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Previously, we reported an agonist antibody to a cytokine receptor, Thrombopoietin receptor (TPOR) that effectively induces cytotoxic killer cells from precursor tumor cells isolated from newly diagnosed AML patients. Here, we show that the TPOR agonist antibody can induce even relapsed AML cells into killer cells more potently than newly diagnosed AML cells. After stimulation by the agonist antibody, these relapsed leukemic cells enter into a differentiation process of killer cells. The antibody-induced killer cells express, Granzyme B and Perforin that assault and kill other members of the AML cell population. Particularly, the agonist antibody showed potent efficacy on the AML xenograft model in mice using the NOD/LtSz-scid IL2Rγc null (NSG) mice. These results show that the TPOR agonist antibody that induces AML cells to kill each other is effective on both relapsed AML cells and in vivo. Therefore, this study suggests a new strategy for the treatment of cancer relapse after chemotherapy.

After decades of nonspecific lymphoma therapy, personalized approaches such as described here are on the horizon., We report the development of a novel platform to enhance the efficacy and safety of follicular lymphoma (FL) treatment. Since lymphoma is a clonal malignancy of a diversity system, every tumor has a different antibody on its cell surface. Combinatorial autocrine-based selection is used to rapidly identify specific ligands for these B cell receptors on the surface of FL tumor cells. The selected ligands are used in a chimeric antigen receptor T cell (CAR-T) format for redirection of human cytotoxic T lymphocytes. Essentially, the format is the inverse of the usual CAR-T protocol. Instead of being a guide molecule, the antibody itself is the target. Thus, these studies raise the possibility of personalized treatment of lymphomas using a private antibody binding ligand that can be obtained in a few weeks.

When combinatorial antibody libraries are rendered infectious for eukaryotic cells, the integrated antibody genotype and cellular phenotype become permanently linked and each cell becomes a selection system unto itself. These systems should be ideal for the identification of proteins and pathways that regulate differentiation so long as selection systems can be devised. Here we use a selection system based on the ability of secreted antibodies to alter the morphology of colonies expressing them when grown in soft agar. Importantly, this approach is different from all previous studies in that it used a pure discovery format where unbiased libraries that were not preselected against any known protein were used as probes. As such, the strategy is analogous to classical forward genetic approaches except that it operates directly at the protein level. This approach led to the identification of integrin-binding agonist antibodies that efficiently converted human stem cells to dendritic cells.

Vectors have been constructed that express the chitin-binding domain (ChBD) on eukaryotic cell surfaces. The ChBD is linked to enhanced green fluorescent protein (EGFP) through a protein that spans the plasma membrane. This binding functionality does not have a counterpart in eukaryotes, thereby endowing the modified cell surface with a property that is orthogonal to animal cells.

Cytoplasmic membrane-associated DNA (cmDNA) is a species of DNA that attaches to the plasma membrane and has physical and chemical properties that differ from those of bulk chromosomal and mitochondrial DNAs. Here, we used deep sequencing to analyze cmDNA and showed that satellite DNAs consisting of both of simple (CCATT) N repeats from the pericentromere regions of the chromosomes and 171-bp α-satellite repeat sequences from centromeres were highly enriched. Importantly, we found there is a special cytoplasmic membrane-associated transcription system in which DNA-dependent RNA polymerase II, which colocalizes with template cmDNA at the plasma membrane, can transcribe the membrane-associated 171-bp α-satellite repeat sequences into RNA. Analysis of phosphorylation patterns indicated that the RNA polymerase II in the plasma membrane is in a different chemical state from its nuclear counterpart.

