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The fibronectin (FN) isoform including the extradomain B (EDB) segment (EDB + FN) is a promising tumor target and is highly expressed in some tumor types, such as breast, head, and neck cancer. To date, mostly immunohistochemistry (IHC) and Western blot have been used for the analysis of EDB + FN. However, complete quantitative measurements of EDB + FN expression in a tumor and circulation are important for the development of anti-EDB therapeutics. To this end, a method using protein enrichment followed by online antipeptide antibody enrichment coupled with a nanoflow LC-MS/MS was developed to quantify EDB + FN in human and cynomolgus plasma, patient-derived xenograft (PDX) tumors, and PDX formalin-fixed paraffin-embedded (FFPE) samples. Mouse plasma EDB + FN was analyzed using a protein immunoaffinity method followed by nanoflow LC-MS/MS. EDB + FN concentrations were 63.1 pmol/g in PDX breast cancer tumor and 49.6 pmol/g in PDX head and neck tumor. Mean plasma concentration was 1.1 nM (pmol/mL, 47.4 ng/mL) in normal healthy humans and 0.35 nM (15.1 ng/mL) in naive cynomolgus. The assay sensitivity was 0.018 nM based on calibration with recombinant human EDB + FN (rhEDB + FN).

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 three Main Workshops and seven 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 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. 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 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

Trastuzumab emtansine (T-DM1) is an antibody–drug conjugate (ADC) that has demonstrated clinical benefit for patients with HER2+ metastatic breast cancer; however, its clinical activity is limited by inherent or acquired drug resistance. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2+ tumors, are not well understood. We used HER2+ cell lines to develop models of T-DM1 resistance using a cyclical dosing schema in which cells received T-DM1 in an “on-off” routine until a T-DM1–resistant population was generated. T-DM1–resistant N87 cells (N87-TM) were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non–cleavable-linked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug transporter protein (e.g., MDR1) expression compared with parental N87 cells. Intriguingly, T-ADCs using auristatin payloads attached via an enzymatically cleavable linker overcome T-DM1 resistance in N87-TM cells. Importantly, N87-TM cells implanted into athymic mice formed T-DM1 refractory tumors that remain sensitive to T-ADCs with cleavable-linked auristatin payloads. Comparative proteomic profiling suggested enrichment in proteins that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells internalize T-ADCs into intracellular caveolin-1 (CAV1)–positive puncta and alter their trafficking to the lysosome compared with N87 cells. T-DM1 colocalization into intracellular CAV1-positive puncta correlated with reduced response to T-DM1 in a panel of HER2+ cell lines. Together, these data suggest that caveolae-mediated endocytosis of T-DM1 may serve as a novel predictive biomarker for patient response to T-DM1. Mol Cancer Ther; 17(1); 243–53. ©2017 AACR.

Supplementary Figure S1: N87 and N87-TM cells are similarly sensitive to unconjugated DM1-SMe; Supplementary Figure S2: Trastuzumab binding to N87 and N87-TM cells; Supplementary Figure S3: Generation and characterization of T-DM1-resistant HCC1954-TM and BT474-TM cells; Supplementary Figure S4: Relative resistance profiles of N87-TM to N87 cells to T-ADCs, unconjugated payloads and standard-of-care chemotherapeutics; Supplementary Figure S5: Distribution of T-ADCs in N87-TM tumors; Supplementary Figure S6: N87-TM cells are cross-resistant to a T-ADC conjugated to a cleavable linker with a cysteine-capped auristatin analog; Supplementary Figure S7: N87-TM cells internalize T-ADC into non-low pH intracellular compartments; Supplementary Figure S8: CAV1-GFP expression causes intracellular caveolae formation capable of internalizing T-ADCs in N87 cells; Supplementary Figure S9: CAV1 knockdown is not sufficient to re-sensitize N87-TM cells to T-DM1; Supplementary Figure S10: Non-cleavable and cleavable T-ADCs co-localize with CAV1 similarly in N87 and N87-TM cells; Supplementary Table S1. Cytotoxicity values of Trastuzumab and isotype-control ADCs in HT29 cells, a HER2-negative cell line

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 covers the recommendations on (Part 2A) BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation and (Part 2B) Regulatory Input. Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 4, and 6 (2021), respectively.

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 three Main Workshops and seven 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 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/OralMultispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. 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, GeneCell Therapy and Vaccine) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9CAR-T Immunogenicity; PCRVaccine Assay Performance; ADA Assay ComparabilityCut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

Although weak lensing (WL) is a powerful method to estimate a galaxy cluster mass without any dynamical assumptions, a model bias can arise when the cluster density profile departs from the assumed model profile. In a merging system, the bias is expected to become most severe because the constituent halos undergo significant structural changes. In this study, we investigate WL mass bias in binary cluster mergers using a suite of idealized hydrodynamical simulations. Realistic WL shear catalogs are generated by matching the source galaxy properties, such as intrinsic shape dispersion, measurement noise, source densities, etc., to those from Subaru and {\it Hubble Space Telescope} observations. We find that, with the typical mass-concentration ($M$-$c$) relation and the Navarro-Frenk-White (NFW) profile, the halo mass bias depends on the time since the first pericenter passage and increases with the mass of the companion cluster. The time evolution of the mass bias is similar to that of the concentration, indicating that, to first order, the mass bias is modulated by the concentration change. For a collision between two $\sim10^{15}~M_{\odot}$ clusters, the maximum bias amounts to $\sim60\%$. This suggests that previous WL studies may have significantly overestimated the mass of the clusters in some of the most massive mergers. Finally, we apply our results to three merger cases: Abell 2034, MACS J1752.0+4440, and ZwCl 1856.8+6616, and report their mass biases at the observed epoch, as well as their pre-merger masses, utilizing their merger shock locations as tracers of the merger phases., Comment: 14 pages, 11 figures, accepted to ApJ

