Tandem-Cleavage Linkers Improve the In Vivo Stability and Tolerability of Antibody-Drug Conjugates.

Although peptide motifs represent the majority of cleavable linkers used in clinical-stage antibody-drug conjugates (ADCs), the sequences are often sensitive to cleavage by extracellular enzymes, such as elastase, which leads to systemic release of the cytotoxic payload. This action reduces the therapeutic index by causing off-target toxicities that can be dose-limiting. For example, a common side-effect of ADCs made using peptide-cleavable linkers is myelosuppression, including neutropenia. Only a few reports describe methods for optimizing peptide linkers to maintain efficient and potent tumor payload delivery while enhancing circulating stability. Herein, we address these critical limitations through the development of a tandem-cleavage linker strategy, where two sequential enzymatic cleavage events mediate payload release. We prepared dipeptides that are protected from degradation in the circulation by a sterically encumbering glucuronide moiety. Upon ADC internalization and lysosomal degradation, the monosaccharide is removed and the exposed dipeptide is degraded, which liberates the attached payload inside the target cell. We used CD79b-targeted monomethyl auristatin E (MMAE) conjugates as our model system and compared the stability, efficacy, and tolerability of ADCs made with tandem-cleavage linkers to ADCs made using standard technology with the vedotin linker. The results, where rat studies showed dramatically improved tolerability in the hematopoietic compartment, highlight the role that linker stability plays in efficacy and tolerability and also offer a means of improving an ADC's therapeutic index for improved patient outcomes.

A Novel HER2-targeted Antibody-drug Conjugate Offers the Possibility of Clinical Dosing at Trastuzumab-equivalent Exposure Levels.

Trastuzumab and the related ADC, ado-trastuzumab emtansine (T-DM1), both target HER2-overexpressing cells. Together, these drugs have treatment indications in both early-stage and metastatic settings for HER2+ breast cancer. T-DM1 retains the antibody functionalities of trastuzumab and adds the potency of a cytotoxic maytansine payload. Interestingly, in the clinic, T-DM1 cannot always replace the use of trastuzumab plus chemotherapy administered together as single agents. We hypothesize that this failure may be due, in part, to the limited systemic exposure achieved by T-DM1 relative to trastuzumab because of toxicity-related dosing constraints on the ADC. We have developed a trastuzumab-based ADC site specifically conjugated to maytansine through a noncleavable linker. This construct, termed CAT-01-106, has a drug-to-antibody ratio (DAR) of 1.8, approximately half the average DAR of T-DM1, which comprises a mixture of antibodies variously conjugated with DARs ranging from 0 to 8. The high DAR species present in T-DM1 contribute to its toxicity and limit its clinical dose. CAT-01-106 showed superior in vivo efficacy compared with T-DM1 at equal payload dosing and was equally or better tolerated compared with T-DM1 at equal payload dosing up to 120 mg/kg in Sprague-Dawley rats and 60 mg/kg in cynomolgus monkeys. CAT-01-106 also showed improved pharmacokinetics in rats relative to T-DM1, with 40% higher ADC exposure levels. Together, the data suggest that CAT-01-106 may be sufficiently tolerable to enable clinical dosing at trastuzumab-equivalent exposure levels, combining the functions of both the antibody and the payload in one drug and potentially improving patient outcomes.

High-Performance Green Light-Emitting Diodes Based on MAPbBr3 with π-Conjugated Ligand.

The morphology, crystal size, and trap density of perovskite films significantly affect the luminescent properties of perovskite light-emitting diodes (PeLEDs). Recently, numerous studies have been conducted on ligands that surround the surface of perovskite crystals and passivate the trap sites to improve the performance of PeLEDs. In this study, a 4-aminobenzonitrile (ABN) ligand improved the performance of methylammonium lead bromide (MAPbBr3)-based PeLEDs by reducing the MAPbBr3 crystal size to the nanoscale and reducing the trap density. Moreover, the properties of PeLEDs with ABN were further improved using a surface-modified hole-transport layer (HTL) with a hydrophilic polymer. Finally, a bright green PeLED was fabricated, which exhibited the maximum luminance of 3350 cd/m2 with an external quantum efficiency of 8.85%. Therefore, it is believed that the use of proper ligands for the perovskite layer and the optimization of the charge-transport layer have great potential for the development of high-performance PeLEDs.

