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WO2025024292A1 - Protéines de fusion d'anticorps huh - Google Patents

Protéines de fusion d'anticorps huh Download PDF

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Publication number
WO2025024292A1
WO2025024292A1 PCT/US2024/038787 US2024038787W WO2025024292A1 WO 2025024292 A1 WO2025024292 A1 WO 2025024292A1 US 2024038787 W US2024038787 W US 2024038787W WO 2025024292 A1 WO2025024292 A1 WO 2025024292A1
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Prior art keywords
huh
tag
protein
fusion protein
affinity moiety
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Wendy R. GORDON
Lidia K. LIMÓN
Joseph M. Muretta
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University of Minnesota Twin Cities
University of Minnesota System
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University of Minnesota Twin Cities
University of Minnesota System
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/10—Oligonucleotides as tagging agents for labelling antibodies

Definitions

  • the present disclosure relates to a fusion protein including an affinity moiety; an HUH tag; and an oligonucleotide, wherein the oligonucleotide is covalently bound by the HUH tag.
  • the affinity moiety includes an IgG antibody, an scFv, or a single-domain antibody.
  • the oligonucleotide includes a barcode sequence, such as a barcode sequence including least 8 nucleotides.
  • the oligonucleotide includes an HUH ori sequence.
  • the HUH tag and the affinity moiety are connected by an amino acid linkage, such as a polypeptide linker.
  • the HUH tag and the affinity moiety are chemically attached, such as via a maleimide linkage, a hydrazide linkage, or an NHS ester linkage.
  • the HUH tag and the affinity moiety are attached via an affinity interaction, such as a biotin-avidin interaction.
  • the affinity moiety includes an intermediate binder protein.
  • the intermediate binder protein includes at least 50 amino acids of protein G (SEQ ID NO: 16).
  • intermediate binder protein includes protein G C3 (SEQ ID NO:51) including an E23BPA mutation.
  • the present disclosure relates to a method of preparing a fusion protein including providing the HUH tag; providing the affinity moiety, wherein the affinity moiety is attached to the HUH tag; providing an uncut oligonucleotide; and reacting the HUH tag with the uncut oligonucleotide to yield the HUH tag attached to the oligonucleotide.
  • providing the HUH tag includes providing a first polynucleotide encoding the HUH tag; contacting a cell with the first polynucleotide encoding the HUH tag; incubating the cell in cell culture media; and purifying the HUH tag.
  • providing the affinity moiety includes providing a second polynucleotide encoding the affinity moiety; contacting a cell with the second polynucleotide encoding the affinity moiety; incubating the cell in cell culture media; and purifying the affinity moiety.
  • a first polynucleotide encodes the HUH tag and the affinity moiety; wherein the HUH tag and the affinity moiety are positioned within a single reading frame; wherein expression of the HUH tag and the affinity moiety are controlled by a promoter; and wherein providing the affinity moiety and providing the HUH tag includes contacting a cell with the first polynucleotide encoding the HUH tag and the affinity moiety; incubating the cell in cell culture media; and purifying the HUH tag and the affinity moiety.
  • providing the affinity moiety includes providing an intermediate binder protein.
  • the intermediate binder protein includes at least a portion of Protein A (SEQ ID NO: 15), Protein L (SEQ ID NO:55), or Protein G (SEQ ID NO: 16).
  • the intermediate binder protein is attached to the HUH tag.
  • the intermediate binder protein includes at least 50 amino acids of protein G (SEQ ID NO: 16).
  • the intermediate binder protein includes an amino acid sequence having at least 80% sequence identity to protein G C3 (SEQ ID NO:51).
  • the intermediate binder protein includes protein G C3 (SEQ ID NO: 51) including an E23BPA mutation.
  • the affinity moiety further includes an antibody of interest.
  • the antibody of interest is noncovalently attached to the intermediate binder protein. In one or more embodiments, the antibody of interest is covalently attached to the intermediate binder protein.
  • the present disclosure relates to a method of detecting presence of a molecule in a sample, the method including contacting a sample with a fusion protein; amplifying at least a portion of the oligonucleotide to provide an amplified oligonucleotide; sequencing the amplified oligonucleotide to obtain sequencing results; and interpreting the sequencing results.
  • interpreting the sequencing results includes detecting the presence of an antigen recognized by the fusion protein.
  • FIG. 1 Plasmid design for HUH-tagged recombinant antibodies.
  • SpI secretion signal
  • BamHI restriction sites for cloning variable domains
  • Either plasmid can be HUH-tagged at the C-terminus of the constant domains. Included in the HUH-tagged expression cassette is an N-terminal rigid linker and a C-terminal HA epitope tag.
  • FIG. 2 Generic full-length antibodies HUH-tagged at the C-terminus of the light or heavy chains. HA epitope tag not shown for clarity.
  • FIG. 3 Adduct formation activity of light chain HUH-tagged antibody versus heavy chain HUH-tagged antibody. Lanes left of the ladder are reaction samples ran on the protein gel under non-reducing conditions and show the full-length antibodies. Lanes to the right of the ladder are reaction samples ran on the protein gel under reducing conditions. Anti-HA antibodies were used to probe the blot, thus only the HUH-tagged chains are observed as the HA epitope tag is bound to WDV.
  • FIG. 4 Coomassie stained SDS-PAGE gel of purification by Protein G Resin.
  • FIG. 5 Chromatograph of an HUH-tagged antibody purification.
  • FIG. 6 Antibody purification analyzed by SDS-PAGE.
  • A Analyzed samples from protein expression through the last step of purification by size exclusion chromatography.
  • B Concentrated pre- and post-purification samples.
  • FIG. 7 Adduct formation reaction with fully purified anti-p l integrin antibody (K20).
  • FIG. 8 SDS-PAGE gels of adduct formation reaction with fully purified anti-pi integrin antibody (K20). Gel under reducing conditions.
  • FIG. 9 SDS-PAGE gels of adduct formation reaction with fully purified anti-pi integrin antibody (K20). Gel under non-reducing conditions.
  • FIG. 10 Overview of HUH-antibody selection.
  • FIG. 11 Schematic cartoon showing potential HUH-antibody configurations.
  • FIG. 12 Schematic cartoon of the mechanism of action of an HUH tag.
  • FIG. 13 Design of the HUH-mAb for DNA conjugation.
  • A The dual plasmid design allows for conjugation of DNA to the antibody at the C-terminus of the light or heavy chain constant region.
  • B A schematic representation of the standard HUH reaction producing the antibody-DNA conjugate.
  • FIG. 14 Characterization of HUH-mAb.
  • A SDS-PAGE of an HUH reaction to link HUH-K20-mAb and HUH-pertuzumab to single- stranded DNA observed under non-reducing conditions as a full-length (FL) antibody-DNA conjugate and under reducing conditions (right) as a heavy-chain (HC)-DNA conjugate.
  • B Binding affinity of the HUH-mAbs are comparable affinities to the commercial antibodies.
  • C Analysis of protein expression across cell lines comparing specificity of the HUH-mAbs and the commercial antibodies to their integrin Bl and HER2 antigens. Western blots of cell lysates probing with HUH-tagged and untagged antibodies produce comparable bands.
  • FIG. 15. Antigen Expression Patterns Analysis of protein expression across cell lines comparing specificity of the HUH-mAbs and commercial antibodies.
  • A Protein and RNA expression for integrin Bl.
  • B Protein and RNA expression for HER2. Protein expression levels were measured by flow cytometry and the RNA expression was reported in the Human Protein Atlas database.
  • FIG. 16 Representative flow cytometry analysis quantifying antigen expression levels across cancer cell lines. Cells were first labeled with the HUH-mAbs, then a secondary HA -tag antibody fluorescently labeled with FITC. The resulting flow cytometry data was gated to include live, single cells. The median fluorescence intensity for FITC was calculated.
  • FIG. 17 A schematic representation of methods of attaching an HUH tag to an antibody. Direct fusion of an antibody to an HUH tag (top), fusion of protein G (PG) and an HUH tag followed by binding to an IgG (middle), and photo-crosslinking an antibody to a BPA- modified protein G (bottom).
  • B The structure of 4-benzoyl-L-phenylalanine.
  • FIG. 18 A PAGE gel showing reaction of each WDV-PG recombinant protein and WDV-PG-x with DNA and an anti-K20 antibody.
  • FIG. 19 Adhesion data from U251 cells treated with WDV-PG or WDV-PG-x and anti- K20. Nuclei and anti- 1 integrin are shown using fluorescence. Cells in the top row were treated with low force, cells in the bottom row were treated with high force.
  • FIG. 20 Design of the HUH-ScF for DNA conjugation.
  • A The plasmid design allows for conjugation of DNA to the ScFv
  • B The split HUH-ScFv fusion
  • C Restriction sites to insert variable fragments of an ScFv of interest.
  • FIG. 21 Characterization of HUH-ScFv fusions.
  • FIG. 22 HUH-ScFv fusions do not alter the specificity of the antibody to its antigen
  • A Analysis of protein expression across cell lines using the normalized median fluorescence intensity from the flow cytometry data with a range of concentration of the HUH-ScFv fusions showing specificity to the HER2 antigen.
  • B Analysis of protein expression across cell lines using the normalized median fluorescence intensity from the flow cytometry data using HUH- pertuzumab fusions (labeled as IgG) showing specificity to their HER2 antigen.
  • FIG. 23 SDS-PAGE gel of WDV-PG fusion protein bound to multiple antibodies.
  • FIG. 24 SDS-PAGE gel of truncated WDV variants. DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
  • the present disclosure relates to a fusion protein including an affinity moiety, an HUH tag, and an oligonucleotide.
  • Antibodies are used for quantifying antigens in a wide range of immuno-assays. These include Western blots, ELISAs, and a variety of related in vitro assays, immuno-histochemical and immuno-fluorescence assays, and flow-cytometry. Immuno-assays are often used in clinical settings as diagnostic tests and tests to characterize or measure progression of a disease. In each of these approaches, the number of distinct antibody-antigen interactions that can be quantitated is limited by the resolution of the readout. As an example, when antibodies are detected via fluorescence, the spectral resolution of the fluorescent probes limits the number of different antibody-antigen interactions that can be detected.
  • CyTOF cytometry time of flight mass spectrometry
  • Antibody barcoding is the process of attaching a unique nucleic acid sequence to an antibody. When multiple antibodies are used, a different barcode can be attached to each different antibody. Barcode presence can be analyzed using sequencing methods. The number of barcodes that can be simultaneously analyzed is effectively limitless, depending on the length of the barcode. Thus, antibody barcoding increases the number of distinct antibody-antigen complexes that can be detected in a single sample. Labeling antibodies with nucleic acid barcodes allows a large number of antibody -antigen interactions to be analyzed in a given experiment.
  • HUH-tags may provide an enzymatic one-step method of barcoding antibodies.
