Tumor Challenge Experiments Research Articles

Abstract 4090: Gut microbiota mimics as a source of cross-reactive tumor rejection antigens

Abstract The crosstalk between gut microbiome and anti-tumor immunity has recently gained attention in clinical settings. The administration of antibiotics during immune checkpoint blockade (ICB) therapies has been correlated with poorer outcomes in cancer patients, suggesting that microbiome alterations affect overall responses to ICB. The establishment of microbiota-specific T cell clones with tumor cross-reactivity potential was proposed as one of the mechanisms linking the microbiome to anti-tumor responses. Vaccine-based strategies aimed at reactivating these dormant pools of T cells to help eliminate tumors have been proposed as an alternate approach, but the parameters required for successful responses remain poorly understood. Here, we performed in silico selection of bacterial peptides derived from healthy mice based on their mimicry of either tumor-associated antigens (TAAs) or neoantigens expressed by murine melanoma cells and not healthy tissue. We show that in silico predicted peptide binding affinity to MHC I correlates with experimental MHC I binding, a key factor associated with strong immune responses. Moreover, we confirmed that a significant proportion of selected peptide candidates can provoke an immune reaction in a vaccination setting. From this group of peptides, we selected candidates which triggered cross-reactive immune responses against TAAs or neoantigens. Data from preliminary tumor challenge experiments with prophylactic peptide vaccination and ICB allowed us to define key parameters such as target gene expression levels which can predict the cross-reactive potential of bacterial peptides that mimic tumor antigens. These findings provide novel insights into the design of gut microbiome peptide-based tumor vaccines and pave the way for establishing combinatorial therapies with immunomodulatory drugs. Citation Format: Vincent Panneton, Mathieu Gigoux, Abderezak Zebboudj, Norah Sadek, Hyejin Choi, Levi M. Mangarin, Mariam M. George, Guillaume Kulakowski, Joao G. Magalhaes, Jean-Marie Carpier, Thibaud Dugat, Camille Gaal, Francesco Strozzi, Lorenzo Tibaldi, Laurent Chene, Jedd D. Wolchok, Vinod P. Balachandran, Taha Merghoub. Gut microbiota mimics as a source of cross-reactive tumor rejection antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4090.

Abstract C006: Loss of the MGAT5 glycosyltransferase sensitizes pancreatic tumor cells to immune clearance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer-related death in the United States. The five-year survival rate of less than nine percent is attributed mainly to a difficulty in early detection and a lack of effective treatments. Tumor-associated glycans represent a potential anti-tumor target for two reasons: (i) protein glycosylation is known to play a role in tumor progression, and (ii) alternatively glycosylated proteins may function as tumor neoantigens. The glycosyltransferase MGAT5 catalyzes the formation of beta-1,6-N-acetylglucosamine branched glycans, and overexpression has been implicated in tumor growth and metastasis in multiple cancers. Using a panel of clonal cell lines that recapitulate the immune heterogeneity of PDAC, we found that knockout of MGAT5 in some clones (“T cell-inflamed" tumors) allows for complete clearance of tumors while in other clones (“non-T cell-inflamed”) MGAT5 deficiency led to a marked decrease in tumor growth. This phenotype was confirmed in orthotopic injection as well as subcutaneous injection into syngeneic mice. By contrast, MGAT5 loss had no impact on tumor cell growth in vitro. To probe immune system involvement in this robust rejection of tumor growth, the MGAT5 KO cells were injected into Nod/Scid mice, resulting in full rescue of the phenotype. Tumor eradication or growth inhibition in vivo was found to be dependent specifically on the presence of CD4/CD8 T cells and dendritic cells. Tumor challenge experiments, in which mice were immunized with MGAT5 KO cells and challenged with wild-type tumors four weeks later, revealed that tumor rejection was associated with a durable immunologic memory. Finally, therapeutic vaccination with the knockout line concomitantly with wild-type tumor cell injection led to decreased tumor growth. These results are consistent with a model in which MGAT5 loss results in the formation of new tumor antigens and/or enhances the immunogenicity of pre-existing antigens. Ongoing work seeks to determine the identity of the responsible antigens and further investigate the relationship between altered glycosylation and anti-tumor immunity. Citation Format: Erin Hollander, Ben Stanger. Loss of the MGAT5 glycosyltransferase sensitizes pancreatic tumor cells to immune clearance [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C006.

Alum Adjuvant and Built-in TLR7 Agonist Synergistically Enhance Anti-MUC1 Immune Responses for Cancer Vaccine.

