Publications by authors named "Giulia Mesiano"

19 Publications

  • Page 1 of 1

CSPG4-Specific CAR.CIK Lymphocytes as a Novel Therapy for the Treatment of Multiple Soft-Tissue Sarcoma Histotypes.

Clin Cancer Res 2020 Dec 8;26(23):6321-6334. Epub 2020 Sep 8.

Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Turin, Italy.

Purpose: No effective therapy is available for unresectable soft-tissue sarcomas (STS). This unmet clinical need prompted us to test whether chondroitin sulfate proteoglycan 4 (CSPG4)-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CAR.CIK) are effective in eliminating tumor cells derived from multiple STS histotypes and in immunodeficient mice.

Experimental Design: The experimental platform included patient-derived CAR.CIK and cell lines established from multiple STS histotypes. CAR.CIK were transduced with a retroviral vector encoding second-generation CSPG4-specific CAR (CSPG4-CAR) with 4-1BB costimulation. The functional activity of CSPG4-CAR.CIK was explored , in two- and three-dimensional STS cultures, and in three STS xenograft models.

Results: CSPG4-CAR.CIK were efficiently generated from patients with STS. CSPG4 was highly expressed in multiple STS histotypes by analysis and on all 16 STS cell lines tested by flow cytometry. CSPG4-CAR.CIK displayed superior cytolytic activity against multiple STS histotypes as compared with paired unmodified control CIK. CSPG4-CAR.CIK also showed strong antitumor activity against STS spheroids; this effect was associated with tumor recruitment, infiltration, and matrix penetration. CSPG4-CAR.CIK significantly delayed or reversed tumor growth in three STS xenograft models (leiomyosarcoma, undifferentiated pleomorphic sarcoma, and fibrosarcoma). Tumor growth inhibition persisted for up to 2 weeks following the last administration of CSPG4-CAR.CIK.

Conclusions: This study has shown that CSPG4-CAR.CIK effectively targets multiple STS histotypes and in immunodeficient mice. These results provide a strong rationale to translate the novel strategy we have developed into a clinical setting.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-0357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710537PMC
December 2020

Cytokine-Induced Killer (CIK) Cells, In Vitro Expanded under Good Manufacturing Process (GMP) Conditions, Remain Stable over Time after Cryopreservation.

Pharmaceuticals (Basel) 2020 May 12;13(5). Epub 2020 May 12.

Department of Public Health and Paediatrics, The University of Turin, piazza Polonia 94, 10126 Torino, Italy.

Cytokine-induced killer (CIK) cells are advanced therapy medicinal products, so their production and freezing process has to be validated before their clinical use, to verify their stability as a drug formulation according to the good manufacturing practice (GMP) guidelines. We designed a stability program for our GMP-manufactured CIK cells, evaluating the viability, identity and potency of cryopreserved CIK cells at varying time periods from freezing, and compared them with fresh CIK cells. We evaluated the effects of the cryopreservation method, transportation, and the length of time of different process phases (pre-freezing, freezing and post-thawing) on the stability of CIK cells. This included a worst case for each stage. The expanded CIK cells were viable for up to 30 min from the addition of the freezing solution, when transported on dry ice within 48 h once frozen, within 60 min from thawing and from 12 months of freezing while preserving their cytotoxic effects. The reference samples, cryopreserved simultaneously in tubes and following the same method, were considered representative of the batch and useful in the case of further analysis. Data obtained from this drug stability program can inform the accurate use of CIK cells in clinical settings.
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http://dx.doi.org/10.3390/ph13050093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281026PMC
May 2020

Cytokine Induced Killer cells are effective against sarcoma cancer stem cells spared by chemotherapy and target therapy.

Oncoimmunology 2018;7(11):e1465161. Epub 2018 Aug 6.

Medical Oncology Division, Candiolo Cancer Institute, FPO-IRCCS. Str. Prov. 142, km 3.95, I-10060, Candiolo (To), Italy.

