The Fc-alpha receptor is a new target antigen for immunotherapy of myeloid leukemia.

Antibody-based immunotherapy of leukemia requires the targeting of specific antigens on the surface of blasts. The Fc gamma receptor (CD64) has been investigated in detail, and CD64-targeting immunotherapy has shown promising efficacy in the targeted ablation of acute myeloid leukemia (AML), acute myelomonocytic leukemia (AMML) and chronic myeloid leukemia cells (CML). Here we investigate for the first time the potential of FcαRI (CD89) as a new target antigen expressed by different myeloid leukemic cell populations. For specific targeting and killing, we generated a recombinant fusion protein comprising an anti-human CD89 single-chain Fragment variable and the well-characterized truncated version of the potent Pseudomonas aeruginosa exotoxin A (ETA'). Our novel therapeutic approach achieved in vitro EC50 values in range 0.2-3 nM depending on the applied stimuli, that is, interferon gamma or tumor necrosis factor alpha. We also observed a dose-dependent apoptosis-mediated cytotoxicity, which resulted in the elimination of up to 90% of the target cells within 72 hr. These findings were also confirmed ex vivo using leukemic primary cells from peripheral blood samples of three previously untreated patients. We conclude that CD89-specific targeting of leukemia cell lines can be achieved in vitro and that the efficient elimination of leukemic primary cells supports the potential of CD89-ETA' as a potent, novel immunotherapeutic agent.

Novel angiogenin mutants with increased cytotoxicity enhance the depletion of pro-inflammatory macrophages and leukemia cells ex vivo.

Immunotoxins are fusion proteins that combine a targeting component such as an antibody fragment or ligand with a cytotoxic effector component that induces apoptosis in specific cell populations displaying the corresponding antigen or receptor. Human cytolytic fusion proteins (hCFPs) are less immunogenic than conventional immunotoxins because they contain human pro-apoptotic enzymes as effectors. However, one drawback of hCFPs is that target cells can protect themselves by expressing endogenous inhibitor proteins. Inhibitor-resistant enzyme mutants that maintain their cytotoxic activity are therefore promising effector domain candidates. We recently developed potent variants of the human ribonuclease angiogenin (Ang) that were either more active than the wild-type enzyme or less susceptible to inhibition because of their lower affinity for the ribonuclease inhibitor RNH1. However, combining the mutations was unsuccessful because although the enzyme retained its higher activity, its susceptibility to RNH1 reverted to wild-type levels. We therefore used molecular dynamic simulations to determine, at the atomic level, why the affinity for RNH1 reverted, and we developed strategies based on the introduction of further mutations to once again reduce the affinity of Ang for RNH1 while retaining its enhanced activity. We were able to generate a novel Ang variant with remarkable in vitro cytotoxicity against HL-60 cells and pro-inflammatory macrophages. We also demonstrated the pro-apoptotic potential of Ang-based hCFPs on cells freshly isolated from leukemia patients.

Inhibition of glutamate/glutamine cycle in vivo results in decreased benzodiazepine binding and differentially regulated GABAergic subunit expression in the rat brain.

PURPOSE: The astrocytic enzyme glutamine synthetase (GS) is a key regulator of glutamate and γ-aminobutyric acid (GABA) metabolism in the glutamate/glutamine cycle (GGC). Inhibition of GS results in changes of neurotransmitter release and recycling. However, little is known about the influence of GGC on neurotransmitter receptor expression. In the pentylenetetrazole model of epilepsy, GS becomes nitrated and partially inhibited, and we demonstrated alterations of neurotransmitter receptor expression in the same model. Therefore, we hypothesized similar changes of neurotransmitter receptor expression when GS is inhibited in vivo. METHODS: Rats were treated with a single dose (100 mg/kg bodyweight) of l-methionine sulfoximine (MSO), an irreversible inhibitor of GS. We used ³H-receptor autoradiography to measure glutamatergic [α-amino-3-hydroxy-5-methyl-4-isoxazol-propionic acid (AMPA), kainate, N-methyl-D-aspartate (NMDA)], GABAergic (GABA(A) , GABA(B) and GABA(A) -associated benzodiazepine (BZ) binding sites], dopamine D1, and adenosine A1 receptor subtypes. In addition, we performed saturation analysis of BZ binding sites on cerebral membrane homogenates and investigated the expression of GABA(A) α1 and γ2 subunits (which primarily mediate BZ binding) by western blot analysis. RESULTS: We demonstrated a significant reduction of BZ binding in the somatosensory, piriform, and entorhinal cortices and in the amygdala, 24 and 72 h after MSO treatment. Saturation analysis revealed decreased BZ binding (B(max)) on cerebral membrane homogenates 72 h after MSO treatment, without changes in binding site affinity (K(D)). Furthermore, we found differential changes of α1 , γ2 , and phosphorylated γ2 subunits following MSO treatment. CONCLUSION: On the basis of our findings, we conclude that the glutamate/glutamine cycle directly influences GABAergic neurotransmission by regulating GABA(A) subunit composition, thereby affecting its modulation by endogenous benzodiazepines.

