Janelle Sharkey

Anti-CD73 antibody therapy inhibits breast tumor growth and metastasis

Extracellular adenosine is a potent immunosuppressor that accumulates during tumor growth. We performed proof-of-concept studies investigating the therapeutic potential and mechanism of action of monoclonal antibody (mAb)-based therapy against CD73, an ecto-enzyme overexpressed on breast-cancer cells that catalyzes the dephosphorylation of adenosine monophosphates into adenosine. We showed that anti-CD73 mAb therapy significantly delayed primary 4T1.2 and E0771 tumor growth in immune-competent mice and significantly inhibited the development of spontaneous 4T1.2 lung metastases. Notably, anti-CD73 mAb therapy was essentially dependent on the induction of adaptive anti-tumor immune responses. Knockdown of CD73 in 4T1.2 tumor cells confirmed the tumor-promoting effects of CD73. In addition to its immunosuppressive effect, CD73 enhanced tumor-cell chemotaxis, suggesting a role for CD73-derived adenosine in tumor metastasis. Accordingly, administration of adenosine-5′-N-ethylcarboxamide to tumor-bearing mice significantly enhanced spontaneous 4T1.2 lung metastasis. Using selective adenosine-receptor antagonists, we showed that activation of A2B adenosine receptors promoted 4T1.2 tumor-cell chemotaxis in vitro and metastasis in vivo. In conclusion, our study identified tumor-derived CD73 as a mechanism of tumor immune escape and tumor metastasis, and it also established the proof of concept that targeted therapy against CD73 can trigger adaptive anti-tumor immunity and inhibit metastasis of breast cancer.

Demonstration of inflammation-induced cancer and cancer immunoediting during primary tumorigenesis

Here we report the effects of loss of the Toll-like receptor-associated signaling adaptor myeloid-differentiation factor 88 (MyD88) on tumor induction in two distinct mouse models of carcinogenesis. The 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA)-induced skin papilloma model depends on proinflammatory processes, whereas the 3′-methylcholanthrene (MCA) induction of fibrosarcoma has been used by tumor immunologists to illustrate innate and adaptive immune surveillance of cancer. When exposed to a combination of DMBA/TPA, mice lacking MyD88 formed fewer skin papillomas than genetically matched WT controls treated in a similar manner. Unexpectedly, however, fewer MyD88−/− mice formed sarcomas than WT controls when exposed to MCA. In contrast, MyD88-deficient mice did not show a defective ability to reject highly immunogenic transplanted tumors, including MCA sarcomas. Despite the reported role of TNF in chronic inflammation, TNF-deficient mice were significantly more susceptible to MCA-induced sarcoma than WT mice. Overall, these data not only confirm the key role that MyD88 plays in promoting tumor development but also demonstrate that inflammation-induced carcinogenesis and cancer immunoediting can indeed occur in the same mouse tumor model.

Combination therapy of established cancer using a histone deacetylase inhibitor and a TRAIL receptor agonist

Histone deacetylase inhibitors (HDACi) and agents such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL receptor (TRAIL-R) antibodies are anticancer agents that have shown promise in preclinical settings and in early phase clinical trials as monotherapies. Although HDACi and activators of the TRAIL pathway have different molecular targets and mechanisms of action, they share the ability to induce tumor cell-selective apoptosis. The ability of HDACi to induce expression of TRAIL-R death receptors 4 and 5 (DR4/DR5), and induce tumor cell death via the intrinsic apoptotic pathway provides a molecular rationale to combine these agents with activators of the TRAIL pathway that activate the alternative (death receptor) apoptotic pathway. Herein, we demonstrate that the HDACi vorinostat synergizes with the mouse DR5-specific monoclonal antibody MD5-1 to induce rapid and robust tumor cell apoptosis in vitro and in vivo. Importantly, using a preclinical mouse breast cancer model, we show that the combination of vorinostat and MD5-1 is safe and induces regression of established tumors, whereas single agent treatment had little or no effect. Functional analyses revealed that rather than mediating enhanced tumor cell apoptosis via the simultaneous activation of the intrinsic and extrinsic apoptotic pathways, vorinostat augmented MD5-1-induced apoptosis concomitant with down-regulation of the intracellular apoptosis inhibitor cellular-FLIP (c-FLIP). These data demonstrate that combination therapies involving HDACi and activators of the TRAIL pathway can be efficacious for the treatment of cancer in experimental mouse models.