  • HUH tags react with unmodified single-stranded DNA (ssDNA) under mild physiological conditions.
  • Unmodified ssDNA barcodes can be synthesized inexpensively at a large scale.
  • HUH tag-attached antibodies may be labeled rapidly, inexpensively, and at virtually any scale.
  • an HUH tag may be used to attach a barcode to an antibody if the HUH tag can be attached to the antibody.
  • Preparing an HUH tag attached to an antibody poses significant technical challenges. Preparing fusion proteins can be unpredictable. The molecular “rules” of attaching two proteins, particularly two proteins that each have a distinct molecular function, are often challenging to discern so that the fusion protein possesses the functions of both proteins. Thus, significant inventive effort is required to design and produce a fusion protein.
  • fusion protein refers to a molecule that includes at least a portion of at least two proteins.
  • the proteins may be attached via an amino acid linkage.
  • a common method of preparing a fusion protein is to prepare a nucleic acid construct (e g., plasmid DNA, mRNA) including a first coding sequence for a first protein and a second coding sequence for a second protein.
  • the first and second coding sequences can be combined such that they occupy the same reading frame.
  • translation of the nucleic acid construct results in a protein including the amino acid sequence of both proteins.
  • Another method of preparing a fusion protein consistent with the present disclosure is post-translational attachment of at least a portion of two proteins.
  • a first protein may be translated having a first free cysteine
  • a second protein may be translated having a second free cysteine.
  • the two free cysteines may be reacted to form a disulfide bond, attaching the two proteins.
  • the two proteins would be attached via a covalent bond.
  • Another method of preparing a fusion protein consistent with the present disclosure is by attaching two proteins via an intermediate binder protein.
  • a first protein and a second protein may each be attached to an intermediate binder protein.
  • One example of such a configuration is the attachment of an antibody to an HUH tag via Protein G.
  • Protein G may be attached to an HUH tag via an amino acid linkage and bound to an IgG antibody via the noncovalent paratope-antigen interaction. This configuration is included in the definition of a “fusion protein” in the present disclosure.
  • Protein G may be covalently crosslinked to the IgG antibody using a chemical crosslinking method, such as BPA, described in greater detail herein.
  • the present disclosure relates to a fusion protein including an affinity moiety, an HUH tag, and an oligonucleotide.
  • the oligonucleotide is covalently bound by the HUH tag.
  • the HUH tag and the oligonucleotide are attached.
  • the affinity moiety and the HUH tag are attached.
  • the HUH tag and the affinity moiety may be directly attached.
  • the HUH tag and the affinity moiety may be attached via an intermediate component (e.g., Protein A or Protein G as described in more detail below).
  • the HUH tag is attached to both the oligonucleotide and the affinity moiety. In this way, the affinity tag is not necessarily directly attached to the oligonucleotide, but is attached to the oligonucleotide via the HUH tag.
  • a schematic of this orientation is depicted in FIG. 11.
  • HUH-tags are a class of small ( ⁇ 40 kDa) DNA-binding proteins derived from HUH endonucleases. HUH-tags form robust, sequence-specific phospho-tyrosine covalent bonds with a short sequence of ssDNA within minutes under physiologic conditions. HUH-tags may be expressed as fusion proteins with any compatible protein of interest.
  • HUH endonucleases are enzymes that cleave and covalently attach to an ssDNA substrate in a sequence specific manner. These endonucleases are characterized by an active site including a pair of histidine (H) residues separated by a bulky hydrophobic residue (U).
  • H histidine
  • U bulky hydrophobic residue
  • An HUH motif may alternately include one histidine, a bulky hydrophobic residue, and a glutamine used for cation coordination in place of the second histidine.
  • Cation coordination triads are completed by a third residue, often glutamic acid or another histidine. This trio or pair of amino acids can coordinate a divalent cation, such as magnesium or manganese.
  • the sequence of ssDNA bound and cleaved by an HUH tag is typically referred to as its “ori” sequence.
  • the ori sequence of the HUH tags described herein are typically know. Examples of full ori sequences include SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:24. After cleavage, a part of the ori sequence is no longer bound by or attached to the HUH tag. Examples of the remaining ori sequence bound by the HUH tag after cleavage include SEQ ID NO:21, SEQ ID NO:23, and SEQ ID NO:25. While these exemplary ori sequences are explicitly described herein, many other ori sequences exist that may be compatible with the fusion proteins of the present disclosure.
  • the HUH tags of the present disclosure are derived from viral replication proteins. In nature, these proteins catalyze nicking of viral genomes to facilitate viral replication. While described herein in the context of a wheat dwarf virus (WDV) replication protein (SEQ ID NO: 17), it should be understood that other HUH tags may be compatible with the fusion protein of the present disclosure.
  • WDV wheat dwarf virus
  • the HUH tag includes an amino acid sequence including SEQ ID NO: 17. In one or more embodiments, the HUH tag includes an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98, or at least 99% sequence identity to SEQ ID NO: 17.
  • the HUH tag includes an amino acid sequence including SEQ ID NO: 17 without the first seven N-terminal amino acids. In one or more embodiments, the HUH tag includes an amino acid sequence including SEQ ID NO: 17 without the last nine C- terminal amino acids. In one or more embodiments, the HUH tag includes an amino acid sequence including SEQ ID NO: 17 without the first seven N-terminal amino acids and without the last nine C-terminal amino acids.
  • the HUH tag includes an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98, or at least 99% sequence identity to SEQ ID NO: 17 without the first seven N-terminal amino acids, without the last nine C-terminal amino acids, or without the first seven N-terminal amino acids and without the last nine C-terminal amino acids.
  • the HUH tag may lack one or more N-terminal or C-terminal residues relative to a wildtype HUH tag.
  • WDV SEQ ID NO: 17
  • WDV is catalytically active when one or more N- terminal residues, C-terminal residues, or both, are removed from the protein (FIG. 24).
  • the HUH tag includes an amino acid sequence including SEQ ID NO:63. In one or more embodiments, the HUH tag includes an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98, or at least 99% sequence identity to SEQ ID NO: 63.
  • the HUH tag includes an amino acid sequence including SEQ ID NO:64. In one or more embodiments, the HUH tag includes an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98, or at least 99% sequence identity to SEQ ID NO:64.
  • the HUH tag includes an amino acid sequence including SEQ ID NO: 65. In one or more embodiments, the HUH tag includes an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98, or at least 99% sequence identity to SEQ ID NO: 65.
  • the HUH tag includes an amino acid sequence including SEQ ID NO:66. In one or more embodiments, the HUH tag includes an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98, or at least 99% sequence identity to SEQ ID NO:66.
  • the HUH tag includes a sequence similar to SEQ ID NO: 17 but lacking one or more N-terminal amino acids. In one or more embodiments, the HUH tag includes an amino acid sequence including SEQ ID NO: 67 or SEQ ID NO:68. In one or more embodiments, the HUH tag includes an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98, or at least 99% sequence identity to SEQ ID NO:67 or SEQ ID NO:68. In one or more embodiments, the HUH tag includes a sequence similar to SEQ ID NO: 17 but lacking one or more C-terminal amino acids.
  • the HUH tag includes an amino acid sequence including any one of SEQ ID NOs:69-71. In one or more embodiments, the HUH tag includes an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98, or at least 99% sequence identity to any one of SEQ ID NOs:69-71.
  • the HUH tag includes an amino acid sequence including SEQ ID NO: 18. In one or more embodiments, the HUH tag includes an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98, or at least 99% sequence identity to SEQ ID NO: 18.
  • the HUH tag includes an amino acid sequence including SEQ ID NO: 19. In one or more embodiments, the HUH tag includes an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98, or at least 99% sequence identity to SEQ ID NO: 19.
  • the HUH tag is structurally similar to at least one of SEQ ID NOs: 17-19.
  • a polypeptide is “structurally similar” to a reference polypeptide if the amino acid sequence of the polypeptide possesses a specified amount of identity compared to the reference polypeptide.
  • Structural similarity of two polypeptides can be determined by aligning the residues of the two polypeptides (for example, a candidate polypeptide and the polypeptide of, for example, SEQ ID NO: 17) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order.
  • a candidate polypeptide is the polypeptide being compared to the reference polypeptide (e.g., SEQ ID NO: 17).
  • a candidate polypeptide can be isolated, for example, from an animal, or can be produced using recombinant techniques, or chemically or enzymatically synthesized.
  • a pair-wise comparison analysis of amino acid sequences can be carried out using the BESTFIT algorithm in the GCG package (version 10.2, Madison WI).
  • polypeptides may be compared using the Blastp program of the BLAST 2 search algorithm, as described by Tatiana et al., (FEMS Microbiol Lett, 174, 247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website.
  • similarity refers to the presence of identical amino acids.
  • similarity refers to the presence of not only identical amino acids but also the presence of conservative substitutions.
  • a conservative substitution for an amino acid in a polypeptide may be selected from other members of the class to which the amino acid belongs. For example, it is well-known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity and hydrophilicity) can be substituted for another amino acid without altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine.
  • Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, or glutamine.
  • the positively charged (basic) amino acids include arginine, lysine, or histidine.
  • the negatively charged (acidic) amino acids include aspartic acid or glutamic acid.
  • Conservative substitutions include, for example, Lys for Arg or vice versa to maintain a positive charge; Glu for Asp or vice versa to maintain a negative charge; Ser for Thr or vice versa so that a free -OH is maintained; or Gin for Asn or vice versa to maintain a free -NH2.
  • biologically active analogs of a polypeptide containing deletions or additions of one or more contiguous or noncontiguous amino acids that do not eliminate a functional activity of the polypeptide are also contemplated.
  • the affinity moiety may be any biomolecule that binds to a biomolecule (e.g., protein, lipid, nucleic acid, carbohydrate, etc.).
  • the affinity moiety may include an antigenbinding site.
  • an antigen-binding site is a paratope.
  • a paratope is the region of an antibody that recognizes and binds to an antigen.
  • the affinity moiety includes a nucleotide aptamer or a glycan.
  • the affinity moiety includes a protein. In one or more embodiments, the affinity moiety is a protein. In embodiments wherein the affinity moiety includes a protein, the HUH tag may be attached directly to the protein. In one or more embodiments, the HUH tag is attached to the N-terminus of the affinity moiety protein. In one or more embodiments, the HUH tag is attached to the C-terminus of the affinity moiety protein. In one or more embodiments, the HUH tag is attached to the affinity moiety protein between the C- terminus and the N-terminus.
  • the affinity moiety includes an antibody.
  • the affinity moiety may be an IgG antibody, an IgM antibody, a single chain variable fragment (scFv), or a single-domain antibody (e.g., a NANOBODY, Ablynx, Ghent, Belgium).
  • the affinity moiety includes an antibody.
  • the antibody may include at least one heavy chain and at least one light chain.
  • the light chain relates to antigen recognition.
  • the light chain may include a paratope.
