Affinity agent

Abstract

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

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 405,301, filed on September 9, 2022, the entire disclosure of which is incorporated herein by reference. Background of the Invention

[0003] The purity of bioproduced therapeutic agents is subject to strict scrutiny and regulation by authorities to ensure safety and efficacy. Accordingly, there remains a need for means to efficiently purify bioproduced therapeutic agents to high purity and in sufficient quantities. Summary of the Invention

[0004] To support the clinical efforts of therapeutic proteins, there is a need for compositions and methods for efficiently purifying proteins from recombinant sources. Affinity purification is a means of separating and / or obtaining the desired protein purity in several steps or a single step. However, the development of affinity agents (e.g., affinity agents comprising an affinity ligand) can be a resource - intensive and time - consuming task. This has led to the development of affinity agents for only a limited number of proteins. In the absence of an affinity agent, purification typically involves inefficient, labor - intensive, and expensive processes (e.g., multi - column processes).

[0005] Exemplary therapeutic proteins include, but are not limited to, bioactive polypeptides / proteins, fusion proteins, enzymes, hormones, antibodies, antibody fragments, and recombinant vaccines. Certain proteins, such as fusion proteins, present additional challenges to purification due to product heterogeneity or the presence of product - related impurities. Some impurities may be generated 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.

[0006] Described herein are affinity agents that bind proteins and can be used for separation and / or affinity purification. In some embodiments, the affinity agent comprises a ligand and a solid support.

[0007] The COVID-19 pandemic has highlighted the benefits of using platform production processes for the rapid development of therapeutics and vaccines. For example, monoclonal antibody therapies were developed and received emergency authorization within an unprecedented time frame because they were able to utilize platform production processes, mainly the Protein A affinity purification platform. In the case of mRNA vaccines, the purification of the mRNA moiety by using existing affinity purification techniques (e.g., Oligo-dT and cellulose chromatography) also helped to accelerate the development. Thus, it is clear that future pandemic preparedness and the facilitation of current global programs for vaccine production would benefit from an expansion of affinity purification tools that provide platform processes, i.e., are applicable to multiple vaccines. This article describes affinity agents that incorporate a trimerization domain and can be used for separation and / or affinity purification. In some embodiments, the affinity agent comprises a ligand and a solid support.

[0008] Some subunit vaccines are based on viral proteins that form trimeric structures. To facilitate the efficient production of the correctly folded trimeric form, a trimerization domain can be fused to the viral protein. The most commonly used trimerization domains are the trimerization domain of bacteriophage T4 fibritin (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 (referred to as the "molecular clamp") has been developed and has been shown to confer conformational stability to vaccine constructs based on envelope virus fusion proteins (see, for example, WO 2018 / 176103, which is incorporated herein by reference in its entirety). Different viral proteins or viral protein variants can be fused to the same trimerization domain to prepare different vaccines. In addition, the trimerization domain can also be used, for example, to trimerize other (i.e., non-vaccine) polypeptides / proteins (especially therapeutic proteins), but is not intended to be limited to any of those mentioned in paragraphs

[0395] -

[0400] of WO 2018 / 176103. An affinity agent that binds to the trimerization domain enables the easy purification of the relevant subunit vaccine independent of the antigenic part and thus provides a platform technology for vaccine production and / or purification.

[0009] In some embodiments, the affinity agent (or the ligand contained in the affinity agent) comprises a three-helix bundle protein (or referred to as a "triple-helix bundle protein"), preferably an antiparallel three-helix bundle protein. In some embodiments, the structure of the three-helix bundle protein can be envisioned as a triangular prism, where each triangular vertex represents a helix, for example, as Figure 1 shown. In some embodiments, any two combinations of the helices define 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:

[0010] 1) Helix 1 and 2 ( Figure 1 faces 1, 2 in

[0011] 2) helices 2 and 3 ( Figure 1 in planes 2, 3);

[0012] 3) helices 1 and 3 ( Figure 1 in planes 1, 3); and

[0013] their combinations.

[0014] In some embodiments, the affinity agent (or the ligand contained in the affinity agent) comprises a plane formed by helices 2 and 3 of a 3 - helix bundle protein. In some embodiments, the main function of helix 1 of the 3 - helix bundle protein is to complete and stabilize the 3 - helix bundle. In some embodiments, helix 1 can be modified to maintain the structure of the 3 - helix bundle protein.

[0015] In some embodiments, provided herein is an affinity agent comprising SEQ ID NO:1 - EQRRNFIENLRWDPSKSARLLARAKRFNDW.

[0016] In some embodiments, the affinity agent provided herein comprising SEQ ID NO:1 is within helices 2 and 3 of a 3 - helix bundle protein.

[0017] In some embodiments, provided herein is an affinity agent comprising SEQ ID NO:2 - VDAKFDKELEEARAEIERLPNLTEEQRRNFIENLRWDPSKSARLLARAKRFNDWQAPK.

[0018] In some embodiments, provided herein is an affinity agent comprising a multimeric polypeptide, the multimeric polypeptide comprising at least two subunits, each subunit being a polypeptide according to the foregoing embodiments.

[0019] In some embodiments, provided herein is an affinity agent comprising a multimeric polypeptide, wherein the subunits are not all the same.

[0020] In some embodiments, provided herein are affinity agents comprising SEQ ID NOs: 3 - 10.

[0021] In some embodiments, provided herein are affinity agents that bind to a trimerization domain.

[0022] In some embodiments, provided herein are affinity agents for purifying a protein containing a trimerization domain.

[0023] In some embodiments, provided herein are affinity agents for purifying a vaccine containing a trimerization domain.

[0024] Definition

[0025] To facilitate a better understanding of the present disclosure, 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.

[0026] About or approximately: As used herein, the term "about" or "approximately" when applied to one or more values of interest refers to a value similar to the stated reference value. In certain embodiments, the term "about" or "approximately" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the stated reference value with increasing preference, unless otherwise stated or otherwise apparent from the context (except in cases where such numbers would exceed 100% of a possible value).

