Affinity agent

Abstract

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

Description

Background Art

[0001] Authorities conduct strict scrutiny and regulation of the purity of biogenically produced therapeutic agents to ensure safety and efficacy. Accordingly, there remains a need for means to efficiently purify biogenically produced therapeutic agents to high purity. Summary of the Invention

[0002] To support the clinical efforts of therapeutic proteins, compositions and methods for efficiently purifying proteins from recombinant sources are needed. Affinity purification is a means to separate and / or achieve the desired purity of a protein in several steps or a single step. However, the development of affinity agents (e.g., those containing an affinity ligand) can be a resource-intensive and time-consuming task. This has led to the development of affinity agents for a limited number of proteins. In the absence of an affinity agent, purification typically involves inefficient, labor-intensive, and expensive processes such as multi-column processes.

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

[0004] Described herein are affinity agents that bind to a protein and can be used for separation 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 area of the biopharmaceutical industry with very promising clinical applications in cancer therapy. Traditional methods for recombinant antibody purification utilize protein A ligand-based affinity chromatography resins. Protein A-based affinity resins provide an efficient, high-yield process for monoclonal antibody purification. The use of protein A affinity resins in industrial antibody production 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 by recombinant protein expression. This therapeutic protein is typically used and produced at a high rate annually (e.g., tons / year). It has also been found that fusions of therapeutic polypeptides or proteins with HSA can be used to extend the serum half-life of the therapeutic polypeptide or protein. In fact, some HSA fusion products have even received FDA approval (e.g., and (Albiglutide), and more products are in clinical development. Due to the therapeutic relevance of HSA and its fusion products, there is a need for an affinity purification solution for HSA and HSA fusion products (e.g., HSA fused with polypeptides and / or proteins) that has a high binding capacity and can be used more than once, for example, after washing with sodium hydroxide under alkaline conditions. Work on developing affinity purification methods for HSA has been based on the natural molecule protein G. Protein G affinity resins have proven to be limited in their low binding capacity and instability when cleaned 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). Therefore, no commercial product of protein G affinity resin has been launched.

[0007] Protein G is derived from Streptococcus sp. G148 protein G and contains three albumin-binding domains (ABD) that comprise a three-helix bundle (3HB). Each wild-type ABD has 46 amino acids (Kraulis et al. 1996), flanked by linkers to the rest of the protein. The binding of protein G to albumin is mediated by contact points in helices 2 and 3 defined by a known core-binding sequence of 25 amino acids (see, for example, U.S. Patent No. 8,937,153). However, the stability of the 3HB protein is affected by all three helices, and thus helix 1 and the flanking sequences are an integral part of the final molecule.

[0008] The binding affinity of the ABD has been improved to the femtomolar concentration, as demonstrated 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 patents partly describe increasing the circulating half-life of the protein by fusing a protein containing the ABD with a small protein drug. Notably, the '887 and '200 patents establish non-natural ABDs with sequences significantly different from those of the earlier U.S. Patent No. 8,937,153. U.S. Patent No. 9,156,887 teaches a protein comprising SEQ ID NO: 1-14 or an isolated albumin-binding domain comprising the amino acid sequence of SEQ ID NO: 1, which amino acid sequence has substitutions at 1, 2, 3, 4, 5, or 6 residues. Additionally, the substitutions are only allowed at certain positions, indicated by X in SEQ ID NO: 2–4. The optimal ligand has an affinity for human albumin (K D ) of 75 pM, k on of 4.04E×10 5 M -1 s -1 and k off of 3.02×10 -5 s -1 .

[0009] Surprisingly, the inventors have found that 7 substitutions can be made to SEQ ID NO: 1, and although the affinity is reduced to below 1 / 40 to 3 nM, the resulting ligand provides an effective affinity purification tool. Additionally, these substitutions can be made at positions outside those taught by U.S. Patent No. 9,156,887.

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

[0011]

[0012] U.S. Patent No. 8,937,153 teaches an engineered albumin-binding polypeptide comprising an albumin-binding motif, wherein the motif consists of the following amino acid sequence: GVSDX5YKX8X9IX11X12AX14TVEGVX 20 ALX23X 24 X25 I (SEQ ID NO:16), wherein, independently of one another,

[0013] X5 is selected from Y and F;

[0014] X8 is selected from N, R, and S;

[0015] X9 is selected from V, I, L, M, F, and Y;

[0016] X 11 is selected from N, S, E, and D;

[0017] X 12 is selected from R, K, and N;

[0018] X 14 is selected from K and R;

[0019] X 20 is selected from D, N, Q, E, H, S, R, and K;

[0020] X 23 is selected from K, I, and T;

[0021] X 24 is selected from A, S, T, G, H, L, and D; and

[0022] X 25 is selected from H, E, and D;

[0023] provided that the amino acid sequence is not GVSDYYKNLI NNAKTVEGVK ALIDEI; or (US8937153, SEQ ID NO:517) GVSDYYKNLI NNAKTVEGVN ALKAEI (U.S. Patent No. 8,937,153, SEQ ID NO:541), and the albumin-binding polypeptide binds to albumin such that the interaction KD value is at most 1×10 -9 M.

[0024] U.S. Patent No. 10,155,792 teaches an albumin-binding polypeptide comprising an albumin-binding motif (BM), wherein the motif consists of the following amino acid sequence: (SEQ ID NO:19) GVSDFYKKLI XaKAKTVEGVEALKXbXcI, wherein, independently of one another,

[0025] Xa is selected from D and E;

[0026] Xb is selected from D and E; and

[0027] Xc is selected from A and E,

[0028] Wherein, compared with the motif of SEQ ID NO:19, the albumin-binding motif has enhanced resistance to cleavage by clostripain, wherein position 8 is 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). The comparison between SEQ ID NO:20 and SEQ ID NOs:1-14 (i.e., the sequences taught in U.S. Patent No. 8,937,153 and U.S. Patent No. 10,155,792) is shown in the following table, and the differences present in SEQ ID NO:20 are highlighted.

[0029]

[0030] Affinity agents that bind to HSA and / or HSA fusion products useful for isolation and / or affinity purification are described herein. In some embodiments, the affinity agent comprises a solid support and a ligand.

[0031] In some embodiments, provided herein is an affinity agent comprising a multimeric polypeptide, wherein the multimeric polypeptide comprises at least two subunits; wherein each subunit comprises a polypeptide according to the foregoing embodiments.

[0032] In some embodiments, provided herein is an affinity agent comprising a multimeric polypeptide, wherein not all of the subunits are identical.

[0033] In some embodiments, provided herein is an affinity agent for purifying HSA and HSA fusion products.

