Immunotherapy For Pancreatic Cancer Research Articles

Adoptive immunotherapy for pancreatic cancer: Cytotoxic T lymphocytes stimulated by the MUC1-expressing human pancreatic cancer cell line YPK-1

MUC1 is a tumor-associated antigen that is overexpressed in invasive ductal carcinomas of the pancreas (PC). MUC1-specific cytotoxic T lymphocytes (CTLs) recognize MUC1 molecules in a HLA-unrestricted manner. In this study, we performed adoptive immunotherapy (AIT) in patients with PC with CTLs stimulated by the MUC1-expressing human PC cell line YPK-1. To induce CTLs, peripheral blood mononuclear cells (PBMCs) were cultured for 3 days with inactivated YPK-1 cells and then stimulated with interleukin (IL)-2 for 7 days. The cytotoxicity of these cells against human cancer cell lines was analyzed, and a variety of antibodies were evaluated for their ability to inhibit cytotoxicity. We treated 8 patients with unresectable PC and 20 patients with resectable PC postsurgically. CTLs were induced as described above, suspended in 100 ml saline and injected intravenously. Induced CTLs were cytotoxic against 5 MUC1-expressing PC cell lines and a breast cancer cell line, regardless of the HLA phenotype. Low cytotoxicity was observed in 7 MUC1-negative cancer cell lines. Anti-CD3 monoclonal antibody (mAb) or anti-CD8 mAb strongly inhibited cytotoxicity against YPK-1, whereas anti-class I mAb showed no inhibition. YPK-1 cells incubated with anti-MUC1 mAb also showed low cytotoxicity. Clinically, the median survival time was 5.0 months for patients with unresectable PC treated with AIT. None of the 5 patients without liver metastasis showed hepatic recurrence. The median survival time was 17.8 months for 18 out of 20 patients with resectable PC who underwent curative surgery, and the 1-, 2- and 3-year survival rates after surgery were 83.3, 32.4, and 19.4%, respectively. Liver metastasis was found in only one patient and no side effects of AIT were observed. CTLs stimulated by a MUC1-expressing human pancreatic cancer cell line showed a strong tumor cytotoxic activity in a MUC1-specific and MHC-unrestricted manner. AIT with stimulated CTLs significantly suppressed the postsurgical hepatic recurrence of PC. Adjuvant immunotherapy with CTLs may be useful in the postsurgical treatment of PC.

Gemcitabine (gem) can enhance immunotherapy (IT) of pancreatic cancer – importance of schedule

With a 5‐year survival rate of only 4%, non‐resectable pancreatic cancer is in urgent need of novel approaches to supplement current therapies. This study assessed parameters for combining gem and IT with the anti‐MUC1 antibody, AR20.5. Upon injection, AR20.5 forms immune complexes with circulating MUC1, leading to enhanced T cell immunity. To examine the effects of gem on immune cells and antigen processing, three parallel approaches were conducted: a) an in vitro co‐culture system of immature DC, tumor and T cells; b) a time course study of gem (60 mg/kg) in Panc02 tumor bearing mice; and c) a combination chemo‐IT study in MUC1‐transgenic mice with established Panc02.MUC1 tumors. In vitro, gem did not affect T cell or DC survival at concentrations ≤5 μg/mL. Interestingly, gem enhanced maturation of DC and T cell proliferation to non‐specific stimuli (anti‐CD3/CD28) as well as to the co‐cultured tumor cells. The in vivo time course study confirmed minor effects of gem on T and B cell survival with rapid recovery. Gem increased DC maturation in the spleen and tumor as well as DC migration to the tumor by Day 5. Further, gem significantly reduced T regs in the draining LN (Day 7) and tumor (Day 10). Reduction in myeloid derived suppressor cells was transient (Day 5–7) in spleen and tumor. Tumor control studies showed synergistic effects for combination gem‐IT over single agents when IT was given 1 week delayed relative to gem.

