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Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG) improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa) and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr-296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr-296 residue in the antibody in the interaction with various Fcγ receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr-296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr-296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa binding, almost all Tyr-296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr-296 in interactions with various Fcγ receptors, and have applications in the modulation of the IgG1-Fc function of therapeutic antibodies.

Removal of the fucose residue from the N-glycans of the Fc portion of immunoglobulin G (IgG) results in a dramatic enhancement of antibody-dependent cellular cytotoxicity (ADCC) through improved affinity for Fcγ receptor IIIa (FcγRIIIa). Here, we present the 2.2-Å structure of the complex formed between nonfucosylated IgG1-Fc and a soluble form of FcγRIIIa (sFcγRIIIa) with two N-glycosylation sites. The crystal structure shows that one of the two N-glycans of sFcγRIIIa mediates the interaction with nonfucosylated Fc, thereby stabilizing the complex. However, fucosylation of the Fc N-glycans inhibits this interaction, because of steric hindrance, and furthermore, negatively affects the dynamics of the receptor binding site. Our results offer a structural basis for improvement in ADCC of therapeutic antibodies by defucosylation., (© 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.)

Background: Antibody-dependent cellular cytotoxicity (ADCC) has recently been identified as one of the critical mechanisms underlying the clinical efficacy of therapeutic antibodies, especially anticancer antibodies. Therapeutic antibodies fully lacking the core fucose of the Fc oligosaccharides have been found to exhibit much higher ADCC in humans than their fucosylated counterparts. However, data which show how fully non-fucosylated antibodies achieve such a high ADCC in human whole blood have not yet been disclosed. The precise mechanisms responsible for the high ADCC mediated by fully non-fucosylated therapeutic antibodies, even in the presence of human plasma, should be explained based on direct evidence of non-fucosylated antibody action in human blood., Methods: Using a human ex vivo B-cell depletion assay with non-fucosylated and fucosylated anti-CD20 IgG1s rituximab, we monitored the binding of the therapeutic agents both to antigens on target cells (target side interaction) and to leukocyte receptors (FcgammaR) on effector cells (effector side interaction), comparing the intensities of ADCC in human blood., Results: In the target side interaction, down-modulation of CD20 on B cells mediated by anti-CD20 was not observed. Simple competition for binding to the antigens on target B cells between fucosylated and non-fucosylated anti-CD20s was detected in human blood to cause inhibition of the enhanced ADCC of non-fucosylated anti-CD20 by fucosylated anti-CD20. In the effector side interaction, non-fucosylated anti-CD20 showed sufficiently high FcgammaRIIIa binding activity to overcome competition from plasma IgG for binding to FcgammaRIIIa on natural killer (NK) cells, whereas the binding of fucosylated anti-CD20 to FcgammaRIIIa was almost abolished in the presence of human plasma and failed to recruit NK cells effectively. The core fucosylation levels of individual serum IgG1 from healthy donors was found to be so slightly different that it did not affect the inhibitory effect on the ADCC of fucosylated anti-CD20., Conclusion: Our results demonstrate that removal of fucosylated antibody ingredients from antibody therapeutics elicits high ADCC in human blood by two mechanisms: namely, by evading the inhibitory effects both of plasma IgG on FcgammaRIIIa binding (effector side interaction) and of fucosylated antibodies on antigen binding (target side interaction).

