Sub-tropical grass pollen allergy: Diagnostic & Immunotherapy

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1 Sub-tropical grass pollen allergy: Diagnostic & Immunotherapy Allergenic components of Bahia and Johnson Grass pollen have been isolated and sequenced Allergens can act as a diagnostic in blood tests or as the active ingredient in immunotherapies for desensitisation $300m opportunity for a subtropical grass sublingual immunotherapy identified in the US Summary of the Opportunity: A team of researchers led by Dr Janet Davies from the University of Queensland (UQ) have identified, isolated and sequenced the key allergenic components of two subtropical grasses, Paspalum notatum (Bahia grass) and Sorghum halepense (Johnson grass). These sequences have five principal commercial applications by generating either recombinant allergenic proteins or monoclonal antibodies that are specific to the allergenic components: 1. The development of a high throughput diagnostic test for Bahia and/or Johnson grass pollen allergy No standardized test for Bahia or Johnson grass allergy is currently approved for use A quantitative immunoassay utilising the major allergic component of Bahia grass (Pas n 1) has been shown to demonstrate outstanding specificity and sensitivity with an area under the ROC curve (AUC) of Adding Bahia and Johnson grass pollen allergen components to a diagnostic range extending beyond detecting serum IgE to Bermuda grass pollens would allow the development of a diagnostic capable of detecting allergy to the most clinically relevant subtropical grass strains. Current desensitisation therapies are suboptimal or ineffective for many pollen allergy sufferers because they are based upon allergens of temperate grasses. Manufacture of a therapy based on whole allergen extract is dependent on a reliable source of pollen. Recent shortages in supply of pollen raw material has resulted in a disruption of immunotherapy supply. An immunotherapy product based on the major allergenic component of Johnson and Bahia grass pollens could be manufactured from recombinant methods avoiding supply chain problems. We estimate the market in the US alone to be approximately $300m per year (see appendix 1) 3. A hypoallergenic mutant for use as an optimised immunotherapeutic for desensitisation. 4. A method of batch standardisation for the potency of sub-tropical allergenic components used in diagnostic tests and immunotherapeutics. 5. A method of environmental monitoring for subtropical grass allergens. 2. The development of an immunotherapy (desensitisation treatment) for the regulation of the allergic response. Currently, marketed immunotherapies for Bahia and Johnson grass pollen rely upon immunisation of allergic individuals using unstandardized whole pollen extract (usually by application of drops/tablet under the tongue or by successive subcutaneous injections). 1 1

2 The Technology and Product Opportunity The Technology Grass allergies are typically diagnosed using serum IgE tests and treated using immunotherapy based on whole pollen extract. The current range of marketed immunodiagnostic tests and immunotherapies are predominantly derived from allergens only produced by temperate grasses. A team of researchers led by Dr Janet Davies from the University of Queensland (UQ) have identified and sequenced the key allergenic pollen components of two sub-tropical grasses, Paspalum notatum (Bahia grass) and Sorghum halepense (Johnson grass). Bahia is endemic to southern regions of North America and is widely distributed in subtropical regions including parts of Australia whilst Johnson grass is found throughout the Mediterranean, Europe, the Middle East as well as parts of Australia, Africa and North America. These regions are currently inadequately served by existing grass allergy tests and immunotherapies based on temperate grass pollens as their allergenic components don t have the sufficient degree of homology with those found in sub-tropical grasses to consistently produce the desired immune response. Bahia grass is phylogenetically distinct from temperate climate grasses (see figure 1). Furthermore, Dr Davies research now demonstrates that allergenic molecules of subtropical grasses differ significantly in their allergenic composition, primary sequences of shared allergens and immunological reactivity. Dr Davies team has shown that sufferers of grass pollen allergy in regions with primary exposure to sub-tropical grasses, such as Bahia and Johnson, develop greater sensitisation to pollen of these local grasses than temperate grasses such as Ryegrass 1,2. The converse was demonstrated in allergy patients residing in temperate regions with primary exposure to Ryegrass. Importantly, only one of three dominant cellular immune determinants is shared between the major Bahia grass pollen allergen and the similar allergens of temperate grass pollens. This indicates that patients living in sub-tropical climates require immunotherapies developed specifically to their environment. The distinct features of sub-tropical grass pollen allergies suggested that their allergenic components should be identified and characterised. Dr Davies and her team found that a recombinant Pas n 1 protein had 85% of the reactivity of a Bahia whole extract 3. The team at UQ also examined the proteome, transcriptome and allergome for Sorghum halepense (Johnson grass) and identified, with a high degree of confidence, that amongst the five allergenic components identified, that Sor h 1 and Sor h 13 are primarily responsible for allergic reactivity 4. Figure 1: Clinically-relevant Poaceae grass phylogenetic tree This has led to the generation of intellectual property that allows the development of a more comprehensive, high throughput diagnostic for sub-tropical grasses and the ability to develop a differentiated recombinant allergen component based immunotherapy for Johnson and Bahia grass allergies. Product Opportunity Almost 70% of allergic rhinitis sufferers indicate that their condition impedes their lifestyle 5. This observation parallels a significant increase in prevalence of allergic disease, including hay fever (seasonal allergic rhinitis) and asthma in recent decades. An increase in grass pollen in the environment is one of the principal contributors to this rise and it is believed to have been triggered by: Global warming resulting in an extended pollen season in many regions 6 ; and Growth of glysophate resistance particularly evident in Johnson grass 7. Johnson grass is regarded as one of the top 10 weed pests in the world 8. In this setting, strong growth in demand for solutions is anticipated from allergy sufferers and healthcare providers. 2 2

