The mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity to virus-infected target cells. I. Identification of the population of effector cells.

Mononuclear cells (MC) from human blood were fractionated by a variety of physical and immunologic techniques, and the cellular subpopulations generated were assessed for their capacity to lyse herpes simplex virus (HSV)-infected target cells in the presence of IgG antibody to HSV. Latex phagocytosis and surface marker studies were performed in parallel in order to identify the major effector cells by their phagocytic properties and their possession of surface immunoglobulin and receptors for either sheep erythrocytes, C3, or the Fc fragment of IgG. Cytotoxic effector cell activity was unaffected or slightly enhanced after the removal of plastic-adherent or carbonyl iron-adherent MC, indicating that the major effector cell is not a classical monocyte. Similar results were obtained after removal of more than 90% of the T cells by depletion of rosette-forming cells. Likewise, effector cell activity was generally unchanged when more than 95% of the B cells were removed by filtering MC on nylon wool columns. Effector cell function was also found to be normal in three patients with B cell-deficient X-linked agammaglobulinemia. These observations strongly suggest that the effector cells are not T cells or B cells. A 4- to 5-fold enrichment in effector cells, however, was consistently found in a subpopulation, consisting of 5% of the unfractionated MC, that was dramatically enriched both for nonphagocytic cells with only Fc receptor (K cells) and for nonphagocytic cells with no detectable surface markers (null cells). Since, as is demonstrated in the accompanying report, effector surface Fc receptors play a critical role in the mediation of antibody-dependent cellular cytotoxicity directed at HSV-infected target cells, the major mononuclear effector cell in human blood is a K cell.     

Antibody-dependent cell-mediated cytotoxicity to target cells infected with type 1 and type 2 herpes simplex virus.

The phenomenon of antibody-dependent cell-mediated cytoxicity (ADCC) has been extended to include target cells acutely infected with herpes simplex type 1 virus (HSV-1) or herpes simplex type 2 virus (HSV-2) in an in vitro system that employs immune human serum and human blood mononuclear cells. The cytotoxic reaction was detectable after 1 hr of incubation and was complete between 4 and 8 hr. The amount of ADCC noted was directly proportional to the logarithm(10) of the effector: target cell ratio (E:T), and ADCC was noted at E:T as low as 1:1. The mononuclear effector cell was present in the blood of both HSV immune and non-immune individuals. The immune serum factor was demonstrated to be an antibody with specificity for HSV membrane antigen(s) and was reactive with target cells infected with either of the two HSV types. The antibody rendered the mononuclear cell cytotoxic by sensitization of the target cell rather than by direct attachment to or "arming" of the mononuclear cell. The physiochemical properties of the antibody as well as its presence in cord blood demonstrated that it is an immunoglobulin on the IgG class.     

Engineered aglycosylated full-length IgG Fc variants exhibiting improved FcγRIIIa binding and tumor cell clearance

FcγRIIIa, which is predominantly expressed on the surface of natural killer cells, plays a key role in antibody-dependent cell-mediated cytotoxicity (ADCC), a major effector function of therapeutic IgG antibodies that results in the death of aberrant cells. Despite the potential uses of aglycosylated IgG antibodies, which can be easily produced in bacteria and do not have complicated glycan heterogeneity issues, they show negligible binding to FcγRIIIa and abolish the activation of immune leukocytes for tumor cell clearance, in sharp contrast to most glycosylated IgG antibodies used in the clinical setting. For directed evolution of aglycosylated Fc variants that bind to FcγRIIIa and, in turn, exert potent ADCC effector function, we randomized the aglycosylated Fc region of full-length IgG expressed on the inner membrane of Escherichia coli. Multiple rounds of high-throughput screening using flow cytometry facilitated the isolation of aglycosylated IgG Fc variants that exhibited higher binding affinity to FcγRIIIa-158V and FcγRIIIa-158F compared with clinical-grade trastuzumab (Herceptin®). The resulting aglycosylated trastuzumab IgG antibody Fc variants could elicit strong peripheral blood mononuclear cell-mediated ADCC without glycosylation in the Fc region.     

