Human IgG Research Articles

Evaluation of a Novel MET-Targeting Camelid-Derived Antibody in Head and Neck Cancer.

In head and neck squamous cell carcinoma (HNSCC), the mesenchymal epithelial transition (MET) receptor drives cancer growth, proliferation, and metastasis. MET is known to be overexpressed in HNSCC and, therefore, is an appealing therapeutic target. In this study, we evaluated MET expression in patients with HNSCC and investigated the potential imaging application of a novel MET-binding single-domain camelid antibody using positron emission tomography/computed tomography (PET/CT) in a preclinical MET-expressing HNSCC model. Multiplex immunostaining for MET protein performed on a tissue microarray from 203 patients with HNSCC found 86% of patients to have MET expression, with 14% having high expression and 53% having low MET expression. Using The Cancer Genome Atlas (TCGA) database, high MET RNA expression was associated with worse progression-free survival and overall survival in patients with HPV-negative HSNCC. Utilizing flow cytometry and immunofluorescence, our novel camelid antibody fused to a human IgG Fc chain (1E7-Fc) showed high binding affinity and specificity to high MET-expressing Detroit 562 cells but not to low MET-expressing HNSCC cells. The efficacy and biodistribution of [89Zr]Zr-1E7-Fc as a PET imaging agent was then investigated in a MET-expressing head and neck xenograft model. [89Zr]Zr-1E7-Fc rapidly localized and showed high tumor uptake in Detroit 562 xenografts (8.4% ID/g at 72 h postinjection), with rapid clearance from the circulatory system (2.7 tumor-to-blood radioactivity ratio at 72 h postinjection). Our preclinical data suggest that the camelid antibody 1E7-Fc could be a potential theranostic agent for HNSCC. Further investigations are warranted to confirm these findings in patients and to evaluate 1E7-Fc as an imaging agent and platform to deliver radionuclide or drug therapy to MET-driven cancers.

Interrogating post-transplant donor HLA-specific antibody characteristics and effector functions using clinical bead assays

Single antigen bead (SAB) assays are the most common and sensitive method used to detect and monitor post-transplant donor specific HLA antibodies (DSA). However, a direct comparison across traditional and modified SAB assays to improve routine DSA monitoring using pre-treated IgG sera to eliminate interference has not been performed. We performed a technical comparison of 251 post-transplant DSA from n = 91 serum samples tested neat (pre-treated, undiluted), at a single 1:16 dilution, in the C1q bead assay, and for IgG subclasses (IgG1, IgG2, IgG3, IgG4) with IgG-enriched sera. We found that DSAs that are detectable by 1:16 dilution and/or C1q are associated with higher IgG MFI values and results could be predicted by testing neat sera. DSA detected at 1:16 dilution correlated with >7000 IgG MFI in neat sera and identified DSA that exceeded the SAB linear range for semiquantitative measurements. C1q positive DSA correlated with >15,000 IgG MFI in neat sera. C1q binding correlated most strongly with total IgG MFI (Spearman r = 0.82, p = 0.002) and not specific subclasses, demonstrating that DSA C1q binding capacity in this cohort is driven by HLA-specific IgG concentration. Evaluation of engineered pan-HLA class I-specific human IgG1 and IgG2 subclass monoclonal antibodies by SAB C1q and C3d assays revealed that IgG2 antibodies can bind complement at higher concentrations. The strengths and limitations of modified SAB assays must be considered to optimize efficient testing and accurate clinical interpretation.

Preclinical development of SGN-CD47M: Protease-activated antibody technology enables selective tumor targeting of the innate immune checkpoint receptor CD47.

