Cell-free synthesis platform for allergen production

Abstract

Disclosed are components, systems, and methods for synthesis of allergen proteins. The components, systems, and methods disclosed herein may be used in synthesizing allergen proteins in a cell-free protein synthesis (CFPS) system.

Description

CELL-FREE SYNTHESIS PLATFORM FOR ALLERGEN PRODUCTION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 508,834, filed on June 16, 2023. The entire contents of which are hereby incorporated by reference. SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (File Name: 702581.02521_SL.xml; Size: 36,525 bytes; Date of Creation: June 14, 2024) submitted herewith is herein incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under grant number 1936789 awarded by the National Science Foundation and grant number AI165279 awarded by the National Institutes of Health and grant number HDTRA1-20-1-0004 awarded by the Department of Defense. The government has certain rights in the invention. BACKGROUND

[0004] 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 an allergen. The immediate allergic response is mediated by mast cells and basophils. SUMMARY

[0005] 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.

[0006] In one embodiment, 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\90543975.1each 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.

[0007] In another embodiment, 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. In yet another embodiment, a lysate prepared from the bacterial cell is provided.

[0008] In another embodiment, 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.

[0009] In one embodiment, 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.

[0010] In another embodiment, 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. In yet another embodiment, a lysate prepared from the bacterial cell is provided.

[0011] In another embodiment, 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\90543975.1expression templates includes a polynucleotide sequence for expression of one or more allergen proteins.

[0012] In yet another embodiment, 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.

[0013] In another embodiment, 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. Even those with lower yields (~<200 μg / mL) are expressed at levels that could readily be used for clinically relevant applications after scale-up of the CFPS reaction. (n=3, error bars = standard deviation).

[0015] 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 α-IgE rabbit IgG and a Ni-chelate acceptor bead associates with dust mite allergen Der p 2. If monoclonal 2G1 or 2F10 IgE recognizes and binds CFPS-expressed Der p 2 then it will bridge the two proteins and their associated beads, leading to an energy transfer between the beads once in close proximity, yielding an emission of light that can be read on a plate reader. (FIG.2B) Titrating CFPS-expressed Der p 2 and each monoclonal IgE reveals a typical binding pattern, where yellow indicates signal and purple indicates no signal (i.e., no binding). In the 0 condition (left most column and bottom most row), when either species is absent, there is no binding detected Page 3 QB\702581.02521\90543975.1as expected. At an optimal concentration of 50 nM Der p 2 and 3-12 nM of IgE, there is optimal bead interactions leading to the highest level of signal, and the signal gradually decreases as the concentration moves away from those optimums. At higher concentrations, excess free binding partner occupies binding sites and disrupts bead interaction attributable to the Hook effect. At lower concentrations, insufficient amounts of either binding partner prevent enough bead interactions to occur to elicit visible signal.

[0016] FIGS.3A-3C. Der p 2 activation of primary human basophils and mast cells. (FIG.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 (top) 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, FcεRIα, 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. These mast cells respond to negative (-) and positive (+) controls as expected (n=2, error bars = standard error) and respond to CFPS-expressed Der p 2 in a dose- dependent manner (n=3, error bars = standard deviation), where 10 nM appears to be optimal for FcεRI crosslinking. Without passive sensitization, 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. Patients sensitized to Der p 2 have Der p 2-specific IgE already occupying the FcεRI receptors on the surface of their basophils and will degranulate upon incubation with Der p 2. Degranulation can be identified and assessed with proper gating strategies without purification for enhanced levels of surface CD63 detected by flow cytometry. Peripheral blood human basophils respond to negative (-) and positive (+) controls as expected and degranulate upon Der p 2 exposure in a dose-dependent manner with >100 pM being the optimal concentration for crosslinking. (n=2 samples, 1 dust mite allergic patient donor, error bars = standard error).

[0017] FIGS.4A-4C. Der p 2 CFPS expression in reducing vs oxidizing conditions. (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\90543975.1reducing conditions. Der p 2 has disulfide bonding at 3 positions (amino acids 25 ↔ 136, 38 ↔ 44, and 90 ↔ 95) and an oxidizing environment likely facilitates protein folding. (n=3, error bars = standard deviation). (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.

[0018] 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) FcεRIα, Siglec-6, Siglec-8.

[0019] 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 μM is optimal for mast cell stimulation as represented by CD63 and CD107a / LAMP1 expression. (FIG. 6B) A titration of α-FcεRI stimulation shows that a volume of 2 μL (0.5 mg / mL stock) is optimal for mast cell stimulation as represented by CD63 and CD107a / LAMP1 expression.

[0020] FIG. 7. Quantification of endotoxin levels in Der p 2 allergen purified from CFPS. Endotoxin quantification using the Pierce Chromogenic Endotoxin Quant Kit (Thermo Fisher, A39552) demonstrated an endotoxin level of 5 EU / mL in purified Der p 2 allergen solutions (where [Der p 2] is 645 μg / mL). The FDA limit of endotoxin is 5 EU / kg. A typical dose of allergen for immunotherapy is 5-20 μg, so only 33 μL (a 30x dilution) of the allergen solution would be needed corresponding to 0.16 EU of endotoxin.

[0021] 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.

[0024] 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. 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. Upon activation, these cells release several preformed and newly generated mediators such as histamine, prostaglandins, leukotrienes, proteases, cytokines, and 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 (FcεRI) whose alpha chain engages with unique sequences in the Fc region of IgE antibodies. An allergic patient will have allergen specific IgE antibodies occupying these surface FcεRI. Allergen exposure to these cell surface IgE antibodies will induce receptor cross-linking, initiating a calcium and kinase-dependent activating cascade through the FcεRI’s immunoreceptor tyrosine-based activating motifs (ITAMs). 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.

[0025] In the clinic, 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. Therapeutically, 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. Page 6 QB\702581.02521\90543975.1

[0026] With increasing interest in molecular diagnosis and treatment of allergy as well as precision allergology, where patients are diagnosed based on the precise allergens to which they are sensitized and treated with personalized immunotherapies containing only those allergens, new tools are needed to produce allergen-based reagents. Allergen extract preparations are often inconsistent with variable amounts of allergen within each suspension and in ratios that are highly dependent upon the organism’s expression. Current methods for allergen extract preparation involve growing colonies of the organism that produces the allergen and formulated into a solution. This yields inconsistent quality, variable doses, and no control over which allergens are present and in what amount. Recombinant approaches for producing allergen have shown promise in leading to effective treatments, but progress is slowed by the rate at which allergen and allergen derivatives can be produced and characterized.

[0027] A cell-free platform for producing recombinant allergen is disclosed that has the potential to overcome some of the existing challenges and enable new diagnostic and therapeutic technologies. Cell-free protein synthesis (CFPS) is a technique that has been applied to protein biologic discovery, glycoprotein synthesis, point-of-care manufacturing, distribution of medicines to resource-limited settings, small molecules, diagnostics, and education. Cell-free protein synthesis enables rapid, on-demand synthesis of specific allergens based on desired allergen identity and dose. The ability to multiplex expression of many allergens or allergen derivatives can speed characterization of IgE-mediated processes to inform molecular understanding of allergy. The ability to express desired allergens and mix and match lysates may help facilitate implementation of personalized allergy treatment and precision allergology.

[0028] 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. These features can be leveraged in the laboratory to rapidly produce several allergen or allergen derivatives that 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 Page 7 QB\702581.02521\90543975.1and treatment. Leveraging these features in the clinic could enable production of a personalized allergen cocktail at the point-of-care by mixing CFPS-expressed allergen-containing lysates or by production of multiple allergens in one-pot, which could facilitate the implementation of precision allergology both diagnostically and therapeutically. Applying this CFPS platform toward the production of allergen makes these features available for the field of allergy and may open new approaches and enhancements for diagnosing and treating allergy, especially at the molecular level.

[0029] Recombinant allergen proteins can be a useful tool in the clinical diagnosis and treatment of allergic disease. 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.

[0030] 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. For example, the term “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. As used herein, 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. If there are uses of the term which are not clear to persons of ordinary skill Page 8 QB\702581.02521\90543975.1in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0034] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0035] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0036] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0037] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 Page 9 QB\702581.02521\90543975.1members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0038] 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. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0039] 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.

[0040] As used herein, 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.^

[0041] Polynucleotides and Synthesis Methods

[0042] The terms “nucleic acid” and “oligonucleotide,” as used herein, 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. There is no intended distinction in length between the terms “nucleic acid”, “oligonucleotide” and “polynucleotide”, and these terms will be used interchangeably. These terms refer 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. For use in the present methods, 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.

[0043] 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\90543975.168:90-99; 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. A review of synthesis methods of conjugates of oligonucleotides and modified nucleotides is provided in Goodchild, 1990, Bioconjugate Chemistry 1(3): 165-187, incorporated herein by reference.

[0044] The term “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. Nos.4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)), and the ligase chain reaction (LCR) (see Barany et al., U.S. Pat. No.5,494,810). 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.

[0045] The terms “target,” “target sequence”, “target region”, and “target nucleic acid,” as used herein, are synonymous and refer to a region or sequence of a nucleic acid which is to be amplified, sequenced, or detected.

[0046] The term “hybridization,” as used herein, 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\90543975.1pair 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. 26(3 / 4):227-259; and Owczarzy et al., 2008, Biochemistry, 47: 5336-5353, which are incorporated herein by reference).

[0047] The term “primer,” as used herein, 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.

[0048] 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.

[0049] 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. For example, 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). The region of the primer that is sufficiently complementary to the template to hybridize is referred to herein as the hybridizing region.

[0050] As used herein, 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\90543975.1the target nucleic acid. Typically, 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. One of skill in the art will recognize that various factors, such as salt conditions as well as base composition of the primer and the location of the mismatches, will affect the specificity of the primer, and that routine experimental confirmation of the primer specificity will be needed in many cases. Hybridization conditions can be chosen under which the primer can form stable duplexes only with a target sequence. Thus, the use of target-specific primers under suitably stringent amplification conditions enables the selective amplification of those target sequences that contain the target primer binding sites.

[0051] As used herein, a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides. “DNA polymerase” catalyzes the polymerization of deoxyribonucleotides. Known 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. Known examples of RNA polymerase (“RNAP”) 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.

[0052] 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.

[0053] As used herein, the term “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

[0054] The polynucleotide sequences contemplated herein may be present in expression vectors. For example, 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.

[0055] As used herein, "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.

[0056] As used herein, “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. As used herein, “expression template” and “transcription template” have the same meaning and are used interchangeably. Page 14 QB\702581.02521\90543975.1

[0057] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably. However, the disclosed methods and compositions are intended to include such other forms of expression vectors, such as viral vectors which serve equivalent functions.

[0058] In certain exemplary embodiments, 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. Within a recombinant expression vector, “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). The term “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).

[0059] Oligonucleotides and polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides. Examples of 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-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5- Page 15 QB\702581.02521\90543975.1oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6- diaminopurine and the like. 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.

[0060] 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. These phrases also refer to DNA 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).

[0061] Regarding polynucleotide sequences, the terms “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. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. 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. 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. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above). Page 16 QB\702581.02521\90543975.1

[0062] Regarding polynucleotide sequences, 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. 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.

[0063] Regarding polynucleotide sequences, “variant,” “mutant,” or “derivative” 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. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250). Such a pair of 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.

[0064] 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. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein. For example, polynucleotide sequences as contemplated herein may encode a protein and may be codon-optimized for expression in a particular host. In the art, 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.

[0065] 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. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic Page 17 QB\702581.02521\90543975.1engineering techniques known in the art. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, 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.

[0066] The 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.

