Linker and Conjugation Site Synergy in Antibody-Drug Conjugates: Impacts on Biological Activity.

Antibody-drug conjugates (ADCs) produced using general conjugation methods yield heterogeneous products containing mixtures of species with different numbers of payloads per antibody (drug-antibody ratios) conjugated at multiple sites. This heterogeneity affects the stability, efficacy, and safety of ADCs. Thus, various site-specific conjugation methods have been developed to achieve homogeneity in ADCs. It was reported that linker structures and conjugation sites generally affected the characteristics of site-specific ADCs such as stability, efficacy, and safety. However, the combined effects of conjugation sites and linker structures on the physicochemical and biological characteristics of site-specific ADCs have remained unclear. In this study, we generated 30 homogeneous site-specific ADCs with a combination of six conjugation sites and five linker structures using THIOMAB technology and evaluated the characteristics of these homogeneous ADCs. We found that both conjugation sites and linker structures affected characteristics unique to ADCs (linker stability as well as target-dependent and target-independent cytotoxicity) in site-specific ADCs. Especially, conjugation to the constant regions of the light chain and the presence of polyethylene glycol structures in the linker are important for those ADC-specific characteristics. Interestingly, we also found that the effects of linker structures on the target-independent cytotoxicity of homogeneous ADCs at certain conjugation sites differed from those seen in conventional heterogeneous ADCs. Our results suggest that optimizing linker structures based on the conjugation site may be necessary for site-specific ADCs.

Strategic design of GalNAc-helical peptide ligands for efficient liver targeting.

There is a growing need for liver-selective drug delivery systems (DDS) in the treatment and diagnosis of liver diseases. The asialoglycoprotein receptor, a trimeric protein specifically expressed in the liver, is a key target for DDS. We hypothesized that peptides with reduced main-chain flexibility and strategically positioned N-acetylgalactosamine (GalNAc) moieties could enhance liver selectivity and uptake efficiency. The helical peptides designed in this study demonstrated superior uptake efficiency and liver selectivity compared with the conventional triantennary GalNAc DDS. These peptides also showed potential in protein delivery. Furthermore, we explored their application in lysosome-targeting chimeras (LYTACs), gaining valuable insights into the requirements for effective LYTAC functionality. This study not only highlights the potential of helical peptides as liver-selective DDS ligands, but also opens avenues for their use in various therapeutic and diagnostic applications, making significant strides in the targeted treatment of liver diseases.

Quality and Safety Considerations for Therapeutic Products Based on Extracellular Vesicles.

Extracellular vesicles (EVs) serve as an intrinsic system for delivering functional molecules within our body, playing significant roles in diverse physiological phenomena and diseases. Both native and engineered EVs are currently the subject of extensive research as promising therapeutics and drug delivery systems, primarily due to their remarkable attributes, such as targeting capabilities, biocompatibility, and low immunogenicity and mutagenicity. Nevertheless, their clinical application is still a long way off owing to multiple limitations. In this context, the Science Board of the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan has conducted a comprehensive assessment to identify the current issues related to the quality and safety of EV-based therapeutic products. Furthermore, we have presented several examples of the state-of-the-art methodologies employed in EV manufacturing, along with guidelines for critical processes, such as production, purification, characterization, quality evaluation and control, safety assessment, and clinical development and evaluation of EV-based therapeutics. These endeavors aim to facilitate the clinical application of EVs and pave the way for their transformative impact in healthcare.

2023 White Paper on Recent Issues in Bioanalysis: Deuterated Drugs; LNP; Tumor/FFPE Biopsy; Targeted Proteomics; Small Molecule Covalent Inhibitors; Chiral Bioanalysis; Remote Regulatory Assessments; Sample Reconciliation/Chain of Custody (PART 1A - Recommendations on Mass Spectrometry, Chromatography, Sample Preparation Latest Developments, Challenges, and Solutions and BMV/Regulated Bioanalysis PART 1B - Regulatory Agencies' Inputs on Regulated Bioanalysis/BMV, Biomarkers/IVD/CDx/BAV, Immunogenicity, Gene & Cell Therapy and Vaccine).

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

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

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

Relationship Between the Lipidome Profile and Disease Activity in Patients with Rheumatoid Arthritis.

