affinity agent

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of U.S. Provisional Patent Application No. 63 / 159,336, filed March 10, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] The purity of biologically produced therapeutics is highly scrutinized and regulated by authorities to ensure safety and efficacy. Therefore, there remains a need for efficient means to purify biologically produced therapeutics to high purity. Summary of the Invention [Means for solving the problem]

[0003] To support clinical efforts in advanced therapeutic pharmaceuticals (ATMPs), compositions and methods for efficient purification of ATMPs from recombinant sources are needed. Affinity purification is a means to isolate proteins and / or achieve a desired purity in a few or even a single step. However, the development of affinity agents (e.g., affinity ligands) can be resource-intensive and time-consuming, and affinity agents exist for only a very small number of proteins. In the absence of affinity agents, purification typically involves inefficient processes such as multi-column processes.

[0004] Exosomes are an emerging ATMP with great therapeutic potential (Madhusoodanan 2020). They are a subclass of extracellular vesicles, which also include microvesicles and apoptotic bodies (Zaborowski et al. 2015). Production of exosomes for therapeutic purposes is difficult and expensive. Cell culture productivity is low, typically around 10 per liter. 11 ~10 14The total amount of exosomes reaches 100 million. Purification is mainly performed by ion exchange chromatography. However, this method is not selective for exosomes, resulting in co-purification with other extracellular vesicles and insufficient removal of host cell proteins. Therefore, there is still a need for selective purification of exosomes.

[0005] Given the recognized ability of affinity purification to selectively purify targets, there is a need for affinity reagents that meet the rigors of modern bioprocessing. Immunoaffinity, i.e., the use of antibodies as affinity ligands, has demonstrated the selectivity required for the purification of exosomes (Kowal et al. 2016). Antibodies selectively bind to surface markers characteristic of exosomes, including tetraspanins, CD9, CD63, and CD81. However, antibodies are unsuitable for bioprocessing and / or lack sufficient stability to be compatible with disinfectants and cleaning agents. Therefore, there is a need for affinity reagents suitable for bioprocessing.

[0006] Described herein are affinity agents that bind to exosomes and are useful for isolating and / or affinity purifying them.

[0007] In some embodiments, provided herein is an affinity agent comprising a cyclic peptide, further comprising the amino acid sequence of SEQ ID NO:1. SEQ ID NO:1:X 1 YWRB 1 VWFPHAQGB 2 VX 2 X 2

[0008] In the formula, X 1 represents H or N, and X 2 represents S or T, and B 1 and B 2 represents the unit at which the peptide is cyclized.

[0009] In some embodiments, the affinity agent comprises a ligand comprising at least one amino acid sequence shown in Table 5, eg, any one of SEQ ID NOs: 2-126.

[0010] In some embodiments, provided herein is an affinity agent comprising at least one ligand comprising the amino acid sequence of SEQ ID NO: 1 that binds to CD81.

[0011] In some embodiments, provided herein is an affinity agent comprising at least one ligand comprising the amino acid sequence of SEQ ID NO: 1 that binds to exosomes.

[0012] In some embodiments, provided herein are affinity agents comprising at least one ligand comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that differs by no more than three, no more than two, or no more than one substitution, addition, or deletion.

[0013] In some embodiments, provided herein are affinity agents that comprise multiple affinity ligands.

[0014] In some embodiments, provided herein are affinity agents for use in purifying exosomes.

[0015] definition In order that the present disclosure may be more readily understood, certain terms are defined below. Unless defined otherwise, technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art.

[0016] Approximately or about: As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about," unless otherwise stated or a different meaning is apparent from the context, refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in either direction (above or below) of the stated reference value (except where such value exceeds 100% of the possible values).

[0017] Biological activity: As used herein, the term "biological activity" refers to a characteristic of any agent that has activity in a biological system, particularly an organism. For example, an agent that, when administered to an organism, has a biological effect on that organism is considered to be biologically active.

[0018] Conservative and Non-Conservative Substitutions: A "conservative" amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, and include basic side chains (e.g., lysine (K), arginine (R), histidine (H)), acidic side chains (e.g., aspartic acid (D), glutamic acid (E)), uncharged polar side chains (e.g., glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C)). Conservative amino acid substitutions include nonpolar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W)), beta-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)), and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)). For example, substitution of phenylalanine for tyrosine is a conservative substitution. In some embodiments, conservative amino acid substitutions in the sequence of the ligand confer or improve specific binding of the ligand to a target of interest. In some embodiments, conservative amino acid substitutions in the sequence of the ligand do not reduce or abolish binding of the ligand to a target of interest. In some embodiments, conservative amino acid substitutions do not significantly affect specific binding of the ligand to a target of interest. Methods for identifying conservative and non-conservative nucleotide and amino acid substitutions that confer, alter, or maintain selective binding affinity are known in the art (see, e.g., Brummell, Biochem. 32:1180-1187 (1993); Kobayashi, Protein Eng. 12(10):879-884 (1999); and Burks, PNAS 94:412-417 (1997)). In some embodiments, non-conservative amino acid substitutions in the ligand's sequence confer or improve specific binding of the ligand to a target of interest. In some embodiments, non-conservative amino acid substitutions in the ligand's sequence do not reduce or abolish binding of the ligand to a target of interest.In some embodiments, the non-conservative amino acid substitutions do not significantly affect specific binding of the ligand to a target of interest.

[0019] Linker: As used herein, "linker" refers to a peptide or other chemical bond that functions to connect otherwise independent functional domains. In some embodiments, the linker is positioned between a ligand and another polypeptide component that comprises an otherwise independent functional domain. In some embodiments, the linker is a peptide or other chemical bond positioned between the ligand and the surface.

[0020] Naturally-occurring: The term "naturally-occurring" when used in reference to biological materials such as nucleic acid molecules, polypeptides, and host cells refers to materials that exist in nature and have not been modified by humans. Conversely, "non-natural" or "synthetic" when used in reference to biological materials refers to materials that are not found in nature and / or that have been modified by humans.

