Active Cancer Immunotherapy Research Articles

Abstract A039: Caspase-2 and oxidative-ER stress crosstalk regulates the exposure of “eat me” signal calreticulin by high hydrostatic pressure treated cancer cells

Abstract Immunogenic cell death (ICD) is a type of cell death induced by various chemotherapeutics, physical modalities such as photodynamic therapy, radiotherapy and high hydrostatic pressure (HHP). HHP induces immunogenic cell death of cancer cell is currently tested in running II. and III. phase of clinical trial for preparation of active cellular cancer immunotherapy (DCVAC). Previous studies have shown that the immunogenicity of ICD mainly relies on surface exposed calreticulin (ecto-CRT); CRT is normally located in the lumen of the endoplasmic reticulum (ER) and may translocate to the cell surface within hours after treatment. Although ICD can be triggers by HHP, underlying danger signaling pathways remain unclear. Therefore ovarian (OV90) cancer cell line was treated by HHP, UV-B and anthracycline (Idarubicin). The kinetics of ICD markers and key components of ER stress-mediated apoptotic pathway were analyzed by flow cytometry, western blotting and confocal microscopy. In this work we described the ability of HHP triggers oxidative ER-stress (the production of reactive oxygen species (ROS) together with ER stress response) which results in phosphorylation of PERK and eIF2α. We also observed a rapid caspase-8 activation subsequently followed by BAP31 cleavage, Bid activation and caspase-3 activation. While caspase-8 activation was necessary for cell surface exposure of calreticulin (ecto-CRT) after HHP treatment, proapoptotic proteins Bax and Bak were dispensable for this process. Moreover, we identified that HHP treatment leads to caspase-2 activation. Whereas Idarubicin treatment has no effect on ecto-CRT exposure in Caspase-2 knockdown tumor cells, ecto-CRT was significantly decreased in these cells after HHP treatment. In addition, phagocytosis of HHP-treated Caspase-2 knockdown tumor cells was compromised in contrast to Idarubicin-treated knockout tumor cells. In conclusion, we characterized the molecular mechanism of ecto-CRT exposure pathway induced by HHP which is analogous to that induced by anthracycline treatment. For the first time we provide evidence that caspase-2 might play an important role in the HHP-mediated ecto-CRT exposure. Citation Format: Irena Kusova Moserova, Iva Truxova, Abhishek G, Garg, Patrizia Agostinis, Piere Francois Cartron, Sarka Vosahlikova, Radek Spisek, Jitka Fucikova. Caspase-2 and oxidative-ER stress crosstalk regulates the exposure of “eat me” signal calreticulin by high hydrostatic pressure treated cancer cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A039.

Active Immunotherapy of Cancer

Clinical progress in the field of cancer immunotherapy has been slow for many years but within the last 5 years, breakthrough successes have brought immunotherapy to the forefront in cancer therapy. Promising results have been observed in a variety of cancers including solid tumors and hematological malignancies with adoptive cell therapy using natural host tumor infiltrating lymphocytes, host cells that have been genetically engineered with antitumor T-cell receptors or chimeric antigen receptors, immune checkpoint inhibitors like anti-CTLA-4, anti-PD-1 or PD-L1 monoclonal antibodies and oncolytic virus-based immunotherapy. However, most treatment modalities have shown limited efficacy with single therapy. The complex nature of cancer with intra- and inter-tumor antigen and genomic heterogeneity coupled with the immune suppressive microenvironment emphasizes the prospect of personalized targeted immunotherapy to manipulate the patient’s own immune system against cancer. For successful, robust and long-lasting cure of cancer, a multi-modal approach is essential, combining anti-tumor cell therapy with manipulation of multiple pathways in the tumor microenvironment to ameliorate tumor-induced immunosuppression.