3 The Product Opportunity (contd) 1 - Diagnostic Current grass allergy diagnostics are primarily based on whole pollen allergen extract and implemented via skin prick test or serum allergen-specific IgE measurement. It is accepted that use of a recombinant in diagnostic tests can achieve higher specificity as they avoid cross-reactive carbohydrate determinants (CCD s) which yield clinically irrelevant IgE binding in some subjects 9. Hence, elimination of post-translational glycosylation of diagnostic markers such as Pas n 1, Sor h 1 and Sor h 13 would avoid false positive responses from patients allergic to other environmental agents (e.g., dust mite, cat dander or other plant allergens). CCD s on natural allergens are known to produce as much as 20% more false positives in IgE tests Recombinant Immunotherapy Currently, there are only two marketed registered products for the treatment of grass pollen allergies available globally: Oralair (Stallergenes, France): A sublingual tablet containing a standardised extract of 5 temperate grasses including sweet vernal grass, orchard grass, perennial rye grass, timothy grass, and Kentucky bluegrass indicated for the treatment of allergic rhinitis with or without conjunctivitis. Territories: US, Australia, Canada, EU & Russia. Grazax / Grazak (Alk-Abello, Denmark & Merck & Co, USA.): A sublingual tablet containing a standardised extract of Timothy grass pollen (Phleum Pratense), indicated for allergic rhinitis and conjunctivitis. Territories: Canada, France, Germany, Italy, Spain, UK, US. house-dust mite allergy resulting in allergic rhinitis, rhinoconjunctivitis and allergic asthma. Merck is screening patients in preparation for a PIII study. All of these products are targeted at patients who are allergic to temperate grasses. 3 - Hypoallergenic Immunotherapy To date, no desensitisation therapies have been developed based upon a hypoallergenic mutant. The technologies to identify both the T-cell and IgE reactive epitopes have been available for some time however the opportunity to develop a specific therapy for subtropical grass pollens with fewer side effects than native IgE binding allergens has not been seized. 4 - Method of Batch Standardisation Manufacturing of diagnostic tests and immunotherapies based on recombinant allergens will require a method to standardise the allergenic potency of each batch of protein. Know-how exists on how this can be achieved by measuring the concentration of group 1 and group 13 allergens from key sub-tropical grasses. 5 - Environmental Monitoring A panel of antibodies specific for the major allergens of Ryegrass 10 or group 1 allergens of subtropical grass pollens 1 can be used to develop an immunoassay for measuring allergen content within environmental volumetric samples of airborne pollen. This would provide a method for measuring the allergenic potency of pollen and biogeographical distribution of airborne pollen of subtropical grasses compared to temperate grasses. Serial subcutaneous injections remain common practice, although adoption of Oralair and Grazax for treatment of temperate grass pollen allergy is steadily increasing. As confidence in sublingual tablets increases, the convenience they offer patients increasingly makes them the administration route of choice. Following this trend, Stallergenes are also developing Actair, a sub-lingual tablet for dust mite allergy. Furthermore, Merck & Co and Alk-Abello are collaborating to develop, Mitivax (MK-8237) for the treatment of 3 3

