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. Summary of the Invention

[0002] 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, developing affinity agents (including, for example, affinity ligands) can be resource-intensive and time-consuming. As a result, affinity agents have been developed for 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.

[0003] Exemplary therapeutic proteins include, but are not limited to, biologically active polypeptides / proteins, fusion proteins, enzymes, hormones, antibodies, and antibody fragments.

[0004] Described herein are affinity agents that bind to proteins and are useful for isolation and / or affinity purification. In some embodiments, the affinity agent comprises a solid support and a ligand.

[0005] Human monoclonal antibody therapy is a rapidly growing sector in the biopharmaceutical industry, with very promising clinical applications in cancer therapy. Traditional methods for recombinant antibody purification utilize affinity chromatography resins based on Protein A ligands. Protein A-based affinity resins offer a highly efficient and high-yielding process for the purification of monoclonal antibodies. The utility of Protein A affinity resins in industrial antibody manufacturing processes highlights the need for suitable affinity purification devices for other classes of molecules.

[0006] Human serum albumin (HSA) is a therapeutic protein that can be purified directly from human plasma or produced via recombinant protein expression. This therapeutic protein is commonly used and is produced in large quantities (e.g., tons / year) annually. It has also been discovered that fusion of a therapeutic polypeptide or protein to HSA can be used to extend the serum half-life of the therapeutic polypeptide or protein. Indeed, some HSA fusion products have been approved by the FDA (e.g., Idelvion® and Tanzeum® (Albiglutide)), and many more are in clinical development. Due to the therapeutic relevance of HSA and its fusion products, there is a need for affinity purification solutions for HSA and its fusion products (e.g., HSA fused to polypeptides and / or proteins) that have high binding capacity and the ability to be used more than once, for example, after washing under alkaline conditions (e.g., with sodium hydroxide). Previous efforts to generate affinity purification methods for HSA have been based on the natural molecule protein G. Protein G affinity resins have been demonstrated to be limited by both their low binding capacity and instability to washing with sodium hydroxide (Gulich et al., "Stability towards alkaline conditions can be engineered into a protein ligand", Journal of Biotechnology 80 (2000) 169-178 and US6613884). As a result, no commercial Protein G affinity resins are available on the market.

[0007] Protein G, including Streptococcus sp. G148 protein G, contains three albumin-binding domains (ABDs) comprising a three-helix bundle (3HB). The wild-type ABDs are each 46 amino acids long (Kraulis et al. 1996), flanked by linkers to the rest of the protein. Protein G binding to albumin is mediated by contact points in the second and third helices, defined by a known core binding sequence of 25 amino acids (see, e.g., U.S. Patent No. 8,937,153). However, the stability of the 3HB protein is affected by all three helices; therefore, helix 1, as well as adjacent sequences, are an integral part of the final molecule.

[0008] The binding affinity of ABDs has been improved to femtomolar concentrations, as evidenced by U.S. Patent No. 8,937,153 (the "153 Patent"), U.S. Patent No. 9,156,887 (the "887 Patent"), and U.S. Patent No. 10,280,200 (the "200 Patent"), which describe, in part, fusing ABD-containing proteins with small protein drugs to increase circulating half-life. Notably, the "887 Patent" and "200 Patent" established non-natural ABDs with sequences significantly different from those of the preceding U.S. Patent No. 8,937,153. U.S. Patent No. 9,156,887 teaches proteins comprising SEQ ID NOS: 1-14, or isolated albumin-binding domains comprising the amino acid sequence of SEQ ID NOS: 1 with substitutions at 1, 2, 3, 4, 5, or 6 residues. Furthermore, substitutions are only permitted at specific positions indicated by X in SEQ ID NOS: 2-4. The best ligands bind to human albumin (K). D ) is 75 pM, and the affinity is 4.04E × 10 5 M -1 s -1 k on and 3.02 × 10 -5 s -1 k off had.

