Immunogenic enhancement and clinical effect by type-I interferon of anti-apoptotic protein, survivin-derived peptide vaccine, in advanced colorectal cancer patients

We previously identified a human leukocyte antigen (HLA)-A24-restricted antigenic peptide, survivin-2B80-88, recognized by CD8+ cytotoxic T lymphocytes (CTL). Subsequently, we attempted clinical trials with this epitope peptide alone for some malignancies, resulting in clinical and immunological responses, although their potential was not strong enough for routine clinical use as a cancer vaccine. In the current study, to assess whether immunogenicity of the survivin-2B80-88 peptide could be enhanced with other vaccination protocols, we performed clinical trials in advanced colon cancer patients with two vaccination protocols: (i) survivin-2B80-88 plus incomplete Freund's adjuvant (IFA); and (ii) survivin-2B80-88 plus IFA and a type-I interferon (IFN), IFNα. Our data clearly indicated that, although the effect of survivin-2B80-88 plus IFA was not significantly different from that with survivin-2B80-88 alone, treatment with the vaccination protocol of survivin-2B80-88 plus IFA and IFNα resulted in clinical improvement and enhanced immunological responses of patients. Tetramer analysis of survivin-2B80-88 peptide-specific CTL demonstrated that such CTL were increased at least twofold after vaccination with this protocol in four of eight patients. In these patients, enzyme-linked immunosorbent spot (ELISPOT) results were also enhanced. Subsequent study of single-cell clone separation by cell sorting of peptide-specific CTL showed that each CTL clone was indeed not only peptide-specific but also cytotoxic against human cancer cells in the context of the expression of both HLA-A24 and survivin molecules. Taken together, these results indicate that vaccination of colon cancer patients with survivin-2B80-88 plus IFA and IFNα can be considered to be a very potent immunotherapeutic regimen, and that this protocol might work for other cancers.     

Randomized phase II trial of survivin 2B peptide vaccination for patients with HLA‐A24‐positive pancreatic adenocarcinoma

The prognosis of advanced pancreatic adenocarcinoma is still extremely poor. This study sought to determine the efficacy of, and immunological response to, peptide vaccination therapy in patients with this disease. In this multicenter randomized phase II study, patients with advanced pancreatic adenocarcinoma after gemcitabine and/or tegafur/gimeracil/oteracil were randomly assigned to 3 groups that each received a 2‐step treatment course. In Step 1, the groups received treatments of: (i) survivin 2B peptide (SVN‐2B) plus interferon‐β (IFNβ); (ii) SVN‐2B only; or (iii) placebo until the patients show progression. In Step 2, all patients who consented to participate received 4 treatments with SVN‐2B plus IFNβ. The primary endpoint was progression‐free survival (PFS) after initiation of Step 1 treatment. Secondary endpoints included immunological effects assessed by analysis of PBMCs after Step 1. Eighty‐three patients were randomly assigned to receive SVN‐2B plus IFNβ (n = 30), SVN‐2B (n = 34), or placebo (n = 19). No significant improvement in PFS was observed. Survivin 2B‐specific CTLs were found to be increased in the SVN‐2B plus IFNβ group by tetramer assay. Among patients who participated in Step 2, those who had received SVN‐2B plus IFNβ in Step 1 showed better overall survival compared with those who had received placebo in Step 1. Patients vaccinated with SVN‐2B plus IFNβ did not have improved PFS, but showed significant immunological reaction after vaccination. Subgroup analysis suggested that a longer SVN‐2B plus IFNβ vaccination protocol might confer survival benefit. (Clinical trial registration number: UMIN 000012146).     

