affinity agent

Abstract

Provided herein are affinity agents that comprise a ligand that specifically binds to a target molecule. The affinity agents are useful for binding, isolation, and / or purification.

Description

[Technical Field]

[0001] The purity of biologically produced therapeutics is closely scrutinized and regulated by health authorities to ensure safety and efficacy. Thus, there remains a need for means to efficiently purify biologically produced therapeutics to high purity.

[0002] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 428,949, filed November 30, 2022, the disclosure of which is incorporated herein by reference in its entirety. Summary of the Invention

[0003] To support clinical efforts for therapeutic proteins, compositions and methods for efficiently purifying proteins from recombinant sources are needed. Affinity purification is a means for isolating and / or achieving a desired degree of purity of a protein in several steps or in a single step. However, the development of affinity agents (including, for example, affinity ligands) can be resource-intensive and time-consuming. As a result, affinity agents have been developed for only a limited number of proteins. In the absence of affinity agents, purification typically involves inefficient, labor-intensive, and expensive processes, such as multi-column processes.

[0004] Exemplary therapeutic proteins include, but are not limited to, biologically active polypeptides / proteins, fusion proteins, enzymes, hormones, antibodies, and antibody fragments.Certain proteins, such as fusion proteins, present additional challenges for purification due to the homogeneity of the product or the presence of product-related impurities.Some impurities may result from incorrectly assembled fusion proteins and proteolytic cleavage, which may be particularly difficult to remove because they are closely related to the desired product.

[0005] Described herein are affinity agents that bind to haptoglobin proteins and are useful for isolating and / or affinity purifying them. In some embodiments, the affinity agent comprises a solid support and a ligand.

[0006] Haptoglobin (Hp) belongs to the acute-phase plasma protein family and is an important scavenger and detoxifier of free hemoglobin. Hp is an acidic tetrameric glycoprotein composed of two α / β dimers and is involved in the body's natural defense system against inflammation and viral infection. 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, which give rise to three distinct genotypes (Hp1-1, Hp1-2, and Hp2-2). The resulting gene product is a heterogeneous mixture of various transcripts. The biological activity of Hp is associated with different phenotypes. The most important biological activity of Hp is its regulation of hemoglobin clearance from the circulation through complex formation with the macrophage CD163 receptor, followed by 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 monocyte / macrophage cells and regulate T helper cell responses, suggesting that it may be an important mediator of various pathogenic disorders, including infectious diseases, diabetes, cardiovascular disease, and cancer.

[0007] Haptoglobin has practical applications as a therapeutic molecule related to its function as a hemoglobin scavenger. Administration of haptoglobin has been shown to improve outcomes in patients experiencing sepsis with high cell-free hemoglobin levels and in patients after burn injury. Traditional methods for haptoglobin purification use the Cohn fractionation process, originally developed during World War II to purify human serum albumin from plasma. The Cohn fractionation process provides a highly efficient process for the purification of plasma proteins, and its continued use since the mid-1940s for industrial production of plasma proteins demonstrates the need for suitable purification equipment.

[0008] A major drawback of using the Cohn Fractionation process for the production of haptoglobin from human plasma is that the method suffers from low purity because the process does not provide a way to separate haptoglobin from human plasma and serum albumin, which is typically concentrated at least 100-fold in Cohn Fractionation Fraction V, which is the starting point for the isolation of haptoglobin from human plasma.

[0009] Described herein are affinity reagents that specifically bind to haptoglobin protein, selectively provide it from serum albumin, and are useful for isolation and / or affinity purification. In some embodiments, the affinity reagent comprises a solid support and a ligand.

[0010] In some embodiments, the affinity agent comprises a three-helix bundle protein. The structure of a three-helix bundle protein can be envisioned as a triangular prism, with each vertex of the triangle representing a helix, as shown, for example, in Figures 1A and 1B. In some embodiments, the combination of any two helices defines a rectangular face. For example, in some embodiments, the three faces of a helix bundle protein are defined by: 1) helices 1 and 2 (faces 1 and 2 in Figure 1B ); 2) helices 2 and 3 (faces 2, 3 in Figure 1B ); 3) helices 1 and 3 (faces 1, 3 in Figure 1B), and A combination of them.

[0011] In some embodiments, provided herein are affinity agents that comprise a face formed from helices 2 and 3 of a three-helix bundle protein (i.e., the residues involved in binding to the target protein are within helices 2 and 3). In some embodiments, the primary function of helix 1 of a three-helix bundle protein is to complete and stabilize the three-helix bundle. In some embodiments, modifications of helix 1 can be made that also maintain helix 1 and the three-helix bundle structure.

[0012] In some embodiments, 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, and X 2 is A, E, G, H, N, Q, S or Y, and X 3 is A, D, E, F, G, H, I, K, L, N, Q, R, S, T, W or Y, and X 4 is A, F, H, L, Q, S, T, V or Y, and X 5 is A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V or Y, and X 6 is A, D, E, G, H, I, K, L, Q, S, T or V, and X 7 is A, E, G, H, I, L, P, S, T, V, W or Y, and X 8 is A, G, I, L, T or V, and X 9 is A, D, E, G, H, K, N, R, S or T, and X 10 is F, H, R, V or W, and X 11is A, D, E, H, K, N, Q, R, S, T), or an affinity agent comprising a ligand comprising an amino acid sequence that differs by no more than three, no more than two, or no more than one substitution.

[0013] In some embodiments, an affinity agent comprising SEQ ID NO: 1 is contained within helix 2 and helix 3, respectively, of a three-helix bundle protein. In some embodiments, provided herein are affinity agents comprising SEQ ID NO: 1 contained within helix 2 and helix 3, respectively, of a three-helix bundle protein.

