Mutant ara h 2 and ara h 6 proteins and uses thereof

Abstract

Provided herein are hypoallergenic mutant Ara h proteins, including Ara h 2 and Ara h 6, and uses thereof in treating allergic or anaphylactic responses to peanut allergens, immunotherapy and diagnostics.

Description

MUTANT ARA H 2 AND ARA H 6 PROTEINS AND USES THEREOFSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made in part with Government support under NIH Grant Number ZIA-ES 102906 and ZIC-ES 102645. The Government has certain rights in this invention.FIELD OF THE INVENTION

[0002] The present invention relates to hypoallergenic mutant Arachis hypogaea (Ara h) proteins (Ara h 2 and Ara h 6) and uses thereof in treating allergic or anaphylactic responses to peanut allergens, immunotherapy and diagnostics.REFERENCE TO SEQUENCE LISTING

[0003] This application contains a Sequence Listing that has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. The file, created on January 24, 2024, is named 060734_782728_Sequence_Listing.xml, and is 17 kb in size.BACKGROUND OF THE INVENTION

[0004] Peanut allergy can be a life-threatening illness, which affects 1-2% of the population and appears to be increasing in the 21st century (1). The reasons for increased prevalence remain unclear (2). Peanut is the most common source of food related anaphylactic reactions (3). This is a major systemic event, however when treated rapidly and appropriately it is rarely fatal (4).Peanut allergy usually emerges in childhood, and in contrast to other food allergies like milk and egg, it does not typically resolve with age (5, 6). Hence, patients and parents frequently prefer active intervention over life-long avoidance.

[0005] Current immunotherapy for peanut allergy typically involves feeding the allergic patient increasing doses of peanut over a period time until the patient tolerates eating a prescribed volume, usually milligrams to grams (7). The timing of the doses, amount of peanut per dose, and route of exposure (e.g., oral, sublingual, dermal, etc.) has been closely studied to optimize the safety and tolerance dose achieved (8). The goal of immunotherapy is to produce lasting unresponsiveness to peanut exposure, however clinical efficacy appears transient (9, 10).Therefore, most oral immunotherapy (OIT) suggests continuous dosing to maintain unresponsiveness (11). This has led some prominent experts to question the value of current OIT, suggesting avoidance as a viable alternative for most people (12, 13). Another problem with current therapy is that feeding a patient who is hypersensitive to peanut has some risk of adverse events including anaphylaxis (12). Factors that increase the risk of an adverse event include fever, menses, sleep deprivation, and high levels of specific IgE antibodies to the peanut allergen Ara h 2 (14, 15). Ara h 6 is a closely related allergen, which has also received considerable attention as a major peanut allergen (Zhuang and Dreskin, 2013; Koid et al. JAFC 2014). Due to the high similarity, many of the same antibodies are likely to be cross-reactive for Ara h 2 and Ara h 6 hence the strategy of this patent is to consider both simultaneously.

[0006] Several strategies have been employed to develop hypoallergens with reduced IgE binding as an alternative to using the native allergen (16). The fundamental goal is to prevent antibody binding to the native allergen. Allergoids are an example of chemical denaturation and modification of the allergen to destroy conformational epitopes (17, 18). While these have met with some success in Europe, the American Food and Drug Administration has concerns about the semi-random nature of the modifications. As an alternative, site directed mutants that destabilize the allergen were tested in examples of Bet v 1 (BM4) or Der p 2 (S47W) (19, 20). These have showed some promise in animal models (21, 22). Given the limited success of such approaches, there is a need in the art for hypoallergens with reduced IgE binding and designs thereof. A better approach would be to make more surgically selective mutations in key epitopes if the epitopes are known.SUMMARY OF THE INVENTION

[0007] Provided herein is a mutant Ara h protein, which may be a mutant Ara h 2 protein or a mutant Ara h 6 protein. The mutant Ara h protein may comprise an amino acid sequence at least 90% identical to a reference Ara h protein, which may be a wild-type Ara h 2 or wild-type Ara h 6 protein or isoform thereof. The reference Ara h protein may comprise the sequence set forth in one of SEQ ID NOs: 1-3. The mutant Ara h protein comprises a substitution at one or more of positions E46, E89 or Q89, E97, El 14, QI 16, R119, Q146, and R147 relative to the reference Ara h protein. The position of each of the one or more substitutions may be relative to the numbering of amino acids in SEQ ID NO: 1.

[0008] Except for the one or more substitutions, the mutant Ara h protein may comprise an amino acid sequence identical to the sequence of the reference Ara h protein, which may comprise the sequence set forth in one of SEQ ID NOs: 1-3. The mutant Ara h protein may comprise substitutions at positions E46, E89 or Q89, E97, El 14, Q146, and R147. The substitutions may be, respectively, E46R, E89R or Q89R, E97R, El 14R, Q146A, and R147E.

[0009] The reference Ara h protein may be an Ara h 2 protein comprising the sequence set forth in SEQ ID NO: 1 or 2. The mutant Ara h 2 protein may comprise the sequence set forth in one of SEQ ID NOs: 5-9. The reference Ara h protein may be an Ara h 6 protein comprising the sequence set forth in SEQ ID NO: 3. The mutant Ara h 6 protein may comprise the sequence set forth in SEQ ID NO: 10. The mutant Ara h 2 protein may comprise a substitution at one or more of positions E46, E89, E97, El 14, QI 46, and R147 relative to a reference Ara h 2 protein comprising the sequence set forth in SEQ ID NO: 1 or 2. The mutant Ara h 2 protein may comprise substitutions comprising E46R, E89R, E97R, El 14R, Q146A, and R147E. The mutant Ara h 6 protein may comprise a substitution at one or more of positions E46, Q89, E97, El 14, Q146, and R147 relative to a reference Ara h 6 protein comprising the sequence set forth in SEQ ID NO: 3. The mutant Ara h 6 protein may comprise substitutions comprising E46R, Q89R, E97R, El 14R, Q146A, and R147E.

[0010] Further provided herein is a nucleic acid encoding the mutant Ara h protein. Also provided herein is a recombinant organism, which may be a genetically modified organism, comprising the nucleic acid. The genetically modified organism may be a plant, which may be Arachis hypogaea. The genetically modified organism may comprise a gene replacement in which, relative to a wild-type organism, a gene or portion thereof encoding a wild-type Ara h protein is replaced by the nucleic acid encoding the mutant Ara h protein.

[0011] Also provided herein is a pharmaceutical composition comprising the mutant Ara h protein and a pharmaceutically acceptable excipient. Further provided is a method of treating or reducing the risk of a medical condition associated with peanuts in a subject in need thereof. The method may comprise administering to the subject the mutant Ara h protein or the pharmaceutical composition. Also provided are use of the mutant Ara h protein or the pharmaceutical composition in the manufacture of a medicament for treating or reducing the risk of the medical condition, and the mutant Ara h protein or pharmaceutical composition for treating or reducing the risk of the medical condition.

[0012] The subject may have or be suspected of having a peanut allergy. The medical condition may be one or more of an immune response, an allergic response, and anaphylaxis. The mutant Ara h protein or pharmaceutical composition may be administered or be intended for administration orally, intravenously, or via injection. The treatment may be an immunotherapy, which may desensitize the subject to one or more peanut allergens. The immunotherapy may be an oral immunotherapy.

[0013] Also provided herein is a method of reducing the binding of a reference Ara h protein to an anti-Ara h antibody, which may comprise contacting the anti-Ara h antibody with the mutant Ara h protein. Further provided is a method of determining the level of an anti-Ara h 2 or an anti- Ara 6 antibody in a sample. The method may comprise contacting a sample comprising or suspected of comprising the anti-Ara h 2 or anti-Ara h 6 antibody with the mutant Ara h protein. The method may also comprise quantifying the amount of binding of the anti-Ara h 2 or anti-Ara h 6 antibody to the mutant Ara h protein. The amount of binding of the anti-Ara h 2 or anti-Ara h 6 antibody to the mutant Ara h protein may be indicative of level of the anti-Ara h 2 or anti-Ara h 6 antibody in the sample. The amount of binding of the anti-Ara h 2 or anti-Ara h 6 antibody to the mutant Ara h protein may be compared to the amount of binding of the anti-Ara h 2 or anti- Ara h 6 antibody to a reference Ara h protein. The degree of reduced binding of the anti-Ara h 2 or anti-Ara h 6 antibody to the mutant Ara h protein as compared to the amount of binding of the anti-Ara h 2 or anti-Ara h 6 antibody to the reference Ara h protein may be indicative of the level of the anti-Ara h 2 or anti-Ara h 6 antibody in the sample.

[0014] The antibody may be T1 and may be representative of bin 1.2-like antibodies and the mutant Ara h protein may comprises substitutions at positions E46 and El 14 relative to the Ara h protein comprising the sequence set forth in one of SEQ ID NOs: 1-3, and the positions of the substitutions may be relative to the numbering of the amino acids in SEQ ID NO: 1. The antibody may be T5 and may be representative of bin 2-like antibodies and the mutant Ara h protein may comprise substitutions at positions E89 and E97 relative to the Ara h protein comprising the sequence set forth in one of SEQ ID NOs: 1-3, and the positions of the substitutions may be relative to the numbering of the amino acids in SEQ ID NO: 1. The antibody may be SI and may be representative of bin 3-like antibodies and the mutant Ara h protein may comprise substitutions at positions Q146 and Q147 relative to the Ara h protein comprising the sequence set forth in one of SEQ ID NOs: 1-3, and the positions of thesubstitutions may be relative to the numbering of the amino acids in SEQ ID NO: 1 . The antibody may be P34 and may be representative of bin 1.1 -like antibodies and the mutant Ara h protein may comprise substitutions at positions QI 16 and Q121 relative to the Ara h protein comprising the sequence set forth in one of SEQ ID NOs: 1-3, and the positions of the substitutions may be relative to the numbering of the amino acids in SEQ ID NO: 1.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The patent or application fde contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office by request and payment of the necessary fee.

