AAV9 affinity ligands

Abstract

The present invention relates to agents for binding adeno-associated virus subtype 9 (AAV9) particles, particularly to affinity ligands useful for selective isolation of an AAV9 particle or capsid or a variant of an AAV9 particle and capsid. Further disclosed are methods for purification and / or isolation of AAV9 using said affinity ligands in combination with affinity chromatography.

Description

[0001] P24-158

[0002] - 1 -

[0003] AAV9 AFFINITY LIGANDS

[0004] FIELD OF THE INVENTION

[0005] 5

[0006] The present invention relates to methods and agents for binding adeno- associated virus subtype 9 (AAV9) particles, particularly to affinity ligands useful for selective isolation of an AAV9 particle or capsid or a variant of an AAV9 particle and capsid.

[0007] BACKGROUND OF THE INVENTION

[0008] Adeno-associated virus (AAV) have been characterized and developed as a potent viral vector to deliver genes in vitro in cultured cells and in vivo. In

[0009] 15 recent years, AAV-based approaches have developed as a leading platform for in vivo delivery of gene therapies.

[0010] AAV is a small, non-enveloped virus containing a single-stranded DNA genome of approximately 4.7 kb, consisting of two inverted terminal repeats (ITRs) that are capable of forming T-shape secondary structure and acting

[0011] 20 as origins of genome replication, one rep region that encodes four overlapping replication proteins, Rep78, Rep68, Rep52, and Rep40, and one cap region that encodes three structural proteins, VP1 , VP2, and VP3, and an assembly activating protein (AAP). Naturally isolated serotypes 1-9 of the AAV viruses share the genomic structure although these serotypes may display different tissue tropism. As the AAVs seem to be nonpathogenic, show an efficient transduction, and a stable expression, they are regarded as being one of the most promising gene delivery vehicles.

[0012] AAV9 is of particular interest to biopharmaceutical developers due to its

[0013] 30 reported ability to cross the blood brain barrier, which is of particular importance to potentially address an array of central nervous system (CNS) P24-158

[0014] - 2 - disorders. In addition to its application for CNS disorders, and due to a broad tropism towards liver, skeletal muscle, and lung tissues, AAV9 is also being used to address non-CNS disorders.

[0015] Recombinant adeno-associated virus serotype 9 (rAAV9) can specifically

[0016] 5 transduce several tissues such as muscle and neuronal tissues, thus, rAAV9 can potentially be used in gene therapy. However, rAAV9 is the most challenging rAAV serotype to purify. Traditionally, rAAV9 has been purified by ultracentrifugation, which is not scalable.

[0017] Affinity ligands for binding AAV viruses are known in the art. Such affinity ligands can for example be used for binding, isolating, coupling, targeting or purifying said AAV particles. Examples of AAV binding ligands are disclosed in WO2020 / 242988 and WO2023 / 212694. To ensure effective and selective binding, it is of high importance that the ligands specifically and effectively

[0018] 15 bind to the aimed target. In recent years only a small number of affinity ligands fulfilling the requirements as stated above have been identified. However, such ligands comprise relatively large proteins, negatively impacting binding affinity of, e.g., chromatography matrixes functionalized with such ligands. Additionally, the production and conjugation of such

[0019] 20 ligands to chromatography matrixes is challenging due to further non-peptide modifications required to provide a reliable and reusable ligand for high efficiency purification of AAV9. Accordingly, there is an increasing interest by the industry to develop affinity ligands with high binding affinity and specificity for AAV9 particles or capsids or a variant of an AAV9 particles and capsids that allow efficient conjugation to desired matrixes.

[0020] 30 P24-158

[0021] - 3 -

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] DEFINITIONS

[0024] 5 Before describing the present invention in detail, it is to be understood that this invention is not limited to specific compositions or process steps, as such may vary.

[0025] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0026] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments; however, it should be

[0027] 15 understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the

[0028] 20 explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. The following terms are defined for purposes of the invention as described herein. P24-158

[0030] - 4 -

[0031] The term "comprise" or variations such as "comprises" or "comprising" according to the present invention means the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The term "consisting essentially of’ according to the present

[0032] 5 invention means the inclusion of a stated integer or group of integers, while excluding modifications or other integers which would materially affect or alter the stated integer. The term "consisting of” or variations such as "consists of” according to the present invention means the inclusion of a stated integer or group of integers and the exclusion of any other integer or group of integers. Embodiments described herein as "comprising" one or more features may also be considered as disclosure of the corresponding embodiments "consisting of" such features.

[0033] The terms "a" and "an" and "the" and similar reference used in the context of

[0034] 15 describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Thus, for example, reference to "a ligand" includes a plurality of ligands and reference to "an antibody" includes a plurality of antibodies and the like.

[0035] 20

[0036] “About” may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. In particular embodiments, the term “about” shall be understood herein as plus or minus (±) 5%, ± 4%, ± 3%, ± 2%, ± 1 %, ± 0.5%, ± 0.1 %, of the numerical value of the number with which it is being used.

[0037] Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format. It is also to be understood that

[0038] 30 such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited P24-158

[0039] - 5 - as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0040] The term "wild-type Sac7d" as used herein refers to a naturally occurring

[0041] 5 Sac7d protein, such as the DNA-binding protein 7d derived from Sulfolobus acidocaldarius. In certain embodiments, the term "wild-type Sac7d" specifically refers to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 (MVKVKFKYKG EEKEVDTSKI KKVWRVGKMV SFTYDDNGKT GRGAVSEKDA PKELLDMLAR AEREKK, UniProt ID: P13123). In alternative embodiments, the term may encompass a variant of this polypeptide in which the N-terminal methionine (M) and the C-terminal lysine (K) residues present in SEQ ID NO: 1 have been removed, thereby yielding the polypeptide sequence set forth in SEQ ID NO: 2 (VKVKFKYKG EEKEVDTSKI KKVWRVGKMV SFTYDDNGKT GRGAVSEKDA

[0042] 15 PKELLDMLAR AEREK).

[0043] The term "viral vector" or “viral particle” is widely used to refer to a viral particle that mediates nucleic acid transfer. Viral particles include various viral components and sometimes also host cell components in addition to

[0044] 20 nucleic acid(s). Illustrative viral vectors include, but are not limited to, retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, vaccinia virus vectors, and herpes simplex virus (HSV) vectors. Preferred viral vectors AAV vectors, most preferred are AAV vectors.

[0045] Adeno-associated virus (AAV) have been characterized and developed as a potent viral vector to deliver genes in vitro in cultured cells and also in vivo. AAV is meanwhile a leading platform for in vivo delivery of gene therapies. Adeno-associated virus (AAV) is a member of the Parvoviridae family. The AAV genome is composed of a linear single-stranded DNA molecule which

[0046] 30 contains approximately 4.7 kilobases (kb) and consists of two major open reading frames encoding the non-structural Rep (replication) and structural Cap (capsid) proteins. Flanking the AAV coding regions are two cis-acting P24-158

[0047] - 6 - inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, with interrupted palindromic sequences that can fold into hairpin structures that function as primers during initiation of DNA replication. In addition to their role in DNA replication, the ITR sequences have been shown

[0048] 5 to be necessary for viral integration, rescue from the host genome, and encapsidation of viral nucleic acid into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129).

[0049] Multiple serotypes of AAV exist and offer varied tissue tropism. Known serotypes include, for example, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11 .

[0050] Vectors derived from AAV are particularly attractive for delivering genetic material because they are able to infect (transduce) a wide variety of nondividing and dividing cell types including muscle fibers and neurons and they

[0051] 15 are devoid of the virus structural genes, thereby eliminating the natural host cell responses to virus infection, e.g., interferon-mediated responses. In addition, wild-type viruses have never been associated with any pathology in humans.

[0052] 20 According to the present invention scAAV are also within the group of AAVs. Self-complementary adeno-associated vectors (scAAV) are viral vectors engineered from the naturally occurring adeno-associated virus (AAV) for use in gene therapy. ScAAV is termed "self-complementary" because the coding region has been designed to form an intramolecular double-stranded DNA template.

[0053] Thus, in some embodiments, by an "AAV " is meant a vector or virus derived from an adeno-associated virus serotype, including without limitation, AAV- 1 , AAV-2, AAV-3, AAV-4, AAV -5, AAV- 6, AAV-7, AAV -8, AAV-9, AAV-10 and AAV-11 . AAV vectors can have one or more of the AAV wild-type genes

[0054] 30 deleted in whole or part, e.g., the rep and / or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the P24-158

[0055] - 7 - rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences that provide for replication and packaging (e.g., functional ITRs) of the virus. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion,

[0056] 5 deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging. In one embodiment, the vector is an AAV-9 vector, with AAV-2 derived ITRs. Also, by an "AAV " is meant the protein shell or capsid, which provides an efficient vehicle for delivery of vector nucleic acid to the nucleus of target cells.

[0057] The term "AAV" as used herein is intended to also encompass recombinant AAV as well as any AAV species deviating from the wild type, like engineered AAVs. Engineered AAVs encompass for example glycosylated, target fused, tagged or sequence modified AAVs. Adeno-associated viruses (AAVs) are herein also called viruses, viral particles or viral vectors.

[0058] 15

[0059] As used herein, the term “cell” or "cell line" refers to a single cell or to a population of cells capable of continuous or prolonged growth and division in vitro. In some embodiments, e.g., the terms "HEK293 cells", "293 cells" or their grammatical equivalents are used interchangeably here and refer to the

[0060] 20 host / packing cell line used in the methods disclosed herein.

[0061] Suitable cells and cell lines have been described for use in production of viral vectors like lentiviral vectors, AAVs and AdVs. The cells themselves may be selected from any biological organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells including insect cells, yeast cells and mammalian cells. Particularly desirable host cells are selected from among any mammalian species, including, without limitation, A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1 , COS 7, BSC 1 , BSC 40, BMT 10, VERO, WI38, HeLa, a HEK 293 cell, Saos, C2C12, L cells, HT1080, HepG2 and primary

[0062] 30 fibroblast, hepatocyte and myoblast cells derived from mammals including human, monkey, mouse, rat, rabbit, and hamster. P24-158

[0063] - 8 -

[0064] During the manufacturing of especially AAVs, a percentage of capsids might not incorporate any of the transgenes and are referred to as empty capsids, empty AAVs or empty AAV particles. Additionally, capsids that contain fragments of the transgene or process related impurities are called partial

[0065] 5 capsids, partial AAVs or partial AAV particles. These undesired impurities are co-produced with the full capsids or full AAVs which contain the full length of the desired transgene.

[0066] Purification means to increase the degree of purity of a target molecule, in this case the viral particles like AAVs, e.g. by removing one or more impurities.

[0067] The term "impurity" or “contaminant” as used herein, refers to any foreign or objectionable molecules or species, including a biological macromolecule such as DNA, RNA, one or more host cell proteins, nucleic acids, endotoxins,

[0068] 15 lipids, impurities of synthetic origin like detergents, partial and / or empty viral particles as well as one or more additives which may be present in a sample containing the viral particles to be purified and thus to be separated from one or more of the impurities. Viral particle impurities include both, process- related impurities and / or product-related impurities. Exemplary process-

[0069] 20 related impurities include, but are not limited to, residual host-cell components (e,g., proteins, DNA - including extra-viral, chromatin- associated DNA - and / or RNA), residual viral production components (e.g., plasmid DNA, and / or helper viruses), residual cell culture components (e.g., antibiotics, supplements, inducers, and / or growth factors), and residual purification components (e.g. buffers, inorganic salts, enzymes, media, and / or detergents), as well as other contaminants. The aim of purifying viral particles is typically to generate viral particles that are substantially free of process related impurities like a host-cell protein, a host-cell DNA, a host-cell RNA, or a combination thereof. Exemplary product-related impurities can

[0070] 30 include, but are not limited to, empty capsids (where undesirable), aggregated viral particles, and degraded viral particles. P24-158

[0071] - 9 -

[0072] It is understood that a sample can be "substantially free" of one or more impurities but continue to have a small amount (e.g, undetectable level, or below an acceptable range) of one or more impurities, and that "substantially free" does not require complete removal of all impurities.

