NONCLINICAL CONSIDERATIONS FOR CELL & …...| Nonclinical Development Considerations for C&G Therapies Cell-based Therapies (Regenerative Medicinal Products) • Process of replacing,

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Copyright © 2019 Covance. All Rights Reserved

NONCLINICAL CONSIDERATIONS FOR CELL & GENE THERAPIESBrian McIntosh, PhDPaul Byrne, PhD

March 11, 2019

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About Us

| Nonclinical Development Considerations for C&G Therapies2

Study Director for Safety Assessment/Toxicology in Madison, WI Trained with stem cell biologist Jamie Thomson and toxicologist Chris Bradfield

at the University of Wisconsin-Madison, and hematopoietic stem cell biologist Michael Cooke at the GNF (Novartis Institute) in San Diego

Subject matter expert in regenerative biology and the development of cellular, gene and regenerative therapies

Member: Covance’s Advanced Therapies Drug and Device Development Group

Principle Scientist for Cell and Gene Therapy, Bioanalytical and BioCMC in Harrogate

20+ years in the CRO industry including Study Director and Head of Molecular Biology

Member: Covance’s Advanced Therapies Drug and Device Development Group

•BRIAN E. McINTOSH, PhD

•PAUL BYRNE, PhD

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AGENDA

Today You’ll Hear About


• How advanced therapies are defined• Preclinical study and regulatory/safety considerations and strategies when

assessing advance therapies• Analytical design and validation strategies• Biodistribution studies and data integrity• Titre assays• CMC considerations• Phase appropriate validations

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What type of Advanced Therapy

are you interested in advancing and where does your product fit?


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Advanced Therapies: Cell vs Gene


Cell-based Therapies(Regenerative Medicinal Products)

• Process of replacing, engineering or regenerating human cells, tissues or organs to restore or establish normal function– Function may be lost due to age,

disease, damage, or congenital defects – Regenerate damaged tissues and

organs in the body by stimulating previously irreparable organs to heal themselves

– Grow tissues and organs in the laboratory and safely implant

Gene Therapies

• Technique for correcting defective genes responsible for disease– Genes as medicine

• A normal gene may be inserted into a non-specific location within the genome to replace a non-functional gene– Viral vectors are the tool typically used to

deliver genetic material into cells– Vectors have included: adenoviruses, herpes

viruses, retroviruses, DNA tumor viruses, RNA tumor viruses, liposomes, and nanotechnology

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Preclinical Program Goals for Advanced Therapeutics


• Data necessary to support development depends on the characteristics of the product

• Preclinical studies are designed to support use of a specific product for a specific clinical indication

• Identification of biologically relevant dose levels to guide clinical dose levels.

• Establish safety in relation to dose level of the clinical product.

• Identification of physiologic endpoints that can be used in the clinic for monitoring.

• Provide safety information to support first-in-human clinical trial.

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Preclinical Program Goals for Advanced Therapeutics (cont.)


• Test system must be appropriate

• Species, disease models, immuno-deficient animals

• Autologous, allogeneic or xenogenic

• Delivery must represent clinical route of administration

• May include device compatibility

• Identity of test article/material is defined

• Identifying local and systemic toxicities following therapeutic administration

• Characterize potential adverse effects with respect to clinical dose, onset, duration

• Characterize derived therapy with respect to tumorigenicity, potential ectopic tissue formation

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•EXPEDITED PROGRAMS FOR REGENERATIVE MEDICINE THERAPIES FOR SERIOUS CONDITIONS

Fast Track and Breakthrough Designation Programs


• Fast Track (designation) – for therapeutics intended to treat a serious condition where data demonstrate the potential to address an unmet medical need– Closer interaction with FDA on the drug development plan– Portions of the Marketing Application can be submitted in advance of the entire application, on a

