing an α-helix domain, wherein the second polypeptide moiety has no binding affinity for the target entity. Furthermore, the presence of the second polypeptide portion optionally improves at least one property of the fusion protein compared to the properties of the first polypeptide portion alone, wherein the improved property is selected from: basic stability, recombinant protein expression, and vector conjugation.

[0337] 2. The fusion protein according to Project 1, wherein the improved basic stability is manifested as the fusion protein as a whole having increased basic stability compared to the basic stability of the first polypeptide moiety alone.

[0338] 3. The fusion protein according to item 1 or 2, wherein the first polypeptide portion is a polypeptide domain.

[0339] 4. The fusion protein according to any one of items 1-3, wherein the first polypeptide portion is not an α-helical bundle domain.

[0340] 5. The fusion protein according to any one of items 1-4, wherein the first polypeptide moiety has a secondary structure comprising a β-sheet.

[0341] 6. The fusion protein according to any one of items 1-5, wherein the first polypeptide moiety is a natural or synthetic antibody domain or antibody fragment or a modified variant thereof.

[0342] 7. The fusion protein according to Item 6, wherein the first polypeptide portion comprises an antibody heavy chain variable domain.

[0343] 8. The fusion protein according to item 6 or 7, wherein the first polypeptide portion lacks the antibody heavy chain constant domain.

[0344] 9. The fusion protein according to any one of items 1-8, wherein the first polypeptide portion is selected from the variable domain of an immunoglobulin heavy chain, a single-domain antibody (sdAb), a variable domain of the heavy chain of a heavy chain antibody (VHH), VNAR, a single-chain variable fragment of immunoglobulin (scFv), and a synthetic variant of an antibody heavy chain variable domain wherein the native VH / VL interaction site has been modified to mimic the naturally occurring sdAb.

[0345] 10 .The fusion protein according to any one of items 1-8, wherein the single-chain polypeptide is a single-chain variable fragment or a single-domain antibody or a variant thereof.

[0346] 11. The fusion protein according to any one of items 1-10, wherein the single-chain polypeptide comprises complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2) and complementarity-determining region 3 (CDR3).

[0347] 12. The fusion protein according to any one of items 1-3, wherein the first polypeptide moiety has a secondary structure comprising at least one β-sheet and at least one α-helix.

[0348] 13. The fusion protein according to Item 12, wherein the first polypeptide portion is or is derived from a protein domain of protein L or protein G.

[0349] 14. The fusion protein according to any one of items 1-3, wherein the first polypeptide portion comprises an α-helical bundle domain.

[0350] 15. The fusion protein according to item 14, wherein the first polypeptide portion comprises a polypeptide as or derived from a protein domain of staphylococcal protein A (SpA).

[0351] 16. The fusion protein according to item 15, wherein the first polypeptide portion comprises an amino acid sequence selected from SEQ ID NO:1-7 and an amino acid sequence having at least 80% identity with any one of SEQ ID NOs:1-7.

[0352] 17. The fusion protein according to Item 14, wherein the first polypeptide portion comprises an albumin-binding domain (ABD).

[0353] 18. The fusion protein according to any one of items 1-17, wherein the first polypeptide moiety alone has lower basic stability than the second polypeptide moiety alone.

[0354] 19. The fusion protein according to any one of items 1-18, wherein after exposure to 0.5 M NaOH for up to 20 hours, for example after 10 hours, or after up to 120 repeated NaOH exposure cycles, for example after 60 cycles, the first polypeptide moiety alone retains 75% or less, for example 50% or less, of its target binding capacity, wherein the NaOH exposure cycle includes contact with 0.5 M NaOH for 10 minutes.

[0355] 20. The fusion protein according to any one of items 1-19, wherein the first polypeptide moiety has binding affinity to a biological entity.

[0356] 21. The fusion protein according to Item 20, wherein the biological entity is a viral particle or viral vector, such as adenovirus, retrovirus (gamma retrovirus and lentivirus), poxvirus, adeno-associated virus (AAV), baculovirus or herpes simplex virus.

[0357] 22. The fusion protein according to Item 21, wherein the biological entity is an adeno-associated virus (AAV), such as AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 (AAVrh10), 11 or 12.

[0358] 23. The fusion protein according to Item 20, wherein the biological entity is an antibody, such as a monoclonal antibody or an antibody fragment.

[0359] 24. The fusion protein according to Item 20, wherein the biological entity is an exosome or a lipid nanoparticle.

[0360] 25. The fusion protein according to any one of items 1-24, wherein the second polypeptide portion is a polypeptide domain.