Strong gravitational lensing of gravitational waves (GWs) occurs when the GWs from a compact binary system travel near a massive object. The mismatch between a lensed signal and unlensed templates determines whether lensing can be identified in a particular GW event. For axisymmetric lens models, the lensed signal is traditionally calculated in terms of model-dependent lens parameters such as the lens mass $M_L$ and source position $y$. We propose that it is useful to parameterize this signal instead in terms of model-independent image parameters: the flux ratio $I$ and time delay $\Delta t_d$ between images. The functional dependence of the lensed signal on these image parameters is far simpler, facilitating data analysis for events with modest signal-to-noise ratios. In the geometrical-optics approximation, constraints on $I$ and $\Delta t_d$ can be inverted to constrain $M_L$ and $y$ for any lens model including the point mass (PM) and singular isothermal sphere (SIS) that we consider. We use our model-independent image parameters to determine the detectability of gravitational lensing in GW signals and find that for GW events with signal-to-noise ratios $\rho$ and total mass $M$, lensing should in principle be identifiable for flux ratios $I \gtrsim 2\rho^{-2}$ and time delays $\Delta t_d \gtrsim M^{-1}$., Comment: 11 pages, 8 figures, submitted to PRD

The 14

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.

As biomarkers continue to become an integral part of drug development and decision-making, there are increased expectations for reliable and quantitative assays. Protein biomarker assay results are directly influenced by the calibrator material. The selection of calibrator material presents many challenges that impact the relative accuracy and performance of the assay. There is an industry-wide challenge finding reliable and well-characterized calibrator material with good documentation. Several case studies are presented that demonstrate some of the challenges involved in selecting appropriate calibrators along with the resolutions that were ultimately applied. From these experiences, we present here a set of recommendations for selecting and characterizing calibrator material based on the intended purpose of the assay. Finally, we introduce a commutability approach, based on common clinical chemistry practices, which can be used to demonstrate inter-changeability with calibrator materials across multiple lots and technology platforms for all types of protein biomarker assays.

// Parul Gupta 1 , Jo-Ann Wentland 2 , Mauricio Leal 2 , Dangshe Ma 3, 6 , Rachel Roach 4 , Antonio Esparza 5 , Lindsay King 2 , Mary E. Spilker 2 , Cedo Bagi 1 , Christopher T. Winkelmann 1 and Anand Giddabasappa 1 1 Global Science and Technology, Comparative Medicine, Pfizer, Inc., La Jolla, CA, USA 2 Pharmacokinetics and Drug Metabolism, Pfizer, Inc., New York NY, USA 3 Oncology Research Unit, Pfizer, Inc., Pearl River, NY, USA 4 Center for Therapeutic Innovation, Pfizer, Inc., La Jolla, CA, USA 5 Comparative Medicine, Pfizer, Inc., La Jolla, CA, USA 6 Current affiliation: Regeneron Pharmaceuticals, Tarrytown, NY, USA Correspondence to: Anand Giddabasappa, email: anand.giddabasappa@pfizer.com Keywords: biotherapeutics, tumor targeting, biodistribution, fluorescence molecular tomography (FMT), molecular imaging Received: January 16, 2017 Accepted: July 03, 2017 Published: July 26, 2017 ABSTRACT Non-invasive imaging using radiolabels is a common technique used to study the biodistribution of biologics. Due to the limited shelf-life of radiolabels and the requirements of specialized labs, non-invasive optical imaging is an attractive alternative for preclinical studies. Previously, we demonstrated the utility of fluorescence molecular tomography (FMT) an optical imaging modality in evaluating the biodistribution of antibody-drug conjugates. As FMT is a relatively new technology, few fluorophores have been validated for in vivo imaging. The goal of this study was to characterize and determine the utility of near-infrared (NIR) fluorophores for biodistribution studies using interleukin-13 receptor subunit alpha-2 antibody (IL13Rα2-Ab). Eight fluorophores ( ex/em : 630/800 nm) with an N-hydroxysuccinimide (NHS) linker were evaluated for Ab conjugation. The resulting antibody-fluorophore (Ab-F) conjugates were evaluated in vitro for degree of conjugation, stability and target-binding, followed by in vivo / ex vivo FMT imaging to determine biodistribution in a xenograft model. The Ab-F conjugates (except Ab-DyLight800) showed good in vitro stability and antigen binding. All Ab-F conjugates (except for Ab-BOD630) resulted in a quantifiable signal in vivo and had similar biodistribution profiles, with peak tumor accumulation between 6 and 24 h post-injection. In vivo / ex vivo FMT imaging showed 17–34% ID/g Ab uptake by the tumor at 96 h. Overall, this is the first study to characterize the biodistribution of an Ab using eight NIR fluorophores. Our results show that 3-dimensional optical imaging is a valuable technology to understand biodistribution and targeting, but a careful selection of the fluorophore for each Ab is warranted.