Potential target antigens for immunotherapy in human pancreatic cancer

Background To be effective and selective, immunotherapy ideally targets specifically tumor cells and spares normal tissues. Identification of tumor specific antigens is a prerequisite to establish an effective immunotherapy. Still very little is known about the expression of tumor-related antigens in pancreatic neoplasms. Cancer Testis antigens (CT) are antigens shared by a variety of malignant tumors, but not by normal tissues with the exception of germ cells in testis. Restricted expression in neoplastic tissues and inherent immunogenic features make CT antigens ideal for use in immunotherapy. We analyzed the expression of a selected panel of nine CT antigens that have been proven to elicit an efficient immunogenic response in other malignancies. In addition we analyzed the expression of HERV-K-MEL, an immunogenic antigen of viral origin. Methods Pancreatic adenocarcinoma tumor samples ( n = 130) were obtained intraoperatively, control tissues ( n = 23) were collected from cadaveric donor and from patients with chronic pancreatitis. Tumor-associated antigen expression of MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, LAGE-1, NY-ESO-1, SCP-1, SSX-2, SSX-4 and HERV-K-MEL was assessed by PCR. Sequencing of PCR products were performed to assess the expression of SSX-4 in neoplastic and normal pancreatic tissues. Results Three of 10 tested antigens were expressed in over 10% of malignant pancreatic tissue samples. SSX-4 was found positive in 30% of cases, SCP-1 in 19% and HERV-K-MEL in 23% of cases. No expression of CT antigens was found in non-malignant pancreatic tissue with the exception of SSX-4 and and SSX-2. Conclusions Fifty two percentage of the analyzed tissues expressed at least one CT antigen. The concomitant expression of SSX-4 in both malignant and non-malignant pancreatic tissue is a new finding which may raise concerns for immunotherapy. However, HERV-K-MEL is expressed with a relatively high prevalence and may be a candidate for specific immunotherapy in a large subgroup of pancreatic cancer patients. This study advocates the analysis of patients with regard to their immunogenic profile before the onset of antigen-specific immunotherapy.

103: Mesothelin: A malignant factor and a novel therapeutic vaccine target for pancreatic cancer

Background: Mesotheline (MSLN) is a GPI-linked glycoprotein, which is overexpressed in several cancer types including mesothelioma, ovarian cancer, and pancreatic cancer, but very little or no expression of MSLN is seen in normal tissues and other cancer types. In this study, we investigated the expression and functions of MSLN in human pancreatic cancer cells, and further studied the effect and mechanism of chimeric virus-like particle (VLP)-hMSLN as a potent vaccine candidate for immunotherapy in pancreatic cancer.

Research advancement on the immunotherapy of pancreatic cancer

随着分子生物学技术和肿瘤免疫学的发展,免疫治疗已成为肿瘤综合治疗模式的重要组成部分.近年来胰腺癌免疫治疗的研究也日趋增多,可望为胰腺癌的治疗开辟新的途径.现综述胰腺癌特异性主动免疫治疗、单克隆抗体治疗、细胞因子治疗和过继性细胞免疫治疗等进展。

New progress in immunotherapy of pancreatic cancer

New progress in immunotherapy of pancreatic cancer

Prostate Stem Cell Antigen Is a Putative Target for Immunotherapy in Pancreatic Cancer

The prostate stem cell antigen (PSCA) is a glycosylphosphatidyl-inositol (GPI)-linked cell surface antigen expressed in normal prostate and overexpressed in the majority of prostate cancers and correlates with tumor grade and disease stage. Because PSCA has been described to be up-regulated in pancreatic cancer, the purpose was to evaluate the expression of PSCA in human pancreatic cancer. Furthermore, the therapeutic efficacy of a monoclonal anti-PSCA antibody in an in vivo pancreatic cancer model was determined. The expression of PSCA in human pancreatic cancer tissues was determined and compared with chronic pancreatitis and normal pancreas by quantitative reverse transcriptase-polymerase chain reaction. Therapeutic efficacy of the monoclonal anti-PSCA antibody 1G8 was examined in Capan-1 pancreatic tumors grown as subcutaneous grafts in athymic nude mice. PSCA was strongly up-regulated in human pancreatic cancer compared with chronic pancreatitis and normal pancreas. In addition, the PSCA protein was expressed on the cell surface of pancreatic cancer cells. Treatment with 1G8 significantly reduced tumor growth initiation in an in vivo pancreatic cancer xenograft model. In addition, antibody treatment of established tumors reduced tumor progression. These results show a potential therapeutic role for anti-PSCA antibodies in the treatment of pancreatic cancer. Furthermore, PSCA might serve as a novel marker in the diagnosis of pancreatic cancer.

