Addition of TLR9 agonist immunotherapy to radiation improves systemic antitumor activity

Radiotherapy (RT) has been used to control tumors by physically damaging DNA and inducing apoptosis; it also promotes antitumor immune responses via neoantigens release and augmenting immune-oncology agents to elicit systemic response. Tumor regression after RT can recruit inflammatory cells, such as tumor-associated macrophages and CD11b⁺ myeloid cell populations, a major subset of which may actually be immunosuppressive. However, these inflammatory cells also express Toll-like receptors (TLRs) that can be stimulated to reverse suppressive characteristics and promote systemic antitumor outcomes. Here, we investigated the effects of adding CMP-001, a CpG-A oligodeoxynucleotide TLR9 agonist delivered in a virus-like particle (VLP), to RT in two murine models (344SQ metastatic lung adenocarcinoma and CT26 colon carcinoma). High-dose RT (12Gy x 3 fractions) significantly increased the percentages of plasmacytoid dendritic cells within the tumor islets 3- and 5-days post-RT; adding CMP-001 after RT also enhanced adaptive immunity by increasing the proportion of CD4⁺ and CD8⁺ T cells. RT plus CMP-001-mediated activation of the immune system led to significant inhibition of tumor growth at both primary and abscopal tumor sites, thereby suggesting a new combinatorial treatment strategy for systemic disease.     

Adoptive immunotherapy combined with intratumoral TLR agonist delivery eradicates established melanoma in mice

Toll-like receptor (TLR) agonists can trigger broad inflammatory responses that elicit rapid innate immunity and promote the activities of lymphocytes, which can potentially enhance adoptive immunotherapy in the tumor-bearing setting. In the present study, we found that Polyinosinic:Polycytidylic Acid [Poly(I:C)] and CpG oligodeoxynucleotide 1826 [CpG], agonists for TLR 3 and 9, respectively, potently activated adoptively transferred T cells against a murine model of established melanoma. Intratumoral injection of Poly(I:C) and CpG, combined with systemic transfer of activated pmel-1 T cells, specific for gp100_25–33, led to enhanced survival and eradication of 9-day established subcutaneous B16F10 melanomas in a proportion of mice. A series of survival studies in knockout mice supported a key mechanistic pathway, whereby TLR agonists acted via host cells to enhance IFN-γ production by adoptively transferred T cells. IFN-γ, in turn, enhanced the immunogenicity of the B16F10 melanoma line, leading to increased killing by adoptively transferred T cells. Thus, this combination approach counteracted tumor escape from immunotherapy via downregulation of immunogenicity. In conclusion, TLR agonists may represent advanced adjuvants within the setting of adoptive T-cell immunotherapy of cancer and hold promise as a safe means of enhancing this approach within the clinic.     

Potentiated autologous tumor cell and peripheral blood lymphocyte lysate vaccination

Immune stimulation is a promising prospect in cancer therapy. Immunotherapy may be local or systemic, aspecific or targeted and may use monoclonal antibodies or vaccines. The aim of using vaccines is to stimulate the body to produce its own antibodies. Autologous tumor-cell vaccination has no contraindications or side-effects, since the patients own materials (lymphocytes, tumor cells) are used. We describe a method for producing an autologous cancer vaccine. The material to be injected as a vaccine derives from a mixed culture of autologous lymphocytes cocultured with autologous cancer cells. Peripheral blood lymphocytes are obtained by lymphocytapheresis. Cancer cells may be obtained from tissue biopsies or biological fluids, or from long-term cultures from the patient who is to be vaccinated. The culture medium (RPMI 1640) is free of fetal calf serum (FCS). The coculture is mixed with autologous plasma in a 1:1 ratio with the addition of 200 IU of recombinant human interleukin-2/mL, and is incubated at 37 degrees C in a humidified 5% CO2-enriched atmosphere for 48 h. The cocultured material is frozen, thawed to lyse cells, aliquoted and stored at -20 degrees C.     

S100A9 interaction with TLR4 promotes tumor growth

By breeding TRAMP mice with S100A9 knock-out (S100A9(-/-)) animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b(+) S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68(+) macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9(-/-) and TLR4(-/-), but not in RAGE(-/-) animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFβ expression in splenic CD11b(+) cells. Lastly, treatment of mice with a small molecule (ABR-215050) that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies.     