[0021] The terms "unnatural amino acid," "amino acid analog," and "non-standard amino acid residue" are used interchangeably herein. Unnatural amino acids that can be substituted in the ligands provided herein are known in the art. In some embodiments, the unnatural amino acid is 4-hydroxyproline, which can be substituted for proline, 5-hydroxylysine, which can be substituted for lysine, 3-methylhistidine, which can be substituted for histidine, homoserine, which can be substituted for serine, and ornithine, which can be substituted for lysine. Additional examples of unnatural amino acids that can be substituted in polypeptide ligands include, but are not limited to, D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, A-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, lanthionine, dehydroalanine, gamma-aminobutyric acid, selenocysteine, and designer amino acids such as pyrrolidine fluoroamino acids, beta-methylamino acids, C alpha-methylamino acids, and N alpha-methylamino acids.

[0022] "Polynucleotide" and "Nucleic Acid Molecule": As used interchangeably herein, polynucleotide and nucleic acid molecule refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include, but are not limited to, DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and / or tRNA.

[0023] Operably linked: As used herein, the term "operably linked" indicates that two molecules are joined in such a way that they retain their respective functional activity. Two molecules are "operably linked" whether they are joined directly or indirectly.

[0024] Peptide tag: As used herein, the term "peptide tag" refers to a peptide sequence that is part of or attached (e.g., by genetic engineering) to another protein to provide a function to the resulting fusion. Peptide tags are typically relatively short compared to the proteins to which they are fused. In some embodiments, peptide tags are 4 or more amino acids in length, such as 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more amino acids. In some embodiments, the ligand is a protein comprising the peptide tag. Numerous peptide tags with uses provided herein are known in the art. Examples of peptide tags that can be components of a ligand fusion protein or targets bound by a ligand (e.g., a ligand fusion protein) include, but are not limited to, HA (hemagglutinin), c-myc, herpes simplex virus glycoprotein D (gD), T7, GST, GFP, MBP, Strep tag, His tag, Myc tag, TAP tag, and FLAG tag (Eastman Kodak, Rochester, NY). Similarly, an antibody epitope directed against the tag allows for detection and localization of the fusion protein in, for example, affinity purification, Western blots, ELISA assays, and immunostaining of cells.

[0025] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, although those skilled in the art will understand that the term is not limited to long chains and can refer to a minimum chain comprising two amino acids linked together via peptide bonds. Polypeptides can be processed and / or modified, as known to those skilled in the art.

[0026] Protein: As used herein, the term "protein" refers to one or more polypeptides that function as separate units. When a single polypeptide is a separate functional unit and does not require permanent or temporary physical association with other polypeptides to form a separate functional unit, the terms "polypeptide" and "protein" may be used interchangeably. When a separate functional unit is composed of multiple polypeptides that are physically associated with each other, the term "protein" refers to the multiple polypeptides that are physically associated and function together as a separate unit.

[0027] Specific Binding: As used herein with respect to a ligand, the terms "specifically bind" or "having selective affinity" mean that the ligand reacts or associates with a particular epitope, protein, or target molecule more frequently, more rapidly, with a longer duration, with greater affinity, or a combination thereof, than with alternative substances, including unrelated proteins. Due to sequence identity between homologous proteins of different species, specific binding may include binding agents that recognize proteins or targets from multiple species. Similarly, due to homology within certain regions of the polypeptide sequences of different proteins, specific binding may include binding agents that recognize multiple proteins or targets. It is understood that in certain embodiments, a binding agent that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require exclusive binding, i.e., binding to a single target (although it can include such binding). Thus, a ligand or affinity agent may, in certain embodiments, specifically bind to multiple targets. In certain embodiments, multiple targets may be bound by the same antigen-binding site on the affinity agent.

[0028] Substantially: As used herein, the term "substantially" refers to the quantitative condition of exhibiting a total or near-total degree or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena, if they exist at all, rarely go to completion and / or rarely proceed perfectly, or rarely achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. The present invention provides, for example, the following items. (Item 1) 1. An affinity agent comprising a ligand that binds to CD81, the ligand comprising a cyclic peptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:1:X 1 YWRB 1 VWFPHAQGB 2 VX 2 X 2 、 In the formula, X 1 represents H or N, and X 2 represents S or T, and B 1 and B 2 represents the unit by which the peptide is cyclized. (Item 2) An affinity agent comprising a ligand comprising at least one amino acid sequence shown in Table 5, for example, any one of SEQ ID NOs: 2 to 126. (Item 3) An affinity agent comprising a ligand comprising the amino acid sequence of SEQ ID NO: 1 that binds to CD81. (Item 4) An affinity agent comprising a ligand comprising the amino acid sequence of SEQ ID NO: 1 that binds to one or more exosomes. (Item 5) An affinity agent comprising a ligand comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that differs by no more than three substitutions, additions, or deletions, no more than two substitutions, additions, or deletions, or no more than one substitution, addition, and / or deletion. (Item 6) An affinity agent comprising a ligand that binds to one or more exosomes, the exosomes comprising a cyclic peptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:1:X 1 YWRB 1 VWFPHAQGB 2 VX 2X 2 、 In the formula, X 1 represents H or N, and X 2 represents S or T, and B 1 and B 2 represents the unit by which the peptide is cyclized. (Item 7) An affinity agent comprising a ligand comprising the amino acid sequence of SEQ ID NO:1. (Item 8) An affinity agent comprising a ligand comprising at least one amino acid sequence of any one of SEQ ID NOs: 2 to 126, or an amino acid sequence that differs by no more than three substitutions, additions, or deletions, no more than two substitutions, additions, or deletions, or no more than one substitution, addition, and / or deletion. (Item 9) 9. The affinity agent according to any one of items 1 to 8, wherein the ligand is bound to a solid surface. (Item 10) 10. The affinity agent according to item 9, wherein the solid surface is a resin or a bead. (Item 11) 10. The affinity agent according to item 9, wherein the solid surface is a membrane. (Item 12) 10. The affinity agent according to item 9, wherein the solid surface is a monolith. (Item 13) 13. The affinity agent according to any one of items 9 to 12, wherein the ligand is conjugated to the solid surface via a linker. (Item 14) 14. The affinity agent according to any one of items 1 to 13, for use in the purification of one or more exosomes. (Item 15) 15. A method for producing an affinity agent, comprising conjugating the ligand according to any one of items 1 to 14 to a solid surface. [Brief explanation of the drawings]

[0029] [Figure 1] A typical standard curve obtained for the CD81 ELISA is shown.

