Methods for purifying albumin fusion proteins

Abstract

To provide a method of purifying albumin-fusion proteins.SOLUTION: Disclosed is a method of purifying albumin-fusion proteins to reduce the level of oxidation of susceptible amino acid residues. The method comprises an affinity matrix chromatography step and an anion exchange chromatography step. The purified albumin-fusion proteins have low levels of oxidation and retain their enhanced half-lives in vivo and retain bioactivity. In some embodiments, the albumin-fusion proteins comprise a scaffold, such as a human Tenascin C scaffold. Compositions comprising the albumin-fusion proteins are further disclosed.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] Sequence Listing Reference This application incorporates by reference the Sequence Listing submitted herewith via EFS-Web as a text file entitled "CD40L-300P1_SL.TXT," created on March 12, 2015, and having a size of 228 kilobytes.

[0002] The present invention generally relates to methods for purifying albumin fusion proteins having low levels of oxidation of tryptophan and / or methionine residues of the albumin fusion protein. Low levels of oxidation of these residues allow the purified albumin fusion protein to retain its relative potency and biological activity. The albumin fusion protein may also include a scaffold, such as one derived from the third fibronectin type III domain of human tenascin-C. The present invention also relates to methods for purifying albumin fusion proteins, purified proteins obtained therefrom, and compositions containing the purified albumin fusion proteins. [Background technology]

[0003] In recent years, there has been growing interest in the use of proteins as potential therapeutic agents. One disadvantage of protein drugs is that they tend to have a short half-life in vivo. To overcome this challenge, proteins and peptides can be conjugated or fused to other molecules. One option for extending half-life is PEGylation, a process in which poly(ethylene glycol), or PEG, is covalently attached to proteins through several available chemical reactions. In addition to extending half-life, PEGylation may also reduce immunogenicity, possibly due to shielding of the protein surface by inert PEG chains. A disadvantage of PEGylation is that it requires a conjugation reaction step and, in many cases, an additional purification step to remove unreacted PEG chains. Despite these challenges, PEGylation technology has been successfully used in several commercially available biopharmaceuticals.

[0004] The second option for extending half-life is fusion protein technology. In this case, a therapeutic protein is genetically fused to a second protein designed to extend its in vivo half-life. This option results in an extended half-life similar to PEGylation, but because the fusion protein is expressed and purified as a single entity, it does not require additional production steps (conjugation and associated purification). Examples of fusion proteins include Fc fusions, transferrin fusions, and albumin fusions. All of these proteins are found at high levels in human plasma, mitigating the effects of elevated levels caused by drugs.

[0005] In addition to the advantages of extended half-life and ease of production, fusion proteins may also be amenable to platform approaches for purification. This is because the carrier protein often constitutes the majority of the fusion protein, and therefore the physiochemical properties of various fusion proteins are similar. For a platform approach to be successful, the purification procedure must be selective for the carrier protein. Summary of the Invention [Means for solving the problem]

[0006] The present invention relates to a method for purifying albumin fusion proteins.

[0007] Citation or discussion of a reference herein shall not be construed as an admission that it is prior art to the present invention.

[0008] In certain aspects, the disclosure herein relates to methods for reducing tryptophan and / or methionine oxidation during the purification of an albumin fusion protein, comprising subjecting a composition comprising the albumin fusion protein to the following purification processes: (a) an affinity matrix; (b) an anion exchange matrix, wherein the albumin fusion protein is eluted from the affinity matrix by applying an elution buffer comprising octanoic acid.

[0009] In a further aspect, disclosed herein is a method for reducing tryptophan and / or methionine oxidation during the purification of an albumin fusion protein, comprising the steps of subjecting a composition comprising an albumin fusion protein to one of the following purification processes: (a) an affinity matrix; (b) an anion exchange matrix, wherein the affinity matrix is: (1) from about 2% to about 20% of 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,6-hexanediol, or (2) 0.05M to 2.0M of a salt selected from sodium chloride, potassium chloride, calcium chloride, lithium chloride, sodium bromide, potassium bromide, and lithium bromide; (3) about 0.02M to about 0.2M sodium sulfate; (4) about 0.01% to about 1% nonionic surfactant; (5) about 0.05M to about 1.0M urea; or (6) about 0.02M to about 0.5M nicotinamide.

[0010] In a further aspect, the disclosure herein relates to a method of obtaining a composition comprising albumin fusion proteins essentially free of oxidized tryptophan residues, comprising the step of subjecting a composition comprising tryptophan-oxidized and non-tryptophan-oxidized albumin fusion proteins to a hydrophobic interaction matrix, wherein the tryptophan-oxidized and non-tryptophan-oxidized albumin fusion proteins are eluted from the hydrophobic interaction matrix at different times, thereby separating the tryptophan-oxidized albumin fusion proteins from the non-tryptophan-oxidized albumin fusion proteins.

[0011] In certain aspects, the disclosure herein relates to methods for isolating albumin fusion proteins essentially free of oxidation of tryptophan / methionine residues, comprising the steps of subjecting a composition comprising the albumin fusion protein to the following purification processes: (a) an affinity matrix chromatography process; (b) an anion exchange chromatography process; and (c) a hydrophobic interaction matrix chromatography process, wherein an elution buffer comprising octanoic acid is applied to the affinity matrix and the tryptophan-oxidized and non-tryptophan-oxidized albumin fusion proteins are eluted from the hydrophobic interaction matrix at different times, thereby separating the tryptophan-oxidized albumin fusion proteins from the non-tryptophan-oxidized albumin fusion proteins.

[0012] In a further aspect, disclosed herein is a method of purifying an albumin fusion protein comprising subjecting a composition comprising the albumin fusion protein to a hydrophobic interaction matrix and one or more of the following purification processes: (a) an affinity matrix to which an elution buffer comprising octanoic acid is applied; and / or (b) an anion exchange matrix, wherein the affinity matrix is ​​(1) from about 2% to about 20% of 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,6-hexanediol, and 2-methyl-2-hexanediol. (2) 0.05M to 2.0M of a salt selected from sodium chloride, potassium chloride, calcium chloride, lithium chloride, sodium bromide, potassium bromide, and lithium bromide; (3) about 0.02M to about 0.2M sodium sulfate; (4) about 0.01% to about 1% non-ionic surfactant; (5) about 0.05M to about 1.0M urea; or (6) about 0.02M to about 0.5M nicotinamide, wherein the resulting purified albumin fusion protein is essentially free of oxidized tryptophan residues.

[0013] In a further aspect, the disclosure herein relates to a method of purifying an albumin fusion protein, comprising the steps of: (a) applying a composition comprising the albumin fusion protein to an affinity matrix; (b) eluting the albumin fusion protein from the affinity matrix of (a) to obtain a first eluate; (c) applying the first eluate to an anion exchange matrix; (d) eluting the albumin fusion protein from the anion exchange matrix to obtain a second eluate; (e) applying the second eluate to an anion exchange membrane; passing the albumin fusion protein through the anion exchange membrane to obtain a flow-through; and (f) applying the flow-through to a hydrophobic interaction matrix; and eluting the albumin fusion protein from the hydrophobic interaction matrix to obtain a third eluate, wherein the third eluate comprises the purified albumin fusion protein.

[0014] The disclosure herein also relates to albumin fusion protein compositions obtained by any of the methods disclosed herein.

[0015] The disclosure herein further relates to compositions comprising albumin fusion proteins having less than 20 ng / mg host cell protein and wherein less than 15% of the tryptophan residues are oxidized.

[0016] Disclosed herein are compositions comprising albumin fusion proteins, the compositions comprising 5×10 -3 The present invention further relates to a composition having less than ng / mg of DNA and less than 15% of the tryptophan residues are oxidized.

[0017] The disclosure herein further relates to compositions comprising albumin fusion proteins, wherein the compositions have less than 20 ng / mg of host cell protein, and wherein the albumin fusion proteins have a relative activity of >90%.

[0018] The disclosure herein also relates to compositions comprising albumin fusion proteins, wherein the compositions have less than 5×10 −3 ng / mg of DNA, and the albumin fusion proteins have a relative activity of >90%.

[0019] The disclosure herein further relates to compositions comprising an albumin fusion protein of SEQ ID NO: 134, 135, 201, 202, 203, 204, 205, 206, 207, or 208, having less than 20 ng / mg host cell protein, wherein the tryptophan at position 46, 151, or both, is not oxidized.

[0020] The disclosure herein also relates to a pharmaceutically acceptable formulation comprising (a) any of the compositions disclosed herein, (b) a buffer, (c) a sugar, and (d) an emulsifier.

[0021] For the purpose of illustrating the invention, there is shown in FIG. 1 specific embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the illustrated embodiments. [Brief explanation of the drawings]

[0022] [Figure 1] 1 shows a flow chart of one embodiment of the rHSA purification process. [Figure 2] A representative chromatogram of Cibacron blue dye chromatography on rHSA operated at 300 cm / hr is shown. [Figure 3] 1 shows a flow chart of one embodiment of an albumin fusion purification process. [Figure 4] A representative chromatogram of Cibacron blue dye chromatography for albumin fusion protein #1 (AFP-1) operated at 300 cm / hr is shown. [Figure 5] A representative Capto Q chromatogram for AFP-1 operated at 300 cm / hr is shown. [Figure 6] A representative Mustang Q membrane chromatogram operated at 10 MV / hr is shown. [Figure 7] A representative Toyopearl PPG-600M chromatogram operated at 130 cm / hr in bind and elute mode is shown. [Figure 8] Cibacron blue dye chromatography of albumin fusion protein using (A) 25 mM octanoic acid and (B) 2 M NaCl elution buffers. [Figure 9] Step yields (Figure 7A) and DNA log reduction values ​​(LRV) (Figure 7B) as a function of pH and NaCl concentration in Mustang Q membrane chromatography. [Figure 10] Figure 1 shows the relative potency of albumin fusion proteins as a function of oxidation. □ represents methionine M498 of AFP-1; open circle represents tryptophan W46 / W151 of AFP-1; ◇ represents methionine residues M74 / M179 of AFP-1; and Δ represents methionine M529 of AFP-1. [Figure 11] 1 shows a summary of tryptophan oxidation over time (days) in process intermediates from the Capto Blue (“Blue”) and Capto Q (“Q”) processes as measured by SEC-HPLC. [Figure 12] Anti-TDO2 Western blot is shown. [Figure 13] Representative HIC chromatograms for AFP-1 are shown, including Capto MMC, Butyl-S Fast Flow, Toyopearl PPG-600M, and Toyopearl Phenyl-650M. [Figure 14] 1 shows the relative potency of purified albumin fusion proteins as a function of HIC-HPLC initial species content in HIC fractions obtained during Butyl-S Fast Flow (closed circles) or PPG-600M (open circles) chromatography runs. DETAILED DESCRIPTION OF THE INVENTION

[0023] Before describing the present invention in detail, it should be understood that the present invention is not limited to specific compositions or process steps, as such may vary. It should be noted that when the singular forms "a," "an," and "the" are used in this specification and the appended claims, they include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an") and "one or more" and "at least one" may be used interchangeably herein.

[0024] Furthermore, when used herein, "and / or" should be interpreted as a specific disclosure of each of two specific features or elements, regardless of the presence or absence of the other. Thus, when the term "and / or" is used herein in phrases such as "A and / or B," it is intended to include "A and B," "A or B," "A" (single) and "B" (single). Similarly, when the term "and / or" is used in phrases such as "A, B, and / or C," it is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0025] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in the present invention.

[0026] Units, prefixes, and symbols are expressed in their Systeme International de Unites (SI) accepted format. Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in the amino to carboxy direction. The headings provided herein are not limitations of the various aspects or embodiments of the invention, which may be included by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0027] Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0028] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides are referred to by their commonly accepted single-letter codes.

[0029] The term "epitope" as used herein refers to a protein determinant capable of binding to the scaffold of the present invention. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0030] The terms "fibronectin type III (FnIII) ​​domain," "FnIII domain," and "FnIII scaffold" refer to a polypeptide homologous to the human fibronectin type III domain, which has at least seven β-strands distributed between two β-sheets, the β-sheets themselves being packed together to form the core of the protein, and further comprising solvent-exposed loops connecting the β-strands to each other. At least three such loops are present at each end of the β-sheet sandwich, and these ends are the boundaries of the protein perpendicular to the direction of the β-strands. In certain embodiments, the FnIII domain comprises seven β-strands designated A, B, C, D, E, F, and G, connected to six loop regions designated AB, BC, CD, DE, EF, and FG, with the loop regions connecting each β-strand.

[0031] As used herein, the term "Tn3 scaffold" refers to a molecule comprising at least one FnIII scaffold, wherein β-strand A comprises SEQ ID NO: 11, β-strand B comprises SEQ ID NO: 12, β-strand C comprises SEQ ID NO: 13 or 14, β-strand D comprises SEQ ID NO: 15, β-strand E comprises SEQ ID NO: 16, β-strand F comprises SEQ ID NO: 17, and β-strand G comprises SEQ ID NO: 18, and wherein at least one loop is a non-naturally occurring variant of a loop in the "parent Tn3 scaffold." In certain embodiments, one or more of the β-strands of a Tn3 module comprise at least one amino acid substitution, except that a cysteine ​​residue in β-strand C (e.g., a cysteine ​​in SEQ ID NO: 13 or 14) and β-strand F (SEQ ID NO: 17) is not substituted.

[0032] The term "parent Tn3" as used herein refers to an FnIII scaffold comprising SEQ ID NO: 3, i.e., a thermostabilized cysteine-engineered FnIII scaffold derived from the third FnIII domain of human tenascin-C.

[0033] The term "multimer" or "multimeric scaffold" refers to a molecule comprising at least two related FnIII scaffolds. The scaffolds forming the multimeric scaffold can be connected via a linker that allows each scaffold to function independently.

[0034] The terms "monomer," "monomeric subunit," or "monomeric scaffold" refer to a molecule that comprises only one FnIII scaffold.

[0035] The term "CD40L-specific monomeric subunit," as used herein, refers to a Tn3 monomer derived from a "parent Tn3," which Tn3 monomer specifically binds to CD40L or a fragment thereof, e.g., a soluble form of CD40L.

[0036] The term "DNA" refers to a sequence of two or more covalently linked, naturally occurring or modified deoxyribonucleotides.

[0037] The term "fusion protein" refers to a protein comprising (i) one or more therapeutic proteins or fragments linked to (ii) a second, different protein (i.e., a "heterologous" protein). Within the scope of the present invention, albumin (HSA, variant HSA, or fragment HSA) is linked to a therapeutic protein or fragment.

[0038] [Table 1]

[0039] The term "heterologous moiety" is used herein to refer to the addition of a composition to the Tn3 scaffold of the present invention, and the composition is not necessarily part of the FnIII domain. Exemplary heterologous moieties include proteins, peptides, protein domains, linkers, drugs, toxins, imaging agents, radioactive compounds, organic and inorganic polymers, and any other compositions that can provide activity that is not inherent in the FnIII domain itself, such as, but not limited to, polyethylene glycol (PEG), cytotoxic drugs, radionuclides, imaging agents, biotin, dimerization domains (e.g., leucine zipper domains), human serum albumin (HSA) or its FcRn-binding portion, antibody domains or fragments (e.g., antibody variable domains, CH1 domains, CK domains, Cλ domains, CH2 or CH3 domains), single-chain antibodies, domain antibodies, albumin-binding domains, IgG molecules, enzymes, ligands, receptors, binding peptides, non-FnIII scaffolds, epitope tags, recombinant polypeptide polymers, cytokines, etc.

[0040] The term "linker," as used herein, refers to any molecular assembly that joins or connects two or more scaffolds. A linker can be a molecule that functions to act as a "spacer" between modules in a scaffold, or it can also be a molecule that has additional functionality (i.e., a "functional moiety"). Molecules included in the definition of "heterologous moiety" can also function as linkers.

[0041] The terms "connected," "conjugated," and "fused" are used interchangeably. These terms refer to the joining of two or more scaffolds, heterologous moieties, or linkers together by any means, including chemical conjugation or recombinant means.

[0042] The term "domain" or "protein domain" refers to a region of a protein that can fold into a stable three-dimensional structure, often independent of the rest of the protein, and that can be endowed with a specific function. This structure maintains the specific function associated with the domain's function within the original protein, such as enzymatic activity, creating a recognition motif for another molecule, or providing a necessary component for the protein to exist in its specific environment. Protein domains can be evolutionarily conserved regions both within a protein family and within related protein superfamilies. When describing components of a multimeric scaffold, the terms "domain," "monomer scaffold," "monomer subunit," and "module" can be used interchangeably. A "native FnIII domain" refers to any non-recombinant FnIII domain encoded by an organism.

[0043] "Protein sequence" or "amino acid sequence" means a linear representation of the amino acid components of a polypeptide from amino- to carboxyl-terminus, in which adjacent residues are contiguous in the primary structure of the polypeptide.

[0044] The term "nucleic acid" refers to any two or more covalently linked nucleotides or nucleotide analogs or derivatives. As used herein, this term includes, but is not limited to, DNA, RNA, and PNA. "Nucleic acid" and "polynucleotide" are used interchangeably herein.

[0045] The term "polynucleotide" is intended to encompass both single and multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The term "isolated" nucleic acid or polynucleotide refers to a nucleic acid molecule, DNA, or RNA that has been removed from its natural environment. For example, a recombinant polynucleotide, such as one encoding a scaffold of the present invention, contained within a vector is considered isolated for purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides purified (partially or substantially) from solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. Furthermore, polynucleotides or nucleic acids can be or include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0046] The term "pharmaceutically acceptable" refers to a compound or protein that can be administered to an animal (eg, a mammal) without significant adverse medical consequences.

[0047] The term "physiologically acceptable carrier" refers to a carrier that has no significant adverse effects on the treated host and that retains the therapeutic properties of the compound with which it is administered. One exemplary physiologically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences, (18th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. (incorporated herein by reference).

[0048] "Polypeptide" refers to any sequence of two or more amino acids linearly linked by amide bonds (peptide bonds), regardless of length, post-translational modification, or function. "Polypeptide," "peptide," and "protein" are used interchangeably herein. Thus, a peptide, dipeptide, tripeptide, or oligopeptide is included within the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the product of post-expression modifications of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides can be derived from natural biological sources or produced by recombinant technology, but are not necessarily translated from a specified nucleic acid sequence. Polypeptides can be made in any manner, for example, by chemical synthesis.

[0049] Polypeptides of the present invention include fragments, derivatives, analogs, or variants of the aforementioned polypeptides, and any combination thereof. Variants can be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. Variant polypeptides can contain conservative or non-conservative amino acid substitutions, deletions, or additions. Also included as "derivatives" are peptides that contain one or more naturally occurring amino acid derivatives of the 20 standard amino acids.

[0050] "Randomized" or "mutated" refers to the inclusion of one or more amino acid modifications, including deletions, substitutions, or additions to a template sequence. "Randomizing" or "mutating" refers to the process of introducing such amino acid modifications into a sequence. Randomization or mutation can generally be achieved through deliberate, blind, or spontaneous sequence variation of a nucleic acid coding sequence and can occur by any technique, for example, PCR, error-prone PCR, or chemical DNA synthesis. The terms "randomizing," "randomized," "mutating," "mutated," and the like are used interchangeably herein.

[0051] "Cologus" or "cognate non-mutated protein" means a protein that is identical in sequence to the variant protein except for amino acid mutations introduced into the variant protein (where the variant protein is randomized or mutated).

[0052] "RNA" means a sequence of two or more covalently linked naturally occurring or modified ribonucleotides. An example of a modified RNA included within the scope of this term is phosphorothioate RNA.

[0053] The term "scaffold of the invention" or "scaffolds of the invention," as used herein, refers to multimeric Tn3 scaffolds and monomeric Tn3 scaffolds. The term "target" refers to a compound recognized by a particular scaffold of the invention. The terms "target" and "antigen" are used interchangeably herein. The term "specificity," as used herein, for example, in the terms "specifically bind" or "specific binding," refers to the relative affinity with which a Tn3 scaffold of the invention binds to one or more antigens via one or more antigen-binding domains, and indicates that the binding involves some complementarity between one or more antigen-binding domains and one or more antigens. According to this definition, a Tn3 scaffold of the invention is said to "specifically bind" to an epitope when it binds to that epitope more readily than it binds to a random, unrelated epitope.

[0054] An "affinity matured" scaffold is one that has one or more modifications, generally in the loops, that result in an improvement in the affinity of the Tn3 scaffold for an epitope compared to the parent Tn3 scaffold without those modifications.

[0055] The term "affinity," as used herein, refers to a measure of the strength of binding of a particular Tn3 scaffold of the invention to a particular epitope.

[0056] The term "avidity," as used herein, refers to the overall stability of the complex between a collection of Tn3 scaffolds of the invention and a particular epitope, i.e., the functional combined strength of binding of multiple Tn3 scaffolds to an antigen. Avidity relates to both the affinity of individual antigen-binding domains to a specific epitope and also to the valency of the scaffolds of the invention.

[0057] The term "target effect" refers to the binding of the Tn3 scaffold of the present invention to one or more targets and the biological effect resulting from such binding. In this context, multiple antigen binding units within the Tn3 scaffold may interact with different targets and / or epitopes, e.g., bringing two targets into physical proximity, triggering metabolic cascades through interaction with distinct targets, etc. With reference to CD40L, "target effect" refers to an effect achieved, for example, by enhancing, stimulating, or activating one or more biological activities of CD40L.

[0058] The term "valency" as used herein refers to the number of potential antigen binding modules, for example, the number of FnIII modules in the scaffold of the present invention. When the Tn3 scaffold of the present invention comprises two or more antigen binding modules, each binding module can specifically bind to, for example, the same epitope or different epitopes on the same target or different targets.

[0059] The term "disulfide bond," as used herein, includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or crosslink with a second thiol group.

[0060] The terms "immunoglobulin" and "antibody" encompass a wide variety of biochemically distinguishable polypeptide classes. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon. It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA, IgG, or IgE, respectively. Modified versions of each of these classes are readily identifiable to those skilled in the art. As used herein, the term "antibody" includes, but is not limited to, intact antibodies, modified antibodies, antibody VL or VL domains, CH1 domains, CK domains, Cλ domains, Fc domains (see below), CH2, or CH3 domains.

[0061] As used herein, the term "Fc domain" refers to a portion of an antibody constant region. Traditionally, the term Fc domain refers to a protease (e.g., papain) cleavage product encompassing the paired CH2, CH3, and hinge regions of an antibody. In the context of this disclosure, the term Fc domain or Fc refers to any polypeptide (or nucleic acid encoding such a polypeptide), regardless of means of production, that includes all or a portion of the CH2, CH3, and hinge regions of an immunoglobulin polypeptide.

[0062] As used herein, the term "modified antibody" includes synthetic forms of antibodies that have been modified so that they do not occur in nature, such as antibodies that contain at least two heavy chain portions rather than two complete heavy chains (e.g., domain-deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) that have been modified to bind to two or more antigens or different epitopes of a single antigen. Additionally, the term "modified antibody" includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies directed against three or more copies of the same antigen) (see, e.g., Antibody Engineering, Kontermann & Dubel, eds., 2010, Springer Protocols, Springer).

[0063] The term "in vivo half-life" is used in its formal sense, i.e., the time during which 50% of the biological activity of a polypeptide is still present in the body / target organ, or the time during which the activity of a polypeptide is 50% of its initial value. As an alternative to measuring functional in vivo half-life, one may measure "serum half-life," i.e., the time during which 50% of the polypeptide molecules circulate in the plasma or bloodstream before being eliminated. Measuring serum half-life is often simpler than measuring functional in vivo half-life, and the magnitude of serum half-life is usually a good indicator of the magnitude of functional in vivo half-life. Alternative terms for serum half-life include "plasma half-life," circulating half-life, circulating half-life, serum clearance, plasma clearance, and clearance half-life. The functionality to be retained is usually selected from procoagulant, proteolytic, cofactor binding, receptor binding activity, or other types of biological activity associated with a particular protein.

[0064] The term "increased" in reference to functional in vivo half-life or plasma half-life is used to indicate a statistically significant increase in the half-life associated with a polypeptide compared to the half-life of a reference molecule (e.g., an unmodified polypeptide) as measured under comparable conditions.

[0065] The term "reduced" in reference to functional in vivo half-life or plasma half-life is used to indicate a statistically significant decrease in the half-life associated with a polypeptide compared to the half-life of a reference molecule (e.g., an unmodified polypeptide) as measured under comparable conditions.

[0066] The term "expression," as used herein, refers to the process by which a gene produces a biochemical, such as a scaffold of the present invention or a fragment thereof. The process includes any expression of the functional presence of a gene inside a cell, including, but not limited to, gene knockdown and both transient and stable expression. It includes, but is not limited to, the transcription of a gene into one or more mRNAs and the translation of such mRNAs into one or more polypeptides. If the final desired product is a biochemical, expression includes the production of that biochemical and any precursors.

[0067] An "expression product" can be either a nucleic acid, e.g., messenger RNA produced by transcription of a gene, or a polypeptide. Expression products as described herein further include nucleic acids with post-transcriptional modifications, e.g., polyadenylation, or polypeptides with post-translational modifications, e.g., methylation, glycosylation, lipid addition (associated with other protein subunits, proteolytic cleavage, etc.).

[0068] The term "vector" or "expression vector" is used herein to mean a vector used in accordance with the present invention as a vehicle for introducing into a host cell and expressing a desired expression product in the host cell. As known to those skilled in the art, such vectors can be easily selected from the group consisting of plasmids, phages, viruses and retroviruses. Generally, vectors compatible with the present invention will contain a selectable marker, appropriate restriction sites to facilitate cloning of the desired nucleic acid and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0069] The term "host cell" refers to a cell that harbors a vector constructed using recombinant DNA technology and that encodes at least one expression product. In describing processes for the isolation of expression products from recombinant hosts, the terms "cells" and "cell culture" are used interchangeably to refer to the source of the expression product unless otherwise clearly specified; i.e., recovery of the expression product from "cells" refers either to recovery from spun-down whole cells or to recovery from a cell culture containing both the medium and suspended cells.

[0070] The terms "treat" or "treatment," as used herein, refer to both therapeutic and prophylactic or preventative treatment, where the purpose is to prevent or slow (alleviate) the progression of an unwanted physiological change or disorder, such as an inflammatory disease or condition, in a subject. Beneficial or desired clinical results include, but are not limited to, relief of symptoms, reduction in the extent of disease, stabilized (i.e., not worsening) disease, delayed or slowed disease progression, amelioration or palliation of the pathology, and remission (partial or total), either detectable or undetectable.

[0071] The term "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in whom the condition or disorder is to be prevented.

[0072] The terms "subject," "individual," "animal," "patient," or "mammal" refer to any individual, patient, or animal, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, domestic animals, livestock, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, and the like.

[0073] The term "CD40L," as used herein, refers to, but is not limited to, CD40L expressed on the surface of T cells, recombinantly expressed CD40L, CD40L expressed and purified from E. coli or other suitable recombinant protein expression systems, deglycosylated CD40L, and soluble fragments of CD40L. As used herein, "CD40L" also refers to MegaCD40L. MegaCD40L™ is a highly active construct in which two trimeric CD40 ligands are artificially linked via the collagen domain of ACRP30 / adiponectin. This construct highly effectively mimics the in vivo membrane-assisted aggregation of CD40L. It offers a simple and equally potent alternative to the CD40L + enhancer combination (Alexis Biochemicals). The term "CD40L" refers to monomeric and oligomeric forms of CD40L, such as trimeric CD40L.

[0074] The term "CD40L" refers to both full-length CD40L and soluble fragments, e.g., the extracellular domain forms of CD40L resulting from proteolysis. The amino acid sequences of the membrane-bound and soluble forms of human CD40L (Swissprot: P29965) are set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0075] The terms "CD40L antagonist" or "antagonist" are used in the broadest sense and include any molecule that partially or completely inhibits, reduces, or inactivates one or more biological activities of CD40L and biologically active variants thereof in vitro, in situ, or in vivo. For example, a CD40L antagonist may function, as a result of its binding to CD40L, to partially or completely inhibit, reduce, or inactivate one or more biological activities of one or more CD40L molecules, or one or more CD40L molecules bound to CD40 or other targets, in vivo, in vitro, or in situ.

[0076] The terms "CD40L agonist" or "agonist" are used in the broadest sense and include any molecule that partially or fully enhances, stimulates, or activates one or more biological activities of CD40L, and biologically active variants thereof, in vitro, in situ, or in vivo. For example, a CD40L agonist may function, as a result of its binding to CD40L, to partially or fully enhance, stimulate, or activate one or more biological activities of one or more CD40L molecules, or one or more CD40L molecules bound to CD40R or other targets, in vivo, in vitro, or in situ.

[0077] The term "crystal", as used herein, refers to a solid-state form of matter in which atoms are arranged in a periodically repeating pattern in three dimensions, typically forming a lattice.