[0034] Definitions

[0035] To facilitate understanding of the present disclosure, certain terms are defined below. Unless otherwise defined, technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art.

[0036] About or approximately: As used herein, when applied to one or more values of interest, the terms "about" or "approximately" refer to a value similar to the reference value. In certain embodiments, the terms "about" or "approximately" refer to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the specified reference value, unless otherwise stated or otherwise apparent from the context (unless such numbers exceed 100% of the possible value).

[0037] Bioactivity: As used herein, the term "bioactive" refers to the property of any agent that is active in a biological system and, in particular, in an organism. For example, an agent that has a biological effect on an organism when administered to the organism is considered to be bioactive.

[0038] Conservative and non-conservative substitutions: A "conservative" amino acid substitution is a substitution in which an amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine (K), arginine (R), histidine (H)); acidic side chains (e.g., aspartic acid (D), glutamic acid (E)); uncharged polar side chains (e.g., glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine (C)); nonpolar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W)); β-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)); and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)). For example, the substitution of tyrosine with phenylalanine is a conservative substitution. In some embodiments, conservative amino acid substitutions in a ligand sequence confer or improve specific binding to a target of interest of the ligand. In some embodiments, conservative amino acid substitutions in a ligand sequence do not reduce or eliminate binding of the ligand to a target of interest. In some embodiments, conservative amino acid substitutions do not significantly affect specific binding of the ligand to a target of interest. Methods for identifying nucleotide and amino acid conservative and non-conservative substitutions that confer, alter, or maintain selective binding affinity are known in the art (see, e.g., Brummell, Biochem. 32:1180-1187 (1993); Kobayashi, Protein Eng. 12(10):879-884 (1999); and Burks, PNAS 94:412-417 (1997)). In some embodiments, non-conservative amino acid substitutions in a ligand sequence confer or improve specific binding to a target of interest of the ligand. In some embodiments, non-conservative amino acid substitutions in a ligand sequence do not reduce or eliminate binding of the ligand to a target of interest. In some embodiments, non-conservative amino acid substitutions do not significantly affect specific binding of the ligand to a target of interest.

[0039] Linker: As used herein, a "linker" refers to a peptide or other chemical linkage used to connect other independent functional domains. In some embodiments, the linker is located between the ligand and another polypeptide component containing other independent functional domains. In some embodiments, the linker is a peptide or other chemical linkage between the ligand and the surface.

[0040] Naturally occurring: When used in connection with biological materials such as nucleic acid molecules, polypeptides, and host cells, the term "naturally occurring" refers to those materials that exist in nature and have not been modified by man. In contrast, when used in connection with biological materials, "non-natural" or "synthetic" refers to those materials that do not exist in nature and / or have been modified by humans.

[0041] "Non-natural amino acid", "amino acid analogue", and "non-standard amino acid residue" are used interchangeably herein. Non-natural amino acids that can be substituted in the ligands provided herein are known in the art. In some embodiments, the non-natural amino acid is 4-hydroxyproline which can substitute for proline; 5-hydroxylysine which can substitute for lysine; 3-methylhistidine which can substitute for histidine; homoserine which can substitute for serine; and ornithine which can substitute for lysine. Additional examples of non-natural amino acids that can be substituted in polypeptide ligands include, but are not limited to, molecules such as: D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, A-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, lanthionine, dehydroalanine, γ-aminobutyric acid, selenocysteine, and pyrrolysine fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids.

[0042] "Polynucleotide" and "nucleic acid molecule": As used interchangeably herein, polynucleotide and nucleic acid molecule refer to polymeric forms of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. These terms include, but are not limited to, DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and / or tRNA.

[0043] Operably linked: As used herein, the term "operably linked" indicates the joining of two molecules such that each retains its functional activity. Whether the two molecules are joined directly or indirectly, they are "operably linked".

[0044] Peptide tag: As used herein, the term "peptide tag" refers to a peptide sequence that is part of or attached to another protein, thereby providing functionality to the resulting fusion. A peptide tag is typically relatively short compared to the protein to which it is fused. In some embodiments, the peptide tag is four or more amino acids in length, such as 5, 6, 7, 8, 9, 10, 15, 20, 25 or more amino acids. In some embodiments, the ligand is a protein that contains a peptide tag. Many peptide tags having the uses provided herein are known in the art. Examples of peptide tags that can be components of ligand fusion proteins or targets that bind to ligands (e.g., ligand fusion proteins) include, but are not limited to, HA (hemagglutinin), c-myc, herpes simplex virus glycoprotein D (gD), T7, GST, GFP, MBP, Strep tag, His tag, Myc tag, TAP tag, and FLAG tag (Eastman Kodak, Rochester, N.Y.). Similarly, antibodies directed against the tag epitope permit detection and localization of the fusion protein in, for example, affinity purification, Western blotting, ELISA assays, and immunostaining of cells.

[0045] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids joined by peptide bonds. The term is used to refer to amino acid chains of any length, but one of ordinary skill in the art will understand that the term is not limited to long chains and can refer to the smallest chain containing two amino acids joined by a peptide bond. As is known to those of skill in the art, polypeptides can be processed and / or modified.

[0046] Protein: As used herein, the term "protein" refers to one or more polypeptides that function as discrete units. The terms "polypeptide" and "protein" can be used interchangeably if a single polypeptide is a discrete functional unit and does not require permanent or transient physical association with other polypeptides to form a discrete functional unit. If the discrete functional unit consists of more than one polypeptide physically associated with each other, the term "protein" refers to the multiple polypeptides that are physically coupled and function together as a discrete unit.

[0047] Specific binding: As used herein with respect to ligands, the term "specifically binds" or "has selective affinity" means that a ligand reacts or associates more frequently, more rapidly, for a longer duration, with greater affinity, or in any combination thereof, with a particular epitope, protein, or target molecule as compared to alternative substances including unrelated proteins. Due to sequence identity between homologous proteins in different species, specific binding can include binders that recognize proteins or targets in more than one species. Similarly, due to homology within certain regions of the polypeptide sequences of different proteins, specific binding can include binders that recognize more than one protein or target. It should be understood that in certain embodiments, a binder that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require (although it can include) exclusive binding (i.e., binding to a single target). Thus, in certain embodiments, a ligand or an affinity agent can specifically bind to more than one target. In certain embodiments, multiple targets can bind to the same antigen-binding site on an affinity agent.

[0048] Substantially: As used herein, the term "substantially" refers to a qualitative condition that exhibits all or nearly all of the range or degree of the characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or continue to completion or achieve or avoid absolute results. Accordingly, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0049] 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, such as by genetic engineering and expression of a fusion gene or chemical ligation methods known in the art (e.g., native chemical ligation, expressed protein ligation, Staudinger ligation, Ser / Thr ligation, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1A Shows exemplary crystal structures of some albumin ligands contemplated herein. The binding interface of the albumin ligand is highlighted by a lighter shading.