[0067] Peptides, Polypeptides, Proteins, and Synthesis Methods

[0068] 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. The term “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. The term “amino acid residue” also may include nonstandard or unnatural amino acids. The term “amino acid residue” may include alpha-, beta-, gamma-, and delta-amino acids.

[0069] In some embodiments, the term “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, β-alanine, β-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, sarcosine, 3-Aminoisobutyric acid, N- Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N- Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3- Page 18 QB\702581.02521\90543975.1Diaminopropionic acid, Ornithine, and N-Ethylglycine. The term “amino acid residue” may include L isomers or D isomers of any of the aforementioned amino acids.

[0070] 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-GlcNAcpβ- serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L- phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an unnatural analogue of a methionine amino acid; an unnatural analogue of a leucine amino acid; an unnatural analogue of a isoleucine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, 19ufa19hor, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or a combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and / or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a keto containing amino acid; an amino acid comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid; a carbon-linked sugar- containing amino acid; a redox-active amino acid; an α-hydroxy containing acid; an amino thio acid; an α,α disubstituted amino acid; a β-amino acid; a γ-amino acid, a cyclic amino acid other than proline or histidine, and an aromatic amino acid other than phenylalanine, tyrosine or tryptophan.

[0071] As used herein, 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). In some embodiments, a peptide Page 19 QB\702581.02521\90543975.1as 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 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or more amino acid residues.

[0072] 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 isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein), glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).

[0073] Modified amino acid sequences that are disclosed herein may include a deletion in one or more amino acids. As utilized herein, 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. As utilized herein, an “insertion” means the addition of one or more amino acids to a native amino acid sequence. The Page 20 QB\702581.02521\90543975.1modified amino acid sequences that are disclosed herein may include a substitution of one or more amino acids. As utilized herein, 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. For example, 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.

[0074] Regarding proteins, 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.

[0075] Regarding proteins, “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. For example, a fragment may comprise from 5 to 1000 contiguous amino acid residues of a reference polypeptide, respectively. In some embodiments, 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.

[0076] Regarding proteins, the words “insertion” and “addition” refer to changes in an amino acid sequence resulting in the addition of one or more amino acid residues. 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\90543975.1protein may have N-terminal insertions, C-terminal insertions, internal insertions, or any combination of N-terminal insertions, C-terminal insertions, and internal insertions.

[0077] Regarding proteins, the phrases “percent identity” and “% identity,” refer 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). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. 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.

[0078] Regarding proteins, 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.

[0079] Regarding proteins, 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. For example, 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\90543975.1acid 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. The following table provides a list of exemplary conservative amino acid substitutions which are contemplated herein: Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

[0080] Conservative amino acid substitutions generally maintain (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. 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

[0081] 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).

[0082] The disclosed proteins may be substantially isolated or purified. The term “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.

[0083] Cell-Free Protein Synthesis (CFPS)

[0084] The components, systems, and methods disclosed herein may be applied to cell-free protein synthesis methods as known in the art. See, for example, U.S. Patent Nos. 5,478,730; 5,556,769; 5,665,563; 6,168,931; 6,548,276; 6,869,774; 6,994,986; 7,118,883; 7,186,525; 7,189,528; 7,235,382; 7,338,789; 7,387,884; 7,399,610; 7,776,535; 7,817,794; 8,703,471; 8,298,759; 8,715,958; 8,734,856; 8,999,668; and 9,005,920. See also U.S. Published Application Nos. 2018 / 0016614, 2018 / 0016612, 2016 / 0060301, 2015-0259757, 2014 / 0349353, 2014- 0295492, 2014-0255987, 2014-0045267, 2012-0171720, 2008-0138857, 2007-0154983, 2005- 0054044, and 2004-0209321. See also U.S Published Application Nos. 2005-0170452; 2006- 0211085; 2006-0234345; 2006-0252672; 2006-0257399; 2006-0286637; 2007-0026485; 2007- 0178551. See also Published PCT International Application Nos. 2003 / 056914; 2004 / 013151; 2004 / 035605; 2006 / 102652; 2006 / 119987; and 2007 / 120932. See also 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. 2014, “Substrate Replenishment and Byproduct Removal Improve Yeast Cell-Free Protein Synthesis,” U.S. Patent Application Serial No.: 61 / 953,275; and Jewett, M.C., Anderson, M.J., Stark, J.C., Hodgman, C.E. 2015, “Methods for activating natural energy metabolism for improved yeast cell-free protein synthesis,” U.S. Patent Application Serial No.: 62 / 098,578. See also Guarino, C., & DeLisa, M. P. (2012). “A prokaryote-based cell-free translation system that Page 24 QB\702581.02521\90543975.1efficiently synthesizes glycoproteins” Glycobiology, 22(5), 596-601. The contents of all of these references are incorporated in the present application by reference in their entireties.

[0085] In some embodiments, 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. In some embodiments, the CFPS reaction mixture can include exogenous RNA translation template. In other aspects, 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. In these other aspects, 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. In these other aspects, 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. It will be understood by one of ordinary skill in the art that 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.

[0086] The disclosed cell-free protein synthesis systems may utilize components that are crude and / or that are at least partially isolated and / or purified. As used herein, 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. The term “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. Page 25 QB\702581.02521\90543975.1

[0087] As used herein, “translation 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.

[0088] The term “reaction mixture,” as used herein, 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. In some embodiments, one or more of the cell-free protein synthesis reagents can be present in the reaction mixture.

[0089] 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. In certain embodiments the platform comprises both the expression template and the translation template. In certain specific embodiments, 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.

[0090] 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. In certain specific embodiments, 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.

[0091] Altering the physicochemical environment of the CFPS reaction to better mimic the cytoplasm can improve protein synthesis activity. The following parameters can be considered alone or in combination with one or more other components to improve robust CFPS reaction platforms based upon crude cellular extracts (for examples, S12, S30 and S60 extracts). Page 26 QB\702581.02521\90543975.1

[0092] 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. In certain aspects, 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.

[0093] The reaction mixture may include any organic anion suitable for CFPS. In certain aspects, the organic anions can be glutamate, acetate, among others. In certain aspects, 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.

[0094] The reaction mixture may include any halide anion suitable for CFPS. In certain aspects the halide anion can be chloride, bromide, iodide, among others. A preferred halide anion is chloride. Generally, 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.

[0095] The reaction mixture may include any organic cation suitable for CFPS. In certain aspects, the organic cation can be a polyamine, such as spermidine or putrescine, among others. Preferably polyamines are present in the CFPS reaction. In certain aspects, 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.

[0096] The reaction mixture may include any inorganic cation suitable for CFPS. For example, suitable inorganic cations can include monovalent cations, such as sodium, potassium, lithium, among others; and divalent cations, such as magnesium, calcium, manganese, among others. In certain aspects, the inorganic cation is magnesium. In such aspects, the magnesium concentration Page 27 QB\702581.02521\90543975.1can 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. In preferred aspects, 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.

[0097] 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). In certain aspects, the reaction uses ATP, GTP, CTP, and UTP. In certain aspects, the concentration of individual NTPs is within the range from about 0.1 mM to about 2 mM.

[0098] The reaction mixture may include any alcohol suitable for CFPS. In certain aspects, the alcohol may be a polyol, and more specifically glycerol. In certain aspects 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.

[0099] In certain exemplary embodiments, one or more of the methods described herein are performed in 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. Examples of 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.

[0100] Producing Allergen Proteins

[0101] The components, systems, and methods disclosed herein may be applied to recombinant cell systems and cell-free protein synthesis methods in order to prepare allergen proteins. In certain embodiments, the 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. In certain embodiments, the expression templates can include a protein-encoding sequence for one or more allergen proteins. In one or more embodiments, the one or more allergen proteins can be any proteins that may elicit an allergic reaction in a subject. In certain embodiments, a protein deemed Page 28 QB\702581.02521\90543975.1to elicit an allergic reaction in a subject can be a protein that can elicit allergen-specific IgE antibodies in a subject.

[0102] In one embodiment, 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. In certain embodiments, 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. In various embodiments, the one or more allergen proteins can be 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.

[0103] In certain embodiments, the one or more allergens proteins can include any amino acid mutations, substitutions, insertions, and the like relative to a wild-type sequence of such allergen proteins. In one embodiment, the encoding sequence for the one or more allergen proteins can include mutations that remove endogenous glycosylation sites, e.g., to prevent off-target glycosylation. For example, in one embodiment, the encoding sequence can include a mutation to substitute a G for N in the allergen protein sequence. In one or more embodiments, the allergen proteins produced according to the systems and methods described herein are not glycosylated.

[0104] In various embodiments, 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. In one example embodiment, the expression templates can include or encode for a ribosome binding site, e.g., to improve expression of the encoded allergen protein. In the same or alternative embodiments, 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. In certain embodiments, the expression templates can encode a purification tag, e.g., for purifying the allergen protein.

[0105] In various embodiments, the expression templates can be present in a vector. In one embodiment, the expression templates can be present in a plasmid vector, e.g., pJL1. Page 29 QB\702581.02521\90543975.1

[0106] In certain aspects, 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.

[0107] In various aspects, the expression templates can comprise a polynucleotide that encodes for one or more allergen proteins. In one or more aspects, the one or more allergen proteins can comprise, consist essentially of, or consist of a polypeptide having at least 80 %, 85 %, 90 %, 95 %, 99 %, or 100% identical to one or more of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.

[0108] One or more of the components of the disclosed systems may be in a preserved form. In some embodiments, one or more components of the disclosed systems are freeze-dried.

[0109] Also disclosed herein are allergen proteins and / or peptides. In one embodiment, the allergen proteins can be expressed using one or more of the expression templates disclosed herein.

[0110] In various embodiments, 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. In some embodiments, suitable CFPS reaction mixtures may include prokaryotic CFPS reaction mixtures comprising a lysate prepared from Escherichia coli. In certain embodiments, the E. coli or other cell can be modified for tailoring and / or optimizing cell-free protein synthesis. For instance, in certain embodiments, the E. coli or other cell can include a genetic modification to facilitate disulfide bond formation in expressed proteins.

[0111] In various embodiments, the CFPS system may may include eukaryotic CFPS reaction mixtures. In some embodiments, suitable CFPS reaction mixtures may include eukaryotic CFPS reaction mixtures comprising a lysate prepared from Saccharomyces cerevisiae or other eukaryote. In certain embodiments, the S. cerevisiae or other cell can be modified for tailoring and / or optimizing cell-free protein synthesis.

[0112] In various embodiments, 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. Page 30 QB\702581.02521\90543975.1

[0113] In certain embodiments, once the allergen protein is expressed, the allergen protein may be purified and / or isolated from the CFPS system using any convenient purification and / or isolation techniques.

[0114] In various embodiments, the systems can include modified cells. For instance, in certain embodiments, the modified cells may be genetically modified bacterial cells. In one or more embodiments, the modified bacterial cells can be modified to express a protein. In various embodiments, the modified cells can include an expression template for the one or more allergen proteins discussed herein. In one embodiment, the modified bacterial cells can produce one or more of the allergen proteins described herein in vivo. In alternative embodiments, a lysate prepared from the modified bacterial cell can be utilized to produce the one or more allergen proteins.

[0115] Methods For Assessment of Allergen Proteins

[0116] In various embodiments, 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.

[0117] In one or more embodiments, 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. For instance, in one embodiment, 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. In various embodiments, 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 tested for allergen function by a basophil acitivation test with primary human basophils, and / or by flow analysis of passively sensitized CD34+ progenitor-derived primary human mast cells.

