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Positional scanning combinatorial libraries: What do they reveal about T cell specificity?

Positional scanning combinatorial libraries: What do they reveal about T cell specificity?. Clemencia Pinilla Torrey Pines Institute for Molecular Studies July 6, 2006 Degeneracy and complexity of the immune system Santa Fe Institute. Genetic Approach Identification of proteins

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Positional scanning combinatorial libraries: What do they reveal about T cell specificity?

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  1. Positional scanning combinatorial libraries: What do they reveal about T cell specificity? Clemencia Pinilla Torrey Pines Institute for Molecular Studies July 6, 2006 Degeneracy and complexity of the immune system Santa Fe Institute

  2. Genetic Approach Identification of proteins Identification of peptides Biochemical Approach Peptide elution Sequence determination Synthetic Combinatorial Libraries one bead one compound (OBOC) Mixture Based Iterative Positional Scanning Identification of T-cell Epitopes

  3. Unbiased peptide collection, made up of systematically arranged mixtures having defined (O) and mixture positions (X) All possible peptides of a given length resulting from all combinations of 19 L-natural amino acids Represent very large numbers of peptides Hexapeptides ~ 50 million 120 mixtures Decapeptides ~ 6 trillion 200 mixtures Positional Scanning Libraries

  4. Substitution analogs Motif analysis Antigen sequence required Substitution analogs (10 mer X 20aa = 200 peptides) Substitution analogs need to be synthesized for each antigen Only sequences of predicted crossreactive peptides are obtained Positional scanning libraries No antigen sequence required All possible decapeptides (1910 = 6 trillion peptides) One library can be used for clones with known and unknown specificity Sequence list of predicted crossreactive peptides and predicted stimulatory activity are obtained T cell cross-reactivity

  5. Screening of Decapeptide PS-SCL for T Cell Response Position • AXXXXXXXXX-NH2CXXXXXXXXX-NH2 • . • . • YXXXXXXXXX-NH2 1 Ac-O1XXXXXXXXX- NH2 2 XO2XXXXXXXX 3 XXO3XXXXXXX 4 XXXO4XXXXXX 5 XXXXO5XXXXX 6 XXXXXO6XXXX 7 XXXXXXO7XXX 8 XXXXXXXO8XX 9 XXXXXXXXO9X 10 XXXXXXXXXXO10 AXXXXXXXXX-NH2 T Cells APC T cell activation: Proliferation Cytokine production Chromium release 20 mixtures/position 200 mixtures

  6. Identification of T Cell Epitopes using PS-SCL PS-SCL screening T cell ligands that do not necessarily correspond to natural epitopes T cell ligands in protein databases: Biometrical Analysis Synthesis of combinations of amino acids in the most active mixtures Synthesis of highest ranking peptides from database analysis Test individual peptides for T cell activity Zhao,et al, 2001, J.Immunol. Review: Borras, et, al.2002 J .Immunol. Methods Review: Nino-Vasquez ,et al, 2004 Mol Immunol

  7. Species TCC Antigen Activity Relative to Native CD4+ Human TL3A6 MBP 87-99 10,000 Human GP5F11 Flu peptide 80 Mouse Tg (BAND) PCC peptide 100 Mouse Tg (BDC2.5) Unknown <10 nM (56 peptides) Mouse 172.10 MPB 1-11 2,500 CD8+ Human 3-3F4 pp65 495-503 20,000 Human LAU 203/1.5 Melan-A 26-35 70 Human LAU 203/17 Melan-A 26-35 14,000 Human LAU50/4D7 SSX-2 41-49 3 Identification of Superagonists UsingPositional Scanning Libraries Borras, et, al.J .Immunol. Methods (2002)

  8. PS-SCL screening data Protein database PS-SCL Based Biometrical Analysis Hemmer, et al, 1999 Nature Medicine Zhao,et al, 2001, J.Immunol.

  9. Ranking of Known Antigens Using Positional Scanning Libraries and Biometrical Analysis * Variants among top 20 Review: Nino-Vasquez Mol Immunol 2004

  10. T Cell Clones of Unknown Specificity

  11. CD4+ T cell clone Specific for myelin basic protein MBP87-99 Screening of decapeptide PS-SCL Proliferative activity Identification of superagonist Data correlation with X-Ray 3A6 T cell Clone

  12. Decapeptide PS-SCL Screening TCC: TL3A6-MBP 89-98 Amino acid in native peptide found in 9 of 10 positions Hemmer, et al, 2000, J.Immunol. Zhao,et al, 2001, J.Immunol.