  • the heavy chain relates to self-recognition and modulation of host immune reaction.
  • the HUH tag is attached to the heavy chain of an antibody.
  • An antibody having an HUH tag attached to the heavy chain is sometimes referred to as “VH(WDV).”
  • the HUH tag is attached to the light chain of an antibody.
  • An antibody having an HUH tag attached to the heavy chain is sometimes referred to as “VL(WDV).”
  • the light chain may be a kappa light chain or a lambda light chain.
  • the HUH tag may be attached to a kappa light chain or a lambda light chain.
  • FIG. 2 A schematic showing a cartoon of an HUH tag attached to the heavy chain or light chain of an IgGl is shown in FIG. 2.
  • the HUH tag is attached between the heavy chain and the light chain of an antibody.
  • An antibody having an HUH tag attached between the heavy chain and the light chain is sometimes referred to as “VH(WDV)VL.”
  • VH(WDV)VL A schematic representation of several possible designs for an HUH tag between the heavy chain and light chain of an antibody is shown in FIG. 20.
  • the term “antibody” refers generally an immunoglobulin or a fragment thereof.
  • the term “antibody” encompasses not only immunoglobulins with an intact Fc region, but also antibody fragments capable of binding to a biological molecule (such as an antigen or receptor) or a portion thereof, including but not limited to Fab, Fab', F(ab')2, pFc', Fd, Fd', Fv, dAB, a single domain antibody (sdAb), a variable fragment (Fv), a single-chain variable fragment (scFv) or a disulfide-linked Fv (sdFv), a diabody or a bivalent diabody, a linear antibody, a single-chain antibody molecule, or a multispecific antibody (e.g., a tribody) formed from antibody fragments.
  • a biological molecule such as an antigen or receptor
  • the antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or subclass.
  • type e.g., IgG, IgE, IgM, IgD, IgA and IgY
  • class e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2
  • subclass e.g., IgG, IgE, IgM, IgD, IgA and IgY
  • class e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2
  • the fusion protein can include a humanized antibody derived from an animal single domain antibody. While an scFv has a heavy variable chain component and a light variable chain component joined by a flanking sequence, a single domain antibody consists of a single monomeric variable chain — i.e., a variable heavy chin or a variable light chain — that is capable of specifically engaging a target.
  • a single domain antibody may be derived from an antibody of any suitable animal such as, for example, a camelid (e.g., a llama or camel) or a cartilaginous fish (e.g., a wobbegong or a nurse shark).
  • a single domain antibody can provide superior physical stability, an ability to bind deep grooves, and increased production yields compared to larger antibody fragments.
  • the affinity moiety may have affinity for any target.
  • the fusion protein described herein may be compatible with an affinity moiety having specificity for nearly any target.
  • the affinity moiety has affinity for a biomolecule, such as a protein, peptide, lipid, glycan, nucleic acid, or a carbohydrate.
  • the affinity moiety has affinity for a protein present on the surface of a cell.
  • the affinity moiety has affinity for HER2, CD4, CD8, CD34, CD44, a cadherin, epidermal growth factor receptor (EGFR), insulin receptor (IR), a histone, dystrophin, an integrin, an ion channel, a peptide-MHC complex, a disease biomarker, or a cytoskeletal protein.
  • Protein G is an immunoglobulin binding protein derived from Streptococcus spp. that includes three IgG binding domains.
  • protein G can be recombinantly expressed in E. coli, either independently or as a fusion protein with another recombinantly expressed protein.
  • the fusion protein includes a recombinantly expressed protein G and a recombinantly expressed HUH tag.
  • the protein G and the HUH tag are covalently attached via an amino acid linker. Any suitable amino acid linker may be used.
  • the protein G may be C-terminal to the HUH tag.
  • the protein G may be N-terminal to the HUH tag.
  • Protein G may be used as an intermediate binder protein (e.g., it may be used to facilitate attachment of an affinity moiety of interest to an HUH tag) or as an affinity moiety itself.
  • an “intermediate binder protein” is defined as a component of an affinity moiety.
  • a fusion protein may include an HUH tag and an affinity moiety, wherein the affinity moiety includes a portion of protein G and an IgG.
  • the affinity moiety includes an intermediate binder protein (a portion of protein G) and a moiety that binds to an epitope of interest (an IgG).
  • the affinity moiety includes all or a portion of protein G (SEQ ID NO: 16). In one or more embodiments, the affinity moiety includes at least 20, at least 30, at least 40, at least 50, at least 60, or at least 70 amino acids of protein G (SEQ ID NO: 16).
  • Protein G includes three IgG domains, referred to herein as Cl (SEQ ID NO:49), C2 (SEQ ID NO:50), and C3 (SEQ ID NO:51).
  • the affinity moiety includes an amino acid sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:49.
  • the affinity moiety includes an amino acid sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:50.
  • the affinity moiety includes an amino acid sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:51. In one or more embodiments, the affinity moiety includes an amino acid sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:51 including an E23BPA mutation.
  • Each IgG domain may independently bind the Fc region of an IgG.
  • a fusion protein includes an HUH tag and Cl, C2, C2, or a combination thereof.
  • the HUH tag and the IgG domain i.e., Cl, C2, or C3 may be recombinantly expressed as a fusion protein bound by an amino acid linker.
  • the HUH tag may be C-terminal to the IgG domain.
  • the HUH tag may be N-terminal to the IgG domain.
  • Protein L is an immunoglobulin binding protein derived from Peptostreptococcus magnus that binds the L chain of immunoglobulins. Protein L binds to all antibody classes, including IgG, IgM, IgA, IgE, and IgD. Protein L may also bind scFvs and Fab fragments.
  • the affinity moiety includes all or a portion of protein L (SEQ ID NO: 55).
  • Protein A is an immunoglobulin binding protein derived from Staphylococcus aureus that binds the Fc region of most immunoglobulins.
  • the affinity moiety includes all or a portion of protein A (SEQ ID NO: 16).
  • the HUH tag and the affinity moiety are directly attached.
  • the HUH tag and the affinity moiety may be directly attached in any suitable manner.
  • the HUH tag and the affinity moiety are connected by an amino acid linkage.
  • the HUH tag and the affinity moiety may be connected by a polypeptide linker.
  • a suitable linker is the rigid linker of SEQ ID NO:48.
  • One example of a suitable linker is the flexible linker of SEQ ID NO:60.
  • Other suitable linkers include SEQ ID NOs:72-78.
  • Another linker is (XP) n , wherein X is any amino acid, preferably Ala, Lys, or Glu.
  • the HUH tag and the affinity moiety may be attached via a chemical linkage.
  • Methods of attaching proteins chemically include, but are limited to, a maleimide linkage, a hydrazide linkage, or an NHS ester linkage.
  • the fusion protein includes one or more modified amino acids.
  • the HUH tag, the affinity moiety, or both the HUH tag and the affinity moiety may include one or more modified amino acids.
  • a “modified” amino acid includes any amino acid other than the 20 common amino acids.
  • a modified amino acid is 4-benzoyl-phenylalanine (BP A), shown in FIG. 17(B).
  • BPA is a photoreactive amino acid that may be incorporated by an unmodified ribosome.
  • UV radiation such as light having a wavelength of 365 nm
  • BPA forms a covalent chemical crosslink with neighboring compounds.
  • BPA may be incorporated into a protein at a location proximate to a second protein.
  • the BPA may bond to a proximate residue of the second protein, covalently attaching the two proteins.
  • the bond formed between BPA and an adjacent amino acid is between the ketone oxygen and a C, S, or O on the amino acid.
  • the bond is an ether.
  • the affinity moiety includes one or more modified amino acids.
  • the affinity moiety includes an intermediate binder protein and an affinity moiety of interest
  • the intermediate binder protein may include one or more modified amino acids.
  • the affinity moiety includes all or a portion of protein G, wherein the protein G includes a modified amino acid.
  • the affinity moiety includes a single IgG binding domain from protein G (i.e., Cl, C2, or C3), wherein the IgG binding domain includes a modified amino acid.
  • the affinity moiety includes C3, wherein C3 includes a modified amino acid.
  • the modified amino acid may be BPA.
  • the modified amino acid may be at residue E23.
  • the affinity moiety may include C3 including E23BPA.
  • Incorporation of BPA into an intermediate binder protein may advantageously increase the strength of attachment of the HUH tag and the affinity moiety.
  • attaching the antibody K20 to an HUH tag via the protein G interaction resulted in a noncovalent bond that was not resistant to strong force.
  • the protein G was modified to include BPA, and when the affinity moiety was exposed to UV radiation when K20 was bound to protein G, the interaction was significantly stronger and held up to rupture force.
  • the strength of the interaction between the HUH tag and the antibody of interest was increased. The strong interaction made the fusion protein suitable for applications such as force sensing.
  • the HUH tag and the affinity moiety are indirectly attached.
  • an indirect attachment is typically noncovalent. Rather, the HUH tag and the affinity moiety may be attached via an intermediate component.
  • the HUH tag may be directly attached to an antibody-binding domain. The antibody-binding domain may bind the antibody, thereby attaching the HUH tag and the affinity moiety indirectly.
  • Any antibody -binding domain may be used in the fusion protein described herein.
  • An antibody -binding domain may include or may be a portion of Protein A (SEQ ID NO: 15).
  • An antibody-binding domain may include or may be a portion of Protein G (SEQ ID NO: 16).
  • the HUH tag includes an amino acid sequence including SEQ ID NO: 15. In one or more embodiments, the HUH tag includes an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98, or at least 99% sequence identity to SEQ ID NO: 15.
  • the HUH tag includes an amino acid sequence including SEQ ID NO: 16. In one or more embodiments, the HUH tag includes an amino acid sequence having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98, or at least 99% sequence identity to SEQ ID NO: 16.
  • An antibody -binding domain may be structurally similar to Protein A (SEQ ID NO: 15) or a portion of Protein A (SEQ ID NO: 15).
  • An antibody-binding domain may be structurally similar Protein G (SEQ ID NO: 16) or a portion of Protein G (SEQ ID NO: 16).
  • the antibody-binding domain is directly attached to the HUH tag.
  • the HUH tag and the antibody -binding domain may be attached via a peptide linkage.
  • the HUH tag and the antibody -binding domain may be attached via a chemical linkage.
  • the HUH tag and the antibody -binding domain may have been expressed from a single coding sequence.
  • the HUH tag and the antibody -binding domain may be encoded by the same polynucleotide and expressed as a fusion protein prior to assembly of the fusion protein.
  • the HUH tag and the affinity moiety may be attached via a protein-based bioconjugation system.
  • a protein-based bioconjugation system forms a covalent linkage, such as an isopeptide linkage.
  • the HUH tag and the affinity moiety may be attached using the SpyTag/SpyCatcher system or the SnoopTag/SnoopCatcher system.
  • the HUH tag is be attached to a first bioconjugation protein and the affinity moiety is attached to a second bioconjugation protein.
