challenges in ada/nabassay design to support gene therapy ...€¦ · gene therapy |emerging...
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Challenges in ADA/NAb Assay Design to Support Gene Therapy Programs
Laboratory Sciences, MPI Research, A Charles River CompanyAmy Smith, BA, Senior Director, Bioanalytical/Analytical Services EBF – Focus WorkshopMay 14-15
Outline
Gene Therapy | Emerging Therapeutic
Gene Therapy | Immunogenicity
Neutralizing Antibodies | Why do they matter?
Immunogenicity and Bioanalysis
Case Study
Points for Consideration
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Gene Therapy | E M E R G I N G T H E R A P E U T I C
• Gene therapy brings the hope of curing monogenic diseases, often rare and without effective treatment
• > 2500 gene and cell based therapy clinical trials have been completed, initiated or underway in the last 30 years
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Gene Therapy | D E L I V E RY
Mechanisms for therapeutic gene/RNA delivery chosen based on target, safety profile of delivered gene, local vs. systemic delivery• Viral vector (AAV)• Engineered virus• Nanoparticle
encapsulation
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Gene Therapy | I M M U N O G E N I C I T Y
• Immune response may be triggered– Against the AAV vector– Against the therapeutic
gene/RNA (transgene)– Against the translated
protein
• Pre-existing AAV may result in immune response with detriment to efficacy and or safety– B cells produce antibodies – Effector T-cells
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Gene Therapy | I M M U N O G E N I C I T Y
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Neutralizing Antibody (NAb)– A type of Anti-drug Antibody
• Subpopulation of binding ADAs
– An antibody that inhibits activity of TA• Directly binds the TA active site • Binds TA and blocks active site via steric hindrance
Neutralizing Antibodies | W H Y D O T H E Y M AT T E R ?
Concern Nonclinical Outcome Clinical OutcomeSafety Potential for animal species
response to interfere with tox end-points; masking clinical safety concerns or off target toxicity
Neutralize activity of endogenous counterpart with unique function causing deficiency syndrome
Hypersensitivity reactions
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Neutralizing Antibodies | W H Y D O T H E Y M AT T E R ?
Concern Nonclinical Outcome Clinical OutcomeSafety Potential for animal species
response to interfere with tox end-points; masking clinical safety concerns or off target toxicity
Neutralize activity of endogenous counterpart with unique function causing deficiency syndrome
Hypersensitivity reactions
Efficacy Enhancing or decreasing efficacy by• Changing half life• Changing biodistribution
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Neutralizing Antibodies | W H Y D O T H E Y M AT T E R ?
Concern Nonclinical Outcome Clinical OutcomeSafety Potential for animal species
response to interfere with tox end-points; masking clinical safety concerns or off target toxicity
Neutralize activity of endogenous counterpart with unique function causing deficiency syndrome
Hypersensitivity reactions
Efficacy Enhancing or decreasing efficacy by• Changing half life• Changing biodistribution
Pharmacokinetics Antibody production may block or diminish exposure
Antibody production may dictate needed changes in subsequent trials
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Neutralizing Antibodies | W H Y D O T H E Y M AT T E R ?