Violet Light-Emitting Diodes Based on p-CuI Thin Film/n-MgZnO Quantum Dot Heterojunction.

As the lighting technology evolves, the need for violet light-emitting diodes (LEDs) is growing for high color rendering index lighting. The present technology for violet LEDs is based on the high-cost GaN materials and metal-organic chemical vapor deposition process; therefore, there have recently been intensive studies on developing low-cost alternative materials and processes. In this study, for the first time, we demonstrated violet LEDs based on low-cost materials and processes using a p-CuI thin film/n-MgZnO quantum dot (QD) heterojunction. The p-CuI thin film layer was prepared by an iodination process of Cu films, and the n-MgZnO layer was deposited by spin-coating presynthesized n-MgZnO QDs. To maximize the performance of the violet LED, an optimizing process was performed for each layer of p- and n-type materials. The optimized LED with 1 × 1 mm2-area pixel fabricated using the p-CuI thin film at the iodination temperature of 15 °C and the n-MgZnO QDs at the Mg alloying concentration of 2.7 at. % exhibited the strongest violet emissions at 6 V.

Antibody-drug conjugate library prepared by scanning insertion of the aldehyde tag into IgG1 constant regions.

The advantages of site-specific over stochastic bioconjugation technologies include homogeneity of product, minimal perturbation of protein structure/function, and - increasingly - the ability to perform structure activity relationship studies at the conjugate level. When selecting the optimal location for site-specific payload placement, many researchers turn to in silico modeling of protein structure to identify regions predicted to offer solvent-exposed conjugatable sites while conserving protein function. Here, using the aldehyde tag as our site-specific technology platform and human IgG1 antibody as our target protein, we demonstrate the power of taking an unbiased scanning approach instead. Scanning insertion of the human formylglycine generating enzyme (FGE) recognition sequence, LCTPSR, at each of the 436 positions in the light and heavy chain antibody constant regions followed by co-expression with FGE yielded a library of antibodies bearing an aldehyde functional group ready for conjugation. Each of the variants was expressed, purified, and conjugated to a cytotoxic payload using the Hydrazinyl Iso-Pictet-Spengler ligation to generate an antibody-drug conjugate (ADC), which was analyzed in terms of conjugatability (assessed by drug-to-antibody ratio, DAR) and percent aggregate. We searched for insertion sites that could generate manufacturable ADCs, defined as those variants yielding reasonable antibody titers, DARs of ≥ 1.3, and ≥ 95% monomeric species. Through this process, we discovered 58 tag insertion sites that met these metrics, including 14 sites in the light chain, a location that had proved refractory to the placement of manufacturable tag sites using in silico modeling/rational approaches.

Effect of Nonionic Surfactant Additive in PEDOT:PSS on PFO Emission Layer in Organic-Inorganic Hybrid Light-Emitting Diode.

Poly(9,9-dioctylfluorene) (PFO) has attracted significant interests owing to its versatility in electronic devices. However, changes in its optical properties caused by its various phases and the formation of oxidation defects limit the application of PFO in light-emitting diodes (LEDs). We investigated the effects of the addition of Triton X-100 (hereinafter shortened as TX) in poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) to induce interlayer diffusion between PEDOT:PSS and PFO to enhance the stability of the PFO phase and suppress its oxidation. Photoluminescence (PL) measurement on PFO/TX-mixed PEDOT:PSS layers revealed that, upon increasing the concentration of TX in the PEDOT:PSS layer, the ß phase of PFO could be suppressed in favor of the glassy phase and the wide PL emission centered at 535 nm caused by ketone defects formed by oxidation was decreased considerably. LEDs were then fabricated using PFO as an emission layer, TX-mixed PEDOT:PSS as hole-transport layer, and zinc oxide (ZnO) nanorods as electron-transport layer. As the TX concentration reached 3 wt %, the devices exhibited dramatic increases in current densities, which were attributed to the enhanced hole injection due to TX addition, along with a shift in the dominant emission wavelength from a green electroluminescence (EL) emission centered at 518 nm to a blue EL emission centered at 448 nm. The addition of TX in PEDOT:PSS induced a better hole injection in the PFO layer, and through interlayer diffusion, stabilized the glassy phase of PFO and limited the formation of oxidation defects.

