Gluten and Celiac Disease Ingrid Swanson Pultz, Ph.D. Institute for Protein Design University of Washington

Gluten is a protein found in wheat, barley, and rye

Gluten “Facts” – True or False?

Gluten “Facts” – True or False? Celiac Disease Prevalence is Increasing because of… True West et al Am J Gastro 2014; 109:757-768

Gluten “Facts” – True or False? CD Prevalence is increasing because of… …increased awareness …increased incidence CD is Both 0.2% 1948-1954 0.8% 2009

Gluten “Facts” – True or False? CD is more prevalent because of genetically-modified wheat

Gluten “Facts” – True or False? CD is more prevalent because of genetically-modified wheat False No GM-wheat is commercially available

Gluten “Facts” – True or False? Gluten can cause symptoms in people without celiac disease

Gluten “Facts” – True or False? Gluten can cause symptoms in people without celiac disease Unclear Non-Celiac Gluten Sensitivity (NCGS)

Gluten “Facts” – True or False? Gluten-free food has no gluten

Gluten “Facts” – True or False? Gluten-free food has no gluten Not Necessarily True FDA Mandate of less than 20 PPM

Gluten “Facts” – True or False? Everyone would benefit from a gluten-free diet False

Gluten “Facts” – True or False? Look, not eating gluten just makes you healthier ok?! True AND False!

Celiac Disease Is a Debilitating Illness Anemia Osteoporosis Malnutrition Neuropathy Headache Neurological Infertility Non-Hodgkin’s Lymphoma Less Common Classical GI Dermatitis Dermatological Abdominal Pain and Diarrhea

Celiac Disease is Characterized by Gluten-Induced Intestinal Damage Sollid et. al. Nature Immunology 2002

The Gluten-Free Diet is Hard Strict: Less than 10-100 mg per day1,2. Difficult to achieve: 60-70% don’t get there3,4. Burdensome: Treatment burden similar to end-stage renal disease5. Expensive: Costs 2-3x more; lost productivity Life-Restricting: Unwillingness to leave home, inability to attend events, etc. 1Catassi et al. Am J Clin Nutr 2007; 85:160-6 2Akobeng & Thomas. Aliment Pharmacol Ther 2008; 27:1044-52 3Midhagen et al. Am J Gastro 2003; 98(9)2023-6 4Hall et al. Appetite 2013; 68:56-62 5Shah et al. Am J Gastro 2014; 109(9):1304-11

Celiac patients and their doctors have indicated that they would place great value in a therapeutic that could prevent symptoms and intestinal damage upon gluten ingestion

Gluten is a protein found in wheat, barley, and rye Gliadin Glutenin a-gliadin g-gliadin w-gliadin

Gliadin is Difficult for Human Digestive Enzymes to Break Down Stomach Intestinal Inflammation, Symptoms Toxic gluten fragment

Essential Characteristics of Oral Enzyme Therapeutics for Celiac Disease Activity in stomach conditions. Target all immunogenic gluten epitopes. Reduce gluten to below disease threshold (10-100 mg). ≥99% Straightforward high-yield production. Specificity for immunogenic gluten.

KumaMax is an Oral Enzyme Therapeutic That Specifically Breaks Down Toxic Gluten Fragments in the Stomach Gliadin KumaMax Stomach Healthy Intestines, Disease Prevented Non-toxic gluten fragment Kuma +

KumaMax Design Strategy: 1. Select Template Kumamolisin from Alicyclobacillus sendaiensis Engineered Enzyme Activity in Acidic Conditions Keep Easy Manufacturability Keep Specificity for PR Specificity for PQ

KumaMax Design Strategy: 2. Computational Redesign 1. Computational design to increase activity 2. Test designed enzymes 3. Combine promising mutations KumaMax Lead Original Kumamolisin

KumaMax Degrades All Immunodominant Epitopes from Wheat, Barley, and Rye 33mer Peptide from a-Gliadin (Wheat) PQQPIPQ||QPQPYPQ||Q QPFPQ||QPEQIIPQ||QP QPFPQPQ||QPFPWQP Hordein (Barley) Secalin (Rye) w-Gliadin (Wheat)

KumaMAX Outcomes Drives Down the Immunogenic Gluten Load in Bread 30 Min 10,000 ppm 84.8% 20 ppm 99.97%

Reduces Gluten Content in Beer to “Gluten Free” Levels KumaMAX Outcomes Reduces Gluten Content in Beer to “Gluten Free” Levels Gluten Concentration in a Wheat Beer at the Indicated Timepoints After Incubation with Gluten-Degrading Enzymes

KumaMAX Outcomes KumaMax Maintains Substantial Anti-Gluten Activity in the Presence of Significant Amounts of Other Dietary Protein Control meal (bun only) Hamburger and Vanilla Shake meal

T Cells from Celiacs Don’t Respond to KumaMax-Treated Gluten KumaMAX Outcomes T Cells from Celiacs Don’t Respond to KumaMax-Treated Gluten Gluten Y T Cell IFN-g IFN-g IFN-g

Summary Celiac disease is a debilitating illness NCGS: more research needed Certain regions of gliadin trigger celiac disease KumaMax is a potential treatment

Life Sciences Discovery Fund Washington Research Foundation Acknowledgements Clancey Wolf David Baker Justin Siegel Christine Tinberg Chia Heng Lee Lauren Carter Sydney Gordon Sarah Wolf Elizabeth Stanley Carmen Gianfrani Alessandra Camarca Life Sciences Discovery Fund Washington Research Foundation