Evaluation of strategies to modify Anti-SARS-CoV-2 monoclonal antibodies for optimal functionality as therapeutics.

The current global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a public health crisis with more than 168 million cases reported globally and more than 4.5 million deaths at the time of writing. In addition to the direct impact of the disease, the economic impact has been significant as public health measures to contain or reduce the spread have led to country wide lockdowns resulting in near closure of many sectors of the economy. Antibodies are a principal determinant of the humoral immune response to COVID-19 infections and may have the potential to reduce disease and spread of the virus. The development of monoclonal antibodies (mAbs) represents a therapeutic option that can be produced at large quantity and high quality. In the present study, a mAb combination mixture therapy was investigated for its capability to specifically neutralize SARS-CoV-2. We demonstrate that each of the antibodies bind the spike protein and neutralize the virus, preventing it from infecting cells in an in vitro cell-based assay, including multiple viral variants that are currently circulating in the human population. In addition, we investigated the effects of two different mutations in the Fc portion (YTE and LALA) of the antibody on Fc effector function and the ability to alleviate potential antibody-dependent enhancement of disease. These data demonstrate the potential of a combination of two mAbs that target two different epitopes on the SARS-CoV2 spike protein to provide protection against SARS-CoV-2 infection in humans while extending serum half-life and preventing antibody-dependent enhancement of disease.

A combination of two human neutralizing antibodies prevents SARS-CoV-2 infection in cynomolgus macaques.

BACKGROUND: Human monoclonal antibody (mAb) treatments are promising for COVID-19 prevention or therapy. The pre-exposure prophylactic efficacy of neutralizing antibodies that are engineered with mutations to extend their persistence in human serum and the neutralizing antibody titer in serum required for protection against SARS-CoV-2 infection remain poorly characterized. METHODS: The Fc region of two neutralizing mAbs (COV2-2130 and COV2-2381) targeting non-overlapping epitopes on the receptor binding domain of SARS-CoV-2 spike protein was engineered to extend their persistence in humans and reduce interactions with Fc gamma receptors. We assessed protection by individual antibodies or a combination of the two antibodies (designated ADM03820) given prophylactically by an intravenous or intramuscular route in a non-human primate (NHP) model of SARS-CoV-2 infection. FINDINGS: Passive transfer of individual mAbs or ADM03820 conferred virological protection in the NHP respiratory tract in a dose-dependent manner, and ADM03820 potently neutralized SARS-CoV-2 variants of concern in vitro. We defined a protective serum-neutralizing antibody titer and concentration in NHPs for passively transferred human antibodies that acted by direct viral neutralization. CONCLUSIONS: In summary, we demonstrate that neutralizing antibodies with extended half-life and lacking Fc-mediated effector functions are efficient for pre-exposure prophylaxis of SARS-CoV-2 infection in NHPs. These results support clinical development of ADM03820 for COVID-19 prevention. FUNDING: This research was supported by a contract from the JPEO-CBRND (W911QY-20-9-003, 20-05); the Joint Sciences and Technology Office and Joint Program Executive Office (MCDC-16-01-002 JSTO, JPEO); a DARPA grant (HR0011-18-2-0001); an NIH grant (R01 AI157155); and the 2019 Future Insight Prize from Merck KGaA.

Neutralizing Concentrations of Anti-Botulinum Toxin Antibodies Positively Correlate with Mouse Neutralization Assay Results in a Guinea Pig Model.