4 Current Status & Development Requirements 1 - Diagnostic Dr Davies and her team have demonstrated that Pas n 1 is the principal allergen in 90% of cases 11. Furthermore Pas n 1 functions as an exceptional diagnostic marker for Bahia Grass allergy. IgE response to purified protein was clinically validated with an area under the ROC curve of 0.96 at a cut off of 0.23 ku/l. Sensitivity (%) Specificity (%) ROC: AUC Table 1. Diagnostic performance of Pas n 1 as a marker for Bahia grass allergy These results indicate that an even higher performing test would be possible if: Direct coupling of the purified Pas n 1 to the ImmunoCAP matrix avoiding the streptavidin link will improve coating capacity and resultant assay sensitivity. A recombinant is used to achieve higher specificity to avoid cross-reactive carbohydrate determinants (CCD s) that can produce false positives. Funding for characterising the diagnostic performance of the three key Johnson grass allergens is currently being sought. Figure 2: 2 - Recombinant Immunotherapy The clinical utility of a desensitisation treatment will rely upon tailoring the specificity of the immunotherapeutic to the desired allergic responses. To this end, the Davies lab has investigated the cross reactivity of sub-tropical grass allergens in a number of studies and has found that only limited cross-reactivity between subtropical grasses (Bahia, Bermuda and Johnson) was observed with temperate grasses (e.g., Timothy and Rye grasses) 12. However, the same study also found that there is a great deal of cross-reactivity between Johnson and Bahia grass Allergens. This suggests that a preferred panel of specific proteins from sub-tropical grasses can be inferred and included in either a broad spectrum or species-specific sublingual immunotherapy tablet. Johnson 1.0 Bahia Bermuda Ryegrass Johnson Bahia Bermuda Ryegrass ( ) ( ) ( ) ( ) ( ) 3 - Hypoallergenic mutant Immunotherapy The three T cell epitopes of highest frequency within the Bahia grass allergen Pas n 1 have been identified by Dr Davies and her team 13. That two of the dominant T cell epitopes identified were unique to Pas n 1, and non-cross reactive with T cells specific to Ryegrass pollen allergen (Lol p 1), further exemplifies the need for specific immunotherapy targeting subtropical grasses. With further work the IgE reactive epitopes of Pas n 1 could be identified and a hypoallergenic mutant engineered with reduced IgE-mediated immune response whilst maintaining T-cell reactivity to achieve desensitisation. 4 - Method of batch standardisation Antibodies specific (Figure 3) could has be utilized in an immunoassay (ie. capture ELISA format) to standardize allergen content of extracts for use in conventional delivery of allergen immunotherapy treatment based on whole pollen extracts. A number of candidate antibodies have ( ) Spearman s r with 95% confidence intervals shown in brackets. All correlations were significant (p < ) based on Gaussian approximations. Table 2. Correlation between plasma IgE reactivity with grass pollens in subjects with grass pollen allergy in Brisbane

5 already been developed. Figure 3 demonstrates the specificity of one proprietary antibody to subtropical grasses. Investigation of a suitable capture platform is continuing whilst funding options are being explored. Davies, J. M. et al. (2011), The dominant 55kDa allergen of the subtropical Bahia grass (Paspalum notatum) pollen is a group 13 pollen allergen, Pas n 13, Molecular Immunology, : Drew, A et al. (2011), Purification of the major group 1 allergen from Bahia grass pollen, Pas n 1, International Archives of Allergy and Immunology, 154 4: Davies, J. M. (2014) Grass pollen allergens globally: the contribution of subtropical grasses to 1 burden of allergic respiratory diseases. Clinical and Experimental Allergy, 44: Figure 3: Proprietary mab 6/C6 is specific to sub-tropical grass pollen allergens 5 - Environmental Monitoring Dr Davies team are interested in developing a prototype for airborne allergen detection assay system for subtropical grass pollens. A similar assay developed for the purpose of allergen standardization would have additional utility in airborne allergen detection. Intellectual Property The foundation Intellectual Property is protected through two published patent families: Applications in the US (14/ ) and Australia (AU ) entitled Immunogenic Protein Pas n1 from Bahia grass pollen, both with an earliest priority date of 22/10/2007. Kliene-Tebbe J, Davies J Grass Pollen Allergens Chapter 3g in in the Global Atlas of Allergy. Published by the European Academy of Allergy and Clinical Immunology, 2014, p What are we looking for? We are seeking a commercial partner to co-develop a sub -tropical portfolio that could include some or all of the following products: Diagnostic: Bahia or Johnson grass pollen test Immunotherapy: Ideally, a combined Bahia and Johnson grass desensitisation therapy Hypoallergenic mutant for an enhanced Immunotherapy: Bahia and Johnson grass pollen allergens Method of Batch Standardisation: Group 1 subtropical grass allergens Environmental Monitor: Subtropical grass pollens A PCT patent application (PCT/AU2014/ ) entitled Johnson grass allergenic pollen proteins, encoding nucleic acids and method of use, filed 18/6/2014. An application in the US (62/ ) entitled Detection and measurement of immunogenic molecules, filed 20/6/2014 In addition to the papers from the Davies lab referenced above, the following publications grant further insight into the know-how retained by them on sub-tropical grass allergy 5 5