Metastatic bone and soft tissue sarcomas often relapse after chemotherapy (CHT) and molecular targeted therapy (mTT), maintaining a severe prognosis. A subset of sarcoma cancer stem cells (sCSC) is hypothesized to resist conventional drugs and sustain disease relapses. We investigated the immunotherapy activity of cytokine induced killer cells (CIK) against autologous sCSC that survived CHT and mTT. The experimental platform included two aggressive bone and soft tissue sarcoma models: osteosarcoma (OS) and undifferentiated-pleomorphic sarcoma (UPS). To visualize putative sCSC we engineered patient-derived sarcoma cultures (2 OS and 3 UPS) with a lentiviral sCSC-detector wherein the promoter of stem-gene Oct4 controls the expression of eGFP. We visualized a fraction of sCSC (mean 24.2 ± 5.2%) and confirmed their tumorigenicity . sCSC resulted relatively resistant to both CHT and mTT . Therapeutic doses of doxorubicin significantly enriched viable eGFPsCSC in both OS (2.6 fold, n = 16) and UPS (2.3 fold, n = 29) compared to untreated controls. Treatment with sorafenib (for OS) and pazopanib (for UPS) also determined enrichment (1.3 fold) of viable eGFPsCSC, even if less intense than what observed after CHT. Sarcoma cells surviving CHT and mTT were efficiently killed by autologous CIK even at minimal effector/target ratios (40:1 = 82%, 1:4 = 29%, n = 13). CIK immunotherapy did not spare sCSC that were killed as efficiently as whole sarcoma cell population. The relative chemo-resistance of sCSC and sensitivity to CIK immunotherapy was confirmed . Our findings support CIK as an innovative, clinically explorable, approach to eradicate chemo-resistant sCSC implicated in tumor relapse.
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http://dx.doi.org/10.1080/2162402X.2018.1465161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6208452PMC
August 2018

Correction to: Cytokines induced killer cells produced in good manufacturing practices conditions: identification of the most advantageous and safest expansion method in terms of viability, cellular growth and identity.

J Transl Med 2018 10 10;16(1):275. Epub 2018 Oct 10.

City of Health and Science Hospital of Turin, Pediatric Oncoematology, Regina Margherita Children's Hospital, Piazza Polonia 94, 10126, Turin, Italy.

Following publication of the original article [1], the authors reported that all of the authors' names were processed incorrectly so that their given and family names were interchanged. In this Correction the correct author names are shown. The original publication of this article has been corrected.
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http://dx.doi.org/10.1186/s12967-018-1656-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6178249PMC
October 2018

Cytokines induced killer cells produced in good manufacturing practices conditions: identification of the most advantageous and safest expansion method in terms of viability, cellular growth and identity.

J Transl Med 2018 08 29;16(1):237. Epub 2018 Aug 29.

City of Health and Science Hospital of Turin, Pediatric Oncoematology, Regina Margherita Children's Hospital, Piazza Polonia 94, 10126, Turin, Italy.

Background: Cytokine-induced killer (CIK) cells are a very promising cell population raising growing interest in the field of cellular antitumor therapy. The aim of our study was to validate the most advantageous expansion method for this advanced therapy medicinal product (ATMP) and to translate it from preclinical field to good manufacturing practices (GMP). GMP ensures that ATMP are consistently produced and controlled to the quality standards required to their intended use. For this reason, the use of the xenogenic sera tended to be minimized by GMP for their high variability and the associated risk of transmitting infectious agents.

Results: We decided to replace Fetal Bovine Serum (FBS), largely used as medium supplement for CIKs expansion, with other culture media. Firstly, Human Serum (HS) and Human Pool Plasma (HPP) were tested as medium supplements giving not compliant results to acceptance criteria, established for CIKs, probably for the great batch to batch variability. Consequently, we decided to test three different serum free expansion media: X-VIVO 15, (largely used by other groups) and Tex Macs and Cell Genix GMP SCGM: two GMP manufactured media. We performed a validation consisting in three run-sand even if the small number of experiments didn't permit us to obtained statistical results we demonstrated that both X-VIVO 15 and Tex Macs fulfilled the quality standards in terms of cellular growth, viability and identity while Cell Genix GMP SCGM resulted not compliant as it caused some technical problems such as high mortality.

Conclusion: In conclusion, these preclinical validation data lay the bases for a GMP-compliant process to improve the CIKs expansion method.
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http://dx.doi.org/10.1186/s12967-018-1613-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6116438PMC
August 2018

Analytic and Dynamic Secretory Profile of Patient-Derived Cytokine-Induced Killer Cells.