Virus-induced enhancement of lymphocyte-mediated antibody-dependent cytotoxicity (ADCC) in vitro

The effect of Parotis virus on antibody-dependent cellular cytotoxicity in vitro (ADCC) of human lymphocytes was investigated in a 51Cr-release assay and, at the effector cell level, in an ADCC plaque assay. Target cells were bovine or chicken erythrocytes, which are not susceptible to natural cytotoxicity (NK) of human lymphocytes. They were not killed when incubated with virus-treated lymphocytes in the absence of antibodies. Treatment of the lymphocytes or the target cells with small amounts of virus, however, resulted in a very significant enhancement of ADCC. The same results were obtained with live or UV-inactivated virus, suggesting that enhancement was a passive phenomenon not requiring infection. Enhancement was already significant after 3 hr of incubation, indicating that it was independent of endogenously released interferon. Enhancement of ADCC by virus was due to effector cell recruitment rather than due to the increase of the cytotoxic potential of the individual K cell. The highest frequency of effector cells was present in Percoll fractions enriched in large granular lymphocytes (LGL). Virus treatment resulted in recruitment of effector cells carrying T cell markers such as the T3 antigen (OKT3+), receptors for sheep erythrocytes, or Fc receptors for IgM. In contrast, the absolute number of K cells carrying the HNK-1 marker (Leu-7) or receptors for C3 fragments was not changed by the virus. It is concluded that Parotis virus enhances ADCC by improving effector cell-target cell contacts, resulting in recruitment of effector cells with T cell characteristics. Recruitment is accompanied by a significant reduction of the antibody concentration needed for ADCC induction. This virus-mediated enhancement of ADCC may be of importance for protection of the host in the early phases of a virus infection in which the amounts of anti-viral IgG antibodies capable of inducing cellular cytotoxicity may yet be very small.     

Exchanging human Fcγ1 with murine Fcγ2a highly potentiates anti-tumor activity of anti-EpCAM antibody adecatumumab in a syngeneic mouse lung metastasis model

An important mode of action shared by human IgG1 antibody therapies is antibody-dependent cellular cytotoxicity (ADCC). ADCC relies on the interaction of the antibody’s Fc portion with Fc-gama receptors (FcγR) on immune effector cells. The anti-tumor activity of human IgG1 antibodies is frequently assessed in mouse models. Binding of human IgG1 to murine FcγRs is however of reduced affinity. We here show that ADCC of adecatumumab (MT201), a fully human IgG1 antibody specific for epithelial cell adhesion molecule (EpCAM/CD326), is drastically lower if human peripheral blood mononuclear cells are replaced by murine splenocytes as effector cells. When the variable domains of adecatumumab were genetically fused to a murine IgG2a backbone (yielding mu-adecatumumab), ADCC with murine effector cells was much improved, but at the same time significantly reduced with human effector cells. The serum half-lives of adecatumumab and mu-adecatumumab were determined in mice and dosing schedules established that gave similar serum trough levels during a 4-week antibody treatment. The anti-tumor activities of adecatumumab and mu-adecatumumab were then compared side-by-side in a lung metastasis mouse model established with a syngeneic B16 melanoma line expressing human EpCAM at physiologically relevant levels. Treatment of mice with mu-adecatumumab led to an almost complete prevention of lung metastases, while the human version of the antibody was much less active. This shows that adecatumumab has high anti-tumor activity when tested in a form that is better compatible with the species’ immune system. Moreover, our data suggest to routinely compare in mouse models human IgG1 and murine IgG2a versions of antibodies to properly assess the contribution of ADCC to overall anti-tumor activity.     

Studies of complement receptors on cytotoxic effector cells in human peripheral blood

