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

[Background technology]

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 405,301, filed September 9, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The purity of biologically produced therapeutics is closely scrutinized and regulated by authorities to ensure safety and efficacy. Thus, there remains a need for means to efficiently purify biologically produced therapeutics in high purity and sufficient quantities. Summary of the Invention

[0003] To support clinical efforts for therapeutic proteins, compositions and methods for efficiently purifying proteins from recombinant sources are needed. Affinity purification is a means to isolate and / or achieve a desired degree of purity of a protein in a few or a single step. However, developing affinity agents (including, for example, affinity ligands) can be resource-intensive and time-consuming. For this reason, the number of proteins for which affinity agents have been developed is limited. In the absence of affinity agents, purification typically involves inefficient, labor-intensive, and expensive processes (e.g., multi-column processes).

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

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

[0006] The COVID-19 pandemic has highlighted the benefits of leveraging platform production processes for the rapid development of both therapeutics and vaccines. For example, monoclonal antibody therapies were developed and emergency approval achieved on an unprecedented timeline thanks to their ability to leverage platform production processes, primarily Protein A affinity purification platforms. In the case of mRNA vaccines, the speed of development was also facilitated by the use of existing affinity purification technologies (e.g., oligo-dT and cellulose chromatography) for purification of the mRNA portion. Clearly, therefore, future pandemic preparedness measures, as well as the acceleration of vaccine production for current global initiatives, will benefit from the expansion of affinity purification tools, i.e., providing platform processes applicable to multiple vaccines. Described herein are affinity agents that bind to trimerization domains and are useful for isolation and / or affinity purification. In some embodiments, the affinity agent comprises a ligand and a solid support.

[0007] Some subunit vaccines are based on viral proteins that form trimer structures. To facilitate efficient production of correctly folded trimeric forms, trimerization domains can be fused to viral proteins. The most commonly used trimerization domains are the T4 phage fibritin trimerization domain (foldon) [Tao Y, Strelkov SV, Mesyanzhinov VV, Rossmann MG, 1997. Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain. Structure 5, 789-798] and the yeast GCN4 trimerization domain [Harbury PB, Zhang T, Kim PS, Alber T, A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science, 1993, 262, 1401-1407]. More recently, another trimerization domain (called a molecular clamp) has been developed and demonstrated to confer stabilizing properties to the conformation of enveloped virus fusion protein-based vaccine constructs (see, e.g., WO 2018 / 176103, incorporated herein by reference in its entirety). Different viral proteins or viral protein variants may be fused to the same trimerization domain to create different vaccines. Furthermore, the trimerization domain can also be utilized to confer trimerization of other (i.e., non-vaccine) polypeptides / proteins, particularly therapeutic proteins (e.g., any of those mentioned in paragraphs

[0395] to

[0400] of WO 2018 / 176103, but are not intended to be limiting). Affinity agents binding to this trimerization domain allow for easy purification of the associated subunit vaccines independently of the antigenic moiety, thus providing a platform technology for vaccine production and / or purification.

[0008] In some embodiments, the affinity agent (or a ligand contained in the affinity agent) comprises a three-helix bundle protein (alternatively referred to as a "three-helix bundle protein"), preferably an antiparallel three-helix bundle protein. In some embodiments, the structure of a three-helix bundle protein can be envisioned as a triangular prism, with the vertices of each triangle representing a helix, for example, as shown in FIG. 1. In some embodiments, the combination of any two helices defines a rectangular face of the three-helix bundle protein. For example, in some embodiments, the three faces of the three-helix bundle protein are defined by: 1) helices 1 and 2 (faces 1 and 2 in Figure 1 ); 2) helices 2 and 3 (faces 2 and 3 in Figure 1 ); 3) helices 1 and 3 (faces 1, 3 in Figure 1 ), and A combination of them.

[0009] In some embodiments, the affinity agent (or a ligand contained therein) comprises a face formed from helices 2 and 3 of a three-helix bundle protein. In some embodiments, the primary function of helix 1 of a three-helix bundle protein is to complete and stabilize the three-helix bundle. In some embodiments, variations of helix 1 can be made that maintain the structure of the three-helix bundle protein.

[0010] In some embodiments, provided herein is an affinity agent comprising SEQ ID NO: 1, EQRRNFIENLRWDPSKSARLLARAKRFNDW.

[0011] In some embodiments, provided herein is an affinity agent comprising SEQ ID NO: 1 contained within helices 2 and 3 of a three-helix bundle protein.

[0012] In some embodiments, provided herein is an affinity agent comprising SEQ ID NO: 2, VDAKFDKELEEARAEIERLPNLTEEQRRNFIENLRWDPSKSARLLARAKRFNDWQAPK.

[0013] In some embodiments, provided herein is an affinity agent comprising a multimeric polypeptide comprising at least two subunits, each subunit being a polypeptide according to the preceding embodiments.

[0014] In some embodiments, provided herein are affinity agents that comprise multimeric polypeptides in which the subunits are not all identical.

[0015] In some embodiments, provided herein are affinity agents comprising SEQ ID NOs: 3-10.

[0016] In some embodiments, provided herein are affinity agents that bind to trimerization domains.

[0017] In some embodiments, provided herein are affinity agents for use in the purification of proteins containing trimerization domains.

[0018] In some embodiments, provided herein are affinity agents for use in the purification of vaccines containing trimerization domains.

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

[0020] Approximately or about: As used herein, the term "approximately" or "about," when applied to one or more subject values, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about," unless otherwise stated or otherwise clear from the context, refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or a smaller percentage, in order of preference, in either direction (greater or lesser than) the stated reference value (except where such number exceeds 100% of the possible values).

