L-Pampo™, a Novel TLR2/3 Agonist, Acts as a Potent Cancer Vaccine Adjuvant by Activating Draining Lymph Node Dendritic Cells.

TLR agonists have emerged as an efficient cancer vaccine adjuvant system that induces robust immune responses. L-pampo™, a proprietary vaccine adjuvant of TLR2 and TLR3 agonists, promotes strong humoral and cellular immune responses against infectious diseases. In this study, we demonstrate that vaccines formulated with L-pampo™ affect the recruitment and activation of dendritic cells (DCs) in draining lymph nodes (dLNs) and leading to antigen-specific T-cell responses and anti-tumor efficacy. We analyzed DC maturation and T-cell proliferation using flow cytometry and ELISA. We determined the effect of L-pampo™ on DCs in dLNs and antigen-specific T-cell responses using flow cytometric analysis and the ELISPOT assay. We employed murine tumor models and analyzed the anti-tumor effect of L-pampo™. We found that L-pampo™ directly enhanced the maturation and cytokine production of DCs and, consequently, T-cell proliferation. OVA or OVA peptide formulated with L-pampo™ promoted DC migration into dLNs and increased activation markers and specific DC subsets within dLNs. In addition, vaccines admixed with L-pampo™ promoted antigen-specific T-cell responses and anti-tumor efficacy. Moreover, the combination of L-pampo™ with an immune checkpoint inhibitor synergistically improved the anti-tumor effect. This study suggests that L-pampo™ can be a potent cancer vaccine adjuvant and a suitable candidate for combination immunotherapy.

pH-dependent structures of ferritin and apoferritin in solution: disassembly and reassembly.

The pH-dependent structures of the ferritin shell (apoferritin, 24-mer) and the ferrihydrite core, under physiological conditions that permit enzymatic activity, were investigated by synchrotron small-angle X-ray scattering (SAXS). The solution structure of apoferritin was found to be nearly identical to the crystal structure. The shell thickness and hollow core volumes were estimated. The intact hollow spherical apoferritin was stable over a wide pH range, 3.40-10.0, and the ferrihydrite core was stable over the pH range 2.10-10.0. The apoferritin subunits underwent aggregation below pH 0.80, whereas the ferrihydrite cores aggregated below pH 2.10 as a result of the disassembly of the ferritin shell under the strongly acidic conditions. As the pH decreased from 3.40 to 0.80, apoferritin underwent stepwise disassembly by first forming a hollow sphere with two holes, then a headset-shaped structure, and, finally, rodlike oligomers. As the pH was increased from pH 1.96, the disassembled rodlike oligomers recovered only to the headset-shaped structure, and the disassembled headset-shaped intermediates recovered only to the hollow spherical structure with two hole defects. The apoferritin hole defects that formed during the disassembly process did not heal as the pH was increased to neutral or slightly basic conditions. The pH-induced apoferritin disassembly and reassembly processes were not fully reversible, although they were pseudoreversible over a limited pH range, between 10.0 and 2.66.

COVID-19 Subunit Vaccine with a Combination of TLR1/2 and TLR3 Agonists Induces Robust and Protective Immunity.

The development of COVID-19 vaccines is critical in controlling global health issues under the COVID-19 pandemic. The subunit vaccines are the safest and most widely used vaccine platform and highly effective against a multitude of infectious diseases. An adjuvant is essential for subunit vaccines to enhance the magnitude and durability of immune responses. In this study, we determined whether a combination of toll-like receptor (TLR)1/2 and TLR3 agonists (L-pampo) can be a potent adjuvant for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine. We measured a neutralizing antibody (nAb) and an angiotensin-converting enzyme 2 (ACE2) receptor-blocking antibody against SARS-CoV-2 receptor-binding domain (RBD). We also detected interferon-gamma (IFN-γ) production by using ELISPOT and ELISA assays. By employing a ferret model, we detected nAbs and IFN-γ producing cells and measured viral load in nasal wash after the challenge of SARS-CoV-2. We found that SARS-CoV-2 antigens with L-pampo stimulated robust humoral and cellular immune responses. The efficacy of L-pampo was higher than the other adjuvants. Furthermore, in the ferret model, SARS-CoV-2 antigens with L-pampo elicited nAb response and antigen-specific cellular immune response against SARS-CoV-2, resulting in substantially decreased viral load in their nasal wash. Our study suggests that SARS-CoV-2 antigens formulated with TLR agonists, L-pampo, can be a potent subunit vaccine to promote sufficient protective immunity against SARS-CoV-2.

pH-dependent structures of an i-motif DNA in solution.

