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Supplementary Table S1

Supplementary Fig. S3. Differentiation of ML NK cells and Preactivated-Expanded CB-NK cells.

Supplementary Materials and Methods

Purpose:Natural killer (NK)-cell recognition and function against NK-resistant cancers remain substantial barriers to the broad application of NK-cell immunotherapy. Potential solutions include bispecific engagers that target NK-cell activity via an NK-activating receptor when simultaneously targeting a tumor-specific antigen, as well as enhancing functionality using IL12/15/18 cytokine pre-activation.Experimental Design:We assessed single-cell NK-cell responses stimulated by the tetravalent bispecific antibody AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on various types of NK cells using mass cytometry and cytotoxicity assays. The combination of AFM13 and IL12/15/18 pre-activation of blood and cord blood–derived NK cells was investigated in vitro and in vivo.Results:We found heterogeneity within AFM13-directed conventional blood NK cell (cNK) responses, as well as consistent AFM13-directed polyfunctional activation of mature NK cells across donors. NK-cell source also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from patients with Hodgkin lymphoma. IL12/15/18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared with cNK cells. Cord-blood NK cells preactivated with IL12/15/18 and ex vivo expanded with K562-based feeders also exhibited enhanced killing with AFM13 stimulation via upregulation of signaling pathways related to NK-cell effector function. AFM13–NK complex cells exhibited enhanced responses to CD30+ lymphomas in vitro and in vivo.Conclusions:We identify AFM13 as a promising combination with cytokine-activated adult blood or cord-blood NK cells to treat CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.

Background Natural killer (NK) cells are a nascent cellular immunotherapy for hematologic malignancies. Target recognition of NK cell-resistant cancers remains a substantial barrier to broad application of NK cell therapy. One solution are bispecific engagers that trigger NK cells via an NK activating receptor when simultaneously engaging a tumor-specific antigen. Methods Here, we investigated single NK cell responses stimulated by the tetravalent bispecific innate cell engager (ICE®) AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on several types of NK cells. Results Multidimensional mass cytometry revealed heterogeneity within AFM13-directed conventional (c)NK cell responses, as well as consistent polyfunctional activation of mature terminally differentiated NK cells across donors. The source of NK cells also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from Hodgkin lymphoma patients. IL-12, IL-15, and IL-18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared to cNK cells. Cord-blood expanded NK cells that were pre-activated with IL-12, IL-15 and IL-18 also exhibited enhanced killing with AFM13 stimulation, via upregulation of signaling pathways related to NK cell effector function. These cells were stably pre-loaded with AFM13 enhancing responses to CD30+ lymphomas in vitro and in vivo in immunodeficient NSG mouse models. Conclusions Collectively, these data identify promising combinations of AFM13 with cytokine-activated adult blood or cord blood NK cells against CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.