IMCT-17STATISTICALLY SIGNIFICANT ASSOCIATION OF GLIOBLASTOMA IMMUNOTHERAPY PHASE II CLINICAL STUDY (ICT-107) TREATMENT AND SURVIVAL TO IMMUNE RESPONSE USING A NOVEL COMPREHENSIVE ELISPOT ANALYSIS

The main objective of active cancer immunotherapy is the activation of a patient's own T cells to attack tumor cells. The accurate measurement of the expansion and functionality of these T cells upon treatment is a key element in establishing the association between treatment and treatment outcomes or survival. While much research and clinical work has been focused on establishing and optimizing immunomonitoring during clinical trials, direct association of patient immune response to patient clinical endpoints remains difficult. IFN-g ELISPOT has been used extensively to monitor the immune response in immunotherapy trials. While there have been significant efforts to harmonize ELISPOT procedures and analysis, a standard methodology for classifying a positive immune response in an immunotherapy context has not emerged. Past studies have used techniques ranging from simple quantification of spot counts to published laboratory ELISPOT statistical analysis on single samples, but these studies lack consensus on establishing response to treatment with pre- and post- treatment samples. Here, novel statistical methods are presented for the direct generation of p-values comparing pre- and post- treatment ELISPOT responses evaluated ex-vivo in patients treated with a multi-peptide loaded dendritic cell-based immunotherapy. Then, a comprehensive scoring system is introduced which incorporates these novel methods, along with previously used metrics, into a single numerical scale. Using an analysis of score distribution, this scoring system is shown to distinguish response from non-response even in the context of low levels of T cell response detection sometimes associated with ex-vivo evaluation. Consequently, binary classification of patients as responders and non-responders is performed. Finally, this classification system is shown to compare favorably to other methodologies with respect to its ability to find statistically significant associations between the immune response detected ex-vivo and treatment group, IL-12 production of dendritic cells, and both overall and progression-free patient survival.

Adjuvant HER2/neu peptide cancer vaccines in breast cancer.

Active cancer immunotherapy remains an exciting and rapidly advancing field in oncology. Peptide cancer vaccines are an attractive therapeutic option as they are safe and easily produced and administered. Peptide cancer vaccines may be most effective in patients with a lower disease burden, when cancer tolerance is minimized. Our experience with three peptide cancer vaccines, E75, GP2 and AE37, in clinically disease-free breast cancer patients provides encouraging results that this method may be effective. Furthermore, the combined results of the initial trials suggest that the vaccine administered may need to be tailored to the specific subtype of cancer and tumor antigen expression level to achieve maximum effectiveness. The results also suggest that combining peptide vaccines with other immunotherapy may lead to a synergistic effect.

Targeting cryptic epitope with modified antigen coupled to the surface of liposomes induces strong antitumor CD8 T-cell immune responses invivo.

Active cancer immunotherapy, such as cancer vaccine, is based on the fundamental knowledge that tumor-associated antigens (TAAs) are presented on MHC molecules for recognition by specific T cells. However, most TAAs are self-antigens and are also expressed on normal tissues, including the thymus. This fact raises the issue of the tolerance of the TAA-specific T-cell repertoire and consequently the inability to trigger a strong and efficient antitumor immune response. In the present study, we used antigens chemically coupled to the surface of liposomes to target telomerase reverse transcriptase (TERT), a widely expressed self/tumor antigen. Taking advantage of the high homology between mouse and human TERT, we investigated immunogenicity and antitumor efficiency of the liposomal TERT peptides in HLA-A*0201 transgenic HHD mice. Using the heteroclitical peptide-modifying approach with antigen-coupled liposomes, we identified a novel cryptic epitope with low affinity for HLA*0201 molecules derived from TERT. The heteroclitical variant derived from this novel low affinity peptide exhibited strong affinity for HLA*0201 molecules. However, it induced only weak CD8 T-cell immune responses in HHD mice when emulsified in IFA. By contrast, when coupled to the surface of the liposomes, it induced powerful CD8 T-cell immune responses which cross-reacted against the original cryptic epitope. The induced CD8 T cells also recognized endogenously TERT-expressing tumor cells and inhibited their growth in HHD mice. These data suggest that heteroclitical antigen derived from low affinity epitope of tumor antigens coupled to the surface of liposome may have a role as an effective cancer vaccine candidate.

