Contributions
Abstract: EP718
Type: E-Poster Presentation
Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research
Background
The overall survival in acute myeloid leukemia (AML) remains poor. New treatment options are needed. Early clinical trials have highlighted that adoptive transfer of natural killer (NK) cells can induce remission in patients with relapsed/refractory AML and high-risk myelodysplastic syndrome (MDS). Recent advances have now enabled investigators to explore the potential of genetically engineered NK cells to improve the efficacy.
Aims
The primary aim was to investigate whether ex vivo expanded human NK cells, engineered to overexpress the gain-of-function bone marrow (BM) homing receptor CXCR4R334X, can be utilized for improved leukemia clearance in vivo. In addition, we further aimed to explore the role for the CXCR4/SDF-1⍺ axis in NK cell BM homing by knocking out the CXCR4 gene.
Methods
A 4D-NucleofectorTM System (Lonza) was used to introduce CXCR4R334X-coding mRNA, or RNP-complex for CRISPR/Cas9 mediated CXCR4 gene-knock-out (KO), into ex vivo expanded human NK cells. RNA transcript levels and cell surface expression of CXCR4 was determined by q-RT-PCR and flow cytometry staining respectively. KO efficacy was evaluated by ddPCR. CD107a degranulation experiments, and Calcein-AM-based killing assays, were used to evaluate the in vitro leukemia targeting potential post electroporation. In vitro migration was assessed by transwell migration assays whereas NSG-SGM3 mice were used to evaluate the in vivo migration capacity. To assess the in vivo anti-leukemic potential, NSG-SMG3 mice were inoculated intrabone with MOLM-14 cells prior to NK cell treatment.
Results
Here, we demonstrate that ex vivo expanded human NK cells, mRNA-transfected to overexpress the gain-of-function bone marrow (BM) homing chemokine receptor CXCR4R334X, have a higher homing propensity for BM compartments in vivo leading to significantly improved leukemia rejection when adoptively infused into AML-bearing mice compared to control NK cells. Further, our data reveal that CXCR4R334X mRNA-transfected NK cells more efficiently delayed leukemia spreading to non-tumor-inoculated BM compartments. Importantly, neither the CRISPR/Cas9 or the mRNA transfection approach used in this study affected the ability of NK cells to target AML cells.
Conclusion
Collectively, these data provide proof-of-concept that genetic engineering of human NK cells to promote homing to BM compartments is an independent modality that can be optimized to further enhance the efficacy of NK cell-based immunotherapy of myeloid leukemia.
Keyword(s): Cancer immunotherapy, CXCR4, Homing, NK cell
Abstract: EP718
Type: E-Poster Presentation
Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research
Background
The overall survival in acute myeloid leukemia (AML) remains poor. New treatment options are needed. Early clinical trials have highlighted that adoptive transfer of natural killer (NK) cells can induce remission in patients with relapsed/refractory AML and high-risk myelodysplastic syndrome (MDS). Recent advances have now enabled investigators to explore the potential of genetically engineered NK cells to improve the efficacy.
Aims
The primary aim was to investigate whether ex vivo expanded human NK cells, engineered to overexpress the gain-of-function bone marrow (BM) homing receptor CXCR4R334X, can be utilized for improved leukemia clearance in vivo. In addition, we further aimed to explore the role for the CXCR4/SDF-1⍺ axis in NK cell BM homing by knocking out the CXCR4 gene.
Methods
A 4D-NucleofectorTM System (Lonza) was used to introduce CXCR4R334X-coding mRNA, or RNP-complex for CRISPR/Cas9 mediated CXCR4 gene-knock-out (KO), into ex vivo expanded human NK cells. RNA transcript levels and cell surface expression of CXCR4 was determined by q-RT-PCR and flow cytometry staining respectively. KO efficacy was evaluated by ddPCR. CD107a degranulation experiments, and Calcein-AM-based killing assays, were used to evaluate the in vitro leukemia targeting potential post electroporation. In vitro migration was assessed by transwell migration assays whereas NSG-SGM3 mice were used to evaluate the in vivo migration capacity. To assess the in vivo anti-leukemic potential, NSG-SMG3 mice were inoculated intrabone with MOLM-14 cells prior to NK cell treatment.
Results
Here, we demonstrate that ex vivo expanded human NK cells, mRNA-transfected to overexpress the gain-of-function bone marrow (BM) homing chemokine receptor CXCR4R334X, have a higher homing propensity for BM compartments in vivo leading to significantly improved leukemia rejection when adoptively infused into AML-bearing mice compared to control NK cells. Further, our data reveal that CXCR4R334X mRNA-transfected NK cells more efficiently delayed leukemia spreading to non-tumor-inoculated BM compartments. Importantly, neither the CRISPR/Cas9 or the mRNA transfection approach used in this study affected the ability of NK cells to target AML cells.
Conclusion
Collectively, these data provide proof-of-concept that genetic engineering of human NK cells to promote homing to BM compartments is an independent modality that can be optimized to further enhance the efficacy of NK cell-based immunotherapy of myeloid leukemia.
Keyword(s): Cancer immunotherapy, CXCR4, Homing, NK cell