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WO2024259349A2 - Plateforme de synthèse acellulaire pour la production d'allergènes - Google Patents

Plateforme de synthèse acellulaire pour la production d'allergènes Download PDF

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WO2024259349A2
WO2024259349A2 PCT/US2024/034170 US2024034170W WO2024259349A2 WO 2024259349 A2 WO2024259349 A2 WO 2024259349A2 US 2024034170 W US2024034170 W US 2024034170W WO 2024259349 A2 WO2024259349 A2 WO 2024259349A2
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allergen
cell
expression
allergen proteins
free
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WO2024259349A3 (fr
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Ariel Nicole THAMES
Michael Christopher Jewett
Bruce Scott Bochner
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Northwestern University
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Northwestern University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43531—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from mites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A61K39/36—Allergens from pollen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the present disclosure generally relates to components, systems, and methods for allergen protein synthesis.
  • the components, systems, and methods herein may be used for therapeutic and/or diagnostic tools for managing allergic disease, in various embodiments.
  • a cell-free protein synthesis system for in vitro production of one or more allergens is provided.
  • the system can include one or more expression templates, where Page 1 QB ⁇ 702581.02521 ⁇ 90543975.1 each of the one or more expression templates includes a polynucleotide sequence for expression of one or more allergen proteins selected from the group consisting of: Der p 1, Der p 2, Bos d 5, Bos d 4, Glad 1, Gal d 2, Gal d 4, Ara h 2, Fel d 1, Amb a 1, Bet v 1, Bla g 2, and Cry j 1; and one or more cell-free protein synthesis reagents for expressing the one or more allergen proteins.
  • a bacterial cell is provided.
  • the bacterial cell includes one or more expression templates, where each of the one or more expression templates includes a polynucleotide sequence for expression of one or more allergen proteins selected from the group consisting of: Der p 1, Der p 2, Bos d 5, Bos d 4, Glad 1, Gal d 2, Gal d 4, Ara h 2, Fel d 1, Amb a 1, Bet v 1, Bla g 2, and Cry j 1.
  • a lysate prepared from the bacterial cell is provided.
  • a method for preparing one or more allergens in a cell-free protein synthesis system is provided.
  • the method can include adding one or more expression templates to one or more cell-free protein synthesis reagents, where each of the one or more expression templates includes a polynucleotide sequence for expression of one or more allergen proteins selected from the group consisting of: Der p 1, Der p2 , Bos d 5, Bos d 4, Glad 1, Gal d 2, Gal d 4, Ara h 2, Fel d 1, Amb a 1, Bet v 1, Bla g 2, and Cry j 1.
  • a cell-free protein synthesis system for in vitro production of one or more allergens is provided.
  • the system can include one or more expression templates, where each of the one or more expression templates includes a polynucleotide sequence for expression of one or more allergen proteins; and one or more cell-free protein synthesis reagents for expressing the one or more allergen proteins.
  • a bacterial cell is provided.
  • the bacterial cell includes one or more expression templates, where each of the one or more expression templates includes a polynucleotide sequence for expression of one or more allergen proteins.
  • a lysate prepared from the bacterial cell is provided.
  • a method for preparing one or more allergens in a cell-free protein synthesis system is provided.
  • the method can include adding one or more expression templates to one or more cell-free protein synthesis reagents, where each of the one or more Page 2 QB ⁇ 702581.02521 ⁇ 90543975.1 expression templates includes a polynucleotide sequence for expression of one or more allergen proteins.
  • a method is provided that can include testing for allergies in a subject by exposing the one or more allergen proteins to the subject or a sample from the subject.
  • a method is provided that can include administering the one or more allergen proteins to a subject. BRIEF DESCRIPTION OF THE FIGURES [0014] FIGS.1A and 1B. Cell-free expression of clinically relevant allergen panel.
  • FIG.1A A schematic depiction of a cell-free protein synthesis (CFPS).
  • CFPS involves isolating the transcription and translation machinery of E. coli cells post-lysis in a test tube. Building blocks, cofactors, and plasmid DNA encoding the desired protein for expression are supplemented. After time for protein expression to occur, the protein of interest can be isolated and used for subsequent purposes.
  • FIG. 1B Expression of each allergen was quantified by incorporation of radioactive 14C-leucine and prepared to measure both total and soluble expression. Green fluorescent protein (GFP) is included as a reference point for high expression. All allergens were amenable to cell- free expression with relatively good solubility.
  • GFP Green fluorescent protein
  • FIGS 2A and 2B CFPS-expressed Der p 2 is recognized by monoclonal IgE.
  • FIG.2A An AlphaLISA assay detects IgE binding of dust mite allergen, Der p 2.
  • the Protein A donor bead associates with ⁇ -IgE rabbit IgG and a Ni-chelate acceptor bead associates with dust mite allergen Der p 2.
  • FIGS.3A-3C Der p 2 activation of primary human basophils and mast cells.
  • FIGS.3A CD34+ progenitors are isolated from human peripheral blood and differentiated into primary human CD34+ progenitor-derived mast cells in culture with cytokines necessary for their selective differentiation.
  • CD34+ progenitors begin as small, round, quickly dividing cells without mature structures. After 7-8 weeks, cells (bottom) appear as large, granular cells with irregular morphology consistent with mast cell features and have surface expression of CD117/KIT, CD33, Fc ⁇ RI ⁇ , Siglec-6, and Siglec-8 (FIG. 5).
  • FIG. 3B CD34+ culture- derived mast cells are then passively sensitized with serum from Der p 2-allergic donors (FIG.8) and incubated with Der p 2 allergen.
  • stimulation with Der p 2 allergen results in background levels of activation indicating that CFPS-expressed Der p 2 effects are IgE-mediated.
  • FIG.3C Peripheral blood human basophils from whole blood are tested for allergen reactivity.
  • FIG.4A Quantification of Der p 2 expression by 14C-leucine incorporation demonstrates significantly improved soluble yield when Der p 2 is expressed in oxidizing conditions when compared to Page 4 QB ⁇ 702581.02521 ⁇ 90543975.1 reducing conditions.