[0030] [Figure 2]Figure 1 shows a CD63 Western blot analysis of purified exosomes as described in Example 6. The description of each lane (from left to right) is as follows: (1) molecular weight marker, (2) CD63-Fc fusion protein, (3) exosome standard, 4E10 particles, (4) exosome standard, 8E9 particles, (5) exosome standard, 3E9 particles, (6) crude feedstream, (7) crude feedstream, 10-fold dilution, (8) column flow-through, (9) elution fraction at the peak apex, (10) strip, and (11) elution pool (entire peak).

[0031] [Figure 3] Chromatogram of purified exosomes described in Example 6.

[0032] [Figure 4] Figure 1 shows a comparison of static binding of resin containing SEQ ID NO: 43 before and after treatment with 0.1 M NaOH for 18 hours. Blank (underivatized) beads were included as a control.

[0033] [Figure 5] 1 shows a sensorgram for the binding of various concentrations of Fc-CD81 fusion protein to immobilized ligand corresponding to SEQ ID NO:58.

[0034] [Figure 6] 1 shows dose-response curves for the binding of several ligands to Fc-CD81. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present disclosure encompasses, inter alia, the recognition that affinity resins prepared from the identified and characterized peptide ligands have been shown to produce highly purified preparations of one or more targets of interest, e.g., in some embodiments, viral particles. In some embodiments, the affinity resins described herein are useful for, inter alia, the removal of protein product-related impurities and host cell-derived contaminants.

[0036] Ligands that bind to a target of interest for use in affinity agents The characteristics of a ligand that binds to a target can be determined using known or modified assays, bioassays, and / or animal models known in the art for assessing such activity.

[0037] As used herein, the terms "binding affinity for a target," "binding to a target," and the like refer to a property of a ligand that can be measured directly, for example, by determining the affinity constant (e.g., the amount of ligand that binds and dissociates at a given antigen concentration). To characterize such molecular interactions, several methods can be used, such as competitive analysis, equilibrium analysis, and microcalorimetry, as well as real-time interaction analysis based on surface plasmon resonance interactions (e.g., using a BIACORE device). These methods are well known to those skilled in the art and are discussed in publications such as Neri D et al. (1996) Tibtech 14:465-470 and Jansson M et al. (1997) J Biol Chem 272:8189-8197.

[0038] The affinity requirements for a given ligand binding event depend on a variety of factors, including, but not limited to, the composition and complexity of the binding matrix, the valency and density of both the ligand and target molecule, and the functional application of the ligand. In some embodiments, the ligand is -3 M, 10 -3 M, 5×10 -4 M, 10 -4 M, 5 x 10 -5 M, or 10 -5 The dissociation constant (K D ) to bind to the target of interest. In some embodiments, the ligand binds to the target of interest at 5×10 -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5 x 10 -8 M, or 10 -8 K below M D Binds to the target of interest at 5×10 -9 M, 10 -9 M, 5 x 10-10 M, 10 -10 M, 5 × 10 -11 M, 10 -11 M, 5 × 10 -12 M, 10 -12 M, 5 × 10 -13 M, 10 -13 M, 5 × 10 -14 M, 10 -14 M, 5 × 10 -15 M or 10 -15 K less than or equal to M D and binds to the target of interest. In some embodiments, the ligand generated by the methods disclosed herein has a dissociation constant of about 10 -4 M to about 10 -5 M, about 10 -5 M to about 10 -6 M, about 10 -6 M to about 10 -7 M, about 10 -7 M to about 10 -8 M, about 10 -8 M to about 10 -9 M, about 10 -9 M to about 10 -10 M, about 10 -10 M to about 10 -11 M, or about 10 -11 M to about 10 -12 M.

[0039] K D And the binding experiments to determine the dissociation rate can be performed under many conditions. The buffer for making these solutions can be readily determined by those skilled in the art and depends greatly on the desired pH of the final solution. Low pH solutions (<pH 5.5) can be made, for example, with citrate buffer, glycine-HCl buffer, or succinate buffer. High pH solutions can be made, for example, with Tris-HCl, phosphate buffer, or sodium bicarbonate buffer. For example, for the purpose of determining the optimal pH and / or salt concentration, many conditions can be used to determine K D and the dissociation rate.

[0040] In some embodiments, the ligand is 0.1 to 10 -7 seconds -1 , 10-2 ~10 -7 seconds -1 , or 0.5 × 10 -2 ~10 -7 seconds -1 k in the range off In some embodiments, the ligand specifically binds the target of interest at a concentration of 5×10 -2 seconds -1 , 10 -2 seconds -1 , 5×10 -3 seconds -1 , or 10 -3 seconds -1 Off-rate (k off ) to bind the target of interest. In some embodiments, the ligand binds at a concentration of 5×10 -4 seconds -1 , 10 -4 seconds -1 , 5×10 -5 seconds -1 , or 10 -5 seconds -1、 5x10 -6 seconds -1 , 10 -6 seconds -1 , 5×10 -7 seconds -1 , or 10 -7 seconds -1 Off-rate (k off ) to bind the desired target. In some embodiments, the ligand is about 10 3 ~10 7 M -1 seconds -1 , 10 3 ~10 6 M -1 seconds -1 , or 10 3 ~10 5 M - 1 second -1 k in the range on In some embodiments, the ligand (e.g., ligand fusion protein) specifically binds to a target of interest at 10 3 M -1 seconds -1 , 5×10 3 M -1 seconds -1 10 4 M-1 seconds -1 , or 5 × 10 4 M -1 seconds -1 Ultra-high on-speed (k on ) to bind the target of interest. In a further embodiment, the ligand is 5 M -1 seconds -1 , 5×10 5 M -1 seconds -1 , 10 6 M -1 seconds -1 , 5×10 6 M -1 seconds -1 , or 10 7 M -1 seconds -1 Super K on to bind the desired target.

[0041] Intended target According to various embodiments, the target of interest specifically bound by the ligand can be any molecule to which it is desirable for the affinity agent's ligand to bind. For example, the target specifically bound by the ligand can be any target of relevance or value for purification, manufacturing, formulation, treatment, diagnosis, or prognosis. Non-limiting applications include therapeutic and diagnostic applications. Some exemplary targets are provided herein by way of example, but these are intended to be illustrative and not limiting. The target of interest can be natural or synthetic. In some embodiments, the target is AAV2 and / or a variant derived from AAV2.

