Compositions and methods for inducing desensitization to peanuts

Abstract

In one embodiment, the present disclosure provides hypoallergenic peanut allergen AraH6 variants, in which at least one epitope recognized by an anti-AraH6 antibody has been altered, thereby reducing or eliminating antibody binding to the peanut allergen. Such hypoallergenic peanut allergen variants retain immunogenicity and can be used in methods for treating and reducing the severity of peanut allergies in subjects (patients) with peanut allergies.

Description

[Technical Field]

[0001] (Sequence Listing Statement) This application contains a Sequence Listing that has been submitted electronically in XML format, and the entirety of this Sequence Listing is incorporated herein by reference. The XML copy, created on October 29, 2023, is named "P-621156-PC_SL.xml" and is 219,755 bytes in size.

[0002] (Technical field) The present disclosure relates generally to the recombinant hypoallergenic peanut allergen AraH6, methods for its production, and uses thereof. [Background technology]

[0003] Peanut allergy is one of the most severe food allergies known, and when exposed to even low concentrations of peanuts, allergic individuals experience a range of symptoms, from mild localized effects to severe, life-threatening effects. Peanuts are 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 US population (Sicherer SH, et al., (2010). J Allergy Clin Immunol. 125(6):1322-6).

[0004] To date, 17 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 major allergens, and when recognized by IgE antibodies, they correlate with more severe symptoms (Palladino, et al., 2018; ibid) (Bernard, et al., (2007) J Agric Food Chem. 55(23):9663-9).

[0005] AraH6, a member of the 2S albumin family, is a major peanut allergen. AraH6 contains 145 amino acids, including a 21-amino acid signal peptide, five predominant α-helices, and five intramolecular disulfide bonds (uniporter A5Z1R0; Q647G9).

[0006] Both AraH2 and AraH6 belong to the conglutin type of 2S-albumin. The conformational models of AraH2 and AraH6 are virtually superimposable, indicating that the tertiary structures of the two proteins are nearly identical. They are currently considered to be the most potent peanut allergens (Kulis, Mike, et al. (2012) Clinical & Experimental Allergy; 42.2: 326-336). AraH2 and AraH6 are the most frequently identified major peanut allergens in children (Flinterman, A.E., et al. (2007) Clinical & Experimental Allergy 37.8: 1221-1228; and van Erp, Francine C., et al. (2017) Journal of Allergy and Clinical Immunology; 139.1: 358-360). Individual reactivity to major peanut allergens remains stable over time (Flinterman AE, et al., (2007) Clin Exp Allergy; 37(8) 1221-1228).

[0007] Due to their similar structures, anti-AraH6 antibodies often cross-react with AraH2 (and vice versa) (Koppelman, SJ, et al. (2005) Clinical & Experimental Allergy; 35.4 (2005): 490-497). Nevertheless, these proteins are not identical, and each is presumed to contain antibody epitopes not present in the other. It has been reported that some allergic patients are sensitized exclusively to peanut 2S albumin protein (Asarnoj A, Glaumann S, Elfstrom L, et al. (2012) Int Arch Allergy Immunol.; 159(2):209-212). These reports suggest that IgE antibodies directed against specific epitopes of AraH6 that are not present in AraH2 are sufficient to induce clinical reactions in at least some patients.

[0008] There remains a need for hypoallergenic peanut proteins and methods of use for standardized immunotherapy treatment in patients with allergies to peanut allergens. Summary of the Invention [Means for solving the problem]

[0009] Described herein are several epitope mapping methods for identifying epitopes on Arah6, as well as protein engineering approaches for designing hypoallergenic Arah6 allergen variants that maintain biophysical and functional characteristics. In one aspect, the present disclosure provides recombinant Arah6 variant polypeptides, wherein at least one epitope recognized by an anti-Arah6 antibody is deleted, resulting in reduced or abolished IgE antibody binding to the variant polypeptide. In some embodiments, the epitope comprises a linear epitope. In some embodiments, the epitope comprises a conformational epitope. In another embodiment, the recombinant Arah6 variant polypeptides of the present disclosure can be used in methods for inducing desensitization and / or immunomodulation to peanut in a human patient allergic to peanut.

[0010] In one aspect, the present disclosure provides a recombinant Arah6 mutant polypeptide.

[0011] In some embodiments, the recombinant Arah6 variant polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 109, and includes an amino acid substitution, deletion, insertion, or any combination thereof, at one or more of positions 3, 5, 8, 19, 45, 46, 86, 89, 90, 98, 106, 108, 110, 114, 116, and 118 of SEQ ID NO: 109 compared to the amino acid residue at the same position in SEQ ID NO: 2.

[0012] In some embodiments, a recombinant AraH6 variant polypeptide of the present disclosure comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, at one or more of positions 2, 3, 5, 7, 8, 10, 12, 16, 19, 22, 24, 33, 37, 38, 40, 41, 42, 45, 46, 47, 74, 78, 81, 82, 83, 86, 89, 90, 97, 98, 99, 106, 108, 110, 113, 114, 116, and 118 of SEQ ID NO: 109 compared to the amino acid residue at the same position in SEQ ID NO: 2.

[0013] In some embodiments, a recombinant AraH6 variant polypeptide of the present disclosure comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, at one or more of positions 2, 3, 5, 7, 8, 10, 12, 16, 19, 22, 24, 33, 37, 38, 40, 41, 42, 45, 46, 47, 63, 74, 78, 81, 82, 83, 86, 89, 90, 97, 98, 99, 106, 108, 109, 110, 113, 114, 116, and 118 of SEQ ID NO: 109 compared to the amino acid residue at the same position in SEQ ID NO: 2.

[0014] In some embodiments, the recombinant Arah6 mutant polypeptide of the present disclosure comprises: (a) At position 2, S; (b) D, or S, at position 3; (c) at position 5, D; (d) D at position 7; (e) A or S at position 8; (f) A, S, or K at position 10; (g) R, D, or N at position 12; (h) S, or D at position 16; (i) Q, L, or R at position 19; (j) at position 22, F; (k) at position 24, D; (l) Q or K at position 33; (m) A, T, or S at position 37; (n) A or S at position 38; (o) S at position 40; (p) D at position 41; (q) K, E, or G at position 42; (r) A or Q at position 45; (t) S, G, or R at position 46; (t) S at position 47; (u) R at position 74; (v) at position 78, L; (w) A or R at position 81; (x) T at position 82; (y) N or K at position 83; (z) D or S at position 86; (aa) N, R, or G at position 89; (bb) at position 90, D; (cc) at position 97, I; (dd) D or L at position 98; (ee) at position 99, M; (ff) K or H at position 106; (gg) P, E, or D at position 108; (hh) E or S at position 110; (ii) D, or I at position 113; (jj) D, H, A, or G at position 114; (kk) K or M at position 116; and (ll) R, or T at position 118; It contains one or more amino acids of:

[0015] In some embodiments, the recombinant AraH6 variant polypeptide of the present disclosure comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, at one or more of positions 15, 17, 20, 28, 35, 57, 59, 61, 64, 91, and 123 of SEQ ID NO: 109 compared to the amino acid residues at the same positions in SEQ ID NO: 2.

[0016] In some embodiments, a recombinant AraH6 variant polypeptide of the present disclosure comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, at one or more of positions 3, 5, 8, 19, 45, 46, 89, 98, 110, 114, 116, and 118 of SEQ ID NO: 109 compared to the amino acid residue at the same position in SEQ ID NO: 2.

[0017] In some embodiments, the amino acid substitutions include one or more of: D or S at position 3; D at position 5; A or S at position 8; Q, L, or R at position 19; A or Q at position 45; S, G, or R at position 46; N, R, or G at position 89; D or L at position 98; E or S at position 110; D, H, A, or G at position 114; K or M at position 116; and R or T at position 118.

[0018] In some embodiments, a recombinant AraH6 variant polypeptide of the present disclosure further comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, at one or more of positions 2, 7, 10, 12, 15, 16, 17, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 47, 57, 59, 61, 63, 64, 74, 78, 81, 82, 83, 86, 90, 91, 97, 99, 106, 108, 109, 113, and 123 of SEQ ID NO: 109 compared to the amino acid residue at the same position in SEQ ID NO: 2.

[0019] In some embodiments, the recombinant Arah6 mutant polypeptide of the present disclosure comprises: (a) At position 2, S; (b) D at position 7; (c) A, S, or K at position 10; (d) R, D, or N at position 12; (e) at position 15, R; (f) S, or D at position 16; (g) at position 17, R; (h) at position 20, D; (i) F at position 22; (j) at position 24, D; (k) at position 28, S; (l) Q or K at position 33; (m) A at position 35; (n) A, T, or S at position 37; (o) A or S at position 38; (p) at position 40, S; (q) D at position 41; (r) K, E, or G at position 42; (t) S at position 47; (t) D at position 57; (u) Y at position 59; (v) F at position 61; (w) S at position 64; (x) at position 74, R; (y) at position 78, L; (z) A or R at position 81; (aa) at position 82, T; (bb) N, or K at position 83; (cc) S or D at position 86; (dd) at position 90, D; (ee) A or S at position 91; (ff) I at position 97; (gg) at position 99, M; (hh) K or H at position 106; (ii) P, E, or D at position 108; (jj) D or I at position 113; and (kk) at position 123, D; It contains one or more amino acids of:

[0020] In some embodiments, the recombinant Arah6 variant polypeptides of the present disclosure comprise one or more amino acid substitutions, deletions, insertions, or any combination thereof located within at least one epitope recognized by an anti-Arah6 antibody.

[0021] In some embodiments, the recombinant Arah6 variant polypeptides of the present disclosure contain one or more amino acid substitutions, deletions, insertions, or any combination thereof located within at least two, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more epitopes recognized by anti-Arah6 antibodies.

[0022] In some embodiments, a recombinant Arah6 variant polypeptide of the present disclosure has at least 80% identity to the amino acid sequence set forth in any of SEQ ID NOs: 3-21 or 24-108.

[0023] In some embodiments, a recombinant Arah6 mutant polypeptide of the present disclosure comprises a sequence set forth in any of SEQ ID NOs: 3-21 or 24-108.

[0024] In some embodiments, a recombinant Arah6 mutant polypeptide of the present disclosure comprises 2 to 30 substitutions, deletions, insertions, or any combination thereof.

[0025] In some embodiments, "SEQ ID NO: 110" and "SEQ ID NO: 109" are used interchangeably herein.

[0026] The present disclosure also provides nucleotides or modified nucleotide sequences encoding any one of the recombinant Arah6 variant polypeptides of the present disclosure, expression vectors containing the nucleotides or modified nucleotide sequences, and cells containing the expression vectors. The present disclosure also provides methods of using the expression vectors to produce any one of the recombinant Arah6 variant polypeptides of the present disclosure.

[0027] In some embodiments, the nucleotide or modified nucleotide sequence is DNA or mRNA. In some embodiments, the mRNA is encapsulated in a nanoparticle. In some embodiments, the mRNA comprises LNP-formulated RNA.

[0028] In some embodiments, the cell comprising the expression vector is a prokaryotic or eukaryotic cell. In some embodiments, the eukaryotic cell comprises a yeast cell, a fungal cell, a plant cell, or a mammalian cell.

[0029] In another aspect, the present disclosure provides compositions comprising a recombinant Arah6 variant polypeptide of the present disclosure and / or an isolated or modified nucleotide sequence encoding a recombinant Arah6 variant polypeptide of the present disclosure. In some embodiments, the compositions of the present disclosure are pharmaceutical compositions that include an acceptable carrier or excipient.

[0030] In another aspect, the present disclosure provides a method for inducing peanut hyposensitization and / or immunomodulation of a response to peanut in a subject allergic to peanut, comprising administering to the subject a composition comprising a recombinant Arah6 variant polypeptide of the present disclosure, thereby inducing peanut hyposensitization and / or immunomodulation of a response to peanut in the subject.

[0031] In another aspect, the present disclosure provides a method for inducing peanut hyposensitization and / or immunomodulation of a response to peanut in a subject allergic to peanut, comprising administering to the subject a composition comprising an isolated nucleotide or modified nucleotide sequence encoding a composition comprising a recombinant Arah6 variant polypeptide of the disclosure, thereby inducing peanut hyposensitization and / or immunomodulation of a response to peanut in the subject.

[0032] In another aspect, the present disclosure provides a transgenic peanut plant that expresses a recombinant Arah6 variant polypeptide of the present disclosure. In some embodiments, the recombinant Arah6 variant polypeptide of the present disclosure is expressed from a heterologous nucleic acid. In some embodiments, the expression level of endogenous wild-type Arah6 allergen is reduced compared to non-transgenic peanuts.

[0033] In another aspect, the present disclosure provides a processed food comprising a recombinant Arah6 variant polypeptide of the present disclosure. In some embodiments, the processed food of the present disclosure has a reduced expression level of the endogenous wild-type Arah6 allergen compared to non-transgenic peanuts. In some embodiments, the processed food of the present disclosure comprises peanuts harvested from a transgenic peanut plant of the present disclosure.

[0034] In some embodiments, compositions of the present disclosure are used to induce peanut hyposensitization and / or immunomodulation of a response to peanut in an allergic subject. In some embodiments, compositions of the present disclosure are used in the preparation of a medicament for inducing peanut hyposensitization and / or immunomodulation of a response to peanut in an allergic subject. [Brief explanation of the drawings]

[0035] The subject matter regarded as the hypoallergenic polypeptide variants described herein, which have reduced allergenicity while maintaining immunogenicity, and methods for their preparation, are particularly pointed out and distinctly claimed in the concluding portion of this specification. However, the Arah6 polypeptide variants of the present invention, both as to their organization and their method of operation, together with their objects, features, and advantages, will best be understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.

