A combination of CAR-NK and CAR-T cells results in rapid ...€¦ · phenotyping and a Violet Cell...
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Contact James Trager, PhD [email protected] www.nkartatx.com Combining CAR19-NK and CAR19-T cells reduces CAR19- T cell proliferation and promotes CAR19-NK cell expansion CAR19-NK and CAR19-T cells were labeled with Violet Cell Trace and then incubated for 6 days with Nalm6 at various ratios. As shown in Figure 4, in the absence of CAR19-NK cells, the majority of CAR19-T cells proliferated regardless of E:T ratio. In the absence of CAR19-T cells, CAR19-NK cells exhibited little to no proliferation. However, when CAR19-T cells and CAR19-NK cells were co-cultured with Nalm6, CAR19-T cell proliferation decreased with increased presence of CAR19-NK cells (A), and CAR19-NK cell proliferation was induced with an increased presence of CAR19-T cells (B). A cellular model is proposed for the mutual impact of CAR19-T and CAR19-NK cells upon encounter with CD19+ Nalm6 tumor cells (C). Introduction CAR-NK cells show rapid and potent anti-tumor cytotoxic activity even at very low effector-to-target ratios; however, they do not typically expand upon encounter with antigen and lack the long-term persistence commonly associated with autologous CAR-T cells. Conversely, CAR-T cells generally exhibit persistent killing activity, even though they are activated more slowly. In addition, the explosive expansion of CAR-T cells is frequently associated with a variety of toxicities (e.g. CRS, CRES, etc.). To address the potential shortcomings associated with either cell type, we have assessed a novel platform that combines CAR-NK and CAR-T cells as a novel cancer therapeutic. Methods NK and T cell were expanded separately to allow evaluation of varying cell ratios. CAR19-NK and –T cells were made by transduction with bicistronic retroviral construct encoding CD19 CAR (αCD19 scFv-CD8αTM-OX40 - CD3ζ) and membrane-bound IL15. To evaluate the overall outcomes of combining CAR-NK and CAR-T cells as an anti-tumor therapy, we have developed two novel in vitro assays: (a) IncuCyte-based matrixed cytotoxicity assay, which allows direct assessment of tumor killing kinetically with different E:T and NK:T ratios; (b) IncuCyte-based tumor re-challenge assay, which provides a convenient and quick assessment of anti-tumor persistence. Additionally, we measured a panel of cytokines from these two assays by LEGENDplex. To further investigate the cellular mechanism(s) underlying the effects of combining CAR-NK and CAR-T cells, we performed flow cytometry-based phenotyping and a Violet Cell Trace-based proliferation assay. Finally, we evaluated the anti-tumor efficacy of the NK plus T platform in vivo using a CDX tumor model in immunodeficient NSG mice. Results Combining of CAR19-NK and CAR19-T cells enhances cytotoxic outcome and kinetics As shown in Figure 1, 20K/well Nalm6-NucRed target cells were seeded in a 96-well plate. Different ratios of CAR19-T and CAR19-NK effector cells to targets cells were established as indicated in (A) and (B). After 7 days of culture in an IncuCyte assay, combinations of NK and T cells that showed greater cytotoxicity than NK or T cells alone are indicated by the boxed green area (A). Additionally, the combination of the two cell types showed more rapid killing even at very low E:T ratios as shown in the red boxed areas (B). Conclusion Proof-of-concept studies using both innate and adaptive immunity with CAR19-NK and CAR19-T cells demonstrate that the combination of both can: • Enhance anti-tumor cytotoxicity (kinetically and cumulatively) and persistence. • Improve the general safety profile of cytokine release. • Promote NK cell expansion but reduce antigen dependent proliferation of T cells • Prevent tumor relapse in a Nalm6 CDX model for at least four months. A combination of CAR-NK and CAR-T cells results in rapid and persistent anti-tumor efficacy while reducing CAR-T cell mediated cytokine release and T-cell proliferation Guangnan Li 1 , Xiumin Wu 1 , Ivan H. Chan 1 , James B. Trager 1 1 Nkarta Therapeutics Inc, South San Francisco, CA, USA Combining of CAR19-NK and CAR19-T cells improves cytotoxic persistence As shown in Figure 2, during the first round of tumor challenge, CAR19-T cells alone at the indicated E:T ratio failed to arrest tumor growth, whereas CAR19-NK cells alone or in combination reduced tumor counts. However, following a second round of tumor challenge, CAR19-NK cells alone at these E:T ratios were no longer able to control tumor growth, whereas only the combination of both could arrest tumor growth after 14 days of culture. Combining CAR19-NK with CAR19-T cells lowers the accumulation of cytokines associated with CRS Different ratios of CAR19-T and CAR19-NK effector cells to Nalm6 targets were established as in Figure 1. At day 5, culture media were collected for cytokine measurement by LEGENDplex. Data from donors #103 (pink) and #452 (green) are shown in Figure 3. The levels of key mediators of CRS (e.g. GM-CSF, IL6) were donor-dependent, CAR19-T:target ratio dependent, and inversely correlated with CAR19-NK:target ratio. Combining CAR19-NK and CAR19-T cells in an in vivo model improves anti-tumor efficacy and survival As shown in Figure 5A, a a xenograft tumor model was established in immunodeficient NSG mice by tail vein injection of Nalm6.ffluc cells (2x10 5 /animal). Treatment was performed at day 3 post tumor injection with CAR19-NK cells (2.5x10 6 /animal) alone, or in combination with either a low dose (5x10 5 /animal) or a high dose of CAR19-T cells (2.5x10 6 /animal). Additional control arms were also included (Figure 5B). At day 121, 4/5 animals in CAR19-NK and high CAR19-T combo group survived and stayed tumor-free, as compared to only 1/5 in the high CAR19-T alone group (Figure 5B-D). T =0 1:16 1:8 1:4 1:2 0 5000 10000 15000 G M -C S F cytokine (pg/m L) (T:target ratio) T =0 1:16 1:8 1:4 1:2 0 5 10 15 20 IL 6 cytokine (pg/m L) (T:target ratio) T =0 1:16 1:8 1:4 1:2 0 100 200 300 400 IL 10 cytokine (pg/m L) (T:target ratio) T =0 1:16 1:8 1:4 1:2 0 10000 20000 30000 40000 IF N g cytokine (pg/m L) (T:target ratio) T =0 1:16 1:8 1:4 1:2 0 50 100 150 200 TN Fa cytokine (pg/m L) (T:target ratio) #103N K =0 #103(N K :target=1:4) #103(N K :target=1:2) #452N K =0 #452(N K :target=1:4) #452(N K :target=1:2) 6 5 4 3 2 1 0 1 2 1 1 1 0 9 8 7 IV Nalm6.ffluc 2e5/animal BLI BLI IV CAR19-NK: 2.5e6 CAR19-T: 5e5 or 2.5e6 NSG TM mice BLI 0 50 100 150 0 20 40 60 80 100 days P ercent survival P B S C A R 19-N K C A R 19-T (low ) C A R 19-N K + C A R 19-T (low ) C A R 19-T (high) C A R 19-N K + C A R 19-T (high) 0 25 10 5 10 6 10 7 10 8 10 9 10 10 50 75 100 125 D ays B LI (photon/sec) P B S C A R 19-N K C A R 19-T (low ) C A R 19-N K + C A R 19-T (low ) C A R 19-T (high) C A R 19-N K + C A R 19-T (high)
Transcript of A combination of CAR-NK and CAR-T cells results in rapid ...€¦ · phenotyping and a Violet Cell...
Contact James Trager, [email protected]
Combining CAR19-NK and CAR19-T cells reduces CAR19-T cell proliferation and promotes CAR19-NK cell expansionCAR19-NK and CAR19-T cells were labeled with Violet Cell Trace and then incubated for 6 days with Nalm6 at various ratios. As shown in Figure 4, in the absence of CAR19-NK cells, the majority of CAR19-T cells proliferated regardless of E:T ratio. In the absence of CAR19-T cells, CAR19-NK cells exhibited little to no proliferation. However, when CAR19-T cells and CAR19-NK cells were co-cultured with Nalm6, CAR19-T cell proliferation decreased with increased presence of CAR19-NK cells (A), and CAR19-NK cell proliferation was induced with an increased presence of CAR19-T cells (B). A cellular model is proposed for the mutual impact of CAR19-T and CAR19-NK cells upon encounter with CD19+ Nalm6 tumor cells (C).
IntroductionCAR-NK cells show rapid and potent anti-tumor cytotoxic activity even at very low effector-to-target ratios; however, they do not typically expand upon encounter with antigen and lack the long-term persistence commonly associated with autologous CAR-T cells. Conversely, CAR-T cells generally exhibit persistent killing activity, even though they are activated more slowly. In addition, the explosive expansion of CAR-T cells is frequently associated with a variety of toxicities (e.g. CRS, CRES, etc.). To address the potential shortcomings associated with either cell type, we have assessed a novel platform that combines CAR-NK and CAR-T cells as a novel cancer therapeutic.
