Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Definitions

• affinity purification is a means to isolate and/or achieve desired purity of a protein in few steps, or a single step.
• affinity agents e.g., comprising an affinity ligand
• affinity agents can be a resource intensive and time-consuming task. This has resulted in the development of affinity agents for a limited number of proteins.
• purification typically involves inefficient, labor intensive, and expensive processes (e.g., a multi-column process).
• Exemplary therapeutic proteins include, but are not limited to, bioactive polypeptides/proteins, fusion proteins, enzymes, hormones, antibodies, antibody fragments and recombinant vaccines.
• an affinity agent comprises a ligand and a solid support.
• an affinity agent comprises a ligand and a solid support.
• Some subunit vaccines are based upon viral proteins that form a trimeric structure. To facilitate efficient production of the correctly folded trimeric form, a trimerization domain can be fused to the viral protein.
• 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.
• said trimerization domain may also be utilized for effecting a trimerization of other (i.e., non-vaccine) polypeptides/proteins, in particular therapeutic proteins, for example, without intending to be limiting, any of those referred to in paragraphs [0395]-[0400] of WO 2018/176103.
• An affinity agent that binds this trimerization domain enables a facile purification of related subunit vaccines independent of the antigen portion and thus provides a platform technology for vaccine production and/or purification.
• an affinity agent (or the ligand comprised in said affinity agent) comprises a 3-helical bundle protein (alternatively referred to as “three-helix bundle protein”), preferably an anti-parallel 3-helical bundle protein.
• the structure of a 3-helical bundle protein can be envisaged as a triangular prism, with each triangle vertex representing a helix, for example, as shown in Figure 1.
• any 2 combinations of helices define a rectangular face of a 3 helical bundle protein.
• the 3 faces of a 3 helical bundle protein are defined by: 1) helix 1 and 2 (Face 1,2 in Figure 1); 2) helix 2 and 3 (Face 2,3 in Figure 1); 3) helix 1 and 3 (Face 1,3 in Figure 1); and combinations thereof.
• an affinity agent or the ligand comprised in said affinity agent
• the primary function of helix 1 of a 3-helical bundle protein is to complete and stabilize the 3-helical bundle.
• variations of helix 1 can be made that maintain the structure of a 3 helical bundle protein.
• the term “approximately” or “about” refers to a range of values that fall within, with increasing preference, 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 unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
• Biologically active refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism.
• 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), phen
• 4 11584315v2 Attorney Docket No.2011039-0112 substitution of a phenylalanine for a tyrosine is a conservative substitution.
• conservative amino acid substitutions in the sequence of a ligand confer or improve specific binding of the ligand to a target of interest.
• conservative amino acid substitutions in the sequence of a ligand do not reduce or abrogate the binding of the ligand to a target of interest.
• conservative amino acid substitutions do not significantly affect specific binding of a ligand to a target of interest.
• non- conservative amino acid substitutions in the sequence of a ligand confer or improve specific binding of the ligand to a target of interest.
• non-conservative amino acid substitutions in the sequences of a ligand do not reduce or abrogate the binding of the ligand to a target of interest.
• Linker refers to a peptide or other chemical linkage that functions to link otherwise independent functional domains, entities, or moieties. In some embodiments, a linker is located between a ligand and another polypeptide component containing an otherwise independent functional domain. In some embodiments, a linker is a peptide or other chemical linkage located between a ligand and a surface, such as a solid surface or solid support.
• the ligand is chemically conjugated (i.e., covalently bound) to the solid surface or solid support through a bond formed between the thiol group of a cysteine of the ligand (preferably, an N- or C-terminal cysteine, more preferably a C-terminal cysteine) and the solid support.
• the ligand may be covalently bound to the solid support through 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.
• ligands e.g., ligands comprising polypeptide(s)
• ligands comprising polypeptide(s)
• solid surface or solid support any of the methods and linkages described in Greg T. Hermanson, “Bioconjugate Techniques”, 3 rd edition, 2013 (which is incorporated herein by reference in its entirety) can be used for covalently binding the ligand to the solid support.
