- 1. Andres H. Gutirrez, Leonard Moise, Frances Terry, Kristen
Dasilva, Chris Bailey-Kellogg, William Martin, Anne S. De Groot
Assessing Unwanted Host Cell Protein Immunogenicity in Therapeutic
Protein Formulations 2013 PDA Europe, 6th Monoclonal Antibodies
Workshop September, 2013
2. How did we get to HCP/CHO/CHOPPI? 2002 Invitation to
Predicting Biologic Protein Immunogenicity Conference at FDA 2011
CHO Genome Published 2006-2007 Immunogenicity scale Tregitopes,
Collaboration 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 . . . 3. Why are we interested in the Impact of species-
specific sequences on immunogenicity? Autoimmune Graves Disease
Graves Disease Example 4. 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 5. 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) 6. 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 7. Genomics Transcriptomics A new
technology for HCP evaluation 8. Pathogen Immune Response? Self/
Microbiome 8 Active area of research - EpiVax/URI 9. HCP
Contamination cancels trial Immune response to HCP (CHO) led to
recent cancellation of phase III clinical trials: Higher than
expected rate of Anti- CHO antibody development (what is
expected????). IB1001 hemophilia (Inspiration Biopharmaceuticals)
10. Danger signals of all sorts Aggregates how do they work?
(probably dont work if no T cell epitopes) Immune complexes
Complement T cell epitope content (absence of) Treg epitope content
Pre-existing T cell response (Tolerance or heterologous immunity)
What drives immunogenicity? 11. 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 T cell epitope drives Immune response 12. Antigen Epitope Drug
or Vaccine How it works 13. 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, function &
previous exposure.** Marc H.V. van Regenmortel, Ph.D., Katia Boven,
M.D., Fred Bader, Ph.D. Immunogenicity of Biopharmaceuticals: An
Example from Erythropoietin: Protein structure, contaminants,
formulation, container, and closure all can affect the
immunogenicity of the product. BioPharm International 2005.
http://www.biopharminternational.com/biopharm/article/articleDetail.jsp?id=174494&sk=&date=&pageID=5
*A.S. De Groot, J. Rayner, W. Martin. Modeling the immunogenicity
of therapeutic proteins using T cell epitope mapping. In:
Immunogenicity of Therapeutic 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-reactive influenza virus-specific CD8+ T cells
contribute to lymphoproliferation in Epstein-Barr virus-associated
infectious mononucleosis. The Journal of clinical investigation
115:3602-3612. CHO are mammalian proteins How can self proteins be
immunogenic? 14. T Cell Epitope Content - Predicted Potential for
Immunogenicity of Selected Proteins -80 -60 -40 -20 0 20 40 60 80
100 HumanFSH beta HumanIgACD HumanIgGCD Human Albumin Human Amylase
De-immunized INF-beta Human Transferrin *Human Gonadotropin Random
Expectation Influenza Hemagglutinin *Human GHRH *Human Gonadotropin
w/signal TetanusToxin Human Erythropoietin BrazilNut Antigen *Human
GHRHw/signal **HumanINF- beta Less Immunogenic Proteins (based on
clinical experience) Have Fewer T cell Epitopes De Groot, As,
Goldberg M, Moise L, Martin W. Evolutionary deimmunization: An
ancillary mechanism for self-tolerance. Cell Immunol. 2007 Apr 17;
Pages 148-153. http://dx.doi.org/10.1016/j.cellimm.2007.02.006 Are
self proteins immunogenic? 15. EpiVax Immunogenicity Hypothesis:
Immune Response = Sum of Epitopes T cell response depends on: T
cell epitope content + HLA of subject Protein Immunogenicity can be
Ranked epitope Protein Therapeutic 1 + 1 + 1 = Response
epitopeepitope De Groot A.S. and L. Moise. Prediction of
immunogenicity for therapeutic 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
16. EpiVax - Immunogenicity Scale Low Neutral High Albumin Tetanus
ToxinProtein 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. Antigen A Antigen B Aggregate immunogenicity drives
Immune response 17. EpiMatrix predicted excess/shortfall in
aggregate immunogenicity relative to a random peptide standard. -
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 Follitropin -Beta A protein score > 20
indicates a significant immunogenic potential. Proteins that have
previously been demonstrated to be immunogenic have higher
potential immunogenicity on the scale. Those that have rarely been
demonstrated to be immunogenicity have lower T cell epitope
content. Immunogenicity scale 18. Some Vaccine Antigens High Scores
(work done for NMRC, Dept. of Defense) 19. - 80 - - 70 - - 60 - -
50 - - 40 - - 30 - - 20 - - 10 - - 00 - - -10 - - -20 - - -30 - -
-40 - - -50 - 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
potential. Other Antigens Extremely Low Scores (Hirudin, Tick
Saliva, Some Parasites) 20. 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 Regulators prefer Leech-like
Proteins And HCPs - Regulatory Perspective 21. 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 22. MHC binding is a prerequisite for
immunogenicity Epitopes are linear and directly derived from
antigen sequence Binding is determined by amino acid side chains
Matrix-based predictor MHCII Mature APC Immunogenicity prediction
23. EpiMatrix EpiVax uses EpiMatrix to predict epitopes matrix
based prediction algorithm Can predict either class I or class II
MHC binding MHC binding is a prerequisite for immunogenicity MHC II
Pocket Peptide Epitope Mature APC MHCII T cell epitopes are linear
and directly derived from antigen sequence Binding is determined by
amino acid side chains (R groups) and encoded in single letter code
23 8/26/2013 Confidential 24. 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. ClustiMer recognizes T-cell epitope clusters as
polypeptides predicted to bind to an unusually large number of HLA
alleles. 8/26/2013 Confidential 25. What Makes Proteins Really
immunogenic? Sequences that Contain EpiBars Confidential Roberts
CGP, Meister GE, Jesdale BM, Lieberman J, Berzofsky JA, A.S. De
Groot, Prediction of HIV peptide epitopes by a novel algorithm,
AIDS Research and Human Retroviruses, 1996, Vol. 12, No. 7, pp.
