In silico selection of therapeutic antibodies for development: viscosity, clearance, and chemical stability.

For mAbs to be viable therapeutics, they must be formulated to have low viscosity, be chemically stable, and have normal in vivo clearance rates. We explored these properties by observing correlations of up to 60 different antibodies of the IgG1 isotype. Unexpectedly, we observe significant correlations with simple physical properties obtainable from antibody sequences and by molecular dynamics simulations of individual antibody molecules. mAbs viscosities increase strongly with hydrophobicity and charge dipole distribution and decrease with net charge. Fast clearance correlates with high hydrophobicities of certain complementarity determining regions and with high positive or high negative net charge. Chemical degradation from tryptophan oxidation correlates with the average solvent exposure time of tryptophan residues. Aspartic acid isomerization rates can be predicted from solvent exposure and flexibility as determined by molecular dynamics simulations. These studies should aid in more rapid screening and selection of mAb candidates during early discovery.

The biodistribution and pharmacokinetic evaluation of choline-bound gold nanoparticles in a human prostate tumor xenograft model.

PURPOSE: Gold nanoparticles (GNPs) have attracted significant attention in the treatment of cancer due to their potential as novel radiation enhancers, particularly when functionalized with various targeting ligands. The aim of this study was to assess the biodistribution and pharmacokinetic characteristics of a novel choline-bound GNP (choline-GNP) stabilized with polyethelenimine (PEI). METHODS: Choline bound to 27 nm diameter GNPs was characterized using transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). Toxicity of choline-GNPs was examined on DU-145 prostate cancer cells using an MTT assay. Using balb/c mice bearing flank DU-145 prostate tumors, choline-GNPs bio-distribution was measured using inductively coupled mass spectroscopy (ICP-MS). Blood, heart, lung, liver, spleen, brain, kidney and tumor gold content were examined at multiple time points over a 24-hour period after tail vein injection. RESULTS: An MTT assay using DU-145 prostate cancer cells yielded a 95% cell viability 72 hours after choline-GNP administration. The tumor GNP area under the concentration-time curve during the first 4 hours (AUC0-4) was 2.2 µg/ml h, representing 13% of the circulating blood GNP concentration over the same time period. The maximum intra-tumor GNP concentration observed was 1.4% of the injected dose per gram of tumor tissue (%ID/g) one hour post injection. CONCLUSIONS: GNPs functionalized with choline demonstrates a viable future nanoparticle platform with increased intra-tumor uptake as compared to unconjugated GNPs. Decreased intra-hepatic accumulation appears to be the reason for the improved systemic bioavailability. The next logical translational investigation will incorporate external beam radiation with the observed maximum intra-tumor uptake.

Effects of Gas Type, Oil, Salts and Detergent on Formation and Stability of Air and Carbon Dioxide Bubbles Produced by Using a Nanobubble Generator.

Recent developments in ultrafine bubble generation have opened up new possibilities for applications in various fields. Herein, we investigated how substances in water affect the size distribution and stability of microbubbles generated by a common nanobubble generator. By combining light scattering techniques with optical microscopy and high-speed imaging, we were able to track the evolution of microbubbles over time during and after bubble generation. Our results showed that air injection generated a higher number of microbubbles (<10 µm) than CO2 injection. Increasing detergent concentration led to a rapid increase in the number of microbubbles generated by both air and CO2 injection and the intensity signal detected by dynamic light scattering (DLS) slightly increased. This suggested that surface-active molecules may inhibit the growth and coalescence of bubbles. In contrast, we found that salts (NaCl and Na2CO3) in water did not significantly affect the number or size distribution of bubbles. Interestingly, the presence of oil in water increased the intensity signal and we observed that the bubbles were coated with an oil layer. This may contribute to the stability of bubbles. Overall, our study sheds light on the effects of common impurities on bubble generation and provides insights for analyzing dispersed bubbles in bulk.

Clonal evolution during metastatic spread in high-risk neuroblastoma.

