Novel immunotherapy compositions and uses thereof

Abstract

The present application relates to novel immunotherapeutic compositions and uses thereof. The present invention relates generally to an immunotherapeutic composition. More specifically, the present invention relates to an immunotherapeutic composition that immunointeracts with T lymphocytes in a subject suffering from peanut allergy or allergy to other tree nuts. Such compositions preferably react immunologically with T cells in a subject suffering from an allergy to an Ara h 1 and / or Ara h 2 allergen. The compositions of the present invention are useful in the therapeutic or prophylactic treatment of conditions characterized by an abnormal, improper or otherwise unwanted immune response to peanut, Ara h1 and / or Ara h2, or a derivative or homolog thereof.

Description

[0001] This application is a divisional application of Chinese patent application No. 201480052398.1 entitled "Novel Immunotherapy Composition and Its Use Therein". The original application was PCT international application PCT / AU2014 / 050249 filed on September 25, 2014, which entered the Chinese national phase on March 23, 2016. Invention Field

[0002] This invention generally relates to an immunotherapeutic composition. More specifically, this invention relates to an immunotherapeutic composition that interacts with T lymphocytes in subjects suffering from peanut allergy or allergy to other tree nuts. This composition preferably induces an immune response in T cells in subjects allergic to Ara h1 and / or Ara h2 allergens. The compositions of this invention can be used for the therapeutic or prophylactic treatment of conditions characterized by an abnormal, inappropriate, or otherwise undesirable immune response to peanut, Ara h1, and / or Ara h2, or their derivatives or homologues. Background of the Invention

[0003] Bibliographical details of the publications mentioned by the author in this instruction manual are listed alphabetically at the end of this instruction manual.

[0004] Any reference to prior art in this specification is not, and should not be construed as, an admission or implication of any kind that such prior art is part of the general public knowledge.

[0005] Peanut allergy is a life-threatening and incurable condition affecting approximately 1% of the general population (Husain et al. J Am Acad Dermatol. 66(1):136-43, 2012, Burks, Lancet. 371(9623):1538-46, 2008). Peanut allergy is characterized by the sudden onset of an allergic reaction, which may occur upon exposure to trace amounts of peanut protein (Hurihane et al., J AllergyClin Immunol 100: 596-600, 1997; Pumphrey, Current Opinion in Allergy & Immunology. 4(4):285-90, 2004). Peanut-induced allergic reactions are most often associated with mortality or life-threatening features (Bock et al., J Allergy Clin Immunol. 119(4):1016-8, 2007; Burks 2008, supra). Peanut protein is often hidden in seemingly safe food sources, resulting in up to 50% of patients being accidentally exposed to peanut protein over a 5-year period (Sicherer et al., Paediatrics 102: e6, 1998). Unsurprisingly, peanut and tree nut allergies are associated with significant psychological morbidity in both victims and caregivers, similar to that experienced by patients with chronic debilitating conditions such as rheumatoid arthritis (Primeau et al., Clin Exp Allergy 30: 1135-43, 2000; Kemp et al., Australian Medical Journal 188(9):503-4, 2008). While a cure is urgently needed to eliminate peanut and tree nut allergies, a cause of mortality, it is also necessary to alleviate the long-term psychological burden borne by peanut allergy sufferers.

[0006] To date, efforts in immunotherapy for peanut allergy have yielded extremely limited results. Nelson et al. demonstrated that a rapid immunotherapy regimen using ungraded peanut extract could induce clinical desensitization to peanuts, but this desensitization disappeared in approximately half of the subjects during maintenance administration. Furthermore, frequent anaphylactic reactions were associated with most subjects during the injection, establishment, and maintenance phases (Nelson et al., J Allergy Clin Immunol 99: 744-51, 1997). Oppenheimer et al. confirmed similar findings in their study, also showing that active therapy using ungraded peanut extract was associated with a high incidence of systemic anaphylactic reactions. The data collection in that study was terminated after randomized subjects who were given peanut extract as a placebo died, highlighting the dangerous nature of the condition (Oppenheimer et al., JAllergy Clin Immunol 90: 256-62, 1992).Recent studies on oral immunotherapy using whole peanut powder encourage the feasibility of desensitization, but observed adverse reactions highlight major safety concerns (Hoffman et al., Journal of Allergy and Clinical Immunology 124, 286, 2009; Jones et al., Journal of Allergy and Clinical Immunology 24, 292, 2009; Clark et al., Allergy 64, 1218, 2009; Vashnie et al., Journal of Allergy and Clinical Immunology 127(3):654-60, 2011; Vashnie et al., Journal of Allergy and Clinical Immunology 124(6):1351-2, 2009; Aninostu et al., Clinical and Experimental Allergy 41(9):1273-81, 2011; Allen and Ohshir, Clinical and Experimental Allergy 41(9):1172-4, 2011; Yu et al., International Archives of Allergy and Immunology 159(2):179-182, 2012; Siyakai et al., Journal of Allergy and Clinical Immunology 126(1):31-2, 2010; Brown et al., Journal of Allergy and Clinical Immunology 126(1):83-91, 2010 (Hofmann et al. J. Allergy Clin. Immunol.124, 286, 2009; Jones et al. J. Allergy Clin. Immunol. 24, 292, 2009; Clark et al. Allergy 64, 1218, 2009; Varshney et al. J Allergy Clin Immunol. 127(3):654-60, 2011; Varshney et al. J Allergy Clin Immunol. 124(6):1351-2, 2009; Anagnostou et al. Clin Exp Allergy. 41(9):1273-81, 2011; Allen & O'Hehir. Clin Exp Allergy. 41(9):1172-4, 2011; Yu et al. Int Arch AllergyImmunol. 159(2):179-182, 2012; Thyagarajan et al. J Allergy Clin Immunol. 126(1):31-2, 2010; Blumchen et al. J Allergy Clin Immunol. 126(1):83-91, 2010)).Even excluding children with severe symptoms or a predisposition to asthma, two studies reported allergic episodes: one during an initial food challenge (Clark et al. Allergy 64, 1218, 2009), and another during treatment of children who had not previously experienced an allergic reaction (Hofmann et al. J. Allergy Clin. Immunol. 124, 286, 2009).

[0007] The development of new strategies to overcome morbidity associated with allergen immunotherapy depends on a precise understanding of the immunological basis of successful immunotherapies and their side effects. It has long been established that morbidity induced by allergen immunotherapy is due to IgE cross-linking, and that this effect is not essential for the efficacy of this treatment (Litwin et al., International Archives of Allergy and Immunology 87: 361-61, 998). It is also known that one of the key factors in the development of tolerance to conventional (subcutaneous, sublingual, or oral ungraded allergen extracts) immunotherapy is its ability to divert the major specific T-cell phenotype from T cells. H 2. They become regulatory phenotypes. These regulatory T cells function by producing the anti-inflammatory cytokines IL-10 and / or TGFβ. (Akdis & Akdis, J Allergy Clin Immunol. 123:735-46, 2009; Akdis & Akdis, Nature Reviews: Drug Discovery 8:645-60. 2009; Akdis & Akdis, J Allergy Clin Immunol. 127:18-27, 2011)

[0008] The key difference between antibody and lymphocyte responses lies in antigen recognition. Antibodies rely on molecular tertiary structure to recognize conformational B-cell epitopes, while CD4+ T cells recognize short, linear peptides. This difference in antigen recognition forms the basis of many novel immunotherapeutic strategies, including those based on T-cell epitopes, B-cell epitope mutants, and altered peptide ligands (Rolland et al. Pharmacology & Therapeutics 121:273-284, 2009). These approaches all depend on alterations or deletions of molecular tertiary structures, thus eliminating IgE crosslinking and effector cell activation. Peptide immunotherapy is an approach with documented efficacy, with cases of cat dander allergy and bee venom allergy.Three different studies have shown that, in the absence of any systemic side effects, clinical and immune tolerance can be achieved using sequences containing T-cell epitopes targeting the major bee venom allergen phospholipase A2 (PLA2) (Muller et al., J Allergy Clin Immunol. 101: 747-54, 1998; Tarzi et al., Clin Exp Allergy. 36: 465-74, 2006; Fellrath et al., J Allergy Clin Immunol. 111: 854-61, 2003). (2003), and some studies have demonstrated that peptides based on the structure of the major feline allergen Fel d1 can be used to induce attenuated clinical responses (Norman et al., American Journal of Respiratory and Critical Care Medicine 154: 1623-8, 1996; Marcotte et al., Journal of Allergy and Clinical Immunology 101: 506-13, 1998; Pene et al., Journal of Allergy and Clinical Immunology 102: 571-8, 1998; Oldfield et al., The Lancet 360: 47-53, 2002; Alexander et al., Clinical and Experimental Allergy 35: 52-8, 2004; Alexander et al., Allergy 60: 1269-74, 2005). 506-13, 1998; Pene et al., J Allergy Clin Immunol 102: 571-8, 1998; Oldfield et al. Lancet 360:47-53, 2002; Alexander et al. Clin Exp Allergy 35: 52-8, 2004; Alexander et al. Allergy 60:1269-74, 2005). Recently, a phase IIa trial confirmed a mixture of heptapeptides derived from Fel d1 (Toleromune cat). © Toleromune Cat, Oxford, UK ©The safety, tolerability, and potential efficacy of [the drug] (Worm et al., J Allergy Clin Immunol. 127: 89-97, 2011) are being investigated, while a Phase IIb trial is underway (Moldaver & Larche. Allergy. 66: 784-91, 2011; Worm et al. Expert Opin. Investig. Drugs. 22(10): 1347-1357, 2013). A key aspect of developing such strategies is preserving T-cell epitopes, which can induce alterations in T-cell phenotype.

[0009] The ability to bind directly to MHC class II molecules allows peptides to be presented by unprofessional or immature APCs without promoting induced responsive T cells (Moldaver & Larche, Allergy 66: 784-91, 2011) and / or pro-inflammatory and co-stimulatory signals of tolerance, non-responsiveness, and / or inhibitory activity in other CD4+ T cells expressing MHC class II. This also allows peptides to be presented at a higher frequency than peptides processed from whole molecules (Santambrogio et al., Proc Natl Acad Sci USA, 1999, 96:15056-61), and because they are also safer than whole allergens, peptides can be delivered at higher concentrations, thus more effectively repolarizing T cell responses.

[0010] Importantly, T cells targeting dominant T cell epitopes specific to major allergens can alter the response to intact allergen extracts (linkage inhibition). Numerous studies reporting the success of peptide immunotherapy in mouse allergy models have shown that administration of dominant T-cell epitope peptides of major allergens not only induces tolerance to these peptides but also to purified allergens and intact allergen extracts (Yang et al., Clinical and Experimental Allergy 40(4):668-78, 2010; Yoshitomi et al., Journal of Peptide Science 13(8):499-503, 2007; Malazuela et al., Journal of Molecular Immunology 45(2):438-45, 2008; Rupa et al., Allergy 67(1):74-82, 2012; Hoehn et al., Journal of Experimental Medicine 178(5):1783-8, 1993; Haller et al., Vaccines 21(5-6):549-61, 2003). Sci. 13(8):499-503, 2007; Marazuela et al. Mol Immunol. 45(2):438-45, 2008; Rupa et al. Allergy. 67(1):74-82, 2012; Hoyne et al. J Exp Med. 178(5):1783-8, 1993; Hall et al. al. Vaccine. 21(5-6):549-61, 2003)).

[0011] Therefore, there is a need to identify both the major peanut allergens and, more importantly, the T-cell epitopes of these allergens. The identification, characterization, and analysis of these T-cell epitopes are crucial for developing specific immunotherapeutic or prophylactic methodologies. To this end, although Ara h 1 and / or Ara h 2 peanut allergen molecules have previously been the subject of analysis, the identification of the core T-cell epitope regions is essential for developing effective vaccines.

[0012] In carrying out the work of this invention, dominant HLA degenerate Ara h 1 and / or Ara h 2 core T cell epitope regions have been identified. The unique feature of this set of core T cell epitope regions is their high efficacy level. Unlike previous studies that identified 20-mer Ara h 1 and / or Ara h 2 peptides solely based on their ability to express a certain level of T cell reactivity, a selected set of core T cell epitope regions has been identified that are immunodominant relative to other Ara h 1 and / or Ara h 2 peptide fragments and are also HLA degenerate due to their binding to two or more HLA types. Furthermore, many of these T cell epitope core regions are presented by HLA-DQ molecules. HLA-DQ molecules are more conserved than HLA-DR molecules in mixed populations. Therefore, peptides presented on HLA-DQ achieve broader population coverage.

[0013] Further research has identified specific subgroups of seven peptides containing both Ara h1 and Ara h2 T-cell epitopes that provide particularly potent immune effects. Therefore, the identification of this unique and potent group of peptides has facilitated the development of particularly effective therapeutic prophylaxis for conditions characterized by allergies to Ara h1 and / or Ara h2 or their derivatives or homologues, other tree nuts, or combinations containing Ara h1 and / or Ara h2 molecules, such as abnormal, inappropriate, or otherwise unwanted immune responses to food allergens. Summary of the Invention

[0014] Throughout this specification and the claims, unless the context otherwise requires, the word "comprise" and its variations such as "comprises" or "comprising" shall be understood to mean including one or more of the mentioned whole or steps, but not excluding any other whole or steps or groups of whole or steps.

[0015] As used herein, the term “derived from” should be understood to mean that a specific whole or group of wholes is derived from the specified species, but not necessarily directly from the specified source. Furthermore, as used herein, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0017] This specification contains amino acid sequence information generated using the program PatentIn version 3.5, which is shown here after the bibliography. Each amino acid sequence is indicated by a number in the sequence listing. <210> This is followed by a sequence identifier (e.g., <210> 1. <210> 2, etc., are used for identification. The length, sequence type (protein, etc.), and biological origin of each sequence are identified by numeric indicator fields. <211> , <212> and <213> The information provided in the specification indicates the amino acid sequence. The amino acid sequence mentioned in the specification is identified by the indicator SEQ ID NO: followed by a sequence identifier (e.g., SEQ ID NO:1, SEQ ID NO:2, etc.). The sequence identifier mentioned in the specification corresponds to the numeric indicator field in the sequence listing. <400> This is followed by a sequence identifier (e.g., <400> 1. <400> The information provided in (e.g., SEQ ID NO:1) is associated with the information indicated in the sequence listing. That is, SEQ ID NO:1 as detailed in this specification is associated with the information indicated in the sequence listing. <400> 1. Sequence correlation.

[0018] One aspect of the present invention relates to an immunomodulatory composition comprising at least five of the Ara h 1 and Ara h 2 T cell epitope regions derived from the list of the following components:

[0019] (i)FQNLQNHR(SEQ ID NO:1)

[0020] (ii)IVQIEA (SEQ ID NO:2)

[0021] (iii)NEGVIVKVSK (SEQ ID NO:3)

[0022] (iv)EVKPDKKNPQLQ (SEQ ID NO:4)

[0023] (v)EGALML (SEQ ID NO:5)

[0024] (vi)IMPAAHP (SEQ ID NO:6)

[0025] (vii)LRPXEQHLM (SEQ ID NO:7)

[0026] (viii)ENNQRXMXEA (SEQ ID NO:8)

[0027] Or its functional derivatives or homologues, wherein residue X is cysteine ​​or serine, and the composition comprises at least one T cell epitope region selected from SEQ ID NOS: 1-6 and at least one T cell epitope region selected from SEQ ID NOS: 7-8.

[0028] In another aspect, the LRPXEQHLM is LRPSEQHLM (SEQ ID NO:137).

[0029] In yet another aspect, the ENNQRXMXEA is ENNQRSMSEA (SEQ ID NO:138).

[0030] According to these aspects and embodiments of the invention, the composition comprises at least six of the T cell epitope regions.

[0031] In another aspect, the composition comprises at least seven of the T cell epitope regions.

[0032] In yet another aspect, the composition comprises each of the eight T-cell epitope regions.