[0078] The term "space group symmetry," as used herein, refers to the total symmetry of a crystal that combines the translational symmetry of the crystal lattice with point group symmetry. A "space group" is designated by a capital letter (P, A, F, etc.) that identifies the lattice group, followed by a point group symbol in which the rotation and reflection elements are expanded to include screw axes and glide planes. It should be noted that the point group symmetry for a given space group can be determined by removing the lattice center symbol of the space group, replacing all screw axes with analogous rotation axes, and replacing all glide planes with mirror planes. The point group symmetry for a space group describes the true symmetry of its reciprocal lattice.

[0079] The term "unit cell," as used herein, refers to atoms in a crystal arranged in a regular repeating pattern, where the smallest repeating unit is called the unit cell. The entire structure can be reconstructed from information about the unit cell, which is characterized by three lengths (a, b, and c) and three angles (α, β, and γ). The quantities a and b are the lengths of the base of the cell, and γ is the angle between these two sides. The quantity c is the height of the unit cell. The angles α and β represent the angles between the base and the vertical side of the unit cell.

[0080] The term "machine-readable data storage medium," as used herein, means a data storage material having machine-readable data encoded thereon, such that a machine can be programmed with instructions for using such data and display the data in a desired format, for example, a graphical three-dimensional representation of a molecule or molecular complex.

[0081] The term "X-ray diffraction pattern" refers to the pattern obtained from the scattering of X-rays off a periodic collection of molecules or atoms in a crystal. X-ray crystallography is a technique that takes advantage of the fact that X-rays are diffracted by crystals. X-rays are of a wavelength appropriate to be scattered by the electron clouds of atoms of comparable size (in the angstrom range, approximately 10 -8 cm). Electron density can be reconstructed based on diffraction patterns obtained from X-ray scattering of molecules or periodic collections of atoms in a crystal. Additional phase information can be extracted from the diffraction data or from supplementing diffraction experiments to complete the reconstruction (the phase problem in crystallography). Models are progressively incorporated into the experimental electron density and refined against the data to generate accurate molecular structures. X-ray structure coordinates define a unique configuration of points in space. Those skilled in the art will understand that a set of structure coordinates for a protein or protein-ligand complex, or portion thereof, defines a relative set of points, which in turn defines a configuration in three dimensions. Similar or identical configurations can be defined by entirely different sets of coordinates, provided that the distances and angles between the coordinates remain essentially the same. Furthermore, a configuration of points can be defined by increasing or decreasing the distances between coordinates by a scalar factor while keeping the angles essentially the same.

[0082] The term "crystal structure" refers to the three-dimensional or lattice spacing arrangement of repeated atomic or molecular units in crystalline material.The crystal structure of crystalline material can be determined by X-ray crystallography, see, for example, "Principles of Protein X-Ray Crystallography" by Jan Drenth, Springer Advanced Texts in Chemistry, Springer Verlag, 2nd ed., February 199, ISBN: 0387985875, and "Introduction to Macromolecular Crystallography" by Alexander McPherson, Wiley-Liss, October 18, 2002, ISBN: 0471251224.

[0083] The term "effector function" refers to a biological activity of an antibody or antibody fragment that can be attributed to the Fc region of an antibody (a native Fc region or an amino acid sequence variant Fc region) and varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0084] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins.

[0085] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. An FcR can be a native sequence human FcR. An FcR can bind an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants or spliced ​​forms of these receptors. The term also includes the neonatal receptor FcRn.

[0086] The term "consensus sequence" refers to a protein sequence that shows the most common amino acid at a particular position after alignment of multiple sequences. A consensus sequence is a way of representing the results of alignment of multiple sequences when related sequences are compared to each other. The consensus sequence indicates which residue is most abundant in the alignment at each position and the degree of variability at each position.

[0087] The term "essentially free" refers to a composition having less than 10% oxidized tryptophan residues relative to the total number of amino acid residues in the protein, less than 8% oxidized tryptophan residues relative to the total number of amino acid residues in the protein, less than 5% oxidized tryptophan residues relative to the total number of amino acid residues in the protein, less than 4% oxidized tryptophan residues relative to the total number of amino acid residues in the protein, less than 3% oxidized tryptophan residues relative to the total number of amino acid residues in the protein, less than 2% oxidized tryptophan residues relative to the total number of amino acid residues in the protein, or less than 1% oxidized tryptophan residues relative to the total number of amino acid residues in the protein. In some embodiments, the term "essentially free" refers to a composition having less than 5% oxidized tryptophan residues relative to the total number of amino acid residues in the protein.

[0088] The term "biological activity" or "activity" refers to the biological activity of a therapeutic protein, e.g., the TN3 scaffold, and its ability to function in its intended manner in vivo, e.g., binding to CD40L. In some embodiments, activity refers to the "relative activity," i.e., the activity of a purified therapeutic protein relative to an unoxidized therapeutic protein. In some embodiments, the relative activity of a purified therapeutic protein is greater than 80%, greater than 85%, greater than 90%, greater than 92%, greater than 94%, greater than 95%, greater than 98%, or greater than 99%.

[0089] Fusion of albumin to a therapeutic protein has been found to increase or extend the in vivo or serum half-life of the fused therapeutic protein. However, it has been found that during purification of such albumin fusion proteins, certain amino acid residues may be susceptible to oxidation, thereby reducing or limiting the biological activity of the albumin fusion protein. The present invention is directed to a method for reducing oxidation of sensitive amino acid residues in albumin fusion proteins and the purification of such albumin fusion proteins. In one embodiment, the albumin fusion protein comprises a scaffold. In another embodiment, the scaffold comprises an Fn3 domain. In yet another embodiment, the scaffold comprises a human tenascin-C (Tn3) scaffold capable of binding to CD40L.

[0090] Process for reducing oxidation of albumin fusion proteins During the purification process of albumin fusion proteins, certain amino acid residues may become susceptible to oxidation, which may inhibit the biological activity and relative potency of the albumin fusion protein. For example, one or more tryptophan and / or methionine residues may be susceptible to oxidation. According to the present invention, oxidation of sensitive amino acid residues of albumin fusion proteins is reduced by subjecting a solution containing the albumin fusion protein to affinity and anion exchange chromatography matrices under appropriate conditions.

[0091] Affinity matrix chromatography The affinity chromatography step utilizes an affinity matrix that preferentially binds albumin. For example, suitable matrices include Cibacron blue dye, Reactive Blue 2, Procion Blue HB, Capto Blue, Capto Blue (high sub), Toyopearl, AF-Blue HC-650M, Blue Sepharose, Blue Trisacryl, Mimetic Blue 1, Mimetic Blue SA, Mimetic Blue SA HL, and other anthraquinone-type compounds, nitrocellulose matrices, antibody-based matrices such as Life Technologies' Capture Select, and fatty acid-based matrices. In one embodiment, Cibacron blue dye chromatography is an ideal choice for purifying albumin fusion proteins from cell culture media due to their affinity for albumin. While numerous Cibacron blue dye chromatography resins are commercially available, many of them are less than ideal for large-scale purification of albumin fusion proteins. For large-scale purification, resins must be made of materials that minimize nonspecific interactions with host-associated impurities, have good pressure-flow characteristics, and be stable at pH extremes (preferably under corrosive conditions) for sanitization purposes. With these properties in mind, several commercially available Cibacron blue dye chromatography resins (Capto Blue and Capto Blue (high sub) from GE Healthcare, and Toyopearl AF-Blue HC-650M from Tosoh Biosciences) have emerged as promising resins for clinical and industrial-scale purification. In some embodiments, the high sub version of the two Capto Blue options is preferred due to its higher ligand density and therefore higher binding capacity.

[0092] In a typical purification process, a Cibacron blue dye column is equilibrated with a buffer (e.g., phosphate, Tris, Bis-Tris, etc.) at near neutral or slightly acidic pH, and then loaded with the clarified cell culture fluid or process intermediate (if the Cibacron blue dye column is not an initial purification step) containing the albumin fusion protein.

[0093] Various amounts of protein can be loaded onto the column. In some embodiments, about 5 g protein / L resin to about 100 g protein / L resin, about 10 g protein / L to about 50 g protein / L resin, or about 25 g protein / L resin can be loaded onto the affinity column.

[0094] After loading the sample, the affinity chromatography column containing the bound albumin fusion protein may optionally be re-equilibrated and then further washed with stronger buffers to further remove host cell impurities that are bound to the column (through non-specific interactions) or to the albumin fusion protein (through protein-protein interactions). The wash buffer may be optimized to remove these impurities. In one embodiment, the wash buffer contains a polyol; salt; sodium sulfate; non-ionic surfactant; urea; and / or nicotinamide.

[0095] In one embodiment, the wash buffer comprises about 2% to about 20% polyol, which may be selected from the group consisting of 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,6-hexanediol, and 2-methyl-2,4-pentanediol.

[0096] Various concentrations of salt may be present in the wash buffer. In some embodiments, the salt is present in an appropriate amount, for example, about 0.05 M to about 2.0 M salt, about 0.1 M to about 1.8 M salt, about 0.2 M to about 1.5 M salt, about 0.3 M to about 1.0 M salt, about 0.4 M to about 0.8 M salt, or about 0.5 M salt. The salt may be selected from those commonly used in the art, such as sodium chloride, potassium chloride, calcium chloride, lithium chloride, sodium bromide, potassium bromide, and lithium bromide.

[0097] Various concentrations of sodium sulfate can be used. Sodium sulfate may be present in an amount of about 0.01M to about 0.5M, 0.02M to about 0.3M, about 0.04M to about 0.2M, or about 0.05M to about 0.1M.

[0098] Various nonionic surfactants can be used. For example, in some embodiments, the nonionic surfactant can be selected from the group consisting of Triton X-100, Tween 80, polysorbate 20, polysorbate 80, nonoxynol 9, poloxamer, stearyl alcohol, or sorbitan monostearate. Various concentrations of nonionic surfactants can be used. For example, in some embodiments, the nonionic surfactant is present in the wash buffer at a concentration of about 0.01% to about 1%, about 0.02%, about 0.4%, about 0.05% to about 0.2%, or about 0.08% to about 0.01%.

[0099] Various chaotropic agents are known in the art. In the present invention, urea is the chaotropic agent to be used in the wash buffer. Urea may be present in the wash buffer in an amount of about 0.02 M to about 1.5 M, about 0.05 M to about 1.0 M, or about 0.08 M to about 1.0 M.

[0100] In some embodiments, nicotinamide is used in the wash buffer. Nicotinamide may be present in the wash buffer in an amount of about 0.01 M to about 1.0 M, about 0.02 M to about 0.5 M, about 0.04 M to about 0.3 M, about 0.06 M to about 0.2 M, or about 0.1 M.

[0101] The wash buffer may have a variety of pH levels. In some embodiments, the pH of the wash buffer is greater than about 5.0, greater than about 5.5, or greater than about 6.0. In some embodiments, the pH of the wash buffer is less than about 8.0, less than about 7.5, less than about 7.0, or less than about 6.5. In some embodiments, the pH of the wash buffer is about 5.0 to about 8.0, about 5.5 to about 7.5, about 5.5 to about 7.0, about 6.0 to about 7.0, or about 6.5 to about 7.0.

[0102] In another embodiment of the present invention, the wash buffer contains about 5% to about 15% polyol, about 0.2 M to about 0.8 M salt, about 0.2 M to about 0.8 M sodium sulfate, about 0.02% to about 0.2% non-ionic surfactant, and / or about 0.2 M to about 1.0 M urea. In one aspect of the present invention, the wash buffer contains polyol, 1,2-propanediol, salt, sodium chloride, and the non-ionic surfactant Triton X-100. In another aspect of the present invention, the wash buffer contains about 0.5 M sodium chloride; about 0.5 M sodium sulfate; or about 10% 1,3-propanediol. According to one aspect of the present invention, the wash buffer has a pH of about 5.5 to about 7.0.

[0103] In some embodiments, the wash buffer contains a DNA concentration of about 5×10 2 Less than ng / mg of DNA, approximately 2 × 10 2 In some embodiments, the wash buffer is suitable for reducing host cell protein (HCP) to less than 50,000 ng / mg, less than 20,000 ng / mg, or less than 10,000 ng / mg.

[0104] In some embodiments, the purified product is eluted from the affinity matrix column by applying a high pH buffer to the column or by adding a high concentration of salt, a mild organic solvent, or a combination to disrupt product binding. In one embodiment, the elution buffer comprises a base, such as Bis-Tris, Tris, or a phosphate base. In another aspect of the invention, the elution buffer base is 50 mM Bis-Tris. In another embodiment, the elution buffer comprises an elution salt, such as octanoate, NaCl, or the sodium and / or potassium salts of caprylic, heptanoic, hexanoic, or nonanoic acid. In some embodiments, the elution buffer comprises sodium caprylate. The salt may be present in the elution buffer in an amount of about 5 mM to about 500 mM, about 20 mM to about 250 mM, about 50 mM to about 200 mM, or about 75 mM to about 150 mM. In another embodiment, the elution buffer comprises EDTA or another chelating agent. In one embodiment, the affinity matrix elution buffer comprises EDTA in a suitable amount, for example, from about 2 mM to about 20 mM EDTA, hi a further embodiment, the affinity matrix elution buffer comprises octanoic acid.

[0105] In accordance with the present invention, affinity chromatography results in low levels of oxidation products. In one embodiment, the intermediate metabolites containing the albumin fusion protein after affinity chromatography have less than about 100%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, or less than about 4% oxidation products relative to the total protein. In another embodiment, the intermediate metabolites containing the albumin fusion protein after affinity chromatography have less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, or less than about 4% oxidized tryptophan residues relative to the total number of tryptophan residues. In another embodiment, the affinity matrix step removes at least 1, 2, 3, 1-2, or 2-3 orders of magnitude of host cell proteins from the original sample. In one embodiment of the invention, the affinity matrix step removes at least 1, 2, 3, 4, 1-2, 2-3, or 3-4 orders of magnitude of DNA impurities from the original sample.

[0106] Virus inactivation In one embodiment of the present invention, fractions or samples containing albumin fusion proteins may be treated to inactivate any viruses that may be present. Thus, the fractions / samples may be treated with a virus inactivating agent, e.g., Triton X-100, Tween 80, Tween 20, tri-n-butyl phosphate, or urea. In one embodiment, a virus inactivation step is performed between the affinity chromatography and anion exchange chromatography steps. Thus, a virus inactivating agent, e.g., Triton X-100, may be added in an amount of about 0.05% to about 3%, about 0.01% to about 1%, or about 0.1% to about 0.5%, for a period of about 1 second to about 10 hours, about 30 seconds to about 5 hours, about 30 minutes to about 3 hours, or about 2 hours. In one embodiment, the virus inactivating agent is 0.5% Triton X-100 (w / w) maintained for about 30 minutes to about 240 minutes, e.g., 130 minutes.

[0107] Anion exchange chromatography In another aspect of the invention, fractions or samples containing the albumin fusion protein are subjected to anion exchange chromatography. Anion exchange may be performed via a bind-and-elute system, a flow-through system, or both. Any suitable anion exchange matrix may be used. In one embodiment, the anion exchange matrix may be a resin, such as agarose or Sepharose, or a synthetic microporous or macroporous membrane. Suitable anion exchange bind-and-elute matrices include, for example, Q-resin, quaternary amines, and DEAE. Commercially available matrices include, for example, Capto Q, Toyopearl SuperQ, ANX, DEAE, Q-Sepharose, Q-Sepharose FF, Q-Sepharose HP, and Q-Sepharose XL, Q-Hyper D, DEAE-cellulose, QAE-cellulose, TMAE, DMAE, or DEAE Fractogel, Mustang Q, Sartobind Q, or Sartobind STIC PA. Such matrices can include highly cross-linked agarose or can be polymers with, for example, polyethersulfone, polypropylene, methacrylate, or polypropylate substrates. Column load challenges range from about 0.1 to about 50 g / L, about 0.5 to about 40 g / L, about 1 to about 30 g / L, or about 5 to about 25 g / L. Membrane loads range from about 0.1 to about 10 g / mL, about 0.2 to about 5.0 g / mL, about 0.5 to about 2.5 g / mL, or about 1.0 to about 2.0 g / mL.

[0108] In another embodiment, the matrix is ​​modified to enhance the purification of albumin fusion proteins. For example, in one embodiment, the matrix is ​​highly cross-linked agarose with a dextran surface extender. In another embodiment, a polyethersulfone-based matrix is ​​modified with a quaternary amine. In another embodiment, a polypropylene-based matrix is ​​modified with a quaternary amine.

[0109] When using a bind-and-elute system, the anion exchange chromatography step may include an equilibration step using buffers such as phosphate, Tris, and Bis-Tris at neutral or slightly acidic pH. The sample is loaded and the matrix is ​​optionally re-equilibrated. The loading buffer is optimized based on the pH and the resin being used, as is known in the art, and to optimize separation of the target albumin fusion protein. Suitable loading buffers include a base, such as Tris or Bis-Tris, in the range of about 5 mM to about 200 mM, about 10 mM to about 150 mM, about 20 mM to about 100 mM, about 30 mM to about 80 mM, or about 50 mM, and a salt, such as NaCl or octanoic acid, in an amount of about 5 mM to about 100 mM, about 10 mM to about 50 mM, or about 20 mM. In one embodiment, a suitable loading buffer for anion exchange comprises 50 mM BisTris, 20 mM NaCl at pH 7.0.

[0110] In a bind-and-elute system, after equilibration of the anion exchange matrix, a sample containing the albumin fusion protein is loaded, and the desired protein is bound to the anion exchange matrix. The affinity chromatography column containing the bound albumin fusion protein is washed with a wash buffer to remove materials present in the solution other than the albumin fusion protein. In some embodiments, the wash buffer is the same as the loading buffer. In some embodiments, the wash buffer comprises 50 mM Bis-Tris, 20 mM NaCl at pH 7.0.

[0111] The bound albumin fusion protein is eluted from the anion exchange matrix by either step elution or gradient elution. In one embodiment, the anion exchange matrix elution buffer contains a salt, such as NaCl, CaCl, or KCl. The salt concentration of the buffer ranges from about 10 mM, about 10 mM to about 150 mM, about 20 mM to about 400 mM, about 50 mM to about 300 mM, about 20 mM to about 140 mM, about 30 mM to about 130 mM, about 40 mM to about 120 mM, or about 50 mM to about 110 mM. The pH range for elution varies from less than about 9, about 6 to about 8, about 6 to about 7.5, about 6 to about 7, or about 6.5 to about 7. In some embodiments, the bound albumin fusion protein is eluted from the matrix using a linear gradient of about 10 mM to about 600 mM salt, e.g., NaCl, or about 20 mM to about 400 mM salt, e.g., NaCl.

[0112] In accordance with the present invention, the anion exchange binding and elution system results in increased monomer content by reducing aggregation products and removes impurities that are involved in the oxidation of the albumin fusion protein and have low levels of oxidation products. In one embodiment, the intermediate metabolites from this step containing the albumin fusion protein have less than about 100%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, or less than about 2% oxidation products relative to total protein. In one embodiment, the intermediate metabolites from this step containing the albumin fusion protein have less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, or less than about 2% oxidized tryptophan residues relative to the total number of tryptophan residues. In another embodiment, the anion exchange binding and elution step removes more than 5, 10, 15, 20, or 30 orders of host cell protein, or at least 5-30, 10-30, 10-40, 15-30, 15-40, 20-30, or 20-40 orders of host cell protein from the original sample. In one embodiment of the invention, the anion exchange binding and elution step removes at least 3, 4, 5, or 6 orders of DNA impurities, or 1-2, 2-3, or 3-4 orders of DNA impurities from the original sample.

[0113] In some embodiments, the anion exchange chromatography matrix is ​​in flow-through mode using a membrane. In some embodiments, the albumin fusion protein is applied to both the anion exchange chromatography matrix and the anion exchange membrane. The membrane may be pre-conditioned and equilibrated prior to loading. Additionally, the pH of the loading buffer may be adjusted to prevent the target albumin fusion protein from binding to the anion exchange matrix. In this manner, any contaminating materials, including DNA, host cell proteins (HCPs), viruses, and small molecule impurities, may be separated from the target albumin fusion protein.

[0114] According to the present invention, in one embodiment, the membrane functions at a pH range of less than about 9, about 6 to about 8, about 6.5 to about 7.5, about 6 to about 7.5, or about 7 to about 7.5, or a pH of about 6, 7, 8, or 9. In another embodiment, the salt concentration of the buffer is greater than 10 mM or in the range of about 10 mM to about 200 mM, about 40 mM to about 180 mM, about 50 mM to about 150 mM, about 60 mM to about 120 mM, or about 60 mM to about 80 mM. In some embodiments, the salt concentration of the buffer is about 50 mM, about 60 mM, or about 70 mM. In other embodiments, the flow-through buffer has a salt concentration of 10 mM to 150 mM and a pH of 6 to 8. In another embodiment, the flow-through buffer has a salt concentration of greater than 10 mM and a pH of less than 8. In particular, both yield and DNA clearance were found to be optimal for albumin fusion proteins at low pH, eg, about 7 to about 7.5, and higher salt concentrations, eg, greater than 60 mM salt.

[0115] According to the present invention, an anion exchange flow-through system results in increased monomer content with low levels of oxidation products and removal of impurities, including HCPs and DNA. In one embodiment, the product from this step containing albumin fusion protein has less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, or less than about 2% oxidation products relative to total protein. In another embodiment, the product from this step containing albumin fusion protein has less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, or less than about 2% oxidized tryptophan residues relative to the total number of tryptophan residues. In another embodiment, the flow-through anion exchange step removes more than one or more orders of magnitude of host cell protein from the original sample. In one embodiment of the invention, the flow-through anion exchange step removes at least 3, 4, 5, or 6, 8, 9, or 10 orders of magnitude, or 3-6, 4-6, or 5-6, 8-10, or 9-10 orders of magnitude of DNA impurities from the original sample.

[0116] According to the present invention, the bind-and-elute mode of anion exchange may be performed in combination with the flow-through mode of anion exchange. Further optional purification steps may be performed before, during, or after the anion exchange step. For example, to inactivate enveloped viruses, a sample containing the albumin fusion protein may be treated with Triton X-100. Alternatively, the sample may undergo diafiltration or ultrafiltration. Salt may be added at a suitable concentration. In one embodiment, the eluate containing the albumin fusion protein is diafiltered against 50 mM Bis-Tris, 20 mM NaCl at pH 7.0.

[0117] Additional purification steps An additional purification step may include subjecting the eluate / fractions containing the albumin fusion protein to a hydrophobic interaction or multimodal matrix. The hydrophobic interaction matrix may be any suitable matrix. Optionally, the hydrophobic interaction matrix comprises phenyl, octyl, or butyl hydrophobic groups. Hydrophobic interaction matrices are commercially available and known to those skilled in the art, such as Capto Butyl, Capto Phenyl, Capto Butyl, Butyl-S Fast Flow (GE Healthcase Life Sciences, Piscataway, NJ), Toyopearl Hexyl, Toyopearl Butyl, Toyopearl Phenyl, Toyopearl PPG, Toyopearl Ether, Toyopearl PPG-600M, and Toyopearl Phenyl-650M, Toyopearl PPG-600M, TSKgel Phenyl, TSKgel Ether (TOSOH Corporations, Tokyo, Japan), and Macro-Prep Methyl (Bio-Rad Laboratories, Hercules, CA). The multimodal matrix may be any suitable matrix. Optionally, the multimodal matrix contains phenyl, octyl, or butyl hydrophobic groups along with cation or anion exchange groups. Multimodal matrices are commercially available and known to those of skill in the art, and include, for example, Capto MMC, Eshmuno HCX, Nuvia cPrime, or Toyopearl MX-Trp-650M. It has been found that albumin fusion proteins are optionally equilibrated with a buffer containing a salt, e.g., ammonium, lithium, potassium, magnesium, calcium, aluminum, or guanidinium salt as the cation, and / or sulfate, phosphate, citrate, tartrate, chloride, bromide, iodide, nitrate, or chlorate as the anion.For example, in some embodiments, the salt is a suitable amount of sodium chloride, sodium sulfate, sodium citrate, or ammonium sulfate, e.g., about 100 mM to about 2 M, about 200 mM to about 1.5 M, about 300 mM to about 1 M, or about 400 mM to about 800 mM salt, e.g., citrate. After equilibration, the sample / fraction containing the albumin fusion protein is loaded onto the column. In one embodiment, the column is re-equilibrated and then eluted using a step or gradient against a buffer having a decreasing salt concentration.

[0118] In another embodiment, the fraction may be further purified by subjecting the eluate / fraction containing the albumin fusion protein to nanofiltration. In some embodiments, nanofiltration may be used to remove potential viral particles and can be performed by standard methods to those skilled in the art.

[0119] In other embodiments, fractions may be subjected to size exclusion chromatography to further purify the albumin fusion protein.

[0120] In another embodiment of the present invention, a method for obtaining a composition comprising albumin fusion proteins essentially free of oxidized tryptophan residues is provided. According to this embodiment, the method comprises the step of subjecting a composition comprising tryptophan-oxidized and non-tryptophan-oxidized albumin fusion proteins to a hydrophobic interaction matrix, such that the tryptophan-oxidized and non-tryptophan-oxidized albumin fusion proteins are eluted from the hydrophobic interaction matrix at different times, thereby separating the tryptophan-oxidized albumin fusion proteins from the non-tryptophan-oxidized albumin fusion proteins.

[0121] Another embodiment of the present invention is directed to a method for isolating an albumin fusion protein essentially free of oxidation of tryptophan and / or methionine residues. According to this embodiment, a composition containing the albumin fusion protein is subjected to the following purification processes: (a) an affinity matrix chromatography process; (b) an anion exchange chromatography process; and (c) a hydrophobic interaction matrix chromatography process. An elution buffer for the affinity matrix chromatography process containing caprylic acid / octanoic acid, and in some embodiments, EDTA, is applied to the affinity matrix. Furthermore, tryptophan-oxidized and non-tryptophan-oxidized albumin fusion proteins are eluted from the hydrophobic interaction matrix at different times, thereby separating the tryptophan-oxidized albumin fusion protein from the non-tryptophan-oxidized albumin fusion protein.

[0122] Another aspect of the invention is a method of purifying an albumin fusion protein, comprising subjecting a composition comprising the albumin fusion protein to a hydrophobic interaction matrix and one or more of the following purification processes: (a) an affinity matrix to which an elution buffer comprising caprylic acid / octanoic acid, and in some embodiments further comprising EDTA, is applied; and / or (b) an anion exchange matrix. According to this embodiment, the affinity matrix may be washed with a wash buffer containing: (1) about 2% to about 20% of a polyol selected from the group consisting of 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,6-hexanediol, and 2-methyl-2,4-pentanediol; (2) 0.05M to 2.0% of a salt selected from sodium chloride, potassium chloride, calcium chloride, lithium chloride, sodium bromide, potassium bromide, and lithium bromide; (3) about 0.02M to about 1M sodium sulfate; (4) about 0.01% to about 1% of a non-ionic surfactant; (5) about 0.05M to about 1.0M urea; and / or (6) about 0.02M to about 0.5M nicotinamide. The resulting purified albumin fusion protein is essentially free of oxidized tryptophan residues.

[0123] A method for purifying an albumin fusion protein comprises applying a composition comprising the albumin fusion protein to an affinity matrix; eluting the albumin fusion protein from the affinity matrix to obtain a first eluate; applying the first eluate to an anion exchange matrix; eluting the albumin fusion protein from the anion exchange matrix to obtain a second eluate; applying the second eluate to an anion exchange membrane; passing the albumin fusion protein through the anion exchange membrane to obtain a flow-through; applying the flow-through to a hydrophobic interaction matrix; and eluting the albumin fusion protein from the hydrophobic interaction matrix to obtain a third eluate, wherein the third eluate comprises purified albumin fusion protein. According to this embodiment, the resulting purified albumin fusion protein has no more than 5% oxidized tryptophan residues.

[0124] Compositions of purified albumin fusion proteins Compositions containing purified albumin fusion proteins are included within the scope of the present invention. These compositions are characterized by low levels of host cell proteins, DNA, and viral activity. In addition, these compositions containing purified albumin fusion proteins have low levels of oxidation and retained biological activity.

[0125] Compositions or fractions comprising albumin fusion proteins purified according to the invention have less than about 1000 ng / mg, 200 ng / mg, 100 ng / mg, 50 ng / mg, 40 ng / mg, 30 ng / mg, 20 ng / mg, or 10 ng / mg of host cell protein. In one embodiment, an albumin fusion protein-containing composition has less than 20 ng / mg of host cell protein. In some embodiments, albumin fusion protein compositions have levels of host cell protein acceptable to governmental organizations, such as the United States Food and Drug Administration, for administration to human subjects.

[0126] Additionally, compositions or fractions containing purified albumin fusion proteins according to the invention may contain up to about 5×10 -2 Less than 1×10 -2 , 5×10 -3 , 1×10 -3 , 5×10 -4 , or 1×10 -4 In one embodiment, the albumin fusion protein purified according to the invention has a 5×10 -3 In some embodiments, the albumin fusion protein compositions have levels of DNA acceptable to governmental organizations, such as the United States Food and Drug Administration, for administration to human subjects.