[0042] Linker The terms "linker" and "spacer" are used interchangeably herein and refer to a peptide or other chemical bond that functions to link otherwise independent functional domains. In some embodiments, the linker is positioned between a ligand and another polypeptide component that comprises an otherwise independent functional domain. A linker suitable for linking two or more linked ligands can generally be any linker used in the art to link peptides, proteins, or other organic molecules. In some embodiments, such linkers are suitable for constructing proteins or polypeptides intended for pharmaceutical use.

[0043] Linkers suitable for operably linking the ligand and additional components of the ligand fusion protein with a single amino acid sequence include, but are not limited to, polypeptide linkers such as glycine linkers, serine linkers, mixed glycine / serine linkers, glycine and serine rich linkers, or linkers composed of predominantly polar polypeptide fragments.

[0044] In some embodiments, the linker comprises a majority of amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In some embodiments, the linker comprises a majority of amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In some embodiments, the ligand linker is composed of a majority of amino acids that are sterically unhindered. In some embodiments, the linker comprises a majority of amino acids selected from glycine, serine, and / or alanine. In some embodiments, the peptide linker is selected from polyglycine (such as (Gly)5 and (Gly)8), poly(Gly-Ala), and polyalanine.

[0045] Linkers can be of any size or composition, so long as they are capable of operably linking the ligands in a manner that allows them to bind to the target of interest. In some embodiments, the linker is about 1-50 amino acids, about 1-20 amino acids, about 1-15 amino acids, about 1-10 amino acids, about 1-5 amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids. It should be apparent that the length, degree of flexibility, and / or other properties of the linker(s) can affect certain properties of the ligand for use in an affinity agent, such as affinity, specificity, or avidity for the target of interest, or for multiple other target proteins of interest, or for proteins that are not of interest (i.e., non-target proteins). In some embodiments, two or more linkers are utilized. In some embodiments, two or more linkers are the same. In some embodiments, two or more linkers are different.

[0046] In some embodiments, the linker is a non-peptide linker such as an alkyl linker or a PEG linker. For example, an alkyl linker such as -NH-(CH2)sC(0)- (where s = 2 to 20) can be used. These alkyl linkers may be further substituted with any non-sterically hindering group, such as lower alkyl (e.g., C1C6) lower acyl, halogen (e.g., Cl, Br), CN, NH2, or phenyl. A representative non-peptide linker is a PEG linker. In some embodiments, the PEG linker has a molecular weight of about 100 to 5,000 kDa, or about 100 to 500 kDa.

[0047] Linkers can be evaluated using techniques described herein and / or otherwise known in the art. In some embodiments, the linker does not alter (e.g., destroy) the ability of the ligand to bind to the target molecule.

[0048] Affinity agents containing conjugated ligands To prepare affinity agents, ligands that promote specific binding to a target of interest can be chemically attached to various chromatographic compositions (e.g., beads, resins, gels, membranes, monoliths, etc.) Affinity agents containing ligands are particularly useful in purification and production applications.

[0049] In some embodiments, the ligand (e.g., a ligand fusion protein) comprises or contains at least one reactive residue. The reactive residue is useful, for example, as a binding site for a conjugate such as a chemotherapeutic drug. An exemplary reactive amino acid residue is lysine. A reactive residue (e.g., lysine) can be added to the ligand at either terminus or within the ligand sequence and / or can be substituted for another amino acid in the ligand sequence. A suitable reactive residue (e.g., lysine, etc.) can also be located within the sequence of an identified ligand without the need for addition or substitution. A further exemplary reactive amino acid residue is cysteine.

[0050] Binding to solid surfaces "Solid surface," "support," or "matrix" are used interchangeably herein and refer, without limitation, to any column (or column material), bead, test tube, microtiter dish, solid particle (e.g., agarose or sepharose), microchip (e.g., silicon, silicon-glass, or gold chip), or membrane (synthetic (e.g., filter) or biological (e.g., liposome or vesicle) origin) to which a ligand, affinity agent, antibody, or other protein may be attached (i.e., coupled, linked, attached), directly or indirectly (e.g., via other binding partner intermediates such as linkers), or into which a ligand may be embedded (e.g., via a receptor or channel). Reagents and techniques for binding polypeptides to solid supports (e.g., matrices, resins, plastics, etc.) are well known in the art. Suitable solid supports include, but are not limited to, chromatography resins or matrices (e.g., SEPHAROSE-4FF agarose beads), the walls or floors of the wells of plastic microtiter dishes, silica-based biochips, polyacrylamide, agarose, silica, nitrocellulose, paper, plastic, nylon, metal, and combinations thereof. Ligands and other compositions can be non-covalently or covalently bound to the support material using reagents and techniques known in the art. In some embodiments, the ligand is attached to the chromatography material using a linker.

[0051] Ligand generation Ligands useful for practicing some embodiments of the provided methods can be generated using a variety of standard techniques for chemical synthesis, semisynthesis, and recombinant DNA methodologies known in the art. Also provided are methods for generating ligands, either individually or as part of multidomain fusion proteins, as soluble agents and cell-associated proteins. In some embodiments, the overall ligand generation scheme involves obtaining a reference protein scaffold and identifying multiple residues within the scaffold for modification. Depending on the embodiment, the reference scaffold may include a protein structure with one or more alpha-helical regions or other tertiary structures. Once identified, any of the multiple residues can be modified, for example, by substitution of one or more amino acids. In some embodiments, one or more conservative substitutions are made. In some embodiments, one or more non-conservative substitutions are made. In some embodiments, a natural amino acid (e.g., one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine) is substituted into the target position of the reference scaffold for modification. In some embodiments, the modification does not involve a substitution at either cysteine ​​or proline. After modifications are made at the identified desired positions in certain embodiments, the resulting modified polypeptides (e.g., candidate ligands) can be recombinantly expressed, for example, in a plasmid, bacteria, phage, or other vector (e.g., to increase the number of each modified polypeptide). The modified polypeptides can then be purified and screened to identify modified polypeptides that specifically bind to a particular target of interest. The modified polypeptides may exhibit enhanced binding specificity for the target of interest compared to the reference scaffold, or may exhibit little or no binding to a given target of interest (or non-target protein).In some embodiments, depending on the target of interest, the reference scaffold may exhibit some interaction (e.g., non-specific interaction) with the target of interest, but particular modified polypeptides will exhibit at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or at least about 100-fold (or more) increased binding specificity for the target of interest. Additional details regarding the generation, selection, and isolation of ligands are provided in more detail below.

