Antibody-dependent cytolysis (ADCC) of tumor cells by activated murine macrophages is a two-step process: Quantification of target binding and subsequent target lysis

To analyze the antibody-dependent cell-mediated cytotoxicity (ADCC) reaction between tumor cells and activated murine macrophages in detail, it must be first determined if physical binding occurred between the two cell types. Over 15–20 min in vitro, antibody-coated HSB neoplastic targets became so firmly attached to the activated macrophages that they resisted removal with 4 vigorous washes. When a quantitative assay of binding was employed, attachment of tumor cells to activated macrophages was found to depend on the concentration of antibody and on the density of the macrophages. These two variables also determined the subsequent extent of cytolysis. Binding of antibody-coated targets by macrophages elicited with thioglycollate broth or activated by bacillus Calmette-Guerin (BCG) was comparable. Lysis by the activated macrophages, however, was far greater. Binding occurred at 4, 22, or 37 °C, while the subsequent lytic reaction occurred only at 37 °C. Thioglycollate broth effectively inhibited lysis but had no effect on binding. A porous filter placed between activated macrophages and targets resulted in abrogation of binding and lysis, even when antibody-coated targets were placed beneath the filters. When labeled, uncoated targets were added to cultures of macrophages in the presence of unlabeled antibody-coated targets, no lysis of the bystander (i.e., uncoated) targets was seen. The data suggest that ADCC is a multistep reaction, that vigorous physical binding of antibody-coated targets by activated macrophages is an initial and necessary step in ADCC, that such binding is not sufficient for ADCC, that such binding controls the selectivity of lysis in ADCC, and that the second step in ADCC results in target lysis.     

Inhibition of antibody-dependent cellular cytotoxicity by protein A from Staphylococcus aureus.

Protein A, a cell wall constituent of several strains of Staphylococcus aureus, binds strongly to the Fc portion of immunoglobulins. This investigation demonstrated that such binding can inhibit antibody-dependent cellular cytotoxicity (ADCC). The degree to which ADCC was inhibited depended upon the relative concentrations of protein A and anti-target cell antiserum. Protein A also inhibited the formation of rosettes between antibody-coated sheep red blood cells and lymphoid cells with Fc receptors. We, therefore, conclude that protein A inhibits ADCC by preventing the binding of antibody-coated target cells to Fc receptors on cytotoxic effector cells.     

Higher ADCC of murine peritoneal cells after immunization with allogenic tumor cells as compared with stimulation by adriamycin, BCG, and thioglycolate

Normal peritoneal cells or spleen cells from C57BL mice could not lyse SRBC in an ADCC assay. After intraperitoneal injection of Adriamycin, BCG or thioglycolate the ADCC of peritoneal cells toward antibody-coated SRBC was elevated to 30% in contrast to the ADCC of spleen cells. However, peritoneal cells but not spleen cells of mice immunized with allogenic tumor cells (DBA SL2) showed ADCC levels at least two times higher than the levels observed after stimulation by other agents. Maximal ADCC levels (55.8%) were observed 10 to 15 days after immunization. Direct cytotoxicity towards SRBC increased to a maximum of 17.7% at 9 days after immunization. The effector cells in this system are thought to be macrophages, for ADCC activity was only present in the plastic-adherent cell fraction. Cell to cell contact was necessary for ADCC to occur; nonsensitized erythrocytes were not lysed when added to a mixture of effector cells and sensitized erythrocytes. Concentrations of antibody of 1 pg/ml were sufficient to induce ADCC, and effector cell to target cell ratios could be as low as 0.05. The finding that macrophages of mice immunized with allogenic tumor cells exhibit higher ADCC levels than macrophages elicited in other ways can contribute to the investigation of combined cancer therapy with antibodies and biological response modifiers.     

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. VII. The effect of immunodeficiency disease.