[0127] It has been found that oxidation of tryptophan / methionine residues in albumin fusion proteins can affect the biological activity and relative potency of the protein. Albumin fusion proteins purified and obtained according to the methods of the present invention have low levels of oxidation. In one embodiment of the present invention, the relative potency of the albumin fusion protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%. In another embodiment, the albumin fusion protein has less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% of oxidized tryptophan residues relative to the total number of tryptophan residues in the protein. In one embodiment, the albumin fusion protein has less than about 20% oxidized tryptophan residues relative to the total number of tryptophan residues in the protein. In another embodiment, the albumin fusion protein has less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% of oxidized tryptophan residues relative to the total number of tryptophan residues in the protein. In one embodiment of the invention, the albumin fusion protein has less than about 5% oxidized tryptophan residues relative to the total number of tryptophan residues in the protein.

[0128] Compositions within the scope of the present invention include albumin fusion proteins, and the compositions contain 5×10 -3 In one embodiment, the composition has less than 5×10 ng / mg of DNA and less than 15% of the tryptophan residues are oxidized. -3 having less than ng / mg of DNA and having albumin fusion proteins in which less than 5% of the tryptophan residues are oxidized.

[0129] Another composition of the invention comprises an albumin fusion protein, wherein the composition has less than 20 ng / mg of host cell protein, and the albumin fusion protein has a relative activity of >90%.

[0130] One embodiment of the present invention is a composition comprising an albumin fusion protein, the composition comprising 5×10 -3 With less than ng / mg of DNA, the albumin fusion protein has a relative activity of >90%.

[0131] Albumin fusion proteins Albumin, such as human serum albumin (HSA), or a fragment or variant thereof, may be fused or conjugated to a therapeutic protein to increase or prolong the half-life of the protein in the bloodstream and / or its tissue penetration. In some embodiments, the property improved by conjugation with an HSA variant is plasma half-life. An improvement in the plasma half-life of an albumin fusion protein can be an alteration in its properties, such as an increase or decrease in plasma half-life, or a change in other pharmacokinetic parameters.

[0132] Fragments or variants of albumin or HSA that extend or increase the in vivo or serum half-life of therapeutic proteins are included within the scope of the present invention. HSA variants, i.e., molecules derived from full-length HSA (SEQ ID NO: 138), containing at least amino acid substitutions, deletions, or sequence truncations have been previously disclosed. For example, the following publications describe HSA variants that may be used: WO 2011 / 103076, WO 2011 / 051489, and WO 2012 / 112188. In one embodiment, the albumin is HSA. In another embodiment, the albumin is a variant HSA.

[0133] In some embodiments, the HSA variant is a mutant derived from full-length HSA (SEQ ID NO: 138). In a specific embodiment, the HSA variant comprises a substitution of cysteine ​​with serine at position 34 (SEQ ID NO: 133). For example, HSA variants that can be used to modify the plasma half-life of the Tn3 scaffold are described, for example, in WO 2011 / 103076 and WO 2011 / 051489 (both of which are incorporated by reference in their entireties). In some embodiments, the plasma half-life of a therapeutic protein of the invention is increased by fusing it to an HSA variant comprising at least one amino acid substitution within domain III of HSA. Another embodiment includes where the amino acid sequence of the variant HSA is SEQ ID NO: 133.

[0134] In some embodiments, the albumin fusion proteins of the invention comprise an HSA variant or fragment thereof comprising the sequence of full-length mature HSA (SEQ ID NO: 138) except for at least one amino acid substitution at a position selected from the group consisting of: 407, 415, 463, 500, 506, 508, 509, 511, 512, 515, 516, 521, 523, 524, 526, 535, 550, 557, 573, 574, and 580, numbered relative to the position in full-length mature HSA; wherein the at least one amino acid substitution does not include lysine (K) to glutamic acid (E) at position 573, and wherein the therapeutic protein has a plasma half-life that is greater than the plasma half-life of the same therapeutic protein not conjugated to the HSA variant.

[0135] In some other embodiments, the at least one amino acid substitution, numbered relative to positions in full-length mature HSA, is at a position selected from the group consisting of 463, 508, 523, and 524, wherein the therapeutic protein has a plasma half-life greater than the plasma half-life of the therapeutic protein unconjugated to the HSA variant.

[0136] In other embodiments, the albumin fusion proteins of the invention comprise: (a) substitution of leucine (L) at position 407 with asparagine (N) or tyrosine (Y); (b) substitution of valine (V) with threonine (T) at position 415; (c) substitution of leucine (L) with asparagine (N) at position 463; (d) substitution of lysine (K) with arginine (R) at position 500; (e) substitution of threonine (T) with tyrosine (Y) at position 506; (f) substitution of threonine (T) with arginine (R) at position 508; (g) substitution of phenylalanine (F) at position 509 with methionine (M) or tryptophan (W); (h) substitution of alanine (A) with phenylalanine (F) at position 511; (i) substitution of aspartic acid (D) with tyrosine (Y) at position 512; (j) substitution of threonine (T) with glutamine (Q) at position 515; (k) substitution of leucine (L) at position 516 with threonine (T) or tryptophan (W); (l) substitution of arginine (R) with tryptophan (W) at position 521; (m) substitution of isoleucine (I) at position 523 with aspartic acid (D), glutamic acid (E), glycine (G), lysine (K), or arginine (R); (n) substitution of lysine (K) with leucine (L) at position 524; (o) substitution of glutamine (Q) with methionine (M) at position 526; (p) substitution of histidine (H) with proline (P) at position 535; (q) substitution of aspartic acid (D) with glutamic acid (E) at position 550; (r) substitution of lysine (K) with glycine (G) at position 557; (s) substitution of lysine (K) at position 573 with phenylalanine (F), histidine (H), proline (P), tryptophan (W), or tyrosine (Y); (t) substitution of lysine (K) with asparagine (N) at position 574; (u) substitution of glutamine (Q) with lysine (K) at position 580; and (v) a combination of two or more of the foregoing substitutions and wherein the therapeutic protein has a plasma half-life greater than the plasma half-life of the same therapeutic protein not conjugated to said HSA variant.

[0137] In some embodiments, the albumin fusion protein comprises: (a) substitution of leucine (L) with asparagine (N) at position 463; (b) substitution of threonine (T) with arginine (R) at position 508; (c) substitution of isoleucine (I) at position 523 with aspartic acid (D), glutamic acid (E), glycine (G), lysine (K), or arginine (R); (d) substitution of lysine (K) with leucine (L) at position 524; and (e) a combination of two or more of the foregoing substitutions and wherein the therapeutic protein has a plasma half-life greater than the plasma half-life of the same therapeutic protein not conjugated to the HSA variant.

[0138] Albumin fusion proteins may be produced by standard techniques, for example, by expression of the fusion protein from a recombinant fusion gene constructed using publicly available gene sequences.

[0139] A therapeutic protein may be any protein that can be fused or conjugated to albumin to increase or extend its half-life. In one embodiment, the therapeutic protein comprises a scaffold moiety containing a tryptophan residue, where oxidation of the tryptophan reduces the biological activity of the albumin fusion protein. In another embodiment, the protein is capable of binding to CD40L. In another embodiment, the therapeutic protein is a scaffold moiety capable of binding to CD40L. Another embodiment provides that the scaffold moiety comprises a third fibronectin type III (FnIII) ​​domain. Scaffolds containing FnIII domains have been previously described, for example, in WO 98 / 56915, WO 2009 / 023184, WO 2009 / 05379, WO 2010 / 051274, and WO 2010 / 093627. In some embodiments, the FnIII domain may be derived from human tenascin-C (Tn3 scaffold), such as those described in WO 2009 / 05379, WO 2010 / 051274, and WO 2013 / 055745.

[0140] Albumin fused to the scaffold In one embodiment of the present invention, the albumin fusion protein comprises a scaffold. For example, the scaffold may comprise CD40L-specific monomer subunits derived from the third FnIII domain of human tenascin-C (Tn3) in which at least one non-naturally occurring intramolecular disulfide bond has been engineered. The monomer subunits forming the Tn3 scaffold of the present invention fold correctly independent of one another, retaining their binding specificity and affinity, and each of the monomer scaffolds retains its functional properties. When the monomer subunits are assembled into a high-valency multimeric Tn3 scaffold, the monomer subunits fold correctly independent of one another, retaining their binding specificity and affinity, and each of the monomers retains its functional properties.

[0141] Scaffolds of the invention comprising two or more monomeric subunits can bind to multiple epitopes, e.g., (i) multiple epitopes in a single target, (ii) a single epitope in multiple targets, (iii) multiple epitopes located on different subunits of a target, or (iv) multiple epitopes on multiple targets, thereby enhancing binding activity.

[0142] Furthermore, due to the possibility of varying the distance between multiple monomers via the linker, the multimeric Tn3 scaffold has the ability to bind to multiple target molecules on a surface (either on the same cell / surface or in different cells / surfaces). As a result of its ability to simultaneously bind two or more targets, the Tn3 multimeric scaffold of the present invention can be used to regulate multiple pathways, cross-link receptors on cell surfaces, bind to cell surface receptors on separate cells, and / or attach target molecules or cells to substrates.

[0143] Additionally, the present invention provides affinity-matured scaffolds, in which the affinity of the scaffold for a specific target is modulated through mutation. Additionally, the present invention provides methods for producing the scaffolds of the present invention, as well as methods for modifying the scaffolds to have desirable physicochemical, pharmacological, or immunological properties. Additionally, the present invention provides methods for the use of such scaffolds, as well as for therapeutic, prophylactic, and diagnostic uses.

[0144] In one embodiment, the albumin fusion protein has a Tn3 scaffold, such as that described in PCT Publication WO 2013 / 055745, filed October 10, 2012, and incorporated herein by reference. It has been found that tryptophan and methionine residues are susceptible to oxidation during the purification of albumin-Tn3 scaffold fusion proteins. For example, when the Tn3 scaffold is selected from the albumin fusion proteins of SEQ ID NOs: 134, 135, 201, 202, 203, 204, 205, 206, 207, or 208, it has been found that oxidation can occur at tryptophan amino acid residues W46 / 151 on the binding loop of Tn3, and at methionine amino acid residues M74 / 179, M498, and M529 on Tn3 and human serum albumin during the purification process. Impact studies have shown that oxidation at W46 / 151, M74 / 179, M498, and M529 of the albumin-Tn3 scaffold protein can affect biological activity. In particular, oxidation at W46 / 151 on the binding loop of Tn3 was found to negatively affect the biological activity and relative potency of MEDI4920. However, oxidation at M74 / 179, M498, and M529 had less of an effect on the biological activity of the fusion protein. Nevertheless, the goal of the present invention is to reduce oxidized species of albumin fusion proteins throughout the purification process, with the intention of controlling the oxidation of sensitive amino acids in albumin fusion proteins.

[0145] FnIII structural motif Suitable scaffolds of the present invention include those based on the structure of the fibronectin type III module (FnIII), which is a domain that is widely found throughout all three domains of living organisms and viruses and in many protein classes. In certain embodiments, the scaffold of the present invention is derived from the third FnIII domain of human tenascin-C (see International Application No. PCT / US2008 / 012398, published as WO2009 / 058379; PCT / US2011 / 032184, published as WO2011 / 130324; and International Application No. PCT / US2011 / 032188, published as WO2011130328).

[0146] In a specific embodiment, the Tn3 scaffold of the present invention comprises a monomeric subunit specific for CD40L derived from the parent Tn3 scaffold. The overall three-dimensional fold of the monomer is closely related to that of the variable region of the heavy chain (VH), the smallest functional antibody fragment that contains the entire antigen recognition unit in single-domain antibodies of camelids and camelids (e.g., llamas).

[0147] The Tn3 monomer subunit of the present invention and the native FnIII domain from tenascin-C are characterized by the same three-dimensional structure: a β-sandwich structure with three β-strands (A, B, and E) on one side and four β-strands (C, D, F, and G) on the other side, connected by six loop regions. These loop regions are named according to the β-strands connected to the N- and C-termini of each loop. Thus, the AB loop is located between β-strands A and B, the BC loop is located between strands B and C, the CD loop is located between β-strands C and D, the DE loop is located between β-strands D and E, the EF loop is located between β-strands E and F, and the FG loop is located between β-strands F and G. FnIII domains have solvent-exposed loops that are resistant to randomization, facilitating the generation of a diverse pool of protein scaffolds capable of binding specific targets with high affinity.

[0148] In one embodiment of the present invention, Tn3 monomer subunits are subjected to directed evolution designed to randomize one or more loops that resemble the complementarity-determining regions (CDRs) of antibody variable regions. Such a directed evolution approach results in the production of antibody-like molecules with high affinity for a target of interest, e.g., CD40L.

[0149] Furthermore, in some embodiments, the Tn3 scaffolds described herein can be used to present defined exposed loops (e.g., loops that have been pre-randomized and selected based on target binding) to guide the evolution of molecules that bind to such introduced loops. This type of selection can be performed to identify recognition molecules for any individual CDR-like loop, or to recognize two or all three CDR-like loops combined with a nonlinear epitope-binding moiety. Three sets of loops (designated BC, DE, and FG) that can confer specific target binding operate between strands B and C; between strands D and E; and between β-strands F and G, respectively. The BC, DE, and FG loops of the third FnIII domain of human tenascin-C are 9, 6, and 10 amino acid residues in length, respectively. The lengths of these loops fall within the narrow range of cognate antigen-recognition loops found in antibody heavy chains, i.e., 7-10, 4-8, and 4-28 amino acids in length, respectively. Similarly, a second set of loops, the AB, CD, and EF loops (7, 7, and 8 amino acids long, respectively), operate between β-strands A and B; between β-strands C and D; and between β-strands E and F, respectively.

[0150] When loops within the Tn3 monomer scaffold are randomized and selected for high-affinity binding to a target, they may contact the target in a manner comparable to that of the cognate CDR loops in an antibody. Thus, in some embodiments, the AB, CD, and EF loops are randomized and selected for high-affinity binding to one or more targets, such as CD40L. In some embodiments, this randomization and selection process may be performed in parallel with the randomization of the BC, DE, and FG loops, while in other embodiments, this randomization and selection process is performed sequentially.

[0151] CD40L-specific monomeric subunit The present invention provides a non-naturally occurring recombinant Tn3 scaffold specific for CD40L comprising multiple β-strand domains linked to multiple loop regions, wherein one or more of said loop regions is altered by the deletion, substitution, or addition of at least one amino acid from its cognate loop in wild-type Tn3 (SEQ ID NO: 3) (see Table 1).

[0152] To generate improved CD40L-specific Tn3 monomer subunits with novel binding properties, amino acid additions, deletions, or substitutions are made to the parent Tn3. It will be understood that the same definitions of β-strands and loops are used when comparing the sequence of a CD40L-specific Tn3 monomer subunit with the sequence of the parent Tn3. In some embodiments, the CD40L-specific Tn3 monomer subunits of the present invention have the amino acid sequence: [ka] where: (a)X AB , X BC , X CD , X DE , X EF , and X FG represent the amino acid residues present in the sequences of the AB, BC, CD, DE, EF, and FG loops, respectively; (b) X1 represents the amino acid residue alanine (A) or threonine (T); and (c) The loop length n is an integer from 2 to 26.

[0153] [Table 2]

[0154] [Table 3]

[0155] In some embodiments, the CD40L-specific Tn3 monomer subunit of the present invention has the amino acid sequence: [ka] where: (a)X AB , X BC , X CD , X DE , X EF , and X FG represent the amino acid residues present in the sequences of the AB, BC, CD, DE, EF, and FG loops, respectively; (b) X1 represents the amino acid residue alanine (A) or threonine (T); and (c) The loop length n is an integer from 2 to 26.

[0156] In one embodiment, the β-strands of the CD40L-specific Tn3 monomer scaffold have at least 90% sequence identity to the β-strands of the parent Tn3 scaffold (SEQ ID NO: 3). To calculate such percentage of sequence identity, the amino acid sequences are aligned using methods known in the art. The percentage of sequence identity is defined as the ratio of (a) the number of amino acids located in the β-strands that are identical in the sequence alignment to (b) the total number of amino acids located in the β-strands.

[0157] In one embodiment, the sequence of the AB loop comprises SEQ ID NO:4 or SEQ ID NO:136. In another embodiment, the sequence of the CD loop comprises SEQ ID NO:6. In another embodiment, the sequence of the EF loop comprises SEQ ID NO:8 or SEQ ID NO:137. In one embodiment, the sequence of the AB loop consists of SEQ ID NO:4 or SEQ ID NO:136. In another embodiment, the sequence of the CD loop consists of SEQ ID NO:6. In another embodiment, the sequence of the EF loop consists of SEQ ID NO:8 or SEQ ID NO:137.

[0158] In one embodiment, the sequence of the BC loop comprises a sequence selected from the group consisting of SEQ ID NOs: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 and 93. In another embodiment, the sequence of the BC loop consists of a sequence selected from the group consisting of SEQ ID NOs: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 and 93.

[0159] In one embodiment, the sequence of the DE loop comprises a sequence selected from the group consisting of SEQ ID NOs: 94, 95, 96, 97, and 98. In another embodiment, the sequence of the DE loop consists of a sequence selected from the group consisting of SEQ ID NOs: 94, 95, 96, 97, and 98.

[0160] In one embodiment, the sequence of the FG loop comprises a sequence selected from the group consisting of SEQ ID NOs: 9, 99, and 139. In another embodiment, the sequence of the FG loop consists of a sequence selected from the group consisting of SEQ ID NOs: 9, 99, and 139.

[0161] In one embodiment, the sequence of the BC loop comprises a sequence selected from the group consisting of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 and 117. In another embodiment, the sequence of the BC loop consists of a sequence selected from the group consisting of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 and 117.

[0162] In some embodiments, the sequence of the DE loop comprises a sequence selected from the group consisting of SEQ ID NOs: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, and 128. In other embodiments, the sequence of the DE loop consists of a sequence selected from the group consisting of SEQ ID NOs: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, and 128.

[0163] In some embodiments, the sequence of the FG loop comprises a sequence selected from the group consisting of SEQ ID NOs: 129 and 130. In other embodiments, the sequence of the FG loop consists of a sequence selected from the group consisting of SEQ ID NOs: 129 and 130.

[0164] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 83, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139. In some embodiments, the sequence of the BC loop consists of SEQ ID NO: 83, the sequence of the DE loop consists of SEQ ID NO: 94, and the sequence of the FG loop consists of SEQ ID NO: 9 or 139.

[0165] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 83, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 99. In other embodiments, the sequence of the BC loop consists of SEQ ID NO: 83, the sequence of the DE loop consists of SEQ ID NO: 94, and the sequence of the FG loop consists of SEQ ID NO: 99.

[0166] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 84, the sequence of the DE loop comprises SEQ ID NO: 95, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139. In other embodiments, the sequence of the BC loop consists of SEQ ID NO: 84, the sequence of the DE loop consists of SEQ ID NO: 95, and the sequence of the FG loop consists of SEQ ID NO: 9 or 139.

[0167] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 85, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139. In other embodiments, the sequence of the BC loop consists of SEQ ID NO: 85, the sequence of the DE loop consists of SEQ ID NO: 94, and the sequence of the FG loop consists of SEQ ID NO: 9 or 139.

[0168] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 86, the sequence of the DE loop comprises SEQ ID NO: 96, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139. In other embodiments, the sequence of the BC loop consists of SEQ ID NO: 86, the sequence of the DE loop consists of SEQ ID NO: 96, and the sequence of the FG loop consists of SEQ ID NO: 9 or 139.

[0169] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 87, the sequence of the DE loop comprises SEQ ID NO: 97, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139. In other embodiments, the sequence of the BC loop consists of SEQ ID NO: 87, the sequence of the DE loop consists of SEQ ID NO: 97, and the sequence of the FG loop consists of SEQ ID NO: 9 or 139.

[0170] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 88, the sequence of the DE loop comprises SEQ ID NO: 95, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139. In other embodiments, the sequence of the BC loop consists of SEQ ID NO: 88, the sequence of the DE loop consists of SEQ ID NO: 95, and the sequence of the FG loop consists of SEQ ID NO: 9 or 139.

[0171] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 89, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139. In other embodiments, the sequence of the BC loop consists of SEQ ID NO: 89, the sequence of the DE loop consists of SEQ ID NO: 94, and the sequence of the FG loop consists of SEQ ID NO: 9 or 139.

[0172] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 90, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139. In other embodiments, the sequence of the BC loop consists of SEQ ID NO: 90, the sequence of the DE loop consists of SEQ ID NO: 94, and the sequence of the FG loop consists of SEQ ID NO: 9 or 139.

[0173] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 91, the sequence of the DE loop comprises SEQ ID NO: 95, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139. In other embodiments, the sequence of the BC loop consists of SEQ ID NO: 91, the sequence of the DE loop consists of SEQ ID NO: 95, and the sequence of the FG loop consists of SEQ ID NO: 9 or 139.

[0174] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 92, the sequence of the DE loop comprises SEQ ID NO: 98, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139. In other embodiments, the sequence of the BC loop consists of SEQ ID NO: 92, the sequence of the DE loop consists of SEQ ID NO: 98, and the sequence of the FG loop consists of SEQ ID NO: 9 or 139.

[0175] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 93, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139. In other embodiments, the sequence of the BC loop consists of SEQ ID NO: 93, the sequence of the DE loop consists of SEQ ID NO: 94, and the sequence of the FG loop consists of SEQ ID NO: 9 or 139.

[0176] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 168, the sequence of the DE loop comprises SEQ ID NO: 169, and the sequence of the FG loop comprises SEQ ID NO: 170. In other embodiments, the sequence of the BC loop consists of SEQ ID NO: 168, the sequence of the DE loop consists of SEQ ID NO: 169, and the sequence of the FG loop consists of SEQ ID NO: 170.

[0177] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 100, the sequence of the DE loop comprises SEQ ID NO: 118, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the BC loop consists of SEQ ID NO: 100, the sequence of the DE loop consists of SEQ ID NO: 118, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0178] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 101, the sequence of the DE loop comprises SEQ ID NO: 119, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 101, the sequence of the DE loop consists of SEQ ID NO: 119, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0179] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 102, the sequence of the DE loop comprises SEQ ID NO: 120, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 102, the sequence of the DE loop consists of SEQ ID NO: 120, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0180] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 103, the sequence of the DE loop comprises SEQ ID NO: 121, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 103, the sequence of the DE loop consists of SEQ ID NO: 121, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0181] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 104, the sequence of the DE loop comprises SEQ ID NO: 122, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 104, the sequence of the DE loop consists of SEQ ID NO: 122, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0182] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 105, the sequence of the DE loop comprises SEQ ID NO: 121, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 105, the sequence of the DE loop consists of SEQ ID NO: 121, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0183] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 106, the sequence of the DE loop comprises SEQ ID NO: 123, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 106, the sequence of the DE loop consists of SEQ ID NO: 123, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0184] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 107, the sequence of the DE loop comprises SEQ ID NO: 123, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 107, the sequence of the DE loop consists of SEQ ID NO: 123, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0185] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 108, the sequence of the DE loop comprises SEQ ID NO: 118, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 108, the sequence of the DE loop consists of SEQ ID NO: 118, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0186] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 109, the sequence of the DE loop comprises SEQ ID NO: 123, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 109, the sequence of the DE loop consists of SEQ ID NO: 123, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0187] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 110, the sequence of the DE loop comprises SEQ ID NO: 121, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 110, the sequence of the DE loop consists of SEQ ID NO: 121, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0188] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 111, the sequence of the DE loop comprises SEQ ID NO: 123, and the sequence of the FG loop comprises SEQ ID NO: 130. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 111, the sequence of the DE loop consists of SEQ ID NO: 123, and the sequence of the FG loop consists of SEQ ID NO: 130.

[0189] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 108, the sequence of the DE loop comprises SEQ ID NO: 121, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 108, the sequence of the DE loop consists of SEQ ID NO: 121, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0190] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 112, the sequence of the DE loop comprises SEQ ID NO: 124, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 112, the sequence of the DE loop consists of SEQ ID NO: 124, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0191] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 113, the sequence of the DE loop comprises SEQ ID NO: 125, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 113, the sequence of the DE loop consists of SEQ ID NO: 125, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0192] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 114, the sequence of the DE loop comprises SEQ ID NO: 118, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 114, the sequence of the DE loop consists of SEQ ID NO: 118, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0193] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 115, the sequence of the DE loop comprises SEQ ID NO: 126, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 115, the sequence of the DE loop consists of SEQ ID NO: 126, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0194] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 116, the sequence of the DE loop comprises SEQ ID NO: 127, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 116, the sequence of the DE loop consists of SEQ ID NO: 127, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0195] In some embodiments, the sequence of the AB loop comprises SEQ ID NO: 136, the sequence of the BC loop comprises SEQ ID NO: 117, the sequence of the DE loop comprises SEQ ID NO: 128, and the sequence of the FG loop comprises SEQ ID NO: 129. In other embodiments, the sequence of the AB loop consists of SEQ ID NO: 136, the sequence of the BC loop consists of SEQ ID NO: 117, the sequence of the DE loop consists of SEQ ID NO: 128, and the sequence of the FG loop consists of SEQ ID NO: 129.

[0196] In some embodiments, the sequence of the BC loop comprises SEQ ID NO: 174, the sequence of the DE loop comprises SEQ ID NO: 175, and the sequence of the FG loop comprises SEQ ID NO: 177. In other embodiments, the sequence of the BC loop consists of SEQ ID NO: 174, the sequence of the DE loop consists of SEQ ID NO: 175, and the sequence of the FG loop consists of SEQ ID NO: 177.

[0197] In some embodiments, the CD40L-specific monomeric subunit comprises a sequence selected from the group consisting of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, and 146. In other embodiments, the CD40L-specific monomeric subunit consists of a sequence selected from the group consisting of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, and 146.

[0198] In some embodiments, the CD40L-specific monomer subunit comprises SEQ ID NO: 28 or 146. In other embodiments, the CD40L-specific monomer subunit consists of SEQ ID NO: 28 or 146.

[0199] In some embodiments, the CD40L-specific Tn3 monomer subunit of the present invention has the amino acid sequence: [ka] where: (a) X1 represents the amino acid residue serine (S) or leucine (L); (b) X2 represents the amino acid residue aspartic acid (D) or glutamic acid (E); (c) X3 represents the amino acid residue histidine (H), isoleucine (I), valine (V), phenylalanine (F), or tryptophan (W); (d) X4 represents the amino acid residue alanine (A), glycine (G), glutamic acid (E), or aspartic acid (D); (e) X5 represents the amino acid residue glutamic acid (E), leucine (L), glutamine (Q), serine (S), aspartic acid (D), or asparagine (N); (f) X6 represents the amino acid residue phenylalanine (F) or tyrosine (Y); (g) X7 represents the amino acid residue isoleucine (I), valine (V), histidine (H), glutamic acid (E), or aspartic acid (D); (h) X8 represents the amino acid residue glycine (G), tryptophan (W), or valine (V); (i) X9 represents the amino acid residue tryptophan (W), phenylalanine (F), or tyrosine (Y); (j)X 10 represents the amino acid residue serine (S), glutamine (Q), methionine (M), or histidine (H); (k)X 11 represents the amino acid residue tryptophan (W) or histidine (H); and (l)X 12 represents the amino acid residue arginine (R) or serine (S).

[0200] In some embodiments, the CD40L-specific Tn3 monomer subunit of the present invention has the amino acid sequence: [ka] where: (a) X1 represents the amino acid residue serine (S) or leucine (L); (b) X2 represents the amino acid residue aspartic acid (D) or glutamic acid (E); (c) X3 represents the amino acid residue histidine (H), isoleucine (I), valine (V), phenylalanine (F), or tryptophan (W); (d) X4 represents the amino acid residue alanine (A), glycine (G), glutamic acid (E), or aspartic acid (D); (e) X5 represents the amino acid residue glutamic acid (E), leucine (L), glutamine (Q), serine (S), aspartic acid (D), or asparagine (N); (f) X6 represents the amino acid residue phenylalanine (F) or tyrosine (Y); (g) X7 represents the amino acid residue isoleucine (I), valine (V), histidine (H), glutamic acid (E), or aspartic acid (D); (h) X8 represents the amino acid residue glycine (G), tryptophan (W), or valine (V); (i) X9 represents the amino acid residue tryptophan (W), phenylalanine (F), or tyrosine (Y); (j)X 10 represents the amino acid residue serine (S), glutamine (Q), methionine (M), or histidine (H); (k)X 11 represents the amino acid residue tryptophan (W) or histidine (H); and (l)X 12 represents the amino acid residue arginine (R) or serine (S).

[0201] In some embodiments, the CD40L-specific monomeric subunit comprises a sequence selected from the group consisting of SEQ ID NOs: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82. In some embodiments, the CD40L-specific monomeric subunit consists of a sequence selected from the group consisting of SEQ ID NOs: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82.