[0014] In some embodiments, provided herein is one or more affinity agents comprising a ligand comprising an amino acid sequence of SEQ ID NO: 2-123, or one that differs by no more than three, no more than two, or no more than one substitution, addition, or deletion.

[0015] In some embodiments, provided herein is an affinity agent comprising a multimeric polypeptide comprising at least two subunits, each subunit comprising a polypeptide according to the preceding embodiments.

[0016] In some embodiments, provided herein are affinity agents that comprise multimeric polypeptides, wherein the subunits are not all the same.

[0017] In some embodiments, provided herein are affinity agents for use in the purification of haptoglobin protein from human plasma.

[0018] In some embodiments, provided herein are affinity agents for use in the purification of human haptoglobin protein from recombinant sources.

[0019] definition In order that the present disclosure may be more readily understood, certain terms are defined below. Unless otherwise defined herein, technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art.

[0020] Approximately or about: As used herein, the term "approximately" or "about," when applied to one or more subject values, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about," unless otherwise specified or clear from the context, refers to a range of values ​​that is 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 or less) of the stated reference value (except when such number exceeds 100% of possible values).

[0021] Biological activity: As used herein, the term "biological activity" refers to a characteristic of any agent that has activity in a biological system, particularly an organism. For example, an agent that, when administered to an organism, has a biological effect on that organism is considered to be biologically active.

[0022] Conservative and Non-Conservative Substitutions: A "conservative" amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains 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), beta-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)), and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W)). Conservative amino acid substitutions are defined in the art, including nucleotide and amino acid substitutions (e.g., valence nucleotides (W), histidine (H)). For example, the substitution of phenylalanine for tyrosine is a conservative substitution. In some embodiments, conservative amino acid substitutions in the sequence of a ligand confer or improve specific binding of the ligand to its intended target. In some embodiments, conservative amino acid substitutions in the sequence of a ligand do not reduce or prevent binding of the ligand to its intended target. In some embodiments, conservative amino acid substitutions do not significantly affect specific binding of the ligand to its intended target. 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 (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 sequence of the ligand confer or improve specific binding of the ligand to its target of interest. In some embodiments, non-conservative amino acid substitutions in the sequence of the ligand do not reduce or prevent binding of the ligand to its target of interest.In some embodiments, the non-conservative amino acid substitutions do not significantly affect the specific binding of the ligand to its intended target.

[0023] Linker: As used herein, "linker" refers to a peptide or other chemical bond that functions to connect otherwise independent functional domains. In some embodiments, the linker is positioned between a ligand and another polypeptide component that contains an otherwise independent functional domain. In some embodiments, the linker is a peptide or other chemical bond positioned between the ligand and the surface.

[0024] Naturally derived: When used in reference to biological materials such as nucleic acid molecules, polypeptides, and host cells, the term "naturally derived" refers to materials found in nature and that have not been modified by humans. Conversely, when used in reference to biological materials, "non-natural" or "synthetic" refers to materials that are not found in nature and / or that have been modified by humans.

[0025] The terms "unnatural amino acid," "amino acid analog," and "non-standard 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 4-hydroxyproline, which can be substituted for proline, 5-hydroxylysine, which can be substituted for lysine, 3-methylhistidine, which can be substituted for histidine, homoserine, which can be substituted for serine, and ornithine, which can be substituted for lysine. Additional examples of unnatural amino acids that can be substituted in polypeptide ligands include molecules that include, but are not limited to, D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, A-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, lanthionine, dehydroalanine, γ-aminobutyric acid, selenocysteine, and pyrrolidine fluoro-amino acids, and designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids.

[0026] "Polynucleotide" and "nucleic acid molecule": As used interchangeably herein, polynucleotide and nucleic acid molecule refer to polymeric forms of nucleotides of any length, either 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 (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and / or tRNA.

[0027] Operably linked: As used herein, the term "operably linked" indicates that two molecules are attached such that each retains functional activity. Two molecules are operably linked regardless of whether they are attached directly or indirectly.

[0028] Peptide tag: As used herein, the term "peptide tag" refers to a peptide sequence that is part of or attached (e.g., via genetic engineering) to another protein to provide a function to the resulting fusion. Peptide tags are typically relatively short compared to the proteins to which they are fused. In some embodiments, peptide tags are 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. Numerous peptide tags having uses provided herein are known in the art. Examples of peptide tags that can be components of a ligand fusion protein or a target bound by a ligand (e.g., a ligand fusion protein) 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, NY). Similarly, an antibody against the tag epitope allows detection and localization of the fusion protein in, for example, affinity purification, Western blots, ELISA assays, and immunostaining of cells.

[0029] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but those skilled in the art will understand that the term is not limited to long chains and can refer to a minimum chain comprising two amino acids linked together via peptide bonds. Polypeptides may be processed and / or modified, as known to those skilled in the art.

[0030] Protein: As used herein, the term "protein" refers to one or more polypeptides that function as individual units. When a single polypeptide is an individual functional unit and does not require permanent or temporary physical association with other polypeptides to form an individual functional unit, the terms "polypeptide" and "protein" may be used interchangeably. When an individual functional unit consists of two or more polypeptides that are physically associated with each other, the term "protein" refers to multiple polypeptides that are physically associated and function together as an individual unit.

