WO2020081537A1 - Method to express, purify, and biotinylate eukaryotic cell-derived major histocompatibility complexes - Google Patents

Method to express, purify, and biotinylate eukaryotic cell-derived major histocompatibility complexes Download PDF

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WO2020081537A1
WO2020081537A1 PCT/US2019/056281 US2019056281W WO2020081537A1 WO 2020081537 A1 WO2020081537 A1 WO 2020081537A1 US 2019056281 W US2019056281 W US 2019056281W WO 2020081537 A1 WO2020081537 A1 WO 2020081537A1
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tag
peptide
mhc
hla
protein
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Devin B. LOWE
Amanda L. WOOSTER
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Texas Tech University TTU
Texas Tech University System
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Texas Tech University System
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification

Definitions

  • the present invention relates in general to the field of immunology, and more particularly to novel methods for expressing, purifying, and post-translationally modifying eukaryotic cell-derived major histocompatibility complexes.
  • MHC major histocompatibility complexes
  • MHC class I and II molecules play an integral role in T cell development and peripheral effector responses (Alcover et al., 2018). MHC class I is retained on the plasma membrane of nucleated cells and consists of a multi-unit heavy chain whose tertiary structure is stabilized by b2 microglobulin through non-covalent forces (Wieczorek et al., 2017). To provide specific binding to antigen specific CD8+ T cells, MHC class I usually retains a short 8-10 amino acid peptide within the MHC peptide binding groove that is derived from degraded intracellular proteins (hereafter referred to as peptide/MHC).
  • peptide/MHC degraded intracellular proteins
  • b2 microglobulin and MHC class I heavy chain (containing a BirA tail) are individually expressed in E. coli and later purified from inclusion bodies through a laborious lysis/solubilization process.
  • a defined MHC class I peptide is then added alongside b2 microglobulin and heavy chain in a precise folding reaction mixture that requires several days to complete prior to affinity chromatography (AC) purification of properly folded peptide/MHC and later biotinylation steps.
  • AC affinity chromatography
  • the standard method is [i] time consuming, [ii] requires substantial levels of raw ingredients (particularly purified MHC class I peptide), and [iii] cannot guarantee large-scale production of properly folded peptide/MHC molecules based on predicted peptide binders. For example, it is extremely difficult to stably produce MHC molecules bearing peptides with low-to-moderate affinity to the MHC peptide-binding groove.
  • peptide/MHC complexes Prior attempts to make peptide/MHC complexes include those of White and colleagues, which designed a soluble HIV-reactive MHC class I molecule (consisting of free heavy chain + linked peptide ⁇ 2 microglobulin) for expression in baculovirus, which was capable of biotinylation/multimerization and identifying a particular T cell hybridoma by flow cytometry (White et al., 1999).
  • An additional approach was by Greten and colleagues performed standard plasmid DNA transfection to produce a peptide ⁇ 2 microglobulin-heavy chain linked protein in J588L cells that was only capable of dimerization due to mouse IgG fusion (Greten et al., 2002).
  • the present invention includes a fusion protein comprising a peptide, a first flexible linker, a b2-h ⁇ op3 ⁇ 4 ⁇ h1 ⁇ h domain, a second flexible linker, a soluble major histocompatibility complex (MHC) heavy chain and a peptide tag.
  • the peptide is an immunogenic peptide epitope.
  • the MHC is a human, mouse, rat, hamster, horse, pig, cow, simian, avian, or chimeric MHC.
  • the MHC is Class I MHC.
  • the peptide tag is selected from wherein the peptide tag is selected from at least one of: a BirA tail, a myc, a FLAG, a glutathione- S-transferase, a His tag, a maltose binding protein, hemagglutinin (HA)-tag, a V5 tag, a T7 tag, a V9 tag, a NusA-tag, a thioredoxin-tag, or a fluorescent protein-tag, a Her2/neu-tag, a CD20-tag, or a GFP-tag.
  • first, the second, or both the first and second flexible linkers comprise glycine, serine or both glycine and serine and comprise 5 to 40 residues, but may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39.
  • the peptide is 8 to 16 residues long.
  • the peptide is selected from at least one of: ABL1; ACPP; Ad5 E1A; AdV 5 Hexon; AdV Hexon; Ag85A; alfa fetoprotein; ASP-2; BA46; BALF4; BAP31; BCL-2; BCL-2A1; BCL-2L1; BCL-X; BCR-ABL; bcr-abl 210 kD fusion protein; Beta-gal; BGLAP; BMI1; BMLF1; BMRF1; BNP; BRAF 27; BRLF1; bZIP factor; BZLF1; Clorf59; CAMEL; Carbonic anhydrase; CB9L2; CD105; CD33; CD59; CEA; CEACAM; Chondromodulin; circadian clock protein PASD1; CP; CPS; cyclin-dependent kinase 4; CYP190; Cytochrome p450; DEP DC1; DLK1; Dutpase; E
  • EGFR EMNA 3 A; EMV-l; Enhanced Green Fluorescent Protein; Env; EphA2; Erkl; ESAL-6; EZH2; FAPa; FLT1; FOLR1; FOXM1; G250; GAD65; Gag; Gag-pol; Glypican 3; gp; gplOO; gp33 (C9M); GPC3; H250; HA-l; HA-2; HA-8; HBB; HBsAg; HBV core; HBV polymerase; HBV surface antigen; HCMV IE1; HCMV pp65; HCV core; HCV E; HCV NS3; HCV NS4b; HCV NS5a; HCV NS5B; Heparanase; HER2; HER-2/neu; Histocompatibility antigen 60; HIV gag p24; HIV nef; HIV pol; HIV-l env gpl20; HIV-l IIIB
  • the MHC is selected from at least one of H-2 Db, H-2 Dd; H-2 Dk; H-2 Kb; H-2 Kd; H-2 Kk; H-2 Ld; HLA- A*0l0l; HLA-A*020l; HLA-A*0301; HLA-A*1101; HLA-A*230l; HLA-A*2402; HLA- A*2902; HLA-A*680l; HLA-B*0702; HLA-B*0801; HLA-BM501; HLA-B*2705; HLA-B*3501; HLA-B*4001; HLA-B*5 l0l; or HLA-E*0101.
  • the peptide is at least one of SEQ ID NO: 1-599
  • the present invention includes a nucleic acid that expresses a fusion protein comprising a peptide, a first flexible linker, a P2-microglobulin domain, a second flexible linker, a soluble major histocompatibility complex (MHC) heavy chain and a peptide tag.
  • the peptide is an immunogenic peptide epitope.
  • the MHC is a human, mouse, rat, hamster, horse, pig, cow, simian, avian, or chimeric MHC.
  • the MHC is Class I MHC.
  • the MHC does not include a transmembrane sequence.
  • the peptide tag is selected from at least one of: a BirA tail, a myc, a FLAG, a glutathione- S-transferase, a His tag, a maltose binding protein, hemagglutinin (HA)-tag, a V5 tag, a T7 tag, a V9 tag, a NusA-tag, a thioredoxin-tag, or a fluorescent protein-tag, a Her2/neu-tag, a CD20-tag, or a GFP-tag.
  • the first, the second, or both the first and second flexible linkers comprise glycine, serine or both glycine and serine and comprise 5 to 40 residues.
  • the peptide is 8 to 16 residues long.
  • the peptide is selected from at least one of: ABL1; ACPP; Ad5 El A; AdV 5 Hexon; AdV Hexon; Ag85A; alfa fetoprotein; ASP-2; BA46; BALF4; BAP31; BCL-2; BCL-2A1; BCL-2L1; BCL-X; BCR-ABL; bcr-abl 210 kD fusion protein; Beta-gal; BGLAP; BMI1; BMLF1; BMRF1; BNP; BRAF 27; BRLF1; bZIP factor; BZLF1; Clorf59; CAMEL; Carbonic anhydrase; CB9L2; CD105; CD33; CD59; CEA; CEACAM; Chondromodulin; circadian clock protein PASD1; CP; CPS; cyclin-dependent kinase 4; CYP190; Cytochrome p450;
  • the MHC is selected from at least one of H-2 Db, H-2 Dd; H-2 Dk; H-2 Kb; H-2 Kd; H-2 Kk; H-2 Ld; HLA-A*0l0l; HLA-A*020l; HLA- A*030l; HLA-A*l l0l; HLA-A*230l; HLA-A*2402; HLA-A*2902; HLA-A*680l; HLA- B*0702; HLA-B*0801; HLA-B*l50l; HLA-B*2705; HLA-B*3501; HLA-B*400l; HLA-B*5 l0l; or HLA-E*0l0l .
  • the peptide is at least one of SEQ ID NO:l-599.
  • the present invention includes a method of making a soluble eukaryotic-derived peptide/MHC complex comprising: expressing in a cell a fusion protein comprising a peptide, a first flexible linker, a 2-microglobulin domain, a second flexible linker, a soluble major histocompatibility complex (MHC) heavy chain and a peptide tag.
  • the method further comprises isolating the fusion protein from a supernatant.
  • the method further comprises forming dimers, trimers, tetramers, or multimers of the fusion protein by mixing the fusion protein with one or more agents that bind to two or more fusion proteins.
  • the agent is selected from an antibody, a cross-linking agent, a ligase, an avidin, a streptavidin, a Protein A, or a J-chain.
  • the peptide is an immunogenic peptide epitope.
  • the MHC is a human, mouse, rat, hamster, horse, pig, cow, simian, avian or chimeric MHC. In another aspect, the MHC is Class I MHC.
  • the MHC does not include a transmembrane sequence
  • the peptide tag is selected from at least one of: a BirA tail, a myc, a FLAG, a glutathione-S-transferase, a His tag, a maltose binding protein, hemagglutinin (HA)-tag, a V5 tag, a T7 tag, a V9 tag, a NusA-tag, a thioredoxin-tag, or a fluorescent protein-tag, a Her2/neu-tag, a CD20-tag, or a GFP-tag.
  • the irst, the second, or both the first and second flexible linkers comprise glycine, serine or both glycine and serine and comprise 5 to 40 residues. In another aspect, the peptide is 8 to 16 residues long.
  • the present invention includes a cell line expressing a fusion protein comprising a fusion protein comprising a peptide, a first flexible linker, a p2-microglobulin domain, a second flexible linker, a soluble major histocompatibility complex (MHC) heavy chain and a peptide tag.
  • the fusion protein is integrated into the genome by co-transfecting a fusion protein expressing vector with a transposase vector that expresses a transposase and wherein the fusion protein expressing vector, the transposase vector, or both further comprise a selectable marker.
  • the peptide is an immunogenic peptide epitope.
  • the MHC is a human, mouse, rat, hamster, horse, pig, cow, simian, avian, or chimeric MHC. In another aspect, the MHC is Class I MHC. In another aspect, the MHC does not include a transmembrane sequence.
  • the peptide tag is selected from at least one of: a BirA tail, a myc, a FLAG, a glutathione-S-transferase, a His tag, a maltose binding protein, hemagglutinin (HA)-tag, a V5 tag, a T7 tag, a V9 tag, a NusA-tag, a thioredoxin-tag, or a fluorescent protein-tag, a Her2/neu- tag, a CD20-tag, or a GFP-tag.
  • the first, the second, or both the first and second flexible linkers comprise glycine, serine or both glycine and serine and comprise 5 to 40 residues.
  • the peptide is 8 to 16 residues long.
  • FIG. 1A shows a representative DNA schematic of the synthetic peptide/MHC complex.
  • the designed peptide/MHC molecule contains distinct peptide, beta-2-microglobulin, and MHC heavy chain regions that are linked via explicit glycine/serine amino acids.
  • the MHC heavy chain lacks a transmembrane domain (designated sHeavy chain), ensuring that properly folded peptide/MHC protein is secreted from cells into the culture medium.
  • the peptide/MHC construct also contains a terminal BirA tail for enzymatic conjugation of biotin.
  • FIG. 1B shows a standard workflow for expressing, purifying, and biotinylating eukaryotic-derived peptide/MHC molecules.
  • a suitable eukaryotic host cell line (such as CHO cells) is transiently transfected with two vectors that allow for transposon-directed integration of genes of interest.
  • Vector 1 encodes the SB transposase (SB100X) while vector 2 is a transposon-compatible plasmid that encodes the synthetic peptide/MHC complex and puromycin resistance transgenes.
  • SB100X SB transposase
  • vector 2 is a transposon-compatible plasmid that encodes the synthetic peptide/MHC complex and puromycin resistance transgenes.
  • a stable cell line secreting peptide/MHC complexes is generated in as little as 2 weeks following antibiotic selection and expansion.
  • Spent culture media is processed through AC (against the sHeavy chain) to selectively purify peptide/MHC complexes.
  • a biotin ligase may then be employed to enzymatically conjugate biotin to the BirA tail of the synthetic peptide/MHC protein.
  • the peptide/MHC reagent can be incorporated into immunologically-relevant assays that, for example, detect antigen-specific T cells.
  • FIGS. 2A and 2B show that stable CHO cells lines were established by a SB transposon system to secrete peptide/MHC molecules into culture media.
  • FIG. 2A Evidence of extracellular peptide/MHC protein was first determined from cell-free supernatants by SDS-PAGE and coomassie blue staining.
  • FIG. 2B Representative chromatogram of small scale AC purification of peptide/MHC-containing media. Cell-free supernatant was passed through an equilibrated agarose bead column containing the MHC class I-reactive antibody Ml/42. After washing away unbound material, peptide/MHC protein was eluted, buffer-exchanged, and concentrated. Protein purity was then assessed using SDS-PAGE and coomassie blue staining. Arrow inset indicating peptide/MHC around the predicted molecular weight. Abbreviation used: protein ladder (L), affinity chromatography (AC), preparation (prep)
  • FIG. 3A shows Purified peptide/MHC (i.e., SIINFEKL/H-2Kb) identify was confirmed by western blot using monoclonal antibodies reactive to the b2 microglobulin and BirA tail regions of the design molecule as depicted in FIG. 1A.