[0118] Methods of Use Page 31 QB\702581.02521\90543975.1

[0119] As discussed above, in various embodiments, the systems and methods described herein can be used to provide allergen proteins for therapeutic and / or diagnostic purposes. For example, in certain embodiments, the allergen proteins produced as described herein can be used to determine whether or not a subject exhibits an allergic disease and / or reaction to such allergens. In one embodiment, a subject or a sample from a subject can be exposed to the one or more allergen proteins. In certain embodiments, the subject can be exposed to the allergen proteins using a skin prick test. In one or more embodiments, a sample from the subject can be exposed to the allergen proteins using a lateral flow assay, ELISA, point-of-care assay, or other assay, e.g., to detect IgE from subject serum binding to the allergen proteins.

[0120] The terms point-of-care (POC) testing, point-of-service (POS) testing, point-of-use (POU) testing, and point-of-incidence (POI) testing are used interchangeably in this disclosure unless otherwise indicated. The term 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.

[0121] 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.

[0122] In various embodiments, the disclosed methods and systems test for allergens in a sample using a point-of service method or device. In some embodiments, the point-of service method or device is performed at a point-of-service location.

[0123] In various embodiments, the allergen proteins produced according to the disclosed methods and systems are used in point-of-use systems, methods, and devices. Use of the disclosed allergen proteins enable production of a personalized allergen cocktail at the point-of-care such as by mixing CFPS-expressed allergen-containing lysates or by production of multiple allergens in one-pot, which could facilitate the implementation of precision allergology both diagnostically and therapeutically. Page 32 QB\702581.02521\90543975.1

[0124] In various embodiments, the allergen proteins produced according to the methods and systems described herein can be used therapeutically. For example, in certain aspects, the one or more allergen proteins can be administered to a subject, e.g., subcutaneously, sublingually, or orally. In one embodiment, the allergen proteins can be added to a food product formulation.

[0125] In various embodiments, 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.

[0126] In various embodiments, a point-of-use test can include a skin prick test. In such an embodimemt, 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. In various embodiments, 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. In the same or alternative embodiments, 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.

[0127] In various embodiments, 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). In such an embodiment, 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. In various embodiments, 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.

[0128] In various embodiments, 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. Page 33 QB\702581.02521\90543975.1

[0129] In various embodiments, one or more allergen proteins, e.g., made using one or more of the CFPS methods and / or systems described herein, can be formulated into a lateral flow assay to detect IgE for an at home or POC test.

[0130] In one or more embodiments, 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.

[0131] Applications

[0132] In certain embodiments, applications of the disclosed technology include, but are not limited to: (i) Skin prick tests for diagnostic allergy testing of patients; (ii) In vitro lab tests for quantification of allergen-specific IgE present in blood (e.g. ELISA); (iii) Administration of subcutaneous, sublingual, or oral immunotherapy; (iv) Formulation in food product(s) for early introduction; (v) Point-of-care or at-home diagnostics; (vi) Paper-based diagnostics; (vii) Lateral flow diagnostics. It is appreciated that the applications mentioned herein would include the use of the allergen proteins prepared according to the methods and system described herein.

[0133] Advantages

[0134] 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-of-care Page 34 QB\702581.02521\90543975.1by mixing CFPS-expressed allergen-containing lysates or by production of multiple allergens in one-pot could facilitate the implementation of precision allergology; and (12) Absence of glycosyltransferases means proteins are not glycosylated, which can be difficult to control in living cells, especially mammalian based expression systems.

[0135] Miscellaneous

[0136] The steps of the methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The steps may be repeated or reiterated any number of times to achieve a desired goal unless otherwise indicated herein or otherwise clearly contradicted by context.

[0137] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0138] Additional Embodiments

[0139] 1. Cell-free expression of allergens. This method may comprise:

[0140] 1.1 DNA design of genetic constructs encoding allergen.

[0141] 1.2 Expression of allergen in cell-free systems.

[0142] 1.3 Expression of allergen in cell-free systems based on bacterial cell-free extracts.

[0143] 1.4 Expression of allergen in genetically cell-free systems based on bacterial modified E. coli cell-free extracts. Page 35 QB\702581.02521\90543975.1

[0144] 1.5 Expression of allergen in genetically modified E. coli extracts to facilitate disulfide bond formation.

[0145] 1.6 Expression of allergen in genetically modified E. coli extracts after lyophilization and rehydration.

[0146] 1.7 Expression of allergen in other bacterial extracts.

[0147] 1.8 Expression of allergen in extracts prepared from other organisms.

[0148] 1.9 Evaluation of human IgE recognition of allergen using an AlphaLISA assay.

[0149] 1.10 Evaluation of allergen expression using radioactivity assay.

[0150] 1.11 Evaluation of allergen function by basophil activation test with primary human basophils.

[0151] 1.12 Evaluation of allergen function by flow analysis of passively sensitized CD34+ progenitor-derived primary human mast cells.

[0152] 2. The use of cell-free expression of allergens for diagnosing and treating allergy. This use may comprise:

[0153] 2.1 Application toward skin prick tests for diagnostic testing of allergy.

[0154] 2.2 Application toward In vitro lab tests for quantification of allergen-specific IgE present in blood (e.g., ELISA).

[0155] 2.3 Application toward administration of subcutaneous, sublingual, or oral immunotherapy.

[0156] 2.4 Application toward formulation in food product(s) for early introduction.

[0157] 2.5 Application toward point-of-care or at-home paper-based diagnostics.

[0158] 2.6 Application toward lateral flow diagnostics. Page 36 QB\702581.02521\90543975.1

[0159] 3. The method of embodiment 1, where synthetic DNA elements are introduced (including but not limited to: purification tags, immune-modulating elements, sites for post- translational modifications, designer epitopes, elements that aid expression, and / or elements that aid folding and assembly).

[0160] 4. The method of embodiment 1, where the protein sequence matches that of what the host organism encodes.

[0161] 5. The method of embodiment 1, used to express Der p 1, Der p 2, Bos d 5, Bos d 4, Ara h 2, Gal d 1, Gal d 2, Gal d 4, Fel d 1, Amb a 1, Bla g 2, Cry j 1, Bet v 1.

[0162] 6. The method of embodiment 1, applied to other allergens.

[0163] 7. The method of embodiment 1, applied to hypoallergenic allergens.

[0164] 8. The method of embodiment 1, applied to allergen epitope constructs.

[0165] 9. The method of embodiment 1, where IgE recognition is assessed by ELISA assay.

[0166] 10. The method of embodiment 1, where IgE recognition is assessed by surface plasmon resonance.

[0167] 11. The method of embodiment 1, where IgE recognition is assessed by biolayer interferometry.

[0168] 12. The method of embodiment 1, where allergen function is assessed by the basophil activation test in human blood basophils.

[0169] 13. The method of embodiment 1, where allergen function is assessed using human skin- derived mast cells.

[0170] 14. The method of embodiment 1, where allergen function is assessed using the human mast cell lines LAD2.

[0171] 15. The method of embodiment 1, where cell-free extracts are derived from other organisms. Page 37 QB\702581.02521\90543975.1EXAMPLES

[0172] The following Examples are illustrative and are not intended to limit the scope of the

[0173] Example 1 - A Cell-Free Protein Synthesis Platform for the Production of a Clinically Relevant Allergen Panel

[0174] Abstract

[0175] Allergens are used in the clinical diagnosis and treatment of allergic disease. Allergen extracts are used for diagnostic skin tests and for in vitro detection of allergen-specific immunoglobulin E (IgE). Allergens are also used in immunotherapy treatments, both via the oral and parenteral route, the latter referred to as “allergy shots”. In some instances, recombinant allergen is used for diagnostic IgE quantification tests and for immunotherapy. With growing interest in molecular allergy diagnosis and precision allergology, new tools are needed for producing allergen-based reagents to enable personalized medicine in the field of allergy. Here, to help address this need, we demonstrate a cell-free protein synthesis approach for 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. We also show that cell-free expressed allergen can activate primary human cells such as peripheral blood basophils, and CD34+ progenitor-derived mast cells in an IgE-dependent manner. We anticipate that diagnostic and therapeutic technologies will be enabled by this cell-free platform for allergen production and will provide useful tools and treatments for both the allergist and the allergic patient.

[0176] 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. 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. Upon activation, these cells release several preformed and newly generated mediators such as histamine, prostaglandins, leukotrienes, proteases, cytokines and Page 38 QB\702581.02521\90543975.1other 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 (FcεRI) whose alpha chain engages with unique sequences in the Fc region of IgE antibodies. An allergic patient will have allergen-specific IgE antibodies occupying these surface FcεRI. Allergen exposure to these cell surface IgE antibodies will induce receptor cross-linking, initiating a calcium and kinase-dependent activating cascade through the FcεRI’s immunoreceptor tyrosine-based activating motifs (ITAMs). 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.

[0177] In the clinic, allergy is diagnosed by detecting allergen-specific IgE with a variety of allergen-based reagents.1By 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. Therapeutically, 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 supplements2to aid in early oral introduction of food allergens, which have been shown to greatly reduce the risk of thedevelopment of food allergy.3, 4

[0178] With increasing interest in molecular diagnosis and treatment of allergy as well as precision allergology, where patients are diagnosed based on the precise allergens to which they are sensitized and treated with personalized immunotherapies containing only those allergens, new tools are needed to produce allergen-based reagents.5, 6Allergen extract preparations are often inconsistent with variable amounts of allergen within each suspension and in ratios that are highly dependent upon the organism’s expression.7Recombinant approaches for producing allergen have shown promise in leading to effective treatments, but progress is slowed by the rate at which allergen and allergen derivatives can be produced and characterized.5Page 39 QB\702581.02521\90543975.1

[0179] Here, we describe a cell-free platform for producing recombinant allergen that has the potential overcome some of the existing challenges and enable new diagnostic and therapeutic technologies. Cell-free protein synthesis (CFPS)8-10is a technique that has been applied to protein biologic discovery,11, 12glycoprotein synthesis,13-18point-of-care manufacturing,19-24distribution of medicines to resource-limited settings,25, 26small molecules,27-29diagnostics,30-32and education.33-35

[0180] 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.36, 37CFPS 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. These features can be leveraged in the laboratory to rapidly produce several allergen or allergen derivatives that 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. Leveraging these features in the clinic could enable production of a personalized allergen cocktail at the point-of-care by mixing CFPS-expressed allergen-containing lysates or by production of multiple allergens in one-pot, which could facilitate the implementation of precision allergology both diagnostically and therapeutically. Applying this CFPS platform toward the production of allergen makes these features available for the field of allergy and may open new approaches and enhancements for diagnosing and treating allergy, especially at the molecular level.

[0181] Cell-free expression of a clinically-relevant allergen panel

[0182] First, we wanted to test whether a bacterial, cell-free system would be amenable to expression of protein allergens from a wide range of taxonomic kingdoms including insects, plants, and mammals (Table 1, Figure 1). We curated a list of common allergens implicated in food allergy and environmental allergy, and we aimed to select allergens with high rates of IgE positivity in sensitized patients. Our list includes the most common allergens, the predominant allergens in several common food allergies as well as the food allergen to which sensitivity is associated with Page 40 QB\702581.02521\90543975.1the highest risk of anaphylaxis, and 5 of the top aeroallergens (Table 1, FIG.4). Addition of14C- leucine into the CFPS reaction enabled quantification of protein yield by detection of radioactive leucine incorporated into the final protein product. Based on this quantification, all allergens expressed in our system, with an average total and soluble yield of 267 ± 132 μg / mL and 174 ± 98 μg / mL, respectively. Even those allergens expressed at the lowest yields would be amenable to production for downstream applications with simple scale-up (FIG.1B, FIG.4).

