Low Picomolar Concentrations Research Articles

Accounting for photophysical processes and specific signal intensity changes in fluorescence-detected sedimentation velocity.

Fluorescence detected sedimentation velocity (FDS-SV) has emerged as a powerful technique for the study of high-affinity protein interactions, with hydrodynamic resolution exceeding that of diffusion-based techniques, and with sufficient sensitivity for binding studies at low picomolar concentrations. For the detailed quantitative analysis of the observed sedimentation boundaries, it is necessary to adjust the conventional sedimentation models to the FDS data structure. A key consideration is the change in the macromolecular fluorescence intensity during the course of the experiment, caused by slow drifts of the excitation laser power, and/or by photophysical processes. In the present work, we demonstrate that FDS-SV data have inherently a reference for the time-dependent macromolecular signal intensity, resting on a geometric link between radial boundary migration and plateau signal. We show how this new time-domain can be exploited to study molecules exhibiting photobleaching and photoactivation. This expands the application of FDS-SV to proteins tagged with photoswitchable fluorescent proteins, organic dyes, or nanoparticles, such as those recently introduced for subdiffraction microscopy and enables FDS-SV studies of their interactions and size distributions. At the same time, we find that conventional fluorophores undergo minimal photobleaching under standard illumination in the FDS. These findings support the application of a high laser power density for the detection, which we demonstrate can further increase the signal quality.

Advances in understanding the role of cardiac glycosides in control of sodium transport in renal tubules.

Cardiotonic steroids have been used for the past 200 years in the treatment of congestive heart failure. As specific inhibitors of membrane-bound Na(+)/K(+) ATPase, they enhance cardiac contractility through increasing myocardial cell calcium concentration in response to the resulting increase in intracellular Na concentration. The half-minimal concentrations of cardiotonic steroids required to inhibit Na(+)/K(+) ATPase range from nanomolar to micromolar concentrations. In contrast, the circulating levels of cardiotonic steroids under physiological conditions are in the low picomolar concentration range in healthy subjects, increasing to high picomolar levels under pathophysiological conditions including chronic kidney disease and heart failure. Little is known about the physiological function of low picomolar concentrations of cardiotonic steroids. Recent studies have indicated that physiological concentrations of cardiotonic steroids acutely stimulate the activity of Na(+)/K(+) ATPase and activate an intracellular signaling pathway that regulates a variety of intracellular functions including cell growth and hypertrophy. The effects of circulating cardiotonic steroids on renal salt handling and total body sodium homeostasis are unknown. This review will focus on the role of low picomolar concentrations of cardiotonic steroids in renal Na(+)/K(+) ATPase activity, cell signaling, and blood pressure regulation.

Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies.

Analysis of High-Affinity Protein Interactions by Fluorescence Optical Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) offers substantial advantages for studying protein complex formation, providing information on size, shape and binding energies for reversible systems from analysis of the sedimentation profiles of molecular mixtures in free solution. By virtue of the hydrodynamic resolution achieved in sedimentation velocity experiments, multiple co-existing complexes can be identified, even in the presence of impurities and aggregates, but until recently the limited sensitivity of AUC has prevented accurate analysis of high affinity interactions. A recently introduced fluorescence optical detection system (FDS) for AUC offers specific advantages for studying high-affinity protein interactions due to the high sensitivity of fluorescence, but the unique characteristics of the optical system in this instrument require new analytical tools. We have developed computational approaches for analysis of FDS data that provide excellent fits to the observed sedimentation boundaries, correcting for effects intrinsic to the use of confocal laser optics. With these tools we have explored the detection limit of FDS, and have now recorded sedimentation signals with hydrodynamic resolution at low picomolar concentrations. This allows us to analyze sedimentation isotherms for interacting systems with Kds in the pM to nM range. Using different systems with high-affinity interaction we will demonstrate how the FDS-AUC system can be reliably applied to study high-affinity protein self- and hetero-associations at orders of magnitude lower concentrations and with much higher accuracy than previously possible.

