Targeting of CD4+CD25high cells while preserving CD4+CD25low cells with low-dose chimeric anti-CD25 antibody in adoptive immunotherapy of cancer.

The CD4+CD25high regulatory T (Treg) cells have been demonstrated to negatively modulate anti-tumor immune responses in cancer patients. In this study, effects of low dose anti-CD25 antibody (Ab) to attenuate Treg cells were investigated in cancer patients in vitro and in vivo. Peripheral blood mononuclear cells (PBMCs) from cancer patients were cultivated in vitro in the presence of a high-affinity chimeric anti-CD25 Ab (basiliximab). The CD4+CD25high population, interferon-gamma (IFN-gamma) production and FOXP3 expression were analyzed using flow cytometry (FCM), enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, respectively. During in vivo studies, basiliximab was administered intravenously on day 1, followed by AIT using autologous activated lymphocytes on day 8, and the treatment cycle was repeated. Subjective and objective effects were observed, and patients' PBMCs were subjected to FCM and RT-PCR analysis. In vitro analysis revealed that a low concentration of 0.01 microg/ml basiliximab reduced almost all of CD4+CD25high cells, but less of the CD4+ CD25low cells, and augmented IFN-gamma production of activated PBMCs. FOXP3 mRNA expression of PBMCs was not affected with or without basiliximab. An in vivo study of 9 metastatic cancer patients (7 colorectal and 2 esophageal) demonstrated no subjective or objective adverse effects, even under repeated administration of basiliximab. The results suggested that low-dose basiliximab can safely be administered repeatedly, and can target CD4+CD25high Treg cells whilst relatively preserving CD4+CD25low activated T cells. The host conditioning with low-dose basiliximab may augment the efficacy of AIT for cancer using activated autologous lymphocytes.

A phase I trial of CPT-11 and S-1 combination chemotherapy in patients with metastatic colorectal cancer.

BACKGROUND/AIMS: To establish a safe and practical chemotherapeutic regimen using CPT-11 in combination with an oral 5FU derivative S-1 for patients with metastatic colorectal cancer, a phase I clinical trial was conducted in an outpatient setting. METHODOLOGY: Nine patients with metastatic colorectal cancer were enrolled. S-1 was administered at approved doses of 80 mg/body/day to eligible patients with a body surface area (BSA) of less than 1.25m2, 100 mg/body/day to those with a BSA of 1.25-1.5m2, and 120 mg/body/day to those with a BSA of more than 1.5m2, for 2 weeks followed by 1 week rest, comprising one treatment cycle of 3 weeks. CPT-11 was administered on day 8 of the S-1 cycle. The dose of CPT-11 was escalated from 60-120 mg/m2 by every 20 mg/m2 for every cohort consisting of at least 3 patients in order to define dose-limiting toxicity (DLT), maximal tolerated dose (MTD), and recommended dose (RD) in preparation for a phase II trial. RESULTS: In regard to the hematologic toxicity, a decrease of WBC to less than grade 2 was observed in 2 patients until the dose was escalated to 100 mg/m2 of CPT-11, which delayed the treatment for 1 week in 1 patient. Regarding non-hematologic toxicity, fatigue and gastrointestinal toxicity, including anorexia and nausea/vomiting, at grades 1 and 2 were commonly observed throughout the dose levels. Diarrhea at grade 3 was observed at the 4th cycle of 100 mg/m2 CPT-11 in 2 of 3 patients, both of whom required hospitalization. All patients were able to complete more than 3 treatment cycles, and 1 patient at 80 mg/m2 of CPT-11 was able to receive 31 treatment cycles. Observed tumor responses included 1 partial response (PR), 2 moderate responses, 4 stable diseases, and 2 progressive diseases. Serum CEA level decreased in 7 of the 9 patients enrolled. CONCLUSIONS: These results suggest that this treatment regimen using CPT-11 in combination with oral S-1 therapy is a safe regimen in an outpatient setting and effective for patients with metastatic colorectal cancer. The DLT is diarrhea at a MTD of 100 mg/m2 of CPT-11, and 80 mg/m2 CPT-11 is recommended for the next phase II trial.

