Patent Grant
11266737
This application is a national stage entry, filed under 35 U.S.C. § 371, of International Application No. PCT/AU2014/050249, filed on Sep. 25, 2014, and claims the benefit of and priority to Australian Application No. 2013903686, filed Sep. 25, 2013, the entire contents of each of which are hereby incorporated herein by reference in their entireties and for all purposes.
The contents of the text file named “Corrected_Sequence_Listing_28616502N01US_121520.TXT”, which was created on Dec. 15, 2020, and is 33,904 bytes in size, is hereby incorporated by reference in its entirety and for all purposes.
The present invention relates generally to an immunotherapeutic composition. More particularly, the present invention relates to an immunotherapeutic composition which interacts immunologically with T lymphocytes in subjects having peanut allergy or allergy to other tree nuts. This composition is preferably immunointeractive with T cells in subjects having an allergy to the Ara h 1 and/or Ara h 2 allergens. The composition of the present invention is useful in the therapeutic or prophylactic treatment of conditions characterised by an aberrant, inappropriate or otherwise unwanted immune response to peanut, Ara hl and/or Ara h 2 or derivative or homologue thereof.
Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge.
Peanut allergy is a life-threatening and incurable disorder, 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). It is characterised by the sudden onset of anaphylaxis, which may occur with exposure to minute quantities of peanut proteins (Hourihane et al., J Allergy Clin Immunol 100: 596-600, 1997; Pumphrey, Current Opinion in Allergy & Immunology. 4(4):285-90, 2004). Peanut induced anaphylaxis is that most frequently associated with mortality or with life-threatening features (Bock et al. J Allergy Clin Immunol. 119(4):1016-8, 2007; Burks 2008, supra). Peanut proteins are frequently concealed within apparently safe food sources, such that accidental contact occurs for up to 50% of sufferers over a 5 year period (Sicherer et al., Paediatrics 102: e6, 1998). Not surprisingly, peanut and tree nut allergy is associated with significant psychological morbidity for sufferers and carers alike, akin to that suffered by those with chronic debilitating illnesses such as rheumatoid arthritis (Primeau et al., Clin Exp Allergy 30: 1135-43, 2000; Kemp et al. Med. J. Aust. 188(9):503-4, 2008). Cure, while being an imperative to remove peanut and tree nut allergy as a cause of mortality, is also necessary to remove the chronic psychological burden that peanut allergic subjects carry.
To date, efforts at immunotherapy for peanut allergy have been met by extremely limited success. Nelson et al. have shown that clinical desensitisation of peanut can be induced using a rush immunotherapy protocol with an unfractionated peanut extract, but that clinical desensitisation is lost in approximately half of the subjects during maintenance dosing and additionally that injections are associated with frequent episodes of anaphylaxis in the majority of subjects during both the buildup and maintenance phases (Nelson et al., J Allergy Clin Immunol 99: 744-51, 1997). Oppenheimer et al. demonstrated similar findings within their study, again showing that active therapy with unfractionated peanut extract is associated with a high rate of systemic anaphylaxis. Data collection in that study was terminated after the administration of peanut extract to a placebo randomised subject resulted in their death, highlighting the dangerous nature of this condition (Oppenheimer et al., J Allergy Clin Immunol 90: 256-62, 1992). Recent studies of oral immunotherapy with whole peanut flour provide encouragement that desensitization is feasible, but the observed adverse reactions highlight major safety concerns (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 Allergy Immunol. 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 with the exclusion of children prone to severe symptoms or asthma, two studies reported an anaphylactic episode, in one case during an initial food challenge (Clark et al. Allergy 64, 1218, 2009) and in the other during treatment of a child who had not previously experienced anaphylaxis (Hofmann et al. J. Allergy Clin. Immunol. 124, 286, 2009).
Development of novel strategies to overcome the morbidity associated with allergen immunotherapy depends on an accurate understanding of the immunological basis to successful immunotherapy, as well as its side-effects. It has long been established that morbidity due to allergen immunotherapy is due to the cross-linking of IgE, and that this action is not required for such therapy to be efficacious (Litwin et al., Int Arch Allergy Appl Immunol 87: 361-61, 998). It is also known that one of the critical actions of conventional (subcutaneous injection or sublingual or oral with unfractionated allergen extract) immunotherapy in producing tolerance is its ability to change the predominant specific T cell phenotype from a TH2 to a regulatory phenotype. These regulatory T cells act via production of 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).
A key difference in antibody and lymphocyte responses is in antigen recognition, antibodies recognising conformational B cell epitopes dependent on molecular tertiary structure, while CD4+ T cells recognise short linear peptides. This difference in antigen recognition is the basis to many novel strategies of immunotherapy, including that using peptides based upon T cell epitopes, B cell epitope mutants and altered peptide ligands (Rolland et al. Pharmacology & Therapeutics 121:273-284, 2009). Such methods all depend on the alteration or absence of molecular tertiary structure, so that IgE cross-linking and effector cell activation is lost. Peptide immunotherapy is a method in respect of which evidence of efficacy exists, being documented for both cat dander allergy and bee venom allergy. Three different studies showed that, in the absence of any systemic side-effects, clinical and immunological tolerance could be achieved for the major bee venom allergen Phospholipase A2 (PLA2) using T cell epitope-containing sequences (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), while several studies have demonstrated that peptides based on the structure of the major cat allergen Fel d 1 can be used to induce diminished clinical responses (Norman et al., Am J Respir Crit Care Med 154: 1623-8, 1996; Marcotte et al., J Allergy Clin Immunol 101: 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). Most recently, a phase IIa trial confirmed the safety, tolerability and potential efficacy of a seven-peptide mixture from Fel d 1 (Toleromune Cat©, Cicassia Ltd., Oxford, UK) (Worm et al. J Allergy Clin Immunol. 127: 89-97, 2011) with Phase IIb trials now underway (Moldaver & Larche. Allergy. 66: 784-91, 2011; Worm et al. Expert Opin. Investig. Drugs. 22(10): 1347-1357, 2013). Crucial to the development of such strategies is the retention of T cell epitopes, so that T cell phenotypic change can be induced.
The ability to bind directly on to MHC class II molecules allows peptides to be presented by non-professional or immature APC without pro-inflammatory and co-stimulatory signals which promotes induction of tolerance, anergy and/or suppressive activity in responding T cells (Moldaver & Larche, Allergy 66: 784-91, 2011) and/or other CD4+ T cells that express MHC class II. This also allows peptides to be presented at higher frequency than peptides processed from the whole molecule (Santambrogio et al. Proc Natl Acad Sci USA, 1999, 96:15056-61), and since they are also safer than whole allergen, peptides can be given at higher concentrations, thus repolarising T cell responses more effectively.
Importantly, targeting T cells specific for dominant T cell epitopes of major allergens can alter responses to whole allergen extracts (linked suppression). Many studies reporting successful peptide immunotherapy in murine models of allergy demonstrated that administration of dominant T-cell epitope peptides of major allergens induced tolerance not only to those peptides, but also to purified allergen and whole allergen extracts (Yang et al. Clin Exp Allergy 40(4):668-78, 2010; Yoshitomi et al. J Pept 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. Vaccine. 21(5-6):549-61, 2003).
Accordingly, there is a need to both identify the major peanut allergens and, further, to identify the T cell epitopes of these allergens. The identification, characterisation, and analysis of these T cell epitopes is critical to the development of specific immunotherapeutic or prophylactic methodology. To this end, although the Ara h 1 and/or Ara h 2 peanut allergen molecules have previously been the subject of analysis, the identification of the T cell core epitopic regions is essential to the development of an effective vaccine.