593-610. ClustiMer - Locates highly immunogenic regions EpiBar : A
common feature of highly immunogenic clusters EpiBar 26. EpiVax
Immunogenicity Scale Confidential - 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-Alpha Non-immunogenic Antibodies
Follitropin-Beta PROTEIN_001 (35.13) Protein Immunogenicity Scale
Proteins Scoring above +20 are considered to be potentially
immunogenic. On the left of the scale we include some well-known
proteins for comparison - 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
Non-immunogenic Antibodies Follitropin-Beta 27. 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) AB06 (EPX Adjusted Score: -47.85) AB07 (EPX
Adjusted Score: -46.99) AB08 (EPX Adjusted Score: -46.30) AB09 (EPX
Adjusted Score: -47.40) AB10 (EPX Adjusted Score: -45.88) AB11 (EPX
Adjusted Score: -47.40) 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 8/26/2013 Confidential Due to the presence
of Tregitopes, antibodies tend to fall lower on the immunogenicity
scale. We have developed a refined method using regression analysis
to predict the immunogenicity of antibody 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. NeoEpitopeContent Tregitope Content High Low Low
Avastin (0%) Herceptin (0%) Mylotarg (3%) Simulect (1%) Synagis
(1%) High Campath (45%) Remicade (26%) Rituxan (27%) 28. CHO genome
Immune Response? Self/ Microbiome 28 Logical Next Step measure
CHO/Self Conservation 29. Databases available Putatively Secreted
(signal peptide) Mouse secreted 165 proteins Transcriptome 32,801
contigs Validated HCP contaminants 25 proteins CHO genome 24,383
predicted genes 30. Key Datasets Genome and transcriptome 31.
Protein databases (UniProtKB/Swiss- Prot, Locate) BLAST SignalP
EpiMatrix BlastiMer - JanusMatrix Tools used for this analysis 32.
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
33. Immunogenicity Scores distribution 34. Immunogenicity Scale
Validated HCP CHO contaminants 35. SL cytokine (84) Lysosomal
protective protein (35) 36. But are human-like proteins
immunogenic? CHO okay? peptides 37. 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 conservation with
Human 38. 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 39. T cell
Receptor Face (epitope) MHC-binding Face (agretope) T cell epitopes
are two-faced 40. 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 41. 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 42. EpiMatrix adjusted immunogenicity
score 43. Determination of conservation with self: JanusMatrix
results 44. 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 45. CEFT
Peptides (immunogenic) 46. 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 47. hTregitope-IGGC-167 hTregitope-IGGC-289
HTREG_IGGC-289 HTREG_IGGC-167 48. CHO: lysosomal protective protein
Lysosomal protective Lysosomal protective 49. SL cytokine CHO: SL
cytokine SL cytokine 50. 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 51. Immune Response = Sum of Epitopes Sum includes + (T
effectors) and (Tregs) scores Protein Therapeutic Host Cell Protein
Contaminant HCP Epitope New Approach 52. 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 = TeffPT + TeffPT + TeffHCP TregPT) = Treg Adjusted
Response Immune response depends on Foreign-ness Potential Tregs
Adjuvant (Danger signal) Proposed adjustment to score 53. Available
now: CHOPPI CHO Protein Predicted Immunogenicity CHOPPI
http://bit.ly/11fZqfJ 54. Formulation (VLP; aggregates) Danger
Signal Route: Subcutaneous delivery? Dose (high/low, persistent,
intermittent) T cell epitope content Differing T cell epitope
content = HCP 55 In Closing Factors affecting Immunogenicity 55.
While CHO are the most commonly used cell lines for mammalian cell
protein expression, Company-specific cell lines may vary.
Furthermore, we cant anticipate Genetic engineering Batch-to-batch
variation Expression (based on above) Which protein will hitchhike
CHO Cell lines may differ 56. Genomics ExpressomeInformatics In the
future Obtain proteins through MS/MS HPLC and Sequence, ID epitopes
57. Thank you! And . . . CHOPPI: http://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 58.
Institute for Immunology and Informatics (iCubed) D. Spero icubed
overview 2011 www.immunome.org URI Alumni Board 2012 59. New
Concept: Tregitopes induce tolerance to protein Therapeutics
(Friday April 20th Session) Epitope may induce different types of
Response 60. CHO Adjustment for Immunogenicity? + + Conserved
epitope Neo-Epitope Neo-Epitope 61. 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 Therapeutic 1 + 1
- Treg = Response epitopeepitope 62. T reg Stimulus 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. 6363Confidential and Copyrighted
EpiVax 63. 64 EpiVax: Immunogenicity scale is 64. 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 NA0.5%12% Neutralizing
Antibodies 5.6%7.8%53%Binding Antibodies -1.761.6234.37EpiMatrix
score FPX 4FPX 3FPX 2Protein Na: not analyzed Negative score
indicates presence of Treg epitope 66. - 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-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*