[0361] 26. The fusion protein according to any one of items 1-25, wherein the second polypeptide portion contains an α-helical domain.

[0362] 27. The fusion protein according to any one of items 1-26, wherein the second polypeptide portion comprises at least one α-helix, for example at least two α-helices, such as three, four, five or six α-helices.

[0363] 28. The fusion protein according to item 27, wherein the second polypeptide portion comprises up to 10 α-helices.

[0364] 29. The fusion protein according to any one of items 1-28, wherein the second polypeptide portion is an α-helical bundle domain.

[0365] 30. The fusion protein according to any one of items 1-29, wherein the second polypeptide comprises a triple-helix bundle domain.

[0366] 31. The fusion protein according to any one of items 1-30, wherein the second polypeptide is a single-domain polypeptide.

[0367] 32. The fusion protein according to any one of items 1-31, wherein the second polypeptide is or is derived from a protein domain of SpA.

[0368] 33. The fusion protein according to item 32, wherein the second polypeptide portion comprises an amino acid sequence selected from SEQ ID NO:7-9, 28-30 and 61-110 and having at least 80% identity with it, preferably at least 85% or at least 90% identity with it.

[0369] 34. The fusion protein according to item 33, wherein the second polypeptide portion comprises an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID NO: 8 or 64.

[0370] 35. The fusion protein according to any one of items 1-34, wherein the second polypeptide portion is capable of binding the Fc domain of immunoglobulin G (IgG) and / or capable of binding the VH3 domain of immunoglobulin G (IgG).

[0371] 36. The fusion protein according to any one of items 32-34, wherein the second polypeptide moiety has a lower binding affinity to the Fc region of trastuzumab than that of SEQ ID NO:8 to the same Fc region.

[0372] 37. The fusion protein according to any one of items 32-34, wherein the second polypeptide moiety has a lower binding affinity to the VH3 region of trastuzumab than that of SEQ ID NO:6 to the same VH3 region.

[0373] 38. The fusion protein according to claims 36 and 37, wherein the second polypeptide moiety has a lower binding affinity to the Fc region of trastuzumab than SEQ ID NO:8 for the same Fc region, and a lower binding affinity to the VH3 region of trastuzumab than SEQ ID NO:6 for the same VH3 region.

[0374] 39. The fusion protein according to item 38, wherein the second polypeptide portion comprises, at position X corresponding to SEQ ID NO:64 33 The amino acid at position X is selected from T, S, G, Q, A, E, H, and R; at position X corresponding to SEQ ID NO:64 40 The amino acids at the position are selected from E, G, R, D, K, Q, H, and S; and at the position corresponding to X 51 The amino acid at position X is selected from the amino acid sequences of L, V, S, I, R, and G; the condition is that when X... 51 When it is L, then X 33 X 40 Selected from AD, HK, EG, ER, GR, AK, AR, PK, RR, and KK, and when X 51 When it is G, then X 33 X40 It's TK.

[0375] 40. The fusion protein according to any one of items 32-39, wherein the second polypeptide moiety comprises an amino acid sequence of a protein domain derived from SpA, and wherein independently, The amino acid residue at position 9 corresponding to SEQ ID NO: 64 is selected from L, E, R, A and Y, and is preferably A; The amino acid residue at position 10 corresponding to SEQ ID NO: 64 is selected from L, E, R, A and Y, and is preferably Y; The amino acid residue at position 11 corresponding to SEQ ID NO: 64 is selected from L, E, R, A and Y, and is preferably R; The amino acid residue at position 13 corresponding to SEQ ID NO: 64 is selected from L, E, R, A and Q, and is preferably L, R, A or Q; The amino acid residue at position 14 corresponding to SEQ ID NO: 64 is selected from L, E, R, A, Q and K, and is preferably A or R; The amino acid residue at position 17 corresponding to SEQ ID NO: 64 is selected from L, E, R and A, and is preferably A; The amino acid residue at position 18 corresponding to SEQ ID NO: 64 is selected from L, E, R and A, and is preferably R; The amino acid residue at position 28 corresponding to SEQ ID NO: 64 is selected from L, E, R and A, and is preferably R or A; The amino acid residue at position 29 corresponding to SEQ ID NO: 64 is selected from L, E, R and A, and is preferably R or A; The amino acid residue at position 33 corresponding to SEQ ID NO: 64 is selected from T, S, G, Q, A, E, H and R, and is preferably S; The amino acid residue at position 40 corresponding to SEQ ID NO: 64 is selected from E, G, R, D, K, Q, H, and S, and is preferably G; and The amino acid residue at position 51 corresponding to SEQ ID NO: 64 is selected from L, V, S, I, R and G, and is preferably V.

