Comprehensive Summary of Safety Data on Nirsevimab in Infants and Children from All Pivotal Randomized Clinical Trials.

BACKGROUND: Nirsevimab is approved in the US for the prevention of respiratory syncytial virus (RSV) lower respiratory tract disease in neonates and infants during their first RSV season and in children aged ≤24 months who remain vulnerable to severe RSV disease through their second RSV season. We summarize a pre-specified analysis of nirsevimab safety data from three randomized controlled trials: Phase 2b (NCT02878330; healthy infants born ≥29 to <35 weeks' gestational age [wGA]); Phase 3 MELODY (NCT03979313; healthy infants born ≥35 wGA); and Phase 2/3 MEDLEY (NCT03959488; infants with congenital heart disease [CHD] and/or chronic lung disease of prematurity [CLD] or born ≤35 wGA). METHODS: Participants (randomized 2:1) received a single intramuscular dose of nirsevimab or comparator (placebo, Phase 2b/MELODY; 5× once-monthly palivizumab, MEDLEY) before their first RSV season (recipients < 5 kg, nirsevimab 50 mg; ≥5 kg, nirsevimab 100 mg). In MEDLEY, children with CHD/CLD continued to a second RSV season: first-season nirsevimab recipients received nirsevimab 200 mg; first-season palivizumab recipients were re-randomized 1:1 to receive nirsevimab 200 mg or 5× once-monthly palivizumab. RESULTS: The incidence, severity, and nature of AEs were similar across treatments (nirsevimab, n = 3184; placebo, n = 1284; palivizumab, n = 304). Most AEs were mild to moderate in severity, with ≥98% unrelated to treatment. AEs of special interest occurred infrequently (<1%): no anaphylaxis or thrombocytopenia were treatment-related, and no immune complex disease was reported. Deaths (incidence < 1.0%) were all unrelated to treatment. CONCLUSIONS: A single dose per season of nirsevimab for the prevention of RSV disease had a favorable safety profile, irrespective of wGA or comorbidities.

2023 White Paper on Recent Issues in Bioanalysis: ISR for ADA Assays, the Rise of dPCR vs qPCR, International Reference Standards for Vaccine Assays, Anti-AAV TAb Post-Dose Assessment, NanoString Validation, ELISpot as Gold Standard (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Biotherapeutics Immunogenicity & Risk Assessment; ADA/NAb Assay/Reporting Harmonization).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19-23, 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) are published in volume 16 of Bioanalysis, issues 8 and 9 (2024), respectively.

Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody-Drug Conjugate.

Deamidation, a common post-translational modification, may impact multiple physiochemical properties of a therapeutic protein. MEDI7247, a pyrrolobenzodiazepine (PBD) antibody-drug conjugate (ADC), contains a unique deamidation site, N102, located within the complementarity-determining region (CDR), impacting the affinity of MEDI7247 to its target. Therefore, it was necessary to monitor MEDI7247 deamidation status in vivo. Due to the low dose, a sensitive absolute quantification method using immunocapture coupled with liquid chromatography-tandem mass spectrometry (LBA-LC-MS/MS) was developed and qualified. We characterized the isomerization via Electron-Activated Dissociation (EAD), revealing that deamidation resulted in iso-aspartic acid. The absolute quantification of deamidation requires careful assay optimization in order not to perturb the balance of the deamidated and nondeamidated forms. Moreover, the selection of capture reagents essential for the correct quantitative assessment of deamidation was evaluated. The final assay was qualified with 50 ng/mL LLOQ for ADC for total and nondeamidated antibody quantification, with qualitative monitoring of the deamidated antibody. The impact of deamidation on the pharmacokinetic characteristics of MEDI7247 from clinical trial NCT03106428 was analyzed, revealing a gradual reduction in the nondeamidated form of MEDI7247 in vivo. Careful quantitative biotransformation analyses of complex biotherapeutic conjugates help us understand changes in product PTMs after administration, thus providing a more complete view of in vivo pharmacology.

Safety of Re-dosing Nirsevimab Prior to RSV Season 2 in Children With Heart or Lung Disease.

In children with congenital heart disease and/or chronic lung disease entering their second respiratory syncytial virus (RSV) season, 200 mg nirsevimab had a similar safety profile to that of palivizumab and resulted in nirsevimab serum exposures associated with efficacy in healthy infants, supporting efficacy in this population at risk of severe RSV disease.

