Granulocyte-macrophage Colony-stimulating Factor Secreting Research Articles

A phase I trial of vaccination with lethally irradiated lymphoma cells admixed with granulocyte-macrophage colony-stimulating factor secreting K562 cells for the treatment of follicular lymphoma

Several vaccine strategies have been tested for the treatment of follicular lymphoma; however, none have proven successful. In a phase I dose-escalation protocol, we developed a vaccine consisting of lethally irradiated whole lymphoma cells admixed with K562 cells that constitutively secreted granulocyte-macrophage colony-stimulating factor (GM-K562)(ClinicalTrials.gov identifier: NCT00487305). Patients with grade 1, 2, or 3 A follicular lymphoma were divided into 2 study tiers based on prior treatment and received a maximum of 6 vaccines. Vaccines contained dose levels of 5 × 106 or 1 × 107 GM-K562 cells admixed with autologous tumor cells at doses ranging from 1 × 105 to 5 × 107.Correlative studies did not demonstrate a significant immune response as assessed by delayed-type hypersensitivity reactions, B and T cell subsets, and natural killer cell subsets. Future vaccine studies should focus on identifying lymphoma-specific immunogenic proteins and modifying the vaccine immune adjuvant.

Phase II trial of vaccination with autologous, irradiated melanoma cells engineered by adenoviral mediated gene transfer to secrete granulocyte-macrophage colony stimulating factor in patients with stage III and IV melanoma.

In the era of immune checkpoint blockade, the role of cancer vaccines in immune priming has provided additional potential for therapeutic improvements. Prior studies have demonstrated delayed type hypersensitivity and anti-tumor immunity with vaccines engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF). The safety, efficacy and anti-tumor immunity of GM-CSF secreting vaccine in patients with previously treated stage III or IV melanoma needs further investigation. In this phase II trial, excised lymph node metastases were processed to single cells, transduced with an adenoviral vector encoding GM-CSF, irradiated, and cryopreserved. Individual vaccines were composed of 1x106, 4x106, or 1x107 tumor cells, and were injected intradermally and subcutaneously at weekly and biweekly intervals. The primary endpoints were feasibility of producing vaccine in stage III patients and determining the proportion of patients alive at two years in stage IV patients. GM-CSF vaccine was successfully developed and administered in all 61 patients. Toxicities were restricted to grade 1-2 local skin reactions. The median OS for stage III patients (n = 20) was 71.1 (95% CI, 43.7 to NR) months and 14.9 (95%CI, 12.1 to 39.7) months for stage IV patients. The median PFS in stage III patients was 50.7 (95%CI, 36.3 to NR) months and 4.1 (95% CI, 3.0-6.3) months in stage IV patients. In the overall population, the disease control rate was 39.3% (95%CI, 27.1 to 52.7%). In stage III patients, higher pre-treatment plasma cytokine levels of MMP-1, TRAIL, CXCL-11, CXCL-13 were associated with improved PFS (p<0.05 for all). An increase in post-vaccination levels of IL-15 and TRAIL for stage III patients was associated with improved PFS (p=0.03 for both). Similarly, an increase in post-vaccination IL-16 level for stage IV patients was associated with improved PFS (p=0.02) and clinical benefit. Vaccination with autologous melanoma cells secreting GM-CSF augments antitumor immunity in stage III and IV patients with melanoma, is safe, and demonstrates disease control. Luminex data suggests that changes in inflammatory cytokines and immune cell infiltration promote tumor antigen presentation and subsequent tumor cell destruction. Additional investigation to administer this vaccine in combination with immune checkpoint inhibitors is needed.

