Differentiation of tracheal basal cells to ciliated cells and tissue reconstruction on the synthesized basement membrane substratum in vitro.

Although lung epithelial cells directly attach to the basement membrane underneath in vivo, harvested epithelial cells are typically cultured on type I collagen gel (Col I-gel) in vitro. Recently we developed new culture substratum, designated as "synthesized Basement Membrane" (sBM), that has bared lamina densa on fibrillar collagen. To validate the usefulness of sBM substratum in airway tissue reconstitution in vitro, we cultured rat tracheal epithelial cells on sBM substratum and Col I-gel. When starting the air-liquid interface culture, most of the epithelial cells were squamous and positive for the basal cell marker cytokeratin 14 (CK14). After 14 days on sBM substratum, CK14-positive cells differentiated not only to Clara and mucous cells, but also to ciliated cells. Those differentiated cells formed pseudostratified-like epithelium and the remaining CK14-positive cells were polarized to the basal side. However, on Col I-gel, the CK14-positive cells were still squamous and not polarized, and ciliated cells did not appear. In conclusion, we established a new culture model on sBM substratum in which basal cells could differentiate to ciliated cells. The application of sBM substratum is useful in the study of the airway epithelial cell differentiation in vitro.

Immunological evaluation of personalized peptide vaccination for patients with pancreatic cancer.

The prognosis of pancreatic cancer is extremely poor, and development of new treatment modalities is needed. One such treatment could be specific immunotherapy. To evaluate safety and immunological responses, we conducted a phase I study of personalized peptide vaccination for pancreatic cancer patients (n=11). Namely, pre-vaccination peripheral blood mononuclear cells were screened for their reactivity in vitro to each of 14 or 16 peptides in HLA-A24(+) or -A2(+) patients, and only the reactive peptides (maximum: 4) were vaccinated in vivo. This regimen was generally well tolerated, although inflammatory reactions at the injection site were observed in 7 patients. Delayed-type hypersensitivity to peptides used for vaccination was observed in 7 patients. Increased cellular and humoral immune responses to at least one of peptides used for vaccination were observed in the post-vaccination PBMCs and sera from 4 of 8 patients and 4 of 10 patients tested, respectively. The 6- and 12-month survival rates for patients who received >3 vaccinations (n=10) were 80% and 20%, respectively. Due to tolerability and capability of inducing specific immunity, further development of personalized peptide-based immunotherapy for pancreatic cancer patients is warranted.

Immunological evaluation of vaccination with pre-designated peptides frequently selected as vaccine candidates in an individualized peptide vaccination regimen.

We previously conducted a trial of an 'individualized' regimen, in which cancer patients were vaccinated with peptides after the confirmation of pre-existing peptide-specific cytotoxic T lymphocyte (CTL) precursors. In this study, we performed a new trial of 'pre-designated' regimen, in which cancer patients were vaccinated with peptides that were frequently selected as vaccine candidates in the preceding individualized regimen. Eighteen cancer patients (10 with uterine cervical cancer and 8 with gastric cancer) were enrolled in the new regimen. The pre-designated regimen was well tolerated by all patients. Although peptide-specific CTL precursors and humoral responses increased in the majority of patients with the pre-designated regimen, neither of the responses correlated with clinical outcome. Three patients had long stable disease, and their pre-vaccination peripheral blood mononuclear cells contained peptide-specific CTL precursors reactive to more than 2 of 4 peptides. With the pre-designated regimen, the levels of pre-existing immunoglobulin G reactive to non-vaccinated peptides decreased in 5 of 15 patients with progressive disease, and their time to progression was very short, whereas such a decrease was rarely observed in the preceding individualized regimen. These results suggest that the pre-designated regimen can elicit a primary immune response, but may incidentally suppress pre-existing immune responses.

Immunological monitoring during combination of patient-oriented peptide vaccination and estramustine phosphate in patients with metastatic hormone refractory prostate cancer.