163 : Recombinant IL-37 inhibits LPS induced inflammation in a SIGIRR- and MAPK-dependent manner

IL-1 family member IL-37 reduces innate inflammation and acquired immune responses. Our published studies demonstrated that over expression of IL-37 in either primary or transfected cell line cultures suppress the production of pro-inflammatory cytokines induced by Toll-like receptor ligands, IL-1b or TNFa. Inhibition of endogenous IL-37 in human monocytes increases IL-1b production. Similar to IL-1 family member IL-1a and IL-33, IL-37 migrates to the nucleus where it affects transcription. Therefore, it remains unclear if IL-37 functions to inhibit innate responses by an intracellular or extracellular mechanism. In the present study, we examined the function of recombinant IL-37 proteins. Recombinant IL-37, either the precursor or mature forms, binds to the alpha chain of the IL-18 receptor, but nevertheless inhibits LPS-induced cytokines. Whereas recombinant IL-37 reduces LPS-induced cytokines in PBMC, IL-37 is particularly effective in reducing (60–70%) LPS-induced cytokines in human monocyte-derived M1 and dendritic cells (DC) and consistently at low picomolar concentrations. In contrast, micromolar or high nanomolar concentrations do not inhibit LPS induced cytokines; hence, IL-37 does not function as an IL-18 receptor antagonist. LPS induced MAPK kinases such as phospho-p38 and ERK are down-regulated by recombinant IL-37. In M1 macrophages, exposure to IL-37 increases mRNA levels of SIGIRR, and down-regulates gene expression of cytokines such as TNF-a. Low picomolar concentrations of IL-37 also reduce LPS-induced cytokines (70–80%) in mouse bone marrow-derived DC, but significantly less reduction in DC derived from SIGIRR deficient mice. Intraperitoneal injection of recombinant IL-37 also reduces circulating and cytokine production in LPS injected mice. This study reveals that in addition to a nuclear function, picomolar concentrations of recombinant IL-37 are active as an extracellular cytokine by first binding to the IL-18 receptor. Furthermore, the anti-inflammatory effects of recombinant IL-37 reduce MAPK activity and require the presence of SIGIRR.

Bispecific antibodies directed to CD4 domain 2 and HIV envelope exhibit exceptional breadth and picomolar potency against HIV-1

In the absence of an effective HIV-1 vaccine, passive immunization using broadly neutralizing Abs or Ab-like molecules could provide an alternative to the daily administration of oral antiretroviral agents that has recently shown promise as preexposure prophylaxis. Currently, no single broadly neutralizing Ab (bNAb) or combination of bNAbs neutralizes all HIV-1 strains at practically achievable concentrations in vivo. To address this problem, we created bispecific Abs that combine the HIV-1 inhibitory activity of ibalizumab (iMab), a humanized mAb directed to domain 2 of human CD4, with that of anti-gp120 bNAbs. These bispecific bNAbs (BibNAbs) exploit iMab's potent anti-HIV-1 activity and demonstrated clinical efficacy and safety to anchor and thereby concentrate a second broadly neutralizing agent at the site of viral entry. Two BibNabs, PG9-iMab and PG16-iMab, exhibit exceptional breadth and potency, neutralizing 100% of the 118 viruses tested at low picomolar concentrations, including viruses resistant to both parental mAbs. The enhanced potency of these BibNAbs was entirely dependent on CD4 anchoring, not on membrane anchoring per se, and required optimal Ab geometry and linker length. We propose that iMab-based BibNAbs, such as PG9-iMab and PG16-iMab, are promising candidates for passive immunization to prevent HIV-1 infection.

Selective Cancer Therapy by Extracellular Activation of a Highly Potent Glycosidic Duocarmycin Analogue

Conventional cancer chemotherapy is limited by systemic toxicity and poor selectivity. Tumor-selective activation of glucuronide prodrugs by beta-glucuronidase in the tumor microenvironment in a monotherapeutic approach is one promising way to increase cancer selectivity. Here we examined the cellular requirement for enzymatic activation as well as the in vivo toxicity and antitumor activity of a glucuronide prodrug of a potent duocarmycin analogue that is active at low picomolar concentrations. Prodrug activation by intracellular and extracellular beta-glucuronidase was investigated by measuring prodrug 2 cytotoxicity against human cancer cell lines that displayed different endogenous levels of beta-glucuronidase, as well as against beta-glucuronidase-deficient fibroblasts and newly established beta-glucuronidase knockdown cancer lines. In all cases, glucuronide prodrug 2 was 1000-5000 times less cytotoxic than the parent duocarmycin analogue regardless of intracellular levels of beta-glucuronidase. By contrast, cancer cells that displayed tethered beta-glucuronidase on their plasma membrane were 80-fold more sensitive to glucuronide prodrug 2, demonstrating that prodrug activation depended primarily on extracellular rather than intracellular beta-glucuronidase activity. Glucuronide prodrug 2 (2.5 mg/kg) displayed greater antitumor activity and less systemic toxicity in vivo than the clinically used drug carboplatin (50 mg/kg) to mice bearing human lung cancer xenografts. Intratumoral injection of an adenoviral vector expressing membrane-tethered beta-glucuronidase dramatically enhanced the in vivo antitumor activity of prodrug 2. Our data provide evidence that increasing extracellular beta-glucuronidase activity in the tumor microenvironment can boost the therapeutic index of a highly potent glucuronide prodrug.

