Affinity agent

Abstract

Provided herein are affinity agents comprising a ligand that specifically binds to a target molecule. The affinity agents can be used for binding, isolation and / or purification.

Description

BACKGROUND OF THE INVENTION

[0001] Authorities closely scrutinize and regulate the purity of biogenerated therapeutic agents to ensure safety and efficacy. Accordingly, there remains a need for means to efficiently purify biogenerated therapeutic agents to high purity.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 428,949, filed on November 30, 2022, the disclosure of which is hereby incorporated herein by reference in its entirety. SUMMARY OF THE INVENTION

[0004] To support the clinical efforts of therapeutic proteins, there is a need for compositions and methods for efficiently purifying proteins from recombinant sources. Affinity purification is a means of separating and / or achieving the desired purity of a protein in several steps or a single step. However, the development of affinity agents (e.g., those comprising an affinity ligand) can be a resource-intensive and time-consuming task. This has led to the development of affinity agents for only a limited number of proteins. In the absence of an affinity agent, purification typically involves inefficient, labor-intensive, and expensive processes such as multi-column processes.

[0005] Exemplary therapeutic proteins include, but are not limited to, bioactive polypeptides / proteins, fusion proteins, enzymes, hormones, antibodies, and antibody fragments. The purification of certain proteins (such as fusion proteins) presents additional challenges due to product homogeneity or the presence of product-related impurities. Some impurities may be caused by misassembled fusion proteins and proteolytic cleavage, which can be particularly difficult to remove because these impurities are closely related to the desired product.

[0006] Described herein are affinity agents that bind to haptoglobin and can be used for separation and / or affinity purification. In some embodiments, the affinity agent comprises a solid support and a ligand.

[0007] Haptoglobin (Hp) belongs to the acute-phase plasma protein family and is an important scavenger and detoxifying agent for free hemoglobin. Hp is an acidic tetrameric glycoprotein composed of two α / β dimers, which participates in the body's natural defense system against inflammation and viral infections. The Hp protein is encoded by a single gene and produced as a single polypeptide chain. After proteolytic processing of the primary amino acid sequence, the α and β chains are covalently linked by multiple disulfide bonds to form the active molecule. In the human population, there are two major alleles, Hp1 and Hp2, that give rise to three different genotypes (Hp1-1, Hp1-2, and Hp2-2), and the resulting gene products are heterogeneous mixtures of various transcripts. The biological activity of Hp is related to different phenotypes. The most important biological activity of Hp is to regulate the clearance of hemoglobin in the circulation by complexing with the macrophage CD163 receptor and then through the endocytosis of the Hp-hemoglobin complex, thereby preventing the severe consequences of oxidative stress. In addition to the antioxidant properties resulting from hemoglobin clearance, Hp also has the ability to stimulate monocytes / macrophages and regulate helper T cell responses, which means that Hp is an important mediator in various pathogenic conditions including infectious diseases, diabetes, cardiovascular diseases, and cancer.

[0008] Haptoglobin has practical applications as a therapeutic molecule, related to its function as a hemoglobin scavenger. Administration of haptoglobin has improved the outcomes of sepsis patients with high cell-free hemoglobin and has been shown to improve the outcomes of patients after burns. Traditional methods for haptoglobin purification use the Cohn fractionation process, which was originally developed for purifying human serum albumin from plasma during World War II. The Cohn fractionation process provides an efficient process for the purification of plasma proteins, and its continuous use in the industrial production of plasma proteins since the mid-1940s has demonstrated the need for suitable purification devices.

[0009] The main drawback of producing haptoglobin from human plasma using the Cohn fractionation process is that the method has low purity problems because the process does not provide a method for separating haptoglobin from serum albumin, where the concentration of serum albumin in human plasma and in Cohn fraction V is at least 100 times higher, which is usually the starting point for separating haptoglobin from human plasma.

[0010] This article describes an affinity reagent that specifically binds to the haptoglobin protein to provide a selection from serum albumin and can be used for separation and / or affinity purification. In some embodiments, the affinity agent comprises a solid support and a ligand.

[0011] In some embodiments, the affinity agent comprises a three-helix bundle protein. The structure of the three-helix bundle protein can be envisioned as a triangular prism, where each triangular vertex represents a helix, as shown in FIG. 1 for example. In some embodiments, the combination of any two helices defines a rectangular face. For example, in some embodiments, the three faces of the helix bundle protein are defined by:

[0012] 1) Helices 1 and 2 ( Figure 1B Face 1, 2 therein);

[0013] 2) Helices 2 and 3 ( Figure 1B Face 2, 3 therein);

[0014] 3) Helices 1 and 3 ( Figure 1B Face 1, 3 therein); and

[0015] their combinations.

[0016] In some embodiments, provided herein is an affinity agent that comprises a face formed by helices 2 and 3 in a three-helix bundle protein (i.e., the residues involved in binding to the target protein are located within helices 2 and 3). In some embodiments, the primary function of helix 1 in the three-helix bundle protein is to complete and stabilize the three-helix bundle. In some embodiments, variations can be made to helix 1 that maintains helix 1 and the three-helix bundle structure.

[0017] In some embodiments, provided herein is an affinity agent that comprises a ligand, the ligand comprising SEQ ID NO:1,

[0018] X 1 QRRX 2 FIX 3 X 4 LRX 5 DPSX 6 SAX 7 LLAX 8 AKX 9 X 10 NDX 11 QAPK, where X 1 is A, D, E, H, I, L, Q, S, T, V or W; X 2 is A, E, G, H, N, Q, S or Y; X 3 is A, D, E, F, G, H, I, K, L, N, Q, R, S, T, W or Y; X 4 is A, F, H, L, Q, S, T, V or Y; X 5 is A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V or Y; X 6 is A, D, E, G, H, I, K, L, Q, S, T or V; X 7is A, E, G, H, I, L, P, S, T, V, W or Y; X 8 is A, G, I, L, T or V; X 9 is A, D, E, G, H, K, N, R, S or T; X 10 is F, H, R, V or W; and X 11 is A, D, E, H, K, N, Q, R, S, T or an amino acid sequence that differs by no more than three, no more than two or no more than one substitution, addition or deletion.

[0019] In some embodiments, the affinity agents provided herein that comprise SEQ ID NO:1 are respectively contained within helix 2 and helix 3 of a three-helix bundle protein. In some embodiments, provided herein is an affinity agent that comprises SEQ ID NO:1 and is respectively contained within helix 2 and helix 3 of a three-helix bundle protein.