[0202] In some embodiments, the CD40L-specific Tn3 monomer subunit of the present invention has the amino acid sequence: [ka] where: (a) X1 represents the amino acid residue lysine (K) or glutamic acid (E); (b) X2 represents the amino acid residue threonine (T) or isoleucine (I); (c) X3 represents the amino acid residue asparagine (N) or alanine (A); (d) X4 represents the amino acid residue serine (S), leucine (L), alanine (A), phenylalanine (F), or tyrosine (Y); (e) X5 represents the amino acid residue tyrosine (Y), alanine (A), glycine (G), valine (V), isoleucine (I), or serine (S); (f) X6 represents the amino acid residue tyrosine (Y), serine (S), alanine (A), or histidine (H); (g) X7 represents the amino acid residue asparagine (N), aspartic acid (D), histidine (H), or tyrosine (Y); (h) X8 represents the amino acid residue leucine (L), phenylalanine (F), histidine (H), or tyrosine (Y); (i) X9 represents the amino acid residue histidine (H), proline (P), serine (S), leucine (L), or aspartic acid (D); (j)X 10 represents the amino acid residue glycine (G), phenylalanine (F), histidine (H), or tyrosine (Y); (k)X 11 represents the amino acid residue alanine (A) or threonine (T); (l)X 12 represents the amino acid residue serine (S), asparagine (N), glutamic acid (E), asparagine (R), or aspartic acid (D); (m)X 13represents the amino acid residue serine (S), glutamine (Q), threonine (T), asparagine (N), or alanine (A); (n)X 14 represents the amino acid residue proline (P), valine (V), isoleucine (I) or alanine (A), or no amino acid; (o)X 15 represents the amino acid residue isoleucine (I) or no amino acid; (p)X 16 represents the amino acid residue glutamic acid (E) or lysine (K); and (q)X 17 represents the amino acid residue serine (S) or asparagine (N).

[0203] In some embodiments, the CD40L-specific Tn3 monomer subunit of the present invention has the amino acid sequence: [ka] where: (a) X1 represents the amino acid residue lysine (K) or glutamic acid (E); (b) X2 represents the amino acid residue threonine (T) or isoleucine (I); (c) X3 represents the amino acid residue asparagine (N) or alanine (A); (d) X4 represents the amino acid residue serine (S), leucine (L), alanine (A), phenylalanine (F), or tyrosine (Y); (e) X5 represents the amino acid residue tyrosine (Y), alanine (A), glycine (G), valine (V), isoleucine (I), or serine (S); (f) X6 represents the amino acid residue tyrosine (Y), serine (S), alanine (A), or histidine (H); (g) X7 represents the amino acid residue asparagine (N), aspartic acid (D), histidine (H), or tyrosine (Y); (h) X8 represents the amino acid residue leucine (L), phenylalanine (F), histidine (H), or tyrosine (Y); (i) X9 represents the amino acid residue histidine (H), proline (P), serine (S), leucine (L), or aspartic acid (D); (j)X 10 represents the amino acid residue glycine (G), phenylalanine (F), histidine (H), or tyrosine (Y); (k)X 11 represents the amino acid residue alanine (A) or threonine (T); (l)X 12 represents the amino acid residue serine (S), asparagine (N), glutamic acid (E), asparagine (R), or aspartic acid (D); (m)X 13 represents the amino acid residue serine (S), glutamine (Q), threonine (T), asparagine (N), or alanine (A); (n)X 14 represents the amino acid residue proline (P), valine (V), isoleucine (I) or alanine (A), or no amino acid; (o)X 15 represents the amino acid residue isoleucine (I) or no amino acid; (p)X 16 represents the amino acid residue glutamic acid (E) or lysine (K); and (q)X 17 represents the amino acid residue serine (S) or asparagine (N).

[0204] In some embodiments, a monomer scaffold specific for CD40L comprises a Tn3 module, wherein one or more of the beta strands comprises at least one amino acid substitution, except that the cysteine ​​residues in beta strand C and beta strand F (SEQ ID NO: 13 or 14, respectively; and SEQ ID NO: 17) may not be substituted.

[0205] The loops connecting the various β-strands of a CD40L-specific monomer subunit can be randomized for length and / or sequence diversity. In one embodiment, a CD40L-specific monomer subunit has at least one loop that is randomized for length and / or sequence diversity. In one embodiment, at least one, at least two, at least three, at least four, at least five, or at least six loops of a CD40L-specific monomer subunit are randomized for length and / or sequence diversity. In one embodiment, at least one loop of a CD40L-specific monomer subunit is kept constant for length and / or sequence diversity, while at least one additional loop is randomized. In another embodiment, at least one, at least two, or all three of loops AB, CD, and EF are kept constant for length and / or sequence diversity, while at least one, at least two, or all three of loops BC, DE, and FG are randomized. In another embodiment, at least one, at least two, or at least three of loops AB, CD, and EF are randomized for length and / or sequence diversity, while at least one, at least two, or all three of loops BC, DE, and FG are randomized. In yet another embodiment, at least one, at least two, at least three, at least four, at least five, or all six of loops AB, CD, EF, BC, DE, and FG are randomized for length or sequence diversity.

[0206] In some embodiments, one or more residues within the loop are kept constant for length and / or sequence diversity, while other residues are randomized. In some embodiments, one or more residues within the loop are kept constant for length and / or sequence diversity with a predetermined limited number of different amino acids, while other residues are randomized. Thus, the CD40L-specific monomeric subunits of the present invention can comprise one or more loops with a degenerate consensus sequence and / or one or more invariant amino acid residues.

[0207] In one embodiment, a CD40L-specific monomer subunit of the present invention comprises a randomized AB loop. In another embodiment, a CD40L-specific monomer subunit of the present invention comprises a randomized BC loop. In one embodiment, a CD40L-specific monomer subunit of the present invention comprises a randomized CD loop. In one embodiment, a CD40L-specific monomer subunit of the present invention comprises a randomized DE loop. In one embodiment, a CD40L-specific monomer subunit of the present invention comprises a randomized EF loop.

[0208] In certain embodiments, CD40L-specific monomeric subunits of the present invention comprise an FG loop that is maintained at least one amino acid residue shorter than the cognate FG loop of the third FnIII domain of human tenascin-C and further randomized at one or more positions.

[0209] In certain embodiments, at least one of loops BC, DE, and FG is randomized, wherein β strand A comprises SEQ ID NO: 10 or 11, β strand B comprises SEQ ID NO: 12, β strand C comprises SEQ ID NO: 13 or 14, β strand D comprises SEQ ID NO: 15, β strand E comprises SEQ ID NO: 16, β strand F comprises SEQ ID NO: 17, and β strand G comprises SEQ ID NO: 18, the AB loop comprises SEQ ID NO: 4 or 136, the CD loop comprises SEQ ID NO: 6, and the EF loop comprises SEQ ID NO: 8 or 137.

[0210] In other specific embodiments, at least one of loops AB, CD, and EF is randomized, wherein β strand A comprises SEQ ID NO: 10 or 11, β strand B comprises SEQ ID NO: 12, β strand C comprises SEQ ID NO: 13 or 14, β strand D comprises SEQ ID NO: 15, β strand E comprises SEQ ID NO: 16, β strand F comprises SEQ ID NO: 17, and β strand G comprises SEQ ID NO: 18, the BC loop comprises SEQ ID NO: 5, the DE loop comprises SEQ ID NO: 7, and the FG loop comprises SEQ ID NO: 9 or 139.

[0211] The stability of the Tn3 scaffold of the present invention may be enhanced by a variety of approaches. In some embodiments, the Tn3 scaffold of the present invention may be stabilized by extending the N- and / or C-terminal regions. The N- and / or C-terminal regions may be extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acids. In other embodiments, the Tn3 scaffold of the present invention may be stabilized by introducing modifications that increase serum half-life, as described herein. In yet another embodiment, the Tn3 scaffold of the present invention comprises the addition, deletion, or substitution of at least one amino acid residue to stabilize the hydrophobic core of the scaffold.

[0212] The Tn3 scaffolds of the present invention can be effectively stabilized by engineering non-native disulfide bonds, as disclosed in International Patent Application No. PCT / US2011 / 032184. In some embodiments, the scaffolds of the present invention comprise non-native disulfide bonds, as described in PCT Publication No. WO2009 / 058379. Bioinformatics methods can be used to identify suitable candidate positions for disulfide bond engineering.

[0213] In one embodiment, the Tn3 monomer subunit of the present invention comprises at least one, at least two, at least three, at least four, or at least five non-naturally occurring intramolecular disulfide bonds. In one embodiment, the Tn3 monomer subunit of the present invention comprises at least one non-naturally occurring intramolecular disulfide bond that stabilizes the monomer. In yet another embodiment, the Tn3 scaffold of the present invention comprises at least one non-naturally occurring disulfide bond, where the bond is located between two different monomeric or multimeric Tn3 scaffolds, i.e., the disulfide bond is an intermolecular disulfide bond. For example, the disulfide bond can link different scaffolds (e.g., two CD40L-specific monomeric scaffolds), a Tn3 scaffold to a linker, a Tn3 scaffold to an Fc domain, or a Tn3 scaffold to its antibody or fragment.

[0214] In some embodiments, the Tn3 scaffold of the invention comprises at least one non-naturally occurring intermolecular disulfide bond linking the Tn3 monomer subunit and the heterologous moiety isolated, fused or conjugated to the same Tn3 scaffold as the Tn3 monomer subunit, or fused or conjugated to a different Tn3 scaffold than the Tn3 monomer subunit.

[0215] In some embodiments, the Tn3 scaffold of the invention comprises disulfide bonds that form a Tn3 multimeric scaffold of at least two, at least three, at least four or more monomeric subunits.

[0216] In another embodiment, the Tn3 scaffold of the present invention may comprise an extension of the N- and / or C-terminal region. In one embodiment, the Tn3 scaffold of the present invention comprises a modification to increase serum half-life, as described herein. In yet another embodiment, the scaffold of the present invention comprises the addition, deletion, or substitution of at least one amino acid residue to stabilize the hydrophobic core of the scaffold.

[0217] Multimeric Tn3 scaffold One aspect of the present invention provides a multimeric Tn3 scaffold comprising at least two Tn3 monomer subunits of the present invention linked in tandem, wherein at least one of the monomers is a CD40L-specific monomer subunit. Such multimeric Tn3 scaffolds can be constructed in multiple formats. In a specific aspect, the present invention provides a multimeric Tn3 scaffold, wherein at least two CD40L-specific monomer subunits are linked in tandem via a peptide linker. In some embodiments, the multimeric Tn3 scaffold exhibits increased target binding valency and / or avidity, or other target activity. In some embodiments, increased target binding valency and / or avidity is achieved when multiple monomer subunits bind to the same target. In some embodiments, increased valency improves target specificity, e.g., enhances target protein dimerization.

[0218] In a specific embodiment, a multimeric Tn3 scaffold of the invention comprises at least two CD40L-specific monomer subunits linked in tandem, wherein each CD40L-specific monomer subunit binds to at least one target, and wherein each CD40L-specific monomer subunit comprises multiple β-strands linked to multiple loop regions, wherein at least one loop is a non-naturally occurring variant of the cognate loop in the parent Tn3 scaffold (SEQ ID NO: 3).

[0219] In one embodiment, the multimeric Tn3 scaffold is generated through a covalent bond between the CD40L-specific monomeric subunits, e.g., by directly linking the CD40L-specific monomeric subunits, or by including a linker, e.g., a peptide linker. In particular examples, the covalently linked Tn3 scaffold is generated by constructing a fusion gene encoding the CD40L-specific monomeric subunits, or by engineering a codon for a cysteine ​​residue into the CD40L-specific monomeric subunits to allow disulfide bond formation to occur between the expression products.

[0220] In one embodiment, the multimeric Tn3 scaffold of the invention comprises at least two CD40L-specific monomeric subunits directly linked to each other without any additional intervening amino acids, hi another embodiment, the multimeric Tn3 scaffold of the invention comprises at least two CD40L-specific monomeric subunits linked in tandem via a linker, e.g., a peptide linker.

[0221] In specific embodiments, the multimeric Tn3 scaffold of the invention comprises at least two CD40L-specific monomeric subunits linked in tandem via a peptide linker, wherein the peptide linker comprises from 1 to about 1000, or from 1 to about 500, or from 1 to about 250, or from 1 to about 100, or from 1 to about 50, or from 1 to about 25 amino acids. In specific embodiments, the multimeric Tn3 scaffold comprises at least two CD40L-specific monomeric subunits linked in tandem via a peptide linker, wherein the peptide linker comprises from 1 to about 20, or from 1 to about 15, or from 1 to about 10, or from 1 to about 5 amino acids.

[0222] In a specific embodiment, the multimeric Tn3 scaffold comprises at least two CD40L-specific monomer subunits linked in tandem via a linker, e.g., a peptide linker, where the linker is a functional moiety. The functional moiety will be selected based on the desired function and / or properties of the multimeric Tn3 scaffold. For example, a functional moiety useful for purification (e.g., a histidine tag) may be used as a linker. Functional moieties useful as linkers include, but are not limited to, polyethylene glycol (PEG), cytotoxic drugs, radionuclides, imaging agents, biotin, dimerization domains, human serum albumin (HSA) or its FcRn-binding portion, antibody domains or fragments, single-chain antibodies, domain antibodies, albumin-binding domains, IgG molecules, enzymes, ligands, receptors, binding peptides, non-Tn3 scaffolds, epitope tags, recombinant polypeptide polymers, cytokines, and the like. Specific peptide linkers and functional moieties that may be used as linkers are disclosed below.

[0223] In certain embodiments, the functional portion is an immunoglobulin or a fragment thereof. In some embodiments, the immunoglobulin or a fragment thereof comprises an Fc domain. In some embodiments, the Fc domain is unable to induce at least one FcγR-mediated effector function, such as ADCC (antibody-dependent cell-mediated cytotoxicity). It is known in the art that an Fc domain may be modified to reduce or eliminate at least one FcγR-mediated effector function; see, for example, U.S. Patent Nos. 5,624,821 and 6,737,056.

[0224] In some embodiments, the multimeric Tn3 scaffold comprises at least two CD40L-specific monomer subunits linked via one or more linkers, where the linker inserted between each CD40L-specific monomer subunit can be the same or different. In some embodiments, the linker can comprise multiple linkers, which can be the same or different. In some embodiments, when multiple linkers are linked together, some or all of the linkers can be functional moieties.

[0225] Scaffold binding stoichiometry In some embodiments, a monomeric or multimeric Tn3 scaffold can comprise monomeric subunits specific for CD40L to different epitopes, which can be different epitopes on a single CD40L molecule or on different CD40L target molecules. In some embodiments, a multimeric Tn3 scaffold can comprise monomeric subunits specific for CD40L, where each subunit targets one or more different epitopes on one or more CD40L molecules.

[0226] In other embodiments, the monomeric or multimeric Tn3 scaffold can bind to two or more different epitopes on the same CD40L molecule. In some embodiments, the different epitopes are non-overlapping epitopes. In other embodiments, the different epitopes are overlapping epitopes.

[0227] In yet another specific embodiment, a monomeric or multimeric Tn3 scaffold can bind to one or more epitopes on one CD40L molecule and further bind to one or more epitopes on a second CD40L molecule. In some embodiments, the different target molecules are part of an oligomeric complex, e.g., a trimeric CD40L complex.

[0228] In yet another specific embodiment, the monomeric or multimeric Tn3 scaffold can bind to a single epitope on a CD40L trimer. In yet another embodiment, the monomeric or multimeric Tn3 scaffold can bind to the same epitope on at least two CD40L trimers.

[0229] In certain embodiments, a monomeric or multimeric Tn3 scaffold can bind to the same epitope on two or more copies of a CD40L molecule on the surface of adjacent cells. In certain embodiments, a monomeric or multimeric Tn3 scaffold can bind to the same epitope on two or more copies of a CD40L molecule in solution. In some embodiments, a monomeric or multimeric Tn3 scaffold can bind to the same or different epitopes on CD40L with the same or different binding affinities and / or avidities.

[0230] In another embodiment, the monomeric or multimeric Tn3 scaffold can bind to an epitope on one or more copies of CD40L to achieve or enhance (e.g., synergistically) a desired effect on the target, e.g., to prevent receptor binding or to prevent oligomerization.

[0231] Furthermore, when the monomeric or multimeric Tn3 scaffolds of the invention comprise multiple CD40L-specific monomer subunits, e.g., different monomers where each monomer targets a different epitope on CD40L, such monomer subunits can be arranged in a specific pattern or with a particular orientation to achieve or enhance a particular biological effect. Such combinations of monomer subunits can be constructed and then evaluated using methods known in the art.

[0232] Furthermore, the Tn3 scaffold of the present invention can be fused to a marker sequence, e.g., a peptide, to facilitate purification. In some embodiments, the marker amino acid sequence is a polyhistidine peptide (His-tag), such as an octahistidine tag (His-8 tag) or a hexahistidine tag (His-6 tag), such as those provided in the pQE expression vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among other vectors, many of which are commercially available. For example, as described in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86:821-824, 1989, polyhistidine provides convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, a hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (see, e.g., Wilson et al., Cell 37:767, 1984), a FLAG tag, a Strep-tag, a myc-tag, a V5 tag, a GFP tag, an AU1 tag, an AU5 tag, an ECS tag, a GST tag, or an OLLAS tag.

[0233] Additional fusion proteins comprising the Tn3 scaffold of the present invention may be generated through techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling").

[0234] DNA shuffling may be used to modify the action of the Tn3 scaffold on its target (e.g., to create a scaffold with higher affinity and lower off-rate). The Tn3 scaffold may be modified by random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. One or more portions of a polynucleotide encoding a scaffold that binds to a specific target may be recombined with one or more components, motifs, segments, portions, domains, fragments, etc., of one or more heterologous molecules.

[0235] Antibodies and Fc domain fusions In some embodiments, the Tn3 scaffold of the invention comprises a monomeric subunit specific for CD40L fused to a domain or fragment of an antibody (e.g., IgG), including but not limited to an Fc domain.

[0236] In some embodiments, only one CD40L-specific monomeric subunit is conjugated or fused to an antibody domain or fragment. For example, a single CD40L-specific monomeric subunit can be fused to the N-terminus of a polypeptide of an antibody domain or fragment (e.g., an antibody heavy or light chain). In other embodiments, a Tn3 scaffold is created by fusing or conjugating one or more CD40L-specific monomeric subunits to the N-terminus and / or C-terminus of a polypeptide of an antibody domain or fragment (e.g., an antibody heavy and / or light chain, or Fc domain).

[0237] In some embodiments, some or all of the CD40L-specific monomeric subunits fused to antibody domains or fragments are identical, while in some other embodiments, some or all of the CD40L-specific monomeric subunits fused to antibody domains or fragments are different.

[0238] In a specific embodiment, a Tn3 scaffold of the invention comprises one CD40L-specific monomer subunit fused to an Fc domain. In other embodiments, a Tn3 scaffold of the invention comprises at least two CD40L-specific monomer subunits fused to an Fc domain. In a specific embodiment, two of the CD40L-specific monomer subunits fused to the Fc domain are identical. In a specific embodiment, two of the CD40L-specific monomer subunits fused to the Fc domain are different. In a specific embodiment, the two CD40L-specific monomer subunits fused to the Fc domain are linked in tandem to each other, and one of the CD40L-specific monomer subunits is fused to the Fc domain.

[0239] In some embodiments, different Tn3 scaffolds of the present invention can be dimerized by using mutations in the Fc domain that support heterodimer formation. It is known in the art that mutations in the Fc region (e.g., amino acid substitutions and / or additions and / or deletions) can enhance or reduce antibody effector functions and alter the pharmacokinetic properties (e.g., half-life) of antibodies. Thus, in certain embodiments, the Tn3 scaffolds of the present invention comprise Fc domains that contain modified Fc regions in which one or more modifications have been made to alter the functional and / or pharmacokinetic properties of the Tn3 scaffold. In certain embodiments, the Tn3 scaffolds of the present invention comprise Fc domains that contain modified Fc regions in which one or more modifications have been made to reduce or eliminate at least one Fc□R-mediated effector function.

[0240] It is also known that glycosylation of the Fc region can be modified to enhance or attenuate effector function and / or anti-inflammatory activity. Thus, in one embodiment, the Tn3 scaffold of the present invention comprises an Fc region with altered glycosylation of amino acid residues to modify the cytotoxic and / or anti-inflammatory properties of the Tn3 scaffold.

[0241] Tn3 scaffold morphology The Tn3 scaffold of the present invention can be fused to the C-terminus of an Fc domain, an antibody light chain, and an antibody heavy chain in any suitable spatial arrangement. For a detailed description of contemplated scaffold configurations, see, e.g., PCT / US2011 / 032184.

[0242] Scaffold fabrication The Tn3 scaffolds described herein may be used in any technique for evolving new or improved target-binding proteins. In one particular example, the target is immobilized on a solid support, such as a column resin or a microtiter plate well, and the target is contacted with a library of binding proteins based on candidate scaffolds. Such libraries may consist of clones constructed from the Tn3 scaffold through randomization of the sequence and / or length of the CDR-like loops.

[0243] In this regard, bacteriophage (phage) display is a well-known technique that allows for screening of large oligopeptide libraries and identifying library members capable of specifically binding to a target. Phage display is a technique in which mutant polypeptides are displayed as fusion proteins on the surface of bacteriophage particles via coat proteins (Scott, JK and Smith, GP (1990) Science 249:386). Bioinformatics methods can be used to determine loop length and diversity preferences for naturally occurring FnIII domains. This analysis can be used to develop "restricted randomization" strategies that incorporate loop length and sequence diversity preferences into the development of library methods. Integrating loop length and sequence diversity analysis into library development results in restricted randomization (i.e., specific positions within randomized loops are restricted so that amino acids can be present at those positions).

[0244] The present invention also provides a recombinant library comprising a diverse population of non-naturally occurring Tn3 scaffolds. In one embodiment, the library comprises non-naturally occurring Tn3 scaffolds comprising multiple β-strand domains linked to multiple loop regions, wherein one or more of the loops are altered by at least one amino acid through deletion, substitution, or addition. In a specific embodiment, the library comprises Tn3 scaffolds derived from a wild-type Tn3 scaffold.

[0245] As detailed above, the loops connecting the various β-strands of the scaffold may be randomized in length and / or sequence diversity. In one embodiment, the library of the present invention comprises a Tn3 scaffold having at least one loop randomized in length and / or sequence diversity. In one embodiment, at least one, at least two, at least three, at least four, at least five, or at least six loops of the Tn3 scaffold are randomized in length and / or sequence diversity. In one embodiment, at least one loop is kept constant in length and / or sequence diversity, while at least one additional loop is randomized. In another embodiment, at least one, at least two, or all three of loops AB, CD, and EF are kept constant in length or sequence diversity, while at least one, at least two, or all three of loops BC, DE, and FG are randomized. In another embodiment, at least one, at least two, or at least three of loops AB, CD, and EF are randomized for length and / or sequence diversity, while at least one, at least two, or all three of loops BC, DE, and FG are randomized.

[0246] In a specific embodiment, a library of the invention comprises an FnIII scaffold, wherein beta strand A comprises SEQ ID NO: 10 or 11, beta strand B comprises SEQ ID NO: 12, beta strand C comprises SEQ ID NO: 13 or 14, beta strand D comprises SEQ ID NO: 15, beta strand E comprises SEQ ID NO: 16, beta strand F comprises SEQ ID NO: 17, and beta strand G comprises SEQ ID NO: 18.

[0247] In a specific embodiment, a library of the invention comprises an FnIII scaffold, wherein beta strand A consists of SEQ ID NO: 10 or 11, beta strand B consists of SEQ ID NO: 12, beta strand C consists of SEQ ID NO: 13 or 14, beta strand D consists of SEQ ID NO: 15, beta strand E consists of SEQ ID NO: 16, beta strand F consists of SEQ ID NO: 17, and beta strand G consists of SEQ ID NO: 18.

[0248] In a specific embodiment, a library of the invention comprises an FnIII scaffold, wherein beta strand A consists essentially of SEQ ID NO: 10 or 11, beta strand B consists essentially of SEQ ID NO: 12, beta strand C consists essentially of SEQ ID NO: 13 or 14, beta strand D consists essentially of SEQ ID NO: 15, beta strand E consists essentially of SEQ ID NO: 16, beta strand F consists essentially of SEQ ID NO: 17, and beta strand G consists essentially of SEQ ID NO: 18.

[0249] As detailed above, one or more residues within the loop can be kept constant for length and / or sequence diversity, while other residues are randomized. Optionally or alternatively, one or more residues within the loop can be kept constant for length and / or sequence diversity with a predetermined limited number of different amino acids, while other residues are randomized. Thus, the libraries of the present invention comprise Tn3 scaffolds that can include one or more loops with a degenerate consensus sequence and / or one or more invariant amino acid residues. In another embodiment, the libraries of the present invention comprise Tn3 scaffolds with randomized BC loops. In another embodiment, the libraries of the present invention comprise Tn3 scaffolds with randomized BC loops. In yet another embodiment, the libraries of the present invention comprise Tn3 scaffolds with randomized BC loops.

[0250] In one embodiment, the library of the present invention comprises a Tn3 scaffold with a randomized DE loop. In one embodiment, the library of the present invention comprises a Tn3 scaffold with a randomized FG loop. In another embodiment, the library of the present invention comprises an FnIII scaffold with a randomized FG loop.

[0251] In a specific embodiment, the library of the invention comprises the amino acid sequence: [ka] a scaffold comprising: (a)X AB , X BC , X CD , X DE , X EF , and X FG represent the amino acid residues present in the sequences of the AB, BC, CD, DE, EF, and FG loops, respectively; (b) X1 represents an amino acid residue A or T; and (c) The loop length n is an integer from 2 to 26.

[0252] In some embodiments, the libraries of the invention comprise the amino acid sequence: [ka] The CD40L-specific Tn3 monomer subunit of the present invention comprises: (a) X1 represents the amino acid residue serine (S) or leucine (L); (b) X2 represents the amino acid residue aspartic acid (D) or glutamic acid (E); (c) X3 represents the amino acid residue histidine (H), isoleucine (I), valine (V), phenylalanine (F), or tryptophan (W); (d) X4 represents the amino acid residue alanine (A), glycine (G), glutamic acid (E), or aspartic acid (D); (e) X5 represents the amino acid residue glutamic acid (E), leucine (L), glutamine (Q), serine (S), aspartic acid (D), or asparagine (N); (f) X6 represents the amino acid residue phenylalanine (F) or tyrosine (Y); (g) X7 represents the amino acid residue isoleucine (I), valine (V), histidine (H), glutamic acid (E), or aspartic acid (D); (h) X8 represents the amino acid residue glycine (G), tryptophan (W), or valine (V); (i) X9 represents the amino acid residue tryptophan (W), phenylalanine (F), or tyrosine (Y); (j)X 10 represents the amino acid residue serine (S), glutamine (Q), methionine (M), or histidine (H); (k)X 11 represents the amino acid residue tryptophan (W) or histidine (H); and (l)X 12 represents the amino acid residue arginine (R) or serine (S).

[0253] In some embodiments, the libraries of the invention comprise the amino acid sequence: [ka] The CD40L-specific Tn3 monomer subunit of the present invention comprises: (a) X1 represents the amino acid residue lysine (K) or glutamic acid (E); (b) X2 represents the amino acid residue threonine (T) or isoleucine (I); (c) X3 represents the amino acid residue asparagine (N) or alanine (A); (d) X4 represents the amino acid residue serine (S), leucine (L), alanine (A), phenylalanine (F), or tyrosine (Y); (e) X5 represents the amino acid residue tyrosine (Y), alanine (A), glycine (G), valine (V), isoleucine (I), or serine (S); (f) X6 represents the amino acid residue tyrosine (Y), serine (S), alanine (A), or histidine (H); (g) X7 represents the amino acid residue asparagine (N), aspartic acid (D), histidine (H), or tyrosine (Y); (h) X8 represents the amino acid residue leucine (L), phenylalanine (F), histidine (H), or tyrosine (Y); (i) X9 represents the amino acid residue histidine (H), proline (P), serine (S), leucine (L), or aspartic acid (D); (j)X 10 represents the amino acid residue glycine (G), phenylalanine (F), histidine (H), or tyrosine (Y); (k)X 11 represents the amino acid residue alanine (A) or threonine (T); (l)X 12 represents the amino acid residue serine (S), asparagine (N), glutamic acid (E), asparagine (R), or aspartic acid (D); (m)X 13 represents the amino acid residue serine (S), glutamine (Q), threonine (T), asparagine (N), or alanine (A); (n)X 14 represents the amino acid residue proline (P), valine (V), isoleucine (I) or alanine (A), or no amino acid; (o)X 15 represents the amino acid residue isoleucine (I) or no amino acid; (p)X 16 represents the amino acid residue glutamic acid (E) or lysine (K); and (q)X 17 represents the amino acid residue serine (S) or asparagine (N).