Type I natural killer T cells suppress tumors caused by p53 loss in mice.

CD1d-restricted T cells are considered to play a host protective effect in tumor immunity, yet the evidence for a role of natural killer T (NKT) cells in tumor immune surveillance has been weak and data from several tumor models has suggested that some (type II) CD1d-restricted T cells may also suppress some types of antitumor immune response. To substantiate an important role for CD1d-restricted T cells in host response to cancer, we have evaluated tumor development in p53(+/-) mice lacking either type I NKT cells (TCR Jalpha18(-/-)) or all CD1d-restricted T cells (CD1d(-/-)). Our findings support a key role for type I NKT cells in suppressing the onset of sarcomas and hematopoietic cancers caused by p53 loss but do not suggest that other CD1d-restricted T cells are critical in regulating the same tumor development.

Multiple Antitumor Mechanisms Downstream of Prophylactic Regulatory T-Cell Depletion

Several reports have shown that prophylactic depletion of regulatory T cells (Treg) using various monoclonal antibodies (mAb) in mice can stimulate potent antitumor immune responses and prevent tumor development. These same depletion methods do not significantly suppress tumor growth in a therapeutic setting. Although different strategies to deplete FoxP3(+) Treg have been used, no study has systematically compared these qualitatively for the effector mechanisms they each liberate. Herein, using prophylactic depletion of FoxP3(+) Tregs with either anti-CD4, anti-CD25, or anti-FR4 mAbs, we have compared the cellular and effector requirements for elimination of the renal carcinoma RENCA and prevention of methylcholanthrene-induced fibrosarcoma. Collectively from these two models, it was clear that CD8(+) T cells and natural killer cells played an important role downstream of Treg depletion. However, whereas all three mAbs quantitatively depleted FoxP3(+) T cells to a similar extent, subtle differences in the downstream mechanisms of tumor control existed for all three approaches. In general, neutralization of any lymphocyte subset or effector mechanism was insufficient to alter tumor suppression initiated by Treg depletion, and in some settings, the neutralization of multiple effector mechanisms failed to prevent tumor rejection. These studies reveal that Tregs control multiple redundant elements of the immune effector response capable of inhibiting tumor initiation and underscore the importance of effectively targeting these cells in any cancer immunotherapy.

Antibodies targeted to TRAIL receptor-2 and ErbB-2 synergize in vivo and induce an antitumor immune response

Despite the development of human epidermal growth factor receptor-2 (ErbB-2/HER2)-targeted therapies, there remains an unmet medical need for breast cancer patients with ErbB-2 overexpression. We investigated the therapeutic activity of an agonist mAb to mouse tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-2 (DR5) against ErbB2-driven breast cancer. Established tumors in BALB/c transgenic mice expressing a constitutively active ErbB-2/neuT were treated with anti-DR5 mAb and/or anti-ErbB-2 mAb and monitored for tumor progression. Treatment with anti-DR5 or anti-ErbB2 mAb as single agents significantly delayed tumor growth, although all tumors eventually progressed. Remarkably, treatment with a combination of anti-DR5 and anti-ErbB-2 mAbs induced complete response in a majority of mice. In vivo blockade of CD11b+ cells, but not natural killer cell depletion, significantly abrogated the early antitumor response. Notably, depletion of CD8+ T cells provoked primary and secondary tumor relapse, revealing the induction of antitumor immunity by the combination treatment. Combined therapy with anti-DR5 and anti-ErbB-2 mAbs further significantly suppressed the growth of advanced spontaneous tumors in ErbB-2/neuT transgenic mice, even when treatment was delayed until tumors were palpable. We thus demonstrated that the combination of anti-DR5 and anti-ErbB2 mAbs might be an effective form of treatment for ErbB-2-overexpressing breast cancer.