Background: Antibody-dependent cellular cytotoxicity (ADCC) is greatly enhanced by the absence of the core fucose of oligosaccharides attached to the Fc, and is closely related to the clinical efficacy of anticancer activity in humans in vivo. Unfortunately, all licensed therapeutic antibodies and almost all currently-developed therapeutic antibodies are heavily fucosylated and fail to optimize ADCC, which leads to a large dose requirement at a very high cost for the administration of antibody therapy to cancer patients. In this study, we explored the possibility of converting already-established antibody-producing cells to cells that produce antibodies fully lacking core fucosylation in order to facilitate the rapid development of next-generation therapeutic antibodies., Results: Firstly, loss-of-function analyses using small interfering RNAs (siRNAs) against the three key genes involved in oligosaccharide fucose modification, i.e. alpha1,6-fucosyltransferase (FUT8), GDP-mannose 4,6-dehydratase (GMD), and GDP-fucose transporter (GFT), revealed that single-gene knockdown of each target was insufficient to completely defucosylate the products in antibody-producing cells, even though the most effective siRNA (>90% depression of the target mRNA) was employed. Interestingly, beyond our expectations, synergistic effects of FUT8 and GMD siRNAs on the reduction in fucosylation were observed, but not when these were used in combination with GFT siRNA. Secondly, we successfully developed an effective short hairpin siRNA tandem expression vector that facilitated the double knockdown of FUT8 and GMD, and we converted antibody-producing Chinese hamster ovary (CHO) cells to fully non-fucosylated antibody producers within two months, and with high converting frequency. Finally, the stable manufacture of fully non-fucosylated antibodies with enhanced ADCC was confirmed using the converted cells in serum-free fed-batch culture., Conclusion: Our results suggest that FUT8 and GMD collaborate synergistically in the process of intracellular oligosaccharide fucosylation. We also demonstrated that double knockdown of FUT8 and GMD in antibody-producing cells could serve as a new strategy for producing next-generation therapeutic antibodies fully lacking core fucosylation and with enhanced ADCC. This approach offers tremendous cost- and time-sparing advantages for the development of next-generation therapeutic antibodies.

Antibody-dependent cellular cytotoxicity (ADCC) is considered to be an important therapeutic function for clinical efficacy of monoclonal antibodies. Recent studies have revealed two methods to increase binding affinity for FcgammaRIIIa and enhance ADCC more efficiently for antibodies: (i) fucose removal from antibody N-linked complex oligosaccharides and (ii) amino acid mutations in the antibody Fc region. In this study, we compare the biological activities of the methods of generating high ADCC antibodies. We used a fucose-negative antibody and two antibodies with sets of mutations, demonstrated previously to optimally enhance ADCC using the chimeric anti-CD20 antibody, rituximab, as the model. Both amino acid mutant antibodies showed a significantly higher affinity for recombinant FcgammaRIIIa than fucose-negative antibody when compared using biosensor analysis. The removal of fucose from the antibodies bearing amino acid mutations exhibited a further enhancement of binding to recombinant FcgammaRIIIa and significantly increased binding to natural killer (NK) cells. Despite the differences manifested in binding for the FcgammaR, ADCCs were indistinguishable between methods and even when the methods were combined. These results indicate that the affinity of binding to FcgammaRIIIa does not predict ADCC beyond a certain threshold and that each method alone is sufficient to induce maximal ADCC of the antibody.

Purpose: Removal of fucose residues from the oligosaccharides of human antibody is a powerful approach to enhance antibody-dependent cellular cytotoxicity (ADCC), a potential important antitumor mechanism of therapeutic antibodies. To provide clinically relevant evidence of this mechanism, we investigated ADCC of a fucose-negative version of trastuzumab [anti-human epidermal growth factor receptor 2 (HER2) humanized antibody] using peripheral blood mononuclear cells (PBMC) from breast cancer patients as effector cells., Experimental Design: Thirty volunteers, including 20 breast cancer patients and 10 normal healthy control donors, were recruited randomly, and aliquots of peripheral blood were collected. ADCC of commercial trastuzumab (fucosylated) and its fucose-negative version were measured using PBMCs drawn from the volunteers as effector cells and two breast cancer cell lines with different HER2 expression levels as target cells. Relationships between cytotoxicity and characteristics of the patients, such as content of natural killer cells in PBMCs, type of therapy, FCGR3A genotypes, etc. were also analyzed., Results: ADCC was significantly enhanced with the fucose-negative antibody compared with the fucose-positive antibody using PBMCs from either normal donors or breast cancer patients. Enhancement of ADCC was observed irrespective of the various clinical backgrounds of the patients, even in the chemotherapy cohort that presented with a reduced number of natural killer cells and weaker ADCC., Conclusions: This preliminary study suggests that the use of fucose-negative antibodies may improve the therapeutic effects of anti-HER2 therapy for patients independent of clinical backgrounds.

