Chronic delivery of antibody fragments using immunoisolated cell implants as a passive vaccination tool

Background: Monoclonal antibodies and antibody fragments are powerful biotherapeutics for various debilitating diseases. However, high production costs, functional limitations such as inadequate pharmacokinetics and tissue accessibility are the current principal disadvantages for broadening their use in clinic. Methodology and Principal Findings: We report a novel method for the long-term delivery of antibody fragments. We designed an allogenous immunoisolated implant consisting of polymer encapsulated myoblasts engineered to chronically release scFv antibodies targeted against the N-terminus of the Aβ peptide. Following a 6-month intracerebral therapy we observed a significant reduction of the production and aggregation of the Aβ peptide in the APP23 transgenic mouse model of Alzheimer's disease. In addition, functional assessment showed prevention of behavioral deficits related to anxiety and memory traits. Conclusions and Significance : The chronic local release of antibodies using immunoisolated polymer cell implants represents an alternative passive vaccination strategy in Alzheimer's disease. This novel technique could potentially benefit other diseases presently treated by local and systemic antibody administration

Chronic Delivery of Antibody Fragments Using Immunoisolated Cell Implants as a Passive Vaccination Tool

BACKGROUND: Monoclonal antibodies and antibody fragments are powerful biotherapeutics for various debilitating diseases. However, high production costs, functional limitations such as inadequate pharmacokinetics and tissue accessibility are the current principal disadvantages for broadening their use in clinic. METHODOLOGY AND PRINCIPAL FINDINGS: We report a novel method for the long-term delivery of antibody fragments. We designed an allogenous immunoisolated implant consisting of polymer encapsulated myoblasts engineered to chronically release scFv antibodies targeted against the N-terminus of the Aβ peptide. Following a 6-month intracerebral therapy we observed a significant reduction of the production and aggregation of the Aβ peptide in the APP23 transgenic mouse model of Alzheimer's disease. In addition, functional assessment showed prevention of behavioral deficits related to anxiety and memory traits. CONCLUSIONS AND SIGNIFICANCE: The chronic local release of antibodies using immunoisolated polymer cell implants represents an alternative passive vaccination strategy in Alzheimer's disease. This novel technique could potentially benefit other diseases presently treated by local and systemic antibody administration

Immunoisolated cell implants for intracerebral delivery of anti-Aß single chain Fv antibodies in the APP23 Alzheimer's mouse model

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the elderly population. AD currently affects approximately 27 million people worldwide. A pathological hallmark of AD is the accumulation and deposition of the amyloid beta (Aβ) peptide in neuritic plaques and cerebral blood vessels in the brain. The amyloid cascade hypothesis postulates that any treatment reducing the production of Aβ or facilitating its clearance is likely to prevent or delay cognitive decline. Passive immunization with anti-Aβ antibodies constitutes a promising therapeutic approach for the treatment of AD. Injected anti-Aβ antibodies can slow cognitive decline and decrease Aβ burden. However, Fc-induced responses and high costs of the treatment remain a concern for its wide use in AD patients. Here we report the use of a novel passive immunotherapy approach based on the intracerebral implantation of encapsulated myoblasts genetically engineered to chronically release single-chain Fv antibody fragments (scFv) targeted to the EFRH N-terminus peptide of Aβ, adjacent to the BACE1 secretase cleavage site. Implantation of permeable capsules containing scFV-secreting myoblasts into the brain prevented the production and aggregation of Aβ in the APP23 transgenic AD mouse model, by binding to soluble Aβ species, and interfering with the BACE1 cleavage of huAPP. Functional effects six-month post-implantation in mice showed significant behavioral recovery in anxiety and memory traits. 1H MRS spectroscopy indicated that reduced levels of phosphocholine in the hippocampus following scFv-capsule therapy were preventing neuronal cell membrane disruption. The present work demonstrates the principle of cell encapsulation technology for in situ chronic delivery of antibodies in the CNS and its potential application for AD, and opens the possibility for its use in peripheral antibody delivery. This novel technique could also benefit the various diseases presently treated by antibody administration

