CN116490516A

CN116490516A - Affinity ligand library of triple helix bundle protein and use thereof

Info

Publication number: CN116490516A
Application number: CN202180068561.3A
Authority: CN
Inventors: B·基尔; M·诺思; S·巴塔查里亚; T·斯坎龙; W·克特
Original assignee: Everbright LLC
Current assignee: Everbright LLC
Priority date: 2020-10-13
Filing date: 2021-10-13
Publication date: 2023-07-25

Abstract

The present disclosure relates to the field of affinity chromatography, and more particularly to providing libraries of nucleic acids and polypeptides encoding triple helix bundle protein domains suitable for selecting affinity ligands that specifically bind to a target molecule of interest. The disclosure also relates to methods of using those libraries to identify and isolate such affinity ligands for target molecules.

Description

Affinity ligand library of triple helix bundle protein and use thereof

Cross Reference to Related Applications

The present application claims the benefit of provisional application U.S. Ser. Nos. 63/091,201 and 63/188,229, filed on 13 months 5 and 2021, each of which is incorporated herein by reference in its entirety.

Technical Field

Background

The purity of biologically produced therapeutic agents is closely reviewed and regulated by authorities to ensure safety and effectiveness. Thus, there remains a need for means to effectively purify biologically produced therapeutic agents to high purity.

Bioprocess affinity chromatography provides a means of separating and purifying proteins by several steps or a single step. However, the development of affinity ligands can be resource intensive and time consuming.

To facilitate the discovery of affinity ligands, affinity libraries have been developed that can be rapidly and efficiently screened to identify ligands for a target of interest. Such libraries include libraries based on protein a domains or Z domains that can be used to purify immunoglobulins, as well as libraries based on antibodies to utilize specific antigen-antibody interactions during purification. Furthermore, the ability to produce high affinity ligands is another important step in the generation of affinity agents for biological process purification. The ligand should also have high stability to withstand the harsh conditions of bioprocessing, especially the NaOH-containing clean-in-place (CIP) protocol. The ability to reuse the resin in many purification cycles has a profound impact on the economics of the purification process. The evolution of protein a-based IgG-binding resins demonstrated this, modern variants were able to withstand repeated 0.5M NaOH CIP cycles. These IgG binding domains are characterized by a high binding affinity to the interface of the CH2 and CH3 domains in the Fc region, typically less than 50nM (Graille et al (2000) Proc Natl Acad Sci USA.97:5399-5404). In addition, there is also a need for affinity ligands that do not bind IgG, but can be engineered to bind other modes that also have high stability. The present disclosure addresses that need. A range of polypeptides are contemplated that can be used as high affinity ligands for modes or molecules other than IgG.

However, many other diverse structures of biological macromolecules are known to have diverse protein-ligand interaction mechanisms, and there remains a need to find affinity ligands for these macromolecules to take advantage of the reduced procedures provided by biological process affinity chromatography.

The diversity and mechanism of molecular interactions is well known (see, e.g., du et al (2016) int. J. Mol. Sci.17.Doi:10.3390/ijms 17020144). Examples of the structural diversity of some protein-ligand interactions encountered in biomacromolecules are shown in fig. 1, and these models emphasize the need and advantage of having a large number of affinity libraries with different ligand structures.

Furthermore, even in known scaffold libraries, it is still necessary to have many different libraries. For example, in one study 48 different scaffold families were queried for ligand discovery, and 62 best ligands were all from a single scaffold library (scaffold 45 in the 48 scaffold library). This remarkable finding underscores the need for diversity in ligand structures, not just high sequence diversity within a particular ligand structure. Efficient handling of ligand diversity among scaffold families is a major technical challenge in ligand discovery work. In further work, sequence similarity between 62 ligands allowed classification of these ligands into 5 clades. While other families produced about 900 ligands with moderate affinities, these ligands lack the selectivity associated with 62 best ligands. Interestingly, scaffolds for 62 best ligands were developed to accommodate biological "lock and key" affinity ligands. The lack of selective ligands from other scaffold types only suggests that this particular scaffold family has the molecular structure required to mimic the cognate receptor (Coyle et al in Approaches to the Purification, analysis and Characterization of Antibody-Based Therapeutics, (edit, mate) Elsevier,2020, pages 55-79 (at page 65).

Thus, the scarcity of affinity ligands and the different utility between libraries has created a need for ligand libraries that are capable of interacting with many different types of structures representing multiple ligand-target structures. The libraries described herein are part of a strategy to address these needs.

Disclosure of Invention

Libraries of affinity ligands can be used as a source of new affinity ligands for target molecules, which meet the need for a simple and economical way to purify those targets using bioprocess affinity chromatography.

In one aspect, the present disclosure provides a nucleic acid library, members of which encode an affinity ligand comprising an amino acid sequence represented by the formula from N-terminus to C-terminus,

[A]-X ₁ QRRX ₂ FIX ₃ X ₄ LRX ₅ DPS-[X ₆ ] _n -SAX ₇ LLAX ₈ AX ₉ X ₁₀ X ₁₁ NDX ₁₂ QAPX ₁₃ -[B](SEQ ID NO.1)，

wherein (a) [ a ] comprises an α -helix forming peptide domain;

(b)X ₁ 、X ₂ 、X ₃ 、X ₄ 、X ₅ 、X ₆ 、X ₇ 、X ₈ 、X ₁₀ 、X ₁₁ and X ₁₂ Is independently any amino acid;

(c) n represents X ₆ The number of residues, and is an integer from one to ten,

(d)X ₉ and X ₁₃ Each of which is independently A, K or R; and is also provided with

(e) [ B ] is absent, VD, or a peptide domain comprising the amino acid sequence:

VDGQAGQGGGSGLNDIFEAQKIEWHEHHHHHH(SEQ ID NO.9)、

GQAGQGGGSGLNDIFEAQKIEWHEHHHHHH(SEQ ID NO.10)、

VDGLNDIFEAQKIEWHEHHHHHH (SEQ ID NO. 11) or

GLNDIFEAQKIEWHEHHHHHH(SEQ ID NO.12)。

In certain embodiments, the α -helix forming peptide domain of [ A ] comprises the base-stable helix 1 of a Staphylococcal Protein A (SPA) domain (such as a Z-domain, A-domain, B-domain, C-domain, D-domain or E-domain, and in some embodiments the Z-domain is preferred). In some embodiments, [ a ] comprises a peptide having the amino acid sequence: VDAKFDKELEEARAEIERLPNLTE (SEQ ID NO. 2), VDAKFDKEL EEARAKIERLPNLTE (SEQ ID NO. 3), VDAKFDKELEEVRAEIER LPNLTE (SEQ ID NO. 4), VDAKFEKELEEARAEIERLPNLTE (SE Q ID NO. 5), VDAKFDKELEEIRAEIERLPNLTE (SEQ ID NO. 6) or VDAKFDKELEEARAEIERLPALTE (SEQ ID NO. 7). For any of these embodiments, [ A ] may be preceded by M or MAQGT (SEQ ID NO. 8) at the N-terminus.

In certain embodiments, the nucleic acid library is any one of the libraries of SEQ ID NOS.13-18 in Table 1. In other embodiments, [ a ] of the nucleic acid libraries of the disclosure]VDAKFDKELEEARAEIERLPNLTE (SEQ ID NO. 2), n is one, and X ₁₃ K is the number.

In some embodiments, any of the nucleic acid libraries of the present disclosure may comprise a peptide tag, optionally wherein the peptide tag is a hemagglutinin, c-Myc, herpes simplex virus glycoprotein D, T, GST, GFP, MBP, strep tag, his tag, myc tag, TAP tag, or FLAG tag.

In certain embodiments, the affinity ligand further comprises a C-terminal lysine or cysteine.

For some embodiments, the nucleic acid library is a phage display library, a yeast display library, an RNA display library, or a DNA display library. Phage display libraries are particularly useful and can comprise about 10 ⁶ To 10 ⁹ A theoretically different nucleic acid sequence.

In another aspect, the present disclosure provides a method of identifying a polypeptide that selectively interacts with a target molecule of interest, comprising (a) exposing the target molecule of interest to a polypeptide produced by expression of a nucleic acid library of the present disclosure; and (b) separating the selectively interacted polypeptide from the polypeptide that does not selectively interact with the target molecule. In this method, embodiments include expressing the target molecule of interest on the surface of a phage, bacterium, or cell, or attached to, tethered to, or otherwise associated with a solid support.

The present disclosure also provides a method of screening a library to obtain polypeptides that specifically bind with high affinity to a target molecule of interest (i.e., affinity ligands), the library comprising a plurality of polypeptides produced by expression of a nucleic acid library of the present disclosure, by (a) incubating a sample of the library with a concentration of the molecules under conditions suitable for specific binding of the polypeptides to the target molecules; (b) Incubating a second sample of the library under the same conditions but without the target molecule; (c) Contacting each of said first and second samples with an immobilized target molecule under conditions suitable for binding of the polypeptide to said immobilized target molecule; (d) Detecting for each sample the polypeptide bound to the immobilized target molecule; and (e) determining the affinity of the polypeptide for the target molecule by calculating the ratio of the amount of bound polypeptide from the first sample to the amount of bound polypeptide from the second sample.

Still other aspects of the disclosure relate to methods of identifying one or more affinity ligands that specifically bind to a target molecule of interest, comprising: (a) contacting the target molecule with a phage display library; (b) Separating phage that specifically bind (or bind) to the target molecule from phage that do not selectively bind to the target molecule to produce an enriched phage library; (c) Repeating steps a) and b) using the enriched phage library to generate a further enriched phage library; (d) Repeating step c) until the further enriched phage library is enriched at least about 10-fold to about 10-fold relative to the original phage library ⁶ Multiple or more; and (e) combining theThe further enriched phage library is plated and individual clones isolated and characterized therefrom to identify one or more affinity ligands that specifically bind to the target molecule of interest.

