A Glyco-Optimized Anti-CD20 Antibody from the Aquatic Plant Lemna: Comparison of Pharmacokinetics, B-Cell Depletion and Complement Activation with Rituxan® in Cynomolgus Monkeys

Monoclonal antibodies represent one of the largest classes of drugs in preclinical and clinical development. For many antibodies, the structure and extent of the N-glycans on the Fc region of the heavy chain plays a significant role in their therapeutic function. A glyco-optimized version of the anti-CD20 antibody (rituximab) was expressed in the clonal aquatic plant Lemna. The optimized glycosylation was accomplished by co-expressing an interfering RNA (RNAi) construct targeting the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes (Cox et al., 2006). The resulting glyco-optimized rituximab contained a single major G0 N-glycan (lacking terminal galactose) without any detectable xylose or fucose. Previous in vitro cell-based studies have shown that the glyco-optimized rituximab had similar CD20 binding affinity and apoptotic effects as Rituxan^® produced in mammalian cells but with significantly enhanced (up to 100- fold) antibody-dependent cellular cytotoxicity (ADCC). Enhanced ADCC activity was found for all FcgRIIIa-158 genotypes. Reported here are the results of expanded studies comparing Rituxan^® with the Lemna–derived glyco-optimized rituximab. B-cell depletion was measured in genotyped human whole blood after ex vivo treatment with both anti-CD20 antibodies. Consistent with prior in vitro ADCC studies, glyco-optimized rituximab showed a significant increase in B-cell depletion for all FcgRIIIa-158 genotypes when compared to Rituxan^®. To extend these findings to an in vivo setting, a comparative monkey study was conducted to evaluate:

1. pharmacokinetic impact of the optimized glycans,

2. B-cell depletion, and

3. complement activation.

The study design included two male Cynomologus monkeys per group in a dose-escalation scheme where each group received a single administration of two dose levels of either Rituxan^® or glycooptimized rituximab. Results showed no difference between the two antibodies in clinical observations and pharmacokinetic profile. Overall the rate of depletion and recovery of the monkey B-cells between Rituxan^® and glyco-optimized rituximab was similar with evidence of an increase in the initial rate of depletion with the glyco-optimized rituximab. The latter finding is not unexpected due to sequence differences between FcgRIIIa in humans and monkeys. Interestingly, 50% higher complement activation (as measured by serum levels of C3a,) was observed with Rituxan^®. This is consistent with our previous observation that glyco-optimized rituximab had up to a ten-fold decrease in complement dependent cytotoxicity (CDC) in Raji cells compared to Rituxan^®. These studies suggest that an optimized anti-CD20 antibody therapeutic can have a similar pharmacokinetic profile with enhanced ADCC activity and decreased CDC activity compared to Rituxan^®. Confirmation that these differences will translate into improved efficacy with decreased side effects associated with CDC activity (Clark and Ledbetter, 2005) will require clinical research. Cox et al (2006). Nat. Biotech. 24: 1591–15197. Clark et al (2005). Ann. Rheum Dis. 64: 77–80.