[0052] Recombinant expression of ligand In some embodiments, a ligand, such as a ligand fusion protein, is "recombinantly produced" (i.e., produced using recombinant DNA technology). Exemplary recombinant methods available for synthesizing a ligand (e.g., a ligand fusion protein) include, but are not limited to, polymerase chain reaction (PCR)-based synthesis, concatemerization, seamless cloning, and recursive directional ligation (RDL) (see, e.g., Meyer et al., Biomacromolecules 3:357-367 (2002), Kurihara et al., Biotechnol. Lett. 27:665-670 (2005), Haider et al., Mol. Pharm. 2:139-150 (2005); and McMillan et al., Macromolecules 32(11):3643-3646 (1999)).

[0053] Nucleic acids containing a polynucleotide sequence encoding a ligand are also provided. Such polynucleotides may further contain one or more expression control elements. For example, the polynucleotide may contain one or more expression control elements, such as a promoter or transcription enhancer, a ribosome binding site, a transcription termination signal, and a polyadenylation signal. The polynucleotide may be inserted into any suitable vector that can be contained in any suitable host cell for expression.

[0054] Expression of a nucleic acid encoding a ligand is typically achieved by operably linking the nucleic acid encoding the ligand to a promoter in an expression vector. Typical expression vectors contain transcription and translation terminators, initiation sequences, and a promoter useful for controlling expression of the desired nucleic acid sequence. Examples of promoters useful for expression in E. coli include, for example, the T7 promoter.

[0055] Methods well known to those skilled in the art can be used to construct expression vectors containing a nucleic acid sequence encoding a ligand along with appropriate transcriptional / translational control signals. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Expression of the polynucleotide can be carried out in any suitable expression host known in the art, including, but not limited to, bacterial cells, yeast cells, insect cells, plant cells, or mammalian cells. In some embodiments, the nucleic acid sequence encoding the ligand is operably linked to a suitable promoter sequence such that the nucleic acid sequence is transcribed and / or translated into the ligand in the host.

[0056] A variety of host-expression vector systems can be utilized to express nucleic acids encoding ligands. Vectors containing nucleic acids encoding ligands (e.g., individual ligand subunits or ligand fusions), or portions or fragments thereof, include plasmid vectors, single-stranded and double-stranded phage vectors, and single-stranded and double-stranded RNA or DNA viral vectors. Phage and viral vectors can also be introduced into host cells in the form of packaged or encapsidated viruses using known techniques for infection and transduction. Furthermore, viral vectors can be replication competent or alternatively replication-defective. Alternatively, cell-free translation systems can be used to produce proteins using RNA derived from DNA expression constructs (see, e.g., WO 86 / 05807 and WO 89 / 01036, and U.S. Pat. No. 5,122,464).

[0057] Generally, any type of cell or cultured cell line can be used to express the ligands provided herein. In some embodiments, the background cell line used to generate the engineered host cell is a phage, bacterial cell, yeast cell, or mammalian cell. A variety of host-expression vector systems can be used to express the nucleic acid sequence encoding the ligand or ligand fusion protein. Mammalian cells can be used as host cell systems transfected with recombinant plasmid DNA or cosmid DNA expression vectors that include or contain a nucleic acid sequence encoding a target of interest and a nucleic acid sequence encoding a polypeptide or fusion polypeptide. The cells can be primary isolates from transformed or transgenic organisms, cultures, or cell lines.

[0058] Suitable host cells include, but are not limited to, microorganisms, such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the ligand coding sequence; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the ligand coding sequence; insect cell lines infected with recombinant viral expression vectors (e.g., Baculovirus) containing the ligand coding sequence; and plant cell lines infected with recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus, CaMV; Tobacco Mosaic Virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti Plasmid) containing the ligand coding sequence.

[0059] Prokaryotes useful as host cells for producing the ligand include gram-negative or gram-positive organisms such as E. coli and B. subtilis. Expression vectors for use in prokaryotic host cells generally contain one or more phenotypic selectable marker genes (e.g., genes encoding proteins that confer antibiotic resistance or proteins that supply autotrophic requirements). Examples of useful prokaryotic host expression vectors include the pKK223-3 (Pharmacia, Uppsala, Sweden), pGEM1 (Promega, Wis., USA), pET (Novagen, Wis., USA), and pRSET (Invitrogen, Calif., USA) vector series (see, e.g., Studier, J. Mol. Biol. 219:37 (1991) and Schoepfer, Gene 124:83 (1993)). Exemplary promoter sequences frequently used in prokaryotic host cell expression vectors include T7 (Rosenberg et al., Gene 56:125-135 (1987)), beta-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615 (1978)), and Goeddel et al., Nature 281:544 (1979)), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, (1980)), and tac promoter (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0060] In some embodiments, exemplary yeast that can be used to generate compositions of the invention that contain or comprise a nucleic acid encoding a ligand include yeast from the genera Saccharomyces, Pichia, Actinomyces, and Kluyveromyces. Yeast vectors typically contain an origin of replication from the 2mu yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Examples of promoter sequences in yeast expression constructs include promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman, J. Biol. Chem. 255:2073 (1980)), and other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Additional suitable vectors and promoters for use in yeast expression and yeast transformation protocols are known in the art, see, e.g., Fleer, Gene 107:285-195 (1991) and Hinnen, PNAS 75:1929 (1978).

[0061] Insect and plant host cell culture systems are also useful for producing the ligands described herein. Such host cell systems include, for example, insect cell systems infected with a recombinant viral expression vector (e.g., baculovirus) that includes or contains a nucleic acid sequence encoding the ligand; plant cell systems infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) that includes a nucleic acid sequence encoding the ligand, including, but not limited to, the expression systems taught in U.S. Patent No. 6,815,184, U.S. Application Nos. 60 / 365,769 and 60 / 368,047, and WO2004 / 057002, WO2004 / 024927, and WO2003 / 078614.