[0254] The present invention further provides methods for identifying recombinant Tn3 scaffolds that bind to a target, e.g., CD40L, and have enhanced stability or improved action on a target, e.g., CD40L, compared to the parent Tn3 scaffold, by screening the libraries of the present invention.

[0255] In certain embodiments, a method for identifying a recombinant Tn3 scaffold that has enhanced protein stability compared to the parent Tn3 scaffold and specifically binds to a target comprises: contacting the target ligand with a library of the invention under conditions suitable for the formation of a scaffold:target ligand complex; obtaining a scaffold from the complex that binds to the target ligand; determining whether the stability of the scaffold obtained in step (b) is higher than the stability of the wild-type Tn3 scaffold; Includes.

[0256] The same method can be used to identify recombinant Tn3 scaffolds with improved binding affinity, avidity, etc. for the target. In one embodiment, in step (a), a scaffold library of the invention is incubated with an immobilized target. In one embodiment, in step (b), the scaffold:target-ligand complex is washed to remove non-specific binders, and the strongest binders are eluted under highly stringent conditions and subjected to PCR to recover sequence information. It is contemplated that the binders and / or sequence information obtained in step (b) can be used to generate new libraries using methods disclosed herein or known to those of skill in the art, and may be used to repeat the selection process, with or without further mutagenesis of the sequences. In some embodiments, several rounds of selection may be performed until binders with sufficient affinity for the antigen are obtained.

[0257] A further embodiment of the present invention is a collection of isolated nucleic acid molecules encoding a library comprising a scaffold of the present invention, as described above.

[0258] The scaffolds of the present invention may undergo affinity maturation. In an art-recognized method, specific binding proteins are subjected to a selection scheme for increased affinity to a specific target (see Wu et al., Proc. Natl. Acad. Sci. USA. 95(11):6037-42). The resulting scaffolds of the present invention may exhibit binding properties at least as high as those of the scaffolds prior to affinity maturation.

[0259] The present invention also provides a method for identifying amino acid sequences of protein scaffolds capable of binding to a target to form a scaffold:target complex. In one embodiment, the method comprises the steps of: (a) contacting a library of the present invention with an immobilized or separable target; (b) separating the scaffold:target complex from free scaffolds; (c) causing replication of the separated scaffolds of (b) to obtain a new polypeptide display library that is distinguished from the library in (a) by having reduced diversity and by being enriched in displayed scaffolds capable of binding to the target; d) optionally repeating steps (a) and (b) with the new library of (c); and e) determining the nucleic acid sequence of the region encoding the displayed scaffolds of the species obtained from (d) and thus predicting peptide sequences capable of binding to the target.

[0260] In another embodiment, the Tn3 scaffolds of the present invention may be further randomized after identification from a library screen. In one embodiment, the method of the present invention includes further randomizing at least one, at least two, at least three, at least four, at least five, or at least six loops of a scaffold identified from a library using the methods described herein. In another embodiment, the further randomized scaffolds are subjected to a subsequent method for identifying scaffolds capable of binding to a target. This method includes: (a) contacting the further randomized scaffold with an immobilized or separable target; (b) separating the further randomized scaffold:target complex from free scaffolds; (c) causing replication of the separated scaffolds of (b), optionally repeating steps (a)-(c); and (d) determining the nucleic acid sequence of the region encoding the further randomized scaffold, thereby predicting peptide sequences capable of binding to the target.

[0261] In further embodiments, the further randomized scaffold comprises at least one, at least two, at least three, at least four, at least five, or at least six randomized loops that were pre-randomized in the initial library. In other further embodiments, the further randomized scaffold comprises at least one, at least two, at least three, at least four, at least five, or at least six randomized loops that were not pre-randomized in the initial library.

[0262] The present invention also provides a method for obtaining at least two Tn3 scaffolds that bind to at least one or more targets. This method allows for the screening of drugs that act cooperatively to elicit a specific response. Such screening can be advantageous when agonist activity requiring the cooperation of two or more scaffolds is required. This method allows for the screening of cooperative drugs without the need for library reorganization to form multimeric complexes. In one embodiment, the method of the present invention comprises the steps of contacting a target ligand with a library of the present invention under conditions that allow the formation of a scaffold:target ligand complex; associating the scaffold with a cross-linking agent (defined as an agent that brings at least two identical or different scaffolds into close proximity) (cross-linking of the scaffolds elicits a detectable response); and obtaining the target-binding scaffold from the complex. In further embodiments, the crosslinker is an antibody specific for the scaffold, or a fragment thereof, an antibody specific for an epitope tag on the fragment, a dimerization domain, such as an Fc region, a coiled-coil motif (such as, but not limited to, a leucine zipper), a chemical crosslinker, or another dimerization domain known in the art.

[0263] affinity maturation The generation of the Tn3 scaffold of the present invention may include one or more in vitro or in vivo affinity maturation steps. In some embodiments, the Tn3 monomer subunit can undergo a single affinity maturation step. In other embodiments, the Tn3 monomer subunit can undergo two or more affinity maturation steps. Any affinity maturation technique can be used that results in amino acid changes in the parent Tn3 scaffold in general, or in the loops of the parent Tn3 scaffold in particular, that improve binding of the affinity-matured Tn3 scaffold to the desired antigen.

[0264] These amino acid changes can be achieved, for example, through random mutagenesis, "walk-through" mutagenesis, and "look-through" mutagenesis, which can be achieved, for example, by using error-prone PCR, yeast or bacterial "mutator" strains, or the incorporation of random or defined nucleic acid changes during ab initio synthesis of all or part of the FnIII-based binding molecule. Methods for performing affinity maturation and / or mutagenesis are described, for example, in U.S. Pat. Nos. 7,195,880; 6,951,725; 7,078,197; 7,022,479; 5,922,545; 5,830,721; 5,605,793; 5,830,650; 6,194,550; 6,699,658; 7,063,943; 5,866,344 and PCT Publication WO 06023144.

[0265] Such affinity maturation methods may further require increasing the stringency of antigen-binding screening assays to select Tn3 scaffolds with improved affinity for the antigen. Art-recognized methods for increasing the stringency of protein-protein interaction assays can be used here. In one embodiment, one or more of the assay conditions are altered (e.g., the salt concentration of the assay buffer) to reduce the affinity of the Tn3 scaffold for the desired antigen. In another embodiment, the length of time allowed for the Tn3 scaffold to bind to the desired antigen is decreased.

[0266] In another embodiment, a competitive binding step can be added to the protein-protein interaction assay. For example, the Tn3 scaffold can first be allowed to bind to the desired immobilized antigen. Then, a specific concentration of non-immobilized antigen is added to compete for binding with the immobilized antigen, so that the Tn3 scaffold with the lowest affinity for the antigen is eluted from the immobilized antigen, resulting in the selection of Tn3 scaffolds with improved antigen binding affinity. The stringency of the assay conditions can be further increased by increasing the concentration of non-immobilized antigen added to the assay.

[0267] The screening method may also require multiple rounds of selection to enrich for one or more Tn3 scaffolds with improved antigen binding. In one embodiment, additional amino acid mutations are introduced into the Tn3 scaffold at each round of selection. In another embodiment, the stringency of binding to the desired antigen is increased at each round of selection to select for Tn3 scaffolds with increased affinity for the antigen.

[0268] In some embodiments, affinity maturation is performed by saturation mutagenesis of the BC, DE, and FG loop portions of Tn3. In some embodiments, saturation mutagenesis is performed using Kunkel mutagenesis. In other embodiments, saturation mutagenesis is performed using PCR.

[0269] In some embodiments, at least one, at least two, at least three, at least four, at least five, or more than five rounds of affinity maturation are applied. In some embodiments, saturation mutagenesis is applied to only one loop, while in some other embodiments, only one loop or a portion of a loop is mutated during one round of affinity maturation. In some embodiments, two or more loops or portions of one or more loops are mutated during the same round of affinity maturation.

[0270] In other embodiments, the BC, DE, and FG loops are mutated simultaneously during the same round of affinity maturation.

[0271] In the case of monomers for constructing multimeric Tn3 scaffolds that bind to different epitopes of the same target, each binding specificity can be screened independently.

[0272] In some embodiments, the loop is randomized using a phage display library. In some embodiments, the binding of the Tn3 scaffold to the desired target can be determined using methods understood in the art. Furthermore, the amino acid sequence of the Tn3 scaffold identified in screening can be determined using methods understood in the art.

[0273] In some embodiments, affinity-matured monomer scaffolds of the invention exhibit at least a 5-fold, at least a 10-fold, at least a 20-fold, at least a 40-fold, at least a 60-fold, at least a 80-fold, or at least a 100-fold or greater increase in affinity for CD40L relative to the same Tn3 scaffold prior to affinity maturation, as measured by surface plasmon resonance or other assays known in the art. In some embodiments, affinity-matured monomer scaffolds of the invention have a dissociation constant (Kd) of less than 5 μM, less than 1 μM, less than 500 μM, less than 250 μM, less than 100 μM, or less than 50 μM, as measured by surface plasmon resonance or other assays known in the art.

[0274] These affinity maturation methods can be applied to develop Tn3 scaffolds with desirable improved binding properties, such as increased affinity or other desirable properties, such as favorable pharmacokinetic properties, high potency, low immunogenicity, increased or reduced cross-reactivity, etc.

[0275] Creation of tandem repeats Tandem construct ligations, i.e., dimers formed by linking two CD40L-specific monomeric subunits, may be generated by ligation of oligonucleotides at restriction sites using restriction enzymes known in the art, including but not limited to type II and type IIS restriction enzymes.

[0276] The multimeric Tn3 scaffolds of the invention may comprise linkers at the C-terminus and / or N-terminus and / or between domains as described herein. Additionally, scaffolds of the invention, including at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or polypeptide scaffolds, include, but are not limited to, (i) a Fab fragment containing the VL, CL, VH and CH1 domains; (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) an Fd fragment having a VH and CH1 domain; (iv) an Fd′ fragment having a VH and a CH1 domain and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) an Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) a dAb fragment consisting of the VH domain; (vii) isolated CDR regions; (viii) F(ab')2 fragment, a bivalent fragment containing two Fab' fragments linked by a disulfide bridge at the hinge region; (ix) single-chain antibody molecules (e.g., single-chain Fv; scFv); (x) "bispecific antibodies" having two antigen-binding sites comprising a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) within the same polypeptide chain; (xi) A "linear antibody" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions; (xii) a full-length antibody; and (xiii) an Fc region comprising CH2-CH3, which may further comprise all or part of a hinge region and / or a CH1 region; The antibody may be fused or conjugated to a dimerization domain comprising an antibody portion selected from:

[0277] Generation of Tn3 scaffold Recombinant expression of the Tn3 scaffold of the present invention requires the construction of an expression vector containing a polynucleotide encoding the Tn3 scaffold. Once a polynucleotide encoding the Tn3 scaffold is obtained, a vector for producing the Tn3 scaffold may be generated by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding a Tn3 scaffold are described herein. Methods well known to those skilled in the art can be used to construct an expression vector containing a sequence encoding a scaffold polypeptide and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination. Thus, the present invention provides a replicable vector containing a nucleotide sequence encoding the Tn3 scaffold of the present invention operably linked to a promoter.

[0278] To produce the Tn3 scaffold of the present invention, the expression vector is transferred into a host cell by conventional techniques, and the transgenic cells are then cultured by conventional techniques. Thus, the present invention includes a host cell containing a polynucleotide encoding the scaffold of the present invention operably linked to a heterologous promoter. Suitable host cells include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis).

[0279] A variety of host-expression vector systems may be utilized to express the Tn3 scaffolds of the present invention. Such host-expression systems represent vehicles by which a coding sequence of interest can be produced and subsequently purified, but also represent cells that, upon transformation or transfection with an appropriate nucleotide coding sequence, can express the scaffolds of the present invention in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) or mammalian cell lines (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors carrying the scaffold coding sequence.

[0280] Methods useful for producing the Tn3 scaffold of the invention are disclosed, for example, in International Patent Application Publication No. WO 2009 / 058379. Once the scaffold of the invention has been produced by recombinant expression, it may be purified by any method known in the art for purifying proteins.

[0281] In some embodiments, the scaffolds of the present invention can be produced in a deglycosylated form by substituting amino acid residues that can be glycosylated during recombinant expression. In a specific embodiment, to prevent glycosylation during recombinant expression, the serine amino acid in the glycine-serine linker (e.g., SEQ ID NO: 131 or SEQ ID NO: 132) can be substituted with other amino acid residues, such as alanine, glycine, leucine, isoleucine, or valine (see, e.g., SEQ ID NOs: 140, 141, 142, and 143). In some specific embodiments, N-glycosylation sites are removed from the Tn3 scaffolds of the present invention. In other embodiments, the scaffolds of the present invention can be deglycosylated after recombinant expression. Methods for in vitro deglycosylation after recombinant expression are known in the art, for example, using enzyme cocktails (e.g., PFGase F, Enodo F Multi, Orela O-linked Glycan Release, Enzymatic CarboRelease, and Enzymatic DeGlycoMx deglycosylation kits marketed by QA-bio, Palm Desert, CA).

[0282] As described in U.S. Patent Publication U.S. Patent Application Publication No. 2010-0298541A1, production of the Tn3 scaffolds of the present invention in the laboratory can be scaled up to produce scaffolds in analytical or production scale reactors.

[0283] Scalable generation of secreted Tn3 scaffolds The Tn3 scaffold of the present invention can be produced intracellularly or as a secreted form. In some embodiments, the secreted scaffold is properly folded and fully functional. The Tn3 scaffold of the present invention can be produced by a scalable process. In some embodiments, the scaffold can be produced by a scalable process of the present invention in a laboratory that can be scaled up to produce the scaffold of the present invention in an analytical scale bioreactor (such as, but not limited to, a 5 L, 10 L, 15 L, 30 L, or 50 L bioreactor). In other embodiments, the Tn3 scaffold can be produced by a scalable process of the present invention in a laboratory that can be scaled up to produce the Tn3 scaffold of the present invention in a production scale bioreactor (such as, but not limited to, a 75 L, 100 L, 150 L, 300 L, or 500 L). In some embodiments, the scalable process of the present invention results in little or no loss in production efficiency compared to a production process performed in a laboratory.

[0284] Linker The monomeric subunits in the multimeric Tn3 scaffold can be linked by protein and / or non-protein linkers, where each linker is fused to at least two monomeric subunits. Suitable linkers can consist of protein linkers, non-protein linkers, and combinations thereof. Linker combinations can be homomeric or heteromeric. In some embodiments, the multimeric Tn3 scaffold of the present invention comprises multiple monomeric subunits, where all linkers are identical. In other embodiments, the multimeric Tn3 scaffold comprises multiple monomeric subunits, where at least one of the linkers is functionally or structurally different from the remaining linkers. In some embodiments, the linker can itself contribute to the activity of the multimeric Tn3 scaffold by directly or indirectly participating in target binding.

[0285] In some embodiments, the protein linker is a polypeptide. The linker polypeptide must be of sufficient length to link two or more monomeric subunits in such a way that they assume the correct conformation relative to each other so as to retain the desired activity.

[0286] In one embodiment, the polypeptide linker comprises 1 to about 1,000 amino acid residues, 1 to about 50 amino acid residues, 1 to 25 amino acid residues, 1 to 20 amino acid residues, 1 to 15 amino acid residues, 1 to 10 amino acid residues, 1 to 5 amino acid residues, or 1 to 3 amino acid residues. The present invention also provides nucleic acids, e.g., DNA, RNA, or a combination of both, encoding polypeptide linker sequences. The amino acid residues selected for inclusion in the polypeptide linker should exhibit properties that do not significantly interfere with the activity or function of the multimeric Tn3 scaffolds of the present invention. Thus, the polypeptide linker as a whole should not exhibit changes that would form bonds or other interactions with amino acid residues within one or more monomer subunits that are inconsistent with the activity or function of the Tn3 multimeric scaffolds of the present invention, interfere with internal folding, or significantly disrupt binding of the multimeric Tn3 scaffolds of the present invention to CD40L.

[0287] The use of natural and artificial peptide linkers to link polypeptides and form novel linked fusion polypeptides is well known in the literature.Therefore, the linker that fuses two or more monomer subunits is natural linker, artificial linker, or a combination thereof.In some embodiments, the amino acid sequences of all peptide linkers present in the Tn3 multimer scaffold of the present invention are identical.In other embodiments, the amino acid sequences of at least two of the peptide linkers present in the multimer Tn3 scaffold of the present invention are different.

[0288] In some embodiments, the polypeptide linker has conformational flexibility. In some embodiments, the polypeptide linker sequence is (GGGGX) mIn a specific embodiment, the polypeptide linker sequence comprises the amino acid sequence (GGGGS) (see, e.g., SEQ ID NO: 209), where x is alanine (A), serine (S), glycine (G), isoleucine (I), leucine (L), or valine (V), and m is a positive integer. m In another specific embodiment, the polypeptide linker sequence comprises the amino acid sequence (GGGGG) m In yet another specific embodiment, the polypeptide linker sequence comprises the amino acid sequence (GGGGA) m wherein m is a positive integer (see, e.g., SEQ ID NO: 149). In some embodiments, the polypeptide linker is an essentially amorphous natural or artificial polypeptide (see, e.g., Schellenberger et al., Nature Biotechnol. 27:1186-1190, 2009; see also Sickmeier et al., Nucleic Acids Res. 35:D786-93, 2007).

[0289] The peptide linker may be modified in such a way that an amino acid residue is introduced that comprises an attachment group for a non-polypeptide moiety. An example of such an amino acid residue may be a cysteine ​​residue (to which the non-polypeptide moiety is subsequently attached), or the amino acid sequence may contain an in vivo N-glycosylation site (by which a sugar moiety (in vivo) is attached to the peptide linker).

[0290] In some embodiments, the amino acid sequences of all peptide linkers present in a polypeptide multimer are identical. Alternatively, the amino acid sequences of all peptide linkers present in a polypeptide multimer may be different.

[0291] The present invention further encompasses the use of Tn3 scaffolds conjugated to therapeutic moieties. The Tn3 scaffolds may be conjugated to therapeutic moieties, such as cytotoxins, e.g., cytostatic or cytocidal agents, therapeutic agents, or radioactive metal ions, e.g., alpha emitters. Cytotoxins or cytotoxic agents include any agent that is detrimental to cells.

[0292] CD40L-specific Tn3 scaffold The present invention provides Tn3 scaffolds that specifically bind to CD40L. In specific embodiments, the scaffolds of the present invention specifically bind to human CD40L. In other specific embodiments, the Tn3 scaffolds of the present invention bind to CD40L homologs from mouse, chicken, rhesus monkey, cynomolgus monkey, rat, or rabbit. In some embodiments, the Tn3 scaffolds of the present invention bind to exposed epitopes of CD40L. Such embodiments include cells transfected to ectopically express CD40L and / or receptors that are endogenously expressed on the cells.

[0293] In some embodiments, the Tn3 scaffold of the present invention recognizes an epitope presented on monomeric CD40L. In other embodiments, the Tn3 scaffold of the present invention recognizes an epitope presented on trimeric CD40L. In other embodiments, the Tn3 scaffold of the present invention recognizes an epitope presented on membrane-bound CD40L. In other embodiments, the Tn3 scaffold of the present invention recognizes an epitope presented on soluble CD40L.

[0294] In yet other embodiments, the Tn3 scaffold of the present invention binds to monomeric CD40L and prevents or interferes with oligomerization of the CD40L molecule. In yet other embodiments, the scaffold of the present invention reduces or inhibits the interaction of CD40L with CD40. In other embodiments, the Tn3 scaffold of the present invention stimulates CD40L-mediated cell signaling. In yet other embodiments, the Tn3 scaffold of the present invention antagonizes CD40L-mediated cell signaling.

[0295] The present invention also provides methods for modulating CD40L activity using the Tn3 scaffolds described herein. In some embodiments, the methods of the present invention comprise contacting CD40L with a CD40L-specific scaffold and blocking the interaction between CD40 and CD40L. In other embodiments, the methods of the present invention comprise contacting a cell expressing CD40L with a CD40L-specific Tn3 scaffold and preventing proteolytic cleavage of CD40L from the cell surface. In other embodiments, the methods of the present invention comprise contacting a CD40L monomer with a CD40L-specific Tn3 scaffold and preventing CD40L oligomerization. In other embodiments, CD40L dimerization or oligomerization may be achieved through the use of a multimeric Tn3 scaffold.

[0296] In some embodiments, the methods of the invention involve administration of a CD40L-specific scaffold that reduces a CD40-mediated immune response (see, e.g., Elqueta et al. 229:152-172, 2009) or downstream signaling pathways initiated by binding of CD40 to CD40L, as measured by routine assays known in the art.

[0297] Without wishing to be bound by any particular theory, the CD40L scaffolds of the present invention may function, for example, by blocking binding of CD40L to CD40, by binding to and sequestering soluble CD40L, by altering the interaction of CD40L with CD40 but not blocking binding, by blocking or enhancing metalloprotease-mediated enzymatic cleavage of CD40L from the cell surface to yield soluble CD40L, or by blocking or enhancing cell surface CD40L endocytosis.

[0298] Specific CD40L-binding sequence In some embodiments, the Tn3 scaffold of the invention comprises a monomeric subunit specific for CD40L that comprises at least one, at least two, at least three, at least four, at least five, or at least six loop sequences that bind to CD40L.

[0299] In some embodiments, the monomeric subunit specific for CD40L is 309 (parental 309 family clone isolated from a naive Tn3 library; SEQ ID NO:20), 309FGwt (parental 309 clone with a humanized FG loop; SEQ ID NO:22), 340 (affinity matured 309 clone; SEQ ID NO:24), 341 (affinity matured 309 clone; SEQ ID NO:26), 342 (affinity matured 309 clone; SEQ ID NO:28 or SEQ ID NO:146), 343 (affinity matured 309 clone; SEQ ID NO:30), 344 (affinity matured 309 clone; SEQ ID NO:32), 345 (affinity matured 309 clone; SEQ ID NO: 34), 346 (affinity matured 309 clone; SEQ ID NO: 36), 347 (affinity matured 309 clone; SEQ ID NO: 38), 348 (affinity matured 309 clone; SEQ ID NO: 40), 349 (affinity matured 309 clone; SEQ ID NO: 42), 311 (parent 311 family clone isolated from a naive Tn3 library; SEQ ID NO: 44), 311K4E (mutant 311 family clone from the first round of affinity maturation; SEQ ID NO: 46); 311K4E_1 (mutant 311 family clone from the second round of affinity maturation) ; SEQ ID NO: 48), 311K4E_2 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 50), 311K4E_3 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 52), 311K4E_4 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 54), 311K4E_5 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 56), 311K4E_7 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 58), 311K4E_8 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 80), 311K4E_9 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 92), 311K4E_10 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 104), 311K4E_11 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 116), 311K4E_12 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 128), 311K4E_13 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 139), 311K4E_14 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 149), 311K4E_15 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 150), 311K4E SEQ ID NO: 60), 311K4E_9 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 62), 311K4E_10 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 64), 311K4E_11 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 66), 311K4E_12 (mutant 311 family clone from the second round of affinity maturation; SEQ ID NO: 68), 311K4E_13 (mutant 311 family clone from the second round of affinity maturation;SEQ ID NO:70), 311K4E_14 (variant 311 family clone from the second round of affinity maturation; SEQ ID NO:72), 311K4E_15 (variant 311 family clone from the second round of affinity maturation; SEQ ID NO:74), 311K4E_16 (variant 311 family clone from the second round of affinity maturation; SEQ ID NO:76), 311K4E_19 (variant 311 family clone from the second round of affinity maturation; SEQ ID NO:78), 311K4E_20 (variant 311 family clone from the second round of affinity maturation; SEQ ID NO:80), and 311K4E_21 (variant 311 family clone from the second round of affinity maturation; SEQ ID NO:82);

[0300] In some embodiments, a monomeric subunit specific for CD40L comprises at least one loop sequence selected from the loop sequences listed in Table 2. In other embodiments, a monomeric subunit specific for CD40L comprises at least one BC loop sequence selected from the BC loop sequences listed in Table 2. In other embodiments, a monomeric subunit specific for CD40L comprises at least one DE loop sequence selected from the DE loop sequences listed in Table 2. In other embodiments, a monomeric subunit specific for CD40L comprises at least one FG loop sequence selected from the FG loop sequences listed in Table 2.

[0301] In some embodiments, a monomeric subunit specific for CD40L comprises a BC loop sequence selected from the BC loop sequences listed in Table 2; and a DE loop sequence selected from the DE loop sequences listed in Table 2. In other embodiments, a monomeric subunit specific for CD40L comprises a BC loop sequence selected from the BC loop sequences listed in Table 2; and an FG loop sequence selected from the FG loop sequences listed in Table 2. In other embodiments, a monomeric subunit specific for CD40L comprises a DE loop sequence selected from the DE loop sequences listed in Table 2; and an FG loop sequence selected from the FG loop sequences listed in Table 2. In some embodiments, a monomeric subunit specific for CD40L comprises loop sequences corresponding to loop sequences from one, two, or three different Tn3 clones.

[0302] In certain embodiments, when a CD40L-specific monomer scaffold sequence includes a linker and / or a histidine tag (e.g., a His-8 tag) at the C-terminus of the sequence, or additional N-terminal amino acids, these C-terminal linkers and / or histidine tags and additional N-terminal amino acids can be removed, such that the corresponding amino acid sequence includes the C-terminal linker and His tag sequence and a deletion of one or more additional amino acids at the N-terminus.

[0303] In some embodiments, the CD40L-specific Tn3 scaffold comprises a single monomer subunit, e.g., the 342 clone sequence (affinity matured 309 clone; SEQ ID NO: 28 and / or SEQ ID NO: 146). In other embodiments, the CD40L-specific scaffold comprises two or more monomer subunits, e.g., two 342 clone monomer subunits (SEQ ID NO: 28 and / or SEQ ID NO: 146) in tandem (see, e.g., SEQ ID NO: 135). In specific embodiments, the Tn3 scaffold of the invention is conjugated to mutant HSA (see, e.g., SEQ ID NO: 134 and SEQ ID NO: 135). In further embodiments, HSA can be conjugated to either the N- or C-terminus of the multimeric Tn3 scaffold.

[0304] In a specific embodiment, a CD40L-specific Tn3 scaffold comprises a single 311K4E_12 monomer subunit, a GS linker, and a C34S HSA mutant (see, e.g., SEQ ID NO: 201). In another specific embodiment, a CD40L-specific Tn3 scaffold comprises a single 311K4E_12 monomer subunit with a β-strand C CELTYG mutant, an all-glycine linker, and a C34S HSA mutant (see, e.g., SEQ ID NO: 202). In another specific embodiment, a CD40L-specific Tn3 scaffold comprises two 311K4E_12 subunits in tandem and two GS linkers, where the first GS linker links the subunits to each other and the second GS linker links one subunit to the C34S HSA mutant (see, e.g., SEQ ID NO: 203). In yet another specific embodiment, a Tn3 scaffold specific for CD40L comprises two 311K4E_12 subunits in tandem and two all-glycine linkers, where a first all-glycine linker connects the subunits to each other and a second all-glycine linker connects one subunit to the C34S HSA mutant (see, e.g., SEQ ID NO: 204).

[0305] In one specific embodiment, a CD40L-specific Tn3 scaffold comprises two 309 subunits linked in tandem via a GS linker (see, e.g., SEQ ID NO: 205). In another specific embodiment, a CD40L-specific Tn3 scaffold comprises a single 309 subunit linked to a C34S HSA mutant (see, e.g., SEQ ID NO: 206). In another specific embodiment, a CD40L-specific Tn3 scaffold comprises two 309 subunits in tandem and two GS linkers, where a first GS linker links the subunits to each other and a second GS linker links one subunit to a C34S HSA mutant (see, e.g., SEQ ID NO: 207).

[0306] In a specific embodiment, a CD40L-specific Tn3 scaffold comprises a single 342 monomer subunit, a GS linker, and a C34S HSA mutant (see, e.g., SEQ ID NO: 134). In another specific embodiment, a CD40L-specific Tn3 scaffold comprises a single 342 monomer subunit, an all-glycine linker, and a C34S HSA mutant (see, e.g., SEQ ID NO: 144). In another specific embodiment, a CD40L-specific Tn3 scaffold comprises two 342 subunits in tandem and two GS linkers, where the first GS linker links the subunits to each other and the second GS linker links one subunit to the C34S HSA mutant (see, e.g., SEQ ID NO: 135). In yet another specific embodiment, a CD40L-specific Tn3 scaffold comprises two 342 subunits in tandem and two all-glycine linkers, where a first all-glycine linker connects the subunits to each other and a second all-glycine linker connects one subunit to the C34S HSA mutant (see, e.g., SEQ ID NO: 145). In yet another specific embodiment, a CD40L-specific Tn3 scaffold comprises two 342 subunits linked in tandem by a GS linker (see, e.g., SEQ ID NO: 208).

