Conditioned Medium of Mesenchymal Stromal Cells Loaded with Paclitaxel Is Effective in Preclinical Models of Triple-Negative Breast Cancer (TNBC).

Triple-negative breast cancer (TNBC) is a very aggressive disease even in its early stages and is characterized by a severe prognosis. Neoadjuvant chemotherapy is one of the milestones of treatment, and paclitaxel (PTX) is among the most active drugs used in this setting. However, despite its efficacy, peripheral neuropathy occurs in approximately 20-25% of cases and represents the dose-limiting toxicity of this drug. New deliverable strategies to ameliorate drug delivery and reduce side effects are keenly awaited to improve patients' outcomes. Mesenchymal stromal cells (MSCs) have recently been demonstrated as promising drug delivery vectors for cancer treatment. The aim of the present preclinical study is to explore the possibility of a cell therapy approach based on the use of MSCs loaded with PTX to treat TNBC-affected patients. For this purpose, we in vitro evaluated the viability, migration and colony formation of two TNBC cell lines, namely, MDA-MB-231 and BT549, treated with MSC-PTX conditioned medium (MSC-CM PTX) in comparison with both CM of MSCs not loaded with PTX (CTRL) and free PTX. We observed stronger inhibitory effects on survival, migration and tumorigenicity for MSC-CM PTX than for CTRL and free PTX in TNBC cell lines. Further studies will provide more information about activity and potentially open the possibility of using this new drug delivery vector in the context of a clinical study.

A comprehensive report of long-term stability data for a range ATMPs: A need to develop guidelines for safe and harmonized stability studies.

BACKGROUND AIMS: Advanced therapy medicinal products (ATMPs) are novel drugs based on genes, cells or tissues developed to treat many different diseases. Stability studies of each new ATMP need to be performed to define its shelf life and guarantee efficacy and safety upon infusion, and these are presently based on guidelines originally drafted for standard pharmaceutical drugs, which have properties and are stored in conditions quite different from cell products. The aim of this report is to provide evidence-based information for stability studies on ATMPs that will facilitate the interlaboratory harmonization of practices in this area. METHODS: We have collected and analyzed the results of stability studies on 19 different cell-based experimental ATMPs, produced by five authorized cell factories forming the Lombardy "Plagencell network" for use in 36 approved phase I/II clinical trials; most were cryopreserved and stored in liquid nitrogen vapors for 1 to 13 years. RESULTS: The cell attributes collected in stability studies included cell viability, immunophenotype and potency assays, in particular immunosuppression, cytotoxicity, cytokine release and proliferation/differentiation capacity. Microbiological attributes including sterility, endotoxin levels and mycoplasma contamination were also analyzed. All drug products (DPs), cryopreserved in various excipients containing 10% DMSO and in different primary containers, were very stable long term at <-150°C and did not show any tendency for diminished viability or efficacy for up to 13.5 years. CONCLUSIONS: Our data indicate that new guidelines for stability studies, specific for ATMPs and based on risk analyses, should be drafted to harmonize practices, significantly reduce the costs of stability studies without diminishing safety. Some specific suggestions are presented in the discussion.

Dendritic Cells and Cancer Immunotherapy: The Adjuvant Effect.

Dendritic cells (DCs) are immune specialized cells playing a critical role in promoting immune response against antigens, and may represent important targets for therapeutic interventions in cancer. DCs can be stimulated ex vivo with pro-inflammatory molecules and loaded with tumor-specific antigen(s). Protocols describing the specific details of DCs vaccination manufacturing vary widely, but regardless of the employed protocol, the DCs vaccination safety and its ability to induce antitumor responses is clearly established. Many years of studies have focused on the ability of DCs to provide overall survival benefits at least for a selection of cancer patients. Lessons learned from early trials lead to the hypothesis that, to improve the efficacy of DCs-based immunotherapy, this should be combined with other treatments. Thus, the vaccine's ultimate role may lie in the combinatorial approaches of DCs-based immunotherapy with chemotherapy and radiotherapy, more than in monotherapy. In this review, we address some key questions regarding the integration of DCs vaccination with multimodality therapy approaches for cancer treatment paradigms.

