Sara Bobisse

A Mutant-p53/Smad Complex Opposes p63 to Empower TGFβ-Induced Metastasis

TGFbeta ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. Here, we show that TGFbeta-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFbeta acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, we identified two candidate metastasis suppressor genes associated with metastasis risk in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFbeta-dependent inhibition of p63 function.

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.

Human TCR That Incorporate CD3ζ Induce Highly Preferred Pairing between TCRα and β Chains following Gene Transfer

TCR gene therapy is adversely affected by newly formed TCRαβ heterodimers comprising exogenous and endogenous TCR chains that dilute expression of transgenic TCRαβ dimers and are potentially self-reactive. We have addressed TCR mispairing by using a modified two-chain TCR that encompasses total human CD3ζ with specificities for three different Ags. Transfer of either TCRα:CD3ζ or β:CD3ζ genes alone does not result in surface expression, whereas transfer of both modified TCR chains results in high surface expression, binding of peptide-MHC complexes and Ag-specific T cell functions. Genetic introduction of TCRαβ:ζ does not compromise surface expression and functions of an endogenous TCRαβ. Flow cytometry fluorescence resonance energy transfer and biochemical analyses demonstrate that TCRαβ:CD3ζ is the first strategy that results in highly preferred pairing between CD3ζ-modified TCRα and β chains as well as absence of TCR mispairing between TCR:CD3ζ and nonmodified TCR chains. Intracellular assembly and surface expression of TCR:CD3ζ chains is independent of endogenous CD3γ, δ, and ε. Taken together, our data support the use of TCRαβ:CD3ζ to prevent TCR mispairing, which may provide an adequate strategy to enhance efficacy and safety of TCR gene transfer.

Reprogramming T Lymphocytes for Melanoma Adoptive Immunotherapy by T-Cell Receptor Gene Transfer with Lentiviral Vectors

T-cell receptor (TCR) gene transfer for cancer immunotherapy is limited by the availability of large numbers of tumor-specific T cells. TCR alpha and beta chains were isolated from a highly lytic HLA-A2-restricted cytotoxic T lymphocyte (CTL) clone recognizing the melanoma-associated Melan-A/MART-1 antigen and inserted into a lentiviral vector carrying a bidirectional promoter capable of robust and coordinated expression of the two transgenes. Lentiviral vector-based gene delivery systems have shown increased transfer efficiency and transgene expression compared with the widely used gamma-retroviral vectors. This vector performed more efficiently than a gamma-retrovirus-based vector containing the same expression cassette, resulting in a T-cell population with 60% to 80% of transgenic TCR expression with mainly CD8(+) intermediate effector phenotype. Transgenic T cells specifically produced cytokine in response to and killed antigen-expressing melanoma cells, retained an overlapping functional avidity in comparison with the TCR donor CTL clone, and exerted significant therapeutic effects in vivo upon adoptive transfer in melanoma-bearing severe combined immunodeficient mice. Optical imaging showed their accumulation in the tumor site. Overall, our results indicate that lentiviral vectors represent a valid tool for stable and high-intensity expression of transgenic TCR and support clinical exploitation of this approach for therapeutic application.

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.

The Scaffold Protein Cybr Is Required for Cytokine-Modulated Trafficking of Leukocytes In Vivo

ABSTRACT Trafficking and cell adhesion are key properties of cells of the immune system. However, the molecular pathways that control these cellular behaviors are still poorly understood. Cybr is a scaffold protein highly expressed in the hematopoietic/immune system whose physiological role is still unknown. In vitro studies have shown it regulates LFA-1, a crucial molecule in lymphocyte attachment and migration. Cybr also binds cytohesin-1, a guanine nucleotide exchange factor for the ARF GTPases, which affects actin cytoskeleton remodeling during cell migration. Here we show that expression of Cybr in vivo is differentially modulated by type 1 cytokines during lymphocyte maturation. In mice, Cybr deficiency negatively affects leukocytes circulating in blood and lymphocytes present in the lymph nodes. Moreover, in a Th1-polarized mouse model, lymphocyte trafficking is impaired by loss of Cybr, and Cybr-deficient mice with aseptic peritonitis have fewer cells than controls present in the peritoneal cavity, as well as fewer leukocytes leaving the bloodstream. Mutant mice injected with Moloney murine sarcoma/leukemia virus develop significantly larger tumors than wild-type mice and have reduced lymph node enlargement, suggesting reduced cytotoxic T-lymphocyte migration. Taken together, these data support a role for Cybr in leukocyte trafficking, especially in response to proinflammatory cytokines in stress conditions.

