Hypoallergenic peanut allergen, its preparation method and use

Abstract

In one embodiment, the present disclosure provides hypoallergenic peanut allergen Arahl or Arahl2 variants, which lack at least one epitope recognized by anti-Arahl or anti-Arahl2 antibodies, resulting in reduced or abolished antibody binding to the peanut allergen variants. Such hypoallergenic peanut allergen variants can be used in methods of inducing hyposensitization to peanut in subjects (patients) allergic to peanut.

Description

[Technical field]

[0001] (Sequence Listing Statement) This application contains a Sequence Listing that has been submitted electronically in XML format, the entirety of which is incorporated herein by reference. The XML copy, created on July 29, 2022, is named "P-605376-PC_SL.xml" and is 421.4 kilobytes in size.

[0002] (Technical field) The present disclosure relates generally to recombinant hypoallergenic peanut allergens Arahl or Arahl2, methods for their production, and uses thereof. [Background technology]

[0003] One of the most severe food allergies currently known is peanut allergy, and allergic individuals can experience symptoms ranging from mild localized effects to severe, life-threatening effects when exposed to even low concentrations of peanut. Peanut is the leading cause of death from food-induced anaphylactic shock in the United States (Finkelman, (2010) Current Opinion in Immunology, 22(6):783-788), and some form of allergic reaction to peanuts has been reported in approximately 1% of the U.S. population (Sicherer SH, et al., (2010). J Allergy Clin Immunol. 125(6):1322-6).

[0004] To date, 16 peanut proteins have been identified as proteins that cause IgE-mediated allergic reactions (Palladino, C., & Breiteneder, H. (2018). Molecular immunology, 100:58-70). Among these proteins, the seed storage proteins Arah1, Arah2, Arah3, and Arah6 are considered to be the major allergens, and when recognized by IgE antibody-mediated responses, they correlate with more severe symptoms (Palladino, et al., 2018; ibid) (Bernard, et al., (2007) J Agric Food Chem. 55(23):9663-9). Of the 16 peanut allergens, AraH2 is considered the most important because it is recognized by approximately 75-80% of serum IgE in 3-6 year old American children (Valcour, et. al., Ann Allergy Asthma Immunol 119 (2017)) (Koppelman et al., (2004). Clin Exp Allergy. 34(4):583-90). AraH2 is a 17 kD monomeric polypeptide and a member of the 2S albumin family, which belongs to the prolamin protein superfamily (Lehmann K, Schweimer K, Reese G, Randow S, Suhr M, Becker WM, et al. (2006) Biochem J.395(3):463-72). Arah2 accounts for 6-10% of the total protein in peanut extracts (Koppelman, SJ, et al. (2001) Allergy 56:2). Arah2 directly induces sensitization via the gastrointestinal tract. The core structure of Arah2 is highly resistant to proteolysis due to a highly stable structure generated from well-conserved cystines that form disulfide bonds. Comparison of folded and unfolded versions of Arah2 revealed that IgE antibodies recognize both linear and conformational epitopes.These are bound by sera only when tested against the folded protein (Bernard et al., (2015) J Allergy Clin Immunol. 135(5):1267-74.e1-8).

[0005] Arah1 is a 63 kDa peanut seed protein that accounts for 12-16% of the total protein in peanut extracts. Arah1 is a heat-stable 7S vicilin-like globulin with a stable homotrimer (Pomes et al. (2003) The Journal of Allergy and Clinical Immunology. 111 (3): 640-5). Arah1 begins as a preproprotein and after two rounds of endoproteolytic cleavage, becomes the mature form found in peanut. The mature form has a flexible region and a core region. The crystal structure of the Arah1 core (residues 170-586) (3S7I.pdb; 3SMH.pdb) shows that the central portion of the allergen has a bicupin fold. Previously, IgE-binding linear epitopes have been mapped to Arahl, and substitution of just one amino acid per epitope abolished IgE binding (Burks et al. (1997). Eur J Biochem 1997; 245(2):334-9). However, conformational epitopes on the heat-stable trimer surface have been less studied.

[0006] Besides completely avoiding exposure to allergens, treatments to control the patient's exposure to increasing amounts of each allergen (i.e., immunotherapy (IT)) have been used. The focus of immunotherapy (IT) is to effectively reduce the exposure to the allergen by increasing the amount of allergens that do not cause an allergic reaction, while potentially preventing allergic reactions due to the ingestion of the allergen by re-educating the immune system to deal with the allergen. Currently, immunotherapy is performed in outpatient clinics. In recent years, companies have developed products standardized with peanut extracts, providing a safer treatment regimen that can be used at home.

[0007] There remains a need for hypoallergenic peanut proteins and methods of use thereof for standardized immunotherapy treatment in patients with allergies to peanut allergens. Summary of the Invention [Means for solving the problem]

[0008] Described herein are several epitope mapping methods for designing hypoallergenic peanut allergens that maintain biophysical and functional properties, e.g., for the generation of Arahl or Arahl2 allergen variants. In one aspect, the disclosure provides hypoallergenic peanut allergen Arahl or Arahl2 variants that lack at least one epitope recognized by anti-Arahl or anti-Arahl2 antibodies, resulting in reduced or abolished antibody binding to the peanut allergen variant.

[0009] In one aspect, the present disclosure provides a recombinant Arah2 variant polypeptide comprising an amino acid sequence having at least 50% identity to the sequence set forth in SEQ ID NO:3, and comprising one or more amino acid substitutions, deletions, insertions, or any combination thereof located within a single epitope recognized by an anti-Arah2 antibody. In another embodiment, the recombinant Arah2 variant polypeptide of the present disclosure comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof located within at least two epitopes recognized by an anti-Arah2 antibody.

[0010] In one embodiment, a recombinant Arah2 variant polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:4, and includes one or more substitutions, deletions, insertions, or any combination thereof, at one or more of positions 12, 15, 16, 22, 24, 46, 53, 65, 80, 83, 86, 87, 90, 104, 115, 123, 127, and 140 of SEQ ID NO:4, compared to the amino acid residues at the same positions in SEQ ID NO:3. (a) N, Q, E, D, T, S, G, P, C, K, H, Y, W, M, I, L, V, or A at position 12; (b) R, E, K, Y, W, F, M, I, V, C, D, G, or A at position 15; (c) R, K, D, Q, T, M, P, C, E, or W at position 16; (d) at position 22, F, Y, W, Q, E, T, S, A, M, I, L, C, R, or H; (e) D, E, H, K, S, T, N, Q, L, I, M, W, Y, F, P, A, or G at position 24; (f) T, V, E, H, S, A, G, Q, N, D, R, P, M, I, L, or C at position 46; (g) T, S, Q, V, A, G, C, P, M, L, I, E, H, R, K, N, or D at position 53; (h) T, A, N, D, Q, R, K, H, I, L, M, V, W, P, G, C, or E at position 65; (i) N, S, T, V, A, I, L, M, F, Y, W, C, E, K, R, or G at position 80; (j) D, A, C, F, I, P, T, V, W, Y, or Q at position 83; (k) Y, F, H, R, E, C, G, I, L, M, V, T, S, or Q at position 86; (l) F, Y, I, L, M, V, A, S, Q, R, K, D, N, E, or P at position 87; (m) at position 90, S, P, Q, or R; (n) L, M, K, R, H, E, D, A, Y, N, S, or W at position 104; (o) V, D, E, I, L, K, M, N, S, T, A, I, W, F, Y, or H at position 115; (p) I, Q, or A at position 123; (q) H, A, D, E, F, G, L, N, P, S, T, W, Y, Q, or V at position 127; or (r) G, A, C, E, Y, F, H, K, L, M, N, P, Q, S, or V at position 140; Contains one or more of the following substitutions:

[0011] In one embodiment, a recombinant Arah2 variant polypeptide of the present disclosure further comprises an additional substitution, deletion, insertion, or any combination thereof, at one or more of positions 28, 44, 48, 51, 55, 63, 67, 107, 108, 109, 124, 125, and 142 of SEQ ID NO:4, compared to the amino acid residues at the same positions in SEQ ID NO:3. (a) S, T, V, N, A, P, I, L, F, Y, H, R, K, E, or D at position 28; (b) I, A, C, G, H, L, F, Y, N, P, Q, K, E, S, T, V, M, or R at position 44; (c) V, G, C, E, H, Q, F, K, L, I, W, Y, N, R, S, T, V, A, or D at position 48; (d) S, G, Y, F, W, M, N, Q, E, R, K, H, T, D, or V at position 51; (e) G, A, D, E, F, Y, H, Q, V, I, L, M, R, K, S, T, C, or W at position 55; (f) P, C, F, V, I, L, M, W, Y, N, S, T, Q, G, H, K, or R at position 63; (g) E, Q, N, R, H, Y, F, W, M, L, V, T, S, A, P, or G at position 67; (h) A, C, F, G, H, I, K, L, M, Q, P, R, S, T, V, W, or Y at position 107; (i) T, V, D, E, R, H, Y, W, I, G, A, Q, or K at position 108; (j) K, C, S, R, G, P, Y, W, L, or I at position 109; (k) D, A, C, F, G, H, I, N, S, T, V, Y, L, E, or Q at position 124; (l) M, I, L, W, Y, G, K, N, T, V, or A at position 125; or (m) M, A, C, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 142; Contains one or more of the following substitutions:

[0012] In one embodiment, a recombinant Arah2 variant polypeptide of the disclosure comprises an amino acid sequence set forth in any of SEQ ID NOs: 10-63, 168, 170, 195-201, 204-210, or 247-249, or an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any of SEQ ID NOs: 10-63, 168, 170, 195-201, 204-210, or 247-249.

[0013] The present disclosure also provides a nucleotide sequence encoding any one of the recombinant Arah2 mutant polypeptides of the present disclosure, an expression vector comprising the nucleotide sequence, and a cell comprising the expression vector. The present disclosure also provides a method for producing the recombinant Arah2 mutant polypeptide of the present disclosure using the expression vector.

[0014] In one embodiment, the present disclosure provides a recombinant Arah1 variant polypeptide comprising an amino acid sequence having at least 50% identity to the sequence set forth in SEQ ID NO: 65, and comprising one or more amino acid substitutions, deletions, insertions, or any combination thereof located within a single epitope recognized by an anti-Arahl antibody. In another embodiment, the recombinant Arah1 variant polypeptide of the present disclosure comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof located within at least two epitopes recognized by an anti-Arahl antibody.

[0015] In one embodiment, a recombinant Arah1 variant polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:67, and comprises one or more substitutions, deletions, insertions, or any combination thereof, at one or more of positions 194, 195, 213, 215, 231, 234, 245, 267, 287, 294, 312, 331, 419, 422, 443, 455, 462, 463, 464, 480, 494, and 500 of SEQ ID NO:67, compared to the amino acid residues at the same positions in SEQ ID NO:65. (a) at position 194, D; (b) A at position 194; (c) at position 213, H; (d) R, D, L, I, F, or A at position 215; (e) A at position 231; (f) E at position 234; (g) R at position 245; (h) E at position 267; (i) D at position 287; (j) E at position 294; (k) at position 312, A or H; (l) H at position 331; (m) E, V, or A at position 419; (n) at position 422, R or A; (o) A at position 443; (p) at position 455, A; (q) A, K, or T at position 462; (r) S at position 463; (s) A or S at position 464; (t) Q at position 480; (u) A, E, or N at position 494; or (v) at position 500, K; Contains one or more of the following substitutions:

[0016] In one embodiment, a recombinant Arah1 variant polypeptide of the present disclosure further comprises an additional substitution, deletion, insertion, or any combination thereof at one or more of positions 12, 24, 27, 30, 42, 57, 58, 73, and 523 of SEQ ID NO: 67, compared to the amino acid residues at the same positions in SEQ ID NO: 65. In one embodiment, a recombinant Arah1 variant polypeptide of the present disclosure further comprises (a) at position 12, K or A; (b) at position 24, V or E; (c) at position 27, A or H; (d) at position 30, E or A; (e) at position 42, L or K; (f) at position 57, D or L; (g) at position 58, S or R; (h) at position 73, A or M; or (i) at position 523, A or K; Contains one or more of the following substitutions:

[0017] In one embodiment, a recombinant Arah1 variant polypeptide of the present disclosure further comprises an additional substitution, deletion, insertion, or any combination thereof at one or more of positions 87, 88, 96, 99, 196, 197, 200, 209, 238, 249, 260, 261, 263, 265, 266, 278, 283, 288, 290, 295, 318, 322, 334, 336, 378, 417, 421, 441, 443, 481, 484, 485, 487, 488, and 491 of SEQ ID NO: 67, compared to the amino acid residues at the same positions in SEQ ID NO: 65. In one embodiment, a recombinant Arah1 variant polypeptide of the present disclosure further comprises (a) A at position 87; (b) A at position 88; (c) A at position 96; (d) A at position 99; (e) at position 196, H; (f) A at position 197; (g) at position 200, V, A, or Q; (h) S at position 209; (i) Q at position 238; (j) N at position 249; (k) at position 260, K; (l) R at position 261; (m) at position 263, K or L; (n) at position 265, S; (o) at position 266, R or L; (p) at position 278, R; (q) E at position 283; (r) Q at position 288; (s) R at position 290; (t) A at position 295; (u) at position 318, H; (v) at position 322, A or K; (w) D, A, or N at position 334; (x) at position 336, R or S; (y) K or E at position 378; (z) R at position 417; (aa) at position 421, E or S; (bb) N at position 441; (cc) A at position 443; (dd) at position 481, A or S; (ee) R, S, A, or M at position 484; (ff) A at position 485; (gg) at position 487, S or K; (hh) A at position 488; or (ii) A, S, or E at position 491; Contains one or more of the following substitutions:

[0018] In one embodiment, the recombinant Arah1 mutant polypeptide of the present disclosure further comprises an A substitution at position 84 of SEQ ID NO:67.

[0019] In one embodiment, a recombinant Arah1 variant polypeptide of the disclosure comprises an amino acid sequence set forth in any of SEQ ID NOs: 68-161, 174, 176, 178, 180, 182, 184, 193, 194, or 211-246, or an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any of SEQ ID NOs: 68-161, 174, 176, 178, 180, 182, 184, 193, 194, or 211-246.

[0020] The present disclosure also provides a nucleotide sequence encoding any one of the recombinant Arah1 mutant polypeptides of the present disclosure, an expression vector comprising the nucleotide sequence, and a cell comprising the expression vector. The present disclosure also provides a method for producing the recombinant Arah1 mutant polypeptide of the present disclosure using the expression vector.

[0021] In another aspect, the present disclosure provides a method of inducing hyposensitization to peanuts in a subject having an allergy to peanuts, comprising administering to the subject a composition comprising a hypoallergenic Arah2 variant of the present disclosure, a hypoallergenic Arah1 variant of the present disclosure, or a combination thereof, thereby inducing hyposensitization to peanuts in the subject.

[0022] In another aspect, the present disclosure provides a method of inducing hyposensitization to peanuts in a subject allergic to peanuts, comprising administering to the subject a composition comprising a nucleotide or modified nucleotide sequence encoding a hypoallergenic Arah2 variant of the disclosure, a nucleotide or modified nucleotide sequence encoding a hypoallergenic Arah1 variant of the disclosure, or a combination thereof, thereby inducing hyposensitization to peanuts in the subject.

[0023] In another aspect, the present disclosure provides a genetically modified peanut plant expressing a hypoallergenic Arah1 variant of the present disclosure, a hypoallergenic Arah2 variant of the present disclosure, or a combination thereof.

[0024] In another aspect, the present disclosure provides a processed food comprising a hypoallergenic Arah1 variant of the present disclosure, a hypoallergenic Arah2 variant of the present disclosure, or a combination thereof. [Brief description of the drawings]

[0025] The subject matter regarded as the hypoallergenic polypeptide variants described herein, and methods for their production, which have reduced allergenicity while maintaining immunogenicity, are particularly pointed out and distinctly claimed in the concluding portion of this specification. However, the Arahl and Arah2 polypeptide variants of the present invention, and methods for their production, both as to their composition and their method of operation, together with their objects, features, and advantages, will best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings.

[0026] [Figure 1] Figure 1: Discovery pipeline for Arah-specific monoclonal antibodies (mAbs). Figure 1 is a flow chart of the epitope mapping procedure based on the discovery of monoclonal antibodies (mAbs) from peanut allergy patients, from patient samples to residue-level mapping of epitopes. The steps shown are from collection of patient PMBCs to purification of mAbs by single-cell sorting (upper panel) or phage display panning (lower panel). [Diagram 2] Figure 2: Three approaches for epitope mapping of Arah purified mAbs. In approach 1, for each isolated Arah-specific mAb, an Arah yeast-displayed single-site saturation mutation library was screened for binding. The screened high- and low-binding populations were sent for deep sequencing, and mutant enrichment analysis identified the Arah region bound by the mAb. In approach 2, peptide arrays were utilized to analyze the identified binding sites. To determine linear epitope binding, two Celluspot™ peptide microarray-based immunoassays were performed for each mAb: a peptide array with the wild-type (WT) Arah sequence and an additional peptide array with point mutations. In approach 3, mutation patch analysis was performed. Structure-based in silico design of surface-exposed patch mutagenesis was followed by indirect ELISA screening of Arah mutants against the specific ArahmAb. Reduced or lost mAb binding to the mutants confirms the epitope location in the sequence. [Figure 3A]Figures 3A and 3B: Point mutants of Arah2 showed reduced binding to serum-derived anti-Arah2 monoclonal antibody (mAb) B701. Figure 3A shows FACS sorting of an Arah2 saturation library based on expression of Arah2 mutants (x-axis) and binding to mAb B701a (y-axis). [Figure 3B] Figure 3B shows the enrichment ratio of library point mutants in the S2low-B701 binding population, expressed as log2(fB701_S2_low / fs1), where fB701_S2_low is the fraction of a given mutation in the sorted library and fs1 is its fraction in the S1 library. Coloring ranges from white (depletion) to blue (enrichment, indicating that the point mutation results in reduced mAb binding). Position numbers (X-axis) are based on SEQ ID NO:1. [Figure 4A] Figures 4A and 4B: Arah1 and Arah2 mutants showed reduced binding to anti-Arah1 and anti-Arah2 mAbs, B536 and B843, respectively. Indirect ELISA titrations with increasing concentrations of anti-Arah mAbs were used to test binding to wild-type (WT) Arah2 or modified Arah2 mutants (Figure 4A), and to WT Arah1 or modified Arah1 mutants (Figure 4B). [Figure 4B] The data presented show that modified Arah1 and Arah2 mutants dramatically reduce binding to anti-Arah2 mAb B536 (FIG. 4A) or anti-Arah1 mAb B843 (FIG. 4B) from serum. Binding of WT Arah2 polypeptide (SEQ ID NO:2) or modified Arah2 mutant polypeptides to increasing concentrations of anti-Arah2 B536 mAb (FIG. 4A) or anti-Arah1 B843 (FIG. 4B) was measured by ELISA assay analysis. Bovine serum albumin (BSA) was used as a negative control. [Figure 5A]Figure 5A and Figure 5B: Linear epitope mapping and de-epitopation revealed mutations that abolished binding to the AraH2 epitope. Figure 5A: Linear epitope mapping of patient P70 revealed IgE binding to AraH1, AraH2, AraH3, and AraH. The black box highlights epitope L3 (peptide derived from positions 42-56 of SEQ ID NO: 3) mapped to AraH2. [Figure 5B] Figure 5B: Linear de-epitope of the P70Arah2 epitope in a patient. The black box highlights the same peptide as in Figure 5A. The box highlights where a point mutation dramatically reduced binding to L3. [Figure 6A] Figures 6A and 6B: Modified Arah2 and Arah1 mutants show reduced activation capacity. Figures 6A and 6B present data showing that modified Arah2 (Figure 6A) and Arah1 (Figure 6B) mutants show reduced activation of basophils. [Figure 6B] Representative results of a rat basophilic leukemia (RBL) SX-38 cell degranulation assay testing serum IgE-mediated cellular responses to either WT (black) or modified (grey) Arah2 variants. Results are shown for eight patient sera: S70, S129, A182, B192, W11, S95, S101, and E282. [Figure 7A] Figures 7A and 7B: Modified Arah2 variants dramatically reduce the ability to activate human basophils compared to native Arah2. Figures 7A and 7B present data from two different peanut allergy patient blood samples showing that modified Arah2 variants dramatically reduce basophil activation. [Figure 7B]Representative results of the Basophil Activation Test (BAT) testing serum IgE-mediated cellular responses to either WT (nArah2 and rArah2) or modified recombinant Arah2 variants B764 (SEQ ID NO: 11) and B1001 (SEQ ID NO: 10). Arah2 is natural Arah2 extracted from peanuts, rArah2 is recombinant Arah2. EC50 values ​​listed below were derived by a three-parameter function (if not listed, reactivity was too low to derive a value). [Figure 8A] Figures 8A and 8B: Activation of peripheral blood T helper cells from allergic patients by recombinant WT and modified Arah2 mutants. Figures 8A and 8B show activation of peripheral blood T helper cells from allergic patients by WT and representative modified Arah2 mutants (B764 and B1001) (Figure 8A is patient SH409, Figure 8B is patient B293). Representative results are shown. [Figure 8B] Cells were stained with "Celltrace" proliferation dye and activated with various allergens (WT or mutants) or left unactivated (untreated) and incubated for 7 days. Cells were harvested and stained for viability and T helper cell markers and live proliferating T helper cells were isolated (CD3+, CD4+, viability dye-, proliferation dye-dim). Graph shows mean and SE of % proliferation of T helper cells per treatment. [Figure 9A] Figures 9A-F: Modified Arah2 mutants maintain high thermal stability. Circular dichroism (CD) analysis of recombinant Arah2 WT (Arah2_B123) and mutants (Arah2_B764 and Arah2_B1001) is shown. Figure 9A (WT) shows the CD spectra of WT and mutants at 25 °C, indicating similar secondary structure composition of the mutants compared to WT. [Figure 9B] FIG. 9B (Arah2_B764) shows the CD spectra of the WT and mutants at 25° C., indicating a similar secondary structure composition of the mutants compared to the WT. [Figure 9C]FIG. 9C (Arah2_B1001) shows the CD spectra of WT and mutants at 25° C., indicating similar secondary structure composition of the mutants compared to the WT. [Figure 9D] Figure 9D (WT) shows the stability of Arah2WT and mutants in the temperature range of 20-90 °C, showing high thermal melting temperatures (TM) above 90 °C, suggesting no significant deviation from the native fold, at least as expected in the WT. [Figure 9E] Figure 9E (Arah2_B764) shows the stability of Arah2WT and mutants in the temperature range of 20-90 °C, showing high thermal melting temperatures (TM) above 90 °C, suggesting no significant deviation from the native fold, at least as expected for the WT. [Figure 9F] Figure 9F (Arah2_B1001) shows the stability of Arah2WT and mutants in the temperature range of 20-90°C, showing high thermal melting temperatures (TM) above 90°C, suggesting no significant deviation from the native fold, at least as expected for the WT (Lehmann, K., et al., (2006). Structure and stability of 2S albumin-type peanut allergens: implications for the severity of peanut allergic reactions. The Biochemical journal, 395(3), 463-472). [Figure 10] Figure 10: Expression and secretion of allergens from transfected cells. Mammalian cells were transfected with vectors encoding wild-type or de-epitope mutants of the peanut allergens Arah2 and Arahl. Secreted allergen proteins were purified and characterized by SDS-PAGE analysis. (Panel a) Wild-type Arahl, (Panel b) Wild-type Arahl, (Panel c) De-epitope Arahl, (Panel d) Two de-epitope mutants of Arahl. [Figure 11]Figure 11: Binding to IgE in serum of allergic patients. Arahl was expressed and secreted from HEK293 cells, purified, bound to serum of allergic patients or control non-allergic serum, and then analyzed for binding to IgE. Binding was compared to native Arahl (nArahl), recombinant E. coli-derived wild type Arahl (rArah1), and recombinant HEK293 cell-derived wild type Arahl (HEKArahl). [Figure 12] Figure 12: Binding to anti-Arah2 monoclonal antibodies. The peanut allergen Arah2 was expressed and secreted from HEK293 cells and purified. Binding to a well-characterized anti-Arah2 monoclonal IgG antibody was assayed and compared between recombinant Arah2 (rArah2) and HEK-derived Arah2 (HEKArah2 wild type). Six IgGs (mAb1-6) were used to demonstrate binding characteristics. HEK293 cell medium was used as a negative control. [Figure 13] FIG. 13 shows an HPLC size-exclusion chromatogram trace of purified Arah1 expressed from transfected mammalian cells, demonstrating the correct trimeric state. [Figure 14] Figure 14 shows the total mass measurement of AraH2 expressed from transfected mammalian cells, showing a mass of 18966.8 Da, which is the predicted mass of the sequence corresponding to the four disulfide bonds of oxidatively folded AraH2: 8 Da. [Figure 15] FIG. 15 shows an overview of a patient sample-based pipeline for allergen de-epitopation. [Figure 16A] Figures 16A-16C show the biochemical characteristics of the Arah2 mutant B1001. Figure 16A: Identification of Arah2B1001 by Western blotting. Proteins were separated by unstained SDS-PAGE and imaged under UV (left panel) as a loading control. Proteins were then transferred to a PVDF membrane and detected using a commercially available polyclonal antibody anti-Arah2 (right panel). Lanes: 1, native Arah2; 2, recombinant WT Arah2; 3, B1001 Arah2 mutant; 4, recombinant Arah1 (peanut negative control); 5, BSA (general negative control). [Figure 16B] Figure 16B: Size Exclusion Chromatography (SEC): HPLC analysis for estimation of molecular size and oligomeric state. Purified native Arah2 (upper panel), recombinant WT Arah2 (middle panel), and Arah2 mutant B1001 (lower panel) were analyzed by SEC-HPLC and the chromatograms are shown. Retention times (RT) and estimated Mw are indicated within the panels. [Figure 16C] Figure 16C: Circular dichroism (CD) analysis of WTArah2 and B1001 mutants. The left panel shows the CD spectra of WTArah2 and B1001 at 25 °C. The right panel shows the CD spectra in the temperature range of 20-90 °C, indicating the stability of the secondary structure (Curves °C are indicated by the colors noted in the legend). [Figure 17] Figure 17 shows that the reduction in binding of patient plasma to B1001 differs between IgE and IgG. ELISA assays were performed on plates coated with Arah2 or B1001. Plasma samples from 24 peanut allergic patients were serially diluted and incubated on the plates to detect the binding of the patients' IgE or IgG to each allergen. Titration curves were derived and used to calculate the area under the curve (AUC) values. Figure 17(A): Relative binding of patients' IgE or IgG to Arah2 or B1001. The figure shows the median and range of AUC. P values ​​of the Wilcoxon rank sum test are shown. Figure 17(B): AUC ratios of B1001 / Arah2 were calculated to show the reduction in binding of the variants. The figure shows the individual AUC ratios, with IgE and IgG ratios for each patient pair shown as thin lines and group medians shown as thick lines. P values ​​of the Wilcoxon rank sum test are shown. [Figure 18A]Figures 18A and 18B show that the allergenicity of B1001 was significantly reduced compared to native Arah2. Figure 18A: RBLSX-38 assay. Cells were incubated overnight with patient plasma, washed and then incubated with increasing concentrations of the protein of interest in Tyrode's buffer. The buffer was then transferred to a separate plate and incubated with a colorimetric substrate for the granule enzyme β-hexosaminidase. OD was measured at 450 nm and net degranulation was calculated by subtracting the OD of untreated wells and dividing by the OD of lysed wells. Reactions were performed in duplicate. Plots show the mean ± SE of 28 patients. [Figure 18B] Figure 18B: BAT assay. Fresh patient blood was induced with at least six concentrations covering the range 1-10,000 ng / ml, varying allergen concentrations depending on the available blood volume. Samples were incubated for 30 min, stained, washed, fixed, and analyzed by flow cytometry. Plots show baseline-subtracted mean values ​​± SE for each concentration, representing 18-44 patients. EC50 values ​​were derived by fitting the resulting curves to a four-parameter logistic regression model. [Figure 19A] Figures 19A and 19B show that B1001 retains partial immunogenicity for peripheral blood T cells of peanut allergic patients. PBMCs were isolated from the blood of peanut allergic patients, stained with Celltrace violet proliferation dye, and incubated for 7 days with DMSO alone or with DMSO-dissolved peptide pools covering the entire sequence of Arah2 or B1001 (4–8 replicates / treatment, 2–2.5×105 cells / well). The media was then removed and saved for cytokine secretion analysis, while cells were stained for Th identification and analyzed by flow cytometry to detect proliferation. Media was analyzed by sandwich ELISA to detect IL-5, IL-13, and IFN-γ secretion. Data were collected only from studies with WT Arah2 responses of [SI>2+M.Wp value<0.1]. Figure 19A: Graph showing mean ± SE, top left indicates number of samples, and above the bars, p values ​​for pairwise comparisons by Wilcoxon rank sum test. [Figure 19B] Figure 19B: Estimates of B1001 overall reactivity are shown. Samples were considered B1001 reactive if at least one test showed a reaction with M.Wp value < 0.1. Samples were estimated to have equal reactivity to B1001 and Arah2 if at least three out of four tests showed reactivity and the majority of tests showed M.Wp value > 0.2 for B1001 vs. Arah2. [Figure 20A] Figures 20A and 20B show that B1001 has a significantly improved safety profile compared to Arah2 and a comparable immunotherapeutic effect to peanut extract in a mouse allergy model. C3H / HeJ mice were orally sensitized with peanut extract (PE) and cholera toxin. Figure 20A: Mice were intraperitoneally administered 30 μg of native Arah2 or B1001. The next day, mice that had been administered B1001 the previous day were randomly divided into two subgroups and re-administered with Arah2 or B1001 at a twice higher dose (up to 240 μg). Upper pane: Anaphylactic scores 120 min after administration. Graphs show individual values ​​and mean ± SE, with Mann-Whitney test significance as above. Lower pane: Body temperature was measured at the indicated times after administration. Mean ± standard deviation is shown. [Figure 20B] Figure 20B: Sensitized mice were subjected to oral immunotherapy with peanut flour extract (PE), B1001, or PBS (sham). Upper pane: Anaphylactic scores 120 min after administration of 35 μg native Arah2. Graphs show individual values ​​and mean ± SE, with Mann-Whitney test significance as above. Lower pane: Mesenteric lymph node cells were isolated from each mouse, plated in 96-well plates, and incubated with 200 μg / ml native Arah2 for 72 h. Cytokine levels in the medium were measured using a ProcartaPlex Luminex panel assay. Mean ± SE is shown, with Mann-Whitney test significance as above (n: 3 controls, 4 sham, 6 PE, 15 B100). [Figure 21]Figure 21 shows SEC-HPLC analysis of ArahlWT and PLP595 (C159) (SEQ ID NO: 156). Chromatograms of native Arahl, recombinant WT Arahl, and Arahl PLP595 are shown. Retention times and estimated MW are shown. All proteins have similar retention times and an estimated MW of approximately 200 kDa, consistent with a trimeric fold. [Figure 22A] Figures 22A and 22B show the assessment of secondary structure using circular dichroism (CD) of Arahl and the PLP595(C159) mutant. Figure 22A: Normalized CD spectra of WT Arah1 (dashed line) and PLP595Ara1 (solid line) both showed similar CD signatures at 2 °C. [Figure 22B] Figure 22B: CD signals normalized to the ellipticity at 205 nm of recombinant WT (circles) and PLP595 (triangles) from 20 to 90 ° C. Both Arah1 variants show secondary structure stability above 85 ° C. [Diagram 23] Figure 23 shows the results of molecular weight analysis of Arahl and PLP595 (C159) mutants using mass spectrophotometry. Overlay histograms of normalized count measurements of recombinant Arahl WT and PLP595 (C159) proteins. The results support the formation of a trimeric fold (expected mass of each monomer is 63 kDa). [Figure 24A] Figure 24A and Figure 24B show a comparison of allergenicity of three different Arah1 variants using the RBLSX-38 degranulation assay. RBLSX-38 cells were sensitized with plasma or serum from allergic patients for 18 h. Cells were then treated with wild-type (WT) Arah1, Combo57 (PLP243), Combo68 (B1305), or Combo159 (PLP595) at concentrations ranging from 5 μg / ml to 0.5 ng / ml for 1 h. Degranulation was measured using a β-hexosaminidase activity assay. Area under the curve (AUC) values ​​were extracted for individual patients (represented by dots, respectively). Figure 24A: Reactivity of 13 Arah1-reactive patients was compared with Combo57 and Combo68. [Figure 24B]Figure 24B: Reactivity of 47 Arahl-reactive patients was compared to Combo57 and Combo159. Combo159 was less allergenic than Combo57 and Combo68, with more than 70% of patients showing no reactivity. The median AUC of Combo159 (1.7) was reduced by 97% compared to WT Arahl (56.3). [Figure 25A] Figure 25A and Figure 25B show the evaluation of allergenicity of different Arah1 variants using RBLSX-38 degranulation assay. RBLSX-38 cells were sensitized with plasma or serum of allergic patients for 18 h. Cells were then treated with wild-type (WT) Arah1, KLH (as a negative control), Combo51 (B1291), Combo52 (B1292), Combo74 (B1309), Combo75 (B1304), or Combo116 (PLP499) at concentrations ranging from 5 μg / ml to 0.05 ng / ml or 0.5 ng / ml for 1 h. Degranulation was measured using a β-hexosaminidase activity assay. Figure 25A: Example of two sera tested with Combo51 and Combo52. [Figure 25B] FIG. 25B: Examples of two sera tested in Combo74. [Figure 26A] Figure 26A and Figure 26B show the evaluation of allergenicity of different Arah1 variants using RBLSX-38 degranulation assay. RBLSX-38 cells were sensitized with plasma or serum of allergic patients for 18 h. Cells were then treated with wild type (WT) Arah1, KLH (as negative control), Combo51, Combo52, Combo74, Combo75, or Combo116 at concentrations ranging from 5 μg / ml to 0.05 ng / ml or 0.5 ng / ml for 1 h. Degranulation was measured using β-hexosaminidase activity assay. Figure 26A: Example of two sera tested with Combo75. [Figure 26B] FIG. 26B: Examples of two sera tested in Combo116. [Figure 27]Figure 27 shows that the allergenicity of C159 (PLP595) was significantly reduced compared to native Arah1. ​​Results were obtained from a BAT assay. Fresh patient blood was induced with 11 allergen concentrations ranging from 6,600 to 0.06 ng / ml (log3 serial dilutions). Samples were then incubated for 30 min, stained, washed, fixed and analyzed by flow cytometry. Plots show baseline-subtracted mean and SE for each concentration, representing 19 patients. EC50 values ​​obtained by fitting the resulting curves to a 4-parameter logistic regression model suggest that C159 was more than 1000-fold less reactive at the population level. [Figure 28] Figure 28 shows examples of back-to-consensus mutants of DEArah2 1001 expressed in HEK293 cells. Mutants 1-23 were transfected in duplicate. Culture supernatants were analyzed by reducing or non-reducing SDS-PAGE (left or right panel, respectively). Black arrows indicate Arah2 double bands. Expression levels were compared to the poorly expressing DE Arah2 1001 (far right lane in all gels). Highly expressing mutants were analyzed for allergenicity and selected for the next round of optimization. In this case, it is the mutants shown as number 2 and number 4 (SEQ ID NO: 208 and SEQ ID NO: 209). [Figure 29]Figure 29 shows a summary of the RBL activation assays (n=18) showing the area under the curve for each assay. The assays measured reactivity to the various proteins at 0.05ng / ml to 5μg / ml, except for the Fc fusion, which was measured at 0.1ng / ml to 10μg / ml to account for dimers presenting two allergens per molecule. Results are shown comparing native Arah2 (nArah2), de-epitope-ed Arahh21001 [SEQ ID NO: 168], Arah2_conbo31 [SEQ ID NO: 247], and 1001-FcIgG4 fusion [SEQ ID NO: 207] (denoted DE Arah 21001, DE Arah2 var.31, DE Arah2-Fc IgG4). The left panel shows a zoomed-in version of the same results to highlight subtle differences between the engineered Arah2 constructs. All de-epitope-ed versions showed a slight reactivity to one serum tested (R567), although this was significantly reduced compared to nArah2. [Diagram 30] Figure 30 shows a comparison of HEK293 expression levels of de-epitope Arah2-1001 [SEQ ID NO: 168] and de-epitope Arah2-Fc fusion [SEQ ID NO: 202]. Constructs were transfected in duplicate into HEK293 cells and culture supernatants were analyzed by SDS-PAGE (left) and Western blot and detected with anti-DEArah2 antibody (right). Each sample was either not reduced or reduced with β-mercaptoethanol (β-ME). Fusion to Fc dramatically increased the secretion levels of de-epitope Arah2-1001. Reduction of the samples prevented detection by Western blot. [Diagram 31]Figure 31 shows the expression of transmembrane fusions of de-epitope-modified Arah2 compared to secreted de-epitope-modified Arah2-1001. HEK293 cells were transfected with either secreted de-epitope-modified 1001-TM Arah2 [SEQ ID NO: 248] or a glycosylation-deficient mutant of the 1001 construct (GM1001-TM) [SEQ ID NO: 249], both fused to the TM domain of HLA-A. Cells were lysed and separated into soluble and membrane fractions by centrifugation. Separated fractions were analyzed by SDS-PAGE (left panel) under non-reducing conditions or under reducing conditions with the addition of β-mercaptoethanol (β-ME). Western blots (right panel) of the same gels were used to detect the presence and cellular location of de-epitope-modified Arah2. The membrane fraction signal was about 4-fold higher compared to the soluble fraction. The signal corresponding to de-epitope-modified Arah2 is indicated by a black arrow. Partial glycosylation of 1001 (a band of approximately 30 kDa indicated by the upper arrow) is observed in the secreted version of this protein and is expected to result in the orientation of the antigen in the extracellular space. The presence of the de-epitope-modified antigen and its orientation on the extracellular surface was confirmed by immunohistochemistry (not shown). [Diagram 32] Figure 32 shows the B cell response to the various constructs monitored after DNA delivery. Naive (untreated) mice were injected with expression plasmids encoding the various potential mRNA therapeutic constructs. Mice were injected 3 times a week and bled 21 days after the first dose (n=5 per group). Allergen-specific antibodies were detected by ELISA. Values ​​shown are subtracted from the KLH negative control. No antibodies were detected at time 0 (before the first DNA injection). Upper panel: Mean IgG titers of wild-type and de-epitope-ed Arah1 constructs. Lower panel: Mean IgG titers of wild-type and de-epitope-ed Arah2. Arah1 and Arah1-derived constructs were significantly more immunogenic compared to the de-epitope-ed Arah2 construct. No antibodies were detected in response to wild-type Arah2 and constructs encoding DEArah2-1001, where 1001 was not fused to an additional protein domain (not shown). [Diagram 33]Figure 33 shows the separation of a peanut extract by a salt gradient on a Q Sepharose column. The chromatogram shows the elution pattern of various peanut proteins expressed as absorbance units (AU) at 280 nm (left axis) versus mobile phase volume (mL). The lines indicate the percentage of the salt reservoir used for the separation. The areas on the chromatogram bounded by black vertical lines are the fractions where the Arah proteins eluted (indicated above each area). [Diagram 34] Figure 34 shows an SDS-PAGE analysis with Coomassie staining of the elution fractions of Figure 33. The different Arah proteins are indicated by arrows and their molecular weights are indicated on the left. The dotted lines drawn between the chromatogram of Figure 33 and the gel of Figure 34 represent the regions where each of the four major peanut allergens (Arah1, Arah2, Arah3, Arah6) eluted. [Diagram 35] FIG. 35 shows a typical elution pattern of nArah2 on a Superdex75SEC column. [Diagram 36] Figure 36 shows the SDS-PAGE pattern of nArah2 on a Superdex75SEC column. Arah2 eluted as a duplet. [Figure 37] Figure 37 shows the hypersensitivity response measured by temperature drop in mice treated sublingually with 5 μg peanut protein (SLIT5 μg) or 50 μg peanut protein (SLIT50 μg) or orally with 500 μg peanut protein (OIT500 μg). No SL / OIT treatment indicates mice that did not receive peanut protein sublingually or orally. [Figure 38A]Figure 38A and Figure 38B show that Arah1 variant C159 (SEQ ID NO: 156) retains partial immunogenicity for peripheral blood T cells from peanut allergic patients, as detected by T cell activation assay. PBMCs were isolated from the blood of peanut allergic patients, stained with Celltrace proliferation dye, and incubated for 7 days with either PBS, recombinant WTArah1 or Arah1 variant C159 (4–8 replicates / treatment, 2–2.5×105 cells / well). The medium was removed and saved for later analysis, and cells were stained for Th identification and analyzed by flow cytometry to detect proliferation. The medium was analyzed by sandwich ELISA to detect secretion of IL-5, IL-13, and IFN-γ. Data were collected only from studies in which WTArah1 responses were [SI>2 + Mann-Whitney p-value<0.1]. FIG. 38A: Graph showing mean values±SE, with sample numbers shown at the top left and p-values ​​for pairwise comparisons by Wilcoxon rank sum test shown above the bars. [Figure 38B] Figure 38B: Estimated C159 overall reactivity. Samples were considered C159 reactive if at least one test showed a reaction with M.Wp value < 0.1. Samples were estimated to have equal reactivity to C159 and Arahl if at least three out of four tests showed reactivity and the majority of tests showed M.Wp value > 0.2 for C159 vs Arahl. [Figure 39A] Figures 39A and 39B show that the reduction in binding of patient plasma to C159 is different for IgE and IgG. ELISA assays were performed on plates coated with Arahl or Arahl mutant C159. Plasma samples from 24 peanut allergic patients were serially diluted and incubated on the plates to detect patient IgE or IgG binding to each allergen. Titration curves were derived and used to calculate area under the curve (AUC) values. Figure 39A: Relative binding of patient IgE or IgG to Arahl or C159. The figure shows the median and range of AUC. P values ​​from Wilcoxon rank sum test are indicated above the bars and the median ratio of Arahl / C159 is indicated below each graph. [Figure 39B] Figure 39B: The AUC ratios of C159 / Arahl were calculated to show the reduced binding of the mutants. The figure shows the individual AUC ratios, with IgE and IgG ratios for each patient pair shown as thin lines and group medians shown as thick lines. P values ​​from the Wilcoxon rank sum test are indicated.

