Similar to Engineering cd19 specific t lymphocytes with interleukin-15 and a suicide gene to enhance their anti lymphoma or leukemia effects and safety
Similar to Engineering cd19 specific t lymphocytes with interleukin-15 and a suicide gene to enhance their anti lymphoma or leukemia effects and safety (20)
2. V Hoyos1 , B Savoldo1,2, C Quintarelli1 , A Mahendravada1 , M Zhang1 , J Vera1 , HE Heslop1,2,3, CM Rooney1,2,3,4,5, MK Brenner1,2,3
and G Dotti1,3,4
Received : 14 January 2013
accepted : 16 March 2013
published : online 29 April 2014
2
6. • Interleukin 15 (IL-15) regulates T cells
activation and proliferation and it ‘s
important for T cells Survival and expansion.
6
7. • A suicide gene, in genetics, will cause a
cell to kill itself through apoptosis.
• Caspases (cysteine-aspartic proteases, cysteine
aspartases or cysteine-dependent aspartate-
directed proteases) are a family of protease
enzymes playing essential roles in
programmed cell death.
• Can be pharmacologically activated with
CID to eliminate cells as required.
7
9. The in vivo survival, expansion and anti-lymphoma activity of CAR.19+ T
cells remain even when the second and third generation of
CARs occurs...
9
10. Engineering a CAR-modified T cells
to enhance their survival and
expansion in vivo , and decrease the
risk of direct toxicity and
uncontrolled proliferation of these
transgenic cells.
10
19. A. Measurement of IL-15 production :
• Control NT T lymphocytes
• CAR.19+ T lymphocytes
• iC9/CAR.19/IL-15+ T lymphocytes
19
20. C. T cells survival in the absence antigen stimulation:
• Neither CAR.CD19+ T cells nor
iC9/CAR.19/IL-15+ T cells
significantly expanded in the
absence of antigen stimulation .
• The viability of iC9/CAR.19/IL-15+ T
cells in the absence of antigen
stimulation was, however, preserved
for long term (4–5 weeks) compared
with control NT or CAR.19 + T cells
20
21. B. The expansion of T cells upon weekly stimulation with CD19+ B-CLL cells:
• IC9/CAR.19/IL-15+ T cell numbers
increased 10-fold compared with
CAR.19+ T cells…..
(157 ×106 vs 15 ×106)
after 5 weeks of culture …
21
22. IL-15 production enhanced the elimination of tumor cells :
We cocultured T cells with Karpas cells modified to stably
express the CD19 molecule (Karpas CD30+ CD19+)
After 4 to 5 days in an initial T cell and tumor cell ratio of 5:1,
residual tumor cells were quantified by FACS analysis
enumerating CD30+ tumor cells.
23
23. IL-15 production enhanced the elimination of tumor cells :
We cocultured T cells with Karpas cells modified to stably
express the CD19 molecule (Karpas CD30+ CD19+)
After 4 to 5 days in an initial T cell and tumor cell ratio of 5:1,
residual tumor cells were quantified by FACS analysis
enumerating CD30+ tumor cells.
24
24. When T-cell lines were maintained in culture for 4 weeks, and stimulated weekly with CD19+ B-CLL
cells, only iC9/CAR.19/IL-15+ T cells maintained their ability to completely eliminate tumor cells from
the culture.
iC9/CAR.19/IL-15+CAR.19+
25
28. T-cell trafficking to the tumor and T-cell persistence in vivo:
• CAR.19 + and iC9/CAR.19/IL-15 + T cells were labeled with eGFP-FFLuc, and infused in
mice previously engrafted with either unlabeled Daudi cells.
• both CAR.19 + and iC9/CAR.19/IL-15 + T cells localized at the tumor site.
31
29. • SCID mice were engrafted with Daudi cells labeled with FFLuc.
• After 7 days, these mice were treated with two weekly of control NT, CAR.19+ or iC9/CAR.19/IL-15+
T cells.
• Tumor growth was monitored by measuring changes in tumor bioluminescence over time.
• Daudi cells rapidly increased in mice receiving control NT T cells.
• Transient control in recipients of CAR.19+ T cells.
• Greatest control in recipients of iC9/CAR.19/IL-15+ T cells. 32
30. • We incorporated in a suicide gene based on the inducible caspase-9 gene.
• The addition of 50 nM CID to cultures of iC9/CAR.19/IL-15 T cells induced apoptosis
/necrosis of > 95% of transgenic cells within 24 h, as assessed by annexin-V-7AAD staining.
iC9/CAR.19/IL-15+T cells are eliminated after activation of the suicide gene by exposure to
the small-molecule CID.
33
31. • The suicide gene was also effective in vivo.
• Mice were engrafted i.v. with Raji tumor cells
and then infused with eGFP-FFLuc labeled
iC9/CAR.19/IL-15+ T cells.
• These cells localized and expanded at the
tumor site by day 14 after infusion as
assessed by bioluminescence measurement.
T-cell bioluminescence drastically reduced
after administration of the CID, consistent
with a significant elimination of the
transgenic cells.
iC9/CAR.19/IL-15+T cells are eliminated after activation of the suicide gene by exposure to
the small-molecule CID
34