USP CHOP Annie De Groot Presentation June 2013

Andres H. Gu,érrez, Leonard Moise, Frances Terry, Kristen Dasilva,

Chris Bailey-‐Kellogg, William Mar,n, Anne S. De Groot

Immunoinforma2c analysis of Chinese Hamster Ovary (CHO)

protein contaminants in therapeu2c protein formula2ons

Measurement of Residual Host Cell Protein and DNA in Biotechnology Products June 3, 2013

How did we get to HCP/CHO/CHOPPI?

2002 Invita,on to

“Predic,ng Biologic Protein Immunogenicity”

Conference at FDA

2011 CHO

Genome Published

2006-‐2007 Immunogenicity scale

Tregitopes, Collabora,on With Gene Koren and others

CHO genome immunogenicity

analysis

Plenary at ECI CCE conference HCP / CHO Cells Host Cell Proteins

Parallels with Graves’ model

2004 Benchmarking Vaccine tools

for Biologics: Clustered

T cell epitopes EpiBars

CHOPPI On line . . .

Why are we interested in the Impact of species-specific sequences on immunogenicity?

HLA DR mice tolerated 2191-‐O, whose core epitope was fully conserved in human and murine FVIII. E16 mice were responsive to this pep,de because they lack endogenous murine FVIII. Epitopes immunogenic in HLA DR mice contain non-‐conserva,ve sequence mismatches.

Autoimmune Graves Disease

Graves Disease Example


“Autoimmune Graves Disease” begins with a response to a single epitope that is mismatched and presented in the context of murine MHC

hTSHR variant 1_NM_000369 and murine TSH-R mTSHR variant 1_NM_011648 alignment

mTSHR_variant_1_NM_011648 PPSTQTLKLIETHLKTIPSLAFSSLPNISRIYLSIDATLQRLEPHSFYNL hTSHR_variant_1_NM_000369 PPSTQTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNL

peptide 5-6 (78-94) (variant)

Graves Disease Example

•  Epitope fully conserved in human and murine FVIII:

•  Tolerated in FVIII-expressing HLA DR mice (have autologous FVIII)

•  Immunogenic in FVIII KO mice (do not have any FVIII)

•  Epitopes containing human/murine FVIII sequence mismatches:

•  immunogenic in FVIII-expressing HLA DR mice (foreign)

•  immunogenic in FVIII KO mice (still foreign)


FVIII KO

Not KO

FVIII Example (murine)

Murine'response'to'TSH/R Mouse'Sequence'same'as'Hu Mouse'Sequence'Different

T'cell'Epitope'Present Tolerance Immunogenicity

T'Cell'Epitope'Absent No'Response'''' Absent'epitope,'no'response

Human'response'to'HCP Human'Sequence'Same'as'CHO Human'Sequence'Different

T'cell'Epitope'Present Tolerance Immunogenicity

T'cell'Epitope'Absent No'Response'''' Absent'epitope,'no'response

Mice immunized with human TSH-‐R

Humans exposed to CHO or other HCP

Important Parallels – HCP effects

Genomics

Transcriptomics Informatics

A new technology for HCP evaluation

Pathogen

Immune Response?

Self/ Microbiome

8

Ac,ve area of research -‐ EpiVax/URI

HCP Contamina,on cancels trial

Immune response to HCP (CHO) led to recent cancella,on of phase III clinical trials: “Higher than expected rate of An,-‐CHO an,body development” (what is expected????).

IB1001 – hemophilia (Inspira3on Biopharmaceu3cals)

•  Danger signals of all sorts •  Aggregates – how do they work?

–  (probably don’t work if no T cell epitopes) –  Immune complexes – Complement

•  T cell epitope content •  (absence of) Treg epitope content •  Pre-‐exis3ng T cell response (Tolerance or heterologous immunity)

What drives immunogenicity?