[0036] [Figure 1A] Figure 1A and Figure 1B: Linear epitope mapping and de-epitope deletion revealed mutations that abolished binding to the AraH6 epitope. Figure 1A: Linear epitope mapping of mAb IgG8 (a mAb isolated from a peanut-allergic patient) revealed IgE binding to the AraH6 peptide. The black box highlights epitope L2 (a peptide derived from positions 61-79 of SEQ ID NO: 1) mapped to AraH6. [Figure 1B]Figure 1B: Linear de-epitope of patient mAb IgG8 AraH6 epitope. The black box highlights the same peptide as in Figure 1A. The gray box highlights where a point mutation dramatically reduced binding to L2. [Figure 2] Figure 2: The two major linear epitope regions for IgE binding were mapped by peptide array using plasma samples from 80 peanut-allergic patients. AraH6 peptide binding by patient plasma or serum at the population level was calculated for each peptide by its relative deviation from the slide median intensity (Z-like score). The distribution of all scores from all slides was plotted and shown as a box plot. The x-axis corresponds to all overlapping peptides, and the y-axis shows the distribution of Z-like scores. The black and gray lines indicate 2 and 3 standard deviations from the slide median intensity, respectively. [Figure 3A] Figures 3A and 3B: Expression and purification of AraH6 wild-type (WT) and epitope-depleted AraH6 mutant D12 (Figure 3A) and AraH6WT and epitope-depleted AraH6 mutants D154, D158, D160, and D179 (Figure 3B). AraH6WT and mutants expressed in E. coli and purified by immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC) were incubated in Laemmli buffer under reducing conditions at 90°C for 5 min. Samples were electrophoresed by TG-SD-SPAGE on a 4-20% polyacrylamide gel and stained with Coomassie. For comparison, native AraH6 is shown in the first lane (Figure 3B). [Figure 3B]Figures 3A and 3B: Expression and purification of AraH6 wild-type (WT) and epitope-depleted AraH6 mutant D12 (Figure 3A) and AraH6WT and epitope-depleted AraH6 mutants D154, D158, D160, and D179 (Figure 3B). AraH6WT and mutants expressed in E. coli and purified by immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC) were incubated in Laemmli buffer under reducing conditions at 90°C for 5 min. Samples were electrophoresed by TG-SD-SPAGE on a 4-20% polyacrylamide gel and stained with Coomassie. For comparison, native AraH6 is shown in the first lane (Figure 3B). [Figure 4A] Figures 4A-4F: Size-exclusion chromatogram (SEC)-HPLC analysis of the purified constructs AraH6 (WT, D12, D154, D158, D160, D160, and D179 variants) demonstrated that the purified constructs were stable and resolved as monomeric forms under the standard conditions tested. Equal volumes of both samples were injected onto an XBridge Protein BEH SEC 200Å column on a UHPLC Arc system (2.5 μm, Waters part number 186009176) at room temperature. Each sample was run at 30°C in a mobile phase of 100 mM sodium phosphate buffer, pH 7.4, 200 mM NaCl at a flow rate of 0.3 mL / min. [Figure 4B] Figures 4A-4F: Size-exclusion chromatogram (SEC)-HPLC analysis of the purified constructs AraH6 (WT, D12, D154, D158, D160, D160, and D179 variants) demonstrated that the purified constructs were stable and resolved as monomeric forms under the standard conditions tested. Equal volumes of both samples were injected onto an XBridge Protein BEH SEC 200Å column on a UHPLC Arc system (2.5 μm, Waters part number 186009176) at room temperature. Each sample was run at 30°C in a mobile phase of 100 mM sodium phosphate buffer, pH 7.4, 200 mM NaCl at a flow rate of 0.3 mL / min. [Figure 4C] Figures 4A-4F: Size-exclusion chromatogram (SEC)-HPLC analysis of the purified constructs AraH6 (WT, D12, D154, D158, D160, D160, and D179 variants) demonstrated that the purified constructs were stable and resolved as monomeric forms under the standard conditions tested. Equal volumes of both samples were injected onto an XBridge Protein BEH SEC 200Å column on a UHPLC Arc system (2.5 μm, Waters part number 186009176) at room temperature. Each sample was run at 30°C in a mobile phase of 100 mM sodium phosphate buffer, pH 7.4, 200 mM NaCl at a flow rate of 0.3 mL / min. [Figure 4D] Figures 4A-4F: Size-exclusion chromatogram (SEC)-HPLC analysis of the purified constructs AraH6 (WT, D12, D154, D158, D160, D160, and D179 variants) demonstrated that the purified constructs were stable and resolved as monomeric forms under the standard conditions tested. Equal volumes of both samples were injected onto an XBridge Protein BEH SEC 200Å column on a UHPLC Arc system (2.5 μm, Waters part number 186009176) at room temperature. Each sample was run at 30°C in a mobile phase of 100 mM sodium phosphate buffer, pH 7.4, 200 mM NaCl at a flow rate of 0.3 mL / min. [Figure 4E]Figures 4A-4F: Size-exclusion chromatogram (SEC)-HPLC analysis of the purified constructs AraH6 (WT, D12, D154, D158, D160, D160, and D179 variants) demonstrated that the purified constructs were stable and resolved as monomeric forms under the standard conditions tested. Equal volumes of both samples were injected onto an XBridge Protein BEH SEC 200Å column on a UHPLC Arc system (2.5 μm, Waters part number 186009176) at room temperature. Each sample was run at 30°C in a mobile phase of 100 mM sodium phosphate buffer, pH 7.4, 200 mM NaCl at a flow rate of 0.3 mL / min. [Figure 4F] Figures 4A-4F: Size-exclusion chromatogram (SEC)-HPLC analysis of the purified constructs AraH6 (WT, D12, D154, D158, D160, D160, and D179 variants) demonstrated that the purified constructs were stable and resolved as monomeric forms under the standard conditions tested. Equal volumes of both samples were injected onto an XBridge Protein BEH SEC 200Å column on a UHPLC Arc system (2.5 μm, Waters part number 186009176) at room temperature. Each sample was run at 30°C in a mobile phase of 100 mM sodium phosphate buffer, pH 7.4, 200 mM NaCl at a flow rate of 0.3 mL / min. [Figure 5A]Figures 5A and 5B: Modified Arah6 variants maintain high thermal stability. Circular dichroism (CD) analysis of recombinant Arah6WT and D12 mutants is shown. CD spectroscopy was performed on the two complete constructs using a Chirascan v.4.7.0.194 CD spectrometer. CD spectra were recorded at far-UV wavelengths of 190-260 nm with a step size of 0.5 nm at 15 temperatures from 20 °C to 90 °C in smooth temperature ramp mode at a heating rate of 1 °C / min. Data for Arah6WT (Figure 5A) and D12 mutant (Figure 5B) are shown at 25 °C and at step temperatures ranging from 20 to 90 °C. The secondary structure of the mutants was shown to be similar to that of the WT, suggesting no significant deviation from the native fold. [Figure 5B] Figures 5A and 5B: Modified Arah6 variants maintain high thermal stability. Circular dichroism (CD) analysis of recombinant Arah6WT and D12 mutants is shown. CD spectroscopy was performed on the two complete constructs using a Chirascan v.4.7.0.194 CD spectrometer. CD spectra were recorded at far-UV wavelengths of 190-260 nm with a step size of 0.5 nm at 15 temperatures from 20 °C to 90 °C in smooth temperature ramp mode at a heating rate of 1 °C / min. Data for Arah6WT (Figure 5A) and D12 mutant (Figure 5B) are shown at 25 °C and at step temperatures ranging from 20 to 90 °C. The secondary structure of the mutants was shown to be similar to that of the WT, suggesting no significant deviation from the native fold. [Figure 6A]Figures 6A-6G: The modified Arah6 mutants dramatically reduced the basophil activation capacity compared with Arah6WT and native Arah6 (nArah6). To evaluate the allergenicity of different Arah6 mutants, we performed a degranulation assay in rat basophilic leukemia (RBL) SX-38 cells. RBLSX-38 cells were sensitized with plasma or serum from allergic patients for 18 h. The cells were then treated with Arah6WT as a negative control, native Arah6 keyhole limpet hemocyanin (KLH), or Arah6 mutants (D12, D75, D76, D77, D154, D158, D160, or D179) at concentrations ranging from 2 μg / ml to 0.02 ng / ml for 1 h. Degranulation was measured using a β-hexosaminidase activity assay. Results for AraH6 variants D12, D75, D76, and D77 are shown for six patient plasma samples: R560 (Figure 6A), R568 (Figure 6B), CL592 (Figure 6C), A601 (Figure 6D), A604 (Figure 6E), and A608 (Figure 6F). Results for AraH6 variants D154, D158, D160, and D179 are shown as the average plasma sample from 11 peanut-allergic patients (Figure 6G). [Figure 6B]Figures 6A-6G: The modified Arah6 mutants dramatically reduced the basophil activation capacity compared with Arah6WT and native Arah6 (nArah6). To evaluate the allergenicity of different Arah6 mutants, we performed a degranulation assay in rat basophilic leukemia (RBL) SX-38 cells. RBLSX-38 cells were sensitized with plasma or serum from allergic patients for 18 h. The cells were then treated with Arah6WT as a negative control, native Arah6 keyhole limpet hemocyanin (KLH), or Arah6 mutants (D12, D75, D76, D77, D154, D158, D160, or D179) at concentrations ranging from 2 μg / ml to 0.02 ng / ml for 1 h. Degranulation was measured using a β-hexosaminidase activity assay. Results for AraH6 variants D12, D75, D76, and D77 are shown for six patient plasma samples: R560 (Figure 6A), R568 (Figure 6B), CL592 (Figure 6C), A601 (Figure 6D), A604 (Figure 6E), and A608 (Figure 6F). Results for AraH6 variants D154, D158, D160, and D179 are shown as the average plasma sample from 11 peanut-allergic patients (Figure 6G). [Figure 6C]Figures 6A-6G: The modified Arah6 mutants dramatically reduced the basophil activation capacity compared with Arah6WT and native Arah6 (nArah6). To evaluate the allergenicity of different Arah6 mutants, we performed a degranulation assay in rat basophilic leukemia (RBL) SX-38 cells. RBLSX-38 cells were sensitized with plasma or serum from allergic patients for 18 h. The cells were then treated with Arah6WT as a negative control, native Arah6 keyhole limpet hemocyanin (KLH), or Arah6 mutants (D12, D75, D76, D77, D154, D158, D160, or D179) at concentrations ranging from 2 μg / ml to 0.02 ng / ml for 1 h. Degranulation was measured using a β-hexosaminidase activity assay. Results for AraH6 variants D12, D75, D76, and D77 are shown for six patient plasma samples: R560 (Figure 6A), R568 (Figure 6B), CL592 (Figure 6C), A601 (Figure 6D), A604 (Figure 6E), and A608 (Figure 6F). Results for AraH6 variants D154, D158, D160, and D179 are shown as the average plasma sample from 11 peanut-allergic patients (Figure 6G). [Figure 6D]Figures 6A-6G: The modified Arah6 mutants dramatically reduced the basophil activation capacity compared with Arah6WT and native Arah6 (nArah6). To evaluate the allergenicity of different Arah6 mutants, we performed a degranulation assay in rat basophilic leukemia (RBL) SX-38 cells. RBLSX-38 cells were sensitized with plasma or serum from allergic patients for 18 h. The cells were then treated with Arah6WT as a negative control, native Arah6 keyhole limpet hemocyanin (KLH), or Arah6 mutants (D12, D75, D76, D77, D154, D158, D160, or D179) at concentrations ranging from 2 μg / ml to 0.02 ng / ml for 1 h. Degranulation was measured using a β-hexosaminidase activity assay. Results for AraH6 variants D12, D75, D76, and D77 are shown for six patient plasma samples: R560 (Figure 6A), R568 (Figure 6B), CL592 (Figure 6C), A601 (Figure 6D), A604 (Figure 6E), and A608 (Figure 6F). Results for AraH6 variants D154, D158, D160, and D179 are shown as the average plasma sample from 11 peanut-allergic patients (Figure 6G). [Figure 6E]Figures 6A-6G: The modified Arah6 mutants dramatically reduced the basophil activation capacity compared with Arah6WT and native Arah6 (nArah6). To evaluate the allergenicity of different Arah6 mutants, we performed a degranulation assay in rat basophilic leukemia (RBL) SX-38 cells. RBLSX-38 cells were sensitized with plasma or serum from allergic patients for 18 h. The cells were then treated with Arah6WT as a negative control, native Arah6 keyhole limpet hemocyanin (KLH), or Arah6 mutants (D12, D75, D76, D77, D154, D158, D160, or D179) at concentrations ranging from 2 μg / ml to 0.02 ng / ml for 1 h. Degranulation was measured using a β-hexosaminidase activity assay. Results for AraH6 variants D12, D75, D76, and D77 are shown for six patient plasma samples: R560 (Figure 6A), R568 (Figure 6B), CL592 (Figure 6C), A601 (Figure 6D), A604 (Figure 6E), and A608 (Figure 6F). Results for AraH6 variants D154, D158, D160, and D179 are shown as the average plasma sample from 11 peanut-allergic patients (Figure 6G). [Figure 6F]Figures 6A-6G: The modified Arah6 mutants dramatically reduced the basophil activation capacity compared with Arah6WT and native Arah6 (nArah6). To evaluate the allergenicity of different Arah6 mutants, we performed a degranulation assay in rat basophilic leukemia (RBL) SX-38 cells. RBLSX-38 cells were sensitized with plasma or serum from allergic patients for 18 h. The cells were then treated with Arah6WT as a negative control, native Arah6 keyhole limpet hemocyanin (KLH), or Arah6 mutants (D12, D75, D76, D77, D154, D158, D160, or D179) at concentrations ranging from 2 μg / ml to 0.02 ng / ml for 1 h. Degranulation was measured using a β-hexosaminidase activity assay. Results for AraH6 variants D12, D75, D76, and D77 are shown for six patient plasma samples: R560 (Figure 6A), R568 (Figure 6B), CL592 (Figure 6C), A601 (Figure 6D), A604 (Figure 6E), and A608 (Figure 6F). Results for AraH6 variants D154, D158, D160, and D179 are shown as the average plasma sample from 11 peanut-allergic patients (Figure 6G). [Figure 6G]Figures 6A-6G: The modified Arah6 mutants dramatically reduced the basophil activation capacity compared with Arah6WT and native Arah6 (nArah6). To evaluate the allergenicity of different Arah6 mutants, we performed a degranulation assay in rat basophilic leukemia (RBL) SX-38 cells. RBLSX-38 cells were sensitized with plasma or serum from allergic patients for 18 h. The cells were then treated with Arah6WT as a negative control, native Arah6 keyhole limpet hemocyanin (KLH), or Arah6 mutants (D12, D75, D76, D77, D154, D158, D160, or D179) at concentrations ranging from 2 μg / ml to 0.02 ng / ml for 1 h. Degranulation was measured using a β-hexosaminidase activity assay. Results for AraH6 variants D12, D75, D76, and D77 are shown for six patient plasma samples: R560 (Figure 6A), R568 (Figure 6B), CL592 (Figure 6C), A601 (Figure 6D), A604 (Figure 6E), and A608 (Figure 6F). Results for AraH6 variants D154, D158, D160, and D179 are shown as the average plasma sample from 11 peanut-allergic patients (Figure 6G). [Figure 7] Figure 7: Arah6WT and epitope-depleted Arah6D12 are expressed, folded, and secreted from mammalian cells. 20 ml of Expi293F cells were transfected with 20 micrograms of plasmid encoding either Arah6WT or Arah6D12, expressed downstream of a human osteonectin leader sequence and carrying a C-terminal 6xHis tag, using Expifectamine293 transfection reagent according to the manufacturer's instructions. Cells were incubated in Expi293 medium at 37°C and 8% CO for 5 days to allow protein expression. Secreted proteins were purified from the expression medium using Ni-NTA Superflow beads, washed, and eluted by adding 350 mM imidazole. Elution fractions were analyzed by SDS-PAGE with or without reduction in β-mercaptoethanol (β-ME). [Figure 8A]Figures 8A-8F: The AraH6 D12 mutant showed reduced binding to anti-AraH6 mAb, four IgGs, and two IgEs. Binding to recombinant AraH6 (SEQ ID NO: 2) or the modified AraH6 D12 mutant (SEQ ID NO: 9) was tested using indirect ELISA titration with increasing concentrations of anti-AraH6 mAb. KLH was used as a negative control. The presented data show that the modified AraH6 D12 mutant dramatically reduced binding to two anti-AraH6 IgEs: E15C2 (Figure 8A) and 7B6 (Figure 8B), and four anti-AraH6 IgGs: IgG5 (Figure 8C), IgG8 (Figure 8D), IgG18 (Figure 8E), and IgG24 (Figure 8F). [Figure 8B] Figures 8A-8F: The AraH6 D12 mutant showed reduced binding to anti-AraH6 mAb, four IgGs, and two IgEs. Binding to recombinant AraH6 (SEQ ID NO: 2) or the modified AraH6 D12 mutant (SEQ ID NO: 9) was tested using indirect ELISA titration with increasing concentrations of anti-AraH6 mAb. KLH was used as a negative control. The presented data show that the modified AraH6 D12 mutant dramatically reduced binding to two anti-AraH6 IgEs: E15C2 (Figure 8A) and 7B6 (Figure 8B), and four anti-AraH6 IgGs: IgG5 (Figure 8C), IgG8 (Figure 8D), IgG18 (Figure 8E), and IgG24 (Figure 8F). [Figure 8C] Figures 8A-8F: The AraH6 D12 mutant showed reduced binding to anti-AraH6 mAb, four IgGs, and two IgEs. Binding to recombinant AraH6 (SEQ ID NO: 2) or the modified AraH6 D12 mutant (SEQ ID NO: 9) was tested using indirect ELISA titration with increasing concentrations of anti-AraH6 mAb. KLH was used as a negative control. The presented data show that the modified AraH6 D12 mutant dramatically reduced binding to two anti-AraH6 IgEs: E15C2 (Figure 8A) and 7B6 (Figure 8B), and four anti-AraH6 IgGs: IgG5 (Figure 8C), IgG8 (Figure 8D), IgG18 (Figure 8E), and IgG24 (Figure 8F). [Figure 8D]Figures 8A-8F: The AraH6 D12 mutant showed reduced binding to anti-AraH6 mAb, four IgGs, and two IgEs. Binding to recombinant AraH6 (SEQ ID NO: 2) or the modified AraH6 D12 mutant (SEQ ID NO: 9) was tested using indirect ELISA titration with increasing concentrations of anti-AraH6 mAb. KLH was used as a negative control. The presented data show that the modified AraH6 D12 mutant dramatically reduced binding to two anti-AraH6 IgEs: E15C2 (Figure 8A) and 7B6 (Figure 8B), and four anti-AraH6 IgGs: IgG5 (Figure 8C), IgG8 (Figure 8D), IgG18 (Figure 8E), and IgG24 (Figure 8F). [Figure 8E] Figures 8A-8F: The AraH6 D12 mutant showed reduced binding to anti-AraH6 mAb, four IgGs, and two IgEs. Binding to recombinant AraH6 (SEQ ID NO: 2) or the modified AraH6 D12 mutant (SEQ ID NO: 9) was tested using indirect ELISA titration with increasing concentrations of anti-AraH6 mAb. KLH was used as a negative control. The presented data show that the modified AraH6 D12 mutant dramatically reduced binding to two anti-AraH6 IgEs: E15C2 (Figure 8A) and 7B6 (Figure 8B), and four anti-AraH6 IgGs: IgG5 (Figure 8C), IgG8 (Figure 8D), IgG18 (Figure 8E), and IgG24 (Figure 8F). [Figure 8F] Figures 8A-8F: The AraH6 D12 mutant showed reduced binding to anti-AraH6 mAb, four IgGs, and two IgEs. Binding to recombinant AraH6 (SEQ ID NO: 2) or the modified AraH6 D12 mutant (SEQ ID NO: 9) was tested using indirect ELISA titration with increasing concentrations of anti-AraH6 mAb. KLH was used as a negative control. The presented data show that the modified AraH6 D12 mutant dramatically reduced binding to two anti-AraH6 IgEs: E15C2 (Figure 8A) and 7B6 (Figure 8B), and four anti-AraH6 IgGs: IgG5 (Figure 8C), IgG8 (Figure 8D), IgG18 (Figure 8E), and IgG24 (Figure 8F). DETAILED DESCRIPTION OF THE INVENTION

