Beneficial modulation of the tumor microenvironment and generation of anti-tumor responses by TLR9 agonist lefitolimod alone and in combination with checkpoint inhibitors.

Activation of Toll-like receptor 9 (TLR9) is known to foster innate and adaptive immune responses and thus improve immune-mediated control of malignant disease. Lefitolimod is a potent TLR9 agonist without chemical modification developed for immunotherapeutic strategies. Modulation of the tumor microenvironment (TME) is a crucial requirement for the response to various immunotherapies: Immunogenic ("hot") tumors, characterized by their T cell-infiltrated TME, respond better compared to non-immunogenic ("cold") tumors. It has been speculated that the mode-of-action of lefitolimod provides the necessary signals for activation of immune cells, their differentiation into anti-tumor effector cells and their recruitment into the TME. We investigated the effect of lefitolimod on TME, and its potency to induce synergistic anti-tumor effects when combined with immune checkpoint inhibitory antibodies (CPI) in a murine model. Indeed, we could show that treatment with single-agent lefitolimod beneficially modulated the TME, via infiltration of activated CD8+ cells and a shift in the macrophage population toward M1 phenotype. The result was a pronounced anti-tumor effect correlated with the magnitude of infiltrated immune cells and tumor-specific T cell responses. In line with this, lefitolimod led to persistent anti-tumor memory in the EMT-6 model after tumor re-challenge. This was accompanied by an increase of tumor-specific T cell responses and cross-reactivity against different tumor cells. Lefitolimod clearly augmented the limited anti-tumor effect of the CPI anti-PD1 in an A20 and anti-PD-L1 in a CT26 model. These properties of potent immune surveillance reactivation render lefitolimod an ideal candidate as therapeutic agent for immuno-oncology, e.g. improving CPI strategies.

TLR9 agonist MGN1703 enhances B cell differentiation and function in lymph nodes.

BACKGROUND: TLR9 agonists are being developed as immunotherapy against malignancies and infections. TLR9 is primarily expressed in B cells and plasmacytoid dendritic cells (pDCs). TLR9 signalling may be critically important for B cell activity in lymph nodes but little is known about the in vivo impact of TLR9 agonism on human lymph node B cells. As a pre-defined sub-study within our clinical trial investigating TLR9 agonist MGN1703 (lefitolimod) treatment in the context of developing HIV cure strategies (NCT02443935), we assessed TLR9 agonist-mediated effects in lymph nodes. METHODS: Participants received MGN1703 for 24 weeks concurrent with antiretroviral therapy. Seven participants completed the sub-study including lymph node resection at baseline and after 24 weeks of treatment. A variety of tissue-based immunologic and virologic parameters were assessed. FINDINGS: MGN1703 dosing increased B cell differentiation; activated pDCs, NK cells, and T cells; and induced a robust interferon response in lymph nodes. Expression of Activation-Induced cytidine Deaminase, an essential regulator of B cell diversification and somatic hypermutation, was highly elevated. During MGN1703 treatment IgG production increased and antibody glycosylation patterns were changed. INTERPRETATION: Our data present novel evidence that the TLR9 agonist MGN1703 modulates human lymph node B cells in vivo. These findings warrant further considerations in the development of TLR9 agonists as immunotherapy against cancers and infectious diseases. FUND: This work was supported by Aarhus University Research Foundation, the Danish Council for Independent Research and the NovoNordisk Foundation. Mologen AG provided study drug free of charge.

Effects of 24-week Toll-like receptor 9 agonist treatment in HIV type 1+ individuals.

DESIGN: This was an exploratory, single-arm clinical trial that tested the immune enhancement effects of 24-weeks of Toll-like receptor 9 (TLR9) agonist (MGN1703; Lefitolimod; 60 mg × 2 weekly) therapy. METHODS: We enrolled HIV-1-infected individuals on suppressive combination antiretroviral therapy. Safety was assessed throughout the study. The primary outcome was reduction in total CD4 T-cell viral DNA levels. Secondary outcomes included safety, detailed immunological and virological analyses, and time to viral rebound (viral load > 5000 copies/ml) after randomization into an analytical treatment interruption (ATI). RESULTS: A total of 12 individuals completed the treatment phase and nine completed the ATI. Adverse events were limited and consistent with previous reports for MGN1703. Although the dosing regimen led to potent T-cell activation and increased HIV-1-specific T-cell responses, there were no cohort-wide changes in persistent virus (total CD4 T cells viral DNA; P = 0.34). No difference in time to rebound was observed between the ATI arms (log rank P = 0.25). One of nine ATI participants, despite harboring a large replication-competent reservoir, controlled viremia for 150 days via both HIV-1-specific cellular and antibody-mediated immune responses. CONCLUSION: A period of 24 weeks of MGN1703 treatment was safe and improved innate as well as HIV-1-specific adaptive immunity in HIV-1+ individuals. These findings support the incorporation of TLR9 agonism into combination HIV-1 cure strategies. TRIAL NAME AND REGISTRATION: TLR9 Enhancement of antiviral immunity in chronic HIV-1 infection: a phase 1B/2A trial; ClinicalTrials.gov NCT02443935.