6 References 1. Davies, JM et al. (2011) Functional immunoglobulin E cross-reactivity between Pas n 1 of Bahia grass pollen and other group 1 grass pollen allergens. Clinical and Experimental Allergy, 41 2: Davies JM, Bright, M. L., Rolland, J. M. and O'Hehir, R. E. (2005) Bahia grass pollen specific IgE is common in seasonal rhinitis patients but has limited cross-reactivity with Ryegrass. Allergy, 60 2: Davies JM et al. (2008) Molecular cloning, expression and immunological characterisation of Pas n 1, the major allergen of Bahia grass Paspalum notatum pollen. Molecular Immunology, 46 2: Campbell B et al. (2014) Total transcriptome, proteome, and allergome of Johnson grass pollen, which is important for allergic rhinitis in subtropical regions. J Allergy Clin Immunol in press; doi: /j.jaci Valovirta E, Myrseth SE, Palkonen S. (2008) The voice of the patients: allergic rhinitis is not a trivial disease. Curr Opin Allergy Clin Immunol 8: Last accessed 10 March Last accessed 10 March Holm LG, Donald P, Pancho JV, Herberger JP (1977) The World's Worst Weeds: Distribution and Biology. The University Press of Hawaii, Honolulu, Hawaii. 609 pp 9. Altmann, F. (2007) The role of protein glycosylation in allergy. International Archives of Allergy and Immunology; 142: Smart I, Heddle RJ, Zola H, Bradley JE. (1983) Development of monoclonal mouse antibodies specific for allergenic components of Ryegrass (Lolium perenne) pollen, Int Arch Allergy Appl Immunol 72: Timbrell V et al., (2014) An immunodiagnostic assay for quantitation of specific IgE to the major pollen allergen component, Pas n 1, of the subtropical Bahia grass. Congress of the European Academy of Allergy and Clinical Immunology, Copenhagen, Abstract in Allergy 69 (Suppl. 99), Davies J.M., et al. (2012), Subtropical grass pollen allergens are important for allergic respiratory diseases in subtropical regions, Clinical and Translational Allergy, 2 4: Etto, T, et al, (2012), Unique and Cross-Reactive T Cell Epitope Peptides of the Major Bahia Grass Pollen Allergen, Pas n 1, International Archives of Allergy and Immunology, 159 4:

7 Appendix 1: A subtropical grass pollen SLIT presents a $300m p.a. opportunity in the US alone Our estimate of the commercial opportunity for a subtropical sublingual immunotherapy (SLIT) in the US has assumed that the target market will be severe hayfever cases in the 16 US states where Bahia Grass is most commonly found. With the launch of Oralair and Grastek in the US earlier this year we expect that the market uptake (prescriptions to patients) for SLITs will, by 2020, reach at least two thirds of the rate currently found in Europe. This suggests an opportunity of approximately $300m exists for a subtropical grass pollen therapy. States Variable with Bahia grass 16 Worst Case 16Likely Case 16Best Case 16 Likely Case Projected 2020 Total Popula on in 161m 161m 161m 172m 16 states 1 Hayfever sufferers 9.5m (11% AR 2 ) Severe cases (30%) 4 benefi ng from SLIT 12.9m (15% AR 3 ) 12.9m (15% AR 3 ) 13.8m (15% AR 3 ) 2.8m 3.9m 3.9m 4.1m Market uptake of 78,000 SLIT 5 (current US rate 2.75%) 464,400 (12%) 700,000 (European rate 18%) 496,800 (12%) Market es mate p.a. $40m (US$500 ea) $233m (US$500 ea) $350m (US$500 ea) 16 states with Paspalum notatum exposure ( $297m ($597ea, 3%CPI) 7 7

