Compositions and methods for inducing desensitization of peanuts

Abstract

In one embodiment, the present disclosure provides a hypoallergenic peanut allergen Ara h 6 variant in which at least one epitope recognized by an anti-Ara h 6 antibody has been modified, thereby having reduced or eliminated binding of the antibody to the peanut allergen. These peanut allergen variants are hypoallergenic, but maintain their immunogenicity, and can be used in methods of treating subjects allergic to peanuts and reducing their allergy severity.

Description

[0001] Sequence Listing

[0002] This application includes a sequence listing, which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The XML copy was created on October 29, 2023, is named P-621156-PC_SL.xml, and is 219,755 bytes in size. Field of the Invention

[0003] The present disclosure generally relates to the recombinant hypoallergenic peanut allergen Ara h 6, methods for its production, and uses thereof.

[0004] background

[0005] One of the most severe food allergies currently known is peanut allergy, in which allergic individuals respond to exposure to peanuts, even at low concentrations, with symptoms ranging from mild local effects to severe life-threatening effects. Peanuts are the leading cause of food-induced anaphylactic shock in the United States (Finkelman, (2010) Current Opinion in Immunology, 22(6):783-788), and some form of allergic reaction to peanuts is reported in approximately 1% of the U.S. population (Sicherer SH et al., (2010). J Allergy Clin Immunol. 125(6):1322-6).

[0006] So far, 17 peanut proteins have been proposed to be involved in IgE-mediated allergic reactions (Palladino, C., & Breiteneder, H. (2018). Molecular immunology, 100: 58-70). Among these proteins, seed storage proteins Ara h 1, Ara h 2, Ara h 3 and Ara h 6 are considered to be major allergens, and their responses mediated by IgE antibody recognition are associated with more severe symptoms (Palladino, et al., 2018; ibid.) (Bernard, et al., (2007) J Agric Food Chem. 55 (23): 9663-9).

[0007] Arah 6 is a member of the 2S albumin family and is the major allergen of peanut (Arachis hypogaea). Arah 6 has 145 amino acids, including a 21-amino acid signal peptide, is dominated by five α-helices, and contains five intramolecular disulfide bonds (uniport A5Z1R0; Q647G9).

[0008] Ara h 2 and Ara h 6 both belong to the conglutin type of 2S-albumin. The conformational models of Ara h 2 and Ara h 6 are actually superimposable, indicating that the tertiary structures of the two proteins are nearly similar. Currently, they are considered to be the most potent peanut allergens (Kulis, Mike et al., (2012) Clinical & Experimental Allergy; 42.2: 326-336). Ara h 2 and Ara h 6 are the most common major peanut allergens in children (Flinterman, AE et al., (2007) Clinical & Experimental Allergy 37.8: 1221-1228; and van Erp, Francine C. et al., (2017) Journal of Allergy and Clinical Immunology; 139.1: 358-360). Individual reactivity to major peanut allergens remains stable over time (Flinterman AE et al. (2007) Clin Exp Allergy; 37(8) 1221-1228).

[0009] Due to their structural similarities, anti-Ara h 6 antibodies often cross-react with Ara h 2 and vice versa (Koppelman, SJ et al., (2005) Clinical & Experimental Allergy: 35.4 (2005): 490-497). However, these proteins are not identical, and in each of them there are putative antibody epitopes that are not present in the other. It has been reported that there are allergic patients who are allergic to only one peanut 2S albumin (Asarnoj A, Glaumann S, Elfstrom L et al., (2012) Int Arch Allergy Immunol.: 159 (2): 209-212). Such reports suggest that IgE antibodies against specific epitopes in Ara h 6 that are not present in Ara h 2 may be sufficient to trigger clinical reactions in at least some patients.

[0010] There remains an unmet need for hypoallergenic peanut proteins and methods for their use in standardized immunotherapy treatments in subjects allergic to peanut allergens.

[0011] Overview

[0012] Described herein are several epitope mapping methods for identifying epitopes on Ara h 6 and protein engineering methods for designing hypoallergenic Ara h 6 allergen variants that maintain biophysical and functional characteristics. In one aspect, disclosed herein are recombinant Ara h 6 variant polypeptides that lack at least one epitope recognized by an anti-Ara h 6 antibody, thereby reducing or eliminating binding of IgE antibodies to the variant polypeptide. In some embodiments, the epitope comprises a linear epitope. In some embodiments, the epitope comprises a conformational epitope. In another aspect, these variant polypeptides can be used in methods for inducing desensitization and / or immunomodulation to peanut in human patients allergic to peanut.

[0013] In one aspect, provided herein are recombinant Ara h 6 variant polypeptides.

[0014] In some embodiments, the recombinant Ara h 6 variant polypeptide comprises an amino acid sequence containing one or more amino acid substitutions, deletions, insertions, or any combination thereof at one or more of positions 3, 5, 8, 19, 45, 46, 86, 89, 90, 98, 106, 108, 110, 114, 116, or 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2.

[0015] In some embodiments, the recombinant Ara h 6 variant polypeptide comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof at one or more of positions 2, 3, 5, 7, 8, 10, 12, 16, 19, 22, 24, 33, 37, 38, 40, 41, 42, 45, 46, 47, 74, 78, 81, 82, 83, 86, 89, 90, 97, 98, 99, 106, 108, 110, 113, 114, 116, or 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2.

[0016] In some embodiments, the recombinant Ara h 6 variant polypeptide comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof at one or more of positions 2, 3, 5, 7, 8, 10, 12, 16, 19, 22, 24, 33, 37, 38, 40, 41, 42, 45, 46, 47, 63, 74, 78, 81, 82, 83, 86, 89, 90, 97, 98, 99, 106, 108, 109, 110, 113, 114, 116, or 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2.

[0017] In some embodiments, the recombinant Ara h 6 variant comprises one or more of the following substitutions:

[0018] (a) S at position 2;

[0019] (b) D or S at position 3;

[0020] (c) D at position 5;

[0021] (d) D at position 7;

[0022] (e) A or S at position 8;

[0023] (f) A, S or K at position 10;

[0024] (g) R, D or N at position 12;

[0025] (h) S or D at position 16;

[0026] (i) Q, L or R at position 19;

[0027] (j) F at position 22;

[0028] (k) D at position 24;

[0029] (l) Q or K at position 33;

[0030] (m) A, T or S at position 37;

[0031] (n) A or S at position 38;

[0032] (o) S at position 40;

[0033] (p) D at position 41;

[0034] (q) K, E or G at position 42;

[0035] (r) A or Q at position 45;

[0036] (s) S, G or R at position 46;

[0037] (t) S at position 47;

[0038] (u) R at position 74;

[0039] (v) L at position 78;

[0040] (w) A or R at position 81;

[0041] (x) T at position 82;

[0042] (y) N or K at position 83;

[0043] (z) D or S at position 86;

[0044] (aa) N, R or G at position 89;

[0045] (bb) D at position 90;

[0046] (cc) I at position 97;

[0047] (dd) D or L at position 98;

[0048] (ee) M at position 99;

[0049] (ff) K or H at position 106;

[0050] (gg) P, E or D at position 108;

[0051] (hh) E or S at position 110;

[0052] (ii) D or I at position 113;

[0053] (jj) D, H, A or G at position 114;

[0054] (kk) K or M at position 116; or

[0055] (ll) R or T at position 118.

[0056] In some embodiments, the variant comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof at positions 15, 17, 20, 28, 35, 57, 59, 61, 64, 91, or 123 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2.

[0057] In some embodiments, the variant comprises an amino acid substitution, deletion, insertion, or any combination thereof at positions 3, 5, 8, 19, 45, 46, 89, 98, 110, 114, 116, and 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2.

[0058] In some embodiments, the substitution includes one or more of: D or S at position 3; D at position 5; or A or S at position 8; Q, L, or R at position 19; A or Q at position 45; S, G, or R at position 46; N, R, or G at position 89; D or L at position 98; E or S at position 110; D, H, A, or G at position 114; K or M at position 116; or R or T at position 118.

[0059] In some embodiments, the variant further comprises an amino acid substitution, deletion, insertion, or any combination thereof at one or more of positions 2, 7, 10, 12, 15, 16, 17, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 47, 57, 59, 61, 63, 64, 74, 78, 81, 82, 83, 86, 90, 91, 97, 99, 106, 108, 109, 113, or 123 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2.

[0060] In some embodiments, substitutions include one or more of the following:

[0061] (a) S at position 2;

[0062] (b) D at position 7;

[0063] (c) A, S or K at position 10;

[0064] (d) R, D or N at position 12;

[0065] (e) R at position 15;

[0066] (f) S or D at position 16;

[0067] (g) R at position 17;

[0068] (h) D at position 20;

[0069] (i) F at position 22;

[0070] (j) D at position 24;

[0071] (k) S at position 28;

[0072] (l) Q or K at position 33;

[0073] (m) A at position 35;

[0074] (n) A, T or S at position 37;

[0075] (o) A or S at position 38;

[0076] (p) S at position 40;

[0077] (q) D at position 41;

[0078] (r) K, E or G at position 42;

[0079] (S) S at position 47;

[0080] (t) D at position 57;

[0081] (u) Y at position 59;

[0082] (v) F at position 61;

[0083] (w) S at position 64;

[0084] (x) R at position 74;

[0085] (y) L at position 78;

[0086] (z) A or R at position 81;

[0087] (aa) T at position 82;

[0088] (bb) N or K at position 83;

[0089] (cc) S or D at position 86

[0090] (dd) D at position 90

[0091] (ee) A or S at position 91;

[0092] (ff) I at position 97;

[0093] (gg) M at position 99;

[0094] (hh) K or H at position 106;

[0095] (ii) P, E or D at position 108;

[0096] (jj) D or I at position 113; and

[0097] (kk)D at position 123.

[0098] In some embodiments, the variant comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, located within at least a single epitope recognized by an anti-Ara h 6 antibody.

[0099] In some embodiments, the variant comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, within at least 2 epitopes, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more epitopes recognized by an anti-Ara h 6 antibody.

[0100] In some embodiments, the variant comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 3-21 or SEQ ID NOs: 24-108.

[0101] In some embodiments, the variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 3-21 or SEQ ID NOs: 24-108.

[0102] In some embodiments, the recombinant Ara h 6 variant comprises between 2-30 substitutions, deletions, insertions, or any combination thereof.

[0103] In some embodiments, "SEQ ID NO: 110" and "SEQ ID NO: 109" are used interchangeably herein.

[0104] Also provided herein are nucleotides or modified nucleotide sequences encoding any of the above-mentioned recombinant Ara h 6 variants, expression vectors comprising the nucleotides or modified nucleotide sequences, and cells comprising the expression vectors. Also provided are methods of using the expression vectors to produce any of the above-mentioned recombinant Ara h 6 variants disclosed herein.

[0105] In some embodiments, the nucleotide or modified nucleotide sequence is DNA or mRNA. In some embodiments, mRNA is encapsulated in nanoparticles. In some embodiments, mRNA comprises mRNA formulated with LNPs.

[0106] In some embodiments, the cell comprising the expression vector is a prokaryotic cell or a eukaryotic cell. In some embodiments, the eukaryotic cell is a yeast cell, a fungal cell, a plant cell or a mammalian cell.

[0107] In another aspect, the present disclosure also provides a composition comprising a recombinant Ara h 6 variant polypeptide and / or an isolated nucleotide or modified nucleotide sequence encoding a recombinant Ara h 6 variant disclosed herein. In some embodiments, the composition is a pharmaceutical composition comprising an acceptable carrier or excipient.

[0108] In another aspect, the present disclosure also provides a method of inducing desensitization to peanut and / or immunomodulation of the response to peanut in a subject allergic to peanut, the method comprising administering to the subject a composition comprising a recombinant Ara h6 variant disclosed herein, thereby inducing desensitization to peanut and / or immunomodulation of the response to peanut in the subject.

[0109] In another aspect, the present disclosure also provides a method of inducing desensitization to peanut and / or immunomodulation of the response to peanut in a subject allergic to peanut, the method comprising administering to the subject a composition comprising an isolated nucleotide or modified nucleotide sequence encoding a recombinant Ara h 6 variant disclosed herein, thereby inducing desensitization to peanut and / or immunomodulation of the response to peanut in the subject.

[0110] In another aspect, the disclosure also provides a genetically modified peanut plant expressing a recombinant Ara h 6 variant polypeptide disclosed herein or a combination thereof. In some embodiments, the expression is from a heterologous nucleic acid. In some embodiments, the expression of endogenous wild-type Ara h 6 allergen is reduced compared to non-genetically modified peanuts.

[0111] In another aspect, the present disclosure also provides processed food products comprising the Ara h 6 variants disclosed herein. In some embodiments, the processed food products comprise a reduced amount of endogenous wild-type peanut Ara h 6 allergens. In some embodiments, the processed food products comprise peanuts harvested from the genetically modified plants disclosed herein.

[0112] In some embodiments, the composition is used to induce desensitization to peanut and / or for immunomodulation in a subject in need thereof. In some embodiments, the composition disclosed herein is used to prepare a medicament for inducing desensitization to peanut and / or for immunomodulation in a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] The subject matter of the hypoallergenic polypeptide variants described herein having reduced allergenicity while maintaining immunogenicity and methods of making the same is specifically noted and expressly claimed in the concluding section of the specification. However, the engineered Ara h 6 polypeptide variants and methods of making the same, both as to their structure and methods of operation, as well as their objects, features and advantages, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings, in which:

[0115] Figure 1A and Figure 1B Linear epitope mapping and de-epitoping revealed mutations that abolished binding to the Ara h 6 epitope. Figure 1A : Linear epitope mapping of mAb IgG8 (mAb isolated from a peanut allergic patient) reveals IgE binding to peptides of Ara h 6. The black box highlights the Ara h 6 mapped epitope L2 (peptide derived from positions 61-79 of SEQ ID NO: 1). Figure 1B : Linear de-epitope of the patient mAb IgG8 Ara h 6 epitope. The black box highlights the Figure 1A Same peptide. Grey boxes highlight points where point mutations significantly reduce binding to L2.

[0116] Figure 2 Two major linear epitope regions of IgE binding mapped by peptide array using plasma samples from 80 peanut allergic patients. For each peptide, Ara h 6 peptide binding in patient plasma or serum at the population level was calculated by the relative deviation from the median intensity of the slide (Z-like score). The distribution of all scores from all slides is plotted and shown as a box plot, where the x-axis corresponds to all overlapping peptides and the y-axis shows the distribution of Z-like scores. The black and gray lines represent 2 and 3 standard deviations from the median slide intensity, respectively.

[0117] Figure 3A-3B Expression and purification of Ara h 6 wild type (WT) and epitope-depleted Ara h 6 variant D12 ( Figure 3A ) and Ara h 6 wild type and epitope-depleted Ara h 6 variants D154, D158, D160 and D179 ( Figure 3B ). Ara h6 WT and variants expressed in E. coli and purified by immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) were incubated in Laemmli buffer at 90°C for 5 minutes under reducing conditions. Samples were run on TG-SDS PAGE using 4%-20% polyacrylamide gels and stained with Coomassie. Native Ara h 6 is shown in the first lane for comparison ( Figure 3B ).

[0118] Figure 4A-4F .Purified construct Ara h 6WT ( Figure 4A )、D12( Figure 4B )、D154( Figure 4C )、D158( Figure 4D )、D160( Figure 4E ) and D179( Figure 4F ) variants showed that both purified constructs were stable and ran as monomers under the standard conditions tested. Equal volumes of both samples were injected at ambient temperature into the XBridge Protein BEH SEC on a UHPLC Arc system. Column (2.5 μm, Waters Cat. No. 186009176). Each sample was run at 30°C in a mobile phase of 100 mM sodium phosphate buffer (pH 7.4), 200 mM NaCl at a flow rate of 0.3 mL / min.