  • FIG. 3B shows that peptide/MHC ligand binding was determined through immunoprecipitation and western blot following incubation of SIINFEKL/H- 2Kb and a TCR-like antibody specific to this particular peptide/MHC class I complex.
  • AC -purified peptide/MHC was also incorporated as a positive control.
  • FIGS. 4A to 4C show that the peptide/MHC was biotinylated as detailed in the Materials and Methods and specific streptavidin binding initially confirmed through (FIG. 4A) western blot and (FIG. 4B) ELISA using wells coated with streptavidin.
  • FIG. 4C Biotinylated peptide/MHC was also incubated with streptavidin-conjugated 5 pm beads and washed extensively. Beads were then exposed to isotype, anti-SIINFEKL/H-2Kb, or anti-MHC monoclonal antibodies followed by washing steps and incubation with relevant secondary PE-conjugated antibodies.
  • FIG. 5 shows that the biotinylated peptide/MHC was incubated with PE-conjugated streptavidin to produce“small-scale” batches of SIINFEKL-reactive tetramers.
  • Purified CD8+ T cells harvested from either wild-type or OT-1 mice were incubated with tetramers, washed, and stained with an anti-CD8 FITC antibody. Cells were again washed, fixed, and analyzed by flow cytometry for ligand binding.
  • WT wild-type
  • SA streptavidin
  • FIG. 6 shows the determination of the ability to construct and express a membrane-bound OYA-specific peptide/MHC molecule (i.e., SIINFEKL/H-2Kb).
  • SIINFEKL/H-2Kb a membrane-bound OYA-specific peptide/MHC molecule
  • 4T1 cells were transiently transfected with two plasmids that allow transposon-directed genomic integration and cultured over a period of two weeks in puromycin-containing culture media (as outlined in FIG. IB).
  • the remaining“stably engineered” cells were then incubated with either an isotype control antibody or antibody that binds the OVA-specific epitope SIINFEKL within the constraints of MHC class I and assessed for reactivity by flow cytometry.
  • SIINFEKL epitope + MHC class I SIINFEKL/H-2Kb
  • MHC major histocompatibility complex
  • FIG. 7 is a DNA coding sequence of the synthetic SIINFEKL/H-2Kb molecule (SEQ ID NO: 600).
  • the present invention includes compositions, vectors, cells, and methods of making MHC class I-specific reagents such as fluorescently-labeled multimers (e.g., tetramers) that can be used to study CD8+ T cells under normal and diseased states.
  • MHC class I-specific reagents such as fluorescently-labeled multimers (e.g., tetramers) that can be used to study CD8+ T cells under normal and diseased states.
  • recombinant MHC class I components comprising MHC class I heavy chain and b2 microglobulin
  • the present inventors have developed an alternative and rapid approach to generating soluble and fully-folded MHC class I molecules in eukaryotic cell lines (such as CHO cells) using, e.g., a Sleeping Beauty transposon system.
  • this method generates stable cell lines that reliably secrete epitope-defined MHC class I molecules into the tissue media for convenient purification and eventual biotinylation/multimerization.
  • MHC class I components are covalently linked, providing the opportunity to produce a diverse set of CD8+ T cell-specific reagents bearing peptides with various affinities to MHC class I.
  • the term“gene” refers to a functional protein, polypeptide or peptide encoding unit.
  • this functional term includes both genomic sequences, cDNA sequences, or fragments or combinations thereof, as well as gene products, including those that may have been altered by the hand of man.
  • Purified genes, nucleic acids, protein and the like are used to refer to these entities when identified and separated from at least one contaminating nucleic acid or protein with which it is ordinarily associated.
  • the term "vector” is used in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another.
  • the vector may be further defined as one designed to propagate specific sequences, or as an expression vector that includes a promoter operatively linked to the specific sequence, or one designed to cause such a promoter to be introduced.
  • the vector may exist in a state independent of the host cell chromosome, or may be integrated into the host cell chromosome
  • the term“host cell” refers to cells that have been engineered to contain nucleic acid segments or altered segments, whether archeal, prokaryotic, or eukaryotic. Thus, engineered, or recombinant cells, are distinguishable from naturally occurring cells that do not contain recombinantly introduced genes through the hand of man.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it effects the transcription of the sequence; or
  • a ribosome binding site is operably linked to e coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” refers to a DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in same reading frame. Enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice.
  • the terms “cell” and “cell culture” are used interchangeably end all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Different designations are will be clear from the contextually clear.
  • plasmid are designated by a lower case p preceded and/or followed by capital letters and/or numbers and refer to self-replicating circular DNA that include an origin of replication, and typically one or more selectable markers.
  • the starting plasmids herein are commercially available, are publicly available on an unrestricted basis, or can be constructed from such available plasmids in accord with published procedures.
  • other equivalent plasmids are known in the art and will be apparent to the ordinary artisan.
  • selectable marker refers to the use of a gene that encodes an enzymatic activity and which confers the ability to grow in medium lacking what would otherwise be an essential nutrient (e.g., the HIS3 gene in yeast cells); in addition, a selectable marker may confer resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed.
  • protein As used herein the terms “protein”, “polypeptide” or “peptide” refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably.
  • fusion protein or“chimeric protein” refer to a hybrid protein, that includes portions of two or more different polypeptides, or fragments thereof, resulting from the expression of a polynucleotide that encodes at least a portion of each of the two polypeptides.
  • transformation refers to a process by which exogenous DNA enters and changes a recipient cell. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed” cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome.
  • transfection refers to the introduction of foreign DNA into eukaryotic cells. Transfection may be accomplished by a variety of methods known to the art including, e.g., calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
  • stable transfection or “stably transfected” refers to the introduction and integration of foreign DNA into the genome of the transfected cell.
  • stable transfectant refers to a cell that has stably integrated foreign DNA into the genomic DNA.
  • transient transfection or “transiently transfected” refers to the introduction of foreign DNA into a cell where the foreign DNA fails to integrate into the genome of the transfected cell.
  • the foreign DNA persists in the nucleus of the transfected cell for several days. During this time the foreign DNA is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes.
  • transient transfectant refers to cells that have taken up foreign DNA but have failed to integrate this DNA.
  • the present invention can be used to circumvent drawbacks in the prior art, in particular, stabilizing peptide binding to the MHC peptide binding groove.
  • Previous efforts have revealed the ability to engineer and produce peptide/MHC molecules in bacteria by covalently joining the MHC class I peptide, b2 microglobulin, and heavy chain with discrete amino acid linkers (designated single-chain trimers [SCTs]) (Yu et al., 2002).
  • SCTs single-chain trimers
  • these engineered proteins fold correctly and specifically engage CD8+ T cells as tetramers (Mitaksov et al., 2007), irrespective of the artificial linker design (Hansen et al., 2009).
  • this particular SCT method still utilizes a bacterial expression system and requires substantial purification and refolding efforts.
  • the present inventors have developed an alternative method to potentially improve the production of peptide/MHC based on the SCT approach.
  • the present invention has the ability to rapidly generate eukaryotic cell lines that stably express and secrete peptide/MHC into the tissue media for purification and biotinylation.
  • This novel protocol provides a much faster/convenient route to generating properly folded peptide/MHC with minimal user intervention, especially for MHC class I targets with high demand (such as the model OVA epitope SIINFEKL).
  • MHC class I targets with high demand (such as the model OVA epitope SIINFEKL).
  • SCT strategy it was possible to generate MHC molecules presenting a range of class I peptides (i.e., low-to-high binding affinity), which can be reliably generated.
  • these eukaryotic- derived peptide/MHC molecules can be used to recapitulate binding dynamics with TCRs in downstream assays (Schmidt and Lill 2018).
  • mice Female 6-8-week-old C57BL/6J (stock #000664) and OT-l (stock #003831) mice were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA) and maintained in micro isolator cages under sterile conditions. Animals were humanely euthanized and spleens/lymph nodes harvested and combined for Ficoll gradient centrifugation (GE Healthcare, Piscataway, NJ). The lymphocyte interphase was then subjected to ACK lysis and eventual CD8+ T cell purification using MACS bead positive selection as instructed by the manufacturer (Miltenyi Biotec, Cambridge, MA). Purified CD8+ T cells were aliquoted in 90% FBS/l0% DMSO and stored in liquid nitrogen until use. All mouse procedures were followed in accordance with TTUHSC IACUC-approved protocols.
  • the leader signal, SIINFEKL epitope, and Gly/Ser amino acid linkers were ultimately added by overhang PCR as previously reported (Hansen et al., 2009) using the Phusion High-Fidelity DNA Polymerase (Thermo Scientific Fisher). PCR fragments were gel excised/purified and ligated (LigaFastTM Rapid DNA Ligation System; Promega, Madison, WI) into pucl9 (NEB, Ipswich, MA) via Sacl/Hindlll restriction enzyme sites and fragments pieced together using the unique Nhel/BamHI sites of the synthetic peptide/MHC sequence.
  • the BirA AviTagTM amino acid sequence (GLNDIFEAQKIEWF1E SEQ ID NO: 600) was subsequently added to the construct’s terminus by overlap-extension PCR and cloned into a separate pucl9 holding vector. Full-length peptide/MHC was then amplified and cloned into the pSBbi-pur transposon vector (kindly provided by Dr. Eric Kowarz [Addgene plasmid # 60523]) using the Sfil restriction enzyme sites (Kowarz et al., 2015). Plasmid transformations were carried out in chemically-competent NEB-5 alpha E. coli (NEB) using ampicillin selection.
  • SB Sleeping Beauty transposon system.
  • Parental cell lines were transiently transfected with transposon-related vectors using Lipofectamine reagent (Thermo Fisher Scientific) as directed by the manufacturer.
  • a plasmid encoding the SB 100X transposase (pCMV[CAT]T7-SB100; designated Vector 1), a gift from Dr. Zsuzsanna Izsvak (Addgene plasmid # 34879), and a transposon plasmid containing the necessary inverted terminal repeats (pSBbi-pur; designated Vector 2) were used.
  • Both vectors were provided concurrently to stably integrate peptide/MHC transgenes into cells.
  • lxlO 5 cells were plated in 24-well plates (Coming, Corning NY) and exposed to Vector 1 (12.5 ng)/Vector 2 (488 ng) in a total volume of 500 pl media as similarly described (Kowarz et al., 2015).
  • Culture media was replenished with 2 ml fresh media after 24 hrs and 48 hrs, whereupon cells were exposed to lethal doses of puromycin (CHO-S - 10 pg/ml; 4T1 - 5 pg/ml) (Invivogen, San Diego, CA).
  • Fresh media containing puromycin was provided every 2-3 days as needed.
  • actively dividing cells were ready for expansion by 2 weeks, with virtually all cell clones expressing peptide/MHC molecules.
  • proteins were resolved on a 4%/ 12% polyacrylamide gel, transferred to a PVDF membrane (Amersham Hybond, 0.2pm; GE Healthcare), and blocked with 5% milk in PBST (0.1% Tween 20 in PBS) for 1 hr at RT.
  • Membranes were then incubated with various primary antibodies (1 pg/ml) in block solution with rocking at 4 °C overnight.
  • Specific primary reagents included anti mouse b 2 microglobulin (clone 893803; R&D Systems, Minneapolis, MN) or anti-BirA tail (clone Abe; Avidity, Aurora, CO) antibodies.
  • Blots were then washed extensively with PBST and incubated in block with secondary HRP-conjugated goat antibodies specific to rat IgG (H+L) or mouse IgG (H+L) (Thermo Scientific Fisher) for 1 hr at RT. Washed blots were finally developed with a SignalFire ECL reagent (Cell Signaling, Danvers, MA) and exposed/imaged on a ChemiDocTM Touch Imaging System (Bio-Rad, Hercules, CA). In separate experiments, blots containing biotinylated protein were probed with a Streptavidin-HRP reagent (Thermo Scientific Fisher) for 1 hr at RT to confirm streptavidin binding potential.
  • a Streptavidin-HRP reagent Thermo Scientific Fisher
  • the anti- SIINFEKL antibody functions as a TCR-like antibody (Lowe et al., 2017), and binds SIINFEKL/H- 2Kb much like SIINFEKL-reactive T cells such as OT-l CD8+ T cells.
  • samples were boiled and analyzed by western blot as described above.
  • Affinity chromatography (AC).
  • the MHC class I-reactive Ml/42 antibody Bio X Cell, West Lebanon, NH was covalently bound to an NHS-activated agarose bead column (HiTrapTM NHS-activated HP; GE Healthcare) manually as directed by the manufacturer.
  • the column was attached to an AKTATM start chromatography system (GE Healthcare) for automatic operation and equilibrated with 20 mM sodium phosphate, pH 7.0 (binding buffer). Cell-free supernatants were first desalted in PBS using PD-10 columns and diluted 1 :2 in binding buffer prior to AC.
  • biotinylated peptide/MHC was incubated with PE-conjugated streptavidin (BD Biosciences, San Jose, CA) at a 4: 1 molar ratio in the dark for 30 min at RT Tetramers were then stored at 4 °C shielded from light.
  • PE-conjugated streptavidin BD Biosciences, San Jose, CA
  • Enzyme-Linked Immunosorbent Assay (ELISA). To investigate the success of peptide/MHC biotinylation, a 96-well high protein binding plate (Corning) was first coated overnight at 4 °C with 4 pg/ml streptavidin (Promega) in 0.1 M sodium carbonate and then blocked (3% BSA/PBS) for 1 hr at RT. Biotinylated protein samples were diluted in block and added to wells at various dilutions and incubated at RT for 1 hr.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • the 4T1 cell line (1x10 s cells) was stained with relevant primary antibodies (anti-SIINFEKL/H-2Kb or mouse IgG isotype antibodies at 2 pg/ml) in FACS buffer (0.5% BSA/0.1% NaN 3 in PBS) for 20 min at 4 °C, washed, incubated with a PE-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch), washed again, and resuspended in FACS buffer for analysis (FIG. 6).