[0183] 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)

[0184] Recognition of CFPS-expressed Der p 2 by monoclonal allergen-specific IgE

[0185] 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. As a prototype, we selected dust mite allergen Der p 2, one of the higher expressing allergens, because of its clinical relevance as one of the most common allergens. To detect binding of IgE to the allergen, we utilized an in-solution, bead-based ELISA assay called the AlphaLISA. Here, a Page 41 QB\702581.02521\90543975.1protein A coated donor bead associates with α-IgE rabbit IgG and a nickel coated acceptor bead associates with the His-tagged Der p 2 allergen. If IgE successfully binds Der p 2 then it will bridge the two species, bringing their associated beads in proximity. Once in close proximity, an energy transfer reaction will take place between the donor and acceptor bead that produces an emission of light that can be read on a plate reader (FIG.2A).

[0186] We performed a co-titration of IgE and CFPS-expressed Der p 2 with the AlphaLISA assay using two monoclonal IgE antibodies, 2G1 and 2F10, that recognize two distinct epitopes on opposite poles of the Der p 2 allergen.38On the final readout of an AlphaLISA, successful binding is indicated by an enhanced level of signal over background at some optimal concentration, with lower signal gradually fanning out as the concentration moves away from the optimum. Background signal is determined by the 0 conditions where either IgE (bottom row) or Der p 2 (left column) is absent. The pattern of signal in both co- titrations, and optimal binding at nM concentrations, is indicative of successful binding by both monoclonal IgE antibodies recognizing either the 2G1 or 2F10 epitope (FIG. 2B). This demonstrates that CFPS-expressed Der p 2 retains its ability to be recognized by IgE, a key interaction underpinning allergic responses and diagnostics.

[0187] Activation of human allergic effector cells by CFPS-expressed Der p 2

[0188] To further confirm bioactivity, it is important to demonstrate that IgE binding and crosslinking of FcεRI 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. These surface activation markers, typically detected by flow cytometry, include CD63 for basophils and mast cells and CD107a (LAMP1) for mast cells.39

[0189] The first cell model utilizes human CD34+ progenitor cells isolated from peripheral blood to generate mast cells in culture. One advantage of this approach is that the mast cells will not have any prior IgE in their receptors, unlike primary mast cells isolated from human tissues. At initial inoculation, cells are small and round and are differentiated into mast cells by culture Page 42 QB\702581.02521\90543975.1with IL-3, IL-6, and stem cell factor as described.40Over time, cells become larger with the appearance of intracellular granules and irregular borders characteristic of mast cell morphology (FIG.3A). They now display surface expression of typical mast cell markers CD117 / KIT, CD33, FcεRIα, Siglec-6, and Siglec-8 (FIG. 5), and are capable of degranulation, as assessed by the appearance of CD107a and CD63 when stimulated (FIG. 6A-6B). At this stage in culture, mast cells can be passively sensitized by incubation with allergen-specific IgE as monoclonals or serum from an allergic donor. 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). Incubation with CFPS- expressed Der p 2 over a logarithmic scale from 1 nM to 1 μM resulted in a positive response at all treatment concentrations with 10 nM being the optimal concentration for crosslinking (FIG. 3B). In contrast, incubation of mast cells with CFPS-expressed Der p 2 at these concentrations in the absence of IgE sensitization yields 3±0.5% CD63 positivity, similar to background activation. This demonstrates that the activating effect of CFPS-expressed Der p 2 is IgE-dependent, as expected and desired.

[0190] The second cell model utilizes basophils circulating in peripheral blood from a Der p 2 allergic donor. 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. To assess activation, levels of CD63 are determined. Under negative control buffer-only conditions, only 9±3% basophils are CD63 positive. Incubation with α-FcεRIα antibody as one positive control leads to CD63 positivity in 76±10% of basophils, while incubation with α-IgE antibody as a second positive control leads to CD63 positivity in 73±17% of basophils. Exposure to a non-IgE- mediated, bacterial tripeptide activator (fMLP) leads to CD63 positivity in 13±4% of basophils. Finally, as hoped, basophils incubated with either HDM extract or CFPS- expressed Der p 2, the latter over a logarithmic range from 1 pM to 1 μM, showed marked CD63 positivity, at all concentrations with >100 pM being optimal for FcεRI crosslinking. The half maximal CFPS-expressed Der p 2 dose is ~10-30 pM. At 3 pM, CD63 positivity remains near background levels (FIG.3C).

[0191] Discussion Page 43 QB\702581.02521\90543975.1

[0192] Recombinant allergen is a useful tool in the clinical diagnosis and treatment of allergic disease.5-7, 41CFPS-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. In our development of a CFPS-based platform for biomanufacturing allergen, we observed soluble yields > 100 μg / mL for 9 out of 13 tested allergens included in our panel and > 50 μg / mL for all allergens. This provides clear proof-of-concept that protein allergens are amenable to cell-free production even across a wide range of phyla.

[0193] Using Der p 2 as a prototype, we show that CFPS-expressed allergen retains its ability to be recognized by IgE and activate human basophils and mast cells in vitro at the picomolar range. More studies need to be done to confirm these important characteristics with other allergens.

[0194] Additional work would need to overcome the presence of endotoxin in E. coli lysates. Since endotoxin paired with allergen is a common approach for oral sensitization in allergy mouse models,42, 43its presence would not be suitable for human use in immunotherapy or skin testing. Here, purification alone was able to achieve endotoxin levels of 0.16 EU per a typical immunotherapy dose, but ideal preparations would achieve undetectable levels (FIG. 7). Optimization of endotoxin removal while minimizing protein loss could overcome this challenge. Also, this would not be a constraint for lab tests such as IgE ELISA’s, nor for in vitro studies of allergen and immune reactions.

[0195] There is a push in the field towards molecular diagnosis and treatment of allergy and precision allergology, and a CFPS-based approach for allergen production makes this more accessible than existing recombinant allergen methods alone. Ultimately, the cell- free platform for allergen production established here has the potential to not only facilitate improved molecular understanding of allergy, but also to open new capabilities and technologies for the clinical allergist and allergic patient.

[0196] Materials and Methods Page 44 QB\702581.02521\90543975.1

[0197] Plasmid design and synthesis

[0198] Allergen protein sequences were retrieved from Uniprot, synthetically modified, and then codon optimized for E. coli K12 strains into a DNA sequence (IDT Codon Optimization Tool). Additional synthetic sequences were added to optimize expression and enable affinity purification (see Tables 2 and 3 below). Inserts were synthesized into a PJL1 backbone at NdeI and SalI restriction sites (Twist Biosciences).

[0199] Plasmid DNA used in CFPS reactions was purified from glycerol stocks provided by Twist Biosciences using the ZymoPURE Midi Kit (Zymo Research D4200).

[0200] Harvest and processing of E. coli lysate for CFPS

[0201] E. coli lysate was prepared using previously published methods44using engineered E. coli strain C321.ΔA.759, a highly productive of MG1655-derived strain.45Briefly, E. coli cells were inoculated at OD 0.08 into 2xYTPG media (yeast extract 10 g / L, tryptone 16 g / L, NaCl 5 g / L, K2HPO47 g / L, KH2PO43 g / L, and glucose 18 g / L, pH = 7.2) in a 10L fermentor (Sartorius Biostat C+) and grown at 34°C with agitation (250 rpm). Cultures were induced at OD 0.6-0.8 with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Sigma-Aldrich I6758) and harvested at OD 3.0. Cells were pelleted in a chilled JLA 8.1 rotor (Beckman Avanti J-25I Refrigerated Centrifuge) for 5 min at 8,000g at 4°C and distributed to twelve 50 mL Falcon tubes. Cells were then washed three times with chilled S30 buffer (10 mM Tris-acetate pH 8.2, 14 mM magnesium acetate, 60 mM potassium acetate) with centrifugation for 2 min at 10,000g at 4°C in a Thermo Heraeus Multifuge X3R Centrifuge in between. Pellets were then weighed and flash frozen in liquid nitrogen. 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 μL) 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 μL), aliquotted, flash frozen in liquid nitrogen, and stored at -80°C for downstream use.

[0202] Cell free protein synthesis of allergen Page 45 QB\702581.02521\90543975.1

[0203] CFPS reactions were assembled based on previously published methods.44In brief, reactions were assembled with the following reagents and concentrations: 6 mM magnesium glutamate; 10 mM ammonium glutamate; 130 mM potassium glutamate; 2.646 mM ATP; 1.874 mM each of GTP, UTP, and CTP; 0.075 mg / mL folinic acid, 0.376 mg / mL E. coli tRNA mixture from strain MRE600 (Roche Applied Science); 0.33 mM nicotinamide adenine dinucleotide (NAD); 0.27 mM coenzyme-A (CoA); 4 mM oxalic acid; 1 mM putrescine; 1.5 mM spermidine; 57 mM HEPES at pH=7.2; 2 mM of each of the 20 standard amino acids; 30 mM phosphoenolpyruvate (PEP) (Roche Applied Science); 13.3 μg / mL of plasmid encoding allergen in pJL1 backbone; and 27% v / v of E. coli crude lysate (prepared above). Reagents sourced from Sigma-Aldrich unless stated otherwise.

[0204] Radioactive quantification of allergen expressed by cell free protein synthesis

[0205] Allergen expressed was quantified by 14C-leucine incorporation according to previously published methods.46In brief, 10.67 μL 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. Then, 4 uL of sample was dispensed onto a Filtermat (Perkin Elmer, 1450-421) and duplicated on a second Filtermat and allowed to dry. One Filtermat was then washed in 5% trichloroacetic acid (Sigma-Aldrich T6399) three times for 15 min each at 4°C and allowed to dry. Scintillation wax was then applied to the Filtermat over a hot plate, cooled, and then signal was read using a Microbeta2 Scintillation Counter. Protein expression was calculated using following formula:

[0206] (((Signal of sample on washed filtermat – signal of no DNA negative control sample) / signal of sample on unwashed filtermat) * [14C-leucine (μM)] * molecular weight of protein (g / mol)) / (# leucine residues x 1000)

[0207] Purification of CFPS-expressed allergens

[0208] Allergen was suspended in Buffer 1 (50 mM NaH2PO4, 300 mM NaCl, pH = 8.0) and isolated by affinity-tag purification with Ni-NTA magnetic beads (Invitrogen Dynabeads His-tag Page 46 QB\702581.02521\90543975.1Isolation and Pulldown, 10104D) according to manufacturer’s instructions, where beads were washed with Buffer 1 and allergen was eluted with 500 mM imidazole (Sigma- Aldrich I5513) in Buffer 1.

[0209] The elution was desalted using Zeba spin columns (Thermo Scientific 89883) according to manufacturer’s instructions and collected in nuclease free water (Ambion, AM9937).

[0210] Detection of IgE binding by AlphaLISA assay

[0211] CFPS-expressed allergen concentration was measured on nanodrop with Protein A280 based on extinction coefficients and molecular weights calculated using Expasy software. 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. Signal was read on a plate reader (Tecan Infinite M1000).

[0212] Activation test of primary human basophils in peripheral blood

[0213] Whole blood was obtained from allergic and non-allergic donors with written informed consent for blood donation (≤ 180 mL) using an institutional review board-approved protocol at Northwestern University Feinberg School of Medicine. Primary human basophils from whole blood were activated with the FlowCast Basophil Activation Test according to manufacturer’s instructions (Bϋhlmann Labs, FK-CCR-U). As positive controls, basophils in whole blood were stimulated with 5 μL anti-IgE of a 0.5 mg / mL stock (BD 555894) and clinical-grade house dust mite extract at a 1:100 dilution.

[0214] Culture and differentiation of human peripheral blood CD34+ progenitor cells into mast cells

[0215] CD34+ progenitor cells from human peripheral blood were purchased from Stemcell Technologies and cultured as previously described.40Page 47 QB\702581.02521\90543975.1

[0216] Passive sensitization and activation of human CD34+ progenitor-derived mast cells

[0217] 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. Patient serum contained 44.2 ng / mL of Der p 2-specific IgE representing 11.9 % of total IgE (890 ng / mL).