Safety, Tolerability and Pharmacokinetics of Half-Life Extended Severe Acute Respiratory Syndrome Coronavirus 2 Neutralizing Monoclonal Antibodies AZD7442 (Tixagevimab-Cilgavimab) in Healthy Adults.

BACKGROUND: AZD7442 is a combination of extended half-life, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific neutralizing monoclonal antibodies (tixagevimab and cilgavimab). METHODS: This phase 1, first-in-human, randomized, double-blind, placebo-controlled, dose-escalation study evaluated AZD7442 administered intramuscularly (300 mg) or intravenously (300, 1000, or 3000 mg) in healthy adults (aged 18-55 years). The primary end point was safety and tolerability. Secondary end points included pharmacokinetics and antidrug antibodies. RESULTS: Between 18 August and 16 October 2020, a total of 60 participants were enrolled; 50 received AZD7442, and 10 received placebo. Adverse events (all of mild or moderate intensity) occurred in 26 participants (52.0%) in the AZD7442 groups and 8 (80.0%) in the placebo group. No infusion or injection site or hypersensitivity reactions occurred. Tixagevimab and cilgavimab had mean half-lives of approximately 90 days (range, 87.0-95.3 days for tixagevimab and 79.8--91.1 days for cilgavimab) and similar pharmacokinetic profiles over the 361-day study period. SARS-CoV-2-specific neutralizing antibody titers provided by AZD7442 were maintained above those in plasma from convalescent patients with coronavirus disease 2019 (COVID-19). CONCLUSIONS: AZD7442 was well tolerated in healthy adults, showing a favorable safety profile across all doses. Depending on the SARS-CoV-2 variant, pharmacokinetic analyses suggest the AZD7442 could offer protection for ≥6 months against symptomatic COVID-19 after a single 300-mg intramuscular administration. CLINICAL TRIALS REGISTRATION: NCT04507256.

Antibodies are proteins produced by the body in response to infections caused by microbes, including viruses. AZD7442 is a combination of 2 human antibodies, with an extended duration of effect, sourced from people who had recovered from coronavirus disease 2019 (COVID-19). These antibodies recognize a specific part (spike protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, and prevent the virus from infecting cells in the body. The current study evaluated the safety of AZD7442 in healthy volunteers. Sixty adults were given AZD7442 or placebo (salt solution) as injections into the muscle (300-mg dose) or infusions into a vein (300­3000-mg doses). The study did not find any safety issues with AZD7442, including at the highest dose. AZD7442 was measured in the blood 12 months after dosing, suggesting a long duration of protection. Following this study, AZD7442 was tested in larger clinical trials to investigate its potential in preventing and treating COVID-19. AZD7442 is currently authorized as treatment for outpatients with COVID-19 and as a preventive drug in people who may not respond well to COVID-19 vaccines and need additional protection (eg, those taking medications that dampen the immune system).

Multiplex Hybrid Antigen-Capture LC-MRM Quantification in Sera and Nasal Lining Fluid of AZD7442, a SARS-CoV-2-Targeting Antibody Combination.

AZD7442 (tixagevimab [AZD8895]/cilgavimab [AZD1061]) is a monoclonal antibody (mAb) combination in development for the prevention and treatment of coronavirus disease 2019. Traditionally, bioanalysis of mAbs is performed using ligand binding assays (LBAs), which offer sensitivity, robustness, and ease of implementation. However, LBAs frequently require generation of critical reagents that typically take several months. Instead, we developed a highly sensitive (5 ng/mL limit of quantification) method using a hybrid LBA-liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) approach for quantification of the two codosed antibodies in serum and nasal lining fluid (NLF), a rare matrix. The method was optimized by careful selection of multiple reaction monitoring, capture reagents, magnetic beads, chromatographic conditions, evaluations of selectivity, and matrix effect. The final assay used viral spike protein receptor-binding domain as capture reagent and signature proteotypic peptides from the complementarity-determining region of each mAb for detection. In contrast to other methods of similar/superior sensitivity, our approach did not require multidimensional separations and can be operated in an analytical flow regime, ensuring high throughput and robustness required for clinical analysis at scale. The sensitivity of this method significantly exceeds typical sensitivity of ∼100 ng/mL for analytical flow 1D LBA-LC-MS/MS methods for large macromolecules, such as antibodies. Furthermore, infection and vaccination status did not impact method performance, ensuring method robustness and applicability to a broad patient population. This report demonstrated the general applicability of the hybrid LBA-LC-MS/MS approach to platform quantification of antibodies with high sensitivity and reproducibility, with specialized extension to matrices of increasing interest, such as NLF.