BACKGROUND: Additive antitumor effects could be achieved by combination of immunotherapy and cytotoxic agents with no or minimum suppression. METHODS: Thirteen patients positive for human leukocyte antigen (HLA)-A24 or -A2 with metastatic hormone refractory prostate cancer (HRPC) who had failed to respond to the prior-peptide vaccination were entered in the combined peptide vaccination and estramustine phosphate. Conducted immune monitoring on those 13 patients were mainly peptide-specific cytotoxic T lymphocyte (CTL) precursor analysis by IFN-gamma productions and peptide-reactive IgG by an enzyme-linked immunosorbent assay (ELISA). RESULTS: Grade 3 arrhythmia or cerebral infarction was observed in two cases, and Grade 1 or 2 dermatologic reaction at the vaccination sites was observed in all 13 cases. Eleven patients who received more than one cycle of treatment were eligible for immunological and clinical evaluation. There was no significant immunosuppression in most cases when the peptide and a half dose (280 mg/day) of estramustine were administrated, whereas severe immunosuppression was observed in the first two patients who received both the peptide and a full dose (560 mg/day) estramustine. Augmentation of peptide-specific CTL precursors or peptide-specific IgG was observed in 6 of 11 or 10 of 11 cases, respectively. Ten of 11 patients showed serum prostate-specific antigen (PSA) level decrease from the baseline including 8 patients with a serum PSA level decrease of > or =50%. CONCLUSIONS: These results encouraged the further evaluation of the combination of peptide vaccination and low-dose estramustine phosphate for metastatic HRPC patients.

Humoral responses to peptides correlate with overall survival in advanced cancer patients vaccinated with peptides based on pre-existing, peptide-specific cellular responses.

PURPOSE: The aim of this study is to find a laboratory marker for overall survival in advanced cancer patients who were vaccinated with peptides based on pre-existing, peptide-specific CTL precursors in the circulation. EXPERIMENTAL DESIGN: A group of 113 patients with advanced cancer (28 colorectal, 22 prostate, 15 lung, 14 gastric, and 34 other cancers) was enrolled in a Phase I clinical study of peptide vaccination in which peptide-specific CTL precursors of prevaccination peripheral blood mononuclear cells were measured, followed by vaccination with these peptides (maximum of four). For cellular responses, pre and postvaccination (sixth) peripheral blood mononuclear cells were provided for measurement of both peptide-specific CTL precursors by IFN-gamma release assay and tumor reactivity by (51)Cr release assay. Delayed type hypersensitivity was also measured. For humoral response, pre and postvaccination (sixth) sera were provided for measurement of peptide-reactive IgG by an ELISA. RESULTS: The median survival time and 1-year survival rate of the total cases were 346 +/- 64.9 days and 44.6%, respectively, and those of patients vaccinated more than six times (n = 91) were 409 +/- 15 days and 54.4%, respectively. In these 91 patients, the overall survival of patients whose sera showed increased levels of peptide-reactive IgG (n = 60) was significantly more prolonged (P = 0.0003) than that of patients whose sera did not (n = 31), whereas none of cellular responses correlated with overall survival. CONCLUSIONS: Peptide-specific IgG in postvaccination sera could be a suitable laboratory maker for the prediction of prolonged survival in advanced cancer patients vaccinated with peptides based on pre-existing CTL precursors.

Identification of peptide vaccine candidates sharing among HLA-A3+, -A11+, -A31+, and -A33+ cancer patients.