The question of whether cells bearing complement receptors (CR) mediate cytotoxicity in vitro against allogeneic Chang liver cell targets was investigated by assessing peripheral blood mononuclear cells (PBMC) from normal humans for cell surface characteristics and cytotoxic capacity before and after depletion of CR+ cells capable of forming rosettes with sheep erythrocytes coated with 19S antibody and mouse complement (EAC) and depletion of Fc receptor-bearing cells capable of forming rosettes with human O+ erythrocytes coated with Ripley antibody (EA-Ripley). PBMC depleted of CR+ cells by density centrifugation contained markedly reduced proportions of phagocytes and sIg + cells and increased proportions of both sIg ?, FcR+ cells as well as cells forming rosettes with sheep erythrocytes (E). PBMC depleted of CR+ cells mediated cytotoxicity to an extent equal to or greater than that mediated by unfractionated PBMC in assays of spontaneous cell-mediated cytotoxicity (SCMC), antibody-dependent cellular cytotoxicity (ADCC), and mitogen-induced cellular cytotoxicity (MICC). Cells harvested from the EAC-rosette enriched pellet mediated cytotoxicity 5- to 10-fold less than unfractionated PBMC; however, the cytotoxic activity of the pellet could not be attributed to CR + effector cells since similar cytotoxic activity was present in cell pellets obtained by density centrifugation of PBMC which had been incubated with E coated with 19S antibody or E alone. PBMC depleted of EA-Ripley rosette-forming cells contained decreased proportions of sIg?, FcR+ cells and increased proportions of CR+ cells; PBMC so depleted contained virtually no SCMC and ADCC effector cell activity. These findings indicate that at least the majority of effector cells which mediate SCMC, ADCC, and MICC do not bear CR.     

Human monocyte-macrophage-mediated antibody-dependent cytotoxicity to herpes simplex virus-infected cells.

Human peripheral blood mononuclear cells which mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus- (HSV) infected target cells consist of both adherent (MA) and nonadherent (MNA) effector cell populations. These two cell populations can be distinguished by their different phagocytic properties and morphologic appearance, their requirement for antibody in the ADCC reaction, and the rapidity with which they lyse target cells in the presence of immune serum. The MA cells are predominantly phagocytic and have the morphologic characteristics of monocyte-macrophages, whereas the MNA cells are nonphagocytic and appear to be small to medium-sized lymphocytes. Optimal expression of ADCC by MA cells requires higher concentrations of immune serum than does MNA cell-mediated ADCC. MA-mediated cell killing is first detectable by 8 hr and reaches completion after 24 hr of incubation. In contrast, MNA-mediated ADCC produces target cell damage by 2 hr and reaches completion at 8 hr of incubation. Unlike MNA effector cells, the MA effector cells are profoundly inhibited after preincubation with either latex or silica particles. The HSV immune status of the donor had no effect on the ability of either cell population to mediate ADCC. These data demonstrate the participation of both nonadherent mononuclear cells, presumably K cells, and monocyte-macrophages, in ADCC directed against HSV-infected target cells.     

Lymphocyte-mediated cytotoxicity to autologous cells infected with measles virus. II. Specificity of the cytotoxic reaction and characterization of the effector cells involved.

The mechanism of lymphocyte-mediated cytotoxicity to cells infected with measles virus was investigated. Cytotoxicity was measured in a direct assay, immediately after the isolation of lymphocytes from human peripheral blood; mononuclear leukocytes, infected with measles virus in vitro, served as autologous target cells. Virus-specific cytotoxicity required the presence of both IgG antibodies against measles virus and of effector lymphocytes. The effector lymphocytes had Fc receptors and were mainly present in a fraction of non-T lymphocytes. Monocytes were not cytotoxic but rather inhibitory. These results indicate that lysis of virus-infected cells in this direct assay is due to antibody-dependent cellular cytotoxicity (ADCC), caused by K cells. Control experiments showed that the virus-infected target cells were sensitive to incubation with human serum or IgG, resulting in a nonspecific increase of ⁵¹Cr release; however, this did not affect the results of K-cell cytotoxicity. Maximal virus-specific lysis by ADCC did not reach the level obtained by complement-dependent cytotoxicity. Possible explanations for this difference are discussed.     

Antibody-dependent cellular cytotoxicity effector cells lack Ia antigens.