Lipid mediators have been suggested to play important roles in the pathogenesis of rheumatoid arthritis (RA). Lipidomics has recently allowed for the comprehensive analysis of lipids and has revealed the potential of lipids as biomarkers for the early diagnosis of RA and prediction of therapeutic responses. However, the relationship between disease activity and the lipid profile in RA remains unclear. In the present study, we performed a plasma lipidomic analysis of 278 patients with RA during treatment and examined relationships with disease activity using the Disease Activity Score in 28 joints (DAS28)-erythrocyte sedimentation rate (ESR). In all patients, five lipids positively correlated and seven lipids negatively correlated with DAS28-ESR. Stearic acid [FA(18:0)] (r = -0.45) and palmitic acid [FA(16:0)] (r = -0.38) showed strong negative correlations. After adjustments for age, body mass index (BMI), and medications, stearic acid, palmitic acid, bilirubin, and lysophosphatidylcholines negatively correlated with disease activity. Stearic acid inhibited osteoclast differentiation from peripheral blood monocytes in in vitro experiments, suggesting its contribution to RA disease activity by affecting bone metabolism. These results indicate that the lipid profile correlates with the disease activity of RA and also that some lipids may be involved in the pathogenesis of RA.

Intracellular polyamine depletion induces N-linked galactosylation of the monoclonal antibody produced by CHO DP-12 cells.

The heterogeneity of the N-linked glycan profile of therapeutic monoclonal antibodies (mAbs) derived from animal cells affects therapeutic efficacy and, therefore, needs to be appropriately controlled during the manufacturing process. In this study, we examined the effects of polyamines on the N-linked glycan profiles of mAbs produced by CHO DP-12 cells. Normal cell growth of CHO DP-12 cells and their growth arrest by α-difluoromethylornithine (DFMO), an inhibitor of the polyamine biosynthetic pathway, was observed when 0.5% fetal bovine serum was added to serum-free medium, despite the presence of cadaverine and aminopropylcadaverine, instead of putrescine and spermidine in cells. Polyamine depletion by DFMO increased IgG galactosylation, accompanied by ß1,4-galactosyl transferase 1 (B4GAT1) mRNA elevation. Additionally, IgG production in polyamine-depleted cells was reduced by 30% compared to that in control cells. Therefore, we examined whether polyamine depletion induces an ER stress response. The results indicated increased expression levels of chaperones for glycoprotein folding in polyamine-depleted cells, suggesting that polyamine depletion causes ER stress related to glycoprotein folding. The effect of tunicamycin, an ER stress inducer that inhibits N-glycosylation, on the expression of B4GALT1 mRNA was examined. Tunicamycin treatment increased B4GALT1 mRNA expression. These results suggest that ER stress caused by polyamine depletion induces B4GALT1 mRNA expression, resulting in increased IgG galactosylation in CHO cells. Thus, introducing polyamines, particularly SPD, to serum-free CHO culture medium for CHO cells may contribute to consistent manufacturing and quality control of antibody production.

Specific peptide conjugation to a therapeutic antibody leads to enhanced therapeutic potency and thermal stability by reduced Fc dynamics.

Antibody-drug conjugates are powerful tools for combatting a wide array of cancers. Drug conjugation to a therapeutic antibody often alters molecular characteristics, such as hydrophobicity and effector function, resulting in quality deterioration. To develop a drug conjugation methodology that maintains the molecular characteristics of the antibody, we engineered a specific peptide for conjugation to the Fc region. We used trastuzumab and the chelator (DOTA) as model antibody and payload, respectively. Interestingly, peptide/DOTA-conjugated trastuzumab exhibited enhanced antibody-dependent cellular cytotoxicity (ADCC) and increased thermal stability. Detailed structural and thermodynamic analysis clarified that the conjugated peptide blocks the Fc dynamics like a "wedge." We revealed that (1) decreased molecular entropy results in enhanced ADCC, and (2) blockade of Fc denaturation results in increased thermal stability. Thus, we believe that our methodology is superior not only for drug conjugation but also as for reinforcing therapeutic antibodies to enhance ADCC and thermal stability.