[0020] In some embodiments, provided herein is one or more affinity agents that comprise a ligand, wherein the ligand comprises SEQ ID NO:2-123, or an amino acid sequence that differs by no more than three, no more than two or no more than one substitution, addition or deletion.

[0021] In some embodiments, provided herein are affinity agents that comprise a multimeric polypeptide, wherein the multimeric polypeptide comprises at least two subunits; and wherein each subunit comprises a polypeptide according to the preceding embodiments.

[0022] In some embodiments, provided herein are affinity agents that comprise a multimeric polypeptide, wherein the subunits are not all the same.

[0023] In some embodiments, provided herein are affinity agents for purifying haptoglobin derived from human plasma.

[0024] In some embodiments, provided herein are affinity agents for purifying human haptoglobin derived from a recombinant source.

[0025] Definitions

[0026] For easier understanding of the present disclosure, certain terms are defined below. Unless otherwise defined, technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art.

[0027] About or approximately: As used herein, when applied to one or more values of interest, the terms "about" or "approximately" refer to a value similar to the reference value. In certain embodiments, the terms "about" or "approximately" refer to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the specified reference value, unless otherwise stated or otherwise apparent from the context (except where such numbers exceed 100% of the possible value).

[0028] Bioactive: As used herein, the term "bioactive" refers to the characteristic of any agent that is active in a biological system and, in particular, in a living organism. For example, an agent that has a biological effect on the living organism when administered to the living organism is considered to be bioactive.

[0029] Conservative and non-conservative substitutions: A "conservative" amino acid substitution is a substitution in which an amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include 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)); nonpolar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W)); β-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 to the target of interest of the ligand. In some embodiments, 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, conservative amino acid substitutions do not significantly affect specific binding of the ligand to the target of interest. Methods for identifying nucleotide and amino acid conservative and non-conservative substitutions that confer, alter, or maintain selective binding affinity are known in the art (see, e.g., Brummell, Biochem. 32:1180-1187 (1993); Kobayashi, Protein Eng. 12(10):879-884 (1999); and Burks, PNAS 94:412-417 (1997)). In some embodiments, non-conservative amino acid substitutions in the ligand sequence confer or improve specific binding to the target of interest of the ligand. In some embodiments, 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 specific binding of the ligand to the target of interest.

[0030] Linker: As used herein, a "linker" refers to a peptide or other chemical linkage used to connect other independent functional domains. In some embodiments, the linker is located between the ligand and another polypeptide component containing other independent functional domains. In some embodiments, the linker is a peptide or other chemical linkage between the ligand and a surface.

[0031] Naturally occurring: When used in connection with biological materials such as nucleic acid molecules, polypeptides, and host cells, the term "naturally occurring" refers to those materials that exist in nature and have not been modified by man. In contrast, when used in connection with biological materials, "non-natural" or "synthetic" refers to those materials that do not exist in nature and / or have been modified by humans.

[0032] "Non-natural amino acid", "amino acid analog", and "non-standard amino acid residue" are used interchangeably herein. Non-natural amino acids that can be substituted in a ligand as provided herein are known in the art. In some embodiments, the non-natural amino acid is 4-hydroxyproline which can substitute proline; 5-hydroxylysine which can substitute lysine; 3-methylhistidine which can substitute histidine; homoserine which can substitute serine; and ornithine which can substitute lysine. Additional examples of non-natural 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, α-aminoisobutyric acid, A-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, lanthionine, dehydroalanine, γ-aminobutyric acid, selenocysteine, and pyrrolysine fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids.

[0033] "Polynucleotide" and "nucleic acid molecule": As used interchangeably herein, polynucleotide and nucleic acid molecule refer to polymeric forms of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. These terms include, but are not limited to, DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and / or tRNA.

[0034] Operably linked: As used herein, the term "operably linked" indicates the joining of two molecules such that each retains its functional activity. Whether the two molecules are joined directly or indirectly, they are "operably linked".

[0035] Peptide tag: As used herein, the term "peptide tag" refers to a peptide sequence that is part of or linked to another protein, thereby providing a function to the resulting fusion. A peptide tag is typically relatively short compared to the protein to which it is fused. 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 provided herein are known in the art. Examples of peptide tags that can be components of ligand fusion proteins or targets that bind to ligands (e.g., ligand fusion proteins) include, but are not limited to, HA (hemagglutinin), c-myc, herpes simplex virus glycoprotein D (gD), T7, GST, GFP, MBP, Strep tag, His tag, Myc tag, TAP tag, and FLAG tag (Eastman Kodak, Rochester, N.Y.). Similarly, antibodies against the tag epitope allow detection and localization of the fusion protein in, for example, affinity purification, Western blotting, ELISA assays, and immunostaining of cells.

[0036] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids linked together by peptide bonds. The term is used to refer to amino acid chains of any length, but one of ordinary skill in the art will understand that the term is not limited to long chains and can refer to the smallest chain containing two amino acids linked together by a peptide bond. As is known to those of skill in the art, polypeptides can be processed and / or modified.

[0037] Protein: As used herein, the term "protein" refers to one or more polypeptides that function as discrete units. The terms "polypeptide" and "protein" can be used interchangeably if a single polypeptide is a discrete functional unit and does not require permanent or transient physical association with other polypeptides to form a discrete functional unit. If the discrete functional unit consists of more than one polypeptide physically associated with each other, the term "protein" refers to the multiple polypeptides that are physically coupled and function together as a discrete unit.

[0038] Specific binding: As used herein with respect to ligands, the term "specifically binds" or "has selective affinity" means that a ligand reacts or associates more frequently, more rapidly, for a longer duration, with greater affinity, or in any combination thereof, with a particular epitope, protein, or target molecule as compared to alternative substances including unrelated proteins. Due to sequence identity between homologous proteins in different species, specific binding can include binders that recognize proteins or targets in more than one species. Similarly, due to homology within certain regions of the polypeptide sequences of different proteins, specific binding can include binders that recognize more than one protein or target. It should be understood that in some embodiments, a binder that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specifically binds" does not necessarily require (although it can include) exclusive binding (i.e., binding to a single target). Thus, in some embodiments, a ligand or affinity agent can specifically bind to more than one target. In some embodiments, multiple targets can bind to the same antigen-binding site on an affinity agent.