In some embodiments of the foregoing methods, the target molecule is bound or attached to a solid support. In other embodiments, the phage display library is bound or attached to a solid support. In either case, the target molecule may be an adeno-associated virus (AAV) or an AAV capsid, and more specifically, AAV is AAV8 or an AAV8 serotype variant.

In another aspect, the present disclosure provides a polypeptide library composition comprising a plurality of synthetic or recombinant polypeptides, each polypeptide comprising an affinity ligand of a nucleic acid library described herein.

In another aspect, the disclosure relates to a method of identifying a polypeptide that specifically binds to a target molecule of interest, comprising: (a) Exposing a target molecule of interest to a polypeptide library composition of the present disclosure; (b) Separating polypeptides that specifically bind to the target molecule from polypeptides that do not selectively bind to the target molecule; and (c) identifying one or more of the polypeptides bound by the target molecule.

Drawings

Figures 1A-1F depict space-filling and banding models of different target-ligand interactions, including (a) large groove interfaces, (B) large plane interfaces, (C) surface-to-surface complementarity, (D) shallow pocket binding, (E) convex/concave (protruding loop) binding, and (F) deep pocket binding.

Fig. 2A and 2B show band diagrams of adding a helix-forming sequence to the N-terminus of a sequence capable of forming a 2-helix domain (a). This addition produced a 3-helix bundle protein (B). In the illustration shown in (a), helices 3 and 2 of the 3-helix bundle protein are shown from left to right, such that the N-terminus of helix 2 is presented to the right. In the illustration shown in (B), the helices are presented in the order of helix 3, helix 1, and helix 2, with the N-terminal end of helix 1 presented in the upper center position of the figure.

Fig. 3 shows a band-shaped illustration of the addition of an elongated loop (highlighted as darker portion) between spiral 3 and spiral 2 of fig. 2B.

FIG. 4 shows a sensor pattern of an exemplary affinity agent.

Fig. 5 shows exemplary stability data for certain affinity agents in the presence of 0.5M NaOH.

Detailed Description

Definition of the definition

For easier understanding of the present disclosure, certain terms are defined below. Unless defined otherwise herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

Units, prefixes, and symbols are expressed in terms of their Syst20 (SI) acceptability. Numerical ranges include the numbers defining the ranges. Unless otherwise specified, amino acid sequences are written from left to right in the amino to carboxyl direction. The headings provided herein are not limitations of the various aspects or implementations of the disclosure which can be had by reference to the specification as a whole. Accordingly, the following directly defined terms are defined in more detail by referring to the specification in its entirety.

It is noted that the term "one or more" entities refers to one or more of the entities; for example, "affinity ligand" is understood to mean one or more affinity ligands. Thus, the terms "one or more", "one or more" and "at least one" are used interchangeably herein.

About or about: as used herein, the term "about" or "approximately" when applied to one or more values of interest refers to a value that is similar to the stated reference value. In certain embodiments, unless stated otherwise or otherwise apparent from the text (except where such numbers would exceed 100% of the possible values), the term "about" or "about" refers to a range of values that fall within either direction (greater than or less than) than 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value.

The term "plurality" as used herein refers to a number of members of a collection, the minimum of which is at least 10, 20, 30, 50, 75, 100, 1000 or more, and the minimum or maximum number may not be readily determinable, but may be indicated by the type of collection or the context in which it is used. For example, a phage display library contains a plurality of phages (which may be the same or different) of equal titer thereto, and thereby encodes a corresponding plurality of polypeptides.

The term "including" is used to mean "including but not limited to. "including" and "including, but not limited to," are used interchangeably.

Biological activity: as used herein, the term "bioactive" refers to the characteristic of any agent that is active in a biological system, particularly in an organism. For example, an agent that has a biological or physiological effect on an organism when administered to the organism is considered to be biologically active.

Variants and mutants: the term "variant" is generally defined in the scientific literature and is used herein to refer to organisms that differ in some way from a recognized standard in inheritance, and "variant" may also be used to describe non-genetic phenotypic differences (King and Stansfield,2002,A dictionary of genetics, 6 th edition, new York, oxford University Press.

The term "mutation" is defined in most dictionaries and is used herein to refer to the process by which a heritable change is introduced into the genetic structure (King and Stansfield, 2002), thereby producing a "mutant". In the scientific and non-scientific literature, the term "variant" is increasingly used instead of the term "mutation". The terms are used interchangeably herein.

Conservative and non-conservative substitutions: a "conservative" amino acid substitution is a substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Amino acid residue families having similar side chains have been defined in the art, including basic side chains (e.g., lysine (K), arginine (R), histidine (H)); acidic side chains (e.g., aspartic acid (D), glutamic acid (E)); uncharged polar side chains (e.g., glycine (G); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine (C)); non-polar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W), beta-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)). And aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)). For example, substitution of tyrosine with phenylalanine is a conservative substitution in some embodiments, conservative amino acid substitutions in the ligand sequence confer or improve specific binding of the ligand to the target of interest. In some embodiments, conservative amino acid substitutions in the ligand sequence do not reduce or eliminate specific binding of the ligand to the target of interest. In some embodiments, conservative amino acid substitutions do not significantly affect specific binding of the ligand to the target of interest. Methods of identifying, altering or maintaining selective binding affinity are known in the art (see, e.g., brmell, biochem.32: biochem. 7 (1993-1187)), amino acid substitutions in the ligand sequence confer or improve specific binding of the ligand to the target of interest (1997; amino acid substitutions in 17; protein in 1997; well-87 (1997) are) in some embodiments, amino acid substitutions in 17-879-well known in the art, non-conservative amino acid substitutions in the ligand sequence do not reduce or eliminate binding of the ligand to the target of interest. In some embodiments, non-conservative amino acid substitutions do not significantly affect the specific binding of the ligand to the target of interest.

Affinity chromatography: as used herein, the term "affinity chromatography" refers to a specific chromatographic mode in which an affinity ligand interacts with a target via biological affinity in a "lock-key" manner. Examples of interactions useful in affinity chromatography are e.g. enzyme-substrate interactions, biotin-avidin interactions, antibody-antigen interactions, etc.

Affinity ligands and ligands: the terms "affinity ligand" and "ligand" are used interchangeably herein. These terms are used herein to refer to molecules capable of reversibly binding with high affinity to a moiety, such as a polypeptide or protein, specific for it.

Protein-based ligands: the term "protein-based ligand" as used herein refers to a ligand comprising a peptide or protein or a portion of a peptide or protein that reversibly binds to a target polypeptide or protein. It should be understood that the "ligands" of the present disclosure are protein-based ligands.

Affinity agent: as used herein, the term "affinity agent" refers to a solid support or matrix to which biospecific affinity ligands are covalently attached. Typically, the solid support or matrix is insoluble in the system for purifying the target molecule. The terms "affinity agent" and "affinity separation matrix" and "separation matrix" are used interchangeably herein.

And (3) joint: as used herein, "linker" refers to a peptide or other chemical linkage that serves to link other independent functional domains. In some embodiments, the linker is located between the ligand and another polypeptide component containing an additional independent functional or structural domain. In some embodiments, the linker is a peptide or other chemical bond between the ligand and the surface.

Naturally occurring: the term "naturally occurring" when used in connection with biological materials such as nucleic acid molecules, polypeptides and host cells refers to those found in nature and not modified by humans. Conversely, when used in connection with biological materials, "non-natural" or "synthetic" refers to those materials that are not found in nature and/or have been modified by humans.

"unnatural amino acid", "amino acid analog" and "nonstandard amino acid residue" are used interchangeably herein. Unnatural amino acids that can be substituted in the ligands provided herein are known in the art. In some embodiments, the unnatural amino acid is a proline-substituted 4-hydroxyproline; 5-hydroxylysine that can be substituted for lysine; 3-methylhistidine, which may be substituted for histidine; homoserine which can replace serine; and ornithine substituted for lysine. Other examples of unnatural amino acids that can be substituted in a polypeptide ligand include, but are not limited to, molecules such as: d-isomers of common amino acids, 2, 4-diaminobutyric acid, alpha-aminoisobutyric acid, A-aminobutyric acid, abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminocaproic acid, aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocysteine, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, lanthionine, dehydroalanine, gamma-aminobutyric acid, selenocysteine and pyrrolysine fluoroamino acids, designer amino acids such as beta-methylamino acid, C alpha-methylamino acid and N alpha-methylamino acid.

"Polynucleotide" and "nucleic acid molecule": as used interchangeably herein, polynucleotide and nucleic acid molecules refer to polymeric forms of nucleotides of any length (ribonucleotides or deoxyribonucleotides). These terms include, but are not limited to DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (microrna), snoRNA (short nucleolar RNA), miRNA (microrna), genomic DNA, synthetic RNA, and/or tRNA (transfer RNA).

Operatively connected to: as used herein, the term "operably connected" means the possibility that two or more components are arranged such that they function properly and that at least one component is permitted to mediate a function imposed on at least one other component. Whether attached directly or indirectly, two molecules are "operably linked".