Local Therapies and Modulation of Tumor Surrounding Stroma in Malignant Pleural Mesothelioma: A Translational Approach.

Malignant Pleural Mesothelioma (MPM) is a rare and aggressive neoplasm of the pleural mesothelium, mainly associated with asbestos exposure and still lacking effective therapies. Modern targeted biological strategies that have revolutionized the therapy of other solid tumors have not had success so far in the MPM. Combination immunotherapy might achieve better results over chemotherapy alone, but there is still a need for more effective therapeutic approaches. Based on the peculiar disease features of MPM, several strategies for local therapeutic delivery have been developed over the past years. The common rationale of these approaches is: (i) to reduce the risk of drug inactivation before reaching the target tumor cells; (ii) to increase the concentration of active drugs in the tumor micro-environment and their bioavailability; (iii) to reduce toxic effects on normal, non-transformed cells, because of much lower drug doses than those used for systemic chemotherapy. The complex interactions between drugs and the local immune-inflammatory micro-environment modulate the subsequent clinical response. In this perspective, the main interest is currently addressed to the development of local drug delivery platforms, both cell therapy and engineered nanotools. We here propose a review aimed at deep investigation of the biologic effects of the current local therapies for MPM, including cell therapies, and the mechanisms of interaction with the tumor micro-environment.

Vascular Remodeling in Moyamoya Angiopathy: From Peripheral Blood Mononuclear Cells to Endothelial Cells.

The pathophysiological mechanisms of Moyamoya angiopathy (MA), which is a rare cerebrovascular condition characterized by recurrent ischemic/hemorrhagic strokes, are still largely unknown. An imbalance of vasculogenic/angiogenic mechanisms has been proposed as one possible disease aspect. Circulating endothelial progenitor cells (cEPCs) have been hypothesized to contribute to vascular remodeling of MA, but it remains unclear whether they might be considered a disease effect or have a role in disease pathogenesis. The aim of the present study was to provide a morphological, phenotypical, and functional characterization of the cEPCs from MA patients to uncover their role in the disease pathophysiology. cEPCs were identified from whole blood as CD45dimCD34+CD133+ mononuclear cells. Morphological, biochemical, and functional assays were performed to characterize cEPCs. A significant reduced level of cEPCs was found in blood samples collected from a homogeneous group of adult (mean age 46.86 ± 11.7; 86.36% females), Caucasian, non-operated MA patients with respect to healthy donors (HD; p = 0.032). Since no difference in cEPC characteristics and functionality was observed between MA patients and HD, a defective recruitment mechanism could be involved in the disease pathophysiology. Collectively, our results suggest that cEPC level more than endothelial progenitor cell (EPC) functionality seems to be a potential marker of MA. The validation of our results on a larger population and the correlation with clinical data as well as the use of more complex cellular model could help our understanding of EPC role in MA pathophysiology.

Generation of donor-derived Wilms tumor antigen 1-specific cytotoxic T lymphocytes with potent anti-leukemia activity for somatic cell therapy in children given haploidentical stem cell transplantation: a feasibility pre-clinical study.

BACKGROUND: The Wilms tumor antigen 1 (WT1) is over-expressed in a vast majority of adult and childhood acute leukemia and myelodysplastic syndromes, being lowly or transiently expressed in normal tissues and hematopoietic stem cells (HSCs). A number of HLA-restricted WT1 epitopes are immunogenic, allowing the in vitro induction of WT1-specific cytotoxic T lymphocytes (CTLs) from patients and healthy donors. AIM: The aim of the study was to investigate the feasibility of producing WT1-specific CTLs suitable for somatic cell therapy to prevent or treat relapse in children with acute myeloid or lymphoblastic leukemia given haploidentical HSC transplantation (haplo-HSCT). METHODS: For WT1-specific CTL production, donor-derived either peripheral blood mononuclear cells (PBMCs) or CD8+ lymphocytes were stimulated with WT1 peptide-loaded donor dendritic cells in the presence of interleukin (IL)-7 and IL-12. Effector cells were re-stimulated once with irradiated donor PBMCs pulsed with WT1-peptides, and then expanded in an antigen-independent way. RESULTS: WT1-specific CTLs, displaying high-level cytotoxicity against patients' leukemia blasts and negligible activity against patients' non-malignant cells, were obtained from both PBMCs and CD8+ lymphocytes. WT1-specific CTLs obtained from PBMCs showed a better expansion capacity and better anti-leukemia activity than those obtained from CD8+ lymphocytes, even though the difference was not statistically significant. In CTLs derived from PBMCs, both CD8+ and CD4+ subpopulations displayed strong anti-leukemia cytotoxic activity. DISCUSSION: Results of this pre-clinical study pave the way to a somatic cell therapy approach aimed at preventing or treating relapse in children given haplo-HSCT for WT1-positive leukemia.