In work leading up to the present invention, dominant, HLA-degenerate Ara h 1 and/or Ara h 2 core T cell epitopic regions have been identified. This group of core T cell epitopic regions is unique in terms of its high level of efficacy. Unlike the previously studied 20mer Ara h 1 and/or Ara h 2 peptides which were identified based only on their ability to exhibit some level of T cell reactivity, there have been identified a selected set of core T cell epitope regions which are both immunodominant, relative to other Ara h 1 and/or Ara h 2 peptide fragments, and are also HLA degenerate in that they bind to two or more HLA types. Still further, many of these T cell epitopic core regions are presented by HLA-DQ molecules. HLA-DQ molecules are more conserved in mixed populations than HLA-DR molecules. Accordingly, peptides presented on HLA-DQ enable broader population coverage.
In a still further finding, it has also been determined that a specific subgroup of seven of these peptides which contain both Ara h 1 and Ara h 2 T cell epitopes provides particularly effective immunological outcomes. Accordingly, the identification of this uniquely effective group of peptides has facilitated the development of particularly effective therapeutic prophylactic methods for the treatment of conditions characterised by aberrant, inappropriate or otherwise unwanted immune responses to Ara h 1 and/or Ara h 2 or derivative or homologue thereof, other tree nut allergies or allergens to a composition, such as foods, containing the Ara h 1 and/or Ara h 2 molecules.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of “a”, “and” and “the” include plural referents unless the context clearly dictates otherwise.
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 belongs.
The subject specification contains amino acid sequence information prepared using the programme PatentIn Version 3.5, presented herein after the bibliography. Each amino acid sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (eg. <210>1, <210>2, etc). The length, type of sequence (protein, etc) and source organism for each sequence is indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Amino acid sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (eg. SEQ ID NO:1, SEQ ID NO:2, etc.). The sequence identifier referred to in the specification correlates to the information provided in numeric indicator field <400> in the sequence listing, which is followed by the sequence identifier (eg. <400>1, <400>2, etc). That is SEQ ID NO:1 as detailed in the specification correlates to the sequence indicated as <400>1 in the sequence listing.
One aspect of the present invention is directed to an immunomodulatory composition comprising at least five of the Ara h 1 and Ara h 2 T cell epitopic regions from the list consisting of:
or functional derivatives or homologues thereof wherein residue X is cysteine or serine and said composition comprises at least one T cell epitopic region selected from SEQ ID NOS: 1-6 and at least one T cell epitopic region selected from SEQ ID NOS: 7-8.
In another aspect, said LRPXEQHLM is LRPSEQHLM (SEQ ID NO;137).
In still another aspect, said ENNQRXMXEA is ENNQRSMSEA (SEQ ID NO:103).
In accordance with these aspects and embodiments of the present invention, said composition comprises at least 6 of said T cell epitopic regions.
In a further aspect, said composition comprises at least 7 of said T cell epitopic regions.
In still a further aspect, said composition comprises each of said 8 T cell epitopic regions.
In another aspect there is provided an immunomodulatory composition comprising each of the Ara h 1 and Ara h 2 T cell epitopic regions from the list consisting of:
or functional derivatives or homologues thereof wherein said residue X is cysteine or serine.
In yet another aspect there is provided an immunomodulatory composition comprising each of the Ara h 1 and Ara h 2 T cell epitopic regions from the list consisting of:
or functional derivatives or homologues thereof.
In a related aspect the present invention is directed to an immunomodulatory composition comprising one or more peptides, each of which peptides is up to 60 contiguous amino acids in length and which peptides include each of the Ara h 1 and Ara h 2 T cell epitopic region combinations detailed hereinbefore.
In a further aspect the present invention is directed to an immunomodulatory composition comprising one or more peptides, each of which peptides is up to 60 contiguous amino acids in length and which peptides include each of the Ara h 1 and Ara h 2 T cell epitopic regions from the list consisting of:
or functional derivatives or homologues thereof.
In yet another aspect said peptides or T cell epitopic regions are capable of modifying T cell function when presented to T cells isolated from subjects having a condition characterised by an aberrant, unwanted or otherwise inappropriate immune response to Ara h 1 and/or Ara h 2 or to an allergen present in a composition, such as food, comprising Ara h 1 and/or Ara h 2 but which peptides are unable to bind to Ara h 1-specific and/or Ara h 2-specific IgE.
In accordance with these aspects, said peptides are selected from the list consisting of:
or functional derivatives or homologues thereof wherein residue X is cysteine or serine.
In one embodiment, said residue X is serine.
In a further asepct said peptides are selected from:
or functional derivatives or homologues thereof.
In another aspect, said immunomodulatory composition comprises each of the Ara h 1 and Ara h 2 T cell peptides from the list consisting of:
or functional derivatives or homologues thereof.
In a yet a further aspect, the inventors have designed a preferred set of seven peptides, five of which comprise Ara h 1 T cell epitopes and two of which comprise Ara h 2 T cell epitopes, which function particularly efficaciously, when administered together, to induce desensitisation or tolerance and thereby either prophylactically or therapeutically treat hypersensitivity to compositions, such as foods, comprising Ara h 1 and/or Ara h 2. These peptides are:
In still yet another aspect there is provided an immunomodulatory composition comprising each of the Ara h 1 and Ara h 2 T cell peptides from the list consisting of:
In a yet still another aspect, there is provided a composition comprising each of the Ara h 1 and Ara h 2 T cell peptides from the list consisting of:
which peptides are capable of reducing Ara h 1 and/or Ara h 2 hypersensitivity or hypersensitivity to a composition comprising Ara h 1 and/or Ara h 2 when administered to a subject having a condition characterised by said hypersensitivity.
The present invention is directed to a composition comprising the peptides hereinbefore defined. It should be understood, though, that the subject composition may comprise additional components, such as additional peptides. Examples of other peptides which may be included in the composition include, but are not limited to:
or functional derivatives or homologues thereof wherein residue X is cysteine or serine.
In another aspect, the present invention provides a nucleic acid molecule composition comprising one or more nucleic acid molecules encoding or complementary to a sequence encoding the T cell epitopes and peptides as hereinbefore defined or a derivative, homologue or analogue thereof.
In still another aspect the present invention provides a method for the treatment and/or prophylaxis of a condition in a subject, which condition is characterised by the aberrant, unwanted or otherwise inappropriate immune response to Ara h 1 and/or Ara h 2 or an allergen in a composition comprising Ara h 1 and/or Ara h 2, said method comprising administering to said subject an effective amount of an immunomodulatory composition as hereinbefore defined for a time and under conditions sufficient to remove or reduce the presence or function in said subject of T cells directed to said Ara h 1 and/or Ara h 2 or other allergen.
In a further aspect said condition is hypersensitivity to peanuts or tree nuts which contain Ara h 1 and Ara h 2 or Ara h 1-like or Ara h 2-like molecules, such as hazelnuts, almonds or Brazil nuts.
In another aspect, said method desensitises or induces immunological tolerance to Ara h 1 and/or Ara h 2 or other allergen of said composition.
In still another aspect, said desensitization or tolerance is achieved by inducing T cell anergy or apoptosis.
In yet still another aspect, said desensitisation or tolerance is achieved by inducing Ara h 1 or Ara h 2-specific Treg cells.
A further aspect of the present invention contemplates the use of an immunomodulatory composition as hereinbefore defined in the manufacture of a medicament for the treatment of a condition in a mammal, which condition is characterised by an aberrant, unwanted or otherwise inappropriate immune response to Ara h 1 and/or Ara h 2.
Preferably said condition is hypersensitivity to peanuts or a tree nut which contains Ara h 1 and/or Ara h 2 or Ara h 1-like and/or Ara h 2-like molecules, such as a hazelnut.
In yet another further aspect, the present invention contemplates a vaccine comprising the composition as hereinbefore defined together with one or more pharmaceutically acceptable carriers and/or diluents. Said composition is referred to as the active ingredient.
