Anti-Gr-1 Antibody Provides Short-Term Depletion of MDSC in Lymphodepleted Mice with Active-Specific Melanoma Therapy.

Lymphodepletion, reconstitution and active-specific tumor cell vaccination (LRAST) enhances the induction of tumor-specific T cells in a murine melanoma model. Myeloid-derived suppressor cells (MDSC) may counteract the induction of tumor-reactive T cells and their therapeutic efficacy. Thus, the aim of the study was to evaluate a possible benefit of MDSC depletion using anti-Gr-1 antibodies (Ab) in combination with LRAST. Female C57BL/6 mice with 3 days established subcutaneous (s.c.) D5 melanoma were lymphodepleted with cyclophosphamide and reconstituted with naive splenocytes. Vaccination was performed with irradiated syngeneic mGM-CSF-secreting D5G6 melanoma cells. MDSC depletion was performed using anti-Gr-1 Ab (clone RB6-8C5). Induction of tumor-specific T cells derived from tumor vaccine draining lymph nodes (TVDLN) was evaluated by the amount of tumor-specific interferon (IFN)-γ release. LRAST combined with anti-Gr-1 mAb administration enhanced the induction of tumor-specific T cells in TVDLN capable of releasing IFN-γ in a tumor-specific manner. Additional anti-Gr-1 mAb administration in LRAST-treated mice delayed growth of D5 melanomas by two weeks. Furthermore, we elucidate the impact of anti-Gr-1-depleting antibodies on the memory T cell compartment. Our data indicate that standard of care treatment regimens against cancer can be improved by implementing agents, e.g., depleting antibodies, which target and eliminate MDSC.

Combination immunotherapy and active-specific tumor cell vaccination augments anti-cancer immunity in a mouse model of gastric cancer.

BACKGROUND: Active-specific immunotherapy used as an adjuvant therapeutic strategy is rather unexplored for cancers with poorly characterized tumor antigens like gastric cancer. The aim of this study was to augment a therapeutic immune response to a low immunogenic tumor cell line derived from a spontaneous gastric tumor of a CEA424-SV40 large T antigen (CEA424-SV40 TAg) transgenic mouse. METHODS: Mice were treated with a lymphodepleting dose of cyclophosphamide prior to reconstitution with syngeneic spleen cells and vaccination with a whole tumor cell vaccine combined with GM-CSF (a treatment strategy abbreviated as LRAST). Anti-tumor activity to subcutaneous tumor challenge was examined in a prophylactic as well as a therapeutic setting and compared to corresponding controls. RESULTS: LRAST enhances tumor-specific T cell responses and efficiently inhibits growth of subsequent transplanted tumor cells. In addition, LRAST tended to slow down growth of established tumors. The improved anti-tumor immune response was accompanied by a transient decrease in the frequency and absolute number of CD4⁺CD25⁺FoxP3⁺ T cells (Tregs). CONCLUSIONS: Our data support the concept that whole tumor cell vaccination in a lymphodepleted and reconstituted host in combination with GM-CSF induces therapeutic tumor-specific T cells. However, the long-term efficacy of the treatment may be dampened by the recurrence of Tregs. Strategies to counteract suppressive immune mechanisms are required to further evaluate this therapeutic vaccination protocol.

Immunotherapy of cancer: key findings and commentary on the third Tegernsee conference.

Cancer immunotherapy broadly includes active immunization, as in the use of cancer vaccines, passive immunization, such as the use of adoptive cell therapy and antibodies that modulate tumor function, and immunostimulation, using antibodies and small molecules to treat malignancy by activating or unleashing an endogenous immune response against tumor cells. Currently, >100 different monoclonal antibodies are in use or under evaluation for use as therapeutic agents in various malignancies. Active stimulation of the host's immune system holds promise for achieving durable remission of malignant disease and represents a nontoxic method of therapy if tumor-specific effector cells can be selectively targeted. However, no active-specific treatment strategy (i.e., a therapeutic cancer vaccine) has yet found its way into the clinical armamentarium, although several promising recent reports suggest that, for follicular lymphoma, prostate cancer, and melanoma, clinical benefit was shown for the first time in randomized trials with a vaccine approach. Here, we report on the key findings of the Third Tegernsee Conference on Immunotherapy of Cancer (Feldafing, Germany, July 2-4, 2009) and provide short commentaries on data presented at this meeting regarding the future role of cancer vaccines, recent developments in adoptive cellular therapy, ways to improve immunotherapeutic treatment modalities (e.g., by manipulating the tumor microenvironment), and some novel targeted therapies that are well advanced in clinical testing, all of which have implications for future oncology practice.

