Efficient Inhibition of Human B-cell Lymphoma in SCID Mice by Synergistic Antitumor Effect of Human 4-1BB Ligand/Anti-CD20 Fusion Proteins and Anti-CD3/Anti-CD20 Diabodies

Here we constructed and produced a recombinant human 4-1BB ligand (4-1BBL)/anti-CD20 fusion protein and examined its antitumor activity, alone and in combination with an anti-CD3/anti-CD20 bispecific diabody. The 4-1BBL/anti-CD20 fusion protein retained both the costimulatory activity of 4-1BBL on T cells and the tumor targeting ability of CD20 antibody on B cells. The fusion protein bound as efficiently to 4-1BB-and CD20-positive cells as its respective parental antibodies, and was capable of cross-linking human T lymphocytes and CD20-positive tumor cells. Combination treatment with 4-1BBL/anti-CD20 fusion protein and anti-CD3/anti-CD20 diabody led to significantly increased T-cell cytotoxicity to human B-lymphoma cells in vitro and drastically more potent tumor inhibitory activity in vivo in xenografted B-cell lymphoma in severe combined immunodeficiency disease mice. Mechanistic studies revealed that the combination treatment remarkably inhibited apoptosis of human peripheral blood lymphocytes, accompanied by upregulation of Bcl-X-L and Bf1-1, perforin and granzyme B mRNA, and increased interleukin-2 production. Taken together, these results suggest that targeted delivery of 4-1BBL to the tumor site, when combined with anti-CD3/anti-CD20 diabody, could strongly potentiate the antitumor activity of the diabody, thus may have significant clinical application in the treatment of human CD20-positive B-cell malignancies

Masson's trichrome analysis of collagen accumulation.

<p>In the control group and the saline-treated group, the collagen fibers are thick, dense and disorganized. In tissues that had been treated with Rg3 (1, 2, 3 and 4 mg/mL) for 28 days, the collagen fibers were arranged more regularly and were more sparse than those in the control group. Original magnification: 400×.</p

The clinical appearance of the wounds and scars.

<p>The top views, side views and full views of the scars that were left untreated and those that had been treated with different concentrations of Rg3 (0, 1, 2, 3 and 4 mg/mL). The wounds that had received injections of Rg3 (1, 2, 3 and 4 mg/mL) are obviously flattened and less reddish than the control and the placebo groups.</p

Scar Elevation Index (SEI) and Epidermal Thickness Index (ETI).

<p>(A) Dermal hypertrophy was measured by SEI. SEI in the Rg3 injection groups was compared with the control group and the saline injection group. SEI 1 depicts a hypertrophic scar. (B) Epidermis thickness was measured by ETI. ETI in the Rg3 injection groups was compared with the control group and the saline injection group. ETI >1 depicts a hypertrophic epidermis. (* indicates significant difference, p<0.05, data represent the mean ± SD of n = 9 wounds.)</p

Effect of different concentrations of Rg3 on HSF apoptosis 48 h after treatment.

<p>Percentage of apoptotic cells was measured by flow cytometry after annexin-V/propidium iodide (PI) staining. Quandrant 1 indicates live cells; quadrant2 indicates cells in early apoptosis; quadrant 3 indicates cells in late apoptosis; quadrant 4 indicates cells in necrosis.</p

The effect of 20(S)-Rg3 on hypertrophic scar fibroblast viability.

<p>Data are expressed as the mean ± SD of triplicate experiments. * <i>p<0.05</i> is considered to be significantly different compared to the control, as determined by the two-tailed Student's t-test.</p

The expression of VEGF mRNA in scars on post-wounding Day 28.

<p>mRNA expressions of VEGF of the scars treated with Rg3 (1, 2, 3 and 4 mg/mL) were significantly down-regulated, in contrast to the control group and the saline-treated group. * <i>p<0.05</i>. Data represent the mean ± SD, for N = 9.</p

Immunohistochemical staining for Col-I on day 28.

<p>(A). Areas of collagen deposition in Immunohistochemical staining histological sections (400× magnification). (B) The relative collagen density. Data represent the mean ± SD for n = 9 (* indicates significant difference, p<0.05).</p