Residue-Level Characterization of Antibody Binding Epitopes Using Carbene Chemical Footprinting.

Characterization of antibody binding epitopes is an important factor in therapeutic drug discovery, as the binding site determines and drives antibody pharmacology and pharmacokinetics. Here, we present a novel application of carbene chemical footprinting with mass spectrometry for identification of antibody binding epitopes at the single-residue level. Two different photoactivated diazirine reagents provide complementary labeling information allowing structural refinement of the antibody binding interface. We applied this technique to map the epitopes of multiple MICA and CTLA-4 antibodies and validated the findings with X-ray crystallography and yeast surface display epitope mapping. The characterized epitopes were used to understand biolayer interferometry-derived competitive binding results at the structural level. We show that carbene footprinting provides fast and high-resolution epitope information critical in the antibody selection process and enables mechanistic understanding of function to accelerate the drug discovery process.

Impact of Drug Conjugation on Thermal and Metabolic Stabilities of Aglycosylated and N-Glycosylated Antibodies.

N-linked glycosylation is one of the most common and complex posttranslational modifications that govern the biological functions and physicochemical properties of therapeutic antibodies. We evaluated thermal and metabolic stabilities of antibody-drug conjugates (ADCs) with payloads attached to the C'E loop in the immunoglobulin G (IgG) Fc CH2 domain, comparing the glycosylated and aglycosylated Fc ADC variants. Our study revealed that introduction of small-molecule drugs into an aglycosylated antibody can compensate for thermal destabilization originating from structural distortions caused by elimination of N-linked glycans. Depending on the conjugation site, glycans had both positive and negative effects on plasma stability of ADCs. The findings highlight the importance of consideration for selection of conjugation site to achieve desirable physicochemical properties and plasma stability.

Atom Interferometry with Floquet Atom Optics.

Floquet engineering offers a compelling approach for designing the time evolution of periodically driven systems. We implement a periodic atom-light coupling to realize Floquet atom optics on the strontium ^{1}S_{0}-^{3}P_{1} transition. These atom optics reach pulse efficiencies above 99.4% over a wide range of frequency offsets between light and atomic resonance, even under strong driving where this detuning is on the order of the Rabi frequency. Moreover, we use Floquet atom optics to compensate for differential Doppler shifts in large momentum transfer atom interferometers and achieve state-of-the-art momentum separation in excess of 400 ℏk. This technique can be applied to any two-level system at arbitrary coupling strength, with broad application in coherent quantum control.

High-Throughput Platform to Identify Antibody Conjugation Sites from Antibody-Drug Conjugate Libraries.

Antibody-drug conjugates (ADCs) are a therapeutic modality that traditionally enable the targeted delivery of highly potent cytotoxic agents to specific cells such as tumor cells. More recently, antibodies have been used to deliver molecules such as antibiotics, antigens, and adjuvants to bacteria or specific immune cell subsets. Site-directed mutagenesis of proteins permits more precise control over the site and stoichiometry of their conjugation, giving rise to homogeneous chemically defined ADCs. Identification of favorable sites for conjugation in antibodies is essential as reaction efficiency and product stability are influenced by the tertiary structure of immunoglobulin G (IgG). Current methods to evaluate potential conjugation sites are time-consuming and labor intensive, involving multistep processes for individually produced reactions. Here, we describe a highly efficient method for identification of conjugatable genetic variants by analyzing pooled ADC libraries using mass spectrometry. This approach provides a versatile platform to rapidly uncover new conjugation sites for site-specific ADCs.

Large Momentum Transfer Clock Atom Interferometry on the 689 nm Intercombination Line of Strontium.

We report the first realization of large momentum transfer (LMT) clock atom interferometry. Using single-photon interactions on the strontium ^{1}S_{0}-^{3}P_{1} transition, we demonstrate Mach-Zehnder interferometers with state-of-the-art momentum separation of up to 141 ℏk and gradiometers of up to 81 ℏk. Moreover, we circumvent excited state decay limitations and extend the gradiometer duration to 50 times the excited state lifetime. Because of the broad velocity acceptance of the interferometry pulses, all experiments are performed with laser-cooled atoms at a temperature of 3 µK. This work has applications in high-precision inertial sensing and paves the way for LMT-enhanced clock atom interferometry on even narrower transitions, a key ingredient in proposals for gravitational wave detection and dark matter searches.

