Tumor-Targeting Anti-CD20 Antibodies Mediate In Vitro Expansion of Memory Natural Killer Cells: Impact of CD16 Affinity Ligation Conditions and In Vivo Priming

Abstract

Natural killer (NK) cells represent a pivotal player of innate anti-tumor immune responses. The impact of environmental factors in shaping the representativity of different NK cell subsets is increasingly appreciated. Human cytomegalovirus (HCMV) infection profoundly affects NK cell compartment, as documented by the presence of a CD94/NKG2C+FcεRIγ- long-lived “memory” NK cell subset, endowed with enhanced CD16-dependent functional capabilities, in a fraction of HCMV-seropositive subjects. However, the requirements for memory NK cell pool establishment/maintenance and activation have not been fully characterized yet. Here, we describe the capability of anti-CD20 tumor-targeting therapeutic monoclonal antibodies (mAbs) to drive the selective in vitro expansion of memory NK cells and we show the impact of donor’ HCMV serostatus and CD16 affinity ligation conditions on this event. In vitro expanded memory NK cells maintain the phenotypic and functional signature of their freshly isolated counterpart; furthermore, our data demonstrate that CD16 affinity ligation conditions differently affect memory NK cell proliferation and functional activation, as rituximab-mediated low-affinity ligation represents a superior proliferative stimulus, while high-affinity aggregation mediated by glycoengineered obinutuzumab results in improved multifunctional responses. Our work also expands the molecular and functional characterization of memory NK cells, and investigates the possible impact of CD16 functional allelic variants on their in vivo and in vitro expansions. These results reveal new insights in Ab-driven memory NK cell responses in a therapeutic setting and may ultimately inspire new NK cell-based intervention strategies against cancer, in which the enhanced responsiveness to mAb-bound target could significantly impact therapeutic efficacy.

Memory-Like NK Cells: Remembering a Previous Activation by Cytokines and NK Cell Receptors

“…Moreover, CD16-induced memory-like NK cells might support T/B cell responses during primary infection or reinfection, when endogenous IgG antibodies are produced e.g., against HCMV ( 71 – 73 ). While (HCMV-specific) antibodies have been shown to promote the expansion of pre-existing HCMV-associated NKG2C + “adaptive” NK cells ( 74 – 76 ), antibodies probably do not mediate in the initial generation of NKG2C + “adaptive” NK cells in HCMV-seronegative individuals ( 76 ). In contrast to the NKG2C + “adaptive” NK cells ( 75 ), CD16-induced memory-like NK cells maintained expression of FcεR1γ and NK cell activating receptors and exerted enhanced IFN-γ in response to IL-12 and enhanced antibody-independent cytotoxicity ( 47 ).…”

Memory NK Cell Features Exploitable in Anticancer Immunotherapy

“…The enhanced response to CD16 stimulation has been well documented in response to antiviral IgG-opsonized infected cells and, of relevance here, to tumor-targeting therapeutic mAb-opsonized tumor cells [19, 21, 33, 34, 74]. Moreover, hyperresponsiveness to anti-CD20 mAb-opsonized tumor cells was also observed in in vitro expanded memory NK cells [74].…”

“…In this context, our recent data [74] demonstrate the unique capability of anti-CD20 therapeutic mAb-opsonized targets to drive the selective in vitro expansion of memory NK cells from HCMV-seropositive healthy donors. Indeed, we developed an effective in vitro culture system, consisting of a 9-day coculture of PBMC with irradiated lymphoblastoid Raji cells opsonized with anti-CD20 therapeutic mAbs, in IL-2-containing medium (Figure 1).…”

Non-Genetically Improving the Natural Cytotoxicity of Natural Killer (NK) Cells

“…Subsequently, they could be transferred to patients lacking them to generate the desired immunity (Figure 1). Direct transfer of anti-HCMV antibodies would probably not work because these antibodies presumably do not mediate in the initial generation of NKG2C + "adaptive" NK cells in HCMV-seronegative individuals (83).…”