[0027] Bioactivity: As used herein, the term "bioactivity" refers to the characteristic of any agent that is active in a biological system, particularly in a living organism. For example, an agent that has a biological effect on a living organism when administered to the organism is considered to be bioactive.

[0028] Conservative and non-conservative substitutions: A "conservative" amino acid substitution is a substitution 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 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., 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), β-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)); and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)). For example, substituting tyrosine with phenylalanine is a conservative substitution. In some embodiments, conservative amino acid substitutions in the ligand sequence confer or improve the specific binding of the ligand to a target of interest. In some embodiments, conservative amino acid substitutions in the ligand sequence do not reduce or eliminate the binding of the ligand to a target of interest. In some embodiments, conservative amino acid substitutions do not significantly affect the specific binding of the ligand to a target of interest. Methods for identifying nucleotide and amino acid conservative and non-conservative substitutions that confer, alter, or maintain selective binding affinity are known in the art (see, e.g., Brummell, Biochem. 32:1180-1187 (1993); Kobayashi, Protein Eng. 12(10):879-884 (1999); and Burks, PNAS 94:412-417 (1997)). In some embodiments, non-conservative amino acid substitutions in the ligand sequence confer or improve the specific binding of the ligand to a target of interest. In some embodiments, non-conservative amino acid substitutions in the ligand sequence do not reduce or eliminate the binding of the ligand to a target of interest. In some embodiments, non-conservative amino acid substitutions do not significantly affect the specific binding of the ligand to a target of interest.

[0029] Linker: As used herein, "linker" refers to a peptide or other chemical linkage that serves to connect originally independent functional domains, entities, or portions. In some embodiments, the linker is located between a ligand and another polypeptide component containing an originally independent functional domain. In some embodiments, the linker is a peptide or other chemical linkage between a ligand and a surface (such as a solid surface or solid support). In particularly preferred embodiments, the ligand is chemically conjugated (i.e., covalently bound) to a solid surface or solid support by forming a bond between the thiol group of a cysteine in the ligand (preferably, an N- or C-terminal cysteine, more preferably a C-terminal cysteine) and the solid support. For example, the ligand can be covalently bound to a solid support by a bond formed by a nucleophilic addition of the thiol group of a cysteine in the ligand (e.g., a C-terminal cysteine) to a maleimide group on the solid surface or solid support. Other methods for covalently or non-covalently linking a ligand (e.g., a ligand comprising a polypeptide) to a solid surface or solid support are well known and routinely employed in the art. For example, any of the methods and linkages described in Greg T. Hermanson, "Bioconjugate Techniques" 3rd Edition (2013) (which is incorporated herein by reference in its entirety) can be used to covalently bind a ligand to a solid support.

[0030] Naturally occurring: When used in connection with biological materials such as nucleic acid molecules, polypeptides, and host cells, the term "naturally occurring" refers to those that exist in nature and have not been modified by humans. In contrast, when used in connection with biological materials, "non-natural" or "synthetic" refers to those that do not exist in nature and / or have been modified by humans.

[0031] "Unnatural amino acids", "amino acid analogs", and "non-standard amino acid residues" are used interchangeably herein. Unnatural amino acids that can be substituted in ligands as provided herein are known in the art. In some embodiments, the unnatural amino acid is 4-hydroxyproline, which can substitute for proline; 5-hydroxylysine, which can substitute for lysine; 3-methylhistidine, which can substitute for histidine; homoserine, which can substitute for serine; and ornithine, which can substitute for lysine. Additional examples of unnatural amino acids that can be substituted in polypeptide ligands include, but are not limited to, molecules such as: D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, A-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, lanthionine, dehydroalanine, γ-aminobutyric acid, selenocysteine, and pyrrolysine fluorinated amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids.

[0032] "Polynucleotide" and "nucleic acid molecule": As used interchangeably herein, polynucleotide and nucleic acid molecule refer to polymeric forms of nucleotides of any length, which contain ribonucleotides or deoxyribonucleotides or both or are composed of them. These terms include, but are not limited to, DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and / or tRNA.

[0033] Operably linked: As used herein, the term "operably linked" means that two molecules are attached so that each retains its functional activity. Whether attached directly or indirectly, two molecules are "operably linked".

[0034] 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 for the resulting fusion. Peptide tags are generally relatively short compared to the proteins 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 a peptide tag. Many peptide tags having the uses provided herein are known in the art. Examples of peptide tags that can be components of ligand fusion proteins or targets (e.g., ligand fusion proteins) that bind ligands include, but are not limited to, HA (hemagglutinin), c-myc, herpes simplex virus glycoprotein D (gD), T7, GST, GFP, MBP, Strep tag, His tag, Myc tag, TAP tag, and FLAG tag (Eastman Kodak, Rochester, N.Y.). Also, antibodies against the tag epitopes allow detection and localization of fusion proteins in, for example, affinity purification, Western blotting, ELISA assays, and cellular immunostaining.

[0035] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids joined together via peptide bonds. The term is used to refer to amino acid chains of any length, but one of ordinary skill in the art will understand that the term is not limited to long chains and can refer to the smallest chain containing two amino acids joined together via peptide bonds. Thus, for the purposes of this disclosure, it should be understood that the terms "peptide" and "polypeptide" are used interchangeably. As is known to those of skill in the art, polypeptides can be processed and / or modified.

[0036] Protein: As used herein, the term "protein" refers to one or more polypeptides that act as discrete units. The terms "polypeptide" and "protein" can be used interchangeably if a single polypeptide is a discrete functional unit and does not require permanent or transient physical association with other polypeptides to form a discrete functional unit. If the discrete functional unit consists of more than one polypeptide physically associated with each other, the term "protein" refers to the multiple polypeptides that are physically coupled and function together as a discrete unit.