Patients with high-risk neuroblastoma generally present with widely metastatic disease and often relapse despite intensive therapy. As most studies to date focused on diagnosis-relapse pairs, our understanding of the genetic and clonal dynamics of metastatic spread and disease progression remain limited. Here, using genomic profiling of 470 sequential and spatially separated samples from 283 patients, we characterize subtype-specific genetic evolutionary trajectories from diagnosis through progression and end-stage metastatic disease. Clonal tracing timed disease initiation to embryogenesis. Continuous acquisition of structural variants at disease-defining loci (MYCN, TERT, MDM2-CDK4) followed by convergent evolution of mutations targeting shared pathways emerged as the predominant feature of progression. At diagnosis metastatic clones were already established at distant sites where they could stay dormant, only to cause relapses years later and spread via metastasis-to-metastasis and polyclonal seeding after therapy.

Process development for robust removal of aggregates using cation exchange chromatography in monoclonal antibody purification with implementation of quality by design.

Aggregate removal is one of the most important aspects in monoclonal antibody (mAb) purification. Cation-exchange chromatography (CEX), a widely used polishing step in mAb purification, is able to clear both process-related impurities and product-related impurities. In this study, with the implementation of quality by design (QbD), a process development approach for robust removal of aggregates using CEX is described. First, resin screening studies were performed and a suitable CEX resin was chosen because of its relatively better selectivity and higher dynamic binding capacity. Second, a pH-conductivity hybrid gradient elution method for the CEX was established, and the risk assessment for the process was carried out. Third, a process characterization study was used to evaluate the impact of the potentially important process parameters on the process performance with respect to aggregate removal. Accordingly, a process design space was established. Aggregate level in load is the critical parameter. Its operating range is set at 0-3% and the acceptable range is set at 0-5%. Equilibration buffer is the key parameter. Its operating range is set at 40 ± 5 mM acetate, pH 5.0 ± 0.1, and acceptable range is set at 40 ± 10 mM acetate, pH 5.0 ± 0.2. Elution buffer, load mass, and gradient elution volume are non-key parameters; their operating ranges and acceptable ranges are equally set at 250 ± 10 mM acetate, pH 6.0 ± 0.2, 45 ± 10 g/L resin, and 10 ± 20% CV respectively. Finally, the process was scaled up 80 times and the impurities removal profiles were revealed. Three scaled-up runs showed that the size-exclusion chromatography (SEC) purity of the CEX pool was 99.8% or above and the step yield was above 92%, thereby proving that the process is both consistent and robust.

High-throughput glycolytic inhibitor discovery targeting glioblastoma by graphite dots-assisted LDI mass spectrometry.

Malignant tumors will become vulnerable if their uncontrolled biosynthesis and energy consumption engaged in metabolic reprogramming can be cut off. Here, we report finding a glycolytic inhibitor targeting glioblastoma with graphite dots-assisted laser desorption/ionization mass spectrometry as an integrated drug screening and pharmacokinetic platform (GLMSD). We have performed high-throughput virtual screening to narrow an initial library of 240,000 compounds down to the docking of 40 compounds and identified five previously unknown chemical scaffolds as promising hexokinase-2 inhibitors. The best inhibitor (Compd 27) can regulate the reprogrammed metabolic pathway in U87 glioma cells (median inhibitory concentration ~ 11.3 µM) for tumor suppression. Highly effective therapy against glioblastoma has been demonstrated in both subcutaneous and orthotopic brain tumors by synergizing Compd 27 and temozolomide. Our glycolytic inhibitor discovery can inspire personalized medicine targeting reprogrammed metabolisms of malignant tumors. GLMSD enables large, high-quality data for next-generation artificial intelligence-aided drug development.

Novel inhibitors of Mycobacterium tuberculosis dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD) identified by virtual screening.

The complex and highly impermeable cell wall of Mycobacterium tuberculosis (Mtb) is largely responsible for the ability of the mycobacterium to resist the action of chemical therapeutics. An L-rhamnosyl residue, which occupies an important anchoring position in the Mtb cell wall, is an attractive target for novel anti-tuberculosis drugs. In this work, we report a virtual screening (VS) study targeting Mtb dTDP-deoxy-L-lyxo-4-hexulose reductase (RmlD), the last enzyme in the L-rhamnosyl synthesis pathway. Through two rounds of VS, we have identified four RmlD inhibitors with half inhibitory concentrations of 0.9-25 µM, and whole-cell minimum inhibitory concentrations of 20-200 µg/ml. Compared with our previous high throughput screening targeting another enzyme involved in L-rhamnosyl synthesis, virtual screening produced higher hit rates, supporting the use of computational methods in future anti-tuberculosis drug discovery efforts.