Effective Inertial Frame in an Atom Interferometric Test of the Equivalence Principle.

In an ideal test of the equivalence principle, the test masses fall in a common inertial frame. A real experiment is affected by gravity gradients, which introduce systematic errors by coupling to initial kinematic differences between the test masses. Here we demonstrate a method that reduces the sensitivity of a dual-species atom interferometer to initial kinematics by using a frequency shift of the mirror pulse to create an effective inertial frame for both atomic species. Using this method, we suppress the gravity-gradient-induced dependence of the differential phase on initial kinematic differences by 2 orders of magnitude and precisely measure these differences. We realize a relative precision of Δg/g≈6×10^{-11} per shot, which improves on the best previous result for a dual-species atom interferometer by more than 3 orders of magnitude. By reducing gravity gradient systematic errors to one part in 10^{13}, these results pave the way for an atomic test of the equivalence principle at an accuracy comparable with state-of-the-art classical tests.

Fucosyl monosialoganglioside: Quantitative analysis of specific potential biomarkers of lung cancer in biological matrices using immunocapture extraction/tandem mass spectrometry.

RATIONALE: Certain lung cancer patients express elevated Fucosyl Monosialoganglioside (Fuc-GM1) in circulation compared to control groups. Several sensitive methods involving characterization of Fuc-GM1 have been reported. However, a highly specific and sensitive method for quantifying multiple potential Fuc-GM1 biomarkers present in various biological matrices has not been reported to date. METHODS: Individual Fuc-GM1 analogs in a commercially obtained standard mixture were characterized using HPLC/UV/MS and high-resolution mass spectrometry (HRMS). Proprietary antibodies, mAb1 and mAb2, were used to selectively capture and pre-concentrate the soluble and drug-bound forms of Fuc-GM1 molecules present in human serum and whole blood, eliminating the background matrix components. Immunocapture extraction (ICE) followed by HPLC/MS/MS was used to quantify specific Fuc-GM1 analogs in biological matrices. RESULTS: The concentration of individual Fuc-GM1 analogs in the standard mixture was estimated to be 7-34%, using HPLC/UV/MS. Using the standard mixture spiked into the biological matrices (100 µL), the lower limit of quantification (LLOQ) of each analog was 0.2-0.4 ng/mL with a dynamic range of up to 200 ng/mL. The applicability of the ICE-HPLC/MS/MS method was demonstrated by detecting endogenous Fuc-GM1 analogs present in rat blood and in several lung cancer cell lines. CONCLUSIONS: This highly specific and sensitive HPLC/MS/MS method for quantifying individual potential Fuc-GM1 biomarkers in serum and whole blood can play a critical role in patient stratification strategies and during drug treatment. This method can be employed for monitoring both free (soluble) form and antibody drug-bound Fuc-GM1.

Phase Shift in an Atom Interferometer due to Spacetime Curvature across its Wave Function.

Spacetime curvature induces tidal forces on the wave function of a single quantum system. Using a dual light-pulse atom interferometer, we measure a phase shift associated with such tidal forces. The macroscopic spatial superposition state in each interferometer (extending over 16 cm) acts as a nonlocal probe of the spacetime manifold. Additionally, we utilize the dual atom interferometer as a gradiometer for precise gravitational measurements.

Matter wave lensing to picokelvin temperatures.

Using a matter wave lens and a long time of flight, we cool an ensemble of ^{87}Rb atoms in two dimensions to an effective temperature of less than 50_{-30}^{+50} pK. A short pulse of red-detuned light generates an optical dipole force that collimates the ensemble. We also report a three-dimensional magnetic lens that substantially reduces the chemical potential of evaporatively cooled ensembles with a high atom number. By observing such low temperatures, we set limits on proposed modifications to quantum mechanics in the macroscopic regime. These cooling techniques yield bright, collimated sources for precision atom interferometry.

Identifying physical therapists' attitudes, beliefs, and barriers toward diagnostic imaging referral: a mixed-methods study.