[0037] In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the recombinant Arah6 variants of the present disclosure and their uses. However, it will be understood by those skilled in the art that the recombinant Arah6 variants of the present disclosure 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 recombinant Arah6 variants of the present disclosure and their uses.

[0038] In some embodiments, the recombinant Arah6 variant polypeptides 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 disrupt one or more epitopes recognized by anti-Arah6 antibodies, either alone or in combination with additional mutations. The allergenicity of Arah6 was assessed using rat basophilic leukemia (RBL) or basophil activation test (BAT) cell-based immunological assays using samples from peanut-allergic patients. The desired immunogenicity, i.e., the ability of recombinant Arah6 to elicit an immune system response without eliciting a mast cell / basophil-mediated allergic reaction, was measured using a T cell activation assay.

[0039] Those skilled in the art will understand that the term "epitope" can be used interchangeably with the term "antigenic determinant," which all have the same meaning and properties, and encompasses a site on an antigen to which an immunoglobulin or antibody (or antigen-binding fragment thereof) specifically binds. Epitopes can be formed from a contiguous sequence of amino acids or from amino acids that are not contiguous but form spatially contiguous patches due to the tertiary structure of the protein. Epitopes formed from a contiguous sequence of amino acids can be linear epitopes. Linear epitopes are epitopes that bind to immunoglobulins as peptides outside the context of a folded protein. Such linear epitopes are typically able to bind to their cognate immunoglobulins upon exposure to denaturing solvents, whereas epitopes formed by tertiary structure (conformational epitopes) typically lose binding upon treatment with denaturing solvents. In some embodiments, the epitope is as small as possible while 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.

[0040] As used herein, "depitope-depleted Arah6 allergen" refers to a modified Arah6 allergen that has reduced or eliminated binding to an anti-Arah6 antibody (compared to antibody binding to the corresponding wild-type Arah6) due to mutations in one or more epitopes recognized by the anti-Arah6 antibody. In one embodiment, the depitope-depleted Arah6 allergen is less allergenic than its wild-type counterpart.

[0041] As used herein, "epitope" refers to the portion of a macromolecule bound by an antibody or antigen-binding fragment thereof (e.g., AraH6 allergen). Protein sequences include linear, contiguous epitopes ("linear epitopes"), which are made up of consecutive amino acids, and discontinuous epitopes ("conformational epitopes"), which are not consecutive but are made up of amino acids grouped together to form contiguous patches within the folded protein.

[0042] As used herein, "allergen" refers to a substance, proteinaceous or non-proteinaceous, that can induce an allergy or specific hypersensitivity.

[0043] As used herein, "allergenic" or "allergenic" refers to the ability of an antigen or allergen to induce an abnormal 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.

[0044] As used herein, "hypoallergenic" refers to a substance that has little or no potential to cause an allergic reaction.

[0045] In some embodiments, the present disclosure provides peanut allergen (e.g., AraH6) 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 or only minimally affect the biophysical and / or functional properties of the peanut allergen. In one aspect, the mutations can be substitutions, deletions, insertions, or any combination thereof. For example, deletions include the removal of a single amino acid important for antibody binding or the removal of the entire mapped epitope region.

[0046] 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, or 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 the class II residue Ala with a class III residue, such as Asp, Asn, Glu, or Gln. Methods for substitution mutagenesis at the nucleotide or amino acid sequence level are well known in the art.

[0047] 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 for a peanut allergen can be altered (e.g., reduced or eliminated) by modifying an antibody, epitope, allergen, or allergen region identified using the methods provided herein.

[0048] AraH6 mutant

[0049] In one embodiment, the present disclosure provides a recombinant Arah6 variant polypeptide comprising an amino acid sequence having at least 50% identity to the sequence set forth in SEQ ID NO:2, wherein the recombinant Arah6 variant polypeptide comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, located within a single epitope recognized by an anti-Arah6 antibody. In another embodiment, the recombinant Arah6 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-Arah6 antibody.

[0050] Those skilled in the art will understand that percent (%) identity provides a numerical value that indicates 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.

[0051] 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).

[0052] In some embodiments, a recombinant Arah6 variant polypeptide of the present disclosure comprises an amino acid sequence having at least 70%, at least 75%, at least 77%, at least 80%, at least 85%, at least 90%, or at least 95% identity to a polypeptide of the present disclosure or a portion thereof as determined using the National Center for Biotechnology Information (NCBI) BlastP software using default parameters.

[0053] In some embodiments, the recombinant Arah6 variant polypeptides of the present disclosure can include deletions, insertions, or amino acid substitution mutations. In one embodiment, the variant polypeptides include conservative substitutions, or deletions, insertions, or substitutions that do not significantly alter the three-dimensional structure of the polypeptides of interest described herein. In some embodiments, the deletions, insertions, or substitutions do not alter the function of the polypeptides of interest of the present disclosure. In some embodiments, the deletions, insertions, or substitutions do not alter the ability to induce an immune system response resulting in hyposensitization to peanut allergens.

[0054] In some embodiments, the recombinant Arah6 variant polypeptide comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, hi some embodiments, the recombinant Arah6 variant polypeptide comprises 2 to 30, e.g., 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 any range therebetween, substitutions, deletions, insertions, or any combination thereof.

[0055] In one embodiment, a recombinant AraH6 variant polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:22 and includes one or more amino acid substitutions, deletions, insertions, or any combination thereof, at one or more of positions 3, 5, 8, 16, 19, 24, 28, 41, 45, 46, 74, 81, 89, 90, 98, 108, 110, 114, 116, o, and 118 of SEQ ID NO:22 compared to the amino acid residue at the same position in SEQ ID NO:2. In one embodiment, the substitution mutation is D or S at position 3. In one embodiment, the substitution mutation is D at position 5. In one embodiment, the substitution mutation is A or S at position 8. In one embodiment, the substitution mutation is S or D at position 16. In one embodiment, the substitution mutation is Q, L, or R at position 19. In one embodiment, the substitution mutation is D at position 24. In one embodiment, the substitution mutation is S at position 28. In one embodiment, the substitution mutation is D at position 41. In one embodiment, the substitution mutation is A or Q at position 45. In one embodiment, the substitution mutation is S, G, or R at position 46. In one embodiment, the substitution mutation is R at position 74. In one embodiment, the substitution mutation is A or R at position 81. In one embodiment, the substitution mutation is N, R, or G at position 89. In one embodiment, the substitution mutation is D at position 90. In one embodiment, the substitution mutation is D or L at position 98. In one embodiment, the substitution mutation is P, E, or D at position 108. In one embodiment, the substitution mutation is E or S at position 110. In one embodiment, the substitution mutation is D, H, A, or G at position 114. In one embodiment, the substitution mutation is K or M at position 116. In one embodiment, the substitution mutation is R or T at position 118.

[0056] In some embodiments, a recombinant AraH6 variant polypeptide of the present disclosure comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitution mutations at one or more of positions 3, 5, 8, 16, 19, 24, 28, 41, 45, 46, 74, 81, 89, 90, 98, 108, 110, 114, 116, and 118 of SEQ ID NO:22 compared to the amino acid residues at the same positions in SEQ ID NO:2.

[0057] In some embodiments, a recombinant AraH6 variant polypeptide of the present disclosure further comprises an additional amino acid substitution, deletion, insertion, or any combination thereof, at one or more of positions 2, 7, 10, 12, 15, 17, 20, 22, 35, 37, 38, 40, 42, 47, 57, 59, 61, 64, 78, 82, 83, 86, 91, 97, 99, 113, and 123 of SEQ ID NO:22, compared to the amino acid residue at the same position in SEQ ID NO:2. In one embodiment, the substitution mutation is S at position 2. In one embodiment, the substitution mutation is D at position 7. In one embodiment, the substitution mutation is A, S, or K at position 10. In one embodiment, the substitution mutation is R, D, or N at position 12. In one embodiment, the substitution mutation is R at position 15. In one embodiment, the substitution mutation is R at position 17. In one embodiment, the substitution mutation is D at position 20. In one embodiment, the substitution mutation is F at position 22. In one embodiment, the substitution mutation is A at position 35. In one embodiment, the substitution mutation is A, T, or S at position 37. In one embodiment, the substitution mutation is A or S at position 38. In one embodiment, the substitution mutation is S at position 40. In one embodiment, the substitution mutation is K, E, or G at position 42. In one embodiment, the substitution mutation is S at position 47. In one embodiment, the substitution mutation is D at position 57. In one embodiment, the substitution mutation is Y at position 59. In one embodiment, the substitution mutation is F at position 61. In one embodiment, the substitution mutation is S at position 64. In one embodiment, the substitution mutation is L at position 78. In one embodiment, the substitution mutation is T at position 82. In one embodiment, the substitution mutation is N or K at position 83. In one embodiment, the substitution mutation is D or S at position 86. In one embodiment, the substitution mutation is A or S at position 91. In one embodiment, the substitution mutation is I at position 97. In one embodiment, the substitution mutation is M at position 99. In one embodiment, the substitution mutation is D or I at position 113.In one embodiment, the substitution mutation is a D at position 123.

[0058] In some embodiments, a recombinant AraH6 variant polypeptide of the present disclosure further comprises 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, or 27 substitution mutations at positions selected from 2, 7, 10, 12, 15, 17, 20, 22, 35, 37, 38, 40, 42, 47, 57, 59, 61, 64, 78, 82, 83, 86, 91, 97, 99, 113, and 123 of SEQ ID NO:22 compared to the amino acid residue at the same position in SEQ ID NO:2.

[0059] In some embodiments, a recombinant AraH6 variant polypeptide of the present disclosure comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, at one or more of positions 2, 3, 5, 7, 8, 10, 12, 16, 19, 22, 24, 33, 37, 38, 40, 41, 42, 45, 46, 47, 63, 74, 78, 81, 82, 83, 86, 89, 90, 97, 98, 99, 106, 108, 109, 110, 113, 114, 116, and 118 of SEQ ID NO: 109 compared to the amino acid residue at the same position in SEQ ID NO: 2.

[0060] In one embodiment, the one or more amino acid substitutions are S at position 2; D or S at position 3; D at position 5; D at position 7; A or S at position 8; A, S, or K at position 10; R, D, or N at position 12; S or D at position 16; Q, L, or R at position 19; F at position 22; D at position 24; Q or K at position 33; A, T, or S at position 37; A or S at position 38; S at position 40; D at position 41; K, E, or G at position 42; A or Q at position 45; S, G, or R at position 46; The amino acid sequence includes one or more of: S at position 47; R at position 74; L at position 78; A or R at position 81; T at position 82; N or K at position 83; D or S at position 86; N, R, or G at position 89; D at position 90; I at position 97; D or L at position 98; M at position 99; K or H at position 106; P, E, or D at position 108; E or S at position 110; D or I at position 113; D, H, A, or G at position 114; K or M at position 116; and R or T at position 118.

[0061] In one embodiment, the substitution mutation is any amino acid at position 63. In one embodiment, the substitution mutation is any amino acid at position 109.

[0062] In one embodiment, the substitution mutation is S at position 2. In one embodiment, the substitution mutation is D or S at position 3. In one embodiment, the substitution mutation is D at position 5. In one embodiment, the substitution mutation is D at position 7. In one embodiment, the substitution mutation is A or S at position 8. In one embodiment, the substitution mutation is A, S, or K at position 10. In one embodiment, the substitution mutation is R, D, or N at position 12. In one embodiment, the substitution mutation is S or D at position 16. In one embodiment, the substitution mutation is Q, L, or R at position 19. In one embodiment, the substitution mutation is F at position 22. In one embodiment, the substitution mutation is D at position 24. In one embodiment, the substitution mutation is Q or K at position 33. In one embodiment, the substitution mutation is A, T, or S at position 37. In one embodiment, the substitution mutation is A or S at position 38. In one embodiment, the substitution mutation is S at position 40. In one embodiment, the substitution mutation is D at position 41. In one embodiment, the substitution mutation is K, E, or G at position 42. In one embodiment, the substitution mutation is A or Q at position 45. In one embodiment, the substitution mutation is S, G, or R at position 46. In one embodiment, the substitution mutation is S at position 47. In one embodiment, the substitution mutation is R at position 74. In one embodiment, the substitution mutation is L at position 78. In one embodiment, the substitution mutation is A or R at position 81. In one embodiment, the substitution mutation is T at position 82. In one embodiment, the substitution mutation is N or K at position 83. In one embodiment, the substitution mutation is D or S at position 86. In one embodiment, the substitution mutation is N, R, or G at position 89. In one embodiment, the substitution mutation is D at position 90. In one embodiment, the substitution mutation is I at position 97. In one embodiment, the substitution mutation is D or L at position 98. In one embodiment, the substitution mutation is M at position 99. In one embodiment, the substitution mutation is K or H at position 106.In one embodiment, the substitution mutation is P, E, or D at position 108. In one embodiment, the substitution mutation is E or S at position 110. In one embodiment, the substitution mutation is D or I at position 113. In one embodiment, the substitution mutation is D, H, A, or G at position 114. In one embodiment, the substitution mutation is K or M at position 116. In one embodiment, the substitution mutation is R or T at position 118.