[0307] In a specific embodiment, the CD40L-specific Tn3 scaffold comprises: In another specific embodiment, the CD40L-specific Tn3 scaffold comprises, in tandem, a 311 subunit, or a 311-derived subunit (e.g., 311K4E_12) and a 309 subunit, or a 309-derived subunit (e.g., 342), and two GS linkers, where the first GS linker links the subunits to each other and the second GS linker links one subunit to the C34S HSA mutant (see, e.g., SEQ ID NO: 135). In yet another specific embodiment, a CD40L-specific Tn3 scaffold comprises a tandem 311 subunit, or a 311-derived subunit (e.g., 311K4E_12), and a 309 subunit, or a 309-derived subunit (e.g., 342), and two all-glycine linkers, where the first all-glycine linker connects the subunits to each other and the second all-glycine linker connects one subunit to the C34S HSA mutant (see, e.g., SEQ ID NO: 145).

[0308] An example of a fusion of a tandem bivalent Tn3 scaffold specific for CD40L with serum albumin (SA) is shown in Figure 2A (see also Figure 9A). A specific linker is provided in Figure 2A, but other linkers are contemplated as provided herein. Wild-type mature SA, such as mouse serum albumin (MSA) or human serum albumin (HSA), may be used, although it is contemplated that one or more cysteine ​​(C) amino acid residues in mature SA may be substituted with, for example, serine (S), alanine (A), glycine (G), etc.

[0309] A representative construct is shown below. The sequence of SA is underlined. The linker is shown in a box. It will be understood that numerous variations are within the scope of the present invention. For example, the linker may be modified (several non-limiting examples are provided herein), the first or second N-terminal amino acid residue (SQ) may be absent and / or substituted with an alternative amino acid residue, a tag (e.g., a 6xHis tag) may be incorporated, an alternative CD40L-specific scaffold (e.g., one based on the 10th Fn3 domain of fibronectin) may be used in a similar construct, etc.

[0310] 342 Monovalent HSA Construct 1 (SEQ ID NO: 134) [342 monomers]-(G4S)2 linker-HSA C34S [ka]

[0311] 342 Monovalent HSA Construct 2 (SEQ ID NO: 144) [342 monomers]-G 10 Linker-HSA C34S : [ka]

[0312] 342 Bivalent HSA Construct 1 (SEQ ID NO: 135) [342 monomers]-(G4S)3 linker-[342 monomers]-(G4S)2 linker-HSA C34S : [ka]

[0313] 342 Bivalent HSA Construct 2 (SEQ ID NO: 145) [342 monomers]-G 15 Linker-[342 monomer]-G 10 Linker-HSA C34S : [ka]

[0314] 311K4E_12 Monovalent HSA Construct 1 (SEQ ID NO: 201) [311K4E_12 monomer]-(G4S)2 linker-HSA C34S : [ka]

[0315] 311K4E_12 Monovalent HSA Construct 2 (SEQ ID NO: 202) [311K4E_12 monomer]-G 10 Linker-HSA C34S : [ka]

[0316] 311K4E_12 Bivalent HSA Construct 1 (SEQ ID NO: 203) [311K4E_12 monomer]-G4S3 linker-[311K4E_12 monomer]-(G4S)2 linker-HSA C34S : [ka]

[0317] 311K4E_12 Bivalent HSA Construct 2 (SEQ ID NO: 204) [311K4E_12 monomer]-G 15 Linker-[311K4E_12 monomer]-G 10 Linker-HSA C34S : [ka]

[0318] Pharmaceutical Composition In another aspect, the present invention provides compositions, including, but not limited to, pharmaceutical compositions, containing one or a combination of albumin fusion proteins of the invention formulated with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises an albumin fusion protein having a scaffold, such as a Tn3 scaffold. In another embodiment, the pharmaceutical composition comprises an albumin fusion protein of SEQ ID NO: 134, 135, 201, 202, 203, 204, 205, 206, 207, or 208, wherein the composition has less than 20 ng / mg host cell protein and the tryptophan at positions 46, 151, or both, is not oxidized. Other embodiments relate to pharmaceutically acceptable formulations comprising albumin fusion proteins purified according to the invention. The formulation may suitably include a buffer, a sugar, and an emulsifier. In one embodiment, the buffer is sodium phosphate buffer, the sugar is sucrose, and the emulsifier is polysorbate 80. The pharmaceutical formulation of claim 100 or 101 is lyophilized.

[0319] The present invention encompasses, for example, the following embodiments:

[0022] Embodiment 1: A method for reducing tryptophan and / or methionine oxidation in an albumin fusion protein during purification, comprising subjecting a composition comprising said albumin fusion protein to the following purification process: (a) Affinity matrix; (b) Anion exchange matrix wherein the albumin fusion protein is eluted from the affinity matrix by applying an elution buffer comprising octanoic acid. [Embodiment 2] The method described in embodiment 1, wherein the elution buffer contains about 2 mM to about 100 mM octanoic acid. [Embodiment 3] The affinity matrix is (1) from about 2% to about 20% of a polyol selected from the group consisting of 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,6-hexanediol, and 2-methyl-2,4-pentanediol; (2) 0.05M to 2.0M of a salt selected from sodium chloride, potassium chloride, calcium chloride, lithium chloride, sodium bromide, potassium bromide, and lithium bromide; (3) about 0.02 M to about 0.2 M sodium sulfate; (4) about 0.01% to about 1% nonionic surfactant; (5) about 0.05M to about 1.0M urea; or (6) about 0.02M to about 0.5M nicotinamide 3. The method of claim 1 or 2, wherein the sequestered ...

[0032] Embodiment 4: A method for reducing tryptophan and / or methionine oxidation in an albumin fusion protein during purification, comprising subjecting a composition comprising said albumin fusion protein to the following purification process: (a) Affinity matrix; (b) Anion exchange matrix wherein the affinity matrix is (1) from about 2% to about 20% of a polyol selected from the group consisting of 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,6-hexanediol, and 2-methyl-2,4-pentanediol; (2) 0.05M to 2.0M of a salt selected from sodium chloride, potassium chloride, calcium chloride, lithium chloride, sodium bromide, potassium bromide, and lithium bromide; (3) about 0.02 M to about 0.2 M sodium sulfate; (4) about 0.01% to about 1% nonionic surfactant; (5) about 0.05M to about 1.0M urea; or (6) about 0.02M to about 0.5M nicotinamide and washing with a washing buffer comprising [Embodiment 5] The method of embodiment 3 or 4, wherein the wash buffer comprises about 5% to about 15% polyol, about 0.2 M to about 0.8 M salt, about 0.2 M to about 0.8 M sodium sulfate, about 0.02% to about 0.4% nonionic surfactant, or about 0.2 M to about 1.0 M urea. [Embodiment 6] The method of any one of embodiments 3, 4, or 5, wherein the polyol is 1,2-propanediol, the salt is sodium chloride, and the nonionic surfactant is Triton X-100. [Embodiment 7] The method of embodiment 3 or 4, wherein the wash buffer comprises: (1) about 0.5 M sodium chloride; (2) about 0.5 M sodium sulfate; or (3) about 10% 1,3-propanediol. [Embodiment 8] The method described in embodiment 1, 2, 3, 5, 6 or 7, wherein the elution buffer comprises BisTris, Tris, phosphate, a salt, a chelating agent, or a combination thereof. [Embodiment 9] The method described in embodiment 8, wherein the elution buffer is 50 mM BisTris. [Embodiment 10] The method described in embodiment 3 or 4, wherein the wash buffer has a pH of 5.5 to 7.0. [Embodiment 11] The method of embodiment 3 or 4, wherein the affinity matrix is ​​equilibrated with a loading buffer. [Embodiment 12] The method according to any one of embodiments 1 to 11, wherein the affinity matrix is ​​a dye affinity matrix. [Embodiment 13] The method described in any one of embodiments 1 to 12, wherein the anion exchange matrix comprises agarose highly cross-linked with a dextran surface extender. [Embodiment 14] The method of any one of embodiments 1-13, wherein the albumin fusion protein is eluted from the anion exchange matrix using a step elution. [Embodiment 13] The method of any one of embodiments 1-13, wherein the albumin fusion protein is eluted from an anion exchanger using gradient elution. [Embodiment 14] The method of embodiment 1, wherein the anion exchange matrix elution buffer comprises a salt selected from the group consisting of NaCl, KCl, CaCl2, HCl, LiCl, NaBr, KBr, and LiBr. [Embodiment 15] The method of embodiment 14, wherein the salt is NaCl and is present in an amount of 20 to 400 mM. [Embodiment 16] The method described in any one of embodiments 1 to 15, wherein the anion exchange matrix is ​​equilibrated with a loading buffer containing 50 mM BisTris and 20 mM NaCl at pH 7.0. [Embodiment 17] The method of any one of embodiments 1 to 16, further comprising passing the composition through an anion exchange membrane. [Embodiment 18] The method described in embodiment 17, wherein the anion exchange membrane is a polyethersulfone-based membrane modified with a quaternary amine. [Embodiment 19] The method described in embodiment 17, wherein the composition is passed through the anion exchange membrane in a flow-through buffer having a salt concentration greater than 10 mM and a pH less than 8. [Embodiment 20] The method described in embodiment 19, wherein the flow-through buffer has a salt concentration of 10 mM to 220 mM and a pH of 6 to 8. [Embodiment 21] The method described in embodiment 20, wherein the flow-through buffer has a pH of 6 to 7.5 and a salt concentration of 50 mM to 220 mM. [Embodiment 22] The method of any one of embodiments 1 to 21, wherein the anion exchange matrix eluate is subjected to diafiltration. [Embodiment 23] The method described in embodiment 22, wherein the diafiltration buffer comprises 50 mM BisTris and 50 mM NaCl at pH 7.0. [Embodiment 24] The method of any one of embodiments 17 to 22, wherein the membrane is pre-conditioned. [Embodiment 25] The method of any one of embodiments 17 to 22, wherein the membrane is equilibrated. [Embodiment 26] The method described in any one of embodiments 1 to 25, further comprising the step of subjecting the composition to a hydrophobic interaction matrix. [Embodiment 27] The method of embodiment 26, wherein the hydrophobic interaction matrix or multimodal matrix is ​​selected from the group consisting of Capto Butyl, Capto Phenyl, Capto Butyl, Butyl-S Fast Flow, Toyopearl Hexyl, Toyopearl Butyl, Toyopearl Phenyl, Toyopearl PPG, Toyopearl Ether, Toyopearl PPG-600M, and Toyopearl Phenyl-650M, Toyopearl PPG-600M, TSKgel Phenyl, TSKgel Ether, Macro-Prep Methyl, Capto MMC, Eshmuno HCX, Nuvia cPrime, or Toyopearl MX-Trp-650M. [Embodiment 28] The method of embodiment 26, wherein the hydrophobic interaction matrix is ​​loaded with a buffer containing citrate, sodium sulfate, or ammonium sulfate. [Embodiment 29] The method of embodiment 26, wherein the hydrophobic interaction matrix is ​​eluted with an elution buffer containing low levels of citrate, sodium sulfate, or ammonium sulfate. [Embodiment 30] The method described in any one of embodiments 1 to 29, further comprising subjecting the composition to ultrafiltration. [Embodiment 31] The method described in any one of embodiments 1 to 30, further comprising subjecting the composition to diafiltration. [Embodiment 32] The method of any one of embodiments 1 to 31, further comprising subjecting the composition to nanofiltration. [Embodiment 33] The method described in any one of embodiments 1 to 32, further comprising subjecting the composition to size exclusion chromatography. [Embodiment 34] A method according to any one of embodiments 1 to 33, further comprising a viral inactivation process. [Embodiment 35] The method of embodiment 34, wherein the viral inactivation process comprises treating the composition comprising the albumin fusion protein with Triton X-100, Tween 80, polysorbate 20, polysorbate 80, nonoxynol-9, poloxamer, stearyl alcohol, or sorbitan monostearate. [Embodiment 36] The method described in embodiment 34, wherein the virus inactivation process is carried out after the process of step (a) and before the process of step (b). [Embodiment 37] The method described in embodiment 38, wherein Triton X-100 is added to a final concentration (w / w) of about 0.01% to about 1% Triton X-100, which is maintained for about 30 to about 240 minutes. Embodiment 38. A method for obtaining a composition comprising an albumin fusion protein essentially free of oxidized tryptophan residues, comprising the step of applying a composition comprising tryptophan-oxidized and non-tryptophan-oxidized albumin fusion proteins to a hydrophobic interaction matrix, wherein the tryptophan-oxidized and non-tryptophan-oxidized albumin fusion proteins are eluted from the hydrophobic interaction matrix at different times, thereby separating the tryptophan-oxidized albumin fusion proteins from the non-tryptophan-oxidized albumin fusion proteins.

[0039] Embodiment 39 is a method for isolating an albumin fusion protein essentially free of oxidation of tryptophan / methionine residues, comprising subjecting a composition comprising the albumin fusion protein to the following purification process: (a) Affinity matrix chromatography process; (b) an anion exchange chromatography process; and (c) Hydrophobic interaction matrix chromatography process wherein an elution buffer comprising octanoic acid is applied to the affinity matrix and the tryptophan-oxidized and non-tryptophan-oxidized albumin fusion proteins are eluted from the hydrophobic interaction matrix at different times, thereby separating the tryptophan-oxidized albumin fusion proteins from the non-tryptophan-oxidized albumin fusion proteins.

[0040] Embodiment 40. A method of purifying an albumin fusion protein, comprising: subjecting a composition comprising an albumin fusion protein to a hydrophobic interaction matrix and the following purification process: (a) an affinity matrix to which an elution buffer containing octanoic acid is applied; and / or (b) Anion exchange matrix wherein the affinity matrix is ​​washed with a wash buffer comprising: (1) about 2% to about 20% of a polyol selected from the group consisting of 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,6 hexanediol, and 2-methyl-2,4-pentanediol; (2) 0.05M to 2.0% of a salt selected from sodium chloride, potassium chloride, calcium chloride, lithium chloride, sodium bromide, potassium bromide, and lithium bromide; (3) about 0.02M to about 0.2M sodium sulfate; (4) about 0.01% to about 1% of a non-ionic surfactant; (5) about 0.05M to about 1.0M urea; or (6) about 0.02M to about 0.5M nicotinamide; wherein the resulting purified albumin fusion protein is essentially free of oxidized tryptophan residues.

[0082] [Embodiment 41] A method for purifying an albumin fusion protein, comprising: (a) applying a composition comprising said albumin fusion protein to an affinity matrix; (b) eluting said albumin fusion protein from the affinity matrix of (a) to obtain a first eluate; (c) applying the first eluate to an anion exchange matrix; (d) eluting the albumin fusion protein from the anion exchange matrix to obtain a second eluate; (e) applying the second eluate to an anion exchange membrane; (f) passing the albumin fusion protein through an anion exchange membrane to obtain a flow-through; (g) applying the flow-through to a hydrophobic interaction matrix; (h) eluting said albumin fusion protein from said hydrophobic interaction matrix to obtain a third eluate; wherein said third eluate comprises said purified albumin fusion protein. [Embodiment 42] The method of any one of embodiments 1 to 41, wherein the albumin in the albumin fusion protein is human serum albumin (HSA). [Embodiment 43] The method described in embodiment 42, wherein the HSA is a mutant HSA. [Embodiment 44] The method described in embodiment 43, wherein the amino acid sequence of the mutant HSA is SEQ ID NO: 133. [Embodiment 45] The method of any one of embodiments 1 to 44, wherein the albumin fusion protein comprises a scaffold portion comprising a third fibronectin type III (FnIII) ​​domain. [Embodiment 46] The method of embodiment 45, wherein the FnIII domain is derived from human tenascin-C (Tn3 scaffold). [Embodiment 47] The method of any one of embodiments 1-46, wherein the albumin fusion protein comprises a scaffold. [Embodiment 48] The method of embodiment 47, wherein the scaffold comprises tryptophan residues. [Embodiment 49] The method of embodiment 48, wherein oxidation of the tryptophan residue reduces the activity of the albumin fusion protein. [Embodiment 50] The method described in any one of embodiments 47 to 49, wherein the scaffold specifically binds to CD40L. [Embodiment 51] The method of embodiment 50, wherein the Tn3 scaffold comprises a single CD40L-specific monomeric subunit. [Embodiment 52] The method of embodiment 51, wherein the Tn3 scaffold comprises two CD40L-specific monomer subunits linked in tandem. [Embodiment 53] The method of embodiment 52, wherein the two CD40L-specific monomer subunits are directly linked. [Embodiment 54] The method of embodiment 52, wherein the two CD40L-specific monomer subunits are linked by a linker.

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] [009 ... m X) n Arrays, where: (a) X is serine (S), alanine (A), glycine (G), Leu (L), isoleucine (I), or valine (V); (b) m and n are integers; (c) m is 1, 2, 3, or 4; and (d) The method of embodiment 55, wherein n is 1, 2, 3, 4, 5, 6, or 7. [Embodiment 57] The method of embodiment 56, wherein the peptide linker comprises SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 142 or SEQ ID NO: 143. [Embodiment 58] The method described in embodiment 45, wherein the Tn3 scaffold comprises a β-strand, and the β-strand of at least one CD40L-specific monomer subunit has at least 90% sequence identity to the β-strand of SEQ ID NO: 3. [Embodiment 59] The method described in embodiment 45, wherein the Tn3 scaffold comprises β strand A, and the β strand A comprises SEQ ID NO: 11 except for at least one mutation. [Embodiment 60] The method described in embodiment 45, wherein the Tn3 scaffold comprises a β-strand B, and the β-strand B comprises SEQ ID NO: 12 except for at least one mutation. [Embodiment 61] The method of embodiment 45, wherein the Tn3 scaffold comprises a β-strand C, wherein the β-strand C comprises SEQ ID NO: 13 or 14 except for at least one mutation, and wherein the cysteine ​​in SEQ ID NO: 13 or 14 is not substituted. [Embodiment 62] The method of embodiment 45, wherein the Tn3 scaffold comprises β-strand D, and the β-strand D comprises SEQ ID NO: 15 except for at least one mutation. [Embodiment 63] The method of embodiment 45, wherein the Tn3 scaffold comprises as Eβ, and the β-strand E comprises SEQ ID NO: 16 except for at least one mutation. [Embodiment 64] The method of embodiment 45, wherein the Tn3 scaffold comprises as Fβ, beta strand D comprises SEQ ID NO: 17 except for at least one mutation, and the cysteine ​​in SEQ ID NO: 17 is not substituted. [Embodiment 65] The method of embodiment 45, wherein the Tn3 scaffold comprises a β-strand G, and the β-strand G comprises SEQ ID NO: 18 except for at least one mutation.

[0066] The CD40L-specific monomeric subunit has the amino acid sequence: [ka] where: (a)X AB , X BC , X CD , X DE , X EF , and X FG represent the amino acid residues present in the sequences of the AB, BC, CD, DE, EF, and FG loops, respectively; (b) X1 represents an amino acid residue A or T; and (c) The method of any one of embodiments 50 to 54, wherein the length n of the loop is an integer between 2 and 26. [Embodiment 67] The method described in embodiment 66, wherein the sequence of the AB loop comprises SEQ ID NO: 4 or SEQ ID NO: 136, the sequence of the CD loop comprises SEQ ID NO: 6, and the sequence of the EF loop comprises SEQ ID NO: 8 or SEQ ID NO: 137. [Embodiment 68] The method described in embodiment 67, wherein the sequence of the BC loop comprises a sequence selected from the group consisting of SEQ ID NOs: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, and 168. [Embodiment 69] The method described in embodiment 67, wherein the sequence of the DE loop comprises a sequence selected from the group consisting of SEQ ID NOs: 94, 95, 96, 97, 98, and 169. [Embodiment 70] The method described in embodiment 67, wherein the sequence of the FG loop comprises a sequence selected from the group consisting of SEQ ID NOs: 9, 99, 139, and 170. [Embodiment 71] The method described in embodiment 67, wherein the sequence of the BC loop comprises a sequence selected from the group consisting of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, and 174. [Embodiment 72] The method described in embodiment 67, wherein the sequence of the DE loop comprises a sequence selected from the group consisting of SEQ ID NOs: 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, and 175. [Embodiment 73] The method described in embodiment 67, wherein the sequence of the FG loop comprises a sequence selected from the group consisting of SEQ ID NOs: 129, 130, and 177. [Embodiment 74] (a) the sequence of the BC loop comprises SEQ ID NO: 83, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139; (b) the sequence of the BC loop comprises SEQ ID NO:83, the sequence of the DE loop comprises SEQ ID NO:94, and the sequence of the FG loop comprises SEQ ID NO:99; (c) the sequence of the BC loop comprises SEQ ID NO: 84, the sequence of the DE loop comprises SEQ ID NO: 95, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139; (d) the sequence of the BC loop comprises SEQ ID NO:85, the sequence of the DE loop comprises SEQ ID NO:94, and the sequence of the FG loop comprises SEQ ID NO:9 or 139; (e) the sequence of the BC loop comprises SEQ ID NO: 86, the sequence of the DE loop comprises SEQ ID NO: 96, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139; (f) the sequence of the BC loop comprises SEQ ID NO:87, the sequence of the DE loop comprises SEQ ID NO:97, and the sequence of the FG loop comprises SEQ ID NO:9 or 139; (g) the sequence of the BC loop comprises SEQ ID NO:88, the sequence of the DE loop comprises SEQ ID NO:95, and the sequence of the FG loop comprises SEQ ID NO:9 or 139; (h) the sequence of the BC loop comprises SEQ ID NO:89, the sequence of the DE loop comprises SEQ ID NO:94, and the sequence of the FG loop comprises SEQ ID NO:9 or 139; (i) the sequence of the BC loop comprises SEQ ID NO: 90, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139; (j) the sequence of the BC loop comprises SEQ ID NO: 91, the sequence of the DE loop comprises SEQ ID NO: 95, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139; (k) the sequence of the BC loop comprises SEQ ID NO: 92, the sequence of the DE loop comprises SEQ ID NO: 98, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139; or (l) The method of embodiment 67, wherein the sequence of the BC loop comprises SEQ ID NO: 93, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139. [Embodiment 75] (a) the sequence of the BC loop comprises SEQ ID NO: 100, the sequence of the DE loop comprises SEQ ID NO: 118, and the sequence of the FG loop comprises SEQ ID NO: 129; (b) the sequence of the BC loop comprises SEQ ID NO:101, the sequence of the DE loop comprises SEQ ID NO:119, and the sequence of the FG loop comprises SEQ ID NO:129; (c) the sequence of the BC loop comprises SEQ ID NO:102, the sequence of the DE loop comprises SEQ ID NO:120, and the sequence of the FG loop comprises SEQ ID NO:129; (d) the sequence of the BC loop comprises SEQ ID NO:103, the sequence of the DE loop comprises SEQ ID NO:121, and the sequence of the FG loop comprises SEQ ID NO:129; (e) the sequence of the BC loop comprises SEQ ID NO:104, the sequence of the DE loop comprises SEQ ID NO:122, and the sequence of the FG loop comprises SEQ ID NO:129; (f) the sequence of the BC loop comprises SEQ ID NO: 105, the sequence of the DE loop comprises SEQ ID NO: 121, and the sequence of the FG loop comprises SEQ ID NO: 129; (g) the sequence of the BC loop comprises SEQ ID NO:106, the sequence of the DE loop comprises SEQ ID NO:123, and the sequence of the FG loop comprises SEQ ID NO:129; (h) the sequence of the BC loop comprises SEQ ID NO: 107, the sequence of the DE loop comprises SEQ ID NO: 123, and the sequence of the FG loop comprises SEQ ID NO: 129; (i) the sequence of the BC loop comprises SEQ ID NO:108, the sequence of the DE loop comprises SEQ ID NO:118, and the sequence of the FG loop comprises SEQ ID NO:129; (j) the sequence of the BC loop comprises SEQ ID NO: 109, the sequence of the DE loop comprises SEQ ID NO: 123, and the sequence of the FG loop comprises SEQ ID NO: 129; (k) the sequence of the BC loop comprises SEQ ID NO:110, the sequence of the DE loop comprises SEQ ID NO:121, and the sequence of the FG loop comprises SEQ ID NO:129; (l) the sequence of the BC loop comprises SEQ ID NO:111, the sequence of the DE loop comprises SEQ ID NO:123, and the sequence of the FG loop comprises SEQ ID NO:130; (m) the sequence of the BC loop comprises SEQ ID NO: 108, the sequence of the DE loop comprises SEQ ID NO: 121, and the sequence of the FG loop comprises SEQ ID NO: 129; (n) the sequence of the BC loop comprises SEQ ID NO: 112, the sequence of the DE loop comprises SEQ ID NO: 124, and the sequence of the FG loop comprises SEQ ID NO: 129; (o) the sequence of the BC loop comprises SEQ ID NO: 113, the sequence of the DE loop comprises SEQ ID NO: 125, and the sequence of the FG loop comprises SEQ ID NO: 129; (p) the sequence of the BC loop comprises SEQ ID NO:114, the sequence of the DE loop comprises SEQ ID NO:118, and the sequence of the FG loop comprises SEQ ID NO:129; (q) the sequence of the BC loop comprises SEQ ID NO:115, the sequence of the DE loop comprises SEQ ID NO:126, and the sequence of the FG loop comprises SEQ ID NO:129; (r) the sequence of the BC loop comprises SEQ ID NO: 116, the sequence of the DE loop comprises SEQ ID NO: 127, and the sequence of the FG loop comprises SEQ ID NO: 129; or (s) the sequence of the BC loop comprises SEQ ID NO: 117, the sequence of the DE loop comprises SEQ ID NO: 128, and the sequence of the FG loop comprises SEQ ID NO: 129. [Embodiment 76] The method described in embodiment 75, wherein the AB loop comprises sequence number 136. [Embodiment 77] The method described in embodiment 75, wherein the CD40L-specific monomer subunit comprises a sequence selected from the group consisting of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 146.

[0078] The CD40L-specific monomeric subunit has the amino acid sequence: [ka] where: (a) X1 represents the amino acid residue serine (S) or leucine (L); (b) X2 represents the amino acid residue aspartic acid (D) or glutamic acid (E); (c) X3 represents the amino acid residue histidine (H), isoleucine (I), valine (V), phenylalanine (F), or tryptophan (W); (d) X4 represents the amino acid residue alanine (A), glycine (G), glutamic acid (E), or aspartic acid (D); (e) X5 represents the amino acid residue glutamic acid (E), leucine (L), glutamine (Q), serine (S), aspartic acid (D), or asparagine (N); (f) X6 represents the amino acid residue phenylalanine (F) or tyrosine (Y); (g) X7 represents the amino acid residue isoleucine (I), valine (V), histidine (H), glutamic acid (E), or aspartic acid (D); (h) X8 represents the amino acid residue glycine (G), tryptophan (W), or valine (V); (i) X9 represents the amino acid residue tryptophan (W), phenylalanine (F), or tyrosine (Y); (j)X 10 represents the amino acid residue serine (S), glutamine (Q), methionine (M), or histidine (H); (k)X 11 represents the amino acid residue tryptophan (W) or histidine (H); and (l)X 12 55. The method of any of embodiments 50-54, wherein represents the amino acid residue arginine (R) or serine (S). [Embodiment 79] The method described in embodiment 78, wherein the CD40L-specific monomer subunit comprises an array selected from the group consisting of SEQ ID NOs: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82.

