Andres H. Gu,érrez, Leonard Moise, Frances Terry, Kristen Dasilva, Chris BaileyKellogg, 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
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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
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” 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
• immunogenic in FVIII-expressing HLA DR mice (foreign)
• immunogenic in FVIII KO mice (still foreign)
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.
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????).
• 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.
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.
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
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.
• 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
as possible immunogenicity trigger.
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
as possible immunogenicity trigger.
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
• 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