ELISA assay for the quantification of ipilimumab in human serum, plasma, milk, and cerebrospinal fluid.

Ipilimumab is an immune checkpoint inhibitor of the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Ipilimumab has become part of the standard of care for different types of cancer. The efficacy of these treatments is limited due to immune-related toxicity and high economic costs. Dose rationalization studies based on pharmacokinetic data may help to address these limitations. For this purpose, more sensitive analytical methods are needed. We report the development and validation of the first enzyme-linked immunosorbent assay (ELISA) for sensitive determination of ipilimumab concentrations in human serum, plasma, cerebrospinal fluid (CSF), and milk. Our assay is based on the specific capture of ipilimumab by immobilized CTLA-4. The lower limit of quantifications of ipilimumab in serum, plasma, and milk are 50 ng/mL and 10 ng/mL in CSF. The ELISA method showed long-term storage stability for at least one year at -80°C and was successfully cross-validated with ultraperformance liquid chromatography coupled with tandem mass spectrometry. The ELISA method is reliable, relatively inexpensive, and can be used in serum, plasma, CSF, and milk from patients treated with ipilimumab, as evidenced by the analysis of real clinical samples.

A multiplex UPLC-MS/MS method for the quantification of three PD-L1 checkpoint inhibitors, atezolizumab, avelumab, and durvalumab, in human serum.

BACKGROUND AND AIM: To support pharmacokinetic studies, a multiplex UPLC-MS/MS assay was developed and validated to quantify PD-L1 checkpoint inhibitors atezolizumab, avelumab, and durvalumab in serum. METHODS: A bottom-up sample pre-treatment procedure was developed to determine atezolizumab, avelumab, and durvalumab in serum. This procedure consisted of (1) precipitation of the monoclonal antibody with ammonium sulfate, (2) reduction with dithiothreitol, (3) denaturation with methanol, and (4) tryptic digestion of the protein. The unique signature peptides resulting after sample pre-treatment of the antibodies were measured using UPLC-MS/MS with a total run time of 11 minutes. The clinical application was evaluated by analyzing 114 atezolizumab patient samples. RESULTS: The developed method was found to be accurate and precise for all three analytes over a concentration range of 3.00-150 µg/mL. No endogenous interference was present in serum samples. Cross-interference experiments showed no cross-analyte interference and acceptable cross-internal standard interference. In addition, no substantial carry-over was observed. The stable isotopically labeled signature peptides were most effective in compensating for matrix effects. Recovery based on back-calculated concentrations of calibration standards and quality control samples was found to be high. The analytes were stable for at least three freeze-thaw cycles, for 42 hours at processing conditions, for at least two days at 2-8°C in the final extract, for five days before re-injection analysis at 4°C, and long-term for at least 11 months at -70°C. The assay was tested for its applicability in clinical practice. For this purpose, 114 atezolizumab patient samples were measured. CONCLUSION: A multiplex UPLC-MS/MS assay was developed and validated to quantify atezolizumab, avelumab, and durvalumab in human serum. The applicability of this method was demonstrated by the analysis of clinical atezolizumab samples. The method is suitable to support clinical pharmacokinetic studies involving atezolizumab, avelumab, or durvalumab.

Continuous sound from a marine vibrator causes behavioural responses of free-ranging, spawning Atlantic cod (Gadus morhua).

Marine vibrators are a new technology being developed for seismic surveys. These devices can transmit continuous instead of impulsive sound and operate over a narrower frequency band and at lower peak pressure than airguns, which is assumed to reduce their environmental impacts. We exposed spawning Atlantic cod (Gadus morhua) to sound produced by a prototype, but full-scale, marine vibrator, and monitored behavioural responses of tagged cod using acoustic telemetry. Fish were exposed to 10 × 3 h continuous sound treatments over a 4-day period using a randomised-block design. Sound exposure levels were comparable to airgun exposure experiments conducted previously with the same set-up ranging from ∼115 to 145 dB re 1 µPa2s during exposure. Telemetry data were used to assess 1) whether marine vibrator exposure displaced cod from the spawning ground, through estimation of residence and survival probabilities, and 2) fine-scale behavioural responses within the test site, namely swimming depth, activity levels, displacement, and home ranges. Forty-two spawning cod were tagged prior to the exposure, with 22 present during the exposure. All 22 tags were equipped with pressure sensors and ten of these additionally with accelerometers. While no premature departure from the spawning site was observed, cod reacted to the exposure by decreasing their activity levels (by up to 50%, SE = 7%) and increasing their swimming depth (by up to 2.5 m, SE = 1.0 m) within the test site during the exposure period. These behavioural responses varied by sex and time of day. Cod reactions to a marine vibrator may be more pronounced than reactions to airguns, possibly because continuous sound is more disturbing to fish than intermittent sound at the same exposure levels. However, given sample size limitations of the present study, further studies with continuous sound are necessary to fully understand its impact and biological significance.