[0376] 41. The fusion protein according to any one of items 32-39, wherein the second polypeptide portion comprises an amino acid sequence according to the following: VDAKFDKEX9X10 X 11 AX 13 X 14 EIX 17 X 18 LPNLTEEQRX 28 X 29 FIQX 33 LKDDPSX 40 SKAILAEAKKX 51 NDAQAPK (SEQ ID NO: 235) Independently, X9 is selected from L, E, R, A and Y, and is preferably A; X 10 The choice is L, E, R, A, and Y, with Y being the most preferred. X 11 The choice is L, E, R, A, and Y, with R being the most preferred. X 13 The choice is L, E, R, A, and Q, and preferably L, R, A, or Q; X 14 The choice is L, E, R, A, Q, and K, with A or R being preferred; X 17 It is selected from L, E, R and A, and preferably A; X 18 It is selected from L, E, R and A, and preferably R; X 28 It is selected from L, E, R and A, and preferably R or A; X 29 It is selected from L, E, R and A, and preferably R or A; X 33 The components are selected from T, S, G, Q, A, E, H, and R, with S being the most preferred. X 40 The selection is made from E, G, R, D, K, Q, H, and S, with G being the most preferred. X 51 It is selected from L, V, S, I, R and G, and preferably V.

[0377] 42. The fusion protein according to item 40 or 41, wherein the second polypeptide portion comprises, or is composed of, an amino acid sequence selected from, or preferably composed of, SEQ ID NO: 7-110 or SEQ ID NO: 64-110.

[0378] 43. The fusion protein according to any one of items 1-42, wherein at least one second polypeptide portion is located at the C-terminus of the first polypeptide.

[0379] 44. The fusion protein according to any one of items 1-43, wherein at least one second polypeptide, including any additional amino acids, is located at the C-terminus of the fusion protein.

[0380] 45. The fusion protein according to any one of items 1-44, wherein the second polypeptide is not located at the N-terminus of the fusion protein.

[0381] 46. ​​The fusion protein according to any one of items 1-45, comprising at least two of the first polypeptide moieties.

[0382] 47. The fusion protein according to item 46 comprises at least 3, for example, at most 6, for example, at most 10 of the first polypeptide moieties.

[0383] 48. The fusion protein according to item 47, wherein the first polypeptide portion has the same amino acid sequence.

[0384] 49. The fusion protein according to item 47, wherein the first polypeptide portion has an amino acid sequence that is distinct from each other.

[0385] 50. The fusion protein according to item 47, wherein the first polypeptide portion is capable of binding to different target entities.

[0386] 51. The fusion protein according to any one of items 1-50, comprising at least two of the second polypeptide moieties, wherein at least one is located at the C-terminus of the fusion protein.

[0387] 52. The fusion protein according to item 51 comprises at least three, for example, up to ten, the second polypeptide motif.

[0388] 53. The fusion protein according to item 51 or 52, wherein the at least two second polypeptide portions are sequentially arranged at the C-terminus of the fusion protein.

[0389] 54. The fusion protein according to any one of items 51-53, wherein the second polypeptide portion has the same amino acid sequence.

[0390] 55. The fusion protein according to any one of items 51-53, wherein the second polypeptide portion has an amino acid sequence that is different from each other.

[0391] 56. The fusion protein according to any one of items 1-55, comprising a further amino acid sequence, optionally selected from: a leader peptide, a signal peptide, an affinity tag, a coupling peptide, a linker peptide, and a spacer peptide.

[0392] 57. The fusion protein according to item 56, wherein the further amino acid sequence is located at the N-terminus or C-terminus of the fusion protein.

[0393] 58. The fusion protein according to item 56, wherein the further amino acid sequence is a leader peptide or signal peptide, such as PelB or OmpA signal peptide.

[0394] 59. The fusion protein according to item 56, wherein the further amino acid sequence comprises a plurality of histidines, such as the (His)6 sequence.

[0395] 60. The fusion protein according to any one of items 1-11 and 18-59, wherein the first polypeptide comprises a framework region (FWR) and a complementarity-determining region in the following order from the N-terminus to the C-terminus: [FWR1]-[CDR1]-[FWR2]-[CDR2]-[FWR3]-[CDR3]-[FWR4].