[0051] Figure 1B Shows exemplary crystal structures of some albumin ligands contemplated herein. The positions at which SEQ ID NO:1 was mutated to produce SEQ ID NO:15 are highlighted by a lighter shading.

[0052] Figure 2Shows the sensorgram of the biotinylated ligand corresponding to SEQ ID NO:21 excited by titration with an HSA-containing solution.

[0053] Figure 3 Shows the superimposed chromatograms before and after repeated cycling of the affinity resin of the present invention.

[0054] Figure 4 Shows the residual HCP levels across 5 purification cycles of the affinity resin of the present invention. Between each cycle, the resin was exposed to 0.5 M NaOH for 6 hours.

[0055] Figure 5 Shows the yields obtained across 5 purification cycles of the affinity resin of the present invention. Between each cycle, the resin was exposed to 0.5 M NaOH for 6 hours.

[0056] Figure 6 Shows the variation of the static binding capacity of the affinity resin of the present invention with pH.

[0057] Figure 7 Shows the effect of elution pH on a specific albumin fusion protein. Overlaid chromatograms of the elution peak (at approximately 26 mL) and the CIP peak (at approximately 36 mL) are shown: elution pH 9 (dark, solid line), pH 8.25 (dashed line), and pH 7.4 (grey line)

[0058] Figure 8 Shows the SDS-PAGE analysis for the removal of albumin from the fraction V paste to isolate minor components. The table below lists the samples loaded onto each lane.

[0059] Figure 9 Shows the breakthrough curves of the resins prepared from the ligands corresponding to SEQ ID No: 29 and 34 - 37 when excited with HSA at 1 mg / mL and a residence time of 7 minutes. Detailed Description

[0060] The present disclosure particularly encompasses the recognition that affinity agents prepared from identified and characterized ligands have been shown to produce highly purified formulations 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 can be particularly useful for removing protein product-related impurities as well as contaminants derived from the host cell.

[0061] The ligand that binds to the target of interest is used in the affinity agent

[0062] The characteristics of the ligand that binds to the target can be determined using known or modified assays, bioassays, and / or animal models known in the art to evaluate such activities.

[0063] As used herein, terms such as "binding affinity for a target", "binding to a target", etc. refer to properties of a ligand that can be directly measured, for example, by determining an affinity constant (e.g., the amount of ligand associating and dissociating at a given antigen concentration). Several methods can be used to characterize such molecular interactions, such as competitive analysis, equilibrium analysis, and microcalorimetry, as well as real-time interaction analysis based on surface plasmon resonance interactions (e.g., using a BIACORE instrument). These methods are well known to those skilled in the art and are discussed in publications such as Neri D et al. (1996) Trends in Biotechnology (Tibtech) 14:465-470 and Jansson M et al. (1997) Journal of Biological Chemistry (J Biol Chem) 272:8189-8197.

[0064] The affinity requirements for a given ligand binding event depend on a variety of factors, including but not limited to: the composition and complexity of the binding matrix, the valency and density of both the ligand and the target molecule, and the functional application of the ligand. In some embodiments, the ligand binds to the target of interest with a dissociation constant (K -3 M, 10 -3 M, 5×10 -4 M, 10 -4 M, 5×10 -5 M, or 10 -5 ) less than or equal to 5×10 D . In some embodiments, the ligand binds to the target of interest with a K -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5×10 -8 M, or 10 -8 less than or equal to 5×10 D . In some embodiments, the ligand binds to the target of interest with a K -9 greater than or equal to 1×10 D . In some embodiments, the ligands generated by the methods disclosed herein have a dissociation constant of from about 10 -4 M to about 10 -5 M, from about 10 -5 M to about 10 -6 M, from about 10 -6 M to about 10 -7 M, from about 10 -7 M to about 10 -8 M, from about 10 -8 M to about 10 -9 M.

[0065] Determining KD The association experiment with the dissociation rate (k off ) can be carried out under many conditions. The buffer for preparing these solutions can be easily determined by those skilled in the art and largely depends on the desired pH of the final solution. Low pH solutions (<pH 5.5) can be prepared, for example, in citrate buffer, glycine-HCl buffer or succinate buffer. High pH solutions can be prepared, for example, in Tris-HCl, phosphate buffer or sodium bicarbonate buffer. Many conditions can be used to determine K D and the dissociation rate to determine, for example, the optimal pH and / or salt concentration.

[0066] In some embodiments, the ligand binds specifically to the target of interest with a k -7 in the range of 0.1 to 10 -1 seconds -2 to 10 -7 seconds -1 or 0.5x10 -2 to 10 -7 seconds -1 . In some embodiments, the ligand binds to the target of interest with a k off less than 5x10 -2 seconds -1 , 10 -2 seconds -1 , 5x10 -3 seconds -1 or 10 -3 seconds -1 . In some embodiments, the ligand binds to the target of interest with a k off less than 5x10 -4 seconds -1 , 10 -4 seconds -1 , 5x10 -5 seconds -1 or 10 -5 seconds -1 , 5x10 -6 seconds -1 , 10 -6 seconds -1 , 5x10 -7 seconds -1 or 10 -7 seconds -1 . In some embodiments, the ligand binds to the target of interest with a k off in the range of about 10 3 to 10 7 M -1 seconds -1 , 10 3 to 10 6 M -1 seconds-1 or 10 3 to 10 5 M -1 seconds -1 within the association rate (k on ) binds specifically to the target of interest. In some embodiments, the ligand (e.g., ligand fusion protein) has an association rate greater than 10 3 M -1 seconds -1 , 5x10 3 M -1 seconds -1 , 10 4 M -1 seconds -1 or 5x10 4 M -1 seconds -1 of k on binds to the target of interest. In a further embodiment, the ligand has an association rate greater than 10 5 M -1 seconds -1 , 5x10 5 M -1 seconds -1 , 10 6 M -1 seconds -1 , 5x10 6 M -1 seconds -1 or 10 7 M -1 seconds -1 of k on binds to the target of interest.

[0067] The target of interest

[0068] According to each embodiment, the target of interest that specifically binds to the ligand can be any molecule to which the ligand of the desired affinity agent binds. For example, the target that specifically binds to the ligand can be any target related to purification, manufacturing, formulation, treatment, diagnosis, or prognosis or its value. Non-limiting uses include therapeutic and diagnostic uses. Many exemplary targets are provided herein by way of example, and the exemplary targets are intended to be illustrative rather than limiting. The target of interest can be naturally occurring or synthetic. In some embodiments, the target comprises human serum albumin (HSA) protein or a fragment thereof or an HSA fusion protein.

