Page 203 - 2021_03-Haematologica-web

Genome editing of donor-derived aβ+ T cells
We hypothesized that aβ haplo-HSCT in combination with CAR T cells generated through genome editing of donor-derived T cells could provide the foundation for an optimal approach that addresses these challenges (Figure 1D). We here show that the T-cell receptor (TCR) aβ+/CD19+ cell fraction that is removed from the graft can be used to engineer non-alloreactive CAR T cells through homologous recombination-mediated genome editing by targeted integration of a CD19-specific CAR in-frame into the TCR alpha chain (TRAC) locus (“aβTCR-CD19 CAR-T”), and demonstrate the antileukemic efficacy of this product in vitro and in vivo. This novel and innovative approach allows for the creation of two different cellular immunotherapy products, with complementary antileukemic mechanisms, from a single apheresis: the aβ haplo-HSCT which provides donor-derived natural killer cells, gδ T cells and the human leukocyte antigen (HLA)- dependent activity of polyclonal T cells, while the “left- over” cell fraction is salvaged to become a therapeutic CAR T-cell product with an HLA-independent mecha- nism and potential to improve cure rates without causing GvHD.
Methods
Plasmid cloning and adeno-associated virus production
Transfer plasmids were cloned between the inverted terminal repeat sequences in pAAV-MCS (Agilent Technologies). The CAR comprises a GM-CSFRa leader sequence, the FMC63 scFv,31 CD28 hinge, transmembrane and intracellular sequences and the CD3ζ intracellular domain. Recombinant adeno-associ- ated virus serotype 6 (rAAV6) production and titration are described in the Online Supplementary Methods.
T-cell culture and genome editing
All human cells were handled according to a protocol approved by the Institutional Review Board at Stanford University. The TCRaβ+/CD19+ cell fraction (non-target fraction from the graft manipulation procedure) was used Fresh or cryo- preserved. T cells were activated for 3 days and beads removed before electroporation. Electroporation and gene targeting were performed as previously described.32
In vitro cytokine measurement and cytotoxicity assay CD19+ Nalm6-GL cells or CD19+ Raji cells (GFP-Luc+) were used in co-culture assays with the CAR T cells or control T cells to determine interleukin-2 and interferon-g production of the
CAR T cells and cytotoxicity. For details see the Online Supplementary Methods.
In vivo xenograft assay
All experiments involving mice were performed according to
a protocol approved by the Administrative Panel on Laboratory Animal Care at Stanford University. CD19+ Nalm6-GL cells (5x105) were transplanted intravenously (i.v.) into 6- to 12-week old male NSG mice. Four days later, tumor burden was evaluat- ed by IVIS bioluminescence imaging (PerkinElmer) and the indi- cated numbers of CAR T cells or control cells injected i.v.. Tumor burden was followed weekly by IVIS imaging.
Off-target analysis
sgRNA target sites were identified and their specificity score calculated by bioinformatics (crispor.tefor.net33). COSMID (crispr.bme.gatech.edu34) was used to identify potential off-target sites in the human genome. For analysis of predicted off-targets, gene editing or mock treatment was performed on T cells from six different donors and predicted off-target sites sequenced using an Illumina MiSeq as described previously.35 For details see the Online Supplementary Methods.
Statistics
Plots show means with error bars representing either the stan- dard deviation or 95% confidence interval (95% CI), as indicat- ed. Groups were compared by statistical tests as described in the figure legends using Prism 7 (GraphPad). Asterisks indicate sta- tistical significance: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. All t tests are two-tailed.
Results
Apheresis and cell processing
aβ haplo-HSCT donors received granulocyte-colony stimulat- ing factor for 4 days at the total dose of 16 mg/kg body weight and apheresis was performed on the fifth day. If the CD34+ cell count was <40/mL on day 4, a CXCR4 antagonist (plerixafor, Mozobil) was given. Manipulations were performed in a closed system according to Good Manufacturing Practice (GMP) stan- dards with clinical grade reagents and instruments from Miltenyi Biotec (Bergisch Gladbach, Germany).
+
Genome editing on TCRaβ cells depleted from the
Depletion by magnetic bead activated cell sorting
We prospectively collected the TCRaβ+/CD19+ cell fraction (non-target fraction) removed from grafts during aβ haplo-HSCT procedures. It has recently been shown that homologous recombination-mediated genome edit- ing using Cas9 ribonucleoprotein (RNP) and AAV6 can mediate targeted integration of a CAR into the TRAC locus,36,37 with up to 50% of cells expressing the CAR. This approach offers the advantages that it establishes TCR knockout in the majority of CAR+ cells, avoids the risk of insertional mutagenesis of randomly-integrating viral vectors, and allows the cells to modulate CAR expression if the CAR is integrated in-frame into the endogenous locus.37
Depletion of TCRaβ+ cells was performed using reagents from Miltenyi according to the manufacturer’s instructions, except after coating with the Streptavidin-microbeads, when the cells were diluted and passed through the column without the wash- ing step. For details see the Online Supplementary Methods.
Antibodies used for flow cytometry
NGFR-APC, NGFR-PE, TCRaβ-FITC, CD19-A488, CD19- A700, CD62L-BV421, CD45RA-PE, CD4-PerCP-Cy5.5, CD8a- APC-Cy7 (all from Biolegend) were used. For CD45RA/CD62L staining, isotype controls as recommended by the manufacturer were used to determine positive and negative populations. The APC-conjugated CD19-CAR idiotype antibody was a gift from Crystal Mackall.
graft during aβ haploidentical hematopoietic stem cell transplanation to create chimeric antigen receptor Tcells
We used homologous recombination-based genome editing32 to integrate a CD19.28.ζ-CAR in-frame into the open-reading frame of the TRAC locus (Figures 1D and 2A), similarly to a recently described approach.37 Disruption of TRAC leads to loss of expression of the
haematologica | 2021; 106(3)
849