Most of the existing therapeutic antibodies that have been licensed and developed as medical agents are of the human IgG1 isotype, the molecular weight of which is approximately 150 kDa. Human IgG1 is a glycoprotein bearing two N-linked biantennary complex-type oligosaccharides bound to the antibody constant region (Fc), in which the majority of the oligosaccharides are core fucosylated, and it exercises the effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity through the interaction of the Fc with either leukocyte receptors (FcgammaRs) or complement. Recently, therapeutic antibodies have been shown to improve overall survival as well as time to disease progression in a variety of human malignancies, such as breast, colon and haematological cancers, and genetic analysis of FcgammaR polymorphisms of cancer patients has demonstrated that ADCC is a major antineoplasm mechanism responsible for clinical efficacy. However, the ADCC of existing licensed therapeutic antibodies has been found to be strongly inhibited by serum due to nonnpecific IgG competing for binding of the therapeutics to FcgammaRIIIa on natural killer cells, which leads to the requirement of a significant amount of drug and very high costs associated with such therapies. Moreover, enhanced ADCC of non-fucosylated forms of therapeutic antibodies through improved FcgammaRIIIa binding is shown to be inhibited by the fucosylated counterparts. In fact, non-fucosylated therapeutic antibodies, not including the fucosylated forms, exhibit the strongest and most saturable in vitro and ex vivo ADCC among such antibody variants with improved FcgammaRIIIa binding as those bearing naturally occurring oligosaccharide heterogeneities and artificial amino acid mutations, even in the presence of plasma IgG. Robust stable production of completely non-fucosylated therapeutic antibodies in a fixed quality has been achieved by the generation of a unique host cell line, in which the endogenous alpha-1,6-fucosyltransferase (FUT8) gene is knocked out. Thus, the application of non-fucosylated antibodies is expected to be a promising approach as next-generation therapeutic antibodies with improved efficacy, even when administrated at low doses in humans in vivo. Clinical trials using non-fucosylated antibody therapeutics are underway at present.

Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC., ((c) 2006 Wiley Periodicals, Inc.)

Purpose: Recent studies have revealed that fucosylated therapeutic IgG1s need high concentrations to compensate for FcgammaRIIIa-competitive inhibition of antibody-dependent cellular cytotoxicity (ADCC) by endogenous human plasma IgG. Here, we investigated whether ADCC of nonfucosylated therapeutic IgG1 is also influenced by plasma IgG in the same way as fucosylated IgG1s., Experimental Design: Ex vivo ADCC upon CD20+ human B cells was induced by incubation of human whole blood with nonfucosylated and/or fucosylated anti-CD20 IgG1s rituximab, and quantified by measuring the remaining CD19+ human B cells using flow cytometry., Results: Nonfucosylated anti-CD20 showed markedly higher (over 100-fold based on EC50) ex vivo B-cell depletion activity than its fucosylated counterpart in the presence of plasma IgG. The efficacy of fucosylated anti-CD20 was greatly diminished in plasma, resulting in the need for a high concentration (over 1.0 microg/mL) to achieve saturated efficacy. In contrast, nonfucosylated anti-CD20 reached saturated ADCC at lower concentrations (0.01-0.1 microg/mL) with much higher efficacy than fucosylated anti-CD20 in all nine donors through improved FcgammaRIIIa binding. Noteworthy, the high efficacy of nonfucosylated anti-CD20 was inhibited by addition of fucosylated anti-CD20. Thus, the efficacy of a 1:9 mixture (10 microg/mL) of nonfucosylated and fucosylated anti-CD20s was inferior to that of a 1,000-fold dilution (0.01 microg/mL) of nonfucosylated anti-CD20 alone., Conclusions: Our data showed that nonfucosylated IgG1, not including fucosylated counterparts, can evade the inhibitory effect of plasma IgG on ADCC through its high FcgammaRIIIa binding. Hence, nonfucosylated IgG1 exhibits strong therapeutic potential through dramatically enhanced ADCC at low doses in humans in vivo.