[0069] Linker

[0070] The terms "linker" and "spacer" are used interchangeably herein to refer to a peptide or other chemical linkage used to connect other independent functional domains. In some embodiments, the linker is located between the ligand and another polypeptide component containing other independent functional domains. Suitable linkers for coupling two or more linked ligands can generally be any linker used in the art for linking peptides, proteins, or other organic molecules. In some embodiments, such linkers are suitable for constructing proteins or polypeptides intended for pharmaceutical use.

[0071] Suitable linkers for operably linking a ligand and additional components of a ligand fusion protein in a single-chain amino acid sequence include, but are not limited to, polypeptide linkers such as glycine linkers, serine linkers, mixed glycine / serine linkers, glycine- and serine-rich linkers, or linkers composed of mostly polar polypeptide segments.

[0072] In some embodiments, the linker comprises mostly amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In some embodiments, the linker comprises mostly amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In some embodiments, the ligand linker is composed of mostly sterically unhindered amino acids. In some embodiments, the linker comprises mostly amino acids selected from glycine, serine, and / or alanine. In some embodiments, the peptide linker is selected from polyglycine (such as (Gly)5 and (Gly)8, poly(Gly-Ala), and polyalanine).

[0073] The linker can have any size or composition, so long as it can operably link the ligand in a manner that permits binding of the ligand to the target of interest. In some embodiments, the linker is about 1 to 50 amino acids, about 1 to 20 amino acids, about 1 to 15 amino acids, about 1 to 10 amino acids, about 1 to 5 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to 10 amino acids, or about 2 to 5 amino acids. It should be appreciated that the length, degree of flexibility, and / or other characteristics of the linker can affect certain properties of the ligand for the affinity agent, such as the affinity, specificity, or avidity for the target of interest, or for one or more other target proteins of interest, or for non-target proteins (i.e., non-target proteins). In some embodiments, two or more linkers are utilized. In some embodiments, the two or more linkers are the same. In some embodiments, the two or more linkers are different.

[0074] In some embodiments, the linker is a non-peptide linker, such as an alkyl linker or a PEG linker. For example, an alkyl linker such as -NH-(CH2) s-C(0)-, where s = 2 - 20. These alkyl linkers can be further substituted by any sterically unhindered group such as lower alkyl (e.g., C1 - C6), lower acyl, halogen (e.g., Cl, I, Br, F), CN, NH2, phenyl, etc. An exemplary non - peptide linker is a PEG linker. In some embodiments, the PEG linker has a molecular weight of about 100 to 5000 kDa, or about 100 to 500 kDa. In some embodiments, the PEG linker has a molecular weight of about 100 to 500 kDa.

[0075] The linker can be evaluated using the techniques described herein and / or otherwise known in the art. In some embodiments, the linker does not alter (e.g., does not disrupt) the ability of the ligand to bind to the target molecule.

[0076] An affinity agent comprising a conjugated ligand

[0077] A ligand that promotes specific binding to a target of interest can be chemically conjugated to a variety of chromatographic compositions (e.g., beads, resins, gels, membranes, monoliths, etc.) to prepare an affinity agent. The affinity agent containing the ligand is particularly useful in purification and manufacturing applications.

[0078] In some embodiments, the ligand (e.g., ligand fusion protein) contains at least one reactive residue. The reactive residue can be used, for example, as a linking site for conjugates such as chemotherapeutic drugs. Exemplary reactive amino acid residues are lysine. The reactive residue (e.g., lysine) can be added to the ligand at either end or within the ligand sequence, and / or can replace another amino acid in the ligand sequence. Suitable reactive residues (e.g., lysine, serine, tyrosine, hydroxytryptophan, etc.) can also be located within the sequence of the identified ligand without the need for addition or replacement. In some embodiments, another exemplary reactive amino acid residue is cysteine. In some embodiments, the reactive amino acid residue is lysine. In some embodiments, the reactive amino acid is serine. In some embodiments, the reactive amino acid residue is tyrosine. In some embodiments, the reactive amino acid residue is hydroxytryptophan.

[0079] Attached to a solid surface

[0080] "Solid surface", "carrier", or "matrix" are used interchangeably herein and refer to, but are not limited to, any column (or column material), bead, test tube, microtiter plate, solid particle (e.g., agarose or agarose gel), microchip (e.g., silicon, silicon-glass, or gold chip), or membrane (synthetic in origin (e.g., filter membrane) or biological (e.g., liposome or vesicle)) to which a ligand, affinity agent, antibody, or other protein can be directly or indirectly (e.g., through other binding partner intermediates such as other antibodies or protein A) linked (coupled, attached, or adhered), or into which a ligand or antibody can be incorporated (e.g., through a receptor or channel). Reagents and techniques for linking polypeptides to solid carriers (e.g., matrices, resins, plastics, etc.) are well known in the art. Suitable solid carriers include, but are not limited to, chromatographic resins or matrices (e.g., SEPHAROSE-4FF agarose beads), the walls or bottom plates of wells in plastic microtiter plates, silica-based biochips, polyacrylamide, agarose, silica, nitrocellulose, paper, plastics, nylon, metals, and combinations thereof. Ligands and other compositions can be linked to the carrier material by non-covalent association or by covalent bonding using reagents and techniques known in the art. In some embodiments, the ligand is coupled to the chromatographic material using a linker.

[0081] Generation of Ligands

[0082] The generation of ligands for several embodiments that can be used to practice the provided methods can be carried out using various standard techniques for chemical synthesis, semi-synthetic methods, and recombinant DNA methods known in the art. Also provided are methods for generating ligands as soluble agents and cell-associated proteins, either individually or as part of a multi-domain fusion protein. In some embodiments, the overall ligand generation scheme involves obtaining a reference protein scaffold and identifying multiple residues within the scaffold for modification. According to an embodiment, the reference scaffold can comprise a protein structure or other tertiary structure having one or more α-helical regions. Once identified, any of the multiple residues can be modified, for example, by substitution of one or more amino acids. In some embodiments, one or more conservative substitutions are made. In some embodiments, one or more non-conservative substitutions are made. In some embodiments, a natural amino acid (e.g., one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine) is substituted into the reference scaffold at the targeted position for modification. In some embodiments, the modification does not include substitution of cysteine or proline. In a particular embodiment, after modification at the desired identified position, the resulting modified polypeptide (e.g., a candidate ligand) can be recombinantly expressed, for example, in a plasmid, bacterium, phage, or other vector (e.g., to increase the number of each of the modified polypeptides). The modified polypeptides can then be purified and screened to identify those modified polypeptides that specifically bind to a particular target of interest. Compared to the reference scaffold, the modified polypeptides can exhibit enhanced binding specificity for the target of interest, or can exhibit little or no binding to a given target of interest (or non-target protein). In some embodiments, depending on the target of interest, the reference scaffold can exhibit some interaction with the target of interest (e.g., non-specific interaction), while certain modified polypeptides will exhibit at least about two-fold, at least about five-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or at least about 100-fold (or more) increased binding specificity for the target of interest. Additional details regarding the generation, selection, and isolation of ligands are provided in more detail below.

