Chimeric single-chain variable fragment-human immunoglobulin G crystallizable fragment antibody against GD2 for neuroblastoma targeted immunotherapy

Aim: The present study aims to generate chimeric mouse single-chain variable fragment (scFv) and immunoglobulin G1 (IgG1) crystallizable fragment (Fc) antibody against disialoganglioside (GD2) for the treatment of neuroblastoma (NB). The generated scFv-IgG Fc antibody, lacking first constant domain of heavy chain (CH1), is of a smaller size than the natural antibody and has anti-tumor activity. Methods: Vector for scFv-IgG Fc antibody was constructed and scFv-IgG Fc antibody was expressed in human embryonic kidney 293T (HEK293T) cell line. Purification of scFv-IgG Fc antibody from the culture supernatant of transfected HEK293T cells was performed by Protein G affinity chromatography. The structure and binding activity of scFv-IgG Fc antibody were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting (WB), and immunofluorescence techniques. Anti-tumor activities by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) were determined. Results: Using plasmid fusion-human IgG1-Fc2 tag vector (pFUSE-hIgG1-Fc2), a plasmid vector encoding chimeric mouse scFv and hIgG1 Fc antibody against GD2 was successfully constructed. This vector was transfected into human HEK293T cells to produce scFv-IgG Fc antibody. The transfected HEK293T cells could produce chimeric scFv-IgG Fc antibody against GD2, which lacks the IgG heavy chain CH1 domain but carries CH2 and CH3 domains. The chimeric antibodies could be purified from the culture supernatant of the transfected HEK293T culture in the presence of zeocin drug. The produced GD2 scFv-IgG Fc antibodies, which are smaller in size than the intact antibody, could trigger the killing of GD2 expressed NB cell line SH-SY5Y by ADCC and ADCP mechanisms. Conclusions: The results indicate that chimeric scFv-hIgG Fc antibody, lacking heavy chain CH1 domain, could mediate antibody induced anti-tumor activities. The small size of this type of chimeric antibody may be employed as anti-GD2 antibody for NB therapy

Association of CD99 short and long forms with MHC class I, MHC class II and tetraspanin CD81 and recruitment into immunological synapses

<p>Abstract</p> <p>Background</p> <p>CD99, a leukocyte surface glycoprotein, is broadly expressed in many cell types. On the cell surface, CD99 is expressed as two distinct isoforms, a long form and a short form. CD99 has been demonstrated to play a key role in several biological processes, including the regulation of T cell activation. However, the molecular mechanisms by which CD99 participates in such processes are unclear. As CD99 contains a short cytoplasmic tail, it is unlikely that CD99 itself takes part in its multi-functions. Association of CD99 with other membrane proteins has been suggested to be necessary for exerting its functions.</p> <p>Results</p> <p>In this study, we analyzed the association of CD99 with other cell surface molecules involved in T cell activation. We demonstrate the association of MHC class I, MHC class II and tetraspanin CD81 with CD99 molecules on the cell surface. Association of CD99 with its partners was observed for both isoforms. In addition, we determined that CD99 is a lipid raft-associated membrane protein and is recruited into the immunologic synapse during T cell activation. The implication of CD99 on T cell activation was investigated. Inhibition of anti-CD3 induced T cell proliferation by an anti-CD99 monoclonal antibody was observed.</p> <p>Conclusions</p> <p>We provide evidence that CD99 directly interact and form the complex with the MHC class I and II, and tetraspanin CD81, and is functionally linked to the formation of the immunologic synapse. Upon T cell activation, CD99 engagement can inhibit T cell proliferation. We speculate that the CD99-MHC-CD81 complex is a tetraspanin web that plays an important role in T cell activation.</p

A Simple Manual Rosetting Method for Absolute CD4(+) Lymphocyte Counting in Resource-Limited Countries

A CD4 monoclonal antibody which reacts with CD4(+) lymphocytes but does not react significantly with monocytes was generated and used to develop a simple method for CD4 count. In a comparison with standard flow cytometry, high correlation was obtained. The developed method can be an alternative to flow cytometry for resource-limited countries

Interaction of CD99 and its ligand upregulates IL-6 and TNF-α upon T cell activation.

CD99 has been reported to be involved in T cell regulation. CD99 ligand involvement in the regulation of T cell activation has been postulated. In this study, recombinant CD99 proteins were produced and used as a tool for determining the role of CD99 and its ligand interaction. Recombinant CD99 proteins induced the upregulation of IL-6 and TNF-α expression, but not IFN-γ, in anti-CD3 monoclonal antibody activated T cells. The cytokine alteration was not observed in unstimulated T cells indicating the cytokine upregulation required the signal from T cell activation. The upregulation of IL-6 and TNF-α was, in addition, observed in CD3- mononuclear cell population including monocytes and NK cells. The recombinant CD99 proteins, however, did not affect either CD25, CD69 or MHC class II expression or T cell proliferation, upon T cell activation. The CD99 ligands were demonstrated to be expressed on monocytes, NK cells and dendritic cells, but not on B and T cells. Our results indicated the presence of CD99 ligands on leukocyte surface. Interaction between CD99 and its ligands involves the regulation of cytokine production

Involvement of CD147 on multidrug resistance through the regulation of P-glycoprotein expression in K562/ADR leukemic cell line

The relationship between P-gp and CD147 in the regulation of MDR in leukemic cells has not been reported. This study aimed to investigate the correlation between CD147 and P-gp in the regulation of drug resistance in the K562/ADR leukemic cell line. The results showed that drug-resistant K562/ADR cells expressed significantly higher P-gp and CD147 levels than drug-free K562/ADR cells. To determine the regulatory effect of CD147 on P-gp expression, anti-CD147 antibody MEM-M6/6 significantly decreased P-gp and CD147 mRNA and protein levels. This is the first report to show that CD147 mediates MDR in leukemia through the regulation of P-gp expression

Immune Alterations in Patients with Anti-Interferon-γ Autoantibodies.

