Myung-Hee Kwon

The mouse 3D8 anti‐DNA antibody can enter cells and localize in the cytoplasm, primarily facilitated by the complementarity‐determining region 1 of the variable light chain (CDR L1) domain. In this study, we grafted the CDR L1 loop from 3D8 onto non‐cell‐penetrating IgG antibodies to investigate whether these IgGs could acquire cytoplasmic localization ability while retaining antigen‐binding activity. One of three IgGs was successfully delivered into the cytoplasm while maintaining antigen‐binding activity. In silico protein modeling suggests that this capability is linked to structural similarity between CDR L1 in the grafted Ab and that in 3D8. This study proposes a strategy to confer cell‐penetrating capability by incorporating a specific CDR loop into an antibody backbone while retaining affinity.

Introduction: The C_H1 domain of IgG antibodies controls assembly and secretion, mediated by the molecular chaperone BiP via the endoplasmic reticulum protein quality control (ERQC) mechanism. However, it is not clear whether the variable domains are necessary for this process.Methods: Here, we generated IgG1 antibodies in which the V domain (V_H and/or V_L) was either removed or replaced, and then assessed expression, assembly, and secretion in HEK293 cells.Results: All Ig variants formed a covalent linkage between the C_γ1 and C_κ, were successfully secreted in an assembled form. Replacement of the cognate V_κ with a non-secretory pseudo V_κ (_ψV_κ) hindered secretion of individual or assembled secretion of neither heavy chains (HCs) nor light chains (LCs). The _ψLC (_ψV_κ-C_κ) exhibited a less folded structure compared to the wild type (wt) LC, as evidenced by enhanced stable binding to the molecular chaperone BiP and susceptibility to proteolytic degradation. Molecular dynamics simulation demonstrated dramatic alterations in overall structure of _ψFab (Fd-_ψLC) from wt Fab.Discussion: These findings suggest that V domains do not initiate HC:LC assembly and secretion; instead, the critical factor governing IgG assembly and secretion is the C_H-C_L pairing. Additionally, the structural integrity of the V_L domain is crucial for IgG secretion. These data offer valuable insight into the design of bioactive molecules based on an IgG backbone.

Human IgE is useful for immunological assays, such as sensitization of FcεRI-positive cells and IgE measurement. In this study, we report the development of a recombinant Ig fragment, designated IgCw-γεκ, as an alternative reagent to human IgE. IgCw-γεκ (∼130 kDa) comprises two hybrid constant H chain regions (Cγ₁-Cε_2–4, each ∼53 kDa) and two constant κ L chains (C_κ, each ∼12 kDa) and lacks a V domain. The presence of Cγ₁ instead of Cε₁ within the H chain increased the production yield and facilitated assembly of the H and L chains. IgCw-γεκ was produced in cultured human embryonic kidney 293F cells, with a yield of ∼27 mg/l. IgCw-γεκ bound to human FcεRIαRs expressed on the surface of rat basophilic leukemia-2H3 cells. A β-hexosaminidase release assay revealed that the biological activity of IgCw-γεκ was comparable with that of IgE. The IgE concentration measured using IgCw-γεκ as a standard was similar to that measured using IgE as a standard. These results suggest that the IgCw-γεκ molecule retains the basic characteristics of IgE, but does not cross-react with Ags, making it an alternative to the IgE isotype references used in a variety of immunological assays. Key Points The Ig fragment IgCw-γεκ comprises the Cγ₁-Cε_2–4 and C_κ domains. IgCw-γεκ binds human FcεRI and inhibits IgE-induced degranulation of RBL-2H3 cells. IgCw-γεκ may be an alternative to the IgE references used for IgE quantification.

Intrabodies, antibodies expressed within cells, offer an interesting way to target intracellular molecules, making them potentially useful for biotechnology and medicine. However, it remains controversial whether full-size IgG intrabodies expressed in the reducing environment of the cytosol of mammalian cells are workable and structurally sound. Herein, we settle this issue with a systematic investigation of the structure and functionality of four chimeric IgG1s with distinct variable (V) domains but identical constant (C) domains. Full-size IgGs expressed in the cytosol of HEK293 cells were either assembly-competent or -incompetent, depending on the intrinsic properties of the V regions. Structural integrity of the C region is required for H:L association and the formation of a functional antigen-binding site. Partial intrachain disulfide bond formation occurs in both H and L chains of cytosolic IgG intrabodies, whereas interchain disulfide bond formation was absent and dispensable for functional assembly. IgG1s expressed in the cytosol and via the ER were shown to assemble differently. Our findings provide insight into the features and possible utilization of full-size IgGs as cytosolic antibodies in biotechnological and medical applications.