Adjuvant therapeutic vaccination in patients with non-small cell lung cancer made lymphopenic and reconstituted with autologous PBMC: first clinical experience and evidence of an immune response.

BACKGROUND: Given the considerable toxicity and modest benefit of adjuvant chemotherapy for non-small cell lung cancer (NSCLC), there is clearly a need for new treatment modalities in the adjuvant setting. Active specific immunotherapy may represent such an option. However, clinical responses have been rare so far. Manipulating the host by inducing lymphopenia before vaccination resulted in a magnification of the immune response in the preclinical setting. To evaluate feasibility and safety of an irradiated, autologous tumor cell vaccine given following induction of lymphopenia by chemotherapy and reinfusion of autologous peripheral blood mononuclear cells (PBMC), we are currently conducting a pilot-phase I clinical trial in patients with NSCLC following surgical resection. This paper reports on the first clinical experience and evidence of an immune response in patients suffering from NSCLC. METHODS: NSCLC patients stages I-IIIA are recruited. Vaccines are generated from their resected lung specimens. Patients undergo leukapheresis to harvest their PBMC prior to or following the surgical procedure. Furthermore, patients receive preparative chemotherapy (cyclophosphamide 350 mg/m2 and fludarabine 20 mg/m2 on 3 consecutive days) for induction of lymphopenia followed by reconstitution with their autologous PBMC. Vaccines are administered intradermally on day 1 following reconstitution and every two weeks for a total of up to five vaccinations. Granulocyte-macrophage-colony-stimulating-factor (GM-CSF) is given continuously (at a rate of 50 microg/24 h) at the site of vaccination via minipump for six consecutive days after each vaccination. RESULTS: To date, vaccines were successfully manufactured for 4 of 4 patients. The most common toxicities were local injection-site reactions and mild constitutional symptoms. Immune responses to chemotherapy, reconstitution and vaccination are measured by vaccine site and delayed type hypersensitivity (DTH) skin reactions. One patient developed positive DTH skin tests so far. Immunohistochemical assessment of punch biopsies taken at the local vaccine site reaction revealed a dense lymphocyte infiltrate. Further immunohistochemical differentiation showed that CD1a+ cells had been attracted to the vaccine site as well as predominantly CD4+ lymphocytes. The 3-day combination chemotherapy consisting of cyclophosphamide and fludarabine induced a profound lymphopenia in all patients. Sequential FACS analysis revealed that different T cell subsets (CD4, CD8, CD4CD25) as well as granulocytes, B cells and NK cells were significantly reduced. Here, we report on clinical safety and feasibility of this vaccination approach during lymphoid recovery and demonstrate a patient example. CONCLUSION: Thus far, all vaccines were well tolerated. The overall trial design seems safe and feasible. Vaccine site reactions associated with infusion of GM-CSF via mini-pump are consistent with the postulated mechanism of action. More detailed immune-monitoring is required to evaluate a potential systemic immune response. Further studies to exploit homeostasis-driven T cell proliferation for the induction of a specific anti-tumor immune response in this clinical setting are warranted.

Tumor-specific T cells signal tumor destruction via the lymphotoxin beta receptor.

BACKGROUND: Previously, we reported that adoptively transferred perforin k/o (PKO), and IFN-gamma k/o (GKO), or perforin/IFN-gamma double k/o (PKO/GKO) effector T cells mediated regression of B16BL6-D5 (D5) pulmonary metastases and showed that TNF receptor signaling played a critical role in mediating tumor regression. In this report we investigated the role of lymphotoxin-alpha (LT-alpha) as a potential effector molecules of tumor-specific effector T cells. METHODS: Effector T cells were generated from tumor vaccine-draining lymph node (TVDLN) of wt, GKO, LT-alpha deficient (LKO), or PKO/GKO mice and tested for their ability to mediate regression of D5 pulmonary metastases in the presence or absence of LT-betaR-Fc fusion protein or anti-IFN-gamma antibody. Chemokine production by D5 tumor cells was determined by ELISA, RT-PCR and Chemotaxis assays. RESULTS: Stimulated effector T cells from wt, GKO, or PKO/GKO mice expressed ligands for LT-beta receptor (LT-betaR). D5 tumor cells were found to constitutively express the LT-betaR. Administration of LT-betaR-Fc fusion protein completely abrogated the therapeutic efficacy of GKO or PKO/GKO but not wt effector T cells (p < 0.05). Consistent with this observation, therapeutic efficacy of effector T cells deficient in LT-alpha, was greatly reduced when IFN-gamma production was neutralized. While recombinant LT-alpha1beta2 did not induce apoptosis of D5 tumor cells in vitro, it induced secretion of chemokines by D5 that promoted migration of macrophages. CONCLUSION: The contribution of LT-alpha expression by effector T cells to anti-tumor activity in vivo was not discernable when wt effector T cells were studied. However, the contribution of LT-beta R signaling was identified for GKO or PKO/GKO effector T cells. Since LT-alpha does not directly induce killing of D5 tumor cells in vitro, but does stimulate D5 tumor cells to secrete chemokines, these data suggest a model where LT-alpha expression by tumor-specific effector T cells interacts via cross-linking of the LT-betaR on tumor cells to induce secretion of chemokines that are chemotactic for macrophages. While the contribution of macrophages to tumor elimination in our system requires additional study, this model provides a possible explanation for the infiltration of inate effector cells that is seen coincident with tumor regression.