CD3 bispecific antibody constructs recruit cytolytic T cells to kill tumor cells, offering a potent approach to treat cancer. T cell activation is driven by the formation of a trimolecular complex (trimer) between drugs, T cells, and tumor cells, mimicking an immune synapse. A translational quantitative systems pharmacology (QSP) model is proposed for CD3 bispecific molecules capable of predicting trimer concentration and linking it to tumor cell killing. The model was used to quantify the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of a CD3 bispecific targeting P-cadherin (PF-06671008). It describes the disposition of PF-06671008 in the central compartment and tumor in mouse xenograft models, including binding to target and T cells in the tumor to form the trimer. The model incorporates T cell distribution to the tumor, proliferation, and contraction. PK/PD parameters were estimated for PF-06671008 and a tumor stasis concentration (TSC) was calculated as an estimate of minimum efficacious trimer concentration. TSC values ranged from 0.0092 to 0.064 pM across mouse tumor models. The model was translated to the clinic and used to predict the disposition of PF-06671008 in patients, including the impact of binding to soluble P-cadherin. The predicted terminal half-life of PF-06671008 in the clinic was approximately 1 day, and P-cadherin expression and number of T cells in the tumor were shown to be sensitive parameters impacting clinical efficacy. A translational QSP model is presented for CD3 bispecific molecules, which integrates in silico, in vitro and in vivo data in a mechanistic framework, to quantify and predict efficacy across species. Electronic supplementary material The online version of this article (10.1208/s12248-019-0332-z) contains supplementary material, which is available to authorized users.

With the growing focus on translational research and the use of biomarkers to drive drug development and approvals, biomarkers have become a significant area of research within the pharmaceutical industry. However, until the US Food and Drug Administration’s (FDA) 2013 draft guidance on bioanalytical method validation included consideration of biomarker assays using LC-MS and LBA, those assays were created, validated, and used without standards of performance. This lack of expectations resulted in the FDA receiving data from assays of varying quality in support of efficacy and safety claims. The AAPS Crystal City VI (CC VI) Workshop in 2015 was held as the first forum for industry-FDA discussion around the general issues of biomarker measurements (e.g., endogenous levels) and specific technology strengths and weaknesses. The 2-day workshop served to develop a common understanding among the industrial scientific community of the issues around biomarkers, informed the FDA of the current state of the science, and will serve as a basis for further dialogue as experience with biomarkers expands with both groups.

Catch a Glimpse of Me is an ongoing project that uses video to help staff deliver more person-centered care for people with dementia living in long-term care. Focus groups consisting of residents, family and staff members were conducted to develop a template for the development of the videos. The five themes they identified as being important to include are: family; interests and hobbies; memories and moments; life space and getting personal. The article describes the process of developing the videos and discusses the ongoing potential of the Catch a Glimpse of Me project.

A mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model was used for preclinical to clinical translation of inotuzumab ozogamicin, a CD22-targeting antibody-drug conjugate (ADC) for B cell malignancies including non-Hodgkin's lymphoma (NHL) and acute lymphocytic leukemia (ALL). Preclinical data was integrated in a PK/PD model which included (1) a plasma PK model characterizing disposition and clearance of inotuzumab ozogamicin and its released payload N-Ac-γ-calicheamicin DMH, (2) a tumor disposition model describing ADC diffusion into the tumor extracellular environment, (3) a cellular model describing inotuzumab ozogamicin binding to CD22, internalization, intracellular N-Ac-γ-calicheamicin DMH release, binding to DNA, or efflux from the tumor cell, and (4) tumor growth and inhibition in mouse xenograft models. The preclinical model was translated to the clinic by incorporating human PK for inotuzumab ozogamicin and clinically relevant tumor volumes, tumor growth rates, and values for CD22 expression in the relevant patient populations. The resulting stochastic models predicted progression-free survival (PFS) rates for inotuzumab ozogamicin in patients comparable to the observed clinical results. The model suggested that a fractionated dosing regimen is superior to a conventional dosing regimen for ALL but not for NHL. Simulations indicated that tumor growth is a highly sensitive parameter and predictive of successful outcome. Inotuzumab ozogamicin PK and N-Ac-γ-calicheamicin DMH efflux are also sensitive parameters and would be considered more useful predictors of outcome than CD22 receptor expression. In summary, a multiscale, mechanism-based model has been developed for inotuzumab ozogamicin, which can integrate preclinical biomeasures and PK/PD data to predict clinical response.

Gastrointestinal malignancies, including colorectal cancer (CRC) remain an area of high unmet need. Here we demonstrate tumor selective and potent in vitro and in vivo efficacy with PF-07062119, a novel T cell engaging CD3 bispecific against tumors expressing the Guanylyl Cyclase C (GUCY2C) receptor, a target expressed in more than 90% of CRC, and in other gastrointestinal cancers. Additionally, to address immune evasion mechanisms, combinations of the GUCY2C-CD3 bispecific are explored with immune checkpoint blockade therapy, as well as with chemotherapeutic and anti-angiogenic agents that could enhance immune infiltration into tumors. Our preclinical in vivo data demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3ϵ bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T cell mediated anti-tumor activity in several human xenograft models of CRC, using adoptive transfer of human T cells, including those with KRAS and BRAF mutations, as well as in models with syngeneic tumors in immunocompetent human CD3 transgenic mice. PF-07062119 activity was further enhanced when combined with anti PD-1/PD-L1 treatment or in combination with chemotherapy or anti-angiogenic therapy. A combination of immunohistochemistry, flow cytometry and CyTOF analyses was used to demonstrate the mechanism of action of PF-07062119 in single agent and combination studies in vivo. Toxicity and pharmacokinetic studies were done in cynomolgus macaques and indicated a monitorable and manageable toxicity profile. These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in CRC and other gastrointestinal malignancies. A clinical Phase I study has been initiated in patients with CRC, gastric and esophageal adenocarcinomas (NCT04171141). Citation Format: Divya Mathur, Adam Root, Bozena Bugaj-Gaweda, Xingzhi Tan, Wei Fang, Stephanie Bisulco, Jonathan Golas, Diane Fernandez, Jessica Kearney, Eric Upeslacis, Johnny Yao, Edward Rosfjord, Chad Stevens, Keith Kobylarz, Lindsay King, Jatin Narula, Kerry Kelleher, David Schaer, Cris Kamperschroer, Bernard Buetow, Cynthia Rohde, Allison Moreau, Gilbert Wong, Puja Sapra. A novel GUCY2C - CD3 bispecific engages T cells to induce cytotoxicity in gastrointestinal tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2283.

