CS1-Specific Chimeric Antigen Receptor Drives Cord Blood NK Cell Maturation and Expansion In Vivo

Chimeric antigen receptors (CAR) have been used extensively to redirect the specificity of T cells against leukemic cells, with remarkable clinical responses in patients with lymphoid malignancies. CAR-transduced natural killer (NK) cells might serve as an alternative to CAR T cells as they lack the potential to cause GVHD, and could be made available as an off-the-shelf allogeneic product for immediate clinical use. Moreover, unlike CAR-T cells, CAR NK cells retain their intrinsic capacity to recognize and target tumor cells through their native receptors, in theory making disease escape through downregulation of the CAR target antigen less likely. We have identified cord blood (CB) as a readily available source of allogeneic NK cells.

Thus, in this study, we reprogrammed CB-NK cells to recognize multiple myeloma (MM) using a retroviral construct encoding a CAR against CS1, a coreceptor expressed on MM cells, IL-15 to enhance their in vivo proliferation and survival, and inducible caspase-9, a suicide gene that can be activated as needed. We consistently achieved a median CAR positivity of >90%, 14 days post transduction and viability of >95% with only limited CS1-mediated fratricide (since CS1 is also expressed on NK cells) during long-term ex vivo culture. CS1-CAR NK cells killed the CS1-expressing MM.1S cell line significantly better than ex vivo expanded non-transduced (NT) CB-NK cells at all effector:target ratios tested (p<0.001). We next tested the in vivo trafficking and antitumor activity of iC9/CAR.CS1/IL-15+ CB-NK cells in a xenograft mouse model of MM. Mice engrafted with MM1.S cells received saline (control) or 1 x10⁷ ex vivo expanded CB-NK cells that were either unmodified or transduced with iC9/CAR.CS1/IL-15; iC9/CAR.CS1/IL15+ CB-NK cells controlled tumor expansion and prolonged survival of mice significantly better than NT CB-NK cells.

This was associated withsignificantly higher frequencies of circulating NK cells in the CAR+ CB-NK cell treated mice (median=19%, range 13.6-31.1%) compared to the NT CB-NK group (median=1.26%, range 0.98-2.49%, p=0.0003). We next used mass cytometry (CyTOF) to interrogate the features that shape NK cell diversity, their maturation and their trafficking in vivo. The expression of CAR.CS1 on NK cells infiltrating tumor sites remained stable over time in vivo (median 86.7%, range 81-89.8% compared to the infused product). This was associated with significantly less tumor infiltration in liver (0.013% vs 33.8%), spleen (0.01% vs 23.4%) and BM (4.3% vs 79.8%) harvested on day 21 post-infusion, compared to mice treated with NT NK cells. Interestingly, the residual MM cells in the BM of iC9/CAR.CS1/IL-15 treated mice lost CS1 expression on their surface, suggesting that target antigen downregulation may also play a part in escape from CAR-mediated immune recognition in MM, as also described with CD19 CAR T cell therapy.

Using a panel of 40 monoclonal antibodies, we profiled the global signature of NK cells harvested from organs of animals sacrificed on day 21 after infusion and compared them to CAR⁺ CB-NK cells prior to infusion. The diversity of iC9/CAR.CS1.IL-15-transduced CB-NK cells, measured using the inverse Simpson index, was similar to that of ex vivo expanded NT CB-NK cells (167 vs 114). Additionally, using t-SNE maps to visualize the high-dimensional data generated by CyTOF, we observed complete overlap of clusters for ex vivo expanded NT CB-NK cells and CS1-CAR-transduced CB-NK cells, indicating, that any potential CS1-related fratricide does not significantly impact on the repertoire of the CAR-transduced CB-NK cells (Figure 1).

Interestingly, there was a profound and significant increase in the diversity of the infiltrating CS1-CAR CB-NK cells in the liver (830), spleen (843) and BM (965) on day 21 after infusion, suggesting in vivo maturation and expansion of NK subpopulations in response to the tumor. t-SNE analysis revealed a clear separation between iC9/CAR.CS1.IL-15 transduced CB-NK cells prior to infusion and tissue-infiltrating CAR+ CB-NK cells, indicating significant shifts in NK subpopulations, with an increase in diversity and maturation after adoptive transfer in vivo (Figure 1). The data from this analysis will have important implications for future studies as we seek to generate CAR+ CB-NK cells with enhanced antitumor potential.

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