Phase I clinical trial of survivin-derived peptide vaccine therapy for patients with advanced or recurrent oral cancer

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is abundantly expressed in most malignancies, but is hardly detectable in normal adult tissues. Previously we have identified a human leukocyte antigen (HLA)-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), recognized by CD8(+) cytotoxic T lymphocytes (CTL). Survivin-2B80-88-specific CTL were induced efficiently from peripheral blood mononuclear cells (PBMC) of oral cancer patients after stimulation with the peptide in vitro. We conducted a phase I clinical study to evaluate the safety and the efficacy of survivin-2B80-88 peptide vaccination in HLA-A24-positive patients with advanced or recurrent oral cancer. The vaccines were given subcutaneously or intratumorally six times at 14-day intervals. Eleven patients were enrolled and 10 patients completed the vaccination protocol. No adverse events were observed in any patients. In two patients, the levels of serum squamous cell carcinoma (SCC) antigen decreased transiently during the period of vaccination. Tumor regression that was compatible with a partial response (PR) was noted in one patient. The remaining nine patients experienced progressive disease (PD). Immunologically, an increase of the peptide-specific CTL frequency was detected in six of the eight patients evaluated by HLA-A24/peptide tetramer analysis. The present clinical trial revealed that survivin-2B peptide vaccination was safe and had therapeutic potential for oral cancer patients. However, subsequent clinical trials in combination with various adjuvant drugs will be required to improve the immunological and therapeutic efficacy. This trial was registered with University Hospital Medical Information Network (UMIN) number UMIN000000976.     

Addition of interferon‐α to the p53‐SLP® vaccine results in increased production of interferon‐γ in vaccinated colorectal cancer patients: A phase I/II clinical trial

We previously established safety and immunogenicity of a p53 synthetic long peptides (p53‐SLP®) vaccine. In the current trial, we investigated whether combination of interferon‐alpha (IFN‐α) with p53‐SLP® is both safe and able to improve the induced p53‐specific IFN‐γ response. Eleven colorectal cancer patients successfully treated for metastatic disease were enrolled in this study. Of these, nine patients completed follow‐up after two injections with p53‐SLP® together with IFN‐α. Safety and p53‐specific immune responses were determined before and after vaccination. Furthermore, cryopreserved PBMCs were compared head‐to‐head to cryopreserved PBMCs obtained in our previous trial with p53‐SLP® only. Toxicity of p53‐SLP® vaccination in combination with IFN‐α was limited to Grade 1 or 2, with predominantly small ongoing swellings at the vaccination site. All patients harbored p53‐specific T cells after vaccination and most patients showed p53‐specific antibodies. Compared to the previous trial, addition of IFN‐α significantly improved the frequency of p53‐specific T cells in IFN‐γ ELISPOT. Moreover, in this trial, p53‐specific T cells were detectable in blood samples of all patients in a direct ex vivo multiparameter flowcytometric assay, opposed to only 2 of 10 patients vaccinated with p53‐SLP® only. Finally, patients in this trial displayed a broader p53‐specific immunoglobulin‐G response, indicating an overall better p53‐specific T‐helper response. Our study shows that p53‐SLP® vaccination combined with IFN‐α injection is safe and capable of inducing p53‐specific immunity. When compared to a similar trial with p53‐SLP® vaccination alone the combination was found to induce significantly more IFN‐γ producing p53‐specific T cells.     

Early phase II study of mixed 19‐peptide vaccine monotherapy for refractory triple‐negative breast cancer

We undertook an early phase II study of mixed 19‐peptide cancer vaccine monotherapy for 14 advanced metastatic triple‐negative breast cancer (mTNBC) patients refractory to systemic chemotherapy to develop a new type of cancer vaccine. The treatment protocol consisted of a weekly vaccination for 6 weeks, and there were no severe adverse events related to the vaccination throughout the trial. Increase of peptide‐specific IgG against the vaccinated human leukocyte antigen‐matched peptides, but not against the nonmatched peptides, was positively correlated with overall survival (OS) (P < .01). The median OS was 11.5 or 24.4 months in all 14 patients or the 10 patients who completed the vaccination. The patients with lower C‐reactive protein levels or 3 or fewer systemic chemotherapies were favorable candidates for this treatment. Advancement of this therapy to the next stage of study could be warranted based on the safety and immune boosting determined herein (clinical trial registration number: UMIN000014616).     