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

[0022] Conservative and Non-Conservative Substitutions: A "conservative" amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including 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., asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C)), nonpolar side chains (e.g., glycine (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)). Conservative amino acid substitutions include phenylalanine (F), tryptophan (W), and histidine (H). For example, substitution of phenylalanine with tyrosine is a conservative substitution. In some embodiments, conservative amino acid substitutions in the sequence of a ligand confer or improve specific binding of the ligand to a target of interest. In some embodiments, conservative amino acid substitutions in the sequence of a 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 substitutions of nucleotides and amino acids that confer, alter, or maintain selective binding affinity are known in the art (e.g., Brummell, Biochem. 32:1180-1187 (1993); Kobayashi, Protein Eng. 12(10):879-884 (1999); and Burks, PNAS 94:412-417 (1997). In some embodiments, non-conservative amino acid substitutions in the sequence of the ligand confer or improve specific binding of the ligand to a target of interest. In some embodiments, non-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, the non-conservative amino acid substitutions do not significantly affect the specific binding of the ligand to its intended target.

[0023] Linker: As used herein, "linker" refers to a peptide or other chemical bond that functions to connect separate, independent functional domains, entities, or moieties. In some embodiments, the linker is positioned between the ligand and another polypeptide component containing another, independent functional domain. In some embodiments, the linker is a peptide or other chemical bond positioned between the ligand and a surface, such as a solid surface or solid support. In particularly preferred embodiments, the ligand is chemically attached (i.e., covalently bound) to the solid surface or solid support via a bond formed between a thiol group of a cysteine ​​of the ligand (preferably an N-terminal or C-terminal cysteine, more preferably a C-terminal cysteine) and the solid support. For example, the ligand can be covalently attached to the solid support via a bond formed by nucleophilic addition of a thiol group of a cysteine ​​(e.g., a C-terminal cysteine) of the ligand to a maleimide group on the solid surface or solid support. Other methods for covalently or non-covalently attaching ligands (e.g., ligands including polypeptides) to solid surfaces or solid supports are well known and routinely used in the art. For example, Greg T. Hermanson, "Bioconjugate Techniques", 3 rd The ligand can be covalently attached to the solid support using any of the methods and linkages described in the "Synthetic Chemistry of Polymers," 2013 edition, which is incorporated herein by reference in its entirety.

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

[0025] The terms "unnatural amino acid," "amino acid analog," and "non-standard amino acid residue" are used interchangeably herein. Unnatural amino acids that can be substituted in the ligands provided herein are known in the art. In some embodiments, the unnatural amino acid is 4-hydroxyproline, which can be substituted for proline, 5-hydroxylysine, which can be substituted for lysine, 3-methylhistidine, which can be substituted for histidine, homoserine, which can be substituted for serine, and ornithine, which can be substituted for lysine. Additional examples of unnatural amino acids that can be substituted in polypeptide ligands include 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 pyrrolidine fluoro-amino acids, engineered amino acids such as beta-methyl amino acids, C alpha-methyl amino acids, and N alpha-methyl amino acids.

[0026] " Polynucleotide " and " nucleic acid molecule ": as used interchangeably herein, polynucleotide and nucleic acid molecule refer to any length of polymeric form of nucleotides, which comprise or consist of either ribonucleotides or deoxyribonucleotides, or both.These terms include but are not limited to DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and / or tRNA.

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

[0028] Peptide tag: As used herein, the term "peptide tag" refers to a peptide sequence that is part of or attached to another protein (e.g., via genetic engineering) to provide a function to the resulting fusion. Peptide tags are usually relatively short compared to the protein to which they are fused. In some embodiments, the peptide tag is four or more amino acids in length, such as 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more amino acids. In some embodiments, the ligand is a protein containing the peptide tag. Numerous peptide tags with uses as provided herein are known in the art. Examples of peptide tags that may be components of a ligand fusion protein or a target of interest 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, GPT, GFP, MBP, Strep tag, His tag, Myc tag, TAP tag, and FLAG tag (Eastman Kodak, Rochester, NY). Similarly, an antibody against the tag epitope allows detection and localization of the fusion protein in, for example, affinity purification, Western blots, ELISA assays, and immunostaining of cells.

[0029] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids linked together via peptide bonds. While the term is used to refer to an amino acid chain of any length, those skilled in the art will understand that the term is not limited to long chains and can refer to a minimum chain containing two amino acids linked together via peptide bonds. Therefore, for purposes of this disclosure, it is understood that the terms "peptide" and "polypeptide" can be used interchangeably. As known to those skilled in the art, polypeptides may be processed and / or modified.

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

[0031] Specific binding: As used herein with respect to a ligand, the term "specifically binds" or "having selective affinity" means that the ligand reacts or associates with a particular epitope, protein, or target molecule more frequently, more rapidly, for a longer period of time, with greater affinity, or a combination thereof, than alternative substances, including unrelated proteins. Due to sequence identity between homologous proteins in different species, specific binding may include a binding agent that recognizes proteins or targets in more than one species. Similarly, due to homology within a particular region of the polypeptide sequence of different proteins, specific binding may include a binding agent that recognizes more than one protein or target. It is understood that in certain embodiments, a binding agent that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require (but can include) exclusive binding, i.e., binding to a single target. Thus, a ligand or affinity agent may, in certain embodiments, specifically bind to more than one target. In certain embodiments, multiple targets may be bound by the same antigen-binding site on the affinity agent.

[0032] Substantially: As used herein, the term "substantially" refers to the qualitative condition indicating the entire or nearly entire extent or degree of a characteristic or property of interest. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or perfection, or 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.

