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WO2025083267A1 - Polypeptides de liaison à aav9 - Google Patents

Polypeptides de liaison à aav9 Download PDF

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Publication number
WO2025083267A1
WO2025083267A1 PCT/EP2024/079595 EP2024079595W WO2025083267A1 WO 2025083267 A1 WO2025083267 A1 WO 2025083267A1 EP 2024079595 W EP2024079595 W EP 2024079595W WO 2025083267 A1 WO2025083267 A1 WO 2025083267A1
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seq
antigen
binding
polypeptide
aav9
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PCT/EP2024/079595
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English (en)
Inventor
Andreas L. M. JONSSON
Gustav MYHRINDER
Ellen MELIN
Peter JÄRVER
Mikael C ÅSTRAND
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Cytiva Bioprocess R&D Ab
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Publication of WO2025083267A1 publication Critical patent/WO2025083267A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • AAV9 BINDING POLYPEPTIDES TECHNICAL FIELD The present invention relates to an antigen-binding polypeptide capable of binding an adeno- associated virus (AAV) and a separation matrix comprising said antigen-binding polypeptide, and to use thereof in the context of affinity capture or affinity separation.
  • AAV adeno- associated virus
  • AAV adeno-associated virus
  • separation matrix comprising said antigen-binding polypeptide
  • Adeno-associated viruses are small, non-enveloped viruses of the Parvoviridae family, and are by nature non-pathogenic and of low immunogenicity.
  • AAV can be engineered to deliver DNA to target cells, and recombinant adeno-associated virus (rAAV) vectors have emerged as one of the most versatile and successful gene therapy delivery vehicles.
  • Adeno-associated viruses have a linear single-stranded DNA (ssDNA) genome contained in a capsid formed of capsid protein subunits.
  • AAV capsid serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 AAVrh10, 11 and 12 have been identified.
  • AAV of different serotypes have different tropism, that is, specificity for infecting a cell or tissue type.
  • AAV serotype 2 AAV2
  • AAV9 AAV9 is considered very promising for many gene therapies targeting the lung, liver, heart, skeletal muscle, or the CNS.
  • AAV vectors, engineered to contain a gene of interest can be produced in mammalian cells, such as human embryonic kidney cells.
  • the AAV vectors are harvested from the cell culture, clarified and purified to remove cell debris, host cell DNA and protein, and any other impurities. Purity, efficacy and safety of clinical grade vectors is crucial, as impurities remaining from the production stage may cause adverse effects, reduce transduction efficiency, and even elicit a systemic immune response or inflammatory response.
  • the vectors also need to retain viral activity throughout the purification process. Normally, several filtration steps and several chromatography steps are used to separate AAV particles from cell cultures. Ultracentrifugation can be efficient, but is not scalable. Chromatographic techniques are used for adenovirus purification, but the results have often been insufficient in respect of purity, yield and capacity.
  • Affinity chromatography is a specific mode of chromatography in which an affinity ligand interacts with a target entity via biological affinity in a "lock-key" fashion.
  • useful interactions in affinity chromatography are e.g., enzyme-substrate interaction, biotin-avidin interaction, antibody-antigen interaction, etc.
  • the affinity ligand is capable of specifically and reversibly binding the desired target entity and is immobilized on a chromatographic support material. When contacted with a sample containing the target entity under binding conditions, the ligand selectively binds the target entity, while other species of the sample can be washed away. The captured target entity can then be eluted, typically by changing buffer conditions, such as conductivity or salt concentration, and/or pH.
  • an object of the invention to provide an AAV affinity capture material that allows more efficient purification of AAV vectors.
  • an antigen-binding polypeptide capable of binding adeno-associated virus serotype 9 (AAV9), the polypeptide comprising a single- domain antibody (sdAb) variant having complementarity determining regions CDR1, CDR2 and CDR3 as defined herein.
  • CDR1 comprises an amino acid sequence selected from the group of sequences defined by SEQ ID NO: 1: X1X2X3SX5X6TMX9 (SEQ ID NO: 1) wherein, independently, X1 is R, L or S, preferably R or L; X2 is T or R, preferably T; X3 is L or F, preferably L; X5 is D, N or E, preferably D; X6 is Y, N or F, preferably Y or F; and X9 is G or A, preferably G.
  • X6 may be F and X9 may be G or A.
  • CDR2 comprises a sequence selected from the group of sequences defined by defined by SEQ ID NO: 2: X1X2SWSGX7X8TX10 (SEQ ID NO: 2) wherein, independently, X1 is A, S, L, V or I, preferably A; X2 is I or V, preferably I; X7 is A or S, preferably A; X8 is Y or F, preferably Y; and X10 is K, F or Y.
  • X1 is A, S, L, V or I, preferably A
  • X2 is I or V, preferably I
  • X7 is A or S, preferably A
  • X8 is Y or F, preferably Y
  • X10 is K, F or Y.
  • X2 is I
  • X7 is A
  • X8 is Y and/or X10 is K.
  • CDR3 comprises a sequence selected from the group of sequences defined by defined by SEQ ID NO: 3: X 1 X 2 TX 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 (SEQ ID NO: 3) wherein, independently, X 1 is G or A, preferably G; X 2 is P or S, preferably P; X 4 is G or P, preferably G; X5 is L, T or P, preferably L or P; X6 is L or I, preferably L; X7 is S, T or A, preferably S or A; X8 is K, R, N or Q; X9 is K, H or R, preferably K or R; X10 is A, S or T, preferably A or T; X11 is T, S, P or A, preferably T; X12 is P, A, T or S, preferably P; X13 is A, P, R or G,
  • the present disclosure provides a multimeric polypeptide comprising at least two moieties, each moiety comprising a single-domain antibody variant as defined herein.
  • a fusion protein comprising at least one antigen-binding polypeptide or a multimeric polypeptide as described herein and a further polypeptide moiety.
  • the present disclosure provides an isolated nucleic acid encoding the antigen-binding polypeptide, the multimeric polypeptide or the fusion protein, as well as an expression vector comprising said nucleic acid, and a recombinant host cell comprising the expression vector.
  • a method of producing the antigen-binding polypeptide, the multimeric polypeptide the fusion protein comprising i) providing recombinant host cells; ii) culturing the host cell under conditions enabling expression of the antigen-binding polypeptide, the multimeric polypeptide or the fusion protein; and iii) isolating the antigen-binding polypeptide, the multimeric polypeptide or the fusion protein.
  • Step iii) may comprise purifying the antigen-binding polypeptide, the multimeric polypeptide or the fusion protein by chromatography, such as immobilized metal affinity chromatography (IMAC) or affinity chromatography.
  • IMAC immobilized metal affinity chromatography
  • the disclosure provides the use of the antigen-binding polypeptide, the multimer or the fusion protein as an affinity ligand for binding of adeno-associated virus serotype 9 (AAV9). Said use may be e.g. for in vitro detection of AAV9 in a sample, or for purification of AAV9 from a sample, optionally in the context of a method of providing a therapeutic composition comprising AAV9 as a viral vector.
  • the disclosure provides a separation matrix and a method of separation.
  • the separation matrix comprises the antigen-binding polypeptide, the multimeric polypeptide or the fusion protein coupled to a support material, thus providing an affinity separation matrix employing the antigen-binding polypeptide as an affinity ligand.
  • the support material may be selected from the group consisting of a particle, a bead, a fiber, a fibrous membrane, a filter, a sheet, a porous monolith, a chip, a plate, and a well.
  • the support material may be a chromatography matrix.
  • the chromatography matrix may comprise a polysaccharide material, such as beads comprising agarose or a derivative thereof.
  • the chromatography matrix may comprise a polymeric fibrous matrix or membrane, such as a nonwoven fibrous matrix.
  • the method of separation comprises the steps of (a) providing a separation matrix of the present disclosure, (b) contacting the separation matrix with a liquid sample comprising said AAV9 viral particles vector under conditions allowing the antigen-binding polypeptide to bind to the AAV9 viral particles, (c) optionally washing the chromatography material, (d) eluting the bound AAV9 viral particles from the separation matrix, and optionally (e) cleaning the separation matrix material with a cleaning liquid.
  • Step (e) typically comprises cleaning the separation matrix with an alkaline cleaning liquid, wherein the cleaning liquid preferably comprises from 0.05 to 0.5 M of NaOH.
  • Fig.1 is a schematic illustration of the arrangement of framework regions and complementarity determining regions of a single-domain antibody.
  • Fig.2a-c are schematic illustrations of various structures of antigen-binding polypeptides according to the present disclosure.
  • Fig.3a-c are schematic illustrations of a fusion protein comprising an antigen-binding polypeptide as disclosed herein fused to another polypeptide moiety.
  • Fig.4a-b are sensorgrams showing the AAV9 binding capacity of two exemplary polypeptides.
  • Fig.5 is a graph showing the alkaline stability of exemplary antigen-binding polypeptides.
  • Fig.6 is a graph showing the alkaline stability of exemplary antigen-binding polypeptides and a commercially available affinity ligand.
  • Figs.7a-h are sensorgrams showing binding to AAV9 and other AAV serotypes for exemplary antigen-binding polypeptides and a commercially available affinity ligand.
  • Fig.8a is a graph plotting the AAV9 binding response of an antigen binding polypeptide having a fusion partner (solid line) or no fusion partner (dashed line), respectively, against the number of binding cycles on Biacore, each cycle involving 0.5 M NaOH exposure.
  • Fig.9a is a picture of a PAGE gel demonstrating stronger protein expression of multimeric fusion proteins according to embodiments of the invention and an antigen binding polypeptide alone (lacking fusion partner).
  • Fig.9b is a graph plotting the AAV9 binding response of the multimeric fusion proteins and the multimer lacking a fusion partner polypeptide, respectively, against the number of binding cycles on Biacore, each cycle involving 0.3 M NaOH exposure.
  • the binding response was normalized against the response of the second cycle (i.e., first cycle excluded).
  • Fig.10 shows AAV9 binding responses obtained for increasing number of cycles involving NaOH exposure for two fusion proteins according to embodiments of the invention.
  • Fig.11a is a chromatogram showing the elution peak from a chromatography column using an AAV9 binding polypeptide according to embodiments of the present invention as affinity ligand immobilized on the chromatography matrix.
  • Fig.11b is a closer view of the elution peak.
  • Fig.12 is a photograph of an SDS-PAGE gel showing the protein content of the eluate fractions from the chromatography run of Example 8.
  • Fig.13 is a graph plotting the dynamic binding capacity as evaluated in Example 8.
  • peptide and “polypeptide” are used synonymously herein to refer to compounds formed of sequences of amino acids, without restriction as to size. “Protein” may be used to refer to the larger compounds of this class. Amino acid sequences are written left to right in the direction from the amino (N) to the carboxy (C) terminus.
  • amino acid residue sequences are denominated by either a three letter or a single letter code as indicated 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).
  • “Peptides” include any oligopeptide, polypeptide, gene product, expression product, or protein.
  • a peptide is comprised of consecutive amino acids and encompasses naturally occurring or synthetic molecules.
  • the term "peptide” refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the 20 gene-encoded amino acids.
  • the peptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art.
  • a “single-chain polypeptide” herein refers to a polypeptide that is formed of a single amino acid sequence, in which the amino acid residues are connected via peptide bonds.
  • a single-chain polypeptide may, upon folding, additionally form other internal bonds such as disulfide bonds.
  • the expression ”antigen-binding polypeptide generally refers to a polypeptide that possesses at least one binding region, and typically at least two, such as three binding regions, such that the polypeptide has a binding affinity for a molecule (referred to as antigen or "target”).
  • An antigen-binding polypeptide may have any protein structure, as long as there is a binding affinity for the antigen.
  • antigen-binding polypeptides that have frequently been exploited and used as a basis for engineered antigen-binding polypeptides often have a scaffold or framework structure that is generally conserved between antigen-binding polypeptides of the same class, but which bind to different antigens.
  • the binding regions of an antigen-binding molecule can typically be engineered or evolved to bind to a particular antigen, while the scaffold or framework structure remains essentially the same.
  • Non-limiting examples of antigen-binding polypeptides include natural or engineered (i) antibodies, such as monoclonal antibodies, (ii) antibody fragments that contain the light chain and/or heavy chain variable region(s) with complementarity determining regions (CDRs), such as Fab (fragment antigen-binding), Fv (variable fragment), scFab (single chain antigen-binding fragment) and scFv (single chain variable fragment), iii) single-domain antibodies, and iv) polypeptides having a scaffold derived from bacteria, such as immunoglobulin-binding bacterial proteins (such as Finegoldia magna protein L, and staphylococcal protein A and protein G) or domains thereof, including the wild types as well as variants in which one or more of the antigen binding regions have been engineered.
  • antibodies such as monoclonal antibodies
  • single-domain polypeptide or “single-domain amino acid sequence” refers to a polypeptide which forms an individual protein domain, and which does not comprise any other individual protein domain.
  • single-domain polypeptides include antibody fragments such as heavy chain variable domain and light chain variable domain, single-domain antibodies (sdAb), albumin binding domain (ABD) and polypeptides derived from a domain of SpA, such as the B, C or Z domain of SpA.
  • Single-domain antibodies are particularly contemplated.
  • a single-domain polypeptide typically consists of a single-chain polypeptide.
  • a single-domain polypeptide is in itself monomeric, but it may form part of a multimer, as described elsewhere herein.
  • a single-domain polypeptide may be joined to another polypeptide, for example via a peptide bond or via a disulfide bond.
  • the antigen-binding polypeptide of the present disclosure may consist of a single polypeptide chain.
  • the term ”single-domain antibody refers to a variable domain which is or is derived from a heavy chain variable domain (VH) of an antibody devoid of light chains.
  • VH heavy chain variable domain
  • a single domain antibody lacks antibody light chains entirely, including the light chain variable domain (VL).
  • a single- domain antibody may also lack such structural features that are needed for a functional VH/VL interaction, and which are present in, e.g., the conventional VH of IgG1.
  • Single domain antibodies do not form part of a dimeric structure with an antibody light chain variable domain.
  • Single-domain antibodies include antibodies naturally devoid of antibody light chains, such as sdAb derived from IgG2 or IgG3 of camelids (Camelidae), e.g. dromedary, camel, llama and alpaca, which are also referred to as VHH (heavy chain variable domain of a heavy chain antibody.
  • Single domain antibodies also include antibodies derived from cartilaginous fishes, such as sharks, usually termed VNARs (variable new antigen receptors).
  • single domain antibodies may be synthetic variants based on an amino acid sequence of an antibody heavy chain variable domain from a species that naturally does not produce sdAb (e.g. cow, rat, mouse or rabbit) and which are modified e.g. with respect to amino acids in the natural VH/VL interaction of such antibodies, so as to mimic an sdAb.
  • Such modification may involve substitution of amino acids of the VH/VL interface region to increase the hydrophilicity or solubility.
  • Single-domain antibody variants of the present invention are synthetic, non-naturally occurring amino acid sequences.
  • Complementary determining regions are hypervariable regions of an antibody or part thereof which participate in binding to a target epitope. The CDRs are typically defined by their separate amino acid sequences, although in an antibody, the CDRs together form a three- dimensional binding site for the target antigen.
  • a variable domain of an antibody heavy chain has three CDRs, referred to as CDR1, CDR2 and CDR3, as positioned from the N terminus of the polypeptide chain.
  • the sequence length of CDRs may vary, and CDR1, CDR2 and especially CDR3 of a VH may be of different lengths.
  • the portions of an antibody VH not forming the CDRs are referred to as framework regions.
  • the framework regions are typically four and referred to as framework region (FW) 1 to 4, as counted from the N terminus.
  • the framework regions are responsible for the general secondary and tertiary structure of the domain and thus for positioning and orientation of the CDR regions.
  • the framework regions may be referred to as a scaffold structure.
  • the framework regions are less variable than the CDRs. Generally, amino acid sequence variations that do not significantly alter the secondary or tertiary structure of the framework may however be tolerated. Some parts or amino acid positions of the framework regions may be conserved. The general stability of the framework regions allows for a high degree of variation in the CDRs.
  • the term “ligand” is a molecule that has a known or unknown affinity for a given entity.
  • the term “ligand” may herein be used interchangeably with the terms ”affinity ligand”, “selective binding molecule”, “selective binding partner”, “capturing molecule” and “capturing agent”.