[0037] Specific binding: As used herein, with respect to a ligand, the term "specifically binds" or "has selective affinity for" means that the ligand reacts or associates with a particular epitope, protein, or target molecule more frequently, more rapidly, for a longer duration, with greater affinity, or in any combination thereof, compared to its reaction or association with alternative substances, including unrelated proteins. Due to sequence identity between homologous proteins in different species, specific binding can include binders that recognize proteins or targets in more than one species. Similarly, due to homology within certain regions of the polypeptide sequences of different proteins, specific binding can include binders that recognize more than one protein or target. It should be understood that in some embodiments, a binder that specifically binds a first target may or may not specifically bind a second target. Thus, "specific binding" does not necessarily require (although it can include) exclusive binding, i.e., binding to a single target. Accordingly, in some embodiments, a ligand or an affinity agent can specifically bind more than one target. In some embodiments, multiple targets can be bound by the same antigen-binding site on the affinity agent.

[0038] Substantially: As used herein, the term "substantially" refers to a qualitative situation that exhibits an overall or near overall range or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological as well as chemical phenomena rarely, if ever, achieve complete and / or proceed to completion or achieve or avoid absolute results. Accordingly, the term "substantially" is used herein to account for the potential lack of completeness inherent in many biological as well as chemical phenomena.

[0039] It should also be understood that when a singular "noun" is referred to herein (e.g., a ligand, a bead, etc.), unless otherwise indicated, the plural form of such terms (e.g., ligands, beads, etc.) is also preferably intended to be included and contemplated herein. Thus, in preferred embodiments, where applicable, referring to a singular noun is also intended to alternatively refer to "at least one", "one or more", or "a plurality" of the terms referred to. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 An exemplary affinity agent containing a three-helix bundle protein is shown, and an example of how the structure of the three-helix bundle protein can be envisioned as a triangular prism, where each triangular vertex represents a helix.

[0041] Figure 2 An example sensorgram of the affinity agent of the present invention is shown. A ligand corresponding to the biotinylated ligand SEQ ID NO:2 was immobilized on the sensor to determine binding to the target analyte trimeric vaccine protein.

[0042] Figure 3Shows the stability of an affinity agent of the present invention. The ligand corresponds to biotinylated SEQ ID NO:7.

[0043] Figure 4 Shows the stability of an affinity agent of the present invention in 0.1M NaOH. The affinity resin contains ligand SEQ ID NO:2.

[0044] Figure 5 Shows the absorbance chromatogram at 280 nm wavelength during the purification of a trimeric vaccine protein using the affinity agent of the present invention. The affinity resin contains ligand SEQ ID NO:7.

[0045] Figure 6 Shows the SDS-PAGE gel of the trimeric vaccine protein purified using the affinity agent of the present invention. The affinity resin contains ligand SEQ ID NO:7. The loading (L), wash (W), elution (E) and stripping (S) fractions were loaded onto the gel. A molecular weight reference standard (Std) is included. The molecular weight of the purified trimeric vaccine protein is 180 kDa. Detailed Description

[0046] The present disclosure particularly encompasses the recognition that affinity agents prepared from 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, trimeric proteins, preferably trimeric vaccine proteins). In some embodiments, the affinity agents described herein (e.g., affinity resins or affinity beads) can be particularly useful for removing impurities associated with protein products and contaminants derived from host cells.

[0047] Ligands for affinity agents that bind to targets of interest

[0048] Known or improved assays, bioassays, and / or animal models known in the art for evaluating such activities can be used to determine the binding characteristics of ligands to targets. For the affinity agents according to the present invention, alternatively, the narrative feature that the affinity agent "binds" to a target of interest (e.g., a trimeric protein, preferably a trimeric vaccine protein) can also be more specifically defined as the ligand contained in the affinity agent "binds" to the target of interest (e.g., a trimeric protein, preferably a trimeric vaccine protein). In other words, it should be understood that the ability of the affinity agent to bind to a target of interest is provided by the ligand contained in the affinity agent. Additionally, as used in the context of binding to a target of interest, the term "binds" is intended to mean and can thus be interchangeably defined as "being able to" bind to the target of interest. 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.

[0049] As used herein, terms such as "binding affinity for a target", "binding to a target" refer to ligand properties that can be directly measured, for example, by determination of an affinity constant (e.g., the amount of ligand associating and dissociating at a given antigen concentration). There are several methods available for characterizing such molecular interactions, e.g., competitive analysis, equilibrium analysis, and microcalorimetry, as well as real-time interaction analysis based on surface plasmon resonance interactions (e.g., using a BIACORE instrument). These methods are well known to those skilled in the art and are discussed in publications such as Neri D et al., (1996) Tibtech 14:465-470 and Jansson M et al., (1997) J Biol Chem 272:8189-8197.

[0050] 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 the ligand and target molecule, and the functional application of the ligand. In some embodiments, the ligand binds to the target of interest (e.g., a trimeric protein, preferably a trimeric vaccine protein) with a dissociation constant (K -3 M) of less than or equal to 5×10 -3 M, 10 -4 M, 5×10 -4 M, 10 -5 M, 5×10 -5 M or 10 D )M. In some embodiments, the ligand binds to the target of interest with a K -6 M of less than or equal to 5×10 -6 M, 10 -7 M, 5×10 -7 M, 10 -8 M, 5×10 -8 M or 10 D M. In some embodiments, the ligand binds to the target of interest with a K -9 M of less than or equal to 5×10 -9 M, 10 -10 M, 5×10 -10 M, 10 -11 M, 5×10 -11 M, 10 -12 M, 5×10 -12 M, 10 -13 M, 5×10 -13 M, 10 -14 M, 5×10 -14 M, 10 -15 M or 10 -15 M. DBind to a target of interest. In some embodiments, the dissociation constant of the ligand generated by the methods disclosed herein binding to the target of interest is: 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.

[0051] Binding experiments to determine K D and the dissociation rate can be carried out under a variety of conditions. The buffer for preparing these solutions can be readily determined by those skilled in the art and mainly depends on the desired pH of the final solution. Low pH solutions (<pH 5.5) can be prepared in, for example, citrate buffer, glycine-HCl buffer or succinate buffer. High pH solutions can be prepared in, for example, Tris-HCl, phosphate buffer or sodium bicarbonate buffer. For the purpose of determining, for example, the optimal pH and / or salt concentration, many conditions can be used to determine K D and the dissociation rate.