Background: Guanylyl cyclase C (GUCY2C) is a regulator of intestinal homeostasis and is expressed in more than 90% of colorectal cancer (CRC), as well as in other gastrointestinal malignancies. Target expression in normal tissues is largely restricted to the apical side of intestinal epithelial tight junctions, which could allow for preferential uptake of GUCY2C-targeted biologics by tumors that have disrupted tight junction architecture. Here we demonstrate tumor-selective uptake and potent efficacy with a half-life extended, GUCY2C-CD3 bispecific molecule that recruits CD3-positive T cells to induce cytotoxicity of GUCY2C-expressing tumors in several CRC models in vivo. Additionally, to address immune evasion mechanisms, rational combinations of the bispecific are explored with immune checkpoint blockade agents, as well as by blocking angiogenesis, which has been reported to enhance T-cell infiltration into tumors. Methods: GUCY2C-CD3 mediated activity was evaluated in vivo in several cell line- and patient derived-xenograft models of CRC, using adoptive transfer of human T cells in established subcutaneous and orthotopic tumors. Immunohistochemistry was performed to demonstrate biodistribution of the drug and recruitment of activated T cells in GUCY2C-expressing tumors vs. normal tissues. We also performed CYTOF analyses on GUCY2C-CD3-treated tumors and tumor-infiltrating lymphocytes (TILs) to identify markers of immune evasion. Combination studies with anti PD-1/PD-L1 checkpoint blockade agents and with an anti VEGF-A antibody were performed using the adoptive transfer model. Results: GUCY2C-CD3 bispecific showed preferential biodistribution to GUCY2C-expressing xenograft tumors compared to normal tissue. The drug demonstrated potent dose-dependent efficacy in several CRC models with no signs of toxicity. Bispecific treated tumors not only showed recruitment of activated T cells to the tumor site, but also upregulated checkpoint mechanisms, such as PD-L1 on tumors and exhaustion markers on TILs. Consequently, anti PD-1/PD-L1 checkpoint blocking antibodies provided enhanced efficacy when combined with GUCY2C-CD3. Additionally, significant combination benefit was observed when GUCY2C-CD3 was combined with an anti VEGF-A blocking antibody. Conclusions: Our preclinical data demonstrate that a GUCY2C-CD3 bispecific can selectively target colorectal tumors, including those with KRAS or BRAF mutations, which are difficult to treat with currently approved therapies. This bispecific shows combination benefits with T-cell checkpoint blockade agents, as well as antiangiogenesis agents, indicating that single-agent activity with GUCY2C-CD3 can be further enhanced with mechanisms that address immune evasion. Citation Format: Divya Mathur, Adam Root, Bozena Bugaj-Gaweda, Xingzhi Tan, Wei Fang, Stephanie Bisulco, Jonathan Golas, Jessica Kearney, Eric Upeslacis, Johnny Yao, Edward Rosfjord, Chad Stevens, Lindsay King, Jatin Narula, Kerry Kelleher, Cynthia Rohde, Lioudmila Tchistiakova, Anhco Nguyen, Puja Sapra. A GUCY2c-CD3 bispecific engages T cells to induce cytotoxicity in gastrointestinal tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A16.

This Land O'Lakes Conference is presented each year by the Division of Pharmacy Professional Development within the School of Pharmacy at the University of Wisconsin-Madison (USA). The purpose of this 3-day conference is to provide an educational forum to discuss issues and applications associated with the analysis of xenobiotics, metabolites, biologics and biomarkers in biological matrices. The conference is designed to include and encourage an open exchange of scientific and methodological applications for bioanalysis. To increase the interactive nature of the conference, the program is a mixture of lectures, interactive discussions and a poster session. This report summarized the presentations at the 16th Annual Conference. 6th Annual Land O'Lakes Bioanalytical Conference, Fluno Center Madison, WI, USA, 13–16 July 2015

Seema Kumar is a Principal Scientist at Pfizer. She leads a group that provides regulated bioanalytical support including assay development, validation and sample analysis for the PK and immunogenicity assessment for preclinical and clinical development of Pfizer's biotherapeutics portfolio. She is also responsible for scientific oversight of regulated studies outsourced at CROs. Prior to Pfizer, Dr Kumar held a similar role as Director of CLIA certified Clinical Bioanalytical Laboratory at XBiotech USA, Inc. She holds a PhD in Biophysical Chemistry from Johns Hopkins University, and has published several publications in peer-reviewed journals, and contributed to book chapters. The objective of antibody–drug conjugate (ADC) bioanalysis at different stages of drug development may vary and so are the associated bioanalytical challenges. While at early drug discovery stage involving candidate selection, optimization and preliminary nonclinical assessments, the goal of ADC bioanalysis is to provide PK, toxicity and efficacy data that assists in the design and selection of potential drug candidates, the late nonclinical and clinical drug development stage typically involves regulated ADC bioanalysis that delivers TK data to define and understand pharmacological and toxicological properties of the lead ADC candidate. Bioanalytical strategies and considerations involved in developing successful ligand binding assays for ADC characterization from early discovery to late nonclinical stages of drug development are presented here.