  • affinity ligand refers to a moiety or molecule that binds reversibly and selectively or preferentially with high affinity to a target entity through a specific interaction with a binding site of the component.
  • an affinity ligand is a polypeptide, and may also be referred to as an “affinity protein”.
  • An affinity ligand may be immobilized to a solid support such as a resin.
  • target entity herein refers to an entity that forms a specific binding partner to the ligand, and may also be referred to as an “analyte”.
  • the analytes or target entities of interest according to the present disclosure are adeno-associated virus vectors, in particular AAV9 vectors.
  • affinity in the context of polypeptides denotes the strength of interaction between two molecules, wherein at least one molecule is a polypeptide. The interaction is selective, i.e. discriminates between the affinity binding partner and other molecules present. Binding affinity is also expressed as the dissociation equilibrium constant (KD).
  • KD dissociation equilibrium constant
  • binding capacity refers to the capability of a ligand to bind the target molecules when the ligand is immobilized on a surface. The binding capacity of a ligand can differ depending on the type of surface on which it is immobilized. For the purpose of the present invention, the binding capacity can be measured by surface plasmon resonance (SPR) technology using e.g.
  • SPR surface plasmon resonance
  • the binding capacity for a certain concentration of analyte is influenced by the density of immobilized ligands on the surface.
  • the binding capacity is preferably determined at a ligand density of at least about 3000 response units (RU), such as in the range of from 3000-4000 RU.
  • RU response units
  • DRC dynamic binding capacity
  • the DBC of a chromatography resin is expressed as the amount of analyte that binds to the resin under given flow conditions before a significant breakthrough of unbound analyte occurs.
  • DBC is determined by loading a sample containing a known concentration of the analyte and monitoring the flow-through. The analyte will bind to the resin to a certain break point before unbound analyte will flow through the column.
  • the DBC can be determined on the breakthrough curve at a loss of, for example, 10% analyte. This is referred to as the QB10% value.
  • a sample is applied to a chromatography resin column during a specific residence time and the dynamic binding capacity for each resin is calculated at 10% of the breakthrough capacity i.e., the amount of analyte sample that is loaded onto the column until the concentration of analyte in the column effluent is 10% of the analyte sample concentration in the feed. If the dynamic binding capacity for each resin is calculated at 80% of the breakthrough capacity, this is referred to as the QB80% value.
  • the term “alkaline stability” as used herein, refers to a property of the antigen-binding polypeptides which relates to its ability to withstand alkali exposure without deleterious effect on structure and/or function of the polypeptide.
  • the alkaline stability is determined based on the affinity for the target entity after exposure to NaOH.
  • the alkaline stability of an antigen-binding polypeptide can be evaluated immobilizing the antigen- binding polypeptide on a support and measuring the target binding capacity before and after one or more cycles of exposure to NaOH, using methods described in the Examples below.
  • the evaluation is made on an SPR chip onto which the antigen-binding polypeptide is immobilized by covalent coupling with thiol, N-hydroxysuccinimide (NHS), streptavidin-biotin binding, or another coupling that is alkali resistant in itself, at a ligand density corresponding to at least 1000 RU, and preferably at least 3000 RU, and for a predetermined analyte concentration.
  • the first alkaline cleaning cycle may have a unique, large impact on binding capacity, e.g. due to removal of non- covalently bound antigen-binding polypeptide from the support surface, and therefore the binding capacity is sometimes represented as normalized to the binding capacity measured after the first cycle of alkali exposure.
  • an antigen-binding polypeptide is considered to be alkali stable if, after 10 cleaning cycles, preferably after 15 cleaning cycles, the immobilized antigen-binding polypeptide retains at least 50 % of the target binding capacity compared to its binding capacity after the first cleaning cycle (i.e. disregarding the binding capacity of the first cycle, prior to the first alkali exposure event), where a cleaning cycle involves 600 s of exposure to at least 0.3 M NaOH, such as at least 0.5 M NaOH.
  • solid support herein refers to a non-aqueous matrix of a solid phase material.
  • Suitable solid phase materials include, but are not limited to, glass, silica (e.g., silica gel), polysaccharides (e.g., a polysaccharide matrix) such as agarose and cellulose, organic polymers such as polyacrylamide, methylmethacrylate, and polystyrenedivinylbenzene copolymers.
  • the solid phase can be of porous or nonporous character and can be compressible or incompressible.
  • the solid phase can be a polymeric matrix or an agarose particle or bead.
  • Preferred solid support materials will be physically and chemically resilient to the conditions employed in a purification process including pumping and cross-flow filtration, and temperatures, pH, and other aspects of the liquids employed.
  • surface herein means all external surfaces of a solid structure.
  • a porous support such as a bead used e.g. in the field of chromatography
  • the term “surface” includes outer surfaces as well as pore surfaces.
  • the term “surface” encompasses the external surface of individual fibers.
  • separation matrix is used herein to denote a material comprising a solid support to which one or more ligand(s) have been coupled.
  • the ligand(s) are capable of binding target entities herein also called analytes, which are to be separated from their surroundings, e.g. a liquid sample, and/or which are to be separated from other components present in the liquid sample.
  • a separation matrix may be employed in various contexts, including analytical assays and purification of target for analytical or preparative purposes.
  • the type of support may be selected depending on the intended use.
  • a separation matrix may further comprise a compound which couples the ligand(s) to the support.
  • spacer extender
  • surface extender may be used to describe such a compound, as further described herein.
  • the term “resin” is sometimes used for a separation matrix in this field.
  • chromatography material” and “chromatography matrix” are used herein to denote a type of separation matrix.
  • An affinity separation matrix is a separation matrix where the ligand is an affinity ligand.
  • spacer refers to a peptide or other chemical linkage or element that extends the structure of an entity.
  • a spacer attached to a polypeptide may be provided at the N terminus or the C terminus of the polypeptide.
  • a spacer may connect a polypeptide to a solid support.
  • Suitable spacers for coupling a polypeptide to a support may generally be any spacer used in the art to connect peptides, proteins or other organic molecules to a support.
  • a spacer may also serve to connect two polypeptides or polypeptide domains.
  • a spacer connecting two polypeptide moieties may also be referred to as a linker.
  • the terms “multimer” and “multimeric protein” as used herein refer to a protein comprising at least two repeating units of the single-domain antibody disclosed herein.
  • the single domain antibody may be regarded as a monomeric polypeptide unit, which may be combined into a multimeric protein, such as a dimer, a trimer, a tetramer, a pentamer etc.
  • the single-domain antibody units of a multimer may be identical, or may differ from one another with regard to their amino acid sequence.
  • a multimer may contain one or more further polypeptide unit(s), such as a fusion partner polypeptide or stabilizing polypeptide unit as described herein, which is not a single-domain antibody.
  • Such a further polypeptide unit may be present in a single copy or at multiple copies in the multimer.
  • a multimeric protein may be formed of a single polypeptide chain.
  • the term “fusion protein” refers to a protein formed of two or more than two separate proteins (fusion partners) produced by recombinant protein expression as a single polypeptide containing both fusion partners. The fusion partners may be joined sequentially one after the other. The genes encoding the respective proteins are joined at the genetic level. The fusion partners typically do not naturally occur as fused to each other. A fusion protein may contain additional amino acids sequences, such as linker in between the fusion partners.
  • linker refers to a peptide or other chemical linkage that functions to link otherwise independent functional domains.
  • a linker may be located between two polypeptide units, monomers or domains, such as between a ligand (e.g., a sdAb) and another polypeptide component containing an otherwise independent functional or structural domain, or between two ligands.
  • Suitable linkers for coupling two or more linked units may generally be any linker used in the art to link peptides, proteins or other organic molecules.
  • a linker peptide may be a stretch of amino acids preferably ranging from 1 to 20 amino acids, such as 2-15, such as 2-8, 2-4 or 4-12 amino acids.
  • vector is herein used to denote a virus particle, normally a recombinant virus particle, which is intended for use to achieve gene transfer to modify specific cell type or tissue.
  • a virus particle can for example be engineered to provide a vector expressing therapeutic genes.
  • virus types are currently being investigated for use to deliver genetic material (e.g., genes) to cells to provide either transient or permanent transgene expression. These include adenoviruses, retroviruses ( ⁇ -retroviruses and lentiviruses), poxviruses, adeno-associated viruses (AAV), baculoviruses, and herpes simplex viruses.
  • capsid means the shell of a virus particle. Sequence identity may be assessed by any convenient method.
  • EMBOSS Needle or EMBOSS stretcher may be used for pairwise sequence alignments while Clustal or MUSCLE may be used for multiple sequence alignments, though any other appropriate programme may be used.
  • the alignment is pairwise or multiple, it must be performed globally (i.e. across the entirety of the reference sequence) rather than locally.
  • Sequence alignments and % identity calculations may be determined using for instance standard Clustal Omega parameters: matrix Gonnet, gap opening penalty 6, gap extension penalty 1.
  • % identity as used throughout the disclosure, may for example be calculated 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)). A comparison is made over the window corresponding to the shortest of the aligned sequences.
  • the shortest of the aligned sequences may in some instances be the target sequence. In other instances, the query sequence may constitute the shortest of the aligned sequences.
  • the amino acid residues at each position are compared and the percentage of positions in the query sequence that have identical correspondences in the target sequence is reported as % identity.
  • the present inventors have found that a novel antigen-binding polypeptide, capable of binding adeno-associated virus (AAV) serotype 9 (AAV9), is useful for affinity capture of AAV9-based vectors.
  • the antigen-binding polypeptide is typically a single-chain polypeptide.
  • the antigen-binding polypeptide is based on a single-domain antibody (sdAb), but may optionally comprise additional amino acids, e.g. positioned N-terminally or C-terminally thereof.
  • the antigen-binding polypeptide of the present invention may be derived from a camelid single-domain antibody, also referred to as a VHH (variable domain of a heavy chain of a heavy chain antibody).
  • the single-domain antibody variant lacks the light chains of conventional antibodies such as IgG1, and also lacks the heavy chain constant domains.
  • a single-domain antibody of the antigen-binding polypeptide disclosed herein comprises only a heavy chain variable domain, including three complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the antigen-binding polypeptide contains no antibody light chain variable region or light chain CDRs.
  • single domain antibodies are easier to express in prokaryotic and eukaryotic cells as compared to full-size antibodies such as IgG, as well as Fab fragments or single-chain fragments including both light and heavy chain portions. Despite having only three CDRs, as opposed to the six CDRs of larger antibodies and antibody fragments, the target binding properties of can be satisfactory.
  • the portions of the sdAb not forming the CDRs are referred to as framework regions.
  • the framework regions of an sdAb which are typically four and referred herein to as framework region (FW) 1 to 4, are responsible for the general secondary and tertiary structure of the sdAb.
  • the framework regions form two beta sheets forming a sandwich and connected via a cysteine bridge.
  • the CDRs are typically presented as three surface loops, one CDR per loop.
  • Another advantage of the sdAb of the present disclosure is that the CDR sequences can be longer than the CDRs of conventional antibodies.
  • the surface loops may thus spatially extend further from the body of the sdAb as have a better ability to bind into pockets of the target epitope.
  • the sdAb variant may comprise framework regions and complementary determining regions in the following order from N-terminus to C-terminus: [FWR1]-[CDR1]-[FWR2]- [CDR2]-[FWR3]-[CDR3]-[FWR4].
  • Fig.1 schematically illustrates the sequence of framework regions and complementarity determining regions (CDRs) of an sdAb, from the direction of the N terminus to the C terminus of the amino acid sequence.
  • Table 1 outlines the FWRs and CDRs as defined herein in relation to the Kabat amino acid numbering system (Kabat et al., 1991, J. Immunol.147(5), 1709-1719).
  • Framework region 1 (FWR1) is formed of the amino acids in positions 1 to 26.
  • amino acids in Kabat positions 27 to 35d form CDR1, Kabat positions 35a-d being optional.
  • Framework region 2 (FWR2) is formed of the amino acids in Kabat positions 36 to 49.
  • CDR2 is formed of the amino acids in Kabat positions 50 to 58 (52a being optional).
  • Framework region 3 (FWR3) is formed of the amino acids in Kabat positions 59 to 94.
  • CDR3 is formed of the amino acids in Kabat positions 95 to 102 (100a-j being optional).
  • framework region 4 is formed of amino acids in Kabat positions 103 to 113. It may be noted that the definition of CDRs and framework regions within the present disclosure does not fully correspond to the definition of CDRs and framework regions of Kabat et al; however, the same amino acid numbering system is used. The framework regions may collectively be referred to as the “framework”.
  • the sdAb of the present invention may have a framework that has a certain degree of sequence identity to a native camelid VHH framework.
  • each of the framework regions of the present sdAb may have a sequence identity in the range of 70-100 % to the respective framework regions of SEQ ID NO: 4, which are represented by SEQ ID NO:5 (FWR1), SEQ ID NO:6 (FWR2), SEQ ID NO:7 (FWR3) and SEQ ID NO:8 (FWR4), respectively.
  • at least one of the framework regions of the present sdAb may comprise an amino acid substitution relative to SEQ ID NO: 4 (i.e. one or more of SEQ ID NO: 5-8, such as preferably one or more of SEQ ID NO: 5-7, such as at least SEQ ID NO: 7), wherein the sdAb exhibits improved alkaline stability (e.g.
  • the framework regions of the sdAbs of the present invention may have a certain degree of sequence identity with the synthetic framework regions of SEQ ID NO: 91 or SEQ ID NO: 92.
  • the individual framework regions of SEQ ID NO: 92 are represented by SEQ ID NO:151 (FWR1), SEQ ID NO:154 (FWR2), SEQ ID NO:157 (FWR3) and SEQ ID NO:8 (FWR4), and the individual framework regions of SEQ ID NO: 91 are represented by SEQ ID NO:149 (FWR1), SEQ ID NO:152 (FWR2), SEQ ID NO:155 (FWR3) and SEQ ID NO:8 (FWR4).
  • the framework regions of the sdAbs of the present invention may have a certain degree of sequence identity with the individual framework regions of SEQ ID NO: 150 (FWR1), SEQ ID NO:153 (FWR2), SEQ ID NO:156 (FWR3) and SEQ ID NO:8 (FWR4).
  • the framework regions of the sdAbs of the present invention may have a certain degree of sequence identity with the synthetic framework regions of any one of SEQ ID NO: 177-181.
  • the individual framework regions for FW1 are defined by SEQ ID NO: 183-186, for FW2 by SEQ ID NO: 187-189, and FW3 by SEQ ID NO: 190-192.
  • each of the framework regions 1, 2 and 3 of the present sdAb may have at least 70% sequence identity with the reference framework region.
  • FWR4 may have less than 70 % sequence identity with the reference framework region.
  • FWR4 may have at least 70% sequence identity with the reference framework region.
  • Parts of the framework regions of an sdAb are relatively conserved and it may be preferable to make only few or no modifications in these parts in relation to e.g. SEQ ID NO: 4, 91 or 92. These parts are herein defined 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.
  • each of FWR1-3, and optionally also FWR4 may have at least 80 %, such as at least 85 %, such as at least 90 %, sequence identity with the corresponding framework region of SEQ ID NO: 4, 91 or 92 over the conserved amino acid positions (i.e., omitting the other positions from the comparison).
  • FWR1 contains 26 aa, whereof 21 amino acids represent the conserved positions as defined above. Substituting 2 of these amino acids may thus result in 90.5 % identity, whereas substituting 3 amino acids may result in 85.7 % identity.
  • the sdAb variant may have an amino acid sequence wherein Kabat positions 1-5, 7-13, 25-26, 36-39, 41- 42, 46, 48-49, 59, 61-75, 77-78, 80-82a and 82c-94 may include up to 15, such as up to 14, such as up to 13, such as 12, 11, or 10 amino acid residues that are substituted in relation to at least one of SEQ ID NOs: 4, 91, 92, 131, 171 and 172.
  • positions 1-5, 7-13 and 25-26 (21 residues) may together have up to 4, such as up to 3, amino acid residues that differ from the corresponding position of SEQ ID NO: 4, 91, 92 or 171.
  • Kabat positions 36-39, 41-42, 46 and 48-49 may together have 1 or 2 amino acid residues that differ from the corresponding position of SEQ ID NO: 4, 91 or 92.