[0031] Specific binding: As used herein with respect to a ligand, the term "specifically binds" or "having selective affinity" means that the ligand reacts or associates with a particular epitope, protein, or target molecule more frequently, more rapidly, with a longer duration, with greater affinity, or a combination of the above, than with alternative substances, including unrelated proteins. Due to sequence identity between homologous proteins in different species, specific binding may involve a binding agent that recognizes more than one protein or target. Similarly, due to homology within a particular region of the polypeptide sequence of different proteins, specific binding may involve a binding agent that recognizes more than one protein or target. It is understood 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 exclusive binding, i.e., binding to a single target (although it can include this). Thus, a ligand or affinity agent may, in certain embodiments, specifically bind to more than one target. In certain embodiments, multiple targets may be bound by the same antigen-binding site on the affinity agent.

[0032] Substantially: As used herein, the term "substantially" refers to the qualitative condition indicating the extent or degree of the totality or near totality of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or progress toward completion, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the lack of completeness inherent in many biological and chemical phenomena. [Brief explanation of the drawings]

[0033] [Figure 1A] FIG. 1A shows the structure of the ligand contemplated herein, depicted as a three-helix bundle protein. [Figure 1B]FIG. 1B shows that the three helices of a three-helix bundle protein can be envisioned as a prism, with each side shown as a face. [Figure 2-1] Figure 2-1 shows sensorgrams of exemplary affinity agents, for biotinylated ligands corresponding to SEQ ID NO: 4 and SEQ ID NO: 5, challenged with titrations of haptoglobin in solution. [Figure 2-2] Figure 2-2 shows sensorgrams of exemplary affinity agents, for biotinylated ligands corresponding to SEQ ID NO: 7 and SEQ ID NO: 8, challenged with titrations of haptoglobin in solution. [Figure 3] Figure 3 shows the equilibrium binding capacity of exemplary resins prepared from affinity agents. The height of the bars in the graph represents the amount of haptoglobin in milligrams that can be captured for each milligram of affinity agent conjugated to the resin. [Figure 4] FIG. 4 shows the dynamic binding capacity of an exemplary resin prepared from an affinity agent corresponding to SEQ ID NO: 118 at residence times of 2, 4, 6, and 8 minutes. [Figure 5] Figure 5 shows the results of a column purification run of haptoglobin purified from fraction V of human plasma using the specific provided affinity agents, analyzed via 4-20% Tris-glycine, SDS-PAGE gel run under reducing conditions. The ligand was SEQ ID NO: 118. The samples loaded in each lane are listed in the table below. [Table 1] [Figure 6] Figure 6 shows the residual HCP and DNA measured over several cycles of purification of human plasma haptoglobin fraction V. The ligand was SEQ ID NO:118. [Figure 7] Figure 7 shows the yield measured over several cycles of purification of haptoglobin as described in Example 8. The ligand was SEQ ID NO:117. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present disclosure includes, inter alia, the recognition that affinity agents prepared from identified and characterized ligands have been shown to produce highly purified preparations of one or more targets of interest, for example, in some embodiments, haptoglobin proteins. In some embodiments, the affinity resins described herein are useful for, inter alia, removing protein product-related impurities as well as host cell-derived contaminants.

[0035] Ligand binding to a target of interest for use in affinity agents The characteristics of a ligand that binds to a target can be determined using assays, bioassays, and / or animal models known or modified in the art for assessing such activity.

[0036] As used herein, terms such as " target binding affinity ", " target binding " refer to the property of ligand that can be measured directly, for example, through determining affinity constant (for example, the amount of ligand that associates and dissociates at a given antigen concentration).To characterize such molecular interaction, several methods are available, for example, competitive analysis, equilibrium analysis and microcalorimetry analysis, and real-time interaction analysis based on surface plasmon resonance interaction (for example, using BIACORE equipment).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.

[0037] The affinity requirements for a given ligand binding event are subject to 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 is 5×10 -3 M, 10 -3 M, 5 x 10 -4 M, 10 -4 M, 5 x 10-5 M, or 10 -5 The dissociation constant (K D ) binds to the target of interest. In some embodiments, the ligand binds to the target of interest at a concentration of 5×10 -6 M, 10 -6 M, 5 x 10 -7 M, 10 -7 M, 5 x 10 -8 M, or 10 -8 K below M D In some embodiments, the ligand binds to the target of interest at a concentration of 5×10 -9 M, 10 -9 M, 5 x 10 -10 M, 10 -10 M, 5 x 10 -11 M, 10 -11 M, 5 x 10 -12 M, 10 -12 M, 5 x 10 -13 M, 10 -13 M, 5 x 10 -14 M, 10 -14 M, 5 x 10 -15 M, or 10 -15 K below M D In some embodiments, the ligands produced by the methods disclosed herein bind to the target of interest at about 10 -4 M~about 10 -5 M, about 10 -5 M~about 10 -6 M, about 10 -6 M~about 10 -7 M, about 10 -7 M~about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M~about 10 -11 M, or about 10 -11 M~about 10 -12 It has a dissociation constant of M.

[0038] K D and dissociation rate (k offThe binding experiment to determine can be conducted under a number of conditions and can be highly dependent on the desired pH of the final solution. Low pH solutions (< pH 5.5) can be made, for example, in citrate buffer, glycine-HCl buffer, or succinate buffer. High pH solutions can be made, for example, in Tris-HCl, phosphate buffer, or sodium bicarbonate buffer. A number of conditions can be used, for example, for the purpose of determining the optimal pH and / or salt concentration, K D and the dissociation rate can be determined.