Constant (C)-region switching of heavy (H) and/or light (L) chains in antibodies (Abs) can affect their affinity and specificity, as demonstrated using mouse, human, and chimeric mouse-human (MH) Abs. However, the consequences of C-region switching between evolutionarily distinct mammalian and avian Abs remain unknown. To explore C-region switching in mouse-chicken (MC) Abs, we investigated antigen-binding parameters and thermal stability of chimeric MC-6C407 and MC-3D8 IgY Abs compared with parental mouse IgGs and chimeric MH Abs (MH-6C407 IgG and MH-3D8 IgG) bearing identical corresponding variable (V) regions. The two MC-IgYs exhibited differences in antigen-binding parameters and thermal stability from their parental mouse Abs. However, changes were similar to or less than those between chimeric MH Abs and their parental mouse Abs. The results demonstrate that mammalian and avian Abs share compatible V-C region interfaces, which may be conducive for the design and utilization of mammalian-avian chimeric Abs.

Anti-DNA autoantibodies are a hallmark of systemic lupus erythematosus (SLE). A subset of anti-DNA IgG autoantibodies is cell-internalizable; thus they can enter living cells in the form of free IgG. However, the contribution made by the Fc region of internalized free-form IgG to the cytokine response has not been studied, despite the recent discovery of tripartite motif-containing 21 (TRIM21), a cytosolic Fc receptor involved in immune signaling. This study used an internalizable IgG anti-DNA antibody (3D8) to examine the cytokine responses of human monocytes to the Fc region of cytosolic free IgG. Internalization of 3D8 IgG and a 3D8 single-chain variable fragment-Fc (scFv-Fc) induced production of IL-8 and TNF-α via activation of NF-κB. By contrast, a 3D8 scFv (comprising variable domains alone) did not. This suggests Fc-dependent cytokine signaling. A 3D8 IgG-N434D mutant that is not recognized by TRIM21 induced greater production of cytokines than 3D8 IgG. Moreover the amounts of cytokines induced by 3D8 IgG in TRIM21-knockdown THP-1 cells were higher than those in control cells, indicating that cytokine signaling is not mediated by TRIM21. The results suggest the existence of a novel Fc-dependent signaling pathway that is activated upon internalization of IgG antibodies by human monocytes.

A subset of monoclonal anti-DNA autoantibodies enters a variety of living cells. Here, we aimed to identify the endocytic receptors recognized by an internalizing anti-nucleic acid autoantibody, the 3D8 single-chain variable fragment (scFv). We found that cell surface binding and internalization of 3D8 scFv were inhibited markedly in soluble heparan sulfate (HS)/chondroitin sulfate (CS)-deficient or -removed cells and in the presence of soluble HS and CS. 3D8 scFv colocalized intracellularly with either HS proteoglycans (HSPGs) or CSPGs in HeLa cells. 3D8 scFv was co-endocytosed and co-precipitated with representative individual HSPG and CSPG molecules: syndecan-2 (a transmembrane HSPG), glypican-3 (a glycosylphosphatidylinositol (GPI)-anchored HSPG); CD44 (a transmembrane CSPG); and brevican (a GPI-anchored CSPG). Collected data indicate that 3D8 scFv binds to the negatively charged sugar chains of both HSPGs and CSPGs and is then internalized along with these molecules, irrespective of how these proteoglycans are associated with the cell membrane. This is the first study to show that anti-DNA antibodies enter cells via both HSPGs and CSPGs simultaneously. The data may aid understanding of endocytic receptors that bind anti-DNA autoantibodies. The study also provides insight into potential cell membrane targets for macromolecular delivery.

Indoor air qualities in smoking banned and smoking free facilities are investigated for figure out the relationship between environmental factors and concentration of smoking pollutants. The KS H ISO 18145 and indoor air quality standard method were used for analysing the nicotine and particulate matter(PM10), respectively. The highest concentration of fine particles and nicotine is recorded in billiard hall. A significant positive relationship between smoking frequency and concentration of indoor pollutants was observed. Among the environmental factors, the ventilation time and frequency was revealed as a main factor influencing on reducing the concentration of fine particles and nicotine which are emitted from indoor smoking.