[0033] It should also be understood that where a singular "noun" (e.g., ligand, bead, etc.) is referred to herein, the plural of such term (e.g., ligand, bead, etc.) is also intended to be included and contemplated herein, unless otherwise specified. Thus, in preferred embodiments, where applicable, reference to a singular noun is also intended to alternatively refer to "at least one," "one or more," or "plurality" of the referred term. [Brief explanation of the drawings]

[0034] [Figure 1] Exemplary affinity agents including three-helix bundle proteins are shown, as well as an example of how the structure of a three-helix bundle protein can be envisioned as a triangular prism, with the vertices of each triangle representing a helix. [Figure 2] 1 shows an exemplary sensorgram of an affinity agent of the present invention. A ligand corresponding to the biotinylated ligand of SEQ ID NO: 2 was immobilized on the sensor and binding to the target analyte, trimeric vaccine protein, was determined. [Figure 3] 1 shows the stability of a type of affinity agent of the present invention. The ligand corresponded to the biotinylated form of SEQ ID NO:7. [Figure 4] 1 shows the stability of one affinity agent of the present invention in 0.1 M NaOH. The affinity resin contained the ligand of SEQ ID NO:2. [Figure 5] 1 shows a chromatogram of absorbance at a wavelength of 280 nm during the purification of a trimeric vaccine protein using an affinity resin of the present invention. The affinity resin contained a ligand of SEQ ID NO:7. [Figure 6] Shown is an SDS-PAGE gel of trimeric vaccine protein purified using the affinity agent of the present invention. The affinity resin contained the ligand of SEQ ID NO: 7. The load (L), wash (W), elution (E), and debris (S) fractions were loaded onto the gel. A molecular weight reference standard (Std) was included. The molecular weight of the purified trimeric vaccine protein is 180 kDa. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present disclosure encompasses, inter alia, the recognition that affinity agents prepared from the identified and characterized peptide ligands have been shown to produce highly purified preparations of one or more targets of interest, such as, in some embodiments, trimeric proteins, preferably trimeric vaccine proteins. In some embodiments, the affinity agents (e.g., affinity resins or affinity beads) described herein are particularly useful for removing protein product-related impurities as well as host cell-derived contaminants.

[0036] Ligand binding to a target of interest for use in affinity agents The target-binding properties of a ligand can be determined using art-known or modified assays, bioassays, and / or animal models for assessing such activity. According to the affinity agents of the present invention, the described characteristic that the affinity agent "binds" to a target of interest (e.g., a trimeric protein, preferably a trimeric vaccine protein) can alternatively and more specifically be defined as the ligand(s) contained in the affinity agent "binding" to the target of interest (e.g., a trimeric protein, preferably a trimeric vaccine protein). In other words, it is understood that the ability of an affinity agent to bind to a target of interest is provided by the ligand contained in the affinity agent. Furthermore, the term "bind," when used in the context of binding to a target of interest, is intended to mean "capable of binding" to the target of interest, and thus can be defined interchangeably. In a preferred embodiment, the affinity agent (or the ligand contained in the affinity agent) "specifically" binds to a trimeric protein, preferably a trimeric vaccine protein, or a trimerization domain contained therein.

[0037] As used herein, terms such as " target binding affinity ", " target binding " refer to the property of ligand that can be directly measured, for example, through determining affinity constant (for example, the amount of ligand that associates and dissociates at a given antigen concentration).There are several methods for characterizing such molecular interactions, for example, competitive analysis, equilibrium analysis, and microcalorimetry analysis, and real-time interaction analysis based on surface plasmon resonance interaction (for example, using BIACORE equipment).These methods are well known to those skilled in the art, and are described 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 binds to a target of interest (e.g., a trimeric protein, preferably a trimeric vaccine protein, or a trimerization domain as referred to herein) at a density of 5×10 -3 M or less, 10 -3 M or less, 5×10 -4 M or less, 10 -4 M or less, 5×10 -5 M or less, or 10 -5 The dissociation constant (K D In some embodiments, the ligand binds to the target of interest at a concentration of 5×10 -6 M or less, 10 -6 M or less, 5×10 -7 M or less, 10 -7 M or less, 5×10 -8 M or less, or 10 -8 K below M D In some embodiments, the ligand binds to the target of interest at a concentration of 5×10 -9 M or less, 10 -9 M or less, 5×10 -10 M or less, 10 -10 M or less, 5×10-11 M or less, 10 -11 M or less, 5×10 -12 M or less, 10 -12 M or less, 5×10 -13 M or less, 10 -13 M or less, 5×10 -14 M or less, 10 -14 M or less, 5×10 -15 M or less, or 10 -15 K below M D In some embodiments, the ligands produced by the methods disclosed herein bind at about 10 -4 M ~ about 10 -5 M, about 10 -5 M~about 10 -6 M, about 10 -6 M~about 10 -7 M, about 10 -7 M ~ about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M ~ about 10 -11 M, or about 10 -11 M~10 -12 It has a dissociation constant of M.

[0039] K D Binding experiments to determine the binding and off-rate can be performed under many conditions. Buffers for making these solutions can be easily determined by those skilled in the art and depend largely on the desired pH of the final solution. Low pH solutions (less than pH 5.5) can be made in, for example, citrate buffer, glycine-HCl buffer, or succinate buffer. High pH solutions can be made in, for example, Tris-HCl, phosphate buffer, or sodium bicarbonate buffer. Several conditions can be used to determine, for example, the optimal pH and / or salt concentration. D and off-rates can be determined.

[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 to the target of interest at a concentration of 5×10 -2 seconds -1 Less than 10 -2 seconds -1 Less than 5 x 10 -3 seconds -1 Less than or equal to 10 -3 seconds -1 Off-rate (k off ) binds to the target of interest. In some embodiments, the ligand binds to the target of interest at a concentration of 5×10 -4 seconds -1 Less than 10 -4 seconds -1 Less than 5 x 10 -5 seconds -1 Less than or equal to 10 -5 seconds -1 Less than 5 x 10 -6 seconds -1 Less than 10 -6 seconds -1 Less than 5 x 10 -7 seconds -1 Less than or equal to 10 -7 seconds -1 Off-rate (k off ) to bind to the target of interest. 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 seconds -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 Super, 5×10 3 M -1 seconds-1 Super, 10 4 M -1 seconds -1 Super, or 5x10 4 M -1 seconds -1 Ultra-high on-speed (k on ) to bind to the target of interest. In a further embodiment, the ligand binds to the target of interest at 10 5 M -1 seconds -1 Super, 5×10 5 M -1 seconds -1 Super, 10 6 M -1 seconds -1 Super, 5×10 6 M -1 seconds -1 Over or 10 7 M -1 seconds -1 Super K on and binds to the target of interest.