Asymmetric Cyclopropanation with 4-Aryloxy-1-sulfonyl-1,2,3-triazoles: Expanding the Range of Rhodium-Stabilized Donor/Acceptor Carbenes to Systems with an Oxygen Donor Group.

Rhodium-catalyzed enantioselective synthesis of 1-phenoxycyclopropane-1-carbaldehydes by intermolecular cyclopropanation of terminal alkenes followed by imine hydrolysis is described. This methodology utilizes 4-aryloxy-1-sulfonyl-1,2,3-triazoles as the carbene precursors and the chiral dirhodium(II) tetracarboxylates Rh2(S-NTTL)4 or Rh2(S-DPCP)4 as the catalysts. These reactions are considered to proceed via rhodium-stabilized donor/acceptor carbene intermediates, and these studies demonstrate that a heteroatom donor group is compatible with an enantioselective transformation.

Comparison of Titer and Signal to Noise (S/N) for Determination of Anti-drug Antibody Magnitude Using Clinical Data from an Industry Consortium.

During biotherapeutic drug development, immunogenicity is evaluated by measuring anti-drug antibodies (ADAs). The presence and magnitude of ADA responses is assessed using a multi-tier workflow where samples are screened, confirmed, and titered. Recent reports suggest that the assay signal to noise ratio (S/N) obtained during the screening tier correlates well with titer. To determine whether S/N could more broadly replace titer, anonymized ADA data from a consortium of sponsors was collected and analyzed. Datasets from clinical programs with therapeutics of varying immunogenicity risk levels (low to high), common ADA assay platforms (ELISA and MSD) and formats (bridging, direct, solid-phase extraction with acid dissociation), and titration approaches (endpoint and interpolated) were included in the analysis. A statistically significant correlation between S/N and titer was observed in all datasets, with a strong correlation (Spearman's r > 0.8) in 11 out of 15 assays (73%). For assays with available data, conclusions regarding ADA impact on pharmacokinetics and pharmacodynamics were similar using S/N or titer. Subject ADA kinetic profiles were also comparable using the two measurements. Determination of antibody boosting in patients with pre-existing responses could be accomplished using similar approaches for titer and S/N. Investigation of factors that impacted the accuracy of ADA magnitude measurements revealed advantages and disadvantages to both approaches. In general, S/N had superior precision and ability to detect potentially low affinity/avidity responses compared to titer. This analysis indicates that S/N could serve as an equivalent and in some cases preferable alternative to titer for assessing ADA magnitude and evaluation of impact on clinical responses.

2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccine Assays; Immunogenicity of Biotherapeutics and Novel Modalities; Integrated Summary of Immunogenicity Harmonization).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.

Critical reagents for ligand-binding assays: process development methodologies to enable high-quality reagents.

Development of biotherapeutics require pharmacokinetic/pharmacodynamic (PK/PD) and immunogenicity assays that are frequently in a ligand-binding assay (LBA) format. Conjugated critical reagents for LBAs are generated conjugation of the biotherapeutic drug or anti-drug molecule with a label. Since conjugated critical reagent quality impacts LBA performance, control of the generation process is essential. Our perspective is that process development methodologies should be integrated into critical reagent production to understand the impact of conjugation reactions, purification techniques and formulation conditions on the quality of the reagent. In this article, case studies highlight our approach to developing process conditions for different molecular classes of critical reagents including antibodies and a peptide. This development approach can be applied to the generation of future conjugated critical reagents.

2020 White Paper on Recent Issues in Bioanalysis: Vaccine Assay Validation, qPCR Assay Validation, QC for CAR-T Flow Cytometry, NAb Assay Harmonization and ELISpot Validation (Part 3 - Recommendations on Immunogenicity Assay Strategies, NAb Assays, Biosimilars and FDA/EMA Immunogenicity Guidance/Guideline, Gene & Cell Therapy and Vaccine Assays).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity). Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation) and Part 2B (Regulatory Input) are published in volume 13 of Bioanalysis, issues 4 and 5 (2020), respectively.