Factors (↑roof Immunogenicity) Immune effect

Glycosylation (↑) Increase presentation? Increase foreign-ness of protein, need T cell epitopes

PEGylation (↓) Slow antigen processing, “mask” T cell epitopes and B cell epitopes

Host Cell-derived Protein (↑) CPG DNA (if bacterial); CHO T cell epitopes Oxidized Form of the Product (↑) Increase foreign-ness, modify presentation Excipients (↑) Increase Danger signal, T cell epitopes Leachates (↑) Increase Danger signal, T cell epitopes Characteristics of Patients (↑or↓) Missing Protein is foreign, T cell epitopes

Frequency, Duration and Route of Administration (↑or↓)

Administration like a vaccine, DAMPs, T cell epitopes

Aggregates (↑) Aggregation increases T cell epitope presentation

In almost every case Mechanism of Action – T cell Response

In almost every case – T cell epitope drives Immune response

An,gen

Epitope

Drug or Vaccine

How it works

In the right context self proteins can be immunogenic. Take Epo†, for example.

T cell epitope content is unequally distributed throughout the human (and CHO) proteome.* Immune response depends on protein prevalence, func,on & previous exposure.**

† Marc H.V. van Regenmortel, Ph.D., Ka,a Boven, M.D., Fred Bader, Ph.D. Immunogenicity of Biopharmaceu,cals: An Example from Erythropoie,n: Protein structure, contaminants, formula,on, container, and closure all can affect the immunogenicity of the product. BioPharm Interna,onal 2005. hmp://www.biopharminterna,onal.com/biopharm/ar,cle/ar,cleDetail.jsp?id=174494&sk=&date=&pageID=5 *A.S. De Groot, J. Rayner, W. Mar,n. Modeling the immunogenicity of therapeu,c proteins using T cell epitope mapping. In: Immunogenicity of Therapeu,c Biological Products. Developments in Biologicals. Fred Brown, Anthony Mire Suis, editors. Basel, Karger, 2003. Vol 112:71-‐80. **Clute, S. C., L. B. Watkin, M. Cornberg, Y. N. Naumov, J. L. Sullivan, K. Luzuriaga, R. M. Welsh, and L. K. Selin. 2005. Cross-‐reac,ve influenza virus-‐specific CD8+ T cells contribute to lymphoprolifera,on in Epstein-‐Barr virus-‐associated infec,ous mononucleosis. The Journal of clinical inves,ga,on 115:3602-‐3612.

CHO are mammalian proteins – How can “self” proteins be immunogenic?

T Cell Epitope Content -‐ Predicted Poten,al for Immunogenicity of Selected Proteins

-‐80 -‐60 -‐40 -‐20 0

20 40 60 80

100

Human FSH

beta

Human IgA CD

Human IgG CD

Human

Albu

min

Human

Amylase

De-‐im

mun

ized

INF-‐be

ta

Human

Transferrin

* Hu

man

Gonado

trop

in

Rand

om

Expe

cta,

on

Influ

enza

Hemagglu,

nin

* Hu

man

GHRH

* Hu

man

Gonado

trop

in

w/signal

Tetanu

s Toxin

Human

Erythrop

oie2

n Brazil Nut

An,g

en

* Hu

man

GHRH

w/signal

** Hum

an IN

F-‐

beta

Less Immunogenic Proteins (based on clinical experience) Have Fewer T cell Epitopes De Groot, As, Goldberg M, Moise L, Mar,n W. Evolu2onary deimmuniza2on: An ancillary mechanism for self-‐tolerance. Cell Immunol.

2007 Apr 17; Pages 148-‐153. hmp://dx.doi.org/10.1016/j.cellimm.2007.02.006

Are self proteins immunogenic?

EpiVax Immunogenicity Hypothesis: Immune Response = Sum of Epitopes

15

T cell response depends on:

T cell epitope content + HLA of subject

Protein Immunogenicity can be Ranked

epitope

Protein Therapeu,c

1 + 1 + 1 = Response

epitope epitope

• De Groot A.S. and L. Moise. Predic,on of immunogenicity for therapeu,c proteins: State of the art. Current Opinions in Drug Development and Discovery. May 2007. 10(3):332-‐40.