CD1d-Based Combination Therapy Eradicates Established Tumors in Mice

The use of Abs that induce tumor cell death together with immunostimulatory reagents to activate innate and adaptive immune cells has emerged as a potent approach for the treatment of cancer. We have previously demonstrated that the use of three mAbs (anti-DR5, anti-CD40, anti-CD137) termed TriMab can induce rejection in a majority of mice with established experimental or carcinogen-induced tumors. However, given the potential toxicity of CD40 agonists in the clinic, we tested an alternative approach to directly activate/mature APCs using anti-CD1d mAbs. In this study, we used a combination of three mAbs (anti-DR5, anti-CD137, anti-CD1d) that we termed 1DMab and demonstrated that this approach suppressed and/or eradicated established experimental renal, breast, and colon carcinomas in mice. Tumor suppression induced by 1DMab therapy required CD8+ T cells, IFN-γ, and CD1d, while NK cells and IL-12 were partially required. Interestingly 1DMab therapy was more effective than TriMab in tumor models regulated by CD1d-restricted type II NKT cells, but less efficacious against tumors where T regulatory cells were critical. Anti-CD1d mAbs could also be relatively effective in combination with anti-CD137 and conventional chemotherapeutics. This is the first study to illustrate the antitumor activity of CD1d-reactive mAbs in combination and our results strongly suggest that rational combination chemoimmunotherapies based on tumor immunoregulation may improve the efficacy of treatment.

CD1d activation and blockade: a new antitumor strategy

CD1d is expressed on APCs and presents glycolipids to CD1d-restricted NKT cells. For the first time, we demonstrate the ability of anti-CD1d mAbs to inhibit the growth of different CD1d-negative experimental carcinomas in mice. Anti-CD1d mAbs systemically activated CD1d+ APC, as measured by production of IFN-γ and IL-12. Tumor growth inhibition was found to be completely dependent on IFN-γ and IL-12 and variably dependent on CD8+ T cells and NK cells, depending upon the tumor model examined. Anti-CD1d mAb induced greater CD8+ T cell-dependent tumor suppression where regulatory CD1d-restricted type II NKT cells have been implicated, and were less effective in a NK cell-dependent manner against tumors where T regulatory cells were immunosuppressive. The ability of anti-CD1d mAbs to coincidently activate CD1d+ APCs to release IL-12 and inhibit CD1d-restricted type II NKT cells makes CD1d an exciting new target for immunotherapy of cancer based on tumor immunoregulation.

Three agonist antibodies in combination with high-dose IL-2 eradicate orthotopic kidney cancer in mice

BackgroundCombination immunotherapies can be effective against subcutaneous tumors in mice but the effect against orthotopic malignant disease is less well characterized. In particular, a combination of three agonist antibodies, termed Tri-mAb, consisting of anti-DR5, anti-CD40 and anti-CD137 has previously been demonstrated to eradicate a large proportion of subcutaneous renal cell carcinoma (Renca) tumors (75% long-term survival), but the effect against orthotopic disease is not known.PurposeTo determine the relative response of orthotopic tumors, we inoculated Renca into the kidney followed by treatment with Tri-mAb.ResultsWe found that orthotopic tumors responded much less to treatment (~13% survival), but a significant improvement in survival was achieved through the addition of IL-2 to the treatment regimen (55% survival). All three agonist antibodies and high dose IL-2, 100,000 IU for up to six doses, were required. CD8+ T cells were also required for optimal anti-tumor responses. Coadministration of IL-2 led to enhanced T cell activity as demonstrated by an increased frequency of IFN-gamma-producing T cells in tumor-draining lymph nodes, which may have contributed to the observed improvement of therapy against kidney tumors.ImplicationsResponses of subcutaneous tumors to immunotherapy do not necessarily reflect how orthotopic tumors respond. The use of combination immunotherapy stimulating multiple facets of immunity and including cytokine support for T cells can induce effective anti-tumor responses against orthotopic and metastatic tumors.

Interleukin 21 Enhances Antibody-Mediated Tumor Rejection

Interleukin-21 (IL-21) is a cytokine with structural and sequence homology to IL-2 and IL-15 that has antitumor activity alone in mouse experimental tumor models and a tolerable safety profile in phase I trials in patients with metastatic melanoma and renal cell carcinoma. Several monoclonal antibodies (mAb) targeted at tumor-associated antigens also have improved antitumor activities in mice when used in combination with IL-21. Recently, we described a rational three antibody-based approach (triple mAb, TrimAb) to eradicating established mouse tumors that required the generation of tumor-reactive CD8(+) T cells and IFN-gamma. Herein, we show that sequentially combining TrimAb with recombinant IL-21 can significantly improve the antitumor activity of this combination against very advanced disease. These data further support the use of IL-21 in adjuvant settings where strong T cell-mediated immune responses to tumors can be generated.