[0119] Figure 5Aand Figure 5B .The modified Ara h 6 variants retain high thermal stability. Circular dichroism (CD) analysis of recombinant Ara h 6WT and D12 variants is shown. CD spectroscopy was performed on both intact constructs using a Chirascan v.4.7.0.194 CD spectrometer. Far-UV CD spectra from 190-260 nm were recorded at 15 temperatures from 20°C to 90°C in smooth ramp mode with a slope of 1°C per minute and a step size of 0.5 nm. Ara h 6WT ( Figure 5A ) and the D12 variant ( Figure 5B ) Data at 25°C and increasing temperatures in the range of 20-90°C show similar secondary structure composition of the variants relative to WT, indicating no significant deviation from the native fold.

[0120] Figure 6A-6G .Compared to Ara h 6WT and Ara h 6 native (nAra h 6), the modified Ara h 6 variants showed significantly reduced basophil activation potential. To evaluate the allergenic potential of different Ara h 6 variants, a rat basophilic leukemia (RBL) SX-38 cell degranulation assay was performed. RBL SX-38 cells were sensitized with plasma or serum from allergic patients for 18 hours. Cells were then treated with Ara h 6WT, Ara h 6 native, keyhole limpet hemocyanin (KLH) as a negative control, Ara h 6 variants (D12, D75, D76, D77, D154, D158, D160 or D179) at concentrations ranging from 2ug / ml to 0.02ng / ml for 1 hour. Degranulation was measured using a β-hexosaminidase activity assay. Results are shown for six patient plasma Ara h 6 variants D12, D75, D76 and D77, denoted R560 ( Fig. 6A )、R568( Figure 6B )、CL592( Figure 6C )、A601( Fig.6D )、A604( Fig. 6E ) and A608( Fig. 6F The results for Ara h 6 variants D154, D158, D160 or D179 are shown as the average of the plasma of 11 peanut allergic patients ( Figure 6G ).

[0121] Figure 7Ara h 6WT and de-epitope Ara h 6D12 were expressed, folded and secreted from mammalian cells. 20 ml of Expi293F cells were transfected with 20 μg of plasmid encoding Ara h 6WT or Ara h 6D12 using Expifectamine293 transfection reagent according to the manufacturer's instructions. Both constructs express the human osteonectin leader sequence downstream and contain a C-terminal 6x his tag. The cells were incubated at 37°C, 8% CO 2 Proteins were expressed in Expi293 medium for 5 days. Secreted proteins were purified from the expression medium using Ni-NTA superflow beads, washed and eluted with the addition of 350 mM imidazole. Eluted fractions were analyzed by SDS PAGE reduced or unreduced with β-mercaptoethanol (β-ME).

[0122] Figure 8A-8F .The Ara h 6D12 variant showed reduced binding to anti-Ara h 6 mAbs (4 IgG and 2 IgE). Indirect ELISA titrations of increasing concentrations of anti-Ara h 6 mAbs were used to test binding to WT recombinant Ara h 6 (SEQ ID NO: 2) or modified Ara h 6D12 variants (SEQ ID NO: 9), with KLH used as a negative control. The data presented show that the modified Ara h 6D12 variant showed reduced binding to 2 anti-Ara h 6 IgEs: E15C2 ( Fig. 8A ) and 7B6( Figure 8B ) and 4 anti-Ara h6IgG: IgG5( Figure 8C )、IgG8( Fig.8D )、IgG18( Fig. 8E ) and IgG24( Figure 8F ) was significantly reduced.

[0123] Details

[0124] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the disclosed recombinant Ara h 6 allergen variants and their uses. However, one skilled in the art will appreciate that the described recombinant Ara h 6 allergen variants and their uses can be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail in order to avoid obscuring the described recombinant Ara h 6 variants and their uses of the present invention.

[0125] In some embodiments, recombinant Arah h 6 variants are mutated based on data collected during the epitope mapping process. The mutation sites are selected based on the likelihood of the mutation, alone or in combination with additional mutations, to alter or disrupt one or more epitopes recognized by the anti-Arah 6 antibody. The allergenicity of the Arah h 6 variants is assessed by rat basophilic leukemia (RBL) and peanut allergy patient samples. The desired immunogenicity, i.e., the ability of the engineered Arah 6 to trigger a response of the immune system without triggering a mast cell / basophil-mediated allergic response, is measured by a T cell activation assay.

[0126] The skilled person will understand that the term "epitope" can be used interchangeably with the term "antigenic determinant" having all the same meanings and properties, and can encompass sites on an antigen that specifically binds to an immunoglobulin or antibody (or its antigen-binding fragment). An epitope can be formed by both sequence-continuous amino acids and sequence-discontinuous amino acids, which form a spatially continuous block (patch) by the tertiary folding of the protein. An epitope formed by sequence-continuous amino acids can also be a linear epitope, which is an epitope that binds to an immunoglobulin as a peptide outside the case of a folded protein. Such a linear epitope is usually able to bind to its cognate immunoglobulin after exposure to a denaturing solvent, while an epitope (conformational epitope) formed by tertiary folding usually loses binding after treatment with a denaturing solvent. In some embodiments, the epitope is as small as possible while still maintaining immunogenicity. Immunogenicity is indicated by the ability to elicit an immune response, as described herein, for example, by the ability to bind to MHC class II molecules and induce T cell responses, for example, by measuring T cell cytokine production.

[0127] As used herein, "de-epitoped Ara h 6 allergen" refers to a modified Ara h 6 allergen that has reduced or eliminated binding to an anti-Ara h 6 antibody (compared to an antibody that binds to wild-type Ara h 6) due to mutations at one or more epitopes recognized by the anti-Ara h 6 antibody. In one embodiment, the de-epitoped Ara h 6 allergen has reduced allergenicity compared to its wild-type counterpart.

[0128] As used herein, "epitope" refers to the portion of a macromolecule (e.g., Ara h6 allergen) that is bound by an antibody or antigen-binding fragment thereof. Within a protein sequence, there are linear and continuous epitopes ("linear epitopes"), which contain amino acids that are continuous in sequence, or there are discontinuous epitopes, which contain amino acids that are not continuous in sequence but come together to create a continuous block in the folded protein ("conformational epitopes").

[0129] As used herein, "allergen" refers to a substance, protein or non-protein, that is capable of inducing an allergic or specific hypersensitivity reaction.

[0130] As used herein, "allergenicity" or "allergenic" refers to the ability of an antigen or allergen to induce an abnormal immune response, which is an excessive reaction and differs from a normal immune response in that it does not result in a protective / preventive effect but rather in physiological dysfunction or tissue damage.

[0131] As used herein, "hypoallergenic" refers to a substance that has little or reduced potential to cause an allergic response.

[0132] In some embodiments, the disclosure provides peanut allergen (e.g., Ara h 6) variants that have been mutated to reduce or eliminate binding of one or more epitopes by anti-peanut allergen antibodies. In one embodiment, the mutations do not affect or only minimally affect the biophysical and / or functional characteristics of the peanut allergen. In one aspect, the mutations can be substitutions, deletions, or insertions, or any combination thereof. For example, deletions can include removal of a single amino acid that is critical for antibody binding, or removal of an entire mapped epitope region.

[0133] Six general classes of amino acid side chains have been classified, and include: Class I (Cys); Class II (Ser, Thr, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). A conservative amino acid substitution is one in which an amino acid in one class is replaced by an amino acid in the same class. For example, replacement of Asp by another Class III residue such as Asn, Gln, or Glu is a conservative substitution. A non-conservative amino acid substitution is one in which an amino acid in one class is replaced by an amino acid from another class: for example, Class II residue Ala is replaced by Class III residues such as Asp, Asn, Glu, or Gln. Methods for substitution mutations at the nucleotide or amino acid sequence level are well known in the art.

[0134] As used herein, the term "modifying" or "modification" refers to changing one or more amino acids in an antigen, epitope, allergen, or allergenic region. Such changes can be made by adding, substituting, or deleting amino acids at one or more positions. Such changes can be made using known techniques such as PCR mutagenesis. For example, in some embodiments, an antigen, epitope, allergen, or allergenic region identified using the methods provided herein can be modified to modify (e.g., reduce or eliminate) the binding affinity of the antibody or antigen binding portion thereof to peanut allergen.

[0135] Ara h 6 variant

[0136] In one embodiment, the present disclosure provides a recombinant Ara h 6 variant polypeptide comprising an amino acid sequence that is at least 75%, such as at least 77%, at least 80% or more identical to the sequence set forth in SEQ ID NO: 2, wherein the Ara h 6 variant comprises one or more substitutions, deletions, insertions, or any combination thereof, located within a single epitope recognized by an anti-Ara h 6 antibody. In another embodiment, the Ara h 6 variant comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, located within at least two epitopes recognized by an anti-Ara h 6 antibody.

[0137] As will be appreciated by those skilled in the art, percent identity (% identity) provides a number that describes how similar a query sequence is to a target sequence (i.e., how many amino acids in each sequence are identical). The higher the percent identity, the more significant the match.

[0138] When used in relation to polypeptide (or protein) sequences, the term "identity" refers to the degree of identity between two or more polypeptide (or protein) sequences or fragments thereof. Typically, the degree of similarity between two or more polypeptide (or protein) sequences refers to the degree of similarity in the composition, order or arrangement of two or more amino acids of the two or more polypeptides (or proteins).

[0139] In some embodiments, the variant Ara h 6 polypeptide comprises an amino acid sequence that is at least 70%, at least 75%, at least 77%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a polypeptide disclosed herein, or a portion thereof, as determined using the BlastP software from the National Center for Biotechnology Information (NCBI) using default parameters.

[0140] In some embodiments, the Ara h 6 variant may comprise a deletion, insertion, or amino acid substitution mutation. In one embodiment, the variant polypeptide comprises a conservative substitution, or a deletion, insertion, or substitution that does not significantly change the three-dimensional structure of the polypeptide of interest described herein. In some embodiments, the deletion, insertion, or substitution does not alter the function of the polypeptide of interest disclosed herein. In some embodiments, the deletion, insertion, or substitution does not alter the potential to induce an immune system response and produce desensitization to peanut allergens.

[0141] In some embodiments, the recombinant Ara h 6 variant polypeptide comprises one or more substitutions, deletions, insertions, or any combination thereof. In some embodiments, the recombinant Ara h 6 variant polypeptide comprises between 2-30 substitutions, deletions, insertions, or any combination thereof, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any range therebetween.

[0142] In one embodiment, the recombinant Ara h 6 variant polypeptide comprises the amino acid sequence set forth in SEQ ID NO:22, wherein the variant comprises a substitution, deletion, insertion, or any combination thereof at one or more positions of SEQ ID NO:22 compared to those of the same positions 3, 5, 8, 16, 19, 24, 28, 41, 45, 46, 74, 81, 89, 90, 98, 108, 110, 114, 116, or 118 in SEQ ID NO:2. In one embodiment, the substitution mutation is D or S at position 3. In one embodiment, the substitution mutation is D at position 5. In one embodiment, the substitution mutation is A or S at position 8. In one embodiment, the substitution mutation is S or D at position 16. In one embodiment, the substitution mutation is Q, L, or R at position 19. In one embodiment, the substitution mutation is D at position 24. In one embodiment, the substitution mutation is S at position 28. In one embodiment, the substitution mutation is D at position 41. In one embodiment, the substitution mutation is A or Q at position 45. In one embodiment, the substitution mutation is S, G or R at position 46. In one embodiment, the substitution mutation is R at position 74. In one embodiment, the substitution mutation is A or R at position 81. In one embodiment, the substitution mutation is N, R or G at position 89. In one embodiment, the substitution mutation is D at position 90. In one embodiment, the substitution mutation is D or L at position 98. In one embodiment, the substitution mutation is P, E or D at position 108. In one embodiment, the substitution mutation is E or S at position 110. In one embodiment, the substitution mutation is D, H, A or G at position 114. In one embodiment, the substitution mutation is K or M at position 116. In one embodiment, the substitution mutation is R or T at position 118.

[0143] In some embodiments of the above-described recombinant Ara h 1 variants, the Ara h 1 variant comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 substitution mutations at at least one position selected from position 3, 5, 8, 16, 19, 24, 28, 41, 45, 46, 74, 81, 89, 90, 98, 108, 110, 114, 116, or 118 of SEQ ID NO: 22 compared to the amino acid residues at those same positions in SEQ ID NO: 2.

[0144] In some embodiments of the above-described recombinant Ara h 6 variants, the Ara h 6 variant further comprises at least one additional substitution, deletion, insertion, or any combination thereof at one or more of positions 2, 7, 10, 12, 15, 17, 20, 22, 35, 37, 38, 40, 42, 47, 57, 59, 61, 64, 78, 82, 83, 86, 91, 97, 99, 113, or 123 of SEQ ID NO: 22 compared to the amino acid residues at those same positions in SEQ ID NO: 2. In one embodiment, the substitution mutation is S at position 2. In one embodiment, the substitution mutation is D at position 7. In one embodiment, the substitution mutation is A, S, or K at position 10. In one embodiment, the substitution mutation is R, D, or N at position 12. In one embodiment, the substitution mutation is R at position 15. In one embodiment, the substitution mutation is R at position 17. In one embodiment, the substitution mutation is D at position 20. In one embodiment, the substitution mutation is F at position 22. In one embodiment, the substitution mutation is A at position 35. In one embodiment, the substitution mutation is A, T or S at position 37. In one embodiment, the substitution mutation is A or S at position 38. In one embodiment, the substitution mutation is S at position 40. In one embodiment, the substitution mutation is K, E or G at position 42. In one embodiment, the substitution mutation is S at position 47. In one embodiment, the substitution mutation is D at position 57. In one embodiment, the substitution mutation is Y at position 59. In one embodiment, the substitution mutation is F at position 61. In one embodiment, the substitution mutation is S at position 64. In one embodiment, the substitution mutation is L at position 78. In one embodiment, the substitution mutation is T at position 82. In one embodiment, the substitution mutation is N or K at position 83. In one embodiment, the substitution mutation is D or S at position 86. In one embodiment, the substitution mutation is A or S at position 91. In one embodiment, the substitution mutation is I at position 97. In one embodiment, the substitution mutation is M at position 99. In one embodiment, the substitution mutation is D or I at position 113. In one embodiment, the substitution mutation is D at position 123.

[0145] In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant further comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 substitution mutations at positions selected from position 2, 7, 10, 12, 15, 17, 20, 22, 35, 37, 38, 40, 42, 47, 57, 59, 61, 64, 78, 82, 83, 86, 91, 97, 99, 113, or 123 of SEQ ID NO: 22 compared to the amino acid residues at those same positions in SEQ ID NO: 2.

[0146] In one embodiment, the recombinant Ara h 6 variant polypeptide comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof at one or more of positions 2, 3, 5, 7, 8, 10, 12, 16, 19, 22, 24, 33, 37, 38, 40, 41, 42, 45, 46, 47, 63, 74, 78, 81, 82, 83, 86, 89, 90, 97, 98, 99, 106, 108, 109, 110, 113, 114, 116, or 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2.

[0147] In one embodiment, the one or more amino acid substitutions comprise one or more of: S at position 2; D or S at position 3; D at position 5; D at position 7; A or S at position 8; A, S or K at position 10; R, D or N at position 12; S or D at position 16; Q, L or R at position 19; F at position 22; D at position 24; Q or K at position 33; A, T or S at position 37; A or S at position 38; S at position 40; D at position 41; K, E or G at position 42; A or Q at position 45; S, G or R at position 46; S at position 47; R at position 74; L at position 78; A or R at position 81; T at position 82; N or K at position 83; D or S at position 86; N, R or G at position 89; D at position 90; I at position 97; D or L at position 98; M at position 99; K or H at position 106; P, E or D at position 108; E or S at position 110; D or I at position 113; D, H, A or G at position 114; K or M at position 116; or R or T at position 118.

[0148] In one embodiment, the substitution mutation is any amino acid at position 63. In one embodiment, the substitution mutation is any amino acid at position 109.