[0009] Surprisingly, the inventors discovered that seven substitutions could be made to SEQ ID NO: 1, reducing affinity by more than 40-fold to 3 nM, yet still provide an effective affinity purification tool for the resulting ligand. Furthermore, these substitutions can be made at positions other than those taught by U.S. Patent No. 9,156,887.

[0010] In some embodiments, provided herein is an affinity agent comprising the sequence of SEQ ID NO: 15, LREAKERAIEELRRAGISSDYYFDLIQKAKTVEGVQALKDEILKA. A comparison of SEQ ID NO: 15 with SEQ ID NOs: 1-14 (i.e., the sequences taught by U.S. Patent No. 9,156,887) is shown in the following table: [Table 1]

[0011] U.S. Patent No. 8,937,153 teaches engineered albumin binding polypeptides containing an albumin binding motif, the motif having the amino acid sequence: GVSDX5YKX8X9I X11X12AX14TVEGVX 20 ALX 23 X 24 X 25 I (SEQ ID NO: 16), which are, independently of each other: X5 is selected from Y and F; X8 is selected from N, R, and S; X9 is selected from V, I, L, M, F, and Y; X 11 is selected from N, S, E, and D, X 12 is selected from R, K, and N; X 14 is selected from K and R; X 20 is selected from D, N, Q, E, H, S, R, and K; X 23 is selected from K, I, and T, X 24 is selected from A, S, T, G, H, L, and D; X 25 is selected from H, E, and D, However, the amino acid sequence is not GVSDYYKNLI NNAKTVEGVK ALIDEI, or (US8937153, SEQ ID NO: 517) GVSDYYKNLI NNAKTVEGVN ALKAEI (US8,937,153, SEQ ID NO: 541), and the albumin binding polypeptide has the K D The maximum value is 1×10 -9 It binds to albumin so that M

[0012] U.S. Patent No. 10,155,792 teaches albumin binding polypeptides containing an albumin binding motif (BM), the motif having the amino acid sequence: (SEQ ID NO: 19) GVSDFYKKLI XaKAKTVEGVE ALKXbXcI, which, independently of each other, Xa is selected from D and E; Xb is selected from D and E; Xc comprises an amino acid sequence selected from A and E; The albumin binding motif has enhanced resistance to clostripain cleavage compared to the motif of SEQ ID NO: 19, which has position 8 substituted with R, S, or N. The amino acid sequence of SEQ ID NO: 19 corresponds to residues 17-44 of SEQ ID NO: 15, i.e., SEQ ID NO: 20. A comparison of SEQ ID NO: 20 with SEQ ID NOs: 1-14 (i.e., the sequences taught by U.S. Pat. Nos. 8,937,153 and 10,155,792) is shown in the table below, highlighting the differences present in SEQ ID NO: 20. [Table 2]

[0013] Described herein are affinity agents that bind to HSA and / or HSA fusion products, useful for isolation and / or affinity purification. In some embodiments, the affinity agent comprises a solid support and a ligand.

[0014] 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.

[0015] In some embodiments, provided herein are affinity agents that comprise multimeric polypeptides, wherein the subunits are not all the same.

[0016] In some embodiments, provided herein are affinity agents for use in the purification of HSA and HSA fusion products.

[0017] 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.

[0018] 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 indicated or apparent from the context, refers to a range of values ​​that falls 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) the stated reference value (except where such number exceeds 100% of possible values).

[0019] 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.

[0020] 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, non-conservative amino acid substitutions do not significantly affect specific binding of the ligand to its target of interest.

[0021] 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.

[0022] 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.

[0023] 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 into 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.

[0024] "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.

[0025] 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.

[0026] 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 cell immunostaining.

[0027] 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.

[0028] 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.

[0029] Specific binding: As used herein with respect to a ligand, the term "specific binding," 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 alternatives, 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.

[0030] 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.