[0073] 5

[0074] As used herein, and unless stated otherwise, the term “sample” refers to any composition or mixture that contains viral particles. Samples may be derived from biological or other sources. Biological sources include eukaryotic and prokaryotic sources, such as plant and animal cells, tissues, and organs. The sample may also include diluents, buffers, detergents, and contaminating species, debris and the like that are found mixed with the target molecule. The sample may be "partially purified" (i.e. , having been subjected to one or more purification steps, such as filtration steps) or may be obtained directly from a host cell producing the viral particles, e.g., the sample may comprise harvested cell culture fluid.

[0075] 15

[0076] The terms "polypeptide", "peptide", and "protein" relate to oligo- and polypeptides and refers to substances which comprise two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more consecutive amino acids linked to one

[0077] 20 another via peptide bonds. Amino acids are referred to herein using the name of the amino acid, the three-letter abbreviation, or the single letter abbreviation. Polypeptides may further comprise substances which contain not only amino acid components but also non-amino acid components such as sugars and phosphate structures, as well as substances containing bonds such as ester, thioether or disulfide bonds. According to the present invention, a nucleic acid such as RNA may encode a polypeptide or protein. Accordingly, a transcribable polynucleotide or a transcript thereof such as mRNAs may contain an open reading frame (ORF) encoding a polypeptide. Said polynucleotide may express the encoded polypeptide or protein. It is

[0078] 30 also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code. As used P24-158

[0079] - 10 - herein, the term “amino acid sequence” is synonymous with the term “polypeptide”, “peptide” and / or the term “protein”.

[0080] Polypeptides as described herein may comprise amino acids with distinct

[0081] 5 biophysical properties, including but not limited to polarity, charge, and structural characteristics. In the context of the present application, and unless indicated otherwise, “X” can be any naturally occurring amino acid, in particular any of the standard amino acids (i.e., the 20 amino acids encoded in the standard genetic code). The term “amino acids with hydrophobic side chains” may refer to amino acids characterized by non-polar, hydrophobic side chains, as understood by those skilled in the art. Non-limiting examples include Alanine (Ala, A), Valine (Vai, V), Isoleucine (lie, I), Leucine (Leu, L), Methionine (Met, M), Phenylalanine (Phe, F), Tyrosine (Tyr, Y), and Tryptophan (Trp, W). The term “amino acids with hydrophilic side chains”

[0082] 15 refers to amino acids with side chains that exhibit a favourable interaction with aqueous environments, typically due to the presence of polar, charged, or hydrogen-bonding functional groups. These amino acids may be broadly classified as follows: Polar, uncharged amino acids, including Asparagine (Asn, N), Glutamine (Gin, Q), Serine (Ser, S), Threonine (Thr, T), Cysteine

[0083] 20 (Cys, C), Tyrosine (Tyr, Y); Positively charged (basic) amino acids, including Arginine (Arg, R), Lysine (Lys, K), Histidine (His, H); Negatively charged (acidic) amino acids, including Aspartic acid (Asp, D), Glutamic acid (Glu, E). The term “amino acids with aromatic side chains” refers to amino acids comprising aromatic structures, as recognized by those skilled in the art. These side chains confer unique chemical properties. Non-limiting examples include Phenylalanine (Phe, F), Tyrosine (Tyr, Y), and Tryptophan (Trp, W). Throughout this disclosure and in the appended claims, the conventional one-letter and three-letter codes for amino acid residues may be utilized, as defined in accordance with the nomenclature guidelines established by the

[0084] 30 IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). P24-158

[0085] - 11 -

[0086] The terms “polynucleotide” and “nucleic acid” are used herein interchangeably. They refer to a polymeric form of nucleotides of any length: Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of

[0087] 5 polynucleotides: coding or non-coding regions of a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, synthetic polynucleotides, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified, such as by

[0088] 15 conjugation with a labelling component. If not stated otherwise, the term “nucleic acid” refers to any nucleic acid such as ribonucleic acid, deoxyribonucleic acid, xeno nucleic acid, single stranded or double stranded.

[0089] As used herein, the term "identity" when used in relation to nucleic acids or polypeptides, describes the degree of similarity between two or more

[0090] 20 nucleotide or polypeptide sequences. The percentage of "sequence identity" between two sequences can be determined by comparing two optimally aligned sequences over a comparison window, such that the portion of the sequence in the comparison window may comprise additions or deletions (gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and

[0091] 30 multiplying the result by 100 to yield the percentage of sequence identity. A sequence that is identical at every position in comparison to a reference P24-158

[0092] - 12 - sequence is said to be identical to the reference sequence and vice-versa. An alignment of two or more sequences may be performed using any suitable computer program. For example, a widely used and accepted computer program for performing sequence alignments is CLUSTALW vl .6

[0093] 5 (Thompson, et al. (1994) Nucl. Acids Res., 22: 4673-4680).

[0094] The “percent sequence identity” between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides I amino acids divided by the total number of nucleotides I amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimise alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such

[0095] 15 as BLAST, which will be familiar to a skilled person.

[0096] AAV9 AFFINITY LIGAND

[0097] One aspect of the present invention refers to an affinity ligand comprising a

[0098] 20 variant of a member of the Sac7d family. Surprisingly, it has been found that said affinity ligand is capable to selectively bind an adeno-associated virus subtype 9 (AAV9) particle or capsid or a variant of an AAV9 particle and capsid. Such affinity ligands are useful for e.g., the isolation and / or affinity purification of AAV9 particles and capsids.

[0099] In a preferred embodiment, the variant of a member of the Sac7d family according to the invention comprise a polypeptide according to SEQ ID NO 5-25.

[0100] In some embodiments, the affinity ligand of the invention binds to an adeno-

[0101] 30 associated virus subtype 9 (AAV9) particle or capsid or a variant of an AAV9 particle and capsid. P24-158

[0102] - 13 -

[0103] In a preferred embodiment, the affinity ligand of the invention specifically binds to an adeno-associated virus subtype 9 (AAV9) particle or capsid or a variant of an AAV9 particle and capsid.

[0104] POLYPEPTIDES

[0105] 5

[0106] One aspect of the present invention relates to an affinity ligand comprising or consisting of a variant of a member of the Sac7d family. In some embodiments, the variant of a member of the Sac7d family comprises or consists of a polypeptide with an amino acid sequence according to formula 1 (SEQ ID NO: 3), from N-terminus to C-terminus:

[0107] VKVKFX1X2X3GE EKEVDTSKIX4 X5VX6RX7GX8X9VXIO FX11YDDNGKX12G X13GX14VX15EKDAP KELLDMLARA EREK

[0108] 15 formula 1 , wherein each of Xi , X2, X3, X4, X5, Xe, X7, Xs, X9, X10, Xu , X12, X13, X14, and X15 represents a single amino acid independently selected from the group consisting of naturally occurring amino acids. In some embodiments, at least

[0109] 1 , 2, 3, 4, or 5 amino acids of Xi , X2, X3, X4, X5, X6, X7, X8, X9, X10, Xu , X12,

[0110] 20 X13, X14, and X15 do not correspond to an amino acid of a wild-type member of the Sac7d family according to SEQ ID NO 2. In embodiments, at least 1 ,

[0111] 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 amino acids of Xi, X2, X3, X4, X5, Xe, X7, Xs, X9, X10, X11 , X12, X13, X14, and Xis do not correspond to an amino acid of a wild-type member of the Sac7d family according to SEQ ID NO 2.

[0112] In certain embodiments, the polypeptide of the invention according to formula 1 (SEQ ID NO: 3) further comprises a N-terminal methionine (M) and a C- terminal lysine (K) according to formula 2 (SEQ ID NO: 4), from N-terminus to C-terminus:

[0113] 30 P24-158

[0114] - 14 -

[0115] MVKVKFX1X2X3G EEKEVDTSKI X4X5VX6RX7GX8X9V X10FX11YDDNGKX12 GX13GX14VX15EKDA PKELLDMLAR AEREKK formula 2

[0116] 5 wherein each of Xi, X2, X3, X4, X5, Xs, X7, Xs, X9, X10, Xu, X12, X13, X14, and X15 represents a single amino acid independently selected from the group consisting of naturally occurring amino acids. In some embodiments, at least

[0117] 1 , 2, 3, 4, or 5 amino acids of Xi , X2, X3, X4, X5, X6, X7, X8, X9, X10, Xu, X12, X13, X14, and X15 do not correspond to an amino acid of a wild-type member of the Sac7d family according to SEQ ID NO 1 . In embodiments, at least 1 ,

[0118] 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 amino acids of Xi, X2, X3, X4, X5, Xe, X7, Xs, X9, X10, X11, X12, X13, X14, and Xis do not correspond to an amino acid of a wild-type member of the Sac7d family according to SEQ ID NO 1.

[0119] 15 In some embodiments, the polypeptide of the invention binds to an adeno- associated virus subtype 9 (AAV9) particle or capsid or a variant of an AAV9 particle and capsid.

[0120] In a preferred embodiment, the polypeptide of the invention specifically binds

[0121] 20 to an adeno-associated virus subtype 9 (AAV9) particle or capsid or a variant of an AAV9 particle and capsid.

[0122] In some embodiments, the polypeptide of the variant of a member of the Sac7d family according to the invention refers to a polypeptide with between 1 -25 amino acids, between 1 -20 amino acids, between 1 -15 amino acids, between 5-15 amino acids, between 10-15 amino acids, or between 12-15 amino acids e.g., about 12, 13, 14, or 15 amino acids, which are mutated, deleted, or inserted, as compared to a wild-type member of the Sac7d family according to SEQ ID NO 1 or 2. In a preferred embodiment, said between 1- 25 between 1-20, between 1 -15 amino acids, between 5-15 amino acids,

[0123] 30 between 10-15 amino acids, or between 12-15 amino acids e.g., about 12, - 15 -

[0124] 13, 14, or 15 amino acids, are mutated as compared to a wild-type member of the Sac7d family according to SEQ ID NO 1 or 2.

[0125] In some embodiments, in the polypeptide with an amino acid sequence according to formula 1 (SEQ ID NO 2) Xi is L, V, or I. In embodiments, X2 is

[0126] 5 W. In embodiments, X3 is Q or L. In embodiments, X4 is D or E. In embodiments, Xs is T, V, or E. In embodiments, Xs is Y. In embodiments, X7 is Q or N. In embodiments, Xs is K or Q. In embodiments, X9 is W or Y. In embodiments, X10 is T. In embodiments, Xu is Y. In embodiments, X12 is F or W. In embodiments, X13 is X, V or A. In embodiments, X14 is H. In embodiments, X15 is X, T, Q, or S.

[0127] In the context of the present application, and unless indicated otherwise, “X” can be any naturally occurring amino acid, in particular any of the standard amino acids (i.e. , the 20 amino acids encoded in the standard genetic code).

[0128] 15

[0129] In embodiments, the mutations, deletions, or insertions may occur at amino acid positions other than Xi , X2, X3, X4, X5, Xs, X7, Xs, X9, X10, Xu, X12, X13, X14, and X15 of the polypeptide with an amino acid sequence according to formula 1 (SEQ ID NO: 3) or amino acid sequence according to formula 2 (SEQ ID NO: 4).