Human CD14+ cells loaded with Paclitaxel inhibit in vitro cell proliferation of glioblastoma.

BACKGROUND AIMS: In attempting to develop new strategies to circumvent the immunosuppression associated with glioblastoma (GB), novel approaches have been designed using dendritic cell (DC)-based vaccination, which is considered a promising strategy to attack high-grade glioma. In previous studies, we demonstrated that human mesenchymal stromal cells without genetic manipulation but primed with Paclitaxel (PTX) acquire a potent anti-tumor activity, providing an interesting new biological approach for drug delivery. On the basis of these results, we here investigated whether both CD14+ and their derived DCs may behave like mesenchymal stromal cells acquiring anti-tumor activity on priming with PTX. METHODS: Human CD14+ cells were isolated from peripheral blood. Fluorescence-activated cell sorter analysis was performed to determine the purity of CD14+ and their differentiation into mature DCs. Cells were primed by incubation with 1 µg/mL of PTX for 24 h, and the PTX released by cells was assessed by mass spectrometry analysis. Anti-tumor activity was checked by testing the conditioned medium (CM) on the proliferation of U87 MG, a GB cell line. RESULTS: Both CD14+ and DCs were able to incorporate PTX and release the drug in the CM in a time-dependent manner (maximal release over 24 h). The addition of CM from CD14+ and DCs loaded with PTX strongly inhibits proliferation of U87 MG cells. CONCLUSIONS: Our results are the first demonstration that peripheral blood-derived CD14+ and DCs, in addition to their application for immunotherapy for GB, could also be used to delivery anti-cancer drugs, such as PTX, to kill GB cells.

In vitro biosafety profile evaluation of multipotent mesenchymal stem cells derived from the bone marrow of sarcoma patients.

BACKGROUND: In osteosarcoma (OS) and most Ewing sarcoma (EWS) patients, the primary tumor originates in the bone. Although tumor resection surgery is commonly used to treat these diseases, it frequently leaves massive bone defects that are particularly difficult to be treated. Due to the therapeutic potential of mesenchymal stem cells (MSCs), OS and EWS patients could benefit from an autologous MSCs-based bone reconstruction. However, safety concerns regarding the in vitro expansion of bone marrow-derived MSCs have been raised. To investigate the possible oncogenic potential of MSCs from OS or EWS patients (MSC-SAR) after expansion, this study focused on a biosafety assessment of MSC-SAR obtained after short- and long-term cultivation compared with MSCs from healthy donors (MSC-CTRL). METHODS: We initially characterized the morphology, immunophenotype, and differentiation multipotency of isolated MSC-SAR. MSC-SAR and MSC-CTRL were subsequently expanded under identical culture conditions. Cells at the early (P3/P4) and late (P10) passages were collected for the in vitro analyses including: sequencing of genes frequently mutated in OS and EWS, evaluation of telomerase activity, assessment of the gene expression profile and activity of major cancer pathways, cytogenetic analysis on synchronous MSCs, and molecular karyotyping using a comparative genomic hybridization (CGH) array. RESULTS: MSC-SAR displayed comparable morphology, immunophenotype, proliferation rate, differentiation potential, and telomerase activity to MSC-CTRL. Both cell types displayed signs of senescence in the late stages of culture with no relevant changes in cancer gene expression. However, cytogenetic analysis detected chromosomal anomalies in the early and late stages of MSC-SAR and MSC-CTRL after culture. CONCLUSIONS: Our results demonstrated that the in vitro expansion of MSCs does not influence or favor malignant transformation since MSC-SAR were not more prone than MSC-CTRL to deleterious changes during culture. However, the presence of chromosomal aberrations supports rigorous phenotypic, functional and genetic evaluation of the biosafety of MSCs, which is important for clinical applications.

