Lymphocyte activation antigen CD70 expressed by renal cell carcinoma is a potential therapeutic target for anti-CD70 antibody-drug conjugates.

Metastatic renal cell carcinoma (RCC) is an aggressive disease refractory to most existing therapeutic modalities. Identifying new markers for disease progression and drug targets for RCC will benefit this unmet medical need. We report a subset of clear cell and papillary cell RCC aberrantly expressing the lymphocyte activation marker CD70, a member of the tumor necrosis factor superfamily. Importantly, CD70 expression was found to be maintained at the metastatic sites of RCC. Anti-CD70 antibody-drug conjugates (ADC) consisting of auristatin phenylalanine phenylenediamine (AFP) or monomethyl auristatin phenylalanine (MMAF), two novel derivatives of the anti-tubulin agent auristatin, mediated potent antigen-dependent cytotoxicity in CD70-expressing RCC cells. Cytotoxic activity of these anti-CD70 ADCs was associated with their internalization and subcellular trafficking through the endosomal-lysosomal pathway, disruption of cellular microtubule network, and G2-M phase cell cycle arrest. The efficiency of drug delivery using anti-CD70 as vehicle was illustrated by the much enhanced cytotoxicity of antibody-conjugated MMAF compared with free MMAF. Hence, ADCs targeted to CD70 can selectively recognize RCC, internalize, and reach the appropriate subcellular compartment(s) for drug release and tumor cell killing. In vitro cytotoxicity of these ADCs was confirmed in xenograft models using RCC cell lines. Our findings provide evidence that CD70 is an attractive target for antibody-based therapeutics against metastatic RCC and suggest that anti-CD70 ADCs can provide a new treatment approach for advanced RCC patients who currently have no chemotherapeutic options.

In vivo drug-linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate.

Effective antibody-drug conjugates (ADC) combine high drug-linker stability in circulation and efficient intratumoral release of drug. Conjugation of monomethyl auristatin E (MMAE) to the anti-CD30 monoclonal antibody (mAb), cAC10, produced a selective and potent ADC against CD30(+) anaplastic large cell lymphoma and Hodgkin's disease models. This ADC, cAC10-valine-citrulline-MMAE, uses a protease-sensitive dipeptide linker designed to release MMAE by lysosomal cathepsin B in target cells but maintain a stable linkage and attenuate drug potency in circulation. To evaluate ADC stability in vivo, we developed methods for measuring drug/mAb ratios at progressive times in plasma from ADC-treated mice and nonhuman primates. Anti-idiotype mAb permitted the capture and quantitation of mAb cAC10, whereas antidrug mAb and MMAE-conjugated horseradish peroxidase reporter provided quantitative detection of conjugated drug following its in vitro release by cathepsin B. These data were validated by an alternative ELISA using anti-idiotype and anti-MMAE mAbs for capture and detection, respectively. Both methods differentiated ADC with variable levels of drug loading and were subsequently applied to stability studies in severe combined immunodeficient mice and cynomolgus monkeys. Evaluation of ADC from mouse circulation showed the linker half-life to be approximately 144 hours (6.0 days), significantly greater than that reported for disulfide- or hydrazone-linked ADCs in mice or human trials. In cynomolgus monkey, the apparent linker half-life was approximately 230 hours (9.6 days), suggesting that the drug-linker will be highly stable in humans. These data represent the longest reported drug-linker half-life to date and provide the basis for the pronounced specificity and antitumor activity of cAC10-valine-citrulline-MMAE.

Enhanced expression of uridine diphosphate-N-acetylglucosaminyl transferase (OGT) in a stable, tetracycline-inducible HeLa cell line using histone deacetylase inhibitors: kinetics of cytosolic OGT accumulation and nuclear translocation.

We have created a stable, tetracycline-inducible HeLa cell line that overexpresses murine uridine diphosphate-N-acetylglucosaminyl transferase (OGT). Tetracycline increased cytosolic OGT activity about 4-fold in a dose-dependent manner (ED(50)=0.03 microg/ml) with enhanced activity observable at 8h and maximal activity observable by 40h. Enhanced OGT activity was due to overexpression of OGT protein as determined by Western analysis. Trichostatin A (TSA), a potent and specific histone deacetylase inhibitor (HDI), markedly enhanced tetracycline-induced OGT gene expression, resulting in a >10-fold increase in OGT activity (>50-fold compared to that of uninduced cells). Other HDIs such as butyrate (ED(50)=1.6mM) and propionate (ED(50)=8mM) were similarly effective, but less potent than TSA (ED(50)=120 nM). We next examined the appearance of recombinant OGT in cytosol and nucleosol at various times (10 min to 6h) after inducing OGT gene. Within 2h, recombinant OGT was detected by Western analysis in both cytosol and nucleosol. This indicates rapid biosynthesis and accumulation of recombinant OGT in the cytosol and subsequent nuclear translocation. Entry of OGT into the nucleus was closely correlated with enhanced O-linked glycosylation of nuclear proteins, indicating that recombinant OGT was enzymatically active. The ability to rapidly induce OGT expression in a stable cell line provides an excellent model system to study the mechanism(s) underlying OGT nuclear translocation and a useful system to elucidate the cascade of signaling events related to O-linked glycosylation.

Role of hexosamine biosynthesis in glucose-mediated up-regulation of lipogenic enzyme mRNA levels: effects of glucose, glutamine, and glucosamine on glycerophosphate dehydrogenase, fatty acid synthase, and acetyl-CoA carboxylase mRNA levels.

Glucose uptake into adipose and liver cells is known to up-regulate mRNA levels for various lipogenic enzymes such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC). To determine whether the hexosamine biosynthesis pathway (HBP) mediates glucose regulation of mRNA expression, we treated primary cultured adipocytes for 18 h with insulin (25 ng/ml) and either glucose (20 mm) or glucosamine (2 mm). A ribonuclease protection assay was used to quantitate mRNA levels for FAS, ACC, and glycerol-3-P dehydrogenase (GPDH). Treatment with insulin and various concentrations of d-glucose increased mRNA levels for FAS (280%), ACC (93%), and GPDH (633%) in a dose-dependent manner (ED50 8-16 mm). Mannose similarly elevated mRNA levels, but galactose and fructose were only partially effective. l-glucose had no effect. Omission of glutamine from the culture medium markedly diminished the stimulatory effect of glucose on mRNA expression. Since glutamine is a crucial amide donor in hexosamine biosynthesis, we interpret these data to mean that glucose flux through the HBP is linked to regulation of lipogenesis through control of gene expression. Further evidence for hexosamine regulation was obtained using glucosamine, which is readily transported into adipocytes where it directly enters the HBP. Glucosamine was 15-30 times more potent than glucose in elevating FAS, ACC, and GPDH mRNA levels (ED50 approximately 0.5 mm). In summary: 1) GPDH, FAS, and ACC mRNA levels are upregulated by glucose; 2) glucose-induced up-regulation requires glutamine; and 3) mRNA levels for lipogenic enzymes are up-regulated by glucosamine. Hyperglycemia is the hallmark of diabetes mellitus and leads to insulin resistance, impaired glucose metabolism, and dyslipidemia. We postulate that disease pathophysiology may have a common underlying factor, excessive glucose flux through the HBP.