In biologics, immunogenicity is related to T cell epitope content

EpiVax -‐ Immunogenicity Scale

Low Neutral High

Albumin Tetanus Toxin Protein X or mAb Y

Proteins ranked by T-‐ Epitope content per Amino Acid

•  De Groot A.S., Drug Discovery Today -‐ 2006; •  De Groot A.S., Mire-‐Sluis, A. Ed.. Dev. Biol. Basel, Karger, 2005. vol 122. pp 137-‐160.

An,gen A An,gen B

Aggregate immunogenicity drives Immune response

EpiMatrix predicted excess/shorwall in aggregate immunogenicity rela,ve to a random pep,de standard.

-‐ 80 -‐ -‐ 70 -‐ -‐ 60 -‐ -‐ 50 -‐ -‐ 40 -‐ -‐ 30 -‐ -‐ 20 -‐ -‐ 10 -‐ -‐ 00 -‐ -‐ -‐ 10 -‐ -‐ -‐ 20 -‐ -‐ -‐ 30 -‐ -‐ -‐ 40 -‐ -‐ -‐ 50 -‐ -‐ -‐ 60 -‐ -‐ -‐ 70 -‐ -‐ -‐ 80 -‐

Thrombopoie2n

Human EPO

Tetanus Toxin Influenza -‐ HA

Albumin

IgG FC Region

EBV -‐ BKRF3

Follitropin -‐ Beta

A protein score > 20 indicates a significant immunogenic poten,al. Proteins that have previously been demonstrated to be immunogenic have higher poten,al immunogenicity on the scale. Those that have rarely been demonstrated to be immunogenicity have lower T cell epitope content.

Immunogenicity scale

Some Vaccine Antigens – High Scores (work done for NMRC, Dept. of Defense)

-  80 -

-  70 -

-  60 -

-  50 -

-  40 -

-  30 -

-  20 -

-  10 -

-  00 -

-  -10 -

-  -20 -

-  -30 -

-  -40 -

-  -50 -

-  -60 -

-  -70 -

-  -80 -

Human EPO Immunogenic Antibodies*

Tetanus Toxin

Influenza-HA

Albumin

IgG FC Region

EBV-BKRF3

Fibrinogen-Alpha Non-immunogenic Antibodies†

Follitropin-Beta

Hirudin(-‐90.41)

See my Blog “Thinking out Loud” for a discussion of Leech proteins and Tick Saliva proteins-‐Tick saliva proteins also have low immunogenicity poten,al.

Hirudin – Very Low Poten,al Immunogenicity -‐ Why? Other Antigens – Extremely Low Scores (Hirudin, Tick Saliva, Some Parasites)

•  Handled on a case-by-case basis •  Consider Source •  Maximum dose (mg biologics/kg body weight) •  Route of administration •  Frequency of dosing •  Pre-clinical and clinical data •  Detection process in evolution

The FDA Prefers Leech-like Proteins And HCPs - Regulatory Perspective

HCP Analytical Technologies

•  Detection – Protein staining –  Immunoblotting

•  Identification –  2D-PAGE/MS –  2D-LC/MS

•  Quantitation – ELISA using anti-HCP antibodies – May need to develop internal processes – Some kits are available

•  Risk assessment – Cytokine release assays

New Approach – Immunogenicity Screening in silico

Analytical Tests for HCP

•  MHC binding is a prerequisite for immunogenicity •  Epitopes are linear and directly derived from an,gen sequence •  Binding is determined by amino acid side chains •  Matrix-‐based predictor

MHC II Mature

APC

Immunogenicity predic,on

EpiMatrix

•  EpiVax uses EpiMatrix to predict epitopes –  matrix based predic,on algorithm

•  Can predict either class I or class II MHC binding –  MHC binding is a prerequisite for immunogenicity

MHC II Pocket

Pep,de Epitope Mature

APC

MHC II

T cell epitopes are linear and directly derived from an,gen sequence Binding is determined by amino acid side chains (R groups) and ‘encoded’ in single lemer code

23 6/3/13 Confidential

Easy easy to deliver as pep,des Clusters of MHC binding drive T cells

DRB1*0101

DRB1*0301

DRB1*0401

DRB1*0701

DRB1*0801

DRB1*1101

DRB1*1301

DRB1*1501

•  T cell epitopes are not randomly distributed but instead tend to cluster in specific regions. –  These clusters can be very powerful, enabling significant immune responses to low scoring

proteins.