• non-natural amino acids that can be substituted in a polypeptide ligand include, but are not limited to molecules such as: D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-amino isobutyric acid, A-aminobutyric acid, Abu, 2-amino butyric acid, gamma-Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, lanthionine, dehydroalanine, ⁇ -aminobutyric acid,
• polynucleotide and nucleic acid molecule refer to a polymeric form of nucleotides of any length, comprising or consisting 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.
• polypeptide refers to a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. Thus, for the sake of the present disclosure, it is understood that the terms “peptide” and “polypeptide” may be used interchangeably.
• binds As used herein in reference to ligands, the term “specifically binds” or “has selective affinity for” means a ligand reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or combinations of the above to a particular epitope, protein, or target molecule than with alternative substances, including unrelated proteins. Because of the sequence identity between homologous proteins in different species, specific binding can include a binding agent that recognizes a protein or target in more than one species. Likewise, because of 7 11584315v2 Attorney Docket No.2011039-0112 homology within certain regions of polypeptide sequences of different proteins, specific binding can include a binding agent that recognizes more than one protein or target.
• a binding agent that specifically binds a first target may or may not specifically bind a second target.
• “specific binding” does not necessarily require (although it can include) exclusive binding, i.e., binding to a single target.
• a ligand or affinity agent may, in certain embodiments, specifically bind more than one target.
• multiple targets may be bound by the same antigen-binding site on an affinity agent.
• Figure 1 shows an exemplary affinity agent comprising a 3 helical bundle protein, as well as examples of how the structure of a 3 helical bundle protein can be envisaged as a triangular prism, with each triangle vertex representing a helix.
• Figure 2 shows an example sensorgram for an affinity agent of the present invention.
• FIG. 1 shows the stability of one affinity agent of the present invention.
• the ligand corresponded to biotinylated SEQ ID NO: 7. 8 11584315v2 Attorney Docket No.2011039-0112
• Figure 4 shows the stability of one affinity agent of the present invention in 0.1 M NaOH.
• the affinity resin contained ligand SEQ ID NO: 2.
• Figure 5 shows a chromatogram of absorbance at wavelength of 280 nm during the purification of a trimeric vaccine protein using an affinity agent of the present invention.
• the present disclosure encompasses, inter alia, the recognition that affinity agents prepared from identified and characterized peptide ligands are shown to generate highly purified preparations of one or more targets of interest, for example, in some embodiments, a trimeric protein, preferably a trimeric vaccine protein.
• affinity agents e.g., affinity resins or affinity beads
• Ligand binding to targets of interest for use in an affinity agent [0041] The characteristics of a ligand binding to a target can be determined using known or modified assays, bioassays, and/or animal models known in the art for evaluating such activity.
• the recited feature that said affinity agent “binds” a target of interest may, alternatively, also be more specifically defined as that the ligand(s) comprised in said affinity agent “binds” said target of interest (e.g., a trimeric protein, preferably a trimeric vaccine protein).
• a target of interest e.g., a trimeric protein, preferably a trimeric vaccine protein
• the ligand(s) comprised in said affinity agent binds” said target of interest (e.g., a trimeric protein, preferably a trimeric vaccine protein).
• the capacity of the affinity agent to bind a target of interest is provided by the ligand comprised in said affinity agent.
• the term “binds”, as used in the context of the binding to a target of interest is intended to mean, and may thus be interchangeably defined as, “capable of binding” to said target of interest.
• Affinity requirements for a given ligand binding event are contingent 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 molecules, and the functional application of the ligand.
• a ligand binds a target of interest (e.g., a trimeric protein, preferably a trimeric vaccine protein, or a trimerization domain as referred to herein) with a dissociation constant (K D ) of less than or equal to 5 ⁇ 10 ⁇ 3 M, 10 ⁇ 3 M, 5 ⁇ 10 ⁇ 4 M, 10 ⁇ 4 M, 5 ⁇ 10 ⁇ 5 M, or 10 ⁇ 5 M.