Serous borderline tumor with distant mediastinal metastasis? An Exceptional presentation of ovarian tumor.

Borderline ovarian tumor is a non-invasive lesion with an excellent prognosis. Here we report a case of 48-year-old woman with distinctive clinical presentation of metastasis of ovarian adenocarcinoma, which was an microinvasive component of a serous borderline tumor. On initial diagnosis patient did not present any clinical manifestation of ovarian tumor. Histological examination of resected ovary showed typical features of the serous borderline tumor with one very diminutive focus of invasive serous adenocarcinoma 4mm in diameter. This exceptional case shows that borderline tumors of ovary with any features of invasion could present an aggressive course with distant metastases.

A new method for identification of outliers in immunogenicity assay cut point data.

The cut point is an important parameter for immunogenicity assay validation and critical to immunogenicity assessment in clinical trials. FDA (2019) recommends using a statistical approach to derive cut point, with an appropriate outlier removal procedure. In general, the industry follows the methods described in Shankar et al. (2008) and Zhang et al. (2013) among others to determine cut point. Outlier removal is a necessary step during the cut point determination exercise to reduce potential false negative classifications. However, the widely used statistical outlier removal method, namely, Tukey's box-plot method (1.5 times inter-quartile range, IQR), is often found to be overly conservative in the sense that it removes too many "outliers". Tukey's box-plot method can be used to flag potential outliers for further investigation, however, it is not a hypothesis testing based statistical method. Removing these suspected "outliers" will lead to lower cut point which might confound immunogenicity assessment due to the presence of many low false positives. Besides, the very nature of assay analytical variability has a non-negligible adverse impact on the reliability of ADA classification in terms of false positive and false negative, demanding as large as possible contribution from biological variability relative to analytical variability. A new outlier removal procedure, which takes into account the relative magnitude between biological variability and analytical variability within the sample population, is proposed and statistically justified. After sequential removal of analytical and biological outliers, a 5% false positive rate and 1% false positive rate in screening and confirmatory assays, respectively, are still targeted without increasing potential false negatives. Internal data shows that this practice has minimal impact on assay sensitivity and has the advantage of selecting true positive samples. It is shown that the new procedure is more appropriate for cut point determination.

Confirmatory cut point has limited ability to make accurate classifications in immunogenicity assays.

Aim: Competitive inhibition with excess unlabeled drug is used to confirm the presence of antidrug antibodies (ADA) in study samples. We evaluated specific and nonspecific responses from both drug-naive and drug-treated subjects to identify conditions required by the confirmatory assay to make accurate ADA classifications. Results: Nonspecific signal measured in drug-naive samples used to determine assay cut points was uniformly low and close to the screening cut point. Confirmatory assays performed on incurred study samples with nonspecific responses significantly above the level observed during cut point determination resulted in incorrect ADA classifications. Conclusion: Intensity of confirmatory response should be proportional to the screening response and therefore, to ensure accurate ADA classifications, the confirmatory responses cannot be considered as independent but need to be evaluated in relation to the screening responses.

2018 White Paper on Recent Issues in Bioanalysis: focus on flow cytometry, gene therapy, cut points and key clarifications on BAV (Part 3 - LBA/cell-based assays: immunogenicity, biomarkers and PK assays).

The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of Bioanalysis, issues 22 and 23 (2018), respectively.

Excessive outlier removal may result in cut points that are not suitable for immunogenicity assessments.

Cut point determination is an important aspect of immunogenicity assay development. The cut point can be influenced by a myriad of factors. Key among those is the analytical variability of the assay itself and biological variation due to test samples. Since a smaller cut point value may result in improved sensitivity, the existing procedures often employ statistical techniques such as outlier removal to produce a conservative cut point. Although such practices are intended to yield acceptable assay sensitivity, they may fail to fully account for biological variability in the data, thus generating higher than expected number of false positive results. In this paper, we introduce the concept of minimum cut point. It is defined as the cut point that is determined in the absence of biological variability. Under the log-normal assumption of the data used for cut point analysis, closed-form formulas are derived for the minimum cut point. This minimum cut point can be used to benchmark whether a cut point derived from a procedure can compromise assay specificity by being too low.