[0130] 20

[0131] In some embodiments, Xi, X3, X4, X5, X7, Xs, X9, X10, X12, and X13 X15, represents a single amino acid independently selected from the group consisting of naturally occurring amino acids; X2 is W; Xs is Y; X10 is T; Xu is Y; and X14 is H. In some embodiments, Xi is L, V, or I; X3 is Q or L; X4 is D or E; X5is T, V, or E, X7is Q or N; X8is K or Q, X9is W or Y; X12 is F or W.

[0132] In a preferred embodiment, in the polypeptide with an amino acid sequence according to formula 1 (SEQ ID NO: 3), or amino acid sequence according to formula 2 (SEQ ID NO: 4), X2 is W, Xs is Y, X10 is T, and Xu is Y. In an even more preferred embodiment, in the polypeptide with an amino acid

[0133] 30 sequence according to formula 1 (SEQ ID NO: 3), or amino acid sequence - 16 - according to formula 2 (SEQ ID NO: 4),X2 is W, Xe is Y, X10 is T, Xu is Y, and X8is K.

[0134] In a preferred embodiment, the polypeptide comprises an amino acid sequence according to SEQ ID NOs: 5-11 :

[0135] 5

[0136] In some embodiments, the polypeptide comprising an amino acid sequence according to SEQ ID NOs: 5-11 further comprises a N-terminal M. In some embodiments, said polypeptide comprising an amino acid sequence according to SEQ ID NOs: 5-11 further comprises a C-terminal C. Preferably, said polypeptide comprising an amino acid sequence according to SEQ ID NOs: 5-11 further comprises a N-terminal M and a C-terminal C.

[0137] 30

[0138] In some embodiments, the polypeptide further comprises an N-terminal affinity tag, such as a histidine tag (His-tag), preferably a polyhistidine tag, P24-158

[0139] - 17 - more preferably an RGS- His6 tag (RGSHHHHHH, SEQ ID NO: 36), even more preferably an RGS- His6 tag comprising an N-terminal methionine (MRGSHHHHHH, SEQ ID NO: 29), and most preferably an N-terminal RGS- His6 tag comprising an N-terminal methionine and a GS linker

[0140] 5 (MRGSHHHHHHGS, SEQ ID NO: 37). In a preferred embodiment, the polypeptide comprising an amino acid sequence according to SEQ ID NOs: 5-11 further comprises an N-terminal RGS- His6 tag comprising an N- terminal methionine and a GS linker and a C-terminal C (SEQ ID NOs: 12- 18).

[0141] In some embodiments, said polypeptide comprising an amino acid sequence referring to residues (from N- to C-terminus) 1 to 57 of SEQ ID NOs: 5-11 (SEQ ID NOs: 19-25).

[0142] In certain embodiments, the polypeptide of the variant of a member of the

[0143] 15 Sac7d family of the affinity ligand according to the invention comprises or consists of a polypeptide with an amino acid sequence according to SEQ ID NOs: 5-25 or a polypeptide with at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at

[0144] 20 least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of an amino acid sequence according to SEQ ID NOs: 5-25.

[0145] In some embodiments, the polypeptide of the invention comprises a sequence comprising amino acids 1 - 54, 1 - 55, 1 -56, 1 - 57, 1 - 58, 1 - 59,

[0146] 1 - 60, 1 - 61 , 1 - 62, 1 - 63, 1 - 64, or 1 - 65 according to SEQ ID NOs 3-11 . In some embodiments, the polypeptide of the invention comprises a sequence comprising amino acids 2 - 54, 2 - 55, 2 -56, 2 - 57, 2 - 58, 2 - 59,

[0147] 2 - 60, 2 - 61 , 2 - 62, 2 - 63, 2 - 64, 2 - 65, or 2 - 66 according to SEQ ID NOs 3-11.

[0148] 30

[0149] Surprisingly, it has been found that certain mutations within the polypeptide according to the invention may not significantly affect the binding affinity of P24-158

[0150] - 18 - the affinity ligand to adeno-associated virus subtype 9 (AAV9) particle or capsid or a variant of an AAV9 particle and capsid. Said tolerated mutations corresponds to positions within the sequence of the polypeptide, which show low conservation within the sequence of the Sac7d-family. In some

[0151] 5 embodiments, the polypeptide according to the invention comprises at least one, at least two, at least tree, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven tolerated mutations at positions selected from the group according to table 1 within the amino acid sequence according to SEQ ID NO 2 or SEQ ID NO: 3.

[0152] Table 1 : Mutations of SEQ ID NO 2

[0153] 30 P24-158

[0154] - 19 -

[0155] TRANSFER OF MUTATION PATTERN

[0156] As previously shown, for instance in WQ2012150314A1 , it is possible to carry certain mutations from one protein of the Sac7d family to the scaffold of another one, these proteins presenting a similar structure and binding.

[0157] 5 Accordingly, one aspect of the present invention refers to an affinity ligand comprising a variant of a member of the Sac7d family binding to an adeno- associated virus subtype 9 (AAV9) particle or capsid or a variant of an AAV9 particle and capsid, wherein the variant comprises from 4 to 20 mutated residues in the interface of binding of the member of the Sac7d family to its natural ligand, and wherein said variants comprises Y8W, W24Y, S31T, T33Y, A44H mutations with the numbering corresponding to the position in the Sac7d amino acid sequence according to SEQ ID NO 1 .

[0158] In some embodiments, said mutated residues in the interface of binding of

[0159] 15 the member of the Sac7d family to its natural ligand are selected from the group consisting of K7, K9, K21 , K22, V26, M29, T40, R42, S46 of Sac7d. Preferably said polypeptide further comprises the mutation K29Q, with the numbering corresponding to the position in the Sac7d sequence SEQ ID NO: 1.

[0160] 20

[0161] Surprisingly, it has been found that certain mutations in the interface of binding of a member of the Sac7d family to its natural ligand are tolerated affecting affinity to a lesser amount. Accordingly, in some embodiments, said polypeptide further comprising at least one mutation selected from

[0162] - K7L, K7V, or K7I,

[0163] - K9Q or K9L,

[0164] - K21 D or K21 E,

[0165] - K22T K22V, or K22E,

[0166] 30

[0167] - V26Q or V26N, P24-158

[0168] - 20 -

[0169] - M29W or M29W,

[0170] - T40Y or T40W,

[0171] - R42V or R42A,

[0172] 5

[0173] - S46T or S46Q, with the numbering corresponding to the position in the Sac7d sequence SEQ ID NO: 1.

[0174] In some embodiments, said polypeptide comprises a sequence selected from SEQ ID NOs: 5-25. In some embodiments, said polypeptide comprises a sequence selected from a sequence comprising amino acids 1 -57, 2-58, or 2-54 of SEQ ID NOs 5-25. In a preferred embodiment, said polypeptide comprises an amino acid sequence according to SEQ ID NOs: 6, 10, 13, 17, 20, and 24.

[0175] 15

[0176] In some embodiments, said polypeptide is selected from the group consisting of Sac7d from Sulfolobus acidocaldarius, Sac7e from Sulfolobus acidocaldarius. SSo7d from Sulfolobus solfataricus, Ssh7b from Sulfolobus shibatae, Ssh7a from Sulfolobus shibatae, DBP7 from Sulfolobus tokodaii,

[0177] 20 Sis7a from Sulfolobus islandicus, Mse7 from Metal losphaera sedula, Mcu7 from Metallosphaera cuprina, Aho7a from Acidianus hospitalis, Aho7b from Acidianus hospitalis, Aho7c from Acidianus hospitalis and Sto7 from Sulfurisphaera tokodaii.

[0178] The skilled person in the art knows how to adapt the wild type sequences of the Sac7d family proteins in a way that the identified mutations as described herein and responsible for the binding affinity to an adeno-associated virus subtype 9 (AAV9) particle or capsid or a variant of an AAV9 particle and capsid correspond to the mutation patterns as shown in the scaffold of formula I.

[0179] 30

[0180] MODIFICATION OF N-TERMINUS P24-158

[0181] - 21 -

[0182] It is to be understood that the amino acid sequences described herein may optionally include an N-terminal methionine residue. While certain sequences are presented with an initiating methionine, the invention may equally be practiced in the absence of such methionine.

[0183] 5

[0184] In various embodiments, the polypeptides of the invention disclosed herein comprise an N-terminal methionine. In other embodiments, the polypeptides lack an N-terminal methionine. In some embodiments, the N-terminus comprises a short leader sequence such as MGS (methionine-glycine- serine).

[0185] In yet further embodiments, the polypeptide comprises an N-terminal affinity tag, such as a histidine tag (His-tag), preferably a polyhistidine tag, more preferably an RGS-His6tag (RGSHHHHHH, SEQ ID NO: 36), and even more preferably an RGS-His6tag comprising an N-terminal methionine

[0186] 15 (MRGSHHHHHH, SEQ ID NO: 29). Such affinity tags may facilitate purification of the polypeptide via affinity chromatography or similar techniques.

[0187] MODIFICATION OF C-TERMINUS

[0188] 20 In certain embodiments, the C-terminus of the polypeptide of the invention comprises a short amino acid sequence that is configured to modify structural characteristics and / or to facilitate functionalization of the polypeptide, for example, to enable immobilization on solid supports, such as chromatography media.

[0189] Such C-terminal extensions may support site-specific coupling, increase surface accessibility, or enhance electrostatic interactions with negatively charged surfaces. For example, positively charged sequences such as polylysine tags (e.g., K, KK, KKK, KKKK (SEQ ID NO: 26), KKKKK (SEQ ID NO: 27), and KKKKKK (SEQ ID NO: 28)) may enhance binding to negatively

[0190] 30 charged materials via ionic interactions. In other cases, terminal cysteine P24-158

[0191] - 22 - residues may be used to enable covalent coupling via thiol-specific chemistries.

[0192] Accordingly, the C-terminus of the polypeptides according to the invention may comprise one or more amino acid sequences selected from the group

[0193] 5 consisting of C, GKK, EKSGKK (SEQ ID NO: 30), EKQKK (SEQ ID NO: 31 ), ARAEREKK (SEQ ID NO: 32), ARAEREK (SEQ ID NO: 33), ARAEKKK (SEQ ID NO: 34), EKQKK (SEQ ID NO: 35), EKK, K, KK, KKK, KKKK (SEQ ID NO: 26), KKKKK (SEQ ID NO: 27), and KKKKKK (SEQ ID NO: 28), or combinations thereof. In a further embodiment, the polypeptide comprises a C-terminal cysteine residue, either alone or in combination with one or more of the aforementioned sequences.

[0194] Similarly, the C-terminus of the polypeptides described herein may comprise a shortened or modified amino acid sequence. In some embodiments, the C-

[0195] 15 terminal portion following residue L57 and L58 of the polypeptide according to formula 1 (SEQ ID NO: 3) and formula 2 (SEQ ID NO: 4) respectively, is removed or replaced with an alternative amino acid sequence.

[0196] In certain embodiments, the substituted C-terminal amino acid sequence is selected from the group consisting of C, GKK, EKSGKK (SEQ ID NO: 30),

[0197] 20 EKQKK (SEQ ID NO: 31 ), ARAEREKK (SEQ ID NO: 32), ARAEREK (SEQ ID NO: 33), ARAEKKK (SEQ ID NO: 34), EKQKK (SEQ ID NO: 35), EKK, K, KK, KKK, KKKK (SEQ ID NO: 26), KKKKK (SEQ ID NO: 27), and KKKKKK (SEQ ID NO: 28), or combinations thereof. In a further embodiment, the polypeptide comprises a C-terminal cysteine residue, either alone or in combination with one or more of the aforementioned sequences. MULTIMERS

[0198] In some embodiments, provided are affinity ligands comprising multimeric polypeptides. As used herein, a multimeric polypeptide refers to a

[0199] 30 polypeptide containing two or more domains. In embodiments, the domains are polypeptides described herein. In some embodiments, the multimeric polypeptide comprises one or more domains having an amino acid sequence P24-158

[0200] - 23 - according to any one of SEQ ID NOs 2-9. In some embodiments, the multimeric polypeptide comprises two or more domains having an amino acid sequence independently selected from any one of SEQ ID NOs 2-9, wherein the two domains are the same. In some embodiments, the multimeric

[0201] 5 polypeptide comprises two or more domains having an amino acid sequence independently selected from any one of SEQ ID NOs 2-9, wherein the two domains are different. In some embodiments, the two or more domains are connected by a linker. In some embodiments, said linker is selected from the group of polypeptides, polynucleotide, nucleosides, chemically modified nucleosides, photo-labile linkers, fatty acid chains, natural and artificial amino-acids at the likes.