Surface immunoglobulin (sIg)-positive and sIg-negative subpopulations of macrophage-depleted murine splenic lymphocytes were obtained by Sephadex anti-Fab immunoabsorbent fractionation. These lymphocyte subpopulations were analyzed for the presence of Thy 1 and Ia alloantigens and also for Fc receptors by fluorescence microscopy. Concurrently, these lymphocyte subpopulations were studied for effector cell activity in antibody-dependent cellular cytotoxicity (ADCC). Effector cells mediating ADCC were contained in the sIg-negative lymphocyte subpopulation and sIg-positive lymphocytes did not mediate cytotoxicity. The majority of sIg-positive lymphocytes were found to bear Ia antigens and Fc receptors, and these cell surface structures were associated in that treatment of these cells with anti-Ia sera inhibited binding of complexed immunoglobulin to Fc receptors. In contrast, most sIg-negative, Thy 1-negative lymphocytes lacked Ia Antigens, and the Fc receptors detected on such cells were not blocked by anti-Ia sera. In addition, a small subpopulation of sIg-negative, Ia antigen-positive, Fc receptor-positive lymphocytes was found. Elimination of this subpopulation of Ia antigen-positive cells from sIg-negative lymphocytes, by treatment with anti-Ia serum and complement, did not diminish ADCC effector cell activity in the resultant cell population when compared with untreated sIg-negative lymphocytes. Thus, in murine spleen, nonphagocytic mononuclear cells that lack both sIg and Ia antigens were shown to mediate ADCC.     

Murine antibody-dependent cellular cytotoxicity to herpes simplex virus-infected target cells.

Freshly collected peritoneal cells (PC) and cultured spleen cells (SC) (but not fresh SC) from nonimmune mice could mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus (HSV)-infected cells in the presence of mouse or human sera containing antibody to HSV. PC also demonstrated variable natural killer cell cytotoxicity to infected cells. Both PC and cultured SC required high concentrations of antibody and high effector to target cell ratios for optimal ADCC. The time kinetics of the reaction appeared to depend on the state of activation of the effector cells. In both PC and SC populations, ADCC activity was limited to adherent cells, and was profoundly inhibited by particulate latex or silica. The murine effector cell found in PC and SC able to mediate ADCC to HSV-infected cells appears to be a macrophage.     

Protection of neonatal mice against herpes simplex virus infection: probable in vivo antibody-dependent cellular cytotoxicity

Infant mice are extremely susceptible to fatal Herpes simplex virus (HSV) infection. They are unable to produce antibody to HSV, and their leukocytes cannot mediate antibody-dependent cellular cytotoxicity (ADCC) to HSV-infected cells. In order to avoid H-2-dependent effector mechanisms and instead analyze possible in vivo ADCC, a murine model employing adoptive transfer of antibody and human leukocytes was developed. Administration of either human immune globulin or leukocytes i.p. from HSV immune or nonimmune humans could not protect infant C57BL/6 mice from fatal HSV infection. In contrast, a combination of a subneutralizing dilution of globulin and leukocytes from nonimmune or immune human donors, given one day before inoculation, was highly protective against lethal HSV infection. The cells involved included lymphocytes or monocyte-macrophages. At least 5 X 10(6) viable leukocytes (or 1 X 10(6) monocyte-macrophages) and immune serum globulin concentrations as low as 10(-8) were protective. Infected cell monolayer adsorption and DEAE column fractionation demonstrated that the protection by globulin was due to specific antiviral IgG antibody. Protection was n ot seen in animals receiving virus before immune transfer. Protection did not involve synergistic viral neutralization by antibody and cells, as shown by in vitro experiments. Animals receiving globulin and cells, unlike normal infant mice, had circulating antiviral antibody and peritoneal leukocytes able to mediate ADCC to HSV-infected cells. This is the first in vivo evidence for the role of human ADCC. This model also allows for the in vivo evaluation of the ability of cells from immunocompromised humans to curb viral infection.     

An Fc engineering approach that modulates antibody-dependent cytokine release without altering cell-killing functions

Cytotoxic therapeutic monoclonal antibodies (mAbs) often mediate target cell-killing by eliciting immune effector functions via Fc region interactions with cellular and humoral components of the immune system. Key functions include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). However, there has been increased appreciation that along with cell-killing functions, the induction of antibody-dependent cytokine release (ADCR) can also influence disease microenvironments and therapeutic outcomes. Historically, most Fc engineering approaches have been aimed toward modulating ADCC, ADCP, or CDC. In the present study, we describe an Fc engineering approach that, while not resulting in impaired ADCC or ADCP, profoundly affects ADCR. As such, when peripheral blood mononuclear cells are used as effector cells against mAb-opsonized tumor cells, the described mAb variants elicit a similar profile and quantity of cytokines as IgG1. In contrast, although the variants elicit similar levels of tumor cell-killing as IgG1 with macrophage effector cells, the variants do not elicit macrophage-mediated ADCR against mAb-opsonized tumor cells. This study demonstrates that Fc engineering approaches can be employed to uncouple macrophage-mediated phagocytic and subsequent cell-killing functions from cytokine release.     

