Phase I trial of interleukin-2 and high-dose arginine butyrate in metastatic colorectal cancer

Interleukin-2 (IL-2) and sodium butyrate allow rats to be cured of peritoneal carcinomatosis from colon cancer. We performed a phase I trial of IL-2 and high-dose arginine butyrate (ArgB) in patients with advanced metastatic colorectal cancer. Patients and methods: From April to July 1997, six patients were included in the trail; they had a median age of 52 years, four had a performance status of 0, two had a performance status of 1 with normal biological functions. All patients had received at least two prior lines of chemotherapy. A fixed dose of 18 MIU/m² IL-2,was administered by subcutaneous injection and ArgB was delivered via continuous intravenous infusion on days 1–6 with escalating doses starting at 2 g kg⁻¹ day⁻¹. Results: The planned dose escalation was not possible because of toxicities. A daily ArgB dose of 2 g/kg was delivered for nine cycles. Level 2 (4 g/kg) could not be delivered in three of the six patients because of liver toxicity. The dose-limiting toxicities were fatigue and liver function disturbances. The maximum tolerated dose for ArgB was 3 g kg⁻¹ day⁻¹, in combination with IL-2 at 12 MIU m² day⁻¹. No clinical response was seen. Pharmacokinetic analysis showed large intra- and interindividual variations. Conclusion: This schedule with a high dose of ArgB proved to be highly toxic with liver insufficiency. We will be running another trial with lower doses of ArgB calculated from the schedule used in the experimental model, starting at a dose of 20 mg kg⁻¹ day⁻¹ for ArgB and 200 000 UI kg⁻¹ day⁻¹ IL-2, every 8 h. Received: 13 May 1999 / Accepted: 28 October 1999     

V gamma 9V delta 2 T cell response to colon carcinoma cells

During analysis of CD8 T cells derived from ascites of a colon cancer patient, we isolated a Vgamma9Vdelta2 T cell clone showing strong reactivity against autologous tumor cell lines. This clone killed a large fraction of allogeneic colon carcinoma and melanoma cell lines, but did not affect a normal colon cell line, colon fibroblasts, or melanocytes. Tumor cell recognition was TCR and NKG2D dependent and induced TNF-alpha and IFN-gamma secretion by the clone; accordingly, tumor targets expressed several NKG2D ligands, such as MHC class I chain-related gene A and UL16-binding protein molecules. Colon tumor recognition by Vgamma9Vdelta2 T cells was highly dependent on isopentenyl pyrophosphate production and ICAM-1 expression by target cells. Finally, similar reactivity patterns against colon carcinoma cell lines were observed using polyclonal Vgamma9Vdelta2 T cells of various origins, and Vgamma9Vdelta2 lymphocytes were present in the majority of colon tumor samples studied. Together, these results suggest that Vgamma9Vdelta2 T cells contribute to the natural immune surveillance against colon cancers. Therefore, this study provides a strong rationale for the use of Vgamma9Vdelta2 T cell agonists in immunotherapies targeting colon tumors.     

Phase-I study of Innacell γδ™, an autologous cell-therapy product highly enriched in γ9δ2 T lymphocytes, in combination with IL-2, in patients with metastatic renal cell carcinoma

PURPOSE: gamma9delta2 T lymphocytes have been shown to be directly cytotoxic against renal carcinoma cells. Lymphocytes T gammadelta can be selectively expanded in vivo with BrHPP (IPH1101, Phosphostim) and interleukin 2 (IL-2). A phase I Study was conducted in patients with metastatic renal cell carcinoma (mRCC) to determine the maximum-tolerated dose and safety of Innacell gammadeltatrade mark, an autologous cell-therapy product based on gamma9delta2 T lymphocytes, in patients with mRCC. EXPERIMENTAL DESIGN: A 1-h intravenous infusion of gamma9delta2 T lymphocytes was administered alone during treatment cycle 1 and combined with a low dose of subcutaneous interleukin-2 (IL-2, 2 MIU/m(2) from Day 1 to Day 7) in the two subsequent cycles (at 3-week intervals). The dose of gamma9delta2 T lymphocytes was escalated from 1 up to 8 x 10(9) cells. RESULTS: Ten patients underwent a total of 27 treatment cycles. Immunomonitoring data demonstrate that gamma9delta2 T lymphocytes are initially cleared from the blood to reappear at the end of IL-2 administration. Dose-limiting toxicity occurred in one patient at the dose of 8 x 10(9) cells (disseminated intravascular coagulation). Other treatment-related adverse events (AEs) included mainly gastrointestinal disorders and flu-like symptoms (fatigue, pyrexia, rigors). Hypotension and tachycardia also occurred, especially with co-administered IL-2. Six patients showed stabilized disease. Time to progression was 25.7 weeks. CONCLUSION: The data collected in ten patients with mRCC indicate that repeated infusions of Innacell gammadeltatrade mark at different dose levels (up to 8 x 10(9) total cells), either alone or with IL-2 is well tolerated. These results are in favor of the therapeutic value of cell therapy with Innacell gammadeltatrade mark for the treatment of cancers.     