[0033] In another aspect, an immunomodulatory composition is provided herein comprising each of the Ara h 1 and Ara h 2 T cell epitope regions derived from the list of the following components:

[0034] (i)FQNLQNHR(SEQ ID NO:1)

[0035] (ii)IVQIEA (SEQ ID NO:2)

[0036] (iii)NEGVIVKVSK (SEQ ID NO:3)

[0037] (iv)EVKPDKKNPQLQ (SEQ ID NO:4)

[0038] (v)EGALML (SEQ ID NO:5)

[0039] (vi)IMPAAHP (SEQ ID NO:6)

[0040] (vii)LRPXEQHLM (SEQ ID NO:7)

[0041] (viii)ENNQRXMXEA (SEQ ID NO:8)

[0042] Or its functional derivatives or homologues, wherein the residue X is cysteine ​​or serine.

[0043] In yet another aspect, an immunomodulatory composition is provided herein comprising each of the Ara h 1 and Ara h 2 T cell epitope regions derived from the list of the following components:

[0044] (i)FQNLQNHR(SEQ ID NO:1)

[0045] (ii)IVQIEA (SEQ ID NO:2)

[0046] (iii)NEGVIVKVSK (SEQ ID NO:3)

[0047] (iv)EVKPDKKNPQLQ (SEQ ID NO:4)

[0048] (v)EGALML (SEQ ID NO:5)

[0049] (vi)IMPAAHP (SEQ ID NO:6)

[0050] (vii)EVKPDKKNPQLQ (SEQ ID NO:137)

[0051] (viii)EVKPDKKNPQLQ (SEQ ID NO:138)

[0052] Or its functional derivatives or homologues.

[0053] In a related aspect, the present invention relates to an immunomodulatory composition comprising one or more peptides, each of which is up to 60 consecutive amino acids in length, and which comprises each of the Ara h 1 and Ara h 2 T cell epitope combinations detailed above.

[0054] In another aspect, the present invention relates to an immunomodulatory composition comprising one or more peptides, each of which is up to 60 consecutive amino acids in length, and which comprises each of the Ara h 1 and Ara h 2 T cell epitope regions derived from the list of the following:

[0055] (i)FQNLQNHR(SEQ ID NO:1)

[0056] (ii)IVQIEA (SEQ ID NO:2)

[0057] (iii)NEGVIVKVSK (SEQ ID NO:3)

[0058] (iv)EVKPDKKNPQLQ (SEQ ID NO:4)

[0059] (v)EGALML (SEQ ID NO:5)

[0060] (vi)IMPAAHP (SEQ ID NO:6)

[0061] (vii)EVKPDKKNPQLQ (SEQ ID NO:7)

[0062] (viii)EVKPDKKNPQLQ (SEQ ID NO:8)

[0063] Or its functional derivatives or homologues.

[0064] In yet another aspect, the peptides or T-cell epitopes can modify T-cell function when presented to T-cells isolated from subjects suffering from conditions characterized by abnormal immune responses to Ara h1 and / or Ara h2 or to combinations containing Ara h1 and / or Ara h2, such as food allergens, but these peptides cannot bind to Ara h1-specific and / or Ara h2-specific IgE.

[0065] Based on these aspects, the peptides are selected from a list consisting of the following:

[0066] (i)FQNLQNHRIVQIEAKPNTLV (SEQ ID NO:11)

[0067] (ii)STRSSENNEGVIVKVSKE (SEQ ID NO:12)

[0068] (iii)EVKPDKKNPQLQ (SEQ ID NO:4)

[0069] (iv)VEIKEGALMLPHFNSKA (SEQ ID NO:13)

[0070] (v)VFIMPAAHPVAINASS (SEQ ID NO:14)

[0071] (vi)ANLRPXEQHLM (SEQ ID NO:15)

[0072] (vii)EFENNQRXMXEALQ (SEQ ID NO:16)

[0073] (viii)NNFGKLFEVKPDKKNPQLQ (SEQ ID NO:17)

[0074] (ix)gdvfimpaahpvainasse (SEQ ID NO:18)

[0075] (x)SQLERANLRPXEQHLM (SEQ ID NO:19)

[0076] (xi)ELNEFENNQRXMXEALQ (SEQ ID NO:20)

[0077] (xii)FQNLQNHRIV (SEQ ID NO:21)

[0078] (xiii)RIVQIEAKPNTLV (SEQ ID NO:22)

[0079] (xiv)ENNEGVIVKVSKE (SEQ ID NO:23)

[0080] (xv)EVKPDKKNPQLQD (SEQ ID NO:24)

[0081] (xvi)EFENNQRXMXEALQQI (SEQ ID NO:25)

[0082] (xvii)NNFGKLFEVKPDKKNPQLQD (SEQ ID NO:26)

[0083] (xviii)ELNEFENNQRXMXEALQQI (SEQ ID NO:27)

[0084] (xx)WSTRSSENNEGVIVKVSKE (SEQ ID NO:28)

[0085] (xxi)GDVFIMPAAHPVAINASS (SEQ ID NO:29)

[0086] Or its functional derivatives or homologues, wherein residue X is cysteine ​​or serine.

[0087] In one embodiment, residue X is serine.

[0088] In another aspect, the peptide is selected from:

[0089] (i)FQNLQNHRIVQIEAKPNTLV (SEQ ID NO:11)

[0090] (ii)STRSSENNEGVIVKVSKE (SEQ ID NO:12)

[0091] (iii)EVKPDKKNPQLQ (SEQ ID NO:4)

[0092] (iv)VEIKEGALMLPHFNSKA (SEQ ID NO:13)

[0093] (v)VFIMPAAHPVAINASS (SEQ ID NO:14)

[0094] (vi)ANLRPSEQHLM (SEQ ID NO:31)

[0095] (vii)EFENNQRSMSEALQ (SEQ ID NO:32)

[0096] (viii)EVKPDKKNPQLQD (SEQ ID NO:24)

[0097] (ix)EFENNQRSMSEALQQI (SEQ ID NO:33)

[0098] Or its functional derivatives or homologues.

[0099] In another aspect, the immunomodulatory composition comprises each of the Ara h1 and Ara h2 T cell peptides from the list of the following components:

[0100] (i) FQNLQNHRIVQIEAKPNTLV (SEQ ID NO: 11);

[0101] (ii)STRSSENNEGVIVKVSKE (SEQ ID NO: 12);

[0102] (iii) EVKPDKKNPQLQ (SEQ ID NO:34) and / or EVKPDKKNPQLQD (SEQ ID NO:24);

[0103] (iv) VEIKEGALMLPHFNSKA (SEQ ID NO: 13);

[0104] (v)VFIMPAAHPVAINASS (SEQ ID NO:14);

[0105] (vi)ANLRPSEQHLM (SEQ ID NO:31); and

[0106] (vii) EFENNQRSMSEALQ (SEQ ID NO:32) and / or EFENNQRSMSEALQQI (SEQ ID NO:33).

[0107] Or its functional derivatives or homologues.

[0108] In another aspect, the inventors have devised a preferred group of seven peptides, five of which contain Ara h1 T cell epitopes and two of which contain Ara h2 T cell epitopes. When administered together, these peptides act particularly effectively to induce desensitization or tolerance and thereby preventively or therapeutically treat hypersensitivity reactions to compositions containing Ara h1 and / or Ara h2, such as foods. These peptides are:

[0109] (i)FQNLQNHRIVQIEAKPNTLV (SEQ ID NO:11)

[0110] (ii)STRSSENNEGVIVKVSKE (SEQ ID NO:12)

[0111] (iii) EVKPDKKNPQLQ (SEQ ID NO:4) and / or EVKPDKKNPQLQD (SEQ ID NO:24)

[0112] (iv)VEIKEGALMLPHFNSKA (SEQ ID NO:13)

[0113] (v)VFIMPAAHPVAINASS (SEQ ID NO:14)

[0114] (vi)ANLRPSEQHLM (SEQ NO:31)

[0115] (vii)EFENNQRSMSEALQ (SEQ NO:32) and / or EFENNQRSMSEALQQI (SEQ ID NO:33)

[0116] In yet another aspect, an immunomodulatory composition is provided herein comprising each of the Ara h 1 and Ara h 2 T cell peptides from the list of the following components:

[0117] (i)FQNLQNHRIVQIEAKPNTLV (SEQ ID NO:11)

[0118] (ii)STRSSENNEGVIVKVSKE (SEQ ID NO:12)

[0119] (iii) EVKPDKKNPQLQ (SEQ ID NO:4) and / or EVKPDKKNPQLQD (SEQ ID NO:24)

[0120] (iv)VEIKEGALMLPHFNSKA (SEQ ID NO:13)

[0121] (v)VFIMPAAHPVAINASS (SEQ ID NO:14)

[0122] (vi)ANLRPSEQHLM (SEQ NO:31)

[0123] (vii)EFENNQRSMSEALQ (SEQ NO:32) and / or EFENNQRSMSEALQQI (SEQ ID NO:33)

[0124] In yet another aspect, a composition is provided herein comprising each of Ara h 1 and Ara h 2 T-cell peptides from the list of the following components:

[0125] (i)FQNLQNHRIVQIEAKPNTLV (SEQ ID NO:11)

[0126] (ii)STRSSENNEGVIVKVSKE (SEQ ID NO:30)

[0127] (iii) EVKPDKKNPQLQ (SEQ ID NO:4) and / or EVKPDKKNPQLD (SEQ ID NO:24)

[0128] (iv)VEIKEGALMLPHFNSKA (SEQ ID NO:13)

[0129] (v)VFIMPAAHPVAINASS (SEQ ID NO:14)

[0130] (vi)ANLRPSEQHLM (SEQ NO:31)

[0131] (vii)EFENNQRSMSEALQ (SEQ NO:32) and / or EFENNQRSMSEALQQI (SEQ ID NO:33)

[0132] When administered to subjects suffering from symptoms characterized by hypersensitivity to Ara h1 and / or Ara h2 or to compositions containing Ara h1 and / or Ara h2, these peptides are able to alleviate said hypersensitivity.

[0133] This invention relates to a composition comprising a peptide as defined above. However, it should be understood that the subject composition may comprise other components, such as additional peptides. Examples of other peptides that may be included in the composition include, but are not limited to:

[0134] (i)ALMLLPHFNSKAMVIVVV (SEQ ID NO:34)

[0135] (ii)NNFGKLFEVKPDKKNPQ (SEQ ID NO:35)

[0136] (iii)SQLERANLRPXEQ (SEQ ID NO:36)

[0137] (iv)ELNEFENNQRXM (SEQ ID NO:37)

[0138] (v)NNFGKLFEVKPDKKNPQLQD (SEQ ID NO:38)

[0139] (vi)NNFGKLFEVKPDKKNPQL (SEQ ID NO:40)

[0140] (vii)SQLERANLRPXEQH (SEQ ID NO:41)

[0141] (viii)KAMVIVVVNKGTGNLELVAV (SEQ ID NO:42)

[0142] (ix)RELRNLPQQXGLRA (SEQ ID NO:43)

[0143] (x)KAMVIVVVNKG (SEQ ID NO:44)

[0144] (xi)AMVIVVVNKGTGNLELV (SEQ ID NO:45)

[0145] (xii)VVNKGTGNLELVAVRK (SEQ ID NO:46)

[0146] Or its functional derivatives or homologues, wherein residue X is cysteine ​​or serine.

[0147] In another aspect, the present invention provides a nucleic acid molecular composition comprising one or more nucleic acid molecules encoding or complementary to sequences encoding or derivatives, homologues or analogues of T-cell epitopes and peptides as defined above.

[0148] In another aspect, the present invention provides a method for treating and / or preventing a condition in a subject characterized by an abnormal, unwanted, or otherwise inappropriate immune response to Ara h 1 and / or Ara h 2 or to an allergen in a composition comprising Ara h 1 and / or Ara h 2, the method comprising administering to the subject an effective amount of an immunomodulatory composition as defined above, for a period of time, provided that the presence or function of T cells in the subject against the Ara h 1 and / or Ara h 2 or other allergens is sufficient to eliminate or reduce such presence or function.

[0149] In another aspect, the symptoms refer to hypersensitivity to peanuts or tree nuts containing Ara h 1 and Ara h 2 or similar molecules, such as hazelnuts, almonds, or Brazil nuts.

[0150] In another aspect, the method enables desensitization to or induction of immune tolerance to Ara h 1 and / or Ara h 2 or other allergens of the composition.

[0151] In another aspect, the desensitization or tolerance is achieved by inducing T cell non-responsiveness or apoptosis.

[0152] In yet another aspect, the desensitization or tolerance is achieved by inducing Ara h 1 or Ara h 2 specific Treg cells.

[0153] Another aspect of the invention covers the use of the immunomodulatory composition as defined above in the manufacture of a medicament for treating a symptom in mammals characterized by an abnormal, unwanted, or otherwise inappropriate immune response to Ara h 1 and / or Ara h 2.

[0154] Preferably, the symptoms are hypersensitivity to peanuts or tree nuts such as hazelnuts containing Ara h 1 and / or Ara h 2 or similar molecules.

[0155] In yet another aspect, the invention covers a vaccine comprising a composition as defined above and one or more pharmaceutically acceptable carriers and / or diluents. The composition is referred to as the active ingredient.

[0156] Another aspect of the invention relates to compositions as defined above when used in the methods of the invention. Attached Figure Description

[0157] Figure 1Graphical representation of CFSE screening for peptide-specific PBMC T cells: (A) CFSE-labeled PBMCs from peanut-allergic subjects incubated with whole peanut extract or Vax (7-peptide compilation). Boxes indicate the percentage of activated and proliferating CD4+ T cells (CD25+CFSElo). SI indicates a multiple increase in T cell activation compared to antigen-free controls. (B) Confirmation of CD4+ T cell activation and proliferation in peanut-allergic donor PBMCs in response to Vax (7-peptide compilation) from 7 subjects (all with positive SI>2.5).

[0158] Figure 2 Graphical representation of the basophil activation test (BAT): (A) FACS plot of blood from peanut-allergic subjects incubated with either whole peanut extract or Vax (7-peptide formulation). Basophils are labeled as IgEhi cells (box, first plot) and activated basophils are labeled as CD63hi (box, plots 2-4). (B) BAT data; and (C) Histamine release data (measured by commercial kit) from peanut-allergic subjects after incubation with increased concentrations (μg / ml) of whole peanut extract or Vax. Positive controls were anti-IgE and fMLP. Whole peanut extract resulted in high levels of basophil activation and histamine release, but Vax did not. Data represent 14 peanut-allergic subjects tested.

[0159] Figure 3 This is a schematic representation of a method for identifying dominant epitopes of the major peanut allergens Ara h 1 and Ara h 2.

[0160] Figure 4 This is a graphical representation of a 7-day CFSE assay designed to detect the ability of dominant 20-mer peptide to induce T cell proliferation in intact PBMCs of peanut-allergic donors. The numbers in the boxes indicate the percentage of dividing (CFSE-low) CD4+ T cells, SI= a multiple increase in dividing cells compared to the unstimulated control.

[0161] Figure 5 This is a graphical representation of the response frequencies of T cell lines to the Ara h1 20-mer peptide. The boxes indicate the nine dominant 20-mers ultimately selected (based on multiple parameters).

[0162] Figure 6 This is a graphical representation of PBMC screening for dominant Ara h 1 20-mer. The ability of the dominant 20-mer to target specific CD4+ T cells in PBMCs from peanut-allergic donors was tested.

[0163] Figure 7This is a graphical representation showing the response frequency of T cell lines to Ara h 2 20-mer peptides and the number of specific TCLs per 20-mer. The boxes indicate the four dominant 20-mer peptides ultimately selected based on multiple parameters.

[0164] Figure 8 It is a graphical representation of the core T cell epitope mapping results.

[0165] Figure 9 This is a graphical representation of HLA restriction of dominant Ara h1 and Ara h2 T cell epitopes.

[0166] Figure 10 This is a graphical representation of the T-cell recognition of peptides in which selected cysteine ​​residues are replaced by serine residues. (e.g., via...) 3 The H-thymidine uptake assay showed the proliferation of TCLs in response to either the "parental" (cysteine-containing) or serine-substituted Ara h 2 peptide. The figure shows representative TCLs (mean cpm repeat wells + SD) for each epitope. A) Ara h 2 (32–44); B) Ara h 2 (37–47); C) Ara h 2 (91–102); D) Ara h 2 (95–107); E) Ara h 2 (128–141).

