BIOTECHNOLOGICAL APPLICATIONS 9

2. Genetically Engineered T Cells
Selecting for high affinity
receptors
Extreme toxicitiesNormal T cell receptor/antigen/MCH
complexes are relatively low affinity
On-target, off tumor

3. Addressing the Issue of Acute Toxicity of
Genetically Engineered T cells

4. N
C
N
C
C
C
N
N
C
O
H2N
H
CH2
O
CH2
CH2HO
ATP ADP
HSV thymidine kinase
N
C
N
C
C
C
N
N
C
O
H2N
H
CH2
O
CH2
CH2OP
O
O-
O-
ACV-TP
cellular kinases
• acyclovir, first specific antiviral drug
• used to treat herpes simplex I and II and varicella zoster virus infections
Antiviral Drugs
promiscuous, broad substrate specificity
active drugcellular TK does not phosphorylate ACV

5. PPP-O-CH2
O
GC4
PPi
HSV DNA polymerase
* * * *
3′
C4
*
P-O-CH2
O
G
chain
termination
Acyclovir: Mechanism of Action
ACV binds with a 30-fold higher affinity to HSV DNA
polymerase relative to cellular polymerase
OH
OH
• ACV selectively phosphorylated by HSV TK
• ACV-TP binds selectively to HSV DNA pol
• ACV functions as a chain terminator
3′ - 5′ phosphodiester bonds

6. Addressing the Issue of Acute Toxicity of
Genetically Engineered T cells

7. Autologous Adoptive Cell Therapy
Immunosuppressive
environment
Even after reinfusion of
expanded and activated T
cells the immunosuppressive
environment of tumors and
natural T cell regulatory
mechanisms limit their
effectiveness, particularly in
solid tumors

8. What if Rather than Go through all the Bioengineering Involved
in this Thought Experiment We Instead Reactivated the Natural
Diverse T Cells Already Present in the Tumor in the Body?

9. An Alternative Approach to T Cell Therapy:
Immuno-Regulation
• Soon after T cells are activated a regulatory mechanism kicks in that begins to
turn them off
CTLA-4 is a member of the CD28 family of co-receptors
except it inhibits rather than activates T cells
CTLA = cytotoxic lymphocyte antigen
Upregulated
48 to 72
hours after
activation
Prevents
pathogenic
autoimmune
disorders

10. T Regulatory Cells Also Use CTLA-4 in Regulating T
Cell Activation
• Increased CTLA-4
• Increased CD25
• No production of IL-2
Tregs
CTLA-4
B7, CD80
Tregs
antigen
presenting
cell
• Interaction results
in internalization
and degradation
of CD80 on
antigen
presenting cells
• APCs can no
longer provide
signal 2 for T cell
activation
Ag
MHC

11. T Regulatory Cells Also Serve as a Sponge for IL-2
Tregs
• Tregs do not express
IL-2 but produce large
amounts of CD25, a
high affinity IL-2
receptor
IL-2
IL-2
IL-2
IL-2
• Binding of IL-2 to Tregs
stimulates Treg
proliferation and
deprives other T cell
types of IL-2

12. Lympho-Depletion Prior to Adoptive T Cell Therapy
Lympho-depletion may work in part by reducing the number of immunosuppressive
lymphocytes

13. Trials Using Ipilimumab, an antibody to CTLA-4, for
Metastatic Melanoma
Gp100 vaccine
Ipi alone
Ipi + gp100 vaccine
It’s not much, but it is a start
676 patients comparing Ipilimumab with and without
vaccination with GP100
GP100, a major TAA in melanoma
Hodi et al NEJM 2010

14. Combining Standard Chemotherapy With and
Without Ipilimumab
Bondarenko et al NEJM 2011
• Immunotherapies often work better with
combined with cytotoxic chemotherapies
• Standard of care chemotherapy for
melanoma is dacarbazine

15. Combining Ipilimumab with Standard Chemotherapy:
Overall Survival
Effect of Ipilimumab on overall survival disappointing

16. Dacarbazine is an Imadazotetrazine Prodrug
Imadazotetrazines are alkylating agents
temozolomide
dacarbazine
Methyldiazonium
ions active
alkylating agents

17. Alkylating Agent Efficacy can be
Influenced by DNA Repair Capacity
alkylating agents
N
C
N
C
C
C
N
N
C
1
2
3
4
5
6
7
8
9
O
H2N
CH3O6-methyl G can
base pair with C or T alkylation at N7 can
lead to depurination
promotes cleavage of N-glycosidic bond
10,000 depurination events per day even in the
absence of alkylating agents
MGMT – methylguanine methyl transferase
Temozolomide – alkylating agent used to treat
glioblastoma
overexpression of MGMT leads to
temozolomide resistance
accepts methyl group from
base inactivating enzyme
gaps can be repaired but if damage too great, the
guardian of the genome, p53, can induce cell
death
Dacarbazine (or temozolomide)

18. There’s More to the Tumor Immunosuppressive
Environment than the Down-Regulation of T Cells by
CTLA-4
• PD-1 is also a
negative
regulator of T
cell activation

19. The SHP Proteins are Tyrosine Phosphatases

20. Activation of SHP Phosphatases Disrupt Numerous
Signaling Pathways

21. Nivolumab Trials for Advanced Melanoma

22. Combining Anti-CTLA-4 and Anti-PD-1 Therapies for
Melanoma

23. Outcomes of the Trial
BRAF a tyrosine kinase involved in melanoma,
which can be targeted by a TKI

24. Adverse Events With New Immuno-modulatory
Agents
Immune related adverse events
Systems affected
• Skin – 3-4 weeks
• GI tract/liver – 6-7 weeks
• endocrine – 9 weeks
Patients who develop anti-CTLA-4
associated endocrinopathies may present
with nonspecific symptoms such as:
• fatigue
• headache
• nausea
• behavioral changes, visual impairments,
memory loss, decreased libido,
• anorexia,
• insomnia,
• cold or heat intolerance.
hypothalamic-pituitary-thyroidal axis