[0083] Recombinant Expression of Ligands

[0084] In some embodiments, ligands such as ligand fusion proteins are "recombinantly produced" (i.e., are produced using recombinant DNA techniques). Exemplary recombinant methods for synthesizing ligand fusion proteins include, but are not limited to, polymerase chain reaction (PCR)-based synthesis, circularization, 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)).

[0085] Also provided are nucleic acids comprising a polynucleotide sequence encoding a ligand. Such polynucleotides optionally further comprise one or more expression control elements. For example, the polynucleotide may comprise one or more promoters or transcriptional enhancers, ribosome binding sites, transcriptional termination signals, and polyadenylation signals, as expression control elements. The polynucleotide may be inserted into any suitable vector, and the vector may be contained within any suitable host cell for expression.

[0086] Expression of the nucleic acid encoding the ligand is generally achieved by operably linking the nucleic acid encoding the ligand to a promoter in an expression vector. Typical expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Exemplary promoters for expression in Escherichia coli (E. coli) include, for example, the T7 promoter.

[0087] Expression vectors containing a nucleic acid sequence encoding a ligand and 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.

[0088] A variety of host expression vector systems can be used 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 can include plasmid vectors, single-stranded phage vectors, double-stranded phage vectors, single-stranded RNA or DNA viral vectors, or double-stranded RNA or DNA viral vectors. Phage and viral vectors can also be introduced into host cells in the form of encapsulated or enveloped viruses using known infection and transduction techniques. Additionally, viral vectors can be replication-competent or, alternatively, replication-defective. Alternatively, cell-free translation systems can also 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. Patent No. 5,122,464).

[0089] 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. A variety of host expression vector systems can be used to express the coding sequence of a ligand fusion protein. Mammalian cells can be used as a host cell system transfected with recombinant plasmid DNA or a cosmid DNA expression vector containing the coding sequence of the target of interest and the coding sequence of the fusion polypeptide. The cells can be primary isolates from organisms, cultures, or cell lines having a transformed or transgenic nature.

[0090] Suitable host cells include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli, Bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, 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 systems infected with recombinant viral expression vectors (e.g., Baculovirus); plant cell systems infected with recombinant viral expression vectors (e.g., Cauliflower mosaic virus, CaMV; Tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing the ligand coding sequence (e.g., Ti plasmid).

[0091] Prokaryotes that can be used as host cells for generating ligands can include Gram-negative or Gram-positive organisms such as Escherichia coli and Bacillus subtilis. Expression vectors for prokaryotic host cells typically contain one or more phenotypically selectable marker genes (e.g., genes encoding proteins that confer antibiotic resistance or provide autotrophic requirements). Examples of useful prokaryotic host expression vectors include pKK223-3 (Pharmacia, Uppsala, Sweden), pGEMl (Promega, Wis., USA), pET (Novagen, Wis., USA), and pRSET (Invitrogen, Calif., USA) series vectors (see, for example, Studier, J. Mol. Biol. 219:37 (1991) and Schoepfer, Gene 124:83 (1993)). Exemplary promoter sequences commonly used for prokaryotic host cell expression vectors include T7 (Rosenberg et al., Gene 56:125-135 (1987)), β-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615 (1978); and Goeddel et al., Nature 281:544 (1979)), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, (1980)), and tac promoter (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

[0092] In some embodiments, a eukaryotic host cell system is used. In some embodiments, the eukaryotic host cell system is a yeast cell transformed with a recombinant yeast expression vector containing the coding sequence of a ligand. Exemplary yeasts that can be used to produce the compositions of the present invention include yeasts from the genus Saccharomyces, Pichia, Actinomycetes, and Kluyveromyces. Yeast vectors typically contain an origin of replication sequence from the 2μ yeast plasmid, an autonomous replication sequence (ARS), a promoter region, a sequence for polyadenylation, a sequence for transcription termination, and a selectable marker gene. Examples of promoter sequences in yeast expression constructs include promoters from metallothionein, 3-phosphoglycerate kinase (Hitzeman, J. Biol. Chem. 255:2073 (1980)), and other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase. Additional suitable vectors and promoters for yeast expression as well as yeast transformation protocols are known in the art. See, for example, Fleer, Gene 107:285-195 (1991) and Hinnen, Proc. Natl. Acad. Sci. USA 75:1929 (1978).

[0093] Insect and plant host cell culture systems can also be used to produce the compositions of the present invention. Such host cell systems include, for example, insect cell systems infected with a recombinant viral expression vector (e.g., baculovirus) containing the 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) containing the ligand coding sequence, including but not limited to the expression systems taught in U.S. Patent No. 6,815,184; U.S. Publications 60 / 365,769 and 60 / 368,047; and WO2004 / 057002, WO2004 / 024927, and WO2003 / 078614.

[0094] In some embodiments, a host cell system can be used. In some embodiments, the host cell system is an animal cell system infected with a recombinant viral expression vector (e.g., adenovirus, retrovirus, adeno-associated virus, herpesvirus, lentivirus). In some embodiments, the host cell system is a cell line that has been engineered to contain multiple copies of DNA encoding a ligand and that stably amplifies (CHO / dhfr) or unstably amplifies in double minute chromosomes (e.g., murine cell lines). In some embodiments, the vector containing the polynucleotide encoding the ligand is polycistronic. Exemplary mammalian cells that can be used to produce these compositions include HEK293 cells (e.g., 293T and 293F), CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 (Crucell, Netherlands) cells VERY, HeLa cells, COS cells, MDCK cells, 3T3 cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT20 cells, T47D cells, CRL7O30 cells, HsS78Bst cells, hybridoma cells, and other mammalian cells. Additional exemplary mammalian host cells that can be used to practice the invention include, but are not limited to, T cells. Exemplary expression systems and selection methods are known in the art and can include those described in the following references and references cited therein: Borth et al., Biotechnol. Bioen. 71(4):266-73 (2000), Werner et al., Arzneimittelforschung / Drug Res. 48(8):870-80 (1998), Andersen et al., Curr. Op. Biotechnol. 13:117-123 (2002), Chadd et al., Curr. Op. Biotechnol. 12:188-194 (2001), and Giddings, Curr. Op. Biotechnol. 12:450-454 (2001). Additional examples of expression systems and selection methods are described in Logan et al., Proc. Natl. Acad. Sci. USA 81:355-359 (1984); Birtner et al., Methods Enzymol. 153:51-544 (1987). Transcriptional and translational control sequences of mammalian host cell expression vectors generally are derived from viral genomes. Promoter sequences and enhancer sequences commonly used in mammalian expression vectors include sequences derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV).Exemplary commercially available expression vectors for mammalian host cells include pCEP4 (Invitrogen) and pcDNA3 (Invitrogen).