[0039] Substantially: As used herein, the term "substantially" refers to a qualitative condition that exhibits all or nearly all of the range or degree of the characteristic or property of interest. Those of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, achieve completion and / or proceed to completion or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of complete integrity inherent in many biological and chemical phenomena. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1A Shows the structure of a ligand contemplated herein depicted as a 3-helix bundle protein.

[0041] Figure 1B Shows that the three helices of the 3-helix bundle protein can be envisioned as a prism, with each side represented as a face.

[0042] Figure 2 Shows a sensorgram of an exemplary affinity agent. The sensorgram is of a biotinylated ligand titrated with a solution containing haptoglobin and corresponding to SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.7, and SEQ ID NO.8.

[0043] Figure 3 Shows the equilibrium binding capacity of an exemplary resin prepared from an affinity agent. The height of the bar graph in the figure represents the amount (in milligrams) of haptoglobin that can be captured per milligram of affinity agent conjugated to the resin.

[0044] Figure 4Shows the dynamic binding capacity of an exemplary resin prepared with an affinity agent corresponding to SEQ ID NO. 118 at residence times of 2 minutes, 4 minutes, 6 minutes, and 8 minutes.

[0045] Figure 5 Shows the results of a column purification run of haptoglobin purified from fraction V of human plasma using certain provided affinity agents, which were analyzed by running on a 4 - 20% Tris - glycine SDS - PAGE gel under reducing conditions. The ligand is SEQ ID NO. 118. The following table lists the samples loaded in each lane.

[0046] Lane Sample Mass (μg) 1 SeeBlue Plus2 Molecular Weight Standard N / A 2 Haptoglobin 1-1 Reference Standard 10 3 Hemin 2-1 Reference Standard 1 4 Seq No.118 Affinity Resin Elution Fraction Pool 10

[0047] Figure 6 Shows the residual HCP and DNA measured over several cycles of purification of haptoglobin fraction V from human plasma. The ligand is SEQ ID NO. 118.

[0048] Figure 7 Shows the yield measured over several cycles of haptoglobin purification as described in Example 8. The ligand is SEQ ID NO. 117. Detailed Description

[0049] The present disclosure particularly encompasses the recognition that affinity agents prepared from identified and characterized peptide ligands are shown to be capable of producing highly purified preparations of one or more targets of interest (e.g., haptoglobin in some embodiments). In some embodiments, the affinity resins described herein can be particularly useful for removing protein - product - related impurities as well as contaminants derived from the host cell.

[0050] Ligands that bind to the target of interest are used in the affinity agent

[0051] The characteristics of ligands that bind to the target can be determined using known or modified assays, bioassays, and / or animal models known in the art to evaluate such activities.

[0052] As used herein, terms such as "binding affinity for a target", "binding to a target", etc. refer to properties of a ligand that can be measured directly, for example, by determining an affinity constant (e.g., the amount of ligand associating and dissociating at a given antigen concentration). Several methods can be used to characterize such molecular interactions, such as competitive assays, equilibrium assays, and microcalorimetry, 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) Trends in Biotechnology (Tibtech) 14:465 - 470 and Jansson M et al. (1997) Journal of Biological Chemistry (J Biol Chem) 272:8189 - 8197.

[0053] The affinity requirements for 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 both the ligand and the target molecule, and the functional application of the ligand. In some embodiments, the ligand binds to the target of interest with a dissociation constant (K -3 M) of less than or equal to 5×10 -3 M, 10 -4 M, 5×10 -4 M, 10 -5 M, 5×10 -5 M, or 10 D ) of less than or equal to 5×10 -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5×10 -8 M, or 10 -8 M. In some embodiments, the ligand binds to the target of interest with a K D ) of less than or equal to 5×10 -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 M. In some embodiments, the ligand binds to the target of interest with a K DBinding to the target of interest. In some embodiments, the dissociation constant of the ligand produced by the methods disclosed herein is from about 10 -4 M to about 10 -5 M, from about 10 -5 M to about 10 -6 M, from about 10 -6 M to about 10 -7 M, from about 10 -7 M to about 10 -8 M, from about 10 -8 M to about 10 -9 M, from about 10 -9 M to about 10 -10 M, from about 10 -10 M to about 10 -11 M, or from about 10 -11 M to about 10 -12 M.

[0054] Determination of K D and the dissociation rate (k off ) binding experiments can be carried out under many conditions. The buffer for preparing these solutions can be readily determined by those skilled in the art and depends largely on the desired pH of the final solution. Low pH solutions (<pH 5.5) can be prepared, for example, in citrate buffer, glycine-HCl buffer, or succinate buffer. High pH solutions can be prepared, for example, in Tris-HCl, phosphate buffer, or sodium bicarbonate buffer. Many conditions can be used to determine K D and the dissociation rate to determine, for example, the optimal pH and / or salt concentration.

[0055] In some embodiments, the ligand binds specifically to the target of interest with a k -7 in the range of 0.1 to 10 -1 seconds, 10 -2 to 10 -7 seconds -1 or 0.5x10 -2 to 10 -7 seconds. -1 In some embodiments, the ligand binds to the target of interest with a k off less than 5x10 -2 seconds, -1 10 -2 seconds, -1 5x10 -3 seconds, -1 or 10 -3 seconds. -1 In some embodiments, the ligand binds to the target of interest with a k off less than 5x10 -4 seconds, -1 10-4 second -1 、5x10 -5 seconds -1 or 10 -5 seconds -1 、5x10 -6 seconds -1 、10 -6 seconds -1 、5x10 -7 seconds -1 or 10 -7 seconds -1 of k off binds to the target of interest. In some embodiments, the ligand binds to the target of interest at an association rate (k 3 from about 10 7 M -1 seconds -1 、10 3 to 10 6 M -1 seconds -1 or 10 3 to 10 5 M -1 seconds -1 ). In some embodiments, the ligand (e.g., ligand fusion protein) binds to the target of interest at a k on greater than 10 3 M -1 seconds -1 、5x10 3 M -1 seconds -1 、10 4 M -1 seconds -1 or 5x10 4 M -1 seconds -1 of k on . In an additional embodiment, the ligand binds to the target of interest at a k 5 greater than 10 -1 M -1 seconds 5 、5x10 -1 M -1 seconds 6 、10 -1 M -1 seconds 6 、5x10 -1 M -1 seconds 7 or 10 -1 M -1 seconds on .

