We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cellmediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation. Mol Cancer Ther; 12(10); 2031-42. Ó2013 AACR.

Growing evidence has raised the important roles of adipocytes as an active player in the tumor microenvironment. In many tumors adipocytes are in close contact with cancer cells. They secrete various factors that can mediate local and systemic effects. The adipocyte-cancer cell crosstalk leads to phenotypical and functional changes of both cell types, which can further enhance tumor progression. Moreover, obesity, which is associated with an increase in adipose mass and an alteration of adipose tissue, has been established as a risk factor for cancer incidence and cancer-related mortality. In this review, we summarize the mechanisms of the adipocyte-cancer cell crosstalk in both obese and lean conditions as well as its impact on cancer cell growth, local invasion, metastatic spread and resistance to treatments. Better characterization of cancer-associated adipocytes and the key molecular events in the adipocyte-cancer cell crosstalk will provide insights into tumor biology and suggest efficient therapeutic opportunities.

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IntroductionTrastuzumab has been used in the treatment of human epidermal growth factor receptor 2 (HER2)-expressing breast cancer, but its efficacy is limited by de novo or acquired resistance. Although many mechanisms have been proposed to explain resistance to trastuzumab, little is known concerning the role of the tumor microenvironment. Given the importance of antibody-dependent cellular cytotoxicity (ADCC) in the antitumor effect of trastuzumab and the abundance of adipose tissue in the breast, we investigated the impact of adipocytes on ADCC.MethodsWe set up a coculture system to study the effect of adipocytes on ADCC in vitro. The results were validated in vivo in a mouse xenograft model.ResultsWe found that adipocytes, as well as preadipocytes, inhibited trastuzumab-mediated ADCC in HER2-expressing breast cancer cells via the secretion of soluble factors. The inhibition of ADCC was not due to titration or degradation of the antibody. We found that adipose cells decreased the secretion of interferon-γ by natural killer cells, but did not alter natural killer cells’ cytotoxicity. Preincubation of breast cancer cells with the conditioned medium derived from adipocytes reduced the sensitivity of cancer cells to ADCC. Using a transcriptomic approach, we found that cancer cells undergo major modifications when exposed to adipocyte-conditioned medium. Importantly, breast tumors grafted next to lipomas displayed resistance to trastuzumab in mouse xenograft models.ConclusionsCollectively, our findings underline the importance of adipose tissue in the resistance to trastuzumab and suggest that approaches targeting the adipocyte–cancer cell crosstalk may help sensitize cancer cells to trastuzumab-based therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0569-0) contains supplementary material, which is available to authorized users.

(S)-1-Phenylethanol dehydrogenase (PED) from the denitrifying bacterium strain EbN1 catalyzes the NAD+-dependent, stereospecific oxidation of (S)-1-phenylethanol to acetophenone and the biotechnologically interesting reverse reaction. This novel enzyme belongs to the short-chain alcohol dehydrogenase/aldehyde reductase family. The coding gene (ped) was heterologously expressed in Escherichia coli and the purified protein was crystallized. The X-ray structures of the apo-form and the NAD+-bound form were solved at a resolution of 2.1 and 2.4 A, respectively, revealing that the enzyme is a tetramer with two types of hydrophobic dimerization interfaces, similar to beta-oxoacyl-[acyl carrier protein] reductase (FabG) from E. coli. NAD+-binding is associated with a conformational shift of the substrate binding loop of PED from a crystallographically unordered "open" to a more ordered "closed" form. Modeling the substrate acetophenone into the active site revealed the structural prerequisites for the strong enantioselectivity of the enzyme and for the catalytic mechanism. Studies on the steady-state kinetics of PED indicated a highly positive cooperativity of both catalytic directions with respect to the substrates. This is contrasted by the behavior of FabG. Moreover, PED exhibits extensive regulation on the enzyme level, being inhibited by elevated concentrations of substrates and products, as well as the wrong enantiomer of 1-phenylethanol. These regulatory properties of PED are consistent with the presence of a putative "transmission module" between the subunits. This module consists of the C-terminal loops of all four subunits, which form a special interconnected structural domain and mediate close contact of the subunits, and of a phenylalanine residue in each subunit that reaches out between substrate-binding loop and C-terminal domain of an adjacent subunit. These elements may transmit the substrate-induced conformational change of the substrate binding loop from one subunit to the others in the tetrameric complex and thus mediate the cooperative behavior of PED.

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Redirecting CD8 T cell immunity with self/tumor-specific affinity-matured T cell receptors (TCRs) is a promising approach for clinical adoptive T cell therapy, with the aim to improve treatment efficacy. Despite numerous functional-based studies, little is known about the characteristics of TCR signaling (i.e., intensity, duration, and amplification) and the regulatory mechanisms underlying optimal therapeutic T cell responses. Using a panel of human SUP-T1 and primary CD8 T cells engineered with incremental affinity TCRs against the cancer-testis antigen NY-ESO-1, we found that upon activation, T cells with optimal-affinity TCRs generated intense and sustained proximal (CD3ζ, LCK) signals associated with distal (ERK1/2) amplification-gain and increased function. In contrast, in T cells with very high affinity TCRs, signal initiation was rapid and strong yet only transient, resulting in poor MAPK activation and low proliferation potential even at high antigen stimulation dose. Under resting conditions, the levels of surface TCR/CD3ε, CD8β, and CD28 expression and of CD3ζ phosphorylation were significantly reduced in those hyporesponsive cells, suggesting the presence of TCR affinity-related activation thresholds. We also show that SHP phosphatases were involved along the TCR affinity gradient, but displayed spatially distinct regulatory roles. While PTPN6/SHP-1 phosphatase activity controlled TCR signaling initiation and subsequent amplification by counteracting CD3ζ and ERK1/2 phosphorylation, PTPN11/SHP-2 augmented MAPK activation without affecting proximal TCR signaling. Together, our findings indicate that optimal TCR signaling can be finely tuned by TCR affinity-dependent SHP-1 and SHP-2 activity, and this may readily be determined at the TCR/CD3 complex level. We propose that these TCR affinity-associated regulations represent potential protective mechanisms preventing high affinity TCR-mediated autoimmune diseases.

Due to its regenerative potential, hyaluronic acid-based hydrogel provides a promising novel therapy to be used alone, or as a scaffold delivering a variety of drugs or cells to combat heart disease in a multifaceted approach.