Mol Med 2017 10 9;23:235-246. Epub 2017 Aug 9.

Department of Life Science and Biotechnology, Sections of Microbiology and Applied Pathology; Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy.

Adoptive immunotherapy with Cytokine Induced Killer (CIK) cells has shown antitumor activity against several kinds of cancers in preclinical models and clinical trials. CIK cells are a subset of ex vivo expanded T lymphocytes with T-NK phenotype and MHC-unrestricted antitumor activity. Literature provides scanty information on cytokines, chemokines and growth factors secreted by CIK cells. Therefore, we investigated the secretory profile of CIK cells generated from tumor patients. The secretome analysis was performed at specific time points (day 1, day 14 and day 21) of CIK cells expansion. Mature CIK cells (day 21) produce a great variety of interleukins and secreted proteins that can be divided into 3 groups based on their secretion quantity: high (IL-13, RANTES, MIP-1α and 1β), medium (IL-1Ra, IL-5, IL-8, IL-10, IL-17, IP-10, INF-γ, VEGF and GMCSF) and low (IL-1β, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, Eotaxin, PDGF-bb, FGF basic, G-CSF and MCP-1) secreted. Moreover, comparing PBMC (day 1) and mature CIK cells (day 14 and 21) secretome, we observed that IL-5, IL-10, IL-13, GM-CSF, VEGF resulted greatly up-regulated, while IL-1β, IL-6, IL-8, IL-15, IL-17, eotaxin, MCP-1, and RANTES were down-regulated. We also performed a gene expression profile analysis of patient-derived CIK cells showing that mRNA for the different cytokines and secreted proteins were modulated during PBMC to CIK differentiation. We highlighted previously unknown secretory properties and provided for the first time a comprehensive molecular characterization of CIK cells. Our findings provide rationale to explore the functional implications and possible therapeutic modulation of CIK secretome.
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http://dx.doi.org/10.2119/molmed.2017.00084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630476PMC
October 2017

Specific Monoclonal Antibody Against Bcr/Abl Out-of-Frame Alternative Proteins as Diagnostic Tool in Chronic Myelogenous Leukemia Patients.

Monoclon Antib Immunodiagn Immunother 2017 Aug 13;36(4):149-156. Epub 2017 Jul 13.

2 Department of Medical Biotechnologies and Translational Medicine, School of Medicine, University of Milan , Milan, Italy .

More recently, alternative splicing of specific genes are investigated for their therapeutic potential. In particular, we reported the existence of BCR-ABL alternative splicing isoforms, in about 80% of Philadelphia-positive patients, which lead to the expression of aberrant proteins. These fusion proteins are characterized by an orphan initial and correct Bcr portion attached to a 112 amino acid sequence, arising from the impairment in the reading frame (reading of ABL exon 4 and 5). We demonstrated that these Abl-out-of-frame (OOF) isoforms could have an immunological role with therapeutic implications. The aim of this study was to characterize a new monoclonal antibody (MAb) specific for Abl-OOF protein portion, for diagnostic use, to detect this biomarker in Philadelphia chromosome-positive chronic myelogenous leukemia (CML) patients and to generate novel approaches in the immunotherapy. 5F11G11 MAb recognizes the OOF protein portion of the native full-length Bcr/Abl-OOF protein expressed in cells transiently transfected, as demonstrated by immunoprecipitation and immunofluorescence. In addition, we demonstrate the MAb's ability to recognize the alternative hybrid Bcr/Abl fusion protein expressed in leukemic cells from CML patients, to support the possible use of 5F11G11 MAb as a diagnostic tool to select patients with Philadelphia chromosome-positive CML that could be eligible for an immunotherapeutic approach with this new antigen.
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http://dx.doi.org/10.1089/mab.2016.0054DOI Listing
August 2017

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

Clin Cancer Res 2017 May 4;23(9):2277-2288. Epub 2016 Nov 4.

Division of Medical Oncology, Experimental Cell Therapy, Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Torino, Italy.

