Lolium perenne peptides for treatment of grass pollen allergy: A randomized, double-blind, placebo-controlled clinical trial
Authors: Mohamed H. Shamji, Jan Ceuppens, Claus Bachert, Peter Hellings, Gaël Placier, Gaëtan Thirion, Nicolas Bovy, Stephen R. Durham, Jean Duchateau, Thierry Legon, Sabine Pirotton
Journal: Journal of Allergy and Clinical Immunology (2017)

Allergen immunotherapy (AIT), first used more than a century ago,1Noon L. Prophylactic inoculation against hayfever.Lancet. 1911; 1: 1572-1573Abstract Scopus (1131) Google Scholar is an effective treatment in patients with seasonal allergic rhinitis. Allergen extract can be administered as subcutaneous or sublingual immunotherapy. It is important to note that allergen extract preparations used for AIT vary between manufacturers' batches and might contain cross-reacting allergens, which can increase the risk of systemic reactions.2Fujimura T. Okamoto Y. Antigen-specific immunotherapy against allergic rhinitis: the state of the art.Allergol Int. 2010; 59: 21-31Abstract Full Text PDF PubMed Scopus (19) Google Scholar Because antigen allergenicity is associated with resistance to proteolysis,3Untersmayr E. Jensen-Jarolim E. The role of protein digestibility and antacids on food allergy outcomes.J Allergy Clin Immunol. 2008; 121: 1301-1310Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar several innovative strategies are being instigated currently, such as replacing whole allergen with allergen-derived peptides. As an advantage over whole-protein allergens, peptides do not bind to IgE and cross-link FcεRI on the surfaces of mast cells and basophils, and therefore they do not release mediators, such as tryptase and histamine. T-cell epitope peptide AIT is being developed for the treatment of several allergies, including cat,4Patel D. Couroux P. Hickey P. Salapatek A.M. Laidler P. Larche M. et al.Fel d 1-derived peptide antigen desensitization shows a persistent treatment effect 1 year after the start of dosing: a randomized, placebo-controlled study.J Allergy Clin Immunol. 2013; 131 (e1-7): 103-109Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar peanut,5Prickett S.R. Voskamp A.L. Phan T. Dacumos-Hill A. Mannering S.I. Rolland J.M. et al.Ara h 1 CD4+ T cell epitope-based peptides: candidates for a peanut allergy therapeutic.Clin Exp Allergy. 2013; 43: 684-697Crossref PubMed Scopus (55) Google Scholar and birch6Spertini F. Perrin Y. Audran R. Pellaton C. Boudousquie C. Barbier N. et al.Safety and immunogenicity of immunotherapy with Bet v 1-derived contiguous overlapping peptides.J Allergy Clin Immunol. 2014; 134 (e13): 239-240Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar allergy. This method uses a mixture of a limited number of synthetic peptides known to contain the immunodominant T-cell epitope of the major allergen component. Another approach involves the use of proteolytic hydrolysates of allergens, which has the advantage over synthetic peptides of providing more epitopes and therefore can be used in a wider population. We have developed a formulation of purified peptidic fragments of rye grass (Lolium perenne peptides [LPPs]) suitable for subcutaneous AIT administration. Because of the extensive cross-reactivity of allergenic components of grass pollen from different species, the L perenne allergen can be used to treat allergic rhinitis induced by other grasses.7Laffer S. Valenta R. Vrtala S. Susani M. van Ree R. Kraft D. et al.Complementary DNA cloning of the major allergen Phl p I from timothy grass (Phleum pratense); recombinant Phl p I inhibits IgE binding to group I allergens from eight different grass species.J Allergy Clin Immunol. 1994; 94: 689-698Abstract Full Text PDF PubMed Scopus (109) Google Scholar The purpose of this study was to assess the safety and immunologic effect of LPPs in adults with grass pollen–induced seasonal allergic rhinitis. In addition, this study examined the adjuvant effect of a bacterial homolog of the heat shock protein 70 family, DnaK.8van Eden W. van der Zee R. Prakken B. Heat-shock proteins induce T-cell regulation of chronic inflammation.Nat Rev Immunol. 