OBJECTIVE: Ten states, including the District of Columbia, have laws that currently permit physical therapists (PTs) to directly order diagnostic imaging (DI) in the United States. Military and civilian PTs order DI judiciously and appropriately demonstrating optimal patient outcomes and satisfaction when compared to other medical professionals. However, no studies have explored perceived attitudes, beliefs, and barriers to PT DI referral specific to North Dakota (ND). Therefore, the purpose of this mixed-methods study was to identify ND PTs' attitudes, beliefs, and barriers toward DI referral. METHODS: A total of 147 participants completed an online survey with a subset of 17 participants agreeing to an interview. Frequency counts of demographic data and perceived barriers were completed. A binary logistic regression was run on demographic data. One-on-one interviews were conducted with a thematic coding process completed within a qualitative analysis. RESULTS: Seventy-four percent of PTs reported not currently referring for DI, although 71% felt that it would improve their patient outcomes. PTs with post-professional training (OR = 4.59), a doctorate degree (OR = 3.84), practicing in an orthopaedic or sports setting (OR = 3.55), and practicing within an urban setting of ND (OR = 3.01) were more likely to refer for DI. The main barriers identified in the survey included: (1) the logistics of performing a DI referral, (2) DI referrals only privileged to other medical providers, (3) provider/work relationship dynamics, (4) the cost of continuing education (CE), (5) and the inability to identify CE. One-on-one interviews further identified five main themes related to DI referral. DISCUSSION/CONCLUSION: Several barriers identified resulted in 74.1% of PTs not directly referring for DI, although certain characteristics (post-professional training, doctorate degree, orthopaedic/sports setting, practicing in an urban area in ND) were more likely to refer for DI. This study may help improve future adoption and implementation of DI referral in current and future states.

New method for gravitational wave detection with atomic sensors.

Laser frequency noise is a dominant noise background for the detection of gravitational waves using long-baseline optical interferometry. Amelioration of this noise requires near simultaneous strain measurements on more than one interferometer baseline, necessitating, for example, more than two satellites for a space-based detector or two interferometer arms for a ground-based detector. We describe a new detection strategy based on recent advances in optical atomic clocks and atom interferometry which can operate at long baselines and which is immune to laser frequency noise. Laser frequency noise is suppressed because the signal arises strictly from the light propagation time between two ensembles of atoms. This new class of sensor allows sensitive gravitational wave detection with only a single baseline. This approach also has practical applications in, for example, the development of ultrasensitive gravimeters and gravity gradiometers.

Enhanced atom interferometer readout through the application of phase shear.

We present a method for determining the phase and contrast of a single shot of an atom interferometer. The application of a phase shear across the atom ensemble yields a spatially varying fringe pattern at each output port, which can be imaged directly. This method is broadly relevant to atom-interferometric precision measurement, as we demonstrate in a 10 m 87Rb atomic fountain by implementing an atom-interferometric gyrocompass with 10 mdeg precision.

Multiaxis inertial sensing with long-time point source atom interferometry.

We show that light-pulse atom interferometry with atomic point sources and spatially resolved detection enables multiaxis (two rotation, one acceleration) precision inertial sensing at long interrogation times. Using this method, we demonstrate a light-pulse atom interferometer for 87Rb with 1.4 cm peak wave packet separation and a duration of 2T=2.3 s. The inferred acceleration sensitivity of each shot is 6.7×10(-12)g, which improves on previous limits by more than 2 orders of magnitude. We also measure Earth's rotation rate with a precision of 200 nrad/s.

Metabolomic profiling of urine: response to a randomised, controlled feeding study of select fruits and vegetables, and application to an observational study.