[0396] 61. The fusion protein according to item 60, wherein the first polypeptide comprises a plurality of frame regions, and each of the frame regions has an amino acid sequence having at least 70%, for example at least 75%, for example at least 80%, for example at least 85% sequence identity with the corresponding frame region of SEQ ID NO: 127, 133 or 137.

[0397] 62. The fusion protein according to any one of items 1-61, wherein basic stability is measured as the ability of the first polypeptide moiety to maintain target binding capacity after exposure to basic conditions.

[0398] 63. The fusion protein according to item 62, wherein exposure to alkaline conditions is a period of repeated exposure to a solution of at least 0.1 M NaOH, for example 0.5 M NaOH, for 10 minutes.

[0399] 64. The fusion protein according to item 62 or 63, wherein the target binding capacity is evaluated after repeated exposure cycles, for example after at least 10 cycles.

[0400] 65. An isolated nucleic acid sequence encoding a fusion protein according to any one of items 1-64.

[0401] 66. An expression vector comprising the nucleic acid sequence described in item 65.

[0402] 67. A recombinant host cell for producing a fusion protein according to any one of items 1-64, comprising the expression vector according to item 66.

[0403] 68. A method for producing a fusion protein according to any one of items 1-64, comprising: i. Provide recombinant host cells as described in item 67; ii. Culture the host cells under conditions that enable the expression of the fusion protein; and iii. Isolate the fusion protein.

[0404] 69. The method according to item 68, wherein the cell is a prokaryotic cell, such as an Escherichia coli cell.

[0405] 70. The method according to item 68, wherein the cell is a eukaryotic cell, such as a mammalian cell, insect cell, or yeast cell.

[0406] 71. The method according to item 70, wherein the mammalian cells are Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells.

[0407] 72. The method according to item 70, wherein the yeast cell is Pichia pastoris or Saccharomyces cerevisiae.

[0408] 73. The method according to any one of items 68-72, wherein the fusion protein comprises a histidine tag, and step iii comprises purifying the fusion protein by immobilized metal affinity chromatography (IMAC).

[0409] 74. The method according to any one of items 68-72, wherein step iii includes purifying the fusion protein by affinity chromatography.

[0410] 75. The method of claim 74, wherein the affinity chromatography uses an affinity ligand that binds to the framework region of the first polypeptide moiety.

[0411] 76. The method of claim 75, wherein the affinity chromatography uses an affinity ligand based on a SpA protein domain, wherein the affinity ligand binds to the VH region of the first polypeptide moiety, such as VH3.

[0412] 77. The method according to item 74, wherein the affinity chromatography uses an affinity ligand that binds to the second polypeptide portion of the fusion protein.

[0413] 78. The method of claim 77, wherein the affinity ligand is bound to an affinity tag included in the amino acid sequence of the second polypeptide moiety or an affinity tag incorporated into the amino acid sequence of the second polypeptide moiety.

[0414] 79. Use of the fusion protein according to any one of items 1-64 for in vitro capture of the target entity.

[0415] 80. For the purpose described in item 79, for detecting the target entity within a sample.

[0416] 81. As described in item 79, for separating the target entity from other components of a sample.

[0417] 82. For analytical separation of target entities, as described in item 81.

[0418] 83. For the preparative purification of target entities, as described in item 81.

[0419] 84. An adsorbent material comprising a fusion protein according to any one of items 1-64 coupled to a carrier.

[0420] 85. The adsorbent material according to item 84, wherein the fusion protein is coupled to the carrier via the second polypeptide.

[0421] 86. The adsorbent material according to item 84 or 85, wherein the carrier is a surface.

[0422] 87. The adsorbent material according to any one of items 84-86, wherein the carrier is selected from chips, plates, pores, sheets, fibers, particles and beads.

[0423] 88. The adsorbent material according to any one of items 84-87, wherein the carrier comprises a polymer material.

[0424] 89. The adsorbent material according to any one of items 84-88, wherein the carrier comprises a polysaccharide-based material, such as agar, agarose, or agarose derivatives.

[0425] 90. The adsorbent material according to item 89, wherein the carrier comprises cross-linked agarose.

[0426] 91. The adsorbent material according to any one of items 84-88, wherein the carrier is selected from fiber matrix, membrane, filter and bulk material.

[0427] 92. The adsorbent material according to item 91, wherein the carrier material is a fibrous material.

[0428] 93. The adsorbent material according to item 92, wherein the carrier material is a fibrous matrix, such as a nonwoven fibrous matrix.

[0429] 94. The adsorbent material according to any one of items 84-93, wherein the carrier is a chromatographic matrix.

[0430] 95. Use of the adsorbent material according to any one of items 84-94 for binding a target entity.