“rolling review” basis• Breakthrough (designation) - for therapies intended to treat a serious condition

where preliminary clinical evidence indicates that the drug may demonstrate a substantial improvement over available therapies– All program features of Fast Track designation, including intensive guidance from FDA, to help

sponsors better tailor their drug development program– Organizational commitment from FDA’s senior managers and experienced review staff to

collaborate in advancing the review of these potentially high-impact drugs

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Issues to Overcome for In vivo Viral Delivery


GENE THERAPY PRODUCTS

• Short Lived– Difficult to rapidly integrate therapeutic DNA into genome.– Rapidly dividing characteristic of certain cells may prevent long time efficacy– May require multiple rounds of therapy

• Immune Response– Foreign material introduced leads to immune response– Increased response when a repeat offender enters– AAV sero-negative screening in NHPs prior to study

• Vector Toxicity– Potential for toxic (gene over-expression), immune, inflammatory response– Potential to cause disease once administered

• Multigene Disorders– Difficult to treat conditions may require more than one gene

• Potential for Malignancy– Insertional mutagenesis may induce a tumor if integrated in a tumor suppressor gene

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Refresher Preclinical Study Considerations


• Test system must be appropriate; may require pre-screen for neutralizing antibodies• Delivery must represent clinical route of administration• Test system may require immune suppression• Study length is dependent on therapeutic and clinical program: 7 days to 6 months• Delivery and virus dependent; shedding studies• Biodistribution/shedding: monitoring tissues for integration and fluids for infectious units• Efficacy assay to determine if the product was delivered and active• Pilot studies to optimize definitive study conditions• Identifying local and systemic toxicities following delivery• Characterize potential adverse effects with respect to clinical dose, onset, duration• Characterize derived GT with respect to potential tumor formation

GENE THERAPY PRODUCTS

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Preclinical Regulatory Considerations


• Safety: acute, tumorigenicity, biodistribution, efficacy

• Open dialogue with the FDA-OTAT, EMA, PMDA…

EX VIVO DERIVED CELL THERAPY

Source: Fink. FDA Regulation of SC-Based Products. Science 2009;324:1662-63.

In vitro properties In vivo concerns

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Perspective Shifts in Early Development


• Safety is primarily shifted to the manufacturing phase (CMC)– Personalized or individualized medicine – Variation in cell source– Limited production, small batch size– Establish minimal criteria to ensure safety efficacy and consistent production

• Assay development to evaluate ex vivo delivery system in relation to therapeutic– Tight control on copy number– Use of insulator elements in the payload– Use of self-inactivating viral vectors– Removal of unneeded/unwanted elements– Phenotype, generation of non-endogenous factors, genotype, karyotype

CAR-T THERAPY PRODUCTS

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Preclinical Study Considerations


• A scientific discussion dictates in vivo safety designs• Controversy: definitive therapeutic is generally short-lived: beware of rise of the clone!

– Safety studies generally focus on long-term tumorigenicity: 3 to 6 months– Biodistribution 14 to 30 days (general lifetime of therapy)– In vitro assay measuring transformation/gain proliferative autonomy may be better measure

• Test system must be appropriate– Immunocompetent mammal would reject cells that were generated from a different species.– Immuno-deficient animals used.

• PDx or Syngenic models may be considered to generate data and be more relevant. • Future: Targeting solid tumors

– Generation of CAR2.0, CARs with kill switch, on/off switch controlled by small molecules, or other therapeutic interventions

CAR-T THERAPY PRODUCTS

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Preclinical Study Considerations


• In vivo Tumorigenicity– Derived CBT/RMP dosed at varied concentrations to an ideally confined location in the

animal. – One group may be required for positive parental cell line or tumorigenic line

– (e.g. iPSC, MCF7, A498, PC3, Hep3B, etc.).

– Looking for ectopic tissue formation or potential migration.– Duration: minimum 3 months or to organism lifetime based on therapy/target population.

• Biodistribution assay– Quantitative PCR time course based analysis of major organs to monitor metastases.– Normally small animal (mouse or rat); may be performed via IHC– Duration:28-days to organism lifetime; time dependent on CBT/RMP target population– Can be in combination with tumorigenicity assay.

