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

Catalytic

Self-assemblyProteins

Mechanicalwork

Therapeutics

Complexmachines

Signaling

GenesOn/off

Structural

Power of engineering

Understand underlying biology such that we can develop design rules to predictably manipulate systems

Basic science Applied science

Linear stringAmino acids

Folds to active protein

… TAC CAG TTA GAG … DNA

proteinY Q F E

I. Designer TCR technologyDiagnostics - track pMHCs in response toDisease & vaccination

Immunotherapeutics - target cancer/ virallyInfected cells, alter autoimmune disease

Basic science- ability to study properties in Vitro; low affinity techniques; design rules

GoalsControl affinity and specificity

ChallengesLow affinity (1-50 uM) - hard to study interactionRules for recognition unclear

Humancell

peptide MHC

T cell

TCR

0

50

100

150

200

250

300

mAU

0.0 5.0 10.0 15.0 ml

S75 SizeExclusion

0

20

40

60

80

100

120

140

160mAU

5.0 10.0 15.0 20.0 25.0 ml

Anion Exchange

DO

11.1

0 sc

TCR

MHC

TCR 1 2 3 4 5 6 7 8 9 10Wild-type C A A S P N Y N V L Y

1721 C A D D Q S N R Y17210 C A A G H R C N Y

1322 C A A * Y T R S P L Y1328 C A A S S F T P R L Y1744 C A A A L N Y H S L Y1746 C A A P R T A F N L Y

1761 C A A S T N WH N L Y2343 C A A V P N Y H L L Y

2144 C A A S Y A Y H Q L Y2146 C A A T P W L S R L Y

176-1, 0.2 nM 235-1, 20 nM DO11.10, 40 nM

Production

Control affinity

1

3

2

OVA 323-339/ I-Ad pMHC

DO11.10scTCR

1

3

2

Ac1-11 MBP/ I-Au pMHC

172.10scTCR

OVA 323-339/ I-Au pMHC

172.10scTCR

with DO11.10CDR 3 and

1

3

2

?

Control specificity

V8.2

V13.1

DO11.10 model T cell/ cancerDemonstrate the utility of

customized TCRs through in vitro competition experiments, in vivo tumor targeting

172.10 is an autoimmune TCRTarget: Myelin Basic Protein Ac1-11

Isolate high affinity mutant for targeted immunosuppression in mouse model of multiple sclerosis

I. Immediate applications

Structural Determination: NIH Structural InitiativeProvide insight to protein mechanism for vaccine and

drug designBioseparations: Control of Down Stream Process (ex Insulin) Controlled Drug Delivery

II. Building protein crystalsGoals:Understanding Lattice contacts:

weak interactions,

H-bonds, 2nd virial

Coeff.Hypercrystallizable Ab for

Scaffold

Challenges:Crystallization: Nucleation and GrowthLow % of proteins crystallized

II. Building protein crystalsCrystal lattice formation Crystal contacts: repeating units

III. Reverse engineering of pathogens

Bacteria Cellinvasin

FACs labeling of functional,surface Expressed Invasin

Model for vaccine Delivery system

III. Whooping coughToxin-mediated disease-- immunostimulatory and inhibitory aspects of PTx?-- neutralizing epitopes?

1B7

PTx-1B7 interaction

Bordetella pertussis Toxin mechanism

Power of engineeringUnderstand 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 techniquesRecombinant TechniquesProtein Expression in Eukaryotes and Bacteria (E. Coli)Protein CharacterizationProtein CrystallizationCell Culture

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.edu

Members of the lab from left to right: Ben Roy, Ryan Myhre, Tarik Khan, Jamie Sutherland and Jen Pai

Protein engineering for crystals

EE

Protein xWith EE tag

Anti-EEscaffold

Anti-EEAnti-EE CDRs onFramework knowcrystallizes

EE

Rakesh -- MS, Minnesota

Immune system Innate immunity

T cell receptors

TCR

Antibodies

Modularity of proteins

Examples

Entanercept: TNF receptor Antibody Fc binds TNF confers favorable PK

Immunotoxin: antibody PEA toxin targets tumor cell toxic to cell

Fine-tuning function

Examples

Affinity: Anti-toxin antibody (Maynard et al, Nat Biotech 2002) 2 AA changes lower Kd from 12 to 0.2 nM

TNF: Dominant negative -> inactivates trimer (Dahiyat et al, Science 2003)

Impact of simple physical insight…

Altman et al, Science 1996

B

BB

B

T cell surface with multiple identical TCRS

Kd 1-100 uM Kd ~4x better