[0041] Target According to various embodiments, the target of interest specifically bound by the ligand can be any molecule to which it is desirable for the ligand of the affinity agent to bind. For example, the target of interest specifically bound by the ligand can be any target of relevance or value for purification, manufacturing, formulation, treatment, diagnosis, or prognosis. Non-limiting uses include therapeutic and diagnostic applications. Some exemplary targets are provided herein by way of example and are intended to be illustrative and not limiting. It is well known in the art that viruses acquire mutations, and exemplary mutations described for targets herein are provided herein by way of example and are intended to be illustrative and not limiting. Targets of interest can be natural or synthetic. In some embodiments, the target comprises the receptor binding domain (RBD) of the CoV-2 viral spike protein (SARS-CoV-2S). In some embodiments, the target comprises the S1 protein of the CoV-2 virus. In some embodiments, the target comprises the spike protein of the CoV-2 virus. In some embodiments, the target comprises a trimeric RBD construct, an S1 protein, or a spike protein of the CoV-2 virus. In some embodiments, the target comprises a CoV-2 viral particle. In a preferred embodiment, the target of interest is a trimeric protein. Particularly preferred trimeric proteins include any of the chimeric polypeptides mentioned in WO 2018 / 176103, the contents of which are incorporated herein by reference in their entirety. In a particularly preferred embodiment, the trimeric protein is a trimeric vaccine protein. As used herein, the term "trimeric vaccine protein" generally refers to a trimeric protein that may provide vaccine utility based on the nature / origin of the polypeptides / proteins contained therein and, consequently, its antigenic potential. Exemplary trimeric vaccine proteins include, in particular, trimeric proteins derived from trimeric viral surface proteins, such as fusion proteins or portions thereof (e.g., extracellular domains or antigenically active portions thereof) of Class I or Class III enveloped viruses.Particularly preferred trimeric vaccine proteins are described in WO 2018 / 176103, the contents of which are incorporated herein by reference in their entirety. In a further preferred embodiment, the target of interest (e.g., a trimeric protein, preferably a trimeric vaccine protein) comprises a trimerization domain. As used herein, the term "trimerization domain" generally refers to a domain that mediates the formation of trimers from three monomeric proteins / polypeptides or portions thereof. In a more specific embodiment, the term "trimerization domain" refers to an amino acid sequence within a polypeptide that promotes self-assembly by associating with two other trimerization domains to form trimers. Various trimerization domains are known in the art, and some particularly preferred representative examples thereof are also mentioned herein above. In a particularly preferred embodiment, the trimerization domain is a polypeptide comprising a "structure-stabilizing moiety" (also known as a molecular clamp) as described in WO 2018 / 176103 (the contents of which are incorporated herein by reference in their entirety), and preferably the trimerization domain is fused to the C-terminus of a heterologous polypeptide (e.g., the extracellular domain (or antigenic portion thereof) of a fusion protein derived from an enveloped virus, such as an enveloped class I or III virus), thereby mediating trimerization of the latter.

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

[0043] Suitable linkers for operably linking a ligand and an additional component of a ligand fusion protein in a single amino acid sequence include, but are not limited to, (poly)peptide linkers such as glycine linkers, serine linkers, mixed glycine / serine linkers, glycine and serine rich linkers, or linkers composed of mostly polar polypeptide fragments.

[0044] In some embodiments, the linker is comprised predominantly of amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In some embodiments, the linker is comprised predominantly of amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In some embodiments, the ligand linker is comprised predominantly of amino acids that are sterically unhindered. In some embodiments, the linker is comprised predominantly of amino acids selected from glycine, serine, and / or alanine. In some embodiments, the peptide linker is selected from polyglycine (e.g., (Gly)5 or (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 ligand (to another ligand, or to a solid support or a molecule bound to a solid support) in a manner that allows the ligand to bind to the target of interest. In some embodiments, the linker is about 1-50 amino acids, about 1-20 amino acids, about 1-15 amino acids, about 1-10 amino acids, about 1-5 amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids. It will be apparent that the length, degree of flexibility, and / or other properties of the linker(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 one or more 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-(CH) s -C(O)- (where s = 2 to 20) can be used. Any such linker can be further substituted with any non-sterically hindering group, such as lower alkyl (e.g., C1-C6 alkyl), lower acyl (e.g., -CO-(C1-C5 alkyl)), halogen (e.g., Cl, Br, F, or I), CN, NH2, phenyl, etc. An exemplary non-peptide linker is a PEG linker. In some embodiments, the PEG linker has a molecular weight of about 100 to 5000 Da, or about 100 to 500 Da. In some embodiments, the PEG linker has a molecular weight of about 100 to 500 Da.

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

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

[0049] In some embodiments, the ligand (e.g., a ligand fusion protein) contains at least one reactive residue. Reactive residues are useful, for example, as attachment sites for conjugates such as chemotherapeutic agents. An exemplary reactive amino acid residue is lysine. A reactive residue (e.g., lysine) can be added to the ligand at either terminus or within the ligand sequence and / or substituted for another amino acid in the ligand sequence. A suitable reactive residue (e.g., lysine, serine, tyrosine, hydroxytryptophan, etc.) can also be located within the sequence of a specified ligand without the need for addition or substitution. In some embodiments, the reactive amino acid residue is cysteine. In some embodiments, the reactive amino acid residue is lysine. In some embodiments, the reactive amino acid residue is serine. In some embodiments, the reactive amino acid residue is tyrosine. In some embodiments, the reactive amino acid residue is hydroxytryptophan.