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. Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter Their stemness potential was confirmed 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, First, we treated MCs 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 two distinct immunodeficient murine models. We visualized eGFP mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate was higher within eGFP MCs (1/42) compared with the eGFP counterpart (1/4,870). mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells ( = 11, 8/11 autologous), with specific lysis ranging from 95% [effector:tumor ratio (E:T), 40:1] to 20% (E:T 1:3). infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP mCSCs ( = 0.001). Sequential CHT-immunotherapy treatment retained antitumor activity ( = 12, = 0.001) reducing mCSC rates ( = 0.01). 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. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-1524DOI Listing
May 2017

Adoptive immunotherapy against sarcomas.

Expert Opin Biol Ther 2015 Apr 16;15(4):517-28. Epub 2014 Dec 16.

Candiolo Cancer Institute-IRCCS, Laboratory of Medical Oncology, Experimental Cell Therapy , Candiolo, Turin , Italy.

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.
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http://dx.doi.org/10.1517/14712598.2015.987121DOI Listing
April 2015

Activity of cytokine-induced killer cells against bone and soft tissue sarcoma.

Oncoimmunology 2014;3:e28269. Epub 2014 Mar 17.

Division and Laboratory of Medical Oncology; Candiolo Cancer Institute-FPO; IRCCS Candiolo; Candiolo, Turin, Italy.

Cytokine-induced killer (CIK) cells are T lymphocytes expanded ex vivo that are endowed with MHC-independent tumoricidal activity. We have recently demonstrated, in a preclinical setting, that CIK cells are active against autologous bone and soft tissue sarcomas. In particular, CIK cells killed a putative sarcoma stem cell population that may underlie disease relapse and chemoresistance.
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http://dx.doi.org/10.4161/onci.28269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091097PMC
March 2014

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

Expert Opin Biol Ther 2014 Sep 16;14(9):1259-70. Epub 2014 May 16.

Candiolo Cancer Institute-FPO, IRCCS , Candiolo, 10060 , Italy.

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.
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http://dx.doi.org/10.1517/14712598.2014.918099DOI Listing
September 2014

Genetically redirected T lymphocytes for adoptive immunotherapy of solid tumors.

Curr Gene Ther 2014 Feb;14(1):52-62

Laboratory of Cell Therapy, Institute for Cancer Research and Treatment, Provinciale 142 - 10060 Candiolo, Torino Italy.

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.
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http://dx.doi.org/10.2174/1566523213666131223130353DOI Listing
February 2014

Cytokine-induced killer cells eradicate bone and soft-tissue sarcomas.

Cancer Res 2014 Jan 19;74(1):119-29. Epub 2013 Dec 19.

Authors' Affiliations: Stem Cell Transplantation and Cell Therapy, Pathology, Sarcoma, Fondazione del Piemonte per l'Oncologia, Laboratory of Molecular Pharmacology, Institute for Cancer Research and Treatment; Department of Oncology, University of Torino Medical School; and Division of Pediatric Onco-Hematology, Sant'Anna OIRM Hospital, (Torino), Italy.

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.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-1559DOI Listing
January 2014

Ex vivo-activated MHC-unrestricted immune effectors for cancer adoptive immunotherapy.

Anticancer Agents Med Chem 2014 Feb;14(2):211-22

Laboratory of Cell Therapy, Institute for Cancer Research and Treatment, Provinciale 142 - 10060 Candiolo, Torino Italy.

Adoptive immunotherapy is considered a promising strategy for the treatment of metastatic tumors and current research efforts are directed to define the optimal approach and facilitate the transferability from preclinical to clinical settings. Among several approaches it is possible to schematically distinguish strategies based on either MHC-restricted or MHC-unrestricted immune effectors. The first are mainly based on the infusion of tumor-specific T lymphocytes capable of recognizing determined MHC-restricted tumor associated antigens (TAA) through their T cell receptor. MHC-unrestricted approaches do not target specific tumor associated antigens and are mainly mediated by effectors of the innate immune system, like natural killer (NK) cells or NKT cells, first barrier against pathogens and tumorigenesis processes, or by ex vivo activated lymphocytes like cytokine-induced killer (CIK) cells. MHC-unrestricted effectors are usually more abundant than TAA-specific precursors and easier to expand. Furthermore their activity is not restricted to precise HLA-haplotypes, not limited to a single tumor histotype and could overcome downregulation of MHC molecules operated by tumor cells as immune escape mechanism. In this review we will discuss the main cancer immunotherapy strategies based on MHC-unrestricted immune effectors. The topic will be approached from the angle of ex vivo expansion protocols in clinical prospective, as well as potential approaches to favorably modulate their functions.
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http://dx.doi.org/10.2174/18715206113136660371DOI Listing
February 2014

Effective activity of cytokine-induced killer cells against autologous metastatic melanoma including cells with stemness features.