[0052] In some embodiments, the ligand binds specifically to the target of interest with a k -7 in the range of 0.1 to 10 -1 sec -2 to 10 -7 sec -1 or 0.5x10 -2 to 10 - 7 sec -1 . In some embodiments, the ligand binds to the target of interest with a dissociation rate (k off ) of less than 5x10 -2 sec -1 , 10 -2 sec -1 , 5x10 -3 sec -1 or 10 -3 sec -1 . In some embodiments, the ligand binds to the target of interest with a dissociation rate of less than 5x10 off -4 ​sec -1 , 10 -4 sec -1 , 5x10 -5 sec -1 or 10 -5 sec -1 , 5x10 -6 sec -1 , 10 -6 sec -1 , 5x10 -7 sec -1 or 10 -7 sec -1 The dissociation rate (k off ) binds to a target of interest.

[0053] In some embodiments, the ligand is present in an amount of about 10 3 Up to 10 7 M -1 sec -1 , 10 3 Up to 10 6 M -1 sec -1 or 10 3 Up to 10 5 M - 1 sec -1 k in the range of on Specifically bind to a target of interest. In some embodiments, the ligand (e.g., ligand fusion protein) is present at a concentration greater than 10 3 M -1 sec -1 , 5x10 3 M -1 sec -1 , 10 4 M -1 sec -1 or 5x10 4 M -1 sec -1 The association rate (k on ) binds to a target of interest. In another embodiment, the ligand is present in an amount greater than 10 5 M -1 sec -1 , 5x10 5 M -1 sec -1 , 10 6 M -1 sec -1 , 5x10 6 M -1 sec -1 or 107 M -1 sec -1 of k on bind to the target of interest.

[0054] target of interest

[0055] According to various embodiments, the target of interest specifically bound by a ligand can be any molecule to which the ligand binds. For example, the target specifically bound by a ligand can be any target of purification, manufacturing, formulation, therapeutic, diagnostic, or prognostic relevance or value. Non-limiting uses include therapeutic and diagnostic uses. As examples, numerous exemplary targets are provided herein, and are intended to be illustrative rather than limiting. It is well known in the art that viruses acquire mutations, and as an example, exemplary mutations described for the targets herein are provided, and are intended to be illustrative rather than limiting. The target of interest can be naturally occurring or synthetic. In some embodiments, the target comprises the receptor binding domain (RBD) of the CoV-2 virus spike protein (SARS-CoV-2 S). 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, S1 protein, or spike protein of the CoV-2 virus. In some embodiments, the target comprises a CoV-2 virus particle. In a preferred embodiment, the target of interest is a trimeric protein. Particularly preferred trimeric proteins include any chimeric polypeptides mentioned in WO 2018 / 176103, the content of which is incorporated herein by reference in its 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 can provide utility as a vaccine based on the nature / origin of the polypeptide / protein contained therein and thus its antigenic ability. In particular, exemplary trimeric vaccine proteins include trimeric proteins derived from trimeric viral surface proteins, such as, for example, fusion proteins of class I or class III enveloped viruses or portions thereof (e.g., extracellular domains or antigenically active portions thereof). Particularly preferred trimeric vaccine proteins are described in WO 2018 / 176103, the content of which is incorporated herein by reference in its 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 a trimer 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 a trimer. Various trimerization domains are known in the art, and some particularly preferred representatives thereof are also mentioned hereinabove.In a particularly preferred embodiment, the trimerization domain is a polypeptide according to the "structurally stabilizing moiety" (also referred to as a molecular clamp) as described in WO 2018 / 176103, the content of which is incorporated herein by reference in its entirety, wherein preferably the trimerization domain is fused to the C-terminus of a heterologous polypeptide (e.g., the extracellular domain (or an antigenic portion thereof) of a fusion protein from an enveloped virus such as a class I or class III enveloped virus) and thereby mediates the trimerization of the latter.

[0056] Linker

[0057] The terms "linker" and "spacer" are used interchangeably herein and refer to a peptide or other chemical linkage that serves to connect otherwise independent functional domains. In some embodiments, the linker is located between the ligand and another polypeptide component containing otherwise independent functional domains. Suitable linkers for coupling two or more ligands can generally be any linker used in the art for connecting peptides, polypeptides, proteins, or other organic molecules. In some embodiments, such linkers are suitable for constructing proteins or polypeptides intended for pharmaceutical use.

[0058] Suitable linkers for operably connecting the ligand and additional components of the ligand fusion protein in a single-chain 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 consisting predominantly of polar polypeptide segments.

[0059] In some embodiments, the linker comprises mostly amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In some embodiments, the linker comprises mostly amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In some embodiments, the ligand linker consists mostly of amino acids that are not sterically hindered. In some embodiments, the linker comprises mostly amino acids selected from glycine, serine, and / or alanine. In some embodiments, the peptide linker is selected from polyglycine (such as, for example, (Gly)5 or (Gly)8), poly(Gly-Ala), and polyalanine.

[0060] Linkers can have any size or composition, so long as they operably link a ligand (to other ligands or to a solid support or to a molecule bound to a solid support) in a manner that permits binding of the ligand to a target of interest. In some embodiments, the linker is about 1 to 50 amino acids, about 1 to 20 amino acids, about 1 to 15 amino acids, about 1 to 10 amino acids, about 1 to 5 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to 10 amino acids, or about 2 to 5 amino acids. It should be appreciated that the length, degree of flexibility, and / or other properties of the linker may affect certain properties of the ligand used for the affinity agent, such as the affinity, specificity, or avidity for the target of interest, or for one or more other target proteins of interest or for proteins of no interest (i.e., non-target proteins). In some embodiments, two or more linkers are used. In some embodiments, the two or more linkers are the same. In some embodiments, the two or more linkers are different.

[0061] In some embodiments, the linker is a non-peptide linker such as an alkyl linker or a PEG linker. For example, an alkyl linker such as -NH-(CH2) s -C(O)- can be used, where s = 2 - 20. Any such linker can also be substituted with any non - sterically - hindering group such as a lower alkyl (e.g., C1 - C6 alkyl), a lower acyl (e.g., -CO-(C1 - C5 alkyl)), a 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.

