Valeria Leuci

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.

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 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.

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.

Immunotherapy of cancer stem cells in solid tumors: initial findings and future prospective

Introduction: Conventional chemotherapies seemed to have reached a therapeutic plateau in the treatment of solid tumors and many metastatic diseases are still incurable. Events of chemo-resistance and relapses appear to be sustained by a subset of putative cancer stem cells (CSCs). New anticancer strategies need to face this new challenge exploring their efficacy against CSCs. Immunotherapy has raised enthusiasms in cancer therapy and its potential against CSCs is an intriguing field of research. Areas covered: In this work we reviewed the immunotherapy approaches directed against CSCs in solid tumors. We schematically divided adaptive immunotherapy strategies, mainly based on dendritic cell-vaccination, and strategies exploiting MHC-unrestricted effectors like natural killer cells, γδ T lymphocytes and cytokine-induced killer cells. Findings, strength and limitations of these models are discussed and compared highlighting their potential clinical relevance. Expert opinion: The important biologic role and clinical relevance of CSCs introduced a ‘noble target’ for immunotherapy and cancer treatments in general. Initial evidences suggest that CSCs may be susceptible to various types of immunotherapy attacks, overcoming their chemo-resistance. Investigation of important safety issues, based on shared features with ‘normal’ stem cells, along with intriguing synergisms with modulatory agents are open challenges for the next future and effective clinical translation.

Efficient Transcriptional Targeting of Human Hematopoietic Stem Cells and Blood Cell Lineages by Lentiviral Vectors Containing the Regulatory Element of the Wiskott-Aldrich Syndrome Gene†‡§

The ability to effectively transduce human hematopoietic stem cells (HSCs) and to ensure adequate but “physiological” levels of transgene expression in different hematopoietic lineages represents some primary features of a gene‐transfer vector. The ability to carry, integrate, and efficiently sustain transgene expression in HSCs strongly depends on the vector. We have constructed lentiviral vectors (LV) containing fragments of different lengths of the hematopoietic‐specific regulatory element of the Wiskott‐Aldrich syndrome (WAS) gene—spanning approximately 1,600 and 170 bp—that direct enhanced green fluorescent protein (EGFP) expression. The performance of vectors carrying the 1,600 and 170 bp fragments of the WAS gene promoter was compared with that of a vector carrying the UbiquitinC promoter in human cord blood CD34+ cells and their differentiated progeny both in vitro and in vivo in non‐obese diabetic mice with severe combined immunodeficiency. All vectors displayed a similar transduction efficiency in CD34+ cells and promoted long‐term EGFP expression in different hematopoietic lineages, with an efficiency comparable to, and in some instances (for example, the 170‐bp promoter) superior to, that of the UbiquitinC promoter. Our results clearly demonstrate that LV containing fragments of the WAS gene promoter/enhancer region can promote long‐term transgene expression in different hematopoietic lineages in vitro and in vivo and represent suitable and highly efficient vectors for gene transfer in gene‐therapy applications for different hematological diseases and for research purposes. In particular, the 170‐bp carrying vector, for its reduced size, could significantly improve the transduction/expression of large‐size genes. STEM CELLS 2009;27:2815–2823

Cytokine-Induced Killer Cells Kill Chemo-surviving Melanoma Cancer Stem Cells

Purpose: The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSC) was explored, as CSCs are considered responsible for chemoresistance and relapses. Experimental Design: Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter oct4. Their stemness potential was confirmed in vivo by limiting dilution assays. We explored the sensitivity of eGFP+ mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence, in vitro. First, we treated MCs in vitro with fotemustine or dabrafenib (BRAF-mutated cases); then, surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed in vivo in two distinct immunodeficient murine models. Results: We visualized eGFP+ mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP+ MCs (1/42) compared with the eGFP− counterpart (1/4,870). In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells (n = 11, 8/11 autologous), with specific lysis ranging from 95% [effector:tumor ratio (E:T), 40:1] to 20% (E:T 1:3). In vivo infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP+ mCSCs (P = 0.001). Sequential CHT–immunotherapy treatment retained antitumor activity (n = 12, P = 0.001) reducing mCSC rates (P = 0.01). Conclusions: These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving CHT or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided. Clin Cancer Res; 23(9); 2277–88. ©2016 AACR.

Adoptive immunotherapy against sarcomas.