Clin Cancer Res 2013 Aug 21;19(16):4347-58. Epub 2013 Jun 21.

Unit of Stem Cell Transplantation and Cell Therapy, Surgical Dermatology, Pathology, and Sarcoma, Fondazione del Piemonte per l'Oncologia, I.R.C.C.S.,Torino, Italy.

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.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-0061DOI Listing
August 2013

Ex vivo allogeneic stimulation significantly improves expansion of cytokine-induced killer cells without increasing their alloreactivity across HLA barriers.

J Immunother 2012 Sep;35(7):579-86

Department of Oncological Sciences, Laboratory of Cell Therapy of Cancer, University of Torino Medical School, Institute for Cancer Research and Treatment, Candiolo, Torino, Italy.

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.
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http://dx.doi.org/10.1097/CJI.0b013e31826b1fd9DOI Listing
September 2012

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

Expert Opin Biol Ther 2012 Jun 14;12(6):673-84. Epub 2012 Apr 14.

Department of Oncological Sciences, Laboratory of Cell Therapy of Cancer, University of Torino Medical School, Institute for Cancer Research and Treatment, Candiolo, Torino, Italy.

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.
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http://dx.doi.org/10.1517/14712598.2012.675323DOI Listing
June 2012

Transient proteasome inhibition as a strategy to enhance lentiviral transduction of hematopoietic CD34(+) cells and T lymphocytes: implications for the use of low viral doses and large-size vectors.

J Biotechnol 2011 Dec 10;156(3):218-26. Epub 2011 Sep 10.

Laboratory of Cell Therapy of Cancer, Department of Oncological Sciences, University of Torino Medical School, Institute for Cancer Research and Treatment, Provinciale 142, 10060 Candiolo, Torino, Italy.

The proteasome system restricts lentiviral transduction of stem cells. We exploited proteasome inhibition as a strategy to enhance transduction of both hematopoietic stem cells (HSC) and T lymphocytes with low dose or large-size lentiviral vectors (LV). HSC showed higher transduction efficiency if transiently exposed to proteasome inhibitor MG132 (41.8% vs 10.7%, p<0.0001). Treatment with MG132 (0.5 μM) retained its beneficial effect with 3 different LV of increasing size up to 10.9 Kb (p<0.01). We extended, for the first time, the application of proteasome inhibition to the transduction of T lymphocytes. A transient exposure to MG132 significantly improved lentiviral T-cell transduction. The mean percentage of transduced T cells progressively increased from 13.5% of untreated cells, to 21% (p=0.3), 30% (p=0.03) and 37% (p=0.01) of T lymphocytes that were pre-treated with MG132 at 0.1, 0.5 and 1 μM, respectively. MG132 did not affect viability or functionality of HSC or T cells, nor significantly increased the number of integrated vector copies. Transient proteasome inhibition appears as a new procedure to safely enhance lentiviral transduction of HSC and T lymphocytes with low viral doses. This approach could be useful in settings where the use of large size vectors may impair optimal viral production.
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http://dx.doi.org/10.1016/j.jbiotec.2011.09.001DOI Listing
December 2011

Ventilatory strategies in the neonatal and paediatric intensive care units.

Paediatr Respir Rev 2008 Dec 6;9(4):281-8; quiz 288-9. Epub 2008 Nov 6.

McGill University Health Center, Montreal Children's Hospital, Montreal, Quebec, Canada.

Mechanical ventilation is a common form of support in the modern day intensive care unit (ICU). In order for the clinician better to understand and apply mechanical ventilation, it is important that they understand the physiological principles of ventilation. This review describes these basic concepts; parameters of mechanical ventilation, high frequency ventilation and non-invasive ventilation. An overview of ventilatory strategies for four common diseases seen in paediatric and neonatal ICUs will be discussed.
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http://dx.doi.org/10.1016/j.prrv.2008.09.001DOI Listing
December 2008