Treatment of malignant pleural mesothelioma by fibroblast activation protein-specific re-directed T cells

INTRODUCTION: Malignant pleural mesothelioma (MPM) is an incurable malignant disease, which results from chronic exposition to asbestos in at least 70% of the cases. Fibroblast activation protein (FAP) is predominantly expressed on the surface of reactive tumor-associated fibroblasts as well as on particular cancer types. Because of its expression on the cell surface, FAP is an attractive target for adoptive T cell therapy. T cells can be re-directed by retroviral transfer of chimeric antigen receptors (CAR) against tumor-associated antigens (TAA) and therefore represent a therapeutic strategy of adoptive immunotherapy. METHODS: To evaluate FAP expression immunohistochemistry was performed in tumor tissue from MPM patients. CD8+ human T cells were retrovirally transduced with an anti-FAP-F19-∆CD28/CD3ζ-CAR. T cell function was evaluated in vitro by cytokine release and cytotoxicity assays. In vivo function was tested with an intraperitoneal xenograft tumor model in immunodeficient mice. RESULTS: FAP was found to be expressed in all subtypes of MPM. Additionally, FAP expression was evaluated in healthy adult tissue samples and was only detected in specific areas in the pancreas, the placenta and very weakly for cervix and uterus. Expression of the anti-FAP-F19-∆CD28/CD3ζ-CAR in CD8+ T cells resulted in antigen-specific IFNγ release. Additionally, FAP-specific re-directed T cells lysed FAP positive mesothelioma cells and inflammatory fibroblasts in an antigen-specific manner in vitro. Furthermore, FAP-specific re-directed T cells inhibited the growth of FAP positive human tumor cells in the peritoneal cavity of mice and significantly prolonged survival of mice. CONCLUSION: FAP re-directed CD8+ T cells showed antigen-specific functionality in vitro and in vivo. Furthermore, FAP expression was verified in all MPM histotypes. Therefore, our data support performing a phase I clinical trial in which MPM patients are treated with adoptively transferred FAP-specific re-directed T cells

Inhibiting HLA-B27 homodimer-driven immune cell inflammation in spondylarthritis

OBJECTIVE: Spondylarthritides (SpA), including ankylosing spondylitis (AS), are common inflammatory rheumatic diseases that are strongly associated with positivity for the HLA class I allotype B27. HLA-B27 normally forms complexes with β(2) -microglobulin (β(2) m) and peptide to form heterotrimers. However, an unusual characteristic of HLA-B27 is its ability to form β(2) m-free heavy chain homodimers (HLA-B27(2) ), which, unlike classic HLA-B27, bind to killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). Binding of HLA-B27(2) to KIR-3DL2-positive CD4+ T and natural killer (NK) cells stimulates cell survival and modulates cytokine production. This study was undertaken to produce an antibody to HLA-B27(2) in order to confirm its expression in SpA and to inhibit its proinflammatory properties. METHODS: We generated monoclonal antibodies by screening a human phage display library positively against B27(2) and negatively against B27 heterotrimers. Specificity was tested by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) assay, and fluorescence-activated cell sorting (FACS) analysis of B27(2) -expressing cell lines and peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with SpA. Functional inhibition of KIR-3DL2-B27(2) interactions was tested using cell lines and PBMCs from patients with SpA. RESULTS: Monoclonal antibody HD6 specifically recognized recombinant HLA-B27(2) by ELISA and by SPR assay. HD6 bound to cell lines expressing B27(2) . FACS revealed binding of HD6 to PBMCs and SFMCs from patients with AS but not from controls. HD6 inhibited both the binding of HLA-B27(2) to KIR-3DL2 and the survival and proliferation of KIR-3DL2-positive NK cells. Finally, HD6 inhibited production of the proinflammatory disease-associated cytokine interleukin-17 by PBMCs from patients with AS. CONCLUSION: These results demonstrate that antibody HD6 has potential for use in both the investigation and the treatment of AS and other B27-associated spondylarthritides

Pro-inflammatory cytokines increase cell-surface B27₂ homodimers.