Botulinum neurotoxins (BoNT) are some of the most toxic proteins known and can induce respiratory failure requiring long-term intensive care. Treatment of botulism includes the administration of antitoxins. Monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics, due to their potency and safety. A three-mAb combination has been developed that specifically neutralizes BoNT serotype A (BoNT/A), and a separate three mAb combination has been developed that specifically neutralizes BoNT serotype B (BoNT/B). A six mAb cocktail, designated G03-52-01, has been developed that combines the anti-BoNT/A and anti-BoNT/B mAbs. The pharmacokinetics and neutralizing antibody concentration (NAC) of G03-52-01 has been determined in guinea pigs, and these parameters were correlated with protection against an inhalation challenge of BoNT/A1 or BoNT/B1. Previously, it was shown that each antibody demonstrated a dose-dependent mAb serum concentration and reached maximum circulating concentrations within 48 h after intramuscular (IM) or intraperitoneal (IP) injection and that a single IM injection of G03-52-01 administered 48 h pre-exposure protected guinea pigs against an inhalation challenge of up to 93 LD50s of BoNT/A1 and 116 LD50s of BoNT/B1. The data presented here advance our understanding of the relationship of the neutralizing NAC to the measured circulating antibody concentration and provide additional support that a single IM or intravenous (IV) administration of G03-52-01 will provide pre-exposure prophylaxis against botulism from an aerosol exposure of BoNT/A and BoNT/B.

Koelreuteria paniculata invasiveness, yielding capacity and harvest date influence on biodiesel feedstock properties.

Due to Koelreuteria paniculata Laxm., high abundance in Novi Sad (Serbia) and previously confirmed biodiesel feedstock suitability, this study aimed to assess generative potential and yield, assess K. paniculata invasive risk, and investigate the influence of harvesting periods on the oil content and quality. Fifty-five specimens present in the Novi Sad inner-city core exhibited very high scores in the conducted invasiveness risk assessment (score 35/39). Determined good overall adaptability, growth and development, vitality and decorativeness of K. paniculata specimens, as well as the absence of phytopathological and entomological damages, due to lack of natural enemies, provided this species high unhindered yielding. The majority of investigated trees reached 5-10 m in height and canopy volumes from 10.1 to 70 m3, with an estimated more than 130 000-700 000 seeds produced per canopy. Seeds from one representative specimen were collected on different harvesting dates, at the end of August, September and October of 2019, and analyzed for oil content and quality for biodiesel production. With the average oil percentage of 22.8 w%, determined in the representative specimen, investigated 55 seed-bearing K. paniculata trees merely in the inner city core of Novi Sad, could produce 115 kg of oil. Since oil properties (<1 w% of free fatty acids and the acid number <2 mgKOH.g-1) and fatty acid profile (high content of monounsaturated fatty acids ≈72 w%) fit the requirements set in EN14214 biodiesel standard, regardless of the harvest date, harvest season can be extended to at least three months without compromising the oil quality for biodiesel production. Thus, intrinsic disservice of K. paniculata - high seed yielding can be shifted into a novel ecosystem service - quality biodiesel production. Aiming to avoid viable seed deposition in the ground and formation of the invasive seed bank, early August harvest can be recommended.

A Monoclonal Antibody Combination against both Serotypes A and B Botulinum Toxin Prevents Inhalational Botulism in a Guinea Pig Model.

Botulinum neurotoxins (BoNT) are extremely potent and can induce respiratory failure, requiring long-term intensive care to prevent death. Recombinant monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics. In contrast, equine antitoxin cannot be used prophylactically and has a short half-life. Two three-mAb combinations are in development that specifically neutralize BoNT serotype A (BoNT/A) and B (BoNT/B). The three-mAb combinations addressing a single serotype provided pre-exposure prophylaxis in the guinea pig inhalation model. A lyophilized co-formulation of six mAbs, designated G03-52-01, that addresses both A and B serotypes is in development. Here, we investigated the efficacy of G03-52-01 to protect guinea pigs against an aerosol exposure challenge of BoNT/A1 or BoNT/B1. Previously, it was found that each antibody demonstrated a dose-dependent exposure and reached maximum circulating concentrations within 48 h after intramuscular (IM) or intravenous (IV) injection. Here we show that G03-52-01, in a single IM injection of G03-52-01 administered 48 h pre-exposure, protected guinea pigs against an aerosol challenge of up to 238 LD50s of BoNT/A1 and 191 LD50s of BoNT/B1. These data suggest that a single IM administration of G03-52-01 provides pre-exposure prophylaxis against botulism from an aerosol exposure of BoNT/A1 or BoNT/B1.

Comparative Study of Subcritical Water and Microwave-Assisted Extraction Techniques Impact on the Phenolic Compounds and 5-Hydroxymethylfurfural Content in Pomegranate Peel.