Peptide tag: the term "peptide tag" as used herein refers to a peptide sequence that is part of or attached (e.g., by genetic engineering) to another protein to provide functionality to the fusion produced thereby. Peptide tags are generally relatively short compared to the proteins they are fused to. In some embodiments, the peptide tag is four or more amino acids in length, such as 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more amino acids. In some embodiments, the ligand is a protein containing a peptide tag. Many peptide tags having the uses as provided herein are known in the art. Examples of peptide tags that may be components of the ligand fusion protein or targets bound by the ligand (e.g., ligand fusion protein) include, but are not limited to, HA (hemagglutinin), c-Myc, herpes simplex virus glycoprotein D (gD), T7, GST, GFP, MBP, strep tags, his tags, myc tags, TAP tags, and FLAG tags (Eastman Kodak, rochester, n.y.). Also, antibodies directed against the tag epitope allow detection and localization of the fusion protein in, for example, affinity purification, western blotting, ELISA assays, and cell immunostaining.

Polypeptide: the term "polypeptide" as used herein refers to a continuous chain of amino acids linked together via peptide bonds. The term is used to refer to chains of amino acids of any length, but one of ordinary skill in the art will appreciate that the term is not limited to long chains and may refer to a smallest chain comprising two amino acids linked together via a peptide bond. The polypeptides may be processed and/or modified as known to those skilled in the art.

Protein: the term "protein" as used herein refers to one or more polypeptides that act as discrete units. The terms "polypeptide" and "protein" are used interchangeably if a single polypeptide is a discrete functional unit and does not require permanent or temporary physical association with other polypeptides to form the discrete functional unit. If a discrete functional unit consists of more than one polypeptide physically associated with each other, the term "protein" refers to a plurality of polypeptides that are physically coupled and function together as a discrete unit.

Specific binding: as used herein, the term "specifically binds" or "has a selective affinity" with respect to a ligand means that the ligand reacts or associates with a particular epitope, protein, or target molecule more frequently, more rapidly, for a longer duration, with greater affinity, or a combination thereof than with an alternative substance (including an unrelated protein). Due to sequence identity between homologous proteins in different species, specific binding may include binding agents that recognize proteins or targets in more than one species, e.g., bispecific or trispecific. Also, due to homology within certain regions of polypeptide sequences of different proteins, specific binding may include binding agents that recognize more than one protein or target. It will be appreciated that in certain embodiments, a binding agent that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require (although it may include) exclusive binding, i.e., binding to a single target. Thus, in certain embodiments, a ligand or affinity agent may specifically bind to more than one target. In certain embodiments, multiple targets may be bound by the same binding site on the affinity agent. "selectively binding" or "selectively interacting" is used interchangeably herein with "specifically binding".

Basically: as used herein, the term "substantially" refers to a qualitative condition that exhibits a feature or characteristic of interest in an overall or near-overall range or degree. It will be appreciated by those of ordinary skill in the biological arts that little, if any, biological and chemical phenomena may be accomplished and/or proceed to completion or achieve or avoid absolute results. Thus, the term "substantially" is used herein to achieve inherent completeness that is potentially lacking in many biological and chemical phenomena.

The present disclosure encompasses, inter alia, nucleic acid and polypeptide libraries for selecting affinity ligands specific for one or more targets of interest (e.g., viral particles in some embodiments).

Affinity ligand libraries, methods of construction and use thereof

Libraries of the present disclosure comprise polypeptide compositions and/or nucleic acid molecules encoding certain triple-helix proteins (fig. 2 and 3), and are useful for identifying and selecting sequences in those libraries that selectively bind one or more target molecules of interest to produce affinity ligands for the biological processing or other uses of the target molecules. Libraries of the present disclosure are capable of generating novel affinity ligands that are, in some embodiments, base stable and that selectively bind to selected targets of interest with high affinity.

Methods of making the libraries disclosed herein include, but are not limited to, direct synthesis, recombinant production, dimer-trimer or codon mutagenesis, site-directed mutagenesis, and the like, and any combination thereof, for nucleic acid libraries, as well as direct chemical synthesis for peptide libraries. All such methods are well known in the art.

Thus, the nucleic acid library of the present disclosure comprises members encoding an affinity ligand comprising an amino acid sequence represented by the formula, from N-terminus to C-terminus,

wherein (a) [ a ] comprises an α -helix forming peptide domain;

VDGQAGQGGGSGLNDIFEAQKIEWHEHHHHHH(SEQ ID NO.9)、

GQAGQGGGSGLNDIFEAQKIEWHEHHHHHH(SEQ ID NO.10)、

VDGLNDIFEAQKIEWHEHHHHHH (SEQ ID NO. 11) or

GLNDIFEAQKIEWHEHHHHHH(SEQ ID NO.12)，

The moiety [ A ] of the formula comprises an alpha-helix forming peptide domain and is preferably base stable. Such peptide domains are well known in the art from a variety of sources. In some embodiments, the α -helix forming peptide domain is a Staphylococcal Protein A (SPA) domain. In certain embodiments, the SPA domain comprises the base-stable helix 1 of the SPA domain found at residues 5-19 (and preferably residues 5-19 of the Z-domain) of any of the SPA Z-domain, A-domain, B-domain, C-domain, D-domain, or E-domain. (see, e.g., nilsson et al (1987) prot. Eng. 1:107-113), and U.S. Pat. Nos. 6534628, 6831161, 7834158, 9187555, 9663558, 9683013, 10308690, 10501557, and 10703774).

In some embodiments, library [ a ] of the present disclosure comprises peptides having the following amino acid sequences: VDAKFDKELEEARAEIERLPNLTE (SEQ ID NO. 2), VDAK FDKELEEARAKIERLPNLTE (SEQ ID NO. 3), VDAKFDKELEEVR AEIERLPNLTE (SEQ ID NO. 4), VDAKFEKELEEARAEIERLPNLT E (SEQ ID NO. 5), VDAKFDKELEEIRAEIERLPNLTE (SEQ ID NO. 6) or VDAKFDKELEEARAEIERLPALTE (SEQ ID NO. 7). The N-terminus of the affinity ligand in the library (i.e.the N-terminus of [ A ]) may be methionine or may comprise the additional amino acid sequence MAQGT (SEQ ID NO. 8).

In embodiments of the nucleic acid library of the present disclosure wherein [ B ] is absent or VD, additional amino acids may be present at the C-terminus of [ B ]. Such additional amino acids include peptide tags and amino acids that facilitate coupling of the affinity ligand to the support matrix.

Thus, any of the nucleic acid libraries of the present disclosure may further comprise peptide tags, including, but not limited to, the following: hemagglutinin, c-Myc, herpes simplex virus glycoprotein D, T7, GST, GFP, MBP, strep tag, his tag, myc tag, TAP tag or FLAG tag.

In certain embodiments, the nucleic acid library is any one of the libraries provided in table 1, i.e., the library comprises members encoding affinity ligands comprising the amino acid sequences listed in table 1, wherein X, [ a ] ]N and [ B ]]As defined above. In some embodiments of the library, n is 1. Because X is ₆ May form part of a ring structure, so X is preferred ₆ Not P or H. In one embodiment, the nucleic acid library of the present disclosure has the formula above, wherein [ a]VDAKFDKELEEARAEIERLPNLTE (SEQ ID NO. 2), n is 1, and X ₁₃ Is K.

TABLE 1

In an exemplary embodiment of the library, [ a]Is VDAKFDKELEEARAE IERLPNLTE; except X ₉ Each X position is, except for, any amino acid; and X is ₉ A, R or K; provided that at least two of the following conditions are satisfied: x is X ₂ Not alanine (A), X ₄ Instead of serine (S), X ₅ Not aspartic acid (D), X ₆ Not glutamine or serine (Q or S), X ₈ Instead of glutamic acid (E), X ₁₀ Not lysine (L) and X ₁₁ Not leucine (L).

In another exemplary embodiment of the library, [ A ]]Is VDAKFDKELEEARAEIERLPNLTE; except X ₉ And X ₁₂ Each X position is, except for, any amino acid; and X is ₉ And X ₁₂ Each of which is A, R or K; provided that at least two of the following conditions are satisfied: x is X ₂ Not alanine (A), X ₄ Instead of serine (S), X ₅ Not aspartic acid (D), X ₆ Not glutamine or serine (Q or S), X ₈ Instead of glutamic acid (E), X ₁₀ Not lysine (L) and X ₁₁ Not leucine (L).

In some embodiments, any of the nucleic acid libraries of the present disclosure may further comprise a peptide tag, including, but not limited to, wherein the peptide tag is a hemagglutinin, a c-Myc, a herpes simplex virus glycoprotein D, T, GST, GFP, MBP, strep tag, a His tag, a Myc tag, a TAP tag, or a FLAG tag. In other embodiments, the affinity ligand in the library comprises a C-terminal lysine or cysteine, whether or not bound to a peptide tag.

In some embodiments, the nucleic acid library of the present disclosure is a phage display library, a yeast display library, an RNA display library, or a DNA display library. In phage display libraries, it is theoretically possible to have 10 ⁶ To 10 ⁹ A different nucleic acid sequence.

According to another aspect of the disclosure, the nucleic acid libraries herein are used in various methods for identifying polypeptides that selectively bind to a target molecule of interest. One embodiment provides a method of identifying a polypeptide that selectively binds to a target molecule of interest, comprising: (a) Exposing a target molecule of interest to a polypeptide resulting from expression of a nucleic acid library of the present disclosure; and (b) separating the selectively bound polypeptide from polypeptides that do not selectively bind to the target molecule. In one embodiment, the target molecule of interest is expressed on the surface of a phage, bacterium, or cell, or is attached to, tethered to, or otherwise associated with a solid support.