GM-CSF secreting leukemia cell vaccination for MDS/AML after allogeneic HSCT: a randomized, double-blinded, phase 2 trial

AbstractVaccination using irradiated, adenovirus transduced autologous myeloblasts to secrete granulocyte–macrophage colony-stimulating factor (GVAX) early after allogeneic hematopoietic stem cell transplantation (HSCT) can induce potent immune responses. We conducted a randomized phase 2 trial of GVAX after HSCT for myelodysplastic syndrome with excess blasts or relapsed/refractory acute myeloid leukemia. Myeloblasts were harvested before HSCT to generate the vaccine. Randomization to GVAX vs placebo (1:1) was stratified according to disease, transplant center, and conditioning. Graft-versus-host disease (GVHD) prophylaxis included tacrolimus and methotrexate. GVAX or placebo vaccination was started between day 30 and 45 if there was engraftment and no GVHD. Vaccines were administered subcutaneously/intradermally weekly × 3, then every 2 weeks × 3. Tacrolimus taper began after vaccine completion. A total of 123 patients were enrolled, 92 proceeded to HSCT, and 57 (GVAX, n = 30; placebo, n = 27) received at least 1 vaccination. No Common Toxicity Criteria grade 3 or worse vaccine-related adverse events were reported, but injection site reactions were more common after GVAX (10 vs 1; P = .006). With a median follow-up of 39 months (range, 9-89 months), 18-month progression-free survival, overall survival, and relapse incidence were 53% vs 55% (P = .79), 63% vs 59% (P = .86), and 30% vs 37% (P = .51) for GVAX and placebo, respectively. Nonrelapse mortality at 18 months was 17% vs 7.7% (P = .18), grade II to IV acute GVHD at 12 months was 34% vs 12% (P = .13), and chronic GVHD at 3 years was 49% vs 57% for GVAX and placebo (P = .26). Reconstitution of T, B, and natural killer cells was not decreased or enhanced by GVAX. There were no differences in serum major histocompatibility chain-related protein A/B or other immune biomarkers between GVAX and placebo. GVAX does not improve survival after HSCT for myelodysplastic syndrome/acute myeloid leukemia. This trial was registered at www.clinicaltrials.gov as #NCT01773395.

RNA-Modified T Cells Mediate Effective Delivery of Immunomodulatory Cytokines to Brain Tumors.

With the presence of the blood-brain barrier (BBB), successful immunotherapeutic drug delivery to CNS malignancies remains a challenge. Immunomodulatory agents, such as cytokines, can reprogram the intratumoral microenvironment; however, systemic cytokine delivery has limited access to the CNS. To bypass the limitations of systemically administered cytokines, we investigated if RNA-modified Tcells could deliver macromolecules directly to brain tumors. The abilities of Tcells to cross the BBB and mediate direct cytotoxic killing of intracranial tumors make them an attractive tool as biological carriers. Using Tcell mRNA electroporation, we demonstrated that activated Tcells can be modified to secrete granulocyte macrophage colony-stimulating factor (GM-CSF) protein while retaining their inherent effector functions invitro. GM-CSF RNA-modified Tcells effectively delivered GM-CSF to intracranial tumors invivo and significantly extended overall survival in an orthotopic treatment model. Importantly, GM-CSF RNA-modified Tcells demonstrated superior anti-tumor efficacy as compared to unmodified Tcells alone or in combination with systemic administration of recombinant GM-CSF. Anti-tumor effects were associated with increased IFN-γ secretion locally within the tumor microenvironment and systemic antigen-specific Tcell expansion. These findings demonstrate that RNA-modified Tcells may serve as a versatile platform for the effective delivery of biological agents to CNS tumors.

PPARγ Contributes to Immunity Induced by Cancer Cell Vaccines That Secrete GM-CSF.

Peroxisome proliferator activated receptor-γ (PPARγ) is a lipid-activated nuclear receptor that promotes immune tolerance through effects on macrophages, dendritic cells (DCs), and regulatory T cells (Tregs). Granulocyte-macrophage colony stimulating factor (GM-CSF) induces PPARγ expression in multiple myeloid cell types. GM-CSF contributes to both immune tolerance and protection, but the role of PPARγ in these pathways is poorly understood. Here, we reveal an unexpected stimulatory role for PPARγ in the generation of antitumor immunity with irradiated, GM-CSF-secreting tumor-cell vaccines (GVAX). Mice harboring a deletion of pparg in lysozyme M (LysM)-expressing myeloid cells (KO) showed a decreased ratio of CD8+ T effectors to Tregs and impaired tumor rejection with GVAX. Diminished tumor protection was associated with altered DC responses and increased production of the Treg attracting chemokines CCL17 and CLL22. Correspondingly, the systemic administration of PPARγ agonists to vaccinated mice elevated the CD8+ T effector to Treg ratio through effects on myeloid cells and intensified the antitumor activity of GVAX combined with cytotoxic T lymphocyte-associated antigen-4 antibody blockade. PPARγ agonists similarly attenuated Treg induction and decreased CCL17 and CCL22 levels in cultures of human peripheral blood mononuclear cells with GM-CSF-secreting tumor cells. Together, these results highlight a key role for myeloid cell PPARγ in GM-CSF-stimulated antitumor immunity and suggest that PPARγ agonists might be useful in cancer immunotherapy. Cancer Immunol Res; 6(6); 723-32. ©2018 AACR.