[A resected case of postoperative liver metastasis of a gastrointestinal stromal tumor showing complete response after imatinib treatment].

A 71-year-old man visited the hospital complaining of nausea in December 2002. Following a diagnosis of a gastrointestinal stromal tumor (GIST), partial resection of the stomach was performed in January 2003. The tumor was immunohistochemically positive for c-kit and CD34. The tumor size was 6.5 x 5.0 x 4.5 cm with a mitotic index of 25 out of 50 in the high-power field. The pathological diagnosis indicated a high-risk GIST. Treatment with imatinib at a dose of 400 mg/day was started because of liver metastasis of the GIST in January 2004. The liver metastasis was gradually reduced and exhibited cystic change. We considered that there was a complete response without accumulation by FDG-PET in June 2007. An hepatic segmentectomy was performed and imatinib was discontinued in July 2007. Most intratumorale in the specimen underwent hyaline degeneration after pathological examination, but there were viable cells in a portion of the tumor border. Imatinib treatment was resumed because of recurrence in the remnant stomach four months postoperatively owing to imatinib withdrawal. In making a diagnosis at the cell level by FDG-PET, it was difficult to determine the effectiveness of imatinib, and therefore, it is suggested that imatinib treatment must be continued after surgical resection.

Postoperative immunosuppression cascade and immunotherapy using lymphokine-activated killer cells for patients with esophageal cancer: possible application for compensatory anti-inflammatory response syndrome.

Immunological parameters were measured in order to elucidate a postoperative immunosuppression mechanism in transthoracic esophagectomy for patients with esophageal cancer. Moreover, lymphokine-activated killer (LAK) cells were transferred just after the surgery to overcome the postoperative immunosuppression. Fifteen consecutive patients who underwent transthoracic esophagectomy were subjected to the postoperative measurement of immunological parameters. Ten patients who underwent open cholecystectomy served as controls. Heparinized venous blood was obtained pre- and postoperatively, and serum levels of cytokines IL-6 and IL-10 and immunosuppressive acidic protein (IAP) were measured. Peripheral blood lymphocytes were harvested and analyzed by flow cytometry for phenotype detection and by a mixed lymphocyte reaction for detecting concanavalin (Con)-A-induced or -non-induced suppressor activity. Another 29 consecutive patients who underwent transthoracic esophagectomy were randomly enrolled in a postoperative immunotherapy trial either with or without lymphokine-activated killer cells. It was found that, in the esophagectomy group, IL-6 and IL-10 increased postoperatively and peaked on day 1, followed by an increase in IAP, peaked again on day 4, with a profound decrease in helper and cytotoxic T-cell subsets, followed by increases in Con-A-induced (on day 7 or later) and spontaneous (on day 10) suppressor activities. These changes were minimal in the cholecystectomy group. LAK cell transfer restored the postoperative decrease in the helper and cytotoxic T-cell population, and there was a trend of reduction for postoperative remote infection such as pneumonia and surgical site infection in the LAK therapy group. Taken together, we would like to propose the existence of a postoperative immunosuppression cascade consisting of increases in cytokines and immunosuppressive proteins, decreases in helper and cytotoxic T-cell populations, and the development of suppressor T-cell activities in surgery for esophageal cancer. Postoperative adoptive transfer of LAK cells may be a novel clinical application in surgery for esophageal cancer as a means of treating this postoperative immunosuppressive condition that may be identical to the status of compensatory anti-inflammatory response syndrome (CARS).

Adoptive immunotherapy using autologous lymphocytes sensitized with HLA class I-matched allogeneic tumor cells.