Structural Basis for Activity and Specificity of an Anticoagulant Anti-FXIa Monoclonal Antibody and a Reversal Agent.

Coagulation factor XIa is a candidate target for anticoagulants that better separate antithrombotic efficacy from bleeding risk. We report a co-crystal structure of the FXIa protease domain with DEF, a human monoclonal antibody that blocks FXIa function and prevents thrombosis in animal models without detectable increased bleeding. The light chain of DEF occludes the FXIa S1 subsite and active site, while the heavy chain provides electrostatic interactions with the surface of FXIa. The structure accounts for the specificity of DEF for FXIa over its zymogen and related proteases, its active-site-dependent binding, and its ability to inhibit substrate cleavage. The inactive FXIa protease domain used to obtain the DEF-FXIa crystal structure reversed anticoagulant activity of DEF in plasma and in vivo and the activity of a small-molecule FXIa active-site inhibitor in vitro. DEF and this reversal agent for FXIa active-site inhibitors may help support clinical development of FXIa-targeting anticoagulants.

Enhanced Light Stability of InGaZnO Thin-Film Transistors by Atomic-Layer-Deposited Y2O3 with Ozone.

We report the effect of Y2O3 passivation by atomic layer deposition (ALD) using various oxidants, such as H2O, O2 plasma, and O3, on In-Ga-Zn-O thin-film transistors (IGZO TFTs). A large negative shift in the threshold voltage (Vth) was observed in the case of the TFT subjected to the H2O-ALD Y2O3 process; this shift was caused by a donor effect of negatively charged chemisorbed H2O molecules. In addition, degradation of the IGZO TFT device performance after the O2 plasma-ALD Y2O3 process (field-effect mobility (µ) = 8.7 cm2/(V·s), subthreshold swing (SS) = 0.77 V/dec, and Vth = 3.7 V) was observed, which was attributed to plasma damage on the IGZO surface adversely affecting the stability of the TFT under light illumination. In contrast, the O3-ALD Y2O3 process led to enhanced device stability under light illumination (ΔVth = -1 V after 3 h of illumination) by passivating the subgap defect states in the IGZO surface region. In addition, TFTs with a thicker IGZO film (55 nm, which was the optimum thickness under the current investigation) showed more stable device performance than TFTs with a thinner IGZO film (30 nm) (ΔVth = -0.4 V after 3 h of light illumination) by triggering the recombination of holes diffusing from the IGZO surface to the insulator-channel interface. Therefore, we envisioned that the O3-ALD Y2O3 passivation layer suggested in this paper can improve the photostability of TFTs under light illumination.

CAT-02-106, a Site-Specifically Conjugated Anti-CD22 Antibody Bearing an MDR1-Resistant Maytansine Payload Yields Excellent Efficacy and Safety in Preclinical Models.

