In response to immunization, B-cells generate a repertoire of antigen-specific antibodies. Antibody-based immunotherapies hold great promise for treating a variety of diseases in humans. Application of antibody-based immunotherapy in cats is limited by the lack of species-specific complete sequences for mRNAs encoding rearranged heavy and light chain immunoglobulins in B cells. To address this barrier, we isolated mRNAs from feline peripheral blood mononuclear cells (PBMCs), and used available immunoglobulin sequences and 5' and 3' RACE to clone and sequence heavy and light chain immunoglobulin mRNAs. We recovered mRNA from PBMCs from two cats, cloned and sequenced the variable and constant domains of the feline heavy chains of IgG1a (IGHG1a), IgG2 (IGHG2), and IgA (IGHA), and the light chains (lambda and kappa). Using these sequences, we prepared two bicistronic vectors for mammalian expression of a representative feline heavy (IGHG1a) together with a light (lambda or kappa) chain. Here we report novel feline Ig sequences, a technique to express antigen-specific felinized monoclonal antibodies, and the initial characterization of a functional felinized monoclonal antibody against feline panleukopenia virus.

Growing evidence indicates that B cells are not the only source of immunoglobulin (Ig). To investigate this discovery further, we used μMT mice, which have a disruption of the first transmembrane exon of the μ heavy chain and do not express the membrane form of IgM. These mice lack mature B cells and thus serve as a good model to explore Ig expression by liver epithelial cells. We found that Ig heavy chains (μ, δ, γ and α) and light chains (κ and λ) were expressed in sorted liver epithelial cells of μMT mice. Surprisingly, each heavy chain class showed its respective variable region sequence characteristics in their variable region, instead of sharing the same VDJ usage, which suggests that class switching does not occur in liver epithelial cells. Moreover, the γ and α chains, but not the μ and δ chains, showed mutations in the variable region, thus indicating that different classes of Ig have different activities. Our findings support the concept that non-B cells, liver epithelial cells here, can produce different classes of Ig.

Keratoconus (KC) is classically considered a non-inflammatory condition caused by central corneal thinning that leads to astigmatism and reduced visual acuity. Previous studies have identified increased systemic levels of pro-inflammatory factors, including interleukin-6, tumor necrosis factor-α, and matrix metalloproteinase-9, suggesting that KC may have an inflammatory component in at least a subset of patients. In this study, we evaluated the levels of different immunoglobulins (light and heavy chains) based on Ig α, Ig λ, Ig κ, Ig µ, and Ig heavy chain subunits in non-KC tears (n = 7 control individuals) and KC tears (n = 7 KC patients) using tandem-liquid chromatography mass spectrometry. The most abundant Ig heavy chains detected in both control individuals and KC patients were Ig α-1 and Ig α-2 likely correlating to the higher IgA levels reported in human tears. We identified significant differences in immunoglobulin κ-chain V-II levels in KC patients compared to control individuals with no significant difference in Ig κ/Ig λ ratios or heavy chain levels. Our study supports previous findings suggesting that KC possesses a systemic component that may contribute to the KC pathology. Further studies are required to define causality and establish a role for systemic immune system-dependent factors and pro-inflammatory processes in KC development or progression.

B-lymphocytes in the bone marrow (BM) must generate a functional B-cell receptor and overcome the negative selection induced by reactivity with autoantigens. Two rounds of DNA recombination are required for the production of functional immunoglobulin heavy (Ig-HCs) and light (LCs) chains necessary for the continuation of B-lymphocyte development in the BM. Both rounds depend on the joint action of recombination activating gene-1 (RAG-1) and RAG-2 endonucleases with the DNA non-homologous end-joining pathway. Loss of the FANC gene leads to the chromosome breakage and cancer predisposition syndrome Fanconi anemia. Because the FANC proteins are involved in certain aspects of the recombination process, we sought to determine the impact of the FANC pathway on the Ig diversification process using Fanca-/- mice. In this work we demonstrated that Fanca-/- animals have a mild B-cell differentiation defect characterized by a specific alteration of the IgM- to IgM+ transition of the B220low B-cell population. Pre-B cells from Fanca-/- mice show evidence of impaired kLC rearrangement at the level of the Vk-Jk junction. Furthermore, Fanca-/- mice showed a skewed Vκ gene usage during formation of the LCs Vk-Jk junctions. Therefore, the Fanca protein appears as a yet unidentified factor involved in the primary diversification of Ig.