[0095] Physical methods for introducing nucleic acids into host cells (e.g., mammalian host cells) include, but are not limited to, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

[0096] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors and especially retroviral vectors have become the most widely used method for inserting genes into mammalian cells (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0097] Methods for introducing DNA and RNA polynucleotides of interest into host cells include, but are not limited to, cell electroporation, where an electric field is applied to the cells to increase the permeability of the cell membrane, thereby allowing the introduction of chemicals, drugs, or polynucleotides into the cells. Ligands containing DNA or RNA constructs can be introduced into mammalian or prokaryotic cells using electroporation.

[0098] In some embodiments, cell electroporation results in the expression of ligand-CAR on the surface of T cells, NK cells, NKT cells. Such expression can be transient or stable over the lifespan of the cells. Electroporation can be accomplished by methods known in the art, including the MaxCyte and Transfection System (MaxCyte, Gaithersburg, MD, USA).

[0099] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle). In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated to use lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). In some embodiments, the nucleic acid is associated with a lipid. The nucleic acid associated with the lipid can be encapsulated in the aqueous interior of a liposome, dispersed in the lipid bilayer of a liposome, linked to a liposome via a linking molecule that associates with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contain micelles or be complexed with micelles, or otherwise associated with a lipid. The composition associated with the lipid, lipid / DNA, or lipid / expression vector is not limited to any particular structure in solution. For example, the composition can exist as a bilayer structure, micelle, or "folded" structure. The composition can also be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids can be naturally occurring fatty substances or synthetic lipids. For example, lipids include fat droplets naturally occurring in the cytoplasm, as well as classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0100] Suitable lipids 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”) 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 about -20°C. Chloroform can be used as the sole solvent because it evaporates more readily than methanol. “Liposome” is a general term encompassing various single and multi-lamellar lipid vehicles formed by the generation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multi-lamellar liposomes have multiple lipid layers separated by aqueous media. When phospholipids are suspended in an excess of aqueous solution, multi-lamellar liposomes form spontaneously. The lipid components undergo self-rearrangement before forming the closed structure, and water and dissolved solutes are trapped between the lipid bilayers (Ghosh et al., Glycobiology 5:505-510 (1991)). However, compositions having structures different from normal vesicular structures in solution are also encompassed. For example, the lipids may assume a micellar structure or exist only as non-uniform aggregates of lipid molecules. Lipid transfection amine-nucleic acid complexes are also contemplated.

[0101] Regardless of the method used to introduce exogenous nucleic acid into a host cell, the presence of a recombinant nucleic acid sequence in the host cell can be routinely confirmed by various assays known in the art. Such assays include, for example, “molecular biology” assays known in the art, such as Southern and Northern blotting, RT-PCR, and PCR; “biochemical” assays, such as detecting the presence or absence of a specific peptide by immunological means (ELISA and Western blotting) or by the assays described herein, to identify agents falling within the scope of the present invention.

[0102] Reporter genes are used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism, tissue or cell and encodes a polypeptide that manifests its expression by some readily detectable property (e.g., enzymatic activity). Expression of the reporter gene is assayed at an appropriate time after the DNA has been introduced into the recipient cell. Suitable reporter genes include, but are not limited to, genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes (e.g., Ui-Tei et al., FEBS Lett. 479:79-82 (2000)). Suitable expression systems are known in the art and can be prepared using known techniques or obtained commercially. Generally, constructs having a minimal 5' flanking region and showing the highest level of reporter gene expression are identified as promoters. Conventionally, such promoter regions can be ligated to a reporter gene and used to evaluate the ability of an agent to modulate promoter-driven transcription.

[0103] A number of selection systems can be used in mammalian host vector expression systems, including but not limited to the herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes. Additionally, antimetabolite resistance can be used as a basis for selection, for example, in the dhfr, gpt, neo, hygro, trpB, hisD, ODC (ornithine decarboxylase) and glutamine synthetase systems.

[0104] Ligand purification

[0105] Once a ligand or ligand fusion protein has been produced by recombinant expression, the ligand or ligand fusion protein can be purified by methods known in the art for purifying recombinant proteins, such as by chromatography (e.g., ion exchange, affinity and size column chromatography), centrifugation, differential solubilization or by any other standard technique for purifying proteins. In some embodiments, the ligand can optionally be fused to a heterologous polypeptide sequence specifically disclosed herein or known in the art to facilitate purification. In some embodiments, prior to the final preparation of the ligand using techniques known in the art, the ligand (e.g., antibody and other affinity matrices) of the ligand affinity column used for affinity purification and optionally other components of the ligand or the composition that bind to these ligands are removed from the ligand fusion composition.

[0106] Chemical synthesis of ligands

[0107] In addition to recombinant methods, organic chemical synthesis of the desired polypeptide using various liquid and solid phase chemical methods known in the art can be used to generate ligands. A variety of automated synthesizers are commercially available and can be used according to known protocols. See, e.g., Tam et al., J. Am. Chem. Soc., 105:6442 (1983); Merrifield, Science, 232:341-347 (1986); Barany and Merrifield, The Peptides, Gross and Meienhofer eds., Academic Press, New York, 1-284; Barany et al., Int. J. Pep. Protein Res., 30:705739 (1987); Kelley et al., Genetic Engineering Principles and Methods, Setlow, J.K. ed., Plenum Press, NY., 1990, Vol. 12, pp. 1-19; Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, 1989. An advantage of these methods is that they allow incorporation of unnatural amino acid residues into the ligand sequence.