[0062] In some embodiments, host cell lines can be used, including animal cell lines infected with recombinant viral expression vectors (e.g., adenovirus, retrovirus, adeno-associated virus, herpes virus, lentivirus), including cell lines engineered to contain multiple copies of DNA encoding either stably amplified ligand (CHO / dhfr) or unstably amplified ligand (e.g., murine cell lines) on bichromosomes. In some embodiments, the vector containing the polynucleotide(s) encoding the ligand is polycistronic. Exemplary mammalian cells useful for producing these compositions include 293 cells (e.g., 293T and 293F), CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 (Crucell, Netherlands) cells VERY, Hela cells, COS cells, MDCK cells, 3T3 cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT20 cells, T47D cells, CRL7O30 cells, HsS78Bst cells, hybridoma cells, and other mammalian cells. Additional exemplary mammalian host cells useful in practicing the present invention include, but are not limited to, T cells. Exemplary expression systems and selection methods are known in the art and include those described in the following references and references cited therein: Borth et al., Biotechnol. Bioen. 71(4):266-73 (2000), in Werner et al., Arzneimittelforschung / Drug Res. 48(8):870-80 (1998), Andersen et al., Curr. Op. Biotechnol. 13:117-123 (2002), Chadd et al., Curr. Op. Biotechnol. 12:188-194 (2001), and Giddings, Curr. Op. Biotechnol. 12:450-454 (2001).Further examples of expression systems and selection methods are described in Logan et al., PNAS 81:355-359 (1984), Birtner et al. Methods Enzymol. 153:51-544 (1987). Transcriptional and translational control sequences for mammalian host cell expression vectors are often derived from viral genomes. Promoter and enhancer sequences commonly used in mammalian expression vectors include sequences derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV). Exemplary commercially available expression vectors for use in mammalian host cells include pCEP4 (Invitrogen) and pcDNA3 (Invitrogen).

[0063] Physical methods for introducing nucleic acids into host cells (e.g., mammalian host cells) include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0064] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0065] Methods for introducing DNA and RNA polynucleotides of interest into host cells include electroporation of the cells, in which an electric field is applied to the cells to increase the permeability of the cell membrane, allowing the introduction of chemicals, drugs, or polynucleotides into the host cells. Ligands, including DNA or RNA constructs, can be introduced into mammalian or prokaryotic cells using electroporation.

[0066] In some embodiments, electroporation of cells results in expression of the ligand-CAR on the surface of T cells, NK cells, and / or NKT cells. Such expression can be transient or stable over the life of the cells. Electroporation can be achieved by methods known in the art, including MaxCyte GT® and STX® Transfection Systems (MaxCyte, Gaithersburg, MD, USA).

[0067] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). When a non-viral delivery system is utilized, the exemplary delivery vehicle may be or include a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In some embodiments, the nucleic acid is associated with a lipid. Nucleic acids associated with lipids may be encapsulated within the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules associated with both the liposome and the oligonucleotide, entrapped in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to a particular structure in solution. For example, they can exist in bilayer structures as micelles or in "collapsed" structures. They may also simply be interspersed in solution or form aggregates that are not uniform in size or shape. Lipids are fatty substances, either natural or synthetic. For example, lipids include lipid droplets that occur naturally in the cytoplasm and classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0068] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristoyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform evaporates more readily than methanol and can therefore be used as the sole solvent. "Liposome" is a generic term encompassing a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as vesicular structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5:505-510 (1991)). However, compositions with structures in solution that differ from the typical vesicle structure are also included. For example, lipids may adopt a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0069] Regardless of the method used to introduce exogenous nucleic acid into a host cell, the presence of the recombinant nucleic acid sequence in the host cell can be routinely confirmed by a variety of assays known in the art. Such assays include "molecular biological" assays known in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot) or the assays described herein to identify agents.

[0070] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. Generally, reporter genes are genes encoding polypeptides that are not present in or expressed by recipient organisms, tissues, or cells, and whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at a suitable time after DNA is introduced into recipient cells. Suitable reporter genes include, but are not limited to, genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., FEBS Lett. 479:79-82 (2000)). Suitable expression systems are well known in the art and can be prepared using known techniques or commercially available. Generally, the construct with the smallest 5' flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such promoter regions can usually be linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0071] A number of selection systems can be used in mammalian host-vector expression systems, including, but not limited to, herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes. Additionally, antimetabolite resistance can be used as the basis for selection for, for example, the dhfr, gpt, neo, hygro, trpB, hisD, ODC (ornithine decarboxylase), and glutamine synthase systems.

[0072] Ligand purification Once the ligand or ligand fusion protein is produced by recombinant expression, it can be purified by methods known in the art for purifying recombinant proteins, for example, by chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for purifying proteins. In some embodiments, the ligand is optionally fused to a heterologous polypeptide sequence, specifically disclosed herein or otherwise known in the art, to facilitate purification. In some embodiments, the ligand (e.g., antibodies and other affinity matrices) is for a ligand affinity column and / or for affinity purification, and optionally other components of the ligand or ligand fusion composition that are bound by these ligands are removed from the composition prior to final preparation of the ligand using techniques known in the art.

[0073] Chemical synthesis of ligands In addition to recombinant methods, ligand production can also be achieved using organic chemical synthesis of the desired polypeptide using a variety of solution and solid phase chemical processes known in the art. A variety of automated synthesizers are commercially available and can be used in accordance with known protocols. See, e.g., Tam et al., J. Am. Chem. Soc., 105:6442 (1983); Merrifield, Science, 232:341-347 (1986); Barany and Merrifield, The Peptides, Gross and Meienhofer, eds., Academic Press, New York, 1-284; Barany et al., Int. J. Pep. Protein Res., 30:705-739 (1987); Kelley et al., in Genetic Engineering Principles and Methods, Setlow, JK, ed. Plenum Press, NY. 1990, vol. 12, pp. 1-19; Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, 1989. One advantage of these methodologies is that they allow for the incorporation of unnatural amino acid residues into the amino acid sequence of the ligand.