  • relevant primary antibodies anti-SIINFEKL/H-2Kb or mouse IgG isotype antibodies at 2 pg/ml
  • FACS buffer (0.5% BSA/0.1% NaN 3 in PBS
  • biotinylated peptide/MHC was incubated with 5 pm PMMA beads conjugated to streptavidin (Poly An, Berlin, Germany), washed, and incubated at 4 °C with correct pairs of primary and PE-conjugated secondary antibodies (as indicated in FIGS. 4A to 4C) prior to analysis in FACS buffer.
  • Frozen CD8+ T cells were thawed, washed, and resuspended in FACS buffer so that each stain consisted of 2xl0 5 viable CD8+ T cells.
  • transmembrane region of MHC class I (designated sHeavychain) to allow for functional and fully- folded secreted protein.
  • This process for eukaryotic expression centered on the use of a SB transposon system to stably integrate transgene content into relevant cell lines (FIG. IB). Therefore, CHO cells would be transiently transfected with transposon-relevant plasmids (encoding separately a SB transposase and synthetic peptide/MHC molecule) and stable cells generated through antibiotic selection. After expanding relevant CHO cell clones, secreted peptide/MHC could be purified by AC, biotinylated, and multimerized to produce, for example, tetramers capable of binding antigen- specific T cells.
  • CHO cells were next transfected with SB-related vectors as described in FIGS. 1A and 1 B to induce stable expression of soluble peptide/MHC
  • Parental CHO cells exhibited enhanced resistance to puromycin, requiring sustained culturing in high concentrations of puromycin at 10 pg/ml.
  • the inventors were able to expand puromycin-resistant clones by 2 weeks post plasmid transfection Cells were then grown to saturating conditions in culture for at least 4 days to generate suitable whole protein concentrations from a total volume of 20 ml media.
  • the choice of serum-free media contained a number of proprietary agents such as surfactants that could potentially interfere with protein purification and validation assays.
  • the inventors therefore, took the precautionary step of desalting and concentrating cell-free supernatants in PBS prior to downstream analysis.
  • the initial assessment of CHO-derived extracellular proteins by SDS-PAGE (under reducing conditions) and coomassie blue staining revealed distinct protein bands around a predicted 51 kDa molecular weight for synthetic peptide/MHC (FIG. 2A) that was not evident from CHO cells expressing a null construct (data not shown). This specific protein band was subsequently confirmed following AC as exhibited in FIG. 2B. That is, cell-free supernatants were passed onto an agarose bead column containing the MHC class I-reactive Ml/42 antibody and bound material eluted and ultimately assessed again by SDS-PAGE.
  • the inventors typically harvested at least 100 ug/ml AC -purified peptide/MHC from small-scale culturing efforts. However, the described protocol can be scaled-up (or down) depending on the desired peptide/MHC total yield. Although beyond the scope of this report, alternative culturing parameters and/or leader signals (Haryadi et al., 2015) may potentially enhance overall CHO secretion of peptide/MHC.
  • the AC procedure appeared to provide substantial protein purity based on obtaining [i] one distinct chromatogram elution peak and [ii] a protein band comprising > 95% of detectable protein by coomassie blue staining.
  • SIINFEKL/H-2Kb was incubated with 2 pg of anti-SIINFEKL/H-2Kb or isotype control antibodies overnight.
  • IgG containing material was purified using protein A/G-complexed agarose and resolved by SDS-PAGE under reducing conditions.
  • the presence of SIINFEKL/H-2Kb was further established through western blot using an anti-mouse b2 microglobulin antibody (FIG. 3B).
  • the inventors assessed the ability of fluorescently-labeled multimers to specifically detect antigen-specific CD8+ T cells.
  • Splenocytes and lymph nodes were harvested from wild-type and OT-1 transgenic (i.e., SIINFEKL-reactive) mice and CD8+ T cells subsequently purified through magnetic bead selection.
  • Biotinylated SIINFEKL/H-2Kb was then incubated with PE- conjugated streptavidin following a 4: 1 molar ratio, thereby, generating tetramers.
  • CD8+ T cells were exposed to dasatinib (as previously described [Dolton et al., 2015]), followed by incubation with tetramers. After suitable wash steps, cells were specifically labeled with a FITC-conjugated anti-mouse CD8 antibody that displays minimal interference with TCR:MHC binding (Clement et al., 2011). Cells were fixed and double positive events (CD8+/tetramer+) assessed by flow cytometry. As detailed in FIG. 5, CD8+ OT-l cells clearly bound PE-labeled tetramers, with most cells displaying CD8 and tetramer positivity (in comparison to wild-type mice).
  • MHC class I peptide candidates can be easily identified through in silico prediction methods (Andreatta and Nielsen 2016), free peptide occupancy of MHC class I molecules tends to be a rate limiting step in successfully generating stable peptide/MHC molecules from bacteria (Altman and Davis 2016).
  • MHC plays in human health (Cho and Sprent 2018)
  • an inability to produce certain peptide/MHC reagents may adversely impact efforts on a number of fronts including diagnostics, therapies, and generalized scientific endeavors.
  • membrane-bound SCTs serve as effective targets for CD8+ T cell priming (Hung et al., 2007) and destruction (Yu et al., 2002).
  • SCTs can be modified for expression/secretion in bacteria and biotinylation by way of an explicit amino acid sequence that directs BirA enzyme function (Mitaksov et al., 2007).
  • eukaryotic cell lines such as CHO suspension cells
  • Soluble peptide/MHC may then be biotinylated and utilized as multimers (via streptavidin), particularly for CD8+ T cell relevant assays.
  • CHO cells are an industry standard in producing FDA approved therapeutic recombinant proteins (Kuo et al., 2018).
  • the inventors sought the advantages of the CHO cell line in order to [i] instigate post-translational modifications and [ii] be easily grown at high density under serum-free conditions in suspension cultures.
  • other common cell line“protein workhorses” e.g., HEK-293 cells
  • HEK-293 cells are amenable to the expression and characterization techniques outlined.
  • One advantage of the present invention is the rapid development of stable CHO cells secreting peptide/MHC using the SB transposon system.
  • SB transposase SB100X which has a high gene insertion efficiency at close-to-random chromosomal sites.
  • the SB approach provides the advantageous properties of viral transduction to insert transgenes without the disadvantages of either maintaining genomic material episomally (e.g., adeno-associated viruses) or near/in proto-oncogenes (e.g., retroviral vectors) (Kebriaei et al., 2017). Additionally, manufacturing high-quality viral particles can be a cumbersome task fraught with regulatory obstacles.
  • the inventors generally experienced minimal difficulty in developing and expanding stable lines from parental cells after transiently transfecting plasmids that propagated the SB transposon system.
  • the protocol is also amenable to freezing material at convenient stopping points. There was no apparent adverse effects to generating multimerized reagents when either cell-free CHO-derived supernatants or biotinylated peptide/MHC was frozen long-term at -80 °C.
  • the method of the present invention is compatible with downstream tetramer production with the added benefits of convenient peptide/MHC expression that can incorporate a range of peptide affinities to the MHC peptide binding groove.
  • tetravalent multimers have been utilized to increase T cell avidity.
  • these soluble peptide/MHC molecules can be utilized for higher order reagents to better discriminate low frequency T cells (or bind“difficult” TCRs) such as fluorochrome-conjugated dextramers since the production process involves incubating biotinylated peptide/MHC with a dextran backbone containing streptavidin (Dolton et al., 2014).
  • the present invention can be used to reliably produce a range of soluble eukaryotic-derived peptide/MHC molecules for diagnostic, therapeutic, and investigative purposes.
  • the words“comprising” (and any form of comprising, such as“comprise” and“comprises”),“having” (and any form of having, such as “have” and“has”),“including” (and any form of including, such as“includes” and“include”) or “containing” (and any form of containing, such as“contains” and“contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps.
  • “comprising” may be replaced with“consisting essentially of’ or“consisting of’.
  • the phrase“consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention.
  • the term“consisting” is used to indicate the presence of the recited integer (e g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only.
  • words of approximation such as, without limitation,“about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present.
  • the extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature.
  • a numerical value herein that is modified by a word of approximation such as“about” may vary from the stated value by at least ⁇ 1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.
  • Anti-CD8 antibodies can trigger CD8+ T cell effector function in the absence of TCR engagement and improve peptide-MHCI tetramer staining. J Immunol. 2011 Jul 15; l87(2):654-63.
  • Hung CF Calizo R, Tsai YC, He L, Wu TC.
  • a DNA vaccine encoding a single-chain trimer of HLA-A2 linked to human mesothelin peptide generates anti-tumor effects against human mesothelin-expressing tumors.
  • Vaccine 2007 Jan 2;25(1): 127-35.
  • CEACAM1 promotes CD8+ T cell responses and improves control of a chronic viral infection. Nat Commun. 2018 Jul 2;9(l):256l.

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Abstract

The present invention includes compositions and methods of making and using a fusion protein comprising a peptide, a first flexible linker, a β2-microglobulin domain, a second flexible linker, a soluble major histocompatibility complex (MHC) heavy chain and a peptide tag.

Description

METHOD TO EXPRESS, PURIFY, AND BIOTINYLATE EUKARYOTIC CELL- DERIVED MAJOR HISTOCOMPATIBILITY COMPLEXES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U S. Provisional Application Serial No. 62/746,198, filed October 16, 2018, the entire contents of which are incorporated herein by reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates in general to the field of immunology, and more particularly to novel methods for expressing, purifying, and post-translationally modifying eukaryotic cell-derived major histocompatibility complexes. STATEMENT OF FEDERALLY FUNDED RESEARCH
[0003] This invention was made with government support under R15 CA215874 and W81XWH- 18-1-0293 awarded by the National Institutes of Health and the Department of Defense, respectively. The government has certain rights in the invention.
INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC
[0004] The present application includes a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 14, 2019, is named TECH213 lWO_SeqList.txt and is 114, kilo bytes in size.
BACKGROUND OF THE INVENTION
[0005] Without limiting the scope of the invention, its background is described in connection with methods of making eukaryotic cell-derived major histocompatibility complexes (MHC).
[0006] The major histocompatibility complex (MHC) class I and II molecules play an integral role in T cell development and peripheral effector responses (Alcover et al., 2018). MHC class I is retained on the plasma membrane of nucleated cells and consists of a multi-unit heavy chain whose tertiary structure is stabilized by b2 microglobulin through non-covalent forces (Wieczorek et al., 2017). To provide specific binding to antigen specific CD8+ T cells, MHC class I usually retains a short 8-10 amino acid peptide within the MHC peptide binding groove that is derived from degraded intracellular proteins (hereafter referred to as peptide/MHC). [0007] The present understanding of basic T cell properties and dynamics under a variety of normal and diseased settings has been greatly advanced by the ability to produce and purify peptide/MHC for use in assays to specifically engage the T cell receptor (TCR). Arguably the most widespread approach incorporates fluorochrome-conjugated peptide/MHC multimers (e.g., tetramers) for analyzing or isolating antigen-specific CD8+ T cells from biological samples (Khaimar et al., 2018; Soen et al., 2003). Peptide/MHC generation has continued similarly to the process outlined in the landmark work by Altman and colleagues (Altman et al., 1996). Briefly, b2 microglobulin and MHC class I heavy chain (containing a BirA tail) are individually expressed in E. coli and later purified from inclusion bodies through a laborious lysis/solubilization process. A defined MHC class I peptide is then added alongside b2 microglobulin and heavy chain in a precise folding reaction mixture that requires several days to complete prior to affinity chromatography (AC) purification of properly folded peptide/MHC and later biotinylation steps. Although this standard production process works to eventually yield excellent reagents for immunologic assays, there exist a number of major disadvantages. Namely, the standard method is [i] time consuming, [ii] requires substantial levels of raw ingredients (particularly purified MHC class I peptide), and [iii] cannot guarantee large-scale production of properly folded peptide/MHC molecules based on predicted peptide binders. For example, it is extremely difficult to stably produce MHC molecules bearing peptides with low-to-moderate affinity to the MHC peptide-binding groove.
[0008] Prior attempts to make peptide/MHC complexes include those of White and colleagues, which designed a soluble HIV-reactive MHC class I molecule (consisting of free heavy chain + linked peptide^2 microglobulin) for expression in baculovirus, which was capable of biotinylation/multimerization and identifying a particular T cell hybridoma by flow cytometry (White et al., 1999). An additional approach was by Greten and colleagues performed standard plasmid DNA transfection to produce a peptide^2 microglobulin-heavy chain linked protein in J588L cells that was only capable of dimerization due to mouse IgG fusion (Greten et al., 2002).
[0009] Thus a need remains for novel compositions, methods, vectors, cells, etc., that include novel constructs that allow for the production of peptide-MHC complexes in eukaryotic cells.
