Jason C. Steel

Interleukin-15 biology and its therapeutic implications in cancer

Cancer immunotherapy is designed to stimulate the immune system to reject and destroy tumors. Recently, interleukin-15 (IL-15), a member of the four α-helix bundle family of cytokines, has emerged as a candidate immunomodulator for the treatment of cancer. IL-15 acts through its specific receptor, IL-15Rα, which is expressed on antigen-presenting dendritic cells, monocytes and macrophages. IL-15 exhibits broad activity and induces the differentiation and proliferation of T, B and natural killer (NK) cells. It also enhances the cytolytic activity of CD8(+) T cells and induces long-lasting antigen-experienced CD8(+)CD44(hi) memory T cells. IL-15 stimulates differentiation and immunoglobulin synthesis by B cells and induces maturation of dendritic cells. It does not stimulate immunosuppressive T regulatory cells (Tregs). Thus, boosting IL-15 activity could enhance innate and specific immunity and fight tumors. Here we review aspects of IL-15 biology that make it a promising agent for anticancer therapy. We also discuss preclinical models in which IL-15 has demonstrated antitumor activity and highlight ongoing clinical trials of IL-15 in patients with cancer and HIV infection.

Simultaneous Blockade of Multiple Immune System Inhibitory Checkpoints Enhances Antitumor Activity Mediated by Interleukin-15 in a Murine Metastatic Colon Carcinoma Model

Purpose: Interleukin 15 (IL-15) is a promising cytokine for immunotherapy of cancer due to its ability to stimulate the immunity of natural killer, B, and T cells. Its effectiveness, however, may be limited by inhibitory checkpoints and pathways that can attenuate immune responses. Finding strategies to abrogate these negative regulators and enhance the efficacy of IL-15 is a critical challenge. Experimental Design: In a preclinical study, we evaluated IL-15 combined with antibodies to block the negative immune regulators cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death ligand 1 (PD-L1) in a metastatic murine CT26 colon carcinoma model. Results: IL-15 treatment resulted in a significant prolongation of survival in mice with metastatic tumor. Administration of IL-15, however, also increased expression of PD-1 on the surface of CD8+ T cells including CD8+CD44high memory phenotype T cells. Moreover, IL-15 also increased the secretion of the immunosuppressive cytokine, IL-10. Combining IL-15 with anti-PD-L1 and anti-CTLA-4 (multiple immune checkpoint blockade) exhibited greater CTL killing and IFNγ secretion. Moreover, this combination resulted in a significant reduction in surface expression of PD-1 on CD8+ T cells, a decrease in IL-10 secretion, and led to significantly longer survival of tumor-bearing animals compared with mice treated with IL-15 alone or combined singularly with anti-PD-L1 or anti-CTLA-4. Conclusions: Combining the immune stimulatory properties of IL-15 with the simultaneous removal of 2 critical immune system inhibitory checkpoints, we showed enhancement of immune responses leading to increased antitumor activity. Clin Cancer Res; 16(24); 6019–28. ©2010 AACR.

Simultaneous inhibition of two regulatory T-cell subsets enhanced Interleukin-15 efficacy in a prostate tumor model

IL-15 has potential as an immunotherapeutic agent for cancer treatment because of its ability to effectively stimulate CD8 T cell, natural killer T cell, and natural killer cell immunity. However, its effectiveness may be limited by negative immunological checkpoints that attenuate immune responses. Recently a clinical trial of IL-15 in cancer immunotherapy was initiated. Finding strategies to conquer negative regulators and enhance efficacy of IL-15 is critical and meaningful for such clinical trials. In a preclinical study, we evaluated IL-15 combined with antibodies to block negative immune regulator cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death ligand 1 (PD-L1) in an established murine transgenic adenocarcinoma of mouse prostate (TRAMP)-C2 prostate tumor model. IL-15 treatment resulted in a significant prolongation of survival in tumor-bearing animals. Coadministration of anti-PD-L1 or anti-CTLA-4 singly with IL-15 did not improve animal survival over that of IL-15 alone. However, simultaneous administration of IL-15 with anti-CTLA-4 and anti-PD-L1 was associated with increased numbers of tumor antigen-specific tetramer-positive CD8 T cells, increased CD8 T-cell tumor lytic activity, augmented antigen-specific IFN-γ release, decreased rates of tumor growth, and improved animal survival compared with IL-15 alone. Furthermore, triple combination therapy was associated with inhibition of suppressive functions of CD4+CD25+ regulatory T cells and CD8+CD122+ regulatory T cells. Thus, simultaneous blockade of CTLA-4 and PD-L1 protected CD4 and/or CD8 T-cell activity from these regulatory T cells. Combining the immune stimulatory properties of IL-15 with simultaneous removal of two critical immune inhibitory checkpoints, we showed enhancement of immune responses, leading to increased antitumor activity.