CD47 is a cell surface glycoprotein that is expressed on normal human tissues and has a key role as a marker of self. Tumor cells have coopted CD47 overexpression to evade immune surveillance and thus blockade of CD47 is a highly active area of clinical exploration in oncology. However, clinical development of CD47-targeted agents has been complicated by its robust expression in normal tissues and the toxicities that arise from blocking this inhibitory signal. Further, pro-phagocytic signals are not uniformly expressed in tumors and antibody blockade alone is often not sufficient to drive antitumor activity. The inclusion of an IgG1 antibody backbone into therapeutic design has been shown to serve as an additional pro-phagocytic signal but also exacerbates toxicities in normal tissues. Therefore, a need persists for more selective therapeutic modalities targeting CD47. To address these challenges, we developed SGN-CD47M, a humanized anti-CD47 IgG1 monoclonal antibody linked to novel masking peptides through linkers designed to be cleaved by active proteases enriched in the tumor microenvironment. Masking technology has the potential to increase the amount of drug that reaches the tumor microenvironment, while concomitantly reducing systemic toxicities. We demonstrate that SGN-CD47M is well tolerated in cynomolgus monkeys and displays a 20-fold improvement in tolerability to hematologic toxicities when compared to the unmasked antibody. SGN-CD47M also displays preferential activation in the tumor microenvironment that leads to robust single-agent antitumor activity. For these reasons, SGN-CD47M may have enhanced antitumor activity and improved tolerability relative to existing therapies that target the CD47-SIRPα interaction.

Vascular endothelial growth factor A inhibition remodels the transcriptional signature of lipid metabolism in psoriasis non‐lesional skin in 12 h ex vivo culture

AbstractBackgroundVascular endothelial growth factor A (VEGF‐A)‐mediated angiogenesis is involved in the pathogenesis of psoriasis. VEGF‐A inhibitors are widely used to treat oncological and ophthalmological diseases but have not been used in psoriasis management. The molecular mechanisms underlying the effects of VEGF‐A inhibition in psoriatic skin remain unknown.ObjectivesTo identify the genes and canonical pathways affected by VEGF‐A inhibition in non‐lesional and plaque skin ex vivo.MethodsTotal RNA sequencing was performed on skin biopsies from patients with psoriasis (n = 6; plaque and non‐lesional skin) and healthy controls (n = 6) incubated with anti‐VEGF‐A monoclonal antibody (bevacizumab, Avastin®) or human IgG1 isotype control for 12 h in serum‐free organ culture. Differentially expressed genes between paired control and treated samples with adjusted p‐values <0.1 were considered significant. Gene ontology and ingenuity pathway analysis was used to identify enriched biological processes, canonical pathways and upstream regulators.ResultsVEGF‐A inhibition upregulated the expression of genes involved in lipid metabolism. Pathway enrichment analysis identified the activation of pathways involved in fatty acids and lipid biosynthesis and degradation in non‐lesional skin and ferroptosis in plaque skin. VEGF‐A inhibition downregulated endothelial cell apoptosis in non‐lesional psoriasis skin and members of the interferon family were identified as potential regulators of the effects of VEGF‐A inhibition in non‐lesional skin.ConclusionEarly response to VEGF‐A inhibition is associated with changes in lipid metabolism in non‐lesional psoriasis skin and cellular stress in psoriasis plaque. More investigation is needed to validate these findings.

Preparation and Construction of Chimeric Humanized Broadly Reactive Antibody 10H10 to Protein E of Tick-Borne Encephalitis Virus.

A full-length humanized chimeric antibody 10H10ch that specifically interacts with the surface glycoprotein E of flaviviruses was obtained. To construct it, we used variable fragments of the heavy and light chains of the monoclonal antibody 10H10 that form the active center of the antibody and a fragment of the constant part of the heavy chain of the human IgG1 antibody. The resulting full-length chimeric humanized antibody 10H10ch specifically interacted with the E protein of flaviviruses pathogenic to humans, such as tick-borne encephalitis, Zika, West Nile, and dengue viruses. An immunochemical assessment of the interaction constants of the 10H10ch antibody with a panel of native and recombinant flavivirus antigens by ELISA and biolayer interferometry showed that the dissociation constant (Kd) of the chimeric antibody is in the nanomolar region and is comparable to that of the high-affinity mouse monoclonal antibody 10H10. The possibility of using the resulting chimeric humanized antibody 10H10ch for the diagnosis, prevention, and treatment of various flavivirus infections is discussed.