SUMMARY OF THE INVENTION
[0010] In one embodiment, the present invention includes a fusion protein comprising a peptide, a first flexible linker, a b2-hΰop¾^h1ΐh domain, a second flexible linker, a soluble major histocompatibility complex (MHC) heavy chain and a peptide tag. In one aspect, the peptide is an immunogenic peptide epitope. In another aspect, the MHC is a human, mouse, rat, hamster, horse, pig, cow, simian, avian, or chimeric MHC. In another aspect, the MHC is Class I MHC. In another aspect, the does not include a transmembrane sequence. In another aspect, the peptide tag is selected from wherein the peptide tag is selected from at least one of: a BirA tail, a myc, a FLAG, a glutathione- S-transferase, a His tag, a maltose binding protein, hemagglutinin (HA)-tag, a V5 tag, a T7 tag, a V9 tag, a NusA-tag, a thioredoxin-tag, or a fluorescent protein-tag, a Her2/neu-tag, a CD20-tag, or a GFP-tag. In another aspect, the first, the second, or both the first and second flexible linkers comprise glycine, serine or both glycine and serine and comprise 5 to 40 residues, but may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39. In another aspect, the peptide is 8 to 16 residues long. In another aspect, the peptide is selected from at least one of: ABL1; ACPP; Ad5 E1A; AdV 5 Hexon; AdV Hexon; Ag85A; alfa fetoprotein; ASP-2; BA46; BALF4; BAP31; BCL-2; BCL-2A1; BCL-2L1; BCL-X; BCR-ABL; bcr-abl 210 kD fusion protein; Beta-gal; BGLAP; BMI1; BMLF1; BMRF1; BNP; BRAF 27; BRLF1; bZIP factor; BZLF1; Clorf59; CAMEL; Carbonic anhydrase; CB9L2; CD105; CD33; CD59; CEA; CEACAM; Chondromodulin; circadian clock protein PASD1; CP; CPS; cyclin-dependent kinase 4; CYP190; Cytochrome p450; DEP DC1; DLK1; Dutpase; E6; E7; EBNA 1; EBNA 3A; EBNA 3B; EBNA 6; EBV BRLF1; EBV EBNA3A; EBV LMP2; EDDR1;
EGFR; EMNA 3 A; EMV-l; Enhanced Green Fluorescent Protein; Env; EphA2; Erkl; ESAL-6; EZH2; FAPa; FLT1; FOLR1; FOXM1; G250; GAD65; Gag; Gag-pol; Glypican 3; gp; gplOO; gp33 (C9M); GPC3; H250; HA-l; HA-2; HA-8; HBB; HBsAg; HBV core; HBV polymerase; HBV surface antigen; HCMV IE1; HCMV pp65; HCV core; HCV E; HCV NS3; HCV NS4b; HCV NS5a; HCV NS5B; Heparanase; HER2; HER-2/neu; Histocompatibility antigen 60; HIV gag p24; HIV nef; HIV pol; HIV-l env gpl20; HIV-l IIIB gpl20; HIV-l nef; HIV-l pl7; HIV-l RT; HIV-l US4 gpl20; HIURP; HLA-A leader sequence peptide; HLA-A2; HLA-Cw3; chlamydia trachomatis MOMP ; HM1.24; HMMR; HMOX1; HO-1; hPSA; HPV 16 E6; HSP105; HSP90 alpha; hTERT; hTOM34p; hTRT; H-Y; IA-2; IAPP; IDO; IE-l; IE62; IGF; IGRP; ILl3r; ILl3Ra; Ilrl; Influenza A (PR8) NP ; Influenza A MP; Influenza A MP1; Influenza A MP2; Influenza A NP; Influenza A PB1; INF /R; Insulin; Interferon gamma inducible protein (GILT) 30; IRS-2; ITGB8; K8.1; KIF20A; KLK; KLK3; Ll; LANA; Large T antigen; Lengsin; Listeria monocytogenes Listeriolysin; LIVIN; LMP-1; LMP-2; LMP-2A; LMP-2A; LY6K; ml39; ml4l; ml45; M164; M2- 1; M38; M45; M57; MAGE-1; MAGE-10; MAGE-3; MAGE-4; MAGE-A1; MAGEA2; MAGE A3; MAGE-A5; MAGE-C1; MAGE-C2, MART-1; MC38 adpgk neoantigen; MC-38; MCL-1; MCMV IE1; MELK; Mena; Middle T antigen; Midkine; miHAg SMCY; MOG precursor; MP; MPP11; MS4A1; MSLN; Mtb 16 kDa; Mtb 19 kDa; Mtb85A; mTERT; Mucin; muFAPa; MuLV env ; Murine Survivin; MYBPC-2; Mycobacterium bovis antigen 85-A; Mycobacterium tuberculosis ESAT-6; Mycobacterium tuberculosis TB10.4; Myelin basic protein; ND; NEF; NEF; Neu/Her- 2/Erbb2 proto-oncoprotein; NG2; Non muscle Myosin-9; Non-muscle Myosin; NP; NP396; NP52; NPM1; NRP-1; NRP-2; NRP-V7 superagonist peptide 8.3 Tg NOD mouse; NS3; Nucleocapsid; Nuf2; NY-ESO-1; OVA; Ovalbumin (subdominant); P1A; P2X5a; p53; p56; P79; PAP-3; PASD1; PAX-5; p-Cadherin; PDGFRbeta; PLAC1; PLAM csp; Plasmodium berghei ANKA acid phosphatase; Plasmodium berghei CSP; Plasmodium CSP; Plasmodium falciparum CSP; Plasmodium falciparum Liver stage antigen; Pol A; Polymerase; Polyprotein; pp65; PPE; Prl; PRAME; Promininl; PSA; PSCA; PSM P2; PSMA; RGS5; RhoC; RNF43; RSV A strain F protein; RSV NP; RT; S protein; SAA; SART3; Sialidases ; SIV gag; SMCY; Spike GP; STEAP1; Surface IgG (sA20-Ig) of A20; Survivin; Survivin-3a; SV40 T antigen; TACE; TARP; TARP 2M; Tax; TB10.3-4; Telomerase; TEM1; tgd057; TGF ; TNP03; topll; TPBG; TPR-protein; TRAG; TRP2; Trypanosoma cruzi ASP-2; Trypanosoma cruzi SP; TTK; Tyrosinase; Tyrosine-3 -hydroxylase; Ubiquitin; UL105; UL138; UL44; USP9Y; V131; Vaccinia virus Copenhagen Protein G5 ; Vaccinia virus Flost range protein 2 ; VACCL3_l00; VEGFR1; VEGFR2; VEGFR2/KDR fragment 1; Vif; Vinculin; VP1; Vpu; VSV N; West Nile virus NY-99 polyprotein precursor; West Nile Virus polyprotein; WT1; Yellow Fever Virus 17D polyprotein; or ZnT-8. In another aspect, the MHC is selected from at least one of H-2 Db, H-2 Dd; H-2 Dk; H-2 Kb; H-2 Kd; H-2 Kk; H-2 Ld; HLA- A*0l0l; HLA-A*020l; HLA-A*0301; HLA-A*1101; HLA-A*230l; HLA-A*2402; HLA- A*2902; HLA-A*680l; HLA-B*0702; HLA-B*0801; HLA-BM501; HLA-B*2705; HLA-B*3501; HLA-B*4001; HLA-B*5 l0l; or HLA-E*0101. In another aspect, the peptide is at least one of SEQ ID NO: 1-599
[0011] In another embodiment, the present invention includes a nucleic acid that expresses a fusion protein comprising a peptide, a first flexible linker, a P2-microglobulin domain, a second flexible linker, a soluble major histocompatibility complex (MHC) heavy chain and a peptide tag. In one aspect, the peptide is an immunogenic peptide epitope. In another aspect, the MHC is a human, mouse, rat, hamster, horse, pig, cow, simian, avian, or chimeric MHC. In another aspect, the MHC is Class I MHC. In another aspect, the MHC does not include a transmembrane sequence. In another aspect, the peptide tag is selected from at least one of: a BirA tail, a myc, a FLAG, a glutathione- S-transferase, a His tag, a maltose binding protein, hemagglutinin (HA)-tag, a V5 tag, a T7 tag, a V9 tag, a NusA-tag, a thioredoxin-tag, or a fluorescent protein-tag, a Her2/neu-tag, a CD20-tag, or a GFP-tag. In another aspect, the first, the second, or both the first and second flexible linkers comprise glycine, serine or both glycine and serine and comprise 5 to 40 residues. In another aspect, the peptide is 8 to 16 residues long. In another aspect, the peptide is selected from at least one of: ABL1; ACPP; Ad5 El A; AdV 5 Hexon; AdV Hexon; Ag85A; alfa fetoprotein; ASP-2; BA46; BALF4; BAP31; BCL-2; BCL-2A1; BCL-2L1; BCL-X; BCR-ABL; bcr-abl 210 kD fusion protein; Beta-gal; BGLAP; BMI1; BMLF1; BMRF1; BNP; BRAF 27; BRLF1; bZIP factor; BZLF1; Clorf59; CAMEL; Carbonic anhydrase; CB9L2; CD105; CD33; CD59; CEA; CEACAM; Chondromodulin; circadian clock protein PASD1; CP; CPS; cyclin-dependent kinase 4; CYP190; Cytochrome p450; DEP DC1; DLK1; Dutpase; E6; E7; EBNA 1; EBNA 3A; EBNA 3B; EBNA 6; EBV BRLF1; EBV EBNA3A; EBV LMP2; EDDR1; EGFR; EMNA 3 A; EMV-l; Enhanced Green Fluorescent Protein; Env; EphA2; Erkl; ESAT-6; EZH2; FAPa; FLT1; FOLR1; FOXM1; G250; GAD65; Gag; Gag-pol; Glypican 3; gp; gplOO; gp33 (C9M); GPC3; H250; HA-1; HA-2; HA-8; HBB; HBsAg; HBV core; HBV polymerase; HBV surface antigen; HCMV IE1; HCMV pp65; HCV core; HCV E; HCV NS3; HCV NS4b; HCV NS5a; HCV NS5B; Heparanase; HER2; HER- 2/neu; Histocompatibility antigen 60; HIV gag p24; HIV nef; HIV pol; HIV-1 env gpl20; HIV-l TUB gpl20; HIV-1 nef; HIV-l pi 7; HIV-l RT; HIV-1 US4 gpl20; HIURP; HLA-A leader sequence peptide; HLA-A2; HLA-Cw3; chlamydia trachomatis MOMP ; HM1.24; HMMR; HMOX1; HO-1; hPSA; HPV 16 E6; HSP105; HSP90 alpha; hLERL; hLOM34p; hLRL; H-Y; IA-2; IAPP; IDO; IE-l; IE62; IGF; IGRP; ILl3r; IL13Ra; Ilrl; Influenza A (PR8) NP ; Influenza A MP; Influenza A MP1; Influenza A MP2; Influenza A NP; Influenza A PB1; INFyR; Insulin; Interferon gamma inducible protein (GILT) 30; IRS-2; ITGB8; K8.1; KIF20A; KLK; KLK3; Ll; LANA; Large T antigen; Lengsin; Listeria monocytogenes Listeriolysin; LIVIN; LMP-l; LMP-2; LMP-2A; LMP-2A; LY6K; ml39; ml4l; ml45; M164; M2-1; M38; M45; M57; MAGE-1; MAGE-10; MAGE-3; MAGE-4; MAGE-A1; MAGEA2; MAGEA3; MAGE-A5; MAGE-C1; MAGE-C2; MART-1; MC38 adpgk neoantigen; MC-38; MCL-l; MCMV IE1; MELK; Mena; Middle T antigen; Midkine; miHAg SMCY; MOG precursor; MP; MPP11; MS4A1; MSLN; Mtb 16 kDa; Mtb 19 kDa; Mtb85A; mTERT; Mucin; muFAPa; MuLV env ; Murine Survivin; MYBPC-2; Mycobacterium bovis antigen 85-A; Mycobacterium tuberculosis ESAT-6; Mycobacterium tuberculosis TB10.4; Myelin basic protein; ND; NEF; NEF; Neu/Fler-2/Erbb2 proto-oncoprotein; NG2; Non muscle Myosin-9; Non-muscle Myosin; NP; NP396; NP52; NPM1; NRP-l; NRP-2; NRP-V7 superagonist peptide 8.3 Tg NOD mouse; NS3; Nucleocapsid; Nuf2; NY-ESO-l; OVA; Ovalbumin (subdominant); P1A, P2X5a; p53; p56; P79; PAP-3; PASD1; PAX-5; p-Cadherin; PDGFRbeta; PLAC1; PLAM csp; Plasmodium berghei ANKA acid phosphatase; Plasmodium berghei CSP; Plasmodium CSP; Plasmodium falciparum CSP; Plasmodium falciparum Liver stage antigen; PolA; Polymerase; Polyprotein; pp65; PPE; Prl; PRAME; Promininl; PSA; PSCA; PSM P2; PSM A; RGS5; RhoC; RNF43; RSV A strain F protein; RSV NP; RT; S protein; SAA; SART3; Sialidases ; SIV gag; SMCY; Spike GP; STEAP1; Surface IgG (sA20-Ig) of A20; Survivin; Survivin-3a; SV40 T antigen; TACE; TARP; TARP 2M; Tax; TB10.3-4; Telomerase; TEM1; tgd057; TGFp; TNP03; topll; TPBG; TPR-protein; TRAG; TRP2; Trypanosoma cruzi ASP-2; Trypanosoma cruzi SP; TTK; Tyrosinase; Tyrosine-3 -hydroxylase; Ubiquitin; UL105; E1L138; UL44; USP9Y; V131; Vaccinia virus Copenhagen Protein G5 ; Vaccinia virus Host range protein 2 ; VACCL3_l00; VEGFR1; VEGFR2; VEGFR2/KDR fragment 1; Vif; Vinculin; VP1; Vpu; VSV N; West Nile virus NY-99 polyprotein precursor; West Nile Virus polyprotein; WT1; Yellow Fever Virus 17D polyprotein; or ZnT-8. In another aspect, the MHC is selected from at least one of H-2 Db, H-2 Dd; H-2 Dk; H-2 Kb; H-2 Kd; H-2 Kk; H-2 Ld; HLA-A*0l0l; HLA-A*020l; HLA- A*030l; HLA-A*l l0l; HLA-A*230l; HLA-A*2402; HLA-A*2902; HLA-A*680l; HLA- B*0702; HLA-B*0801; HLA-B*l50l; HLA-B*2705; HLA-B*3501; HLA-B*400l; HLA-B*5 l0l; or HLA-E*0l0l . In another aspect, the peptide is at least one of SEQ ID NO:l-599.
[0012] In another embodiment, the present invention includes a method of making a soluble eukaryotic-derived peptide/MHC complex comprising: expressing in a cell a fusion protein comprising a peptide, a first flexible linker, a 2-microglobulin domain, a second flexible linker, a soluble major histocompatibility complex (MHC) heavy chain and a peptide tag. In another aspect, the method further comprises isolating the fusion protein from a supernatant. In another aspect, the method further comprises forming dimers, trimers, tetramers, or multimers of the fusion protein by mixing the fusion protein with one or more agents that bind to two or more fusion proteins. In another aspect, the agent is selected from an antibody, a cross-linking agent, a ligase, an avidin, a streptavidin, a Protein A, or a J-chain. In another aspect, the peptide is an immunogenic peptide epitope. In another aspect, the MHC is a human, mouse, rat, hamster, horse, pig, cow, simian, avian or chimeric MHC. In another aspect, the MHC is Class I MHC. In another aspect, the MHC does not include a transmembrane sequence In another aspect, the peptide tag is selected from at least one of: a BirA tail, a myc, a FLAG, a glutathione-S-transferase, a His tag, a maltose binding protein, hemagglutinin (HA)-tag, a V5 tag, a T7 tag, a V9 tag, a NusA-tag, a thioredoxin-tag, or a fluorescent protein-tag, a Her2/neu-tag, a CD20-tag, or a GFP-tag. In another aspect, the irst, the second, or both the first and second flexible linkers comprise glycine, serine or both glycine and serine and comprise 5 to 40 residues. In another aspect, the peptide is 8 to 16 residues long.