  • FIG. 4B An SDS-PAGE gel post-purification of Der p 2 reveals higher recovery after Der p 2 expression in oxidizing conditions (left) compared to reducing conditions (right) as indicated by the higher intensity band. Gel is representative of three independent experiments.
  • FIG. 4C Monoclonal IgE binding of Der p 2 expressed in oxidizing conditions compared to binding when expressed in reducing conditions at equivalent concentrations.
  • FIG.5. Phenotypic characterization of culture-derived human mast cells from CD34+ cells. After 8 weeks of culture, CD34+ progenitor-derived mast cells have surface expression of CD117/KIT, CD33, and (weakly) Fc ⁇ RI ⁇ , Siglec-6, Siglec-8.
  • FIGS.6A and 6B Optimization of CD34+ progenitor-derived mast cell stimulation with positive controls.
  • FIG.6A A titration of ionomycin stimulation shows that a concentration of 2 ⁇ M is optimal for mast cell stimulation as represented by CD63 and CD107a/LAMP1 expression.
  • FIG. 6B A titration of ⁇ -Fc ⁇ RI stimulation shows that a volume of 2 ⁇ L (0.5 mg/mL stock) is optimal for mast cell stimulation as represented by CD63 and CD107a/LAMP1 expression.
  • FIG. 7 Quantification of endotoxin levels in Der p 2 allergen purified from CFPS.
  • FIG. 8 Characteristics of Der p 2-allergic donor serum used for passive sensitization of CD34+ culture-derived mast cells.
  • CD34+ culture derived mast cells were incubated with Der p-allergic patient serum overnight at a dilution of 1:10 before stimulation with CF-expressed Der p 2 allergen.
  • Patient serum contained 44.2 ng/mL of Der p 2-specific IgE representing 11.9 % of total IgE (890 ng/mL).
  • DETAILED DESCRIPTION [0022] Overview Page 5 QB ⁇ 702581.02521 ⁇ 90543975.1 [0023] The present disclosure generally relates to components, systems, and methods for allergen synthesis and/or for therapeutic and/or diagnostic tools for managing allergic disease.
  • the allergic response requires the presence of three key players: production by plasma cells of allergen-specific immunoglobulin E (IgE), allergen-responsive cells by virtue of their surface expression of IgE receptors, and allergen.
  • IgE immunoglobulin E
  • allergen-responsive cells by virtue of their surface expression of IgE receptors
  • allergen The immediate allergic response is mediated by mast cells and basophils. Mast cells are tissue-resident cells while basophils circulate in the blood. One of the distinguishing features of both mast cells and basophils is the presence of metachromatic-staining granules.
  • Both mast cells and basophils express high-affinity IgE receptors on their surface (Fc ⁇ RI) whose alpha chain engages with unique sequences in the Fc region of IgE antibodies.
  • Fc ⁇ RI high-affinity IgE receptors on their surface
  • IgE antibodies Allergen exposure to these cell surface IgE antibodies will induce receptor cross-linking, initiating a calcium and kinase-dependent activating cascade through the Fc ⁇ RI’s immunoreceptor tyrosine-based activating motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activating motifs
  • This activating cascade leads to the fusion of mast cell and basophil preformed granules and release of the mediators they contain in a process called degranulation.
  • an allergy is diagnosed by detecting allergen-specific IgE with a variety of allergen-based reagents. By performing skin testing, a positive test result can be determined from mast cell activation that results in the generation of a histamine-dependent wheal and flare response.
  • Extracts from allergen-producing organisms, plants, foods, and drugs are introduced into the skin to screen for the development of localized allergic reactions.
  • Purified and recombinant allergens are used in ELISA lab tests of patient sera to quantify allergen specific IgE in a manner that also aids in diagnosis and prognosis.
  • allergen extracts are used to desensitize the patient to the allergen with repeated small exposures over time given orally or in “allergy shots”. More recently, allergen extracts have been formulated as supplements to aid in early oral introduction of food allergens, which have been shown to greatly reduce the risk of the development of food allergy.
  • CFPS involves the isolation of transcription and translation machinery from lysed E. coli cells and collection in a test tube, where supplementation of necessary building blocks, buffers, and cofactors in addition to plasmid encoding a protein of interest enables expression of desired proteins, obviating the need for live cells in culture.
  • CFPS offers several unique features including an open reaction environment, production of one protein per reaction, and the opportunity to multiplex individual production of many components.
  • CFPS-based production possesses several features that can be particularly useful for allergen production and can aid in the implementation of molecular diagnosis and treatment of allergy as well as precision allergology. These features include an open reaction environment, portability that could enable point-of-care use, single allergen production with direct control over dosing, and the ability to rapidly multiplex multiple protein allergens.
  • the platform invented here can catalyze innovation in the diagnostic and therapeutic allergy space and equip the clinical allergist and allergic patient with new tools for managing allergic disease.
  • Definitions and Terminology [0031] The disclosed components, systems, and methods for allergen synthesis and/or for therapeutic and/or diagnostic tools for managing allergic disease may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. [0032] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise.
  • a polynucleotide or an “allergen protein” should be interpreted to mean “one or more polynucleotides” and “one or more allergen proteins,” respectively, unless the context clearly dictates otherwise.
  • the term “plurality” means “two or more.” [0033] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used.
  • the modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same.
  • the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”
  • the term “disease” refers to any deviation from the normal health of a mammal and includes a state when disease symptoms are present, as well as conditions in which a deviation (e.g., dysbiosis, infection, gene mutation, genetic defect, etc.) has occurred, but symptoms are not yet manifested.
  • the terms “disease”, “condition”, and “disorder” are used interchangeably unless otherwise indicated.
  • a “symptom” of a disease includes and clinical or laboratory manifestation associated with the disease, and is not limited to what a subject can feel or observe.
  • Polynucleotides and Synthesis Methods refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D- ribose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base.
  • nucleic acid refers only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.
  • an oligonucleotide also can comprise nucleotide analogs in which the base, sugar, or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs.
  • Oligonucleotides can be prepared by any suitable method, including direct chemical synthesis by a method such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. Page 10 QB ⁇ 702581.02521 ⁇ 90543975.1 68:90-99; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage et al., 1981, Tetrahedron Letters 22:1859-1862; and the solid support method of U.S. Pat. No. 4,458,066, each incorporated herein by reference.