Vaccination with autologous myeloblasts admixed with GM-K562 cells in patients with advanced MDS or AML after allogeneic HSCT.

We report a clinical trial testing vaccination of autologous myeloblasts admixed with granulocyte-macrophage colony-stimulating factor secreting K562 cells after allogeneic hematopoietic stem cell transplantation (HSCT). Patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with ≥5% marrow blasts underwent myeloblast collection before HSCT. At approximately day +30, 6 vaccines composed of irradiated autologous myeloblasts mixed with GM-K562 were administered. Tacrolimus-based graft-versus-host disease (GVHD) prophylaxis was not tapered until vaccine completion (∼day 100). Thirty-three patients with AML (25) and MDS (8) enrolled, 16 (48%) had ≥5% marrow blasts at transplantation. The most common vaccine toxicity was injection site reactions. One patient developed severe eosinophilia and died of eosinophilic myocarditis. With a median follow-up of 67 months, cumulative incidence of grade 2-4 acute and chronic GVHD were 24% and 33%, respectively. Relapse and nonrelapse mortality were 48% and 9%, respectively. Progression-free survival (PFS) and overall survival (OS) at 5 years were 39% and 39%. Vaccinated patients who were transplanted with active disease (≥5% marrow blasts) had similar OS and PFS at 5 years compared with vaccinated patients transplanted with <5% marrow blasts (OS, 44% vs 35%, respectively, P = .81; PFS, 44% vs 35%, respectively, P = .34). Postvaccination antibody responses to angiopoietin-2 was associated with superior OS (hazard ratio [HR], 0.43; P = .031) and PFS (HR, 0.5; P = .036). Patients transplanted with active disease had more frequent angiopoeitin-2 antibody responses (62.5% vs 20%, P = .029) than those transplanted in remission. GM-K562/leukemia cell vaccination induces biologic activity, even in patients transplanted with active MDS/AML. This study is registered at www.clinicaltrials.gov as #NCT 00809250.

Abstract IA13: The interplay between tumor cells, cancer associated fibroblasts, and immature myeloid cells in the esophageal tumor microenvironment

Abstract Esophageal cancer has two major subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). ESCC is more common worldwide and illustrative of other SCCs (e.g. head/neck, lung) from a molecular pathogenesis viewpoint. EAC is rising in incidence in the United States and Western Europe. Unfortunately, 5-year survival remains dismal for both types, at about 15%. Interrogation of the esophageal tumor microenvironment, replete with desmoplasia, CAFs and diverse immune cells, may afford opportunities to reveal new therapeutic targets, especially in a combinatorial fashion. We have generated previously a conditional knockout model of p120-catenin whereby mice develop preneoplastic and neoplastic lesions in the esophagus. Tumor-derived cells secrete granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNFα). The tumors contain significant desmoplasia and immune cell infiltration. Immature myeloid cells (myeloid deriver suppressor cells or MDSCs) comprise a significant percentage of the immune cells present and likely participate in fostering a favorable tumor microenvironment, including the activation of fibroblasts. We have identified CD38 as playing a vital role in MDSC biology. CD38 belongs to the ADP-ribosyl cyclase family and possesses both ectoenzyme and receptor functions. It has been described to function in lymphoid and early myeloid cell differentiation, cell activation, and neutrophil chemotaxis. CD38high MDSCs are more immature than MDSCs lacking CD38 expression, suggesting a potential role for CD38 in the maturation halt found in MDSC populations. CD38high MDSCs also possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree than CD38low MDSCs, likely as a result of increased iNOS production. In addition, we have identified novel tumor–derived factors, specifically IL-6, IGFBP3, and CXCL16, which induce CD38 expression by MDSCs ex vivo. Finally, we have detected an expansion of CD38+ MDSCs in peripheral blood of advanced-stage cancer patients. Additional work reveals cross-talk between CAFs and tumor cells through the induction of IL-6 and RANTES, suggesting there is interaction between tumor cells, CAFs and MDSCs. In aggregate, we find that CD38 (on MDSCs), IL-6 and RANTES (secreted by tumor cells and CAFs) serve to provide novel approaches in therapy. Citation Format: Tatiana Karakasheva, Eric Lin, Phil Hicks, Todd Waldron, Anil K. Rustgi. The interplay between tumor cells, cancer associated fibroblasts, and immature myeloid cells in the esophageal tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA13.