A 29-year-old female breast cancer patient with multiple bone metastases (HLA-A2) was treated with adoptive transfer using autologous peripheral blood mononuclear cells (PBMCs) activated with the HLA-A2-matched allogeneic GC022588 gastric cancer cell line and interleukin-2 plus an immobilized anti-CD3 antibody culture system. The relief of bone pain in parallel with a decrease of serum carcinoembryonic antigen levels was obtained just after the administration of GC022588-activated effector lymphocytes, and a good quality of life was accomplished for 4 months. The GC022588-activated effector lymphocytes included 44% CD4+, 77% CD8+, and 26% CD4+CD8+ phenotypes, and expressed 25% killing activity against GC022588 stimulator cells at an E/T ratio of 50:1. T cell receptor (TCR) usage analysis for the effector cells showed oligoclonal expression of TCRVbeta1, 3, 9, and 11, especially TCRVbeta5.2, 12, 13.1 and 17, and their killing activity was significantly inhibited in the presence of anti-TCRalphabeta antibody and anti-TCRVbeta12 antibody. SSCP analysis revealed clonotypic bands of TCRVbeta12. These results suggest that shared antigens exist between breast and gastric adenocarcinomas. Allogeneic tumor cells can stimulate PBMCs to generate effector cells with selected TCRCDR3 usages that recognize tumor antigens. These effector lymphocytes may be good candidates for the adoptive immunotherapy of cancer.

Essential requirement of toll-like receptor 4 expression on CD11c+ cells for locoregional immunotherapy of malignant ascites using a streptococcal preparation OK-432.

Toll-like receptors (TLRs) are important molecules that stimulate the innate immunity in order to eradicate microbial pathogens, after which the adaptive immunity emerges. The involvement of TLRs in the action mechanism of OK-432, a bacterial preparation, was investigated in the locoregional treatment of malignant ascites from gastric cancer. The expression of TLRs in ascites cells was analyzed using reverse-transcription polymerase chain reaction specific for TLRs and by flow cytometry using anti-TLR2, -TLR4, -CD4, -CD8, and -CD11c antibodies. These measurements were compared with the locoregional response of OK-432 immunotherapy for malignant ascites, as well as TNF-alpha producing potential, which was measured by ELISA, of ascites cells stimulated in vitro with OK-432. It was observed that OK-432 immunotherapy for malignant ascites showed 8 positive (67%) and 4 negative responses with the tolerable adverse effects of fever elevation and abdominal pain. The TNF-alpha production of ascites cells by in vitro OK-432 stimulation was significantly higher in responder patients than in non-responders. The clinical responses were correlated with the expression of the TLR4 gene of ascites cells. The TNF-alpha-producing potential of ascites cells by in vitro OK-432 stimulation was dependent on the existence of a CD11c + TLR-4+ cell population in ascites cells. OK-432 was highly stimulatory for TNF-alpha production of ascites cells compared with other biological response modifiers of PSK and LEM. These results suggest that TLR-4 expression on ascites cells of a macrophage lineage is essential for ascites cells to produce TNF-alpha in relation to OK-432 stimulation and for subsequent positive clinical responses in locoregional immunotherapy using OK-432 for malignant ascites from gastric cancer.

HER2-specific cytotoxic activity of lymphokine-activated killer cells in the presence of trastuzumab.