[0016] FIG. 1 shows an amino acid sequence alignment of wild-type Ara h 2.0101 (SEQ ID NO: 1), Ara h 2.0201 (SEQ ID NO: 2), and Ara h 6 (SEQ ID NO: 3). It has been reported that Ara h 2.0102 has an E and position highlighted in red and Ara h 2.0202 has a D at the position highlighted in red.

[0017] FIG. 2A-D show data related to the structure of Ara h 2 bound to antibodies. FIGS. 2A-C show ternary crystal structures of Ara h 2 with T1 (cyan, bin 1.2) and SI (red, bin 3) (FIG. 2A, PDB code 8DB4) and with Tl(cyan) and T5 (green, bin 2) (FIG. 2B, PDB code 8G4P). FIG. 2C shows a ternary structure of Ara h 2 with SI (red) and P34 (yellow, bin 1.1). FIG. 2D shows the epitopes on Ara h 2 colored by the corresponding antibodies (rendered as transparent cartoons) Epitope colors are green (T5), cyan (Tl), red (SI), and yellow (P34). Residues that are common to two epitopes are colored teal for T1 / T5, dark grey S1 / T5, and light grey T1 / P34. A detailed legend accompanies FIG. 3.

[0018] FIG. 3A-B show amino acids interacting with anti-Ara h protein antibodies. FIG. 3A shows an alignment of Ara 6.0101 (SEQ ID NO: 3), Ara h 2.0101 (SEQ ID NO: 1), and Ara h 2.0201 (SEQ ID NO: 2), and indicates residues interacting with SI (Bin 3), Ara h 2 residues interacting with Tl (Bin 1.2), Ara h 2 residues interacting with T5 (Bin 2), Ara h 2 residues common to Tl and T5, Ara h 2 residues common to SI and T5, Ara h 2 residues interacting with P34 (Bin 1.1), Ara h 2 residues common to Tl and P34, disordered loops, DPYSPS (SEQ ID NO: 4) repeated motifs, Ara h protein hexamutant mutations (black box), and linear epitopes of Ara h proteins outside the repeated motifs. FIG. 3B shows the structure of Ara h 2 labeled to show the residues indicated in FIG. 3 A.

[0019] FIG. 4 shows an amino acid sequence alignment of wild-type Ara h 2.0101 (SEQ ID NO: 1), Ara h 2.0201 (SEQ ID NO: 2), and Ara h 6 (SEQ ID NO: 3). The focus of this figure is the highlighted residues which can be mutated to reduce antibody binding to Ara h proteins.

[0020] FIG. 5A-E show epitope-paratope interactions for all three major conformation epitope bins shown in FIG. 1. (FIG. 5A) T1 (heavy chain magenta, light chain yellow) and Ara h 2 (white) (FIG. 5B) T5 (heavy chain peach, light chain orange) and Ara h 2 (white) (FIG. 5C) SI (heavy chain green, light chain cyan) and Ara h 2 (white). Key residue interactions are indicated. FIG. 5D. Key residues in Ara h 2 (colored orange) are identified in the Ara h 2 : P34 interface. P34 interface residues are colored white. FIG. 5E. The same residues colored orange are shown with respect to the Ara h 2: T1 interface. T1 is yellow and magenta. Ara h 2 is colored peach.

[0021] FIG. 6A-C show antibody binding of various mutant Ara h 2 proteins. Direct ELISAs coating the plate with T1 (FIG. 6A), T5 (FIG. 6B), and SI (FIG. 6C) and probing for the binding of the indicated Ara h 2 mutants. The EC50 and fold change are reported for the mutants relative to Maltose Binding Protein (MBP)-tagged WT Ara h 2 adjacent to each graph. FIG. 6D-E. The epitope maps for T1 (cyan), T5 (green), and SI (red) are colored on the surface of Ara h 2 as determined from the program PISA. FIG. 6D and E are 180 degree rotations of Ara h 2. The locations of mutation sites are indicated with a stick model of the residue.

[0022] FIG. 7A and 7B show antibody binding of various Ara h 2 proteins. Direct ELISAs coating the plate with P34 (FIG. 7A) or T1 (FIG. 7B) and probing for the binding of the indicated Ara h 2 mutants.

[0023] FIG. 8A-E show the binding of a hexamutant Ara h 2 protein to a panel of IgG monoclonal antibodies. Affinity measurements of mAb for WT or hexamutant Ara h 2 using BLI. Biotinylated Ara h 2 or hexamutant were loaded on separate streptavidin (SA) sensors. Antibodies were added in serial dilutions to measure affinities. The top concentration 5 Lig / mL is shown in the sensogram for P34 (FIG. 8 A), T1 (FIG. 8B), T5 (FIG. 8C), and SI (FIG. 8D) comparing the WT (blue line) to the hexamutant (red line). FIG. 8E. The negative log of the dissociation constants (KD) is plotted with standard deviations for replicate measurements. See also the tabulated data in Table 1.

[0024] FIG. 9A-P show the results of competitive binding of a hexamutant Ara h 2 protein vs. wild-type Ara h 2 to sera from 16 peanut allergic patients from previous clinical trial cohorts PNOIT1, PNOIT2, and PU. FIG. 9A-P show the %IgE inhibition of binding to fixed MBP- WTAra h 2 by either soluble MBP-WT Ara h 2 (blue line) or soluble MBP-hexamutant (red line) for 16 different peanut allergic patients. Standard deviation error bars are indicated. FIG. 9Q shows an averaged curve utilizing data from all 16 patients. FIG. 9R summarizes the change in IgE inhibition for Ara h 2 versus the Hexamutant at the highest dose level for all patients studied.

[0025] FIG. 10A-B show images of tissue in a humanized FcERI mouse model of passive cutaneous anaphylaxis demonstrating that the extent of dye leakage is greater for wild-type MBP-Ara h 2 (WT) protein compared to a MBP-hexamutant Ara h 2 (HM) protein or a PB S control. FIG. 10 A. Representative ear images after PCA in mice sensitized with PBS or Pooled Serum from peanut allergic patients (n=6) and challenged with PBS, WT, or HM Ara h 2.Strikingly, HM-challenged mouse ears exhibited markedly reduced blue coloration, indicative of attenuated anaphylaxis compared to WT Ara h 2 challenge (FIG. 10B). OD measurements of extracted blue dye. Error bars represent SEMs for 9-10 humanized FcERI mouse ears per group. Student’s t-test shows **P < .006.

[0026] FIG. 11 Peptide mapping of important linear epitope residues of the DPYSPS (SEQ ID NO: 4) motif. A heatmap of relative binding of an array of peptides to a selection of monoclonal antibodies known to bind the linear epitope in the vicinity of DPYSPS (SEQ ID NO: 4).

[0027] FIG 12. Concept for a diagnostic assay. Competitive inhibition experiments were performed exactly as in FIG. 9, at 100 p.g / ml using the hexamutant (orange bars) and the double mutant Q146A / R147E (blue bars) for 4 patients. By comparison with WT, the relative contribution of the bin 3 antibodies (blue bars) or the bin 3+bin 2+bin 1.2 (orange bars) to the patient antibody repertoire can be assessed.DETAILED DESCRIPTION

[0028] The inventors have examined key conformational IgG and IgA epitopes on Ara h 2 identified in patients undergoing immunotherapy for peanuts. The inventors reasoned that these reasonably represent IgE epitopes and designed mutants which abrogated binding to the mAb. They have demonstrated that, surprisingly, these mutants reduced IgE binding from allergic sera using ELISA, and a mouse model of passive cutaneous anaphylaxis. This represents a significant first step in hypoallergen design. The inventors’ insight to use a combination of structural analysis from cloned human antibodies and empirical data led to the discovery of improved hypoallergenic mutant Ara h proteins disclosed herein.

[0029] In particular, the inventors have discovered hypoallergenic forms of the major peanut allergen Ara h 2. The hypoallergens may be used either in immunotherapy or as a diagnostic in the treatment of peanut allergy. The inventors identified four immunodominant conformational epitopes on the major peanut allergen Ara h 2, and one important linear epitope called DPYSPS (SEQ ID NO: 4). They accomplished this by solving the crystal structures of Ara h 2, the major peanut allergy protein, in complex with representative IgG antibody constructs derived from all immunodominant conformational epitopes. The inventors identified select amino acid residues that when mutated would abrogate binding of the monoclonal antibodies. In one example, an Ara h 2 mutant with all 6 mutations that were shown to eliminate monoclonal antibody binding was generated. Using ELISA, the hexamutant showed reduced ability to inhibit IgE from sera binding to the wildtype Ara h 2. In a mouse model of passive cutaneous anaphylaxis, there was a reduction in anaphylactic response using the hexamutant compared to the wild type Ara h 2, when the mouse was primed with pooled human allergic sera. This led to the discovery by the inventors that hypoallergenic forms of Ara h 2 and Ara h 6 can be used in a therapy with a reduced risk of an allergic and / or anaphylactic response. Mutations of key residues in the DPYSPS (SEQ ID NO: 4) (also denoted as DPYSXS (SEQ ID NO: 14) where X may be 3- hydroxyproline (3Hyp) or 4-hydroxyproline (4Hyp)) linear epitope reduce IgE binding as well. Designer mutant Ara h 2 and Ara h 6 may be used as a therapeutic and provide a safer alternative to current therapy with native allergen. Other applications include using mutants selective for certain epitopes as diagnostics of therapeutic progress or outcome.1. Definitions.

