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Preclinical Immunogenicity Assessment
BioProcess International Webinar
slide 36-Oct-10
Philippe Stas, Head of Applied Protein Services / Lonza Gent / September 2010
Applied Protein Services: Risk Assessment & Optimization
Di D l t M f t Di t ib tibasic
research disease
discoverydrug
discoverydrug
developmentclinicaltrials production packaging
marketingsales
distribution
Discovery Development Manufacture Distribution
Quality / Comparability
EarlyRisk Assessment
ImproveLead Properties Desired Outcome
leaddiscovery
leadselection
leadoptimization
process development
& formulation
Mitigate risksImprove quality & safetyR d tt itiIn silico Assessment
ImmunogenicityStability
In vitro AssessmentImmunogenicity
Stability
Reduce attrition
slide 46-Oct-10
Applied Protein Services:Overview
A S l ™
Immunogenicity Screening Aggregation Removal
Epibase IV™ AggreSolve™Epibase™Tripole™
Deimmunization Humanization Deaggregation
In vitro Validation• Immunogenicity
A ti• Aggregation• Activity
• Expression
slide 56-Oct-10
Cell Line / Strain Construction
Immunogenicity Drivers
Intrinsic DriversSelf/nonselfMonomeric/multimeric
Extrinsic Drivers Mode of administrationDoseMonomeric/multimeric
Fusion proteinsGlycosylation
DoseFormulationAggregatesD d ti d tDegradation productsProduction contaminantsIndication
slide 66-Oct-10
Immunogenicity of Antibody Therapeutics
Therapeutic protein Type Target Indication Assay AR (%)
Pop Supr(%) r
OKT3 murine CD3 Graft rejection ELISA 54 82 +
Bexxar/tositumomab murine CD20 Non-Hodgkin's lymphoma
ELISA 9 55 +
Reopro/abciximab chimeric GPIIb/IIIa Coronary angioplasty ELISA 21 500
Remicade/infliximab chimeric TNF Crohn's disease 9 199 +Remicade/infliximab chimeric TNFα Crohn's disease 9 199 +
Remicade/infliximab chimeric TNFα Crohn's disease ELISA 61 125 +
Remicade/infliximab chimeric TNFα Rheumatoid arthritis ELISA 8 60 +
Rituxan/rituximab chimeric CD20 Non-Hodgkin's lymphoma
ELISA 0 37
Rituxan/rituximab chimeric CD20 Systemic lupus erythematosus
ELISA 65 17 +
Rituxan/rituximab chimeric CD20 Primary Sjogren's syndrome
RIA 27 15 +
Raptiva/efalizumab humanised CD11a Psoriasis 2.3 501
Raptiva/efalizumab humanised CD11a Psoriasis 4 292 +Raptiva/efalizumab humanised CD11a Psoriasis 4 292 +
Raptiva/efalizumab humanised CD11a Psoriasis ELISA 6 1063
Campath/alemtuzumab humanised CD52 Rheumatoid arthritis 63 40
Campath/alemtuzumab humanised CD52 Rheumatoid arthritis 29 31
Campath/alemtuzumab humanised CD52 Rheumatoid arthritis 53 30
slide 76-Oct-10
Campath/alemtuzumab humanised CD52 Rheumatoid arthritis 53 30
Campath/alemtuzumab humanised CD52 B-cell lymphoma 1.9 211
Humira/adalimumab human TNFα Rheumatoid arthritis ELISA 5 1062 +
Therapeutic Antibodies
Rituxan/rituximab chimeric CD20 NHL 0 37 multiple ivRituxan/rituximab chimeric CD20 SLE 65 17 multiple ivRituxan/rituximab chimeric CD20 RA 27 15 multiple iv
Humira human TNFa RA
slide 86-Oct-10 Hwang & Foote, 2005
Immunogenicity Assessment
leaddiscovery
leadselection
leadoptimization
process development
& formulationpreclinical clinical
trials
In silico T-Epitope Identification
In vitro T-cell Assays
Anti-Drug Antibody Screening
y
slide 106-Oct-10
Preclinical Immunogenicity Assessment
GUIDELINE ON IMMUNOGENICITY ASSESSMENT OF BIOTECHNOLOGY-DERIVED THERAPEUTIC PROTEINS
Doc. Ref. EMEA/CHMP/BMWP/14327/2006
4.2 Non-clinical assessment of immunogenicity and its consequencesTherapeutic proteins show species differences in most cases. Thus, human proteins will be recognized as foreign proteins by animals. For this reason, the predictivity of non-g g p y , p y
clinical studies for evaluation of immunogenicity is considered low.