Concern Nonclinical Outcome Clinical OutcomeSafety Potential for animal species
response to interfere with tox end-points; masking clinical safety concerns or off target toxicity
Neutralize activity of endogenous counterpart with unique function causing deficiency syndrome
Hypersensitivity reactions
Efficacy Enhancing or decreasing efficacy by• Changing half life• Changing biodistribution
Pharmacokinetics Antibody production may block or diminish exposure
Antibody production may dictate needed changes in subsequent trials
Translation Species specific response, or lack there of, is not indicative of potential response in the clinic
Mechanism for eliciting NAb response• Delivery• Naked gene• Translated protein
Potential ADA must be closely monitored in clinical population
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Immunogenicity and Bioanalysis
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• Draft Guidance (2016): Assay Development and Validation for Immunogenicity Testing of Therapeutic Protein Products
• EMA – (2008): Guideline on Immunogenicity assessments of biotech-derived therapeutic proteins
• EMA – (2017): Guideline on Immunogenicity assessment of therapeutic proteins
• Predominant clinical focus
Immunogenicity to Bioanalysis | T I E R E D A P P R O A C H
• Screening assay using ligand binding immunoassays to detect the presence of any ADA
• Confirmatory assay used to confirm samples screening positive for ADA
• Titer assay• Characterization of ADA
– Neutralizing capacity– Binding affinity– Isotyping
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Immunogenicity and Bioanalysis | C E L L - B A S E D N A B A S S AY S
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• Cell based NAb assays are preferred by the regulators over plate based ligand binding assays
• Provide mechanistic insight into immunogenicity• Recapitulate in-vivo biology• Plate based assays can be used when an appropriate
target is available and the binding mechanism is well understood
Immunogenicity and Bioanalysis | C E L L - B A S E D N A B A S S AY S
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Cell based NAb assay challenges:• Cell maintenance is time/budget consuming• Time required for development and validation can be
extensive• Assays are more susceptible to matrix effects and drug
interference• Assays are generally less precise/robust than ligand
binding assays due to biological variation
Immunogenicity and Bioanalysis | N A b A S S AY S
• Positive Control – must be capable of neutralizing the drug of interest to assess assay sensitivity
• Negative Control• Cell Line – appropriate viral tropism as target tissue for
therapeutic delivery. Must provide appropriate sensitivity and dynamic range
• Endpoint Readout – ability to detect a change in the assay based on a specific cellular response typically resulting in either a fluorescence or luminescence signal. Ex., GFP or Luciferase production
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Immunogenicity and Bioanalysis | C A S E S T U D Y
Retinitis Pigmentosa • Rare; <20,000 US cases annually• Disease that often begins in childhood and results in progressive loss of
vision• Treatments currently exist, but not a cure• Genetic mutation cause rods and cones in retina to degenerate
Immunogenicity and Bioanalysis | C A S E S T U D Y
Gene therapy indicated for Retinitis Pigmentosa• Therapeutic: Adeno-Associated Viral Vector 2 (AAV2) encoding the transgene,
mVChR1, Modified Volvox Channel Rhodopsin-1 • Dose Route: Direct Injection • Study Design: Single Dose Intravitreal Tox Study in Monkey• Bioanalysis: Assay developed and validated to assess NAbs against the AAV2
vector– Serum samples collected at pre-dose and termination
Recombinanthumangene
AAV2viralvector
Immunogenicity and Bioanalysis | C A S E S T U D Y
Assay Component In Vivo Assay Homolog Cell Line Retinal Pigment
Epithelium HEK293 from ATCC
Delivery Modified AAV2 viral vector
AAV2-Luciferase control virus
Transgene mVChR1(ModifiedVolvoxChannelRhodopsin-1)
Firefly Luciferase gene
Transgene expression Rhodopsin Luciferase
Positive Control Antibody Mouse Monoclonal Antibody to AAV2
Negative Control Monkey Serum
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Immunogenicity and Bioanalysis | C A S E S T U D Y
Assay Component In Vivo Assay Homolog Cell Line Retinal Pigment
Epithelium HEK293 from ATCC
Delivery Modified AAV2 viral vector
AAV2-Luciferase control virus
Transgene mVChR1(ModifiedVolvoxChannelRhodopsin-1)
Firefly Luciferase gene
Transgene expression Rhodopsin Luciferase
Positive Control Antibody Mouse Monoclonal Antibody to AAV2
Negative Control Monkey Serum
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Immunogenicity and Bioanalysis | C A S E S T U D Y
Assay Component In Vivo Assay Homolog Cell Line Retinal Pigment
Epithelium HEK293 from ATCC
Delivery Modified AAV2 viral vector
AAV2-Luciferase control virus
Transgene mVChR1(ModifiedVolvoxChannelRhodopsin-1)
Firefly Luciferase gene
Transgene expression Rhodopsin Luciferase