Dendritic Cells as Pharmacological Tools for Cancer Immunotherapy.

Although the earliest—rudimentary—attempts at exploiting the immune system for cancer therapy can be traced back to the late 18th Century, it was not until the past decade that cancer immunotherapeutics have truly entered mainstream clinical practice. Given their potential to stimulate both adaptive and innate antitumor immune responses, dendritic cells (DCs) have come under intense scrutiny in recent years as pharmacological tools for cancer immunotherapy. Conceptually, the clinical effectiveness of this form of active immunotherapy relies on the completion of three critical steps: 1) the DCs used as immunotherapeutic vehicles must properly activate the antitumor immune effector cells of the host, 2) these immune effector cells must be receptive to stimulation by the DCs and be competent to mediate their antitumor effects, which 3) requires overcoming the various immune-inhibitory mechanisms used by the tumor cells. In this review, following a brief overview of the pivotal milestones in the history of cancer immunotherapy, we will introduce the reader to the basic immunobiological and pharmacological principles of active cancer immunotherapy using DCs. We will then discuss how current research is trying to define the optimal parameters for each of the above steps to realize the full clinical potential of DC therapeutics. Given its high suitability for immune interventions, acute myeloid leukemia was chosen here to showcase the latest research trends driving the field of DC-based cancer immunotherapy.

Cancer immunotherapy in mice and humans: personal observations.

An experiment involving active immunotherapy of leukemia in mice and its extension to human patients with chronic lymphocytic leukemia is described. First, anticancer activation of the immune system, how it is achieved, and observations of the central blockade of the immune system in leukemia are discussed. Clinical results of antileukemia immune activation are then presented, along with a description of new attempts to remove the immune checkpoint blockade. Personal observations are presented on possible further steps to achieving more effective active immunotherapy of cancer.

Mucin 1-specific active cancer immunotherapy with tecemotide (L-BLP25) in patients with multiple myeloma: An exploratory study

Patients (n = 34) with previously untreated, slowly progressive asymptomatic stage I/II multiple myeloma or with stage II/III multiple myeloma in stable response/plateau phase following conventional anti-tumor therapy were immunized repeatedly with the antigen-specific cancer immunotherapeutic agent tecemotide (L-BLP25). Additionally, patients were randomly allocated to either single or multiple low doses of cyclophosphamide to inhibit regulatory T cells (Treg). Immunization with tecemotide resulted in the induction/augmentation of a mucin 1-specific immune response in 47% of patients. The immune responses appeared to involve a Th1-like cellular immune response involving CD4 and CD8 T cells. The rate of immune responses was similar with single versus multiple dosing of cyclophosphamide and in patients with vs. without pre-existing mucin 1 immunity. On-treatment reductions in the slope of M-protein concentration over time (but not fulfilling clinical criteria for responses with conventional anti-tumor agents) were observed in 45% of evaluable patients, predominantly in those without versus with pre-existing mucin 1 immunity and in patients with early stage disease. No differences were seen in patients receiving single or multiple cyclophosphamide dosing. Treatment with tecemotide was generally well tolerated. Repeated vs. single dosing of cyclophosphamide had no impact on Treg numbers and was stopped after a case of fatal encephalitis that was assessed as possibly study-related. Tecemotide immunotherapy induces mucin 1-specific cellular immune responses in a substantial proportion of patients, with preliminary evidence of changes in the M-protein concentration time curve in a subset of patients.

Epitope mapping of epidermal growth factor receptor (EGFR) monoclonal antibody and induction of growth-inhibitory polyclonal antibodies by vaccination with EGFR mimotope

One of the proposed approaches in cancer therapy is to induce and direct the patient’s own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.

Telomerase (GV1001) vaccination together with gemcitabine in advanced pancreatic cancer patients.