We investigated whether trastuzumab, a humanized anti-HER2 monoclonal antibody, could induce HER2-specific cytotoxic activity on lymphokine-activated killer (LAK) cells. Trastuzumab alone was not toxic to the HER2-positive breast cancer cell lines MDA-MB453 and ZR75-1, nor to the HER2-negative breast cancer cell lines MDA-MB468 and MCF-7. LAK cells, which were activated with 1000 U/ml IL-2 for 4 days (4-day LAK), showed cytotoxic activity against the MDA-MB453, ZR75-1 and MCF-7 cells, but not against MDA-MB468 cells. LAK cell cytotoxic activity against the HER2-positive breast cancer cell lines MDA-MB453 and ZR75-1 was significantly augmented in the presence of 10 nM trastuzumab, but that against the HER2-negative breast cancer cell lines MDA-MB468 and MCF-7 was not. The cytotoxic activity of LAK cells plus trastuzumab against the MDA-MB453 cells was significantly inhibited by the addition of cold MDA-MB453 cells or cold ZR75-1 cells, but not by addition of cold MDA-MB468 cells. Twenty-nine percent of the 4-day LAK cells were CD16+, and the cytotoxicity of LAK cells plus trastuzumab was abrogated with the anti-CD16 antibody treatment of the LAK cells in the cytotoxicity assay. Only 7% of the 10-day LAK cells were CD16+, and the 10-day LAK cells failed to exhibit cytotoxicity even with trastuzumab. These results suggest that HER2-specific cytotoxic activity, which is mediated by an antibody-dependent cellular cytotoxicity (ADCC) mechanism, can be induced on LAK cells by the addition of trastuzumab.

Feasibility study of adoptive immunotherapy for metastatic lung tumors using peptide-pulsed dendritic cell-activated killer (PDAK) cells.

We have established a novel culture system to generate effector lymphocytes designated as peptide-pulsed dendritic cell-activated killer (PDAK) cells using cultured dendritic cells (DCs), synthetic peptide, peripheral blood lymphocytes, and interleukin-2 plus immobilized anti-CD3 antibody. A feasibility study of an adoptive immunotherapy trial using PDAK cells was conducted on HLA-A2 and HLA-A24 cancer patients with antigen-positive lung metastasis that was defined by serological analysis or PCR analysis. Eleven patients with lung metastasis participated in the study: 6 with colorectal cancer, 2 with pancreatic cancer, 1 each with breast and lung cancer, and 1 with melanoma. The patients received either Muc-1, CEA, gpl00, Her-2 or SART-3-PDAK cells generated in vitro, intravenously in combination with 350,000 U IL-2 weekly for 9 weeks, together with a planned dose-escalation schedule of three transfers each of 1 x 10(7), 3 x 10(7) and 1 x 10(8) PDAK cells/kg for 6 patients, and with a uniform dose of 3 x 10(7) PDAK cells/kg for the remaining 5 patients. Peptide/HLA-specific cytotoxic activity and TCRVbeta gene usage of PDAK cells were analyzed. All transfers of PDAK cells, which showed peptide/HLA-specific lysis, were well-tolerated in all patients, and adverse effects (elevation of transaminase, fever, and headache) were observed primarily at grade 1, but in no case greater than grade 2. The generation of sufficient cells to treat the patients with 3 x 10(7) PDAK cells/kg was feasible using our culture system, but we were able to generate and administer the dose of 1 x 10(8) PDAK cells/kg in only one patient. One partial response (PR) of lung metastasis occurred in a pancreatic cancer patient who received 3 x 10(7) Muc-1-PDAK cells/kg. The cytolytic units of PDAK cells in this patient appeared to be substantially higher compared to those in PD patients. TCR gene usage analysis on PDAK cells revealed preferential usage of TCRVbeta segments. These results suggest that adoptive immunotherapy using PDAK cells for cancer patients with antigen-positive lung metastasis is safe and feasible, and tumor response should be examined in a future clinical trial

Neoadjuvant therapy of rectal cancer using oral tegaful-uracil (UFT) plus concomitant radiotherapy--a case report.