Evaluating efficacy and safety of ocrelizumab biosimilar (Xacrel) compared to the originator (Ocrevus) in relapsing multiple sclerosis: a phase III, randomized, equivalency, clinical trial

Multiple sclerosis is an inflammatory demyelinating disease and represents a global health concern. Ocrelizumab, a humanized IgG monoclonal antibody, selectively targets CD20 on B cells and CD20-expressing T cells. This study aimed to compare the efficacy and safety of the biosimilar ocrelizumab candidate (Xacrel) to the originator product (Ocrevus) in Relapsing Multiple Sclerosis (RMS) patients. In this randomized trial, patients received either Xacrel or Ocrevus for 96 weeks. The primary endpoint was the equivalency of the medications in reducing the annualized relapse rate (ARR) at week 48. The secondary endpoints included time to the onset of disability progression confirmed at 12 and 24 weeks, the proportion of relapse-free patients, magnetic resonance imaging (MRI) evaluations, safety assessments, and immunogenicity over 96 weeks. A total of 170 patients were randomized (1:1 ratio). In the per protocol analysis, the upper and lower limits of 95% two-sided confidence intervals of difference between treatments in the 48-week ARR rate were in the predefined margin of − 0.2 to 0.2 (− 0.002; 95% CI − 0.080 to 0.075). The two products were also comparable in terms of other efficacy parameters, safety, and immunogenicity. The results confirmed that Xacrel is equivalent to Ocrevus in terms of 48-week ARR in RMS patients, with no considerable difference in other efficacy parameters and the safety profile during the 96 weeks. The trial was registered in Iranian registry of clinical trials (IRCT) on 10/06/2019 with the registration number of IRCT20150303021315N13 and in Clinicaltrials.gov on 19/07/2021 with the registration code of NCT04966338.

The mouse monoclonal antibody 4E3 is specific for the G1m17 allotype of human IgG1

Allotype is an amino acid variation within the immunoglobulin isotypes. Four allotypes have been described for human IgG1 and two of them (G1m3 and G1m17) are located at position 214 in the CH1 region of the gamma chain. Various diseases have been associated with allotype expression, making the allotype research an interesting field in immunology. However, allotype-specific reagents are rare. In the present study the specificity of a widely used and commercially available IgG1-specific monoclonal antibody named 4E3, described as binding to the hinge region of IgG1, was evaluated. Using the ImmunoCAP™ assay technology, surprisingly no IgG1 was measured in 13 of 23 human serum and plasma samples when 4E3 was used in an antibody-enzyme conjugate as detection reagent. Further evaluation of 4E3 using eight IgG1 myeloma paraproteins revealed that 4E3 did not bind to three of them. No association was seen between the binding pattern and myeloma light chain glycosylation or the kappa or light chain use. By comparing the myeloma paraprotein binding patterns of 4E3 and TM14 (a monoclonal antibody with known G1m3 specificity), it was indicated that 4E3 only bound to myeloma paraproteins expressing the G1m17 allotype. This was confirmed using recombinant human antibodies expressing either the G1m3 or G1m17 allotype. The G1m17 bias of 4E3 seen using ImmunoCAP was also observed in microtiter plate-based enzyme-linked immunosorbent assays. The antibody 4E3 has a G1m17 bias limiting its use in assays to measure IgG1 antibodies. However, it may be a valuable allotype-specific reagent.

A single-dose circular RNA vaccine prevents Zika virus infection without enhancing dengue severity in mice

Antibody-dependent enhancement (ADE) is a potential concern for the development of Zika virus (ZIKV) vaccines. Cross-reactive but poorly neutralizing antibodies, usually targeting viral pre-membrane or envelope (E) proteins, can potentially enhance dengue virus (DENV) infection. Although E domain III (EDIII) contains ZIKV-specific epitopes, its immunogenicity is poor. Here, we show that dimeric EDIII, fused to human IgG1 Fc fragment (EDIII-Fc) and encoded by circular RNA (circRNA), induces better germinal center reactions and higher neutralizing antibodies compared to circRNAs encoding monomeric or trimeric EDIII. Two doses of circRNAs encoding EDIII-Fc and ZIKV nonstructural protein NS1, another protective antigen, prevent lethal ZIKV infection in neonates born to immunized C57BL/6 mice and in interferon-α/β receptor knockout adult C57BL/6 mice. Importantly, a single-dose optimized circRNA vaccine with improved antigen expression confers potent and durable protection without inducing obvious DENV ADE in mice, laying the groundwork for developing flavivirus vaccines based on circRNAs encoding EDIII-Fc and NS1.