Telomerase is expressed in 85-90 % of pancreatic adenocarcinomas and might be a target for active cancer immunotherapy. A study was conducted to investigate safety and immunogenicity in non-resectable pancreatic carcinoma patients using a 16-amino acid telomerase peptide (GV1001) for vaccination in combination with GM-CSF and gemcitabine as first line treatment. Three different vaccine treatment schedules were used; [A (n=6), B (n=6) and C (n=5)]. Groups A/B received GV1001, GM-CSF and gemcitabine concurrently. Group C received initially GV1001 and GM-CSF while gemcitabine was added at disease progression. Group D (n=4) was treated with gemcitabine alone. Adverse events (AE) related to vaccination were mild (grades I-II). Grade III AEs were few and transient. An induced GV 1001‑specific immune response was defined as an increase ≥2 above the baseline value in one of the assays (DTH, proliferation, ELISPOT and cytokine secretion assays, respectively). A telomerase‑specific immune response was noted in 4/6 patients in group A, 4/6 patients in group B and 2/5 patients in group C. An induced ras‑specific immune response (antigenic spreading) was seen in 5 of the 17 patients. The cytokine pattern was that of a Th1-like profile. A treatment induced telomerase or ras response was also noted in group D. All responses were weak and transient. A significant decrease in regulatory T-cells over time was noted in patients in groups A and B (p<0.05). Telomerase vaccination (GV1001) in combination with chemotherapy appeared to be safe but the immune responses were weak and transient. Measures have to be taken to optimize immune responses of GV1001 for it to be considered of clinical interest.

Expression of tolerogenic potential-representing markers on clinical-grade therapeutic dendritic cell-based cancer vaccines

Background. Currently a particular interest is given to specific active cancer immunotherapy (therapeutic cancer vaccination) strategies such as treatment with specially “educated” autologous dendritic cells (DCs). The purpose of these vaccines is to enhance antitumor immune responses against the existing tumor. DC vaccines are composed of tumor antigen-loaded mature DCs, which are produced for each cancer patient individually. However, recent data indicate that there are at least two types of mature DCs – immunogenic, which activate antitumor immune responses, and tolerogenic, which suppress immune responses, thereby interfering with effector mechanisms of protective antitumor immunity. Hence, the aim of our study was to assess the expression of immunogenic and tolerogenic potential-representing markers on the surface of therapeutic DC vaccines generated using two different maturation approaches. Materials and methods. Two different DC maturation approaches were investigated: Cocktail 1, composed of lipopolysaccharide (200 ng/ mL) and interferon-γ (50 ng/mL), and Cocktail 2, composed of IL1β (10 ng/mL), IL-6 (10 ng/mL), TNF-α (10 ng/mL), PGE2 (1 μg/mL). Secretion of two basic cytokines – the immunostimulatory IL-12p70 and the immunosuppressive IL-10 – has also been investigated. Results. We show that a subset of immature DCs expressed tolerogenic markers. Importantly their expression profile considerably differed on mature DCs, depending on the maturation approach used. Conclusions. In particular, our results indicate that Cocktail 1 is superior to Cocktail 2 for the production of clinical-grade therapeutic cancer DC vaccines, both in terms of immunophenotypical attributes of DC tolerogenicity and their cytokine secretory profile.

Active (Patient Specific) Immunotherapy of Colon Cancer: A Transition from Preclinical Studies to Successful Clinical Trials

Active (Patient Specific) Immunotherapy of Colon Cancer: A Transition from Preclinical Studies to Successful Clinical Trials

Clinical experience with live-attenuated, double-deleted (LADD) listeria monocytogenes targeting mesothelin-expressing tumors

Meeting abstracts Immune-based cancer therapies have demonstrated benefit in multiple clinical trials. The interest in using recombinant bacteria as vaccine vectors for active cancer immunotherapy derives in part from their ability to stimulate multiple innate immune pathways and, at the same time

Vascular normalization: a strategy to recondition the tumor immune microenvironment

Meeting abstracts Malignant tumors escape from host immune surveillance through multiple mechanisms. Of these, abnormal tumor vasculature and hypoxia are critical in establishing an immunosuppressive tumor microenvironment, and consequently, impeding an active cancer immunotherapy. Thus, we

A randomized, double-blind, placebo-controlled, multicenter, multinational, phase II trial immunotherapy with L-BLP25 (tecemotide) in patients with colorectal carcinoma following R0/R1 hepatic metastasectomy.