Biological activity validation of a computationally designed Rituximab/CD3 T cell engager targeting CD20+ cancers with multiple mechanisms of action.

BACKGROUND: Bispecific T cell engaging antibodies (TEAs) with one arm targeting a cancer antigen and another arm binding to CD3 have demonstrated impressive efficacy in multiple clinical studies. However, establishing a safety/efficacy balance remains challenging. For instance, some TEAs have severe safety issues. Additionally, not all patients or all cancer cells of one patient respond equally to TEAs. METHODS: Here, we developed a next-generation bispecific TEA with better safety/efficacy balance and expanded mechanisms of action. Using the computer-aided antibody design strategy, we replaced heavy chain complementarity-determining regions (HCDRs) in one Rituximab arm with HCDRs from a CD3 antibody and generated a novel CD20/CD3 bispecific antibody. RESULTS: After series of computer-aided sequence optimization, the lead molecule, GB261, showed great safety/efficacy balance both in vitro and in animal studies. GB261 exhibited high affinity to CD20 and ultra-low affinity to CD3. It showed comparable T cell activation and reduced cytokine secretion compared with a benchmark antibody (BM). ADCC and CDC caused by GB261 only killed CD20+ cells but not CD3+ cells. It exhibited better RRCL cell killing than the BM in a PBMC-engrafted, therapeutic treatment mouse model and good safety in cynomolgus monkeys. CONCLUSIONS: Thus, GB261 is a promising novel TEA against CD20+ cancers.

New Zealand dental practitioners' experience of patient allergies to dental alloys used for prosthodontics.

UNLABELLED: The biocompatibility of metal alloys has generated much concern for practitioners and patients alike over recent years. OBJECTIVES: To investigate dentists' experience of patient allergies to metal alloys used in prosthodontic restorations. DESIGN: Cross-sectional survey of New Zealand practising general dentists. METHODS: A random sample of 700 was taken from the New Zealand dental register. The questionnaire asked dentists whether any of their patients have encountered any allergic reactions to metal alloys. It also sought information on what alloys were being prescribed for use in different types of prosthodontic restorations. RESULTS: A response rate of 71.4% was obtained (N = 476). Some 83 dentists (17.4%) reported having encountered suspected metal allergies in patients; of those, 70 had had the allergies confirmed with a biopsy. Of the entire sample, 327 dentists (72.2%) were aware of the metals used in their restorations, and 201 (44.8%) specified the alloys used in their restorations. For cast removable prosthodontic restorations (such as removable partial dentures), base metal alloys were the most preferred choice; for full cast crowns, high noble alloys were the most favoured; noble alloys were the most favoured for both porcelain-fused-to-metal crowns and fixed-bridge restorations. CONCLUSION: As many as one in six general practising dentists have encountered allergic reactions to metal alloys in their patients. General practising dentists' awareness of the indications for the various metal alloys used in prosthodontic restorations should be raised, and biocompatibility issues should be clarified, so that dentists prescribe the optimum metal alloy for each type of restoration.

A novel anti-HER2 antibody GB235 reverses Trastuzumab resistance in HER2-expressing tumor cells in vitro and in vivo.

HER2 overexpression is frequently associated with tumor metastasis and poor prognosis of breast cancer. More evidence indicates that HER3 is involved in HER2-resistant therapies. Combination treatments with two or more different monoclonal antibodies are a promising strategy to overcome resistance to HER2 therapies. We presented a novel fully human HER2-targeted monoclonal antibody, GB235, screened from a phage-display library against the HER2 antigen. GB235 in combination with Trastuzumab overcomes resistance in HER2-positive tumors and results in more sustained inhibition of tumor growth over time. The competition binding assay showed that the epitopes of GB235 do not overlap with those of Pertuzumab and Trastuzumab on HER2. Further HER2 mutagenesis results revealed that the binding epitopes of GB235 were located in the domain III of HER2. The mechanism of action of GB235 in blocking HER2-driven tumors is different from the mechanisms of Trastuzumab or Pertuzumab. GB235 does not affect the heterodimerization of HER2 and HER3, whereas the GB235 combined treatment with Trastuzumab significantly inhibited heregulin-induced HER3 phosphorylation and downstream signaling. Moreover, GB235 in combination with Trastuzumab reversed the resistance to heregulin-induced proliferation in HER2-overexpressing cancer cell lines. GB235 combined with Trastuzumab treatment in xenograft models resulted in improved antitumor activity. Complete tumor suppression was observed in the HER2-positive NCI-N87 xenograft model treated with the combination treatment with GB235 and Trastuzumab. In a Trastuzumab-resistant patient-derived tumor xenograft model GA0060, GB235 plus Trastuzumab reversed the resistance to Trastuzumab monotherapy. Because GB235 showed a different working mechanism with Pertuzumab and Trastuzumab, these agents can be considered complementary therapy against HER2 overexpression tumors.