[0063] In some embodiments, the recombinant AraH6 variant polypeptide of the present disclosure comprises a sequence selected from positions 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, and 40 of SEQ ID NO: 109. At positions where the amino acid residues are present, the amino acid sequence contains 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 substitution mutations compared to the amino acid residues at the same positions in SEQ ID NO:2.

[0064] In one embodiment, the recombinant Arah6 variant polypeptide comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof at positions 3, 5, 8, 19, 45, 46, 89, 98, 110, 114, 116, and 118 of SEQ ID NO: 109 compared to the amino acid residues at the same positions in SEQ ID NO: 2.

[0065] In one embodiment, the one or more amino acid substitutions include one or more of: D or S at position 3; D at position 5; A or S at position 8; Q, L, or R at position 19; A or Q at position 45; S, G, or R at position 46; N, R, or G at position 89; D or L at position 98; E or S at position 110; D, H, A, or G at position 114; K or M at position 116; and R or T at position 118.

[0066] In one embodiment, the recombinant Arah6 variant polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 109 and includes one or more amino acid substitutions, deletions, insertions, or any combination thereof, at one or more of positions 3, 5, 8, 19, 45, 46, 86, 89, 90, 98, 106, 108, 110, 114, 116, and 118 of SEQ ID NO: 109 compared to the amino acid residue at the same position in SEQ ID NO: 2.

[0067] In some embodiments, a recombinant AraH6 variant polypeptide of the present disclosure comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 substitution mutations at positions selected from positions 3, 5, 8, 19, 45, 46, 86, 89, 90, 98, 106, 108, 110, 114, 116, and 118 of SEQ ID NO: 109 compared to the amino acid residues at the same positions in SEQ ID NO: 2.

[0068] In some embodiments, a recombinant AraH6 variant polypeptide of the present disclosure comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, at one or more of positions 2, 7, 10, 12, 15, 16, 17, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 47, 57, 59, 61, 63, 64, 74, 78, 81, 82, 83, 86, 90, 91, 97, 99, 106, 108, 109, 113, and 123 of SEQ ID NO: 109 compared to the amino acid residue at the same position in SEQ ID NO: 2.

[0069] In one embodiment, the one or more amino acid substitutions are S at position 2; D at position 7; A, S or K at position 10; R, D or N at position 12; R at position 15; S or D at position 16; R at position 17; D at position 20; F at position 22; D at position 24; S at position 28; Q or K at position 33; A at position 35; A, T or S at position 37; A or S at position 38; S at position 40; D at position 41; K, E or G at position 42; The amino acid sequence includes one or more of: S at position 47; D at position 57; Y at position 59; F at position 61; S at position 64; R at position 74; L at position 78; A or R at position 81; T at position 82; N or K at position 83; S or D at position 86; D at position 90; A or S at position 91; I at position 97; M at position 99; K or H at position 106; P, E or D at position 108; D or I at position 113; and D at position 123.

[0070] In one embodiment, the substitution mutation is any amino acid at position 63. In one embodiment, the substitution mutation is any amino acid at position 109.

[0071] In some embodiments, a recombinant AraH6 variant polypeptide of the present disclosure comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, at one or more of positions 2, 7, 10, 12, 15, 16, 17, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 47, 57, 59, 61, 64, 74, 78, 81, 82, 83, 91, 97, 99, 113, and 123 of SEQ ID NO: 109 compared to the amino acid residue at the same position in SEQ ID NO: 2. In one embodiment, the substitution mutation is S at position 2. In one embodiment, the substitution mutation is D at position 7. In one embodiment, the substitution mutation is A, S, or K at position 10. In one embodiment, the substitution mutation is R, D, or N at position 12. In one embodiment, the substitution mutation is R at position 15. In one embodiment, the substitution mutation is S or D at position 16. In one embodiment, the substitution mutation is R at position 17. In one embodiment, the substitution mutation is D at position 20. In one embodiment, the substitution mutation is F at position 22. In one embodiment, the substitution mutation is D at position 24. In one embodiment, the substitution mutation is S at position 28. In one embodiment, the substitution mutation is Q or K at position 33. In one embodiment, the substitution mutation is A at position 35. In one embodiment, the substitution mutation is A, T, or S at position 37. In one embodiment, the substitution mutation is A or S at position 38. In one embodiment, the substitution mutation is S at position 40. In one embodiment, the substitution mutation is D at position 41. In one embodiment, the substitution mutation is K, E, or G at position 42. In one embodiment, the substitution mutation is S at position 47. In one embodiment, the substitution mutation is D at position 57. In one embodiment, the substitution mutation is Y at position 59. In one embodiment, the substitution mutation is F at position 61. In one embodiment, the substitution mutation is S at position 64. In one embodiment, the substitution mutation is R at position 74. In one embodiment, the substitution mutation is L at position 78. In one embodiment, the substitution mutation is A or R at position 81.In one embodiment, the substitution mutation is T at position 82. In one embodiment, the substitution mutation is N or K at position 83. In one embodiment, the substitution mutation is A or S at position 91. In one embodiment, the substitution mutation is I at position 97. In one embodiment, the substitution mutation is M at position 99. In one embodiment, the substitution mutation is D or I at position 113. In one embodiment, the substitution mutation is D at position 123.

[0072] In some embodiments, a recombinant AraH6 variant polypeptide of the present disclosure comprises 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, or 33 substitution mutations at positions selected from 2, 7, 10, 12, 15, 16, 17, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 47, 57, 59, 61, 64, 74, 78, 81, 82, 83, 91, 97, 99, 113, and 123 of SEQ ID NO: 109 compared to the amino acid residue at the same position in SEQ ID NO: 2.

[0073] In one embodiment, a recombinant AraH6 variant polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 109, and includes a substitution mutation, deletion, insertion, or any combination thereof, at one or more of positions 2, 3, 5, 7, 8, 10, 12, 15, 16, 17, 19, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 45, 46, 47, 57, 59, 61, 64, 74, 78, 81, 82, 83, 86, 89, 90, 91, 97, 98, 99, 106, 108, 110, 113, 114, 116, 118, and 12 of SEQ ID NO: 109 compared to the amino acid residue at the same position in SEQ ID NO: 2. In one embodiment, the substitution mutation is S at position 2. In one embodiment, the substitution mutation is D or S at position 3. In one embodiment, the substitution mutation is D at position 5. In one embodiment, the substitution mutation is D at position 7. In one embodiment, the substitution mutation is A or S at position 8. In one embodiment, the substitution mutation is A, S, or K at position 10. In one embodiment, the substitution mutation is R, D, or N at position 12. In one embodiment, the substitution mutation is R at position 15. In one embodiment, the substitution mutation is S or D at position 16. In one embodiment, the substitution mutation is R at position 17. In one embodiment, the substitution mutation is Q, L, or R at position 19. In one embodiment, the substitution mutation is D at position 20. In one embodiment, the substitution mutation is F at position 22. In one embodiment, the substitution mutation is D at position 24. In one embodiment, the substitution mutation is S at position 28. In one embodiment, the substitution mutation is Q or K at position 33. In one embodiment, the substitution mutation is A at position 35. In one embodiment, the substitution mutation is A, T, or S at position 37. In one embodiment, the substitution mutation is A or S at position 38. In one embodiment, the substitution mutation is S at position 40. In one embodiment, the substitution mutation is D at position 41. In one embodiment, the substitution mutation is K, E, or G at position 42. In one embodiment, the substitution mutation is A or Q at position 45.In one embodiment, the substitution mutation is S, G, or R at position 46. In one embodiment, the substitution mutation is S at position 47. In one embodiment, the substitution mutation is D at position 57. In one embodiment, the substitution mutation is Y at position 59. In one embodiment, the substitution mutation is F at position 61. In one embodiment, the substitution mutation is S at position 64. In one embodiment, the substitution mutation is R at position 74. In one embodiment, the substitution mutation is L at position 78. In one embodiment, the substitution mutation is A or R at position 81. In one embodiment, the substitution mutation is T at position 82. In one embodiment, the substitution mutation is N or K at position 83. In one embodiment, the substitution mutation is D or S at position 86. In one embodiment, the substitution mutation is N, R, or G at position 89. In one embodiment, the substitution mutation is D at position 90. In one embodiment, the substitution mutation is A or S at position 91. In one embodiment, the substitution mutation is I at position 97. In one embodiment, the substitution mutation is D or L at position 98. In one embodiment, the substitution mutation is M at position 99. In one embodiment, the substitution mutation is K or H at position 106. In one embodiment, the substitution mutation is P, E, or D at position 108. In one embodiment, the substitution mutation is E or S at position 110. In one embodiment, the substitution mutation is D or I at position 113. In one embodiment, the substitution mutation is D, H, A, or G at position 114. In one embodiment, the substitution mutation is K or M at position 116. In one embodiment, the substitution mutation is R or T at position 118. In one embodiment, the substitution mutation is D at position 123.

[0074] In some embodiments, the recombinant AraH6 variant polypeptide of the present disclosure comprises a sequence identical to that at positions 2, 3, 5, 7, 8, 10, 12, 15, 16, 17, 19, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 45, 46, 47, 57, 59, 61, 64, 74, 78, 81, 82, 83, 86, 89, 90, 91, 97, 98, 99, 106, 108, 110, 113, 114, 116, 118, 119, 220, 221, 222, 224, 2 ... , and 123, and contain 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, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 substitution mutations compared to the amino acid residue at the same position in SEQ ID NO:2.

[0075] In some embodiments, the recombinant AraH6 variant polypeptide of the present disclosure comprises a sequence identical to that at positions 2, 3, 5, 7, 8, 10, 12, 15, 16, 17, 19, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 45, 46, 47, 57, 59, 61, 63, 64, 74, 78, 81, 82, 83, 86, 89, 90, 91, 97, 98, 99, 106, 108, 109, 110, 113, 114, 116, 118, 119, 220, 221, 222, 224, 228, 33, 35, 37, 38, 40, 41, 42, 45, 46, 47, 57, 59, 61, 63, 64, 74, 78, 81, 82, 83, 86, 89, 90, 91, 97, 98, 99, 106, 108, 109, 110, 113, 114, 116, 118, 119, 221, 222, 224, 228, 33, 35, 37, 38, 40, 41, 42, 45, 46, 47, 57, 59, 61, 63, 64, 74, 78, 81, 82, 83, , and 123, contain 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, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 substitution mutations compared to the amino acid residue at the same position in SEQ ID NO:2.

[0076] In some embodiments, a recombinant AraH6 variant polypeptide of the present disclosure comprises between 2 and 51 substitutions, deletions, insertions, or any combination thereof, e.g., 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, 45, 46, 47, 48, 49, 50, 51, or any range therebetween.

[0077] In some embodiments, a recombinant AraH6 variant polypeptide of the present disclosure comprises at least one amino acid mutation, deletion, insertion, or any combination thereof, at one or more of positions 15, 16, 17, 19, 20, 22, 24, 28, 57, 59, 61, 64, 74, 78, 81, 82, 83, 86, 98, and 116 of SEQ ID NO: 109 compared to the amino acid residue at the same position in SEQ ID NO: 2.

[0078] In some embodiments, a recombinant Arah6 mutant polypeptide of the present disclosure comprises an amino acid sequence set forth in any of SEQ ID NOs: 3-21.

[0079] In some embodiments, a recombinant Arah6 mutant polypeptide of the present disclosure comprises an amino acid sequence set forth in any of SEQ ID NOs: 24-108.

[0080] In some embodiments, the recombinant AraH6 variant of the present disclosure comprises an amino acid sequence set forth in any of SEQ ID NOs: 3-21, or an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any of SEQ ID NOs: 3-21.

[0081] In some embodiments, the recombinant AraH6 variant of the present disclosure comprises an amino acid sequence set forth in any of SEQ ID NOs: 3-21, or an amino acid sequence having at least 77% identity to an amino acid sequence set forth in any of SEQ ID NOs: 3-21.

[0082] In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid sequence set forth in any of SEQ ID NOs: 3-21 or 24-108.

[0083] In some embodiments, a recombinant AraH6 variant of the present disclosure comprises an amino acid sequence having at least 70%, at least 75%, at least 77%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence set forth in any of SEQ ID NOs: 3-21 or 24-108.

[0084] In some embodiments, a recombinant AraH6 variant of the present disclosure comprises an amino acid sequence set forth in any of SEQ ID NOs: 24-108, or an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any of SEQ ID NOs: 24-108.

[0085] In some embodiments, a recombinant AraH6 variant of the present disclosure comprises an amino acid sequence set forth in any of SEQ ID NOs: 24-108, or an amino acid sequence having at least 77% identity to an amino acid sequence set forth in any of SEQ ID NOs: 24-108.

[0086] In some embodiments, a recombinant Arah6 variant of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:46. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:76. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:80. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:82. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:101. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:108.

[0087] In some embodiments, basophil degranulation release induced by a recombinant Arah6 variant of the present disclosure is at least 10-fold lower than that induced by an Arah6 wild-type polypeptide.

[0088] In some embodiments, the recombinant Arah6 variant has a binding EC50 or KD reduced by 50% or more compared to the Arah6 wild-type polypeptide.

[0089] In some embodiments, the recombinant Arah6 variant comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof located within at least one epitope recognized by an anti-Arah6 antibody.

[0090] In some embodiments, the AraH6 epitope comprises a linear epitope (L2) comprising amino acids at positions 36-54 of SEQ ID NO:2. In some embodiments, the AraH6 epitope comprises a linear epitope (L3) comprising amino acids at positions 76-90 of SEQ ID NO:2. In some embodiments, the AraH6 epitope comprises a linear epitope (L4) comprising amino acids at positions 92-102 of SEQ ID NO:2. In some embodiments, the AraH6 epitope comprises a linear epitope (L5) comprising amino acids at positions 104-118 of SEQ ID NO:2.

[0091] In some embodiments, the AraH6 epitope comprises a conformational epitope (C1) comprising amino acids at positions 16, 19, 74, and 81 of SEQ ID NO:2. In some embodiments, the AraH6 epitope comprises a conformational epitope (C2) comprising amino acids at positions 19, 22, and 24 of SEQ ID NO:2. In some embodiments, the AraH6 epitope comprises a conformational epitope (C3) comprising amino acids at positions 33 and 106 of SEQ ID NO:2. In some embodiments, the AraH6 epitope comprises a conformational epitope (C4) comprising amino acids at positions 63, 108, and 109 of SEQ ID NO:2. In some embodiments, the AraH6 epitope comprises a conformational epitope (C5) comprising amino acids at positions 114 and 116 of SEQ ID NO:2.

[0092] In some embodiments, a recombinant Arah6 variant of the present disclosure comprises at least one, e.g., at least two or more, amino acid substitutions, deletions, insertions, or any combination thereof located within at least one epitope. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises at least two amino acid substitutions, deletions, insertions, or any combination thereof located within at least one conformational epitope selected from C1, C2, C3, C4, and C5. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises at least two amino acid substitutions, deletions, insertions, or any combination thereof located within at least two conformational epitopes selected from C1, C2, C3, C4, and C5. In some embodiments, the conformational epitope (C1) comprises amino acids located at positions 16, 19, 74, and 81 of SEQ ID NO:2. In some embodiments, the conformational epitope (C2) comprises amino acids located at positions 19, 22, and 24 of SEQ ID NO:2. In some embodiments, conformational epitope (C3) comprises amino acids located at positions 33 and 106 of SEQ ID NO:2. In some embodiments, conformational epitope (C4) comprises amino acids located at positions 63, 108, and 109 of SEQ ID NO:2. In some embodiments, conformational epitope (C5) comprises amino acids located at positions 114 and 116 of SEQ ID NO:2.

