The IL-15-Based ALT-803 Complex Enhances FcγRIIIa-Triggered NK Cell Responses and In Vivo Clearance of B Cell Lymphomas.

PURPOSE: Anti-CD20 monoclonal antibodies (mAb) are an important immunotherapy for B-cell lymphoma, and provide evidence that the immune system may be harnessed as an effective lymphoma treatment approach. ALT-803 is a superagonist IL-15 mutant and IL-15Rα-Fc fusion complex that activates the IL-15 receptor constitutively expressed on natural killer (NK) cells. We hypothesized that ALT-803 would enhance anti-CD20 mAb-directed NK-cell responses and antibody-dependent cellular cytotoxicity (ADCC). EXPERIMENTAL DESIGN: We tested this hypothesis by adding ALT-803 immunostimulation to anti-CD20 mAb triggering of NK cells in vitro and in vivo. Cell lines and primary human lymphoma cells were utilized as targets for primary human NK cells. Two complementary in vivo mouse models were used, which included human NK-cell xenografts in NOD/SCID-γc (-/-) mice. RESULTS: We demonstrate that short-term ALT-803 stimulation significantly increased degranulation, IFNγ production, and ADCC by human NK cells against B-cell lymphoma cell lines or primary follicular lymphoma cells. ALT-803 augmented cytotoxicity and the expression of granzyme B and perforin, providing one potential mechanism for this enhanced functionality. Moreover, in two distinct in vivo B-cell lymphoma models, the addition of ALT-803 to anti-CD20 mAb therapy resulted in significantly reduced tumor cell burden and increased survival. Long-term ALT-803 stimulation of human NK cells induced proliferation and NK-cell subset changes with preserved ADCC. CONCLUSIONS: ALT-803 represents a novel immunostimulatory drug that enhances NK-cell antilymphoma responses in vitro and in vivo, thereby supporting the clinical investigation of ALT-803 plus anti-CD20 mAbs in patients with indolent B-cell lymphoma.

Antagonism of inhibitor of apoptosis proteins increases bone metastasis via unexpected osteoclast activation.

UNLABELLED: Inhibitor of apoptosis (IAP) proteins play a central role in many types of cancer, and IAP antagonists are in development as anticancer agents. IAP antagonists cause apoptosis in many cells, but they also activate alternative NF-κB signaling through NF-κB-inducing kinase (NIK), which regulates osteoclasts. In bone metastasis, a positive feedback loop between tumors and osteoclasts promotes tumor growth and osteolysis. We therefore tested the effect of IAP antagonists on the bone microenvironment for metastasis. In both drug-sensitive and drug-resistant tumors, growth in bone was favored, as compared with other sites during IAP antagonist treatment. These drugs also caused osteoporosis and increased osteoclastogenesis, mediated by NIK, and enhanced tumor-associated osteolysis. Cotreatment with zoledronic acid, a potent osteoclast inhibitor, reduced IAP antagonist-enhanced tumor growth in bone and osteolysis. Thus, IAP antagonist-based cancer treatment may be compromised by osteoporosis and enhanced skeletal metastasis, which may be prevented by antiresorptive agents. SIGNIFICANCE: Although IAP antagonists are a class of anticancer agents with proven efficacy in multiple cancers, we show that these agents can paradoxically increase tumor growth and metastasis in the bone by stabilizing NIK and activating the alternative NF-κB pathway in osteoclasts. Future clinical trials of IAP antagonist-based therapy may require detailed examination of this potential for enhanced bone metastasis and osteoporosis, as well as possible combination with antiresorptive agents.

Interleukin 12 stimulates IFN-gamma-mediated inhibition of tumor-induced regulatory T-cell proliferation and enhances tumor clearance.

To define the factors that modulate regulatory T (Treg) cells in the tumor setting, we cocultured various tumor cells with either purified Treg cells, or with unfractionated splenocytes. We found that Treg expansion occurred only with unfractionated splenocytes, suggesting that accessory cells and/or factors produced by them play an essential role in tumor-induced Treg expansion. We performed gene expression profiling on tumor-associated Treg cells to identify candidate signaling molecules and studied their effects on tumor-induced Treg expansion. We inadvertently discovered that interleukin (IL)-12 treatment blocked Treg expansion in an IL-12 receptor-dependent fashion. Additional studies showed that IL-12 acts by stimulating IFN-gamma mediated inhibition of Treg cell proliferation, which may partially account for the antitumor effects of IL-12. Furthermore, IL-12 treatment was found to decrease IL-2 production, which may lead to IFN-gamma-independent inhibition of Treg cells, as IL-2 is required for their survival and expansion. Mechanistic studies revealed that IFN-gamma signaling directly causes cell cycle arrest in Treg cells. This study shows that an IL-12-IFN-gamma axis can suppress tumor-induced Treg proliferation. This mechanism may counteract the ability of Treg cells to promote tumor growth in vivo.

Granzyme B and perforin are important for regulatory T cell-mediated suppression of tumor clearance.

Granzyme B is important for the ability of NK cells and CD8(+) T cells to kill their targets. However, we showed here that granzyme B-deficient mice clear both allogeneic and syngeneic tumor cell lines more efficiently than do wild-type (WT) mice. To determine whether regulatory T (Treg) cells utilize granzyme B to suppress immune responses against these tumors, we examined the expression and function of granzyme B in Treg cells. Granzyme B was not expressed in naive Treg cells but was highly expressed in 5%-30% of CD4(+)Foxp3(+) Treg cells in the tumor environment. Adoptive transfer of WT Treg cells, but not granzyme B- or perforin-deficient Treg cells, into granzyme B-deficient mice partially restored susceptibility to tumor growth; Treg cells derived from the tumor environment could induce NK and CD8(+) T cell death in a granzyme B- and perforin-dependent fashion. Granzyme B and perforin are therefore relevant for Treg cell-mediated suppression of tumor clearance in vivo.