Identification of CDCA1‐derived long peptides bearing both CD4+ and CD8+ T‐cell epitopes: CDCA1‐specific CD4+ T‐cell immunity in cancer patients

We recently identified a novel cancer‐testis antigen, cell division cycle associated 1 (CDCA1) using genome‐wide cDNA microarray analysis, and CDCA1‐derived cytotoxic T lymphocyte (CTL)‐epitopes. In this study, we attempted to identify CDCA1‐derived long peptides (LPs) that induce both CD4⁺ helper T (Th) cells and CTLs. We combined information from a recently developed computer algorithm predicting HLA class II‐binding peptides with CDCA1‐derived CTL‐epitope sequences presented by HLA‐A2 (A*02:01) or HLA‐A24 (A*24:02) to select candidate CDCA1‐LPs encompassing both Th cell epitopes and CTL‐epitopes. We studied the immunogenicity of CDCA1‐LPs and the cross‐priming potential of LPs bearing CTL‐epitopes in both human in vitro and HLA‐class I transgenic mice in vivo. Then we analyzed the Th cell response to CDCA1 in head‐and‐neck cancer (HNC) patients before and after vaccination with a CDCA1‐derived CTL‐epitope peptide using IFN‐γ enzyme‐linked immunospot assays. We identified two CDCA1‐LPs, CDCA1_39–64‐LP and CDCA1_55–78‐LP, which encompass naturally processed epitopes recognized by Th cells and CTLs. CDCA1‐specific CTLs were induced through cross‐presentation of CDCA1‐LPs in vitro and in vivo. In addition, CDCA1‐specific Th cells enhanced induction of CDCA1‐specific CTLs. Furthermore, significant frequencies of CDCA1‐specific Th cell responses were detected after short‐term in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with CDCA1‐LPs in HNC patients (CDCA1_39–64‐LP, 74%; CDCA1_55–78‐LP, 68%), but not in healthy donors. These are the first results demonstrating the presence of CDCA1‐specific Th cell responses in HNC patients and underline the possible utility of CDCA1‐LPs for propagation of both CDCA1‐specific Th cells and CTLs.     

A potent immunogenic general cancer vaccine that targets survivin, an inhibitor of apoptosis proteins.

We reported previously a HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), recognized by CD8(+) CTL. This peptide was derived from survivin protein, an inhibitor of apoptosis proteins, expressed in a variety of tumors, such as adenocarcinoma, squamous cell carcinoma, and malignant melanoma. In this report, we provide further evidence that survivin-2B80-88 peptide might serve as a potent immunogenic cancer vaccine for various cancer patients. Overexpression of survivin was detected in surgically resected primary tumor specimens of most breast and colorectal cancers and some gastric cancers as assessed by immunohistochemical study. HLA-A24/survivin-2B80-88 tetramer analysis revealed that there existed an increased number of CTL precursors in peripheral blood mononuclear cells (PBMC) of HLA-A24(+) cancer patients, and in vitro stimulation of PBMCs from six breast cancer patients with survivin-2B80-88 peptide could lead to increases of the CTL precursor frequency. Furthermore, CTLs specific for this peptide were successfully induced from PBMCs in all 7 (100%) patients with breast cancers, 6 of 7 (83%) patients with colorectal cancers, and 4 of 7 (57%) patients with gastric cancers. These data indicate that survivin expressed in tumor tissues is antigenic in cancer patients, and survivin-2B80-88-specific CTLs are present in PBMCs of various cancer patients. Our study raises the possibility that this peptide may be applicable as a general cancer vaccine to a large proportion of HLA-A24(+) cancer patients.     