[0218] Mast cells were then collected and suspended in Tyrode’s buffer (135 mM NaCl, 5 mM KCl, 5.6 mM glucose, 1.8 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, and 0.5 mg / mL bovine serum albumin (BSA), pH = 7.3). Allergen and control stimulants (10 μL of 10x stock) were distributed to 1.5 mL Eppendorf tubes and 200k-250k cells (suspended in 90 μL Tyrode’s buffer) were distributed to each stimulant sample. Cells were incubated with stimulant for 20 minutes at 37°C at 170 rpm. Optimal positive control concentrations were determined to be 2 μM ionomycin (Thermo Scientific, J60628LB0) and 2 uL of a 50 mg / mL stock anti-FcεRIα (Biolegend, 334602) (FIG.5).

[0219] For flow cytometry, cells were incubated at 4°C for 30 minutes with live / dead staining (Invitrogen L23105) suspended in PBS according to manufacturer’s instructions. Cells were then blocked with α-human CD32 (Biolegend 334602) in FACS buffer (PBS, 2.5% BSA) according to manufacturer’s instructions (10-minute incubation) then labeled with fluorescent antibodies FITC α-human FcεRIα (Biolegend 334608), APC α-human CD107a / LAMP1 (BD 641581), Bv421 α- human CD117 / KIT (BD 562434), PE α-human CD63 (BD 557305) all suspended in FACS buffer according to manufacturer’s instructions and incubated at 45 minutes at 4°C.