[0074] The ligands used in the methods of the invention may be modified during or after synthesis or translation, for example, by glycosylation, acetylation, benzylation, phosphorylation, amidation, pegylation, formylation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to an antibody molecule, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, ubiquitination, and the like. (See, e.g., Creighton, Proteins: Structures and Molecular Properties, 2nd Ed. (W.H. Freeman and Co., NY, 1992); Postranslational Covalent Modification of Proteins, Johnson, ed. (Academic Press, New York, 1983), pp. 1-12; Seifter, Meth. Enzymol., 182:626-646 (1990); Rattan, Ann. NY Acad. Sci., 663:48-62 (1992).) In some embodiments, the ligand is acetylated at the N-terminus and / or amidated at the C-terminus.

[0075] Any of a number of chemical modifications can be made by known techniques, including, but not limited to, acetylation, formylation, etc. Additionally, the derivative can include one or more non-classical amino acids.

[0076] In some embodiments, cyclization or macrocyclization of the peptide backbone is achieved by side chain to side chain bond formation. Methods for achieving this are well known in the art and can involve natural and unnatural amino acids. Approaches include disulfide formation, lanthionine formation or thiol alkylation (e.g., Michael addition), amidation between amino and carboxylate side chains, click chemistry (e.g., azide-alkyne condensation), peptide stapling, ring-closing metathesis, and the use of enzymes.

[0077] Affinity agent for purification In affinity chromatography-based purification, targets of interest (e.g., proteins or molecules) are selectively isolated according to their ability to specifically and reversibly bind to ligands that are typically covalently attached to a chromatography matrix. In some embodiments, the ligands can be used as reagents for affinity purification of targets of interest from recombinant sources or natural sources, such as biological samples (e.g., serum).

[0078] In some embodiments, a ligand that specifically binds to a target of interest is immobilized on beads and then used to affinity purify the target.

[0079] Methods for covalently binding proteins to surfaces are known to those skilled in the art, and peptide tags that can be used to bind ligands to solid surfaces are known to those skilled in the art. Furthermore, ligands can be bound (i.e., coupled, linked, and / or attached) to solid surfaces using any reagent or technique known in the art. In some embodiments, the solid support comprises beads, glass, slides, chips, and / or gelatin. Thus, arrays can be created on solid surfaces using a series of ligands using techniques known in the art. For example, U.S. Application Publication No. 2004 / 0009530 discloses methods for preparing arrays.

[0080] In some embodiments, the ligand is used to isolate the target of interest by affinity chromatography. In some embodiments, the ligand is immobilized on a solid support. The ligand can be immobilized on the solid support using techniques and reagents described herein or otherwise known in the art. Suitable solid supports are described herein or otherwise known in the art, and in certain embodiments, are suitable for packing a chromatography column. The immobilized ligand can then be loaded or contacted with a solution under conditions suitable for forming a complex between the ligand and the target of interest. Unbound material can be washed away. One of skill in the art can easily determine appropriate wash conditions. Examples of suitable wash conditions are described in Shukla and Hinckley, Biotechnol Prog. 2008 Sep-Oct;24(5):1115-21. doi:10.1002 / btpr.50.

[0081] In some embodiments, chromatography is performed by mixing a solution containing the target of interest and the ligand, and then isolating the complex of the target of interest and the ligand. For example, the ligand is immobilized on a solid support such as beads and then separated from the solution along with the target of interest by filtration. In some embodiments, the ligand is or includes a fusion protein containing a peptide tag, such as a poly-His tail or a streptavidin-binding region, that can be used to isolate the ligand after complex formation using an immobilized metal affinity chromatography resin or a streptavidin-coated substrate. Once separated, the target of interest is released from the ligand under elution conditions and recovered in a purified form. [Example]

[0082] Example 1 Peptides were synthesized by standard Fmoc solid-phase peptide synthesis techniques and purified by preparative reverse-phase HPLC. Peptide purity was assessed by RPUPLC equipped with both UV and quadrupole time-of-flight mass spectrometry detection.

[0083] Example 2 This example demonstrates the binding of a biotinylated ligand to the CD81 capsid using Biolayer Interferometry (ForteBio, Menlo Park, CA). The biotinylated ligand was immobilized on a sensor and incubated with a solution containing different concentrations of Fc-CD81 (R&D Systems, Minneapolis, MN) in PBS containing 0.01% (w / v) bovine serum albumin and 0.1% (v / v) Tween® 20, pH 7.4. A blank sensor was included as a control. An example sensorgram is shown in Figure 5, and example data are shown in Figure 6.

[0084] Example 3 This example describes assays used to monitor the tetraspanin exosome markers CD9, CD63, and CD81. Reagents for Western blotting are listed in Table 1. Gels were blotted onto PVDF membranes and processed in a GO Blot Processor (Cytoskeleton, Denver, CO). Blots were developed with Super Signal™ West Pico PLUS chemiluminescent substrate (Thermo Scientific, Waltham, MA) and imaged with a BioRad Chemi Doc MP system (BioRad, Hercules, CA). [Table 1]

[0085] CD81 was also assayed by applying the PSCapture™ Exosome ELISA Kit (Fujifilm, Richmond, VA). Anti-CD81 antibody MA5-13548 (Invitrogen, Waltham, MA) diluted 1:500 was used instead of the anti-CD63 antibody included in the kit. An example of a standard curve is shown in Figure 1.

[0086] Example 4 This example demonstrates the generation and characterization of affinity resins containing the ligands identified and described herein. Affinity resins were prepared by conjugating aminated ligands to agarose beads. RAPIDRUN 6% agarose beads (ABT, Madrid, Spain) and Praesto® Jetted A50 beads (Purolite, King of Prussia, PA) were activated with disuccinimidyl carbonate and coupled with the ligand at ligand densities of 1-8 mg / mL resin. The actual ligand densities of all resins were measured using a subtractive RP-HPLC method according to the following equation: Actual ligand density = ([ligand] measured in feed - [ligand] measured in effluent).

[0087] To prepare affinity resins with thiolated ligands conjugated to agarose beads, Praesto® Jetted A50 beads (Purolite, King of Prussia, PA) were activated with disuccinimidyl carbonate and combined with excess ethylenediamine. After washing, bromoacetate was conjugated to the aminated beads using EDC activation. After washing, the ligands were conjugated to the beads at room temperature. After washing, the beads were deactivated with excess thioglycerol. Ligand density was determined as described above.