TPS3124^ Background: 15-20% of all patients (pts) diagnosed with colorectal cancer (crc) develop metastases (mets) surgical resection remains the only potentially curative treatment available. Current 5-year survival rate following R0 resection of liver mets lies between 28-39%, recurrence occurs in up to 70% of pts. To date, adjuvant chemotherapy has not significantly improved clinical outcomes. The primary objective of the ongoing LICC trial (L-BLP25 In Colorectal Cancer) is to determine whether L-BLP25, an active MUC1-specific cancer immunotherapy, extends recurrence-free survival (RFS) time over placebo in crc pts following R0/R1 resection of liver mets known to highly express MUC1 glycoprotein. Phase III data from L-BLP25 in NSCLC will be reported at this meeting. Methods: This is a multinational, phase II, multicenter, randomized, double-blind, placebo-controlled trial with a sample size of 159 pts from 20 centers in 3 countries. Pts must have stage IV cr adenocarcinoma limited to liver mets. Following curative-intent complete resection of the primary tumor and of all synchronous/metachronous mets, eligible pts are randomized 2:1 to receive either L-BLP25 or placebo. L-BLP25 arm receives a single dose of 300 mg/m2 cyclophosphamide (CPA) 3 d before 1st L-BLP25 dose, then primary treatment with sc L-BLP25 930 μg weekly for 8 weeks, followed by sc L-BLP25 930 μg maintenance doses at 6-week (year 1 and 2) and 12-week (year 3) intervals until recurrence. Control arm: CPA is replaced by saline solution and L-BLP25 by placebo. Primary endpoint (PE) is RFS time. Secondary endpoints: Overall survival (OS), safety, tolerance, RFS/OS in MUC-1 positive cancers. Exploratory immune response analyses are planned. Study start was in Q3 2011. 19 centers were initialized and 36 patients recruited, no SUSARs occurred. Study recruitment will end Q3 2013: follow-up until Q3 2017. PE assessment is in Q3 2016. Interim analyses are not planned.No major practical issues were identified during setup and early conduct of the study. Clinical trial information: 2011-000218-20.

A randomized, double-blind, placebo-controlled, multicenter, multinational, phase II trial of L-BLP25 in patients with colorectal carcinoma following R0/R1 hepatic metastasectomy.

TPS3641^ Background: Approximately 15-20% of patients diagnosed with colorectal cancer (crc) develop metastatic disease. Surgical resection remains the only potentially curative treatment. 5-year survival following R0-resection of liver metastases lies ~28 -39%. Recurrence occurs in ~70% of pts. Adjuvant chemotherapy has not significantly improved clinical outcomes. The primary objective of the LICC trial (L-BLP25 in Colorectal Cancer) is to analyze whether L-BLP25, an active cancer immunotherapy, extends recurrence-free survival (RFS) time over placebo in colorectal cancer pts following R0/R1 resection of hepatic metastases. L-BLP25 targets MUC1 glycoprotein, which is highly expressed in hepatic metastases from crc. In a phase IIB trial, L-BLP25 showed acceptable tolerability and a trend toward longer survival in pts with stage IIIB NSCLC. Methods: This is a multinational, phase II, multicenter, randomized, double-blind, placebo-controlled trial with a sample size of 159 pts from 20 centers in 3 countries. Pts must have stage IV cr adenocarcinoma limited to liver metastases. Following complete resection of the primary tumor and all syn-/metachronous metastases, eligible pts are randomized 2:1 to receive either L-BLP25 or placebo. Those allocated to L-BLP25 receive a single dose of 300 mg/m2 cyclophosphamide (CP) 3 days before first L-BLP25 dose, then primary treatment with sc L-BLP25 930 μg once weekly for 8 weeks, followed by maintenance doses at 6-week (years 1 and 2) and 12-week (year 3) intervals until recurrence. In the control arm, CP is replaced by saline solution and L-BLP25 by placebo. Primary endpoint: RFS time. Secondary endpoints: OS time, safety status, tolerability, RFS/OS in MUC-1 positive cancers. Exploratory immune response analyses are planned. First recruitment was of Q3 2011. To date, 8 of 20 centers are initiated and 4 pts recruited. Completion of recruitment is scheduled for Q3 2013. Primary endpoint will be assessed in Q3 2016: Follow-up will end Q3 2017. No interim analysis is planned. Design and implementation of this vaccination study in colorectal cancer is feasible. No major issues identified during setup of the study.