Therapeutic Strategies to Improve the Treatment of Pleural Mesothelioma.

Pleural mesothelioma is a rare neoplastic disease with aggressive features. Patient survival is poor due to the lack of early symptoms and the absence of effective therapeutic strategies. The development of pleural mesothelioma is mainly associated with asbestos exposure and related chronic inflammation. From a molecular-based perspective, this disease is a heterogeneous tumor lacking actionable alterations. The median overall survival of patients affected by this tumor does not exceed 16 months from diagnosis. Molecular and biochemical approaches have shown that this disease is characterized by resistance to drug-induced apoptosis associated with the activation of cell survival pathways and expression of anti-apoptotic proteins. Thus, there is an urgent need to develop efficient and safe therapeutic strategies. Here, we review the pharmacological options available for the treatment of this disease with specific reference to the antitumor agents used in systemic therapies. In addition, novel pharmacological approaches, such as drug delivery tools, to improve pleural mesothelioma treatment are discussed.

Potency assays and biomarkers for cell-based advanced therapy medicinal products.

Advanced Therapy Medicinal Products (ATMPs) based on somatic cells expanded in vitro, with or without genetic modification, is a rapidly growing area of drug development, even more so following the marketing approval of several such products. ATMPs are produced according to Good Manufacturing Practice (GMP) in authorized laboratories. Potency assays are a fundamental aspect of the quality control of the end cell products and ideally could become useful biomarkers of efficacy in vivo. Here we summarize the state of the art with regard to potency assays used for the assessment of the quality of the major ATMPs used clinic settings. We also review the data available on biomarkers that may substitute more complex functional potency tests and predict the efficacy in vivo of these cell-based drugs.

In vitro evaluation of graft-versus-graft alloreactivity as a tool to identify the predominant cord blood unit before double cord blood transplantation.

The transplantation of two cord blood (CB) units obtained from unrelated donors (double CBT) is an effective strategy for adult patients with hematologic malignancies. Sustained hematopoiesis after double CBT is usually derived from a single donor, and only a few transplantation recipients displaying a stable mixed donor-donor chimerism have been reported. We investigated the mechanisms underlying single-donor predominance in double CBT by studying in vitro the role of the graft-versus-graft cell-mediated immune effect in two-way mixed-lymphocyte culture, along with the contribution of differential hematopoietic progenitor (HP) potency in HP mixed cultures. Results for the two-way mixed-lymphocyte culture showed that despite the weak and variable alloantigen-specific cytotoxic potential displayed by CB mononuclear cells, an immune-mediated dominance for one of the two CB units was detected in the majority of experiments. Alloantigen-induced cytotoxic activity was directed toward both CB-HP and phytohemagglutinin (PHA)-activated T lymphoblastoid cells. The CB unit with the higher fold expansion of CD34(+) cells in single-expansion culture was prevalent in the HP mixed-expansion culture, as shown by DNA chimerism evaluation. Based on these data, we hypothesize that the dominant CB unit is able to develop prevalent cytotoxic activity toward activated lymphocytes of the other CB unit, thereby preventing them from exerting alloantigen-specific cytotoxic potential against both activated lymphocytes and HPs of the dominant unit. In accordance with this hypothesis, we propose the evaluation of alloantigen-induced cytotoxic activity generated in two-way mixed-lymphocyte culture and directed toward PHA-activated T lymphoblastoid cells as a tool to identify the potentially predominant CB unit before double CBT.