A GM-CSF and CD40L bystander vaccine is effective in a murine breast cancer model.

BackgroundThere is increasing interest in using cancer vaccines to treat breast cancer patients in the adjuvant setting to prevent recurrence in high risk situations or in combination with other immunomodulators in the advanced setting. Current peptide vaccines are limited by immunologic compatibility issues, and engineered autologous cellular vaccines are difficult to produce on a large scale. Using standardized bystander cell lines modified to secrete immune stimulating adjuvant substances can greatly enhance the ability to produce immunogenic cellular vaccines using unmodified autologous cells or allogeneic medical grade tumor cell lines as targets. We investigated the efficacy of a cellular vaccine using B78H1 bystander cell lines engineered to secrete granulocyte macrophage-colony stimulating factor and CD40 ligand (BCG) in a murine model of breast cancer.MethodsFive-week-old female BALB/c mice were injected orthotopically in the mammary fat pad with 4T1 tumor cells. Treatment consisted of irradiated 4T1 ± BCG cells given subcutaneously every 4 days and was repeated three times per mouse when tumors became palpable. Tumors were measured two to three times per week for 25 days. The vaccine’s activity was confirmed in a second experiment using Lewis lung carcinoma (LLC) cells in C57BL/6 mice to exclude a model specific effect. Interferon-γ (IFN-γ) and interleukin-2 (IL-2) enzyme-linked immunospots (ELISPOTS) were performed on splenic lymphocytes incubated with 4T1 lysates along with immunohistochemistry for CD3 on tumor sections.ResultsTumor growth was significantly inhibited in the 4T1-BCG and LLC-BCG treatment groups when compared to 4T1 and LLC treatment groups. There were higher levels of IL-2 and IFN-γ secreting T-cells on ELISPOT for BCG treated groups, and a trend for higher numbers of tumor infiltrating CD3+ lymphocytes. Some tumors in the 4T1-BCG demonstrated organized lymphoid structures within the tumor microenvironment as well.ConclusionThe use of BCG bystander cell lines demonstrates proof of concept for anti-tumor activity and immunogenicity in an immunocompetent murine model of breast cancer. This vaccine is being evaluated in lung cancer and should be explored further in clinical trials of breast cancer patients at high risk of recurrence or in combination with other immunomodulatory agents.

Galectin-3 Shapes Antitumor Immune Responses by Suppressing CD8+ T Cells via LAG-3 and Inhibiting Expansion of Plasmacytoid Dendritic Cells.

Galectin-3 is a 31-kDa lectin that modulates T-cell responses through several mechanisms, including apoptosis, T-cell receptor (TCR) cross-linking, and TCR downregulation. We found that patients with pancreatic ductal adenocarcinoma (PDA) who responded to a granulocyte-macrophage colony-stimulating factor-secreting allogeneic PDA vaccine developed neutralizing antibodies to galectin-3 after immunization. We show that galectin-3 binds activated antigen-committed CD8(+) T cells only in the tumor microenvironment. Galectin-3-deficient mice exhibit improved CD8(+) T-cell effector function and increased expression of several inflammatory genes. Galectin-3 binds to LAG-3, and LAG-3 expression is necessary for galectin-3-mediated suppression of CD8(+) T cells in vitro. Lastly, galectin-3-deficient mice have elevated levels of circulating plasmacytoid dendritic cells, which are superior to conventional dendritic cells in activating CD8(+) T cells. Thus, inhibiting galectin-3 in conjunction with CD8(+) T-cell-directed immunotherapies should enhance the tumor-specific immune response.

