Dynamic Contrast-Enhanced Magnetic Resonance Imaging for the Prediction of Monoclonal Antibody Tumor Disposition.

The prediction of monoclonal antibody (mAb) disposition within solid tumors for individual patients is difficult due to inter-patient variability in tumor physiology. Improved a priori prediction of mAb pharmacokinetics in tumors may facilitate the development of patient-specific dosing protocols and facilitate improved selection of patients for treatment with anti-cancer mAb. Here, we report the use of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), with tumor penetration of the contrast agent gadobutrol used as a surrogate, to improve physiologically based pharmacokinetic model (PBPK) predictions of cetuximab pharmacokinetics in epidermal growth factor receptor (EGFR) positive xenografts. In the initial investigations, mice bearing Panc-1, NCI-N87, and LS174T xenografts underwent DCE-MRI imaging with the contrast agent gadobutrol, followed by intravenous dosing of an 125Iodine-labeled, non-binding mAb (8C2). Tumor concentrations of 8C2 were determined following the euthanasia of mice (3 h-6 days after 8C2 dosing). Potential predictor relationships between DCE-MRI kinetic parameters and 8C2 PBPK parameters were evaluated through covariate modeling. The addition of the DCE-MRI parameter Ktrans alone or Ktrans in combination with the DCE-MRI parameter Vp on the PBPK parameters for tumor blood flow (QTU) and tumor vasculature permeability (σTUV) led to the most significant improvement in the characterization of 8C2 pharmacokinetics in individual tumors. To test the utility of the DCE-MRI covariates on a priori prediction of the disposition of mAb with high-affinity tumor binding, a second group of tumor-bearing mice underwent DCE-MRI imaging with gadobutrol, followed by the administration of 125Iodine-labeled cetuximab (a high-affinity anti-EGFR mAb). The MRI-PBPK covariate relationships, which were established with the untargeted antibody 8C2, were implemented into the PBPK model with considerations for EGFR expression and cetuximab-EGFR interaction to predict the disposition of cetuximab in individual tumors (a priori). The incorporation of the Ktrans MRI parameter as a covariate on the PBPK parameters QTU and σTUV decreased the PBPK model prediction error for cetuximab tumor pharmacokinetics from 223.71 to 65.02%. DCE-MRI may be a useful clinical tool in improving the prediction of antibody pharmacokinetics in solid tumors. Further studies are warranted to evaluate the utility of the DCE-MRI approach to additional mAbs and additional drug modalities.

Nonclinical Development of Biologics: Integrating Safety, Pharmacokinetics, and Pharmacodynamics to Create Smarter and More Flexible Nonclinical Safety Programs Optimizing Animal Use.

Safety assessment of biological drugs has its challenges due to the multiple new different modalities, for example, antibody-drug conjugates, bispecifics, nanobodies, fusion proteins and advanced therapy medicinal products (ATMPs), their different pharmacokinetic and pharmacodynamic properties, and their ability to trigger immunogenicity and toxicity. In the public and in the pharmaceutical industry, there is a strong and general desire to reduce the number of animals used in research and development of drugs and in particular reducing the use of nonhuman primates. Important discussions and activities are ongoing investigating the smarter designs of early research and dose range finding studies, reuse of animals, and replacing animal experiments with in vitro studies. Other important challenges include absence of a relevant species and design of studies and developing genetically modified animals for special investigative toxicology studies. Then, the learnings and challenges from the development of the first ATMPs are available providing valuable insights in the development path for these new potentially transformative treatments. Finally, development of strategies for assessment of immunogenicity and prediction of translation of immunogenicity and associated findings to the clinic. On this, the eighth meeting for the European BioSafe members, these challenges served as the basis for the presentations and discussions during the meeting. This article serves as the workshop report reviewing the presentations and discussions at the meeting.

High-Throughput, Sensitive LC-MS Quantification of Biotherapeutics and Biomarkers Using Antibody-Free, Peptide-Level, Multiple-Mechanism Enrichment via Strategic Regulation of pH and Ionic and Solvent Strengths.