[0149] In one embodiment, the substitution mutation is S at position 2. In one embodiment, the substitution mutation is D or S at position 3. In one embodiment, the substitution mutation is D at position 5. In one embodiment, the substitution mutation is D at position 7. In one embodiment, the substitution mutation is A or S at position 8. In one embodiment, the substitution mutation is A, S or K at position 10. In one embodiment, the substitution mutation is R, D or N at position 12. In one embodiment, the substitution mutation is S or D at position 16. In one embodiment, the substitution mutation is Q, L or R at position 19. In one embodiment, the substitution mutation is F at position 22. In one embodiment, the substitution mutation is D at position 24. In one embodiment, the substitution mutation is Q or K at position 33. In one embodiment, the substitution mutation is A, T or S at position 37. In one embodiment, the substitution mutation is A or S at position 38. In one embodiment, the substitution mutation is S at position 40. In one embodiment, the substitution mutation is D at position 41. In one embodiment, the substitution mutation is K, E or G at position 42. In one embodiment, the substitution mutation is A or Q at position 45. In one embodiment, the substitution mutation is S, G or R at position 46. In one embodiment, the substitution mutation is S at position 47. In one embodiment, the substitution mutation is R at position 74. In one embodiment, the substitution mutation is L at position 78. In one embodiment, the substitution mutation is A or R at position 81. In one embodiment, the substitution mutation is T at position 82. In one embodiment, the substitution mutation is N or K at position 83. In one embodiment, the substitution mutation is D or S at position 86. In one embodiment, the substitution mutation is N, R or G at position 89. In one embodiment, the substitution mutation is D at position 90. In one embodiment, the substitution mutation is I at position 97. In one embodiment, the substitution mutation is D or L at position 98. In one embodiment, the substitution mutation is M at position 99. In one embodiment, the substitution mutation is K or H at position 106. In one embodiment, the substitution mutation is P, E or D at position 108. In one embodiment, the substitution mutation is E or S at position 110. In one embodiment, the substitution mutation is D or I at position 113. In one embodiment, the substitution mutation is D, H, A or G at position 114. In one embodiment, the substitution mutation is K or M at position 116. In one embodiment, the substitution mutation is R or T at position 118.

[0150] In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 of the sequences selected from SEQ ID Substitution mutations at position 2, 3, 5, 7, 8, 10, 12, 16, 19, 22, 24, 33, 37, 38, 40, 41, 42, 45, 46, 47, 63, 74, 78, 81, 82, 83, 86, 89, 90, 97, 98, 99, 106, 108, 109, 110, 113, 114, 116 or 118 of NO: 109 compared to the amino acid residues at the same positions in SEQ ID NO: 2.

[0151] In one embodiment, the recombinant Ara h 6 variant polypeptide comprises an amino acid substitution, deletion, insertion, or any combination thereof at positions 3, 5, 8, 19, 45, 46, 89, 98, 110, 114, 116, and 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2.

[0152] In one embodiment, the one or more amino acid substitutions comprise one or more of: D or S at position 3; D at position 5; A or S at position 8; Q, L or R at position 19; A or Q at position 45; S, G or R at position 46; N, R or G at position 89; D or L at position 98; E or S at position 110; D, H, A or G at position 114; K or M at position 116; or R or T at position 118.

[0153] In one embodiment, a recombinant Ara h 6 variant polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 109, wherein the variant comprises a substitution, deletion, insertion, or any combination thereof, at one or more of positions 3, 5, 8, 19, 45, 46, 86, 89, 90, 98, 106, 108, 110, 114, 116, or 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2.

[0154] In some embodiments of the above-described recombinant Ara h 6 variants, the Ara h 6 variant comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 substitution mutations at positions 3, 5, 8, 19, 45, 46, 86, 89, 90, 98, 106, 108, 110, 114, 116, or 118 selected from SEQ ID NO: 110 compared to the amino acid residues at those same positions in SEQ ID NO: 2.

[0155] In some embodiments, the recombinant Ara h 6 variant further comprises an amino acid substitution, deletion, insertion, or any combination thereof at one or more of positions 2, 7, 10, 12, 15, 16, 17, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 47, 57, 59, 61, 63, 64, 74, 78, 81, 82, 83, 86, 90, 91, 97, 99, 106, 108, 109, 113, or 123 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2.

[0156] In some embodiments, the one or more amino acid substitutions comprise one or more of: S at position 2; D at position 7; A, S, or K at position 10; R, D, or N at position 12; R at position 15; S or D at position 16; R at position 17; D at position 20; F at position 22; D at position 24; S at position 28; Q or K at position 33; A at position 35; A, T, or S at position 37; A or S at position 38; S at position 40; D at position 41; K, E or G at position 42; S at position 47; D at position 57; Y at position 59; F at position 61; S at position 64; R at position 74; L at position 78; A or R at position 81; T at position 82; N or K at position 83; S or D at position 86; D at position 90; A or S at position 91; I at position 97; M at position 99; K or H at position 106; P, E or D at position 108; D or I at position 113; and D at position 123.

[0157] In one embodiment, the substitution mutation is any amino acid at position 63. In one embodiment, the substitution mutation is any amino acid at position 109.

[0158] In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant further comprises an additional substitution, deletion, insertion, or any combination thereof at one or more of positions 2, 7, 10, 12, 15, 16, 17, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 47, 57, 59, 61, 64, 74, 78, 81, 82, 83, 91, 97, 99, 113, or 123 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2. In one embodiment, the substitution mutation is S at position 2. In one embodiment, the substitution mutation is D at position 7. In one embodiment, the substitution mutation is A, S, or K at position 10. In one embodiment, the substitution mutation is R, D, or N at position 12. In one embodiment, the substitution mutation is R at position 15. In one embodiment, the substitution mutation is S or D at position 16. In one embodiment, the substitution mutation is R at position 17. In one embodiment, the substitution mutation is D at position 20. In one embodiment, the substitution mutation is F at position 22. In one embodiment, the substitution mutation is D at position 24. In one embodiment, the substitution mutation is S at position 28. In one embodiment, the substitution mutation is Q or K at position 33. In one embodiment, the substitution mutation is A at position 35. In one embodiment, the substitution mutation is A, T or S at position 37. In one embodiment, the substitution mutation is A or S at position 38. In one embodiment, the substitution mutation is S at position 40. In one embodiment, the substitution mutation is D at position 41. In one embodiment, the substitution mutation is K, E or G at position 42. In one embodiment, the substitution mutation is S at position 47. In one embodiment, the substitution mutation is D at position 57. In one embodiment, the substitution mutation is Y at position 59. In one embodiment, the substitution mutation is F at position 61. In one embodiment, the substitution mutation is S at position 64. In one embodiment, the substitution mutation is R at position 74. In one embodiment, the substitution mutation is L at position 78. In one embodiment, the substitution mutation is A or R at position 81. In one embodiment, the substitution mutation is T at position 82. In one embodiment, the substitution mutation is N or K at position 83. In one embodiment, the substitution mutation is A or S at position 91. In one embodiment, the substitution mutation is I at position 97. In one embodiment, the substitution mutation is M at position 99. In one embodiment, the substitution mutation is D or I at position 113. In one embodiment, the substitution mutation is D at position 123.

[0159] In some embodiments of the above recombinant Ara h 2 variants, the Ara h 6 variant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 of the sequences selected from SEQ ID NO: Substitution mutations at positions 2, 7, 10, 12, 15, 16, 17, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 47, 57, 59, 61, 64, 74, 78, 81, 82, 83, 91, 97, 99, 113 or 123 of NO:109 compared to the amino acid residues at those same positions in SEQ ID NO:2.

[0160] In one embodiment, the recombinant Ara h 6 variant polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 109, wherein the variant comprises a substitution, deletion, insertion, or any combination thereof at one or more of positions 2, 3, 5, 7, 8, 10, 12, 15, 16, 17, 19, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 45, 46, 47, 57, 59, 61, 64, 74, 78, 81, 82, 83, 86, 89, 90, 91, 97, 98, 99, 106, 108, 110, 113, 114, 116, 118, or 123 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2. In one embodiment, the substitution mutation is S at position 2. In one embodiment, the substitution mutation is D or S at position 3. In one embodiment, the substitution mutation is D at position 5. In one embodiment, the substitution mutation is D at position 7. In one embodiment, the substitution mutation is A or S at position 8. In one embodiment, the substitution mutation is A, S or K at position 10. In one embodiment, the substitution mutation is R, D or N at position 12. In one embodiment, the substitution mutation is R at position 15. In one embodiment, the substitution mutation is S or D at position 16. In one embodiment, the substitution mutation is R at position 17. In one embodiment, the substitution mutation is Q, L or R at position 19. In one embodiment, the substitution mutation is D at position 20. In one embodiment, the substitution mutation is F at position 22. In one embodiment, the substitution mutation is D at position 24. In one embodiment, the substitution mutation is S at position 28. In one embodiment, the substitution mutation is Q or K at position 33. In one embodiment, the substitution mutation is A at position 35. In one embodiment, the substitution mutation is A, T or S at position 37. In one embodiment, the substitution mutation is A or S at position 38. In one embodiment, the substitution mutation is S at position 40. In one embodiment, the substitution mutation is D at position 41. In one embodiment, the substitution mutation is K, E or G at position 42. In one embodiment, the substitution mutation is A or Q at position 45. In one embodiment, the substitution mutation is S, G or R at position 46. In one embodiment, the substitution mutation is S at position 47. In one embodiment, the substitution mutation is D at position 57. In one embodiment, the substitution mutation is Y at position 59. In one embodiment, the substitution mutation is F at position 61. In one embodiment, the substitution mutation is S at position 64. In one embodiment, the substitution mutation is R at position 74. In one embodiment, the substitution mutation is L at position 78.In one embodiment, the substitution mutation is A or R at position 81. In one embodiment, the substitution mutation is T at position 82. In one embodiment, the substitution mutation is N or K at position 83. In one embodiment, the substitution mutation is D or S at position 86. In one embodiment, the substitution mutation is N, R or G at position 89. In one embodiment, the substitution mutation is D at position 90. In one embodiment, the substitution mutation is A or S at position 91. In one embodiment, the substitution mutation is I at position 97. In one embodiment, the substitution mutation is D or L at position 98. In one embodiment, the substitution mutation is M at position 99. In one embodiment, the substitution mutation is K or H at position 106. In one embodiment, the substitution mutation is P, E or D at position 108. In one embodiment, the substitution mutation is E or S at position 110. In one embodiment, the substitution mutation is D or I at position 113. In one embodiment, the substitution mutation is D, H, A or G at position 114. In one embodiment, the substitution mutation is K or M at position 116. In one embodiment, the substitution mutation is R or T at position 118. In one embodiment, the substitution mutation is D at position 123.

[0161] In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 of the sequences selected from SEQ ID NO: NO:109 at position 2, 3, 5, 7, 8, 10, 12, 15, 16, 17, 19, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 45, 46, 47, 57, 59, 61, 64, 74, 78, 81, 82, 83, 86, 89, 90, 91, 97, 98, 99, 106, 108, 110, 113, 114, 116, 118 or 123, a substitution mutation compared to the amino acid residues at those same positions in SEQ ID NO:2.

[0162] In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 of the sequences selected from SEQ ID NO:109 at position 2, 3, 5, 7, 8, 10, 12, 15, 16, 17, 19, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 45, 46, 47, 57, 59, 61, 63, 64, 74, 78, 81, 82, 83, 86, 89, 90, 91, 97, 98, 99, 106, 108, 109, 110, 113, 114, 116, 118 or 123, a substitution mutation compared to the amino acid residues at those same positions in SEQ ID NO:2.

[0163] In some embodiments, the recombinant Ara h 6 variant polypeptide comprises between 2-51 substitutions, deletions, insertions, or any combination thereof, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or any range therebetween.

[0164] In some embodiments of the above-described recombinant Ara h 6 variants, the Ara h 6 variant comprises at least one or more amino acid substitutions, deletions, insertions, or any combination thereof at one or more of positions 15, 16, 17, 19, 20, 22, 24, 28, 57, 59, 61, 64, 74, 78, 81, 82, 83, 86, 98, or 116 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2.

[0165] In some embodiments, the variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 3-21.

[0166] In some embodiments, the variant comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 24-108.

[0167] In some embodiments of the above-described recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3-21 or comprises an amino acid sequence that is at least 80% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 3-21.

[0168] In some embodiments of the above-described recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3-21 or comprises an amino acid sequence that is at least 77% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 3-21.

[0169] In some embodiments of the above-described recombinant Ara h 6 variants, the Ara h 6 variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 3-21 or SEQ ID NOs: 24-108.

[0170] In some embodiments of the above-described recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid sequence that is at least 70%, at least 75%, at least 77%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 3-21 or SEQ ID NOs: 24-108.

[0171] In some embodiments of the above-described recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 24-108 or comprises an amino acid sequence that is at least 80% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 24-108.

[0172] In some embodiments of the above-described recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 24-108 or comprises an amino acid sequence that is at least 77% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 24-108.

[0173] In some embodiments, the recombinant Ara h 6 variant comprises the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the recombinant Ara h 6 variant comprises the amino acid sequence set forth in SEQ ID NO:46. In some embodiments, the recombinant Ara h 6 variant comprises the amino acid sequence set forth in SEQ ID NO:76. In some embodiments, the recombinant Ara h 6 variant comprises the amino acid sequence set forth in SEQ ID NO:80. In some embodiments, the recombinant Ara h 6 variant comprises the amino acid sequence set forth in SEQ ID NO:82. In some embodiments, the recombinant Ara h 6 variant comprises the amino acid sequence set forth in SEQ ID NO:101. In some embodiments, the recombinant Ara h 6 variant comprises the amino acid sequence set forth in SEQ ID NO:108.

[0174] In some embodiments of the above-described recombinant Ara h 6 variants, the basophil degranulation release induced by the variant is at least 10-fold lower than the release induced by the Ara h 6 wild-type polypeptide.

[0175] In some embodiments of the above recombinant Ara h 6 variants, the EC50 or KD of the recombinant Arah 6 variant binding to at least one patient-derived Ara h 6 IgE is reduced by 50% or more compared to the Ara h 6 wild-type polypeptide.

[0176] In some embodiments, the recombinant Ara h 6 variants described above comprise one or more amino acid substitutions, deletions, insertions, or any combination thereof located within at least a single epitope recognized by an anti-Ara h 6 antibody.

[0177] In some embodiments, the Ara h 6 epitope comprises a linear epitope (L1) comprising the amino acids at positions 2-14 of SEQ ID NO:2. In some embodiments, the Ara h 6 epitope comprises a linear epitope (L2) comprising the amino acids at positions 36-54 of SEQ ID NO:2. In some embodiments, the Ara h 6 epitope comprises a linear epitope (L3) comprising the amino acids at positions 76-90 of SEQ ID NO:2. In some embodiments, the Ara h 6 epitope comprises a linear epitope (L4) comprising the amino acids at positions 92-102 of SEQ ID NO:2. In some embodiments, the Ara h 6 epitope comprises a linear epitope (L5) comprising the amino acids at positions 104-118 of SEQ ID NO:2.

[0178] In some embodiments, the Ara h 6 epitope comprises a conformational epitope (C1) comprising amino acids at positions 16, 19, 74, and 81 of SEQ ID NO:2. In some embodiments, the Ara h 6 epitope comprises a conformational epitope (C2) comprising amino acids at positions 19, 22, and 24 of SEQ ID NO:2. In some embodiments, the Ara h 6 epitope comprises a conformational epitope (C3) comprising amino acids at positions 33 and 106 of SEQ ID NO:2. In some embodiments, the Ara h 6 epitope comprises a conformational epitope (C4) comprising amino acids at positions 63, 108, and 109 of SEQ ID NO:2. In some embodiments, the Ara h 6 epitope comprises a conformational epitope (C5) comprising amino acids at positions 114 and 116 of SEQ ID NO:2.