[0013] In another embodiment, the present invention includes a cell line expressing a fusion protein comprising a fusion protein comprising a peptide, a first flexible linker, a p2-microglobulin domain, a second flexible linker, a soluble major histocompatibility complex (MHC) heavy chain and a peptide tag. In one aspect, the fusion protein is integrated into the genome by co-transfecting a fusion protein expressing vector with a transposase vector that expresses a transposase and wherein the fusion protein expressing vector, the transposase vector, or both further comprise a selectable marker. In one aspect, the peptide is an immunogenic peptide epitope. In another aspect, the MHC is a human, mouse, rat, hamster, horse, pig, cow, simian, avian, or chimeric MHC. In another aspect, the MHC is Class I MHC. In another aspect, the MHC does not include a transmembrane sequence. In another aspect, the peptide tag is selected from at least one of: a BirA tail, a myc, a FLAG, a glutathione-S-transferase, a His tag, a maltose binding protein, hemagglutinin (HA)-tag, a V5 tag, a T7 tag, a V9 tag, a NusA-tag, a thioredoxin-tag, or a fluorescent protein-tag, a Her2/neu- tag, a CD20-tag, or a GFP-tag. In another aspect, the first, the second, or both the first and second flexible linkers comprise glycine, serine or both glycine and serine and comprise 5 to 40 residues. In another aspect, the peptide is 8 to 16 residues long.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:
[0015] FIG. 1A shows a representative DNA schematic of the synthetic peptide/MHC complex. The designed peptide/MHC molecule contains distinct peptide, beta-2-microglobulin, and MHC heavy chain regions that are linked via explicit glycine/serine amino acids. The MHC heavy chain lacks a transmembrane domain (designated sHeavy chain), ensuring that properly folded peptide/MHC protein is secreted from cells into the culture medium. The peptide/MHC construct also contains a terminal BirA tail for enzymatic conjugation of biotin. FIG. 1B shows a standard workflow for expressing, purifying, and biotinylating eukaryotic-derived peptide/MHC molecules. [1] A suitable eukaryotic host cell line (such as CHO cells) is transiently transfected with two vectors that allow for transposon-directed integration of genes of interest. Vector 1 encodes the SB transposase (SB100X) while vector 2 is a transposon-compatible plasmid that encodes the synthetic peptide/MHC complex and puromycin resistance transgenes. [2] A stable cell line secreting peptide/MHC complexes is generated in as little as 2 weeks following antibiotic selection and expansion. [3] Spent culture media is processed through AC (against the sHeavy chain) to selectively purify peptide/MHC complexes. [4] A biotin ligase may then be employed to enzymatically conjugate biotin to the BirA tail of the synthetic peptide/MHC protein. [5] Following an additional polishing step such as size exclusion chromatography and [6] multimerization steps, the peptide/MHC reagent can be incorporated into immunologically-relevant assays that, for example, detect antigen-specific T cells. Abbreviation used: secretory MHC class I heavy chain (sHeavy chain), major histocompatibility complex (MHC)
[0016] FIGS. 2A and 2B show that stable CHO cells lines were established by a SB transposon system to secrete peptide/MHC molecules into culture media. (FIG. 2A) Evidence of extracellular peptide/MHC protein was first determined from cell-free supernatants by SDS-PAGE and coomassie blue staining. (FIG. 2B) Representative chromatogram of small scale AC purification of peptide/MHC-containing media. Cell-free supernatant was passed through an equilibrated agarose bead column containing the MHC class I-reactive antibody Ml/42. After washing away unbound material, peptide/MHC protein was eluted, buffer-exchanged, and concentrated. Protein purity was then assessed using SDS-PAGE and coomassie blue staining. Arrow inset indicating peptide/MHC around the predicted molecular weight. Abbreviation used: protein ladder (L), affinity chromatography (AC), preparation (prep)
[0017] FIG. 3A shows Purified peptide/MHC (i.e., SIINFEKL/H-2Kb) identify was confirmed by western blot using monoclonal antibodies reactive to the b2 microglobulin and BirA tail regions of the design molecule as depicted in FIG. 1A. FIG. 3B shows that peptide/MHC ligand binding was determined through immunoprecipitation and western blot following incubation of SIINFEKL/H- 2Kb and a TCR-like antibody specific to this particular peptide/MHC class I complex. AC -purified peptide/MHC was also incorporated as a positive control. Abbreviations used: protein ladder (L), SIINFEKL epitope + MHC class I (SIINFEKL/H-2Kb), isotype (Iso), positive (Pos), control (ctrl)
[0018] FIGS. 4A to 4C show that the peptide/MHC was biotinylated as detailed in the Materials and Methods and specific streptavidin binding initially confirmed through (FIG. 4A) western blot and (FIG. 4B) ELISA using wells coated with streptavidin. (FIG. 4C) Biotinylated peptide/MHC was also incubated with streptavidin-conjugated 5 pm beads and washed extensively. Beads were then exposed to isotype, anti-SIINFEKL/H-2Kb, or anti-MHC monoclonal antibodies followed by washing steps and incubation with relevant secondary PE-conjugated antibodies. Specific ligand reactivity was subsequently determined by flow cytometry. Abbreviations used: protein ladder (L), SIINFEKL epitope + MHC class I (SIINFEKL/H-2Kb), biotinylated SIINFEKL/H-2Kb (b- SIINFEKL/H-2Kb), positive (Pos), isotype (Iso), control (ctrl), major histocompatibility complex (MHC), horseradish peroxidase (HRP), streptavidin (SA), primary antibody (1° Ab)
[0019] FIG. 5 shows that the biotinylated peptide/MHC was incubated with PE-conjugated streptavidin to produce“small-scale” batches of SIINFEKL-reactive tetramers. Purified CD8+ T cells harvested from either wild-type or OT-1 mice were incubated with tetramers, washed, and stained with an anti-CD8 FITC antibody. Cells were again washed, fixed, and analyzed by flow cytometry for ligand binding. Abbreviations used: wild-type (WT), streptavidin (SA)
[0020] FIG. 6 shows the determination of the ability to construct and express a membrane-bound OYA-specific peptide/MHC molecule (i.e., SIINFEKL/H-2Kb). 4T1 cells were transiently transfected with two plasmids that allow transposon-directed genomic integration and cultured over a period of two weeks in puromycin-containing culture media (as outlined in FIG. IB). The remaining“stably engineered” cells were then incubated with either an isotype control antibody or antibody that binds the OVA-specific epitope SIINFEKL within the constraints of MHC class I and assessed for reactivity by flow cytometry. Abbreviations used: SIINFEKL epitope + MHC class I (SIINFEKL/H-2Kb), major histocompatibility complex (MHC)
[0021] FIG. 7 is a DNA coding sequence of the synthetic SIINFEKL/H-2Kb molecule (SEQ ID NO: 600).
DETAILED DESCRIPTION OF THE INVENTION
[0022] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
[0023] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as“a”,“an” and“the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.
[0024] The present invention includes compositions, vectors, cells, and methods of making MHC class I-specific reagents such as fluorescently-labeled multimers (e.g., tetramers) that can be used to study CD8+ T cells under normal and diseased states. However, recombinant MHC class I components (comprising MHC class I heavy chain and b2 microglobulin) are usually produced in bacteria following a lengthy purification protocol that requires additional non-covalent folding steps with exogenous peptide for complete molecular assembly. The present inventors have developed an alternative and rapid approach to generating soluble and fully-folded MHC class I molecules in eukaryotic cell lines (such as CHO cells) using, e.g., a Sleeping Beauty transposon system. Importantly, this method generates stable cell lines that reliably secrete epitope-defined MHC class I molecules into the tissue media for convenient purification and eventual biotinylation/multimerization. Additionally, MHC class I components are covalently linked, providing the opportunity to produce a diverse set of CD8+ T cell-specific reagents bearing peptides with various affinities to MHC class I.
[0025] As used herein, the term“gene” refers to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences, or fragments or combinations thereof, as well as gene products, including those that may have been altered by the hand of man. Purified genes, nucleic acids, protein and the like are used to refer to these entities when identified and separated from at least one contaminating nucleic acid or protein with which it is ordinarily associated.
[0026] As used herein, the term "vector" is used in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The vector may be further defined as one designed to propagate specific sequences, or as an expression vector that includes a promoter operatively linked to the specific sequence, or one designed to cause such a promoter to be introduced. The vector may exist in a state independent of the host cell chromosome, or may be integrated into the host cell chromosome
[0027] As used herein, the term“host cell” refers to cells that have been engineered to contain nucleic acid segments or altered segments, whether archeal, prokaryotic, or eukaryotic. Thus, engineered, or recombinant cells, are distinguishable from naturally occurring cells that do not contain recombinantly introduced genes through the hand of man.
[0028] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it effects the transcription of the sequence; or a ribosome binding site is operably linked to e coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" refers to a DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in same reading frame. Enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice.
[0029] As used herein, the terms "cell" and "cell culture" are used interchangeably end all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Different designations are will be clear from the contextually clear.
[0030] As used herein, the term "plasmid" are designated by a lower case p preceded and/or followed by capital letters and/or numbers and refer to self-replicating circular DNA that include an origin of replication, and typically one or more selectable markers. The starting plasmids herein are commercially available, are publicly available on an unrestricted basis, or can be constructed from such available plasmids in accord with published procedures. In addition, other equivalent plasmids are known in the art and will be apparent to the ordinary artisan.
[0031] As used herein, the term "selectable marker" refers to the use of a gene that encodes an enzymatic activity and which confers the ability to grow in medium lacking what would otherwise be an essential nutrient (e.g., the HIS3 gene in yeast cells); in addition, a selectable marker may confer resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed. A review of the use of selectable markers in mammalian cell lines is provided in Sambrook, J., et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) pp.16.9-16.15.
[0032] As used herein the terms "protein", "polypeptide" or “peptide” refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably.
[0033] As used herein, the terms "fusion protein" or“chimeric protein” refer to a hybrid protein, that includes portions of two or more different polypeptides, or fragments thereof, resulting from the expression of a polynucleotide that encodes at least a portion of each of the two polypeptides.
[0034] As used herein, the term "transformation," refers to a process by which exogenous DNA enters and changes a recipient cell. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome.
[0035] As used herein, the term“transfection” refers to the introduction of foreign DNA into eukaryotic cells. Transfection may be accomplished by a variety of methods known to the art including, e.g., calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics. Thus, the term "stable transfection" or "stably transfected" refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has stably integrated foreign DNA into the genomic DNA. The term also encompasses cells that transiently express the inserted DNA or RNA for limited periods of time. Thus, the term "transient transfection" or "transiently transfected" refers to the introduction of foreign DNA into a cell where the foreign DNA fails to integrate into the genome of the transfected cell. The foreign DNA persists in the nucleus of the transfected cell for several days. During this time the foreign DNA is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes. As used herein, the term "transient transfectant" refers to cells that have taken up foreign DNA but have failed to integrate this DNA.
[0036] The present invention can be used to circumvent drawbacks in the prior art, in particular, stabilizing peptide binding to the MHC peptide binding groove. Previous efforts have revealed the ability to engineer and produce peptide/MHC molecules in bacteria by covalently joining the MHC class I peptide, b2 microglobulin, and heavy chain with discrete amino acid linkers (designated single-chain trimers [SCTs]) (Yu et al., 2002). For most SCTs reported, these engineered proteins fold correctly and specifically engage CD8+ T cells as tetramers (Mitaksov et al., 2007), irrespective of the artificial linker design (Hansen et al., 2009). However, this particular SCT method still utilizes a bacterial expression system and requires substantial purification and refolding efforts.
[0037] The present inventors have developed an alternative method to potentially improve the production of peptide/MHC based on the SCT approach. The present invention has the ability to rapidly generate eukaryotic cell lines that stably express and secrete peptide/MHC into the tissue media for purification and biotinylation. This novel protocol provides a much faster/convenient route to generating properly folded peptide/MHC with minimal user intervention, especially for MHC class I targets with high demand (such as the model OVA epitope SIINFEKL). By using a SCT strategy, it was possible to generate MHC molecules presenting a range of class I peptides (i.e., low-to-high binding affinity), which can be reliably generated. Additionally, these eukaryotic- derived peptide/MHC molecules can be used to recapitulate binding dynamics with TCRs in downstream assays (Schmidt and Lill 2018).
[0038] Mice. Female 6-8-week-old C57BL/6J (stock #000664) and OT-l (stock #003831) mice were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA) and maintained in micro isolator cages under sterile conditions. Animals were humanely euthanized and spleens/lymph nodes harvested and combined for Ficoll gradient centrifugation (GE Healthcare, Piscataway, NJ). The lymphocyte interphase was then subjected to ACK lysis and eventual CD8+ T cell purification using MACS bead positive selection as instructed by the manufacturer (Miltenyi Biotec, Cambridge, MA). Purified CD8+ T cells were aliquoted in 90% FBS/l0% DMSO and stored in liquid nitrogen until use. All mouse procedures were followed in accordance with TTUHSC IACUC-approved protocols.
[0039] Cell lines and culture. FreeStyle™ Chinese Flamster Ovary (CFlO-S) (Thermo Fisher Scientific, Waltham, MA) and 4T1 (ATCC, Manassas, VA) cells were utilized for in vitro studies. CHO-S cells were passaged in FreeStyle™ CHO Expression Medium (Thermo Fisher Scientific) according to the manufacturer’s recommendations. 4T1 cells are naturally deficient in FI-2Kb expression and were grown in RPMI 1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 pg/ml streptomycin, and 10 mmol/l L-glutamine (all from Thermo Fisher Scientific). All cell lines were maintained in vented flasks at 37 °C with 5% C02.