Another embodiment provides a method of screening a library to obtain polypeptides that selectively bind to a target molecule of interest with high affinity, the library comprising a plurality of polypeptides produced by expression of a nucleic acid library of the present disclosure, and comprising: (a) Incubating a sample of the library with a concentration of the molecule under conditions suitable for specific binding of the polypeptide to the target molecule; (b) Incubating a second sample of the library under the same conditions but without the target molecule; (c) Contacting each of said first and second samples with an immobilized target molecule under conditions suitable for binding of the polypeptide to said immobilized target molecule; (d) Detecting for each sample the polypeptide bound to the immobilized target molecule; (e) Determining the affinity of the polypeptide for the target molecule by calculating the ratio of the amount of bound polypeptide from the first sample to the amount of bound polypeptide from the second sample.

Yet another embodiment provides a method of identifying one or more affinity ligands that selectively bind to a target molecule of interest, comprising: (a) Contacting the target molecule with a phage display library of the present disclosure; (b) Separating phage that selectively bind to the target molecule from phage that do not selectively bind to the target molecule to produce an enriched phage library; (c) Repeating steps (a) and (b) with the enriched phage library to produce a further enriched phage library; (d) Repeating step (c) until the further enriched phage library is enriched by at least about 10-fold to about 10-fold relative to the original phage library ⁶ Multiple or more; and (e) plating the further enriched phage library and isolating and characterizing individual clones therefrom, thereby identifying one or more affinity ligands that selectively bind the target molecule of interest. Obtaining a sufficiently further enriched phage library to facilitate isolation of the desired individual clonesThe number of cycles is typically three to eight rounds of selection and more typically can be accomplished by 3-4 rounds of selection. In this method, the target molecule or phage display library can be bound or attached to a solid support to facilitate selective binding (and simplify washing conditions, the stringency of which can vary in successive rounds (see examples). Any method known in the art for eluting and recovering bound phage can be used.

In a preferred embodiment of any of these methods, the target molecule is an AAV virus or capsid, and preferably an AAV8 virus or serotype variant thereof.

Still other aspects relate to polypeptide library compositions comprising a plurality of synthetic or recombinant polypeptides, each polypeptide comprising an affinity ligand as defined above for any one of the nucleic acid libraries of the present disclosure. In some embodiments, the plurality is 50 or more or as defined herein (see above). In those embodiments of the polypeptide composition produced from the phage display library, the composition may have a length of 100 to 10 ¹⁰ Individual polypeptides, as determined by phage titer.

Yet another aspect relates to a method of identifying a polypeptide (affinity ligand) that selectively binds to a target molecule of interest, comprising (a) exposing the target molecule of interest to a polypeptide library composition of the present disclosure; and (b) separating the polypeptides that selectively bind to the target molecule from polypeptides that do not selectively bind to the target molecule.

Accordingly, the present disclosure provides an effective method for screening and selecting affinity ligands with binding specificity for one or more of any number of desired molecular target molecules. Libraries of the present disclosure can be screened, and clones comprising putative binding moiety sequences (polypeptides and/or nucleic acids) can be enriched, purified, and tested in any in vitro or in vivo biological assay known and available in the art for a particular molecular target molecule of interest. Once clones that bind the molecular targets are isolated, the polypeptide and/or nucleic acid molecules encoding the affinity ligand can be identified and optionally isolated. Those skilled in the art can use standard genetic and molecular engineering, such as affinity maturation and other well known techniques to optimize the characteristics of the binding moiety for its intended purpose, such as the generation of affinity ligands that interact with the target of interest with high affinity and bind reversibly to facilitate purification and biological process manufacturing of the target. In general, the affinity ligands of the present disclosure are base stable.

Ligands for affinity agents that bind to targets of interest

The characteristics of ligand binding to the target may be determined using known or improved assays, bioassays, and/or animal models known in the art for assessing such activity.

As used herein, terms such as "binding affinity to a target," "binding to a target," and the like refer to a property of a ligand that can be measured directly, for example, by an assay of an affinity constant (e.g., the amount of ligand that associates and dissociates at a given antigen concentration). There are several methods available for characterizing such molecular interactions, for example, competition analysis, equilibrium analysis and micro-thermal analysis, as well as real-time interaction analysis based on surface plasmon resonance interactions (e.g., using a BIACORE instrument). These methods are well known to those skilled in the art and are discussed in publications such as Neri D et al (1996) Tibtech 14:465-470 and Jansson M et al (1997) J Biol Chem 272:8189-8197.

The affinity requirements of a given ligand binding event depend on a variety of factors including, but not limited to, the composition and complexity of the binding matrix, the valency and density of the ligand and target molecule, and the functional application of the ligand. In some embodiments, the ligand is present in an amount of less than or equal to 5X 10 ^-3 M、10 ^-3 M、5×10 ^-4 M、10 ^-4 M、5×10 ^-5 M or 10 ^-5 Dissociation constant of M (K _D ) Bind to a target of interest. In some embodiments, the ligand is present in an amount of less than or equal to 5X 10 ^-6 M、10 ^-6 M、5×10 ^-7 M、10 ^-7 M、5×10 ^-8 M or 10 ^-8 K of M _D Bind to a target of interest. In some embodiments, the ligand is present in an amount of less than or equal to 5X 10 ^-9 M、10 ^-9 M、5×10 ^- ¹⁰ M、10 ^-10 M、5×10 ^-11 M、10 ^-11 M、5×10 ^-12 M、10 ^-12 M、5×10 ^-13 M、10 ^-13 M、5×10 ^-14 M、10 ^-14 M、5×10 ^-15 M or 10 ^-15 K of M _D Bind to a target of interest. In some embodiments, the ligand produced by the methods disclosed herein has the following dissociation constants: about 10 ^-4 M to about 10 ^-5 M, about 10 ^-5 M to about 10 ^-6 M, about 10 ^-6 M to about 10 ^-7 M, about 10 ^-7 M to about 10 ^- ⁸ M, about 10 ^-8 M to about 10 ^-9 M, about 10 ^-9 M to about 10 ^-10 M, about 10 ^-10 M to about 10 ^-11 M, or about 10 ^-11 M to about 10 ^-12 M。

Determination of K can be performed under a variety of conditions _D Binding experiments to dissociation rates. Buffers for preparing these solutions can be readily determined by those skilled in the art and are primarily dependent on the desired pH of the final solution. The low pH solution may be prepared in, for example, citrate buffer, glycine-HCl buffer or succinic acid buffer<pH 5.5). The high pH solution may be prepared in, for example, tris-HCl, phosphate buffer or sodium bicarbonate buffer. For the purpose of determining, for example, the optimal pH and/or salt concentration, a number of conditions may be used to determine K _D And dissociation rate.

In some embodiments, the ligand is at k in the following range _off Specifically bind to a target of interest: 0.1 to 10 ^-7 Second of ^-1 、10 ^-2 To 10 ^-7 Second of ^-1 Or 0.5X10 ^-2 To 10 ^-7 Second of ^-1 . In some embodiments, the ligand is cleaved at a rate (k) less than _off ) Binding to a target of interest: 5X 10 ^-2 Second of ^-1 、10 ^-2 Second of ^-1 、5×10 ^-3 Second of ^-1 Or 10 ^-3 Second of ^-1 . In some embodiments, the ligand is cleaved at a rate (k) less than _off ) Binding to a target of interest: 5X 10 ^-4 Second of ^-1 、10 ^-4 Second of ^-1 、5×10 ^-5 Second of ^-1 Or 10 ^-5 Second of ^-1 、5×10 ^-6 Second of ^-1 、10 ^-6 Second of ^-1 、5×10 ^-7 Second of ^-1 Or 10 ^-7 Second of ^-1 。

In some embodiments, the ligand is at k in the following range _on Specifically bind to a target of interest: about 10 ³ To 10 ⁷ M ^-1 Second of ^-1 、10 ³ To 10 ⁶ M ^-1 Second of ^-1 Or 10 ³ To 10 ⁵ M ^-1 Second of ^-1 . In some embodiments, the ligand (e.g., ligand fusion protein) associates at a rate (k) greater than _on ) Binding to a target of interest: 10 ³ M ^-1 Second of ^-1 、5×10 ³ M ^-1 Second of ^-1 、10 ⁴ M ^-1 Second of ^-1 Or 5X 10 ⁴ M ^-1 Second of ^-1 . In a further embodiment, the ligand is present at a k greater than _on Binding to a target of interest: 10 ⁵ M ^-1 Second of ^-1 、5×10 ⁵ M ^-1 Second of ^-1 、10 ⁶ M ^-1 Second of ^-1 、5×10 ⁶ M ^-1 Second of ^-1 Or 10 ⁷ M ^-1 Second of ^-1 。

Target of interest

According to various embodiments, the target of interest to which the ligand specifically binds may be any molecule for which ligand binding of the affinity agent is desired. For example, the target specifically bound by a ligand may be any target of purification, manufacture, formulation, treatment, diagnostic or prognostic relevance or value. Non-limiting uses include therapeutic and diagnostic uses. For example, a number of exemplary targets are provided herein, and are intended to be illustrative and not limiting. The target of interest may be naturally occurring or synthetic. In some embodiments, the target is a biologically active protein. In some embodiments, the target of interest is an extracellular or intracellular component, a soluble factor (e.g., enzyme, hormone, cytokine, growth factor, antibody, etc.), or a transmembrane protein (e.g., cell surface receptor). In some embodiments, the target of interest to which the ligand specifically binds is itself a ligand having a different sequence.