Therapeutic T cells induce tumor-directed chemotaxis of innate immune cells through tumor-specific secretion of chemokines and stimulation of B16BL6 melanoma to secrete chemokines.

BACKGROUND: The mechanisms by which tumor-specific T cells induce regression of established metastases are not fully characterized. In using the poorly immunogenic B16BL6-D5 (D5) melanoma model we reported that T cell-mediated tumor regression can occur independently of perforin, IFN-gamma or the combination of both. Characterization of regressing pulmonary metastases identified macrophages as a major component of the cells infiltrating the tumor after adoptive transfer of effector T cells. This led us to hypothesize that macrophages played a central role in tumor regression following T-cell transfer. Here, we sought to determine the factors responsible for the infiltration of macrophages at the tumor site. METHODS: These studies used the poorly immunogenic D5 melanoma model. Tumor-specific effector T cells, generated from tumor vaccine-draining lymph nodes (TVDLN), were used for adoptive immunotherapy and in vitro analysis of chemokine expression. Cellular infiltrates into pulmonary metastases were determined by immunohistochemistry. Chemokine expression by the D5 melanoma following co-culture with T cells, IFN-gamma or TNF-alpha was determined by RT-PCR and ELISA. Functional activity of chemokines was confirmed using a macrophage migration assay. T cell activation of macrophages to release nitric oxide (NO) was determined using GRIES reagent. RESULTS: We observed that tumor-specific T cells with a type 1 cytokine profile also expressed message for and secreted RANTES, MIP-1alpha and MIP-1beta following stimulation with specific tumor. Unexpectedly, D5 melanoma cells cultured with IFN-gamma or TNF-alpha, two type 1 cytokines expressed by therapeutic T cells, secreted Keratinocyte Chemoattractant (KC), MCP-1, IP-10 and RANTES and expressed mRNA for MIG. The chemokines released by T cells and cytokine-stimulated tumor cells were functional and induced migration of the DJ2PM macrophage cell line. Additionally, tumor-specific stimulation of wt or perforin-deficient (PKO) effector T cells induced macrophages to secrete nitric oxide (NO), providing an additional effector mechanism for T cell-mediated tumor regression. CONCLUSION: These data suggest two possible sources for chemokine secretion that stimulates macrophage recruitment to the site of tumor metastases. Both appear to be initiated by T cell recognition of specific antigen, but one is dependent on the tumor cells to produce the chemokines that recruit macrophages.

Characterization of gastric adenocarcinoma cell lines established from CEA424/SV40 T antigen-transgenic mice with or without a human CEA transgene.

