TARGETING FLT3 WITH CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS CONFERS POTENT REACTIVITY AGAINST ACUTE MYELOID LEUKEMIA (AML)
(Abstract release date: 05/18/17)
EHA Library. JETANI H. 06/23/17; 181432; S145
HARDIKKUMAR JETANI
Contributions
Contributions
Abstract
Abstract: S145
Type: Oral Presentation
Presentation during EHA22: On Friday, June 23, 2017 from 12:30 - 12:45
Location: Room N109
Background
Adoptive immunotherapy with chimeric antigen receptor (CAR)-modified T cells has therapeutic potential in hematologic malignancies. We are pursuing FMS like tyrosine kinase 3 (FLT3) as a novel CAR target in acute myeloid leukemia (AML). FLT3 is a homodimeric transmembrane protein with uniform expression on AML, irrespective of cytogenetic and histomorphologic subtype. FLT3 provides survival signals to AML blasts and is a key driver of leukemia-genesis in AML cases with internal tandem duplication (FLT3-ITD). These attributes suggest FLT3 may be an ‘Achilles heel’, making AML blasts susceptible to CAR T-cell mediated recognition and elimination.
Aims
We therefore explored the anti-leukemia efficacy of FLT3-CAR modified T cells against FLT3-ITD+ and FLT3 wild type AML in pre-clinical models in vitro and in vivo.
Methods
A FLT3-CAR comprising a single-chain variable fragment (4G8), fused to an IgG-Fc spacer, and signaling module with CD3 zeta and CD28 was encoded in a lentiviral vector (epHIV7) for gene-transfer into CD8+ and CD4+ T cells of healthy donors (n>4) and AML patients. CAR T-cell mediated cytolytic activity was evaluated in FACS-/luminescence-based assays, cytokine production analyzed by ELISA and proliferation assessed by CFSE dye dilution. Immunodeficient NSG mice were engrafted with AML cell line (Molm-13) or primary AML blasts and treated with 5x106 CAR-modified or control T cells (CD8:CD4 ratio=1:1).
Results
We confirmed specific recognition and high-level cytolytic activity of CD8+ FLT3-CAR T cells against a panel of AML cell lines including THP-1 (FLT3 wild type), and Molm-13 (FLT3-ITD heterozygous). Both CD8+ and CD4+ FLT3-CAR T cells produced IFN-γ and IL-2, and underwent proliferation after antigen stimulation. FLT3-CAR T cells that we prepared from AML patients exerted specific anti-leukemia reactivity against autologous primary AML blasts, with near-complete cytolysis within 24 hours of co-culture. Further, FLT3-CAR T cells conferred a potent anti-leukemia effect in in vivo models of systemic leukemia, both with AML cell lines (Molm-13) and primary AML blasts. A single dose of FLT3-CAR T cells conferred complete eradication of leukemia from peripheral blood, bone marrow and spleen, as confirmed by bioluminescence imaging and flow cytometry.
FLT3 is not expressed in any normal solid tissues and mature hematopoietic cells, but shows limited expression in hematopoietic progenitors and hematopoietic stem cells (HSCs). Preliminary data show that FLT3-CAR T cells recognize FLT3+/high normal HSCs and interfere with normal hematopoiesis, but preserve a proportion of HSCs capable of reconstituting hematopoietic lineages. Studies to assess recognition of normal HSCs in vivo are ongoing.
Conclusion
Collectively, our data demonstrate that T cells expressing a FLT3-specific CAR mediate potent reactivity against FLT3 wild type and FLT3-ITD+ AML in vitro and in vivo, and establish FLT3 as a novel CAR target in AML. FLT3-ITD positivity identifies a high-risk AML subgroup that may particularly benefit from adoptive therapy with FLT3-CAR T cells, e.g. in order to achieve ‘minimal residual disease’ (MRD) negativity prior to allogeneic HSC transplantation. Our data further suggest that in contrast to CD33 and CD123, which are pursued as alternative CAR targets in AML, targeting of FLT3 may preserve a fraction of normal HSC and enable the implementation of CAR therapy outside the transplant setting.
