A Novel Mutation in the Upstream Open Reading Frame of the CDKN1B Gene Causes a MEN4 Phenotype

PubMed ID: 23555276This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Ridirezionamento dell'immunità anti-tumorale in terapia cellulare adottiva: trasferimento genico del T-Cell Receptor mediato da vettori lentivirali

Genetic modification of T cells with genes encoding a tumor-specific T-cell Receptor (TCR) represents a novel strategy to obtain large quantities of tumor-reactive T lymphocytes to be employed in adoptive cell therapy protocols. As a transfer method, lentiviral vectors might represent an appealing alternative to the most widely used oncoretroviral vectors, because they do not require cell division for nuclear uptake. We have characterized the TCR of a highly cytotoxic T lymphocyte (CTL) clone recognizing the HLA-A2-restricted Melan-A/MART-1 melanocyte differentiation antigen on both pulsed T2 cells and SK-23 MEL melanoma tumor cells. The ? (V?2.2) e ? (V?14) chains of the TCR were cloned and used to construct a lentiviral vector carrying a bidirectional promoter and capable of a robust and coordinated expression of the two transgenes. Transduction of Jurkat T leukemia cells showed that more than 60% of cells could be transduced without selection at a low MOI, as assessed by antigen-specific tetramer staining. High expression Jurkat clones disclosed that the new assembled TCR was at high intensity, very stable over time, and fully functional, as demonstrated by intracellular signaling upon TCR triggering. Transduction of activated PBMC produced a highly expressed transgenic TCR in around 5-10% of cells. This initial low, but clearly detectable, fraction of transduced lymphocytes could be quickly expanded (4-6 weeks) upon subsequent antigen-specific in vitro restimulation. The resulting population had a 50-70% of transgenic TCR expression and mainly a CD8+ phenotype, specifically recognized antigen-expressing melanoma cells (cytokine production and cytotoxic activity), and exerted relevant therapeutic effects in vitro upon adoptive transfer in SK-23 MEL-bearing mice. Our results indicate that LV constitute a valid tool for stable and high-intensity expression of transgenic TCR, and support further studies to address potential feasibility of this approach for clinical application

Biotechnologies to tackle the challenge of neoantigen identification

Among immune correlates of clinical responses, tumor-specificneoantigens took the spotlight as relevant targets for cancerimmunotherapy. The implementation of pipelines forpersonalized cancer therapy remains challenging due to theprivacy, that is patient-specificity, of neoantigens and the low-frequency of neoantigen-specific T cells in blood and tumorsamples. To overcome these obstacles, recent developmentsin the field of biotechnology have allowed the multiplexedidentification of neoepitope-specific T cells. This reviewaddresses the pros and cons of conventional neoantigenscreening methodologies and highlights the current as well asthe prospective biotechnological opportunities in the field

Personalized approaches to active immunotherapy in cancer

Immunotherapy is emerging as a promising anti-cancer curative modality. However, in contrast to recent advances obtained employing checkpoint blockade agents and T cell therapies, clinical efficacy of therapeutic cancer vaccines is still limited. Most vaccination attempts in the clinic represent "off-the shelf" approaches since they target common "self" tumor antigens, shared among different patients. In contrast, personalized approaches of vaccination are tailor-made for each patient and in spite being laborious, hold great potential. Recent technical advancement enabled the first steps in the clinic of personalized vaccines that target patient-specific mutated neo-antigens. Such vaccines could induce enhanced tumor-specific immune response since neo-antigens are mutation-derived antigens that can be recognized by high affinity T cells, not limited by central tolerance. Alternatively, the use of personalized vaccines based on whole autologous tumor cells, overcome the need for the identification of specific tumor antigens. Whole autologous tumor cells could be administered alone, pulsed on dendritic cells as lysate, DNA, RNA or delivered to dendritic cells in-vivo through encapsulation in nanoparticle vehicles. Such vaccines may provide a source for the full repertoire of the patient-specific tumor antigens, including its private neo-antigens. Furthermore, combining next-generation personalized vaccination with other immunotherapy modalities might be the key for achieving significant therapeutic outcome

Microfluidic device performing on flow study of serial cell–cell interactions of two cell populations

In this study we present a novel microfluidic hydrodynamic trapping device to probe the cell-cell interaction between all cell samples of two distinct populations. We have exploited an hydrodynamic trapping method using microfluidics to immobilize a batch of cells from the first population at specific locations, then relied on hydrodynamic filtering principles, the flowing cells from the second cell population are placed in contact with the trapped ones, through a roll-over mechanism. The rolling cells interact with the serially trapped cells one after the other. The proposed microfluidic phenomenon was characterized with beads. We have shown the validity of our method by detecting the capacity of olfactory receptors to induce adhesion of cell doublets overexpressing these receptors. We report here the first controlled on-flow single cell resolution cell-cell interaction assay in a microfluidic device for future application in cell-cell interactions-based cell library screenings