2005; 5: 318-330Crossref PubMed Scopus (431) Google Scholar DnaK has been shown to suppress inflammation in animal models of arthritis through induction of IL-10–producing cells and has been tested in clinical trials.8van Eden W. van der Zee R. Prakken B. Heat-shock proteins induce T-cell regulation of chronic inflammation.Nat Rev Immunol. 2005; 5: 318-330Crossref PubMed Scopus (431) Google Scholar LPPs (ranging between 1 and 10 kDa) were generated by means of enzymatic hydrolysis of purified proteins from L perenne (see the Methods section in this article's Online Repository at www.jacionline.org for further details). Recombinant DnaK was produced in Escherichia coli and purified as an aqueous solution.9Castanie M.P. Berges H. Oreglia J. Prere M.F. Fayet O. A set of pBR322-compatible plasmids allowing the testing of chaperone-assisted folding of proteins overexpressed in Escherichia coli.Anal Biochem. 1997; 254: 150-152Crossref PubMed Scopus (53) Google Scholar LPPs and DnaK were reproducibly manufactured in gram quantities according to Good Manufacturing Practice guidelines. The study was approved by local ethics committees (Leuven University Hospital), and all subjects provided written informed consent. The binding capacity of LPPs to IgE was assessed by means of immunoblot analysis with IgE-rich serum (L perenne–specific IgE >100 kU/L) obtained from patients with grass pollen allergy (see the Methods section in this article's Online Repository for further details). Purified pollen proteins contained major (Lol p 1 and p 5) and minor (Lol p 11, Lol p 2, and Lol p 3) allergens from L perenne (Fig 1, A, lane 2). These proteins were not detectable in the corresponding peptide preparations, and no IgE binding was detectable (Fig 1, A, lane 1). Purified L perenne proteins inhibited serum IgE binding to proteins with a concentration at which the inhibitor response is reduced by half [IC50] of 0.1 μg/mL (Fig 1, B). In contrast, the IC50 for the corresponding peptides was 100-fold higher (10 μg/mL). Reproducibility of LPP batch-to-batch variability was also assessed by using ELISA (Fig 1, C). Allergenicity of LPPs was investigated by using blood samples from 12 allergic and 12 nonallergic subjects (see Table E1 in this article's Online Repository at www.jacionline.org). Dose-dependent expression of CD63, a marker of basophil activation and degranulation, was observed in allergic patients (Fig 1, D) but not in nonallergic subjects (Fig 1, E and see Table E2 in this article's Online Repository at www.jacionline.org) when basophils were stimulated with L perenne proteins. This stimulation was significantly greater than that induced by the same concentration of LPP. A 100-fold higher LPP concentration was required to elicit basophil degranulation. DnaK alone had no effect on basophil activation and degranulation. Adding DnaK to the peptides (1:1 wt/wt) reduced their ability to induce degranulation (P < .01 at 1000 ng/mL compared with LPPs; Fig 1, D). Moreover, LPP immunogenicity was demonstrated when PBMCs obtained from patients with grass pollen allergy and nonallergic subjects were stimulated with L perenne proteins, LPPs, DnaK, and LPPs/Dnak. LPPs induced dose-dependent proliferation at levels less than that seen in whole proteins and restricted to allergic subjects (see Fig E1, A, and Table E3 in this article's Online Repository at www.jacionline.org). L perenne proteins and LPPs both stimulated, to a similar extent, the production of IL-5 in PBMCs from allergic subjects (see Fig E1, B and C, and see Table E3). We tested the safety of LPPs and LPP/DnaK in a randomized, double-blind, placebo-controlled, parallel-group trial of AIT (www.Clinicaltrials.gov: NCT01111279). Study participants (n = 27) were sequentially randomized 1:1:1 to receive 5 subcutaneous injections (weekly) of placebo, increasing doses of LPPs, or LPP/DnaK (see Figs E2 and E3 and Table E4 in this article's Online Repository at www.jacionline.org). The primary end point was to assess the safety of LPPs and LPP/DnaK. In the placebo group all local reactions were mild (≤5 cm). No clinically relevant differences were observed in local reactions between the LPP and LPP/DnaK groups, and reaction size did not increase with a successive increase of LPP or LPP/DnaK dose (see Fig E4 and Table E5 in this article's Online Repository at www.jacionline.org). During