321 Analysis of Combinatorial Effect of Multikinase Inhibitor (Sorafenib, Sunitinib) or Cetuximab with MEK or mTOR Inhibitors in Human Head and Neck Cancer Cells

Background: Prostate cancer is the most common cancer in men. Although screening, radiation, surgery and hormone ablation therapy have greatly improved the detection and treatment of early stage prostate cancer, few options exist to treat metastatic, castrate-resistant prostate cancer (CRPC). We have developed a therapeutic approach that uses a bispecific SCORPION (multi-specific protein therapeutic) molecule that redirects T cell cytotoxicity against cells expressing a common prostate cancer antigen, PSMA (Prostate Specific Membrane Antigen). SCORPION molecules are antibody-like therapeutics containing two sets of binding domains linked to immunoglobulin Fc domains, which extends the half-life of the molecule in vivo. Materials and Methods: Cytotoxic activity was examined in vitro by treating PSMA(+) cell lines (LNCaP, C4−2B, MDA-PCa-2b) and PSMA(−) cell lines (DU-145, BxPC-3) with SCORPION molecules in the presence of purified human T cells or human peripheral blood mononuclear cells (PBMCs). Cytotoxic activity was determined by chromium release assays or by incorporation of a label such as propidium iodide or 7-aminoactinomycin D. CFSE-labeled T cells were assessed for activation and proliferation in a similar setting using multi-color flow cytometry. To evaluate serum stability, SCORPION proteins were incubated in mouse serum and monitored for functional activity. Results: SCORPION molecules targeting PSMA and CD3 efficiently redirected T cell cytotoxicity against PSMA(+) cell lines in vitro at low picomolar concentrations. Cytotoxic activity was target dependent and required the presence of T cells. T cells were activated and proliferated in response to the SCORPION therapeutic in the presence of PSMA(+) target cells; no proliferation was observed in response to PSMA(−) cells. SCORPION proteins were stable to freeze/thaw in mouse serum and showed stable functional activity in mouse serum when incubated at 37oC for up to one week. Conclusions: These studies show that anti-PSMA× anti-CD3 SCORPION therapeutics may be efficient agents for redirecting T cell cytotoxicity and show promise as potential therapeutics in CRPC.

320 Comparative Pharmacokinetics of Trastuzumab-US and Trastuzumab-EU and the Potential Biosimilar Trastuzumab-Pfizer in Male CD-1 Mice

Background: Prostate cancer is the most common cancer in men. Although screening, radiation, surgery and hormone ablation therapy have greatly improved the detection and treatment of early stage prostate cancer, few options exist to treat metastatic, castrate-resistant prostate cancer (CRPC). We have developed a therapeutic approach that uses a bispecific SCORPION (multi-specific protein therapeutic) molecule that redirects T cell cytotoxicity against cells expressing a common prostate cancer antigen, PSMA (Prostate Specific Membrane Antigen). SCORPION molecules are antibody-like therapeutics containing two sets of binding domains linked to immunoglobulin Fc domains, which extends the half-life of the molecule in vivo. Materials and Methods: Cytotoxic activity was examined in vitro by treating PSMA(+) cell lines (LNCaP, C4−2B, MDA-PCa-2b) and PSMA(−) cell lines (DU-145, BxPC-3) with SCORPION molecules in the presence of purified human T cells or human peripheral blood mononuclear cells (PBMCs). Cytotoxic activity was determined by chromium release assays or by incorporation of a label such as propidium iodide or 7-aminoactinomycin D. CFSE-labeled T cells were assessed for activation and proliferation in a similar setting using multi-color flow cytometry. To evaluate serum stability, SCORPION proteins were incubated in mouse serum and monitored for functional activity. Results: SCORPION molecules targeting PSMA and CD3 efficiently redirected T cell cytotoxicity against PSMA(+) cell lines in vitro at low picomolar concentrations. Cytotoxic activity was target dependent and required the presence of T cells. T cells were activated and proliferated in response to the SCORPION therapeutic in the presence of PSMA(+) target cells; no proliferation was observed in response to PSMA(−) cells. SCORPION proteins were stable to freeze/thaw in mouse serum and showed stable functional activity in mouse serum when incubated at 37oC for up to one week. Conclusions: These studies show that anti-PSMA× anti-CD3 SCORPION therapeutics may be efficient agents for redirecting T cell cytotoxicity and show promise as potential therapeutics in CRPC.

