Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity

Abstract

Chimeric antigen receptors (CARs) have been used to redirect the specificity of autologous T cells against leukemia and lymphoma with promising clinical results. Extending this approach to allogeneic T cells is problematic as they carry a significant risk of graft-versus-host disease (GVHD). Natural killer (NK) cells are highly cytotoxic effectors, killing their targets in a non-antigen-specific manner without causing GVHD. Cord blood (CB) offers an attractive, allogeneic, off-the-self source of NK cells for immunotherapy. We transduced CB-derived NK cells with a retroviral vector incorporating the genes for CAR-CD19, IL-15 and inducible caspase-9-based suicide gene (iC9), and demonstrated efficient killing of CD19-expressing cell lines and primary leukemia cells in vitro, with marked prolongation of survival in a xenograft Raji lymphoma murine model. Interleukin-15 (IL-15) production by the transduced CB-NK cells critically improved their function. Moreover, iC9/CAR.19/IL-15 CB-NK cells were readily eliminated upon pharmacologic activation of the iC9 suicide gene. In conclusion, we have developed a novel approach to immunotherapy using engineered CB-derived NK cells, which are easy to produce, exhibit striking efficacy and incorporate safety measures to limit toxicity. This approach should greatly improve the logistics of delivering this therapy to large numbers of patients, a major limitation to current CAR-T-cell therapies.

Fig. 1. Anti-tumor function of CB-NK cells transduced with the iC9/CAR.19/IL15 vector

Panels A and B summarize the cytotoxic activity of iC9/CAR.19/IL15-transduced CB-NK cells (CAR, solid lines) vs. NT CB-NK cells (broken lines), as measured by ⁵¹Cr release assay, against Raji (n=18) (A) and primary CLL cells (n=6) (B). CAR transduced NK cell kill CD19 expressing targets more efficiently than non-transduced (NT) ex vivo expanded and activated NK cells (p<0.001, all comparisons). CAR-transduced NK cells (solid blue line) were equally efficient as NT NK cells (broken blue line) in killing K562 targets. Data are presented as specific lysis relative to K562 targets(20) to correct for inter-donor variability in killing. (C) Cytokine production and CD107a degranulation by flow cytometry in gated CAR positive vs. CAR negative NK cells in response to different stimuli in 8 independent experiments, showing that CAR transduction of CB-NK cells significantly increased their cytokine effector response (IFN-γ and TNF-α production) and CD107a degranulation to the CD19-expressing Raji cell line and primary CLL cells. The effector function of both iC9/CAR.19/IL15-transduced CB-NK-CAR and non-transduced NK cells against K562 was similar.

Fig. 1. Anti-tumor function of CB-NK cells transduced with the iC9/CAR.19/IL15 vector

Panels A and B summarize the cytotoxic activity of iC9/CAR.19/IL15-transduced CB-NK cells (CAR, solid lines) vs. NT CB-NK cells (broken lines), as measured by ⁵¹Cr release assay, against Raji (n=18) (A) and primary CLL cells (n=6) (B). CAR transduced NK cell kill CD19 expressing targets more efficiently than non-transduced (NT) ex vivo expanded and activated NK cells (p<0.001, all comparisons). CAR-transduced NK cells (solid blue line) were equally efficient as NT NK cells (broken blue line) in killing K562 targets. Data are presented as specific lysis relative to K562 targets(20) to correct for inter-donor variability in killing. (C) Cytokine production and CD107a degranulation by flow cytometry in gated CAR positive vs. CAR negative NK cells in response to different stimuli in 8 independent experiments, showing that CAR transduction of CB-NK cells significantly increased their cytokine effector response (IFN-γ and TNF-α production) and CD107a degranulation to the CD19-expressing Raji cell line and primary CLL cells. The effector function of both iC9/CAR.19/IL15-transduced CB-NK-CAR and non-transduced NK cells against K562 was similar.

Fig. 1. Anti-tumor function of CB-NK cells transduced with the iC9/CAR.19/IL15 vector

Panels A and B summarize the cytotoxic activity of iC9/CAR.19/IL15-transduced CB-NK cells (CAR, solid lines) vs. NT CB-NK cells (broken lines), as measured by ⁵¹Cr release assay, against Raji (n=18) (A) and primary CLL cells (n=6) (B). CAR transduced NK cell kill CD19 expressing targets more efficiently than non-transduced (NT) ex vivo expanded and activated NK cells (p<0.001, all comparisons). CAR-transduced NK cells (solid blue line) were equally efficient as NT NK cells (broken blue line) in killing K562 targets. Data are presented as specific lysis relative to K562 targets(20) to correct for inter-donor variability in killing. (C) Cytokine production and CD107a degranulation by flow cytometry in gated CAR positive vs. CAR negative NK cells in response to different stimuli in 8 independent experiments, showing that CAR transduction of CB-NK cells significantly increased their cytokine effector response (IFN-γ and TNF-α production) and CD107a degranulation to the CD19-expressing Raji cell line and primary CLL cells. The effector function of both iC9/CAR.19/IL15-transduced CB-NK-CAR and non-transduced NK cells against K562 was similar.