Updates in the Development of ImmunoRNases for the Selective Killing of Tumor Cells.

Targeted cancer therapy includes, amongst others, antibody-based delivery of toxic payloads to selectively eliminate tumor cells. This payload can be either a synthetic small molecule drug composing an antibody-drug conjugate (ADC) or a cytotoxic protein composing an immunotoxin (IT). Non-human cytotoxic proteins, while potent, have limited clinical efficacy due to their immunogenicity and potential off-target toxicity. Humanization of the cytotoxic payload is essential and requires harnessing of potent apoptosis-inducing human proteins with conditional activity, which rely on targeted delivery to contact their substrate. Ribonucleases are attractive candidates, due to their ability to induce apoptosis by abrogating protein biosynthesis via tRNA degradation. In fact, several RNases of the pancreatic RNase A superfamily have shown potential as anti-cancer agents. Coupling of a human RNase to a humanized antibody or antibody derivative putatively eliminates the immunogenicity of an IT (now known as a human cytolytic fusion protein, hCFP). However, RNases are tightly regulated in vivo by endogenous inhibitors, controlling the ribonucleolytic balance subject to the cell's metabolic requirements. Endogenous inhibition limits the efficacy with which RNase-based hCFPs induce apoptosis. However, abrogating the natural interaction with the natural inhibitors by mutation has been shown to significantly enhance RNase activity, paving the way toward achieving cytolytic potency comparable to that of bacterial immunotoxins. Here, we review the immunoRNases that have undergone preclinical studies as anti-cancer therapeutic agents.

Engineered human angiogenin mutations in the placental ribonuclease inhibitor complex for anticancer therapy: Insights from enhanced sampling simulations.

Targeted human cytolytic fusion proteins (hCFPs) represent a new generation of immunotoxins (ITs) for the specific targeting and elimination of malignant cell populations. Unlike conventional ITs, hCFPs comprise a human/humanized target cell-specific binding moiety (e.g., an antibody or a fragment thereof) fused to a human proapoptotic protein as the cytotoxic domain (effector domain). Therefore, hCFPs are humanized ITs expected to have low immunogenicity. This reduces side effects and allows long-term application. The human ribonuclease angiogenin (Ang) has been shown to be a promising effector domain candidate. However, the application of Ang-based hCFPs is largely hampered by the intracellular placental ribonuclease inhibitor (RNH1). It rapidly binds and inactivates Ang. Mutations altering Ang's affinity for RNH1 modulate the cytotoxicity of Ang-based hCFPs. Here we perform in total 2.7 µs replica-exchange molecular dynamics simulations to investigate some of these mutations-G85R/G86R (GGRRmut ), Q117G (QGmut ), and G85R/G86R/Q117G (GGRR/QGmut ). GGRRmut turns out to perturb greatly the overall Ang-RNH1 interactions, whereas QGmut optimizes them. Combining QGmut with GGRRmut compensates the effects of the latter. Our results explain the in vitro finding that, while Ang GGRRmut -based hCFPs resist RNH1 inhibition remarkably, Ang WT- and Ang QGmut -based ones are similarly sensitive to RNH1 inhibition, whereas Ang GGRR/QGmut -based ones are only slightly resistant. This work may help design novel Ang mutants with reduced affinity for RNH1 and improved cytotoxicity.

CD64-directed microtubule associated protein tau kills leukemic blasts ex vivo.