  • Kabat positions 59, 61-75, 77-78, 80-82a, 82c-94 may have up to 6 amino acid residues that differ from the corresponding position of SEQ ID NO: 4, 91 or 92.
  • the amino acid in Kabat position 1 may advantageously be selected from Q, V, D and E, and may in particular be Q or E.
  • any sdAb amino acid sequence disclosed herein in which Kabat position 1 is not E (e.g., is Q), may instead have E in Kabat position 1.
  • the framework regions of sdAbs typically have a secondary structure comprising ⁇ -strands and/or ⁇ -sheets.
  • the framework regions typically form a ⁇ -sandwich structure often comprising a cysteine bridge.
  • the CDRs are typically presented as three surface loops at one end of the structure, one CDR per loop.
  • An advantage of using sdAbs as affinity binders is that the CDR sequences can be more diverse with regard to their length than the CDRs of conventional antibodies.
  • the surface loops formed of the CDRs can thus spatially extend further from the main body of the sdAb and thus have a better ability to bind into pockets of a target epitope, or the surface loops may be shorter, forming flatter surfaces which may facilitate binding to other epitopes.
  • the present inventors have identified novel amino acid sequences which have excellent AAV9 binding properties when used as CDRs in an sdAb framework.
  • these binding properties provide surprisingly good capacity for binding AAV9.
  • the present sdAbs can differentiate different AAV serotypes such that they bind selectively to AAV9, while other AAV sdAbs bind several serotypes. The binding of the present sdAbs is thus more specific compared to the prior art.
  • CDR sequences disclosed herein have been found to contribute to the overall alkaline stability of the sdAb.
  • sdAbs also known as “nanobodies” have poor alkaline stability. It is also a common feature of previous AAV sdAb ligands, as expressed e.g. in patent application WO2020242988A2, which relates to affinity agents comprising ligands that bind AAV, where it in [0005] is stated “Currently, there are only 3 affinity resins available for the purification of AAV, POROSTM, CaptureSelectTM AAV9, POROSTM CaptureSelectTM AAVX and AVB Sepharose. These resins have 2 major shortcomings, they cannot be cleaned with sodium hydroxide and can only be reused for a few cycles.
  • the present sdAb not only bind AAV selectively and with high capacity (good affinity), but that they also are alkaline stable. It was identified that certain CDR sequences greatly increased alkali stability especially when combined with a modified sdAb framework, where the resulting sdAbs became even more stable. In addition, when fusing the sdAbs with a further polypeptide moiety derived from a protein domain of staphylococcal protein A (SpA), the resulting fusion protein showed even better properties.
  • SpA staphylococcal protein A
  • sdAbs capable of binding AAV9 exist in the art, such as disclosed in CN116751284, but they are not specific for AAV9, but most importantly, they are not alkali stable as the present sdAbs.
  • CN116396381 discloses sdAbs alleged as capable of binding a plurality of serotypes, including AAV9, and having acid base tolerance. However, when tested, the sdAbs has very low affinity towards AAV9.
  • CBS buffer is used (the buffer is not further specified, only pH is noted), presumably a sodium carbonate bicarbonate buffer at different pH. It is well known that the type of buffer, ionic strength and molarity greatly affect the stability of a polypeptide.
  • the buffer used in CN116396381 is not corresponding or similar to the NaOH buffer used for cleaning in place (CIP), and to establish alkaline stability of a protein. It may be noted that for achieving the alkaline stability as defined in the present disclosure, the polypeptide should be able to undergo CIP with NaOH or KOH of at least 0.05 M, such as 0.05-1 M.
  • CDR1 may include a sequence of between 9 and 13 amino acids.
  • CDR1 is formed of 9 amino acids.
  • the amino acid sequences for CDR1 described herein may be supplemented with up to 4 additional amino acids, especially at the C- terminal.
  • the CDR1 may be formed of amino acid positions 27, 28, 29, 30, 31, 32, 33, 34 and 35 (9 amino acids) or the positions from 27-35 and up to 35d (thus up to 13 amino acids).
  • CDR2 may include between 10 and 13 amino acids.
  • CDR2 is formed of 10 amino acids.
  • the CDR2 may constitute positions 50, 51, 52, 52a, 53, 54, 55, 56, 57 and 58.
  • CDR3 may include up to 18 amino acids.
  • CDR3 is formed of 17 amino acids.
  • the CDR3 may be formed of amino acid positions 95, 96, 97, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, 100g, 100h, 100i, 101 and 102.
  • CDR1 may be constituted by amino acid positions according to the Kabat numbering system as outlined in the Table 2a below.
  • Table 2a CDR1 positions Position (Kabat) 27 28 29 30 31 32 33 34 35 35a 35b 35c 35d Position in SEQ ID 1 2 3 4 5 6 7 8 9 - - - - NO: 1
  • Useful amino acids R, L, T, R L, F S D, Y, T M A, G according to the S N, E N, F present disclosure Preferred amino R T L S D Y, F T M G - - - - acid(s)
  • amino acids may be selected, for each position independently, from the useful and preferred amino acids indicated in Table 2a.
  • the present inventors found that the amino acid sequence of CDR1 had a substantial influence on the binding properties and stability of the antigen-binding polypeptide, with the potential to improve one or both of these characteristics.
  • Amino acid substitutions in CDR1 were in general found to impact binding capacity and stability to a greater extent than amino acid substitutions in CDR3, which appears to tolerate an even higher degree of variation.
  • the amino acids in Kabat positions 30, 33 and 34 were advantageously fixed as serine (S), threonine (T) and methionine (M), respectively.
  • other positions of CDR1 e.g. Kabat position 32 were found to allow some variability, with acceptable or further improved binding capacity.
  • CDR1 may be selected from the group consisting of SEQ ID NOs:9-17, such as SEQ ID NOs:13, 14, 16 and 17.
  • CDR2 may be formed of amino acid positions according to the Kabat numbering system as outlined in the Table 3a below. Table 3a. CDR2 positions Position (Kabat) 50 51 52 52a 52b 52c 52d 53 54 55 56 57 58 Position in 1 2 3 4 - - - 5 6 7 8 9 10 SEQ ID NO: 2
  • Preferred amino A I S W - - - S G A Y T K, Y, acid(s) F It was found that certain amino acid substitutions in CDR2 had an influence on the binding properties and stability of the antigen-binding polypeptide, with the potential to improve one or both of these characteristics.
  • amino acids may be selected, for each position independently, from the useful and preferred amino acids indicated in Table 2a.
  • SEQ ID NO: 2 X 2 may be I; X 7 may be A; X 8 may be Y; and/or X 10 may be K.
  • CDR2/SEQ ID NO: 2 X 2 is I and X 8 is Y.
  • X 1 is A
  • X 1 is S, L or I
  • X 7 is A.
  • Table 3b shows exemplary CDR2 sequences of the sdAb variants disclosed herein. Table 3b.
  • CDR2 amino acid sequences CDR2 variant SEQ ID NO AVSWSGSFTY 21 AISWSGSFTY 22 SISWSGAYTY 23 LISWSGAYTY 24 IISWSGAYTY 25 AISWSGAYTY 26 AVSWSGAYTY 27 AVSWSGAYTK 28 AISWSGAYTK 29 AISWSGAYTH 30 AISWSGAYTF 31
  • CDR2 may be selected from the group consisting of SEQ ID NOs: 21-31, such as selected from the group consisting of SEQ ID NOs: 26, 28, 29, and 31.
  • CDR3 may be constituted by amino acid positions according to the Kabat numbering system as outlined in the Table 4a below.
  • the present sdAbs tolerate a relatively high degree of variability in the CDR3 amino acid sequence without deterioration of binding capacity or alkaline stability. Nevertheless, it was found that for some positions, certain amino acids are particularly advantageous for the purpose of enhancing binding capacity or alkaline stability. Hence, for CDR3, amino acids are selected (useful and preferred, respectively) as indicated in Table 4a. Table 4a.
  • SEQ ID NO: 3 is P; X7 is S; X8 is K, N or R, such as K or R; X9 is K; X10 is A or T; X11 is T; X13 is A; and/or X17 is Y.
  • at least one of X8 and X9 may be K.
  • at least one of X5 and X6 may be L, and preferably both X5 and X6 are L.
  • SEQ ID NO: 3 X 13 is A and, preferably, X 10 is A.
  • SEQ ID NO: 3 X 14 is D and X 16 is D.
  • Table 4b shows exemplary CDR3 sequences of the sdAb disclosed herein. Table 4b.
  • Exemplary CDR3 sequences CDR3 Sequence SEQ ID NO CDR3 Sequence SEQ ID NO GPTGPLSRRSSPPDYDY 34 GPTGLLSRKSTPADYDY 54 GSTGPLTRRSTPPDYDY 35 GPTGLLSKKATPADYDY 55 APTGLLSKKATPADYDY 36 GPTGLLSKKTTPADYDY 56 GSTGLLSKKATPADYDY 37 GPTGLLSKKAAPADYDYDY 57 GPTGLLSKKSTPADYDY 38 GPTGLLSKKATSADYDY 58 GPTGPLSKKATPADYDY 39 GPTGLLSKKATTADYDY 59 GPTGTLSKKATPADYDY 40 GPTGLLSKKATPRDYDY 60 GPTGLISKKATPADYDY 41 GPTGLLSKKATPANYDY 61 GPTGLLAKKATPADYDY 42 GPTGLLSKKATPAEYDY 62 GPTGLLSNKATPADYDY 43 GPTGLLSKKATPAQ
  • Exemplary combinations of CDR sequences are listed in Table 5.
  • the table also refers to exemplary sdAb variant sequences having said combination of CDRs. All of the listed polypeptides exhibited good or excellent binding to AAV9, as demonstrated in the Examples.
  • Table 5 CDR1 sequence CDR2 sequence CDR3 sequence Exemplary sdAb LISWSGAYTY GPTGLLSKKSTPADYDY (SEQ ID NO:24) (SEQ ID NO:38) vh29 (SEQ ID NO:78) IISWSGAYTY GPTGLLSKKSTPADYDY (SEQ ID NO:25) (SEQ ID NO:38) vh30 (SEQ ID NO:79) IISWSGAYTY GPTGLLSRKSTPADYDY (SEQ ID NO:25) (SEQ ID NO:54) vh33 (SEQ ID NO:82) LISWSGAYTY GPTGLLSKKATPADYDY (SEQ ID NO:24) (SEQ ID NO:55) vh35 (SEQ ID NO:
  • the sdAb variant may comprise a) an amino acid sequence selected from SEQ ID NOs: 75-93, 95-98, 101, 102, 105-108, 109-124,126-146, 174, such as selected from SEQ ID NOs: SEQ ID NO: 92, 97, 109, 116, 131,140-146 and 174; or b) an amino acid sequences having at least 90 %, such as at least 95 %, identity to a sequence as defined in a), provided that the CDRs are as defined for SEQ ID NO:1-3.
  • an sdAb variant according to option b) which has less than 100 % identity to any one of said sequences may differ with regard to the amino acid residues of the CDRs (still being within the definition of SEQ ID NOs: 1-3), and/or the framework amino acid residues of Kabat positions 6, 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b and/or 89.
  • the terms “capable of binding AAV9”, “AAV9 binding”, “capable of interacting with AAV9” and ”interaction with AAV9” as used in this disclosure refer to an event or a property of a polypeptide which may be tested for example by ELISA or by surface plasmon resonance (SPR) technology.
  • AAV9 interaction may be tested in an experiment in which the polypeptide to be tested is immobilized on a sensor chip of the surface plasmon resonance (SPR) instrument, and a liquid sample containing AAV9 (the target or analyte) is passed over the chip.
  • the target here AAV9
  • the target here AAV9
  • the polypeptide to be tested is contained in a liquid sample that is passed over the surface. The skilled person may then interpret the results obtained by such experiments to establish at least a qualitative measure of the binding affinity of the polypeptide for AAV9.
  • the AAV9 binding polypeptide may bind AAV9 “selectively”, meaning that it may bind to AAV9, while it does not bind to other AAV serotypes, or binds to other AAV serotypes to a much lower degree.
  • the term “selectivity”, sometimes referred to as “specificity”, of the binding polypeptide for a target refers to a binding polypeptide which will bind to the target with high affinity, but typically not to other antigens.
  • a selective or specific binding polypeptide/single domain antibody will not, or to a low extent, cross-react with other targets than the intended antigen.
  • binding “specifically” it is meant that the binding protein binds to its target (i.e.
  • AAV9 in a manner that can be distinguished from binding to non-target molecules, more particularly that the binding polypeptide binds its target (AAV9) with greater binding affinity than with which it binds other molecules. That is, the binding polypeptide does not bind to other, non-target, molecules, or does not do so to an appreciable or significant degree, or binds with lower affinity to such other molecules than with which it binds AAV9.
  • the biding polypeptides bind AAV9, but not other serotypes (or to a very low degree).
  • the interaction with other AAV serotypes may be less than 20 %, such as less than 10 % or less than 5 %, of the AAV9 interaction response as measured on a Biacore instrument as detailed in the examples below.
  • serotypes is referred to other distinct serotypes, such as for example AAV2, AAV5, and AAV8.
  • the present polypeptides selectively bind AAV9, with no or very low affinity for other serotypes. It may be noted that closely related sequence variants, such as AAV-PHP.eB, are not seen as a distinct serotype in this sense, and hence not included in the “other serotypes” not bound.
  • the AAV9 binding polypeptide disclosed herein exhibit an improved AAV9 binding property in relation to known sdAb based affinity ligands to AAV9.
  • a binding property that may be improved is binding capacity.
  • the present AAV9 binding polypeptides may have improved AAV9 binding capacity.
  • the inventors found that in addition to having an initial high binding capacity for AAV9, the polypeptides can retain a high AAV9 binding capacity also after alkali treatment. Improved alkaline stability is a great benefit in the context of affinity chromatography, as the chromatography material is conventionally subjected to cleaning using an alkaline agent (such as NaOH or KOH) between the purification cycles.
  • an alkaline agent such as NaOH or KOH
  • Increased alkaline stability of the affinity ligand means that the chromatography material can be used for a higher number of purification cycles before binding capacity becomes unacceptably low.
  • the alkaline stability is usually determined based on the binding capacity after a certain number of alkali treatment cycles.
  • the terms “treatment”, “exposure” and “cleaning” are used interchangeably.
  • “improved alkaline stability” can mean that the polypeptide can withstand a higher number of alkali cleaning cycles, or the same number of cycles but with harsher alkali conditions, without a deterioration in binding capacity.
  • “improved alkaline stability” can mean that the polypeptide will retain a higher percentage of the initial binding capacity after an equal number of cleaning cycles using the same conditions.
  • the alkaline stability may be particularly relevant for a polypeptide intended to be immobilized on a solid support.
  • the alkali treatment may involve contact or incubation with 0.05-1 M NaOH or 0.1-0.5 M NaOH, such as 0.3-0.5 M NaOH for a time period of 5-15 minutes, such as 10 minutes (600 seconds) or about 10 minutes.
  • the AAV9 polypeptides of the invention retain a high AAV9 binding capacity after repeated cleaning cycles involving 600 s exposure to 0.3 or 0.5 M NaOH.
  • the AAV9 binding polypeptide as disclosed herein may retain at least 50 % of the AAV9 binding capability after at least 8 repeated binding cycles followed by cleaning with 0.5 M NaOH.
  • the AAV9 binding polypeptide may retain at least 50 % binding capability after at least 10 cycles, such as after at least 12 cycles of alkali exposure. More preferably, the AAV9 binding polypeptide retains at least 50 % binding capability after at least 20, 22 or 24 cycles of exposure to 0.5 M NaOH. A higher number of cycles with at least 50 % binding capability retained indicates an improved alkaline stability.
  • a higher retained binding capacity after the same number of cycles of exposure to cleaning liquid indicates an improved alkaline stability.
  • the polypeptide may retain at least 50 %, such as at least 60 %, at least 70 %, at least 80 %or at least 90 %, of its initial target entity binding capacity, and/or after 20 cycles of contact with cleaning liquid the polypeptide retains at least 50 %, at least 60 %, such as at least 70 % or at least 80 % of its initial target entity binding capacity.
  • the polypeptide may retain at least 60 %, at least 70 %, at least 80 %, at least 90 %, such as at least 95 %, of the target entity binding capacity of the 2 nd cycle, and/or after 20 cycles of contact with alkaline cleaning liquid, the polypeptide may retains at least 60 %, at least 70 %, at least 80 %, at least 90 %, such as at least 95 %, of the target entity binding capacity of the 2 nd cycle.
