epivax host cell protein immunogenicity tool: choppi may 13 2014, dubrovnik
DESCRIPTION
EpiVax Host Cell Protein Immunogenicity tool: CHOPPI May 13 2014, DubrovnikTRANSCRIPT
Chris Bailey-Kellogg, Andres H. Gutiérrez, Leonard Moise, Frances Terry, William Martin,
Anne S. De Groot
Immunoinformatic analysis of Chinese Hamster Ovary (CHO)
protein contaminants in therapeutic protein formulations
European meeting on HCPsMay, 2014
How we got to CHO?
2002Invitation to
Immunogenicity Conference at FDA
2011CHO
Genome Published
2006Immunogenicity scale
Tregitopes
CHO genome immunogenicity
analysis
ECI conferenceHCP / CHO Cells
Host Cell ProteinsParallels with Graves’ model
2004Clustered
T cell epitopesEpiBars
CHOPPIOn line . . .
Why examine CHO HCP Immunogenicity?
• What contaminants could be there?• Are they likely to be immunogenic?
IL-6 (pdb id 2l3y) Cathepsin Z (pdb id 1deu)
Immunoinformatics analysis
Databases available
Mousesecreted
165 proteins
Transcriptome32,801 contigs
Validated HCP contaminants
26 proteins
CHO genome24,383
predicted genes
Proteome6,164 proteins
Putatively Secreted
(signal peptide)
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
• 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
Peptide epitope
`M
HC II Mature
APC
MHC II
Easy easy to deliver as peptidesClusters 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.
What Makes Proteins Really immunogenic?Sequences that Contain EpiBars
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
EpiVax Immunogenicity Scale- 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
Immunogenic Antibodies*
Tetanus Toxin
Influenza-HA
Albumin
IgG FC Region
EBV-BKRF3
Non-immunogenic Antibodies†
Follitropin-Beta
≥ 20: potentially immunogenic
Immunogenicity Scores distribution
≥ 20: potentially immunogenic
But what about conservation with Human?
What is the impact of the cross-conservation between CHO and Human?
Human
The God of Two Faces: JanusMatrix
MHC/HLA
TCR
MHC
T cell epitop
e
T cell receptor
Identifies cross-reactive peptides:• Identical T cell-facing residues• Same MHC allele, but …• OK if different MHC-facing
residues
Moise L et al. Hum Vaccin Immunother. 2013 Jul;9(7):1577-86
Source epitope
Human protein with cross-reactive epitopes
Cross-reactive human epitope
Source (pathogen) protein
pH1N1 - HA Hepatitis C virus
Effector T cells Regulatory T cells
Visualizing cross-reactivity patterns
Moise L et al. Hum Vaccin Immunother. 2013 Jul;9(7):1577-86. He L et al. BMC Bioinformatics 2014, 15(Suppl 4):S1
CHO-unique epitope content
CHOPPICHO Protein Predicted Immunogenicity
Biotechnology and Bioengineering (In press)
CHOPPI results
≥ 20: potentially immunogenic
`
C-X-C motif chemokine 3
Lysosomal alpha-mannosidase
CHOP cross-reactivity
Lysosomal protective protein
• Exploring immunogenicity adjustment for conservation with self – JanusMatrix, cross-reactive networks
• Define ratio of non cross-reactive vs. cross-reactive associated with immunogenicity
• Further in vitro studies required to determine the impact and implications of findings
• Add additional genomes.. Such as NS0 mouse? E. Coli --- the website is set up so we can add genomes as they become available.
Next Steps
How to do a Risk Assessment on a single HCP?
• Go into CHOPPI• Paste in the protein you are looking for• Run CHOPPI• Look at the overall score: If over 20, possibly a problem.• Look at the conservation with human: if high, then less
of a problem. • We don’t yet know what “ratio” of CHO unique vs.
conserved is clinically meaningful. • We need HCP immunogenicity data – if you have that
information, we will build it into the tool.