BACKGROUND: Gastric carcinoma is one of the most frequent cancers worldwide. Patients with gastric cancer at an advanced disease stage have a poor prognosis, due to the limited efficacy of available therapies. Therefore, the development of new therapies, like immunotherapy for the treatment of gastric cancer is of utmost importance. Since the usability of existing preclinical models for the evaluation of immunotherapies for gastric adenocarcinomas is limited, the goal of the present study was to establish murine in vivo models which allow the stepwise improvement of immunotherapies for gastric cancer. METHODS: Since no murine gastric adenocarcinoma cell lines are available we established four cell lines (424GC, mGC3, mGC5, mGC8) from spontaneously developing tumors of CEA424/SV40 T antigen (CEA424/Tag) mice and three cell lines derived from double-transgenic offsprings of CEA424/Tag mice mated with human carcinoembryonic antigen (CEA)-transgenic (CEA424/Tag-CEA) mice (mGC2CEA, mGC4CEA, mGC11CEA). CEA424/Tag is a transgenic C57BL/6 mouse strain harboring the Tag under the control of a -424/-8 bp CEA gene promoter which leads to the development of invasive adenocarcinoma in the glandular stomach. Tumor cell lines established from CEA424/Tag-CEA mice express the well defined tumor antigen CEA under the control of its natural regulatory elements. RESULTS: The epithelial origin of the tumor cells was proven by morphological criteria including the presence of mucin within the cells and the expression of the cell adhesion molecules EpCAM and CEACAM1. All cell lines consistently express the transgenes CEA and/or Tag and MHC class I molecules leading to their susceptibility to lysis by Tag-specific CTL in vitro. Despite the presentation of CTL-epitopes derived from the transgene products the tumor cell lines were tumorigenic when grafted into C57BL/6, CEA424/Tag or CEA424/Tag-CEA-transgenic hosts and no significant differences in tumor take and tumor growth were observed in the different hosts. Although no spontaneous tumor rejection was observed, vaccination of C57BL/6 mice with lysates from gastric carcinoma cell lines protected C57BL/6 mice from tumor challenge, demonstrating the tumorigenicity of the tumor cell lines in nontransgenic mice of the H-2b haplotype. CONCLUSION: These tumor cell lines grafted in different syngeneic hosts should prove to be very useful to optimize immunotherapy regimens to be finally tested in transgenic animals developing primary gastric carcinomas.

Oral DNA vaccination with a plasmid encoding soluble ICAM-1 modulates cytokine expression profiles in nonobese diabetic mice.

Adhesion molecules are important for leukocyte extravasation and for the delivery of costimulatory signals in T cell activation. We therefore interfered in the immune process leading to islet inflammation in diabetes prone NOD mice by oral vaccination with plasmid DNA encoding soluble ICAM-1. Female NOD mice were treated orally with ICAM-1, TGF-beta, or control plasmid DNA and received a single injection of cyclophosphamide for synchronization and acceleration of the disease process in the pancreas. Quantitative RT-PCR analysis of pancreatic mRNA showed that cyclophosphamide induced the expression of Th1 cytokines (IFN-gamma and IL-12p40) in vehicle- or control plasmid-treated mice. Treatment with ICAM-1 and TGF-beta DNA resulted in increased levels of IL-10 mRNA in the pancreas, indicating an anti-inflammatory regulatory immune response. Histological analysis of pancreatic islets showed that the DNA treatment did not alter islet infiltration in response to cyclophosphamide. Hence vaccination with the ICAM-1 plasmid had not suppressed leukocyte migration but rather modulated lymphocyte activity, similarly as seen for the TGF-beta-encoding plasmid. Neither of the three plasmids caused recognizable changes in cytokine expression in the small intestine, Peyer's patches, or mesenteric lymph nodes. We conclude that oral vaccination with DNA encoding immunoregulatory molecules such as ICAM-1 and TGF-beta represents an approach for modulating the ongoing inflammatory process in the pancreas of diabetes prone NOD mice.

Active-specific immunotherapy for non-small cell lung cancer.

Non-small cell lung cancer constitutes about 85% of all newly diagnosed cases of lung cancer and continues to be the leading cause of cancer-related deaths worldwide. Standard treatment for this devastating disease, such as systemic chemotherapy, has reached a plateau in effectiveness and comes with considerable toxicities. For all stages of disease fewer than 20% of patients are alive 5 years after diagnosis; for metastatic disease the median survival is less than one year. Until now, the success of active-specific immunotherapy for all tumor types has been sporadic and unpredictable. However, the active-specific stimulation of the host's own immune system still holds great promise for achieving non-toxic and durable antitumor responses. Recently, sipuleucel-T (Provenge(®); Dendreon Corp., Seattle, WA) was the first therapeutic cancer vaccine to receive market approval, in this case for advanced prostate cancer. Other phase III clinical trials using time-dependent endpoints, e.g. in melanoma and follicular lymphoma, have recently turned out positive. More sophisticated specific vaccines have now also been developed for lung cancer, which, for long, was not considered an immune-sensitive malignancy. This may explain why advances in active-specific immunotherapy for lung cancer lag behind similar efforts in renal cell cancer, melanoma or prostate cancer. However, various vaccines are now being evaluated in controlled phase III clinical trials, raising hopes that active-specific immunotherapy may become an additional effective therapy for patients with lung cancer. This article reviews the most prominent active-specific immunotherapeutic approaches using protein/peptide, whole tumor cells, and dendritic cells as vaccines for lung cancer.

