No abstract

Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem-like cells (sCSC) that are likely responsible for relapses and drug resistance. In this study, we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS, including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma. To identify putative sCSCs, autologous bone sarcoma and STS cells were engineered with a CSC detector vector encoding eGFP under the control of the human promoter for OCT4, a stem cell gene activated in putative sCSCs. Using CIK cells expanded from 21 patients, we found that CIK cells efficiently killed allogeneic and autologous sarcoma cells in vitro. Intravenous infusion of CIK cells delayed autologous tumor growth in immunodeficient mice. Further in vivo analyses established that CIK cells could infiltrate tumors and that tumor growth inhibition occurred without an enrichment of sCSCs relative to control-treated animals. These results provide preclinical proof-of-concept for an effective strategy to attack autologous sarcomas, including putative sCSCs, supporting the clinical development of CIK cells as a novel class of immunotherapy for use in settings of untreatable metastatic disease. Cancer Res; 74(1); 119-29. Ó2013 AACR.

Donor-derived cytokine-induced killer (CIK) can be infused as adoptive immunotherapy after hematopoietic cell transplant (HCT). Promising results were recently reported in HLA-identical HCT, where mild grafts versus host (GVH) events were observed. To extend this strategy across major HLA barriers (e.g. HLA-haploidentical HCT), further studies on CIK cells' alloreactivity are needed. We hypothesized that alloreactivity and anti-tumor activity of CIK cells segregate within two different cell subsets and could consequently be separated according to CD56 and CD3 expression. We tested CIK cells expanded from seven patients who underwent HCT as treatment of metastatic colorectal cancer. We found that CIK cells maintained their alloreactivity across major HLA barriers when tested as bulk population; after CD56-positive selection, anti-tumor activity was restricted to the CD3+/CD56+ cell fraction and alloreactivity versus HLA-mismatched PBMC was restricted to the CD3+/CD56- cell fraction. Bulk CIK cells from engrafted patients did not exhibit alloreactivity in response to host- or donor-derived PBMC, confirming their low potential for GVH across minor HLA barriers. Moreover, we tested if CIK cells expanded from engrafted patients after HCT were as effective as donor-derived ones and could be considered as an alternative option. The expansion rate and tumor cell killing was comparable to that observed in sibling donors. In conclusion, depletion of CD3+/CD56- cells might reduce the risk of GVH without affecting the tumor-killing capacity and could help extending CIK infusions across major HLA barriers. Engrafted patients after HCT could also be considered as an effective alternative option to donor-derived CIK cells.

For the first time, the intense killing activity of CIK cells against autologous metastatic melanoma, including mCSCs, has been shown. These findings move clinical investigation of a new immunotherapy for metastatic melanoma, including mCSCs, closer.

We investigated the possibility of introducing exogenous T cell receptor (TCR) genes into T cells by lentiviral transduction, without prior stimulation of endogenous TCR with anti-CD3. TCR transfer is used to impose tumor antigen specificity on recipient T cells, but sustained activation required for retroviral transduction may affect the clinical efficacy of engineered T cells. Cytokine stimulation makes T cells susceptible to lentiviral transduction in the absence of TCR triggering, but this advantage has never been exploited for TCR transfer. Autoimmune diseases are a source of high-affinity TCRs specific for self/tumor antigens. We selected, from a patient with vitiligo, a Mart1-specific TCR based on intrinsic interchain pairing properties and functional avidity. After lentiviral transduction of human peripheral blood mononuclear cells, preferential pairing of exogenous alpha and beta chains was observed, together with effective recognition of Mart1(+) melanoma cells. We tested transduction efficiency on various T cell subsets prestimulated with interleukin (IL)-2, IL-7, IL-15, and IL-21 (alone or in combination). Both naive and unfractionated CD8(+) T cells could be transduced without requiring endogenous TCR triggering. IL-7 plus IL-15 was the most powerful combination, allowing high levels of transgene expression without inducing T cell differentiation (34 +/- 5% Mart1-TCR(+) cells in naive CD8(+) and 16 +/- 6% in unfractionated CD8(+)). Cytokine-prestimulated, Mart1-redirected naive and unfractionated CD8(+) cells expanded better than CD3-CD28-prestimulated counterparts in response to both peptide-pulsed antigen-presenting cells and Mart1(+) melanoma cells. This strategy allows the generation of tumor-specific T cells encompassing truly naive T cells, endowed with an intact proliferative potential and a preserved differentiation stage.