2022 White Paper on Recent Issues in Bioanalysis: FDA Draft Guidance on Immunogenicity Information in Prescription Drug Labeling, LNP & Viral Vectors Therapeutics/Vaccines Immunogenicity, Prolongation Effect, ADA Affinity, Risk-based Approaches, NGS, qPCR, ddPCR Assays (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Immunogenicity & Risk Assessment of Biotherapeutics and Novel Modalities; NAb Assays Integrated Approach).

The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th 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 ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 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 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1 (Mass Spectrometry and ICH M10) and Part 2 (LBA, Biomarkers/CDx and Cytometry) are published in volume 15 of Bioanalysis, issues 16 and 15 (2023), respectively.

2022 White Paper on Recent Issues in Bioanalysis: ICH M10 BMV Guideline & Global Harmonization; Hybrid Assays; Oligonucleotides & ADC; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A - Recommendations on Mass Spectrometry, Chromatography and Sample Preparation, Novel Technologies, Novel Modalities, and Novel Challenges, ICH M10 BMV Guideline & Global Harmonization Part 1B - Regulatory Agencies' Inputs on Regulated Bioanalysis/BMV, Biomarkers/CDx/BAV, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th 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 the ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 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 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Mass Spectrometry and ICH M10. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (LBA, Biomarkers/CDx and Cytometry) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 15 and 14 (2023), respectively.

Target-independent Immune-cell Activation by Aggregates of T Cell-redirecting Bispecific Antibodies.

T cell-redirecting bispecific antibodies (bsAbs) have been under development as a new class of biotherapeutics for cancer immunotherapy. T cell-redirecting bsAbs simultaneously bind tumor-associated antigens on tumor cells and CD3 on T cells, resulting in T cell-mediated cytotoxicity against tumor cells. In this study, we prepared a tandem scFv-typed bsAb targeting HER2 and CD3 (HER2-CD3), and evaluated the impact of aggregation of HER2-CD3 on the in vitro immunotoxicity. A cell-based assay using CD3-expressing reporter cells revealed that the aggregates of HER2-CD3 directly activated CD3-expressing immune cells in the absence of target antigen (HER2)-expressing cells. Comparison of the aggregates generated under various stress conditions indicated the possibility that insoluble protein particles, which were detected by qLD analysis and contained non-denatured functional domains, contributed to the activation of CD3-expressing immune cells. In addition, HER2-CD3 aggregates stimulated hPBMCs and strongly induced the secretion of inflammatory cytokines and chemokines. The cytokine/chemokine-release profiles suggested that the aggregates could induce inflammatory responses not only by CD3-mediated T cell activation but also by other immune cell activations. These results indicated the potential risk of aggregation of T cell-redirecting bsAbs, which could induce unwanted immune cell activation and inflammation and thereby immune-mediated adverse reactions.

Characterization of anti-SARS-CoV-2 monoclonal antibodies focusing on antigen binding, neutralization, and FcγR activation via formation of immune complex.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Antibodies induced by SARS-CoV-2 infection or vaccination play pivotal roles in the body's defense against the virus; many monoclonal antibodies (mAbs) against SARS-CoV-2 have been cloned, and some neutralizing mAbs have been used as therapeutic drugs. In this study, we prepared an antibody panel consisting of 31 clones of anti-SARS-CoV-2 mAbs and analyzed and compared their biological activities. The mAbs used in this study were classified into different binding classes based on their binding epitopes and showed binding to the SARS-CoV-2 spike protein in different binding kinetics. A multiplex assay using the spike proteins of Alpha, Beta, Gamma, Delta, and Omicron variants clearly showed the different effects of variant mutations on the binding and neutralization activities of different binding classes of mAbs. In addition, we evaluated Fcγ receptor (FcγR) activation by immune complexes consisting of anti-SARS-CoV-2 mAb and SARS-CoV-2 pseudo-typed virus, and revealed differences in the FcγR activation properties among the binding classes of anti-SARS-CoV-2 mAbs. It has been reported that FcγR-mediated immune-cell activation by immune complexes is involved in the promotion of immunopathology of COVID-19; therefore, differences in the FcγR-activation properties of anti-SARS-CoV-2 mAbs are among the most important characteristics when considering the clinical impacts of anti-SARS-CoV-2 mAbs.

Pharmacokinetics of Biopharmaceuticals: Their Critical Role in Molecular Design.