Safety and Survival With GVAX Pancreas Prime and Listeria Monocytogenes–Expressing Mesothelin (CRS-207) Boost Vaccines for Metastatic Pancreatic Cancer

GVAX pancreas, granulocyte-macrophage colony-stimulating factor-secreting allogeneic pancreatic tumor cells, induces T-cell immunity to cancer antigens, including mesothelin. GVAX is administered with low-dose cyclophosphamide (Cy) to inhibit regulatory T cells. CRS-207, live-attenuated Listeria monocytogenes-expressing mesothelin, induces innate and adaptive immunity. On the basis of preclinical synergy, we tested prime/boost vaccination with GVAX and CRS-207 in pancreatic adenocarcinoma. Previously treated patients with metastatic pancreatic adenocarcinoma were randomly assigned at a ratio of 2:1 to two doses of Cy/GVAX followed by four doses of CRS-207 (arm A) or six doses of Cy/GVAX (arm B) every 3 weeks. Stable patients were offered additional courses. The primary end point was overall survival (OS) between arms. Secondary end points were safety and clinical response. A total of 90 patients were treated (arm A, n = 61; arm B, n = 29); 97% had received prior chemotherapy; 51% had received ≥ two regimens for metastatic disease. Mean number of doses (± standard deviation) administered in arms A and B were 5.5 ± 4.5 and 3.7 ± 2.2, respectively. The most frequent grade 3 to 4 related toxicities were transient fevers, lymphopenia, elevated liver enzymes, and fatigue. OS was 6.1 months in arm A versus 3.9 months in arm B (hazard ratio [HR], 0.59; P = .02). In a prespecified per-protocol analysis of patients who received at least three doses (two doses of Cy/GVAX plus one of CRS-207 or three of Cy/GVAX), OS was 9.7 versus 4.6 months (arm A v B; HR, 0.53; P = .02). Enhanced mesothelin-specific CD8 T-cell responses were associated with longer OS, regardless of treatment arm. Heterologous prime/boost with Cy/GVAX and CRS-207 extended survival for patients with pancreatic cancer, with minimal toxicity.

PD-1/PD-L1 blockade together with vaccine therapy facilitates effector T-cell infiltration into pancreatic tumors.

Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis due to late detection and resistance to conventional therapies. Published studies show that the PDA tumor microenvironment is predominantly infiltrated with immune suppressive cells and signals that if altered, would allow effective immunotherapy. However, single-agent checkpoint inhibitors including agents that alter immune suppressive signals in other human cancers such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1), and its ligand PD-L1, have failed to demonstrate objective responses when given as single agents to PDA patients. We recently reported that inhibition of the CTLA-4 pathway when given together with a T cell inducing vaccine gives objective responses in metastatic PDA patients. In this study, we evaluated blockade of the PD-1/PD-L1 pathway. We found that PD-L1 is weakly expressed at a low frequency in untreated human and murine PDAs but treatment with a granulocyte macrophage colony-stimulating factor secreting PDA vaccine (GVAX) significantly upregulates PD-L1 membranous expression after treatment of tumor-bearing mice. In addition, combination therapy with vaccine and PD-1 antibody blockade improved murine survival compared with PD-1 antibody monotherapy or GVAX therapy alone. Furthermore, PD-1 blockade increased effector CD8 T lymphocytes and tumor-specific interferon-γ production of CD8 T cells in the tumor microenvironment. Immunosuppressive pathways, including regulatory T cells and CTLA-4 expression on T cells were overcome by the addition of vaccine and low-dose cyclophosphamide to PD-1 blockade. Collectively, our study supports combining PD-1 or PD-L1 antibody therapy with a T cell inducing agent for PDA treatment.

