pre-conference eip immunogenicity course · • 13:30 neutralizing antibodies, emerging trends in...

EIP Immunogenicity course

Pre-conference EIP immunogenicity course

Wireless: tivolihotel


EIP background• The European Immunogenicity Platform acts as a central

meeting place for European biopharmaceutical companies and scientific experts active in the field of immunogenicity.

• Its scope is: • To build a broad knowledge-base related to

immunogenicity, and to stimulate interactions and developments in the broad field of immunogenicity

• Through its working-group structure, the EIP can react in a focused way on regulatory and scientific evolutions in the immunogenicity-field

• This is the first EIP introduction course to the field of immunogenicity


Pre-conference Immunogenicity course - Agenda• 10.00 Course introduction. Immunogenicity a growing field within basic

research and drug development. Basic introduction.• Christian Ross, Novo Nordisk• 10:30 The immunology and major mechanisms affecting immunogenicity• Huub Schellekens, University of Utrecht• 11:30 Binding antibodies, Assay methodologies, Comparison of different

methods, Screening Confirmation, Characterization of anti-drug-antibodies• Daniel Kramer, Merck-Serono• 12:30 Lunch break• 13:30 Neutralizing antibodies, Emerging trends in the development of Nab

assays, Clinical implementation of an established Nab assay• Arno Kromminga, IPM Biotech• 14:30 Predictive Immunogenicity tools, different methods, are they predictive,

guideline status on predictive tools• Philippe Stas, Lonza• 15:30 Critical review of previous immunogenicity issues: Interferons, EPO,

Coagulation factors and mAbs. Learnings and future perspectives from research to clinical consequence.

• Daniel Kramer, Christian Ross, Huub Schellekens


”Immunogenicity”:

The ability of a substance to provoke an immune response or the degree to which it provokes a response

(medical dictionary)

.


Traditional antibody response


Not always as single Ig-molecules


Two fundamental terms


B cell development


Ag+

Danger

maturation

Mature DC

Steady State Inflammation:

Immature DC take up Ag

Ag release

Lymph Node

Treg

Tr

Tr

CD4CD8

Maintenanceof

tolerance

Lymph Node

CD4 (autoreactive IL-17)

CD8

Loss of tolerance

Th2/FH B

Adapted from Roncarolo 2007


Clinical consequence of ADA?

Insulins

EPO

G-CSF

Interferons

mAbs

Koag. factors

MGDF


Formulation changes can induce significant

immunogenicity of protein drugs


Clinical aspect: PRCA related to EPO antibodies


In contrast anti-insulins are frequent but not dangerous Schnabel 2006


Planning of the immunogenicity program

RISK BASED APPROACH

LIKELIHOOD CONSEQUENCE


RISK BASED APProach (EPO)

LIKELIHOOD

Consequence


RISK BASED APProach (Insulin)

LIKELIHOOD

Consequence


BackgroundReported clinical immunogenicities of

therapeutic mAbs

Source of data: Baker et al. ,Landes Biosci. 2010

0 10 20 30 40 50Muromab

ArcitumomabFanolesomab

ImciromabCapromab

IbritumomabTositumomabNofetumomab

AbciximabRituximab

BasiliximabInfliximab

CetuximabDaclizumab

TrastuzumabPalivizumab

CanakinumabGemtuzumab

CampathOmalizumab

EfalizumabBevacizumabRanibizumab

EculizumabNatalizumab

Cetolizumab pegolOfatumumabTocilizumabAdalimumab

PanitumumabGolimumab

UstekinomabDenosumab

Murine

HumanizedChimeric

Human

Reported immunogenicity

Ther

apeu

tic a

ntib

ody Significant clinical

immunogenicity occurs for many mAbs


RISK BASED APProach (mAbs)

LIKELIHOOD

Consequence


The immunology and major mechanisms affecting immunogenicity

• Dr. Huub Schellekens, professor of Pharmaceutical Biotechnology, Utrecht University.

• Teaches Medical Biotechnology at the Department of Innovation Studies and has a research position at the Faculty of Pharmaceutical Sciences.

• Member of the Dutch Medicine Evaluation Board and National Expert of the European Medicine Agency and also a member of the Board of the European Immunogenicity Platform.