[0027] It will be understood that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, dimensions of some elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals have been repeated among the figures to indicate corresponding or similar elements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the disclosed recombinant Arah1 and Arah2 allergen variants and their uses. However, it will be understood by those skilled in the art that the disclosed recombinant Arah1 and Arah2 allergen variants and their uses can be practiced without the specific details set forth below. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the disclosed recombinant Arah1 and Arah2 allergen variants and their uses.

[0029] In some embodiments, the recombinant Arah1 and Arah2 variants of the present disclosure were mutated based on data collected during the epitope mapping process. Mutation sites were selected based on their potential to alter or destroy one or more epitopes recognized by anti-Arah1 or anti-Arah2 antibodies, either alone or in combination with additional mutations. The allergenicity of Arah1 and Arah2 variants was evaluated by rat basophilic leukemia (RBL) or basophil activation test (BAT) cell-based immunological assays using peanut allergy patient samples. The desired immunogenicity, i.e., the ability of recombinant Arah1 or Arah2 to induce a response of the immune system without inducing a mast cell / basophil mediated allergic reaction, was measured by T cell activation assay.

[0030] Those skilled in the art will understand that the term "epitope" can be used interchangeably with the term "antigenic determinant," all of which have the same meaning and properties, and encompasses a site on an antigen to which an immunoglobulin or antibody (or an antigen-binding fragment thereof) specifically binds. Epitopes can be formed from both contiguous and non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids (linear epitopes) typically are retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding (conformational epitopes) typically disappear on treatment with denaturing solvents. Epitopes generally include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. In some embodiments, epitopes are as small as possible while still maintaining immunogenicity. Immunogenicity, as described herein, is indicated by the ability to elicit an immune response, e.g., the ability to bind to an MHC class II molecule and induce a T cell response, e.g., by measuring T cell cytokine production.

[0031] As used herein, "depitope-depleted polypeptide X" refers to a modified polypeptide X that has reduced or abolished binding to an anti-polypeptide X antibody (compared to antibody binding to the corresponding wild type) due to a mutation in one or more epitopes recognized by the anti-polypeptide X antibody.

[0032] As used herein, "depitope-depleted Arahl allergen" refers to a modified Arahl allergen that has reduced or abolished binding to anti-Arahl antibodies (compared to the corresponding antibody binding to wild-type Arahl) due to mutations in one or more epitopes recognized by the anti-Arahl antibody. In one embodiment, the depitope-depleted Arahl allergen is reduced in allergenicity compared to its wild-type counterpart.

[0033] As used herein, "depitope-depleted Arah2 allergen" refers to a modified Arah2 allergen that has reduced or abolished binding to anti-Arah2 antibodies (compared to the corresponding antibody binding to wild-type Arah2) due to mutations in one or more epitopes recognized by anti-Arah2 antibodies. In one embodiment, the depitope-depleted Arah2 allergen is less allergenic compared to its wild-type counterpart.

[0034] As used herein, "epitope" refers to the portion of a macromolecule that is bound by an antibody or an antigen-binding fragment thereof (e.g., Arahl allergen or Arahl2 allergen). In a protein sequence, there are continuous epitopes, which are linear sequences of amino acids to which an antibody binds, and discontinuous epitopes, which are only present when the protein is folded into a particular conformation.

[0035] As used herein, "allergen" refers to a substance, proteinaceous or non-proteinaceous, that can induce an allergy or specific hypersensitivity.

[0036] As used herein, "allergenic" or "allergenic" refers to the ability of an antigen or allergen to induce an aberrant immune response that differs from a normal immune response in that it is an exaggerated response, does not provide a protective / preventative effect, and instead causes physiological dysfunction or tissue damage.

[0037] As used herein, "hypoallergenic" refers to a substance that has little or no potential to cause an allergic reaction.

[0038] In some embodiments, the present disclosure provides peanut allergen (e.g., Arahl, Arahl2) variants that have been mutated to reduce or eliminate one or more epitopes bound by anti-peanut allergen antibodies. In one embodiment, the mutations do not affect the biophysical and / or functional properties of the peanut allergen. The mutations in one aspect can be substitutions, deletions, insertions, or any combination thereof. For example, deletions include removal of a single amino acid important for antibody binding, or removal of the entire mapped epitope region.

[0039] Amino acid side chains are classified into six types: class I (Cys); class II (Ser, Thr, Ala, Gly); class III (Asn, Asp, Gln, Glu); class IV (His, Arg, Lys); class V (Ile, Leu, Val, Met); and class VI (Phe, Tyr, Trp). In addition, Pro in the mutant structure may be substituted. Conservative amino acid substitution refers to the substitution of an amino acid of one class with an amino acid of the same class. For example, the substitution of Asp for another class III residue such as Asn, Gln, Glu is a conservative substitution. Non-conservative amino acid substitution refers to the substitution of an amino acid of one class with an amino acid of another class; for example, the substitution of class II residue Ala with class III residue such as Asp, Asn, Glu, Gln. Methods for substitution mutations at the nucleotide or amino acid sequence level are well known in the art.

[0040] As used herein, the term "modified" or "recombinant" refers to changing one or more amino acids in an antibody or antigen-binding portion thereof. The change can be made by adding, substituting, or deleting amino acids at one or more positions. The change can also be made using known techniques such as PCR mutagenesis. For example, in some embodiments, the binding affinity of an antibody or antigen-binding portion thereof to a peanut allergen can be modified by modifying an antibody or antigen-binding portion thereof identified using the methods provided herein.

[0041] AraH1 mutant

[0042] In one embodiment, the present disclosure provides a recombinant Arah1 variant polypeptide comprising an amino acid sequence having at least 50% identity to the sequence set forth in SEQ ID NO: 65, the recombinant Arah1 variant polypeptide comprising one or more amino acid substitutions, deletions, insertions, or any combination thereof located within a single epitope recognized by an anti-Arah1 antibody. In another embodiment, the recombinant Arah1 variant polypeptide of the present disclosure comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof located within at least two epitopes recognized by an anti-Arah1 antibody.

[0043] In one embodiment, the recombinant Arah1 variant polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:67, and includes one or more substitutions, deletions, insertions, or any combination thereof at one or more of positions 194, 195, 213, 215, 231, 234, 245, 267, 287, 294, 312, 331, 419, 422, 443, 455, 462, 463, 464, 480, 494, and 500 of SEQ ID NO:67, compared to the amino acid residue at the same position in SEQ ID NO:65. In one embodiment, the substitution mutation is D at position 194. In one embodiment, the substitution mutation is A at position 195. In one embodiment, the substitution mutation is H at position 213. In one embodiment, the substitution mutation is R, D, L, I, F, or A at position 215. In one embodiment, the substitution mutation is A at position 231. In one embodiment, the substitution mutation is an E at position 234. In one embodiment, the substitution mutation is an R at position 245. In one embodiment, the substitution mutation is an E at position 267. In one embodiment, the substitution mutation is a D at position 287. In one embodiment, the substitution mutation is an E at position 294. In one embodiment, the substitution mutation is an A or H at position 312. In one embodiment, the substitution mutation is an H at position 331. In one embodiment, the substitution mutation is an E, V, or A at position 419. In one embodiment, the substitution mutation is an R or A at position 422. In one embodiment, the substitution mutation is an A at position 443. In one embodiment, the substitution mutation is an A at position 455. In one embodiment, the substitution mutation is an A, K, or T at position 462. In one embodiment, the substitution mutation is an S at position 463. In one embodiment, the substitution mutation is an A or S at position 464. In one embodiment, the substitution mutation is a Q at position 480. In one embodiment, the substitution mutation is an A, E, or N at position 494. In one embodiment, the substitution mutation is a K at position 500.

[0044] One of skill in the art will understand that the percent identity (%) provides a numerical value indicating how similar a query sequence is to a target sequence (i.e., how many identical amino acids are in each sequence). The higher the percent identity, the better the match.

[0045] The term "identity," when used in reference to polypeptide (or protein) sequences, refers to the degree of identity between two or more polypeptide (or protein) sequences or fragments thereof. Generally, the degree of similarity between two or more polypeptide (or protein) sequences refers to the similarity in the composition, order, or sequence of two or more amino acids of the two or more polypeptides (or proteins).

[0046] In some embodiments, a recombinant Arah1 mutant polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identity to a polypeptide or portion thereof of the present disclosure as determined using the BlastP software of the National Center for Biotechnology Information (NCBI) using default parameters.

[0047] In some embodiments, the recombinant Arah1 variant polypeptide of the present disclosure may include deletions, insertions, or amino acid substitution mutations. In one embodiment, the variant polypeptide includes conservative substitutions, or deletions, insertions, or substitutions that do not significantly change the three-dimensional structure of the polypeptide of interest described herein. In some embodiments, the deletions, insertions, or substitutions do not change the function of the polypeptide of interest of the present disclosure. In some embodiments, the deletions, insertions, or substitutions do not change the ability to induce a response of the immune system to cause hyposensitization to peanut allergens.

[0048] In some embodiments of the recombinant Arah1 variant described above, the recombinant Arah1 variant comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 substitution mutations at one or more of positions 194, 195, 213, 215, 231, 234, 245, 267, 287, 294, 312, 331, 419, 422, 443, 455, 462, 463, 464, 480, 494, and 500 of SEQ ID NO:67 compared to the amino acid residues at the same positions in SEQ ID NO:65.

[0049] In some embodiments of the recombinant Arah1 variant described above, the recombinant Arah1 variant further comprises an additional substitution, deletion, insertion, or any combination thereof at one or more of positions 12, 24, 27, 30, 42, 57, 58, 73, and 523 of SEQ ID NO:67, compared to the amino acid residue at the same position in SEQ ID NO:65. In one embodiment, the substitution mutation is K or A at position 12. In one embodiment, the substitution mutation is V or E at position 24. In one embodiment, the substitution mutation is A or H at position 27. In one embodiment, the substitution mutation is E or A at position 30. In one embodiment, the substitution mutation is L or K at position 42. In one embodiment, the substitution mutation is D or L at position 57. In one embodiment, the substitution mutation is S or R at position 58. In one embodiment, the substitution mutation is A or M at position 73. In one embodiment, the substitution mutation is A or K at position 523. In some embodiments of the recombinant Arah1 variants described above, the recombinant Arah1 variants further comprise an additional substitution, deletion, insertion, or any combination thereof at one or more of positions 87, 88, 96, 99, 196, 197, 200, 209, 238, 249, 260, 261, 263, 265, 266, 278, 283, 288, 290, 295, 318, 322, 334, 336, 378, 417, 421, 441, 443, 481, 484, 485, 487, 488, and 491 of SEQ ID NO: 67, compared to the amino acid residue at the same position in SEQ ID NO: 65. In one embodiment, the substitution mutation is an A at position 87. In one embodiment, the substitution mutation is an A at position 88. In one embodiment, the substitution mutation is an A at position 96. In one embodiment, the substitution mutation is an A at position 99. In one embodiment, the substitution mutation is an H at position 196. In one embodiment, the substitution mutation is an A at position 197. In one embodiment, the substitution mutation is a V, A, or Q at position 200. In one embodiment, the substitution mutation is an S at position 209. In one embodiment, the substitution mutation is a Q at position 238. In one embodiment, the substitution mutation is an N at position 249.In one embodiment, the substitution mutation is a K at position 260. In one embodiment, the substitution mutation is an R at position 261. In one embodiment, the substitution mutation is a K or L at position 263. In one embodiment, the substitution mutation is an S at position 265. In one embodiment, the substitution mutation is an R or L at position 266. In one embodiment, the substitution mutation is an R at position 278. In one embodiment, the substitution mutation is an E at position 283. In one embodiment, the substitution mutation is a Q at position 288. In one embodiment, the substitution mutation is an R at position 290. In one embodiment, the substitution mutation is an A at position 295. In one embodiment, the substitution mutation is an H at position 318. In one embodiment, the substitution mutation is an A or K at position 322. In one embodiment, the substitution mutation is a D, A, or N at position 334. In one embodiment, the substitution mutation is an R or S at position 336. In one embodiment, the substitution mutation is a K or E at position 378. In one embodiment, the substitution mutation is an R at position 417. In one embodiment, the substitution mutation is an E or S at position 421. In one embodiment, the substitution mutation is an N at position 441. In one embodiment, the substitution mutation is an A at position 443. In one embodiment, the substitution mutation is an A or S at position 481. In one embodiment, the substitution mutation is an R, S, A, or M at position 484. In one embodiment, the substitution mutation is an A at position 485. In one embodiment, the substitution mutation is an S or K at position 487. In one embodiment, the substitution mutation is an A at position 488. In one embodiment, the substitution mutation is an A, S, or E at position 491.

[0050] In some embodiments of the recombinant Arah1 variant described above, the recombinant Arah1 variant further comprises a substitution mutation at position 84 of SEQ ID NO: 67, compared to the amino acid residue at the same position in SEQ ID NO: 65. In one embodiment, the substitution mutation is an A at position 84.

[0051] In some embodiments of the recombinant Arah1 variant described above, the recombinant Arah1 variant is selected from the group consisting of those at positions 12, 24, 27, 30, 42, 52, 57, 58, 73, 84, 87, 88, 96, 99, 194, 195, 196, 197, 200, 209, 213, 215, 231, 234, 238, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, , 249, 260, 261, 263, 265, 266, 267, 278, 283, 287, 288, 290, 294, 295, 312, 318, 322, 331, 334, 336, 378, 417, 419, 421, 422, 441, 443, 445, 455, 462, 463, 464, 480, 481, 4 84, 485, 487, 488, 491, 494, 500, and 523, compared to the amino acid residues at the same positions in SEQ ID NO: 65, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68 substitution mutations.

[0052] In some embodiments of the above recombinant Arah1 variant, the recombinant Arah1 variant comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, or 90% identity to the sequence set forth in SEQ ID NO:65.

[0053] In some embodiments of the recombinant Arah1 variant described above, the recombinant Arah1 variant is selected from the group consisting of the variants at positions 12, 24, 27, 30, 42, 52, 57, 58, 73, 84, 87, 88, 96, 99, 194-197, 200, 209, 213, 215, 231, 234, 238, 245, 249, 260, 261, 263, 265, 266, 267, 278, 283, 287, 288, 290, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, In one or more of SEQ ID NO:65, the amino acid residues at the same positions in SEQ ID NO:65 further include one or more substitutions, deletions, insertions, or any combination thereof, in one or more of SEQ ID NO:65, the amino acid residues at the same positions in SEQ ID NO:65.

[0054] In some embodiments of the above recombinant Arah1 variant, the recombinant Arah1 variant comprises an amino acid sequence set forth in any of SEQ ID NOs: 68-161, 174, 176, 178, 180, 182, 184, 193, 194, or 211-246, or comprises an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any of SEQ ID NOs: 68-161, 174, 176, 178, 180, 182, 184, 193, 194, or 211-246.

[0055] In some embodiments of the recombinant Arah1 mutants described above, basophil degranulation release induced by the mutants is at least three-fold lower than that induced by the Arah1 wild-type polypeptide.

[0056] In some embodiments of the recombinant Arah1 variants described above, the recombinant Arah1 variant has a binding EC50 or KD reduced by 50% or more compared to the Arah1 wild-type polypeptide.

[0057] AraH2 mutant

[0058] In one embodiment, the present disclosure provides a recombinant Arah2 variant polypeptide comprising an amino acid sequence having at least 50% identity to the sequence set forth in SEQ ID NO:3, the recombinant Arah2 variant polypeptide comprising one or more amino acid substitutions, deletions, insertions, or any combination thereof located within a single epitope recognized by an anti-Arah2 antibody. In another embodiment, the recombinant Arah2 variant polypeptide of the present disclosure comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof located within at least two epitopes recognized by an anti-Arah2 antibody.

[0059] In one embodiment, a recombinant Arah2 variant polypeptide of the present disclosure comprises an amino acid sequence as set forth in SEQ ID NO:4, and includes one or more substitutions, deletions, insertions, or any combination thereof, at one or more of positions 12, 15, 16, 22, 24, 46, 53, 65, 80, 83, 86, 87, 90, 104, 115, 123, 127, and 140 of SEQ ID NO:4, compared to the amino acid residue at the same position in SEQ ID NO:3. In one embodiment, the substitution mutation is N, Q, E, D, T, S, G, P, C, K, H, Y, W, M, I, L, V, or A at position 12. In one embodiment, the substitution mutation is R, E, K, Y, W, F, M, I, V, C, D, G, or A at position 15. In one embodiment, the substitution mutation is R, K, D, Q, T, M, P, C, E, or W at position 16. In one embodiment, the substitution mutation is F, Y, W, Q, E, T, S, A, M, I, L, C, R, or H at position 22. In one embodiment, the substitution mutation is D, E, H, K, S, T, N, Q, L, I, M, W, Y, F, P, A, or G at position 24. In one embodiment, the substitution mutation is T, V, E, H, S, A, G, Q, N, D, R, P, M, I, L, or C at position 46. In one embodiment, the substitution mutation is T, S, Q, V, A, G, C, P, M, L, I, E, H, R, K, N, or D at position 53. In one embodiment, the substitution mutation is T, A, N, D, Q, R, K, H, I, L, M, V, W, P, G, C, or E at position 65. In one embodiment, the substitution mutation is N, S, T, V, A, I, L, M, F, Y, W, C, E, K, R, or G at position 80. In one embodiment, the substitution mutation is D, A, C, F, I, P, T, V, W, Y, or Q at position 83. In one embodiment, the substitution mutation is Y, F, H, R, E, C, G, I, L, M, V, T, S, or Q at position 86. In one embodiment, the substitution mutation is F, Y, I, L, M, V, A, S, Q, R, K, D, N, E, or P at position 87. In one embodiment, the substitution mutation is S, P, Q, or R at position 90. In one embodiment, the substitution mutation is L, M, K, R, H, E, D, A, Y, N, S, or W at position 104.In one embodiment, the substitution mutation is V, D, E, I, L, K, M, N, S, T, A, I, W, F, Y, or H at position 115. In one embodiment, the substitution mutation is I, Q, or A at position 123. In one embodiment, the substitution mutation is H, A, D, E, F, G, L, N, P, S, T, W, Y, Q, or V at position 127. In one embodiment, the substitution mutation is G, A, C, E, Y, F, H, K, L, M, N, P, Q, S, or V at position 140.

[0060] In some embodiments, a recombinant Arah2 variant polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identity to a polypeptide or portion thereof of the present disclosure as determined using the BlastP software of the National Center for Biotechnology Information (NCBI) using default parameters.

[0061] In some embodiments, the recombinant Arah2 variant polypeptide of the present disclosure may include deletions, insertions, or amino acid substitution mutations. In one embodiment, the variant polypeptide includes conservative substitutions, or deletions, insertions, or substitutions that do not significantly change the three-dimensional structure of the polypeptide of interest described herein. In some embodiments, the deletions, insertions, or substitutions do not change the function of the polypeptide of interest of the present disclosure. In some embodiments, the deletions, insertions, or substitutions do not change the ability to induce a response of the immune system to cause hyposensitization to peanut allergens.

[0062] In some embodiments of the above recombinant Arah2 variants, the recombinant Arah2 variants comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 substitution mutations at one or more of positions 12, 15, 16, 22, 24, 46, 53, 65, 80, 83, 86, 87, 90, 104, 115, 123, 127, and 140 of SEQ ID NO:4 compared to the amino acid residues at the same positions in SEQ ID NO:3.

[0063] In some embodiments of the recombinant Arah2 variant described above, the amino acids at positions 12-16 of SEQ ID NO:4 comprise the sequence set forth in SEQ ID NO:5.

[0064] In some embodiments of the recombinant Arah2 variant described above, the amino acids at positions 44-65 of SEQ ID NO:4 comprise the sequence set forth in SEQ ID NO:6.

[0065] In some embodiments of the recombinant Arah2 variant described above, the amino acids at positions 44-67 of SEQ ID NO:4 comprise the sequence set forth in SEQ ID NO:9.

[0066] In some embodiments of the recombinant Arah2 variants described above, the amino acids at positions 11-90 of SEQ ID NO:4 comprise the sequence set forth in SEQ ID NO:7 or SEQ ID NO:8.