Fig. 2. Assessments of immunologic synapse formation and function in human iC9/CAR.19/IL15-transduced CB-NK cells

(A) Confocal microscopy showing representative synapse images of CB-NK cells (transduced with iC9/CAR.19/IL15) conjugated to primary CLL cells. Conjugates were stained with anti-perforin (green), phalloidin-F-actin (red) and anti-CD19-CAR (yellow). Note formation of immunological synapse (black arrow; left panels). (B- top panel) Confocal representative images (original magnification ×100) demonstrating that CD19-specific CAR on NK cells preferentially accumulates at the CLL (target) cell synapse and not at the K562 (non-target) cell synapse. Cells were imaged in Z stacks covering the entire volume of the immunological synapse. Imaging was performed on a Leica TCS SP8 confocal microscope using a 100X oil objective. Images were acquired with Imaris software (Bitplane). Transmitted light (TL) overlay, single-color anti–CD19 CAR (blue), anti-perforin (red) and an overlay of all stains are shown. (B- bottom panel) summarizes data on the accumulation of CD19-specific CARs at the immunologic synapse between CB-NK cells transduced with iC9/CAR.19/IL15 vector with CLL cells (CD19 positive) vs. K562 targets (CD19 negative). *p= 0.02. There was significantly more accumulation of CD19-specific CARs at the immunologic synapse with CLL cells compared to K562 targets. (C) iC9/CAR.19/IL15-transduced CB NK cells, CAR.19-transduced CB NK cells (without IL-15) and CLL patient-derived NK cells transduced with iC9/CAR.19/IL15 were assessed and compared with non-transduced NK cells for their ability to polarize lytic granules and MTOC to CLL targets (left panel) vs. K562 cells (right) (measured by distance from the MTOC to the immune synapse). Results from two independent experiments are shown; each data point represents a single immunologic synapse. Cells were imaged as a Z stack on a Leica TCS SP8 laser scanning microscope. Images were acquired with Volocity software (PerkinElmer). The asterisk indicates statistical significance (P < 0.05 by Student’s t test) vs. the control or another CAR construct.

Fig. 2. Assessments of immunologic synapse formation and function in human iC9/CAR.19/IL15-transduced CB-NK cells

(A) Confocal microscopy showing representative synapse images of CB-NK cells (transduced with iC9/CAR.19/IL15) conjugated to primary CLL cells. Conjugates were stained with anti-perforin (green), phalloidin-F-actin (red) and anti-CD19-CAR (yellow). Note formation of immunological synapse (black arrow; left panels). (B- top panel) Confocal representative images (original magnification ×100) demonstrating that CD19-specific CAR on NK cells preferentially accumulates at the CLL (target) cell synapse and not at the K562 (non-target) cell synapse. Cells were imaged in Z stacks covering the entire volume of the immunological synapse. Imaging was performed on a Leica TCS SP8 confocal microscope using a 100X oil objective. Images were acquired with Imaris software (Bitplane). Transmitted light (TL) overlay, single-color anti–CD19 CAR (blue), anti-perforin (red) and an overlay of all stains are shown. (B- bottom panel) summarizes data on the accumulation of CD19-specific CARs at the immunologic synapse between CB-NK cells transduced with iC9/CAR.19/IL15 vector with CLL cells (CD19 positive) vs. K562 targets (CD19 negative). *p= 0.02. There was significantly more accumulation of CD19-specific CARs at the immunologic synapse with CLL cells compared to K562 targets. (C) iC9/CAR.19/IL15-transduced CB NK cells, CAR.19-transduced CB NK cells (without IL-15) and CLL patient-derived NK cells transduced with iC9/CAR.19/IL15 were assessed and compared with non-transduced NK cells for their ability to polarize lytic granules and MTOC to CLL targets (left panel) vs. K562 cells (right) (measured by distance from the MTOC to the immune synapse). Results from two independent experiments are shown; each data point represents a single immunologic synapse. Cells were imaged as a Z stack on a Leica TCS SP8 laser scanning microscope. Images were acquired with Volocity software (PerkinElmer). The asterisk indicates statistical significance (P < 0.05 by Student’s t test) vs. the control or another CAR construct.