Antibody Targeting of Cell-Bound MUC1 SEA Domain Kills Tumor Cells

The cell-surface glycoprotein MUC1 is a particularly appealing target for antibody targeting, being selectively overexpressed in many types of cancers and a high proportion of cancer stem-like cells. However the occurrence of MUC1 cleavage, which leads to the release of the extracellular α subunit into the circulation where it can sequester many anti-MUC1 antibodies, renders the target problematic to some degree. To address this issue, we generated a set of unique MUC1 monoclonal antibodies that target a region termed the SEA domain that remains tethered to the cell surface after MUC1 cleavage. In breast cancer cell populations, these antibodies bound the cancer cells with high picomolar affinity. Starting with a partially humanized antibody, DMB5F3, we created a recombinant chimeric antibody that bound a panel of MUC1+ cancer cells with higher affinities relative to cetuximab (anti-EGFR1) or tratuzumab (anti-erbB2) control antibodies. DMB5F3 internalization from the cell surface occurred in an efficient temperature-dependent manner. Linkage to toxin rendered these DMB5F3 antibodies to be cytotoxic against MUC1+ cancer cells at low picomolar concentrations. Our findings show that high-affinity antibodies to cell-bound MUC1 SEA domain exert specific cytotoxicity against cancer cells, and they point to the SEA domain as a potential immunogen to generate MUC1 vaccines.

The novel tribody [(CD20)2xCD16] efficiently triggers effector cell-mediated lysis of malignant B cells

Bispecific antibodies (bsab) offer a promising approach for optimizing antibody-based therapies. In the present study, [(CD20)(2)xCD16], a recombinant CD20- and CD16-directed bsab in the tribody format, was designed to optimize recruitment of FcγRIII (CD16)-positive effector cells. [(CD20)(2)xCD16] retained the antigen specificities of the parental monoclonal antibodies and binding to FcγRIIIa was not compromised by the F/V polymorphism at amino-acid position 158. [(CD20)(2)xCD16] mediated potent lysis of lymphoma cell lines and freshly isolated tumor cells from patients, even at low picomolar concentrations (∼10 pM). Irrespective of the CD16a allotype, potency as well as efficacy of lysis obtained with the tribody was significantly higher than lysis triggered by rituximab. Tumor cell killing also occurred when autologous NK cells were used as effector cells. Compared with rituximab, the tribody demonstrated depletion of autologous B cells in ex vivo whole blood assays at 100-fold lower antibody concentration. In mice with a reconstituted humanized hematopoietic system, established by transplantation of human CD34-positive cord blood cells, this novel tribody significantly depleted autologous human B cells. Thus, tribodies such as [(CD20)(2)xCD16], recruiting CD16-positive effector cells, may represent promising candidates for clinical development.

Abstract 4622: A novel CD33/CD3-bispecific BiTE antibody can effectively recruit autologous T cells from AML-patients for in vitro cell lysis of CD33+ blasts

