Human Anti-CCR4 Minibody Gene Transfer for the Treatment of Cutaneous T-Cell Lymphoma

Background: Although several therapeutic options have become available for patients with Cutaneous T-cell Lymphoma (CTCL), no therapy has been curative. Recent studies have demonstrated that CTCL cells overexpress the CC chemokine receptor 4 (CCR4). Methodology/Principal Findings In this study, a xenograft model of CTCL was established and a recombinant adeno-associated viral serotype 8 (AAV8) vector expressing a humanized single-chain variable fragment (scFv)-Fc fusion (scFvFc or “minibody”) of anti-CCR4 monoclonal antibody (mAb) h1567 was evaluated for curative treatment. Human CCR4+ tumor-bearing mice treated once with intravenous infusion of AAV8 virions encoding the h1567 (AAV8-h1567) minibody showed anti-tumor activity in vivo and increased survival. The AAV8-h1567 minibody notably increased the number of tumor-infiltrating Ly-6G+ FcγRIIIa(CD16A)+ murine neutrophils in the tumor xenografts over that of AAV8-control minibody treated mice. Furthermore, in CCR4+ tumor-bearing mice co-treated with AAV8-h1567 minibody and infused with human peripheral blood mononuclear cells (PBMCs), marked tumor infiltration of human CD16A+ CD56+ NK cells was observed. The h1567 minibody also induced in vitro ADCC activity through both mouse neutrophils and human NK cells. Conclusions/Significance: Overall, our data demonstrate that the in vivo anti-tumor activity of h1567 minibody is mediated, at least in part, through CD16A+ immune effector cell ADCC mechanisms. These data further demonstrate the utility of the AAV-minibody gene transfer system in the rapid evaluation of candidate anti-tumor mAbs and the potency of h1567 as a potential novel therapy for CTCL

PBMC-mediated antitumor activity of the AAV8-derived h1567 minibody in a xenograft SCID-BEIGE mouse model.

<p>(<b>a</b>) Growth in tumor volume was quantified by caliper measurements. Tumor progression was significantly inhibited in the AAV8-h1567-treated group compared with the AAV8-11A control group. Mice were given a single intravenous injection of AAV vectors 11 days after inoculation of the tumor cells, which was followed by a single injection of PBMC on day 18. *P<0.05; **P<0.01. (<b>b</b>) Tumor growth was monitored <i>in vivo</i> by optical imaging and quantified weekly by bioluminescent imaging. *P<0.05; **P<0.01. (<b>c</b>) Sequential <i>in vivo</i> imaging of tumor growth over time in the tumor mouse model. Panels depict a representative mouse from each group. (<b>d</b>) Micro-CT/PET fusion images of representative mice 28 days after tumor inoculation. Representative coronal (left), sagittal (right), and transverse sections (below) are shown for both controls and treated mice. Arrows indicate tumor location. FDG PET revealed a decrease in glucose metabolism in AAV8-h1567-treated mice. Data shown are mean values ± SD.</p

AAV vector construction and the expression of human mAbs in AAV-transduced mice.

<p>(<b>a</b>) Schematic representation of AAV-single chain variable region antibody (scFv) - human IgG1 Fc fusion (scFvFc) or “minibody” construct. Human mAb 11A (control) and “humanized” h1567 genes encoding the V domains of heavy (VH) and light (VL) chains were cloned between the AAV internal terminal repeats (ITRs) contained in vector pTRUF and expressed as a minibody protein. (<b>b</b>) <i>In vivo</i> transduction with AAV8-h1567-scFvFc and AAV8-11A-scFvFc in SCID-BEIGE mice after administering 2×10¹¹ vg (viral genome) units per mouse by a single intravenous (i.v.) tail vein injection and in a final volume of 150 ul PBS. Serum levels were measured over time by human IgG ELISA. (<b>c</b>) SDS-PAGE confirming the molecular weight and disulfide-bond integrity of the 11A and h1567 minibodies. (<b>d</b>) Western blotting analysis of the monomer and dimer forms of the 11A and h1567 minibodies using an anti-human IgG1-Fc antibody and processed under reducing and non-reducing conditions. (c & d) minibody proteins recovered from <i>in vitro</i> culture (left) and serum following <i>in vivo</i> transduction (right) are shown. (<b>e</b>) Binding specificity of the AAV8-derived h1567 minibody. The specific binding of h1567 scFv-Fc in serum was shown using CCR4-positive cell lines, Mac-1 and 293T-CCR4 by flow cytometry. An equivalent concentration of the control 11A minibody did not show any binding. 293T cells serve as CCR4 negative control cells.</p