Non-clinical studies aiming at predicting immunogenicityin humans are normally not requiredin humans are normally not required.
However, ongoing consideration should be given to the use of emerging technologies (novel in vivo, in vitro and in silico models),
slide 116-Oct-10
which might be used as tools.
Preclinical Immunogenicity Assessment
CONCEPT PAPER ON IMMUNOGENICITY ASSESSMENT OF MONOCLONAL ANTIBODIES INTENDED FOR IN VIVO CLINICAL USE
Doc. Ref. EMEA/CHMP/BMWP/114720/2009 (DRAFT)
1. Discussion
Recently developed combinations of in-silico and T-cell based procedures are showingRecently developed combinations of in silico and T cell based procedures are showing promise for predicting potential immunogenicity with some biologicals including mAbs.
Identification of epitopes associated with induction or suppression of immune responses has been possible.
4. Recommendations
The main topics to be addressed include: (6 topics)
slide 126-Oct-10
The main topics to be addressed include: (6 topics) …
Approaches which may be helpful in predicting unwanted immunogenicity of mAbs.
Early Stage Immunogenicity Assessment
leaddiscovery
leadselection
leadoptimization
process development
& formulationpreclinical clinical
trials
Epibase IV™Epibase™
In vitro T-cell Assays
Epibase IV™Epibase™
In silico T-Epitope Identification
y
Anti-Drug Antibody Screening
Generating a risk profile of the potential immunogenicityAnalysis of the probability to observe immunogenicity
slide 136-Oct-10
Analysis of the severity of the observed immunogenicity
Preclinical Strategies
In vivo strategiesAnimal studies exploring ADA response
In vitro strategiesT- cell epitope binding assays (HLA binding assays)p
Transgenic animal studies exploring T-cell responsesTolerized animal
( g y )T-cell activation and proliferation assays
Tolerized animal models/humanized animals In silico strategies
T-cell epitope mapping tools
slide 146-Oct-10
Predict and Reduce Immunogenicity
B cells need T-cell help to produce high affinity antibodies (i.e. IgG against
B-cell epitopes: The surface patches required for B-cell response
protein / antibody drugs)
Unlike B-cell eptitopes, T-helper epitopes can be predicted and tested
Eliminate T-helper epitopes will potentially reduce / diminish immunogenicity
T cell epitopes: Peptide
slide 166-Oct-10
T-cell epitopes: Peptide sequence of the protein needed for effective T-cell help
Epibase™ In Silico Immunoprofiling
leaddiscovery
leadselection
leadoptimization
process development
& formulationpreclinical clinical
trials
Epibase™ Epibase IV™
Epibase™ Epibase IV™
In silico T-Epitope Identification
In vitro T-cell Assaysy
Anti-Drug Antibody Screening
T-cell Epitope PredictionLead ranking and selectionD i i ti i bi ti ith th t f t i
slide 176-Oct-10
Deimmunization in combination with the support of protein modelling
Classical Predictive Methods
Score each position in the peptide for “likelihood” to fit in “pockets”Sum those scores => epitope vs. non-epitope
slide 186-Oct-10
Epibase™
Model BuildingTemplate Identification: Retrieve HLA subtypes of known 3-D structure that are at least 50% identical to a given HLA subtypeg ypBuild a 3-D structure
Run the Proprietary FASTER Program1Run the Proprietary FASTER ProgramSelect relevant part of the receptor
Include the flexibility of side chainsy
Determine2
Binding affinity
slide 196-Oct-10
Binding affinity
Promiscuity(1) EP 1226528, Proteins, 2002; (2) Proteins, 2005
Population frequencies from Doolan et al. 2000
MHCII Population Frequencies
20
25
30
5
10