Epithelial-to-mesenchymal plasticity of cancer stem cells: therapeutic targets in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) remains one of the most common and lethal malignancies worldwide despite the development of various therapeutic strategies. A better understanding of the mechanisms responsible for HCC initiation and progression is essential for the development of more effective therapies. The cancer stem cell (CSC) model has provided new insights into the development and progression of HCC. CSCs are specialized tumor cells that are capable of self-renewal and have long-term repopulation potential. As they are important mediators of tumor proliferation, invasion, metastasis, therapy resistance, and cancer relapse, the selective targeting of this crucial population of cells has the potential to improve HCC patient outcomes and survival. In recent years, the role of epithelial-to-mesenchymal transition (EMT) in the advancement of HCC has gained increasing attention. This multi-step reprograming process resulting in a phenotype switch from an epithelial to a mesenchymal cellular state has been closely associated with the acquisition of stem cell-like attributes in tumors. Moreover, CSC mediates tumor metastasis by maintaining plasticity to transition between epithelial or mesenchymal states. Therefore, understanding the molecular mechanisms of the reprograming switches that determine the progression through EMT and generation of CSC is essential for developing clinically relevant drug targets. This review provides an overview of the proposed roles of CSC in HCC and discusses recent results supporting the emerging role of EMT in facilitating hepatic CSC plasticity. In particular, we discuss how these important new insights may facilitate rational development of combining CSC- and EMT-targeted therapies in the future.

Therapy of Advanced Established Murine Breast Cancer with a Recombinant Adenoviral ErbB-2/neu Vaccine

ErbB-2 (HER-2/neu) is a transforming oncogene expressed by a substantial fraction of breast cancers, and monoclonal antibody therapy directed toward this antigen is an established treatment modality. However, not all tumors respond, and with a monoclonal antibody directed to a single epitope, there is always the risk of tumor escape. Furthermore, passive antibody therapy requires continual treatment. Whereas cancer vaccines have prevented the growth of tumors, it has been far more difficult to treat large established tumors. Here, we show that vaccination with a recombinant adenovirus expressing a truncated ErbB-2 antigen can cure large established subcutaneous ErbB-2-expressing breast cancers in mice, and can also cure extensive established lung metastatic disease. We also show that the mechanism of protection involves antibody-mediated blockade of ErbB-2 function, independent of Fc receptors. We conclude that a vaccine inducing antibodies to a functional oncogenic receptor could have tremendous therapeutic potential against cancers overexpressing such molecules.

Sphere Culture of Murine Lung Cancer Cell Lines Are Enriched with Cancer Initiating Cells

Cancer initiating cells (CICs) represent a unique cell population essential for the maintenance and growth of tumors. Most in vivo studies of CICs utilize human tumor xenografts in immunodeficient mice. These models provide limited information on the interaction of CICs with the host immune system and are of limited value in assessing therapies targeting CICs, especially immune-based therapies. To assess this, a syngeneic cancer model is needed. We examined the sphere-forming capacity of thirteen murine lung cancer cell lines and identified TC-1 and a metastatic subclone of Lewis lung carcinoma (HM-LLC) as cell lines that readily formed and maintained spheres over multiple passages. TC-1 tumorspheres were not enriched for expression of CD133 or CD44, putative CIC markers, nor did they demonstrate Hoechst 33342 side population staining or Aldefluor activity compared to adherent TC-1 cells. However, in tumorsphere culture, these cells exhibited self-renewal and long-term symmetric division capacity and expressed more Oct-4 compared to adherent cells. HM-LLC sphere-derived cells exhibited increased Oct-4, CD133, and CD44 expression, demonstrated a Hoechst 33342 side population and Aldefluor activity compared to adherent cells or a low metastatic subclone of LLC (LM-LLC). In syngeneic mice, HM-LLC sphere-derived cells required fewer cells to initiate tumorigenesis compared to adherent or LM-LLC cells. Similarly TC-1 sphere-derived cells were more tumorigenic than adherent cells in syngeneic mice. In contrast, in immunocompromised mice, less than 500 sphere or adherent TC-1 cells and less than 1,000 sphere or adherent LLC cells were required to initiate a tumor. We suggest that no single phenotypic marker can identify CICs in murine lung cancer cell lines. Tumorsphere culture may provide an alternative approach to identify and enrich for murine lung CICs. Furthermore, we propose that assessing tumorigenicity of murine lung CICs in syngeneic mice better models the interaction of CICs with the host immune system.