Fc gamma receptor I (FcγRI, CD64) is a well-known target antigen for passive immunotherapy against acute myeloid leukemia and chronic myelomonocytic leukemia. We recently reported the preclinical immunotherapeutic potential of microtubule associated protein tau (MAP) against a variety of cancer types including breast carcinoma and Hodgkin's lymphoma. Here we demonstrate that the CD64-directed human cytolytic fusion protein H22(scFv)-MAP kills ex vivo 15-50% of CD64+ leukemic blasts derived from seven myeloid leukemia patients. Furthermore, in contrast to the nonspecific cytostatic agent paclitaxel, H22(scFv)-MAP showed no cytotoxicity towards healthy CD64+ PBMC-derived cells and macrophages. The targeted delivery of this microtubule stabilizing agent therefore offers a promising new strategy for specific treatment of CD64+ leukemia.

Angiogenin mutants as novel effector molecules for the generation of fusion proteins with increased cytotoxic potential.

Human cytolytic fusion proteins (hCFPs) are therapeutically efficacious recombinant polypeptides comprising a target cell-specific binding component and a human effector domain that induces apoptosis. Compared with former generations of immunotoxins, which contain immunogenic cytotoxic domains derived from bacteria or plants, hCFPs contain solely human proteins that do not induce an immune response, thus avoiding the development of neutralizing antibodies. Here, we investigated the suitability of human angiogenin (Ang) mutants as effector domains. We engineered 3 different Ang variants that outperformed the wild-type enzyme by replacing amino acid residues with key roles in the protein's catalytic activity and its interaction with the ribonuclease inhibitor RNH1. The cytotoxic potential of these mutants was compared with wild-type Ang by fusing each to the CD64-specific single-chain variable fragment H22. All hCFPs were successfully expressed in HEK293T cells and purified from the cell culture supernatant by immobilized metal ion affinity chromatography. The Ang mutant-based hCFPs showed normal binding activity towards human interferon-γ-stimulated CD64 HL-60 cells and activated human macrophages isolated from peripheral blood mononuclear cells, but increased cytotoxicity based on reduced affinity towards RNH1 and higher ribonucleolytic activity.

Laminar distribution of neurotransmitter receptors in different reeler mouse brain regions.

Mapping of multiple receptors of neurotransmitters provides insight into the spatial distribution of neurotransmission-relevant molecules in the cerebral cortex. During development, lack of reelin leads to impaired migration, disturbed lamination of the hippocampus and inverted neocortical layering. In the adult, reelin may regulate synaptic plasticity by modulating neurotransmitter receptor function. Using quantitative in vitro receptor autoradiography, different receptors, in particular, the binding site densities and laminar distribution of various glutamate, GABA, muscarinic and nicotinic acetylcholine, serotonin, dopamine and adenosine receptors, were analyzed in cortical and subcortical structures of reeler and wild-type brains. Differential changes in the laminar distribution, maximum binding capacity (B (max)) and regional density of neurotransmitter receptors were found in the reeler brain. A decrease of whole brain B (max) was found for adenosine A(1) and GABA(A) receptors. In the forebrain, several binding sites were differentially up- or down-regulated (kainate, A(1), benzodiazepine, 5-HT(1), M(2), α(1) and α(2)). In the hippocampus, a significant decrease of GABA(B), 5-HT(1) and A'1 receptors were observed. The density of M(2) receptors increased, while other receptors remained unchanged. In the neocortex, some receptors demonstrated an obviously inverted laminar distribution (AMPA, kainate, NMDA, GABA(B), 5-HT(1), M(1), M(3), nAch), while the distribution of others (A(1), GABA(A), benzodiazepine, 5-HT(2), muscarinic M(2), adrenergic α(1), α(2)) seemed to be less affected. Thus, the laminar receptor distribution is modulated by the developmental impairment and suggests and reflects partially the laminar inversion in reeler mice.

Fast, quantitative in situ hybridization of rare mRNAs using 14C-standards and phosphorus imaging.