Joint

The terms "linker" and "spacer" are used interchangeably herein to refer to a peptide or other chemical linkage that serves to link other independent functional domains. In some embodiments, the linker is located between the ligand and another polypeptide component comprising an additional independent functional domain. Suitable linkers for coupling two or more linked ligands may generally be any linker used in the art for linking peptides, proteins or other organic molecules. In some embodiments, this linker is suitable for use in constructing proteins or polypeptides intended for pharmaceutical use.

Suitable linkers for operably linking the ligand and additional components of the ligand fusion protein in a single chain amino acid sequence include, but are not limited to, polypeptide linkers, such as glycine linkers, serine linkers, mixed glycine/serine linkers, glycine and serine rich linkers, or linkers composed primarily of polar polypeptide fragments.

In some embodiments, the linker comprises a majority of amino acids selected from the group consisting of: glycine, alanine, proline, asparagine, glutamine and lysine. In some embodiments, the linker comprises a majority of amino acids selected from the group consisting of: glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In some embodiments, the ligand linker consists of a majority of amino acids that are not sterically hindered. In some embodiments, the linker comprises a majority of amino acids selected from glycine, serine, and/or alanine. In some embodiments, the peptide linker is selected from the group consisting of poly glycine (such as (Gly) ₅ Sum (Gly) ₈ ) Poly (Gly-Ala) and polyalanine.

The linkers can have any size or composition so long as they are capable of operably linking the ligand in a manner that allows the ligand to bind to the target of interest. In some embodiments, the linker is about 1 to 50 amino acids, about 1 to 20 amino acids, about 1 to 15 amino acids, about 1 to 10 amino acids, about 1 to 5 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to 10 amino acids, or about 2 to 5 amino acids. It should be clear that the length, degree of flexibility and/or other properties of the linker may affect certain properties of the ligand for the affinity agent, such as affinity, specificity or avidity for the target of interest, or one or more other target proteins of interest, or proteins not of interest (i.e. non-target proteins). In some embodiments, two or more linkers are used. In some embodiments, two or more linkers are the same. In some embodiments, two or more linkers are different.

In some embodiments, the linker is a non-peptide linker such as an alkyl linker or a PEG linker. For example, alkyl linkers such as-NH- (CH 2) s-C (0) -, where s=2-20, can be used. These alkyl linkers may also be substituted with any non-sterically hindered group, such as lower alkyl (e.g., C1C 6), lower acyl, halogen (e.g., CI, br), CN, NH2, phenyl, and the like. An exemplary non-peptide linker is a PEG linker. In some embodiments, the PEG linker has a molecular weight of about 100 to 5000kDa or about 100 to 500 kDa.

Other techniques described herein and/or known in the art may be used to evaluate the linker. In some embodiments, the linker does not alter (e.g., does not disrupt) the ability of the ligand to bind to the target molecule.

Affinity agents comprising conjugated ligands: affinity separation matrix

Ligands that promote specific binding to the target of interest may be chemically conjugated to a variety of surfaces used in chromatography, such as beads, resins, gels, membranes, monoliths, etc., to prepare affinity agents. Affinity agents comprising ligands directed against a target of interest may be used for purification and manufacturing applications.

In some embodiments, the ligand (e.g., ligand fusion protein) contains at least one reactive residue. The reactive residues may be used as attachment sites for, for example, conjugates, such as chemotherapeutic drugs or diagnostic agents. Exemplary reactive amino acid residues include, for example, lysine and cysteine. The reactive residue may be added to either end of the ligand or within the ligand sequence, and/or may replace another amino acid in the ligand sequence. Suitable reactive residues (e.g., lysine, cysteine, etc.) may also be located within the sequence of the identified ligand without the need for additions or substitutions.

Attachment to solid surfaces

"solid surface," "support," or "matrix" are used interchangeably herein and refer to, but are not limited to, any column (or column material), bead, tube, microtiter plate, solid particle (e.g., agarose or Sepharose), microchip (e.g., silicon, silica glass, or gold chip), or membrane (synthetic (e.g., filter) or biological (e.g., liposome or vesicle)), to which a ligand or other protein can be attached (i.e., coupled, linked, or adhered) directly or indirectly (e.g., through other binding partner intermediates, such as linkers), or into which a ligand can be embedded (e.g., through receptors or channels). Reagents and techniques for attaching polypeptides to solid supports are well known in the art, such as carbamate or thiol ether coupling. Suitable solid supports include, but are not limited to, chromatographic resins or matrices (e.g., agarose beads such as Sepharose-4FF agarose beads), well walls or bottoms in plastic microtiter plates, silica-based biochips, polyacrylamides, agarose, silica, nitrocellulose, paper, plastics, nylon, metals, and combinations thereof. The ligands and other compositions may be attached to the support material by non-covalent association or by covalent bonding using reagents and techniques known in the art. In some embodiments, the ligand is coupled to the chromatographic material using a linker.

In one aspect, the present disclosure provides an affinity agent (affinity separation matrix) consisting of a library of the present disclosure, a ligand or a multimer coupled to an insoluble support. The support may be one or more particles, such as beads; a membrane; a filter; a capillary tube; a monolithic column; as well as any other form commonly used in chromatography. In an advantageous embodiment of the affinity separation matrix, the support consists of substantially spherical particles (also called beads). Suitable particle sizes may be in the range of 5-500 μm, such as 10-100 μm, e.g. 20-80 μm in diameter. In an alternative embodiment, the support is a membrane. In order to obtain a high adsorption capacity, the support is preferably porous, and the ligand may be coupled to the outer surface as well as to the pore surface. In an advantageous embodiment of this aspect, the support is porous.

In another aspect, the present disclosure relates to a method of preparing a chromatographic affinity agent, the method comprising providing a ligand as described above, and coupling the ligand to a support. For example, coupling may be via the nitrogen or sulfur atom of the ligand. The ligand may be coupled to the support directly or indirectly via a spacer element providing a suitable distance between the support surface and the ligand. Methods for immobilizing protein ligands to porous or non-porous surfaces are well known in the art.

Ligand production

A variety of standard techniques known in the art for chemical synthesis, semisynthetic methods, and recombinant DNA methods can be used to generate ligands useful in practicing the provided methods. Methods for producing ligands as soluble agents and cell-associated proteins are also provided, either alone or as part of a multi-domain fusion protein. In some embodiments, the overall production scheme of the ligand includes obtaining a reference protein scaffold and identifying multiple residues within the scaffold for modification. According to embodiments, the reference scaffold may comprise a protein structure or other tertiary structure having one or more alpha-helical regions. Once identified, any of a number of residues may be modified, for example by substitution of one or more amino acids. In some embodiments, one or more conservative substitutions are made. In some embodiments, one or more non-conservative substitutions are made. In some embodiments, a natural amino acid (e.g., one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine) is substituted into the reference scaffold at the target site. In some embodiments, the modification does not include substitution of cysteine or proline. After modification at the identified location desired for a particular embodiment, the resulting modified polypeptides (e.g., candidate ligands) may be recombinantly expressed, e.g., in a plasmid, bacteria, phage, or other vector (e.g., to increase the number of polypeptides per modification). The modified polypeptides may then be purified and screened to identify those modified polypeptides that have specific binding to a particular target of interest. The modified polypeptide may exhibit enhanced binding specificity for a target of interest as compared to a reference scaffold, or may exhibit little or no binding to a given target of interest (or non-target protein). In some embodiments, depending on the target of interest, the reference scaffold may exhibit some interactions (e.g., non-specific interactions) with the target of interest, while certain modified polypeptides will exhibit at least about two-fold, at least about five-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or at least about 100-fold (or more) increased binding specificity to the target of interest. Additional details regarding the generation, selection, and isolation of ligands are provided in more detail below.

Recombinant expression of ligands

In some embodiments, the ligand, such as a ligand fusion protein, is "recombinantly produced" (i.e., produced using recombinant DNA techniques). Exemplary recombinant methods that can be used to synthesize the ligand fusion protein include, but are not limited to, polymerase Chain Reaction (PCR) -based synthesis, concatamerization, seamless cloning, and Recursive Directed Ligation (RDL) (see, e.g., meyer et al, biomacromolecules 3:357-367 (2002); kurihara et al, biotechnol. Lett.27:665-670 (2005); haider et al, mol. Pharm.2:139-150 (2005); and McMillan et al, macromolecules 32 (11): 3643-3646 (1999).

In another aspect, there is also provided a nucleic acid comprising a polynucleotide sequence encoding a ligand or multimer according to the embodiments disclosed above. Thus, the present disclosure encompasses all forms of the nucleic acid sequences of the invention, such as RNA and DNA encoding polypeptides (ligands). The present disclosure provides vectors, such as plasmids, that contain, in addition to the coding sequence, the signal sequences required for expression of a polypeptide or multimer according to the present disclosure. Such polynucleotides optionally further comprise one or more expression control elements. For example, a polynucleotide may comprise one or more promoters or transcription enhancers, ribosome binding sites, transcription termination signals and polyadenylation signals as expression control elements. The polynucleotide may be inserted into any suitable vector, which may be contained within any suitable host cell for expression.

Expression of the nucleic acid encoding the ligand is typically achieved by operably linking the nucleic acid encoding the ligand to a promoter in an expression vector. Typical expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence. Exemplary promoters useful for expression in E.coli include, for example, the T7 promoter.

Methods known in the art can be used to construct expression vectors containing ligand-encoding nucleic acid sequences and appropriate transcriptional/translational control signals. These methods include, but are not limited to, recombinant DNA techniques in vitro, synthetic techniques, and recombinant/gene recombination in vivo. Expression of the polynucleotide may be carried out in any suitable expression host known in the art, including but not limited to bacterial cells, yeast cells, insect cells, plant cells, or mammalian cells. In some embodiments, the nucleic acid sequence encoding the ligand is operably linked to a suitable promoter sequence such that the nucleic acid sequence is transcribed and/or translated into the ligand in the host.