[0167] Figure 11 This is a graphical representation of T cell cytokine production in response to peptides in which selected cysteine ​​residues are replaced by serine residues. Cytokine secretion in response to the “parental” or cysteine-substituted Ara h 2 peptide, as measured by ELISPOT. The figure shows representative TCLs (mean repeat pore spots + SD) for each epitope. IL-4, black bars; IL-5, shaded bars; IFN-γ, white bars; A) Ara h 2 (32–44); B) Ara h 2 (37–47); C) Ara h 2 (91–102); D) Ara h 2 (95–107); E) Ara h 2 (128–141).

[0168] Figure 12 This is a graphical representation of the PBMC responses of peptide libraries and whole peanuts. The peptides contained in each library are shown in Table 23. The p-values ​​represent the Wilcoxon paired signed-rank test (for nonparametric data).

[0169] Figure 13 This is a graphical representation of the PBMC responses of peptide libraries and whole peanuts. The peptides contained in each library are shown in Table 23. The p-values ​​represent the Wilcoxon paired signed-rank test (for nonparametric data).

[0170] Figure 14It is a graphical representation of PBMC T cell responses to a preferred 7-peptide library. Statistics: Kruskal-Wallis test for nonparametric data and post-hoc correction for testing differences between multiple groups. The data were normalized to the percentage of responses to whole peanuts due to variations in the magnitude of responses from different subjects in different groups analyzed for different libraries.

[0171] Figure 15 This is a graphical representation of the inhibition of T cell proliferation induced by Ara h2 peptide.

[0172] Figure 16 This is a diagram illustrating the inhibition of T cell proliferation induced by Ara h1 peptide. Detailed Implementation Plan

[0173] This invention is based in part on the identification of the Ara h 1 and Ara h 2 epitope sets, which, when administered together in groups of at least five, produce an immune effect more potent than that produced by any of these epitopes alone or in combination with these or other Ara h 1 or Ara h 2 peptides. Specifically, it has been determined that the use of all eight epitopes produces particularly and exceptionally potent functional results, especially when administered in the context of the heptapeptides exemplified herein. This composition is designed to enable the development of significantly more potent therapeutic and preventative compositions and treatments than those available to date for conditions such as, but not limited to, peanut allergy.

[0174] Therefore, one aspect of the present invention relates to an immunomodulatory composition comprising at least five of the Ara h 1 and Ara h 2 T cell epitope regions derived from the list of the following components:

[0175] (i)FQNLQNHR(SEQ ID NO:1)

[0176] (ii)IVQIEA (SEQ ID NO:2)

[0177] (iii)NEGVIVKVSK (SEQ ID NO:3)

[0178] (iv)EVKPDKKNPQLQ (SEQ ID NO:4)

[0179] (v)EGALML (SEQ ID NO:5)

[0180] (vi)IMPAAHP (SEQ ID NO:6)

[0181] (vii)LRPXEQHLM (SEQ ID NO:7)

[0182] (viii)ENNQRXMXEA (SEQ ID NO:8)

[0183] Or its functional derivatives or homologues, wherein residue X is cysteine ​​or serine, and the composition comprises at least one T cell epitope region selected from SEQ ID NOS: 1-6 and at least one T cell epitope region selected from SEQ ID NOS: 7-8.

[0184] In one embodiment, the LRPXEQHLM is LRPSEQHLM (SEQ ID NO:137).

[0185] In another embodiment, the ENNQRXMXEA is ENNQRSMSEA (SEQ ID NO:138).

[0186] According to these aspects and embodiments of the invention, the composition comprises at least six of the T cell epitope regions.

[0187] In another embodiment, the composition comprises at least seven of the T cell epitope regions.

[0188] In yet another embodiment, the composition comprises each of the eight T-cell epitope regions.

[0189] According to this embodiment, an immunomodulatory composition is provided comprising each of the Ara h 1 and Ara h 2 T cell epitope regions derived from the list of the following components:

[0190] (i)FQNLQNHR(SEQ ID NO:1)

[0191] (ii)IVQIEA (SEQ ID NO:2)

[0192] (iii)NEGVIVKVSK (SEQ ID NO:3)

[0193] (iv)EVKPDKKNPQLQ (SEQ ID NO:4)

[0194] (v)EGALML (SEQ ID NO:5)

[0195] (vi)IMPAAHP (SEQ ID NO:6)

[0196] (vii)LRPXEQHLM (SEQ ID NO:7)

[0197] (viii)ENNQRXMXEA (SEQ ID NO:8)

[0198] Or its functional derivatives or homologues, wherein the residue X is cysteine ​​or serine.

[0199] In another embodiment, an immunomodulatory composition is provided herein comprising each of the Ara h 1 and Ara h 2 T cell epitope regions derived from the list of the following components:

[0200] (i)FQNLQNHR(SEQ ID NO:1)

[0201] (ii)IVQIEA (SEQ ID NO:2)

[0202] (iii)NEGVIVKVSK (SEQ ID NO:3)

[0203] (iv)EVKPDKKNPQLQ (SEQ ID NO:4)

[0204] (v)EGALML (SEQ ID NO:5)

[0205] (vi)IMPAAHP (SEQ ID NO:6)

[0206] (vii)EVKPDKKNPQLQ (SEQ ID NO:137)

[0207] (viii)EVKPDKKNPQLQ (SEQ ID NO:138)

[0208] Or its functional derivatives or homologues.

[0209] In a related aspect, the present invention relates to an immunomodulatory composition comprising one or more peptides, each of which is up to 60 consecutive amino acids in length, and which comprises each of the Ara h 1 and Ara h 2 T cell epitope combinations detailed above.

[0210] In this respect, the present invention relates to an immunomodulatory composition comprising one or more peptides, each of which is up to 60 consecutive amino acids in length, and which comprises each of the Ara h1 and Ara h2 T cell epitope regions derived from the list of the following:

[0211] (i)FQNLQNHR(SEQ ID NO:1)

[0212] (ii)IVQIEA (SEQ ID NO:2)

[0213] (iii)NEGVIVKVSK (SEQ ID NO:3)

[0214] (iv)EVKPDKKNPQLQ (SEQ ID NO:4)

[0215] (v)EGALML (SEQ ID NO:5)

[0216] (vi)IMPAAHP (SEQ ID NO:6)

[0217] (vii)EVKPDKKNPQLQ (SEQ ID NO:7)

[0218] (viii)EVKPDKKNPQLQ (SEQ ID NO:8)

[0219] Or its functional derivatives or homologues.

[0220] In another embodiment of the foregoing aspects of the invention, the peptides or T-cell epitope regions, when presented to T cells isolated from a subject suffering from symptoms characterized by abnormal immune responses to Ara h1 and / or Ara h2 or to compositions containing Ara h1 and / or Ara h2, such as food allergens, are capable of modifying T-cell function, but these peptides cannot bind to Ara h1-specific and / or Ara h2-specific IgE.

[0221] This invention is not intended to limit the scope of the invention. Peanuts contain a number of proteins, the number of which, when viewed on SDS-PAGE, are characteristic bands depending on the method used. Up to 53 bands are visible after high-performance liquid chromatography (de Jong et al., Clin Exp Allergy 28: 743-51, 1998). Of these proteins, only two have been confirmed as major allergens using standard criteria, hence IgE reactivity is observed in more than 50% of peanut allergy populations; these proteins are referred to as Ara h 1 and Ara h 2 (Burks et al., Allergy 53: 725-30, 1998).Although many studies have shown that Ara h2 is stronger of the two allergens (Blanc et al., Clinical and Experimental Allergy. 2009; 39(8):1277-85; Koppelman et al., Clinical and Experimental Allergy. 2004; 34(4):583-90; Palmer et al., Clinical Immunology. 2005;115(3):302-12), Ara h1 also plays a major role in the pathogenesis of peanut allergy. Numerous studies have reported that symptom severity is related to the activity of Ara h1 and Ara h2. 2. Strong correlation between the IgE reactivity of the two (Glaumann et al. Allergy. 2012; 67(2):242-7; Jiang et al. Pediatr Allergy Immunol. 2009; 21(2 Pt 2):e429-38; Asarnoj et al. Allergy. 2010, 65(9):1189-95; Muwula et al. International Archives of Allergy and Immunology 2011; 156(3):282-90; Lin et al. Journal of Microbiology and Immunology 2012; Peters et al. Clinical and Experimental Allergy 2007; 37(1):108-15) Allergy. 2010, 65(9):1189-95; Moverare et al. Int Arch Allergy Immunol 2011; 156(3):282-90; Lin et al. J Microbiol Immunol Infect. 2012; Peeters et al. Clin Exp Allergy. 2007; 37(1):108-15). Ara h 1 is the most abundant major allergen in peanuts, accounting for 12%-16% of the total peanut protein (Koppelman et al. Allergy. 2001; 56(2):132-7).

[0222] This invention is not limited in any way. The Ara h 1 allergen is a 7S seed storage glycoprotein or pea globulin. The concentration of Ara h 1 in peanuts increases with nucleolus size (4-16 mg of extracted Ara h 1 / g peanut), and therefore the protein expression is associated with peanut maturity (Pomés et al., 2006, Clin. Exp. Allergy 36(6):824-30). Ara h 1 is a homotrimer held together by a hydrophobic region at the distal end of the monomer, in which most IgE-binding B-cell epitopes are located. Each 64.5 kD monomer has a cupin motif consisting of two core β-barrels, each β-barrel associated with a loop domain of an α-helix.

[0223] Ara h 2 is a glycoprotein identified as a member of the lupin seed storage family. 20% of the molecular weight of Ara h 2 represents the carbohydrate side chain, and it migrates as a duplex on SDS-PAGE with an average molecular weight of 17.5 kDa (Burks et al, Int ArchAllergy Immunol 119:165-172, 1992). Based on the reactivity of Ara h 2 with 6 / 6 test serum, Ara h 2 has been characterized as a major allergen (Burks et al, 1992, ibid.). The importance of Ara h 2 has also been confirmed by others; Clarke demonstrated that, based on Western blotting of crude peanut extract, 71% of subjects had Ara h 2-specific IgE. Kleber-Janke et al. have demonstrated that 85% of subjects have IgE specific to its recombinant form based on Western blotting, while de Jong's group has shown that approximately 78% of their subjects exhibit IgE specific to purified native Ara h 2 (Clarke et al., Clin Exp Allergy 28: 1251-7, 1998; de Jong et al., 1998 ibid.; Kleber-Janke et al., International Archives of Allergy and Immunology 119: 265-274, 1999).

[0224] The reference to “Ara h 1” should be understood to refer to all forms of this molecule, including any isoform that can be produced by alternative splicing of Ara h 1 mRNA, or functional mutants or polymorphic forms of Ara h 1. This should be further understood to extend to any protein encoded by the Ara h 1 gene, any subunit polypeptide, such as the precursor forms that may be produced, whether as monomers, multimers, or fusion proteins. It also includes references to Ara h 1 analogs or equivalents, which may arise in cases where products naturally containing Ara h 1 are synthesized for the purpose of producing products such as food additives. The present invention therefore provides T-cell epitopes and methods for diagnosing and treating any condition characterized by hypersensitivity to Ara h 1 or Ara h 1 analogs (such as peanut allergy or tree nut allergy) or allergy to allergens present in compositions such as foods (which also contain Ara h 1). Preferably, Ara h 1 comprises the sequence listed in SEQ ID NO:9, and Ara h 2 comprises the sequence listed in SEQ ID NO:10.

[0225] The term "T cell" should be understood to refer to any cell containing a T cell receptor. In this regard, the T cell receptor may comprise any one or more of the α, β, γ, or δ chains. This invention is not intended to be limited to any specific functional subclass of T cells, but in preferred embodiments, the subject T cell is a T helper cell, and still more preferably a Th2 cell and / or a Treg cell. In this regard, the term "modified T cell function" should be understood to refer to any one or more functions that a modified T cell can perform. For example, the subject function may be proliferation, differentiation, or other forms of cellular functional activity, such as cytokine production. In one embodiment, the subject functional activity is proliferation.

[0226] Regarding the “functional modification” of T cells isolated from subjects suffering from conditions characterized by an abnormal, unwanted, or inappropriate immune response to Ara h1 and / or Ara h2 or to compositions containing Ara h1 and / or Ara h2, it should be understood that this does not necessarily refer to modification of the function of all T cells in a given biological sample, but may actually reflect functional modifications of only some T cells in the sample. For example, only a portion of the T helper cells in a given T cell sample may be functionally responsive to contact with the subject peptide. Such a portion should be understood to be within the scope of the invention. It should also be understood that the T cells derived from the subject may be freshly harvested T cells or they may have undergone some form of in vitro or in vivo manipulation prior to testing. For example, T cell lines may be generated from cell samples, and it is these T cell lines that subsequently form the subject-derived T cell population tested according to the invention. With regard to the subject functional activity being T cell proliferation, T cell proliferation assays are preferably performed as disclosed herein. Still more preferably, the subject modification of T cell function is the induction of proliferation. In this regard, references to “Ara h 1-responsive” or “Arah 2-responsive” T cells should be understood as referring to HLA-presenting T cells that are functionally responsive to Ara h 1 and / or Ara h 2 T cell epitopes, respectively. Similarly, references to “Ara h 1-specific” or “Arah 2-specific” IgE should be understood as referring to IgE targeting Ara h 1 or Ara h 2 B cell epitopes, respectively.

[0227] The reference to "abnormal, unnecessary, or otherwise inappropriate" immune response should be understood as referring to any form of physiological activity involving the activation and / or functionalization of one or more immune cells, where the inappropriateness lies in the type or degree of the immune cell activity. Abnormality may lie in its failure to occur when it should, or its occurrence when it should not, according to known immunological principles. In another instance, inappropriateness of the immune response may lie in its physiologically normal nature, but its unnecessary and / or undesirable nature, such as a type I hypersensitivity reaction to a harmless allergen. In the context of this invention, such an immune response may be directed against Ara h1 and / or Ara h2, or it may be directed against different allergens present in the composition together with Ara h1 and / or Ara h2. Without limiting the invention to any particular theory or mode of action, it has been determined that even in cases of hypersensitivity reactions against allergens other than Ara h1 and / or Ara h2, where the allergen is present in the composition still containing Ara h1 and / or Ara h2, treatment via the method of the present invention targeting Ara h1 and / or Ara h2 still induces beneficial regulation of Th2 and Treg function, thereby reducing any existing hypersensitivity to irrelevant allergens. Preferably, the immune response is peanut hypersensitivity.

[0228] “Peanut hypersensitivity” refers to the clinical symptoms of IgE-induced peanut hypersensitivity. However, it should be understood that while clinical symptoms may be apparent, not all such individuals will necessarily exhibit detectable levels of peanut-specific serum IgE as measured using the Kallestad Allercoat EAST system (Sanofi-Pasteur Diagnostics, USA), though such individuals should still be understood as falling within the definition of “peanut hypersensitivity.” Alternatively, testing can be performed using any EAST, Pharmacia, or UniCap system or an allergen prick test. The reference to “Ara h 1 and / or Ara h 2 hypersensitivity” should be understood to have a corresponding meaning in the context of reactivity to Ara h 1 and / or Ara h 2 proteins.