[0056] the target of interest

[0057] According to various embodiments, a target of interest that specifically binds to a ligand can be any molecule to which the ligand of the desired affinity agent binds. For example, a target that specifically binds to a ligand can be any target that is relevant to purification, manufacture, formulation, treatment, diagnosis, or prognosis or a value thereof. Non-limiting uses include therapeutic and diagnostic uses. Numerous exemplary targets are provided herein by way of example, and the exemplary targets are intended to be illustrative rather than limiting. The target of interest can be naturally occurring or synthetic. In some embodiments, the target comprises haptoglobin. In some embodiments, the target comprises haptoglobin from human plasma. In some embodiments, the target comprises haptoglobin from Cohn fraction IV of human plasma. In some embodiments, the target comprises haptoglobin from Cohn fraction V of human plasma. In some embodiments, the target comprises haptoglobin from a recombinant source well known to those skilled in the art.

[0058] Linker

[0059] The terms "linker" and "spacer" are used interchangeably herein to refer to a peptide or other chemical linkage used to connect other independent functional domains. In some embodiments, the linker is located between the ligand and another polypeptide component containing other independent functional domains. Suitable linkers for coupling two or more linked ligands can generally be any linker used in the art for linking peptides, proteins, or other organic molecules. In some embodiments, such linkers are suitable for constructing proteins or polypeptides intended for pharmaceutical use.

[0060] Suitable linkers for operably linking a ligand and additional components of a 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 of mostly polar polypeptide segments.

[0061] In some embodiments, the linker comprises mostly amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In some embodiments, the linker comprises mostly amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In some embodiments, the ligand linker is composed of mostly sterically unhindered amino acids. In some embodiments, the linker comprises mostly amino acids selected from glycine, serine, and / or alanine. In some embodiments, the peptide linker is selected from polyglycine (such as (Gly)5 and (Gly)8, poly(Gly-Ala), and polyalanine).

[0062] The linker can have any size or composition, so long as it is operably linked to the ligand in a manner that permits binding of the ligand 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 appreciated that the length, degree of flexibility, and / or other characteristics of the linker can affect certain properties of the ligand used in the affinity agent, such as the affinity, specificity, or avidity for the target of interest, or for one or more other target proteins of interest, or for non-target proteins (i.e., non-target proteins). In some embodiments, two or more linkers are utilized. In some embodiments, the two or more linkers are the same. In some embodiments, the two or more linkers are different.

[0063] In some embodiments, the linker is a non-peptide linker, such as an alkyl linker or a PEG linker. For example, an alkyl linker such as -NH-(CH2)s-C(0)- can be used, where s = 2 - 20. These alkyl linkers can be further substituted with any sterically unhindered group, such as lower alkyl (e.g., C1, C6), lower acyl, halogen (e.g., Cl, I, Br, F), CN, NH2, phenyl, etc. An exemplary non-peptide linker is a PEG linker. In some embodiments, the PEG linker has a molecular weight of about 100 to 5000 kDa, or about 100 to 500 kDa. In some embodiments, the PEG linker has a molecular weight of about 100 to 500 kDa.

[0064] Techniques described herein and / or otherwise known in the art can 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.

[0065] An affinity agent comprising a conjugated ligand

[0066] A ligand that promotes specific binding to the target of interest can be chemically conjugated to a variety of chromatographic compositions (e.g., beads, resins, gels, membranes, monoliths, etc.) to prepare an affinity agent. Affinity agents comprising the ligand are particularly useful in purification and manufacturing applications.

[0067] In some embodiments, the ligand (e.g., ligand fusion protein) contains at least one reactive residue. The reactive residue can be used, for example, as a linking site for conjugates such as chemotherapeutic agents. Exemplary reactive amino acid residues are lysine. The reactive residue (e.g., lysine) can be added to the ligand at either end or within the ligand sequence, and / or can replace another amino acid in the ligand sequence. Suitable reactive residues (e.g., lysine) can also be located within the sequence of the identified ligand without the need for addition or replacement. In some embodiments, additional exemplary reactive amino acid residues are cysteine. In some embodiments, the reactive amino acid residue is lysine.

[0068] Attached to a solid surface

[0069] The terms “solid surface,” “carrier,” or “matrix” are used interchangeably herein and refer to, but are not limited to, any column (or column material), bead, test tube, microtiter plate, solid particle (e.g., agarose or agarose gel), microchip (e.g., silicon, silicon-glass, or gold chip), or membrane (synthetic in origin (e.g., filter membrane) or biological (e.g., liposome or vesicle)) to which a ligand, affinity agent, antibody, or other protein can be directly or indirectly (e.g., through other binding partner intermediates such as other antibodies or protein A) linked (coupled, joined, or adhered), or into which a ligand or antibody can be incorporated (e.g., through a receptor or channel). Reagents and techniques for linking polypeptides to solid supports (e.g., matrices, resins, plastics, etc.) are well known in the art. Suitable solid supports include, but are not limited to, chromatographic resins or matrices (e.g., SEPHAROSE-4FF agarose beads), the walls or bottom plates of wells in plastic microtiter plates, silica-based biochips, polyacrylamide, agarose, silica, nitrocellulose, paper, plastics, nylon, metals, and combinations thereof. Ligands and other compositions can be linked 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.

[0070] Generation of ligands

[0071] The generation of ligands for several embodiments that can be used to practice the provided methods can be carried out using a variety of standard techniques for chemical synthesis, semi-synthetic methods, and recombinant DNA methods known in the art. Also provided are methods for generating ligands as soluble agents and cell-associated proteins, either alone or as part of a multi-domain fusion protein. In some embodiments, the overall ligand generation protocol involves obtaining a reference protein scaffold and identifying multiple residues within the scaffold for modification. According to embodiments, the reference scaffold can comprise a protein structure or other tertiary structure having one or more α-helical regions. Once identified, any of the multiple residues can 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 targeted position for modification. In some embodiments, the modification does not include substitution of cysteine or proline. In certain embodiments, after modification at the desired identified positions, the resulting modified polypeptides (e.g., candidate ligands) can be recombinantly expressed (e.g., to increase the number of each of the modified polypeptides) in, for example, a plasmid, bacterium, phage, or other vector. The modified polypeptides can then be purified and screened to identify those modified polypeptides that specifically bind to a particular target of interest. Compared to the reference scaffold, the modified polypeptides can exhibit enhanced binding specificity for the target of interest, or can 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 can exhibit some interaction with the target of interest (e.g., non-specific interaction), 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 for the target of interest. Additional details regarding ligand generation, selection, and isolation are provided in more detail below.