Sodium butyrate sensitises human pancreatic cancer cells to both the intrinsic and the extrinsic apoptotic pathways

Pancreatic cancer is characterised by a highly malignant phenotype with a marked resistance to conventional therapies and to apoptotic activators. Here, we demonstrate that sodium butyrate (NaBt), an inhibitor of histone deacetylases, sensitises human pancreatic cancer cell lines to both mitochondria- and Fas-mediated apoptosis. The analysis of anti-apoptotic and pro-apoptotic members of the Bcl-2 family in untreated pancreatic cancer cell lines shows a generalised low expression of Bcl-2 and a strong expression of Bcl-x L. NaBt treatment results in a marked down-regulation of Bcl-x L expression, mitochondrial membrane depolarization, cytochrome c release from mitochondria, activation of caspase-9 and -3 and apoptosis induction. Furthermore, NaBt sensitises pancreatic cancer cells to Fas-mediated apoptosis as well. In fact, the combined treatment with NaBt and the agonistic antibody anti-Fas (CH11) is able to induce apoptosis at an early time, in which neither NaBt nor CH11 alone induce apoptosis. Down-regulation of FLIP and activation of caspase-8 allow apoptosis to occur. These findings suggest that sodium butyrate could represent a good candidate for the development of new therapeutic strategies aimed at improving chemotherapy and immunotherapy in pancreatic cancer.

Immunotherapy for pancreatic cancer — science driving clinical progress

The identification of key signalling pathways involved in immune-system regulation, along with the development of early pancreatic tumours in mouse models have provided new opportunities for pancreatic cancer treatment and prevention. Immunotherapy for pancreatic cancer is one approach that is at a crucial crossroads, as therapeutics that are designed to target pancreatic-cancer-associated antigens and regulatory signalling molecules are entering clinical trials.

Patients Undergoing Treatment for Pancreatic Adenocarcinoma Can Mount an Effective Immune Response to Vaccinations

Background: Immunotherapy has been proposed as a novel treatment for pancreatic cancer. However, patients with pancreatic cancer have been observed to have depressed immune responses, suggesting that immunotherapy might have limited utility in this group of patients. We sought to determine whether patients undergoing postresection or primary medical treatment for pancreatic adenocarcinoma were immunocompetent. Methods: We enrolled patients with pancreatic adenocarcinoma scheduled for postresection or primary chemotherapy and/or radiation therapy. At the initiation of therapy, the patients had an anergy panel placed and baseline blood work performed. During the first week of treatment, patients received tetanus toxoid (TT), Haemophilus influenzae and Pneumococcus vaccines. Twelve weeks after vaccine administration, IgG titers against the 3 administered vaccines were done, and lymphocyte proliferation assays in response to TT were performed. Results: Eighteen patients were originally enrolled, and 14 patients completed all elements of the trial. Anergy panel responses were obtained for 15 patients who comprised the final study group; both pre- and postvaccination data were available for 14 patients. Nine of 15 patients demonstrated at least a 10-mm induration in response to mumps or Candida antigen (60% response rate, 95% confidence interval (CI) 32–84%). Thirteen of 14 patients demonstrated a ≧3-fold increase in IgG against one or more vaccines (93% response rate, 95% CI 66–100%). Nine of 14 patients (64% response rate, 95% CI 35–87%) demonstrated at least a 3-fold rise of lymphocyte proliferation against TT. Conclusions: Patients with pancreatic cancer were capable of mounting effective cellular and humoral responses to standard vaccines. These data suggest that immunotherapy for pancreatic cancer may be feasible and merits further investigation.