Yet another aspect of the present invention relates to the compositions, as hereinbefore defined, when used in the method of the present invention.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The present invention is predicated, in part, on the identification of a group of Ara h 1 and Ara h 2 epitopes which, when administered together, in a group of at least five, produce more efficacious immunological outcomes than any of these epitopes used either alone or together with other combinations of these or other Ara h 1 or Ara h 2 peptides. In particular, it has been determined that the use of all eight epitopes produces particularly and exceptionally efficacious functional outcomes, most particularly when these eight epitopes are administered in the context of the seven peptides exemplified herein. The design of this composition has enabled the development of significantly more efficacious therapeutic and prophylactic compositions and treatment approaches, than have been available to date, for conditions such as, but not limited to, peanut allergy.
Accordingly, one aspect of the present invention is directed to an immunomodulatory composition comprising at least five of the Ara h 1 and Ara h 2 T cell epitopic regions from the list consisting of:
or functional derivatives or homologues thereof wherein residue X is cysteine or serine and said composition comprises at least one T cell epitopic region selected from SEQ ID NOS: 1-6 and at least one T cell epitopic region selected from SEQ ID NOS: 7-8.
In one embodiment, said LRPXEQHLM is LRPSEQHLM (SEQ ID NO;137).
In another embodiment, said ENNQRXMXEA is ENNQRSMSEA (SEQ ID NO:103).
In accordance with these aspects and embodiments of the present invention, said composition comprises at least 6 of said T cell epitopic regions.
In a further embodiment, said composition comprises at least 7 of said T cell epitopic regions.
In still a further embodiment, said composition comprises each of said 8 T cell epitopic regions.
According to this embodiment there is therefore provided an immunomodulatory composition comprising each of the Ara h 1 and Ara h 2 T cell epitopic regions from the list consisting of:
or functional derivatives or homologues thereof wherein said residue X is cysteine or serine.
In another embodiment there is provided an immunomodulatory composition comprising each of the Ara h 1 and Ara h 2 T cell epitopic regions from the list consisting of:
or functional derivatives or homologues thereof.
In a related aspect the present invention is directed to an immunomodulatory composition comprising one or more peptides, each of which peptides is up to 60 contiguous amino acids in length and which peptides include each of the Ara h 1 and Ara h 2 T cell epitopic region combinations detailed hereinbefore.
In accordance with this aspect the present invention is directed to an immunomodulatory composition comprising one or more peptides, each of which peptides is up to 60 contiguous amino acids in length and which peptides include each of the Ara h 1 and Ara h 2 T cell epitopic regions from the list consisting of:
or functional derivatives or homologues thereof.
In another embodiment of the preceding aspects of the invention, said peptides or T cell epitopic regions are capable of modifying T cell function when presented to T cells isolated from subjects having a condition characterised by an aberrant, unwanted or otherwise inappropriate immune response to Ara h 1 and/or Ara h 2 or to an allergen present in a composition, such as food, comprising Ara h 1 and/or Ara h 2 but which peptides are unable to bind to Ara h 1-specific and/or Ara h 2-specific IgE.
Without limiting the present invention in any way, peanuts contain many proteins, with the number of distinct bands visible on SDS-PAGE depending on the methodology used. Up to 53 bands are visible following high pressure liquid chromatography (de Jong et al., Clin Exp Allergy 28: 743-51, 1998). Only two of these proteins warrant classification as major allergens using standard criteria, whereby IgE reactivity occurs within greater than 50% of the peanut allergic population; these proteins are termed Ara h 1 and Ara h 2 (Burks et al., Allergy 53: 725-30, 1998). Although a number of studies have indicated Ara h 2 to be the more potent of these two allergens (Blanc et al. Clin Exp Allergy. 2009; 39(8):1277-85; Koppelman et al. Clin Exp Allergy. 2004; 34(4):583-90; Palmer et al. Clin Immunol. 2005; 115(3):302-12), Ara h 1 also plays a major role in the pathogenesis of peanut allergy, with numerous studies reporting strong correlations between symptom severity and IgE reactivity to both Ara h 1 and Ara h 2 (Glaumann et al. Allergy. 2012; 67(2):242-7; Chiang et al. Pediatr Allergy Immunol. 2009; 21(2 Pt 2):e429-38; Asarnoj et al. 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 peanut, accounting for 12-16% of total peanut protein (Koppelman et al. Allergy. 2001; 56(2):132-7).
Still without limiting the present invention in any way, the Ara h 1 allergen is a 7S seed storage glycoprotein or vicilin. The concentration of Ara h 1 in peanuts increases with the size of the kernel (4-16 mg extracted Ara h 1/g peanut), so expression of the protein is associated with peanut maturity (Pomés et al. 2006, Clin. Exp. Allergy 36(6):824-30). Ara h 1 is a homotrimer held together through hydrophobic areas at the distal ends of the monomers, where most of the IgE binding B cell epitopes are located. Each 64.5 kD monomer has a cupin motif which consists of two core β-barrels, each associated to a loop domain of α-helices.
Ara h 2 is a glycoprotein which has been identified as a member of the conglutin seed storage family. 20% of the Ara h 2 molecular mass represents carbohydrate side chains and it migrates as a doublet on SDS-PAGE with an average molecular mass of 17.5 kDa (Burks et al, Int Arch Allergy Immunol 119:165-172, 1992). It has been characterised as a major allergen, on the basis of its reactivity with 6 out of 6 sera tested (Burks et al, 1992, supra). Others have also confirmed its importance; Clarke demonstrated that 71% of subjects possessed specific IgE to Ara h 2 upon western blotting of crude peanut extract. Kleber-Janke et al. have demonstrated that 85% of subjects possessed IgE towards their recombinant form upon western blotting, and de Jong's group have shown that approximately 78% of their group demonstrate specific IgE to purified natural Ara h 2 (Clarke et al., Clin Exp Allergy 28: 1251-7, 1998; de Jong et al, 1998 supra; Kleber-Janke et al., Int Arch Allergy Immunol 119: 265-274, 1999).
Reference to “Ara h 1” should be understood as a reference to all forms of this molecule including reference to any isoforms which may arise from alternative splicing of Ara h 1 mRNA or functional mutant or polymorphic forms of Ara h 1. It should still further be understood to extend to any protein encoded by the Ara h 1 gene, any subunit polypeptide, such as precursor forms which may be generated, whether existing as a monomer, multimer or fusion protein. It also includes reference to analogues or equivalents of Ara h 1 such as may occur where a product which naturally comprises Ara h 1 is synthetically generated for the purpose of generating a product such as a food additive. The present invention thereby provides T cell epitopes and methods for their use in the diagnosis and treatment of any condition characterised by hypersensitivity to an Ara h 1 or Ara h 1-like molecule, such as peanut allergy or a tree-nut allergy, or an allergy to an antigen present in a composition, such as food, which composition also comprises Ara h 1. Preferably, said Ara h 1 comprises the sequence set forth in SEQ ID NO:9 and Ara h 2 comprises the sequence set forth in SEQ ID NO: 10.
Reference to “T cells” should be understood as a reference to any cell comprising 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. The present invention is not intended to be limited to any particular functional sub-class of T cells although in a preferred embodiment the subject T cell is a T helper cell and still more preferably a Th2-type cell and/or Treg cell. In this regard, reference to “modifying T cell function” should be understood as a reference to modifying any one or more functions which a T cell is capable of performing. For example, the subject function may be proliferation, differentiation or other form of cellular functional activity such as the production of cytokines. In one embodiment the subject functional activity is proliferation.