15CaucasianJapaneseChinese
0
5
DR1(DRB1*0101)
DR3(DBR1*0301)
DR7(DBR1*0701)
DR8(DBR1*0802)
DR9(DBR1*0901)
Epibase™
Global
Epibase™
Caucasian
Epibase™
Asian
Epibase™
Hispanic
Epibase™
Afro-Am.DR 45 27 29 26 24
slide 206-Oct-1020
DQ 23 14 14 N/A N/ADP 10 7 8 N/A N/A
Epibase™ In Vitro Immunoprofiling
leaddiscovery
leadselection
leadoptimization
process development
& formulationpreclinical clinical
trials
Epibase™ Epibase IV™
In silico T-Epitope Identification
In vitro T-cell Assays
Epibase™ Epibase IV™
y
Anti-Drug Antibody Screening
Characterize T-Cell Epitopes Guided by In SilicoAt individual donor and population level
slide 226-Oct-10
Epibase™ In Vitro Immunoprofiling
leaddiscovery
leadselection
leadoptimization
process development
& formulationpreclinical clinical
trials
Epibase™ Epibase IV™
In silico T-Epitope Identification
In vitro T-cell Assays
Epibase™ Epibase IV™
An Indication of External Factors on Drug ImmunogenicityF l ti
y
Anti-Drug Antibody Screening
FormulationAggregatesDegradation products
slide 236-Oct-10
Production contaminants
Epibase™ In Vitro Immunoprofiling
leaddiscovery
leadselection
leadoptimization
process development
& formulationpreclinical clinical
trials
Epibase™ Epibase IV™
In silico T-Epitope Identification
In vitro T-cell Assays
Epibase™ Epibase IV™
C i f I i it b t F ll /S d
y
Anti-Drug Antibody Screening
Comparison of Immunogenicity between Follow-on /Second-Generation Products and Reference ProductsHealthy population/Patient population
slide 246-Oct-10
T-Cell Activation Assays: PBMC from Healthy Individuals / Patients
leaddiscovery
leadselection
leadoptimization
process development
& formulationpreclinical clinical
trials
Epibase™ Epibase IV™
In silico T-Epitope Identification
In vitro T-cell Assays
Epibase™ Epibase IV™
y
Anti-Drug Antibody Screening
slide 256-Oct-10
PBMC Isolation
within 3 hours after collection
Automated cell counting by Guava
Automated cell freezing by Nicool+
Sample storage management in LIMS
slide 266-Oct-10
T-Cell Activation Assays:Different Read Out Parameters / Assay Formats
leaddiscovery
leadselection
leadoptimization
process development
& formulationpreclinical clinical
trials
Epibase™ Epibase IV™
In silico T-Epitope Identification
In vitro T-cell Assays
Epibase™ Epibase IV™
y
Anti-Drug Antibody Screening
slide 286-Oct-10
Identify T-Cell Responses: Overview Read Out Parameters
Cell surface markers (CD3/CD4)
Proliferation (EDU)
Proliferating cells
Cytokine analysis
Intracellular cytokine stainingProliferating cells
Blank
staining
CBA
ELISAA ti
CD4/CD8 T-cell ELISPOT
Antigen-specific T cells
HLA class I tetramers
HLA class II tetramers
Antigen
slide 296-Oct-10
HLA class II tetramers
T-Cell Assays:
PBMC
Whole PBMC Format
Analyze T‐Cells
+ Antigen
D 1 7
PBMC
Day 0 Day 1‐7
Flow CytometryDissect cell subtypesMultiparameter approach
Proliferating cells
p ppSurface markersActivation markersFunctional assays
slide 306-Oct-10
Functional assays (proliferation, cytokine expression)
T-Cell Assays: DC/CD4+ Format
GM‐CSF IL4
Day 0 Day 4 5
1. Antigen2. Maturation cocktail
Day 0 Day 4‐5
Wash, count & incubate
Day 6‐7
count & incubate
1. CD14 isolation
2. CD4 isolation
Day 12‐15Readout
slide 316-Oct-10
Readout
Analyze T‐Cells
Analyze T-Cells: Ensure Quality
PARTICIPATE IN PROFICIENCY PANELS ELISPOT PROFICIENCY PANEL IV CVC-2009, REPORT FOR LAB 22, DATE: September 20, 2009
Reference value
slide 326-Oct-10
Screening StrategySSP-PCR Check by independent
reviewerHLA typingor InnoLiPa
50 PBMC preparation
reviewer