The strange case of the lost NRAS mutation in a child with juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative disorder of early childhood characterized by mutations of the RAS-RAF-MAP kinase signaling pathway. We report the case of a child with a diagnosis of JMML carrying two mutations of NRAS gene (c.37G>C and c.38G>A) independently occurring in long-term culture initiating cells. However, only the former was consistently found in more mature hematopoietic cells, suggesting that cancer transformation may lead to the loss of a mutation. This case also indicates that molecular analysis on cell types other than peripheral blood leukocytes may be useful to obtain relevant biological information on JMML pathogenesis.

Inhibition of Human Malignant Pleural Mesothelioma Growth by Mesenchymal Stromal Cells.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is an aggressive tumor that has a significant incidence related to asbestos exposure with no effective therapy and poor prognosis. The role of mesenchymal stromal cells (MSCs) in cancer is controversial due to their opposite effects on tumor growth and in particular, only a few data are reported on MSCs and MPM. METHODS: We investigated the in vitro efficacy of adipose tissue-derived MSCs, their lysates and secretome against different MPM cell lines. After large-scale production of MSCs in a bioreactor, their efficacy was also evaluated on a human MPM xenograft in mice. RESULTS: MSCs, their lysate and secretome inhibited MPM cell proliferation in vitro with S or G0/G1 arrest of the cell cycle, respectively. MSC lysate induced cell death by apoptosis. The efficacy of MSC was confirmed in vivo by a significant inhibition of tumor growth, similar to that produced by systemic administration of paclitaxel. Interestingly, no tumor progression was observed after the last MSC treatment, while tumors started to grow again after stopping chemotherapeutic treatment. CONCLUSIONS: These data demonstrated for the first time that MSCs, both through paracrine and cell-to-cell interaction mechanisms, induced a significant inhibition of human mesothelioma growth. Since the prognosis for MPM patients is poor and the options of care are limited to chemotherapy, MSCs could provide a potential new therapeutic approach for this malignancy.

PGE2 Is Crucial for the Generation of FAST Whole- Tumor-Antigens Loaded Dendritic Cells Suitable for Immunotherapy in Glioblastoma.

Dendritic cells (DC) are the most potent antigen-presenting cells, strongly inducers of T cell-mediated immune responses and, as such, broadly used as vaccine adjuvant in experimental clinical settings. DC are widely generated from human monocytes following in vitro protocols which require 5-7 days of differentiation with GM-CSF and IL-4 followed by 2-3 days of activation/maturation. In attempts to shorten the vaccine's production, Fast-DC protocols have been developed. Here we reported a Fast-DC method in compliance with good manufacturing practices for the production of autologous mature dendritic cells loaded with antigens derived from whole tumor lysate, suitable for the immunotherapy in glioblastoma patients. The feasibility of generating Fast-DC pulsed with whole tumor lysate was assessed using a series of small-scale cultures performed in parallel with clinical grade large scale standard method preparations. Our results demonstrate that this Fast protocol is effective only in the presence of PGE2 in the maturation cocktail to guarantee that Fast-DC cells exhibit a mature phenotype and fulfill all requirements for in vivo use in immunotherapy approaches. Fast-DC generated following this protocol were equally potent to standard DC in inducing Ag-specific T cell proliferation in vitro. Generation of Fast-DC not only reduces labor, cost, and time required for in vitro clinical grade DC development, but can also minimizes inter-preparations variability and the risk of contamination.

Automated Large-Scale Production of Paclitaxel Loaded Mesenchymal Stromal Cells for Cell Therapy Applications.

Mesenchymal stromal cells (MSCs) prepared as advanced therapies medicinal products (ATMPs) have been widely used for the treatment of different diseases. The latest developments concern the possibility to use MSCs as carrier of molecules, including chemotherapeutic drugs. Taking advantage of their intrinsic homing feature, MSCs may improve drugs localization in the disease area. However, for cell therapy applications, a significant number of MSCs loaded with the drug is required. We here investigate the possibility to produce a large amount of Good Manufacturing Practice (GMP)-compliant MSCs loaded with the chemotherapeutic drug Paclitaxel (MSCs-PTX), using a closed bioreactor system. Cells were obtained starting from 13 adipose tissue lipoaspirates. All samples were characterized in terms of number/viability, morphology, growth kinetics, and immunophenotype. The ability of MSCs to internalize PTX as well as the antiproliferative activity of the MSCs-PTX in vitro was also assessed. The results demonstrate that our approach allows a large scale expansion of cells within a week; the MSCs-PTX, despite a different morphology from MSCs, displayed the typical features of MSCs in terms of viability, adhesion capacity, and phenotype. In addition, MSCs showed the ability to internalize PTX and finally to kill cancer cells, inhibiting the proliferation of tumor lines in vitro. In summary our results demonstrate for the first time that it is possible to obtain, in a short time, large amounts of MSCs loaded with PTX to be used in clinical trials for the treatment of patients with oncological diseases.