Sweet potato leaf curl virus (SPLCV) causes yield losses in sweet potato cultivation. Diagnostic techniques such as serological detection have been developed because these plant viruses are difficult to treat. Serological assays have been used extensively with recombinant antibodies such as whole immunoglobulin or single-chain variable fragments (scFv). An scFv consists of variable heavy (VH) and variable light (VL) chains joined with a short, flexible peptide linker. An scFv can serve as a diagnostic application using various combinations of variable chains. Two SPLCV-specific scFv clones, F7 and G7, were screened by bio-panning process with a yeast cell which expressed coat protein (CP) of SPLCV. The scFv genes were subcloned and expressed in Escherichia coli. The binding affinity and characteristics of the expressed proteins were confirmed by enzyme-linked immunosorbent assay using SPLCV-infected plant leaves. Virus-specific scFv selection by a combination of yeast-surface display and scFv-phage display can be applied to detection of any virus.

Shiga toxin is a major virulence factor of food-poisoning caused by Escherichia coli such as O157:H7. Secretory immunoglobulin (Ig) A (SIgA) is supposed to prevent infection of the mucosal surface and is a candidate agent for oral immunotherapy. We previously established a recombinant monoclonal antibody (mAb) consisting of variable regions from a mouse IgG mAb specific for the binding subunit of Shiga toxin 1 (Stx1) and the Fc region of mouse IgA. Here we produced a secretory form of the recombinant IgA (S-hyIgA) with transgenic Arabidopsis thaliana plant. All the S-hyIgA cDNAs (heavy, light, J chain and secretory component) were expressed under the control of a bidirectional promoter of a chlorophyll a/b-binding protein of A. thaliana without using a viral promoter. The plant-based S-hyIgA exhibited antigen binding, and was modified with plant-specific N-linked sugar chains. The Ig heavy chain and secretory components were observed in an intracellular protein body-like structure of the transgenic leaves on immuno-electron microscopy. An extract of the transgenic leaves neutralized the cytotoxicity of Stx1 toward butyrate-treated Caco-2 cells, a human colon carcinoma cell line. These results will contribute to the development of edible therapeutic antibodies such as those for the treatment of mucosal infection.

A major class of bispecific antibodies (BsAbs) utilizes heterodimeric Fc to produce the native immunoglobulin G (IgG) structure. Because appropriate pairing of heavy and light chains is required, the design of BsAbs produced through recombination or reassembly of two separately-expressed antigen-binding fragments is advantageous. One such method uses intein-mediated protein trans-splicing (IMPTS) to produce an IgG1-based structure. An extra Cys residue is incorporated as a consensus sequence for IMPTS in successful examples, but this may lead to potential destabilization or disturbance of the assay system. In this study, we designed a BsAb linked by IMPTS, without the extra Cys residue. A BsAb binding to both TNFR2 and CD30 was successfully produced. Cleaved side product formation was inevitable, but it was minimized under the optimized conditions. The fine-tuned design is suitable for the production of IgG-like BsAb with high symmetry between the two antigen-binding fragments that is advantageous for screening BsAbs.

The engineering of immunoglobulin-G molecules (IgGs) is of wide interest for improving therapeutics, for example by modulating the activity or multiplexing the specificity of IgGs to recognize more than one antigen. Optimization of engineered IgG requires knowledge of three-dimensional (3D) structure of synthetic IgG. However, due to flexible nature of the molecules, their structural characterization is challenging. Here, we use our reported individual-particle electron tomography (IPET) method with optimized negative-staining (OpNS) for direct 3D reconstruction of individual IgG hole-hole homodimer molecules. The hole-hole homodimer is an undesired variant generated during the production of a bispecific antibody using the knob-into-hole heterodimer technology. A total of 64 IPET 3D density maps at ~15 Å resolutions were reconstructed from 64 individual molecules, revealing 64 unique conformations. In addition to the known Y-shaped conformation, we also observed an unusual X-shaped conformation. The 3D structure of the X-shaped conformation contributes to our understanding of the structural details of the interaction between two heavy chains in the Fc domain. The IPET approach, as an orthogonal technique to characterize the 3D structure of therapeutic antibodies, provides insight into the 3D structural variety and dynamics of heterogeneous IgG molecules.