[0062] Techniques described herein and / or otherwise known in the art can be used to evaluate the linker. In some embodiments, the linker does not alter (e.g., does not disrupt) the ability of the ligand to bind to the target molecule.

[0063] Affinity agents comprising a conjugated ligand

[0064] Ligands that promote specific binding to a target of interest can be chemically conjugated to a variety of chromatographic compositions (e.g., beads, resins, gels, membranes, monoliths, etc.) to prepare affinity agents. Affinity agents comprising the ligand can be particularly useful for purification and manufacturing applications.

[0065] In some embodiments, a ligand (e.g., a ligand fusion protein) contains at least one reactive residue. The reactive residue can, for example, serve as an attachment site for conjugates such as chemotherapeutic agents. An exemplary reactive amino acid residue is lysine. The reactive residue (e.g., lysine) can be added to either end of the ligand or within the ligand sequence, and / or can replace another amino acid in the ligand sequence. Suitable reactive residues (e.g., lysine, serine, tyrosine, hydroxytryptophan, etc.) can also be located within the identified ligand sequence without addition or replacement. 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.

[0066] Attached to a solid surface

[0067] The terms "solid surface", "support", "solid support", or "matrix" are used interchangeably herein and refer to, but are not limited to, any column (or column material), resin, bead (e.g., an agarose bead or Sepharose TM bead), test tube, microtiter plate, solid particle (e.g., agarose or Sepharose TM ), microchip (e.g., a silicon, silicon-glass, or gold chip), or membrane of synthetic (e.g., a filter) or biological (e.g., a liposome or vesicle) origin to which a ligand, an affinity agent, an antibody, or other protein can be directly or indirectly (e.g., through other binding partner intermediates such as an antibody or protein A or G) attached (i.e., coupled, conjugated, linked, or adhered), or into which a ligand or antibody can be incorporated (e.g., through 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, chromatographic resins or matrices (e.g., agarose or Sepharose TM(such as Sepharose 4 Fast Flow) beads), the walls or bottoms of the wells in plastic microtiter plates, 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 bonding using reagents and techniques known in the art. In an exemplary preferred embodiment of the non-covalent association of a ligand with a solid support, the ligand comprises one member of a binding pair (i.e., the first member) (e.g., an affinity tag), and the solid support comprises the corresponding other member of the binding pair (i.e., the second member) (e.g., an affinity matrix that is specific for binding to 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 chelator, 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., Avi-tag TM ), and the solid support comprises avidin (and / or streptavidin and / or neutravidin). In other embodiments, the ligand is attached to the solid support by means of an antibody that specifically binds the ligand. In the latter embodiment, the antibody can be attached to the solid support by means of protein A and / or protein G contained in the solid support (or attached or conjugated to the solid support itself). A large number of other suitable binding pairs are well known in the art, and each of them can be used for the purposes disclosed herein. In some embodiments, the ligand is coupled to the solid surface or solid support via a linker (e.g., a chromatographic material such as beads or resins made of agarose or Sepharose TM ).

[0068] Generation of Ligands

[0069] The generation of ligands useful for practicing several embodiments of the provided methods and uses can be carried out using a variety of standard techniques known in the art for chemical synthesis, semi-synthetic methods, as well as recombinant DNA and protein expression and purification methods. Also provided are methods for generating ligands as soluble agents and cell-associated proteins, either alone or as part of a multi-domain fusion protein. In some embodiments, the overall ligand generation protocol includes obtaining a reference protein scaffold and identifying multiple residues within the scaffold for modification. Depending on the embodiment, the reference scaffold can comprise a protein structure or other tertiary structure having one or more α-helical regions. Once identified, any of the multiple residues can be modified, for example, by substitution of one or more amino acids. In some embodiments, one or more conservative substitutions are made. In some embodiments, one or more non-conservative substitutions are made. In some embodiments, a natural amino acid (e.g., one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine) is substituted into the reference scaffold at the target position for modification. In some embodiments, the modification does not include substitution of cysteine or proline. After modification at the identified positions required in a particular embodiment, the resulting modified polypeptide (e.g., a candidate ligand) can be recombinantly expressed, for example, in a plasmid, bacterium, phage, or other vector (e.g., to increase the number of each of the modified polypeptides). The modified polypeptides can then be purified and screened to identify those modified polypeptides that specifically bind to a particular target of interest. Compared to the reference scaffold, the modified polypeptides can exhibit enhanced binding specificity to the target of interest, or can exhibit little or no binding to a given target of interest (or to non-target proteins). In some embodiments, depending on the target of interest, the reference scaffold can exhibit some interaction (e.g., non-specific interaction) with the target of interest, while certain modified polypeptides will exhibit at least about two-fold, at least about five-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or at least about 100-fold (or more) increased binding specificity to the target of interest. Additional details regarding ligand generation, selection, and isolation are provided in more detail below.

[0070] Recombinant Expression of Ligands

[0071] In some embodiments, ligands such as ligand fusion proteins are "recombinantly produced" (i.e., produced using recombinant DNA techniques). Exemplary recombinant methods that can be used to synthesize ligand fusion proteins include, but are not limited to, polymerase chain reaction (PCR)-based synthesis, concatenation, seamless cloning, and recursive directional ligation (RDL) (see, for example, 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)).

[0072] Also provided are nucleic acids comprising a polynucleotide sequence encoding a ligand. Such polynucleotides optionally comprise one or more expression control elements. For example, the polynucleotide can comprise one or more promoters or transcriptional enhancers, ribosome binding sites, transcriptional termination signals, and polyadenylation signals as expression control elements. The polynucleotide can be inserted into any suitable vector, and the vector can be contained within any suitable host cell for expression.

[0073] Expression of the 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 transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Exemplary promoters that can be used for expression in Escherichia coli include, for example, the T7 promoter.

[0074] Expression vectors containing a nucleic acid sequence encoding a ligand and appropriate transcriptional / translational control signals can be constructed using methods known in the art. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / gene rearrangement. 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.