[0050] Mounting to a solid surface The terms "solid surface," "support," "solid support," or "matrix" are used interchangeably herein and refer, without limitation, to any column (or column material), resin, bead (e.g., agarose beads or Sepharose™ beads), test tube, microtiter dish, solid particle (e.g., agarose or Sepharose™), microchip (e.g., silicon, silicon-glass, or gold chip), or membrane (e.g., filter) or biological (e.g., liposome or vesicle) origin to which a ligand, affinity agent, antibody, or other protein can be attached (bound, conjugated, linked, or attached) directly or indirectly (e.g., via an antibody or other binding partner intermediate such as Protein A or G), or to which a ligand or antibody can be embedded (e.g., via a receptor or channel). Reagents and techniques for attaching polypeptides to solid supports (e.g., matrices, resins, plastics, etc.) are well known in the art. Suitable solid supports include, but are not limited to, chromatography resins or matrices (e.g., agarose or Sepharose® (such as Sepharose 4 Fast Flow) beads), the walls or floors of wells in plastic microtiter dishes, silica-based biochips, polyacrylamide, agarose, silica, nitrocellulose, paper, plastic, nylon, metal, and combinations thereof. Ligands and other compositions can be attached to the support material by non-covalent association or by covalent attachment using reagents and techniques known in the art. In an exemplary preferred embodiment of non-covalent association of a ligand to a solid support, the ligand comprises one member (i.e., the first member) of a binding pair (e.g., an affinity tag) and the solid support comprises the corresponding other member (i.e., the second member) of the binding pair (e.g., an affinity matrix specific for binding of the affinity tag).For example, in a preferred aspect of the latter embodiment, the ligand comprises a polyhistidine tag (e.g., a hexahistidine tag (6xHis tag or His6 tag)) and the solid support comprises a chelating agent, preferably nitrilotriacetic acid (NTA) agarose resin or a derivative thereof. In a further preferred embodiment, the ligand comprises, or additionally comprises, a biotinylated tag (e.g., AviTag™) and the solid support comprises avidin (and / or streptavidin and / or neutravidin). In other embodiments, the ligand is attached to the solid support by an antibody that specifically binds to the ligand. In the latter embodiment, the antibody may be attached to the solid support by protein A and / or protein G contained within (or itself attached to or bound to) the solid support. Numerous additional suitable binding pairs are well known in the art, each of which may be used for the purposes disclosed herein. In some embodiments, the ligand is attached to a solid surface or solid support (eg, beads or resins, eg, chromatographic materials made from agarose or Sepharose™) via a linker.

[0051] Ligand generation Ligands useful in practicing some embodiments of the provided methods and uses can be produced using a variety of standard techniques for chemical synthesis, semi-synthesis, and recombinant DNA and protein expression and purification methodologies known in the art. Also provided are methods for producing ligands as soluble drugs and cell-associated proteins, either individually or as part of a multidomain fusion protein. In some embodiments, the overall ligand production scheme involves obtaining a reference protein scaffold and identifying multiple residues within the scaffold for modification. Depending on the embodiment, the reference scaffold may include one or more alpha-helical regions or protein structures with other tertiary structure. Once identified, any of the multiple residues may be modified, for example, by substitution of one or more amino acids. In some embodiments, one or more conservative substitutions are made. In some embodiments, one or more non-conservative substitutions are made. In some embodiments, a natural amino acid (e.g., one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine) is substituted into the reference scaffold at the target position for modification. In some embodiments, the modification does not include substitution with either cysteine ​​or proline. After modifications are made at the identified positions desired in certain embodiments, the resulting modified polypeptides (e.g., candidate ligands) can be recombinantly expressed (e.g., to increase the number of each modified polypeptide) in, for example, a plasmid, bacteria, phage, or other vector. The modified polypeptides can then be purified and screened to identify modified polypeptides 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 proteins).In some embodiments, depending on the target of interest, the canonical scaffold may exhibit some degree of interaction (e.g., non-specific interaction) with the target of interest, while certain modified polypeptides exhibit at least about a 2-fold, at least about a 5-fold, at least about a 10-fold, at least about a 20-fold, at least about a 50-fold, or at least about a 100-fold (or more) increase in binding specificity for the target of interest. Further details regarding the 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 ligand fusion proteins include, but are not limited to, polymerase chain reaction (PCR)-based synthesis, concatemerization, seamless cloning, and recursive directional ligation (RDL) (see, e.g., Meyer et al., Biomacromolecules 3:357-367 (2002), Kurihara et al., Biotechnol. Lett. 27:665-670 (2005), Haider et al., Mol. Pharm. 2:139-150 (2005); and McMillan et al., Macromolecules 32(11):3643-3646 (1999)).

[0053] Nucleic acids comprising polynucleotide sequences encoding ligands are also provided. Such polynucleotides may optionally comprise one or more expression control elements. For example, the polynucleotide may comprise one or more promoters or transcription enhancers, ribosome binding sites, transcription termination signals, and polyadenylation signals as expression control elements. The polynucleotide may be inserted into any suitable vector, which may 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 promoters useful for regulating expression of the desired nucleic acid sequence. Exemplary promoters useful for expression in E. coli include, for example, the T7 promoter.

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

[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 can include plasmid vectors, single-stranded phage vectors, double-stranded phage vectors, single-stranded RNA or DNA viral vectors, or double-stranded RNA or DNA viral vectors. Phage and viral vectors can also be introduced into host cells in the form of packaged or enclosed viruses using known infection and transduction techniques. Furthermore, viral vectors can be replication-competent or replication-incompetent. Alternatively, cell-free translation systems can be used to produce ligands 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 recombinant host cell is a bacterial cell, a yeast cell, or a mammalian cell. A variety of host-expression vector systems can be used to express the coding sequence of the ligand fusion protein. Mammalian cells can be used as host cell systems transfected with recombinant plasmid DNA or cosmid DNA expression vectors containing the coding sequence of the target of interest and the coding sequence of the fusion polypeptide. The cells can 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 a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vector containing the ligand coding sequence, yeast (e.g., Saccharomyces, Pichia) transformed with a recombinant yeast expression vector containing the ligand coding sequence, insect cell lines infected with a recombinant viral expression vector (e.g., baculovirus) containing the ligand coding sequence, and plant cell lines infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) containing the ligand coding sequence or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid).