Trastuzumab emtansine (T-DM1) is an antibody–drug conjugate (ADC) that has demonstrated clinical benefit for patients with HER2+ metastatic breast cancer; however, its clinical activity is limited by inherent or acquired drug resistance. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2+ tumors, are not well understood. We used HER2+ cell lines to develop models of T-DM1 resistance using a cyclical dosing schema in which cells received T-DM1 in an “on-off” routine until a T-DM1–resistant population was generated. T-DM1–resistant N87 cells (N87-TM) were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non–cleavable-linked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug transporter protein (e.g., MDR1) expression compared with parental N87 cells. Intriguingly, T-ADCs using auristatin payloads attached via an enzymatically cleavable linker overcome T-DM1 resistance in N87-TM cells. Importantly, N87-TM cells implanted into athymic mice formed T-DM1 refractory tumors that remain sensitive to T-ADCs with cleavable-linked auristatin payloads. Comparative proteomic profiling suggested enrichment in proteins that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells internalize T-ADCs into intracellular caveolin-1 (CAV1)–positive puncta and alter their trafficking to the lysosome compared with N87 cells. T-DM1 colocalization into intracellular CAV1-positive puncta correlated with reduced response to T-DM1 in a panel of HER2+ cell lines. Together, these data suggest that caveolae-mediated endocytosis of T-DM1 may serve as a novel predictive biomarker for patient response to T-DM1. Mol Cancer Ther; 17(1); 243–53. ©2017 AACR.

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years’ editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies’ Input. Part 1 (Small molecules bioanalysis using LCMS) was published in the Bioanalysis issue 6(22) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the Bioanalysis issue 6(24).

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years’ editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for Large molecules bioanalysis using LBA and Immunogenicity. Part 1 (Small molecules bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' Input) were published in the Bioanalysis issues 6(22) and 6(23), respectively.

The objective of this research was to develop an in vitro dendritic cell based assay to assess if differences in internalization of biotherapeutic drugs (BioTx) and/or intra-cellular trafficking could be measured and used to help predict clinical immunogenicity risk. An unwanted immune response can adversely impact pharmacokinetics, safety and efficacy. Since immunogenicity in preclinical species is not clinically predictive there is a strong need for in vitro tools to enable BioTx drug design. Immature dendritic cells (iDCs) were selected because of their high fluid phase pinocytosis rates, ability to recycle IgGs and route immunogenic molecules to MHC-II compartments. Initial assay development evaluated iDC differentiated from a monocytic human cell line vs primary monocytes from fresh whole blood using IL4 and GM-CSF. Fluid phase internalization rates of Ovalbumin and Lucifer yellow decreased from day 3 to day 5 and CD80, CD86 and HLA-DR expression increased consistent with DC maturation. Imaging cytometry was used to measure internalization extent, rate and LAMP-1 co-localization of fluorescently conjugated ovalbumin and BioTx with known clinical immunogenicity incidence. Primary monocytes derived iDC were selected based on extent of internalization for further assay characterization in multiple donors. No target expression was observed. The extent of Adalimumab, Traztuzumab, and Utomilumab internalization was correlated with clinical immunogenicity incidence suggesting the assay may be able to detect frame work differences. Statistical power calculation of fold change set required donor numbers at 10. The assay can be used in combination with other in vitro immunogenicity assays to support BioTx design.

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer (OVCA), and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor recurrence and metastasis. We demonstrate that protein tyrosine kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase in the Wnt signaling pathway, is enriched on TICs in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). To deliver a potent anticancer drug to PTK7-expressing TICs, we generated a targeted antibody-drug conjugate (ADC) composed of a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker, and Aur0101, an auristatin microtubule inhibitor. The PTK7-targeted ADC induced sustained tumor regressions and outperformed standard-of-care chemotherapy. Moreover, the ADC specifically reduced the frequency of TICs, as determined by serial transplantation experiments. In addition to reducing the TIC frequency, the PTK7-targeted ADC may have additional antitumor mechanisms of action, including the inhibition of angiogenesis and the stimulation of immune cells. Together, these preclinical data demonstrate the potential for the PTK7-targeted ADC to improve the long-term survival of cancer patients.

Aim: Complex nature of bioconjugates require multiple bioanalytical approaches to support PK and absorption, distribution, metabolism and excretion characterization. For antibody-drug conjugate (ADC) bioanalysis both LC–MS and ligand-binding assays (LBAs) are employed. Results: A method consisting of immunocapture extraction of ADC from biomatrices followed by LC–MS analysis of light and heavy chain is described. Drug antibody ratio (DAR) profiles of ADC Tras-mcVC-PF06380101 dosed at 0.3, 1 and 3 mg/kg in Sprague Dawley rats were obtained. Combined with total antibody (monoclonal antibody) measurement by LBA, conjugated payload concentration was calculated. Conclusion: PK profiles from LBA, ADC and calculated conjugated payload (DAR × monoclonal antibody) were in good agreement. We present a new tool for PK assessment of ADCs while also exploring ADC metabolism and DAR sensitivity of LBA ADC assay.