Immunological evaluation of personalized peptide vaccination monotherapy in patients with castration‐resistant prostate cancer

(Cancer Sci 2010; 101: 601–608) We previously reported that personalized peptide vaccine (PPV) therapy in combination with leutenizing hormone‐releasing hormone (LH‐RH) analog and estramustine phosphate in certain cases is safe and capable of inducing both immune responses and clinical responses for metastatic castration‐resistant prostate cancer (CRPC) patients. In the present study, PPV monotherapy was given to CRPC patients. Twenty‐three patients with metastatic CRPC were treated with PPV without any additional treatment modalities, including LH‐RH analogs. Samples were analyzed for peptide‐specific cytotoxic T‐lymphocyte (CTL) precursor analysis and peptide‐reactive IgG. Toxicity and immunological and clinical responses were assessed on a three‐monthly basis. Seventeen patients were available for immunological and clinical evaluation. The vaccines were well tolerated, with grade 3 erythema at injection sites in only one patient. Augmentation of CTL or IgG responses to at least one of the peptides was observed in six of 17 (35%) and 15 of 17 (88%) patients tested, respectively. Among 57 peptides used, 9 and 36 peptides induced CTL and IgG responses, respectively. Delayed‐type hypersensitivity reaction was observed in eight of 17 patients. More than 30% prostate‐specific antigen (PSA) decline was observed in four of 17 patients. Of these, one patient achieved a complete PSA response and another patient showed a partial PSA response with profound shrinking of lymph node metastases and prostate. The overall median survival time was 24 months (range, 5–37 months). These results suggest that PPV monotherapy appears to be safe and capable of inducing peptide‐specific immune responses and clinical responses in CRPC patients. This trial was registered with University Hospital Medical Information Network (UMIN) number R000003339.     

Whole blood interferon‐γ levels predict the therapeutic effects of adoptive T‐cell therapy in patients with advanced pancreatic cancer

A core challenge in administering immune‐based treatments for cancer is the establishment of easily accessible immunological assays that can predict patients' clinical responses to immunotherapy. In this study, our aim was to predict the therapeutic effects of adoptive T‐cell therapy in patients with advanced pancreatic cancer. To do this, we evaluated whole blood cytokine levels and peripheral regulatory T cells (Tregs) in 46 patients with unresectable or recurrent pancreatic cancer who received adoptive T‐cell therapy at 2‐week intervals. To test immune function, venous blood was obtained from patients before the start of therapy and 2 weeks after the 4th treatment. Whole blood interferon (IFN)‐α levels (after stimulation with the Sendai virus) were evaluated, as well as the levels of 9 cytokines stimulated with phytohemagglutinin [interleukin (IL)?2, IL‐4, IL‐5, IL‐10, IL‐12(p70), IL‐13, tumor necrosis factor‐α, IFN‐γ, and granulocyte‐monocyte colony‐stimulating factor]. Peripheral Tregs were analyzed by flow cytometry. Using the obtained data, we then observed the relationship between these immunological parameters and clinical outcome of patients. We found that the whole blood production of IFN‐γ, IL‐2, IL‐4, IL‐5 and IL‐13 significantly increased after adoptive T‐cell therapy, whereas the number of peripheral Tregs did not change. Multivariate Cox proportional hazards analyses indicated that the number of peripheral Tregs before receiving adoptive T‐cell therapy and the change in IFN‐γ levels after adoptive T‐cell therapy were independent variables predicting overall survival. The findings of this study indicate that the assay of whole blood IFN‐γ production offers promise for evaluating the clinical response of patients to cancer immunotherapy.     

Induction of human tumor‐associated differentially expressed gene‐12 (TADG‐12/TMPRSS3)‐specific cytotoxic T lymphocytes in human lymphocyte antigen‐A2.1–positive healthy donors and patients with advanced ovarian cancer

BACKGROUND:

Tumor‐associated differentially expressed gene‐12 (TADG‐12) is a serine protease recently found highly differentially expressed in epithelial ovarian cancer. The goal of this study was to identify potential immunogenic peptides derived from TADG‐12 for immunotherapy of ovarian carcinoma.

METHODS:

A bioinformatics approach (ie, the BIMAS algorithm, National Institutes of Health, http://bimas.dcrt.nih.gov/molbio/hla_bind ) was used to identify multiple immunogenic peptides derived from TADG‐12 that bind to human leukocyte antigen‐A2.1 and elicit peptide‐specific human cytotoxic T lymphocyte (CTL) responses in healthy individuals and in patients with advanced stage ovarian cancer.