[0067] In some embodiments of the recombinant Arah2 variants described above, the recombinant Arah2 variants further comprise an additional substitution, deletion, insertion, or any combination thereof, at one or more of positions 28, 44, 48, 51, 55, 63, 67, 107, 108, 109, 124, 125, and 142 of SEQ ID NO:4, compared to the amino acid residue at the same position in SEQ ID NO:4. In one embodiment, the substitution mutation is S, T, V, N, A, P, I, L, F, Y, H, R, K, E, or D at position 28. In one embodiment, the substitution mutation is I, A, C, G, H, L, F, Y, N, P, Q, K, E, S, T, V, M, or R at position 44. In one embodiment, the substitution mutation is V, G, C, E, H, Q, F, K, L, I, W, Y, N, R, S, T, V, A, or D at position 48. In one embodiment, the substitution mutation is S, G, Y, F, W, M, N, Q, E, R, K, H, T, D, or V at position 51. In one embodiment, the substitution mutation is G, A, D, E, F, Y, H, Q, V, I, L, M, R, K, S, T, C, or W at position 55. In one embodiment, the substitution mutation is P, C, F, V, I, L, M, W, Y, N, S, T, Q, G, H, K, or R at position 63. In one embodiment, the substitution mutation is E, Q, N, R, H, Y, F, W, M, L, V, T, S, A, P, or G at position 67. In one embodiment, the substitution mutation is A, C, F, G, H, I, K, L, M, Q, P, R, S, T, V, W, or Y at position 107. In one embodiment, the substitution mutation is T, V, D, E, R, H, Y, W, I, G, A, Q, or K at position 108. In one embodiment, the substitution mutation is K, C, S, R, G, P, Y, W, L, or I at position 109. In one embodiment, the substitution mutation is D, A, C, F, G, H, I, N, S, T, V, Y, L, E, or Q at position 124. In one embodiment, the substitution mutation is M, I, L, W, Y, G, K, N, T, V, or A at position 125. In one embodiment, the substitution mutation is M, A, C, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 142.

[0068] In some embodiments of the recombinant Arah2 variants described above, the recombinant Arah2 variants comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 substitution mutations at one or more of positions 12, 15, 16, 22, 24, 28, 48, 51, 53, 55, 63, 65, 67, 80, 83, 86, 87, 90, 104, 107, 108, 109, 115, 123, 124, 125, 127, 140, or 142 of SEQ ID NO:4, compared to the amino acid residues at the same positions in SEQ ID NO:3.

[0069] In one embodiment of the above recombinant Arah2 variant, the recombinant Arah2 variant comprises the amino acid sequence set forth in SEQ ID NO:4 and comprises one or more substitution mutations at one or more of positions 44, 48, 51, 55, 63, and 67 of SEQ ID NO:4 compared to the amino acid residues at the same positions in SEQ ID NO:3.

[0070] In some embodiments of the above recombinant Arah2 variants, the recombinant Arah2 variants comprise an amino acid sequence having at least 70%, 75%, 80%, 85%, or 90% identity to the sequence set forth in SEQ ID NO:3.

[0071] In some embodiments of the recombinant Arah2 variants described above, the recombinant Arah2 variants further comprise one or more substitutions, deletions, insertions, or any combination thereof, at one or more of positions 6, 11-28, 32, 39, 44-56, 58, 60, 63, 69, 80-87, 89-90, 92, 96-97, 99, 100, 102-105, 107-119, 123, 125, 127-131, 133, 134, 136-144, 146, and 148-153 of SEQ ID NO:4, compared to the amino acid residues at the same positions in SEQ ID NO:3.

[0072] In some embodiments of the recombinant Arah2 variants described above, the recombinant Arah2 variant comprises an amino acid sequence set forth in any of SEQ ID NOs: 10-63, 168, 170, 195-201, 204-210, or 247-249, or an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any of SEQ ID NOs: 10-63, 168, 170, 195-201, 204-210, or 247-249.

[0073] In some embodiments of the recombinant Arah2 mutants described above, basophil degranulation release induced by the mutants is at least 10-fold lower than that induced by the Arah2 wild-type polypeptide.

[0074] In some embodiments of the recombinant Arah2 variants described above, the recombinant Arah2 variant has a binding EC50 or KD reduced by 50% or more compared to the Arah2 wild-type polypeptide.

[0075] Nucleotides, Vectors, and Host Cells

[0076] As used herein, the term "nucleotide", "nucleotide sequence", or "nucleic acid molecule" is intended to include DNA molecules and RNA molecules or modified RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded. In some embodiments, the nucleotide comprises a modified nucleotide. In some embodiments, the nucleotide comprises an mRNA. In some embodiments, the nucleotide comprises a modified mRNA. In some embodiments, the nucleotide comprises a modified mRNA, the modified mRNA comprises a 5'-capped mRNA. In some embodiments, the modified mRNA comprises a molecule in which a portion of the nucleosides are replaced by either naturally modified nucleosides or synthetic nucleosides. In some embodiments, the modified nucleotide comprises a modified mRNA, the modified mRNA comprises a 5'-capped mRNA, the modified mRNA comprises a molecule in which a portion of the nucleosides are replaced by either naturally modified nucleosides or synthetic nucleosides.

[0077] As used herein, the term "isolated nucleotide" or "isolated nucleic acid molecule" refers to a nucleic acid encoding a peanut allergen variant of the disclosure (e.g., Arahl variant, Arahl2 variant), wherein the nucleotide sequence is essentially free of other genomic nucleotide sequences which naturally flank the nucleic acid in genomic DNA.

[0078] One aspect of the disclosure is a nucleotide or nucleic acid sequence encoding a peanut allergen variant of the disclosure (eg, Arahl variant, Arahl2 variant).

[0079] As used herein, the term "vector" refers to a discrete element used to introduce heterologous nucleic acid into a cell for expression or replication. Expression vectors include vectors capable of expressing a nucleic acid operably linked to a regulatory sequence, such as a promoter region, capable of affecting the expression of such a nucleic acid. Thus, an expression vector may refer to a DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector that results in expression of a nucleic acid when introduced into a suitable host cell. Suitable expression vectors are well known to those skilled in the art and include, for example, those that are replicable in prokaryotic and / or eukaryotic cells, those that remain episomal, or those that integrate into the host cell genome.

[0080] One aspect of the present disclosure is an expression vector comprising a nucleic acid construct encoding a peanut allergen variant of the present disclosure (e.g., Arahl variant, Arahl2 variant).

[0081] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It should be understood that this term is intended to refer not only to a particular cell, but to the progeny of that cell. Because certain modifications may occur in progeny, either due to mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are included within the scope of the term "host cell" as used herein.

[0082] One aspect of the disclosure is a host cell comprising an expression vector carrying a nucleic acid construct encoding a peanut allergen variant of the disclosure (e.g., Arah1 variant, Arah2 variant). In one embodiment, the cell or host cell is a prokaryotic or eukaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, a fungal cell, an algae cell, a plant cell, or a mammalian cell. In some embodiments, the peanut allergen variant may be produced in bacteria, such as E. coli. In some other embodiments, the peanut allergen variant may be produced in yeast or fungi, such as Saccharomyces cerevisiae, Aspergillus, Trichoderma, Pichia pastoris.

[0083] Nucleic acids encoding Arah1 variants

[0084] In one embodiment, the present disclosure provides a nucleic acid or modified nucleic acid molecule encoding a recombinant Arah1 variant polypeptide comprising an amino acid sequence having at least 50% identity to the sequence set forth in SEQ ID NO:65, wherein the recombinant Arah1 variant comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, located within a single epitope recognized by an anti-Arah1 antibody.

[0085] In another embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah1 variant comprising an amino acid sequence having at least 50% identity to the sequence set forth in SEQ ID NO:65, and comprising one or more amino acid substitutions, deletions, insertions, or any combination thereof located within at least two epitopes recognized by an anti-Arah1 antibody.

[0086] One of skill in the art will understand that the percent identity (%) provides a numerical value indicating how similar a query sequence is to a target sequence (i.e., how many identical amino acids are in each sequence). The higher the percent identity, the better the match.

[0087] The term "identity," when used in reference to polypeptide (or protein) sequences, refers to the degree of identity between two or more polypeptide (or protein) sequences or fragments thereof. Generally, the degree of similarity between two or more polypeptide (or protein) sequences refers to the similarity in the composition, order, or sequence of two or more amino acids of the two or more polypeptides (or proteins).

[0088] In some embodiments, a recombinant Arah1 mutant polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identity to a polypeptide or portion thereof of the present disclosure as determined using the BlastP software of the National Center for Biotechnology Information (NCBI) using default parameters.

[0089] In some embodiments, the recombinant Arah1 variant polypeptide of the present disclosure may include deletions, insertions, or amino acid substitution mutations. In one embodiment, the variant polypeptide includes conservative substitutions, or deletions, insertions, or substitutions that do not significantly change the three-dimensional structure of the polypeptide of interest described herein. In some embodiments, the deletions, insertions, or substitutions do not change the function of the polypeptide of interest of the present disclosure. In some embodiments, the deletions, insertions, or substitutions do not change the ability to induce a response of the immune system to cause hyposensitization to peanut allergens.

[0090] In one embodiment, the nucleic acid or modified nucleic acid is DNA or mRNA. In one embodiment, the mRNA comprises a UTR, a leader sequence, or both a UTR and a leader sequence. In one embodiment, the UTR comprises a chimeric or novel sequence that is superior to the native UTR sequence and can promote higher overall protein expression.

[0091] In one embodiment, the mRNA comprises (i) a UTR having the sequence set forth in SEQ ID NO: 162 or 163, and (ii) a leader sequence having the sequence set forth in SEQ ID NO: 185, 187, 189, or 191.

[0092] In one embodiment, the mRNA comprises an optimized sequence. As used herein, "optimized sequence" encompasses an mRNA sequence that comprises a computationally modified nucleotide sequence that promotes higher expression levels in human cells compared to the unmodified sequence, while maintaining favorable properties for in vitro transcription (IVT) and enzymatic capping.

[0093] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah1 variant comprising an amino acid sequence set forth in any of SEQ ID NOs: 68-161, 174, 176, 178, 180, 182, 184, 193, 194, or 211-246, or an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any of SEQ ID NOs: 68-161, 174, 176, 178, 180, 182, 184, 193, 194, or 211-246.

[0094] In one embodiment, the nucleic acid or modified nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 173. In one embodiment, the nucleic acid or modified nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 175. In one embodiment, the nucleic acid or modified nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 177. In one embodiment, the nucleic acid or modified nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 179. In one embodiment, the nucleic acid or modified nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 181. In one embodiment, the nucleic acid or modified nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 183.

[0095] In one embodiment, the nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah1 variant polypeptide comprising the amino acid sequence set forth in SEQ ID NO:67, comprising a substitution, deletion, insertion, or any combination thereof at one or more of positions 194, 195, 213, 215, 231, 234, 245, 267, 287, 294, 312, 331, 419, 422, 443, 455, 462, 463, 464, 480, 494, and 500 of SEQ ID NO:67, compared to the amino acid residue at the same position in SEQ ID NO:65. In one embodiment, the substitution mutation is D at position 194. In one embodiment, the substitution mutation is A at position 195. In one embodiment, the substitution mutation is H at position 213. In one embodiment, the substitution mutation is R, D, L, I, F, or A at position 215. In one embodiment, the substitution mutation is an A at position 231. In one embodiment, the substitution mutation is an E at position 234. In one embodiment, the substitution mutation is an R at position 245. In one embodiment, the substitution mutation is an E at position 267. In one embodiment, the substitution mutation is a D at position 287. In one embodiment, the substitution mutation is an E at position 294. In one embodiment, the substitution mutation is an A or H at position 312. In one embodiment, the substitution mutation is an H at position 331. In one embodiment, the substitution mutation is an E, V, or A at position 419. In one embodiment, the substitution mutation is an R or A at position 422. In one embodiment, the substitution mutation is an A at position 443. In one embodiment, the substitution mutation is an A at position 455. In one embodiment, the substitution mutation is an A, K, or T at position 462. In one embodiment, the substitution mutation is an S at position 463. In one embodiment, the substitution mutation is an A or S at position 464. In one embodiment, the substitution mutation is a Q at position 480. In one embodiment, the substitution mutation is an A, E, or N at position 494. In one embodiment, the substitution mutation is a K at position 500.

[0096] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah1 variant comprising the amino acid sequence set forth in SEQ ID NO:67, comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 substitution mutations at one or more of positions 194, 195, 213, 215, 231, 234, 245, 267, 287, 294, 312, 331, 419, 422, 443, 455, 462, 463, 464, 480, 494, and 500 of SEQ ID NO:67 compared to the amino acid residues at the same positions in SEQ ID NO:65.

[0097] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah1 variant comprising the amino acid sequence set forth in SEQ ID NO:67, which in addition to the substitution mutations described above, further comprises a substitution, deletion, insertion, or any combination thereof at one or more of positions 24, 27, and 30 of SEQ ID NO:67 compared to the amino acid residue at the same position in SEQ ID NO:65. In one embodiment, the substitution mutation is a V at position 24. In one embodiment, the substitution mutation is an A at position 27. In one embodiment, the substitution mutation is an E at position 30.

[0098] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah1 variant comprising the amino acid sequence set forth in SEQ ID NO:67, further comprising, in addition to the substitution mutations described above, additional substitution mutations at one or more of positions 87, 88, 96, 99, 196, 197, 209, 288, 290, 295, 322, 334, 336, 481, 484, 485, 487, 488, and 491 of SEQ ID NO:67 compared to the amino acid residues at the same positions in SEQ ID NO:65. In one embodiment, the substitution mutation is an A at position 87. In one embodiment, the substitution mutation is an A at position 88. In one embodiment, the substitution mutation is an A at position 96. In one embodiment, the substitution mutation is an A at position 99. In one embodiment, the substitution mutation is an H at position 196. In one embodiment, the substitution mutation is an A at position 197. In one embodiment, the substitution mutation is an S at position 209. In one embodiment, the substitution mutation is a Q at position 288. In one embodiment, the substitution mutation is an R at position 290. In one embodiment, the substitution mutation is an A at position 295. In one embodiment, the substitution mutation is an A or a K at position 322. In one embodiment, the substitution mutation is a D or an N at position 334. In one embodiment, the substitution mutation is an R at position 336. In one embodiment, the substitution mutation is an A or an S at position 481. In one embodiment, the substitution mutation is an R, S, A, or M at position 484. In one embodiment, the substitution mutation is an A at position 485. In one embodiment, the substitution mutation is an S or a K at position 487. In one embodiment, the substitution mutation is an A at position 488. In one embodiment, the substitution mutation is an A or an E at position 491.

[0099] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah1 variant comprising the amino acid sequence set forth in SEQ ID NO:67, which in addition to the substitution mutations described above, further comprises an additional substitution mutation at position 84 of SEQ ID NO:67 compared to the amino acid residue at the same position in SEQ ID NO:65. In one embodiment, the substitution mutation is an A at position 84.

[0100] In one embodiment, the nucleic acid or modified nucleic acid molecule of the present disclosure is a recombinant Arah1 mutant comprising the amino acid sequence set forth in SEQ ID NO:67, wherein the ARAH1 mutant is selected from the group consisting of ARAH1 at positions 24, 27, 30, 84, 87, 88, 96, 99, 194, 195, 196, 197, 209, 213, 215, 287, 288, 290, 294, 295, 322, 331, 334, 336, 419, 422, 455, 462, 464, 480, 481, 484, 485, 487, 488, 489, 490, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 491, and 494 encode a recombinant Arah1 variant comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 substitution mutations compared to the amino acid residues at the same positions in SEQ ID NO:65.

[0101] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah1 variant comprising an amino acid sequence having at least 70%, 75%, or 80% identity to the sequence set forth in SEQ ID NO:65.

[0102] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah1 variant comprising the amino acid sequence set forth in SEQ ID NO:67, comprising one or more substitution mutations at one or more of positions 24, 27, 30, 84, 87, 88, 96, 99, 194-197, 200, 209, 213, 215, 263, 267, 271, 287, 288, 290, 294, 295, 322, 331, 334, 336, 378, 417, 419, 421, 422, 439, 455, 462-464, 468, 480, 481, 484, 485, 487, 488, 491, 494, 500, and 502 of SEQ ID NO:67 compared to the amino acid residues at the same positions in SEQ ID NO:65.

[0103] Nucleic acids encoding Arah2 variants

[0104] In one embodiment, the present disclosure provides a nucleic acid or modified nucleic acid molecule encoding a recombinant Arah2 variant polypeptide comprising an amino acid sequence having at least 50% identity to the sequence set forth in SEQ ID NO:3, wherein the recombinant Arah2 variant comprises one or more substitutions, deletions, insertions, or any combination thereof located within a single epitope recognized by an anti-Arah2 antibody.

[0105] In another embodiment, the nucleic acid or modified nucleic acid molecule encodes a recombinant Arah2 variant comprising an amino acid sequence having at least 50% identity to the sequence set forth in SEQ ID NO:3, and comprising one or more amino acid substitutions, deletions, insertions, or any combination thereof located within at least two epitopes recognized by an anti-Arah2 antibody.

[0106] A person skilled in the art would be able to easily understand the above percent identity. In some embodiments, a recombinant Arah2 variant polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identity to a polypeptide of the present disclosure or a portion thereof, as determined using the BlastP software of the National Center for Biotechnology Information (NCBI) using default parameters.

[0107] In some embodiments, the recombinant Arah2 variant polypeptide of the present disclosure may include deletions, insertions, or amino acid substitution mutations. In one embodiment, the variant polypeptide includes conservative substitutions, or deletions, insertions, or substitutions that do not significantly change the three-dimensional structure of the polypeptide of interest described herein. In some embodiments, the deletions, insertions, or substitutions do not change the function of the polypeptide of interest of the present disclosure. In some embodiments, the deletions, insertions, or substitutions do not change the ability to induce a response of the immune system to cause hyposensitization to peanut allergens.

[0108] In one embodiment, the nucleic acid or modified nucleic acid is DNA or mRNA. In one embodiment, the mRNA comprises a UTR, a leader sequence, or both a UTR and a leader sequence. In one embodiment, the UTR comprises a chimeric or novel sequence that is superior to the native UTR sequence and can promote higher overall protein expression.

[0109] In one embodiment, the mRNA comprises (i) a UTR having the sequence set forth in SEQ ID NO: 162 or 163, and (ii) a leader sequence having the sequence set forth in SEQ ID NO: 185, 187, 189, or 191.

[0110] In one embodiment, the mRNA comprises an optimized sequence. As used herein, "optimized sequence" encompasses an mRNA sequence that comprises a computationally modified nucleotide sequence that promotes higher expression levels in human cells compared to the unmodified sequence, while maintaining favorable properties for in vitro transcription (IVT) and enzymatic capping.

[0111] In one embodiment, a nucleic acid or modified nucleic acid molecule of the disclosure encodes a recombinant Arah2 variant comprising an amino acid sequence set forth in any of SEQ ID NOs: 10-63, 168, 170, 195-201, 204-210, or 247-249, or an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any of SEQ ID NOs: 10-63, 168, 170, 195-201, 204-210, or 247-249.

[0112] In one embodiment, the nucleic acid or modified nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 167. In one embodiment, the nucleic acid or modified nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO:169.

[0113] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah2 variant polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4, comprising a substitution mutation at one or more of positions 12, 15, 16, 22, 24, 46, 53, 65, 80, 83, 86, 87, 90, 104, 115, 123, 127, and 140 of SEQ ID NO:4, compared to the amino acid residue at the same position in SEQ ID NO:3. In one embodiment, the substitution mutation is N, Q, E, D, T, S, G, P, C, K, H, Y, W, M, I, L, V, or A at position 12. In one embodiment, the substitution mutation is R, E, K, Y, W, F, M, I, V, C, D, G, or A at position 15. In one embodiment, the substitution mutation is R, K, D, Q, T, M, P, C, E, or W at position 16. In one embodiment, the substitution mutation is F, Y, W, Q, E, T, S, A, M, I, L, C, R, or H at position 22. In one embodiment, the substitution mutation is D, E, H, K, S, T, N, Q, L, I, M, W, Y, F, P, A, or G at position 24. In one embodiment, the substitution mutation is T, V, E, H, S, A, G, Q, N, D, R, P, M, I, L, or C at position 46. In one embodiment, the substitution mutation is T, S, Q, V, A, G, C, P, M, L, I, E, H, R, K, N, or D at position 53. In one embodiment, the substitution mutation is T, A, N, D, Q, R, K, H, I, L, M, V, W, P, G, C, or E at position 65. In one embodiment, the substitution mutation is N, S, T, V, A, I, L, M, F, Y, W, C, E, K, R, or G at position 80. In one embodiment, the substitution mutation is D, A, C, F, I, P, T, V, W, Y, or Q at position 83. In one embodiment, the substitution mutation is Y, F, H, R, E, C, G, I, L, M, V, T, S, or Q at position 86. In one embodiment, the substitution mutation is F, Y, I, L, M, V, A, S, Q, R, K, D, N, E, or P at position 87. In one embodiment, the substitution mutation is S, P, Q, or R at position 90. In one embodiment, the substitution mutation is L, M, K, R, H, E, D, A, Y, N, S, or W at position 104.In one embodiment, the substitution mutation is V, D, E, I, L, K, M, N, S, T, A, I, W, F, Y, or H at position 115. In one embodiment, the substitution mutation is I, Q, or A at position 123. In one embodiment, the substitution mutation is H, A, D, E, F, G, L, N, P, S, T, W, Y, Q, or V at position 127. In one embodiment, the substitution mutation is G, A, C, E, Y, F, H, K, L, M, N, P, Q, S, or V at position 140.

[0114] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah2 variant polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4, comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 substitution mutations at one or more of positions 12, 15, 16, 22, 24, 46, 53, 65, 80, 83, 86, 87, 90, 104, 115, 123, 127, and 140 of SEQ ID NO:4 compared to the amino acid residues at the same positions in SEQ ID NO:3.

[0115] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah2 variant comprising the amino acid sequence set forth in SEQ ID NO:4, wherein amino acids at positions 12 to 16 of SEQ ID NO:4 comprise the sequence set forth in SEQ ID NO:5.

[0116] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah2 variant comprising the amino acid sequence set forth in SEQ ID NO:4, wherein amino acids at positions 44 to 65 of SEQ ID NO:4 comprise the sequence set forth in SEQ ID NO:6.

[0117] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah2 variant comprising the amino acid sequence set forth in SEQ ID NO:4, wherein amino acids at positions 44 to 67 of SEQ ID NO:4 comprise the sequence set forth in SEQ ID NO:9.

[0118] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah2 variant comprising the amino acid sequence set forth in SEQ ID NO:4, wherein amino acids at positions 11 to 90 of SEQ ID NO:4 comprise the sequence set forth in SEQ ID NO:7 or SEQ ID NO:8.

[0119] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah2 variant comprising the amino acid sequence set forth in SEQ ID NO:4, further comprising, in addition to the substitution mutations described above, additional substitution mutations at one or more of positions 28, 44, 48, 51, 55, 63, 67, 107, 108, 109, 124, 125, and 142 of SEQ ID NO:4 compared to the amino acid residues at the same positions in SEQ ID NO:3. In one embodiment, the substitution mutation is S, T, V, N, A, P, I, L, F, Y, H, R, K, E, or D at position 28. In one embodiment, the substitution mutation is I, A, C, G, H, L, F, Y, N, P, Q, K, E, S, T, V, M, or R at position 44. In one embodiment, the substitution mutation is V, G, C, E, H, Q, F, K, L, I, W, Y, N, R, S, T, V, A, or D at position 48. In one embodiment, the substitution mutation is S, G, Y, F, W, M, N, Q, E, R, K, H, T, D, or V at position 51. In one embodiment, the substitution mutation is G, A, D, E, F, Y, H, Q, V, I, L, M, R, K, S, T, C, or W at position 55. In one embodiment, the substitution mutation is P, C, F, V, I, L, M, W, Y, N, S, T, Q, G, H, K, or R at position 63. In one embodiment, the substitution mutation is E, Q, N, R, H, Y, F, W, M, L, V, T, S, A, P, or G at position 67. In one embodiment, the substitution mutation is A, C, F, G, H, I, K, L, M, Q, P, R, S, T, V, W, or Y at position 107. In one embodiment, the substitution mutation is T, V, D, E, R, H, Y, W, I, G, A, Q, or K at position 108. In one embodiment, the substitution mutation is K, C, S, R, G, P, Y, W, L, or I at position 109. In one embodiment, the substitution mutation is D, A, C, F, G, H, I, N, S, T, V, Y, L, E, or Q at position 124. In one embodiment, the substitution mutation is M, I, L, W, Y, G, K, N, T, V, or A at position 125. In one embodiment, the substitution mutation is M, A, C, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y at position 142.

[0120] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah2 variant polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:4, comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 substitution mutations at one or more of 12, 15, 16, 22, 24, 28, 44, 46, 48, 51, 53, 55, 63, 65, 67, 80, 83, 86, 87, 90, 104, 107, 108, 109, 115, 123, 124, 125, 127, 140, and 142 of SEQ ID NO:4 compared to the amino acid residues at the same positions in SEQ ID NO:65.

[0121] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah2 variant comprising the amino acid sequence of SEQ ID NO:4, comprising one or more substitution mutations at one or more of positions 44, 48, 51, 55, 63, and 67 of SEQ ID NO:4 compared to the amino acid residues at the same positions in SEQ ID NO:3.

[0122] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah2 variant comprising an amino acid sequence having at least 70%, 75%, or 80% identity to the sequence set forth in SEQ ID NO:3.

[0123] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah2 variant polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4, comprising one or more substitution mutations at one or more of positions 6, 11-28, 32, 39, 44-56, 58, 60, 63, 69, 80-87, 89-90, 92, 96-97, 99, 100, 102-105, 107-119, 123, 125, 127-131, 133, 134, 136-144, 146, and 148-153 of SEQ ID NO:4 compared to the amino acid residues at the same positions in SEQ ID NO:3.

[0124] Manufacturing method

[0125] In some embodiments, the variant polypeptides of the present disclosure can be produced using a cell-free in vitro translation system, such as, for example, methods well known in the art, including, but not limited to, those reviewed in Dondapati et al. (2020) BioDrugs 34(3):327-348. In one embodiment, the present disclosure provides a method for producing a hypoallergenic peanut allergen comprising an Arah1 variant of the present disclosure, comprising culturing a cell comprising the expression vector described above under conditions to express the Arah1 variant. In one embodiment, the cell is a prokaryotic or eukaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, a fungal cell, a plant cell, or a mammalian cell.

[0126] In one embodiment, the present disclosure provides a method for producing a hypoallergenic peanut allergen comprising the Arah2 variant of the present disclosure, comprising culturing a cell comprising the expression vector described above under conditions for expressing the Arah2 variant. In one embodiment, the cell is a prokaryotic or eukaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, a fungal cell, a plant cell, or a mammalian cell.

[0127] In some embodiments, the nucleic acid or modified nucleic acid molecule of the present disclosure is transcribed in an in vitro transcription system (IVT), and the transcribed nucleic acid or modified nucleic acid can then be used for immunotherapy by gene delivery. Administration of RNA results in the in vivo production of peanut allergens or peanut allergen variants.

[0128] In some embodiments, the nucleic acid molecule encodes a wild-type (WT) peanut allergen. In some embodiments, the nucleic acid molecule encodes a peanut allergen variant that contains one or more amino acid substitutions, deletions, insertions, or any combination thereof located within a single epitope recognized by an antibody to the allergen.

[0129] In some embodiments of the method of manufacture of the present disclosure, the nucleic acid molecule encodes a WTArahl polypeptide. In some embodiments of the method of manufacture of the present disclosure, the nucleic acid molecule encoding a WTArahl polypeptide is selected from the sequence set forth in SEQ ID NO: 171 or 172. In some embodiments of the method of manufacture of the present disclosure, the nucleic acid molecule encodes a WTArahl polypeptide. In some embodiments of the method of manufacture of the present disclosure, the nucleic acid molecule encoding a WTArahl polypeptide is selected from the sequence set forth in SEQ ID NO: 164 or 165.

[0130] In some embodiments of the method of manufacture of the present disclosure, the nucleic acid molecule encodes an Arah1 variant polypeptide comprising one or more amino acid substitutions, deletions, insertions, or any combination thereof located within a single epitope recognized by an anti-Arah1 antibody. In some embodiments, the nucleic acid comprises a modified nucleic acid encoding an Arah1 variant polypeptide comprising one or more amino acid mutations located within a single epitope recognized by an anti-Arah1 antibody. In some embodiments of the method of manufacture of the present disclosure, the nucleic acid molecule encoding an Arah1 variant polypeptide comprises a sequence set forth in any of SEQ ID NOs: 173, 175, 177, 179, 181, or 183. In some embodiments of the method of manufacture of the present disclosure, the nucleic acid molecule encodes an Arah1 variant polypeptide comprising an amino acid sequence set forth in any of SEQ ID NOs: 68-161, 174, 176, 178, 180, 182, 184, 193, 194, or 211-246.

[0131] In some embodiments of the method of manufacture of the present disclosure, the nucleic acid molecule encodes an Arah2 variant polypeptide comprising one or more amino acid substitution mutations located within a single epitope recognized by an anti-Arah2 antibody. In some embodiments, the nucleic acid comprises a modified nucleic acid encoding an Arah2 variant polypeptide comprising one or more amino acid mutations located within a single epitope recognized by an anti-Arah2 antibody. In some embodiments of the method of manufacture of the present disclosure, the nucleic acid molecule encoding the Arah2 variant polypeptide comprises a sequence set forth in any of SEQ ID NOs: 167 or 169. In some embodiments of the method of manufacture of the present disclosure, the nucleic acid molecule encodes an Arah2 variant polypeptide comprising an amino acid sequence set forth in any of SEQ ID NOs: 10-63, 168, 170, 195-201, 204-210, or 247-249.

[0132] Chemical synthesis or synthesis and capping of RNA molecules by enzymatic processes such as bacteriophage RNA polymerase are well-established methods in the art for mRNA production and are described, for example, in "Elain T. Schenborn Methods in Molecular Biology, Vol. 37: In Vitro Transcript / on and Translation Protocols pages 1-12 DOI: 10.1385 / 0-89603-288-4:1".

[0133] One of skill in the art will appreciate that other known IVT systems can be used to transcribe the nucleic acids or modified nucleic acid molecules described herein. In some embodiments, mRNA molecules are transcribed in vitro using an IVT system.

[0134] The production of the peanut allergen variants, Arahl variants and Ara2 variants may involve in vivo translation, where transcribed mRNA is administered to a subject (patient).