Sensitive and high-throughput measurement of biotherapeutics and biomarkers in plasma and tissues is critical for protein-drug development. Enrichment of target signature peptide (SP) after sample digestion permits sensitive LC-MS-based protein quantification and carries several prominent advantages over protein-level enrichment; however, developing high-quality antipeptide antibodies is challenging. Here we describe a novel, antibody-free, peptide-level-enrichment technique enabling high-throughput, sensitive, and robust quantification of proteins in biomatrices, by highly selective removal of matrix peptides and components via cation-exchange (CX) reversed-phase (RP) SPE with strategically regulated pH and ionic and organic strengths. Multiple-mechanism washing and elution achieved highly selective separation despite the low plate number of the SPE cartridge. We first investigated the adsorption-desorption behaviors of peptides on CX-RP sorbent and the coexisting, perplexing effects of pH, and ionic and organic strengths on the selectivity for SP enrichment, which has not been previously characterized. We demonstrated that the selectivity for separating target SPs from matrix peptides was closely associated with buffer pH relative to the pI of the SP, and pH values of pI - 2, pI, and pI + 2 respectively provided exceptional specificity for the ionic wash, the hydrophobic wash, and selective elution. Furthermore, desorption of peptides from the mixed-mode sorbent showed exponential and linear dependence, respectively, on organic-solvent percentage and salt percentage. On the basis of these findings, we established a streamlined procedure for rapid and robust method development. Quantification of biotherapeutics, targets, and biomarkers in plasma and tissues was used as the model system. Selective enrichment of target SPs was achieved along with elimination of 87-95% of matrix peptides, which improved the LOQ by 20-fold (e.g., 2 ng per gram of tissue). Application was demonstrated by sensitive quantification of time courses of mAb (T84.66) and target (CEA) in plasma and tumor tissues from a low-dose mouse PK study. For the first time, down-regulation of membrane-associated antigen following mAb treatment was observed. The CX-RP enrichment is robust, high-throughput, and universally applicable and thus is highly valuable for ultrasensitive, large-scale measurement of target protein in plasma and tissues.

Are Biotransformation Studies of Therapeutic Proteins Needed? Scientific Considerations and Technical Challenges.

For therapeutic proteins, the currently established standard development path generally does not foresee biotransformation studies by default because it is well known that the clearance of therapeutic proteins proceeds via degradation to small peptides and individual amino acids. In contrast to small molecules, there is no general need to identify enzymes involved in biotransformation because this information is not relevant for drug-drug interaction assessment and for understanding the clearance of a therapeutic protein. Nevertheless, there are good reasons to embark on biotransformation studies, especially for complex therapeutic proteins. Typical triggers are unexpected rapid clearance, species differences in clearance not following the typical allometric relationship, a mismatch in the pharmacokinetics/pharmacodynamics (PK/PD) relationship, and the need to understand observed differences between the results of multiple bioanalytical methods (e.g., total vs. target-binding competent antibody concentrations). Early on during compound optimization, knowledge on protein biotransformation may help to design more stable drug candidates with favorable in vivo PK properties. Understanding the biotransformation of a therapeutic protein may also support designing and understanding the bioanalytical assay and ultimately the PK/PD assessment. Especially in cases where biotransformation products are pharmacologically active, quantification and assessment of their contribution to the overall pharmacological effect can be important for establishing a PK/PD relationship and extrapolation to humans. With the increasing number of complex therapeutic protein formats, the need for understanding the biotransformation of therapeutic proteins becomes more urgent. This article provides an overview on biotransformation processes, proteases involved, strategic considerations, regulatory guidelines, literature examples for in vitro and in vivo biotransformation, and technical approaches to study protein biotransformation. SIGNIFICANCE STATEMENT: Understanding the biotransformation of complex therapeutic proteins can be crucial for establishing a pharmacokinetic/pharmacodynamic relationship. This article will highlight scientific, strategic, regulatory, and technological features of protein biotransformation.

Prediction of Human Pharmacokinetics of Fomepizole from Preclinical Species Pharmacokinetics Based on Normalizing Time Course Profiles.