Immunotherapy of lung cancer: an update.

In Germany lung cancer is the leading cause of cancer-associated death in men. Surgery, chemotherapy and radiation may enhance survival of patients suffering from lung cancer but the enhancement is typically transient and mostly absent with advanced disease; eventually more than 90% of lung cancer patients will die of disease. New approaches to the treatment of lung cancer are urgently needed. Immunotherapy may represent one new approach with low toxicity and high specificity but implementation has been a challenge because of the poor antigenic characterization of these tumors and their ability to escape immune responses. Several different immunotherapeutic treatment strategies have been developed. This review examines the current state of development and recent advances with respect to non-specific immune stimulation, cellular immunotherapy (specific and non-specific), therapeutic cancer vaccines and gene therapy for lung cancer. The focus is primarily placed on immunotherapeutic cancer treatments that are already in clinical trial or well progressed in preclinical studies. Although there seems to be a promising future for immunotherapy in lung cancer, presently there is not standard immunotherapy available for clinical routine.

Characterization of immune responses in gastric cancer patients: a possible impact of H. pylori to polarize a tumor-specific type 1 response?

Recently, we were able to show that Helicobacter pylori-positive gastric cancer (GC) patients have a significantly better survival after the complete resection of their tumor compared to H. pylori-negative GC patients. H. pylori is known to polarize an immune response towards a type 1 cytokine profile and tumor-specific type 1 cytokine responses are associated with protection from tumor challenge and T-cell-mediated tumor regression. Therefore, we hypothesized that the improved survival in H. pylori-positive patients may be secondary to the induction of a GC-specific type 1 T cell response. To characterize the anti-tumor immune response in GC patients we analyzed tumor-infiltrating lymphocytes (TIL) isolated from primary tumors. The CD3+ T cell population contained 50% CD4+ (range 0.4-81%) and 39% CD8+ cells (range 22-53%). The number of B cells (CD19+, P = 0.03) was significantly increased and the number of T cells (CD3+, P = 0.02) significantly decreased in intestinal compared to diffuse type of tumors. Four tumor cell lines were established from primary GCs and three from lymph node metastases. T cell cultures were established from isolated TIL from four H. pylori-positive and one H. pylori-negative GC patients and tested for tumor-specific cytokine secretion. Eight of ten T cell cultures derived from H. pylori-positive patients secreted both IFN-gamma and IL-5 after restimulation with autologous tumor cells. The only tumor-specific TIL line expressing a dominant IL-5 response was derived from an H. pylori-negative patient.

Dehydroepiandrosterone restores depressed peripheral blood mononuclear cell function following major abdominal surgery via the estrogen receptors.

OBJECTIVE: Peripheral blood mononuclear cell (PBMC) dysfunction occurs following major abdominal surgery and correlates with an increased rate of septic complications. Studies have shown that dehydroepiandrosterone (DHEA) restores cell-mediated immune responses after trauma-hemorrhage in mice. Nonetheless, it remains unknown whether DHEA has any salutary effects on depressed PBMC function in surgical patients. DESIGN: Laboratory experiment. SETTING: University laboratory. PATIENTS: Fifteen patients undergoing major abdominal surgery. INTERVENTIONS: Blood samples were obtained preoperatively and 2 hrs postoperatively. MEASUREMENTS AND MAIN RESULTS: PBMCs were cultured with 33% plasma in the presence or absence of DHEA (10(-10) M, 10(-8) M physiologic concentration, 10(-6) M, 10(-5) M). In an additional set of samples, the estrogen receptor antagonist tamoxifen (10(-6) M) was added. The release of proinflammatory cytokines (interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha) was measured in the supernatants by enzyme-linked immunosorbent assay. Abdominal surgery resulted in depressed interleukin-1beta and tumor necrosis factor-alpha release by PBMC. Addition of DHEA to the culture medium, however, significantly improved the release of interleukin-1beta and tumor necrosis factor-alpha and stimulated the interleukin-6 release capacity of PBMC. This effect was most pronounced for a concentration of 10(-5)M DHEA. The immunomodulatory effect of DHEA on PBMC cytokine release was completely blocked by tamoxifen. In contrast, the modulatory effect of DHEA was enhanced by the addition of postoperative plasma. CONCLUSIONS: DHEA stimulates proinflammatory cytokine release capacities of human PBMCs following major abdominal surgery. The estrogen receptor appears to be involved in mediating the immunomodulatory effect of DHEA. Thus, DHEA might be a useful adjunct for preventing immunosuppression in surgical patients.