treatment, 2 unsolicited adverse events (AEs) were reported in the placebo group as related to treatment, 16 AEs were reported in the LPP group, and 12 AEs were reported in the LPP/DnaK group. The frequency of AEs of special interest (especially headache) was greater in the LPP/DnaK group. All AEs were mild or moderate, and those of special interest were mild and resolved without corrective therapy. No differences in hematologic and biochemistry parameters were observed between groups (see Table E6 in this article's Online Repository at www.jacionline.org). The first patient who received LPPs had anaphylaxis at the first injection (50 μg). After this event, the treatment schedule was reviewed, and the initiation dose was reduced to 5 μg. The new revised treatment schedule was tolerated by all patients (see the Methods section in this article's Online Repository for further details). This study was not powered to demonstrate the clinical efficacy of LPPs, LPP/DnaK, or both. No difference was observed between the treated and placebo groups when considering the full analysis set. However, when considering patients receiving at least a cumulative dose of 105 μg of LPPs (78% of the cumulative dose per protocol), a reduction of 35% was observed for average combined scores (ACS), 30% for rhinoconjunctivitis total symptom scores, 43% for rescue medication scores, and 32% for total symptom scores, and the mean number of well days was greater in patients treated with LPPs compared with placebo over the grass pollen season (see Table E7 in this article's Online Repository at www.jacionline.org). In contrast, ACSs, rhinoconjunctivitis total symptom scores, rescue medication scores, and total symptom scores were higher, and the mean number of well days was lower in the LPP/DnaK group than in the placebo group (see Table E7). The higher frequency of AEs in the LPP/DnaK group and the apparent lack of efficacy suggested that the proportion of LPPs and DnaK might be not optimal. A post hoc analysis of the relation between cumulative LPP dose and clinical score showed that the LPP dose was positively correlated with the number of well days (Fig 1, I and J) and negatively correlated with the ACS (data not shown). An inverse relationship was observed in the LPP/DnaK group. These results suggest that DnaK might have an adjuvant effect at very low cumulative doses (45 μg). Further investigations are required to confirm this hypothesis. The secondary endpoint was to assess the effect of LPPs and LPP/DnaK on IgE, IgG4, and blocking antibody responses. Grass pollen–specific IgE levels were increased in the LPP- and LPP/DnaK-treated groups at weeks 5, 14, and 25 compared with baseline (Fig 1, F). A natural course of specific IgE induction was observed in the placebo-treated group at week 14 (pollen season) and persisted until week 25. Grass pollen–specific IgG4 levels were increased in the LPP and LPP/DnaK groups after treatment and remained increased until week 25 (all P < .01). No significant change in the placebo groups was observed (Fig 1, G). IgG-associated blocking antibodies, as measured by using the IgE-FAB assay (see the Methods section in this article's Online Repository for further details) were induced in both treated groups. Allergen-IgE binding to B cells was reduced after treatment with LPPs and LPP/DnaK and reached a −30.1% decrease compared with baseline (P < .01) in the LPP group and −21.3% (not significant) in the LPP/DnaK group at the end of the study (Fig 1, I). In summary, for the first time, we report the characterization of LPPs tested as candidates for AIT. The administration of only 5 subcutaneous injections of increasing doses of LPP was safe and well tolerated. The treatment was associated with the induction of immune-reactive and IgG-associated blocking antibodies that persisted for at least 24 weeks. Consequently, LPPs alone might be a potential candidate for AIT and are being further validated in AIT trials (NCT01111279). Download .docx (.03 MB) Help with docx files Online Repository text Download .docx (.05 MB) Help with docx files Tables E1-E7Fig E2View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4View Large Image Figure ViewerDownload Hi-res image Download (PPT)