An Fc engineering approach that modulates antibody-dependent cytokine release without altering cell-killing functions

Cytotoxic therapeutic monoclonal antibodies (mAbs) often mediate target cell-killing by eliciting immune effector functions via Fc region interactions with cellular and humoral components of the immune system. Key functions include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). However, there has been increased appreciation that along with cell-killing functions, the induction of antibody-dependent cytokine release (ADCR) can also influence disease microenvironments and therapeutic outcomes. Historically, most Fc engineering approaches have been aimed toward modulating ADCC, ADCP, or CDC. In the present study, we describe an Fc engineering approach that, while not resulting in impaired ADCC or ADCP, profoundly affects ADCR. As such, when peripheral blood mononuclear cells are used as effector cells against mAb-opsonized tumor cells, the described mAb variants elicit a similar profile and quantity of cytokines as IgG1. In contrast, although the variants elicit similar levels of tumor cell-killing as IgG1 with macrophage effector cells, the variants do not elicit macrophage-mediated ADCR against mAb-opsonized tumor cells. This study demonstrates that Fc engineering approaches can be employed to uncouple macrophage-mediated phagocytic and subsequent cell-killing functions from cytokine release.     

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.     

E-rosette receptors induced by phytohemagglutinin on human K cells expressing T-cell surface antigens.

Killer cells (K cells) enriched from human blood mononuclear cells which mediate antibody-dependent cellular cytotoxicity (ADCC) were examined for surface markers. Sixty-seven percent of the E-rosette-negative, sIg-negative cells reacted with anti-T cell serum (AMT) previously shown to react with immunochemically defined T-cell antigens. Phytohemagglutinin induced 25% of K cells to express an E-rosette receptor. When these induced cells were isolated, greater than 98% reacted with AMT and 17% expressed the Fc receptor for IgG. Furthermore, they retained their functional capacity in ADCC. These findings demonstrate that an E-rosette receptor can be induced on human K cells. The data suggest the K-cell fraction included a population of thymus-dependent lymphocytes which can function as effector cells in ADCC.     

The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity

Therapeutic monoclonal antibodies are the fastest growing class of biological therapeutics for the treatment of various cancers and inflammatory disorders. In cancer immunotherapy, some IgG1 antibodies rely on the Fc-mediated immune effector function, antibody-dependent cellular cytotoxicity (ADCC), as the major mode of action to deplete tumor cells. It is well-known that this effector function is modulated by the N-linked glycosylation in the Fc region of the antibody. In particular, absence of core fucose on the Fc N-glycan has been shown to increase IgG1 Fc binding affinity to the FcγRIIIa present on immune effector cells such as natural killer cells and lead to enhanced ADCC activity. As such, various strategies have focused on producing afucosylated antibodies to improve therapeutic efficacy. This review discusses the relevance of antibody core fucosylation to ADCC, different strategies to produce afucosylated antibodies, and an update of afucosylated antibody drugs currently undergoing clinical trials as well as those that have been approved.     

Antibody-mediated cell cytotoxicity in a defined system: regulation by antigen, antibody, and complement.

The use of a serum-free environment and target cells carrying defined amounts of radiolabeled antigen allowed a quantitative study of the role of antigen, antibody, and complement on antibody-mediated cell cytotoxicity (AbMC). For lysis to occure, a minimum number of antigen molecules must be present on the target cell. 51Cr release from target cells with lower antigen density requires larger concentration of effector cells and antibodies. Target cell-bound complement, itself unable to mediate cytotoxicity, reduces the number of IgG molecules required for lysis. The antibody and complement, however, have to be bound to the same target cell. Bystander complement-coated erythrocytes, present in the same reaction mixture with IgG-coated targets, are not lysed. Blocking of AbMC is effected only by antigen, either soluble or in immune complexes prepared in antigen excess. Antigen competes at the level of the target cell. Blocking at the level of the effector cell, by use of immune complexes prepared at equivalence or in antibody excess, is difficult to achieve. The large number of cells with Fc receptors contained in mouse spleens may explain this finding. Arming of effector cells by passive binding of immune complexes is poorly effective as a means of obtaining lysis of the target cells. In all situations, the outcome of the reaction is determined by the presence of free antibody-combining sites, alone, or in immune complexes, that are able to combine with the target cell membrane antigen. The requirements for lysis are rather stringent.     