• Medical microbiologist by training and works on the preclinical development of biopharmaceuticals.

• Published more than 300 papers in peer reviewed international journals concerning many aspects of the development of therapeutic proteins. During recent years his work has included the immunogenicity of protein drugs and the problems related to biosimilars.


Binding antibodies, Assay methodologies, Comparison of different methods, Screening Confirmation, Characterization of anti-drug-antibodies

• Dr. Daniel Kramer, Ph.D• Corporate Expert Immunogenicity,• Merck Serono Research and Development, EIP board member• Institute of Drug Metabolism and Pharmacokinetics • Published significantly in the field of immunogenicity and numerous

high quality lectures in the field


Patient samples taken at appropriate time-points

Screening AssayNeg. samples rejected Positive samples

Confirmatory Assay

Neutralization assayor Functional assay

Confirmed positive samples Characterization

Assess correlation of characterized antibodieswith clinical responses to biological therapeutic

Assays for clinical markers and assessment of clinical response in patients

Antibody Detection and Characterization


Neutralizing antibodies, Emerging trends in the development of Nab assays, Clinical implementation of an established Nab assay

• Arno Kromminga CEO at GLP-certified IPM BIOTECH, Hamburg, Germany. • Studied Biochemistry and Immunology and has more than 15 years of

experience in assay development and validation. • Additional position as Head of Immunology at the Clinical Laboratory

Lademannbogen, Hamburg, and faculty member of the University of Kiel. • Co-founder of the European Immunogenicity Platform (EIP) and a member of

several scientific societies. • He is the author of multiple publications in peer-reviewed journals and books.

He has gained substantial industrial experience in various Biotech companies and has been involved in the development and establishment of novel immunological and cellular assays for immunogenicity testing and PK/PD analyses for pre-clinical and clinical studies. Most recently, he extended his activities at IPM BIOTECH to functional assays such as ADCC/CDC and apoptosis.


Patient samples taken at appropriate time-points

Screening AssayNeg. samples rejected Positive samples

Confirmatory Assay

Neutralization assayor Functional assay

Confirmed positive samples Characterization

Assess correlation of characterized antibodieswith clinical responses to biological therapeutic

Assays for clinical markers and assessment of clinical response in patients

Antibody Detection and Characterization

Neg. samples ?? !!

Positive samplesAction needed !!!


Predictive Immunogenicitytools, different methods, are they predictive, guideline status on predictive tools

• Philippe Stas, Head, Applied Protein Sciences, Lonza, UK• From 1990 to 1997, he attended the Free University of Brussels (Belgium)

where he conducted applied research for projects involving antibody engineering and bioinformatics.

• Co-founder of Algonomics in 1999, where he was in charge of operations and business development.

• Since Lonza’s acquisition of Algomonics in 2009, Philippe has been leading the Applied Protein Services group in Cambridge, UK and Gent, BE. Philippe Stas holds an MBA, a M.Sc.E. in biotechnology and a M.Sc. in Information Technology.

• Chairman of the Belgian Association for Bioindustries (Bio.be) and President of the European Immunogenicity Platform (EIP).


Preclinical immunogenicity

testing

From the EMEA workshop on Immunogenicity, London, 2007


IFNEPO


Different tools for immunogenicity prediction

• In silico prediction of MHCII-peptide binding

• MHCII-peptide binding assays

• In vitro T-cell assays


Indication of correlation between in vitro T-cell response and clinical immunogenicity reported by Antitope and others

Baker et al. 2008


Once again take home message:

”PROTEIN DRUGS ARE

IMMUNOGENIC”


Vussow and Borden 1989• Clinical research has defined the effectiveness of interferons (IFNs)

in both viral and neoplastic diseases.• Attention has been paid not only to the efficacy of IFNs, but also to

their clinical safety and side effects.• It was anticipated initially that IFN, as a human protein, would not be

antigenic when administered to the human host. • In early clinical trials, there was little evidence of antigenicity;

however, with broader trials, steps were taken to assess any antibody response that a patient might develop.

• As more sensitive techniques have been applied over the past 7-8 years, it has become apparent that a minority of patients develop an

• antibody specific to human IFN.• No commonly agreed methodologies or definitions of titer for

antibody to IFNs exist at the present time.