Abstract Bispecific T cell-engaging (BiTE®) antibodies are single-chain antibody constructs which combine dual specifity against CD3 on T cells and against a tumor-associated antigen. Phase 1 and 2 clinical trials with the CD19/CD3-bispecific BiTE antibody blinatumomab have shown very high response rates in patients suffering from non-Hodgkin's lymphoma and acute lymphoblastic leukemia. Here, we report on the potential of a novel BiTE antibody targeting CD33 in redirecting autologous T cells for in vitro lysis of blasts from acute myeloid leukemia (AML) patients. Initial antibody titration experiments showed that CD33 BiTE concentrations at low picomolar ranges mediate effective lysis of leukemic cell lines. We next analysed primary samples from AML patients, which were co-cultured with mononuclear cells (MNC) from healthy donors in presence or absence of specific BiTE antibodies at an E:T ratio of 1:1. After 48h of incubation in the presence of 1ng/ml CD33 BiTE we observed a decrease of both CD33+ AML blasts as well as CD33+ monocytes when compared to samples with control BiTE or without BiTE. 1ng/ml CD33 BiTE specifically induced strong upregulation of activation markers CD25 and CD69 on the vast majority of all CD4+ and CD8+ T cells. We furthermore investigated whether T cells from AML patients were able to mediate lysis of CD33+ leukemia cells by CD33 BiTE. T cells purified from peripheral blood or bone marrow of newly diagnosed AML patients were tested for BiTE mediated lysis of U-937 target cells. Redirected T cells from AML patients were capable of eliminating leukemic cells in the presence of CD33 BiTE as effectively as T cells from healthy controls. In consequence of this we studied blast lysis and T cell behaviour in a completely autologous setting using cryo-conserved samples from AML patients. Despite strong activation of T cells only moderate lysis of myeloid cells was observed after 48h, possibly due to low E:T ratios in these samples. Therefore, to allow autologous T cell expansion, cells were cultured for up to 144h. After this prolonged incubation, CD33 BiTE-mediated redirected lysis of AML blasts and myeloid cells was observed in most but those patient samples that had a very low initial effector-to-target (E:T) cell ratio. To adress this problem, we stimulated autologous T cells in a first step by incubating AML samples in presence of 1ng/ml CD33 BiTE for 7 days. After purification of the expanded T cells we performed autologous cultures with high initial E:T ratio (1:1). This led to fast, effective and almost complete lysis of CD33+ target cells. Taken together, autologous T cells from AML patients are effectively engaged and activated by the novel CD33 BiTE and redirected for the elimination of myeloblasts at low picomolar concentrations in vitro and may thereby constitute a promising novel therapeutic option for the treatment of patients with CD33+ acute myeloid leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4622. doi:1538-7445.AM2012-4622

Abstract 5699: Mesenchymal stem cells (MSCs) as a selective delivery vehicle for a PSA-activated protoxin for advanced prostate cancer

Abstract Circulating bone marrow-derived Mesenchymal Stem Cells (MSCs) can differentiate into cells of the mesoderm lineage and have an innate tropism for tumor tissue in response to the inflammatory microenvironment present in malignant lesions. MSCs have been detected in the perivascular space of many tumors, including prostate. MSCs are inherently non-immunogenic, which prevents allogeneic MSCs from being rejected by host defense mechanisms. This immune-privileged status, together with their oncotropic properties, makes possible the infusion of allogeneic MSCs into patients for therapeutic purposes. PRX302 is a PSA-activated aerolysin-based protoxin that forms membrane pores and leads to necrosis by a proliferation-independent mechanism at low picomolar concentrations. Active PSA is bound to inhibitors, such as A2M, in the plasma, and therefore, is only present in an active form at sites of local and metastatic prostate cancer. Consequently, PRX302 will only be converted to a toxic form in the tumor microenvironment and thereby, limit off-target toxicity. Based upon this rationale we hypothesize that MSCs can be used as a cell-based targeting vehicle to selectively deliver therapeutic agents, such as PRX302, to primary and metastatic sites of prostate cancer, and thus spare host toxicity. We have demonstrated that fluorescently-labeled MSCs “home” to sites of prostate cancer in nude mice bearing castration-resistant CWR-22RH xenografts and a significant fraction are localized to the perivascular space. This co-localization with the tumor vasculature suggests that a MSC-delivered cytotoxin would have profound anti-tumor efficacy through effects on both cancer and endothelial cells. Furthermore, we have confirmed that MSCs and stromal cells derived from human prostate cancers (PrCSCs) in men older than 50 (>10 tested), but not those isolated from normal prostates in men under 30 (>7 tested), can differentiate into adipocytes, osteoblasts, and chondrocytes under the appropriate conditions. Furthermore, these PrCSCs are positive for CD90, CD73, CD105, and FAP, but do not express CD34, CD45, CD11b, CD19, or HLA-DR, which is consistent with the hMSC phenotype and suggests that MSCs migrate to cancerous prostates in the human as well. Importantly, PRX302 binds with low nanomolar affinity to GPI-anchor proteins, which are highly expressed on the surface of all mammalian cells. Using PIG-A-targeted zinc finger nucleases (ZFNs) to knockout GPI-anchor synthesis, we have demonstrated that GPI-anchor-deficient MSCs retain their tumor homing properties. Therefore, MSCs can be genetically modified to endogenously express PRX302 from the PIG-A locus as a ‘safe harbor’ and prevent self-sterilization due to protoxin secretion. The therapeutic efficacy and host toxicity of these PRX302-expressing MSCs will be evaluated against a series of human prostate cancer xenograft models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5699. doi:1538-7445.AM2012-5699