In terms of “modifying the function” of T cells isolated from subjects having a condition characterised by an aberrant, unwanted or inappropriate immune response to Ara h 1 and/or Ara h 2 or to a composition which comprises Ara h 1 and/or Ara h 2, it should be understood that this is not necessarily a reference to modifying the function of all the T cells in a given biological sample but is likely, in fact, to reflect the modification of functioning of only some of the T cells in the sample. For example, only a portion of the T helper cells in a given T cell sample may functionally respond to contact with the subject peptide. Such a partial response should be understood to fall within the scope of the present invention. It should also be understood that the T cells which are 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 have been generated from the cell sample and it is these T cell lines which then form the subject derived T cell population which is tested in accordance with the present invention. To the extent that the subject functional activity is T cell proliferation, the T cell proliferation assay is preferably performed as disclosed herein. Still more preferably, the subject modification of T cell function is the induction of proliferation. In this regard, reference to “Ara h 1-reactive” or “Arah 2-reactive” T cells should be understood as a reference to a T cell which responds functionally to HLA presentation of an Ara h 1 and/or Ara h 2 T cell epitope, respectively. Similarly, reference to “Ara h 1-specific” or “Ara h 2-specific” IgE should be understood as a reference to IgE directed to Ara h 1 or Ara h 2 B cell epitopes, respectively.
Reference to an “aberrant, unwanted or otherwise inappropriate” immune response should be understood as a reference to any form of physiological activity which involves the activation and/or functioning of one or more immune cells where that activity is inappropriate in that it is of an inappropriate type or proceeds to an inappropriate degree. It may be aberrant in that according to known immunological principles it either should not occur when it does so or else should occur when it does not do so. In another example, the immune response may be inappropriate in that it is a physiologically normal response but which is unnecessary and/or unwanted, such as occurs with respect to type-I hypersensitivity responses to innocuous allergens. In the context of the present invention, this immune response may be directed to Ara h 1 and/or Ara h 2 or it may be directed to a different allergen which is present in a composition together with Ara h 1 and/or Ara h 2. Without limiting the present invention to any one theory or mode of action, it has been determined that even where the hypersensitivity response is directed to an allergen other than Ara h 1 and/or Ara h 2, which allergen is present in a composition which nevertheless comprises Ara h 1 and/or Ara h 2, treatment via the method of the present invention which is directed to Ara h 1 and/or Ara h 2 nevertheless induces beneficial modulation of Th2 and Treg functionality such that the hypersensitivity which exists to the unrelated allergen is nevertheless reduced. Preferably said immune response is peanut hypersensitivity.
By “peanut hypersensitivity” is meant the induction of clinical symptoms of IgE mediated peanut hypersensitivity. However, it should be understood that although clinical symptoms may be evident, not all such individuals would necessarily exhibit detectable levels of peanut specific serum IgE which is measured using the Kallestad Allercoat EAST System (Sanofi-Pasteur Diagnostics, USA), although such individuals should nevertheless be understood to fall within the scope of the definition of those exhibiting “peanut hypersensitivity”. Alternatively, testing may proceed utilising any of the EAST, Pharmacia or the UniCap systems or allergen skin prick testing. 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 the Ara h 1 and/or Ara h 2 protein.
In accordance with the preceding aspects, said peptides are selected from the list consisting of:
or functional derivatives or homologues thereof wherein residue X is cysteine or serine.
In one embodiment, said residue X is serine.
Preferably, said peptides are selected from:
or functional derivatives or homologues thereof.
In another embodiment, said immunomodulatory composition comprises each of the Ara h 1 and Ara h 2 T cell peptides from the list consisting of:
or functional derivatives or homologues thereof.
The reduction of peanut, Ara h 1 and Ara h 2 hypersensitivity (and allergen hypersensitivity more generally) is discussed in more detail hereafter. Briefly, however, this may take the form of either partially or completely desensitising or tolerising an individual to Ara h 1 and Ara h 2 specifically or peanut or other proteins more generally.
Reference to a “peptide” includes reference to a peptide, polypeptide or protein or parts thereof. The peptide may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins. Reference hereinafter to a “peptide” includes a peptide comprising a sequence of amino acids as well as a peptide associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
“Derivatives” include fragments, parts, portions and variants from natural, synthetic or recombinant sources including fusion proteins. Parts or fragments include, for example, active regions of the subject peptide. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence.
Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. An example of substitutional amino acid variants are conservative amino acid substitutions. Conservative amino acid substitutions typically include substitutions within the following groups: 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. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins. In one embodiment, cysteine residues are substituted with serine, as exemplified herein.
Chemical and functional equivalents of the subject peptide should be understood as molecules exhibiting any one or more of the functional activities of these molecules and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening.
Homologues include peptides derived from varieties other than peanuts, such as peptides derived from other tree nuts.
Analogues contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogues. Mutants include molecules which exhibit modified functional activity (for example, Ara h 1 peptides which express one or more T cell epitopes but lack B cell reactivity).
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide. Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH. Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated herein is shown in Table 1.
Crosslinkers can be used, for example, to stabilise 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety.
It is possible to modify the structure of a peptide according to the invention for various purposes such as for increasing solubility, enhancing therapeutic or preventative efficacy, enhancing stability or increasing resistance to proteolytic degradation. A modified peptide may be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion or addition, to modify immunogenicity and/or reduce allergenicity. Similarly components may be added to peptides of the invention to produce the same result.
For example, a peptide can be modified so that it exhibits the ability to induce T cell anergy. In this instance, critical binding residues for the T cell receptor can be determined using known techniques (for example substitution of each residue and determination of the presence or absence of T cell reactivity) In one example, those residues shown to be essential to interact with the T cell receptor can be modified by replacing the essential amino acid with another, preferably similar amino acid residue (a conservative substitution) whose presence is shown to alter T cell reactivity or T cell functioning. In addition, those amino acid residues which are not essential for T cell receptor interaction can be modified by being replaced by another amino acid whose incorporation may then alter T cell reactivity or T cell functioning but does not, for example, eliminate binding to relevant MHC proteins. In yet another example, mutant peptides may be created which exhibit normal T cell binding but abrogated IgE binding.
Exemplary conservative substitutions are detailed, below, and include:
Such modifications will result in the production of molecules falling within the scope of “mutants” of the subject peptide as herein defined. “Mutants” should be understood as a reference to peptides which exhibit one or more structural features or functional activities which are distinct from those exhibited by the non-mutated peptide counterpart.
Peptides of the invention may also be modified to incorporate one or more polymorphisms resulting from natural allelic variation and D-amino acids, non-natural amino acids or amino acid analogues may be substituted into the peptides to produce modified peptides which fall within the scope of the invention. Peptides may also be modified by conjugation with polyethylene glycol (PEG) by known techniques. Reporter groups may also be added to facilitate purification and potentially increase solubility of the peptides according to the invention. Other well known types of modification including insertion of specific endoprotease cleavage sites, addition of functional groups or replacement of hydrophobic residues with less hydrophobic residues as well as site-directed mutagenesis of DNA encoding the peptides of the invention may also be used to introduce modifications which could be useful for a wide range of purposes. The various modifications to peptides according to the invention which have been mentioned above are mentioned by way of example only and are merely intended to be indicative of the broad range of modifications which can be effected.
As detailed hereinbefore, the present invention provides peptides which retain all or some of their capacity to interact with T cells but exhibit partially or completely inhibited, abrogated or otherwise down-regulated antibody reactivity. Effecting the down-regulation of antibody reactivity can be achieved by any suitable method, which methods would be well known to those skilled in the art. For example, to the extent that a B cell epitope is defined by its linear amino acid sequence, one may add, delete or substitute one or more amino acid residues in order to render the mutated linear sequence distinct from the naturally occurring sequence. To the extent that an epitope may be additionally, or alternatively, defined by a conformational epitope, one may seek to disrupt that conformation by disrupting a 2° or, to the extent that homodimers or heterodimers exist, a 3° structure of the peptide. This may be achieved, for example, by disrupting the formation of bonds, such as disulphide bonds, which are known to stabilise 2° and/or 3° structures. In terms of the T cell epitopes hereinbefore defined, these T cells epitopic regions do not comprise B cell epitopes.
