Immunogenicity Testing Strategies for a Novel Modality: Lipid Nanoparticles Containing Small Interfering RNA Payloads

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1 Immunogenicity Testing Strategies for a Novel Modality: Lipid Nanoparticles Containing Small Interfering RNA Payloads Renuka C. Pillutla, PhD Bioanalytical Sciences Translational Medicine

2 Biological Drugs and Immunogenicity Traditional Paradigm Biologic Types Immunogenicity Assays Reporter Non-neutralizing Ab o o o o Monoclonal antibodies Domain antibodies Antibody drug conjugates Proteins Target receptor Neutralizing Ab Reporter cell v Biological drugs or protein therapeutics often trigger unwanted immune responses against themselves. v The immune response may involve the innate and adaptive arms, leading to toxic cytokine storms, cytotoxic T cell and Natural Killer cell activation, T helper responses and B cell activation. v The immune response that is most frequently monitored is antibody (Ab) production by B cells. v Antibodies (Abs) bind to drugs and reduce or eliminate their therapeutic effects. v Such Abs can also cause complications and adversely impact safety of the drug. ADA Assay nab Assay 2

3 Lipid Nanoparticle Drugs Unknown Paradigm for Immunogenicity Yi C. Tam et. al Pharmaceutics 2013 vincreased drug complexity. vmultiple components with immunostimulatory potential. vantibodies against more than one component. vin addition to antibody (Ab) response, innate immune responses are anticipated. vcytokine production and complement activation may have an impact on safety and efficacy. What assays do we develop/prioritize given the complexity of the drug and its potential to activate various arms of the immune system? 3

4 Lipid Nanoparticle Drugs Potential for Immune Responses Synthetic lipids can promote immune responses at high doses Anti-lipid Abs Inflammatory cytokines Potentiation of an anti-sirna Ab response (adjuvant effect) Nucleic acids are potent stimulators of the innate immune system Potential for cytokine secretion and related adverse events Potentiation of an anti-sirna Ab response (adjuvant effect) Potentiation of an anti-lipid Ab response (adjuvant effect) Nucleic acids can generate antibodies anti-sirna Ab response Activation of the complement system by cationic lipids Potential Ab response to PEG Hypersensitivity reactions from preexisting anti-peg Abs Complement and opsonin activation by anti-peg IgM and IgG Abs 4

5 BMS Immunogenicity Mitigating Factors v Size of the nanoparticle particle is small (100±25 nm) reducing immunogenic potential. v Under physiologic ph conditions, surface charge of the drug is neutral, mitigating potential for complement activation. Metabolized cationic lipids may still activate the complement system. v sirna sequence was selected following in vitro cell-based cytotoxicity and immune-toxicity assays (TLR response and cytokine induction to assess innate immune system activation). v Naked or unformulated sirna is highly unstable and rapidly degraded by nucleases in biological fluids, does not accumulate in target tissues and cannot readily cross target cell membranes. v Vitamin A is a small hydrophobic molecule that is coated with retinol binding protein with only the OH group making contact with the aqueous environment. Chances of exposure to the immune system and anti-vitamin A ADA generation are low. 5

6 BMS Immunogenicity Monitoring Plans Innate Immune System v sirna mediated inflammation. Measure IL-1b, TNF-a, IFN-b, IFN-g, IL-6, and IP-10 within 2 hours following drug infusion. v Lipid mediated inflammation. Include leukotrienes and prostaglandins in chemistry panel. v Complement activation by cationic lipids. Measure key complement factors from the alternative and classical pathways e.g. CH50, C3a, C5a and factor Bb. Adaptive Immune System v T independent Ab responses are a major concern. v Detect Abs to whole drug, Vitamin A- PEG conjugate and vitamin A. v Deprioritize Ab assay to sirna. Non-encapsulated sirna has a short half-life in plasma (0.6 hours). PK profiles of sirna following repeated drug administration were not affected in Phase I trials, indicating that there is no Ab mediated clearance of the drug. 6

7 Monitoring ADA Responses Whole LNP Ab Detection Multi-component Ab Detection The 3 ADA assays shown on the left will assess antibody (Ab) responses to various surface-exposed components of the LNP. Each assay is a direct ELISA format. Whole LNP or components are coated on ELISA plates to measure Ab levels in human plasma to each individual component. Generic anti-human k + l Abs with reporters are used to detect all Abs in each assay type. In each case, specific binding of the Abs to the drug may be checked by using the whole drug or individual components as a competing reagent. Individual component Ab Detection 7

8 Technical Challenges of an ADA Assay Lack of positive controls for individual components. Example: anti-lipid, anti-targeting agent, anti-peg etc., Lack of knowledge of assay conditions that enable the detection of various antibodies S to ra g e o f F o r m u la te d D ru g a t 4 0 C F o llo w in g T h a w fro m C P la te C o a tin g a n d E ffe c t o n A s s a y S ig n a l 1 D a y Example: Tween-free assay buffers for PEG Ab detection may or may not capture other Abs. Until controls are available, assay conditions cannot be tested D a y s 1 5 D a y s 2 0 D a y s 2 7 D a y s Stability of formulated drug product used for plate coating. Stability has to be monitored over time for liquid formulations. Lyophilized material needs to be weighed and resuspended accurately each time. S ig n a l/n o is e D a y s 4 1 D a y s 4 8 D a y s 7 0 D a y s 7 2 D a y S to ra g e fo llo w in g firs t th a w 2.7 fo ld d e c re a s e a t c u rre n t s e n s itiv ity le v e l Lack of Ab controls limits stability data that is accrued over time. Example stability data is shown for an anti-peg control on the right a n ti-p E G A b C o n tr o l