[0135] In some embodiments, the nucleic acid molecules or modified nucleic acid molecules of the present disclosure can be used to produce peanut allergen variant polypeptides in vivo, including administration of the nucleic acid or modified nucleic acid molecule to a subject (patient) by viral, non-viral, or physical means, such as liposomes, cationic lipids, cationic polymers or hybrid lipid-polymer systems, retroviral or DNA viral delivery (e.g., lentivirus, fomivirus, adenovirus, etc.), ultrasound delivery, electroporation, hydrodynamic delivery, etc. In some embodiments, the nucleic acid molecules of the present disclosure can be used to produce peanut allergen WT polypeptides in vivo, including administration of the nucleic acid molecule to a subject (patient) by viral, non-viral, or physical means, such as liposomes, cationic lipids, cationic polymers or hybrid lipid-polymer systems, retroviral or DNA viral delivery (e.g., lentivirus, fomivirus, adenovirus, etc.), ultrasound delivery, electroporation, hydrodynamic delivery, etc. Methods for in vivo administration of nucleic acid molecules, e.g., mRNA molecules of the present disclosure encoding Arah1 or Arah2 variants, are well known in the art and are described, for example and without limitation, in Jones et al., Overcoming Nonviral Gene Delivery Barriers: Perspective and Future. Mol. Pharmaceutics 2013, 10, 11, 4082-4098; Kamimura et al. Advances in Gene Delivery Systems. Pharmaceut Med. 25(5):293-306; and Nayerossadat et al., Viral and nonviral delivery systems for gene delivery. Adv Biomed Res 2012;1:27, which are incorporated herein by reference in their entirety.

[0136] In some embodiments, the subject comprises a human subject. In certain embodiments, the human subject comprises a baby, a child, an adolescent, a young adult, or a mature adult. In some embodiments, the human subject comprises a baby.

[0137] In some embodiments, the subject includes a subject in need of induction of hyposensitization to peanuts. In some embodiments, the subject has an allergy to peanuts. In some embodiments, the subject suffers from other food allergies. In some embodiments, the subject is prone to developing a peanut allergy.

[0138] How to use

[0139] In one embodiment, the present disclosure provides a method of inducing hyposensitization to peanut in a subject having an allergy to peanut, comprising administering to the subject a composition comprising a hypoallergenic Arah1 variant of the present disclosure, thereby inducing hyposensitization to peanut in the subject.

[0140] In one embodiment, the present disclosure provides a method of inducing hyposensitization to peanut in a subject having an allergy to peanut, comprising administering to the subject a composition comprising a hypoallergenic Arah2 variant of the present disclosure, thereby inducing hyposensitization to peanut in the subject.

[0141] In one embodiment, the present disclosure provides a method of inducing hyposensitization to peanut in a subject having an allergy to peanut, comprising administering to the subject a composition comprising a combination of the hypoallergenic Arah1 and Arah2 variants of the present disclosure, thereby inducing hyposensitization to peanut in the subject.

[0142] In some embodiments, the methods of the disclosure include the use of an adjuvant. According to the present invention, "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. Adjuvants can also function as tissue depots that slowly release antigens. Examples of adjuvants include, but are not limited to, monophosphoryl lipid A (MPL-A), microcrystalline tyrosine (MCT), calcium phosphate, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, levamisole, CpG-DNA, oil or hydrocarbon emulsions, and potentially useful adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum. In some embodiments, the Arah1 and Arah2 variants are adsorbed to MCT and administered with or without MPL-A. Both MCT and MPL-A improve the efficacy of allergy immunotherapy and are believed to have synergistic effects when used in combination. Specifically, administration of the adjuvant reduces the number of injections required, reduces the dose, and promotes the production of protective IgG antibodies. In addition, MCT adsorption improves the safety of the product due to the depot effect and sustained release of the protein.

[0143] In one embodiment, the disclosure provides a method of inducing hyposensitization to peanuts in a subject allergic to peanuts, comprising administering to the subject a composition comprising a nucleotide or modified nucleotide sequence encoding a recombinant hypoallergenic Arah1 variant of the disclosure, thereby inducing hyposensitization to peanuts in the subject. In one embodiment, the disclosure provides a method of inducing hyposensitization to peanuts in a subject allergic to peanuts, comprising administering to the subject a composition comprising a nucleotide or modified nucleotide sequence encoding a recombinant hypoallergenic Arah2 variant of the disclosure, thereby inducing hyposensitization to peanuts in the subject. In one embodiment, the disclosure provides a method of inducing hyposensitization to peanuts in a subject allergic to peanuts, comprising administering to the subject a composition comprising a nucleotide or modified nucleotide sequence encoding a combination of recombinant hypoallergenic Arah1 variants and Arah2 variants of the disclosure, thereby inducing hyposensitization to peanuts in the subject. In one embodiment, the composition comprises a bacterium having a nucleotide sequence. In one embodiment, the nucleotide sequence is in the form of DNA or RNA.

[0144] In one embodiment, the composition in the above method is administered orally. In another embodiment, the composition in the above method is administered by a route selected from subcutaneous, intramuscular, intranasal, sublingual, topical, rectal, and inhalation. In one embodiment, the subject in the above method is an infant. In one embodiment, the composition in the above method comprises powdered milk (baby milk) or baby food.

[0145] In one embodiment, the present disclosure provides a method for inducing hyposensitization to peanuts in a subject allergic to peanuts, comprising administering to the subject a composition comprising a nucleic acid molecule encoding a recombinant Arah1 polypeptide, thereby inducing hyposensitization to peanuts in the subject. In some embodiments, the nucleic acid molecule used in the method for inducing hyposensitization to peanuts in a subject allergic to peanuts comprises a nucleic acid molecule encoding a WT recombinant Arah1 polypeptide. In some embodiments, the nucleic acid molecule used in the method for inducing hyposensitization to peanuts in a subject allergic to peanuts comprises a nucleic acid molecule encoding a mutant recombinant Arah1 polypeptide or a modified nucleic acid molecule comprising one or more amino acid substitution mutations located within a single epitope recognized by an anti-Arahl antibody. In some embodiments, the nucleic acid molecule used in the method for inducing hyposensitization to peanuts in a subject allergic to peanuts comprises a nucleic acid molecule encoding a WT recombinant Arah2 polypeptide. In some embodiments, a nucleic acid molecule used in a method for inducing hyposensitization to peanut in a subject allergic to peanut comprises a nucleic acid molecule or a modified nucleic acid molecule encoding a mutant recombinant Arah2 polypeptide that contains one or more amino acid substitution mutations located within a single epitope recognized by an anti-Arah2 antibody.

[0146] In one embodiment, the present disclosure provides a method of inducing hyposensitization to peanuts in a subject allergic to peanuts, comprising administering to the subject a composition comprising a nucleic acid or modified nucleic acid molecule encoding a recombinant hypoallergenic Arah1 variant of the present disclosure, thereby inducing hyposensitization to peanuts in the subject.

[0147] In one embodiment, the present disclosure provides a method of inducing hyposensitization to peanuts in a subject having an allergy to peanuts, comprising administering to the subject a composition comprising a nucleic acid or modified nucleic acid molecule encoding a recombinant hypoallergenic Arah2 variant of the present disclosure, thereby inducing hyposensitization to peanuts in the subject.

[0148] In one embodiment, the present disclosure provides a method of inducing hyposensitization to peanut in a subject allergic to peanut, comprising administering to the subject a composition comprising a nucleic acid or modified nucleic acid molecule encoding a combination of recombinant hypoallergenic Arahl and Arahl2 variants of the present disclosure, thereby inducing hyposensitization to peanut in the subject.

[0149] In one embodiment, the composition in the above method comprises a bacterium having the nucleic acid or modified nucleic acid molecule of the present disclosure.In one embodiment, the nucleic acid or modified nucleic acid molecule is DNA or mRNA.Examples of DNA or mRNA are as described above.

[0150] In some embodiments of the method of inducing hyposensitization to peanuts in a subject allergic to peanuts, the nucleic acid molecule encodes a WTArahl polypeptide. In some embodiments of the method of inducing hyposensitization to peanuts in a subject allergic to peanuts, the nucleic acid molecule encoding a WTArahl polypeptide comprises a sequence set forth in any of SEQ ID NOs: 171 or 172. In some embodiments of the method of inducing hyposensitization to peanuts in a subject allergic to peanuts, the nucleic acid molecule encodes a WTArahl polypeptide. In some embodiments of the method of inducing hyposensitization to peanuts in a subject allergic to peanuts, the nucleic acid molecule encoding a WTArahl polypeptide comprises a sequence set forth in any of SEQ ID NOs: 164 or 165.

[0151] In some embodiments of the method of inducing hyposensitization to peanuts in a subject allergic to peanuts, the nucleic acid molecule encodes an Arah1 variant polypeptide comprising one or more amino acid substitution mutations located within a single epitope recognized by an anti-Arah1 antibody. In some embodiments, the nucleic acid comprises a modified nucleic acid encoding an Arah1 variant polypeptide comprising one or more amino acid substitution mutations located within a single epitope recognized by an anti-Arah1 antibody. In some embodiments of the method of inducing hyposensitization to peanuts in a subject allergic to peanuts, the nucleic acid molecule encoding an Arah1 variant polypeptide comprises a sequence set forth in any of SEQ ID NOs: 173, 175, 177, 179, 181, or 183. In some embodiments of the method of inducing hyposensitization to peanuts in a subject allergic to peanuts, the nucleic acid molecule encoding an Arah1 variant polypeptide comprises an amino acid sequence set forth in any of SEQ ID NOs: 68-161, 174, 176, 178, 180, 182, 184, 193, 194, or 211-246.

[0152] In some embodiments of the method of inducing hyposensitization to peanuts in a subject allergic to peanuts, the nucleic acid molecule encodes an Arah2 variant polypeptide comprising one or more amino acid substitution mutations located within a single epitope recognized by an anti-Arah2 antibody. In some embodiments, the nucleic acid comprises a modified nucleic acid encoding an Arah2 variant polypeptide comprising one or more amino acid substitution mutations located within a single epitope recognized by an anti-Arah2 antibody. In some embodiments of the method of inducing hyposensitization to peanuts in a subject allergic to peanuts, the nucleic acid molecule encoding the Arah2 variant polypeptide comprises a sequence set forth in any of SEQ ID NOs: 167 or 169. In some embodiments of the method of inducing hyposensitization to peanuts in a subject allergic to peanuts, the nucleic acid molecule encodes an Arah2 variant polypeptide comprising an amino acid sequence set forth in any of SEQ ID NOs: 10-63, 168, 170, 195-201, 204-210, or 247-249.

[0153] In one embodiment, the composition in the above method is administered orally. In another embodiment, the composition in the above method is administered by a route selected from subcutaneous, intramuscular, intravenous, intranasal, sublingual, topical, rectal, and inhalation. In one embodiment, the subject in the above method is an infant.

[0154] Sublingual immunotherapy using peanut allergens

[0155] There are two main approaches to the treatment of allergies. The first approach is based on reducing allergic inflammation by pharmacological therapy and / or biological agents. The second approach is based on an allergen-specific form of intervention, i.e. allergen-specific immunotherapy (AIT). The main advantages of AIT are its relatively low cost, its high efficacy if performed with high-quality allergens, its long-lasting therapeutic effect even after treatment has been stopped if treatment is performed for more than two years, and its disease-modifying effect that prevents the progression of symptoms from mild to severe.

[0156] Immunotherapy involves administering small amounts of the allergens that cause allergies, which can increase "immunity" or tolerance to the allergens and reduce allergy symptoms. Sublingual immunotherapy (SLIT) is a type of immunotherapy in which droplets or tablets of an allergen extract are placed under the tongue. Many people refer to this procedure as "allergy drops" and it is an alternative to allergy shots. SLIT has been used in Europe for many years and has recently gained interest in the United States.

[0157] Only a few allergy drops are approved by the U.S. Food and Drug Administration (FDA). In 2014, three SLIT products in tablet form were approved by the FDA for the treatment of grass or ragweed allergies. More recently, a SLIT product was approved by the FDA for treating allergic rhinitis and conjunctivitis caused by house dust mites. SLIT is being studied as a potential treatment for peanut allergy. The main drawback of using peanut extract (PE) in SLIT is that it is not as effective as oral immunotherapy (OIT) in achieving desensitization. The amount of protein used in OIT is approximately 100-500 times higher (300-1000 mg per day) compared to the amount of protein used in SLIT (limited to 2-4 mg per tablet). Such a difference in dosage is thought to be the reason why SLIT is not as effective as oral immunotherapy in achieving desensitization of peanut allergy.

[0158] Molecular allergen-specific immunotherapy (AIT) can take several forms, including (i) production of wild-type recombinant allergens that resemble all the properties of the corresponding natural allergens, (ii) synthesis of peptides containing allergen-derived T cell epitopes without IgE reactivity, (iii) use of nucleic acids encoding allergens, and (iv) recombinant and synthetic hypoallergenic derivatives that exhibit significantly reduced IgE binding capacity and allergenic activity but at the same time contain allergen-specific T cell epitopes (e.g. long synthetic peptides, recombinant hypoallergenic allergen derivatives) or contain T cell-helping carrier elements (e.g. B cell epitopes based on peptide carriers) instead of allergen-specific T cell epitopes.

[0159] As used herein, "allergenic" or "allergenic" refers to the ability of an antigen or allergen to induce an aberrant immune response that differs from a normal immune response in that it is an exaggerated response, does not provide a protective / preventative effect, and instead causes physiological dysfunction or tissue damage.

[0160] An important difference between the SLIT method using peanut extract and the SLIT method disclosed herein is the amount of protein that can theoretically be given to a patient. It is well known that the amount of protein applied in immunotherapy by the sublingual route is significantly less (10-100 times less) than that of the oral route. Peanut extract is composed of lipids, carbohydrates, and various proteins, which account for only about 25% of the net weight of the peanut extract. Therefore, the amount of a single protein in the peanut extract is small (e.g., Arah2 accounts for only 6-9% of the total protein). Thus, a SLIT tablet of 2-4 mg of peanut extract contains only about 60 μg of Arah2. In contrast, an orally administered peanut extract in the range of 300 mg to 1000 mg contains about 4-12 mg of Arah2. As a result, even if natural peanut extract is used, it is not possible to inoculate a sufficient amount of Arah2 (about 0.1-1 mg).

[0161] The method of the present disclosure circumvents this obstacle by using recombinant pure protein. In one embodiment, the method of the present disclosure can deliver up to 4 mg of peanut allergen (e.g., Arahl, Arahl2, or variants thereof in a QD or BID regimen), thereby greatly increasing the amount of a particular protein in a SLIT tablet and obtaining much better efficacy without the safety issues of the unique route of administration. The realization that increasing the amount of peanut allergen improves efficacy is not trivial and is considered an innovative step that no one has tried before. In one embodiment, the dose of SLIT for Arahl is about 0.2 mg to about 4 mg. In one embodiment, the dose of SLIT for Arahl is about 0.1 mg to about 4 mg.

[0162] The data presented herein comparing SLIT with OIT (oral immunotherapy) showed that SLIT showed similar clinical allergy desensitization effects as OIT, but with 10-fold less peanut protein. Non-sensitized mice showed a strong anaphylactic hypothermia in response to peanut challenge, but OIT with 500 μg Arah2 or SLIT with 50 μg Arah2 prevented this anaphylactic event.

[0163] This disclosure presents experiments with AraH2 as an example. Those skilled in the art will readily recognize that the methods described herein are equally applicable to other peanut allergens, such as AraH1.

[0164] In one embodiment, the disclosure provides a method of inducing hyposensitization to peanut in a subject allergic to peanut, comprising sublingually administering to the subject a composition comprising about 0.2 mg to about 4 mg of Arahl, thereby inducing hyposensitization to peanut in the subject. In one embodiment, the subject is peanut allergic. In another embodiment, the subject is at risk for peanut allergy. In one embodiment, Arahl is purified from peanut according to methods commonly known in the art. In another embodiment, Arahl is produced by recombinant techniques commonly known in the art. In one embodiment, Arahl comprises an amino acid sequence set forth in any of SEQ ID NOs: 64-67. In another embodiment, the Arahl variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 68-161, 174, 176, 178, 180, 182, 184, 193, 194, or 211-246, or an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 68-161, 174, 176, 178, 180, 182, 184, 193, 194, or 211-246. In one embodiment, the sublingually administered composition is a tablet. In one embodiment, the tablet comprises about 0.2 mg to about 4 mg of Arahl.

[0165] In one embodiment, the disclosure provides a method of inducing hyposensitization to peanut in a subject allergic to peanut, comprising sublingually administering to the subject a composition comprising about 0.2 mg to about 4 mg of Arah2, thereby inducing hyposensitization to peanut in the subject. In one embodiment, the subject is peanut allergic. In another embodiment, the subject is at risk for peanut allergy. In one embodiment, Arah2 is purified from peanut according to methods commonly known in the art. In another embodiment, Arah2 is produced by recombinant techniques commonly known in the art. In one embodiment, Arah2 comprises an amino acid sequence set forth in any of SEQ ID NOs: 1-4. In another embodiment, the Arah2 variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 10-63, 168, 170, 195-201, 204-210, or 247-249, or comprises an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 10-63, 168, 170, 195-201, 204-210, or 247-249. In one embodiment, the sublingually administered composition is a tablet. In one embodiment, the tablet comprises about 0.1 mg to about 4 mg of Arah2.

[0166] In one embodiment, the disclosure provides a method of inducing hyposensitization to peanut in a subject allergic to peanut, comprising sublingually administering to the subject a composition comprising a combination of about 0.2 mg to about 4 mg of Arahl and about 0.1 mg to about 4 mg of Arahl, thereby inducing hyposensitization to peanut in the subject. In one embodiment, the subject is allergic to peanut. In another embodiment, the subject is at risk for peanut allergy. In one embodiment, Arahl and Arahl are purified from peanut according to methods commonly known in the art. In another embodiment, Arahl and Arahl are produced by recombinant techniques commonly known in the art. In one embodiment, Arahl comprises an amino acid sequence set forth in any of SEQ ID NOs: 64-67. In one embodiment, Arahl comprises an amino acid sequence set forth in any of SEQ ID NOs: 1-4. In one embodiment, the composition administered sublingually is a tablet. In one embodiment, the tablet comprises about 0.1 mg to about 4 mg of Arahl.

[0167] In one embodiment, the disclosure provides a method of inducing hyposensitization to peanuts in a subject allergic to peanuts, comprising sublingually administering to the subject a composition comprising about 0.2 mg to about 4 mg of Arahl, thereby reducing an allergic response to peanuts in the subject. In one embodiment, Arahl is purified from peanuts according to methods commonly known in the art. In another embodiment, Arahl is produced by recombinant techniques commonly known in the art. In one embodiment, Arahl comprises an amino acid sequence set forth in any of SEQ ID NOs: 64-67. In one embodiment, the composition administered sublingually is a tablet. In one embodiment, the tablet comprises about 0.2 mg to about 4 mg of Arahl.

[0168] In one embodiment, the disclosure provides a method of reducing an allergic response to peanuts in a subject, comprising sublingually administering to the subject a composition comprising about 0.1 mg to about 4 mg of Arah2, thereby reducing an allergic response to peanuts in the subject. In one embodiment, Arah2 is purified from peanuts according to methods commonly known in the art. In another embodiment, Arah2 is produced by recombinant techniques commonly known in the art. In one embodiment, Arah2 comprises an amino acid sequence set forth in any of SEQ ID NOs: 1-4. In one embodiment, the composition administered sublingually is a tablet. In one embodiment, the tablet comprises about 0.1 mg to about 4 mg of Arah2.

[0169] In one embodiment, the disclosure provides a method for reducing an allergic response to peanuts in a subject, comprising sublingually administering to the subject a composition comprising a combination of about 0.2 mg to about 4 mg of Arahl and about 0.1 mg to about 4 mg of Arahl, thereby reducing an allergic response to peanuts in the subject. In one embodiment, Arahl and Arahl are purified from peanuts according to methods generally known in the art. In another embodiment, Arahl and Arahl are produced by recombinant techniques generally known in the art. In one embodiment, Arahl comprises an amino acid sequence set forth in any of SEQ ID NOs: 64-67. In one embodiment, Arahl comprises an amino acid sequence set forth in any of SEQ ID NOs: 1-4. In one embodiment, the composition administered sublingually is a tablet. In one embodiment, the tablet comprises about 0.1 mg to about 4 mg of Arahl.

[0170] In another embodiment, the present disclosure provides a tablet for sublingual immunotherapy of peanut allergy, the tablet comprising about 0.2 mg to about 4 mg of Arahl. In one embodiment, Arahl is purified from peanuts according to methods generally known in the art. In another embodiment, Arahl is produced by recombinant techniques generally known in the art. In one embodiment, Arahl comprises an amino acid sequence set forth in any of SEQ ID NOs: 64-67.

[0171] In another embodiment, the present disclosure provides a tablet for sublingual immunotherapy of peanut allergy, the tablet comprising about 0.1 mg to about 4 mg of Arah2. In one embodiment, Arah2 is purified from peanuts according to methods generally known in the art. In another embodiment, Arah2 is produced by recombinant techniques generally known in the art. In one embodiment, Arah2 comprises an amino acid sequence set forth in any of SEQ ID NOs: 1-4.

[0172] In another embodiment, the present disclosure provides a tablet for sublingual immunotherapy of peanut allergy, the tablet comprising a combination of about 0.2 mg to about 4 mg of Arahl and about 0.1 mg to about 4 mg of Arahl. In one embodiment, Arahl and Arahl are purified from peanuts according to methods generally known in the art. In another embodiment, Arahl and Arahl are produced by recombinant techniques generally known in the art. In one embodiment, Arahl comprises an amino acid sequence set forth in any of SEQ ID NOs: 64-67. In one embodiment, Arahl comprises an amino acid sequence set forth in any of SEQ ID NOs: 1-4.

[0173] In one embodiment, the present disclosure provides the above tablet for inducing hyposensitization to peanuts in a subject. In one embodiment, the subject has an allergy to peanuts. In another embodiment, the subject is at risk of peanut allergy.

[0174] In one embodiment, the present disclosure provides a tablet as described above for reducing an allergic response to peanuts in a subject.

[0175] In another embodiment, the compositions of the present disclosure can be formulated into a nucleic acid vaccine composition for inducing hyposensitization to peanuts in a subject or reducing an allergic response to peanuts in a subject.

[0176] As used herein, "nucleic acid vaccine" refers to a vaccine or vaccine composition that includes a nucleic acid or nucleic acid molecule (e.g., a polynucleotide) that encodes an allergen or a derivative thereof (e.g., a variant of an Arahl and / or Arah2 protein or polypeptide). In exemplary embodiments, a nucleic acid vaccine includes a ribonucleic acid ("RNA") polynucleotide, a ribonucleic acid ("RNA"), or a ribonucleic acid ("RNA") molecule. Such embodiments may be referred to as a ribonucleic acid ("RNA") vaccine. In some embodiments, a nucleic acid vaccine includes a messenger RNA ("mRNA") polynucleotide, a messenger RNA ("mRNA"), or a messenger RNA ("mRNA") molecule as described herein. Such embodiments may be referred to as a messenger RNA ("mRNA") vaccine. Such a vaccine may include other substances and molecules (e.g., pharmaceutical excipients) that are required or advantageous when it is administered to a patient.

[0177] In one embodiment, the RNA vaccine comprises an RNA sequence encoding an allergen. This RNA sequence may be the sequence of the allergen or may be adapted in terms of its codon usage. By adapting the codon usage, the translational efficiency and half-life of the RNA can be increased. In one embodiment, a poly-A tail containing at least 30 adenosine residues is attached to the 3' end of the RNA to increase the half-life of the RNA. In one embodiment, the 5' end of the RNA is capped with a modified ribonucleotide having the structure m7G(5')ppp(5')N (Cap 0 structure) or its derivatives, which can be incorporated during RNA synthesis or can be enzymatically engineered after RNA transcription using vaccinia virus capping enzyme (VCE; consisting of mRNA triphosphatase, guanylate transferase and guanine-7-methyltransferase), which catalyzes the construction of the N7-monomethylated Cap 0 structure. The Cap 0 structure plays an important role in maintaining the stability and translational efficiency of the RNA vaccine. The 5' cap of the RNA vaccine can be further modified by 2'-O-methyltransferase to generate the Cap 1 structure (m7Gppp[m2'-O]N) to further enhance translation efficiency. A vaccine or vaccine formulation according to the invention can further comprise an adjuvant.

[0178] Plants and products

[0179] In one embodiment, the present disclosure provides a transgenic peanut plant, including a peanut that expresses an Arah1 mutant of the present disclosure.

[0180] In one embodiment, the present disclosure provides a transgenic peanut plant, including a peanut that expresses an Arah2 variant of the present disclosure.

[0181] In one embodiment, a transgenic peanut plant is provided, comprising a peanut expressing a combination of the hypoallergenic Arah1 and Arah2 variants of the present disclosure.

[0182] In one embodiment, the Arah1 variant, the Arah2 variant, or a combination thereof expressed in the transgenic peanut plant described above is expressed from a heterologous nucleic acid.

[0183] In one embodiment, the Arah1 mutant, the Arah2 mutant, or a combination thereof expressed in the transgenic peanut plant described above is endogenously expressed from the transgenic chromosome.

[0184] In some embodiments of the above transgenic peanut plants, expression of the endogenous wild-type Arahl allergen, the endogenous wild-type Arahl2 allergen, or a combination thereof is reduced compared to a non-transgenic peanut plant.

[0185] In some embodiments of the above-described genetically modified peanut plants, the genetically modified plants further express at least one RNA silencing molecule that (i) reduces expression of the endogenous Arah1 allergen, the endogenous Arah2 allergen, or a combination thereof, and (ii) does not reduce expression of the Arah1 variant, the Arah2 variant, or a combination thereof.

[0186] In some embodiments of the above-described transgenic peanut plants, the transgenic plants further express a DNA editing system aimed at reducing expression of the endogenous Arahl allergen, the endogenous Arahl2 allergen, or a combination thereof.

[0187] In one embodiment, the present disclosure provides a processed food product comprising an Arah1 variant of the present disclosure.

[0188] In one embodiment, the present disclosure provides a processed food product comprising an Arah2 variant of the present disclosure.

[0189] In one embodiment, the present disclosure provides a processed food product comprising a combination of the Arah1 and Arah2 variants of the present disclosure.

[0190] In one embodiment, the processed food contains low amounts of endogenous peanut Arahl allergens, endogenous Arahl2 allergens, or a combination thereof.

[0191] In one embodiment, the processed food product comprises peanuts harvested from the genetically modified plant.

[0192] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms of their referents unless the context clearly dictates otherwise. For example, the terms "a compound" or "at least one compound" can include a plurality of compounds, including combinations thereof.

[0193] As used herein, the term "about" indicates a deviation from the stated value of up to 1%, more specifically up to 5%, more specifically up to 10%, more specifically up to 15%, and in some cases up to 20%, the range of deviation including integer values ​​and, where applicable, non-integer values, constituting a continuous range.

[0194] Throughout this application, various embodiments of Arah1 and Arah2 variants and their mutation and / or epitope locations may be presented in a range format. It should be understood that the range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the various embodiments of Arah1 and Arah2 variants and their mutation and / or epitope locations. Thus, any description of a range should be considered to have specifically disclosed all possible subranges within that range as well as each individual numerical value contained within the range. For example, description of a range of 1 to 6 should be considered to have specifically disclosed the subranges of 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, ... as well as each individual numerical value (e.g., 1, 2, 3, 4, 5, and 6) contained within the range. This applies regardless of the breadth of the range.

[0195] Whenever a numerical range is specified herein, it is meant to include all numerical values ​​(fractional or integer) contained within the specified numerical range. The phrases "ranging between" a first specified number and a second specified number, and "ranging from" a first specified number to a second specified number, are used interchangeably herein and are meant to include the first specified number and the second specified number, and all fractional and integer numbers therebetween.

[0196] Working Example

[0197] Example 1: Materials and Methods

[0198] Peptide microarray assay

[0199] To determine the anti-Arahl and anti-Arahl2 epitopes, a Celluspot™ peptide microarray-based immunoassay (Intavis, Cologne, Germany) was performed (Winkler, Dirk FH, Peptide microarrays. Humana Press, 2009). Peptides of 15 amino acids in length and a 4 amino acid offset derived from the primary sequences of the peanut allergens Arahl (UniProt entry P43238, positions 25-626; SEQ ID NO: 64), Arahl2 (UniProt entry Q6PSU2; SEQ ID NO: 1), Arahl3 (UniProt entry O82580), Arahl6 (UniProt entry A5Z1R0), and Arahl8 (UniProt entry Q6VT83) were synthesized and spotted (in duplicate) onto the microarray. Slides were washed overnight at 4°C in blocking buffer (150 mM NaCl, 0.05% Tween, 2.5% skim milk, 50 mM Tris pH 7.5). Slides were then washed and incubated with 3 ml of 6.2 μg / ml single chain variable fragment (scFv) in blocking buffer for 4 h at 4°C on a rotator. For detection, slides were incubated with 3 ml of horseradish peroxidase (HRP)-conjugated goat anti-human IgE (abcam, Cambridge, United Kingdom), diluted 1:10,000 in blocking buffer, and incubated for 2 h at 25°C on a rotator. After washing, a Femtogram HRP Substrate Kit (Azure Biosystem, Dublin, California) was added and chemiluminescence was read on a ChemiDoc (BioRad, Hercules, CA). Peptide array images were processed by an in-house python script that detected peptide spots, normalized their intensities, and reported series of at least two duplicate spots that showed a mean signal across replicates higher than two standard deviations from the slide mean.

[0200] Generation of a human scFv phage display library

[0201] Whole blood samples of 5–20 ml were collected from patients with a clinical diagnosis of peanut allergy using heparin- or EDTA-treated tubes (BD). Peripheral blood mononuclear cells (PBMCs) were extracted from blood samples using Sepmate tubes (STEMCELL) according to the manufacturer's instructions. RNA was extracted from 5–15 × 10 cells using the RNAeasy extraction kit (Qiagen; Hilden, Germany). 6 RNA was purified from PBMCs. cDNA was made from 1–5 μg of RNA (depending on the amount of RNA obtained).

[0202] The entire cDNA reaction was split into PCR reactions to amplify the antibody hypervariable domains of each patient's variable genes. Light chains were amplified using a gene subfamily-specific forward primer with an unstructured, nonspecific overhang followed by a NotI restriction site and a reverse primer specific for the IGLK and IGLL isotypes with homology to the 5' portion of the unstructured linker. Heavy chains were amplified using a gene subfamily-specific forward primer with homology to the 3' portion of the unstructured linker and a reverse primer specific for the IGHG and IGHE genes with an unstructured, nonspecific overhang followed by a NotI restriction site. Primers were described in "Phage display: Methods and Protocols'' (2018) Hust M and List T eds. Springer Protocols. PCR 50 μl reactions were performed using the Phusion Hot Start Taq Polymerase Kit (200 μM dNPT, 2% DMSO, 1.25 M betaine, 1-5 μg cDNA, and 0.5 μM of each primer). Reactions were performed using the following PCR program: 98°C for 3 min, 30 cycles of 98°C for 20 s + 60°C for 60 s + 72°C for 45 s, and a final extension step at 72°C for 10 min.