Positive Control Antibody Mouse Monoclonal Antibody to AAV2
Negative Control Monkey Serum
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Immunogenicity and Bioanalysis | C A S E S T U D Y
Assay Component In Vivo Assay Homolog Cell Line Retinal Pigment
Epithelium HEK293 from ATCC
Delivery Modified AAV2 viral vector
AAV2-Luciferase control virus
Transgene mVChR1(ModifiedVolvoxChannelRhodopsin-1)
Firefly Luciferase gene
Transgene expression Rhodopsin Luciferase
Positive Control Antibody Mouse Monoclonal Antibody to AAV2
Negative Control Monkey Serum
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Immunogenicity and Bioanalysis | C A S E S T U D Y
Assay Component In Vivo Assay Homolog Cell Line Retinal Pigment
Epithelium HEK293 from ATCC
Delivery Modified AAV2 viral vector
AAV2-Luciferase control virus
Transgene mVChR1(ModifiedVolvoxChannelRhodopsin-1)
Firefly Luciferase gene
Transgene expression Rhodopsin Luciferase
Positive Control Antibody Mouse Monoclonal Antibody to AAV2
Negative Control Monkey Serum
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Immunogenicity and Bioanalysis | C A S E S T U D Y
Assay Component In Vivo Assay Homolog Cell Line Retinal Pigment
Epithelium HEK293 from ATCC
Delivery Modified AAV2 viral vector
AAV2-Luciferase control virus
Transgene mVChR1(ModifiedVolvoxChannelRhodopsin-1)
Firefly Luciferase gene
Transgene expression Rhodopsin Luciferase
Positive Control Antibody Mouse Monoclonal Antibody to AAV2
Negative Control Monkey Serum
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Immunogenicity and Bioanalysis | CASE STUDY
Validation Results– Acceptance criteria for the assay was met– Validation Fixed Cut Point 5,997– Confirmatory Cut Point = 43.1%– NHP Average Sensitivity = 714 ng/mL
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Immunogenicity and Bioanalysis | C A S E S T U D Y
Sample Analysis Results• Screening Analysis: 26/60 positive and 1/60
indeterminate (replicates spanned cut point)– Animals were not pre-screened prior to being placed on
study• 9 pretest samples and 1 control animal screened positive
(indicates prior anti-AAV2 antibody exposure). These data highlight the importance of robust NAb screening pre-study as a basis for animal selection
• Confirmatory Analysis: 27/60 samples underwent confirmatory, of which 26 confirmed positive
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Immunogenicity and Bioanalysis | C A S E S T U D Y
Assay met all validation criteria, but:• Were HEK293 cells a good homolog for NHP Retinal Pigment Epithelium
cells? • Does the AAV2-Luciferace Control Virus instigate a comparable immune
response as the mAAV2 proprietary vector?• Is the mouse monoclonal antibody to AAV2 an appropriate positive control
for NHP antibody production?• Is Luciferase indicative of transgene gene transcription and protein
translation?• Was the cut-point appropriate?
– In-study cut-point could not be established because there were an insufficient number of control animals that screened negative
• Was the assay sensitivity appropriate at 714 ng/mL?
Ultimately the study was repeated, using a pre-screened animal population because the clinical signs became the only study endpoint
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Immunogenicity and Bioanalysis | CHALLENGES
• Cell-line can be troublesome, as it must reflect TA MoA and viral tropism
• Appropriate cell bank size to support study: cell passage effects on assay performance
• Surrogate AAV control vectors with assay readout• Longer timelines for cell-based assay development based on
optimized biological assay readout• Identification of appropriate critical reagents: must have a
neutralizing PC antibody• Therapeutic development support• Relevant regulatory guidelines
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Topics for Consideration
• Are we answering the right questions to support gene therapy development?
• Are we designing the right assays?
• Or are we answering questions to meet regulatory guidance that may or may not be relevant?
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Acknowledgments
Arkadeep Sinha, Ph.D.Elaina Breznau, Ph.D.Jessica St. Charles, Ph.D.Kalia Bared FreyreRachel AkunaRoger Hayes, Ph.D.
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Additional Slides
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NAbs and Bioanalysis | O P T I O N S
Cell-Based NAb Assay – based on the MoA• Direct Binding Assay: TA is an agonist (initiates)• Indirect Binding Assay: TA is an antagonist (inhibits)
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NeutralizingactivityNosignal
Direct Binding Assay: TA induces direct cellular effect via receptor binding
Targetpathwayactivation
Drug
Nab
Receptor
NAbs and Bioanalysis | O P T I O N S
TAtargetsligandsuppressessignal
NabbindsTA,ligandbinding
stimulatessignal
Indirect Binding Assay: TA indirectly blocks cellular effect via ligand binding
NAbs and Bioanalysis | O P T I O N S
Drug
Nab
Receptor
Ligand
Ligandbindingstimulatessignal