R. macrophages. 2B8T2M activates natural killer cells to enhance antibody-dependent cellular cytotoxicity, mediates complement-dependent cytotoxicity, and induces apoptosis of B-lymphoma cells. Compared with rituximab, 2B8T2M exhibits significantly stronger antitumor activity in a xenograft SCID mouse model and depletes B cells in cynomolgus monkeys more efficiently. Thus, ALT-803 can be modified as a functional scaffold for creating multispecific, targeted IL-15-based immunotherapeutic agents and may serve as a novel platform to improve the antitumor activity and clinical efficacy of therapeutic antibodies. the IL-15N72DIL-15RSuFc complex) could also function as a protein scaffold to create multispecific IL-15-based targeted immunotherapeutic agents. To test this, we converted the variable regions of the heavy and light chains of rituximab into an scFv (sc2B8) (9) and genetically fused sc2B8 to the N termini of IL-15N72D and IL-15RSuFc proteins of ALT-803. Based on the high binding affinity between the IL-15N72D and IL-15RSu domains, we expected the fusion proteins to form a heterodimeric complex between sc2B8-IL-15N72D and sc2B8-IL-15RSuFc. In addition, sc2B8-IL-15RSuFc was expected to form a covalent dimer using the disulfide bonds provided by the Fc domain. Therefore, this novel fusion protein complex (designated 2B8T2M) was predicted to consist of two sc2B8-IL-15N72D and two sc2B8-IL-15RSuFc proteins (Fig. 1milli absorption units. 2B8T2M Retains Rabbit Polyclonal to NOTCH2 (Cleaved-Val1697) CD20-binding, Fc Receptor-binding, and IL-15 Biological Activities To verify the CD20-binding properties, FITC-labeled 2B8T2M and rituximab were generated and used to stain human HLA-DR+ B cells. Our results indicate that human B cells were able to bind FITC-labeled rituximab (Fig. 2apoptotic activity) (10). In contrast, type II antibodies do not stabilize CD20 in lipid rafts and thus exhibit reduced CDC compared with Bis-PEG4-acid type I antibodies, but these antibodies potently induce lysosomal cell death. Rituximab is a type I anti-CD20 mAb that exhibits higher CDC activity but a lower ability to induce apoptosis of B-lymphoma cells than type II anti-CD20 mAbs such as tositumomab (11). 2B8T2M has the same binding domain as rituximab and is predicted to have similar properties. To investigate this, we first assessed the ability of 2B8T2M to mediate CDC against CD20+ Daudi cells. As shown in Fig. 3250 nm) was required to induce comparable apoptotic activity against Daudi cells. This activity was also observed with Fc-mutant 2B8T2M-LA and IL-15-mutant 2B8T2M-D8N complexes (Fig. 3= 3). and = 5) or purified NK cells, CD4+ T cells Bis-PEG4-acid or CD8+ T cells (= 2 donors) were used as effector cells. The effector cells were plated with violet-labeled target cells at the indicated effector:target (E:T) ratios with rituximab or 2B8T2M at the indicated concentrations. Target cell viability was assessed on day 2 for PBMCs (< 0.01 (10 nm) and < 0.05 (1 nm) compared with 2B8T2M. Values represent the mean S.E. and and and and compared with IL-15 (4). To evaluate the immunostimulatory activity of 2B8T2M in comparison with ALT-803, IL-15, and other fusion protein complexes < 0.001) but significantly less CD8+ T cells than the ALT-803 treatment group (< 0.001). Mice that received 2B8T2M also showed a larger percentage of NK cells in the spleen compared with IL-15- or PBS-treated mice (< 0.001, Fig. 6= 5 or 6/group). On day 2 post-transfer, 2B8T2M (5 mg/kg), 2B8T2M-LA (5 mg/kg), 2B8T2M-D8N (5 mg/kg), IL-15 (0.056 mg/kg), and Bis-PEG4-acid PBS were injected i.v. and > 0.05, IL-15 Bis-PEG4-acid 2B8T2M-D8N and 2B8T2M 2B8T2M-LA; < 0.01 among other groups. Data represent the mean S.D. We further examined the proliferation of donor CD8+ T cells and NK cells in spleens of recipient mice. As shown in Fig. 6, and antitumor activities of 2B8T2M and rituximab, we employed the Daudi B-lymphoma/SCID mouse model. Daudi cells (1 107) were injected i.v. into female SCID mice, and 15 days post-inoculation, the presence of tumor cells in the bone marrow was verified by flow cytometry using PE-conjugated anti-human HLA-DR antibody (two mice showed 0.5% and 2.8% Daudi cells in the bone marrow). The remaining Daudi-bearing mice were randomized into three groups and treated on days 15 and 18 with rituximab at 10 mg/kg (equivalent to a clinical dose of 375 mg/m2 for non-Hodgkin's lymphoma (NHL) patients), 2B8T2M at 5 mg/kg, or PBS as a vehicle control. Hind leg paralysis was used as survival end point for Bis-PEG4-acid this study. As shown in Fig. 7= 0.001), 2B8T2M treatment further prolonged survival relative to rituximab (= 0.006). Open in a separate window FIGURE 7. Prolonged survival of tumor-bearing mice treated with 2B8T2M and efficacy of 2B8T2M antitumor activity. = 6) and treated.