[0031] HSA fusion product: As used herein, the term "HSA fusion product" refers to a human serum albumin protein fused to one or more polypeptides and / or proteins. HSA fusion products can be produced by any of a variety of means, for example, by genetic engineering and expression of a fusion gene, or by chemical ligation methods commonly known in the art (e.g., native chemical ligation, expressed protein ligation, Staudinger ligation, Ser / Thr ligation, etc.). [Brief explanation of the drawings]

[0032] [Figure 1A] 1A shows exemplary crystal structures of some of the albumin ligands contemplated herein, with the binding interface of the albumin ligands highlighted by lighter shading. [Figure 1B] 1B shows exemplary crystal structures of some of the albumin ligands contemplated herein, with the positions of mutations made to SEQ ID NO:1 to create SEQ ID NO:15 highlighted by lighter shading. [Figure 2] FIG. 2 shows a sensorgram of the biotinylated ligand corresponding to SEQ ID NO: 21 challenged with a titration of HSA in solution. [Figure 3] FIG. 3 shows overlay chromatograms before and after repeated cycling of an affinity resin of the present invention. [Figure 4]Figure 4 shows the residual HCP levels over five purification cycles of an affinity resin of the invention, with the resin exposed to 0.5 M NaOH for 6 hours between each cycle. [Figure 5] Figure 5 shows the yields obtained over five purification cycles of the affinity resin of the invention, with the resin exposed to 0.5 M NaOH for 6 hours between each cycle. [Figure 6] FIG. 6 shows the static binding capacity of an affinity resin of the present invention as a function of pH. [Figure 7] Figure 7 shows the effect of elution pH on a particular albumin fusion protein. Overlay chromatograms of the elution peak (approximately 26 mL) and CIP peak (approximately 36 mL) are shown for elution pH 9 (dark solid line), pH 8.25 (dashed line), and pH 7.4 (gray line). [Figure 8] Figure 8 shows the SDS-PAGE analysis of the capture of albumin from Fraction V paste to isolate minor components. The samples loaded in each lane are listed in the table below. [Figure 9] FIG. 9 shows the breakthrough curves for resins prepared from ligands corresponding to SEQ ID NOs: 29 and 34-37 when challenged with HSA at 1 mg / mL and a 7 minute residence time. DETAILED DESCRIPTION OF THE INVENTION

[0033] 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, e.g., in some embodiments, HSA and / or HSA fusion products. In some embodiments, the affinity resins described herein are useful for, inter alia, the removal of protein product-related impurities as well as host cell-derived contaminants.

[0034] Ligand binding to a target of interest for use in affinity agents The properties of a ligand that binds to a target can be determined using assays, bioassays, and / or animal models known in the art or modified to assess such activity.

[0035] As used herein, terms such as " target binding affinity ", " target binding " refer to the property of ligand that can be directly measured, 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, such as 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 described in Neri D et al. (1996) Tibtech 14:465-470 and Jansson M et al. (1997) J Biol Chem 272:8189-8197.

[0036] 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 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 1×10 -9 K over M DIt binds to the target. In some embodiments, the ligand generated by the methods disclosed herein has a dissociation constant of about 10 -4 M to about 10 -5 M, about 10 -5 M to about 10 -6 M, about 10 -6 M to about 10 -7 M, about 10 -7 M to about 10 -8 M, about 10 -8 M to about 10 -9 M.

[0037] K D and the dissociation rate (k off ) can be determined in many conditions. The buffer used to make these solutions can be easily determined by those skilled in the art and depends greatly 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. Using some conditions, for example, for the purpose of determining the optimal pH and / or salt concentration, K D and the dissociation rate can be determined.

[0038] In some embodiments, the ligand specifically binds to the target with a k -7 in the range of 0.1 to 10 -1 seconds, 10 2 to 10 -7 seconds, or 0.5×10 -1 to 10 -2 seconds. -7 In some embodiments, the ligand has a k -1 less than 5×10 off seconds, 10 -2 seconds, 5×10 -1 seconds, or 10 -2 seconds, -1 and specifically binds to the target. -3 In some embodiments, the ligand has a k -1 less than 10 -3 seconds -1 off ​In some embodiments, the ligand binds to the target of interest at a concentration of 5×10 -4 seconds -1 , 10 -4 seconds -1 , 5×10 -5 seconds -1 , or 10 -5 seconds -1 , 5×10 -6 seconds -1 , 10 -6 seconds -1 , 5×10 -7 seconds -1 , or 10 -7 seconds -1 Less than k off In some embodiments, the ligand binds to the target of interest at about 10 3 ~10 7 M -1 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 107 M -1 seconds -1 exceeding k on and binds to the target of interest.