[0088] Example 5 This example demonstrates the binding capacity of affinity agents containing the binding ligands described herein for affinity capture of exosomes. Filter plate binding experiments using Durapore membranes. Cat# MSHVS4510 (Millipore, Burlington MA) were operated as shown in Table 2. [Table 2]

[0089] The resins were prepared by conjugating the ligands to A50 beads. The ligand density and capture efficiency of each resin are shown in Table 3. [Table 3]

[0090] Example 6 This example demonstrates the use of affinity agents containing the binding ligands described herein for the purification of exosomes. A 0.18 mL glass column (3 x 25 mm) was packed with resin containing the ligand corresponding to SEQ ID NO: 43 at a ligand density of 11.2 mg / mL and operated as described in Table 4. [Table 4]

[0091] Fractions from the column run were analyzed by Western blotting for the exosome marker CD63, as shown in Figure 2, and the chromatogram is shown in Figure 3. The Western blot clearly demonstrates that the resin is effective at capturing and eluting exosomes.

[0092] Example 7 This example demonstrates the stability of affinity agents containing the ligands described herein to sodium hydroxide. Resin containing SEQ ID NO:43 was incubated with 0.1 M NaOH for 18 hours, washed, and subjected to static binding experiments as described in Example 5. Resin not incubated with 0.1 M NaOH was included as a control, and a comparison of binding is shown in Figure 4, clearly showing that the resin is stable to 0.1 M NaOH.

[0093] It is contemplated that various combinations or subcombinations of the specific features and aspects of the above-disclosed embodiments may be made and are within the scope of the present invention. Furthermore, any specific feature, aspect, method, property, characteristic, quality, attribute, element, etc. of the disclosure herein associated with one embodiment may be used in all other embodiments set forth herein. Accordingly, it should be understood that various features and aspects of the disclosed embodiments may be combined with or substituted for one another. Therefore, it is not intended that the scope of the invention as described herein should be limited by the specific disclosed embodiments set forth above. Furthermore, while the invention is susceptible to various modifications and alternative forms, specific examples thereof are shown in the drawings and described in detail herein. However, it should be understood that the invention is not limited to the specific forms or methods disclosed, but rather the invention is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the described embodiments.

[0094] The methods disclosed herein do not have to be performed in the order listed. The methods disclosed herein involve specific actions performed by a practitioner, but may also include, explicitly or implicitly, third-party instructions regarding those actions. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] X 1 =H or N X 2 -=S or T · Ac- means N-terminal acetylation ·-amide means C-terminal amidation 5 represents an aspartic acid residue whose carboxyl side chain is attached to either the ε-amino group of the lysine residue represented by 4 or the β-amino group of the 2,3-diaminopropionic acid residue represented by 2. Suc stands for succinic acid (butanedioic acid) 3 means 3-(4-hydroxyphenyl)propionate (Peg)3- means 12-amino-4,7,10-trioxadodecanoic acid subunit Cysteine ​​residues can form intramolecular disulfide bonds

Claims

1. 1. An affinity agent comprising a ligand that binds to CD81, the ligand comprising a cyclic peptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:1:X １ YWRB １ VWFPHAQGB ２ VX ２ X ２ , In the formula, X １ represents H or N, and X ２ represents S or T, B １ and B ２ represents the unit by which the peptide is cyclized.

2. The affinity agent according to claim 1, wherein B 1 of the cyclic peptide is aspartic acid and B 2 is either lysine or 2,3-diaminopropionic acid.

3. An affinity agent described in claim 1 or 2, wherein the amino acid sequence of the cyclic peptide comprises an N-terminal or C-terminal modification selected from glycosylation, acetylation, benzylation, phosphorylation, amidation, PEGylation, or formylation, and preferably the cyclic peptide is acetylated at the N-terminus and / or amidated at the C-terminus.

4. An affinity agent described in any one of claims 1 to 3, wherein the amino acid sequence of the cyclic peptide includes a C-terminal linker peptide of 1 to 10 amino acids.

5. The affinity agent described in claim 4, wherein the linker peptide contains a reactive residue selected from lysine or cysteine.

6. An affinity agent described in claim 4 or 5, wherein the cyclic peptide comprises an amino acid sequence selected from SEQ ID NOs: 77, 78, 82, 83, 87 and 88.

7. An affinity agent described in any one of claims 1 to 5, wherein the amino acid sequence of the cyclic peptide includes a C-terminal non-peptide linker.

8. The affinity agent described in claim 7, wherein the C-terminal non-peptide linker is selected from an alkyl linker or a polyethylene glycol (PEG) linker.

9. The affinity agent described in claim 8, wherein the alkyl linker comprises 12-amino-4,7,10-trioxadodecanoic acid.

10. The affinity agent described in claim 1, wherein the cyclic peptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 3, 7, 8, 12, 13, 17, 18, 22, 23, 27, 28, 32, 33, 37, 38, 42, 43, 47, 48, 52, 53, 57 and 58.

11. 11. The affinity agent of any one of claims 1 to 10, wherein the ligand is bound to a solid surface, optionally the solid surface is a resin, a bead, a membrane or a monolith, and further optionally the ligand is conjugated to the solid surface via a linker.

12. 12. The affinity agent according to any one of claims 1 to 11 for use in a method for purifying one or more exosomes.

13. An affinity agent comprising a ligand that binds to CD81, wherein the ligand comprises a cyclic peptide having an amino acid sequence selected from SEQ ID NOs: 4-6, 9-11, 14-16, 19-21, 24-26, 29-31, 34-36, 39-41, 44-46, 49-51, 54-56, 59-61, 79-81, 84-86 and 89-91.

14. An affinity agent comprising a ligand that binds to CD81, wherein the ligand comprises a cyclic peptide comprising an amino acid sequence selected from SEQ ID NOs: 62 to 76 and 107 to 121.

15. An affinity agent comprising a ligand that binds to CD81, wherein the ligand comprises a cyclic peptide comprising an amino acid sequence selected from SEQ ID NOs: 92 to 106 and 122.

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