Virus-specific cytotoxic CD4+ T cells for the treatment of EBV-related tumors

Although adoptive immunotherapy with CD8(+) CTL is providing clinically relevant results against EBV-driven malignancies, the effector role of CD4(+) T cells has been poorly investigated. We addressed this issue in a lymphoblastoid cell line-induced mouse model of posttransplant lymphoproliferative disease (PTLD) by comparing the therapeutic efficacy of EBV-specific CD4(+) and CD8(+) T cell lines upon adoptive transfer. CD4(+) T cells disclosed a long-lasting and stronger proliferative potential than CD8(+) T cells, had a similar activation and differentiation marker profile, efficiently killed their targets in a MHC class II-restricted manner, and displayed a lytic machinery comparable to that of cognate CD8(+) T cells. A detailed analysis of Ag specificity revealed that CD4(+) T cells potentially target EBV early lytic cycle proteins. Nonetheless, when assessed for the relative therapeutic impact after in vivo transfer, CD4(+) T cells showed a reduced activity compared with the CD8(+) CTL counterpart. This feature was apparently due to a strong and selective downmodulation of MHC class II expression on the tumor cells surface, a phenomenon that could be reverted by the demethylating agent 5-aza-2'-deoxycytidine, thus leading to restoration of lymphoblastoid cell line recognition and killing by CD4(+) T cells, as well as to a more pronounced therapeutic activity. Conversely, immunohistochemical analysis disclosed that HLA-II expression is fully retained in human PTLD samples. Our data indicate that EBV-specific cytotoxic CD4(+) T cells are therapeutic in mice bearing PTLD-like tumors, even in the absence of CD8(+) T cells. These findings pave the way to use cultures of pure CD4(+) T cells in immunotherapeutic approaches for EBV-related malignancies

PSMA-Specific CAR-Engineered T Cells Eradicate Disseminated Prostate Cancer in Preclinical Models

Immunology-based interventions have been proposed as a promising curative chance to effectively attack postoperative minimal residual disease and distant metastatic localizations of prostate tumors. We developed a chimeric antigen receptor (CAR) construct targeting the human prostate-specific membrane antigen (hPSMA), based on a novel and high affinity specific mAb. As a transfer method, we employed last-generation lentiviral vectors (LV) carrying a synthetic bidirectional promoter capable of robust and coordinated expression of the CAR molecule, and a bioluminescent reporter gene to allow the tracking of transgenic T cells after in vivo adoptive transfer. Overall, we demonstrated that CAR-expressing LV efficiently transduced short-term activated PBMC, which in turn were readily stimulated to produce cytokines and to exert a relevant cytotoxic activity by engagement with PSMA+ prostate tumor cells. Upon in vivo transfer in tumor-bearing mice, CAR-transduced T cells were capable to completely eradicate a disseminated neoplasia in the majority of treated animals, thus supporting the translation of such approach in the clinical setting

A paclitaxel-hyaluronan bioconjugate targeting ovarian cancer affords a potent in vivo therapeutic activity.

PURPOSE: This study was designed to evaluate the pharmacologic and biological properties of a paclitaxel-hyaluronan bioconjugate (ONCOFID-P) against IGROV-1 and OVCAR-3 human ovarian cancer xenografts following i.p. administration. EXPERIMENTAL DESIGN: In vitro tumor sensitivity to ONCOFID-P was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, whereas bioconjugate interaction with cells was studied cytofluorimetrically and by confocal microscopy. In vivo toxicity was assessed by a single-dose maximum-tolerated dose, peripheral blood cell count determination and by histologic analysis. Biodistribution of the compound was evaluated with a small animal-dedicated scintigraphy gamma camera following injection of 99mTc-labeled ONCOFID-P. Pharmacokinetic analysis was also carried out. Female severe combined immunodeficiency mice implanted with ovarian cancer cells underwent treatment with ONCOFID-P or free paclitaxel starting from day 7 or 14 after tumor injection, and survivals were compared. RESULTS: ONCOFID-P interacted with CD44, entered cells through a receptor-mediated mechanism, and exerted a concentration-dependent inhibitory effect against tumor cell growth. After i.p. administration, the bioconjugate distributed quite uniformly within the peritoneal cavity, was well-tolerated, and was not associated with local histologic toxicity. Pharmacokinetic studies revealed that blood levels of bioconjugate-derived paclitaxel were much higher and persisted longer than those obtained with the unconjugated free drug. Intraperitoneal treatment of tumor-bearing mice with the bioconjugate revealed that ONCOFID-P exerted a relevant increase in therapeutic activity compared with free drug. CONCLUSIONS: ONCOFID-P significantly improved results obtained with conventional paclitaxel, in terms of in vivo tolerability and therapeutic efficacy; these data strongly support its development for locoregional treatment of ovarian cancer