[0075] A variety of host expression vector systems can be utilized to express nucleic acids encoding ligands. Vectors containing nucleic acids encoding ligands (e.g., a single ligand subunit or a ligand fusion) 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 encapsulated viruses using known infection and transduction techniques. Additionally, viral vectors can be replication-competent or, alternatively, replication-deficient. Alternatively, cell-free translation systems can also be used to produce ligands using RNA derived from DNA expression constructs (see, e.g., WO86 / 05807 and WO89 / 01036; and U.S. Patent No. 5,122,464).

[0076] 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 engineered host cells is a bacterial cell, a yeast cell, or a mammalian cell. A variety of host expression vector systems can be used to express the coding sequence of a ligand fusion protein. Mammalian cells can be used as host cell systems transfected with recombinant plasmid DNA or cosmid DNA expression vectors containing the coding sequence of a target of interest and the coding sequence of a fusion polypeptide. The cells can be primary isolates from an organism, culture, or cell line having a transformed or transgenic nature.

[0077] Suitable host cells include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli, Bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing the ligand coding sequence; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the ligand coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus); plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing the ligand coding sequence (e.g., Ti plasmid).

[0078] Prokaryotes that can be used as host cells for the production of ligands include Gram-negative or Gram-positive organisms such as Escherichia coli and Bacillus subtilis. Expression vectors for use in prokaryotic host cells typically contain one or more phenotypic selection marker genes (e.g., genes encoding proteins that confer antibiotic resistance or provide autotrophic requirements). Examples of available prokaryotic host expression vectors include the pKK223-3 (Pharmacia, Uppsala, Sweden), pGEM1 (Promega, Wis., USA), pET (Novagen, Wis., USA), and pRSET (Invitrogen, Calif., USA) series of vectors (see, e.g., Studier, J. Mol. Biol. 219:37 (1991) and Schoepfer, Gene 124:83 (1993)). Exemplary promoter sequences commonly used in prokaryotic host cell expression vectors include T7 (Rosenberg et al., Gene 56:125-135 (1987)), beta-lactamase (penicillinase), the lactose promoter system (Chang et al., Nature 275:615 (1978)); and Goeddel et al., Nature 281:544 (1979)), the tryptophan (trp) promoter system (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, N.Y.).

[0079] 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 a coding sequence for a ligand. Exemplary yeasts that can be used to produce the compositions of the present invention include yeasts from the genera Saccharomyces, Pichia, Actinomyces, and Kluyveromyces. Yeast vectors typically contain a replication origin sequence from the 2mu yeast plasmid, an autonomous replication sequence (ARS), a promoter region, a polyadenylation sequence, a transcription termination sequence, and a selectable marker gene. Examples of promoter sequences in yeast expression constructs include promoters from metallothionein, 3-phosphoglycerate kinase (Hitzeman, J. Biol. Chem. 255:2073 (1980)), and other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other suitable vectors and promoters for yeast expression, as well as yeast transformation protocols, are known in the art. See, for example, Fleer, Gene 107:285-195 (1991) and Hinnen, PNAS 75:1929 (1978).

[0080] Insect and plant host cell culture systems can also be used to produce 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 a coding sequence for a 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) containing a coding sequence for a ligand, including but not limited to the expression systems taught in U.S. Patent No. 6,815,184; U.S. Publication Nos. 60 / 365,769 and 60 / 368,047; and WO2004 / 057002, WO2004 / 024927, and WO2003 / 078614.

[0081] In some embodiments, a host cell system can be used. In some embodiments, the host cell system is an animal cell system infected with a recombinant viral expression vector (e.g., adenovirus, retrovirus, adeno-associated virus, herpesvirus, lentivirus). In some embodiments, the host cell system is a cell line (e.g., a murine cell line) engineered to contain multiple copies of DNA encoding a ligand that is stably amplified (CHO / dhfr) or unstably amplified in double minute chromosomes. In some embodiments, the vector containing the polynucleotide encoding the ligand is polycistronic. Exemplary mammalian cells that can be used to produce these compositions include HEK293 cells (e.g., 293T and 293F), CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 (Crucell, 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 that can be used to practice 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); 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)). Transcriptional and translational control sequences of mammalian host cell expression vectors generally are derived from viral genomes. Promoter sequences and enhancer sequences commonly used in mammalian expression vectors include sequences derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV). Exemplary commercially available expression vectors for mammalian host cells include pCEP4 (Invitrogen) and pcDNA3 (Invitrogen).

[0082] Physical methods for introducing nucleic acids into host cells (e.g., mammalian host cells) include, but are not limited to, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. Methods for generating cells containing a vector and / or exogenous nucleic acid are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0083] Biological methods for introducing polynucleotides of interest into host cells 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 cells (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0084] Methods for introducing DNA and RNA polynucleotides of interest into host cells include, but are not limited to, electroporation of cells, wherein an electric field is applied to the cells to increase the permeability of the cell membrane, thereby allowing the introduction of chemicals, drugs, or polynucleotides into the cells. Electroporation can be used to introduce ligands containing DNA or RNA constructs into mammalian cells or prokaryotic cells.

[0085] In some embodiments, electroporation of the 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 throughout the life cycle of the cells. Electroporation can be accomplished by methods known in the art, including the MaxCyte and Transfection System (MaxCyte, Gaithersburg, MD, USA).

[0086] Chemical means for introducing polynucleotides into a host cell can include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle). In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In some embodiments, the nucleic acid is associated with a lipid. The nucleic acid associated with the lipid can be encapsulated in the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that associates with both the liposome and the oligonucleotide, entrapped within a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension within a lipid, contain micelles or complexed with micelles, or otherwise associated with a lipid. Compositions associated with lipids, lipid / DNA, or lipid / expression vector are not limited to any particular structure in solution. For example, they can exist in bilayer structures, micelles, or "collapsed" structures. They can also be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic lipids. For example, lipids include the fat droplets naturally present in the cytoplasm and a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0087] 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; 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 about -20 °C. Chloroform can be used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term encompassing various single and multi-layered lipid mediators formed by generating closed lipid bilayers or aggregates. Liposomes can be characterized by having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multi-lamellar liposomes have multiple lipid layers separated by aqueous media. When phospholipids are suspended in an excess of aqueous solution, they spontaneously form. The lipid components rearrange themselves before forming the closed structure and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5:505-510 (1991)). However, compositions having structures different from the normal vesicular structure in solution are also encompassed. For example, the lipids can assume a micellar structure or exist only as non-uniform aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0088] Regardless of the method used to introduce exogenous nucleic acid into a host cell, the presence of a recombinant nucleic acid sequence in the host cell can be routinely confirmed by a variety of assays known in the art. Such assays include, for example, "molecular biology" assays known in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a specific peptide, e.g., by immunological means (ELISA and Western blotting) or by the assays described herein to identify agents falling within the scope of the present invention.

