Protein Aggregation and Immunogenicity

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Protein Aggregation and Immunogenicity

Protein Aggregation and Immunogenicity

Critical Role of the Formulation

Wim Jiskoot

Division of Drug Delivery Technology

Leiden/Amsterdam Center for Drug Research (LACDR)

PSWC-AAPS

New Orleans

17 November 2010


Practically all proteins are immunogenic

Proteins of animal origin (> 1920s)

e.g. porcine/bovine insulin

• Human derived proteins (> 1950s)

growth hormone, factor VIII

• Recombinant human proteins (> 1980s)

insulin, interferons, GM-CSF, epo

• Monoclonal antibodies (> 1980s)

mouse, chimeric, humanized, human

• Follow-on biologics / biosimilars (> 2000s)

growth hormone, epoetin, filgrastim ...

Impurities,

contaminants

Product quality


Immunogenicity - definition

T cell

activation

Innate immunity

CDC

(complement

dependent

cytotoxicity)

Cell mediated

cytotoxicity

The ability of a

substance (e.g. antigen

or vaccine) to elicit an

immune response

Hypersensitivity

ADCC

(antibody-dependent cellmediated

cytotoxicity )

Cytokine

storm

Anti-drug antibodies


Immunogenicity - definition

T cell

activation

Innate immunity

CDC

(complement

dependent

cytotoxicity)

Cell mediated

cytotoxicity

The ability of a

substance (e.g. antigen

or vaccine) to elicit an

immune response

Hypersensitivity

ADCC

(antibody-dependent cellmediated

cytotoxicity )

Anti-Drug Antibodies

Cytokine

storm

Anti-drug antibodies


Clinical consequences of immunogenicity vary and are

highly unpredictable

Loss of therapeutic efficacy (common)

IFN-beta, IFN-alfa, anti-TNF proteins, insulin, monoclonals…

Increase of therapeutic efficacy (rare)

growth hormone

Neutralization of endogenous protein (rare)

epoetin, MDGF

General immune effects

allergy, anaphylaxis, serum sickness, etc.

None (common)


Formation of anti-drug antibodies in patient receiving

an anti-TNFα MAb

MAb conc. (ng/ml)

10000

1000

100

30

20

10

Anti-MAb (% binding)

10

0

0 10 20 30 40 50 60

Week

Courtesy of Lucien Aarden


Factors influencing immunogenicity of proteins


Factors influencing immunogenicity of proteins

So, THE immunogenicity of a protein does not exist

Product Characteristics Indication Immunogenicity (%)

Rituxan/rituximab chimeric/ CD20 NHL 0

Rituxan/rituximab chimeric / CD20 SLE 65

Rituxan/rituximab chimeric / CD20 PSS 27


Factors influencing immunogenicity

Product Quality

&

Formulation


Simplified summary:

protein aggregates are immunogenic


Aggregates and immunogenicity: clinical evidence

• Intravenous immunoglobulins (1950s, 1960s)

– Aggregate removal resulted in less immunogenic products

• Human growth hormone purified from formalin-fixed

pituitary glands (1950s)

– The higher the aggregate levels (up to 70%!) the more

immunogenic

• Betaseron

– Contains substantial aggregate levels and is more immunogenic

than other interferon-beta products

• Interleukin 2

– Aggregated formulation induced antibodies in most patients

• Interferon-alfa

– Formulation with oxidized and aggregated products was more

immunogenic than other formulations

Hermeling et al., Pharm Res, 2004; Rosenberg, AAPS J, 2006


Antibody response to human growth hormone: aggregate

level determines antibody response and persistence

(Moore and Leppert 1980)

% 125 I-hGH Bound

50

40

30

Highly aggregated HGH

Minimally aggregated HGH

0.28

0.24

0.20

0.16

0.12

0.08

0.04

0

40

60

Monomer

80

100

20

10

0.28

0.24

0.20

0.16

0.12

0.08

0.04

Monomer

0

3

6 9 12

Months of therapy

15

28

0

40

60 80 100

Courtesy of Amy Rosenberg


Immunogenicity: the formulation matters

Neutralizing antibodies

Immunogenicity differences between IFN-a2 formulations

2000

1800

1600

1400

1200

1000

800

600

400

200

0

0 1 2 3 4 5 6 7 8

Duration of treatment (months)

B (n = 86)

C (n = 110)

D (n = 81)

E (n = 74)

A (n = 190)

Ryff, J Interferon Cytokine Res, 1997


Immunogenicity: the formulation matters

Reformulated REBIF (interferon beta) shows reduced

immunogenicity in phase IIIb clinical trial

260 patients - persistent neutralizing antibodies:

Old = 14%, high titers, 58% relapse free

RNF2 = 2.5%, low titers, 67% relapse free

Courtesy of Matthew Baker


Immunogenicity: the formulation matters

Number of Epoetin Alfa

PRCA Cases

Anti-epoetin antibody-related pure red cell aplasia

Formulation change

Replacement of albumin by Tween 80/glycine in epoetin alfa (outside USA)

80

70

60

50

40

30

20

10

0


Why are recombinant human proteins immunogenic

Simpliflied scheme of the immune system


M. Salmon, 1994

Courtesy of Matthew Baker


Fate of auto-reactive B cells after encountering

self-protein arrays on virus like particles (VLPs)

Monomeric B-cell receptor /

self-Ag complexes

Oligomerization of B-cell receptor /

self-Ag signaling complexes

Toleragenic Signals

Proliferative Signals


Any substance can be rendered immunogenic!

Soluble molecules:

B-cell tolerance

Repetitive epitopes on polymeric or

particulate drug carriers: B-cell stimulation

Prerequisites:

• At least 10-20 repetitive epitopes ()

• Inter-epitope distance between 5-10 nm

Jiskoot et al., Pharm Res 26: 1303-1314 (2009)


B-cell stimulation by epitope arrays

Wouldn’t protein aggregates act similarly

= protein molecule

And particles with adsorbed proteins

= protein molecule

Poliovirus

FMD Virus


Protein immunogenicity – our current knowledge

• Practically all rh therapeutic proteins and antibodies are

immunogenic

• We do not exactly know why

• Besides patient- and treatment-related factors,

formulation and product quality are important

• Aggregates are suspicious as risk factors

• We do not know which aggregates

• We do not know the critical aggregate dose


Protein immunogenicity – our current knowledge

Product quality

Formulation and

characterization tools,

PAT, QbD, …

Clinical immunogenicity

Immunogenicity assays

THE GAP:

Predictive models to link the two;

understanding immune mechanisms


Tools to predict immunogenicity

• Process / formulation characterization

(including aggregates, particles…)

• Animal studies - transgenics

• In silico B-cell prediction

• In silico T-cell prediction

• T-cell epitope screening assays

uncapable of

predicting

formulationrelated

factors

• T-cell activation assays

• Clinical studies

• Postmarketing surveillance / pharmacovigilance


Tools to predict immunogenicity

• Process / formulation characterization

(including aggregates, particles…)

• Animal studies - transgenics

• In silico B-cell prediction

• In silico T-cell prediction

• T-cell epitope screening assays

• T-cell activation assays

THE MAIN CHALLENGE NOW:

• Clinical How studies to bridge the gap

• Postmarketing surveillance / pharmacovigilance


Tools to predict immunogenicity

Transgenic immune tolerant mouse

models to correlate

Product Quality & Formulation

to

Immunogenicity


Human interferon-a2 immune tolerant mice

IgG Titer

100000

rhIFN a2b

ovalbumin

10000

1000

Titers on

day 22

100

10

Wildtypes

Transgenics

Week 1 Week 2 Week 3

Day 1 Day 8

Day 15

Day 22

5 μg / injection

Blood sampling

(s.c. or i.p.)


Preparation of aggregated rhIFNa2

optical density

Fluorescence intensity

OD 214 nm

SDS-PAGE (non-reducing)

HPLC-SEC

0.20 Native

Hydrogenperoxide

0.15

Metal-catalyzed

Glutaraldehyde

0.10

Boiled

0.05

0.00

10 20 30 40

Time (min)

0.30

0.25

0.20

0.15

0.10

0.05

UV/Vis spectroscopy

Native

Hydrogen peroxide

Metal-catalyzed

Glutaraldehyde

Boiled

Trp fluorescence spectroscopy

3.0×10 7 Native

Hydrogenperoxide

2.0×10 7

Metal-catalyzed

Glutaraldehyde

1.0×10 7

Boiled

0.00

200 250 300 350 400 450

Wavelength (nm)

0

300 325 350 375 400 425 450

Emission wavelength (nm)

Hermeling et al., Pharm Res 22, 1997-2006 (2005)


Only “native like” aggregates induce antibodies against native

recombinant human IFNa2a in transgenic mice

anti-rhIFNa2b IgG titer

1000

t=14 days t=21 days

100

10

5/5

4/5

N M G H B

N= untreated

M= metal-catalyzed oxidized

G= glutaraldehyde treated

H= oxidized by H 2 O 2

B= boiled

Hermeling et al., Pharm Res 22, 1997-2006 (2005)


