Antibody-drug conjugates are targeted anticancer agents consisting of a cytotoxic drug covalently linked to a monoclonal antibody for tumor antigen-specific activity. Once bound to the target cell-surface antigen, the conjugate must be processed to release an active form of the drug, which can reach its intracellular target. Here, we used both biological and biochemical methods to better define this process for antibody-maytansinoid conjugates. In particular, we examined the metabolic fate in cells of huC242-maytansinoid conjugates containing either a disulfide linker (huC242-SPDB-DM4) or a thioether linker (huC242-SMCC-DM1). Using cell cycle analysis combined with lysosomal inhibitors, we showed that lysosomal processing is required for the activity of antibody-maytansinoid conjugates, irrespective of the linker. We also identified and characterized the released maytansinoid molecules from these conjugates, and measured their rate of release compared with the kinetics of cell cycle arrest. Both conjugates are efficiently degraded in lysosomes to yield metabolites consisting of the intact maytansinoid drug and linker attached to lysine. The lysine adduct is the sole metabolite from the thioether-linked conjugate. However, the lysine metabolite generated from the disulfide-linked conjugate is reduced and S-methylated to yield the lipophilic and potently cytotoxic metabolite, S-methyl-DM4. These findings provide insight into the mechanism of action of antibodymaytansinoid conjugates in general, and more specifically, identify a biochemical mechanism that may account for the significantly enhanced antitumor efficacy observed with disulfide-linked conjugates. (Cancer Res 2006; 66(8): 4426-33)

Purpose: The CD38 cell surface antigen is expressed in diverse hematologic malignancies including multiple myeloma, B-cell non-Hodgkin lymphoma (NHL), B-cell chronic lymphocytic leukemia, B-cell acute lymphoblastic leukemia (ALL), and T-cell ALL. Here, we assessed the antitumor activity of the anti-CD38 antibody SAR650984.Experimental Design: Activity of SAR650984 was examined on lymphoma, leukemia and multiple myeloma cell lines, primary multiple myeloma samples, and multiple myeloma xenograft models in immunodeficient mice.Results: We identified a humanized anti-CD38 antibody with strong proapoptotic activity independent of cross-linking agents, and potent effector functions including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis (ADCP), equivalent in vitro to rituximab in CD20 þ and CD38 þ models. This unique antibody, termed SAR650984, inhibited the ADP-ribosyl cyclase activity of CD38, likely through an allosteric antagonism as suggested by 3D structure analysis of the complex. In vivo, SAR650984 was active in diverse NHL, ALL, and multiple myeloma CD38 þ tumor xenograft models. SAR650984 demonstrated single-agent activity comparable with rituximab or cyclophosphamide in Daudi or SU-DHL-8 lymphoma xenograft models with induction of the proapoptotic marker cleaved capase-7. In addition, SAR650984 had more potent antitumor activity than bortezomib in NCI-H929 and Molp-8 multiple myeloma xenograft studies.

We report that the cytomegalovirus-encoded cell death suppressor vMIA binds Bax and prevents Bax-mediated mitochondrial membrane permeabilization by sequestering Bax at mitochondria in the form of a vMIA–Bax complex. vMIA mutants with a defective mitochondria-targeting domain retain their Bax-binding function but not their ability to suppress mitochondrial membrane permeabilization or cell death. vMIA does not seem to either specifically associate with Bak or suppress Bak-mediated mitochondrial membrane permeabilization. Recent evidence suggests that the contribution of Bax and Bak in the mitochondrial apoptotic signaling pathway depends on the distinct phenotypes of cells, and it appears from our data that vMIA is capable of suppressing apoptosis in cells in which this pathway is dominated by Bax, but not in cells where Bak also plays a role.

Depletion of CD4 1 CD25 1 FoxP3 1 Treg using PC61 mAb (anti-murine CD25 rat IgG1) is widely used to characterize Treg function in vivo. However, the mechanism of Treg depletion remains largely unknown. Herein, we report the PC61 mAb's mechanism of action. In peripheral blood, a single injection of PC61 mAb eliminated $70% of CD4 1 FoxP3 1 cells with the remaining Treg expressing low or no CD25. Functional blockade of Fcc receptors with 2.4G2 mAb significantly inhibited PC61 mAb activity. Furthermore, Fcc receptor (FccR)III À/À mice were resistant to Treg depletion. FccRIII is expressed on immune cells including NK cells and macrophages that are the major effector cells for Ab-dependentcellular-cytotoxicity and Ab-dependent-cellular-phagocytosis, respectively. Depletion of NK cells had no effect, whereas depletion of phagocytes, including macrophages, by clodronate liposome significantly inhibited Treg depletion. Furthermore, in vitro, PC61 mAb can mediate Ab-dependent-cellular-phagocytosis of CD25 1 cells by WT or FccRIIB À/À , but not FccRIII À/À , macrophages. Altogether these data demonstrate the critical role of FccRIII 1 phagocytes in mediating Treg depletion by PC61 mAb. This finding may be useful in guiding the development of human Treg targeting therapy.Key words: CD25 . Fcc receptor . PC61 Ab . Phagocytes . Treg Supporting Information available online IntroductionNaturally occurring Treg play a critical role in the maintenance of peripheral tolerance. They can suppress not only CD4 1 and CD8 1 T cells, but also NK cells, macrophages and dendritic cells [1]. Treg are marked by expression of FoxP3 1 transcription factor (Foxp3), which confers the Treg's regulatory activity. Induction of Foxp3 expression in naïve T cells can convert them into Treg [2,3]. Before the discovery of Foxp3, Treg were identified by expression of CD25, an IL-2 receptor a subunit. Initially, CD25 was simply considered a Treg marker that was not associated with Treg function because of its expression on activated T cells without suppressive activity. More recently, however, it became clear that CD25, thus also IL-2, is essential for the generation, peripheral expansion and maintenance of Treg. CD25-deficient mice and mice lacking IL-2 or Foxp3 have a reduced Treg number and spontaneously develop a fatal lymphoproliferative autoimmune syndrome that can be rescued by adoptive transfer of Treg from WT mice [1]. Temporary neutralization of IL-2 using anti-IL-2 mAb also reduces Treg number, induces autoimmune gastritis in BALB/c mice and accelerates autoimmune diabetes in NOD mice [4]. Additionally, ectopic expression of IL-2 receptor b in the thymus increases Treg production [5]. A recent study using transgenic mice expressing GFP-linked Foxp3 shows that the peripheral expansion of CD25 À Treg is significantly reduced compared with that of CD25 Results and discussion Treg depletion by PC61 mAbWe first tested the extent of Treg depletion after CD25 mAb injection in C57BL/6 mice. In naïve mice (Fig. 1A left panel), around 70% of CD4 1 Fo...

When subjected to selective conditions that impose starvation, a bacterial population can accumulate mutations, called adaptive, that allow colony formation. Here, the reversion of a lac allele under selective conditions, in a model system using Escherichia coli with the lac mutation on an F' plasmid, was shown to require the conjugational capacity of the plasmid. Reversion associated with transfer was shown, and when the same lac allele was chromosomal, reversion to Lac+ was 25 to 50 times less frequent. Postplating reversion was 25 times less when mating was inhibited by the presence of detergent. Mutability associated with conjugation provides new ways of thinking about the origin of adaptive mutations.