[0229] Based on the foregoing, the peptide is selected from a list consisting of the following:

[0230] (i)FQNLQNHRIVQIEAKPNTLV (SEQ ID NO:11)

[0231] (ii)STRSSENNEGVIVKVSKE (SEQ ID NO:12)

[0232] (iii)EVKPDKKNPQLQ (SEQ ID NO:4)

[0233] (iv)VEIKEGALMLPHFNSKA (SEQ ID NO:13)

[0234] (v)VFIMPAAHPVAINASS (SEQ ID NO:14)

[0235] (vi)ANLRPXEQHLM (SEQ ID NO:15)

[0236] (vii)EFENNQRXMXEALQ (SEQ ID NO:16)

[0237] (viii)NNFGKLFEVKPDKKNPQLQ (SEQ ID NO:17)

[0238] (ix)gdvfimpaahpvainasse (SEQ ID NO:18)

[0239] (x)SQLERANLRPXEQHLM (SEQ ID NO:19)

[0240] (xi)ELNEFENNQRXMXEALQ (SEQ ID NO:20)

[0241] (xii)FQNLQNHRIV (SEQ ID NO:21)

[0242] (xiii)RIVQIEAKPNTLV (SEQ ID NO:22)

[0243] (xiv)ENNEGVIVKVSKE (SEQ ID NO:23)

[0244] (xv)EVKPDKKNPQLQD (SEQ ID NO:24)

[0245] (xvi)EFENNQRXMXEALQQI (SEQ ID NO:25)

[0246] (xvii)NNFGKLFEVKPDKKNPQLQD (SEQ ID NO:26)

[0247] (xviii)ELNEFENNQRXMXEALQQI (SEQ ID NO:27)

[0248] (xx)WSTRSSENNEGVIVKVSKE (SEQ ID NO:28)

[0249] (xxi)GDVFIMPAAHPVAINASS (SEQ ID NO:29)

[0250] Or its functional derivatives or homologues, wherein residue X is cysteine ​​or serine.

[0251] In one embodiment, residue X is serine.

[0252] Preferably, the peptide is selected from:

[0253] (i)FQNLQNHRIVQIEAKPNTLV (SEQ ID NO:11)

[0254] (ii)STRSSENNEGVIVKVSKE (SEQ ID NO:12)

[0255] (iii)EVKPDKKNPQLQ (SEQ ID NO:4)

[0256] (iv)VEIKEGALMLPHFNSKA (SEQ ID NO:13)

[0257] (v)VFIMPAAHPVAINASS (SEQ ID NO:14)

[0258] (vi)ANLRPSEQHLM (SEQ ID NO:31)

[0259] (vii)EFENNQRSMSEALQ (SEQ ID NO:32)

[0260] (viii)EVKPDKKNPQLQD (SEQ ID NO:24)

[0261] (ix)EFENNQRSMSEALQQI (SEQ ID NO:33)

[0262] Or its functional derivatives or homologues.

[0263] In another embodiment, the immunomodulatory composition comprises each of the Arah 1 and Arah 2 T cell peptides from the list of the following components:

[0264] (i) FQNLQNHRIVQIEAKPNTLV (SEQ ID NO: 11);

[0265] (ii)STRSSENNEGVIVKVSKE (SEQ ID NO: 12);

[0266] (iii) EVKPDKKNPQLQ (SEQ ID NO:34) and / or EVKPDKKNPQLQD (SEQ ID NO:24);

[0267] (iv) VEIKEGALMLPHFNSKA (SEQ ID NO: 13);

[0268] (v)VFIMPAAHPVAINASS (SEQ ID NO:14);

[0269] (vi)ANLRPSEQHLM (SEQ ID NO:31); and

[0270] (vii) EFENNQRSMSEALQ (SEQ ID NO:32) and / or EFENNQRSMSEALQQI (SEQ ID NO:33).

[0271] Or its functional derivatives or homologues.

[0272] The reduction of hypersensitivity to peanuts, Ara h1, and Ara h2 (or more generally, allergen hypersensitivity) will be discussed in more detail below. Simply put, however, this can take the form of partially or completely desensitizing or tolerating an individual to Ara h1 and Ara h2, or more generally to peanuts or other proteins.

[0273] The term "peptide" includes references to peptides, polypeptides, or proteins, or portions thereof. Peptides can be glycosylated or non-glycosylated and / or may contain a series of other molecules, such as amino acids, lipids, carbohydrates, or other peptides, polypeptides, or proteins, that are fused, linked, bound, or otherwise associated with the protein. The term "peptide" as used below includes peptides comprising an amino acid sequence as well as peptides associated with other molecules, such as amino acids, lipids, carbohydrates, or other peptides, polypeptides, or proteins.

[0274] "Derivatives" include fragments, parts / portions, and variants from natural, synthetic, or recombinant sources, including fusion proteins. Parts or fragments include, for example, the active region of a subject peptide. Derivatives can originate from the insertion, deletion, or substitution of amino acids. Amino acid insertion derivatives include amino and / or carboxyl terminal fusions and intra-sequence insertions of one or more amino acids. Insertion-type amino acid sequence variants are variants that introduce one or more amino acid residues into predetermined sites in a protein, although random insertion is also possible with suitable selection of the resulting product. Deletion-type variants are characterized by the removal of one or more amino acids from the sequence.

[0275] A substituted amino acid variant is a variant in which at least one residue in the sequence has been removed and a different residue has been inserted at its position. Examples of substituted amino acid variants are conserved amino acid substitutions. Conserved amino acid substitutions typically include substitutions within the following group: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. The addition of amino acid sequences includes fusion with other peptides, polypeptides, or proteins. In one embodiment, as illustrated herein, a cysteine ​​residue is substituted with serine.

[0276] The chemical and functional equivalents of the subject peptides should be understood as molecules that exhibit the functional activity of any one or more of these molecules and can be derived from any source, such as chemical synthesis or identification via screening processes such as natural product screening.

[0277] Homologous peptides include peptides derived from varieties other than peanuts, such as peptides derived from other tree nuts.

[0278] The analogues covered herein include, but are not limited to, modifications of side chains, incorporation of non-natural amino acids and / or their derivatives during peptide, polypeptide, or protein synthesis, and the use of cross-linking agents and other methods that impose conformational restrictions on protein molecules or their analogues. Mutants include molecules that exhibit modified functional activity (e.g., the Ara h1 peptide expressing one or more T-cell epitopes but lacking B-cell responsiveness).

[0279] Examples of side-chain modifications covered by this invention include modifications of amino groups such as by reacting with an aldehyde followed by NaBH2. 4 Reductive alkylation was performed; amidation was carried out with acetylinium methyl ester; acylation was carried out with acetic anhydride; carbamylation of the amino group was carried out with cyanate ester; trinitrobenzylation of the amino group was carried out with 2,4,6-trinitrobenzenesulfonic acid (TNBS); acylation of the amino group was carried out with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxalation of lysine phosphoric acid was carried out with pyridoxal-5-phosphate, followed by NaBH2O. 4 reduction.

[0280] The guanidinyl group of arginine residues can be modified by forming heterocyclic condensation products with reagents such as 2,3-butanedione, benzoylcarboxaldehyde, and glyoxal. The carboxyl group can be modified by forming an O-acylisourea followed by derivatization to, for example, the corresponding amide followed by carbodiimide activation. The thiol group can be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; oxidation of performic acid to sulfoalanine; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride, or other substituted maleimide; formation of mercury derivatives using 4-chloromercuric benzoate, 4-chloromercuric benzyl acid, phenylmercuric chloride, 2-chloromercuryl-4-nitrophenol, and other mercuric agents; and carbamylation with cyanate esters at alkaline pH. Tryptophan residues can be modified, for example, by oxidation with N-bromosuccinimide or by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or thiobenzyl chloride. On the other hand, tyrosine can be altered by nitration with tetranitromethane to form 3-nitrotyrosine derivatives.

[0281] Modification of the imidazole ring of histidine residues can be achieved by alkylation with iodoacetic acid derivatives or by N-carboxymethylation with diethyl pyrocarbonate.

[0282] Examples of incorporation of non-natural amino acids and derivatives during protein synthesis include, but are not limited to, the use of D-isomers of leucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, tert-butylglycine, valine, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thiophene alanine, and / or amino acids. A list of non-natural amino acids covered herein is shown in Table 1.

[0283]

[0284]

[0285]

[0286] 3D conformations can be stabilized using, for example, crosslinking agents, such as those with (CH) 2 ) n Bifunctional imine esters, glutaraldehyde, and N-hydroxysuccinimide esters with spacer groups (where n=1 to n=6) are homobifunctional crosslinking agents, and heterobifunctional agents typically containing an amino reactive moiety such as N-hydroxysuccinimide and another group-specific reactive moiety.

[0287] The structure of the peptides according to the invention can be modified for various purposes, such as increasing solubility, enhancing therapeutic or preventative effects, improving stability, or increasing resistance to protein degradation. Modified peptides, in which the amino acid sequence is altered, can be produced by means of amino acid substitution, deletion, or addition, thereby modifying immunogenicity and / or reducing allergenicity. Similarly, components can be added to the peptides of the invention to produce the same results.

[0288] For example, peptides can be modified to induce T cell unresponsiveness. In this case, known techniques (e.g., substituting each residue and determining the presence or absence of T cell responsiveness) can be used to identify key binding residues for the T cell receptor. In one instance, it has been shown that residues essential for T cell receptor response can be modified by replacing essential amino acids with another amino acid residue, preferably a similar amino acid residue (conservative substitution), the presence of which is shown to alter T cell responsiveness or T cell function. Furthermore, amino acid residues not essential for T cell receptor interaction can be modified by replacing them with another amino acid, the incorporation of which can then alter T cell responsiveness or T cell function without, for example, eliminating binding to the associated MHC protein. In yet another instance, mutant peptides exhibiting normal T cell binding but eliminating IgE binding can be produced.

[0289] Exemplary conservative substitutions are detailed in Table 2 below and include:

[0290]

[0291] Such modifications will result in molecules falling within the scope of “mutants” of the subject peptide as defined herein. “Mutant” should be understood to refer to peptides exhibiting one or more structural features or functional activities that differ from their non-mutant counterparts.

[0292] The peptides of the present invention can also be modified to incorporate one or more polymorphisms arising from natural allelic variations, and non-natural amino acids or amino acid analogs can be substituted into these peptides to produce modified peptides falling within the scope of the present invention. The peptides can also be modified by conjugation with polyethylene glycol (PEG) using known techniques. A reporter group can also be added to facilitate purification and potentially increase the solubility of the peptides according to the present invention. Other well-known types of modifications can also be used, including insertion of specific endonuclease cleavage sites, addition of functional groups or replacement of hydrophobic residues with less hydrophobic residues, and site-directed mutagenesis of the DNA encoding the peptides of the present invention to introduce modifications usable for a wide range of purposes. The various modifications to the peptides according to the present invention mentioned above are only by way of example and are intended to illustrate only the wide range of modifications that can be achieved.

[0293] As detailed above, the present invention provides peptides that retain all or some of the ability to interact with T cells but exhibit partial or complete inhibition, elimination, or other downregulation of antibody reactivity. Downregulation of antibody reactivity can be achieved by any suitable method, which will be well known to those skilled in the art. For example, in the case where a B cell epitope is defined by its linear amino acid sequence, one or more amino acid residues can be added, deleted, or substituted to make the mutated linear sequence different from the naturally occurring sequence. Where the epitope may additionally or alternatively be defined by a conformational epitope, it can be achieved by disrupting the peptide's 2... o The structure or the disruption of the peptide's 3' in the presence of homodimers or heterodimers may disrupt the peptide's structure. o The structure is used to disrupt that conformation. This can be done, for example, by disrupting what is known to be stable. o and / or 3 o The structure is achieved through the formation of bonds such as disulfide bonds. Regarding the T-cell epitopes defined above, these T-cell epitope regions do not contain B-cell epitopes.

[0294] In a related aspect, the inventors have devised a preferred group of seven peptides, five of which contain Ara h1 T cell epitopes and two of which contain Ara h2 T cell epitopes. When administered together, these peptides act particularly effectively to induce desensitization or tolerance and thereby preventively or therapeutically treat hypersensitivity reactions to compositions containing Ara h1 and / or Ara h2, such as foods. These peptides are:

[0295] (i)FQNLQNHRIVQIEAKPNTLV (SEQ ID NO:11)

[0296] (ii)STRSSENNEGVIVKVSKE (SEQ ID NO:12)

[0297] (iii) EVKPDKKNPQLQ (SEQ ID NO:4) and / or EVKPDKKNPQLQD (SEQ ID NO:24)

[0298] (iv)VEIKEGALMLPHFNSKA (SEQ ID NO:13)

[0299] (v)VFIMPAAHPVAINASS (SEQ ID NO:14)

[0300] (vi)ANLRPSEQHLM (SEQ NO:31)

[0301] (vii)EFENNQRSMSEALQ (SEQ NO:32) and / or EFENNQRSMSEALQQI (SEQ ID NO:33)

[0302] Therefore, in a preferred embodiment, an immunomodulatory composition is provided herein comprising each of the Ara h 1 and Ara h 2 T cell peptides from the list of the following components:

[0303] (i)FQNLQNHRIVQIEAKPNTLV (SEQ ID NO:11)

[0304] (ii)STRSSENNEGVIVKVSKE (SEQ ID NO:12)

[0305] (iii) EVKPDKKNPQLQ (SEQ ID NO:4) and / or EVKPDKKNPQLQD (SEQ ID NO:24)

[0306] (iv)VEIKEGALMLPHFNSKA (SEQ ID NO:13)

[0307] (v)VFIMPAAHPVAINASS (SEQ ID NO:14)

[0308] (vi)ANLRPSEQHLM (SEQ NO:31)

[0309] (vii)EFENNQRSMSEALQ (SEQ NO:32) and / or EFENNQRSMSEALQQI (SEQ ID NO:33)

[0310] In another aspect, a composition is provided herein comprising each of Ara h 1 and Ara h 2 T-cell peptides from the list of the following components:

[0311] (i)FQNLQNHRIVQIEAKPNTLV (SEQ ID NO:11)

[0312] (ii)STRSSENNEGVIVKVSKE (SEQ ID NO:12)

[0313] (iii) EVKPDKKNPQLQ (SEQ ID NO:4) and / or EVKPDKKNPQLQD (SEQ ID NO:24)

[0314] (iv)VEIKEGALMLPHFNSKA (SEQ ID NO:13)

[0315] (v)VFIMPAAHPVAINASS (SEQ ID NO:14)

[0316] (vi)ANLRPSEQHLM (SEQ NO:31)

[0317] (vii)EFENNQRSMSEALQ (SEQ NO:32) and / or EFENNQRSMSEALQQI (SEQ ID NO:33)

[0318] When administered to subjects suffering from symptoms characterized by hypersensitivity to Ara h1 and / or Ara h2 or to compositions containing Ara h1 and / or Ara h2, these peptides are able to alleviate said hypersensitivity.

[0319] The peptides of the present invention can be prepared by recombinant or chemical synthesis. According to a preferred aspect of the invention, a recombinant peptide or a mutant thereof is provided herein that preferentially elicits an immune response to T cells from an individual with peanut hypersensitivity, expressed by expression in host cells transformed with a vector encoding the peptide sequence of the present invention. The peptide can be fused with another peptide, polypeptide, or protein. Alternatively, the peptide can be prepared by chemical synthesis techniques, such as by a Merrifield solid-phase synthesis procedure. Furthermore, although the synthetic peptides with the sequences given above represent preferred embodiments, the invention extends to biologically pure formulations of naturally occurring peptides and fragments thereof. "Bio-pure" means a formulation containing at least about 60%, preferably at least about 70%, or preferably at least about 80%, and still more preferably at least about 90% or more, as determined by weight, activity, or other suitable means.

[0320] Therefore, this invention should be understood to encompass peptides comprising at least one T-cell core epitope region of Ara h1 and / or Ara h2 as defined above, along with other amino acids (which may or may not be naturally occurring) or other chemical substances. In a preferred aspect of the invention, such peptides may comprise one or more epitopes of Ara h1 and / or Ara h2, which are T-cell core epitope regions. Peptides having one or more T-cell epitopes of Ara h1 and / or Ara h2 are desirable for increased therapeutic efficacy.

