Outline. o Immunogenicity of therapeutic protein aggregates. o Clinical evidence. o Models to study the immunogenicity of aggregates

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1 Immunogenicity of Therapeutic Protein Aggregates Ehab Moussa

2 Outline o Immunogenicity of therapeutic protein aggregates o Clinical evidence o Models to study the immunogenicity of aggregates o Mechanisms of the immunogenicity of aggregates o Results with aggregated IGIV o Gaps in our understanding 1

3 Immune response Components Response Innate 1. Skin and mucous membranes 2. Phagocytes 3. Dendritic cells 4. Natural killer cells 5. Complement Immediate, limited potency Adaptive 1. Humoral response (Bcells) 2. Cellular response (Tcells) Delayed, potent Specificity Non-specific Highly-specific Memory Same on 2 nd exposure Fast and more potent on 2 nd exposure 2

4 Hypotheses of the antigenicity of protein aggregates 3 Felipe et al. Aggregation of Therapeutic Proteins

5 Clinical evidence Human gamma globulin Human growth hormone Recombinant human erythropoietin Interferons 4 Moussa et al. J Pharm Sci

6 Utility of Clinical Information Product- and host-specific factors can influence immunogenicity Healthy vs. immune-suppressed populations The immune state and medication regimen of the population tested Receipt of multiple doses from different therapeutic lots Post-marketing immunogenicity data are limited and are not typically tracked or reported by physicians The lack of standardization of assays for detecting ADAs 5 Moussa et al. J Pharm Sci

7 In vivo models Wild-type mouse models o Human proteins?! o Murine proteins Transgenic mouse models 6

8 In vitro models PBMC o Innate inflammatory response o DC activation o T-cell proliferation o Peptide mapping Cell lines o Innate inflammatory response o DC activation 3D models 7

9 Aggregate interactions with APCs 8 Moussa et al. J Pharm Sci

10 T-cell dependent mechanism 9 Moussa et al. J Pharm Sci

11 T-cell independent mechanism 10 Moussa et al. J Pharm Sci

12 Effect of aggregate structure: IGIV 11 Moussa et al. Pharm Res

13 Effect of aggregate structure: IGIV 12 Moussa et al. Pharm Res

14 Effect of aggregate structure: monocytes 13 Moussa et al. Pharm Res

15 Effect of aggregate structure: THP-1 cells 14 Moussa et al. Pharm Res

16 Effect of aggregate structure: MM6 cells 15 Moussa et al. Pharm Res

17 Interactions with innate receptors Monocytes THP-1 16 Moussa et al. Pharm Res

18 Interactions with innate receptors 17 Moussa et al. Pharm Res

19 Interactions with innate receptors 18 Moussa et al. Pharm Res

20 Aggregate structure? 19 Zhang and Topp. Molecular Pharmaceutics

21 Points to consider Aggregate attributes may work synergistically to enhance immunogenicity The potential immunogenicity of an aggregate may be exacerbated by other intrinsic and extrinsic factors and/or external stresses The potential impact of any particular attribute of an aggregate may vary with the disease state and co-medication of the patient population Aggregate attributes, even if well-characterized in vitro and/or in the drug product, may change once the drug is administered. 20 Moussa et al. J Pharm Sci

22 Gaps in our understanding The relative importance of receptor-mediated pathways and endocytic pathways in stimulating immune cells to respond to protein aggregates The potential immune response to aggregate amounts and attributes similar to those in marketed therapeutics is unknown Do aggregates bind other proteins or lipids in vivo to alter their biological function? What analytical methods should be used to monitor aggregate levels in protein therapeutics? 21 Moussa et al. J Pharm Sci

23 Recommendations for future research 1. Mining the data of the different lots and of immune responses in clinical trials 2. Pooling such data across products and manufacturers Challenges: o Changes to aggregate attributes and particle numbers over the product shelflife o Changes to attributes after administration, as well as a host of patient-related factors such as exposure to multiple medications and product lots varying in their attributes 22 Moussa et al. J Pharm Sci

24 Recommendations for future research 3. Assessing contributions of surface chemistry and epitope presentation to immunogenicity 4. Analytical approaches to assess the ability of protein aggregates to bind to receptors of interest and to map the presentation of epitopes on the aggregate surface 5. Standardized threshold measurements 6. Validated animal models 7. Models for SC space 23 Moussa et al. J Pharm Sci

25 Acknowledgements Purdue University: Prof. Elizabeth Topp Dr. Shenbaga Moorthy (Pfizer-Hospira) Dr. Jainik Panchal (Merck) Indiana University: Prof. Janice Blum Amgen, Inc: Dr. Linda Narhi Dr. Marisa Joubert FDA CBER: Dr. Ewa Marszal Dr. Joseph Kotarek (Brand Institute, Inc.) 24