Spontaneous lymphocyte-mediated cytotoxicity (SLMC) and antibody-dependent cellular cytotoxicity (ADCC) was assessed in 13 patients with immunodeficiency diseases—immunodeficiency-thymoma syndrome (1), Bruton type agammaglobulinemia (3), and common variable hypogammaglobulinemia (9). SLMC and ADCC function were intact (and possibly enhanced) in the patient with immunodeficiency thymoma. Both ADCC and SLMC were detectable in the three patients with X-linked agammaglobulinemia, one of whom had lower than expected SLMC. In all of the immunodeficient patients, the relative inability of B lymphocytes to produce immunoglobulin in vivo or in vitro did not consistently affect the ability of (presumably) other lymphocytes to mediate SLMC and ADCC, although in three of the CVH patients this was lower than normal. In every case, removal of Fc receptor-bearing cells from the patients' lymphocyte preparations severely depleted SLMC (and ADCC when tested), but cytotoxicity was either unchanged or enhanced by depletion of E rosette forming T cells. The effects of Fc receptor-positive cell depletion, T-cell depletion, culture serum variation, or the addition of antibody-coated erythrocytes to the assay were similar on both SLMC and ADCC effector cells (“NK” and “K” cells), and whether patients' or normal lymphocytes were tested. The possible significance of the results with respect to surveillance against cancer is discussed.     

The effects of corticosteroids on immunoregulation in sarcoidosis.

The effects of in vivo hydrocortisone administration on the kinetics and functional capabilities of cells involved in the immune response in sarcoidosis were examined. Untreated sarcoidosis patients have a decrease in the absolute numbers of circulating T lymphocytes (P < 0.05). However, with regard to the proportions of T lymphocyte subpopulations, there is an increase in the relative proportions of IgG Fc receptor positive T cells (T_G) (P < 0.01), which have suppressor capabilities in certain in vitro systems of mitogen-induced antibody production, and a relative decrease in IgM Fc receptor positive T lymphocytes (T^M) which have helper effects in this system (P < 0.05). Additionally, sarcoidosis patients have circulating “suppressor” monocytes capable of suppressing anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) responses by pokeweed mitogen (PWM)-stimulated lymphocytes. The in vitro removal of this cell abrogated this depressed response (P < 0.01). Intravenous administration of hydrocortisone produced a transient absolute T lymphocytopenia (P < 0.01) accompanied by a relative increase in T^G cells (P < 0.01) and a relative decrease in T_M cells (P < 0.02). Four hours after hydrocortisone therapy, at the point of maximal hydrocortisone-induced monocytopenia (P < 0.01), the suppressed ability of sarcoidosis lymphocytes to synthesize and secrete in vitro anti-SRBC antibody after polyclonal activation was corrected (P < 0.01), and PFC responses comparable to those seen in untreated normal subjects were obtained. These studies demonstrate that corticosteroid administration has profound effects on certain in vitro demonstrable immunoregulatory abnormalities in sarcoidosis.     

Further characterization of suppressor lymphocytes induced by fetal calf serum in murine lymphoid cell cultures: comparison with in vitro-generated cytotoxic lymphocytes.

Nonspecific suppressor cell (SPC) activity has been induced in vitro by preculturing splenocytes from normal mice in the presence of fetal calf serum (FCS) for 3 days or more. In adoptive transfer experiments in vivo, these precultured SPC were shown to reduce the humoral response of mice to SRBC and the cell-mediated cytotoxic (CMC) response to allogeneic tumor cells. In mixing experiments in vitro, using freshly explanted splenocytes, the precultured splenocytes abrogated the generation of specific cytotoxic lymphocytes (CL) in primary mixed lymphocyte-tumor cell cultures (MLTC). By contrast, secondary cytotoxic response was only marginally affected. Supernatants of precultured cells were also inhibitory, although to a lesser degree than whole cells. The induction of suppressive activity was abolished by addition of mitogenic amounts of concanavalin A to the preculturing medium.By the use of cell fractionation techniques it was found that both specific CL and nonspecific SPC lack an Fc receptor, do not adhere to nylon wool, and cannot be separated from each other by density sedimentation on a discontinuous BSA gradient. However, precursors of SPC and CL differed in their susceptibility to cyclophosphamide, hydrocortisone, and irradiation. The data presented does not exclude the possibility that suppressive activity exerted by FCS-induced SPC is mediated through a cytotoxic effect.     