Uptake routes of tumor-antigen MAGE-A3 by dendritic cells determine priming of naïve T-cell subtypes.

Induction of tumor-antigen-specific T cells in active cancer immunotherapy is generally difficult due to the very low anti-tumoral precursor cytotoxic T cells. By improving tumor-antigen uptake and presentation by dendritic cells (DCs), this problem can be overcome. Focusing on MAGE-A3 protein, frequently expressed in many types of tumors, we analyzed different DC-uptake routes after additional coating the recombinant MAGE-A3 protein with either a specific monoclonal antibody or an immune complex formulation. Opsonization of the protein with antibody resulted in increased DC-uptake compared to the uncoated rhMAGE-A3 protein. This was partly due to Fcγ receptor-dependent internalization. However, unspecific antigen internalization via macropinocytosis also played a role. When analyzing DC-uptake of MAGE-A3 antigen expressed in multiple myeloma cell line U266, pretreatment with proteasome inhibitor bortezomib resulted in increased apoptosis compared to γ-irradiation. Bortezomib-mediated immunogenic apoptosis, characterized by elevated surface expression of hsp90, triggered higher phagocytosis of U266 cells by DCs involving specific DC-derived receptors. We further investigated the impact of antigen delivery on T-cell priming. Induction of CD8(+) T-cell response was favored by stimulating naïve T cells with either antibody-opsonized MAGE-A3 protein or with the bortezomib-pretreated U266 cells, indicating that receptor-mediated uptake favors cross-presentation of antigens. In contrast, CD4(+) T cells were preferentially induced after stimulation with the uncoated protein or protein in the immune complex, both antigen formulations were preferentially internalized by DCs via macropinocytosis. In summary, receptor-mediated DC-uptake mechanisms favored the induction of CD8(+) T cells, relevant for clinical anti-tumor response.

Cancer and Innate Immune System Interactions

Passive immunotherapy, including adoptive T-cell therapy and antibody therapy, has shown encouraging results in cancer treatment lately. However, active immunotherapy of solid cancers remains an elusive goal. It is now known that the human innate immune system recognizes pathogen-associated molecular patterns conserved among microbes or damage-associated molecular patterns released from tissue injuries to initiate adaptive immune responses during infection and tissue inflammation, respectively. In contrast, how the innate immune system recognizes endogenously arising cancer remains poorly understood at the molecular level, which poses a significant roadblock to the development of active cancer immunotherapy. We hereby review the current knowledge of how solid cancers directly and indirectly interact with cells of the human innate immune system, with a focus on the potential effect of such interactions to the resultant adaptive immune responses against cancer. We believe that understanding cancer and innate immune system interactions may allow us to better manipulate the adaptive immune system at the molecular level to develop effective active immunotherapy against cancer. Current and future perspectives in clinical development that exploits these molecular interactions are discussed.