[0072] Recombinant Expression of Ligands

[0073] In some embodiments, ligands such as ligand fusion proteins are “recombinant produced” (i.e., are produced using recombinant DNA techniques). Exemplary recombinant methods that can be used to synthesize ligand fusion proteins include, but are not limited to, polymerase chain reaction (PCR)-based synthesis, ligation, seamless cloning, and recursive directional ligation (RDL) (see, for example, 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)).

[0074] Nucleic acids comprising a polynucleotide sequence encoding a ligand are also provided. Such polynucleotides optionally further comprise one or more expression control elements. For example, the polynucleotide may comprise one or more promoters or transcriptional enhancers, ribosome binding sites, transcription termination signals, and polyadenylation signals as expression control elements. The polynucleotide can be inserted into any suitable vector, and the vector can be contained within any suitable host cell for expression.

[0075] Expression of the nucleic acid encoding a ligand is typically achieved by operably linking the nucleic acid encoding the ligand to a promoter in an expression vector. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Exemplary promoters that can be used for expression in E. coli include, for example, the T7 promoter.

[0076] Expression vectors containing a nucleic acid sequence encoding a ligand and appropriate transcriptional / translation control signals can be constructed using methods known in the art. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. Expression of the polynucleotide can 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 a ligand is operably linked to a suitable promoter sequence such that the nucleic acid sequence is transcribed and / or translated into a ligand in the host.

[0077] A variety of host expression vector systems can be used to express nucleic acids encoding ligands. Vectors containing one or more nucleic acids encoding a ligand (e.g., individual ligand subunits or ligand fusions) or portions or fragments thereof can include plasmid vectors, single-stranded phage vectors, double-stranded phage vectors, single-stranded RNA or DNA viral vectors, or double-stranded RNA or DNA viral vectors. Phage and viral vectors can also be introduced into host cells in the form of encapsulated or enveloped viruses using known infection and transduction techniques. In addition, viral vectors can be replication-competent or, alternatively, replication-deficient. Alternatively, cell-free translation systems can also be used to produce proteins and / or ligands using RNA derived from a DNA expression construct (see, e.g., WO86 / 05807 and WO89 / 01036; and U.S. Patent No. 5,122,464).

[0078] Generally, 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 engineered host cells is a bacterial cell, a yeast cell, or a mammalian cell. A variety of host expression vector systems can be used to express the coding sequence of a ligand fusion protein. Mammalian cells can be used as a host cell system transfected with recombinant plasmid DNA or a cosmid DNA expression vector containing the coding sequence of a target of interest and the coding sequence of a fusion polypeptide. The cells can be primary isolates from an organism, culture, or cell line having a transformed or transgenic nature.

[0079] Suitable host cells include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli or Bacillus subtilis) transformed with a recombinant phage DNA, plasmid DNA, or cosmid DNA expression vector containing the ligand coding sequence; yeast (e.g., Saccharomyces or Pichia) transformed with a recombinant yeast expression vector containing the ligand coding sequence; insect cell systems infected with a recombinant viral expression vector (e.g., Baculovirus); plant cell systems infected with a recombinant viral expression vector (e.g., Cauliflower mosaic virus (CaMV) or Tobacco mosaic virus (TMV)) or transformed with a recombinant plasmid expression vector containing the ligand coding sequence (e.g., Ti plasmid).

[0080] Prokaryotes that can be used as host cells for generating ligands can include Gram-negative or Gram-positive organisms, such as Escherichia coli and Bacillus subtilis. Expression vectors for prokaryotic host cells typically contain one or more phenotypically selectable 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 commonly used for prokaryotic host cell expression vectors include T7 (Rosenberg et al., Gene 56:125-135 (1987)), β-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615 (1978); and Goeddel et al., Nature 281:544 (1979)), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, (1980)), and tac promoter (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

[0081] In some embodiments, a eukaryotic host cell system is used. In some embodiments, the eukaryotic host cell system is a yeast cell transformed with a recombinant yeast expression vector containing the coding sequence of a ligand. Exemplary yeasts that can be used to produce the compositions of the present invention include yeasts from the genus Saccharomyces, Pichia, Actinomycetes, and Kluyveromyces. Yeast vectors generally contain an origin of replication sequence from the 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, a sequence for polyadenylation, a sequence for transcription termination, and a selectable marker gene. 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. Additional suitable vectors and promoters for yeast expression as well as yeast transformation protocols are known in the art. See, for example, Fleer, Gene 107:285-195 (1991) and Hinnen, Proc. Natl. Acad. Sci. USA 75:1929 (1978).

[0082] Insect and plant host cell culture systems can also be used to produce the compositions of the present invention. Such host cell systems include, for example, insect cell systems infected with a recombinant viral expression vector (e.g., baculovirus) containing the coding sequence of a ligand; plant cell systems infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing the coding sequence of a ligand, including but not limited to those taught in U.S. Patent No. 6,815,184; U.S. Patent Application Nos. 60 / 365,769 and 60 / 368,047; and WO2004 / 057002, WO2004 / 024927, and WO2003 / 078614.

[0083] In some embodiments, a host cell system can be used. In some embodiments, the host cell system is an animal cell system infected with a recombinant viral expression vector (e.g., adenovirus, retrovirus, adeno-associated virus, herpes virus, or lentivirus). In some embodiments, the host cell system is a cell line that has been engineered to contain multiple copies of DNA encoding a ligand and that stably amplifies (CHO / dhfr) or unstably amplifies in double minute chromosomes (e.g., murine cell lines). In some embodiments, the vector containing the polynucleotide encoding the ligand is polycistronic. Exemplary mammalian cells that can be used to produce these compositions include HEK293 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, HsS78Bst cells, hybridoma cells, and other mammalian cells. Additional exemplary mammalian host cells that can be used to practice the present invention include, but are not limited to, T cells. Exemplary expression systems and selection methods are known in the art and can 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). Additional examples of expression systems and selection methods are described in Logan et al., Proc. Natl. Acad. Sci. USA 81:355-359 (1984); Birtner et al. Methods Enzymol. 153:51-544 (1987). Transcriptional and translational control sequences for mammalian host cell expression vectors generally are derived from viral genomes. Promoter sequences and enhancer sequences commonly used in mammalian expression vectors include sequences derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV).Exemplary commercially available expression vectors for mammalian host cells include pCEP4 (Invitrogen) and pcDNA3 (Invitrogen).