RESULTS:

CD8+ CTL populations generated against 5 TADG‐12–derived peptides were consistently able to induce lysis of autologous peptide‐loaded target cells above background. Importantly, TADG‐12 YLPKSWTIQV peptide‐specific CTLs from healthy donors and ovarian cancer patients were found to effectively kill ovarian cancer cells naturally expressing TADG‐12. Cytotoxicity was significantly inhibited by anti–human lymphocyte antigen (HLA)‐A2.1 (BB7‐2) and anti–HLA class I (W6 of 32) monoclonal antibodies, whereas natural killer–sensitive K562 cells were not lysed. TADG‐12 YLPKSWTIQV peptide‐specific CTL precursor frequency was low in peripheral blood leukocytes of normal donors and ovarian cancer patients, as determined by interferon‐γ production in enzyme‐linked immunosorbent spot‐forming cell assays. Intracellular cytokine expression measured by flow cytometry after OKT‐3 monoclonal antibody stimulation showed a type 1 cytokine profile in YLPKSWTIQV peptide‐specific CTLs.

CONCLUSIONS:

The TADG‐12 YLPKSWTIQV peptide is an immunogenic epitope in ovarian tumors and may represent an attractive target for immunotherapy of ovarian cancer. These data may pave the way for TADG‐12 peptide‐derived cell‐based therapy, including dendritic cell immunotherapy, for the vaccination of ovarian cancer patients harboring chemotherapy‐resistant or residual disease. Cancer 2009. © 2008 American Cancer Society.     

Phase I pilot study of Wilms tumor gene 1 peptide‐pulsed dendritic cell vaccination combined with gemcitabine in pancreatic cancer

This study aimed to evaluate the feasibility of and immune response to Wilms tumor gene 1 (WT1) peptide‐pulsed dendritic cell vaccination combined with gemcitabine (DCGEM) as a first‐line therapy among patients with advanced pancreatic cancer. Ten HLA‐A*2402 patients were treated with WT1 peptide‐pulsed DC vaccination (1 × 10⁷ cells) on days 8 and 22 and gemcitabine (1000 mg/m²) on days 1, 8 and 15. Induction of a WT1‐specific immune response was evaluated using the delayed‐type hypersensitivity (DTH) skin test, interferon‐γ enzyme‐linked immunospot and HLA tetramer assays, along with assays for various immunological factors. DCGEM was well‐tolerated, and the relative dose intensity of gemcitabine was 87%. Disease control associated with a low neutrophil/lymphocyte ratio was observed in all three patients with DTH positivity; it was also correlated with a low percentage of granulocytic myeloid derived suppressor cells in the pretreatment peripheral blood (P = 0.017). Patients with liver metastases and high levels of inflammatory markers such as C‐reactive protein and interleukin‐8 (IL‐8) showed poor survival even though a WT1‐specific immune response was induced in them. WT1 peptide‐pulsed DCGEM is feasible and effective for inducing anti‐tumor T‐cell responses. Our results support future investigations for pancreatic cancer patients with non‐liver metastases and favorable immunological conditions. This trial was registered with the University hospital Medical Information Network (UMIN) Clinical Trials Registry ( http://www.umin.ac.jp/ctr/ number: UMIN‐000004855).     

Children with endemic Burkitt lymphoma are deficient in EBNA1‐specific IFN‐γ T cell responses