[0179] In some embodiments of the above-mentioned recombinant Ara h 6 variants, the Ara h 6 variant comprises at least one, for example, at least two or more amino acid substitutions, deletions, insertions, or any combination thereof, located within at least one epitope. In some embodiments of the above-mentioned recombinant Ara h 6 variants, the Ara h 6 variant comprises at least two amino acid substitutions, deletions, insertions, or any combination thereof, located within at least one conformational epitope selected from C1, C2, C3, C4, or C5. In some embodiments of the above-mentioned recombinant Ara h 6 variants, the Ara h 6 variant comprises at least two amino acid substitutions, deletions, insertions, or any combination thereof, located within at least two conformational epitopes selected from C1, C2, C3, C4, or C5. In some embodiments, the conformational epitope (C1) comprises amino acids located at positions 16, 19, 74, and 81 of SEQ ID NO: 2. In some embodiments, the conformational epitope (C2) comprises amino acids located at positions 19, 22, and 24 of SEQ ID NO: 2. In some embodiments, the conformational epitope (C3) comprises amino acids located at positions 33 and 106 of SEQ ID NO: 2. In some embodiments, the conformational epitope (C4) comprises the amino acids at positions 63, 108, and 109 of SEQ ID NO: 2. In some embodiments, the conformational epitope (C5) comprises the amino acids at positions 114 and 116 of SEQ ID NO:2.

[0180] In some embodiments of the above-mentioned recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid substitution, deletion, insertion, or any combination thereof at positions 3, 5, 8, 19, 45, 46, 89, 98, 110, 114, 116, and 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2. In some embodiments, the variant comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in any one of SEQ ID NO: 46, 76, 80, 82, 101. In some embodiments, the variant comprises an amino acid sequence that is at least 77% identical to the amino acid sequence set forth in any one of SEQ ID NO: 46, 76, 80, 82, 101. In some embodiments, the variant comprises an amino acid sequence that is identical to the amino acid sequence set forth in any one of SEQ ID NO: 46, 76, 80, 82, 101. In some embodiments, the Ara h 6 variant comprises a conformational epitope recognized by an anti-Ara h 6 IgG antibody. In some embodiments, the Ara h 6 variant has reduced or eliminated binding to anti-Ara h 6 IgE antibodies, for example within a conformational epitope. In some embodiments, reduction refers to a reduction in binding affinity of the variant by at least 10-fold compared to the binding of native or wild-type Ara h 6 (SEQ ID NOs: 1 and 2, respectively).

[0181] In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid substitution at position 3, 5, 8, 19, 35, 37, 38, 45, 46, 89, 90, 98, 108, 110, 114, 116, 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2. In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 46.

[0182] In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid substitution at position 3, 5, 8, 19, 35, 37, 38, 45, 46, 86, 89, 90, 98, 106, 108, 110, 114, 116, 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2. In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 76.

[0183] In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid substitution at position 3, 5, 8, 19, 45, 46, 86, 89, 90, 98, 106, 108, 110, 114, 116, 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2. In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 80.

[0184] In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid substitution at position 3, 5, 8, 19, 37, 45, 46, 86, 89, 90, 98, 106, 108, 110, 114, 116, 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2. In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 82.

[0185] In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid substitution at position 3, 5, 8, 19, 45, 46, 86, 89, 98, 106, 110, 114, 116, 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO: 2. In some embodiments of the above recombinant Ara h 6 variants, the Ara h 6 variant comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 101.

[0186] In some embodiments of the above-mentioned recombinant Ara h 6 variants, the Ara h 6 variant comprises a methionine located upstream of the N-terminus of the amino acid sequence (or protein sequence) described herein. In this embodiment, the methionine thus forms the N-terminus of the amino acid sequence. Such methionine is generally derived from the translation of protein-coding RNA, where it is encoded by the nucleic acid triplet ATG. Unless the ATG triplet is already present in the nucleic acid sequence, the ATG triplet can be attached to the 5' end of the nucleic acid molecule by methods known to those skilled in the art.

[0187] In some embodiments of Ara h 6 variants, the amino acid sequence set forth in any one of SEQ ID NOs: 3-21 further comprises a methionine located upstream of the N-terminus of the amino acid sequence set forth in any one of SEQ ID NOs: 3-21. In some embodiments of Ara h 6 variants, the amino acid sequence set forth in any one of SEQ ID NOs: 24-108 further comprises a methionine located upstream of the N-terminus of the amino acid sequence set forth in any one of SEQ ID NOs: 24-108.

[0188] In some embodiments of Ara h 6 variants, the amino acid sequence further comprises a tag or label (purification tag or stability tag) at its N-terminus or at its C-terminus. In some embodiments, the tag is selected from a His tag, an HA tag, or other suitable tags known in the art.

[0189] In some embodiments, "SEQ ID NO: 110" and "SEQ ID NO: 109" are used interchangeably herein.

[0190] Nucleotides, vectors and host cells

[0191] In one embodiment, the present disclosure provides an isolated nucleotide or modified nucleotide sequence encoding a recombinant Ara h 6 variant described in detail herein. In some embodiments, the nucleotide or modified nucleotide sequence is DNA or mRNA. In some embodiments of the nucleotide or modified nucleotide sequence, the mRNA comprises an mRNA formulated with a liquid nanoparticle (LNP).

[0192] In one embodiment, the present disclosure provides an expression vector comprising an isolated nucleotide or modified nucleotide sequence described herein.

[0193] In one embodiment, the disclosure provides a prokaryotic cell or a eukaryotic cell comprising an expression vector described herein. In some embodiments, the eukaryotic cell is a yeast cell, a fungal cell, a plant cell, or a mammalian cell.

[0194] In one embodiment, the present disclosure provides a composition comprising a recombinant Ara h 6 variant polypeptide as described in detail herein.

[0195] As used herein, the terms "nucleotide", "nucleotide sequence" or "nucleic acid molecule" are intended to include DNA molecules and RNA molecules or modified RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded. In some embodiments, nucleotides include modified nucleotides. In some embodiments, nucleotides include mRNA. In some embodiments, nucleotides include modified mRNA. In some embodiments, nucleotides include modified mRNA, wherein the modified mRNA includes 5'-capped mRNA. In some embodiments, the modified mRNA includes molecules in which some nucleosides have been replaced by naturally modified nucleosides or synthetic nucleosides. In some embodiments, modified nucleotides include modified mRNA, the modified mRNA includes 5'-capped mRNA, and some of the nucleosides have been replaced by naturally modified nucleosides or synthetic nucleosides.

[0196] As used herein, the term "isolated nucleotide" or "isolated nucleic acid molecule" refers to a nucleic acid encoding a peanut allergen variant disclosed herein (e.g., Ara h 6 variant), wherein the nucleotide sequence is substantially free of other genomic nucleotide sequences that naturally flank the nucleic acid in genomic DNA.

[0197] In one aspect, disclosed herein are nucleotide or nucleic acid sequences encoding the peanut allergen variants disclosed herein (eg, Ara h 6 variants).

[0198] As used herein, the term "vector" refers to a discrete element for introducing a heterologous nucleic acid into a cell for its expression or replication. Expression vectors include vectors capable of expressing nucleic acids, which are operably linked to regulatory sequences such as promoter regions that can affect the expression of such nucleic acids. Therefore, expression vectors can refer to DNA or RNA constructs, such as plasmids, phages, recombinant viruses or other vectors, which, when introduced into appropriate host cells, result in the expression of nucleic acids. Suitable expression vectors are well known to those skilled in the art, and include those that are replicable in prokaryotic cells and / or eukaryotic cells, as well as those that remain episomal or those that are integrated into the host cell genome.

[0199] In one aspect, disclosed herein are expression vectors comprising a nucleic acid construct encoding a peanut allergen variant disclosed herein (eg, an Ara h 6 variant).

[0200] The term "recombinant host cell" (or simply "host cell") as used herein refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the specific subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0201] In one aspect, disclosed herein is a host cell comprising an expression vector carrying a nucleic acid construct encoding a peanut allergen variant disclosed herein (e.g., an Ara h 6 variant). In one embodiment, the cell or host cell is a prokaryotic cell or a eukaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, a fungal cell, an algae cell, a plant cell, or a mammalian cell. In some embodiments, the peanut allergen variant can be produced in bacteria such as Escherichia coli. In some other embodiments, the peanut allergen variant can be produced in yeast or fungi, such as Saccharomyces cerevisiae, Aspergillus, Trichoderma, or Pichia pastoris.

[0202] Nucleic acid encoding Ara h 6 variant

[0203] In one embodiment, provided herein is a nucleic acid or modified nucleic acid molecule encoding a recombinant Ara h 6 variant polypeptide comprising an amino acid sequence at least 80% identical to the sequence set forth in SEQ ID NO: 2, wherein the Ara h 6 variant comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof, located within a single epitope recognized by an anti-Ara h 1 antibody.

[0204] In another embodiment, the nucleic acid or modified nucleic acid molecule encodes a recombinant Ara h 6 variant comprising an amino acid sequence at least 80% identical to the sequence set forth in SEQ ID NO: 2, wherein the Ara h 6 variant comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof located within at least two epitopes recognized by anti-Ara h 2 antibodies.

[0205] As will be appreciated by those skilled in the art, percent identity (% identity) provides a number that describes how similar a query sequence is to a target sequence (i.e., how many amino acids in each sequence are identical). The higher the percent identity, the more significant the match.

[0206] When used in relation to polypeptide (or protein) sequences, the term "identity" refers to the degree of identity between two or more polypeptide (or protein) sequences or fragments thereof. Typically, the degree of similarity between two or more polypeptide (or protein) sequences refers to the degree of similarity in the composition, order or arrangement of two or more amino acids of the two or more polypeptides (or proteins).

[0207] In some embodiments, the variant Ara h 6 polypeptide comprises an amino acid sequence that is at least 70%, at least 75%, at least 77%, at least 80%, at least 85%, or at least 90% identical to the amino acid sequence SEQ ID NO: 2 disclosed herein, or a portion thereof, as determined using the BlastP software from the National Center for Biotechnology Information (NCBI) using default parameters.

[0208] In some embodiments, the Ara h 6 variants described herein may comprise deletions, insertions, or amino acid substitution mutations. In one embodiment, the variant polypeptide comprises a conservative substitution, or a deletion, insertion, or substitution that does not significantly change the three-dimensional structure of the polypeptide of interest described herein. In some embodiments, the deletion, insertion, or substitution does not alter the function of the polypeptide of interest disclosed herein. In some embodiments, the deletion, insertion, or substitution does not alter the potential to induce an immune system response and produce desensitization to peanut allergens.

[0209] In one embodiment, the nucleic acid or modified nucleic acid is DNA or mRNA. In one embodiment, the mRNA comprises a UTR, or the mRNA comprises a leader sequence, or the mRNA comprises a UTR and a leader sequence. In one embodiment, the UTR comprises a chimeric sequence or a new sequence, which may be superior to the native UTR sequence, promoting overall higher protein expression.

[0210] In one embodiment, the mRNA comprises an optimized sequence. As used herein, an "optimized sequence" includes an mRNA sequence comprising a computationally altered nucleotide sequence that promotes higher expression levels in human cells compared to the unaltered sequence while maintaining features that facilitate in vitro transcription (IVT) and enzymatic capping.

[0211] In one embodiment, a nucleic acid or modified nucleic acid molecule disclosed herein encodes an Ara h 6 variant comprising an amino acid sequence set forth in any one of SEQ ID NOs: 3-21 or comprising an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 3-21.

[0212] In one embodiment, a nucleic acid or modified nucleic acid molecule disclosed herein encodes an Ara h 6 variant comprising an amino acid sequence set forth in any one of SEQ ID NOs: 24-108, or comprising an amino acid sequence having at least 80% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 24-108.

[0213] In some embodiments, the nucleic acid or modified nucleic acid molecules disclosed herein further comprise a nucleic acid sequence encoding a marker or tag (purification tag or stability tag) at the 5' or 3' end. In some embodiments, the tag is selected from a His tag, an HA tag, or other tags known in the art. In some embodiments, the nucleic acid or modified nucleic acid molecules disclosed herein further comprise a restriction endonuclease sequence.

[0214] Generation method

[0215] In some embodiments, the variant polypeptides disclosed herein can be produced using a cell-free in vitro translation system, as is well known in the art, such as, but not limited to, the methods reviewed in Dondapati et al. (2020) BioDrugs 34(3):327-348. In one embodiment, the present disclosure provides a method for producing a hypoallergenic peanut allergen comprising an Ara h 6 variant disclosed herein, the method comprising culturing a cell comprising an expression vector described above under conditions that express the Ara h 6 variant. In one embodiment, the cell is a prokaryotic cell or a eukaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, a fungal cell, a plant cell, or a mammalian cell.

[0216] In some embodiments, the nucleic acids or modified nucleic acid molecules disclosed herein are transcribed in an in vitro transcription system (IVT), wherein the transcribed nucleic acids or modified nucleic acids can then be used for immunotherapy by gene delivery, wherein administration of the mRNA results in the in vivo production of peanut allergens or peanut allergen variants.

[0217] In some embodiments of the production methods, the nucleic acid molecule encodes a variant Ara h 6 polypeptide comprising one or more amino acid substitutions, deletions, insertions, or any combination thereof, located within a single epitope recognized by an anti-Ara h 1 antibody. In some embodiments, the nucleic acid comprises a modified nucleic acid encoding a variant Ara h 6 polypeptide comprising one or more amino acid mutations located within a single epitope recognized by an anti-Ara h 2 antibody.

[0218] Synthesis and capping of RNA molecules by chemical synthesis or by enzymatic processes such as bacteriophage RNA polymerase are recognized methods in the art for mRNA production as described in Elain T. Schenborn Methods in Molecular Biology, Vol. 37: In Vitro Transcript / on and Translation Protocols, pp. 1-12 DOI: 10.1385 / 0-89603-288-4:1.

[0219] Those skilled in the art will appreciate that other known IVT systems can be used to transcribe the nucleic acids or modified nucleic acid molecules described herein. In some embodiments, an mRNA molecule is transcribed in vitro using an IVT system.

[0220] The production of the peanut allergen variant, Ara h 6 variant, may involve in vivo translation, wherein the transcribed mRNA is administered to a subject (in vivo translation).

[0221] In some embodiments, the nucleic acids or modified nucleic acid molecules disclosed herein can be used to produce peanut allergen variant polypeptides in vivo, including administration of the nucleic acid or modified nucleic acid molecules to a subject by viral, non-viral, or physical means such as liposomes, cationic lipids, cationic polymers, or mixed lipid polymer systems, retroviral or DNA viral delivery, e.g., lentivirus, foamyviral, adenovirus, etc., sonoporation, electroporation, hydrodynamic delivery. In some embodiments, the nucleic acid molecules disclosed herein can be used to produce peanut allergen WT polypeptides in vivo, including administration of the nucleic acid molecules to a subject by viral, non-viral, or physical means such as liposomes, cationic lipids, cationic polymers, or mixed lipid polymer systems, retroviral or DNA viral delivery, e.g., lentivirus, foamyviral, adenovirus, etc., sonoporation, electroporation, hydrodynamic delivery. Methods for in vivo administration of nucleic acid molecules, such as mRNA molecules encoding Ara h 6 variants described herein, are well known in the art, such as, but not limited to, the methods reviewed in Jones et al., Overcoming Nonviral Gene Delivery Barriers: Perspective and Future. Mol. Pharmaceutics 2013, 10, 11, 4082-4098; Kamimura et al., Advances in Gene Delivery Systems. Pharmaceut Med. 25(5): 293-306; and Nayerossadat et al., Viral and nonviral delivery systems for gene delivery. Adv Biomed Res 2012; 1: 27, which are incorporated herein in their entireties.

[0222] In some embodiments, the subject comprises a human subject. In certain embodiments, the subject comprises an infant, a child, a teenager, a young adult, or a mature adult. In some embodiments, the subject comprises an infant.