•  Clus,Mer recognizes T-‐cell epitope clusters as polypep,des predicted to bind to an unusually large number of HLA alleles.

6/3/13 Confidential

What Makes Proteins Really immunogenic? Sequences that Contain EpiBars

Confiden,al

Roberts CGP, Meister GE, Jesdale BM, Lieberman J, Berzofsky JA, A.S. De Groot, Predic,on of HIV pep,de epitopes by a novel algorithm, AIDS Research and Human Retroviruses, 1996, Vol. 12, No. 7, pp. 593-‐610.

Clus,Mer -‐ Locates highly immunogenic regions

EpiBar : A common feature of highly

immunogenic clusters

EpiBar

EpiVax Immunogenicity Scale

Confiden,al

- 80 -

- 70 -

- 60 -

- 50 -

- 40 -

- 30 -

- 20 -

- 10 -

- 00 -

- -10 -

- -20 -

- -30 -

- -40 -

- -50 -

- -60 -

- -70 -

- -80 -

Thrombopoietin

Human EPO

Immunogenic Antibodies*

Tetanus Toxin

Influenza-HA

Albumin

IgG FC Region

EBV-BKRF3

Fibrinogen-AlphaNon-immunogenic Antibodies†

Follitropin-Beta

PROTEIN_001 (35.13)

Protein Immunogenicity Scale

Proteins Scoring above +20 areconsidered to be potentiallyimmunogenic.

On the left of the scale weinclude some well-knownproteins for comparison

- 80 - - 70 - - 60 - - 50 - - 40 - - 30 - - 20 - - 10 - - 00 - - - 10 - - - 20 - - - 30 - - - 40 - - - 50 - - - 60 - - - 70 - - - 80 -

Thrombopoietin

Human EPO


Tetanus Toxin Influenza - HA

Albumin

IgG FC Region

EBV - BKRF3

Non - immunogenic Antibodies†

Follitropin - Beta

EpiMatrix mAb Immunogenicity Scale - 80 -

- 70 -

- 60 -

- 50 -

- 40 -

- 30 -

- 20 -

- 10 -

- 00 -

- -10 -

- -20 -

- -30 -

- -40 -

- -50 -

- -60 -

- -70 -

- -80 -

IgG FC Region

Nuvion (0%)

Avastin (0%)

AB01 (EPX Adjusted Score: -46.98)

AB02 (EPX Adjusted Score: -44.48)AB03 (EPX Adjusted Score: -44.81)AB04 (EPX Adjusted Score: -45.81)AB05 (EPX Adjusted Score: -45.88)







Synagis (1%)

Simulect (1.4%)Humira (12%)

Bivatuzumab (6.7%)

Remicade (26%) Rituxan (27%)Campath (45%)

Humicade (7%)

Reopro (5.8%)Tysabri (7%)

LeukArrest (0%)

Herceptin (0.1%)

Compare with:

27 6/3/13 Confidential

Due to the presence of Tregitopes, an,bodies tend to fall lower on the immunogenicity scale.

We have developed a refined method using regression analysis to predict the immunogenicity of an,body sequences based on observed clinical responses (next slide).

We have found that a balance in favor of Tregitope (regulatory) content over neo-‐epitope (effector) content is correlated with reduced clinical immunogenicity.

Neo

Epi

tope

Con

tent

Tregitope Content High Low

Low

Avastin (0%) Herceptin (0%)

Mylotarg (3%) Simulect (1%) Synagis (1%)

Hig

h

Campath (45%) Remicade (26%) Rituxan (27%)

CHO genome

Immune Response?