Cytokine-induced killer cells (CIKs) are ex vivo expanded T-NK lymphocytes capable of HLA-unrestricted antitumor activity. CIKs are promising candidates for adoptive cancer immunotherapies; they can be generated and infused in autologous settings of cancer patients, or from donors, after allogeneic hematopoietic cell transplant. Ex vivo expansion rates of CIKs are greatly variable among patients, with consequent potential clinical limitations for "poor expanders." We compared the standard expansion protocol with a new one, which included the timed addition of irradiated allogeneic peripheral blood mononuclear cells. Our hypothesis is that allogeneic stimulation might provide CIK cells with a proliferative boost and simultaneously decrease their alloreactivity versus third parties, if HLA-mismatched from the allogeneic stimulators. Allo-stimulated CIKs (AS-CIK) reached significantly higher expansion rates compared with standard controls, regardless if generated form healthy donors (131- vs. 32-fold) or cancer patients (117- vs. 14-fold). The expansion of the CD3CD56 subset was 2243-fold for AS-CIKs compared with 362 for standard CIKs. AS-CIKs efficiently killed osteosarcoma targets in vitro, results were comparable with that of standard CIKs. Standard and AS-CIKs did not show differences in phenotype and telomere length. The alloreactivity of AS-CIKs against third party HLA-mismatched peripheral blood mononuclear cells was reduced compared with standard CIKs (37% vs. 23%). In conclusion, alloreactivity of CIK cells may be exploited enhancing their final ex vivo expansion. In clinical perspective these findings may facilitate the extension of CIK-based immunotherapy to larger numbers of patients and, translated into hematopoietic cell transplant settings, contribute to reduce the risk of graft versus host disease in the hypothesis of infusions across HLA barriers.

No abstract

Receptor tyrosine kinases (RTKs) inhibitors’ activity in advanced osteosarcoma is significant but short-lived. To prevent or at least delay drug resistance, we explored a vertical inhibition by combining drugs acting at different levels of the RTK pathways (pazopanib + trametinib). We studied pazopanib + trametinib antitumor activity both in vitro and in vivo (MNNG-HOS and KHOS xenografts in NOD/SCID mice) investigating the molecular mechanisms and potential escapes. The involvement of MAPK-PI3K pathways was validated by Nanostring technology, western blot and by silencing/overexpression experiments. Pazopanib targets were expressed on seven osteosarcoma cell lines and their pathways were activated. Pazopanib + trametinib exhibited synergistic antitumor activity by inducing apoptosis and inhibiting ERK1/2 and Akt. In vivo antitumor activity was shown in osteosarcoma-bearing mice. The drug combination significantly down-modulated RTK Ephrin Type-A Receptor 2 (EphA2) and Interleukin-7 Receptor (IL-7R), whereas induced mitogen-activated protein-kinase kinase (MAPKK) MEK6. EphA2 silencing significantly reduced osteosarcoma cell proliferation and migration, while impeding MEK6 up-regulation in the treated cells significantly increased the antitumor effect of the studied drugs. Moreover, the up-regulation of MEK6 reduced combination activity. Pazopanib + trametinib demonstrated synergistic antitumor effects in osteosarcoma models through ERK and Akt inhibition and EphA2 and IL-7R down-modulation. MEK6 up-regulation might evoke escaping mechanism.