Check quality PBMC by polyclonal T-cell activation
assay
yp g
Donor
50 donor cell preps
Assay development
S iScreening
Peptides/proteins
Following project-Readout & data QC/QA f d t d
Flow cytometry ELISpot Technology platform
Using statistical program « R »
Following projectspecific flow
charts
Readout & data management
Data analysis
QC/QA of raw data and manipulations
slide 336-Oct-10
Final report
T-Cell Activation Assays: Population Analysis
slide 346-Oct-10
From single donor to population level. Compare/rank immunogenicity of test products (benchmark protein: KLH)
Case Study 1: Adalimumab
Human antibody recognizing TNF-α isolated by phage-display
Patients were tested for: HAHA response (low, high)
Determined from the binding of technology
109 RA patients enrolled for
the Humira Fab fragment to protein A absorbed patient IgG
DQ DR high resolution typingthe study (collaboration with Sanquin and Genmab)
DQ, DR high resolution typingNo DP typing was done as no strong epitopes were identified by Epibase™
slide 356-Oct-10
Immunoprofiling of Adalimumab
Epibase ProfilingEpitope identification on full sequenceRemoval of epitopes present in the human germlineRemoval of epitopes present in the human germlineCritical epitopes are identified as the strong and medium binders to DRB1, and the strong binders to DRB3/4/5, DQ and DP
7 Strong Epitopes Found5 strong epitopes in the VH
2 in the FwR2 HCDR2 region2 in the FwR2-HCDR2 region3 in the FwR3-HCDR3 region
2 strong epitopes in the VL:LCDR1 d F R3 LCDR3
slide 366-Oct-10
LCDR1 and FwR3-LCDR3
Patient Data
Level of HAHA Response19 patients show a HAHA response, i.e.17 6% of the patients are HAHA +17.6% of the patients are HAHA +
RA-associated HLA Allotypes:
Allotype Caucasian RA group
DRB1*0101 17.2% 28.4%
DRB1*0401 9.8% 52.3%
DRB1*0404 5.9% 9.2%
slide 376-Oct-10
Epitopes and HAHA ResponseHAHA+
Epitopes Region HLA allotypes HAHA+ patients
1 FwR2-HCDR2 DRB1*0701 1
2FwR2-HCDR2 DQA1*0201|DQB1*0303 1
The 7 strong epitopes explain 17/19 HAHA+
2 HCDR2 DQA1*0201|DQB1*0303 1 DQA1*0401|DQB1*0402 DQA1*0501|DQB1*0301 3 DRB1*0101 4 DRB1*0401 7
patients
Epitopes are directed against the DRB1*0405 1
DRB1*0407 DRB1*0901 1
3FwR3-HCDR3 DRB5*0101 5
directed against the RA associated allotypes
4 FwR3-HCDR3 DRB1*0407
5 FwR3-HCDR3 DRB1*0801
6 LCDR1 DQA1*0501|DQB1*0201 3
slide 386-Oct-10
6 LCDR1 DQA1 0501|DQB1 0201 3
7 FwR3-LCDR3 DRB5*0101 5
Case Study 2:Ofatumumab and Rituximab
Targeting CD20, a B-cell Differentiation Antigen
Observed Immunogenicity of Rituximab<1% in B-CLL
Treatment of Cancer: e.g. Follicular lymphoma.
1% in B CLL35-60% in SLE4.3-23% in RAChimeric antibody
y pInflammatory disease: e.g. Rheumatoid arthritis, SLE
Chimeric antibody
OfatumumabPh III t i l i B CLLPhase III trial in B-CLLPhase II in RAFully human antibody
slide 396-Oct-10
Immunoprofile: Ofatumumab and Rituximab
Strong Epitopes
12
16HuMax-CD20 Rituximab
Medium Epitopes
30
40
50Ofatumumab is very clean in epitopes as compared to rituximab
0
4
8
DRB1 DRB3/4/5 DQ DP 0
10
20Ofatumumab contains no epitopes for HLA allotypes associated
HLA class II gene RA Risk ratio Epitopes in Epitopes in
DRB1 DRB3/4/5 DQ DPDRB1
ypwith RA
rituximab ofatumumabDRB1*0401 1 in 35 2 strong no
DRB1*0404 1 in 20 no no
slide 406-Oct-10
DRB1*0101 1 in 80 4 strong no
0401 and 0404 1 in 7 2 strong no
Thank you for your attention
LonzaApplied Protein Services
Technologiepark 4Technologiepark 4B-9052 Gent-Belgium
Tel.: +32 (0) 9 241 11 00Fax: +32 (0) 9 241 11 02
http://www immunogenicity com
slide 416-Oct-10
http://www.immunogenicity.com