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

[0060] In some embodiments, a eukaryotic host cell system is used. In some embodiments, the eukaryotic host cell system is a yeast cell transformed with a recombinant yeast expression vector containing the coding sequence of the ligand. Exemplary yeast that can be used to produce the compositions of the present invention include yeast from the genera Saccharomyces, Pichia, Actinomycetes, and Kluyveromyces. Yeast vectors typically contain an origin of replication sequence from the 2mu yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, a sequence for polyadenylation, a sequence for transcription termination, and a selectable marker gene. Examples of promoter sequences in yeast expression constructs include those encoding 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 of the present invention. Such host cell systems include, for example, insect cell systems infected with a recombinant viral expression vector (e.g., baculovirus) containing the ligand coding sequence, plant cell systems infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) containing the ligand coding sequence, or plant cell systems transformed with a recombinant plasmid expression vector (e.g., Ti plasmid), including, but not limited to, the expression systems described in U.S. Patent No. 6,815,184, U.S. Patent Application Publication Nos. 60 / 365,769 and 60 / 368,047, and International Publication Nos. 2004 / 057002, 2004 / 024927, and WO2003 / 078614.

[0062] In some embodiments, a host cell system may be used. In some embodiments, the host cell system is an animal cell system infected with a recombinant viral expression vector (e.g., adenovirus, retrovirus, adeno-associated virus, herpesvirus, lentivirus). In some embodiments, the host cell system is a cell line engineered to contain multiple copies of the DNA encoding the ligand, stably amplified (CHO / dhfr) or unstably amplified in double minute chromosomes (e.g., mouse cell lines). In some embodiments, the vector containing the polynucleotide encoding the ligand is polycistronic. Exemplary mammalian cells useful for producing these compositions include HEK293 cells (e.g., 293T and 293F), CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 (Crucell, The Netherlands) cells VERY, HeLa cells, COS cells, MDCK cells, 3T3 cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT20 cells, T47D cells, CRL7O30 cells, HsS78Bst cells, hybridoma cells, and other mammalian cells. Additional exemplary mammalian host cells useful in the practice of the present invention include, but are not limited to, T cells. Exemplary expression systems and selection methods are known in the art and may 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). Additional examples of expression systems and selection methods are described in Logan et al., PNAS 81:355-359 (1984), Birtner et al. Methods Enzymol. 153:51-544 (1987). The transcriptional and translational regulatory sequences of mammalian host cell expression vectors are often derived from viral genomes.The 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 acid into host cells (e.g., mammalian host cells) include, but are not limited to, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation.Methods for producing cells containing vectors and / or foreign nucleic acids are well known in the art.See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0064] The biological method for introducing target polynucleotide into host cell includes the use of DNA vector and RNA vector.Virus vector, especially retrovirus vector, has become the most widely used method for inserting gene into mammalian cell (e.g., human cell).Other virus vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus and adeno-associated virus.See, for example, U.S. Patent No. 5,350,674 and 5,585,362.

[0065] The method of introducing target DNA and RNA polynucleotide into host cell includes, but is not limited to, electroporation of cell, in which an electric field is applied to cell to increase the permeability of cell membrane, allowing chemical substance, drug or polynucleotide to be introduced into cell.The ligand containing DNA or RNA construct can be introduced into mammalian or prokaryotic cell by using electroporation.

[0066] In some embodiments, electroporation of cells results in the expression of ligand-CAR on the surface of T cells, NK cells, NKT cells. Such expression can be transient or stable over the life of the cell. Electroporation can be achieved using methods known in the art, such as MaxCyte GT® and STX® transfection system (MaxCyte, Gaithersburg, Maryland, USA).

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

[0068] Suitable lipids for use are commercially available. For example, dimyristoyl phosphatidylcholine (DMPC) is available from Sigma (St. Louis, MO), dicetyl phosphate (DCP) is available from K&K Laboratories (Plainview, NY), cholesterol (Choi) is available from Calbiochem-Behring, and dimyristoyl phosphatidylglycerol (DMPG) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform evaporates more readily than methanol and can therefore be used as the sole solvent. "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Phospholipids spontaneously form when suspended in excess aqueous solution. The lipid components rearrange themselves before forming a closed structure, incorporating 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 vesicular structure are also encompassed. For example, lipids can be in 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 foreign 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, including, for example, "molecular biological" assays well known in the art (such as Southern blotting, Northern blotting, RT-PCR, and PCR), and "biochemical" assays (e.g., assays detecting the presence or absence of specific peptides by immunological techniques (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the present invention).

[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 (e.g., 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 known in the art and can be prepared using known techniques or commercially available. Generally, the construct with the minimal 5'-flanking region that maximizes reporter gene expression is identified as the promoter. Such promoter regions are routinely linked to reporter genes and can be used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0071] Several 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 genes (Lowy et al., Cell 22:817 (1980)). In addition, 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 the purification of recombinant proteins, for example, by chromatography (e.g., ion exchange, affinity, and size exclusion chromatography (SEC)), centrifugation, differential solubility, or any other standard technique for purifying proteins. In some embodiments, the ligand is optionally fused to a heterologous peptide or polypeptide sequence specifically disclosed herein or otherwise known in the art (e.g., a His tag (e.g., a 6xHis tag) or a biotinylation tag (e.g., an Avi tag) to facilitate purification). In some embodiments, ligands (e.g., antibodies and other affinity matrices) for ligand affinity columns in affinity purification are removed from the composition prior to final preparation of the ligand using techniques known in the art. In some embodiments, the ligands or other components of the 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 carried out using organic chemical synthesis of the desired polypeptide using a variety of liquid and solid phase chemical processes known in the art. A variety of automated synthesizers are commercially available and can be used in accordance with known protocols. For example, Tam et al., J. Am. Chem. Soc., 105:6442 (1983); Merrifield, Science, 232:341-347 (1986); Barany and Merrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, New York, 1- 284; Barany et al., Int. J. Pep. Protein Res., 30:705 739 (1987); Kelley et al. in Genetic Engineering Principles and Methods, Setlow, JK, ed. Plenum Press, NY. 1990, vol. 12, pp. 1-19; Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, 1989. One advantage of these methodologies is that they allow the incorporation of unnatural amino acid residues into the 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, iodation, methylation, myristoylation, oxidation, prenylation, racemization, selenoylation, sulfation, ubiquitination, and the like. (See, e.g., Creighton, Proteins: Structures and Molecular Properties, 2nd Ed. (WH Freeman and Co., NY, 1992); Posttranslational Covalent Modification of Proteins, Johnson, ed. (Academic Press, New York, 1983), pp. 1-12; Seifter, Meth. Enzymol., 182:626-646 (1990); Rattan, Ann. NY Acad. Sci., 663:48-62 (1992).) In some embodiments, the peptides are acetylated at the N-terminus and / or amidated at the C-terminus.