[0321] As detailed above, this invention relates to a composition comprising the peptides defined above. However, it should be understood that the subject composition may comprise additional components, such as additional peptides. These peptides may cover, for example, a portion of a core minimal epitope. Alternatively, they may not include any portion of a T-cell epitope as disclosed herein but may be incorporated for other reasons. Examples of other peptides that may be included in the composition include, but are not limited to:

[0322] (i)ALMLLPHFNSKAMVIVVV (SEQ ID NO:34)

[0323] (ii)NNFGKLFEVKPDKKNPQ (SEQ ID NO:35)

[0324] (iii)SQLERANLRPXEQ (SEQ ID NO:36)

[0325] (iv)ELNEFENNQRXM (SEQ ID NO:37)

[0326] (v)NNFGKLFEVKPDKKNPQLQD (SEQ ID NO:38)

[0327] (vi)NNFGKLFEVKPDKKNPQL (SEQ ID NO:40)

[0328] (vii)SQLERANLRPXEQH (SEQ ID NO:41)

[0329] (viii)KAMVIVVVNKGTGNLELVAV (SEQ ID NO:42)

[0330] (ix)RELRNLPQQXGLRA (SEQ ID NO:43)

[0331] (x)KAMVIVVVNKG (SEQ ID NO:44)

[0332] (xi)AMVIVVVNKGTGNLELV (SEQ ID NO:45)

[0333] (xii)VVNKGTGNLELVAVRK (SEQ ID NO:46)

[0334] Or its functional derivatives or homologues, wherein residue X is cysteine ​​or serine.

[0335] Depending on the specific circumstances, other potentially beneficial peptides or molecules may also be included.

[0336] In another aspect, the present invention provides a nucleic acid molecular composition comprising one or more nucleic acid molecules encoding or complementary to sequences encoding or derivatives, homologues or analogues of T-cell epitopes and peptides as defined above.

[0337] It should be understood that references to "peptide" include references to peptides containing one or more T-cell epitopes. The nucleic acid molecule encoding the subject peptide is preferably a sequence of deoxyribonucleic acid (such as cDNA) or a genomic sequence. The genomic sequence may contain exons and introns. The genomic sequence may also contain promoter regions or other regulatory regions.

[0338] Nucleic acid molecules can be conjugated to expression vectors capable of expression in prokaryotic cells (e.g., *E. coli*) or eukaryotic cells (e.g., yeast cells, fungal cells, insect cells, mammalian cells, or plant cells). Nucleic acid molecules can be conjugated, fused to, or otherwise associated with nucleic acid molecules encoding another entity (e.g., a signal peptide). Nucleic acid molecules may also contain additional nucleotide sequence information fused to, conjugated to, or otherwise associated with the 3' or 5' terminal portions. Nucleic acid molecules can also be part of a vector such as an expression vector. The latter embodiment facilitates the production of recombinant forms of the subject peptide, which are covered by this invention.

[0339] These nucleic acids can be used to recombinantly generate Arah 1 and / or Arah 2, or T-cell epitopes containing their proteins, by inserting them into appropriate vectors and transfecting them into suitable cell lines. Such expression vectors and host cell lines also form an aspect of this invention.

[0340] In the process of generating peptides via recombinant technology, host cells transformed with nucleic acids having sequences encoding the peptides according to the invention or functional equivalents of nucleic acid sequences are cultured in a culture medium suitable for the relevant specific cells. The peptides can then be purified from the cell culture medium, host cells, or both using techniques well known in the art, such as ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, or immunopurification using peptide-specific antibodies.

[0341] Nucleic acids encoding Ara h1 and / or Ara h2, or peptides containing T-cell core epitope regions of Ara h1 and / or Ara h2, can be expressed in bacterial cells (such as *Escherichia coli*), insect cells, yeast, or mammalian cells (such as Chinese hamster ovary cells (CHO)). Suitable expression vectors, promoters, enhancers, and other expression control elements have been mentioned in Sambruck et al. (1989). Other suitable expression vectors, promoters, enhancers, and other expression control elements are well known to those skilled in the art. Examples of suitable expression vectors in yeast include Yep Sec 1 (Balderi et al., 1987, Embo J., 6:229-234); pMFa (Kurjan and Herskowitz., 1982, Cell., 30:933-943); JRY88 (Schultz et al., 1987, Gene., 54:113-123); and pYES2 (Invitrogen Corporation, San Diego, CA). These vectors can be freely used as baculovirus and mammalian expression systems. For example, the baculovirus system (ParMingen, San Diego, CA) is commercially available for expression in insect cells, while the pMsg vector (Pharmacia, Piscataway, NJ) is commercially available for expression in mammalian cells.

[0342] For expression in *E. coli*, suitable expression vectors include pTrc (Amann et al., 1998, *Gene.*, 69:301-315)); pGex (Amrad Corporation, Melbourne, Australia); pMal (NE Biolabs, Beverley, MA); pRit5 (Pharmacia, Piscataway, New Jersey); pEt-11d (Novagen, Maddison, WI) (Jameel et al., 1990, *J. Virol.*, 64:3963-3966) and pSem (Nap et al., 1990, *Biotechnology*, 64:3963-3966) and pSem (Nap et al., 1990, *Biotechnology*, 64:3963-3966). 8:280-281 (Knapp et al., 1990, BioTechniques., 8:280-281)). The use of pTRC and pEt-11d will, for example, lead to the expression of non-fusion proteins. The use of pMal, pRit5, pSem, and pGex will enable the expression of allergens fused to maltose E-binding protein (pMal), protein A (pRit5), truncated galactosidase (PSEM), or glutathione S-transferase (pGex). When the T-cell epitope of Ara h1 or the peptide containing it is expressed as a fusion protein, it is particularly advantageous to introduce an enzyme cleavage site at the fusion junction between the carrier protein and the associated peptide. The peptides of the present invention can then be recovered from the fusion protein by enzymatic cleavage at the enzyme site and biochemical purification using conventional techniques for purifying proteins and peptides. Different carriers also have different promoter regions, thereby allowing constitutive or inducible expression or temperature-induced expression. Furthermore, expressing the recombinant peptide in different *E. coli* hosts may be appropriate, as these hosts possess the ability to alter the recombinant protein to cause degradation. Alternatively, modifying the nucleic acid sequence to use codons preferentially utilized by *E. coli* may be advantageous, where such nucleic acid modification will not affect the amino acid sequence of the expressed protein.

[0343] Host cells can be transformed to express the nucleic acids of the present invention using conventional techniques such as co-precipitation with calcium phosphate or calcium chloride, DEAE-dextran-mediated transfection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al. (1989) and other laboratory texts. The nucleic acid sequences of the present invention can also be chemically synthesized using standard techniques.

[0344] In addition to recombining to produce the peptides according to the invention, nucleic acids can be used as probes for experimental or purification purposes.

[0345] The identification and synthesis of peptides disclosed herein now facilitate the development of a range of preventative and therapeutic treatments for peanut-related immunological conditions. It also aids in the development of the reagents used therein. Therefore, this invention should be understood to extend to the use of peptides or their functional derivatives, homologues, or analogues in the therapeutic and / or preventative treatment of patients. Such treatments include, but are not limited to:

[0346] (i) The subject peptide is administered to the subject as a means of desensitizing or inducing immune tolerance to peanuts, Ara h 1 and / or Ara h 2, or Ara h 1 and / or Ara h 2 analogues. This can be achieved, for example, by inducing Ara h 1 and / or Ara h 2-directed Th2 non-responsiveness or apoptosis. In a preferred embodiment, such an outcome is obtained by using a peptide that maintains T cell epitope reactivity but is not susceptible to IgE binding. Alternatively, a treatment regimen can be used that is based on administering a specific concentration of a given peptide according to a specific regimen in order to induce tolerance. This approach can eliminate Ara h 1 and / or Ara h 2 hypersensitivity or it can reduce the severity of Ara h 1 and / or Ara h 2 hypersensitivity or sensitivity to allergens present in compositions containing Ara h 1 and / or Ara h 2 (such as peanut allergy). The reference to treatment for Ara h 1 and / or Ara h 2 sensitivity should be understood to encompass, within its scope, the treatment of symptoms characterized by sensitivity to compositions containing Ara h 1 and / or Ara h 2 (such as, generally, peanuts), even if such sensitivity is to allergens other than Ara h 1 and / or Ara h 2.

[0347] Preferably, such treatment regimens modify the T-cell response or both B-cell and T-cell responses in the relevant individual. As used herein, modification of the anaphylactic response in an individual with peanut hypersensitivity can be defined as inducing non-responsiveness to the Ara h1 molecule or reducing symptoms, as determined by standard clinical procedures (Varney et al., 1991 British Medical Journal 302:265-269). Symptom reduction includes any reduction in the individual's anaphylactic response to Ara h1 after completion of the treatment regimen. This reduction can be subjective or clinically determined, for example, by using standard food challenge tests or standard skin tests known in the art.

[0348] Individuals exposed to the peptides of the present invention may tolerate appropriate T cell subsets or render appropriate T cell subsets unresponsive, such that they are unresponsive to Ara h1 and / or Ara h2 after such exposure and do not participate in stimulating an immune response. Preferably, the peptides according to the invention will retain immunodominant T cell epitopes but have eliminated IgE binding. Furthermore, even if the allergen in question is not Ara h1 and / or Ara h2, but a different allergen present in the same composition as Ara h1 and / or Ara h2 (such as different peanut allergens), immunization with Ara h1 and / or Ara h2 can still induce a bystander inhibition effect, which reduces the degree of hypersensitivity to that allergen.

[0349] Compared to exposure to naturally occurring Ara h 1 and / or Ara h 2 allergens, administration of the peptides of this invention can modify the cytokine secretion pattern. This exposure may also affect T cell subsets normally involved in allergic responses, causing them to migrate away from sites of normal allergen exposure and toward sites of therapeutic administration. This redistribution of T cell subsets can improve or reduce an individual's immune system's ability to stimulate a normal immune response at sites of normal allergen exposure, thereby leading to a reduction in allergy symptoms.

[0350] As detailed above, modifications to B cell responses can be achieved, for example, by regulating the morphology of cytokines produced by T cells. Specifically, reducing the production of IL-4 and IL-13 derived from T cells reduces IgE synthesis.

[0351] (ii) The peptides of the present invention can be used in the ability of adsorbents to remove Ara h1 and / or Ara h2-oriented T cells from biological samples or from patients.

[0352] Therefore, in another aspect, the present invention provides a method for treating and / or preventing a condition in a subject characterized by an abnormal, unwanted, or otherwise inappropriate immune response to Ara h 1 and / or Ara h 2 or to an allergen in a composition containing Ara h 1 and / or Ara h 2, the method comprising administering to the subject an effective amount of an immunomodulatory composition as defined above, for a period of time, provided that the presence or function of T cells in the subject against the Ara h 1 and / or Ara h 2 or other allergens is sufficient to eliminate or reduce them.

[0353] Preferably, the symptoms are hypersensitivity to peanuts or tree nuts containing Ara h 1 and Ara h 2 or similar molecules, such as hazelnuts, almonds or Brazil nuts.

[0354] In one embodiment, the method enables desensitization to or induction of immune tolerance to Ara h 1 and / or Ara h 2 or other allergens of the composition.

[0355] In another embodiment, the desensitization or tolerance is achieved by inducing T cell non-responsiveness or apoptosis.

[0356] In yet another embodiment, the desensitization or tolerance is achieved by inducing Ara h 1 or Ara h 2 specific Treg cells.

[0357] "Effective dose" means the amount necessary to at least partially achieve the desired immune response or delay the onset of a specific symptom to be treated, or to inhibit its progression or to completely stop its onset or progression. This dose varies depending on the health and physical condition of the individual being treated, the individual's taxonomy, the desired level of protection, the formulation of the composition, assessment of the medical condition, and other relevant factors. It is anticipated that this dose will fall within a relatively broad range that can be determined through routine testing.

[0358] Subjects undergoing treatment or prevention are typically mammals, such as, but not limited to, humans, primates, livestock (e.g., sheep, cows, horses, donkeys, pigs), companion animals (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), and captive wild animals (e.g., foxes, deer). Preferably, the mammal is a human or a primate. Most preferably, the mammal is a human.

[0359] The terms "treatment" and "prevention" are used here in their broadest sense. The term "treatment" does not necessarily mean treating a subject until full recovery. Similarly, "prevention" does not necessarily mean that a subject will ultimately not develop disease symptoms. Therefore, treatment and prevention include improving the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prevention" can be considered as reducing the severity or onset of a particular condition. "Treatment" also reduces the severity of existing symptoms.

[0360] The compositions of the present invention (referred to herein as “pharmaceuticals”) may be administered in the form of pharmaceutical compositions by any convenient means. It is anticipated that the pharmaceuticals of the pharmaceutical compositions will exhibit therapeutic activity when administered in amounts appropriate to the specific circumstances. Variations depend, for example, on the selected human or animal and the pharmaceutical. A wide range of dosages may be applicable. Considering the patient, for example, the pharmaceuticals may be administered in doses of about 0.01 μg to about 1 mg. The dosing regimen can be adjusted to provide the most suitable therapeutic response. For example, several separate doses may be taken daily, weekly, monthly, or at other suitable intervals, or the dose may be reduced proportionally according to the urgency of the situation. In another instance, the composition may be initially administered to induce tolerance, and subsequently, if necessary, a booster dose may be given to maintain tolerance. These boosters may be given monthly, for example, and may be administered continuously for any period of time, including a patient’s lifetime.

[0361] The pharmaceutical preparation can be administered in convenient manners, such as via: oral, intravenous (when water-soluble), intraperitoneal, intramuscular, subcutaneous, intradermal (with or without conventional needle or other percutaneous delivery devices), percutaneous, intranasal, sublingual, or suppository routes, or implantation (e.g., using sustained-release molecules). Preferably, the composition is administered intradermally. The pharmaceutical preparation can be administered as a pharmaceutically acceptable non-toxic salt, such as an acid addition salt or a metal complex, for example, formed with zinc, iron, etc. (which is considered a salt for the purposes of this application). Examples of such acid addition salts are hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate, etc. If the active ingredient is administered in tablet form, the tablet may contain a binder, such as astragalus gum, corn starch, or gelatin; a disintegrant, such as alginate; and a lubricant, such as magnesium stearate.

[0362] According to these methods, the pharmaceutical agents defined according to the present invention can be administered together with one or more other compounds or molecules. "Co-administered" means administered simultaneously in the same formulation or in two different formulations via the same or different routes, or sequentially via the same or different routes. "Sequentially administered" means that there is a time difference of seconds, minutes, hours, or days between the administration of the two types of molecules. These molecules can be administered in any order. It should also be understood that the peptides of the present invention can themselves be administered simultaneously or sequentially. They can be administered simultaneously or sequentially as one or more compositions. For example, some of these peptides can be formulated into one formulation and other peptides into another separate formulation; wherein one of these two formulations is administered on each arm. Alternatively, additional separate formulations can be generated and administered simultaneously to different sites or sequentially. Designing and producing suitable formulations or mixtures of formulations is entirely within the capabilities of those skilled in the art.

[0363] Another aspect of the invention covers the use of the immunomodulatory composition as defined above in the manufacture of a medicament for treating a symptom in mammals characterized by an abnormal, unwanted, or otherwise inappropriate immune response to Ara h 1 and / or Ara h 2.

[0364] Preferably, the symptoms are hypersensitivity to peanuts or tree nuts such as hazelnuts containing Ara h 1 and / or Ara h 2 or similar molecules.

[0365] In yet another aspect, the invention covers a vaccine comprising a composition as defined above and one or more pharmaceutically acceptable carriers and / or diluents. The composition is referred to as the active ingredient.

[0366] Suitable drug forms for injection include sterile aqueous solutions (when soluble in water) or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions, or may be in the form of creams or other forms suitable for topical application. These drug forms must be stable under manufacturing and storage conditions and must be preserved against microbial contamination such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, in the case of dispersions by maintaining the required particle size, and by using surfactants. Microbial action can be prevented by various antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. Tonic modifiers can be used to maintain the isotonicity of the formulation with human plasma and thereby prevent tissue damage. Commonly used tonic modifiers include dextran, trehalose, glycerol, and mannitol. Glycerol and sodium chloride are other options but are generally less commonly used. In many cases, isotonic agents, such as sugar or sodium chloride, are preferably included. Extended absorption of the injectable composition can be achieved by using a combination of agents with delayed absorption, such as aluminum monostearate and gelatin.