Self/ Microbiome

28

Logical Next Step measure CHO/Self Conservation

Databases available

Puta,vely Secreted

(signal pep3de)

Mouse secreted

165 proteins

Transcriptome 32,801 con,gs

Validated HCP contaminants 25 proteins

CHO genome 24,383

predicted genes

Key Datasets Genome and transcriptome

•  Protein databases (UniProtKB/Swiss-Prot, Locate) •  BLAST •  SignalP •  EpiMatrix •  BlastiMer - JanusMatrix

Tools used for this analysis

•  Identify secreted CHO proteins •  Collect published HCP from CHO •  Evaluate potential immunogenicity •  Evaluate sequence homology •  Identify clustered regions – compare to CHO; •  Are human/CHO different at the cluster? Count

as possible immunogenicity trigger.

Approach

Immunogenicity Scores distribu,on

Immunogenicity Scale Validated HCP CHO contaminants

Other potential contaminants SL cytokine (84)

Lysosomal protective protein (35)

But are human-‐like proteins immunogenic?

CHO

okay?

peptides

Putatively Secreted

(signal peptide)

Mouse secreted

165 proteins

Transcriptome 32,801 contigs

Validated HCP contaminants 25 proteins

CHO genome 24,383

predicted genes

Human proteome 20,238 proteins

Approach to conserva,on with Human

•  Identify secreted CHO proteins •  Evaluate potential immunogenicity •  Evaluate sequence homology •  Identify clustered regions – compare to CHO; •  Are human/CHO different at the cluster? Count


Approach

T cell Receptor Face (epitope)

MHC-‐binding Face (agretope)

T cell epitopes are two-‐faced

Identifies cross-reactive peptides: •  Identical T cell-facing residues •  Same HLA allele but . . •  OK if different MHC-facing residues

The God of Two Faces: JanusMatrix

TCR face vs. MHC binding face

MHC/HLA

TCR

The most conservative approach: •  Identical T cell-facing residues •  Same HLA allele and minimally different

MHC-facing residues

EpiMatrix adjusted immunogenicity score

Determina,on of conserva,on with self: JanusMatrix results

Cross-‐reactivity visualization

Predicted 9-‐mer epitope from a source protein

Human protein where cross-‐reactive epitopes are present

9-‐mer from human prevalent proteome, 100% TCR face identical to source epitope

Source protein

HCV_G1_NS2_794

CEFT Pep,des (immunogenic)

Flu and Tet tox epitopes

SNF2 histone linker PHD RING

helicase

ETAA16 protein

Ankyrin repeat domain 18A

Flu HA308-318

Ubiquitin specific

protease 1

Poly ADP ribose polymerase

family, member 9

Poly ADP ribose polymerase

family, member 9

Tetanus Toxin830-844

Olfactory receptor, family 5, subfamily D,

member 14

hTregitope-‐IGGC-‐167 hTregitope-‐IGGC-‐289

HTREG_IGGC-289

HTREG_IGGC-167

CHO: lysosomal protective protein

Lysosomal

protective

Lysosomal

protective

SL cytokine

CHO: SL cytokine

SL cytokine

•  Identify secreted CHO proteins •  Evaluate potential immunogenicity •  Evaluate sequence homology •  Identify clustered regions – compare to CHO; •  Are human/CHO different at the cluster? Count


New Approach for CHO

Immune Response = Sum of Epitopes Sum includes + (T effectors) and – (Tregs) scores

Protein Therapeu,c

Host Cell Protein Contaminant

HCP Epitope

New Approach

For an individual, T cell response depends on: T cell epitope content x HLA – Treg Epitope content x HLA

Vaccine or Foreign Protein = (TeffPT1+ TeffPT2 . . . ) = Response CHO = Σ ( TeffPT + TeffPT + TeffHCP – TregPT) = Treg Adjusted Response

Immune response depends on Foreign-‐ness Potential Tregs Adjuvant (Danger signal)