HLA class I expression in metastatic melanoma correlates with tumor development during autologous vaccination

Our knowledge of the mechanisms underlying tumor-specific immune response and tumor escape has considerably increased. HLA class I antigen defects remain an important tumor escape mechanism since they influence the interactions between tumor cells and specific T and NK cells in the course of malignant disease. We have studied here HLA class I expression in six subcutaneous metastases obtained from a melanoma patient immunized with an autologous melanoma cell vaccine (M-VAX). We report in this paper that HLA class I antigen expression on these metastatic lesions strongly correlated with the course of the disease. The three metastases that were partially regressing at the time of their excision showed a strong HLA class I expression, whereas the progressing ones showed a very weak or negative staining with most of the anti-HLA class I mAbs used. Real-time quantitative PCR of the samples obtained from microdissected tumor tissue revealed a significant difference in the mRNA levels of HLA-ABC heavy chain and beta2m between the two types of metastases, i.e., lower levels in progressing metastases and high levels in regressing ones, confirming the immunohistological findings. This is, to our knowledge, the first report where the clinical outcome of different HLA class I positive and negative melanoma metastases can be clearly correlated with the regression and progression of the disease, respectively.     

TLR1/TLR2 agonist induces tumor regression by reciprocal modulation of effector and regulatory T cells

Using TLR agonists in cancer treatment can have either beneficial or detrimental effects. Therefore, it is important to determine their effect on the tumor growth and understand the underlying mechanisms in animal tumor models. In this study, we report a general immunotherapeutic activity of a synthetic bacterial lipoprotein (BLP), a TLR1/TLR2 agonist, on established lung carcinoma, leukemia, and melanoma in mice. Systemic treatment of 3LL tumor-bearing mice with BLP, but not LPS, led to a dose-dependent tumor regression and a long-lasting protective response against tumor rechallenge. The BLP-mediated tumor remission was neither mediated by a direct tumoricidal activity nor by innate immune cells, because it lacked therapeutic effect in immunodeficient SCID mice. Instead, BLP treatment reduced the suppressive function of Foxp3(+) regulatory T cells (Tregs) and enhanced the cytotoxicity of tumor-specific CTL in vitro and in vivo. Furthermore, adoptive cotransfer of BLP-pretreated but not untreated CTL and Tregs from wild-type but not from TLR2(-/-) mice was sufficient to restore antitumor immunity in SCID mice by reciprocally modulating Treg and CTL function. These results demonstrate that the TLR1/TLR2 agonist BLP may have a general tumor therapeutic property involving reciprocal downregulation of Treg and upregulation of CTL function. This property may play an important role in the development of novel antitumor strategies.     

IL-6 and maturation govern TLR2 and TLR4 induced TLR agonist tolerance and cross-tolerance in dendritic cells

Stimulation of naive mouse dendritic cells (DC) with LPS or Pam(3)CSK(4) (P3C) induces production of TNF-alpha via TLR4- or TLR2-signaling. Although tolerance in macrophages has been studied in detail, we investigated the role of TLR agonist concentration and IL-6 for tolerance in DC. P3C- or LPS-primed DC were nonresponsive to P3C or LPS restimulation in terms of TNF-alpha but not IL-6 production. The mechanisms involved in tolerance were dependent on the concentration of the TLR ligand used for DC priming. DC primed with LPS or P3C at high concentrations developed a maturation dependent, IL-6 independent tolerance associated with inhibition of TLR signaling upstream of IkappaB as indicated by decreased IkappaB degradation. In contrast, priming of DC with LPS or P3C at low concentrations resulted in IL-6-dependent tolerance, which was abolished in IL-6 deficient DC, and was not accompanied by maturation of DC or by down-regulation of TLR2 or TLR4. In homotolerogenic DC primed with LPS or P3C at high concentrations, degradation of IkappaB upon restimulation with LPS or P3C was inhibited suggesting tolerance mechanism(s) upstream of IkappaB; in contrast, cross-tolerance in DC primed with LPS or P3C at low concentrations was not associated with reduced IkappaB degradation suggesting tolerance mechanisms downstream of IkappaB. Our data indicate that in naive DC TLR4- and TLR2-stimulation results in homo- and cross-tolerance; the mechanisms involved in tolerance depend on the concentration of the TLR agonist used for DC priming and are governed by IL-6 and maturation.     

Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice

The Lyme disease vaccine is based on the outer-surface lipoprotein (OspA) of the pathogen Borrelia burgdorferi, and 95% of vaccine recipients develop substantial titers of antibodies against OspA. Here, we identified seven individuals with very low antibody titers after vaccination (low responders). The macrophages of low responders produced less tumor necrosis factor-alpha and interleukin-6 after OspA stimulation and had lower cell-surface expression of Toll-like receptor (TLR) 1 as compared to normal cells, but normal expression of TLR2. TLRs activate innate responses to pathogens, and TLR2 recognizes lipoproteins and peptidoglycan (PGN). After OspA immunization, mice genetically deficient in either TLR2 (TLR2(-/-)) or TLR1 (TLR1(-/-)) produced low titers of antibodies against OspA. Notably, macrophages from TLR2(-/-) mice were unresponsive to OspA and PGN, whereas those from TLR1(-/-) mice responded normally to PGN but not to OspA. These data indicate that TLR1 and TLR2 are required for lipoprotein recognition and that defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination.     