Antigen-specific helper activity in serum of mice primed with sheep red cells. I. Definition of the test system and comparison with other systems

An adoptive transfer system is described to measure serum helper activity in the primary antibody response to sheep red blood cells (SRBC). Mice injected with a high dose of cyclophosphamide and reconstituted with rabbit anti-thymocyte serum-treated spleen cells were used as recipients. Serum obtained 9 hr after ip injection of normal mice with 2 × 10⁸ SRBC (S(SRBC)) injected i.v. in the recipients caused a significant enhancement of the antibody response to 2 × 10⁷ SRBC. The serum helper activity was not generated in thymectomized animals and could be absorbed from S(SRBC) by normal and formalinized SRBC. The SRBC-specific serum helper activity (SSHA) is heat labile (30 min 56 °C) and shows allogeneic restriction. Another test system described in literature for measuring T-cell help in vivo was less suited to measure SSHA in the response to 2 × 10⁷ SRBC. A system using normal mice injected with 10⁵ SRBC for determining specific immune response-enhancing factor (SIREF), demonstrated SIREF activity in S(SRBC). It did, however, not measure SSHA, as absorption of S(SRBC) with formalinized SRBC did not abolish the activity in that system.     

Identification of five human lymphocyte subpopulations by their differential binding of various strains of bacteria.

The ability of human peripheral blood lymphocytes to kill antibody-coated Chang liver cells in antibody-dependent cell-mediated cytotoxicity (ADCC) can be blocked with aggregated IgG (agg-IgG) or by soluble immune complexes. Dissociation of aggregates of IgG or immune complexes from the cell surface, however, resulted in partial recovery of the ability both to bind agg-IgG and to kill in the ADCC assay. Our results indicate that “unblocking” of effector cells could occur in vivo when the concentration of circulating immune complexes is lowered.     

Modification of regression of virally xenogenized tumor cells by cyclophosphamide and busulfan

Summary Rat fibrosarcoma cells infected with Friend leukemia virus (FV-KMT-17) grow for a short time and then regress spontaneously in syngeneic hosts. This regression mechanism was examined by analyzing the immunomodulating action of the antitumor drugs busulfan (BU) and cyclophosphamide (CY). In preliminary experiments, the optimum dosages of BU and CY for the enhancement of DTH responses to SRBC were 10 mg/kg and 40 mg/kg respectively. Treatment of rats with BU (10 mg/kg) on day 5 induced the regression of KMT-17 cells, while in contrast, the same drug delayed the spontaneous regression of FV-KMT-17 cells. Pretreatment with CY (40 mg/kg) on day 5 did not affect the growth of KMT-17 or FV-KMT-17 cells. After the same treatment schedule, BU inhibited humoral antibody formation against SRBC and against virus-associated antigen (VAA), NK cell activity, and ADCC effector cell activity. On the other hand, CY did not affect the activities of NK cells or ADCC effector cells, although it significantly augmented the DTH responses to SRBC and the production of antibody to VAA but had no effect on production of antibodies to SRBC. These results suggest that NK cells and ADCC may play an important role in the initial stage of the spontaneous regression of FV-KMT-17 cells.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education Abbreviations used: BU, busulfan; CY, cyclophosphamide; PFC assay, plaque forming cell assay; VAA, virus-associated antigen; NK cell, natural killer cell; ADCC, antibody dependent cellular cytotoxicity; MuLV, murine leukemia virus; DTH, delayed type hypersensitivity; SRBC, sheep red blood cells; C.I., cytotoxic index; CRBC, chicken red blood cells; IL-1, interleukin 1; IL-2, interleukin 2; IFN, interferon     

In Vitro Induction of Antibody-Dependent Cytotoxic Macrophages by the Local Anesthetic Lidocaine

Normal macrophages were activated to antibody-dependent cytotoxic effector cells by in vitro treatment with the local anesthetic lidocaine. Experiments on the dose-response and time course of the effect of lidocaine showed that incubation of normal macrophages with 10 mM lidocaine for 10 min at 28 C was enough for induction of antibody-dependent cellular cytotoxicity. The activation by lidocaine was accompanied by enhanced phagocytosis of sheep red blood cells (SRBC) sensitized with anti-SRBC antiserum, but not enhanced ingestion of polystyrene latex particles (PLP). These findings suggest that lidocaine, which has various effects on cell membranes, induces some perturbation of macrophage membranes, resulting in activation of Fc receptor functions in antibody-dependent cytotoxicity and phagocytosis.     