Hematologically derived tumors make up ∼10% of all newly diagnosed cancer cases in the United States. Of these, the non-Hodgkin lymphoma (NHL) designation describes a diverse group of cancers that collectively rank among the top 10 most commonly diagnosed cancers worldwide. Although long-term survival trends are improving, there remains a significant unmet clinical need for treatments to help patients with relapsed or refractory disease, one cause of which is drug efflux through upregulation of xenobiotic pumps, such as MDR1. CD22 is a clinically validated target for the treatment of NHL, but no anti-CD22 agents have yet been approved for this indication. Recent approval of an anti-CD22 antibody-drug conjugate (ADC) for the treatment of relapsed/refractory ALL supports the rationale for targeting this protein. An opportunity exists for a next-generation anti-CD22 antibody-drug conjugate (ADC) to address unmet medical needs in the relapsed/refractory NHL population. We describe a site-specifically conjugated antibody-drug conjugate, made using aldehyde tag technology, targeted against CD22 and bearing a noncleavable maytansine payload that is resistant to MDR1-mediated efflux. The construct was efficacious against CD22+ NHL xenografts and could be repeatedly dosed in cynomolgus monkeys at 60 mg/kg with no observed significantly adverse effects. Exposure to total ADC at these doses (as assessed by AUC0-inf) indicated that the exposure needed to achieve efficacy was below tolerable limits. Together, the data suggest that this drug has the potential to be used effectively in patients with CD22+ tumors that have developed MDR1-related resistance to prior therapies. Mol Cancer Ther; 17(1); 161-8. ©2017 AACR.

High-Performance Green Light-Emitting Diodes Based on MAPbBr3-Polymer Composite Films Prepared by Gas-Assisted Crystallization.

The morphology of perovskite films has a significant impact on luminous characteristics of perovskite light-emitting diodes (PeLEDs). To obtain a highly uniform methylammonium lead tribromide (MAPbBr3) film, a gas-assisted crystallization method is introduced with a mixed solution of MAPbBr3 precursor and polymer matrix. The ultrafast evaporation of the solvent causes a high degree of supersaturation which expedites the generation of a large number of nuclei to form a MAPbBr3-polymer composite film with full surface coverage and nano-sized grains. The addition of the polymer matrix significantly affects the optical properties and morphology of MAPbBr3 films. The PeLED made of the MAPbBr3-polymer composite film exhibits an outstanding device performance of a maximum luminance of 6800 cd·m-2 and a maximum current efficiency of 1.12 cd·A-1. Furthermore, 1 cm2 area pixel of PeLED displays full coverage of a strong green electroluminescence, implying that the high-quality perovskite film can be useful for large-area applications in perovskite-based optoelectronic devices.

Factor XIa-specific IgG and a reversal agent to probe factor XI function in thrombosis and hemostasis.

Thrombosis is a major cause of morbidity and mortality. Current antithrombotic drugs are not ideal in that they must balance prevention of thrombosis against bleeding risk. Inhibition of coagulation factor XI (FXI) may offer an improvement over existing antithrombotic strategies by preventing some forms of thrombosis with lower bleeding risk. To permit exploration of this hypothesis in humans, we generated and characterized a series of human immunoglobulin Gs (IgGs) that blocked FXIa active-site function but did not bind FXI zymogen or other coagulation proteases. The most potent of these IgGs, C24 and DEF, inhibited clotting in whole human blood and prevented FeCl3-induced carotid artery occlusion in FXI-deficient mice reconstituted with human FXI and in thread-induced venous thrombosis in rabbits at clinically relevant doses. At doses substantially higher than those required for inhibition of intravascular thrombus formation in these models, DEF did not increase cuticle bleeding in rabbits or cause spontaneous bleeding in macaques over a 2-week study. Anticipating the desirability of a reversal agent, we also generated a human IgG that rapidly reversed DEF activity ex vivo in human plasma and in vivo in rabbits. Thus, an active site-directed FXIa-specific antibody can block thrombosis in animal models and, together with the reversal agent, may facilitate exploration of the roles of FXIa in human disease.

Low-Temperature Facile Synthesis of Sb-Doped p-Type ZnO Nanodisks and Its Application in Homojunction Light-Emitting Diode.

This study explores low-temperature solution-process-based seed-layer-free ZnO p-n homojunction light-emitting diode (LED). In order to obtain p-type ZnO nanodisks (NDs), antimony (Sb) was doped into ZnO by using a facile chemical route at 120 °C. The X-ray photoelectron spectra indicated the presence of (SbZn-2VZn) acceptor complex in the Sb-doped ZnO NDs. Using these NDs as freestanding templates, undoped n-type ZnO nanorods (NRs) were epitaxially grown at 95 °C to form ZnO p-n homojunction. The homojunction with a turn-on voltage of 2.5 V was found to be significantly stable up to 100 s under a constant voltage stress of 5 V. A strong orange-red emission was observed by the naked eye under a forward bias of 5 V. The electroluminescence spectra revealed three major peaks at 400, 612, and 742 nm which were attributed to the transitions from Zni to VBM, from Zni to Oi, and from VO to VBM, respectively. The presence of these deep-level defects was confirmed by the photoluminescence of ZnO NRs. This study paves the way for future applications of ZnO homojunction LEDs using low-temperature and low-cost solution processes with the controlled use of native defects.

