A cDNA encoding a multispecific organic anion transporter 3 (hOAT3) was isolated from a human kidney cDNA library. The hOAT3 cDNA consisted of 2179 base pairs that encoded a 543-amino-acid residue protein with 12 putative transmembrane domains. The deduced amino acid sequence of hOAT3 showed 36 to 51% identity to those of other members of the OAT family. Northern blot analysis revealed that hOAT3 mRNA is expressed in the kidney, brain, and skeletal muscle. When expressed in Xenopus laevis oocytes, hOAT3 mediated the transport of estrone sulfate (K(m) = 3.1 microM), p-aminohippurate (K(m) = 87.2 microM), methotrexate (K(m) = 10.9 microM), and cimetidine (K(m) = 57.4 microM) in a sodium-independent manner. hOAT3 also mediated the transport of dehydroepiandrosterone sulfate, ochratoxin A, PGE(2), estradiol glucuronide, taurocholate, glutarate, cAMP and uric acid. Estrone sulfate did not show any trans-stimulatory effects on either influx or efflux of [(3)H]estrone sulfate via hOAT3. hOAT3 interacted with chemically heterogeneous anionic compounds, such as nonsteroidal anti-inflammatory drugs, diuretics, sulfobromophthalein, penicillin G, bile salts and tetraethyl ammonium bromide. The hOAT3 protein was shown to be localized in the basolateral membrane of renal proximal tubules and the hOAT3 gene was determined to be located on the human chromosome 11q12-q13.3 by fluorescent in situ hybridization analysis. These results suggest an important role of hOAT3 in the excretion/detoxification of endogenous and exogenous organic anions in the kidney.

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Results: We found KM3065-mediated antibody-dependent cellular cytotoxicity was increased 10 to 100-fold compared with rituximab for each of the 20 donors. In contrast to rituximab, KM3065 antibody-dependent cellular cytotoxicity enhancement was similar for both FCGR3A alleles and thus independent of genotype. In addition, antibody-dependent cellular cytotoxicity of both KM3065 and rituximab requires natural killer cells but not monocytes nor polymorphonuclear cells. The antibody-dependent cellular cytotoxicity (ADCC) of each of the 20 donors correlated with the natural killer cell numbers present in the PBMCs. Importantly, using KM3065, the ADCC mediated by effector cells bearing the lower affinity variant Fc␥RIIIa-158F was significantly increased compared with rituximab-mediated ADCC using effector cells bearing the higher affinity Fc␥RIIIa-158V receptors.Conclusions: The use of low-fucose antibodies might improve the therapeutic effects of anti-CD20 therapy for all patients independent of Fc␥RIIIa phenotype beyond that currently seen with even the most responsive patients.

Purpose: Recent studies have revealed that fucose removal from the oligosaccharides of human IgG1 antibodies results in a significant enhancement of antibody-dependent cellular cytotoxicity (ADCC) via improved IgG1 binding to Fc;RIIIa. In this report, we investigated the relationship between enhanced ADCC and antigen density on target cells using IgG1 antibodies with reduced fucose.Experimental Design: Using EL4 cell-derived transfectants with differential expression levels of exogenous human CC chemokine receptor 4 or human CD20 as target cells, ADCC of fucose variants of chimeric IgG1 antibodies specific for these antigens were measured. We further investigated IgG1 binding to natural killer (NK) cells and NK cell activation during ADCC induction to elucidate the mechanism by which low-fucose IgG1 induces ADCC upon target cells with low antigen expression.Results: Low-fucose IgG1s showed potent ADCC at low antigen densities at which their corresponding highfucose counterparts could not induce measurable ADCC. The quantitative analysis revealed that fucose depletion could reduce the antigen amount on target cells required for constant degrees of ADCC induction by 10-fold for CC chemokine receptor 4 and 3-fold for CD20. IgG1 binding to NK cells was increased by ligating IgG1 with clustered antigen, especially for low-fucose IgG1. Up-regulation of an activation marker, CD69, on NK cells, particularly the CD56 dim subset, in the presence of both the antibody and target cells was much greater for the low-fucose antibodies.Conclusions: Our data showed that fucose removal from IgG1 could reduce the antigen amount required for ADCC induction via efficient recruitment and activation of NK cells.