Abstract B21: Mesenchymal stem cells (MSCs) as a selective delivery vehicle for a PSA-activated protoxin for advanced prostate cancer

Abstract Circulating bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into cells of the mesoderm lineage and have an innate tropism for tumor tissue in response to the inflammatory microenvironment present in malignant lesions. We have demonstrated that endogenous MSCs migrate to sites of prostate cancer with many of these cells located in the perivascular space similar to observations in multiple other tumor types. MSCs are inherently non-immunogenic, which prevents rejection of allogeneic MSCs by host defense mechanisms. This immune-privileged status, together with their oncotropic properties, makes possible the infusion of allogeneic MSCs into patients for therapeutic purposes. Currently, MSCs are being tested in numerous FDA-approved clinical trials for a variety of human diseases and have been safely administered to thousands of patients. PRX302 is a PSA-activated aerolysin-based protoxin that forms membrane pores and leads to necrosis by a proliferationindependent mechanism at low picomolar concentrations. Active PSA is bound to inhibitors in the plasma, and therefore, is only active at sites of local and metastatic prostate cancer. Consequently, PRX302 will only be converted to a toxic form in the tumor microenvironment and thereby, limit off-target toxicity. Based upon this rationale we hypothesize that allogeneic MSCs can be used as a cell-based targeting vehicle to selectively deliver therapeutic agents, such as PRX302, to sites of prostate cancer, and thus spare host toxicity. To achieve optimal delivery of therapeutic concentrations, we are genetically engineering MSCs to endogenously express and secrete the protoxin from a defined genomic locus, or “safe harbor”, using zinc finger nucleases (ZFNs). ZFNs are hybrid proteins generated by fusing the sequence-specific DNA-binding domain of zinc-finger proteins to the non-specific endonuclease domain of the Fok1 restriction enzyme. The nuclease is fused to either the sense or antisense sequence-specific DNA-binding domain to create an obligate heterodimer at the safe harbor gene, which results in a double strand break. When combined with a third vector containing a gene of interest (PRX302) flanked by linkers encoding a complementary sequence to the “safe harbor” gene, homologous recombination occurs. Importantly, PRX302 binds with low nanomolar affinity to GPI-anchor proteins, which are highly expressed on the surface of all mammalian cells. PIG-A is the first enzyme in GPI-anchor biosynthesis. Using PIGA-targeted ZFNs, we have demonstrated that GPI-anchor-deficient MSCs retain their tumor homing properties. Therefore, MSCs can be genetically modified to endogenously express PRX302 from the PIG-A locus as a “safe harbor” and prevent self-sterilization due to protoxin secretion. The therapeutic efficacy and host toxicity of these PRX302-expressing MSCs are initially being evaluated in preclinical proof-of-principle studies against a series of human prostate cancer xenograft models. Translation of gene therapy strategies into the clinic has been slow due to legitimate concerns surrounding their safety, largely as a result of random vector insertion, which can be alleviated by the targeted insertion of genes into “safe harbors” using ZFNs. Additionally, clinical translation of this approach will be further expedited due to the fact that PRX302 has already been given safely to more than 120 patients and has undergone extensive toxicology profiling during its clinical development as a local therapy for symptomatic BPH, for which it is currently entering phase III trials. Therefore, we propose that a systemic delivery platform for PRX302 can be created through the use of allogeneic MSCs as a vehicle for the selective delivery of PRX302 to sites of local and metastatic prostate cancer. Citation Format: W. Nathaniel Brennen, Samuel R. Denmeade, John T. Isaacs. Mesenchymal stem cells (MSCs) as a selective delivery vehicle for a PSA-activated protoxin for advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B21.