• K D dissociation constant
• a ligand binds a target of interest with a KD of less than or equal to 5 ⁇ 10 ⁇ 6 M, 10 ⁇ 6 M, 5 ⁇ 10 ⁇ 7 M, 10 ⁇ 7 M, 5 ⁇ 10 ⁇ 8 M, or 10 ⁇ 8 M.
• a ligand generated by methods disclosed herein has a dissociation constant with respect to the binding to a target of interest of from about 10 -4 M to about 10 -5 M, from about 10 -5 M to about 10 -6 M, from about 10 -6 M to about 10 -7 M, from about 10 -7 M to about 10 -8 M, from about 10 -8 M to about 10 -9 M, from about 10 -9 M to about 10 -10 M, from about 10 -10 M to about 10 -11 M, or from about 10 -11 M to about 10 -12 M.
• Binding experiments to determine K D and off-rates can be performed in a number of conditions.
• the buffers in which to make these solutions can readily be determined by one of skill in the art and depend largely on the desired pH of the final solution.
• Low pH solutions ⁇ pH 5.5
• High pH solutions can be made, for example, in Tris-HCl, phosphate buffers, or sodium bicarbonate buffers.
• a number of 10 11584315v2 Attorney Docket No.2011039-0112 conditions may be used to determine K D and off-rates for the purpose of determining, for example, optimal pH and/or salt concentrations.
• a ligand specifically binds a target of interest with a koff ranging from 0.1 to 10 -7 sec -1 , 10 -2 to 10 -7 sec -1 , or 0.5 x 10 -2 to 10 -7 sec -1 . In some embodiments, a ligand binds a target of interest with an off rate (k off ) of less than 5 x10 -2 sec -1 , 10 -2 sec -1 , 5 x10 -3 sec -1 , or 10 -3 sec -1 .
• a ligand binds the target of interest with an on rate (kon) of greater than 10 3 M -1 sec -1 , 5 x10 3 M -1 sec -1 , 10 4 M -1 sec -1 , or 5 x10 4 M -1 sec -1 .
• a ligand binds a target of interest with a kon of greater than 10 5 M -1 sec -1 , 5 x10 5 M -1 sec -1 , 10 6 M -1 sec -1 , 5 x10 6 M -1 sec -1 , or 10 7 M -1 sec -1 .
• a target comprises the receptor binding domain (RBD) of the CoV-2 virus spike protein (SARS-CoV-2S).
• RBD receptor binding domain
• a target comprises the S1 protein of the CoV-2 virus.
• a target comprises the spike protein of the CoV-2 virus.
• a target comprises a trimeric RBD construct, S1 protein or spike protein of the CoV-2 virus.
• a target comprises a CoV-2 virus particle.
• a target of interest is a trimeric protein.
• 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.
• trimerization domains are known in the art and some particularly preferred representatives thereof are also referred to herein above.
• Linkers [0047]
• the terms “linker” and “spacer” are used interchangeably herein to refer to a peptide or other chemical linkage that functions to link otherwise independent functional domains.
• a linker is located between a ligand and another polypeptide component containing an otherwise independent functional domain.
• Suitable linkers for coupling two or more ligands may generally be any linker used in the art to link peptides, polypeptides, proteins or other organic molecules. In some embodiments, such a linker is suitable for constructing proteins or polypeptides that are intended for pharmaceutical use.
• a linker comprises a majority of amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine.
• a ligand linker is made up of a majority of amino acids that are sterically unhindered.
• a linker comprises a majority of amino acids selected from glycine, serine, and/or alanine.
• a peptide linker is selected from polyglycines (such as, e.g., (Gly) 5 or (Gly) 8 ), poly(Gly-Ala), and polyalanines.
• Linkers can be of any size or composition so long as they are able to operably link a ligand (with other ligands or with the solid support or molecules bound to the solid support) in a manner that permits the ligand to bind a target of interest.
• linkers are from about 1 to 50 amino acids, from about 1 to 20 amino acids, from about 1 to 15 amino acids, from about 1 to 10 amino acids, from about 1 to 5 amino acids, from about 2 to 20 amino acids, from about 2 to 15 amino acids, from about 2 to 10 amino acids, or from about 2 to 5 amino acids.