  • Amplification reaction refers to any chemical reaction, including an enzymatic reaction, which results in increased copies of a template nucleic acid sequence or results in transcription of a template nucleic acid.
  • Amplification reactions include reverse transcription, the polymerase chain reaction (PCR), including Real Time PCR (see U.S. Pat.
  • Exemplary “amplification reactions conditions” or “amplification conditions” typically comprise either two or three step cycles. Two-step cycles have a high temperature denaturation step followed by a hybridization/elongation (or ligation) step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step.
  • target refers to a region or sequence of a nucleic acid which is to be amplified, sequenced, or detected.
  • target refers to the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between “substantially complementary” nucleic acid strands that contain minor regions of mismatch. Conditions under which hybridization of fully complementary nucleic acid strands is strongly preferred are referred to as “stringent hybridization conditions” or “sequence-specific hybridization conditions”.
  • Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of mismatch tolerated can be controlled by suitable adjustment of the hybridization conditions.
  • Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length and base Page 11 QB ⁇ 702581.02521 ⁇ 90543975.1 pair composition of the oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art (see, e.g., Sambrook et al., 1989, Molecular Cloning– A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol.
  • primer refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under suitable conditions. Such conditions include those in which synthesis of a primer extension product complementary to a nucleic acid strand is induced in the presence of four different nucleoside triphosphates and an agent for extension (for example, a DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
  • a primer is preferably a single-stranded DNA.
  • the appropriate length of a primer depends on the intended use of the primer but typically ranges from about 6 to about 225 nucleotides, including intermediate ranges, such as from 15 to 35 nucleotides, from 18 to 75 nucleotides and from 25 to 150 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template. The design of suitable primers for the amplification of a given target sequence is well known in the art and described in the literature cited herein.
  • Primers can incorporate additional features which allow for the detection or immobilization of the primer but do not alter the basic property of the primer, that of acting as a point of initiation of DNA synthesis.
  • primers may contain an additional nucleic acid sequence at the 5' end which does not hybridize to the target nucleic acid, but which facilitates cloning or detection of the amplified product, or which enables transcription of RNA (for example, by inclusion of a promoter) or translation of protein (for example, by inclusion of a 5’-UTR, such as an Internal Ribosome Entry Site (IRES) or a 3’-UTR element, such as a poly(A)n sequence, where n is in the range from about 20 to about 200).
  • a 5’-UTR such as an Internal Ribosome Entry Site (IRES)
  • a 3’-UTR element such as a poly(A)n sequence, where n is in the range from about 20 to about 200).
  • a primer is “specific,” for a target sequence if, when used in an amplification reaction under sufficiently stringent conditions, the primer hybridizes primarily to Page 12 QB ⁇ 702581.02521 ⁇ 90543975.1 the target nucleic acid.
  • a primer is specific for a target sequence if the primer-target duplex stability is greater than the stability of a duplex formed between the primer and any other sequence found in the sample.
  • a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides.
  • DNA polymerase catalyzes the polymerization of deoxyribonucleotides.
  • DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNA polymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNA polymerase, among others.
  • RNA polymerase catalyzes the polymerization of ribonucleotides.
  • the foregoing examples of DNA polymerases are also known as DNA-dependent DNA polymerases.
  • RNA-dependent DNA polymerases also fall within the scope of DNA polymerases.
  • Reverse transcriptase which includes viral polymerases encoded by retroviruses, is an example of an RNA-dependent DNA polymerase.
  • RNA polymerase examples include, for example, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase and E. coli RNA polymerase, among others.
  • the foregoing examples of RNA polymerases are also known as DNA-dependent RNA polymerase.
  • the polymerase activity of any of the above enzymes can be determined by means well known in the art.
  • the term “promoter” refers to a cis-acting DNA sequence that directs RNA polymerase and other trans-acting transcription factors to initiate RNA transcription from the DNA template that includes the cis-acting DNA sequence.
  • sequence defined biopolymer refers to a biopolymer having a specific primary sequence.
  • a sequence defined biopolymer can be equivalent to a genetically- encoded defined biopolymer in cases where a gene encodes the biopolymer having a specific primary sequence.
  • Page 13 QB ⁇ 702581.02521 ⁇ 90543975.1 The polynucleotide sequences contemplated herein may be present in expression vectors.
  • the vectors may comprise: (a) a polynucleotide encoding an ORF of a protein; (b) a polynucleotide that expresses an RNA that directs RNA-mediated binding, nicking, and/or cleaving of a target DNA sequence; and both (a) and (b).
  • the polynucleotide present in the vector may be operably linked to a prokaryotic or eukaryotic promoter. “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • Vectors contemplated herein may comprise a heterologous promoter (e.g., a eukaryotic or prokaryotic promoter) operably linked to a polynucleotide that encodes a protein.
  • a “heterologous promoter” refers to a promoter that is not the native or endogenous promoter for the protein or RNA that is being expressed.
  • Vectors as disclosed herein may include plasmid vectors.
  • expression refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.
  • Transcripts and encoded polypeptides may be collectively referred to as "gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • expression template refers to a nucleic acid that serves as substrate for transcribing at least one RNA that can be translated into a sequence defined biopolymer (e.g., a polypeptide or protein).
  • Expression templates include nucleic acids composed of DNA or RNA. Suitable sources of DNA for use a nucleic acid for an expression template include genomic DNA, cDNA and RNA that can be converted into cDNA. Genomic DNA, cDNA and RNA can be from any biological source, such as a tissue sample, a biopsy, a swab, sputum, a blood sample, a fecal sample, a urine sample, a scraping, among others.
  • the genomic DNA, cDNA and RNA can be from host cell or virus origins and from any species, including extant and extinct organisms.
  • expression template and “transcription template” have the same meaning and are used interchangeably.
  • Page 14 QB ⁇ 702581.02521 ⁇ 90543975.1 the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid the term “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • expression vectors are referred to herein as “expression vectors.”
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” can be used interchangeably.
  • the disclosed methods and compositions are intended to include such other forms of expression vectors, such as viral vectors which serve equivalent functions.