Fomepizole is used as an antidote to treat methanol poisoning due to its selectivity towards alcohol dehydrogenase. In the present study, the goal is to develop a method to predict the fomepizole human plasma concentration versus time profile based on the preclinical pharmacokinetics using the assumption of superimposability on simulated time course profiles of animals and humans. Standard allometric equations with/without correction factors were also assimilated in the prediction. The volume of distribution at steady state (Vss) predicted by simple allometry (57.55 L) was very close to the reported value (42.17 L). However, clearance (CL) prediction by simple allometry was at least 3-fold higher to the reported value (33.86 mL/min); hence, multiple correction factors were used to predict the clearance. Both brain weight and maximum life span potential could predict the CL with 1.22- and 1.01-fold difference. Specifically, the predicted Vss and CL values via interspecies scaling were used in the prediction of series of human intravenous pharmacokinetic parameters, while the simulation of human oral profile was done by the use of absorption rate constant (Ka) from dog following the applicability of human bioavailability value scaled from dog data. In summary, the findings indicate that the utility of diverse allometry approaches to derive the human pharmacokinetics of fomepizole after intravenous/oral dosing.

Synthesis of a tubugi-1-toxin conjugate by a modulizable disulfide linker system with a neuropeptide Y analogue showing selectivity for hY1R-overexpressing tumor cells.

Tubugi-1 is a small cytotoxic peptide with picomolar cytotoxicity. To improve its cancer cell targeting, it was conjugated using a universal, modular disulfide derivative. This allowed conjugation to a neuropeptide-Y (NPY)-inspired peptide [K4(C-ßA-),F7,L17,P34]-hNPY, acting as NPY Y1 receptor (hY1R)-targeting peptide, to form a tubugi-1-SS-NPY disulfide-linked conjugate. The cytotoxic impacts of the novel tubugi-1-NPY peptide-toxin conjugate, as well as of free tubugi-1, and tubugi-1 bearing the thiol spacer (liberated from tubugi-1-NPY conjugate), and native tubulysin A as reference were investigated by in vitro cell viability and proliferation screenings. The tumor cell lines HT-29, Colo320 (both colon cancer), PC-3 (prostate cancer), and in conjunction with RT-qPCR analyses of the hY1R expression, the cell lines SK-N-MC (Ewing`s sarcoma), MDA-MB-468, MDA-MB-231 (both breast cancer) and 184B5 (normal breast; chemically transformed) were investigated. As hoped, the toxicity of tubugi-1 was masked, with IC50 values decreased by ca. 1,000-fold compared to the free toxin. Due to intracellular linker cleavage, the cytotoxic potency of the liberated tubugi-1 that, however, still bears the thiol spacer (tubugi-1-SH) was restored and up to 10-fold higher compared to the entire peptide-toxin conjugate. The conjugate shows toxic selectivity to tumor cell lines overexpressing the hY1R receptor subtype like, e.g., the hard to treat triple-negative breast cancer MDA-MB-468 cells.

Biotherapeutics in non-clinical development: Strengthening the interface between safety, pharmacokinetics-pharmacodynamics and manufacturing.

Biological drugs comprise a wide field of different modalities with respect to structure, pharmacokinetics and pharmacological function. Considerable non-clinical experience in the development of proteins (e.g. insulin) and antibodies has been accumulated over the past thirty years. In order to improve the efficacy and the safety of these biotherapeutics, Fc modifications (e.g. Fc silent antibody versions), combinations (antibody-drug conjugates, protein-nanoparticle combinations), and new constructs (darpins, fynomers) have been introduced. In the last decade, advanced therapy medicinal products (ATMPs) in research and development have become a considerable and strongly growing part of the biotherapeutic portfolio. ATMPs consisting of gene and cell therapy modalities or even combinations of them, further expand the level of complexity, which already exists in non-clinical development strategies for biological drugs and has thereby led to a further diversification of expertise in safety and PKPD assessment of biological drugs. It is the fundamental rationale of the BioSafe meetings, held yearly in the EU and in the US, to convene experts on a regular basis and foster knowledge exchange and mutual understanding in this fast growing area. In order to reflect at least partially the variety of the biotherapeutics field, the 2016 EU BioSafe meeting addressed the following topics in six sessions: (i) In vitro Meets in vivo to Leverage Biologics Development (ii) New developments and regulatory considerations in the cell and gene therapy field (iii) CMC Challenges with Biologics development (iv) Minipigs in non-clinical safety assessment (v) Opportunities of PKPD Assessment in Less Common Administration Routes In the breakout sessions the following questions were discussed: (i) Cynomolgus monkey as a reprotoxicology Species: Impact of Immunomodulators on Early Pregnancy Maintenance (ii) Safety Risk of Inflammation and Autoimmunity Induced by Immunomodulators (iii) Experience with non-GMP Material in Pivotal Non-clinical Safety Studies to Support First in Man (FiM) Trials (iv) Safety Assessment of Combination Products for Non-oncology.