8 Appendix 2: An increasing burden of subtropical grass pollen allergy in USA Key point Evidence Inference References The burden of seasonal allergic rhinitis is high. Increasing population in southern states Subtropical grass pollens are extending range Patients in subtropical regions are primarily sensitised to subtropical grass pollens Subtropical grass pollens are immunologically distinct from temperate grass pollens Population based studies in USA show a high prevalence ( %) of allergic sensitization (SPT and IgE) to grass pollens (temperate Ryegrass and subtropical Bermuda grass). This prevalence of medically diagnosed allergic rhinitis in 11% of children is underestimated based on sensitization rates and frequency of seasonal symptoms. The population in southern states such as Florida, Texas, Louisiana and Mississippi, increased by 18.3% to 52.3 million between 2000 and Global warming increases in temperature and CO 2 favor the growth of subtropical grasses. Exposure to subtropical grass pollens as secondary seasonal peak in temperate regions. Highest sensitisation to Bahia grass pollen in children with allergic rhinitis of military personnel in Texas. Highest sensitisation to Bahia grass pollen, Louisana. Lack of in vivo cross-reactivity between Bahia and Timothy grass pollen by nasal challenge in grass pollen-allergic patients, Florida. Distinct asymmetric patterns of IgE cross-reactivity between subtropical and temperate grass pollens using pooled patient sera of unknown origin. Two of three immunodominant T cell epitopes of major allergens of Bahia (Pas n 1) and Bermuda (Cyn d 1) grass pollens are not represented in temperate grasses (eg Phl p 1 or Lol p 1). Grass pollens are an important trigger for symptoms of allergic rhinitis in USA. Increasing allergic sensitisation to subtropical grass pollens. Increasing exposure of patients to subtropical grass pollens. Primary sensitivity with subtropical grass pollens in subtropical regions. Subtropical grass pollen allergens should be specifically targeted in desensitisation therapy. [1-3] [4] US Census Bureau [5, 6] [7, 8] [9] [10] [11] [12, 13] [14, 15] (Dr Davies, School of Medicine, UQ, unpublished) References 1. Meltzer, E.O., et al., Burden of allergic rhinitis: results from the Pediatric Allergies in America survey. J Allergy Clin Immunol, (3 Suppl): p. S Arbes, S.J., Jr., et al., Prevalences of positive skin test responses to 10 common allergens in the US population: results from the third National Health and Nutrition Examination Survey. J Allergy Clin Immunol, (2): p Salo, P.M., et al., Allergy-related outcomes in relation to serum IgE: results from the National Health and Nutrition Examination Survey J Allergy Clin Immunol, (5): p e7. 4. Gupta, A., Geoindicators for tropical urbanization. Environmental Geology, : p Morgan, J.A., et al., C4 grasses prosper as carbon dioxide eliminates desiccation in warmed semi-arid grassland. Nature, (7359): p Seidel, D.J., et al., Widening of the tropical belt in a changing climate. Nature Geoscience, : p Kosisky, S.E., M.S. Marks, and M.R. Nelson, Pollen aeroallergens in the Washington, DC, metropolitan area: a 10-year volumetric survey ( ). Ann Allergy Asthma Immunol, (3): p Frenguelli, G., et al., Bridging allergologic and botanical knowledge in seasonal allergy: a role for phenology. Ann Allergy Asthma Immunol, (3): p Calabria, C.W. and J. Dice, Aeroallergen sensitization rates in military children with rhinitis symptoms. Ann Allergy Asthma Immunol, (2): p Hensel, A.E., Jr. and R.C. Griffith, Clinical experiences with Paspalum notatum (Bahia grass): a new grass antigen. South Med J, (6): p Phillips, J.W., et al., Bahia grass pollen, a significant aeroallergen: evidence for the lack of clinical cross-reactivity with timothy grass pollen. Ann Allergy, (6 Pt 1): p Leiferman, K.M. and G.J. Gleich, The cross-reactivity of IgE antibodies with pollen allergens. I. Analyses of various species of grass pollens. J Allergy Clin Immunol, (1 PT. 2): p Martin, B.G., L.E. Mansfield, and H.S. Nelson, Cross-allergenicity among the grasses. Ann Allergy, (2): p Etto, T., et al., Unique and cross-reactive T cell epitope peptides of the major Bahia grass pollen allergen, Pas n 1. International archives of allergy and immunology, (4): p Eusebius, N.P., et al., Oligoclonal analysis of the atopic T cell response to the group 1 allergen of Cynodon dactylon (bermuda grass) pollen: pre- and post-allergen -specific immunotherapy. Int Arch Allergy Immunol, (3): p

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