Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/35—Allergens
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• 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.
• 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.
• 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.
• 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 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.
• 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.
• nucleic acid encoding the mutant Ara h protein.
• 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.
• a pharmaceutical composition comprising the mutant Ara h protein and a pharmaceutically acceptable excipient.
• a method of treating or reducing the risk of a medical condition associated with peanuts in a subject in need thereof may comprise administering to the subject the mutant Ara h protein or the pharmaceutical composition.
• 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.
• 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.
• 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 the substitutions 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.
• 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.
• 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).
• 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.
• 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.
• SI Ara 6.0101
• FIG. 3B shows the structure of Ara h 2 labeled to show the residues indicated in FIG. 3 A.
• 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.
• 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.
• 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.
• FIG. 6A-C show antibody binding of various mutant Ara h 2 proteins. Direct ELISAs coating the plate with T1 (FIG.
• 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.
• 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.
• FIG. 8A-E show the binding of a hexamutant Ara h 2 protein to a panel of IgG monoclonal antibodies.
• 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).
• the negative log of the dissociation constants (KD) is plotted with standard deviations for replicate measurements. See also the tabulated data in Table 1.
• 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- WT Ara 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. 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.
• 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.
• FIG. 11 Peptide mapping of important linear epitope residues of the DPYSPS (SEQ ID NO: 4) motif.
• 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.
• 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.
• 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.
• 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.
• 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.
• DPYSXS 3- hydroxyproline
• 4Hyp 4-hydroxyproline
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• FIG. 1 An alignment of reference Ara h 2.0101, Ara h 2.0201, and Ara h 6 proteins is shown in FIG. 1.
• Ara h 2.0101 SEQ ID NO: 1
• FIG. 2 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.
• 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.
• 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.
• the mutant Ara h protein comprises E46R, E89R/Q89R, E97R, El 14R, Q146A, and R147E substitutions.
• 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 protein comprises 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.
• the mutant Ara h protein comprises one or more substitutions at QI 16R, R119A, R119L, R119W, Q121R, and may have reduced affinity for P34.
• 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.
• 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:
• the mutant Ara h 2.0101 protein may comprise the following sequence:
• 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)
• the mutant Ara h 2.0101 protein comprises the following sequence: MAKLTILVALALFLLAAHASARQQWELQGDRRCQSQLERANLRPCRQHLMQKIQRDED SYERDPYSPSQDPYSPSPYDRRGAGSSQHQRRCCNELNRFENNQRCMCEALQQIMRNQ SDRLQGRQQEQQFKRELRNLPQQCGLRAPAECDLDVESGG (hexamutant Ara h 2.0101, SEQ ID NO: 8)
• the mutant Ara h 2.0201 protein comprises the following sequence: MAKLTILVALALFLLAAHASARQQWELQGDRRCQSQLERANLRPCRQHLMQKIQRDED SYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQRRCCNELNRFENNQRCM CEALQQIMRNQSDRLQGRQQEQQFKRELRNLPQQCGLRAPAECDLEVESGGRDRY (hexamutant Ara h 2.0201, SEQ ID NO: 9)
• an amino acid may be substituted by any other amino acid, which may be indicated by an X.
• the mutant Ara h 2.0101 protein comprises the following sequence:
• the mutant Ara h 2.0201 protein comprises the following sequence:
• the mutant Ara h 6 protein comprises the following sequence:
• 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.
• compositions comprising the mutant Ara h protein.
• the mutant Ara h protein is freeze dried and suitable for reconstitution in an aqueous solution.
• 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).
• BSA bovine serum albumin
• SPR surface plasm
• 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.
• 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.
• 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.
• the mutant Ara h protein may be used as an immunotherapy.
• 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.
• 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.
• the mutant Ara h protein may act as a hypoallergen and may reduce the subject’s immune response to one or more peanut allergens.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 protein described herein.
• the reference protein is a hexamutant Ara h protein disclosed herein.
• 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.
• 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).
• 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.
• the present invention has multiple aspects, illustrated by the following non-limiting examples.
• 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.
• 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.
• 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.
• FIG. 8 shows two important results.
• Conformation epitope 1.1 is still recognized indicating that the overall protein structure of the hexamutant is similar to the native.
• 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.
• 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.
• the mice 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.
• 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.
• WT Ara h 2 and hexamutant.
• the conclusion is that the hexamutant has reduced anaphylactic potential.
• FIG. 12 A diagnostic method idea is demonstrated in FIG. 12.
• Competitive inhibition curves were performed exactly as previously described for FIG. 9.
• 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.
• the hexamutant decreased IgE binding the most in Pt 6, but this was the least relative contribution of the bin 3-related antibodies.
• this type of measurement could instead be targeted at IgG (using a different detect antibody). That would be useful in diagnosis of 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.
• 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 for successful 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.
• 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).
• MBP(TEV)A2 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).
• 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 for HisTRX(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.
• 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 GROWTHTM flasks (Oceanside, CA) with 8% CO2 and 80% humidity at 37°C while shaking at 130 rpm at a density of 6* 10 6 cells/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.
• ExpiFectamine 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* 10 6 cells/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).
• 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 M NaCl. 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.
• 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.
• 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 Center for Structure Genomics, Anatrace) 1 -4 at 4°C and RT using sitting drop vapor diffusion.
• MCSG Middle Center for Structure Genomics, Anatrace
• 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).
• ELISA assay was performed at room temperature using recombinant IgG antibodies and MBP(TEV)A2.
• IgGs 13T1, 13T5, or 22S1
• 50 mM Carbonate/bicarbonate buffer pH 9.6 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.
• PBST 137 mM NaCl, 2.7 mM KC1, 10 mM Na2HPO4, and 1.8 mM KH2PO4 pH 7.4, and 0.05 % Tween 20
• the plate was blocked with 100 pL PBST containing 1 % BSA (Sigma) (PBST-BSA) for 1 hour followed by washing.
• PBST-BSA 100 pL PBST containing 1 % BSA (Sigma)
• 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.
• 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.
• pH6-MBP-TEV-BirA (Addgene plasmid # 179694) was transformed into E. coli BL21 cells in the presence of 100 pg/mL ampicillin.
• 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.
• PBS buffer 137 mM NaCl, 2.7 mM KC1, 10 mM Na2HPO4, and 1.8 mM KH2PO4
• 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.
• 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.
• a competitive ELISA was conducted to demonstrate the percent inhibition or binding of inhibitor to patient serum IgE at varying inhibitor concentrations.
• 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.
• 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.
• 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 of substrate 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.
• 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).
• 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.
• Custom-synthesized, freeze-dried, biotinylated peptides labeled with a biotin and a hydrophilic linker (TTDS) on the N-terminus 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 63DPYSP OH SDPYS72.
• TTDS hydrophilic linker

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