[0030] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0031] For recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitly contemplated.2. Mutant Ara h proteins

[0032] Provided herein is a mutant Ara h protein, which has an altered amino acid sequence as compared to a reference Ara h protein. The reference Ara h protein may be derived from Arachis hypogaea (peanut). The reference Ara h protein may be a wild-type Ara h 2 protein or isoform thereof, which may be Ara h 2.0101 or Ara h 2.0201, and which may be as defined in the WHO / IUIS Allergen Nomenclature Database. The reference Ara h protein may be a wild-type Ara h 6 protein, which may be Ara 6.0101, and which may be as defined in the WHO / IUIS Allergen Nomenclature Database.

[0033] The reference Ara h 2.0101 protein may have the sequence as set forth in GenBank Accession No. AAK96887.1. The amino acid sequence of the reference Ara h 2.0101 protein may be as follows, an isoform thereof, a fragment of the foregoing, or a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% identical thereto.MAKLTILVALALFLLAAHASARQQWELQGDRRCQSQLERANLRPCEQHLMQKIQRDED SYERDPYSPSQDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQRCMCEALQQIMENQS DRLQGRQQEQQFKRELRNLPQQCGLRAPQRCDLDVESGG (SEQ ID NO: 1)

[0034] The reference Ara h 2.0201 protein have the sequence as set forth in GenBank Accession No. AAN77576.1. The amino acid sequence of the reference Ara h 2.0201 protein may be as follows, an isoform thereof, a fragment of the foregoing, or a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% identical thereto.MAKLTILVALALFLLAAHASARQQWELQGDRRCQSQLERANLRPCEQHLMQKIQRDED SYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQRCM CEALQQIMENQSDRLQGRQQEQQFKRELRNLPQQCGLRAPQRCDLEVESGGRDRY (SEQ ID NO: 2)

[0035] The reference Ara h 6 protein may have the sequence a set forth in GenBank Accession No. AAD56337.1. The amino acid sequence of the reference Ara h 6 protein may be as follows, an isoform thereof, a fragment of the foregoing, or a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% identical thereto.MRRERGRQGD S S SCERQ VDGVNLKPCEQHIMQRIMGEQEQ YD S YNFGSTRS SDQQQRC CDELNEMENTQRCMCEALQQIMENQCDGLQDRQMVQHFKRELMNLPQQCNFGAPQR CDLDVSGGRC (SEQ ID NO: 3)

[0036] The mutant Ara h protein may comprise one or more mutations which may be in epitopes that are immunodominant in a subject. The one or more mutations may reduce the binding of the mutant Ara h protein to one or more IgE antibodies, which may be relative to a corresponding reference Ara h 2 protein. In one example, the residues that are mutated in the mutant Ara h protein may interact with one or more anti-Ara h protein IgG or IgE antibodies. The one or more IgG antibodies may be one or more of SI, Tl, T5, and P34. The one or more mutations in the mutant Ara h protein may reduce the allergenicity of the Ara h 2 protein relative to a corresponding reference Ara h protein, and may represent hypoallergenic mutations.

[0037] An alignment of reference Ara h 2.0101, Ara h 2.0201, and Ara h 6 proteins is shown in FIG. 1. When referring to mutations and / or mutant forms of reference Ara h 2 and Ara h 6 proteins, numbering is in reference to Ara h 2.0101 (SEQ ID NO: 1) as shown in FIG. 1, unless otherwise stated. Crystal structures showing interactions among IgG antibodies SI and Tl, and Ara h 2; IgG antibodies SI and P34, and Ara h 2; and IgG antibodies Tl and T5, and Ara h 2 are provided in FIG. 2. FIG. 3 shows an alignment of reference proteins Ara h 6, Ara h 2.0101, and Ara h 2.0201. The mutant Ara h protein may comprise one or more substitutions within one or more linear epitopes relative to a reference Ara h protein (marked by boxes in FIG. 3), which may be at one or more of positions 30-39, 121-128, and 130-140. The mutant Ara h 2 protein may comprise one or more substitutions within one or more positions within a DPYSPS (SEQ ID NO: 4) linear epitope relative to a reference Ara h 2 protein. In particular, the substitution may be at one or more of D, Y, and P (with or without a hydroxylation on the proline (such as DPYSXS (SEQ ID NO: 14), where X is 3Hyp or 4Hyp) within the linear epitope. The substitution may be as indicated in FIG. 4.

[0038] The mutant Ara h protein may comprise one or more substitutions at E46, E89 or Q89, E97, El 14, QI 16, R119, QI 46, and R147 relative to a reference Ara h protein. The one or more substitutions may comprise one or more of E46R, E89R / Q89R, E97R, El 14R, QI 16R, R119A, R119L, R119W, Q121R, Q146A, and R147E relative to a reference Ara h protein. In one example, the mutant Ara h protein comprises E46R, E89R / Q89R, E97R, El 14R, Q146A, and R147E substitutions. In one example, the mutant Ara h protein comprises substitutions at E46 and El 14 and has reduced binding to Tl relative to a reference Ara h protein. In another example, the mutant Ara h protein has substitutions at E89 / Q89 and E97 and has reduced binding to T5 relative to a reference Ara h protein. In another example, the mutant Ara h proteincomprises substitutions at Q146 and R147 and has reduced binding to SI relative to a reference Ara h protein. In one example, the mutant Ara h protein comprises a substitution at E46 and has reduced binding to T1 relative to a reference Ara h protein. The mutant Ara h protein may comprise substitutions E46R and El 14R. The mutant Ara h protein may comprise substitutions E89R / Q89R and E97. The mutant Ara h protein may comprise substitutions Q146A and R147E. In other examples the mutant Ara h protein comprises one or more substitutions at QI 16R, R119A, R119L, R119W, Q121R, and may have reduced affinity for P34. In other examples the mutant Ara h protein contains one or more substitutions at R119A, R119L, R119W, and may have a reduced affinity for Tl. In many cases, other residues substitutions at these sites may be equally successful in abrogating antibody binding.

[0039] In one example, the one or more substitutions in the mutant Ara h protein are relative to a truncated reference Ara h 2.0101 protein which may comprise the following sequence:RRCQ SQLERANLRPCEQHLMQKIQRDED S YERDP YSP SQDP YSP SP YDRRGAGS SQHQE RCCNELNEFENNQRCMCEALQQIMENQSDRLQGRQQEQQFKRELRNLPQQCGLRAPQ RCDLDVESGGRDRY (SEQ ID NO: 5)

[0040] The mutant Ara h 2.0101 protein may comprise the following sequence:RRCQ SQLERANLRPCRQHLMQKIQRDED S YERDP YSP SQDP YSP SP YDRRGAGS SQHQR RCCNELNRFENNQRCMCEALQQIMRNQSDRLQGRQQEQQFKRELRNLPQQCGLRAPA ECDLDVESGG (SEQ ID NO: 6)

[0041] In another example, the mutant Ara h 2 protein comprises the following sequence: RRCQ SQLERANLRPCRQHLMQKIQRDED S YERDP YSP SQDP YSP SP YDRRGAGS SQHQR RCCNELNRFENNQRCMCEALQQIMRNQSDRLQGRQQEQQFKRELRNLPQQCGLRAPA ECDLDVESGGRDRY (SEQ ID NO: 7)

[0042] In one example, the mutant Ara h 2.0101 protein comprises the following sequence: MAKLTILVALALFLLAAHASARQQWELQGDRRCQSQLERANLRPCRQHLMQKIQRDED SYERDPYSPSQDPYSPSPYDRRGAGSSQHQRRCCNELNRFENNQRCMCEALQQIMRNQ SDRLQGRQQEQQFKRELRNLPQQCGLRAPAECDLDVESGG (hexamutant Ara h 2.0101, SEQ ID NO: 8)

[0043] In one example, the mutant Ara h 2.0201 protein comprises the following sequence:MAKLTILVALALFLLAAHASARQQWELQGDRRCQSQLERANLRPCRQHLMQKIQRDED SYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQRRCCNELNRFENNQRCM CEALQQIMRNQSDRLQGRQQEQQFKRELRNLPQQCGLRAPAECDLEVESGGRDRY (hexamutant Ara h 2.0201, SEQ ID NO: 9)

[0044] In one example, the mutant Ara h 6 protein comprises the following sequence:MRRERGRQGDSSSCERQVDGVNLKPCRQHIMQRIMGEQEQYDSYNFGSTRSSDQQRRC CDELNRMENTORCMCEALOOIMRNOCDGLODROMVOHFKRELMNLPOQCNFGAPAE CDLDVSGGRC (hexamutant Ara h 6, SEQ ID NO: 10)

[0045] In some examples, an amino acid may be substituted by any other amino acid, which may be indicated by an X. In one example, the mutant Ara h 2.0101 protein comprises the following sequence:MAKLTILVALALFLLAAHASARQQWELQGDRRCQSQLERANLRPCXQHLMQKIQRDED SYERDPYSPSQDPYSPSPYDRRGAGSSQHQXRCCNELNXFENNQRCMCEALQQIMXNQ SDRLQGRQQEQQFKRELRNLPQQCGLRAPXXCDLDVESGG (hexamutant Ara h 2.0101, SEQ ID NO: 11)