[0220] 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. Page 48 QB\702581.02521\90543975.1DNA Sequences of allergens (in pJL1 backbone): Cat linker – Protein - BamHI linker -Glyctag -His tag- Stop codon Der p 1 (Adapted from Uniprot ID: P08176) (SEQ ID NO: 1) ATGGAGAAAAAAATCCGTCCCAGCTCGATTAAAACGTTTGAGGAGTACAAGAAAGCATTT AATAAGTCTTACGCCACCTTTGAGGACGAAGAAGCAGCCCGCAAAAACTTTCTGGAGTCC GTTAAGTATGTACAGAGCAATGGCGGTGCTATTAACCACTTGTCCGACTTATCCTTGGATG AGTTTAAGAACCGCTTCCTTATGAGCGCCGAAGCATTTGAGCACCTTAAGACACAGTTTGA CCTGAACGCAGAGACTAACGCATGTTCCATCAACGGTAATGCCCCTGCGGAAATTGACCT GCGTCAAATGCGCACTGTAACACCCATCCGTATGCAAGGTGGATGCGGATCCTGCTGGGC ATTTTCAGGCGTCGCGGCGACTGAGTCAGCTTACTTGGCTTATCGTAACCAGTCGTTAGAC CTGGCTGAGCAAGAATTGGTGGACTGTGCTTCGCAACACGGGTGTCATGGGGATACGATT CCGCGCGGAATCGAATACATCCAGCACAACGGCGTCGTACAAGAATCATACTATCGCTAC GTGGCTCGTGAGCAATCTTGTCGTCGCCCCAACGCGCAACGTTTCGGGATTTCTAACTATT GCCAAATTTACCCACCAAATGTGAATAAAATCCGTGAAGCATTAGCCCAGACCCATTCTG CGATCGCTGTAATCATTGGAATTAAAGATTTAGACGCTTTCCGTCATTACGATGGGCGTAC AATCATTCAACGCGACAATGGTTACCAGCCAAACTATCACGCGGTGAACATTGTCGGATA TTCTAATGCGCAGGGGGTAGATTATTGGATCGTGCGCAACTCTTGGGACACAAATTGGGG TGATAACGGGTACGGCTATTTTGCAGCCAACATTGACTTAATGATGATCGAAGAGTATCCC TATGTGGTTATCTTAGGATCCGGTCGTGCAACCACAGGTGGTAATTGGACAACCCGTGCCG GTGGTCATCACCATCATCACCATTAA Der p 2 (Uniprot ID: P49278) (SEQ ID NO: 2) ATGGAGAAAAAAATCGATCAGGTGGATGTTAAAGATTGCGCCAACCACGAAATCAAAAA AGTTCTGGTTCCGGGTTGTCATGGTAGCGAACCGTGTATTATTCATCGTGGTAAACCGTTT CAGCTGGAAGCAGTTTTTGAAGCAAATCAGAATACCAAAACCGCCAAGATTGAAATCAAA GCCAGCATTGATGGTCTGGAAGTTGATGTTCCGGGTATTGATCCGAATGCATGCCACTATA TGAAATGTCCGCTGGTTAAAGGTCAGCAGTATGATATCAAATATACCTGGAACGTGCCGA AAATTGCACCGAAAAGCGAAAATGTTGTTGTGACCGTTAAAGTGATGGGTGATGATGGTG TTCTGGCATGTGCCATTGCAACCCATGCAAAAATTCGTGATGGATCCGGTCGTGCAACCAC AGGTGGTAATTGGACAACCCGTGCCGGTGGTCATCACCATCATCACCATTAA Bos d 5 (Adapted from Uniprot ID: P02754) (SEQ ID NO: 3) ATGGAGAAAAAAATCTTAATCGTCACTCAAACCATGAAGGGATTGGATATCCAGAAAGTT GCCGGAACGTGGTACTCTCTGGCTATGGCTGCATCGGATATTAGTTTACTGGATGCACAGA GCGCTCCGCTTCGTGTCTACGTGGAAGAGCTGAAGCCAACACCGGAAGGTGACTTGGAGA TTCTTTTGCAAAAATGGGAGAACGGCGAATGCGCTCAAAAGAAAATTATTGCCGAAAAAA CCAAAATCCCGGCTGTATTCAAAATTGACGCTTTGAACGAGAACAAAGTTTTGGTATTGG ATACCGATTACAAGAAATATCTGTTATTCTGTATGGAAAATTCAGCGGAACCTGAGCAGT CGTTAGCCTGTCAGTGCTTGGTACGCACCCCGGAGGTCGACGATGAAGCTCTTGAAAAGT TTGACAAGGCCCTTAAAGCCTTGCCTATGCACATTCGTTTGTCATTCAATCCTACTCAGTTG GAGGAGCAGTGTCACATTGGATCCGGTCGTGCAACCACAGGTGGTAATTGGACAACCCGT GCCGGTGGTCATCACCATCATCACCATTAA Bos d 4 (Adapted from Uniprot ID: P00711) (SEQ ID NO: 4) ATGGAGAAAAAAATCGAACAACTGACTAAATGTGAAGTTTTTCGTGAATTAAAAGACCTT AAGGGGTATGGAGGCGTGAGCCTGCCTGAATGGGTTTGCACCACATTTCATACTAGCGGA TATGATACCCAGGCAATCGTACAGAATAACGACAGCACTGAATACGGCTTGTTCCAAATC AATAATAAGATTTGGTGCAAGGATGACCAGAACCCACACAGCTCTAACATCTGCAACATT AGCTGTGATAAGTTTTTAGACGACGATCTTACTGATGATATTATGTGCGTCAAGAAAATTC TTGACAAGGTTGGCATTAACTACTGGTTAGCCCACAAGGCGTTGTGCTCAGAGAAATTGG Page 49 QB\702581.02521\90543975.1ACCAATGGTTGTGTGAGAAGTTGGGATCCGGTCGTGCAACCACAGGTGGTAATTGGACAA CCCGTGCCGGTGGTCATCACCATCATCACCATTAA Gal d 1 (Adapted from Uniprot ID: P01005) (SEQ ID NO: 5) ATGGAGAAAAAAATCGCCGAAGTGGACTGTTCCCGTTTTCCTGGGGCAACAGATAAAGAG GGCAAAGACGTTCTGGTCTGTAATAAGGACCTTCGTCCTATTTGTGGTACGGATGGCGTAA CGTATACCAATGACTGTCTTTTGTGTGCCTACTCTATCGAGTTCGGCACAGGCATCTCCAA GGAACACGATGGGGAATGTAAAGAGACTGTTCCAATGGGCTGTTCCTCCTACGCAGGAAC AACGTCTGAAGATGGCAAGGTCATGGTTTTATGCAACCGCGCCTTCAATCCCGTATGCGG GACTGACGGTGTGACATACGACAACGAATGCCTGTTGTGCGCTCATAAGGTTGAACAAGG AGCTTCCGTTGATAAACGCCATGACGGAGGCTGCCGTAAAGAGCTGGCCGCAGTGAGCGT GGATTGTTCGGAATATCCAAAACCTGACTGCACGGCAGAAGACCGTCCCTTGTGTGGCAG TGACAATAAAACCTACGGCAATAAGTGTAATTTTTGCAACGCAGTTGTTGAGTCCGGCGG TACGCTGACATTAAGTCATTTCGGTAAGTGCGGATCCGGTCGTGCAACCACAGGTGGTAA TTGGACAACCCGTGCCGGTGGTCATCACCATCATCACCATTAA Gal d 2 (Adapted from Uniprot ID: P01012) (SEQ ID NO: 6) ATGGAGAAAAAAATCTCTACCCGTACACAGATCAATAAGGTGGTCCGCTTCGATAAGTTA CCTGGGTTCGGCGATTCAATCGAAGCCCAATGTGGAACAAGCGTCAACGTTCATTCAAGT CTTCGTGACATTTTGAATCAGATCACTAAACCGAATGATGTCTACTCTTTCAGTCTTGCGTC GCGTTTATATGCTGAAGAGCGTTACCCGATCTTACCCGAGTATTTACAGTGCGTCAAAGAA TTGTACCGCGGAGGACTGGAACCAATCAATTTCCAAACGGCCGCCGACCAAGCCCGTGAA TTAATTAATAGTTGGGTGGAGTCACAGACTAACGGGATCATCCGTAACGTGTTGCAGCCCT CATCGGTCGATTCCCAAACTGCCATGGTATTGGTAAATGCCATTGTGTTTAAGGGGTTGTG GGAGAAAGCCTTTAAGGATGAGGACACGCAGGCGATGCCATTCCGCGTAACGGAGCAGG AAAGCAAGCCTGTGCAGATGATGTACCAGATCGGACTTTTCCGCGTCGCTTCGATGGCGTC TGAGAAGATGAAAATCTTGGAACTTCCCTTTGCATCCGGAACCATGTCGATGTTAGTGCTT CTTCCGGATGAAGTCTCGGGATTAGAGCAGTTGGAAAGTATTATTAATTTCGAGAAATTG ACTGAATGGACCTCGTCTAACGTTATGGAAGAACGTAAGATTAAGGTATATTTACCCCGC ATGAAAATGGAGGAGAAATATAACTTGACATCAGTCCTTATGGCGATGGGTATTACAGAC GTGTTTTCATCATCCGCAAACTTATCAGGAATTTCAAGCGCGGAGTCTTTAAAGATTTCCC AGGCAGTCCACGCTGCACATGCAGAAATTAATGAGGCCGGGCGCGAGGTCGTCGGCAGC GCTGAGGCGGGCGTTGATGCAGCATCTGTTAGCGAGGAATTTCGTGCAGACCACCCTTTTT TGTTCTGCATCAAACACATTGCCACCAACGCGGTATTGTTTTTCGGTCGTTGTGTTTCCCCC GGATCCGGTCGTGCAACCACAGGTGGTAATTGGACAACCCGTGCCGGTGGTCATCACCAT CATCACCATTAA Gal d 4 (Adapted from Uniprot ID: P00698) (SEQ ID NO: 7) ATGGAGAAAAAAATCAAGGTTTTCGGACGCTGCGAATTAGCGGCTGCTATGAAGCGCCAT GGGTTAGATAATTATCGCGGGTACTCGTTAGGTAATTGGGTCTGTGCGGCTAAGTTTGAAT CTAACTTCAATACTCAAGCTACGAATCGCAACACGGATGGTAGCACTGATTACGGAATTTT GCAGATTAACTCTCGTTGGTGGTGCAATGATGGACGTACACCTGGAAGTCGTAACCTTTGC AACATTCCGTGCAGCGCCCTGTTGTCGTCCGATATTACAGCCTCGGTAAACTGCGCGAAGA AAATTGTAAGCGACGGGAATGGGATGAACGCCTGGGTAGCATGGCGCAACCGCTGTAAA GGAACCGACGTACAAGCCTGGATTCGCGGATGCCGTCTGGGATCCGGTCGTGCAACCACA GGTGGTAATTGGACAACCCGTGCCGGTGGTCATCACCATCATCACCATTAA Ara h 2 (Adapted from Uniprot ID: Q6PSU2 / A5Z1Q9) (SEQ ID NO: 8) ATGGAGAAAAAAATCCGTCAGCAGTGGGAATTACAAGGAGACCGCCGCTGCCAAAGTCA ACTGGAGCGCGCCAATCTGCGTCCCTGCGAGCAGCATTTAATGCAGAAGATCCAGCGTGA CGAGGATTCGTACGGGCGTGACCCCTATTCTCCGTCTCAGGACCCATATTCGCCGTCACAA GACCCTGATCGTCGCGATCCGTATAGCCCTAGCCCGTATGACCGCCGTGGAGCGGGTAGC TCACAACACCAAGAGCGTTGTTGTAATGAATTGAATGAGTTCGAGAACAACCAGCGTTGT ATGTGTGAGGCTTTGCAGCAAATCATGGAGAACCAGTCTGATCGCCTTCAAGGACGTCAG Page 50 QB\702581.02521\90543975.1CAAGAGCAGCAATTCAAACGCGAACTGCGTAATCTGCCGCAACAGTGTGGTCTTCGTGCA CCGCAGCGTTGTGACTTAGAGGTTGAATCTGGAGGACGTGATCGTTATGGATCCGGTCGT GCAACCACAGGTGGTAATTGGACAACCCGTGCCGGTGGTCATCACCATCATCACCATTAA Fel d 1 (Adapted from Uniprot ID: P30440 + P30438) (SEQ ID NO: 9) ATGGAGAAAAAAATCGTTAAGATGGCCGAAACCTGCCCGATCTTTTACGACGTCTTTTTCG CTGTAGCGAACGGAAACGAATTGTTATTAGACCTGTCATTGACCAAAGTGGGGGCAACGG AACCTGAACGCACTGCGATGAAAAAAATCCAGGACTGCTATGTGGAAAACGGTCTGATTT CGCGCGTCCTTGATGGGCTTGTTATGACAACTATTAGCTCCAGTAAAGACTGTATGGGAGA AGCTGTTCAGAACACGGTAGAAGACTTGAAGTTGAATACCCTGGGACGTGAAATCTGTCC TGCCGTCAAACGCGATGTCGACTTGTTCTTGACGGGTACGCCCGATGAATATGTCGAACA GGTCGCTCAGTACAAGGCCCTTCCAGTAGTCTTGGAAAACGCCCGCATTCTGAAAAACTG TGTTGATGCTAAGATGACGGAAGAAGACAAGGAGAATGCTTTAAGTGTTTTGGATAAAAT CTACACATCTCCGCTGTGCGGATCCGGTCGTGCAACCACAGGTGGTAATTGGACAACCCG TGCCGGTGGTCATCACCATCATCACCATTAA Amb a 1 (Adapted from Uniprot ID: E1XUL2) (SEQ ID NO: 10) ATGGAGAAAAAAATCGCGGAGGATCTTCAAGAGATCCTTCCCGTTAACGAGACTCGTCGC TTGACGACATCGGGGGCGTATAATATCATCGACGGGTGCTGGCGTGGGAAAGCTGATTGG GCAGAAAACCGTAAGGCGCTGGCGGACTGCGCACAGGGGTTCGGTAAAGGCACTGTGGG CGGCAAGGACGGCGACATCTACACTGTTACGTCCGACTTGGACGACGATGTAGCCAACCC CAAGGAAGGCACGCTGCGTTTTGGTGCAGCGCAAAATCGTCCGCTGTGGATTATTTTCGA ACGCGACATGGTGATTCGTTTGGATAAAGAAATGGTAGTGAACTCGGATAAGACTATCGA CGGACGCGGGGCGAAGGTGGAGATTATCAATGCTGGATTTACGTTAAACGGTGTGAAGAA CGTGATTATTCACAATATCAATATGCACGACGTGAAGGTTAATCCTGGCGGATTGATTAAA AGCAACGATGGTCCGGCTGCCCCCCGTGCGGGGTCTGACGGGGACGCCATCTCAATTAGC GGAAGCTCTCAAATCTGGATTGATCACTGCAGTTTGTCTAAGAGCGTAGACGGCTTGGTA GATGCTAAGTTGGGCACGACGCGTCTTACCGTTTCGAATAGCTTGTTCACTCAACACCAAT TCGTACTTCTTTTTGGTGCTGGGGACGAAAATATCGAGGATCGTGGAATGCTGGCAACGGT AGCATTCAATACATTTACCGACAACGTGGATCAGCGTATGCCTCGCTGTCGTCATGGGTTT TTTCAAGTAGTTAATAACAACTATGACAAGTGGGGGAGCTACGCTATTGGCGGCTCGGCG TCACCAACTATCTTATCGCAGGGGAATCGTTTTTGTGCACCCGATGAGCGTTCAAAAAAGA ATGTTCTGGGACGTCATGGTGAGGCCGCTGCTGAGTCCATGAAGTGGAACTGGCGCACGA ACAAAGACGTGCTGGAGAACGGCGCCATTTTCGTCGCTTCAGGTGTGGACCCCGTGCTGA CACCAGAGCAAAGCGCCGGGATGATCCCAGCAGAGCCTGGGGAGAGTGCACTGAGTCTG ACCAGCAGCGCCGGCGTGCTGAGTTGCCAACCGGGTGCTCCCTGTGGATCCGGTCGTGCA ACCACAGGTGGTAATTGGACAACCCGTGCCGGTGGTCATCACCATCATCACCATTAA Bet v 1 (Adapted from Uniprot ID: O23748) (SEQ ID NO: 11) ATGGAGAAAAAAATCGAATTTGGCGTGTTCAACTATGAAACCGAAACCACCAGTGTTATT CCGGCAGCACGTCTGTTTAAAGCATTTATTCTGGATGGCGATAACCTGTTTCCGAAAGTTG CACCGCAGGCAATTAGCAGCGTTGAAAATATTGAAGGTAATGGTGGTCCGGGTACGATCA AAAAAATCAGCTTTCCGGAAGGTCTGCCGTTCAAATATGTTAAAGATCGTGTTGATGAAG TGGACCACACCAACTTCAAATATAACTATAGCGTGATTGAAGGTGGTCCGATTGGTGATA CCCTGGAAAAAATCTCCAACGAGATTAAAATCGTTGCAACACCGGATGGTGGTAGCATTC TGAAAATTAGCAACAAGTATCACACCAAAGGCGATCATGAAGTTAAAGCCGAACAGGTTA AAGCCAGCAAAGAAATGGGTGAAACCCTGCTGCGTGCAGTTGAAAGCTATCTGCTGGCAC ATAGTGATGCATATAATCTGCAAGGATCCGGTCGTGCAACCACAGGTGGTAATTGGACAA CCCGTGCCGGTGGTCATCACCATCATCACCATTAA Bla g 2 (Adapted from Uniprot ID: P54958) (SEQ ID NO: 12) ATGGAGAAAAAAATCGTTCCATTATATAAATTAGTTCACGTCTTTATTAATACCCAATATG CAGGCATTACCAAGATTGGTAACCAAAATTTTCTGACCGTTTTTGACAGTACGTCTTGCAA TGTAGTCGTGGCATCACAAGAATGTGTTGGCGGTGCTTGCGTATGCCCGAATTTGCAAAA Page 51 QB\702581.02521\90543975.1ATACGAAAAACTGAAACCTAAGTATATCAGTGATGGAAATGTCCAAGTTAAATTTTTTGA CACAGGATCAGCTGTGGGCCGTGGTATTGAGGATTCTTTGACAATTAGTGGTCTGACCACC TCACAGCAAGACATCGTATTAGCAGATGAGCTGAGCCAGGAAGTGTGCATCTTAAGCGCC GATGTGGTCGTAGGTATCGCTGCACCTGGCTGCCCCAACGCTCTGAAGGGAAAGACCGTT CTTGAAAACTTCGTCGAAGAAAACCTTATTGCACCCGTATTCTCTATTCACCACGCCCGCT TCCAAGATGGCGAGCATTTCGGTGAAATCATTTTTGGTGGGTCAGATTGGAAATATGTTGA TGGAGAGTTTACTTATGTGCCTCTGGTGGGGGACGATAGCTGGAAATTCCGCCTTGACGGT GTGAAAATTGGGGATACTACCGTTGCCCCGGCAGGTACCCAAGCCATCATCGACACTAGC AAAGCGATTATCGTCGGTCCGAAAGCGTACGTGAACCCCATCAACGAAGCAATCGGTTGC GTAGTAGAGAAGACCACGACACGCCGTATCTGTAAACTGGACTGCTCTAAGATTCCGTCC TTACCAGATGTCACCTTTGTCATTAATGGGCGTAACTTTGGCATTTCATCGCAGTACTATAT TCAGCAAAATGGAAATCTGTGCTACTCAGGATTTCAGCCTTGCGGCCACAGCGACCATTTC TTCATCGGGGACTTCTTTGTGGATCACTACTATTCTGAGTTTAACTGGGAGGGCAAAACCA TGGGTTTTGGCCGCTCCGTAGAAAGCGTAGGATCCGGTCGTGCAACCACAGGTGGTAATT GGACAACCCGTGCCGGTGGTCATCACCATCATCACCATTAA Cry j 1 (Adapted from Uniprot ID: P18632) (SEQ ID NO: 13) ATGGAGAAAAAAATCGACAACCCTATCGACAGTTGCTGGCGCGGAGATAGCAACTGGGCT CAGAATCGCATGAAGCTGGCTGATTGCGCCGTTGGCTTTGGTAGTTCAACCATGGGGGGC AAAGGGGGGGACCTTTACACAGTAACAAATAGTGACGACGATCCCGTTAACCCGGCGCCT GGGACCTTACGTTATGGTGCCACTCGTGATCGCCCTTTATGGATCATCTTCTCAGGAAATA TGAACATTAAATTGAAGATGCCAATGTATATTGCTGGATACAAAACATTCGATGGACGTG GAGCCCAGGTATATATTGGAAATGGAGGCCCATGTGTCTTTATTAAGCGCGTATCCAATGT GATCATCCATGGGTTATACTTGTATGGATGCAGTACAAGCGTGTTGGGGAACGTGTTAATT AATGAAAGTTTCGGCGTTGAACCTGTCCACCCCCAGGACGGAGATGCCCTTACACTGCGT ACAGCCACAAACATTTGGATCGATCACAATAGTTTCTCTGGATCATCAGATGGACTTGTAG ACGTGACTCTTACCTCGACAGGAGTGACCATTAGTAACAATCTGTTTTTTAACCACCATAA GGTAATGTTGTTAGGCCACGATGATGCATATAGCGATGATAAATCTATGAAAGTAACAGT TGCTTTTAACCAATTTGGACCGAACTGCGGTCAACGTATGCCCCGTGCACGCTACGGCTTG GTTCATGTTGCGAATAACAACTACGATCCTTGGACTATCTACGCTATCGGCGGATCGAGTA ATCCCACAATTCTGAGCGAAGGCAACAGCTTCACTGCTCCGGGGGAGTCCTATAAGAAGC AGGTTACCATCCGCATTGGCTGCAAAACCTCATCATCATGTAGCAATTGGGTGTGGCAAA GCACACAGGATGTTTTTTACAATGGGGCCTACTTCGTCAGTAGCGGCAAGTATGAGGGAG GCAACATTTATACAAAGAAAGAAGCGTTTAACGTAGAAAACGGAGGAGCCACTCCACAA TTGACGAAAAATGCAGGAGTCCTTACGTGCAGTTTAAGCAAACGTTGTGGATCCGGTCGT GCAACCACAGGTGGTAATTGGACAACCCGTGCCGGTGGTCATCACCATCATCACCATTAA