  • a cycle of exposure to alkaline cleaning liquid may be a chromatography cycle as described herein, or a cycle of an alkaline stability assessment as described elsewhere herein. It will be understood that each binding cycle is followed by a cleaning step.
  • a cleaning step may be for example approximately 5-15 minutes incubation in contact with 0.1-0.5 M NaOH, such as for example approximately 5 minutes or 10 minutes or 15 minutes.
  • the time of incubation in contact with 0.5 M NaOH may be 5 ⁇ 0.5 minutes, 6 ⁇ 0.5 minutes, 7 ⁇ 0.5 minutes, or 8 ⁇ 0.5 minutes, 9 ⁇ 0.5 minutes 10 ⁇ 0.5 minutes, 11 ⁇ 0.5 minutes, 13 ⁇ 0.5 minutes, 14 ⁇ 0.5 minutes, or 15 ⁇ 0.5 minutes.
  • Said incubation may be for example at 22 +/- 2 °C.
  • the polypeptides of the present invention may have CDRs which contribute to improved alkaline stability.
  • polypeptides disclosed herein may have a stabilized framework, meaning that at least one of the framework regions is modified relative to the corresponding framework region of SEQ ID NO: 4 in such a way that it contributes to an increased alkaline stability of the polypeptide.
  • the polypeptide as a whole may have an improved alkaline stability.
  • certain mutations of the amino acid residue in that position are not only tolerated without compromising functionality, but may even lead to improved performance, especially an improved alkaline stability, of the resulting sdAb variant.
  • the antigen-binding polypeptide may comprise a single-domain antibody variant comprising an amino acid sequence in which at least one of the amino acid positions 6, 14, 19, 23, 24, 40, 43, 44, 45, 47, 60, 76, 79, 82b and 89 is modified in relation to the corresponding amino acid of SEQ ID NO: 4 and where the sdAb framework has increased alkaline stability compared to SEQ ID NO:4.
  • Particularly advantageous modifications are outlined in Table 6.
  • the antigen-binding polypeptide may optionally comprise a single-domain antibody variant comprising an amino acid sequence in which at least one, such as two, such as three, four five, six, seven, eight, nine or ten, of the residues in Kabat positions 19, 23, 24, 40, 43, 44, 45, 76, 79 and/or 89 are selected from the stabilizing amino acids indicated in Table 6. For example, at least eight, such as at least nine, of these positions may have amino acids selected accordingly. Table 6.
  • an antigen-binding polypeptide 200 comprises an N-terminal amino acid sequence 201, an sdAb variant 202 and a C-terminal amino acid sequence 203.
  • N- terminal amino acid sequence 201 and the C-terminal sequence 203 are optional.
  • the antigen-binding polypeptide may optionally be provided in the form of a multimeric antigen-binding polypeptide (“multimer”) schematically illustrated in Fig.2b-c.
  • a multimeric polypeptide 220 comprises multiple single-domain antibody variants 202 as disclosed herein, which may optionally be joined by a linker sequence 204.
  • N-terminal amino acid sequence 201’ is optional and can be a leader peptide of e.g.2-6, such as 4, amino acids.
  • C-terminal sequence 203 is optional and may comprise a purification tag, optionally including a linker.
  • the sdAb variant units 202 may be identical, or may differ from one another with regard to their amino acid sequence.
  • the polypeptide may comprise two sdAb variants (“dimer”), or three sdAb variants (“trimer”).
  • Fig.2c shows an exemplary trimer 230, comprising three sdAb variants 202.
  • a multimeric antigen-binding polypeptide may contain at least one additional amino acid sequence which does not represent an sdAb variant, for example a fusion partner polypeptide as described in connection with the fusion protein elsewhere herein.
  • Multimeric variants of the present polypeptide may be advantageous in that they can provide an increased binding capacity, and/or an improved stability, in relation to a polypeptide comprising a single copy of an sdAb variant.
  • the antigen-binding polypeptide or multimer may, in addition to the sdAb variant(s), optionally comprise additional amino acids located N- and/or C-terminally thereof, such as illustrated in Fig.2a-d.
  • Such a polypeptide or multimer should be understood as a polypeptide or multimer having one or more additional amino acid residues at the very first and/or the very last position in the polypeptide chain, i.e. at the N- and/or C-terminus of the polypeptide or multimer.
  • the antigen-binding polypeptides as defined herein may comprise any suitable number of additional amino acid residues, for example one, two, three, four, five, six, seven, eight, nine, ten or more, such as up to 20, additional amino acid residues optionally in a consecutive sequence.
  • a sequence of additional amino acids may consist of from 1 to 12 amino acids, and antigen binding polypeptide may comprise several such sequences.
  • the additional amino acid may be a legacy from recombinant protein expression, a leader peptide, a signal peptide (e.g. PelB or OmpA), a purification tag, a peptide intended for coupling to support, or the like.
  • the additional amino acid may be a spacer or linker as defined elsewhere herein, or have the ability to serve as spacer or linker.
  • the additional amino acid residues may individually or collectively improve production, purification, stabilization in vitro or coupling of the polypeptide to substrates of interest, for example to a solid support, such as a solid support described in connection to the aspect of a separation matrix.
  • Such additional N-terminal or C-terminal amino acid residues do typically not affect the functional properties, such as binding affinity, of the antigen-binding polypeptide or the single domain antibody as such.
  • the antigen-binding polypeptide when provided in the form of a multimer, comprising a plurality of single-domain antibody units, said additional amino acid residues may for example not be repeated for each occurrence of the single-domain antibody.
  • said additional amino acids may occur only at the N-terminus or the C-terminus of the multimer.
  • Said additional amino residues may be coupled to the sdAb or multimer by means of chemical conjugation (using known organic chemistry methods) or by any other means, such as expression as a fusion protein or joined in any other fashion, either directly or via a linker, for example a peptide linker as described above.
  • the polypeptide and/or multimer further comprises at the C-terminal or N-terminal end one or more tags or coupling elements, selected from the group consisting of a cysteine residue, a plurality of lysine residues and a plurality of histidine residues, and combinations thereof.
  • tags or coupling peptides include (His) 6 , (His) 6 -Cys and AEAAAKHHHHHHC (SEQ ID NO: 159).
  • the coupling element may e.g. be a single cysteine at the C-terminal end.
  • the coupling element(s) may be directly linked to the C- or N-terminal end, or it/they may be linked via a linker comprising up to 15 amino acids, such as 1-5, 1-10 or 5-10 amino acids.
  • This stretch should preferably also be sufficiently stable in alkaline environments not to impair the properties of the protein. For this purpose, it is advantageous if the stretch does not contain asparagine. It can additionally be advantageous if the stretch does not contain glutamine.
  • An advantage of having a C-terminal cysteine is that endpoint coupling of the protein can be achieved through reaction of the cysteine thiol with an electrophilic group on a support. This provides excellent mobility of the coupled protein.
  • a monomer moiety may refer to a single-domain polypeptide as described herein.
  • linkers may be used in order to for example increase stability or improve folding of fusion proteins.
  • a linker may also ensure a certain spatial distance between two sdAb variants, or between the sdAb variant and a fusion partner polypeptide, which in either case may be beneficial for the access of the target to the binding surface of the sdAb variant.
  • a multimer as defined herein may further comprise at least one linker.
  • a linker is present between each monomer within the multimer.
  • the linker may for example be selected from the group consisting of flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers.
  • Peptide linkers may be structured or unstructured. Typically, unstructured linkers are more flexible and less rigid than structured linkers. Alternatively, the linker may be a non-peptidic linker.
  • polypeptides disclosed herein, or sub-units thereof may be linked to each directly by peptide bonds between the C-terminal and N-terminal ends of the polypeptides.
  • two or more monomers, in other words monomer units or moieties, within the multimer can be linked by elements comprising oligomeric or polymeric species, such as elements comprising up to 15 or 30 amino acids, such as 1-5, 1-10 or 5-10 amino acids.
  • said linker comprises up to 15 amino acid residues.
  • the nature of such a link should preferably not destabilize the spatial conformation of the protein units, which is of the monomers within the multimer. This can e.g. be achieved by avoiding the presence of proline in the linkers.
  • linkers should preferably also be sufficiently stable in alkaline environments not to impair the properties of the protein units.
  • linkers do not contain asparagine.
  • linker does not contain glutamine.
  • the multimer may further at the N-terminal end comprise additional amino acid residues as described above, e.g. originating from the cloning process or constituting a residue from a cleaved off signaling sequence.
  • the number of additional amino acid residues may e.g. be 15 or less, such as 10 or less or 5 or less.
  • the antigen-binding polypeptide may be provided in the form of a fusion protein.
  • the antigen-binding polypeptide may be a fusion protein containing at least one sdAb variant as disclosed herein and a further polypeptide moiety, also referred to as a fusion partner polypeptide.
  • Fig.3a schematically illustrates a fusion protein 300 comprising, in the N- to C-terminal direction, a first polypeptide moiety 200 and a second polypeptide moiety 301, joined by an optional peptide linker 204.
  • the second polypeptide moiety 301 is located C- terminally of the first polypeptide moiety 200.
  • the second polypeptide moiety may be located N-terminally of the first polypeptide moiety.
  • the first polypeptide moiety 200 may comprise or consist of only a single-chain polypeptide 202 as illustrated in Fig.2a, or may comprise or consist of a single-chain polypeptide 202 and additional amino acids, such as the additional amino acid sequence 201 and/or linker 204.
  • the second polypeptide moiety 301 may be referred to as a fusion partner polypeptide.
  • the fusion partner polypeptide 301 may be a polypeptide that improves or adds at least one functionality or desirable property.
  • the fusion partner polypeptide may be a stabilizing polypeptide, which is a polypeptide that further improves stability, such as alkaline stability, of the antigen-binding polypeptide.
  • the fusion partner polypeptide may be a polypeptide that improves expression of the antigen-polypeptide in a recombinant cell, such as E. coli.
  • a fusion partner polypeptide may provide an opportunity for purification of the antigen-binding polypeptide by affinity chromatography.
  • a fusion protein of the present disclosure may in particular comprise an sdAb variant, or a plurality of sdAb variants, fused to an ⁇ -helix-containing polypeptide, such as a protein domain.
  • the ⁇ -helix containing polypeptide may contain at least one ⁇ -helix, such as at least 2 ⁇ -helices, such as 3, 4, 5 or 6 ⁇ -helices, such as up 10 ⁇ -helices. In embodiments, it may be an ⁇ -helix bundle domain, such as a three-helix bundle domain. In an ⁇ -helix bundle domain, the ⁇ -helices are spatially grouped together and form the major part of the protein domain, which may lack other prominent unstructured or beta-structured regions. Examples of ⁇ -helix bundle domains include protein domains of Staphylococcus aureus protein A (SpA) and albumin binding domain (ABD).
  • SpA Staphylococcus aureus protein A
  • ABS albumin binding domain
  • the fusion partner polypeptide of the fusion protein of the present disclosure may comprise an amino acid sequence derived from SpA domain A, domain B, domain C, domain D or domain E.
  • the fusion partner polypeptide may comprise an amino acid sequence having at least 80 % identity, such as at least 85 % or at least 90 % identity, to SEQ ID NO: 161 or 162.
  • the fusion protein of the present disclosure may be a multimeric protein, meaning that it may comprise more than one sdAb, or more than one fusion partner polypeptide. Multimeric variants containing multiple copies of an sdAb may be advantageous in that they can provide an increased binding capacity in relation to a fusion protein containing only one copy.
  • a multimeric fusion protein containing multiple fusion partner polypeptides may provide an improved stability.
  • the fusion protein may optionally be provided in the form of a multimeric fusion protein (“multimer”) containing at least two antigen-binding polypeptides, and/or at least two fusion partner polypeptides, as schematically illustrated in Fig.3b-c.
  • a multimeric fusion protein 310 comprises multiple units of the antigen-binding polypeptide 200 arranged sequentially in the N- to C-terminal direction, and which may optionally be joined to one another by linker sequences 204, which may be the same or different for each occurrence.
  • the antigen-binding polypeptides 200 may be identical, or may be different from one another with regard to their amino acid sequence.
  • Fig.3b shows three antigen-binding polypeptides 200 (“trimer”), but it is envisaged that the fusion protein may comprise any suitable number of said antigen-binding polypeptide 200.
  • the fusion protein may contain at least two, or at least 3, 4, 5 or 6 units of the antigen-binding polypeptide.
  • the fusion protein may contain e.g. up to 10 antigen-binding polypeptides 200.
  • the fusion protein may contain at least 2, such as 3, 4, 5 or 6 units, e.g. up to 10 units, of the fusion partner polypeptide.
  • Fig.3c illustrates a fusion protein 320 containing one first antigen-binding polypeptide 200 and three polypeptide moieties 301.
  • Linkers 204 are optional, and may be the same or different for each occurrence.
  • the polypeptide moieties 301 may be identical, or may be different with regard to their amino acid sequences.
  • the fusion protein may contain two fusion partner polypeptides and any suitable number of the antigen-binding polypeptide 200 as described above.
  • the fusion protein may contain one antigen- binding polypeptide flanked by two fusion partner polypeptides.
  • a fusion protein comprises multiple antigen-binding polypeptides and multiple fusion partner polypeptides, for example 2 or 3 of each
  • the different polypeptide moieties 200, 301 may be arranged in an alternating fashion instead of polypeptides of the same kind being arranged sequentially as depicted in Fig.3b (for the antigen-binding polypeptide 200) and Fig.3c (for the fusion partner polypeptide 301).
  • a multimeric fusion protein containing two units of each polypeptide may have the following general structure: [antigen-binding polypeptide]-[fusion partner polypeptide]-[ antigen- binding polypeptide]-[fusion partner polypeptide].
  • a fusion protein according to the present disclosure may have a structure as follows: ([A-L1] m -[Z-L2] n ) p wherein A represents a polypeptide as described herein; Z represents a further polypeptide moiety (fusion partner polypeptide) as described herein; L1 for each occurrence may be present or absent, and where present, represents a linker or spacer; L2 for each occurrence may be present or absent, and where present, represents a linker or spacer; m represents an integer of from 1 to 4; n represents an integer of from 1 to 4, or when m ⁇ 2, n represents 0 or an integer of from 1 to 4; and p represents an integer of from 1 to 4.
  • (m+n)*p that is, the total number of polypeptide moieties A and Z in the fusion protein, may be up to 10, such as up to 8.
  • m is 2 or 3
  • n is 1 and p is 1.
  • m is 1, n is 1 and p is 2 or 3.
  • fusion protein comprises at least two fusion partner polypeptides
  • at least one is located at the C terminus of the fusion protein.
  • at least two fusion partner polypeptides may be arranged sequentially at the C terminus of the fusion protein.
  • a short amino acid sequence such as a leader or signal peptide.
  • a fusion protein such as a multimeric fusion protein, a tag or spacer, or another short amino acid sequence may optionally be provided as desired.
  • the fusion partner polypeptide is not located at or near the N-terminus of the fusion protein.
  • any reference herein to the antigen-binding polypeptide of the present disclosure encompasses also multimeric variants and fusion proteins thereof, unless stated otherwise.
  • the antigen-binding polypeptide of the present invention can be produced by conventional biotechnological methods, by recombinant protein production using genetically engineered host cells.
  • the present disclosure provides an isolated nucleic acid encoding an antigen-binding polypeptide as described herein; an expression vector comprising said nucleic acid; and a host cell comprising said expression vector.
  • the host cells may be prokaryotic cells, such as bacteria, or eukaryotic cells.
  • exemplary prokaryotic host cells are Escherichia coli.
  • Suitable eukaryotic cells may be yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris, or animal cells, such as insect cells or mammalian cells commonly used in the field of biotechnology for the production of proteins, e.g.
  • the antigen-binding polypeptide can be purified, e.g. by conventional means known in the art.
  • a step of purifying the antigen-binding polypeptide may comprise immobilized metal affinity chromatography (IMAC).
  • IMAC immobilized metal affinity chromatography
  • Other methods of purification that may be used include affinity chromatography.