In a related aspect, the inventors have designed a preferred set of seven peptides, five of which comprise Ara h 1 T cell epitopes and two of which comprise Ara h 2 T cell epitopes, which function particularly efficaciously, when administered together, to induce desensitisation or tolerance and thereby either prophylactically or therapeutically treat hypersensitivity to compositions, such as foods, comprising Ara h 1 and/or Ara h 2. These peptides are:
Accordingly, in a preferred embodiment there is provided an immunomodulatory composition comprising each of the Ara h 1 and Ara h 2 T cell peptides from the list consisting of:
In a further aspect, there is provided a composition comprising each of the Ara h 1 and Ara h 2 T cell peptides from the list consisting of:
which peptides are capable of reducing Ara h 1 and/or Ara h 2 hypersensitivity or hypersensitivity to a composition comprising Ara h 1 and/or Ara h 2 when administered to a subject having a condition characterised by said hypersensitivity.
The peptides of the present invention may be prepared by recombinant or chemical synthetic means. According to a preferred aspect of the present invention, there is provided a recombinant peptide or mutant thereof which is preferentially immunologically reactive with T cells from individuals with peanut hypersensitivity, which is expressed by the expression of a host cell transformed with a vector coding for the peptide sequence of the present invention. The peptide may be fused to another peptide, polypeptide or protein. Alternatively, the peptide may be prepared by chemical synthetic techniques, such as by the Merrifield solid phase synthesis procedure. Furthermore, although synthetic peptides of the sequence given above represent a preferred embodiment, the present invention also extends to biologically pure preparations of the naturally occurring peptides or fragments thereof. By “biologically pure” is meant a preparation comprising at least about 60%, preferably at least about 70%, or preferably at least about 80% and still more preferably at least about 90% or greater as determined by weight, activity or other suitable means.
The present invention should therefore be understood to encompass peptides that comprise at least one T cell core epitopic region of Ara h 1 and/or Ara h 2, as hereinbefore defined, in conjunction with other amino acids (which may or may not be naturally occurring) or other chemical species. In a preferred aspect of the invention such peptides may comprise one or more epitopes of Ara h 1 and/or Ara h 2, which epitopes are T cell core epitopic regions. Peptides with one or more T cell epitopes of Ara h 1 and/or Ara h 2 are desirable for increased therapeutic effectiveness.
As detailed hereinbefore, the present invention is directed to a composition comprising the peptides hereinbefore defined. It should be understood, though, that the subject composition may comprise additional components, such as additional peptides. These peptides may encompass, for example, partial regions of the core minimal epitope. Alternatively, they may not comprise any part of a T cell epitope as disclosed herein but may be incorporated for either reasons. Examples of other peptides which may be included in the composition include, but are not limited to:
or functional derivatives or homologues thereof wherein residue X is cysteine or serine.
One may also include still other peptides or molecules which may be advantageous given the particulars of a specific situation.
In another aspect, the present invention provides a nucleic acid molecule composition comprising one or more nucleic acid molecules encoding or complementary to a sequence encoding the T cell epitopes and peptides as hereinbefore defined or a derivative, homologue or analogue thereof.
It should be understood that reference to “peptides” includes reference to peptides comprising one or more T cell epitopes. A nucleic acid molecule encoding the subject peptide is preferably a sequence of deoxyribonucleic acids such as cDNA or a genomic sequence. A genomic sequence may comprise exons and introns. A genomic sequence may also include a promoter region or other regulatory regions.
The nucleic acid molecule may be ligated to an expression vector capable of expression in a prokaryotic cell (eg. E. coli) or a eukaryotic cell (eg. yeast cells, fungal cells, insect cells, mammalian cells or plant cells). The nucleic acid molecule may be ligated or fused or otherwise associated with a nucleic acid molecule encoding another entity such as, for example, a signal peptide. It may also comprise additional nucleotide sequence information fused, linked or otherwise associated with it either at the 3′ or 5′ terminal portions or at both the 3′ and 5′ terminal portions. The nucleic acid molecule may also be part of a vector, such as an expression vector. The latter embodiment facilitates production of recombinant forms of the subject peptide which forms are encompassed by the present invention.
Such nucleic acids may be useful for recombinant production of T cell epitopes of Ara h 1 and/or Ara h 2 or proteins comprising them by insertion into an appropriate vector and transfection into a suitable cell line. Such expression vectors and host cell lines also form an aspect of the invention.
In producing peptides by recombinant techniques, host cells transformed with a nucleic acid having a sequence encoding a peptide according to the invention or a functional equivalent of the nucleic acid sequence are cultured in a medium suitable for the particular cells concerned. Peptides can then be purified from cell culture medium, the host cells or both using techniques well known in the art such as ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis or immunopurification with antibodies specific for the peptide.
Nucleic acids encoding Ara h 1 and/or Ara h 2 or peptides comprising T cell core epitopic regions of Ara h 1 and/or Ara h 2 may be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells such as Chinese hamster ovary cells (CHO). Suitable expression vectors, promoters, enhancers and other expression control elements are referred to in Sambruck et al (1989). Other suitable expression vectors, promoters, enhancers and other expression 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, Calif.). These vectors are freely available as are baculovirus and mammalian expression systems. For example, a baculovirus system is commercially available (ParMingen, San Diego, Calif.) for expression in insect cells while the pMsg vector is commercially available (Pharmacia, Piscataway, N.J.) for expression in mammalian cells.
For expression in E. coli suitable expression vectors include among others, pTrc (Amann et al., 1998, Gene., 69:301-315) pGex (Amrad Corporation, Melbourne, Australia); pMal (N.E. Biolabs, Beverley, Mass.); pRit5 (Pharmacia, Piscataway, N.J.); pEt-11d (Novagen, Maddison, Wis.) (Jameel et al., 1990, J. Virol., 64:3963-3966) and pSem (Knapp et al., 1990, Bio Techniques., 8:280-281). The use of pTRC, and pEt-11d, for example, will lead to the expression of unfused protein. The use of pMal, pRit5, pSem and pGex will lead to the expression of allergen fused to maltose E binding protein (pMal), protein A (pRit5), truncated -galactosidase (PSEM) or glutathione S-transferase (pGex). When a T cell epitope of Ara h 1 or a peptide comprising it is expressed as a fusion protein, it is particularly advantageous to introduce an enzymatic cleavage site at the fusion junction between the carrier protein and the peptide concerned. The peptide of the invention may then be recovered from the fusion protein through enzymatic cleavage at the enzymatic site and biochemical purification using conventional techniques for purification of proteins and peptides. The different vectors also have different promoter regions allowing constitutive or inducible expression or temperature induction. It may additionally be appropriate to express recombinant peptides in different E. coli hosts that have an altered capacity to degrade recombinantly expressed proteins. Alternatively, it may be advantageous to alter the nucleic acid sequence to use codons preferentially utilised by E. coli, where such nucleic acid alteration would not effect the amino acid sequence of the expressed proteins.
Host cells can be transformed to express the nucleic acids of the invention using conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection or electroporation. Suitable methods for transforming the host cells may be found in Sambruck et al. (1989), and other laboratory texts. The nucleic acid sequence of the invention may also be chemically synthesised using standard techniques.
In addition to recombinant production of peptides according to the invention, the nucleic acids may be utilised as probes for experimental or purification purposes.
Identification and synthesis of the peptides as disclosed herein now facilitates the development of a range of prophylactic and therapeutic treatment protocols for use with respect to peanut related immune conditions. Also facilitated is the development of reagents for use therein. Accordingly, the present invention should be understood to extend to the use of the peptides or functional derivatives, homologues or analogues thereof in the therapeutic and/or prophylactic treatment of patients. Such methods of treatment include, but are not limited to:
Accordingly, in another aspect the present invention provides a method for the treatment and/or prophylaxis of a condition in a subject, which condition is characterised by the aberrant, unwanted or otherwise inappropriate immune response to Ara h 1 and/or Ara h 2 or an allergen in a composition comprising Ara h 1 and/or Ara h 2, said method comprising administering to said subject an effective amount of an immunomodulatory composition as hereinbefore defined for a time and under conditions sufficient to remove or reduce the presence or function in said subject of T cells directed to said Ara h 1 and/or Ara h 2 or other allergen.