Use of large combinatorial antibody libraries and next-generation sequencing of nucleic acids are two of the most powerful methods in modern molecular biology. The libraries are screened using the principles of evolutionary selection, albeit in real time, to enrich for members with a particular phenotype. This selective process necessarily results in the loss of information about less-fit molecules. On the other hand, sequencing of the library, by itself, gives information that is mostly unrelated to phenotype. If the two methods could be combined, the full potential of very large molecular libraries could be realized. Here we report the implementation of a phenotype-information-phenotype cycle that integrates information and gene recovery. After selection for phage-encoded antibodies that bind to targets expressed on the surface of Escherichia coli , the information content of the selected pool is obtained by pyrosequencing. Sequences that encode specific antibodies are identified by a bioinformatic analysis and recovered by a stringent affinity method that is uniquely suited for gene isolation from a highly degenerate collection of nucleic acids. This approach can be generalized for selection of antibodies against targets that are present as minor components of complex systems.

An attractive, but as yet generally unrealized, approach to cancer therapy concerns discovering agents that change the state of differentiation of the cancer cells. Recently, we discovered a phenomenon that we call “receptor pleiotropism” in which agonist antibodies against known receptors induce cell fates that are very different from those induced by the natural agonist to the same receptor. Here, we show that one can take advantage of this phenomenon to convert acute myeloblastic leukemic cells into natural killer cells. Upon induction with the antibody, these leukemic cells enter into a differentiation cascade in which as many as 80% of the starting leukemic cells can be differentiated. The antibody-induced killer cells make large amounts of perforin, IFN-γ, and granzyme B and attack and kill other members of the leukemic cell population. Importantly, induction of killer cells is confined to transformed cells, in that normal bone marrow cells are not induced to form killer cells. Thus, it seems possible to use agonist antibodies to change the differentiation state of cancer cells into those that attack and kill other members of the malignant clone from which they originate.

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists have emerged as treatment options for type 2 diabetes mellitus (T2DM). GLP-1R signals through G-protein-dependent, and G-protein-independent pathways by engaging the scaffold protein β-arrestin; preferential signalling of ligands through one or the other of these branches is known as ‘ligand bias'. Here we report the discovery of the potent and selective GLP-1R G-protein-biased agonist, P5. We identified P5 in a high-throughput autocrine-based screening of large combinatorial peptide libraries, and show that P5 promotes G-protein signalling comparable to GLP-1 and Exendin-4, but exhibited a significantly reduced β-arrestin response. Preclinical studies using different mouse models of T2DM demonstrate that P5 is a weak insulin secretagogue. Nevertheless, chronic treatment of diabetic mice with P5 increased adipogenesis, reduced adipose tissue inflammation as well as hepatic steatosis and was more effective at correcting hyperglycaemia and lowering haemoglobin A1c levels than Exendin-4, suggesting that GLP-1R G-protein-biased agonists may provide a novel therapeutic approach to T2DM., GLP-1 is a gut hormone with glucose-lowering activity. Here the authors report the peptide, P5, a variant of the GLP-1 receptor agonist exendin-4, with 'biased' signalling activity, and show that P5 improves glucose homeostasis in diabetic mice by increasing adipose tissue hyperplasia.

Antibodies that selectively bind to N-formylmethionyl leucyl phenylalanine (fMLF, also known as fMLP) have been generated. These antibodies bound to fMLF with higher affinity than to non-formylated peptide MLF: the differences in the binding energies between fMLF and MLF were 1.4->2.1 kcal/mol.

The use of copper radioisotopes in imaging and therapy applications has created a greater need for bifunctional chelates (BFCs) for complexing copper radioisotopes to biomolecules. It has been demonstrated that the charge and lipophilicity of the Cu-BFC complex has a significant effect on the in vivo behavior of the radiolabeled Cu-BFC-biomolecule conjugate. To evaluate the effects of charge, stability, and macrocyclic backbone size on the biological behavior of 64Cu complexes, a series of macrocyclic 64Cu complexes have been prepared, and the biodistributions of these agents were evaluated in normal Sprague-Dawley rats. Two macrocyclic backbones, dodecane and tetradecane, were evaluated; cyclen, DOTA, and DO2A were dodecane backbone derivatives, and cyclam, TETA, and et-cyclam were tetradecane backbone derivatives. The biodistributions of the 64Cu-labeled complexes correlated with differences in the size of the macrocycle backbone and the formal charge of the complex. All compounds showed uptake and clearance through the liver and kidneys; however, the positively charged 64Cu complexes showed significantly higher uptake in both of these organs than did the negatively charged or neutral complexes. 64Cu-TETA, a negatively charged complex with the tetradecane backbone, had the most efficient clearance by 24 hours' postinjection. These data suggest that negatively charged complexes may have more favorable clearance properties when used as BFCs.