  • the HUH tag may be attached to the SpyTag protein and the affinity moiety may be attached to the SnoopTag protein.
  • the first protein of the protein-based bioconjugation system is directly attached to the HUH tag.
  • the HUH tag and the first protein may be attached via a peptide linkage.
  • the HUH tag and the first protein may be attached via a chemical linkage.
  • the HUH tag and the first protein may have been expressed from a single coding sequence.
  • the HUH tag and the first protein may be encoded by the same polynucleotide and expressed as a fusion protein prior to assembly of the fusion protein.
  • the second protein of the protein-based bioconjugation system is directly attached to the affinity moiety.
  • the affinity moiety and the second protein may be attached via a peptide linkage.
  • the affinity moiety and the second protein may be attached via a chemical linkage.
  • the affinity moiety and the second protein may have been expressed from a single coding sequence.
  • the affinity moiety and the second protein may be encoded by the same polynucleotide and expressed as a fusion protein prior to assembly of the fusion protein.
  • the HUH tag and the affinity moiety may be attached via an affinity interaction.
  • An affinity interaction is typically a strong, noncovalent interaction.
  • the affinity interaction may be, for example, a biotin-avidin interaction.
  • a “fusion protein” may refer to an affinity moiety attached to an HUH tag via a noncovalent affinity interaction protein.
  • the fusion protein includes an oligonucleotide.
  • nucleic acid or “oligonucleotide” refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • Nucleic acids include but are not limited to genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules such as aptamers, plasmids, anti-sense DNA strands, shRNA, ribozymes, nucleic acids conjugates, and oligonucleotides.
  • a nucleic acid may be single-stranded, double-stranded, linear, or covalently circularly closed molecule.
  • a nucleic acid can be isolated.
  • isolated nucleic acid means that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, (iv) was synthesized, for example, by chemical synthesis, or (vi) extracted from a sample.
  • a nucleic acid might be introduced — i.e., transfected — into cells. When RNA is used to transfect cells, the RNA may be modified by stabilizing modifications, capping, or polyadenylation.
  • nucleic acid can be extracted, isolated, amplified, or analyzed by a variety of techniques such as those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, NY 2,028 pages (2012); or as described in U.S. Patent No. 7,957,913; U.S. Patent No. 7,776,616; U.S. Patent No. 5,234,809; and U.S. Patent No. 9,012,208.
  • Examples of nucleic acid analysis include, but are not limited to, sequencing and DNA-protein interaction. Sequencing may be by any method known in the art.
  • DNA sequencing techniques include classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, and next generation sequencing methods such as sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, Illumina/Solexa sequencing, allele specific hybridization to a library of labeled oligonucleotide probes, sequencing by synthesis using allele specific hybridization to a library of labeled clones that is followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, polony sequencing, and SOLiD sequencing.
  • Separated molecules may be sequenced by sequential or single extension reactions using polymerases or ligases as well as by single or sequential differential hybridizations with libraries of probes.
  • the oligonucleotide may include three nucleotides to 100 nucleotides, such as five nucleotides to 50 nucleotides, or 10 nucleotides to 30 nucleotides.
  • the oligonucleotide may include at least three nucleotides, at least four nucleotides, at least five nucleotides, at least six nucleotides, at least seven nucleotides, at least eight nucleotides, at least nine nucleotides, or at least 10 nucleotides.
  • the oligonucleotide may include at most 100 nucleotides, at most 90 nucleotides, at most 80 nucleotides, at most 70 nucleotides, at most 60 nucleotides, at most 50 nucleotides, at most 45 nucleotides, at most 40 nucleotides, at most 35 nucleotides, at most 30 nucleotides, at most 25 nucleotides, or at most 20 nucleotides.
  • the oligonucleotide includes a barcode.
  • the barcode may include five nucleotides to 50 nucleotides.
  • the barcode may include at least four nucleotides, at least five nucleotides, at least six nucleotides, at least seven nucleotides, or at least eight nucleotides.
  • the barcode may include at most 50 nucleotides, at most 45 nucleotides, at most 40 nucleotides, at most 35 nucleotides, at most 30 nucleotides, at most 25 nucleotides, at most 24 nucleotides, at most 23 nucleotides, at most 22 nucleotides, at most 21 nucleotides, at most 20 nucleotides, at most 19 nucleotides, at most 18 nucleotides, at most 17 nucleotides, at most 16 nucleotides, at most 15 nucleotides, at most 14 nucleotides at most 13 nucleotides, at most 12 nucleotides, at most 11 nucleotides, at most 10 nucleotides, or at most nine nucleotides.
  • the oligonucleotide includes an HUH ori sequence or a portion of an HUH ori sequence.
  • the oligonucleotide is attached to the fusion protein by reacting an oligonucleotide with the HUH tag. After reacting with the HUH tag, a 5' region of the HUH ori sequence is cleaved and may diffuse away. A 3' region of the HUH ori sequence remains attached to the HUH tag via a covalent bond between the newly formed 3' hydroxyl and the catalytic tyrosine (FIG. 12).
  • the fusion protein includes a region of nucleotides attached to the HUH tag.
  • the fusion protein includes an oligonucleotide sequence including SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25.
  • oligonucleotides with modified nucleotides is typically more expensive than synthesis of oligonucleotides without modified nucleotides.
  • modified nucleotides are often required for commonly used methods of conjugating an oligonucleotide to a protein.
  • HUH tags react with oligonucleotides not including modified nucleotides.
  • the fusion proteins of the present disclosure and methods of preparation thereof may advantageously not require oligonucleotides with modified nucleotides.
  • the oligonucleotide includes DNA nucleotides, RNA nucleotides, or a combination thereof.
  • the oligonucleotide comprises at most 5, at most 4, at most 3, at most 2, or at most 1 modified nucleotides. In one or more of these embodiments, the oligonucleotide does not comprise any modified nucleotides.
  • a “modified” nucleotide is any nucleotide other than the standard four DNA nucleotides and the standard four RNA nucleotides.
  • a modified nucleotide is any nucleotide other than cytidylate (CMP, cytidine 5 '-monophosphate), deoxycytidylate (dCMP, deoxy cytidine 5 '-monophosphate), adenylate (AMP, adenosine 5 '-monophosphate), deoxyadenylate (dAMP, deoxy adenosine 5 '-monophosphate), guanylate (GMP, guanosine 5'- monophosphate), deoxy guanyl ate (dGMP, deoxy guanosine 5 '-monophosphate), uridylate (UMP, uridine 5 '-monophosphate), or thymidylate (dTMP, thymidylate (
  • the nucleobase e.g., adenine, cytosine, guanine, thymine, or uracil
  • the nucleotide backbone e.g., the sugar phosphate backbone
  • the oligonucleotide may be modified to include an affinity moiety, such as a biotin.
  • the oligonucleotide may include a fluorophore, such as a CY molecule, an ALEXA FLUOR molecule (Molecular Probes, Inc., Eugene, OR), an ATTO molecule (Atto-tec, GmbH, Siegen, Germany), an IRDYE molecule (Li-Cor Biotech, LLC, Lincoln, NE), a rhodamine-based dye, ROX, FITC, FAM, fluorescein, or HEX.
  • a fluorophore such as a CY molecule, an ALEXA FLUOR molecule (Molecular Probes, Inc., Eugene, OR), an ATTO molecule (Atto-tec, GmbH, Siegen, Germany), an IRDYE molecule (Li-Cor Biotech, LLC, Lincoln, NE), a rhodamine-based dye, ROX, FITC,
  • the oligonucleotide includes one or more nucleotides having a modified backbone.
  • the oligonucleotide includes one or more nucleotides having a peptide nucleic acid (PNA) backbone. PNA nucleotides may be advantageous because they are resistant to degradation by endonucleases and exonucleases.
  • the oligonucleotide includes one or more phosphorothioate modifications.
  • the fusion protein may include additional components.
  • the additional components may confer additional activities or abilities. Any additional component may be on a N-terminus of the fusion protein, a C-terminus of the fusion protein, or between an N-terminus and a C-terminus of the fusion protein.
  • a fusion protein may include more than one additional component.
  • a fusion protein may include an affinity tag and a secretion signal, described in greater detail below.
  • the fusion protein includes an affinity tag.
  • the affinity tag may be a HIS-tag, FLAG tag, SUMO tag, hemagglutinin (HA) tag, a MYC-tag, a SpyTag, or a SnoopTag.
  • the fusion protein includes a fluorescent or colorimetric label.
  • the fluorescent label may include a fluorescent protein.
  • the fluorescent protein may include GFP, YFP, RFP, mCherry, mKate2, mVenus, tdTomato, or CFP.
  • the fluorescent label may include a small molecule fluorophore.
  • the small molecule fluorophore may include a CY molecule, an ALEXA FLUOR molecule (Molecular Probes, Inc., Eugene, OR), an ATTO molecule (Atto-tec, GmbH, Siegen, Germany), an IRDYE molecule (Li-Cor Biotech, LLC, Lincoln, NE), a rhodamine-based dye, ROX, FITC, FAM, fluorescein, or HEX.
  • the fusion protein includes a secretion signal.
  • the secretion signal is on the N-terminus of the fusion protein.
  • the secretion signal is typically between five and 20 amino acids long.
  • the secretion signal includes five amino acids to 20 amino acids, such as 5 amino acids to 16 amino acids, 8 amino acids to 14 amino acids, or 10 amino acids to 12 amino acids.
  • this disclosure describes an isolated nucleic acid sequence that encodes any embodiment of the fusion protein, or any component fragment of the fusion protein, such as the amino acid sequence of any one of SEQ ID NO:6 and SEQ ID NOs: 15-19. Given the amino acid sequence of any fusion protein, or one or more component fragments of the fusion protein, a person of ordinary skill in the art can determine the full scope of polynucleotides that encode that amino acid sequence using conventional, routine methods.
  • a “coding region” refers to a nucleotide sequence that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end.
  • a “regulatory sequence” is a nucleotide sequence that regulates expression of a coding sequence to which it is operably linked. Regulatory sequences include, for example, promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and transcription terminators.
  • the term “operably linked” refers to a juxtaposition of components such that they are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence is “operably linked” to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.
  • the present disclosure relates to a polynucleotide encoding all or a portion of a fusion protein described herein.
  • a polynucleotide encodes an HUH tag and an affinity moiety.
  • the HUH tag and the affinity moiety may be encoded to be expressed as a single recombinant fusion protein.
  • the HUH tag and the affinity moiety may be encoded to be expressed as two separate recombinant proteins.
  • a first polynucleotide encodes an HUH tag and a second polynucleotide encodes an affinity moiety. The separate recombinant proteins may later be attached as described herein.
  • the present disclosure relates to a plate including the fusion protein of the present disclosure.
  • the plate may be a multi-well plate, such as a 48-well plate, a 96-well plate, or a 384-well plate.