Introduction: Conventional treatments reached an unsatisfactory therapeutic plateau in the treatment of advanced unresectable bone and soft tissue sarcomas that remain an unsolved medical need. Several evidences support the concept that adoptive immunotherapy may effectively integrate within the complex and multidisciplinary treatment of sarcomas. Areas covered: In this work we reviewed adoptive immunotherapy strategies that have been explored in sarcoma settings, with specific focus on issues related to their clinic transferability. We schematically divided approaches based on T lymphocytes specific for MHC-restricted tumor-associated antigens or relying on MHC-independent immune effectors such as natural killer (NK), cytokine-induced killer (CIK) or γδ T cells. Expert opinion: Preclinical findings and initial clinical reports showed the potentialities and drawbacks of different adoptive immunotherapy strategies. The expansion of tumor infiltrating lymphocytes is difficult to be reproduced outside melanoma. Genetically redirected T cells appear to be a promising option and initial reports are encouraging against patients with sarcomas. Adoptive immunotherapy with MHC-unrestricted effectors such as NK, CIK or γδ T cells has recently shown great preclinical potential in sarcoma setting and biologic features that may favor clinical transferability. Combination of different immunotherapy approaches and integration with conventional treatments appear to be key issues for successful designing of next clinical trials.

BRAF and MEK inhibitors increase PD1-positive melanoma cells leading to a potential lymphocyte-independent synergism with anti-PD1 antibody

Purpose: BRAF and MEK inhibitors (BRAF/MEKi) favor melanoma-infiltrating lymphocytes, providing the rationale for current combinatorial trials with anti–PD-1 antibody. A portion of melanoma cells may express PD-1, and anti–PD-1 antibody could have a direct antitumor effect. Here, we explore whether BRAF/MEKi modulate rates of PD-1+ melanoma cells, supporting an additional—lymphocyte-independent—basis for their therapeutic combination with anti–PD-1 antibody. Experimental Design: With data mining and flow cytometry, we assessed PD-1, PD-L1/2 expression on melanoma cell lines (CCLE, N = 61; validation cell lines, N = 7) and melanoma tumors (TCGA, N = 214). We explored in vitro how BRAF/MEKi affect rates of PD-1+, PD-L1/2+ melanoma cells, and characterized the proliferative and putative stemness features of PD-1+ melanoma cells. We tested the functional lymphocyte-independent effect of anti–PD-1 antibody alone and in combination with BRAF/MEKi in vitro and in an in vivo immunodeficient murine model. Results: PD-1 is consistently expressed on a small subset of melanoma cells, but PD-1+ cells increase to relevant rates during BRAF/MEKi treatment [7.3% (5.6–14.2) vs. 1.5% (0.7–3.2), P = 0.0156; N = 7], together with PD-L2+ melanoma cells [8.5% (0.0–63.0) vs. 1.5% (0.2–43.3), P = 0.0312; N = 7]. PD-1+ cells proliferate less than PD-1− cells (avg. 65% less; t = 7 days) and are preferentially endowed with stemness features. In vivo, the direct anti-melanoma activity of PD-1 blockage as monotherapy was negligible, but its association with BRAF/MEKi significantly delayed the development of drug resistance and tumor relapse. Conclusions: BRAF/MEKi increase the rates of PD-1+ melanoma cells that may sustain tumor relapse, providing a lymphocyte-independent rationale to explore combinatory strategies with anti–PD-1 antibody. Clin Cancer Res; 24(14); 3377–85. ©2018 AACR.

Cytokine Induced Killer cells effectively kill chemo-resistant melanoma cancer stem cells

Background Metastatic Melanoma (mMel) is considered refractory to conventional chemotherapies. New molecular targeted approaches, inhibiting mutated forms of the serinethreonine kinase B-RAF, significantly increased the response rate but patients almost invariably relapse and prognosis remains severe [1,2]. Open challenge is the characterization and targeting of cancer stem cells (CSCs), considered responsible for chemoresistance and disease relapse. Adoptive immunotherapy holds great promises for the treatment of mMel and research efforts are ongoing to explore its potential activity against melanoma CSCs (mCSCs). Cytokine Induced Killer (CIK) cells are a subset of ex vivo expanded T lymphocytes with mixed CD3CD56 phenotype and endowed with HLA-unrestricted tumor killing activity. We and others recently reported the preclinical activity of CIK cells against several solid tumors including mMel [3]. Aim of our research is to explore the preclinical activity of CIK cells against autologous mCSCs surviving treatments with chemo or molecular targeted therapies.