Low Total Lymphocyte Count Is Associated with Poor Survival in Patients with Resected Pancreatic Adenocarcinoma Receiving a GM-CSF Secreting Pancreatic Tumor Vaccine

Low total lymphocyte count (TLC) and lymphocyte-to-neutrophil ratio have been found to be poor prognostic indicators in several different tumor types at various stages. Although immune-based therapies are under rapid development, it is not known whether baseline complete blood counts, particularly lymphocytes, are associated with the clinical outcomes of patients receiving immunotherapies. We performed a retrospective analysis of complete blood count for 59 patients enrolled onto a phase II trial evaluating the integration of an adjuvant immunotherapy-irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting allogeneic pancreatic tumor vaccine (GVAX)-with standard chemoradiation. After adjusting for nodal status, individuals with a TLC of <1,500 cells/mm(3) (10 patients) had significantly higher risk, both in terms of overall survival (OS) [adjusted hazard ratio 2.63, 95 % confidence interval (CI) 1.22-5.67, p = 0.013] and progression-free survival (adjusted hazard ratio 3.07, 95 % CI 1.03-6.93, p = 0.003), compared to those with a TLC of ≤ 1,500 cells/mm(3) (49 patients). Adjuvant chemoradiation significantly reduced lymphocyte counts from baseline values. Patients with suppression of their lymphocytes to <500 cells/mm(3) after chemoradiation also had shorter disease-free and OS. Immunosuppressive conditions associated with surgical procedures and chemoradiation may affect the efficacy of immunotherapy.

A bystander cell-based GM-CSF secreting vaccine synergized with a low dose of cyclophosphamide presents therapeutic immune responses against murine hepatocellular carcinoma

Granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting cellular tumor vaccines contribute to the induction of potent antitumor immune responses in murine models and patients suffering from cancers. Hepatocellular carcinoma (HCC) is one of the most frequent and malignant cancers in China. We describe, for the first time, a GM-CSF releasing vaccine strategy that represents a step toward combating this type of cancer. In this study, a bystander cell-based GM-CSF secreting vaccine against murine HCC, Hepa1-6/B78H1-GM-CSF, was co-administered with a low dose of cyclophosphamide (CY). After challenging with tumor and vaccination, immunological assays demonstrated that the cellular antitumor immune responses were efficiently activated and that tumor development was significantly retarded, which was dependent on synergy with CY. The promising outcome of the anti-HCC vaccine in the murine model demonstrates the feasibility of a future clinical application for this treatment in HCC patients.

Abstract PR9: Granulocyte macrophage colony stimulating factor (GM-CSF) pancreas tumor vaccine in combination with blockade of PD-1 in a preclinical model of pancreatic cancer.

Abstract Introduction: Our phase I/II human clinical trials utilizing a GM-CSF secreting allogeneic pancreas tumor vaccine (GVAX) have been shown to be safe and effective in inducing anti-tumor immune response in pancreatic adenocarcinoma patients. GVAX treated patients have demonstrated infiltration of PD-1+ T cells which is a major immunosuppressive mechanism in the tumor microenvironment. We hypothesized that the use of a PD-1 blocking antibody and GVAX will improve vaccine therapy and pancreatic cancer survival. Methods: Mice were orthotopically transplanted with 2 x 106 Panc02 pancreatic tumor cells to form liver metastases by a hemisplenectomy technique on day 0. Following tumor transplantation, wild-type or PD-1 knockout mice were treated subcutaneously with a mouse GM-CSF secreting pancreatic tumor vaccine (mouse GVAX) in combination with anti-PD-1 antibodies or IgG isotype control. GVAX was given subcutaneously to mice on days 4, 7, 14, and 21 following tumor transplantation, together with a single low-dose of Cytoxan on day 3. Anti-PD-1 antibodies or IgG were administered twice weekly starting post-operative day 3. Results: PD-1 knockout mice challenged with pancreatic liver metastases had a significant survival advantage over wild-type mice (p=0.0002). Wild-type mice showed improved survival both with anti-PD-1 antibody alone (p=0.008) or in combination with vaccine (p&amp;lt;0.0001) versus IgG controls. GVAX in combination with anti-PD-1 antibodies versus anti-PD-1 antibodies alone shows a statistically significant improved survival (p&amp;lt;0.05). Memory T cell analysis demonstrated a strong trend toward increased tumor infiltration of CD8+ effector memory T cells in the combinatorial treatment group versus vaccine alone (66.67% vs 26.47%, p= 0.1). Tumor antigen (mesothelin)-specific cytotoxic CD8+ tumor infiltrating lymphocytes were increased in the combination treatment group (GVAX and anti-PD1 therapy) versus GVAX alone (13.4% vs 5.29%). Conclusions: PD-1 blockade through monoclonal antibodies or genetic knockout in combination with vaccine result in a synergistic anti-tumor effect in a preclinical model of pancreatic cancer versus treatment with anti-PD1 alone. Additionally, anti-PD1 treatment facilitates tumor infiltration of functionally activated mesothelin specific CD8+ T cells. Anti-PD-1 blockade antibody is currently being tested in phase II clinical trials for treating chemotherapy-refractory solid tumor patients. Our study provides a strong rationale for combining PD-1 antibody with GVAX therapy for pancreatic cancer treatment. This abstract is also presented as Poster B28. Citation Format: Kevin C. Soares, Kelly Olino, Barish Edil, Donger Zhou, Anthony Wamwea, Ashley Leubner, Richard Schulick, Drew Pardoll, Elizabeth Jaffee, Lei Zheng. Granulocyte macrophage colony stimulating factor (GM-CSF) pancreas tumor vaccine in combination with blockade of PD-1 in a preclinical model of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR9.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Deletion of p120-Catenin Results in a Tumor Microenvironment with Inflammation and Cancer that Establishes It as a Tumor Suppressor Gene