[0039] In some embodiments, the ligand binds specifically to the target at a k -7 in the range of 0.1 to 10 -1 seconds, 10- 2 to 10 -7 seconds, -1 or 0.5 × 10- 2 to 10- 7 seconds. -1 In some embodiments, the ligand binds to the target at a k off of 5 × 10 -2 seconds, 10 -1 seconds, 5 × 10 -2 seconds, -1 or 10- -3 seconds -1 less than. 3 In some embodiments, the ligand binds to the target at a k -1 of 5 × 10 off seconds, 10 -4 seconds, 5 × 10 -1 seconds, -4 or 10 -1 seconds, 5 × 10 [[ID=,51]] -5 seconds, 10 -1 seconds, 5 × 10 -5 seconds, 10 -1 seconds, 5 × 10 -6 seconds, 10 -1 seconds, 5 × 10 -6 seconds, 10 -1 seconds, 5 × -7 seconds, -1 or 10 -7 seconds -1 less than. off In some embodiments, the ligand binds to the target at a k 3 of about 10 7 to 10 -1 M seconds.-1 , 10 3 ~10 6 M -1 seconds -1 , or 10 3 ~10 5 M -1 seconds -1 The binding rate (k on ) specifically binds to a target of interest. In some embodiments, the ligand (e.g., a ligand fusion protein) 3 M -1 seconds -1 , 5×10 3 M -1 seconds -1 , 10 4 M -1 seconds -1 , or 5 × 10 4 M -1 seconds -1 exceeding k on In a further embodiment, the ligand binds to the target of interest at 10 5 M -1 seconds -1 , 5×10 5 M -1 seconds -1 , 10 6 M -1 seconds -1 , 5×10 6 M -1 seconds -1 , or 10 7 M -1 seconds -1 exceeding k on and binds to the target of interest.

[0040] Target According to various embodiments, the target of interest specifically bound by the ligand can be any molecule to which it is desirable for the ligand of the affinity agent to bind. For example, the target of interest specifically bound by the ligand can be any target of purification, manufacture, formulation, therapeutic, diagnostic, or prognostic relevance or value. Non-limiting uses include therapeutic and diagnostic applications. A number of exemplary targets are provided herein by way of illustration and not by way of limitation. The target of interest can be naturally occurring or synthetic. In some embodiments, the target comprises a haptoglobin protein. In some embodiments, the target comprises haptoglobin from human plasma. In some embodiments, the target comprises haptoglobin from fraction IV of the Cohn fractionation of human plasma. In some embodiments, the target comprises haptoglobin from fraction V of the Cohn fractionation of human plasma. In some embodiments, the target comprises haptoglobin from a recombinant source known to those of skill in the art.

[0041] Linker The terms "linker" and "spacer" are used interchangeably herein and refer to a peptide or other chemical bond that functions to link otherwise independent functional domains. In some embodiments, the linker is located between a ligand and another polypeptide component containing an otherwise independent functional domain. A suitable linker for linking two or more linked ligands may generally be any linker used in the art to link peptides, proteins, or other organic molecules. In some embodiments, such linkers are suitable for constructing proteins or polypeptides intended for pharmaceutical use.

[0042] Suitable linkers for operably linking the ligand and additional components of the ligand fusion protein in a single amino acid sequence include, but are not limited to, polypeptide linkers such as glycine linkers, serine linkers, mixed glycine / serine linkers, glycine-rich linkers and serine-rich linkers, or linkers composed of predominantly polar polypeptide fragments.

[0043] In some embodiments, the linker comprises a majority of amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In some embodiments, the linker comprises a majority of amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In some embodiments, the ligand linker is composed of a majority of amino acids that are sterically unhindered. 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 polyglycine (e.g., (Gly)5 and (Gly)8), poly(Gly-Ala), and polyalanine.

[0044] The linker can be of any size or composition, so long as it is 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 1-50 amino acids, about 1-20 amino acids, about 1-15 amino acids, about 1-10 amino acids, about 1-5 amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids. It will be apparent that the length, degree of flexibility, and / or other properties of the linker can affect certain properties of the ligand for use in the affinity agent, such as 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, two or more linkers are the same. In some embodiments, two or more linkers are different.

[0045] 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)sC(0)- (where s = 2-20) can be used. These alkyl linkers can be further substituted with any non-sterically hindering group, such as lower alkyl (e.g., C1-C6) lower acyl, halogen (e.g., Cl, I, Br, F), CN, NH2, or phenyl. An exemplary non-peptide linker is a PEG linker. In some embodiments, the PEG linker has a molecular weight of about 100-5000 kDa or about 100-500 kDa. In some embodiments, the PEG linker has a molecular weight of about 100-500 kDa.

[0046] Linkers can be evaluated using techniques described herein and / or otherwise known in the art. In some embodiments, the linker does not alter (e.g., destroy) the ability of the ligand to bind to the target molecule.

[0047] Affinity Agents Containing Conjugated Ligands Ligands that promote specific binding to a target of interest can be chemically conjugated to various chromatographic compositions (e.g., beads, resins, gels, membranes, monoliths, etc.) to prepare affinity agents. Affinity agents containing ligands are particularly useful in purification and manufacturing applications.

[0048] In some embodiments, the ligand (e.g., a ligand fusion protein) contains at least one reactive residue. The reactive residue is useful, for example, as a binding site for a conjugate such as a chemotherapeutic drug. An exemplary reactive amino acid residue is lysine. A reactive residue (e.g., lysine) can be added to the ligand at either terminus or within the ligand sequence and / or substituted for another amino acid within the ligand sequence. A suitable reactive residue (e.g., lysine, etc.) can also be located within the sequence of a specified ligand without the need for addition or substitution. In some embodiments, an additional exemplary reactive amino acid residue is cysteine. In some embodiments, the reactive amino acid residue is lysine.