The tumor-associated antigen mucin 1 (MUC1) is an attractive target of antitumor vaccine, but its weak immunogenicity is a big challenge for the development of vaccine. In order to enhance immune responses against MUC1, herein, we conjugated small molecular toll-like receptor 7 agonist (TLR7a) to carrier protein BSA via MUC1 glycopeptide to form a three-component conjugate (BSA-MUC1-TLR7a). Furthermore, we combined the three-component conjugate with Alum adjuvant to explore their synergistic effects. The immunological studies indicated that Alum adjuvant and built-in TLR7a synergistically enhanced anti-MUC1 antibody responses and showed Th1-biased immune responses. Meanwhile, antibodies elicited by the vaccine candidate effectively recognized tumor cells and induced complement-dependent cytotoxicity. In addition, Alum adjuvant and built-in TLR7a synergistically enhanced MUC1 glycopeptide-specific memory CD8+ T-cell immune responses. More importantly, the vaccine with the binary adjuvant can significantly inhibit tumor growth and prolong the survival time of mice in the tumor challenge experiment. This novel vaccine construct provides an effective strategy to develop antitumor vaccines.

MUC1 expressing tumor growth was retarded after human mucin 1 (MUC1) plasmid DNA immunization.

IntroductionNaked DNA is one of the attractive tools for vaccination studies. We studied naked DNA vaccination against the human tumor antigen, mucin, which is encoded by the MUC1 gene.MethodsWe constructed the pcDNA3.0-MUC1 (pcDNA-MUC1) plasmid expressing an underglycosylated MUC1 protein. BALB/c mice were immunized intradermally thrice at 2-weeks intervals with pcDNA-MUC1. Two weeks after the last immunization, tumor challenge experiments were performed using either the CT26 or TA3HA tumor cell lines, both of which transduce human MUC1.ResultsImmune cell population monitoring from pcDNA-MUC1-immunized animals indicated that immune cell activation was induced by MUC1-specific immunization. Using intracellular fluorescence activated cell sorting and enzyme-linked immunosorbent spot assay, we reported that interferon-γ secreting CD8+ T cells were mainly involved in MUC1-specific immunization. In all mice immunized with MUC1 DNA, tumor growth inhibition was observed, whereas control mice developed tumors (p < 0.001).ConclusionOur results suggest that intradermal immunization with MUC1 DNA induces MUC1-specific CD8+ T cell infiltration into tumors, elicits tumor-specific Th1-type immune response, and inhibits tumor growth.

Lyotropic liquid crystal-based transcutaneous peptide delivery system: Evaluation of skin permeability and potential for transcutaneous vaccination

Transcutaneous drug delivery is a promising method in terms of drug repositioning and reformulation because of its non-invasive and easy-to-use features. To overcome the skin barrier, which is the biggest challenge in transcutaneous drug delivery, a number of techniques, such as microemulsion, solid-in-oil dispersions and liposomes, have been studied extensively. However, the low viscosity of these formulations limits drug retention on the skin and reduces patient acceptability. Although viscosity can be increased by adding a thickening reagent, such an addition often alters formulation nanostructures and drug solubility, and importantly, decreases skin permeability. In this study, a gel-like lyotropic liquid crystal (LLC) was used as a tool to enhance skin permeability. In particular, we prepared 1-monolinolein (ML)-based LLCs with different water contents. All LLCs significantly enhanced skin permeation of a peptide drug, an epitope peptide of melanoma, despite their high viscoelasticity. Fourier transform infra-red spectroscopic analysis of the skin surface treated with the LLCs revealed that the gyroid geometry more strongly interacted with the lamellar structure inside the stratum corneum (SC) than the diamond geometry. Finally, as the result of the in vivo tumor challenge experiment using B16F10 melanoma-bearing mice, the LLC with the gyroid geometry showed stronger vaccine effect against tumor than a subcutaneous injection. Collectively, ML-based LLCs, especially with the gyroid geometry, are a promising strategy to deliver biomacromolecules into skin. Statement of significanceTranscutaneous drug delivery is a promising method for drug repositioning and reformulation because of its non-invasive and easy-to-use features. To overcome the skin barrier, which is the biggest challenge in transcutaneous drug delivery, we used a gel-like lyotropic liquid crystal (LLC) as a novel tool to enhance skin permeability. In this paper, we demonstrated that an LLC with a specific liquid crystalline structure has the highest skin permeation enhancement effect for a peptide antigen as a model drug. Moreover, the peptide antigen-loaded LLC showed a vaccine effect that was comparable to a subcutaneous injection in vivo. This study provides a basis for designing a transcutaneous delivery system of peptide drugs with LLC.