[0046] In one example, the mutant Ara h 2.0201 protein comprises the following sequence:MAKLTILVALALFLLAAHASARQQWELQGDRRCQSQLERANLRPCXQHLMQKIQRDED SYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQXRCCNELNXFENNQRCM CEALQQIMXNQSDRLQGRQQEQQFKRELRNLPQQCGLRAPXXCDLEVESGGRDRY (hexamutant Ara h 2.0201, SEQ ID NO: 12)

[0047] In one example, the mutant Ara h 6 protein comprises the following sequence:MRRERGRQGDSSSCERQVDGVNLKPCXQHIMQRIMGEQEQYDSYNFGSTRSSDQQXRC CDELNXMENTQRCMCEALQQIMXNQCDGLQDRQMVQHFKRELMNLPQQCNFGAPXX CDLDVSGGRC (hexamutant Ara h 6, SEQ ID NO: 13)

[0048] Also provided herein are nucleic acids that encode one or more mutant Ara h proteins disclosed herein. The nucleic acids may be contained in a vector, which may be a bacterial, yeast, viral, or animal vector, or may be integrated into a genome of a genetically modified organism.3. Formulations

[0049] Also provided are compositions comprising the mutant Ara h protein. In one example, the mutant Ara h protein is freeze dried and suitable for reconstitution in an aqueous solution. In another example, the composition comprises an aqueous solution. The aqueous solution may comprise the mutant Ara h protein and a pharmaceutically acceptable carrier or excipient. The composition may be suitable for oral, sublingual, or intravenous administration, or via injection. The injection may be epi cutaneous, intramuscular, or subcutaneous. Intravenous or injectable compositions may comprise one or more of a saline solution, bovine serum albumin (BSA), a buffer, and a sugar (e.g., dextrose). The composition may have a pharmaceutically acceptable pH. Diagnostics may also be provided with biotinylation or other methodologies for ease of coupling to sensor arrays like surface plasmon resonance (SPR), biolayer interferometry (BLI), IMMUNOCAP technologies, or similar.4. Recombinant organisms

[0050] Further provided herein is a recombinant organism or cell that expresses one or more mutant Ara h proteins disclosed herein. The organism may be, or the cell may be derived from, a plant, a yeast, a plant, or a mammal. The recombinant organism may be a genetically modified organism. The genetically modified organism may be a plant, which may be Arachis hypogaea. The genetically modified plant may comprise one or more nucleic acids encoding the one or more Ara h proteins. The genetically modified plant may comprise one or more gene replacements in which each gene replacement comprises a gene encoding a mutant Ara h protein. The gene encoding the mutant Ara h protein may replace an endogenous Ara h protein-encoding gene. Methods of making genetically modified plants are well known in the art. In one example, CRISPR-Cas9 technology is used to edit a plant genome to introduce a gene encoding the mutant Ara h protein, or a portion thereof, which may be all or part of a coding region that encodes a mutant portion of the Ara h protein. The composition comprising the mutant Ara h protein may comprise a genetically modified peanut from the genetically modified plant. The genetically modified peanut may be raw, roasted, extracted, or used as a food product. The genetically modified peanut may be combined with wild-type peanuts and / or wild-type peanut food products.5. Methods of treatment

[0051] Provided herein is a method of treating or reducing the risk of a medical condition associated with peanuts in a subject in need thereof. The method may comprise administering a mutant Ara h protein or a composition comprising a mutant Ara h protein to the subject. Further provided are use of a mutant Ara h protein in the manufacture of a medicament for treating a medical condition associated with peanuts in a subject, and a composition for use in treating a medical condition associated with peanuts in a subject. The medical condition may be an immune response, an allergic response or anaphylaxis. The subject may have or be suspected of having a peanut allergy.

[0052] The mutant Ara h protein may be used as an immunotherapy. Provided herein are a method of immunotherapy in a subject in need thereof described herein comprising administering the mutant Ara h protein, use of the mutant Ara h protein in the manufacture of a medicament for immunotherapy, and the mutant Ara h protein for use in immunotherapy. In one example, the immunotherapy is oral immunotherapy, but administration via sublingual, dermal, intravenous, and injection means may also be used. The mutant Ara h protein may be administered to the subject at a dose which the subject can tolerate without serious adverse effects, at frequency and duration sufficient to reduce the subject’s immune response to one or more peanut allergens.

[0053] The mutant Ara h protein may act as a hypoallergen and may reduce the subject’s immune response to one or more peanut allergens. In such applications, the mutant Ara h protein may be administered in combination with one or more reference Ara h proteins. The dose and timing of each of the one or more mutant Ara h proteins, and each of the one or more reference Ara h proteins, may be different or the same.

[0054] In one example, the mutant Ara h protein modulates the subject’s B-cell response. The mutant Ara h protein may comprise one or more substitutions relative to a reference Ara h protein that result in reduced binding of an antibody disclosed herein in the subject to specific epitopes in the mutant Ara h protein and also reinforce binding of other antibodies in the subject to other epitopes in Ara h protein. In one example, the mutant Ara h protein has reduced binding to the epitope of bin 3 (an example is mAb SI) and increases production of antibodies to bin 1.2 (e.g., Tl), bin 2 (e.g., T5), and bin 1.1 (e.g., P34) in the subject. In another example, the mutant Ara h protein has reduced binding to bin 1.2 epitopes and increases production of bin 1.1, bin 2,and bin 3 antibodies in the subject. In a further example, the Ara h protein has reduced binding to bin 1.2 and bin 2 epitopes and increases production of bin 1.1 and bin 3 antibodies in the subject. All possible permutations and combinations are envisioned of the mutations. The mutant Ara h protein administered could be tailored to the needs of a subject.

[0055] The mutant Ara h protein may be administered at a dose of about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 g, or a range of the foregoing. The mutant Ara h protein may also be administered at a dose of about 0.01, 0.05, 1, 2, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 pg, or a range of the foregoing. The mutant Ara h protein may be administered 1, 2, 3, 4, 5, 6, 7, or 8 times, which may be daily. The mutant Ara h protein may be administered for a duration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or a range of the foregoing. Any of the mutant Ara h proteins described herein may be used in combination, or in series, or in parallel with other immunotherapy approaches or therapeutics.6. Diagnostic methods

[0056] Provided herein are uses of the mutant Ara h protein in molecular assays and diagnostics. The mutant Ara h protein may be used in comparative or competitive assays to determine the levels of one or more anti-Ara h IgG antibodies disclosed herein. The mutant Ara h protein may be useful for determining the level of an antibody, which may be an IgG antibody or an IgE antibody, that represents bin 1.1 (e.g., P34), bin 1.2 (e.g., Tl), bin 2 (e.g. T5) or bin 3 (e.g., SI) in a sample. For example, the mutant Ara h protein E46R / E114R has reduced binding to Tl and may be used to determine levels of bin 1.2-like antibodies in a sample. The mutant Ara h protein E89R / E97R has reduced binding to T5 and may be used to determine levels of bin 2-like antibodies in sample. The mutant Ara h protein Q146A / R147E has reduced binding to SI and may be used to determine the levels of bin 3-like antibodies in sample. The mutant Ara h proteins QI 16R or Q121R have reduced binding to P34 and may be used to determine the levels of bin 1.1 -like antibodies in a sample. The sample may be a blood sample from a subject. The subject may have or suspected have having a peanut allergy. The level of the antibody may be indicative of the efficacy of an immunotherapy described herein.

[0057] The assays may comprise direct, or indirect ELISA, BLI, or bead-based immunoassays, and may comprise comparing mutant protein binding to that of a reference protein. The reference protein may be a wild-type Ara h 2 or Ara h 6 protein, or a mutant Ara h 2 or Ara h 6 proteindescribed herein. In one example, the reference protein is a hexamutant Ara h protein disclosed herein. In one example, the method comprises a multiplex assay in which all or a plurality of bins is probed in a single assay. In direct comparisons, levels of IgG binding to a bin being probed may be compared to reference protein binding. The reduction in levels of IgG is representative of the fraction of IgG for that bin. In an indirect experiment, or competitive inhibition experiment, a reference protein may be attached to an immobilized phase (which may be either a plate in an ELISA, or a chip in SPR or BLI). The ability of the reference protein versus the mutant protein to inhibit soluble IgE or IgG binding to the immobilized phase may be compared. Weaker ability to inhibit antibody binding may be indicative of the fraction of antibodies to that epitope in the sample.

[0058] The present invention has multiple aspects, illustrated by the following non-limiting examples.Example 1Design of an Ara h 2 hypoallergen from immunodominant epitopes

[0059] This example demonstrates that hypoallergens can be designed based on mutating the dominant public epitopes to achieve reduced IgE binding. Hypoallergens have been suggested as an improvement to current allergy immunotherapy to reduce the risk of adverse events during treatment, which is common in peanut oral immunotherapy. One class of proposed hypoallergens relies on mutating residues in immunodominant epitopes that reduce IgE binding, but this is predicated on specific knowledge of those epitopes. Recently cloned human IgG from peanut immunotherapy patients suggested three public immunodominant epitopes for the major peanut allergen Ara h 2. As discussed below, X-ray crystallography was used to characterize public epitopes in detail, followed by mutational analysis of key residues to modify mAb and serum IgE binding. These were assessed by ELISA and BLI. A designed Ara h 2 hypoallergen was tested for reduced vascularization in a mouse model of passive cutaneous anaphylaxis using pooled peanut allergic patient serum.

