Trastuzumab-MMAU Antibody-Auristatin Conjugates: Valine-Glucoserine Linker with Stabilized Maleimide Conjugation Improves In Vivo Efficacy and Tolerability.

PURPOSE: Antibody-drug conjugates (ADCs) have shown impressive clinical activity with approval of many agents in hematological and solid tumors. However, challenges remain with both efficacy and safety of ADCs. This study describes novel trastuzumab-auristatin conjugates with the hydrophilic MMAE prodrug MMAU, and optimization of a glycopeptide linker leading to a wider therapeutic window. EXPERIMENTAL DESIGN: Trastuzumab was conjugated with auristatin payloads via a series of linkers using a stabilized maleimide handle. The ADCs were characterized in vitro and their relative in vivo anti-tumor efficacies were assessed in HER2+ xenograft models. Relative linker stabilities and the mechanism of linker cleavage were studied using in vitro assays. Toxicity and toxicokinetics of the best performing ADC were evaluated in cynomolgus monkey (cyno). RESULTS: The trastuzumab-MMAU ADC with stabilized glycopeptide linker showed maleimide stabilization and higher resistance to cleavage by serum and lysosomal enzymes compared to a valine-citrulline conjugated trastuzumab ADC (trastuzumab-vc-MMAE). A single dose of 1 or 2 mg/kg of trastuzumab-MMAU at drug-to-antibody ratios (DAR) of 8 and 4 respectively resulted in xenograft tumor growth inhibition, with superior efficacy to trastuzumab-vc-MMAE. Trastuzumab-MMAU DAR4 was tolerated at doses up to 12 mg/kg in cyno, which represents 2- to 4-fold higher dose than that observed with vedotin ADCs, and had increased terminal half-life and exposure. CONCLUSIONS: The optimized trastuzumab-MMAU ADC showed potent antitumor activity and was well tolerated with excellent pharmacokinetics in non-human primates, leading to a superior preclinical therapeutic window. The data supports potential utility of trastuzumab-MMAU for treatment of HER2+ tumors.

A long-acting GDF15 analog causes robust, sustained weight loss and reduction of food intake in an obese nonhuman primate model.

Growth Differentiation Factor-15 (GDF15) is a circulating polypeptide linked to cellular stress and metabolic adaptation. GDF15's half-life is ~3 h and activates the glial cell line-derived neurotrophic factor family receptor alpha-like (GFRAL) receptor expressed in the area postrema. To characterize sustained GFRAL agonism on food intake (FI) and body weight (BW), we tested a half-life extended analog of GDF15 (Compound H [CpdH]) suitable for reduced dosing frequency in obese cynomolgus monkeys. Animals were chronically treated once weekly (q.w.) with CpdH or long-acting GLP-1 analog dulaglutide. Mechanism-based longitudinal exposure-response modeling characterized effects of CpdH and dulaglutide on FI and BW. The novel model accounts for both acute, exposure-dependent effects reducing FI and compensatory changes in energy expenditure (EE) and FI occurring over time with weight loss. CpdH had linear, dose-proportional pharmacokinetics (terminal half-life ~8 days) and treatment caused exposure-dependent reductions in FI and BW. The 1.6 mg/kg CpdH reduced mean FI by 57.5% at 1 week and sustained FI reductions of 31.5% from weeks 9-12, resulting in peak reduction in BW of 16 ± 5%. Dulaglutide had more modest effects on FI and peak BW loss was 3.8 ± 4.0%. Longitudinal modeling of both the FI and BW profiles suggested reductions in BW observed with both CpdH and dulaglutide were fully explained by exposure-dependent reductions in FI without increase in EE. Upon verification of the pharmacokinetic/pharmacodynamic relationship established in monkeys and humans for dulaglutide, we predicted that CpdH could reach double digit BW loss in humans. In summary, a long-acting GDF15 analog led to sustained reductions in FI in overweight monkeys and holds potential for effective clinical obesity pharmacotherapy.

Enhanced delivery of antibodies across the blood-brain barrier via TEMs with inherent receptor-mediated phagocytosis.