Two environmentally friendly innovative extraction techniques - subcritical water (SWE) and microwave-assisted extraction (MAE) were applied for the extraction of phenolics from pomegranate peel. The impact of process conditions (SWE: temperature 100-220 °C, extraction time 5-30 min; MAE: solvent water and 50% ethanol, irradiation power 470 and 800 W) on the quality of extracts in terms of the content of total phenolics, total flavonoids, major phenolic constituents (gallic acid, ellagic acid, punicalin, punicalagin), as well as 5-hydroxymethylfurfural(HMF) amount was investigated. For SWE, temperature of 130 °C and 20 min extraction time were found optimal for obtaining high content of bioactive compounds and minimizing the yield of HMF. During MAE, phenolic compounds were effectively extracted by using lower microwave power and 50% ethanol. Comparing two techniques, MAE is more efficient than SWE for the extraction of phenolics from pomegranate peel while obtaining a HMF-free extracts.

Valorization of Yarrow (Achillea millefolium L.) By-Product through Application of Subcritical Water Extraction.

In the present study, valorization of yarrow (Achillea millefolium) by-product from the filter tea industry was investigated through the application of subcritical water for the extraction of bioactive compounds. The influence of different process parameters (temperature 120-200 °C, extraction time 10-30 min, and HCl concentration in extraction solvent 0-1.5%) on extract quality in terms of content of bioactive compounds and antioxidant activity was investigated. Optimal conditions of the extraction process (temperature 198 °C, extraction time 16.5 min, and without acidifer) were determined and, when applied, the most efficient exploitation of by-products is achieved, that is, attainment of extracts rich in total phenols and flavonoids and high antioxidant activity. In addition, by applying the high performance liquid chromatographic analysis, the content of chlorogenic acid was determined as well as the hydroxymethylfurfural content in obtained extracts. The results demonstrated that subcritical water can be successfully used for utilization of yarrow by-products for obtaining extracts rich in antioxidants.

Spray Drying of a Subcritical Extract Using Marrubium vulgare as a Method of Choice for Obtaining High Quality Powder.

White horehound (Marrubium vulgare L.), is a grey-leaved perennial herb, belonging to Lamiaceae family, distributed in Eurasia and northern Africa. Despite the fact that M. vulgare has been used since ancient times in treating diverse diseases, it is only in the last decade or so that scientists have been able to lay the foundation for its potential pharmacological actions from the results observed through the prism of ethnopharmacological use of this species. The novelty of this study was that subcritical water extraction, acknowledged as a powerful extraction technology to recover phenolic compounds, was coupled with spray drying. The subcritical horehound extract, obtained using optimal process parameters, was used as a liquid feed in spray drying. Maltodextrin was used as a carrier in a concentration of 10%. Thus, two M. vulgare powders, carrier-free and 10% MD, were produced. Comprehensive powders characterization was conducted in order to evaluate their quality. Results confirmed that spray drying can be used as a method of choice for obtaining high quality horehound powders which kept the amorphous structure constant after 6 months.

Safety and Pharmacokinetics of a Four Monoclonal Antibody Combination Against Botulinum C and D Neurotoxins.

Botulism is caused by botulinum neurotoxin (BoNT), the most poisonous substance known. BoNTs are also classified as Tier 1 biothreat agents due to their high potency and lethality. The existence of seven BoNT serotypes (A-G), which differ between 35% to 68% in amino acid sequence, necessitates the development of serotype specific countermeasures. We present results of a Phase 1 clinical study of an anti-toxin to BoNT serotypes C and D, NTM-1634, which consists of an equimolar mixture of four fully human IgG1 monoclonal antibodies (mAbs), each binding to non-overlapping epitopes on BoNT serotypes C and D resulting in potent toxin neutralization in rodents. This first-in-human study evaluated the safety and pharmacokinetics of escalating doses of NTM-1634 administered intravenously to healthy adults (NCT03046550). Three cohorts of eight healthy subjects received a single intravenous dose of NTM-1634 or placebo at 0.33 mg/kg, 0.66 mg/kg or 1 mg/kg. Follow-up examinations and pharmacokinetic evaluations were continued up to 121 days post-infusion. Subjects were monitored using physical examinations, hematology and chemistry blood tests, and electrocardiograms. Pharmacokinetic parameters were estimated using noncompartmental methods. The results demonstrated that the materials were safe and well-tolerated with the expected half-lives for human mAbs and with minimal anti-drug antibodies detected over the dose ranges and duration of the study.