Preclinical evaluation of a fully human, quadrivalent-hantavirus polyclonal antibody derived from a non-human source.

This candidate polyclonal human IgG product was produced using synthetic gene-based vaccines and transgenic cows. Having now gone through cGMP production, GLP safety testing, and efficacy testing in animals, SAB-163 is the world's most advanced anti-hantavirus antibody-based medical countermeasure, aside from convalescent human plasma. Importantly, SAB-163 targets the most prevalent hantaviruses on four continents.

A novel non-catalytic plasmonic immunoabsorbant assay amplification mechanism

The amplification of signals is dependent upon enzyme/ catalyst. Enzyme activity, specificity, pH condition, enzyme linkers, stability, storage and efficiency are some very important factors that can eventually affect the detection results. We aimed to amplify signals without the assistance of enzyme/ catalyst so that the strategy can be used in the wide range of signal detection including immune-assay. Silver nanocrystals (Ag NCs) grown from self-nucleation are used as chromogens due to their strong plasmon band at ∼ 400 nm. During 140 s of reaction time in presence of 6.10 mM CTAB and 0.61 mM of AgNO3, the 0.57 mM of Ascorbic acid allows Ag over growth on antibody-tagged gold nanospheres (10±0.5 nm) that competes silver atoms from the self-nucleation growth and thus affects the growth kinetics of the latter. By utilizing the opened kinetic window, sensitive detection has been achieved from 6.67 × 10–14 M (1 pg) to 3.33 × 10–9 M (50 ng) using human IgG as a model protein. The non-catalytic plasmonic immunoabsorbant assay we developed herein adds a new possibility of plasmonic NPs for sensitive detection. The detection slope and resolution is higher and better than those via signal amplification through the assistance of enzymes/ catalysts. This work supports the concept that nanoparticles mediated reactions are sensitive enough that they have the potential to go independently without enzymes/ catalyst assistance.

Immuno-PET Imaging of EGFR with 64Cu-NOTA Panitumumab in Subcutaneous and Metastatic Nonsmall Cell Lung Cancer Xenografts.