Changes in erythrocyte morphology induced by imipramine and chlorpromazine

Chlorpromazine (CPZ), a phenothiazine derivative, is a potent antipsychotic agent and imipramine (IP) is a widely used tricyclic antidepressant. The interaction between these molecules and erythrocyte membranes is of particular interest considering the role of these cells in the transport and release of these drugs at the central nervous system. In the present paper, we intend to study the effects of IP on erythrocyte membranes and to compare these effects with those of CPZ. Erythrocytes from adult Sprague-Dawley rats were incubated separately with different concentrations of IP or CPZ for lh at room temperature, fixed and stained by Giemsa. Changes in erythrocyte morphology were quantified by an image analysis system. The interaction of both drugs, CPZ and IP, with the erythrocyte membrane causes similar changes in cell shape. Increasing concentrations of both drugs induces the formation of stomatocytes, spherostomatocytes and spherocytes, because of an irreversible loss of area and volume, probably due to endovesiculation. Our results also show that the CPZ is more potent than IP.     

Phase I study of bromohydrin pyrophosphate (BrHPP, IPH 1101), a Vγ9Vδ2 T lymphocyte agonist in patients with solid tumors

Purpose

Vγ9Vδ2 (γδ) T lymphocytes, a critical peripheral blood lymphocyte subset, are directly cytotoxic against many solid and hematologic tumor types. Vγ9Vδ2 T lymphocytes can be selectively expanded in vivo with BrHPP (IPH1101) and IL-2. The present phase I trial was conducted with the aim of determining the maximum-tolerated dose (MTD) and safety of IPH1101 combined with a low dose of IL-2 in patients with solid tumors.

Experimental design

A 1-h intravenous infusion of IPH11 was administered alone at cycle 1, combined with a low dose of SC IL-2 (1 MIU/M² d1 to d7) in the subsequent cycles (day 1 every 3 weeks). The dose of IPH1101 was escalated from 200 to 1,800 mg/m².

Results

As much as 28 patients with solid tumors underwent a total of 109 treatment cycles. Pharmacodynamics data demonstrate that γδ T lymphocyte amplification in humans requires the co-administration of IL-2 and is dependent on IPH 1101 dose. Dose-limiting toxicity occurred in two patients at a dose of 1,800 mg/m²: one grade 3 fever (1 patient) and one grade 3 hypotension (1 patient) suggesting cytokine release syndrome immediately following the first infusion. At lower doses the treatment was well tolerated; the most frequent adverse events were mild fever, chills and abdominal pain, without exacerbation in the IL-2 combined cycles.

Conclusion

IPH1101 in combination with SC low-dose IL-2 is safe, well tolerated and induces a potent γδ T lymphocyte expansion in patients. Its clinical activity will be evaluated in phase II clinical trials.     

Neutrophils, dendritic cells and Toxoplasma

Toxoplasma gondii rapidly elicits strong Type 1 cytokine-based immunity. The necessity for this response is well illustrated by the example of IFN-gamma and IL-12 gene knockout mice that rapidly succumb to the effects of acute infection. The parasite itself is skilled at sparking complex interactions in the innate immune system that lead to protective immunity. Neutrophils are one of the first cell types to arrive at the site of infection, and the cells release several proinflammatory cytokines and chemokines in response to Toxoplasma. Dendritic cells are an important source of IL-12 during infection with T. gondii and other microbial pathogens, and they are also specialized for high-level antigen presentation to T lymphocytes. Tachyzoites express at least two types of molecules that trigger innate immune cell cytokine production. One of these involves Toll-like receptor/MyD88 pathways common to many microbial pathogens. The second pathway is less conventional and involves molecular mimicry between a parasite cyclophilin and host CC chemokine receptor 5-binding ligands. Neutrophils, dendritic cells and Toxoplasma work together to elicit the immune response required for host survival. Cytokine and chemokine cross-talk between parasite-triggered neutrophils and dendritic cells results in recruitment, maturation and activation of the latter. Neutrophil-empowered dendritic cells possess properties expected of highly potent antigen presenting cells that drive T helper 1 generation.     

