Obinutuzumab-mediated high-affinity ligation of FcγRIIIA/CD16 primes NK cells for IFNγ production

Abstract

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), based on the recognition of IgG-opsonized targets by the low-affinity receptor for IgG FcγRIIIA/CD16, represents one of the main mechanisms by which therapeutic antibodies (mAbs) mediate their antitumor effects. Besides ADCC, CD16 ligation also results in cytokine production, in particular, NK-derived IFNγ is endowed with a well-recognized role in the shaping of adaptive immune responses.Obinutuzumab is a glycoengineered anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for CD16 and consequently the killing of mAb-opsonized targets. However, the impact of CD16 ligation in optimized affinity conditions on NK functional program is not completely understood.Herein, we demonstrate that the interaction of NK cells with obinutuzumab-opsonized cells results in enhanced IFNγ production as compared with parental non-glycoengineered mAb or the reference molecule rituximab. We observed that affinity ligation conditions strictly correlate with the ability to induce CD16 down-modulation and lysosomal targeting of receptor-associated signaling elements. Indeed, a preferential degradation of FcεRIγ chain and Syk kinase was observed upon obinutuzumab stimulation independently from CD16-V158F polymorphism. Although the downregulation of FcεRIγ/Syk module leads to the impairment of cytotoxic function induced by NKp46 and NKp30 receptors, obinutuzumab-experienced cells exhibit an increased ability to produce IFNγ in response to different stimuli.These data highlight a relationship between CD16 aggregation conditions and the ability to promote a degradative pathway of CD16-coupled signaling elements associated to the shift of NK functional program.

CD16A Activation of NK Cells Promotes NK Cell Proliferation and Memory-Like Cytotoxicity against Cancer Cells

“…Following CD16A engagement, we observed a transient impaired function in CD16A-dependent as well as "natural" NK cell cytotoxicity and IFNg expression toward tumor cells. The impairment of CD16A-dependent activity may be explained by the nearly complete loss of CD16A expression upon CD16A engagement, which, at least in part, involved matrix metalloproteinase-mediated cleavage, consistent with previous studies, or receptor internalization, as additionally described (24,28,(41)(42)(43). The transient lower responsiveness in "natural" NK cell antitumor reactivity toward K562 cells suggested a desensitization of other NK cell-activating receptors, such as NKp30 and NKG2D, that have previously been shown to be involved in K562 lysis (44,45).…”

“…PKC activation can mediate IFNg production and is important for K562 lysis but dispensable for ADCC, which, in turn, requires PI3K activation (48,49). The observed impairment in our study after 20-hour exposure to AFM13 or rituximab may exceed the inhibitory effect of shortterm (1.5-hour) CD16A engagement, which was reported to not affect IFNg production or responsiveness to PMA/ionomycin, while resulting in defective degranulation due to SHP-1 recruitment, inhibition of PLCg2/Vav-1/SLP-76 phosphorylation, and both FceR1g and CD3z degradation (42,43,50).…”

“…This may be explained by the upregulation of the high-affinity IL12 and IL18 receptors, which potently induce IFNg. Similarly, IFNg production in response to IL12 has recently been shown to be functional and enhanced after Fc-mediated antibody engagement of CD16A (43). It has been reported that CD16A and IL12 receptor activation can synergistically promote IFNg production (51).…”

Boosting therapeutic potency of antibodies by taming Fc domain functions

“…Fc-engineered antibodies with improved affinity to FcγRIIIa show higher therapeutic efficacy relative to native Fc because they are capable of priming and activating NK cells more efficiently 14,15 . However, several studies showed that although the depletion of NK cells or neutrophils did not significantly reduce tumor suppression activity, exhaustion of macrophages abrogated the Fig.…”

Prognostic Impact of Natural Killer Cell Count in Follicular Lymphoma and Diffuse Large B-cell Lymphoma Patients Treated with Immunochemotherapy

“…Obinutuzumab (GA101) is a type II anti-CD20 mAb with enhanced direct cell death activity and a glycoengineered Fc region that enhances its binding affinity to FcgRIIIa on NK and other effector cells, thereby resulting in stronger ADCC compared with rituximab (11)(12)(13)(14). Such high affinity ligation of FcgRIIIa has been shown to prime NK cells for IFNg production (15).…”