We have investigated for the first time the structure of i-motif DNA in solution at various pH conditions by using synchrotron small-angle X-ray scattering technique. To facilitate direct structural comparison between solution structures of i-motif DNA at various pH values, we created atomic coordinates of i-motif DNA from a fully folded to unfolded atomic model. Under mild acidic conditions, the conformations for i-motif DNA appeared to be similar to that of the partially unfolded i-motif atomic model in overall shape, rather than the fully folded i-motif atomic model. Collectively, our observations indicate that i-motif DNA molecule is structurally dynamic over a wide pH range, adopting multiple conformations ranging from the folded i-motif structure to a random coil conformation. As the i-motif structure has been used as an important component in nanomachines, we can therefore believe that the structural evidence presented herein will promote the development of future DNA-based molecular-actuator devices.

Order-order and order-disorder transitions in thin films of an amphiphilic liquid crystalline diblock copolymer.

In this study, we quantitatively investigated the temperature-dependent phase transition behaviors of thin films of an interesting amphiphilic diblock copolymer, poly(ethylene oxide)-b-poly(11-[4-(4-butylphenylazo)phenoxy]undecyl methacrylate) (p(EO)-b-p(MAAZ)) and the resulting morphological structures by using synchrotron grazing incidence X-ray scattering (GIXS) and differential scanning calorimetry. The quantitative GIXS analysis showed that the diblock copolymer in the homogeneous, isotropic melt state undergoes phase-separation near 190 degrees C and then forms a body-centered cubic (BCC) structure of spherical p(EO) domains in the p(MAAZ) matrix, at which point the p(EO) domains and the p(MAAZ) matrix are both in amorphous, liquid states. The BCC structure of spherical p(EO) domains is converted to a hexagonal cylinder structure near 120 degrees C, which is induced by the transformation of the isotropic phase of the p(MAAZ) matrix to the smectic A phase, which is composed of a laterally ordered structure of p(MAAZ) blocks with fully extended side groups. The resulting hexagonal cylinder structure is very stable below 120 degrees C. This microscopic hexagonal cylinder structure is retained as the smectic A phase of the p(MAAZ) matrix undergoes further transitions to smectic C near 104 degrees C and to a smectic X phase near 76 degrees C, while the amorphous, liquid phase of the p(EO) cylinders undergoes crystallization near -15 degrees C. These complicated temperature-dependent disorder-order and order-order phase transitions in the films were found to take place reversibly during the heating run. A face-centered orthorhombic structure of p(EO) domains was also found during the heating run and is an intermediate structure in the hexagonal cylinder structure to BCC structure transformation. We use these structural analysis results to propose molecular structure models at various temperatures for thin films of the diblock polymer.

Non-genetic engineering of cytotoxic T cells to target IL-4 receptor enhances tumor homing and therapeutic efficacy against melanoma.

Adoptive transfer of cytotoxic T lymphocytes (CTLs) has been used as an immunotherapy in melanoma. However, the tumor homing and therapeutic efficacy of transferred CTLs against melanoma remain unsatisfactory. Interleukin-4 receptor (IL-4R) is commonly up-regulated in tumors including melanoma. Here, we studied whether IL-4R-targeted CTLs exhibit enhanced tumor homing and therapeutic efficacy against melanoma. CTLs isolated from mice bearing melanomas were non-genetically engineered with IL4RPep-1, an IL-4R-binding peptide, using a membrane anchor composed of dioleylphosphatidylethanolamine. Compared to control CTLs, IL-4R-targeted CTLs showed higher binding to melanoma cells and in vivo tumor homing. They also exerted a more rapid and robust effector response, including increased cytokine secretion and cytotoxicity against melanoma cells and enhanced reprogramming of M2-type macrophages to M1-type macrophages. Moreover, IL-4R-targeted CTLs efficiently inhibited melanoma growth and reversed the immunosuppressive tumor microenvironment. These results suggest that non-genetically engineered CTLs targeting IL-4R have potential as an adoptive T cell therapy against melanoma.