Pharmacokinetics of Human Recombinant Anti-Botulinum Toxin Antibodies in Rats.

Botulinum neurotoxins (BoNT) are potential biothreat agents due to their high lethality, potency, and ease of distribution, thus the development of antitoxins is a high priority to the US government. This study examined pre-clinical pharmacokinetic studies in rats of four oligoclonal anti-BoNT mAb-based therapeutics (NTM-1631, NTM-1632, NTM-1633, NTM-1634) for five BoNT serotypes (A, B, E, C, and D). NTM-1631, NTM-1632, and NTM-1633 each consist of three IgG1 mAbs, each with a distinct human or humanized variable region which bind to distinct epitopes on BoNT serotype A, B, or E respectively. NTM-1634 consists of four human immunoglobulin G1 (IgG1) mAbs binding BoNT C/D mosaic toxins. The mechanism of these antitoxins requires that three antibodies simultaneously bind toxin to achieve rapid clearance. Rats (total 378) displayed no adverse clinical signs attributed to antibody treatment from any of the antitoxins. Pharmacokinetic evaluation demonstrated that the individual mAbs are slowly eliminated, exhibiting dose-dependent exposure and long elimination half-lives ranging from 6.5 days to 10 days. There were no consistent differences observed between males and females or among the individual antibodies in each formulation in half-life. Anti-drug antibodies (ADA) were observed, as expected for human antibodies administered to rats. The results presented were used to support the clinical investigation of antibody-based botulism antitoxins.

Monoclonal Antibody Combinations Prevent Serotype A and Serotype B Inhalational Botulism in a Guinea Pig Model.

Botulinum neurotoxins (BoNT) are some of the most toxic proteins known, with a human LD50 of ~1 ng/kg. Equine antitoxin has a half-life in circulation of less than 1 day and is limited to a treatment rather than a prevention indication. The development of monoclonal antibodies (mAbs) may represent an alternative therapeutic option that can be produced at high quantities and of high quality and with half-lives of >10 days. Two different three mAb combinations are being developed that specifically neutralize BoNT serotypes A (BoNT/A) and B (BoNT/B). We investigated the pharmacokinetics of the anti-BoNT/A and anti-BoNT/B antibodies in guinea pigs (Cavia porcellus) and their ability to protect guinea pigs against an aerosol challenge of BoNT/A1 or BoNT/B1. Each antibody exhibited dose-dependent exposure and reached maximum circulating concentrations within 48 h post intraperitoneal or intramuscular injection. A single intramuscular dose of the three mAb combination protected guinea pigs against an aerosol challenge dose of 93 LD50 of BoNT/A1 and 116 LD50 of BoNT/B1 at 48 h post antibody administration. These mAbs are effective in preventing botulism after an aerosol challenge of BoNT/A1 and BoNT/B1 and may represent an alternative to vaccination to prevent type A or B botulism in those at risk of BoNT exposure.

An ambient temperature-stable antitoxin of nine co-formulated antibodies for botulism caused by serotypes A, B and E.