  13. Amino Acids of Most Active MixturesTCC: TL3A6 MBP 89-98 MHC-binding residues, X-Ray Li et al J.Mol.Biol 2000 TCR binding residues, X-Ray Li et al EMBO J 2005

  14. Peptide # subs. nM Ac-FFKNIVTPRT-NH2 0 34 Ac-WFKLITTTKL-NH2 6 0.003 Ac-WFKLITTPKL-NH2 5 0.120 Ac-WFKLILTKKG-NH2 6 0.130 Ac-WFKLIPTKKL-NH2 6 0.170 Ac-WFKLITTTKG-NH2 6 0.520 Ac-WFKLILTPKL-NH2 5 0.600 Ac-WFKLITTPKG-NH2 5 1.2 Ac-WFKLILTTKL-NH2 6 1.3 Ac-WFKLITTKKL-NH2 6 1.4 Ac-WFKLIPTPKL-NH2 5 1.8 Activity of Individual PeptidesTL3A6 Clone WFKLILTTKL T K P P Motif Superagonists

  15. Interaction of TCR 3A6 with HLA-DR2a and MBP peptide TL3A6-a chain TL3A6-b chain WFKLILTTKL T K P P Motif Superagonists Yi et al EMBO J 2005

  16. A-scan analogs of TL3A6 SuperagonistK38-27 Proliferation MPB89-98 FFKNIVTPRT K38-27 WFKLITTTKL MHC Binding GM-CSF production

  17. CD4+ T cell clone Specific for Influenza hemagglutinin HA 309-318 Screening of decapeptide PS-SCL Proliferative activity Matrix PS-SCL based biometrical analysis Virus GP5F11 T cell Clone

  18. Decapeptide PS-SCL Screening TCC: GP5F11-Flu-HA308-317 Amino acid in native peptide found in 7 of 10 positions Zhao,et al, 2001, J.Immunol. Markovic-Plese, et al,2005,J.Neuroimmunology

  19. Pos. Q V 4 * 6 I N T I N T 7 G G G G G G P P P P P P 9 FR FR FR FR FR FR FR FR FR FR FR FR Comparison of Multiple and Single Substitution AnalogsTCR GP5F11 Native Single Substitutions of Native 24 Peptides

  20. Natural Cross-reactive AnalogsTCR GP5F11

  21. CD8+ T cell clone Specific for tumor associated antigen: Melan-A 26-35 Screening of decapeptide PS-SCL Cytolytic activity Positional Scanning based biometrical database analysis Human Viral Bacterial Melan-A T cell Clone Pinilla et al, Cancer Research (2001) Rubio-Godoy, J.Immunol (2002)

  22. Decapeptide PS-SCL Screening Melan-A Specific T Cell Clone 1.5

  23. Viral peptides 100 75 50 Human peptides 100 25 75 0 23 24 25 26 27 28 29 30 32 33 34 35 36 37 38 39 40 42 43 44 45 46 47 31 41 50 % Specific lysis Peptide number MSI 44- 25 Bacterial peptides 100 0 1 2 6 7 8 9 3 4 5 12 16 11 13 14 15 17 18 19 20 21 22 10 75 Peptide number MSI 44- Clone LAU 203/1.5 50 % Specific lysis 25 0 48 49 50 52 53 54 55 56 57 58 59 60 62 63 64 65 66 67 68 69 70 72 51 61 71 Peptide number MSI 44- Peptide Recognition by T Cell Clone LAU 203/1.5 Clone NM/55-FLU

  24. Activity of cross reactive peptides relative to the native peptide Melan-A26-35

  25. Sequences of Melan-A Crossreactive Peptides

  26. Number of Active Peptides Based on Activity of Individual Peptides

  27. T cell are activated by mixtures of billions of peptides Degeneracy overcomes the fM concentration of each peptide Specificity explains the clear differentiation among active and not active mixtures Cross reactive peptides have been identified for all T cells studied Range of activities: Better than native to less than native Sequences: Natural and non natural occurring having single or multiple substitutions 1 to 4 residues are maintained among cross reactive peptides Combination of amino acids at certain positions are important Coexistence and balance of degeneracy and T cell specificity is clear Conclusions

  28. TPIMS/MSI Jon Appel Marc Giulianotti Adel Nefzi John Ostresh Lisa Osthues Patricia Norori Richard Houghten Biometrical analysis Roland Martin NIH Mireia Sospedra Silva Markovic-Plese Yindong Zhao NCI Richard Simon Melan Specific CTL Jean-Charles Cerottini Ludwig Pedro Romero Danila Valmori Acknowledgments Funding MSNRI and NIH

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