[0431] 96. For the purpose described in item 95, for detecting the target entity within a sample.

[0432] 97. As described in item 95, for separating the target entity from other components of a sample.

[0433] 98. For analytical separation of target entities, as described in item 97.

[0434] 100. For the preparative purification of target entities, as described in item 97.

[0435] 101. Use of an α-helical polypeptide domain to improve the in vitro basic stability of an antigen-binding polypeptide, wherein the α-helical polypeptide is fused to the C-terminus of the antigen-binding polypeptide.

[0436] 102. The use as described in item 101, wherein the α-helical polypeptide domain is a second polypeptide moiety as defined in any of items 1-64.

[0437] 103. The use as described in item 101, wherein the antigen-binding polypeptide is a first polypeptide portion as defined in any of items 1-64.

[0438] 104. The use according to any one of items 101-103, wherein after exposure to 0.5 M NaOH for up to 20 hours, for example after 10 hours, or after up to 120 repeated NaOH exposure cycles, for example after 60 cycles, the antigen-binding polypeptide alone retains 75% or less, for example 50% or less, of its target binding capacity, wherein the NaOH exposure cycle includes contact with 0.5 M NaOH for 10 minutes.

[0439] 105. A separation method, comprising the following steps: (a) Providing an adsorbent material according to any one of items 84-94, wherein the fusion protein has a binding capacity for the target entity. (b) Contacting the adsorbent material with a liquid sample containing the target entity, under conditions that allow the target entity to bind to the first polypeptide of the fusion protein. (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.

[0440] 106. The method of using a cleaning liquid according to the project, wherein the cleaning liquid is alkaline and preferably contains 0.05-0.5 M NaOH or KOH.

[0441] 107. The method according to item 105 or 106, wherein steps (a)-(e) are repeated at least 10 times, for example at least 20 times.

[0442] 108. The method according to item 107, wherein after 10 cycles of contact with an alkaline cleaning liquid, the 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.

[0443] 109. The method according to item 107, wherein after 20 cycles of contact with an alkaline cleaning liquid, the 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.

[0444] 110. The method according to item 107, wherein after 10 cycles of contact with an alkaline cleaning liquid, the 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.

[0445] 111. The method according to Item 107, wherein after 20 cycles of contact with an alkaline cleaning liquid, the 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.

[0446] Table 19: Exemplary amino acid sequences References WO2003080655A1 WO 2016 / 079033A1 WO2022 / 013272 WO 2023 / 046886 PCT / EP23 / 056401 S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964) US6602990 US7396467 WO2019 / 137869 WO2018 / 011600 Schmitz et al, Structure 21 , 1214-1224 (2013) Fleetwood et al., Cell.Mol. Life Sci. 70:1081-1093 (2013) Adams R. Et al, MABS VOL. 8, NO. 7, 1336–1346 (2016)。

Claims

1. A fusion protein comprising at least one first polypeptide moiety and at least one second polypeptide moiety, wherein the first polypeptide moiety is a single-chain polypeptide capable of binding to a target entity, and the second polypeptide moiety comprises a single-chain α-helical domain, wherein the second polypeptide moiety has no binding affinity to the target entity.

2. The fusion protein of claim 1, wherein the presence of the second polypeptide portion improves at least one property of the fusion protein compared to the properties of the first polypeptide portion alone, wherein the improved property is selected from: basic stability, recombinant protein expression, and vector conjugation.

3. The fusion protein of claim 2, wherein the improved basic stability is manifested in the fusion protein as a whole having increased basic stability compared to the basic stability of the first polypeptide moiety alone.

4. The fusion protein according to any one of claims 1-3, wherein the second polypeptide portion is a polypeptide domain.

5. The fusion protein according to any one of claims 1-4, wherein the second polypeptide portion contains an α-helix domain.

6. The fusion protein according to any one of claims 1-5, wherein the second polypeptide portion comprises at least one α-helix, for example at least two α-helices, such as three, four, five or six α-helices.

7. The fusion protein according to any one of claims 1-6, wherein the second polypeptide portion is an α-helical bundle domain.

8. The fusion protein according to any one of claims 1-7, wherein the second polypeptide comprises a triple-helix bundle domain.

9. The fusion protein according to any one of claims 1-8, wherein the second polypeptide is a single-domain polypeptide.

10. The fusion protein according to any one of claims 1-9, wherein the second polypeptide is or is derived from a protein domain of SpA.