Hochuli 1997• Studies with interferon-alpha 2a (IFN-alpha 2a) have shown the

presence of neutralizing antibodies in a proportion of patients. • A number of technical aspects of its production and storage were

investigated• Presence of both interferon-interferon (IFN-IFN) aggregates and

aggregates of interferon with human serum albumin (HSA), the excipient of the galenical form of IFN-alpha 2a (IFN-HSA) aggregates.

• The amount of aggregates was temperature dependent. The relative immunogenicity of IFN-alpha 2a increased when the vials were stored at ambient temperature but not when stored at 4 degrees C.

• These findings demonstrate that the immunogenicity of IFN-alpha 2a is likely to be related to the storage temperature. Storage of IFN-alpha 2a vials at 2-8 degrees C is now recommended. A new formulation has been introduced that does not contain HSA as an excipient, removing the possibility of IFN-HSA aggregation.


Development and specificities of anti-interferon neutralizing antibodies in patients with chronic hepatitis C treated with

pegylated interferon-Scagnolari et al 2011

Clinical Microbiology and Infectionpages no-no, 22 DEC 2011 DOI: 10.1111/j.1469-0691.2011.03729.xhttp://onlinelibrary.wiley.com/doi/10.1111/j.1469-0691.2011.03729.x/full#f2


Scagnolari – conclusion - 2011

• The results indicate that a change to LE-IFN-therapy can be associated with restoration of clinical responses in NAB-positive patients who had become resistant after showing an initial response to PEG-IFN- treatment.

• This study emphasizes the importance of evaluating NAB development in CHC patients who become resistant to PEG-IFN- treatment, and suggests management alternatives for patients who develop NAB.


Millela et al. hepatitis, 1998

0

20

40

60

80

100

120

0 3 6 9 12

Time (months)

Vir

em

ia

Init.Resp. Non Cont.resp.


Remember statistics !

Responders

NON-responders


Remember statistics !

Responders

NON-responders

NAb-pos NAb-neg


Route of administration does affect Immunogenicity of different IFN-beta

preparations in MSThis image cannot currently be displayed.This image cannot currently be displayed.

Time (Months)0 3 6 9 12 15 18 21 24 27 30 33 36

Num

ber o

f Ab-

posi

tive

pts.

(%)

0

20

40

60

80

100

IFN-ß1A (Rebif, 1.5MIU x1IFN-ß1B IFN-ß 1A (Rebif, 6MIU x 3)IFN-ß 1A (12MIE x 3) Placebo IFN-ß 1A (Avonex)IFN-ß 1A (Rebif, 6MIU x 3)


Clinical consequence of Nabs ?This image cannot currently be displayed.This image cannot currently be displayed.This image cannot currently be displayed.

Time (Months)0 3 6 9 12 15 18 21 24

Bin

ding

(%)

0

10

20

30

40

50

60

70

80

Neu

tral

izat

ion

(%)

0

20

40

60

80

Binding 1IU/ml 3IU/ml 10LU/ml


Clinical consequence of Nabs ?This image cannot currently be displayed.This image cannot currently be displayed.This image cannot currently be displayed.This image cannot currently be displayed.

Time (Months)0 3 6 9 12 15 18 21 24

Bin

ding

(%)

0

10

20

30

40

50

60

70

80

Neu

tral

izat

ion

(%)

0

20

40

60

80

Dis

soci

atio

n (h

ours

)

0

5

10

15

20

25

30

35

Binding 1IU/ml 3IU/ml 10LU/ml Diss.


Significant variation between laboratories and assaysThis image cannot currently be displayed.This image cannot currently be displayed.This image cannot currently be displayed.

Protein-G binding capacity (%)

0 20 40 60 80 100

Ant

i-IFN

-ß E

IA (T

iter)

050

100150200250300350400450

High

HighLowLow

Ross et al


NAb frequency depending on definition


Relapse odds ratio: Ab pos/neg


Relapse free vs nAb

Soelberg-S et al 2003


Anti-IFN-beta NAbs affect clinical activity !