  • the recombinant expression vectors comprise a nucleic acid sequence in a form suitable for expression of the nucleic acid sequence in one or more of the methods described herein, which means that the recombinant expression vectors include one or more regulatory sequences which is operatively linked to the nucleic acid sequence to be expressed.
  • operably linked is intended to mean that the nucleotide sequence encoding one or more rRNAs or reporter polypeptides and/or proteins described herein is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription and/or translation system).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
  • Oligonucleotides and polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleotides.
  • modified nucleotides include, but are not limited to diaminopurine, S2T, 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-a
  • Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and/or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone.
  • the terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof.
  • RNA or RNA of genomic, natural, or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand.
  • percent identity and “% identity” refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Patent No.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • NCBI National Center for Biotechnology Information
  • the BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
  • blastn a tool that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
  • BLAST 2 Sequences also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website.
  • percent identity may be measured over the length of an entire defined polynucleotide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • variants may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information’s website. (See Tatiana A. Tatusova, Thomas L.
  • nucleic acids may show, for example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
  • Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code where multiple codons may encode for a single amino acid.
  • nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
  • polynucleotide sequences as contemplated herein may encode a protein and may be codon-optimized for expression in a particular host.
  • codon usage frequency tables have been prepared for a number of host organisms including humans, mouse, rat, pig, E. coli, plants, and other host cells.
  • a “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence.
  • recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence.
  • Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
  • nucleic acids disclosed herein may be “substantially isolated or purified.”
  • the term “substantially isolated or purified” refers to a nucleic acid that is removed from its natural environment, and is at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which it is naturally associated.
  • Peptides, Polypeptides, Proteins, and Synthesis Methods As used herein, the terms “peptide,” “polypeptide,” and “protein,” refer to molecules comprising a chain a polymer of amino acid residues joined by amide linkages.
  • amino acid residue includes but is not limited to amino acid residues contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues.
  • alanine Al or A
  • cysteine cysteine
  • Asp or D aspartic acid
  • Glu or E
  • amino acid residue also may include nonstandard or unnatural amino acids.
  • amino acid residue may include alpha-, beta-, gamma-, and delta-amino acids.
  • amino acid residue may include nonstandard or unnatural amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, ⁇ -alanine, ⁇ -Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4- Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine
  • amino acid residue may include L isomers or D isomers of any of the aforementioned amino acids.
  • Other examples of nonstandard or unnatural amino acids include, but are not limited, to a p-acetyl-L-phenylalanine, a p-iodo-L-phenylalanine, an O-methyl-L-tyrosine, a p- propargyloxyphenylalanine, a p-propargyl-phenylalanine, an L-3-(2-naphthyl)alanine, a 3- methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcp ⁇ - serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azid
  • a “peptide” is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).
  • a peptide Page 19 QB ⁇ 702581.02521 ⁇ 90543975.1 as contemplated herein may include no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.
  • a polypeptide also referred to as a protein, is typically of length > 100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).
  • a polypeptide, as contemplated herein, may comprise, but is not limited to, 100, 101, 102, 103, 104, 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1
  • a peptide or polypeptide as contemplated herein may be further modified to include non-amino acid moieties. Modifications may include but are not limited to acylation (e.g., O- acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an alkyl
  • Modified amino acid sequences that are disclosed herein may include a deletion in one or more amino acids.
  • a “deletion” means the removal of one or more amino acids relative to the native amino acid sequence.
  • the modified amino acid sequences that are disclosed herein may include an insertion of one or more amino acids.
  • an “insertion” means the addition of one or more amino acids to a native amino acid sequence.
  • the Page 20 QB ⁇ 702581.02521 ⁇ 90543975.1 modified amino acid sequences that are disclosed herein may include a substitution of one or more amino acids.
  • a “substitution” means replacement of an amino acid of a native amino acid sequence with an amino acid that is not native to the amino acid sequence.
  • the modified amino sequences disclosed herein may include one or more deletions, insertions, and/or substitutions in order modified the native amino acid sequence of a target protein to include one or more heterologous amino acid motifs that are glycosylated by an N- glycosyltransferase.
  • a “deletion” refers to a change in the amino acid sequence that results in the absence of one or more amino acid residues.
  • a deletion may remove at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, or more amino acids residues.
  • a deletion may include an internal deletion and/or a terminal deletion (e.g., an N-terminal truncation, a C-terminal truncation or both of a reference polypeptide).
  • a “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include a deletion relative to the reference polypeptide sequence.
  • “fragment” is a portion of an amino acid sequence which is identical in sequence to but shorter in length than a reference sequence.
  • a fragment may comprise up to the entire length of the reference sequence, minus at least one amino acid residue.
  • a fragment may comprise from 5 to 1000 contiguous amino acid residues of a reference polypeptide, respectively.
  • a fragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous amino acid residues of a reference polypeptide.
  • Fragments may be preferentially selected from certain regions of a molecule.
  • the term “at least a fragment” encompasses the full-length polypeptide.
  • a fragment may include an N-terminal truncation, a C-terminal truncation, or both truncations relative to the full-length protein.
  • a “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include a fragment of the reference polypeptide sequence.
  • An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or more amino acid residues.
  • a “variant,” “mutant,” or “derivative” of a reference polypeptide sequence may include an insertion or addition relative to the reference polypeptide sequence.
  • a variant of a Page 21 QB ⁇ 702581.02521 ⁇ 90543975.1 protein may have N-terminal insertions, C-terminal insertions, internal insertions, or any combination of N-terminal insertions, C-terminal insertions, and internal insertions.
  • percent identity refers to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail below, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Patent No.7,396,664, which is incorporated herein by reference in its entirety).
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • the BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.
  • percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • the amino acid sequences of variants, mutants, or derivatives as contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence.
  • a variant, mutant, or derivative protein may include conservative amino acid substitutions relative to a reference molecule.
  • conservative amino acid substitutions are those substitutions that are a substitution of an amino acid for a different amino Page 22 QB ⁇ 702581.02521 ⁇ 90543975.1 acid where the substitution is predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference polypeptide.