A novel recombinant protein vaccine containing the different E7 proteins of the HPV16, 18, 6, 11 E7 linked to the HIV-1 Tat (47-57) improve cytotoxic immune responses.

Human papillomavirus infection (HPV) is the most common viral infection which is causes of cervical, penal, vulvar, anal and, oropharyngeal cancer. E7 protein of HPV is a suitable target for induction of T cell responses and controlling HPV-related cancer. The aim of the current study was to designed and evaluated a novel fusion protein containing the different E7 proteins of the HPV 16, 18, 6 and 11, linked to the cell-penetrating peptide HIV-1 Tat 49-57, in order to improve cytotoxic immune responses in in-vitro and in-vivo. In this study whole sequence of HPV16,18,6,11 E7-Tat (47-57) and HPV16,18,6,11 E7 cloned into the vector and expressed in E. coli (BL21). The purified protein was confirmed by SDS page and western blotting and then injected into the C57BL/6 mice. The efficiency of the fusion protein vaccine was assessed by antibody response assay, cytokine assay (IL-4 and IFN-γ), CD + 8 cytotoxicity assay and tumor challenge experiment. Result showed that fusion proteins containing Adjuvant (IFA,CFA) could express higher titer of antibody. Also, we showed that vaccination with E7-Tat and, E7-Tat-ADJ induced high frequencies of E7-specific CD8 + T cells and CD107a expression as well as IFN-γ level and enhanced long-term survival in the therapeutic animal models. Our finding suggested that this novel fusion protein vaccine was able to induce therapeutic efficacy and immunogenicity by improving CD8 + T cell in TC-1 tumor bearing mice; so this vaccine may be appreciated for research against HPV and tumor immunotherapies.

Inflammation-induced tumor antigen expression on epithelial cells reveals the mechanism of their generation and provides a system for identification of new tumor targets

Abstract Tumor-associated antigens (TAAs) are self-molecules abnormally expressed on tumor cells that elicit humoral and cellular immunity and are targets of effective cancer immunosurveillance. Surprisingly, immunity to TAAs is found in many healthy individuals with no history of cancer. We previously showed that other events in addition to cancer, such as viral and bacterial infections, can cause the expression of some TAAs and correlate with effective tumor immunosurveillance. We are now testing in mice the ability of two LCMV strains, one that causes acute (Armstrong) and one that causes chronic (Cl-13) infection, to elicit expression of and immune memory to TAAs and protect from a later tumor challenge. We observed that infection with the acute strain elicits immunity to specific TAAs expressed by two tumor cell lines that were then used in tumor challenge experiments. So far, we were able to show a protective effect of a previous Armstrong infection against our model of lymphoid tumor (EL4). We also developed a highly reproducible in vitro model of acute inflammation of primary epithelial cells and confirmed that short-term exposure to pro-inflammatory cytokines IL-1β, IL-6 and TNF-α leads to abnormal expression of three well known TAAs, MUC1, CEA and HER2/neu. These cells were found to activate macrophages, which should promote antigen uptake and presentation of these and other TAAs. We have used this system and 2D Difference Gel Electrophoresis (2D-DIGE) combined with mass spectrometry to identified so far seven new TAAs. Because these TAAs are generated first as disease-associated antigens (DAAs), they represent highly predictable shared antigens for safe and effective preventative cancer vaccines.

Preparation of a new combination nanoemulsion-encapsulated MAGE1-MAGE3-MAGEn/HSP70 vaccine and study of its immunotherapeutic effect