Germ-line mutation of the NRAS gene may be responsible for the development of juvenile myelomonocytic leukaemia.

We report the case of a child with clinical and haematological features indicative of juvenile myelomonocytic leukaemia (JMML). The patient showed dysmorphic features: high forehead, bilateral epicanthal folds, long eyebrows, low nasal bridge and slightly low-set ears. A 38G>A (G13D) mutation in exon 1 of the NRAS gene was first demonstrated on peripheral blood cells, and then confirmed on granulocyte-macrophage colony-forming units. The same mutation was also found in buccal swab, hair bulbs, endothelial cells, skin fibroblasts. This case suggests for the first time that constitutional mutations of NRAS may be responsible for development of a myeloproliferative/myelodysplastic disorder in children.

Expansion of effector and memory T cells is associated with increased survival in recurrent glioblastomas treated with dendritic cell immunotherapy.

BACKGROUND: The efficacy of dendritic cell (DC) immunotherapy as a single therapeutic modality for the treatment of glioblastoma (GBM) patients remains limited. In this study, we evaluated in patients with GBM recurrence the immune-mediated effects of DC loaded with autologous tumor lysate combined with temozolomide (TMZ) or tetanus toxoid (TT). METHODS: In the phase I-II clinical study DENDR2, 12 patients were treated with 5 DC vaccinations combined with dose-dense TMZ. Subsequently, in eight patients, here defined as Variant (V)-DENDR2, the vaccine site was preconditioned with TT 24 hours before DC vaccination and TMZ was avoided. As a survival endpoint for these studies, we considered overall survival 9 months (OS9) after second surgery. Patients were analyzed for the generation of effector, memory, and T helper immune response. RESULTS: Four of 12 DENDR2 patients reached OS9, but all failed to show an immunological response. Five of eight V-DENDR2 patients (62%) reached OS9, and one patient is still alive (OS >30 months). A robust CD8+ T-cell activation and memory T-cell formation were observed in V-DENDR2 OS>9. Only in these patients, the vaccine-specific CD4+ T-cell activation (CD38+/HLA-DR+) was paralleled by an increase in TT-induced CD4+/CD38low/CD127high memory T cells. Only V-DENDR2 patients showed the formation of a nodule at the DC injection site infiltrated by CCL3-expressing CD4+ T cells. CONCLUSIONS: TT preconditioning of the vaccine site and lack of TMZ could contribute to the efficacy of DC immunotherapy by inducing an effector response, memory, and helper T-cell generation.

Donor/recipient mixed chimerism does not predict graft failure in children with beta-thalassemia given an allogeneic cord blood transplant from an HLA-identical sibling.