[0049] Adhesion to solid surfaces "Solid surface," "support," or "matrix," used interchangeably herein, refer to, but are not limited to, any column (or column material), bead, test tube, microtiter dish, solid particle (e.g., agarose or sepharose), microchip (e.g., silicon, glass, silicon, or gold chip), or membrane (of synthetic (e.g., filter) or biological (e.g., liposome or vesicle) origin) to which a ligand, affinity agent, antibody, or other protein can be directly or indirectly attached (i.e., bound, linked, or adhered) (e.g., via other antibodies or other binding partner intermediates such as protein A) or to which a ligand or antibody can be embedded (e.g., via a receptor or channel). Reagents and techniques for attaching 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, chromatography resins or matrices (e.g., SEPHAROSE-4 FF agarose beads), the walls or floors of wells in plastic microtiter dishes, silica-based biochips, polyacrylamide, agarose, silica, nitrocellulose, paper, plastic, nylon, metal, and combinations thereof. 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 attached to the chromatography material using a linker.

[0050] Ligand production Ligands useful for practicing some embodiments of the provided methods can be produced using a variety of standard techniques for chemical synthesis, semisynthetic methods, and recombinant DNA methodologies known in the art. Also provided are methods for producing ligands as soluble agents and cell-associated proteins, individually or as part of multidomain fusion proteins. In some embodiments, the overall ligand production scheme involves obtaining a reference protein scaffold and identifying multiple residues within the scaffold for modification. Depending on the embodiment, the reference scaffold may include one or more α-helical regions or protein structures with other tertiary structures. Once identified, any of the multiple 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 position for modification. In some embodiments, the modification does not include substitution with either cysteine ​​or proline. In certain embodiments, after modifications are made 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 modified polypeptide) in, for example, a plasmid, bacteria, phage, or other vector. The modified polypeptides can then be purified and screened to identify modified polypeptides with specific binding to a particular target of interest. The modified polypeptides may exhibit enhanced binding specificity for the target of interest compared to the reference scaffold, or may exhibit little or no binding to a given target of interest (or non-target proteins).In some embodiments, depending on the target of interest, the reference scaffold may exhibit some interaction (e.g., non-specific interaction) with the target of interest, while the particular modified polypeptide exhibits at least about a 2-fold, at least about a 5-fold, at least about a 10-fold, at least about a 20-fold, at least about a 50-fold, or at least about a 100-fold (or more) increase in binding specificity for the target of interest. Further details regarding the production, selection, and isolation of ligands are provided in more detail below.

[0051] Recombinant expression of ligand In some embodiments, a ligand, such as a ligand fusion protein, is "recombinantly produced" (i.e., produced using recombinant DNA technology). Exemplary recombinant methods available for synthesizing ligand fusion proteins include, but are not limited to, polymerase chain reaction (PCR)-based synthesis, concatemerization, seamless cloning, and recursive directional 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)).

[0052] Also provided is a nucleic acid comprising a polynucleotide sequence encoding a ligand.These polynucleotides optionally further comprise one or more expression control elements.For example, polynucleotides can comprise one or more promoters or transcription enhancers, ribosome binding sites, transcription termination signals, and polyadenylation signals as expression control elements.Polynucleotides can be inserted into any suitable vector, which can be contained in any suitable host cell for expression.

[0053] Expression of a 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 useful for regulating expression of the desired nucleic acid sequence. Exemplary promoters useful for expression in E. coli include, for example, the T7 promoter.

[0054] Expression vectors containing a nucleic acid sequence encoding a ligand, along with appropriate transcriptional / translational 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 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.

[0055] A variety of host-expression vector systems can be utilized to express nucleic acids encoding ligands. Vectors containing one or more nucleic acids encoding ligands (e.g., individual ligand subunits or ligand fusions) or portions or fragments thereof may 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 may also be introduced into host cells in the form of packaged or enclosed viruses using known techniques for infection and transduction. Furthermore, viral vectors may be replication-competent or, alternatively, replication-defective. Alternatively, cell-free translation systems may be used to produce proteins and / or ligands using RNA derived from DNA expression constructs (see, e.g., WO86 / 05807 and WO89 / 01036, and U.S. Pat. No. 5,122,464).

[0056] 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 the engineered host cell is a bacterial cell, a yeast cell, or a mammalian cell. Various host-expression vector systems can 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 coding sequence of the target of interest and the coding sequence of the fusion polypeptide. The cells can also be primary isolates from transformed or transgenic organisms, cultures, or cell lines.

[0057] Suitable host cells include, but are not limited to, microorganisms such as bacteria (e.g., E. coli or B. subtilis) transformed with recombinant bacteriophage DNA, plasmid or cosmid DNA expression vectors 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 lines infected with a recombinant viral expression vector (e.g., baculovirus) containing the ligand coding sequence, and plant cell lines infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV)) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing the ligand coding sequence.

[0058] Prokaryotes useful as host cells in the production of ligands can include gram-negative or gram-positive organisms, such as Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis). Expression vectors for use in prokaryotic host cells generally 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 the series of vectors pKK223-3 (Pharmacia, Uppsala, Sweden), pGEM1 (Promega, Wisconsin, USA), pET (Novagen, Wisconsin), and pRSET (Invitrogen, California, USA) (see, for example, 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., Nucl. Acids Res. 8:4057, (1980)), and the tac promoter (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0059] 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 the ligand. Exemplary yeast that can be used to produce the compositions of the present invention include yeast from the genera Saccharomyces, Pichia, Actinomycetes, and Kluyveromyces. Yeast vectors typically contain an origin of replication sequence from the 2mu 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, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Additional suitable vectors and promoters for use in yeast expression and yeast transformation protocols are known in the art. See, for example, Fleer, Gene 107:285-195 (1991) and Hinnen, PNAS 75:1929 (1978).