Elevated circulating Retinol-binding protein 4 (RBP4) has been associated with insulin resistance, dyslipidemia, and hypertension. However, many commonly used RBP4 ELISAs have limited dynamic range. We therefore developed an enzyme-linked immunosorbent sandwich assay (ELISA) employing a novel immunoglobulin A (IgA)-type capture mAb called AG102 instead of IgG subtypes, which was selected for its stability, capture efficiency, and specificity for human RBP 4. These features of RBP4 have hampered the development of quantitative immunological assays. Molecular analysis of AG102 revealed IgA heavy and light chains and a J chain, as expected. AG102 demonstrated notable detection of both bacterial- and HEK293-expressed RBP4 in Western blots. Serial and internal deletion experiments suggested that a putative epitope may be located in the first 35 amino acids of the mature RBP4. Compared with commercial ELISAs, the AG102-based system exhibited more significant recovery of RBP4 from serum or urine at any given dilution factor. To substantiate its quantitation capacity, comparison between RBP4 measurements from quantitative western blots and the AG102-based ELISA demonstrated a significant correlation (R2 = 0.859). After measurement for those analytes, our data suggested that IgA-based ELISA could be adapted for quantitative measurement of those analytes existing as major serum proteins or as multi-protein complexes like RBP4.

Anthrax is a highly lethal infectious disease caused by the bacterium Bacillus anthracis, and the associated shock is closely related to the lethal toxin (LeTx) produced by the bacterium. The central role played by the 63 kDa protective antigen (PA63) region of LeTx in the pathophysiology of anthrax makes it an excellent therapeutic target. In the present study, a human/murine chimeric IgG mAb, hmPA6, was developed by inserting murine antibody variable regions into human constant regions using antibody engineering technology. hmPA6 expressed in 293F cells could neutralize LeTx both in vitro and in vivo. At a dose of 0.3 mg/kg, it could protect all tested rats from a lethal dose of LeTx. Even administration of 0.6 mg/kg hmPA6 48 h before LeTx challenge protected all tested rats. The results indicate that hmPA6 is a potential candidate for clinical application in anthrax treatment.

We have previously shown that glioblastoma stem cells (GSCs) are enriched in the hypoxic tumor microenvironment, and that monocarboxylate transporter-4 (MCT4) is critical for mediating GSC signaling in hypoxia. Basigin is involved in many physiological functions during early stages of development and in cancer and is required for functional plasma membrane expression of MCT4. We sought to determine if disruption of the MCT-Basigin interaction may be achieved with a small molecule. Using a cell-based drug-screening assay, we identified Acriflavine (ACF), a small molecule that inhibits the binding between Basigin and MCT4. Surface plasmon resonance and cellular thermal-shift-assays confirmed ACF binding to basigin in vitro and in live glioblastoma cells, respectively. ACF significantly inhibited growth and self-renewal potential of several glioblastoma neurosphere lines in vitro, and this activity was further augmented by hypoxia. Finally, treatment of mice bearing GSC-derived xenografts resulted in significant inhibition of tumor progression in early and late-stage disease. ACF treatment inhibited intratumoral expression of VEGF and tumor vascularization. Our work serves as a proof-of-concept as it shows, for the first time, that disruption of MCT binding to their chaperon, Basigin, may be an effective approach to target GSC and to inhibit angiogenesis and tumor progression.

4CMenB is the first broad coverage vaccine for the prevention of invasive meningococcal disease caused by serogroup B strains. To gain a comprehensive picture of the antibody response induced upon 4CMenB vaccination and to obtain relevant translational information directly from human studies, we have isolated a panel of human monoclonal antibodies from adult vaccinees. Based on the Ig-gene sequence of the variable region, 37 antigen-specific monoclonal antibodies were identified and produced as recombinant Fab fragments, and a subset also produced as full length recombinant IgG1 and functionally characterized. We found that the monoclonal antibodies were cross-reactive against different antigen variants and recognized multiple epitopes on each of the antigens. Interestingly, synergy between antibodies targeting different epitopes enhanced the potency of the bactericidal response. This work represents the first extensive characterization of monoclonal antibodies generated in humans upon 4CMenB immunization and contributes to further unraveling the immunological and functional properties of the vaccine antigens. Moreover, understanding the mechanistic nature of protection induced by vaccination paves the way to more rational vaccine design and implementation.