11. The fusion protein according to any one of claims 1-10, wherein the second polypeptide portion (i) Has a lower binding affinity for the Fc region of trastuzumab compared to the binding affinity for the same Fc region of SEQ ID NO:8, and / or (ii) It has a lower binding affinity for the VH3 region of trastuzumab compared to the binding of SEQ ID NO:6 to the same VH3 region.

12. The fusion protein according to any one of claims 1-11, wherein the second polypeptide portion is derived from the protein domain of SpA of the Fc and / or VH3 regions of naturally occurring immunoglobulins, and the amino acid sequence of the second polypeptide has been modified to eliminate binding to both the Fc and VH3 regions, such that it has a lower binding affinity to the Fc region of trastuzumab compared to the binding of SEQ ID NO:8 to the same Fc region, and a lower binding affinity to the VH3 region of trastuzumab compared to the binding of SEQ ID NO:6 to the same VH3 region.

13. The fusion protein of claim 11, wherein the second polypeptide moiety having a lower affinity for the Fc region of trastuzumab comprises amino acids selected from SEQ ID NO: 64-110, 31-40 and 59-63.

14. The fusion protein of claim 11, wherein the second polypeptide moiety having a lower affinity for the VH3 region of trastuzumab comprises an amino acid sequence selected from SEQ ID NO: 64-110 and 9-30.

15. The fusion protein of claim 12, wherein the second polypeptide moiety having a lower binding affinity for the Fc region of trastuzumab comprises an amino acid sequence selected from SEQ ID NO: 64-110.

16. The fusion protein of claim 11, wherein the second polypeptide moiety having a lower binding affinity for the Fc region of trastuzumab comprises an amino acid sequence, wherein at position X corresponding to SEQ ID NO:64 33 The amino acid at position X is selected from T, S, G, Q, A, E, H, and R; at position X corresponding to SEQ ID NO:64 40 The amino acid at the position is selected from E, G, R, D, K, Q, H, and S; and corresponding to X 51 The amino acid at position X is selected from L, V, S, I, R, and G; the condition is that when X... 51 When it is L, then X 33 X 40 Selected from AD, HK, EG, ER, GR, AK, AR, PK, RR, and KK, and when X 51 When it is G, then X 33 X 40 It's TK.

17. The fusion protein according to any one of claims 7-16, wherein the second polypeptide moiety comprises an amino acid sequence of a protein domain derived from SpA, and wherein... The amino acid residue at position 9 corresponding to SEQ ID NO: 64 is selected from L, E, R, A and Y, and is preferably A; The amino acid residue at position 10 corresponding to SEQ ID NO: 64 is selected from L, E, R, A and Y, and is preferably Y; The amino acid residue at position 11 corresponding to SEQ ID NO: 64 is selected from L, E, R, A and Y, and is preferably R; The amino acid residue at position 13 corresponding to SEQ ID NO: 64 is selected from L, E, R, A and Q, and is preferably L, R, A or Q; The amino acid residue at position 14 corresponding to SEQ ID NO: 64 is selected from L, E, R, A, Q and K, and is preferably A or R; The amino acid residue at position 17 corresponding to SEQ ID NO: 64 is selected from L, E, R and A, and is preferably A; The amino acid residue at position 18 corresponding to SEQ ID NO: 64 is selected from L, E, R and A, and is preferably R; The amino acid residue at position 28 corresponding to SEQ ID NO: 64 is selected from L, E, R and A, and is preferably R or A; The amino acid residue at position 29 corresponding to SEQ ID NO: 64 is selected from L, E, R and A, and is preferably R or A; The amino acid residue at position 33 corresponding to SEQ ID NO: 64 is selected from T, S, G, Q, A, E, H and R, and is preferably S; The amino acid residue at position 40 corresponding to SEQ ID NO: 64 is selected from E, G, R, D, K, Q, H and S, and is preferably G; The amino acid residue at position 51 corresponding to SEQ ID NO: 64 is selected from L, V, S, I, R and G, and is preferably V.

18. The fusion protein according to claim 16 or 17, wherein the second polypeptide portion comprises or is composed of: The amino acid sequence is selected from SEQ ID NO: 64-110 and 7-63, preferably SEQ ID NO: 64-110, and has at least 80% identity with it, preferably at least 85% or at least 90% identity with it.

19. The fusion protein according to any one of the preceding claims, wherein the second polypeptide portion comprises an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID NO: 64, 8, 103, or 110.

20. The fusion protein according to any one of the preceding claims, wherein the second polypeptide portion is capable of binding the Fc domain of immunoglobulin G (IgG) and / or capable of binding the VH3 domain of immunoglobulin G (IgG).