Toxoplasma gondii triggers myeloid differentiation factor 88-dependent IL-12 and chemokine ligand 2 (monocyte chemoattractant protein 1) responses using distinct parasite molecules and host receptors

Toll-like receptors (TLR) that signal through the common adaptor molecule myeloid differentiation factor 88 (MyD88) are essential in proinflammatory cytokine responses to many microbial pathogens. In this study we report that Toxoplasma gondii triggers neutrophil IL-12 and chemokine ligand 2 (CCL2; monocyte chemoattractant protein 1) production in strict dependence upon functional MyD88. Nevertheless, the responses are distinct. Although we identify TLR2 as the receptor triggering CCL2 production, parasite-induced IL-12 release did not involve this TLR. The production of both IL-12 and CCL2 was increased after neutrophil activation with IFN-gamma. However, the synergistic effect of IFN-gamma on IL-12, but not CCL2, was dependent upon Stat1 signal transduction. Although IL-10 was a potent down-regulator of Toxoplasma-triggered neutrophil IL-12 release, the cytokine had no effect on parasite-induced CCL2 production. Soluble tachyzoite Ag fractionation demonstrated that CCL2- and IL-12 inducing activities are biochemically distinct. Importantly, Toxoplasma cyclophilin-18, a molecule previously shown to induce dendritic cell IL-12, was not involved in neutrophil IL-12 production. Our results show for the first time that T. gondii possesses multiple molecules triggering distinct MyD88-dependent signaling cascades, that these pathways are independently regulated, and that they lead to distinct profiles of cytokine production.     

Cross-talk in the innate immune system: neutrophils instruct recruitment and activation of dendritic cells during microbial infection

Type I inflammatory cytokines are essential for immunity to many microbial pathogens, including Toxoplasma gondii. Dendritic cells (DC) are key to initiating type 1 immunity, but neutrophils are also a source of chemokines and cytokines involved in Th1 response ignition. We found that T. gondii triggered neutrophil synthesis of CC chemokine ligand (CCL)3, CCL4, CCL5, and CCL20, chemokines that were strongly chemotactic for immature DC. Moreover, supernatants obtained from parasite-stimulated polymorphonuclear leukocytes induced DC IL-12(p40) and TNF-alpha production. Parasite-triggered neutrophils also released factors that induced DC CD40 and CD86 up-regulation, and this response was dependent upon parasite-triggered neutrophil TNF-alpha production. In vivo evidence that polymorphonuclear leukocytes exert an important influence on DC activation was obtained by examining splenic DC cytokine production following infection of neutrophil-depleted mice. These animals displayed severely curtailed splenic DC IL-12 and TNF-alpha production, as revealed by ex vivo flow cytometric analysis and in vitro culture assay. Our results reveal a previously unrecognized regulatory role for neutrophils in DC function during microbial infection, and suggest that cross-talk between these cell populations is an important component of the innate immune response to infection.     

pH-dependent effects of chlorpromazine on liposomes and erythrocyte membranes

Chlorpromazine (CPZ) is an amphipathic antipsychotic drug that binds to erythrocytes reaching in this way the central nervous system. CPZ is a basic molecule with pK=8.6. This paper reports on CPZ-induced lysis of red blood cells and liposomes. Haemolysis was tested under hypotonic conditions, in the pH range 5.0-10.0. Cell sensitivity towards CPZ increased with increasing pH. Increasing pH caused also a decrease in the critical micellar concentrations of CPZ. These results are interpreted in terms of a competition between repulsive electrostatic forces and attractive hydrophobic forces, that would act both in pure CPZ and in mixed CPZ-phospholipid micelles. In order to eliminate possible pH effects mediated by red blood cell proteins, experiments were carried out in which CPZ induced release of a fluorescent dye from liposomes (large unilamellar vesicles). The latter observations confirmed that membrane sensitivity towards CPZ was increased at higher pH.