Mechanistic Modeling of Central Nervous System Pharmacokinetics and Target Engagement of HER2 Tyrosine Kinase Inhibitors to Inform Treatment of Breast Cancer Brain Metastases.

PURPOSE: This study evaluated the central nervous system (CNS) pharmacokinetics and target engagement of lapatinib, neratinib, and tucatinib in patients with cancer, using a physiologically based pharmacokinetic (PBPK) modeling approach. EXPERIMENTAL DESIGN: Drug-specific parameters for in vitro metabolism, binding to plasma proteins and brain tissues, transcellular passive permeability, and interactions with efflux transporters were determined. Whole-body PBPK models integrated with a 4-compartment permeability-limited brain model was developed and verified for predicting plasma and CNS pharmacokinetics. Target engagement ratio (TER), defined as the ratio of the average steady-state unbound drug brain concentration (Css,ave,br) to in vitro IC50 for HER2 inhibition, was used as a predictor of intracranial efficacy. RESULTS: PBPK models predicted that following 1 cycle of standard dosing, tucatinib and lapatinib achieved similar Css,ave,br (14.5 vs. 16.8 nmol/L), while neratinib Css,ave,br (0.68 nmol/L) was 20-fold lower. Tucatinib and neratinib were equally potent for HER2 inhibition (IC50, 6.9 vs. 5.6 nmol/L), while lapatinib was less potent (IC50, 109 nmol/L). The model-predicted population mean TER in the human normal brain was 2.1 for tucatinib, but < 0.20 for lapatinib and neratinib. CONCLUSIONS: The PBPK modeling suggests that tucatinib induces sufficient HER2 inhibition (TER > 2.0) in not only brain metastases with a disrupted blood-brain barrier (BBB), but also micrometastases where the BBB largely remains intact. These findings, in line with available clinical pharmacokinetics and efficacy data, support the therapeutic value of tucatinib for treatment of brain metastases and warrant further clinical investigation for the prevention of brain metastases in patients with HER2-positive breast cancer.

2021 White Paper on Recent Issues in Bioanalysis: Mass Spec of Proteins, Extracellular Vesicles, CRISPR, Chiral Assays, Oligos; Nanomedicines Bioanalysis; ICH M10 Section 7.1; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A - Recommendations on 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).

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 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

Elimination of tucatinib, a small molecule kinase inhibitor of HER2, is primarily governed by CYP2C8 enantioselective oxidation of gem-dimethyl.

PURPOSE: Tucatinib, a small molecule for the treatment of metastatic HER2-positive breast cancer, was extensively metabolized in humans to multiple oxidative metabolites. To fully understand the elimination and biotransformation pathways of tucatinib, we investigated the in vitro and in vivo metabolism of tucatinib, and also conducted a Phase I trial using [14C]tucatinib. METHODS: To identify the responsible enzymes for tucatinib clearance, we investigated the in vitro metabolism of tucatinib including enzyme phenotyping, which facilitated the discovery of several metabolites in human and monkey plasma and excreta, in particular M1 (ONT-993, an aliphatic hydroxylated metabolite). Stereoselective formation of M1 was further investigated in vitro, in vivo, and in silico. RESULTS: In humans, approximately 86% of the total radiolabeled dose was recovered in feces and 4% in urine; in plasma, approximately 76% of radioactivity circulated as parent drug, with 19% attributed to multiple metabolites. The primary isoforms responsible for the elimination of tucatinib were CYP2C8 and CYP3A4/5. CYP2C8 was shown to possess sole catalytic activity for the formation of M1, whereas CYP3A4/5 and aldehyde oxidase catalyzed the formation of the remaining metabolites. Subsequent investigation revealed that M1 was formed in a stereoselective manner. Examination of the enantiomeric ratio of M1 stereoisomers observed in humans relative to cynomolgus monkeys revealed comparable results, suggesting that the enantiomers that comprise M1 were not considered to be unique or disproportionately high in human. CONCLUSION: CYP2C8 and CYP3A4/5 are the primary drug-metabolizing enzymes involved in the in vitro metabolism of tucatinib, which provided the basis to describe human disposition of tucatinib and formation of the observed metabolites.

2020 White Paper on Recent Issues in Bioanalysis: BMV of Hybrid Assays, Acoustic MS, HRMS, Data Integrity, Endogenous Compounds, Microsampling and Microbiome (Part 1 - Recommendations on Industry/Regulators Consensus on BMV of Biotherapeutics by LCMS, Advanced Application in Hybrid Assays, Regulatory Challenges in Mass Spec, Innovation in Small Molecules, Peptides and Oligos).

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, cell therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by Mass Spectrometry (hybrid assays, LCMS and HRMS) 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 1) Hybrid Assays, Innovation in Small Molecules, & Regulated Bioanalysis. Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation), Part 2B (Regulatory Input) and Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 5, and 6 (2021), respectively.