Metabolomic profiles were used to characterise the effects of consuming a high-phytochemical diet compared with a diet devoid of fruits and vegetables (F&V) in a randomised trial and cross-sectional study. In the trial, 8 h fasting urine from healthy men (n 5) and women (n 5) was collected after a 2-week randomised, controlled trial of two diet periods: a diet rich in cruciferous vegetables, citrus and soya (F&V), and a fruit- and vegetable-free (basal) diet. Among the ions found to differentiate the diets, 176 were putatively annotated with compound identifications, with forty-six supported by MS/MS fragment evidence. Metabolites more abundant in the F&V diet included markers of the dietary intervention (e.g. crucifers, citrus and soya), fatty acids and niacin metabolites. Ions more abundant in the basal diet included riboflavin, several acylcarnitines and amino acid metabolites. In the cross-sectional study, we compared the participants based on the tertiles of crucifers, citrus and soya from 3 d food records (n 36) and FFQ (n 57); intake was separately divided into the tertiles of total fruit and vegetable intake for FFQ. As a group, ions individually differential between the experimental diets differentiated the observational study participants. However, only four ions were significant individually, differentiating the third v. first tertile of crucifer, citrus and soya intake based on 3 d food records. One of these ions was putatively annotated: proline betaine, a marker of citrus consumption. There were no ions significantly distinguishing tertiles by FFQ. The metabolomic assessment of controlled dietary interventions provides a more accurate and stronger characterisation of the diet than observational data.

Chthonic severance: dinosaur eggs of the Mesozoic, the significance of partially buried eggs and contact incubation precursors.

For most dinosaurs, clutches consisted of a single layer of spherical to sub-spherical, highly porous eggs that were probably fully buried. Both eggs and clutch form change drastically with pennaraptoran theropods, the clade that includes birds. Here, far less porous, more elongate eggs are arranged with additional complexity, and only partially buried. While partial egg burial seems to be effective for an extremely small group of modern birds, the behaviour's overall rarity complicates our understanding of Mesozoic analogies. Recent experimental examination of pennaraptoran nesting thermodynamics suggests that partial egg burial, combined with contact incubation, may be more efficacious than has been presumed. We propose that nest guarding behaviour by endothermic archosaurs may have led to an indirect form of contact incubation using metabolic energy to affect temperature change in a buried clutch through a barrier of sediment, which in turn may have selected for shallower clutch burial to increasingly benefit from adult-generated energy until partial egg exposure. Once partially exposed, continued selection pressure may have aided a transition to fully subaerial eggs. This hypothesis connects the presence of partially buried dinosaurian clutches with the transition from basal, crocodile-like nesting (buried clutches guarded by adults) to the dominant avian habit of contact incubating fully exposed eggs. This article is part of the theme issue 'The evolutionary ecology of nests: a cross-taxon approach'.

Generation of 43 W of quasi-continuous 780 nm laser light via high-efficiency, single-pass frequency doubling in periodically poled lithium niobate crystals.

We demonstrate high-efficiency frequency doubling of the combined output of two 1560 nm 30 W fiber amplifiers via single pass through periodically poled lithium niobate (PPLN) crystals. The temporal profile of the 780 nm output is controlled by adjusting the relative phase between the seeds of the amplifiers. We obtain a peak power of 34 W of 780 nm light by passing the combined output through one PPLN crystal, and a peak power of 43 W by passing through two cascading PPLN crystals. This source provides high optical power, excellent beam quality and spectral purity, and agile frequency and amplitude control in a simple and compact setup, which is ideal for applications such as atom optics using Rb atoms.

Preclinical Characterization of Relatlimab, a Human LAG-3-Blocking Antibody, Alone or in Combination with Nivolumab.

Novel therapeutic approaches combining immune-checkpoint inhibitors are needed to improve clinical outcomes for patients with cancer. Lymphocyte-activation gene 3 (LAG-3) is an immune-checkpoint molecule that inhibits T-cell activity and antitumor immune responses, acting through an independent mechanism from that of programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). Here, we describe the development and preclinical characterization of relatlimab, a human antibody that binds to human LAG-3 with high affinity and specificity to block the interaction of LAG-3 with the ligands MHC II and fibrinogen-like protein-1, and to reverse LAG-3-mediated inhibition of T-cell function in vitro. Consistent with previous reports, in mouse models, the combined blockade of LAG-3 and PD-1 with surrogate antibodies resulted in enhanced antitumor activity greater than the individual blockade of either receptor. In toxicity studies in cynomolgus monkeys, relatlimab was generally well tolerated when combined with nivolumab. These results are consistent with findings from the RELATIVITY-047 phase II/III trial showing that relatlimab combined with nivolumab is a well-tolerated regimen that demonstrates superior progression-free survival compared with nivolumab monotherapy in patients with unresectable or metastatic melanoma.