• linker(s) may influence certain properties of a ligand for use in an affinity agent, such as affinity, specificity or avidity for a target of interest, or for one or more other target proteins of interest, or for proteins not of interest (i.e., non-target proteins).
• two or more linkers are utilized.
• two or more linkers are the same.
• two or more linkers are different.
• a linker is a non-peptide linker such as an alkyl linker, or a PEG linker.
• An exemplary non-peptide linker is a PEG linker. In some embodiments, a PEG linker has a molecular weight of from about 100 to 5000 Da, or from about 100 to 500 Da.
• a ligand e.g., a ligand fusion protein
• Reactive residues are useful, for example, as sites for the attachment of conjugates such as chemotherapeutic drugs.
• An exemplary reactive amino acid residue is lysine.
• a reactive residue e.g., lysine
• a suitable reactive residue (e.g., lysine, serine, tyrosine, hydroxytryptophan, etc.) can also be located within the sequence of an identified ligand without need for addition or substitution.
• a reactive amino acid residue is cysteine.
• a reactive amino acid residue is lysine.
• a reactive amino acid residue is serine.
• a reactive amino acid residue is tyrosine.
• a reactive amino acid residue is hydroxytryptophan.
• solid surface refers to, without limitation, any column (or column material), resin, bead (e.g., agarose bead or Sepharose TM bead), test tube, microtiter dish, solid particle (for example, agarose or Sepharose TM ), microchip (for example, silicon, silicon-glass, or gold chip), or membrane of synthetic (e.g., a filter) or biological (e.g., liposome or vesicle) origin to which a ligand, affinity agent, antibody, or other protein may be attached (i.e., coupled, conjugated, linked, or adhered), either directly or indirectly (for example, through other binding partner intermediates, such as antibodies or Protein A or G), or in which a ligand or antibody may be embedded (for example, through a receptor or channel).
• synthetic e.g., a filter
• biological e.g., liposome or vesicle
• Solid supports e.g., matrices, resins, plastic, etc.
• Suitable solid supports include, but are not limited to, a chromatographic resin or matrix (e.g., agarose or Sepharose TM (such as Sepharose 4 Fast Flow) beads), the wall or floor of a well in a plastic microtiter dish, a silica-based biochip, polyacrylamide, agarose, silica, nitrocellulose, paper, plastic, nylon, metal, and combinations thereof.
• Ligands and other compositions may be attached on a support material by a non-covalent association or by covalent bonding, using reagents and techniques known in the art.
• the ligand comprises one member (i.e., a first member) of a binding pair (e.g., an affinity tag) and the solid support comprises the corresponding other member (i.e., a second member) of the binding pair (e.g., an affinity matrix specific for binding of the affinity tag).
• the antibody may be attached to the solid support by means of Protein A and/or Protein G comprised in (or itself attached or conjugated to) the solid support.
• Protein A and/or Protein G comprised in (or itself attached or conjugated to) the solid support.
• a multitude of further suitable binding pairs are well known in the art, each of which may be employed for the herein disclosed purposes.
• a ligand is coupled to a solid surface or solid support (e.g., a chromatography material, such as a bead or resin made of, e.g., agarose or Sepharose TM ) via a linker.
• the reference scaffold 15 11584315v2 Attorney Docket No.2011039-0112 may comprise a protein structure with one or more alpha-helical regions, or other tertiary structure.
• any of a plurality of residues can be modified, for example by substitution of one or more amino acids.
• one or more conservative substitutions are made.
• one or more non-conservative substitutions are made.
• 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
• modifications do not include substituting in either a cysteine or a proline.
• the resulting modified polypeptides can be recombinantly expressed, for example in a plasmid, bacteria, 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 have specific binding to a particular target of interest. Modified polypeptides may show enhanced binding specificity for a target of interest as compared to a reference scaffold, or may exhibit little or no binding to a given target of interest (or to a non-target protein).
• the reference scaffold may show some interaction (e.g., nonspecific interaction) with a 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 for the target of interest. Additional details regarding production, selection, and isolation of ligand are provided in more detail below.
• Recombinant expression of ligands [0057]
• 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)).