[0093] In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid substitution, deletion, insertion, or any combination thereof, at positions 3, 5, 8, 19, 45, 46, 89, 98, 110, 114, 116, and 118 of SEQ ID NO: 109 compared to the amino acid residues at the same positions in SEQ ID NO: 2. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in any of SEQ ID NOs: 46, 76, 80, 82, and 101. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid sequence having at least 77% identity to the amino acid sequence set forth in any of SEQ ID NOs: 46, 76, 80, 82, and 101. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid sequence identical to the amino acid sequence set forth in any of SEQ ID NOs: 46, 76, 80, 82, and 101. In some embodiments, recombinant Arah6 variants of the present disclosure comprise a conformational epitope recognized by anti-Arah6 IgG antibodies. In some embodiments, recombinant Arah6 variants of the present disclosure reduce or eliminate binding to anti-Arah6 IgE antibodies, e.g., within the conformational epitope. In some embodiments, reduction refers to at least a 10-fold decrease in the binding affinity of the variant compared to binding to native or wild-type (WT) Arah6 (SEQ ID NOs: 1 and 2).

[0094] In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid substitution at positions 3, 5, 8, 19, 35, 37, 38, 45, 46, 89, 90, 98, 108, 110, 114, 116, 118 of SEQ ID NO: 109 compared to the amino acid residues at the same positions in SEQ ID NO: 2. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 46.

[0095] In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid substitution at positions 3, 5, 8, 19, 35, 37, 38, 45, 46, 86, 89, 90, 98, 106, 108, 110, 114, 116, 118 of SEQ ID NO: 109 compared to the amino acid residue at the same position in SEQ ID NO: 2. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 76.

[0096] In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid substitution at positions 3, 5, 8, 19, 45, 46, 86, 89, 90, 98, 106, 108, 110, 114, 116, 118 of SEQ ID NO: 109 compared to the amino acid residues at the same positions in SEQ ID NO: 2. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 80.

[0097] In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid substitution at positions 3, 5, 8, 19, 37, 45, 46, 86, 89, 90, 98, 106, 108, 110, 114, 116, 118 of SEQ ID NO: 109 compared to the amino acid residues at the same positions in SEQ ID NO: 2. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 82.

[0098] In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid substitution at positions 3, 5, 8, 19, 45, 46, 86, 89, 98, 106, 110, 114, 116, 118 of SEQ ID NO: 109 compared to the amino acid residues at the same positions in SEQ ID NO: 2. In some embodiments, a recombinant Arah6 variant of the present disclosure comprises an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 101.

[0099] In some embodiments, the recombinant Arah6 variants of the present disclosure include a methionine located upstream of the N-terminus of the amino acid sequence (or protein sequence) described herein. In this embodiment, the methionine forms the N-terminus of the amino acid sequence. Such a methionine is typically derived from translation of the RNA encoding the protein and is encoded by the nucleic acid triplet ATG. Unless already present in the nucleic acid sequence, the ATG triplet can be attached to the 5' end of the nucleic acid molecule by methods known to those skilled in the art.

[0100] In some embodiments of the recombinant Arah6 variants of the present disclosure, the amino acid sequence set forth in any one of SEQ ID NOs: 3-21 further comprises a methionine located upstream of the N-terminus of the amino acid sequence set forth in any one of SEQ ID NOs: 3-21. In some embodiments of the recombinant Arah6 variants of the present disclosure, the amino acid sequence set forth in any one of SEQ ID NOs: 24-108 further comprises a methionine located upstream of the N-terminus of the amino acid sequence set forth in any one of SEQ ID NOs: 24-108.

[0101] In some embodiments of the recombinant AraH6 variants of the present disclosure, the amino acid sequence further comprises a label or tag (purification tag or stability tag) at its N-terminus or its C-terminus. In some embodiments, the tag is selected from a His tag, an HA tag, or other suitable tag known in the art.

[0102] In some embodiments, "SEQ ID NO: 110" and "SEQ ID NO: 109" are used interchangeably herein.

[0103] Nucleotides, vectors, and host cells

[0104] In one embodiment, the present disclosure provides an isolated nucleotide or modified nucleotide sequence encoding the recombinant Arah6 variant described herein. In some embodiments, the nucleotide or modified nucleotide sequence is DNA or mRNA. In some embodiments of the nucleotide or modified nucleotide sequence of the present disclosure, the mRNA comprises lipid nanoparticle (LNP)-formulated mRNA.

[0105] In one embodiment, the present disclosure provides an expression vector comprising an isolated nucleotide or modified nucleotide sequence described herein.

[0106] In one embodiment, the disclosure provides a prokaryotic or eukaryotic cell comprising an expression vector described herein. In some embodiments, the eukaryotic cell is a yeast cell, a fungal cell, a plant cell, or a mammalian cell.

[0107] In one embodiment, the present disclosure provides a composition comprising a recombinant Arah6 mutant polypeptide as described in detail herein.

[0108] As used herein, the terms "nucleotide," "nucleotide sequence," or "nucleic acid molecule" are 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 nucleotides comprise modified nucleotides. In some embodiments, the nucleotides comprise mRNA. In some embodiments, the nucleotides comprise modified mRNA. In some embodiments, the nucleotides comprise modified mRNA, and the modified mRNA comprises a 5'-capped mRNA. In some embodiments, the modified mRNA comprises a molecule in which some of the nucleosides are replaced with either naturally occurring modified nucleosides or synthetic nucleosides. In some embodiments, the modified nucleotides comprise modified mRNA, and the modified mRNA comprises a 5'-capped mRNA, and some of the nucleosides are replaced with either naturally occurring modified nucleosides or synthetic nucleosides.

[0109] As used herein, the term "isolated nucleotide" or "isolated nucleic acid molecule" refers to a nucleic acid encoding a peanut allergen variant of the present disclosure (e.g., an AraH6 variant), wherein the nucleotide sequence is essentially free of other genomic nucleotide sequences that naturally flank the nucleic acid in genomic DNA.

[0110] One aspect of the present disclosure is a nucleotide or nucleic acid sequence encoding a peanut allergen variant (e.g., an AraH6 variant) of the present disclosure.

[0111] 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 nucleic acids operably linked to regulatory sequences, such as promoter regions, that can affect the expression of such nucleic acids. Thus, an expression vector can 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 an appropriate host cell. Suitable expression vectors are well known to those of skill 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.

[0112] One aspect of the present disclosure is an expression vector comprising a nucleic acid construct encoding a peanut allergen variant (e.g., an AraH6 variant) of the present disclosure.

[0113] 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 the particular cell but to the progeny of that cell. Because certain modifications may occur in progeny, due either to mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0114] One aspect of the present disclosure is a host cell comprising an expression vector carrying a nucleic acid construct encoding a peanut allergen variant (e.g., AraH6) of the present disclosure. 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 can be produced in bacteria such as Escherichia coli. In other embodiments, the peanut allergen variant can be produced in yeast or fungi, such as Saccharomyces cerevisiae, Aspergillus, Trichoderma, or Pichia pastoris.

[0115] Nucleic acids encoding AraH6 variants

[0116] In one embodiment, the present disclosure provides a nucleic acid or modified nucleic acid molecule encoding a recombinant Arah6 variant polypeptide comprising an amino acid sequence having at least 50% identity to the sequence set forth in SEQ ID NO:2, wherein the recombinant Arah6 variant comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, located within a single epitope recognized by an anti-Arah6 antibody.

[0117] In another embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes a recombinant Arah6 variant comprising an amino acid sequence having at least 50% identity to the sequence set forth in SEQ ID NO:2, and comprising one or more amino acid substitutions, deletions, insertions, or any combination thereof, located within at least two epitopes recognized by anti-Arah6 antibodies.

[0118] Those skilled in the art will understand that percent (%) identity provides a numerical value that indicates 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.

[0119] 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).

[0120] In some embodiments, a recombinant Arah6 variant polypeptide of the present disclosure comprises an amino acid sequence having at least 70%, at least 75%, at least 77%, at least 80%, at least 85%, or at least 90% identity to the sequence set forth in SEQ ID NO:2, or a portion thereof, as determined using the National Center for Biotechnology Information (NCBI) BlastP software using default parameters.

[0121] In some embodiments, the recombinant Arah6 variant polypeptides of the present disclosure can include deletions, insertions, or amino acid substitution mutations. In one embodiment, the variant polypeptides include conservative substitutions, or deletions, insertions, or substitutions that do not significantly alter the three-dimensional structure of the polypeptides of interest described herein. In some embodiments, the deletions, insertions, or substitutions do not alter the function of the polypeptides of interest of the present disclosure. In some embodiments, the deletions, insertions, or substitutions do not alter the ability to induce an immune system response resulting in hyposensitization to peanut allergens.

[0122] 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 may promote higher overall protein expression.

[0123] In one embodiment, the mRNA comprises an optimized sequence. As used herein, "optimized sequence" encompasses mRNA sequences that contain computationally modified nucleotide sequences that promote higher expression levels in human cells compared to the unmodified sequence, while maintaining favorable properties for in vitro transcription (IVT) and enzymatic capping.

[0124] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes an Arah6 variant comprising an amino acid sequence set forth in any of SEQ ID NOs: 3-21, or an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any of SEQ ID NOs: 3-21.

[0125] In one embodiment, a nucleic acid or modified nucleic acid molecule of the present disclosure encodes an Arah6 variant comprising an amino acid sequence set forth in any of SEQ ID NOs: 24-108, or an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any of SEQ ID NOs: 24-108.

[0126] In some embodiments, the nucleic acids or modified nucleic acid molecules disclosed herein further comprise a nucleic acid sequence encoding a label or tag (such as a purification tag or a stability tag) at the 5' or 3' end. In some embodiments, the tag is selected from a His tag, an HA tag, or other tags known in the art. In some embodiments, the nucleic acids or modified nucleic acid molecules disclosed herein further comprise a restriction endonuclease sequence.

[0127] Manufacturing method

[0128] In some embodiments, the variant polypeptides of the present disclosure can be produced using a cell-free in vitro translation system, such as those 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 Arah6 variant of the present disclosure, the method comprising culturing a cell comprising an expression vector of the present disclosure described above under conditions for expression of the Arah6 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.

[0129] In some embodiments, the nucleic acids or modified nucleic acid molecules of the present disclosure are transcribed in an in vitro transcription system (IVT), and the transcribed nucleic acids or modified nucleic acids can then be used for immunotherapy by gene delivery. Administration of the RNA results in the in vivo production of peanut allergens or peanut allergen variants.

[0130] In some embodiments of the disclosed manufacturing methods, the nucleic acid molecule encodes an Arah6 variant polypeptide containing one or more amino acid substitutions, deletions, insertions, or any combination thereof, located within a single epitope recognized by an anti-Arah6 antibody. In some embodiments, the nucleic acid comprises a modified nucleic acid encoding an Arah6 variant polypeptide containing one or more amino acid mutations located within a single epitope recognized by an anti-Arah6 antibody.

[0131] 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, as 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".

[0132] Those skilled 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.

[0133] The production of the peanut allergen variant, AraH6 variant, may involve in vivo translation in which transcribed mRNA is administered to a subject (patient).

[0134] 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 administering 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, fomv virus, 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 administering 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, fomv virus, 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 Arah6 variants, are well known in the art and are described, for example, but not limited to, 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 entireties.

[0135] 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.

[0136] In some embodiments, the subject includes a subject in need of induction of hyposensitivity 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.

[0137] How to use

[0138] 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 hypoallergenic Arah6 variant of the present disclosure, thereby inducing hyposensitization to peanuts in the subject.

[0139] In one embodiment, the present disclosure provides a method for inducing immunomodulation of a response to peanuts in a subject allergic to peanuts, comprising administering to the subject a composition comprising a hypoallergenic Arah6 variant of the present disclosure, thereby increasing the subject's ability to tolerate peanuts.

[0140] As used herein, peanut allergy desensitization or peanut desensitization, also referred to as allergy immunotherapy, allergy immunomodulation, immunomodulation of peanut responses, or allergen-specific immunotherapy, refers to a treatment aimed at reducing the severity of clinical reactions to peanut, increasing the tolerable dose of peanut, and / or increasing long-term tolerance to peanut. Peanut immunotherapy can be tested using methods known in the art, such as food challenge testing. Peanut immunotherapy may be partial, in which a patient can tolerate increased amounts of food allergen compared to before treatment but still respond to high doses of food allergen, or it may be complete desensitization, in which a patient can tolerate all doses of food allergen tested. In some embodiments, peanut desensitization involves reducing the activation capacity of basophils and / or mast cells compared to before treatment.

[0141] In some embodiments, "immunomodulation of a response to peanut" comprises a decrease in the allergic response to peanut. In some embodiments, the allergic response to peanut in the patient is reduced (decreased) compared to the patient's allergic response to peanut before treatment or compared to an earlier time point during the course of treatment. In some embodiments, the allergic response to peanut is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, a decrease in the allergic response to peanut in the patient comprises a decrease in the ability to activate basophils and / or mast cells compared to before treatment.

[0142] As used herein, "allergy immunomodulation," also known as "allergy desensitization," "allergy immunotherapy," or "allergen-specific immunotherapy," refers to a treatment aimed at reducing the severity of clinical reactions to peanut or increasing the tolerable dose of peanut. Peanut immunotherapy can be tested using methods known in the art, such as food challenge testing. Peanut immunotherapy can be partial, meaning that the patient can tolerate increased amounts of food allergen compared to before treatment but still reacts to high doses of food allergen, or it can be complete desensitization, meaning that the patient can tolerate all doses of food allergen tested.

[0143] In some embodiments, the methods of the present 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, surfactants such as lysolecithin, Pluronic® polyols, polyanions, peptides, levamisole, CpG-DNA, oil or hydrocarbon emulsions, and potentially useful adjuvants such as Bacillus Calmette-Guerin (BCG) and Corynebacterium parvum. In some embodiments, the Arah6 variant is adsorbed to MCT and administered with or without MPL-A. Both MCT and MPL-A improve the efficacy of allergy immunotherapy and are thought to exert 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 product safety due to the depot effect and sustained release of the protein.

[0144] 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 nucleotide or modified nucleotide sequence encoding a recombinant hypoallergenic Arah6 variant of the present disclosure, thereby inducing hyposensitization to peanuts in the subject. In one embodiment, the composition comprises a bacterium carrying the nucleotide sequence. In one embodiment, the nucleotide sequence is in the form of DNA or RNA.

[0145] In one embodiment, the present disclosure provides a method for inducing immunomodulation of a response to peanuts in a subject allergic to peanuts, the method comprising administering to the subject a composition comprising a nucleotide or modified nucleotide sequence encoding a recombinant hypoallergenic Arah6 variant of the present disclosure, thereby inducing immunomodulation of a response to peanuts in the subject. In one embodiment, the composition comprises a bacterium carrying the nucleotide sequence. In one embodiment, the nucleotide sequence is in the form of DNA or RNA.

[0146] In one embodiment, the composition in the method of the present disclosure is administered orally. In another embodiment, the composition in the method of the present disclosure is administered by a route selected from subcutaneous, intramuscular, intranasal, sublingual, topical, rectal, and inhalation. In one embodiment, the subject in the method of the present disclosure is an infant. In one embodiment, the composition in the method of the present disclosure comprises powdered milk (baby milk) or baby food.

