challenges in ada/nabassay design to support gene therapy ...€¦ · gene therapy |emerging...

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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Efficacy Enhancing or decreasing efficacy by• Changing half life• Changing biodistribution

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Pharmacokinetics Antibody production may block or diminish exposure

Antibody production may dictate needed changes in subsequent trials

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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


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


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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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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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


TAtargetsligandsuppressessignal

NabbindsTA,ligandbinding

stimulatessignal

Indirect Binding Assay: TA indirectly blocks cellular effect via ligand binding


Drug

Nab

Receptor

Ligand

Ligandbindingstimulatessignal

challenges in ada/nabassay design to support gene therapy ...€¦ · gene therapy |emerging...

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