Immunogenicity of “native like” aggregates of recombinant

human IFNa2a in transgenic mice is dose dependent

IgG titer

b

Wildtype

Transgenic

10000

1000

**

***

***

5/5

4/5

5/5

100

2/5

10

ND

A B C D E

Increasing aggregate content

Hermeling et al., J Pharm Sci 95, 1084-1096 (2006)


Memory study - injection and blood sampling scheme

Week 1 Week 2 Week 3

t = 0 t = 7 t = 14 t = 21 days

… Week 9

Week 4

Wash out

Week 10 Week 11

Boost

Blood sampling

Injection (i.p.) with

5 μg protein


Transgenic immune tolerant mice do not show immunological

memory against aggregated rh IFNa2a

Anti-rhIFNa2a antibody titers before and after a

6-week wash-out period

Sauerborn et al. (2010) Manuscript in preparation


Antibody response against aggregated rhIFNa2a is T cell

dependent – in both wildtype and transgenic mice

Anti-rhIFNa2a antibody titers after a 3-week

injection protocol

– CD4 + T cells

depleted

+ undepleted

Sauerborn et al. (2010) Manuscript in preparation


Antibody response against aggregated rhIFNa2a is T cell

dependent – in both wildtype and transgenic mice

Anti-rhIFNa2a antibody titers after a 3-week

injection protocol

Pneumovax

(TI antigen)

– CD4 + T cells

blocked

+ not blocked

Sauerborn et al. (2010) Manuscript in preparation


Aggregated rhIFNb does not act as an adjuvant for native

rhIFNa2a in transgenic mice

Anti-rhIFNa2a antibody titers after a 3-week

injection protocol

rhIFNb

Sauerborn et al. (2010) Manuscript in preparation


IgG titer

IgG titer

Adsorption of rhIFNb to metal (but not glass or polystyrene)

beads enhances its immunogenicity in transgenic mice

Anti-rhIFNb antibody titers after a 3-week injection

protocol

10000

10000

p


Conclusions from studies with transgenic mice

Transgenic immune tolerant mice are useful models to

study product-related factors of immunogenicity:

• Some aggregates were shown to be immunogenic, others not

Immunogenicity of aggregates is dose dependent

Protein adsorption to non-proteinaceous subvisible particles can

promote antibody formation

• Immune mechanism differs from a classical vaccination reaction:

- No memory

- CD4 + T-cell dependent


Proposed mechanisms of antibody induction by

protein aggregates

V. Filipe et al. In: Aggregation of Therapeutic Proteins (W. Wang and C.J. Roberts, Eds.),

John Wiley & Sons, Hoboken, NJ, pp. 403-433 (2010)


Protein immunogenicity – wrap up

Still work to do!


Acknowledgments

The immunogenicity team

Miranda van Beers

Melody Sauerborn

Suzanne Hermeling

Vasco Filipe

Riccardo Torosantucci

Andrea Hawe

Ruediporn (Sun) Tantipolphan

Vera Brinks

Basak Kukrer

UIPS

Utrecht Institute for

Pharmaceutical Sciences

Ted Randolph

Jared Bee

Daan Crommelin

Huub Schellekens

And may others – students, technicians, collaborators…


Thank you !

Hope to see you again at the

Third Open Scientific EIP Symposium

Immunogenicity of Biopharmaceuticals

8-9 December 2010, Gent, Belgium

Info & registration: http://e-i-p.eu


Epitope presentation in protein aggregates

Native

protein molecules

V. Filipe et al. In: Aggregation of Therapeutic Proteins (W. Wang and C.J. Roberts, Eds.),

John Wiley & Sons, Hoboken, NJ, pp. 403-433 (2010)


IgG titer

Transgenic immune tolerant mice do not show immunological

memory against aggregated rhIFNb

Anti-rhIFNb antibody titers before and after a 6-week

wash-out period

100000

10000

1000

100

10

Before After Before After

Wildtype

Transgenic

Cf clinical data: Perini et al., (2001)

Millonig et al. (2009)

Van Beers et al.

Pharm Res 27: 1812–1824 (2010)


Antibody response against aggregated rhIFNb is T cell

dependent – in both wildtype and transgenic mice

Anti-rhIFNb antibody titers after a 3-week injection

protocol

– CD4 + T cells

depleted

+ undepleted

Sauerborn et al. (2010) Manuscript in preparation


Switching from IFNβ-1b to IFNβ-1a

• Decrease in BAB levels after switch to Avonex®

• No immunological memory

Total

IgG

3 months wash-out

(Betaferon®)

(Avonex®)

Patient A B C D

Perini et al. 2001

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