<p><b>(A-E)</b> Cell cultures of leukocyte populations were analyzed by flow cytometry for presence of cell-surface B27_<b>2</b> homodimers after stimulation with cytokines. HD6-biotinylated and streptavidin APC were used for detection. Mean fluorescence intensity (MFI) values were plotted from data analysis. Statistical analysis was plotted against “without cytokines” data values. Values are expressed as mean±SEM. *p<0.05, **p<0.01, ***p<0.005 as determined by one-way ANOVA followed by Bonferroni post-hoc analysis.</p

HD5 binds specifically to cell-surface B27₂ homodimers expressed in human .220 B cell lines.

<p>Representative flow cytometry analysis of HD5 and control antibodies (IgG1 Isotype, W6/32, ME1, HC10, and HD6) binding to LBL721.220 cells either untransfected (.220), or transfected with <i>HLA–B7</i>, <i>B27</i>.<i>C67S</i> (<i>HLA-B27</i> with a mutation of Cys-67 to serine that abrogates cell-surface B27_<b>2</b> expression), <i>B27</i>.<i>hutpn</i> (<i>HLA-B27</i> together with transfection of human tapasin, that reduces B27_<b>2</b> expression and increases HLA-B27 heterotrimer formation), or <i>HLA-B27</i> (<i>HLA-B27</i> transfected cells expressing high levels of cell-surface B27_<b>2</b>). RU = responsive units. ELISA values are expressed as mean±SEM.</p

Detection of cell-surface B27₂ in leukocyte populations of <i>HLA-B27</i> transgenic rats at different ages.

<p><b>(A)</b> Representative flow cytometry analysis of cell-surface B27_<b>2</b> expression in leukocytes populations of Tg rats at different ages (6, 15, 23 and 30 weeks) from spleen and MLNs. WT leukocytes represent the control population where B27_<b>2</b> is absent. <b>(B)</b> MFI values plotted of positive B27_<b>2</b> stains from splenocytes. <b>(C)</b> MFI values plotted of positive B27_<b>2</b> stains from MLNs. Detection of cell-surface B27_<b>2</b> homodimers was performed using HD6-biotinylated and detected by streptavidin-APC. HD6 had been previously assessed as an antibody capable of recognizing cell-surface B27_<b>2</b> in human [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130811#pone.0130811.ref012" target="_blank">12</a>] and rat [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130811#pone.0130811.ref041" target="_blank">41</a>] leukocyte populations. Antibody panels: CD4+ T-cells (+CD3, +CD4), CD8+ T-cells (+CD3, +CD8), NK (+CD161a,—lineage), B cells (+CD45RA,-lineage) and Monocytes (+CD172a,—RP-1).</p

HD5 mAb blocks interaction of B27₂ to leukocytes and modulates activation of CD4+ T-cells by B27₂.

<p><b>(A)</b> B27_<b>2</b> (1x)-tetramers were shown to bind to CD4+ T-cells, B-cells (CD45RA,-lineage) and NK (CD161a,-lineage) populations. Pre-incubation of HD5 or HC10 with B27_<b>2</b>(1x)-tetramers partially blocked interactions with cells when compared to control antibodies (W6/32 and isotype). <b>(B)</b> Tg and WT CD4+ T-cells produce TNF after incubation with B27_<b>2</b> (1x)-tetramers. Pre-incubation of HD5 or HC10 with B27_<b>2</b> (1x)-tetramers inhibited the interaction to CD4+ T-cells and the production of TNF. <b>(C)</b> Tg and WT CD4+ T-cells produce TNF after incubation with .220 B27cells expressing B27_<b>2</b> dimers, but not with .220 and .220 B7 cells. Pre-incubation of HD5 or HC10 with .220 B27 cells blocked the interaction of B27_<b>2</b> to CD4+ T-cells and the production of TNF. Representative flow cytometry plots belong to leukocytes populations of Tg rats. Tests were performed in triplicates. Tet = tetramer. Values are expressed as mean±SEM. *p<0.05, **p<0.01, as determined by one-way ANOVA followed by Bonferroni post-hoc analysis.</p