Biopharmaceuticals have developed rapidly in recent years due to the remarkable progress in gene recombination and cell culture technologies. Since the basic structure of biopharmaceuticals can be designed and modified, it is possible to control the duration of action and target specific tissues and cells by kinetic modification. Amino acid sequence modifications, albumin fusion proteins, polyethylene glycol (PEG) modifications, and fatty acid modifications have been utilized to modify the duration of action control and targeting. This review first describes the position of biopharmaceuticals, and then the kinetics (absorption, distribution, metabolism, elimination, and pharmacokinetics) of classical biopharmaceuticals and methods of drug quantification. The kinetic innovations of biopharmaceuticals are outlined, including insulin analog, antibody-related drugs (monoclonal antibodies, Fab analogs, Fc analogs, Fab-PEG conjugated proteins, antibody-drug conjugates, etc.), blood coagulation factors, interferons, and other related drugs. We hope that this review will be of use to many researchers interested in pharmaceuticals derived from biological components, and that it aids in their knowledge of the latest developments in this field.

Comparison of Immunochemical Reactions of Infliximab Innovator and Biosimilars on an Infliximab Detection Kit Used for Therapeutic Drug Monitoring.

Monitoring serum infliximab (INF) concentrations is crucial for designing appropriate doses for patients with rheumatoid arthritis. It is recommended to maintain the serum trough INF level at least 1.0 µg/mL. In Japan, an in vitro diagnostic kit using immunochromatography has been approved to determine whether the serum INF concentration is over 1.0 µg/mL or not, and to support the determination of the necessity of increasing the dose or switching to another drug. Biosimilars (BS) of INF may have immunochemical properties different from those of its innovator product, which may show different reactivities on the diagnostic kit. In this study, the responses of the innovator and five BS products on the kit were compared. Based on visually comparing the intensity of color development between the test and control samples, differences were found in the judgment results depending on the analyst. In particular, 1.0 µg/mL was not determined as positive in some cases, whereas 2.0 µg/mL was reliably determined as positive. Overall, no significant difference in reactivity was found between the innovator and five BS products. To further compare the differences in immunochemical properties, the reactivity of these products with three enzyme-linked immunosorbent assay (ELISA) kits was compared. The results confirmed that there were no significant differences among the innovator and BS products in reactivity with the examined kits. When using that diagnostic kit, the users need to be aware that the judgement around 1.0 µg/mL INF may differ depending on the test conditions, including the analyst.

Antibody feedback contributes to facilitating the development of Omicron-reactive memory B cells in SARS-CoV-2 mRNA vaccinees.

In contrast to a second dose of the SARS-CoV-2 mRNA vaccine, a third dose elicits potent neutralizing activity against the Omicron variant. To address the underlying mechanism for this differential antibody response, we examined spike receptor-binding domain (RBD)-specific memory B cells in vaccinated individuals. Frequency of Omicron-reactive memory B cells increased ∼9 mo after the second vaccine dose. These memory B cells show an altered distribution of epitopes from pre-second memory B cells, presumably due to an antibody feedback mechanism. This hypothesis was tested using mouse models, showing that an addition or a depletion of RBD-induced serum antibodies results in a concomitant increase or decrease, respectively, of Omicron-reactive germinal center (GC) and memory B cells. Our data suggest that pre-generated antibodies modulate the selection of GC and subsequent memory B cells after the second vaccine dose, accumulating more Omicron-reactive memory B cells over time, which contributes to the generation of Omicron-neutralizing antibodies elicited by the third vaccine dose.

Biophysical Characterization of the Contribution of the Fab Region to the IgG-FcγRIIIa Interaction.