Lymphoma immunotherapy with CpG oligodeoxynucleotides requires TLR9 either in the host or in the tumor itself

Established widely metastatic tumor was cured in a transplanted mouse B cell lymphoma model, by the combination of chemotherapy plus intratumoral injection of oligodeoxynucleotides containing unmethylated C-G motifs (CpG). This therapeutic effect required that the CpG be injected directly into the tumor and was dependent on CD8 T cells. Although the efficacy of CpG oligodeoxynucleotides has been thought to depend on the expression of TLR9, we unexpectedly found that tumor rejection did not require host expression of TLR9. By using a TLR9-deficient tumor and a TLR9KO host, we demonstrate that TLR9 expression either by the host or the tumor is required. These results indicate that activation of Ag presentation by cells within the tumor via TLR9 stimulation can be an effective form of immunotherapy. This study forms the basis of an ongoing clinical trial in patients with lymphoma.     

Study of TLR3, TLR4, and TLR9 in prostate carcinomas and their association with biochemical recurrence

Background  

Toll-like receptors (TLRs) have garnered an extraordinary amount of interest in cancer research due to their role in tumor progression. By activating the production of several biological factors, TLRs induce type I interferons and other cytokines, which drive an inflammatory response and activate the adaptive immune system. The aim of this study was to investigate the expression and clinical relevance of TLR3, 4, and 9 in prostate cancer.     

WT1 peptide vaccination combined with BCG-CWS is more efficient for tumor eradication than WT1 peptide vaccination alone

A Wilms tumor gene WT1 is expressed at high levels not only in most types of leukemia but also in various types of solid tumors, including lung and breast cancer. WT1 protein has been reported to serve as a target antigen for tumor-specific immunotherapy both in vitro in human systems and in vivo in murine models. We have shown that mice immunized with WT1 peptide or WT1 cDNA could reject a challenge from WT1-expressing tumor cells (a prophylactic model). However, it was not examined whether WT1 peptide vaccination had the potency to reject tumor cells in a therapeutic setting. In the present study, we demonstrated for the first time that WT1 peptide vaccination combined with Mycobacterium bovis bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) was more effective for eradication of WT1-expressing tumor cells that had been implanted into mice before vaccination (a therapeutic model) compared with WT1 peptide vaccination alone. An intradermal injection of BCG-CWS into mice, followed by that of WT1 peptide at the same site on the next day, generated WT1-specific cytotoxic T lymphocytes (CTLs) and led to rejection of WT1-expressing leukemia or lung cancer cells. These results showed that BCG-CWS, which was well known to enhance innate immunity, could enhance WT1-specific immune responses (acquired immunity) in combination with WT1 peptide vaccination. Therefore, WT1 peptide vaccination combined with BCG-CWS may be applied to cancer immunotherapy in clinical settings.H. Nakajima and K. Kawasaki contributed equally to this study.     

A bivalent CMV vaccine formulated with human compatible TLR9 agonist CpG1018 elicits potent cellular and humoral immunity in HLA expressing mice

There is now convincing evidence that the successful development of an effective CMV vaccine will require improved formulation and adjuvant selection that is capable of inducing both humoral and cellular immune responses. Here, we have designed a novel bivalent subunit vaccine formulation based on CMV-encoded oligomeric glycoprotein B (gB) and polyepitope protein in combination with human compatible TLR9 agonist CpG1018. The polyepitope protein includes multiple minimal HLA class I-restricted CD8⁺ T cell epitopes from different antigens of CMV. This subunit vaccine generated durable anti-viral antibodies, CMV-specific CD4⁺ and CD8⁺ T cell responses in multiple HLA expressing mice. Antibody responses included broad T_H1 isotypes (IgG2a, IgG2b and IgG3) and potently neutralized CMV infection in fibroblasts and epithelial cells. Furthermore, polyfunctional antigen-specific T cell immunity and antiviral antibody responses showed long-term memory maintenance. These observations argue that this novel vaccine strategy, if applied to humans, could facilitate the generation of robust humoral and cellular immune responses which may be more effective in preventing CMV-associated complications in various clinical settings.     