[0060] Insect and plant host cell culture systems are also useful for producing 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 ligand coding sequence, plant cell systems infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid), including, but not limited to, the expression systems taught in U.S. Patent No. 6,815,184, U.S. Patent Publication Nos. 60 / 365,769 and 60 / 368,047, and WO2004 / 057002, WO2004 / 024927, and WO2003 / 078614.

[0061] In some embodiments, a host cell line may be used. In some embodiments, the host cell line is an animal cell line infected with a recombinant viral expression vector (e.g., adenovirus, retrovirus, adeno-associated virus, herpes virus, or lentivirus). In some embodiments, the host cell line is a cell line engineered to contain multiple copies of the DNA encoding the ligand, either stably amplified (CHO / dhfr) or unstably amplified in double minute chromosomes (e.g., mouse cell lines). In some embodiments, the vector containing the polynucleotide encoding the ligand is polycistronic. Exemplary mammalian cells useful for producing 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, The 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 useful in the practice of 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).Further 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 often derived from viral genomes. Commonly used promoter and enhancer sequences in mammalian expression vectors include sequences derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV). Exemplary commercially available expression vectors for use in mammalian host cells include pCEP4 (Invitrogen) and pcDNA3 (Invitrogen).

[0062] Physical methods for introducing nucleic acid 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).

[0063] Biological methods for introducing a target polynucleotide into a host cell include the use of DNA vectors and RNA vectors. Viral vectors, particularly 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 type I, adenoviruses, and adeno-associated viruses. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0064] Methods for introducing target DNA and RNA polynucleotides into host cells include, but are not limited to, electroporation of cells, in which an electric field is applied to cells to increase the permeability of the cell membrane, allowing chemicals, drugs, or polynucleotides to be introduced into the cells. Ligands containing DNA constructs or RNA constructs can be introduced into mammalian cells or prokaryotic cells using electroporation.

[0065] In some embodiments, electroporation of cells results in the expression of ligand-CAR on the surface of T cells, NK cells, and NKT cells. Such expression can be transient or stable over the life of the cell. Electroporation can be achieved using methods known in the art, including MaxCyte GT® and STX® Transfection Systems (MaxCyte, Gaithersburg, MD, USA).

[0066] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle). When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is intended for the introduction of nucleic acid into host cells (in vitro, ex vivo, or in vivo). In some embodiments, the nucleic acid is associated with a lipid. Lipid-associated nucleic acids may be encapsulated within the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, bound to liposomes via linking molecules associated with both the liposomes and oligonucleotides, entrapped in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in micelles, complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they may exist as micelles or in bilayer structures with a "folded" structure. They may also simply be interspersed in solution or may form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0067] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") can be obtained from Sigma (St. Louis, MO), dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY), cholesterol ("Choi") can be obtained from Calbiochem-Behring, and dimyristoyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform evaporates more readily than methanol and can therefore be used as the sole solvent. "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5:505-510 (1991)). However, compositions with structures different from normal vesicle structures in solution are also encompassed. For example, lipids can take the form of micellar structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0068] Regardless of the method used to introduce exogenous nucleic acid into a host cell, the presence of the recombinant nucleic acid sequence in the host cell can be routinely confirmed through a variety of assays known in the art, including "molecular biological" assays known in the art, such as Southern and Northern blots, RT-PCR, and PCR, as well as "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the present invention.

[0069] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. Generally, reporter genes are genes encoding polypeptides that are absent from or expressed by recipient organisms, tissues, or cells, and whose expression is manifested by some easily detectable property, such as enzymatic activity. The expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes include, but are not limited to, genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (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, the construct with the smallest 5'-flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be routinely linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0070] A number of 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 the basis for selection for, for example, the dhfr, gpt, neo, hygro, trpB, hisD, ODC (ornithine decarboxylase), and glutamine synthase systems.

[0071] Ligand purification Once the ligand or ligand fusion protein is produced by recombinant expression, it can be purified by methods known in the art for the purification of recombinant proteins, for example, by chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. In some embodiments, the ligand is optionally fused to a heterologous polypeptide sequence, specifically disclosed herein or otherwise known in the art, to facilitate purification. In some embodiments, the ligands (e.g., antibodies and other affinity matrices) of the ligand affinity column for affinity purification, 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 ligand using techniques known in the art.

[0072] Chemical synthesis of ligands In addition to recombinant methods, ligand production can also be carried out using organic chemical synthesis of the desired polypeptide using a variety of liquid and solid phase chemical processes known in the art. A variety of automated synthesizers are commercially available and can be used in accordance with known protocols. 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, JK, ed. Plenum Press, NY. 1990, vol. 12, pp. 1-19, Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, 1989. One advantage of these methodologies is that they allow for the incorporation of unnatural amino acid residues into the sequence of the ligand.

[0073] The ligands used in the methods of the invention may be modified during or after synthesis or translation, for example, by glycosylation, acetylation, benzylation, phosphorylation, amidation, pegylation, formylation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to an antibody molecule, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, ubiquitination, etc. (See, e.g., Creighton, Proteins: Structures and Molecular Properties, 2nd Ed. (WH Freeman and Co., NY, 1992); Postranslational Covalent Modification of Proteins, Johnson, ed. (Academic Press, New York, 1983), pp. 1-12; Seifter, Meth. Enzymol., 182:626-646 (1990); Rattan, Ann. NY Acad. Sci., 663:48-62 (1992).) In some embodiments, the peptides are acetylated at the N-terminus and / or amidated at the C-terminus.