Characterization of novel neutralizing mouse monoclonal antibody JM1-24-3 developed against MUC18 in metastatic melanoma.

BACKGROUND: MUC18 is a glycoprotein highly expressed on the surface of melanoma and other cancers which promotes tumor progression and metastasis. However, its mechanism of action and suitability as a therapeutic target are unknown. METHODS: A monoclonal antibody (mAb) (JM1-24-3) was generated from metastatic melanoma tumor live cell immunization, and high-throughput screening identified MUC18 as the target. RESULTS: Analysis of molecular interactions between MUC18 and JM1-24-3 revealed that the downstream signaling events depended on binding of the mAb to a conformational epitope on the extracellular domain of MUC18. JM1-24-3 inhibited melanoma cell proliferation, migration and invasion in vitro and reduced tumor growth and metastasis in vivo. CONCLUSION: These results confirm that MUC18 is mechanistically important in melanoma growth and metastasis, suggest that the MUC18 epitope identified is a promising therapeutic target, and that the JM1-24-3 mAb may serve as the basis for a potential therapeutic agent.

Structure-directed combinatorial library design.

In recent years pharmaceutical companies have utilized structure-based drug design and combinatorial library design techniques to speed up their drug discovery efforts. Both approaches are routinely used in the lead discovery and lead optimization stages of the drug discovery process. Fragment-based drug design, a new power tool in the drug design toolbox, is also gaining acceptance across the pharmaceutical industry. This review will focus on the interplay between these three design techniques and recent developments in computational methodologies that enhance their integration. Examples of successful synergistic applications of these three techniques will be highlighted. Opinion regarding possible future directions of the field will be given.

Combinatorial library-based design with Basis Products.

Uncovering useful lead compounds from a vast virtual library of synthesizable compounds continues to be of tremendous interest to pharmaceutical researchers. Here we present the concept of Basis Products (BPs), a new and broadly applicable method for achieving efficient selections from a combinatorial library. By definition, Basis Products are a strategically selected subset of compounds from a potentially very large combinatorial library, and any compound in a combinatorial library can represented by its BPs. In this article we will show how to use BP docking scores to find the top compounds of a combinatorial library. Compared with the brute-force docking of an entire virtual library, docking with BPs are much more efficient because of the substantial size reduction, saving both time and resources. We will also demonstrate how BPs can be used for property-based combinatorial library designs. Furthermore, BPs can also be considered as fragments carrying chemistry knowledge, hence they can potentially be used in combination with any fragment-based design method. Therefore, BPs can be used to integrate combinatorial design with structure-based design and/or fragment-based design. Other potential applications of BPs include lead hopping and consensus core building, which we will describe briefly as well in this report.

Viral clearance using disposable systems in monoclonal antibody commercial downstream processing.

Once highly selective protein A affinity is chosen for robust mAb downstream processing, the major role of polishing steps is to remove product related impurities, trace amounts of host cell proteins, DNA/RNA, and potential viral contaminants. Disposable systems can act as powerful options either to replace or in addition to polishing column chromatography to ensure product purity and excellent viral clearance power for patients' safety. In this presentation, the implementation of three disposable systems such as depth filtration, membrane chromatography, and nanometer filtration technology in a commercial process are introduced. The data set of viral clearance with these systems is presented. Application advantages and disadvantages including cost analysis are further discussed.

Dihydroxylphenyl amides as inhibitors of the Hsp90 molecular chaperone.

Information from X-ray crystal structures were used to optimize the potency of a HTS hit in a Hsp90 competitive binding assay. A class of novel and potent small molecule Hsp90 inhibitors were thereby identified. Enantio-pure compounds 31 and 33 were potent in PGA-based competitive binding assay and inhibited proliferation of various human cancer cell lines in vitro, with IC(50) values averaging 20 nM.

pH-conductivity hybrid gradient cation-exchange chromatography for process-scale monoclonal antibody purification.