  • the plate may include a surface coating.
  • the surface coating may include a fusion protein of the present disclosure.
  • the surface coating may include a blocking protein, such as albumin.
  • the plate may be used for a method of the present disclosure.
  • the plate may be suitable for cell culture.
  • the plate may be sterile.
  • the plate may include any suitable material, such as glass or plastic, such as polystyrene.
  • kits including the fusion protein of the present disclosure relate to a kit including the fusion protein of the present disclosure.
  • a kit of the present disclosure is used to determine the presence of one or more epitopes in a sample.
  • a kit of the present disclosure is used to determine the abundance of one or more epitopes in a sample.
  • a kit includes at least one fusion of the present disclosure, including an HUH tag, an oligonucleotide, and an affinity moiety, and one or more other components in a suitable packaging material in an amount sufficient for at least one reaction.
  • the kit may also include reagents for binding and nucleotide amplification (e.g., PCR) and suitable buffers as described herein. Additional examples of other components include a positive control fusion protein, such as a fusion protein of a known concentration, or a negative control fusion protein, such as an affinity moiety not including an oligonucleotide. Optionally, other reagents such as buffers and solutions needed to fusion protein are also included. Instructions for use of the packaged components are also typically included.
  • the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit.
  • the packaging material is constructed by known methods, preferably to provide a sterile, contaminant-free environment.
  • the packaging material has a label which indicates that the components can be used for determining the presence and/or abundance of an epitope.
  • the packaging material contains instructions indicating how the materials within the kit are employed to practice a reaction with a fusion protein.
  • the term “package” refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits the polypeptides.
  • Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.
  • a method of preparing the fusion protein includes providing the HUH tag, providing the affinity moiety, wherein the affinity moiety is attached to the HUH tag, providing an uncut oligonucleotide, and reacting the HUH tag with the uncut oligonucleotide to yield the HUH tag attached to the oligonucleotide.
  • the HUH tag may be provided in any suitable manner.
  • the HUH tag is provided as a purified protein.
  • the HUH tag may be expressed as a recombinant protein prior to preparation of a fusion protein.
  • the HUH tag may be expressed using bacterial cell culture, which is typically scalable and relatively inexpensive. However, many HUH tags are compatible with expression in cells other than bacterial cells, such as insect cells (e g., Sf9 cells) or mammalian cells (e.g., HEK293 cells).
  • providing the HUH tag includes recombinantly expressing the HUH tag.
  • Recombinantly expressing the HUH tag may include providing a polynucleotide encoding the HUH tag.
  • a polynucleotide encoding an HUH tag typically includes expression elements such as a promoter and polyadenylation signal.
  • a polynucleotide encoding an HUH tag includes DNA nucleotides.
  • a polynucleotide encoding an HUH tag includes a plasmid.
  • a polynucleotide encoding an HUH tag includes RNA nucleotides.
  • a polynucleotide encoding an HUH tag includes an mRNA transcript. In one or more embodiments, a polynucleotide encoding an HUH tag includes a selectable marker, such as an antibiotic resistance cassette.
  • the affinity moiety may be provided in any suitable manner.
  • the affinity moiety is provided as a purified protein.
  • the affinity moiety may be expressed as a recombinant protein prior to preparation of a fusion protein.
  • Many affinity moi eties are available commercially.
  • One possible advantage of the fusion proteins and methods described herein is their compatibility with commercially available affinity moieties, such as antibodies.
  • providing the affinity moiety includes recombinantly expressing the affinity moiety.
  • Recombinantly expressing the affinity moiety may include providing a polynucleotide encoding the affinity moiety.
  • a polynucleotide encoding an affinity moiety typically includes expression elements such as a promoter and polyadenylation signal.
  • a polynucleotide encoding an affinity moiety includes DNA nucleotides.
  • a polynucleotide encoding an affinity moiety includes a plasmid.
  • a polynucleotide encoding an affinity moiety includes RNA nucleotides.
  • a polynucleotide encoding an affinity moiety includes an mRNA transcript. In one or more embodiments, a polynucleotide encoding an affinity moiety includes a selectable marker, such as an antibiotic resistance cassette.
  • each polynucleotide may include a selectable marker.
  • the selectable markers may be the same.
  • Each selectable marker may be different. Use of a separate selectable marker for each polynucleotide may enable selection with multiple markers to ensure that all producer cells include each polynucleotide.
  • the HUH tag and the affinity moiety are provided by recombinantly expressing a polynucleotide encoding both the affinity moiety and the HUH tag.
  • the HUH tag and the affinity moiety may be encoded as a fusion protein attached by an amino acid linker on a polynucleotide.
  • the polynucleotide encoding the HUH tag and the affinity moiety may be contacted with a cell.
  • the cell may be incubated in cell culture media to facilitate expression of the HUH tag and the affinity moiety as a fusion protein attached via an amino acid linker.
  • the affinity moiety may be a relatively small affinity moiety, such as an scFv or a single-domain antibody.
  • a polynucleotide encoding the HUH tag and the antibody attached via a polypeptide linker may be provided.
  • a polynucleotide encoding an HUH tag attached to the heavy chain of an antibody is sometimes referred to herein as “VH-CH1-CH2-CH3-HUH tag.”
  • a polynucleotide encoding an HUH tag attached to the light chain of an antibody is sometimes referred to herein as “VL-CL-HUH tag.”
  • “express” and variations thereof refer to the ability of a cell to transcribe a structural gene, resulting in an mRNA, then translating the mRNA to form a protein that provides a detectable biological function to the cell.
  • Transformation of a host cell may be stable or transient.
  • transient transformation or “transiently transformed” refers to the introduction of one or more transgenes into a cell in the absence of integration of the transgene into the host cell's genome. Transient transformation may be detected by, for example, enzyme-linked immunosorbent assay (ELISA) that detects the presence of a polypeptide encoded by one or more of the transgenes. Alternatively, transient transformation may be detected by detecting the activity of the protein encoded by the transgene.
  • ELISA enzyme-linked immunosorbent assay
  • transient transformation may be detected by detecting the activity of the protein encoded by the transgene.
  • transient transformant refers to a cell that has transiently incorporated one or more transgenes.
  • stable transformation or “stably transformed” refers to the introduction and integration of one or more transgenes into the genome of a cell.
  • stable transformant refers to a cell that has stably integrated one or more transgenes into the genomic DNA.
  • a stable transformant is distinguished from a transient transformant in that, whereas genomic DNA from the stable transformant contains one or more transgenes, genomic DNA from the transient transformant does not contain a transgene.
  • the cell may include a mammalian cell, a bacterial cell, an insect cell, or any other suitable cell type
  • the fusion protein is expressed in a mammalian cell.
  • the mammalian cell may be a CHO cell (e.g., ExpoCHO cell), HEK293 cell (e.g., Expo293 cell, 6E HEK cell), or a HeLa cell.
  • a method includes lysing the cell.
  • the cell may be lysed using any suitable method, such as detergent lysis, hyperosmotic lysis, hypoosmotic lysis, sonication, or pressure lysis (e.g., use of a French press).
  • the method does not include lysing the cell.
  • cell lysis may not be required to isolate and purify the fusion protein.
  • the HUH tag, the affinity moiety, or both may be purified from cell culture media, such as conditioned cell culture media.
  • conditioned cell culture media is media in which cells have been cultured for at least 12 hours, such as 18 hours, 20 hours, 24 hours, or 48 hours.
  • Methods including recombinant expression of the HUH tag typically include purifying the HUH tag, the affinity moiety, or both.
  • purifying includes affinity purification.
  • the affinity tag may be used to purify the fusion protein.
  • Affinity purification may include use of an affinity column, affinity resin, magnetic affinity beads, or any other suitable substrate.
  • a Ni-NTA resin may be used to purify the fusion protein.
  • purifying includes chromatography.
  • purifying may include column chromatography. Ion exchange chromatography, size exclusion chromatography, and affinity chromatography may be used independently or in combination.
  • a fusion protein includes an intermediate binder protein.
  • a method of preparing a fusion protein further includes providing an intermediate binder protein.
  • the intermediate binder protein may include at least a portion of Protein A (SEQ ID NO: 15), protein L (SEQ ID NO: 525 or Protein G (SEQ ID NO: 16).
  • the intermediate binder protein may include protein G Cl (SEQ ID NO:49), protein G C2 (SEQ ID NO:50), or protein G C3 (SEQ ID NO:51).
  • the intermediate binder protein is attached to the HUH tag.
  • the intermediate binder protein may be covalently attached to the HUH tag.
  • the intermediate binder protein may be noncovalently attached to the HUH tag.
  • the attachment between the HUH tag and the intermediate binder protein is a strong molecular interaction.
  • the method includes crosslinking the intermediate binder protein to an antibody of interest.
  • crosslinking includes exposing the intermediate binder protein to light, such as UV radiation.
  • UV radiation includes light having a wavelength of 100 nm to 400 nm, such as a wavelength of 365 nm.
  • An intermediate binder protein attached to an HUH tag may be provided as an isolated protein.
  • an intermediate binder protein attached to an HUH tag may be recombinantly expressed in a cell.
  • a polynucleotide encoding an HUH tag and an intermediate binder protein attached via an amino acid linkage may be provided.
  • the HUH tag may be expressed fused to the intermediate binder protein using methods of protein expression described herein.
  • the present disclosure relates to a method of using the fusion protein described herein.
  • the fusion proteins of the present disclosure enable identification of tens or hundreds of antibodies via unique oligonucleotide barcodes attached to each antibody.
  • any method including identification of antibodies may be compatible with the fusion proteins described herein.
  • a method of using the fusion protein described herein includes contacting a sample with the fusion protein of the present disclosure, amplifying at least a portion of the oligonucleotide to provide an amplified oligonucleotide, sequencing the amplified oligonucleotide to obtain sequencing results, and interpreting the sequencing data.
  • the method further includes detecting the presence of an antigen recognized by the fusion protein.
  • amplifying at least a portion of the oligonucleotide includes polymerase chain reaction (PCR). PCR methods with low amplification bias, such as digital droplet PCR (ddPCR) may be used. It is typically desirable for the sequence of the barcode to be preserved with high accuracy during amplification. Thus, a high-fidelity polymerase may be used to amplify at least a portion of the oligonucleotide.
  • PCR polymerase chain reaction
  • a sample may include more than one fusion protein.
  • Each fusion protein may have the same barcode.
  • different fusion proteins may have different barcodes.
  • each affinity moiety may be attached to a unique barcode.
  • interpreting the sequencing data includes identifying which barcodes are present in the sequencing results and correlating the presence of each barcode to an affinity moiety.
  • each fusion protein typically includes exactly one oligonucleotide.
  • the method further includes adding a control oligonucleotide to the sample prior to amplification.
  • the control oligonucleotide may help provide a normalized quantification of each barcode present in the sample, and therefore, each antigen present.