Tucatinib Inhibits Renal Transporters OCT2 and MATE Without Impacting Renal Function in Healthy Subjects.

Tucatinib is a potent tyrosine kinase inhibitor selective for human epidermal growth factor receptor 2 (HER2) approved by the US Food and Drug Administration for the treatment of HER2-positive metastatic breast cancer and in development for other HER2-positive solid tumors. Modest, reversible serum creatinine (SCr) elevations have been observed in tucatinib clinical trials. SCr is conveyed by the renal drug transporters organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1) and 2-K (MATE2-K) and can increase in the presence of inhibitors of these transporters. In vitro, tucatinib inhibited OCT2-, MATE1-, and MATE2-K-mediated transport of metformin, with IC50 values of 14.7, 0.340, and 0.135 µM, respectively. Tucatinib also inhibited OCT2- and MATE1-mediated transport of creatinine, with IC50 values of 0.107 and 0.0855 µM, respectively. A phase 1 study with metformin administered orally in the absence and presence of tucatinib was conducted in 18 healthy subjects. Renal function was assessed by measuring glomerular filtration rate (GFR; based on iohexol plasma clearance) and endogenous markers (SCr, cystatin C-based estimated glomerular filtration rate [eGFR]) with and without tucatinib. Metformin exposure increased (1.4-fold) and renal clearance decreased (29.99-17.64 L/h) with tucatinib, with no effect on metformin maximum concentration. Creatinine clearance transiently decreased 23% with tucatinib. GFR and eGFR, which are unaffected by OCT2 and/or MATE1/2-K transport, were unchanged with tucatinib. These data demonstrate that tucatinib inhibits OCT2- and MATE1/2-K-mediated tubular secretion of creatinine, which may manifest as mild SCr elevations that are not indicative of renal impairment.

2018 White Paper on Recent Issues in Bioanalysis: focus on immunogenicity assays by hybrid LBA/LCMS and regulatory feedback (Part 2 - PK, PD & ADA assays by hybrid LBA/LCMS & regulatory agencies' inputs on bioanalysis, biomarkers and immunogenicity).

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, week-long event - a full immersion week of 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 2) covers the recommendations for PK, PD and ADA assays by hybrid LBA/LCMS and regulatory agencies' input. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 3 (LBA/cell-based assays: immunogenicity, biomarkers and PK assays) are published in volume 10 of Bioanalysis, issues 22 and 24 (2018), respectively.

2017 White Paper on recent issues in bioanalysis: aren't BMV guidance/guidelines 'Scientific'? (Part 1 - LCMS: small molecules, peptides and small molecule biomarkers).

The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California from 3 April 2017 to 7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of 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 analysis involving LCMS, hybrid LBA/LCMS and ligand-binding assay (LBA) approaches. This 2017 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 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for Small Molecules, Peptides and Small Molecule Biomarkers using LCMS. Part 2 (Biotherapeutics, Biomarkers and Immunogenicity Assays using Hybrid LBA/LCMS and Regulatory Agencies' Inputs) and Part 3 (LBA: Immunogenicity, Biomarkers and PK Assays) are published in volume 9 of Bioanalysis, issues 23 and 24 (2017), respectively.

Antibody-conjugated drug assay for protease-cleavable antibody-drug conjugates.

BACKGROUND: Antibody-drug conjugates (ADCs) require multiple assays to characterize their PK. These assays can separately evaluate the ADC by quantifying the antibody or the conjugated drug and may give different answers due to assay measurement differences, heterogeneous nature of ADCs and potential biotransformations that occur in vivo. RESULTS: We present a new version of the antibody-conjugated drug assay for valine-citrulline-linked monomethylauristatin E (vcMMAE) ADCs. A stable isotope-labeled internal standard, protein A affinity capture and solid-phase cleavage of MMAE using papain was used prior to LC-MS/MS analysis. CONCLUSION: The assay was used to assess the difference in ex vivo drug-linker stability of native-cysteine versus engineered cysteine ADCs and to determine the number of drugs per antibody of a native-cysteine ADC in vivo.

2015 White Paper on recent issues in bioanalysis: focus on new technologies and biomarkers (Part 2 - hybrid LBA/LCMS and input from regulatory agencies).

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of over 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. It is once again a 5-day week long event - a full immersion bioanalytical week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations that emerged from the extensive discussions held during the workshop, and is aimed at providing the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to advance scientific excellence, improve quality and deliver better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 2 covers the recommendations for hybrid LBA/LCMS and regulatory agencies' inputs. Part 1 (small molecule bioanalysis using LCMS) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) will be published in volume 7 of Bioanalysis, issues 22 and 24, respectively.

2014 White Paper on recent issues in bioanalysis: a full immersion in bioanalysis (Part 3 - LBA and immunogenicity).

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.