[0203] The PCR products of each family (VHγ, VHκ, VLκ, and VLλ) were combined and each pool was concentrated by ethanol precipitation, loaded onto a 1% agarose gel, extracted using a gel extraction kit (Qiagen), and washed using Amicon ultra 30K centrifugal filters (Sigma-Aldrich Merck, Israel). A DNA mixture of amplified V gene segments was generated in the following proportions: 45% Vγ, 5% Vε, 25% Vκ, and 25% Vλ. The generation of the combinatorial light-weight scFv library was performed by PCR reactions using the same reagents as the first PCR, but with 100 μl and 100 ng of the V gene mixture per reaction, with a "pull-through" primer (complementary to the overhang adjacent to the restriction site of each product from the first PCR) at a concentration of 250 nM. Multiplex recombination reactions (18-24) were made without primers and PCR was performed using the following program: 98°C for 3 min, 5 cycles of 98°C for 20 s + 60°C for 60 s + 72°C for 60 s. Primers were then added and the reaction was performed using the following program: 98°C for 1 min, 30 cycles of 98°C for 20 s + 67°C for 60 s + 72°C for 45 s, and a final extension step at 72°C for 3 min.

[0204] The PCR product was concentrated by ethanol precipitation, loaded onto a 1% agarose gel, extracted using a gel extraction kit (Qiagen), and cleaned using an Amicon Ultra 30K centrifugal filter (Sigma-Aldrich Merck, Israel). The pLibGD vector (described below) and purified scFv DNA (at least 4 μg of vector and 2 μm of scFv) were restricted using high fidelity NcoI and NotI enzymes (NEB; MA, USA) according to the manufacturer's instructions. The vector was further treated with QuickCIP (NEB) according to the manufacturer's instructions. The restricted vector was extracted from a 1% agarose gel and cleaned by centrifugal filter, similar to the previous steps. The restricted scFv was purified using a PCR purification column (Qiagen).

[0205] A 20 μl ligation reaction was set up according to the manufacturer's instructions using 130 ng of vector and 70 ng of insert (3:1 ratio) and run overnight at 10° C. Ligations of at least 3 μg of DNA in total were performed.

[0206] Ligation libraries were transformed into SS320 electrocompetent bacteria (Lucigen; WI, USA) according to the manufacturer's instructions. Each library was split into two transformations and plated onto three 15 cm 2YT-agar dishes containing 100 μg / ml carbenicillin and 2% glucose. The agar plates were incubated overnight at 30°C. Serial dilutions of the transformations were plated onto separate kanamycin and ampicillin dishes to estimate transformation efficiency. Libraries <107 were considered of sufficient quality and were used further.

[0207] The next day, SS320 were scraped off the agar plate with 6 ml of 2YT, diluted to OD=0.1 in 60 ml of 2YT supplemented with 100 μg / ml carbenicillin and 2% glucose, grown to OD=0.5, and infected with 1:1000 diluted KO7 helper phage (NEB) for 30 minutes at 37° C. The bacteria were then centrifuged at 3000 g for 10 minutes, resuspended in 200 ml of 2YT + 100 μg / ml carbenicillin + 25 μg / ml kanamycin, and grown at 30° C. in a baffled flask with shaking at 250 RPM for at least overnight or up to 24 hours to produce scFv-displaying phage.

[0208] The next day, the cells were centrifuged at 18,000g and 16,000g for 10 min. The supernatant was transferred to a fresh tube and phages were precipitated by adding PEG / NaCl stock (20% PEG-8000, 2.5M NaCl) to a final concentration of 20% (1:4 ratio of PEG-NaCl stock to supernatant). The samples were incubated on ice for 20 min and centrifuged at 18,000g for 30 min at 4°C. The supernatant was discarded and the pellet was centrifuged again for 2 min and the remaining supernatant was removed. The pellet was resuspended in 10 ml PBS / 100 ml culture medium and centrifuged at 18,000g for 10 min to remove remaining bacterial cell debris. The samples were then subjected to a second identical PEG-NaCl precipitation and resuspended in 4 ml PBS / 100 ml culture medium. Samples were centrifuged at 20,000 g for 15 min to remove remaining debris, and purified phages were supplemented with 50% glycerol and 2 mM EDTA and stored at −80° C. until use.

[0209] Screening of phage display libraries for allergen-specific scFv

[0210] Isolation of allergen-specific scFvs was performed by panning the phage libraries with either native purified allergen or recombinant allergen variants with modified suspected epitopes. Maxisorp high-binding 96-well plates (Nunc) were coated (8 wells per library) with 100 μl of 5 μg / ml allergen solution (in PBS) or 2% BSA solution (in PBS). OmniMAX™ bacteria (Thermo Fisher Scientific; MA, USA) were plated in 2YT + tetracycline (5 μg / ml) and grown overnight at 37°C with shaking at 250 RPM.

[0211] The next day, OmniMAX™ bacteria were diluted to 0.1 OD in 2YT+tetracycline and grown at 37°C with shaking at 250 RPM to an OD=0.6-0.8, then stored on ice until use. Phage stocks (2-4 ml) were thawed, purified by PEG-NaCl purification (as above), and resuspended in 1 ml PBST (PBS+0.05% Tween). A sample of the unpanned phage stock was set aside for input measurement. If negative selection was performed, the maxisorp plate was washed with 200 μl / well of PBST x 3, then the phage solution was incubated in BSA-coated wells at 100 μl / well with gentle shaking to remove non-specific binders. The phage solution was then transferred to the allergen-coated wells and incubated at 4°C for 1 hour with gentle shaking. If negative selection was not performed, the phage-PBST solution was added directly to the allergen-coated wells. Plates were then washed twice with 200 μl / well PBST to remove unbound phage. Bound phage were eluted by incubation with 100 μl / well 100 mM HCl for 5 min at room temperature with gentle shaking. The elution reaction was stopped with 12.5 μl / well of Tris 1M, pH 11.

[0212] The eluted samples were added to 5 ml of OmniMAX™ at the required OD and incubated for 30 min at 37°C with shaking at 250 RPM. Panning output titrations were assessed by making serial 10-fold dilutions on samples of the infected stock and plating 5 μl drops in triplicate on LB agar dishes supplemented with carbenicillin, kanamycin, or tetracycline. The remaining output was grown by superinfecting 1:1000 with 1:100 KO7 helper stock and incubating for 45 min at 37°C with shaking at 250 RPM. Superinfected bacterial stocks were passaged into 50 ml of 2YT supplemented with carbenicillin and kanamycin and grown overnight at 37°C with shaking at 250 RPM to produce phage for the next panning. Panning input titration was assessed by infecting serial 10-fold dilutions of the input sample with OmniMAX™ bacteria and incubating at 37°C with shaking at 250 RPM for 30 minutes before plating in triplicate on carbenicillin and kanamycin LB agar plates.

[0213] Subsequent panning rounds were performed by performing a single PEG-NaCl precipitation of the overnight output amplification and using it as input. From one panning round to the next, the stringency of panning was increased (3-4 panning cycles per library) by increasing the number of wash cycles and decreasing the number of panning wells.

[0214] To isolate individual allergen-specific scFvs, output serial dilutions of selected rounds were plated on LB-agar-carbenicillin dishes and grown overnight at 37°C. The next day, individual colonies were inoculated into minitubes containing 300 μl 2YT+carbenicillin+KO7 1:1000 and grown overnight at 37°C with shaking at 250 RPM. The next day, supernatants from the minitubes were analyzed by ELISA using plates coated with allergen or BSA. Supernatant scFvs that specifically bound to allergen but not BSA were amplified by PCR using primers flanking the scFv region of the pLibGD plasmid. PCR products corresponding to full-length scFvs were subjected to standard PCR cleanup with ExoI and rSAP restriction enzymes (NEB) and sequenced by standard Sanger reaction (Hylabs). Unique full-length monoclones were used to generate purified scFvs.

[0215] scFv purification

[0216] The variable regions of the monoclonal antibodies were introduced into scFv polypeptide chains that can be easily expressed in a bacterial expression system. For scFv expression, the scFv was cloned into a LibG plasmid encoding a periplasmic secretion signal and a Flag tag at the N´-terminus and a His tag at the C´-terminus (ST2 secretion signal-scFv-His tag) under the transcriptional control of the Tac promoter. The construct was grown at 37 °C and induction was performed at 20 °C overnight by adding 1 mM IPTG when the cells reached an OD of 0.8-1.0. The cells were harvested (4800g, 20 min) and the cell pellet was resuspended in PBS lysis buffer (1% v / v Triton X-100, 250 U benzonase, 0.2 mM PMSF, 1 mg / ml lysozyme, 10 mM imidazole). The cells were lysed by shaking at 4 °C for 1 h. Lysates were then separated by centrifugation (15000g, 30 min). The supernatant was loaded onto prewashed Ni-NTA beads (PBS containing 10 mM imidazole) and incubated for 1 h at 4°C. The beads were washed with PBS containing increasing concentrations of imidazole (up to 250 mM). The beads were dialyzed overnight at 4°C using SnakeSkin dialysis tubing 3.5 kDa (Thermo Fisher Scientific) and the buffer was exchanged into PBS. ScFvs were concentrated with a 3 kDa centricone (Amicon, Mercury) and their concentration was measured by absorbance at 280 nm.

[0217] Single-cell sorting of allergen-specific B cells

[0218] Peanut allergic patient PBMCs were thawed, washed with PBS and stained for viability analysis (LIVE / DEAD near infrared kit (Thermo-Fisher)) according to the manufacturer's instructions. The cells were then incubated for 1 h on ice with various concentrations of target allergens depending on the type of allergen. The allergens used were either native purified allergens fluorescently labeled with the Alexa-Flor Protein Labeling Kit (a mixture of allergens labeled with two different fluorophores according to the manufacturer's instructions (Thermo-Fisher)), ORwt recombinant allergens HA-tagged at either the C-terminus or N-terminus, or biotin-avidin-labeled wt recombinant allergens (a mixture of allergens labeled with two different fluorophores). The cells were then washed and stained with fluorochrome-conjugated antibodies for the following markers: CD14, CD16, IgM, IgD, CD3, CD19, IgG1. When HA-tagged allergens were used, two anti-HA antibodies with different fluorochrome conjugates were also added. Cells were then washed and sorted by ARIA-III sorting flow cytometry. Single allergen-specific B cells (LIVE / DEADdimCD14-CD16-IgD-IgM-CD3-CD19+IgG1+allergen fluorescent dye double positive) were sorted into 96-well plates containing 4 μl / well of ice-cold lysis buffer (PBS×0.5, 10 mM DTT, 8 U RNase inhibitor). As PCR negative controls, some wells of each plate were left empty.

[0219] Isolation of antibody genes from selected cells and antibody expression

[0220] Single-sorted allergen-specific B cell lysates were directly reverse transcribed (SSIV, Invitrogen, according to the manufacturer's instructions). Two sequential PCR reactions (2nd PCR nested) were performed to amplify the heavy chain genes (Hot Start TAK polymerase, NEB) and the light chain genes (Kapa Hot Start PCRF mix) using a mixture of primers covering the majority of known antibody gene alleles. PCR products were sequenced and aligned to the genome. If a cell had reliable sequences for both the heavy and light chains, the sequences were cloned into a mammalian expression plasmid (pSF) and expressed in HEK-293t cells.

[0221] Generation of yeast surface-displayed mutant saturation libraries and flow cytometric cell sorting

[0222] A library of Arah2 mutants with single mutations at each residue was ordered from TWIST Bioscience (CA, USA) and cloned into the YSD vector (pETCON). To display the Arah2 library on the yeast surface, designated S1, the library was grown in SDCAA selection medium (2% glucose, 0.67% Difco Yeast Nitrogen Base, 0.5% Bactocasa Amino Acids, 0.52% Na2HPO4, 0.856% NaH2PO4·H2O) and induced in galactose medium (for SDCAA, 2% galactose was used instead of glucose) according to established protocols (Chao, G., Lau, W., Hackel, B. et al. Isolating and engineering human antibodies using yeast surface display. Nat Protoc 1, 755-768 (2006)). Arah2 expression was detected by anti-Myc antibody conjugated to FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), and anti-Arah2scFv binding was detected by a secondary anti-FLAG antibody conjugated to APC (Miltenyi Biotec, Bergisch Gladbach, Germany). For pairwise selection screening, approximately 1 × 10 6 Yeast cells were incubated with different anti-Arah2scFv in binding buffer (100 mM Tris, pH=8.0, 1 mM CaCl2, 1% BSA) for 1 h at room temperature. The cells were then washed with binding buffer and incubated with anti-Myc-FITC and anti-FLAG-APC antibodies for 30 min. The cells were then washed again with binding buffer and high and low selectivity mutants were selected by performing several independent sortings using a FACSAria. Arah2 mutants showing high or low binding affinity to anti-Arah2scFv, i.e., mutants that represent the lowest and highest 1% of the total population, were selected and designated mAb_S2_low and mAb_S2_high.

[0223] High-throughput sequencing library preparation

[0224] YSD vectors (pETCON) containing the Arah2 gene were isolated from the naive and selected libraries using Zymoprep Yeast Plasmid Miniprep II (Zymo research, Irvine, CA) according to the manufacturer's protocol. Approximately 200 ng of pETCON was isolated from each yeast library using this kit. The extracted pETCON was sent to the NGS laboratory at Hy Laboratories (Hylabs, Rehovot, Israel) for 20 and 8 cycles of primary and secondary PCR using Fluidigm Access Array primers to add adapters and barcodes. DNA library samples were then purified with AmpureXP beads (Beckman Coulter, Brea, CA) and the concentration of the samples was measured on Qubit using a DNA high sensitivity assay. Samples were pooled and loaded onto a TapeStation (Agilent, Santa Clara, CA) to confirm the size of the PCR products. As a final quality test, the pools were subjected to qRT-PCR to determine the concentration of sequenceable DNA. The pool was then loaded for sequencing on an Illumina Miseq using the 600v2 kit.

[0225] Deep sequencing read analysis

[0226] Paired-end reads were analyzed and filtered for quality using the fastp command line preprocessing tool (Chen, S., Zhou, Y., Chen, Y., & Gu, J. (2018). fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics (Oxford, England), 34(17), i884-i890). Depending on the quality of the whole library, all sequences with a Phred quality score below 20 for more than 10% or 20% of the sequences were excluded from further analysis. Reads were then aligned based on a probability model of overlapping regions implemented in the pandaseq assembler (Masella, AP, Bartram, AK, Truszkowski, JM et al. PANDAseq: paired-end assembler for illumina sequences. BMC Bioinformatics 13, 31 (2012)). Translated sequences were filtered for the occurrence of expected mutations (single mutation per sequence, i.e., single mutation per mutation) and analyzed for sequence enrichment.

[0227]

number

[0228] In the formula, aa i where f is a particular amino acid at position i, f is the proportion of reads of a given amino acid at position i in the sorted library, and f is the same proportion in the input library. This calculation gives the enrichment of each specific Arah2 point variant.

[0229] For convenience, the following can also be expressed as an enrichment index for a particular amino acid at position i:

[0230]

number

[0231] Computing the Shannon entropy for each position provides a synthesis of information about all mutations at a given position.

[0232]

number

[0233] where i is a given position and INaaz represents the augmentation index of the given amino acid, normalized by the augmentation index of all amino acids.

[0234] Purification of AraH1 and AraH2

[0235] For purification of Arah2 and Arah1 mutants, Arah2WT (SEQ ID NO: 2) and its mutants were cloned into the pET28 plasmid, as were Arah1WT (SEQ ID NO: 65) and its mutants. Arah2 was fused to DNA encoding His-tagged Trx and a TEV protease cleavage sequence (Trx-His * 6-TEV site-AraH2). For the AraH1 mutant DNA, Met-TEV-His * A 6-tag DNA sequence was added to the N-terminus, and for some mutants, Met was added as the N-terminus start codon and His was added to the C-terminus. *6 was added. Additional restriction sites were incorporated, if necessary, for restriction cloning. All mutants were expressed under the transcriptional control of the T7 promoter. Cells were grown at 37 °C until an OD of 0.5-0.8 was reached, and induction was performed by adding 1 mM IPTG overnight at 20 °C or for 3 h at 37 °C. Cells were harvested (4800 g, 30 min) and cell pellets were resuspended in lysis buffer (50 mM Tris pH 8.0, 350 mM NaCl, 10% v / v glycerol, 0.2% Triton X-100, 250 U benzonase, 0.2 mM PMSF, and 1 mg / ml lysozyme) and lysed by sonication (35% amplitude, 10 s on, 30 s off, 2 min). The lysate was centrifuged (15000g, 45 min) and the supernatant was loaded onto Ni-NTA beads prewashed with binding buffer (50 mM Tris pH 8.0, 350 mM NaCl, and 10% v / v glycerol) and incubated at 4 °C for 1 h. The beads were washed with binding buffer with increasing imidazole concentrations. For Arah2 purification, the buffer was exchanged into PBS by adding TEV protease to the sample containing Trx-Arah2 protein and dialyzing overnight at 4 °C using SnakeSkin dialysis tubing 3.5 kDa (Thermo Fisher scientific). After TEV cleavage, the Trx-His tagged moiety and TEV protease (also containing the His tag) were removed by loading the solution onto a Ni-NTA column. The flow-through containing Arah2 was collected and concentrated by a 3 kDa centricone (Amicon, Mercury) and the protein concentration was measured by absorbance at 280 nm. For purification of Arah1, an additional gel filtration step on Superdex200 was performed.

[0236] Analysis of binding to monoclonal antibodies by ELISA

[0237] The concentrations of Anti-Arah1 and Anti-Arah2 scFv required to give 50% of the maximum binding to WT-Arah and Arah variants (EC50) were determined by ELISA. Briefly, wells of a 96-well microtiter plate (Thermo Fisher Scientific, Waltham, MA) were coated with 200 ng of Arah2 or Arah1 overnight at 4°C. Plates were blocked with 0.5% BSA in PBS (200 μl / well) for 1 h at room temperature. Anti-Arah scFv variants were made by serial dilution in PBS at a starting concentration of 4 μM, added to the Arah-coated wells and incubated for 1 h at 37°C. After washing, the amount of bound scFv was detected by incubation with goat anti-FLAG conjugated to HRP polyclonal antibody (Abcam, Cambridge, United Kingdom) and TMB substrate.

[0238] All incubation steps were performed in PBS containing 0.5% BSA and 0.05% Tween 20. The highest concentration of Anti-Arah scFv was saturated, and the amount of binding to Arahl or Arahl2 reached a maximum at this concentration.

[0239] Computational design of mutants with multiple mutations

[0240] Based on experimental results that identified point mutations that reduced binding to mAbs and / or patient sera, mutants with combinations of mutations predicted to maintain their stability were generated using the Schrodinger Maestro software suite (Schrodinger, LLC "The Maestro suite of programs: A powerful, all-purpose molecular modeling environment." New York: Schroedinger LLC (2005)). A solved crystal structure of Arah2 (PDB accession 3ob4) was generated for analysis (residues belonging to the MBP protein fused to Arah2 were removed, and the protein creation wizard was used to remove water, optimize hydrogen bonds, and minimize the protein backbone). A residue scanning tool was then used to perform Monte Carlo sampling of up to 5 simultaneous mutations when mutations were combined at the epitope level, and up to 25 simultaneous mutations when mutations were combined at the protein level, allowing for backbone minimization upon side chain mutations and the generation of 250 structures. Mutations were evaluated by ΔG, the change in free energy of the protein upon mutation. To eliminate experimental testing of nearly identical protein sequences, sequences were ranked by ΔG, structures with ΔG>10 were eliminated, and ranked by sequence diversity.

[0241] Degranulation assay of RBL SX-38 cells

[0242] RBL SX-38 cells were received from Professor Stephen Dreskin, University of California, Denver, with permission from BIDMC, Boston. Cells were cultured at 37°C, 5% CO2 in maintenance medium containing 80% MEM, 20% RPMI-1640, 5% FCS (non-heat inactivated) supplemented with L-glutamine, penicillin-streptomycin, and G418 at 1mg / ml (all from Gibco-Thermo fisher, USA). At least 48 hours prior to the assay, cells were split and cultured in assay medium (maintenance medium without RPMI and G418). On the day of the assay, cells were detached using 0.05% trypsin-EDTA (Gibco), centrifuged at 300g for 10 min, and then cultured at 3 × 10 in assay medium supplemented with 5–10% clinical sample (plasma / serum from peanut allergic patients, dilution rate varies from sample to sample). 6The cells were resuspended to a final concentration of 10,000 cells / ml. If plasma was produced with an anticoagulant other than heparin, 30 U / ml heparin (Sodium-Heparin, Sigma) was added to the samples and incubated for 10 min at room temperature before being added to the cells. The cells were then seeded in 96-well flat-bottom tissue culture plates (Greiner bio-one, Austria) at 50 μl / well (final 150,000 cells / well) and cultured overnight. The following day, activation solutions were made by diluting the allergen or an irrelevant protein negative control at various concentrations in Tyrode's buffer. The next day, activation solutions were made by diluting allergens or unrelated protein negative controls at different concentrations in Tyrode's buffer (137 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 0.5 mM MgCl2, 1.4 mM CaCl2, 10 mM Hepes, pH 7.3, 5.6 mM glucose, 0.1% BSA, pH adjusted to 7.4, 80% ddw water composition and 20% D2O deuterium oxide, Merck-Sigma Aldrich, Israel). Cells were then washed three times with Tyrode's buffer made with ddw only and 100 μl of allergen activation solution was added to the appropriate wells (performed in duplicate). For each allergen, 5-6 concentrations were used with 10-fold dilutions. Each clinical sample was tested for WT allergen, mutant allergen and an unrelated protein (KLH, Sigma) as a negative control. Duplicate wells were also prepared with lysis buffer (Tyrode's buffer containing 1% Tritonx-100, Fisher Scientific) to measure total degranulation, and with Tyrode's buffer alone to measure background degranulation. Cells were then incubated for 1 hour at 37°C, 5% CO2. Immediately after incubation, 30 μl of each well was transferred to the corresponding well in a clear non-binding 96-well plate (Greiner Bio-one) and 50 μl of PNAG colorimetric substrate (4-nitrophenyl N-acetyl-β-D-glucosaminide made up in 0.1 M citric acid to a final concentration of 1.368 mg / ml pH 4.5) was added.Reactions were incubated for 1 h at 37°C with gentle shaking in the dark, after which 100 μl of stop solution (0.2 M glycine, pH 10.7) was added to stop the reaction and develop color. Optical density was read at 405 nm for signal and 630 nm for background absorbance using a SynergyLX microplate spectrophotometer reader (Biotek, Vermont). After subtracting background absorbance, net degranulation was calculated by dividing the OD of each cell by the OD of the corresponding lysis buffer well (total degranulation) and subtracting the OD of the buffer only well (background degranulation). EC50 values ​​were calculated for each allergen, and the relative allergenicity of each allergen variant was calculated by dividing its EC50 by the EC50 of the WT allergen. Where an EC50 could not be derived due to low signal, a qualitative analysis was performed.

[0243] BAT assay

[0244] Fresh whole blood samples were dispensed in 100 μl aliquots into heparinized tubes (Biological Industries). Allergens and controls were diluted to 2x stocks in RPMI-1640 (Biological Industries) and added 1:1 to the tubes (final volume 200 μl) and incubated for 30 min in a humidified incubator at 37°C and 5% CO2. The dose range used for each allergen was 1-10000 ng / ml. Crude peanut extract (CPE), fMLP, and anti-human IgE antibodies were used as positive controls. KLH protein was used as a negative control. The reaction was stopped by incubation on ice for 5 min. A cocktail of fluorochrome-conjugated antibodies was added directly to the samples to detect the following markers: CD203c, CD63, HLA-DR, CD45, and CD123. Cells were incubated on ice for 30 min. Red blood cell lysis was performed with a kit (BD FACS lysate) according to the manufacturer's instructions, and cells were washed and analyzed by flow cytometry. Cells were gated for basophil detection and activation percentage (% CD63 positive basophils) was measured. At least 500 basophils were analyzed per tube. EC50 values ​​were calculated for each allergen, and the relative allergenic activity of each allergen variant was calculated by dividing its EC50 by the EC50 of the WT allergen.

[0245] T cell activation assay

[0246] PBMCs were isolated from heparinized peanut-allergic patient blood samples. Cells were washed with PBS, stained with Celltrace violet (Thermo-Fisher) according to the manufacturer's instructions, and plated at 0.2–0.5 × 10 cells / well in 96-well round-bottom plates in X-vivo15 medium supplemented with 5% human AB serum (Biotag) and 1% penicillin-streptomycin solution (Biological Industries). 6Cells were seeded at 100 cells / well (depending on the number of cells available after purification and staining). Recombinant WT and mutant allergens were purified with the Rapid Endotoxin Removal Kit (Abcam), tested for residual endotoxin contamination (LAL Chromogenic Endotoxin Quantitation Kit, Pierce), diluted in the same medium as the cells, sterilized by 0.22 μM filtration, and added to the cells at 200 μl per well to a final concentration of 50 μg / ml. Unactivated wells (baseline, medium only) and each allergen were tested in at least three replicate wells per patient. Each assay included healthy donor samples along with the patients as negative controls for assay quality assurance. Final endotoxin concentrations in wells for all allergens were less than 0.5 EU. Cells were incubated at 37°C in a humidified incubator with 5% CO2 for 7 days. If the medium in any well turned yellow during the culture period, half of the medium was replaced with new medium for all wells. After 7 days, cells were harvested, stained for viability (LIVE / DEAD stain, Thermo-Fisher), stained with anti-CD3 and anti-CD4 fluorochrome-conjugated antibodies (Biolegend; USA), and analyzed by flow cytometry. Live T helper cells were gated (LIVE / DEADlowCD4+CD3+) and the percentage of proliferating cells (Celltracedim / total T helper cells) was measured. A positive result (allergen causes activation of patient T cells) was determined when the mean of allergen-stimulated wells was greater than the mean + 3xSD of unstimulated wells.

[0247] Circular dichroism spectroscopy is a useful technique to analyze the secondary structure and folding properties of proteins in solution using very small amounts of protein. It is based on the difference in absorbance of left and right circularly polarized light by chromophores. CD analysis of proteins is based on information from amide chromophores in the far-UV region (below 240 nm) and aromatic side chains (260-320 nm). For example, α-helical proteins have negative bands at 222 nm and 208 nm and a positive band at 193 nm, while proteins with well-defined antiparallel β-pleated sheets (β-sheets) have negative bands at 218 nm and positive bands at 195 nm.

[0248] Circular dichroism spectra of recombinant Arah proteins were measured on a Chirascan CD spectrometer (Applied Photophysics) at Bar-Ilan University. Far-UVCD spectra were acquired from 200-260 nm wavelengths using a 10 mm path length cuvette. Arah recombinant WT and mutants were measured in PBS buffer and concentrations were measured at 280 nm. To assess stability, spectra were acquired at 25°C and elevated temperatures from 20 to 90°C.

[0249] Strains, plasmids, and growth conditions

[0250] E. coli stable strains (New England Biolabs) were routinely used for all cloning procedures, E. coli OmniMAX™ (Thermo Fisher scientific) were used for screening the phage display library, E. coli BL21(DE3) cells were used for scFv purification, and E. coli Origami or BL21De3 (Novagen) were used for purification of Arah2 and Arah1. ​​All strains were grown at 37°C on 2YT broth and LB agar plates. Phagemids were used for scFvs phage display library and scFv purification from peanut allergic patients. tPCR was used to insert a non-specific scFv derived from a healthy donor and designed with an unstructured GGGSx4 linker, adding restriction sites (NcoI at the 5´ end and NotI at the 3´ end) at either end of the scFv segment (modified plasmids were internally marked LibGD). Plasmid pET28 (Invitrogen) was used for recombinant purification of AraH2, AraH1, and mutants. Transformation for scFv display was performed using SS320 electrocompetent E. coli (Lucigen).

[0251] Example 2: Epitope mapping and de-epitopylation of Arah1 and Arah2 polypeptides

[0252] Objectives: The overall objective is to lay the foundation for defined targeted mutations of allergenic polypeptides that are stable and retain T cell activating activity but reduce binding to IgE allergen antibodies. For immunotherapy purposes, the functionality of the Arah1 and Arah2 mutant polypeptides includes maintaining immunogenicity, for example by the ability to activate T cells. This series of experiments was performed to identify and map conformational and linear epitopes on the peanut allergens Arah1 and Arah2 based on the binding of specific monoclonal antibodies from peanut allergic patient samples. This series of experiments was also performed to identify amino acid residues within the Arah1 and Arah2 mAb binding epitopes that contribute to binding and are not predicted to destabilize the protein when mutated.

[0253] result

[0254] The pipeline for single epitope mapping and de-epitope of the peanut allergens AraH2 and AraH1 includes two steps: (1) discovery of AraH1 and AraH2 specific monoclonal antibodies (mAbs) from peanut allergy patient samples that show specific IgE binding to AraH1 or AraH2 as measured by ELISA assays and peptide arrays (Figure 1), and (2) mapping of the epitopes bound by each antibody (Figure 2). The first step, mAb discovery, was performed using scFv phage display libraries by amplification of variable genes and construction of scFvs fused to pIII protein and displayed on phage, or by single cell sorting of AraH-specific B cells, followed by sequencing of the variable regions and production of recombinant mAbs. Figure 1 is a schematic of the process for identifying the AraH2 antibody. A similar method was used to identify the AraH1 antibody.

[0255] Briefly, scFv phage display libraries were generated from PBMCs of 37 peanut allergic patients as described in Example 1, and after a panning process of these libraries, 35 Arah1-specific mAbs and 42 Arah2-specific mAbs were identified. The scFvmAbs were expressed in E. coli and purified. The epitope mapping procedure shown in Figure 2 below was performed on 19 Arah1 and 10 Arah2 scFVmAbs. From single cell sorting analysis of one patient PBMCs, 14 Arah2-specific mAbs were identified, expressed in HEK293 cells as IgG, and their epitopes were mapped to Arah2.

[0256] In the second step, anti-Arah1 or anti-Arah2 specific purified mAbs were used for epitope mapping in three complementary approaches.

[0257] Approach A: Site saturation mutagenesis by yeast surface display YSD (Siloto and Weselake (2012) Site saturation mutagenesis: Methods and applications in protein engineering. Biocatalysis and Agricultural Biotechnology, Volume 1(3):181-189) (Cherf GM, Cochran JR. (2015) Applications of Yeast Surface Display for Protein Engineering. Methods Mol Biol. 1319:155-75).

[0258] Epitope mapping with the Arah2YSD mutagenesis library: For the purpose of epitope mapping, a two-step procedure was performed. First, the Arah2 point mutant library was sorted for expression only, and mutants that successfully YSDed were collected, resulting in a sorted library called S1. The expression threshold was defined as a fluorescence value higher than unstained cells (background). Each cell with a fluorescence signal higher than background was collected (S1 lib). Next, binding of the S1 library to 56 mAbs was evaluated. Arah2 yeast cells that exhibited Arah2 mutants and showed mAb binding signals (APC) in the bottom and top 1% of the population were sorted (libraries were assigned as S2-mAb-low or S2-mAb-high). See sorting example in Figure 3A. Here, the shaded regions R8 and R9 are FACS gates that define which Arah2-expressing yeast cells to collect based on expression and binding levels (R9: Arah2 point mutants showing high Arah2 binding, R8: mutants showing high expression but low Arah2 binding). In some embodiments, variants that exhibit low Arah2 binding comprise technical features of interest.