BACKGROUND: The near impermeability of the blood-brain barrier (BBB) and the unique neuroimmune environment of the CNS prevents the effective use of antibodies in neurological diseases. Delivery of biotherapeutics to the brain can be enabled through receptor-mediated transcytosis via proteins such as the transferrin receptor, although limitations such as the ability to use Fc-mediated effector function to clear pathogenic targets can introduce safety liabilities. Hence, novel delivery approaches with alternative clearance mechanisms are warranted. METHODS: Binders that optimized transport across the BBB, known as transcytosis-enabling modules (TEMs), were identified using a combination of antibody discovery techniques and pharmacokinetic analyses. Functional activity of TEMs were subsequently evaluated by imaging for the ability of myeloid cells to phagocytose target proteins and cells. FINDINGS: We demonstrated significantly enhanced brain exposure of therapeutic antibodies using optimal transferrin receptor or CD98 TEMs. We found that these modules also mediated efficient clearance of tau aggregates and HER2+ tumor cells via a non-classical phagocytosis mechanism through direct engagement of myeloid cells. This mode of clearance potentially avoids the known drawbacks of FcγR-mediated antibody mechanisms in the brain such as the neurotoxic release of proinflammatory cytokines and immune cell exhaustion. CONCLUSIONS: Our study reports a new brain delivery platform that harnesses receptor-mediated transcytosis to maximize brain uptake and uses a non-classical phagocytosis mechanism to efficiently clear pathologic proteins and cells. We believe these findings will transform therapeutic approaches to treat CNS diseases. FUNDING: This research was funded by Janssen, Pharmaceutical Companies of Johnson & Johnson.

Bifunctional molecules targeting SARS-CoV-2 spike and the polymeric Ig receptor display neutralization activity and mucosal enrichment.

The global health crisis and economic tolls of COVID-19 necessitate a panoply of strategies to treat SARS-CoV-2 infection. To date, few treatment options exist, although neutralizing antibodies against the spike glycoprotein have proven to be effective. Because infection is initiated at the mucosa and propagates mainly at this site throughout the course of the disease, blocking the virus at the mucosal milieu should be effective. However, administration of biologics to the mucosa presents a substantial challenge. Here, we describe bifunctional molecules combining single-domain variable regions that bind to the polymeric Ig receptor (pIgR) and to the SARS-CoV-2 spike protein via addition of the ACE2 extracellular domain (ECD). The hypothesis behind this design is that pIgR will transport the molecule from the circulation to the mucosal surface where the ACE ECD would act as a decoy receptor for the nCoV2. The bifunctional molecules bind SARS-Cov-2 spike glycoprotein in vitro and efficiently transcytose across the lung epithelium in human tissue-based analyses. Designs featuring ACE2 tethered to the C-terminus of the Fc do not induce antibody-dependent cytotoxicity against pIgR-expressing cells. These molecules thus represent a potential therapeutic modality for systemic administration of neutralizing anti-SARS-CoV-2 molecules to the mucosa.

Translational PK/PD and model-informed development of JNJ-67842125, a Fab reversal agent for JNJ-64179375, a long-acting thrombin inhibitor.

BACKGROUND AND PURPOSE: Antigen-binding fragment (Fab ) reversal agents were developed to reverse, in bleeding emergency, the long-acting anticoagulant effect of JNJ-64179375 (JNJ-9375), a monoclonal antibody that binds exosite-1 on thrombin. EXPERIMENTAL APPROACH: The pharmacokinetic and pharmacodynamic (PK/PD) activities of three reversal agents of varying in vitro binding affinities to JNJ-9375 were characterised in cynomolgus monkeys. The time course of JNJ-9375 anticoagulant activity and reversal effects of each agent were evaluated. A mechanism-based PK/PD model, which integrated free serum concentrations of reversal agent, total and free serum concentrations of JNJ-9375, and thrombin time, was developed to quantitatively relate JNJ-9375 neutralisation to reversal of induced thrombin time prolongation. Model-based allometric scale-up of the lead reversal agent and the PK/PD relationship of JNJ-9375 in healthy volunteers were utilised to predict clinical dosing regimens. KEY RESULTS: Lowering of free JNJ-9375 by the reversal agents corresponded with reversal of thrombin time prolongation. Total JNJ-9375 displayed typical mAb clearance at 2.75 ml·day-1 ·kg-1 , whereas reversal agents cleared faster between 1400 and 2400 ml·day-1 ·kg-1 . The model-estimated in vivo KD values for JNJ-9375 reversal agents were 9 nM (ICHB-256), 0.4 nM (ICHB-281) and 13.7 pM (ICHB-164), in rank-ordered agreement of their KD values determined in vitro. The three reversal agents exhibited different neutralisation characteristics in vivo, governed primarily by their binding kinetics to JNJ-9375. The model predicted a priori free JNJ-9375 kinetics after dosing ICHB-164 (JNJ-67842125) and JNJ-9375 under a different regimen. CONCLUSION AND IMPLICATIONS: The results enabled selection of JNJ-67842125 as the reversal agent for JNJ-9375.

A minimal physiologically based pharmacokinetic model to characterize colon TNF suppression and treatment effects of an anti-TNF monoclonal antibody in a mouse inflammatory bowel disease model.

Biotherapeutic drugs against tumor necrosis factor (TNF) are effective treatments for moderate to severe inflammatory bowel disease (IBD). Here, we evaluated CNTO 5048, an antimurine TNF surrogate monoclonal antibody (mAb), in a CD45RBhigh adoptive T cell transfer mouse colitis model, which allows examination of the early immunological events associated with gut inflammation and the therapeutic effects. The study was designed to quantitatively understand the effects of IBD on CNTO 5048 disposition, the ability of CNTO 5048 to neutralize pathogenic TNF at the colon under disease conditions, and the impact of dosing regimen on CNTO 5048 treatment effect. CNTO 5048 and TNF concentrations in both mice serum and colon homogenate were also measured. Free TNF concentrations in colon, but not in serum, were shown to correlate well with the colon pharmacodynamic readout, such as the summed histopathology score and neutrophil score. A minimal physiologically based pharmacokinetic (mPBPK) model was developed to characterize CNTO 5048 PK and disposition, as well as colon soluble TNF target engagement (TE). The mPBPK/TE model reasonably captured the observed data and provided a quantitative understanding of an anti-TNF mAb on its colon TNF suppression and therapeutic effect in a physiologically relevant IBD animal model. These results also provided insights into the potential benefits of using induction doses for the treatment of IBD patients.

Characterization of concurrent target suppression by JNJ-61178104, a bispecific antibody against human tumor necrosis factor and interleukin-17A.

Tumor necrosis factor (TNF) and interleukin (IL)-17A are pleiotropic cytokines implicated in the pathogenesis of several autoimmune diseases including rheumatoid arthritis (RA) and psoriatic arthritis (PsA). JNJ-61178104 is a novel human anti-TNF and anti-IL-17A monovalent, bispecific antibody that binds to both human TNF and human IL-17A with high affinities and blocks the binding of TNF and IL-17A to their receptors in vitro. JNJ-61178104 also potently neutralizes TNF and IL-17A-mediated downstream effects in multiple cell-based assays. In vivo, treatment with JNJ-61178104 resulted in dose-dependent inhibition of cellular influx in a human IL-17A/TNF-induced murine lung neutrophilia model and the inhibitory effects of JNJ-61178104 were more potent than the treatment with bivalent parental anti-TNF or anti-IL-17A antibodies. JNJ-61178104 was shown to engage its targets, TNF and IL-17A, in systemic circulation measured as drug/target complex formation in normal cynomolgus monkeys (cyno). Surprisingly, quantitative target engagement assessment suggested lower apparent in vivo target-binding affinities for JNJ-61178104 compared to its bivalent parental antibodies, despite their similar in vitro target-binding affinities. The target engagement profiles of JNJ-61178104 in humans were in general agreement with the predicted profiles based on cyno data, suggesting similar differences in the apparent in vivo target-binding affinities. These findings show that in vivo target engagement of monovalent bispecific antibody does not necessarily recapitulate that of the molar-equivalent dose of its bivalent parental antibody. Our results also offer valuable insights into the understanding of the pharmacokinetics/pharmacodynamics and target engagement of other bispecific biologics against dimeric and/or trimeric soluble targets in vivo.

Discovery and characterization of single-domain antibodies for polymeric Ig receptor-mediated mucosal delivery of biologics.

Mucosal immunity is dominated by secretory IgA and IgM, although these are less favorable compared to IgG molecules for therapeutic development. Polymeric IgA and IgM are actively transported across the epithelial barrier via engagement of the polymeric Ig receptor (pIgR), but IgG molecules lack a lumen-targeted active transport mechanism, resulting in poor biodistribution of IgG therapeutics in mucosal tissues. In this work, we describe the discovery and characterization of single-domain antibodies (VHH) that engage pIgR and undergo transepithelial transport across the mucosal epithelium. The anti-pIgR VHH panel displayed a broad range of biophysical characteristics, epitope diversity, IgA competition profiles and transcytosis activity in cell and human primary lung tissue models. Making use of this diverse VHH panel, we studied the relationship between biophysical and functional properties of anti-pIgR binders targeting different domains and epitopes of pIgR. These VHH molecules will serve as excellent tools for studying pIgR-mediated transport of biologics and for delivering multispecific IgG antibodies into mucosal lumen, where they can target and neutralize mucosal antigens.

Application of multiplexed pharmacokinetic immunoassay to quantify in vivo drug forms and coadministered biologics.

Aim: Meso Scale Discovery U-PLEX® provides an opportunity to develop multiplexed pharmacokinetic (PK) immunoassays. Two case studies demonstrate the utility of multiplexed PK methods. Materials & methods: Development of PK ligand-binding assays quantify of nonclinical plasma concentrations of a biotherapeutic that has degraded due to in vivo biotransformation, and clinical serum concentrations from two biotherapeutics spiked into a single sample. Results: Data from multiplexed U-PLEX PK methods are comparable to results from single-readout streptavidin Meso Scale Discovery gold PK methods. Multiplex measurement of a nonclinical study showed acceptable performance for accuracy, precision and dilutional linearity while a clinical study additionally passed selectivity, specificity and stability. Conclusion: Regulated, validation-ready multiplex PK methods for both nonclinical and clinical studies allow opportunities for high-throughput bioanalysis.

Development and Translational Application of a Minimal Physiologically Based Pharmacokinetic Model for a Monoclonal Antibody against Interleukin 23 (IL-23) in IL-23-Induced Psoriasis-Like Mice.

The interleukin (IL)-23/Th17/IL-17 immune pathway has been identified to play an important role in the pathogenesis of psoriasis. Many therapeutic proteins targeting IL-23 or IL-17 are currently under development for the treatment of psoriasis. In the present study, a mechanistic pharmacokinetics (PK)/pharmacodynamics (PD) study was conducted to assess the target-binding and disposition kinetics of a monoclonal antibody (mAb), CNTO 3723, and its soluble target, mouse IL-23, in an IL-23-induced psoriasis-like mouse model. A minimal physiologically based pharmacokinetic model with target-mediated drug disposition features was developed to quantitatively assess the kinetics and interrelationship between CNTO 3723 and exogenously administered, recombinant mouse IL-23 in both serum and lesional skin site. Furthermore, translational applications of the developed model were evaluated with incorporation of human PK for ustekinumab, an anti-human IL-23/IL-12 mAb developed for treatment of psoriasis, and human disease pathophysiology information in psoriatic patients. The results agreed well with the observed clinical data for ustekinumab. Our work provides an example on how mechanism-based PK/PD modeling can be applied during early drug discovery and how preclinical data can be used for human efficacious dose projection and guide decision making during early clinical development of therapeutic proteins.

Radiation stability of visible and near-infrared optical and magneto-optical properties of terbium gallium garnet crystals.

Perspectives of terbium gallium garnet, Tb3Ga5O12 (TGG), for the use of radiation-resistant high magnetic field sensing are studied. Long-term radiation stability of the TGG crystals was analyzed by comparing the optical and magneto-optical properties of a radiation-exposed TGG crystal (equivalent neutron dose 6.3×10¹³ n/cm²) to the properties of TGG control samples. Simulations were also performed to predict radiation damage mechanisms in the TGG crystal. Radiation-induced increase in the absorbance at shorter wavelengths was observed as well as a reduction in the Faraday effect while no degradation of magneto-optical effect was observed when at wavelengths above 600 nm. This suggests that TGG crystal would be a good candidate for use in magneto-optical radiation-resistant magnetic field sensors.

A novel approach to evaluate the pharmacokinetic biocomparability of a monoclonal antibody derived from two different cell lines using simultaneous crossover design.

A parallel study design with a large number of subjects has been a typical path for pharmacokinetic (PK) biocomparability assessment of biotherapeutics with long half-lives and immunogenic propensity, for example, monoclonal antibodies (mAb). A recently published innovative bioanalytical method that can quantify mAb produced from two different cell lines in the same sample opened an avenue to exploring a simultaneous crossover study design for PK biocomparability assessment of biotherapeutics. Siltuximab, a chimeric IgG1 mAb-targeting interleukin-6, was studied as an example. The pharmacokinetic biocomparability of siltuximab derived from mouse myeloma (Sp2/0) cells and Chinese hamster ovary cells was previously assessed and demonstrated in a clinical PK biocomparability study that enrolled more than 140 healthy subjects using a parallel trial design. The biocomparability was successfully shown in six cynomolgus monkeys in a preclinical proof-of-concept study using the new crossover study design supported by the analytical method. The impact of antidrug antibodies on the assessment of biocomparability was minimal. This novel approach opened up a new arena for the evaluation of PK biocomparability of biotherapeutics with unique molecular signatures such as a mAb derived from different cell lines.

Characterization of critical reagents in ligand-binding assays: enabling robust bioanalytical methods and lifecycle management.

The effective management of validated ligand-binding assays used for PK, PD and immunogenicity assessments of biotherapeutics is vital to ensuring robust and consistent assay performance throughout the lifetime of the method. The structural integrity and functional quality of critical reagents is often linked to ligand-binding assay performance; therefore, physicochemical and biophysical characterization coupled with assessment of assay performance can enable the highest degree of reagent quality. The implementation of a systematic characterization process for monitoring critical reagent attributes, utilizing detailed analytical techniques such as LC-MS, can expedite assay troubleshooting and identify deleterious trends. In addition, this minimizes the potential for costly delays in drug development due to reagent instability or batch-to-batch variability. This article provides our perspectives on a proactive critical reagent QC process. Case studies highlight the analytical techniques used to identify chemical and molecular factors and the interdependencies that can contribute to protein heterogeneity and integrity.

A novel approach for the simultaneous quantification of a therapeutic monoclonal antibody in serum produced from two distinct host cell lines.

Therapeutic monoclonal antibodies (mAbs) possess a high degree of heterogeneity associated with the cell expression system employed in manufacturing, most notably glycosylation. Traditional immunoassay formats used to quantify therapeutic mAbs are unable to discriminate between different glycosylation patterns that may exist on the same protein amino acid sequence. Mass spectrometry provides a technique to distinguish specific glycosylation patterns of the therapeutic antibody within the same sample, thereby allowing for simultaneous quantification of the same mAb with different glycosylation patterns. Here we demonstrate a two-step approach to successfully differentiate and quantify serum mixtures of a recombinant therapeutic mAb produced in two different host cell lines (CHO vs. Sp2/0) with distinct glycosylation profiles. Glycosylation analysis of the therapeutic mAb, CNTO 328 (siltuximab), was accomplished through sample pretreatment consisting of immunoaffinity purification (IAP) and enrichment, followed by liquid chromatography (LC) and mass spectrometry (MS). LC-MS analysis was used to determine the percentage of CNTO 328 in the sample derived from either cell line based on the N-linked G1F oligosaccharide on the mAb. The relative amount of G1F derived from each cell line was compared with ratios of CNTO 328 reference standards prepared in buffer. Glycoform ratios were converted to concentrations using an immunoassay measuring total CNTO 328 that does not distinguish between the different glycoforms. Validation of the IAP/LC-MS method included intra-run and inter-run variability, method sensitivity and freeze-thaw stability. The method was accurate (%bias range = -7.30-13.68%) and reproducible (%CV range = 1.49-10.81%) with a LOQ of 2.5 µg/mL.