Fucose depletion from oligosaccharides of human IgG1-type antibodies results in a great enhancement of antibody-dependent cellular cytotoxicity (ADCC). The aim of this study was to clarify the effect of fucose removal on effector functions of all human IgG subclasses. A panel of anti-CD20 chimeric antibodies having a matched set of human heavy chain subclasses with different fucose contents in their oligosaccharides was constructed using wild-type and fucosyltransferase-knockout Chinese hamster ovary cells as host cells. As found previously for IgG1, fucose-negative variant of IgG2, IgG3, and IgG4 exhibited enhanced ADCC and FcgammaRIIIa binding compared with their highly fucosylated counterparts. In contrast, fucose removal did not affect complement-dependent cytotoxicity (CDC) of any IgGs. Consequently, fucose removal from IgG2 and IgG4 resulted in a unique effector function profile; they had potent ADCC and no CDC. In conclusion fucose depletion can provide a panel of IgGs with enhanced ADCC without an impact on other inherent properties specific for each IgG subclass, such as CDC.

To generate industrially applicable new host cell lines for antibody production with optimizing antibody-dependent cellular cytotoxicity (ADCC) we disrupted both FUT8 alleles in a Chinese hamster ovary (CHO)/DG44 cell line by sequential homologous recombination. FUT8 encodes an alpha-1,6-fucosyltransferase that catalyzes the transfer of fucose from GDP-fucose to N-acetylglucosamine (GlcNAc) in an alpha-1,6 linkage. FUT8(-/-) cell lines have morphology and growth kinetics similar to those of the parent, and produce completely defucosylated recombinant antibodies. FUT8(-/-)-produced chimeric anti-CD20 IgG1 shows the same level of antigen-binding activity and complement-dependent cytotoxicity (CDC) as the FUT8(+/+)-produced, comparable antibody, Rituxan. In contrast, FUT8(-/-)-produced anti-CD20 IgG1 strongly binds to human Fcgamma-receptor IIIa (FcgammaRIIIa) and dramatically enhances ADCC to approximately 100-fold that of Rituxan. Our results demonstrate that FUT8(-/-) cells are ideal host cell lines to stably produce completely defucosylated high-ADCC antibodies with fixed quality and efficacy for therapeutic use.

Human immunoglobulin G isotype 4 (IgG4) antibodies are suitable for use in either the antagonist or agonist format because their low effector functions prevent target cytotoxicity or unwanted cytokine secretion. However, while manufacturing therapeutic antibodies, they are exposed to low pH during purification, and IgG4 is more susceptible to low-pH-induced aggregation than IgG1. Therefore, we investigated the underlying mechanisms of IgG4 aggregation at low pH and engineered an IgG4 with enhanced stability. By swapping the constant regions of IgG1 and IgG4, we determined that the constant heavy chain (CH3) domain is critical for aggregate formation, but a core-hinge-stabilizing S228P mutation in IgG4 is insufficient for preventing aggregation. To identify the aggregation-prone amino acid, we substituted the CH3 domain of IgG4 with that of IgG1, changing IgG4 Arg409 to a Lys, thereby preventing the aggregation of the IgG4 variant as effectively as in IgG1. A stabilizing effect was also recorded with other variable-region variants. Analysis of thermal stability using differential scanning calorimetry revealed that the R409K substitution increased the Tm value of CH3, suggesting that the R409K mutation contributed to the structural strengthening of the CH3-CH3 interaction. The R409K mutation did not influence the binding to antigens/human Fcγ receptors; whereas, the concurrent S228P and R409K mutations in IgG4 suppressed Fab-arm exchange drastically and as effectively as in IgG1, in both in vitro and in vivo in mice models. Our findings suggest that the IgG4 R409K variant represents a potential therapeutic IgG for use in low-effector-activity format that exhibits increased stability. [ABSTRACT FROM AUTHOR]