Nonmyeloablative allogeneic stem cell transplantation as immunotherapy for pancreatic cancer and other solid tumors

2539 Background: For the treatment of advanced and metastatic disease, conventional anti-tumor therapies such as chemotherapy and radiotherapy have been largely unsuccessful. Particularly in cases of advanced pancreatic cancer, prognosis is extremely poor. Recently, nonmyeloablative allogeneic stem cell transplantation (NST) has been successfully applied for treatment of solid tumors such as renal cell carcinoma. We performed NST in order to establish the role of this immunotherapy in the treatment strategy for Japanese patients with pancreatic cancer and other solid tumors. Methods: Japanese patients under the age of 65 with advanced disease resistant against chemotherapy and/or radiotherapy were enrolled in this pilot study. Fludarabine (Flu) based reduced intensity conditioning was performed prior to the infusion of G-CSF mobilized peripheral blood stem cells obtained from HLA- matched sibling donors. Donor chimerism of both T cells and mononuclear cells (MNC) was determined by PCR-based analysis of polymorphic short tandem repeat markers. In order to investigate the mechanism by which NST elicits an anti-tumor effect, serum levels of TNFα were measured by ELISA in four cases of pancreatic cancer. Results: Outcome of patients is given in the table below. Complete T cell chimerism was achieved within 100 days in all cases. Elevation of TNFα compared to baseline level was noted in patients #5 and #7, who achieved CR and SD, respectively. Serum levels of TNFα were unchanged in patients #6 and #8, who had progressive disease.Conclusions: Successful engraftment and anti-tumor effect of NST were observed. TNFα has a possible immunological role during the clinical course of NST. No significant financial relationships to disclose.

Nonmyeloablative allogeneic stem cell transplantation as immunotherapy for pancreatic cancer and other solid tumors

Nonmyeloablative allogeneic stem cell transplantation as immunotherapy for pancreatic cancer and other solid tumors

Development of immunotherapy for pancreatic cancer.

: Human tumor antigens recognized by T cells have been recently identified in various cancers, including pancreatic cancer. With the identified antigens, new immunotherapies can be developed using more efficient immunologic intervention (due to sufficient amounts of antigens in a more immunogenic form), as well as more quantitative and qualitative immunomonitoring. Various immunotherapies for patients with various cancers, including pancreatic cancer, are currently under evaluation in clinical trials. These include adoptive transfer of tumor reactive T cells and LAK cells; nonmyeloablative stem cell transplantation; active immunization with the identified tumor antigens, various tumor-derived products, dendritic cells pulsed with tumor antigens, and gene-modified tumor cells. Although these efforts in the realm of pancreatic cancer are still limited, various groups in Japan continue to be actively involved in this field of research.

Autoantibody-mediated inhibition of pancreatic cancer cell growth in an athymic (nude) mouse model.

Antiribosomal P autoantibodies are detectable in 12-16% of patients with systemic lupus erythematosus. To assess whether antiribosomal P autoantibodies could be useful as a novel form of immunotherapy for pancreatic cancer. Three pancreatic cancer cell lines were incubated with antiribosomal P or normal human immunoglobulin. Viability was assayed with MTT, and apoptosis was detected by TUNEL. MIA PaCa-2 cells were injected into athymic mice. Animals were treated with intraperitoneal antibody or control immunoglobulin. Serum antibody levels were measured by ELISA. Tumor nodule size was measured weekly. Binding of the antibody to tumors was demonstrated with fluorescent microscopy. Antiribosomal P antibody inhibited pancreatic cancer cell proliferation up to 54.6% (p < 0.01) and was associated with a threefold increase in the rate of apoptosis (p < 0.05). Tumor volume after 4 weeks of treatment was 23.2 mm3, versus 141.5 mm3 for the control group (p < 0.05). Apoptosis rate within the nodules was increased twofold, to 11.4%, in comparison with control (p < 0.05). Antiribosomal P autoantibody at levels similar to those that can exist in SLE inhibits the growth of pancreatic cancer cells, in vitro and in vivo. The mechanism involves surface binding and apoptosis.

Detection of peptide-specific cytotoxic T-lymphocyte precursors used for specific immunotherapy of pancreatic cancer.

The prognosis of pancreatic cancer is extremely poor with a 5-year survival of approximately 3%. Thus, the development of new treatment modalities, including a specific immunotherapy, is required. Our study investigated whether cytotoxic T-lymphocyte (CTL) precursors reacting to peptides with vaccine candidates (13 peptides for HLA-A2+ or -A24+ patients, respectively) were detectable in the prevaccination peripheral blood mononuclear cells (PBMCs) of 15 pancreatic cancer patients. Peptide-specific CTL precursors were detectable in the majority (11 of 15, 73%) of patients, with a mean positive number of 1.5 peptides (ranging from 0-5 peptides) per patient. Positive peptide profiles varied among patients. These results may provide a scientific basis for a new kind of cancer immunotherapy, namely, a CTL precursor-oriented peptide vaccine, for pancreatic cancer patients.

