sebastian spindeldreher novartis institutes for biomedical research / dmpk-biologics aggregation of...

Sebastian SpindeldreherNovartis Institutes for Biomedical Research / DMPK-Biologics

Aggregation of Human Recombinant Monoclonal Antibodies Enhances Their Presentation by Dendritic Cells In Vitro

7th Open Scientific EIP Symposium, Lisbon, February 24, 2015

Aims of this study

Do proteinaceous SVP have increased immunogenic potential?

Why (mechanism)?

Which factors influence immunogenicity:• Size?

• Type of stress/structure?

2 | EIP Symposium | S. Spindeldreher | Feb 24, 2015

Acknowledgements

DC data:

Andrea Kiessling

Babette Wolf

Jennifer Krieg*

Stewart Jones*

T cell data

Aida Baban

MAPPs data

Anette Karle

Sascha Gottlieb

Stephan Koepke

Laetitia Sordé

Xavier Charles Leber

Steffen Hartmann

Aggregates generation and phys/chem characterization:

Verena Rombach-Riegraf

Atanas Koulov*

Bahman Ossuli

Dolores Tinhan

Philippe Simeoni

Marie-Claude Djidja

Kamal Egodage

Markus Blümel

Margit Jeschke

Maria Brauchle

* Left Novartis


Selected stress conditions

2 model antibodies: mAb1 and mAb2 (terminated projects)

Both IgG1 subclass targeting soluble plasma proteins

Very different biophysical properties from one another with regard to isoelectric point, melting temperature, surface hydrophobicity, colloidal stability

Three stress conditions• Heat/shake (HS)

- Unstressed

- Stress level 1 (sl1) 10 min at 65°C and 1400 rpm

- Stress level 2 (sl2) 6 min at 80°C and 1400 rpm

• Shear stress (S)- Unstressed

- Stress level 1 (sl1) draw/empty syringe once

- Stress level 2 (sl2) draw/empty syringe four times

• Freeze Thaw (FT)- Unstressed

- Stress level 1 (sl1) 3 FT cycles

- Stress level 2 (sl2) 10 FT cycles


Applied technologies

Phys/Chem analytics• Microflow Imaging

• Size Exclusion Chromatography

• Dynamic Light Scattering

• Capillary Electrophoresis Sodium Dodecyl Sulfate

• LC/MS peptide mapping

Biological assays:• DC activation assay

• MAPPs assay

• T cell assay


Particle size distributionDetermined via Microflow imaging

unstressed FT stress level 1 FT stress level 2 unstressed FT stress level 1 FT stress level 2mAb1 mAb2

mAb1 mAb2HS

FT

S


Immune cell interplay

T-cell assay

MAPPs

DC-assay


DC maturation assayAssay procedure

stain

HealthyBlood Donor

Buffy Coat PBMC Monocyte ImmatureDendritic Cell

GM-CSFIL-4

5d

Ficoll

CD14 isolationwith magnetic

beads

drug

Maturation

3dCD86

CD83CD80

CD11c

72h maturation cocktail72h medium control72h DPBS

cytokines


Induction of DC maturation by stressed mAb materials

Response index: response relative to unstressed condition


Relative number of positive responder with response index at least 1.5


Identification of naturally processed HLA peptides MHC-associated Peptide Proteomics (MAPPs)

Cell isolation

Differentiation

Maturation

Immuno-precipitation

Washing and peptide elution

Nano LC-MS and database

search

mAb

Monocyte

ImmatureDC

Mature DC

mass spectraBuffy Coat

peptide


MAPPsexample: clusters in human interferon beta

1 111

2 11 1

1 1

Donor 1Donor 2Donor 3Donor 4Donor 5Donor 6Donor 7Donor 8Donor 9Donor 10Donor 11Donor 12Donor 13Donor 14

Cluster Cluster

Different Peptides

One Donor

Heat map of different peptides


Peptide clusters presented on DCs from heat stressed mAb1 and mAb2

Donor HS 1Donor HS 2Donor HS 3Donor HS 4Donor HS 5Donor HS 6Donor HS 7Donor HS 8Donor HS 9