2014 White Paper on recent issues in bioanalysis: a full immersion in bioanalysis (Part 2 - hybrid LBA/LCMS, ELN & regulatory agencies' input).

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).

Measurement of in vivo drug load distribution of cysteine-linked antibody-drug conjugates using microscale liquid chromatography mass spectrometry.

Analysis of samples containing intact antibody-drug conjugates (ADC) using mass spectrometry provides a direct measurement of the drug-load distribution. Once dosed, the drug load distribution changes due to a combination of biological and chemical factors. Liquid chromatography-mass spectrometry (LC-MS) methods to measure the in vivo drug load distribution have been established for ADCs containing native disulfide bonds (lysine-linked or cysteine-linked). However, because of an IgG reduction step in conjugation processes, using LC-MS to analyze intact cysteine-linked ADCs requires native conditions, thus limiting sensitivity. While this limitation has been overcome at the analytical scale, to date, these methods have not been translated to a smaller scale that is required for animal or clinical doses/sampling. In this manuscript, we describe the development of ADC specific affinity capture reagents for processing in vivo samples and optimization of native LC-MS methods at a microscale. These methods are then used to detect the changing drug load distribution over time from a set of in vivo samples, representing to our knowledge the first native mass spectra of cysteine-linked ADCs from an in vivo source.

Advancing antibody drug conjugation: from the laboratory to a clinically approved anticancer drug.

The concept of using monoclonal antibodies for delivering drugs to cancer cells has been explored for decades, with early work surrounding nonspecific targets and drugs with low potencies. These studies underscored the importance of critical parameters, such as antigen and tumor target selection, linker stability, drug potency, pharmacokinetics, and conjugation methodology, in developing effective antibody drug conjugates with acceptable safety profiles. Brentuximab vedotin represents the culmination of much research and development activities in which many of these parameters were addressed. This article provides an overview of many studies that led to the development of this highly active antibody drug conjugate.

Metabolic engineering of monoclonal antibody carbohydrates for antibody-drug conjugation.

The role that carbohydrates play in antibody function and pharmacokinetics has made them important targets for modification. The terminal fucose of the N-linked glycan structure, which has been shown to be involved in modulation of antibody-directed cellular cytotoxicity, is a particularly interesting location for potential modification through incorporation of alternative sugar structures. A library of fucose analogues was evaluated for their ability to incorporate into antibody carbohydrates in place of the native fucose. A number of efficiently incorporated molecules were identified, demonstrating the ability of fucosyltransferase VIII to utilize a variety of non-natural sugars as substrates. Among these structures was a thiolated analogue, 6-thiofucose, which was incorporated into the antibody carbohydrate with good efficiency. This unnatural thio-sugar could then be used for conjugation using maleimide chemistry to produce antibody-drug conjugates with pronounced cytotoxic activities and improved homogeneity compared to drug attachment through hinge disulfides.

CYP3A-mediated drug-drug interaction potential and excretion of brentuximab vedotin, an antibody-drug conjugate, in patients with CD30-positive hematologic malignancies.

Brentuximab vedotin is an antibody-drug conjugate (ADC) that selectively delivers monomethyl auristatin E (MMAE) into CD30-expressing cells. This study evaluated the CYP3A-mediated drug-drug interaction potential of brentuximab vedotin and the excretion of MMAE. Two 21-day cycles of brentuximab vedotin (1.2 or 1.8 mg/kg intravenously) were administered to 56 patients with CD30-positive hematologic malignancies. Each patient also received either a sensitive CYP3A substrate (midazolam), an effective inducer (rifampin), or a strong inhibitor (ketoconazole). Brentuximab vedotin did not affect midazolam exposures. ADC exposures were unaffected by concomitant rifampin or ketoconazole; however, MMAE exposures were lower with rifampin and higher with ketoconazole. The short-term safety profile of brentuximab vedotin in this study was generally consistent with historic clinical observations. The most common adverse events were nausea, fatigue, diarrhea, headache, pyrexia, and neutropenia. Over a 1-week period, ∼23.5% of intact MMAE was recovered after administration of brentuximab vedotin; all other species were below the limit of quantitation. The primary excretion route is via feces (median 72% of the recovered MMAE). These results suggest that brentuximab vedotin (1.8 mg/kg) and MMAE are neither inhibitors nor inducers of CYP3A; however, MMAE is a substrate of CYP3A.

Bioanalysis of antibody-drug conjugates: American Association of Pharmaceutical Scientists Antibody-Drug Conjugate Working Group position paper.