9 Whole Nanoparticle ADA Assay A Fit-for-Purpose Assay for IM E C L U n i t s A n t i - W h o l e N a n o p a r t i c l e A n t i b o d y A s s a y H u m a n P l a s m a P o o l D i l u t i o n F a c t o r = ( H P P ) - a n t i - h u m a n A b b a c k g r o u n d N e g a t i v e Q u a l i t y C o n t r o l A s s a y R a n g e n g / m L R a b b i t a n t i - P E G A n t i b o d y R a b b i t A b i n H P P H i g h Q u a l i t y C o n t r o l R a b b i t A b i n H P P L o w Q u a l i t y C o n t r o l F u t u r e C h a l l e n g e : D e c r e a s e h u m a n b a c k g r o u n d H u m a n P l a s m a P o o l ( H P P ) - a n t i - r a b b i t A b b a c k g r o u n d v v v v A FFP assay has been developed to analyze all Abs generated to surface antigens of the whole nanoparticle. The assay was developed using a rabbit monoclonal Ab control specific for the PEG component of the drug. The assay has 3 tiers: screening, confirmatory and titer. Cut points for screen, confirmatory and titer positives were set following a statistical analysis of data from approximately 50 individual normal human plasma samples. 9

10 Whole LNP ADA Assays Assay Sensitivity and Drug Tolerance FDA 2016 Guidance on Immunogenicity Assay Development and Validation states that The assays should have sufficient sensitivity to enable detection of low levels of ADA. Recommendations on sensitivity based on clinical events is 100 ng/ml or ng/ml. For LNP based drugs, which ADA concentrations are associated with altered pharmacokinetic, pharmacodynamic, safety, or efficacy profiles? Lack of data for LNP drugs. Are previous sensitivity recommendations applicable? 10

11 Drug Tolerance Thought Experiment PK data (C trough ) is available for 2 excipients and the API (encapsulated oligonucleotide payload). Which of the three C trough measurements are to be used to build ADA assay drug tolerance? Hints: Consider if API ADA are relevant to drug exposure and efficacy. Consider the specificity of the ADA positive control. Does the PK data for all 3 analytes hint at intactness of the drug product? 11

12 Immunogenicity Assessment Guidelines Our Recommendations Consider the structural complexity/diversity of components. Investigate the immunogenic potential of individual components. Evaluate innate and/or adaptive immune response potential of components. Be holistic to start with. Organize your strategy around immune response potential and known physical characteristics (e.g. size, surface charge). Adopt a risk-based strategy to assay development. Identify the most important and informative assays for safety, exposure and efficacy assessments. Limit the number of assays. Adapt to maximize information (e.g. confirmatory assay competitors). Harness available PK data. What is the half-life of the oligonucleotide cargo? Is an Ab assay necessary? Do previous PK profiles show alterations in a multi-dose study? Consider time needed for generating positive (Ab) control for ADA assays. Select the most relevant and useful controls (e.g. anti-target Ab control). Data analysis. Correlation with safety. Evaluate all available data sets for impact (e.g. complement, cytokines). antibodies. Correlation with exposure/efficacy. Evaluate relevant data sets only (e.g. anti-peg Abs and complement). 12

13 Immunogenicity and PD Correlations A well defined strategy for drug exposure levels and efficacy. API (sirna) in serum/target tissue vs. mrna decrease or other biomarkers for efficacy. Immunogenicity has safety and efficacy implications (PD). Categorize the immunogenicity read-outs for safety or efficacy (or both) assessments as appropriate. Examples: Complement activation can impact both safety and drug exposure levels. Same with anti- PEG or anti-lipid ADA. anti-sirna ADA? Impact on efficacy? Unlikely. Cytokines are important for safety, but look for correlations with ADA production (potentiation of the adaptive immune system). 13

14 Acknowledgements Members of the BMS drug manufacturing, regulatory, pharmacology and clinical teams Members of the BMS bioanalytical sciences team o Wendy Miller o Qin Ji o Renuka Pillutla Our partners and colleagues at Nitto Denko o Liping Wang o Sonya Zabludoff o Annie Pham 14

15 Recommended Reading Ilinskaya, A.N., Dobrovolskaia, M.A., Understanding the immunogenicity and antigenicity of nanomaterials: Past, present and future. Toxicology and Applied Pharmacology 299, Moghimi, S.M., Hunter, A.C., Andresen, T.L., Factors Controlling Nanoparticle Pharmacokinetics: An Integrated Analysis and Perspective. Annu Rev Pharmacol Toxicol 52, Marlowe, J.L., Akopian, V., Karmali, P., Kornbrust, D., Lockridge, J., Semple, S., Recommendations of the Oligonucleotide Safety Working Group s Formulated Oligonucleotide Subcommittee for the Safety Assessment of Formulated Oligonucleotide-Based Therapeutics. Nucleic Acid Therapeutics 27 (4), Zatsepin, T.S., Kotelevtsev, Y.V., Koteliansky, V., Lipid nanoparticles for targeted sirna delivery-going from bench to bedside. International Journal of Nanomedicine 11, Tremblay, G.A., and Oldfield, P.R Bioanalysis of sirna and Oligonucleotide Therapeutics in Biological Fluids and Tissues. Bioanalysis 1(3),