[0221]

[0222] Table 3: Allergen accession ID and polypeptide sequences Cat linker – Protein - BamHI linker – Glyctag - His tag Der p 1 (Adapted from Uniprot ID: P08176) (SEQ ID NO: 14) MEKKIRPSSIKTFEEYKKAFNKSYATFEDEEAARKNFLESVKYVQSNGGAINHLSDLSLDEFKN RFLMSAEAFEHLKTQFDLNAETNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVA ATESAYLAYRNQSLDLAEQELVDCASQHGCHGDTIPRGIEYIQHNGVVQESYYRYVAREQSC RRPNAQRFGISNYCQIYPPNVNKIREALAQTHSAIAVIIGIKDLDAFRHYDGRTIIQRDNGYQPN Page 52 QB\702581.02521\90543975.1YHAVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANIDLMMIEEYPYVVILGSGRATT GGNWTTRAGGHHHHHH Der p 2 (Uniprot ID: P49278) (SEQ ID NO: 15) MEKKIDQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGKPFQLEAVFEANQNTKTAKIEIKASID GLEVDVPGIDPNACHYMKCPLVKGQQYDIKYTWNVPKIAPKSENVVVTVKVMGDDGVLAC AIATHAKIRDGSGRATTGGNWTTRAGGHHHHHH Bos d 5 (Adapted from Uniprot ID: P02754) (SEQ ID NO: 16) MEKKILIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSAPLRVYVEELKPTPEGDLEILL QKWENGECAQKKIIAEKTKIPAVFKIDALNENKVLVLDTDYKKYLLFCMENSAEPEQSLACQC LVRTPEVDDEALEKFDKALKALPMHIRLSFNPTQLEEQCHIGSGRATTGGNWTTRAGGHHHH HH Bos d 4 (Adapted from Uniprot ID: P00711) (SEQ ID NO: 17) MEKKIEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINN KIWCKDDQNPHSSNICNISCDKFLDDDLTDDIMCVKKILDKVGINYWLAHKALCSEKLDQWL CEKLGSGRATTGGNWTTRAGGHHHHHH Gal d 1 (Adapted from Uniprot ID: P01005) (SEQ ID NO: 18) MEKKIAEVDCSRFPGATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTGISKEH DGECKETVPMGCSSYAGTTSEDGKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQGASV DKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESGGTLTLS HFGKCGSGRATTGGNWTTRAGGHHHHHH Gal d 2 (Adapted from Uniprot ID: P01012) (SEQ ID NO: 19) MEKKISTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYA EERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTA MVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILEL PFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTS VLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFR ADHPFLFCIKHIATNAVLFFGRCVSPGSGRATTGGNWTTRAGGHHHHHH Gal d 4 (Adapted from Uniprot ID: P00698) (SEQ ID NO: 20) MEKKIKVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGIL QINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKG TDVQAWIRGCRLGSGRATTGGNWTTRAGGHHHHHH Ara h 2 (Adapted from Uniprot ID: Q6PSU2 / A5Z1Q9) (SEQ ID NO: 21) MEKKIRQQWELQGDRRCQSQLERANLRPCEQHLMQKIQRDEDSYGRDPYSPSQDPYSPSQDP DRRDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQRCMCEALQQIMENQSDRLQGRQQEQQ FKRELRNLPQQCGLRAPQRCDLEVESGGRDRYGSGRATTGGNWTTRAGGHHHHHH Fel d 1 (Adapted from Uniprot ID: P30440 + P30438) (SEQ ID NO: 22) MEKKIVKMAETCPIFYDVFFAVANGNELLLDLSLTKVGATEPERTAMKKIQDCYVENGLISRV LDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGREICPAVKRDVDLFLTGTPDEYVEQVAQY KALPVVLENARILKNCVDAKMTEEDKENALSVLDKIYTSPLCGSGRATTGGNWTTRAGGHHH HHH Amb a 1 (Adapted from Uniprot ID: E1XUL2) (SEQ ID NO: 23) MEKKIAEDLQEILPVNETRRLTTSGAYNIIDGCWRGKADWAENRKALADCAQGFGKGTVGG KDGDIYTVTSDLDDDVANPKEGTLRFGAAQNRPLWIIFERDMVIRLDKEMVVNSDKTIDGRG AKVEIINAGFTLNGVKNVIIHNINMHDVKVNPGGLIKSNDGPAAPRAGSDGDAISISGSSQIWID HCSLSKSVDGLVDAKLGTTRLTVSNSLFTQHQFVLLFGAGDENIEDRGMLATVAFNTFTDNV DQRMPRCRHGFFQVVNNNYDKWGSYAIGGSASPTILSQGNRFCAPDERSKKNVLGRHGEAA Page 53 QB\702581.02521\90543975.1AESMKWNWRTNKDVLENGAIFVASGVDPVLTPEQSAGMIPAEPGESALSLTSSAGVLSCQPG APCGSGRATTGGNWTTRAGGHHHHHH Bet v 1 (Adapted from Uniprot ID: O23748) (SEQ ID NO: 24) MEKKIEFGVFNYETETTSVIPAARLFKAFILDGDNLFPKVAPQAISSVENIEGNGGPGTIKKISFP EGLPFKYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLEKISNEIKIVATPDGGSILKISNKYHTKG DHEVKAEQVKASKEMGETLLRAVESYLLAHSDAYNLQGSGRATTGGNWTTRAGGHHHHHH Bla g 2 (Adapted from Uniprot ID: P54958) (SEQ ID NO: 25) MEKKIVPLYKLVHVFINTQYAGITKIGNQNFLTVFDSTSCNVVVASQECVGGACVCPNLQKYE KLKPKYISDGNVQVKFFDTGSAVGRGIEDSLTISGLTTSQQDIVLADELSQEVCILSADVVVGIA APGCPNALKGKTVLENFVEENLIAPVFSIHHARFQDGEHFGEIIFGGSDWKYVDGEFTYVPLVG DDSWKFRLDGVKIGDTTVAPAGTQAIIDTSKAIIVGPKAYVNPINEAIGCVVEKTTTRRICKLDC SKIPSLPDVTFVINGRNFGISSQYYIQQNGNLCYSGFQPCGHSDHFFIGDFFVDHYYSEFNWEGK TMGFGRSVESVGSGRATTGGNWTTRAGGHHHHHH Cry j 1 (Adapted from Uniprot ID: P18632) (SEQ ID NO: 26) MEKKIDNPIDSCWRGDSNWAQNRMKLADCAVGFGSSTMGGKGGDLYTVTNSDDDPVNPAP GTLRYGATRDRPLWIIFSGNMNIKLKMPMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIH GLYLYGCSTSVLGNVLINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSGSSDGLVDVTLTST GVTISNNLFFNHHKVMLLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGLVHVANN NYDPWTIYAIGGSSNPTILSEGNSFTAPGESYKKQVTIRIGCKTSSSCSNWVWQSTQDVFYNGA YFVSSGKYEGGNIYTKKEAFNVENGGATPQLTKNAGVLTCSLSKRCGSGRATTGGNWTTRAG GHHHHHH

[0223] References Cited in Example 1

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[0267] 36. Jewett, M. C., and Swartz, J. R. (2004) Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis, Biotechnol Bioeng 86, 19-26.

[0268] 37. Jewett, M. C., Calhoun, K. A., Voloshin, A., Wuu, J. J., and Swartz, J. R. (2008) An integrated cell-free metabolic platform for protein production and synthetic biology, Mol Syst Biol 4, 220.

[0269] 38. Mueller, G. A., Glesner, J., Daniel, J. L., Zhang, J., Hyduke, N., Richardson, C. M., DeRose, E. F., Chapman, M. D., Peebles, R. S., Jr., S, A. S., and Pomes, A. (2020) Mapping Human Monoclonal IgE Epitopes on the Major Dust Mite Allergen Der p 2, J Immunol 205, 1999- 2007.

[0270] 39. Grutzkau, A., Smorodchenko, A., Lippert, U., Kirchhof, L., Artuc, M., and Henz, B. M. (2004) LAMP-1 and LAMP-2, but not LAMP-3, are reliable markers for activation- induced secretion of human mast cells, Cytometry A 61, 62-68.

[0271] 40. Radinger, M., Jensen, B. M., Kuehn, H. S., Kirshenbaum, A., and Gilfillan, A. M. (2010) Generation, isolation, and maintenance of human mast cells and mast cell lines derived from peripheral blood or cord blood, Curr Protoc Immunol Chapter 7, Unit 737.

[0272] 41. Zhernov, Y., Curin, M., Khaitov, M., Karaulov, A., and Valenta, R. (2019) Recombinant allergens for immunotherapy: state of the art, Curr Opin Allergy Clin Immunol 19, 402-414.

[0273] 42. Eisenbarth, S. C., Piggott, D. A., Huleatt, J. W., Visintin, I., Herrick, C. A., and Bottomly, K. (2002) Lipopolysaccharide-enhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen, J Exp Med 196, 1645-1651.

[0274] 43. Tsuchiya, K., Siddiqui, S., Risse, P. A., Hirota, N., and Martin, J. G. (2012) The presence of LPS in OVA inhalations affects airway inflammation and AHR but not remodeling in a rodent model of asthma, Am J Physiol Lung Cell Mol Physiol 303, L54-63.

[0275] 44. Martin, R. W., Des Soye, B. J., Kwon, Y. C., Kay, J., Davis, R. G., Thomas, P. M., Majewska, N. I., Chen, C. X., Marcum, R. D., Weiss, M. G., Stoddart, A. E., Amiram, M., Ranji Page 60 QB\702581.02521\90543975.1Charna, A. K., Patel, J. R., Isaacs, F. J., Kelleher, N. L., Hong, S. H., and Jewett, M. C. (2018) Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids, Nat Commun 9, 1203.

[0276] 45. Des Soye, B. J., Gerbasi, V. R., Thomas, P. M., Kelleher, N. L., and Jewett, M. C. (2019) A Highly Productive, One-Pot Cell-Free Protein Synthesis Platform Based on Genomically Recoded Escherichia coli, Cell Chem Biol 26, 1743-1754 e1749.

[0277] 46. Swartz, J. R., Jewett, M. C., and Woodrow, K. A. (2004) Cell-Free Protein Synthesis With Prokaryotic Combined Transcription-Translation, In Methods in Molecular Biology (Balbás, P., and Lorence, A., Eds.), pp 169-182, Humana Press, Totowa, NJ.

[0278] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0279] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0280] Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an Page 61 QB\702581.02521\90543975.1inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification. Page 62 QB\702581.02521\90543975.1

Claims

CLAIMS We claim:

1. A cell-free protein synthesis system for in vitro production of one or more allergen proteins, comprising: one or more expression templates, wherein each of the one or more expression templates comprises 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.

2. The system of claim 1, wherein the polynucleotide sequence comprises a sequence at least 80 %, 85 %, 90 %, 95 % or 99 % identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

3. The system of claim 1 or 2, wherein the one or more cell-free synthesis reagents comprises a lysate prepared from a prokaryote.