[0084] Physical methods for introducing nucleic acids into host cells (e.g., mammalian host cells) include, but are not limited to, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0085] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors and especially retroviral vectors have become the most widely used method for inserting genes into mammalian cells (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0086] Methods for introducing DNA and RNA polynucleotides of interest into host cells include, but are not limited to, cell electroporation, where an electric field is applied to the cells to increase the permeability of the cell membrane, thereby allowing the introduction of chemicals, drugs, or polynucleotides into the cells. Ligands containing DNA or RNA constructs can be introduced into mammalian or prokaryotic cells using electroporation.

[0087] In some embodiments, cell electroporation results in the expression of ligand-CAR on the surface of T cells, NK cells, NKT cells. Such expression can be transient or stable over the lifespan of the cell. Electroporation can be accomplished by methods known in the art, including the MaxCyte and Transfection System (MaxCyte, Gaithersburg, MD, USA).

[0088] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle). In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated to use lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). In some embodiments, the nucleic acid is associated with a lipid. The nucleic acid associated with a lipid can be encapsulated in the aqueous interior of a liposome, dispersed in the lipid bilayer of a liposome, linked to a liposome via a linking molecule that associates with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contain micelles or be complexed with micelles, or otherwise associated with a lipid. The composition associated with a lipid, lipid / DNA, or lipid / expression vector is not limited to any particular structure in solution. For example, the composition can exist as a bilayer structure, micelle, or "folded" structure. The composition can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids can be naturally occurring fatty substances or synthetic lipids. For example, lipids include the fat droplets that occur naturally in the cytoplasm, as well as classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0089] Suitable lipids are commercially available. For example, dimyristoyl phosphatidylcholine ("DMPC") is available from Sigma (St. Louis, MO); dicetyl phosphate ("DCP") is available from K&K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristoyl phosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform can be used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term encompassing a variety of unilamellar and multilamellar lipid agents formed by the generation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous media. When phospholipids are suspended in an excess of aqueous solution, multilamellar liposomes form spontaneously. The lipid components undergo self-rearrangement before forming a closed structure, and water and dissolved solutes are trapped between the lipid bilayers (Ghosh et al., Glycobiology 5:505-510 (1991)). However, compositions having structures different from normal vesicular structures in solution are also encompassed. For example, the lipids may assume a micellar structure or exist only as an inhomogeneous aggregate of lipid molecules. Lipid transfection amine-nucleic acid complexes are also contemplated.

[0090] Regardless of the method used to introduce exogenous nucleic acid into a host cell, the presence of a 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 Southern and Northern blotting, RT-PCR, and PCR; "biochemical" assays, such as detecting the presence or absence of a specific peptide by immunological means (ELISA and Western blotting) or by the assays described herein, to identify agents falling within the scope of the present invention.

[0091] Reporter genes are used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism, tissue or cell and encodes a polypeptide that manifests its expression by some readily detectable property (e.g., enzymatic activity). The expression of the reporter gene is assayed at an appropriate time after the DNA has been introduced into the recipient cell. Suitable reporter genes include, but are not limited to, genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, 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 using known techniques or obtained commercially. Generally, constructs having a minimal 5' flanking region and showing the highest level of reporter gene expression are identified as promoters. Conventionally, such promoter regions can be ligated to a reporter gene and used to evaluate the ability of an agent to modulate promoter-driven transcription.

[0092] Many selection systems can be used in mammalian host vector expression systems, including but not limited to herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes. Additionally, antimetabolite resistance can be used as a basis for selection in, for example, dhfr, gpt, neo, hygro, trpB, hisD, ODC (ornithine decarboxylase) and glutamine synthetase systems.

[0093] Ligand purification

[0094] Once a ligand or ligand fusion protein has been produced by recombinant expression, the ligand or ligand fusion protein can be purified by methods known in the art for purifying recombinant proteins, such as by chromatography (e.g., ion exchange, affinity and size column chromatography), centrifugation, differential solubilization or by any other standard technique for purifying proteins. In some embodiments, the ligand can optionally be fused to a heterologous polypeptide sequence specifically disclosed herein or known in the art to facilitate purification. In some embodiments, prior to the final preparation of the ligand using techniques known in the art, the ligand (e.g., antibody and other affinity matrices) of the ligand affinity column used for affinity purification and optionally the ligand or other components of the composition that bind to these ligands are removed from the ligand fusion composition.

[0095] Chemical synthesis of ligands

[0096] In addition to recombinant methods, organic chemical synthesis of the desired polypeptides using various liquid and solid phase chemical methods known in the art can be used to generate ligands. A variety of automated synthesizers are commercially available and can be used according to known protocols. See, for example, Tam et al., J. Am. Chem. Soc., 105:6442 (1983); Merrifield, Science, 232:341-347 (1986); Barany and Merrifield, The Peptides, Gross and Meienhofer eds., 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. ed., Plenum Press, NY., 1990, Vol. 12, pp. 1-19; Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman & Co., San Francisco, 1989. An advantage of these methods is that they allow incorporation of unnatural amino acid residues into the ligand sequence.

[0097] The ligands used in the methods of the present invention can be modified during or after synthesis or translation, for example, by glycosylation, acetylation, benzylation, phosphorylation, amidation, polyethylene glycolylation, formylation, derivatization with known protecting / blocking groups, proteolytic cleavage, ligation to antibody molecules, hydroxylation, iodination, methylation, myristoylation, oxidation, polyethylene glycolylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, ubiquitination, etc. (see, for example, Creighton, Proteins: Structures and Molecular Properties, 2nd ed. (W.H. Freeman and Co., N.Y.), 1992); Postranslational Covalent Modification of Proteins, edited by Johnson (Academic Press, N.Y.), 1983, pp. 1-12; Seifter, Meth. Enzymol., 182:626-646 (1990); Rattan, Ann. NY Acad. Sci., 663:48-62 (1992)). In some embodiments, the peptide is acetylated at the N-terminus and / or amidated at the C-terminus.

[0098] Any of a number of chemical modifications can be carried out by known techniques, including but not limited to acetylation, formylation, etc. Additionally, the derivatives can contain one or more non-classical amino acids.

