Human NK cells lyse Ab-coated target cells through the process of Ab-dependent cellular cytotoxicity (ADCC). Improving ADCC responses is desirable because it is thought to be an important antitumor mechanism for some Abs. NK cell inhibitory receptors, such as killer cell Ig-like receptors, engage with MHC class I molecules on self-cells to block NK cell activation. Accordingly, we enhanced ADCC responses by blocking NK cell inhibitory receptors, thus perturbing induction of the self-recognition signal. In a cell line model of anti-lymphoma therapy, the combination of rituximab with an Ab that blocks inhibitory self-recognition yielded increased NK cell-mediated target cell lysis when compared with rituximab alone. To validate this proof-of-concept, we then used a more representative approach in which an individual’s fresh primary NK cells encountered autologous, EBV-transformed B cells. In this system, rituximab and a combination of Abs that block NK cell inhibitory receptors yielded improved NK cell-mediated lysis over rituximab alone. The results show, for the first time, that disruption of inhibitory self-recognition can efficiently promote ADCC in a human model, applying an autologous system in which physiologic checkpoints are in place. This method provides an alternative approach to potentiate the therapeutic benefit of antitumor Abs that mediate ADCC.

To better understand the genetic controls of leaf senescence, a tobacco (Nicotiana tabacum L. cv. SR1) mRNA that is up-regulated during senescence was isolated by the cDNA-amplified restriction fragment polymorphism method and the cDNA was cloned. The mRNA coded for the early light-induced protein (ELIP), a member of the chlorophyll a/b-binding protein family that has been implicated in assembly or repair of the photosynthetic machinery during early chloroplast development and abiotic stress. A protein antigenically recognized by antibodies to ELIP appeared during senescence with kinetics similar to those of its mRNA. The mRNA, designated ELIP-TOB, was detected earlier when senescence was enhanced by leaf detachment and treatment with 1-amino-cyclopropane-1-carboxylic acid, and was detected later when senescence was retarded by benzyladenine. However, no ELIP-TOB mRNA was seen in the dark even though senescence was accelerated under these conditions. Furthermore, water stress and anaerobiosis stimulated the appearance of ELIP-TOB mRNA before losses of chlorophyll could be detected. We discuss the conditions that may lead to the up-regulation of ELIP-TOB during senescence and speculate as to the role of the gene product in this terminal phase of leaf development.

Inherent and acquired multidrug resistance (MDR) is characterized by a simultaneous resistance to diverse anticancer drugs and is a major impediment towards curative chemotherapy of cancer. Hence one important goal is to develop strategies aimed at specific targeting of major anticancer drug efflux transporters of the ATP-binding cassette (ABC) superfamily including multidrug resistance protein 1 -MRP1 (ABCC1). To date, no monoclonal antibody has been isolated that can target an extracellular MRP1 epitope. Using a phage display approach, we have isolated a recombinant singlechain Fv (scFv) antibody that specifically reacts with the extracellular N-terminus of the human MRP1. Flow cytometric analysis revealed that this scFv fragment binds specifically to various viable human tumor cells that display variable Key words: multi drug resistance (MDR); scFv antibody; MRP1; phage displayCombination chemotherapy continues to play a major role in the treatment of various human malignancies. However, the efficacy of various chemotherapeutics has been limited by the frequent emergence of various anticancer drug resistance phenomena. The best-studied mechanisms of multidrug resistance (MDR) in malignant cells involve the overexpression of ATP-driven anticancer drug efflux pumps of the ABC superfamily. 1-3 These include the multidrug transporter, P-glycoprotein (Pgp), which extrudes various hydrophobic cytotoxic agents, as well as members of the multidrug resistance protein (MRP) family, currently comprising 9 exporters (MRP1 through MRP9). 3-5 MRP1 was discovered during a differential hybridization screen aimed at identifying mRNAs overexpressed in doxorubicin-resistant lung cancer cells that lack P-glycoprotein overexpression. 6 The 100 -200-fold MRP1 mRNA overexpression was a result of gene amplification. Analysis of the 1,531 amino acid sequence of MRP1 sequence identified this protein as a member of the ATP-binding cassette (ABC) superfamily of transporter proteins. 6 Importantly, transfection of the MRP1 cDNA conferred upon sensitive cells MDR to anthracyclines, Vinca alkaloids, etoposide, arsenical and antimonial oxyanions but not to cisplatinum and mitoxantrone. 7 These studies paved the way for the isolation of additional MRPs, MRP2 through MRP9, which have been shown to confer resistance to various toxic compounds that are either initially hydrophilic or that undergo intracellular conjugation to glutathione, sulfate and glucuronate. 5 Furthermore, another ABC transporter recently shown to mediate MDR in various tumor cells of epithelial origin is the breast cancer resistance protein (BCRP). 8 In order to study anticancer drug transporter proteins such as MRP1 in clinical specimens and to reveal their physiological functions, specific monoclonal antibodies (MAbs) were recently developed. 9 -11 Monoclonal antibodies against MDR proteins were used for 2 main purposes: a) detection and quantification of MRP and P-glycoprotein expression in drug-resistant cancer cells and b) reversal of MDR in cancer cells by abolishin...