Objective: About 65-90% of nonsmall cell lung cancer (NSCLC) express the epithelial growth factor receptor (EGFR) as a transmembrane protein that is activated by binding of specific ligands, including epidermal growth factor and transforming growth factor α (TGFα). Identifying EGFR as an oncogene has led to the development of anticancer therapeutics directed against EGFR, including the full-length human IgG2 monoclonal antibody panitumumab. The main goal of the present study was to investigate 64Cu-labeled panitumumab with immuno-PET in subcutaneous and metastatic EGFR-positive NSCLC xenografts. Methods: Bifunctional chelating agent 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclo-nonane-1,4,7-triacetic acid (NOTA-NCS) was attached to panitumumab. The number of chelators per panitumumab was determined using matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy. The incorporation efficiency of 64Cu into NOTA-panitumumab was measured by using radio-TLC. EGFR-expressing epithelial-like H1299-luc+ NSCLC cells were used for in vitro and in vivo experiments. Cell uptake of [64Cu]Cu-NOTA-panitumumab was measured in the presence and absence of panitumumab. Subcutaneous and metastatic H1299-luc tumor models were grown in male NSG mice. The presence of tumors at lung and metastatic sites was analyzed by [18F]FLT PET. Immuno-PET with [64Cu]Cu-NOTA-panitumumab was performed as static PET imaging at 2, 24, and 48 h postinjection in both tumor models. Proof-of-target was confirmed by blocking experiments with panitumumab. Detailed ex vivo biodistribution experiments were performed in both animal tumor models to confirm biodistribution profiles obtained by immuno-PET imaging. Results: MALDI analysis confirmed the attachment of ∼1.5 NOTA per antibody. Radiolabeling efficiency with [64Cu]CuCl2 was 93.8 ± 5.7% and a molar activity of 0.65 MBq/μg. Cellular uptake studies with [64Cu]Cu-NOTA-panitumumab in H1299 cells demonstrated increasing uptake over time, reaching 29.1 ± 2.9% radioactivity(Bq)/mg protein (n = 3) and plateauing at 45 min. Addition of 25 μg of panitumumab reduced radioligand uptake to 1.22 ± 0.06% radioactivity/mg protein (n = 3). PET imaging revealed high uptake of [64Cu]Cu-NOTA-panitumumab in subcutaneous tumors: Standardized uptake values (SUV)mean reached 4.70 ± 0.42 and 5.37 ± 0.40 (n = 5) after 24 and 48 h postinjection, respectively. Administration of 1 mg panitumumab reduced tumor uptake significantly to 1.94 ± 0.22 and 1.66 ± 0.08 (n = 4; p < 0.001). In the metastatic model, the following SUVmean were analyzed from liver and lung lesions: 5.55 ± 0.34 and 6.28 ± 0.46 (both n = 23 lesions from 6 mice) after 24 and 48 h postinjection, which was also significantly reduced to 2.53 ± 0.39 and 2.31 ± 0.15 (both n = 16 lesions from 4 mice; p < 0.001) after injection of 1 mg panitumumab. Detailed ex vivo biodistribution confirmed immuno-PET analysis in both models. Panitumumab reduced radioactivity uptake into subcutaneous tumors from 11.01 ± 0.72 (n = 4) to 3.67 ± 0.33% ID/g (n = 5; p < 0.001), and in metastatic liver lesions from 29.44 ± 8.14 (n = 4) to 8.35 ± 1.30% ID/g (n = 5; p < 0.001), respectively. Conclusions: [64Cu]Cu-NOTA-panitumumab was successfully used for immuno-PET imaging of EGFR-expressing subcutaneous and metastatic NSCLC tumors. This result represents the basis for developing radiotheranostics for targeting EGFR in cancers and for selecting the right patients for the right treatment at the right time.

The impact of tildrakizumab on quality of life in patients suffering from moderate-to-severe psoriasis: a 36-week prospective, monocentric, real-life, observational study.

Psoriasis may significantly impact on patients' health related quality of life (HRQoL). Clinical assessment has been combined with HRQoL scores to evaluate the ways that cutaneous disease is a burden to patients. Tildrakizumab is a humanized IgG1 monoclonal antibody targeting interleukin-23 p19 and is approved for the management of moderate-to-severe plaque psoriasis. The aim of our study was to evaluate the impact of tildrakizumab treatment on psychological field in patients affected by moderate-to-severe plaque psoriasis. a 36-weeks observational study was carried out enrolling psoriasis patients who initiated treatment with tildrakizumab 100 mg. DLQI and Skindex-16 questionnaire were administered at baseline, week 12, week 24 and week 36. PASI, BSA and pruritus (p)-VAS were also assessed at baseline and at each follow-up. A total of 34 patients were enrolled. Baseline PASI and BSA score were 28.4±5.6 and 38.8±21.4, respectively. Similarly, the impact of psoriasis on HRQoL was collected (DLQI: 26.4±3.2, Skindex-16: 68±5.8, p-VAS: 8.2). Clinical improvement was assessed since week 12 (PASI: 12.4±4.2; BSA: 16.5±7.3), continuing to week 24 (PASI: 4.2±2.8; BSA: 6.1±3.1) and 36 (PASI: 3.6±3.2; BSA: 4.2±1.37). Clinical improvement was accompanied by an improvement in quality of life at week 16 (DLQI: 15.5±2.9; Skindex-16: 28.2±4.2; p-VAS: 3.8) , 24 (DLQI: 8.2±1.4, Skindex-16: 16.2; p-VAS: 2.6) and 36 (DLQI: 3.1±2.4; Skindex-16: 9.3±2.8; p-VAS: 2.8). Our study also confirmed the safety of tildrakizumab in real life settings, with no treatment discontinuation for inefficacy or adverse events reported during the study. our results confirm tildrakizumab as an effective option for the improvement of psoriasis patients' HRQoL.