[0060] The results show that using the crystal structures of ternary Ara h 2- patient derived IgG, site specific mutants were designed that reduced mAb binding by orders of magnitude. By combining designed mutations from the 3 major conformational bins, a hexamutant (Ara h 2 E46R, E89R, E97R, El 14R, Q146A, R147E was created that reduced IgE binding in serum fromallergic patients. Further, in a mouse model of passive cutaneous anaphylaxis where mice were primed with peanut allergic patient serum, vascularization upon allergen challenge was greatly decreased using the hexamutant.Results

[0061] Previous studies from patients undergoing oral immunotherapy for peanuts established that there were 3 major conformational epitope bins using competitive antibody binding (23). Bins 1 and 3 were observed in a ternary crystal structure, published previously (23). The antibodies T1 (bin 1.2) and SI (bin 3) bind to epitopes on Ara h 2 approximately 180 degrees apart, as seen in FIG. 2A. Herein, we report a second ternary crystal structure of Ara h 2, T1 and T5 (bin 2), shown in FIG. 2B. There are several novel features of this ternary structure. T1 and T5 make an acute angle with respect to the epitopes they recognize, approximately 90 degrees. The T1 and T5 antibodies contact each other via the heavy chain of T5 contacting the light chain of Tl. This requires some minor conformational changes of the Tl light chain compared to complex of Tl :Ara h 2: SI. Another interesting observation of the T5:Ara h 2 interactions is that the paratope is composed completely of the T5 heavy chain. This is unique among the anti-Ara h 2 human antibodies studied so far. The vast majority of the T5 / Bin 2 epitope is constituted by residues within helix 2, although a few discontinuous residues make contact with CDRs.

[0062] Herein, we also report a ternary crystal structure of P34, SI, and Ara h 2. This defines a sub-bin of bin 1 called bin 1.1 (23). This is shown in FIG. 2C.

[0063] The precise interactions between the allergen and antibodies can be studied for residues that might be modified to reduce binding in a hypoallergen. FIG. 5 shows a close examination of the epitope-paratope interactions for all 3 bins. Ara h 2 residues El 14 and E46 appeared to interact with tyrosine residues in the heavy chain that were frequently conserved in bin 1 antibodies (FIG. 5A). And in bin 2 Ara h 2 residues E97 and E89 formed 2 salt bridges or 2 hydrogen bonds, respectively, with T5 (FIG. 5B). In bin 3 we identified Ara h 2 residues R147 and Q146 as making multiple hydrogen bond interactions with SI (FIG. 5C). These were identified as candidate residues for mutagenesis in hypoallergen design. FIG. 5D zooms in on 3 residues identified to likely strongly influence binding of Ara h 2 to P34 in the crystal structure with salt bridges and hydrogen bonds. These are QI 16, R119, and Q121. Bin 1.1 overlaps slightly with Bin 1.2 and therefore we examined the interface of Tl and Ara h 2 in FIG. 5E. From this analysis we would predict that only the R119 mutations might affect Tl binding.

[0064] Six double mutations were designed to either reduce the Ara h 2 sidechain to an alanine or to change the charge to influence Bins 1.2, 2, and 3. These mutant proteins were tested for antibody binding in a direct ELISA using the mAbs. FIG. 6 shows that in each case, the designed mutations reduced mAb binding of the affected epitope but did not perturb binding to the other epitopes. First, this confirms the location of the epitopes as determined by crystallography. Second, it also confirms that the site directed mutations do not affect the conformation of the protein substantially, since two other monoclonal antibodies which recognize conformational epitopes will still bind. For mutants binding to Tl and T5 only one double mutant showed a >1000 fold change in EC50 hence those respective mutations were selected for further study. In the case of SI both double mutants had equivalent effects. We arbitrarily selected the change in charge mutation (Q146A / R147E) and proceeded with further studies on this in creating a hexamutant, described below.

[0065] FIG. 7 shows the results of direct ELISA experiments probing the binding of P34 and T1 to single mutations. In 7A the results indicate that Q121R, followed by QI 16R, and R119W substantially reduced the affinity of P34. The other mutations R119A and R119L were not as successful. FIG. 7B shows that binding of the mutations at 119 caused a reduction in affinity for Tl, but not the mutations at 116 and 121. This indicates that the 119 site is not selective for P34, and hence mutations at the sites 116 and 121 should be used for probing the P34 epitope.

[0066] For a general purpose hypoallergen that reduces IgE binding to 3 bins we created a hexamutant of Ara h 2 (E46R, E89R, E97R, El 14R, QI 46 A, R147E) selected from the mutations discussed above and probed to determine whether the hexamutant binding would be reduced to a larger panel of IgG mAb. Dissociation constants were measured by biolayer interferometry (BLI) and plotted in FIG. 8 and tabulated in Table 1.Table 1

[0067] In a previous study of these antibodies, bin 1 was subdivided into bins 1. 1 and 1.2 that could be differentiated by peptides which blocked Ab binding (23). FIG. 8 shows two important results. First, conformation epitope 1.1 is still recognized indicating that the overall protein structure of the hexamutant is similar to the native. Second, antibodies in the same bins show a consistent response to the targeted mutations. This makes sense since antibodies belonging to the different bins that were cloned from different patients had highly similar CDR sequences suggesting that the antibodies converge on immunodominant epitopes with similar paratopes.

[0068] We hypothesized that the Ara h 2 mutations may reduce IgE binding if the IgE paratopes were similar to those found in the IgG. To test if IgE binding to the hexamutant would be reduced a competitive ELISA was designed to compare inhibition of serum IgE binding to either WT or hexamutant. The results are shown in FIG. 9 for 16 peanut allergic patient sera, plotted as percent inhibition. In each case, the hexamutant showed a decreased ability to inhibit soluble IgE binding to the immobilized wild type, albeit with varying success depending on the patient. There was individual variation in the antibody repertoire to Ara h 2 from patient to patient, but a consistent reduction was observed. This is summarized in FIG. 9Q as an average of all the presented curves. FIG. 9R shows the reduction in IgE inhibition at the highest concentration for each patient, demonstrating a consistent reduction.

[0069] The next important question for a hypoallergen is whether it will reduce the potential for anaphylaxis. A mouse model of passive cutaneous anaphylaxis (PCA) was designed to test if the hexamutant would be less effective at stimulating anaphylaxis. Transgenic mice with a human FcsRl were intradermally injected with pooled peanut allergic serum in their ears, effectively creating a peanut allergic mouse with a human-like repertoire of IgE localized to their ears. Themice were subsequently challenged with intradermal injection of WT rAra h 2, hexamutant Ara h 2, or PBS and vascular leakage was measured by the amount of Evan’s blue dye that extravasated into the surrounding tissue. This experiment is a surrogate for human histamine- mediated vascular leakage during anaphylaxis. FIG. 10A shows images of the mouse tissue demonstrating that the extent of dye leakage is greater for WT compared to the hexamutant or the PBS control. The amount of dye was extracted and quantified as shown in panel B for ear tissue confirming the photographic evidence. In both cases there was a significant difference between WT Ara h 2 and hexamutant. The conclusion is that the hexamutant has reduced anaphylactic potential.

[0070] To understand previously published work on the DPYSPS (SEQ ID NO: 4) motif that repeats in Ara h 2 (23), a number of antibodies that were shown to bind this region were tested for binding to a variety of peptides with designed mutations. The results in FIG. 11 for the different antibodies could be categorized in 4 ways based on the patterns of binding to the various mutations. At the bottom of the figure are the 4 control antibodies that were known not to bind the DPYSPS motif. At the top are 8 antibodies that appear to require the DxYxP / Zx, where Z is a hydroxylated proline. The clearest example for this group is the short peptide DAY AZA, which still shows good binding (dark blue color) to all 8 of these antibodies. The next 3 antibodies appear to need a longer peptide, but the same residues appear critical. Lastly 93C9 is more difficult to categorize the specificity as it binds the short and long peptides with similar relative affinity. The conclusion is that a hypoallergen will likely need mutations at the D, Y, and Z sites of this motif.

[0071] A diagnostic method idea is demonstrated in FIG. 12. Competitive inhibition curves were performed exactly as previously described for FIG. 9. However, the double mutant Q146A / R147E was included for comparison with the hexamutant, and only the data at 100 pg / ml is shown, where the biggest contrast in hexamutant versus WT was previously found. The data for these three patients shows the difference in contribution of the bin 3 or SI -like antibodies (orange bars) or the hexamutant in total (blue bars) subtracted from WT. This data suggests that between 17 and 24% of the reduction in patient IgE was targeted to bin 3. In terms of relative contributions, the hexamutant decreased IgE binding the most in Pt 6, but this was the least relative contribution of the bin 3-related antibodies. In concept this type of measurement could instead be targeted at IgG (using a different detect antibody). That would be useful in diagnosisof the amount of the important bin 3 antibodies during immunotherapy. It was previously shown (23) that the sustained unresponsive patients have bin 3 and bin 1.2 antibodies. In concept therefore, the levels could be measured during immunotherapy to determine if the therapy was likely to be sustained after treatment.Discussion

[0072] Immunotherapy to treat hay fever was first suggested by Noon in 1911 (24). In these experiments patients were treated with doses of grass pollen extract. Surprisingly, 100 years later, immunotherapy is very similar. It still carries the obvious risk that one is exposing a patient to something that makes them ill. The use of hypoallergens has been proposed as a way to alleviate this risk as a compromise between presenting the allergen that makes the patient ill, versus the therapeutic benefit of retraining the immune system with the native allergen (25, 26). In this example, we focused on one class of proposed hypoallergens, whereby minimal mutations are proposed, which reduce the risk of anaphylaxis, but maintain enough of the native structure that the immune system will recognize the antigen and retrain for therapeutic benefit (16).