Fig. 2. Assessments of immunologic synapse formation and function in human iC9/CAR.19/IL15-transduced CB-NK cells

(A) Confocal microscopy showing representative synapse images of CB-NK cells (transduced with iC9/CAR.19/IL15) conjugated to primary CLL cells. Conjugates were stained with anti-perforin (green), phalloidin-F-actin (red) and anti-CD19-CAR (yellow). Note formation of immunological synapse (black arrow; left panels). (B- top panel) Confocal representative images (original magnification ×100) demonstrating that CD19-specific CAR on NK cells preferentially accumulates at the CLL (target) cell synapse and not at the K562 (non-target) cell synapse. Cells were imaged in Z stacks covering the entire volume of the immunological synapse. Imaging was performed on a Leica TCS SP8 confocal microscope using a 100X oil objective. Images were acquired with Imaris software (Bitplane). Transmitted light (TL) overlay, single-color anti–CD19 CAR (blue), anti-perforin (red) and an overlay of all stains are shown. (B- bottom panel) summarizes data on the accumulation of CD19-specific CARs at the immunologic synapse between CB-NK cells transduced with iC9/CAR.19/IL15 vector with CLL cells (CD19 positive) vs. K562 targets (CD19 negative). *p= 0.02. There was significantly more accumulation of CD19-specific CARs at the immunologic synapse with CLL cells compared to K562 targets. (C) iC9/CAR.19/IL15-transduced CB NK cells, CAR.19-transduced CB NK cells (without IL-15) and CLL patient-derived NK cells transduced with iC9/CAR.19/IL15 were assessed and compared with non-transduced NK cells for their ability to polarize lytic granules and MTOC to CLL targets (left panel) vs. K562 cells (right) (measured by distance from the MTOC to the immune synapse). Results from two independent experiments are shown; each data point represents a single immunologic synapse. Cells were imaged as a Z stack on a Leica TCS SP8 laser scanning microscope. Images were acquired with Volocity software (PerkinElmer). The asterisk indicates statistical significance (P < 0.05 by Student’s t test) vs. the control or another CAR construct.

Fig. 3. IL-15 production and phenotype of iC9/CAR.19/IL15-transduced CB-NK cells

(A) IL-15 production by NT-NK cells or CAR transduced NK cells cultured in the presence or absence of CLL targets for 24h, 48h or 72hs in 4 independent experiments. For each time point, secretion of IL-15 by iC9/CAR.19/IL15-transduced CB NK cells was greater in the presence of antigenic stimulation in the form of CLL targets compared to iC9/CAR.19/IL15-transduced CB NK cells cultured alone (p<0.05 in all 3 cases). (B) CB-NK cell phenotype based on the average expression of 25 markers, including NK cell receptors, transcription factors, adaptor molecules, homing receptors and markers of exhaustion, in triplicate experiments. MFI or the percentages of positive cells were submitted to a hierarchical clustering program to generate a global view of marker expression in iC9/CAR.19/IL15-transduced NK vs. non-transduced (NT) CB-NK cells (n=3 independent NK expansion and transduction experiments using different CB units). The transduced cells lacked any phenotypic evidence of exhaustion and maintained a phenotype similar to that of NT CB-NK cells. (C) Proliferative capacity of CAR-transduced vs. NT CB-NK expansion in response to in vitro stimulation with clone 9 and IL-2 (200 iU/mL). The kinetics of iC9/CAR.19/IL15 NK fold expansion in vitro was similar to NT-NKs (starting from 2 x10⁶ CB-NK cells; N=5).

Fig. 3. IL-15 production and phenotype of iC9/CAR.19/IL15-transduced CB-NK cells

(A) IL-15 production by NT-NK cells or CAR transduced NK cells cultured in the presence or absence of CLL targets for 24h, 48h or 72hs in 4 independent experiments. For each time point, secretion of IL-15 by iC9/CAR.19/IL15-transduced CB NK cells was greater in the presence of antigenic stimulation in the form of CLL targets compared to iC9/CAR.19/IL15-transduced CB NK cells cultured alone (p<0.05 in all 3 cases). (B) CB-NK cell phenotype based on the average expression of 25 markers, including NK cell receptors, transcription factors, adaptor molecules, homing receptors and markers of exhaustion, in triplicate experiments. MFI or the percentages of positive cells were submitted to a hierarchical clustering program to generate a global view of marker expression in iC9/CAR.19/IL15-transduced NK vs. non-transduced (NT) CB-NK cells (n=3 independent NK expansion and transduction experiments using different CB units). The transduced cells lacked any phenotypic evidence of exhaustion and maintained a phenotype similar to that of NT CB-NK cells. (C) Proliferative capacity of CAR-transduced vs. NT CB-NK expansion in response to in vitro stimulation with clone 9 and IL-2 (200 iU/mL). The kinetics of iC9/CAR.19/IL15 NK fold expansion in vitro was similar to NT-NKs (starting from 2 x10⁶ CB-NK cells; N=5).