  • an affinity ligand that binds to a framework region of the sdAb variant may be used, or the affinity chromatography may use an affinity ligand based on a domain of SpA, wherein the affinity ligand binds to an VH region, such as VH3, of the antigen-binding polypeptide.
  • the sdAb variant of the present disclosure may be capable of VH3 interaction with SpA domain or variant thereof.
  • the affinity ligand may bind to a fusion partner, such as to an affinity tag included in the amino acid sequence of a fusion partner polypeptide.
  • the antigen-binding polypeptide as disclosed herein may alternatively 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 comprise: - step-wise coupling of the amino acids and/or the amino acid derivatives to form a polypeptide having protected reactive side-chains, - removal of the protecting groups from the reactive side-chains of the, and - folding of the polypeptide in aqueous solution.
  • the present antigen-binding polypeptide may be used for capture of the target entity for which it has an affinity.
  • the antigen-binding polypeptide as such is not intended for use as a therapeutic compound, and is preferably not intended for use in vivo.
  • the present antigen-binding polypeptide may be used for in vitro capture of the target entity.
  • the capture may be for the detection of the target entity within a sample, or for separation of the target entity from other components in a sample.
  • the antigen-binding polypeptide may be used for analytical separation of the target entity, or it may be used for preparative purification of the target entity.
  • the term “preparative” refers to the process of preparing a purified target entity, in particular during the manufacture of a product, such as a pharmaceutical product or a therapeutic composition, which comprises the target entity.
  • An in vitro method of detecting AAV9 viral particles present in a sample may comprise: providing a sample containing AAV9 viral particles; contacting the sample with an antigen-binding polypeptide, multimer or fusion protein as described herein, under conditions allowing the sdAb variant to bind to the AAV9 viral particles, wherein the binding event generates a detectable signal; and detecting the detectable signal.
  • Suitable means for generation of a detectable signal in response to a binding event are known in the art.
  • the antigen-binding polypeptide of the present invention may be used in applications involving interaction between antigen-binding polypeptide and its target entity, where either the antigen-binding polypeptide or the target entity is coupled to a support.
  • the antigen-binding polypeptide is coupled or immobilized to a support, it is still capable of binding its target entity.
  • this may be referred to as an adsorbent material or an affinity capture material.
  • Adsorbent materials may be used in various industrial or laboratory applications, including separation, purification, detection and/or quantification of the target entity.
  • a separation matrix as described herein is an adsorbent material intended for use in separation of a target entity from other components.
  • the coupling of the antigen- binding polypeptide to the support may optionally be provided via a C-terminal amino acid sequence suitable for coupling to a support.
  • a tag or spacer ending with a cysteine may be provided for coupling via a thioether bond, as described in more detail below.
  • a C-terminal fusion partner polypeptide may have such a C-terminal amino acid sequence suitable for coupling to a support as described below.
  • the antigen-binding polypeptide is not coupled to a support, it may be used e.g.
  • a reporter entity may be used for generating a detectable signal, wherein the reporter entity is capable of binding to the antigen binding polypeptide, or capable of binding to the fusion partner in the case of a fusion protein.
  • the reporter entity may be e.g. a fluorescent labelled or radiolabelled entity as known in the art. If the antigen binding polypeptide or fusion protein is capable of binding to IgG, the reporter entity may be based on IgG.
  • the antigen-binding polypeptide may be used in applications where it may be exposed to alkaline conditions, such as treatment with NaOH. Such treatments are frequently used in the field of chromatography, to strip a chromatography matrix of bound impurities before the next purification cycle (cleaning-in-place, as described above). However, alkali exposure or cleaning of a binding surface may be used also in other contexts.
  • the support may be a solid support and may optionally be a porous material.
  • the support may be or comprise a surface onto which the antigen-binding polypeptide is coupled. Examples of such support materials include conventional protein-binding support and surfaces such as a chip, a plate, a well, and a sheet.
  • the support may optionally be provided in other forms such as a fiber, a membrane, a fibrous matrix, a filter, a porous monolith, a particle or a bead, such as a gel bead as used in chromatography resins.
  • Particles or beads can be porous or non-porous. Particles or beads may include magnetic beads. Supports in the form of beads or particles can be used as a packed bed or in a suspended form. Suspended forms include those known as expanded beds and pure suspensions, in which the particles or beads are free to move. In case of monoliths, packed bed and expanded beds, a separation procedure commonly follows conventional chromatography with a concentration gradient. In case of pure suspension, batch-wise mode will be used.
  • the support may be made of any suitable material, as outlined in more detail below.
  • a conventional affinity separation matrix is often of organic nature and based on polymers that expose a hydrophilic surface to the aqueous media used, i.e. expose hydroxy (-OH), carboxy (-COOH), carboxamido (-CONH 2 , possibly in N- substituted forms), amino (-NH 2 , possibly in substituted form), oligo- or polyethylenoxy groups on their external and, if present, also on internal surfaces.
  • the antigen-binding polypeptide may be attached to the support via known coupling techniques utilizing e.g. thiol, amino and/or carboxy groups present in the antigen-binding polypeptide.
  • the coupling may be a random or multipoint coupling (e.g. via a plurality of lysines or histidines) or a single point coupling (e.g. via a single cysteine).
  • the sdAb variant as such does not comprise any histidine residues.
  • the antigen- binding polypeptide as a whole may comprise a histidine tag, such as a (His) 6 tag.
  • the antigen-binding polypeptide may be attached to the support by non-covalent bonding, such as physical adsorption or biospecific adsorption.
  • the antigen-binding polypeptide may be coupled to the support via thioether bonds. Methods for performing such coupling are well-known in this field and easily performed by the skilled person in this field using standard techniques and equipment. Thioether bonds are flexible and stabile and generally suited for use in affinity chromatography.
  • the thioether bond is via a terminal or near-terminal cysteine residue on the antigen-binding polypeptide
  • the mobility of the coupled antigen-binding polypeptide is enhanced which provides improved binding capacity and binding kinetics.
  • the antigen-binding polypeptide is coupled via a C-terminal cysteine provided on the protein as described above. This allows for efficient coupling of the cysteine thiol to electrophilic groups, e.g. epoxide groups, halohydrin groups etc. on a support, resulting in a thioether bridge coupling.
  • the support may comprise a polymeric material. Polymeric materials include materials of natural or synthetic polymers, and combinations thereof.
  • the support may comprise a polyhydroxy polymer, such as a polysaccharide.
  • polysaccharides include e.g. dextran, starch, cellulose, pullulan, agar, agarose etc, including derivatives thereof.
  • the beneficial features of these polysaccharides could further be improved by means of covalent cross-linking, rendering them particularly suitable for enzyme immobilization with a wide range of derivatization methods taking advantage of chemical modification of a fraction of the polymer hydroxyls, where the hydroxyl functions could be partially or totally derivatized.
  • agarose derivatives include e.g.
  • the support may comprise agar or agarose, such as crosslinked agarose.
  • Such supports used in the present invention can easily be prepared according to standard methods, such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964)).
  • the base matrices are commercially available products, such as crosslinked agarose beads sold under the name of SEPHAROSETM FF (CytivaTM).
  • the support has been adapted to increase its rigidity using the methods described in US6602990 or US7396467, which are hereby incorporated by reference in their entirety, and hence renders the matrix more suitable for high flow rates.
  • Synthetic polymers useful as material for the support include polyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides etc.
  • the surface of the matrix can be hydrophilized to expose hydrophilic groups as defined above to a surrounding aqueous liquid.
  • hydrophobic polymers such as matrices based on divinyl and monovinyl-substituted benzenes
  • the surface of the matrix can be hydrophilized to expose hydrophilic groups as defined above to a surrounding aqueous liquid.
  • Such polymers are easily produced according to standard methods.
  • a commercially available product such as SOURCETM (CytivaTM) may be used.
  • the solid support according to the invention comprises a support of inorganic nature, e.g. silica, zirconium oxide etc.
  • the antigen-binding polypeptide may be coupled to a support which is a convection-based chromatography matrix.
  • Such convection-based chromatography matrix may comprise a porous polymer membrane, a filter, a fibrous matrix, or a porous monolith.
  • a porous polymer membrane examples include Mustang TM membranes (Cytiva) and Sartobind TM membranes (Sartorius).
  • a fibrous support may be based on electrospun polymeric fibers or cellulose fibers, optionally non-woven fibers.
  • a fibrous matrix may thus be a non-woven fibrous matrix.
  • the fibers may have a cross-sectional diameter of 10-1000 nm, such as 200-800 nm, 200-400 nm or 300-400 nm.
  • Such a fibrous support can be found in a HiTrap FibroTM unit (CytivaTM).
  • adsorbent material or separation matrix as described herein may be used for the same purposes as mentioned above for the antigen-binding polypeptide.
  • the invention provides a chromatography material comprising a separation matrix as disclosed herein, as well as chromatography columns or devices incorporating such separation matrix.
  • the adsorbent material or separation matrix may be a sensor surface intended for use in a sensor or other detection or quantification devices.
  • the present invention provides a method of separating or isolating a target entity, wherein a separation matrix as disclosed herein is used.
  • the method comprises contacting a liquid sample comprising the target entity with a separation matrix as disclosed herein.
  • the contacting is performed under conditions under which the target entity can bind to the first polypeptide moiety.
  • the method may furthermore comprise washing said separation matrix with a washing liquid, eluting the target entity from the separation matrix with an elution liquid, and optionally cleaning the separation matrix with a cleaning liquid.
  • the cleaning liquid can alternatively be called a cleaning-in-place (CIP) liquid.
  • the cleaning liquid typically is an alkaline solution, such as comprising NaOH or KOH of at least 0.05 M, such as 0.05-1 M, such as 0.05-0.5 M, such as at least 0.1 M, such as 0.1-0.5 M, e.g.0.3 M or 0.5 M.
  • the contact (incubation) time may be at least 10 min.
  • the binding, eluting and cleaning steps may advantageously be repeated at least 10 times, such as at least 20 times.
  • the liquid sample to be purified may be any sample comprising the target entity in an environment from which it is to be purified or separated.
  • the sample may be obtained from a cell culture, such as a clarified cell culture harvest, and may have been subjected to one or more conventional steps of filtration, concentration, dilution and/or buffer exchange, and optionally one of more initial chromatography steps, in particular a step which is not based on affinity chromatography, such as ion exchange chromatography, hydrophobic interaction chromatography, multimodal chromatography, or size exclusion chromatography.
  • the sample may be a clarified and filtered cell culture harvest.
  • the sample may have been subjected to one or more steps of filtration by tangential flow filtration (TFF).
  • TFF tangential flow filtration
  • liquid sample Prior to contacting with the present separation matrix, liquid sample contains the target entity and at least one impurity, such as host cell protein (HCP) or host cell nucleic acids.
  • HCP host cell protein
  • the present separation matrix is useful for separating the target entity from such impurities, and after performing the above process, the eluate containing the target entity has a reduced level of at least one such impurity.
  • the eluate containing purified target entity may be subjected to one or more steps of filtration, concentration, dilution or buffer exchange, or chromatography step(s) not based on affinity chromatography, such as ion exchange chromatography, hydrophobic interaction chromatography, multimodal chromatography, or size exclusion chromatography.
  • a further chromatography step performed after the affinity separation of the present disclosure may be referred to as a polishing step. While the invention is described herein with respect to exemplary embodiments, the skilled person will appreciate that the invention is not limited to these. As the skilled person will realize, the function of any polypeptide is dependent on the tertiary structure of the polypeptide.
  • the disclosure encompasses modified variants of the antigen-binding polypeptide.
  • modified variants are such that their affinity for the target AAV vector, in particular AAV9, is at least retained.
  • Use of the verb “to comprise” and its conjugations does not exclude the presence of elements or steps other than those stated.
  • a polypeptide “comprising” a complementarity determining region 1 (CDR1) can also have further CDRs, such as a CDR2 and a CDR3, and may have other regions, such as FWRs, and optionally other structures or sequences.
  • Example 1 Production of polypeptides This example describes the generation of glycerol stock solution for the production of candidate polypeptides used in Examples 3-6 and 8. Materials and equipment used were as follows: plasmid DNA for the candidate polypeptides to be produced; chemically competent BL21(DE3) E.
  • coli prepared in-house, KCM buffer (5X) 0.5M KCl, 0.15M CaCl2, 0.25M MgCl2, LB culture medium (InvitrogenTM), carbenicillin (100 mg/ml), agar plates (BD BACTOTM Agar) supplemented with carbenicillin (100 ⁇ g/ml) (PanReac AppliChemTM), 14 ml FalconTM Round-Bottom (CorningTM), Infors HT Shaking incubator. Desired amino acid sequences of candidate polypeptides were back-translated to DNA and optimized to remove rare codons.
  • Plasmid DNA was used for transformation into chemically competent BL21(DE3) E. coli, plated on agar plate supplemented with 100 ⁇ g/ml carbenicillin and grown overnight at 37°C. For each candidate, a single colony was used to inoculate 4 mL LB media supplemented with 100 ⁇ g/ml carbenicillin in a 14 ml round-bottom tube and grown over night at 37°C, 200 rpm agitation.
  • Example 2 AAV9 binding of sdAb variants having CDR2/CDR3 variations The ability of the candidate polypeptides to bind AAV9 was investigated by surface plasmon resonance analysis using a BiacoreTM instrument (Cytiva, Sweden). Each candidate polypeptide contained an sdAb with variations in the CDR2 and CDR3 amino acid sequences as outlined in Table 7. All candidates had the same CDR1 amino acid sequence RTLSDYTMG (SEQ ID NO: 16).
  • Constructs were expressed with pelB signal peptide and a C-terminal tag (AEAAAKHHHHHHC, SEQ ID NO: 161) in E. coli. Protein purification was performed using immobilized metal affinity chromatography (IMAC). Purified candidate polypeptides were shipped to Cytiva, Sweden. Biotinylation of purified candidate polypeptides Purified candidate polypeptides were biotinylated at the C-terminal cysteine using EZ-LinkTM Maleimide-PEG2-Biotin, No-WeighTM Format (ThermoFisher Scientific) with a 5X molar excess of biotin. After biotinylation, buffer exchange to PBS was performed using PD MidiTrapTM G-25 columns (Cytiva).
  • BiacoreTM Series S SA chip AAV9 (2E12 viral particles (vp)/ml) was used as analyte. Materials and equipment used were as follows: BiacoreTM Series S SA sensor chips, BiacoreTM 8K+ instrument (all from Cytiva, Sweden); AAV9 (prepared in-house); candidate polypeptides generated as described above. Immobilization was performed using a standard method in BiacoreTM software with coupling of biotinylated polypeptides in Flow Cell 2 (FC2) and activation/inactivation in Flow cell 1 (FC1).
  • Biotinylated AAV9 binding polypeptide variants were diluted in PBS at a concentration of 100 ⁇ g/ml. The immobilization levels obtained were 5503 +/- 324 RU. In each run the polypeptide was immobilized in FC2. Multiple sensor chips were used until all candidates were tested.
  • BiacoreTM method for binding analysis Running buffer: PBS-P+; Flow rate: 5 ⁇ l/min; Sample injection: 2400 s/40 min over both Flow Cells (FC1 and FC2); Dissociation time: 2400 s/40 min; Regeneration: 10 mM Glycine-HCl pH 1.5, 30 ⁇ l/min, 2x30 s.
  • Injections of analyte (AAV9) for each channel (cycles) were as follows: running buffer, AAV92.5E11 vp/ml, AAV91E12 vp/ml, AAV92E12 vp/ml. All sensorgrams were generated as reference subtracted and the responses as the difference between baseline before injection and signal just before end of injection. Results Table 8 below the reports the immobilization level and maximum binding responses recorded for the respective analyte concentrations. All candidates showed binding to AAV9 and in particular vh29, vh30, vh33, and vh35-41 demonstrated excellent binding capacity, and the binding capacity was higher for the two highest AAV9 concentrations tested.
  • Example 3A Binding and stability of further candidate polypeptides with CDR point modifications This example further investigated the effect of single-point amino acid modifications in the CDR regions of the candidate polypeptides on alkaline stability and target binding.
  • the candidate polypeptides tested were expressed as fusion protein in which the respective sdAb had SEQ ID NO:161 fused to its C-terminal via a linker. All candidates had an N-terminal OmpA signal peptide and a C-terminal (His) 6 tag.
  • the sdAb sequences are presented in Tables 9a-c.