A variety of host expression vector systems may be utilized to express the nucleic acid encoding the ligand. Vectors containing nucleic acids encoding a ligand (e.g., a single ligand subunit or ligand fusion) or a portion or fragment thereof include plasmid vectors, single-and double-stranded phage vectors, and single-and double-stranded RNA or DNA viral vectors. Phage and viral vectors can also be introduced into host cells in packaged or packaged virus form using known infection and transduction techniques. Furthermore, the viral vector may be replication competent or replication defective. Alternatively, cell-free translation systems may also be used to produce proteins using RNA derived from DNA expression constructs (see, e.g., WO86/05807 and WO89/01036; and U.S. Pat. No. 5,122,464).

In general, any type of cell or cultured cell line can be used to express the ligands provided herein. In some embodiments, the background cell line used to generate the engineered host cell is a phage, bacterial cell, yeast cell, or mammalian cell. A variety of host expression vector systems may be used to express the coding sequence of the ligand fusion protein. Mammalian cells can be used as host cell systems transfected with recombinant plasmid DNA or cosmid DNA expression vectors containing the target coding sequences of interest and the fusion polypeptide coding sequences. The cells may be primary isolates from organisms, cultures or cell lines having transformed or transgenic properties.

Suitable host cells include, but are not limited to, microorganisms, such as bacteria (e.g., E.coli, B.subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing ligand-encoding sequences; yeasts transformed with recombinant yeast expression vectors containing ligand coding sequences (e.g., saccharomyces, pichia (Pichia)); insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing ligand coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, caMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., ti plasmid) containing ligand coding sequences.

Prokaryotes that can be used as host cells in the production of ligands include gram-negative or gram-positive organisms such as E.coli and B.subtilis. Expression vectors for prokaryotic host cells typically contain one or more phenotypic selection marker genes (e.g., genes encoding proteins that confer antibiotic resistance or provide autotrophic requirements). Examples of useful prokaryotic host expression vectors include pKK223-3 (Pharmacia, uppsala, sweden), pGEMl (Promega, wis., USA), pET (Novagen, wis., USA) and pRSET (Invitrogen, calif., USA) series vectors (see, e.g., studier, J.mol. Biol.219:37 (1991) and Schoepfer, gene 124:83 (1993)). Exemplary promoter sequences frequently used in prokaryotic host cell expression vectors include T7 (Rosenberg et al, gene 56:125-135 (1987)), beta-lactamase (penicillinase), lactose promoter systems (Chang et al, nature 275:615 (1978)); and Goeddel et al, nature 281:544 (1979)), tryptophan (trp) promoter systems (Goeddel et al, nucleic acids Res.8:4057 (1980)) and tac promoters (Sambrook et al, 1990,Molecular Cloning,A Laboratory Manual, 2 nd edition, cold Spring Harbor Laboratory, cold Spring Harbor, N.Y.).

In some embodiments, eukaryotic host cell systems are used, including yeast cells transformed with recombinant yeast expression vectors containing ligand-encoding sequences. Exemplary yeasts useful for producing the compositions of the present disclosure include yeasts from Saccharomyces, pichia, actinomyces, and Kluyveromyces (Kluyveromyces). Yeast vectors typically contain an origin of replication sequence, an Autonomously Replicating Sequence (ARS), a promoter region, a polyadenylation sequence, a transcription termination sequence and a selectable marker gene from a 2mu yeast plasmid. Examples of promoter sequences in yeast expression constructs include promoters from: metallothionein, 3-phosphoglycerate kinase (Hitzeman, J.biol. Chem.255:2073 (1980)) and other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase and glucokinase. Other suitable vectors and promoters for yeast expression and yeast transformation protocols are known in the art. See, e.g., fleer, gene 107:285-195 (1991) and Hinnen, PNAS 75:1929 (1978).

Insect and plant host cell culture systems may also be used to produce the compositions of the present disclosure. Such host cell systems include, for example, insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing ligand-encoding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, caMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing ligand coding sequences (e.g., ti plasmid), including but not limited to U.S. Pat. No. 6,815,184; U.S. publication nos. 60/365,769 and 60/368,047; and expression systems as taught in WO2004/057002, WO2004/024927 and WO 2003/078614.

In some embodiments, host cell systems, including animal cell systems infected with recombinant viral expression vectors (e.g., adenovirus, retrovirus, adeno-associated virus, herpes virus, lentivirus), including cell lines engineered to contain multiple copies of DNA encoding stably amplified (CHO/dhfr) or unstably amplified ligands in double minichromosomes (e.g., murine cell lines) may be used. In some embodiments, the vector comprising the polynucleotide encoding the ligand is polycistronic. Exemplary mammalian cells that can be used to produce these compositions include 293 cells (e.g., 293T and 293F), CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, per.c6 (Crucell, netherlands) cells VERY, hela cells, COS cells, MDCK cells, 3T3 cells, W138 cells, BT483 cells, hs578T cells, HTB2 cells, BT20 cells, T47D cells, CRL7O30 cells, hsS Bst cells, hybridoma cells, and other mammalian cells. Other exemplary mammalian host cells useful in practicing the present disclosure include, but are not limited to, T cells. Exemplary expression systems and selection methods are known in the art and include those described in the following references and references cited therein: borth et al, biotechnol. Bioen.71 (4): 266-73 (2000); werner et al, arzneimittelforschung/Drug Res.48 (8): 870-80 (1998); andersen et al, curr.op.Biotechnol.13:117-123 (2002); chadd et al, curr.op, biotechnol.12:188-194 (2001), and Giddings, curr.op.Biotechnol.12:450-454 (2001). Other examples of expression systems and selection methods are described in Logan et al, PNAS 81:355-359 (1984); birtner et al Methods enzymol.153:51-544 (1987)). The transcriptional and translational control sequences of mammalian host cell expression vectors are typically derived from the viral genome. Promoter sequences and enhancer sequences commonly used in mammalian expression vectors include sequences derived from polyoma virus, adenovirus 2, simian virus 40 (SV 40) and human Cytomegalovirus (CMV). Exemplary commercially available expression vectors for mammalian host cells include pCEP4 (Invitrogen) and pcDNA3 (Invitrogen).

Physical methods for introducing nucleic acids into host cells (e.g., mammalian host cells) include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York).

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and in particular retroviral vectors, have become the most widely used method of inserting genes into mammalian (e.g., human) cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.

Methods for introducing DNA and RNA polynucleotides of interest into a host cell include electroporation of the cell, wherein an electric field is applied to the cell to increase the permeability of the cell membrane, thereby allowing chemicals, drugs, or polynucleotides to be introduced into the cell. Electroporation may be used to introduce ligands containing DNA or RNA constructs into mammalian cells or prokaryotic cells.

In some embodiments, electroporation of the cells results in expression of the ligand-CAR on the surface of T cells, NK cells, NKT cells. Such expression may be transient or stable throughout the life cycle of the cell. Electroporation can be accomplished by methods known in the art, including MaxCyteAnd->Transfection System (MaxCyte, gaithersburg, md., USA).

Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle). In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. The use of lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo or in vivo) is contemplated. In some embodiments, the nucleic acid is associated with a lipid. Nucleic acids associated with a lipid can be encapsulated within the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing the lipid, mixed with the lipid, bound to the lipid, contained in the lipid as a suspension, contained or complexed with a micelle, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector associated composition is not limited to any particular structure in solution. For example, they may exist in bilayer structures, micelles, or "collapsed" structures. They may also be simply dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include aliphatic droplets naturally occurring in the cytoplasm and a class of compounds containing long chain aliphatic hydrocarbons and derivatives thereof such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use are available from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") is available from Sigma, st.louis, MO; dicetyl phosphate ("DCP") is available from K & K Laboratories (Plainview, N.Y.); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristoyl phosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, inc (Birmingham, AL.). A stock solution of lipids in chloroform or chloroform/methanol may be stored at about-20 ℃. Chloroform can be used as the only solvent because it evaporates more readily than methanol. "liposome" is a generic term that encompasses various unilamellar and multilamellar lipid vehicles formed by the production of a closed lipid bilayer or aggregate. Liposomes are characterized by a vesicle structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. Phospholipids spontaneously form when suspended in excess aqueous solution. The lipid components self-rearrange before forming a closed structure and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al, glycobiology5:505-510 (1991)). However, compositions having a structure in solution that is different from the normal vesicle structure are also contemplated. For example, lipids may be assumed to have a micelle structure or exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

Regardless of the method used to introduce the exogenous nucleic acid into the host cell, the presence of the recombinant nucleic acid sequence in the host cell can be routinely confirmed by a variety of assays known in the art. Such assays include, for example, "molecular biology" assays known in the art, such as DNA and northern blots, RT-PCR, and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, identify agents that fall within the scope of the present disclosure, for example, by immunological means (ELISA and western blot) or by the assays described herein.

Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by a recipient organism, tissue or cell and encodes a polypeptide whose expression is evidenced by some readily detectable property, such as enzymatic activity. The expression of the reporter gene is determined at a suitable time after introduction of the DNA into the recipient cell. Suitable reporter genes include, but are not limited to, genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., ui-Tei et al, FEBS Lett.479:79-82 (2000)). Suitable expression systems are known in the art and can be prepared or commercially available using known techniques. In general, constructs with minimal 5' flanking regions that show the highest expression levels of the reporter gene are identified as promoters. Such promoter regions may be routinely linked to reporter genes and used to assess the ability of an agent to modulate promoter-driven transcription.

