Immunoinformatic analysis ofChinese Hamster Ovary (CHO)protein contaminants intherapeutic protein formulationsAndres H. Gu...
Therapeutic proteinsTrastuzumab (Herceptin)   -omab       -ximab       -zumab      -umabbound to Her2 receptor    http://w...
Therapeutic proteinsS. simulans lysostaphin(chops S. aureus cell wall)deimmunization:Monday @ 1:30
Producing the proteinWurm, Nat Biotech, 2004                            R. Carbonell, NC StateWikimedia commons
Extracting the proteinR. Carbonell, “Downstream Processing of Biopharmaceuticals: Challenges and Prospects”
Therapeutic protein, ready to go?                                        +BioRadProblem: host cell contamination   IL-6 (p...
Host cell protein contaminationImmune response to HCP (CHO) led to recent cancellationof two phase III clinical trialsIB10...
Immunoinformatic analysis• What contaminants could be there?• Are they likely to be immunogenic?  IL-6 (pdb id 2l3y)    Ca...
What’s predicted to be there?  Chinese  hamster  genome      CHO transcriptome             Mouse secreted proteins        ...
What’s known to be there?2D LC/MS                     2D DIGE                              Jin et al., Biotechnol Bioeng. ...
Immunogenicity prediction: EpiMatrix•    MHC binding is a prerequisite for immunogenicity•    Epitopes are linear and dire...
Promiscuous epitope clusters: ClustiMer • T cell epitopes not randomly distributed • Clusters enable significant immune re...
Overall protein immunogenicity                                 -    80   -              Thrombopoietin                    ...
What’s there & immunogenic?       43%   27%       28%   18%       25%   14%
Some potential contaminants              SL cytokine (84)              Lysosomal protective protein (35)
But are human-like peptides immunogenic?                        CHO                    okay?
Presentation and recognition T cell Receptor face            (epitope)    MHC-binding face         (agretope)
Two-faced scoring: JanusMatrixIdentifies cross-reactive peptides:• Identical T cell-facing residues• Same HLA allele, but ...
Two-faced peptide evaluation                The most conservative approach:                • Identical T cell-facing resid...
Cross-reactivity visualization Source protein Predicted 9-mer epitope from a source protein 9-mer from human prevalent pro...
CEFT Peptides (immunogenic)
Flu and Tet tox epitopes                        Poly ADP ribose                          polymerase                       ...
hTregitope-IGGC-167hTregitope-IGGC-289                      HTREG_IGGC-167                          HTREG_IGGC-289
CHO: lysosomal protective protein                                     Lysosomal                                     protec...
CHO: SL cytokine                     SL cytokine       SL cytokine
Self assessment                                                                                                           ...
Summary• HCPs may contribute to adverse clinical side effects• Can score immunogenicity of entire  genomes/transcriptomes/...
Coming soon: CHOPPI
ThanksTranslational Immunology Research   and Accelerated [Vaccine] DevelopmentInstitute for Immunology and InformaticsUni...
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Cho Hcp Immunogenicity Iciw Bailey Kellog

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

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  • 2) hTreg vs. Plasma proteome, which includes 2 peptides: - HTEG-IGGC-167 - HTEG-IGGC-289Legend:Light blue rombo: Source (protein, cluster, peptide)Gray square: 9-mer epitope predicted from the source.Blue triangle: 9-mer from a determined database (Plasma proteome database), 100% TCR face identical to the source-derived epitope.Lavender(?) circle: Protein from the database where the epitope is present.
  • Cho Hcp Immunogenicity Iciw Bailey Kellog

    1. 1. Immunoinformatic analysis ofChinese Hamster Ovary (CHO)protein contaminants intherapeutic protein formulationsAndres H. Gutierrez, Leonard Moise,Frances Terry, Kristen Dasilva,Chris Bailey-Kellogg, William Martin,Anne S. De Groot
    2. 2. Therapeutic proteinsTrastuzumab (Herceptin) -omab -ximab -zumab -umabbound to Her2 receptor http://www2.mrc-lmb.cam.ac.uk/antibody/(pdb id 1n8z)
    3. 3. Therapeutic proteinsS. simulans lysostaphin(chops S. aureus cell wall)deimmunization:Monday @ 1:30
    4. 4. Producing the proteinWurm, Nat Biotech, 2004 R. Carbonell, NC StateWikimedia commons
    5. 5. Extracting the proteinR. Carbonell, “Downstream Processing of Biopharmaceuticals: Challenges and Prospects”
    6. 6. Therapeutic protein, ready to go? +BioRadProblem: host cell contamination IL-6 (pdb id 2l3y)
    7. 7. Host cell protein contaminationImmune response to HCP (CHO) led to recent cancellationof two phase III clinical trialsIB1001 – hemophilia(InspirationBiopharmaceuticals)
    8. 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. 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. 10. What’s known to be there?2D LC/MS 2D DIGE Jin et al., Biotechnol Bioeng. 2010Doneanu et al., mAbs, 2012 28 proteins
    11. 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. 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 allelesDRB1*0101DRB1*0301DRB1*0401DRB1*0701DRB1*0801DRB1*1101DRB1*1301DRB1*1501
    13. 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. 14. What’s there & immunogenic? 43% 27% 28% 18% 25% 14%
    15. 15. Some potential contaminants SL cytokine (84) Lysosomal protective protein (35)
    16. 16. But are human-like peptides immunogenic? CHO okay?
    17. 17. Presentation and recognition T cell Receptor face (epitope) MHC-binding face (agretope)
    18. 18. Two-faced scoring: JanusMatrixIdentifies cross-reactive peptides:• Identical T cell-facing residues• Same HLA allele, but …• OK if different MHC-facing residues
    19. 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. 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. 21. CEFT Peptides (immunogenic)
    22. 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. 23. hTregitope-IGGC-167hTregitope-IGGC-289 HTREG_IGGC-167 HTREG_IGGC-289
    24. 24. CHO: lysosomal protective protein Lysosomal protective Lysosomal protective
    25. 25. CHO: SL cytokine SL cytokine SL cytokine
    26. 26. Self assessment 100% Adjusted Immunogenicity 100% TCR Non Protein Immunogenicity 9-mers Epitopea score identityb face identicald score identitycSL cytokine 83.7 -92.68 222 54(24%) 7(13%) 67% 33%Putative bifunctionalmethylenetetrahydrofolate 47.93 -83.16 271 57(21%) 16(28%) 28(49%) 13(23%)dehydrogenase/cyclohydrolase 2ADP-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. 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. 28. Coming soon: CHOPPI
    29. 29. ThanksTranslational Immunology Research and Accelerated [Vaccine] DevelopmentInstitute for Immunology and InformaticsUniversity of Rhode IslandDartmouth CollegeEpiVax, Inc.

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