Kazuko Katagiri

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-γ release assay and tumor reactivity by 51Cr 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.

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

Abstract. 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.

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.

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.

Peptide Vaccination for Patients With Melanoma and Other Types of Cancer Based on Pre-existing Peptide-Specific Cytotoxic 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.

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. (Cancer Sci 2004; 95: 77–84)

A phase I trial of cytotoxic T-lymphocyte precursor-oriented peptide vaccines for colorectal carcinoma patients.

In most protocols of peptide-based vaccination, no consideration has been paid to whether or not peptide-specific cytotoxic T-lymphocyte (CTL) precursors are pre-existent in cancer patients. Initiation of immune boosting through vaccination is better than that of immune priming to induce prompt and strong immunity. In this study, 10 human histocompatibility leukocyte antigen-A24+ patients with advanced colorectal carcinomas were treated with up to four peptides that had been positive for pre-vaccination measurement of peptide-specific CTL precursors in the circulation (CTL precursor-oriented peptide vaccine). No severe adverse effect was observed, although local pain and fever of grade I or II were observed. Post-vaccination peripheral blood mononuclear cells (PBMCs) from five patients demonstrated an increased peptide-specific immune response to the peptides. Increased CTL response to cancer cells was detected in post-vaccination PBMCs of five patients. Antipeptide immunoglobulin G became detectable in post-vaccination sera of seven patients. Three patients developed a positive delayed-type hypersensitivity response to at least one of the peptides administrated. One patient was found to have a partial response; another had a stable disease, sustained through 6 months. These results encourage further development of CTL precursor-oriented vaccine for colorectal cancer patients.

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 β-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.

Detection of peptide-specific CTL-precursors in peripheral blood lymphocytes of cancer patients.

Development of therapeutic vaccines is one of the major areas of tumour immunotherapy today. However, clinical trials of peptide-based cancer vaccines have rarely resulted in tumour regression. This failure might be due to an insufficient induction of cytotoxic T lymphocytes in the current regimes, in which cytotoxic T lymphocytes-precursors in pre-vaccination peripheral blood mononuclear cells are not measured. Initiation of immune-boosting through vaccination could be better than that of immune-priming with regard to induction of prompt and strong immunity. If this is also the case for therapeutic vaccines, pre-vaccination measurement of peptide-specific cytotoxic T lymphocytes-precursors will be important. In the present study, we investigated whether cytotoxic T lymphocytes-precursors reacting to 28 kinds of peptides of vaccine candidates (13 and 15 peptides for HLA-A24+ and HLA-A2+ patients, respectively) were detectable in pre-vaccination peripheral blood mononuclear cells of 80 cancer patients. Peptide-specific cytotoxic T lymphocytes-precursors were found to be detectable in peripheral blood mononuclear cells of the majority of cancer patients (57 out of 80 cases, 71%). The mean numbers of positive peptides were 2.0 peptides per positive case. Peripheral blood mononuclear cells incubated with positive peptides, not with negative peptides, showed significant levels of HLA-class-I-restricted cytotoxicity to cancer cells. The profiles of positive peptides entirely varied among patients, and were not influenced by the cancer origin. These results may provide a scientific basis for the development of a new approach to cancer immunotherapy, e.g.) cytotoxic T lymphocytes-precursor-oriented peptide vaccine.

In Vitro Effects of Immunosuppressive Agents on Cytokine Production by HTLV-I-Infected T Cell Clones Derived from the Ocular Fluid of Patients with HTLV-I Uveitis

The present study was designed to investigate the in vitro effects of potential therapeutic agents on cytokine production by five HTVL‐I‐infected T cell clones (TCC) established from the ocular fluid of patients with HTLV‐I uveitis. Each of the five HTLV‐I‐infected TCC was cultured at 1 × 106 cells/ml with or without an immunosuppressive agent (hydrocortisone, FK506, rapamycin, indomethacin, or prostaglandin E2) for 22 hr in humidified 5% CO2 in air at 37 C. The production of various cytokines in the culture supernatant from each TCC was measured by ELISA. The HTLV‐I‐infected TCC produced high amounts of IL‐1α, IL‐3, IL‐6, IL‐8, TNF‐α, IFN‐γ, and GM‐CSF, and low but significant levels of IL‐2 and IL‐10 without any stimuli. Hydrocortisone severely depressed the production by these TCC of all the cytokines except for IL‐2, which was slightly increased. Prostaglandin E2 depressed the production of IL‐1α, while it up‐regulated the production of IL‐6, TNF‐α, and IFN‐γ. Rapamycin depressed the production of IL‐6 and TNF‐α, and FK506 depressed the production of TNF‐α. Hydrocortisone also severely depressed the cytokine production by PHA‐stimulated peripheral blood mononuclear cells obtained from healthy volunteers. Of the immunosuppressive agents tested, hydrocortisone exhibited the strongest suppression of cytokine production by HTLV‐I‐infected TCC. This result was in agreement with the in vivo effects of hydrocortisone in patients with HTLV‐I uveitis. These TCC will be useful in investigating the effects of potential therapeutic agents for HTLV‐I uveitis in vitro.