[0099] In some embodiments, cyclization or macrocyclization of the peptide backbone is achieved by formation of side-chain to side-chain linkages. Methods for achieving this are well known in the art and may involve natural and non-natural amino acids. Such methods include disulfide formation, lanthionine formation or thiol alkylation (e.g., Michael addition), amidation between an amino group and a carboxylic acid ester side-chain, click chemistry (e.g., azide-alkyne condensation), peptide stapling, ring-closing metathesis, and the use of enzymes.

[0100] Affinity agents for purification

[0101] In purification based on affinity chromatography, the target of interest (e.g., a protein or molecule) is selectively separated according to its ability to specifically and reversibly bind to a ligand that can be covalently coupled to a chromatographic matrix. In some embodiments, the ligand can be used as a reagent for affinity purification of the target of interest from recombinant or natural sources such as biological samples (e.g., serum or cells).

[0102] In some embodiments, a ligand that specifically binds to a target of interest is immobilized on beads and then used for affinity purification of the target.

[0103] Methods for covalently coupling proteins to surfaces are known to those of skill in the art. Peptide tags that can be used to link ligands to solid surfaces are known to those of skill in the art. Additionally, any reagent or technique known in the art can be used to link (i.e., couple, attach, or adhere) a ligand to a solid surface. In some embodiments, the solid support comprises beads, glass, slides, chips, and / or gelatin. Accordingly, arrays can be prepared on solid surfaces using a range of ligands with techniques known in the art. For example, U.S. Publication No. 2004 / 0009530 discloses methods for preparing arrays.

[0104] In some embodiments, a ligand is used to separate a target of interest (e.g., haptoglobin) by affinity chromatography. In some embodiments, the ligand is immobilized on a solid support. The ligand can be immobilized on the solid support using techniques and reagents described herein or known in the art. Suitable solid supports are described herein or known in the art and, in specific embodiments, are suitable for packing a chromatography column. The immobilized ligand can be loaded or contacted with a solution under conditions that favor formation of a complex between the ligand and the target of interest. Unbound material can be washed away. Those of skill in the art can readily determine suitable washing conditions. Examples of suitable washing conditions are described in Shukla and Hinckley, Biotechnol Prog. September - October 2008; 24(5):1115 - 21. doi:10.1002 / btpr.50.

[0105] In some embodiments, chromatography is performed by mixing a solution containing the target of interest and the ligand and then separating the complex of the target of interest and the ligand. For example, the ligand is immobilized on a solid support such as beads and then separated from the solution along with the target of interest by filtration. In some embodiments, the ligand is a fusion protein containing a peptide tag, such as a poly - HIS tail or a streptavidin - binding region, which can be used to separate the ligand after formation of a complex using immobilized metal affinity chromatography resin or a streptavidin - coated substrate. Once separated, the target of interest can be released from the ligand under elution conditions and recovered in purified form.

[0106] In some embodiments, ligands comprising the initiating N-terminal methionine are separated, as the initiator is a protein sequence encoded by DNA. In some embodiments, ligands without an N-terminal methionine residue are separated. In some embodiments, a mixture is obtained using only a portion of the purified ligand containing an N-terminal methionine. It will be apparent to those skilled in the art that the presence or absence of the N-terminal methionine does not affect the conclusions herein.

[0107] Example

[0108] Example 1. Purification of recombinant protein ligands.

[0109] Recombinant protein ligands are expressed in Escherichia coli and / or Pichia Pastoris using standard techniques. Ligands are purified using multi-column chromatography. For his-tagged ligands, IMAC is used as the primary capture step. The Avtag TM system (Avidity, Aurora, CO) is used to generate biotinylated ligands. Non-biotinylated ligands containing the Avtag TM sequence are prepared by omitting exogenous biotin. The purity and identity of the recombinant protein ligands are evaluated by a combination of SDS-PAGE, RP UPLC, quadrupole time-of-flight mass spectrometry, and SEC. In many cases, ligands without an N-terminal methionine residue are separated, which are thought to be cleaved during expression. In many cases, a mixture is obtained using only a portion of the purified ligand containing an N-terminal methionine. It will be apparent to those skilled in the art that the presence or absence of the N-terminal methionine does not affect the conclusions herein. For clarity, the N-terminal methionine is included.

[0110] Example 2. Binding of biotinylated ligands to haptoglobin.

[0111] This example demonstrates the binding of biotinylated ligands to haptoglobin using biolayer interferometry (ForteBio, Menlo Park, CA). The biotinylated ligands are immobilized on sensors and incubated with solutions containing various concentrations of haptoglobin. The binding curves are fit to a 1:1 binding model using ForteBio software to determine the affinity, and the resulting data are shown in the table below. As expected, all the affinity agents bind to haptoglobin with high affinity. In most cases, the binding affinity is less than 10 nM, and in many cases, less than 1 nM.

[0112] Table 1 Measured affinities (K D ) of selected ligands for haptoglobin

[0113]

[0114]

[0115] Example 3. Sodium hydroxide stability of the affinity ligand.

[0116] This example demonstrates the sodium hydroxide stability of the affinity ligand. The ligand was incubated in 0.25 M NaOH for 8 hours and then neutralized. The binding of the NaOH-treated ligand was measured as described in Example 1 and compared to the untreated ligand. The retained binding was calculated according to the following formula:

[0117] % Retained binding = (Response measured after NaOH treatment) ÷ (Response measured untreated) × 100

[0118] The data are shown in the table below and show that all tested affinity ligands exhibited high stability under the test conditions.

[0119] Table 2 Binding activity of affinity ligand to haptoglobin after long-term exposure to sodium hydroxide

[0120]

[0121]

[0122] Example 4. Generation and characterization of the affinity agent.

[0123] This example demonstrates the generation and characterization of an affinity agent comprising the ligands identified and described herein. Affinity resin was prepared by conjugating the ligand to agarose beads. The Jetted A50 beads (Purolite, King of Prussia, PA) were activated with disuccinimidyl carbonate and coupled to excess ethylenediamine. After washing, the bromoacetate was conjugated to the aminated beads using EDC activation. After washing, the ligand was conjugated to the beads at room temperature. The targeted ligand density varied between 5 - 12 g / L. After washing, the beads were inactivated with excess thioglycerol. The actual ligand density of all resins was measured using a subtractive RP-HPLC method according to the following formula:

[0124] Actual ligand density = ([Ligand] measured in the feed - [Ligand] measured in the effluent).