[0040] Cloning strategy and construction of transposon expression vectors. Full-length mouse b2 microglobulin (NCBI Reference Sequence: NM 009735.3) and MHC class I heavy chain (H-2Kb) (NCBI Reference Sequence: NM_001001892.2), relevant sequences incorporated herein by reference, cDNA were synthesized from C57BL/6J splenocytes following TRIzol lysis (Thermo Fisher Scientific) and RT-PCR using oligo(dT) primers (Superscript IV First-Strand Synthesis Kit; Thermo Fisher Scientific). Secretory MHC class I heavy chain was designed to not include the transmembrane domain. The leader signal, SIINFEKL epitope, and Gly/Ser amino acid linkers were ultimately added by overhang PCR as previously reported (Hansen et al., 2009) using the Phusion High-Fidelity DNA Polymerase (Thermo Scientific Fisher). PCR fragments were gel excised/purified and ligated (LigaFast™ Rapid DNA Ligation System; Promega, Madison, WI) into pucl9 (NEB, Ipswich, MA) via Sacl/Hindlll restriction enzyme sites and fragments pieced together using the unique Nhel/BamHI sites of the synthetic peptide/MHC sequence. The BirA AviTag™ amino acid sequence (GLNDIFEAQKIEWF1E SEQ ID NO: 600) was subsequently added to the construct’s terminus by overlap-extension PCR and cloned into a separate pucl9 holding vector. Full-length peptide/MHC was then amplified and cloned into the pSBbi-pur transposon vector (kindly provided by Dr. Eric Kowarz [Addgene plasmid # 60523]) using the Sfil restriction enzyme sites (Kowarz et al., 2015). Plasmid transformations were carried out in chemically-competent NEB-5 alpha E. coli (NEB) using ampicillin selection. All vector constructs were confirmed by restriction enzyme digestion and DNA sequencing [0041] Sleeping Beauty (SB) transposon system. Parental cell lines were transiently transfected with transposon-related vectors using Lipofectamine reagent (Thermo Fisher Scientific) as directed by the manufacturer. A plasmid encoding the SB 100X transposase (pCMV[CAT]T7-SB100; designated Vector 1), a gift from Dr. Zsuzsanna Izsvak (Addgene plasmid # 34879), and a transposon plasmid containing the necessary inverted terminal repeats (pSBbi-pur; designated Vector 2) were used. Both vectors were provided concurrently to stably integrate peptide/MHC transgenes into cells. Briefly, lxlO5 cells were plated in 24-well plates (Coming, Corning NY) and exposed to Vector 1 (12.5 ng)/Vector 2 (488 ng) in a total volume of 500 pl media as similarly described (Kowarz et al., 2015). Culture media was replenished with 2 ml fresh media after 24 hrs and 48 hrs, whereupon cells were exposed to lethal doses of puromycin (CHO-S - 10 pg/ml; 4T1 - 5 pg/ml) (Invivogen, San Diego, CA). Fresh media containing puromycin was provided every 2-3 days as needed. Generally, actively dividing cells were ready for expansion by 2 weeks, with virtually all cell clones expressing peptide/MHC molecules.
[0042] SDS-PAGE and western blot. To remove extraneous tissue media components such as surfactants, CHO cell supernatants were passed onto PD 10 columns (GE Healthcare) and concentrated/washed in PBS using a 30 MWCO Amicon centrifugal unit (MilliporeSigma, Burlington, MA). Protein concentration was determined using a BCA protein assay kit (Thermo Scientific Fisher) and sample aliquots stored at -80 °C until use. Protein purity was assessed with SDS-PAGE and coomassie blue staining while protein identity was confirmed through western blotting. Briefly, proteins were resolved on a 4%/ 12% polyacrylamide gel, transferred to a PVDF membrane (Amersham Hybond, 0.2pm; GE Healthcare), and blocked with 5% milk in PBST (0.1% Tween 20 in PBS) for 1 hr at RT. Membranes were then incubated with various primary antibodies (1 pg/ml) in block solution with rocking at 4 °C overnight. Specific primary reagents included anti mouse b2 microglobulin (clone 893803; R&D Systems, Minneapolis, MN) or anti-BirA tail (clone Abe; Avidity, Aurora, CO) antibodies. Blots were then washed extensively with PBST and incubated in block with secondary HRP-conjugated goat antibodies specific to rat IgG (H+L) or mouse IgG (H+L) (Thermo Scientific Fisher) for 1 hr at RT. Washed blots were finally developed with a SignalFire ECL reagent (Cell Signaling, Danvers, MA) and exposed/imaged on a ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA). In separate experiments, blots containing biotinylated protein were probed with a Streptavidin-HRP reagent (Thermo Scientific Fisher) for 1 hr at RT to confirm streptavidin binding potential. [0043] Immunoprecipitation. A Pierce Classic IP kit (Thermo Scientific Fisher) was used to investigate ligand binding of soluble peptide/MHC protein. Peptide/MHC protein was incubated overnight at 4 °C with 2 ug of the anti-SIINFEKL (clone eBio25-Dl 16; Thermo Scientific Fisher) or mouse IgG isotype (clone MOPC-21; MP Biomedicals, Santa Ana, CA) antibodies. The anti- SIINFEKL antibody functions as a TCR-like antibody (Lowe et al., 2017), and binds SIINFEKL/H- 2Kb much like SIINFEKL-reactive T cells such as OT-l CD8+ T cells. Following purification of IgG containing complexes, samples were boiled and analyzed by western blot as described above.
[0044] Affinity chromatography (AC). The MHC class I-reactive Ml/42 antibody (Bio X Cell, West Lebanon, NH) was covalently bound to an NHS-activated agarose bead column (HiTrap™ NHS-activated HP; GE Healthcare) manually as directed by the manufacturer. The column was attached to an AKTA™ start chromatography system (GE Healthcare) for automatic operation and equilibrated with 20 mM sodium phosphate, pH 7.0 (binding buffer). Cell-free supernatants were first desalted in PBS using PD-10 columns and diluted 1 :2 in binding buffer prior to AC. Samples were then applied to the Ml/42 column, unbound material washed away with binding buffer, and peptide/MHC molecules eluted using 0.1 M glycine, pH 2.7. Eluates were dispensed in tubes containing 1 M Tris, pH 9 and relevant fractions combined and concentrated/washed in PBS as explained above.
[0045] Biotinylation and tetramerization. Peptide/MHC was biotinylated using 2.5 pg BirA ligase (Avidity) overnight at 4 °C according to the manufacturer’s guidelines. Reaction components were removed by successive washes in PBS using a 30 MWCO centrifugal filter (MilliporeSigma). Alternatively, larger reaction mixtures could be exclusively polished through size exclusion chromatography (Altman and Davis 2016) using a HiPrep™ 16/60 Sephacryl™ S-200 HR column (GE Healthcare) as indicated in FIG. IB. In order to produce small-scale tetramer batches, biotinylated peptide/MHC was incubated with PE-conjugated streptavidin (BD Biosciences, San Jose, CA) at a 4: 1 molar ratio in the dark for 30 min at RT Tetramers were then stored at 4 °C shielded from light.
[0046] Enzyme-Linked Immunosorbent Assay (ELISA). To investigate the success of peptide/MHC biotinylation, a 96-well high protein binding plate (Corning) was first coated overnight at 4 °C with 4 pg/ml streptavidin (Promega) in 0.1 M sodium carbonate and then blocked (3% BSA/PBS) for 1 hr at RT. Biotinylated protein samples were diluted in block and added to wells at various dilutions and incubated at RT for 1 hr. Wells were washed extensively with PBST and then exposed to 4 pg/ml of either a MHC class I-reactive (clone Ml/42; Biolegend) or rat isotype control IgG (Biolegend) antibody in block. In separate wells, a positive control biotinylated irrelevant rat antibody (Biolegend) was incorporated to validate the extent of streptavidin binding. The plate was again washed with PBST and relevant wells provided a goat anti-rat HRP (Thermo Scientific Fisher) or goat anti-mouse FcyR-specific HRP (Jackson ImmunoRe search, West Grove, PA) antibody in block for 1 hr at RT. Wells were washed with PBST and developed for 5 min after the addition of 200 pi 1-Step Ultra TMB (Thermo Scientific Fisher). Reactions were terminated with 100 pi TMB stop solution (KPL), and the absorbance immediately read at 450 nm using a Cytation 5 Imaging Reader (Biotek, Winooski, VT).
[0047] Flow cytometry. To confirm gene expression, the 4T1 cell line (1x10s cells) was stained with relevant primary antibodies (anti-SIINFEKL/H-2Kb or mouse IgG isotype antibodies at 2 pg/ml) in FACS buffer (0.5% BSA/0.1% NaN3 in PBS) for 20 min at 4 °C, washed, incubated with a PE-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch), washed again, and resuspended in FACS buffer for analysis (FIG. 6). To ensure proper orientation, biotinylated peptide/MHC was incubated with 5 pm PMMA beads conjugated to streptavidin (Poly An, Berlin, Germany), washed, and incubated at 4 °C with correct pairs of primary and PE-conjugated secondary antibodies (as indicated in FIGS. 4A to 4C) prior to analysis in FACS buffer. Frozen CD8+ T cells were thawed, washed, and resuspended in FACS buffer so that each stain consisted of 2xl05 viable CD8+ T cells. Cells were exposed to 50 nM dasatinib (Selleck Chemicals, Houston, TX) for 30 min at 37 °C and promptly incubated with peptide/MHC tetramers for 20 min at RT in the dark (Dolton et al., 2015). Cells were washed with FACS buffer and labeled with an anti-CD8- FITC antibody (clone 53-6.7; BioLegend, San Diego, CA) at 4 °C for 20 min shielded from light. After additional wash steps, cells were resuspended in fix solution (1% FBS, 2.5% formaldehyde in PBS) and assessed by flow cytometry using a BD LSRFortessa. Single and double color stain analysis was carried out using FlowJo software. In the case of tetramer analysis, PE/FITC compensated events were first gated based on FSC/SSC profiles and subsequently evaluated by PE and FITC fluorescence.
[0048] Cloning, expression, and purification strategy for soluble eukaryotic-derived peptide/MHC. The design of linked peptide/MHC class I molecules closely followed the previously reported generation of SCTs in bacteria (Hansen et al., 2009). Essentially, as presented in FIG. 1A, flexible glycine/serine linkers join a particular peptide epitope, b2 microglobulin, and MHC class I heavy chain A BirA tail was also genetically encoded to the 3' end of the molecule for enzyme-directed biotinylation of secreted protein. Yet, a notable difference of this design involved omitting the transmembrane region of MHC class I (designated sHeavychain) to allow for functional and fully- folded secreted protein. This process for eukaryotic expression centered on the use of a SB transposon system to stably integrate transgene content into relevant cell lines (FIG. IB). Therefore, CHO cells would be transiently transfected with transposon-relevant plasmids (encoding separately a SB transposase and synthetic peptide/MHC molecule) and stable cells generated through antibiotic selection. After expanding relevant CHO cell clones, secreted peptide/MHC could be purified by AC, biotinylated, and multimerized to produce, for example, tetramers capable of binding antigen- specific T cells.
[0049] Purification of secreted and fully-folded peptide/MHC protein. The suitability of the SB transposon system to stably integrate the peptide/MHC transgene was first determined. However, this particular peptide/MHC construct differed from the aforementioned soluble design in FIG. 1A by encoding full-length MHC class I heavy chain (i.e., the murine MHC haplotype H-2Kb), thereby, ensuring cell surface expression for relative ease of detection. Briefly, 4T1 (H-2Kb null) cells were exposed to transposon vectors that expressed either a null or SIINFEKL/H-2Kb construct and puromycin resistant clones selected and expanded in culture. Cells were then stained with isotype or anti-SIINFEKL/H-2Kb antibodies and analyzed by flow cytometry using a PE-conjugated secondary antibody. In comparison to 4Tl-null cells, 4Tl-SIINFEKL H-2Kb cells expressed clear and robust levels of synthetic peptide/MHC (FIG. 7). These confirmatory results were expected, given the widespread use of the SB approach to induce expression of various protein classes in established cell lines and primary cells (Kebriaei et al., 2017).
[0050] CHO cells were next transfected with SB-related vectors as described in FIGS. 1A and 1 B to induce stable expression of soluble peptide/MHC Parental CHO cells exhibited enhanced resistance to puromycin, requiring sustained culturing in high concentrations of puromycin at 10 pg/ml. However, the inventors were able to expand puromycin-resistant clones by 2 weeks post plasmid transfection Cells were then grown to saturating conditions in culture for at least 4 days to generate suitable whole protein concentrations from a total volume of 20 ml media. Yet, the choice of serum-free media contained a number of proprietary agents such as surfactants that could potentially interfere with protein purification and validation assays. The inventors, therefore, took the precautionary step of desalting and concentrating cell-free supernatants in PBS prior to downstream analysis. The initial assessment of CHO-derived extracellular proteins by SDS-PAGE (under reducing conditions) and coomassie blue staining revealed distinct protein bands around a predicted 51 kDa molecular weight for synthetic peptide/MHC (FIG. 2A) that was not evident from CHO cells expressing a null construct (data not shown). This specific protein band was subsequently confirmed following AC as exhibited in FIG. 2B. That is, cell-free supernatants were passed onto an agarose bead column containing the MHC class I-reactive Ml/42 antibody and bound material eluted and ultimately assessed again by SDS-PAGE. The inventors typically harvested at least 100 ug/ml AC -purified peptide/MHC from small-scale culturing efforts. However, the described protocol can be scaled-up (or down) depending on the desired peptide/MHC total yield. Although beyond the scope of this report, alternative culturing parameters and/or leader signals (Haryadi et al., 2015) may potentially enhance overall CHO secretion of peptide/MHC. The AC procedure appeared to provide substantial protein purity based on obtaining [i] one distinct chromatogram elution peak and [ii] a protein band comprising > 95% of detectable protein by coomassie blue staining.