Malignant pleural mesothelioma ( MPM) is a rare and highly aggressive neoplasm that arises from the pleural, pericardial, or peritoneal lining. Although surgery, chemotherapy, radiotherapy, and combinations of these therapies are used to treat MPM, the median survival of such patients is dismal. Therefore, there is a compelling need to develop novel therapeutics with different modes of action. Ganglioside GM2 is a glycolipid that has been shown to be overexpressed in various types of cancer. However, there are no published reports regarding the use of GM2 as a potential therapeutic target in cases of MPM. In this study, we evaluated the efficacy of the anti- GM2 antibody BIW-8962 as an anti- MPM therapeutic using in vitro and in vivo assays. Consequently, the GM2 expression in the MPM cell lines was confirmed using flow cytometry. In addition, eight of 11 cell lines were GM2-positive (73%), although the GM2 expression was variable. BIW-8962 showed a significant antibody-dependent cellular cytotoxicity activity against the GM2-expressing MPM cell line MSTO-211H, the effect of which depended on the antibody concentration and effector/target ratio. In an in vivo orthotropic mouse model using MSTO-211H cells, BIW-8962 significantly decreased the incidence and size of tumors. Additionally, the GM2 expression was confirmed in the MPM clinical specimens. Fifty-eight percent of the MPM tumors were positive for GM2, with individual variation in the intensity and frequency of staining. These data suggest that anti- GM2 antibodies may become a therapeutic option for MPM patients. [ABSTRACT FROM AUTHOR]

Objective: Antibody-dependent cellular cytotoxicity mediated by natural killer cells via leukocyte receptor IIIa (FcγRIIIa) is greatly enhanced by the absence of the core fucose of Fc oligosaccharides, and is closely related to the clinical efficacy of anticancer processes in humans in vivo. Here, we focused on the physiological functions of nonfucosylated anti-CD20 IgG1 rituximab, in particular those functions mediated by human neutrophils, which highly express FcγRIIIb, a highly homologous FcγR to FcγRIIIa. Materials and Methods: After treatment with anti-CD20, the response of neutrophils to fluorescently labeled CD20+ B-cell lymphoma in human whole blood was quantitatively analyzed by measuring their activities of antibody-dependent phagocytosis and major histocompatibility complex (MHC) class II expression on the phagocytotic neutrophils using flow cytometry. Results: In human whole blood, most of the added CD20+ B-cell lymphoma died shortly, within 4 hours, irrespective of the presence or absence of anti-CD20. Neutrophils were not directly concerned in the death because depletion of neutrophils from human whole blood did not affect the phenomenon. However, neutrophils aggressively phagocytosed newly dead lymphoma cells, and the nonfucosylated anti-CD20 effectively enhanced neutrophil phagocytosis solely by enhancing binding for the phagocytosis coreceptor FcγRIIIb. Noteworthy, more increased expression of MHC class II was also observed on the phagocytotic neutrophils than those observed on spontaneous and fucosylated anti-CD20 stimulated phagocytotic neutrophils. Conclusions: Our data showed that antibody therapy composed of nonfucosylated rituximab can activate human neutrophil functions involving phagocytosis and MHC class II expression, which may favorably potentiate the adaptive immune response in cancer patients. [Copyright &y& Elsevier]

