Abstract
The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation
chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now
on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further
engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in
other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma
model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with
MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using
recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells
to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also
described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are
described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of
established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of
mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other
therapeutic agents or adapted for the use of CAR T cells against different target antigens.
chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now
on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further
engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in
other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma
model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with
MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using
recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells
to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also
described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are
described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of
established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of
mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other
therapeutic agents or adapted for the use of CAR T cells against different target antigens.
| Original language | English |
|---|---|
| Journal | Journal of Visualized Experiments |
| Volume | 140 |
| Early online date | 16 Oct 2018 |
| DOIs | |
| Publication status | Published - 2018 |
Research Beacons, Institutes and Platforms
- Manchester Cancer Research Centre