[0074] Any of a number of chemical modifications may be made by known techniques, including, but not limited to, acetylation, formylation, etc. Additionally, the derivative may contain one or more non-classical amino acids.

[0075] In some embodiments, cyclization or macrocyclization of the peptide backbone is achieved by side chain bond formation. Methods for achieving this are well known in the art and can involve natural as well as unnatural amino acids. Approaches include disulfide formation, lanthionine formation, or thiol alkylation (e.g., Michael addition), amidation between amino and carboxylate side chains, click chemistry (e.g., azide-alkyne condensation), peptide stapling, ring-closing metathesis, and the use of enzymes.

[0076] Affinity Agents for Purification In affinity chromatography-based purification, targets of interest (e.g., proteins or molecules) are selectively isolated according to their ability to specifically and reversibly bind to ligands, which can be covalently attached to a chromatography matrix. In some embodiments, the ligands can be used as reagents for affinity purification of targets of interest from either recombinant sources or natural sources, such as biological samples (e.g., serum or cells).

[0077] In some embodiments, a ligand that specifically binds to a target of interest is immobilized on beads and then used to affinity purify the target.

[0078] Methods for covalently binding proteins to surfaces are known to those skilled in the art. Peptide tags that can be used to attach ligands to solid surfaces are known to those skilled in the art. Furthermore, ligands can be attached (i.e., bound, linked, or adhered) to solid surfaces using any reagent or technique known in the art. In some embodiments, the solid support comprises beads, glass, slides, chips, and / or gelatin. Thus, a series of ligands can be used to create arrays on solid surfaces using techniques known in the art. For example, U.S. Patent Publication No. 2004 / 0009530 discloses a method for preparing arrays.

[0079] In some embodiments, the ligand is used to isolate a target of interest (e.g., a haptoglobin protein) by affinity chromatography. In some embodiments, the ligand is immobilized on a solid support. The ligand can be immobilized on a solid support using techniques and reagents described herein or otherwise known in the art. Suitable solid supports are described herein or otherwise known in the art, and in certain embodiments, are suitable for packing a chromatography column. The immobilized ligand may be loaded or contacted with a solution under conditions favorable for forming a complex between the ligand and the target of interest. Unbound materials may be washed away. Suitable washing conditions can be easily determined by one of skill in the art. Examples of suitable washing conditions are described in Shukla and Hinckley, Biotechnol Prog. 2008 Sep-Oct;24(5):1115-21. doi: 10.1002 / btpr.50.

[0080] In some embodiments, chromatography is performed by mixing a solution containing the target of interest and the ligand, followed by isolating 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 isolate the ligand after complex formation using an 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 a purified form.

[0081] In some embodiments, the ligand is isolated containing an initial N-terminal methionine because it is a protein sequence encoded by DNA. In some embodiments, the ligand is isolated without an N-terminal methionine residue. In some embodiments, the mixture is obtained using only a portion of the purified ligand containing an N-terminal methionine. It is clear to those skilled in the art that the presence or absence of an N-terminal methionine does not affect the conclusions herein. [Example]

[0082] Example 1. Purification of recombinant protein ligand. Recombinant protein ligands were expressed in Escherichia coli (E. coli) and / or Pichia pastoris using standard techniques. Ligands were purified using multicolumn chromatography. For his-tagged ligands, IMAC was used as the primary capture step. Biotinylated ligands were produced using the Avitag™ system (Avidity, Aurora, CO). Non-biotinylated ligands with the Avitag™ sequence were prepared by omitting exogenous biotin. The purity and identity of the recombinant protein ligands were assessed by a combination of SDS-PAGE, RP UPLC, quadrupole time-of-flight mass spectrometry, and SEC. In many cases, ligands are isolated without the N-terminal methionine residue, which is thought to be cleaved during expression. In many cases, mixtures are obtained using only a portion of the purified ligand that contains the N-terminal methionine. It will be clear to those skilled in the art that the presence or absence of the N-terminal methionine does not affect the conclusions herein. For clarity, we include the N-terminal methionine.

[0083] Example 2. Binding of biotinylated ligands to haptoglobin proteins. This example demonstrates the binding of biotinylated ligands to haptoglobin protein using biolayer interferometry (ForteBio, Menlo Park, CA). The biotinylated ligands were immobilized on a sensor and incubated with a solution containing haptoglobin protein at various concentrations. Affinity was determined by fitting a 1:1 binding model to the binding curves using ForteBio software, and the resulting data are shown in the table below. As expected, all affinity agents bound to haptoglobin protein with high affinity. In most cases, the binding affinity was less than 10 nM, and in many cases, less than 1 nM. [Table 2-1] [Table 2-2]

[0084] Example 3. Sodium hydroxide stability of affinity ligands. This example demonstrates the sodium hydroxide stability of affinity ligands. Ligands were incubated in 0.25 M NaOH for 8 hours and then neutralized. Binding of the NaOH-treated ligands was measured as described in Example 1 and compared to untreated ligands. Retained binding was calculated according to the following formula: % Retained Binding = (Measured Response After NaOH Treatment) ÷ (Untreated Measured Response) × 100

[0085] The data are presented in the table below and show that all of the affinity ligands tested exhibit high stability under the conditions tested. [Table 3-1] [Table 3-2]

[0086] Example 4. Generation and characterization of affinity agents. This example demonstrates the generation and characterization of affinity agents containing the ligands identified and described herein. Affinity resins were prepared by conjugating the ligands to agarose beads. Praesto® Jet A50 beads (Purolite, King of Prussia, PA) were activated with disuccinimidyl carbonate and coupled with excess ethylenediamine. After washing, bromoacetate was conjugated to the aminated beads using EDC activation. After washing, the ligands were conjugated to the beads at room temperature. Target ligand densities varied from 5 to 12 g / L. After washing, the beads were deactivated with excess thioglycerol. The actual ligand density of all resins was measured using a subtractive RP-HPLC method according to the following equation: Actual Ligand Density = (Measured in Feed [Ligand] - Measured in Eluate [Ligand]).