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.

T-cell receptor gene transfer by lentiviral vectors in adoptive cell therapy.

Adoptive cell therapy can be envisioned as a promising strategy for tumour immunotherapy. However, existing protocols of adoptive cell therapy still require optimisation as many factors, such as specificity, avidity, level of differentiation and amount of transferred T lymphocytes, can influence their immunocompetence and in vivo functionality. In particular, the need to reduce the in vitro expansion phase and to obtain large numbers of tumour-reactive T cells, as a favourable condition for cancer regression, make TCR gene transfer a potentially ideal tool to overcome the limits of adoptive cell therapy strategies. Here, the authors review the state-of-the-art and recent advances in TCR transfer with particular emphasis on lentiviral vector systems. Initial data from preclinical models and recent clinical trials encourage optimisation of a safe, simplified and stable transfer system. In this regard, HIV-based vectors are emerging as good alternative candidates over the most widely used oncoretroviral vectors due to their peculiar molecular features that fit the ideal conditions for donor T cell in vitro manipulation.

Functional Avidity–Driven Activation-Induced Cell Death Shapes CTL Immunodominance

Immunodominance is a complex phenomenon that relies on a mere numerical concept, while being potentially influenced at every step of the immune response. We investigated the mechanisms leading to the establishment of CTL immunodominance in a retroviral model and found that the previously defined subdominant Env-specific CD8+ T cells are endowed with an unexpectedly higher functional avidity than is the immunodominant Gag-recognizing counterpart. This high avidity, along with the Env Ag overload, results in a supraoptimal TCR engagement. The overstimulation makes Env-specific T lymphocytes more susceptible to apoptosis, thus hampering their expansion and leading to an unintentional “immune kamikazing.” Therefore, Ag-dependent, hyperactivation-induced cell death can be regarded as a novel mechanism in the establishment of the immunodominance that restrains and opposes the expansion of high-avidity T cells in favor of lower-affinity populations.

Feasibility of Telomerase-Specific Adoptive T-cell Therapy for B-cell Chronic Lymphocytic Leukemia and Solid Malignancies

Telomerase (TERT) is overexpressed in 80% to 90% of primary tumors and contributes to sustaining the transformed phenotype. The identification of several TERT epitopes in tumor cells has elevated the status of TERT as a potential universal target for selective and broad adoptive immunotherapy. TERT-specific cytotoxic T lymphocytes (CTL) have been detected in the peripheral blood of B-cell chronic lymphocytic leukemia (B-CLL) patients, but display low functional avidity, which limits their clinical utility in adoptive cell transfer approaches. To overcome this key obstacle hindering effective immunotherapy, we isolated an HLA-A2-restricted T-cell receptor (TCR) with high avidity for human TERT from vaccinated HLA-A*0201 transgenic mice. Using several relevant humanized mouse models, we demonstrate that TCR-transduced T cells were able to control human B-CLL progression in vivo and limited tumor growth in several human, solid transplantable cancers. TERT-based adoptive immunotherapy selectively eliminated tumor cells, failed to trigger a self-MHC-restricted fratricide of T cells, and was associated with toxicity against mature granulocytes, but not toward human hematopoietic progenitors in humanized immune reconstituted mice. These data support the feasibility of TERT-based adoptive immunotherapy in clinical oncology, highlighting, for the first time, the possibility of utilizing a high-avidity TCR specific for human TERT. Cancer Res; 76(9); 2540-51. ©2016 AACR.