BIOTECHNOLOGICAL APPLICATIONS 8

1. Adoptive T Cell Therapy: Harnessing the Other Arm
of the Adaptive Immune System to Fight Cancer
and Other Disease
Provides the specificity for
T Cell Antigen presenting
cell interactions
CD28
B7 proteins, CD80
and CD86
Signal 1
Signal 2
• Signal 2 amplifies
intracellular signals
initiated by signal
1
• Both signals
needed for T cell
activation,
redundancy likely
contributes to
mechanisms of
tolerance to self
antigens where
only signal 1 may
occur Upregulated by
pathogens

2. The CD3 Complex is Required for Signal 1
CD3

3. After Activation T Cell Respond through an Autocrine
Loop Involving IL-2
T cell cytokine (growth
factor)

4. Summary of Accessory Proteins

5. Integrins Contribute to the Strength of T Cell Antigen
Presentation Cell Interactions
weak initial
interaction between
integrin and ICAM
MHC/antigen-receptor
interaction sends signal
to activate integrin
strengthened
interaction between
integrin and ICAM
leukocyte adhesion
deficiency
beta subunit -LFA1 integrin
repeated bacterial
infections
inside-out signaling

6. Many Things have to Come Together to Activate a T
Cell, which BTW is Probably a Good Thing

7. Autologous Adoptive Cell Therapy
Tumor infiltrating
lymphocytes

8. Lympho-Depletion Prior to Adoptive T Cell Therapy

9. H OH
HOH
O
N
CH
N
C
C
C
N
C
N
HOCH2
H
adenine
arabinose
NH2
F
Fludarabine
• Fludarabine inhibits DNA polymerase
• Fludarabine is a chain terminator if
incorporated into a growing DNA chain
• Fludarabine inhibits ribonucleotide
reductase

10. O
OHOH
BP-P-O-CH2
H H
O
HOH
BP-P-O-CH2
H H
ribonucleotide
reductase
Ribonucleotide reductase Converts Ribonucleotides to
Deoxyribonucleotides

11. Ribonucleotide Reductase: Properties
• Class I enzymes widely distributed
• Tetramer, R1 and R2 subunits
• 2 active sites at subunit interface
O
OH + 2Fe2+ + O2 + H+ + e
Fe3+
O2-
Fe3+
+ H2O
C
+
H
H
ENZ
C
H
H
ENZ
generation of tyrosine radical
on/off switch
substrate choice
• Multiple allosteric sites to regulate
enzyme activity and specificiy

12. Ribonucleotide Reductase: Insights into Reaction
Mechanism
OC
H
H
ENZ OHC
H
H
ENZ
C N OH
H
O
H2N
hydroxyurea
active enzyme inactive enzyme
tyrosine radical is distant from the enzyme’s
active site

13. OC
H
H
ENZ
OC
H
H
OC
H
H
OC
H
H
X active site
OC
H
H
ENZ
OC
H
H
OC
H
H
OC
H
H
S active site
Relay Brings the Tyrosine Radical to the
Ribonucleotide Reductase Active Site
H
H
H
H
H
H
H
H
NDP
dNDP
S

14. O
OHOH
BP-P-O-CH2
H H
3′ H exchanges
with solvent
Ribonucleotide Reductase: Insights into Reaction
Mechanism
O
HOH
BP-P-O-CH2
H H
ribonucleotide
reductase

15. freeenergy(G)
reaction progress
S
P
-ΔG (P-S)
activation energy
(uncatalyzed reaction)
activation energy
(catalyzed reaction)
energy state
of substrates
energy state
of products
T*
transition
state
S P
reaction spontaneous
as written
Enzyme Energetics
[ES]

16. O
OHOH
O
+OH2OH
O
OH
+
O
OH H
O
OH
BP-P-O-CH2
H
H
O
OHOH
BP-P-O-CH2
H H
H H H
H H
Enz-S
Enz-S
E
S H
S H
E
S -
S H
H2O
O
OH
+
H
E
S -
S H
E
S
S
H+
H -
Enz-S H
Enz-S
Ribonucleotide Reductase:
Reaction Mechanism
439
225
462
H
resonance
stabilization

17. 754
759
754
759
Regenerating Active Ribonucleotide Reductase
R1 R1
R2 R2
thioredoxin

18. E
S
S
E
S H
S H
NDP
dNDP
225/462
RR
E
S H
S H
E
S
S
754/759
RR
E
S H
S H
E
S
S
thioredoxin
NADPH
NADP+
thioredoxin
reductase
reducing equivalents
derived from nutrients
Regeneration of Free Thiol Groups in the
Ribonucleotide Reductase Reaction Cycle

19. Use of Tumor Infiltrating Lymphocytes to Treat
Melanoma
• Melanoma is particularly responsive to immune-based therapies
• IL-2 alone can show significant responses in melanoma
• Melanoma tumor associated antigens – MART1 and gp100???
• Exomic sequencing in 3,000 tumor/normal pairs reveals a mutation rate
in melanoma of ~ 100 non-synonymous mutations/Mb compared to 0.1
in certain pediatric tumors

20. (Thought Experiment) Use the Mutational Spectrum of
Melanoma as a Selection Tool

21. Genetically Engineered T Cells
Selecting for high affinity
receptors
Extreme toxicities

22. T Cell Receptor Accessory Proteins
CD3

23. LAT = linker of activated T
cells – links to downstream
signaling pathways
Comparison of Different T Cell Strategies

24. The Tumor Associated Antigen for CAR-T Studies
Reported Here is CD19

25. The Manufacturing Process for CAR-T Therapy
Can be performed in 10 days from blood
draw to reinfusion

26. James N. Kochenderfer et al. JCO 2015;33:540-549
CAR expression
central
memory T
cells
cytotoxicity
Anti-CD19 Chimeric Antigen Receptor (CAR) Design
and Function

27. Response to CAR-T Against CD19 in Patients with
Refractory Diffuse B Cell Lymphoma
mediastinal tumor
liver metastasis
splenic mass

28. CAR-T Toxicities