Fig. 3. IL-15 production and phenotype of iC9/CAR.19/IL15-transduced CB-NK cells

(A) IL-15 production by NT-NK cells or CAR transduced NK cells cultured in the presence or absence of CLL targets for 24h, 48h or 72hs in 4 independent experiments. For each time point, secretion of IL-15 by iC9/CAR.19/IL15-transduced CB NK cells was greater in the presence of antigenic stimulation in the form of CLL targets compared to iC9/CAR.19/IL15-transduced CB NK cells cultured alone (p<0.05 in all 3 cases). (B) CB-NK cell phenotype based on the average expression of 25 markers, including NK cell receptors, transcription factors, adaptor molecules, homing receptors and markers of exhaustion, in triplicate experiments. MFI or the percentages of positive cells were submitted to a hierarchical clustering program to generate a global view of marker expression in iC9/CAR.19/IL15-transduced NK vs. non-transduced (NT) CB-NK cells (n=3 independent NK expansion and transduction experiments using different CB units). The transduced cells lacked any phenotypic evidence of exhaustion and maintained a phenotype similar to that of NT CB-NK cells. (C) Proliferative capacity of CAR-transduced vs. NT CB-NK expansion in response to in vitro stimulation with clone 9 and IL-2 (200 iU/mL). The kinetics of iC9/CAR.19/IL15 NK fold expansion in vitro was similar to NT-NKs (starting from 2 x10⁶ CB-NK cells; N=5).

Fig. 4. In vivo homing, proliferation and antitumor activity of iC9/CAR.19/IL15-transduced CB-NK cells in NSG Raji mouse model

(A) Bioluminescence imaging was used to monitor the growth of FFluc-labeled Raji tumor cells in NSG mice. The plot summarizes the bioluminescence data from 4 groups of mice treated with Raji alone, or Raji plus one dose (10 × 10⁶) of iC9/CAR.19/IL15 CB-NK cells, CAR.19 (no IL-15) CB-NK cells or NT CB-NK cells (5 mice per group). Infusion of one dose of 10 × 10⁶ iC9/CAR.19/IL15-transduced CB-NK cells into NSG mice engrafted with FFluc-labeled Raji cells results in superior control of tumor (blue line) compared with NT CB-NK cells (green line) and NK cells transduced with CAR.19 lacking IL-15 (pink line). (B) BLI figures of the experiments described in panel A. Colors indicate intensity of luminescence (red, highest; blue, lowest). (C) Kaplan Meier plots showing the probability of survival for the 4 groups of mice described in Panel A (5 mice per group). Mice treated with a single dose of 10 × 10⁶ iC9/CAR.19/IL15-transduced CB NK cells (blue line) had significantly better survival than mice receiving CB-NK cells that were either not transduced (green line) (p=0.001) or transduced with a CAR.CD19 construct lacking IL-15 (pink line) (p=0.044). (D) Kaplan Meier plots showing that infusion of two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15-transduced CB-NK cells in NSG mice engrafted with Raji cells resulted in better survival, but was associated with early toxicity. P values were computed using the log rank test. (E) NSG mice were treated with Raji cells alone or Raji plus two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15 CB-NK cells, CAR.19 (no IL-15) CB-NK cells or NT CB-NK cells (n=5 mice per group). Mice were sacrificed on day +21 post-infusion and peripheral blood, bone marrow (BM), liver and spleen were harvested and analyzed by flow cytometry for expression of human (h)CD45, hCD19, hCD56 and CAR. Representative FACS plots are presented. iC9/CAR.19/IL15 transduced CB-NK cells home to sites of disease (liver, spleen, BM) more efficiently than CAR.19 transduced CB-NK cells or NT-NK cells and control disease. (F) Mice that received Raji cells plus two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15 CB-NK cells were monitored over time by weekly blood collection for expansion of CAR-expressing NK cells. Serial measurement of CAR expressing NK cells in the peripheral blood of mice by flow cytometry shows iC9/CAR.19/IL15⁺ CB-NK cells expand over time and could be detected up to 68 days post infusion.

Fig. 4. In vivo homing, proliferation and antitumor activity of iC9/CAR.19/IL15-transduced CB-NK cells in NSG Raji mouse model