Tunable Film Morphologies of Brush-Linear Diblock Copolymer Bearing Difluorene Moieties Yield a Variety of Digital Memory Properties.

An amphiphilic brush-linear diblock copolymer bearing a rigid difluorene moiety was synthesized, yielding a copolymer with a high thermal stability and excellent processability. The immiscibility of the blocks induced the formation of a variety of nanostructures, depending on the fabrication conditions, which differed significantly from the nanostructures observed among common diblock copolymers in similar composition. Interestingly, the orientations of the nanostructures could be controlled. The nanostructured polymer displayed a variety of tunable morphologies that yielded distinct electrical memory properties when incorporated as the active layer into a digital memory device. The memory devices could be operated under very low power consumption levels and displayed excellent unipolar switching properties.

High-performance triisopropylsilylethynyl pentacene transistors via spin coating with a crystallization-assisting layer.

The effects of spin speed and an amorphous fluoropolymer (CYTOP)-patterned substrate on the crystalline structures and device performance of triisopropylsilylethynyl pentacene (TIPS-PEN) organic field-effect transistors (OFETs) were investigated. The crystallinity of the TIPS-PEN film was enhanced by decreasing the spin speed, because slow evaporation of the solvent provided a sufficient time for the formation of thermodynamically stable crystalline structures. In addition, the adoption of a CYTOP-patterned substrate induced the three-dimensional (3D) growth of the TIPS-PEN crystals, because the patterned substrate confined the TIPS-PEN molecules and allowed sufficient time for the self-organization of TIPS-PEN. TIPS-PEN OFETs fabricated at a spin speed of 300 rpm with a CYTOP-patterned substrate showed a field-effect mobility of 0.131 cm(2) V(-1) s(-1), which is a remarkable improvement over previous spin-coated TIPS-PEN OFETs.

Effects of side-chain interdigitation on stability: an environmentally, electrically, and thermally stable semiconducting polymer.

A polymeric semiconductor, poly(3,6-dihexyl-[2,2']bi[thieno[3,2-b]thiophene]) (PDHTT), was synthesized and tested as an active layer in organic thin film transistors (OTFTs). This semiconductor showed considerable potential for use in commercial electronic devices because of its superior characteristics, particularly its good stability. PDHTT-based OTFTs exhibited high stability in air, retaining their initial performance after exposure to 70% relative humidity for 50 days; they were also stable under repeated electrical stress and even after exposure to temperatures as high as 250 °C. We attribute the remarkable stability of PDHTT OTFTs to the relatively low highest occupied molecular orbital (5.1 eV) level of the polymer and its highly interdigitated structure in the thin film state.

Synchrotron grazing incidence X-ray scattering study of the morphological structures in thin films of a polymethacrylate diblock copolymer bearing POSS moieties.

The morphological structures in thin films of a diblock copolymer of methyl methacrylate and polyhedral oligomeric silsesquioxane (POSS) functionalized methacrylate (PMMA-b-PMAPOSS) with a volume ratio of 13/87 were investigated in detail by using synchrotron grazing incidence small and wide-angle X-ray scattering (GISAXS and GIWAXS). In addition, its thermal properties were studied. Thin films of this diblock copolymer were found to undergo phase-separation during solvent-annealing with carbon disulfide and post thermal annealing. To quantitatively analyze the scattering data, GISAXS and GIWAXS formulas were derived and applied. Our detailed analysis found that cylinders of PMMA blocks are induced to form in the diblock copolymer films by solvent-annealing and are hexagonally packed in the PMAPOSS matrix, in which the cylinders are oriented vertically with respect to the film plane. In the solvent-annealed films, both the PMMA cylinders and the PMAPOSS matrix are featureless, i.e., amorphous. However, the post thermal annealing process induces aggregation of the POSS moieties, which results in the formation of crystals with an orthorhombic lattice unit cell. These crystals were found to consist of PMAPOSS block chains in a helical conformation in which the molecular PMAPOSS cylinders are aligned in the film plane. The formation of these crystals is induced by the ordering ability of the POSS moieties. The crystals were found to melt above 190 degrees C during heating and subsequent cooling. In contrast, the hexagonally packed structure of the PMMA cylinders in the solvent-annealed and post thermally annealed films was found to be retained during the heating and the subsequent cooling. In addition, the scattering analysis provides detailed structural parameters. The 2D GISAXS and GIWAXS patterns were reconstructed from the determined structural parameters by using the derived scattering formulas, and found to be in good agreement with the experimental patterns. Moreover, a model for the structure of the films of the diblock copolymer is proposed.