[0073] Reducing IgE is important in peanut allergy where therapy is occasionally dangerous (12). The approach described herein is differentiated from the others, in that the immunodominant epitopes were studied and structurally characterized first, as determined from mAb patient IgG (23). Other studies have inferred dominant epitopes from peptide arrays (27), murine antibodies (28), mutational analysis (29), or chimeric allergens (30). Based on our analysis 3 bins of conformation IgG epitopes were identified. A significant question is whether these are the same or different from IgE epitopes. Some researchers have proposed that IgE and IgG epitopes to allergens may be different and proposed immune mechanisms that may account for this (31). However, mutations that reduce binding to the IgG cloned antibodies analyzed herein, clearly also reduce IgE in the competitive ELISA and well as the PCA. In addition, IgE sequences from other researchers appear to closely match the IgG sequences of T1 and 13A4 (23). We conclude that there is substantial overlap in the IgE and IgG paratopes, mostly likely due to class switching.

[0074] In considering hypoallergens for future therapy there remain many questions. Among them, will the mutated hypoallergen induce appropriate blocking antibodies? For example, the bin 1.2 antibodies were suggested to be important for sustained tolerance. Therefore, it may be advantageous to mutate other residues adjacent to these epitopes so that key epitopes forsuccessful therapy are created. So instead, perhaps the very common bin 1 .1 should be targeted for reduced IgE binding. Crafting the therapy so that the patient develops the ‘right’ antibodies is related to the discussion of how to induce patients to make highly specific yet broadly neutralizing antibodies to HIV.(32) Another question is will the hypoallergen produce the appropriate T-cell signals for developing sustained unresponsiveness (33)? A previous proposal for a hypoallergen of Der p 2 showed reduced skin-prick test size in the hypoallergen yet equivalent T-cell proliferation (34). In other words, knocking out the B-cell response did not diminish the regulatory T-cell response. This example demonstrates that structural knowledge of the important B-cell epitopes can be used to tailor IgE reactivity to Ara h 2 in experimental models of peanut allergic patients. This allows for developing site specific hypoallergens for peanut and other allergic diseases.Materials and Methods

[0075] Recombinant Ara h 2 cloning, expression and purification

[0076] For crystallography, Ara h 2.01 was fused with an N-terminal 6His-thioredoxin tag and a TEV cleavage site to create a gene construct called HisTRX(TEV)A2 that was bacterially expressed and purified. HisTRX(TEV)A2 was transformed into A. coli Origami B cells in the presence of antibiotics (100 pg / mL ampicillin, 50 pg / mL kanamycin, and 12.5 pg / mL tetracycline). For ELISA, Ara h 2.01 was cloned into pMal vector with the Notl and EcoRI restriction sites to create a construct called MBP(TEV)A2 having N-terminal maltose binding fusion protein followed by a TEV cleavage site. MBP(TEV)A2 was transformed into E. coli Origami B cells harboring a TRX plasmid in the presence of antibiotics (100 pg / mL ampicillin, 50 pg / mL kanamycin, 35 pg / mL chloramphenicol, and 12.5 pg / mL tetracycline). Glycerol stocks were prepared and inoculated into 25 mL Luria broth containing the antibiotics for overnight culture, which was transferred into 1 L Terrific broth with the corresponding antibiotics. Cells were grown at 37°C until the OD600 reached 0.6 when 500 pM IPTG was added to induce protein expression, and cells were incubated at 18°C for overnight. Cells were harvested by centrifugation at 4,000g for 15 minutes, and the pellet was lysed by sonication in the resuspension buffer (25 mM Tris pH 8.0, 500 mM NaCl). The soluble fraction was separated by centrifugation at 47,900g and loaded onto 5 mL Ni-NTA for HisTRX(TEV)A2 and 5 mL amylose resin for MBP(TEV)A2 in batch at 4°C. The resins were washed with the buffer three times, followed by elution by resuspension buffer containing 400 mM imidazole forHisTRX(TEV)A2 and 40 mM maltose for MBP(TEV)A2, respectively. Concentrated proteins were loaded onto Superdex 200 26 / 60 equlibrated with the resuspension buffer, and the peak fractions were pooled, frozen, and kept at -80°C until used.

[0077] Recombinant antibody expression

[0078] Heavy and light chain antibody plasmids (22S1, 13T1, 23P34 and 13T5) were prepared using QIAGEN® Plasmid plus Giga Kit as described in the manufacturer’s manual.Recombinant antibodies were expressed using the ExpiCHO expression system (Thermo Fisher Scientific, Carlsbad, CA) with Max titer protocol. 500 mL of ExpiCHO-STM cells were prepared in ExpiCHO expression media as a suspension culture using Thomson OPTIMUM GROWTH™ flasks (Oceanside, CA) with 8% CO2 and 80% humidity at 37°C while shaking at 130 rpm at a density of 6* 106cells / mL. An equal amount (0.5 mg each) of heavy and light chain vectors were added into the final 20 mL of OptiPRO SFM media and incubated for 5 min at RT. 1.6 mL of ExpiFectamine were added to 18.4 mL of OptiPRO SFM media and incubated for 5 min at RT. These two media containing DNAs and ExpiFectamine were mixed and slowly added to the ExpiCHO-STM cells (density of 6* 106cells / mL) while swirling the flask gently. Cultures were incubated in a shaker for 22 hr (Day 0). To enhance the antibody expression, 3 mL ExpiCHO enhancer mixed with 80 mL ExpiCHO feed media was added to the culture gently. The cells were incubated with 5% CO2 and 80% humidity at 32°C while shaking at 130 rpm (Day 1). On the 5thday, an additional 80 mL ExpiCHO feed media was added to the culture and continued growth. On the 14thday, cells were harvested by centrifugation at 500 g for 10 min. The supernatant was then mixed with diatomaceous earth, passing through a 0.2 pm filter.

[0079] Recombinant antibody purification and Fab preparation

[0080] The secreted antibodies were captured by Protein A resin (Gold bio) equilibrated with 0.1 M Tris pH 8.0, 500 mM NaCl, and eluted with 0.1 M glycine pH 2.5, 500 mM NaCl, and neutralized with 1 / 10 volume of 1 M Tris pH 8.0. Fabs were prepared as follows. The buffer of antibodies was exchanged using a Hitrap desalting column (Cytiva) equilibrated with the fresh digestion buffer (1XPBS with 0.02 M EDTA and 0.02 M L-cysteine pH 7.0). Antibodies were digested with papain-immobilized resin (ThermoFisher, 32 mg AB / mL settled resin) in the digestion buffer overnight at 37°C. Fabs were collected from the flowthrough fractions after passing through the Hitrap rProtein A FF column equilibrated with 0.1 M Tris PH 7.5, 0.5 MNaCl. Full-length antibodies and Fabs were analyzed using SDS-PAGE gel. The concentration of antibodies was estimated using an extinction coefficient 1.4 (mg / mL)-l.

[0081] Crystallization of 13T1 / 13T5 / Ara h 2 and structure determination

[0082] HisTRX(TEV)A2 protein was mixed with 13T5 or 22S1 Fab at a molar ratio of 1.2: 1.0 and allowed to form binary complexes at RT for 30 min. The TRX tag in the complex was digested with TEV protease at 4°C while dialyzing against the buffer 25 mM HEPES pH 7.4, 150 mM NaCl for 18 hours. The tagless binary complexes 13T5 / Ara h 2 and 22S1 / Ara h 2 were purified with 16 / 60 Superdex 200 equilibrated with the dialysis buffer and confirmed with SDS- PAGE analysis. These binary compexes were mixed with an equal molar of 13T1 to make 13T1 / 13T5 / Ara h 2 respectively, and purified with Superdex 200 16 / 60. 10 mg / mL of 13T1 / 13T5 / Ara h 2 complexes were used for the crystal screening with MCSG (Midwest Center for Structure Genomics, Anatrace) 1-4 at 4°C and room temperature using sitting drop vapor diffusion. Diffraction quality crystals were obtained from the condition containing 13T1 / 13T5 / Ara h 2 in 0. 1 M Tris pH 8.5, 0.2 M Lithium sulfate, 40 % (w / w) PEG400. We added 5 to 15% ethylene glycol as a cryo-protectant for data collection. Data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory (wavelength 1.0 A and temperature 100 K) (41). Molecular replacement was performed using the previously reported ternary complex 22S1 / 13T1 / Ara h 2 (PDB ID: 8D12) as a search model using the Phaser module in Phenix. The model was refined using iterative cycles of refinement in Phenix and manual building in Coot. The 3-D cartoons were made with PyMol (The PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC.).

[0083] Crystallization of 23P34 / 22S1 / Ara h 2 and structure determination

[0084] HisTRX(TEV)A2 protein was mixed with 23P34 Fab at a molar ratio of 1.1 : 1.0 and allowed to form a binary complexes at RT for 30 min. The TRX tag on Ara h 2 was removed by digestion with TEV protease at 4°C while dialyzing against the buffer 25 mM HEPES pH 7.4, 150 mM NaCl for 18 hours. The tagless binary complexes 23P34 / Ara h 2 was purified with a 16 / 60 Superdex 200 equilibrated with the dialysis buffer. The binary complex was mixed with 22S1 Fab at a molar ratio of 1 : 1.3 (binary / 22Sl), and the ternary complex was purified with a Superdex 200 16 / 60 equilibrated in 25 mM HEPES pH 7.4, 150 mM NaCl. 10 mg / mL of 23P34 / 22S1 / Ara h 2 complex was used for the crystal screening against MCSG (Midwest Centerfor Structure Genomics, Anatrace) 1 -4 at 4°C and RT using sitting drop vapor diffusion. Diffraction quality crystals were obtained from the condition containing 23P34 / 22S1 / Ara h 2 in 0.1 M BisTrisPropane pH 7.5, 0.2 M potassium sodium tartrate tetrahydrate, 20% PEG3350. We added 15% ethylene glycol as a cryo-protectant for data collection. Data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory (wavelength 1.0 A and temperature 100 K).