Easy M Our immune systems can produce a vastly diverse repertoire of antibody molecules that each recognize and bind to a specific foreign antigen via a hypervariable region. However, there are a few bacterial antigens—such as Protein A, Protein G, and Protein L—that instead bind to the antibody's conserved regions and can bind to a large number of different antibodies. These high-affinity broad-spectrum antibody-binding properties have been widely exploited both in the laboratory and in industry for purifying, immobilizing, and detecting antibodies. Grover et al. (p. 656 ) have now identified Protein M found on the surface of human mycoplasma, which displays even broader antibody-binding specificity. The crystal structure of Protein M revealed how Protein-M binding blocks the antibody's antigen binding site. This mechanism may be exploited by mycoplasma to escape the humoral immune response.

The 67Ga(III) and/or 111In(III) complexes of four new hexadentate ligands have been prepared and evaluated in vitro and in vivo. These substituted triazacyclononane ligands bind the metal ion through three tertiary ring nitrogens and three oxygens from pendant phenolic or hydroxypyridyl arms. The hydroxypyridyl moieties increase the aqueous solubility of the metal complexes while retaining a lipophilic character. As indicated by their large positive partition coefficients, the phenolic ligands proved to be significantly more lipophilic than the hydroxypyridyl ligands. Biodistribution in Sprague-Dawley rats indicated that the more lipophilic phenolic complexes cleared the body primarily through the liver, while the less lipophilic hydroxypyridyl complexes cleared rapidly, primarily through the kidney. To differentiate the clearance characteristics of these radiolabeled compounds, radiochemical purity of selected complexes in vivo was measured. The complexes were evaluated for overall charge in vitro and in vivo, in plasma samples. In addition, plasma and urine were analyzed for possible metabolites. With one exception, each complex was unmetabolized in vivo. All complexes and metabolites formed were neutral in vitro and in vivo. Extended stability in serum of selected radiometal complexes has been measured. Each complex measured was stable to exchange with transferrin, up to 72 h, as expected from the large stability constants of the complexes. The clearance characteristics of the hydroxypyridyl and phenolic ligands, however, were markedly different. The rapid hepatic clearance of the phenolic ligands indicates potential as bifunctional chelates for Ga(III) or In(III).

The mechanism of chronic rejection of transplanted human kidneys is unknown. An understanding of this process is important because, chronic rejection ultimately leads to loss of the kidney allograft in most transplants. One feature of chronic rejection is the infiltration of ectopic B-cell clusters that are clonal into the transplanted kidney. We now show that the antibodies produced by these B-cells react strongly with the core carbohydrate region of LPS. Since LPS is a costimulatory immunogen that can react with both the B-cell receptor (BCR) and the Toll-like receptor 4 (TLR4), these results suggest a mechanism for the selective pressure that leads to clonality of these B-cell clusters and opens the possibility that infection and the attendant exposure to LPS plays a role in the chronic rejection of human kidney transplants. If confirmed by clinical studies, these results suggest that treating patients with signs of chronic rejection with antibiotics may improve kidney allograft survival.