[0202] MODIFICATIONS

[0203] It was shown by the applicant that the modification of the N- or C-terminus of the polypeptide comprising the affinity ligand according to the invention may

[0204] 15 be modified. Surprisingly, N- or C-terminal modification of the polypeptide does not significantly affect affinity properties of the ligand thus enabling the attachment of moieties useful within the scope of the invention. For instance, in certain embodiments, the polypeptide according to the invention may be N- and / or C-terminally modified with e.g., peptide-tags, small molecules,

[0205] 20 antibodies or other modifications known by the skilled person in the art such as, for instance, useful for the production of the ligand, purification of the ligand, increasing the stability of the ligand, enabling coupling to a chromatography matrix and the likes.

[0206] In some embodiments, the affinity ligand according to the invention is conjugated to at least one agent operably linked to said ligand. In a preferred embodiment, the ligand is selected from the group of polypeptide, DNA, RNA, small molecule, antibody, nanobody, single chain variable domain, and immunoglobulin fragment.

[0207] 30

[0208] In some embodiments, the polypeptides described herein comprise a protein tag, preferably a polyhistidine tag. In a preferred embodiment, the - 24 - polyhistidine tag is located at the N-terminus of the polypeptide sequence. In embodiments, the polyhistidine tag is located at the C-terminus of the polypeptide sequence. In embodiments, the polypeptides are biotinylated. In embodiments, the polypeptides described are extended at the N- or C-

[0209] 5 terminus. In embodiments, the extension comprises a polypeptide linker.

[0210] As known by the skilled person in the art, any method or approach for the modification of the polypeptide of the invention may be utilized. For instance, when the modification relates to a second polypeptide, said second polypeptide may be encoded in frame on the same vector as the first polypeptide. Small molecules and the likes may be coupled, for instance, directly to the polypeptide by means of chemical synthesis approaches, such as click chemistry.

[0211] VECTORS

[0212] 15

[0213] The sequences of the polypeptides comprising the affinity ligand of the invention can be cloned in any appropriate vector by any molecular genetic methods known in the art. Accordingly, a further aspect of the present invention relates to a nucleic acid molecule coding for the polypeptide of the invention.

[0214] 20

[0215] These recombinant DNA constructs comprising a nucleotide sequence, coding for a polypeptide comprising a variant as described above, are used in connection with a vector, such as a plasmid, phagemid, phage or viral vector.

[0216] These recombinant nucleic acid molecules can be produced by techniques described in Sambrook et al. ,1989 (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, New York). Alternatively, the DNA sequences may be chemically synthesized using, for example, synthesizers.

[0217] 30

[0218] Recombinant constructs of the invention comprise the expression vectors that can express the RNA and thus lead to production of proteins from the - 25 - above genetic sequences. The vector may thus further comprise regulatory sequences, including a suitable promoter operably linked to the open reading frame (ORF) of the genetic sequences herein disclosed. The vector may further comprise a selectable marker sequence such as an antibiotic

[0219] 5 resistance gene. Specific initiation and bacterial secretory signals also may be required for efficient translation of the coding sequences when bacteria as used as the expression host.

[0220] PRODUCTION OF THE POLYPEPTIDE

[0221] Cells are transfected or transformed with vectors containing the sequences coding for the polypeptides comprising the affinity ligand as disclosed above. The cells are then cultured in such conditions as to have the protein expressed and favourably secreted. The conditions of culture of the cells are the conditions generally used for recombinant antibody production and are

[0222] 15 known in the art. Such conditions that are known in the art can also be optimized by the person skilled in the art if needed. Kunert and Reinhart (Appl Microbiol Biotechnol. 2016; 100: 3451- 3461 ) review such methods and provide ample references thereto. One can use bacterial, phage (Shukra et al, Eur J Microbiol Immunol (Bp). 2014; 4(2): 91 -98) or eukaryotic systems of

[0223] 20 production. One shall prefer to use eukaryotic cells in order to obtain proper post- translational modifications such as glycosylation. One can use CHO (Chinese Hamster Ovary) cells, PER.C6 cells (human cell line, Pau et al, Vaccine. 2001 21 ; 19(17-19):2716-21 ), HEK 293b cells (Human embryonic kidney cells 293), NS0 cells (cell line derived from the non-secreting murine myeloma) or EB66 cells (a duck cell line Valneva, Lyons, France).

[0224] Also provided by the present disclosure are host cells containing at least one of the DNAs constructs coding for a polypeptide comprising the variant as disclosed herein. The host cell can be any cell for which expression vectors are available. As indicated above, it may be a higher eukaryotic host cell,

[0225] 30 such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or a prokaryotic cell, such as a bacterial cell. P24-158

[0226] - 26 -

[0227] Introduction of the recombinant construct into the host cell is performed by any method known in the art (such as calcium phosphate transfection, lipofection, DEAE, dextran mediated transfection, electroporation or phage infection). The vectors can be inserted within the genome of the host cell or

[0228] 5 be maintained as an extragenomic vector (such as a Bacterial Artificial Chromosome or a Yeast Artificial Chromosome). When introduced within the cell genome, such introduction may be random or targeted using methods known in the art (homologous recombination or the like).

[0229] BACTERIAL HOSTS AND EXPRESSION

[0230] Useful expression vectors for bacterial use are constructed by inserting the recombinant DNA sequence together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin

[0231] 15 of replication to ensure maintenance of the vector and, if desirable, to provide amplification within the host.

[0232] Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0233] 20

[0234] EUKARYOTIC HOSTS AND EXPRESSION

[0235] Examples of the eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. In particular, one can use the cells mentioned above.

[0236] The transformed or transfected cells are cultured according to methods known in the art and the polypeptide are recovered from intracellular or extracellular fractions (depending on whether it is secreted or not).

[0237] ISOLATION OF THE MOLECULES

[0238] 30 The recombinant protein produced can be separated and purified by any of various known separation methods utilizing the physical or chemical property of the protein, from the intracellular or extracellular fraction. P24-158

[0239] - 27 -

[0240] In particular, one can use methods such as precipitation, ultrafiltration, various types of liquid chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, dialysis, and a combination

[0241] 5 thereof.

[0242] In general, any method known and used to purify recombinant polypeptides is adapted for the purification of the molecules herein disclosed.

[0243] If a tag has been introduced within the recombinant sequence (such as a poly- Histidine tag), one can purify the molecules using this tag. However, in certain embodiments, it is preferred to use affinity to purify the molecules.

[0244] One can, in particular, use the fact that the molecule herein produced binds to a specific target and use any affinity method (affinity column, FACS, beads) to isolate such molecules.

[0245] 15

[0246] One particular advantage of the molecules herein disclosed is that they don’t need to be glycosylated to be active and can thus be produced in any type of cells, and not necessarily eukaryotic cells. They are particularly well produced in bacterial cells.

[0247] 20

[0248] USE OF THE AFFINITY LIGANDS

[0249] The affinity ligands according to the present invention may be used for multiple purposes requiring affinity ligands for an adeno-associated virus subtype 9 (AAV9) particle or capsid or a variant of an AAV9 particle and capsid such as, but not limited to, immunofluorescence applications, immunoassays such as ELISA, and the likes as known by the skilled person in the art.

[0250] TARGET PRODUCTION

[0251] 30 Viral vector production is known to the person skilled in the art. In the following, the production of AAV vectors is described in more detail as an example. P24-158

[0252] - 28 -

[0253] Suitable compositions of the liquid sample comprising AAV vectors, the washing buffers and the elution buffers, as well as the general conditions for performing the separation are well known in the art of affinity chromatography. For instance, AAV vectors can be produced in various cell

[0254] 5 lines in adherent or suspension cell culture formats using transient transfection or co-infection methods. Preferably, AAV vectors are produced in suspension cell lines. Depending on specific serotypes and production times, viral particles including full, partial and empty species can be secreted out of cells into culture medium or contained inside cells at various ratios.

[0255] Initially, stable AAV producer cells were generated by transfection and selection of human-derived cells, like HeLa or HEK293 cells, with an rAAV transfer vector containing the ITR cassette and a packaging construct containing Rep and Cap. Production of recombinant AAV vectors (rAAV) was then achieved by infection with auxiliary viruses such as adenoviruses (AdV)

[0256] 15 that provide the helper function. After identification of AdV genes required for AAV vector packaging, a helper virus-free method was established using a duo or triple transfection protocol consisting of two or three plasmids including a constructed helper plasmid instead of an auxiliary virus. This system is widely used in research and drug development. In addition,

[0257] 20 development of baculovirus expression vectors provides another method to produce rAAV viruses in insect Sf9 cells. These different technologies are shown to be able to produce sufficient quantities of rAAV viruses for use in laboratories and clinical trials.

[0258] A cell lysis step is generally required at harvest to release viral particles into the supernatant if the viral particles are not secreted by the cells. Suitable methods are known to the experts. For this application, typical cell lysis reagents such as Triton® X-100, Tween® 20, Deviron® C16, Deviron®13- S9 and NaCI are suitable.

[0259] 30

[0260] After cell lysis or secretion by the cells, the AAVs need to be purified. Typical AAV purification processes include clarification, concentration and P24-158

[0261] - 29 - diafiltration using tangential flow filtration, chromatography purification by using affinity chromatography and ion exchange chromatography. In some processes, ultracentrifugation and gradient ultracentrifugation are used instead of chromatography or in addition to chromatography. Final steps in

[0262] 5 AAV purification typically involve concentration and diafiltration into suitable excipient buffer composition and sterile filtration.

[0263] In the following an exemplary process is described in more detail.

[0264] Typically, the selected cells are expanded in suitable culture media in a bioreactor under suitable conditions. Typically, the cells are grown as suspension culture. For example, in suspension culture of HEK293 cells suitable seeding numbers before transfection are 0.5 to 1 .1 10A6 viable cells per ml.

[0265] 15 Suitable methods for the transduction are known in the art. In one embodiment, cells can be transduced in vitro by combining a rAAV with the cells, e.g., in appropriate media, and screening for those cells harboring the DNA of interest using conventional techniques such as Southern blots and / or PCR, or by using selectable markers.

[0266] 20

[0267] Transfection can be performed using any of the techniques known in the art, including but not limited to electroporation, lipofection, e.g. with a lipofectamine, cationic polymers and cationic lipids. Any suitable transfection media may be used. In one embodiment of the transfection process, adherent or suspension human embryonic kidney (HEK293) cells are transfected with a dual or triple DNA plasmid polyethylenimine (PEI) coprecipitation.

[0268] After a suitable virus production period post transfection or infection, the cells are optionally lysed and the viral particles, like AAVs or any other viral

[0269] 30 particles that have been produced, are harvested. In some embodiments, the cells are dissociated from the bioreactor before the cell lysis process is initiated. In some embodiments, the cells are lysed in situ. P24-158

[0270] - 30 -

[0271] The released viral particles can then be isolated and / or purified, whereby preferably a chromatographic purification on an affinity matrix is included.