[0075] Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, acetylation, formylation, etc. In addition, the derivatives may contain one or more non-classical amino acids.

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

[0077] Affinity Agents for Purification In affinity chromatography-based purification, a target of interest (preferably a trimeric protein or trimeric vaccine molecule) is selectively isolated according to its ability to specifically and reversibly bind to a ligand, which can be covalently attached to a chromatography matrix. In some embodiments, the ligand can be used as a reagent to affinity purify the target of interest from either recombinant sources or natural sources such as biological samples (e.g., serum, cells).

[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 a surface are known to those skilled in the art. Peptide tags that can be used to attach ligands to solid surfaces are known to those skilled in the art. Furthermore, ligands can be attached (i.e., bound, conjugated, linked, 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, a series of ligands can be used to create an array on a solid surface using techniques known in the art. For example, U.S. Patent Application Publication No. 2004 / 0009530 discloses a method for preparing an array.

[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 be loaded or contacted with a solution (e.g., a sample containing the target of interest) under conditions favorable for forming a complex between the ligand and the target of interest. Unbound materials may be washed away. Suitable washing conditions can be easily determined by one of skill in the art. Examples of suitable washing conditions are disclosed 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, followed by isolating the complex of the target of interest and the ligand. For example, the ligand is immobilized on a solid support such as beads and then separated from the solution along with the target of interest by filtration. In some embodiments, the ligand is a fusion protein (i.e., a ligand fusion protein) containing a peptide tag such as a poly-HIS tail or a streptavidin-binding region (e.g., a biotinylated tag such as an Avi tag), which can be used to isolate the ligand after the complex is formed using an immobilized metal affinity chromatography resin or a streptavidin-coated substrate. Once separated, the target of interest can be released from the ligand under elution conditions and recovered in a purified form.

[0082] In some embodiments, the ligand containing the initiator N-terminal methionine is isolated because it is a protein sequence encoded by DNA. In some embodiments, the ligand is isolated without an N-terminal methionine residue. In some embodiments, a mixture is obtained in which only a portion of the purified ligand contains an N-terminal methionine. It will be understood by those skilled in the art that the presence or absence of an N-terminal methionine does not affect the suitability of the ligand for the purposes disclosed herein. [Example]

[0083] Example 1 Recombinant protein ligands were expressed in E. coli and / or Pichia pastoris using standard techniques. Ligands were purified using multi-column chromatography. For his-tagged ligands, IMAC was used as the primary capture step. Biotinylated ligands were generated using the Avitag™ system (Avidity, Aurora, CO). Non-biotinylated ligands with the Avitag™ sequence were prepared by excluding exogenous biotin. The purity and identity of the recombinant protein ligands were assessed by a combination of SDS-PAGE, RP UPLC, quadrupole time-of-flight mass spectrometry, and SEC. In many cases, ligands are isolated without the N-terminal methionine residue, which is thought to be cleaved during expression. Often, mixtures are obtained in which only a portion of the ligands contain the N-terminal methionine. It will be clear to those skilled in the art that the presence or absence of the N-terminal methionine does not affect the conclusions herein. For clarity, the N-terminal methionine is included.

[0084] Example 2 This example demonstrates the binding of a biotinylated ligand to a target protein using Biolayer Interferometry (ForteBio, Menlo Park, CA). The biotinylated ligand was immobilized on a sensor and incubated with a solution containing a trimeric vaccine protein at various concentrations. An example sensorgram is shown in Figure 2.

[0085] Example 3 This example demonstrates the stability of affinity ligands to sodium hydroxide (NaOH). Ligands were incubated in 0.1 M NaOH for a given time and then neutralized. Binding of the NaOH-treated ligands was measured as described in Example 2 and compared to untreated ligands. Retained binding was calculated according to the following formula: % Retained Binding = (Measured Response After NaOH Treatment) ÷ (Untreated Measured Response) × 100 An example of stability is shown in Figure 3.

[0086] Example 4 This example demonstrates the production and characterization of affinity agents containing the ligands identified and described herein. Affinity resins were prepared by coupling the ligands to activated agarose beads. After washing, the ligands were coupled to the beads at room temperature. Target ligand densities ranged from about 2 g / L to about 20 g / L. After washing, the beads were deactivated with excess thioglycerol. The actual ligand densities of all resins were determined using a subtractive RP-HPLC method according to the following formula: Actual Ligand Density = (Measured in Feed [Ligand] - Measured in Effluent [Ligand]).

[0087] Example 5 This example demonstrates the stability of a test resin after clean-in-place (CIP) with 0.1 M sodium hydroxide (NaOH). The resin was prepared from a ligand corresponding to SEQ ID NO: 7. The binding capacity of the resin before and after incubation in 0.1 M NaOH was measured in a binding capacity assay, and the results are shown in Figure 4.

[0088] Example 6 This example demonstrates the use of affinity agents containing binding ligands as described herein for the affinity purification of trimeric proteins. Clarified cell culture feedstream (CCCF) obtained from the production of trimeric vaccine proteins was applied to a 30 mm internal diameter (ID) x 100 mm column packed with resin prepared from the ligand corresponding to SEQ ID NO: 7. The chromatographic method is shown in the table below, and the resulting chromatogram is shown in Figure 5.

[0089] [Table 1] The purity of the eluted material was demonstrated using a Coomassie blue stained SDS-PAGE gel and is shown in FIG.

[0090] The above examples demonstrate that affinity resins can be fine-tuned to achieve different performance characteristics that different applications and users may require.