[0125] Example 5. Binding characteristics of the affinity agent.

[0126] This example demonstrates the binding characteristics of an affinity chromatography resin prepared from the affinity ligand described herein. Briefly, 5 μL of the resin was aliquoted into duplicate wells of a 96-well filter plate. The resin was pre-sterilized by washing with 0.1 M sodium hydroxide for 5 minutes and then equilibrated with a neutral pH buffer, and binding was probed in batch form. After equilibration with PBS, the resin was challenged with haptoglobin at 1.5 mg / mL for 60 minutes to allow complete binding to the resin. After the binding challenge, the microtiter plate was centrifuged to separate the unbound haptoglobin from the resin, and the amount of unbound haptoglobin was quantified by absorbance measurement at the effluent at 280 nm. The procedure is detailed in the table below:

[0127]

[0128] The equilibrium binding capacity was calculated by measuring the amount of haptoglobin remaining in solution after incubation with the resin relative to the amount of haptoglobin loaded. The binding capacity was calculated by comparing the amount of haptoglobin bound to the resin divided by the amount of ligand immobilized on the resin, as measured according to the following equation based on the method outlined in Example 5:

[0129] Binding capacity = (Haptoglobin in feed - Haptoglobin in effluent) ÷ (Ligand on resin)

[0130] Data are shown in Figure 3 and demonstrate efficient capture of haptoglobin by all the affinity agents tested.

[0131] Example 6. Dynamic binding capacity of the affinity agent.

[0132] This example demonstrates the dynamic binding capacity of the affinity ligand described herein for purifying haptoglobin. Haptoglobin was diluted to a concentration of 1 mg / mL in PBS. The resin prepared from the ligand corresponding to SEQ ID NO. 118 was packed into a column. A 0.3 x 10 cm (0.707 mL) column was operated according to the table below.

[0133]

[0134]

[0135] To evaluate the effect of residence time on the dynamic binding capacity, the linear velocity of the loading solution was varied. Different dynamic binding capacity measurements were made using linear velocities of 75 cm / hr (8-minute residence time), 100 cm / hr (6-minute residence time), 150 cm / hr (4-minute residence time), and 300 cm / hr (2-minute residence time).

[0136] The material was analyzed by measuring the absorbance of each effluent fraction compared to the absorbance of the loaded solution. The mass of the haptoglobin-activated aggregates at 10% of the loaded solution when measured in the effluent was reported as the dynamic binding capacity of the resin. At Figure 4 Data for an exemplary affinity resin produced from the affinity ligand corresponding to SEQ ID NO. 118 are shown, and it is demonstrated that the dynamic binding capacity of the resin increases with increasing residence time in the range of 2 - 6 minutes; increasing the residence time to 8 minutes does not increase the binding capacity.

[0137] Example 7. Reuse of the Affinity Agent.

[0138] This example demonstrates the reuse of an affinity agent containing the binding ligand described herein for the affinity purification of haptoglobin. Haptoglobin was purified from Cohn fraction V of human plasma using a packed column, and the measured titer of haptoglobin was 0.9 mg / mL. The resin was prepared from the ligand corresponding to SEQ ID NO. 118. The resin was packed into a 0.66 x 11.5 cm (3.9 mL) column and operated as shown in the table below.

[0139]

[0140] Between each cycle, the resin was exposed to the CIP agent for a total of 3 hours to simulate 6 CIP sanitization cycles.

[0141] The eluate fractions from each purification cycle were collected and the purity was analyzed by SDS-PAGE. The data are shown in Figure 5 and confirm the high purity provided by the affinity agent, where serum albumin was clearly absent in the eluate fraction. The purity was also evaluated by analytical RP-HPLC. The purity percentage was calculated by comparing the peak area of haptoglobin to the total area of all peaks in the analysis. The data are shown in Figure 6 and confirm the consistent purification performance across dozens of simulated cycles after exposure to 0.25 M sodium hydroxide for > 25 hours. The yield was determined by analytical RP-HPLC by comparing the peak area of haptoglobin in the first cycle to all subsequent cycles. The data are shown in Figure 7 and confirm that the resin can be cleaned with NaOH and reused in multiple cycles.

[0142] Table 3. Sequences

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

Claims

1. An affinity agent, which comprises a ligand that binds to haptoglobin, wherein the ligand comprises the sequence of SEQ ID NO: 1 X 1 QRRX 2 FIX 3 X 4 LRX 5 DPSX 6 SAX 7 LLAX 8 AKX 9 X 10 NDX 11 QAPK, where X 1 is A, D, E, H, I, L, Q, S, T, V or W; X 2 is A, E, G, H, N, Q, S or Y; X 3 is A, D, E, F, G, H, I, K, L, N, Q, R, S, T, W or Y; X 4 is A, F, H, L, Q, S, T, V or Y; X 5 is A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V or Y; X 6 is A, D, E, G, H, I, K, L, Q, S, T or V; X 7 is A, E, G, H, I, L, P, S, T, V, W or Y; X 8 is A, G, I, L, T or V; X 9 is A, D, E, G, H, K, N, R, S or T; X 10 is F, H, R, V or W; and X 11 is A, D, E, H, K, N, Q, R, S or T.

2. An affinity agent, which comprises a ligand that binds to haptoglobin, wherein the ligand comprises at least one sequence of any one of SEQ ID NOs: 2-123, or an amino acid sequence with no more than three, no more than two, or no more than one substitution, addition, or deletion.

3. The affinity agent according to claim 1 or claim 2, which comprises a multimeric polypeptide, wherein the multimeric polypeptide comprises at least two subunits; and wherein each subunit comprises the polypeptide according to claim 1 or claim 2.

4. The affinity agent according to claim 3, wherein the subunits are not all the same.

5. The affinity agent according to any one of claims 1 to 4, wherein the ligand is linked to a solid surface.

6. The affinity agent according to claim 5, wherein the solid surface is a resin or a bead.

7. The affinity agent according to claim 5, wherein the solid surface is a membrane.

8. The affinity agent according to claim 5, wherein the solid surface is a monolith.

9. The affinity agent according to any one of claims 5 to 9, wherein the ligand is conjugated to the solid surface through a linker.

10. The affinity agent according to any one of claims 1 to 9, which is used for the purification of haptoglobin.

11. A method for preparing an affinity agent, the method comprising conjugating the ligand according to any one of claims 1 to 4 to a solid surface.

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