No abstract

Inherent and acquired MDR is characterized by simultaneous resistance to diverse anticancer drugs and continues to be a major impediment in the curative chemotherapy of cancer. The MDR1 gene product, Pgp, is an ATP-driven efflux pump, which extrudes a variety of dissimilar hydrophobic cytotoxic compounds from MDR cells. Pgp overexpression results in MDR of tumor cell lines in vitro as well as of a variety of human malignancies. Thus, one major goal is to develop strategies aimed at specifically disrupting Pgp drugefflux activity. To this end, we have developed a small recombinant antibody capable of potent reversal of MDR, by disrupting Pgp drug-efflux activity. Using a phage display approach, we isolated a small scFv recombinant antibody fragment that specifically reacts with the first extracellular loop of human Pgp. This scFv fragment binds specifically to various Pgp-overexpressing human MDR carcinoma cell lines, consequently disrupts Pgp drug-efflux function and thereby reverses the MDR phenotype. We have successfully disrupted anticancer drug-extrusion pump activity in MDR cells using a small recombinant scFv fragment. We propose that these novel small Fv-based recombinant antibody molecules may lead to the development of a new class of antibody fragment-based agents that specifically inhibit Pgp drug extrusion. Hence, these small recombinant antibody fragments may be applied in combination chemotherapy to overcome MDR in various human cancers. © 2004 Wiley-Liss, Inc. Key words: P-glycoprotein; multidrug resistance; single-chain FvCombination chemotherapy continues to play a major role in the treatment of various human malignancies. MDR, whereby tumor cells simultaneously possess intrinsic or acquired cross-resistance to diverse anticancer drugs, hampers the efficacy of the chemotherapeutic treatment of various human cancers. 1-3 Intense molecular investigations of the MDR phenomenon over the past 2 decades have resulted in the isolation and characterization of genes encoding for several plasma membrane glycoproteins associated with MDR. The best-studied mechanism of MDR in cancer cells is overexpression of an energy-dependent efflux pump, the multidrug transporter Pgp. 4 Pgp is a member of a large ABC superfamily of transport proteins. 5 Pgp-mediated MDR plays an important clinical role in the resistance of various tumor cells to chemotherapy, as judged by the high incidence of MDR1 expression in tumors of various types and the correlations between MDR1 expression and lack of response to chemotherapy in various human malignancies. 6,7 These findings prompted a major effort aimed at identifying strategies and agents capable of reversing Pgp-mediated MDR. 3 Inhibitors of the MDR phenotype in cancer cells may function in 2 main modes: they can either modify the expression (i.e., decrease it) or disrupt the drug-efflux function of the transporter proteins involved in MDR. 3 The search for "chemosensitizers" that disrupt the function of these drug exporters and thereby reverse MDR has grown in parallel with th...