Immune Monitoring of CD34+ Placental Cell Derived Natural Killer Cell Therapy (PNK-007) in Phase I Study of Multiple Myeloma

Background: Natural Killer (NK) cells are innate immune lymphocytes with cytotoxic function and are critical for immune surveillance. Unlike T cells which rely on antigen-specific responses, NK cells recognize transformed cells through a variety of NK cell-specific receptor-ligand interactions. Clinical studies have highlighted the therapeutic potential of NK cell-based therapies and Celularity has developed a GMP procedure for generating PNK-007: an off-the-shelf and allogeneic NK cell product culture-expanded and differentiated from human placental CD34+ stem cells. PNK-007 was evaluated as a single-dose allogeneic cell therapy in a phase I dose-escalation study for multiple myeloma (MM) patients receiving autologous stem cell transplant (ASCT) (NCT02955550). 12 of 15 patients received subcutaneous IL-2 every 48 hours for 10 days following PNK-007 cell infusion while 3 patients in a separate cohort did not receive IL-2. Here, we report translational studies evaluating post-treatment immune reconstitution, minimal residual disease (MRD) detection at 10^-5 threshold, and serum profiling of cytokines and chemokines.

Methods: Patient bone marrow aspirate was collected for EuroFlow MRD assessment and immune phenotyping by flow cytometry at baseline, 90-100 days, 6 months and 1 year post-ASCT. Peripheral blood was collected at baseline, then weekly for six weeks following PNK-007 infusion, at 6 months and at 1 year post-ASCT. PNK-007 persistence, leukocyte populations, and HLA antibody panels were determined by flow cytometry. Serum was analyzed by Luminex for panel cytokines, chemokines and soluble cytokine receptors. All patients and PNK-007 drug product were typed for HLA and killer-cell immunoglobulin-like (KIR) genotype.

Results: PNK-007 infusion did not interfere with immune reconstitution kinetics post-ASCT. Cohorts receiving PNK infusion 14 days post-ASCT had already recovered white blood cell counts to normal levels. One cohort receiving 3x10⁷ PNK cells/kg 7 days post-ASCT showed an immune deficient environment (0.05x10³ ± 0.004x10³ leukocytes/ml, n=3), but recovered their white blood cell counts by day 21 (6.8x10³ ± 1.8x10³ leukocytes/ml, n=3). Using a validated Euro-flow MRD assay, 4/15 patients were MRD(-) at pre-ASCT baseline, and by day 90, 10/15 pts were MRD(-). PNK-007 persistence was not detected in patients by flow cytometry with the earliest timepoint tested being 7 days post-infusion. Panel HLA serum antibodies were not detected at any timepoint indicating the absence of alloantibodies. Withholding IL-2 administration in one cohort allowed us to evaluate its potential effects on NK cell recovery and immune reconstitution. We found that subcutaneous IL-2 q.o.d. for 10 days following PNK-007 infusion did not affect recovery kinetics and concentration of endogenous NK cells. However, these patients showed a 5-10 fold increase in serum soluble IL-2RA from baseline and elevated regulatory T cell (T_reg) count in the blood versus baseline (167 ± 10⁷ T_reg/mL vs. 61 ± 33 T_reg/mL, p=0.0075). Patients not receiving IL-2 saw no change in serum soluble IL-2RA or blood T_reg level from baseline. CD4 and CD8 T cells from all patients retained their ability to become activated in response to ex vivo stimulation and favored release of IL-2 and IFNg. T cell effector function was maintained in all patients post-ASCT but was lost in a subset of patients at 1 year. Serum analysis showed low levels of free IL-15 at the time of PNK dosing despite the transient lymphodepleted state associated with ASCT. Cytokines associated with myeloid inflammation and T cell immunity in the month post dosing were within normal homeostatic level. Serum TGFβ was significantly lower at the day of PNK infusion compared to normal homeostatic levels.

Conclusion: Translational data from a Phase I study of PNK-007 in post-ASCT MM established that dosing up to 3x10⁷ cells/kg at 14 or 7 days post-transplant did not impair engraftment or immune reconstitution. EuroFlow MRD assessment of the bone marrow showed conversion of 6/11 patients from MRD(+) to MRD(-) by day 90 post transplant. The administration of IL-2 in this clinical study did not appear to benefit NK cell reconstitution but instead favored soluble IL2RA antagonism and increased systemic levels of T_reg. These results demonstrate the feasibility of allogeneic NK cell therapy in the MM + ASCT setting and will help inform the design of further clinical studies.