Many selection systems are available for use in mammalian host-vector expression systems, including but not limited to the herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyl transferase, and adenine phosphoribosyl transferase (Lowy et al, cell 22:817 (1980)) genes. In addition, antimetabolite resistance can be used as a basis for selection of, for example, dhfr, gpt, neo, hygro, trpB, hisD, ODC (ornithine decarboxylase) and glutamine synthase systems.

In some embodiments, the initiator N-terminal methionine is contained at the NH terminus of one or more ligands of the library of the present disclosure. In many cases, the isolated ligand has no N-terminal methionine residue, which is presumed to be cleaved during expression. In many cases, only a portion of the purified ligand contained an N-terminal methionine in the resulting mixture. It will be apparent to those skilled in the art that the presence or absence of an N-terminal methionine does not affect the functionality of the libraries, ligands and affinity agents described herein.

Ligand purification

Once the ligand or ligand fusion protein is produced by recombinant expression, it can be purified by recombinant protein purification methods known in the art, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. In some embodiments, the ligand is optionally fused to a heterologous polypeptide sequence specifically disclosed herein or known in the art to facilitate purification. In some embodiments, the ligands (e.g., antibodies and other affinity matrices) and optionally the ligands or other components of the ligand fusion composition bound by these ligands are removed from the composition prior to final preparation of the ligands using techniques known in the art.

Chemical synthesis of ligands

In addition to recombinant methods, ligand production can be performed using a variety of liquid and solid phase chemical methods known in the art, using organic chemical synthesis of the desired polypeptide. Various automated synthesizers are commercially available and can be used according to known protocols. See, e.g., tam et al, j.am.chem.soc.,105:6442 (1983); merrifield, science,232:341-347 (1986); barany and Merrifield, the Peptides, gross and Meienhofer, editions, academic Press, new York,1-284; barany et al, int.J.pep.protein Res., 30:705:739 (1987); kelley et al Genetic Engineering Principles and Methods, setlow, J.K., edit Plenum Press, NY.1990, vol 12, pages 1-19; stewart et al, solid-Phase Peptide Synthesis, W.H. Freeman Co., san Francisco,1989. One of the advantages of these methods is that they allow for the incorporation of unnatural amino acid residues into ligand sequences.

The ligands used in the methods of the present disclosure may be modified during or after synthesis or translation, for example by glycosylation, acetylation, benzylation, phosphorylation, amidation, pegylation, formylation, derivatization by known protecting/blocking groups, proteolytic cleavage, conjugation to antibody molecules, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, ubiquitination, etc. (see e.g., creghton, proteins: structures and Molecular Properties, 2 nd edition (w.h.freeman and co., n.y., 1992), postranslational Covalent Modification of Proteins, johnson, editions (Academic Press, new York, 1983), pages 1-12; seiter, meth. Enzymol.,182:626-646 (1990)), ttan.n.y acad. Sci.,663:48-62 (1992)). In some embodiments, the peptide is acetylated at the N-terminus and/or amidated at the C-terminus.

Any of a number of chemical modifications may be made by known techniques including, but not limited to, acetylation, formylation, and the like. In addition, the derivative may contain one or more non-classical amino acids.

In some embodiments, cyclization or macrocyclization of the peptide backbone is achieved by formation of side chains to side chain linkages. Methods for achieving this are well known in the art and may involve natural and unnatural amino acids. Pathways include disulfide formation, lanthionine formation or thiol alkylation (e.g., michael addition), amidation between amino and carboxylic acid side chains, click chemistry (e.g., azide-alkyne condensation), peptide binding, ring closure metathesis, and use of enzymes.

Affinity agent for purification

In affinity chromatography-based purification, a target of interest (e.g., a protein or molecule) is selectively isolated according to its ability to specifically and reversibly bind to a ligand, which is typically covalently coupled to a chromatography matrix. In some embodiments, the ligands identified from the libraries of the present disclosure can be used as affinity purification reagents for a target of interest from recombinant sources or natural sources such as biological samples (e.g., serum).

In some embodiments, a ligand that specifically binds to a target of interest is immobilized on a bead, and then used for affinity purification of the target.

Methods of covalently coupling proteins to surfaces are known to those skilled in the art, and peptide tags useful for attaching ligands to solid surfaces are known to those skilled in the art. In addition, the ligand may be attached (i.e., coupled, linked, or adhered) to the solid surface using any agent or technique known in the art. In some embodiments, the solid support comprises a bead, glass slide, chip, and/or gelatin. Thus, a range of ligands can be used to fabricate arrays on solid surfaces using techniques known in the art. For example, U.S. publication No. 2004/0009530 discloses a method for preparing an array.

In some embodiments, ligands derived from the libraries of the present disclosure are used to isolate their cognate targets of interest by affinity chromatography. In some embodiments, such ligands are immobilized on a solid support. The ligands may be immobilized on a solid support using other techniques and reagents described herein or known in the art. Suitable solid supports are described herein or are known in the art and are suitable for packing chromatography columns in particular embodiments. The affinity agent may be packed in columns of various sizes and run at various linear velocities, or the immobilized affinity ligand may be loaded or contacted with a solution under conditions conducive to the formation of a complex between the ligand and the target of interest. Unbound material can be washed away. Suitable wash conditions can be readily determined by those skilled in the art. Examples of suitable washing conditions are described in Shukla and Hinckley, biotechnol prog.2008, 9 months to 10 months; 24 1115-21.Doi:10.1002/btpr.50.

In some embodiments, chromatography is performed by mixing solutions containing the target of interest and the ligand, and then separating the complex of target of interest and ligand. For example, the ligand is immobilized on a solid support such as a bead, and then separated from the solution by filtration along with the target of interest. In some embodiments, the ligand is a fusion protein containing a peptide tag, such as a polyHis tail or streptavidin binding region, which can be used to isolate the ligand after complex formation using an immobilized metal affinity chromatography resin or streptavidin coated substrate. Once isolated, the target of interest can be released from the ligand under elution conditions and recovered in purified form.

Elution of the target of interest can be accomplished by techniques well known in the art, including by lowering the pH and increasing the salt concentration or otherwise altering the salt conditions. For example, elution of the virus particles is typically achieved by lowering the pH, e.g., 2.0-3.0, although higher pH may be used. The optimal conditions for eluting AAV8 and variants and mutants thereof can be readily determined by those skilled in the art.

The affinity agents of the present disclosure are alkali resistant and can be cleaned using NaOH at concentrations up to 0.5M. In certain embodiments, a CIP regimen, for example, of up to 30 to 60 minutes of exposure to 0.5M NaOH per cycle, can ensure chromatographic performance uniformity over several cycles (e.g., 15-30 cycles), including up to 70% -90% initial AAV8 binding capacity and low residual DNA and HCP levels, as well as substantially unchanged flow capacity.

Although some embodiments of the present disclosure have been described by way of illustration, it will be apparent that the invention herein may be practiced with many modifications, variations and adaptations, and with the use of many equivalents or alternative solutions that are within the scope of those skilled in the art, without departing from the spirit of the present invention or exceeding the scope of the claims.

All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Examples

Example 1: library construction and screening

Phage display libraries were designed based on the facial variant positions in Z domain helices 2 and 3 (see fig. 2 and 3) using the following scaffolds:

[A]-X ₁ QRRX ₂ FIX ₃ X ₄ LRX ₅ DPSX ₆ SAX ₇ LLAX ₈ AX ₉ X ₁₀ X ₁₁ NDX ₁₂ -[B]。

scaffolds for phage libraries were constructed using trimeric codon phosphoramidite mutagenesis.

For a library, [ A ]]Is VDAKFDKELEEARAEIERLPNLTE; except X ₉ Each X position is, except for, any amino acid; and X is ₉ A, R or K; provided that at least two of the following conditions are satisfied: x is X ₂ Not alanine (A), X ₄ Instead of serine (S), X ₅ Not aspartic acid (D), X ₆ Not glutamine or serine (Q or S), X ₈ Instead of glutamic acid (E), X ₁₀ Not lysine (L) and X ₁₁ Not leucine (L).

And at least two of the following are satisfied as true.

For another library, [ A ]]Is VDAKFDKELEEARAEIERLPNLTE; except X ₉ And X ₁₂ Each X position is, except for, any amino acid; and X is ₉ And X ₁₂ Is A, R or K; provided that at least two of the following conditions are satisfied: x is X ₂ Not alanine (A), X ₄ Instead of serine (S), X ₅ Not aspartic acid (D), X ₆ Not glutamine or serine (Q or S), X ₈ Instead of glutamic acid (E), X ₁₀ Not lysine (L) and X ₁₁ Not leucine (L).

Phage library panning is typically performed as described in Griffiths et al 1994, EMBO J., 13:3245-3260. Multiple rounds of panning were performed as needed for targets of interest (including, e.g., AAV8 capsids).

Individual phage clones can be tested for binding to a target of interest in a phage ELISA. Briefly describedWill be 1×10 ¹² Each phage was incubated in 96-well plates coated with 1. Mu.g/mL of target of interest and negative control. After one hour incubation at room temperature, unbound particles were removed by washing the wells three times in PBS-0.1% Tween-20. Bound phage are detected using specific anti-M13 antibodies, isolated and sequenced to identify affinity ligands that bind to the target of interest. After identification, the affinity ligand may be prepared as a peptide or recombinantly produced.