LC-ESI-MS/MS determination of 4-methylpyrazole in dog plasma and its application to a pharmacokinetic study in dogs.

A simple, specific, sensitive and rapid LC-ESI-MS/MS method has been developed and validated for the quantification of 4-methylpyrazole in dog plasma using N-methylnicotinamide-d4 as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a simple protein precipitation. Chromatographic separation of 4-methylpyrazole and the IS was performed on a monolithic (Chromolith RP18e ) column using an isocratic mobile phase comprising 0.2% formic acid in water and acetonitrile (20:80, v/v) at a flow rate of 1.0 mL/min. Elution of 4-methylpyrazole and the IS occurred at ~1.60 and 1.56 min, respectively. The total chromatographic run time was 3.2 min. A linear response function was established in the concentration range of 4.96-4955 ng/mL. The intra- and inter-day accuracy and precision were in the ranges 1.81-12.9 and 3.80-11.1%, respectively. This novel method has been applied to a pharmacokinetic study in dogs.

Intramedullary nailing in opening wedge high tibial osteotomy-in vitro test for validation of a method of fixation.

PURPOSE: Opening wedge high tibial osteotomy (HTO) as a treatment in unicompartimental osteoarthritis of the knee can significantly relieve pain and prevent or at least delay an early joint replacement. The fixation of the osteotomy has undergone development and refinements during the last years. The angle-stable plate fixator is currently one of the most commonly used plates in HTOs. The angular stable fixation between screws and the plate offers a high primary stability to retain the correction with early weight-bearing protocols. This surgical technique is performed as a standard of care and generally well tolerated by the patients. Nevertheless, some studies observed that many patients complained about discomfort related to the implant. METHODS: Therefore, the stability of two different intramedullary nails, a short implant used in humeral fractures and a long device used in tibial fractures for stabilization in valgus HTOs, was investigated as an alternative fixation technique. The plate fixator was defined as reference standard. Nine synthetic tibia models were standardly osteotomized and stabilized by one of the fixation devices. Axial compression was realized using a special testing machine and two protocols were performed: a multi-step fatigue test and a load-to-failure test. RESULTS: Overall motion, medial, and lateral displacements were documented. Fractures always occurred at the lateral cortex. Axial cyclic loading up to 800 N was tolerated by all implants without failure. The tibia nail provided highest fatigue strength under the load-to-failure conditions. CONCLUSIONS: The results suggest that intramedullary nailing might be used as an alternative concept in HTO.

In Vivo Biotransformation of the Fusion Protein Tetranectin-Apolipoprotein A1 Analyzed by Ligand-Binding Mass Spectrometry Combined with Quantitation by ELISA.

The in vivo biotransformation of a novel fusion protein tetranectin/apolipoprotein A1 (TN-ApoA1) was investigated by ligand-binding mass spectrometry (LB-MS) in support of enzyme-linked immunosorbent assays (ELISA). The main focus was on catabolites formed by proteolysis of the fusion protein in rabbit following intravenous administration of lipidated TN-ApoA1. The drug and its catabolites were isolated from rabbit plasma by immunocapture with a monoclonal antibody (mAb) binding to the fusion region of TN-ApoA1. The captured drug and catabolites were released from the streptavidin-coated magnetic beads, separated by monolithic RP capillary HPLC, and online detected by high-resolution mass spectrometry. In addition, the same extract was digested with LysN to confirm or further narrow down the structure of the found catabolites. Two pharmacologically active catabolites were identified with conserved fusion region. The major catabolite [3-285] was formed by truncation of AP at the N-terminus and the minor catabolite [29-270] by truncations of either side of the TN-ApoA1 sequence. Since the ELISA determined the sum of TN-ApoA1, along with its two main catabolites, the individual PK profiles of all three components could be derived by applying their mass peak composition for each sampling point. Parent drug accounted for 25% of drug-related material, whereas that of the catabolites [3-285] and [29-270] accounted for 66% and 9%, respectively. This result could be obtained without catabolite specific ELISAs or quantitative LC-MS assays. It was also confirmed that all relevant functional molecules of TN-ApoA1 in the plasma samples were quantified by the ELISA, which provided a good relationship for pharmacokinetic/pharmacodynamic evaluations.