[0091] It is contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments disclosed above may be made and still be included within the present invention. Furthermore, any specific feature, aspect, method, property, characteristic, quality, attribute, element, etc. disclosed herein in connection with an embodiment may be used in all other embodiments described herein. Accordingly, it should be understood that various features and aspects of the disclosed embodiments may be combined with or substituted for one another. Thus, it is intended that the scope of the invention described herein should not be limited by the specific disclosed embodiments above. Moreover, while the invention is susceptible to various modifications and alternative forms, specific examples thereof have been shown in the drawings and are described in detail herein. However, the invention is not limited to the specific forms or methods disclosed; on the contrary, it should be understood that the invention encompasses all modifications, equivalents, and alternatives falling within the spirit and scope of the various embodiments described.

[0092] Any methods disclosed herein do not have to be performed in the order listed. The methods disclosed herein include specific acts to be performed by a practitioner, but may also include any third-party direction of those acts, either express or implied.

[0093] Table 6 Sequence [Table 2-1] [Table 2-2]

[0094] [Item 1] 1. An affinity agent comprising a ligand that binds to a trimeric protein, said ligand comprising at least one polypeptide, said polypeptide comprising: (i) the amino acid sequence defined by SEQ ID NO: 1, and / or (ii) An affinity agent that comprises or consists of an amino acid sequence that differs from the amino acid sequence defined by SEQ ID NO: 1 by no more than three, no more than two, or no more than one amino acid substitution, addition, or deletion. [Item 2] the at least one polypeptide (i) an amino acid sequence defined by any one of SEQ ID NOs: 2 to 10, and / or (ii) The affinity agent according to item 1, which comprises or consists of an amino acid sequence that differs from the amino acid sequence defined by any one of SEQ ID NOs: 2 to 10 by not more than three, not more than two, or not more than one amino acid substitution, addition, or deletion. [Item 3] The ligand is 3. The affinity agent according to item 1 or 2, comprising a multimeric polypeptide comprising at least two subunits, each subunit comprising a polypeptide as defined in item 1 or 2. [Item 4] 4. The affinity agent according to item 3, wherein the polypeptides contained in the subunits are not identical in amino acid sequence. [Item 5] 5. The affinity agent according to any one of items 1 to 4, wherein the trimeric protein is a trimeric vaccine protein. [Item 6] 6. The affinity agent according to any one of items 1 to 5, wherein the trimeric protein or the trimeric vaccine protein comprises a trimerization domain. [Item 7] 7. The affinity agent according to any one of items 1 to 6, wherein the ligand is attached to a solid surface. [Item 8] 8. The affinity agent according to item 7, wherein the solid surface comprises or consists of a resin or beads. [Item 9] 8. The affinity agent according to item 7, wherein the solid surface comprises or consists of a membrane. [Item 10] 8. The affinity agent according to item 7, wherein the solid surface comprises or consists of a monolith. [Item 11] 11. The affinity agent according to any one of items 7 to 10, wherein the ligand is bound to the solid surface by a covalent or non-covalent bond. [Item 12] 11. The affinity agent according to any one of items 7 to 10, wherein the ligand is covalently bound to the solid surface via a linker. [Item 13] 13. Use of the affinity agent according to any one of items 1 to 12 for purifying one or more trimeric proteins from a sample containing the one or more trimeric proteins. [Item 14] 14. The use according to item 13, wherein the trimeric protein is a trimeric vaccine protein. [Item 15] 10. A method of making an affinity agent, the method comprising attaching a ligand as defined in any one of items 1 to 6 to a solid surface. [Item 16] 16. The method of claim 15, wherein the ligand is attached to the solid surface via a linker.

Claims

1. 1. An affinity agent comprising a ligand that binds to a trimeric protein, said ligand comprising at least one polypeptide, said polypeptide comprising: (i) the amino acid sequence defined by SEQ ID NO: 1, and / or (ii) An affinity agent that comprises or consists of an amino acid sequence that differs from the amino acid sequence defined by SEQ ID NO: 1 by no more than three, no more than two, or no more than one amino acid substitution, addition, or deletion.

2. the at least one polypeptide (i) an amino acid sequence defined by any one of SEQ ID NOs: 2 to 10, and / or (ii) The affinity agent of claim 1, which comprises or consists of an amino acid sequence that differs from the amino acid sequence defined by any one of SEQ ID NOs: 2-10 by no more than three, no more than two, or no more than one amino acid substitution, addition, or deletion.

3. The ligand is 3. An affinity agent according to claim 1 or claim 2, comprising a multimeric polypeptide comprising at least two subunits, each subunit comprising a polypeptide as defined in claim 1 or claim 2.

4. The affinity agent of claim 3 , wherein the polypeptides contained in the subunits are not identical in amino acid sequence.

5. The affinity agent according to any one of claims 1 to 4, wherein the trimeric protein is a trimeric vaccine protein.

6. The affinity agent according to any one of claims 1 to 5, wherein the trimeric protein or the trimeric vaccine protein comprises a trimerization domain.

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

8. 8. The affinity agent of claim 7, wherein the solid surface comprises or consists of a resin or beads.

9. 8. The affinity agent of claim 7, wherein the solid surface comprises or consists of a membrane.

10. 8. The affinity agent of claim 7, wherein the solid surface comprises or consists of a monolith.

11. The affinity agent according to any one of claims 7 to 10, wherein the ligand is bound to the solid surface by a covalent or non-covalent bond.

12. The affinity agent of any one of claims 7 to 10, wherein the ligand is covalently attached to the solid surface via a linker.

13. Use of the affinity agent according to any one of claims 1 to 12 for purifying one or more trimeric proteins from a sample containing said one or more trimeric proteins.

14. The use according to claim 13, wherein the trimeric protein is a trimeric vaccine protein.

15. A method of making an affinity agent, comprising attaching a ligand as defined in any one of claims 1 to 6 to a solid surface.

16. The method of claim 15 , wherein the ligand is attached to the solid surface via a linker.

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