Enhanced Potency of Glycoengineered Anti-CD52 Monoclonal Antibodies (MAbs).

The CD52 MAb alemtuzumab (Campath) has been licensed for Fludarabine-refractory CLL. Like most commercial therapeutic MAbs, it is produced in mammalian cells (Chinese Hamster Ovary cells) and contains fucosylated carbohydrates attached to the Fc region. In contrast, a fraction of natural human serum IgG lacks fucose on their Fc-oligosaccharides and such antibodies bind with higher affinity to the FcgRIII receptors of immune effector cells. We have produced glycoengineered versions of alemtuzumab with high levels of non-fucosylated Fc oligosaccharides, increased binding affinity to FcgRIII and increased target-cell killing potency via ADCC. Glycoengineering was achieved by stable over-expression of genes encoding carbohydrate-modifying enzymes in an industrial-grade CHO cell line. Two glycovariants were produced: CAMGlyco1, enriched in non-fucosylated, hybrid-type Fc carbohydrates and CAMGlyco2, carrying non-fucosylated oligosaccharides both of hybrid- and complex-type (in approximately equal proportions). Both glycovariants had a proportion of non-fucosylated oligosaccharides leading to markedly enhanced antibody-dependent cellular cytotoxicity (ADCC) in comparison with regular alemtuzumab. CAMGlyco1 mediated lower complement-dependent cytotoxicity (CDC) than CAMGlyco2. The activity of the two glycoforms was assessed in cynomolgus monkeys. The pharmacokinetics of both glycoforms were not significantly different from wild-type alemtuzumab. There was no increased cytokine release with either of the glycoforms. Both CAMGlyco1 and CAMGlyco2 appeared to deplete lymphocytes from both blood and lymph node better than wild-type alemtuzumab. Mean lymphocyte loss from peripheral blood following low dose treatment was 33.7%, 55.9% and 55.0% for alemtuzumab, CAMGlycol1 and CAMGlyco2, respectively (p<0.003). Mean lymphocyte counts per mm³ of lymph node biopsy were 4.6x10⁵, 3.2x10⁵ and 3.6x10⁵ for alemtuzumab, CAMGlycol1 and CAMGlyco2, respectively. A Phase I/II clinical trial comparing CAMGlyco1 directly to alemtuzumab in patients with relapsed CLL has been initiated. Patients receive alternating doses of wild-type alemtuzumab and CAMGlyco1 and rates of decrease in peripheral blood lymphocyte count are monitored. Preliminary results show no increased toxicity. Glycoengineering of CD52 MAbs may allow for enhanced cytolytic efficacy in tumor therapy.