BackgroundMAGE family genes have been studied as targets for tumor immunotherapy for a long time. Here, we combined MAGE1-, MAGE3- and MAGEn-derived peptides as a cancer vaccine and tested whether a new combination nanoemulsion-encapsulated vaccine could be used to inhibit the growth of tumor cells in humanized SCID mice. MethodsThe nanoemulsion-encapsulated complex protein vaccine (MAGE1, MAGE3, and MAGEn/HSP70 fusion protein; M1M3MnH) was prepared using a magnetic ultrasonic technique. After screening, human PBMCs were injected into SCID mice to mimic the human immune system. Then, the humanized SCID mice were challenged with M3-HHCC cells and immunized with nanoemulsion-encapsulated MAGE1-MAGE3-MAGEn/HSP70 [NE(M1M3MnH)] or M1M3MnH. The cellular immune responses were detected by IFN-γ ELISPOT and cytotoxicity assays. Therapeutic and tumor challenge experiments were also performed. ResultsThe results showed that the immune responses elicited by NE(M1M3MnH) were apparently stronger than those elicited by M1M3MnH, NE(-) or PBS, suggesting that this novel nanoemulsion carrier induces potent antitumor immunity against the encapsulated antigens. The results of the therapeutic and tumor challenge experiments also indicated that the new vaccine had a definite effect on SCID mice bearing human hepatic cancer. ConclusionOur study indicated that the combination of several tumor antigen-derived peptides may be a relatively good strategy for peptide-based cancer immunotherapy. These results suggest that the complex nanoemulsion vaccine could have broader applications for both therapy and prevention mediated by antitumor effects in the future.

Genetically engineered recombinant adenovirus expressing interleukin‑2 for hepatocellular carcinoma therapy.

Regulatory and effector T cells possess immunological cytotoxicity for tumor cells in the tumor microenvironment during tumor progression and are the primary suppressors inhuman cancer therapy. Interleukin‑2 (IL‑2) is an anticancer cytokine, which triggers human innate and adaptive immunity by stimulating T cell propagation and lymphocyte infiltration into tumor sites. IL‑2 has been used successfully for cancer therapy. Recombinant adenovirus expressing IL‑2 (rAd‑IL‑2) injection is a gene therapy agent that may improve prognosis of hepatocellular carcinoma (HCC) patients. In the present study, the ability of IL‑2 to stimulate an immune response and the ability of recombinant adenovirus to inhibit tumor cell growth in HCC was investigated in a HCC tumor model. It was demonstrated that the regulatory and effector cell‑mediated tumor suppression by antitumor cluster of differentiation (CD)4+ and CD8+ Tcells stimulated by rAd‑IL‑2 is tumor‑specific. Furthermore, rAd‑IL‑2 significantly stimulated tumor‑specific cytotoxic T lymphocyte responses, increased interferon‑γ release and enhanced antitumor immunity by inducing CD4+ and CD8+ T cell recruitment into the tumor, and additionally induced memory to protect tumor‑bearing mice against tumor challenge. Treatment with rAd‑IL‑2 led to tumor regression and long‑term survival of mice in the 120‑day treatment period. Tumor challenge experiments demonstrated that rAd‑IL‑2 induced memory, protecting against reinfection. In conclusion, rAd‑IL‑2 may promote tumor‑associated effector and regulatory T cell expansion and may be a potential therapeutic agent for clinical immunotherapy application in the treatment of cancer.

Multifaceted Immunomodulatory Nanoliposomes: Reshaping Tumors into Vaccines for Enhanced Cancer Immunotherapy

The design and synthesis of nanoparticles mimicking the key features in size, shape, and surface molecular organization of biological objects that provide danger‐signals are essential in the formulation of effective vaccine. Here, multifaceted immunomodulatory nanoliposomes (denoted as “tumosomes”) that can turn tumors into vaccines and induce enhanced antitumor immune response are suggested. Multifaceted tumosomes are synthesized by assembling tumor cell membrane proteins as tumor‐associated antigens with two lipid‐based adjuvants as pathogen characters (i.e., monophosphoryl lipid A as a danger signal, dimethyldioctadecylammonium as a cell‐invasion moiety). The enhanced antitumor immunity of tumosomes afforded by the improved antigenicity and antigen uptake efficiency is also evaluated in a tumor challenge experiment after their image‐guided intralymph node injection. Tumosomes are able to provide tumor antigens and molecular adjuvants for the priming of a long‐term adaptive immune response in tumor draining lymph nodes, as well as in the spleen. Tumosome injection leads to an inhibition of tumor growth, and the survival rate of mice treated with the tumosomes is higher than those of other groups. Taken together, the tumosomes are expected to be used for the reshaping the immune response in the lymph node and enhanced antitumor immunity.