[0051] Determination of peptide/MHC identity and upstream binding potential. Next, the inventors confirmed the identity of AC purified protein by western blot separate from MHC class I reactivity. Based on the linked design of polychain peptide/MHC molecules (FIG. 1A), the inventors incorporated monoclonal antibodies specific to mouse b2 microglobulin and the BirA tail peptide GLNDIFEAQKIEWHE (SEQ ID NO: 600). Developed blots clearly verified the integrity of synthetic peptide/MHC molecule expression, indicating no observable issues in CHO cells secreting these artificial proteins (FIG. 3A). An immunoprecipitation reaction was subsequently attempted in order to demonstrate ligand binding potential of linked SIINFEKL to the MHC class I binding groove. SIINFEKL/H-2Kb was incubated with 2 pg of anti-SIINFEKL/H-2Kb or isotype control antibodies overnight. As detailed in the Materials and Methods, IgG containing material was purified using protein A/G-complexed agarose and resolved by SDS-PAGE under reducing conditions. The presence of SIINFEKL/H-2Kb was further established through western blot using an anti-mouse b2 microglobulin antibody (FIG. 3B). Overall, these results help further validate the identity of soluble peptide/MHC from CHO cells and establish that these secreted proteins retain ligand binding specificity at the peptide/MHC class I interface. [0052] Multimerization and functional assessment of eukaryotic-derived peptide/MHC. Biotinylated peptide/MHC arguably provides the greatest convenience to users for downstream assays. The inventors, therefore, designed a BirA tail to the terminal end of the peptide/MHC transgene by using a particular BirA tail peptide, GLNDIFEAQKIEWHE (SEQ ID NO: 600), which offers a highly targeted site for enzymatic conjugation of biotin with minimal footprint (Beckett et al., 1999). AC purified peptide/MHC was subjected to BirA ligase activity in the presence of free biotin overnight. Excess biotin and other reaction components were removed by excessive washing in PBS using a 30 MWCO centrifugal unit, and the extent of biotinylation first assessed by western blot and ELISA. In the case of western blot analysis, SDS-PAGE-resolved biotinylated protein was incubated with HRP-conjugated streptavidin to generate a specific peptide/MHC signal (FIG. 4A). Likewise, ELISA determination of biotinylated peptide/MHC clearly confirmed the functionality of the BirA tail region (FIG. 4B). Briefly, wells were coated overnight with streptavidin followed by various concentrations of peptide/MHC and biotinylated peptide/MHC. Detection was finally determined indirectly by MHC class I reactivity using the Ml/42 antibody. Biotinylated SIINFEKL/H-2Kb (as low as 0.25 pg/ml protein) was clearly evident in appropriate wells, with negligible reactivity occurring for conditions containing either unbiotinylated SIINFEKL/H-2Kb or biotinylated SIINFEKL/H-2Kb incubated with an isotype control. Next, the ability of immobilized/properly oriented biontinylated peptide/MHC to specifically engage ligands through the MHC class I peptide binding site was confirmed. Streptavidin was covalently attached to PMMA 5 mM beads and incubated with saturating conditions of biotinylated SIINFEKL H-2Kb. Beads were washed, incubated with appropriate primary reagents (i.e., anti-SIINFEKL/H-2Kb and anti-MHC class I agents), and the extent of MHC ligand binding determined by flow cytometry using secondary PE-conjugated antibodies. Ultimately, as initially validated by the immunoprecipitation shown in FIG. 3B, biotinylated peptide/MHC bound streptavidin and specifically interacted with a TCR-like antibody that recapitulates CD8+ T cell interactions (FIG. 4C), indicating no observable functional issues with peptide/MHC post biotinylation and multimerization.
[0053] Next, the inventors assessed the ability of fluorescently-labeled multimers to specifically detect antigen-specific CD8+ T cells. Splenocytes and lymph nodes were harvested from wild-type and OT-1 transgenic (i.e., SIINFEKL-reactive) mice and CD8+ T cells subsequently purified through magnetic bead selection. Biotinylated SIINFEKL/H-2Kb was then incubated with PE- conjugated streptavidin following a 4: 1 molar ratio, thereby, generating tetramers. In order to stabilize membrane dynamics of TCRs, CD8+ T cells were exposed to dasatinib (as previously described [Dolton et al., 2015]), followed by incubation with tetramers. After suitable wash steps, cells were specifically labeled with a FITC-conjugated anti-mouse CD8 antibody that displays minimal interference with TCR:MHC binding (Clement et al., 2011). Cells were fixed and double positive events (CD8+/tetramer+) assessed by flow cytometry. As detailed in FIG. 5, CD8+ OT-l cells clearly bound PE-labeled tetramers, with most cells displaying CD8 and tetramer positivity (in comparison to wild-type mice). Altogether, these data show the overarching strategy (as outlined in FIG. 1B) of stably producing eukaryotic-derived peptide/MHC that can be multimerized and used for immunologically-relevant assays such as CD8+ T cell identification.
[0054] Although MHC class I peptide candidates can be easily identified through in silico prediction methods (Andreatta and Nielsen 2016), free peptide occupancy of MHC class I molecules tends to be a rate limiting step in successfully generating stable peptide/MHC molecules from bacteria (Altman and Davis 2016). Considering the vital role MHC plays in human health (Cho and Sprent 2018), an inability to produce certain peptide/MHC reagents may adversely impact efforts on a number of fronts including diagnostics, therapies, and generalized scientific endeavors. For example, the burgeoning field of neoepitope identification for personalized cancer therapy could be stalled by those unique patient epitopes that exhibit high dissociation rates from MHC class I (Hu et al., 2018). One workaround to the issue of peptide occupancy has been in designing and expressing polychain SCTs that ensure full assembly of peptide, b2 microglobulin, and MHC class I heavy chain through flexible linkers (Hansen et al., 2009). SCTs retained on the plasma membrane of eukaryotic cells maintain their native conformation and are highly resistant to exogenous peptide binding (Yu et al., 2002). Additionally, membrane-bound SCTs serve as effective targets for CD8+ T cell priming (Hung et al., 2007) and destruction (Yu et al., 2002). In the case of diagnostic determination of CD8+ T cell frequencies by tetramers, SCTs can be modified for expression/secretion in bacteria and biotinylation by way of an explicit amino acid sequence that directs BirA enzyme function (Mitaksov et al., 2007).
[0055] Previously, the present inventors developed a unique protocol to establish eukaryotic cell lines (such as CHO suspension cells) in as little as two weeks that stably secrete peptide/MHC molecules through transposon-directed delivery. Soluble peptide/MHC may then be biotinylated and utilized as multimers (via streptavidin), particularly for CD8+ T cell relevant assays. Currently, CHO cells are an industry standard in producing FDA approved therapeutic recombinant proteins (Kuo et al., 2018). The inventors sought the advantages of the CHO cell line in order to [i] instigate post-translational modifications and [ii] be easily grown at high density under serum-free conditions in suspension cultures. However, other common cell line“protein workhorses” (e.g., HEK-293 cells) are amenable to the expression and characterization techniques outlined.
[0056] One advantage of the present invention is the rapid development of stable CHO cells secreting peptide/MHC using the SB transposon system. These studies incorporated the SB transposase SB100X, which has a high gene insertion efficiency at close-to-random chromosomal sites (Mates et al., 2009). The SB approach provides the advantageous properties of viral transduction to insert transgenes without the disadvantages of either maintaining genomic material episomally (e.g., adeno-associated viruses) or near/in proto-oncogenes (e.g., retroviral vectors) (Kebriaei et al., 2017). Additionally, manufacturing high-quality viral particles can be a cumbersome task fraught with regulatory obstacles. The inventors generally experienced minimal difficulty in developing and expanding stable lines from parental cells after transiently transfecting plasmids that propagated the SB transposon system. The protocol is also amenable to freezing material at convenient stopping points. There was no apparent adverse effects to generating multimerized reagents when either cell-free CHO-derived supernatants or biotinylated peptide/MHC was frozen long-term at -80 °C. The method of the present invention is compatible with downstream tetramer production with the added benefits of convenient peptide/MHC expression that can incorporate a range of peptide affinities to the MHC peptide binding groove. Traditionally, to circumvent the low intrinsic affinity of the TCR with peptide/MHC, tetravalent multimers have been utilized to increase T cell avidity. However, these soluble peptide/MHC molecules can be utilized for higher order reagents to better discriminate low frequency T cells (or bind“difficult” TCRs) such as fluorochrome-conjugated dextramers since the production process involves incubating biotinylated peptide/MHC with a dextran backbone containing streptavidin (Dolton et al., 2014). Thus, the present invention can be used to reliably produce a range of soluble eukaryotic-derived peptide/MHC molecules for diagnostic, therapeutic, and investigative purposes.
[0057] Table 1. MHC, Peptide Sequences, and Target.
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
[0058] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
[0059] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
[0060] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
[0061] The use of the word“a” or“an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean“one,” but it is also consistent with the meaning of“one or more,”“at least one,” and“one or more than one.” The use of the term“or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term“about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects
[0062] As used in this specification and claim(s), the words“comprising” (and any form of comprising, such as“comprise” and“comprises”),“having” (and any form of having, such as “have” and“has”),“including” (and any form of including, such as“includes” and“include”) or “containing” (and any form of containing, such as“contains” and“contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein,“comprising” may be replaced with“consisting essentially of’ or“consisting of’. As used herein, the phrase“consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term“consisting” is used to indicate the presence of the recited integer (e g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only.
[0063] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example,“A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
[0064] As used herein, words of approximation such as, without limitation,“about”, "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as“about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
[0065] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
[0066] To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words“means for” or“step for” are explicitly used in the particular claim.
[0067] For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.
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Claims

What is claimed is:
1. A fusion protein comprising a peptide, a first flexible linker, a b 2-microglobulin domain, a second flexible linker, a soluble major histocompatibility complex (MHC) heavy chain and a peptide tag.
2. The fusion protein of claim 1, wherein the peptide is an immunogenic peptide epitope.
3. The fusion protein of claim 1, wherein the MHC is a human, mouse, rat, hamster, horse, pig, cow, simian, avian, or chimeric MHC.
4. The fusion protein of claim 1, wherein the MHC is Class I MHC.
5. The fusion protein of claim 1, wherein the MHC does not include a transmembrane sequence.
6. The fusion protein of claim 1, wherein the peptide tag is selected from wherein the peptide tag is selected from at least one of: a BirA tail, a myc, a FLAG, a glutathione-S-transferase, a His tag, a maltose binding protein, hemagglutinin (HA)-tag, a V5 tag, a T7 tag, a V9 tag, a NusA-tag, a thioredoxin-tag, or a fluorescent protein-tag, a Her2/neu-tag, a CD20-tag, or a GFP-tag.
7. The fusion protein of claim 1, wherein the first, the second, or both the first and second flexible linkers comprise glycine, serine or both glycine and serine and comprise 5 to 40 residues.
8. The fusion protein of claim 1, wherein the peptide is 8 to 16 residues long.
9. The fusion protein of claim 1, wherein the peptide is selected from at least one of: ABL1;
ACPP; Ad5 El A; AdV 5 Hexon; AdV Hexon; Ag85A; alfa fetoprotein; ASP-2; BA46; BALF4; BAP31; BCL-2; BCL-2A1; BCL-2L1; BCL-X; BCR-ABL; bcr-abl 210 kD fusion protein; Beta-gal; BGLAP; BMI1; BMLF1; BMRF1; BNP; BRAF 27; BRLF1; bZIP factor; BZLF1; Clorf59;
CAMEL; Carbonic anhydrase; CB9L2; CD105; CD33; CD59; CEA; CEACAM; Chondromodulin; circadian clock protein PASD1; CP; CPS; cyclin-dependent kinase 4; CYP190; Cytochrome p450; DEP DC1; DLK1; Dutpase; E6; E7; EBNA 1; EBNA 3A; EBNA 3B; EBNA 6; EBV BRLF1; EBV EBNA3A; EBV LMP2; EDDR1; EGFR; EMNA 3A; EMV-1; Enhanced Green Fluorescent Protein; Env; EphA2; Erkl; ESAT-6; EZH2; FAPa; FLT1; FOLR1; FOXM1; G250; GAD65; Gag; Gag-pol; Glypican 3; gp; gplOO; gp33 (C9M); GPC3; H250; HA-l; HA-2; HA-8; HBB; HBsAg; HBV core; HBV polymerase, HBV surface antigen; HCMV IE1; HCMV pp65; HCV core; HCV E; HCV NS3; HCV NS4b; HCV NS5a; HCV NS5B; Heparanase; HER2; HER-2/neu; Histocompatibility antigen 60; HIV gag p24; HIV nef, HIV pol; HIV-l env gpl20; HIV-l IIIB gpl20; HIV-1 nef; HIV-l pl7; HIV-l RT; HIV-l US4 gpl20; HJURP; HLA-A leader sequence peptide; HLA-A2; HLA-Cw3; chlamydia trachomatis MOMP ; HM1.24; HMMR; HMOX1; HO-l; hPSA; HPV 16 E6; HSP105; HSP90 alpha; hTERT; hTOM34p; hTRT; H-Y; IA-2; IAPP; IDO, IE-1; IE62; IGF; IGRP; IL13r; IL13Ra; Ilrl; Influenza A (PR8) NP ; Influenza A MP; Influenza A MP1; Influenza A MP2;
Influenza A NP; Influenza A PB1; INFyR; Insulin; Interferon gamma inducible protein (GILT) 30; IRS-2; ITGB8; K8.1; KIF20A; KLK; KLK3; Ll; LANA; Large T antigen; Lengsin; Listeria monocytogenes Listeriolysin; LIVIN; LMP-l; LMP-2; LMP-2A; LMP-2A; LY6K; ml39; ml4l; ml45; M164; M2-1; M38; M45; M57; MAGE-l; MAGE-10; MAGE-3; MAGE-4; MAGE-A1; MAGEA2; MAGE A3; MAGE-A5; MAGE-C1; MAGE-C2; MART-l; MC38 adpgk neoantigen; MC-38; MCL-1; MCMV IE1; MELK; Mena; Middle T antigen; Midkine; miHAg SMCY; MOG precursor; MP; MPP11; MS4A1; MSLN; Mtb 16 kDa; Mtb 19 kDa; Mtb85A; mTERT; Mucin; muFAPa; MuLV env ; Murine Survivin; MYBPC-2; Mycobacterium bovis antigen 85-A;
Mycobacterium tuberculosis ESAT-6; Mycobacterium tuberculosis TB10.4; Myelin basic protein; ND; NEF; NEF; Neu/Her-2/Erbb2 proto-oncoprotein; NG2; Non muscle Myosin-9; Non-muscle Myosin; NP; NP396; NP52; NPM1; NRP-l; NRP-2; NRP-V7 superagonist peptide 8.3 Tg NOD mouse; NS3; Nucleocapsid; Nuf2; NY-ESO-1; OVA; Ovalbumin (subdominant); P1A; P2X5a; p53; p56; P79; PAP-3; PASD1; PAX-5; p-Cadherin; PDGFRbeta; PLAC1; PLAM csp; Plasmodium berghei ANKA acid phosphatase; Plasmodium berghei CSP; Plasmodium CSP; Plasmodium falciparum CSP; Plasmodium falciparum Liver stage antigen; PolA; Polymerase; Polyprotein; pp65; PPE; Prl; PRAME; Promininl; PSA; PSCA; PSM P2; PSMA; RGS5; RhoC; RNF43; RSV A strain F protein; RSV NP; RT; S protein; SAA; SART3; Sialidases ; SIV gag; SMCY; Spike GP;
Surface IgG (sA20-Ig) of A20; Survivin; Survivin-3a; SV40 T antigen; TACE; TARP; TARP 2M; Tax; TB10.3-4; Telomerase; TEM1; tgd057; TGFfi: TNP03; topll; TPBG; TPR-protein; TRAG; TRP2; Trypanosoma cruzi ASP-2; Trypanosoma cruzi SP; TTK; Tyrosinase; Tyrosine-3- hydroxylase; Ubiquitin; UL105; UL138; UL44; USP9Y; V131; Vaccinia virus Copenhagen Protein G5 ; Vaccinia virus Host range protein 2 ; VACCL3 100; VEGFR1; VEGFR2; VEGFR2/KDR fragment 1; Vif; Vinculin; VP1; Vpu; VSV N; West Nile virus NY-99 polyprotein precursor; West Nile Virus polyprotein; WT1; Yellow Fever Virus 17D polyprotein; or ZnT-8.
