Combining Cytokine-Induced Memory-like NK Cells with Tafasitamab Demonstrates Efficacy Against Acute B-Cell Leukemia

Introduction: Acute B-cell lymphoblastic leukemia (B-ALL) can often be cured with multiagent chemotherapy but prognosis following relapse remains poor with conventional chemotherapy alone. While chimeric antigen receptor (CAR)-T cells and bispecific T cell engager therapy can be effective in treating acute B-ALL, NK cell therapies may emerge as an alternative approach as they have a lower risk of adverse effects such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANs). Notably, when briefly stimulated with the cytokines IL-12, IL-15 and IL-18, NK cells acquire memory-like properties and demonstrate enhanced anti-cancer responses. Tafasitamab (TAFA) is an Fc-enhanced humanized anti-CD19 monoclonal antibody, that has been approved for use in diffuse large B cell lymphoma. TAFA exerts its effects through antibody-derived cellular cytotoxicity (ADCC), which is a critical anti-tumor function mediated by NK cells.

Purpose: To evaluate whether the combination of memory-like NK cells (CIMLNK) with TAFA can augment cytotoxicity in B-ALL.

Methods: NK cells were isolated from peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors (n=8). For the generation of CIMLNK, NK cells were incubated overnight (12-16 hours) with IL-12 10 ng/ml, IL-15 50 ng/ml and IL-18 50 ng/ml. For the in vitro cytotoxicity assays, target cells (NALM-6 or RS4;11- both B-ALL cell lines) were labeled with CD71-FITC mAb and co-incubated with the effectors (NK or CIMLNK) at 2.5 effector: target (E: T) ratio, with or without TAFA (0.5 - 1 ug/ml) for 4 hours. PI was added just before analysis by flow cytometry (BD-LSR Fortessaâ) . The percentage (%) of target cell cytolysis in all conditions was calculated using the following formula: %CD71+PI+ exp - %CD71+PI+ target cell only.

For the in vivo experiments, irradiated immunodeficient female NSG mice received 10,000 Nalm6-luc, via teil vein injection (IV) on day 0. On day 1 TAFA was administered at 0.3 - 0.5 mg/kg, IV or IP respectively. 1 X 10⁶ CIMLNK derived from healthy donors (n=3), were injected IV either 4h or 24h after the tumor injection. The luciferase bioluminescence imaging (BLI) system was used to determine leukemia burden.

Statistics: One-way ANOVA tests with Tukey's test for multiple comparisons were used to detect differences among the culture conditions. For the in vivo studies, Kaplan-Meier survival curves were analyzed using the Gehan-Breslow-Wilcoxon test. BLI data were compared with multiple unpaired t-tests. P-values of < 0.05 were considered significant.

Results: CIMLNK + TAFA resulted in significantly enhanced cytotoxicity against the NALM6 compared with the CIMLNK alone (84.2% vs 24.3%, p=0.004) as well as against RS4;11, (63.6% vs 10.1%, p=0.0094). Notably, the combination of TAFA+ CIMLNK significantly increased cytotoxicity when compared to NK+TAFA against NALM6 (84.2% vs 38.9%, p=0.005) but not in the RS4;11 cells (63.6% vs 28.3%, p=0.11). CIMLNK alone demonstrated a trend toward higher cytotoxicity when compared to NK cells against NALM6 (24.3% vs 1.1%, p=0.06) and RS4;11 (10.1% vs 0.9%, p=0.14). Cytotoxicity was not observed against these cell lines when TAFA was used alone.

In the in vivo experiments, the mean BLI in the mice treated with the combination therapy at week 3 was reduced by 1 log compared with that of untreated mice (p = 0.01) when CIMLNK were administered the same day as the tumor. In terms of survival, combining CIMLNK with TAFA resulted in a median survival of 35 days, which was significantly higher than that seen in untreated mice (28 days, p= 0.01), CIMLNK (26.5 days, p=0.009) and TAFA monotherapy (28 days, p= 0.003).

Conclusion: The combination of CIMLNK + TAFA exerts synergistic effects against acute B-cell leukemia and may prove to be an alternative therapeutic approach for patients with relapsed refractory disease.

No relevant conflicts of interest to declare.