[0223] In some embodiments, the subject includes a subject in need of induction of desensitization to peanut. In some embodiments, the subject is allergic to peanuts. In some embodiments, the subject suffers from other food allergies. In some embodiments, the subject may be prone to peanut allergies.

[0224] How to use

[0225] In one embodiment, the disclosure provides a method of inducing desensitization to peanut in a subject allergic to peanut, the method comprising administering to the subject a composition comprising a hypoallergenic Ara h 6 variant disclosed herein, thereby increasing the subject's ability to tolerate peanut.

[0226] In one embodiment, the disclosure provides a method of inducing immunomodulation of a response to peanut in a subject allergic to peanut, the method comprising administering to the subject a composition comprising a hypoallergenic Ara h 6 variant disclosed herein, thereby increasing the subject's ability to tolerate peanut.

[0227] As used herein, allergy desensitization to peanut or desensitization to peanut, also referred to as allergy immunotherapy, allergy immunomodulation, immunomodulation of the response to peanut, or allergen-specific immunotherapy, is a treatment aimed at reducing the severity of clinical reactions to peanut and / or increasing the tolerated dose of peanut and / or long-term tolerance to peanut. Peanut immunotherapy can be tested using methods known in the art, including food challenges. Peanut immunotherapy can be partial, in which the subject tolerates increased amounts of the food allergen compared to before treatment, but still reacts to higher doses of the food allergen; or desensitization can be complete, in which the patient tolerates all tested doses of the food allergen. In some embodiments, desensitization to peanut includes a reduction in the activation potential of basophils and / or mast cells compared to before treatment.

[0228] In some embodiments, "immunomodulation of the response to peanut" comprises a reduction in an allergic response to peanut. In some embodiments, the reduced allergic response to peanut in a subject is a reduction relative to the subject's allergic response to peanut prior to treatment or compared to an earlier time point during treatment (decreased). In some embodiments, the reduced allergic response to peanut provides a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in an allergic response to peanut. In some embodiments, the reduced allergic response to peanut in a subject comprises a reduced activation potential of basophils and / or mast cells compared to prior to treatment.

[0229] As used herein, "allergy immunomodulation," also known as "allergy desensitization," "allergy immunotherapy," or "allergen-specific immunotherapy," is a treatment designed to reduce the severity of a clinical reaction to peanut or to increase the tolerated dose of peanut. Peanut immunotherapy can be tested using methods known in the art, including food challenges. Peanut immunotherapy can be partial, in which the subject tolerates increased amounts of a food allergen compared to before treatment, but still reacts to higher doses of the food allergen, or desensitization can be complete, in which the patient tolerates all tested doses of the food allergen.

[0230] In some embodiments, the methods described herein include the use of an adjuvant. According to the present invention, "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant can also be used as a tissue reservoir for slowly releasing an antigen. Examples of adjuvants include, but are not limited to, monophosphoryl lipid A (MPL-A), microcrystalline tyrosine (MCT), calcium phosphate, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, levamisole, CpG-DNA, oil or hydrocarbon emulsions, and potentially useful adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. In some embodiments, Ara h 6 variants are adsorbed to MCT and administered with or without MPL-A. Both MCT and MPL-A should improve the efficacy of allergy immunotherapy and can have a synergistic effect when combined. Specifically, the administration of adjuvants can reduce the number of injections required, reduce the dosage, and lead to increased production of protective IgG antibodies. In addition, MCT adsorption can improve the safety of the product due to the depot effect and gradual release of the protein.

[0231] In one embodiment, the present disclosure provides a method of inducing desensitization to peanut in a subject allergic to peanut, the method comprising administering to the subject a composition comprising a nucleotide sequence encoding a recombinant hypoallergenic Ara h 6 variant disclosed herein or a modified nucleotide sequence, thereby inducing desensitization of the subject to peanut. In one embodiment, the present disclosure provides a method of inducing desensitization to peanut in a subject allergic to peanut, the method comprising administering to the subject a composition comprising a nucleotide sequence encoding a recombinant hypoallergenic Ara h 6 variant disclosed herein or a modified nucleotide sequence, thereby inducing desensitization of the subject to peanut. In one embodiment, the above composition comprises bacteria carrying the nucleotide sequence. In one embodiment, the nucleotide sequence is in the form of DNA or RNA.

[0232] In one embodiment, the present disclosure provides a method of inducing immunomodulation of a response to peanut in a subject allergic to peanut, the method comprising administering to the subject a composition comprising a nucleotide sequence encoding a recombinant hypoallergenic Ara h 6 variant or a modified nucleotide sequence disclosed herein, thereby inducing immunomodulation of a response to peanut in the subject. In one embodiment, the present disclosure provides a method of inducing immunomodulation of a response to peanut in a subject allergic to peanut, the method comprising administering to the subject a composition comprising a nucleotide sequence encoding a recombinant hypoallergenic Ara h 6 variant or a modified nucleotide sequence disclosed herein, thereby inducing immunomodulation of a response to peanut in the subject. In one embodiment, the above composition comprises bacteria carrying the nucleotide sequence. In one embodiment, the nucleotide sequence is in the form of DNA or RNA.

[0233] In one embodiment, the composition in the above method is administered orally. In another embodiment, the composition is administered by a route selected from subcutaneous, intramuscular, intradermal, intranasal, sublingual, topical, rectal or inhaled. In one embodiment, the subject in the above method is an infant. In one embodiment, the composition in the above method includes formula milk (milk formula) or baby food.

[0234] In one embodiment, the present disclosure provides a method for inducing desensitization to peanut in a subject allergic to peanut, the method comprising administering to the subject a composition comprising a nucleic acid molecule encoding a recombinant Ara h 6 polypeptide, thereby inducing desensitization to peanut in the subject. In some embodiments, the nucleic acid molecule used in the method for inducing desensitization to peanut in a subject allergic to peanut comprises a nucleic acid molecule encoding a WT recombinant Ara h 6 polypeptide. In some embodiments, the nucleic acid molecule used in the method for inducing desensitization to peanut in a subject allergic to peanut comprises a nucleic acid molecule encoding a variant recombinant Ara h 6 polypeptide or a modified nucleic acid molecule, the variant recombinant Ara h 6 polypeptide comprising one or more amino acid substitution mutations located within a single epitope recognized by an anti-Ara h 2 antibody.

[0235] In one embodiment, the present disclosure provides a method of inducing desensitization to peanut in a subject allergic to peanut, the method comprising administering to the subject a composition comprising a nucleic acid or modified nucleic acid molecule encoding a recombinant hypoallergenic Ara h 6 variant disclosed herein, thereby inducing desensitization to peanut in the subject.

[0236] In one embodiment, the present disclosure provides a method of inducing immunomodulation of a response to peanut in a subject allergic to peanut, the method comprising administering to the subject a composition comprising a nucleic acid molecule encoding a recombinant Ara h 6 polypeptide, thereby inducing immunomodulation of a response to peanut in the subject. In some embodiments, the nucleic acid molecule used in the method of inducing immunomodulation of a response to peanut in a subject allergic to peanut comprises a nucleic acid molecule encoding a WT recombinant Ara h 6 polypeptide. In some embodiments, the nucleic acid molecule used in the method of inducing immunomodulation of a response to peanut in a subject allergic to peanut comprises a nucleic acid molecule encoding a variant recombinant Ara h 6 polypeptide or a modified nucleic acid molecule, the variant recombinant Ara h 6 polypeptide comprising one or more amino acid substitution mutations located within a single epitope recognized by an anti-Ara h 2 antibody.

[0237] In one embodiment, the present disclosure provides a method of inducing immunomodulation of a response to peanut in a subject allergic to peanut, the method comprising administering to the subject a composition comprising a nucleic acid or modified nucleic acid molecule encoding a recombinant hypoallergenic Ara h 6 variant disclosed herein, thereby inducing immunomodulation of a response to peanut in the subject.

[0238] In some embodiments, the compositions comprising the isolated nucleotides or modified nucleotide sequences encoding the recombinant Ara h 6 variants described herein are used to induce desensitization to peanut in a subject allergic to peanut. In some embodiments, the compositions comprising the isolated nucleotides or modified nucleotide sequences encoding the recombinant Ara h 6 variants described herein are used to induce immunomodulation of the response to peanut in a subject allergic to peanut.

[0239] In some embodiments, the compositions comprising the recombinant Ara h 6 variant polypeptides described herein are used to induce desensitization to peanut in a subject allergic to peanut. In some embodiments, the compositions comprising the recombinant Ara h 6 variant polypeptides described herein are used to induce immunomodulation of the response to peanut in a subject allergic to peanut.

[0240] In one embodiment, the composition in the above method comprises bacteria carrying a nucleic acid or modified nucleic acid molecule disclosed herein. In one embodiment, the nucleic acid or modified nucleic acid molecule is DNA or mRNA. Examples of DNA or mRNA have been described above.

[0241] In one embodiment, the composition in the above method is administered orally. In another embodiment, the composition is administered by a route selected from subcutaneous, intramuscular, intravenous, intranasal, sublingual, topical, rectal or inhaled. In one embodiment, the subject in the above method is an infant.

[0242] As used herein, a "nucleic acid composition" refers to a composition comprising a nucleic acid or nucleic acid molecule (e.g., a polynucleotide) encoding an allergen or a derivative thereof (e.g., a variant of an Ara h 6 protein or polypeptide). In an exemplary embodiment, the nucleic acid composition comprises a ribonucleic acid ("RNA") polynucleotide, a ribonucleic acid ("RNA"), or a ribonucleic acid ("RNA") molecule. Such embodiments may be referred to as ribonucleic acid ("RNA") compositions. In some embodiments, the nucleic acid composition comprises a messenger RNA ("mRNA") polynucleotide, a messenger RNA ("mRNA"), or a messenger RNA ("mRNA") molecule as described herein. Such embodiments may be referred to as a messenger RNA ("mRNA") composition. The composition may include other substances and molecules (e.g., pharmaceutical excipients) that are required or advantageous when the composition is administered to an individual.

[0243] In one embodiment, the RNA composition comprises an RNA sequence encoding an allergen. The RNA sequence may be the sequence of an allergen, or may be adjusted with respect to its codon usage. The adjustment of codon usage may increase the translation efficiency and half-life of the RNA. In one embodiment, a poly A tail comprising at least 30 adenosine residues is attached to the 3' end of the RNA to increase the half-life of the RNA. In one embodiment, the 5' end of the RNA is capped with a modified ribonucleotide or derivative thereof having the structure m7G(5')ppp(5')N (cap 0 structure), which may be incorporated during RNA synthesis, or may be enzymatically engineered after RNA transcription using a vaccinia virus capping enzyme (VCE, composed of mRNA triphosphatase, guanylate transferase, and guanine-7-methyltransferase), which catalyzes the construction of the N7-monomethylated cap 0 structure. The cap 0 structure plays a vital role in maintaining the stability and translation efficiency of the RNA composition. The 5' cap of the RNA composition may be further modified by 2'-O-methyltransferase, which results in the generation of the cap 1 structure (m7Gppp[m2'-O]N), which further improves translation efficiency. The composition or formulation according to the invention may also comprise an adjuvant.

[0244] In one embodiment, Ara h 6 variants and / or nucleic acids or modified nucleic acid molecules encoding recombinant Ara h 6 variants disclosed herein are combined for inducing desensitization to peanut in a subject allergic to peanut. In one embodiment, several variants and / or several nucleic acids or modified nucleic acid molecules encoding recombinant Ara h 6 variants disclosed herein are combined for inducing desensitization to peanut in a subject allergic to peanut. In one embodiment, Ara h 6 variants and / or nucleic acids or modified nucleic acid molecules encoding recombinant Ara h 6 variants disclosed herein are combined with other compositions and / or treatments, e.g., other Ara hx allergens and variants thereof (wild type and / or mutant), for inducing desensitization to peanut in a subject allergic to peanut.

[0245] Plants and products

[0246] In one embodiment, the present disclosure provides a genetically modified peanut plant comprising peanut expressing an Ara h 6 variant disclosed herein.

[0247] In one embodiment, the Ara h 6 variant expressed in the genetically modified peanut plants described above is expressed from a heterologous nucleic acid.

[0248] In one embodiment, the Ara h 6 variant expressed in the genetically modified peanut plant described above is endogenously expressed from the genetically modified chromosome.

[0249] In some embodiments of the above genetically modified peanut plants, expression of endogenous wild-type Ara h 6 allergen is reduced compared to a non-genetically modified peanut plant.

[0250] In some embodiments, the reduced expression of endogenous wild-type Ara h 6 allergen in the genetically modified peanut plant is compared to the amount of endogenous wild-type Ara h 6 allergen in a corresponding non-genetically modified peanut plant. In some embodiments, the reduced expression of endogenous wild-type Ara h 6 allergen in the genetically modified peanut plant comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or up to 99% less expression when compared to the expression of endogenous wild-type Ara h 6 allergen in a corresponding non-genetically modified peanut plant.

[0251] In some embodiments of the genetically modified peanut plants described above, the modified plant further expresses at least one RNA silencing molecule that (i) reduces expression of endogenous Ara h 6 allergens, and (ii) does not reduce expression of Ara h 6 variants.

[0252] In some embodiments of the genetically modified peanut plants described above, the modified plants further express a DNA editing system designed to reduce the expression of endogenous Ara h 6 allergens.

[0253] In one embodiment, the present disclosure provides a processed food product comprising an Ara h 6 variant disclosed herein.

[0254] In one embodiment, the processed food product described above comprises a reduced amount of endogenous wild-type peanut Arah 6 allergen. In some embodiments, the reduced amount of endogenous wild-type Ara h 6 allergen is compared to the amount of endogenous wild-type Ara h 6 allergen in a corresponding processed food product that does not comprise the Ara h 6 variants described in detail herein. In some embodiments, the reduced amount of endogenous wild-type Ara h 6 allergen comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or up to 99% less endogenous wild-type Ara h 6 allergen when compared to the endogenous peanut Ara h 6 allergen in a corresponding processed food product that does not comprise the Ara h 6 variants described in detail herein.

[0255] In one embodiment, the above processed food product comprises peanuts harvested from the genetically modified plants described above.

[0256] The terms "comprises," "comprising," "includes," "including," "having" and their cognates mean "including but not limited to."

[0257] Unless the context clearly indicates otherwise, as used herein, the singular forms "a", "an", and "the" include plural referents. For example, the term "recombinant Ara h 6 variant" may include more than one variant, including mixtures thereof. Similarly, the term "isolated nucleotide or modified nucleotide sequence encoding a recombinant Ara h 6 variant" may include more than one nucleotide or modified nucleotide sequence, including mixtures thereof.

[0258] It is assumed herein that all numerical values ​​are modified by the term "about". As used herein, the term "about" indicates that the value may deviate from the indicated value by up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the indicated value, and the deviation range includes integer values ​​and, if applicable, also non-integer values, constituting a continuous range.

[0259] The word “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any implementation described as “exemplary” is not necessarily to be construed as preferred or advantageous over other implementations and / or to exclude the incorporation of features from other implementations.

[0260] In the present application, various embodiments of Ara h 6 variants and mutations and / or epitope positions thereof may be presented in a range format. It should be understood that the description in range format is only for convenience and brevity, and should not be interpreted as an unchangeable limitation on the range of Ara h 6 variants and mutations and / or epitope positions thereof. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within the range. For example, a description such as a range from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within the range such as 1, 2, 3, 4, 5 and 6. This applies regardless of the width of the range.