A 59-year-old male patient with rectal cancer 2 cm in diameter (T2) at the peritoneal reflection with suspicious left lateral node metastasis was treated with 400 mg of preoperative oral uracil and tegaful (UFT) for 5 weeks, 5 days a week in combination with concomitant radiotherapy of 45 Gy per 25 fractions for 5 weeks. After resting for another 5 weeks, colon fiberscopy, barium enema, and computed tomography revealed a trace of the primary tumor and a 40% shrinkage of the lateral metastasis. The serum CEA level decreased to the normal range during treatment. The adverse effects were nausea, bloody stool and elevation of transaminase, all at grade 1. Low anterior resection with a left hemi-lateral lymphadenectomy was performed through a suprapubic, one hand-size incision without laparoscopy. The preoperative treatment did not affect any operative procedures, and no postoperative complications occurred. The surgical specimen showed that the rectal tumor had been remarkably shrunk by the preoperative treatment, to the level of a superficial type tumor. Histological analysis indicated moderately differentiated adenocarcinoma cells that were present at only 2 mm in diameter in the mucosal layer, 6 mm in the submucosal layer, and 1 mm or less in the muscular layer with scar formation. No metastasis was detected in the 16 lymph nodes dissected, but an organizing tumor thrombus, which had preoperatively been diagnosed as lateral node metastasis, was detected. These results suggest that preoperative oral UFT plus concomitant radiotherapy may be a feasible, tolerable and effective treatment for patients with rectal cancer.

FOLFIRI plus bevacizumab as a first-line treatment for Japanese patients with metastatic colorectal cancer: a JACCRO CC-03 multicenter phase II study.

PURPOSE: The purpose of this multicenter phase II study was to evaluate the efficacy and safety of a combination of irinotecan, 5-fluorouracil (5-FU), and leucovorin (FOLFIRI) plus bevacizumab as first-line chemotherapy in Japanese patients with metastatic colorectal cancer. METHODS: Patients with metastatic colorectal cancer were eligible for enrollment. On day 1 of a 14-day cycle, patients received bevacizumab 5 mg/kg, irinotecan 150 mg/m², and L-leucovorin 200 mg/m² as an intravenous infusion, followed by 5-FU 400 mg/m² as an intravenous bolus and then 5-FU 2,400 mg/m² as an 46-h intravenous infusion. This treatment cycle was repeated. The primary endpoint was progression-free survival (PFS). RESULTS: We enrolled 40 patients, but one withdrew consent before starting treatment. The remaining 39 patients received a total of 509 cycles of FOLFIRI plus bevacizumab (median 11 per patient; range 1-30). The median PFS was 11.5 months, the median overall survival (OS) was 22.0 months, and the 1-year OS rate was 81.8 %. All 39 patients had adverse events. Grade 3 or 4 neutropenia and stomatitis occurred in 21 (53.9 %) and 4 (10.3 %) patients, respectively. CONCLUSION: Our results suggest that FOLFIRI plus bevacizumab is a clinically effective regimen with a manageable toxicity profile as first-line chemotherapy in patients with metastatic colorectal cancer.

Phase I trial of oxaliplatin plus S-1 chemotherapy in patients with metastatic colorectal cancer.

Advanced colorectal cancer can be effectively treated with S-1, as well as with a combination of oxaliplatin, 5-fluorouracil (5FU) and leucovorin (LV). However, S-1 together with oxaliplatin may provide a more convenient alternative to 5FU/LV. To evaluate the performance of S-1 combined with oxaliplatin for patients with colorectal cancer, we conducted a Phase I clinical trial in an outpatient setting. We administered S-1 to 15 patients with advanced colorectal adenocarcinoma for two weeks followed by one week of rest. Oxaliplatin was also administered on day 1 of the S-1 cycle. The dose of oxaliplatin was increased from 40 to 85 mg/m(2) to define the maximum tolerated dose and recommended dose in preparation for a Phase II trial. We administered 102 courses of treatment to 15 patients. Grade 3 thrombocytopenia developed in only 1 patient at a dose of 85 mg/m(2) of oxaliplatin plus oral S-1. No other grade 3-4 toxicities developed. No dose-limiting toxicity developed at level 4 of our regimen (oxaliplatin 85 mg/m(2)), and the recommended dose for a Phase II trial was 85 mg/m(2) of oxaliplatin in an outpatient setting.