PURPOSE: Only a few studies have been reported on CTL epitope peptides restricted with alleles other than HLA-A2 and -A24. The HLA-A11, -A31, and -A33 alleles share similar binding motifs with HLA-A3 and -A68 alleles, and, thus, are classified as an HLA-A3 supertype. This study tried to identify CTL epitope peptides as vaccine candidates sharing by HLA-A3(+), -A11(+), -A31(+), and -A33(+) cancer patients. EXPERIMENTAL DESIGN: Seven peptides possessing the ability to induce HLA-A31-restricted and tumor-reactive CTLs were examined for their ability to induce HLA-A3-, -A11-, and -A33-restricted and tumor-reactive CTLs from peripheral blood mononuclear cells (PBMCs) of 18 epithelial cancer patients. The five reference peptides all have the ability to induce CTL activity restricted with one of the HLA-A3 supertypes, and, thus, were also examined as positive controls. RESULTS: Three peptides (2 from beta-tublin5- and 1 from CGI37-derived peptides) induced tumor-reactive CTLs in PBMCs of HLA-A3(+), -A11(+), and -A33(+) cancer patients with various frequencies (17-50%). One RLI- or KIAA0036-derived peptide induced tumor-reactive CTLs in PBMCs of HLA-A3(+) and -A11(+) or HLA-A11(+) and -A33(+) cancer patients also with various frequencies (22-67%), respectively, whereas the other peptide induced CTL activity in only HLA-A33(+) patients. Among the five reference peptides tested, one peptide, TRP2-197, induced CTL activity in both HLA-A11(+)- and -A33(+)-restricted manners. CONCLUSIONS: We identified new peptide vaccine candidates for HLA-A3, -A11, -A31, and -A33 positive cancer patients. This study may facilitate the development of both basic and clinical studies of peptide-based immunotherapy for cancer patients with other alleles of HLA-A2 and -A24.

Phase I trial of patient-oriented vaccination in HLA-A2-positive patients with metastatic hormone-refractory prostate cancer.

To evaluate the safety and toxicity of peptide vaccination for patients with metastatic hormone-refractory prostate cancer (HRPC) based on pre-existing peptide-specific cytotoxic T-lymphocyte (CTL) precursors in the circulation, 10 patients positive for human leukocyte antigen (HLA)-A2 with metastatic HRPC were enrolled in a phase I study. Peptide-specific CTL-precursors reactive to 16 kinds of vaccine candidates in the pre-vaccination peripheral blood mononuclear cells (PBMCs) were measured, and patients were followed by vaccination with only positive peptides (up to 4 kinds of peptides). Serum prostate-specific antigen (PSA) levels were monitored regularly. The peptide vaccination was safe and well tolerated with no major adverse effects. The most common toxicities were dermatologic reactions at the injection site. Increased CTL response to peptides was observed in 4 of 10 patients. Anti-peptide IgG was also detected in post-vaccination sera of 7 of 10 patients. One patient showed the disappearance of a pelvic bone metastasis after five vaccinations. Three patients showed a decrease of serum PSA level from the baseline after the vaccination, but no patients showed a serum PSA level decrease of >/= 50%. The median survival duration of study patients was 22 months with follow-up from 3 to 27 months. We consider that the increase in cellular and humoral immune responses, and decrease in PSA level in some patients justify further development of peptide vaccination for metastatic HRPC patients.

Immunological evaluation of peptide vaccination for patients with gastric cancer based on pre-existing cellular response to peptide.

There is no standard treatment modality for advanced gastric cancer (GC) at the present time. To develop a new treatment modality, we investigated the immunological responses of advanced GC patients (n = 13, 9 non-scirrhous and 4 scirrhous types) vaccinated with peptides to a regimen under which pre-vaccination peripheral blood mononuclear cells (PBMCs) were screened for their reactivity in vitro to each of 14 peptides on HLA-A24 or 16 peptides on -A2 allele, then only the reactive peptides (maximum: 4) were administered in vivo. This regimen was generally well tolerated, although grade I levels of fever and local skin reactions were observed in several patients. Delayed-type hypersensitivity (DTH) to the vaccinated peptides was observed in 4 patients. Increased cellular and humoral immune responses to the vaccinated peptides were observed in post-vaccination PBMCs from 4 of 8 patients and in post-vaccination sera of 8 of 10 patients tested, respectively. Prolonged survival was observed in patients showing cellular and humoral immune responses to the vaccinated peptides in the post-vaccination samples, including all 4 patients with the scirrhous type. These results encourage further development of peptide-based immunotherapy for GC patients.

Peptide vaccination for patients with melanoma and other types of cancer based on pre-existing peptide-specific ctotoxic T-lymphocyte precursors in the periphery.