Safe and effective antitoxins to treat and prevent botulism are needed for biodefense. We have developed recombinant antibody-based therapeutics for botulinum neurotoxin (BoNT) serotypes A, B, and E. The mechanism of action of this antitoxin requires that three mAbs bind one toxin molecule to achieve clearance. Here we present a co-formulation of an antitoxin to the three most important serotypes. Combining these antibodies obviates the need to identify the serotype causing intoxication prior to drug administration, which would facilitate administration. The lyophilized powder formulation contains nine mAbs, three mAbs for each of the three serotypes (A, B, E). The formulation was stored as a liquid and lyophilized powder for up to one year, and characterized by binding affinity and multiple physicochemical methods. No significant increase in soluble higher order aggregates, cleavage products, or change in charge isoforms was measured after storage as a lyophilized powder at 50°C for one year. Furthermore, toxin-domain binding ELISA data indicated that each of the individual antibodies in the lyophilized drug product showed essentially full binding capability to their respective toxin domains after being stored at 50°C for one year. Physicochemical characterization of the formulation demonstrated the nine individual mAbs were remarkably stable. This work demonstrates feasibility of lyophilized, oligoclonal antibody therapies for biodefense with ambient temperature stability, that would facilitate stockpiling, distribution, and administration.

Antibody engineering to improve manufacturability.

Expression variation among antibodies produced by stably transfected Chinese Hamster Ovary (CHO) cells is well established. While developing CHO-K1 cell lines, we encountered a human monoclonal antibody, mAb B-c, with severe manufacturability issues, including very poor expression and high levels of heavy chain (HC) dimer and high molecular weight aggregates. Using transient expression in CHO-K1 cells, we identified light chain (LC) as the source of the manufacturability issues for this antibody. While other antibodies achieved optimal expression at 1:1 or 2:1 LC to HC ratios, mAb B-c required up to a 6:1 LC:HC for maximal expression, which was still significantly lower than that for other tested antibodies. To overcome the manufacturability issues, LC shuffling was performed with the original HC to select antibodies with unique LCs which retained acceptable binding affinities. Transient CHO-K1 expression evaluation of the new LCs co-expressed with the original HC identified antibodies with high expression at a 1:1 or 2:1 LC:HC; the expression levels of these new antibodies were comparable to those of other well-expressed antibodies. Expression of these new antibodies in stably transfected CHO-K1 cells confirmed these results. In addition, antibodies containing the new LCs had very low levels of high molecular weight aggregates and HC dimer. These results demonstrate that certain antibody manufacturability issues can be attributed to LC and that engineering antibodies by pairing HCs with alternate LCs can improve antibody expression and product quality while maintaining or improving affinity.

Pressure-Induced Conductivity in a Neutral Nonplanar Spin-Localized Radical.

There is a growing interest in the development of single-component molecular conductors based on neutral organic radicals that are mainly formed by delocalized planar radicals, such as phenalenyl or thiazolyl radicals. However, there are no examples of systems based on nonplanar and spin-localized C-centered radicals exhibiting electrical conductivity due to their large Coulomb energy (U) repulsion and narrow electronic bandwidth (W) that give rise to a Mott insulator behavior. Here we present a new type of nonplanar neutral radical conductor attained by linking a tetrathiafulvalene (TTF) donor unit to a neutral polychlorotriphenylmethyl radical (PTM) with the important feature that the TTF unit enhances the overlap between the radical molecules as a consequence of short intermolecular S···S interactions. This system becomes semiconducting upon the application of high pressure thanks to increased electronic bandwidth and charge reorganization opening the way to develop a new family of neutral radical conductors.

Charge transfer tuning by chemical substitution and uniaxial pressure in the organic complex tetramethoxypyrene-tetracyanoquinodimethane.

In the search for novel organic charge transfer salts with variable degrees of charge transfer we have studied the effects of two modifications of the recently synthesized donor-acceptor system [tetramethoxypyrene (TMP)]-[tetracyanoquinodimethane (TCNQ)]. One is of chemical nature by substituting the acceptor TCNQ molecules by F4TCNQ molecules. The second consists in simulating the application of uniaxial pressure along the stacking axis of the system. In order to test the chemical substitution, we have grown single crystals of the TMP-F4TCNQ complex and analyzed its electronic structure via electronic transport measurements, ab initio density functional theory (DFT) calculations and UV/VIS/IR absorption spectroscopy. This system shows an almost ideal geometrical overlap of nearly planar molecules stacked alternately (mixed stack) and this arrangement is echoed by a semiconductor-like transport behavior with an increased conductivity along the stacking direction. This is in contrast to TMP-TCNQ which shows a less pronounced anisotropy and a smaller conductivity response. Our band structure calculations confirm the one-dimensional behavior of TMP-F4TCNQ with pronounced dispersion only along the stacking axis. Infrared measurements illustrating the C≡N vibration frequency shift in F4TCNQ suggest however no improvement in the degree of charge transfer in TMP-F4TCNQ with respect to TMP-TCNQ. In both complexes about 0.1e is transferred from TMP to the acceptor. Concerning the pressure effect, our DFT calculations on the designed TMP-TCNQ and TMP-F4TCNQ structures under different pressure conditions show that application of uniaxial pressure along the stacking axis of TMP-TCNQ may be the route to follow in order to obtain a much more pronounced charge transfer.