[0039] 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 affinity agent's ligand 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. Some exemplary targets are provided herein for illustrative purposes and are not intended to be limiting. The target of interest can be naturally occurring or synthetic. In some embodiments, the target comprises human serum albumin (HSA) protein or a fragment thereof, or an HSA fusion protein.

[0040] 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 positioned between a ligand and another polypeptide component containing an otherwise independent functional domain. A suitable linker for joining 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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-(CH) s-C(0)- can be used, where s=2-20. 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, phenyl, etc. 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.

[0045] 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.

[0046] 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.

[0047] 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, serine, tyrosine, hydroxytryptophan, 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. In some embodiments, the reactive amino acid residue is serine. In some embodiments, the reactive amino acid residue is tyrosine. In some embodiments, the reactive amino acid residue is hydroxytryptophan.

[0048] Adhesion to solid surfaces "Solid surface," "support," or "matrix" are used interchangeably herein and refer to, but are not limited to, any column (or column material), bead, 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.

[0049] Ligand production Ligands useful for practicing some embodiments of the provided methods can be produced using a variety of standard techniques for chemical synthesis, semi-synthetic 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 alpha-helical regions or protein structures with other tertiary structure. 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.

[0050] 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)).

[0051] Nucleic acids containing 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 expression control elements, such as a promoter or transcription enhancer, a ribosome binding site, a transcription termination signal, and a polyadenylation signal. The polynucleotide can be inserted into any suitable vector, which can be contained in any suitable host cell for expression.

[0052] 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.

[0053] 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.

[0054] A variety of host-expression vector systems can be utilized to express nucleic acids encoding ligands. Vectors containing 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 infection and transduction techniques. 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).

[0055] 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.

[0056] Suitable host cells include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid or cosmid DNA expression vectors containing the ligand coding sequence, yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the ligand coding sequence, insect cell lines infected with recombinant viral expression vectors (e.g., baculovirus) containing the ligand coding sequence, and plant cell lines infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the ligand coding sequence.

[0057] Prokaryotes useful as host cells in producing the ligand can include gram-negative or gram-positive organisms, such as E. coli and B. subtilis. Expression vectors for use in prokaryotic host cells generally contain one or more phenotypic selectable marker genes (e.g., genes encoding proteins that confer antibiotic resistance or provide autotrophic requirements). Examples of useful prokaryotic host expression vectors include the pKK223-3 (Pharmacia, Uppsala, Sweden), pGEM1 (Promega, Wisconsin, USA), pET (Novagen, Wisconsin), and pRSET (Invitrogen, California, USA) series of vectors (see, e.g., Studier, J. Mol. Biol. 219:37 (1991) and Schoepfer, Gene 124:83 (1993)). Exemplary promoter sequences frequently used in prokaryotic host cell expression vectors include T7 (Rosenberg et al., Gene 56:125-135 (1987)), beta-lactamase (penicillinase), lactose promoter systems (Chang et al., Nature 275:615 (1978)), and Goeddel et al., Nature 281:544 (1979)), tryptophan (trp) promoter systems (Goeddel et al., 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).

[0058] 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).

[0059] 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.

[0060] 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, 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).

[0061] 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).

[0062] Biological methods for introducing a polynucleotide of interest 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.

[0063] 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.

[0064] In some embodiments, electroporation of cells 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 life of the cell. Electroporation can be achieved using methods known in the art, including MaxCyte GT® and STX® Transfection Systems (MaxCyte, Gaithersburg, Maryland, USA).