  • the sdAb of SEQ ID NO:92 was used as the reference, into which selected single point mutations were introduced. As indicated in Tables 9a-c below, each candidate differed from SEQ ID NO:92 in one amino acid position, either in CDR1, CDR2 or CDR3. The following aspects were evaluated: (1) Affinity assessment of AAV9 Interaction (tested through high concentration injection of AAV9). (2) Alkaline stability (tested by reduction of AAV9 binding after increasing number of treatments with 0.5 M NaOH).
  • Each flask was inoculated with approximately 500 ⁇ l from the previous overnight culture to obtain a starting OD600 of 0.05.
  • the flasks were incubated in Infors HT shaking incubator for approximately 3.5 hours at 37°C and 140 rpm shaking until OD600 reached 1.0. Thereafter 50 ⁇ l IPTG (1 M) was added to a final concentration of 1 mM and the flasks were incubated in Infors HT shaking incubator at 27°C and 140 rpm shaking for 18 hours, whereafter the cultures were transferred to Falcon tubes (50 ml).
  • Protein was eluted using 50 mM sodium acetate pH 3.5 in a step gradient over 5 CV. Concentration of the purified samples was measured using NanoDrop (Thermo Scientific) according to manufacturer’s instructions. Following purification, samples were kept in refrigerator in 41ntarctic tubes until all measurements had been performed and were then kept in freezer until Biacore analysis. Protein mass was determined by liquid chromatography–mass spectrometry on a ACQUITY Rda Detector (WatersTM) using a BioRessolve column (WatersTM). Biacore analysis of binding affinity for AAV9 To assess binding to AAV9, the candidate polypeptides were immobilized on a BiacoreTM Series S CM5 chip.
  • AAV9 (2E12 viral particles (vp)/ml) was used as analyte.
  • Materials and equipment used were as follows: BiacoreTM Series S CM5 sensor chips, BiacoreTM Amine coupling kit, BiacoreTM Acetate buffer pH 5.0, BiacoreTM 8K+ instrument (all from Cytiva, Sweden); AAV9 (prepared in-house); candidate polypeptides generated as described above. Immobilization was performed using a standard method in BiacoreTM software with coupling of AAV9 binding polypeptide variants in Flow Cell 2 (FC2) and activation/inactivation in Flow cell 1 (FC1). AAV9 binding polypeptide variants were diluted in BiacoreTM Acetate buffer pH 5.0 at a concentration of 25 ⁇ g/ml.
  • the immobilization levels obtained were 3484 +/- 197 RU. In each run the polypeptide was immobilized in FC2. Multiple sensor chips were used until all candidates were tested.
  • Running buffer PBS-P+; Flow rate: 5 ⁇ l/min; Sample injection: 2400 s/40 min over both Flow Cells (FC1 and FC2); Dissociation time: 2400 s/40 min; Regeneration: 10 mM Glycine-HCl pH 1.5, 30 ⁇ l/min, 2x30 s.
  • Injections of analyte (AAV9) for each channel (cycles) were as follows: running buffer, 2E11 vp/ml, 5E11 vp/ml, 2E12 vp/ml.
  • Biacore method for alkaline stability (per cycle): Running buffer: PBS-P+; Flow rate: 10 ⁇ l/min; Sample injection 1 (AAV95E11 vp/ml): 300 s over both Flow Cells; Dissociation time 1: 30 s; Sample injection 2 (0.5 M NaOH): 600 s over both flow cells; Dissociation time 2: 0 s; Regeneration: 10 mM Glycine-HCl pH 1.5, 30 ⁇ l/min, 2x30 s. This cycle was repeated 40 times to follow stability of AAV9 response values. All sensorgrams were generated as reference subtracted and the responses as the difference between baseline before injection and signal just before end of injection.
  • a binding capacity of at least 80 % of that of vh118 is considered satisfactory.
  • some point mutations in the CDR regions cf. SEQ ID NOs:13, 14, 37, 39, 42-44, 56
  • maintaining a binding capacity of at least 50 % after 10 cycles is considered acceptable; a retained binding capacity of at least 50 % after 15 cycles being preferred.
  • Particularly preferred are those variants that exhibited a normalized binding capacity of at least 100 % and maintained at least 50 % binding capacity for 20 cycles or more such as at least 24 cycles.
  • the candidate polypeptides tested were expressed as fusion protein in which the respective sdAb had SEQ ID NO: 161 fused to its C-terminal via a linker. All candidates had an N-terminal OmpA signal peptide and a C-terminal (His)6 tag.
  • the sdAb sequences tested are presented in Table 9d.
  • the sdAb of SEQ ID NO:92 (vh118) was used as the reference, into which point mutations were introduced in amino acid positions 100b and/or 100c. The following aspects were evaluated: (1) Affinity assessment of AAV9 interaction (tested through high concentration injection of AAV9). (2) Alkaline stability (tested by reduction of AAV9 binding after increasing number of treatments with 0.5 M NaOH).
  • Candidate polypeptides were designed, generated and evaluated for binding affinity and alkaline stability on a Biacore instrument, as described in Example 3A. Results The immobilization levels obtained were 3394 +/- 183 RU. The response levels of AAV9 interactions of the candidate polypeptides using the maximum AAV9 concentration (2E12 vp/ml) was normalized to the response of the candidate vh118 and summarized in Table 9d. It was seen that a satisfactory AAV9 binding capacity was achieved for many of the investigated point mutations. Alkaline stability was recorded as the number of cycles with at least 50 % binding capacity retained. The first cycle was excluded, and binding capacity values were normalized against the binding capacity recorded for the second cycle.
  • the candidate polypeptides tested were expressed as fusion protein in which the respective sdAb had SEQ ID NO: 161 fused to its C-terminal via a linker, and had a C-terminal (His) 6 - tag.
  • the sdAb sequences variants are presented in Tables 10a-b. The following aspects were evaluated: (1) Affinity assessment of AAV9 interaction (tested through high concentration injection of AAV9). (2) Alkaline stability (tested by reduction of AAV9 binding after increasing number of treatments with 0.5 M NaOH).
  • Table 10b FW FWR1 FWR2 FWR3 FWR4 variant 1 QVQLQQSGGGSVQ WFRQRPGKEREFVA YADSVKGRFTISRDNAKNTVY WGQGTQVTVSS AGGSLRLSCTISG (SEQ ID NO:152) LQMNSLKPEDTAVYYCAA (SEQ ID NO: 8) (SEQ ID NO:149) (S
  • Protein expression culture medium (TB medium supplemented with 100 ⁇ g/ml carbenicillin and 2 mM MgCl 2 ) was prepared and added to filled 500 ml baffled glass shake flasks (50 ml per flask, 7 flasks in total). Each flask was inoculated with approximately 500 ⁇ l from the previous overnight culture to obtain a starting OD600 of 0.05. The flasks were incubated in Infors HT shaking incubator for approximately 3.5 hours at 37°C and 140 rpm shaking until OD600 reached 1.0.
  • IPTG 1 M
  • HT shaking incubator 27°C and 140 rpm shaking for 18 hours
  • the cultures were transferred to Falcon tubes (50 mL).
  • the Falcon tubes were incubated in 48°C water bath for 2 hours followed by pelleting of cell debris by centrifugation at 8000 g for 10 min and filtration of supernatant with a 0.45 ⁇ m filter.
  • Clarified lysate was loaded on HisTrapTM FF 1 mL chromatography columns (CytivaTM), equilibrated in 50 mM sodium phosphate pH 7.5, 500 mM NaCl, allowing a residence time of about 2 min. The column was then washed with 15 column volumes (CV) of 50 mM sodium phosphate pH 7.5, 500 mM NaCl. Protein was eluted using 0.5 M imidazole pH 7.5-8, linear gradient (0-100 %) over 10 CV. Eluted protein was buffer-exchanged into phosphate buffered saline (Medicago) pH 7.4 using gravity flow columns prepacked with SephadexTM G-25 resin (Cytiva) prior to further analysis.
  • BiacoreTM Series S CM5 sensor chips BiacoreTM Amine coupling kit, BiacoreTM Acetate buffer pH 5.0, BiacoreTM 8K+ instrument (all from Cytiva, Sweden); AAV9 (prepared in-house); candidate polypeptides generated as described above.
  • Immobilization was performed using a standard method in BiacoreTM software with coupling of AAV9 binding polypeptide variants in Flow Cell 2 (FC2) and activation/inactivation in Flow cell 1 (FC1).
  • AAV9 binding polypeptide variants were diluted in acetate buffer pH 5.0 at a concentration of 25 ⁇ g/ml. The immobilization levels obtained were 3001 +/- 159 RU.
  • Biacore analysis of alkaline stability was done on the BiacoreTM chips prepared above. After evaluation of AAV9 binding (see above), the samples were subjected to followed by repeated cycles of binding to AAV9 (5E11 vp/ml) followed by injection of NaOH (0.5 M).
  • Biacore method for alkaline stability (per cycle): Running buffer: PBS-P+; Flow rate: 10 ⁇ l/min; Sample injection 1 (AAV95E11 vp/ml): 300 s over both Flow Cells; Dissociation time 1: 30 s; Sample injection 2 (0.5 M NaOH): 600 s over both flow cells; Dissociation time 2: 0 s; Regeneration: 10 mM Glycine-HCl pH 1.5, 30 ⁇ l/min, 2x30 s. This cycle was repeated 40 times to follow stability of AAV9 response values. All sensorgrams were generated as reference subtracted and the responses as the difference between baseline before injection and signal just before end of injection.
  • results The response levels of AAV9 interactions of the candidate polypeptides using the maximum AAV9 concentration (2E12 vp/ml) was normalized to the response of vh118 and summarized in Table 11, recording alkaline stability as the number of cycles with at least 50 % binding capacity retained.
  • Fig.5 presents the result of the alkaline stability assessment, plotting the binding capacity against the no. of cycles.
  • the binding response values were normalized against the binding capacity recorded for the second cycle (i.e., excluding the first cycle). In each cycle, 0.5 M NaOH was injected with a contact time of 10 min at 10 ⁇ l/min.
  • Example 5A Comparative study of AAV9 binding and stability
  • 7 candidate polypeptides were tested and compared to a commercially available affinity ligand (CaptureSelectTM Biotin Anti-AAV9 Conjugate, ThermoFischer Scientific).
  • the candidate polypeptides were expressed as fusion proteins in which the respective sdAb had SEQ ID NO: 161 fused to its C-terminal via a peptide linker (AA), and all constructs had a C-terminal tag (HHHHHHC).
  • the sdAb sequences variants are presented in Table 12.
  • Biotinylation of purified candidate polypeptides Purified candidate polypeptides were biotinylated at the C-terminal cysteine using EZ-LinkTM Maleimide-PEG2-Biotin, No-WeighTM Format (ThermoFisher Scientific) with a 2X molar excess of biotin. SEC purification of biotinylated candidate polypeptides Proteins were additionally purified by size exclusion chromatography (SEC) for separation from excess biotin, non-biotinylated protein dimers and other impurities.
  • SEC size exclusion chromatography
  • AAV9 (2E12 viral particles (vp)/ml) was used as analyte.
  • Materials and equipment used were as follows: BiacoreTM Series S SA sensor chips, BiacoreTM 8K+ instrument (all from Cytiva, Sweden); AAV9 (prepared in-house); candidate polypeptides generated as described above; CaptureSelectTM Biotin Anti-AAV9 Conjugate (Thermo ScientificTM). Immobilization was performed using a standard method in BiacoreTM software with coupling of biotinylated AAV9 binding polypeptide variants and CaptureSelectTM Biotin Anti-AAV9 Conjugate (Thermo ScientificTM) in Flow Cell 2 (FC2) and activation/inactivation in Flow cell 1 (FC1).
  • Biotinylated AAV9 binding polypeptide variants were diluted in PBS at a concentration of 100 ⁇ g/ml and CaptureSelectTM Biotin Anti-AAV9 Conjugate (Thermo ScientificTM) was diluted in PBS at a concentration of 10 ⁇ g/ml.
  • BiacoreTM method for binding analysis Running buffer: PBS-P+; Flow rate: 5 ⁇ l/min; Sample injection: 2400 s/40 min over both Flow Cells (FC1 and FC2); Dissociation time: 2400 s/40 min; Regeneration: 10 mM Glycine-HCl pH 1.5, 30 ⁇ l/min, 2x30 s.
  • Injections of analyte (AAV9) for each channel (cycles) were as follows: running buffer, 2E11 vp/ml, 5E11 vp/ml, 2E12 vp/ml. All sensorgrams were generated as reference subtracted and the responses as the difference between baseline before injection and signal just before end of injection.
  • Biacore analysis of alkaline stability Assessment of alkaline stability was done on the same BiacoreTM chips as used for binding analysis as described above. After evaluation of AAV9 binding (see above), the samples were subjected to 88 repeating cycles of exposure to NaOH (0.3 M).
  • Biacore method for alkaline stability (per cycle): Running buffer: PBS-P+; Flow rate: 10 ⁇ l/min; Sample injection 1 (AAV95E11 vp/ml or running buffer PBS-P+ every second cycle to reduce analyte consumption): 300 s over both flow cells; Dissociation time 1: 30 s; Sample injection 2 (0.3 M NaOH): 600 s over both flow cells; Dissociation time 2: 0 s; Regeneration: 10 mM Glycine-HCl pH 1.5, 30 ⁇ l/min, 2x30 s.
  • the alkaline stability of the inventive candidates was exceedingly high. In fact, for one candidate, vh377, the AAV9 response never dropped below 50 % during the 88 cycles.
  • the result of the alkaline stability assessment is also shown in Fig.6.
  • the Biacore run stopped between cycle 28 and 30, hence the data point at cycle 30 was not measured. The run was continued, and next binding data was measured at cycle 32.
  • Example 5B Comparative study of target selectivity
  • the binding selectivity for AAV9 was tested for the 7 candidate polypeptides of Example 5A and a commercially available AAV9 binding affinity ligand.
  • the candidate polypeptides were expressed as fusion proteins in which the respective sdAb had SEQ ID NO: 161 fused to its C- terminal via a peptide linker (AA), and all constructs had a C-terminal tag (HHHHHHC).
  • Materials and methods IMAC purified candidate polypeptides generated in Example 5A were used. Purified candidate polypeptides were biotinylated at the C-terminal cysteine using EZ-LinkTM Maleimide-PEG2-Biotin, No-WeighTM Format (ThermoFisher Scientific) with a 2X molar excess of biotin. Proteins were separated from excess biotin by dialysis.
  • Dialysis of biotinylated protein samples was performed according to Slide-A-LyzerTM G2 Dialysis Cassette 3 mL 10 kDa MWCO (Thermo ScientificTM) protocol using PBS as dialysis buffer. Concentration of dialyzed samples were measured using NanoDrop (Thermo Scientific) according to manufacturer’s instructions and were then kept in freezer until further analysis. Biacore for different AAV To assess binding to different AAV serotypes, the candidate polypeptides as well as CaptureSelectTM Biotin Anti-AAV9 Conjugate (Thermo ScientificTM) were immobilized on a BiacoreTM Series S SA chip.
  • Tangential flow filtrated (TFF) AAV material for serotype AAV2, AAV5, AAV8 and AAV9 were used as analytes.
  • Materials and equipment used were as follows: BiacoreTM Series S SA sensor chips, BiacoreTM 8K+ instrument (all from Cytiva, Sweden); TFF material of AAV2, AAV5, AAV8, AAV9 (around 1E12 vp/ml for all AAV serotypes) (prepared in-house); candidate polypeptides generated as described above; CaptureSelectTM Biotin Anti-AAV9 Conjugate (Thermo ScientificTM).
  • Immobilization was performed using a standard method in BiacoreTM software with coupling of biotinylated AAV9 binding polypeptide variants and CaptureSelectTM Biotin Anti-AAV9 Conjugate in Flow Cell 2 (FC2) and activation/inactivation in Flow cell 1 (FC1).
  • Biotinylated AAV9 binding polypeptide variants were diluted in PBS at a concentration of 100 ⁇ g/ml and CaptureSelectTM Biotin Anti-AAV9 Conjugate was diluted in PBS at a concentration of 10 ⁇ g/ml. Immobilization was performed to get similar molar amounts of AAV9-binding polypeptides, hence different immobilization levels depending on molecular weight.