  • amplified oligonucleotide or “PCR product” refers to an amplified fragment of DNA of defined size.
  • PCR product detection methods include, but are not restricted to, gel electrophoresis using agarose or polyacrylamide gel and adding ethidium bromide staining (a DNA intercalant), labeled probes (radioactive or non-radioactive labels, southern blotting), labeled deoxyribonucleotides (for the direct incorporation of radioactive or non-radioactive labels) or silver staining for the direct visualization of the amplified PCR products; restriction endonuclease digestion, which relies on agarose gel electrophoresis, polyacrylamide gel electrophoresis, or high-performance liquid chromatography (HPLC); dot blots, using the hybridization of the amplified DNA on specific labeled probes (radioactive or non-radioactive labels); high-pressure liquid
  • Fusion proteins of the present disclosure may be applied, for example, in any method wherein multiple antigens are simultaneously detected.
  • the fusion protein of the present disclosure may be used to profile the proteins present on the surface of a cell.
  • the fusion protein of the present disclosure may be used to profile antigens in lineage tracing methods.
  • the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended — i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
  • the steps may be performed in any feasible order. And, as appropriate, any combination of two or more steps may be performed simultaneously.
  • Embodiment l is a fusion protein including an affinity moiety; an HUH tag; and an oligonucleotide, wherein the oligonucleotide is covalently bound by the HUH tag.
  • Embodiment 2 is the fusion protein of embodiment 1, wherein the affinity moiety includes an IgG antibody, an scFv, or a single-domain antibody.
  • Embodiment 3 is the fusion protein of embodiment 1, wherein the oligonucleotide includes a barcode sequence.
  • Embodiment 4 is the fusion protein of embodiment 3, wherein the barcode sequence includes at least 8 nucleotides.
  • Embodiment 5 is the fusion protein of embodiment 1, wherein the oligonucleotide includes DNA nucleotides, RNA nucleotides, PNA nucleotides, or a combination thereof.
  • Embodiment 6 is the fusion protein of embodiment 1, wherein the oligonucleotide includes an HUH ori sequence.
  • Embodiment 7 is the fusion protein of embodiment 6, wherein the oligonucleotide includes SEQ ID N0:21.
  • Embodiment 8 is the fusion protein of any preceding embodiment, wherein the HUH tag and the affinity moiety are connected by an amino acid linkage.
  • Embodiment 9 is the fusion protein of embodiment 8, wherein the HUH tag and the affinity moiety are connected by a polypeptide linker.
  • Embodiment 10 is the fusion protein of any preceding embodiment, wherein the HUH tag and the affinity moiety are chemically attached.
  • Embodiment 11 is the fusion protein of embodiment 10, wherein the HUH tag and the affinity moiety are chemically attached via a maleimide linkage, a hydrazide linkage, or an NHS ester linkage.
  • Embodiment 12 is the fusion protein of any preceding embodiment, wherein the HUH tag and the affinity moiety are connected by an ether bond.
  • Embodiment 13 is the fusion protein of any preceding embodiment, wherein the HUH tag and the affinity moiety are attached via an affinity interaction.
  • Embodiment 14 is the fusion protein of embodiment 13, wherein the affinity interaction includes a biotin-avidin interaction.
  • Embodiment 15 is the fusion protein of any preceding embodiment, wherein the affinity moiety is an antibody including a heavy chain and a light chain.
  • Embodiment 16 is the fusion protein of embodiment 15, wherein the HUH tag is attached to the light chain.
  • Embodiment 17 is the fusion protein of embodiment 15, wherein the HUH tag is attached to the heavy chain.
  • Embodiment 18 is the fusion protein of any preceding embodiment, wherein the affinity moiety includes a protein having an N-terminus and a C-terminus.
  • Embodiment 19 is the fusion protein of embodiment 18, wherein the HUH tag is attached to the N-terminus of the affinity moiety.
  • Embodiment 20 is the fusion protein of embodiment 18, wherein the HUH tag is attached to the C-terminus of the affinity moiety.
  • Embodiment 21 is the fusion protein of embodiment 18, wherein the HUH tag is attached to the affinity moiety between the N-terminus and the C-terminus.
  • Embodiment 22 is the fusion protein of any preceding embodiment, wherein the affinity moiety includes an intermediate binder protein.
  • Embodiment 23 is the fusion protein of embodiment 22, wherein the intermediate binder protein includes at least 50 amino acids of protein G (SEQ ID NO: 16).
  • Embodiment 24 is the fusion protein of embodiment 23, wherein the intermediate binder protein includes an amino acid sequence having at least 80% sequence identity to protein G C3 (SEQ ID NO:51).
  • Embodiment 25 is the fusion protein of embodiment 22, wherein the intermediate binder protein includes protein G C3 (SEQ ID NO:51) including an E23BPA mutation.
  • 26 is the fusion protein of any one of embodiments 22-25, wherein the affinity moiety further includes an antibody of interest.
  • Embodiment 27 is the fusion protein of embodiment 26, wherein the antibody of interest is noncovalently attached to the intermediate binder protein.
  • Embodiment 28 is the fusion protein of embodiment 26, wherein the antibody of interest is covalently attached to the intermediate binder protein.
  • Embodiment 29 is the fusion protein of any preceding embodiment, further including an affinity tag.
  • Embodiment 30 is the fusion protein of embodiment 29, wherein the affinity tag includes a HIS-tag, FLAG tag, SUMO tag, hemagglutinin (HA) tag, a SpyTag, a SnoopTag, or a MYC- tag.
  • the affinity tag includes a HIS-tag, FLAG tag, SUMO tag, hemagglutinin (HA) tag, a SpyTag, a SnoopTag, or a MYC- tag.
  • Embodiment 31 is the fusion protein of any preceding embodiment, further including a
  • Embodiment 32 is the fusion protein of embodiment 31, wherein the fluorescent label includes a fluorescent protein.
  • Embodiment 33 is the fusion protein of embodiment 32, wherein the fluorescent protein includes GFP, YFP, RFP, mCherry, mKate2, mVenus, tdTomato, or CFP.
  • fO Embodiment 34 is the fusion protein of embodiment 31, wherein the fluorescent label includes a small molecule fluorophore.
  • Embodiment 35 is the fusion protein of embodiment 34, wherein the small molecule fluorophore includes a CY molecule, an ALEXA FLUOR molecule, an ATTO molecule, an IRDYE molecule, a rhodamine-based dye, ROX, FITC, FAM, fluorescein, or HEX.
  • the small molecule fluorophore includes a CY molecule, an ALEXA FLUOR molecule, an ATTO molecule, an IRDYE molecule, a rhodamine-based dye, ROX, FITC, FAM, fluorescein, or HEX.
  • Embodiment 36 is the fusion protein of any preceding embodiment, further including a secretion signal.
  • Embodiment 37 is a method of preparing the fusion protein of any preceding embodiment including providing the HUH tag; providing the affinity moiety, wherein the affinity moiety is attached to the HUH tag; providing an uncut oligonucleotide; and reacting the HUH tag with the
  • Embodiment 38 is the method of embodiment 37, wherein providing the HUH tag includes providing a first polynucleotide encoding the HUH tag; contacting a cell with the first polynucleotide encoding the HUH tag; incubating the cell in cell culture media; and purifying the HUH tag.
  • Embodiment 39 is the method of embodiment 37 or 38, wherein providing the affinity moiety includes providing a second polynucleotide encoding the affinity moiety; contacting a cell with the second polynucleotide encoding the affinity moiety; incubating the cell in cell culture media; and purifying the affinity moiety.
  • Embodiment 40 is the method of embodiment 37, wherein a first polynucleotide encodes
  • the HUH tag and the affinity moiety wherein the HUH tag and the affinity moiety are positioned within a single reading frame; wherein expression of the HUH tag and the affinity moiety are controlled by a promoter; and wherein providing the affinity moiety and providing the HUH tag includes contacting a cell with the first polynucleotide encoding the HUH tag and the affinity moiety; incubating the cell in cell culture media; and purifying the HUH tag and the affinity moiety.
  • Embodiment 41 is the method of any one of embodiments 38 to 40, further including lysing the cell.
  • Embodiment 42 is the method of any one of embodiments 38 to 41, wherein the HUH tag and the affinity molecular are purified from the cell culture media.
  • Embodiment 43 is the method of any one of embodiments 38 to 42, wherein purifying includes affinity purification.
  • Embodiment 44 is the method of any one of embodiments 38 to 42, wherein purifying includes chromatography.
  • Embodiment 45 is the method of embodiment 44, wherein chromatography includes size exclusion chromatography.
  • Embodiment 46 is the method of any one of embodiments 38 to 45, wherein the cell includes a mammalian cell.
  • Embodiment 47 is the method of any one of embodiments 38 to 45, wherein the cell includes a bacterial cell.
  • Embodiment 48 is the method of any one of embodiments 38 to 47, wherein the first polynucleotide includes a first selectable marker.
  • Embodiment 49 is the method of embodiment 48, wherein the second polynucleotide includes a second selectable marker.
  • Embodiment 50 is the method of embodiment 49, wherein the first selectable marker includes a polynucleotide encoding resistance to an antibiotic and the selectable marker includes a polynucleotide encoding resistance to an antibiotic.
  • Embodiment 51 is the method of any one of embodiments 37 to 50, wherein providing the affinity moiety includes providing an intermediate binder protein.
  • Embodiment 52 is the method of embodiment 51, wherein the intermediate binder protein includes at least a portion of Protein A (SEQ ID NO: 15), Protein L (SEQ ID NO:55), or Protein G (SEQ ID NO: 16).
  • Embodiment 53 is the method of embodiment 51 or 52, wherein the intermediate binder protein is attached to the HUH tag.
  • Embodiment 54 is the method of embodiment 53, wherein the intermediate binder protein is covalently attached to the HUH tag via an amino acid linkage.
  • Embodiment 55 is the method of any one of embodiments 51 to 54, wherein the intermediate binder protein is noncovalently attached to the affinity moiety.
  • Embodiment 56 is the method of embodiment 51, wherein the intermediate binder protein includes at least 50 amino acids of protein G (SEQ ID NO: 16).
  • Embodiment 57 is the method of embodiment 56, wherein the intermediate binder protein includes an amino acid sequence having at least 80% sequence identity to protein G C3 (SEQ ID NO:51).
  • Embodiment 58 is the method of embodiment 51, wherein the intermediate binder protein includes protein G C3 (SEQ ID NO: 51) including an E23BPA mutation.
  • Embodiment 59 is the method of any one of embodiments 51-58, wherein the affinity moiety further includes an antibody of interest.
  • Embodiment 60 is the method of embodiment 59, wherein the antibody of interest is noncovalently attached to the intermediate binder protein.
  • Embodiment 61 is the method of embodiment 59, wherein the antibody of interest is covalently attached to the intermediate binder protein.