Identification of antigenic peptides expressed on cancer cells enables us to treat cancer patients with peptide-based immunotherapy. Although optimal protocols for peptide-based vaccines have not yet been elucidated, boosting the immune system could be a better approach than priming the immune system to elicit prompt and potent peptide-specific T-cell responses in cancer patients. With this possibility in mind, the authors undertook a clinical trial in which cancer patients were vaccinated with peptides (maximum 4) after confirmation of pre-existing peptide-specific cytotoxic T-lymphocyte (CTL) precursors in the periphery. Fourteen patients (seven with melanoma and seven with other types of cancer) positive for either HLA-A24 or HLA-A2 were enrolled in this study. Fourteen and 16 peptides were used to screen for HLA-A24+ and HLA-A2+ patients, respectively. The vaccination was well tolerated, and the only adverse effects were local pain and fever. Kinetic analysis revealed that peptide-reactive CTLs increased after peptide vaccination in 7 of 14 patients. Immunoglobulin G (IgG) reactive to the administered peptides was detected in 2 patients before vaccination, although it became detectable in 8 of the other 12 patients after the peptide vaccination. Stable disease for more than 6 months was observed in five patients (one with melanoma and four with other types of cancer); all of these patients showed increased levels of peptide-specific IgG. These results indicate that peptide vaccination of patients showing evidence of pre-existing peptide-specific CTL precursors can be applied in further clinical trials aimed at the treatment of melanoma and other types of cancer.

Immunological evaluation of CTL precursor-oriented vaccines for advanced lung cancer patients.

Recent clinical trials of peptide vaccine for cancer patients have rarely resulted in tumor regression. One of the reasons for this failure could be an insufficient induction of anti-tumor responses in these regimens, in which peptide-specific memory cytotoxic T lymphocytes (CTLs) were not measured prior to vaccination. We investigated in this study whether pre-vaccination measurement of peptide-specific CTLs can provide any advantages in lung cancer patients receiving peptide vaccination with regard to safety and immunological responses. Ten patients with advanced lung cancer received vaccination with peptides under a regimen of CTL precursor-oriented vaccination, in which pre-vaccination peripheral blood mononuclear cells (PBMCs) were at first screened for reactivity in vitro to each of 14 peptides, followed by in vivo administration of only the reactive peptides. Profiles of the vaccinated peptides varied markedly among the 10 patients. This regimen was generally well-tolerated, although local skin reactions, diarrhea, and colitis were observed in 8, 2, and 1 patient, respectively. Increased CTL responses against the immunized peptides and tumor cells were observed in the post-vaccination PBMCs from 4 of 8 and 3 of 10 patients tested, respectively. Peptide-specific IgG became detectable in post-vaccination sera in 4 of 10 patients tested, and these 4 patients had a long progression-free survival. Furthermore, the median survival time of 9 patients with non-small cell lung cancer was 668.0 +/- 164.2 days. These results encourage further development of CTL precursor-oriented peptide vaccination for lung cancer patients.

A simple culture protocol to detect peptide-specific cytotoxic T lymphocyte precursors in the circulation.

The detection and monitoring of peptide-specific cytotoxic T lymphocyte (CTL) precursors is essential for successful peptide-based immunotherapy against cancers. In contrast to the development of effective methods of detecting antigen-specific CTL, such as ELISpot and HLA-class I tetramer assay, stimulation with peptide-pulsed antigen-presenting cells (APC) has for some time been conventionally employed to induce peptide-specific CTL from peripheral blood mononuclear cells (PBMC). This culture protocol, however, needs a substantial number of PBMC to test the reactivity against a panel of peptides. In the present study, we established a simple culture protocol which has no need of additional APC. Addition of a corresponding peptide every 3 days was found to induce not only Epstein-Barr virus (EBV)-specific CTL from healthy donors, but also tumor antigen-derived peptide-specific CTL from cancer patients. A 10-ml blood sample was almost sufficient to test the presence of CTL precursors against 20 different peptides in triplicate assays. Overall, this culture protocol can be useful in detecting and monitoring peptide-specific CTL precursors in the circulation in peptide-based immunotherapy against cancer.