Giant pressure-induced volume collapse in the pyrite mineral MnS2.

Dramatic volume collapses under pressure are fundamental to geochemistry and of increasing importance to fields as diverse as hydrogen storage and high-temperature superconductivity. In transition metal materials, collapses are usually driven by so-called spin-state transitions, the interplay between the single-ion crystal field and the size of the magnetic moment. Here we show that the classical S = 5/2 mineral hauerite (MnS2) undergoes an unprecedented (ΔV ~ 22%) collapse driven by a conceptually different magnetic mechanism. Using synchrotron X-ray diffraction we show that cold compression induces the formation of a disordered intermediate. However, using an evolutionary algorithm we predict a new structure with edge-sharing chains. This is confirmed as the thermodynamic ground state using in situ laser heating. We show that magnetism is globally absent in the new phase, as low-spin quantum S = 1/2 moments are quenched by dimerization. Our results show how the emergence of metal-metal bonding can stabilize giant spin-lattice coupling in Earth's minerals.

Absence of metallicity in K-doped picene: importance of electronic correlations.

Potassium-doped picene (K(x)picene) has recently been reported to be a superconductor at x=3 with critical temperatures up to 18 K. Here we study the electronic structure of K-doped picene films by photoelectron spectroscopy and ab initio density functional theory combined with dynamical mean-field theory (DFT+DMFT). Experimentally we observe that, except for spurious spectral weight due to the lack of a homogeneous chemical potential at low K concentrations (x≈1), the spectra always display a finite energy gap. This result is supported by our DFT+DMFT calculations which provide clear evidence that K(x)picene is a Mott insulator for integer doping concentrations x=1, 2, and 3. We discuss various scenarios to understand the discrepancies with previous reports of superconductivity and metallic behavior.

Separation of oxidized variants of a monoclonal antibody by anion-exchange.

Monoclonal antibodies are subject to a variety of degradation mechanisms, therefore orthogonal techniques are required to demonstrate product quality. In this study, the three individual antibodies comprising a multi-antibody drug product, XOMA 3AB were evaluated by both cation-exchange (CEX) and anion-exchange chromatography (AEX). In contrast to CEX analysis which showed only a single, broad peak for the force-oxidized antibodies, AEX analysis of Ab-A (pI=7.6) revealed two more basic peaks. Ab-B (pI=6.7) bound but exhibited only a single major peak while Ab-C (pI=8.6) flowed through. Peptide mapping LC/MS analysis of the isolated Ab-A fractions demonstrated that the basic peaks resulted from oxidation in a complementary determining region (CDR). Differential scanning calorimetry (DSC) analysis of the oxidized Ab-A species showed a decrease in the Fab melting point for the oxidized species consistent with unfolding of the molecule. Greater/lesser surface exposure of ionic residues resulting from a conformational change provides a likely explanation for the dramatic shift in retention behavior for the Ab-A oxidized variants. Peptide mapping analysis of the Ab-B antibody showed, in contrast to Ab-A, no detectable CDR oxidation. Hence, the lack of separation of oxidized variants in Ab-B can be explained by the absence of CDR oxidation and the associated changes in secondary/tertiary structure which were observed for oxidized Ab-A. In summary, anion-exchange HPLC shows potential as an orthogonal analytical technique for assessing product quality of monoclonal antibody therapeutics. In the case of the XOMA 3AB drug product, two of the antibodies bound and one, Ab-A, exhibited separation of CDR oxidized variants.