BPI Lonza October 2010 - bioprocessintl.com · r OKT3 murine CD3 Graft rejection ELISA 54 82 +...

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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

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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

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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

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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



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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

According to the EMEA …

slide 96-Oct-10

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

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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.


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


trials

Epibase IV™Epibase™


Epibase IV™Epibase™


y


Generating a risk profile of the potential immunogenicityAnalysis of the probability to observe immunogenicity

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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

Immune Response

slide 156-Oct-10 Winslow, 2001

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

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T-cell epitopes: Peptide sequence of the protein needed for effective T-cell help

Epibase™ In Silico Immunoprofiling

leaddiscovery

leadselection

leadoptimization

process development


trials

Epibase™ Epibase IV™



In vitro T-cell Assaysy


T-cell Epitope PredictionLead ranking and selectionD i i ti i bi ti ith th t f t i

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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

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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

Comparison

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21 April 18 2007

Van Walle et al, Expert Opin. Biol. Ther., 7(3) 2007

Epibase™ In Vitro Immunoprofiling

leaddiscovery

leadselection

leadoptimization

process development


trials





y


Characterize T-Cell Epitopes Guided by In SilicoAt individual donor and population level

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leaddiscovery

leadselection

leadoptimization

process development


trials





An Indication of External Factors on Drug ImmunogenicityF l ti

y


FormulationAggregatesDegradation products

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Production contaminants


leaddiscovery

leadselection

leadoptimization

process development


trials





C i f I i it b t F ll /S d

y


Comparison of Immunogenicity between Follow-on /Second-Generation Products and Reference ProductsHealthy population/Patient population

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T-Cell Activation Assays: PBMC from Healthy Individuals / Patients

leaddiscovery

leadselection

leadoptimization

process development


trials





y


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

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Donor Population

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T-Cell Activation Assays:Different Read Out Parameters / Assay Formats

leaddiscovery

leadselection

leadoptimization

process development


trials





y


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

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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

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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

Send questions to:

BPI Seminars@propelmg [email protected]

BPI Lonza October 2010 - bioprocessintl.com · r OKT3 murine CD3 Graft rejection ELISA 54 82 +...

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