p120-catenin (p120ctn) interacts with E-cadherin, but to our knowledge, no formal proof that p120ctn functions as a bona fide tumor suppressor gene has emerged to date. We report herein that p120ctn loss leads to tumor development in mice. We have generated a conditional knockout model of p120ctn whereby mice develop preneoplastic and neoplastic lesions in the oral cavity, esophagus, and squamous forestomach. Tumor-derived cells secrete granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNFα). The tumors contain significant desmoplasia and immune cell infiltration. Immature myeloid cells comprise a significant percentage of the immune cells present and likely participate in fostering a favorable tumor microenvironment, including the activation of fibroblasts.

Treatment with GM-CSF Secreting Myeloid Leukemia Cell Vaccine Prior to Autologous-BMT Improves the Survival of Leukemia-Challenged Mice

Vaccination with irradiated autologous tumor cells, engineered to secrete granulocyte macrophage-colony stimulating factor (GM-CSF) (GM tumor), can generate potent antitumor effects when combined with autologous bone marrow transplantation (BMT). That notwithstanding, the post-BMT milieu, characterized by marked cytopenia, can pose a challenge to the implementation of vaccine immunotherapies. To bypass this problem, partial post-BMT immune reconstitution has been allowed to develop prior to vaccination. However, delaying vaccination can also potentially allow the expansion of residual tumor cells. Other approaches have used reinfusion of "primed" autologous lymphocytes and multiple administrations of GM tumor cells, which required the processing of large amounts of tumor. Utilizing the MMB3.19 murine myeloid leukemia model, we tested whether a single dose of GM tumor cells, 7 days prior to syngeneic BMT, could be a curative treatment in MMB3.19-challenged recipient mice. This vaccination protocol significantly improved survival of mice by eliciting long-lasting host immune responses that survived lethal irradiation, and were even protective against post-BMT tumor rechallenge. Furthermore, we demonstrated that mature donor lymphocytes can also play a limited role in mounting the antitumor response, but our pre-BMT vaccination strategy obviated the need for either established de novo immune reconstitution or the use of multiple post-BMT immunizations.

Antibody responses to galectin-8, TARP and TRAP1 in prostate cancer patients treated with a GM-CSF-secreting cellular immunotherapy.