Autoantibodies against interferon-gamma (IFN-γ) can cause immunodeficiency and are associated with various opportunistic infections. In the present study, we investigated other cellular immune parameters for a better understanding of the immunodeficiency condition in the patients. The numbers of WBC, monocytes and NK cells were increased in patients with anti-IFN-γ autoantibodies (AAbs). Upon TCR activation, T cell proliferation and IL-2 receptor of the patients remained intact. Nonetheless, the Th1 cytokine (IFN-γ and TNF-α) production was up-regulated. The production of Th2 (IL-4) and Th17 (IL-17) cytokines was unchanged. We suggest that, in addition to the presence of anti-IFN-γ autoantibodies, alterations in the cellular immune functions may also contribute to this immunodeficiency

Ligation of Na, K ATPase β3 subunit on monocytes by a specific monoclonal antibody mediates T cell hypofunction

<div><p>T cells play a crucial role in orchestrating body immune responses. T cell hyperfunction, however, leads to inflammation and induction of autoimmune diseases. Understanding of T cell regulation mechanisms and successful modulation of T cell responses is beneficial in treatment of disease associated to T cell hyperresponsiveness. Our previous study indicated that monoclonal antibody (mAb) P-3E10, a mAb to Na, K ATPase β3 subunit, inhibited anti-CD3-induced PBMC proliferation. In the current study, we further investigated the mechanism of mAb P-3E10 in the induction of T cell hypofunction. We demonstrated that mAb P-3E10 decreased T cell proliferation and Th1, Th2 and Th17 cytokine production. Monocytes were the cells playing a key role in mediation of mAb P-3E10 induced T cell hypofunction. The inhibition of T cell activation by mAb P-3E10 required cell contact between monocytes and T cells. The mAb P-3E10 induced the down-expression level of MHC class II and CD86 and increased IL-6, IL-10 and TNF-α production of monocytes. We concluded that ligation of the Na, K ATPase β3 subunit on monocytes by mAb P-3E10 arbitrated T cell hypofunction. This mAb might be a promising novel immunotherapeutic antibody for the treatment of hyperresponsive T cell associated diseases.</p></div

Ligation of Na, K ATPase β3 subunit on monocytes downregulates MHC class II and CD86 expressions and upregulates IL-6, IL-10 and TNF-α production.

<p>(A) PBMCs were stimulated with anti-CD3 mAb in the absence (medium control) or presence of mAb P-3E10 or isotype-matched control mAb. The surface expression levels of MHC class I (HLA-ABC) (<i>n</i> = 4), MHC class II (HLA-DR) (<i>n</i> = 3) and CD86 (<i>n</i> = 4) on CD14⁺ monocytes were determined by flow cytometry. The relative geometric mean fluorescence intensity (GeoMFI of specific marker mAb staining/GeoMFI of isotype-matched control mAb staining) was normalized to medium control as 1. Unpaired t-test was used in the comparison. *P<0.05. **P<0.01. (B) PBMCs were stimulated with anti-CD3 mAb or kept unstimulated in the absence (medium control) or presence of mAb P-3E10 or isotype-matched control mAb. The % IL-6, % IL-10 and % TNF-α producing CD14⁺ monocytes in the indicated conditions are shown as the representative result from one of the four individuals. (C) The data were analyzed as the percentage of cytokine positive cells (% specific cytokine staining—% isotype-matched control staining). The percentage of cytokine positive cells were normalized to isotype-matched mAb treated control of each activation condition as 100%. The data are expressed as mean ± SE (<i>n</i> = 4). Unpaired t-test was performed. *P<0.05. ***P<0.001. The flow cytometric data were illustrated in the Supporting Information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199717#pone.0199717.s004" target="_blank">S4 Fig</a>).</p

The inhibition of T cell activation by mAb P-3E10.

<p>(A) PBMCs were activated with anti-CD3 mAb OKT3 or kept unstimulated in the presence of mAb P-3E10 or mAb 13M (isotype-matched control mAb) or medium alone. Flow cytometric data were expressed in histograms showing the percentage of divided cells in each condition using CFSE proliferation assay. (B) The percentage of divided cells upon OKT3-induced PBMC proliferation in the presence of indicated concentrations of mAb P-3E10 or isotype-matched control mAb is shown. Each individual data was normalized relative to its medium control as 100%. Unpaired <i>t</i>-test was used for comparison, *<i>P</i><0.05. ***<i>P</i><0.001. (C) After stimulation as was described in (A), CD3⁺ T cells were gated. The percentage of the indicated cytokine producing T cells was determined and is shown as mean ± SE from three individuals. The data were exhibited as the percentage of cytokine positive cells by normalization to medium control of OKT3-stimulated condition as 100%. Two-way ANOVA followed by Tukey’s test was used for comparison, *<i>P</i><0.05. **<i>P</i><0.01. ***<i>P</i><0.001. The flow cytometric data were illustrated in the Supporting Information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199717#pone.0199717.s002" target="_blank">S2 Fig</a>).</p