[0089] Reporter genes are used to identify potential transfected cells and to evaluate the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present or not expressed in the recipient organism, tissue or cell, and the expression of the polypeptide encoded by it is manifested by some easily detectable properties such as enzyme activity. The expression of the reporter gene is assayed at an appropriate time after introducing the DNA into the recipient cell. Suitable reporter genes include, but are not limited to, genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes (e.g., Ui-Tei et al., FEBS Lett. 479:79-82 (2000)). Suitable expression systems are known in the art and can be prepared using known techniques or obtained commercially. In general, constructs having the smallest 5' flanking region that shows the highest expression level of the reporter gene are identified as promoters. Such promoter regions can be routinely ligated to the reporter gene and used to evaluate the ability of an agent to modulate promoter-driven transcription.

[0090] Many 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 of, for example, dhfr, gpt, neo, hygro, trpB, hisD, ODC (ornithine decarboxylase) and glutamine synthetase systems.

[0091] Ligand purification

[0092] Once a ligand or ligand fusion protein has been produced by recombinant expression, it can be purified by recombinant protein purification methods known in the art, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography and size exclusion chromatography (SEC)), centrifugation, differential solubility or by 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 (such as, for example, a His tag (e.g., 6xHis tag) or a biotinylation tag (e.g., Avi tag)) or a heterologous peptide or polypeptide sequence otherwise known in the art to facilitate purification. In some embodiments, the ligand of the ligand affinity column for affinity purification (e.g., antibodies and other affinity matrices) is removed from the composition before the ligand is finally prepared using techniques known in the art. In some embodiments, other components of the ligand or ligand fusion composition bound by these ligands are removed from the composition before the ligand is finally prepared using techniques known in the art.

[0093] Chemical synthesis of ligands

[0094] In addition to recombinant methods, ligand generation can be carried out using organic chemical synthesis of the desired polypeptide using a variety of liquid and solid phase chemical methods known in the art. Various automated synthesizers are commercially available and can be used according to known protocols. See, e.g., Tam et al., J. Am. Chem. Soc., 105:6442 (1983); Merrifield, Science, 232:341-347 (1986); Barany and Merrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, New York, 1-284; Barany et al., Int. J. Pep. Protein Res., 30:705 739 (1987); Kelley et al., Genetic Engineering Principles and Methods, Setlow, J.K., ed. Plenum Press, NY. 1990, vol. 12, pp. 1-19; Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, 1989. One advantage of these methods is that they allow incorporation of non-natural amino acid residues into the ligand sequence.

[0095] The ligands used in the methods of the invention can be modified during or after synthesis or translation, e.g., by glycosylation, acetylation, benzylation, phosphorylation, amidation, polyethylene glycolylation, formylation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to antibody molecules, hydroxylation, iodination, methylation, myristoylation, oxidation, isoprenylation, racemization, selenylation, sulfation, ubiquitination, etc. (See, e.g., Creighton, Proteins: Structures and Molecular Properties, 2nd ed. (W.H. Freeman and Co., N.Y., 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 peptide is acetylated at the N-terminus and / or amidated at the C-terminus.

[0096] Any of a number of chemical modifications can be carried out by known techniques, including but not limited to acetylation, formylation, etc. Additionally, the derivatives can contain one or more non-classical amino acids.

[0097] In some embodiments, cyclization or macrocyclization of the peptide backbone is achieved through the formation of side-chain to side-chain linkages. Methods for accomplishing this are well known in the art and may involve natural as well as non-natural amino acids. Pathways include disulfide formation, lanthionine formation or thiol alkylation (e.g., Michael addition), amidation between amino and carboxylic acid side chains, click chemistry (e.g., azide-alkyne condensation), peptide stapling, ring-closing metathesis, and the use of enzymes.

[0098] Affinity agents for purification

[0099] In purification based on affinity chromatography, the target of interest (preferably a trimeric protein or a trimeric vaccine molecule) is selectively separated according to its ability to specifically and reversibly bind to a ligand, which can be covalently coupled to a chromatographic matrix. In some embodiments, the ligand can be used as a reagent for affinity purification of the target of interest from recombinant or natural sources such as biological samples (e.g., serum, cells).

[0100] In some embodiments, a ligand that specifically binds to the target of interest is immobilized on beads and then used for affinity purification of the target.

[0101] Methods for covalently coupling proteins to surfaces are known to those skilled in the art. Peptide tags that can be used to attach ligands to solid surfaces are known to those skilled in the art. Additionally, any reagent or technique known in the art can be used to attach (e.g., couple, conjugate, link, or adhere) a ligand to a solid surface. In some embodiments, the solid support includes beads, glass, slides, chips, and / or gelatin. Thus, a range of ligands can be used to fabricate arrays on solid surfaces using techniques known in the art. For example, U.S. Publication No. 2004 / 0009530 discloses methods for preparing arrays.

[0102] In some embodiments, a ligand is used to separate a 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 that favor formation of a complex between the ligand and the target of interest. Unbound material can be washed away. Those skilled in the art can readily determine suitable washing conditions. Examples of suitable washing conditions are described in Shukla and Hinckley, Biotechnol Prog. 2008 Sep - Oct;24(5):1115 - 21.doi:10.1002 / btpr.50.