[0272] Preferably, the mixture obtained from the cell culture or after lysis is first filtered or centrifuged.

[0273] 5

[0274] In one embodiment the mixture is filtered through a filter that removes large molecule contaminants and cellular debris but that permits viral particles to pass therethrough.

[0275] In a preferred embodiment, the released viral particles can be separated and purified from the cell culture medium using clarification. Clarification is a process, which includes at least one filtration step. It might involve one or more steps selected from centrifugation, tangential flow filtration, depth filtration, and sterile filtration. Clarification is a process in which relatively

[0276] 15 larger components such as cells, lysed cells, cellular debris, protein aggregates and other bulky mainly process related impurities are removed from a solution, typically the solution resulting from culturing cells and optionally lysing them. Clarification filters include depth filtration, charged depth filtration and similar microfiltration techniques. The resulting sample is

[0277] 20 a clarified sample.

[0278] Tangential flow filtration can be used to concentrate the mixture of purified viral particles and to remove salts and process related contaminants like proteins or nucleic acids. Tangential flow filtration (TFF) refers to a generally rapid and efficient method for filtration or purification of a solution containing target product and / or impurities during which a solution or liquid stream flows parallel to a filtering membrane.

[0279] Centrifugation can, for example, be a low-speed centrifugation to remove larger particles like cellular debris. This can be for example done at 10000 to

[0280] 30 12000 g for 10 to 30 minutes. The released viral particles can be found in the supernatant. P24-158

[0281] - 31 -

[0282] In some embodiments, a nuclease, typically an endonuclease, is added, e.g. to reduce the amount of host cell DNA. It can be added directly to the mixture in the bioreactor before, while or after lysis. The nuclease may be one that degrades both DNA and RNA. In one embodiment, the endonuclease is a

[0283] 5 genetically engineered endonuclease from Serratia marcescens that is sold under the name Benzonase(R) (EMD Millipore, US). If the activity of the nuclease depends on the presence of certain divalent cations it might be favorable to do the nuclease treatment separately, e.g. prior to the addition of a chelator.

[0284] AFFINITY PURIFICATION

[0285] The affinity ligands of the invention are, however, particularly useful for the isolation and / or purification of adeno-associated virus subtype 9 (AAV9) particles or capsids or variants of an AAV9 particle and capsid by e.g., affinity

[0286] 15 chromatography. In purification based on affinity chromatography, a target of interest (e.g. protein or molecule) is selectively isolated according to its ability to specifically and reversibly bind to a ligand that has typically been covalently coupled to a chromatography matrix.

[0287] It has surprisingly been found that the affinity ligands according to the

[0288] 20 invention allow the selective binding as well as subsequent elution of AAV9 to the affinity ligand according to the invention conjugated to a chromatography support. The affinity ligands of the disclosure can be used as ligands for affinity purification of adeno-associated virus subtype 9 (AAV9) particles or capsids or variants of an AAV9 particle and capsid from e.g., clarified cell culture solutions or other natural sources such as biological samples (e.g., serum).

[0289] Accordingly, a further aspect of the present invention refers to an affinity ligand conjugated to a chromatography matrix useful for the purification of

[0290] 30 adeno-associated virus subtype 9 (AAV9) particles or capsids or variants of an AAV9 particle and capsid. In some embodiments, the chromatography P24-158

[0291] - 32 - matrix is a chromatographic resin or membrane. In certain embodiments, the chromatography matrix is a hydrophilic polymer.

[0292] The terms "purifying," "separating," or "isolating," as used interchangeably herein, refer to increasing the degree of purity of the target viral particles from

[0293] 5 a composition or sample comprising the target viral particles and one or more impurities.

[0294] The term "chromatography" refers to any kind of technique which separates an analyte of interest (e.g. a target viral particle) from other molecules present in a sample. Usually, the target viral particle is separated from other molecules as a result of differences in rates at which the individual molecules of the mixture bind to and / or migrate through a chromatography matrix under the influence of a moving phase.

[0295] 15 The term "matrix", "chromatography matrix", or “chromatography support” are used interchangeably herein and refers to a solid phase through which the sample migrates in the course of a chromatographic separation. The matrix typically comprises a base material and ligands covalently bound to the base material. The matrix of the present invention may comprise or

[0296] 20 consists e.g. of resin particles, membranes or monoliths. Suitable base materials are described herein or otherwise known in the art.

[0297] A “ligand” is a functional group that is part of the chromatography matrix, typically it is attached to the base material of the matrix, and that determines the binding properties and interaction properties of the matrix. According to the present invention, the chromatography matrix comprises at least affinity ligands. An affinity ligand is a ligand attached to a base material at least comprising a variant of a member of the Sac7d family as defined above. Said affinity ligand is capable to selectively bind an adeno-associated virus subtype 9 (AAV9) particle or capsid or a variant of an AAV9 particle and

[0298] 30 capsid. In a preferred embodiment, the variant of a member of the Sac7d P24-158

[0299] - 33 - family according to the invention comprise a polypeptide according to SEQ ID NO 5-25.

[0300] The affinity ligand may comprise further functionalities for attachment, purification or further binding properties.

[0301] 5

[0302] Preferably, the affinity ligands are covalently bound to the porous base material, that means covalent bonds are formed between the affinity ligands and the functional groups on the base material.

[0303] Coupling may be realized via one or more functional groups on the affinity ligand, like functional groups at the N-terminus, the C-terminus or at any other position within the peptide sequence, preferably coupling is realized via one or more functional groups at the C-terminus.

[0304] The affinity ligands or a multimers according to the invention may be attached to the base material of the matrix by any type of covalent attachment.

[0305] 15 Methods of covalently coupling polypeptides to a surface are known by those of skill in the art, and peptide tags that can be used to attach a ligand to a matrix are known to those of skill in the art. Further, ligands can be attached (i.e., coupled, linked, or adhered) to a solid surface using any reagents or techniques known in the art. A detailed overview of techniques as well as

[0306] 20 protocols suitable to attach ligands on base materials can be found in Greg T. Hermanson, Bioconjugate Techniques, Third Edition, Academic Press, 2013, especially in Chapter 15 (Greg T. Hermanson, Chapter 15 - Immobilization of Ligands on Chromatography Supports, Editor(s): Greg T. Hermanson, Bioconjugate Techniques (Third Edition), Academic Press, 2013, Pages 589-740, ISBN 9780123822390, https: / / doi.org / 10.1016 / B978- 0-12-382239-0.00015-7). In the following exemplary functional groups and techniques are listed. Further details and suitable attachment conditions can be found in Greg T. Hermanson, Bioconjugate Techniques, Chapter 15.

[0307] 30 Covalent attachment can for example be performed by directly bonding the functional groups of the affinity ligand to suitable residues on the base material like OH, NH2, carboxyl, phenol, anhydride, aldehyde, epoxide or P24-158

[0308] - 34 - thiol etc. Preferably, the attachment of the affinity ligands to the base material is performed by reacting affinity ligands comprising at least one thiol group with a base material that comprises epoxy groups.

[0309] In another preferred embodiment, the attachment of the affinity ligands to the

[0310] 5 base material is performed by reacting affinity ligands comprising at least one amino group with a membrane that comprises aldehyde groups by reductive amination.

[0311] As used herein, the term “ligand density” refers to the amount of affinity ligand immobilized on the chromatography matrix, normalised to the volume of the matrix. In case of membranes it is normalized to the membrane volume and typically expressed as milligrams of ligand per millilitre of membrane volume (mg / ml).

[0312] 15 In some embodiments, the ligand density of the chromatographic matrix is at least about 0.1 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 12 mg / ml, 15 mg / ml, or 20 mg / ml. In certain embodiments, ligand densities are between 0.1 and 10 mg / ml, ligand densities between about 3 mg / ml and 10 mg / ml are preferred.

[0313] 20

[0314] It is also possible to attach the ligands via suitable linkers. It is also possible to generate the matrix by polymerizing monomers comprising the affinity ligands and a polymerizable moiety. Examples of matrices generated by polymerization of suitable monomers are polystyrene, polymethacrylamide or polyacrylamide-based matrices generated by polymerizing suitable styrole or acryloyl monomers.

[0315] In another embodiment the chromatography matrix can be generated by grafting the affinity ligands onto the base material or from the base material. For grafting from processes with controlled free-radical polymerisation, such

[0316] 30 as, for example, the method of atom-transfer free-radical polymerisation (ATRP), are suitable. A very preferred one-step grafting from polymerisation reaction of acrylamides, methacrylates, acrylates, methacrylates etc. which P24-158

[0317] - 35 - are functionalized e.g. with ionic, hydrophilic or hydrophobic groups can be initiated by cerium (IV) on a hydroxyl-containing support, without the support having to be activated.

[0318] Particulate base materials, also called resins, can be prepared, for example,

[0319] 5 from organic polymers. Organic polymers of this type can be polysaccharides, such as agarose, dextranes, starch, cellulose, etc., or synthetic polymers, such as poly(acrylamides), poly(methacrylamides), poly(acrylates), poly(methacrylates), hydrophilically substituted poly(alkyl allyl ethers), hydrophilically substituted poly(alkyl vinyl ethers), poly(vinyl alcohols), poly(styrenes) and copolymers of the corresponding monomers. These organic polymers can preferably also be employed in the form of a crosslinked hydrophilic network. This also includes polymers made from styrene and divinylbenzene, which can preferably be employed, like other hydrophobic polymers, in a hydrophilized form.

[0320] 15

[0321] Alternatively, inorganic materials, such as silica, zirconium oxide, titanium dioxide, aluminium oxide, etc., can be employed as particulate base materials. It is equally possible to employ composite materials, i.e., for example, particles which can themselves be magnetised by copolymerisation

[0322] 20 of magnetisable particles or of a magnetisable core. It is also possible to use core shell materials whereby the shell, i.e. at least the surface or a coating, has OH groups.

[0323] However, preference is given to the use of hydrophilic base materials which are stable to hydrolysis or can only be hydrolysed with difficulty since the materials according to the invention should preferably withstand alkaline cleaning or regeneration at e.g. basic pH over an extended use duration.

[0324] The base matrix may consist of irregularly shaped or spherical particles, whose particle size can be between 2 and 1000 pm. Preference is given to

[0325] 30 average particle sizes between 3 and 300 pm, in a most preferred embodiment the average particle size is between 20 - 63 pm. P24-158

[0326] - 36 -

[0327] The particulate base material may, in particular, be in the form of non-porous or preferably porous particles. The average pore sizes can be between 2 and 300 nm. Preference is given to pore sizes between 5 and 200 nm, most preferred average pore size is between 40 - 110 nm.

[0328] 5

[0329] In a very preferred embodiment, the particulate base material is formed by copolymerisation of a hydrophilically substituted alkyl vinyl ether selected from the group of 1 ,4-butanediol monovinyl ether, 1 ,5-pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclo-'hexane-'dimethanol monovinyl ether and divinylethyleneurea (1 ,3-divinylimidazolin-2-one) as crosslinking agent.

[0330] An example of a suitable commercially available vinylether based base material is Eshmuno®, Merck KGaA, Germany.

[0331] 15 A membrane or membrane matrix according to the invention can be distinguished from particle-based chromatography systems by the fact that the interaction between a fluid sample, e.g. the target structure and / or contaminants, and the matrix does not take place in the pores of a particle, but mainly in the throughpores of the membrane. Accordingly, in certain

[0332] 20 embodiments, the affinity membrane comprises or consists of a porous membrane matrix having a surface comprising a plurality of functional groups. Exemplary types of membranes according to the invention are flat sheet systems, stacks of membranes, microporous polymer sheets with incorporated cellulose, polystyrene or silica-based membranes as well as radial flow cartridges, hollow fiber modules and hydrogel membranes. Membranes may also be produced by 3D printing processes.