Efficient identification of human immunodeficiency virus type 1 mutants resistant to a protease inhibitor by using a random mutant library.

Emergence of drug-resistant mutant viruses during the course of antiretroviral therapy is a major hurdle that limits the success of chemotherapeutic treatment to suppress human immunodeficiency virus type 1 (HIV-1) replication and AIDS progression. Development of new drugs and careful patient management based on resistance genotyping data are important for enhancing therapeutic efficacy. However, identifying changes leading to drug resistance can take years of clinical studies, and conventional in vitro assays are limited in generating reliable drug resistance data. Here we present an efficient in vitro screening assay for selecting drug-resistant variants from a library of randomly mutated HIV-1 strains generated by transposon-directed base-exchange mutagenesis. As a test of principle, we screened a library of mutant HIV-1 strains containing random mutations in the protease gene by using a reporter T-cell line in the presence of the protease inhibitor (PI) nelfinavir (NFV). Analysis of replicating viruses from a single round of infection identified 50 amino acid substitutions at 35 HIV-1 protease residue positions. The selected mutant viruses showed specific resistance to NFV and included most of the known NFV resistance mutations. Therefore, the new assay is efficient for identifying changes leading to drug resistance. The data also provide insights into the molecular mechanisms underlying the development of drug resistance.

Introducing predetermined mutations throughout a target gene using TDEM (transposon-directed base-exchange mutagenesis).

Transposon-directed base-exchange mutagenesis (TDEM) is an efficient and controllable method for introducing a mutation(s) into a gene. Each round of TDEM removes a predetermined number of bases (up to 11 base pairs) from a randomly selected site within the target gene and replaces them with any length of DNA of predetermined sequence. Therefore, the number of bases to be deleted and inserted can be precisely regulated. Because each round of TDEM generates mutation(s) at a single site, the number of mutations introduced can be determined by the number of cycles of TDEM. Furthermore, using a novel frame-checking procedure, non-functional mutants containing a frameshift or stop codon can be minimized. Thus, TDEM can be used to introduce a limited and predetermined change at each round of mutagenesis, thereby providing a useful tool for studying protein structure and function.

A rapid and economic in-house DNA purification method using glass syringe filters.

BACKGROUND: Purity, yield, speed and cost are important considerations in plasmid purification, but it is difficult to achieve all of these at the same time. Currently, there are many protocols and kits for DNA purification, however none maximize all four considerations. METHODOLOGY/PRINCIPAL FINDINGS: We now describe a fast, efficient and economic in-house protocol for plasmid preparation using glass syringe filters. Plasmid yield and quality as determined by enzyme digestion and transfection efficiency were equivalent to the expensive commercial kits. Importantly, the time required for purification was much less than that required using a commercial kit. CONCLUSIONS/SIGNIFICANCE: This method provides DNA yield and quality similar to that obtained with commercial kits, but is more rapid and less costly.

Transposon-directed base-exchange mutagenesis (TDEM): a novel method for multiple-nucleotide substitutions within a target gene.

In this report we describe transposon-directed base-exchange mutagenesis (TDEM), an efficient and controllable method for introducing a mutation into a gene. Each round of TDEM can remove up to 11 base pairs from a randomly selected site within the target gene and replace them with any length of DNA of predetermined sequence. Therefore, the number of bases to be deleted and inserted can be independently regulated providing greater versatility than existing methods of transposon-based mutagenesis. Subsequently, multiple rounds of mutagenesis will provide a diverse mutant library that contains multiple mutations throughout the gene. Additionally, we developed a simple frame-checking procedure that eliminates nonfunctional mutants containing frameshifts or stop codons. As a proof of principle, we used TDEM to generate mutant lacZalpha lacking alpha-complementation activity and recovered active revertants using a second round of TDEM. Furthermore, a single round of TDEM yielded unique, inactive mutants of ccdB.