[0147] In one embodiment, the present disclosure provides a method for inducing peanut hyposensitization in a peanut-allergic subject, the method comprising administering to the subject a composition comprising a nucleic acid molecule encoding a recombinant Arah6 polypeptide, thereby inducing peanut hyposensitization in the subject. In some embodiments, the nucleic acid molecule used in the method for inducing peanut hyposensitization in a peanut-allergic subject comprises a nucleic acid molecule encoding a wild-type recombinant Arah6 polypeptide. In some embodiments, the nucleic acid molecule used in the method for inducing peanut hyposensitization in a peanut-allergic subject comprises a nucleic acid molecule or a modified nucleic acid molecule encoding a mutant recombinant Arah6 polypeptide containing one or more amino acid substitution mutations located within a single epitope recognized by an anti-Arah6 antibody.

[0148] 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 or modified nucleic acid molecule encoding a recombinant hypoallergenic Arah6 variant of the disclosure, thereby inducing hyposensitization to peanuts in the subject.

[0149] In one embodiment, the present disclosure provides a method for inducing immunomodulation of a response to peanuts in a subject with peanut allergy, comprising administering to the subject a composition comprising a nucleic acid molecule encoding a recombinant Arah6 polypeptide, thereby inducing immunomodulation of a response to peanuts in the subject. In some embodiments, the nucleic acid molecule used in the method for inducing immunomodulation of a response to peanuts in a subject with peanut allergy comprises a nucleic acid molecule encoding a wild-type recombinant Arah6 polypeptide. In some embodiments, the nucleic acid molecule used in the method for inducing immunomodulation of a response to peanuts in a subject with peanut allergy comprises a nucleic acid molecule or a modified nucleic acid molecule encoding a mutant recombinant Arah6 polypeptide containing one or more amino acid substitution mutations located within a single epitope recognized by anti-Arah6 antibodies.

[0150] In one embodiment, the present disclosure provides a method for inducing immunomodulation of a response to peanuts in a subject having a peanut allergy, comprising administering to the subject a composition comprising a nucleic acid or modified nucleic acid molecule encoding a recombinant hypoallergenic Arah6 variant disclosed herein, thereby inducing immunomodulation of a response to peanuts in the subject.

[0151] In some embodiments, compositions comprising an isolated nucleotide or modified nucleotide sequence encoding a recombinant Arah6 variant described herein are for use in inducing desensitization to peanuts in a subject with a peanut allergy. In some embodiments, compositions comprising an isolated nucleotide or modified nucleotide sequence encoding a recombinant Arah6 variant described herein are for use in inducing immunomodulation of a response to peanuts in a subject with a peanut allergy.

[0152] In some embodiments, compositions comprising a recombinant Arah6 mutant polypeptide described herein are for use in inducing desensitization to peanuts in a subject with a peanut allergy. In some embodiments, compositions comprising a recombinant Arah6 mutant polypeptide described herein are for use in inducing immunomodulation of a response to peanuts in a subject with a peanut allergy.

[0153] In one embodiment, the composition of the method of the present disclosure comprises a bacterium having a 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.

[0154] In one embodiment, the composition in the disclosed method is administered orally. In another embodiment, the composition in the disclosed method is administered by a route selected from subcutaneous, intramuscular, intravenous, intranasal, sublingual, topical, rectal, and inhalation. In one embodiment, the subject in the disclosed method is an infant.

[0155] As used herein, "nucleic acid composition" refers to a composition comprising a nucleic acid or nucleic acid molecule (e.g., a polynucleotide) encoding an allergen or a derivative thereof (e.g., a variant of an AraH6 protein or polypeptide). In exemplary embodiments, the nucleic acid composition comprises 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") composition. In some embodiments, the nucleic acid composition comprises 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") composition. Such a composition may also include other substances and molecules (e.g., pharmaceutical excipients) that are required or advantageous when it is administered to a patient.

[0156] In one embodiment, the RNA composition 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. Adapting the codon usage can increase the translation efficiency and half-life of the RNA. In one embodiment, a polyA 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 derivative, which can be incorporated during RNA synthesis or 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 translation efficiency of the RNA composition. The 5' cap of the RNA composition can be further modified by 2'-O-methyltransferase to generate the Cap 1 structure (m7Gppp[m2'-O]N), further enhancing translation efficiency. The composition or formulation according to the present invention can further comprise an adjuvant.

[0157] In one embodiment, nucleic acids or modified nucleic acid molecules encoding the Arah6 variants and / or recombinant Arah6 variants disclosed herein are combined to induce desensitization to peanuts in a subject with a peanut allergy. In one embodiment, several variants and / or several nucleic acids or modified nucleic acid molecules encoding the recombinant Arah6 variants disclosed herein are combined to induce desensitization to peanuts in a subject with a peanut allergy. In one embodiment, nucleic acids or modified nucleic acid molecules encoding the Arah6 variants and / or recombinant Arah6 variants disclosed herein are combined with other compositions and / or treatments, such as other Arahx allergens and variants thereof (WT and / or mutant forms), to induce desensitization to peanuts in a subject with a peanut allergy.

[0158] Plants and products

[0159] In one embodiment, the present disclosure provides a transgenic peanut plant, wherein the transgenic peanut plant expresses an Arah6 variant of the present disclosure.

[0160] In one embodiment, the Arah6 variant expressed in the transgenic peanut plant of the present disclosure is expressed from a heterologous nucleic acid.

[0161] In one embodiment, the Arah6 variant expressed in the transgenic peanut plant of the present disclosure is endogenously expressed from the transgenic chromosome.

[0162] In some embodiments of the transgenic peanut plants of the present disclosure, the expression level of endogenous wild-type Arah6 is reduced compared to a non-transgenic peanut plant.

[0163] In some embodiments, the expression level of the endogenous wild-type AraH6 allergen in the transgenic peanut plant is reduced compared to the endogenous wild-type AraH6 allergen in a corresponding non-transgenic peanut plant, hi some embodiments, the expression level of the endogenous wild-type AraH6 allergen in the transgenic peanut plant is reduced by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to the endogenous wild-type AraH6 allergen in a corresponding non-transgenic peanut plant.

[0164] In some embodiments, the genetically modified peanut plants of the present disclosure further express at least one RNA silencing molecule that (i) reduces the endogenous AraH6 allergen, and (ii) does not reduce expression of AraH6.

[0165] In some embodiments, the transgenic peanut plants of the present disclosure further express a DNA editing system aimed at reducing expression of the endogenous AraH6 allergen.

[0166] In one embodiment, the present disclosure provides a processed food product comprising an AraH6 variant of the present disclosure.

[0167] In one embodiment, the processed food of the present disclosure comprises a reduced amount of endogenous wild-type peanut AraH6 allergen. In some embodiments, the amount of endogenous wild-type AraH6 allergen is reduced compared to the amount of endogenous wild-type AraH6 allergen in a corresponding processed food that does not contain the AraH6 variants described in detail herein. In some embodiments, the amount of endogenous wild-type AraH6 allergen is reduced by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to the amount of endogenous peanut AraH6 allergen in a corresponding processed food that does not contain the AraH6 variants described in detail herein.

[0168] In one embodiment, the processed food product of the present disclosure comprises peanuts harvested from the genetically modified peanut plants of the present disclosure described above.

[0169] As used herein, the words "comprises," "comprising," "includes," "including," or "having," and their conjugations, mean "including, but not limited to."

[0170] As used herein, the singular forms "a," "an," and "the" are intended to include the plural of their referents unless the context clearly indicates otherwise. For example, the term "a recombinant Arah6 variant" can include multiple variants, including combinations thereof. Similarly, the term "an isolated nucleotide or modified nucleotide sequence encoding a recombinant Arah6 variant" can include multiple nucleotide or modified nucleotide sequences, including combinations thereof.

[0171] All numerical values ​​herein are assumed to be modified by the term "about." 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 and, where applicable, non-integer values, constituting a continuous range.

[0172] The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any aspect or embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other aspects or embodiments.

[0173] Throughout this application, various embodiments of Arah6 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 Arah6 variants, and their mutation and / or epitope locations. Accordingly, the description of a range should be considered to have specifically disclosed not only each individual numerical value subsumed within that range, but also all the possible subranges subsumed within that range. For example, a 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 subsumed within that range (e.g., 1, 2, 3, 4, 5, and 6). This applies regardless of the breadth of the range.

[0174] Whenever a range of values ​​is specified herein, it is meant to include all numbers (fractional or integer) subsumed within the specified range of values. 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.

[0175] Example

[0176] Example 1: Materials and Methods

[0177] Peptide microarray assay

[0178] To determine the anti-Arah6 epitope, a Celluspot™ peptide microarray-based immunoassay (Intavis, Cologne, Germany) was performed (Winkler, Dirk FH, Peptide microarrays. Humana Press, 2009). Peptides consisting of 15 amino acids in length and a 4-amino acid offset derived from the primary sequences of peanut allergens Arah1 (UniProt entry P43238, positions 25-626), Arah2 (UniProt entry Q6PSU2), Arah3 (UniProt entry O82580), Arah6 (UniProt entry A5Z1R0, positions 13-147), and Arah8 (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). Then, slides were washed and incubated with plasma in blocking buffer for 4 hours 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) or diluted 1:10,000 in blocking buffer and incubated for 2 hours at 25°C on a rotator. After washing, a Femtogram HRP Substrate Kit (Azure Biosystems, Dublin, California) was added, and chemiluminescence was read on a ChemiDoc (BioRad, Hercules, California). 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.

[0179] Generation of a human scFv phage display library

[0180] Five to 20 ml of whole blood samples were collected from patients clinically diagnosed with peanut allergy using heparin- or EDTA-treated tubes (BD). Peripheral blood mononuclear cells (PBMCs) were extracted from the blood samples using Sepmate tubes (STEMCELL) according to the manufacturer's instructions. RNA was extracted from 5 to 15 × 10 cells using the RNAeasy extraction kit (Qiagen; Hilden, Germany). 6 RNA was purified from PBMCs. cDNA was prepared from 1–5 μg of RNA (depending on the amount of RNA obtained).

[0181] The entire cDNA reaction was divided 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 reactions were performed in 50 μl using the Phusion Hot Start Taq Polymerase Kit (200 μM dNPT, 2% DMSO, 1.25 M betaine, 1–5 μg of cDNA, and 0.5 μM of each primer) 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 of 72°C for 10 min.

[0182] The PCR products from 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 an Amicon Ultra 30K centrifugal filter (Sigma-Aldrich Merck, Israel). A DNA mixture of amplified V gene segments was generated at a ratio of 45% Vγ, 5% Vε, 25% Vκ, and 25% Vλ. Combinatorial light-weight scFv libraries were generated by PCR reactions using the same reagents as the first PCR, but with 100 μl of 100 ng of the V gene mixture per reaction at a concentration of 250 nM, along with a "pull-through" primer (complementary to the overhang adjacent to the restriction site of each product from the first PCR). Multiplex recombination reactions (18-24) were generated without primers and PCR was performed using the following program: 98°C for 3 min, followed by 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, followed by 30 cycles of 98°C for 20 s + 67°C for 60 s + 72°C for 45 s, and a final extension step of 72°C for 3 min.

[0183] The PCR product was concentrated by ethanol precipitation, loaded onto a 1% agarose gel, extracted using a gel extraction kit (Qiagen), and washed 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 with 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 washed using a centrifugal filter, similar to the previous steps. The restricted scFv was purified using a PCR purification column (Qiagen).

[0184] 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. At least 3 μg of DNA was ligated in total. The ligation reaction was heat inactivated, washed with a PCR cleanup column, and concentrated with an Amicon 30K centrifugal filter.

[0185] The ligation library was transformed into SS320 electrocompetent bacteria (Lucigen; WI, USA) according to the manufacturer's instructions. Each library was divided into two transformations and plated onto three 15 cm 2YT-agar plates 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 plates to estimate transformation efficiency. 10 7 The library was deemed of sufficient quality for further use.

[0186] The next day, SS320 were scraped from the agar plates using 6 ml of 2YT, diluted to an OD of 0.1 in 60 ml of 2YT supplemented with 100 μg / ml carbenicillin and 2% glucose, grown to an OD of 0.5, and infected with a 1:1000 dilution of 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 in a baffled flask with shaking at 250 RPM at 30°C for at least overnight or up to 24 hours to produce scFv-displaying phage.

[0187] The next day, the cells were centrifuged at 18,000g and 16,000g for 10 minutes. The supernatant was transferred to a fresh tube, and phage was 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 sample was incubated on ice for 20 minutes and then centrifuged at 18,000g for 30 minutes at 4°C. The supernatant was discarded, and the pellet was centrifuged again for 2 minutes, after which the remaining supernatant was removed. The pellet was resuspended in 10 ml of PBS / 100 ml culture medium and centrifuged at 16,000g for 10 minutes to remove any remaining bacterial cell debris. The sample was then subjected to a second, identical PEG-NaCl precipitation and resuspended in 4 ml of PBS / 100 ml culture medium. The sample was centrifuged at 20,000 g for 15 min to remove any remaining debris, and the purified phage was supplemented with 50% glycerol and 2 mM EDTA and stored at −80° C. until use.

[0188] Screening of phage display libraries for allergen-specific scFvs

[0189] Allergen-specific scFvs were isolated by panning phage libraries with either native purified allergens or recombinant allergen variants with altered epitopes. Maxisorp high-binding 96-well plates (Nunc) were coated with 100 μl of 5 μg / ml allergen solution in PBS or 2% BSA solution in PBS (8 wells per library). 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.

[0190] 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 of 0.6-0.8, then stored on ice until use. Phage stocks (2-4 ml) were thawed, purified by PEG-NaCl purification (as described above), and resuspended in 1 ml of 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 three times with 200 μl / well of PBST, and then the phage solution was incubated in BSA-coated wells at 100 μl / well with gentle shaking to remove nonspecific 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. The plate was then washed twice with 200 μl / well PBST to remove unbound phage. Bound phage were eluted by incubation with 100 μl / well of 100 mM HCl for 5 minutes at room temperature with gentle shaking. The elution reaction was stopped with 12.5 μl / well of Tris 1 M, pH 11.

[0191] The eluted sample was added to 5 ml of OmniMAX™ at the required OD and incubated at 37°C for 30 minutes with shaking at 250 RPM. Panning output titration was assessed by performing serial 10-fold dilutions on samples of the infected strain and plating 5 μl drops in triplicate onto LB agar plates supplemented with carbenicillin, kanamycin, or tetracycline. The remaining output was grown by superinfecting 1:1000 with 1:100 KO7 helper stock and incubating at 37°C for 45 minutes with shaking at 250 RPM. The superinfected bacterial strain was 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 round of 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.

[0192] 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 by increasing the number of wash cycles and decreasing the number of panning wells (3–4 panning cycles per library).

[0193] To isolate individual allergen-specific scFvs, serial dilutions of the output from the selected round were plated onto LB-agar-carbenicillin plates and grown overnight at 37°C. The following day, individual colonies were inoculated into minitubes containing 300 μl of 2YT + carbenicillin + 1:1000 KO7 and grown overnight at 37°C with shaking at 250 RPM. The following day, supernatants from the minitubes were analyzed by ELISA using plates coated with allergen or BSA. Supernatant scFvs that specifically bound to the 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. Full-length monoclones were cloned into a mammalian expression plasmid (pSF) and expressed as IgG in HEK-293T cells.