[0261] Whenever a numerical range is indicated herein, it is intended to include any cited numerical value (fractional or integer) within the indicated range. The phrases "in the range between a first indicated number and a second indicated number" and "in the range from a first indicated number to a second indicated number" are used interchangeably herein and are intended to include the first indicated number and the second indicated number and all fractional and integer values ​​therebetween. Example

[0262] Example 1: Materials and Methods

[0263] Peptide microarray assay

[0264] To identify the epitope of Ara h 6, a Celluspot-based TMImmunoassay of peptide microarrays (Intavis, Cologne, Germany) (Winkler, Dirk FH, Peptide microarrays. Humana Press, 2009). Peptides of 15 amino acids in length and 4 amino acids offset were synthesized, derived from the primary sequences of the peanut allergens Ara h 1 (uniprot entry P43238 positions 25-626), Ara h 2 (uniprot entry Q6PSU2), Ara h 3 (uniprot entry O82580), Ara h 6 (uniprot entry A5Z1R0 positions 13-147) and Ara h 8 (uniprot entry Q6VT83) and spotted in duplicate on the microarray. The slides were rinsed with blocking buffer (150 mM NaCl, 0.05% Tween, 2.5% skim milk, 50 mM Tris pH 7.5) at 4°C overnight. Then, the slides were washed and incubated on a rotator at 4 ° C for 4 hours with plasma in blocking buffer. For detection, the slides were incubated with 3 ml of goat anti-human IgE (abcam, Cambridge, UK) labeled with horseradish peroxidase (HRP), or diluted 1:10,000 in blocking buffer for 2 hours on a rotator at 25 ° C. After washing, femtogram HRP substrate kit [Azure Biosystem, Dublin, California] was added and chemiluminescence was read via ChemiDoc [BioRad, Hercules, CA]. Peptide array images are processed by an internal python script that detects peptide spots, normalizes their intensities, and reports any series of at least two overlapping spots, showing an average signal above the slide mean of two standard deviations on duplicates.

[0265] Generation of human scFv phage display library

[0266] Whole blood samples of 5 ml to 20 ml were collected from clinically diagnosed peanut allergic patients using heparin or EDTA-treated tubes (BD). Peripheral blood mononuclear cells (PBMCs) were extracted from blood samples using Sepmate tubes (STEMCELL) according to the manufacturer's instructions. RNAeasy extraction kit (Qiagen; Hilden, Germany) was used to extract PBMCs from 5 × 10 6 -15×10 6 RNA was purified from each PBMC, and cDNA was prepared from 1 μg-5 μg RNA (depending on the amount of RNA obtained).

[0267] The entire cDNA reaction was divided into PCR reactions to amplify the antibody hypervariable domains of each patient's variable gene. Light chains were amplified using a gene subfamily-specific forward primer with an unstructured, nonspecific overhang followed by a NotI restriction site and a reverse primer specific for the IGLK and IGLL isoforms with homology to the 5' portion of the unstructured linker. Heavy chains were amplified using a gene subfamily-specific forward primer with homology to the 3' portion of the unstructured linker and a reverse primer specific for the IGHG and IGHE genes with an unstructured, nonspecific overhang followed by a NcoI restriction site. Primers adapted from "Phage display: Methods and Protocols" (2018) Hust M and List T, eds. Springer Protocols. PCR 50 μl reactions were performed using Phusion hot start Taq polymerase kit, 200 μM dNPT, 2% DMSO, 1.25 M betaine, 1 μg-5 μg cDNA and 0.5 μM of each primer. The following PCR program was used for the reaction: 98°C for 3 minutes, 30 cycles of 98°C for 20 seconds + 60°C for 60 seconds + 72°C for 45 seconds, and a final extension phase of 72°C for 10 minutes.

[0268] The PCR products of each family (VHγ, VHε, VLκ and VLλ) were combined, each pool was concentrated by ethanol precipitation, run on 1% agarose gel, extracted using a gel extraction kit (Qiagen), and cleaned using an Amicon ultra 30K centrifugal filter (Sigma-Aldrich Merck, Israel). A DNA mixture of amplified V gene segments was prepared at a ratio of 45% Vγ, 5% Vε, 25% Vκ and 25% Vλ. The generation of the combined light-heavy scFv library was performed by PCR reaction, using the same reagents as the first PCR, but 100 μl per reaction, using 100 ng of V-gene mixture, using a concentration of 250 nM "pull-through" primer (complementary to the overhangs flanking the restriction sites of each product from the first PCR). Multiple recombination reactions (18-24) were prepared for connection without primers, and PCR was performed using the following program: 98°C for 3 minutes, 98°C for 20 seconds + 60°C for 60 seconds + 72°C for 60 seconds for 5 cycles. Primers were then added and the reaction was performed using the following program: 98°C for 1 min, 30 cycles of 98°C for 20 sec + 67°C for 60 sec + 72°C for 45 sec and a final extension phase of 72°C for 3 min.

[0269] The PCR product was concentrated by ethanol precipitation, run on a 1% agarose gel, extracted using a gel extraction kit (Qiagen), and cleaned using an Amicon ultra 30K centrifugal filter (Sigma-Aldrich Merck). According to the manufacturer's instructions, high-fidelity NcoI and NotI enzymes (NEB; MA, USA) were used to restrict the pLibGD vector (described below) and purified scFv DNA (at least 4 μg of vector and 2 μm scFv). According to the manufacturer's instructions, the vector was further processed by QuickCIP (NEB). As in the previous step, the restricted vector was cleaned by extraction from a 1% agarose gel and a centrifugal filter. The restricted scFv was purified using a PCR cleanup column (Qiagen).

[0270] According to the manufacturer's instructions, a 20 μl ligation reaction was set up using 130 ng of vector and 70 ng of insert (yielding a 3:1 ratio) and performed overnight at 10°C. At least 3 μg of DNA was ligated in total. The ligation was heat inactivated, cleaned up by PCR cleanup columns, and concentrated by Amicon 30K centrifugal filters.

[0271] The ligated libraries were transformed into SS320 electrocompetent bacteria (Lucigen; WI, USA) according to the manufacturer's instructions. Each library was split into two transformants and plated on three 15 cm 2YT agar plates containing 100 μg / ml carbenicillin and 2% glucose. The plates were incubated overnight at 30°C. Serial dilutions of the transformants were plated on separate kanamycin and ampicillin plates to assess transformation efficiency. >10 7 The library was considered to be of sufficient quality and was used further.

[0272] The next day, SS320 were scraped from the culture dishes using 6 ml 2YT, diluted to OD = 0.1 in 60 ml 2YT supplemented with 100 μg / ml carbenicillin and 2% glucose, grown to OD = 0.5, and infected with 1:1000 diluted KO7 helper phage (NEB) for 30 minutes at 37° C. The bacteria were then centrifuged at 3000 g for 10 minutes, resuspended in 200 ml 2YT + 100 μg / ml carbenicillin + 25 μg / ml kanamycin, and grown at 30° C. in baffled flasks with shaking at 250 RPM for at least overnight or up to 24 hours to produce phage displaying scFv.

[0273] The next day, the bacteria were centrifuged at 16,000g for 10 minutes. The supernatant was transferred to a fresh tube, and the phage was precipitated by adding PEG / NaCl stock solution (PEG-8000 20%, NaCl 2.5M) to a final concentration of 20% (the ratio of PEG-NaCl stock solution to supernatant was 1:4). The sample was incubated on ice for 20 minutes, and centrifuged at 4 ° C for 30 minutes at 16,000g. The supernatant was discarded, and the precipitate was centrifuged again for 2 minutes to remove the remaining supernatant. The precipitate was resuspended with 10ml PBS / 100ml culture medium, and centrifuged at 16,000g for 10 minutes to remove the remaining bacterial cell debris. The sample was then subjected to a second round of identical PEG-NaCl precipitation, and resuspended with 4ml PBS / 100ml culture medium. Samples were centrifuged at 20,000 g for 15 min to remove residual debris, and purified phages were supplemented with 50% glycerol and 2 mM EDTA and stored at -80°C until use.

[0274] Screening of phage display libraries for allergen-specific scFv

[0275] Isolation of allergen-specific scFvs was performed by panning phage libraries using either native purified allergens or recombinant allergen variants with modified putative epitopes. Maxisorp high binding 96-well plates (Nunc) were coated with 100 μl of 5 μg / ml allergen solution in PBS or with 2% BSA solution in PBS (8 wells per library). TM Bacteria (Thermo Fisher Scientific; MA, USA) were inoculated in 2YT + tetracycline (5 ug / ml) and grown overnight at 37°C with shaking at 250 RPM.

[0276] The next day, OmniMAX TMBacteria were diluted to 0.1 OD in 2YT + tetracycline, grown to OD = 0.6-0.8 at 37°C with shaking at 250 RPM, and kept on ice until use. Phage stocks (2ml-4ml) were thawed, purified by PEG-NaCl purification (as above), and resuspended in 1ml PBST (PBS + 0.05% Tween). A sample of the unpanned phage stock was set aside for input measurement. If negative selection was performed, the maxisorp plate was washed with 200μl / well PBSTx3, and the phage solution was then incubated at 100μl / well in BSA-coated wells at 4°C with gentle shaking for 1 hour to remove non-specific binders. The phage solution was then transferred to the allergen-coated wells and incubated at 4°C with gentle shaking for 1 hour. If negative selection was not performed, the phage-PBST solution was added directly to the allergen-coated wells. The plate was then washed twice with 200 μl / well PBST to remove unbound phage. Bound phage were eluted by incubation with 100 μl / well 100 mM HCl for 5 minutes at room temperature with gentle shaking. The elution reaction was terminated with 12.5 μl / well Tris 1M, pH 11.

[0277] Add the eluted sample to 5 ml of OmniMAX at the desired OD. TM , and incubate at 37°C with shaking at 250 RPM for 30 minutes. Panning output titration was assessed by making serial 10-fold dilutions of samples of the infected stock and plating in triplicate 5 μl droplets on LB-agar plates with carbenicillin or kanamycin or tetracycline. The remaining output was multiplied by superinfection with 1:100 KO7 auxiliary stock at 1:1000 at 37°C with shaking at 250 RPM for 45 minutes. The superinfected bacterial stock was added to 50 ml 2YT supplemented with carbenicillin and kanamycin and grown overnight at 37°C with shaking at 250 RPM to generate phage for the next round of panning. Panning input titration was assessed by making serial 10-fold dilutions of the input sample, infecting the OmniMAX at 37°C with shaking at 250 RPM TM Bacteria were grown for 30 min and plated in triplicate on carbenicillin and kanamycin LB-agar plates.

[0278] Subsequent panning rounds were performed by performing a single PEG-NaCl precipitation of the overnight output amplification and using it as input. From one round of panning to the next, the number of wash cycles increased and the number of panning wells decreased to increase the stringency of the panning (3 to 4 panning cycles per library).

[0279] To isolate individual allergen-specific scFvs, serial dilutions of the output from the selected rounds were plated onto LB-agar-carbenicillin plates and grown overnight at 37°C. The next day, single colonies were plated into microtubes containing 300 μl 2YT + carbenicillin + 1:1000 KO7 and grown overnight at 37°C with shaking at 250 RPM. The next day, supernatants from the microtubes were assayed by ELISA using plates coated with allergens or BSA. scFvs from the supernatants that specifically bound to allergens but not BSA were amplified by PCR using primers flanking the scFv region of the pLibGD plasmid. PCR products consistent with the full-length scFv were subjected to standard PCR cleanup by ExoI and rSAP restriction endonucleases (NEB) and sequenced by standard sanger reactions (Hylabs). Full-length monoclonal clones were cloned into mammalian expression plasmids (pSF) and expressed as IgG in HEK-293T cells.

[0280] Single-cell isolation of allergen-specific B cells

[0281] Peanut allergic patient PBMCs were thawed, washed with PBS, and stained for viability (LIVE / DEAD near infrared kit, Thermo-fisher) according to the manufacturer's instructions. The cells were then incubated on ice for 1 hour with different concentrations of the target allergen depending on the allergen type. The allergens used were natural purified allergens fluorescently labeled with the alexa-fluor protein labeling kit (Thermo-fisher, a mixture of allergens labeled with 2 different fluorophores according to the manufacturer's instructions), or WT recombinant allergens with an HA tag at the C-terminus or N-terminus, or biotin-avidin labeled WT recombinant allergens (a mixture of allergens labeled with 2 different fluorophores). The cells were then washed and stained with fluorophore-conjugated antibodies for the following markers: CD14, CD16, IgM, IgD, CD3, CD19, IgG1. If HA-tagged allergens were used, two anti-HA antibodies with different fluorophore conjugates were also added. The cells were then washed and sorted on an ARIA-III sorting flow cytometer. Single allergen-specific B cells (LIVE / DEADdim CD14-CD16-IgD-IgM-CD3-CD19+IgG1+allergen fluorophore double positive) were sorted into 96-well plates containing 4 μl / well ice-cold lysis buffer (PBSx0.5, 10 mM DTT, 8 U RNase inhibitor). Several wells in each plate were kept empty as negative controls for PCR.

[0282] Isolation of antibody genes from sorted cells and antibody expression

[0283] Reverse transcription was performed directly on single sorted allergen-specific B cell lysates (SSIV, Invitrogen, according to the manufacturer's instructions). Two consecutive PCR reactions (second nested PCR) were performed to amplify the heavy chain gene (Hotstart taq polymerase, NEB) and the light chain gene (Kapa hot-start PCRF mixture) using a mixture of primers covering most known antibody gene alleles. The PCR products were sequenced and aligned to the genome. When the cells had authentic sequences for both heavy and light chains, the sequences were cloned into a mammalian expression plasmid (pSF) and expressed as IgG in HEK-293T cells.

[0284] Preparation of a saturation library of yeast surface-displayed mutants and flow cytometry sorting

[0285] A library consisting of Arah 6 variants with a single mutation in each residue was ordered from TWIST Bioscience (CA, USA) and cloned into a YSD vector similar to pCHA. To display the Arah 6 library on the surface of yeast denoted S0, the library was grown in SDCAA selective medium (2% dextrose, 0.67% Difco yeast nitrogen base, 0.5% Bacto casamino acids, 0.52% Na 2 HPO 4 and 0.856% NaH 2 PO 4 ·H 2 O) and induced expression with galactose medium (same as SDCAA, but with 2% galactose instead of dextrose) according to established protocols (Chao, G., Lau, W., Hackel, B. et al. Isolating and engineering human antibodies using yeast surface display. Nat Protoc 1, 755-768 (2006)). Ara h 6 expression was detected by anti-Myc antibody conjugated with FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), and anti-Ara h 6 IgG binding was detected by secondary affinipure donkey anti-human IgG (H+L) antibody conjugated with APC (Jackson ImmunoResearch Laboratories, PA, USA). For paired selective screening, ~1×10 6 Yeast cells were incubated with different anti-Ara h 6 IgG at room temperature in binding buffer (100 mM Tris, pH = 8.0, 1 mM CaCl 2, 1% BSA) for 1 h. Then, the cells were washed with binding buffer and incubated with anti-Myc-FITC and anti-human IgG-APC antibodies for 30 minutes. Then, the cells were washed again with binding buffer and low selectivity variants were sorted by performing several independent sortings using an S3E cell sorter (Bio-Rad). Ara h 6 variants that showed low binding affinity to anti-Ara h 6 IgG were selected, i.e., the first few lowest Ara h 6 variants as low as 3% in the entire population.

[0286] High-throughput sequencing library preparation

[0287] According to the manufacturer's protocol, the YSD vector containing the Ara h 6 gene was isolated from the initial library and the sorting library using Zymoprep yeast plasmid Miniprep II (Zymo research, Irvine, CA). Using this kit, 200ng of DNA was isolated from each yeast library. The extracted vector was sent to the NGS laboratory of Hy Laboratories (Hylabs, Rehovot, Israel), and the first PCR and the second PCR of 20 and 8 cycles were performed respectively using Fluidigm Access Array primers to add adapters and barcodes. Then, the DNA library samples were purified with AmpureXP beads (Beckman Coulter, Brea, CA), and the concentration of the samples was determined in Qubit by using DNA high sensitivity determination. The samples were pooled and then run on TapeStation (Agilent, Santa Clara, CA) to verify the size of the PCR product. As a final quality test, qRT-PCR was performed to the pool to determine the concentration of the DNA that can be sequenced. The pool was then loaded for sequencing on an Illumina Miseq using the 600v2 kit.

