EpiVax Host Cell Protein Immunogenicity tool: CHOPPI May 13 2014, Dubrovnik

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EpiVax Host Cell Protein Immunogenicity tool: CHOPPI May 13 2014, Dubrovnik

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EpiVax Host Cell Protein Immunogenicity tool: CHOPPI May 13 2014, Dubrovnik

  1. 1. 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 HCPs May, 2014
  2. 2. How we got to CHO? 2002 Invitation to Immunogenicity Conference at FDA 2011 CHO Genome Published 2006 Immunogenicity scale Tregitopes CHO genome immunogenicity analysis ECI conference HCP / CHO Cells Host Cell Proteins Parallels with Graves’ model 2004 Clustered T cell epitopes EpiBars CHOPPI On line . . .
  3. 3. Why examine CHO HCP Immunogenicity?
  4. 4. • What contaminants could be there? • Are they likely to be immunogenic? IL-6 (pdb id 2l3y) Cathepsin Z (pdb id 1deu) Immunoinformatics analysis
  5. 5. Databases available Mouse secreted 165 proteins Transcriptome 32,801 contigs Validated HCP contaminants 26 proteins CHO genome 24,383 predicted genes Proteome 6,164 proteins Putatively Secreted (signal peptide)
  6. 6. 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 ` MHCII Mature APC MHC II
  7. 7. 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.
  8. 8. 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
  9. 9. 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-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 ≥ 20: potentially immunogenic
  10. 10. Immunogenicity Scores distribution −100 −50 0 50 100 0.0000.0050.0100.015 immunogenicity score densityofproteins ≥ 20: potentially immunogenic
  11. 11. But what about conservation with Human? What is the impact of the cross-conservation between CHO and Human? Human CHO
  12. 12. The God of Two Faces: JanusMatrix MHC/HLA TCR MHC T cell epitope 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
  13. 13. 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
  14. 14. 0 20 40 60 80 100 0.000.020.040.060.08 # CHO−unique epitopes densityofproteins CHO-unique epitope content
  15. 15. CHOPPI CHO Protein Predicted Immunogenicity Biotechnology and Bioengineering (In press)
  16. 16. CHOPPI results ≥ 20: potentially immunogenic
  17. 17. ` C-X-C motif chemokine 3 Lysosomal alpha-mannosidase CHOP cross-reactivity Cross-reac ve epitope Human protein with cross- reac ve epitopes Cross-reac ve human epitope Source (pathogen) protein No cross-reac ve epitope Lysosomal protective protein
  18. 18. • 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
  19. 19. 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.
  20. 20. Thank you! Dartmouth College EpiVax, Inc. Contact information: Dr. Anne De Groot dr.annie.degroot@gmail.com

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