[0194] Single-cell sorting of allergen-specific B cells

[0195] 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. Next, cells were incubated on ice for 1 hour with various concentrations of target allergens, depending on the allergen type. 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)), HA-tagged at either the C-terminus or N-terminus, or wild-type recombinant allergens, or biotin-avidin-labeled wild-type recombinant allergens (a mixture of allergens labeled with two different fluorophores). Cells were then washed and stained with fluorochrome-conjugated antibodies for the following markers: CD14, CD16, IgM, IgD, CD3, CD19, and IgG1. When using HA-labeled allergens, two anti-HA antibodies with different fluorochrome conjugates were also added. Cells were then washed and sorted using the ARIA-III sorting flow cytometer. 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 (PBSx 0.5, 10 mM DTT, 8 U RNase inhibitor). Several wells of each plate were left empty as PCR negative controls.

[0196] Isolation of antibody genes from selected cells and antibody expression

[0197] Lysates of single-selected allergen-specific B cells were directly reverse-transcribed (SSIV, Invitrogen, according to the manufacturer's instructions). Two nested sequential PCR reactions (2nd PCR) 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 most of the known antibody gene alleles. The PCR products were sequenced and aligned to the genome. If cells contained 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.

[0198] Generation of a yeast surface-displayed mutant saturation library and flow cytometric cell sorting

[0199] A library of Arah6 variants with single mutations at each residue was ordered from TWIST Bioscience (CA, USA) and cloned into the YSD vector, which is similar to pCHA. To display the Arah6 library on the yeast surface (designated S1), the library was grown in SDCAA selective medium (2% glucose, 0.67% Difco Yeast Nitrogen Base, 0.5% BactoCasa Amino Acids, 0.52% NaHPO, 0.856% NaHPO·H2O) and induced in galactose medium (2% galactose was used instead of glucose for SDCAA) 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)). Arah6 expression was detected with an anti-Myc antibody conjugated to FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), and anti-Arah6 IgG binding was detected with 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-Arah6 IgGs in binding buffer (100 mM Tris, pH 8.0, 1 mM CaCl2, 1% BSA) at room temperature for 1 hour. The cells were then washed with binding buffer and incubated with anti-Myc-FITC and anti-IgG-APC antibodies for 30 minutes. Next, the cells were washed again with binding buffer, and several independent sorting runs were performed using an S3E cell sorter (Bio-Rad) to select low-selectivity mutants. Arah6 mutants exhibiting low binding affinity to anti-Arah6 IgG, i.e., mutants representing at least 3% of the total population, were selected.

[0200] High-throughput sequencing library preparation

[0201] The YSD vector (pETCON) containing the Arah6 gene was 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), where 20 and 8 cycles of primary and secondary PCR were performed using Fluidigm Access Array primers to add adapters and barcodes. The DNA library samples were then purified with AmpureXP beads (Beckman Coulter, Brea, CA), and the sample concentrations were measured on a Qubit™ system using a DNA high-sensitivity assay. The 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.

[0202] Deep sequencing read analysis

[0203] Paired-end reads were analyzed and quality filtered 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, or 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.

[0204]

number

[0205] In the formula, aa i is the specific amino acid at position i, fS1 is the proportion of reads of a given amino acid at position i in the sorted library, and fS0 is the same proportion in the input library. This calculation gives the enrichment of each specific AraH6 point variant.

[0206] For convenience, the following can also be expressed as an increasing index of a particular amino acid at position i:

[0207]

number

[0208] Computing the Shannon entropy for each position provides a synthesis of information about all mutations at a given position.

[0209]

number

[0210] where i is a given position and INaaz represents the growth index of the given amino acid, normalized by the growth index of all amino acids.

[0211] Purification of AraH6

[0212] For the purification of Arah6 mutants, Arah6WT (SEQ ID NO: 2) and its mutants were cloned into pET28 plasmid. AraH6 was fused to DNA encoding N-terminal His-tagged Trx (AraH6-His *6). 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 either overnight at 20°C or for 3 hours at 37°C. Cells were harvested (4800 x g for 30 min), and the cell pellet was 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 (15,000 g, 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 for 1 h at 4°C. The beads were washed with binding buffer containing increasing concentrations of imidazole. To improve the purity of AraH6, the imidazole concentration was then diluted by size-exclusion chromatography (SEC). Fractions containing AraH6 were collected and concentrated using a 3 kDa centricone (Amicon, Mercury), and the protein concentration was measured by absorbance at 280 nm.

[0213] Analysis of binding to monoclonal antibodies by ELISA

[0214] The concentration of anti-Arah6 IgG required to confer 50% of maximal binding to wild-type Arah6 and Arah6 mutants (EC50) was determined by ELISA. Briefly, wells of a 96-well microtiter plate (Thermo Fisher Scientific, Waltham, MA) were coated with 200 ng of Arah6 overnight at 4°C. The plate was blocked with 0.5% BSA in PBS (200 μl / well) for 1 hour at room temperature. Anti-Arah6 IgG was prepared by serial dilution in PBS, added to the Arah6-coated wells, and incubated for 1 hour at room temperature. After washing, the amount of bound IgG was detected by incubation with goat anti-human IgG conjugated to an HRP polyclonal antibody (Abcam, Cambridge, United Kingdom) followed by TMB substrate.

[0215] All incubation steps were carried out in PBS containing 0.5% BSA and 0.05% Tween 20. The highest concentration of anti-Arah6 IgG was saturated, and the amount of binding to Arah6 reached a maximum at this concentration.

[0216] Computational design of mutants with multiple mutations

[0217] Based on experimental results identifying point mutations that reduce binding to mAbs and / or patient sera, we used computational protein analysis tools to create mutants with combinations of mutations predicted to maintain their stability. The NMR structure of AraH6 was energetically optimized (for each NMR state). Next, a combinatorial mutagenesis scanning tool was used to perform Monte Carlo sampling of up to five simultaneous mutations when mutations were combined at the epitope level, and up to 25 simultaneous mutations when mutations were combined at the protein level. This allowed for backbone minimization during side-chain mutations, resulting in the generation of 250 structures. Mutations were evaluated by ΔG, the change in protein free energy upon mutation. To eliminate experimental testing of nearly identical protein sequences, sequences were ranked by ΔG, structures with significantly increased ΔG were eliminated, and then ranked by sequence diversity.

[0218] Degranulation assay of RBL SX-38 cells

[0219] RBL SX-38 cells were received from Professor Stephen Dreskin of the University of California, Denver, with permission from BIDMC, Boston. Cells were cultured at 37°C and 5% CO2 in maintenance medium containing 80% MEM, 20% RPMI-1640, and 5% FCS (non-heat-inactivated) supplemented with L-glutamine, penicillin-streptomycin, and G418 at 1 mg / ml (all from Gibco-Thermo Fisher, USA). At least 48 h 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 300 g for 10 min, and then diluted to 2.5 × 10 cells in assay medium supplemented with 5–10% clinical sample (plasma / serum from peanut-allergic patients, dilution rate varied for each sample). 6The cells were resuspended to a final concentration of 100,000 cells / ml. If the plasma was produced with an anticoagulant other than heparin, 30 U / ml of heparin (Sodium-Heparin, Sigma) was added to the sample and incubated at room temperature for 10 minutes before adding to the cells. The cells were then seeded into 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 prepared by diluting varying concentrations of allergen or unrelated protein negative control 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, 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 (in duplicate). Five to six concentrations were used for each allergen, with 10-fold dilutions. Each clinical sample was tested for the wild-type allergen, the mutant allergen, and an unrelated protein (KLH, Sigma) as a negative control. Duplicate wells were prepared using lysis buffer (Tyrode's buffer containing 1% Triton x-100, Fisher Scientific) to measure total degranulation, and Tyrode's buffer alone to measure background degranulation. Cells were then incubated for 1 hour at 37°C and 5% CO. Immediately after incubation, 30 μl of each well was transferred to a 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 at 37°C for 1 hour with gentle shaking in the dark, followed by the addition of 100 μl of stop solution (0.2 M glycine, pH 10.7) to terminate 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 wild-type allergen.

[0220] BAT assay

[0221] Fresh whole blood samples were dispensed into heparinized tubes (Biological Industries) in 100 μl aliquots. Allergens and controls were diluted 2x with RPMI-1640 (Biological Industries) and added 1:1 to the tubes (final volume: 200 μl). The allergens were incubated at 37°C in a humidified incubator with 5% CO2 for 30 minutes. The dose range for each allergen was 1–10,000 ng / ml. Crude peanut extract (CPE), fMLP, and anti-human IgE antibody were used as positive controls. KLH protein was used as a negative control. The reaction was stopped by incubation on ice for 5 minutes. A cocktail of fluorochrome-conjugated antibodies was added directly to the samples to detect the following markers: CD203c, CD63, HLA-DR, CD45, and CD123. The cells were incubated on ice for 30 minutes. Red blood cells were lysed using a kit (BD FACS Lysing Solution) according to the manufacturer's instructions, washed, and analyzed by flow cytometry. Cells were gated for basophil detection and the activation rate (% CD63-positive basophils) was measured. At least 500 basophils were analyzed per tube.

[0222] T cell activation assay

[0223] PBMCs were isolated from heparinized blood samples from peanut-allergic patients. 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 per well in a 96-well round-bottom plate in X-vivo15 medium supplemented with 5% human AB serum (Biotag) and 1% penicillin-streptomycin solution (Biological Industries). 6 Recombinant wild-type and mutant allergens were seeded at 100 cells / well (based on the number of cells available after purification and staining). Recombinant wild-type and mutant allergens were purified using a 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 patient as negative controls for assay quality assurance. The final endotoxin concentration in all allergen wells was 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 in all wells was replaced with fresh medium. 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-induced activation of patient T cells) was determined when the mean of allergen-stimulated wells was greater than the mean + 3xSD of unstimulated wells.

[0224] Circular dichroism spectroscopy (CD) of AraH6WT and mutants

[0225] Circular dichroism spectroscopy (CD) is a useful technique for analyzing the secondary structure and folding properties of proteins in solution using very small amounts of protein. It is based on the differential absorbance of left and right circularly polarized light by a chromophore. CD analysis of proteins is based on information from amide chromophores in the far-UV region (below 260 nm) and aromatic side chains (260-320 nm). For example, α-helical proteins have negative bands at 222 and 208 nm and a positive band at 193 nm, whereas proteins with well-defined antiparallel β-pleated sheets (β-sheets) have a negative band at 218 nm and a positive band at 195 nm. Circular dichroism spectra of recombinant Arah6 proteins were measured using a Chirascan CD spectrometer (Applied Photophysics) at Bar-Ilan University. Far-UV CD spectra were acquired from 200 to 260 nm using a 10 mm pathlength cuvette. Purified Arah6 recombinant WT and D12 mutants were analyzed in PBS buffer, and concentrations were measured using SEC-HPLC and compared with those of the native protein. To assess stability, spectra were acquired at 25°C and elevated temperatures from 20 to 90°C.

[0226] Strains, plasmids, and growth conditions

[0227] 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 AraH6 purification. All strains were grown at 37°C in 2YT broth and on LB agar plates. Phagemids were used to generate a phage display library of scFvs derived from peanut-allergic patients. tPCR was used to insert a nonspecific 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) to either end of the scFv segment (the modified plasmid was internally marked LibGD). Plasmid pET28 (Invitrogen) was used for recombinant purification of AraH6 and variants. Transformation for scFv display was performed using SS320 electrocompetent E. coli (Lucigen).

[0228] Example 2: Epitope mapping and de-epitope of AraH6 polypeptide

[0229] Objective: The overall goal is to lay the foundation for defined, targeted mutations of allergenic polypeptides that retain stable and T cell-activating activity but reduce binding to IgE allergen antibodies. For immunotherapy purposes, functionality of the Arah6 mutant polypeptides includes maintaining immunogenicity, e.g., through the ability to activate T cells. This series of experiments was performed to identify and map conformational and linear epitopes on the peanut allergen Arah6 based on binding of specific monoclonal antibodies from peanut-allergic patient samples. This series of experiments was also performed to identify amino acid residues within the Arah6 mAb-binding epitope that contribute to binding and are not predicted to destabilize the protein when mutated.

[0230] result

[0231] The pipeline for single epitope mapping and epitope depletion of the peanut allergen Arah6 involves two steps: (1) discovery of Arah6-specific monoclonal antibodies (i.e., serum or isolated mAbs) from peanut-allergic patient samples that exhibit specific IgE binding to Arah6 as measured by ELISA assays and peptide arrays, and (2) mapping of the epitopes bound by each antibody. The first step, mAb discovery, was performed by amplifying the variable genes and constructing scFvs fused to pIII protein and displayed on phage, or by using an scFv phage-display library by single-cell sorting of Arah6-specific B cells, followed by sequencing of the variable regions and production of recombinant mAbs.

[0232] Briefly, an scFv phage display library was generated from the PBMCs of one peanut-allergic patient as described in Example 1, and three Arah6-specific mAbs were identified after the panning process of these libraries. Single-selected allergen-specific B cell lysates of 11 peanut-allergic patients were generated as described in Example 1, and 15 Arah6-specific mAbs were identified. All 18 mAbs were cloned into a mammalian expression plasmid (pSF) and expressed as IgG in HEK-293T cells. The epitope mapping procedure described below was performed on the 14 Arah6 IgG mAbs.

[0233] In the second step, anti-AraH6 specific purified mAbs were used for epitope mapping in two complementary approaches.

[0234] Approach A: Screening a site-saturation mutant library using 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).

[0235] Epitope mapping using the Arah6 YSD mutagenesis library: For epitope mapping, a two-step procedure was performed. First, the Arah6 point mutant library was sorted for expression only, and mutants that successfully underwent YSD were collected to obtain a sorted library called S1. The expression threshold was defined as a fluorescence value higher than that of unstained cells (background). Cells with a fluorescence signal higher than background were collected (S1 library). Next, binding of the S1 library to 14 mAbs was evaluated. Arah6 yeast cells that showed mAb binding signals (APC) in the bottom and top 1% of the population were sorted.

[0236] To identify positions that influence binding to specific mAbs, we performed detailed sequencing on each mAb. Because the library underwent selection for expression and low mAb binding, we analyzed the sequencing results by enrichment calculations. Each unique DNA sequence encoding a point mutation was counted, and the fold change in its relative abundance was calculated to indirectly estimate changes in mAb binding.

[0237] At least two conformational epitopes were identified in AraH6.

[0238] Approach B: Peptide microarray assays were performed as described in Example 1 using purified mAbs (commercially available scFv or IgG) to map several continuous epitopes on the allergen Arah6. This method was also used to validate the data from the YSD saturation method for linear epitopes. Five linear epitopes were identified in Arah6 and confirmed by six mAbs analyzed by peptide array. The mAbs mapped to Arah6 were then assayed using arrays containing mutated "depitope" spots to screen for peptides that showed the most significant reduction in binding. Representative array results are shown for linear epitope mapping (FIG. 1A) and de-epitope (FIG. 1B) of Arah6mAb.

[0239] IgE epitope mapping and de-epitopization of AraH6 based on plasma of allergic patients (see Example 3). Critical positions in the three epitopes were identified using peptide microarrays, similar to the process in Approach B. However, instead of mapping isolated monoclonal antibodies, IgE repertoires derived from plasma of allergic patients were used, as described in Example 3.

[0240] summary

[0241] Table 1 summarizes embodiments of WT Arah6, amino acid mutations, and Arah6 variants with mutations at positions relative to their epitopes. The details of the mutations / epitopes shown in Table 1 were collated from the results of Examples 2 and 3.

[0242] [Table 1-1]

[0243] [Table 1-2]

[0244] [Table 1-3]

[0245] [Table 1-4]

[0246] [Table 1-5]

[0247] [Table 1-6]

[0248] Example 3: IgE epitope mapping and de-epitope analysis based on serum samples from allergic patients

[0249] Objective: Following the overall objective of laying the foundation for defined targeted mutations of allergenic polypeptides that are stable and retain their functional characteristics but have reduced binding to IgE allergen antibodies, the aim of these experiments is to identify continuous (linear) IgE epitopes for peanut patient serum and plasma and to analyze these mutants.