Limited Biomarker Potential for IgG Autoantibodies Reactive to Linear Epitopes in Systemic Lupus Erythematosus or Spondyloarthropathy.

Autoantibodies are commonly used as biomarkers in autoimmune diseases, but there are limitations. For example, autoantibody biomarkers have poor sensitivity or specificity in systemic lupus erythematosus and do not exist in the spondyloarthropathies, impairing diagnosis and treatment. While autoantibodies suitable for strong biomarkers may not exist in these conditions, another possibility is that technology has limited their discovery. The purpose of this study was to use a novel high-density peptide array that enables the evaluation of IgG binding to every possible linear antigen in the entire human peptidome, as well as a novel machine learning approach that incorporates ELISA validation predictability in order to discover autoantibodies that could be developed into sensitive and specific markers of lupus or spondyloarthropathy. We used a peptide array containing the human peptidome, several viral peptidomes, and key post-translational modifications (6 million peptides) to quantify IgG binding in lupus, spondyloarthropathy, rheumatoid arthritis, Sjögren's disease, and control sera. Using ELISA data for 70 peptides, we performed a random forest analysis that evaluated multiple array features to predict which peptides might be good biomarkers, as confirmed by ELISA. We validated the peptide prediction methodology in rheumatoid arthritis and COVID-19, conditions for which the antibody repertoire is well-understood, and then evaluated IgG binding by ELISA to peptides that we predicted would be highly bound specifically in lupus or spondyloarthropathy. Our methodology performed well in validation studies, but peptides predicted to be highly and specifically bound in lupus or spondyloarthropathy could not be confirmed by ELISA. In a comprehensive evaluation of the entire human peptidome, highly sensitive and specific IgG autoantibodies were not identified in lupus or spondyloarthropathy. Thus, the pathogenesis of lupus and spondyloarthropathy may not depend upon unique autoantigens, and other types of molecules should be sought as optimal biomarkers in these conditions.

De novo protein sequencing of antibodies for identification of neutralizing antibodies in human plasma post SARS-CoV-2 vaccination

The antibody response to vaccination and infection is a key component of the immune response to pathogens. Sequencing of peripheral B cells may not represent the complete B cell receptor repertoire. Here we present a method for sequencing human plasma-derived polyclonal IgG using a combination of mass spectrometry and B-cell sequencing. We investigate the IgG response to the Moderna Spikevax COVID-19 vaccine. From the sequencing data of the natural polyclonal response to vaccination, we generate 12 recombinant antibodies. Six derived recombinant antibodies, including four generated with de novo protein sequencing, exhibit similar or higher binding affinities than the original natural polyclonal antibody. Neutralization tests reveal that the six antibodies possess neutralizing capabilities against the target antigen. This research provides insights into sequencing polyclonal IgG antibodies and the potential of our approach in generating recombinant antibodies with robust binding affinity and neutralization capabilities. Directly examining the circulating IgG pool is crucial due to potential misrepresentations by B-cell analysis alone.

Discovery of a common light chain bispecific antibody targeting PD-1 and PD-L1 by Hybridoma-to-phage-to-yeast (H2PtY) platform