Non-clinical Safety Evaluation of Biotherapeutics - Challenges, Opportunities and new Insights.

New challenges and opportunities in nonclinical safety testing of biotherapeutics were presented and discussed at the 5th European BioSafe Annual General Membership meeting in November 2015 in Ludwigshafen. This article summarizes the presentations and discussions from both the main and the breakout sessions. The following topics were covered in six main sessions: The following questions were discussed across 4 breakout sessions (i-iv) and a case-study based general discussion (v).

Subcutaneous absorption of biotherapeutics: knowns and unknowns.

Subcutaneous administration of biotherapeutics offers several potential advantages compared with intravenous administration. Many biotherapeutics, both marketed or in development, are administered via the subcutaneous route. This minireview provides an overview of the presystemic absorption processes following subcutaneous administration, the resulting pharmacokinetics after subcutaneous administration, and provides recent case examples of the development of subcutaneous administered drugs with a focus on monoclonal antibodies. Subcutaneous absorption of biotherapeutics is relatively slow and mostly incomplete. Knowledge of the subcutaneous tissue is important to understand the absorption kinetics after subcutaneous administration. Transport in the subcutis to the absorbing blood or lymph capillaries appears to be a major contributor to the slow subcutaneous absorption. Larger proteins (>20 kDa) are mostly absorbed via the lymphatic system, although potential species differences are not fully understood yet. Also, the presystemic catabolism leading to incomplete bioavailability is little understood, both the involved enzymes and its translation across species. For IgGs, binding to the neonatal Fc receptor is important to obtain a high bioavailability. Overall, several aspects of subcutaneous absorption are still poorly understood, which hampers, e.g., translation across species. Further research in this area is warranted.

New challenges and opportunities in nonclinical safety testing of biologics.

New challenges and opportunities in nonclinical safety testing of biologics were discussed at the 3rd European BioSafe Annual General Membership meeting in November 2013 in Berlin: (i)Approaches to refine use of non-human primates in non-clinical safety testing of biologics and current experience on the use of minipigs as alternative non-rodent species.(ii)Tissue distribution studies as a useful tool to support pharmacokinetic/pharmacodynamic (PKPD) assessment of biologics, in that they provide valuable mechanistic insights at drug levels at the site of action.(iii)Mechanisms of nonspecific toxicity of antibody drug conjugates (ADC) and ways to increase the safety margins.(iv)Although biologics toxicity typically manifests as exaggerated pharmacology there are some reported case studies on unexpected toxicity.(v)Specifics of non-clinical development approaches of noncanonical monoclonal antibodies (mAbs), like bispecifics and nanobodies.

Effects of feeding diets containing increasing proportions of bunt-infected wheat (Tilletia caries) on performance and health of pigs.

Tilletia caries is a fungus that mainly infects wheat, causing the disease bunt. Wheat bunt had been one of the most dangerous plant diseases before seed dressing was a common practice. However, it is still of importance in organic farming, since seed dressing is not permitted. Trimethylamine (TMA) is a metabolic product of T. caries known to cause a fishy odour of highly contaminated wheat. Therefore, contamination of feed is supposed to decrease feed intake. However, systematic studies on the effect of practically relevant proportions of bunt-infected wheat on performance and health of pigs are not available. Therefore, the aim of the present study was to investigate the effects of increasing proportions of bunt-infected wheat (0%, 10%, 20% and 30% of the diet) corresponding to a total spore content of 0%, 0.2%, 0.4% and 0.6% on the performance and health of piglets during a 5-week experiment starting after weaning. TMA was not detectable in the contaminated wheat and the feed intake of piglets remained unaffected. However, live weight gain decreased linearly and feed-to-gain ratio significantly increased as the proportion of contaminated wheat increased. Haematological and serum clinical-chemical characteristics as well as the viability of peripheral blood mononuclear cells were not influenced by dietary treatments. Based on the limited data of the present experiment it might be concluded from the adverse effects of bunt-infected wheat in piglet diets on the feed-to-gain ratio that a spore content of 0.2% of the complete diet should not be exceeded.

