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Cho Hcp Immunogenicity Iciw Bailey Kellog

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Annie De Groot
Annie De Groot

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

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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
Therapeutic proteins Trastuzumab (Herceptin) -omab -ximab -zumab -umab bound to Her2 receptor http://www2.mrc-lmb.cam.ac.uk/antibody/ (pdb id 1n8z)
Therapeutic proteins S. simulans lysostaphin (chops S. aureus cell wall) deimmunization: Monday @ 1:30
Producing the protein Wurm, Nat Biotech, 2004 R. Carbonell, NC State Wikimedia commons
Extracting the protein R. Carbonell, Downstream Processing of Biopharmaceuticals: Challenges and Prospects
Therapeutic protein, ready to go? + BioRad Problem: host cell contamination IL-6 (pdb id 2l3y)
Host cell protein contamination Immune response to HCP (CHO) led to recent cancellation of two phase III clinical trials IB1001 C hemophilia (Inspiration Biopharmaceuticals)
Immunoinformatic analysis ? What contaminants could be there? ? Are they likely to be immunogenic? IL-6 (pdb id 2l3y) Cathepsin Z (pdb id 1deu)
Whats 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
Whats known to be there? 2D LC/MS 2D DIGE Jin et al., Biotechnol Bioeng. 2010 Doneanu et al., mAbs, 2012 28 proteins
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
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
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 -
Whats 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: JanusMatrix Identifies cross-reactive peptides: ? Identical T cell-facing residues ? Same HLA allele, but ? OK if different MHC-facing residues
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
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
CEFT Peptides (immunogenic)
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
hTregitope-IGGC-167 hTregitope-IGGC-289 HTREG_IGGC-167 HTREG_IGGC-289
CHO: lysosomal protective protein Lysosomal protective Lysosomal protective
CHO: SL cytokine SL cytokine SL cytokine
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.
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 C JanusMatrix, cross- reactive networks ? Further in vitro studies required to determine the impact and implications of findings
Coming soon: CHOPPI
Thanks Translational Immunology Research and Accelerated [Vaccine] Development Institute for Immunology and Informatics University of Rhode Island Dartmouth College EpiVax, Inc.

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Cho Hcp Immunogenicity Iciw Bailey Kellog

  • 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 C 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. Whats 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. Whats 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. Whats 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
  • 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 C JanusMatrix, cross- reactive networks ? Further in vitro studies required to determine the impact and implications of findings
  • 29. Thanks Translational Immunology Research and Accelerated [Vaccine] Development Institute for Immunology and Informatics University of Rhode Island Dartmouth College EpiVax, Inc.

Editor's Notes

  • #24: 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.