Purpose: Antibody drug conjugates (ADCs) combine the ideal properties of both antibodies and cytotoxic drugs by targeting potent drugs to the antigen-expressing tumor cells, thereby enhancing their antitumor activity. Successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency, and mode of linker-drug conjugation to the antibody. Here, we systematically examined the in vitro potency as well as in vivo preclinical efficacy and safety profiles of a heterogeneous preparation of conventional trastuzumab-mcc-DM1 (TMAb-mcc-DM1) ADC with that of a homogeneous engineered thio-trastuzumab-mpeo-DM1 (thioTMAb-mpeo-DM1) conjugate.Experimental Design and Results: To generate thioTMAb-mpeo-DM1, one drug maytansinoid 1 (DM1) molecule was conjugated to an engineered cysteine residue at Ala114 (Kabat numbering) on each trastuzumab-heavy chain, resulting in two DM1 molecules per antibody. ThioTMAb-mpeo-DM1 retained similar in vitro anti-cell proliferation activity and human epidermal growth factor receptor 2 (HER2) binding properties to that of the conventional ADC. Furthermore, it showed improved efficacy over the conventional ADC at DM1-equivalent doses (μg/m 2 ) and retained efficacy at equivalent antibody doses (mg/kg). An improved safety profile of >2-fold was observed in a short-term target-independent rat safety study. In cynomolgus monkey safety studies, thioTMAb-mpeo-DM1 was tolerated at higher antibody doses (up to 48 mg/kg or 6,000 μg DM1/m 2 ) compared with the conventional ADC that had dose-limiting toxicity at 30 mg/kg (6,000 μg DM1/m 2 ). Conclusions:The engineered thioTMAb-mpeo-DM1 with broadened therapeutic index represents a promising antibody drug conjugate for future clinical development of HER2-positive targeted breast cancer therapies.

Human T cell leukemia virus type 1 encodes an ''accessory'' protein named p13 II that is targeted to mitochondria and triggers a rapid flux of K ؉ and Ca 2؉ across the inner membrane. In this study, we investigated the effects of p13 II on tumorigenicity in vivo and on cell growth in vitro. Results showed that p13 II significantly reduced the incidence and growth rate of tumors arising from c-myc and Ha-ras-cotransfected rat embryo fibroblasts. Consistent with these findings, HeLa-derived cell lines stably expressing p13 II exhibited markedly reduced tumorigenicity, as well as reduced proliferation at high density in vitro. Mixed culture assays revealed that the phenotype of the p13 II cell lines was dominant over that of control lines and was mediated by a heat-labile soluble factor. The p13 II cell lines exhibited an enhanced response to Ca 2؉ -mediated stimuli, as measured by increased sensitivity to C2-ceramide-induced apoptosis and by cAMP-responsive element-binding protein (CREB) phosphorylation in response to histamine. p13 II -expressing Jurkat T cells also exhibited reduced proliferation, suggesting that the protein might exert similar effects in T cells, the primary target of HTLV-1 infection. These findings provide clues into the function of p13 II as a negative regulator of cell growth and underscore a link between mitochondria, Ca 2؉ signaling, and tumorigenicity.apoptosis ͉ calcium signaling ͉ tumorigenicity ͉ retrovirus H uman T cell leukemia virus type 1 (HTLV-1) possesses a complex genome that codes for Gag, Pol, Env, Tax, Rex, and a number of ''accessory'' proteins (1-4). Although the function of the accessory proteins has not yet been elucidated completely, they elicit an immune response in HTLV-1 infected individuals (5) and are required for efficient viral propagation in an animal model (6).One of the HTLV-1 accessory proteins, p13 II , accumulates in mitochondria by means of an amphipathic mitochondrial targeting signal and disrupts mitochondrial morphology (7). Biochemical analyses showed that p13 II is inserted in the inner mitochondrial membrane and alters mitochondrial conductance to Ca 2ϩ and K ϩ , leading to swelling and collapse of inner mitochondrial membrane potential (8).These effects suggest that p13 II might alter key mitochondrial functions such as energy production, redox status, and apoptosis, which could in turn disrupt the balance between cell death and proliferation. In this study, we investigated the impact of p13 II on cell growth in vitro and tumor growth in vivo by using the rat embryo fibroblast (REF) transformation model and cell lines expressing p13 II . Results showed that p13 II -expressing cells exhibit reduced tumorigenicity in vivo and slower proliferation at high density in vitro. p13 II -expressing cells also display perturbations in signal transduction pathways depending on Ca 2ϩ homeostasis. Materials and MethodsGeneration of HeLa Tet-On Cell Lines Expressing p13 II . The doxycyclininducible p13 II expression plasmid pTRE-p13 II -AU1 was constructed by inse...

BACKGROUND AND PURPOSE rhuMAb Beta7 is a humanized anti‐human β7 monoclonal antibody currently in phase I in inflammatory bowel disease. rhuMAb Beta7 binds the β7 subunit of the integrins α4β7 and αEβ7, blocking interaction with their ligands. These integrins play key roles in immune cell homing to and retention in mucosal sites, and are associated with chronic inflammatory diseases of the gastrointestinal tract. The goal of this study was to evaluate the mucosal specificity of rhuMAb Beta7. EXPERIMENTAL APPROACH We assessed the effect of murine anti‐Beta7 on lymphocyte homing in mouse models of autoimmune disease. We also compared the effect of rhuMAb Beta7 on circulating mucosal‐homing versus peripheral‐homing T cells in naïve non‐human primates. KEY RESULTS In cynomolgus monkeys, occupancy of β7 integrin receptors by rhuMAb Beta7 correlated with an increase in circulating β7+ mucosal‐homing lymphocytes, with no apparent effect on levels of circulating β7‐ peripheral‐homing lymphocytes. rhuMAb Beta7 also inhibited lymphocyte homing to the inflamed colons of severe combined immunodeficient mice in CD45RBhigh CD4+ T‐cell transfer models. Consistent with a lack of effect on peripheral homing, in a mouse model of experimental autoimmune encephalomyelitis, anti‐β7 treatment resulted in no amelioration of CNS inflammation. CONCLUSIONS AND IMPLICATIONS The results presented here suggest that rhuMAb Beta7 selectively blocks lymphocyte homing to the gastrointestinal tract without affecting lymphocyte trafficking to non‐mucosal tissues. rhuMAb Beta7 provides a targeted therapeutic approach with the potential for a more attractive benefit : risk ratio than currently available inflammatory bowel disease therapies.

Background: Human T-lymphotropic virus type-1 (HTLV-1) causes adult T-cell leukemia/ lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Taxresponsive element-mediated transcription through its interaction with CREB-binding protein/ p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation.