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.

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.

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.

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.

FcγRIIIa-158V/F polymorphism affects the performance of FcγRIIIa-related bioassay.

Bioassays are important for estimating biosimilarity between biological products. Comparability studies including bioassays are needed to demonstrate that a biosimilar product has no meaningful differences that affect safety and efficacy compared with the reference product. Among the most important biological characteristics of therapeutic mAbs are Fc-mediated functions, which induce immune-cell activation which can affect both efficacy and safety. Thus, when developing biosimilar products of therapeutic mAbs, it is necessary to compare the Fc-mediated functions by using various bioassays. Though it is reported that polymorphism of Fcγ receptors (FcγRs) affects Fc-mediated cellular activations of therapeutic mAbs, the impacts of the polymorphism of FcγRs on bioassay performance are still unclear. In this study, we evaluated the impact of FcγRIIIa-158V/F polymorphism on assay performance in distinguishing differences in the biological activities of therapeutic mAbs. The results showed that different bioassay methods produced different assessments of biological activities of mAbs, and that the FcγRIIIa-158V/F polymorphism clearly affected the performance of the FcγRIIIa-binding assay using recombinant proteins and FcγRIIIa-expressing reporter assays. That is, the assays using the FcγRIIIa-158F variant were superior to those using the FcγRIIIa-158V variant in distinguishing the difference in FcγRIIIa-binding and -activation properties. These results indicate that we should evaluate the comparability of biosimilars by considering the impacts of FcγRIIIa-158V/F polymorphism on bioassay performance.

Fcγ Receptor-Dependent Internalization and Off-Target Cytotoxicity of Antibody-Drug Conjugate Aggregates.

PURPOSE: Antibody-drug conjugates (ADCs), which are monoclonal antibodies (mAbs) conjugated with highly toxic payloads, achieve high tumor killing efficacy due to the specific delivery of payloads in accordance with mAbs' function. On the other hand, the conjugation of payloads often increases the hydrophobicity of mAbs, resulting in reduced stability and increased aggregation. It is considered that mAb aggregates have potential risk for activating Fcγ receptors (FcγRs) on immune cells, and are internalized into cells via FcγRs. Based on the mechanism of action of ADCs, the internalization of ADCs into target-negative cells may cause the off-target toxicity. However, the impacts of aggregation on the safety of ADCs including off-target cytotoxicity have been unclear. In this study, we investigated the cytotoxicity of ADC aggregates in target-negative cells. METHODS: The ADC aggregates were generated by stirring stress or thermal stress. The off-target cytotoxicity of ADC aggregates was evaluated in several target-negative cell lines, and FcγR-activation properties of ADC aggregates were characterized using a reporter cell assay. RESULTS: Aggregation of ADCs enhanced the off-target cytotoxicity in several target-negative cell lines compared with non-stressed ADCs. Notably, ADC aggregates with FcγR-activation properties showed dramatically enhanced cytotoxicity in FcγR-expressing cells. The FcγR-mediated off-target cytotoxicity of ADC aggregates was reduced by using a FcγR-blocking antibody or Fc-engineering for silencing Fc-mediated effector functions. CONCLUSIONS: These results indicated that FcγRs play an important role for internalization of ADC aggregates into non-target cells, and the aggregation of ADCs increases the potential risk for off-target toxicity.

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.

Development of anti-drug monoclonal antibody panels against adalimumab and infliximab.

The generation of anti-drug antibodies (ADAs) is one of the most serious problems in therapy using monoclonal antibodies (mAbs), because ADAs can impact the pharmacokinetics, efficacy, and safety of mAbs. It is therefore important to detect the generated ADAs in patients. For the appropriate detection of ADAs, methods that detect various types of ADAs (e.g., low- and high-affinity ADAs) are needed, but since there are no adequate reference preparations of ADAs relevant to human ADAs in most cases, it is difficult to determine whether or not the developed methods have enough analytical performance. Here, we developed human-rat chimeric ADA panels against the anti-TNF-α therapeutic antibodies infliximab and adalimumab. The developed ADA panels consist of 7 (for infliximab) and 11 (for adalimumab) ADAs with various binding characters, and most of the ADAs are neutralizing antibodies. Using these ADA panels, we compared the detectability of model methods, i.e., binding assays using SPR, BLI, and ECL, and a cell-based assay to detect neutralization activity. Since we obtained ADAs showing low and high responses with the various methods, the ADA panels we developed were shown to be useful for the development of ADA assays.

Human-rat chimeric anti-occludin monoclonal antibodies inhibit hepatitis C virus infection.

Occludin (OCLN), an integral tetra-spanning plasma membrane protein, is a host entry factor essential for hepatitis C virus (HCV) infection, making it a promising host-targeting molecule for HCV therapeutic intervention. We previously generated rat anti-OCLN monoclonal antibodies (mAbs) that strongly prevented HCV infection in vitro and in vivo. In the present study, we attempted to improve the druggability of the extracellular loop domain-recognizing anti-OCLN mAbs, namely clones 1-3 and 37-5, using genetic engineering. To avoid adverse reactions induced by antibody-dependent cellular cytotoxicity and enhance the antibody stability, we developed human-rat chimeric immunoglobulin G4 S228P mutant (IgG4m) forms of clones 1-3 and 37-5 (named Xi 1-3 and Xi 37-5, respectively) by grafting the variable regions of the light and heavy chains of each rat anti-OCLN mAb into those of human IgG4m. The constructed Xi 1-3 and Xi 37-5 chimeras demonstrated levels of affinity and specificity similar to each parental rat anti-OCLN mAb, and the Fcγ receptor Ⅲa was not activated by the antigen-bound chimeric mAbs, as expected. Both chimeric mAbs inhibited in vitro infection with various HCV genotypes. These results indicate that the IgG4m forms of human-rat chimeric anti-OCLN mAbs may be potential candidate molecules of host-targeting antivirals with pan-genotypic anti-HCV activity.

In-depth site-specific O-Glycosylation analysis of therapeutic Fc-fusion protein by electron-transfer/higher-energy collisional dissociation mass spectrometry.

Unexpected O-glycosylations, including O-xylosylations and mucin-type O-glycosylations, have been reported in recent glycosylation analyses of Fc-fusion proteins produced in mammalian cell expression systems. This observation suggests that therapeutic proteins with novel structures can undergo unintended O-glycosylations, having implications regarding their efficacy and safety. Therefore, the implementation of O-glycosylation analysis during product developmental is essential. However, detail site-specific O-glycosylation analysis is difficult because no consensus sequence for mucin-type O-glycosylations is known, and O-glycopeptides often contain multiple or continuous glycosylation sites. Recently, a new mass spectrometric fragmentation method called electron-transfer/higher-energy collisional dissociation (EThcD) has been used for site-specific glycosylation analysis. In this study, we conducted site-specific O-glycosylation analysis of commercially available GLP1-Fc fusion protein with (G4S)3 linker peptide using liquid chromatography/mass spectrometry (LC/MS) with EThcD and a glycoproteomic database search. We successfully identified unexpected O-xylosylations at Ser residues in the (G4S)3 linker peptide, mucin-type O-glycosylations at Thr and Ser residues in the GLP-1 peptide, and Ser residues in the (G4S)3 linker peptide. This study is the first to report these unexpected O-xylosylations and mucin-type O-glycosylations in this therapeutic fusion protein. Mammalian-cell production of therapeutic fusion proteins that contain novel structures may require exhaustive O-glycosylation analysis to ensure their quality, efficacy, and safety.

Interlaboratory comparison about feasibility of insoluble particulate matter test for injections with reduced test volume in light obscuration method.

Insoluble particulate matter test for injections in pharmacopoeia is mandatory for parenteral drug products. In this test using light obscuration, four measurements of at least 5-mL are required. Since therapeutic protein injections of low dosage volumes are getting more popular, reduction of test volumes is desired. In this collaborative study, the impact of lower measurement volume on the accuracy and precision of particle count was evaluated using 2, 5, 10, and 25-µm polystyrene count standards for the validity of test with reduced sample volumes. Good accuracy (3000 particles/mL ±â€¯10%) was obtained at all measurement volumes, and the inter-run variability (RSD) was the same levels between 5 and 1 mL. Although the inter-run variability increased at 0.2 mL, it was below 5%. These results indicated that light obscuration method can be used with 5 mL-0.2 mL, and that it is feasible for monitoring particles ≥2 µm.

Occludin as a functional marker of vascular endothelial cells on tube-forming activity.

Cell therapy using endothelial progenitor cells (EPCs) is a promising strategy for the treatment of ischemic diseases. Two types of EPCs have been identified: early EPCs and late EPCs. Late EPCs are able to form tube structure by themselves, and have a high proliferative ability. The functional marker(s) of late EPCs, which relate to their therapeutic potential, have not been fully elucidated. Here we compared the gene expression profiles of several human cord blood derived late EPC lines which exhibit different tube formation activity, and we observed that the expression of occludin (OCLN) in these lines correlated with the tube formation ability, suggesting that OCLN is a candidate functional marker of late EPCs. When OCLN was knocked down by transfecting siRNA, the tube formation on Matrigel, the S phase + G2 /M phase in the cell cycle, and the spheroid-based sprouting of late EPCs were markedly reduced, suggesting the critical role of OCLN in tube formation, sprouting, and proliferation. These results indicated that OCLN plays a novel role in neovascularization and angiogenesis.

Effects of amino acid substitutions on the biological activity of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori).

Recombinant monoclonal antibodies (mAbs) have been used in various therapeutic applications including cancer therapy. Fc-mediated effector functions play a pivotal role in the tumor-killing activities of some tumor-targeting mAbs, and Fc-engineering technologies with glyco-engineering or amino acid substitutions at the antibody Fc region have been used to enhance cytotoxic activities including antibody-dependent cellular cytotoxicity (ADCC). We previously reported that the mAbs produced using transgenic silkworms showed stronger ADCC activity and lower complement-dependent cytotoxicity (CDC) activity than mAbs derived from Chinese hamster ovary (CHO) cells due to their unique N-glycan structure (lack of core-fucose and non-reducing terminal galactose). In this study, we generated anti-CD20 mAbs with amino acid substitutions using transgenic silkworms and analyzed their biological activities to assess the effect of the combination of glyco-engineering and amino acid substitutions on the Fc-mediated function of mAbs. Three types of amino acid substitutions at the Fc region (G236A/S239D/I332E, L234A/L235A, and K326W/E333S) modified the Fc-mediated biological activities of silkworm-derived mAbs as in the case of CHO-derived mAbs, resulting in the generation of Fc-engineered mAbs with characteristic Fc-mediated functions. The combination of amino acid substitutions at the Fc region and glyco-engineering using transgenic silkworm made it possible to generate Fc-engineered mAbs with suitable Fc-mediated biological functions depending on the pharmacological mechanism of their actions. Transgenic silkworms were shown to be a promising system for the production of Fc-engineered mAbs.

Glycosylation of IgG-Fc: a molecular perspective.

Antibodies of the IgG class carry a pair of oligosaccharides (N-glycans) in the Fc region. The importance of the N-glycan is clearly demonstrated by its profound effect in the physicochemical and biological properties of antibodies. The term 'glycoengineering' has been coined to describe contemporary strategies to improve the performance of therapeutic monoclonal antibodies on the basis of modifications in the structure and composition of the N-glycan. These methodologies have resulted in the approval and commercialization of a new generation of antibodies with improved therapeutic efficacy. So far, these advances have been driven by herculean efforts in a process of trial-and-error. The collective work of researchers in this field is progressively revealing the molecular basis of N-glycans for the function of antibodies. This knowledge will ultimately be conducive to the application of rational approaches for the successful manipulation of antibodies using glycoengineering strategies. Herein, we review advances in our understanding of the role of the N-glycan in the structural and dynamic integrity, and biological activity, of antibodies. Since the N-glycan has a multifaceted effect in antibodies, in this review we have emphasized the importance of integrating various techniques that address this problem from multiple points of view. In particular, the combination of X-ray crystallography with nuclear magnetic resonance, molecular dynamics simulations and biophysical approaches based on thermodynamic principles, has emerged as a powerful combination that is deepened our understanding of this unique system with critical implications for human well-being.

Development of cell-based assay for predictively evaluating the FcγR-mediated human immune cell activation by therapeutic monoclonal antibodies.

Therapeutic monoclonal antibodies (mAbs) have important roles in treatments for various cancers and inflammatory diseases. Their highly target specificities provide controlled safety profiles. However, therapeutic mAbs commonly pose a risk of the induction of the release of cytokines, which may result in adverse events including infusion reaction and cytokine release syndrome. Several mechanisms are involved in the cytokine releases induced by therapeutic mAbs, and the activation of immune effector cells via Fcγ receptors (FcγRs) is one of the putative mechanisms for most IgG-subclass mAbs. The relationship between cytokine releases and mAbs' Fc functions is not fully understood. Here we developed a simple reporter cell-based assay for estimating the FcγR-mediated activation of human immune effector cells by mAbs. Our use of the cell-based assay to compare Fc-engineered mAbs with different FcγR-activation profiles revealed that the releases of inflammatory cytokines and chemokines from human peripheral blood mononuclear cells (hPBMCs) induced by the mAbs were elevated by treatment with Fc-engineered mAbs with higher FcγR-activation properties. Our results also suggested the involvement of monocytic effector cells in the activation of hPBMCs as sources of released cytokines and chemokines, which may lead to the immune cell-mediated adverse events. Our new reporter cell assay is a promising tool for evaluating and predicting the activation of human immune cells by novel Fc-engineered mAbs.

Hinge-Deficient IgG1 Fc Fusion: Application to Human Lactoferrin.

Fusion of therapeutic proteins with the antibody Fc domain is a strategy widely applied to increase protein half-life in plasma. In our previous study, we generated a recombinant human lactoferrin (hLF)-immunoglobulin G1 Fc fusion protein (hLF-hinge-CH2-CH3) with improved stability, biological activity, and pharmacokinetics ( Shiga , Y. et al. Eur J Pharm Sci. , 2015 , 67 , 136 - -143 ). However, the Fc domain in fusion proteins can potentially induce antibody-dependent and complement-dependent cytotoxicity and serious side effects. To overcome these drawbacks, we engineered an hLF-Fc fusion protein (hLF-CH2-CH3) without the Fc hinge region which is essential for engaging Fc receptors on immune cells and inducing complement-mediated cell lysis. The hLF-CH2-CH3 protein was stably expressed in Chinese hamster ovary (CHO) DG44 cells and compared for in vitro activities, thermal stability, pharmacokinetics, and attenuation of Fc-mediated immune effector functions with the conventional hinge-containing Fc fusion protein. Both hLF-hinge-CH2-CH3 and hLF-CH2-CH3 exhibited iron-binding activity, superior uptake by Caco-2 cells, similar thermal stability, and longer plasma half-life compared to recombinant hLF. However, in contrast to conventional hLF-hinge-CH2-CH3, hinge-deficient hLF-CH2-CH3 did not elicit Fc-mediated effector response potentially damaging for the target cells. Our findings demonstrate that conjugation of hinge-deficient Fc to therapeutic proteins is a promising strategy for improving their pharmacokinetic properties without enhancing effector functions. Cell-expressed hinge-deficient hLF-CH2-CH3 is a potential drug candidate with improved plasma half-life for parenteral administration.

Comparative study of the number of report and time-to-onset of the reported adverse event between the biosimilars and the originator of filgrastim.

PURPOSE: The objective of this study is to specify the most reported adverse events as preferred terms (PTs) and to compare the reported adverse events about some properties including the number of report and time-to-onset (TTO) distribution of the originator of filgrastim Neupogen® and its biosimilars in Europe, using VigiBase®. METHODS: We identified the biosimilar which was reported as the suspected drug in more than 100 individual case safety reports in Europe. Then, we specified the top ranking 10 PTs in the cases reported with Neupogen® or each biosimilar as the suspected drug. We also compared the TTO of the most reported PTs using the data about the onset date of the PT and the start date of filgrastim. We used Kolmogorov-Smirnov method to detect significant difference. RESULTS: The total ICSR numbers with Neupogen® and 3 biosimilars, Zarzio®, Nivestim®, and Tevagrastim® were 1,301, 295, 156, and 127, respectively, in Europe. The most reported PTs with Neupogen® were bone pain, pyrexia, and dyspnoea. The TTO of bone pain and pyrexia with Zarzio® (N: 22 and 16, median: 1 and 0.5 days) were significantly shorter than those with Neupogen® (P < 0.01, N: 72 and 33, median: 3.5 and 3 days), respectively. The most reported PTs with biosimilars were drug ineffective and neutropenia. CONCLUSION: The difference in the TTO was identified between originator filgrastim Neupogen and its biosimilar regarding some PTs, which may suggest the difference in their safety profile. Copyright © 2017 John Wiley & Sons, Ltd.

The possible interaction between periostin expressed by cancer stroma and tumor-associated macrophages in developing mycosis fungoides.

Mycosis fungoides (MF) starts as an indolent disease, progresses from a patch stage to confluent plaques and ultimately develops skin tumors. Tumor-associated macrophages (TAMs) play roles in maintaining the tumor microenvironment in MF. The purpose of this study was to elucidate the involvement of TAMs in the lesional skin of different stages of MF. First, we immunohistologically examined the percentage of CD163+ macrophages and CD206+ cells, as well as the levels of periostin and IL-4 in cancer stroma. The percentage of CD206+ cells increased in parallel with tumor progression, while there was no significant difference in the percentage of CD163+ cells. Periostin was prominent in the stromal area at the patch and plaque stages but decreased at the tumor stage. In contrast, IL-4 was prominently stained at both plaque and tumor stages. To further elucidate the molecular mechanisms of the effects of these stromal factors on TAMs, we examined their effects on mRNA expression in monocyte-derived macrophages in vitro. Based on microarray analysis and gene ontology, we examined a series of chemokines and MMPs whose expression was strongly connected with periostin stimulation. The DNA microarray results were verified in M2 macrophages using real-time PCR. We further examined the mRNA expression of these chemokines and MMPs in the presence of periostin and IL-4 to simulate the advanced stages of MF and validated their protein expression by ELISA. Our present report suggests possible roles of periostin on TAMs in establishing the tumor microenvironment in MF.

Successful Treatment of Segmental Vitiligo in Children with the Combination of 1-mm Minigrafts and Phototherapy.

BACKGROUND: Minigrafts using a 1-mm biopsy punch (1-mm minigrafts) are being increasingly used to treat vitiligo. However, there have been few reports of the use of 1-mm minigrafts in pediatric patients. OBJECTIVE: To examine the effectiveness of combination therapy with 1-mm minigrafts and phototherapy in children with segmental vitiligo. METHODS: Minigrafts were placed in 13 patients aged ≤16 years with segmental vitiligo. Following surgery, 11 patients underwent irradiation with excimer laser light and 2 with narrow-band ultraviolet B light. RESULTS: A mean repigmentation of 81.6% was obtained. A particularly high mean repigmentation of 87.9% was seen in patients aged ≤12 years, indicating greater efficacy in these patients than in patients aged ≥13 years (mean, 67.5%). Although a transient cobblestone appearance occurred as an adverse effect, it improved over time. CONCLUSIONS: Combined treatment of segmental vitiligo with 1-mm minigrafts and phototherapy can be performed safely and is highly effective in young patients.

Different Results of IgE Binding- and Crosslinking-Based Allergy Tests Caused by Allergen Immobilization.

The physicochemical nature of allergen molecules differ from the liquid phase to the solid phase. However, conventional allergy tests are based on the detection of immunoglobulin (Ig)E binding to immobilized allergens. We recently developed an in vitro allergy testing method using a luciferase-reporting humanized rat mast cell line to detect IgE crosslinking-induced luciferase expression (EXiLE test). The aim of the present study was to evaluate the effects of antigen immobilization on the results of different in vitro allergy tests using two anti-ovalbumin (OVA) antibodies (Abs), E-C1 and E-G5, with different properties in the OVA-induced allergic reaction. Both Abs showed clear binding to OVA with an enzyme-linked immunosorbent assay and by BIAcore analysis. However, only E-C1 potentiated EXiLE response for the liquid-phase OVA. On the other hand, OVA immobilized on solid-phase induced EXiLE responses in both E-C1 Ab- and E-G5 Ab-sensitized mast cells. Western blotting of OVA indicated that E-C1 Ab binds both to OVA monomers and dimers, unlike E-G5 Ab, which probably binds only to the OVA dimer. These results suggest that antigen immobilization enhanced IgE crosslinking ability through multimerization of allergen molecules in the solid phase, resulting in an increase in false positives in IgE binding-based conventional in vitro allergy tests. These findings shed light on the physicochemical nature of antigens as an important factor for the development and evaluation of in vitro allergy tests and suggest that mast cell activation-based allergy testing with liquid-phase allergens is a promising strategy to evaluate the physiological interactions of IgE and allergens.