Induction of allogeneic mixed chimerism by immature dendritic cells and bone marrow transplantation leads to prolonged tolerance to major histocompatibility complex disparate allografts

Mixed chimerism has been shown to lead to prolonged major histocompatibility complex (MHC) disparate allograft survival and immune‐specific tolerance; however, traditional conditioning regimes often involve myeloablation, which may pose a significant safety risk. In this study we examined the use of donor C57BL/6 (H‐2b) immature dendritic cells (imDCs) to tolerize the BALB/c (H‐2d) recipient to bone marrow transplantation (BMT), allowing the induction of mixed chimerism without immunosuppression or myeloablation. We showed that successful mismatched bone marrow engraftment can be achieved using imDCs given up to 3 days prior to BMT and that mixed chimerism can be established and detected in excess of 100 days post‐BMT without evidence of graft‐versus‐host disease. Furthermore, we showed that imDCs can suppress lymphocyte proliferation in response to mismatched MHC stimulation, leading to increased expression of interleukin (IL)‐4 and IL‐10 and decreased expression of IL‐2 and interferon‐γ (IFN‐γ). The induction of stable chimeras through pre‐conditioning of mice with donor imDCs followed by BMT led to tolerance, allowing the long‐term survival (> 110 days) of mismatched cardiac allografts and the prolonged survival of mismatched skin allografts without the need for immunosuppression or myeloablation. Transplantation with third‐party C3H allografts were rapidly rejected in this model, suggesting that immune‐specific tolerance was achieved. The induction of immune‐specific tolerance without the need for immunosuppression or myeloablation represents a significant advance in transplant immunology and may provide clinicians with a plausible alternative in combating organ rejection following transplantation.

Microsphere‐liposome complexes protect adenoviral vectors from neutralising antibody without losses in transfection efficiency, in‐vitro

Adenoviral vectors have been commonly used in gene therapy protocols but the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral‐specific neutralising antibodies are produced, which limits further administration. This study examines the effectiveness of a novel combination of microspheres and liposomes for the shielding of adenovirus from neutralising antibodies in an in‐vitro setting. We show that liposomes are effective in the protection of adenovirus from neutralising antibody and that the conjugation of these complexes to microspheres augments the level of protection. This study further reveals that previously neutralised adenovirus may still be transported into the cell via liposome‐cell interactions and is still capable of expressing its genes, making this vector an effective tool for circumvention of the humoral immune response. We also looked at possible side effects of using the complexes, namely increases in cytotoxicity and reductions in transfection efficiency. Our results showed that varying the liposome:adenovirus ratio can reduce the cytotoxicity of the vector as well as increase the transfection efficiency. In addition, in cell lines that are adenoviral competent, transfection efficiencies on par with uncomplexed adenoviral vectors were achievable with the combination vector.

Vaccination with tumor cells expressing IL-15 and IL-15Rα inhibits murine breast and prostate cancer

A number of antitumor vaccines have recently shown promise in upregulating immune responses against tumor antigens and improving patient survival. In this study, we examine the effectiveness of vaccination using interleukin (IL)-15-expressing tumor cells and also examine their ability to upregulate immune responses to tumor antigens. We demonstrated that the coexpression of IL-15 with its receptor, IL-15Rα, increased the cell-surface expression and secretion of IL-15. We show that a gene transfer approach using recombinant adenovirus to express IL-15 and IL-15Rα in murine TRAMP-C2 prostate or TS/A breast tumors induced antitumor immune responses. From this, we developed a vaccine platform, consisting of TRAMP-C2 prostate cancer cells or TS/A breast cancer cells coexpressing IL-15 and IL-15Rα that inhibited tumor formation when mice were challenged with tumor. Inhibition of tumor growth led to improved survival when compared with animals receiving cells expressing IL-15 alone or unmodified tumor cells. Animals vaccinated with tumor cells coexpressing IL-15 and IL-15Rα showed greater tumor infiltration with CD8+ T and natural killer (NK) cells, as well as increased antitumor CD8+ T-cell responses. Vaccination with IL-15/IL-15Rα-modified TS/A breast cancer cells provided a survival advantage to mice challenged with unrelated murine TUBO breast cancer cells, indicating the potential for allogeneic IL-15/IL-15Rα-expressing vaccines.

Lung cancer-initiating cells: a novel target for cancer therapy

Lung cancer is a major public health problem causing more deaths than any other cancer. A better understanding of the biology of this disease and improvements in treatment are greatly needed. Increasing evidence supports the concept that a rare and specialized population of cancer cells, so-called cancer-initiating cells with stem cell-like characteristics, is responsible for tumor growth, maintenance, and recurrence. Cancer-initiating cells also exhibit characteristics that render them resistant to both radiation and chemotherapy, and therefore they are believed to play a role in treatment failure. This has led to the hypothesis that traditional therapies that indiscriminately kill tumor cells will not be as effective as therapies that selectively target cancer-initiating cells. Investigating putative cancer-initiating cells in lung cancer will greatly benefit the understanding of the origins of this disease and may lead to novel approaches to therapy by suggesting markers for use in either further isolating this population for study or for selectively targeting these cells. This review will discuss (1) lung cancer, (2) stem cells, and the role of cancer-initiating cells in tumorigenesis; (3) markers and functional characteristics associated with lung cancer-initiating cells; and (4) the potential to selectively target this subpopulation of tumor cells.