A critical factor in clinical development of cancer immunotherapies is the identification of tumor-associated antigens that may be related to immunotherapy potency. In this study, protein microarrays containing >8,000 human proteins were screened with serum from prostate cancer patients (N = 13) before and after treatment with a granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting whole cell immunotherapy. Thirty-three proteins were identified that displayed significantly elevated (P <or= 0.05) signals in post-treatment samples, including three proteins that have previously been associated with prostate carcinogenesis, galectin-8, T-cell alternative reading frame protein (TARP) and TNF-receptor-associated protein 1 (TRAP1). Expanded analysis of antibody induction in metastatic, castration-resistant prostate cancer (mCRPC) patients (N = 92) from two phase 1/2 trials of prostate cancer immunotherapy, G-9803 and G-0010, indicated a significant (P = 0.03) association of TARP antibody induction and median survival time (MST). Antibody induction to TARP was also significantly correlated (P = 0.036) with an increase in prostate-specific antigen doubling time (PSADT) in patients with a biochemical (PSA) recurrence following prostatectomy or radiation therapy (N = 19) from in a previous phase 1/2 trial of prostate cancer immunotherapy, G-9802. RNA and protein encoding TARP and TRAP1 was up-regulated in prostate cancer tissue compared to matched normal controls. These preliminary findings suggest that antibody induction to TARP may represent a possible biomarker for treatment response to GM-CSF secreting cellular immunotherapy in prostate cancer patients and demonstrates the utility of using protein microarrays for the high-throughput screening of patient-derived antibody responses.

Timed Sequential Treatment With Cyclophosphamide, Doxorubicin, and an Allogeneic Granulocyte-Macrophage Colony-Stimulating Factor–Secreting Breast Tumor Vaccine: A Chemotherapy Dose-Ranging Factorial Study of Safety and Immune Activation

Granulocyte-macrophage colony-stimulating factor (GM-CSF) -secreting tumor vaccines have demonstrated bioactivity but may be limited by disease burdens and immune tolerance. We tested the hypothesis that cyclophosphamide (CY) and doxorubicin (DOX) can enhance vaccine-induced immunity in patients with breast cancer. We conducted a 3 x 3 factorial (response surface) dose-ranging study of CY, DOX, and an HER2-positive, allogeneic, GM-CSF-secreting tumor vaccine in 28 patients with metastatic breast cancer. Patients received three monthly immunizations, with a boost 6 to 8 months from study entry. Primary objectives included safety and determination of the chemotherapy doses that maximize HER2-specific immunity. Twenty-eight patients received at least one immunization, and 16 patients received four immunizations. No dose-limiting toxicities were observed. HER2-specific delayed-type hypersensitivity developed in most patients who received vaccine alone or with 200 mg/m(2) CY. HER2-specific antibody responses were enhanced by 200 mg/m(2) CY and 35 mg/m(2) DOX, but higher CY doses suppressed immunity. Analyses revealed that CY at 200 mg/m(2) and DOX at 35 mg/m(2) is the combination that produced the highest antibody responses. First, immunotherapy with an allogeneic, HER2-positive, GM-CSF-secreting breast tumor vaccine alone or with CY and DOX is safe and induces HER2-specific immunity in patients with metastatic breast cancer. Second, the immunomodulatory activity of low-dose CY has a narrow therapeutic window, with an optimal dose not exceeding 200 mg/m(2). Third, factorial designs provide an opportunity to identify the most active combination of interacting drugs in patients. Further investigation of the impact of chemotherapy on vaccine-induced immunity is warranted.

Cyclophosphamide Augments Antitumor Immunity: Studies in an Autochthonous Prostate Cancer Model

To study the immune response to prostate cancer, we developed an autochthonous animal model based on the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse in which spontaneously developing tumors express influenza hemagglutinin as a unique, tumor-associated antigen. Our prior studies in these animals showed immunologic tolerance to hemagglutinin, mirroring the clinical situation in patients with cancer who are generally nonresponsive to their disease. We used this physiologically relevant animal model to assess the immunomodulatory effects of cyclophosphamide when administered in combination with an allogeneic, cell-based granulocyte-macrophage colony-stimulating factor-secreting cancer immunotherapy. Through adoptive transfer of prostate/prostate cancer-specific CD8 T cells as well as through studies of the endogenous T-cell repertoire, we found that cyclophosphamide induced a marked augmentation of the antitumor immune response. This effect was strongly dependent on both the dose and the timing of cyclophosphamide administration. Mechanistic studies showed that immune augmentation by cyclophosphamide was associated with a transient depletion of regulatory T cells in the tumor draining lymph nodes but not in the peripheral circulation. Interestingly, we also noted effects on dendritic cell phenotype; low-dose cyclophosphamide was associated with increased expression of dendritic cell maturation markers. Taken together, these data clarify the dose, timing, and mechanism of action by which immunomodulatory cyclophosphamide can be translated to a clinical setting in a combinatorial cancer treatment strategy.