(A) Bioluminescence imaging was used to monitor the growth of FFluc-labeled Raji tumor cells in NSG mice. The plot summarizes the bioluminescence data from 4 groups of mice treated with Raji alone, or Raji plus one dose (10 × 10⁶) of iC9/CAR.19/IL15 CB-NK cells, CAR.19 (no IL-15) CB-NK cells or NT CB-NK cells (5 mice per group). Infusion of one dose of 10 × 10⁶ iC9/CAR.19/IL15-transduced CB-NK cells into NSG mice engrafted with FFluc-labeled Raji cells results in superior control of tumor (blue line) compared with NT CB-NK cells (green line) and NK cells transduced with CAR.19 lacking IL-15 (pink line). (B) BLI figures of the experiments described in panel A. Colors indicate intensity of luminescence (red, highest; blue, lowest). (C) Kaplan Meier plots showing the probability of survival for the 4 groups of mice described in Panel A (5 mice per group). Mice treated with a single dose of 10 × 10⁶ iC9/CAR.19/IL15-transduced CB NK cells (blue line) had significantly better survival than mice receiving CB-NK cells that were either not transduced (green line) (p=0.001) or transduced with a CAR.CD19 construct lacking IL-15 (pink line) (p=0.044). (D) Kaplan Meier plots showing that infusion of two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15-transduced CB-NK cells in NSG mice engrafted with Raji cells resulted in better survival, but was associated with early toxicity. P values were computed using the log rank test. (E) NSG mice were treated with Raji cells alone or Raji plus two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15 CB-NK cells, CAR.19 (no IL-15) CB-NK cells or NT CB-NK cells (n=5 mice per group). Mice were sacrificed on day +21 post-infusion and peripheral blood, bone marrow (BM), liver and spleen were harvested and analyzed by flow cytometry for expression of human (h)CD45, hCD19, hCD56 and CAR. Representative FACS plots are presented. iC9/CAR.19/IL15 transduced CB-NK cells home to sites of disease (liver, spleen, BM) more efficiently than CAR.19 transduced CB-NK cells or NT-NK cells and control disease. (F) Mice that received Raji cells plus two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15 CB-NK cells were monitored over time by weekly blood collection for expansion of CAR-expressing NK cells. Serial measurement of CAR expressing NK cells in the peripheral blood of mice by flow cytometry shows iC9/CAR.19/IL15⁺ CB-NK cells expand over time and could be detected up to 68 days post infusion.

Fig. 4. In vivo homing, proliferation and antitumor activity of iC9/CAR.19/IL15-transduced CB-NK cells in NSG Raji mouse model

(A) Bioluminescence imaging was used to monitor the growth of FFluc-labeled Raji tumor cells in NSG mice. The plot summarizes the bioluminescence data from 4 groups of mice treated with Raji alone, or Raji plus one dose (10 × 10⁶) of iC9/CAR.19/IL15 CB-NK cells, CAR.19 (no IL-15) CB-NK cells or NT CB-NK cells (5 mice per group). Infusion of one dose of 10 × 10⁶ iC9/CAR.19/IL15-transduced CB-NK cells into NSG mice engrafted with FFluc-labeled Raji cells results in superior control of tumor (blue line) compared with NT CB-NK cells (green line) and NK cells transduced with CAR.19 lacking IL-15 (pink line). (B) BLI figures of the experiments described in panel A. Colors indicate intensity of luminescence (red, highest; blue, lowest). (C) Kaplan Meier plots showing the probability of survival for the 4 groups of mice described in Panel A (5 mice per group). Mice treated with a single dose of 10 × 10⁶ iC9/CAR.19/IL15-transduced CB NK cells (blue line) had significantly better survival than mice receiving CB-NK cells that were either not transduced (green line) (p=0.001) or transduced with a CAR.CD19 construct lacking IL-15 (pink line) (p=0.044). (D) Kaplan Meier plots showing that infusion of two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15-transduced CB-NK cells in NSG mice engrafted with Raji cells resulted in better survival, but was associated with early toxicity. P values were computed using the log rank test. (E) NSG mice were treated with Raji cells alone or Raji plus two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15 CB-NK cells, CAR.19 (no IL-15) CB-NK cells or NT CB-NK cells (n=5 mice per group). Mice were sacrificed on day +21 post-infusion and peripheral blood, bone marrow (BM), liver and spleen were harvested and analyzed by flow cytometry for expression of human (h)CD45, hCD19, hCD56 and CAR. Representative FACS plots are presented. iC9/CAR.19/IL15 transduced CB-NK cells home to sites of disease (liver, spleen, BM) more efficiently than CAR.19 transduced CB-NK cells or NT-NK cells and control disease. (F) Mice that received Raji cells plus two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15 CB-NK cells were monitored over time by weekly blood collection for expansion of CAR-expressing NK cells. Serial measurement of CAR expressing NK cells in the peripheral blood of mice by flow cytometry shows iC9/CAR.19/IL15⁺ CB-NK cells expand over time and could be detected up to 68 days post infusion.

Fig. 4. In vivo homing, proliferation and antitumor activity of iC9/CAR.19/IL15-transduced CB-NK cells in NSG Raji mouse model