Potentiation of cell-mediated lysis of xenogemeic tumor cells: the role of antiserum and effector cells.

The development of the immune response to xenogeneic tumor cells and the mechanism of potentiation of cell-mediated cytotoxicity (CMC) by xenoantiserum were investigated. The kinetics of potentiation of CMC resembled, both qualitatively and quantitatively, the kinetics of antibody-dependent cellular cytotoxicity (ADCC) of target cells treated with the same xenoantisera. Varying proportions of immune and nonimmune effector cells did not influence the amount of lysis of antibody-treated tumor cells. It would appear, therefore, that spleens from immunized animals contained cell populations that were capable of mediating both CMC and ADCC. Potentiation of CMC would appear to result from the preferential expression of ADCC effector cells; interaction of CMC effector cells was apparently hindered by the presence of antibody on the tumor cell surface. Immune complexes formed in antibody excess may also modify ADCC and the potentiation of CMC.     

Reversal of SV40 tumor-mediated suppression of spleen cell cytotoxicity by antibody.

Spleen cells obtained from hamsters bearing PARA-7 tumors greater than 1.0 cm were not reactive in microcytotoxicity assays unless preincubated overnight. The events occurring during in vitro incubation which lead to reversal of tumor-mediated suppression of cellular immunity were investigated. After 24 hr of incubation, supernatants overlying spleen cells from tumor-bearing hosts contained a factor which blocked cytotoxicity of simian virus 40 (SV40)3-sensitized spleen cells at the PARA-7 target cell level but not at the effector cell level. The preparations did not mediate antibody-dependent cellular cytotoxicity (ADCC). Opposite results were obtained in assays of culture medium overlying spleen cells from hosts with a tumor burden less than 0.1 cm. Although ADCC activity was present, no significant blocking was detectable. Treatment of inactive spleen cells with anti-hamster gamma-globlin in the presence of complement (anti-HGG + C) prevented activation and formation of blocking factor but did not impair the cytotoxic activity of already activated cells. Addition of SV40 antiserum to anti-HGG + C-treated cells led to effector cell activation, whereas heterologous virus-immune sera did not. Control studies established that the antibody-mediated recovery of cytotoxicity was not due to arming. Further studies showed that PARA-7 tumor antigen extract blocked at the effector cell level, not at the target cell level. Addition of PARA-7 extract to spleen cell supernatants mediating ADCC resulted in formation of a factor which blocked at the target cell level but not at the effector cell level. These data are compatible with the following interpretation. Spleen cell unresponsiveness is due to antigen blockade. Recovery of cytotoxicity occurs because antibody synthesized during the incubation period promotes elution of antigen from the effector cell surface. Thus, activation is accompanied by the generation of tumor antigen-antibody complexes.     

Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation

For some antibodies intended for use as human therapeutics, reduced effector function is desired to avoid toxicities that might be associated with depletion of target cells. Since effector function(s), including antibody-dependent cell-mediated cytotoxicity (ADCC), require the Fc portion to be glycosylated, reduced ADCC activity antibodies can be obtained through aglycosylation of the human IgG1 isotype. An alternative is to switch to an IgG4 isotype in which the glycosylated antibody is known to have reduced effector function relative to glycosylated IgG1 antibody. ADCC activity of glycosylated IgG1 antibodies is sensitive to the fucosylation status of the Fc glycan, with both in vitro and in vivo ADCC activity increased upon fucose removal (“afucosylation”). The effect of afucosylation on activity of IgG4 antibodies is less well characterized, but it has been shown to increase the in vitro ADCC activity of an anti-CD20 antibody. Here, we show that both in vitro and in vivo activity of anti-CD20 IgG4 isotype antibodies is increased via afucosylation. Using blends of material made in Chinese hamster ovary (CHO) and Fut8KO-CHO cells, we show that ADCC activity of an IgG4 version of an anti-human CD20 antibody is directly proportional to the fucose content. In mice transgenic for human FcγRIIIa, afucosylation of an IgG4 anti-mouse CD20 antibody increases the B cell depletion activity to a level approaching that of the mIgG2a antibody.