Session topic: 24. Gene therapy, cellular immunotherapy and vaccination
Keyword(s): FLT3, Cancer immunotherapy, Acute Myeloid Leukemia
Abstract: S145
Type: Oral Presentation
Presentation during EHA22: On Friday, June 23, 2017 from 12:30 - 12:45
Location: Room N109
Background
Adoptive immunotherapy with chimeric antigen receptor (CAR)-modified T cells has therapeutic potential in hematologic malignancies. We are pursuing FMS like tyrosine kinase 3 (FLT3) as a novel CAR target in acute myeloid leukemia (AML). FLT3 is a homodimeric transmembrane protein with uniform expression on AML, irrespective of cytogenetic and histomorphologic subtype. FLT3 provides survival signals to AML blasts and is a key driver of leukemia-genesis in AML cases with internal tandem duplication (FLT3-ITD). These attributes suggest FLT3 may be an ‘Achilles heel’, making AML blasts susceptible to CAR T-cell mediated recognition and elimination.
Aims
We therefore explored the anti-leukemia efficacy of FLT3-CAR modified T cells against FLT3-ITD+ and FLT3 wild type AML in pre-clinical models in vitro and in vivo.
Methods
A FLT3-CAR comprising a single-chain variable fragment (4G8), fused to an IgG-Fc spacer, and signaling module with CD3 zeta and CD28 was encoded in a lentiviral vector (epHIV7) for gene-transfer into CD8+ and CD4+ T cells of healthy donors (n>4) and AML patients. CAR T-cell mediated cytolytic activity was evaluated in FACS-/luminescence-based assays, cytokine production analyzed by ELISA and proliferation assessed by CFSE dye dilution. Immunodeficient NSG mice were engrafted with AML cell line (Molm-13) or primary AML blasts and treated with 5x106 CAR-modified or control T cells (CD8:CD4 ratio=1:1).
Results
We confirmed specific recognition and high-level cytolytic activity of CD8+ FLT3-CAR T cells against a panel of AML cell lines including THP-1 (FLT3 wild type), and Molm-13 (FLT3-ITD heterozygous). Both CD8+ and CD4+ FLT3-CAR T cells produced IFN-γ and IL-2, and underwent proliferation after antigen stimulation. FLT3-CAR T cells that we prepared from AML patients exerted specific anti-leukemia reactivity against autologous primary AML blasts, with near-complete cytolysis within 24 hours of co-culture. Further, FLT3-CAR T cells conferred a potent anti-leukemia effect in in vivo models of systemic leukemia, both with AML cell lines (Molm-13) and primary AML blasts. A single dose of FLT3-CAR T cells conferred complete eradication of leukemia from peripheral blood, bone marrow and spleen, as confirmed by bioluminescence imaging and flow cytometry.
FLT3 is not expressed in any normal solid tissues and mature hematopoietic cells, but shows limited expression in hematopoietic progenitors and hematopoietic stem cells (HSCs). Preliminary data show that FLT3-CAR T cells recognize FLT3+/high normal HSCs and interfere with normal hematopoiesis, but preserve a proportion of HSCs capable of reconstituting hematopoietic lineages. Studies to assess recognition of normal HSCs in vivo are ongoing.
Conclusion
Collectively, our data demonstrate that T cells expressing a FLT3-specific CAR mediate potent reactivity against FLT3 wild type and FLT3-ITD+ AML in vitro and in vivo, and establish FLT3 as a novel CAR target in AML. FLT3-ITD positivity identifies a high-risk AML subgroup that may particularly benefit from adoptive therapy with FLT3-CAR T cells, e.g. in order to achieve ‘minimal residual disease’ (MRD) negativity prior to allogeneic HSC transplantation. Our data further suggest that in contrast to CD33 and CD123, which are pursued as alternative CAR targets in AML, targeting of FLT3 may preserve a fraction of normal HSC and enable the implementation of CAR therapy outside the transplant setting.
Session topic: 24. Gene therapy, cellular immunotherapy and vaccination
Keyword(s): FLT3, Cancer immunotherapy, Acute Myeloid Leukemia
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