[0333] In certain embodiments, the porous membrane matrix comprises a polymeric support impregnated with a (crosslinked) hydrogel network, also known as

[0334] 30 hydrogel membranes. In a preferred embodiment, the porous membrane matrix comprises or consists of a macroporous polymeric support P24-158

[0335] - 37 - impregnated with a crosslinked hydrogel network and the plurality of functional groups is present on the surface of the hydrogel network.

[0336] Such membranes thus comprise a membrane polymeric support and a

[0337] 5 hydrogel formed within the pores of said support. The membrane polymeric support provides mechanical strength to the hydrogel. The hydrogel determines the properties of the final product, like pore size and binding chemistry. The membrane polymeric support can consist of any porous membrane like polymeric membranes, ceramic based membranes and woven or non-woven fibrous material. Suitable polymeric materials for membrane polymeric supports are cellulose or cellulose derivatives as well as other preferably inert polymers like polyethylene, polypropylene, polybutylenterephthalate or polyvinylidene-difluoride.

[0338] The hydrogels can be formed through in-situ reaction of one or more

[0339] 15 polymerizable monomers with one or more crosslinkers and / or one or more cross-linkable polymers to form a cross-linked gel that has preferably macropores. Suitable polymerizable monomers include monomers containing vinyl or acryl groups. Preferred are monomers comprising an additional functional group that either directly forms the ligand of the matrix

[0340] 20 or is suitable for attaching the ligands. Suitable crosslinkers are compounds containing at least two vinyl or acryl groups. In a preferred embodiment, the hydrogel network comprises or consists of a polyacrylamide or copolymers of acrylamide formed in situ within the support.

[0341] Further details about suitable polymeric supports, monomers, crosslinkers etc. as well as suitable production conditions can be found in WO04073843 and WO2010 / 027955. WO2014134147 discloses membranes mixed mode membranes that optionally comprise more than one group like hydrophobic groups, hydrophilic groups or ion exchange groups. Especially preferred are

[0342] 30 membranes made of an inert, flexible fiber web support comprising assembly within and around the fiber web support a porous polyacrylamide hydrogel P24-158

[0343] - 38 - comprising hydroxyl groups like Natrix® type membranes, Merck KGaA, Darmstadt, Germany.

[0344] Examples of suitable membranes of the present invention are

[0345] 5

[0346] - Membranes with a polyethersulfone (PES)-based support and a crosslinked polymeric coating, functionalized with suitable ligands, like Mustang® type membranes, Pall.

[0347] - Membranes made of stabilized reinforced cellulose, functionalized with suitable ligands, like Sartobind® type membranes, Sartorius.

[0348] - Membranes made of stabilized reinforced cellulose, comprising a hydrogel with suitable ligands, like Sartobind® Jumbo Membranes, Sartorius, made of stabilized reinforced cellulose

[0349] 15

[0350] - Membranes made of a fine fiber non-woven scaffold comprising a hydrogel with suitable ligands, like 3MTMEmphaze™ Hybrid Purifier type membranes, 3M.

[0351] 20 - Membranes made of an inert, flexible fiber web support comprising within and around the fiber web support a porous polyacrylamide hydrogel with suitable cation exchange and hydrophobic ligands , like Natrix® type Chromatography membranes, Merck KGaA, Darmstadt, Germany.

[0352] The membranes to be used in the present invention preferably have mean flow pore diameters of 0.3 to 2 pm, more preferably, between 0.6 and 1.2 pm. Mean Flow Pore (MFP) Diameter refers to the average diameter of the pores in a membrane through which fluid can flow. MFP diameter is determined by capillary flow porometry which is used to construct a pore size

[0353] 30 distribution curve that can then be used to determine the mean flow pore diameter. P24-158

[0354] - 39 -

[0355] The membranes may have different sizes and formats depending on the application or the equipment that is used. Typically, the thickness of the membranes is between 10 and 2000 pm, preferably between 50 and 500 pm, most preferred between 150 and 250 pm.

[0356] 5

[0357] A monolith or a monolithic sorbent, similar to a membrane, has throughpores, like interconnected channels, so that liquid can flow from one side of the monolith, through the monolith, to the other side of the monolith.

[0358] Since the mobile phase is flowing through these throughpores, molecules to be separated are transported by convection rather than by diffusion. Due to their structure monolithic sorbents show flow rate independent separation efficiency and dynamic capacity.

[0359] The monolith is typically formed in situ from reactant solutions and can have

[0360] 15 any shape or confined geometry, typically with frit-free construction, which guarantees convenience of operation. Preferably, monolithic materials have a binary porous structure, mesopores and macropores. The micron-sized macropores are the throughpores and ensure fast dynamic transport and low backpressure in applications; mesopores contribute to sufficient surface area

[0361] 20 and thus high loading capacity.

[0362] The monoliths can be made of organic, inorganic or organic / inorganic hybrid materials. Preferred are organic polymer-based monoliths.

[0363] The synthesis of organic polymer monoliths is typically done by a one-step polymerization providing a tunable porous structure with tailored functional groups. Generally, a pre-polymerization mixture consisting of the monomers, crosslinkers, porogenic solvents, and initiators in an appropriate ratio is polymerized in a suitable container, also called mould, determining the format

[0364] 30 of the monolith. Polymerization is typically initiated by heating, use of UV radiation, microwave or y-ray radiation in the presence of initiators. After reaction for the prescribed time at an appropriate temperature, the resulting P24-158

[0365] - 40 - material is typically washed with solvents to remove unreacted components and porogenic solvents.

[0366] Suitable organic polymers are polymethacrylates, polyacrylamides,

[0367] 5 polystyrenes, polyurethanes, etc., like Poly(methacrylic acid-ethylene dimethacrylate), Poly(glycidyl methacrylate-ethylene dimethacrylate) or Poly(acrylamide-vinylpyridine-N,N'-methylene bisacrylamide).

[0368] Inorganic monoliths can be made of silica or other inorganic oxides. Preferably they are made of silica. Silica monoliths are normally prepared via a sol-gel method with phase separation. This mainly includes hydrolysis, condensation, and polycondensation of silica precursors. Typically, tetraethoxysilane (TEOS) or tetramethylorthosilicate (TMOS) is distributed in a suitable solvent in the presence of a porogen (e.g. polyethylene glycol) (PEG)), followed by the addition of a catalyst, acid or base, or a binary

[0369] 15 catalyst, acid and base in sequence. After reaction for a prescribed time, the resulting gel-like product is washed with solvents to remove unreacted precursor, porogen, and catalyst, followed by the proper post treatment, typically a heat treatment.

[0370] 20

[0371] The monoliths can be modified with suitable functional groups, preferably at least ion exchange groups, to generate the targeted interaction with the sample comprising the target molecule and thus the targeted separation.

[0372] Membranes and monoliths can also be produced by 3D printing processes.

[0373] When the chromatography matrix is used in a chromatographic separation it is typically used in a separation device, also called housing, as a means for holding the matrix. Suitable housings are known to the skilled person.

[0374] The affinity ligand conjugated to the matrix may be packed in columns of

[0375] 30 various sizes and operated at various linear velocities or immobilized affinity ligand can be contacted with a solution under conditions favourable to form - 41 - a complex between the ligand and the adeno-associated virus subtype 9 (AAV9) particles or capsids or variants of an AAV9 particle and capsid. Nonbinding materials can be washed away. Suitable wash conditions and buffers can readily be determined by one of skill in the art.

[0376] 5

[0377] According to the present invention the term “buffer” or “solvent” is used for any liquid composition that is used to load, wash, elute, re-equilibrate, strip, clean and / or sanitize a chromatography matrix.

[0378] A "buffer" is a solution that resists changes in pH by the action of its acidbase conjugate components. Various buffers which can be employed depending, for example, on the desired pH of the buffer are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). Non- limiting examples of buffers include MES, MOPS, MOPSO, Tris, HEPES, phosphate,

[0379] 15 acetate, citrate, succinate, and ammonium buffers, as well as combinations of these.

[0380] METHOD FOR AFFINITY CHROMATOGRAPHY

[0381] Another aspect of the present invention relates to an affinity chromatography

[0382] 20 method for isolating adeno-associated virus subtype 9 (AAV9) particles or capsids or variants of an AAV9 particle and capsid, wherein an affinity ligand conjugated to a chromatography matrix as disclosed above is utilized.

[0383] In certain embodiments, the method comprises consists, or essentially consists of the steps of

[0384] (a) providing a liquid sample comprising adeno-associated virus subtype 9 (AAV9) particles and / or capsids or variants of an AAV9 particle and capsid, an affinity ligand conjugated to a chromatography matrix according to the invention, at least one wash buffer, and at least one

[0385] 30 elution buffer, - 42 -

[0386] (b) contacting said liquid sample with the affinity ligand conjugated to a chromatography matrix,

[0387] (c) optionally but preferably washing the chromatography matrix with at least one washing buffer,

[0388] 5

[0389] (d) eluting the adeno-associated virus subtype 9 (AAV9) particles and / or capsids or variants of an AAV9 particle and capsid from the affinity ligand conjugated to a chromatography matrix with at least one elution buffer, and

[0390] (e) optionally cleaning the chromatography matrix with a cleaning liquid.

[0391] The affinity chromatography method for isolating AAV9 vectors according to the invention may be performed in bind-elute. When doing affinity chromatography, the target viral particle is bound to the affinity

[0392] 15 chromatography matrix while the impurities do not bind and flow through. After optional washing of the matrix bound viral particles, the particles are eluted from the matrix with a suitable elution buffer.

[0393] In some embodiments, the method for isolation of the viral particles may

[0394] 20 include one or more of the following process steps:

[0395] - clarification

[0396] - filtration

[0397] - dialysis / diafiltration

[0398] - tangential flow filtration

[0399] - treatment with nuclease, e.g. RNase and / or DNase

[0400] - treatment with chloroform

[0401] 30

[0402] - ion exchange chromatography P24-158

[0403] - 43 -

[0404] - affinity chromatography

[0405] - multimodal chromatography

[0406] - hydrophobic interaction chromatography

[0407] 5

[0408] - centrifugation

[0409] - ultracentrifugation

[0410] - (PEG) precipitation

[0411] - flocculation

[0412] Affinity chromatography may be performed by a) loading the sample comprising the viral particles and process related impurities onto an affinity chromatography matrix targeted against the viral

[0413] 15 particles under conditions that allow binding between the viral particles and the affinity matrix; b) undertaking at least one wash step, preferably undertaking 1 to 3 wash steps; and

[0414] 20 c) eluting the viral particles from the affinity matrix. some embodiments the affinity matrix comprises or consists of the affinity ligand of the invention, wherein the ligand is conjugated to the chromatography matrix. In some embodiments, said matrix is a resin or a membrane. In some embodiments the matrix is a hydrophilic polymer.

[0415] Preferably, in step a), prior to loading the target structure to the affinity chromatography matrix, the matrix is conditioned (equilibrated) with a suitable starting buffer to facilitate binding between the target structure and

[0416] 30 the affinity chromatography matrix. Typically, the starting buffer has a pH P24-158

[0417] - 44 - between 6 and 9. In one embodiment the starting buffer, also called equilibration buffer, is the same buffer as the loading buffer.

[0418] The loading of the (liquid) sample comprising the target structure onto the

[0419] 5 affinity matrix is performed at a pH which is preferably in the range between 6 and 9. PBS pH 7.0 to 7.5 is a preferred loading buffer. However, other standard neutral pH buffers such as 10-50 mM sodium phosphate or Tris can also be used. Adding 0.1 -0.5 M NaCI or KCI may prevent nonspecific adsorption due to protein / protein interactions.

