Zhengming Xiong

Superior Efficacy of Tumor Cell Vaccines Grown In Physiologic Oxygen

Purpose: Atmospheric oxygen (∼20% O2) has been the universal condition employed to culture tumor cells used as vaccine antigen. We tested the hypothesis that reducing oxygen tension would increase the efficacy of tumor cell lysate vaccines. Experimental Design: GL261 glioma cells and EMT6 breast carcinoma cells were grown in 5% or 20% O2. Syngeneic tumor-bearing mice were vaccinated with these tumor cell lysates mixed with CpG oligodeoxynucleotides as an adjuvant. Tumor infiltrating T cells and apoptotic GL261 cells were quantified by immunohistochemistry. Tumor-reactive immunoglobulin was detected by Western blot. Ovalbumin and gp100-derived peptides were mixed with GL261 lysates as marker antigens to detect changes in presentation of exogenous antigen on MHC class I in vitro, and in vivo following adoptive transfer of gp100-specific CD8+ T cells. Results: Mice bearing orthotopic glioma and breast carcinoma survived significantly longer when vaccinated with 5% O2 lysates. Antigen-specific CTL activation was significantly enhanced following stimulation with lysates derived from GL261 cells grown in 5% O2 versus 20% O2 through a mechanism that involved enhanced cross-presentation of exogenous antigen on MHC I. Vaccination with 5% O2 GL261 cell lysates caused a significant increase in CTL proliferation, tumoricidal function, and trafficking into brain tumor sites, whereas 20% O2 lysate vaccines predominantly evoked an antibody response. Conclusions: Tissue culture oxygen functions as an “immunologic switch” by dictating the cellular and humoral immune responses elicited by tumor cell lysates. These results have profound implications for cancer vaccines that utilize tumor cells as the source of antigen. Clin Cancer Res; 16(19); 4800–8. ©2010 AACR.

Topical Imiquimod has Therapeutic and Immunomodulatory Effects Against Intracranial Tumors

Topical imiquimod cream (trade name: Aldara) is a Toll-like receptor (TLR) 7 agonist that is approved for the treatment of cutaneous tumors. Aldara is also used as a vaccine adjuvant in clinical trials in patients with glioma and other tumors. The main mechanism of action ascribed to Aldara has been the local activation of TLR7+ cells near the application site. Here we report the unexpected finding that Aldara has therapeutic and immunomodulatory activity as a single agent in mice bearing intracranial tumors. Repeated administration of Aldara onto the skin significantly increased the survival of mice bearing intracranial GL261 glioma and EMT6 breast carcinoma. Aldara treatment was associated with a reduction in the number CD4+Foxp3+ regulatory T cells in the blood and brain tumor site. Mice treated with Aldara exhibited a generalized lymphopenia in the blood amidst an increase in brain tumor infiltrating CD4+ and CD8+ T cells and dendritic cells. Brain-infiltrating CD8+ T cells were tumor reactive as demonstrated by degranulation and interferon-&ggr; secretion in a GL261-dependent manner. In addition, soluble imiquimod directly inhibited the proliferation of GL261 cells in a TLR7-independent manner. This is the first report demonstrating that topical application of imiquimod can enhance T-cell responses to intracranial tumors as a single agent. The results must be interpreted with caution considering anatomical and biological differences between mice and humans. Nevertheless, Aldara that is being used as a vaccine adjuvant in clinical trials may have direct antitumor effects that are independent of exogenous antigen. Further studies in humans are warranted.

Structure–Activity Relationship Analysis of Imidazoquinolines with Toll-like Receptors 7 and 8 Selectivity and Enhanced Cytokine Induction

Toll-like receptors 7 and 8 (TLRs) have emerged as key targets in the design of small molecule adjuvants and stimulants for use in immunotherapies. This study examines the structure-activity relationship of a series of C2- and N1-substituted C7-methoxycarbonylimidazoquinolines to gain insight to the structural basis to TLR-7 and -8 selective activity. The analysis is further applied to evaluate the induction of multiple cytokines, including IL-10, IL-12, IL-1β, TNF-α, IFN-α, and IFN-γ, using murine BMDCs and human PBMCs. The results show TLR-7/8 activity is correlated to the C2-alkyl chain length, with peak activity occurring for the butyl (TLR-7) and pentyl (TLR-8) derivatives. A similar SAR is identified in the production of IL-1β, IL-12, and IFN-γ, which are shown to depend on both the C2-alkyl chain length and substitution to the N1-position. The compounds were also potent stimulators of IFN-α and IL-10 production but with less pronounced structure-based correlations.