[0250] result

[0251] Using the same peptide array technique as used for purified mAb analysis, we identified all continuous epitopes on the allergen Arah6 in polyclonal IgE derived from allergic patient sera. These arrays were analyzed with sera from 216 peanut-allergic patients to examine serum-derived IgE binding to Arah6-derived peptides. Of the sera tested, 80 slides each identified IgE binding to at least one peptide derived from Arah6. Analysis and clustering of the peptide arrays allowed us to map all linear epitopes of the protein (Figure 2).

[0252] Based on the mapped epitopes, two additional arrays were synthesized, and for each epitope mapped to AraH6, wild-type peptides were spotted alongside computer-designed mutant peptides that reduced IgE binding. The peptides were 15 amino acids long and contained either point mutations or double substitution mutations. Sera mapped to AraH6 were then assayed using arrays containing the mutated "depitope" spots to screen for peptides that showed the most significant reduction in binding (data not shown). Furthermore, the mutation / epitope details shown in Table 1 in Example 2 were collated from the results of both Examples 2 and 3.

[0253] Using plasma from 80 allergic patients, two major linear epitope regions for IgE binding were mapped by peptide microarray. The linear epitope of AraH6 at the population level was calculated for each peptide by its relative deviation from the median intensity of the slide (Z-like score). The distribution of all scores from all slides was plotted and shown as a box plot. Here, the x-axis corresponds to all overlapping peptides, and the y-axis shows the distribution of Z-like scores. The black and gray lines indicate 2 and 3 standard deviations from the median intensity of the slide, respectively (Figure 2).

[0254] Example 4: Mutation of single or multiple epitopes

[0255] Objective: Using the data collected in Examples 2 and 3, mutants were designed with combinations of mutations.

[0256] Results: Mutation combinations were based on computational predictions of the energetic effects of mutations on protein stability. Calculations were performed starting from the solved structure of Arah6 (PDB accession 1W2Q). Several epitopes could be mutated within a single mutant. Mutations included one to seven substitution mutations within the epitope. The designed mutants were produced in Escherichia coli and tested to verify reduced binding to anti-Arah6 mAb by indirect enzyme-linked immunosorbent assay (ELISA).

[0257] Table 2-1

[0258] Table 2-2

[0259] Table 2-3

[0260] Table 2-4

[0261] Table 2-5

[0262] Table 2-6

[0263] Table 2-7

[0264] Table 2-8

[0265] Table 2-9

[0266] Table 2-10

[0267] [Table 2-11]

[0268] [Table 2-12]

[0269] [Table 2-13]

[0270] summary

[0271] According to the above method, seven AraH6 epitopes were found.

[0272] The Arah6 D12 mutant showed reduced binding to anti-Arah6 mAb, 10 IgG, and 2 IgE. Binding to wild-type (WT) Arah6 (SEQ ID NO: 2) or the modified Arah6 D12 mutant (SEQ ID NO: 9) was tested using indirect ELISA titration with increasing concentrations of anti-Arah6 mAb. Keyhole limpet hemocyanin (KLH) was used as a negative control. The presented data show that the modified Arah6 D12 mutant dramatically reduced binding to two anti-Arah6 IgEs (E15C2 and 7B6) and four anti-Arah6 IgGs (IgG5, IgG8, IgG18, and IgG24) (Figures 8A-8F).

[0273] Example 5: Evaluation of allergenicity of modified proteins by ex vivo basophil degranulation assay

[0274] Objective: To evaluate the allergenicity of modified AraH6 compared to the wild-type protein.

[0275] result

[0276] Based on the results of single-site linear and conformational de-epitope analysis shown in Examples 2-4, mutations that abolish binding to each epitope were combined to construct AraH6 mutants mutated at multiple binding sites (SEQ ID NOS: 3-21 and 24-108; details are shown in Table 2). Alternatively, additional sequences were computationally combined using Monte Carlo methods to generate multi-site mutated protein mutants starting from residue-level data. The mutations shown in Table 1 above summarize the individual mutation sites.

[0277] In some embodiments of the Arah6 variant, the amino acid sequence set forth in any of SEQ ID NOs: 3 to 21 further comprises a methionine located upstream of the N-terminus of the amino acid sequence set forth in any of SEQ ID NOs: 3 to 21. In some embodiments of the Arah6 variant, the amino acid sequence set forth in any of SEQ ID NOs: 24 to 108 further comprises a methionine located upstream of the N-terminus of the amino acid sequence set forth in any of SEQ ID NOs: 24 to 108.

[0278] 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 were tested by cell degranulation assay using a humanized rat basophilic leukemia cell line (RBLSX-38) sensitized with peanut-allergic patient serum. Representative results of the RBL assay for Arah6 are shown in Figures 6A-6G. Cell degranulation of the mutant allergens was clearly reduced compared to the WT and native allergens.

[0279] summary

[0280] Based on ex vivo assays in RBL and BAT, a potential loss of allergenicity was observed in several AraH6 variants with combinations of mutations in one or more epitopes.

[0281] Example 6: Immunogenicity assessment by T cell activation

[0282] Objective: To evaluate the immunogenicity of representative AraH6 variants.

[0283] To ensure the efficacy of immunotherapy, the genetically engineered hypoallergenic variants must substantially retain their immunogenicity, allowing for reprogramming of the immune response. To assess the immunogenicity level of the Arah6 variants, we used various methods known in the art, including T cell assays, animal models, and IgE / IgG binding ratios.

[0284] Example 7: Biophysical properties of mutants

[0285] Objective: To ensure correct 3D folding in the mutants, it is important to maintain the same oligomerization level as the native protein (i.e., AraH6 monomers). To verify the oligomerization state of the protein, 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).

[0286] Both the wild-type and D12 variants of AraH6 with a 6x histidine tag expressed in E. coli BL21(DE3) were purified by standard immobilized metal affinity chromatography (IMAC) and SEC. The two purified AraH6 proteins with a 6x histidine tag, the wild-type and D12 variant, measured approximately 17 kDa in size on SDS-PAGE under reducing conditions (Figure 3A). The AraH6 variants D154, D158, D160, and D179 (Figure 3B), and D119 (data not shown), were stable and monomeric under the standard conditions tested, as confirmed by SEC-HPLC analysis (Figures 4A–4F).

[0287] Several of the key Arah6 mutants were further analyzed for thermal stability using circular dichroism. The thermal melting midpoints (TM) of both the D12 mutant and the WT were above 90 °C (Figures 5A and 5B). The spectral minima of the unmodified structures were near 205-210 nm and 220 nm, respectively, indicating that both the WT and D12 Arah6 mutants are primarily α-helical and share similar thermal stability up to 90 °C, suggesting high stability and correct folding.

[0288] summary

[0289] The major AraH6 variants exhibited high melting points in CD, suggesting similar thermal stability to the WT allergen. Combinatorial AraH6 variants were tested by SEC-HPLC and showed a monomeric mass (approximately 17 kDa) suggesting correct folding.

[0290] While certain features of the variant hypoallergenic peanut allergen AraH6 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 fall within the true spirit of these variations and uses.

[0291] Example 8: Expression and secretion of allergen variants from mammalian cells

[0292] Objective: To demonstrate that AraH6 peanut protein and de-epitope (DE) allergens can be expressed, folded, and secreted from mammalian cells.

[0293] method

[0294] Twenty milliliters of Expi293F (ThermoFisher Scientific) cells were transfected with 20 μg of plasmid encoding either Arah6WT or Arah6D12 using Expifectamine 293 transfection reagent according to the manufacturer's instructions. Both Arah6WT and Arah6D12 constructs express downstream of the human osteonectin leader sequence and contain a C-terminal 6x histidine tag. Cells were incubated at 37°C and 8% CO2 in Expi293 medium for 5 days to allow protein expression. Secreted proteins were purified from the expression medium using Ni-NTA Superflow beads, washed, and eluted with 350 mM imidazole. Eluted fractions were analyzed by SDS-PAGE with or without reduction with β-mercaptoethanol (β-ME).

[0295] result

[0296] The following results demonstrate that the peanut allergen AraH6 and its de-epitope variants are expressed at high levels. Figure 7 shows that wild-type or de-epitope variants of the peanut allergen AraH6 were expressed and secreted from transfected mammalian cells. AraH6 purified from the transfected mammalian cells was found to have the correct size.

Claims

1. 1. A recombinant Arah6 mutant polypeptide comprising: comprising the amino acid sequence set forth in SEQ ID NO: 109, A recombinant Arah6 mutant polypeptide comprising one or more amino acid substitutions, deletions, insertions, or any combination thereof, at one or more of positions 2, 3, 5, 7, 8, 10, 12, 16, 19, 22, 24, 33, 37, 38, 40, 41, 42, 45, 46, 47, 63, 74, 78, 81, 82, 83, 86, 89, 90, 97, 98, 99, 106, 108, 109, 110, 113, 114, 116, and 118 of SEQ ID NO: 109, compared to the amino acid residues at the same positions in SEQ ID NO:

2.

2. 2. The recombinant Arah6 mutant polypeptide of claim 1, The amino acid substitution is (a) at position 2, S; (b) D or S at position 3; (c) at position 5, D; (d) at position 7, D; (e) A or S at position 8; (f) A, S, or K at position 10; (g) at position 12, R, D, or N; (h) at position 16, S, or D; (i) Q, L, or R at position 19; (j) at position 22, F; (k) at position 24, D; (l) Q or K at position 33; (m) A, T, or S at position 37; (n) A or S at position 38; (o) S at position 40; (p) at position 41, D; (q) K, E, or G at position 42; (r) A or Q at position 45; (t) S, G, or R at position 46; (t) S at position 47; (u) at position 74, R; (v) at position 78, L; (w) A or R at position 81; (x) at position 82, T; (y) N or K at position 83; (z) D or S at position 86; (aa) at position 89, N, R, or G; (bb) at position 90, D; (cc) I at position 97; (dd) D or L at position 98; (ee) M at position 99; (ff) K or H at position 106; (gg) P, E, or D at position 108; (hh) E or S at position 110; (ii) D or I at position 113; (jj) at position 114, D, H, A, or G; (kk) K, or M at position 116; and (ll) R, or T at position 118; A recombinant Arah6 mutant polypeptide comprising one or more of:

3. 3. The recombinant Arah6 mutant polypeptide of claim 1 or 2, A recombinant Arah6 mutant polypeptide comprising an amino acid substitution, deletion, insertion, or any combination thereof at one or more of positions 3, 5, 8, 19, 45, 46, 89, 98, 110, 114, 116, and 118 of SEQ ID NO: 109 compared to the amino acid residues at the same positions in SEQ ID NO:

2.

4. 4. The recombinant Arah6 mutant polypeptide of claim 3, The amino acid substitutions in the recombinant Arah6 mutant polypeptide include one or more of: D or S at position 3; D at position 5; A or S at position 8; Q, L, or R at position 19; A or Q at position 45; S, G, or R at position 46; N, R, or G at position 89; D or L at position 98; E or S at position 110; D, H, A, or G at position 114; K or M at position 116; and R or T at position 118.

5. 5. The recombinant Arah6 mutant polypeptide of claim 3 or 4, A recombinant Arah6 mutant polypeptide further comprising an amino acid substitution, deletion, insertion, or any combination thereof, at one or more of positions 2, 7, 10, 12, 15, 16, 17, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 47, 57, 59, 61, 63, 64, 74, 78, 81, 82, 83, 86, 90, 91, 97, 99, 106, 108, 109, 113, and 123 of SEQ ID NO: 109 compared to the amino acid residues at the same positions in SEQ ID NO:

2.

6. 6. The recombinant Arah6 mutant polypeptide of claim 5, The amino acid substitution is (a) at position 2, S; (b) D at position 7; (c) A, S, or K at position 10; (d) R, D, or N at position 12; (e) R at position 15; (f) S, or D at position 16; (g) R at position 17; (h) at position 20, D; (i) at position 22, F; (j) at position 24, D; (k) at position 28, S; (l) Q or K at position 33; (m) A at position 35; (n) A, T, or S at position 37; (o) A or S at position 38; (p) S at position 40; (q) D at position 41; (r) K, E, or G at position 42; (t) S at position 47; (t) at position 57, D; (u) Y at position 59; (v) F at position 61; (w) S at position 64; (x) at position 74, R; (y) at position 78, L; (z) A or R at position 81; (aa) at position 82, T; (bb) N or K at position 83; (cc) S or D at position 86; (dd) D at position 90; (ee) A or S at position 91; (ff) I at position 97; (gg) M at position 99; (hh) K or H at position 106; (ii) P, E, or D at position 108; (jj) D or I at position 113; and (kk) at position 123, D; A recombinant Arah6 mutant polypeptide comprising one or more of:

7. A recombinant Arah6 mutant polypeptide according to any one of claims 1 to 6, comprising: A recombinant Arah6 mutant polypeptide comprising one or more amino acid substitutions, deletions, insertions, or any combination thereof, located within at least one epitope recognized by an anti-Arah6 antibody.

8. 2. The recombinant Arah6 mutant polypeptide of claim 1, A recombinant Arah6 mutant polypeptide having at least 80% identity to the amino acid sequence set forth in any of SEQ ID NOs: 3-21 or 24-108.

9. 2. The recombinant Arah6 mutant polypeptide of claim 1, A recombinant Arah6 mutant polypeptide comprising a sequence set forth in any of SEQ ID NOs: 3-21 or 24-108.

10. An isolated or modified nucleotide sequence encoding a recombinant Arah6 mutant polypeptide according to any one of claims 1 to 9, The nucleotide or modified nucleotide sequence comprises DNA or mRNA.

11. 11. A nucleotide or modified nucleotide sequence according to claim 10, The mRNA is a nucleotide or modified nucleotide sequence comprising an LNP-formulated RNA.

12. 12. An expression vector comprising the isolated or modified nucleotide sequence of claim 10 or 11.

13. A prokaryotic or eukaryotic cell comprising the expression vector of claim 12, The eukaryotic cell may be a prokaryotic or eukaryotic cell, including a yeast cell, a fungal cell, a plant cell, or a mammalian cell.

14. A composition comprising a recombinant Arah6 mutant polypeptide according to any one of claims 1 to 9.

15. 15. The composition of claim 14, for use in inducing hyposensitization to peanuts and / or immunomodulation of the response to peanuts in a subject allergic to peanuts.

16. 12. A composition comprising the isolated nucleotide or modified nucleotide sequence of claim 10 or 11.

17. 17. The composition of claim 16 for use in inducing hyposensitization to peanuts and / or immunomodulation of the response to peanuts in a subject allergic to peanuts.

18. A transgenic peanut plant, wherein the transgenic peanut plant expresses the recombinant Arah6 mutant polypeptide of any one of claims 1 to 9.

19. 19. The transgenic peanut plant of claim 18, A transgenic peanut plant, wherein said recombinant Arah6 mutant polypeptide is expressed from a heterologous nucleic acid.

20. 20. The genetically modified peanut plant of claim 18 or 19, A genetically modified peanut plant in which the expression level of the endogenous wild-type AraH6 allergen is reduced compared to non-genetically modified peanuts.

21. A processed food comprising the recombinant Arah6 mutant polypeptide of any one of claims 1 to 9.

22. 22. The processed food according to claim 21, A processed food in which the expression level of endogenous wild-type AraH6 allergen is reduced compared to non-genetically modified peanuts.

23. 23. The processed food according to claim 21 or 22, 20. A processed food product comprising peanuts harvested from the genetically modified peanut plant of claim 18.

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