BACKGROUND: Donor/recipient mixed chimerism has been reported to be associated with an increased risk of graft failure in patients with beta-thalassemia given a bone marrow transplant. We investigated the relationship between the degree of mixed chimerism over time and clinical outcome of children undergoing cord blood transplantation for beta-thalassemia. DESIGN AND METHODS: Twenty-seven consecutive children given a cord blood transplant from a related donor were analyzed by short tandem repeat polymerase chain reaction and their chimerism results were compared with those of 79 consecutive patients who received a bone marrow transplant from either a relative (RD-BMT, n=42) or an unrelated donor (UD-BMT, n=37). Cord blood and bone marrow recipients received comparable preparative regimens. RESULTS: All cord blood recipients engrafted and displayed mixed chimerism early after transplantation; 13/27 converted to full donor chimerism over time, while 14 maintained stable mixed chimerism; all patients are alive and transfusion-independent. Twenty-four of the 79 bone marrow-recipients (12 UD- and 12 RD-BMT) exhibited full donor chimerism at all time points examined, 4/79 (2 UD- and 2 RD-BMT) did not engraft and 51/79 (23 UD- and 28 RD-BMT) displayed mixed chimerism at the time of hematologic reconstitution. Forty of 51 bone marrow recipients with mixed chimerism converted to full donor chimerism (17 UD- and 23 RD-BMT), 3/51 maintained stable mixed chimerism (1 UD- and 2 RD-BMT), while 8/51 (5 UD- and 3 RD-BMT) progressively lost the graft, and became transfusion-dependent again. CONCLUSIONS: Mixed chimerism is a frequent event and does not predict the occurrence of graft failure in children with beta-thalassemia given a cord blood transplant from a relative.

Single-cell cloning of human, donor-derived antileukemia T-cell lines for in vitro separation of graft-versus-leukemia effect from graft-versus-host reaction.

In previous studies, we showed the possibility of expanding in vitro polyclonal CTL lines directed against patient leukemia cells using effector cells derived from both HLA-matched and HLA-mismatched hematopoietic stem cell donors. Some CTL lines, especially those derived from an HLA-disparate donor, displayed residual alloreactivity against patient nonmalignant cells. In this study, we evaluated the possibility of separating in vitro CTLs with selective graft-versus-leukemia (GVL) activity from those potentially involved in the development of graft-versus-host disease (GVHD) through single T-cell cloning of antileukemia polyclonal CTL lines. We showed that CTLs that were expanded from a single T-cell clone (TCC), able to selectively kill leukemia blasts and devoid of alloreactivity towards nonmalignant cells, can be obtained from antileukemia alloreactive polyclonal CTL lines. TCCs expressed a wide repertoire of different T-cell receptor (TCR)-Vbeta families, mainly produced IFNgamma and interleukin 2, irrespective of CD8 or CD4 phenotype, and could be extensively expanded in vitro without losing their peculiar functional features. The feasibility of our approach for in vitro separation of GVL from GVH reaction opens perspectives for using TCCs, which are selectively reactive towards leukemia blasts, for antileukemia adoptive immune therapy approaches after hematopoietic stem cell transplantation, in particular from HLA-mismatched donors.

In Vitro Killing of Colorectal Carcinoma Cells by Autologous Activated NK Cells is Boosted by Anti-Epidermal Growth Factor Receptor-induced ADCC Regardless of RAS Mutation Status.

Treatment of advanced metastatic colorectal cancer (mCRC) patients is associated with a poor prognosis and significant morbidity. Moreover, targeted therapies such as anti-epidermal growth factor receptor (EGFR) have no effect in metastatic patients with tumors harboring a mutation in the RAS gene. The failure of conventional treatment to improve outcomes in mCRC patients has prompted the development of adoptive immunotherapy approaches including natural killer (NK)-based therapies. In this study, after confirmation that patients' NK cells were not impaired in their cytotoxic activity, evaluated against long-term tumor cell lines, we evaluated their interactions with autologous mCRC cells. Molecular and phenotypical evaluation of mCRC cells, expanded in vitro from liver metastasis, showed that they expressed high levels of polio virus receptor and Nectin-2, whereas UL16-binding proteins were less expressed in all tumor samples evaluated. Two different patterns of MICA/B and HLA class I expression on the membrane of mCRC were documented; approximately half of mCRC patients expressed high levels of these molecules on the membrane surface, whereas, in the remaining, very low levels were documented. Resting NK cells were unable to display sizeable levels of cytotoxic activity against mCRC cells, whereas their cytotoxic activity was enhanced after overnight or 5-day incubation with IL-2 or IL-15. The susceptibility of NK-mediated mCRC lysis was further significantly enhanced after coating with cetuximab, irrespective of their RAS mutation and HLA class I expression. These data open perspectives for combined NK-based immunotherapy with anti-epidermal growth factor receptor antibodies in a cohort of mCRC patients with a poor prognosis refractory to conventional therapies.