High accumulation of nivolumab in human breast milk: A case report.

Nivolumab is an immunotherapeutic monoclonal antibody (mAb) that is used for the treatment of several types of cancer. The evidence on its use during lactation is lacking. Here, we report on a 39-year-old woman with metastasized melanoma who was treated with 480 mg nivolumab every four weeks during lactation. Breast milk samples were collected over the course of 34 days, including two cycles of nivolumab. The highest measured concentration of nivolumab during the first cycle was 503 ng/mL at day 13. The cumulative relative infant dose (RID) over the first cycle (28 days) was 9.8 %. The highest overall measured nivolumab concentration was 519 ng/mL at day 33, five days after administration of the second nivolumab cycle. Nivolumab seems to accumulate in breast milk over two consecutive cycles, hence the RIDs of consecutive cycles are expected to be higher. To draw further conclusions regarding safety of breastfeeding during nivolumab therapy, more information about the oral bioavailability of nivolumab in newborns, the nivolumab steady-state concentrations in breast milk and its pharmacodynamic effects are needed.

Effects of airgun discharges used in seismic surveys on development and mortality in nauplii of the copepod Acartia tonsa.

Seismic surveys are conducted worldwide to explore for oil and gas deposits and to map subsea formations. The airguns used in these surveys emit low-frequency sound waves. Studies on zooplankton responses to airguns report a range of effects, from none to substantial mortality. A field experiment was conducted to assess mortality and naupliar body length of the calanoid copepod Acartia tonsa when exposed to the discharge of two 40-inch airguns. Nauplii were placed in plastic bags and attached to a line at a depth of 6 m. For each treatment, three bags of nauplii were exposed to one of three treatments for 2.5 h: Airgun array discharge, a boat control, or a silent control. After exposure, nauplii were kept in filtered seawater in the laboratory without food. Immediate mortality in the nauplii was approximately 14% compared to less than 4% in the silent and boat control. Similarly, there was higher mortality in the airgun exposed nauplii up to six days after exposure compared to the control treatments. Nearly all of the airgun exposed nauplii were dead after four days, while >50% of the nauplii in the control treatments were alive at six days post-exposure. There was an interaction between treatment and time on naupliar body length, indicating lower growth in the nauplii exposed to the airgun discharge (growth rates after 4 days: 1.7, 5.4, and 6.1 µm d-1 in the airgun exposed, silent control, and boat control, respectively). These experiments indicate that the output of two small airguns affected mortality and growth of the naupliar stages of Acartia tonsa in close vicinity to the array.

Optimized sample pre-treatment procedure for the simultaneous UPLC-MS/MS quantification of ipilimumab, nivolumab, and pembrolizumab in human serum.

Ipilimumab, nivolumab, and pembrolizumab are immune checkpoint inhibiting monoclonal antibodies. Their efficacy has been proven to be correlated with clearance, and hence, bioanalytical assays to study their pharmacokinetics are of pivotal importance. We present the first kit-free sample pre-treatment procedure of only three hours for the Ultra-Performance Liquid Chromatography - tandem Mass Spectrometry (UPLC-MS/MS) simultaneous quantification of ipilimumab, nivolumab, and pembrolizumab in human serum. The conventional bottom-up sample pre-treatment steps for protein MS bioanalysis including pre-digestion purification, denaturation, reduction, alkylation, and digestion were optimized in terms of sensitivity and reproducibility. In the final, optimal sample pre-treatment procedure, samples were purified by protein precipitation with saturated ammonium sulfate solution, reduced with dithiothreitol, denatured with methanol, and digested with trypsin. The method was then validated according to European Medicines Agency (EMA) and the United States Food and Drug Administration (FDA) guidelines for bioanalytical method validation, and 4-6-20 acceptance criteria were applied. This method was selective, accurate, and precise within the range of 3-200 µg/mL for all analytes. The validated developed assay was applied to determine ipilimumab, nivolumab, and pembrolizumab concentrations in patient serum, and the results were compared to enzyme-linked immunosorbent assay (ELISA) results.

Quantification of anti-SARS-CoV-2 antibodies in human serum with LC-QTOF-MS.