(A) Bioluminescence imaging was used to monitor the growth of FFluc-labeled Raji tumor cells in NSG mice. The plot summarizes the bioluminescence data from 4 groups of mice treated with Raji alone, or Raji plus one dose (10 × 10⁶) of iC9/CAR.19/IL15 CB-NK cells, CAR.19 (no IL-15) CB-NK cells or NT CB-NK cells (5 mice per group). Infusion of one dose of 10 × 10⁶ iC9/CAR.19/IL15-transduced CB-NK cells into NSG mice engrafted with FFluc-labeled Raji cells results in superior control of tumor (blue line) compared with NT CB-NK cells (green line) and NK cells transduced with CAR.19 lacking IL-15 (pink line). (B) BLI figures of the experiments described in panel A. Colors indicate intensity of luminescence (red, highest; blue, lowest). (C) Kaplan Meier plots showing the probability of survival for the 4 groups of mice described in Panel A (5 mice per group). Mice treated with a single dose of 10 × 10⁶ iC9/CAR.19/IL15-transduced CB NK cells (blue line) had significantly better survival than mice receiving CB-NK cells that were either not transduced (green line) (p=0.001) or transduced with a CAR.CD19 construct lacking IL-15 (pink line) (p=0.044). (D) Kaplan Meier plots showing that infusion of two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15-transduced CB-NK cells in NSG mice engrafted with Raji cells resulted in better survival, but was associated with early toxicity. P values were computed using the log rank test. (E) NSG mice were treated with Raji cells alone or Raji plus two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15 CB-NK cells, CAR.19 (no IL-15) CB-NK cells or NT CB-NK cells (n=5 mice per group). Mice were sacrificed on day +21 post-infusion and peripheral blood, bone marrow (BM), liver and spleen were harvested and analyzed by flow cytometry for expression of human (h)CD45, hCD19, hCD56 and CAR. Representative FACS plots are presented. iC9/CAR.19/IL15 transduced CB-NK cells home to sites of disease (liver, spleen, BM) more efficiently than CAR.19 transduced CB-NK cells or NT-NK cells and control disease. (F) Mice that received Raji cells plus two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15 CB-NK cells were monitored over time by weekly blood collection for expansion of CAR-expressing NK cells. Serial measurement of CAR expressing NK cells in the peripheral blood of mice by flow cytometry shows iC9/CAR.19/IL15⁺ CB-NK cells expand over time and could be detected up to 68 days post infusion.

Fig. 4. In vivo homing, proliferation and antitumor activity of iC9/CAR.19/IL15-transduced CB-NK cells in NSG Raji mouse model

(A) Bioluminescence imaging was used to monitor the growth of FFluc-labeled Raji tumor cells in NSG mice. The plot summarizes the bioluminescence data from 4 groups of mice treated with Raji alone, or Raji plus one dose (10 × 10⁶) of iC9/CAR.19/IL15 CB-NK cells, CAR.19 (no IL-15) CB-NK cells or NT CB-NK cells (5 mice per group). Infusion of one dose of 10 × 10⁶ iC9/CAR.19/IL15-transduced CB-NK cells into NSG mice engrafted with FFluc-labeled Raji cells results in superior control of tumor (blue line) compared with NT CB-NK cells (green line) and NK cells transduced with CAR.19 lacking IL-15 (pink line). (B) BLI figures of the experiments described in panel A. Colors indicate intensity of luminescence (red, highest; blue, lowest). (C) Kaplan Meier plots showing the probability of survival for the 4 groups of mice described in Panel A (5 mice per group). Mice treated with a single dose of 10 × 10⁶ iC9/CAR.19/IL15-transduced CB NK cells (blue line) had significantly better survival than mice receiving CB-NK cells that were either not transduced (green line) (p=0.001) or transduced with a CAR.CD19 construct lacking IL-15 (pink line) (p=0.044). (D) Kaplan Meier plots showing that infusion of two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15-transduced CB-NK cells in NSG mice engrafted with Raji cells resulted in better survival, but was associated with early toxicity. P values were computed using the log rank test. (E) NSG mice were treated with Raji cells alone or Raji plus two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15 CB-NK cells, CAR.19 (no IL-15) CB-NK cells or NT CB-NK cells (n=5 mice per group). Mice were sacrificed on day +21 post-infusion and peripheral blood, bone marrow (BM), liver and spleen were harvested and analyzed by flow cytometry for expression of human (h)CD45, hCD19, hCD56 and CAR. Representative FACS plots are presented. iC9/CAR.19/IL15 transduced CB-NK cells home to sites of disease (liver, spleen, BM) more efficiently than CAR.19 transduced CB-NK cells or NT-NK cells and control disease. (F) Mice that received Raji cells plus two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15 CB-NK cells were monitored over time by weekly blood collection for expansion of CAR-expressing NK cells. Serial measurement of CAR expressing NK cells in the peripheral blood of mice by flow cytometry shows iC9/CAR.19/IL15⁺ CB-NK cells expand over time and could be detected up to 68 days post infusion.

Fig. 4. In vivo homing, proliferation and antitumor activity of iC9/CAR.19/IL15-transduced CB-NK cells in NSG Raji mouse model