EnanDIM - a novel family of L-nucleotide-protected TLR9 agonists for cancer immunotherapy.

BACKGROUND: Toll-like receptor 9 agonists are potent activators of the immune system. Their clinical potential in immunotherapy against metastatic cancers is being evaluated across a number of clinical trials. TLR9 agonists are DNA-based molecules that contain several non-methylated CG-motifs for TLR9 recognition. Chemical modifications of DNA backbones are usually employed to prevent degradation by nucleases. These, however, can promote undesirable off-target effects and therapeutic restrictions. METHODS: Within the EnanDIM® family members of TLR9 agonists described here, D-deoxyribose nucleotides at the nuclease-accessible 3'-ends are replaced by nuclease-resistant L-deoxyribose nucleotides. EnanDIM® molecules with varying sequences were screened for their activation of human peripheral blood mononuclear cells based on secretion of IFN-alpha and IP-10 as well as activation of immune cells. Selected molecules were evaluated in mice in a maximum feasible dose study and for analysis of immune activation. The ability to modulate the tumor-microenvironment and anti-tumor responses after EnanDIM® administration was analyzed in syngeneic murine tumor models. RESULTS: The presence of L-deoxyribose containing nucleotides at their 3'-ends is sufficient to prevent EnanDIM® molecules from nucleolytic degradation. EnanDIM® molecules show broad immune activation targeting specific components of both the innate and adaptive immune systems. Activation was strictly dependent on the presence of CG-motifs, known to be recognized by TLR9. The absence of off-target effects may enable a wide therapeutic window. This advantageous anti-tumoral immune profile also promotes increased T cell infiltration into CT26 colon carcinoma tumors, which translates into reduced tumor growth. EnanDIM® molecules also drove regression of multiple other murine syngeneic tumors including MC38 colon carcinoma, B16 melanoma, A20 lymphoma, and EMT-6 breast cancer. In A20 and EMT-6, EnanDIM® immunotherapy cured a majority of mice and established persistent anti-tumor immune memory as evidenced by the complete immunity of these mice to subsequent tumor re-challenge. CONCLUSIONS: In summary, EnanDIM® comprise a novel family of TLR9 agonists that facilitate an efficacious activation of both innate and adaptive immunity. Their proven potential in onco-immunotherapy, as shown by cytotoxic activity, beneficial modulation of the tumor microenvironment, inhibition of tumor growth, and induction of long-lasting, tumor-specific memory, supports EnanDIM® molecules for further preclinical and clinical development.

Short-Course Toll-Like Receptor 9 Agonist Treatment Impacts Innate Immunity and Plasma Viremia in Individuals With Human Immunodeficiency Virus Infection.