There are many sources of analytical variability in ligand binding assays (LBA). One strategy to reduce variability has been duplicate analyses. With recent advances in LBA technologies, it is conceivable that singlet analysis is possible. We retrospectively evaluated singlet analysis using Gyrolab data. Relative precision of duplicates compared to singlets was evaluated using 60 datasets from toxicokinetic (TK) or pharmacokinetic (PK) studies which contained over 23,000 replicate pairs composed of standards, quality control (QC), and animal samples measured with 23 different bioanalytical assays. The comparison was first done with standard curve and QCs followed by PK parameters (i.e., Cmax and AUC). Statistical analyses were performed on combined duplicate versus singlets using a concordance correlation coefficient (CCC), a measurement used to assess agreement. Variance component analyses were conducted on PK estimates to assess the relative analytical and biological variability. Overall, 97.5% of replicate pairs had a %CV of11% and 50% of the results had a %CV of ≤1.38%. There was no observable bias in concentration comparing the first replicate with the second (CCC of 0.99746 and accuracy value of 1). The comparison of AUC and Cmax showed no observable difference between singlet and duplicate (CCC for AUC and Cmax0.99999). Analysis of variance indicated an AUC inter-subject variability 35.3-fold greater than replicate variability and 8.5-fold greater for Cmax. Running replicates from the same sample will not significantly reduce variation or change PK parameters. These analyses indicated the majority of variance was inter-subject and supported the use of a singlet strategy.

Bio-layer interferometry (BLI) is a label-free technology that can be used for kinetic characterization of proteins. Although other label-free platforms have been used for quantitation purposes (most notably surface plasmon resonance), little work has been done using BLI. Here we present rationale and strategies for the development and analytical qualification of a BLI assay for the quantitation of a humanized antibody therapeutic in cynomolgus monkey plasma. Results of the qualification were compared to those of a validated ELISA used to quantitate the same therapeutic. Selectivity, matrix effect, and precision and accuracy were similar between the two methods. Target interference was more pronounced in the BLI assay compared to the ELISA. The main difference between the two assays was in the dynamic range (0.1–10 μg/mL for ELISA vs. 0.4–50 μg/mL for BLI). The monkey plasma BLI assay was applied to rat plasma for the comparison of study samples generated in the same matrix by ELISA. A direct quantitation comparison of sample results for the two methods shows a high degree of agreement (r2 = 0.979, slope = 1.017). However, an evaluation of low concentration samples showed a bias of over-recovery in the BLI compared to the ELISA. In addition to utilizing the quantitative capabilities of the platform, we evaluated the utility of using the kinetic properties of the quantitative assay to detect anti-drug antibodies (ADA) and illustrated the potential for ADA to cause either over recovery (non-neutralizing ADA) or under recovery (neutralizing ADA) of a biotherapeutic using the BLI assay.

We propose convergent plasma lenses, possibly from current sheets, as a generic solution to strong interstellar scattering. These lenses resolve the overpressure problem by geometric alignment as noted by Goldreich and Shridhar (2006). They further quantitatively explain properties of extreme scattering events, and pulsar parabolic arcs. This model makes quantitative predictions testable by VLBI on scattering events. It differs conceptually from previous models by occurring through rare, localized underdense sheets. Such sheets are thermally and kinematically stable, and could be consequences of reconnection. The apparent diffractive effects are a result of coherent interference of refractive images. We propose that these lenses can be used for precision distance determination to pulsars, enabling accurate gravity source localization.

Most therapeutic monoclonal antibodies are designed to bind a specific antigen to elicit pharmacological effects. Accurate quantification of a therapeutic monoclonal antibody in biological matrices is essential for assessing its pharmacokinetics and selecting an effective dosing regimen. Therapeutic antibodies may exist in free, partially bound and fully bound forms in the bloodstream. The choice of which form(s) to measure and how to measure them is gaining much attention with the increase in the number of soluble therapeutic targets. This article will review the bioanalytical methods used in supporting the clinical development of the US FDA-approved therapeutic monoclonal antibodies and also discuss how different factors, such as assay format, target and antibody concentrations, and sample dilutions, can have an impact on the measurement of each form of antibody. Appreciation of which form of drug is being measured and what factors may impact measurement under different conditions are important for interpretation of the pharmacokinetics of therapeutic antibodies.