• PCR polymerase chain reaction
• RDL recursive directional ligation
• Nucleic acids comprising a polynucleotide sequence encoding a ligand are also provided.
• Such polynucleotides optionally comprise one or more expression control elements.
• a polynucleotide can comprise one or more promoters or transcriptional enhancers, ribosomal binding sites, transcription termination signals, and polyadenylation signals, as expression control elements.
• a polynucleotide can be inserted within any suitable vector, which can be contained within any suitable host cell for expression.
• nucleic acids encoding ligands is typically achieved by operably linking a 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 regulation of the expression of the desired nucleic acid sequence.
• Exemplary promoters useful for expression in E. coli include, for example, the T7 promoter.
• Methods known in the art can be used to construct expression vectors containing the nucleic acid sequence encoding a ligand along with appropriate transcriptional/ translational control signals.
• a nucleic acid sequence encoding a ligand is operably linked to a suitable promoter sequence such that the nucleic acid sequence is transcribed and/or translated into ligand in a host.
• a variety of host-expression vector systems can be utilized to express a nucleic acid encoding a ligand.
• a vector containing the nucleic acid encoding a ligand may include a plasmid vector, a single-stranded phage vector, a double-stranded phage vector, a single-stranded RNA or DNA viral vector, or a double- stranded RNA or DNA viral vector.
• Phage and viral vectors may also be introduced into host cells in the form of packaged or encapsulated virus using known techniques for infection and transduction.
• viral vectors may be replication competent or alternatively, replication defective.
• cell-free translation systems may also be used to produce the ligand using RNAs derived from the DNA expression constructs (see, e.g., WO86/05807 and WO89/01036; and U.S. Pat. No. 5,122,464). 17 11584315v2 Attorney Docket No.2011039-0112 [0062]
• a background cell line used to generate an engineered host cell is a bacterial cell, a yeast cell or a mammalian cell.
• a variety of host-expression vector systems may be used to express the coding sequence of a ligand fusion protein.
• a mammalian cell can be used as a host cell system transfected with recombinant plasmid DNA or a cosmid DNA expression vector containing the coding sequence of the target of interest and the coding sequence of the fusion polypeptide.
• a cell can be a primary isolate from an organism, culture, or cell line of transformed or transgenic nature.
• Suitable host cells include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B.
• subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing ligand coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing ligand coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., Baculovirus) containing ligand coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing ligand coding sequences.
• yeast e.g., Saccharomyces, Pichia
• insect cell systems infected with recombinant virus expression vectors e.g., Baculovirus
• plant cell systems infected with recombinant virus expression vectors e.
• Prokaryotes useful as host cells in producing a ligand may include gram-negative or gram- positive organisms, such as E. coli and B. subtilis.
• Expression vectors for use in prokaryotic host cells generally contain one or more phenotypic selectable marker genes (e.g., genes encoding proteins that confer antibiotic resistance or that supply an autotrophic requirement).
• prokaryotic host expression vectors examples include the pKK223-3 (Pharmacia, Uppsala, Sweden), pGEMl (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 frequently used in prokaryotic host cell expression vectors include T7 (Rosenberg et al., Gene 56:125-135 (1987)), beta-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615 (1978)); and Goeddel et al., Nature 281 :544 (1979)), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, (1980)), and tac promoter (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
• a eukaryotic host cell system is used.
• the eukaryotic host cell system is a yeast cell transformed with a recombinant yeast expression vector 18 11584315v2 Attorney Docket No.2011039-0112 containing the coding sequence of a ligand.
• Exemplary yeast that can be used to produce compositions of the invention include yeast from the genera Saccharomyces, Pichia, Actinomycetes and Kluyveromyces.
• Yeast vectors typically contain an origin of replication sequence from a 2mu yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
• ARS autonomously replicating sequence
• 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-phospho glycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
• promoters from metallothionein 3- phosphoglycerate kinase (Hitzeman, J. Biol. Chem.255:2073 (1980)) and other glycolytic enzymes, such as, enolase, glyceraldehyde-3
• yeast transformation protocols are known in the art. See, e.g., Fleer, Gene 107:285-195 (1991) and Hinnen, PNAS 75:1929 (1978). [0066] Insect and plant host cell culture systems are also useful for producing the ligands of the invention.
