Jean-Yves Douillard

An interleukin 2/sodium butyrate combination as immunotherapy for rat colon cancer peritoneal carcinomatosis

BACKGROUND/AIMS Immunotherapy using interleukin 2 has had disappointing results in the treatment of colon cancer. Overcoming escape mechanisms, such as lack of antigen presentation and absence of accessory adhesion molecules on cancer cells, may increase its efficiency. We tried to do so by modifying the phenotype of the weakly immunogenic rat colon cancer PROb cells with sodium butyrate. METHODS After in vitro treatment with butyrate, PROb cells were tested for lymphokine-activated killer cell sensitivity and, using cytofluorometry, expression of adhesion molecules. We then treated established PROb peritoneal carcinomatoses with intraperitoneal injections of interleukin 2 and butyrate. Tumors were studied histologically and immunohistochemically. We tested the specificity of the immune protection by subsequent subcutaneous challenges with either PROb or glioma cells and by Winns assay. RESULTS Butyrate increased lymphokine-activated killer cell sensitivity and expression of major histocompatibility complex class I and intercellular adhesion molecule 1 in vitro. Interleukin 2/butyrate combination resulted in cases of complete cure of carcinomatosis with specific protection against PROb cells. We noticed a complex stroma reaction with numerous functional antigen presenting cells close to PROb cells. CONCLUSIONS The complete regression of tumor masses may be attributed, at least in part, to a butyrate-induced increase in immunogenicity of the cancer cells. This new combined immunotherapy may be of interest in the treatment of colon cancer.

Apoptosis induced by sodium butyrate treatment increases immunogenicity of a rat colon tumor cell line

We have recently demonstrated that a treatment combining the cell differentiating agent sodium butyrate (NaBut) and interleukin-2 (IL2) resulted in a remission of established peritoneal colorectal carcinomatosis in rats. NaBut or IL2 treatment alone, never cured these tumour-bearing rats. In the present investigation, we report that NaBut-treatments induce apoptosis in the colonic cancer cells both in vitro and in vivo. We postulated that the significant therapeutic effect of NaBut/IL2 treatment can be mainly attributed to a NaBut-induced apoptosis of the tumoural cells increasing their immunogenicity. Indeed, treatment which combined apoptotic bodies (apobodies) as cell vaccine, plus IL2 immunotherapy significantly increased tumour remission and survival rate of the vaccinated rats, whereas IL2 treatment alone did not. We observed that the cured rats presented long-term protection against subsequent challenge with the parental tumour cells. This latter result suggests that these treatments generate an immune protection. This was confirmed by the presence, in the sera of the cured rats, of anti-tumoural antibodies directed against both the apobodies and the tumour cells, but not against normal colonocytes. In addition, we show that injections of apobodies before administration of the parental tumour cells results in a partial protection. We provide the first evidence that apobodies, derived from cancer cells after NaBut-treatment, induce a specific immune response against parental tumours cells. These data suggest that the distinctive immunologic properties of apobodies could provide a valuable tool in colorectal cancer immunotherapy.

Focus on the Controversial Activation of Human iNKT Cells by 4-Deoxy Analogue of KRN7000

4-Deoxy-alpha-GalCer analogues are considered weaker agonists than KRN7000 for the stimulation of human iNKT cells, but this remains strongly debated. In this work, we described a strategy toward 4-deoxy-alpha-GalCers with, as a key step, a metathesis reaction allowing sphingosine modifications from a single ethylenic alpha-galactoside precursor. The 4-deoxy-KRN7000 derivative 2, described here, induced potent cytokinic responses, comparable to those of KRN7000, both from human iNKT cells in vitro and from their murine counterpart in vivo.

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

Abstract Introduction: 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.

Defect in lectin-induce interleukin 2 (IL-2) production by peripheral blood lymphocytes of patients with Hodgkin's Disease

Abstract Peripheral blood lymphocytes (PBL) of patients with Hodgkins disease were studied for their capacity to produce interleukin 2 upon in vitro phytohemaglutinin stimulation in the presence or absence of either interleukin 1 or indomethacin (2 μ g/ml ); eight patients were studied at the discovery of their disease before receiving any therapy (onset HD; OHD). Seventeen patients were tested in long-term (>3 yr remission (remission HD; RHD); most RHD were treated with both chemotherapy and irradiation. Fourteen healthy individuals served as controls. PBL from OHD have a significant ( P P

3-Fluoro- and 3,3-Difluoro-3,4-dideoxy-KRN7000 Analogues as New Potent Immunostimulator Agents: Total Synthesis and Biological Evaluation in Human Invariant Natural Killer T Cells and Mice

We propose here the synthesis and biological evaluation of 3,4-dideoxy-GalCer derivatives. The absence of the 3- and 4-hydroxyls on the sphingoid base is combined with the introduction of mono or difluoro substituent at C3 (analogues 8 and 9, respectively) to evaluate their effect on the stability of the ternary CD1d/GalCer/TCR complex which strongly modulate the immune responses. Biological evaluations were performed in vitro on human cells and in vivo in mice and results discussed with support of modeling studies. The fluoro 3,4-dideoxy-GalCer analogues appears as partial agonists compared to KRN7000 for iNKT cell activation, inducing T(H)1 or T(H)2 biases that strongly depend of the mode of antigen presentation, including human vs mouse differences. We evidenced that if a sole fluorine atom is not able to balance the loss of the 3-OH, the presence of a difluorine group at C3 of the sphingosine can significantly restore human iNKT activation.