Summary Objective To examine whether the baseline ratio of a type II collagen breakdown marker to a synthesis marker, or the level of these markers individually, is associated with the likelihood of knee osteoarthritis (OA) progression between baseline and 18 months. Methods Participants were recruited from community sources and had knee OA. Blood was drawn at baseline. Collagen synthesis was measured by commercial enzyme-linked immunosorbent assay (ELISA) assay that detects c-propeptide of type II procollagen (CPII). Serum markers of collagenase cleavage of cartilage type II collagen [C2C epitope (COL2-3/4Clong mono) and C1,2C epitope (COL2-3/4Cshort)] were also assayed. Knee radiographs (semi-flexed with fluoro confirmation) were obtained at baseline and 18 months. OA progression was examined using worsening of joint space grade and worsening of Kellgren/Lawrence grade. The relationship between baseline serum markers and subsequent progression was analyzed from logistic regression. Results Baseline levels of these markers, considered individually, were not associated with a change in the odds of progression. Belonging to the low synthesis tertile was associated with a greater likelihood of progression, approaching significance (adjusted odds ratio [OR] 1.86, 95% confidence interval [CI] 0.96, 3.63). A greater C2C:CPII ratio and C1,2C:CPII ratio were each associated with an increase in the odds of joint space grade progression, which approached significance (e.g., adjusted OR of C2C:CPII ratio was 3.15, 95% CI 0.91, 10.85). Conclusion While the degradation markers individually, considered as continuous variables, did not predict OA progression, belonging to the lower synthesis marker tertile and greater degradation/synthesis marker ratios were associated with an elevation in the odds of progression albeit not achieving significance.

Although weight concerns and smoking are related, anorexic and bulimic women, both of whom have elevated weight concerns, have significant differences in smoking status. Fewer anorexic women smoke, compared with bulimic women, suggesting that weight concerns do not fully explain smoking status. This study investigated the contribution of one factor, cognitive style, to differences in smoking status among college women with different levels of weight concerns: Anorexic tendencies (n = 47), bulimic tendencies (n = 62), weight concerns (n = 56), and no weight concerns (n = 76). Nearly 47% of women with bulimic tendencies, 37.5% of women with weight concerns, and 27.7% of women with anorexic tendencies were current smokers. A total of 22% of women without weight concerns were current smokers. A logistic regression model revealed that race, age, the Personal Standards subscale of the Frost Multidimensional Perfectionism Scale, the Self-Control and Self-Esteem subscale of the Brief Mizes Anorectic Cognitions Questionnaire, and membership in the bulimic tendencies category were significant independent predictors of smoking status. Both weight concerns and cognitive-style variables, including perfectionism and self-control, carried "weight" in the prediction of smoking among college women. In light of these findings, treatment research should explore both the behavioral and cognitive factors associated with weight-concerned college smokers.

A mathematical model capable of accurately characterizing intracellular disposition of ADCs is essential for a priori predicting unconjugated drug concentrations inside the tumor. Towards this goal, the objectives of this manuscript were to: (1) evolve previously published cellular disposition model of ADC with more intracellular details to characterize the disposition of T-DM1 in different HER2 expressing cell lines, (2) integrate the improved cellular model with the ADC tumor disposition model to a priori predict DM1 concentrations in a preclinical tumor model, and (3) identify prominent pathways and sensitive parameters associated with intracellular activation of ADCs. The cellular disposition model was augmented by incorporating intracellular ADC degradation and passive diffusion of unconjugated drug across tumor cells. Different biomeasures and chemomeasures for T-DM1, quantified in the companion manuscript, were incorporated into the modified model of ADC to characterize in vitro pharmacokinetics of T-DM1 in three HER2+ cell lines. When the cellular model was integrated with the tumor disposition model, the model was able to a priori predict tumor DM1 concentrations in xenograft mice. Pathway analysis suggested different contribution of antigen-mediated and passive diffusion pathways for intracellular unconjugated drug exposure between in vitro and in vivo systems. Global and local sensitivity analyses revealed that non-specific deconjugation and passive diffusion of the drug across tumor cell membrane are key parameters for drug exposure inside a cell. Finally, a systems pharmacokinetic model for intracellular processing of ADCs has been proposed to highlight our current understanding about the determinants of ADC activation inside a cell.

Objective Ankylosing spondylitis (AS) is a progressive disease in which chronic inflammation can lead to extensive new bone formation throughout the spine. At present, few measures of the activity or extent of the disease are available. In this study, we sought to determine whether markers of cartilage synthesis and degradation could provide such quantitative measures. Methods Serum samples from 23 patients receiving infliximab treatment for AS were obtained at baseline and at weeks 2, 6, 14, and 22. Patients were stratified with respect to joint involvement and baseline levels of inflammatory markers, and responders were defined according to the Assessments in Ankylosing Spondylitis 20% criteria. Serial measurements of interferon-γ, tumor necrosis factor α, transforming growth factor β (TGFβ), interleukin-10 (IL-10), and IL-1 were done at each time point. The following biomarkers were measured by enzyme-linked immunosorbent assay: the proteoglycan aggrecan 846 epitope, a marker of cartilage turnover; C-propeptide of type II collagen (CPII), a biosynthesis marker; and the Col2-3/4long mono (C2C) and Col2-3/4short (C1–2C) neoepitopes, reflecting collagen cleavage of type II collagen and type I/type II collagen, respectively. Results At baseline, patients with AS demonstrated significant elevations in serum levels of CPII, the 846 epitope, and the CPII-to-C2C (CPII:C2C) ratio (but not C2C or C1–2C) compared with normal controls. Of the biomarkers examined, only CPII:C2C showed a correlation with the C-reactive protein (CRP) level. Among the biomarker–cytokine relationships, TGFβ demonstrated a trend toward a positive correlation with the 846 epitope. Conclusion In AS, elevated serum levels of CPII and the 846 epitope may be related to biosynthetic turnover of hyaline cartilage and the intervertebral discs but may also reflect progressive bone formation as a result of endochondral ossification. The correlation of the CPII:C2C ratio with CRP suggests that the CPII:C2C ratio might prove to be a useful marker of disease activity in AS.