The commercial production of recombinant human monoclonal antibody therapeutics demands robust processes. In this article we describe the development of a pH-conductivity hybrid gradient for a cation-exchange chromatography step to obtain high binding capacity and consistent purification resolution in scale process. Operational parameters and their ranges were characterized with DOE statistical method. Aggregate, DNA and leached protein A removal were examined during development. The advantages and disadvantages of hybrid gradient elution compared to sodium chloride gradient elution were explored. As this step was designed as a good fit for the compatibility of the feed and operating pH/conductivity conditions for next step, the effects of elution by either changing sodium chloride concentration or changing pH of elution buffers on overall separation efficiency were compared. The operation condition was further confirmed in six 2000 L scale runs. The thorough evaluation demonstrated process reliability of hybrid gradient cation-exchange chromatography with high step purity and yield.

TIP, a T-cell factor identified using high-throughput screening increases survival in a graft-versus-host disease model.

A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins.

Identification of JL1037 as a novel, specific, reversible lysine-specific demethylase 1 inhibitor that induce apoptosis and autophagy of AML cells.

Lysine-specific demethylase 1 (LSD1) has been recognized as a potential therapeutic target for acute myeloid leukemia (AML). Herein, we identified a novel LSD1 inhibitor, JL1037, via Computer Aided Drug Design technology. JL1037 is a potent, selective and reversible LSD1 inhibitor with IC50s of 0.1 µM and >1.5 µM for LSD1 and monoamine oxidases A/B (MAO-A/B), respectively. Treatment of THP-1 and Kasumi-1 cell lines with JL1037 resulted in dose dependent accumulation of H3K4me1 and H3K4me2, the major substrates of LSD1, as well as inhibition of cell proliferation, blockade of cell cycle and induction of apoptosis. Further investigations demonstrated that JL1037 could upregulate cell cycle-related proteins P21, P57, pro-apoptotic protein Bax and downregulate anti-apoptosis proteins Bcl-2 and Bcl-XL. JL1037 appeared to activate autophage response in AML cell lines as well as primary cells from AML patients by increasing LC3-II expression and the formation of autophagosomes and autolysosomes in cytoplasm. Co-treatment with autophagy inhibitor chloroquine (CQ) enhanced JL1037-induced cell apoptosis. Moreover, daily intravenous administration of JL1037 tended to reduce tumor burden and prolong the survival of t(8;21) leukemia mice. In conclusion, JL1037 exhibited potent anti-leukemia effect and could be a potential therapeutic agent for AML treatment.

New Q membrane scale-down model for process-scale antibody purification.

Process-scale antibody production requires polishing steps with extremely high product throughput and robust operation. In this communication, the Sartobind Q membrane adsorber for process-scale antibody production is evaluated as an alternative to Q column chromatography. Although the capacity seen with large-scale membrane adsorbers is competitive with column chromatography, the same throughput is not achieved with the current scale-down models. The operational issues currently found in membrane scale-down models, including backpressure, which significantly compromises the membrane's capacity, were examined. A new scale-down model was designed to mimic the liquid flow path found in the large-scale capsule, and a new process capacity equivalent at both small and large scale was successfully achieved. Results of a 4-model virus study with a redesigned Sartobind Q absorber scale-down model at the new process capacity are presented.

Basic concepts in Q membrane chromatography for large-scale antibody production.

The large-scale production of recombinant human monoclonal antibodies demands economical purification processes with high throughputs. In this article we briefly describe a common antibody process and evaluate the Q membrane adsorber for process-scale antibody production as an alternative to a Q-packed-bed column in a flow-through mode. The scientific concepts underlining Q membrane technology and its application are reviewed. The disadvantages and advantages of using Q membrane chromatography as a purification unit in large-scale production are discussed, including problems initially seen with the Q membrane scale-down model but solved with the invention of a new scale-down model. The new Q-membrane unit operation has a process capacity greater than 3,000 g/m(2) or 10.7 kg/L with a LRV over 5 for four model viruses. In this Review, a cost analysis illustrates that Q membrane chromatography is a viable alternative to Q column chromatography as a polishing step in a flow-through mode for process-scale antibody production.