[0065] 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 be interspersed in solution or 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.

[0066] 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, dimyristoyl phosphatidylglycerol ("DMPG") 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.

[0067] 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. Such assays include "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.

[0068] 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 not present in 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.

[0069] 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.

[0070] 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.

[0071] 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. in 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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, cells).

[0076] 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.

[0077] 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.

[0078] In some embodiments, the ligand is used to isolate a target of interest (e.g., HSA or an HSA fusion 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 ordinary 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.

[0079] 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.

[0080] 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]

[0081] [Example 1] Recombinant protein ligands were expressed in 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. 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. Biotinylated ligands were generated by conjugation to maleimide-PEG2-biotin. Ligands are often isolated without the N-terminal methionine residue, which is thought to be cleaved during expression. Often, mixtures are obtained using only a portion of the purified ligand that contains the N-terminal methionine. Those skilled in the art will understand that the presence or absence of the N-terminal methionine does not affect the conclusions herein. For clarity, we include the N-terminal methionine.

[0082] [Example 2] This example demonstrates the binding of a biotinylated ligand to albumin molecules using biolayer interferometry (ForteBio, Menlo Park, CA). A biotinylated ligand corresponding to SEQ ID NO: 21 was immobilized on a sensor and incubated with a solution containing albumin protein at various concentrations. An exemplary sensorgram for binding to HSA is shown in Figure 2. Affinity was determined by fitting a 1:1 binding model to the binding curve using ForteBio software, and the resulting data are shown in Table 1. [Table 3]

[0083] [Example 3] 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 either bromoacetyl-activated or epoxy-activated Praesto® Jet A50 beads (Purolite, King of Prussia, PA) or ABT700 (Agarose Bead Technologies, Madrid, Spain) agarose beads.

[0084] Bromoacetyl-activated beads 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 ligand was conjugated to the beads at room temperature. Epoxy-activated beads were obtained from vendors or prepared using standard methods known in the art. Ligands were coupled to the epoxy-activated beads at 37-40°C. The density of the target ligands varied from 5 to 20 g / L. After washing, the beads were deactivated with excess thioglycerol. The actual ligand density of all resins was determined using a subtractive RP-HPLC method according to the following equation: Actual Ligand Density = (Measured in Feed [Ligand] - Measured in Eluate [Ligand]).

[0085] [Example 4] This example demonstrates the binding properties of affinity chromatography resins prepared from the affinity ligands described herein. Monomeric and multimeric ligands, including monomers containing multimers with different domains, were conjugated to bromoacetyl-activated resin and the static binding capacity was determined according to the following process. [Table 4] [Table 5]

[0086] [Example 5] This example demonstrates the repeated use of affinity agents containing binding ligands described herein for the affinity purification of albumin fusion proteins and the stability of the resin against NaOH. Clarified cell culture feedstream (CCCF) from a CHO cell line was spiked with albumin fusion protein at a titer of 1.0 g / L. Resin prepared from the ligand corresponding to SEQ ID NO:21 was packed into a 0.3 x 10 cm (0.707 mL) column. The column was operated for five cycles, each cycle comprising the steps shown in the table below. [Table 6]

[0087] Overlaid chromatograms from the first and fifth cycles are shown in Figure 3. The residual HCP content of the purified protein was measured over all five cycles using the Cygnus™ CHO Host Cell Protein Third Generation Assay. The results, shown in Figure 4, indicate that high purity was achieved and maintained over all cycles. Consistent yields were obtained over all five cycles and are shown in Figure 5. In summary, the resin performed well after 30 hours of exposure to 0.5 M NaOH, similar to the first cycle.

[0088] [Example 6] This example demonstrates that the pH of the binding solution can be manipulated to increase binding capacity. Resins prepared from the ligand corresponding to SEQ ID NO: 21 were used in static binding (filter plate) experiments. As shown in Figure 6, a higher binding capacity was achieved at pH 5 compared to higher pHs.