Peptides were synthesized by standard Fmoc solid phase peptide synthesis techniques and purified by preparative reverse phase HPLC. The purity of the peptides was assessed by RP-UPLC with UV and quadrupole time-of-flight mass spectrometry.

Recombinant affinity ligands are expressed in E.coli or Pichia using standard techniques. The ligand may be purified using multi-column chromatography. For His-tagged ligands, immobilized Metal Affinity Chromatography (IMAC) was used as the primary capture step. Using AviTag ^TM The system (density, aurora, CO) produces biotinylated ligands. Preparation of AviTag-bearing by omitting exogenous Biotin ^TM Non-biotinylated ligands of the sequences. Purity and identity of recombinant protein ligands by SDS-PAGE, RP UPLC, quadrupole time-of-flight mass spectrometry, and Size Exclusion Chromatography (SEC); the combination of Sephadex S75 (Cytiva, marlborough, mass.) was evaluated. In many cases, the isolated ligand has no N-terminal methionine residue, which is presumed to be cleaved during expression.

Example 2 exemplary AAV8 affinity ligands

This example demonstrates the binding of biotinylated ligand to AAV8 capsids based on affinity ligand obtained from the library described in example 1 using biolayer interferometry (ForteBio, menlo Park, CA). The biotinylated ligand was immobilized on the sensor and was mixed with a solution containing 5X 10 in 100mM sodium phosphate, 100mM sodium chloride, 0.01% (w/v) bovine serum albumin and 0.1% (v/v) Triton X-100 ¹¹ Each pellet/mL of solution (pH 7.0) was incubated. Blank sensors and unbound ligand were included as controls. The association phase shows an initial linear increase in response, which is a typical feature of AAV. As the sensor becomes saturated, the sensor map shows a greater curvature (fig. 4, ligand 1).Responses were measured after 4000 seconds incubation time and are shown in table 2 below for exemplary AAV8 ligands.

TABLE 2

Ligand	Response to
		1	7.31
2	4.47
		3	6.48
4	5.99
		5	6.19
6	6.29
		7	5.56
8	6.00
		9	6.16
10	6.21
		11	5.91
12	6.23
		13	4.94
14	4.47
		15	6.06
16	5.85
		17	6.18
18	5.79
		19	4.95
20	5.60
		Blank sensor	0.05
Non-binding ligands	0.03

EXAMPLE 3 alkaline stability of library affinity ligands

This example demonstrates the sodium hydroxide stability of the affinity ligand. The ligand was incubated in 0.5M NaOH for 16 hours and then neutralized. Binding of NaOH-treated ligand was measured and compared to untreated ligand as described in example 2. The retained binding is calculated according to the following formula:

binding = (measured response after NaOH treatment)/(measured response without treatment) ×100% retention

The data demonstrate that the affinity ligands of the library can have high base stability (fig. 5).

Example 4 construction of exemplary affinity resins

This example demonstrates the generation and characterization of affinity agents comprising the ligands identified and described herein. Affinity resins were prepared by conjugating ligands to agarose beads. RAPID RUN 6% agarose beads (ABT, madrid, spain) and Jetted a50 beads (Purolite, king of Prussia, PA) were activated with disuccinimidyl carbonate and conjugated to peptide ligands at ligand densities of 1-8mg/mL resin. The actual ligand density of all resins was measured using the subtractive RP-HPLC method according to the following formula:

actual ligand density =

(amount of ligand in feed-amount of ligand in effluent)/(amount of ligand in effluent)

Resin volume.

Many modifications and other embodiments of the disclosure set forth herein will come to mind to one skilled in the art to which these disclosure pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosure is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims and list of embodiments disclosed herein. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Any of the methods disclosed herein need not be performed in the order recited. The methods disclosed herein include certain actions taken by a practitioner; however, they may also include any third party indication of such behavior, whether explicit or implicit.

Claims

1. A nucleic acid library, the members of which encode an affinity ligand comprising an amino acid sequence represented by the formula from N-terminus to C-terminus,

[A]-X ₁ QRRX ₂ FIX ₃ X ₄ LRX ₅ DPS-[X ₆ ] _n -SAX ₇ LLAX ₈ AX ₉ X ₁₀ X ₁₁ NDX ₁₂ QAPX ₁₃ -[B]

(SEQ ID NO.1)，

wherein (a) [ a ] comprises an α -helix forming peptide domain;

(b)X ₁ 、X ₂ 、X ₃ 、X ₄ 、X ₅ 、X ₆ 、X ₇ 、X _8、 X ₁₀ 、X ₁₁ and X ₁₂ Is independently any amino acid;

VDGQAGQGGGSGLNDIFEAQKIEWHEHHHHHH(SEQ ID NO.9)、

GQAGQGGGSGLNDIFEAQKIEWHEHHHHHH(SEQ ID NO.10)、

VDGLNDIFEAQKIEWHEHHHHHH (SEQ ID NO. 11) or

GLNDIFEAQKIEWHEHHHHHH(SEQ ID NO.12)。

2. The nucleic acid library of claim 1, wherein the alpha-helix-forming peptide domain comprises the base-stable helix 1 of the Staphylococcal Protein A (SPA) domain found at residues 5-19 of any of the SPA Z-domain, a-domain, B-domain, C-domain, D-domain and E-domain, and preferably the Z-domain.

3. The nucleic acid library of claim 1 or 2, wherein [ a ] comprises a peptide having the amino acid sequence:

VDAKFDKELEEARAEIERLPNLTE(SEQ ID NO.2)、

VDAKFDKELEEARAKIERLPNLTE(SEQ ID NO.3)、

VDAKFDKELEEVRAEIERLPNLTE(SEQ ID NO.4)、

VDAKFEKELEEARAEIERLPNLTE(SEQ ID NO.5)、

VDAKFDKELEEIRAEIERLPNLTE (SEQ ID NO. 6) or

VDAKFDKELEEARAEIERLPALTE(SEQ ID NO.7)。

4. The nucleic acid library of claim 3, wherein [ A ] is N-terminally preceded by M or MAQGT (SEQ ID NO. 8).

5. The nucleic acid library of any one of claims 1-4, comprising a peptide tag, optionally wherein the tag is a hemagglutinin, c-Myc, herpes simplex virus glycoprotein D, T, GST, GFP, MBP, strep tag, his tag, myc tag, TAP tag, or FLAG tag.

6. The nucleic acid library of claims 1-5, wherein the ligand comprises a C-terminal lysine or cysteine.

7. The nucleic acid library of any one of claims 1-6, wherein [ a]VDAKFDKELEEARAEIERLPNLTE (SEQ ID NO. 2), n is 1, and X ₁₃ Is K.

8. The nucleic acid library of claim 1, wherein the library comprises any one of SEQ ID nos. 13-18.

9. The nucleic acid library of any one of claims 1-8, wherein the library is a phage display library, a yeast display library, an RNA display library, or a DNA display library.

10. A method of identifying a polypeptide that selectively interacts with a target molecule of interest, comprising:

a) Exposing a target molecule of interest to a polypeptide resulting from expression of the nucleic acid library of any one of claims 1-9; and

b) Separating the polypeptides that selectively interact with the target molecule from the polypeptides that do not selectively interact with the target molecule.

11. The method of claim 10, wherein the target molecule of interest is expressed on the surface of a phage, bacteria, or cell, or is attached to, tethered to, or otherwise associated with a solid support.

12. A method of screening a library to obtain polypeptides that specifically bind to a target molecule of interest with high affinity, the library comprising a plurality of polypeptides resulting from the expression of the nucleic acid library of any one of claims 1-9, the method comprising:

(a) Incubating a sample of the library with a concentration of the molecule under conditions suitable for specific binding of the polypeptide to the target molecule;

(b) Incubating a second sample of the library under the same conditions but in the absence of the target molecule;

(c) Contacting each of said first and second samples with an immobilized target molecule under conditions suitable for binding of said polypeptide to said immobilized target molecule;

(d) Detecting for each sample the polypeptide bound to the immobilized target molecule; and

(e) Determining the affinity of the polypeptide for the target molecule by calculating the ratio of the amount of bound polypeptide from the first sample to the amount of bound polypeptide from the second sample.

13. A method of identifying one or more affinity ligands that specifically bind to a target molecule of interest, comprising:

(a) Contacting the target molecule with the phage display library of claim 9;

(b) Separating phage that specifically bind to the target molecule from phage that do not selectively interact with the target molecule to produce an enriched phage library;

(c) Repeating steps a) and b) with the enriched phage library to produce a further enriched phage library;

(d) Repeating step c) until the further enriched phage library is enriched at least about 10-fold to about 10-fold relative to the original phage library ⁶ Multiple or more; and

(e) The further enriched phage library is plated and individual clones isolated and characterized therefrom to identify one or more affinity ligands that specifically bind to the target molecule of interest.

14. The method of claim 13, wherein the target molecule or the phage display library is bound or attached to a solid support.

15. The method of claim 13 or 14, wherein the target molecule is an adeno-associated virus (AAV) or AAV capsid.

16. The method of claim 15, wherein the AAV is AAV8 or an AAV8 serotype variant.

17. A composition comprising a plurality of synthetic or recombinant polypeptides, each polypeptide comprising an affinity ligand according to any one of claims 1-8.

18. A method of identifying a polypeptide that specifically binds to a target molecule of interest, comprising:

(a) Exposing a target molecule of interest to the composition of claim 17;

(b) Separating polypeptides that specifically bind to the target molecule from polypeptides that do not selectively bind to the target molecule; and

(c) Identifying one or more polypeptides bound by the target molecule.