Preferably said condition is hypersensitivity to peanuts or tree nuts which contain Ara h 1 and Ara h 2 or Ara h 1-like or Ara h 2-like molecules, such as hazelnuts, almonds or Brazil nuts.
In one embodiment, said method desensitises or induces immunological tolerance to Ara h 1 and/or Ara h 2 or other allergen of said composition.
In another embodiment, said desensitization or tolerance is achieved by inducing T cell anergy or apoptosis.
In still another embodiment, said desensitisation or tolerance is achieved by inducing Ara h 1 or Ara h 2-specific Treg cells.
An “effective amount” means an amount necessary at least partly to attain the desired immune response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated. The amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
The subject of the treatment or prophylaxis is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer). Preferably the mammal is a human or primate. Most preferably the mammal is a human.
Reference herein to “treatment” and “prophylaxis” is to be considered in its broadest context. The term “treatment” does not necessarily imply that a subject is treated until total recovery. Similarly, “prophylaxis” does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term “prophylaxis” may be considered as reducing the severity or onset of a particular condition. “Treatment” may also reduce the severity of an existing condition.
Administration of the composition of the present invention (herein referred to as “agent”) in the form of a pharmaceutical composition, may be performed by any convenient means. The agent of the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the agent chosen. A broad range of doses may be applicable. Considering a patient, for example, from about 0.01 μg to about 1 mg of an agent may be administered per dose. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. In another example, said composition is administered initially to induce tolerance and then, if necessary, booster administrations of the composition are administered to maintain tolerance. These boosters may be administered monthly, for example, and may be administered for any period of time, including the life of the patient.
The agent may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal (with or without using a traditional needle or other transdermal delivery device), transdermal, intranasal, sublingual or suppository routes or implanting (e.g. using slow release molecules). Preferably, said composition is administered intradermally. The agent may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g. with zinc, iron or the like (which are considered as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, the tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.
In accordance with these methods, the agent defined in accordance with the present invention may be coadministered with one or more other compounds or molecules. By “coadministered” is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. By “sequential” administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order. It should also be understood that the peptides of the present invention may be themselves administered simultaneously or sequentially. They may be administered as one or more compositions, either simultaneously or sequentially. For example, one may formulate some of the peptides in one formulation and the others in a separate formulation; with these two formulations being given one in each arm. Alternatively, additional separate formulations could be generated and administered, either simultaneously to different sites or sequentially. It is well within the skill of the person in the art to design and generate the production of an appropriate formulation or mix of formulations.
Another aspect of the present invention contemplates the use of an immunomodulatory composition as hereinbefore defined in the manufacture of a medicament for the treatment of a condition in a mammal, which condition is characterised by an aberrant, unwanted or otherwise inappropriate immune response to Ara h 1 and/or Ara h 2.
Preferably said condition is hypersensitivity to peanuts or a tree nut which contains Ara h 1 and/or Ara h 2 or Ara h 1-like and/or Ara h 2-like molecules, such as a hazelnut.
In yet another further aspect, the present invention contemplates a vaccine comprising the composition as hereinbefore defined together with one or more pharmaceutically acceptable carriers and/or diluents. Said composition is referred to as the active ingredient.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. Tonicity adjusting agents are useful to keep the preparation isotonic with human plasma and thus avoid tissue damage. Commonly used tonicity agents include Dextrose, Trehalose, Glycerin and Mannitol. Glycerol and sodium chloride are other options but are less commonly used. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 1000 μs of active compound.
The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.
The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule encoding a modulatory agent. The vector may, for example, be a viral vector.
Routes of administration include, but are not limited to, respiratorally (eg. intranasally or orally via aerosol), intratracheally, nasopharyngeally, intravenously, intraperitoneally, subcutaneously, intracranially, intradermally, transdermally, intramuscularly, intraoccularly, intrathecally, intracereberally, intranasally, infusion, orally, rectally, via IV drip patch, implant and sublingual. Preferably, said route of administration is subcutaneously, intradermally, transdermally or intranasally.
Yet another aspect of the present invention relates to the compositions, as hereinbefore defined, when used in the method of the present invention.
The present invention is further described by reference to the following non-limiting examples.
Ara h 1 and Ara h 2 are the most allergenic and abundant proteins in peanut, making peptides comprising their dominant T-cell epitopes essential for inclusion in a therapy. 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 be suitable for treating a genetically diverse human population. The HLA-restriction of peptide presentation to T cells was tested using blocking antibodies and HLA-genotyping and showed that every T cell epitope identified could be presented on two or more different HLA-molecules. Furthermore, it was demonstrated that the identified T cell epitopes were collectively presented on a combination of HLA-DR, HLA-DQ and HLA-DP molecules (
Adjacent or overlapping T cell epitopes were combined into single peptides (<20 aa long) to minimise the number of peptides in the final therapeutic set resulting in three candidate peptides from Ara h 2 and seven from Ara h 1. Since cysteine residues can be problematic for peptide stability and biological reactivity, cysteine residues were substituted with structurally conserved but less reactive serine residues. Minor changes were also made to two Ara h 1 peptides to improve stability and/or solubility (
Preclinical screening of these peptides confirms PBMC T-cell reactivity (
Brief Overview of Steps
Materials and Methods
Subjects: Peanut-allergic adult subjects were recruited from The Alfred Allergy Clinic, Melbourne, Australia. Peanut-allergic subjects had clinical symptoms of IgE-mediated peanut allergy and peanut-specific IgE CAP score≥2 (≥1.16 kUA/1; Pharmacia CAP System™, Pharmacia Diagnostics, Uppsala, Sweden) and many had a history of anaphylaxis. Some subjects were genotyped (HLA-DRB1, -DQB1 and -DPB1, exon 2) by the Victorian Transplantation and Immunogenetics Service. The study was approved by The Alfred and Monash University Ethics Committees and informed written consent obtained from each subject.
Antigens: Crude peanut extract (CPE) was prepared from commercial unsalted, dry-roasted peanuts as described elsewhere, (de Leon et al. Clin Exp Allergy. 2003; 33(9):1273-80) dialyzed against phosphate-buffered saline (PBS) and filter-sterilized (0.2 μm). Natural Ara h 1 and Ara h 2 were enriched from CPE based on published methodology. (de Jong E C et al. Clin Exp Allergy. 1998; 28(6):743-51) Briefly, CPE was buffer exchanged into 20 mM TRIS-bis-propane (TBP), pH 7.2, using Vivaspin columns (Sartorius Stedim Biotech S.A., Aubagne, France) and applied onto 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 bound proteins (1 mL/min). Fractions, 0.5 mL, were analyzed by SDS-PAGE and those containing Ara h 1 or Ara h 2 with minimal other proteins pooled and dialyzed against PBS. Endotoxin contents were 1.7, 4.0 and 78.0 EU/mg for CPE, Ara h 1 and Ara h 2 respectively (Endpoint Chromogenic LAL assay, 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-mers and truncated peptide sets) or PBS, 1-2% acetic acid or 0.1M ammonium bicarbonate buffers as specified (custom-synthesized core epitope peptides). All antigens were confirmed to be neither mitogenic nor toxic as described (Eusebius N P et al., Int Arch Allergy Immunol. 2002; 127(3):234-44).