[0103] In some embodiments, chromatography is performed by mixing a solution containing the target of interest and the ligand and then separating the complex of the target of interest and the ligand. For example, the ligand is immobilized on a solid support such as beads and then separated from the solution together 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 separate the ligand using immobilized metal affinity chromatography resin or a streptavidin - coated substrate after complex formation. Once separated, the target of interest can be released from the ligand under elution conditions and recovered in purified form.

[0104] In some embodiments, a ligand including the initiator N - terminal methionine is separated, since this is the protein sequence encoded by the DNA. In some embodiments, a ligand lacking the N - terminal methionine residue is separated. In some embodiments, a mixture is obtained in which only a portion of the purified ligand contains the N - terminal methionine. Those skilled in the art will understand that the presence or absence of the N - terminal methionine does not affect the suitability of the ligand for the purposes disclosed herein.

[0105] Examples

[0106] Example 1

[0107] Recombinant protein ligands were expressed in Escherichia coli and / or Pichia pastoris using standard techniques. The ligands were purified using multi - column chromatography. For his - tagged ligands, IMAC was used as the primary capture step. The biotinylated ligand was generated using the Avitag TM system (Avidity, Aurora, CO). The Avitag TMA non-biotinylated ligand of the sequence. The purity and characteristics of the recombinant protein ligand were evaluated by a combination of SDS-PAGE, RP UPLC, quadrupole time-of-flight mass spectrometry, and SEC. In many cases, the isolated ligand does not contain an N-terminal methionine residue, which is presumably cleaved during expression. In many cases, a mixture is obtained in which only a portion of the purified ligand contains an N-terminal methionine. It will be apparent to those skilled in the art that the presence or absence of the N-terminal methionine does not affect the conclusions herein. For clarity, we included the N-terminal methionine.

[0108] Example 2

[0109] This example confirmed the binding of the biotinylated ligand to the target protein using biolayer interferometry (ForteBio, Menlo Park, CA). The biotinylated ligand was immobilized on the sensor and incubated with solutions containing various concentrations of the trimeric vaccine protein. Figure 2 Example sensorgrams are shown.

[0110] Example 3

[0111] This example confirmed the sodium hydroxide (NaOH) stability of the affinity ligand. The ligand was incubated in 0.1 M NaOH for a predetermined time and then neutralized. The binding of the NaOH-treated ligand was measured as described in Example 2 and compared to the untreated ligand. The retained binding was calculated according to the following formula:

[0112] % Retained binding = (response measured after NaOH treatment) ÷ (response measured without treatment) × 100

[0113] Figure 3 An example of the stability is shown.

[0114] Example 4

[0115] This example demonstrated the production and characterization of an affinity agent comprising the ligand identified and described herein. The affinity resin was prepared by conjugating the ligand to activated agarose beads. After washing, the ligand was conjugated to the beads at room temperature. The targeted ligand density varied between about 2 g / L and about 20 g / L. After washing, the beads were inactivated with an excess of thioglycerol. The actual ligand density of all resins was measured using a subtractive RP-HPLC method according to the following formula:

[0116] Actual ligand density = ([ligand] measured in the feed – [ligand] measured in the effluent).

[0117] Example 5

[0118] This example demonstrates the stability of the test resin after an in-place cleaning (CIP) challenge with 0.1 M sodium hydroxide (NaOH). The resin was prepared from the 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 below.

[0119] Example 6

[0120] This example demonstrates the use of an affinity agent comprising the binding ligand described herein for the affinity purification of a trimeric protein. A clarified cell culture feed stream (CCCF) from trimeric vaccine protein production was applied to a 30 mm inner 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 below.

[0121]

[0122] The purity of the eluted material was confirmed using an SDS-PAGE gel stained with Coomassie blue and is shown in Figure 6 below.

[0123] The above examples demonstrate that the affinity resin can be fine-tuned to achieve different performance characteristics for different applications and as may be required by the user.

[0124] It is contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments disclosed above can be made and still fall within the scope of the present invention. Additionally, any specific feature, aspect, method, property, characteristic, quality, attribute, element, etc. disclosed in connection with one embodiment can be used in all other embodiments set forth herein. Accordingly, it should be understood that the various features and aspects of the disclosed embodiments can be combined with or substituted for one another. Therefore, the scope of the present invention as described herein is not intended to be limited by the specific embodiments disclosed above. Moreover, although the present invention admits of various modifications and alternative forms, specific examples thereof have been shown in the drawings and described in detail herein. However, it should be understood that the present invention is not limited to the specific forms or methods disclosed, but rather the present invention encompasses all modifications, equivalents, and alternatives falling within the spirit and scope of the various embodiments described.

[0125] Any method disclosed herein need not be performed in the recited order. The methods disclosed herein include certain actions taken by a practitioner; however, they can also include any third-party instructions for these actions, whether explicit or implicit.

[0126] Table 6. Sequences

[0127]

[0128]

Claims

1. An affinity agent comprising a ligand that binds to a trimeric protein, wherein the ligand comprises at least one polypeptide comprising or consisting of the following: (i) The amino acid sequence defined as SEQ ID NO:1; and / or (ii) An amino acid sequence that differs from the amino acid sequence defined as 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 affinity agent according to claim 1, wherein the at least one polypeptide comprises or consists of the following: (i) The amino acid sequence defined as any one of SEQ ID NOs: 2-10; and / or (ii) An amino acid sequence that differs from the amino acid sequence defined as 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 affinity agent according to claim 1 or 2, wherein the ligand comprises: A multimeric polypeptide comprising at least two subunits, wherein each subunit comprises a polypeptide as defined in claim 1 or 2.

4. The affinity agent according to claim 3, wherein the polypeptides comprised 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 according to any one of claims 1 to 6, wherein the ligand is attached to a solid surface.

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

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

10. The affinity agent according to 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 covalently or non-covalently conjugated to the solid surface.

12. The affinity agent according to any one of claims 7 to 10, wherein the ligand is covalently conjugated 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 comprising the one or more trimeric proteins.

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

15. A method for preparing an affinity agent, the method comprising conjugating a ligand as defined in any one of claims 1 to 6 to a solid surface.

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

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