Awesome presentation combining efforts from Chris B-K, EpiVax, and iCubed on CHO/HCP immunogenicity prediction

1. Immunoinformatic analysis of
Chinese Hamster Ovary (CHO)
protein contaminants in
therapeutic protein formulations
Andres H. Gutierrez, Leonard Moise,
Frances Terry, Kristen Dasilva,
Chris Bailey-Kellogg, William Martin,
Anne S. De Groot

2. Therapeutic proteins
Trastuzumab (Herceptin) -omab -ximab -zumab -umab
bound to Her2 receptor http://www2.mrc-lmb.cam.ac.uk/antibody/
(pdb id 1n8z)

3. Therapeutic proteins
S. simulans lysostaphin
(chops S. aureus cell wall)
deimmunization:
Monday @ 1:30

4. Producing the protein
Wurm, Nat Biotech, 2004
R. Carbonell, NC State
Wikimedia commons

5. Extracting the protein
R. Carbonell, “Downstream Processing of Biopharmaceuticals: Challenges and Prospects”

6. Therapeutic protein, ready to go?
+
BioRad
Problem: host cell contamination IL-6 (pdb id 2l3y)

7. Host cell protein contamination
Immune response to HCP (CHO) led to recent cancellation
of two phase III clinical trials
IB1001 – hemophilia
(Inspiration
Biopharmaceuticals)

8. Immunoinformatic analysis
• What contaminants could be there?
• Are they likely to be immunogenic?
IL-6 (pdb id 2l3y) Cathepsin Z (pdb id 1deu)

9. What’s predicted to be there?
Chinese
hamster
genome CHO transcriptome Mouse secreted proteins
Signal
24,383 32,801 contigs peptide LOCATE (382)
predicted UniProt (76)
genes
39 proteins

10. What’s known to be there?
2D LC/MS 2D DIGE
Jin et al., Biotechnol Bioeng. 2010
Doneanu et al., mAbs, 2012 28 proteins

11. Immunogenicity prediction: EpiMatrix
• 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
MHC II
Mature
APC

12. Promiscuous epitope clusters: ClustiMer
• T cell epitopes not randomly distributed
• Clusters enable significant immune responses
• => Identify peptides predicted to bind an unusually large number of HLA alleles
DRB1*0101
DRB1*0301
DRB1*0401
DRB1*0701
DRB1*0801
DRB1*1101
DRB1*1301
DRB1*1501

13. Overall protein immunogenicity
- 80 -
Thrombopoietin
- 70 -
- 60 -
Human EPO
- 50 -
EBV-BKRF3
- 40 - ≥ 20: potentially immunogenic
- 30 -
Tetanus Toxin
Influenza-HA - 20 -
- 10 -
- 00 -
Albumin
Immunogenic Antibodies* - -10 -
- -20 -
IgG FC Region - -30 -
Non-immunogenic Antibodies† - -40 -
- -50 -
- -60 -
Follitropin-Beta - -70 -
- -80 -

14. What’s there & immunogenic?
43% 27%
28% 18%
25% 14%

15. Some potential contaminants
SL cytokine (84)
Lysosomal protective protein (35)

16. But are human-like peptides immunogenic?
CHO
okay?

17. Presentation and recognition
T cell Receptor face
(epitope)
MHC-binding face
(agretope)

18. Two-faced scoring: JanusMatrix
Identifies cross-reactive peptides:
• Identical T cell-facing residues
• Same HLA allele, but …
• OK if different MHC-facing
residues

19. Two-faced peptide evaluation
The most conservative approach:
• Identical T cell-facing residues
• Same HLA allele and minimally different
MHC-facing residues
TCR
MHC/HLA

20. Cross-reactivity visualization
Source protein
Predicted 9-mer epitope
from a source protein
9-mer from human
prevalent proteome,
100% TCR face identical to
source epitope
Human protein where
cross-reactive epitopes
are present

21. CEFT Peptides (immunogenic)

22. Flu and Tet tox epitopes
Poly ADP ribose
polymerase
family, member
9
Poly ADP ribose
polymerase
family, member
Ankyrin repeat
9 domain 18A
Ubiquitin
specific
protease 1
Tetanus
Toxin830-844
Flu HA308-318
ETAA16 protein
Olfactory
receptor, family
5, subfamily D,
member 14
SNF2 histone
linker PHD RING
helicase

23. hTregitope-IGGC-167
hTregitope-IGGC-289
HTREG_IGGC-167
HTREG_IGGC-289

24. CHO: lysosomal protective protein
Lysosomal
protective
Lysosomal
protective

25. CHO: SL cytokine
SL cytokine
SL cytokine

26. Self assessment
100%
Adjusted
Immunogenicity 100% TCR Non
Protein Immunogenicity 9-mers Epitopea
score identityb face identicald
score
identityc
SL cytokine 83.7 -92.68 222 54(24%) 7(13%) 67% 33%
Putative bifunctional
methylenetetrahydrofolate 47.93 -83.16 271 57(21%) 16(28%) 28(49%) 13(23%)
dehydrogenase/cyclohydrolase 2
ADP-ribosyl cyclase 2 46.47 -58.11 253 56(22%) 5(9%) 27(48%) 24(43%)
Metalloproteinase inhibitor 1 43.21 -71.57 195 40(21%) 2(5%) 22(55%) 16(40%)
Lysosomal protective protein 34.82 -85.24 467 109(23%) 42(39%) 39% 60%
Kit ligand 24.62 -85.16 265 55(21%) 17(31%) 21(38%) 17(31%)
Epididymal secretory protein E1 23.46 -105.09 141 27(19%) 7(26%) 13(48%) 7(26%)
aNumber of predicted epitopes.
bNumber of predicted epitopes 100% identical to 9-mers from Human UniProt reviewed database.
cDifferent MHC-facing residues, identical TCR-facing residues, but the epitope was predicted to bind to at least one common allele.
dDifferent MHC-facing residues, one or more mutations at the TCR-facing residues.

27. Summary
• HCPs may contribute to adverse clinical side effects
• Can score immunogenicity of entire
genomes/transcriptomes/secretomes
• Here secretome, common to all CHO cultures; in
future, also internal proteins (hitch-hikers)
• Exploring immunogenicity adjustment for
conservation with self – JanusMatrix, cross-
reactive networks
• Further in vitro studies required to determine the
impact and implications of findings

28. Coming soon: CHOPPI

29. Thanks
Translational Immunology Research
and Accelerated [Vaccine] Development
Institute for Immunology and Informatics
University of Rhode Island
Dartmouth College
EpiVax, Inc.