4. The system of claim 3, wherein the prokaryote is E. coli.

5. The system of claim 4, wherein the lysate was prepared from genetically modified E. coli, wherein the genetic modification comprises a genetic modification to facilitate disulfide bond formation.

6. The system of any one of claims 1-5, wherein the one or more expression templates and / or the one or more cell-free synthesis reagents is in a freeze-dried format.

7. The system of any one of claims 1-6, wherein the one or more cell-free synthesis reagents comprises one or more of ATP, GTP, UTP, CTP, or a tRNA mixture from E. coli. Page 63 QB\702581.02521\90543975.

18. A bacterial cell that comprises one or more expression templates, wherein each of the one or more expression templates comprise 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.

9. The bacterial cell of claim 8, wherein the polynucleotide sequence comprises a sequence at least 80 %, 85 %, 90 %, 95 % or 99 % identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

10. The bacterial cell of claim 8 or 9, wherein the bacterial cell is genetically modified to facilitate disulfide bond formation.

11. A lysate prepared from the bacterial cell of any one of claims 8-10.

12. The lysate of claim 11, wherein the lysate is freeze-dried.

13. A method for preparing one or more allergens in a cell-free protein synthesis system, comprising: adding one or more expression templates to one or more cell-free protein synthesis reagents, wherein each of the one or more expression templates comprises 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.

14. The method of claim 13, wherein the polynucleotide sequence comprises a sequence at least 80 %, 85 %, 90 %, 95 % or 99 % identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

15. The method of claim 13 or 14, wherein the one or more cell-free synthesis reagents comprises a lysate prepared from a prokaryote. Page 64 QB\702581.02521\90543975.

116. The method of claim 15, wherein the prokaryote is E. coli.

17. The method of claim 16, wherein the lysate was prepared from genetically modified E. coli, wherein the genetic modification comprises a genetic modification to facilitate disfulfide bond formation.

18. The method of any one of claims 13-17, wherein the one or more expression templates and / or the one or more cell-free synthesis reagents was freeze-dried prior to the adding one or more expression templates to one or more cell-free protein synthesis reagents.

19. The method of any one of claims 13-18, wherein the one or more cell-free synthesis reagents comprises one or more of ATP, GTP, UTP, CTP, or a tRNA mixture from E. coli.

20. The method of any one of claims 13-19, further comprising purifying the one or more allergen proteins.

21. The method of claim 20, further comprising, subsequent to the purifying, quantifying the one or more allergen proteins.

22. The method of claim 21, wherein the one or more allergen proteins comprise14C.

23. The method of claim 22, wherein the one or more allergen proteins comprise14C- leucine.

24. The method of claim 20, further comprising exposing the one or more allergen proteins to human IgE to detect binding of the one or more allergens to the human IgE.

25. The method of claim 24, wherein the exposing the one or more allergen proteins to human IgE to detect binding of the one or more allergen proteins to the human IgE is performed using one or more of: an AlphaLISA assay, an ELISA, surface plasmon resonance, or biolayer interferometry. Page 65 QB\702581.02521\90543975.

126. The method of claim 20, further comprising evaluating allergen function of the one or more allergen proteins by: a basophil activation test with primary human basophils, flow analysis of passively sensitized CD34+ progenitor-derived primary human mast cells, or both.

27. A method, comprising: testing for allergies in a subject by exposing the one or more allergen proteins prepared according to the method of any one of claims 13-20, 78 to the subject or a sample from the subject.

28. The method of claim 27, wherein the exposing comprises a skin prick test.

29. The method of claim 27, wherein the exposing comprises quantifying allergen- specific IgE present in the sample from the subject, e.g., using an ELISA.

30. The method of claim 27, wherein the exposing comprises utilizing a lateral flow assay on the sample from the subject.

31. A method, comprising: administering the one or more allergen proteins prepared according to the method of any one of claims 13-20, 78 to a subject.

32. The method of claim 31, wherein the administering is subcutaneous, sublingual, or oral.

33. The method of claim 31, wherein the one or more allergen proteins are formulated into one or more food products.

34. The system of any one of claims 1-7, wherein the polynucleotide sequence further comprises one or more elements for a purification tag, immune-modulating elements, sites for post-translational modifications, designer epitopes, expression aiding elements, elements that aid in folding and / or assembly. Page 66 QB\702581.02521\90543975.

135. The system of any of claims 1-7 and 34, wherein the expression template is present in a plasmid vector.

36. The method of claim 24, wherein the exposing the one or more allergen proteins to human IgE to detect binding of the one or more allergen proteins to the human IgE is performed using a point-of-care testing device.

37. The method of claim 27, wherein the exposing comprises utilizing a point-of-care assay on the sample from the subject.

38. A method of testing for allergies in a subject comprising performing a point-of-care method using the one or more allergen proteins prepared according to the method of any one of claims 13-20, 78 on a sample from the subject at a point-of-care location.

39. A cell-free protein synthesis system for in vitro production of one or more allergen proteins, comprising: one or more expression templates, wherein each of the one or more expression templates comprises 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.

40. The system of claim 39, wherein each of the one or more expression templates comprises 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 / or wherein the polynucleotide sequence comprises a sequence at least 80 %, 85 %, 90 %, 95 % or 99 % identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

41. The system of claim 39 or 40, wherein the one or more cell-free synthesis reagents comprises a lysate prepared from a prokaryote. Page 67 QB\702581.02521\90543975.

142. The system of claim 41, wherein the prokaryote is E. coli.

43. The system of claim 42, wherein the lysate was prepared from genetically modified E. coli, wherein the genetic modification comprises a genetic modification to facilitate disulfide bond formation.

44. The system of any one of claims 39-43, wherein the one or more expression templates and / or the one or more cell-free synthesis reagents is in a freeze-dried format.

45. The system of any one of claims 39-44, wherein the one or more cell-free synthesis reagents comprises one or more of ATP, GTP, UTP, CTP, or a tRNA mixture from E. coli.

46. A bacterial cell that comprises one or more expression templates, wherein each of the one or more expression templates comprise a polynucleotide sequence for expression of one or more allergen proteins.

47. The bacterial cell of claim 46, wherein each of the one or more expression templates comprise 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, or wherein the polynucleotide sequence comprises a sequence at least 80 %, 85 %, 90 %, 95 % or 99 % identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

48. The bacterial cell of claim 46 or 47, wherein the bacterial cell is genetically modified to facilitate disulfide bond formation.

49. A lysate prepared from the bacterial cell of any one of claims 46-48.

50. The lysate of claim 49, wherein the lysate is freeze-dried. Page 68 QB\702581.02521\90543975.

151. A method for preparing one or more allergens in a cell-free protein synthesis system, comprising: adding one or more expression templates to one or more cell-free protein synthesis reagents, wherein each of the one or more expression templates comprises a polynucleotide sequence for expression of one or more allergen proteins.

52. The method of claim 51, wherein each of the one or more expression templates comprises 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, or wherein the polynucleotide sequence comprises a sequence at least 80 %, 85 %, 90 %, 95 % or 99 % identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

53. The method of claim 51 or 52, wherein the one or more cell-free synthesis reagents comprises a lysate prepared from a prokaryote.

54. The method of claim 53, wherein the prokaryote is E. coli.

55. The method of claim 54, wherein the lysate was prepared from genetically modified E. coli, wherein the genetic modification comprises a genetic modification to facilitate disfulfide bond formation.

56. The method of any one of claims 51-55, wherein the one or more expression templates and / or the one or more cell-free synthesis reagents was freeze-dried prior to the adding one or more expression templates to one or more cell-free protein synthesis reagents.

57. The method of any one of claims 51-56, wherein the one or more cell-free synthesis reagents comprises one or more of ATP, GTP, UTP, CTP, or a tRNA mixture from E. coli.

58. The method of any one of claims 51-57, further comprising purifying the one or more allergen proteins. Page 69 QB\702581.02521\90543975.

159. The method of claim 58, further comprising, subsequent to the purifying, quantifying the one or more allergen proteins.

60. The method of claim 59, wherein the one or more allergen proteins comprise14C.

61. The method of claim 60, wherein the one or more allergen proteins comprise14C- leucine.

62. The method of claim 58, further comprising exposing the one or more allergen proteins to human IgE to detect binding of the one or more allergens to the human IgE.

63. The method of claim 62, wherein the exposing the one or more allergen proteins to human IgE to detect binding of the one or more allergen proteins to the human IgE is performed using one or more of: an AlphaLISA assay, an ELISA, surface plasmon resonance, or biolayer interferometry.

64. The method of claim 58, further comprising evaluating allergen function of the one or more allergen proteins by: a basophil activation test with primary human basophils, flow analysis of passively sensitized CD34+ progenitor-derived primary human mast cells, or both.

65. A method, comprising: testing for allergies in a subject by exposing the one or more allergen proteins prepared according to the method of any one of claims 51-58, 80 to the subject or a sample from the subject.

66. The method of claim 65, wherein the exposing comprises a skin prick test.

67. The method of claim 65, further comprising quantifying allergen-specific IgE present in the sample from the subject, e.g., using an ELISA. Page 70 QB\702581.02521\90543975.

168. The method of claim 65, wherein the exposing comprises utilizing a lateral flow assay on the sample from the subject.

69. A method, comprising: administering the one or more allergen proteins prepared according to the method of any one of claims 51-58, 80 to a subject.

70. The method of claim 69, wherein the administering is subcutaneous, sublingual, or oral.

71. The method of claim 69, wherein the one or more allergen proteins are formulated into one or more food products.

72. The system of any one of claims 39-45, wherein the polynucleotide sequence further comprises one or more elements for a purification tag, immune-modulating elements, sites for post-translational modifications, designer epitopes, expression aiding elements, elements that aid in folding and / or assembly.

73. The system of any of claims 39-45 and 72, wherein the expression template is present in a plasmid vector.

74. The method of claim 62, wherein the exposing the one or more allergen proteins to human IgE to detect binding of the one or more allergen proteins to the human IgE is performed using a point-of-care testing device.

75. The method of claim 65, wherein the exposing comprises utilizing a point-of-care assay on the sample from the subject.

76. A method of testing for allergies in a subject comprising performing a point-of-care method using the one or more allergen proteins prepared according to the method of any one of claims 51-58 on a sample from the subject at a point-of-care location. Page 71 QB\702581.02521\90543975.

177. The system of claim 1 or 2, wherein the one or more cell-free synthesis reagents comprises a lysate prepared from a eukaryote.

78. The method of claim 13 or 14, wherein the one or more cell-free synthesis reagents comprises a lysate prepared from a eukaryote.

79. The system of claim 39 or 40, wherein the one or more cell-free synthesis reagents comprises a lysate prepared from a eukaryote.

80. The method of claim 51 or 52, wherein the one or more cell-free synthesis reagents comprises a lysate prepared from a eukaryote.

81. The system of claim 2 or claim 40, wherein the one or more allergen proteins comprise or consist of an amino acid sequence that is at least 80 %, 85 %, 90 %, 95 % or 99 % identical to SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.

82. The method of caim 9 or 47, wherein the one or more allergen proteins comprise or consist of an amino acid sequence that is at least 80 %, 85 %, 90 %, 95 % or 99 % identical to SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.

83. The method of claim 14 or 52, wherein the one or more allergen proteins comprise or consist of an amino acid sequence that is at least 80 %, 85 %, 90 %, 95 % or 99 % identical to SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26. Page 72 QB\702581.02521\90543975.1ABSTRACT OF THE DISCLOSURE are components, systems, and methods for synthesis of allergen proteins. The components, systems, and methods disclosed herein may be used in synthesizing allergen proteins in a cell-free protein synthesis (CFPS) system. Page 73 QB\702581.02521\90543975.1

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