[0288] Deep sequencing read analysis

[0289] Paired end reads were analyzed and filtered for quality using the fastp command-line preprocessing tool (Chen, S., Zhou, Y., Chen, Y., & Gu, J. (2018). fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics (Oxford, England), 34 (17), i884-i890.). All sequences with more than 10% or 20% of the sequences having a Phred quality score of less than 20 were discarded from subsequent analysis, depending on the quality of the entire library. The reads were then aligned based on a probabilistic model of the overlapping regions of the reads, implemented in the pandaseq assembler (Masella, AP, Bartram, AK, Truszkowski, JM et al. PANDAseq: paired-end assembler for illumina sequences. BMC Bioinformatics 13, 31 (2012)). The translated sequences were filtered for the occurrence of expected mutations (single mutation per sequence, i.e. single mutation per variant) and analyzed for sequence enrichment:

[0290] Enrichment

[0291] Among them aa i is the specific amino acid at position i, fS 1 is the fraction of reads for a given amino acid at position i in the sorted library and fS 0 is the same fraction in the input library. This calculation provides the enrichment of each specific Ara h 6 point mutant.

[0292] For convenience, the following may also be used to express the increasing index of a particular amino acid at position i.

[0293]

[0294] This information is integrated for all mutations at a given position by calculating the Shannon entropy at each position:

[0295]

[0296] where i is the given position, INaa z represents the increase index of a given amino acid, normalized by the increase index of all amino acids.

[0297] Ara h 6 purification

[0298] For Ara h 6 variant purification, Ara h 6WT (SEQ ID NO: 2) and mutants were cloned into pET28 plasmid. Ara h 6 was fused to DNA (Ara h 6-His*6) encoding His tag at the N-terminus. All variants were expressed under the transcriptional control of T7 promoter. Cells were grown at 37 ° C until an OD of 0.5-0.8 was reached, and induced at 37 ° C for 3 hours by adding 0.5 mM IPTG. Harvest (4800 g for 30 min) cells, and cell pellets were resuspended with lysis buffer (50 mM Tris pH 8.0, 350 mM NaCl, 10% v / v glycerol, 0.2% Triton X-100, 250 U Benzonase, 0.2 mM PMSF and 1 mg / ml lysozyme), and lysed by sonication (35% amplitude, open for 10 seconds and close for 30 seconds, for 2 min). The lysate was centrifuged (15000 g, 45 min) and the supernatant was loaded onto Ni-NTA beads pre-washed with binding buffer (50 mM Tris pH 8.0, 350 mM NaCl and 10% v / v glycerol) and incubated at 4 ° C for 1 hour. The beads were washed with binding buffer containing increasing imidazole concentrations. The purity of Ara h 6 was then increased and the imidazole concentration was diluted by size exclusion chromatography (SEC). The fractions containing Ara h 6 were collected and concentrated by a 3 kDa microcentrifugal concentrator (Amicon, Mercury) and the protein concentration was measured by absorbance at 220 nm.

[0299] Analysis of binding to monoclonal antibodies by ELISA

[0300] ELISA was used to determine the concentration (EC50) of anti-Ara h 6 IgG required for 50% maximum binding to WT-Ara h 6 and Ara h 6 variants. In brief, 200 ng of Ara h 6 was coated overnight in a 96-well microtiter plate (Thermo Fisher Scientific, Waltham, MA) at 4 ° C. The plate was sealed with 0.5% BSA in PBS (200 μl / well) at room temperature for 1 hour. Anti-Ara h 6 IgG was prepared by serial dilution in PBS, added to the wells coated with Ara h 6 and incubated at room temperature for 1 hour. After the washing step, the amount of bound IgG was detected by incubation with goat anti-human IgG (Jackson ImmunoResearch Laboratories, PA, USA) conjugated with HRP polyclonal antibody and then TMB substrate.

[0301] All incubation steps were performed in PBS containing 0.5% BSA and 0.05% Tween 20. The highest concentrations of anti-Ara h 6 IgG were saturating, and the amount of binding to Ara h 6 was maximal at these levels.

[0302] Computational design of variants with mutations at multiple sites

[0303] Based on the experimental results of identifying point mutations that reduce binding to mAbs and / or to patient plasma, computational protein design tools are used to generate variants with combinations of mutations that are predicted to maintain their stability. The NMR structure of Ara h 6 is energy optimized (for each NMR state). Next, Monte-Carlo sampling is performed for up to 5 simultaneous mutations (combined mutations at the epitope level) or up to 25 simultaneous mutations (combined mutations at the protein level) using a combined mutagenesis scanning tool. This allows the main chain to be minimized during side chain mutagenesis, generating up to 250 structures. Mutations are evaluated by calculated ΔG (changes in protein free energy during mutation). The sequences are sorted by their ΔG, eliminating any structures with significantly increased ΔG, and sorted by their sequence diversity to eliminate experimental tests on almost identical protein sequences.

[0304] RBL SX-38 cell degranulation assay

[0305] RBL SX-38 cells were obtained from Professor Stephen Dreskin of UC Denver with permission from BIDMC, Boston. The cells were maintained at 37°C with 5% CO 2 Cultured in maintenance medium containing 80% MEM, 20% RPMI 1640, 5% FCS (not heat inactivated), supplemented with 1 mg / ml L-glutamine, penicillin-streptomycin and G418 (all from Gibco-Thermo fisher, USA). At least 48 hours before the assay, cells were split and expanded in assay medium (maintenance medium without RPMI and G418). On the day of the assay, cells were detached using 0.05% trypsin-EDTA (Gibco), centrifuged at 300 g for 10 minutes, and resuspended in assay medium supplemented with 5%-10% clinical sample (plasma / serum from peanut allergic patients, dilution varied between samples) to 2.5×10 6The final concentration of cells / ml. If the plasma is produced with any anticoagulant other than heparin, the sample is first supplemented with 30U / ml heparin (heparin sodium, Sigma) and incubated at room temperature for 10 minutes before adding the cells. The cells are then seeded in 96-well flat-bottom tissue culture plates (Greiner bio-one, Austria) at 50 μl per well (final 125,000 cells / well) and cultured overnight. The next day, the cells are cultured in Tyrode buffer (137mM NaCl, 2.7mM KCl, 0.4mM NaH 2 PO 4 , 0.5 mM MgCl 2 , 1.4 mM CaCl 2 , 10 mM Hepes pH 7.3, 5.6 mM glucose, 0.1% BSA, pH adjusted to 7.4, in 80% ddw and 20% D 2 Activation solution was prepared by diluting allergens or irrelevant protein negative controls at different concentrations in Tyrode buffer prepared with ddw alone, Merck-Sigma Aldrich, Israel. The cells were then washed 3 times with Tyrode buffer prepared with ddw alone, and 100 μl of allergen activation solution was added to the appropriate wells in duplicate. For each allergen, 5-6 concentrations were used at 10-fold dilutions. Each clinical sample was tested against WT allergen, variant allergen, and an irrelevant protein (KLH, Sigma) as a negative control. Duplicate wells were also prepared with lysis buffer (Tyrode buffer with 1% Triton x-100, Fisher Scientific) for measuring total degranulation, and duplicate wells were prepared with Tyrode buffer alone for measuring background degranulation. The cells were then incubated at 37°C, 5% CO 2Incubation lasts for 1 hour. Immediately after incubation, 30 μl of each well is transferred to the corresponding well in a transparent non-binding 96-well plate (Greiner Bio-one), and 50 μl PNAG colorimetric substrate (4-nitrophenyl N-acetyl-β-D-glucosamine prepared in 0.1M citric acid, to a final concentration of 1.368 mg / ml pH 4.5) is supplemented. The reaction is incubated at 37 ° C for 1 hour, gently shaken in the dark, and then 100 μl stop solution (0.2M glycine, pH 10.7) is added to stop the reaction and develop the color. The optical density of the signal is read at 405nm using a Synergy LX microplate spectrophotometer reader (Biotek, Vermont), and background absorbance is read at 630nm. After subtracting background absorbance, net degranulation is calculated by dividing the OD of each cell by the OD in the corresponding lysis buffer well (total degranulation) and subtracting the OD (background degranulation) of only the buffer well.

[0306] BAT assay

[0307] Fresh whole blood samples in heparinized tubes (BD biosciences) were divided into 100 ul per tube. Allergens and controls were diluted to x2 stock solutions in RPMI1640 (Biological Industries), added 1:1 to the tubes (final volume 200 ul), and incubated at 37°C, 5% CO 2 Incubate in a humidified incubator for 30 minutes. The dose range used was 0.1-10000 ng / ml. Crude peanut extract (CPE), fMLP and anti-human IgE antibodies were used as positive controls. KLH protein was used as a negative control. The reaction was terminated by incubating on ice for 5 minutes. A mixture of fluorophore-conjugated antibodies was added directly to the sample to detect the following markers: CD203c, CD63, HLA-DR, CD45, CD123. The cells were incubated on ice for 30 min. RBC lysis was performed with a kit according to the manufacturer's instructions (BD FACS lysis solution), and the cells were washed and analyzed by flow cytometry. Cells were gated for basophil detection and activation rate (% CD63 positive basophils) was measured. At least 500 basophils were analyzed per tube.

[0308] T cell activation assay

[0309] PBMCs were isolated from heparinized blood samples of patients with peanut allergy. The cells were washed with PBS, stained with Celltrace violet (Thermo-fisher) according to the manufacturer's instructions, and cultured at 0.2 × 10 in X-vivo15 medium supplemented with 5% human AB serum (Biotag) and 1% penicillin-streptomycin solution (Biological industries). 6 -0.5×10 6 Cells / well (based on the number of available cells after purification and staining) were seeded in 96-well round-bottom plates. Recombinant WT and variant allergens were purified by Rapid Endotoxin Removal Kit (Abcam), tested for residual endotoxin contamination (LAL chromogenic endotoxin quantification kit, Pierce), diluted in the same culture medium as the cells, sterilized by 0.22 μM filtration, and added to the cells at 200 μl per well to a final concentration of 50 μg / ml. Unactivated wells (baseline, culture medium only) and each allergen from each patient were tested by 3 or more replicate wells. Each assay included healthy donor samples along with patient samples as negative controls for assay quality assurance. The final endotoxin level for all allergens in the wells was <0.5 EU. Cells were incubated at 37°C, 5% CO 2 Incubate in a humidified incubator for 7 days. If the culture medium in any well turns yellow during the incubation period, half of the culture medium in all wells is replaced with fresh culture medium. After 7 days, cells are harvested, stained for viability (LIVE / DEAD staining, Thermo-fisher), stained with fluorophore-conjugated anti-CD3 and anti-CD4 antibodies (Biolegend; USA) and analyzed by flow cytometry. Live helper T cells are gated (LIVE / DEAD low CD4+CD3+), and the percentage of proliferating cells is measured (Celltracedim / total helper T cells). Positive results (allergens cause patient T cell activation) are determined, where the mean value of the wells stimulated by the allergen is greater than the mean value+3xSD of the unstimulated wells.

[0310] CD of Ara h 6WT and variants

[0311] Circular dichroism (CD) spectroscopy is a useful technique for analyzing the secondary structure and folding properties of purified proteins in solution using very small amounts of protein. It is based on the different absorbances of chromophores to left and right circularly polarized light. The CD analysis of proteins is based on amide chromophores in the far UV region (below 260nm). For example, alpha helical proteins have negative bands at 222nm and 208nm, and positive bands at 193nm, while proteins with clear antiparallel β pleated sheets (β pleated sheets) have negative bands at 218nm, and positive bands at 195nm. The circular dichroism spectra of recombinant Ara h 6 protein were measured on a Chirascan CD chromatograph (Applied Photophysics) at Bar Ilan University. Far UV CD spectra of 200-260nm were obtained with a cuvette of 10mm path length. Purified Ara h 6 recombinant WT and D12 variants were measured in PBS buffer, and the concentration compared to the reference native protein was determined using SEC-HPLC. Spectra were acquired at 25 °C and in the temperature range of 20 °C–90 °C to assess protein stability.

[0312] Strains, plasmids and growth conditions

[0313] E. coli subcloning efficiency DH5α competent cells (Invitrogen) were routinely used for all cloning procedures, E. coli OmniMAX TM (Thermo Fisher scientific) was used for phage display library screening, and E. coli BL21 (DE3) cells were used for Ara h 6 purification. All strains were grown at 37°C in 2YT broth and LB agar plates. Phagemids were used for scFv phage display libraries derived from peanut allergy patients. tPCR was used to insert non-specific scFvs (modified plasmids were internally labeled as pLibGD) derived from healthy donors and designed with unstructured GGGSx4 linkers and restriction sites added at both ends of the scFv segment (NcoI at the 5' end and NotI at the 3' end). Plasmid pET28 (Invitrogen) was used for recombinant purification of Ara h 6 and mutants. Transformation for scFv display was performed using SS320 electrocompetent E. coli (Lucigen).

[0314] Example 2: Epitope Mapping and Epitope Removal of Ara h 6 Polypeptide

[0315] Objective: The overall objective is to lay the foundation for well-defined targeted mutagenesis of allergen polypeptides that are stable, retain their T cell activating activity but have reduced binding to IgE antibodies. For the purpose of immunotherapy, the functionality of these Ara h 6 variant polypeptides includes maintaining immunogenicity, for example, through the ability to activate T cells. This series of experiments was performed to identify and map conformational and linear epitopes on the peanut allergen Ara h 6 based on the binding of patient sera or specific monoclonal antibodies isolated from peanut allergic patient samples; as well as to identify amino acid residues within the Ara h 6 mAb binding epitope that contribute to binding and are not expected to destabilize the protein when mutated.

[0316] result

[0317] The approach to individual epitope mapping and de-epitoping of the peanut allergen Ara h 6 involves two stages—(1) discovery of Ara h 6-specific antibodies (i.e., serum or isolated mAbs) from peanut-allergic patient samples that exhibit binding to Ara h 6-specific IgE, as measured by ELISA assays and peptide arrays, and (2) mapping of the epitope to which each antibody binds. Stage 1, mAb discovery, was performed using scFv phage display libraries, either by amplifying variable genes and constructing scFvs fused to pill protein and displayed on phage, or by single-cell sorting of Ara h 6-specific B cells, followed by sequencing of the variable regions and generation of recombinant mAbs.

[0318] Briefly, scFv phage display libraries from PBMCs of a peanut allergic patient were generated as described in Example 1, and these libraries were subsequently subjected to a panning process, resulting in the identification of three Ara h 6 specific mAbs. Single sorted allergen-specific B cell lysates from 11 peanut allergic patients were generated as described in Example 1, and 15 Ara h 6 specific mAbs were identified. All 18 mAbs were cloned into a mammalian expression plasmid (pSF) and expressed as IgG in HEK-293T cells. As described below, epitope mapping procedures were completed for the 14 Ara h 6 IgG mAbs.

[0319] In the second phase, anti-Ara h 6 specific purified mAbs were used for epitope mapping in two complementary approaches:

[0320] Method A. Screening of site saturation variant libraries using yeast surface display (YSD) (Siloto and Weselake (2012) Site saturation mutagenesis: Methods and applications in protein engineering. Biocatalysis and Agricultural Biotechnology, Vol. 1(3): 181-189) (Cherf GM, Cochran JR. (2015) Applications of Yeast Surface Display for Protein Engineering. Methods Mol Biol. 1319: 155-75).

[0321] Epitope mapping using the Ara h 6 YSD saturation library: For the purpose of epitope mapping, a two-step procedure was performed. First, the Ara h 6 point mutant library was sorted for expression only, and those variants that underwent successful YSD were collected, resulting in a sorted library called S1. The expression threshold was defined as the fluorescence value above that of unstained cells (background). Each cell with a fluorescence signal above background was collected (S1 library). Next, the S1 library was evaluated for binding to 14 mAbs. Yeast cells that displayed Ara h 6 variants and exhibited mAb binding signals (APC) in the lower 3% of the population were sorted.