Background: Antibody-dependent cellular cytotoxicity (ADCC) is greatly enhanced by the absence of the core fucose of oligosaccharides attached to the Fc, and is closely related to the clinical efficacy of anticancer activity in humans in vivo. Unfortunately, all licensed therapeutic antibodies and almost all currently-developed therapeutic antibodies are heavily fucosylated and fail to optimize ADCC, which leads to a large dose requirement at a very high cost for the administration of antibody therapy to cancer patients. In this study, we explored the possibility of converting already-established antibody-producing cells to cells that produce antibodies fully lacking core fucosylation in order to facilitate the rapid development of next-generation therapeutic antibodies. Results: Firstly, loss-of-function analyses using small interfering RNAs (siRNAs) against the three key genes involved in oligosaccharide fucose modification, i.e. α1,6-fucosyltransferase (FUT8), GDP-mannose 4,6-dehydratase (GMD), and GDP-fucose transporter (GFT), revealed that single-gene knockdown of each target was insufficient to completely defucosylate the products in antibody-producing cells, even though the most effective siRNA (>90% depression of the target mRNA) was employed. Interestingly, beyond our expectations, synergistic effects of FUT8 and GMD siRNAs on the reduction in fucosylation were observed, but not when these were used in combination with GFT siRNA. Secondly, we successfully developed an effective short hairpin siRNA tandem expression vector that facilitated the double knockdown of FUT8 and GMD, and we converted antibody-producing Chinese hamster ovary (CHO) cells to fully non-fucosylated antibody producers within two months, and with high converting frequency. Finally, the stable manufacture of fully non-fucosylated antibodies with enhanced ADCC was confirmed using the converted cells in serum-free fed-batch culture. Conclusion: Our results suggest that FUT8 and GMD collaborate synergistically in the process of intracellular oligosaccharide fucosylation. We also demonstrated that double knockdown of FUT8 and GMD in antibody-producing cells could serve as a new strategy for producing next-generation therapeutic antibodies fully lacking core fucosylation and with enhanced ADCC. This approach offers tremendous cost- and time-sparing advantages for the development of next-generation therapeutic antibodies. [ABSTRACT FROM AUTHOR]

Abstract: Fucose depletion from oligosaccharides of human IgG1-type antibodies results in a great enhancement of antibody-dependent cellular cytotoxicity (ADCC). The aim of this study was to clarify the effect of fucose removal on effector functions of all human IgG subclasses. A panel of anti-CD20 chimeric antibodies having a matched set of human heavy chain subclasses with different fucose contents in their oligosaccharides was constructed using wild-type and fucosyltransferase-knockout Chinese hamster ovary cells as host cells. As found previously for IgG1, fucose-negative variant of IgG2, IgG3, and IgG4 exhibited enhanced ADCC and FcγRIIIa binding compared with their highly fucosylated counterparts. In contrast, fucose removal did not affect complement-dependent cytotoxicity (CDC) of any IgGs. Consequently, fucose removal from IgG2 and IgG4 resulted in a unique effector function profile; they had potent ADCC and no CDC. In conclusion fucose depletion can provide a panel of IgGs with enhanced ADCC without an impact on other inherent properties specific for each IgG subclass, such as CDC. [Copyright &y& Elsevier]

Eliciting neutralizing antibodies is thought to be a key activity of a vaccine against human immunodeficiency virus (HIV). However, a number of studies have suggested that in addition to neutralization, interaction of IgG with Fc gamma receptors (Fc?R) may play an important role in antibody-mediated protection. We have previously obtained evidence that the protective activity of the broadly neutralizing human IgG1 anti-HIV monoclonal antibody (MAb) b12 in macaques is diminished in the absence of Fc?R binding capacity. To investigate antibody-dependent cellular cytotoxicity (ADCC) as a contributor to Fc?R-associated protection, we developed a nonfucosylated variant of b12 (NFb12). We showed that, compared to fully fucosylated (referred to as wild-type in the text) b12, NFb12 had higher affinity for human and rhesus macaque Fc?RIIIa and was more efficient in inhibiting viral replication and more effective in killing HIV-infected cells in an ADCC assay. Despite these more potent in vitro antiviral activities, NFb12 did not enhance protection in vivo against repeated low-dose vaginal challenge in the simian-human immunodeficiency virus (SHIV)/macaque model compared to wild-type b12. No difference in protection, viral load, or infection susceptibility was observed between animals given NFb12 and those given fully fucosylated b12, indicating that Fc?R-mediated activities distinct from Fc?RIIIa-mediated ADCC may be important in the observed protection against SHIV challenge. [ABSTRACT FROM AUTHOR]