The Complete Guide to CAR Cell in Vitro Analysis - PowerPoint PPT Presentation

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The Complete Guide to CAR Cell in Vitro Analysis

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As a revolution in the area of cancer treatment in recent years, immunotherapy is more specific and less toxic to patients compared to the traditional methods, such as invasive surgeries, radiation and chemotherapy. Chimeric antigen receptor (CAR)-engineered T cell therapy is the most promising approach with the remarkable ability to eliminate various kinds of tumors, especially for B cell malignancies, with up to 95% response rates and durable complete remission. – PowerPoint PPT presentation

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Title: The Complete Guide to CAR Cell in Vitro Analysis


1
The Complete Guide to CAR Cell in Vitro Analysis
2
CAR Cell in Vitro Analysis
CAR Expression Test
A
Cytokine Release Test
B
In Vitro Cytotoxicity Test
C
CAR Cell Proliferation Test
D
Viability and Bio-distribution Analysis
E
3
Part 1
4
CAR Expression Test
CAR modified T cell or Natural killer (NK) cell
has been becoming an attractive immunotherapy for
treating many types of cancer. In order to obtain
high-quality CAR-T cells prior to scientific
studies, preclinical researches, and clinical
trials, transgene expression is evaluated for
gene transfection efficiency and the reactivity
of engineered lymphocytes to antigen. Ideal gene
transfer means that exogenous DNA can be
incorporated into the host genome and stable
long-term expression. To ensure the best outcome,
the expression level of CAR gene should be tested
at different stages during the whole process of
CAR-T cell therapy, including post-transfection
with CAR gene, cell preparation, and clinical
treatment period. Generally, the methods include
western blot (WB), enzyme-linked immunosorbent
assay (ELISA), flow cytometry (FCM),
immune-histochemical (IHC), etc.
5
Part 3
6
In Vitro Cytotoxicity Test
CAR engrafted T cell or NK cell may be toxic to
normal cells that also express some tumor
associated antigens on cell surface, however,
this possibility can be evaluated via
cytotoxicity analysis. Meanwhile, as an efficient
alternative strategy, CARs can be expressed with
an appropriate suicide gene that can regulate
this potential cytotoxicity in order to kill the
cancer cells without influencing the normal ones.
7
Part 4
8
CAR Cell Proliferation Test
The second and the third generation CAR products
can enhance the persistence of CAR-T cells or -NK
cells proliferation. Meanwhile, this activation
and proliferation process can also be evaluated
via different proliferation assay approaches such
as MTT, MTS, Cell Counting Kit-8 (CCK-8), 5,
6-carboxyfluorescein diacetate succinimidyl ester
(CFSE) in combination with flow cytometric
analysis, 3H thymidine incorporation and other
techniques. At the same time, cytokines
concentration (e.g. IL-2) assay is also available
for determining T cell proliferation.
9
Part 5
10
Viability and Bio-distribution Analysis
Since a major obstacle associated with current
CAR-T cell therapy is that the limited
replicative lifespan of CAR-T cells prohibits the
in vivo expansion and long-term persistence of
these cells, potentially hindering the long-term
therapeutic efficacy of CAR-T cells, verification
of the normal ability of CAR-T cells to
proliferate in cultural and physiological
conditions manifests as the first critical
evaluation parameter.
11
Part 2
12
CAR Expression Test
The second and the third generation CAR products
can enhance the persistence of CAR-T cells or -NK
cells proliferation. Meanwhile, this activation
and proliferation process can also be evaluated
via different proliferation assay approaches such
as MTT, MTS, Cell Counting Kit-8 (CCK-8), 5,
6-carboxyfluorescein diacetate succinimidyl ester
(CFSE) in combination with flow cytometric
analysis, 3H thymidine incorporation and other
techniques. At the same time, cytokines
concentration (e.g. IL-2) assay is also available
for determining T cell proliferation.
13
Part 2
14
CAR Expression Test
The second and the third generation CAR products
can enhance the persistence of CAR-T cells or -NK
cells proliferation. Meanwhile, this activation
and proliferation process can also be evaluated
via different proliferation assay approaches such
as MTT, MTS, Cell Counting Kit-8 (CCK-8), 5,
6-carboxyfluorescein diacetate succinimidyl ester
(CFSE) in combination with flow cytometric
analysis, 3H thymidine incorporation and other
techniques. At the same time, cytokines
concentration (e.g. IL-2) assay is also available
for determining T cell proliferation.
15
As a global company, we have more than 200
talented and well-trained scientists located in
different continents working closely with
partners from the entire world to develop and
produce medicines of tomorrow. Specifically, we
are the established leading expert in TCR and CAR
TNK cell immune therapy development, as we offer
the one-stop custom services that cover the
entire new drug development pipeline.
Creative Biolabs
16
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17
Thanks
Creative Biolabs
www.creative-biolabs.com/car-t
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