  • Non-conservative amino acids typically disrupt (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • Page 23 QB ⁇ 702581.02521 ⁇ 90543975.1 The disclosed proteins, mutants, variants, or described herein may have one or more functional or biological activities exhibited by a reference polypeptide (e.g., one or more functional or biological activities exhibited by wild-type protein).
  • the disclosed proteins may be substantially isolated or purified.
  • substantially isolated or purified refers to proteins that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.
  • CFPS Cell-Free Protein Synthesis
  • Jewett M.C., Hong, S.H., Kwon, Y.C., Martin, R.W., and Des Soye, B.J. 2014, “Methods for improved in vitro protein synthesis with proteins containing non standard amino acids,” U.S. Patent Application Serial No.: 62/044,221; Jewett, M.C., Hodgman, C.E., and Gan, R.2013, “Methods for yeast cell-free protein synthesis,” U.S. Patent Application Serial No.: 61/792,290; Jewett, M.C., J.A. Schoborg, and C.E. Hodgman.
  • a CFPS reaction mixture or cell-free protein synthesis (CFPS) reagents may contain one or more of a crude or partially-purified cell extract, an RNA translation template, and a suitable reaction buffer for promoting cell-free protein synthesis from the RNA translation template.
  • the CFPS reaction mixture can include exogenous RNA translation template.
  • the CFPS reaction mixture can include a DNA expression template encoding an open reading frame operably linked to a promoter element for a DNA-dependent RNA polymerase.
  • the CFPS reaction mixture can also include a DNA-dependent RNA polymerase to direct transcription of an RNA translation template encoding the open reading frame.
  • additional NTP’s and divalent cation cofactor can be included in the CFPS reaction mixture.
  • a reaction mixture is referred to as complete if it contains all reagents necessary to enable the reaction, and incomplete if it contains only a subset of the necessary reagents.
  • reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for application-dependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete reaction mixture. Furthermore, it will be understood by one of ordinary skill in the art that reaction components are packaged separately for commercialization and that useful commercial kits may contain any subset of the reaction components of the invention.
  • the disclosed cell-free protein synthesis systems may utilize components that are crude and/or that are at least partially isolated and/or purified.
  • the term “crude” may mean components obtained by disrupting and lysing cells and, at best, minimally purifying the crude components from the disrupted and lysed cells, for example by centrifuging the disrupted and lysed cells and collecting the crude components from the supernatant and/or pellet after centrifugation.
  • isolated or purified refers to components that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.
  • reaction template for a polypeptide refers to an RNA product of transcription from an expression template that can be used by ribosomes to synthesize polypeptides or proteins.
  • reaction mixture refers to a solution containing reagents necessary to carry out a given reaction. A reaction mixture is referred to as complete if it contains all reagents necessary to perform the reaction. Components for a reaction mixture may be stored separately in separate container, each containing one or more of the total components. Components may be packaged separately for commercialization and useful commercial kits may contain one or more of the reaction components for a reaction mixture.
  • a reaction mixture may include an expression template, a translation template, or both an expression template and a translation template.
  • the expression template serves as a substrate for transcribing at least one RNA that can be translated into a sequence defined biopolymer (e.g., a polypeptide or protein).
  • the translation template is an RNA product that can be used by ribosomes to synthesize the sequence defined biopolymer.
  • the platform comprises both the expression template and the translation template.
  • the reaction mixture may comprise a coupled transcription/translation (“Tx/Tl”) system where synthesis of translation template and a sequence defined biopolymer from the same cellular extract.
  • the reaction mixture may comprise one or more polymerases capable of generating a translation template from an expression template.
  • the polymerase may be supplied exogenously or may be supplied from the organism used to prepare the extract.
  • the polymerase is expressed from a plasmid present in the organism used to prepare the extract and/or an integration site in the genome of the organism used to prepare the extract.
  • Altering the physicochemical environment of the CFPS reaction to better mimic the cytoplasm can improve protein synthesis activity.
  • the temperature may be any temperature suitable for CFPS. Temperature may be in the general range from about 10o C. to about 40o C., including intermediate specific ranges within this general range, include from about 15o C. to about 35o C., from about 15o C. to about 30o C., from about 15o C. to about 25o C.
  • the reaction temperature can be about 15o C., about 16o C., about 17o C., about 18o C., about 19o C., about 20o C., about 21o C., about 22o C., about 23o C., about 24o C., about 25o C.
  • the reaction mixture may include any organic anion suitable for CFPS.
  • the organic anions can be glutamate, acetate, among others.
  • the concentration for the organic anions is independently in the general range from about 0 mM to about 200 mM, including intermediate specific values within this general range, such as about 0 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM and about 200 mM, among others.
  • the reaction mixture may include any halide anion suitable for CFPS.
  • the halide anion can be chloride, bromide, iodide, among others.
  • a preferred halide anion is chloride.
  • the concentration of halide anions, if present in the reaction is within the general range from about 0 mM to about 200 mM, including intermediate specific values within this general range, such as those disclosed for organic anions generally herein.
  • the reaction mixture may include any organic cation suitable for CFPS.
  • the organic cation can be a polyamine, such as spermidine or putrescine, among others. Preferably polyamines are present in the CFPS reaction.
  • the concentration of organic cations in the reaction can be in the general about 0 mM to about 3 mM, about 0.5 mM to about 2.5 mM, about 1 mM to about 2 mM. In certain aspects, more than one organic cation can be present.
  • the reaction mixture may include any inorganic cation suitable for CFPS.
  • suitable inorganic cations can include monovalent cations, such as sodium, potassium, lithium, among others; and divalent cations, such as magnesium, calcium, manganese, among others.
  • the inorganic cation is magnesium.
  • the magnesium concentration Page 27 QB ⁇ 702581.02521 ⁇ 90543975.1 can be within the general range from about 1 mM to about 50 mM, including intermediate specific values within this general range, such as about 1 mM, about 2 mM, about 3 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, among others.
  • the concentration of inorganic cations can be within the specific range from about 4 mM to about 9 mM and more preferably, within the range from about 5 mM to about 7 mM.
  • the reaction mixture may include endogenous NTPs (i.e., NTPs that are present in the cell extract) and or exogenous NTPs (i.e., NTPs that are added to the reaction mixture).
  • the reaction uses ATP, GTP, CTP, and UTP.