CELL THERAPY PRODUCTS

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Preclinical Study Considerations (cont.)


• Acute/Efficacy/Dose Response– Employ disease or injury model test system– Adverse effects with respect to dose, onset, duration– Dependent on cellular response and physiology to detect signal and time course.– Combination: may be performed with biodistribution and/or tumorigenicity studies– Duration: may be 2 weeks up to 9 months

• Test article analysis– Viability – Compatibility with dosing apparatus – Characterization of cell type (flow)– Stability time course established: usually pre-dose and post-dose– GLP: pre-study validations required for enumeration, flow, antibody clones per cell type

CELL THERAPY PRODUCTS

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Closing remarks


• Changing SA landscape with Cell and Gene Therapeutics

• Therapeutic dictates the study design

• Seek advice from a regulatory body and knowledgeable source prior to initiating work

• And the process is the product…

CELL AND GENE THERAPY STUDY DESIGNS

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“What are the design considerations for bioanalytical assays to support

biodistribution studies?”

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Biodistribution Studies


• Assay design is critical– Span the junction of modifications– Theoretical specificity– Feasibility– Multiple applications

– CMC– Clinical

• Validation considerations– LOQ, LOD, Linearity, Range, Specificity, Inhibition, Robustness,

Precision and Stability

QPCR / RT-QPCR ASSAY DESIGN CONSIDERATIONS

Biodistribution

Toxicology

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• Sensitivity is critical – LOQ – 50 copies / µg of genomic DNA

– Chemistry is important – e.g. TaqMan or SYBR Green

• Specificity– Importance of assay design

• Extraction considerations– Challenge selections of tissues, biofluids and blood– Recovery of plasmid/vector/stem cell DNA

– 50%-80% (20%-80% recovery)

•QPCR ASSAY DESIGN CONSIDERATIONS

Biodistribution

Toxicology

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“What are the key analytical and design considerations for preclinical

biodistribution studies?”

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• Combined with toxicology study: separate biodistribution groups/ cohorts• Route of administration: dependent on therapy• Timepoints – 1wk, 1M and 3M• Assessment of distribution and persistence of vector/cell

– Tissues– Depends on route and MOA

– Blood– Biofluids

– Viral shedding• DNA and transgene (mRNA) expression• Focus on preventing contamination

•QPCR ASSAY DESIGN CONSIDERATIONS

Biodistribution

Toxicology

Dispensary AnimalRoom Necropsy Extraction qPCR

Analyses

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Biodistribution

Toxicology

Dispensary

Animalroom

Necropsy

Extraction

qPCRAnalyses

Sterile, biological handlingSpecific procedures and aliquots

Specialized dosingHandling, separation and housing

Precautions and proceduresDisposable equipment and sample collection

Purpose build laboratoriesAutomated high throughput and controls

PCR clean areasHigh throughput and controls

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Biodistribution Studies - Responsibilities


Biodistribution

Toxicology

Dispensary

Animalroom

Necropsy

Extraction

qPCRAnalyses

Sterile, biological handlingSpecific procedures and aliquots

Specialized dosingHandling, separation and housing

Precautions and proceduresDisposable equipment and sample collection

Purpose build laboratoriesAutomated high throughput and controls

PCR clean areasHigh throughput and controls

SME SD PI

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• Significant risks for compromised studies– Cost of repeats– Delays to submissions– Impact to clinical trial design

• Important to have alignment between in-life, necropsy and analytical– Sample collection

– Guided by analytical team– Handling, storage and shipping

– Sample management, labels and barcodes– Reporting and analytical validation

– Collaboration between SD and PI

CONFIDENCE IN DATA

Biodistribution

Toxicology

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• CMC Information for Human Gene Therapy Investigational INDs –FDA Draft Guidance for Industry, July 2018

– Ensure ability to compare the preclinical dose to the clinical dose– Use the same qualified method to quantitate preclinical and clinical lots

• Human Gene Therapy for Hemophilia –FDA Draft Guidance for Industry, July 2018

• The assay for vector titre determination of the preclinical lots should be identical to the assay used for clinical lots

• QPCR or ddPCR?