Conference report: Bioanalysis highlights from the 2012 American Association of Pharmaceutical Scientists National Biotechnology Conference.

The American Association of Pharmaceutical Scientists (AAPS) is an international forum for the exchange of knowledge among scientists to enhance their contributions to drug development. The annual National Biotechnology Conference, organized by the AAPS on 21-23 May 2012 in San Diego, CA, USA, brings together experts from various disciplines representing private industry, academia and governing institutions dedicated toward advancing the scientific and technological progress related to discovery, development and manufacture of medical biotechnology products. Over 300 scientific poster presentations and approximately 50 oral presentation and discussion sessions examined a breadth of topics pertaining to biotechnology drug development, such as the advancement of vaccines and biosimilars, emerging and innovative technologies, nonclinical and clinical bioanalysis, and regulatory updates. This conference report highlights the existing challenges with ligand-binding assays, emerging challenges, innovative integration of various technology platforms and applicable regulatory considerations as they relate to immunogenicity and pharmacokinetic bioanalytical assessments.

Preliminary dielectric measurement and analysis protocol for determining the melting temperature and binding energy of short sequences of DNA in solution.

Measurement of the real dielectric constant of bulk buffer solutions containing short sequences of DNA as a function of temperature through the DNA melting or denaturiztion transition can be used to determine melting temperatures, T(m), and to estimate the binding energy of the complimentary strands. We describe a preliminary dielectric measurement and analysis protocol to determine these parameters and its application to two known short sequences. The relative real dielectric constant for the bulk solutions was determined over the frequency range of 50 Hz-20 kHz and temperature range of <40-65 degrees C. The measurements were performed on dilute solutions and utilized low electric field strengths. Based on fits to the data by modified sigmoid functions, the melting temperatures, width of transition, and binding energy for the two sequences in solution were estimated. It was observed that the order of the transition appeared to be second order. The results were then compared against predictions of a number of models from the literature that provide theoretical estimates for the melting temperatures of known short sequences of DNA.

Canine cancer screening via ultraviolet absorbance and fluorescence spectroscopy of serum proteins.

A cost-effective optical cancer screening and monitoring technique was demonstrated in a pilot study of canine serum samples and was patented for commercialization. Compared to conventional blood chemistry analysis methods, more accurate estimations of the concentrations of albumin, globulins, and hemoglobin in serum were obtained by fitting the near UV absorbance and photoluminescence spectra of diluted serum as a linear combination of component reference spectra. Tracking these serum proteins over the course of treatment helped to monitor patient immune response to carcinoma and therapy. For cancer screening, 70% of dogs with clinical presentation of cancer displayed suppressed serum hemoglobin levels (below 20 mg/dL) in combination with atypical serum protein compositions, that is, albumin levels outside of a safe range (from 4 to 8 g/dL) and globulin levels above or below a more normal range (from 1.7 to 3.7 g/dL). Of the dogs that met these criteria, only 20% were given a false positive label by this cancer screening test.

Thermal cycling and the optical and electrical characterization of self-assembled multilayer nile blue A-gold thin films.

Some laser applications produce high power densities that can be dangerous to equipment and operators. We have fabricated thin-film coatings by using molecular electrostatic self-assembly to create a spectrally selective absorbing coating that is able to withstand thermal fluctuations from -20 degrees C to 120 degrees C. We made the thin-film coatings by alternating deposition of an organic dye and gold colloidal nanoparticles onto glass substrates. Nile Blue A perchlorate, with a maximum absorbance slightly above 632 nm, was chosen as the organic dye. Strong coupling between the dye molecules and the gold nanoparticles provides a redshift that increases as the film's thickness is increased. The incorporation of the gold colloidal nanoparticles also decreases the resistivity of the film. The resistivity of the film was measured with a four-point probe and found to be approximately 10 omega/cm for the two samples measured. Atomic-force microscopy was used to show that film thickness increased 2.4 nm per bilayer. The optical properties of the film were measured at the end of every 5 thermal cycles from -20 degrees C to 120 degrees C, and negligible degradation was observed after 30 cycles.