[0259] To identify positions that affect binding to specific mAbs, detailed sequencing was performed for each S2-mAb. As the library underwent selection for expression and low mAb binding, the sequencing results were analyzed by enrichment calculations. Each unique DNA sequence encoding a point mutant was counted and the fold change in its relative abundance was calculated to indirectly estimate the change in mAb binding. Representative results of a mapping example are shown in Figure 3B.

[0260] The population of high-affinity Arah2 point mutants was compared to the population of low-affinity mutants, allowing the identification of mutations that are enriched in the low- but not the high-binding population.

[0261] Of the 56 mAbs evaluated, only 22 were successfully mapped. A similar approach using YSD was performed for the Arah1 mutants and mAbs.

[0262] Approach B: Structure-based in silico design of surface-exposed patch mutagenesis (the patch method was used for Arah1 but not Arah2). The core domain of Arah1 (SEQ ID NO:66; amino acids 87-503 of SEQ ID NO:65) has a well-defined trimeric structure. Data on surface exposure (calculated by FreeSASA software, an open-source C library for solvent-accessible surface area calculations (Simon Mitternacht (2016))) were combined with evolutionary conservation to mutate surface-exposed positions without disrupting the trimeric structure. For each mutant generated, a set of 4-7 structurally close surface positions was selected and mutated to alanine if the position was less evolutionarily conserved in multiple sequence alignments, or to amino acids identified between homologs if they were more evolutionarily conserved. Conservation was assessed by BLAST collection of Arah1 homologs using default parameters (Altschul, Stephen F., et al. "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic acids research 25.17 (1997): 3389-3402) and by generating multiple sequence alignments using Clustal Omega (Sievers, Fabian, et al. "Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega." Molecular systems biology 7.1 (2011): 539). Using this surface patch mutagenesis method, conformational epitopes of Arah1 were mapped. All patches were mutated and recombinant mutants were tested for binding to Arah1mAbs by ELISA.

[0263] Conformational epitopes

[0264] At least five conformational epitopes in Arahl were identified: C4: comprising at least residues 84, 87, 88, 96, 99, 419, and 422 of SEQ ID NO:65. C3: comprising at least residues 322, 334, 455, and 464 of SEQ ID NO:65. C1: comprising at least residues 462, 484, 485, 488, 491, and 494 of SEQ ID NO:65. L1: comprising at least residues 194-197 of SEQ ID NO:65. L2: comprising at least residues 287-295 of SEQ ID NO:65.

[0265] At least five conformational epitopes in AraH2 were identified: C3: comprising at least residues 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 80, 97, 99, 100, 102, 103, 104, 105, 107, 108, 109, 110, 111, 112, and 113 of SEQ ID NO:3. C1: comprising at least residues 82, 83, 86, 87, 90, and 92 of SEQ ID NO:3. C2: comprises at least residues 97, 99, 100, 102, 103, 104, 105, 107, 108, 127, 128, 129, 130, 134, 136, 137, 138, 139, 140, 141, 142, and 143 of SEQ ID NO: 3. C4: comprises at least residues 123, 124, 125, 127, 138, 139, 140, 141, 142, 143, and 144 of SEQ ID NO: 3. L4: comprises at least residues 109-115 of SEQ ID NO: 3.

[0266] Approach C: Peptide microarray assays were performed as described in Example 1 using purified mAbs (scFv or IgG) to map some of the continuous epitopes on allergens Arahl and Arahl2 (Figure 2). This method was also used to validate the data from the YSD saturation or patch method for linear epitopes. In Arahl2, two linear epitopes were identified (L1 residues 12-20 of SEQ ID NO:3 and L3 residues 44-69 of SEQ ID NO:3) and confirmed in 14 mAbs analyzed by peptide array. Six linear epitopes were identified in AraH1 (L7: residues 24-30 of SEQ ID NO:65, L6: residues 209-215 of SEQ ID NO:65, L3: residues 331-336 of SEQ ID NO:65, L4: residues 41-422 of SEQ ID NO:65, L5: residues 480-487 of SEQ ID NO:65, and L8: residues 260-267 of SEQ ID NO:65), all of which were confirmed in 13 mAbs analyzed using peptide microarrays. Additional AraH singularity mutation peptide arrays were used to find hot spots of 15mer peptides important for mAb binding.

[0267] IgE epitope mapping and de-epitopation of Arah1 based on sera of allergic patients (see Example 3). Critical position X in 16 epitopes was identified using peptide microarrays, similar to the process of approach C. However, instead of mapping isolated monoclonal antibodies, IgE repertoires from sera of allergic patients were used, as described in Example 3. The linear epitopes identified include: La9: comprises at least residue 12 of SEQ ID NO:65; La16: comprises at least residue 42 of SEQ ID NO:65; La23: comprises at least residue 52 of SEQ ID NO:65; La13: comprises at least residues 57 and 58 of SEQ ID NO:65; La17: comprises at least residue 73 of SEQ ID NO:65; La10: comprises at least residues 231, 234, 238, and 249 of SEQ ID NO:65; La11: comprises at least residue 245 of SEQ ID NO:65; La21: comprises at least residues 278 and 283 of SEQ ID NO:65. La12: comprises at least residues 312 and 318 of SEQ ID NO:65. La22: comprises at least residue 378 of SEQ ID NO:65. La24: comprises at least residue 441 of SEQ ID NO:65. La18: comprises at least residue 443 of SEQ ID NO:58. La14: comprises at least residue 445 of SEQ ID NO:73. La19: comprises at least residue 463 of SEQ ID NO:65. La15: comprises at least residue 500 of SEQ ID NO:65. La20: comprises at least residue 523 of SEQ ID NO:65.

[0268] summary

[0269] Table 1 summarizes embodiments of WT Arah1, amino acid mutations, and Arah1 mutants with mutations at positions relative to their epitopes. Bold text in the left-most column and in the mutation column indicates primary hotspots, and italic text indicates secondary hotspots. Details of the mutations / epitopes shown in Table 1 were collated from the results of Examples 2 and 3.

[0270] [Table 1-1]

[0271]

Table 1-2

[0272]

Table 1-3

[0273]

Table 1-4

[0274]

Table 1-5

[0275]

Table 1-6

[0276]

Table 1-7

[0277]

Table 1-8

[0278]

Table 1-9

[0279]

Table 1-10

[0280]

Table 1-11

[0281] [Table 1-12]

[0282] [Table 1-13]

[0283] Table 2 summarizes embodiments of WT Arah2, amino acid mutations, and Arah2 mutants with mutations at positions relative to their epitopes. Bold in the left-most column indicates primary hotspots, and italicized letters indicate secondary hotspots. Bold in the "Mutation" column indicates mutations in Arah2 mutant B1001.

[0284] [Table 2-1]

[0285] [Table 2-2]

[0286] [Table 2-3]

[0287] [Table 2-4]

[0288] [Table 2-5]

[0289] [Table 2-6]

[0290] Example 3: IgE epitope mapping and de-epitope synthesis based on serum samples from allergy patients

[0291] Objectives: Following the overall objective of laying the foundation for defined targeted mutations of allergenic polypeptides that are stable and retain their functional characteristics but reduce binding to IgE allergen antibodies, the aim of these experiments is to identify continuous (linear) IgE epitopes for peanut patient serum / plasma and to analyze their mutants.

[0292] result

[0293] Using the same peptide arrays as in the purified mAbs analysis, we identified all continuous epitopes on the allergens AraH1 and AraH2 for polyclonal IgE from allergy patient sera. These arrays were analyzed with 250 peanut allergic patient sera and serum-derived IgE binding to AraH1- and AraH2-derived peptides was examined. Of the sera examined, 192 and 168 slides identified IgE binding to at least one peptide derived from AraH1 or AraH2, respectively. Analysis of the peptide arrays and clustering results allowed mapping of all linear epitopes of the protein (data not shown).

[0294] Based on the mapped epitopes, two additional arrays were synthesized and for each epitope mapped to Arahl or Arah2, WT peptide was spotted together with mutated peptides computationally designed to reduce IgE binding. Peptides were 15 amino acids long and contained either point mutations or double substitution mutations. Sera mapped to Arahl or Arah2 were then assayed using arrays containing mutated "de-epitope" spots to screen for peptides that showed the most significant reduction in binding. Representative array results are shown for linear epitope mapping (Figure 5A) and de-epitope (Figure 5B) of Arah2 serum P70. Additionally, the mutation / epitope details shown in Table 1 of Example 2 were collated from the results of both Example 2 and Example 3.

[0295] Example 4: Mutation of single or multiple epitopes

[0296] Objective: Using the data collected in Examples 2 and 3, design mutants with combinations of mutations.

[0297] Results: The combination of mutations was based on computational predictions of the energetic effect of the mutations on the stability of the protein. The calculations were performed starting from the solved structures of Arah2 (PDB accession 3ob4) and Arah1 (PDB accession 3s7i). At this stage, each mutant is mutated in one epitope. In other embodiments, several epitopes can be mutated in a single mutant. In other embodiments, a single mutant has multiple epitopes mutated at once. The mutations include one to seven substitution mutations in an epitope. The designed mutants were produced in E. coli and tested to verify the reduction of binding to the Arah protein by indirect enzyme-linked immunosorbent assay (ELISA). Representative results are shown for Arah1mAbB843 (Figure 4B) and Arah2mAbB536 (Figure 4A), tested against three Arah1 or Arah2 mutants, respectively, mutated in the mapped epitope regions.

[0298] Tables 3-5 show single epitope de-epitopeated Arah2 variants and their effect on specific mAbs. Table 6 shows multiple epitope de-epitopeated Arah2 variants. Table 7 shows single epitope (SEQ ID NO: 68-87) and multiple epitope de-epitopeed variants at once (SEQ ID NO: 88-161, 174, 176, 178, 180, 182, 184, 193, 194, 211-246).

[0299] [Table 3]

[0300] [Table 4]

[0301] [Table 5]

[0302] [Table 6-1]

[0303] [Table 6-2]

[0304] [Table 6-3]

[0305] [Table 7-1]

[0306] [Table 7-2]

[0307] [Table 7-3]

[0308] [Table 7-4]

[0309] summary

[0310] According to the above method, 7 AraH2 epitopes and at least 27 AraH1 epitopes were found. 20 AraH1 and 50 AraH2 single epitope de-epitope mutants were verified by indirect ELISA to show at least a 50% reduction in binding EC50 compared to WT AraH.

[0311] Example 5: Evaluation of allergenicity of modified proteins by ex vivo basophil degranulation assay

[0312] Objective: To evaluate the allergenicity of modified Arah1 and Arah2 variants in comparison with the wild-type protein.

[0313] result

[0314] Based on the results of single-site linear and conformational de-epitope shown in Examples 2-4, mutations that abolish binding to each epitope were combined to construct Arahl (SEQ ID NOs: 68-161, 174, 176, 178, 180, 182, 184, 193, 194, 211-246) and Arah2 (SEQ ID NOs: 10-63, 168, 170, 195-201, 204-210, 247-249) mutants mutated at multiple binding sites (details shown in Table 6). Alternatively, additional sequences were computationally combined by a Monte Carlo method starting from residue-level data to generate protein mutants mutated at multiple sites. The mutations shown in Tables 1-2 above summarize the individual mutation sites.

[0315] This process yielded mutants with reduced allergenicity compared to the WT protein. These recombinant mutants were expressed in E. coli, purified, and tested for allergenicity. First, a wide range of mutants was tested by cell degranulation assays using a humanized rat basophilic leukemia cell line (RBLSX-38) sensitized with peanut allergy patient serum. Representative results of the RBL assay for Arah1 and Arah2 are shown in Figure 6A (Arah2) and Figure 6B (Arah1). The mutant allergens showed a clear reduction in cell degranulation compared to the WT allergen, and it was dramatically reduced for the Arah2 mutant.

[0316] The most promising variants were then evaluated in the Basophil Activation Test (BAT) and compared to the actual native allergen purified from lightly roasted peanut flour. The BAT is a clinical grade test that uses fresh blood from allergy patients to detect and assess the severity of allergies and is becoming the gold standard for allergy diagnosis. Representative BAT results are shown in Figures 7A and 7B. The results show that basophil activation by the major Arah2 variants B764 and B1001 variants is reduced compared to the native allergen in Israeli and American patients, respectively.

[0317] summary

[0318] Based on ex vivo assays in RBL and BAT, a potential loss of allergenicity was observed for several Arah1 and Arah2 mutants with combinations of mutations in multiple epitopes.

[0319] Example 6: Immunogenicity assessment by T cell activation

[0320] Objective: To evaluate the immunogenicity of representative Arahl and Arahl2 variants.

[0321] result

[0322] To ensure the efficacy of immunotherapy, genetically engineered hypoallergenic mutants must retain T cell immunogenicity that allows reprogramming of immune responses. To ensure retention of immunogenicity, we tested the ability of Arah2 mutants to induce allergen-specific proliferation of T helper cells from peripheral blood of peanut allergic patients. Similar analysis was performed for Arah1 mutants. Examples of T cell proliferation assays performed on PBMCs collected from two peanut allergic patients with two representative mutants are shown in Figure 8A and Figure 8B (patients SH409 and B293). Both WT and mutant treated cells showed proliferation above background of untreated cells, suggesting that the mutants retain T cell activation capacity.

[0323] summary

[0324] T cell proliferation assays demonstrated that the two T cell activation properties of Arah2 mutants were preserved, suggesting that immunotherapy with these mutants may be successful.

[0325] Example 7: Biophysical characterization of mutants

[0326] Objective: To ensure correct 3D folding in the mutants, it is important to maintain the same oligomerization level of the native protein (i.e., Arah1 is trimer and Arah2 is monomeric). To verify the oligomerization state of the proteins, size exclusion chromatography (SEC) HPLC was performed for each mutant, and only mutants with the correct oligomerization state were considered valid candidates for the development of hypoallergenic mutants (data not shown).

[0327] Some of the major AraH2 mutants were further analyzed for thermal stability using circular dichroism. Both mutants tested (B764 and B1001) and the WT showed peaks at 208 and 222 nm, characteristic of α-helical content. The thermal melting midpoints (TM) of both mutants and the WT were above 90°C, suggesting high stability and correct folding (Figure 9A-F).

[0328] summary

[0329] Two of the major Arah2 variants showed high melting points in CD, suggesting similar thermal stability to the WT allergen. All combinatorial variants of Arah2 and Arah1 were tested by SEC-HPLC, and the Arah2 variants showed a monomeric mass (~19 kDa) and the Arah1 variants a trimeric mass (~180 kDa).

[0330] Although certain features of the variant hypoallergenic peanut allergens Arahl and Arahl2 have been illustrated and described herein, various modifications, substitutions and equivalents will occur to those skilled in the art. It is therefore to be understood that the appended claims are intended to cover all such modifications and alterations that come within the true spirit of these variations and their uses.

[0331] Example 8: Expression and secretion of allergen variants from mammalian cells

[0332] Objective: To demonstrate that peanut proteins can be expressed, folded, and secreted from mammalian cells based on DNA vectors, and to demonstrate that recombinant de-epitope allergens can be expressed, folded, and secreted from mammalian cells.

[0333] method

[0334] Cloning: DNA vectors for wt peanut allergens Arah2 and Arah1, and de-epitope (DE) Arah2 and Arah1 were synthesized with codon optimization for mammalian cell expression and cloned into pTwistCMVpuro plasmid with HMM+38 leader sequence. For mRNA template vectors, sequences were optimized for in vitro transcription and mammalian expression and synthesized and cloned into their own plasmids. The coding sequence of the mRNA template is flanked by SP6 transcription sites for IVT, TEV' leader UTR, Xenopus β-globin 3' UTR, and a 120-mer polyA templated in the plasmid. Each sequence was cloned with leader sequences from either human IgG kappa light chain, human IgE heavy chain, or human osteonectin (basement membrane protein 40).

[0335] Production of mRNA: All mRNA constructs were produced by Vernal Bioscience Inc. The mRNA used for animal studies was enzymatically capped with cap1 and had all uridines replaced with N1-methyl-pseudouridine.

[0336] Transient cell transfection: Expi293 cells (ThermoFisher Scientific) were transfected according to the manufacturer's protocol. Briefly, cells were transfected at 2.5 × 10 in 25 ml of Expi293 expression medium. 6 Cells were split into 125 ml flasks at 4 x 10 cells / ml. Cells were transfected with 25 μg of DNA complexed with ExpiFectamine complexed in Opti-MEM. The day after transfection, Enhancer 1 and Enhancer 2 were added to the growth medium according to the manufacturer's recommended ratio. ExpiCHO cells (ThermoFisher Scientific) were transfected according to the manufacturer's protocol. Briefly, cells were transfected at 4 x 10 cells / ml in 15 ml of ExpiCHO expression medium. 6 Cells were then transfected with 7.5 μg of DNA complexed with ExpiFectamineCHO reagent. The day after transfection, the growth medium was supplemented with ExpiCHO Enhancer and ExpiCHO Feed according to the manufacturer's recommended ratios.

[0337] Protein purification: Peanut allergens Arah2 and Arah1, as well as de-epitope variants of Arah2 and Arah1, were expressed in transiently transfected cells as described above for 4-5 days, after which the cells were spun down and the clarified medium was dialyzed overnight against 20 mM Tris (pH 8.0), 350 mM NaCl, 5% glycerol. The dialyzed proteins were loaded onto a Ni-NTA resin column equilibrated with Buffer A: 20 mM Tris (pH 8.0), 350 mM NaCl, 10 mM imidazole, washed with Buffer A, and eluted with Buffer A plus 240 mM imidazole. The eluted proteins were then concentrated in a centrifugal concentrator and loaded onto an appropriate size-exclusion column (Superdex75 or Superdex200 for Arahh2 and Arahh1, respectively) equilibrated with PBS. The eluted proteins were analyzed by SDS-PAGE, and appropriate fractions were pooled and concentrated in a centrifugal concentrator.

[0338] Analytical HPLC: Purified recombinant Arah1 and Arah2 were subjected to analytical size-exclusion HPLC and compared to a native peanut allergen standard (INDOOR Biotechnologies) to confirm the correct oligomerization and oxidative folding state. Briefly, approximately 10 μg of protein in 10 μl was injected into a Waters Acquity Arc UHPLC equipped with a BEH200 Å analytical SEC column equilibrated with PBS, and eluted protein was monitored by UV absorbance. Purity and concentration were calculated from the resulting chromatogram traces and used in subsequent experiments.

[0339] Total Mass Spectrometry: For purified recombinant Arah2, the protein was subjected to total mass spectrometry to determine the exact composition and oxidation state. This was performed at the Hebrew University Core Facility Mass Spectrometry Unit. Samples of recombinant Arah2 were buffer exchanged into 20 mM ammonium bicarbonate at pH 9.0 and subjected to ESIMS methodology for accurate mass quantification.

[0340] Allergen antibody binding assay: The ability of allergy sufferer sera or anti-Arah1 or anti-Arah2 antibodies to bind the panel of purified mammalian expressed recombinant peanut allergens was assessed by ELISA. Briefly, plates were coated with 100 μL of 2 μg / ml antigen in PBS and 0.5% BSA in PBS as a negative control. Plates were sealed and incubated overnight at 4°C on a shaker. The coating solution was discarded and 200 μl of PBS + 0.5% BSA blocking solution was added to each well and incubated for 2 hours with shaking. 50 μl of allergy sufferer serum or antibody solution was added to each well and incubated for 1 hour at room temperature. Wells were washed 3 times with PBST and then treated with secondary antibodies: HRP-conjugated anti-human IgE for samples tested with human serum and HRP-conjugated anti-FLAG or HRP-conjugated anti-IgG secondary antibodies for samples assayed with ScFv or IgG antibodies. After incubation with the secondary antibody for 30 min, the wells were washed three times with PBST and reacted with TMB solution. The TMB reaction was quenched and binding was quantified by absorbance at 450 nm.

[0341] result

[0342] The results below demonstrate that peanut allergens Arahl, Arahl2, and their de-epitope variants are expressed at high levels. Figure 10 shows that wild-type or de-epitope peanut allergens Arahl2 and Arahl were expressed and secreted from transfected mammalian cells. Arahl purified from transfected mammalian cells was found to have the correct trimeric fold as shown by HPLC analysis (Figure 13). Total mass measurements shown in Figure 14 show that Arahl from transfected mammalian cells has the exact mass expected from the sequence of transfected Arahl2.

[0343] Mammalian cell-derived allergens retain the ability to bind anti-allergen antibodies, both purified monoclonal antibodies and IgE from allergy patient sera. Figure 11 shows that native Arahl, recombinant E. coli-derived wild-type Arahl, and recombinant HEK cell-derived wild-type Arahl have comparable binding to IgE in allergy patient sera. Figure 12 shows that recombinant Arahl2 and HEK-derived wild-type Arahl have comparable binding to a number of well-characterized anti-Arahl2 monoclonal IgG antibodies.

[0344] Example 9: Preliminary Animal Studies

[0345] Objective: To determine the feasibility of generating an immune response from peanut allergen delivered by mRNA gene therapy and to assay the leader sequence for increased allergen protein secretion.

[0346] Study design

[0347] Thirty-five BALB / c mice were fed exclusively peanut-free diet to prevent the formation of anti-peanut antibodies, and divided into seven groups of five mice each, and each group was intravenously injected six times weekly with 10 μg of a specific mRNA construct (see Table 8) made with Trans-IT-mRNA (Mirus Bio) and DMEM according to the manufacturer's instructions.

[0348] [Table 8]

[0349] Mouse sera were collected at weeks 1, 3, and 5, and the mice were sacrificed at week 7. Serum from each group was analyzed by ELISA for anti-peanut allergen antibody production and for peanut protein itself.

[0350] result

[0351] Delivery of mRNA encoding wild-type peanut allergens generated a B cell response and induced the production of IgG antibodies against WTArah1, but not against WTArah2, as detected by an ELISA assay using native peanut allergens. The detection of such antibodies indicated a B cell response to the secreted allergen proteins and demonstrated that mRNA delivery of peanut allergens is a promising strategy for subsequent experiments with de-epitope-ed allergens for desensitization. Blood serum levels of peanut proteins, as well as anti-allergen IgG levels, were compared among the various leader sequences to indicate the secretion efficiency of each leader sequence. This information was used to determine which leader sequence promoted the most efficient secretion of each peanut allergen. The BM-40 leader sequence showed the highest antibody titers, in good agreement with the expression level pattern observed in mammalian cells using the same constructs.

[0352] Example 10: Allergy model animal test

[0353] Objective: To determine the feasibility and extent of desensitization in sensitized mice by mRNA delivery of de-epitope-modified Arah2.

[0354] Study design

[0355] Mice (female C3H / HeJ70) were first sensitized to peanut by intraperitoneal injection of peanut extract, then divided into 14 cohorts of 5 mice (see Table 9) and intravenously injected weekly or every 3 weeks with 30 μg of wild-type or one of the two major de-epitope Arah2 mutants mRNAs made in Trans-IT-mRNA (Mirus Bio) and DMEM according to the manufacturer's instructions, or with controls.

[0356] [Table 9]

[0357] Serum was collected at weeks 3, 5, and 7 after the first mRNA injection. After termination of mRNA administration (week 7), mice were challenged with peanut extract or purified native Arah2 and the subsequent allergic response was monitored and scored (behavioral, physiological, and serological measures).

[0358] Expected Results

[0359] Hyposensitization was considered successful if, after administration of de-epitope-modified Arah2, mice showed a statistically significant reduction in clinical parameter scores, e.g., anaphylaxis, reduced levels of mouse mast cell proteases, and reduced relative levels of anti-Arah2 IgE.

[0360] While certain features of the nucleic acids encoding the variant hypoallergenic peanut allergens Arahl and Arahl2 have been illustrated and described herein, various modifications, substitutions and equivalents will occur to those skilled in the art. It is therefore to be understood that the appended claims are intended to cover all modifications and alterations that come within the true spirit of these variations and their uses.

[0361] Example 11: Characterization of the biological activity of AraH2 variant B1001 (SEQ ID NO: 10)

[0362] Objective: To demonstrate the biological activity of the AraH2 mutant B1001

[0363] method:

[0364] Expression and purification of recombinant Arah2

[0365] Recombinant Ara h 2 sequences (WT or B1001) were cloned into pET28 plasmid and fused to a sequence encoding His-tagged TRX protein and a TEV protease cleavage site (N-Trx-His X6-TEV site-Ara h 2-C). Plasmids were transformed into ORIGAMI™ cells (New England Labs) to express the protein under the transcriptional control of the T7 promoter. Cells were grown at 37 °C with shaking at 250 RPM until an OD of 0.5-0.8 was reached, and induction was performed by addition of 1 mM IPTG and incubation at 37 °C for an additional 3 h. Cells were pelleted (4800g for 30 min) and resuspended in 10x (w / v) lysis buffer (50mM Tris pH 8.0, 350mM NaCl, 10% v / v glycerol, 0.2% TritonX-100, 5U / ml benzonase (Sigma), 0.2m MPMSF (Thermos-Fisher Scientific), 1mg / ml lysozyme (Angene)). Cells were disrupted by sonication (60% amplitude, 10s on, 30s off, 2min). The lysate was centrifuged (15000g, 45min) and the supernatant was loaded onto a Ni-NTA column pre-washed with binding buffer (50mM Tris pH 8.0, 350mM NaCl, 10% v / v glycerol). The beads were washed with binding buffer containing increasing concentrations of imidazole, and individual fractions were collected and analyzed by SDS-PAGE. Fractions containing the protein of interest were pooled. The buffer was exchanged into imidazole-free binding buffer by dialysis overnight at room temperature using SnakeSkin dialysis tubing 3.5 kDa (Thermo Fisher Scientific). The next day, His-tagged TEV protease (TEV:rArah2) was added to the sample at a 1:30 molar ratio and incubated at room temperature for 3 h. The solution was loaded onto a Ni-NTA column prewashed with binding buffer to remove the Trx-His tagged moiety and TEV protease.Flow-through and 20 mM imidazole wash fractions containing Arah2 were collected, concentrated to approximately 5 mg / ml by a 3 kDa centricone (Amicon, Mercury) and loaded onto a Superdex 75 pgSEC column prewashed with PBS buffer (Cytiva). Fractions containing monomeric Arah2 were pooled and the concentration was measured and calculated from the absorbance at 280 nm using the elongation factor (0.817 for WT and 0.672 for B1001). Proteins were flash frozen in liquid nitrogen and stored at -80 °C until use.

[0366] Circular dichroism

[0367] Purified Arah2 proteins (WT and B1001) were diluted to 0.3 mg / ml in PBS buffer and subjected to circular dichroism (CD) analysis (Chirascan™-plus CD Spectrometer) for the spectrum from 200 to 260 nm at room temperature. For the thermal stability analysis of the secondary structure, CD spectra (200 to 260 nm) were recorded under the following conditions: temperature was increased from 20 to 90 °C at a rate of 1 °C / min and a path length of 1 mm.

[0368] Western blot analysis

[0369] 2 μg of purified protein was mixed with Leammeli sample buffer containing β-mercaptoethanol (Bio Rad) and loaded onto unstained Min-Protean TGX gels (Bio Rad). Prior to transfer, gels were visualized on a Molecular Imager® ChemiDoc™ XRS+ (Bio Rad) and transferred to a Transblot turbo PVDF membrane (Bio Rad). Blocking was performed with 5% nonfat milk (Sigma) in PBST for 1 h at room temperature. Detection of Arah2 was performed using PAb rabbit anti-Arah2 (Indoor) at 1:1000 and secondary antibody anti-rabbit HRP at 1:10000. Femtogram ECL substrate was used for visualization of bands.

[0370] SEC- and RP-HPLC

[0371] Purified native Arah2 (Indoor), WTArah2 and B1001Arah2 were analyzed by SEC-HPLC at 30°C (UHPLC Arc System, Waters; Column XBrige Protein BEH SEC 200A, 2.5 μm in 0.1 M sodium phosphate buffer as mobile phase). Molecular weights were estimated based on gel filtration molecular weight standards (Biorad). Proteins were also analyzed by RP-HPLC at 50°C using a C18 column, 0.1% TFA in HPLC grade water as mobile phase A and 0.1% TFA in acetonitrile as mobile phase B (UHPLC Arc System, Waters; Column Jupiter 5um C18 300A, 250x4.6 mm). For both, analytical detection was performed at UV 220 nm.

[0372] Samples for allergy sufferers

[0373] All samples were obtained from patients with a recent history of allergic reactions to peanuts and clinically diagnosed with peanut allergy. All collaborating medical centers obtained local institutional review board approval to donate samples for this study. Whole blood was collected from peanut-allergic patients at collaborating medical centers in Israel using heparinized tubes. Plasma was isolated by centrifugation at 800 g for 10 min and separation of the upper phase. Peripheral blood mononuclear cells (PBMCs) were extracted from blood samples using Sepmate tubes (Stemcell, Canada) according to the manufacturer's instructions and cryopreserved using endotoxin-free materials: FBS (Biological industries, ISR), PBSx10 pH 7.4 (Gibco, US), ultrapure ddw (Bioline, ISR), and Lymphoprep (Stemcell). Fresh whole blood for basophil activation testing was also obtained from clinical centers across the United States by Amerimmune.

[0374] Additional plasma, serum (separated by gel-phase lock tubes), and PBMCs were obtained using equivalent isolation techniques from the following partners and providers: Nadeau Laboratory, Stanford University (CA, USA), Abbaltis (UK), Ebisawa Laboratory, Jikei University (Tokyo, Japan), Niño Jesus University Hospital (Madrid, Spain), Access biologicals (CA, USA), Amerimmune (VA, USA), and Mie University Hospital (Japan). Additional information on clinical samples is provided in the Supplementary Data.

[0375] Analysis of IgE and IgG levels by ELISA

[0376] Maxisorp 96-well plates (Thermo Fisher Scientific) were coated overnight at 4°C with 100 μl of native Arah2 or B1001 at 2 μg / ml in PBS. All subsequent steps were performed at room temperature with PBST washes (PBS+0.05% Tween20) between steps. Titration curves were generated for each serum or plasma sample at a 10-fold dilution followed by serial 2.1-fold dilutions (for IgE detection) or a 25-fold dilution followed by serial 2.5-fold dilutions (for IgG). Plates were blocked with PBST+2%BSA (Sigma) for 2 hours, incubated with titrated samples or no sample (blank) for 2 hours, then incubated with HRP-goat anti-human IgE at 1:5,000 (Abcam) or HRP-donkey anti-human IgG at 1:20,000 (Jackson labs) for 1 hour. Finally, the plates were incubated with 100 μl of 1-Step Ultra TMB (Thermofisher) until color development and the reaction was stopped by adding 100 μl of 0.5 M H2SO4. The optical density at 450 nm was recorded using a Synergy LX microplate spectrophotometer (Biotek, Vermont), the OD of blank wells (no sample) was subtracted, and the area under the curve was calculated using Prism Graphpad.