Generation of Ara h 1 and Ara h 2-specific CD4+ T-cell lines (TCL): Ara h 1 or Ara h 2-specific oligoclonal TCL were generated from peripheral blood mononuclear cells (PBMC) of peanut-allergic subjects using 5,6-carboxyfluorescein diacetate succinimidylester (CFSE)-based methodology. (Mannering S I et al., J Immunol Methods. 2005;298(1-2):83-92; Prickett S R, et al., J Allergy Clin Immunol. 2011; 127(3):608-15 el-5). Briefly, culturing was performed in RPMI-1640 containing 2 mM L-glutamine, 100 IU/mL penicillin-streptomycin and 5% human AB serum (Sigma-Aldrich, St Louis, USA) (cRPMI). PBMC were labelled with 0.1 μM CFSE (Molecular Probes, Eugene, USA) and cultured (2.5×106/mL) 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 pools (10 μg/mL/peptide) or as a control, tetanus toxoid (TT; 10 LfU/mL; Statens Serum Institute, Copenhagen, Denmark) for 7 days at 37° C. After staining with CD4-PE and 7AAD (BD Pharmingen, San Diego, USA), CD4+CFSEdim7AAD− cells were sorted (10 cells/well) into 96-U-well plates containing irradiated allogeneic feeder-cells, anti-CD3 (OKT-3), rIL-2 (Cetus, Emeryville, USA) and Fungizone (Invitrogen, Carlsbad, USA) as described. Cells were fed with rIL-2 as required and after 10-14 days, transferred to 48-well plates and tested for proliferation to Ara h 1 or Ara h 2 (10 μg/mL). Ara h 1 or Ara h 2-positive TCL were expanded with anti-CD3 and rIL-2 in T25 culture flasks (BD, Franklin Lakes, USA) for 10-12 days then tested for specificity (proliferation) to overlapping 20-mer peptides spanning the respective sequence (10 μg/mL). Core epitope sequences were mapped within selected 20-mers using peptide sets truncated from the N- or C-terminus of the 20-mer as described (Prickett S R, et alc. J Allergy Clin Immunol. 2011; 127(3):608-15 el-5). T-cell assays:All culturing was performed in RPMI-1640 containing 2 mM L-glutamine, 100 IU/mL penicillin-streptomycin and 5% heat-inactivated human AB serum (Sigma-Aldrich, St Louis, USA) (cRPMI). Antigen-induced TCL proliferation was assessed by 3H-thymidine (3H-TdR) uptake assays as follows: assays were performed on 72-hour duplicate or triplicate cultures in 96-U-well plates containing 1×104 T cells/well, 1×104 irradiated (5000 rads) autologous EBV-transformed PBMC (EBV-B cells) as antigen presenting cells and antigens as specified. Negative control was cRPMI alone. Cells were pulsed with 3H-thymidine (3H-TdR; 0.5 μCi/well) for the last 16 hours and uptake recorded as mean counts per minute (cpm) of replicate cultures. A stimulation index (SI; cpm antigen-stimulated T cells/cpm unstimulated T cells)≥2.5 was considered positive and all positive responses confirmed in ≥2 assays. To allow detection of peptide-induced CD4+ T-cell proliferation within whole PBMC, 7-day cultures of CFSE-labelled PBMC were set up as described for TCL generation, with addition of anti-CD25 antibodies (BD) to assess T cell activation in addition to proliferation. At least 10,000 CD4+ T cells were analyzed per sample and SI calculated as percentage of CD4+CFSElo (proliferated), CD4+CD25+(activated) or CD4+CD25+CFSElo (activated and proliferated) cells with antigen divided by the percentage of the same population without antigen (background). Analysing CD4+CD25+CFSElo (activated and proliferated) cells provided the most sensitive method for detection of T cell responses with designation of an SI≥1.5 as positive.
HLA class II blocking assays: T cells and irradiated EBV-B cells (1×104 of each) were incubated with 0.1-10 μg/mL blocking monoclonal antibody (mAb) against HLA-DR (L243, BD Pharmingen), HLA-DQ (SVP-L3) or HLA-DP (B7/21) or isotype-control antibodies (IgG2a: BD Pharmingen; IgG1: BioLegend, San Diego, USA) for 1 hour at 37° C. prior to addition of peptides (2-10 μg/mL) or CPE (100 μg/mL) and testing proliferative response as above. Cytokine ELISPOT assays: MAW ELISPOT plates (Millipore, Billerica, USA) were coated overnight at 4° C. with 10 μg/mL IL-4, IFN-γ or IL-5 antibodies (eBioscience, San Diego, USA) in PBS. Wells were blocked (cRPMI, 1 hour, 37° C.) then PBMC (3.5×105) or T cells and irradiated EBV-B cells (1×104 of each) added in duplicate 100 μL cultures with CPE (100 μg/mL), nAra h 2 (10 μg/mL) or peptides (10 μg/mL). Controls were cRPMI alone, TT (10 lfU/ml) and phytohaemagglutinin (1 μg/mL; Sigma-Aldrich). After 48 hours culture at 37° C., plates were incubated with biotinylated IL-4, IL-5 or IFN-γ antibodies (eBioscience) (1 μg/ml PBS, 2 hours) followed by ExtrAvidin®-alkaline phosphatase (Sigma-Aldrich) (1/3,000 PBS, 2 hours) before developing with alkaline phosphatase substrate (Bio-Rad). When spots appeared in positive-control wells, plates were washed, air dried and read (AID ELISPOT 4.0 h reader, Autoimmun Diagnostika, Strassberg, Germany).
Basophil activation test: Basophil activation was assessed by CD63 upregulation detected by flow cytometry as described (Drew A C, 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 over a 3 log concentration range (50, 5 and 0.5 μg/mL) and the peptide pool was tested over a 4-log concentration range (50, 5, 0.5 and 0.05 μg/mL). Histamine release was assessed using Histamine Release and the Histamine ELISA kits (IBL International GmbH, Hamburg, Germany) as per manufacturer's instructions.
Results
The factors considered in dominant 20-mer selection included:
145 Ara h 1-specific T cells lines (TCL) were generated from 18 peanut-allergic donors and 65/69 overlapping 20-mer peptides spanning Ara h 1 were recognised by these TCL (see
These selections were made based on number of specific TCL per subject, magnitude of TCL response, reproducibility of TCL response and ability to target specific T cells in PBMC. The 9 20-mers which were selected were:
T cell responses in 18/20 additional subjects with 8-16 responders (40-80%) per 20-mer. One or more of the nine 20-mers was recognised by T cells in 35 (92%) of 38 subjects analysed by TCL isolation and/or CFSE-screening (
The 9 dominant peptides ultimately selected are shown in Table 2.
PBMC Screening with Dominant Ara h 1 20-Mers
Ara h 2 Dominant 20-Mer Selection
69 Ara h 2-specific T cells lines (TCL) were generated from 16 peanut-allergic donors and 16/17 overlapping 20-mer peptides spanning Ara h 2 were recognised by these TCL (
One or more of the four 20-mers was recognised by T cells in 16/16 (100%) of subjects analysed by TCL isolation and/or CFSE-screening (
The dominant 20-mers are indicated in Table 3.
