Dario Sangiolo

Cytokine-induced killer (CIK) cells as feasible and effective adoptive immunotherapy for the treatment of solid tumors

Introduction: Cytokine-induced killer (CIK) cells are heterogeneous ex vivo-expanded T lymphocytes with mixed T-NK phenotype and endowed with a wide MHC-unrestricted antitumor activity. CIK cells can be expanded from peripheral blood mononuclear cells (PBMC) cultured with the timed addition of IFN-γ, Ab anti-CD3 and IL2. A consistent subset of mature CIK cells presents a CD3+CD56+ phenotype. The CD3+CD56+ cellular subset is the main responsible for the tumor-killing activity, mostly mediated by the interaction of NKG2D receptor with MHC-unrestricted ligands (MIC A/B; ULBPs) on tumor cells. Areas covered: In the present work, we described the biologic characteristics of CIK cells, focusing on those aspects that may favor their clinical translation. We reviewed preclinical data and analyzed reports from clinical trials. A specific paragraph is dedicated to future research perspectives in the field. Expert opinion: CIK cells represent a realistic new option in the field of cancer immunotherapy. Crucial issues, favoring their clinical translation, are the easy availability of large amounts of expanded CIK cells and their MHC-unrestricted tumor killing, potentially effective against many tumor types. Intriguing future perspectives and open challenges are the investigation of synergisms with other immunotherapy approaches, targeted therapies or even conventional chemotherapy.

The Combination of Sorafenib and Everolimus Abrogates mTORC1 and mTORC2 Upregulation in Osteosarcoma Preclinical Models

Purpose: The multikinase inhibitor sorafenib displays antitumor activity in preclinical models of osteosarcoma. However, in sorafenib-treated patients with metastatic-relapsed osteosarcoma, disease stabilization and tumor shrinkage were short-lived and drug resistance occurred. We explored the sorafenib treatment escape mechanisms to overcome their drawbacks. Experimental Design: Immunoprecipitation, Western blotting, and immunohistochemistry were used to analyze the mTOR pathway [mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2)]. Cell viability, colony growth, and cell migration were evaluated in different osteosarcoma cell lines (MNNG-HOS, HOS, KHOS/NP, MG63, U-2OS, SJSA-1, and SAOS-2) after scalar dose treatment with sorafenib (10–0.625 μmol/L), rapamycin-analog everolimus (100–6.25 nmol/L), and combinations of the two. Cell cycle, reactive oxygen species (ROS) production, and apoptosis were assessed by flow cytometry. Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice injected with MNNG-HOS cells were used to determine antitumor and antimetastatic effects. Angiogenesis and vascularization were evaluated in vitro by exploiting endothelial branching morphogenesis assays and in vivo in xenografted mice and chorioallantoic membranes. Results: After sorafenib treatment, mTORC1 signaling was reduced (downstream target P-S6), whereas mTORC2 was increased (phospho-mTOR Ser2481) in MNNG-HOS xenografts compared with vehicle-treated mice. Combining sorafenib with everolimus resulted in complete abrogation of both mTORC1 [through ROS-mediated AMP-activated kinase (AMPK) activation] and mTORC2 (through complex disassembly). The sorafenib/everolimus combination yielded: (i) enhanced antiproliferative and proapoptotic effects, (ii) impaired tumor growth, (iii) potentiated antiangiogenesis, and (iv) reduced migratory and metastatic potential. Conclusion: mTORC2 activation is an escape mechanism from sorafenib treatment. When sorafenib is combined with everolimus, its antitumor activity is increased by complete inhibition of the mTOR pathway in the preclinical setting. Clin Cancer Res; 19(8); 2117–31. ©2013 AACR.

Active immunotherapy in HER2 overexpressing breast cancer: current status and future perspectives