  • the concentration of individual NTPs is within the range from about 0.1 mM to about 2 mM.
  • the reaction mixture may include any alcohol suitable for CFPS.
  • the alcohol may be a polyol, and more specifically glycerol.
  • the alcohol is between the general range from about 0% (v/v) to about 25% (v/v), including specific intermediate values of about 5% (v/v), about 10% (v/v) and about 15% (v/v), and about 20% (v/v), among others.
  • a vessel e.g., a single, vessel.
  • the term “vessel,” as used herein, refers to any container suitable for holding on or more of the reactants (e.g., for use in one or more transcription, translation, and/or glycosylation steps) described herein.
  • vessels include, but are not limited to, a microtitre plate, a test tube, a microfuge tube, a beaker, a flask, a multi-well plate, a cuvette, a flow system, a microfiber, a microscope slide and the like.
  • CFPS synthesis systems can include one or more expression templates and one or more cell-free protein synthesis reagents and/or a CFPS reaction mixture.
  • the expression templates can include a protein-encoding sequence for one or more allergen proteins.
  • the one or more allergen proteins can be any proteins that may elicit an allergic reaction in a subject.
  • a protein deemed Page 28 QB ⁇ 702581.02521 ⁇ 90543975.1 to elicit an allergic reaction in a subject can be a protein that can elicit allergen-specific IgE antibodies in a subject.
  • the one or more allergen proteins can include Der p 1, Der p 2, Bos d 5, Bos d 4, Glad 1, Gal d 2, Gal d 4, Ara h 2, Fel d 1, Amb a 1, Bet v 1, Bla g 2, and/or Cry j 1, or other allergens, or one or more hypoallergenic allergen proteins.
  • the one or more allergen proteins can comprise or consist of one or more of Der p 1, Der p 2, Bos d 5, Bos d 4, Glad 1, Gal d 2, Gal d 4, Ara h 2, Fel d 1, Amb a 1, Bet v 1, Bla g 2, or Cry j 1.
  • the encoding sequence can include a mutation to substitute a G for N in the allergen protein sequence.
  • the allergen proteins produced according to the systems and methods described herein are not glycosylated.
  • the expression templates can also include additional elements, including but not limited to, purification tags, immune-modulating elements, sites for post- translational modifications, designer epitopes, elements that aid expression, elements that aid folding and assembly.
  • the expression templates can include or encode for a ribosome binding site, e.g., to improve expression of the encoded allergen protein.
  • the expression templates can include or encode for a synthetic glycosylation site (e.g., Glyctag) to install one or more ligands, e.g., Siglec ligands, onto the allergen protein.
  • the expression templates can encode a purification tag, e.g., for purifying the allergen protein.
  • the expression templates can be present in a vector.
  • the expression templates can be present in a plasmid vector, e.g., pJL1.
  • the expression templates can include or comprise a polynucleotide sequence that is at least 80 %, 85 %, 90 %, 95 %, 99 %, or 100% identical to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.
  • the expression templates can comprise a polynucleotide that encodes for one or more allergen proteins.
  • the CFPS system can include one or more CFPS reagents or reaction mixtures for expressing the one or more allergen proteins.
  • Suitable CFPS reaction mixtures for the disclosed methods may include prokaryotic CFPS reaction mixtures.
  • suitable CFPS reaction mixtures may include prokaryotic CFPS reaction mixtures comprising a lysate prepared from Escherichia coli.
  • the E. coli or other cell can be modified for tailoring and/or optimizing cell-free protein synthesis.
  • the E. coli or other cell can include a genetic modification to facilitate disulfide bond formation in expressed proteins.
  • the CFPS system may include eukaryotic CFPS reaction mixtures.
  • suitable CFPS reaction mixtures may include eukaryotic CFPS reaction mixtures comprising a lysate prepared from Saccharomyces cerevisiae or other eukaryote.
  • the S. cerevisiae or other cell can be modified for tailoring and/or optimizing cell-free protein synthesis.
  • the allergen proteins are expressed by combining the one or more expression vectors with one or more cell-free protein synthesis reagents and/or a CFPS reaction mixture, as discussed above and in the Examples below.
  • the allergen protein may be purified and/or isolated from the CFPS system using any convenient purification and/or isolation techniques.
  • the systems can include modified cells.
  • the modified cells may be genetically modified bacterial cells.
  • the modified bacterial cells can be modified to express a protein.
  • the modified cells can include an expression template for the one or more allergen proteins discussed herein.
  • the modified bacterial cells can produce one or more of the allergen proteins described herein in vivo.
  • a lysate prepared from the modified bacterial cell can be utilized to produce the one or more allergen proteins.
  • the systems and methods described herein can include methods for assessing the function of the allergen proteins produced according to the systems and methods described herein. In certain embodiments, the methods can include assessing whether or not the allergen proteins bind to IgE from a sample of a subject. In various embodiments, such binding can be assessed using any convenient technique, include but not limited to the use of an AlphaLISA, an ELISA, point-of-use assay, surface plasmon resonance, or biolayer interferometry.
  • the allergen proteins produced according to the systems and methods described herein can additionally, or alternatively, be tested for allergen function by use of any other convenient allergen function test.
  • the allergen proteins can be evaluated for allergen function by a basophil activation test with primary human basophils, and/or by flow analysis of passively sensitized CD34+ progenitor-derived primary human mast cells.
  • point-of-care testing point-of-service testing
  • point-of-use testing point-of-use testing
  • POI point-of-incidence testing
  • point-of-use testing also known as on-site or near-subject testing, refers to diagnostic testing performed at or near a subject where a sample is obtained and/or at the site where service, care, or treatment is provided.
  • the disclosed methods and systems may use point-of-use systems, methods, and devices.
  • Point-of-use systems, methods, and devices are configured for use at a point-of-use location, where a point-of-use location may be a location at which a sample is obtained, such as a facility (e.g., hospital, clinic, pharmacy, or clinician’s office), room or other area. Any point-of- care testing system known in the art may be used, including a single-use testing device or a portable analyzer device.
  • the disclosed methods and systems test for allergens in a sample using a point-of service method or device.
  • the point-of service method or device is performed at a point-of-service location.