Objective To characterize the time course of aggrecan and type II collagen degradation in the rat iodoacetate model of cartilage degeneration in relationship to the temporal sequence that has been described in human osteoarthritis (OA). Design Rats were injected intra-articularly in one knee joint with iodoacetate and damage to the tibial plateau was assessed from digitized images captured using an image analyzer. The articular cartilage from the tibial plateau was harvested, extracted and glycosaminoglycan (GAG) content was measured using the dimethylmethylene blue (DMMB) assay. Cartilage aggrecan neoepitopes were detected in cartilage extracts by Western blotting using antibodies recognizing the aggrecanase-generated C-terminal neoepitope NITEGE (BC-13) and the MMP-generated C-terminal neoepitope DIPEN (BC-4). A type II collagen collagenase-generated neoepitope was detected in cartilage extracts by ELISA using the Col2-3/4Cshort antibody; denatured collagen was detected using the Col2-3/4m antibody. Results Degenerative joint changes and proteoglycan (GAG) loss progressed with time after iodoacetate injection. Western blotting of cartilage extracts of iodoacetate treated rats demonstrated an increase in both aggrecanase- and MMP-generated epitopes with the NITEGE aggrecanase neoepitope being significantly elevated on days 7, 14 and 21 while DIPEN the MMP neoepitope was significantly elevated on days 7 and 14. The type II collagen neoepitope recognized by Col2-3/4Cshort was significantly increased in cartilage extracts of rats at days 14 and 21 after iodoacetate injection. Conclusion The proteoglycan fragments extracted from the knee cartilage of rats after the intra-articular injection of iodoacetate appeared to result from cleavage at both aggrecanase and MMP sites. Cleavage of type II collagen by collagenase was also detected after iodoacetate injection and occurred subsequent to the initiation of aggrecan loss. These observations serve to demonstrate similarities in the mechanisms of cartilage degeneration induced by iodoacetate to those seen in articular cartilage in OA.

This paper uses results of two research projects investigating the pressures on volunteers in UK sport to illustrate the implications of partnerships between national governing bodies of sport and the central government. National governing bodies of sport receive funding from the central government via Sport England and UK Sport. This funding has conditions attached. The conditions will affect volunteers at the national level of the NGB, but will also cascade down to volunteers in the sports clubs. Thus, they add to the complexity and scale of tasks performed by club level volunteers, who require additional support to perform them. For most NGBs, this external funding is a very significant proportion of their income, so it may have a corresponding impact on the development of the sports and the work of volunteers. The conditions attached to support can be understood in the broader context of a professionalisation of sport in the voluntary sector. As such, the pressures from the central government ...

Increasingly, commercial immunoassay kits are used to support drug discovery and development. Longitudinally consistent kit performance is crucial, but the degree to which kits and reagents are characterized by manufacturers is not standardized, nor are the approaches by users to adapt them and evaluate their performance through validation prior to use. These factors can negatively impact data quality. This paper offers a systematic approach to assessment, method adaptation and validation of commercial immunoassay kits for quantification of biomarkers in drug development, expanding upon previous publications and guidance. These recommendations aim to standardize and harmonize user practices, contributing to reliable biomarker data from commercial immunoassays, thus, enabling properly informed decisions during drug development.

This article shows how the concept of a recruitment niche can be valuable in understanding the difficulties the Guide Association in the United Kingdom has in recruiting new volunteers. Understanding Guiding as career volunteering, within serious leisure, shows how the distinctive ethos of the existing volunteers contributes to the social construction of the recruitment niche. The defining boundaries of the niche restrict the ability to recruit new volunteers. Thus the article gives an example of how a recruitment niche for a voluntary organization can be defined using the socially constructed ethos of volunteers involved in career volunteering rather than by characteristics such as level of educational attainment. It also demonstrates the implications of this for voluntary organizations wishing to increase recruitment.

The L4 Global Harmonization Team on reagents and their stability focused on the management of critical reagents for pharmacokinetic, immunogenicity, and biomarker ligand binding assays. Regulatory guidance recognizes that reagents are important for ligand binding assays but do not address numerous aspects of critical reagent life cycle management. Reagents can be obtained from external vendors or developed internally, but regardless of their source, there are numerous considerations for their reliable long-term use. The authors have identified current best practices and provided recommendations for critical reagent lot changes, stability management, and documentation.

Author for correspondence: Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, Eastern Point Rd, Groton, CT 06340, USA Tel.: +1 860 715 0641 Fax: +1 860 715 9501 E-mail: tracey.clark@pfizer.com Xiaogang Han Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, CT, USA Lindsay King Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, CT, USA Frank Barletta Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer, NY, USA Antibody–drug conjugate (ADC) therapeutics utilize the specificity of monoclonal antibodies (mAbs) and potency of highly toxic small molecules. ADCs are typically composed of a mAb with a cytotoxin conjugated to it, resulting in a heterogeneous mixture of mAb with various numbers of toxins. Due to this heterogeneity, characterized by the therapeutic drug-to-antibody ratio (DAR), the selection of bioanalytical tools, biomeasure assays and analytes used to understand and develop ADCs can be challenging [1]. Since the therapeutic has both largeand small-molecule components, one can use bioanalytical tools in both spaces. Questions for ADC programs are typically: what should be measured, when, and by which method? Since all assays have limitations [1], a combination of bioanalytical tools is typically used to understand the ADC in vitro/in vivo, understand payload delivery to the site of action and to establish an exposure–response relationship.