Effect of C(60) fullerene on the duplex formation of i-motif DNA with complementary DNA in solution.

The structural effects of fullerene on i-motif DNA were investigated by characterizing the structures of fullerene-free and fullerene-bound i-motif DNA, in the presence of cDNA and in solutions of varying pH, using circular dichroism and synchrotron small-angle X-ray scattering. To facilitate a direct structural comparison between the i-motif and duplex structures in response to pH stimulus, we developed atomic scale structural models for the duplex and i-motif DNA structures, and for the C(60)/i-motif DNA hybrid associated with the cDNA strand, assuming that the DNA strands are present in an ideal right-handed helical conformation. We found that fullerene shifted the pH-induced conformational transition between the i-motif and the duplex structure, possibly due to the hydrophobic interactions between the terminal fullerenes and between the terminal fullerenes and an internal TAA loop in the DNA strand. The hybrid structure showed a dramatic reduction in cyclic hysteresis.

Synchrotron X-ray scattering characterization of the molecular structures of star polystyrenes with varying numbers of arms.

We have synthesized well-defined multiarmed star polystyrenes, with 6, 9, 17, 33, and 57 arms, and studied their molecular shapes and structural characteristics in a good solvent (tetrahydrofuran at 25 degrees C) and in a theta (Theta) solvent (cyclohexane at 35 degrees C) by small-angle X-ray scattering (SAXS) using a synchrotron radiation source. Analysis of the SAXS data provided a detailed characterization of the molecular shapes, including the contributions of the blob morphology of the arms, the radius of gyration, the paired distance distribution, the radial electron density distribution, and the Zimm-Stockmayer and Roovers g-factor, for the multiarmed star polystyrenes. In particular, the molecular shapes of the star polystyrenes were found to change from a fuzzy ellipsoid, for the 6-armed polystyrene, to a fuzzy sphere, for the 57-armed polystyrene, with an increasing number of arms. The ellipsoidal character of the star polystyrenes with fewer arms may originate from the extended anisotropically branched architecture at the center of the molecule. The arms of the star polystyrenes were found to be more extended than those of the linear polystyrenes. Furthermore, the degree of chain extension in the arms increased with the number of arms.

Direct binding of recombinant plasminogen kringle 1-3 to angiogenin inhibits angiogenin-induced angiogenesis in the chick embryo CAM.

Angiogenin is one of the most potent angiogenesis-inducing proteins. Angiostatin is one of the most potent angiogenesis inhibitors, and it contains the first four kringle domains of plasminogen (K1-4). Recombinant human plasminogen kringle 1-3 (rK1-3) was expressed in Escherichia coli and purified to homogeneity. The binding of t-4-aminomethylcyclohexanecarboxylic acid with the purified kringle 1-3 was determined by changes in intrinsic fluorescence. rK1-3 exhibits comparable ligand-binding properties as native human plasminogen kringle 1-3. The purified rK1-3 inhibits neovascularization in the chick embryo chorioallantoic membrane (CAM) assay. Interaction of angiogenin with rK1-3 was examined by immunological binding assay and surface plasmon resonance kinetic analysis, and the equilibrium dissociation constants for the complex, Kd, are 0.89 and 0.18 microM, respectively. rK1-3 inhibits angiogenin-induced angiogenesis in the chick embryo CAM in a concentration-dependent manner. These results indicate that rK1-3 directly binds to angiogenin and thus rK1-3 inhibits the angiogenic activity of angiogenin.