[0420] Suitable conditions and buffers to undertake one or more wash steps, preferably 1 to 3 wash steps, might vary between the pH 5 and 9. PBS pH 7.0 to 7.5 is a preferred wash buffer. However, other standard neutral pH buffers such as 10-50 mM sodium phosphate or Tris can be used. Adding 0.05 to 0.5 M NaCI and / or KCI and / or chelator and / or detergents / surfactants

[0421] 15 and / or amino acids may prevent nonspecific adsorption due to protein / protein interactions and improve impurity removal.

[0422] Conditions and buffers to elute the target structure from the affinity chromatographic matrix may vary. A preferred elution buffer comprises a pH

[0423] 20 in the range between 2 and 3. Glycine buffer at a pH of 2.5 is a preferred elution buffer. However, other buffers in a pH range of 2 to 3, such as 50-100 mM citrate or phosphate can be used. Adding 0.1 -0.5 M NaCI or KCI and / or chelator and / or detergents / surfactants and / or amino acids may enhance elution efficiency of the target.

[0424] The affinity chromatography matrices according to the present invention allow for binding capacities above 1 x 10A14 vp / ml matrix, preferably above 1 x 10A15 vp / ml matrix evaluated by enzyme-linked immunosorbent assay (ELISA).

[0425] 30 For membrane based matrices, the residence time is preferably below 1 minute, most preferred between 0.1 and 0.5 minutes. For particle based P24-158

[0426] - 45 - chromatography resins the residence time is preferably between 1 and 3 minutes. Residence time refers to the average time that the target AAV spends in the chromatography matrix during the separation process.

[0427] In another embodiment, another chromatographic method is used in

[0428] 5 combination with affinity chromatography. Suitable chromatographic methods are for example ion exchange chromatography or multimodal chromatography. In one embodiment, only a cation- or only an anion exchange or only a multimodal chromatography is performed prior or after affinity chromatography. In another embodiment first an affinity chromatography and then at least one other chromatography selected from anion exchange, cation exchange or multimodal chromatography is performed.

[0429] A further aspect of the present invention relates to the use of the affinity

[0430] 15 ligand according to the disclosure for isolation of an adeno-associated virus subtype 9 (AAV9) particle or capsid or a variant of an AAV9 particle and capsid.

[0431] For the sake of good order, it is emphasized that all preferred embodiments

[0432] 20 mentioned above also apply to the use as claimed. Therefore, their repetition is not necessary.

[0433] PHARMACEUTICAL COMPOSITION

[0434] It has been surprisingly found that affinity ligands according to the present invention are able to e.g., deliver cargo to target cells or tissues such as cells infected with an AAV9. Accordingly, a further aspect of the present invention relates to a pharmaceutical composition comprising the affinity ligand, or the nucleic acid encoding said ligand of the invention, and a pharmaceutically acceptable carrier.

[0435] 30

[0436] In some embodiments the pharmaceutical composition comprises a therapeutically effective amount of an affinity ligand provided by the P24-158

[0437] - 46 - invention, optionally attached to at least one cargo molecule, or a salt thereof, and a pharmaceutically acceptable carrier or diluent. Relatedly, the present disclosure provides a method of treating or ameliorating a disease or disorder, comprising administering the pharmaceutical composition to a

[0438] 5 subject in need thereof.

[0439] In a preferred embodiment, cargo is considered any therapeutic agent, diagnostic agent, imaging agent, or toxin. Affinity ligands according to the invention can be fused to cargo in many ways known in the art. For example, nucleic acid cargo can be covalently attached to the lignad by click chemistry. Additionally, multiple identical or different molecules of cargo can be attached to the affinity ligand. For instance, but not limiting, two molecules of heterologous cargo can be attached to an affinity ligand such as an aptamer obtainable by the method according to the present invention

[0440] 15

[0441] Administering a therapeutically effective amount of the pharmaceutical composition according to the invention can be achieved by any means known such as intravenously, subcutaneously, intraperitoneally, orally, or through inhalation.

[0442] 20 As used herein “therapeutically effective amount” refers to an amount of a composition that relieves (to some extent, as judged by a skilled medical practitioner) one or more symptoms of the disease or condition in a mammal. Additionally, by “therapeutically effective amount” of a composition is meant an amount that returns to normal, either partially or completely, physiological or biochemical parameters associated with or causative of a disease or condition. A clinician skilled in the art can determine the therapeutically effective amount of a composition in order to treat or prevent a particular disease condition, or disorder when it is administered, such as intravenously, subcutaneously, intraperitoneally, orally, or through inhalation. The precise

[0443] 30 amount of the composition required to be therapeutically effective will depend upon numerous factors, e.g., such as the specific activity of the active agent, the delivery device employed, physical characteristics of the agent, purpose P24-158

[0444] - 47 - for the administration, in addition to many patient-specific considerations. But a determination of a therapeutically effective amount is within the skill of an ordinarily skilled clinician upon the appreciation of the disclosure set forth herein.

[0445] 5

[0446] The terms “treating,” “treatment,” “therapy,” and “therapeutic treatment” as used herein refer to curative therapy, prophylactic therapy, or preventative therapy. An example of “preventative therapy” is the prevention or lessening the chance of a targeted disease (e.g., cancer or other proliferative diseases) or related condition thereto. Those in need of treatment include those already with the disease or condition as well as those prone to have the disease or condition to be prevented. The terms “treating,” “treatment,” “therapy,” and “therapeutic treatment” as used herein also describe the management and care of a mammal for the purpose of combating a disease, or related condition, and include the administration of a composition to alleviate the

[0447] 15 symptoms, side effects, or other complications of the disease, condition. Therapeutic treatment for cancer includes, but is not limited to, surgery, chemotherapy, radiation therapy, gene therapy, and immunotherapy.

[0448] The invention will be explained in more detail by virtue of the examples set

[0449] 20 forth herein below. While at least one exemplary embodiment is presented, it should be appreciated that a vast number of variations exist. It should also be appreciated that the exemplary embodiments are only examples, and are not intended to limit the scope, applicability, or configuration in any way. Rather, the forgoing description will provide those with ordinary skill in the art with the essential characteristics of this invention for implementing at least one exemplary embodiment, it being understood that various changes may be made without departing from the scope as set forth in the appended claims.

[0450] 30 P24-158

[0451] - 48 -

[0452] BRIEF DESCRIPTION OF THE DRAWINGS

[0453] Figure 1 shows a bar chart representing the results of an ELISA assay, wherein affinity ligands were incubated with AAV9 capsid. Depicted is the

[0454] 5 OD450 signal after incubation.

[0455] Figure 2 shows results of a BLI assay depicting increasing layer thickness on a biotin labelled GFP fusion of affinity ligands of the invention caused by the association with purified AAV9 capsids.

[0456] Figure 3 shows the evaluation of binding of alanine variants (single amino acid mutations) of the affinity ligand NF08.

[0457] Figure 4 shows results of a BLI assay depicting increasing layer thickness on biotin labelled NF08 ligand baring sensors caused by the association with: a)

[0458] 15 purified empty AAV9 capsids, b) purified mixture of empty and full AAV9 capsids, c) purified mixture of empty and full AAV9 capsids there 500nM, 250nM and 125nM concentrations of capsids in BLI working buffer (TBS-T- B).

[0459] Figure 5 shows results of a BLI assay depicting increasing layer thickness on

[0460] 20 biotin labelled NF08 ligand baring sensors caused by the association with AAV9 capsids in clarified cell lysate. 500nM, 250nM and 125nM concentrations of capsids were used thereby diluting the solution with BLI working buffer (TBS-T-B). In a) the non-diluted clarified cell lysate was containing 0.1 % Tween 20 and 500mM NaCI, b) the non-diluted clarified cell lysate was containing 0.1 % TritonX-100.

[0461] Figure 6 shows results of a dynamic chromatography run using NF08 ligand baring Eshmuno® resin and AAV9 containing HEK293T cell lysate. The chromatogram demonstrated the UV signal at 280 nm as well as the pH signal curve. During the loading phase, in total 2.56 x 10A14 particles were

[0462] 30 loaded onto a 0.2mL column prototype. After washing out remaining impurities and not loaded particles using PBS washing buffer, 2.08 x 10A14 P24-158

[0463] - 49 -

[0464] AAV9 particles could be eluted at pH 2.5, resulting in a elution amount of 1.06 x 10A15 vp / mL resin. The column was sanitized and cleaned using 100mM Tris and 2M NaCI (Strip) and 0.1 M NaOH (CIP).

[0465] 5

[0466] 15

[0467] 20

[0468] 30 P24-158

[0469] - 50 -

[0470] EXAMPLES

[0471] Example 1 : Characterization of affinity ligands binding AAV9

[0472] The binding capacity of different affinity ligands according to the invention was evaluated by enzyme-linked immunosorbent assay (ELISA). This

[0473] 5 method is based on the detection of a complex which is built from the immobilized target, tag containing affinity ligand interacting with it and a molecule, which specifically interacts with a tag containing affinity ligand and horseradish peroxidase, which then can be visualized through chromogenic reactions.

[0474] For this method first MaxiSorp™ F96 plate (VWR International, GmbH, Darmstadt, Germany) was coated with purified AAV9 capsids (Sirion Biotech GmbH, Graefelfing, Gaermany). Usually this involves incubating the purified AAV9 capsids in the MaxiSorp™ plate overnight under 600 rpm stirring and 4°C temperature conditions, washing the plate several times after incubation

[0475] 15 and booking the plate with blocking agent.

[0476] In parallel the affinity ligands to be tested (NF01 , NF08, NF04, NF06, NF03, NF09, and NF10) were prepared by N-terminal fusion of Methionine + RGS- HHHHHH-tag + GS Linker (SEQ ID NO: 37) to the polypeptides corresponding to amino acid positions 2-65 (SEQ ID NOs 5-11 ) and a C-

[0477] 20 terminal Cysteine = SEQ ID NOs: 12-18. The described affinity ligands are then purified and dialysed in the TBS buffer containing 0.1 % Tween.

[0478] Next, affinity ligands are given to the MaxiSorp™ plate with immobilized AAV9 capsids and after 1 hour incubation under ambient temperature and 600 rpm stirring are removed by washing the plate with TBS buffer containing 0.1 % Tween. For the detection of this complex, anti-RGS-His-Tag antibody (QIAGEN GmbH, Hilden, Germany) is subjected to the MaxiSorp™ plates and interacts with RGS-HHHHHH tag (SEQ ID NO: 36) on the affinity ligand. Additionally, this antibody is specific to horseradish peroxidase HRP (BioRad Laboratories, Inc., Hercules, CA, USA), which is then used through

[0479] 30 chromogenic reaction to visualise the complexes consisting of the immobilized AAV9 capsid, tag containing affinity ligand, anti-RGS-His-Tag P24-158

[0480] - 51 - antibody and HRP. The visualization follows in the ultraviolet light adsorption of 450 nm.

[0481] 5

[0482] As depicted in Figure 1 , all tested ligands have built a complex with the immobilized AAV9 capsids compared to a control construct, while in particular NF08 and NF09 affinity ligands showed superior quantities of the built complex. The measured adsorption values for the control were below 0.15, whereby for all of the affinity ligand and immobilized AAV9 capsids the adsorption values were > 0.3 and for the NF08 and NF09 affinity ligands the adsorption values were >0.9.