[0080] The CD40L-specific monomeric subunit has the amino acid sequence: [ka] where: (a) X1 represents the amino acid residue lysine (K) or glutamic acid (E); (b) X2 represents the amino acid residue threonine (T) or isoleucine (I); (c) X3 represents the amino acid residue asparagine (N) or alanine (A); (d) X4 represents the amino acid residue serine (S), leucine (L), alanine (A), phenylalanine (F), or tyrosine (Y); (e) X5 represents the amino acid residue tyrosine (Y), alanine (A), glycine (G), valine (V), isoleucine (I), or serine (S); (f) X6 represents the amino acid residue tyrosine (Y), serine (S), alanine (A), or histidine (H); (g) X7 represents the amino acid residue asparagine (N), aspartic acid (D), histidine (H), or tyrosine (Y); (h) X8 represents the amino acid residue leucine (L), phenylalanine (F), histidine (H), or tyrosine (Y); (i) X9 represents the amino acid residue histidine (H), proline (P), serine (S), leucine (L), or aspartic acid (D); (j)X 10 represents the amino acid residue glycine (G), phenylalanine (F), histidine (H), or tyrosine (Y); (k) (l)X 11 represents the amino acid residue alanine (A) or threonine (T); (m)X 12 represents the amino acid residue serine (S), asparagine (N), glutamic acid (E), asparagine (R), or aspartic acid (D); (n)X 13represents the amino acid residue serine (S), glutamine (Q), threonine (T), asparagine (N), or alanine (A); (o)X 14 represents the amino acid residue proline (P), valine (V), isoleucine (I) or alanine (A), or no amino acid; (p)X 15 represents the amino acid residue isoleucine (I) or no amino acid; (q)X 16 represents the amino acid residue glutamic acid (E) or lysine (K); and (r)X 17 79. The method of embodiment 78, wherein represents the amino acid residue serine (S) or asparagine (N). [Embodiment 81] The method of embodiment 46, wherein the Tn3 scaffold comprises a sequence selected from the group consisting of SEQ ID NOs: 134, 135, 205, 206, 207 and 208. [Embodiment 82] The method of embodiment 46, wherein the Tn3 scaffold comprises a sequence selected from the group consisting of SEQ ID NOs: 201, 202, 203, and 204. [Embodiment 83] The method of embodiment 50, wherein the CD40L is human CD40L. [Embodiment 84] The method of embodiment 83, wherein the CD40L is membrane-bound CD40L (SEQ ID NO: 1), soluble CD40L (SEQ ID NO: 2), or a fragment thereof. [Embodiment 85] The method of any one of embodiments 50 to 84, wherein the scaffold binds to CD40L and blocks binding of CD40L to CD40. [Embodiment 86] The method of any one of embodiments 50 to 85, wherein the scaffold binds to CD40L and disrupts CD40-mediated signaling. [Embodiment 87] The method of any one of embodiments 50 to 86, wherein the scaffold binds to CD40L with an affinity (Kd) of about 1 μM or less, or about 500 nM or less, or about 100 nM or less, or about 50 nM or less, or about 25 nM or less, or about 10 nM or less, or about 5 nM or less, or about 2 nM or less. [Embodiment 88] A method described in any one of embodiments 50 to 87, wherein the CD40L-specific monomer subunit specifically binds to a CD40L epitope comprising amino acids located at positions 142 to 155, 200 to 230, or 247 to 251 of SEQ ID NO: 2. [Embodiment 89] An albumin fusion protein composition obtained by the method of any one of embodiments 1 to 88. [Embodiment 90] The composition of embodiment 89, wherein the purified albumin fusion protein has a relative potency of >90%. [Embodiment 91] A composition comprising an albumin fusion protein having less than 20 ng / mg of host cell protein and having less than 15% of the tryptophan residues oxidized. [Embodiment 92] The composition described in embodiment 91, wherein less than 5% of the tryptophan residues are oxidized. [Embodiment 93] The composition described in embodiment 94, wherein less than 5% of the amino acid residues in the total protein are oxidized. [Embodiment 94] A composition comprising an albumin fusion protein, comprising 5 x 10 -3 A composition having less than ng / mg of DNA and less than 15% of the tryptophan residues are oxidized. [Embodiment 95] The composition described in embodiment 94, wherein less than 5% of the tryptophan residues are oxidized. [Embodiment 96] The composition described in embodiment 95, wherein less than 20% of the amino acid residues in the total protein are oxidized. [Embodiment 97] A composition comprising an albumin fusion protein having less than 20 ng / mg of host cell protein, wherein the albumin fusion protein has a relative activity of >90%.

[0099] Embodiment 98. A composition comprising an albumin fusion protein, comprising 5 x 10 -3 1. A composition having less than ng / mg of DNA, wherein the albumin fusion protein has a relative activity of >90%. [Embodiment 99] A composition comprising an albumin fusion protein of SEQ ID NO: 134, 135, 201, 202, 203, 204, 205, 206, 207, or 208, having less than 20 ng / mg host cell protein, wherein the tryptophan at positions 46, 151, or both, is not oxidized. [Embodiment 100] (a) the composition according to any one of embodiments 90 to 99; (b) buffer; (c) sugar; and (d) Emulsifier A pharmaceutically acceptable formulation comprising: [Embodiment 101] The formulation of embodiment 100, wherein the buffer is a sodium phosphate buffer, the sugar is sucrose, and the emulsifier is polysorbate 80. [Embodiment 102] The formulation of embodiment 100 or 101, which is lyophilized. equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

[0320] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. [Example]

[0321] The invention will now be described with reference to the following examples, which are merely illustrative and the invention should in no way be construed as being limited to these examples, but rather as encompassing any and all variations that become evident as a result of the teachings provided herein.

[0322] Example 1 chemicals Propylene glycol was obtained from Alfa Aesar (Ward Hill, MA, USA). Sucrose was obtained from Pfanstiehl (Waukegan, IL, USA). Triton X-100 and sodium sulfate were obtained from EMD Millipore (Billerica, MA, USA). Bis-Tris, Bis-Tris HCl, and nicotinamide were obtained from Sigma-Aldrich (St. Louis, MO, USA). Glacial acetic acid, arginine, glycine, sodium acetate, sodium caprylate, sodium chloride, sodium citrate, sodium hydroxide, sodium phosphate, Tris, and urea were obtained from JT Baker (Center Valley, PA, USA).

[0323] protein The protein used in this study, albumin fusion protein #1 (AFP-1) (SEQ ID NO: 145), is a CD40L antagonist consisting of two identical tenascin-C (TnC) domains derived from the human fibronectin type III protein domain fused to human serum albumin. Each TnC domain (derived from the third fibronectin type III protein domain of human TnC) binds to human CD40L and inhibits its interaction with human CD40. The human serum albumin fusion ensures favorable pharmacokinetic properties of the molecule. The protein is expressed in Chinese hamster ovary (CHO) cells using techniques familiar to those skilled in the art. Recombinant human albumin (rHSA; expressed in rice) was purchased as a lyophilized powder from Sigma-Aldrich (catalog number A9731). Table 3 summarizes the properties of rHSA and AFP-1.

[0324] [Table 4]

[0325] Measurement of total protein concentration Protein concentrations in all process intermediates (except for clarified media and, optionally, Cibacron blue dye chromatography pools) were measured by absorbance at 280 nm using standard spectrophotometric procedures common in the industry, with an extinction coefficient of 0.98 (mg / mL) for AFP-1. -1 cm -1 , 0.531 (mg / mL) for rHSA -1 cm -1 was used.

[0326] HSA affinity high-performance liquid chromatography Analytical high-performance HSA affinity chromatography (HSA-HPLC) was performed using an Agilent 1200 HPLC system (Palo Alto, CA, USA) with a Poros CaptureSelect HSA column from Life Technologies (Grand Island, NY, USA). The equilibration buffer phase was 10–50 mM sodium phosphate, pH 7.2, at 3.5 mL / min, and the product was eluted with 100 mM glycine, pH 2.0 buffer. Samples of 10–100 μg were injected appropriately, and the elution profile was monitored spectrophotometrically at 280 nm. Data were collected and analyzed using Agilent ChemStation software, and product-specific concentrations were determined from a calibration curve generated using purified protein.

[0327] Dye affinity chromatography Cibacron blue dye affinity chromatography was performed under typical binding and elution conditions in a small-scale chromatography column with a 20 cm bed height. All runs were performed using an AKTA Explorer liquid chromatography system from GE Healthcare (Piscataway, NJ, USA), with the column operated at 300 cm / h. Under baseline conditions, the column was equilibrated with 50 mM Bis-Tris (or phosphate), 50 mM NaCl, pH 6.0, and then loaded with up to 25 g of protein per L of resin (based on the HSA-HPLC titer in the clarified cell culture fluid). After loading, the column was re-equilibrated, washed with 50 mM Bis-Tris (or phosphate), pH 7.0, and then eluted with 50 mM Bis-Tris (or phosphate), 25 mM sodium octanoate, 10 mM EDTA, pH 7.0. Product peaks were collected based on absorbance criteria of 100 mAU on the leading and trailing sides of the product peak. During optimization (see Example 2), additional washes were applied to the column during re-equilibration and a 50 mM phosphate, pH 7 wash. Capto Blue (high sub) resin was obtained from GE Healthcare (Piscataway, NJ, USA). Toyopearl AF-Blue HC-650M resin was obtained from Tosho Biosciences (King of Prussia, PA, USA).

[0328] Anion exchange chromatography Anion exchange chromatography (AEX) was performed under typical binding and elution conditions in a miniaturized chromatography column packed to a 20 cm bed height. All runs were performed using a GE Healthcare AKTA Explorer liquid chromatography system, with the column operated at 300 cm / h. Under baseline conditions, the column was equilibrated with 50 mM Bis-Tris, 20 mM sodium chloride, pH 7.0, and the protein was loaded and then washed with the equilibration buffer. The column was eluted using a stepwise or 10-column-volume (CV) linear gradient from 20 to 400 mM sodium chloride in Bis-Tris buffer at pH 7.0. Product peaks were collected based on an absorbance criterion of 100 mAU for the leading and trailing edges of the product peak. Capto Q resin was obtained from GE Healthcare (Piscataway, NJ, USA).

[0329] Anion-exchange membrane chromatography Anion exchange membrane chromatography (AEMC) was performed under typical flow-through conditions. All runs were performed using a GE Healthcare AKTA Explorer liquid chromatography system, with the column operated at 10 MV / min. Under baseline conditions, the membrane was equilibrated with 50 mM Bis-Tris, 50 mM sodium chloride, pH 7.0, and then the load material was passed through the membrane. Flow-through product peaks were collected based on an absorbance criterion of 100 mAU for the leading and trailing sides of the product peak. During optimization (see Example 2), buffer conditions ranging from 10 to 220 mM NaCl and pH 6 to 8 were used. Mustang Q membranes were obtained from Pall Life Sciences (Port Washington, NY, USA).

[0330] Hydrophobic interaction chromatography Hydrophobic interaction chromatography (HIC) was performed under typical binding and elution conditions in a small-scale chromatography column with a 20 cm bed height. All runs were performed using an AKTA Explorer liquid chromatography system from GE Healthcare (Piscataway, NJ). The column was operated at 130–300 cm / h. Under baseline conditions, the column was equilibrated with 50 mM Bis-Tris, 1 M sodium citrate, pH 7.0. The load was prepared by diluting 1 part (by weight) of protein solution with 2 parts (by weight) of 50 mM Bis-Tris, 2 M sodium citrate, pH 7.0, and then loading the column with up to 25 g of protein per L of resin. After loading, the column was re-equilibrated with equilibration buffer and then eluted with a linear gradient of sodium citrate from 1 M to 0 mM over 20 column volumes. The product peak was collected in fractions, while early-eluting material was enriched in oxidation products. Toyopearl PPG 600M and Toyopearl Phenyl 650M resins were from Tosoh Bioscience (King of Prussia, PA, USA); Capto MMC and Butyl-S Fast Flow resins were from GE Healthcare (Piscataway, NJ, USA).

[0331] Analytical Size Exclusion Chromatography Analytical high-performance size-exclusion chromatography (SEC-HPLC) was performed using an Agilent 1200 HPLC system (Palo Alto, CA, USA) with a TSK-GEL G3000SWXL column (7.8 mm x 30 cm) from Tosoh Biosciences (King of Prussia, PA, USA). The mobile phase was 0.1 M sodium phosphate, 0.1 M sodium sulfate, 10% isopropanol, pH 6.8, at 30°C and 0.8 mL / min for 22 min. A 250 μg sample was injected appropriately, and the column was calibrated using molecular weight standards from Bio-Rad (Hercules, CA, USA). Elution profiles were monitored using a spectrophotometer at 280 nm, and data were collected and analyzed using Agilent's ChemStation software. Results are reported as the area percent of the product monomer peak relative to all other peaks, excluding a buffer-related peak observed after approximately 12 min. When the SEC-HPLC method is run without 10% isopropanol in the mobile phase, a front shoulder (not fully resolved) to the monomer peak is observed, which is identified as the tryptophan oxidation monomer. Therefore, it can be used to measure monomer and aggregates or to estimate tryptophan oxidation, depending on the SEC-HPLC assay procedure.

[0332] Example 2 Albumin fusion protein purification (500L scale) Recombinant human albumin (rHSA) was purified using a process involving three bind-and-elute chromatography columns: a flow-through chromatography membrane, a Triton virus inactivation step, and an ultrafiltration / diafiltration step. To generate the starting material for the rHSA process, cell culture supernatant from a monoclonal antibody (mAb) process was depleted of antibody by collecting unbound material during Protein A chromatography, and then lyophilized rHSA powder was dissolved in the antibody-free supernatant. This starting material contained host cell proteins, DNA, and small molecule impurities that are typically present in cell culture supernatants of albumin fusion proteins expressed in CHO cell culture.

[0333] The rHSA purification process shown in Figure 1 was used to purify approximately 700 mL of supernatant. Table 4 shows performance parameters from a 1 L-scale purification. As can be seen in the table, step yields for the chromatography unit operations were generally high, with the exception of Capto Blue (high sub). Figure 2 shows the Cibacron blue (high sub) dye affinity chromatogram for rHSA. As can be seen, large absorbance peaks are observed during the load and 0.5 M NaCl wash. From this run alone, it is unknown whether the low yield was due to overloading the column (i.e., the column was saturated with rHSA and not all of the rHSA was captured from the supernatant) or the 0.5 M NaCl wash. In either case, it is likely that the yield loss could be minimized by optimizing the column load and wash step conditions.

[0334] [Table 5]

[0335] A summary of the product quality attributes of the rHSA process intermediates is shown in Table 5. As can be seen, HCPs and DNA were well controlled along with the purification process to low levels, with HCPs measured at <10 ng / mg and DNA at 1.7 x 10-4 Measured in ng / mg. HCP reduction is greater than 2 logs over the Capto Blue column and an additional log (or more) from the CaptoQ and PPG-600M columns. Greater than 5 logs of DNA are removed by the CaptoQ column, and an additional log (or more) is removed by the Capto Blue and Mustang Q steps. Further aggregate removal was observed over multiple steps during the purification process. Overall, the process was very successful for purifying rHSA and could be used as a starting point for the purification of albumin fusion proteins.

[0336] [Table 6]

[0337] Example 3 Albumin fusion protein purification (500L scale) Recombinant human serum albumin fusion protein AFP-1 (HSA fusion or albumin fusion) is expressed in CHO cells and purified using a process that includes a flow-through chromatography membrane, a Triton virus inactivation step, three bind-and-elute chromatography columns including a nanofilter, and two intermediate ultrafiltration / diafiltration steps.

[0338] The albumin fusion purification process is shown in Figure 3. The purification process shown in Figure 3 was scaled up to two 500 L bioreactors. Table 6 shows the performance parameters obtained from the two 500 L bioreactor-scale purifications. As can be seen, the step yields and pool volumes for the chromatography unit operations are consistent between lots. The overall process yields (including UF / DF and nanofiltration) for Lot 1 and Lot 2 were 48% and 53%, respectively. A summary of the product quality attributes of the process intermediates is shown in Table 7. The overall performance (i.e., process yield) and product quality (absolute levels and LRVs) are very comparable to the purification process used for rHSA purification. This is particularly true for the steps responsible for the majority of HCP removal (Capto Blue (high sub) and Capto Q) and DNA clearance (Capto Q and Mustang Q).

[0339] [Table 7]

[0340] [Table 8]

[0341] Cibacron blue dye affinity chromatography using Capto Blue (high sub) resin is used as the capture column for the AFP-1 purification process. Figure 4 shows a representative chromatogram of Cibacron blue dye chromatography on AFP-1 operated at 300 cm / hr. As can be seen in Figure 4, a large flow-through peak is observed during loading, as indicated by the large OD signal beginning at approximately 6 column volumes (CV). This flow-through peak contains the majority of process-related impurities present in the conditioned medium, including host cell proteins (HCPs) and DNA. After loading, the column is re-equilibrated and then washed with different buffers: first with a buffer containing 0.5 M NaCl at pH 6.0, followed by re-equilibration and then with a buffer containing 10% propylene glycol at pH 7.0. The washes reduce HCP and DNA levels in the product pool by dissociating these impurities from the Cibacron blue dye ligand and / or albumin fusion protein. The column is then eluted with a buffer containing sodium octanoate and EDTA. As can be seen in Table 7, after Capto Blue(high sub) chromatography, the product intermediate has less DNA (0.6-0.7 log clearance) and fewer HCPs (2.7-2.9 log clearance) compared to the conditioned medium. Additionally, the Capto Blue(high sub) product has a high monomer content (≥98.0%) and low levels of oxidation products.

[0342] After initial capture by Cibacron blue dye chromatography, the product is treated with Triton X-100 to inactivate potential enveloped viruses. In this example, 10% Triton X-100 is added to the Capto Blue (high sub) pool to a final concentration of 0.5% Triton X-100 (w / w) and maintained at room temperature for 130 minutes. Under these conditions, efficient virus inactivation is achieved (see Example 5 for details).

[0343] After Triton X-100 treatment, the albumin fusion protein is purified by anion exchange chromatography using a Capto Q column in bind-and-elute mode. Figure 5 shows a representative Capto Q chromatogram for AFP-1 operated at 300 cm / hr. As can be seen in Figure 5, a large flow-through peak is observed during loading, as indicated by the large OD signal starting at approximately 7 CV. This flow-through peak contains Triton X-100 from the previous step. After loading, the column is re-equilibrated and then eluted with a linear NaCl gradient to 0.4 M NaCl over 10 CV (at pH 7.0). As can be seen in Table 7, the Capto Q intermediate has fewer HCPs (1.3-1.5 log clearance) and significantly less DNA (6-6.7 log clearance) than the Capto Blue (high sub) pool. It can also be seen that Capto Q has the ability to increase the monomer content (by reducing aggregation products) and also remove impurities involved in the oxidation of AFP-1 (see Example 7 for details).

[0344] To prepare for purification using a Mustang Q membrane chromatography step, the Capto Q product is diafiltered against 50 mM Bis-Tris, 50 mM NaCl, pH 7.0. Figure 6 shows a representative Mustang Q membrane chromatogram operated at 10 MV / hr. After preconditioning and equilibration of the membrane, the product is applied to the membrane and collected in the flow-through, while impurities bind to the membrane. Upon stripping with 2 M NaCl, a large peak containing both impurities and some product is observed (the strip peak is not included in the chromatogram in Figure 6). As can be seen in Table 7, DNA is further reduced by the Mustang Q membrane (0.6-1 log clearance).

[0345] The final chromatography step is a hydrophobic interaction column using Toyopearl PPG-600M. Figure 7 shows a representative Toyopearl PPG-600M chromatogram for AFP-1 operated at 130 cm / hr. After equilibration with a buffer containing 1 M citrate, the product is loaded onto the column. No flow-through peak is observed, as the product is fairly pure up to this stage in the purification process. After loading, the column is re-equilibrated and then eluted with a gradient against a buffer containing no citrate. The product elutes in a sharp peak and is collected in fractions that are assayed for oxidation product content by HIC-HPLC. All fractions containing less than 15% oxidized species by HIC-HPLC are pooled and subjected to prior nanofiltration.

[0346] Nanofiltration using Viresolve Vpro+ is performed using techniques standard to those skilled in the art of protein purification. The goal of nanofiltration is to remove potential viral particles. After nanofiltration, the product is concentrated, diafiltered, and formulated in 10 mM phosphate, 250 mM sucrose, 0.02% polysorbate 80. After completion (or formulation) of nanofiltration, the product pool is tested for additional impurities introduced into the product during the purification process. Table 8 summarizes the process-related impurity testing. As can be seen in Table 8, buffer components and Cibacron blue dye ligand introduced into the process in the early process steps (blue dye chromatography and viral inactivation steps) are reduced to very low levels by the subsequent purification steps.

[0347] [Table 9]

[0348] Example 4 Cibacron blue dye affinity chromatography Capto Blue (high sub) and Toyopearl AF-Blue HC-650M were compared in terms of binding capacity to remove impurities from clarified cell culture fluid. Table 9 summarizes the dynamic binding capacity of AFP-1 in clarified cell culture fluid. For Capto Blue (high sub), the dynamic binding capacity showed an indirect correlation with pH, ​​with pH 5 having the highest binding capacity (37.4 g AFP-1 per L of resin) and pH 8 having the lowest binding capacity (12.3 g / L). While high binding capacity is desirable, operation at pH 5 is less desirable due to increased aggregation rates of the molecule below pH 6 (data not shown). Therefore, pH 6 was selected as the optimal pH to balance high binding capacity and product stability. Comparison of the dynamic binding capacities of Capto Blue (high sub) and Toyopearl AF-Blue HC-650M showed that the dynamic binding capacity of Capto Blue (high sub) at pH 6 was nearly doubled.

[0349] [Table 10]

[0350] To compare these two resins for impurity clearance from clarified cell culture fluid, each column was operated under baseline conditions, loaded to 75-80% of its dynamic binding capacity (17.5 g / L for Capto Blue (high sub) and 10.0 g / L for Toyopearl AF-Blue HC-650M), and eluted from the column with 25 mM sodium octanoate. Table 10 shows a comparison of the two Cibacron blue dye resins used for capture and purification of AFP-1 from clarified cell culture fluid. As can be seen in Table 10, yields for both resins were similar (when eluted with 25 mM octanoic acid), with typical yields >90%. Furthermore, although both resins effectively reduced HCP and DNA levels from clarified cell culture fluid, Toyopearl AF-Blue HC-650M chromatography demonstrated slightly better HCP and DNA clearance compared to Capto Blue (high sub).

[0351] [Table 11]

[0352] For the AFP-1 production process, Capto Blue (high sub) resin was selected for its higher binding capacity. To optimize the capture step using Capto Blue (high sub), several factors were considered, including column load and wash and elution buffer composition. As can be seen in Table 10, column load had no effect on DNA but showed a small effect on HCP clearance. HCP clearance at the extremes of the column load tested (10 g / L or 25 g / L load) was more effective than at the intermediate load (17.5 g / L load). Therefore, it is advantageous to operate the column at or near its dynamic binding capacity to increase throughput and further increase HCP removal.

[0353] The elution buffer composition was optimized for the Cibacron blue dye chromatography capture step. Figure 8 shows a comparison of elution buffers for Cibacron blue dye chromatography. As can be seen in Figure 8 and Table 10, octanoic acid is a much more effective choice for elution from a Cibacron blue dye column compared to 2M NaCl. In this example, 2M NaCl demonstrated incomplete elution of AFP-1 from the column based on a 60% yield and the broad elution profile seen in the chromatogram. The 2M NaCl product quality run showed much higher HCP levels and much lower monomer levels compared to the elution using 25 mM octanoic acid. Furthermore, using 2M NaCl (alone or in combination with a solvent) can be less desirable from a manufacturing standpoint, as these elution buffers are costly and the process may require a buffer exchange step to facilitate binding to the next chromatography column in the process.

[0354] The final step in the development of the Cibacron blue dye capture step was wash optimization. It is well known that albumin can bind a wide variety of molecules, and the same is true for albumin fusion proteins. Therefore, it is anticipated that impurities (e.g., HCP and DNA) may interact with the albumin fusion protein and co-purify with the desired product. To improve impurity clearance, several washes were tested, taking advantage of the strong binding of albumin fusion proteins to the Cibacron blue dye chromatography resin, by disrupting the interaction between the impurity and the Cibacron blue dye ligand or between the impurity and the ligand-bound albumin fusion protein. Various types of washes were used, including ionic agents, chaotropic agents, kosmotropic agents, detergents, and weak solvents. Furthermore, each wash was tested at pH 6 and pH 7 to determine any significant effect of pH.

[0355] Table 11 summarizes the washes tested for Cibacron blue dye chromatography. As can be seen in Table 11, the pH of the wash is critical for HCP removal. At pH 7, all washes tested were more effective at HCP reduction, with most reducing HCPs by more than two-fold compared to the same wash at pH 6. Of the washes tested, 0.5 M NaCl at pH 7 was the most effective at HCP reduction, demonstrating a greater than seven-fold reduction in HCPs compared to the control run. Unlike HCP clearance, DNA clearance was not affected by pH between pH 6 and 7. Of all washes tested, only the ionic washes (NaCl and Na2SO4) demonstrated improved DNA clearance compared to the control run. In these cases, a four- to five-fold reduction in DNA was observed compared to the control run. It is also noteworthy that although higher monomer levels were observed for runs containing 10% propylene glycol, a slight increase in monomer levels was observed for some of the washes tested. No effect on monomer purity with pH was observed.

[0356] Based on the data in Table 11, 0.5M NaCl is a very effective wash in terms of HCP and DNA clearance, and 10% propylene glycol is an effective wash for increasing monomer purity. It should be noted that the 10% propylene glycol wash was also effective in reducing the likelihood of product oxidation in the Capto Blue pool (see Example 7 for further details). Yield was most negatively impacted by the 0.5M NaCl wash, with yields decreasing by 5% and 14% at pH 6 and 7, respectively. Interestingly, no yield loss was observed with the other washes tested. Based on these results, a wash containing 0.5M NaCl at pH 6 and a wash containing 10% propylene glycol at pH 7 were incorporated into the production process in Example 3.

[0357] [Table 12]

[0358] Example 5 Triton X-100 viral inactivation For AFP-1, an albumin fusion molecule with an isoelectric point in the range of 5.4–5.5, low pH treatment was determined to be detrimental to the product quality of the molecule (as evidenced by aggregation and precipitation). Therefore, Triton X-100 treatment was chosen for virus inactivation. Similar to low pH treatment, the addition of Triton X-100 disrupts the envelope around the virus, inactivating it. Unlike low pH treatment, Triton X-100 has no measurable effect on the product quality of AFP-1.

[0359] Viral inactivation by Triton X-100 was tested using xenotropic murine leukemia virus (XMuLV) as an enveloped virus model. Material purified by Cibacron blue dye chromatography was added with 10% (w / w) Triton X-100 to a final Triton X-100 concentration of 0.5% (w / w), incubated for the indicated time, and then tested for infectivity using a plate-based method known to those skilled in the art. Log reduction values ​​(LRVs) were calculated based on XMuLV titers from infectivity assays performed on samples before and after Triton X-100 treatment.

[0360] Table 12 summarizes the LRVs obtained in XMuLV virus inactivation. As can be seen, treatment with 0.5% (w / w) Triton X-100 is an effective method for XMuLV inactivation. Samples measured immediately after Triton X-100 treatment yielded LRV values ​​of 4.73 and >5.15 in duplicate experiments. By the end of the 120-minute incubation, both tests showed >5.15 log inactivation of XMuLV. These LRVs are within the same range as those obtained with low-pH treatment of monoclonal antibodies.

[0361] [Table 13]

[0362] After Triton X-100 treatment, the product was purified using anion exchange chromatography (see Figure 3 for the purification process). During anion exchange chromatography, the albumin fusion was strongly bound to the stationary phase, while some impurities flowed through the column. Triton X-100 was not retained by the anion exchange column and could be detected in the anion exchange column flow-through due to its absorbance at 280 nm. Figure 5 shows a representative anion exchange chromatogram with a strong 280 nm absorbance signal during column loading. Although further clearance of Triton X-100 may be achieved during hydrophobic interaction chromatography, it is assumed that Triton X-100 binds to the hydrophobic column along with the product. Therefore, removal of Triton X-100 from the product was not very robust and depended on the selectivity between Triton X-100 and the albumin fusion protein. After purification by the process shown in Figure 3, Triton X-100 levels were measured at less than 0.1 μg / mL (see Table 5).

[0363] Example 6 Anion-exchange membrane chromatography Anion exchange (AEX) membranes operated in flow-through mode can provide excellent removal of host cell impurities such as host cell proteins (HCPs), DNA, and viruses. For monoclonal antibodies (mAbs), whose pIs are typically in the range of 7.5–9.5, product binding is of little importance when flow-through AEX membranes are operated at approximately neutral pH, and yields are often >95% (independent of salt concentration or conductivity). When the pH approaches the pI of the mAb, binding can occur and yields may decrease. For mAbs, host cell impurity clearance is achieved under conditions of high pH and low salt (or conductivity). Therefore, for a typical mAb, operating conditions are optimized to minimize conductivity and maintain the pH as high as possible while maintaining it below the pI.

[0364] For albumin fusion proteins with low pI, the development and optimization of the flow-through AEX membrane chromatography step is more complex and cannot be predicted in advance. Unlike typical mAbs, there is likely some binding of the target molecule to the AEX membrane at all pH values ​​near neutral, with reduced binding expected at lower pH (due to less protein modification) and higher salt concentrations (which would mask the interaction between the albumin fusion protein and the chromatography ligand). Therefore, higher yields are expected at lower pH and higher salt concentrations. On the other hand, trends in impurity clearance related to salt and pH are expected to mirror those seen with mAbs, where higher pH and lower salt concentrations result in higher impurity clearance. Therefore, a balance must be struck between high yield (low pH and high salt) and high purity (high pH and low salt), and the pH, salt concentration, and membrane load must be optimized for a given product.

