Tumor-Localized Costimulatory T-Cell Engagement by the 4-1BB/HER2 Bispecific Antibody-Anticalin Fusion PRS-343.

PURPOSE: 4-1BB (CD137) is a key costimulatory immunoreceptor and promising therapeutic target in cancer. To overcome limitations of current 4-1BB-targeting antibodies, we have developed PRS-343, a 4-1BB/HER2 bispecific molecule. PRS-343 is designed to facilitate T-cell costimulation by tumor-localized, HER2-dependent 4-1BB clustering and activation. EXPERIMENTAL DESIGN: PRS-343 was generated by the genetic fusion of 4-1BB-specific Anticalin proteins to a variant of trastuzumab with an engineered IgG4 isotype. Its activity was characterized using a panel of in vitro assays and humanized mouse models. The safety was assessed using ex vivo human cell assays and a toxicity study in cynomolgus monkeys. RESULTS: PRS-343 targets 4-1BB and HER2 with high affinity and binds both targets simultaneously. 4-1BB-expressing T cells are efficiently costimulated when incubated with PRS-343 in the presence of cancer cells expressing HER2, as evidenced by increased production of proinflammatory cytokines (IL2, GM-CSF, TNFα, and IFNγ). In a humanized mouse model engrafted with HER2-positive SK-OV-3 tumor cells and human peripheral blood mononuclear cells, PRS-343 leads to tumor growth inhibition and a dose-dependent increase of tumor-infiltrating lymphocytes. In IND-enabling studies, PRS-343 was found to be well tolerated, with no overt toxicity and no relevant drug-related toxicologic findings. CONCLUSIONS: PRS-343 facilitates tumor-localized targeting of T cells by bispecific engagement of HER2 and 4-1BB. This approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity compared with current monospecific approaches. The reported data led to initiation of a phase I clinical trial with this first-in-class molecule.See related commentary by Su et al., p. 5732.

Getting across the cell membrane: an overview for small molecules, peptides, and proteins.

The ability to efficiently access cytosolic proteins is desired in both biological research and medicine. However, targeting intracellular proteins is often challenging, because to reach the cytosol, exogenous molecules must first traverse the cell membrane. This review provides a broad overview of how certain molecules are thought to cross this barrier, and what kinds of approaches are being made to enhance the intracellular delivery of those that are impermeable. We first discuss rules that govern the passive permeability of small molecules across the lipid membrane, and mechanisms of membrane transport that have evolved in nature for certain metabolites, peptides, and proteins. Then, we introduce design strategies that have emerged in the development of small molecules and peptides with improved permeability. Finally, intracellular delivery systems that have been engineered for protein payloads are surveyed. Viewpoints from varying disciplines have been brought together to provide a cohesive overview of how the membrane barrier is being overcome.

In vivo visualization of MET tumor expression and anticalin biodistribution with the MET-specific anticalin 89Zr-PRS-110 PET tracer.

UNLABELLED: Anticalins are a novel class of biopharmaceuticals, displaying highly desirable attributes as imaging agents. The anticalin PRS-110 was rationally engineered to target the oncogene MET with high affinity and specificity. The aim of this study was to visualize MET expression and analyze biodistribution of (89)Zr-labeled PRS-110 in human tumor-bearing mice. METHODS: (89)Zr-PRS-110 was generated. For biodistribution studies (96 h after injection of tracer) 10 µg of (89)Zr-PRS-110 (with 0-490 µg of unlabeled PRS-110) were injected into BALB/c mice bearing high MET-expressing H441 non-small cell lung cancer xenografts. Further characterization with PET imaging was performed at 6, 24, 48, and 96 h after injection of 50 µg of (89)Zr-PRS-110 into mice bearing H441, primary glioblastoma U87-MG (intermediate MET), or ovarian cancer A2780 (low MET) xenografts. Drug distribution was also analyzed ex vivo using fluorescently labeled PRS-110. RESULTS: Biodistribution analyses showed a dose-dependent tumor uptake of (89)Zr-PRS-110, with the highest fractional tumor uptake at 10 µg of (89)Zr-PRS-110, with no unlabeled PRS-110. Small-animal PET imaging supported by biodistribution data revealed specific tumor uptake of (89)Zr-PRS-110 in the MET-expressing H441 and U87-MG tumors whereas the MET-negative A2780 tumor model showed a lower uptake similar to a non-MET binder anticalin control. Tumor uptake increased up to 24 h after tracer injection and remained high, whereas uptake in other organs decreased over time. Ex vivo fluorescence revealed intracellular presence of PRS-110. CONCLUSION: (89)Zr-PRS-110 specifically accumulates in MET-expressing tumors in a receptor density-dependent manner. PET imaging provides real-time noninvasive information about PRS-110 distribution and tumor accumulation in preclinical models.