[0377] Degranulation assay of RBL SX-38 cells

[0378] RBL SX-38 cells were received from Professor Stephen Dreskin, University of California, Denver, with permission from BIDMC, Boston. Cells were cultured in ventilated flasks (Greiner) in maintenance medium containing 80% MEM (Gibco, US), 20% RPMI-1640, 5% FCS (non-heat inactivated), 2 mM L-glutamine, penicillin-streptomycin (Biological industries, ISR), and 1 mg / ml G418 (Formedium, UK) at 37°C and 5% CO2. Cells were split and cultured in assay medium (without RPMI and G418) for 48 h. On the day of the assay, cells were detached using 0.05% trypsin-EDTA (Gibco), centrifuged at 300 g for 10 min, and aliquots of 250 × 10 cells were cultured in assay medium supplemented with 10% clinical sample (plasma or serum from peanut allergy patients). 5The cells were resuspended to 10 cells / ml. Non-heparinized plasma was supplemented with 30 U / ml sodium-heparin (Sigma) and incubated at room temperature for 10 min before being added to the cells to prevent clotting. The next day, native Arah2, B1001, or negative control (KLH, Sigma) were serially diluted 10-fold in Tyrode's buffer to create the activation solution. Buffer composition: 137 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 0.5 mM MgCl2, 1.4 mM CaCl2, 10 mM Hepes (pH 7.3), 5.6 mM glucose, 0.1% BSA (Sigma Aldrich, ISR), pH adjusted to 7.4, 80% ddw, 20% D2O deuterium oxide (Sigma Aldrich) water composition. Cells were washed three times with Tyrode's buffer made up of ddw only, and 100 μl of activation solution was added to the appropriate wells (in duplicate). Duplicate wells were also made up with lysis buffer (Tyrode's buffer containing 1% Tritonx-100, Fisher Scientific) to measure total degranulation, and with Tyrode's buffer alone to measure baseline degranulation. Cells were then incubated for 1 hour at 37°C and 5% CO2. Immediately after incubation, 30 μl of each well was transferred to the corresponding well in a clear non-binding 96-well plate (Greiner Bio-one) and 50 μl of the colorimetric substrate 4-nitrophenyl N-acetyl-β-D-glucosaminide (Sigma) made up to a final concentration of 1.368 mg / ml in 0.1 M citric acid, pH 4.5 was added. After gentle shaking for 1 hour at 37°C, the reaction was stopped with 100 μl of glycine, pH 10.7. Optical density was read at 405 nm for signal and 630 nm for background absorbance. % net degranulation was calculated by subtracting background absorbance, subtracting baseline degranulation, and dividing by total degranulation count.

[0379] Basophil activation test

[0380] Fresh whole blood was dispensed in 100 μl aliquots into heparinized tubes (individual FACS tubes or 96-well plates at 2 ml depth). Allergens and controls were diluted in RPMI-1640 (Biological Industries) to 2x stocks and added 1:1 to the tubes (final volume 200 μl). Doses used ranged from 0.03 to 105 ng / ml, with doses at 10x or 3x intermediate steps (1, 3, 10, 30 etc.) depending on available volume, but at 6-10x or higher concentrations. Crude peanut extract (CPE), fMLP (Sigma), and goat serum anti-human IgE antibody (kindly provided by Dreskin Laboratory) were used as positive controls, and KLH (Sigma) was used as a negative control at 105 ng / ml (6 concentrations of CPE if volume was available). Samples were incubated for 30 min in a humidified incubator at 37 °C, 5% CO2, followed by 5 min incubation on ice to stop the reaction. Samples were then stained for 30 min on ice with fluorochrome-conjugated antibodies against the following markers: CD203c, CD63, HLA-DR, CD45, CD123 (Biolegend). Red blood cell lysis was performed using lysis solution (BDFACS) according to the manufacturer's instructions, cells were washed with PBSx1 and analyzed by flow cytometry. Cells were gated for basophil detection (cells > singlets > CD45 high / SSC low > CD123 high / HLA-DR low > CD203c high) and activation percentage (% CD63 positive basophils) was measured. At least 500 basophils were analyzed per tube, and a baseline was set by gating on non-activated wells. Only samples that showed 5% or more activation at at least one concentration of Arah2 or CPE were included in the analysis. Patient averaging and curve fitting was performed in Prism Graphpad.

[0381] T cell activation assays: proliferation assay and cytokine ELISA

[0382] All materials used were confirmed to be endotoxin-free. Peptide pools covering the entire sequence of WTArah2 or B1001 (35–41 mers with 20 AA overlap, Peptide 2.0, VA, USA) were generated in DMSO (Alfa Aesar, MA, USA). Peanut-allergic patient PBMCs were stained with 10 μM Celltrace violet (Thermo-Fisher) in PBS+0.5% FBS for 20 min at 37°C, then quenched with RPMI+5% FBS for 5 min. Cells were washed, resuspended in X-vivo15 medium (Lonza, Switzerland) supplemented with 1% penicillin-streptomycin (Biological industries), and plated at 2–2.5 × 10 cells / ml in 96-well round-bottom plates. 5 Cells were seeded per well and 4–8 replicates (according to the number of cells available). Peptide pools were added to wells at 200 μl / well to a final concentration of 10 μg / ml per peptide. Equivalent dilutions were added to non-stimulated wells and CPE was used as a positive control. Cells were incubated for 7 days in a humidified incubator at 37°C and 5% CO2. Afterwards, cells were pelleted, medium was removed and kept for cytokine ELISA. Collected cells were stained with a viability dye (near-infrared LIVE / DEAD stain, Thermo-Fisher) and then stained for CD3 and CD4 with fluorescent dye-conjugated antibodies (Biolegend, USA) and analyzed by flow cytometry. Cells were gated for live and proliferating T helper (singlet>LIVE / DEADlow>CD4high / CD3high>Celltrace dim, final proliferation gating was derived by baseline and positive control samples). Proliferation %, Stimulation Index (SI, mean allergen activation / mean baseline) Mann-Whitney U test (MW) significance was calculated (Graphpad Prism). Each sample was considered as true activation and included in the data only if WT SI ≥ 2 and MW p-value ≤ 0.1. For final mean values, data for each patient were normalized to the value of one of the baseline replicates.

[0383] For detection of IL5, IL13, and IFNγ levels in the maintenance medium, ELISAs were performed using unconjugated / biotinylated antibody pairs (Mabtech, Sweden) optimized for sandwich ELISA. Maxisorp plates were coated overnight at 4°C with 50 μl of unconjugated capture antibody at 1 μg / ml in bicarbonate buffer (Sigma). The next day, standard curves were generated with recombinant IL5, IL13, or IFNγ (Peprotech, ISR) in PBST-2% BSA. Plates were blocked with PBST+2% BSA for 2 h at room temperature and then incubated overnight at 4°C with 50 μl of assay medium or appropriate standards. On the final day, plates were incubated for 1 h with biotin-conjugated detection antibody at 1 μg / ml in PBST+2% BSA, followed by incubation for 1 h with HRP-conjugated streptavidin. Finally, the plates were incubated with 100 μl of 1-Step Ultra TMB until color development, the reaction was stopped by adding 100 μl of 0.5 M H2SO4, and the optical density was recorded at 450 nm. The standard curve was fitted with a nonlinear regression model and used to interpolate the individual values. SI of WT and p-values ​​of MW were calculated, and only truly activated samples (SI ≥ 2, p-value ≤ 0.1) were included in the data. For the final mean values, data of each patient were normalized to the value of one of the baseline replicates.

[0384] Mouse allergy model research

[0385] All mouse studies were performed as a contract study of Porsolt SAS (France). Naive female C3H / HeJ mice (Jackson Laboratory, Bar Harbor, US) were maintained on a peanut- and soy-free diet until 3 weeks of age. They were orally administered 2 mg of defatted peanut flour (50% protein) mixed with 10 μg of the mucosal adjuvant cholera toxin (List Laboratories, CA, USA) in 250 μl of PBS once a week for 4 weeks, with a final dose of 4 mg, doubled. Safety studies: Mice were administered native Arah2 or B1001 in a final volume of 250 μl by intraperitoneal injection. The next day, B1001-altered mice were randomized into two subgroups and were again administered a higher dose of Arah2 or B1001. Body temperature was measured rectally at baseline and 10, 20, 30, 45, 60, and 120 min after each dose. Anaphylactic symptoms were assessed 120 min after each dose using the following common clinical scoring system (0 - no clinical signs; 1: edema / swelling around eyes and mouth; 2: decreased activity; 3: prone position, immobile for >1 min; 4: no response to whisker stimulation, little or no response to prodding; 5: endpoint: tremors, convulsions, death).

[0386] Oral immunotherapy (OIT) study: Mice were sensitized as in the safety study, and another control was left untreated. After sensitization, mice were given 350 μg peanut flour in 250 μl PBS ip, and mice with clinical scores of 2 or more or whose body temperature did not fall by 1.5°C or more were excluded from the study. Starting 2 weeks after the last sensitization dose, mice were desensitized by orally administering PBS (sham OIT), 15 mg peanut flour in 250 μl PBS, or 1000 μg B1001 in 1000 μl PBS (divided twice a day to avoid single doses of more than 500 μl) 5 times a week for 3 weeks. Twelve days after the last desensitization dose, mice were given 35 μg native Arah2 in 250 μl PBS by ip injection, and anaphylaxis scoring and body temperature were recorded as above. Surviving mice were sacrificed 5 days later, and mesenteric lymph nodes (MLNs) were harvested and transferred to ice-cold PBS supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin (Pen / strp mix). MLNs were cut into small pieces, homogenized using a GentleMACS dissociator, and cells were isolated by passing the homogenate through a 70 μM cell strainer pre-wetted with TexMACS medium (Miltenyi Biotec). MLN cells were then seeded (400,000 cells / 100 μl) in 96-well U-bottom plates for 72 h in TexMACS medium containing pen / strep mix with 10% FBS and 200 μg / ml native Arah2. Culture medium was harvested and IL-4, IL-5, and IL-13 levels were measured using a Luminex panel assay (ProcartaPlex, ThermoFisher Scientific) according to the manufacturer's instructions. Data were analyzed with Bio-Plex Manager software (Biorad) and concentrations were calculated using standard curves of the corresponding cytokines (values ​​within the detection range were corrected to 0). All data were analyzed for significance by the Mann-Whitney U test.

[0387] result

[0388] Figure 15 shows a general overview of a patient sample-based pipeline for allergen de-epitope. In one embodiment, peripheral blood mononuclear cells (PBMCs) and plasma or serum are isolated from blood samples of clinically confirmed allergy patients of diverse backgrounds. Fresh blood is provided by collaborating Israeli medical facilities, and processed or frozen isolates are obtained from various locations around the world (obtained through collaborations with academic and clinical institutions or purchased from licensed commercial clinical sample donors). Naturally occurring allergen-specific B cell clones were isolated from patient PBMCs by generating and screening combinatorial scFv antibody phage display libraries or by single cell sorting flow cytometry. These clones were used to generate patient-derived allergen-specific monoclonal antibodies (mAbs). Confirmatory epitopes were then mapped by generating yeast-displayed allergen mutant saturation libraries and screening with allergen-specific mAbs. Linear epitope mapping was performed by analyzing the binding of IgE or mAbs in patient serum / plasma to peptide arrays representing sliding window coverage or the entire sequence of the allergen. The comprehensive mapping process and proprietary bioinformatics process described herein are applied to carefully designed allergen variants with the least possible modifications. These variants were recombinantly expressed and biochemically characterized to verify stability and overall structural similarity to the native allergen. The IgE binding and allergenicity of well-folded variants and WT allergen were then compared by ELISA and RBL-SX38 assays using patient serum / plasma. The lead candidate variants were then used as input for further iterations of the design-validation process until variants with substantially reduced allergenicity were obtained.

[0389] Biochemical characterization of the Arah2 mutant B1001

[0390] After mapping and de-epitope-ization of AraH2, we determined the minimal number and identity of mutations required to substantially increase safety while retaining the basic identity. These mutations were combined to ultimately design and express a mutant with 80% sequence identity to WT AraH2. The biochemical properties of this novel mutant, designated B1001, were evaluated.

[0391] Its primary identity to Arah2 was confirmed by Western blot analysis (Figure 16A) using a commercially available Arah2-specific rabbit polyclonal antibody (pAb). Indeed, the pAb bound specifically to native Arah2 (two bands corresponding to known isomers), recombinantly expressed WTArah2 and B1001 alike (Figure 16A, right panel). Recombinant WTArah1 was also used as a peanut-related negative control, and BSA was used as a general negative control (lanes 4, 5), and the pAbs did not bind to either. Loading of separated protein controls prior to Western blot analysis confirmed the visible presence of all five proteins on the membrane (Figure 16A, left panel).

[0392] Arah2 is a 17 kDa monomeric protein, composed mostly of α-helices, with four disulfide bonds, and showed a remarkably high thermal stability. Using size-exclusion HPLC, the molecular weight and oligomeric state of B1001 were estimated and its profile was compared with recombinant WT and native Arah2. All three proteins had similar retention times and estimated molecular weights of 17-18 kDa (Figure 16B). The secondary structure of B1001 was then examined by circular dichroism (CD) and compared with the WT protein. Both proteins showed typical α-helical spectra (Figure 16C, left panel), similar to the native protein. The thermal stability of the secondary structure of B1001 and WTArah2 was investigated by performing CD at gradually increasing temperatures (20-90 °C). Both proteins retained their secondary structure even at 90 °C (Figure 16C, right panel) (as previously shown for Arah2).

[0393] In summary, we found that the B1001 mutant folds into a stable monomer with molecular weight, secondary structure, and thermal stability comparable to that of the WT Arah2 protein. Furthermore, we found that B1001 exhibits clear immunocross-reactivity to Arah2 by binding to an Arah2-specific pAb. These results imply that B1001 retains the essential identity of the Arah2 mutant.

[0394] Differentially reduced patient antibody binding to the Arah2 mutant B1001

[0395] Engineering an allergen to make it less allergenic for immunotherapy is of course likely to compromise its immunogenicity, and therefore requires a compromise between these opposing outcomes. At the level of epitope-antibody interactions, this means striking a balance between reducing binding of pathogenic IgE and retaining essential identity to the native allergen such that IgG binding capacity is preserved.

[0396] To investigate how the modifications altered IgE and IgG binding, we performed ELISA assays. Plates coated with native Arah2 or B1001 were incubated with serially diluted plasma or serum from 24 peanut-allergic patients. The resulting curves were significantly different in shape, which was expected considering the complex interactions between the multiple factors that shape each patient's antibody repertoire. This suggests that comparing binding at a single dilution or deriving an EC50 value may provide a partial and potentially misleading indication. Therefore, we compared the differences in area-under-the-curve (AUC) values. Although the AUC values ​​are not clinically interpretable, they are not subject to local bias or distortion due to fitting of a regression model.

[0397] We found that both IgE and IgG fractions had a significant reduction in binding to B1001, although this reduction was significantly slower in the IgG fraction (Figure 17A, Wilcoxon rank-sum test P value < 0.0001). Indeed, when observing the individual [AUC of B1001 / AUC of Arah2] ratios, the reduction was more pronounced in the IgE fraction than in the IgG fraction in all patients, with median ratios of 0.173 for the IgE fraction and 0.593 for the IgG fraction (Figure 17B, Wilcoxon P value < 0.0001). These results indicate that the Arah2 de-epitopation process was successful in selectively reducing IgE binding sites over IgG binding sites.

[0398] The allergens in B1001 were significantly reduced compared to the native AraH2.

[0399] The overall binding strength of a patient's IgE repertoire to an allergen is shaped by multiple factors, including antigen-specific titers, clonal diversity, and the binding strength of individual clones. However, the allergenicity of a molecule may be affected to different degrees by these factors and is a separate property that cannot be easily predicted from simple binding assays. Other important factors that influence allergenicity include, among others, the patient's relative allergen-specific IgE titers and the binding of specific epitopes that are sterically compatible with effector cell activation. Thus, reduced IgE binding may or may not indicate reduced allergenicity and must be considered separately.

[0400] We tested the ability of B1001 to activate the humanized rat basophil-like RBLSX-38 cell line. This widely used cell line can be sensitized with human patient samples to respond to allergen stimuli by cell degranulation. The rate of degranulation is proportional to the allergenicity of the stimulating molecule and can be measured by an enzymatic reaction with a colorimetric substrate of the granulocyte enzyme β-hexosaminidase. After sensitizing RBLSX-38 cells overnight with 1:10 plasma or serum from 28 clinically validated peanut allergic patients of various backgrounds, we stimulated the cells with either 0.01–10,000 ng / ml of AraH2, B1001, or the unrelated negative control protein keyhole limpet hemocyanin (KLH). Surprisingly, plotting the point-wise average values ​​showed that B1001 essentially lost all its ability to induce RBL degranulation across the entire concentration range tested, displaying a similar unresponsiveness to KLH (Figure 18A). In fact, in all samples tested, the response to B1001 was reduced by more than 1000-fold compared to native AraH2 (data not shown). These results demonstrated that the de-epitope process of B1001 dramatically reduced its allergenicity.

[0401] The RBL assay allows for high-throughput comparison of multiple variants using multiple patient samples and is a powerful tool for engineering and validation of modified allergens. However, the sensitivity and accuracy of this assay in predicting patient responses may be limited by several factors, such as human serum cytotoxicity on rat cells, variability in the number of surface FcεRI molecules and the absence of human FcεRIβ subunits, the absence of human IgG receptors, and the lack of an individual's immune background. On the other hand, the Basophil Activation Test (BAT) is a well-established cytometric assay favored by physicians and researchers for its accuracy, sensitivity, and ability to provide clinical predictive data. To test the safety of B1001 compared to Arah2, the BAT assay was performed in a cohort of 44 peanut-allergic patients from Israel and the United States, using a commonly accepted protocol, with allergen concentrations ranging from 0.03 to 10,000 ng / ml (the range and number of test points per patient will vary depending on the amount of blood available). The relative allergenicity of both proteins was estimated by plotting the average values ​​per point, fitting the resulting curves to a four-parameter logistic regression model, and extracting the EC50 value for each curve. The EC50 of Arah2 was 39.3, and the EC50 of B1001 was 11,986, indicating that on average, B1001 is approximately 300 times less allergenic than Arah2 (Figure 18B). These results indicated that the safety of B1001 is higher than that of Arah2, supporting the application of B1001 in immunotherapy.

[0402] T cell immunogenicity of B1001

[0403] It is well established that immunotherapy relies on the reprogramming of pre-existing allergen-specific T helper clones from a Th2A to a Th1 / iTreg phenotype. This is thought to be achieved by careful exposure to suballergenic doses that trigger chronic activation of these clones without the original Th2A skewing. Thus, for a modified allergen to be an effective immunotherapeutic drug, it must retain at least some immunogenicity for pre-existing allergen-specific Th clones. Thus far, in vitro T cell activation assays have not been calibrated to reliably correlate to any predictable degree with clinical efficacy. However, such assays remain a robust approach to assess whether a molecule's immunogenicity has been definitively compromised.

[0404] To confirm that the modification does not abolish the immunogenicity of B1001 T cells, peripheral blood T cells from peanut allergic patients were treated with a proliferation detection dye and stimulated with a pool of overlapping peptides containing the entire sequence of either unmodified Arah2 or B1001. Both cells were then kept for cell proliferation analysis, and the medium was kept for sandwich ELISA analysis of secretion of Th2 cytokines IL-5 and IL-13 and Th1 cytokine IFNγ (Figure 19A). Data were collected only from samples that cleared a predefined threshold for Arah2. As a result, the reactivity of peanut allergenic T cells to B1001 was reduced compared to WT, but activation was still clearly and significantly preserved in all parameters tested (SI for B1001 / SI for WT: proliferation 61%, IL-5 39%, IL-13 50%, IFNγ 71%). To assess the overall reactivity of B1001, a predefined threshold was used to classify each patient sample as non-reactive or as having partial or comparable reactivity compared to AraH2. Of the total 21 samples tested, 3 samples were estimated as equivalent, 13 as partial, and 5 as non-reactive (Figure 19B). These results indicate that IgE de-epitope only partially impairs the T cell immunogenicity of the allergen. This finding suggests that complete loss of immunogenicity occurred in only a small proportion of patients.

[0405] Safety and immunotherapeutic efficacy of B1001 in a mouse peanut allergy model

[0406] Current mouse food allergy models provide only tentative clinical insights due to several important differences from humans, including prominent IgG-mediated anaphylaxis, different clinical responses (e.g., systemic hypothermia), differences in epitope specificity, and the lack of an IgG4 mouse homolog. Allergens have been specifically de-epitoped in a manner tailored to humans, further limiting the clinical predictability of mouse studies. Nonetheless, numerous studies on peanut allergy and immunotherapy using the C3H / HeJ model have been published, showing that remarkable clinical responses can be obtained. Using this model, we established protocols and performed two studies that provide evidence supporting the potential safety and efficacy of B1001 for immunotherapy in humans.

[0407] First, C3H / HeJ mice were sensitized by orally administering defatted peanut flour mixed with cholera toxin, a mucosal adjuvant, in PBS four times a week. Mice were then randomly divided into two groups and challenged sequentially with increasing doses of native Arah2 or B1001 by intraperitoneal (IP) injection. Mice were assessed for anaphylactic reaction after 120 min by a common clinical scoring index (Figure 20A, upper panel, 0 = no reaction to 5 = severe reaction and death) and by rectal recording of body temperature over 120 min (Figure 20A, lower panel). After the initial 30 μg challenge, all Arah2-loaded mice showed obvious symptoms (n = 12, mean clinical score 2.3 ± 0.2, mean maximum body temperature drop -7 °C ± 0.7), whereas B1001-challenged mice (n = 11) showed no reaction at all. The next day, surviving mice were challenged with 60 μg of either protein, but this time none of the mice showed any reaction (data not shown). This is consistent with previous findings suggesting that repeated exposure above a certain dose results in unresponsiveness, likely due to exhaustion of effector cells. To avoid this effect, the next day, the challenged groups were further randomized with B1001 and rechallenged with 120 μg of Arah2 (n=5, score 3.2 ± 0.2, maximum temperature drop -9.8 °C ± 1.45) or B1001 (n=6, score 0.3 ± 0.2, no temperature drop). This process was repeated on the final day, and a similar trend was observed for Arah2 (n=3, score 3.2 ± 0.2, maximum temperature drop -9.8 °C ± 1.45) and B1001 (n=3, score 0, maximum temperature drop -0.3 °C ± 0.1).

[0408] Peanut OIT (oral immunotherapy) was performed in parallel with B1001 OIT as standard to evaluate its immunotherapeutic potential. Mice were sensitized with the same protocol as above, and a group of non-sensitized mice was reserved as a control and underwent OIT with either peanut flour extract (PE), B1001, or vehicle PBS 5 times daily for 3 weeks. After a 12-day recovery period, mice were IP challenged with 35 μg native Arah2 and anaphylaxis scores were recorded (Figure 20B, top pane). Peanut sensitization was evident (mean scores: control=0, sham=3.5±0.5, MW p-value=0.01). In both the PE and B1001 treatment groups, anaphylaxis scores were significantly improved compared to the sham treatment group (sham) (PE mean value 2.7±0.4 (p=0.19 vs. sham), B1001 mean value 2.4±0.4 (p=0.08 vs. sham)).

[0409] The effect of OIT on cytokine secretion in Arah2-stimulated mesenteric lymph node cells was further analyzed. MLNs from surviving mice were harvested and isolated 5 days after challenge, then seeded and stimulated for 72 hours. The levels of Th2 cytokines IL-4, IL-5, and IL-13 were then detected in the cell culture medium using a Luminex panel assay (Figure 20B, bottom panel). For all three cytokines, the PE-treated group showed a significant decrease in secretion compared to the sham-treated group (p-values: IL-4=0.18, IL-5=0.005, IL-0.02). A similar decrease was observed in the B1001-treated group, but to a smaller extent and with less significance (p-values: IL-4=0.26, IL-5=0.087, IL-0.095).

[0410] In summary, findings from mouse studies demonstrated that B1001 has a clearly superior in vivo safety profile compared to AraH2 in allergy models and supported the potential of B1001 as an immunotherapeutic agent.

[0411] Example 12: Characterization of Arahl mutant PLP595 (C159) (SEQ ID NO: 156) and other Arahl mutants

[0412] Biochemical characterization of the Arah1 mutant PLP595

[0413] Objective: Characterization of the Arah1 mutant PLP595 compared to WT Arah1

[0414] The Arah1 protein is a trimeric protein, and each monomer weighs about 62 kDa, so the native molecular weight is about 200 kDa. Size-exclusion HPLC was used to estimate the molecular weight and oligomeric state of Arah1 mutant PLP595, and its profile was compared with that of recombinant WT and native Arah1 proteins. As shown in Figure 21, all three proteins had similar retention times and estimated molecular weights of about 200 kDa. This was further verified by mass spectrophotometry, which showed that the molecular weights of the WT and Arah1 mutant PLP595 proteins were about 200 kDa, as shown in Figure 23. Therefore, we inferred that Arah1 mutant PLP595 has a similar molecular weight to WT Arah1 protein and forms a trimer.

[0415] Next, the secondary structure profile of Arah1 mutant PLP595 was examined by circular dichroism (CD) and compared to the WT Arah1 protein. Both proteins showed similar CD profiles, as shown in Figure 22A. The thermal stability of the secondary structure of Arah1 mutant PLP595 was also assessed and compared to the WT Arah1 protein by performing CD at gradually increasing temperatures (20-90 °C). Arah1 mutant PLP595 was stable up to 85 °C, while the WT was stable up to 90 °C, as indicated by the higher ellipticity at 205 nm (Figure 22B).

[0416] Differentially reduced patient antibody binding to Arahl mutant C159

[0417] To investigate how the modifications altered IgE and IgG binding, ELISA assays were performed. Plates coated with native Arah1 or PLP159 (Combo 159) were incubated with serially diluted plasma or serum from 16 peanut-allergic patients. As observed with Arah2, the curves obtained were significantly different in shape, so the differences in the area under the curve (AUC) values ​​were compared. The results showed that the binding to C159 was significantly reduced in both the IgE and IgG fractions. However, this reduction was significantly more gradual in the IgG fraction (Figure 39A, Wilcoxon rank sum test P value < 0.0001). Indeed, when observing the individual [C159AUC / Arah1AUC] ratios, it was clear that the reduction was more pronounced in the IgE fraction than in the IgG fraction in all patients (Figure 39B, Wilcoxon P value < 0.0001). These results indicate that the Arah1 de-epitope process was successful in selectively reducing IgE-binding sites over IgG-binding sites.

[0418] The allergenicity of C159 was significantly reduced compared to native Arahl.

[0419] We tested the ability of C57, C68, and C159 to activate the humanized rat basophil-like RBLSX-38 cell line. This widely used cell line can be sensitized with human patient samples to respond to allergen stimulation by cell degranulation. After overnight sensitization of RBLSX-38 cells with 1:10 plasma or serum from 13 clinically validated peanut allergy patients of various backgrounds, we stimulated the cells with 0.5–5000 ng / ml of either AraH1, C57, or C68. To compare each patient's response to the different allergens, individual AUC values ​​were calculated. Overall, all patients showed reactivity to AraH1 (median AUC 45.7) and better with C68 (median AUC 9.3) compared to C57 (median AUC 22.5) (Figure 24A). Using the same method, RBLSX-38 cells were sensitized overnight with 1:10 plasma or serum from 47 different clinically validated peanut allergic patients of various backgrounds, after which the cells were stimulated with 0.5–5000 ng / ml of either AraH1, C57, or C159. To compare each patient's response to the different allergens, individual AUC values ​​were calculated. Overall, all patients showed reactivity to AraH1 (median AUC 56.3), with reduced reactivity to both variants, with C159 (median AUC 1.7) being better compared to C57 (median AUC 7.5) (Figure 24A).

[0420] These results indicate that de-epitope treatment of B1001 dramatically reduced its allergenicity.

[0421] During the Arahl de-epitope process, we tested the performance in the SX-38RBL degranulation assay of additional Arahl mutants, such as Combo51 (B1291), 52 (B1292), 74 (B1309), 75 (B1304), or 116 (PLP499). Examples of some tests from individual patients carrying these mutations are shown in Figures 25-26.

[0422] The RBL assay allows for high-throughput comparison of multiple variants using multiple patient samples and is a powerful tool for engineering and validation of modified allergens. However, the sensitivity and accuracy of this assay in predicting patient responses may be limited. To further validate the safety of C159 compared to Arah1, we performed the BAT assay with allergen concentrations ranging from 0.06 to 6,600 ng / ml using a commonly accepted protocol in a cohort of 19 peanut allergy patients from Israel and the United States. EC50 values ​​fitted to a four-parameter logistic regression model from the resulting curves suggested that C159 had a more than 1000-fold reduction in reactivity at the population level (Figure 27). These results indicated that the safety of C159 is higher than Arah1 and supported the application of C159 in immunotherapy.

[0423] T cell immunogenicity of C159

[0424] To confirm that the modification does not abolish the immunogenicity of C159 T cells, peripheral blood T cells from peanut allergic patients were treated with a proliferation detection dye and stimulated with either PBS, recombinant WT Arah1, or C159. Cells were then retained for cytometric proliferation analysis, and media was retained for sandwich ELISA analysis of secretion of Th2 cytokines IL-5 and IL-13 and Th1 cytokine IFNγ (Figure 38A). Data were collected only from samples that cleared a predefined threshold for Arah1. ​​As a result, the reactivity of peanut allergenic T cells to C159 was reduced compared to WT, yet activation was clearly and significantly retained in all parameters tested. To evaluate the overall reactivity of C159, each patient sample was classified as non-reactive or as having partial or comparable reactivity compared to Arah1 using a predefined threshold. Of the total 19 samples tested, two samples were estimated to be equivalent, 14 samples to be partial, and three samples to be non-reactive (Figure 38B). These results indicate that de-epitopation of IgE only partially impairs the T cell immunogenicity of an allergen.

[0425] Example 13: Increasing the half-life of de-epitope-modified AraH2 for mRNA therapy

[0426] Aim: To increase the half-life of de-epitope-modified Arah2 and improve its therapeutic potential.

[0427] De-epitope-ed Arah2 (designated as 1001) was initially designed for bacterial expression, but is poorly expressed in mammalian cells. The lack of expression and secretion of this protein from mammalian cells may preclude its use as part of an mRNA therapeutic. In addition to its poor expression in mammalian cells, de-epitope-ed Arah2 is a small monomeric protein with a molecular weight of less than 19 kDa, and is therefore expected to be rapidly cleared by the renal pathway. Increasing the half-life of this protein improves its therapeutic potential by effectively extending its exposure to the immune system, i.e., the opportunity to generate a desired immune response.

[0428] We also observed that Arah2 and its de-epitope derivatives were pseudo-O-glycosylated in a manner that inhibited protein expression through the mammalian secretory pathway (verified by ETD mass spectrometry, data not shown). Elimination of the glycosylation site improved the expression levels of wild-type Arah2 but was insufficient to increase the expression levels of the de-epitope derivatives.

[0429] To address the above issues, we designed several constructs in which de-epitopeized Arah2-1001 was tailored to function as part of an mRNA therapy.

[0430] method

[0431] Cell transfection: Expi293 cells (Thermo-Fisher) were cultured in Expi293 expression medium and transfected according to the manufacturer's protocol. Briefly, prior to transfection, cells were grown to a viable cell density of 4–5 million cells / ml, diluted to 3 million cells / ml, and transfected with 1 μg of DNA per ml of medium. DNA was diluted in OptiMEM (Thermo-Fisher) to 6.1% of the expression volume. In a separate tube, ExpiFectamine293 was diluted 1:18.5 in OptiMEM to 6% of the expression volume. After 5 min of incubation, the diluted Expifectamine293 (Gibco) and DNA were mixed and added to the cell culture after 10 min of incubation.

