In an antibody-dependent cell-mediated viral inhibition (ADCVI) assay (16) using a SHIVSF162P3-infected human T-cell line as Panitumumab (anti-EGFR) custom synthesis Panitumumab (anti-EGFR) In Vivo target cells, NFb12 gave a Anti-Mouse CD28 Antibody Panitumumab (anti-EGFR) web Formula 10-fold increase in viral inhibition for human PBMCs (Fig. We next evaluated NFb12 in a Anti-Mouse CD11a Antibody Protocol series of Fc-dependent effector function assays for which activity of wild-type b12 could be shown (see Discussion below). FUT8 catalyzes the transfer of fucose to N-linked oligosaccharides, and antibodies generated in these cells therefore lack fucose (5, 49). A MALDITOF-MS analysis showed no evidence of fucose for the NFb12 antibody compared to 94 fucose for wild-type b12 generated in FUT8-proficient CHO cells. Binding to human and rhesus macaque Fc Rs. To evaluate the affinity of NFb12 for Fc RIIIa, we performed surface plasmon resonance (SPR) and ELISA studies comparing the interactions of NFb12 and wild-type b12 with recombinant human (genotypes F158 and V158) and rhesus macaque I212 (genotypes 1 or 2) and V212 (genotype 3) Fc RIIIa. NFb12 showed PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/25762297?dopt=Abstract increased affinity to all Fc RIIIa proteins (6- to 8-fold by SPR) compared to wild-type b12 (Fig. 1; see also Fig. S1A in the supplemental material). Binding to the other activating Fc gamma receptors was largely unaffected by the absence of fucose (see Fig. S1B and C). As expected from previous studies (14, 39, 41), these experiments demonstrate that IgG fucosylation largely impacts binding to Fc RIIIa exclusively, with NFb12 showing a substantially increased affinity for this receptor. In vitro antiviral activities of NFb12. As expected, the antigen specificity, binding to cell surface-expressed envelope, and neutralization (HIV strains JRFL and JRCSF were tested in addition [data not shown]) properties of NFb12 were equivalent to those of wild-type b12 (Fig. 2A, B, and C). We next evaluated NFb12 in a series of Fc-dependent effector function assays for which activity of wild-type b12 could be shown (see Discussion below). In an antibody-dependent cell-mediated viral inhibition (ADCVI) assay (16) using a SHIVSF162P3-infected human T-cell line as target cells, NFb12 gave a 10-fold increase in viral inhibition for human PBMCs (Fig. 3A) as effector cells relative to b12 but no increase for human monocytes (Fig. 3B) as effector cells in a comparable experiment. As freshly isolated monocytes mainly express Fc RIIa (9), these results nicely demonstrate the importance of Fc RIIIa and NK cells in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/25843419?dopt=Abstract the enhanced ADCVI activity of NFb12 compared to wild-type b12. We next performed an ADCC assay using engineered human NK cells as effector cells and showed that NFb12 mediated killing of HIV-infected target cells 10-fold more efficiently than wild-type b12, as seen by a decrease in luciferase signal from target cells (Fig. 3C). Finally, we performed an NK cell degranulation assay (1, 8), and, as seen in Fig. 3D, NFb12 possessed an increased ability to induce NK degranulation compared toJune 2012 Volume 86 Numberjvi.asm.orgMoldt et al.FIG 3 In vitro Fc-dependent activities of wild-type b12 and NFb12.