Endemic Burkitt lymphoma (eBL) is the most common childhood cancer in equatorial Africa and is linked to Epstein–Barr virus (EBV) and Plasmodium falciparum coinfections early in life. Epstein–Barr nuclear antigen 1 (EBNA1) is the sole viral latent antigen expressed in BL tumors. Loss of EBNA1‐specific immune surveillance could allow eBL emergence. Therefore, EBNA1‐specific T cell responses were analyzed by IFN‐γ ELISPOT in Kenyan children with eBL and compared to healthy children with divergent malaria exposure. Significantly fewer children with eBL, 16% (7/44) had EBNA1‐specific IFN‐γ responses in contrast to healthy children living in a malaria holoendemic area or in an area with sporadic malaria transmission, 67% (40/60) and 72% (43/60) responders, respectively (p < 0.003). Children with eBL maintained IgG₁ dominated antibody responses to EBNA1 similar to healthy children suggesting a selective loss of IFN‐γ secreting EBNA1‐specific T cells in the presence of intact humoral immunity. CD8⁺ T cell responses to EBV lytic and latent antigens not expressed in the tumors were similarly robust in eBL patients compared to healthy children. In addition, CD4⁺ T cell responses to a malaria protein, merozoite surface protein 1, were present in lymphoma patients. This study demonstrates a selective loss of EBNA1‐specific T cell responses in children with eBL and suggests a potential immunotherapeutic target for this EBV‐associated lymphoma. © 2008 Wiley‐Liss, Inc.     

Immunohistological analysis of pancreatic carcinoma after vaccination with survivin 2B peptide: Analysis of an autopsy series

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer by providing new options in addition to existing therapies. However, peptide vaccination therapies still represent an attractive approach, because of the antigen specificity. We identified survivin 2B peptide (SVN‐2B), a 9‐mer antigenic peptide encoded by survivin, and an SVN‐2B peptide vaccine‐based phase II randomized clinical trial targeting unresectable and refractory pancreatic carcinoma was undertaken. The SVN‐2B peptide vaccine did not have any statistically significant clinical benefits in that study. Therefore, we undertook an autopsy study to analyze the immune status of the pancreatic cancer lesions at the histological level. Autopsies were carried out in 13 patients who had died of pancreatic cancer, including 7 who had received SVN‐2B peptide vaccination and 6 who had not, as negative controls. The expression of immune‐related molecules was analyzed by immunohistochemical staining. Cytotoxic T lymphocytes were analyzed by tetramer staining and enzyme‐linked immunospot assay. Histological analysis revealed dense infiltration of CD8⁺ T cells in some lesions in patients who had received the SVN‐2B peptide vaccine. A high rate of programmed cell death ligand 1 expression in cancer cells was observed in these cases, indicating that CTLs were induced by SVN‐2B peptide vaccination and had infiltrated the lesions. The lack of a significant antitumor effect was most likely attributable to the expression of immune checkpoint molecules. These findings suggest that the combination of a tumor‐specific peptide vaccine and an ICI might be a promising approach to the treatment of pancreatic carcinoma in the future.     

Personalized peptide vaccination as second‐line treatment for metastatic upper tract urothelial carcinoma

This study investigated the applicability of personalized peptide vaccination (PPV) for patients with metastatic upper tract urothelial cancer (mUTUC) after failure of platinum‐based chemotherapy. In this single arm, open‐label, phase II clinical trial, patients with mUTUC received PPV at a single institution. Personalized peptide vaccination treatment used a maximum of four peptides chosen from 27 candidate peptides according to human leukocyte antigen types and peptide‐reactive IgG titers, for six s.c. injections weekly as one cycle. The safety of PPV, as well as its influence on host immunity and effect on overall survival were assessed. Forty‐eight patients were enrolled in this study. Personalized peptide vaccinations were well tolerated without severe adverse events. Median survival time was 7.3 months (95% confidence interval [CI], 5.3–13.1) with 13.0 months for patients receiving combined salvage chemotherapy (95% CI, 5.7–17.5) and 4.5 months for patients receiving PPV alone (95% CI, 1.7–10.1) (P = 0.080). Patients with positive CTL responses showed a significantly longer survival than patients with negative CTL responses (hazard ratio, 0.37; 95% CI, 0.16–0.85; P = 0.019). Multivariate Cox regression analysis showed that lower numbers of Bellmunt risk factors and lower levels of B‐cell activating factor were significantly associated with favorable overall survival for patients under PPV treatment. This study indicated that PPV for patients with mUTUC after failure of platinum‐based chemotherapy induced substantial peptide‐specific CTL responses without severe adverse events and has the potential to prolong survival when combined with salvage chemotherapy. UMIN Clinical Trials Registry ID: 000001854.     

