1. Lab for engineering in infection & immunity Engineering proteins to control immune responses and bacterial pathogenesis Jennifer Maynard, Dept. Chemical Engineering Bioreactor Cell as reactor Products Interactions

2. Catalytic Self- assembly Proteins Mechanical work Therapeutics Complex machines Signaling Genes On/off Structural

3. Power of engineering Understand underlying biology such that we can develop design rules to predictably manipulate systems Basic science Applied science

4. Linear string Amino acids Folds to active protein … TAC CAG TTA GAG … DNA protein YQFE

5. I. Designer TCR technology Diagnostics - track pMHCs in response to Disease & vaccination Immunotherapeutics - target cancer/ virally Infected cells, alter autoimmune disease Basic science- ability to study properties in Vitro; low affinity techniques; design rules Goals Control affinity and specificity Challenges Low affinity (1-50 uM) - hard to study interaction Rules for recognition unclear Human cell peptide MHC T cell TCR

6. MHC TCR 176-1, 0.2 nM 235-1, 20 nM DO11.10, 40 nM Production Control affinity Control specificity

7. V  8.2 V  13.1 DO11.10 model T cell/ cancer Demonstrate the utility of customized TCRs through in vitro competition experiments, in vivo tumor targeting 172.10 is an autoimmune TCR Target: Myelin Basic Protein Ac1-11 Isolate high affinity mutant for targeted immunosuppression in mouse model of multiple sclerosis I. Immediate applications

8. Structural Determination: NIH Structural Initiative Provide insight to protein mechanism for vaccine and drug design Bioseparations: Control of Down Stream Process (ex Insulin) Controlled Drug Delivery II. Building protein crystals Goals: Understanding Lattice contacts: weak interactions, H-bonds, 2nd virial Coeff. Hypercrystallizable Ab for Scaffold Challenges: Crystallization: Nucleation and Growth Low % of proteins crystallized

9. II. Building protein crystals Crystal lattice formationCrystal contacts: repeating units

10. III. Reverse engineering of pathogens Bacteria Cell invasin FACs labeling of functional,surface Expressed Invasin Model for vaccine Delivery system

11. III. Whooping cough Toxin-mediated disease -- immunostimulatory and inhibitory aspects of PTx? -- neutralizing epitopes? 1B7 PTx-1B7 interaction Bordetella pertussis Toxin mechanism

12. Power of engineering Understand underlying biology such that we can develop design rules to predictably manipulate systems Basic science Applied science Focus on TCRs and Antibody fragments with the following techniques Recombinant Techniques Protein Expression in Eukaryotes and Bacteria (E. Coli) Protein Characterization Protein Crystallization Cell Culture

13. Maynard laboratory will be offering 2 graduate research assistant Positions in Fall 2009 in the areas of T cell receptors and protein expression Contact: maynard@che.utexas.edumaynard@che.utexas.edu Members of the lab from left to right: Ben Roy, Ryan Myhre, Tarik Khan, Jamie Sutherland and Jen Pai

15. Protein engineering for crystals EE Protein x With EE tag Anti-EE scaffold Anti-EE Anti-EE CDRs on Framework know crystallizes EE Rakesh -- MS, Minnesota

16. Immune system Innate immunity T cell receptors TCR Antibodies

17. Modularity of proteins Examples Entanercept: TNF  receptorAntibody Fc binds TNF  confers favorable PK Immunotoxin: antibodyPEA toxin targets tumor celltoxic to cell

18. Fine-tuning function Examples Affinity: Anti-toxin antibody (Maynard et al, Nat Biotech 2002) 2 AA changes lower K d from 12 to 0.2 nM TNF  : Dominant negative -> inactivates trimer (Dahiyat et al, Science 2003)

19. Impact of simple physical insight… Altman et al, Science 1996 B BB B T cell surface with multiple identical TCRS K d 1-100 uM K d ~4x better