[0432] Protein expression: Expi293 cells were cultured at 37°C, 5% CO2. The day after transfection, Expifectamine 293 Enhancer 1 and 2 were added to the cells at 1:160 and 1:16, respectively. Cells were left for a total of 5 days to allow protein expression.

[0433] Protein purification: Media supernatants were clarified by centrifugation at 300xg for 10 min and filtered through a 0.45 μm PES filter. His-tagged constructs were dialyzed overnight against 100 volumes of 20 mM tris pH 8.0, 200 mM NaCl. The dialyzed supernatant was agitated with Ni-NTA Superflow resin (Thermo-Fisher) for 1 h at 4°. The resin was washed with 20 mM tris pH 8.0, 200 mM NaCl, 10 mM imidazole. Proteins were eluted with 20 mM tris pH 8.0, 200 mM NaCl, 250 mM imidazole. For Fc-fusion proteins, clarified media supernatants were incubated with Protein A-bound resin (Toyopearl, HC-650F) for 1 h. The resin was washed with 100 resin volumes of PBS. Proteins were eluted by the addition of 0.1 M sodium citrate buffer (pH 3.0). The eluted fractions were neutralized by the addition of 0.33 elution volumes of 1 M Tris (pH 9.0). For further purification, the eluted fractions were concentrated with Amicon centrifugal filters (Merk Millipore) of appropriate MWCO of 10 kDa or 50 kDa and loaded onto Superdex75PG or Superdex200PG16 / 600 (Cytiva) equilibrated with PBS for size exclusion chromatography separation.

[0434] ELISA assay: Serum was collected from mice before dosing and 21 days after the first DNA injection. Antigen-specific antibodies were detected in mouse serum by ELISA assay. Briefly, 96-well ELISA plates (MaxiSorp, Nunc) were coated with 50 μl (1 μg / ml) of purified proteins in phosphate-buffered saline at 4 °C according to the following scheme: Native Arah1 was used to detect α-Arah1 and α-DEArah1 Combo 68 antibodies (both soluble and transmembrane fusion). Native Arah2 was used to detect α-Arah2 antibodies and recombinant DE Arah2 1001 was used to detect α-DE Arah2 1001 antibodies (both Fc fusion and transmembrane fusion). 1 μg / ml of keyhole limpet hemocyanin (KLH, Sigma Aldrich) was used as a negative control. All conditions were performed in duplicate. After coating, the plates were blocked by incubation with PBS 0.1% Tween 20 (PBST), 2% BSA for 1 h at room temperature, and then washed once with 200 μl PBST. Sera were diluted 1:200 in PBST 2% BSA, and 50 μl / well were transferred to the ELISA plate according to the above scheme and incubated for 1.5 h at room temperature. The plates were washed three times with 200 μl / well PBST. All wells were incubated with 50 μl of 1:10,000 HRP-conjugated α-mouse IgG (Jackson ImmunoResearch) secondary antibody. The wells were washed three times with 200 μl / well PBST, followed by TMB reaction (Promega) and quenching by addition of H2SO4. Optical density values ​​were subtracted from those of the KLH control.

[0435] In vivo transfection: 6-8 week old female C3H / HeNHsd mice were purchased from Envigo (Envigo, Israel) and treated with a DNA construct (´pTwistCMV´, Twist Bioscience) consisting of a plasmid encoding the protein of interest flanked by a CMV promoter and an SV40 polyadenylation signal. Mice were treated by injection of 10 µg of DNA in PBS or by PEI transfection. Briefly, to generate 840 µl of DNA for PEI transfection, DNA was diluted in 400 µl to a final concentration of 0.42 mg / ml in 5% glucose. In another tube, 70.4 µl of 1 mg / ml 25 kDa linear PEI (Polyscience) was diluted in 440 µl of 5% glucose. The two tubes were mixed by pipetting and incubated for 15 min before injection. The final DNA n / p ratio was 6. Mice were injected intramuscularly (IM) into the femoral tail muscle at 50 μl, 0.2 mg / ml, three times a week and bled 21 days after the first dose.

[0436] result

[0437] Consensus mutations

[0438] To address the poor expression of de-epitope-ed Arah2 1001 in mammalian cells, a series of back-to-consensus mutations were designed with the intention of restoring the stability of the wild-type protein while avoiding reintroduction of IgE epitopes. The designs were iteratively tested for both expression levels and RBL activation. After several design iterations, a version that expressed de-epitope-ed Arah2 well without significant reintroduction of IgE epitopes was achieved (Arah2_conbo31). Figure 28 shows examples of back-to-consensus mutants of DE Arah2 1001 expressed in HEK293 cells, showing that the secretion levels of a subset of the back-to-consensus mutants were increased compared to the DE Arah2 1001 from which they were derived (right lane). Non-reducing gels showed that the mutants were monomeric, not misfolded, and formed intermolecular disulfide bonds, a phenomenon observed with some recombinant forms of Arah2.

[0439] As shown in Figure 29, the final back-to-consensus mutant (var31) and DE Arah2 1001-Fc fusion protein were significantly less allergenic compared to native Arah2 as measured by the RBL assay.

[0440] Fc fusion

[0441] To address the poor expression, short half-life, and fast clearance of de-epitope-ed Arah2, de-epitope-ed Arah2 was fused to antibody Fc. The Fc portion serves two functions, acting as a carrier in the secretory pathway and increasing the half-life of the fused therapeutic moiety. In addition to the above functions, fusion of de-epitope-ed allergens to Fc of IgG4 is expected to inhibit allergic reactions by binding to FcγR. Figure 30 shows that fusion to Fc dramatically increased the secretion level of de-epitope-ed Arah2 1001. Figure 30 also shows that the secretion level of de-epitope-ed Arah2Fc fusion (and assembly of Fc dimers) was significantly increased compared to the monomeric protein. The monomeric protein is barely detectable by Western blot, while the Fc fusion is clearly overexpressed and secreted into the medium as seen in the SDS-PAGE gel. Western blot confirmed that the overexpressed protein band contained the depitopised Arah2 fusion.

[0442] transmembrane fusion

[0443] To further address the poor expression, short half-life, and fast clearance of de-epitope-ed Arah2, we designed a membrane-anchored version of de-epitope-ed Arah2. In addition to promoting increased expression, the membrane-fusogen is not cleared by the renal system as occurs with the soluble version. In addition, the membrane-fusogen can induce antibody production but cannot induce FcεRI cross-linking because it is anchored to the cell membrane, and therefore is less likely to cause allergic reactions.

[0444] As shown in Figure 31, DE Arah2 1001 is overexpressed in a membrane-anchored manner, but only trace amounts of protein are found in the soluble fraction. Figure 32 shows the antibody response to the various constructs when delivered as part of gene therapy. This response confirms that the constructs can indeed be expressed and secreted in vivo, and that they induce the immune response expected to be essential for immunotherapy.

[0445] Example 14: Sublingual immunotherapy for peanut allergy

[0446] Preparation of peanut extract

[0447] 100 g of defatted peanut flour (Shaked Tavor, ~48% protein, ~80% defatted from lightly roasted peanuts) was mixed with 500 ml of extraction buffer (20 mM Tris, pH 8.0), homogenized using a hand homogenizer mixer, and stirred at room temperature for 2 h. The mixture was then centrifuged at 5000 g for 5 min and the supernatant was centrifuged again at 20,000 g for 50 min at 4 °C. The resulting supernatant was centrifuged again at 20,000 g for 50 min at 4 °C and filtered through a 0.45 μm filter. The filtered peanut extract (PE) was stored at -80 °C until the purification step.

[0448] Isolation of native Arah2

[0449] 100 ml of PE was loaded onto a 70 ml Q Sepharose HP column (Cytiva) and pre-equilibrated with extraction buffer. Peanut proteins were eluted using a linear gradient of 0-0.4 M NaCl over 18 column volumes in extraction buffer (Figure 33A). Native Arah2 (nArah2)-containing fractions (see Figure 33B) were pooled, concentrated to 2-5 mg / ml by a 3 kDa centricone (Amicon, Mercury) and loaded onto a Hiload 16 / 600 Superdex75 PGSEC column (Cytiva) equilibrated with PBS. Pure monomeric nArah2-containing fractions were pooled and concentrated to approximately 2 mg / ml (see Figures 3 and 4). The concentration was measured using absorbance at 280 nm (ε=14940). The final concentration of material was measured by SEC-HPLC using a BEH SEC200A column (Waters) and a myoglobin standard curve.

[0450] Sublingual immunotherapy for peanut allergy in a mouse model

[0451] Experimental procedure

[0452] Thirty-six 3-week-old naïve female C3H / HeJ mice were ordered. The weight range of the mice on day 1 was 14-18 g. The mice were identified by marking the tails of the mice with an indelible marker. The mice were supplied by The Jackson Laboratory, Bar Harbor, USA.

[0453] Sensitization phase

[0454] Thirty-six mice (including sham animals) were orally sensitized as follows.

[0455] Weeks 1, 2, and 3: Once a week, 2 mg (50% protein) of peanut extract was mixed in 0.250 mL of PBS, 10 μg of mucosal adjuvant cholera toxin (List Laboratories, Campbell, Calif., reference 100B).

[0456] Week 4: 4 mg of peanut extract (50% protein) was mixed with 0.250 mL of PBS, 10 μg of mucosal adjuvant cholera toxin (List Laboratories, Campbell, Calif.).

[0457] Mice were food-deprived for 3 hours prior to each oral gavage.

[0458] On day 29, all mice were given 350 μg of peanut extract intraperitoneally. The body temperatures of the mice were measured with a temperature probe inserted into the rectum before, 30 minutes after, and 40 minutes after intraperitoneal administration. A decrease in body temperature of 1.5°C or more was considered positive.

[0459] Forty minutes after intraperitoneal administration, anaphylactic symptoms were evaluated using the following scoring system. 0: no clinical symptoms; 1: Repeated scratching of mouth or ears or ear canal with hind legs; 2: Decreased activity, edema / swelling around the eyes and mouth; 3: prone position, motionless for 1 minute or more; 4: No reaction to whisker stimulation, little or no reaction when poked. 5: Endpoint: tremors, convulsions, death.

[0460] Next, approximately 100 μL of blood samples were collected without anesthesia at the submandibular vein level (polypropylene serum tubes containing a clot activator) for the measurement of total immunoglobulins with a Porsolt enzyme immunoassay kit. The tubes were inverted several times to mix the whole blood with the clot activator. The vials were kept at room temperature for 20-30 min (tubes were kept upright). The blood was then centrifuged at 1000 g, room temperature, for 10 min. The serum samples (one serum sample of 25 μL + one serum sample of the remaining volume) were transferred to polypropylene tubes and stored frozen at -80 °C until analysis.

[0461] Quantification of total immunoglobulins (IgA, IgE, IgG1, IgG2b, IgG3, IgM, and IgG2c) was performed using clarified plasma samples and the antibody isotyping 7-Plex Mouse ProcartaPlex™ Panel (reference EPX070-20816-901, ThermoFisher). Total IgG2a quantification was performed using the ProcartaPlex Mouse Basic Kit for IgG2a (reference EPX010-20440-901, ThermoFisher).

[0462] Twenty-eight mice were selected for inclusion with data (body temperature and clinical scores) obtained on day 29 after intraperitoneal administration of 350 μg peanut extract. No additional examination was performed on day 33.

[0463] treatment period

[0464] From day 36, oral or sublingual immunotherapy was started (5 times a week for 3 consecutive weeks). For sublingual administration (i.e., Sham, Group 3 and Group 4, see Table 1), mice were briefly anesthetized with a mixture of ketamine / medetomidine (50 / 1 mg / kg, 10 mL / kg ip) during the first week of treatment. After about 10 minutes, the depth of anesthesia of the mice was checked to ensure that they were sufficiently anesthetized.

[0465] Sublingual administration: Mice were held upright and 10 μL of solution per mouse was administered sublingually using a micropipette.

[0466] Tongue Rolling: After dosing, mice were subjected to gentle tongue rolling for approximately 1 min, which can be performed with the tip of a micropipette to simulate normal tongue movements in conscious animals.

[0467] Recovery position: Mice were then placed in a hunched position (sitting with head tucked over lower legs) for approximately 20 minutes after sublingual administration to minimize the possibility of the mice swallowing the solution.

[0468] The two groups of mice were orally administered and then briefly anesthetized with a mixture of ketamine / medetomidine (50 / 1 mg / kg, 10 mL / kg ip) as in the other groups, thus all animals were tested under the same experimental conditions (i.e., short-term anesthesia).

[0469] Due to anesthesia-related deaths during the first week of treatment, the anesthesia protocol was modified: mice were briefly anesthetized with a mixture of ketamine / medetomidine (25 / 2 mg / kg, 10 mL / kg ip).

[0470] Approximately 30 min after anesthesia, atipamezole (1 mg / kg, 10 ml / kg ip) was used to reverse the anesthetic effects of ketamine / medetomidine.

[0471] [Table 10]

[0472] Challenge(ip)

[0473] Mice were intraperitoneally injected with 35 μg of native Arah2 protein / 250 μL on day 67. Core body temperatures of mice were measured using a rectally inserted temperature probe before, 10, 20, 30, 45, 60, 120 min, and 24 h after intraperitoneal injection. A decrease in body temperature of 1.5°C or more was considered positive.

[0474] Cytokine secretion from cells of the spleen and mesenteric lymph nodes

[0475] After blood samples were taken (day 72), spleens and mesenteric lymph nodes (MLNs) were harvested and transferred to 1XPBS containing 100U / mL penicillin and 100μg / mL streptomycin in separate Falcon tubes placed on ice. MLNs were cut into small pieces using sterile instruments. Spleens were freshly homogenized using a GentleMACS dissociator. They were then transferred onto a 70μM cell strainer pre-wetted with TexMACS medium (ref. 130-097-196, Miltenyi Biotec).

[0476] Splenocytes were isolated and then centrifuged at 450g for 8 min. Red blood cells were lysed using lysis buffer (ref 555899, BD Biosciences). The reaction was stopped with 5 volumes of 2% FBS in PBS and cells were washed once with PBS. MLN cells were isolated by gently squeezing the tissue with a syringe plunger while repeatedly adding medium, then centrifuged at 450g for 8 min.

[0477] Splenocytes and MLN cells were seeded in 96-well U-bottom plates (400,000 cells / 100 μL) in TexMACS medium (ref. 130-097-196, Miltenyi Biotec) and 10% FBS containing 100 U / mL penicillin and 100 μg / mL streptomycin and treated with cell culture medium (group 1) or native Arah2 (groups 1–4) at a final concentration of 200 μg / mL. Cells from sham and sensitized mice were also treated with concanavalin A (final concentration 2.5 μg / mL) or stimulated with CD3-CD28 beads using the Mouse T Cell Activation / Culture Kit (ref. 130-093-627, Miltenyi Biotec) as a control. Supernatants were harvested 24 and 72 h after treatment and stored at -80 °C until analysis.

[0478] Cytokine (IL-4, IL-5, IL-10, IL-13, INFγ, IL-12, IL-9, TGFβ) levels were measured using Luminex panel assays according to the manufacturer's instructions (ProcartaPlex 7 plex Assay, ThermoFisher Scientific, reference no. EPX010-20440-901 and TGF beta1 Mouse ProcartaPlex(tm) Simplex Kit, ThermoFisher Scientific, reference no. EPX01A-20608-901). Data were analyzed using Bio-Plex Manager software (Biorad) and concentrations were calculated using standard curves of the corresponding cytokines.

[0479] result

[0480] Challenge (ip): Assessment of hypersensitivity reactions

[0481] The hypersensitivity reaction measured by the change in body temperature is shown in Figure 5. In sham mice, a gradual decrease in body temperature was observed over time (maximum of -11.5±1.3°C at 120 minutes after intraperitoneal administration).

[0482] In mice treated with peanut protein (400 μg / mouse, orally administered), the decrease in body temperature was less pronounced than in sham mice (maximum of -3.9 ± 1.3 °C 60 min after intraperitoneal administration and -1.7 ± 0.6 °C 120 min after intraperitoneal administration). The difference between the two groups reached statistical significance 20 to 120 min after administration.

[0483] In mice administered peanut protein (5 μg / mouse sublingually), the decrease in body temperature was not significantly different compared to sham mice.

[0484] In mice administered peanut protein (50 μg / mouse sublingually), the decrease in body temperature was less pronounced than in sham mice (maximum of -4.9 ± 1.2 °C 60 min after intraperitoneal administration and -2.77 ± 1.3 °C 120 min after administration). The difference between the two groups reached statistical significance 20 to 120 min after administration.

[0485] In all mice, the clinical score measured 30 minutes after intraperitoneal administration was 2. Therefore, no differences were observed between the groups.

[0486] Analysis of cytokine production

[0487] Positive control

[0488] Positive controls induced increased cytokine secretion for most of the cytokines tested from splenocytes and mesenteric lymph node cells, but at low levels in mesenteric lymph node cells. Spleens and mesenteric lymph nodes were harvested from non-responding animals and not from naive animals.

[0489] splenocytes

[0490] In the supernatants of splenocytes from sham control mice, the levels of IL-4, IL-5, IL-10, IL-13, INFγ, and IL-12 increased between 24 and 72 hours. IL-9 levels were below the lower limit of quantification, and TGFβ levels remained stable over time. As a negative control, splenocytes from sham control mice treated with culture medium were used, and the levels of cytokines were very low or below the lower limit of quantification, except for TGFβ (basal level of approximately 350 pg / mL).

[0491] In the supernatants of splenocytes from orally sensitized mice (400 μg / mouse orally administered), IL-4, IL-5, IL-10, IL-13, INFγ, and IL-12 levels were not significantly altered compared to sham control mice. TGFβ levels were significantly increased at 24 h (+81%, p<0.01) and 72 h (+80%, p<0.05) compared to sham control mice. However, this difference seems to lack biological relevance considering the basal levels measured under control conditions.

[0492] In the supernatants of splenocytes from sublingually sensitized mice (5 μg / mouse or 50 μg / mouse), no significant changes were observed in IL-4, IL-5, IL-10, IL-13, INFγ, IL-12, and TGFβ levels compared with sham control mice.

[0493] [Table 11]

[0494] Mesenteric lymph node cells

[0495] In the supernatants of mesenteric lymph node cells from sham control mice, the levels of IL-4, IL-5, IL-10, IL-13, INFγ, and IL-12 increased between 24 and 72 hours. IL-9 levels were below the lower limit of quantification. The kinetics of TGFβ were different in the two wells tested. In mesenteric lymph node cells from sham control mice treated with medium as a negative control, the levels of cytokines were very low or below the lower limit of quantification, except for TGFβ (basal level of about 400 pg / mL).

[0496] In the supernatants of mesenteric lymph node cells from orally sensitized mice (400 μg / mouse orally administered), IL-4, IL-5, IL-10, and IL-13 levels were decreased compared to sham control mice. IFNγ, IL-12, and IL-9 levels were zero or below the lower limit of quantification. TGFβ levels showed no significant change compared to sham control mice.

[0497] In the supernatants of mesenteric lymph node cells from sublingually sensitized mice (5 μg / mouse or 50 μg / mouse), the levels of IL-4, IL-5, IL-10, and IL-13 were decreased compared to sham control mice. The levels of IFNγ, IL-12, and IL-9 were zero or below the lower limit of quantification. The TGFβ levels were not significantly changed compared to sham control mice. The effect appeared to be more pronounced at the highest concentration and at 72 hours.

[0498] [Table 12]

[0499] In conclusion, these results suggest that oral (400 μg / mouse) or sublingual (50 μg / mouse) administration of peanut protein reduces anaphylactic reactions reflected by a significant decrease in body temperature and an increase in clinical scores in female C3H / HeJ mice previously sensitized with peanut extract. The lowest sublingual dose (5 μg / mouse) did not affect anaphylactic reactions. Allergic skin reactions (ear swelling) were not altered by any dose.

[0500] Mice treated with peanut protein showed similar elevations in IgG at all doses compared to sham control mice. Total IgE and IgA were elevated after peanut protein treatment (oral and sublingual at 5 μg / mouse), but not after SLIT treatment at 50 μg / mouse.

[0501] These treatments also modified the increase in cytokine release in the supernatants of splenocytes or mesenteric lymph node cells after ex vivo stimulation with peanut protein, with a non-statistical trend towards a decrease for some cytokines and an increase for TNF.

[0502] In the supernatants of native Arah2 (5 μg / mouse sublingually or 50 μg / mouse sublingually) stimulated (200 μg / mL native Arah2) mesenteric lymph node cells, the levels of IL-4, IL-5, IL-10, and IL-13 were decreased compared to sham-stimulated mesenteric lymph node cells, and the levels of TGFβ were significantly reduced (-31%, p<0.05) in the 5 μg / mouse treatment group compared to sham-stimulated mesenteric lymph node cells.

Claims

1. A recombinant Ara h2 variant polypeptide, at positions selected from positions 12, 15, 16, 22, 24, 46, 53, 65, 80, 83, 86, 87, 90, 104, 115, 123, 127, and 140 of SEQ ID NO: 4, containing at least two substitutions as compared with the amino acid residues at the same positions in SEQ ID NO: 3, and the recombinant Ara h2 variant polypeptide has at least 80% identity to the sequence set forth in SEQ ID NO:

3.

2. The recombinant Ara h2 variant polypeptide according to claim 1, wherein the substitutions are a) at position 12: N, Q, E, D, T, S, G, P, C, K, H, Y, W, M, I, L, V, or A; b) at position 15: R, E, K, Y, W, F, M, I, V, C, D, G, or A; c) at position 16: R, K, D, Q, T, M, P, C, E, or W; d) at position 22: F, Y, W, Q, E, T, S, A, M, I, L, C, R, or H; e) at position 24: D, E, H, K, S, T, N, Q, L, I, M, W, Y, F, P, A, or G; f) at position 46: T, V, E, H, S, A, G, Q, N, D, R, P, M, I, L, or C; g) at position 53: T, S, Q, V, A, G, C, P, M, L, I, E, H, R, K, N, or D; h) at position 65: T, A, N, D, Q, R, K, H, I, L, M, V, W, P, G, C, or E; i) at position 80: N, S, T, V, A, I, L, M, F, Y, W, C, E, K, R, or G; j) at position 83: D, A, C, F, I, P, T, V, W, Y, or Q; k) at position 86: Y, F, H, R, E, C, G, I, L, M, V, T, S, or Q; l) at position 87: F, Y, I, L, M, V, A, S, Q, R, K, D, N, E, or P; m) at position 90: S, P, Q, or R; n) at position 104: L, M, K, R, H, E, D, A, Y, N, S, or W; o) at position 115: V, D, E, I, L, K, M, N, S, T, A, I, W, F, Y, or H; p) at position 123: I, Q, or A; q) at position 127: H, A, D, E, F, G, L, N, P, S, T, W, Y, Q, or V; and, at position 140, G, A, C, E, Y, F, H, K, L, M, N, P, Q, S, or V; a recombinant Ara h2 variant polypeptide comprising one or more of the following. **Claim 3** The recombinant Ara h2 variant polypeptide according to claim 1, wherein at one or more of positions 28, 44, 48, 51, 55, 63, 67, 107, 108, 109, 124, 125, and 142 of SEQ ID NO: 4, compared to the amino acid residue at the same position in SEQ ID NO: 3, further comprises additional substitutions. **Claim 4** The recombinant Ara h2 variant polypeptide according to claim 3, wherein the additional substitutions are a) at position 28, S, T, V, N, A, P, I, L, F, Y, H, R, K, E, or D; b) at position 44, I, A, C, G, H, L, F, Y, N, P, Q, K, E, S, T, V, M, or R; c) at position 48, V, G, C, E, H, Q, F, K, L, I, W, Y, N, R, S, T, V, A, or D; d) at position 51, S, G, Y, F, W, M, N, Q, E, R, K, H, T, D, or V; e) at position 55, G, A, D, E, F, Y, H, Q, V, I, L, M, R, K, S, T, C, or W; f) at position 63, P, C, F, V, I, L, M, W, Y, N, S, T, Q, G, H, K, or R; g) at position 67, E, Q, N, R, H, Y, F, W, M, L, V, T, S, A, P, or G; h) at position 107, A, C, F, G, H, I, K, L, M, Q, P, R, S, T, V, W, or Y; i) at position 108, T, V, D, E, R, H, Y, W, I, G, A, Q, or K; j) at position 109, K, C, S, R, G, P, Y, W, L, or I; k) at position 124, D, A, C, F, G, H, I, N, S, T, V, Y, L, E, or Q; l) at position 125, M, I, L, W, Y, G, K, N, T, V, or A; and m) at position 142, M, A, C, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; a recombinant Ara h2 variant polypeptide comprising one or more of the following. **Claim 5** The recombinant Ara h2 variant polypeptide according to claim 1, wherein comprising the amino acid sequence set forth in any of SEQ ID NOs: 168, 10-63, 170, 195-201, 204-210, or 247-249, or a recombinant Ara h2 variant polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence set forth in any of SEQ ID NOs: 168, 10-63, 170, 195-201, 204-210, or 247-249. **Claim 6** An isolated nucleotide or modified nucleotide sequence encoding the recombinant Ara h2 variant polypeptide according to claim 1, wherein the nucleotide or modified nucleotide sequence is a nucleotide or modified nucleotide sequence comprising DNA or mRNA. **Claim 7** A nucleotide or modified nucleotide sequence according to claim 6, wherein the nucleotide or modified nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 167 or 169, or a nucleotide or modified nucleotide sequence comprising a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 167 or 169. **Claim 8** An expression vector comprising the nucleotide or modified nucleotide sequence according to claim 6. **Claim 9** A prokaryotic or eukaryotic cell comprising the expression vector according to claim 8, wherein the eukaryotic cell is a prokaryotic or eukaryotic cell comprising a yeast cell, a fungal cell, a plant cell, or a mammalian cell. **Claim 10** A composition comprising the recombinant Ara h2 variant polypeptide according to claim 1, or the nucleotide or modified nucleotide sequence according to claim 6. **Claim 11** The composition according to claim 10 for inducing desensitization to peanuts in a subject allergic to peanuts. **Claim 12** A recombinant Ara h1 variant polypeptide, wherein at least two substitutions are included at positions selected from positions 194, 195, 213, 215, 231, 234, 245, 267, 287, 294, 312, 331, 419, 422, 443, 455, 462, 463, 464, 480, 494, and 500 of SEQ ID NO: 67 as compared to the amino acid residues at the same positions in SEQ ID NO: 65, and the recombinant Ara h1 variant polypeptide has at least 80% identity to the sequence set forth in SEQ ID NO:

65.

13. The recombinant Arah1 variant polypeptide according to claim 12, wherein the substitution is (a) D at position 194; (b) A at position 194; (c) H at position 213; (d) R, D, L, I, F, or A at position 215; (e) A at position 231; (f) E at position 234; (g) R at position 245; (h) E at position 267; (i) D at position 287; (j) E at position 294; (k) A or H at position 312; (l) H at position 331; (m) E, V, or A at position 419; (n) R or A at position 422; (o) A at position 443; (p) A at position 455; (q) A, K, or T at position 462; (r) S at position 463; (s) A or S at position 464; (t) Q at position 480; (u) A, E, or N at position 494; and (v) K at position 500; a recombinant Arah1 variant polypeptide comprising one or more of the above.

14. The recombinant Arah1 variant polypeptide according to claim 12, wherein at one or more of positions 12, 24, 27, 30, 42, 57, 58, 73, and 523 of SEQ ID NO: 67, further comprising additional substitutions as compared to the amino acid residues at the same positions in SEQ ID NO: 65, a recombinant Arah1 variant polypeptide.

15. The recombinant Arah1 variant polypeptide according to claim 14, wherein the additional substitution is (a) K or A at position 12; (b) V or E at position 24; (c) A or H at position 27; (d) E or A at position 30; (e) L or K at position 42; (f) D or L at position 57; (g) S or R at position 58; (h) A or M at position 73; and (i) A or K at position 523; a recombinant Arah1 variant polypeptide comprising one or more of the above.

16. The recombinant Arah1 variant polypeptide according to claim 14, wherein A recombinant Ara h1 variant polypeptide that, at one or more of positions 87, 88, 96, 99, 196, 197, 200, 209, 238, 249, 260, 261, 263, 265, 266, 278, 283, 288, 290, 295, 318, 322, 334, 336, 378, 417, 421, 441, 443, 481, 484, 485, 487, 488, and 491 of SEQ ID NO: 67, further comprises additional substitutions compared to the amino acid residues at the same positions in SEQ ID NO:

65. **Claim 17** The recombinant Ara h1 variant polypeptide according to claim 16, wherein the additional substitutions are (a) A at position 84; (b) A at position 87; (c) A at position 88; (d) A at position 96; (e) A at position 99; (f) H at position 196; (g) A at position 197; (h) V, A, or Q at position 200; (i) S at position 209; (j) Q at position 238; (k) N at position 249; (l) K at position 260; (m) R at position 261; (n) K or L at position 263; (o) S at position 265; (p) R or L at position 266; (q) R at position 278; (r) E at position 283; (s) Q at position 288; (t) R at position 290; (u) A at position 295; (v) H at position 318; (w) A or K at position 322; (x) D, A, or N at position 334; (y) R or S at position 336; (z) K or E at position 378; (aa) R at position 417; (bb) E or S at position 421; (cc) N at position 441; (dd) A at position 443; (ee) A or S at position 481; (ff) R, S, A, or M at position 484; (gg) A at position 485; (hh) S or K at position 487; (ii) A at position 488; and (jj) A, S, or E at position 491 and comprises one or more of the above, the recombinant Ara h1 variant polypeptide. **Claim 18** The recombinant Ara h1 variant polypeptide according to claim 12, wherein comprising the amino acid sequence set forth in any of SEQ ID NOs: 156, 68-155, 157-161, 174, 176, 178, 180, 182, 184, 193, 194, or 211-246, or a recombinant Ara h1 variant polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence set forth in any of SEQ ID NOs: 156, 68-155, 157-161, 174, 176, 178, 180, 182, 184, 193, 194, or 211-246. **Claim 19** An isolated nucleotide or modified nucleotide sequence encoding the recombinant Ara h1 variant polypeptide according to claim 12, wherein the nucleotide or modified nucleotide sequence is a nucleotide or modified nucleotide sequence comprising DNA or mRNA. **Claim 20** The nucleotide or modified nucleotide sequence according to claim 19, wherein the nucleotide or modified nucleotide sequence comprises the sequence of SEQ ID NO: 173, 175, 177, 179, 181, or 183, or a nucleotide or modified nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 173, 175, 177, 179, 181, or 183. **Claim 21** An expression vector comprising the nucleotide or modified nucleotide sequence according to claim 19. **Claim 22** The expression vector according to claim 21, wherein the eukaryotic cell is a prokaryotic or eukaryotic cell including a yeast cell, a fungal cell, a plant cell, or a mammalian cell. **Claim 23** A composition comprising the recombinant Ara h1 variant polypeptide according to claim 12, or the nucleotide or modified nucleotide sequence according to claim 19. **Claim 24** The composition according to claim 23 for inducing desensitization to peanuts in a subject allergic to peanuts.

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