(A) Bioluminescence imaging was used to monitor the growth of FFluc-labeled Raji tumor cells in NSG mice. The plot summarizes the bioluminescence data from 4 groups of mice treated with Raji alone, or Raji plus one dose (10 × 10⁶) of iC9/CAR.19/IL15 CB-NK cells, CAR.19 (no IL-15) CB-NK cells or NT CB-NK cells (5 mice per group). Infusion of one dose of 10 × 10⁶ iC9/CAR.19/IL15-transduced CB-NK cells into NSG mice engrafted with FFluc-labeled Raji cells results in superior control of tumor (blue line) compared with NT CB-NK cells (green line) and NK cells transduced with CAR.19 lacking IL-15 (pink line). (B) BLI figures of the experiments described in panel A. Colors indicate intensity of luminescence (red, highest; blue, lowest). (C) Kaplan Meier plots showing the probability of survival for the 4 groups of mice described in Panel A (5 mice per group). Mice treated with a single dose of 10 × 10⁶ iC9/CAR.19/IL15-transduced CB NK cells (blue line) had significantly better survival than mice receiving CB-NK cells that were either not transduced (green line) (p=0.001) or transduced with a CAR.CD19 construct lacking IL-15 (pink line) (p=0.044). (D) Kaplan Meier plots showing that infusion of two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15-transduced CB-NK cells in NSG mice engrafted with Raji cells resulted in better survival, but was associated with early toxicity. P values were computed using the log rank test. (E) NSG mice were treated with Raji cells alone or Raji plus two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15 CB-NK cells, CAR.19 (no IL-15) CB-NK cells or NT CB-NK cells (n=5 mice per group). Mice were sacrificed on day +21 post-infusion and peripheral blood, bone marrow (BM), liver and spleen were harvested and analyzed by flow cytometry for expression of human (h)CD45, hCD19, hCD56 and CAR. Representative FACS plots are presented. iC9/CAR.19/IL15 transduced CB-NK cells home to sites of disease (liver, spleen, BM) more efficiently than CAR.19 transduced CB-NK cells or NT-NK cells and control disease. (F) Mice that received Raji cells plus two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15 CB-NK cells were monitored over time by weekly blood collection for expansion of CAR-expressing NK cells. Serial measurement of CAR expressing NK cells in the peripheral blood of mice by flow cytometry shows iC9/CAR.19/IL15⁺ CB-NK cells expand over time and could be detected up to 68 days post infusion.

Fig. 4. In vivo homing, proliferation and antitumor activity of iC9/CAR.19/IL15-transduced CB-NK cells in NSG Raji mouse model

(A) Bioluminescence imaging was used to monitor the growth of FFluc-labeled Raji tumor cells in NSG mice. The plot summarizes the bioluminescence data from 4 groups of mice treated with Raji alone, or Raji plus one dose (10 × 10⁶) of iC9/CAR.19/IL15 CB-NK cells, CAR.19 (no IL-15) CB-NK cells or NT CB-NK cells (5 mice per group). Infusion of one dose of 10 × 10⁶ iC9/CAR.19/IL15-transduced CB-NK cells into NSG mice engrafted with FFluc-labeled Raji cells results in superior control of tumor (blue line) compared with NT CB-NK cells (green line) and NK cells transduced with CAR.19 lacking IL-15 (pink line). (B) BLI figures of the experiments described in panel A. Colors indicate intensity of luminescence (red, highest; blue, lowest). (C) Kaplan Meier plots showing the probability of survival for the 4 groups of mice described in Panel A (5 mice per group). Mice treated with a single dose of 10 × 10⁶ iC9/CAR.19/IL15-transduced CB NK cells (blue line) had significantly better survival than mice receiving CB-NK cells that were either not transduced (green line) (p=0.001) or transduced with a CAR.CD19 construct lacking IL-15 (pink line) (p=0.044). (D) Kaplan Meier plots showing that infusion of two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15-transduced CB-NK cells in NSG mice engrafted with Raji cells resulted in better survival, but was associated with early toxicity. P values were computed using the log rank test. (E) NSG mice were treated with Raji cells alone or Raji plus two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15 CB-NK cells, CAR.19 (no IL-15) CB-NK cells or NT CB-NK cells (n=5 mice per group). Mice were sacrificed on day +21 post-infusion and peripheral blood, bone marrow (BM), liver and spleen were harvested and analyzed by flow cytometry for expression of human (h)CD45, hCD19, hCD56 and CAR. Representative FACS plots are presented. iC9/CAR.19/IL15 transduced CB-NK cells home to sites of disease (liver, spleen, BM) more efficiently than CAR.19 transduced CB-NK cells or NT-NK cells and control disease. (F) Mice that received Raji cells plus two doses (10 × 10⁶ each, 5 – 7 days apart) of iC9/CAR.19/IL15 CB-NK cells were monitored over time by weekly blood collection for expansion of CAR-expressing NK cells. Serial measurement of CAR expressing NK cells in the peripheral blood of mice by flow cytometry shows iC9/CAR.19/IL15⁺ CB-NK cells expand over time and could be detected up to 68 days post infusion.