  • Embodiment 62 is a method of detecting presence of a molecule in a sample, the method including contacting a sample with the fusion protein of any one of embodiments 1-36; amplifying at least a portion of the oligonucleotide to provide an amplified oligonucleotide; sequencing the amplified oligonucleotide to obtain sequencing results; and interpreting the sequencing results.
  • Embodiment 63 is the method of embodiment 62, wherein interpreting the sequencing results includes detecting the presence of an antigen recognized by the fusion protein.
  • Preparation of HUH-tagged recombinant antibodies included 1) cloning HUH-tag at the C-terminus of heavy /light chains, obtaining sequences of desired antibody variable regions, cloning variable regions of heavy and light chains into mammalian expression plasmids, 2) cotransfection of antibody plasmids to EXPI293 cells (Thermo Fisher Scientific, Inc., Waltham, MA), 3) purification of antibodies with Protein G or Protein A resin, 4) Purification of antibodies by size exclusion chromatography, 5) Testing activity of HUH-fused antibodies via reaction with DNA oligos.
  • the present examples describe preparation of fusion protein including a humanized HER2 -targeting monoclonal antibody (pertuzumab). A pi integrin antibody (clone K20) was also prepared.
  • Example 1 Cloning of HUH-tag at C-terminus of heavy /light chain and variable regions
  • the rigid linker-WDV-HA-tag-stop sequence was amplified with forward primer (SEQ ID NO:7) and reverse primer (SEQ ID NO:8).
  • the amplified fragment was inserted into the C- terminus of the constant heavy chain 3 using BamHI restriction site using a DNA assembly kit (NEBUILDER HiFi, New England Biolabs, Inc., Ipswich, MA) per the manufacturer’s protocol.
  • the rigid linker-WDV-HA-tag-stop sequence was amplified with forward primer (SEQ ID NO:9) and reverse primer (SEQ ID NO: 10).
  • the amplified fragment was inserted into the C- terminus of the constant light chain using BamHI restriction site using a DNA assembly kit (NEBUILDER HiFi, New England Biolabs, Inc., Ipswich, MA) per the manufacturer’s protocol.
  • variable fragment of the heavy chain was amplified with forward primer (SEQ ID NO: 11) and reverse primer (SEQ ID NO: 12) and inserted into RVL5 (Plasmid #104583) between the secretion signal sequence and the heavy chain constant region using SapI restriction enzyme.
  • variable fragment of the light chain was amplified with forward primer (SEQ ID NO: 13) and reverse primer (SEQ ID NO: 14) and inserted into RVL4 (Plasmid #104582) between the secretion signal sequence and the light chain constant region using SapI restriction enzyme using a DNA assembly kit (NEBUILDER HiFi, New England Biolabs, Inc., Ipswich, MA)per the manufacturer's protocol.
  • Plasmids were transformed into Escherichia coli (E. coll) STELLAR cells and plated onto 100 pg/mL ampicillin Luria-Bertani (LB) agar plates. Plasmids were then isolated and purified from individual colonies using the Qiagen miniprep kit. Plasmids were sequence verified by Sanger sequencing.
  • Plasmids were transiently transfected into EXPI293F (Thermo Fisher Scientific, Inc., Waltham, MA) suspension cells at a 1 :1 ratio using a transfection kit (EXPIFECT AMINE 293, Life Technologies, Corp., Carlsbad, CA) per the manufacturer's protocol in a vented flask at a cell density of 3 million cells/mL at 37 °C.
  • EXPIFECT AMINE 293, Life Technologies, Corp., Carlsbad, CA a transfection kit
  • Conditioned media was harvested 5 days to 7 days post transfection and clarified at 4,000*g for 25 minutes at 4 °C.
  • the supernatant was dialyzed overnight at 4 °C into a buffer of 20 mM Na2HPO4, 150 mM NaCl, pH 7.0, using a 10 kDa dialysis tube.
  • the dialyzed supernatant was filtered prior to purification using a 0.22 pM sterile polyethersul
  • Protein expression was first analyzed by western blotting using anti-HA tag antibody prior to protein purification.
  • Conditioned media samples were first run on a 4-12% SDS-PAGE gel and transferred to a nitrocellulose membrane.
  • the membrane was incubated in 5% skim milk in TBST blocking buffer for one hour on a shaker.
  • the membrane was first incubated overnight at 4 °C in a 1:2000 solution of anti-HA.l 1 antibody in 5% nonfat milk in Tris Buffered Saline with 0.01% Tween-20 (TBST) pH 7.4, followed by an hour incubation in a 1 :2000 solution of HRP Goat anti -Mouse 2° antibody.
  • the membrane was then washed 4x with TBST.
  • the membrane was incubated with a chemiluminescent substrate (Western Lightning Plus ECL, Revvity, Inc., Waltham, MA) solution and chemiluminescence imaged using a chemiluminescence imager (AMERSHAM 600UV, GE Life Sciences, Marlboro, MA).
  • chemiluminescent substrate Western Lightning Plus ECL, Revvity, Inc., Waltham, MA
  • AMERSHAM 600UV GE Life Sciences, Marlboro, MA
  • HUH-ScFv fusions were purified from the dialyzed supernatant using Ni 2+ -NTA resin.
  • 0.5 mL of Ni 2+ -NTA slurry was equilibrated in equilibration buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.5).
  • equilibration buffer 20 mM sodium phosphate, 500 mM NaCl, pH 7.5.
  • Approximately -100 ml of supernatant was incubated with the prepared resin at 4 °C on a rotator for one hour.
  • the protein-bound resin was then washed with 1 mL of Wash 1 buffer (20 mM Sodium phosphate, 500 mM NaCl, 20 mM Imidazole, pH 7.5.
  • the protein fusions were eluted with 1.5 mL of elution buffer (20 mM Sodium phosphate, 500 mM NaCl, 500 mM Imidazole, pH 7.5.) The eluted proteins were then buffer exchanged and concentrated into a storage buffer (20 mM Sodium phosphate, 150 mM NaCl buffer, pH 7.5) using an ultracentrifugation unit (AMICON Ultra- 15, Merck KGAA, Darmstadt, Germany) with a 3 kDa molecular cut off.
  • elution buffer 20 mM Sodium phosphate, 500 mM NaCl, 500 mM Imidazole, pH 7.5.
  • the eluted proteins were then buffer exchanged and concentrated into a storage buffer (20 mM Sodium phosphate, 150 mM NaCl buffer, pH 7.5) using an ultracentrifugation unit (AMICON Ultra- 15, Merck KGAA, Darmstadt, Germany) with a 3 kDa mo
  • Antibodies were purified from the dialyzed supernatant using Protein G resin (GenScript, Piscataway, NJ) or Protein A resin (Protein A resin (GenScript, Piscataway, NJ). 1 mL of resin was equilibrated in 20 mM Na2HPO4, 150 mM NaCl, pH 7.0 buffer prior to incubation with the dialyzed supernatant. Incubation of approximately 25 mL of supernatant with the prepared resin was done at 4 °C on a rotator for one hour. The antibody -bound resin was then washed with 30 mL of a 20 mM Na2HPC>4, 150 mM NaCl pH 7.0 buffer.
  • the antibodies were eluted 2X with 5 mL each of 100 mM glycine buffer pH 2 (Protein G resin) or pH 3 (Protein G resin) and immediately neutralized with approximately 1/10 volume of a 1 M Tris-HCl pH 9 buffer.
  • the eluted antibodies were then buffer exchanged and concentrated into 50 mM HEPES pH 8.0, 300 mM NaCl buffer using an ultracentrifugation unit (AMICON Ultra-15, Merck KGAA, Darmstadt, Germany) with a 3 kDa molecular weight cut-off.
  • a gel showing purification of the integrin antibody K20 with Protein G beads is shown in FIG. 6.
  • FIG. 4 is a gel showing purification of the integrin K20 VH (WDV) full-length antibodies using Protein G Resin as described herein. Elution from the resin under acidic conditions shows two clean peaks corresponding to the heavy and light chains of the full-length antibody under reducing conditions.
  • WDV integrin K20 VH
  • Antibodies were purified using size exclusion chromatography with a 24 mL column (SUPEROSE 6 10/300 GL, Cytiva Bioprocess R&D AB, Uppsala, Sweden) in a running buffer of 300 mM NaCl, 50 mM HEPES, pH 7.5. About 500 pL of protein sample concentrated to approximately 1 mg/mL was purified in a single purification run. Multiple purification runs were performed. An example of a chromatograph from one of the purification runs is shown in FIG. 5. Pooled fractions were then exchanged and concentrated into a 50 mM HEPES pH 8, 50 mM NaCl buffer.
  • FIG. 8 is a gel showing purification of the pertuzumab monoclonal antibody using Protein A or Protein G resin followed by size-exclusion chromatography.
  • FIG. 8 is a gel showing purification of the integrin K20 antibody using Protein A or Protein G resin followed by sizeexclusion chromatography.
  • HUH adduct formation reactions were performed with single- stranded oligos, containing the WDV DNA recognition motif (SEQ ID NO:20). Reactions were prepared with 3 pM of HUH-tagged antibody proteins and 10 pM of DNA oligo in a buffer solution of 50 mM HEPES pH 8, 50 mM NaCl, and 1 mM MnCh and incubated at 37 °C for 30 minutes. The reaction was stopped by the addition of 4/ Laemmli buffer with or without 5% beta-mercaptoethanol for reducing or non-reducing conditions. For reduced conditions, reactions were boiled at 100 °C for five minutes. Protein-DNA covalent product was analyzed using a 4-12% SDS-PAGE gel.
  • the upper molecular band shift of the protein-DNA covalent product was calculated by band intensity using imaging software (IMAGE LAB, Bio-Rad laboratories, Inc., Hercules, CA) with background subtraction. Antibodies were run under reducing and non-reducing conditions.
  • pertuzumab antibodies with fusions of the WDV HUH-tag at the C- terminus of the light chain or heavy chain were transfected in EXPI293 cells (Thermo Fisher Scientific, Inc., Waltham, MA). Reaction of a 30-nucleotide oligonucleotide including the WDV recognition sequence (SEQ ID NO: 19) was reacted directly with 4 pL of the conditioned media containing the secreted antibodies at 37 °C extended to one hour. A Western blot was run using anti -HA antibodies to visualize the HA epitope tag on the C-terminus of WDV (FIG. 3).
  • HUH-mAbs were prepared as described using recombinant anti-HER2 antibody (Thermo Fisher Scientific, Inc., Waltham, MA) and recombinant anti-integrin betal antibody (Abnova Corp., Taipei, Taiwan). Western blots of cell lysate were treated with recombinant HUH-mAbs to detect the HER2 receptor and the pi-integrin across four cancer cell lines. Briefly, cell lysates were prepared from cells cultured on 10 cm plates. Cells were washed twice with cold PBS and incubated for 30 minutes at 4 °C in a RIPA buffer containing a 1 : 100 dilution of a protease inhibitor (P8340).