Investigating neoplastic progression of ulcerative colitis with label-free comparative proteomics.

Patients with extensive ulcerative colitis (UC) have an increased risk of colorectal cancer. Although UC patients generally undergo lifelong colonoscopic surveillance to detect dysplasia or cancer in the colon, detection of cancer in this manner is expensive and invasive. An objective biomarker of dysplasia would vastly improve the clinical management of cancer risk in UC patients. In the current study, accurate mass and time methods with ion intensity-based label-free proteomics are applied to profile individual rectal and colon samples from UC patients with dysplasia or cancer (UC progressors) compared to rectal samples from patients that are dysplasia/cancer free (UC nonprogressors) to identify a set of proteins in the rectum mucosa that differentiate the two groups. In addition to the identification of proteins in UC dysplastic colon tissue, we for the first time identified differentially expressed proteins in nondysplastic rectal tissue from UC progressors. This provides a candidate pool of biomarkers for dysplasia/cancer that could be detected in a random nondysplastic rectal biopsy. Mitochondrial proteins, cytoskeletal proteins, RAS superfamily, proteins relating to apoptosis and metabolism were important protein clusters differentially expressed in the nondysplastic and dysplastic tissues of UC progressors, suggesting their importance in the early stages of UC neoplastic progression. Among the differentially expressed proteins, immunohistochemistry analysis confirmed that TRAP1 displayed increased IHC staining in UC progressors, in both dysplastic and nondysplastic tissue, and CPS1 showed a statistically significant difference in IHC staining between the nonprogressor and progressor groups. Furthermore, rectal CPS1 staining could be used to predict dysplasia or cancer in the colon with 87% sensitivity and 45% specificity, demonstrating the feasibility of using surrogate biomarkers in rectal biopsies to predict dysplasia and/or cancer in the colon.

A proteomics platform combining depletion, multi-lectin affinity chromatography (M-LAC), and isoelectric focusing to study the breast cancer proteome.

The discovery of breast cancer associated plasma/serum biomarkers is important for early diagnosis, disease mechanism elucidation, and determination of treatment strategy for the disease. In this study of serum samples, a multidimensional fractionation platform combined with mass spectrometric analysis were used to achieve the identification of medium to lower abundance proteins, as well as to simultaneously detect glycan and abundance changes. Immuno-affinity depletion and multi-lectin chromatography (M-LAC) were integrated into an automated HPLC platform to remove high abundance protein and fractionate glycoproteins. The collected glycoproteomes were then subjected to isoelectric focusing (IEF) separation by a digital ProteomeChip (dPC), followed by in-gel digestion and LC-MS analysis using an Orbitrap mass spectrometer. As a result, the total number of identified proteins increased significantly when the IEF fractionation step was included as part of the platform. Relevant proteins with biological and disease significance were observed and the dynamic range of the serum proteome measurement was extended. In addition, potential glycan changes were indicated by comparing proteins in control and cancer samples in terms of their affinity to the multi-lectin column (M-LAC) and the pI profiles in IEF separation. In conclusion, a proteomics platform including high abundance protein depletion, lectin affinity fractionation, IEF separation, and LC-MS analysis has been applied to discover breast cancer-associated proteins. The following candidates, thrombospondin-1 and 5, alpha-1B-glycoprotein, serum amyloid P-component, and tenascin-X, were selected as promising examples of the use of this platform. They show potential abundance and glycan changes and will be further investigated in future studies.

Antibody blockade of CD96 by distinct molecular mechanisms.

The molecular interactions of mouse CD96 to CD155 ligand and to two surrogate antibodies have been investigated. Biophysical and structural studies demonstrate that CD96 forms a homodimer but assembles as 1:1 heterodimeric complexes with CD155 or with one of the surrogate antibodies, which compete for the same binding interface. In comparison, the other surrogate antibody binds across the mouse CD96 dimer and recognizes a quaternary epitope spanning both protomers to block exposure of the ligand-binding site. This study reveals different blocking mechanisms and modalities of these two antibodies and may provide insight into the functional effects of antibodies against CD96.