The aim of this study was to develop the first quantitative serological test for anti-SARS-CoV-2 antibodies in human serum with liquid chromatography - quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Other assays, mostly immunoassays, are only qualitative or semi-quantitative, and hence, actual antibody concentrations after SARS-CoV-2 infection are unknown. In our assay, anti-SARS-CoV-2 antibodies were isolated with spike protein subunit 1 (S1) coupled to magnetic beads. IgG1 signature peptide GPSVFPLAPSSK was selected for quantification using ipilimumab calibration standards and SILuMAb K1 as the stable-isotope labeled internal standard. The anti-SARS-CoV-2 IgG1 calibration range was from 1.35 to 135 nM. Inter-assay accuracies were between 98.8%- 107% with inter-assay precisions between 8.37%- 13.5% measured at 3 concentration levels on three separate occasions. Anti-SARS-CoV-2 IgG1 antibodies were quantified in PCR-positive patients with mild to severe symptoms. IgM signature peptide DGFFGVPR was detected in patients that recently recovered from COVID-19. A unique and quantitative LC-QTOF-MS method to quantify anti-SARS-CoV-2 IgG1 in serum was successfully developed and its clinical applicability has been demonstrated.

Simultaneous quantification of abemaciclib and its active metabolites in human and mouse plasma by UHPLC-MS/MS.

Abemaciclib is the third cyclin-dependent kinase 4 and 6 inhibitor approved for the treatment of advanced or metastatic breast cancer. In humans, abemaciclib is extensively metabolized by CYP3A4 with the formation of three active metabolites: N-desethylabemaciclib (M2), hydroxyabemaciclib (M20) and hydroxy-N-desethylabemaciclib (M18). These metabolites showed similar potency compared to the parent drug and were significantly abundant in plasma circulation. Thus, M2, M20, and M18 may contribute to the clinical activity of abemaciclib. For this reason, an UHPLC-MS/MS method for the simultaneous quantification of abemaciclib and its active metabolites in human and mouse plasma was developed and validated to support further clinical or preclinical investigations on this drug. Samples were processed by protein precipitation with acetonitrile, followed by supernatant dilution and filtration. Chromatographic separation was performed on a Kinetex C18 column (150 × 2.1 mm ID, 2.6 µm) using gradient elution with 10 mM ammonium bicarbonate in water (eluent A) and in methanol-water (9:1, v/v, eluent B). This method was selective, linear, accurate and precise within the range of 1-600 ng/mL for abemaciclib, 0.5-300 ng/mL for M2 and M20, and 0.2-120 ng/mL for M18. Furthermore, stability of the analytes in human and mouse plasma samples in several conditions was demonstrated. Finally, this assay was successfully used in a preclinical pharmacokinetic study, where abemaciclib and its active metabolites were identified and quantified. Inter-species differences between human and mouse samples were encountered, especially in the formation of M20, where isomers of this compound were detected in mouse plasma, but not in human plasma. This was confirmed by high resolution-mass spectrometry (HR-MS) measurements.

Trained Immunity-Promoting Nanobiologic Therapy Suppresses Tumor Growth and Potentiates Checkpoint Inhibition.

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.

Bottom-up sample preparation for the LC-MS/MS quantification of anti-cancer monoclonal antibodies in bio matrices.

Therapeutic monoclonal antibodies (mAbs) are rapidly taking over the treatment of many malignancies, and an astonishing number of mAbs is in development. This causes a high demand for quantification of mAbs in biomatrices both for measuring therapeutic mAb concentrations and to support pharmacokinetics and pharmacodynamics studies. Conventionally, ligand-binding assays are used for these purposes, but LC-MS is gaining popularity. Although intact (top-down) and subunit (middle-down) mAb quantification is reported, signature peptide (bottom-up) quantification is currently most advantageous. This review provides an overview of the reported bottom-up mAb quantification methods in biomatrices as well as general recommendations regarding signature peptide and internal standard selection, reagent use and optimization of digestion in bottom-up quantification methods.

Electric and magnetic senses in marine animals, and potential behavioral effects of electromagnetic surveys.

Electromagnetic surveys generate electromagnetic fields to map petroleum deposits under the seabed with unknown consequences for marine animals. The electric and magnetic fields induced by electromagnetic surveys can be detected by many marine animals, and the generated fields may potentially affect the behavior of perceptive animals. Animals using magnetic cues for migration or local orientation, especially during a restricted time-window, risk being affected by electromagnetic surveys. In electrosensitive animals, anthropogenic electric fields could disrupt a range of behaviors. The lack of studies on effects of the electromagnetic fields induced by electromagnetic surveys on the behavior of magneto- and electrosensitive animals is a reason for concern. Here, we review the use of electric and magnetic fields among marine animals, present data on survey generated and natural electromagnetic fields, and discuss potential effects of electromagnetic surveys on the behavior of marine animals.

Noise can affect acoustic communication and subsequent spawning success in fish.