In situ activation of murine macrophages by liposomes containing lymphokines

Murine alveolar and peritoneal macrophages harvested after injection of lymphokines encapsulated within multilamellar phospholipid vesicles (liposomes) were tumoricidal in vitro. The state and degree of activation depended on the route of liposome administration. Activation of peritoneal macrophages was achieved by intraperitoneal injection of liposomes and alveolar macrophages were activated by injecting liposomes intravenously but not intraperitoneally. The in vivo rendering of macrophages with tumoricidal properties might be useful toward destruction of tumor cells in vivo.     

Detection of Fc receptors for IgA on rat alveolar and peritoneal macrophages.

The presence of Fc receptors for IgA on alveolar macrophages was determined by rosette assay and immunogold labeling. IgA-mediated phagocytosis by alveolar macrophages was observed. Results of these assays were compared between rats receiving no treatment and those receiving long-term cortisone administration. Sheep erythrocytes coated with dextran and an IgA monoclonal antibody specific for the alpha 1,3 linkages of dextran bound to 16% of alveolar macrophages. However, peritoneal macrophages did not form rosettes with dextran-IgA-coated erythrocytes. Immunogold labeling by transmission electron microscopy revealed that most Fc receptors for IgA were found on the membrane of pseudopodia of activated alveolar macrophages. Long-term cortisone administration diminished the phagocytosis and phagocytic index of alveolar macrophages, thereby contributing to decreased host resistance to infection (e.g., Pneumocystis carinii pneumonia).     

Localization of receptors and early events of phagocytosis in the macrophage

We studied the attachment of MRBC, 1.1 μm latex (foreign surface receptor), hemocyanin-DNP antiDNP Ab and sensitized SRBC (Fc receptor) to mouse peritoneal macrophages in culture, and the phagocytosis of 1.1 μm latex, AbAg complex, and sensitized SRBC by macrophages. Both types of receptors appeared over the whole cell surface. The particles attached mostly in the central area of the cell, but peripheral attachment was also observed. Internalization of 1.1 μm latex beads appeared to be mostly perinuclear, by sinking in of the cytoplasm with the attached particles, rather than a flow of membrane process over the beads. Also in the case of the AbAg complex attached to the Fc receptor internalization seemed mostly perinuclear and similar in mode to the latex ingestion. When attachment occurred in the periphery, active ruffling was observed. Sensitized SRBC internalization was seen mostly in the extreme periphery of the cells after the attached SRBC were moved from the perinuclear area where many of them first attached.     

Generation and Preclinical Characterization of an NKp80-Fc Fusion Protein for Redirected Cytolysis of Natural Killer (NK) Cells against Leukemia

The capacity of natural killer (NK) cells to mediate Fc receptor-dependent effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), largely contributes to their clinical application. Given that activation-induced C-type lectin (AICL), an identified ligand for the NK-activating receptor NKp80, is frequently highly expressed on leukemia cells, the lack of therapeutic AICL-specific antibodies limits clinical application. Here we explore a strategy to reinforce NK anti-leukemia reactivity by combining targeting AICL-expressing leukemia cells with the induction of NK cell ADCC using NKp80-Fc fusion proteins. The NKp80-Fc fusion protein we generated bound specifically to leukemia cells in an AICL-specific manner. Cell binding assays between NK and leukemia cells showed that NKp80-Fc significantly increased NK target cell conjugation. In functional analyses, treatment with NKp80-Fc clearly induced the ADCC effect of NK cells. NKp80-Fc not only promoted NK-mediated leukemia cell apoptosis in the early stage of cell conjugation but also enhanced NK cell degranulation and cytotoxicity activity in the late stage. The bifunctional NKp80-Fc could redirect NK cells toward leukemia cells and triggered NK cell killing in vitro. Moreover, NKp80-Fc enhanced the lysis of NK cells against tumors in leukemia xenograft non-obese diabetic/severe combined immunodeficiency mice. Taken together, our results demonstrate that NKp80-Fc potently amplifies NK cell anti-leukemia effects in vitro and in vivo through induction of the NK cell ADCC effect. This method could potentially be useful for molecular targeted therapy, and the fusion proteins may be a promising drug for immunotherapy of leukemia.     