21. The fusion protein according to any one of the preceding claims, wherein the at least one second polypeptide portion is located at the C-terminus of the first polypeptide.

22. The fusion protein according to any one of the preceding claims, wherein the at least one second polypeptide, comprising any additional amino acids, is located at the C-terminus of the fusion protein.

23. The fusion protein according to any one of the preceding claims, wherein the first polypeptide portion is a polypeptide domain.

24. The fusion protein according to any one of the preceding claims, wherein the first polypeptide portion is not an α-helical bundle domain.

25. The fusion protein according to any one of the preceding claims, wherein the first polypeptide portion has a secondary structure comprising a β-sheet.

26. The fusion protein according to any one of the preceding claims, wherein the first polypeptide portion is a natural or synthetic antibody domain or antibody fragment or a modified variant thereof.

27. The fusion protein of claim 26, wherein the first polypeptide portion comprises an antibody heavy chain variable domain.

28. The fusion protein according to claim 26 or 27, wherein the first polypeptide moiety lacks the antibody heavy chain constant domain.

29. The fusion protein according to any one of the preceding claims, wherein the first polypeptide portion is selected from a synthetic variant of an immunoglobulin heavy chain variable domain, a single-domain antibody (sdAb), a variable domain of the heavy chain of a heavy chain antibody (VHH), VNAR, an immunoglobulin single-chain variable fragment (scFv), and an antibody heavy chain variable domain, wherein the native VH / VL interaction site has been modified to mimic the naturally occurring sdAb.

30. The fusion protein according to any one of the preceding claims, wherein the single-chain polypeptide is a single-chain variable fragment or a single-domain antibody or a variant thereof.

31. The fusion protein according to any one of the preceding claims, wherein the single-chain polypeptide comprises complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3).

32. The fusion protein according to any one of claims 1-23, wherein the first polypeptide moiety has a secondary structure comprising at least one β-sheet and at least one α-helix.

33. The fusion protein of claim 32, wherein the first polypeptide portion is or is derived from a protein domain of protein L or protein G.

34. The fusion protein according to any one of claims 1-23, wherein the first polypeptide portion comprises an α-helical bundle domain.

35. The fusion protein of claim 34, wherein the first polypeptide portion comprises a polypeptide as or derived from a protein domain of staphylococcal protein A (SpA).

36. The fusion protein of claim 35, wherein the first polypeptide portion comprises an amino acid sequence selected from SEQ ID NO:1-7 and an amino acid sequence having at least 80% identity with any one of SEQ ID NO:1-7.

37. The fusion protein of claim 34, wherein the first polypeptide portion comprises an albumin-binding domain (ABD).

38. The fusion protein according to any one of the preceding claims, wherein the first polypeptide moiety alone has lower basicity stability than the second polypeptide moiety alone.

39. The fusion protein according to any one of the preceding claims, wherein the first polypeptide moiety alone retains 75% or less, for example 50% or less, of its target binding capacity after exposure to 0.5 M NaOH for up to 20 hours, such as 10 hours, or after up to 120 cycles, such as 60 cycles of repeated NaOH exposure, wherein one NaOH exposure cycle includes contact with 0.5 M NaOH for 10 minutes.

40. The fusion protein according to any one of the preceding claims, wherein the first polypeptide portion has binding affinity to a biological entity.

41. The fusion protein of claim 40, wherein the biological entity is a viral particle or viral vector, such as adenovirus, retrovirus (γ-retrovirus and lentivirus), poxvirus, adeno-associated virus (AAV), baculovirus or herpes simplex virus.

42. The fusion protein of claim 41, wherein the biological entity is an adeno-associated virus (AAV), such as AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 (AAVrh10), 11 or 12.

43. The fusion protein of claim 40, wherein the biological entity is an antibody, such as a monoclonal antibody or an antibody fragment.

44. The fusion protein according to any one of the preceding claims is a multimeric fusion protein comprising at least two of the first polypeptide moieties.

45. The fusion protein of any one of claims 44, wherein the first polypeptide moieties have amino acid sequences distinct from each other, and wherein the first polypeptide moieties are optionally capable of binding to different target entities.

46. ​​The fusion protein according to any one of the preceding claims is a multimeric fusion protein comprising at least two of the second polypeptide moieties.

47. The fusion protein according to any one of claims 46, wherein at least one second polypeptide portion is located at the C-terminus of the fusion protein.