  • BiacoreTM method for binding analysis Running buffer: PBS-P+; Flow rate: 10 ⁇ l/min; Sample injection: 500 s over both Flow Cells (FC1 and FC2); Dissociation time: 500 s; Regeneration: 10 mM Glycine-HCl pH 1.5, 30 ⁇ l/min, 2x30 s. Injections of analyte for each channel (cycles) were as follows: running buffer, AAV2 TFF, AAV5 TFF, AAV8 TFF, AAV9 TFF. All sensorgrams were generated as reference subtracted and the responses as the difference between baseline before injection and signal just before end of injection. Results Immobilization levels obtained are shown in Table 14.
  • Fig.7a- h are sensorgrams showing the binding response to TFF samples of different AAV serotypes: AAV9 (dashed line), AAV8 (dotted line), AAV2 (solid line), AAV5 (solid line).
  • the running buffer response is shown in solid line.
  • the candidates vh97 (Fig.7a), vh118 (Fig.7b), vh324 (Fig.7c), vh376 (Fig.7d), vh377 (Fig.7e), vh378 (Fig.7f) and vh379 (Fig.7g) were selective for AAV9, with no binding to AAV2, AAV5 or AAV8 samples.
  • the reference affinity ligand also showed high binding to both AAV9 and AAV8 samples (Fig.7h).
  • Example 6A AAV9 binding polypeptide with or without C-terminal fusion partner
  • a single-domain antibody capable of binding AAV9 was fused to a stabilizing polypeptide and the impact on protein expression, antigen binding capacity and alkaline stability was investigated.
  • the polypeptides tested are identified in Table 15.
  • a single-domain antibody (sdAb) was compared to a fusion of the same single-domain antibody with a stabilizing polypeptide.
  • the single domain antibody (denoted “sdAb”) was an AAV9-binding VHH variant (SEQ ID NO:92).
  • the stabilizing polypeptide (denoted “Z”) was an SpA domain Z variant (SEQ ID NO: 161) in which binding to the Fc and VH3 portions of IgG had been abolished.
  • Each flask was inoculated with approximately 500 ⁇ l from the previous overnight culture to obtain a starting OD600 of 0.05.
  • the flasks were incubated in an Infors HT shaking incubator for approximately 3.5 hours at 37°C and 140 rpm shaking until OD600 reached 1.0.
  • 50 ⁇ l of IPTG (1 M) was added to a final concentration of 1 mM and the flasks were incubated in Infors HT shaking incubator at 27°C and 140 rpm shaking for 18 hours, whereafter the cultures were transferred to Falcon tubes (50 ml).
  • Protein was eluted using 50 mM sodium acetate pH 3.5 in a step gradient over 5 CV. Concentration of the purified samples was measured using NanoDrop (ThermoFisher Scientific) according to manufacturer’s instructions. Following purification, samples were kept in refrigerator in Eppendorf tubes until all measurements had been performed and were then kept in freezer until Biacore analysis. Protein mass was determined by liquid chromatography–mass spectrometry on a ACQUITY Rda Detector (WatersTM) using a BioRessolve column (WatersTM).
  • Biacore analysis of binding affinity for AAV9 To assess binding to AAV9, the candidates were immobilized on a BiacoreTM Series S CM5 chip using Amine coupling kit (CytivaTM) and analyzed using BiacoreTM 8K+ instrument (CytivaTM). A sample containing AAV9 viral particles (vp) at a concentration of 2E12 vp/ml was used as analyte. Immobilization was performed using a standard method in Biacore software with coupling of the polypeptides in Flow Cell 2 (FC2) and activation/inactivation in Flow cell 1 (FC1).
  • FC2 Flow Cell 2
  • FC1 activation/inactivation in Flow cell 1
  • Polypeptides were diluted in BiacoreTM Acetate buffer pH 5.0 (CytivaTM) at a concentration of 17-30 ⁇ g/ml, depending on their molecular weight. The immobilization levels varied between different polypeptide variants corresponding to their molecular weight. In each run the polypeptide was immobilized in FC2.
  • BiacoreTM method for binding analysis Running buffer: PBS-P+; Flow rate: 5 ⁇ l/min; Sample injection: 2400 s/40 min over both FC1 and FC2; Dissociation time: 2400 s/40 min; Regeneration: 10 mM Glycine-HCl pH 1.5, 30 ⁇ l/min, 2x30 s.
  • Injections of analyte (AAV9) for each channel (cycles) were as follows: running buffer, AAV92E11 vp/ml, 5E11 vp/ml, 2E12 vp/ml. All sensorgrams were generated as reference subtracted and the responses as the difference between baseline before injection and signal just before end of injection. Biacore analysis of alkaline After evaluation of AAV9 binding (see above) the same chips were subjected to repeated cycles of binding to AAV9 (5E11 vp/ml) followed by injection of NaOH (0.5 M) for the assessment of alkaline stability.
  • AAV9 analyte
  • Biacore method for alkaline stability (per cycle): Running buffer: PBS-P+; Flow rate: 10 ⁇ l/min; Sample injection 1 (AAV95E11 vp/ml): 300 s over both flow cells; Dissociation time 1: 30 s; Sample injection 2 (0.5 M NaOH): 600 s over both flow cells; Dissociation time 2: 0 s ; Regeneration: 10 mM Glycine-HCl pH 1.5, 30 ⁇ l/min, 2x30 s. This cycle was repeated 40 times to follow stability of AAV9 response values. All sensorgrams were generated as reference subtracted and the responses as the difference between baseline before injection and signal just before end of injection.
  • the fusion protein 6A-2 comprising a single- domain antibody variant fused at its C-terminal to a stabilizing polypeptide, shows improved alkaline stability over the single-domain antibody 6A-1 alone.
  • the 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 Fig.6B.
  • the drop in AAV9 binding capacity experienced between the first and second cycles is merely 20 % for fusion protein, compared to the 60 % reduction in response for the sdAb which is not part of a fusion protein.
  • Table 16 summarizes the results of the AAV9 binding capacity and alkaline stability measurements.
  • a single-domain antibody (sdAb) trimer was compared to trimeric fusion proteins containing one or three stabilizing polypeptides, respectively.
  • the single domain antibody (denoted “sdAb”) was an AAV9-binding VHH variant (SEQ ID NO: 92).
  • the stabilizing polypeptide (denoted “Z”) was a SpA domain Z variant (SEQ ID NO: 161) in which binding to the Fc and VH3 portions of IgG had been abolished.
  • Each construct had a C-terminal linker followed by a (His) 6 -Cys tag. Table 17 Candidate no.
  • EZ-LinkTM Maleimide-PEG2-Biotin, No weightTM format (Thermo Scientific) was dissolved in 190 ⁇ l PBS according to protocol and 5 ⁇ l dissolved biotin was added to each 1 ml eluate, to reach a final 2-5 molar excess of biotin. Samples were incubated on ice for 2 hours, followed by dialysis according to Slide-A-LyzerTM G2 Dialysis Cassette (Thermo Scientific) protocol. The concentration of dialyzed samples was measured using NanoDrop (Thermo Scientific) according to manufacturer’s instructions and kept in refrigerator in Eppendorf tubes until all measurements had been performed and were then kept in freezer until further analysis.
  • Protein mass was determined by liquid chromatography–mass spectrometry on a ACQUITY Rda Detector (WatersTM) using a BioRessolve column (WatersTM).
  • Bis-Tris PAGE analysis To evaluate the protein expression levels, a Bis-Tris PAGE was performed on filtered supernatant post heat treatment. Materials and equipment used were as follows: NuPAGE LDS sample buffer (x4), Page Ruler prestained, 4-12% Bis-Tris minigel, Invitrogen NP0321BOX, Mini-gel tank (Invitrogen), 20x MES buffer (Invitrogen), QuickBlue Dye. 15 ⁇ l of filtered supernatant from post heat treatment was mixed with 5 ⁇ l LDS sample buffer.
  • the sample mix was heated to 70°C for 10 minutes and lightly centrifuged to collect condensate from the tube cap.15 ⁇ l sample mix for each expressed construct was loaded on the gel. 5 ⁇ l PageRuler was loaded as marker. The gel was run at 200 V for 35 minutes and stained in QuickBlue dye for 2 hours. The gel was destained in deionized water overnight. of binding affinity for AAV9
  • the candidates were immobilized on a BiacoreTM Series S SA chip (CytivaTM) and analyzed using BiacoreTM 8K+ instrument (CytivaTM).
  • a sample containing AAV9 viral particles (vp) at a concentration of 2E12 vp/ml was used as analyte.
  • Immobilization was performed using a standard SA coupling method in Biacore software with coupling of the polypeptides in Flow Cell 2 (FC2) and activation/inactivation in Flow cell 1 (FC1).
  • the polypeptides were diluted in PBS-P+ (CytivaTM) at a concentration of approximately 25 ⁇ g/ml.
  • the immobilization levels varied somewhat between different polypeptide variants corresponding to their molecular weight. In each run the polypeptide was immobilized in FC2.
  • BiacoreTM method for binding analysis Running buffer: PBS-P+ ; Flow rate: 5 ⁇ l/min ; Sample injection: 2400 s/40 min over both FC1 and FC2; Dissociation time: 2400 s/40 min ; Regeneration: 10 mM Glycine-HCl pH 1.5, 30 ⁇ l/min, 2x30 s.
  • Injections of analyte (AAV9) for each channel (cycles) were as follows: running buffer, AAV92E11 vp/ml, 5E11 vp/ml, 2E12 vp/ml. All sensorgrams were generated as reference subtracted and the responses as the difference between baseline before injection and signal just before end of injection.
  • BiacoreTM method for alkaline stability (per cycle): Running buffer: PBS-P+ ; Flow rate: 10 ⁇ l/min ; Sample injection 1 (AAV95E11 vp/ml): 300 s over both flow cells ; Dissociation time 1: 30 s ; Sample injection 2 (0.3 M NaOH): 600 s over both flow cells ; Dissociation time 2: 0 s ; Regeneration: 10 mM Glycine-HCl pH 1.5, 30 ⁇ l/min, 2x30 s.
  • Fig.9B plots the AAV9 binding capacity for increasing number of cycles involving 0.3 M NaOH exposure (sample injection 2 above) normalized to the response after completing the first cycle (i.e., excluding the first cycle).
  • the first cycle may remove loosely bound polypeptides from the surface.
  • the fusion proteins 6B-2 and 6B-3 both show notable improvement in alkaline stability over the sdAb trimer 6B-1.
  • AAV9 binding polypeptide with C-terminal fusion partner This example compares single domain antibodies having different framework sequences, wherein one candidate had a framework that was modified to enhance alkaline stability. The following aspects were evaluated: (1) Affinity assessment of AAV9 interaction (tested through high concentration injection of AAV9). (2) Alkaline stability (tested by reduction of target binding after increasing number of treatments with 0.5 M NaOH). The single domain antibodies were VHH variants directed against AAV9.
  • VHH was expressed as a fusion protein, fused C-terminally to an SpA domain Z variant (SEQ ID NO: 162) capable of binding to immunoglobulin Fc but not VH3.
  • SEQ ID NO: 162 an SpA domain Z variant capable of binding to immunoglobulin Fc but not VH3.
  • the tested polypeptide candidates (fusion proteins) are outlined in Table 19.
  • Table 19 Candidate VHH variant Type of VHH VHH sequence Full protein sequence no.
  • coli cells (ThermoFisherTM); chemically competent BL21(DE3) E. coli cells (prepared in-house); chemically competent K12-017 E. coli cells (prepared in-house); SOC-media (InvitrogenTM); stock of kanamycin 50 mg/ml (PanReac AppliChemTM); KCM buffer (5X) 0.5M KCl, 0.15M CaCl 2 , 0.25M MgCl 2 ; Luria-Bertani (LB) culture medium (InvitrogenTM); agar plate supplemented with kanamycin (50 ⁇ g/ml); 14 ml FalconTM Round-Bottom tube (CorningTM); PlasmidPrep Mini Spin Kit (Cytiva TM); Infors HT Shaking incubator.
  • KCM buffer 5X
  • 0.5M KCl 0.15M CaCl 2 , 0.25M MgCl 2
  • Luria-Bertani (LB) culture medium InvitrogenTM
  • the vector plasmid and G-blocks containing the DNA sequences for all candidates were digested with restriction enzymes KpnI-HF and HindIII-HF.
  • the plasmid was dephosphorylated using Antarctic phosphatase and gel purified according to manufacturer’s protocols.
  • Restriction enzyme digested G-blocks were ligated into the gel purified plasmid using T4 DNA ligase for 2h at room temperature and transformed into Top10 E. coli cells. Briefly, cells and ligation mix were incubated for 20 min on ice and 10 min at room temperature in KCM-buffer.
  • Cells and plasmid were incubated for 20 min on ice and 10 min at room temperature in KCM-buffer. Cells were then transferred to SOC media and incubated at 37°C for 1h and then seeded on agar plates containing kanamycin 50 ⁇ g/ml and incubated at 37°C over night. A single colony was used to inoculate 4 mL LB media supplemented with 50 ⁇ g/ml kanamycin in a 14 ml round-bottom tube and grown over night at 37°C, 200 rpm. A glycerol stock of each variant was prepared with 900 ⁇ l overnight culture and 500 ⁇ l 50% glycerol and stored at -80°C.
  • Protein expression and purification For each candidate 5 ⁇ l BL21(DE3) glycerol stock produced as described above was inoculated in 4 ml LB culture medium supplemented with 100 ⁇ g/ml carbenicillin in 14 ml round- bottom tube and incubated at 37°C overnight at 160 rpm. Protein expression culture medium (TB medium supplemented with 50 ⁇ g/ml kanamycin and 2 mM MgCl 2 ) was prepared and added to filled 100 ml baffled glass shake flasks (20 ml per flask). Each flask was inoculated with approximately 100 ⁇ l from the previous overnight culture to obtain a starting OD600 of 0.05.
  • the flasks were incubated in Infors HT shaking incubator for approximately 3 hours at 37°C and 140 rpm shaking until OD600 reached 1.0. Thereafter 20 ⁇ l IPTG (1 M) was added to a final concentration of 1 mM and the flasks were incubated in Infors HT shaking incubator at 27°C and 140 rpm shaking for 18 hours, whereafter the cultures were transferred to Falcon tubes (50 mL). To generate crude variant lysates the Falcon tubes were incubated in 48°C water bath for 2 hours followed by pelleting of cell debris by centrifugation at 8000 g for 10 min and filtration of supernatant with a 0.22 ⁇ m filter.
  • the samples were purified using gravity flow in PD-10 columns (CytivaTM) packed in-house with IgG Sepharose 6FF chromatography resin (CytivaTM). Clarified lysate was loaded on the column, equilibrated in 50 mM Tris, 150 mM NaCl, 0.05% Tween20 (TST buffer). The column was washed with 10 column volumes (CV) of TST buffer followed by 2 CV of 2 CV 5 mM NH4Ac pH 5. Protein was eluted using 2.5 ml 0.5 M Hac. Eluted protein was buffer-exchanged into phosphate buffered saline (Medicago) pH 7.4 using gravity flow columns PD-10 (Cytiva) prior to further analysis.
  • IgG Sepharose purified samples were measured using NanoDrop (Thermo Scientific) according to manufacturer’s instructions. Following purification, samples were kept in refrigerator in Eppendorf tubes until all measurements were performed and were then kept in freezer until Biacore analysis. Protein mass was determined by liquid chromatography–mass spectrometry on a ACQUITY Rda Detector (WatersTM) using a BioRessolve column (WatersTM). Biacore analysis of AAV9 binding affinity To assess binding to AAV9, candidate polypeptides were immobilized on a BiacoreTM Series S CM5 chip using Amine coupling kit and analyzed with a BiacoreTM 8K+ instrument (all from Cytiva).
  • the analytes used was AAV9 (prepared in-house). Immobilization was performed using a standard method in Biacore software with coupling of polypeptide variants in Flow Cell 2 (FC2) and activation/inactivation in Flow cell 1 (FC1). AAV9 binding candidates were diluted in BiacoreTM Acetate buffer pH 5.0 (CytivaTM) at a concentration of 25 ⁇ g/ml, and the average immobilization levels was 4319 RU +/-640 RU. The polypeptides were immobilized in FC2.