[0322] Deep sequencing was performed on each mAb to identify positions that affect binding to a specific mAb. Since the library had already undergone selection for expression and lower mAb binding, the sequencing results were analyzed by enrichment calculations. Each unique DNA sequence encoding a point mutant was counted and the fold change in its relative abundance was calculated as an indirect estimate of the change in mAb binding.

[0323] At least two (2) conformational epitopes were identified in Ara h 6.

[0324] Method B. Peptide microarray assays were performed as described in Example 1 with purified mAbs (commercial IgE or IgG) to map some continuous epitopes on the allergen Ara h 6. This method was also used to validate the linear epitopes from the YSD saturated data. Five linear epitopes were identified and confirmed in Ara h 6 using peptide array analysis with the six mAbs. Next, the mAbs mapped with Ara h 6 were assayed with arrays containing mutated "de-epitope" spots to screen for those peptides that showed the most significant reduction in binding. Linear epitope mapping for Ara h 6 mAbs ( Figure 1A ) and epitope removal ( Figure 1B), showing the results of a representative array.

[0325] IgE epitope mapping and de-epitoping of Ara h 6 based on plasma from allergic patients (see Example 3). Key positions in the three epitopes were identified using peptide microarrays similar to the process in Method B. However, instead of mapping isolated monoclonal antibodies, an IgE repertoire from plasma of allergic patients was used, as described in Example 3.

[0326] Summarize

[0327] Table 1 summarizes embodiments of Ara h 6 variants having mutations at positions relative to WT Ara h 6, amino acid mutations and epitopes thereof. The mutation details presented in Table 1 were collated from the results of Examples 2 and 3.

[0328] Table 1: Ara h 6 variants*

[0329]

[0330]

[0331]

[0332]

[0333]

[0334] *Highlighted positions refer to consensus substitutions occurring in Ara h 6 variants D119, D154, D158, D160, D179 (SEQ ID NOs: 46, 76, 80, 82, and 101, respectively).

[0335] Example 3: IgE epitope mapping and de-epitope analysis based on plasma samples from allergic patients

[0336] Purpose: Following the overall aim of laying the foundation for well-defined targeted mutagenesis of allergen peptides that are stable, retain their functional characteristics but have reduced binding to IgE allergen antibodies, the aim of these experiments was to identify continuous linear IgE epitopes in plasma of peanut-allergic patients and to analyze their mutant variants.

[0337] result

[0338] The same peptide arrays as in the purified mAb analysis procedure were used to identify all linear epitopes on the allergen Ara h 6 from polyclonal IgE from allergic patient plasma. These arrays were assayed with plasma from 216 peanut allergic patients, testing plasma-derived IgE binding of Ara h 6-derived peptides. In the plasma tested, 80 slides identified IgE binding to at least one peptide from Ara h 6. Analysis and clustering of the peptide array results allowed mapping of all linear epitopes of the protein ( Figure 2 ).

[0339] Based on the mapped epitopes, additional arrays were synthesized in which, for each Ara h 6 mapped epitope, the WT peptide was spotted with mutated peptides that were computationally designed to reduce IgE binding. The peptides were 15 amino acids long and included point mutations or double substitution mutations. Next, the plasma mapped to Ara h 6 was analyzed with arrays containing the mutated "de-epitope" spots to screen for those peptides that showed the most significant reduction in binding (data not shown). In addition, the mutation / epitope details presented in Table 1 of Example 2 were collated from the results of both Example 2 and Example 3.

[0340] The two main linear epitope regions of IgE binding were mapped by peptide microarray using plasma from 80 allergic patients. The Ara h 6 linear epitope at the population level was calculated for each peptide by the relative deviation from the median intensity of the slide (Z-score). The distribution of all scores from all slides was plotted and displayed as a box plot, where the x-axis corresponds to all overlapping peptides and the y-axis shows the distribution of Z-scores. The black and gray lines represent 2 and 3 standard deviations from the median intensity of the slide, respectively ( Figure 2 ).

[0341] Example 4: Mutation of single or multiple epitopes

[0342] Objective: Using the data collected in Examples 2 and 3, design variants using combinations of mutations.

[0343] Results: Mutations were combined based on computational predictions of their energetic effects on protein stability. Calculations were performed starting from the solved structure of Ara h 6 (PDB accession number 1W2Q). Several epitopes could be mutated within a single variant. Mutations included 1-7 substitution mutations within an epitope. The designed variants were produced in E. coli and tested by indirect enzyme-linked immunosorbent assay (ELISA) to verify reduced binding to anti-Ara h 6 mAb.

[0344] Table 2: Amino acid sequences of Ara h 6 variants

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353] Summarize

[0354] Following the above procedure, seven Ara h 6 epitopes were discovered.

[0355] The Ara h 6D12 variant showed reduced binding to anti-Ara h 6 mAbs (10 IgG and 2 IgE). Indirect ELISA titrations of increasing concentrations of anti-Ara h 6 mAbs were used to test binding to WT recombinant Ara h 6 (SEQ ID NO: 2) or modified Ara h 6D12 variants (SEQ ID NO: 9), with keyhole limpet hemocyanin (KLH) used as a negative control. The data presented show that the modified Ara h 6D12 variant showed significantly reduced binding to 2 anti-Ara h 6 IgEs (E15C2 and 7B6) and 4 anti-Ara h 6 IgGs (IgG5, IgG8, IgG18, and IgG24) ( Fig. 8A -Figure→8F).

[0356] Example 5: Evaluation of the allergenicity of engineered proteins by ex vivo basophil degranulation assay →

[0357] Objective: To evaluate the allergenicity of engineered Ara h 6 variants relative to the wild-type protein.

[0358] result

[0359] Based on the results of single-site linear and conformational de-epitoping seen in Examples 2-4, mutations that eliminated binding to each epitope were combined to construct Ara h 6 variants mutated at more than one binding site (SEQ ID NO: 3-21 and SEQ ID NO: 24-108; see Table 2 for details). Alternatively, additional sequences were computationally combined starting from residue-level data by a Monte-Carlo program, and protein variants mutated at multiple sites were generated. The mutations listed in Table 1 above summarize the individual mutation sites.

[0360] In some embodiments of Ara h 6 variants, the amino acid sequence set forth in any one of SEQ ID NOs: 3-21 further comprises a methionine located upstream of the N-terminus of the amino acid sequence set forth in any one of SEQ ID NOs: 3-21. In some embodiments of Ara h 6 variants, the amino acid sequence set forth in any one of SEQ ID NOs: 24-108 further comprises a methionine located upstream of the N-terminus of the amino acid sequence set forth in any one of SEQ ID NOs: 24-108.

[0361] The variants generated by this process showed reduced allergenic potential compared to the WT protein. These engineered recombinant variants were expressed in E. coli, purified and tested for allergenicity. The extensive variant collection was first tested by cell degranulation assay using a humanized rat basophilic leukemia cell line (RBL SX-38) sensitized with plasma from patients with peanut allergy. Representative results of the RBL assay for Ara h 6 variants are shown in Fig. 6A - Figure → 6G, Middle. Variant allergens caused significantly reduced cell degranulation compared to WT and natural allergens.

[0362] Summarize

[0363] Based on the RBL ex vivo assay, potential abrogation of allergenicity was observed for multiple Ara h 6 mutant variants with combinations of mutations at more than one epitope.

[0364] Example 6: Immunogenicity Assessment →

[0365] Objective: To evaluate the immunogenicity of representative Ara h 6 variants.

[0366] To ensure immunotherapeutic effect, recombinant engineered hypoallergenic variants should substantially retain immunogenicity, which can achieve reprogramming of immune response. To evaluate the immunogenicity level of Ara h 6 variants, various techniques known in the art can be used, such as T cell assays, animal models, IgE / IgG binding ratios.

[0367] Example 7: Biophysical characteristics of variants →→

[0368] Purpose: It was important to maintain the same oligomerization level of the native protein (i.e. monomers for Ara h 6) to ensure the correct 3D folding of the mutant variants. To verify the oligomerization state of the protein, size exclusion chromatography (SEC) HPLC was performed on each variant and only variants with the correct oligomerization state were considered valid candidates for the development of hypoallergenic variants (data not shown).

[0369] Both WT and D12 variants of Ara h 6 with a 6× histidine tag expressed in E. coli BL21 (DE3) were purified using standard immobilized metal affinity chromatography (IMAC) and SEC. Both purified Ara h 6 proteins with a 6× histidine tag, WT and D12 variants, ran at a size of ˜17 kDa on SDS-PAGE under reducing conditions ( Figure 3A ), and Arah 6 variants D154, D158, D160 and D179 ( Figure 3B ) and D119 (data not shown) were stable and ran as monomers under standard test conditions, as seen in SEC-HPLC analysis ( Figure 4A-4F ).

[0370] Circular dichroism was used to further analyze the thermal stability of some major Ara h 6 variants. The midpoint of the thermal melting temperature (TM) of both the D12 variant and the WT was >90°C ( Figure 5A-Figure 5B ). The spectral minima at -205-210 nm and 220 nm for the intact constructs indicated that both Ara h 6 WT and D12 were predominantly α-helical and had similar thermal stabilities at temperatures up to 90°C, indicating high stability and correct folding.

[0371] Summarize

[0372] The major Ara h 6 variant exhibited a high melting point in CD, indicating thermal stability similar to the WT allergen.Combinatorial variants of Ara h 6 were tested in SEC HPLC and displayed a monomeric mass (-17 kDa) indicative of correct folding.

[0373] While certain features of the variant hypoallergenic peanut allergen Ara h 6 have been illustrated and described herein, numerous modifications, substitutions, changes and equivalents will now occur to those of ordinary skill in the art. It should therefore be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of such variations and uses thereof.

[0374] Example 8: Expression and secretion of allergen variants from mammalian cells

[0375] Objective: To demonstrate that Ara h 6 peanut protein and de-epitope (DE) allergens can be expressed, folded, and secreted from mammalian cells.

[0376] method

[0377] 20 ml of Expi293F (ThermoFisher Scientific) cells were transfected with 20 μg of plasmid encoding Ara h6WT or Ara h 6D12 using Expifectamine 293 transfection reagent according to the manufacturer's instructions. Both constructs were expressed downstream of the human osteonectin leader sequence and contained a C-terminal 6×his tag. The cells were incubated at 37°C, 8% CO 2 Proteins were expressed in Expi293 medium for 5 days. Secreted proteins were purified from the expression medium using Ni-NTA superflow beads, washed and eluted with the addition of 350 mM imidazole. Eluted fractions were analyzed by SDS PAGE reduced or non-reduced with β-mercaptoethanol (β-ME).

[0378] result

[0379] The results showed that the peanut allergen Ara h 6 and its epitope-depleted variants could be expressed at high levels. Figure 7 It is shown that wild type or de-epitope peanut allergen Ara h 6 is expressed and secreted from transfected mammalian cells. Purified Ara h 6 from transfected mammalian cells was found to be of the correct size.

Claims

1. A recombinant Ara h 6 variant polypeptide comprising one or more amino acid substitutions, deletions, insertions, or any combination thereof at one or more of positions 2, 3, 5, 7, 8, 10, 12, 16, 19, 22, 24, 33, 37, 38, 40, 41, 42, 45, 46, 47, 63, 74, 78, 81, 82, 83, 86, 89, 90, 97, 98, 99, 106, 108, 109, 110, 113, 114, 116, or 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO:

2.

2. The recombinant Ara h 6 variant of claim 1, wherein the substitution comprises one or more of the following: (a) S at position 2; (b) D or S at position 3; (c) D at position 5; (d) D at position 7; (e) A or S at position 8; (f) A, S or K at position 10; (g) R, D or N at position 12; (h) S or D at position 16; (i) Q, L or R at position 19; (j) F at position 22; (k) D at position 24; (l) Q or K at position 33; (m) A, T or S at position 37; (n) A or S at position 38; (o) S at position 40; (p) D at position 41; (q) K, E or G at position 42; (r) A or Q at position 45; (s) S, G or R at position 46; (t) S at position 47; (u) R at position 74; (v) L at position 78; (w) A or R at position 81; (x) T at position 82; (y) N or K at position 83; (z) D or S at position 86; (aa) N, R or G at position 89; (bb) D at position 90; (cc) I at position 97; (dd) D or L at position 98; (ee) M at position 99; (ff) K or H at position 106; (gg) P, E or D at position 108; (hh) E or S at position 110; (ii) D or I at position 113; (jj) D, H, A or G at position 114; (kk) K or M at position 116; or (ll) R or T at position 118.

3. The recombinant Ara h 6 variant of claim 1 or 2, wherein the variant comprises an amino acid substitution, deletion, insertion, or any combination thereof at positions 3, 5, 8, 19, 45, 46, 89, 98, 110, 114, 116, and 118 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO:

2.

4. The recombinant Ara h 6 variant of claim 3, wherein the substitution comprises one or more of: D or S at position 3; D at position 5; A or S at position 8; Q, L or R at position 19; A or Q at position 45; S, G or R at position 46; N, R or G at position 89; D or L at position 98; E or S at position 110; D, H, A or G at position 114; K or M at position 116; or R or T at position 118.

5. The recombinant Ara h 6 variant of claim 3 or 4, wherein the variant further comprises an amino acid substitution, deletion, insertion, or any combination thereof at one or more of positions 2, 7, 10, 12, 15, 16, 17, 20, 22, 24, 28, 33, 35, 37, 38, 40, 41, 42, 47, 57, 59, 61, 63, 64, 74, 78, 81, 82, 83, 86, 90, 91, 97, 99, 106, 108, 109, 113, or 123 of SEQ ID NO: 109 compared to the amino acid residues at those same positions in SEQ ID NO:

2.

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

7. The recombinant Ara h 6 variant according to any one of claims 1-6, wherein the variant comprises one or more amino acid substitutions, deletions, insertions, or any combination thereof located within at least a single epitope recognized by an anti-Ara h 6 antibody.

8. The recombinant Ara h 6 variant of claim 1, wherein the variant comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 3-21 or SEQ ID NOs: 24-108.

9. The recombinant Ara h 6 variant of claim 1, wherein the variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 3-21 or SEQ ID NOs: 24-108.

10. An isolated nucleotide or modified nucleotide sequence encoding the recombinant Ara h 6 variant according to any one of claims 1 to 9, wherein the nucleotide or modified nucleotide sequence is DNA or mRNA.

11. The isolated nucleotide or modified nucleotide sequence of claim 10, wherein the mRNA comprises an LNP-formulated mRNA.

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

13. A prokaryotic cell or eukaryotic cell comprising the expression vector according to claim 12, wherein the eukaryotic cell is a yeast cell, a fungal cell, a plant cell or a mammalian cell.

14. A composition comprising the recombinant Ara h 6 variant polypeptide of any one of claims 1-9.

15. A composition according to claim 14 for use in inducing desensitization to peanut and / or immunomodulation of the response to peanut in a subject allergic to peanut.

16. A composition comprising the isolated nucleotide or modified nucleotide sequence according to claim 10 or 11.

17. A composition according to claim 16 for use in inducing desensitization to peanut and / or immunomodulation of the response to peanut in a subject allergic to peanut.

18. A genetically modified peanut plant expressing the recombinant Ara h 6 variant polypeptide of any one of claims 1-9.

19. The genetically modified peanut plant of claim 18, wherein the Ara h 6 variant is expressed from a heterologous nucleic acid.

20. The genetically modified peanut plant of claim 18 or 19, wherein expression of endogenous wild-type Ara h 6 allergen is reduced compared to non-genetically modified peanut.

21. A processed food product comprising the Ara h 6 variant of any one of claims 1-9.

22. The processed food product of claim 21 comprising a reduced amount of endogenous wild-type peanut Ara h6 allergen.

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

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