140 160 180120 380 400320 340 360260 280 300200 220 2400 20 420

sl1

sl2

HS

mA

b1

he

avy

ch

ain

un

HS

mA

b2

he

avy

ch

ain

un

sl1

sl2

0 20 360 380300 320 340240 260 280180 200 220120 140 16060 1600 20 40 60 80 100 120 140400 180

40 60 80 100

mA

b2

lig

ht c

ha

in

80 10040

0 20 40 60

420

mA

b1

lig

ht c

ha

in

180 200100 120 140 16080

color coding

number of different peptides 0 1 5 10 15 20 25 30 35 40 45 >50


Naturally processed peptides: MAPPs assay Number of different peptides and clusters

mAb1 mAb2


Correlation of peptide presentation and protein amount in particles

r2 = 0.994

r2 = 0.993

r2 = 0.86

r2 = 0.943

Linear regression analyses of increase of HLA-DR associated peptides/clusters as functions of the calculated amount of protein present in subvisible particles.


NaïveCD4+

T cell

APC

ActivatedCD4+

T cell

ActivatedCD4+

T cell

ActivatedCD4+

T cell

T cell proliferation

T cell expansion(7 days)

T cell assay

Aim: Link to other studies that show enhanced T cell activation

PBMCs prepared from buffy coats of healthy human donors by density gradient centrifugation.

On day 0, challenge with different preparations of biotherapeutics at 200 mg/mL.

IFN-y ELISpot after 7 days in culture

Limited no. of donors tested (8 blood donors tested for FT and 13 different for HS)

MAb2 not tested due to direct interference in the assay

Shear stress not tested


T cell assay results

mAb1 only due to direct interference of mAb2 in T cell assay


Summary

Aggregates can induce DC maturation. More particles induce stronger DC maturation in more donors

Aggregates lead to increased HLA-restricted antigen presentation

Extent of presentation correlates exceptionally well with calculated amount of protein contained within the subvisible particles

Aggregates can lead to increased T cell responses

Thus, highly aggregated proteins are able to induce adaptive immune responses


Summary cont’d

Aggregates and subvisible particles are present, to a limited extent, in every biopharmaceutical product sold on the market today.

The aggregation levels typically observed for marketed therapeutic antibodies are much lower than the levels generated for this study.

To shed further light on the mechanistic involvement of aggregates during induction of immunogenicity additional investigations have been initiated as part of ABIRISK: Additional aggregated monoclonal antibodies (marketed formulations) which

are also part of clinical investigations in ABIRISK

Advanced assays performed by multiple labs


Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the RISK

Evaluation and development of technologies for predicting immunogenicity

Aim 1: Evaluate clinical relevance and gain a greater understanding of technologies of prediction of immunogenicity

Aim 2: Develop and assess novel prediction methodsAim 3: Assess effects of aggregation on immunogenicity

Co-leadersBernard Maillere, CEA Christian Pedersen Ross, Novo NordiskSebastian Spindeldreher, Novartis Pharma

WP 3

Aggregate generation and characterization Focus on antibodies (rituximab, infliximab, natalizumab, adalimumab) Syringe stress:

• SS-L1: 3x "up and down“• SS-L2: 10x "up and down“

Heat stress:• HS-L1: 24 h @55 °C• HS-L2: 72 h @55°C

Physicochemical analytics : SEC, DLS, MFI and Turbidity

Applied methods MAPPs DC activation assays T cell assay

Aim 3: Effect of aggregatesA sneak preview

Natalizumab:Changes in peptide presentation

1 5 10 15 20 25 30 35 40 45 50 58heatmap coloring based on peptide numbers

donor 1

donor 3donor 4donor 5donor 6donor 7donor 8donor 9

donor 10donor 11donor 12donor 13donor 14

donor 16

donor 1



donor 16

donor 1



donor 1donor 2donor 3donor 4donor 5donor 6donor 7donor 8donor 9


donor 1


donor 10

donor 12donor 13donor 14

donor 16

unst

ress

edSS

-SL1

donor 15

donor 2

donor 15

donor 2

nata

lizum

ab

120 140 320

SS-S

L2H

S-SL

1H

S-SL

2

donor 15

donor 11

donor 2

donor 2

0 20 40 60 80 100

CH 2+3

420

CH 1

340 360 380 400160 180 200 220 240 260 280 300 0 20 40 60 80 100CL

120 140 160 180

1 5 10 15 20 25 30 35 40 45 50 59heatmap coloring based on peptide numbers

Constant framework Hinge

Variable framework CDR

Legend for sequence coloring:

Infliximab: Changes in peptide presentation



donor 13donor 14


donor 7donor 8

donor 10donor 11

donor 13donor 14



donor 13donor 14

donor 1donor 2donor 3

donor 8donor 9

donor 11

donor 13donor 14


donor 8donor 9

donor 11donor 12

donor 7

donor 10

donor 13donor 14donor 15donor 16

donor 12

donor 15donor 16

donor 6

unst

ress

edSS

-L1

SS-L

2H

S-L1

donor 6

donor 12

donor 15donor 16

donor 6

donor 9

donor 12

donor 15donor 16

donor 6

donor 12

donor 15donor 16

donor 4donor 5donor 6donor 7

donor 10

HS-

L2

420

infli

xim

ab

CH1 H CH2+3

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

CL

1800 20 40 60 80 100 120 140 160

1 5 10 15 20 30 40 50 60 70 80 87

Legend for heatmap coloring:

1 5 10 15 20 30 40 50 60 70 80 90 93

Legend for heatmap coloring:

Constant framework Hinge

Variable framework CDR

Legend for sequence coloring:

Increased stress leads to increased peptide presentation

0

2

4

6

8

10

unstressed L1 L2

aver

age c

luste

rs pe

r don

orstress level

FT HSrituximab

0

2

4

6

8

10

12

unstressed L1 L2

aver

age c

luste

rs p

er d

onor

stress level

FT HSadalimumab

0

2

4

6

8

10

12

unstressed L1 L2

aver

age c

luste

rs pe

r don

or

stress level

FT HSinfliximab

0

1

2

3

4

5

6

unstressed L1 L2

aver

age c

luste

rs pe

r don

or

stress level

FT HSnatalizumab

0.02.04.06.08.0

10.012.014.016.0

un sl1 sl2

cluste

rs pe

r don

or

stress level

S FT HSmAb 1

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

un sl1 sl2

cluste

rs p

er d

onor

stress level

S FT HSmAb 2

New IMI studyNovartis internal study

MAPPs on 4 marketed antibodies confirmed the findings of the Novartis study The stronger the stress, the more peptides are being presented on HLA

class II The baseline presentation as well as the degree of change in peptide

presentation depends on the molecule.

Ongoing: DC activation assays with multiple endpoints:

• Cell surface activation markers• Cytokine secretion• Chemokine and cytokine transcription• Cell signaling (phosphorylation)

Selection of T cell assay setup for aggregate study

Conclusion

NovartisSebastian SpindeldreherAnette KarleAndrea KiesslingAnja MatterHannah Morgan Sascha GottliebStephan KoepkeVerena Rombach-Riegraf

INSERM U996Marc PallardySophie Tourdot Isabelle Turbica

INSERM U872Sebastien Lacroix-DesmazesAnastas PashovSandrine DelignatatM. Zhu

INSERM U1014Yu-Chun Lone

CEABernard MaillereMoustafa HamzeSylvain Meunier

DRK-BSDPeter MilanovStefanie Roth

IRBAntonio LanzavecchiaFederica Sallusto

SANOFIVincent MikolCatherine PradesDaniel KramerLaurent DuhauMagali AgnelPierre LafarguetteStephane Coren

Novo NordiskChristian Ross PedersenThomas BäckströmAnne Månsson Kvarnhammar

BayerHans-Werner VohrJeannette LoMargret LeineweberFred Aswad Pedro Paz

SciCrossPierre Dönnes

WP3 partners

sebastian spindeldreher novartis institutes for biomedical research / dmpk-biologics aggregation of...

Documents

rpm stress level

ft cycles stress level

selected stress conditions

induction of dc maturation

rpm shear stress s unstressed

thaw ft unstressed

acknowledgements dc

h maturation cocktail