[0365] To optimize the AEX membrane chromatography step in AFP-1, pH (pH 6.0–8.0), NaCl concentration (10–220 mM), and membrane load (0.5–2.5 g / mL membrane) were explored in a multivariate design of experiments (DoE). This study used a screening design where the corners of the design space were tested along with two central point conditions and two additional points along the edges to determine the effect on step yield and impurity clearance. Table 13 summarizes the AEX membrane optimization experiment in AFP-1. As can be seen in Table 13, yield was affected by all three factors and generally followed the expected trend of higher yields being obtained with low pH, low salt, and higher loads. On the other hand, DNA clearance did not follow the expected trend of better clearance being obtained with higher pH and lower salt. Instead, DNA clearance was observed to be worst at 10 mM NaCl, pH 8. Interestingly, the effect was not driven by a single factor. For example, increased impurity clearance was observed under weak (pH 6, 220 mM NaCl) and moderate (pH 6, 10 mM NaCl or pH 8, 110 mM NaCl) binding conditions compared to strong binding conditions (pH 8.0, 10 mM NaCl). Impurity clearance was negatively affected only under the strongest binding conditions tested. One explanation for this result may be competitive binding between the albumin fusion protein and impurities. Under strong binding conditions, the albumin fusion protein may gain an advantage in competition for binding sites, resulting in lower binding capacity for DNA and lower yields of product. It is also noteworthy that no HCP clearance was observed and aggregate levels remained relatively unchanged under all conditions tested.

[0366] Before completing the above optimization studies for the Mustang Q step, it was anticipated that a balance would need to be struck between yield and impurity clearance. However, the opposite was observed during testing. Figure 9 summarizes the step yield and DNA log reduction value (LRV) as a function of pH and NaCl concentration. As can be seen, both yield and DNA clearance were found to be optimal at low pH and higher NaCl concentrations (indicated by the red outline). While this was an unexpected finding for DNA, a similar effect may be observed for viral clearance.

[0367] [Table 14]

[0368] Example 7 Control of oxidation variants using hydrophobic interaction chromatography The protein used in this study is an albumin fusion protein containing two Tn3 scaffolds linked to recombinant human serum albumin. Each Tn3 scaffold contains an active site capable of binding to the CD40L ligand. The albumin fusion protein contains eight methionine residues (six in the albumin portion of the molecule and one in each Tn3 scaffold) and seven tryptophan residues (five in the albumin portion of the molecule and one in each Tn3 scaffold). Methionine and tryptophan residues near the surface region can be oxidized during cell culture and / or the purification process. Figure 10 shows the relative potency as a function of oxidation as determined by peptide mapping mass spectrometry. As can be seen, methionine oxidation on the albumin (M498 and M529) or Tn3 (M74 and M17) portions of the molecule does not contribute to a loss of potency. On the other hand, tryptophan oxidation (W46 and W151) occurring on the Tn3 scaffold near the active site (on the BC loop) of the molecule leads to a decrease in the potency of the molecule. Therefore, tryptophan oxidation needs to be well controlled throughout the entire production process.

[0369] To monitor tryptophan oxidation during the development and production of AFP-1, peptide mapping mass spectrometry, SEC-HPLC, and HIC-HPLC were used at various stages of development. While mass spectrometry can be used to measure methionine and tryptophan levels fairly accurately, it is typically reserved for characterization of critical samples due to its low throughput and the resulting increased time and resources. While more rapid HPLC methods are available for in-process testing, both HPLC assays have drawbacks. For example, SEC-HPLC can measure tryptophan oxidation but only estimates it because the tryptophan shoulder is not completely resolved from the native molecule, whereas HIC-HPLC can accurately measure oxidation levels but cannot distinguish between methionine and tryptophan oxidation. To gain a fuller understanding of AFP-1 oxidation, we utilized all three methods during development and production. With the intention of improving the specificity of AFP oxidation quantification, we developed an RP-HPLC method to monitor the detection of peptides containing TN3 tryptophan (W46 and W151) oxidation. This method can be used for future in-process testing and quality control of AFP-1.

[0370] Successful oxidation control methods involve both inhibition of oxidation and removal of oxidized species that form during the production process. Figure 11 shows tryptophan oxidation as a function of time in the Capto Blue and Capto Q pools. In these runs, the pools were nominally at pH 7–8, which is representative of the operating pH in the purification process. As can be seen in Figure 11, tryptophan oxidation in the Capto Blue pool (measured by SEC-HPLC) varied depending on the bioreactor and decreased when a 10% propylene glycol wash was used and when the pool was stored at a lower temperature. Furthermore, the addition of 10 mM EDTA to the Capto Blue pool also slowed tryptophan oxidation (data not shown). As can be seen in Figure 9, tryptophan oxidation in the Capto Q pool appears negligible, and the Capto Q pool is fairly stable even at room temperature.

[0371] To further examine the underlying cause of AFP-1 tryptophan oxidation, experiments were performed to demonstrate that the presence of Cibcron Blue dye and / or high salt concentration did not cause oxidation. It was observed that AFP-1 tryptophan oxidation requires a unique combination of and exposure to components found in the initial process sample (conditioned medium or Capto Blue pool). One likely source of tryptophan oxidation is an enzyme, such as tryptophan 2,3-dioxygenase (TDO2) or tryptophan hydroxylase (TPH), both of which can specifically oxidize tryptophan. It is noteworthy that TDO2 was positively identified in CM using anti-TDO2 Western blot (see Figure 12), potentially causing AFP-1 tryptophan oxidation.

[0372] If oxidation occurs during an early process step, the level of oxidized variants may need to be controlled later in downstream processing. For AFP-1, a hydrophobic interaction chromatography step was utilized to remove excess oxidation, including tryptophan oxidation. Figure 13 shows a representative HIC chromatogram for AFP-1. As can be seen, several HIC resins and multimodal (cation exchange / HIC) resins were investigated for AFP-1 purification. On all resins tested, AFP-1 eluted near the middle of the gradient, which was suitable for removing tryptophan oxidation. When HIC was used during gradient elution of AFP-1, oxidation products (including methionine and tryptophan oxidation) eluted earlier than the native product and were concentrated at the front of the peak. Under preparative-scale conditions, the oxidized species were concentrated at the front of the peak, but the resolution was insufficient to see distinct oxidation product peaks.

[0373] During development, both Butyl-S Fast Flow (Butyl-S) and Toyopearl PPG-600M (PPG-600M) columns were scaled up and operated in linear gradient mode to determine whether each column could be used to remove oxidation products. In each case, the elution pool was fractionated in 0.5 column-volume fractions to peak maximum, and the remaining product peak was then collected in a single final fraction. In both runs, the fractions were tested for oxidized content (by HIC-HPLC) and potency. Figure 14 shows the relative potency versus HIC-HPLC initial species content in fractions collected during Butyl-S or PPG-600M chromatography runs. In both cases, early-eluting fractions (starting on the right side of the figure) contained more oxidation product (as measured by HIC-HPLC) and had lower potency than later-eluting fractions (left side of the figure). Under these conditions, preparative HIC can be used to control oxidation levels and, consequently, product potency.

[0374] conclusion The above outlines various approaches for purifying recombinant human albumin (rHSA) and albumin fusion proteins using scalable technologies suitable for clinical or commercial manufacturing. Initial steps in the process were optimized to reduce host-related impurities, such as HCPs, DNA, and viruses. The Cibacron blue dye chromatography capture step included extensive washes to reduce HCPs and employed selective elution with octanoic acid. Triton X-100 viral inactivation was shown to be a robust method for inactivating enveloped viruses without affecting product quality. The AEX column and membrane chromatography steps were optimized to reduce DNA to extremely low levels. Interestingly, the membrane chromatography step was shown to be optimal at the low pH and high salt conditions anticipated prior to this study. Finally, the purification process was designed to control the levels of oxidized variants, which were shown to be less potent. Control methods included a propylene glycol wash during the Cibacron blue chromatography step and the addition of EDTA to the elution buffer to help limit tryptophan oxidation. Furthermore, hydrophobic interaction chromatography was used as an effective option for removal of oxidation products. The purification process was scaled up to purify a 500 L bioreactor and shown to be consistent in terms of batch-to-batch yield and product quality.

[0375] The examples set forth above illustrate various aspects of the present invention and the practice of the methods of the present invention. These examples are not intended to provide a detailed description of the various embodiments of the present invention. Thus, while the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will readily appreciate that numerous changes and modifications can be made therein without departing from the spirit or scope of the appended claims.

[0376] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein.

[0377] [Sequence table] SEQUENCE LISTING <110> MedImmune LLC <120> METHOD OF PURIFYING ALBUMIN-FUSION PROTEINS <130> PA23-216 <150> US62 / 132198 <151> 2015-03-12 <160> 210 <170> PatentIn version 3.5 <210> 1 <211> 261 <212> PRT <213> Homo sapiens <400> 1 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145 150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255 Gly Leu Leu Lys Leu 260 <210> 2 <211> 149 <212> PRT <213> Homo sapiens <400> 2 Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 1 5 10 15 Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 20 25 30 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 35 40 45 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 50 55 60 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 65 70 75 80 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 85 90 95 Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 100 105 110 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 115 120 125 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 130 135 140 Gly Leu Leu Lys Leu 145 <210> 3 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 3 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Phe 1 5 10 15 Lys Pro Leu Ala Glu Ile Asp Gly Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu Asp Glu Asn 35 40 45 Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55 60 Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr 65 70 75 80 Phe Thr Thr <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 4 Lys Asp Val Thr Asp Thr Thr 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 5 Phe Lys Pro Leu Ala Glu Ile Asp Gly 1 5 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 6 Lys Asp Val Pro Gly Asp Arg 1 5 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 7 Thr Glu Asp Glu Asn Gln 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 8 Gly Asn Leu Lys Pro Asp Thr Glu 1 5 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 9 Arg Arg Gly Asp Met Ser Ser Asn Pro Ala 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 10 Arg Leu Asp Ala Pro Ser Gln Ile Glu Val 1 5 10 <210> 11 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 11 Ile Glu Val 1 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 12 Ala Leu Ile Thr Trp 1 5 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 13 Cys Glu Leu Ala Tyr Gly Ile 1 5 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 14 Cys Glu Leu Thr Tyr Gly Ile 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 15 Thr Thr Ile Asp Leu 1 5 <210> 16 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 16 Tyr Ser Ile 1 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 17 Tyr Glu Val Ser Leu Ile Cys 1 5 <210> 18 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 18 Lys Glu Thr Phe Thr Thr 1 5 <210> 19 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 19 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ser Asp Glu Phe Gly His Tyr Asp Gly Cys Glu Leu Thr Tyr Gly Ile 20 25 30 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Trp His Ser 35 40 45 Ala Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 50 55 60 Ser Leu Ile Cys Tyr Thr Asp Gln Glu Ala Gly Asn Pro Ala Lys Glu 65 70 75 80 Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His His His His 85 90 95 His His <210> 20 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 20 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser 1 5 10 15 Asp Glu Phe Gly His Tyr Asp Gly Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Trp His Ser Ala 35 40 45 Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55 60 Leu Ile Cys Tyr Thr Asp Gln Glu Ala Gly Asn Pro Ala Lys Glu Thr 65 70 75 80 Phe Thr Thr <210> 21 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 21 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ser Asp Glu Phe Gly His Tyr Asp Gly Cys Glu Leu Thr Tyr Gly Ile 20 25 30 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Trp His Ser 35 40 45 Ala Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 50 55 60 Ser Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu 65 70 75 80 Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His His His His 85 90 95 His His <210> 22 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 22 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser 1 5 10 15 Asp Glu Phe Gly His Tyr Asp Gly Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Trp His Ser Ala 35 40 45 Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55 60 Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr 65 70 75 80 Phe Thr Thr <210> 23 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 23 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ser Asp Asp Phe Asp Asn Tyr Glu Trp Cys Glu Leu Thr Tyr Gly Ile 20 25 30 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Tyr His Met 35 40 45 Ala Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 50 55 60 Ser Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu 65 70 75 80 Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His His His His 85 90 95 His His <210> 24 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 24 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser 1 5 10 15 Asp Asp Phe Asp Asn Tyr Glu Trp Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Tyr His Met Ala 35 40 45 Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55 60 Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr 65 70 75 80 Phe Thr Thr <210> 25 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 25 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ser Asp Asp Phe Ala Asp Tyr Val Trp Cys Glu Leu Thr Tyr Gly Ile 20 25 30 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Trp His Ser 35 40 45 Ala Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 50 55 60 Ser Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu 65 70 75 80 Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His His His His 85 90 95 His His <210> 26 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 26 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser 1 5 10 15 Asp Asp Phe Ala Asp Tyr Val Trp Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Trp His Ser Ala 35 40 45 Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55 60 Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr 65 70 75 80 Phe Thr Thr <210> 27 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 27 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ser Asp Asp Phe Gly Glu Tyr Val Trp Cys Glu Leu Thr Tyr Gly Ile 20 25 30 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Tyr His His 35 40 45 Ala His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 50 55 60 Ser Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu 65 70 75 80 Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His His His His 85 90 95 His His <210> 28 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 28 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser 1 5 10 15 Asp Asp Phe Gly Glu Tyr Val Trp Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Tyr His His Ala 35 40 45 His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55 60 Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr 65 70 75 80 Phe Thr Thr <210> 29 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 29 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Leu Asp Asp Trp Gly Ser Tyr His Val Cys Glu Leu Thr Tyr Gly Ile 20 25 30 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Tyr His Gln 35 40 45 Ala Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 50 55 60 Ser Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu 65 70 75 80 Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His His His His 85 90 95 His His <210> 30 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 30 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Leu 1 5 10 15 Asp Asp Trp Gly Ser Tyr His Val Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Tyr His Gln Ala 35 40 45 Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55 60 Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr 65 70 75 80 Phe Thr Thr <210> 31 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 31 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ser Asp Glu Val Gly Asp Tyr Val Val Cys Glu Leu Thr Tyr Gly Ile 20 25 30 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Tyr His Met 35 40 45 Ala Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 50 55 60 Ser Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu 65 70 75 80 Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His His His His 85 90 95 His His <210> 32 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 32 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser 1 5 10 15 Asp Glu Val Gly Asp Tyr Val Val Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Tyr His Met Ala 35 40 45 Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55 60 Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr 65 70 75 80 Phe Thr Thr <210> 33 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 33 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ser Asp Asp Phe Ala Glu Tyr Val Gly Cys Glu Leu Thr Tyr Gly Ile 20 25 30 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Trp His Ser 35 40 45 Ala Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 50 55 60 Ser Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu 65 70 75 80 Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His His His His 85 90 95 His His <210> 34 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 34 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser 1 5 10 15 Asp Asp Phe Ala Glu Tyr Val Gly Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Trp His Ser Ala 35 40 45 Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55 60 Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr 65 70 75 80 Phe Thr Thr <210> 35 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 35 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ser Asp Asp Phe Glu Glu Tyr Val Val Cys Glu Leu Thr Tyr Gly Ile 20 25 30 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Trp His Ser 35 40 45 Ala Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 50 55 60 Ser Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu 65 70 75 80 Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His His His His 85 90 95 His His <210> 36 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 36 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser 1 5 10 15 Asp Asp Phe Glu Glu Tyr Val Val Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Trp His Ser Ala 35 40 45 Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55 60 Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr 65 70 75 80 Phe Thr Thr <210> 37 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 37 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ser Asp Glu Val Gly Gln Tyr Val Gly Cys Glu Leu Thr Tyr Gly Ile 20 25 30 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Tyr His Met 35 40 45 Ala Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 50 55 60 Ser Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu 65 70 75 80 Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His His His His 85 90 95 His His <210> 38 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 38 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser 1 5 10 15 Asp Glu Val Gly Gln Tyr Val Gly Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Tyr His Met Ala 35 40 45 Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55 60 Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr 65 70 75 80 Phe Thr Thr <210> 39 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 39 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ser Asp Asp Ile Gly Leu Tyr Val Trp Cys Glu Leu Thr Tyr Gly Ile 20 25 30 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Phe His Gln 35 40 45 Ala Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 50 55 60 Ser Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu 65 70 75 80 Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His His His His 85 90 95 His His <210> 40 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 40 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser 1 5 10 15 Asp Asp Ile Gly Leu Tyr Val Trp Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Phe His Gln Ala 35 40 45 Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55 60 Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr 65 70 75 80 Phe Thr Thr <210> 41 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 41 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ser Asp Glu His Ala Glu Phe Ile Gly Cys Glu Leu Thr Tyr Gly Ile 20 25 30 Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Trp His Ser 35 40 45 Ala Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 50 55 60 Ser Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu 65 70 75 80 Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His His His His 85 90 95 His His <210> 42 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 42 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser 1 5 10 15 Asp Glu His Ala Glu Phe Ile Gly Cys Glu Leu Thr Tyr Gly Ile Lys 20 25 30 Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Trp Trp His Ser Ala 35 40 45 Trp Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55 60 Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr 65 70 75 80 Phe Thr Thr <210> 43 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 43 Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Thr Asn Arg Ser Ser Tyr Tyr Asn Leu His Gly Cys Glu Leu Thr Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Ser Ser 35 40 45 Pro Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85 90 95 His His His His His 100 <210> 44 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 44 Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Thr 1 5 10 15 Asn Arg Ser Ser Tyr Tyr Asn Leu His Gly Cys Glu Leu Thr Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Ser Ser Pro 35 40 45 Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60 Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro Ala 65 70 75 80 Lys Glu Thr Phe Thr Thr 85 <210> 45 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 45 Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Thr Asn Arg Ser Ser Tyr Tyr Asn Leu His Gly Cys Glu Leu Thr Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Ser Ser 35 40 45 Pro Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85 90 95 His His His His His 100 <210> 46 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 46 Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Thr 1 5 10 15 Asn Arg Ser Ser Tyr Tyr Asn Leu His Gly Cys Glu Leu Thr Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Ser Ser Pro 35 40 45 Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60 Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro Ala 65 70 75 80 Lys Glu Thr Phe Thr Thr 85 <210> 47 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 47 Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ile Asn Arg Ser Tyr Tyr Ala Asp Leu His Gly Cys Glu Leu Thr Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asp Gln 35 40 45 Ile Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Lys Tyr 50 55 60 Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85 90 95 His His His His His 100 <210> 48 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 48 Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ile 1 5 10 15 Asn Arg Ser Tyr Tyr Ala Asp Leu His Gly Cys Glu Leu Thr Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asp Gln Ile 35 40 45 Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Lys Tyr Glu 50 55 60 Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro Ala 65 70 75 80 Lys Glu Thr Phe Thr Thr 85 <210> 49 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 49 Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Thr Asn Arg Ser Ser Tyr Ser His Leu Asp Gly Cys Glu Leu Thr Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Ser Ala 35 40 45 Ala Ile Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu 50 55 60 Tyr Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn 65 70 75 80 Pro Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His 85 90 95 His His His His His His 100 <210> 50 <211> 87 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 50 Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Thr 1 5 10 15 Asn Arg Ser Ser Tyr Ser His Leu Asp Gly Cys Glu Leu Thr Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Ser Ala Ala 35 40 45 Ile Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr 85 <210> 51 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 51 Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ile Asn Arg Ser Ser Tyr His Asn Phe Pro His Cys Glu Leu Ala Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Ser 35 40 45 Pro Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85 90 95 His His His His His 100 <210> 52 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 52 Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ile 1 5 10 15 Asn Arg Ser Ser Tyr His Asn Phe Pro His Cys Glu Leu Ala Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Ser Pro 35 40 45 Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60 Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro Ala 65 70 75 80 Lys Glu Thr Phe Thr Thr 85 <210> 53 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 53 Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Thr Asn Arg Ser Ser Tyr Ser Asn His Leu Gly Cys Glu Leu Ala Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Asn 35 40 45 Ile Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85 90 95 His His His His His 100 <210> 54 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 54 Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Thr 1 5 10 15 Asn Arg Ser Ser Tyr Ser Asn His Leu Gly Cys Glu Leu Ala Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Asn Ile 35 40 45 Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60 Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro Ala 65 70 75 80 Lys Glu Thr Phe Thr Thr 85 <210> 55 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 55 Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Thr Asn Arg Ser Ser Tyr Ser Asn Phe His Gly Cys Glu Leu Ala Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Ser 35 40 45 Pro Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85 90 95 His His His His His 100 <210> 56 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 56 Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Thr 1 5 10 15 Asn Arg Ser Ser Tyr Ser Asn Phe His Gly Cys Glu Leu Ala Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Ser Pro 35 40 45 Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60 Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro Ala 65 70 75 80 Lys Glu Thr Phe Thr Thr 85 <210> 57 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 57 Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Thr Asn Arg Ser Phe Tyr Ser Asn Leu His Gly Cys Glu Leu Thr Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Gln 35 40 45 Pro Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85 90 95 His His His His His 100 <210> 58 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 58 Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Thr 1 5 10 15 Asn Arg Ser Phe Tyr Ser Asn Leu His Gly Cys Glu Leu Thr Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Gln Pro 35 40 45 Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60 Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro Ala 65 70 75 80 Lys Glu Thr Phe Thr Thr 85 <210> 59 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 59 Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Thr Asn Arg Ser Ser Tyr Ala Tyr Leu His Gly Cys Glu Leu Ala Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Gln 35 40 45 Pro Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85 90 95 His His His His His 100 <210> 60 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 60 Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Thr 1 5 10 15 Asn Arg Ser Ser Tyr Ala Tyr Leu His Gly Cys Glu Leu Ala Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Gln Pro 35 40 45 Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60 Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro Ala 65 70 75 80 Lys Glu Thr Phe Thr Thr 85 <210> 61 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 61 Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ile Asn Arg Ser Ser Tyr Ala Asn Leu His Gly Cys Glu Leu Thr Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Ser Ser 35 40 45 Pro Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85 90 95 His His His His His 100 <210> 62 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 62 Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ile 1 5 10 15 Asn Arg Ser Ser Tyr Ala Asn Leu His Gly Cys Glu Leu Thr Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Ser Ser Pro 35 40 45 Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60 Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro Ala 65 70 75 80 Lys Glu Thr Phe Thr Thr 85 <210> 63 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 63 Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Thr Asn Arg Ser Ser Tyr Ala Asn Tyr His Gly Cys Glu Leu Ala Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Gln 35 40 45 Pro Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85 90 95 His His His His His 100 <210> 64 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 64 Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Thr 1 5 10 15 Asn Arg Ser Ser Tyr Ala Asn Tyr His Gly Cys Glu Leu Ala Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Gln Pro 35 40 45 Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60 Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro Ala 65 70 75 80 Lys Glu Thr Phe Thr Thr 85 <210> 65 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 65 Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Thr Asn Arg Ser Ser Tyr Ala Asn Leu Pro Gly Cys Glu Leu Thr Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Ser 35 40 45 Pro Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85 90 95 His His His His His 100 <210> 66 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 66 Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Thr 1 5 10 15 Asn Arg Ser Ser Tyr Ala Asn Leu Pro Gly Cys Glu Leu Thr Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Ser Pro 35 40 45 Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60 Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro Ala 65 70 75 80 Lys Glu Thr Phe Thr Thr 85 <210> 67 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 67 Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Thr Asn Arg Ser Ser Tyr Ser Asn Leu His Gly Cys Glu Leu Ala Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Gln 35 40 45 Pro Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Asn Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85 90 95 His His His His His 100 <210> 68 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 68 Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Thr 1 5 10 15 Asn Arg Ser Ser Tyr Ser Asn Leu His Gly Cys Glu Leu Ala Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Gln Pro 35 40 45 Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60 Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Asn Asn Pro Ala 65 70 75 80 Lys Glu Thr Phe Thr Thr 85 <210> 69 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 69 Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Ile Asn Arg Ser Ser Tyr Ala Asn Leu His Gly Cys Glu Leu Thr Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Ser 35 40 45 Pro Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr 50 55 60 Glu Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro 65 70 75 80 Ala Lys Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Gly His His His 85 90 95 His His His His His 100 <210> 70 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 70 Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Ile 1 5 10 15 Asn Arg Ser Ser Tyr Ala Asn Leu His Gly Cys Glu Leu Thr Tyr Gly 20 25 30 Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Asn Ser Pro 35 40 45 Tyr Val His Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60 Val Ser Leu Ile Cys Leu Thr Thr Asp Gly Thr Tyr Ser Asn Pro Ala 65 70 75 80 Lys Glu Thr Phe Thr Thr 85 <210> 71 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 71 Ala Ile Glu Val Glu Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp 1 5 10 15 Thr Ala Arg Ser Ala Tyr Ser His His His Tyr Cys Glu Leu Thr Tyr 20 25 30 Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Arg Gln 35 40 45 Pro Tyr Val...

Claims

1. 1. A composition comprising a Tn3 scaffold comprising a monomeric subunit specific for CD40L, wherein the monomeric subunit comprises SEQ ID NO: 146, the Tn3 scaffold comprises oxidized and non-oxidized tryptophan residues, the Tn3 scaffold has less than 5% oxidized tryptophan residues relative to the total number of tryptophan residues in the Tn3 scaffold, the CD40L-specific monomeric subunit is linked to a heterologous moiety, and the heterologous moiety is human serum albumin (HSA).

2. 2. The composition of claim 1, wherein the non-oxidized tryptophan residue is W46 of SEQ ID NO:

145.

3. 2. The composition of claim 1, wherein the non-oxidized tryptophan residue is W151 of SEQ ID NO:

145.

4. 2. The composition of claim 1, wherein the Tn3 scaffold comprises a second CD40L-specific monomer subunit, and the second CD40L-specific monomer subunit comprises the sequence of SEQ ID NO:

146.

5. 5. The composition of claim 4, wherein the non-oxidized tryptophan residue is W46 and / or W151 of SEQ ID NO:

145.

6. The composition of claim 4, wherein the CD40L-specific monomeric subunit and the second CD40L-specific monomeric subunit are linked in tandem by a polypeptide linker.

7. 7. The composition of claim 6, wherein the polypeptide linker comprises a sequence selected from the group consisting of SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:142 and SEQ ID NO:

143.

8. The composition of claim 7, wherein the polypeptide linker comprises the sequence of SEQ ID NO:

142.

9. The composition of claim 7, wherein the polypeptide linker comprises the sequence of SEQ ID NO:

143.

10. The composition described in claim 1, wherein the HSA is a mutant HSA, and the mutant HSA comprises sequence number 133.

11. The composition of claim 4 , wherein the CD40L-specific monomer subunit and the second CD40L-specific monomer subunit have the same sequence.

12. 11. The composition of claim 10, wherein the composition comprises a Tn3 scaffold consisting of SEQ ID NO:

145.

13. below: (a) a Tn3 scaffold comprising a CD40L-specific monomeric subunit, the monomeric subunit comprising SEQ ID NO: 146, the Tn3 scaffold having less than 5% oxidized tryptophan residues relative to the total number of tryptophan residues in the Tn3 scaffold, the Tn3 scaffold comprising non-oxidized tryptophan residues contained in a DE loop sequence of the CD40L-specific monomeric subunit, the CD40L-specific monomeric subunit being linked to a heterologous moiety, and the heterologous moiety being human serum albumin (HSA); and (b) a pharmaceutically acceptable carrier A composition comprising:

14. 1. A composition comprising a Tn3 scaffold comprising two CD40L-specific monomeric subunits, wherein the monomeric subunits comprise SEQ ID NO: 146, the Tn3 scaffold has less than 5% oxidized tryptophan residues relative to the total number of tryptophan residues in the Tn3 scaffold, the Tn3 scaffold comprises an unoxidized tryptophan at W46 and / or W151 of SEQ ID NO: 145, and one of the CD40L-specific monomeric subunits is linked to a heterologous moiety, and the heterologous moiety is human serum albumin (HSA).

15. Use of a Tn3 scaffold in the manufacture of a medicament for treating a CD40-mediated immune response, the treatment comprising administering a composition comprising a Tn3 scaffold comprising a CD40L-specific monomeric subunit, the monomeric subunit comprising SEQ ID NO: 146, the Tn3 scaffold having less than 5% oxidized tryptophan residues relative to the total number of tryptophan residues in the Tn3 scaffold, the Tn3 scaffold comprising non-oxidized tryptophan residues contained in the DE loop sequence of the CD40L-specific monomeric subunit, the CD40L-specific monomeric subunit being linked to a heterologous moiety, the heterologous moiety being human serum albumin (HSA), and the administration is effective in reducing a CD40-mediated immune response.

16. 16. The use of claim 15, wherein the non-oxidized tryptophan residue is W46 of SEQ ID NO:

145.

17. 16. The use of claim 15, wherein the non-oxidized tryptophan residue is W151 of SEQ ID NO:

145.

18. The use of claim 15, wherein the Tn3 scaffold comprises a second CD40L-specific monomer subunit, and the second CD40L-specific monomer subunit comprises the sequence of SEQ ID NO:

146.

19. 19. The use of claim 18, wherein the non-oxidized tryptophan residues are W46 and W151 of SEQ ID NO:

145.

20. The use of claim 18, wherein the CD40L-specific monomer subunit and the second CD40L-specific monomer subunit are linked in tandem by a polypeptide linker.

21. 21. The use of claim 20, wherein the polypeptide linker comprises a sequence selected from the group consisting of SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 142, SEQ ID NO: 143, and combinations thereof.

22. 22. The use according to claim 21, wherein the polypeptide linker comprises the sequence of SEQ ID NO:

142.

23. 22. The use according to claim 21, wherein the polypeptide linker comprises the sequence of SEQ ID NO:

143.

24. The use described in claim 15, wherein the HSA is a mutant HSA, and the mutant HSA comprises sequence number 133.

25. The use of claim 18, wherein the CD40L-specific monomer subunit and the second CD40L-specific monomer subunit have the same sequence.

26. 25. The use of claim 24, wherein the composition comprises a Tn3 scaffold consisting of SEQ ID NO: 145.

Images