The effects of interleukin-6 signal blockade on immune system, reproductive and skeletal development in juvenile mice.

Interleukin-6 (IL-6) is involved in the pathogenesis of multiple disorders, including juvenile autoimmune diseases. IL-6 participates in a broad spectrum of physiological events, and the IL-6 receptor (IL-6R) is widely distributed across multiple organs. The interrelationship of development phases in juveniles together with organs involved in IL-6 signaling called for evaluations of anti-IL-6R antibody induced effects in a juvenile mouse model to assess the safety of such an approach in human juvenile arthritis. Here we show that naive mice in which IL-6 signals have been transiently blocked during the juvenile period develop normally. The fatal immunogenic reactions recorded earlier by repeated administration of the chosen rat anti-mouse IL-6R antibody, MR16-1, to mice were avoided successfully by application of a high loading dose followed by lower maintenance doses, with the support of modeling data. The high loading-dose regimen enabled us to conduct assessments without any major interference due to immunogenicity. Transient and complete inhibition of IL-6 signals from postnatal days 22 to 79 in mice exhibited no biologically important changes in sexual maturation or development of immune and skeletal systems. Although tendencies toward reductions of peripheral blood T-cell counts were observed, normal levels of antigen-specific IgG/IgM antibody productions indicating sufficient immunological functions were confirmed. Our results demonstrate that blockage of IL-6R by the neutralizing antibody does not affect juvenile development. This may be in part due to the generation or existence of compensatory pathways in the whole body system.

Click Chemistry-Generated Auristatin F-Linker-Benzylguanine for a SNAP-Tag-Based Recombinant Antibody-Drug Conjugate Demonstrating Selective Cytotoxicity toward EGFR-Overexpressing Tumor Cells.

Antibody-drug conjugates (ADCs) are bifunctional molecules combining the targeting potential of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs. This simple yet intelligently designed system directly addresses the lack of specificity encountered with conventional anti-cancer treatment regimes. However, despite their initial success, the generation of clinically sustainable and effective ADCs has been plagued by poor tumor penetration, undefined chemical linkages, unpredictable pharmacokinetic profiles, and heterogeneous mixtures of products. To this end, we generated a SNAP-tag-based fusion protein targeting the epidermal growth factor receptor (EGFR)-a biomarker of aggressive and drug-resistant cancers. Here, we demonstrate the use of a novel click coupling strategy to engineer a benzylguanine (BG)-linker-auristatin F (AuriF) piece that can be covalently tethered to the EGFR-targeting SNAP-tag-based fusion protein in an irreversible 1:1 stoichiometric reaction to form a homogeneous product. Furthermore, using these recombinant ADCs to target EGFR-overexpressing tumor cells, we provide a proof-of-principle for generating biologically active antimitotic therapeutic proteins capable of inducing cell death in a dose-dependent manner, thus alleviating some of the challenges of early ADC development.

CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate.

PURPOSE: Triple-negative breast cancer (TNBC) is phenotypic of breast tumors lacking expression of the estrogen receptor (ER), the progesterone receptor (PgR), and the human epidermal growth factor receptor 2 (HER2). The paucity of well-defined molecular targets in TNBC, coupled with the increasing burden of breast cancer-related mortality, emphasizes the need to develop targeted diagnostics and therapeutics. While antibody-drug conjugates (ADCs) have emerged as revolutionary tools in the selective delivery of drugs to malignant cells, their widespread clinical use has been hampered by traditional strategies which often give rise to heterogeneous mixtures of ADC products. METHODS: Utilizing SNAP-tag technology as a cutting-edge site-specific conjugation method, a chondroitin sulfate proteoglycan 4 (CSPG4)-targeting ADC was engineered, encompassing a single-chain antibody fragment (scFv) conjugated to auristatin F (AURIF) via a click chemistry strategy. RESULTS: After showcasing the self-labeling potential of the SNAP-tag component, surface binding and internalization of the fluorescently labeled product were demonstrated on CSPG4-positive TNBC cell lines through confocal microscopy and flow cytometry. The cell-killing ability of the novel AURIF-based recombinant ADC was illustrated by the induction of a 50% reduction in cell viability at nanomolar to micromolar concentrations on target cell lines. CONCLUSION: This research underscores the applicability of SNAP-tag in the unambiguous generation of homogeneous and pharmaceutically relevant immunoconjugates that could potentially be instrumental in the management of a daunting disease like TNBC.