[0108] The ligands used in the methods of the present invention can be modified during or after synthesis or translation, for example, by glycosylation, acetylation, benzylation, phosphorylation, amidation, polyethylene glycolylation, formylation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation with antibody molecules, hydroxylation, iodination, methylation, myristoylation, oxidation, polyethylene glycolylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, ubiquitination, etc. (see, e.g., Creighton, Proteins: Structures and Molecular Properties, 2nd ed. (W.H. Freeman and Co., N.Y., 1992); Postranslational Covalent Modification of Proteins, edited by Johnson (Academic Press, N.Y., 1983), pp. 1-12; Seifter, Meth. Enzymol., 182:626-646 (1990); Rattan, Ann. NY Acad. Sci., 663:48-62 (1992)). In some embodiments, the peptide is acetylated at the N-terminus and / or amidated at the C-terminus.

[0109] Any of a number of chemical modifications can be carried out by known techniques, including but not limited to acetylation, formylation, etc. Additionally, the derivatives can contain one or more non-classical amino acids.

[0110] In some embodiments, cyclization or macrocyclization of the peptide backbone is achieved by formation of side-chain to side-chain linkages. Methods for achieving this are well known in the art and may involve natural and non-natural amino acids. Such methods include disulfide formation, lanthionine formation or thiol alkylation (e.g., Michael addition), amidation between an amino and a carboxylic acid ester side chain, click chemistry (e.g., azide-alkyne condensation), peptide stapling, ring-closing metathesis, and the use of enzymes.

[0111] Affinity agents for purification

[0112] In purification based on affinity chromatography, the target of interest (e.g., a protein or molecule) is selectively separated based on its ability to specifically and reversibly bind to a ligand that can be covalently coupled to a chromatographic matrix. In some embodiments, the ligand can be used as a reagent for affinity purification of the target of interest from recombinant or natural sources such as biological samples (e.g., serum, cells).

[0113] In some embodiments, a ligand that specifically binds to a target of interest is immobilized on beads and then used for affinity purification of the target.

[0114] Methods for covalently coupling proteins to surfaces are known to those skilled in the art. Peptide tags that can be used to link ligands to solid surfaces are known to those skilled in the art. In addition, any reagent or technique known in the art can be used to link (i.e., couple, attach, or adhere) a ligand to a solid surface. In some embodiments, the solid support comprises beads, glass, slides, chips, and / or gelatin. Thus, arrays can be prepared on solid surfaces using a range of ligands with techniques known in the art. For example, U.S. Publication No. 2004 / 0009530 discloses methods for preparing arrays.

[0115] In some embodiments, a ligand is used to separate 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 the solid support using the techniques and reagents described herein or known in the art. Suitable solid supports are described herein or are known in the art and, in specific embodiments, are suitable for packing a chromatography column. The immobilized ligand can be loaded or contacted with a solution under conditions that favor the formation of a complex between the ligand and the target of interest. Unbound material can be washed away. Those skilled in the art can readily determine suitable washing conditions. Examples of suitable washing conditions are described in Shukla and Hinckley, Biotechnol Prog. September - October 2008; 24(5):1115 - 21. doi:10.1002 / btpr.50.

[0116] In some embodiments, chromatography is performed by mixing a solution containing the target of interest and the ligand and then separating the complex of the target of interest and the ligand. For example, the ligand is immobilized on a solid support such as beads and then separated from the solution along with the target of interest by filtration. In some embodiments, the ligand is a fusion protein containing a peptide tag, such as a poly - HIS tail or a streptavidin - binding region, which can be used to separate the ligand after formation of a complex using immobilized metal affinity chromatography resin or a streptavidin - coated substrate. Once separated, the target of interest can be released from the ligand under elution conditions and recovered in purified form.

[0117] In some embodiments, ligands including the initiating N-terminal methionine are separated, as the initiator is a protein sequence encoded by DNA. In some embodiments, ligands without an N-terminal methionine residue are separated. In some embodiments, a mixture is obtained using only a portion of the purified ligand containing an N-terminal methionine. It will be apparent to those skilled in the art that the presence or absence of the N-terminal methionine does not affect the conclusions herein.

[0118] Example

[0119] Example 1

[0120] Recombinant protein ligands are expressed in Escherichia coli and / or Pichia Pastoris using standard techniques. Ligands are purified using multi-column chromatography. For HIS-tagged ligands, IMAC is used as the primary capture step. The purity and identity of the recombinant protein ligands are evaluated by a combination of SDS-PAGE, RP UPLC, quadrupole time-of-flight mass spectrometry, and SEC. Biotinylated ligands are generated by conjugation with Maleimide-PEG2-biotin. In many cases, ligands without an N-terminal methionine residue are separated, which are thought to be cleaved during expression. In many cases, a mixture is obtained using only a portion of the purified ligand containing an N-terminal methionine. 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, the N-terminal methionine is included.

[0121] Example 2

[0122] This example demonstrates the binding of a biotinylated ligand to albumin molecules using biolayer interferometry (ForteBio, Menlo park, CA). The biotinylated ligand corresponding to SEQ ID NO:21 is immobilized on a sensor and incubated with solutions containing various concentrations of albumin protein. Figure 2 An exemplary sensorgram of the binding to HSA is shown. The binding curve is fit to a 1:1 binding model using ForteBio software to determine the affinity, and the resulting data are shown in Table 1.

[0123] Table 1. Affinity data for the binding of albumin and albumin fusion protein (AFP) to the ligand corresponding to SEQ ID NO:21:

[0124] Target Fusion <![CDATA[Affinity (K D )]]> <![CDATA[k on (1 / M·s)]]> <![CDATA[k off (1 / s)]]> HSA - 3nM 2.9E+04 9.7E-05 AFP#1 N-terminus <1 nM 8.0E+04 4.2E-05 AFP#2 C-terminus 2nM 2.4E+04 6.0E-05 AFP#3 C-terminus <1 nM 4.0E+04 1.9E-05 AFP#4 C-terminus 1nM 3.5E+04 5.0E-05 AFP#5 C-terminus <1 nM 5.4E+04 1.5E-05

[0125] Example 3

[0126] This example demonstrates the generation and characterization of an affinity agent comprising the ligands identified and described herein. By conjugating the ligand to bromoacetyl- or epoxy-activated Jetted A50 beads (Purolite, King of Prussia, PA) or ABT700 (Agarose Bead Technologies, Madrid, Spain) agarose beads to prepare the affinity resin.

[0127] The bromoacetyl-activated beads were activated with disuccinimidyl carbonate and coupled with excess ethylenediamine. After washing, the bromoacetate was conjugated to the aminated beads using EDC activation. After washing, the ligand was conjugated to the beads at room temperature. The epoxy-activated beads were obtained from a supplier or prepared using standard methods well known in the art. The ligand was coupled to the epoxy-activated beads at 37 - 40 °C. The targeted ligand density varied between 5 - 20 g / L. After washing, the beads were inactivated with excess thioglycerol. The actual ligand density of all resins was measured using a subtractive RP-HPLC method according to the following formula:

[0128] Actual ligand density = ([ligand] measured in the feed - [ligand] measured in the effluent).