[0367] Sterile injectable solutions are prepared by incorporating the active compound, in the required amount, with various other components listed above into a suitable solvent, followed by filtration and sterilization. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile carrier containing a base dispersion medium and any other desired components from those listed above. For sterile powders used in the preparation of sterile injectable solutions, preferred methods are vacuum drying and freeze-drying techniques, which produce powders of the active ingredient and any additional desired components from its previous sterile filtered solution.

[0368] When the active ingredients are properly protected, they can be administered orally, for example, with an inert diluent or with an absorbable, edible carrier, or encapsulated in hard or soft-shell capsules, or compressed into tablets, or directly incorporated into food. For oral therapeutic administration, the active compound can be combined with excipients and used in the form of ingestible tablets, sublingual tablets, lozenges, capsules, elixirs, suspensions, syrups, rice paper capsules, etc. Such compositions and formulations should contain at least 1% by weight of the active compound. Of course, the percentage of the composition and formulation can be varied and is preferably between about 5% to about 80% of the unit weight. The amount of the active compound in such therapeutically useful compositions should enable the attainment of an appropriate dose. Preferred compositions or formulations according to the invention are prepared such that the oral unit dosage form contains between about 0.1 µg and 1000 μg of the active compound.

[0369] Tablets, sublingual tablets, pills, capsules, etc., may also contain the following components: binders, such as gum arabic, corn starch, or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, lactose, or saccharin; or flavorings, such as peppermint, wintergreen oil, or cherry flavoring. When the unit dosage form is a capsule, it may contain a liquid carrier in addition to the materials of the types mentioned above. Various other materials may be present as coatings or otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrups or elixirs may contain active compounds, sucrose as a sweetener, methylparaben and propylparaben as preservatives, dyes, and flavorings such as cherry or orange flavoring. Of course, any materials used in the preparation of any unit dosage form should be pharmaceutically pure and substantially non-toxic when used in large quantities. In addition, active compounds can be incorporated into sustained-release formulations and preparations.

[0370] Pharmaceutical compositions may also contain gene molecules, such as vectors capable of transfecting target cells, wherein the vector carries a nucleic acid molecule encoding a regulator. For example, the vector may be a viral vector.

[0371] Routes of administration include, but are not limited to: inhalation (e.g., intranasal or oral administration via aerosol), intratracheal, nasopharyngeal, intravenous, intraperitoneal, subcutaneous, intracranial, intradermal, percutaneous, intramuscular, intraocular, intrathecal, intracerebral, intranasal, infusion, oral, rectal, via IV drip, implantation, and sublingual. Preferably, the route of administration is subcutaneous, intradermal, percutaneous, or intranasal.

[0372] Another aspect of the invention relates to compositions as defined above when used in the methods of the invention.

[0373] The invention will be further described with reference to the following non-limiting examples.

[0374] Example 1

[0375] Ara h 1 and Ara h 2 are the most allergenic and abundant proteins in peanuts, making the inclusion of peptides containing their dominant T-cell epitopes essential for treatment. Another important consideration when selecting peptides for immunotherapy is whether they can be presented by different MHC class II molecules (HLA molecules in humans) and therefore are suitable for treating genetically diverse human populations. HLA restriction of peptide presentation to T cells was tested using blocking antibodies and HLA genotyping, and it was shown that each identified T-cell epitope can be presented on two or more different HLA molecules. Furthermore, the identified T-cell epitopes were confirmed to be presented in combination of HLA-DR, HLA-DQ, and HLA-DP molecules (Table 2). Inclusion of HLA-DQ and HLA-DP restricted T-cell epitopes is particularly advantageous for treatment because these HLA types are more conserved than HLA-DR molecules in mixed populations, enabling broader population coverage with fewer T-cell epitope sequences.

[0376] Adjacent or overlapping T-cell epitopes were combined into single peptides (length <20 aa) to minimize the number of peptides in the final treatment group, resulting in three candidate peptides from Ara h 2 and seven candidate peptides from Ara h 1. Cysteine ​​residues were substituted with structurally conserved but less reactive serine residues because cysteine ​​residues can be problematic in terms of peptide stability and bioactivity. Minor modifications were also made to two Ara h 1 peptides to improve stability and / or solubility (Table 2). In all cases, T-cell responsiveness to the variant peptides was confirmed to be preserved.

[0377] Table 2: Therapeutic candidate peptides of Ara h 1 and Ara h 2

[0378]

[0379] Ara h 1 peptide, shaded; Ara h 2 peptide, unshaded. HLA lists the HLA types known to present intrapeptide T-cell epitopes. Modifying peptides to improve performance; 1 'W', omitted from the N-end; 2 'E' (from the natural sequence) is added to the C-terminus. 3 In bold, serine replaces cysteine.

[0380] Clinical screening of these peptides has confirmed the T-cell responsiveness of PBMCs. Figure 1 ), lack of inflammatory cell activation ( Figure 2Serum stability in another peanut allergy cohort (n = 40) was also observed. PBMC T cells have been confirmed to recognize one or more of these ten peptides in 100% of the subjects analyzed (n = 20), with 50%–90% responding to each peptide. The data to date clearly demonstrate that the ten peptides listed in Table 2 above provide a sufficient, feasible, and suitable mixture.

[0381] Example 2

[0382]

[0383] Materials and Methods

[0384] Subjects: Adult subjects with peanut allergy were recruited from The Alfred Allergy Clinic, Melbourne, Australia. Subjects had clinical symptoms of IgE-mediated peanut allergy and a peanut-specific IgE CAP score ≥2 (≥1.16 kUA / l; Pharmacia CAPSystem™, Pharmacia Diagnostics, Uppsala, Sweden), and many had a history of allergic reactions. Genotyping (HLA-DRB1, HLA-DQB1, and HLA-DPB1, exon 2) was performed on some subjects by the Victorian Transplantation and Immunogenetics Service. This study was approved by The Alfred and Monash University Ethics Committees, and written consent was obtained from each subject.

[0385] Antigens: Crude peanut extract (CPE) was prepared from commercially available unsalted, dry-roasted peanuts as described elsewhere (de Leon et al., Clin Exp Allergy. 2003;33(9):1273-80), dialyzed with phosphate-buffered saline (PBS) and sterile filtered (0.2 μm). Natural Ara h 1 and Ara h 2 were enriched from CPE based on the published method. (de Jong EC et al. Clin Exp Allergy. 1998;28(6):743-51) In short, CPE buffer was replaced with 20 mM TRIS-bis-propane (TBP) (pH 7.2) using a Vivaspin column (Sartorius Stedim Biotech SA, Aubagne, France) and applied to a 5 mL Mono-Q 10 / 10 column (Pharmacia FPLC system, St Albans, UK) equilibrated with TBP. After washing with TBP, a linear gradient of 30 mL 0–1 M NaCl / TBP was applied to elute the bound protein (1 mL / min). 0.5 mL fractions were analyzed by SDS-PAGE, and fractions containing Ara h 1 or Ara h 2 were pooled with minimal amounts of other proteins and dialyzed with PBS. Endotoxin concentrations for CPE, Ara h 1, and Ara h 2 were 1.7, 4.0, and 78.0 EU / mg, respectively (Endpoint Chromogenic LAL test, Lonza, Walkersville, USA). Peptides (Mimotopes, Victoria, Australia and GenScript USA Inc, New Jersey, USA) were reconstituted at 1–4 mg / mL in 10% dimethyl sulfoxide / PBS (20-mer and truncated peptide groups) or PBS, 1%–2% acetic acid, or 0.1 M ammonium bicarbonate buffer (custom-synthesized core epitope peptides).As described, all antigens were confirmed to be neither mitotic nor toxic (Eusebius NP et al., IntArch Allergy Immunol. 2002;127(3):234-44).

[0386] Generation of Ara h 1 and Ara h 2 specific CD4+ T cell lines (TCLs): Ara h 1 or Ara h 2 specific oligoclonal TCLs were generated from peripheral blood mononuclear cells (PBMCs) of peanut-allergic subjects using a 5,6-carboxyfluorescein diacetate succinimide (CFSE)-based method. (Mannering SI et al., J Immunol Methods. 2005;298(1-2):83-92); Prickett SR, et al., J Allergy Clin Immunol. 2011;127(3):608-15 e1-5. In summary, PBMCs were cultured in RPMI-1640 (Sigma-Aldrich, St Louis, USA) (cRPMI) containing 2 mM L-glutamine, 100 IU / mL penicillin-streptomycin, and 5% human AB serum. PBMCs were labeled with 0.1 µM CFSE (Molecular Probes, Eugene, USA) and cultured at 37˚C with cRPMI alone, CPE (100 µg / mL), Ara h 1 or Ara h 2 (10 µg / mL), Ara h 1 or Ara h 2 20-mer peptide libraries (10 μg / mL / peptide), or tetanus toxoid (TT; 10 LfU / mL; Statens Serum Institute, Copenhagen, Denmark) as a control (2.5 × 10⁶ / mL) for 7 days. After staining with CD4-PE and 7AAD (BD Pharmingen, San Diego, USA), CD4+CFSEdim7AAD cells were sorted (10 cells / well) into 96-well plates containing irradiated allogeneic feeder cells, anti-CD3 (OKT-3), rIL-2 (Cetus, Emeryville, USA), and amphotericin B (Invitrogen, Carlsbad, USA).Cells were fed with rIL-2 as needed, and after 10–14 days, they were transferred to 48-well plates and tested for proliferation against Ara h 1 or Ara h 2 (10 μg / mL). Ara h 1 or Ara h 2-positive TCLs were amplified in T25 culture flasks (BD, Franklin Lakes, USA) for 10–12 days with anti-CD3 and rIL-2, and then tested for specificity (proliferation) against overlapping 20-mer peptides (10 μg / mL) across the corresponding sequence. As described, core epitope sequences were mapped within a selected 20-mer using peptide groups truncated from the N- or C-terminus of the 20-mer (Prickett SR, et al. J Allergy ClinImmunol. 2011;127(3):608-15 e1-5).

[0387] T cell assay: All cultures were performed in RPMI-1640 (Sigma-Aldrich, St. Louis, USA) (cRPMI) containing 2 mM L-glutamine, 100 IU / mL penicillin-streptomycin, and 5% heat-inactivated human AB serum. The results were obtained via 3H-thymidine (…). 3 H-TdR uptake assay was performed to assess antigen-induced TCL proliferation as follows: Divide or triple-replicate culture media were measured in 96-U wells containing 1 × 10⁻⁶ H⁺ dr. 4 1 T cells / well, 1 × 10⁸ as antigen-presenting cells 4 One irradiated (5000 rads) autologous EBV-transformed PBMCs (EBV-B cells) and a specified antigen. The negative control was cRPMI alone. During the last 16 hours... 3 H-thymidine ( 3Cells were pulsed at H-TdR (0.5 μCi / well) and uptake was recorded as the average count per minute (cpm) for replication culture. Stimulation index (SI; cpm antigen-stimulated T cells / cpm unstimulated T cells) 2.5 was considered positive and all positive responses were confirmed in ≥2 assays. To allow detection of peptide-induced CD4+ T cell proliferation throughout PBMCs, 7-day cultures of CFSE-labeled PBMCs were established as described for TCL production, with the addition of anti-CD25 antibody (BD) to assess T cell activation in addition to proliferation. At least 10,000 CD4+ T cells were analyzed per sample, and SI was calculated as the percentage of antigen-containing CD4+CFSElo (proliferating), CD4+CD25+ (activated), or CD4+CD25+CFSElo (activated and proliferating) cells / the percentage of the same population without antigen (background). Analyzing CD4+CD25+CFSElo (activated and proliferating) cells provides the most sensitive method for detecting T cell responses, with SI 1.5 specified as positive.

[0388] HLA class II blocking assay: T cells and irradiated EBV-B cells (1 × 10⁻⁶ each) were used. 4 (1) IgG2a was incubated with 0.1-10 μg / mL blocking monoclonal antibody (mAb) or isotype control antibody (IgG2a: BD Pharmingen; IgG1: BioLegend, San Diego, USA) against HLA-DR (L243, BD Pharmingen), HLA-DQ (SVP-L3) or HLA-DP (B7 / 21) at 37˚C for 1 hour, followed by the addition of peptide (2-10 μg / mL) or CPE (100 μg / mL) and the proliferation reaction was tested as above.

[0389] Cytokine ELISPOT assay: MAIP ELISPOT plates (Millipore, Billerica, USA) were coated overnight at 4˚C with 10 μg / mL IL-4, IFN-γ, or IL-5 antibody (eBioscience, San Diego, USA) in PBS. The wells were then blocked (cRPMI, 1 h, 37˚C) and PBMCs (3.5 × 10⁻⁶) were then used. 5100 μL of duplicate cultures containing either T cells or irradiated EBV-B cells (1 × 10⁴ cells each) were added to a 100 μL duplicate culture containing CPE (100 μg / mL), nAra h 2 (10 μg / mL), or peptide (10 μg / mL). Controls were cRPMI, TT (10 lfU / mL), and phytohemagglutinin (1 μg / mL; Sigma-Aldrich) alone. After culturing at 37˚C for 48 hours, the plates were incubated with biotinylated IL-4, IL-5, or IFN-γ antibody (eBioscience) (1 μg / mL PBS, 2 h), followed by incubation with ExtrAvidin®-alkaline phosphatase (Sigma-Aldrich) (1 / 3,000 PBS, 2 h), and then developed with alkaline phosphatase substrate (Bio-Rad). When a spot appears in the positive control well, the plate is washed, air-dried, and read (AID ELISPOT 4.0 h reader, AutoimmunDiagnostika, Strassberg, Germany).

[0390] Basophil activation assay: Basophil activation was assessed as described by CD63 upregulation detected by flow cytometry (Drew AC, et al., J Immunol. 2004;173(9):5872-9). Positive controls were rabbit anti-human IgE antibody (7.5 µg / mL; DAKO Corporation, CA, USA), N-formyl-methionine-leucine-phenylalanine (fMLP) (0.4 µg / mL; Sigma), and CPE. CPE was tested at 3-log concentrations (50, 5, and 0.5 µg / mL) and peptide libraries were tested at 4-log concentrations (50, 5, 0.5, and 0.05 µg / mL). Histamine release was assessed using the Histamine Release and Histamine ELISA Kit (IBL International GmbH, Hamburg, Germany) following the manufacturer’s instructions.

[0391] result

[0392] Factors considered in dominant 20-mer selection include:

[0393] • Response frequency

[0394] • Number of specific TCLs generated per patient / Incidence of specific T cells in patient PBMCs

[0395] • Magnitude of T cell response

[0396] • Patterns of T-cell responses (combinations of peptides recognized within or between subjects).

[0397] • The ability to directly target specific T cells throughout the entire PBMC population with peptides (CFSE screening)

[0398] • Consistency of T cell responses

[0399] • Identification of core T-cell epitopes within the 20-mer peptide

[0400] Ara h 1 dominant 20-mer selection

[0401] 145 Ara h 1-specific T cell lines (TCLs) were generated from 18 peanut-allergic donors, and these TCLs recognized a 65 / 69 overlapping 20-mer peptide across Ara h 1 (see Table 3 and...). Figure 5 Of the 65 20-mer peptides, 14 were selected as the most frequently identified (4–6 of 18 respondents; 22%–33%) (peptide numbers 23, 24, 26, 38, 40, 44–51, and 57). Nine of these 14 peptides were selected for further analysis (peptide numbers 23, 24, 40, 46, 47, 49, 50, 51, and 57) (Table 4).

[0402] These selections were made based on the number of subject-specific TCLs, the magnitude of the TCL response, the reproducibility of the TCL response, and the ability to target specific T cells in PBMCs. The nine selected 20-mers were:

[0403] • Identified by 16 out of 18 subjects (89%) with TCL in this group.

[0404] • Typically induces a strong and consistent response in specific TCLs.

[0405] • Multiple TCL identifiers originating from numerous respondents

[0406] • Each can target specific T cells in donor PBMCs (co-inducing detectable PBMC T cell responses in 18 / 20 additional subjects, with 8-16 responders (40-80%) in each 20-mer).