Antibody-drug conjugates (ADCs) typically consist of a cytotoxic drug covalently bound to an antibody by a linker. These conjugates have the potential to substantially improve efficacy and reduce toxicity compared with cytotoxic small-molecule drugs. Since ADCs are generally complex heterogeneous mixtures of multiple species, these novel therapeutic products present unique bioanalytical challenges. The growing number of ADCs being developed across the industry suggests the need for alignment of the bioanalytical methods or approaches used to assess the multiple species and facilitate consistent interpretation of the bioanalytical data. With limited clinical data, the current strategies that can be used to provide insight into the relationship between the multiple species and the observed clinical safety and efficacy are still evolving. Considerations of the bioanalytical strategies for ADCs based on the current industry practices that take into account the complexity and heterogeneity of ADCs are discussed.

Analytical and bioanalytical technologies for characterizing antibody-drug conjugates.

Antibody-drug conjugates (ADCs) require multiple assays for analytical and bioanalytical characterization due to their heterogeneous and dynamic nature. These assays can help address questions from the drug-loading distribution following conjugation to exposure-response relationships after dosing in vivo. This review describes new assay technologies that have been developed for physiochemical characterization and determination of pharmacokinetic parameters of ADCs.

Development of orally active inhibitors of protein and cellular fucosylation.

The key role played by fucose in glycoprotein and cellular function has prompted significant research toward identifying recombinant and biochemical strategies for blocking its incorporation into proteins and membrane structures. Technologies surrounding engineered cell lines have evolved for the inhibition of in vitro fucosylation, but they are not applicable for in vivo use and drug development. To address this, we screened a panel of fucose analogues and identified 2-fluorofucose and 5-alkynylfucose derivatives that depleted cells of GDP-fucose, the substrate used by fucosyltransferases to incorporate fucose into protein and cellular glycans. The inhibitors were used in vitro to generate fucose-deficient antibodies with enhanced antibody-dependent cellular cytotoxicity activities. When given orally to mice, 2-fluorofucose inhibited fucosylation of endogenously produced antibodies, tumor xenograft membranes, and neutrophil adhesion glycans. We show that oral 2-fluorofucose treatment afforded complete protection from tumor engraftment in a syngeneic tumor vaccine model, inhibited neutrophil extravasation, and delayed the outgrowth of tumor xenografts in immune-deficient mice. The results point to several potential therapeutic applications for molecules that selectively block the endogenous generation of fucosylated glycan structures.

Antibody-drug conjugates: targeted drug delivery for cancer.

The antibody-drug conjugate field has made significant progress recently owing to careful optimization of several parameters, including mAb specificity, drug potency, linker technology, and the stoichiometry and placement of conjugated drugs. The underlying reason for this has been obtained in pre-clinical biodistribution and pharmacokinetics studies showing that targeted delivery leads to high intratumoral free drug concentrations, while non-target tissues are largely spared from chemotherapeutic exposure. Recent developments in the field have led to an increase in the number of ADCs being tested clinically, with 3 in late stage clinical trials: brentuximab vedotin (also referred to as SGN-35) for Hodgkin lymphoma; Trastuzumab-DM1 for breast cancer; and Inotuzumab ozogamicin for non-Hodgkin lymphoma. This review highlights the recent pre-clinical and clinical advances that have been made.

Intracellular activation of SGN-35, a potent anti-CD30 antibody-drug conjugate.

PURPOSE: SGN-35 is an antibody-drug conjugate (ADC) containing the potent antimitotic drug, monomethylauristatin E (MMAE), linked to the anti-CD30 monoclonal antibody, cAC10. As previously shown, SGN-35 treatment regresses and cures established Hodgkin lymphoma and anaplastic large cell lymphoma xenografts. Recently, the ADC has been shown to possess pronounced activity in clinical trials. Here, we investigate the molecular basis for the activities of SGN-35 by determining the extent of targeted intracellular drug release and retention, and bystander activities. EXPERIMENTAL DESIGN: SGN-35 was prepared with (14)C-labeled MMAE. Intracellular ADC activation on CD30(+) and negative cell lines was determined using a combination of radiometric and liquid chromatograhpy/mass spectrometry-based assays. The bystander activity of SGN-35 was determined using mixed tumor cell cultures consisting of CD30(+) and CD30(-) lines. RESULTS: SGN-35 treatment of CD30(+) cells leads to efficient intracellular release of chemically unmodified MMAE, with intracellular concentrations of MMAE in the range of 500 nmol/L. This was due to specific ADC binding, uptake, MMAE retention, and receptor recycling or resynthesis. MMAE accounts for the total detectable released drug from CD30(+) cells, and has a half-life of retention of 15 to 20 h. Cytotoxicity studies with mixtures of CD30(+) and CD30(-) cell lines indicated that diffusible released MMAE from CD30(+) cells was able to kill cocultivated CD30(-) cells. CONCLUSIONS: MMAE is efficiently released from SGN-35 within CD30(+) cancer cells and, due to its membrane permeability, is able to exert cytotoxic activity on bystander cells. This provides mechanistic insight into the pronounced preclinical and clinical antitumor activities observed with SGN-35.