Abstract 5065: Role of EpCAM in EGFR signaling and tumorigenesis

Abstract EpCAM is a transmembrane glycoprotein that functions as a homophilic cell adhesion molecule. EpCAM is expressed in normal epithelium and at even higher levels in epithelial cancers, including breast, pancreatic and lung cancers. Since its discovery in the 1980s, EpCAM has been targeted in several different epithelial cancers. Even though numerous novel therapeutic strategies targeting EpCAM have been investigated, the biological role and functional significance of EpCAM expression in cancer remains unclear. EpCAM expression appears to be associated with prognosis in several epithelial cancers -in some cancers it is associated with a favorable prognosis (renal cell, rectal) and in others it is associated with a poor prognosis (lung, pancreatic, breast). Thus, the function of EpCAM appears to be context-dependent in epithelial cancers. To understand the impact of EpCAM in human cancer, we specifically ablated EpCAM expression in over 40 cancer cell lines, and assessed the impact on invasion. We identified the A431 epidermoid cancer cell line as a cell line that overexpresses EpCAM, but is exquisitely sensitive to manipulation of EpCAM expression. A431 is known to overexpress EGFR and serves as a model cell line for EGFR signaling. Specific ablation of EpCAM significantly enhanced the migration and invasion of the A431 cell line. Specific ablation of EpCAM was also associated with increased EGFR activity, as demonstrated by phospho-immunoblot. Immunoprecipitation studies demonstrate that EpCAM binds directly to EGFR. Using specific EpCAM mutant constructs, we showed that the N-terminal domain of EpCAM is required for binding and suppression of EGFR activity. Using a soft agar colony growth assay, we were able to confirm that N-terminal EpCAM domain mutants (NTD288) inhibited colony growth in 3T3-transfected cells relative to the wildtype. In addition, a transformation assay showed that the same N-terminal mutant was able to inhibit transformation of 3T3 cells. To assess the effects of EpCAM ablation on in vivo tumor growth, we specifically ablated EpCAM and then performed tumor challenge experiments using immunocompromised mice. Specific ablation of EpCAM increased tumor growth relative to control. These data suggest that EpCAM may modulate EGFR signaling. Further studies are ongoing to define the mechanisms by which EpCAM inhibits EGFR activity. Citation Format: Lincoln Muhoro, Timothy Fleming, Narendra Sankpal, William Gillanders. Role of EpCAM in EGFR signaling and tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5065.

Gene Gun Her2/neu DNA Vaccination: Evaluation of Vaccine Efficacy in a Syngeneic Her2/neu Mouse Tumor Model.

Genetic vaccination using naked plasmid DNA is an immunization strategy both against infectious diseases and cancer. In order to improve the efficacy of DNA vaccines, particularly in large animals and humans, different strategies have been pursued. These vaccination strategies are based on different application routes, schedules, and coexpression of immunomodulatory molecules as adjuvants. Our mouse tumor model offers the possibility to investigate Her2/neu DNA vaccines in different settings, i.e., intramuscular or intradermal application with or without coexpression of adjuvants. Protection from tumor growth in tumor challenge experiments and both T cell and humoral immune responses against Her2/neu peptides are used as surrogate parameters for vaccine efficacy.

Combined Treatment of Murine Fibrosarcoma with Chemotherapy (Paclitaxel), Radiotherapy, and Intratumoral Injection of Dendritic Cells

BackgroundNew antitumor therapeutic strategies aim to combine different approaches that are able to induce tumor-specific effector and memory T cell responses that might control tumor growth. Dendritic cells (DCs) have the capacity to induce antigen-specific cytotoxic T lymphocytes. We have previously shown that the combined treatment of paclitaxel chemotherapy (Chemo) and injection of DCs led to complete tumor regression.ObjectiveThe goal of this study was to evaluate synergistic antitumor effect of a triple combination treatment comprising radiotherapy, paclitaxel Chemo and intratumoral injection of syngeneic bone marrow-derived DCs on murine fibrosarcoma, compared to other single or double combination treatments.MethodsFor the murine fibrosarcoma model, naïve C57BL/6 mice were inoculated intradermally with 2×103 MCA102 cells in the right upper flank. Mice were assigned to five groups (untreatedcontrol, RT alone, RT+Chemo, RT+DC, and RT+Chemo+DC), with eight mice in each group. In vitro cytotoxicity assays were performed to assess the immune activity. The persistence of tumor-specific immunity was determined by second tumor challenge in mice with complete tumor regression.ResultsThe triple combination treatment showed a significantly enhanced therapeutic efficacy by decreasing tumor size and inducing complete tumor regression, resulting in a cure of 50% of mice. The results of in vitro cytotoxicity assays and the second tumor challenge experiment strongly indicated the induction of a tumor-specific cytotoxic T lymphocyte response and acquisition of prolonged tumor immunity.ConclusionThese findings suggest that the triple combination treatment can be a promising strategy for the treatment of murine fibrosarcoma.