[0407] In 35 out of 38 subjects (92%) analyzed by TCL isolation and / or CFSE screening, T cells recognized one or more of the nine 20-mers (Table 3).

[0408] Table 3:

[0409] TCL proliferative response (thymidine uptake) to the Ara h 1 20-mer peptide. The table shows the SI values ​​(= multiple increase in TCL proliferation with the presence of the peptide compared to unstimulated TCL). Only stimulation indices (SI) ≥ 2.5 are shown. The highest SI is shown for subjects with multiple TCLs specific to a given 20-mer. Dark gray, SI ≥ 2.5 < 5.0; SI ≥ 5.0. The nine dominant peptides ultimately selected are shown in Table 4.

[0410] Table 3:

[0411]

[0412] Table 4: Overview of Dominant Ara h 1 20-mer Selection

[0413]

[0414] PBMC screening for dominant Ara h1 20-mer

[0415] Table 5 shows that ≥ 1 dominant 20-mer peptides were identified by 18 / 20 (or 22 / 24 of all data). Each 20-mer peptide was identified by at least 7 subjects. Combined CFSE and TCL data: identification confirmed in 43 / 45 subjects.

[0416] Table 5:

[0417] SI of peptide-induced proliferation in 24 subjects. The upper part shows the new peanut allergy donor cohort (different from the cohort used for TCL). The lower part shows 4 subjects from the cohort used for TCL production, along with the total from the upper and lower parts.

[0418] CPE, crude peanut extract; +VE, positive; nt, not tested (peptide stock not available at the time of testing); gray, irritation index ≥ 1.1 < 2.5; black, irritation index ≥ 2.5.

[0419]

[0420] Ara h 2 dominant 20-mer selection

[0421] Sixty-nine Ara h 2-specific T cell lines (TCLs) were generated from 16 peanut-allergic donors, and these TCLs recognized 16 / 17 overlapping 20-mer peptides across Ara h 2 (Table 6). Four of these 16 20-mers were selected as the most frequently recognized (each in 7–9 of the 16 donors; 44%–46%) (peptide numbers 4, 5, 11, and 15). Figure 7These selections were made based on the number of subject-specific TCLs, the magnitude of the TCL response, the reproducibility of the TCL response, and the ability to target specific T cells in PBMCs. The four 20-mer peptides selected were:

[0422] • Identified by TCL from all 16 subjects (100%) in this group.

[0423] • Typically induces a strong and consistent response in specific TCLs.

[0424] • Multiple TCL identifiers originating from numerous respondents

[0425] • Jointly identified by approximately 80% of all 69 TCL companies.

[0426] • Capable of targeting specific T cells in donor PBMCs (detectable PBMC T cell responses were demonstrated in the 6 subjects tested).

[0427] In 16 out of 16 (100%) subjects analyzed by TCL isolation and / or CFSE screening, T cells recognized one or more of the four 20-mers (Table 6).

[0428] Table 6:

[0429] TCL proliferative response to the Ara h 2 20-mer peptide (thymidine uptake). The table shows the SI values ​​(= multiple increase in TCL proliferation in the presence of the peptide compared to unstimulated TCL). Only the positive stimulation index (≥2.5) is shown: gray, ≥2.5 ≤ 5.0; black, > 5.0. A) Allergen-driven TCL; B) Peptide-driven TCL. Dominant 20-mer is shown in Table 7.

[0430] Table 6:

[0431]

[0432] Table 7: Overview of Dominant Ara h2 20-mer Selection

[0433]

[0434] Core T cell epitope mapping

[0435] Technical methods

[0436] TCLs from different subjects were used to map core T cell epitopes. The exact T cell epitopes varied between TCLs and subjects. The reactivity of TCLs from different subjects was tested for proliferation of truncated peptide groups. Figure 8The minimal T cell stimulating sequence (core epitope) within each selected 20-mer was used. The number of residues required to induce maximum T cell proliferation varied from 6 to 19 amino acids among different TCLs and / or subjects (Tables 8 and 9). Due to the variation in the number of side residues required for optimal epitope recognition, if peptides containing a common core sequence induce recognition, then the TCL is considered to recognize the same epitope. Based on this criterion, ten different Ara h 1 and five different Ara h 2 CD4 were identified. + T-cell epitopes (“uniform epitopes”, Tables 8 and 9), in which the common “minimum core epitope” sequence varies from 5 to 12 aa (underlined sequences, Tables 8 and 9). The “uniform epitope” sequence is the minimum sequence that covers all the residues required for optimal T-cell responsiveness across different subjects to ensure the broadest possible recognition.

[0437] (i) Core T cell epitopes found in dominant Ara h 1 20-mer

[0438] Mapping of the core T-cell epitope sequence within the dominant Ara h 1 20-mer peptide. Ten Ara h 1 T-cell epitopes (“unified T-cell epitopes”) were identified, including four pairs of overlapping T-cell epitopes. (Table 8)

[0439] Table 8:

[0440]

[0441] (ii) Core T cell epitopes found in dominant Ara h 2 20-mer

[0442] Core T-cell epitope sequences were mapped within the dominant Ara h 2 20-mer peptide. Five Ara h 2 T-cell epitopes (“unified T-cell epitopes”) were identified, including two pairs of overlapping T-cell epitopes (Table 9).

[0443] Table 9:

[0444]

[0445] HLA restriction of Ara h1 and Ara h2 T cell epitopes

[0446] Blocking T cell recognition of dominant T cell epitopes using monoclonal antibodies against HLA-DP, HLA-DQ, or HLA-DR ( Figure 9 When T cell epitopes are presented on HLA-DP, HLA-DQ, and HLA-DR, some T cell epitopes are presented on both HLA-DR and HLA-DQ molecules (Table 10).

[0447] Table 10:

[0448]

[0449] HLA restriction of Ara h1 and Ara h2 T cell epitope presentation

[0450] HLA typing of subjects was performed using TCL to identify dominant T-cell epitopes in order to assess potential HLA subtypes that could be used for T-cell presenting epitopes. The absence of common HLA alleles used to identify T-cell epitopes with confirmed HLA-DR / DQ / DP restriction in subjects indicates HLA-binding degeneracy of T-cell epitopes. Ara h 1 results are shown in Table 11 and Ara h 2 results are shown in Table 12.

[0451] Table 11:

[0452] The gray shading indicates the T-cell epitopes contained in the current 7-peptide mixture.

[0453]

[0454] Nt = HLA limitation not tested

[0455] HLA restriction of Ara h2 epitope presentation

[0456] Table 12:

[0457]

[0458] The absence of a shared HLA-DQB1 allele among all subjects blocked by anti-HLA-DQ at its Ara h 2 T-cell epitope (95–107) suggests that this T-cell epitope must be presented by multiple HLA-DQB1 molecules. Similarly, the diversity in HLA-DRB1 alleles among all subjects blocked by anti-HLA-DR at its Ara h 2 T-cell epitopes (127–141) or (37–47) suggests binding degeneracy of T-cell epitopes for multiple HLA-DRB1 molecules.

[0459] In addition to being presented by at least two HLA-DR molecules, Ara h 2 T cell epitopes (37-47) are also presented by HLA-DQB1. Presented at 06:09, the reason being that subjects who identified this T-cell epitope in the HLA-DQ case had this allele, and for subject 9, only the DQB1 allele was present.

[0460] Due to DPB1 04:01 or DRB1 The 15:01 allele was present in all subjects who recognized Ara h 2 T cell epitopes (32-44) (blocked by anti-HLA-DP) or (95-107) (blocked by anti-HLA-DR), respectively, therefore the degeneracy of these T cell epitopes could not be determined. However, due to DPB1 0401 and DRB1 1501 is prevalent in global populations, so T-cell epitopes presented by these HLA molecules can still be widely identified.

[0461] There is no shared allele between two or more subjects who identified dominant uniform Ara h 1 T cell epitopes for a given HLA type, which indicates that each of the identified Ara h 1 T cell epitopes is also presented by two or more different HLA molecules.

[0462] Predicted HLA binding motif: Ara h1 20-mer peptide

[0463] Table 13 provides an overview of the results of the HLA-DR prediction algorithm for binding motifs within the dominant Ara h 1 20-mer.

[0464] Table 13:

[0465]

[0466] Predicted HLA binding motif: Ara h2 20-mer peptide

[0467] Table 14 provides an overview of the results of the 2 HLA-DR prediction algorithm for binding motifs within the three dominant Ara h 2 20-mers (NB dominant '20-mer 5' was not shown as expected and the actual epitope overlaps with '20-mer 4').

[0468] Table 14:

[0469]

[0470] Table 15:

[0471] The shading indicates alleles that are particularly common in Caucasian populations.

[0472]

[0473] Refined peptides for therapeutic delivery

[0474] Replacing potentially problematic cysteine ​​residues with structurally conserved but chemically less reactive serine residues. Confirming preserved T cell responsiveness. Figure 10 and 11T-cell epitope peptides containing serine exhibit T-cell responses comparable to those of naturally occurring cysteine-containing peptides.

[0475] Combining overlapping Ara h 1 T cell epitopes into single peptides with a length ≤20 aa

[0476] Table 16:

[0477]

[0478] The gray shading indicates overlapping, uniform T-cell epitope pairs that combine to form single peptides for further analysis as described herein. The asterisks and boxes indicate seven Ara h 1 candidate peptides recommended for therapeutic use.

[0479] Combining overlapping Ara h 2 T cell epitopes into single peptides with a length ≤20 aa

[0480] Table 17:

[0481]

[0482] Overview of Ara h 1 and Ara h 2 candidate peptides

[0483] Ten candidate peptides were identified: seven from Ara h 1 and three from Ara h 2 (Table 18).

[0484] Table 18:

[0485]

[0486] Thirteen additional shorter peptide variants (based on a single T-cell epitope) were designed for comparison. Some sequences were lengthened or shortened (according to the native sequence and key residues for T-cell recognition) to improve peptide production performance and solubility. This resulted in a group of 23 candidate peptides for comparison. Peptide details are summarized in Table 19.

[0487] All peptides in Table 18 were produced with a purity of 95%–99.9% and their solubility in solution was determined. T-cell responses to two doses of each of these peptides were then compared in PBMCs from 25 peanut-allergic subjects to select the final treatment combination.

[0488]

[0489] • NB buffer: First test all peptides in PBS; then use 0.1M NH4HCO3 if the sequence shows a preference for high pH, ​​or use 1% acetic acid if a preference for low pH; if insoluble in 1% acetic acid, increase to 2%, 5%, 10%, etc.

[0490] • Omitting the N-terminal 'W' from 'peptide 2' improves stability and facilitates synthesis.

[0491] • Add the C-terminal 'E' to 'peptide 7' to improve solubility (otherwise the peptide is insoluble unless in a toxic buffer).

[0492] Considerations for selecting the final peptide

[0493] Target:

[0494] Maximize population coverage and / or T cell responsiveness while minimizing sequence number and / or length.

[0495] A comprehensive consideration for peptide selection:

[0496] • Comparison of T cell responses in 23-peptide screening

[0497] • Previous T cell responsiveness data (for individual T cell epitopes / peptides)

[0498] • Sequence (production convenience / solubility)

[0499] • HLA restriction (most degenerate and HLA-DQ restricted T cell epitopes)

[0500] Selection considerations based on data from the 23-peptide screening:

[0501] • Key assessment criteria based on SI values ​​of CD25+CFSE-low cells

[0502] • Donor response frequency / peptide (one or two concentrations)

[0503] • Compare responses to long and short variants

[0504] • The strength / consistency of the response (i.e., those subjects who responded to both concentrations versus those who responded to only one concentration).

[0505] • Response Mode

[0506] Table 20 shows the analysis of PBMC T cell responses to all 23 candidate peptides in 34 subjects with peanut allergy. These data show the SI values ​​of the percentage of CD25+CFSElow CD4+ T cells present / unstimulated with the peptide.

[0507] The data were grouped into long and short formats for each region containing a T-cell epitope (see column borders; for example, the first "group" = peptides 1, 23, and 24 [peptide 1 combines overlapping peptides 23 and 24, each containing a separate T-cell epitope]). A summary at the bottom of each group reviews the best peptide selected from that group. (Data show the SI value of % CD25+CFSElow CD4+ T cells with / without the peptide).

[0508] Data are categorized by descending order of values ​​for each peptide "group" in long format: 50 μg sample (or 10 μg sample, if the response is better than this dose). Each row within a peptide group shows data for an individual subject, but the order of subjects varies within each peptide group.

[0509]

[0510]

[0511]

[0512]

[0513]

[0514] Overview of responses to the 23-peptide group in 34 groups

[0515] The data indicates that the selected 7 peptides are the optimal combination, but the boxes indicate groups containing other feasible peptides that could serve as alternatives (or supplements) to the current library:

[0516] For example:

[0517] 1) Peptide 3 can replace peptide 15

[0518] 2) Peptide 8 can replace peptide 21

[0519] 3) Peptide 9 can replace peptide 23

[0520]

[0521] Overview of the responses to each peptide in the 7-peptide mixture in the 39 groups

[0522] • All subjects identified one or more peptides

[0523] • 13 / 39 (33%) identified 100% of the peptides

[0524] • 21 / 39 (54%) identified >85% (6 or more) peptides

[0525] • 31 / 39 (79%) identified >70% (5 or more) peptides

[0526] • Each peptide was identified by at least 25 / 39 subjects (64%).

[0527] Of the 74 participants tested, all React to at least one of the seven selected peptides.

[0528] Response to different peptide libraries

[0529] Table 23:

[0530]

[0531] NB: Table 19 provides the sequences of peptides 1-23.

[0532] Library 1 = 7 × original Ara h 1 "candidates"

[0533] Library2 = 3 × original Ara h 2 "candidates"

[0534] Library 3 = 10 × the mixture of the two libraries mentioned above (Ara h 1 and Ara h 2)

[0535] Library 4 = 3 × Ara h 1 “candidates” + 5 × shorter variants (Ara h 1 sequence coverage equals Library 1)

[0536] Library 5 = 5 × shorter (single epitope) variants of Ara h 2 candidates (Ara h 2 sequence coverage equals Library 2)

[0537] Table 24: Refined Peptide Library

[0538]

[0539]

[0540] Basophilic response to the 7-peptide library (Library 7b)

[0541] Basophil reactivity data were collected from 14 peanut-allergic subjects after incubation with peanut (CPE) or a 7-peptide library (librium 7b) at concentrations spanning 3–4 log (g / ml). Figure 2 In these subjects, basophil activation and histamine release were induced by intact peanuts and positive controls but not by the 7-peptide mixture.

[0542] Before selecting libraries 7a and 7b, libraries 7a and 7b were then designed and tested.

[0543] Compare PBMC T cell responses to intact peanuts or peptide libraries 1-5 from Table 23. Figure 12 and 13Compared to libraries 1-5, none of these libraries induced a positive T-cell response in all tested subjects. Almost all responses to the peptide libraries were significantly lower than those to whole peanuts (at the tested concentrations), and only one subject each of libraries 2, 3, and 4 showed a peptide response greater than or equal to that to whole peanuts. Regarding libraries 7a and 7b, 100% responses (SI > 1.5) were recorded for libraries 7a and 7b. Libraries 7a and 7b induced responses comparable to or greater than those to whole peanuts in many subjects, with 6 / 30 = ≥100 CPE responses and 6 / 30 = 50%–80% CPE responses.

[0544] When comparing PBMC T cell responses to the 7-peptide library ( Figure 14 There were no significant differences between libraries 7a and 7b (comparing paired data; n = 15 / group; no advantage from adding the third Ara h 2 peptide). When comparing the complete dataset of library 7b (n = 30) with the cohort of library 7a using the unpaired Man Whitney test for nonparametric data, there were still no significant differences (p = 0.9).

[0545] In summary, both libraries 7a and 7b were significantly better than the other five libraries tested. There was no significant difference between library 7a and library 7b. Library 7b was recognized by 100% of the tested subjects and induced a PBMC T-cell response comparable to or greater than that of a peanut in over 33% of the subjects. Libraries 1-5 were not recognized by 100% of the subjects and rarely induced a response comparable to that of a whole peanut.