The use of radiolabelled probes for in situ hybridization (ISH) bears the advantage of high sensitivity and quantifiability. The crucial disadvantages are laborious hybridization protocols, exposition of hybridized sections to film for up to several weeks and the time consuming need to prepare tissue standards with relatively short-lived isotopes like (33)P or (35)S for each experiment. The quantification of rare mRNAs like those encoding for subunits of neurotransmitter receptors is therefore a challenge in ISH. Here, we describe a method for fast, quantitative in situ hybridization (qISH) of mRNAs using (33)P-labelled oligonucleotides together with (14)C-polymer standards (Microscales, Amersham Biosciences) and a phosphorus imaging system (BAS 5000 BioImage Analyzer, Raytest-Fuji). It enables a complete analysis of rare mRNAs by ISH. The preparation of short-lived (33)P-standards for each experiment was replaced by co-exposition and calibration of long-lived (14)C-standards together with (33)P-labelled brain paste standards. The use of a phosphorus imaging system allowed a reduction of exposition time following hybridization from several weeks to a few hours or days. We used this approach as an example for applications to quantify the expression of GluR1 and GluR2 subunit mRNAs of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the hippocampus of untreated rats, and after intraperitoneal application of the organo-arsenic compound dimethyl arsenic acid.

Magnetic properties of cationic tungsten(IV) half sandwich compounds: experimental and theoretical study of a solvent and ligand stabilized singlet ground state leading to a thermally induced singlet-triplet spin state interconversion.

A series of novel neutral tungsten(III) and cationic tungsten(IV) complexes with disubstituted 4,4'-R,R-2,2'-bipyridyl (R(2)-bpy) ligands of the type [CpW(R(2)-bpy)Cl(2)](n+) (n = 0,1) were prepared and characterized by X-ray crystallography. Susceptibility measurements of the tungsten(IV) complexes revealed an intrinsic paramagnetism of these compounds and evidenced different magnetic properties of the dimethylamino and methyl (R = NMe(2), Me) substituted tungsten(IV) compounds in solution and in the solid state. In dichloromethane solution, singlet ground states with thermally populated triplet states were observed, whereas triplet (R = Me) and singlet ground states (R = NMe(2)) were observed in the solid state. Using both experimental and theoretical techniques (DFT) allowed to establish solvation and ligand effects to account for the different magnetic behavior. Thermodynamic parameters were derived for the spin equlibria in solution by fits of the temperature dependent (1)H NMR shifts to the Van Vleck equation and were found to be in excellent agreement with the DFT calculations.

Tuning the redox potentials of dinuclear tungsten oxo complexes [(Cp*W(4,4'-R,R-2,2'-bpy)(mu-O))2][PF6]2 toward photochemical water splitting.

A series of novel dinuclear tungsten(IV) oxo complexes with disubstituted 4,4'-R,R-2,2'-bipyridyl (R(2)bpy) ligands of the type [(Cp*W(R(2)bpy)(mu-O))(2)][PF(6)](2) (R=NMe(2), tBu, Me, H, Cl) was prepared by hydrolysis of the tungsten(IV) trichloro complexes [Cp*W(R(2)bpy)Cl(3)]. Cyclic voltammetry measurements for the tungsten(IV) oxo compounds provided evidence for one reversible oxidation and two reversible reductions leading to the oxidation states W(V)W(IV), W(IV)W(III) and W(III)W(III). The corresponding complexes [(Cp*W(R(2)bpy)(mu-O))(2)](n+) [PF(6)](n) (n=0 for R=Me, tBu, and 1, 3 for both R=Me) could be isolated after chemical oxidation/reduction of the tungsten(IV) oxo complexes. The crystal structures of the complexes [(Cp*W(R(2)bpy)(mu-O))(2)][BPh(4)](2) (R=NMe(2), tBu) and [(Cp*W(Me(2)bpy)(mu-O))(2)](n+)[PF(6)](n) (n=0, 1, 2, 3) show a cis geometry with a puckered W(2)O(2) four-membered ring for all compounds except [(Cp*W(Me(2)bpy)(mu-O))(2)] which displays a trans geometry with a planar W(2)O(2) ring. Examining the interaction of these novel tungsten oxo complexes with protons, we were able to show that the W(IV)W(IV) complexes [(Cp*W(R(2)bpy)(mu-O))(2)][PF(6) (-)](2) (R=NMe(2), tBu) undergo reversible protonation, while the W(III)W(III) complexes [(Cp*W(R(2)bpy)(mu-O))(2)] transfer two electrons forming the W(IV)W(IV) complex and molecular hydrogen.