A combined opiate agonist and antagonist treatment reduces prolactin secreting pituitary tumor growth

Prolactin secreting pituitary adenomas (prolactinomas) is the most common pituitary tumors in humans. Animal studies have identified aggressive prolactinoma development in fetal alcohol exposed rats. We have recently identified a combination treatment of a μ opioid receptor antagonist naltrexone and a δ opioid receptor agonist D-Ala2-,N-Me-Phe4,Gly-ol Enkephalin (DPDPE) increases innate immune function. In this study, we tested whether naltrexone and DPDPE combination therapy is useful to control pituitary tumor growth. Fetal alcohol exposed and control Fischer 344 female rats at 60 days of age were ovariectomized and received an estrogen implant to induce prolactinomas. Six weeks after the estrogen implant, these animals received treatments of naltrexone and DPDPE or saline. The growth of the pituitary tumor prior to and after opioidergic agent treatments was visualized using magnetic resonance imaging (MRI). At the end of the treatment, pituitary weights, plasma prolactin and splenic levels of cytotoxic factors were determined. Both imaging data and weight data indicated that the volume and the weight of the pituitary were increased more after estrogen treatment in animals exposed to fetal alcohol than control. Naltrexone and DPDPE treatment reduced the weight and volume of the pituitary gland and plasma levels of prolactin in both fetal alcohol exposed and control-fed animals. The treatment of opioidergic agents also increased the levels of cytotoxic factors in the spleen. These data provide a novel possibility in treating pituitary tumors using a combination therapy of naltrexone and DPDPE.     

TLR agonist rMBP-NAP inhibits B16 melanoma tumor growth via induction of DCs maturation and T-cells cytotoxic response

Cytotechnology - Melanoma is the most aggressive skin cancer with increasing incidence and poor prognosis all over the world. Recent research has found that immunological abnormalities played a key...     

Activation of innate immunity, inflammation, and potentiation of DNA vaccination through mammalian expression of the TLR5 agonist flagellin

Improving DNA vaccination remains a fundamental goal in vaccine research. Theoretically, this could be achieved by molecules encoded by DNA capable of activating TLRs to mimic inflammatory responses generated by infection. Therefore, we constructed an expression vector that allows mammalian cells to express the TLR5 agonist flagellin (FliC) at the cell surface. In vitro, cell lines expressing FliC stimulated production of proinflammatory cytokines and the up-regulation of costimulatory molecules on monocytes. Mice given the FliC expression vector intradermally exhibited site-specific inflammation and, in combination with vectors expressing Ags, developed dramatic increases in Ag-specific IgG as well as IgA. Surprisingly, mice also developed strong Ag-specific MHC class I-restricted cellular immunity. To determine whether vaccination using FliC vectors could elicit protective immunity to an infectious agent, mice were given dermal injections of FliC expression vector together with a vector encoding the influenza A virus nucleoprotein. This vaccination strategy elicited protective immunity to lethal influenza A virus infection. These results demonstrate that expression of DNA-encoded TLR agonists by mammalian cells greatly enhance and broaden immune responses, imposing new possibilities on DNA vaccination to infectious agents and cancer.     

Association of human TLR1 and TLR6 deficiency with altered immune responses to BCG vaccination in South African infants

The development of effective immunoprophylaxis against tuberculosis (TB) remains a global priority, but is hampered by a partially protective Bacillus Calmette-Guérin (BCG) vaccine and an incomplete understanding of the mechanisms of immunity to Mycobacterium tuberculosis. Although host genetic factors may be a primary reason for BCG''s variable and inadequate efficacy, this possibility has not been intensively examined. We hypothesized that Toll-like receptor (TLR) variation is associated with altered in vivo immune responses to BCG. We examined whether functionally defined TLR pathway polymorphisms were associated with T cell cytokine responses in whole blood stimulated ex vivo with BCG 10 weeks after newborn BCG vaccination of South African infants. In the primary analysis, polymorphism TLR6_C745T (P249S) was associated with increased BCG-induced IFN-γ in both discovery (n = 240) and validation (n = 240) cohorts. In secondary analyses of the combined cohort, TLR1_T1805G (I602S) and TLR6_G1083C (synonymous) were associated with increased IFN-γ, TLR6_G1083C and TLR6_C745T were associated with increased IL-2, and TLR1_A1188T was associated with increased IFN-γ and IL-2. For each of these polymorphisms, the hypo-responsive allele, as defined by innate immunity signaling assays, was associated with increased production of TH1-type T cell cytokines (IFN-γ or IL-2). After stimulation with TLR1/6 lipopeptide ligands, PBMCs from TLR1/6-deficient individuals (stratified by TLR1_T1805G and TLR6_C745T hyporesponsive genotypes) secreted lower amounts of IL-6 and IL-10 compared to those with responsive TLR1/6 genotypes. In contrast, no IL-12p70 was secreted by PBMCs or monocytes. These data support a mechanism where TLR1/6 polymorphisms modulate TH1 T-cell polarization through genetic regulation of monocyte IL-10 secretion in the absence of IL-12. These studies provide evidence that functionally defined innate immune gene variants are associated with the development of adaptive immune responses after in vivo vaccination against a bacterial pathogen in humans. These findings could potentially guide novel adjuvant vaccine strategies as well as have implications for IFN-γ-based diagnostic testing for TB.