Stability of YREE complexes with the trihydroxamate siderophore desferrioxamine B at seawater ionic strength

Organic complexation of yttrium and the rare earth elements (YREEs), although generally believed to be important, is an understudied aspect of YREE solution speciation in the open ocean. We report the first series of stability constants for complexes of YREEs (except Ce and Pm) with the trihydroxamate siderophore desferrioxamine B (DFOB), representing a class of small organic ligands that have an extraordinary selectivity for Fe(III) and are found in surface seawater at low-picomolar concentrations. Constants were measured by potentiometric titration of DFOB (pH 3–10) in the presence of single YREEs, in simple media at seawater ionic strength (NaClO 4 or NaCl, I = 0.7 M). Under these circumstances, the terminal amine of DFOB does not deprotonate. The four acid dissociation constants of the siderophore were determined separately by potentiometric titration of DFOB alone. Values for the bidentate (log β 1), tetradentate (log β 2), and hexadentate (log β 3) complexes of La–Lu range from 4.88 to 6.53, 7.70 to 11.27, and 10.09 to 15.19, respectively, while Y falls between Gd and Tb in each case. Linear free-energy relations of the three stability constants with the first YREE hydrolysis constant, log β 1 ∗ , yield regression coefficients of >0.97. On the other hand, plots of the constants vs. the radius of the inner hydration sphere display an increasing deviation from linearity for the lightest REEs (La > Pr > Nd). This may signify steric constraints in DFOB folding around bulkier cations, a larger mismatch in coordination number, or a substantial degree of covalence in the YREE–hydroxamate bond. Complexes of the YREEs with DFOB are many orders of magnitude more stable than those with carbonate, the dominant inorganic YREE ligand in seawater. Speciation modeling with MINEQL indicates that, for an average seawater composition, the hexadentate complex could constitute as much as 28% of dissolved Lu at free DFOB concentrations as low as 10 −13 M. Such conditions might occur when DFOB or other siderophores are present in excess over metals for which they have high affinity, like Fe(III) and Co(III), for example during plankton blooms. Even if it turns out that trihydroxamate siderophores are not the dominant organic YREE ligand in seawater, our results establish a benchmark for producing effects on YREE solution speciation comparable to that of DFOB: the free concentration of any weaker organic ligand L must exceed that of DFOB by a factor β 3/ L β 1, assuming its first order complex is formed in greatest abundance.

HER2-affitoxin: a potent therapeutic agent for the treatment of HER2-overexpressing tumors.

Cancers overexpressing the HER2/neu gene are usually more aggressive and are associated with poor prognosis. Although trastuzumab has significantly improved the outcome, many tumors do not respond or acquire resistance to current therapies. To provide an alternative HER2-targeted therapy, we have developed and characterized a novel recombinant protein combining an HER2-specific Affibody and modified Pseudomonas aeruginosa exotoxin A (PE 38), which, after binding to HER2, is internalized and delivered to the cytosol of the tumor cell, where it blocks protein synthesis by ADP ribosylation of eEF-2. The effect of the Affitoxin on cell viability was assessed using CellTiter-Glo (Promega). To assess HER2-specific efficacy, athymic nude mice bearing BT-474 breast cancer, SK-OV-3 ovarian cancer, and NCI-N87 gastric carcinoma xenografts were treated with the Affitoxin (HER2- or Tag-specific), which was injected every third day. Affitoxin immunogenicity in female BALB/c mice was investigated using standard antibody production and splenocyte proliferation assays. In vitro experiments proved that HER2-Affitoxin is a potent agent that eliminates HER2-overexpressing cells at low picomolar concentrations. Therapeutic efficacy studies showed complete eradication of relatively large BT-474 tumors and significant effects on SK-OV-3 and NCI-N87 tumors. HER2-Affitoxin cleared quickly from circulation (T(1/2) < 10 minutes) and was well tolerated by mice at doses of 0.5 mg/kg and below. Immunogenicity studies indicated that HER2-Affitoxin induced antibody development after the third injected dose. Our findings showed that HER2-Affitoxin is an effective anticancer agent and a potential candidate for clinical studies.

Quantification of a Male Sea Lamprey Pheromone in Tributaries of Laurentian Great Lakes by Liquid Chromatography–Tandem Mass Spectrometry