Fig. 5. IL-15-transduced CB-NK cells lack signs of autonomous or dysregulated growth

(A) iC9/CAR.19/IL15-transduced CB NK and NT CB NK cells were put in culture without cytokines or exogenous stimulation to assess their growth over time. iC9/CAR.19/IL15-transduced CB NK cells stop expanding within 6 weeks of in vitro culture with no evidence of autonomous growth. (B) NSG mice 10 months after treatment with CB-NK cells transduced with iC9/CAR.19/IL15 or CAR.19 (no IL15) were sacrificed and examined for evidence of NK dysregulated growth or leukemia/lymphoma. Photomicrographs of mesenteric lymph nodes show vestigial lymphoid tissue with no lymphocytic infiltration. Images of the spleen show rudimentary periarteriolar lymphoid tissue devoid of lymphocytes (black arrows) and is surrounded by hematopoietic tissue comprising of erythroid and myeloid series cells in different stages of development, including megakaryocytes and hemosiderin-laden macrophages. Bone marrow contains normal hematopoietic cells and no abnormal lymphocytes. H&E stain, magnification x200. The micrographs are from two representative groups of NSG mice treated with iC9/CAR.19/IL15-transduced CB-NK cells.

Fig. 5. IL-15-transduced CB-NK cells lack signs of autonomous or dysregulated growth

(A) iC9/CAR.19/IL15-transduced CB NK and NT CB NK cells were put in culture without cytokines or exogenous stimulation to assess their growth over time. iC9/CAR.19/IL15-transduced CB NK cells stop expanding within 6 weeks of in vitro culture with no evidence of autonomous growth. (B) NSG mice 10 months after treatment with CB-NK cells transduced with iC9/CAR.19/IL15 or CAR.19 (no IL15) were sacrificed and examined for evidence of NK dysregulated growth or leukemia/lymphoma. Photomicrographs of mesenteric lymph nodes show vestigial lymphoid tissue with no lymphocytic infiltration. Images of the spleen show rudimentary periarteriolar lymphoid tissue devoid of lymphocytes (black arrows) and is surrounded by hematopoietic tissue comprising of erythroid and myeloid series cells in different stages of development, including megakaryocytes and hemosiderin-laden macrophages. Bone marrow contains normal hematopoietic cells and no abnormal lymphocytes. H&E stain, magnification x200. The micrographs are from two representative groups of NSG mice treated with iC9/CAR.19/IL15-transduced CB-NK cells.

Fig. 6. Activation of inducible caspase-9 suicide gene eliminates iC9/CAR.19/IL15⁺ CB-NK cells

(A) Addition of 10 nM of AP1903 to cultures of iC9-CAR-IL15+ CB-NK cells induced apoptosis/necrosis of transgenic cells within 4 hours as assessed by annexin-V-7AAD staining in 4 independent experiments. The dimerizer did not induce apoptosis in NT NK cells. (B) A representative FACS plot of the experiment described in Panel A is presented. (C) NSG mice engrafted with Raji cells and infused with iC9/CAR.19/IL15⁺ CB-NK cells were treated 10–14 days later with two doses of the AP1903 dimerizer (50 μg) i.p. 2 days apart. FACS plots from a representative experiment are presented. iC9/CAR.19/IL15-expressing NK cells were substantially reduced in all organs tested 3 days later as measured by the frequencies of CAR-positive NK cells in blood, bone marrow, spleen and liver of animals by flow cytometry.

Fig. 6. Activation of inducible caspase-9 suicide gene eliminates iC9/CAR.19/IL15⁺ CB-NK cells

(A) Addition of 10 nM of AP1903 to cultures of iC9-CAR-IL15+ CB-NK cells induced apoptosis/necrosis of transgenic cells within 4 hours as assessed by annexin-V-7AAD staining in 4 independent experiments. The dimerizer did not induce apoptosis in NT NK cells. (B) A representative FACS plot of the experiment described in Panel A is presented. (C) NSG mice engrafted with Raji cells and infused with iC9/CAR.19/IL15⁺ CB-NK cells were treated 10–14 days later with two doses of the AP1903 dimerizer (50 μg) i.p. 2 days apart. FACS plots from a representative experiment are presented. iC9/CAR.19/IL15-expressing NK cells were substantially reduced in all organs tested 3 days later as measured by the frequencies of CAR-positive NK cells in blood, bone marrow, spleen and liver of animals by flow cytometry.

Fig. 6. Activation of inducible caspase-9 suicide gene eliminates iC9/CAR.19/IL15⁺ CB-NK cells

(A) Addition of 10 nM of AP1903 to cultures of iC9-CAR-IL15+ CB-NK cells induced apoptosis/necrosis of transgenic cells within 4 hours as assessed by annexin-V-7AAD staining in 4 independent experiments. The dimerizer did not induce apoptosis in NT NK cells. (B) A representative FACS plot of the experiment described in Panel A is presented. (C) NSG mice engrafted with Raji cells and infused with iC9/CAR.19/IL15⁺ CB-NK cells were treated 10–14 days later with two doses of the AP1903 dimerizer (50 μg) i.p. 2 days apart. FACS plots from a representative experiment are presented. iC9/CAR.19/IL15-expressing NK cells were substantially reduced in all organs tested 3 days later as measured by the frequencies of CAR-positive NK cells in blood, bone marrow, spleen and liver of animals by flow cytometry.