[0087] Example 5. Binding properties of affinity agents. This example demonstrates the binding properties of affinity chromatography resins prepared from the affinity ligands described herein. Briefly, 5 μL of resin was dispensed into replicate wells of a 96-well filter plate. The resin was pre-sanitized by washing with 0.1 M sodium hydroxide for 5 minutes, then equilibrated with a neutral pH buffer, and tested for binding in a batch format. After equilibration with PBS, the resin was challenged with 1.5 mg / mL haptoglobin protein for 60 minutes to allow complete binding to the resin. After the binding challenge, the multi-well filter plate was centrifuged to separate unbound haptoglobin from the resin, and the amount of unbound haptoglobin was quantified by measuring the absorbance of the effluent at 280 nm. The procedure is detailed in the table below. [Table 4]

[0088] 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 dividing the amount of haptoglobin bound to the resin, measured according to the method outlined in Example 5, by the amount of ligand immobilized on the resin according to the following formula: Binding capacity = (haptoglobin in feed - haptoglobin in effluent) ÷ (ligand on resin)

[0089] The data are presented in Figure 3 and demonstrate the effective capture of haptoglobin protein for all affinity agents tested.

[0090] Example 6. Dynamic binding capacity of affinity agents. This example demonstrates the dynamic binding capacity of the affinity ligands described herein for the purification of haptoglobin. Haptoglobin protein was diluted in PBS to a concentration of 1 mg / mL. 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 following table: [Table 5]

[0091] To assess the effect of residence time on dynamic binding capacity, the linear velocity of the loading solution was varied: separate dynamic binding capacity measurements were performed using linear velocities of 75 cm / hr (8 min residence time), 100 cm / hr (6 min residence time), 150 cm / hr (4 min residence time), and 300 cm / hr (2 min residence time).

[0092] The material was analyzed by measuring the absorbance of each effluent fraction compared to the absorbance of the loading solution. The total mass of haptoglobin challenge at which the effluent measured 10% of the loading solution is reported as the dynamic binding capacity of the resin. Data for an exemplary affinity resin generated from the affinity ligand corresponding to SEQ ID NO: 118 are shown in Figure 4 and show that the dynamic binding capacity of the resin increased with increasing residence time over the range of 2 to 6 minutes, while increasing the residence time up to 8 minutes did not increase binding capacity.

[0093] Example 7. Repeated use of affinity agents. This example demonstrates the repeated use of affinity agents containing the binding ligands described herein for the affinity purification of haptoglobin protein. The packed column was used to purify haptoglobin protein from fraction V of the Cohn fractionation of human plasma, with a measured haptoglobin titer of 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. [Table 6]

[0094] Six CIP sanitizing cycles were simulated with the resin exposed to the CIP agent for a total of three hours between each cycle.

[0095] Elution fractions from each purification cycle were collected and analyzed for purity via SDS-PAGE. The data, shown in Figure 5, confirm the high purity provided by the affinity agent and the significant absence of serum albumin in the elution fractions. Purity was also assessed by analytical RP-HPLC. Percent purity was calculated by comparing the haptoglobin protein peak area to the total area of ​​all peaks in the analysis. The data, shown in Figure 6, confirm consistent purification performance over dozens of simulated cycles after over 25 hours of exposure to 0.25 M sodium hydroxide. Yield was determined using analytical RP-HPLC by comparing the haptoglobin peak area in the first cycle with all subsequent cycles. The data, shown in Figure 7, confirm that the resin can be cleaned with NaOH and reused for multiple cycles. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

Claims

1. 1. An affinity agent comprising a ligand that binds to a haptoglobin protein, wherein the ligand comprises the sequence of SEQ ID NO: 1; ︸ １ 1.2) ２ ︦︸ ３ ︸ ４ ️ ５ ..︸ ６ 3.1 ７ ﹁️ ８ _: ９ ︸ １０ ︤ １１ !.P, In the formula, X １ is A, D, E, H, I, L, Q, S, T, V, or W, and X ２ is A, E, G, H, N, Q, S, or Y, and X ３ is A, D, E, F, G, H, I, K, L, N, Q, R, S, T, W, or Y, and X ４ is A, F, H, L, Q, S, T, V, or Y, and X ５ is A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, or Y, and X ６ is A, D, E, G, H, I, K, L, Q, S, T, or V, and X ７ is A, E, G, H, I, L, P, S, T, V, W, or Y, and X ８ is A, G, I, L, T, or V, and X ９ is A, D, E, G, H, K, N, R, S, or T, and 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 comprising a ligand that binds to a haptoglobin protein, wherein the ligand comprises at least one amino acid sequence of any one of SEQ ID NOs: 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.

3. a multimeric polypeptide, the multimeric polypeptide comprising at least two subunits; The affinity agent of claim 1 or 2, wherein each subunit comprises the ligand.

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

5. The affinity agent of claim 1 or 2, wherein the ligand is attached to a solid surface.

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

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

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

9. The affinity agent of claim 5 , wherein the ligand is conjugated to the solid surface via a linker.

10. The affinity agent according to claim 1 or 2, which is used for purifying a haptoglobin protein.

11. A method for making an affinity agent according to claim 1 or 2, comprising conjugating the ligand to a solid surface.

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