BACKGROUND.: Treatment with latency reversing agents (LRAs) enhances human immunodeficiency virus type 1 (HIV-1) transcription in vivo but leads to only modest reductions in the size of the reservoir, possibly due to insufficient immune-mediated elimination of infected cells. We hypothesized that a single drug molecule-a novel Toll-like receptor 9 (TLR9) agonist, MGN1703-could function as an enhancer of innate immunity and an LRA in vivo. METHODS.: We conducted a single-arm, open-label study in which 15 virologically suppressed HIV-1-infected individuals on antiretroviral therapy received 60 mg MGN1703 subcutaneously twice weekly for 4 weeks. We characterized plasmacytoid dendritic cell, natural killer (NK), and T-cell activation using flow cytometry on baseline and after 4 weeks of treatment. HIV-1 transcription was quantified by measuring plasma HIV-1 RNA during MGN1703 administration. RESULTS.: In accordance with the cell type-specific expression of TLR9, MGN1703 treatment led to pronounced activation of plasmacytoid dendritic cells and substantial increases in plasma interferon-α2 levels (P < .0001). Consistently, transcription of interferon-stimulated genes (eg, OAS1, ISG15, Mx1; each P < .0001) were upregulated in CD4+ T cells as demonstrated by RNA sequencing. Further, proportions of activated cytotoxic NK cells and CD8+ T cells increased significantly during MGN1703 dosing, suggesting an enhancement of cellular immune responses. In 6 of 15 participants, plasma HIV-1 RNA increased from <20 copies/mL to >1500 copies/mL (range, 21-1571 copies/mL) during treatment. CONCLUSIONS.: TLR9 agonist treatment in HIV infection has a dual potential by increasing HIV-1 transcription and enhancing cytotoxic NK cell activation, both of which are key outcomes in HIV-1 eradication therapy. CLINICAL TRIALS REGISTRATION.: NCT02443935.

Distinct immunological activation profiles of dSLIM® and ProMune® depend on their different structural context.

INTRODUCTION: DNA-based TLR9 agonists are potent activators of the immune system. ProMune® and dSLIM® belong to different families of TLR9 agonists and both have been established as cancer immunotherapeutics in clinical proof-of-concept studies. Unfortunately, ProMune® failed in pivotal oncological trials. dSLIM®, the active ingredient of Lefitolimod (MGN1703), successfully finished a double-blinded, placebo-controlled phase II study in patients with advanced colorectal cancer, exhibiting improved progression-free survival and durable disease control. METHODS: To explain the different systemic efficacies of dSLIM® and ProMune®, both TLR9 agonists and chimeric molecules thereof are analyzed side-by-side in a panel of in vitro assays for immune activation. RESULTS AND CONCLUSIONS: Indeed, dSLIM® exposure results in an IFN-α dependent broad activation of immune cells whereas ProMune® strongly stimulates B cells. Moreover, all functional effects of dSLIM® strictly depend on the presence of CG-motifs within its dumbbell-shaped, covalently closed structural context. Conversely, several immunological effects of ProMune® like IL-8 secretion are independent of CG-motifs and could be ascribed to the phosphorothioate-modifications of its DNA backbone, which may have caused the side effects of ProMune® in clinical trials. Finally, we showed that the implementation of ProMune® (ODN2006) base sequence into the characteristic dSLIM® dumbbell form resulted in dSLIM2006 with all beneficial effects for immunostimulation combined from both TLR9 classes without any CG-independent effects.

Design and characterization of the tumor vaccine MGN1601, allogeneic fourfold gene-modified vaccine cells combined with a TLR-9 agonist.

The tumor vaccine MGN1601 was designed and developed for treatment of metastatic renal cell carcinoma (mRCC). MGN1601 consists of a combination of fourfold gene-modified cells with the toll-like receptor 9 agonist dSLIM, a powerful connector of innate and adaptive immunity. Vaccine cells originate from a renal cell carcinoma cell line (grown from renal cell carcinoma tissue), express a variety of known tumor-associated antigens (TAA), and are gene modified to transiently express two co-stimulatory molecules, CD80 and CD154, and two cytokines, GM-CSF and IL-7, aimed to support immune response. Proof of concept of the designed vaccine was shown in mice: The murine homologue of the vaccine efficiently (100%) prevented tumor growth when used as prophylactic vaccine in a syngeneic setting. Use of the vaccine in a therapeutic setting showed complete response in 92% of mice as well as synergistic action and necessity of the components. In addition, specific cellular and humoral immune responses in mice were found when used in an allogeneic setting. Immune response to the vaccine was also shown in mRCC patients treated with MGN1601: Peptide array analysis revealed humoral CD4-based immune response to TAA expressed on vaccine cells, including survivin, cyclin D1, and stromelysin.

MIDGE Technology for the Production of a Fourfold Gene-Modified, Allogenic Cell-Based Vaccine for Cancer Therapy.

Gene modification of eukaryotic cells by electroporation is a widely used method to express selected genes in a defined cell population for various purposes, like gene correction or production of therapeutics. Here, we describe the generation of a cell-based tumor vaccine via fourfold transient gene modification of a human renal cell carcinoma (RCC) cell line for high expression of CD80, CD154, GM-CSF, and IL-7 by use of MIDGE(®) vectors. The two co-stimulatory molecules CD80 and CD154 are expressed at the cell surface, whereas the two cytokines GM-CSF and IL-7 are secreted yielding cells with enhanced immunological properties. These fourfold gene-modified cells have been used as a cell-based tumor vaccine for the treatment of RCC.