How to obtain labeled proteins and what to do with them.

We review new and established methods for the chemical modification of proteins in living cells and highlight recent applications. The review focuses on tag-mediated protein labeling methods, such as the tetracysteine tag and SNAP-tag, and new developments in this field such as intracellular labeling with lipoic acid ligase. Recent promising advances in the incorporation of unnatural amino acids into proteins are also briefly discussed. We describe new tools using tag-mediated labeling methods including the super-resolution microscopy of tagged proteins, the study of the interactions of proteins and protein domains, the subcellular targeting of synthetic ion sensors, and the generation of new semisynthetic metabolite sensors. We conclude with a view on necessary future developments, with one example being the selective labeling of non-tagged, native proteins in complex protein mixtures.

Benzothiazinones: prodrugs that covalently modify the decaprenylphosphoryl-ß-D-ribose 2'-epimerase DprE1 of Mycobacterium tuberculosis.

Benzothiazinones (BTZs) form a new class of potent antimycobacterial agents. Although the target of BTZs has been identified as decaprenylphosphoryl-ß-D-ribose 2'-epimerase (DprE1), their detailed mechanism of action remains obscure. Here we demonstrate that BTZs are activated in the bacterium by reduction of an essential nitro group to a nitroso derivative, which then specifically reacts with a cysteine residue in the active site of DprE1.

Location, tilt, and binding: a molecular dynamics study of voltage-sensitive dyes in biomembranes.

We present a molecular dynamics study on the interaction of styryl-type voltage-sensitive dyes with a lipid membrane. In this work, voltage-sensitive dyes are proposed as interesting model amphiphiles for biomolecular simulation, due to the wealth of biophysical and thermodynamical data available on their behavior and their binding to lipid membranes. Taking this data as a basis, we tested the recently developed MARTINI coarse-grained model (J. Phys. Chem. B 2007, 111, 7812). The focus was on the fast computation of the free energy of membrane binding. As a first step, we investigated the tilt and location of a coarse-grained representation of the dye Di-4-ASPBS in a lipid membrane, and found good agreement with atomistic simulations and experimental data. Then, we performed umbrella sampling to obtain the theoretical binding free energy for a number of Di-4-ASPBS derivates. In most cases, simulation and experimental binding data were in good agreement regarding the impact of structural changes in the amphiphile on binding. The work yields a general molecular picture of how such structural variations lead to changes of the binding mode and binding strength of amphiphiles to lipid membranes. Further, it provides insights into the possibilities and current limitations of rapid free energy computation for membrane binding with the coarse-grained MARTINI model. The results suggest that the MARTINI model may be a generally useful tool for the study and optimization of molecules interacting with membranes, such as biophysical probes or pharmaceutical compounds.

Molecular view of cholesterol flip-flop and chemical potential in different membrane environments.

The relative stability of cholesterol in cellular membranes and the thermodynamics of fluctuations from equilibrium have important consequences for sterol trafficking and lateral domain formation. We used molecular dynamics computer simulations to investigate the partitioning of cholesterol in a systematic set of lipid bilayers. In addition to atomistic simulations, we undertook a large set of coarse grained simulations, which allowed longer time and length scales to be sampled. Our results agree with recent experiments (Steck, T. L.; et al. Biophys. J. 2002, 83, 2118-2125) that the rate of cholesterol flip-flop can be fast on physiological time scales, while extending our understanding of this process to a range of lipids. We predicted that the rate of flip-flop is strongly dependent on the composition of the bilayer. In polyunsaturated bilayers, cholesterol undergoes flip-flop on a submicrosecond time scale, while flip-flop occurs in the second range in saturated bilayers with high cholesterol content. We also calculated the free energy of cholesterol desorption, which can be equated to the excess chemical potential of cholesterol in the bilayer compared to water. The free energy of cholesterol desorption from a DPPC bilayer is 80 kJ/mol, compared to 67 kJ/mol for a DAPC bilayer. In general, cholesterol prefers more ordered and rigid bilayers and has the lowest affinity for bilayers with two polyunsaturated chains. Overall, the simulations provide a detailed molecular level thermodynamic description of cholesterol interactions with lipid bilayers, of fundamental importance to eukaryotic life.

Semisynthetic fluorescent sensor proteins based on self-labeling protein tags.

