Positively charged cyclodextrins as effective molecular transporters of active phosphorylated forms of gemcitabine into cancer cells.

Positively charged cyclodextrins (PCCDs) are molecular carriers of particular interest for their ability to readily enter into cancer cells. Of main interest, guanidino- and aminoalkyl- PCCDs can be conveniently synthesized and form stable and strong inclusion complexes with various active molecules bearing phosphate groups. We have addressed here the challenge to deliver into cancer cells phosphorylated gemcitabine drugs well known for their instability and inability to permeate cell membranes. NMR data corroborated by semiempirical theoretical calculations have shown that aminoalkyl-CDs form sufficiently stable complexes with both mono- and tri-phosphate forms of gemcitabine by simple mixing of the compounds in aqueous solution at physiological pH. Confocal microscopy and radioactivity counting experiments revealed that the developed systems enabled phosphorylated gemcitabine to penetrate efficiently into aggressive human breast cancer cells (MCF7), eventually leading to a substantial reduction of IC50 values. Moreover, compared to free drugs, phosphorylated metabolites of gemcitabine encapsulated in PCCDs displayed improved in vitro activities also on the aggressive human cancer cells CCRF-CEM Ara-C/8 C, a nucleoside transport-deficient T leukemia cell line. The current study offers the proof-of-principle that phosphorylated nucleoside drugs could be efficiently transported by PCCDs into cancer cells.

Efficient "green" encapsulation of a highly hydrophilic anticancer drug in metal-organic framework nanoparticles.

Metal-organic frameworks (MOFs) are coordination polymers of interest for biomedical applications. Of particular importance, nanoparticles made of iron(III) trimesate (MIL-100, MIL standing for Material Institut Lavoisier) (nanoMOFs) can be conveniently synthesised under mild and green conditions. They were shown to be biodegradable, biocompatible and efficient to encapsulate a variety of active molecules. We have addressed here the challenges to encapsulate a highly hydrophilic anticancer prodrug, phosphated gemcitabin (Gem-MP) known for its instability and inability to bypass cell membranes. MIL-100 nanoMOFs acted as efficient "nanosponges", soaking Gem-MP from its aqueous solution with almost perfect efficiency (>98%). Maximal loadings reached ∼30 wt% reflecting the strong interaction between the drug and the iron trimesate matrices. Neither degradation nor loss of crystalline structure was observed after the loading process. Storage of the loaded nanoMOFs in water did not result in drug release over three days. However, Gem-MP was released in media containing phosphates, as a consequence to particle degradation. Drug-loaded nanoMOFs were effective against pancreatic PANC-1 cells, in contrast to free drug and empty nanoMOFs. However, an efflux phenomenon could contribute to reduce the efficacy of the nanocarriers. Size optimization and surface modification of the nanoMOFs are expected to further improve these findings.

A "ship in a bottle" strategy to load a hydrophilic anticancer drug in porous metal organic framework nanoparticles: efficient encapsulation, matrix stabilization, and photodelivery.

An essential challenge in the development of nanosized metal organic framework (nanoMOF) materials in biomedicine is to develop a strategy to stabilize their supramolecular structure in biological media while being able to control drug encapsulation and release. We have developed a method to efficiently encapsulate topotecan (TPT, 1), an important cytotoxic drug, in biodegradable nanoMOFs. Once inside the pores, 1 monomers aggregate in a "ship in a bottle" fashion, thus filling practically all of the nanoMOFs' available free volume and stabilizing their crystalline supramolecular structures. Highly efficient results have been found with the human pancreatic cell line PANC1, in contrast with free 1. We also demonstrate that one- and two-photon light irradiation emerges as a highly promising strategy to promote stimuli-dependent 1 release from the nanoMOFs, hence opening new standpoints for further developments in triggered drug delivery.

Upregulation of adhesion molecules on leukemia targets improves the efficacy of cytotoxic T cells transduced with chimeric anti-CD19 receptor.

