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
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
Therapeutic proteinsTrastuzumab (Herceptin) -omab -ximab -zumab -umabbound to Her2 receptor http://www2.mrc-lmb.cam.ac.uk/antibody/(pdb id 1n8z)
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 (pdb id 2l3y)
Host cell protein contaminationImmune response to HCP (CHO) led to recent cancellationof two phase III clinical trialsIB1001 – hemophilia(InspirationBiopharmaceuticals)
Immunoinformatic analysis• What contaminants could be there?• Are they likely to be immunogenic? IL-6 (pdb id 2l3y) Cathepsin Z (pdb id 1deu)
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
What’s known to be there?2D LC/MS 2D DIGE Jin et al., Biotechnol Bioeng. 2010Doneanu 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 allelesDRB1*0101DRB1*0301DRB1*0401DRB1*0701DRB1*0801DRB1*1101DRB1*1301DRB1*1501
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 …• 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
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.
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