B cells and their immunoglobulin products participate in allograft rejection of transplanted human kidneys in which an interesting feature is the presence of a germinal center like B-cell clusters in the allograft. We report here that the immunoglobulin repertoires of these infiltrating B cells are highly restricted and the B cells within a cluster are clonal. Antibody libraries made from the infiltrating B cells of individual patients unexpectedly revealed that each patient utilizes a particular set of dominant germ line genes as well as dominant complementarity determining region 3. Comparison of kidney and peripheral blood from the same patient showed that the immunoglobulin genes from both compartments had dominant clones, but they differed. The lymphocytes that infiltrate the kidneys express the immunoglobulin gene somatic recombination machinery usually restricted to highly activated lymphocytes in germinal centers and lymphomas. An analogy can be made between the inescapable antigenic drive in chronic infection versus that in an allograft, both of which may lead to emergence of dominant B-cell clones and even lymphoid malignancy.

Antibodies are among the most highly selective tight-binding ligands for proteins. Because the human genome project has deciphered the proteome, there is an opportunity to use combinatorial antibody libraries to select high-affinity antibodies to every protein encoded by the genome. However, this is a large task because the selection formats used today for combinatorial antibody libraries are geared toward generating antibodies to one antigen at a time. Here, we describe a method that accelerates the identification of antibodies to a multitude of antigens simultaneously by matching combinatorial antibody libraries against eukaryotic antigen libraries so that replication-competent cognate antigen–antibody pairs can be directly selected. Phage and yeast display systems are used because they each link genotype to phenotype and can be replicated individually. When combined with cell sorting, the two libraries can be selected against each other for recovery of cognate antigen–antibody clones in a single experiment.

Recently, we showed that antibodies catalyze the generation of hydrogen peroxide (H 2 O 2 ) from singlet molecular oxygen ( 1 O 2 *) and water. Here, we show that this process can lead to efficient killing of bacteria, regardless of the antigen specificity of the antibody. H 2 O 2 production by antibodies alone was found to be not sufficient for bacterial killing. Our studies suggested that the antibody-catalyzed water-oxidation pathway produced an additional molecular species with a chemical signature similar to that of ozone. This species is also generated during the oxidative burst of activated human neutrophils and during inflammation. These observations suggest that alternative pathways may exist for biological killing of bacteria that are mediated by potent oxidants previously unknown to biology.

Much has been "written of late regarding the advent of new information technologies, new weapons systems and platforms, and intelligence architectures offering to fuse real-time information that will achieve total battlefield awareness The excitement that these advances and the ongoing Revolution in Military Affairs bring to the military strategist is palpable While acknowledging we are on the cusp of a wondrous new technologic landscape, however, much uncertainty is expressed as to what exactly these technology breakthroughs will bring what they will do, how they will change how we live and wage war, and how they will shape our world Concurrently, the end of the Cold War shattered the comfortable bi-polar world the US inhabited, sending everyone, the military strategist included, scurrying to find a new roadmap New challenges are destabilizing the international arena, while at home the burgeoning information culture encourages citizens and politicians to demand better performance and insist on more involvement in military decision-making Modern technologies are expensive to build and deploy and the military is increasingly asked to contribute on the full spectrum of engagement, yet budgets are tight and will get tighter Societal and epistemological changes are driving the tide of Post-modernism, questioning the very validity of "truth", and are just beginning to send out ripples which will affect the fabric of society which undergirds the military In short, the world is in flux on multiple levels, familiar terrain has been left behind and many anxiously lament that the future is shrouded by a fog of uncertainty.

Carl von Clausewitz's remarkable grasp of the fundamental aspects of war have stood the test of time. Clausewitz remains relevant in the post-modern era despite advances in technology, precision weapons, communication architectures and computers--developments which have rendered other theorists passe. His writings provide a bedrock of understanding on which to overlay current capabilities and conceptual frameworks. The freshness and relevance of Clausewitz's insights stem in large part from the non-linearity of his cognitive approach. Alan Beyerchen makes a most persuasive case that Clausewitz was a non-linear thinker, whose work is best approached from the perspective of the relatively new non-linear sciences. This paper investigates how Clausewitz's classic work "On War" addresses the issue of intelligence, explores the value of understanding the importance of non-linearity in fully comprehending his thought processes, and posits some implications for the Intelligence Community.