ADCC activity of h1567 minibody in a xenograft human PBMC-SCID/BEIGE mouse model.

<p>(<b>a</b>) Immunohistochemical staining of a representative tumor section with mAb directed against human NK cell surface marker CD56. The immunostaining shows highly positive CD56 tumor-infiltrating human NK cells (brown stain) in tumor from the SCID/BEIGE mice treated with AAV8-h1567 and hPBMCs (upper panel). Negative CD56 staining was seen in the tumor treated with control vector AAV8-11A plus hPBMCs (lower panel). Images are shown from whole tumor cut sections (left panels) and tumor sections at 20× magnifications (right panels). (<b>b</b>) The percentage of immunohistochemically detected tumor-infiltrating natural killer cells was plotted. A significantly higher percentage of tumor-infiltrating human CD56-positive cells were detected in the AAV8-h1567-treated mice group. **, p<0.01. (<b>c</b>) NK cell-mediated cytotoxicity was observed in a dose-dependent manner. Minibody concentrations from 0.0001 to 0.1 ug/ml were tested at an E:T ratio of 2∶1. The average and error bars (mean + SD) shown were calculated from triplicate wells of one experiment. The figures shown are representative of three independent experiments. *P<0.05, **P<0.01 when comparing h1567 minibody-treated and 11A control minibody-treated group. All data is shown as the mean ± SD.</p

Anti-tumor effect of AAV8-derived h1567 minibody.

<p>(<b>a</b>) The tumor volume of each individual tumor plotted as a function of times (days post inoculation). AAV vectors were delivered intravenously by tail vein injection 7 days after the inoculation of 2.5×10⁶ Mac-1 tumor cells. **P<0.01, ***P<0.0005 when comparing tumor mass in AAV8-h1567-treated and control vector AAV8-11A-treated group on Day 18 and Day 21, respectively. (<b>b</b>) Survival analysis of AAV8-h1567 or control AAV8-11A-treated tumor-bearing mice (engrafted with 2.5×10⁶ Mac-1 tumor cells). Tumor-bearing PBS-treated mice were used as background controls. Statistically significant difference was observed between 1567 minibody-treated group and control groups (P<0.01). (<b>c</b>) Immunohistochemical analysis of representative tumor sections with anti-Ly-6G, a specific mAb recognizing murine neutrophils. The immunostaining shows tumor-infiltrating neutrophils (brown stain) in tumor from the SCID-BEIGE mice 21 days after administration of AAV8-h1567 encoding anti-CCR4 minibody (upper-left for entire tumor section and center-panel for magnified section). No staining was seen in the tumor from the mice treated with control vector AAV8-11A (lower-left and right panel). (<b>d</b>) Quantification of neutrophil infiltration from panel C. Entire tumor sections were captured using the Aperio ImageScope instrument, and the percentage of positively stained cells were quantitated by using a color deconvolution algorithm. *P<0.05. (<b>e</b>) <i>In vitro</i> ADCC activity against Mac-1 cells in the presence of h1567 minibody. The ADCC activity was assessed using purified SCID-BEIGE neutrophils as effector cells and CCR4+ Mac-1 cells as target cells. Neutrophil-mediated lysis of target cells was induced at an E:T ratio of 80∶1 in the presence of 50 ug/ml purified h1567 minobodies. The figure shown is representative of three independent experiments. *P<0.05, **P<0.01, ***P<0.0005. All data are represented as the mean ± SD.</p