MethodsNK and T cell were expanded separately to allow evaluation of varying cell ratios. CAR19-NK and –T cells were made by transduction with bicistronicretroviral construct encoding CD19 CAR (αCD19 scFv-CD8αTM-OX40 -CD3ζ) and membrane-bound IL15.
To evaluate the overall outcomes of combining CAR-NK and CAR-T cells as an anti-tumor therapy, we have developed two novel in vitro assays:
(a) IncuCyte-based matrixed cytotoxicity assay, which allows direct assessment of tumor killing kinetically with different E:T and NK:T ratios; (b) IncuCyte-based tumor re-challenge assay, which provides a convenient and quick assessment of anti-tumor persistence. Additionally, we measured a panel of cytokines from these two assays by LEGENDplex.
To further investigate the cellular mechanism(s) underlying the effects of combining CAR-NK and CAR-T cells, we performed flow cytometry-based phenotyping and a Violet Cell Trace-based proliferation assay.
Finally, we evaluated the anti-tumor efficacy of the NK plus T platform in
vivo using a CDX tumor model in immunodeficient NSG mice.
ResultsCombining of CAR19-NK and CAR19-T cells enhances cytotoxic outcome and kineticsAs shown in Figure 1, 20K/well Nalm6-NucRed target cells were seeded in a 96-well plate. Different ratios of CAR19-T and CAR19-NK effector cells to targets cells were established as indicated in (A) and (B). After 7 days of culture in an IncuCyte assay, combinations of NK and T cells that showed greater cytotoxicity than NK or T cells alone are indicated by the boxed green area (A). Additionally, the combination of the two cell types showed more rapid killing even at very low E:T ratios as shown in the red boxed areas (B).
ConclusionProof-of-concept studies using both innate and adaptive immunity with CAR19-NK and CAR19-T cells demonstrate that the combination of both can:
• Enhance anti-tumor cytotoxicity (kinetically and cumulatively) and persistence.
• Improve the general safety profile of cytokine release.
• Promote NK cell expansion but reduce antigen dependent proliferation of T cells
• Prevent tumor relapse in a Nalm6 CDX model for at least four months.
A combination of CAR-NK and CAR-T cells results in rapid and persistent anti-tumor efficacy while reducing CAR-T cell mediated cytokine release and T-cell proliferationGuangnan Li1, Xiumin Wu1, Ivan H. Chan1, James B. Trager1
1Nkarta Therapeutics Inc, South San Francisco, CA, USA
Combining of CAR19-NK and CAR19-T cells improves cytotoxic persistenceAs shown in Figure 2, during the first round of tumor challenge, CAR19-T cells alone at the indicated E:T ratio failed to arrest tumor growth, whereas CAR19-NK cells alone or in combination reduced tumor counts. However, following a second round of tumor challenge, CAR19-NK cells alone at these E:T ratios were no longer able to control tumor growth, whereas only the combination of both could arrest tumor growth after 14 days of culture.
Combining CAR19-NK with CAR19-T cells lowers the accumulation of cytokines associated with CRSDifferent ratios of CAR19-T and CAR19-NK effector cells to Nalm6 targets were established as in Figure 1. At day 5, culture media were collected for cytokine measurement by LEGENDplex. Data from donors #103 (pink) and #452 (green) are shown in Figure 3. The levels of key mediators of CRS (e.g. GM-CSF, IL6) were donor-dependent, CAR19-T:target ratio dependent, and inversely correlated with CAR19-NK:target ratio. Combining CAR19-NK and CAR19-T cells in an in vivo
model improves anti-tumor efficacy and survivalAs shown in Figure 5A, a a xenograft tumor model was established in immunodeficient NSG mice by tail vein injection of Nalm6.ffluc cells (2x105/animal). Treatment was performed at day 3 post tumor injection with CAR19-NK cells (2.5x106 /animal) alone, or in combination with either a low dose (5x105 /animal) or a high dose of CAR19-T cells (2.5x106/animal). Additional control arms were also included (Figure 5B). At day 121, 4/5 animals in CAR19-NK and high CAR19-T combo group survived and stayed tumor-free, as compared to only 1/5 in the high CAR19-T alone group (Figure 5B-D).
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