Core T-Cell Epitope Mapping
Technical Approach
TCL from different subjects were used to map each core T cell epitope. Exact T cell epitopes varied between TCL and subjects. The minimum T-cell stimulatory sequence (core epitope) within each selected 20-mer was determined by testing proliferation of reactive TCL from different subjects to truncated peptide sets (
(i) Core T Cell Epitopes Found in Dominant Ara h 1 20-Mers
Core T-cell epitope sequences were mapped within the dominant Ara h 1 20-mer peptides. 10 Ara h 1 T cell epitopes were identified (‘consolidated T cell epitope’), including 4 pairs of overlapping T cell epitopes. (
(ii) Core T Cell Epitopes Found in Dominant Ara h 2 20-Mers
Core T-cell epitope sequences were mapped within dominant Ara h 2 20-mer peptides. 5 Ara h 2 T cell epitopes were identified (‘consolidated T cell epitopes’), including 2 pairs of overlapping T cell epitopes (
HLA-Restriction of Ara h 1 and Ara h 2 T Cell Epitopes
T cell recognition of dominant T cell epitopes was blocked with monoclonal antibodies against HLA-DP, HLA-DQ or HLA-DR (
HLA-Restriction of Ara h 1 and Ara h 2 T Cell Epitope Presentation
HLA-typing was performed on subjects, with TCL recognising dominant T cell epitopes, in order to assess HLA-subtypes potentially able to T cell present epitopes. The absence of shared HLA alleles for subjects recognising a T cell epitope with confirmed HLA-DR/DQ/DP restriction indicated T cell epitope HLA-binding degeneracy. The Ara h 1 results are shown in
The absence of a shared HLA-DQB1 allele between all subjects from whom recognition of Ara h 2 T cell epitope (95-107) was blocked by anti-HLA-DQ indicated that this T cell epitope must be presented by multiple HLA-DQB1 molecules. Similarly, the diversity in HLA-DRB1 alleles between subjects for whom recognition of Ara h 2 T cell epitopes (127-141) or (37-47) was blocked by anti-HLA-DR indicated binding-degeneracy of both T-cell epitopes for multiple HLA-DRB1 molecules.
In addition to presentation by at least 2 HLA-DR molecules, Ara h 2 T cell epitope (37-47) was also presented by HLA-DQB1*06:09 as both subjects who recognised this T cell epitope in the context of HLA-DQ had this allele, and for subject 9 it was the only DQB1 allele present.
As DPB1*04:01 or DRB1*15:01 alleles were present in all subjects recognising Ara h 2 T cell epitopes (32-44) (blocked by anti-HLA-DP) or (95-107) (blocked by anti-HLA-DR) respectively, degeneracy of these T cell epitopes could not be determined. However, as DPB1*0401 and DRB1*1501 are prevalent in populations worldwide, T cell epitopes presented by these HLA-molecules would still be broadly recognized.
There were no shared alleles between two or more subjects recognising the dominant consolidated Ara h 1 T cell epitopes on a given HLA-type, thus demonstrating that each of the identified Ara h 1 T cell epitopes was also presented by 2 or more different HLA-molecules.
Predicting HLA-Binding Motifs: Ara h 1 20-Mer Peptides
Predicting HLA-Binding Motifs: Ara h 2 20-Mer Peptides
Table 5 provides a results summary for 2 HLA-DR prediction algorithms for binding motifs within 3 dominant Ara h 2 20-mers (NB dominant ‘20-mer 5’ not shown as predicted and actual epitopes fall in overlap with ‘20-mer 4’).
Refining Peptides for Therapeutic Delivery
Potentially problematic cysteine residues were replaced with structurally conserved, but less chemically reactive serine residues. Retained T cell reactivity was confirmed (
Combining Overlapping Ara h 2 T Cell Epitopes into Single Peptides≤20 Aa Long
Ara h 1 and Ara h 2 Candidate Peptides Summarised
There are 10 candidate peptides: 7 from Ara h 1 and 3 from Ara h 2 (Table 7)
Also designed are 13 additional shorter peptide variants (based on single T cell epitopes) for comparison. Some sequences have been lengthened or shortened (in line with native sequences and critical residues for T cell recognition) to improve peptide properties for production and solubility. This resulted in a panel of 23 candidate peptides for comparison. Peptide details are summarised in
All of the peptides in Table 7 were produced at 95-99.9% purity and solutions determined for solubility. T cell responses were then compared to each of these peptides at 2 doses in PBMC from 25 peanut-allergic subjects in order to select a final therapeutic combination.
NB Buffers: all peptides tried in PBS first; then 0.1M NH4HCO3 if sequence suggested preference for high pH, or 1% acetic acid for low pH; if not soluble in 1% acetic acid, increased to 2, 5, 10% etc.
Maximise population coverage and/or T cell reactivity whilst minimising sequence number and/or length.
Broad Considerations for Peptide Selection:
Data are grouped into a long and short version of each T cell epitope-containing region (see column borders; e.g. 1st ‘group’=peptides 1, 23 and 24 [Pep 1 combines overlapping peps 23 and 24 which each contain a separate T cell epitope]). The summary at the bottom of each group comments on the optimal peptide selected from that group. (Data show SI values for % CD25+CFSElow CD4+ T cells with peptide/unstimulated).
Data are sorted by descending value for 50 μg sample of long version for each peptide ‘group’ (or 10 μg sample where responses are better to this dose). Each row within in a peptide group shows data for a single subject, but the order of subjects varies in each peptide group.
Summary of Responses to 23-Peptide Panel in Cohort of 34
The data indicate that the selected 7 peptides are the best combination, but boxes indicate groups containing other viable peptides as substitutions (or additions) to the current pool:
For example:
1) Peptide 3 could replace peptide 15
2) Peptide 8 could replace peptide 21
3) Peptide 9 could replace peptide 23
Summary of Responses to Each Peptide of 7-Peptide Mix in Cohort of 39
Of the 74 subjects tested, all reacted to at least 1 peptide of the 7 selected peptides.
Responses to Different Peptide Pools
NB:
Basophil reactivity data was collected from 14 peanut-allergic subjects following incubation with peanut (CPE) or the 7-peptide pool (pool 7b) over a 3-4 log concentration range (m/m1)(
Prior to selection of pools 7a and 7b. Pools 7a and 7b were subsequently designed and tested.
PBMC T cell responses were compared to whole peanut and peptide pools 1-5 of
When comparing PBMC T cell responses to the 7-peptide pool (
In summary, pools 7a and 7b were both significantly better than the other 5 pools tested. There was no significant difference in pool 7a over pool 7b. Pool 7b was recognised by 100% of subjects tested and induced comparable or greater PBMC T cell responses than peanut in over 33% of subjects. Pools 1-5 were not recognised by 100% of subjects and very rarely induced responses equal to whole peanut.
Ara h 2 Peptide-Induced T Cell Anergy in an Ara h 2 Epitope Specific TCC (
T cells (1×106/ml) of an Ara h 2 peptide specific human T cell clone were cultured for 16 hours in the presence of Ara h 2 peptide (ANLRPSEQHLM (SEQ ID NO:30; hatched) at 100 μg/ml in the absence of accessory cells or in complete medium* alone (No Ag). The T cells were then washed thoroughly and rechallenged (104/well) with complete medium alone, IL-2 50 U/ml or an immunogenic concentration of the Ara h 2 peptide (10 μg/ml) in the presence of irradiated autologous PBMC (105/well) as accessory cells. Proliferation as correlated with tritiated thymidine incorporation was determined at 72 hours. Results are expressed as mean cpm+SD for triplicate cultures. *Complete medium: RPMI+5% AB serum+Pen/Strep/L-glutamine+10 U/mL of IL-2.
Ara h 1 Peptide-Induced Anergy in an Ara h 1 Epitope-Specific TCC (
T cells (1×106/ml) of an Ara h 1 peptide specific human T cell clone were cultured for 16 hours in the presence of Ara h I peptide (STRSSENNEGVIVKVSKE (SEQ ID NO:12; hatched) or an irrelevant Bahia grass Pas n 1 peptide (stippled) at 100 μg/ml in the absence of accessory cells or in complete medium* alone (No Ag). The T cells were then washed thoroughly and rechallenged (104/well) with complete medium alone, IL-2 50 U/ml or an immunogenic concentration of the Ara h I peptide (10 μg/ml) in the presence of irradiated autologous PBMC (105/well) as accessory cells. Proliferation as correlated with tritiated thymidine incorporation was determined at 72 hours. Results are expressed as mean cpm for triplicate cultures. *Complete medium: RPMI+5% AB serum+Pen/Strep/L-glutamine+10 U/mL of IL-2.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
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| Number | Date | Country | |
|---|---|---|---|
| 20160375130 A1 | Dec 2016 | US |