Pharmacokinetic study of radiolabeled anti-colorectal carcinoma monoclonal antibodies in tumor-bearing nude mice

Monoclonal antibodies (MoAbs) 17-1A and 19-9, which specifically bind human colorectal carcinoma (CRC) cells, were tested for their usefulness in localizing colorectal tumors in nude mice. One of the 131I-labeled MoAbs and an irrelevant 125I-labeled immunoglobulin of the same isotype were injected into nude mice simultaneously bearing a human CRC and a human melanoma. The percentage of the injected dose of antibody per gram of tissue, the CRC/tissue ratios of antibody distribution, and the localization indices were calculated at various time intervals (2 h to 9 days). For both MoAbs, labeling to a specific activity of 10 μCi/μg by the iodogen method gave optimum immunoreactivity. The accumulation of MoAb 17-1A in CRC reached its maximum at 5 days and remained at this level for up to 9 days postinjection. For MoAb 19-9, which detects a circulating antigen shed by the tumor into the serum, the accumulation in the CRC was maximum at 24 h, and decreased thereafter. The CRC/organ ratios and localization indices for both MoAbs increased with time in the CRC tissue, but remained low and unchanged in the melanoma and normal tissues. Using F(ab′)2 antibody fragments, faster kinetics with earlier maximum accumulation, higher tumor/organ ratios, and better localization indices were achieved than with intact MoAbs. The data obtained was useful in defining parameters which must be considered before radiolabeled MoAbs are used in cancer patients for diagnostic purposes.

Immunization against a rat colon carcinoma by sodium butyrate-treated cells but not by interleukin 2-secreting cells

BACKGROUND & AIMS Vaccination of patients with colon cancer with irradiated autologous tumor cells and bacille Calmette-Guérin (BCG) was reported to augment mean survival. It was recently observed that a local treatment combining recombinant interleukin 2 and the differentiation agent sodium butyrate cured rats with colon cancer peritoneal carcinomatosis. To optimize vaccination protocols, the comparison of the efficacy of irradiated tumor cells mixed with BCG with that of interleukin 2-gene-transfected cells and of tumor cells pretreated with sodium butyrate was performed. METHODS The poorly immunogenic rat colon carcinoma cells PROb were used in a vaccination assay. Interleukin 2-transfected PROb cells, either proliferating or irradiated, were used. The efficiency of irradiated PROb cells mixed with BCG, of interleukin 2-transfected cells, or of cells pretreated with sodium butyrate was tested. RESULTS Vaccination with irradiated parental cells and BCG did not provide protection. Irradiated interleukin 2-transfected cells were poorly efficient in the vaccination assay. Conversely, vaccination with irradiated parental cells pretreated with sodium butyrate before injection provided good protection. CONCLUSIONS Interleukin 2-secreting cells efficiently vaccinated animals when injected while replicating but not after irradiation. Conversely, sodium butyrate pretreatment provided a simple and efficient vaccination scheme that generated a long-term immune memory and allowed the use of irradiated cells.

Immunotherapy of gastrointestinal cancer with monoclonal antibodies

Twenty patients with widespread metastatic colorectal carcinoma were infused with 500 mg of the cytotoxic IgG2a monoclonal antibody 17-1A preincubated with autologous peripheral blood leukocytes. Ten patients showed no benefit from such therapy and ten died, with a mean survival time of 7.6±4.5 months after treatment and a median survival of 6 months. In ten additional patients, the sourse of disease was modified by antibody therapy; disease in five of these patients stabilized, while tumor size in the other five patients decreased after therapy. Median actual survival in this group of ten patients is presently 24 months; four of these patients died of disease progression within a mean of 15±5 months. Duration of response was 10.5±6.7 months after antibody treatment. Treatment tolerance for these 20 patients was excellent in all but one patient, who experienced an anaphylactic reaction during a second infusion with 17-1A.

Interleukin‐2/sodium butyrate treatment cures rats bearing liver tumors after acquired 5‐fluorouracil resistance

We studied the effect of immunotherapy using recombinant interleukin‐2 (rIL‐2) in combination with a differentiating agent, sodium butyrate (NaBut), on experimental 5‐fluorouracil (5‐FU)‐resistant liver metastasis from colorectal cancer in rats. For this purpose, we used direct liver injection of 5‐FU resistant cells, PRObR1, in syngeneic BDIX rats to establish liver tumors. The growth of liver metastasis was followed before and after NaBut/rIL‐2 treatment by magnetic resonance imaging (MRI). The presence of liver tumors was checked by MRI 7 days after tumor cell injection. Evaluable rats were then assigned randomly to a control and an experimental group. The different treatments were started on day 10 and administered intraperitoneally (i.p.). Combined NaBut/rIL‐2 treatment followed by MRI on days 56 and 91 was shown both to significantly reduce the growth of liver tumors and to prevent extrahepatic spread. In addition, NaBut/rIL‐2 treatment induced a complete regression in 50% of the rats which remained free of disease. Int. J. Cancer 78:735–739, 1998.