[0546] Table 25: More detailed steps and data overview

[0547]

[0548]

[0549] Example 3

[0550] In Ara h 2 epitope-specific TCC, Ara h 2 peptide-induced T cell unresponsiveness ( Figure 15 )

[0551] In the presence of 100 µg / ml Ara h 2 peptide (ANLRPSEQHLM (SEQ ID NO:31; shaded)), in the absence of helper cells or in complete culture medium alone. In (Ag-free) T cells cloned from Ara h 2 peptide-specific human T cells (1 × 10⁻⁶) 6 The cells were cultured for 16 hours at 100 ml / ml. The T cells were then thoroughly washed and irradiated with autologous PBMCs (100 ml / ml) as helper cells. 5In the presence of (cells / well), re-stimulation was performed with complete culture medium alone (IL-2, 50 U / ml) or immunogenic concentration of Arah 2 peptide (10 µg / ml). 4 / well). Proliferation associated with tritium-labeled thymidine incorporation was determined at 72 hours. Results are expressed as mean cpm + SD of triplicate cultures. Complete culture medium: RPMI + 5% AB serum + Pen / Strep / L-glutamine + 10U / mL IL-2.

[0552] In Ara h 1 epitope-specific TCC, Ara h 1 peptide-induced unresponsiveness ( Figure 16 )

[0553] In the presence of 100 µg / ml Ara h 1 peptide (STRSSENNEGVIVKVSKE (SEQ ID NO:12; shaded)) or unrelated Bahia grass Pas n 1 peptide (dotted), in the absence of helper cells or in complete culture medium alone. In (Ag-free) T cells cloned from Ara h1 peptide-specific human T cells (1 × 10⁻⁶) 6 The cells were cultured for 16 hours at 10⁵ / ml. The T cells were then thoroughly washed and re-elicited (10⁵ cells / well) in the presence of irradiated autologous PBMCs (10⁵ cells / well) as helper cells, with either complete culture medium alone (IL-2, 50 U / ml) or an immunogenic concentration of Ara h I peptide (10 µg / ml). 4 / well). Proliferation associated with tritium-labeled thymidine incorporation was determined at 72 hours. Results are expressed as mean cpm of triplicate cultures. Complete culture medium: RPMI + 5% AB serum + Pen / Strep / L-glutamine + 10U / mL IL-2.

[0554] Those skilled in the art will understand that variations and modifications may be made to the invention described herein, other than those specifically described. It should be understood that the invention includes all such variations and modifications. The invention also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, as well as any combination of any two or more of said steps or features.

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[0635] The technical solutions corresponding to the original claims of the parent application are hereby incorporated in this specification:

[0636] 1. An immunomodulatory composition comprising at least five of the Ara h 1 and Ara h 2 T cell epitope regions derived from the list of the following components:

[0637] (i)FQNLQNHR(SEQ ID NO:1)

[0638] (ii)IVQIEA (SEQ ID NO:2)

[0639] (iii)NEGVIVKVXK (SEQ ID NO:3)

[0640] (iv)EVKPDKKNPQLQ (SEQ ID NO:4)

[0641] (v)EGALML (SEQ ID NO:5)

[0642] (vi)IMPAAHP (SEQ ID NO:6)

[0643] (vii)LRPXEQHLM (SEQ ID NO:7)

[0644] (viii)ENNQRXMXEA (SEQ ID NO:8)

[0645] Or its functional derivatives or homologues, wherein residue X is cysteine ​​or serine, and the composition comprises at least one epitope region selected from SEQ ID NOS: 1-6 and at least one epitope region selected from SEQ ID NOS: 7-8.

[0646] 2. The composition according to item 1, wherein the epitope region LRPXEQHLM is LRPSEQHLM (SEQ ID NO:137).

[0647] 3. The composition according to item 1 or 2, wherein the epitope region ENNQRXMXEA is ENNQRSMSEA (SEQ ID NO: 138).

[0648] 4. The composition according to any one of items 1 to 4, wherein the composition comprises at least 6 of the epitope regions.

[0649] 5. The composition according to any one of items 1 to 4, wherein the composition comprises at least 7 epitope regions.

[0650] 6. The composition according to any one of items 1 to 4, wherein the composition comprises each of the eight epitope regions.

[0651] 7. The composition according to any one of items 1 to 6, wherein the composition comprises one or more peptides, each of which has a length of up to 60 consecutive amino acids, and the peptides comprise one or more of the Ara h 1 and Ara h 2 T cell epitope regions as defined by SEQ ID NOS:1-8.

[0652] 8. The composition according to any one of items 1 to 7, wherein the composition comprises Ara h 1 and Ara h 2 T-cell peptides selected from the list of the following:

[0653] (i)FQNLQNHRIVQIEAKPNTLV(SEQ ID NO:11)

[0654] (ii)STRSSENNEGVIVKVSKE(SEQ ID NO:12)

[0655] (iii)EVKPDKKNPQLQ(SEQ ID NO:4)

[0656] (iv)VEIKEGALMLPHFNSKA(SEQ ID NO:13)

[0657] (v)VFIMPAAHPVAINASS(SEQ ID NO:14)

[0658] (vi)ANLRPXEQHLM(SEQ ID NO:15)

[0659] (vii)EFENNQRXMXEALQ(SEQ ID NO:16)

[0660] (viii)NNFGKLFEVKPDKKNPQLQ(SEQ ID NO:17)

[0661] (ix)gdvfimpaahpvainasse(SEQ ID NO:18)

[0662] (x)SQLERANLRPXEQHLM(SEQ ID NO:19)

[0663] (xi)ELNEFENNQRXMXEALQ(SEQ ID NO:20)

[0664] (xii)FQNLQNHRIV(SEQ ID NO:21)

[0665] (xiii)RIVQIEAKPNTLV(SEQ ID NO:22)

[0666] (xiv)ENNEGVIVKVSKE(SEQ ID NO:23)

[0667] (xv)EVKPDKKNPQLQD(SEQ ID NO:24)

[0668] (xvi)EFENNQRXMXEALQQI(SEQ ID NO:25)

[0669] (xvii)NNFGKLFEVKPDKKNPQLQD (SEQ ID NO:26)

[0670] (xviii)ELNEFENNQRXMXEALQQI (SEQ ID NO:27)

[0671] (xx)WSTRSSENNEGVIVKVSKE (SEQ ID NO:28)

[0672] (xxi)GDVFIMPAAHPVAINASS (SEQ ID NO:29)

[0673] Or its functional derivatives or homologues, wherein residue X is cysteine ​​or serine.

[0674] 9. The composition according to item 8, wherein the residue X is serine.

[0675] 10. The composition according to item 8, wherein the composition comprises Ara h 1 and Ara h 2 T-cell peptides selected from the list of the following:

[0676] (i)FQNLQNHRIVQIEAKPNTLV (SEQ ID NO:11)

[0677] (ii)STRSSENNEGVIVKVSKE (SEQ ID NO:12)

[0678] (iii)EVKPDKKNPQLQ (SEQ ID NO:4)

[0679] (iv)VEIKEGALMLPHFNSKA (SEQ ID NO:13)

[0680] (v)VFIMPAAHPVAINASS (SEQ ID NO:14)

[0681] (vi)ANLRPSEQHLM (SEQ ID NO:31)

[0682] (vii)EFENNQRSMSEALQ (SEQ ID NO:32)

[0683] (viii)EVKPDKKNPQLQD (SEQ ID NO:24)

[0684] (ix)EFENNQRSMSEALQQI (SEQ ID NO:33)

[0685] Or its functional derivatives or homologues.

[0686] 11. The composition according to item 10, wherein the composition comprises each of the Ara h 1 and Ara h 2 T cell peptides selected from the list of the following:

[0687] (i) FQNLQNHRIVQIEAKPNTLV (SEQ ID NO: 11);

[0688] (ii)STRSSENNEGVIVKVSKE (SEQ ID NO: 12);

[0689] (iii) EVKPDKKNPQLQ (SEQ ID NO:4) and / or EVKPDKKNPQLQD (SEQ ID NO:24);

[0690] (iv) VEIKEGALMLPHFNSKA (SEQ ID NO: 13);

[0691] (v)VFIMPAAHPVAINASS (SEQ ID NO:14);

[0692] (vi)ANLRPSEQHLM (SEQ ID NO:31); and

[0693] (vii)EFENNQRSMSEALQ (SEQ ID NO:32) and / or EFENNQRSMSEALQQI (SEQ ID NO:33)

[0694] Or its functional derivatives or homologues.

[0695] 12. The composition according to any one of items 1 to 11, wherein the peptide, when administered to a subject suffering from symptoms characterized by Arah 1 and / or Ara h 2 hypersensitivity or hypersensitivity to compositions containing Arah 1 and / or Ara h 2, is capable of alleviating said hypersensitivity.

[0696] 13. The composition according to any one of items 1 to 12, wherein the composition further comprises one or more peptides selected from the list of the following:

[0697] (i)ALMLLPHFNSKAMVIVVV (SEQ ID NO:34)

[0698] (ii)NNFGKLFEVKPDKKNPQ (SEQ ID NO:35)

[0699] (iii)SQLERANLRPXEQ (SEQ ID NO:36)

[0700] (iv)ELNEFENNQRXM (SEQ ID NO:37)

[0701] (v)NNFGKLFEVKPDKKNPQLQD (SEQ ID NO:38)

[0702] (vi)NNFGKLFEVKPDKKNPQL (SEQ ID NO:40)

[0703] (vii)SQLERANLRPXEQH (SEQ ID NO:41)

[0704] (viii)KAMVIVVVNKGTGNLELVAV (SEQ ID NO:42)

[0705] (ix)RELRNLPQQXGLRA (SEQ ID NO:43)

[0706] (x)KAMVIVVVNKG (SEQ ID NO:44)

[0707] (xi)AMVIVVVNKGTGNLELV (SEQ ID NO:45)

[0708] (xii)VVNKGTGNLELVAVRK (SEQ ID NO:46)

[0709] Or its functional derivatives or homologues, wherein residue X is cysteine ​​or serine.

[0710] 14. A composition comprising one or more nucleic acid molecules encoding or complementary to a sequence encoding an epitope and a peptide according to any one of items 1 to 10 or 13.

[0711] 15. A method for treating and / or preventing a condition in a subject, characterized by an abnormal, unwanted, or otherwise inappropriate immune response to Ara h 1 and / or Ara h 2 or to an allergen in a composition comprising Ara h 1 and / or Ara h 2, the method comprising administering to the subject an effective amount of the immunomodulatory composition according to any one of items 1 to 14 for a period of time, provided that the presence or function of T cells in the subject against said Ara h 1 and / or Ara h 2 or other allergens is sufficient to eliminate or reduce them.

[0712] 16. Use of the immunomodulatory composition according to any one of items 1 to 14 in the manufacture of a medicament for treating a symptom in mammals characterized by an abnormal, unwanted, or otherwise inappropriate immune response to Ara h 1 and / or Ara h 2.

[0713] 17. The method or use according to any one of items 15 or 16, wherein the symptom is hypersensitivity to peanuts or tree nuts containing Ara h1 and Ara h2 or similar molecules.

[0714] 18. The method or use according to item 17, wherein the nut is a hazelnut, almond, or Brazil nut.

[0715] 19. The method or use according to any one of items 15 to 18, wherein the method causes desensitization to or induction of immune tolerance to Ara h 1 and / or Ara h 2 or other allergens of the composition.

[0716] 20. The method or use according to item 19, wherein the desensitization or tolerance is achieved by inducing T cell non-responsiveness or apoptosis.

[0717] 21. The method or use according to item 19, wherein the desensitization or tolerance is achieved by inducing Ara h 1 or Arah 2 specific Treg cells.

[0718] 22. The method or use according to any one of items 15 to 21, wherein the composition is administered intradermally or transdermally.

[0719] 23. The method or use according to any one of items 15 to 22, wherein the subject is a human.

Claims

1. An immunomodulatory composition comprising peptides, wherein the peptides in the composition are composed of six peptides selected from (i) to (vi) and one or two peptides selected from (vii) to (viii): (i)FQNLQNHRIVQIEAKPNTLV (SEQ ID NO:11) (ii)EVKPDKKNPQLQ (SEQ ID NO:4) (iii)VEIKEGALMLPHFNSKA (SEQ ID NO:13) (iv)VFIMPAAHPVAINASS (SEQ ID NO:14) (v)ANLRPSEQHLM (SEQ ID NO:31) (vi) EFENNQRSMSEALQ (SEQ ID NO:32) (vii) STRSSENNEGVIVKVSKE (SEQ ID NO:12) (viii) RELRNLPQQXGLRA (SEQ ID NO:43), wherein residue X is a serine.

2. The composition according to claim 1, wherein the peptide comprises the following peptides: (i) FQNLQNHRIVQIEAKPNTLV (SEQ ID NO: 11); (ii) EVKPDKKNPQLQ (SEQ ID NO: 4); (iii) VEIKEGALMLPHFNSKA (SEQ ID NO: 13); (iv)VFIMPAAHPVAINASS (SEQ ID NO:14); (v)ANLRPSEQHLM (SEQ ID NO:31); (vi) EFENNQRSMSEALQ (SEQ ID NO:32); and (vii) STRSSENNEGVIVKVSKE (SEQ ID NO:12).

3. The composition according to claim 1, wherein the peptide comprises the following peptides: (i) FQNLQNHRIVQIEAKPNTLV (SEQ ID NO: 11); (ii)EVKPDKKNPQLQ (SEQ ID NO:4); (iii) VEIKEGALMLPHFNSKA (SEQ ID NO:13); (iv)VFIMPAAHPVAINASS (SEQ ID NO:14); (v) ANLRPSEQHLM (SEQ ID NO:31); (vi) EFENNQRSMSEALQ (SEQ ID NO:32); and (vii) RELRNLPQQXGLRA (SEQ ID NO:43), wherein residue X is serine.

4. The composition according to claim 1, wherein the peptide comprises the following peptides: (i) FQNLQNHRIVQIEAKPNTLV (SEQ ID NO: 11); (ii)EVKPDKKNPQLQ (SEQ ID NO:4); (iii) VEIKEGALMLPHFNSKA (SEQ ID NO:13); (iv)VFIMPAAHPVAINASS (SEQ ID NO:14); (v) ANLRPSEQHLM (SEQ ID NO:31); (vi) EFENNQRSMSEALQ (SEQ ID NO:32); (vii) STRSSENNEGVIVKVSKE (SEQ ID NO:12); and (viii) RELRNLPQQXGLRA (SEQ ID NO:43), where X is serine.

5. The composition according to any one of claims 1 to 4, wherein the peptide, when administered to a subject suffering from symptoms characterized by Ara h1 and / or Ara h2 hypersensitivity or hypersensitivity to compositions containing Ara h1 and / or Ara h2, is capable of alleviating said hypersensitivity.

6. A composition comprising one or more nucleic acid molecules encoding or complementary to a sequence encoding a peptide according to any one of claims 1 to 4.

7. Use of the immunomodulatory composition according to any one of claims 1 to 6 in the manufacture of a medicament for treating a symptom in a mammal characterized by an abnormal, unwanted, or otherwise inappropriate immune response to Ara h 1 and / or Ara h 2.

8. The use according to claim 7, wherein the condition is hypersensitivity to peanuts or tree nuts containing Ara h 1 and Ara h 2 or similar molecules of Ara h 1 or Ara h 2.

9. The use according to claim 8, wherein the nut is a hazelnut, almond, or Brazil nut.

10. The use according to any one of claims 7 to 9, wherein the use results in desensitization to Ara h 1 and / or Ara h 2 or other allergens of the composition or induction of immune tolerance to these allergens.

11. The use according to claim 10, wherein the desensitization or tolerance is achieved by inducing T cell non-responsiveness or apoptosis.

12. The use according to claim 10, wherein the desensitization or tolerance is achieved by inducing Ara h 1 or Ara h 2 specific Treg cells.

13. The use according to any one of claims 7 to 12, wherein the drug is suitable for intradermal or transdermal administration.

14. The use according to any one of claims 7 to 13, wherein the mammal is a human.

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