Genetically encoded fluorescent sensor proteins offer the possibility to probe the concentration of key metabolites in living cells. The approaches currently used to generate such fluorescent sensor proteins lack generality, as they require a protein that undergoes a conformational change upon metabolite binding. Here we present an approach that overcomes this limitation. Our biosensors consist of SNAP-tag, a fluorescent protein and a metabolite-binding protein. SNAP-tag is specifically labeled with a synthetic molecule containing a ligand of the metabolite-binding protein and a fluorophore. In the labeled sensor, the metabolite of interest displaces the intramolecular ligand from the binding protein, thereby shifting the sensor protein from a closed to an open conformation. The readout is a concomitant ratiometric change in the fluorescence intensities of the fluorescent protein and the tethered fluorophore. The observed ratiometric changes compare favorably with those achieved in genetically encoded fluorescent sensor proteins. Furthermore, the modular design of our sensors permits the facile generation of ratiometric fluorescent sensors at wavelengths not covered by autofluorescent proteins. These features should allow semisynthetic fluorescent sensor proteins based on SNAP-tag to become important tools for probing previously inaccessible metabolites.

ANNINE-6plus, a voltage-sensitive dye with good solubility, strong membrane binding and high sensitivity.

We present a novel voltage-sensitive hemicyanine dye ANNINE-6plus and describe its synthesis, its properties and its voltage-sensitivity in neurons. The dye ANNINE-6plus is a salt with a double positively charged chromophore and two bromide counterions. It is derived from the zwitterionic dye ANNINE-6. While ANNINE-6 is insoluble in water, ANNINE-6plus exhibits a high solubility of around 1 mM. Nonetheless, it displays a strong binding to lipid membranes. In contrast to ANNINE-6, the novel dye can be used to stain cells from aqueous solution without surfactants or organic solvents. In neuronal membranes, ANNINE-6plus exhibits the same molecular Stark effect as ANNINE-6. As a consequence, a high voltage-sensitivity is achieved with illumination and detection in the red end of the excitation and emission spectra, respectively. ANNINE-6plus will be particularly useful for sensitive optical recording of neuronal excitation when organic solvents and surfactants must be avoided as with intracellular or extracellular staining of brain tissue.

Genetic targeting of individual cells with a voltage-sensitive dye through enzymatic activation of membrane binding.

Optical recording of the electrical activity of individual neurons in culture or in a tissue requires cell-selective staining with a fluorescent voltage-sensitive dye. In a proof-of-principle experiment, we implement a novel approach to genetically targeted staining. The method relies on a water-soluble precursor dye and an overexpressed cell-surface enzyme that transforms the precursor into a hydrophobic dye that binds to the targeted cell. We fused an alkaline phosphatase to a specifically designed general-purpose membrane anchor, and the fusion protein was expressed on the surface of HEK293 cells, as was corroborated by immuno- and histochemical staining. We next synthesised an amphiphilic hemicyanine dye containing two enzymatically cleavable phosphate groups at its hydrocarbon tails. When the phosphate groups were removed, the binding to membranes was enhanced by a factor of a thousand, as shown by titration with lipid vesicles. We observed selective staining of enzymatically active cells by fluorescence microscopy in a mixed population of phosphatase-transfected and untransfected HEK293 cells. The critical parameters of enzyme-induced cell-selective staining were elucidated by a simple kinetic model to guide further developments of the method.

Enzyme-induced staining of biomembranes with voltage-sensitive fluorescent dyes.

We consider the physicochemical basis for enzyme-induced staining of cell membranes by fluorescent voltage-sensitive dyes, a method that may lead to selective labeling of genetically encoded nerve cells in brain for studies of neuronal signal processing. The approach relies on the induction of membrane binding by enzymatic conversion of a water-soluble precursor dye. We synthesized an amphiphilic hemicyanine dye with and without an additional phosphate appendix at its polar headgroup. The fluorescence of these dyes is negligible in water but high when bound to lipid membranes. By fluorescence titration with lipid vesicles it was shown that the phosphate group lowers the partition coefficient from water to membrane by more than an order of magnitude. By isothermal titration calorimetry, we showed that the dye phosphate was a substrate for a water-soluble alkaline phosphatase following MichaelisMenten kinetics. In a suspension of lipid vesicles, the enzyme reaction led to a fluorescence increase due to enhanced membrane binding of the product dye in accord with the MichaelisMenten kinetics of the reaction and the partition coefficients of substrate and product. We successfully tested the staining method by fluorescence microscopy with individual giant lipid vesicles and with individual red blood cells. In both systems, the membrane fluorescence due to bound hemicyanine was enhanced by an order of magnitude, proving the feasibility of enzyme-induced staining with voltage-sensitive dyes.