BACKGROUND The use of anti-HER2 monoclonal antibodies (mAbs) has improved the clinical outcome of HER2-overexpressing breast cancers (BCs). Unfortunately, often these tumors tend to relapse and, when metastatic, the duration of clinical benefit is limited over time and almost invariably followed by tumor progression. Alternative approaches to this essentially passive immunotherapy are therefore needed in HER2-overexpressing BC patients. As HER2 is one of the most suitable targets for active immunotherapy in BC, manipulating the immune system is a highly attractive approach. MATERIAL AND METHODS A computer-based literature search was carried out using PubMed (keywords: breast neoplasm, HER2 vaccine, immunology); data reported at international meetings were included. RESULTS This review provides a focus on the following active vaccinal approaches under clinical investigation against HER2-overexpressing BC: (i) peptide and protein based; (ii) DNA based; (iii) whole tumor cell based; (iv) dendritic cell based. Moreover, the review discuss future challenges in the field, trying to define the best setting for the development of this innovative strategy, considering both immunological and clinical aspects of HER2 targeting. CONCLUSIONS Development of effective vaccines for BC remains a distinct challenge but is likely to become a substantial advance for patients with HER2-overexpressing BCs.BACKGROUND The use of anti-HER2 monoclonal antibodies (mAbs) has improved the clinical outcome of HER2-overexpressing breast cancers (BCs). Unfortunately, often these tumors tend to relapse and, when metastatic, the duration of clinical benefit is limited over time and almost invariably followed by tumor progression. Alternative approaches to this essentially passive immunotherapy are therefore needed in HER2-overexpressing BC patients. As HER2 is one of the most suitable targets for active immunotherapy in BC, manipulating the immune system is a highly attractive approach. MATERIAL AND METHODS A computer-based literature search was carried out using PubMed (keywords: breast neoplasm, HER2 vaccine, immunology); data reported at international meetings were included. RESULTS This review provides a focus on the following active vaccinal approaches under clinical investigation against HER2-overexpressing BC: (i) peptide and protein based; (ii) DNA based; (iii) whole tumor cell based; (iv) dendritic cell based. Moreover, the review discuss future challenges in the field, trying to define the best setting for the development of this innovative strategy, considering both immunological and clinical aspects of HER2 targeting. CONCLUSIONS Development of effective vaccines for BC remains a distinct challenge but is likely to become a substantial advance for patients with HER2-overexpressing BCs.

Effective Activity of Cytokine-Induced Killer Cells against Autologous Metastatic Melanoma Including Cells with Stemness Features

Purpose: We investigate the unknown tumor-killing activity of cytokine-induced killer (CIK) cells against autologous metastatic melanoma and the elusive subset of putative cancer stem cells (mCSC). Experimental Design: We developed a preclinical autologous model using same patient-generated CIK cells and tumor targets to consider the unique biology of each patient/tumor pairing. In primary tumor cell cultures, we visualized and immunophenotypically defined a putative mCSC subset using a novel gene transfer strategy that exploited their exclusive ability to activate the promoter of stemness gene Oct4. Results: The CIK cells from 10 patients with metastatic melanoma were successfully expanded (median, 23-fold; range, 11–117). Primary tumor cell cultures established and characterized from the same patients were used as autologous targets. Patient-derived CIK cells efficiently killed autologous metastatic melanoma [up to 71% specific killing (n = 26)]. CIK cells were active in vivo against autologous melanoma, resulting in delayed tumor growth, increased necrotic areas, and lymphocyte infiltration at tumor sites. The metastatic melanoma cultures presented an average of 11.5% ± 2.5% putative mCSCs, which was assessed by Oct4 promoter activity and stemness marker expression (Oct4, ABCG2, ALDH, MITF). Expression was confirmed on mCSC target molecules recognized by CIK cells (MIC A/B; ULBPs). CIK tumor killing activity against mCSCs was intense (up to 71%, n = 4) and comparable with results reported against differentiated metastatic melanoma cells (P = 0.8). Conclusions: 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. Clin Cancer Res; 19(16); 4347–58. ©2013 AACR.

Cytokine induced killer cells as adoptive immunotherapy strategy to augment graft versus tumor after hematopoietic cell transplantation

Donor lymphocyte infusion (DLI) is used to increase the graft versus tumor (GVT) effect after allogeneic hematopoietic cell transplant (HCT). The limited spectrum of activity and high risk of graft versus host disease (GVHD) remain major limitations of this approach. The finding of new cell populations for adoptive immunotherapy, with the ability to separate GVT from GVHD, would be useful. Here we review the main basic, preclinical and clinical research on cytokine-induced killer (CIK) cells, highlighting the aspects of their antitumor and alloreactive potentials that might favourably affect the balance between GVT and GVHD. CIK cells are ex vivo-expanded T lymphocytes sharing NK markers and endowed with a potent MHC-unrestricted antitumor activity against haematological and solid malignancies. Studies in preclinical animal models have demonstrated their low GVHD potential when infused across MHC-barriers, and recent clinical studies seem to confirm these findings in patients with hematological malignances relapsing after HCT. If consolidated with larger clinical trials, adoptive immunotherapy with CIK cells might represent an effective alternative to classic DLI, helping HCT to succesfully meet current challenges like the extension across major HLA-barriers and application to solid tumors.