  • BiacoreTM method for binding analysis Running buffer: PBS-P+ ; Flow rate: 5 ⁇ l/min ; Sample injection: 2400 s/40 min over both FC1 and FC2; Dissociation time: 2400 s/40 min ; Regeneration: 10 mM Glycine-HCl pH 1.5, 30 ⁇ l/min, 2x30 s.
  • Injections of analyte (AAV9) for each channel (cycles) were as follows: running buffer, 1.25E11 vp/ml, 5E11 vp/ml, 2E12 vp/ml. All sensorgrams were generated as reference subtracted and the responses as the difference between baseline before sample injection and signal just before end of injection.
  • Biacore analysis of alkaline stability After the binding analysis (see above) the same CM5 chips having immobilized polypeptides were subjected to repeated cycles of binding to AAV9 followed by injection of NaOH for the assessment of alkaline stability (0.5 M).
  • Biacore method (per cycle): Running buffer: PBS-P+ ; Flow rate: 10 ⁇ l/min ; Sample injection 1 (AAV93E11 vp/ml): 300 s over both flow cells ; Dissociation time 1: 30 s; Sample injection 2 (0.5 M NaOH): 600 s over both flow cells; Dissociation time 2: 0 s ; Regeneration: 10 mM Glycine-HCl pH 1.5, 30 ⁇ l/min, 2x30 s.
  • Fig.10 shows the binding capacities obtained for increasing number of cycles involving NaOH exposure (sample injection 2) as normalized to the response of the first cycle (i.e., including the first cycle).
  • the AAV9-binding polypeptide which was based on stabilized VHH framework (7-2) showed improved alkaline stability in relation to the corresponding candidate (7-1) having a VHH framework that had not been subject to stabilizing modifications to the same extent.
  • Example 8 Functional evaluation – affinity chromatography This example demonstrates with a basic experiment the functionality of an AAV9 binding polypeptide as an affinity ligand immobilized on a separation matrix.
  • Materials and methods AAV9 binding candidate vh118 (SEQ ID NO:92) was produced as a fusion protein with SEQ ID NO: 161 and purified as described in Example 6A.
  • the fusion protein (SEQ ID NO:170) was immobilized on epoxy activated chromatography resin (highly crosslinked agarose beads) using cysteine coupling.0.2 ml of the resin was packed in Tricorn 5/20 columns (Cytiva).
  • Fig.11b shows only the region of the elution peak.
  • Fig.12 shows the photograph of the gel.
  • VP1, VP2 and VP3 denotes the AAV9 virion proteins 1, 2 and 3, respectively.
  • the results show that the present candidate was able to successfully purify AAV9 viral particles, with an estimated dynamic binding capacity (QB10%) of about 3E+14 viral particles/ml resin and estimated recovery of loaded virus particles of 70-80%.
  • the result of the alkaline stability assessment is shown in Fig.13 which plots the binding capacity normalized to the binding capacity before the first NaOH exposure. Over the 40 cycles of exposure to 0.5 M NaOH, which corresponded to an accumulated exposure time of 20 hours, the AAV9 binding capacity was reduced by less than 20 %.
  • Example 9 AAV9 binding polypeptides in different frameworks and two sets of CDRs This example further investigated five additional distinct sdAb frameworks with the same sdAb CDR sequences. One framework was also tested with two different sets of CDR sequences. All sdAbs have the same CDR1 and CDR2 sequences, while vh516 had a distinct CDR3 from vh511-vh515. Vh511-515 have the same CDRs as vh324 but have different frameworks. Vh324 has framework variant 1, while vh511-515 has five new framework permutations, as shown in the tables below.
  • the candidate polypeptides tested were expressed as fusion protein in which the respective sdAb had SEQ ID NO: 161 fused to its C-terminal via a linker, and had a C-terminal (His)6-tag.
  • the sdAb sequences variants are presented in Tables 21 and 22. The following aspects were evaluated: (1) Affinity assessment of AAV9 interaction (tested through high concentration injection of AAV9). (2) Alkaline stability (tested by reduction of AAV9 binding after increasing number of treatments with 0.3 M NaOH).
  • ZVH3 hexamer was provided as described in WO2023174900A1, the hexamer having the sequence according to SEQ ID NO: 177 of WO2023174900A1, which is a hexamer of the monomer as defined by SEQ ID NO: 15 of WO2023174900A1, which is incorporated by reference herein.
  • Biacore analysis of alkali stability Materials and equipment used were as follows: BiacoreTM Series S CM5 sensor chips, BiacoreTM Amine coupling kit, BiacoreTM Acetate buffer pH 5.0, BiacoreTM 8K+ instrument (all from Cytiva, Sweden); 0.5 M NaOH; ZVH3-ligand; candidate polypeptides generated as described above.
  • the polypeptide CDR AAV9 binding is compared to the AAV9 ZVH3 backbone binding for the AAV9 binding polypeptides vh260-268. It may be seen that the alkaline stability is increased also for this target.
  • the unstable polypeptides vh267 and vh268 are unstable for both bindings.
  • ⁇ CDR1 comprises an amino acid sequence selected from the group of sequences defined by SEQ ID NO: 1: X 1 X 2 X 3 SX 5 X 6 TMX 9 (SEQ ID NO: 1) wherein, independently, X1 is R, L or S, preferably R or L; X2 is T or R, preferably T; X 3 is L or F, preferably L; X 5 is D, N or E, preferably D; X 6 is Y, N or F, preferably Y or F; X9 is G or A, preferably G; ⁇ CDR2 comprises a sequence selected from the group of sequences defined by defined by SEQ ID NO: 2: X1X2SWSGX7X8TX10 (SEQ ID NO: 2) wherein, independently, X1 is R, L or S, preferably R or L; X2 is T or R, preferably T; X 3 is L or F, preferably L; X 5 is D, N or E, preferably D; X 6 is Y
  • the antigen-binding polypeptide according to item 1 which is a single-chain antigen- binding polypeptide.
  • the antigen-binding polypeptide according to item 1 or 2 wherein the polypeptide lacks antibody heavy chain constant domains.
  • said sdAb variant is a variable domain of a heavy chain of a heavy chain antibody (VHH) variant.
  • VHH heavy chain antibody
  • the antigen-binding polypeptide according to any one of the preceding items wherein the sdAb variant is capable of VH3 interaction with staphylococcal protein A domain or a variant thereof.
  • SEQ ID NO: 1 X1 is R. 13.
  • CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 9-17, such as selected from the group consisting of SEQ ID NOs: 13, 14, 16 and 17.
  • 20 The antigen-binding polypeptide according to any one of the preceding items wherein i) in SEQ ID NO: 2 X 1 is A, or ii) in SEQ ID NO: 2 X 1 is S, L or I, and in SEQ ID NO: 3 X 10 is A. 21.
  • the antigen-binding polypeptide according to any one of the preceding items wherein in SEQ ID NO: 2 X 10 is K. 25.
  • CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 21-31, such as selected from the group consisting of SEQ ID NOs: 26, 28, 29, and 31. 27.
  • SEQ ID NO: 3 at least one of X8 and X9 is K. 28.
  • SEQ ID NO: 3 X2 is P. 29.
  • the antigen-binding polypeptide according to any one of the preceding items wherein in SEQ ID NO: 3 X8 is K, N or R, such as K or R. 32.
  • the antigen-binding polypeptide according to any one of the preceding items wherein in SEQ ID NO: 3 X9 is K. 33.
  • the antigen-binding polypeptide according to any one of the preceding items wherein in SEQ ID NO: 3 X 10 is A or T. 34.
  • the antigen-binding polypeptide according to any one of the preceding items wherein in SEQ ID NO: 3 X11 is T.
  • the antigen-binding polypeptide according to any one of the preceding items wherein in SEQ ID NO: 3 X13 is A and, preferably, X10 is A.
  • the antigen-binding polypeptide according to any one of the preceding items wherein in SEQ ID NO: 3 X 14 is D and X 16 is D.
  • the antigen-binding polypeptide according to any one of the preceding items wherein in SEQ ID NO: 3 X17 is Y.
  • CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 34-72, such as selected from SEQ ID NO: 36, 37, 39, 40, 42-46, 48-50, 55, 56, 62, 64, 68 and 70-72. 39.
  • CDR1 is selected from SEQ ID NOs: 9-17, such as selected from SEQ ID NO: 13, 1416 and 17
  • CDR2 is selected from SEQ ID NOs: 21-31, such as selected from SEQ ID NO: 26, 28, 29 and 31
  • CDR3 is selected from SEQ ID NOs: 34-72, such as selected from SEQ ID NO: 36, 37, 39, 40, 42-46, 48-50, 55, 56, 62, 64, 68 and 70-72. 40.
  • the antigen-binding polypeptide according to any one of items 1-7, wherein the sdAb variant has a) an amino acid sequence selected from SEQ ID NOs: 75-93, 95-98, 101, 102, 105-107, 109- 124 and 126-146, such as selected from SEQ ID NOs: SEQ ID NO: 92, 97, 109, 116, 131 and 140-146; or b) an amino acid sequences having at least 90 % identity, such as at least 95 % identity, to a sequence as defined in a) provided that the CDRs are as defined in item 1. 41.
  • the antigen-binding polypeptide according to any one of the preceding items comprising at least one further amino acid sequence optionally selected from the group consisting of a leader peptide, a signal peptide, a purification tag, a coupling peptide, a spacer peptide and a linker peptide, each further amino acid sequence comprising up to 20 amino acids.
  • the antigen-binding polypeptide according to item 43 wherein said further amino acid sequence is a PelB or OmpA signal peptide.
  • said further amino acid sequence comprises a plurality of histidines, such as a (His)6 sequence, and optionally a C-terminal cysteine.
  • said further amino acid sequence comprises a C-terminal tag, such as selected from the group consisting of HHHHHH, HHHHHHC and AEAAAKHHHHHHC (SEQ ID NO: 159). 47.
  • a multimeric polypeptide comprising at least two moieties, each moiety comprising a single-domain antibody variant as defined in any one of the preceding items, said moieties optionally being joined by a peptide linker.
  • a fusion protein comprising at least one antigen-binding polypeptide according to any one of items 1-46 or a multimeric polypeptide according to item 47 and a further polypeptide moiety.
  • the fusion protein according to item 48 wherein the further polypeptide moiety comprises an ⁇ -helix-containing polypeptide domain.
  • the fusion protein according to item 50 wherein the further polypeptide moiety is or is derived from a protein domain of staphylococcal protein A (SpA).
  • SpA staphylococcal protein A
  • 52. The fusion protein according to item 50 or 51, wherein the further polypeptide moiety comprises an amino acid sequence having least at least 80 % identity, such as at least 85 % or at least 90 % identity to SEQ ID NO: 161 or 162.
  • 53. The fusion protein according to any one of items 48-52, wherein said further polypeptide moiety is positioned C-terminally of said antigen-binding polypeptide.
  • 54. The fusion protein according to any one of items 48-52, comprising two or more copies of said further polypeptide moiety, optionally separated by a linker peptide. 55.
  • the fusion protein according to item 48 comprising the following structure ([A-L1] m -[Z-L2] n ) p wherein A represents an antigen-binding polypeptide as defined in any one of items 1-46, L1 for each occurrence may be present or absent, and where present, represents a linker or spacer, Z represents a further polypeptide moiety according to any one of items 49-52, L2 for each occurrence may be present or absent, and where present, represents a linker or spacer, m represents an integer of from 1 to 4, n represents an integer of from 1 to 4, or when m ⁇ 2, n represents 0 or an integer of from 1 to 4, and p represents an integer of from 1 to 4. 56.
  • the fusion protein according to any one of items 55-57 comprising a C-terminal tag comprising a plurality of histidine residues, preferably at least 6, histidine residues, optionally wherein the fusion protein except for the tag does not contain any histidine residues.
  • 59. An isolated nucleic acid encoding the antigen-binding polypeptide of any one of items 1- 46, the multimeric polypeptide of item 47 or the fusion protein of any one of items 48-58. 60.
  • An expression vector comprising the nucleic acid of item 59. 61.
  • the method of item 62, wherein the cells are prokaryotic cells, such as E. coli cells.
  • the cells are eukaryotic cells.
  • the method of item 64, wherein the eukaryotic cells are yeast cells, such as Saccharomyces cerevisiae or Pichia pastoris cells.
  • the method of 64, wherein the eukaryotic cells are animal cells, such as mammalian cells, such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells. 67.
  • step iii comprises purifying the antigen- binding polypeptide, the multimeric polypeptide or the fusion protein by affinity chromatography.
  • step iii comprises purifying the antigen- binding polypeptide, the multimeric polypeptide or the fusion protein by affinity chromatography.
  • the affinity chromatography uses an affinity ligand that binds to a framework region of the single-domain antibody variant.
  • AAV9 adeno-associated virus serotype 9
  • a separation matrix comprising the antigen-binding polypeptide of any one of items 1-46 or the multimeric polypeptide of item 47 or the fusion protein of any one of items 48-58 as an affinity ligand coupled to a support material.
  • the support material is a surface.
  • the support material is selected from a particle, a bead, a fiber, a fibrous membrane, a filter, a sheet, a porous monolith, a chip, a plate, and a well.
  • the separation matrix according to item 74 wherein the support material is a chromatography matrix.
  • the matrix comprises a polysaccharide-based material, such as agarose and derivatives thereof, or cellulose and derivatives thereof.
  • the matrix comprises crosslinked agarose.
  • the support material is a chromatography matrix selected from the group consisting of a fibrous matrix, a membrane, a filter and a porous monolith.
  • the support material is a nonwoven fibrous matrix. 80.
  • An in vitro method of detecting AAV9 viral particles present in a sample comprising (a) providing a sample containing AAV9 viral particles; (b) contacting the sample with the antigen- binding polypeptide of any one of items 1-46, the multimer of item 47 or the fusion protein of any one of items 48-58, under conditions allowing the sdAb variant to bind to the AAV9 viral particles, wherein the binding event generates a detectable signal; and (c) detecting the detectable signal.
  • a method of chromatographic separation of AAV9 viral particles comprising the steps of (a) providing a separation matrix according to any one of items 72-79, (b) contacting the separation matrix with a liquid sample comprising said AAV9 viral particles under conditions allowing the antigen-binding polypeptide to bind to the AAV9 viral particles, (c) optionally washing the separation matrix, (d) eluting the bound AAV9 viral particles from the separation matrix, and (e) cleaning the separation matrix material with a cleaning liquid.
  • step (e) comprises cleaning the separation matrix with an alkaline cleaning liquid, wherein the cleaning liquid preferably comprises from 0.05 to 0.5 M of NaOH.
  • steps (a)-(e) are repeated at least 10 times, such as at least 20 times.
  • steps (a)-(e) are repeated at least 10 times, such as at least 20 times.
  • steps (a)-(e) are repeated at least 10 times, such as at least 20 times.
  • steps (a)-(e) are repeated at least 10 times, such as at least 20 times.
  • 84. The method of any one of items 81-83 wherein after 10 cycles of contact with cleaning liquid the polypeptide retains at least 50 %, such as at least 60 %, at least 70 %, at least 80 %or at least 90 %, of its initial target entity binding capacity.
  • 85 The method of any one of items 81-84, wherein after 20 cycles of contact with cleaning liquid the polypeptide retains at least 50 %, at least 60 %, such as at least 70 % or at least 80 % of its initial target entity binding capacity.
  • any one of items 81-83 wherein, after 10 cycles of contact with alkaline cleaning liquid, the polypeptide retains at least 60 %, at least 70 %, at least 80 %, at least 90 %, such as at least 95 %, of the target entity binding capacity of the 2 nd cycle.
  • the polypeptide retains at least 60 %, at least 70 %, at least 80 %, at least 90 %, such as at least 95 %, of the target entity binding capacity of the 2 nd cycle.

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Abstract

L'invention concerne un polypeptide de liaison à l'antigène capable de se lier au sérotype 9 du virus adéno-associé (AAV9), le polypeptide comprenant un variant d'anticorps à domaine unique (sdAb) ayant des régions déterminant la complémentarité CDR1, CDR2 et CDR3 telles que décrites dans la description. Le polypeptide de liaison à l'antigène est utile en tant que ligand d'affinité pour des applications de capture d'affinité, telles que la séparation par affinité.
PCT/EP2024/079595 2023-10-20 2024-10-18 Polypeptides de liaison à aav9 WO2025083267A1 (fr)

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