HAMA, HACA, HAHA – Mean frequency

(Hwang et al 2005)

0

10

20

30

40

50

60

70

AAR

HAMA n=44HACA n=16HAHA n=22


Reported clinical immunogenicities of therapeutic mAbs

Baker et al. , Landes Biosci. 2010

0 10 20 30 40 50Muromab

ArcitumomabFanolesomab

ImciromabCapromab

IbritumomabTositumomabNofetumomab

AbciximabRituximab

BasiliximabInfliximab

CetuximabDaclizumab

TrastuzumabPalivizumab

CanakinumabGemtuzumab

CampathOmalizumab

EfalizumabBevacizumabRanibizumab

EculizumabNatalizumab

Cetolizumab pegolOfatumumabTocilizumabAdalimumab

PanitumumabGolimumab

UstekinomabDenosumab

Murine

HumanizedChimeric

Human

Reported immunogenicity

Ther

apeu

tic a

ntib

ody Significant clinical

immunogenicity occurs for many mAbs


Natalizumab: humanized monoclonal antibody against

alpha-4 ( 4) integrin (Calabresi 2007)


Natalizumab: humanized monoclonal antibody against alpha-4 ( 4) integrin (Calabresi 2007)


Drug concentration vs HACA,Aarden et al 2008, Curr Op Immunol


De Vries 2009, Ann. Rheum., Adalimumab in ancylosing spondylitis


Copyright ©2001 American Association for Cancer Research

Ritter, G. et al. Cancer Res 2001;61:6851-6859

Fig. 2HAHA response pattern of patients with colon cancer after

treatment with huAb A33, (Ritter et al 2001)


Infliximab phase 1a+B, Elliott et al, Lancet, 1994,

• In phase 1A, 20 pts. 1Ab pos after SD• In phase 1B, 4 patients had no HACA responses when

tested at least 6 weeks after the last infusion. • The remaining 4 patients developed HACAs at varying

times after retreatment (titres 10, 20, 80, and 640 in patients 3, 1, 5, and 9, respectively), all specific for the murine variable region of cA2. Of these, 2 patients completed all four cycles, 1 completed two cycles, and 1 was withdrawn during cycle 2.

• Some patients with HACAs showed a reduction in response duration in cycles 2-4.


Infliximab, 26 week, 101 RA pts, W 0,2,6,10,14 – HACA measured 12 weeks later,

Maini et al 1998, Arthr Rheum

0

10

20

30

40

50

60

1mg 3mg 10mg

Monother.+MTX


Anti-Antibody Responsesare important

Inverse relationship between drug levels and AAR

Response failure related to AAR in subgroups of patients

AAR persistant vs transient

Concomitant medication and dosing can affect AAR

AAR rarely related to severe adverse events


Antibody-mediated PRCA per patient-years (rate per 100,000 patient years) for different EPO products


Conclusion EPO• Treatment with subcutaneous rhEPO may induce an immune

response and lead to the formation of anti-EPO antibodies. • These antibodies may in rare cases lead to the clinical manifestation

of PRCA • The rise of EPO-induced PRCA cases in the period of 1998–2004

has made health authorities, manufacturers and clinicians aware of the risk of adverse immune reactions and the importance of the analysis of anti-EPO antibodies.

• Reliable and sensitive screening assays for the detection of anti-EPO

• antibodies are available, although attempts to generate a common calibrator

• for those assays are still not successful. • The neutralizing capacity of anti-EPO antibodies has been shown by

a cellular assay using cell lines or bone marrow derived cells that are unequivocally dependent on EPO.


Conclusion Insulin• Protein drugs such as insulin are immunogenic.

The development of insulin antibodies with the use of subcutaneous administration appears to be common, although the antibody level varies dependent on factors such as:

• the preparation, route of administration and age of the patient.

• Despite initial increases in antibody formation, there appears to be no consistent relationship between antibody formation and glycaemic control or between antibody formation and safety in terms of adverse events.


CONCLUSIONS:•The clinical consequence of anti-drug antibody development varies significantly based on the antigen

•The clinical consequence of drug induced antibodies is not well defined

•A risk based approach is requested but not validated

•Short term demonstration of only bAbs does not exclude the long term development of nAbs

•Be careful in study design

•Optimize antibody analyses in order to make the correct conclusions

•Lessons can be learned from previous studies

pre-conference eip immunogenicity course · • 13:30 neutralizing antibodies, emerging trends in...

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