[0129] Example 4

[0130] This example demonstrates the binding characteristics of an affinity chromatography resin prepared from the affinity ligands described herein. Monomeric and polymeric ligands, including polymers with different domains, were conjugated to bromoacetyl-activated resins, and the static binding capacity was determined according to the following method.

[0131] 1. Wash (water, shake 3 x 5 minutes)

[0132] 2. Pretreatment with 0.1 M NaOH: shake for 5 minutes

[0133] 3. Neutralize and wash with PBS: shake 2 x 5 minutes

[0134] 4. Incubate the HSA target at 20 mg / mL with shaking for 1 hour

[0135] 5. Drain and assay

[0136] 6. Wash 3 times with PBS and assay

[0137] Table 2. Static binding capacity of monomeric and polymeric ligands. The domain containing SEQ ID NO:15 is represented by a circle (“O”), and “X” represents a non-binding domain.

[0138] SEQ ID NO Polymodularity Polymeric form Ligand density mg / mL SBC mg / mL 21 Monomer O 8.8 22.8 22 Dimer O-O 8.5 16.5 23 Tetramer O-O-O-O 8.8 20.3 24 Trimer X-O-X 8.3 13.6 25 Tetramer X-O-O-X 9.1 20.1 26 Pentamer O-O-O-O-X 10.4 19.9 27 Pentamer X-O-O-O-X 8 19.1

[0139] Example 5

[0140] This example demonstrates the reuse of an affinity agent comprising a binding ligand described herein for the affinity purification of an albumin fusion protein, as well as the stability of the resin against NaOH. A clarified cell culture feed stream (CCCF) from a CHO cell line was spiked with an 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 run for 5 cycles, each cycle comprising the procedure shown in the table below.

[0141] Step Retention time Details Loading 4 minutes 1g / L Tracking 4 minutes PBS (equilibration buffer) Washing 3 minutes 1M NaCl, 50mM phosphate, pH 7.4 Elution 8 minutes 15% hexanediol, 0.5M proline, 50mM glycine, pH 9 CIP 4 minutes 0.5M NaOH, static hold for 6 hours

[0142] Overlaid chromatograms from the first and 5th cycles are shown in Figure 3 Using a Cygnus TM CHO Host Cell Protein 3rd generation assay to measure the residual HCP content of the purified protein, for all 5 cycles. The results showed that high purity was achieved and maintained in all cycles, and are shown in Figure 4 A consistent yield was obtained across all 5 cycles, and is shown in Figure 5 In summary, after exposure to 0.5 M NaOH for 30 hours, the resin performed as well as in the first cycle.

[0143] Example 6

[0144] This example demonstrates that the binding capacity can be increased by manipulating the pH of the binding solution. Resin prepared from the ligand corresponding to SEQ ID NO:21 was used in a static binding (filter plate) experiment. As shown in Figure 6 a higher binding capacity was achieved at pH 5 relative to higher pHs.

[0145] Example 7

[0146] This example demonstrates that an affinity resin with high binding capacity can be achieved with the ligands of the present invention. An affinity resin was prepared by conjugating the ligand corresponding to SEQ ID NO:29 to bromoacetyl-activated or epoxy-activated Jetted A50 agarose beads and packed into a 3 x 50 mm column to determine the dynamic binding capacity (DBC) of the resin using HSA as a test substrate.

[0147] Activation SEQ ID NO Ligand density DBC Bromoacetyl 21 18.0mg / mL 30g / L Epoxy 29 18.6mg / mL 34.7g / L

[0148] Example 8

[0149] This example demonstrates the effect of elution pH on a specific albumin fusion protein. The elution solution contained 0.5 M proline, 20% hexylene glycol, at pH 9, 8.25, and 7.4. For pH 9, 50 mM glycine was included as a buffer, for pH 8.25, 50 mM HEPES was included as a buffer, and for pH 7.4, phosphate was included as a buffer. The sharpest peak and highest yield were obtained at pH 9, as Figure 7 shown.

[0150] Example 9

[0151] This example demonstrates that the resin can be used to selectively remove albumin from a protein preparation. The albumin content of fraction V paste from the Cohn process was greater than 95% of the total protein with a small amount of other serum proteins. The fraction V paste (Seraplex, Pasadena, CA) was redissolved in 10 mM acetate buffer at pH 4.7 and 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 Jetted A50 agarose beads. The equivalent of approximately 10 g of paste was loaded onto the column and traced with PBS. The flow-through was collected and analyzed by SDS PAGE. As Figure 8 shown, the column effectively removed albumin and provided a protein mixture free of albumin. The lanes loaded as follows Figure 8 are:

[0152] Lane Sample 1 SeeBlue Plus2 2 SeeBlue Plus2 3 10x-diluted loading (redissolved fraction V paste) 4 Flow-through fraction cell

[0153] Example 10

[0154] This example demonstrates that the linker affects the binding capacity of the resin. Five ligands were conjugated to epoxy-activated A50 beads at the ligand densities indicated in the following examples. The resulting resin was packed into a 0.3 x 5 cm column, and the DBC was determined using HSA at 1 mg / mL with a 7-minute residence time. Figure 9 and the breakthrough curves shown in the following table indicate that the resins prepared from SEQ ID NO:34, 36, and 37 have the highest binding capacity.

[0155] Ligand Ligand density (g / L) DBC (g / L) at 10% breakthrough SEQ ID NO:29 16.2 34 SEQ ID NO:34 16.8 41 SEQ ID NO:35 17.2 34 SEQ ID NO:36 18.4 44 SEQ ID NO:37 18.1 41

[0156] Table 3. Sequences

[0157]

[0158]

[0159]

Claims

1. An affinity agent comprising a ligand, said ligand comprising the sequence SEQ ID No: 15 LREAKERAIEELRRAGISSDYYFDLIQKAKTVEGVQALKDEILKA, 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. wherein the ligand binds to albumin such that the K D value of the interaction is at most 1×10 -9 M.

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

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

4. An affinity agent comprising a ligand that binds to human serum albumin and / or a human albumin fusion protein, said ligand comprising at least one sequence of any one of SEQ ID NO: 21 - 37, 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.

5. The affinity agent according to any one of claims 1 to 4, which comprises a multimeric polypeptide, wherein the multimeric polypeptide comprises at least two subunits; and 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 according to claim 5, wherein the subunits are not all the same.

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

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

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

10. The affinity agent according to 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 according to any one of claims 1 to 11, which is used for the purification of human serum albumin or a human albumin fusion protein.

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

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