[0483] Example 2: Characterization of affinity ligands binding AAV9

[0484] In order to further characterize the binding properties of the affinity ligands NF01 , NF08, NF04, NF06, NF03, NF09, and NF10, further measurements were performed. Binding parameters of the AAV9-specific affinity ligands

[0485] 30 were measured by biolayer-interferometry (BLI) technology on an Octet RED96 system (ForteBio). P24-158

[0486] - 52 -

[0487] BLI is based on the irreversible interaction between the streptavidin modified sensors and biotin labelled affinity ligands, where such sensors, after modification with labelled ligand, are subjected to solution containing a AAV9 capsid target. The binding of the labelled ligand and the target is then

[0488] 5 detected by the shift of the passing light through such biosensor, and this shift is based on the layer thickness on the sensor surface. This allows to determine the association (binding) and dissociation (elution) of the selected labelled ligand and target under selected solution conditions, such as addition of detergents, salts, or using clarified cell cultures containing targets. Streptavidin biosensors were first functionalized with biotinylated GFP tagged affinity ligands (10pg / mL in TBS containing 0.002% Tween 20 and 0.01 % BSA) at 1.5 nm. Biosensors were allowed to equilibrate for 60 s and binding kinetic was then evaluated by exposing simultaneously biosensors to various concentrations (5, 2.5, 1.25, 0.625, 0.3125, 0.1563, and 0.0781

[0489] 15 nM) of AAV9 in TBS containing 0.002% Tween 20 and 0.01 %. Association was measured for 180s each. The results are shown in Figure 2. As already shown in the ELISA-based assay, all tested affinity ligands bound the intended AAV9 target, while a control affinity ligand (control NF) was unable to bind. In particular, NF08 and NF09 showed increased affinity properties.

[0490] 20

[0491] Example 3: Identification of essential residues of affinity ligands

[0492] The affinity ligands according to SEQ ID NOs 3-9 were generated by mutating the wildtype amino acid sequence of the Sac7d protein according to SEQ ID NO 1 . In order to further characterize the nature of the impact of the identified mutations, the affinity ligand presenting most promising affinity properties for AAV9, NF08, was systematically mutated at defined positions corresponding to variable regions of the affinity ligands according to the invention in comparison to the wildtype sequence of Sac7d of SEQ ID NO 1 . In this process, the variable amino acids at positions 7, 8, 9, 21 , 22, 24, 26,

[0493] 30 29, 31 , 33, 40, 42, 44, and 46 (positions within the polypeptide according to SEQ ID NO 4) were individually substituted with an alanine residue P24-158

[0494] - 53 - generating candidates comprising single amino acid mutations compared to NF08.

[0495] The binding affinity to AAV9 of these different variants was then evaluated by ELISA as previously described in example 1 . As shown in Figure 3 it was

[0496] 5 found that residues 42 and 46 were more permissible for changes in amino acid residue. Accordingly, mutations within the scaffold of the Sac7d amino acids identified herein are required in order to allow AAV9-specific binding and random mutations at the identified residues significantly reduces AAV9- binding affinity.

[0497] Example 4: Binding (association) of biotin labelled AAV9 capsid

[0498] Binding affinity for multiple AAV9 targets was measured by BLI as described above in order to evaluate the binding affinity for empty AAV9 capsid, and

[0499] 15 mixtures of empty and full capsids.

[0500] Three lots of purified AAV9 capsids were used to measure the association of the biotinylated affinity ligand NF08 with AAV9 capsid at various concentration (500, 250, and 125nM). The results (Figure 4) show an increase of the layer thickness proportional to the quantity of the AAV9 capsid

[0501] 20 concentration (from 125nM to 500nM) and is a proof, that biotin labelled affinity ligands of the invention can bind purified AAV9 full and empty capsids.

[0502] The same set-up was used with two different clarified HEK293T cell lysates containing AAV9 capsids. The results (Figure 5) show an increase of the layer thickness proportional to the quantity of the AAV9 capsid concentration (from 125nM to 500nM), while the layer thickness is smaller than the purified AAV9 capsid samples.

[0503] 30 P24-158

[0504] - 54 -

[0505] Example 5: Static binding (association) of AAV9 to immobilized affinity ligand to chromatographic stationary phase

[0506] To test the performance of covalently immobilized affinity ligand (NF08), epoxy activated Eshmuno® resin was modified with purified affinity ligand.

[0507] 5 The modification of epoxy activated Eshmuno® resin was then caried out in phosphate buffer with salt addition pH 9.0 at 40°C temperature for 6 hours in the ratio of 16mg affinity ligand quantity for the 1 ml of Eshmuno® resin after reducing the dimeric polypeptides with DTT for at least 30 minutes.

[0508] After modification the obtained affinity ligand baring Eshmuno® resin is washed to remove non-covalently bound ligand and stored in 20% ethanol and 150mM NaCI containing solution.

[0509] Two additional experiments were performed thereby increasing the salt concentration used and reducing the quantity of DTT via dialysis prior the coupling under increased salt concentration. The obtained affinity ligand

[0510] 15 densities ranged from 5.74mg / ml resin (initial coupling protocol) to 6.66mg / ml resin (protocol with increasing salt concentration and to 8.91 mg / ml (protocol with increasing salt concentration nd dialysis prior coupling).

[0511] The static binding performance was measured by subjecting the resins to

[0512] 20 clarified HEK293T cell culture containing AAV9 capsids. The resins were able to bind all the AAV9 capsids present in the clarified HEK293T cell culture, and upon pH induced elution (100mM Glycine, 250mM NaCI, 0.001 % Pluronic, pH 2.5), ~60% of the bond capsids were eluted. The amount of the binding capacity, calculated for each resin was >2 x 10A14 vp / ml resin.

[0513] To investigate this even further, Eshmuno® NF08 affinity ligand baring resin with highest ligand density was subjected to clarified HEK293T cell culture solution containing 0.5% Tween 20 and 500mM NaCI and to the PBS solution containing purified AAV9 capsids. The resins were able to bind AAV9 capsids

[0514] 30 to the maximum level of 2 x 10A15 viral particles(v) / ml resin with the capsid recovery in the elution fraction between 40-60% (Table 3). P24-158

[0515] - 55 -

[0516] Table 3: Static binding capacity summary table

[0517] 5

[0518] Example 6: Dynamic binding (association) of AAV9 to immobilized affinity ligand to chromatographic stationary phase

[0519] The performance of covalently immobilized affinity ligand (NF08) was demonstrated in a dynamic purification process. Therefore, epoxy activated

[0520] 15 Eshmuno® resin was modified with purified affinity ligand in a parallel approach to Example 5.. The modification of epoxy activated Eshmuno® resin was then caried out in phosphate buffer pH 9.0 at 40°C temperature for 6 hours in the ratio of 16mg affinity ligand quantity for the 1 ml of Eshmuno® resin after reducing the dimeric polypeptides with DTT for at least 30 minutes

[0521] 20 (referring to the initial coupling protocol).

[0522] After modification, the obtained affinity ligand baring Eshmuno® resin is washed to remove non-covalently bound ligand and stored in 20% ethanol and 150mM NaCI containing solution. The affinity ligand density was calculated to be 4.04 mg / ml. The Eshmuno® NF08 affinity ligand baring resin was used to manufacture a 0.196 mL column prototype with 0.5cm x 1 cm column dimensions. The dynamic binding performance was measured by loading 324 mL of clarified HEK293T cell culture containing 7.9 x 10A11vp / mL AAV9 capsids concentration to the resin prototype. The AAV9

[0523] 30 capsids present in the clarified HEK293T cell culture could be bound within neutral pH conditions (PBS, pH 7.4) to the in PBS, pH 4 equilibrated resin, followed by an additional wash step (PBS, pH 7.4). The pH induced elution P24-158

[0524] - 56 - took place using 100mM Glycine, 250mM NaCI, 0.001 % Pluronic, pH 2.5. The resin was able to bind and elute 3.21 x 10A13vp / mL with a respecting 82% elution step recovery. The dynamic binding capacity at 22% breakthrough was extrapolated to 1.06 x 10A15vp / mL resin. The overall

[0525] 5 recovery of all AAV9 particles was calculated to be 94%. Further, the resin has been sanitized and cleaned using 25 column volumes Strip buffer (100mM Tris, 2M NaCI) followed by 25 column volumes CIP buffer (0.1 M NaOH). The belonging chromatography run is demonstrated using the UV signal curve tracked at 280nm and a respective pH signal curve in Figure 6, whereas the AAV9 yield values are summarized in Table 4.

[0526] Table 4: AAV9 dynamic purification run summary

[0527] 30

Claims

P24-158- 57 -CLAIMS1 . An affinity ligand comprising or consisting of a variant of a member of the Sac7d family comprising a polypeptide with an amino acid sequence according to formula 1 (SEQ ID NO 3), from N-terminus to C-5 terminus:VKVKFX1X2X3GE EKEVDTSKIX4 X5VX6RX7GX8X9VXIO FX11YDDNGKX12G X13GX14VX15EKDAP KELLDMLARA EREK formula 1 , whereinXi , X3, X4, X5, X7, Xs, X9, X10, X12, and XisXis, represents a single amino acid independently selected from the group consisting of naturally occurring amino acids;X2is W;15X6is Y;Xio is T;Xn is Y;Xi4is H;20 and wherein said affinity ligand specifically binds with an adeno- associated virus subtype 9 (AAV9) particle or capsid or a variant of an AAV9 particle and capsid.

2. The affinity ligand of claim 1 , whereinXi is L, V, or I;X3 is Q or L;X4 is D or E;X5is T, V, or E30Xy is Q or N;- 58 -Xs is K or Q,X9is W or Y;X12 is F or W.5 3. The affinity ligand of any of the preceding claims, wherein X13 is V or A.

4. The affinity ligand of any of the preceding claims, wherein X15 is T, Q, or S.

5. The affinity ligand of any of the preceding claims, wherein Xs is Q.

6. The affinity ligand of any of the preceding claims, wherein the polypeptide comprises an amino acid sequence having at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence according to SEQ ID NOs: 5-25.

157. The affinity ligand of any of the preceding claims, wherein polypeptide has an amino acid sequence according to SEQ ID NOs: 5-25.

8. The affinity ligand of any of the preceding claims, wherein the polypeptide has an amino acid sequence selected from SEQ ID NOs:20 6, 10, 13, 17, 20, and 24.

9. A nucleic acid molecule coding for the polypeptide of any one of claims 1 to 8.

10. The affinity ligand of any of the preceding claims, wherein the ligand is conjugated to a chromatography matrix.11 . The affinity ligand according to any of the preceding claims, wherein the chromatography matrix is a chromatographic resin.

12. The affinity ligand according to any of the preceding claims, wherein the chromatography matrix is a hydrophilic polymer.30P24-158- 59 -13. A method for isolating adeno-associated virus subtype 9 (AAV9) particles or capsids or variants of an AAV9 particle and capsid, the method comprising, consisting, or essentially consisting of the steps:(a) providing a liquid sample comprising adeno-associated virus5 subtype 9 (AAV9) particles and / or capsids or variants of an AAV9 particle and capsid, an affinity ligand conjugated to a chromatography matrix according to any of claims 10 to 12, at least one wash buffer, and at least one elution buffer,(b) contacting said liquid sample with the affinity ligand conjugated to a chromatography matrix,(c) optionally washing the chromatography matrix with at least one washing buffer,(d) eluting the adeno-associated virus subtype 9 (AAV9) particles15 and / or capsids or variants of an AAV9 particle and capsid from the affinity ligand conjugated to a chromatography matrix with at least one elution buffer, and(e) optionally cleaning the chromatography matrix with a cleaning20 liquid.

14. The use of the affinity ligand of any of claims 10 to 12 for isolation of an adeno-associated virus subtype 9 (AAV9) particle or capsid or a variant of an AAV9 particle and capsid.

15. A method for producing the polypeptide of any one of claims 1 to 8, comprising the steps consisting of a) culturing a cell culture wherein the cells have been transformed by the nucleic acid of claim 9, and b) recovering the polypeptide.

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