  • the one or more allergen proteins can be administered to a subject, e.g., subcutaneously, sublingually, or orally.
  • the allergen proteins can be added to a food product formulation.
  • the allergen proteins can be made at point-of use, e.g., using one or more of the CFPS methods and/or systems described herein, and applied to patient whole blood for a basophil acitivation test, such as for example, with primary human basophils, and/or by flow analysis of passively sensitized CD34+ progenitor-derived primary human mast cells.
  • a point-of-use test can include a skin prick test.
  • the skin prick test can include preparing the allergen proteins at point-of use, e.g., using one or more of the CFPS methods and/or systems described herein, and exposing a subject in a skin prick test to the allergen proteins.
  • the skin prick test can include introducing the allergen proteins to the subject, e.g., under the subject’s skin, by scratching the skin, breaking the skin, and/or intradermal application.
  • the allergen proteins once the allergen proteins are introduced to the subject, the presence of a localized wheal and flare response could help in the diagnosis of allergen sensitivity.
  • an immunotherapy cocktail can be made and utilized at the POC based on the patient’s sensitization profile (e.g., to one or more allergen proteins).
  • the immunotherapy cocktail can be made by producing the one or more allergen proteins for the immunotherapy cocktail at the point-of-use, e.g., using one or more of the CFPS methods and/or systems described herein.
  • the allergen proteins a patient has sensitivity for could be made and mixed in an immunotherpapy that could be given as allergey shots, e.g., over repeated doses, which can be done at the point-of-use.
  • the allergen proteins can be prepared at point-of use, e.g., using one or more of the CFPS methods and/or systems described herein, and then an IgE binding assay from patient serum can be performed, e.g., at the point-of-care or elsewhere, using an ELISA, AlphaLISA, surface plasmon resonance (SPR), biolayer interferometry (BLI), or flow cytometry assay.
  • an IgE binding assay from patient serum can be performed, e.g., at the point-of-care or elsewhere, using an ELISA, AlphaLISA, surface plasmon resonance (SPR), biolayer interferometry (BLI), or flow cytometry assay.
  • one or more allergen proteins can be formulated into a lateral flow assay to detect IgE for an at home or POC test.
  • the allergen proteins can be made in a centralized facility using CFPS (this overcomes cell cytotoxicity constraints of other cell-based systems but retains the precision allergy capability since it’s one allergen and not an extract), in a clinical laboratory using CFPS, or at the point-of-care using CFPS.
  • the systems and methods disclosed herein offer one or more of the following advantages over conventional technologies: (1) On-site production of allergen circumvents need for cold stain storage; (2) On-demand expression and purification expand access of allergen testing and treatment to resource-limited settings; (3) Does not require living cells during the workflow; (4) Plug-in-play method where whichever allergen is needed can be chosen by addition of the corresponding plasmid; (5) Multiplexed expression of individual allergens; (6) Enables personalized immunotherapies containing only allergens to which patient is sensitized; (7) Control of molar ratio of different allergens; (8) Enables optimization of molar ratios for best efficacy; (9) Can express allergen at time of need; (10) Rapid production of several allergen or allergen derivatives would enable high-throughput characterization of IgE-mediated responses that would enhance our understanding of allergy at a molecular level and lead to improved molecular allergy diagnosis and treatment; (11) Production of a personalized allergen cocktail at the point-
  • [00151] 1.12 Evaluation of allergen function by flow analysis of passively sensitized CD34+ progenitor-derived primary human mast cells.
  • allergens are also used in immunotherapy treatments, both via the oral and parenteral route, the latter referred to as “allergy shots”.
  • recombinant allergen is used for diagnostic IgE quantification tests and for immunotherapy.
  • diagnostic IgE quantification tests and for immunotherapy.
  • new tools are needed for producing allergen-based reagents to enable personalized medicine in the field of allergy.
  • allergen production of a clinically relevant allergen panel spanning a wide range of phylogenetic kingdoms and representing a range of the most common allergens. We show that allergen produced with this approach can be recognized by allergen-specific IgE, either monoclonal or in patient sera.
  • the allergic response requires the presence of three key players: production by plasma cells of allergen-specific immunoglobulin E (IgE), allergen-responsive cells by virtue of their surface expression of IgE receptors, and allergen.
  • IgE immunoglobulin E
  • allergen-responsive cells by virtue of their surface expression of IgE receptors
  • allergen The immediate allergic response is mediated by mast cells and basophils. Mast cells are tissue-resident cells while basophils circulate in the blood.
  • mast cells and basophils are distinct and newly generated mediators such as histamine, prostaglandins, leukotrienes, proteases, cytokines and Page 38 QB ⁇ 702581.02521 ⁇ 90543975.1 other substances that cause inflammation, vasodilation, bronchoconstriction, diarrhea, sneeze and itch observed during an allergic response.
  • Both mast cells and basophils express high-affinity IgE receptors on their surface (Fc ⁇ RI) whose alpha chain engages with unique sequences in the Fc region of IgE antibodies.
  • allergen extracts have been formulated as supplements 2 to aid in early oral introduction of food allergens, which have been shown to greatly reduce the risk of the development of food allergy.
  • Table 1 Panel of Allergens Allergen Panel Allergen Source Common Indoor Allergens Der p 1 Dust mite Der p 2 Dust mite Major Milk and Egg Allergens Bos d 5 Milk Bos d 4 Milk Gal d 1 Egg (ovomucoid) Gal d 2 Egg (ovalbumin) Gal d 4 Egg (lysozyme) Major Peanut Allergen Ara h 2 Peanut Other Common Aeroallergens Fel d 1 Cat (dander) Amb a 1 Ragweed (pollen) Bet v 1 Birch (pollen) Bla g 2 Cockroach Cry j 1 Japanese cedar (pollen) [00184] Recognition of CFPS-expressed Der p 2 by monoclonal allergen-specific IgE [00185] After demonstrating soluble synthesis of numerous allergens, we next sought to determine whether the CFPS-expressed allergen retains its function insofar as being able to bind IgE.