In Vitro Glycoengineering of IgG1 and Its Effect on Fc Receptor Binding and ADCC Activity

The importance and effect of Fc glycosylation of monoclonal antibodies with regard to biological activity is widely discussed and has been investigated in numerous studies. Fc glycosylation of monoclonal antibodies from current production systems is subject to batch-to-batch variability. If there are glycosylation changes between different batches, these changes are observed not only for one but multiple glycan species. Therefore, studying the effect of distinct Fc glycan species such as galactosylated and sialylated structures is challenging due to the lack of well-defined differences in glycan patterns of samples used. In this study, the influence of IgG1 Fc galactosylation and sialylation on its effector functions has been investigated using five different samples which were produced from one single drug substance batch by in vitro glycoengineering. This sample set comprises preparations with minimal and maximal galactosylation and different levels of sialylation of fully galactosylated Fc glycans. Among others, Roche developed the glycosyltransferase enzyme sialyltransferase which was used for the in vitro glycoengineering activities at medium scale. A variety of analytical assays, including Surface Plasmon Resonance and recently developed FcγR affinity chromatography, as well as an optimized cell-based ADCC assay were applied to investigate the effect of Fc galactosylation and sialylation on the in vitro FcγRI, IIa, and IIIa receptor binding and ADCC activity of IgG1. The results of our studies do not show an impact, neither positive nor negative, of sialic acid- containing Fc glycans of IgG1 on ADCC activity, FcγRI, and RIIIa receptors, but a slightly improved binding to FcγRIIa. Furthermore, we demonstrate a galactosylation-induced positive impact on the binding activity of the IgG1 to FcγRIIa and FcγRIIIa receptors and ADCC activity.     

Effect of recombinant IFN-gamma (rIFN-gamma) on the mechanism of human macrophage IgG FcRI-mediated cytotoxicity. rIFN-gamma decreases inhibition by cytophilic human IgG and changes the cytolytic mechanism.

Different classes of receptors for the Fc moiety of IgG (Fc gamma R) have been defined on human monocytes and macrophages: Fc gamma RI, Fc gamma RII, and Fc gamma RIII. All three classes are capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Fc gamma RI, which binds monomeric human IgG (hIgG) with high affinity, was shown an effective cytotoxic trigger molecule on different types of cells. In vitro, the inhibition of Fc gamma RI-mediated ADCC by hIgG is well documented. The low affinity receptor classes, Fc gamma RII and Fc gamma RIII, are not blocked by monomeric hIgG. Because monomeric hIgG is present at high concentrations in plasma and interstitial fluids it has been postulated inhibitory in vivo. We investigated the effect of rIFN-gamma on macrophage Fc gamma RI-mediated ADCC in the presence of low doses hIgG. With human E sensitized with hIgG as target cells, Fc gamma RI was studied selectively. We found that rIFN-gamma enhances both expression and cell surface density of Fc gamma RI on cultured peripheral blood monocytes. Furthermore, this cytokine partially reversed the inhibitory effect of monomeric hIgG on ADCC. More interestingly, we found that the cytolytic mechanism of monocyte-derived macrophages changed completely after prolonged culture with rIFN-gamma. Monocytes cultured for 9 days in control medium mediate predominantly phagocytosis. After long term rIFN-gamma stimulation (9 days), monocyte-derived macrophages almost completely lost the capacity to perform phagocytosis. Interestingly, they became highly efficient in mediating extracellular lysis of human E sensitized with hIgG. Short term rIFN-gamma stimulated monocyte-derived macrophages (for the last 40 h of culture) were found to mediate both phagocytosis and extracellular lysis. Our findings suggest that in vivo rIFN-gamma-stimulated macrophages may be most efficient in Fc gamma RI-mediated cytolysis as a consequence of a changed cytolytic mechanism in combination with enhanced Fc gamma RI density.     