Rhodium-Catalyzed Intermolecular C-H Functionalization as a Key Step in the Synthesis of Complex Stereodefined ß-Arylpyrrolidines.

The synthesis of ß-arylpyrrolidines via a catalytic enantioselective intermolecular allylic C(sp)3-H functionalization of trans-alkenes followed by immediate reduction, ozonolysis, and then in situ diversification of the resulting cyclic hemiaminal to furnish highly substituted, stereoenriched ß-arylpyrrolidines is reported. This methodology utilizes 4-aryl-1-sulfonyl-1,2,3-triazoles as carbene precursors and the dirhodium tetracarboxylate catalyst Rh2( S-NTTL)4. A variety of ß-arylpyrrolidines were prepared in good yields with high levels of diastereo- and enantioselectivity over four linear steps, requiring only a single purification procedure.

Storage Conditions of Conjugated Reagents Can Impact Results of Immunogenicity Assays.

Consistent performance of anti-drug antibody (ADA) assays through all stages of clinical development is critical for the assessment of immunogenicity and interpretation of PK, PD, safety, and efficacy. The electrochemiluminescent assays commonly employed for ADA measurement use drug conjugated with ruthenium and biotin to bind ADA in samples. Here we report an association between high nonspecific ADA responses in certain drug-naïve individuals and the storage buffer of the conjugated reagents used in a monoclonal antibody ADA assay. Ruthenylated reagents stored in phosphate-buffered saline (PBS) buffer had increased levels of aggregate and produced variable and high baseline responses in some subjects. Reagents stored in a histidine-sucrose buffer (HSB) had lower aggregate levels and produced low sample responses. In contrast to PBS, conjugated reagents formulated in HSB remained low in aggregate content and in sample response variability after 5 freeze/thaw cycles. A reagent monitoring control (RMC) serum was prepared for the real-time evaluation of conjugated reagent quality. Using appropriate buffers for storage of conjugated reagents together with RMCs capable of monitoring of reagent aggregation status can help ensure consistent, long-term performance of ADA methods.

Enantioselective Intermolecular C-H Functionalization of Allylic and Benzylic sp(3) C-H Bonds Using N-Sulfonyl-1,2,3-triazoles.

The enantioselective intermolecular sp(3) C-H functionalization at the allylic and benzylic positions was achieved using rhodium-catalyzed reactions with 4-phenyl-N-(methanesulfonyl)-1,2,3-triazole. The optimum dirhodium tetracarboxylate catalyst for these reactions was Rh2(S-NTTL)4. The rhodium-bound α-imino carbene intermediates preferentially reacted with tertiary over primary C-H bonds in good yields and moderate levels of enantioselectivity (66-82% ee). This work demonstrates that N-sulfonyltriazoles can be applied to the effective C-H functionalization at sp(3) C-H bonds of substrates containing additional functionality.

Lack of CD151/integrin α3ß1 complex is predictive of poor outcome in node-negative lobular breast carcinoma: opposing roles of CD151 in invasive lobular and ductal breast cancers.

BACKGROUND: The proposed involvement of CD151 in breast cancer (BCa) progression is based on findings from studies in invasive ductal carcinoma (IDC). The IDC and invasive lobular carcinoma (ILC) represent distinct disease entities. Here we evaluated clinical significance of CD151 alone and in association with integrin α3ß1 in patients with ILC in context of the data of our recent IDC study. METHODS: Expression of CD151 and/or integrin α3ß1 was evaluated in ILC samples (N=117) using immunohistochemistry. The findings were analysed in relation to our results from an IDC cohort (N=182) demonstrating a prognostic value of an expression of CD151/integrin α3ß1 complex in patients with HER2-negative tumours. RESULTS: Unlike in the IDCs, neither CD151 nor CD151/α3ß1 complex showed any correlation with any of the ILC characteristics. Lack of both CD151 and α3ß1 was significantly correlated with poor survival (P=0.034) in lymph node-negative ILC N(-) cases. The CD151(-)/α3ß1(-) patients had 3.12-fold higher risk of death from BCa in comparison with the rest of the ILC N(-) patients. CONCLUSIONS: Biological role of CD151/α3ß1 varies between ILC and IDC. Assessment of CD151/α3ß1 might help to identify ILC N(-) patients with increased risk of distant metastases.