We developed an assay for measuring 7α,12α,24-trihydroxy-5a-cholan-3-one-24-sulfate (3kPZS), a mating pheromone released by male sea lampreys (Petromyzon marinus), at low picomolar concentrations in natural waters to assess the presence of invasive populations. 3kPZS was extracted from streamwater at a rate of recovery up to 90% using a single cation-exchange and reversed-phase mixed-mode cartridge, along with [(2)H(5)]3kPZS as an internal standard, and quantified using ultrahigh performance liquid chromatography-tandem mass spectrometry. The limit of detection was below 0.1 ng L(-1) (210 fM), which was the lowest concentration tested. Intra- and interday coefficients of variation were between 0.3-11.6% and 4.8-9.8%, respectively, at 1 ng 3kPZS L(-1) and 5 ng 3kPZS L(-1). This assay was validated by repeat measurements of water samples from a stream spiked with synthesized 3kPZS to reach 4.74 ng L(-1) or 0.24 ng L(-1). We further verified the utility of this assay to detect spawning populations of lampreys; in the seven tributaries to the Laurentian Great Lakes sampled, 3kPZS concentrations were found to range between 0.15 and 2.85 ng L(-1) during the spawning season in known sea lamprey infested segments and were not detectable in uninfested segments. The 3kPZS assay may be useful for the integrated management of sea lamprey, an invasive species in the Great Lakes where pheromone-based control and assessment techniques are desired.

Nongenomic Response to Aldosterone

To the Editor: It is with great interest that we read the article titled, “GPR30 Expression Is Required for the Mineralocorticoid Receptor–Independent Rapid Vascular Effects of Aldosterone,” by Gros et al,1 showing that aldosterone at low picomolar concentrations can act rapidly via both GPR30 and classic mineralocorticoid receptors over a range of parameters. These included extracellular signal-regulated kinase 1/2 activation and myosin light chain phosphorylation in rat aortic vascular smooth muscle cells in vitro; for extracellular signal-regulated kinase activation, aldosterone had equivalent action via both receptors at low picomolar concentrations, whereas for myosin light chain phosphorylation, 10 nmol/L …

Fractal-like Silver Aggregates Enhance the Brightness and Stability of Single-Molecule Fluorescence

This paper describes a method to significantly enhance single-molecule fluorescence detection in confocal microscopy, demonstrating that fractal-like silver structures significantly improve dye stability and brightness. The experiments compared two immunoassay models based on the deposition of rabbit IgG on silver structures. The experiments were performed with the fluorophore-labeled protein at low picomolar concentrations. Well-separated bright spots were still easily distinguishable. Under standardized conditions we observed increased photostability and brightness enhancement for the dyes that were immobilized on the surface using a primary antibody. In contrast, when the unlabeled primary antibody was immobilized on the surface and the labeled secondary antibody was placed at a larger distance, we observed only a modest enhancement of fluorescence. Furthermore, based on backscattered reflected light images, it was proven that the observed fluorescence enhancements originate from the areas with deposited silver nanostructures. Fractal-like substrates are relatively easy to prepare. We believe that with their superior performance, they should find wide applications in single-molecule studies in which a longer observation time is required.

GPR30, Mineralocorticoid Receptors, and the Rapid Vascular Effects of Aldosterone

See related article, pp 442–451 It is now generally accepted that in common with other steroid hormones aldosterone has rapid nongenomic effects, in addition to those mediated via DNA-directed, RNA-mediated protein synthesis. The currently accepted physiology of aldosterone was primarily charted by nephrologists, who understandably focused on the epithelial (and genomic) effects of aldosterone on urinary electrolytes, and the homeostatic changes in aldosterone secretion in response to sodium deficiency or potassium loading. The most acute physiological stimulus to aldosterone secretion, however, is assumption of the upright posture. Given this, it is not inappropriate to seek responses, similarly acute, to this rapid change in plasma aldosterone levels. In the paper by Gros et al,1 the authors conclusively show that aldosterone at low picomolar concentrations can act rapidly via both GPR30 (originally an “orphan” G protein–coupled receptor, subsequently an erstwhile membrane estrogen receptor) and classic mineralocorticoid receptors (MR) over a range of parameters. These include extracellular signal-regulated kinase (ERK) 1/2 activation and myosin light chain phosphorylation in rat aortic vascular smooth muscle cells (VSMC) in vitro; for ERK activation, aldosterone has equivalent action via both receptors at low picomolar concentrations. The effects on GPR30 appear mineralocorticoid specific: in rat aortic endothelial cells, with MR expression below detection levels by Western blotting, aldosterone increased ERK activation with an EC50 < 10 pM, an action abrogated by the GPR30 antagonist G15. In contrast, the effect of estradiol on inhibition of ERK activation was unaffected by G15 but blocked by the estrogen receptor α (ERα) antagonist ICI-182780. The first point to be made is that these studies firmly establish GPR30 as a bona fide receptor for aldosterone, given the low picomolar concentrations used (except for myosin light chain phosphorylation, where inexplicably 10 nmol/L aldosterone was used, and Figures 8 and 9 point in …