10. The fusion protein of claim 1, wherein the MHC is selected from at least one of H-2 Db, H-2 Dd; H-2 Dk; H-2 Kb; H-2 Kd; H-2 Kk; H-2 Ld; HLA-A*0101; HLA-A*0201; HLA-A*0301; HLA- A* 1101; HLA-A*2301; HLA-A*2402; HLA-A*2902; HLA-A*6801; HLA-B*0702; HLA-B*0801; HLA-BH501; HLA-B*2705; HLA-B*3501; HLA-B 001; HLA-B*510l; or HLA-E*0l0l .
11. The fusion protein of claim 1, wherein the peptide is at least one of SEQ ID NO: 1-599.
12. A nucleic acid that expresses a fusion protein comprising a peptide, a first flexible linker, a p2-microglobulin domain, a second flexible linker, a soluble major histocompatibility complex (MHC) heavy chain and a peptide tag.
13. The nucleic acid of claim 12, wherein the peptide is an immunogenic peptide epitope.
14. The nucleic acid of claim 12, wherein the MHC is a human, mouse, rat, hamster, horse, pig, cow, simian, avian, or chimeric MHC.
15. The nucleic acid of claim 12, wherein the MHC is Class I MHC.
16. The nucleic acid of claim 12, wherein the MHC does not include a transmembrane sequence.
17. The nucleic acid of claim 12, wherein the peptide tag is selected from at least one of: a BirA tail, a myc, a FLAG, a glutathione-S-transferase, a His tag, a maltose binding protein,
hemagglutinin (HA)-tag, a V5 tag, a T7 tag, a V9 tag, a NusA-tag, a thioredoxin-tag, or a fluorescent protein-tag, a Her2/neu-tag, a CD20-tag, or a GFP-tag.
18. The nucleic acid of claim 12, wherein the first, the second, or both the first and second flexible linkers comprise glycine, serine or both glycine and serine and comprise 5 to 40 residues.
19. The nucleic acid of claim 12, wherein the peptide is 8 to 16 residues long.
20. The nucleic acid of claim 12, wherein the peptide is selected from at least one of: ABL1;
ACPP; Ad5 El A; AdV 5 Hexon; AdV Hexon; Ag85A; alfa fetoprotein; ASP-2; BA46; BALF4; BAP31; BCL-2; BCL-2A1; BCL-2L1; BCL-X; BCR-ABL; bcr-abl 210 kD fusion protein; Beta-gal; BGLAP; BMI1; BMLF1; BMRF1; BNP; BRAF 27; BRLF1; bZIP factor; BZLF1; Clorf59;
CAMEL; Carbonic anhydrase; CB9L2; CD105; CD33; CD59; CEA; CEACAM; Chondromodulin; circadian clock protein PASD1; CP; CPS; cyclin-dependent kinase 4; CYP190; Cytochrome p450; DEP DC1; DLK1; Dutpase; E6; E7; EBNA 1; EBNA 3A; EBNA 3B; EBNA 6; EBV BRLF1; EBV EBNA3A; EBV LMP2; EDDR1; EGFR; EMNA 3A; EMV-1; Enhanced Green Fluorescent Protein; Env; EphA2; Erkl; ESAT-6; EZH2; FAPa; FLT1; FOLR1; FOXM1; G250; GAD65; Gag; Gag-pol; Glypican 3; gp; gplOO, gp33 (C9M); GPC3; H250; HA-l; HA-2; HA-8; HBB; HBsAg; HBV core; HBV polymerase; HBV surface antigen; HCMV IE1; HCMV pp65; HCV core; HCV E; HCV NS3; HCV NS4b; HCV NS5a; HCV NS5B; Heparanase; HER2; HER-2/neu; Histocompatibility antigen 60; HIV gag p24; HIV nef, HIV pol; HIV-l env gpl20; HIV-l IIIB gpl20; HIV-1 nef; HIV-l pl7; HIV-l RT; HIV-l US4 gpl20; HJURP; HLA-A leader sequence peptide; HLA-A2; HLA-Cw3; chlamydia trachomatis MOMP ; HM1.24; HMMR; HMOX1; HO-l; hPSA; HPV 16 E6; HSP105; HSP90 alpha; hTERT; hTOM34p; hTRT; H-Y; IA-2; IAPP; IDO; IE-l; IE62; IGF; IGRP; ILl3r; ILl3Ra; Ilrl; Influenza A (PR8) NP ; Influenza A MP; Influenza A MP1; Influenza A MP2;
Influenza A NP; Influenza A PB1; INFyR; Insulin; Interferon gamma inducible protein (GILT) 30; IRS-2; ITGB8; K8.1; KIF20A; KLK; KLK3; Ll; LANA; Large T antigen; Lengsin; Listeria monocytogenes Listeriolysin; LIVIN; LMP-l; LMP-2; LMP-2A; LMP-2A; LY6K; ml39; ml4l; ml45; M164; M2-1; M38; M45; M57; MAGE-l; MAGE-10; MAGE-3; MAGE-4; MAGE-A1; MAGEA2; MAGE A3; MAGE-A5; MAGE-C1; MAGE-C2; MART-l; MC38 adpgk neoantigen; MC-38; MCL-l; MCMV IE1; MELK; Mena; Middle T antigen; Midkine; miHAg SMCY; MOG precursor; MP; MPP11; MS4A1; MSLN; Mtb 16 kDa; Mtb 19 kDa; Mtb85A; mTERT; Mucin; muFAPa; MuLV env ; Murine Survivin; MYBPC-2; Mycobacterium bovis antigen 85-A;
Mycobacterium tuberculosis ESAT-6; Mycobacterium tuberculosis TB10.4; Myelin basic protein; ND; NEF; NEF; Neu/Her-2/Erbb2 proto-oncoprotein; NG2; Non muscle Myosin-9; Non-muscle Myosin; NP; NP396; NP52; NPM1; NRP-l; NRP-2; NRP-V7 superagonist peptide 8.3 Tg NOD mouse; NS3; Nucleocapsid; Nuf2; NY-ESO-1; OVA; Ovalbumin (subdominant); P1A; P2X5a; p53; p56; P79; PAP-3; PASD1; PAX-5; p-Cadherin; PDGFRbeta; PLAC1; PLAM csp; Plasmodium berghei ANKA acid phosphatase; Plasmodium berghei CSP; Plasmodium CSP; Plasmodium falciparum CSP; Plasmodium falciparum Liver stage antigen; PolA; Polymerase; Polyprotein; pp65; PPE; Prl; PRAME; Promininl; PSA; PSCA; PSM P2; PSMA; RGS5; RhoC; RNF43; RSV A strain F protein; RSV NP; RT; S protein; SAA; SART3; Sialidases ; SIV gag; SMCY; Spike GP;
Surface IgG (sA20-Ig) of A20; Survivin; Survivin-3a; SV40 T antigen; TACE; TARP; TARP 2M; Tax; TB10.3-4; Telomerase; TEM1; tgd057; TGFfl: TNP03; topll; TPBG; TPR-protein; TRAG; TRP2; Trypanosoma cruzi ASP-2; Trypanosoma cruzi SP; TTK; Tyrosinase; Tyrosine-3- hydroxylase; Ubiquitin; UL105; UL138; UL44; USP9Y; V131; Vaccinia virus Copenhagen Protein G5 ; Vaccinia virus Host range protein 2 ; VACCL3 100; VEGFR1; VEGFR2; VEGFR2/KDR fragment 1; Vif; Vinculin; VP1; Vpu; VSV N; West Nile virus NY-99 polyprotein precursor; West Nile Virus polyprotein; WT1; Yellow Fever Virus 17D polyprotein; or ZnT-8.
21. The nucleic acid of claim 12, wherein the MHC is selected from at least one of H-2 Db, H-2 Dd; H-2 Dk; H-2 Kb; H-2 Kd; H-2 Kk; H-2 Ld; HLA-A*0l0l; HLA-A*020l; HLA-A*0301; HLA- A* 1101; HLA-A*2301; HLA-A*2402; HLA-A*2902; HLA-A*680l; HLA-B*0702; HLA-B*0801;
HLA-BM501; HLA-B*2705; HLA-B*3501; HLA-BM001; HLA-B*510l; or HLA-E*0l0l .
22. The nucleic acid of claim 12, wherein the peptide is SEQ ID NO: 1-599.
23. A method of making a soluble eukaryotic-derived peptide/MHC complex comprising:
expressing in a cell a fusion protein comprising a peptide, a first flexible linker, a b2- microglobulin domain, a second flexible linker, a soluble major histocompatibility complex (MHC) heavy chain and a peptide tag.
24. The method of claim 23, further comprising isolating the fusion protein from a supernatant.
25. The method of claim 23, further comprising forming dimers, trimers, tetramers, or multimers of the fusion protein by mixing the fusion protein with one or more agents that bind to two or more fusion proteins.
26. The method of claim 25, wherein the agent is selected from an antibody, a cross-linking agent, a ligase, an avidin, a streptavidin, a Protein A, or a J-chain.
27. The method of claim 23, wherein the peptide is an immunogenic peptide epitope.
28. The method of claim 23, wherein the MHC is a human, mouse, rat, hamster, horse, pig, cow, simian, avian or chimeric MHC.
29. The method of claim 23, wherein the MHC is Class I MHC.
30. The method of claim 23, wherein the MHC does not include a transmembrane sequence.
31. The method of claim 23, wherein the peptide tag is selected from at least one of: a BirA tail, a myc, a FLAG, a glutathione-S-transferase, a His tag, a maltose binding protein, hemagglutinin (HA)-tag, a V5 tag, a T7 tag, a V9 tag, a NusA-tag, a thioredoxin-tag, or a fluorescent protein-tag, a Her2/neu-tag, a CD20-tag, or a GFP-tag.
32. The method of claim 23, wherein the first, the second, or both the first and second flexible linkers comprise glycine, serine or both glycine and serine and comprise 5 to 40 residues.
33. The method of claim 23, wherein the peptide is 8 to 16 residues long.
34. A cell line expressing a fusion protein comprising a fusion protein comprising a peptide, a first flexible linker, a f32-microglobulin domain, a second flexible linker, a soluble major histocompatibility complex (MHC) heavy chain and a peptide tag.
35. The cell line of claim 34, wherein the fusion protein is integrated into the genome by co transfecting a fusion protein expressing vector with a transposase vector that expresses a transposase and wherein the fusion protein expressing vector, the transposase vector, or both further comprise a selectable marker.
36. The cell line of claim 34, wherein the peptide is an immunogenic peptide epitope.
37. The cell line of claim 34, wherein the MHC is a human, mouse, rat, hamster, horse, pig, cow, simian, avian, or chimeric MHC .
38. The cell line of claim 34, wherein the MHC is Class I MHC.
39. The cell line of claim 34, wherein the MHC does not include a transmembrane sequence.
40. The cell line of claim 34, wherein the peptide tag is selected from at least one of: a BirA tail, a myc, a FLAG, a glutathione-S-transferase, a His tag, a maltose binding protein, hemagglutinin
(HA)-tag, a V5 tag, a T7 tag, a V9 tag, a NusA-tag, a thioredoxin-tag, or a fluorescent protein-tag, a Her2/neu-tag, a CD20-tag, or a GFP-tag.
41. The cell line of claim 34, wherein the first, the second, or both the first and second flexible linkers comprise glycine, serine or both glycine and serine and comprise 5 to 40 residues.
42. The cell line of claim 34, wherein the peptide is 8 to 16 residues long.
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