Evaluation of a new oil adjuvant for use in peptide‐based cancer vaccination

Vaccine therapies are increasingly being used for the treatment of various diseases, and the antigen molecules themselves are being expanded from whole microorganisms to fine molecules such as peptides. Accordingly, there is a need for new adjuvants to support these new applications. In this paper, we used pharmaceutical grade mineral oil and sorbitan monooleate to develop a new oil adjuvant formula, NH₂, and investigated its effects on peptide vaccination at both the pre‐clinical and clinical levels. The adjuvant effect of NH₂ on peptide‐induced cellular immunity in mice was superior to that of Montanide ISA51VG, a commercially available incomplete Freund’s adjuvant for clinical use, although no significant difference was observed between the two adjuvants on peptide‐induced humoral immunity. The adjuvant effects of NH₂ were also confirmed in a Phase‐I clinical trial of peptide vaccines for patients with advanced cancers. These results suggest that NH₂ is a suitable adjuvant for peptide vaccination, particularly for cancer vaccines (Phase‐I clinical trial of pan‐HLA type personalized peptide vaccine for advanced cancer patients, UMIN clinical trial registry number: UMIN 000000619). (Cancer Sci 2010)     

Fowlpox‐based survivin vaccination for malignant mesothelioma therapy

Survivin protein is an attractive candidate for cancer immunotherapy since it is abundantly expressed in most common human cancers and mostly absent in normal adult tissues. Malignant mesothelioma (MM) is a deadly cancer associated with asbestos or erionite exposure for which no successful therapies are currently available. In this study, we evaluated the therapeutic efficacy of a novel survivin‐based vaccine by subcutaneous or intraperitoneum injection of BALB/c mice with murine fiber‐induced MM tumor cells followed by vaccination with recombinant Fowlpox virus replicons encoding survivin. Vaccination generated significant immune responses in both models, leading to delayed tumor growth and improved animal survival. Flow cytometry and immunofluorescence analyses of tumors from vaccinated mice showed CD8⁺ T‐cell infiltration, and real‐time PCR demonstrated increased mRNA and protein levels of immunostimulatory cytokines. Analyses of survivin peptide‐pulsed spleen and lymph node cells from vaccinated mice using ELISPOT and intracellular cytokine staining confirmed antigen‐specific, interferon‐γ‐producing CD8⁺ T‐cell responses. In addition pentamer‐based flow cytometry showed that vaccination generated survivin‐specific CD8⁺ T cells. Importantly, vaccination did not affect fertility or induce autoimmune abnormalities in mice. Our results demonstrate that vaccination with recombinant Fowlpox expressing survivin improves T‐cell responses against aggressive MM tumors and may form the basis for promising clinical applications.     

Interleukin‐10 gene transfer activates interferon‐γ and the interferon‐γ‐inducible genes Gbp‐1/Mag‐1 and Mig‐1 in mammary tumors

Expression of IL‐10 as a transgene inhibits murine mammary tumor growth and metastasis. Using differential display methodology, we sought genes whose expression was modulated by IL‐10. We compared mRNA isolated from parental murine mammary 66.1 tumors, as well as tumors derived from neo^r‐transfected cells and 6 different IL‐10‐expressing cell lines. We identified 2 cDNA products that were up‐regulated in all 6 IL‐10‐expressing tumors in comparison to parental and 66‐neo tumors. One cDNA corresponds to the murine guanylate‐binding protein gene Gbp‐1/Mag‐1. The other cDNA corresponds to the chemokine Mig‐1 (monokine induced by IFN‐γ). Both genes were originally identified in IFN‐γ‐activated macrophages or macrophage cell lines. We now report that cultured mammary epithelial tumor cell lines also express both genes in response to treatment with IFN‐γ and LPS. Furthermore, IFN‐γ mRNA is elevated in IL‐10‐expressing tumors in comparison with parental or neo‐transfected tumors. Thus, high‐level expression of IL‐10 as a transgene results in activation rather than suppression of IFN‐γ as well as 2 IFN‐γ‐inducible genes. Up‐regulation of host IFN‐γ is critical to anti‐tumor activity since IL‐10 no longer inhibits tumor growth in hosts with a deletion in the IFN‐γ gene. Additionally, Gbp‐1/Mag‐1 and Mig‐1 gene induction no longer occur in IFN‐γ mutant mice. Int. J. Cancer 80:624–629, 1999. © 1999 Wiley‐Liss, Inc.     