There are substantial concerns that increasing levels of anthropogenic noise in the oceans may impact aquatic animals. Noise can affect animals physically, physiologically and behaviourally, but one of the most obvious effects is interference with acoustic communication. Acoustic communication often plays a crucial role in reproductive interactions and over 800 species of fish have been found to communicate acoustically. There is very little data on whether noise affects reproduction in aquatic animals, and none in relation to acoustic communication. In this study we tested the effect of continuous noise on courtship behaviour in two closely-related marine fishes: the two-spotted goby (Gobiusculus flavescens) and the painted goby (Pomatoschistus pictus) in aquarium experiments. Both species use visual and acoustic signals during courtship. In the two-spotted goby we used a repeated-measures design testing the same individuals in the noise and the control treatment, in alternating order. For the painted goby we allowed females to spawn, precluding a repeated-measures design, but permitting a test of the effect of noise on female spawning decisions. Males of both species reduced acoustic courtship, but only painted gobies also showed less visual courtship in the noise treatment compared to the control. Female painted gobies were less likely to spawn in the noise treatment. Thus, our results provide experimental evidence for negative effects of noise on acoustic communication and spawning success. Spawning is a crucial component of reproduction. Therefore, even though laboratory results should not be extrapolated directly to field populations, our results suggest that reproductive success may be sensitive to noise pollution, potentially reducing fitness.

Evidence for ontogenetically and morphologically distinct alternative reproductive tactics in the invasive Round Goby Neogobius melanostomus.

Alternative reproductive tactics are characterized by the occurrence of discrete alternative morphs that differ in behavioural, morphological and physiological traits within the same sex. Although much effort has been made to describe the behaviour, morphology and physiology of such alternative morphs, less effort has been invested investigating how much overlap there is in the characteristics of such morphs in natural populations. We studied random population samples of the invasive Round Goby Neogobius melanostomus from five different localities in the river Rhine system in the Netherlands. We found two morphologically and physiologically distinct male morphs which likely represent alternative reproductive tactics. Almost all mature males under 9.35 cm total length had a gonadosomatic index > 3%, suggestive of a sneaker tactic, while nearly all males above 9.35 cm has a gonadosomatic index of < 3%, suggestive of a parental tactic. Cheek size and eye diameter alone were sufficient to distinguish the two morphs. Gonads had a different relationship with size in the two morphs, indicating separate growth trajectories. The gonad mass of sneaker morphs would be ca. 7.5 times as high as the gonad mass of parental morphs of the same total length after extrapolation. Few (9%) intermediates were found, suggesting that the expression of alternative reproductive tactics is determined before the first breeding season. This contrasts with studies on other goby species, which show evidence of plastic tactics that can be affected by social circumstances. We conclude that it is possible to distinguish two alternative male morphs in the Dutch Round Goby population using morphological measurements alone. Although behavioural observations are needed to provide conclusive evidence, the difference in GSI between these morphs indicates that these morphs reflect alternative reproductive tactics.

Operational sex ratio but not density affects sexual selection in a fish.

The operational sex ratio (OSR) and density are considered important factors affecting the strength of sexual selection. Although there is increasing evidence that OSR and density affect the potential for sexual selection, few studies have addressed whether this is realized in phenotypic selection and how the two factors interact. We manipulated OSR (three levels) and male density (two levels) in 36 experimental breeding populations of Gobiusculus flavescens-a fish with paternal care. We measured mating competition behavior, the opportunity for selection (I), and selection on four morphological traits in males. We found sexual selection on two male traits, with the strongest selection being 20% of I. As predicted from OSR theory, increasing female scarcity caused males to become more competitive, concomitant with an increase in I and selection on morphological traits. Model simulations of I based on random mating (Imin ) and maximum mate monopolization (Imax ) demonstrated that the potential for sexual selection was close to its theoretical maximum across the range of OSRs. However, male density and its interaction with the OSR did not affect sexual selection. We argue that a multifaceted approach, combining mating behavior and selection analyses, can help us to understand how ecological factors affect sexual selection.

Sex roles and mutual mate choice matter during mate sampling.

The roles of females and males in mating competition and mate choice have lately proven more variable, between and within species, than previously thought. In nature, mating competition occurs during mate search and is expected to be regulated by the numbers of potential mates and same-sex competitors. Here, we present the first study to test how a temporal change in sex roles affects mating competition and mate choice during mate sampling. Our model system (the marine fish Gobiusculus flavescens) is uniquely suitable because of its change in sex roles, from conventional to reversed, over the breeding season. As predicted from sex role theory, courtship was typically initiated by males and terminated by females early in the breeding season. The opposite pattern was observed late in the season, at which time several females often simultaneously courted the same male. Mate-searching females visited more males early than late in the breeding season. Our study shows that mutual mate choice and mating competition can have profound effects on female and male behavior. Future work needs to consider the dynamic nature of mating competition and mate choice if we aim to fully understand sexual selection in the wild.