Proposed adjustment to score

Available now: CHOPPI CHO Protein Predicted Immunogenicity

CHOPPI hmp://bit.ly/11fZqfJ

•  Formula,on (VLP; aggregates) •  “Danger Signal” •  Route: Subcutaneous delivery? •  Dose (high/low, persistent, intermiment) •  T cell epitope content •  Differing T cell epitope content = HCP

55

In Closing Factors affec,ng Immunogenicity

•  While CHO are the most commonly used cell lines for mammalian cell protein expression, Company-specific cell lines may vary. Furthermore, we can’t anticipate

•  Genetic engineering •  Batch-to-batch variation •  Expression (based on above) •  Which protein will ‘hitchhike’

CHO Cell lines may differ

Genomics

“Expressome” Informatics

In the future – Obtain proteins through MS/MS HPLC – and Sequence, ID epitopes

Thank you! And . . . CHOPPI: hmp://bit.ly/11fZqfJ or contact me.

Translational Immunology Research and Accelerated [Vaccine] Development Institute for Immunology and Informatics University of Rhode Island

Dartmouth College

EpiVax, Inc. SL cytokine

Institute for Immunology and Informatics (iCubed)

D. Spero icubed overview 2011

www.immunome.org URI Alumni Board 2012

New Concept:

Tregitopes induce

tolerance to

protein

Therapeu,cs

(Friday April 20th Session)

Epitope may induce different types of Response

CHO Adjustment for Immunogenicity ?

+ +

Conserved epitope Neo-Epitope

Neo-Epitope

Immune Response = Sum of Epitopes Sum includes + (T effectors) and – (Tregs) scores

ISPRI approach to analyzing mAbs . . .

T cell response depends on:

T cell epitope content x HLA – Treg Epitope content x HLA

Protein Immunogenicity can be Ranked

Treg epitope

Protein Therapeu,c

1 + 1 -‐ Treg = Response

epitope epitope

T reg S,mulus

IL 10, TNF alpha Additional Treg Epitope

Modify Effector T cell response: Reduce T effector Stimulus

Current Hypothesis: More Tregitopes Lower Immunogenicity

De Groot A.S. and D. Scott. Immunogenicity of Protein Therapeutics. Trends in Immunology. Invited Review. Trends Immunol. 2007 Nov;28(11):482-90.

63 1/29/11 63 Confiden,al and Copyrighted EpiVax

64

EpiVax: Immunogenicity scale is

Correlation of antibody immunogenicity with Tregitope adjusted EPX Scores

65

Correlation of EpiMatrix Scores and Immunogenicity in Human studies

40%

37%

21.97

FPX 1

0%

9.3%

-‐111.25

FPX 5

NA 0.5% 12% Neutralizing An,bodies

5.6% 7.8% 53% Binding An,bodies

-‐1.76 1.62 34.37 EpiMatrix score

FPX 4 FPX 3 FPX 2 Protein

Na: not analyzed Nega,ve score indicates presence of

Treg epitope

-  80 -

-  70 -

-  60 -

-  50 -

-  40 -

-  30 -

-  20 -

-  10 -

-  00 -

-  -10 -

-  -20 -

-  -30 -

-  -40 -

-  -50 -

-  -60 -

-  -70 -

-  -80 -

Thrombopoietin

Human EPO


Tetanus Toxin

Influenza-HA

Albumin

IgG FC Region

EBV-BKRF3

Fibrinogen-Alpha Non-immunogenic Antibodies†

Follitropin-Beta

Ab K (-38.23)

Ab E (-16.03)

Ab N (-53.88)

Ab P (-70.14)

Ab B (-00.32)

Ab A (13.82)

Ab D (-08.87)

Ab F (-22.13)

Ab I (-25.77)

Ab O (-54.26)

Ab L (-48.49)

Ab C (-02.03)

Ab M (-52.25)

Ab H (-24.99)

Ab J (-28.94)

Ab G (-24.33)

*Tregitope adjusted

Application – Germline Abs*

USP CHOP Annie De Groot Presentation June 2013

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