Antibody-dependent cytotoxicity mediated by chimeric monoclonal antibody Nd2 and experimental immunotherapy for pancreatic cancer.

In a previous study, mouse monoclonal antibody (MoAb) Nd2 (m‐Nd2, mouse IgGl) labeled with 131 I exhibited efficacy in in vivo radioimmunotherapy against pancreatic cancer. In this study we prepared mouse/human chimeric antibody Nd2 (c‐Nd2, human IgG1) for clinical use and examined whether c‐Nd2 induced antibody‐dependent cell‐mediated cytotoxicity (ADCC). Cytotoxicity to pancreatic cancer (PC) cell lines, including Nd2 antigen‐positive (SW1990, RWP‐1, Capan‐1) and Nd2 antigen‐negative (Panc‐1, MiaPaca‐2, Capan‐2) lines, was evaluated by mixed human leukocyte and tumor cell culture (MLTC) at an effector cell to target cell (E/T) ratio of 50 with or without Nd2. Cytotoxicities to SW1990 with no antibody, m‐Nd2 and c‐Nd2 (1 μg/ml) were 26.7%, 38.0% and 55%, respectively; to RWP‐1, 28%, 41% and 70%; to Capan‐1, 26%, 30% and 52%; to Panc‐1, 24%, 28% and 30%; to MiaPaca‐2, 18%, 20% and 27% and to Capan‐2, 29.7%, 35.0% and 40.6%. Cytotoxic capacity during MLTC with c‐Nd2 was significantly higher than during MLTC with m‐Nd2 or with no antibody. These findings indicated that cytotoxicity to Nd2‐positive PC cells during MLTC is induced by ADCC. Intraperitoneal injection of c‐Nd2 inhibited the tumor growth of SW1990 xenografted subcutaneously in nude mice and prolonged the survival of nude mice in which SW1990 tumor was transplanted orthotopically at the tail of the pancreas. These findings suggested that, because of its ability to induce ADCC, c‐Nd2 may be clinically useful for the immunotherapeutic treatment of pancreatic cancer.

A3--a novel colon and pancreatic cancer reactive antibody from a primate phage library selected using intact tumour cells.

The identification of novel tumour-associated antigens (TAAs) is pivotal for progression in the fields of tumour immunotherapy and diagnosis. In the present study, we have developed, based on flow cytometric evaluation and use of a mini-library composed of specific antibody clones linked to different antibiotic resistance markers, methods for positive and subtractive selection of phage antibodies employing intact cells as the antigen source. An scFv phage library (2.7 x 10(7)) was constructed from a primate (Macaca fascicularis) immunised with pooled human colon carcinomas. This library was selected for 3 rounds by binding to Colo 205 colon adenocarcinoma cells and proteolytic elution followed by phage amplification. Several antibodies reactive with colon carcinomas and with restricted reactivity to a few epithelial normal tissues were identified by immunohistochemistry. One clone, A3 scFv, recognised an epitope that was homogeneously expressed in 11/11 of colon and 4/4 pancreatic carcinomas studied and in normal tissue restricted to subtypes of epithelia in the gastrointestinal tract. The A3 scFv had an apparent overall affinity approximately 100-fold higher than an A3 Fab, suggesting binding of scFv homodimers. The cell surface density of the A3 epitope, calculated on the basis of Fab binding, was exceptionally high, approaching 3 million per cell. We also demonstrate efficient T-cell-mediated killing of colon cancer cells coated with A3 scFv fused to the low MHC class II binding superantigen mutant SEA(D227A). The identified A3 molecule thus represents a TAA with properties that suggest its use for immunotherapy of colon and pancreatic cancer.