Heat shock protein 105 peptide vaccine could induce antitumor immune reactions in a phase I clinical trial

Heat shock protein 105 (HSP105) is overexpressed in many cancers, including colorectal cancer (CRC) and esophageal cancer (EC). We carried out a phase I clinical trial of HLA‐A24‐ and HLA‐A2‐restricted HSP105 peptide vaccines in patients with CRC or EC. In this additional study of the trial, we examined the immunological efficacy of the novel vaccine. Thirty patients with advanced CRC or EC underwent HSP105 peptide vaccination. Immunological responses were evaluated by ex vivo and in vitro γ‐interferon enzyme‐linked immunospot assays and their correlation with patients’ prognosis was analyzed. The HSP105 peptide vaccines induced peptide‐specific CTLs in 15 of 30 patients. Among HLA‐A24 patients (n = 15), 7 showed induction of CTLs only ex vivo, whereas among HLA‐A2 patients (n = 15), 4 showed the induction ex vivo and 6 in vitro. Heat shock protein 105‐specific CTL induction correlated with suppression of cancer progression and was revealed as a potential predictive biomarker for progression‐free survival (P = .008; hazard ratio = 3.03; 95% confidence interval, 1.34‐6.85) and overall survival (P = .025; hazard ratio = 2.72; 95% confidence interval, 1.13‐6.52). Production of cytokines by HSP105 peptide‐specific CTLs was observed at the injection sites (skin) and tumor tissues, suggesting that HSP105‐specific CTLs not only accumulated at vaccination sites but also infiltrated tumors. Furthermore, we established 2 HSP105 peptide‐specific CTL clones, which showed HSP105‐specific cytokine secretion and cytotoxicity. Our results suggest that the HSP105 peptide vaccine could induce immunological effects in cancer patients and improve their prognosis.     

Epithelial–mesenchymal transition in human gastric cancer cell lines induced by TNF‐α‐inducing protein of Helicobacter pylori

Helicobacter pylori strains produce tumor necrosis factor‐α (TNF‐α)‐inducing protein, Tipα as a carcinogenic factor in the gastric epithelium. Tipα acts as a homodimer with 38‐kDa protein, whereas del‐Tipα is an inactive monomer. H. pylori isolated from gastric cancer patients secreted large amounts of Tipα, which are incorporated into gastric cancer cells by directly binding to nucleolin on the cell surface, which is a receptor of Tipα. The binding complex induces expression of TNF‐α and chemokine genes, and activates NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells). To understand the mechanisms of Tipα in tumor progression, we looked at numerous effects of Tipα on human gastric cancer cell lines. Induction of cell migration and elongation was found to be mediated through the binding to surface nucleolin, which was inhibited by the nucleolin‐targeted siRNAs. Tipα induced formation of filopodia in MKN‐1 cells, suggesting invasive morphological changes. Tipα enhanced the phosphorylation of 11 cancer‐related proteins in serine, threonine and tyrosine, indicating activation of MEK‐ERK signal cascade. Although the downregulation of E‐cadherin was not shown in MKN‐1 cells, Tipα induced the expression of vimentin, a significant marker of the epithelial–mesenchymal transition (EMT). It is of great importance to note that Tipα reduced the Young's modulus of MKN‐1 cells determined by atomic force microscopy: This shows lower cell stiffness and increased cell motility. The morphological changes induced in human gastric cancer cells by Tipα are significant phenotypes of EMT. This is the first report that Tipα is a new inducer of EMT, probably associated with tumor progression in human gastric carcinogenesis.