Cytokine-Induced Killer Cells Eradicate Bone and Soft-Tissue Sarcomas

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.

Complete Resolution of Life-Threatening Bleomycin-Induced Pneumonitis After Treatment With Imatinib Mesylate in a Patient With Hodgkin's Lymphoma: Hope for Severe Chemotherapy-Induced Toxicity?

Introduction Bleomycin-induced pneumonitis (BIP) is a scarcely manageable pulmonary toxicity that occurs in approximately 20% of patients who are affected with cancers that are treated with bleomycin-containing regimens. Generally, the clinical picture is extremely complicated and therapy is based on steroids. The overall response rate to such therapy is limited. Recent insights in BIP pathogenesis have indicated that a key feature is deregulated mechanisms of tissue repair that are driven by profibrotic cytokines. Here we describe a patient with lifethreatening BIP who was completely cured with imatinib mesylate (IM) after steroids and all other therapies had failed.

T Cell Receptor (TCR) Gene Transfer with Lentiviral Vectors Allows Efficient Redirection of Tumor Specificity in Naive and Memory T Cells Without Prior Stimulation of Endogenous TCR

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.

PARP1 expression drives the synergistic antitumor activity of trabectedin and PARP1 inhibitors in sarcoma preclinical models

BackgroundEnhancing the antitumor activity of the DNA-damaging drugs is an attractive strategy to improve current treatment options. Trabectedin is an isoquinoline alkylating agent with a peculiar mechanism of action. It binds to minor groove of DNA inducing single- and double-strand-breaks. These kinds of damage lead to the activation of PARP1, a first-line enzyme in DNA-damage response pathways. We hypothesized that PARP1 targeting could perpetuate trabectedin-induced DNA damage in tumor cells leading finally to cell death.MethodsWe investigated trabectedin and PARP1 inhibitor synergism in several tumor histotypes both in vitro and in vivo (subcutaneous and orthotopic tumor xenografts in mice). We searched for key determinants of drug synergism by comparative genomic hybridization (aCGH) and gene expression profiling (GEP) and validated their functional role.ResultsTrabectedin activated PARP1 enzyme and the combination with PARP1 inhibitors potentiated DNA damage, cell cycle arrest at G2/M checkpoint and apoptosis, if compared to single agents. Olaparib was the most active PARP1 inhibitor to combine with trabectedin and we confirmed the antitumor and antimetastatic activity of trabectedin/olaparib combination in mice models. However, we observed different degree of trabectedin/olaparib synergism among different cell lines. Namely, in DMR leiomyosarcoma models the combination was significantly more active than single agents, while in SJSA-1 osteosarcoma models no further advantage was obtained if compared to trabectedin alone. aCGH and GEP revealed that key components of DNA-repair pathways were involved in trabectedin/olaparib synergism. In particular, PARP1 expression dictated the degree of the synergism. Indeed, trabectedin/olaparib synergism was increased after PARP1 overexpression and reduced after PARP1 silencing.ConclusionsPARP1 inhibition potentiated trabectedin activity in a PARP1-dependent manner and PARP1 expression in tumor cells might be a useful predictive biomarker that deserves clinical evaluation.

Genetically redirected T lymphocytes for adoptive immunotherapy of solid tumors.

Genetic engineering of T lymphocytes to confer new antitumor specificities is a fascinating approach that may help the successful clinical translation of adoptive immunotherapy strategies. The recognition of tumor-specific antigens may be obtained inducing the membrane expression of transgene encoded antitumor T cell receptors (TCR) or chimeric antigen receptors (CAR). Few but very informative clinical trials with TCR or CAR redirected T lymphocytes have been attempted in the last years, reporting important clinical results along with disappointing failures and important warnings. In this work, we will focus on TCR and CAR redirected T lymphocytes as adoptive immunotherapy for solid tumors. We will review the main topics of these strategies from the angle of clinical applications, discussing the main issues that emerged from early clinical trials and their impact on next study designs.