  • P8340 protease inhibitor
  • Cell lysates were collected in 1.5 mL tubes and centrifuged at 13,000*g for 30 minutes at 4 °C. Supernatant samples on a 4-20% protein gel (Mini-PROTEAN TGX, Bio-Rad Laboratories, Inc., Hercules, CA) and blotted onto a nitrocellulose membrane. All antibody solutions used for the cell lysate analysis were prepared in the standard 5 % milk in TBST solution, washed and imaged as described above. The integrin pi antigen was detected by the commercial K20 antibody at a 1 :500 solution, followed by a 1 :4000 solution of the secondary anti-mouse-HRP antibody.
  • a 1 :500 solution was used and followed with a 1 : 10,000 solution of an HRP-conjugated antihuman IgG (H+L) antibody (#109-035-088, Jackson ImmunoResearch Laboratories, West Grove, PA).
  • HRP-conjugated antihuman IgG (H+L) antibody #109-035-088, Jackson ImmunoResearch Laboratories, West Grove, PA.
  • HRP-conjugated anti-human antibody H+L antibody
  • a 1 :1000 solution was used and followed with the same HRP-conjugated anti-human antibody previously described.
  • BLI biolayer interferometry
  • His-tagged recombinant human integrin aVpi heterodimer and His-tagged human ErbB2/Her2 protein ligands were purchased from R&D Systems (Minneapolis, MN) and were independently immobilized onto biosensors at a concentration of 25 pg/mL.
  • binding affinities of commercial pertuzumab and HUH-pertuzumab mAbs with HER2-ECD II were measured using the recombinant human HER2 protein (rHER2, R&D Systems, Minneapolis, MN).
  • Antibody solutions of 1 : 1 dilutions ranging from 5 pM to 0.039 pM concentrations were prepared in a buffer of PBS with 0.05% Tween-20 and 0.1% BSA. Following ligand immobilization and after the initial baseline, the BLI signal was monitored for association for 120 seconds, then dissociation for another 120 seconds. Binding affinities (Kd) were calculated using non-linear regression analysis for association then dissociation curves using PRISM10 software (GraphPad Software Inc., San Diego, CA).
  • the binding affinity of commercial K20 was found to be 212 nM.
  • the binding affinity of HUH-K20 was found to be 134 nM (FIG. 14B). These binding affinities were considered relatively comparable. These results suggest that the HUH fusion does not affect binding affinity.
  • the binding affinity of commercial pertuzumab was found to be of 3.9 nM.
  • the binding affinity of HUH-pertuzumab was found to be 5 nM (FIG. 14B). These binding affinities were considered relatively comparable, additionally suggesting that HUH fusion did not affect binding affinity.
  • Example 10 Cell-surface protein expression by flow cytometry
  • cell surface protein expression was characterized using flow cytometry. Independently, used the HUH-pertuzumab as the primary antibody to bind to the HER2 antigen and the HUH-K20 mAb to bind the pi -integrin antigen. Each HUH-mAb was detected by an anti-HA FITC-conjugated antibody against the HA tag located at the C-terminus. Briefly, cell surface expression of the HER2 and integrin 01 proteins were quantified from four cancer cell lines harvested from culture plates using TRYPLETM dissociation reagent, washed once in cold PBS with 1% BSA (w/v) and centrifuged at 300*g for five minutes.
  • Cells were resuspended in PBS with 1% BSA and aliquoted into separate four tubes of equal volumes for incubation with one of the following labeling conditions: HUH-K20 mAbs, HUH- pertuzumab-mAbs, fluorescently labeled secondary antibody (antiHA-FITC) and unlabeled. Labeling solutions were prepared in PBS with 1% BSA at 1 :200 antibody dilutions, with no antibody in the unlabeled solution. For each cell line, two cell suspensions were separately labeled with the prepared labeling solutions of the HUH-K20 mAb and the HUH-pertuzumab- mAb for 30 minutes at 4 °C and mixed intermittently by inverting the tube.
  • a third tube of cells were then labeled with the secondary antibody (anti-HA-FITC) for 30 minutes at 4 °C and mixed intermittently by inverting the tube. All labeled and unlabeled cells were then washed twice with cold PBS with 1% BSA and once with the flow buffer containing PBS with 1% BSA and 1 mM EDTA. After the final centrifugation step, cells were resuspended in 200 pL of flow buffer and kept covered and on ice until analyzed by flow cytometry (FIG. 16).
  • Example 11 WDV-Protein G (WDV-PG) recombinant expression, purification, and characterization.
  • Plasmid constructs encoding WDV-PG Cl (SEQ ID NO:49), WDV-PG C2 (SEQ ID NO:50), and WDV-PG C3 (SEQ ID NO:51) were prepared. Each protein was separately recombinantly expressed in E. coli and purified using Ni-NTA resin. Each fusion protein was incubated with a DNA duplex including an HUH binding site on one strand and a biotin on the other strand. The biotin was included to facilitate future adhesion to a neutravidin coated plate. Following incubation with the DNA duplex, each fusion protein was incubated with the anti- 1 integrin antibody K20. A PAGE gel was used to determine binding efficiency to the DNA and the K20 antibody.
  • FIG. 18 shows the bands observed for WDV-PG K20 including BPA incorporated in the C3 domain at E23 reacted with DNA in lane three.
  • the fusion protein bound to K20 was plated onto a neutravidin-coated cell culture plate to produce a WDV-PG K20 surface.
  • a second cell culture plate was prepared by incubating an otherwise identical fusion protein bound to an anti-YAP antibody rather than K20. Unbound protein was removed by washing.
  • U251 cells were plated on the surface incubated with WDV- PG K20 and the surface incubated with WDV-PG anti-YAP. It was observed that cells plated on the anti-YAP surface did not adhere, while cells plated on the K20 surface adhered. From this example, it was determined that the WDV-PG fusion protein could bind DNA without impairing antibody binding. It was also determined that the WDV-PG fusion protein could bind to multiple antibodies and was not limited to mammalian antibodies (FIG. 23).
  • a LASIC Protein G-WDV fusion protein (SEQ ID NO:61) was expressed recombinantly.
  • a PAGE gel was used to determine efficiency of the BPA crosslinking reaction and reaction of the WDV-PG-x with the K20 antibody.
  • FIG. 18 shows the bands observed for WDV-PG-x with the K20 antibody and DNA in lane four.
  • Example 13 HUH covalent adduct reactions of split ScFv-WDV fusions
  • pertuzumab split ScFv-RLFL-WDV (SEQ ID NO: 59) and ScFv-RL- WDV (SEQ ID NO:58) were independently transfected in EXPI293 cells (Thermo Fisher Scientific, Inc., Waltham, MA).
  • a 30-nucleotide oligonucleotide including the WDV recognition sequence (SEQ ID NO:62) was reacted directly with the conditioned media, which contained the secreted protein.
  • conditioned media which contained the secreted protein.
  • a volume of 15 pL of conditioned media samples were used and incubation at 37°C.
  • This Western blot used anti- HisTag-HRP antibodies to probe the 6/-Histidine epitope tag C-terminal to the variable fragment (FIG. 21). Non-reducing conditions are on the left and reducing on the right. This blot shows that the HUH-tag WDV reacts with oligo whether it is in the fusion with the rigid linkers or the rigid and flexible linkers. Both fusion proteins appear to form a similar percentage of DNA covalent adduct. A lower adduct formation is observed for the split ScFv-RLFL-WDV when analyzed under reducing conditions, but that may be a result of the transfer from the SDS-PAGE gel to the western blot membrane.
  • the DNA covalent adduct was observed to be 75.5% for the split ScFv-RL-WDV and 61.3% for the split ScFv-RLFL-WDV. Expression levels were found to be comparable, but not directly measured, therefore, this is not a definitive result.
  • SEQ ID NO: 6 Pertuzumab variable heavy chain fragment amino acid sequence: EVQLVESGGG LVQPGGSLRL SCAASGFTFT DYTMDWVRQA PGKGLEWVAD VNPNSGGS IY NQRFKGRFTL SVDRSKNTLY LQMNSLRAED TAVYYCARNL GPS FYFDYWG QGTLVTVSS
  • X is BPA.

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  • Urology & Nephrology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Selon un aspect, la présente divulgation concerne une protéine de fusion comprenant une fraction d'affinité ; une étiquette HUH ; et un oligonucléotide, l'oligonucléotide étant lié de manière covalente par l'étiquette HUH. L'étiquette HUH et la fraction d'affinité peuvent être liées par une liaison d'acides aminés ou liées chimiquement. Dans un ou plusieurs modes de réalisation, la fraction d'affinité comprend une protéine de liant intermédiaire et une fraction d'affinité d'intérêt. Selon un autre aspect, la présente divulgation concerne un procédé de préparation d'une telle protéine de fusion. Selon un autre aspect, la présente divulgation concerne un procédé de détection de la présence d'une molécule dans un échantillon.
PCT/US2024/038787 2023-07-21 2024-07-19 Protéines de fusion d'anticorps huh Pending WO2025024292A1 (fr)

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US202363528140P 2023-07-21 2023-07-21
US63/528,140 2023-07-21
US202463638184P 2024-04-24 2024-04-24
US63/638,184 2024-04-24

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140004565A1 (en) * 2009-07-06 2014-01-02 Alnylam Pharmaceuticals, Inc. Cell-based bioprocessing
WO2018231999A1 (fr) * 2017-06-13 2018-12-20 Regents Of The University Of Minnesota Matériaux et méthodes permettant d'accroître la fréquence d'édition de gènes
US20200087657A1 (en) * 2018-09-19 2020-03-19 California Institute Of Technology High-throughput identificaton of intermolecular interactions
CN112778426A (zh) * 2021-01-06 2021-05-11 深圳伯生生物传感技术有限公司 一种精准抗体核酸定向连接方法
WO2024010907A1 (fr) * 2022-07-08 2024-01-11 Regents Of The University Of Minnesota Capteur de tension moléculaire avec acides nucléiques et marqueurs huh

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140004565A1 (en) * 2009-07-06 2014-01-02 Alnylam Pharmaceuticals, Inc. Cell-based bioprocessing
WO2018231999A1 (fr) * 2017-06-13 2018-12-20 Regents Of The University Of Minnesota Matériaux et méthodes permettant d'accroître la fréquence d'édition de gènes
US20200087657A1 (en) * 2018-09-19 2020-03-19 California Institute Of Technology High-throughput identificaton of intermolecular interactions
CN112778426A (zh) * 2021-01-06 2021-05-11 深圳伯生生物传感技术有限公司 一种精准抗体核酸定向连接方法
WO2024010907A1 (fr) * 2022-07-08 2024-01-11 Regents Of The University Of Minnesota Capteur de tension moléculaire avec acides nucléiques et marqueurs huh

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