Abstract Background Therapeutic antibody drugs targeting the PD-1 pathway are generally characterized by relatively low response rates and susceptibility to drug resistance during clinical application. Therefore, there is an urgent need for alternative therapeutic strategies to increase the immune response rate. Bispecific antibodies co-targeting PD-1 and PD-L1 may have greater potential to improve the efficacy of the immune checkpoint pathway. Method In this study, we developed a potent humanized common light chain IgG shape bispecific antibody, named JMB2005, based on Hybridoma-to-Phage-to-Yeast platform. The platform allowed us to discover common light chain bispecific antibody from traditional mice for any pair of given targets. Results JMB2005 exhibited favorable developability, good manufacturing property, and satisfactory efficacy, which could be given via subcutaneous injection at the concentration of 120 mg/mL. Mechanistically, JMB2005 could bridge tumor cells and T cells with both Fab arms and promote T-cells to function as direct tumor cell killers. It could also promote T cell activation by blocking the binding of PD-L1 to CD80. Furthermore, JMB2005 has exhibited a favorable half-life and has demonstrated promising anti-tumor therapeutic efficacy in vivo. Conclusion Consequently, the present study showed that the novel humanized common light chain bispecific antibody JMB2005 may represent a novel therapeutic agent of great clinical potential.

Carrier-Guided Proteome Analysis in a High Protein Background: An Improved Approach to Host Cell Protein Identification.

Many shotgun proteomics experiments are negatively influenced by highly abundant proteins, such as those measuring residual host cell proteins (HCP) amidst highly abundant recombinant biotherapeutic or plasma proteins amidst albumin and immunoglobulins. While western blotting and ELISAs can reveal the presence of specific low abundance proteins from highly abundant background proteins, mass spectrometry approaches are required to define the low abundance protein composition in these scenarios. The challenge in detecting low abundance proteins in a high protein background by standard shotgun approaches is that spectra are often not triggered on their peptides in data dependent acquisition methods but rather on the highly abundant background peptides. Here, we use tandem mass tags (TMT) to introduce a carrier proteome approach to enhance the detection of proteins, such as from residual host cell proteomes amidst a highly abundant background. Using a mixture of bovine serum albumin (BSA) and E. coli as a mock high background/low abundance target protein formulation, we demonstrate proof-of-principle experiments allowing the improved detection of target proteins amidst a high protein background. While we observed significant coisolation interference, we mitigated it by using a spike-in interference detection TMT channel. Finally, we use the approach to identify 300 residual E. coli proteins from a protein A pulldown of a human IgG antibody, demonstrating that it may be applicable to analysis of HCPs in biotherapeutic protein formulations.

The novel and potent CD40 agonist KHK2840 augments the antitumor efficacy of anti-PD-1 antibody and paclitaxel.

Lack of tumor-reactive cytotoxic T lymphocytes (CTLs)limits the antitumor efficacy of immune checkpoint inhibitors (ICIs). CD40 agonists have been expected to overcome this limitation by generating tumor-reactive CTLs. However, the clinical efficacy of CD40 agonistic antibodies is not as good as in non-clinical studies. The novel human CD40 (hCD40) agonist KHK2840 is a fully human anti-CD40 IgG2 agonistic antibody that is Fc-engineered to minimize complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. Compared to other hCD40 agonists, KHK2840 exhibited the most potent hCD40 agonistic signal in tumor-bearing hCD40 transgenic mice and human peripheral blood B cells. Moreover, KHK2840 enhanced the antitumor efficacy of the antiprogrammed cell death 1 antibody and paclitaxel. Comprehensive immune profiling revealed that the antitumor immune response of the triple combination involved tumor-draining lymph nodes in addition to tumor microenvironments. This suggests that a coordinated antitumor immune response between tumors and lymph nodes may underlie the synergistic antitumor efficacy of the triple combination therapy. Finally, a toxicology study in cynomolgus monkeys demonstrated that KHK2840 activated the CD40 signal with tolerable toxicological properties. These results indicate that KHK2840 is a novel and potent hCD40 agonistic antibody for cancer immunotherapy, which is expected to augment the antitumor efficacy of ICIs and chemotherapy.

Immunogenicity dynamics and covariate effects after satralizumab administration predicted with a hidden Markov model.