N-terminal domain-deleted mu transposase exhibits increased transposition activity with low target site preference in modified buffers.

BACKGROUND: Transposition using MuA and the Mu-transposon is frequently used for insertional and deletional mutagenesis and to introduce primer sequences into DNA of unknown sequence. However, mutagenesis studies are often limited by the number of transposition events taking place within a small target sequence. Although the intrinsic target site preference of MuA is low, it nevertheless may limit the recovery of desired events. METHODS: In the current study, we have compared the transposition activity of wild-type, C-terminally deleted, N-terminally deleted and both C- and N-terminally deleted MuA. RESULTS: We found that N-terminally deleted MuA was most active with an about sevenfold increase in transposition efficiency compared to wild-type. In addition, we identified a novel reaction condition in which the transposition activity of N-terminally deleted MuA was increased about 45-fold. CONCLUSION: The use of the N-terminally deleted MuA with the newly established condition enhanced the relative transposition activity about 280-fold compared to that of wild-type MuA with the conventional buffer. In addition, the optimized buffer decreased the target site preference of N-terminally deleted MuA resulting in a more even distribution of integration events throughout the target gene.

Maspin and p53 protein expression in gastric adenocarcinoma and its clinical applications.

OBJECTIVES: To investigate the role of maspin and p53 expression in the progression of gastric cancer, and its value as a prognostic indicator. MATERIALS AND METHODS: The expression of maspin and p53 in 152 cases of gastric cancer was detected by immunohistochemistry and compared with the clinicopathologic tumor parameters. The relationship between maspin and p53 expression was also analyzed in the gastric cancers. RESULTS: The positive expression rates for maspin and p53 in the cancers were 71.7% (109 of 152 cases) and 56.6% (86 of 152 cases), respectively. Two patterns of immunostaining for maspin were seen in the maspin-positive gastric cancer cases: cytoplasm-only staining (67.0%, 73 of 109 cases) and staining of both cytoplasm and nucleus (33.0%, 36 of 109 cases). Maspin expression showed a negative association with histologic grade, depth of invasion, metastasis, and TNM stage (all P<0.05). p53 expression showed an association with node metastasis, and TNM stage (both P<0.05). Maspin expression was negatively correlated with p53 expression (P<0.001, r=-0.291). In univariate log-rank analysis, loss of maspin expression, histologic grade, distant metastasis, and TNM stage were associated with patient survival. Interestingly, patients with nuclear and cytoplasmic maspin expression survived longer than those with only cytoplasmic expression. However, in multivariate analysis TNM stage and regional node metastasis were the only independent prognostic factors. CONCLUSIONS: Maspin expression might be an important factor in tumor progression and patient prognosis, but is not an independent prognostic factor. Maspin expression is inversely correlated with mutant p53 expression in gastric cancer, which suggests that maspin expression is regulated by the p53 pathway.

Hypotensive effect of butein via the inhibition of angiotensin converting enzyme.

Butein (3,4,2',4'-tetrahydroxychalcone), a plant polyphenol, has been known to elucidate endothelium-dependent vasodilation. In the present study, the hypotensive effect of butein and its possible mechanism, especially an angiotensin converting enzyme (ACE) inhibitory effect, were investigated. Intravenous injection of butein lowered the arterial blood pressure of anesthetized rats in a dose-dependent manner. The plasma ACE activities were significantly inhibited by the addition of butein in a dose-dependent manner, the IC(50) value of which was 198 microg/ml (730 microM). Moreover, angiotensin I-induced contraction was markedly attenuated by prior exposure of endothelium-intact aortic rings to butein, but angiotensin II-induced contraction was not altered. These results suggest that butein has a hypotensive effect, at least in part, via the inhibition of angiotensin converting enzyme.