LICC: L-BLP25 in patients with colorectal carcinoma after curative resection of hepatic metastases--a randomized, placebo-controlled, multicenter, multinational, double-blinded phase II trial

Background15-20% of all patients initially diagnosed with colorectal cancer develop metastatic disease and surgical resection remains the only potentially curative treatment available. Current 5-year survival following R0-resection of liver metastases is 28-39%, but recurrence eventually occurs in up to 70%. To date, adjuvant chemotherapy has not improved clinical outcomes significantly. The primary objective of the ongoing LICC trial (L-BLP25 In Colorectal Cancer) is to determine whether L-BLP25, an active cancer immunotherapy, extends recurrence-free survival (RFS) time over placebo in colorectal cancer patients following R0/R1 resection of hepatic metastases. L-BLP25 targets MUC1 glycoprotein, which is highly expressed in hepatic metastases from colorectal cancer. In a phase IIB trial, L-BLP25 has shown acceptable tolerability and a trend towards longer survival in patients with stage IIIB locoregional NSCLC.Methods/DesignThis is a multinational, phase II, multicenter, randomized, double-blind, placebo-controlled trial with a sample size of 159 patients from 20 centers in 3 countries. Patients with stage IV colorectal adenocarcinoma limited to liver metastases are included. Following curative-intent complete resection of the primary tumor and of all synchronous/metachronous metastases, eligible patients are randomized 2:1 to receive either L-BLP25 or placebo. Those allocated to L-BLP25 receive a single dose of 300 mg/m2 cyclophosphamide (CP) 3 days before first L-BLP25 dose, then primary treatment with s.c. L-BLP25 930 μg once weekly for 8 weeks, followed by s.c. L-BLP25 930 μg maintenance doses at 6-week (years 1&2) and 12-week (year 3) intervals unless recurrence occurs. In the control arm, CP is replaced by saline solution and L-BLP25 by placebo. Primary endpoint is the comparison of recurrence-free survival (RFS) time between groups. Secondary endpoints are overall survival (OS) time, safety, tolerability, RFS/OS in MUC-1 positive cancers. Exploratory immune response analyses are planned. The primary endpoint will be assessed in Q3 2016. Follow-up will end Q3 2017. Interim analyses are not planned.DiscussionThe design and implementation of such a vaccination study in colorectal cancer is feasible. The study will provide recurrence-free and overall survival rates of groups in an unbiased fashion.Trial RegistrationEudraCT Number 2011-000218-20

A Novel Therapeutic Vaccine of Mouse GM-CSF Surface Modified MB49 Cells Against Metastatic Bladder Cancer

Immunotherapy is considered effective for muscle invasive bladder cancer mini metastasis. We developed what is to our knowledge a novel technology by which streptavidin tagged mouse GM-CSF was displayed on the surface of biotinylated bladder cancer cells to induce antitumor immunity. Mouse subcutaneous and lung metastasis bladder cancer models were established. Mice were injected subcutaneously with 1 × 10(6) mouse GM-CSF surface modified MB49 bladder cancer cells and monitored for tumor growth and survival. Immunohistochemical and flow cytometric assay were done to assess the proportion of T lymphocytes. The T-lymphocyte cytotoxicity assay was performed to assess MB49 specific cytotoxicity. On day 60 after MB49 implantation the vaccine cured mice were injected subcutaneously with MB49 or RM-1 cells in the left or right hind leg, respectively. They were monitored for survival and T-lymphocyte cytotoxicity. Mouse GM-CSF surface modified vaccine significantly inhibited tumor growth in the subcutaneous model and extended survival in the lung model. More CD4 and CD8 T cells appeared at tumor sites and in peripheral blood in the vaccine treated group than in other control groups. Splenocytes from the vaccine treated group showed the most potent cytotoxicity on MB49 cells. Cured mice in the vaccine treated group resisted the second injection of MB49 bladder cancer cells but not the RM-1 prostate cancer cell challenge. Mouse GM-CSF surface modified MB49 bladder cancer cell vaccine induced specific antitumor immunity and was efficient for metastatic bladder cancer.