Glycosylation in the Fc region of antibodies has been shown to play an important role in antibody function. In the current study, glycosylation of human monoclonal antibodies was metabolically modulated using a potent alpha-mannosidase I inhibitor, kifunensine, resulting in the production of antibodies with oligomannose-type N-glycans. Growing Chinese hamster ovary cells for 11 days in batch culture with a single treatment of kifunensine was sufficient to elicit this effect without any significant impact on cell viability or antibody production. Antibodies expressed in the presence of kifunensine at a concentration as low as 60 ng/mL contained mainly oligomannose-type glycans and demonstrated increased ADCC activity and affinity for FcgammaRIIIA, but reduced C1q binding. Although the kifunensine-mediated shift to oligomannose-type glycans could, in theory, result in rapid clearance of the antibody through increased mannose receptor binding, the serum levels of antibody in mice were not significantly altered up to 168 h following injection. The use of kifunensine provides a simple and rapid method for the production of antibodies with increased ADCC without the time-consuming need to re-engineer either the antibody molecule or the host cell line.

Alemtuzumab is a monoclonal antibody that targets cell surface CD52 and is effective in depleting lymphocytes by cytolytic effects in vivo. Although the cytolytic effects of alemtuzumab are dependent on the density of CD52 antigen on cells, there is scant information regarding the expression levels of CD52 on different cell types. In this study, CD52 expression was assessed on phenotypically distinct subsets of lymphoid and myeloid cells in peripheral blood mononuclear cells (PBMCs) from normal donors. Results demonstrate that subsets of PBMCs express differing levels of CD52. Quantitative analysis showed that memory B cells and myeloid dendritic cells (mDCs) display the highest number while natural killer (NK) cells, plasmacytoid dendritic cells (pDCs) and basophils have the lowest number of CD52 molecules per cell amongst lymphoid and myeloid cell populations respectively. Results of complement dependent cytolysis (CDC) studies indicated that alemtuzumab mediated profound cytolytic effects on B and T cells with minimal effect on NK cells, basophils and pDCs, correlating with the density of CD52 on these cells. Interestingly, despite high CD52 levels, mDCs and monocytes were less susceptible to alemtuzumab-mediated CDC indicating that antigen density alone does not define susceptibility. Additional studies indicated that higher expression levels of complement inhibitory proteins (CIPs) on these cells partially contributes to their resistance to alemtuzumab mediated CDC. These results indicate that alemtuzumab is most effective in depleting cells of the adaptive immune system while leaving innate immune cells relatively intact.

NY-ESO-1 elicits frequent antibody responses in cancer patientsN Y-ESO-1 is a member of the cancer͞testis family of genes (1). Expression of NY-ESO-1 in normal tissues is limited to germ cells, but in cancer, NY-ESO-1 is expressed in a broad range of different tumor types. NY-ESO-1 was discovered by serological analysis of a recombinant cDNA expression library obtained from an esophageal cancer (1). NY-ESO-1 is one of the most immunogenic tumor antigens defined to date, eliciting a humoral response in nearly half of patients with advanced cancers expressing NY-ESO-1 (2) along with a strong CD8 ϩ T cell (3-6) and CD4 ϩ T cell (7) response. Using optimized techniques for analyzing antibodies and newer methodologies for CD8 ϩ T cell responses, such as HLAA2͞peptide tetramer complexes and enzyme-linked immunospot (Elispot) assays, we recently obtained a comprehensive picture of the spontaneous immune response against NY-ESO-1 in a cohort of patients with advanced tumors (4). The study showed that humoral and cellular responses occurred simultaneously against NY-ESO-1. Antibodies appeared to be good indicators of CD8 ϩ T cell responses in this system. Studies of T cell reactivity against NY-ESO-1, however, remain limited because of the few epitopes described thus far. With regard to HLA-A2-restricted epitopes, three overlapping NY-ESO-1 peptides are known (3). Wang et al. (8) defined epitopes presented by the HLA-A31 molecule, encoded by both the normal and an alternative reading frame of NY-ESO-1.In the present study, we devised an assay to monitor CD8 ϩ T lymphocyte responses to NY-ESO-1 in cancer patients that does not require knowledge of predefined peptide epitopes or particular HLA profiles. Recombinant adenovirus and vaccinia vectors were constructed to express the full-length NY-ESO-1 gene. The first vector was used to transduce antigen-presenting cells (APCs) with NY-ESO-1 and stimulate memory effector T cells from cancer patients, and the second was used to induce antigen expression in B cells, the targets for measuring T cell responses. Using this set of non-cross-reactive recombinant viruses, we recalled and characterized CD8 ϩ T cell responses against naturally processed NY-ESO-1 epitopes. Materials and MethodsTumor Typing for NY-ESO-1 mRNA. Expression of NY-ESO-1 mRNA in tumor specimens was assessed by reverse transcription-PCR, using previously described primers (1).Assays for NY-ESO-1 Antibody. NY-ESO-1 serum antibodies were assayed by ELISA and Western blots, using NY-ESO-1 recombinant protein purified from Escherichia coli (2).Generation of Viral Vectors. Adenoviral constructs Ad2͞EV (empty vector) and Ad2͞EGFP (encoding green fluorescent protein) have been described previously (9). Adenovirus recombinant for NY-ESO-1 (Ad2͞ESO) was generated with a cloning method described previously (10). Briefly, pBK-CMV NY-ESO-1 (1) was digested with EcoRI and XbaI, and an 0.8-kb NY-ESO-1 fragment was isolated and cloned into EcoRI and XbaI sites of pSV2-ICEU-1 pAd Quick shuttle vector. The shuttle plasmid pSV2-...