Immunogenicity is the propensity of a therapeutic protein to generate an immune response to itself. While reporting of antidrug antibodies (ADAs) is increasing, model-based analysis of such data is seldom performed. Model-based characterization of factors affecting the emergence and dissipation of ADAs may inform drug development and/or improve understanding in clinical practice. This analysis aimed to predict ADA dynamics, including the potential influence of individual covariates, following subcutaneous satralizumab administration. Satralizumab is a humanized IgG2 monoclonal recycling IL-6 receptor antagonist antibody approved for treating neuromyelitis optica spectrum disorder (NMOSD). Longitudinal pharmacokinetic (PK) and ADA data from 154 NMOSD patients in two pivotal Phase 3 studies (NCT02028884, NCT02073279) and PK data from one Phase 1 study (SA-001JP) in 72 healthy volunteers were available for this analysis. An existing population PK model was adapted to derive steady-state concentration without ADA for each patient. A mixed hidden Markov model (mHMM) was developed whereby three different states were identified: one absorbing Markov state for non-ADA developer, and two dynamic and inter-connected Markov states-transient ADA negative and positive. Satralizumab exposure and body mass index impacted transition probabilities and, therefore, the likelihood of developing ADAs. In conclusion, the mHMM model was able to describe the time course of ADA development and identify patterns of ADA development in NMOSD patients following treatment with satralizumab, which may allow for the formulation of strategies to reduce the emergence or limit the impact of ADA in the clinical setting.

Comparison of protein immobilization methods with covalent bonding on paper for paper-based enzyme-linked immunosorbent assay

In this study, two methods were examined to optimize the immobilization of antibodies on paper when conducting a paper-based enzyme-linked immunosorbent assay (P-ELISA). Human IgG, as a test-capture protein, was immobilized on paper via the formation of Schiff bases. Aldehyde groups were introduced onto the surface of the paper via two methods: NaIO4 and 3-aminopropyltriethoxysilane (APTS) with glutaraldehyde (APTS-glutaraldehyde). In the assay, horseradish peroxidase-conjugated anti-human IgG (HRP-anti-IgG) binds to the immobilized human IgG, and the colorimetric reaction of 3,3′,5,5′-tetramethylbenzyzine (TMB) produces a blue color in the presence of H2O2 and HRP-anti-IgG as a model analyte. The immobilization of human IgG, the enzymatic reaction conditions, and the reduction of the chemical bond between the paper surface and immobilized human IgG all were optimized in order to improve both the analytical performance and the stability. In addition, the thickness of the paper was examined to stabilize the analytical signal. Consequently, the APTS-glutaraldehyde method was superior to the NaIO4 method in terms of sensitivity and reproducibility. Conversely, the reduction of imine to amine with NaBH4 proved to exert only minimal influence on sensitivity and stability, although it tended to degrade reproducibility. We also found that thick paper was preferential when using P-ELISA because a rigid paper substrate prevents distortion of the paper surface that is often caused by repeated washing processes.

Post-marketing safety of tralokinumab: a real-world pharmacovigilance study based on the FDA adverse event reporting system

ABSTRACT Background Tralokinumab is a fully human IgG4 monoclonal antibody targeting IL-13, used for treating atopic dermatitis. This study analyzed tralokinumab-related adverse drug events by mining the Food and Drug Administration Adverse Event Reporting System (FAERS) database to provide a safety reference for clinical application. Methods Adverse drug event reports from Q1 2022 to Q2 2024 were extracted from the FAERS database. After standardizing the data, various signal detection methods were used for analysis, including ROR, PRR, BCPNN, and MGPS. Results A total of 1,820 reports of adverse events (AEs) with tralokinumab as the primary suspected drug were identified. 70 preferred terms (PTs) met the criteria across four signal detection methods, involving 11 system organ classes (SOCs). These included known adverse reactions like conjunctivitis and injection site reactions, and signals not previously reported in clinical trials, such as eye pruritus, dry eye, eye swelling, pneumonia pneumococcal, and cutaneous T-cell lymphoma. Most AEs occurred within one month of initiating tralokinumab treatment. Conclusions Based on the FAERS database, this study comprehensively and systematically analyzed AE signals in tralokinumab treatment. The results enhance the understanding of tralokinumab’s safety and serve as valuable references for reducing the risk of adverse reactions during clinical use.