Abstract 1261: T cell responses against mutations in oncoproteins/tumor suppressor proteins and their induction by vaccination with long peptides.

Abstract Vaccination with peptides comprising tumor-associated antigens (TAAs) is a promising approach in cancer immunotherapy. TAAs derived from mutated genes are exclusively expressed in the tumor and are not shared with normal tissue. This reduces the risk of autoimmunity compared to non-mutated proteins when employed for cancer peptide vaccination. Moreover, the effectiveness of the vaccine can be enhanced by combining epitopes to induce cytolytic CD8+ as well as T helper (CD4+) cell responses. We therefore use long peptides (28-35 amino acids) to facilitate a presentation of MHC I and II epitopes. More precisely, we designed a panel of long peptides, with sequences derived from the most frequent mutated variants of the tumor suppressor Trp53 and the oncoproteins Kras and Braf described for colorectal (CRC) and pancreatic carcinomas. To investigate the relevance of immune responses against the chosen mutations we screened blood and bone marrow from 26 CRC patients for T cell responses against the long peptides using IFNγ ELISpot analysis. We found a tendency towards stronger responses against the mutated peptides compared to those against corresponding wild-type (wt) peptides. Furthermore, we correlated the ELISpot results with the abundance of Trp53 and Kras mutations identified in the patients’ primary tumors and metastases. This revealed that patients carrying mutations were more likely to be responsive against wt and mutated peptides than patients with no detectable mutation. In order to analyze the potency of our long peptides for active vaccination, we utilized C57BL/6J mice as well as a MHC-Class I/II humanized mouse strain (β2m-deficient, HLA-A2/Db/hβ2m [HHD chimera] and HLA-DR1 double transgenic) in a multi-peptide vaccination setting. Immune responses were monitored with flow cytometry by measuring cytokine secretion after in vitro restimulation of T cells from immunized mice. Thereby we observed responses for the majority of the long peptides tested. Interestingly, some of the mutated peptides showed a significantly higher induction of cytokine levels than corresponding wt sequences suggesting mutation-specific responses. In addition we were able to monitor both CD4+ and CD8+ T cell responses, in which the presence of CD4+ T cells enhanced CD8+ T cell responsiveness. This observation was made for several peptides in different combinations suggesting the induction of a multi-epitope response. As a final goal we plan to investigate the tumor-protective capacity of our vaccination approach. Therefore, the MHC-Class I/II transgenic mice were treated with the carcinogen 3-methylcholanthrene to generate a syngeneic tumor cell line. We were able to establish a fibrosarcoma cell line growing in vivo, which is engineered to express the most-immunogenic mutations from our vaccination panel to be tested in tumor challenge experiments. Citation Format: Jasmin Quandt, Christoph Schlude, Michael Bartoschek, Angel Cid-Arregui, Philipp Beckhove, Frank Momburg. T cell responses against mutations in oncoproteins/tumor suppressor proteins and their induction by vaccination with long peptides. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1261. doi:10.1158/1538-7445.AM2013-1261

Abstract LB-346: The Oxysterol-CXCR2 axis plays a key role in the recruitment of tumor promoting neutrophils.