Effective CpG Immunotherapy of Breast Carcinoma Prevents but Fails to Eradicate Established Brain Metastasis

Purpose: Breast cancer patients with brain metastasis have a dismal prognosis. We determined the ability of immunostimulatory CpG oligodeoxynucleotides (ODN) to treat or prevent brain metastasis in a mouse model. Experimental Design: Mice bearing orthotopic breast carcinoma with or without concurrent i.c. tumors were treated by injections of CpG ODN at the primary tumor. Immunologic memory was tested by tumor rechallenge and immune responses were assessed by flow cytometry, delayed-type hypersensitivity, and CTL assays. Results: Orthotopic tumors regressed in treated mice regardless of whether concurrent i.c. disease was present. In mice bearing only orthotopic tumors, CpG ODN rendered 50% tumor-free and they rejected tumor rechallenge in breast and brain. In mice with concurrent i.c. disease, there was no difference in brain tumor growth compared with saline controls, despite regression of the primary tumor. Flow cytometry revealed that treated mice that died from i.c. disease exhibited a significant increase in brain-infiltrating T and natural killer cells relative to saline controls. CTLs from these mice were able to kill tumor in vitro and extend survival of naive mice bearing less-established brain tumors by adoptive transfer. Conclusions: The lack of survival benefit in mice with appreciable brain metastasis was not explained by a deficit in lymphocyte trafficking or function because CTLs from these mice killed tumor and inhibited microscopic brain metastasis by adoptive transfer. These results indicate that CpG ODN might be beneficial as a preventative adjuvant to initial therapy preceding brain metastasis or to inhibit progression of microscopic brain metastases.

Design and Synthesis of N1-Modified Imidazoquinoline Agonists for Selective Activation of Toll-like Receptors 7 and 8

A series of N1-modified imidazoquinolines were synthesized and screened for Toll-like receptors (TLR) 7 and 8 activities to identify recognition elements that confer high affinity binding and selectivity. These receptors are key targets in the development of immunomodulatory agents that signal the NF-κB mediated transcription of pro-inflammatory chemokines and cytokines. Results are presented showing both TLR7/8 activations are highly correlated to N1-substitution, with TLR8 selectivity achieved through inclusion of an ethyl-, propyl-, or butylamino group at this position. While the structure-activity relationship analysis indicates TLR7 activity is less sensitive to N1-modification, extension of the aminoalkyl chain length to pentyl and p-methylbenzyl elicited high affinity TLR7 binding. Cytokine profiles are also reported that show the pure TLR8 agonist [4-amino-2-butyl-1-(2-aminoethyl)-7-methoxycarbonyl-1H-imidazo[4,5-c]quinoline] induces higher levels of IL-1β, IL-12, and IFNγ when compared with TLR7 selective or mixed TLR7/8 agonists. The results are consistent with previous work suggesting TLR8 agonists are Th1 polarizing and may help promote cell-mediated immunity.

Tumor-derived vaccines containing CD200 inhibit immune activation: implications for immunotherapy

There are over 400 ongoing clinical trials using tumor-derived vaccines. This approach is especially attractive for many types of brain tumors, including glioblastoma, yet so far the clinical response is highly variable. One contributor to poor response is CD200, which acts as a checkpoint blockade, inducing immune tolerance. We demonstrate that, in response to vaccination, glioma-derived CD200 suppresses the anti-tumor immune response. In contrast, a CD200 peptide inhibitor that activates antigen-presenting cells overcomes immune tolerance. The addition of the CD200 inhibitor significantly increased leukocyte infiltration into the vaccine site, cytokine and chemokine production, and cytolytic activity. Our data therefore suggest that CD200 suppresses the immune systems response to vaccines, and that blocking CD200 could improve the efficacy of cancer immunotherapy.

Use of CD200 blockade inhibitor to enhance glioma immunotherapy.

Meeting abstracts Utilizing tumors as a source of vaccine antigens in immunotherapy has demonstrated promising results. However, to date, researchers have failed to overcome the complex interplay between the tumor and its surrounding microenvironment resulting in T cell tolerance. The progression

Abstract B32: Preclinical testing of three immune adjuvants in vaccination therapy for invasive canine meningioma.

Malignant and atypical meningiomas are resistant to standard therapies, but therapeutic vaccines have not been tested preclinically or clinically. Pet dogs with naturally occurring meningioma are an underutilized model for aggressive human meningioma and an outstanding opportunity for assessing experimental therapeutic approaches. We treated 11 meningioma-bearing dogs with surgery and vaccine immunotherapy consisting of autologous tumor cell lysate combined with CpG or imiquimod. Interferon gamma-elaborating T cells were detected in the peripheral blood of two of cases, but vaccine-induced tumor-reactive antibody responses were found in the sera of all dogs. Systemic antibody responses were polyclonal, recognizing intracellular and cell surface antigens, and heat shock protein 60 was identified as one common antigen. Tumor-reactive antibodies bound allogeneic canine and human meningiomas, demonstrating common antigens across breed and species. Histological analysis revealed robust infiltration of antibody-secreting plasma cells into the brain around the tumor in treated compared to pre-treatment samples. Tumor-reactive antibodies were capable of inducing antibody dependent cell-mediated cytotoxicity to autologous and allogeneic tumor cells. Moreover, median survival for lysate/CpG and lysate/imiquimod vaccination was 646 days versus 222 days in historic surgery controls (p Citation Format: Brian M. Andersen, G Elizabeth Pluhar, Charles E. Seiler, Zhengming Xiong, Michelle R. Goulart, Matthew Gerry O9Sullivan, Matthew A. Hunt, Charles E. Schiaffo, David M. Ferguson, John R. Ohlfest. Preclinical testing of three immune adjuvants in vaccination therapy for invasive canine meningioma. [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 B32.