TITRE / DOSE DETERMINATION

Biodistribution

Toxicology

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“What questions do you need to address to ensure safety, efficacy and consistency for

IND/IMPD submissions?”

Chemistry, Manufacturing and Controls (CMC)

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Critical Quality Attributes


• How much do you have?• How potent is it?• How safe is it?• How pure is your product?• Is it what you think it is?• Is it stable?

CMC

CMC

Clinical

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How Much Do You Have?


Part 1 – retroviral/lentiviral vectorPart 2 – active substance (CAR-T)

CRITICAL QUALITY ATTRIBUTES

CMC

Clinical

AAV Vectors CAR-TPart 1 Part 2

Genomic Titre (QPCR) Vector titre (QPCR) Viability (Flow)Capsid Titre (ELISA) Cell count (Flow)

Quantity and Titre

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How Potent Is It?



CMC

Clinical


Cell-based assay with ELISA endpoint

PBMC transduction Cell based assay with ELISA endpoint

Infectious titre (cell-based QPCR endpoint)

Transduction efficiency (QPCR)

CAR expression (flow)

Biological Activity, Transduction and Potency

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How Safe Is It?



CMC

Clinical


Safety testing ICH Q5A Safety testing ICH Q5A

Sterility

rcAAV RCR/RCL Mycoplasma

Endotoxin

Safety Testing

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How Pure Is It?



CMC

Clinical


Host Cell Proteins and DNA(ELISA and QPCR)

HCP and HCDNA (ELISA and QPCR)

Vector (QPCR)

BSA, Benzonase (ELISA) RCR/RCL Beads (Microscopy)

Capsid purity (CE)

Empty vs full

Purity and Impurities

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Is It What You Think It Is?



CMC

Clinical


Sequencing, Mass Spec Sequencing QPCR

Identity

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“How can I rely on the data for submission, and what does a phase appropriate

validation plan look like?”

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Regulatory perspective


• CMC Information for Human Gene Therapy Investigational INDs –FDA Draft Guidance for Industry, July 2018

– Validation not required for IND phase 1– Appropriately controlled

– Complete validation before BLA– Evaluate performance through development– Have a plan in place

– Consistent with European guidance

VALIDATIONS

Biodistribution

Toxicology

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Development Pathway


CMC

Product Understanding

PreclinicalPhase I

Phase IIPhase III

Commercial

BLA / MAA IND / IMPD

CMC: Validation

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Assay Validation


Fit for purpose / controlled / suitability• Compendial

– 1 run to qualify (additional for small volumes)• Identity / Purity (QPCR, ELISA, CE-SDS, MS, FACS)

– 2-3 runs• Titre (QPCR, ELISA)

– 3-4 runs• Potency (Cell based)

– 15 runs

DEFINITIONS

CMC

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Assay Validation


Limited validation• Identity / Purity / Titre (QPCR, ELISA, CE-SDS, MS)

– 7-8 runs• Potency (cell-based)

– 9 runsFull validation (robustness)

– Design of experiment (DOE)– 8 runs– QPCR included with limited validation

DEFINITIONS

CMC

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Validation Pathway


CMC

Phase IPhase II

Phase IIICommercial

Validation requirement

Compendial

Fit for purpose

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Validation Pathway


CMC

Phase IPhase II

Phase IIICommercial


Identity / Purity / Potency

Fit for purpose Limited validation Full validation

Limited validation Full validation

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Validation Pathway


CMC

Phase IPhase II

Phase IIICommercial


Titre Assays

Fit for purpose Limited validation Full validation

Preclinical??Full validation

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Copyright © 2019 Covance. All Rights Reserved

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NONCLINICAL CONSIDERATIONS FOR CELL & …...| Nonclinical Development Considerations for C&G Therapies Cell-based Therapies (Regenerative Medicinal Products) • Process of replacing,