A3—a novel colon and pancreatic cancer reactive antibody from a primate phage library selected using intact tumour cells

The identification of novel tumour-associated antigens (TAAs) is pivotal for progression in the fields of tumour immunotherapy and diagnosis. In the present study, we have developed, based on flow cytometric evaluation and use of a mini-library composed of specific antibody clones linked to different antibiotic resistance markers, methods for positive and subtractive selection of phage antibodies employing intact cells as the antigen source. An scFv phage library (2.7 x 10(7)) was constructed from a primate (Macaca fascicularis) immunised with pooled human colon carcinomas. This library was selected for 3 rounds by binding to Colo 205 colon adenocarcinoma cells and proteolytic elution followed by phage amplification. Several antibodies reactive with colon carcinomas and with restricted reactivity to a few epithelial normal tissues were identified by immunohistochemistry. One clone, A3 scFv, recognised an epitope that was homogeneously expressed in 11/11 of colon and 4/4 pancreatic carcinomas studied and in normal tissue restricted to subtypes of epithelia in the gastrointestinal tract. The A3 scFv had an apparent overall affinity approximately 100-fold higher than an A3 Fab, suggesting binding of scFv homodimers. The cell surface density of the A3 epitope, calculated on the basis of Fab binding, was exceptionally high, approaching 3 million per cell. We also demonstrate efficient T-cell-mediated killing of colon cancer cells coated with A3 scFv fused to the low MHC class II binding superantigen mutant SEA(D227A). The identified A3 molecule thus represents a TAA with properties that suggest its use for immunotherapy of colon and pancreatic cancer.

Immunotherapy in pancreatic cancer - current status and future.

In pancreatic cancer, multimodal protocols, involving chemotherapy, radiation, or regional treatment, are initiated to improve oncological outcome. Since pancreatic adenocarcinoma has been shown to be susceptible to immune stimulation, several immunotherapy approaches have been investigated to define the role of immunotherapy in pancreatic cancer. A review of current and past data concerning experimental and clinical immunotherapy in pancreatic cancer is presented in the context of basic immunotherapeutic principles. Past pitfalls and future developments are analyzed and a synthesis of immune stimulation and immune suppression is deduced on the basis of published data. Preclinical and initial clinical studies with monoclonal antibodies CO17-1A, BW494/32 and anti-epidermal growth factor receptor (EGFR) have been conducted, and various targets suitable for immunotherapy have been identified involving new molecular and gene technology. Targets on pancreatic cancer cells currently under investigation are mucins (MUC-1), glycoproteins (GA733), ras peptides and EGFRs. Side effects are minor and rarely auto-immune reactive. Another approach combines randomized regional with systemic chemoimmunotherapy (mitomycin C, 5-fluorouracil, folinic acid, carboplatin, epirubicin; interferon-gamma, interleukin-2) in nonresectable pancreatic cancer and obtains significant differences in median survival rates (14 months vs 4.5 months in controls) and quality of life. Although single remarkable improvements in the immunological approach to treatment of pancreatic cancer have been made, immunotherapy in pancreatic cancer is still experimental. On the basis of reliable preclinical data, new immunotherapy protocols will have to be evaluated clinically. Careful monitoring of immune responses and side effects, and assessment of quality of life will ensure identification of effective immunotherapy protocols for human pancreatic cancer in the near future.

Adjuvant therapy of pancreatic cancer using monoclonal antibodies and immune response modifiers.

Pancreatic cancer is a devastating disease with poor survival. At present, no effective adjuvant or palliative therapies are available. Unresponsiveness to chemotherapy, radiotherapy, and antihormonal treatment is one of the reasons that pancreatic cancer patients have an overall median survival time of 4-6 mo. This article summarizes clinical trials on immunotherapy of pancreatic cancer using the murine monoclonal antibodies (MAbs) 17-1A and BW 494. In addition, the use of MAb treatment in combination with immune response modifiers is discussed. In four clinical trials, MAb 17-1A was given by iv infusion to 100 patients with pancreatic cancer. In 30 of the these patents, antibody treatment was accompanied by gamma-interferon, also given intravenously. Complete response, partial response, and stable disease were reported in 1,5, and 23 patients, respectively. Passive immunotherapy using the MAb BW 494 was carried out in 148 pancreatic cancer patients in two phase I and two phase II trials. In 1 out of 75 patients a partial response and in 25 out of 74 patients stable disease were reported. However, in a controlled randomized trial enrolling 61 patients following Whipple resection, comparable survival times in patients with and without MAb BW 494 treatment led to the termination of further clinical trials with this antibody. New clinical studies using humanized MAbs in combination with immune response modifiers should be initiated to further evaluate immunotherapy as a treatment option in pancreatic cancer.