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

49. The fusion protein according to any one of claims 1-31 and 33-48, wherein the first polypeptide comprises a framework region (FWR) and a complementarity-determining region in the following order from the N-terminus to the C-terminus: [FWR1]-[CDR1]-[FWR2]-[CDR2]-[FWR3]-[CDR3]-[FWR4].

50. The fusion protein of claim 49, wherein the first polypeptide comprises a plurality of frame regions, and each frame region has an amino acid sequence having at least 70%, for example at least 75%, for example at least 80%, for example at least 85% sequence identity with the corresponding frame region of SEQ ID NO: 127, 133, 137, 139 or 239-243.

51. The fusion protein of claim 50, wherein the fusion protein comprises an amino acid sequence selected from SEQ ID NO: 141-142, 155, 238, 260, 262-264, 266-268 and 270-272, preferably SEQ ID NO: 142, 155, 260, 262, -262 and 238, and an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 141-142, 155, 238, 260, 262-264, 266-268 and 270-272.

52. The fusion protein according to any one of claims 2-51, wherein basic stability is measured as the ability of the first polypeptide moiety to maintain target binding capacity after exposure to basic conditions.

53. The fusion protein of claim 52, wherein exposure to alkaline conditions is a period of repeated exposure to a solution of at least 0.1 M NaOH, for example 0.5 M NaOH, for 10 minutes.

54. The fusion protein according to claim 52 or 53, wherein the target binding capacity is evaluated after repeated cyclic exposure, for example after at least 10 cycles.

55. An isolated nucleic acid sequence encoding a fusion protein according to any one of the preceding claims.

56. An expression vector comprising the nucleic acid sequence according to claim 55.

57. A recombinant host cell for producing a fusion protein according to any one of claims 1-54, comprising the expression vector according to claim 56.

58. A method for producing a fusion protein according to any one of claims 1-54, comprising: i. Providing the recombinant host cell according to claim 57; ii. Culture the host cells under conditions that enable the expression of the fusion protein; and iii. Isolate the fusion protein.

59. The method of claim 58, wherein the fusion protein comprises a histidine tag, and step iii comprises purifying the fusion protein by immobilized metal affinity chromatography (IMAC).

60. The method of claim 58, wherein step iii comprises purifying the fusion protein by affinity chromatography.

61. The method of claim 60, wherein the affinity chromatography uses an affinity ligand that binds to the framework region of the first polypeptide moiety.

62. The method of claim 60, wherein the affinity chromatography uses an affinity ligand that binds to a second polypeptide moiety of the fusion protein.

63. The method of claim 62, wherein the affinity ligand binding comprises an affinity tag in or added to the amino acid sequence of the second polypeptide moiety.

64. The fusion protein according to any one of claims 1-54 is used for in vitro capture of the target entity, for example for detecting the target entity within a sample.

65. The use according to claim 64, for separating the target entity from other components of a sample.

66. An adsorbent material comprising a fusion protein coupled to a carrier according to any one of claims 1-54.

67. The adsorbent material according to claim 15, wherein the carrier is selected from chips, plates, pores, sheets, fibers, particles, beads, fiber matrices, membranes, filters, and porous materials.

68. The adsorbent material according to any one of claims 66-67, wherein the carrier comprises a polysaccharide-based material, such as agar, agarose, or an agarose derivative.

69. The adsorbent material according to any one of claims 66-67, wherein the carrier material is a fiber matrix, such as a nonwoven fiber matrix.

70. The adsorbent material according to any one of claims 66-69, wherein the support is a chromatographic matrix.

71. Use of an α-helical polypeptide domain that improves the in vitro basic stability of an antigen-binding polypeptide by fusing an α-helical polypeptide to the C-terminus of the antigen-binding polypeptide.

72. The use according to claim 71, wherein the α-helical polypeptide domain is a second polypeptide portion as defined in any one of claims 1-54.

73. The use according to claim 71 or 72, wherein the antigen-binding polypeptide is the first polypeptide portion as defined in any one of claims 1-54.

74. A separation method, comprising the following steps: (a) Providing an adsorbent material according to any one of claims 66-70, wherein the fusion protein has a binding capacity for the target entity. (b) Contacting the adsorbent material with a liquid sample containing the target entity, under conditions that allow the target entity to bind to the first polypeptide of the fusion protein. (c) Optionally, wash the adsorbent material. (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.

75. The method of claim 74, wherein steps (a)-(e) are repeated at least 10 times, for example at least 20 times.

76. The method of claim 75, wherein after 10 cycles of contact with an alkaline cleaning liquid, the 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.

77. The method of claim 75, wherein after 10 cycles of contact with an alkaline cleaning liquid, the 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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