Non-human primates in the PKPD evaluation of biologics: Needs and options to reduce, refine, and replace. A BioSafe White Paper.

Monoclonal antibodies (mAbs) deliver great benefits to patients with chronic and/or severe diseases thanks to their strong specificity to the therapeutic target. As a result of this specificity, non-human primates (NHP) are often the only preclinical species in which therapeutic antibodies cross-react with the target. Here, we highlight the value and limitations that NHP studies bring to the design of safe and efficient early clinical trials. Indeed, data generated in NHPs are integrated with in vitro information to predict the concentration/effect relationship in human, and therefore the doses to be tested in first-in-human trials. The similarities and differences in the systems defining the pharmacokinetics and pharmacodynamics (PKPD) of mAbs in NHP and human define the nature and the potential of the preclinical investigations performed in NHPs. Examples have been collated where the use of NHP was either pivotal to the design of the first-in-human trial or, inversely, led to the termination of a project prior to clinical development. The potential impact of immunogenicity on the results generated in NHPs is discussed. Strategies to optimize the use of NHPs for PKPD purposes include the addition of PD endpoints in safety assessment studies and the potential re-use of NHPs after non-terminal studies or cassette dosing several therapeutic agents of interest. Efforts are also made to reduce the use of NHPs in the industry through the use of in vitro systems, alternative in vivo models, and in silico approaches. In the case of prediction of ocular PK, the body of evidence gathered over the last two decades renders the use of NHPs obsolete. Expert perspectives, advantages, and pitfalls with these alternative approaches are shared in this review.

Novel in Vivo and in Vitro Pharmacokinetic/Pharmacodynamic-Based Human Starting Dose Selection for Glofitamab.

We present a novel approach for first-in-human (FIH) dose selection of the CD20xCD3 bispecific antibody, glofitamab, based on pharmacokinetic/pharmacodynamic (PKPD) assessment in cynomolgus monkeys to select a high, safe starting dose, with cytokine release (CR) as the PD endpoint. Glofitamab pharmacokinetics were studied in mice and cynomolgus monkeys; PKPD of IL-6, TNF-α and interferon-γ release following glofitamab, with/without obinutuzumab pretreatment (Gpt) was studied in cynomolgus monkeys. Potency differences for CR between cynomolgus monkeys and humans were determined by glofitamab incubation in whole blood of both species. The PKPD model for CR was translated to humans to project a starting dose that did not induce CR exceeding a clinically-predefined threshold. In cynomolgus monkeys, glofitamab showed a species-specific atypical high clearance, with and without B-cell debulking by Gpt. CR was related to glofitamab serum levels and B-cell counts. B-cell reduction by Gpt led to a marked decrease in CR. FIH starting dose (5 µg) was selected based on IL-6 release considering the markedly higher glofitamab in vitro potency in human vs monkey blood. This is a novel PKPD-based approach for selection of FIH starting dose for a CD20xCD3 bispecific antibody in B-cell lymphoma, evidenced in the glofitamab study, NP30179 (NCT03075696).

Protein engineering of a stable and potent anti-inflammatory IL-37-Fc fusion with enhanced therapeutic potential.

Harnessing the immunomodulatory activity of cytokines is a focus of therapies targeting inflammatory disease. The interleukin (IL)-1 superfamily contains pro-inflammatory and anti-inflammatory members that help orchestrate the immune response in adaptive and innate immunity. Of these molecules, IL-37 has robust anti-inflammatory activity across a range of disease models through inhibition of pro-inflammatory signaling cascades downstream of tumor necrosis factor, IL-1, and toll-like receptor pathways. We find that IL-37 is unstable with a poor pharmacokinetic and manufacturing profile. Here, we present the engineering of IL-37 from an unstable cytokine into an anti-inflammatory molecule with an excellent therapeutic likeness. We overcame these shortcomings through site-directed mutagenesis, the addition of a non-native disulfide bond, and the engineering of IL-37 as an Fc-fusion protein. Our results provide a platform for preclinical testing of IL-37 Fc-fusion proteins. The engineering approaches undertaken herein will apply to the conversion of similar potent yet short-acting cytokines into therapeutics.