Human peritoneal macrophages possess two populations of IgG Fc receptors

To characterize the binding properties of the Fc receptors on human macrophages, the binding of radiolabeled human IgG1 to peritoneal macrophages was assessed. Cells were obtained at the time of diagnostic laparoscopy from women undergoing evaluation of infertility. Macrophages bound on the average more IgG1 monomer than monocytes but the avidity with which both types of cells bound IgG1 monomer was comparable. By contrast, macrophages bound much more IgG1 dimers than monocytes. Scatchard plots of the binding of dimer to monocytes were linear, but plots of binding to macrophages were markedly curvilinear. This curvilinearity was not an artifact of extensive ligand internalization or catabolism by cells, since 80% of binding was reversible and there was very little catabolism of ligand in the medium. Assuming that the observed curvilinearity was due to the presence of two independent subpopulations of receptors, an objective estimate for the number of receptors per cell and of the avidity with which each subpopulation bound IgG1 dimer was obtained using a previously described computer program (Scatfit). The analysis of the binding of dimer to macrophages from six donors suggested the presence of 42,000 +/- 33,000 high avidity receptors per cell which bind IgG1 dimer with a mean Ka of 2.7 X 10(9) M-1 and 218,000 +/- 127,000 low avidity receptors which bind the same ligand with a Ka of 1.1 X 10(7) M-1. ADCC of IgG antibody-coated sheep red blood cells mediated by macrophages was less readily inhibited by soluble IgG1 monomer than ADCC mediated by peripheral blood monocytes. This provides further evidence for the presence of low avidity receptors which bind monomeric IgG1 poorly and also suggests that these sites are functionally active in triggering antibody-dependent immune clearance.     

Nuclear disintegration of target cells by killer B lymphocytes from tumor-bearing mice

Normal murine B lymphocytes are not known to be effectors of the Fc receptor-mediated, antibody-dependent cellular cytotoxicity (ADCC). In contrast, we report here that highly purified splenic B cells from mammary tumor-bearing mice develop the potential of lysing antibody-coated target cells. These lymphocytes are characterized by being G-10 nonadherent, nylon wool adherent, sIg+, FcR+, Thy 1.2-, asialo GM1-, and the immunoglobulin heavy-chain genes of both chromosomes are rearranged. The lytic reaction is characterized by a noninterdigitating binding and by the appearance of endocytotic vesicles in the target cells. Nuclear disintegration occurs 18 h after initial effector-target cell conjugate formation. At such time, only minor cytoplasmic membrane alterations are evident. The emergence of killer B cells in tumor-bearing hosts indicates that all lymphoreticular cell types bearing Fc receptors are capable of mediating ADCC.     

Development of surface membrane characteristics of "premedullary" macrophages in organ cultures of embryonic rat and hamster lungs

A replicating population of non-monocyte-derived free cells appears in organ-cultured embryonic rat lungs, indistinguishable from alveolar macrophages by classical criteria such as ultrastructure, lysosomal enzyme cytochemistry, and phagocytic behavior. We demonstrate similar events in cultured embryonic hamster lungs and development of macrophage-associated properties on the plasmalemma of these cells in both species. Immunoperoxidase localizations were obtained using monoclonal antibodies against alveolar macrophage antigen (HAM1) in hamsters, and rat macrophage antigen (ED1) and leukocyte-common antigen (OX1) in rats. Fc and C3b receptors were identified in both species by immune rosetting. HAM1 staining, perinuclear in rare cells at explantation, gains definitive surface localization 3-4 days later as cells prepare to emerge through the pleura. ED1 and OX1 cytoplasmic staining first occurs after 24 hr, increases as macrophages multiply and congregate beneath the pleura, and translocates to the plasmalemma of emerged cells. Some glass-adherent cells from lung explants have Fc receptors. The proportion rises sharply for 24 hr and equals fully emerged cells (90-95%) by days 3-4. At first phagocytosis is slow to follow Fc receptor binding, but ingestion time decreases to 3-10 min as macrophages mature. A minority of emerged macrophages bind complement-opsonized erythrocytes, which are rarely taken up. These properties are shared by alveolar macrophages of adults.     

Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities

The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its “regular” fucosylated counterpart and a series of mixtures containing varying proportions of “regular” and afucosylated materials. Compared with the “regular” fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC₅₀ values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.