• Such host cell systems include for example, insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the coding sequence of a ligand; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequence of a ligand, including, but not limited to, the expression systems taught in U.S. Pat. No. 6,815,184; U.S. Publ. Nos.
• recombinant virus expression vectors e.g., baculovirus
• plant cell systems infected with recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
• recombinant plasmid expression vectors e.g., Ti plasmid
• a host cell system may be used.
• the host cell system is an animal cell system infected with recombinant virus expression vectors (e.g., adenoviruses, retroviruses, adeno-associated viruses, herpes viruses, lentiviruses).
• recombinant virus expression vectors e.g., adenoviruses, retroviruses, adeno-associated viruses, herpes viruses, lentiviruses.
• the host cell system is a cell line engineered to contain multiple copies of the DNA encoding a ligand either stably amplified (CHO/dhfr) or unstably amplified in double-minute chromosomes (e.g., murine cell lines).
• a vector comprising a polynucleotide(s) encoding a 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, Netherlands) cells VERY, HeLa cells, COS cells, MDCK cells, 3T3 cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT20 cells, T47D cells, 19 11584315v2 Attorney Docket No.2011039-0112 CRL7O30 cells, HsS78Bst cells, hybridoma cells, and other mammalian cells.
• HEK293 cells e.g., 293T and 293F
• CHO cells e.g., 293T and 293F
• BHK cells e.g., NS0 cells, SP2/0 cells
• Transcriptional and translational control sequences for mammalian host cell expression vectors are frequently derived from viral genomes.
• Commonly used promoter sequences and enhancer sequences 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).
• a nucleic acid into a host cell include, but are not limited to, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).
• Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
• Viral vectors and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells (e.g., human cells).
• Other viral vectors may be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses, and adeno- associated viruses. See, for example, U.S. Pat, Nos.5,350,674 and 5,585,362.
• Methods for introducing DNA and RNA polynucleotides of interest into a host cell include, but are not limited to, the electroporation of a cell, in which an electrical field is applied to a cell in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or polynucleotides to be introduced into the cell.
• Ligand containing DNA or RNA constructs may be introduced into a mammalian or a prokaryotic cell using electroporation. 20 11584315v2 Attorney Docket No.2011039-0112 [0071]
• electroporation of cells results in the expression of a ligand-CAR on the surface of T cells, NK cells, NKT cells.
• a ligand is optionally fused to a heterologous peptide or polypeptide sequence specifically disclosed herein (such as, for example, a His-tag (e.g., a 6xHis-tag) or a biotinylation-tag (e.g., an Avi-tag)) or otherwise known in the art to facilitate purification.
• a His-tag e.g., a 6xHis-tag
• a biotinylation-tag e.g., an Avi-tag
• ligands e.g., antibodies and other affinity matrices
• affinity matrices for ligand affinity columns for affinity purification are removed from the composition prior to final preparation of the ligand using techniques known in the art.
• the ligands that are used in the methods of the present invention may be modified during or after synthesis or translation, e.g., by glycosylation, acetylation, benzylation, phosphorylation, amidation, pegylation, formylation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule, hydroxylation, iodination, methylation, myristoylation, oxidation, prenylation, racemization, selenoylation, sulfation, ubiquitination, etc.
• cyclization, or macrocyclization of the peptide backbone is achieved by sidechain-to-sidechain linkage formation. Methods for achieving this are well known in the art and may involve natural as well as unnatural amino acids.
• Affinity agents for purification [0082] In purification based on affinity chromatography, 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 that may be covalently coupled to a chromatographic matrix.
• a target of interest preferably a trimeric protein or trimeric vaccine molecule
• a ligand may be attached (i.e., coupled, conjugated, linked, or adhered) to a solid surface using any reagents or techniques known in the art.
• a solid support comprises beads, glass, slides, chips and/or gelatin.
• a series of ligands can be used to make an array on a solid surface using techniques known in the art.
