edited by : Qussai Abbas

1. Qussay Abbas
‫السورية‬ ‫العربية‬ ‫الجمهورية‬
‫للمتميزين‬ ‫الوطني‬ ‫المركز‬ ‫خريجي‬
‫ّة‬‫ي‬‫الحيو‬ ‫الطبية‬ ‫العلوم‬ ‫برنامج‬

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
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4. 4

5. CD19 is expressed on
the surface of most B
cell malignancies
5

6. • Interleukin 15 (IL-15) regulates T cells
activation and proliferation and it ‘s
important for T cells Survival and expansion.
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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.
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8. (iC9/CAR.19/IL-15)
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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...
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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.
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12. 12

13. Daudi and Raji (CD19+ Burkitt lymphoma cell
lines)
HDLM-2 (CD30+ CD19- Hodgkin’s lymphoma cell
line)
Karpas-299 (CD30+ CD19- anaplastic lymphoma
cell line)
K562 (chronic erythroid leukemia cell line)
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14. CAR.19
retroviral vector
(iC9/CAR.19/IL-15)
retroviral vector
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15. Isolation.
Transduction on day 3
Expansion using IL-2 and then used
for the experiments
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16. 1- Cytokine production
2- T-cell expansion.
3- T-cell division and death.
4- Antitumor effects.
5- Activation of the suicide
gene.
6-Cytotoxic activity.
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17. Trafficking and expansion of
CAR+ cells.
Antitumor effect of CAR+ T
cells.
Validation of the suicide gene.
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19. A. Measurement of IL-15 production :
• Control NT T lymphocytes
• CAR.19+ T lymphocytes
• iC9/CAR.19/IL-15+ T lymphocytes
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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
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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 …
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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.
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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.
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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+
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25. SCID (severe combined immunodeficiency) mouse
lymphoma xenograft.
I.V inoculation of CD19+ lymphoma cell lines,
labeled with FFLuc.
bioluminescence imaging system.
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26. Daudi had engrafted diffusely in
bone marrow, lymphnodes and
spleen .
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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.
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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.
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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
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