Abstract
The function of the low-affinity IgG-receptor FcγRIIIb (CD16b), which is uniquely and abundantly expressed on human granulocytes, is not clear. Unlike the other Fcγ receptors (FcγR), it is a glycophosphatidyl inositol (GPI) -anchored molecule and does not have intracellular signaling motifs. Nevertheless, FcγRIIIb can cooperate with other FcγR to promote phagocytosis of antibody-opsonized microbes by human neutrophils. Here we have investigated the role of FcγRIIIb during antibody-dependent cellular cytotoxicity (ADCC) by neutrophils toward solid cancer cells coated with either trastuzumab (anti-HER2) or cetuximab (anti-EGFR). Inhibiting FcγRIIIb using CD16-F(ab')2 blocking antibodies resulted in substantially enhanced ADCC. ADCC was completely dependent on FcγRIIa (CD32a) and the enhanced ADCC seen after FcγRIIIb blockade therefore suggested that FcγRIIIb was competing with FcγRIIa for IgG on the opsonized target cells. Interestingly, the function of neutrophil FcγRIIIb as a decoy receptor was further supported by using neutrophils from individuals with different gene copy numbers of FCGR3B causing different levels of surface FcγRIIIb expression. Individuals with one copy of FCGR3B showed higher levels of ADCC compared to those with two or more copies. Finally, we show that therapeutic antibodies intended to improve FcγRIIIa (CD16a)-dependent natural killer (NK) cell ADCC due to the lack of fucosylation on the N-linked glycan at position N297 of the IgG1 heavy chain Fc-region, show decreased ADCC as compared to regularly fucosylated antibodies. Together, these data confirm FcγRIIIb as a negative regulator of neutrophil ADCC toward tumor cells and a potential target for enhancing tumor cell destruction by neutrophils.
Highlights
Fc-receptors play a vital role in cancer immunotherapy by inducing antibody-dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP)
As mentioned before FcγRIIIb is subject to considerable gene copy number variation (CNV) [9], and the expression levels of FcγRIIIb are directly linked to the number of copies present in the genome (Figure 1B)
We did not find any significant correlations when comparing FcγRIIa expression levels and killing (Supplementary Figure 6). These findings show that in non-stimulated neutrophils FCGR3B CNV is an important determinant of ADCC capacity, with higher levels of CNV and concurrent FcγRIIIb surface expression negatively affecting neutrophil ADCC, thereby providing genetic evidence for a role of FcγRIIIb as a decoy receptor
Summary
Fc-receptors play a vital role in cancer immunotherapy by inducing ADCC and antibody dependent cellular phagocytosis (ADCP). The high affinity receptor FcγRI (CD64) is only present on activated neutrophils, but does generally not contribute to ADCC of solid cancer cells even when expressed [3] Both FcγRI and FcγRIIa signal via immunoreceptor tyrosinebased activation motifs (ITAM), encoded in the cytoplasmic tail of the receptors (FcγRIIa) or in the associated γ-chain (FcγRI). Neutrophils express the highly abundant, 100– 200-thousand copies per cell, low affinity receptor FcγRIIIb, which is a GPI-linked Fc-receptor that lacks intrinsic intracellular signaling capacity [4]. On the level of neutrophil-mediated ADCC of cancer cells all polymorphic variants appear effective [3], but Abbreviations: FcγR, Fcγ receptor; ADCC, antibody dependent cellular cytotoxicity; NK cell, natural killer cell; ADCP, antibody dependent cellular phagocytosis; ITAM, immunoreceptor tyrosine-based activation motif; CNV, copy number variation; G-CSF, granulocyte-colony stimulating factor; IFNγ, interferon-γ
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