• U.S. Publ. No. 2004/0009530 discloses methods for preparing arrays.
• a ligand is used to isolate a target of interest by affinity chromatography.
• a 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 specific embodiments, are suitable for packing a chromatography column.
• the immobilized ligand may be loaded or contacted with a solution (e.g., a sample comprising the target of interest) under conditions favorable to form a complex between the ligand and the target of interest. Non-binding materials may be washed away.
• Suitable wash conditions can readily be determined by one of skill in the art. Examples of suitable wash conditions are described in Shukla and Hinckley, Biotechnol Prog.2008 Sep-Oct;24(5):1115-21. doi: 10.1002/btpr.50.
• chromatography is carried out by mixing a solution containing a target of interest and a ligand followed by isolation of complexes of a target of interest and a ligand.
• a ligand is immobilized on a solid support such as beads, then separated from a solution along with a target of interest by filtration.
• a ligand is a fusion protein (i.e., a ligand fusion protein) that contains a peptide tag, such as a poly-HIS tail or streptavidin binding region (e.g., a biotinylation tag, such as an Avi-tag), which can be used to isolate the ligand after complexes have formed using an immobilized metal affinity chromatographic resin or streptavidin-coated substrate. Once separated, a target of interest can be released from the ligand under elution conditions and recovered in a purified form.
• a ligand is isolated that includes the initiator N-terminal methionine since that is the protein sequence encoded by the DNA.
• a ligand is isolated without the N-terminal methionine residue. In some embodiments, a mixture is obtained with only a proportion of the purified ligand containing the N-terminal methionine. It is understood by those 25 11584315v2 Attorney Docket No.2011039-0112 skilled in the art that the presence or absence of the N-terminal methionine does not affect the suitability of the ligands for the herein disclosed purposes.
• Biotinylated ligands were generated with the AvitagTM system (Avidity, Aurora, CO). Non-biotinylated ligands bearing the AvitagTM sequence were prepared by omitting exogenous biotin. The purity and identity of recombinant protein ligands was assessed by a combination of SDS-PAGE, RP UPLC, quadrupole time-of-flight mass spectrometry and SEC. In many instances, the ligand is isolated without the N-terminal methionine residue, which is presumed to be cleaved during expression. In many instances, a mixture is obtained with only a proportion of the purified ligand containing the N-terminal methionine.
• Example 2 This example demonstrates the binding of biotinylated ligands to target protein using biolayer interferometry (ForteBio, Menlo Park, CA). Biotinylated ligands were immobilized on sensors and incubated with solutions containing trimeric vaccine protein at various concentrations. An example sensorgram is shown in Figure 2.
• Example 3 This example demonstrates the sodium hydroxide (NaOH) stability of the affinity ligands. Ligands were incubated in 0.1 M NaOH for a predetermined time and then neutralized.
• NaOH sodium hydroxide
• Example 4 This example demonstrates the production and characterization of affinity agents comprising ligands identified and described herein. Affinity resins were prepared by conjugating ligands to activated agarose beads. After washing, ligands were conjugated to the beads at room temperature. Targeted ligand densities were varied from about 2 g/L to about 20 g/L.
• Example 6 This example demonstrates use of affinity agents comprising binding ligands described herein for affinity purification of trimeric proteins.
• Clarified cell culture feed stream (CCCF) from the production of a trimeric vaccine protein was applied to a 30 mm internal diameter (ID) x 100 mm column packed with resin prepared from a ligand corresponding to SEQ ID NO: 7.
• ID internal diameter
• the chromatographic method is shown in the following table and the resulting chromatogram is shown in Figure 5.
• Step Buffer Residence Column 27 11584315v2 Attorney Docket No.2011039-0112 Wash 100 mM Na-Octanoate, 25 mM 4 7.1 HEPES pH 8 e pu y o e e u e a e a was e o s a e us g a - ge s a e with Coomassie Blue and is shown in Figure 6.
• the above examples demonstrate that the affinity resins can be fine-tuned to achieve different performance features that different applications and users may require.
• various combinations or sub-combinations of the specific features and aspects of the embodiments disclosed above may be made and still fall within the invention.

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