Abstract Tumor formation depends on molecular alterations responsible for neoplastic transformation of normal cells, as well as on the ability of tumor cells to co-opt inflammatory/immune cells to create a tumor growth-permissive microenvironment. Targeted therapies inhibiting molecular alterations in tumor cells have improved antitumor responses. However, overall survival has been enhanced only slightly. Tumor-infiltrating immune cells can be conditioned by molecules released within the microenvironment to thwart antitumor immune responses. Among immune cells, neutrophils are recognized as playing an important pro-tumorigenic role, by favoring neoangiogenesis and/or by suppressing antitumor immune responses. We have recently shown that tumor-derived Liver X Receptor (LXR) ligands/oxysterols, known to be involved in cholesterol homeostasis and in modulating physiologic immune responses, favor tumor growth by inhibiting dendritic cell migration towards lymphoid organs. Here, we identify an unanticipated function of tumor-derived LXR ligands/oxysterols, which contribute to the recruitment of neutrophils within tumor microenvironment in an LXR-independent, CXCR2-dependent manner, ultimately favoring tumor growth. In particular, we show a continuous recruitment of neutrophils in oxysterol-releasing tumors, as evaluated by parabiosis experiments. We demonstrate by mass spectrometry and high-performance liquid chromatography analyses that the lymphoma RMA, the Lewis Lung Carcinoma (LLC) and the mesothelioma AB1 produce some oxysterols (22R-HC, 27-HC and 24S-HC), which are able to induce CD11b+Ly6G+ neutrophil migration in vitro and in vivo. Neutrophil migration is independent of LXR signaling both in vitro and in vivo, as demonstrated by using Lxrαβ-/- mice, while it depends on the oxysterol-CXCR2 interaction, as evaluated in vitro by migration experiments with Cxcr2-/- neutrophils, GTPγS binding assays, measurements of cAMP levels, and, in vivo by tumor challenge experiments in Cxcr2-/- bone marrow chimera mice. Tumor-recruited neutrophils are then able to favor tumor growth by promoting neoangiogenesis or immunosuppression of antitumor responses. Furthermore, we demonstrate that RMA, LLC and AB1 tumors engineered to express the oxysterol-inactivating enzyme sulfotransferase 2B1b (SULT2B1b), show a reduced number of tumor-infiltrating neutrophils as well as of CD31+CD45- endothelial cells, as compared to mock-expressing tumors. This reduction parallels tumor growth delay and prolonged survival of SULT2B1b-tumor-bearing mice. Antitumor responses are also obtained with genetic or pharmacologic inactivation of CXCR2, the latter mediated by the CXCR2 antagonist SB225002. These results, along with the observation that some human tumors release oxysterols, identify an unanticipated pro-tumor function of the oxysterol-CXCR2 axis and a possible new target for cancer therapy. Citation Format: Vincenzo Russo, Laura Raccosta, Raffaella Fontana, Daniela Maggioni, Claudia Lanterna, Aida Paniccia, Andrea Musumeci, Claudio Doglioni, Andrea Leiva, Eduardo J. Villablanca, Rodrigo Mora, Elena Chiricozzi, Maria Grazia Ciampa, Laura Mauri, Alessandro Prinetti, Sandro Sonino, Maria Letizia Trincavelli, Simona Daniele, Claudia Martini, Knut Steffensen, Jan-Ake Gustafsson, Safiyè Gozalvo Feo, Claudio Bordignon, Catia Traversari, Silvano Sozzani. The Oxysterol-CXCR2 axis plays a key role in the recruitment of tumor promoting neutrophils. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-346. doi:10.1158/1538-7445.AM2013-LB-346

Antitumor Activity of Human Hydatid Cyst Fluid in a Murine Model of Colon Cancer

This study evaluates the antitumor immune response induced by human hydatic cyst fluid (HCF) in an animal model of colon carcinoma. We found that anti-HCF antibodies were able to identify cell surface and intracellular antigens in CT26 colon cancer cells. In prophylactic tumor challenge experiments, HCF vaccination was found to be protective against tumor formation for 40% of the mice (P = 0.01). In the therapeutic setting, HCF vaccination induced tumor regression in 40% of vaccinated mice (P = 0.05). This vaccination generated memory immune responses that protected surviving mice from tumor rechallenge, implicating the development of an adaptive immune response in this process. We performed a proteomic analysis of CT26 antigens recognized by anti-HCF antibodies to analyze the immune cross-reactivity between E. granulosus (HCF) and CT26 colon cancer cells. We identified two proteins: mortalin and creatine kinase M-type. Interestingly, CT26 mortalin displays 60% homology with E. granulosus hsp70. In conclusion, our data demonstrate the capacity of HCF vaccination to induce antitumor immunity which protects from tumor growth in an animal model. This new antitumor strategy could open new horizons in the development of highly immunogenic anticancer vaccines.

Chimeric Infectious Bursal Disease Virus-Like Particles as Potent Vaccines for Eradication of Established HPV-16 E7–Dependent Tumors

Cervical cancer is caused by persistent high-risk human papillomavirus (HR-HPV) infection and represents the second most frequent gynecological malignancy in the world. The HPV-16 type accounts for up to 55% of all cervical cancers. The HPV-16 oncoproteins E6 and E7 are necessary for induction and maintenance of malignant transformation and represent tumor-specific antigens for targeted cytotoxic T lymphocyte–mediated immunotherapy. Therapeutic cancer vaccines have become a challenging area of oncology research in recent decades. Among current cancer immunotherapy strategies, virus-like particle (VLP)–based vaccines have emerged as a potent and safe approach. We generated a vaccine (VLP-E7) incorporating a long C-terminal fragment of HPV-16 E7 protein into the infectious bursal disease virus VLP and tested its therapeutic potential in HLA-A2 humanized transgenic mice grafted with TC1/A2 tumor cells. We performed a series of tumor challenge experiments demonstrating a strong immune response against already-formed tumors (complete eradication). Remarkably, therapeutic efficacy was obtained with a single dose without adjuvant and against two injections of tumor cells, indicating a potent and long-lasting immune response.