[0085] Generation of Ara h 2 mutants

[0086] Site directed mutagenesis of Ara h 2 proceeded using QuickChange mutagenesis kit from ThermoFischer according to the manufacturer protocols, after interface analysis using PISA server.

[0087] ELISA assay

[0088] ELISA assay was performed at room temperature using recombinant IgG antibodies and MBP(TEV)A2. One day before conducting the assay, 1 mg / mL of IgGs (13T1, 13T5, or 22S1) were diluted with 50 mM Carbonate / bicarbonate buffer pH 9.6 at a ratio of 1: 1000 (v / v). The antibodies were plated onto 96 well plates (Thermofisher) with a volume of 100 pL in each well overnight at 4°C. On the next day, the plate was washed with PBST (137 mM NaCl, 2.7 mM KC1, 10 mM Na2HPO4, and 1.8 mM KH2PO4 pH 7.4, and 0.05 % Tween 20) 3 times with blotting onto paper towels, and this washing step was repeated for every reagent treatment. The plate was blocked with 100 pL PBST containing 1 % BSA (Sigma) (PBST-BSA) for 1 hour followed by washing. The antibodies were allowed to bind MBP:Ara h 2 with 100 pL of the protein with a range of concentration from 0 to 102.4 pg / mL for 1 hour at room temperature followed by washing. 100 pL of Anti-MBP-biotin antibody (1: 1000 dilution, Rockland inc.) in PBST-BSA were added to the plated, which was incubated for 1 hour at room temperature followed by washing. 100 pL strep-avidin-HRP (1 mg / mL in dH2O) in PBST-BSA was added to bind the biotin in the plate, which was incubated for 1 hour at room temperature. After washing, 100 pL 3% H2O2 in 10 mL ABTS (Sigma) was added to the plate and waited until the green color developed. The absorbance at 405 nm was measured with a Polarstar Omega plate reader (BMG Labtech). Data were analyzed and plotted with the GraphPad prism.

[0089] Protein prep for biotinylation

[0090] An avitag coding the amino acid sequence GLNDIFEAQKIEWHE (SEQ ID NO: 15) (GGACTAAATGATATATTTGAAGCGCAGAAGATCGAATGGCATGAA; SEQ ID NO: 16)was engineered into a pMalMBP(TEV)A2 vector using restriction sites so that the construct express avitagMBP(TEV)A2. To prepare biotin ligase, pH6-MBP-TEV-BirA (Addgene plasmid # 179694) was transformed into E. coli BL21 cells in the presence of 100 pg / mL ampicillin. The pMal-avitagMBP(TEV)A2 and avitag-MBP(TEV)HEXA2 were transformation into E. coli Origami B cells and plated onto LB agar plates containing antibiotics (100 pg / mL ampicillin, 50 pg / mL kanamycin, and 12.5 pg / mL tetracycline). AvitagMBP(TEV)A2, avitagMBP(TEV)HEXA2, and pH6-MBP-TEV-BirA were expressed and purified as described previously / Briefly, a glycerol stock was prepared and inoculated into 25 mL Luria broth containing relevant antibiotics for overnight culture, which was transferred into 1 L Terrific broth with the same antibiotics. Cells were grown at 37°C until the OD600 reached 0.6 when 500 pM IPTG was added to induce protein expression, and cells were incubated at 18°C for overnight. Cells were harvested by centrifugation at 4,000g for 15 minutes, and the pellet was lysed by sonication in the resuspension buffer (500 mM NaCl in 25 mM Tris at pH 8.0). The soluble fraction was separated by centrifugation at 47,900g and loaded onto 5 mL amylose resin in batch at 4°C. The resin was washed with the buffer three times, followed by batch elution with resuspension buffer containing 40 mM maltose. Concentrated protein was loaded onto Superdex 200 26 / 60 equilibrated with PBS buffer (137 mM NaCl, 2.7 mM KC1, 10 mM Na2HPO4, and 1.8 mM KH2PO4), and the peak fractions, and kept at -80°C until used.

[0091] Biotinylation and evaluation

[0092] The biotinylation of proteins was performed as described (Fairhead and Howart 2015). In a total volume of 1 mL of PBS buffer, final 100 pM of avitagMBP(TEV)A2 or avitagMBP(TEV)HEXA2 were prepared in the presence of 5 pL of 1 M MgCh, 20 pL of 100 mM ATP, 20 pL of 190 pM of biotin ligase, and 3 pL of 50 mM D-Biotin (Sigma A14207) dissolved in DMSO. For control, the same mixture was prepared in the absence of biotin lagase. The mixture was incubated for 1 hr at 30°C in a waterbath with a gentle homogenization every 10 min. Then fresh 20 pL of 190 pM biotin ligase and 3 pL of 50 mM D-Biotin was added in the mixture. The mixture was incubate for another hour at 30°C with a gentle homogenization every 10 min. To remove the biotin ligase, 50 % slurry of 100 pL Ni-NTA resin in PBS was added to the mixture and placed on the rocker for 30 min at RT. The mixture was dialyzed against PBS to remove the excess biotin overnight. The efficiency of the biotinylation reaction was evaluate using a streptavidin gel-shift assay. Briefly, 1 pL of 10 pM biotin-AviMBPTEVArah2 wasmixed with 17.5 pL 2X SDS sample buffer and 1 .5 pL of PBS containing 50 pM Strepavidin (Thermofisher). 1 pL of the mixture was used for the SDS-PAGE analysis.

[0093] Competitive ELISA Protocol

[0094] Sixteen peanut-allergic patient sera from three different cohorts PNOIT 1 (n=4), PNOIT 2 (n=8) and PU (n=4) were used. All patients had a history of clinical reactivity to peanut and elevated peanut specific IgE levels by ImmunoCAP. Patient sera pre-OIT was used. Patients with a high threshold of peanut allergy (PU) were selected after passing a double-blind placebo controlled oral food challenge to 443 mg peanut. Protein G columns were washed with distilled water 2x and equilibrated with 3 column volumes of PBS pH 7.4. The columns were sealed with parafilm after 2 ml of serum was added and placed at 4°C overnight on rotator table. The flow through and 3 column volumes of PBS wash was pooled. Protein G columns were regenerated by eluting the IgG with 1 M glycine pH 2.7 into tubes containing a neutralization buffer of IM Tris- HC1 pH 9 at a 1 : 10 ratio of buffer to eluate. The OD was checked to determine volume needed for elution of the IgG from the Protein G column. The process was repeated twice more but with shorter gravity flow the depleted serum past the protein G resin. The flow through and wash containing IgE was concentrated to 2 ml and stored with 0.02% sodium azide at 4°C.

[0095] A competitive ELISA was conducted to demonstrate the percent inhibition or binding of inhibitor to patient serum IgE at varying inhibitor concentrations. In this experiment, a 1 : 1000 dilution of [concentration] MBP-TEV-Ara h 2, MBP:Hexa2 to 50 mM carbonate and bicarbonate pH 9.6 coating buffer was plated onto Thermo Scientific clear Flat-Bottom Immuno Non-Sterile 96-well plates. For a negative control, a 1 :1000 dilution of 1% bovine serum albumin (BSA) in PBS with 0.05% of Tween 20 pH 7.4 (PBS-T) blocking buffer to coating buffer was plated. ELISA plates were incubated overnight at 4C. Wells were washed 3x with PBS-T and incubated with a blocking buffer of 1% BSA in PBS-T for 1 hr at room temperature. WtMBP-Ara h 2 and hexamutant dilutions were prepared by making a 100 pg / mL stock in PBS pH 7.4. 1 :5 dilutions of concentrated IgE serum were incubated with serial dilutions of both wildtype and hexamutant for 1 hr at room temperature. For the positive and negative controls, diluted IgE serum with no inhibitor was pipetted into respective wells and incubated for 1 hr at room temperature before washing 3x with PBS. Detection of IgE used a 1 :2000 dilution of mouse monoclonal anti-human IgE HRP from abeam (ab99806) in 1% BSA blocking buffer that incubated for 1 hr at room temperature. After washing 3x, the substrate solution was then made by adding equal parts ofsubstrate reagent A to substrate reagent B from BD Biosciences TMB substrate reagent set (55124). One-hundred microliters of the substrate solution was pipetted into each well of the washed ELISA plate resulting in a color change from clear to blue. Absorbance was measured on a SpectraMax ID5 plate reader until the OD 605 reached approximately 1 and the reaction was stopped by adding 100 pL of 1 M HC1 to each well and one last reading was taken at 450 nm. Percent inhibition was scaled from 0 to 100% using the positive control (IgE) as 100% and no inhibitor (background) as 0%.

[0096] Biolayer interferometry (BLI) assays

[0097] BLI assays were performed at a plate temperature of 30°C and a shaking speed of 1000 rpm on an Octet K2 Protein Analysis System (Sartorius).

[0098] For measurement of affinity, monoclonal antibodies were diluted in kinetics buffer (DPBS + 1% (w / v) BSA + 0.02% (v / v) Tween 20 (Amresco) into a black flat bottom 96 well plate (Greiner Bio-One). Either biotinylated recombinant Ara h 2.0201 (0.5 pg / mL in kinetics buffer) or biotinylated hexamutant was loaded onto streptavidin sensors (Sartorius) for 100 seconds. Affinities were measured using a minimum of 5 curves with reference well with a Chi- squared (x2) value <3 to and R-squared value (R2) >0.95. Streptavidin sensors were regenerated at most 10 times with a 30 second cycle of regeneration in glycine 1.5 pH (Bio-Rad) and neutralization in kinetics buffer.