[0089] [Example 7] This example demonstrates that affinity resins with high binding capacities can be achieved with the ligands of the present invention. Affinity resins were prepared with the ligand corresponding to SEQ ID NO: 29 conjugated to either bromoacetyl-activated or epoxy-activated Jet A50 agarose beads, packed into 3 x 50 mm columns, and the dynamic binding capacity (DBC) of the resins was determined using HSA as the test substrate. [Table 7]

[0090] [Example 8] This example demonstrates the effect of elution pH on a specific albumin fusion protein. The elution solution contained 0.5 M proline and 20% hexanediol at pH 9, 8.25, and 7.4. For pH 9, 50 mM glycine was included as the buffer, for pH 8.25, 50 mM HEPES, and for pH 7.4, phosphate was included. As shown in Figure 7, the sharpest peak and highest yield were obtained at pH 9.

[0091] [Example 9] This example demonstrates that a resin can be used to selectively remove albumin from a protein preparation. The albumin content of Fraction V paste from the Cohn process exceeds 95% of the total protein, with small amounts of other serum proteins. Fraction V paste (Seraplex, Pasadena, CA) was resolubilized in 10 mM acetate buffer, pH 4.7, then adjusted to pH 7.4 with 1 M Tris and filtered. The solution was applied to a 100 mL column containing resin prepared with a ligand corresponding to SEQ ID NO: 29 conjugated to epoxy-activated Jet A50 agarose beads. The equivalent of approximately 10 g of paste was loaded onto the column and chased with PBS. The flow-through was collected and analyzed by SDS-PAGE. As shown in Figure 8, the column effectively removed albumin, yielding an albumin-free protein mixture. The lanes in Figure 8 were loaded as follows: [Table 8]

[0092] [Example 10] This example shows that the linker influences the binding capacity of the resin. Five ligands were conjugated to epoxy-activated A50 beads at the ligand densities shown in the following examples. The resulting resins were packed into 0.3 x 5 cm columns, and DBC was determined using 1 mg / mL HSA at a 7-minute retention time. The breakthrough curves shown in Figure 9 and the table below indicate that the resins prepared from SEQ ID NOs: 34, 36, and 37 had the highest binding capacity. [Table 9] [Table 10-1] [Table 10-2] [Table 10-3]

Claims

1. an affinity agent, comprising a ligand comprising the sequence LREAKERAIEEELRRAGISSDYYFDLIQKAKTVEGVQALKDEILKA of SEQ ID NO: 15, 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; The ligand is the K Ｄ The maximum value is 1 x 10 －９ An affinity agent that binds to albumin so that M.

2. 10. The affinity agent of claim 1, which binds to serum albumin and / or one or more serum albumin fusion proteins.

3. 3. The affinity agent of claim 1 or claim 2, which binds to human serum albumin and / or one or more human albumin fusion proteins.

4. 1. An affinity agent comprising a ligand that binds to human serum albumin and / or a human albumin fusion protein comprising at least one amino acid sequence of any one of SEQ ID NOs:21-37, or a sequence that differs by no more than three, no more than two, or no more than one substitution, addition, or deletion.

5. a multimeric polypeptide, said multimeric polypeptide comprising at least two subunits; An affinity agent according to any one of claims 1 to 4, wherein each subunit comprises or consists of a polypeptide (e.g. a ligand) according to any one of claims 1 to 4.

6. The affinity agent of claim 5 , wherein the subunits are not all the same.

7. The affinity agent of any one of claims 1 to 6, wherein the ligand is attached to a solid surface.

8. The affinity agent of claim 7 , wherein the solid surface is a resin or a bead.

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

10. The affinity agent of claim 7 , wherein the solid surface is a monolith.

11. The affinity agent according to any one of claims 7 to 10, wherein the ligand is conjugated to the solid surface via a linker.

12. The affinity agent of any one of claims 1 to 11, for use in the purification of human serum albumin or a human albumin fusion protein.

13. A method of making an affinity agent, comprising conjugating a ligand according to any one of claims 1 to 11 to a solid surface.

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