Enhanced anti-tumor effects of HPV16E749–57-based vaccine by combined immunization with poly(I:C) and oxygen-regulated protein 150

Background: It is well known that both heat shock protein (HSP) and Toll-like receptor (TLR)3 agonist polyinosinic:polycytidylic acid (poly(I:C)) are capable of promoting the antigen-specific immune responses. In the current study, we assessed whether the anti-tumor effects of the HPV16E749–57-based vaccine can be elevated by combined applications of poly(I:C) and oxygen-regulated protein 150 (ORP150) in a mouse cervical cancer model. Methods: Recombinant mouse ORP150 and HPV E749–57 peptide were combined to passively form the ORP150–E749–57 complex under heat shock conditions. The effects of ORP150–E749–57 complex plus poly(I:C) adjuvant on lymphocyte proliferation and functional cytotoxic T cells were investigated by methyl thiazolyl tetrazolium (MTT), ELISPOT, and non-radioactive cytotoxicity assays. Finally, the complex's therapeutic anti-tumor effects with and without adjuvant therapy were observed in a tumor challenge experiment. Results: This combination vaccine approach significantly enhanced the proliferation of splenocytes and induced strong E749–57-specific CTL responses. More importantly, the ORP150–E749–57 complex plus poly(I:C) vaccine format demonstrated more potent anti-tumor effects than ORP150–E749–57 complex alone or E749–57 plus poly(I:C) in TC-1 tumor-bearing mice. Conclusion: Both poly(I:C) and ORP150 chaperone can synergistically enhance the anti-tumor effects of the HPV16E749–57-based vaccine in vitro and in vivo. This strategy provides a platform for the design of a tumor therapeutic vaccine capable of inducing an effective anti-tumor immune response.

G1-4 A, an arabinogalactan polysaccharide from Tinospora cordifolia increases dendritic cell immunogenicity in a murine lymphoma model

The immunogenicity of dendritic cells (DC) is known to increase with their maturation state and both are induced by microbial products like LPS. In this study, we have investigated the effect of G1-4A, a polysaccharide isolated from Indian medicinal plant, Tinospora cordifolia on phenotypic and functional maturation of murine bone marrow derived dendritic cells (BMDC) and its ability to be used as an adjuvant in immunotherapy. G1-4A, enhanced surface expression of CD40, CD80, CD86, MHCII by BMDC in vitro and splenic DC in vivo. T cell allostimulatory activity and secretion of IL-12 and TNFα by BMDC were also increased. Treatment with G1-4A resulted in decreased phagocytosis and increased antigen processing that are characteristic of mature DC. G1-4A treated DC cross presented exogenous antigens on a MHC I background which resulted in the activation of cytotoxic T cells. These cells thus activated could cause lysis of target tumor cells in vitro. Administration of tumor lysate pulsed G1-4A treated DC resulted in decreased tumor burden in preventive as well as therapeutic tumor challenge experiments in a murine lymphoma model. These results thus confirm that G1-4A could be a promising nontoxic maturation agent to be potentially used in DC based immunotherapy of tumor.

Antitumor activity of novel chimeric peptides derived from cyclinD/CDK4 and the protein transduction domain 4

CyclinD1/CDK4 and cyclinD3/CDK4 complexes are key regulators of the cell progression and therefore constitute promising targets for the design of anticancer agents. In the present study, the key peptide motifs were selected from these two complexes. Chimeric peptides with these peptides conjugated to the protein transduction domain 4 (PTD4) were designed and synthesized. The chimeric peptides, PTD4-D1, PTD4-D3, PTD4-K4 exhibited significant anti-proliferation effects on cancer cell lines. These peptides could compete with the cyclinD/CDK4 complex and induce the G1/S phase arrest and apoptosis of cancer cells. In the tumor challenge experiment, these peptides showed potent antitumor effects with no significant side effects. Our results suggested that these peptides could be served as novel leading compounds with potent antitumor activity.