[0099] Assessment of monoclonal antibody binding to double mutated Ara h 2 was performed by capture of the antibody on anti-human Fab-CHl (FAB2G, Sartorius) followed by association with either native Ara h 2 (Indoor Biotechnologies) or double mutated Ara h 2.

[0100] Analysis was performed using Octet® Analysis Studio Software version 12.2 and GraphPad Prism (version 9.3.1).

[0101] Murine passive cutaneous anaphylaxis

[0102] In a model of passive cutaneous anaphylaxis (PCA), hFcsRIa mice (gifted from Robert Anthony) were used to assess the compare IgE-mediated activation by Hexamutant and native Ara h 2. The ears of hFcsRIa mice were sensitized either with 20uL pooled, IgG-depleted human serum from peanut allergic individuals (IRB number) or PBS in the ears through intradermal injection. After 4 hours, the mice were intravenously challenged with 50 pg of rAra h 2.0201 or Hexamutant, in Evan’s blue dye. Forty minutes after challenge, the amount of blue dye in the ear was quantified as a surrogate of histamine-mediated vascular leakage after overnight incubationin extraction buffer and measurement at the A680 nm using the spectrophotometer. All experiments using animals were approved by the Institutional Animal Care and Use Committee of Massachusetts General Hospital.

[0103] Peptide Experiments

[0104] Custom-synthesized, freeze-dried, biotinylated peptides labeled with a biotin and a hydrophilic linker (TTDS) on the N-terminus (BioTides, JPT Peptide Technologies, Berlin, Germany) were diluted in 100 microliters of dimethyl sulfoxide (Sigma-Aldrich), aliquoted, and stored at -80°C. Immediately prior to usage, they were diluted to a concentration of 8 nM in PBS and then further diluted to 0.034 nM in kinetics buffer (DPBS + 1% (w / v) BSA + 0.02% Tween 20 (Amresco)) for the 63DPYSPOHSDPYS72. For all peptide experiments, monoclonal antibodies binding to the 63DPYSPOHSDPYS72 epitope were associated for 600 seconds and monoclonal antibodies binding to all other epitopes were associated for 300-400 seconds after 60-second baseline steps in kinetics buffer. The sensors were regenerated in between each antibody association unless otherwise noted. Sensors were regenerated no more than twenty times through a 30-second cycle alternating between glycine 1.5 pH (Bio-Rad) and kinetics buffer. Antibody binding to each peptide was measured by response rate (nm) and aligned to the immediately preceding baseline step. All BLI assays were run on an Octet R2 Protein Analysis System (Sartorius) at a plate temperature of 30°C with a shaking speed of lOOOrpm. Data were analyzed using Octet® Analysis Studio Software version 12.2 (Sartorius).REFERENCES FOR EXAMPLE AND BACKGROUND

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Claims

CLAIMSWhat is claimed is:

1. A mutant Ara h protein, comprising an amino acid sequence at least 90% identical to a reference Ara h protein comprising the sequence set forth in one of SEQ ID NOs: 1-3, and comprising a substitution at one or more of positions E46, E89 or Q89, E97, El 14, QI 16, R119, QI 46, and R147 relative to the reference Ara h protein, wherein the position of each of the one or more substitutions is relative to the numbering of the amino acids in SEQ ID NO: 1.

2. The mutant Ara h 2 protein of claim 1, comprising an amino acid sequence identical to the sequence set forth in one of SEQ ID NOs: 1-3 other than the one or more substitutions.

3. The mutant Ara h protein of claim 1 or 2, comprising substitutions at positions E46, E89 or Q89, E97, El 14, Q146, and R147.

4. The mutant Ara h protein of any one of claims 1-3, wherein the E46, E89 or Q89, E97, El 14, Q146, and R147 substitutions are respectively E46R, E89R or Q89R, E97R, El 14R, Q146A, and R147E.

5. The mutant Ara h protein of any one of claims 1-4, wherein the reference Ara h protein is an Ara h 2 protein comprising the sequence set forth in SEQ ID NO: 1 or 2.

6. The mutant Ara h 2 protein of claim 5, comprising the sequence set forth in one of SEQ ID NOs: 5-9.

7. The mutant Ara h protein of any one of claims 1-4, wherein the reference Ara h protein is an Ara h 6 protein comprising the sequence set forth in SEQ ID NO: 3.

8. The mutant Ara h 6 protein of claim 7, comprising the sequence set forth in SEQ ID NO: 10.

9. A mutant Ara h 2 protein, comprising a substitution at one or more of positions E46, E89, E97, El 14, Q146, and R147 relative to a reference Ara h 2 protein comprising the sequence set forth in SEQ ID NO: 1 or 2.

10. The mutant Ara h 2 protein of claim 9, comprising substitutions comprising E46R, E89R, E97R, El 14R, Q146A, and R147E.

11. The mutant Ara h 2 protein of claim 10, comprising the sequence set forth in one of SEQ ID NOs: 5-9.

12. A mutant Ara h 6 protein, comprising a substitution at one or more of positions E46, Q89, E97, El 14, Q146, and R147 relative to a reference Ara h 6 protein comprising the sequence set forth in SEQ ID NO: 3.

13. The mutant Ara h 6 protein of claim 12, comprising substitutions comprising E46R, Q89R, E97R, E114R, Q146A, and R147E.

14. A pharmaceutical composition comprising the mutant Ara h protein of any one of claims 1-13 and a pharmaceutically acceptable excipient.

15. A method of treating or reducing the risk of a medical condition associated with peanuts in a subject in need thereof, comprising administering to the subject the mutant Ara h protein of any one of claims 1-13 or the pharmaceutical composition of claim 14.

16. The method of claim 15, wherein the subject has or is suspected of having a peanut allergy.

17. The method of claim 15 or 16, wherein the medical condition is one or more of an immune response, an allergic response, and anaphylaxis.

18. The method of any one of claims 15-17, wherein the mutant Ara h protein or pharmaceutical composition is administered orally, intravenously, or via injection.

19. The method of any one of claims 15-18, wherein the method of treating is an immunotherapy that desensitizes the subject to one or more peanut allergens.

20. The method of claim 19, wherein the immunotherapy is oral immunotherapy.21 . A method of reducing binding of a reference Ara h protein to an anti-Ara h antibody, comprising contacting the anti-Ara h antibody with the mutant Ara h protein of any one of claims 1-13.

22. A method of determining the level of an anti-Ara h 2 or anti-Ara h 6 antibody in a sample, comprising:(a) contacting a sample comprising or suspected of comprising the anti-Ara h 2 or anti-Ara h 6 antibody with the mutant Ara h protein of claim 1;(b) quantifying the amount of binding of the anti-Ara h 2 or anti-Ara h 6 antibody to the mutant Ara h protein, wherein the amount of binding of the anti-Ara h 2 or anti-Ara h 6 antibody to the mutant Ara h protein is indicative of the level of the anti-Ara h 2 or anti-Ara h 6 antibody in the sample.

23. The method of claim 22, wherein the amount of binding of the anti-Ara h 2 or anti-Ara h 6 antibody to the mutant Ara h protein is compared to the amount of binding of the anti-Ara h 2 or anti-Ara h 6 antibody to a reference Ara h protein, and wherein the degree of reduced binding of the anti-Ara h 2 or anti-Ara h 6 antibody to the mutant Ara h protein as compared to the amount of binding of the anti-Ara h 2 or anti-Ara h 6 antibody to the reference Ara h protein is indicative of the level of the anti-Ara h 2 or anti-Ara h 6 antibody in the sample.

24. The method of claim 22 or 23, wherein the antibody is T1 and is representative of bin 1.2-like antibodies and the mutant Ara h protein comprises substitutions at positions E46 and El 14 relative to an Ara h protein comprising the sequence set forth in one of SEQ ID NOs: 1-3,wherein the positions of the substitutions are relative to the numbering of the amino acids in SEQID NO: 1.

25. The method of claim 22 or 23, wherein the antibody is T5 and is representative of bin 2-like antibodies and the mutant Ara h protein comprises substitutions at positions E89 and E97 relative to an Ara h protein comprising the sequence set forth in one of SEQ ID NOs: 1-3, wherein the positions of the substitutions are relative to the numbering of the amino acids in SEQ ID NO: 1.

26. The method of claim 22 or 23, wherein the antibody is SI and is representative of bin 3-like antibodies and the mutant Ara h protein comprises substitutions at positions Q146 and Q147 relative to an Ara h protein comprising the sequence set forth in one of SEQ ID NOs: 1-3, wherein the positions of the substitutions are relative to the numbering of the amino acids in SEQ ID NO: 1.

27. The method of claim 22 or 23, wherein the antibody is P34 and is representative of bin 1.1 -like antibodies and the mutant Ara h protein comprises substitutions at positions QI 16 and QI 21 relative to an Ara h protein comprising the sequence set forth in one of SEQ ID NOs: 1-3, wherein the positions of the substitutions are relative to the numbering of the amino acids in SEQ ID NO: 1.

28. A nucleic acid encoding the mutant Ara h protein of any one of claims 1-13.

29. A genetically modified organism comprising the nucleic acid of claim 28.

30. The genetically modified organism of claim 29, wherein the organism is a plant.31 . The genetically modified organism of claim 30, wherein the plant is Arachis hypogaea.

32. The genetically modified organism of claim 31, comprising a gene replacement wherein a gene or portion thereof encoding a wild-type Ara h protein is replaced by the nucleic acid encoding the mutant Ara h protein.

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