The cell-surface receptor FcγRIIIa is crucial to the efficacy of therapeutic antibodies as well as the immune response. The interaction of the Fc region of IgG molecules with FcγRIIIa has been characterized, but until recently, it was thought that the Fab regions were not involved in the interaction. To evaluate the influence of the Fab regions in a biophysical context, we carried out surface plasmon resonance analyses using recombinant FcγRIIIa ligands. A van't Hoff analysis revealed that compared to the interaction of the papain-digested Fc fragment with FcγRIIIa, the interaction of commercially available, full-length rituximab with FcγRIIIa had a more favorable binding enthalpy, a less favorable binding entropy, and a slower off rate. Similar results were obtained from analyses of IgG1 molecules and an IgG1-Fc fragment produced by Expi293 cells. For further validation, we also prepared a maltose-binding protein-linked IgG1-Fc fragment (MBP-Fc). The binding enthalpy of MBP-Fc was nearly equal to that of the IgG1-Fc fragment for the interaction with FcγRIIIa, indicating that such alternatives to the Fab domains as MBP do not positively contribute to the IgG-FcγRIIIa interactions. Our investigation strongly suggests that the Fab region directly interacts with FcγRIIIa, resulting in an increase in the binding enthalpy and a decrease in the dissociation rate, at the expense of favorable binding entropy.

Development of a penetratin-conjugated stapled peptide that inhibits Wnt/ß-catenin signaling.

Wnt/ß-catenin pathway triggers the formation of a complex between ß-catenin and T cell-specific transcription factor (TCF), which induces transcriptional activation. Excessive transcriptional activation of this pathway is associated with the development, cause, and deterioration of various cancers. Therefore, the Wnt/ß-catenin pathway is an attractive drug target for cancer therapeutics and small molecule- and peptide-based protein-protein interaction (PPI) inhibitors have been developed. However, peptide-based PPI inhibitors generally have low cell-membrane permeability because of their large molecular size. To improve cell-membrane permeability, conjugating cell-penetrating peptides (CPPs) to PPI-inhibiting peptides is a useful method for developing intracellularly targeted PPI inhibitors. In this study, we focused on the interaction between ß-catenin and liver receptor homologue-1 (LRH-1) and designed and synthesized a series of LRH-1-derived peptides to develop inhibitors against Wnt/ß-catenin signaling. The results showed that a penetratin-conjugated LRH-1-derived peptide (Penetratin-st7) predominantly inhibited DLD-1 cell growth at 20 µM treatment via inhibition of the Wnt signaling pathway. This result suggests that Penetratin-st7 is one of promising PPI inhibitors between TCF and ß-catenin.

[Quality Evaluation of Therapeutic Antibodies by Multi-attribute Method].

In the development of therapeutic monoclonal antibodies (mAbs), it is essential to characterize the modifications causing structural heterogeneity because certain modifications are associated with safety and efficacy. However, the rapid structural analysis of mAbs remains challenging due to their structural complexity. The multi-attribute method (MAM) is a structural analytical method based on peptide mapping using LC/MS, and has drawn attention as a new quality control method for therapeutic mAbs instead of conventional structural heterogeneity analyses using several chromatographic techniques. Peptide mapping, which is regarded as an identification test method, is used to confirm that the amino acid sequence corresponds to that deduced from the gene sequence for the desired product. In contrast, MAM is used for simultaneously monitoring the modification rates of individual amino acid residues of therapeutic mAbs, indicating that MAM is used as quantitative test rather than identification test. In this review, we summarized the typical structural heterogeneities of mAbs and the general scheme of MAM. We also introduced our optimized sample preparation method for MAM, and examples of simultaneous monitoring of several modifications including deamidation, oxidation, N-terminal pyroglutamination, C-terminal clipping and glycosylation by our MAM system.

[Investigation of Foreign Particles in Moderna COVID-19 Vaccine].

Particular batches of Moderna mRNA Coronavirus Disease 2019 (COVID-19) vaccine were recalled after foreign particles were found in some vaccine vials at the vaccination site in Japan in August 2021. We investigated the foreign particles at the request of the Ministry of Health, Labour and Welfare. Energy dispersive X-ray spectroscopy analysis suggested that the foreign particles found in the vials recalled from the vaccination sites were from stainless steel SUS 316L, which was in line with the findings of the root cause investigation by the manufacturer. The sizes of the observed particles ranged from <50 µm to 548 µm in the major axis. Similar foreign particles were also detected in 2 of the 5 vaccine vials of the same lot stored by the manufacturer, indicating that the foreign particles have already been administered to some people via vaccine. Observation of the vials of the same lot by digital microscope found smaller particles those were not detected by visual inspection, suggesting that more vials were affected. Contrarily, visual inspection and subvisible particulate matter test indicated no foreign particles in the vials of normal lots. Possible root cause and strategies to prevent such a deviation were discussed from technical and regulatory aspects.