T lymphocytes engineered to express chimeric antigen receptors (CARs) interact directly with cell surface molecules, bypassing MHC antigen presentation dependence. We generated human anti-CD19ζ CAR cytotoxic T lymphocytes and cytokine-induced killer cells and studied their sensitivity to the expression of adhesion molecules for the killing of primary B-lineage acute lymphoblastic leukemia (B-ALL) targets. Despite a very low basal expression of surface adhesion molecules, B-ALL blasts were lysed by the anti-CD19ζ-CAR transduced effectors as expected. We next investigated the regulatory role of adhesion molecules during CAR-mediated cytolysis. The blocking of these accessory molecules strongly limited the chimeric effector's cytotoxicity. Thereafter, B-ALL cells surface adhesion molecule level expression was induced by IFN-γ or by the combined use of CD40L and IL-4 and the cells were submitted to anti-CD19ζ-CAR transduced effectors lysis. Upregulation of adhesion molecules expression by blasts potentiated their killing. The improved cytotoxicity observed was dependent on target surface expression of adhesion molecules, particularly CD54. Taken together, these results indicate that adhesion molecules, and principally CD54, are involved in the efficiency of recognition by effector chimeric ζ. These observations have potential implications for the design of immunotherapy treatment approaches for hematological malignancies and tumors based on the adoption of CAR effector cells.

Impact of phosphorylation on the encapsulation of nucleoside analogues within porous iron(iii) metal-organic framework MIL-100(Fe) nanoparticles.

Encapsulation of azidothymidine (AZT) or its phosphorylated derivatives (AZT-MP and AZT-TP) has been performed using nanoparticles of the porous crystalline iron(iii) trimesate metal-organic framework MIL-100(Fe). The number of phosphate groups per nucleoside analogue has a high impact on the drug loading capacity, and their interaction with the Lewis acid sites from the nanoMOFs is also discussed through a combination of techniques such as UV-vis absorption, circular dichroism, isothermal titration calorimetry, HPLC and molecular simulations. Finally, the effect of the differences in terms of host-guest interactions is discussed through the release in physiological buffers of AZT, AZT-MP and AZT-TP. New perspectives for the nanoencapsulation of monophosphorylated nucleoside analogues for effective anti-cancer and anti-viral therapies are then discussed.

In vitro comparison of three different chimeric receptor-modified effector T-cell populations for leukemia cell therapy.

The identification of the optimal T-cell effector subtype is a crucial issue for adoptive cell therapy with chimeric receptor-modified T cells. The ideal T cell population must be able to home toward tumor site, exert prolonged antitumoral activity, and display minimal toxicity against normal tissues. Therefore, we characterized the in vitro antitumoral properties of three effector T-cell populations: Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs), cytokine-induced killer (CIK) cells, and γ9δ2 T (GDT) cells, after transduction with a chimeric receptor specific for the CD33 antigen, broadly expressed on acute myeloid leukemia cells. EBV-CTLs, CIK, and GDT cells were generated and transduced with high efficiency with a retroviral vector coding for an anti-CD33-ζ chimeric receptor without alterations of their native phenotype. Anti-CD33-ζ chimeric receptor-redirected T cells displayed analogous in vitro chemotactic activity toward CXCL12. In addition, anti-CD33-ζ chimeric receptor-expressing EBV-CTLs, CIK, and GDT cells showed potent and similar cytotoxicity against several CD33⁺ leukemic targets both in short-term 4-hours-5¹chromium-release assays (mean killing vs primary leukemic cells at effector:target ratio of 5:1; 50%, 61%, and 50% for EBV-CTLs, CIK, and GDT cells, respectively) and in long-term assays, where they were cocultured with leukemic cells for 6 days on stromal mesenchymal cells (mean survival of primary leukemic cells at effector:target ratio of 1:100; 18%, 16%, and 29% for EBV-CTLs, CIK, and GDT cells, respectively). Moreover, all effector cells acquired consistent capability to proliferate in vitro after contact with CD33⁺ cells and to release high and comparable levels of immunostimulatory cytokines, while secreting similar low amount of immunoregulatory cytokines as the unmanipulated counterpart. Our results indicate that expression of an anti-CD33-ζ chimeric receptor potently and similarly increase the antileukemic functions of different effector T-cell subtypes, underlying the impossibility to identify a more potent T-cell population through in vitro analysis, and consistently with recent observations that have emerged from clinical trials with chimeric receptor-modified T cells, suggesting the need to perform such type of studies in the human setting.

In vitro and in vivo model of a novel immunotherapy approach for chronic lymphocytic leukemia by anti-CD23 chimeric antigen receptor.

Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19(+)CD5(+)CD20(dim) B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23.CAR). CD23.CAR(+) T cells showed specific cytotoxic activity against CD23(+) tumor cell lines (average lysis 42%) and primary CD23(+) CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR(+) T cells released inflammatory cytokines (1445-fold more TNF-ß, 20-fold more TNF-α, and 4-fold more IFN-γ). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR(+) T cells. Redirected T cells were also effective in vivo in a CLL Rag2(-/-)γ(c)(-/-) xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23.CAR(+) T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23.CAR(+) T cells represent a selective immunotherapy for the elimination of CD23(+) leukemic cells in patients with CLL.

Cytokine-induced killer cells for cell therapy of acute myeloid leukemia: improvement of their immune activity by expression of CD33-specific chimeric receptors.

BACKGROUND: Cytokine-induced killer cells are ex vivo-expanded cells with potent antitumor activity. The infusion of cytokine-induced killer cells in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplant is well tolerated, but limited clinical responses have been observed. To improve their effector functions against acute myeloid leukemia, we genetically modified cytokine-induced killer cells with chimeric receptors specific for the CD33 myeloid antigen. DESIGN AND METHODS: SFG-retroviral vectors coding for anti-CD33-ζ and anti-CD33-CD28-OX40-ζ chimeric receptors were used to transduce cytokine-induced killer cells. Transduced cells were characterized in vitro for their ability to lyse leukemic targets (4-hour (51)chromium-release and 6-day co-cultures assays on human stromal mesenchymal cells), to proliferate ((3)H-thymidine-incorporation assay) and to secrete cytokines (flow cytomix assay) after contact with acute myeloid leukemia cells. Their activity against normal CD34(+) hematopoietic progenitor cells was evaluated by analyzing the colony-forming unit capacity after co-incubation. RESULTS: Cytokine-induced killer cells were efficiently transduced with the anti-CD33 chimeric receptors, maintaining their native phenotype and functions and acquiring potent cytotoxicity (up to 80% lysis after 4-hour incubation) against different acute myeloid leukemia targets, as also confirmed in long-term killing experiments. Moreover, introduction of the anti-CD33 chimeric receptors was accompanied by prominent CD33-specific proliferative activity, with the release of high levels of immunostimulatory cytokines. The presence of CD28-OX40 in chimeric receptor endodomain was associated with a significant amelioration of the anti-leukemic activity of cytokine-induced killer cells. Importantly, even though the cytokine-induced killer cells transduced with anti-CD33 chimeric receptors showed toxicity against normal hematopoietic CD34(+) progenitor cells, residual clonogenic activity was preserved. CONCLUSIONS: Our results indicate that anti-CD33 chimeric receptors strongly enhance anti-leukemic cytokine-induced killer cell functions, suggesting that cytokine-induced killer cells transduced with these molecules might represent a promising optimized tool for acute myeloid leukemia immunotherapy.

Exploration of the lysis mechanisms of leukaemic blasts by chimaeric T-cells.

Adoptive transfer of specific cytotoxic T lymphocytes (CTL) and Cytokine Induced Killer Cells (CIK) following genetic engineering of T-cell receptor zeta hold promising perspective in immunotherapy. In the present work we focused on the mechanisms of anti-tumor action of effectors transduced with an anti-CD19 chimaeric receptor in the context of B-lineage acute lymphoblastic leukemia (B-ALL). Primary B-ALL blasts were efficiently killed by both z-CD19 CTL and z-CD19 CIK effectors. The use of death receptor mediated apoptosis of target cells was excluded since agonists molecules of Fas and TRAIL-receptors failed to induce cell death. Perforin/granzyme pathway was found to be the mechanism of chimaeric effectors mediated killing. Indeed, cytolytic effector molecules perforin as well as granzymes were highly expressed by CTL and CIK. CD19 specific stimulation of transduced effectors was associated with degranulation as attested by CD107 membrane expression and high IFN-gamma and TNF-alpha release. Moreover inhibitors of the perforin-based cytotoxic pathway, Ca(2+)-chelating agent EGTA and Concanamycin A, almost completely abrogated B-ALL blast killing. In conclusion we show that the cytolysis response of z-CD19 chimaeric effectors is predominantly mediated via perforin/granzyme pathway and is independent of death receptors signaling in primary B-ALL.