  • Activation of human allergic effector cells by CFPS-expressed Der p 2 [00188] To further confirm bioactivity, it is important to demonstrate that IgE binding and crosslinking of Fc ⁇ RI translates to a cellular response against our CFPS-expressed allergen. To do this, we utilized two human allergic effector cell models involving culture- derived mast cells and primary basophils to assess activation and degranulation upon exposure to relevant concentrations of CFPS-expressed Der p 2. Degranulation can be assessed by detecting proteins associated with intracellular granules that appear on the cell surface during granule membrane fusion and mediator release.
  • the first cell model utilizes human CD34+ progenitor cells isolated from peripheral blood to generate mast cells in culture.
  • the mast cells will not have any prior IgE in their receptors, unlike primary mast cells isolated from human tissues.
  • cells are small and round and are differentiated into mast cells by culture Page 42 QB ⁇ 702581.02521 ⁇ 90543975.1 with IL-3, IL-6, and stem cell factor as described.
  • cells become larger with the appearance of intracellular granules and irregular borders characteristic of mast cell morphology (FIG.3A).
  • mast cells can be passively sensitized by incubation with allergen-specific IgE as monoclonals or serum from an allergic donor.
  • IgE allergen-specific IgE
  • CD34+ progenitor-derived human mast cells were sensitized with human serum from a dust mite allergic donor at a dilution ratio of 1:10 (FIG.8).
  • Anticoagulated whole blood is used in the FlowCAST Basophil Activation Test where allergen is added to the sample and basophils are gated on by both light scatter and the basophil-selective marker, CCR3.
  • CCR3 basophil-selective marker
  • levels of CD63 are determined. Under negative control buffer-only conditions, only 9 ⁇ 3% basophils are CD63 positive. Incubation with ⁇ -Fc ⁇ RI ⁇ antibody as one positive control leads to CD63 positivity in 76 ⁇ 10% of basophils, while incubation with ⁇ -IgE antibody as a second positive control leads to CD63 positivity in 73 ⁇ 17% of basophils.
  • Recombinant allergen is a useful tool in the clinical diagnosis and treatment of allergic disease.
  • 41 CFPS-based production possesses several features that can be particularly useful for allergen production and can aid in the implementation of molecular diagnosis and treatment of allergy as well as precision allergology. These features include an open reaction environment, portability that could enable point-of-care use, single allergen production with direct control over dosing, and the ability to rapidly multiplex multiple protein allergens.
  • lysis For lysis, thawed cells were resuspended in 0.8 mL per gram of wet cell mass S30 buffer and aliquotted in 1.4 mL increments. Cells were kept on ice and underwent sonication for 45 s on, 59 s at 50% amplitude until 950 joules was reached. Supernatant (820 ⁇ L) was collected and underwent a 1 hour runoff reaction at 37°C with agitation (250 rpm). Lysate was then centrifuged at 12,000g for 10 min at 4°C and the supernatant was collected (500 ⁇ L), aliquotted, flash frozen in liquid nitrogen, and stored at -80°C for downstream use.
  • PEP phosphoenolpyruvate
  • Radioactive quantification of allergen expressed by cell free protein synthesis was quantified by 14C-leucine incorporation according to previously published methods. 46 In brief, 10.67 ⁇ L 14C-leucine was supplemented into assembled CFPS reactions described above. After 20 hours of overnight expression, protein was precipitated by addition of 0.5N KOH in a 1:1 ratio to samples containing total protein and samples containing soluble protein (supernatant remaining after centrifugation at 12,000g for 5 min). Samples were incubated at 37°C for 20 min.
  • Allergen and IgE were diluted in AlphaLISA buffer (50 mM HEPES pH 7.4 with 150 mM NaCl, 0.015% v/v TritonX-100, and 1 g/L BSA) and a 2x serial dilution was prepared by adding half volume of the previous dilution to the next dilution and mixing by pipette in a serial fashion.
  • AlphaLISA reactions were assembled using an acoustic liquid handler (Echo 525). Protein components were distributed to a 384-well plate (PerkinElmer, 6008280) and allowed to incubate for at least 1 hour. Bead components were then distributed according to manufacturer’s instructions and allowed to incubate for at least 1 additional hour.
  • CD34+ progenitor cells were purchased from Stemcell Technologies and cultured as previously described. 40 Page 47 QB ⁇ 702581.02521 ⁇ 90543975.1 [00216] Passive sensitization and activation of human CD34+ progenitor-derived mast cells [00217] Cultured CD34+ progenitor-derived mast cells were incubated overnight with allergic patient serum (FIG.8) at a 1:10 dilution. For the data of FIG.8, CD34+ culture derived mast cells were incubated with Der p-allergic patient serum overnight at a dilution of 1:10 before stimulation with CF-expressed Der p 2 allergen.
  • Table 2 Allergen accession ID and polynucleotide sequences Allergens were designed with synthetic sequences as annotated below and with endogenous glycosylation sites mutated (N->G) to prevent off-target glycosylation.
  • Cat linker (orange) standardizes the ribosome binding site to improve expression.
  • BamHI restriction site linker separates the allergen sequence from a synthetic glycosylation site (Glyctag, purple) that can be used to install Siglec ligands and a downstream affinity His tag (brown) for purification and analysis. Sequences were cloned into a pJL1 backbone at NdeI (5’) and SalI (3’) restriction sites.
  • Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice, 14, eabn1252. [00236] 12. Hunt, A. C., Vögeli, B., Kightlinger, W. K., Yoesep, D. J., Krüger, A., and Jewett, M. [00237] C. (2021) A high-throughput, automated, cell-free expression and screening platform for antibody discovery, bioRxiv. [00238] 13. Hershewe, J., Kightlinger, W., and Jewett, M. C. (2020) Cell-free systems for accelerating glycoprotein expression and biomanufacturing, J Ind Microbiol Biotechnol 47, 977- 991.

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Abstract

L'invention concerne des composants, des systèmes et des procédés de synthèse de protéines allergènes. Les composants, les systèmes et les procédés de l'invention peuvent être utilisés dans la synthèse de protéines allergènes dans un système de synthèse de protéines acellulaire (CFPS).
PCT/US2024/034170 2023-06-16 2024-06-14 Plateforme de synthèse acellulaire pour la production d'allergènes Pending WO2024259349A2 (fr)

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