Design and Structural Requirements of the Potent and Safe TLR-9 Agonistic Immunomodulator MGN1703.

Single-stranded oligodeoxynucleotides (ODN), containing nonmethylated cytosine-guanine motifs (CpG ODN), are recognized by the innate immune system as "danger signals." CpG ODN are efficacious immunomodulators but require phosphorothioate (PT) or other backbone modifications for metabolic stability, which cause toxicities in mice and primates. We therefore designed a covalently closed DNA molecule (dSLIM(®)) where two single-stranded loops containing CG motifs are connected through a double-stranded stem in the absence of any nonnatural DNA component. The most promising immunomodulator, MGN1703, comprises two loops of 30 nucleotides containing three CG motifs each, and a connecting stem stem of 28 base pairs. MGN1703 stimulates cytokine secretion [interferon (IFN)-α, IFN-γ, interleukin (IL)-12, IL-6, and IL-2] and activates immune cells by increased expression of CD80, CD40, human leukocyte antigen (HLA)-DR and ICAM-1. Efficacy of immunomodulation strictly depends on the descriptive dumbbell shape and size of the molecule. Variations in stem length and loop size lead to reduced potency of the respective members of the dSLIM(®) class. In a representative mouse model, toxicities from injections of high amounts of a CpG ODN-PT and of MGN1703 were evaluated. The CpG ODN-PT group showed severe organ damage, whereas no such or other pathologies were found in the MGN1703 group. Oncological clinical trials of MGN1703 already confirmed our design.

MGN1703, an immunomodulator and toll-like receptor 9 (TLR-9) agonist: from bench to bedside.

The adaptive immune system has been the main focus of immunological strategies in oncology with only more recent approaches targeting innate immunity. Endosomal toll-like receptors (TLR-7, TLR-9) activate innate immune responses by signaling damage-associated molecular patterns (DAMP) from decaying tumor cells. This has led to the development of DNA-based TLR-9 agonists, which induce antitumor activity through innate and subsequent adaptive immune responses. Early clinical trials with CpG-ODN as TLR-9 agonists were associated with unfavorable tolerability and narrow clinical efficacy, leading to failure in pivotal trials. dSLIM, the active ingredient of MGN1703, is a DNA-based, radically different molecular alternative to CpG-ODN, which results in genuine antitumor immunomodulation. Preclinical and clinical studies of MGN1703 have confirmed that this TLR-9 agonist has therapeutic potential in a variety of solid tumors, while long-term treatment with high doses was very well tolerated. A pivotal trial of first-line maintenance treatment with MGN1703 in patients with metastatic colorectal cancer is underway.

Phase I clinical study of the toll-like receptor 9 agonist MGN1703 in patients with metastatic solid tumours.

PURPOSE: This study was initiated to evaluate safety, toxicity, pharmacokinetics, and pharmacodynamics of treatment with MGN1703, a novel synthetic DNA-based toll-like receptor 9 (TLR9)-immunomodulator. METHODS: The study consisted of an escalating single dose regimen followed by a multiple dose part. Dose levels of 0.25, 2, 10, 30, and 60 mg of MGN1703 were administered subcutaneously over 6 weeks twice weekly. Patients with at least stable disease (SD) could participate in the extension phase of the study for six further weeks. Effects on the immune status were monitored. RESULTS: 28 patients with metastatic solid tumours were included. Fatigue and activated partial thromboplastin time (aPTT) prolongation were the only two cases of drug-related grade 3 Common Terminology Criteria adverse events (CTCAE). The most frequently reported drug-related adverse events were of CTC Grade ⩽2. There was no relationship between toxicity and dose and no patient was withdrawn from the study due to drug-related AE. No drug-related serious AE (SAE) were reported. Six out of 24 patients had SD after 6 weeks of treatment and three of those remained in SD after a total of 12 weeks. Four patients were further treated in a compassionate use programme showing long-term disease stabilisation for up to 18 months. Immune assessment of cell compartments showed a non-significant increase of TLR9 expressing naïve B cells during therapy. CONCLUSION: Twice weekly subcutaneous applications of MGN1703 in a dose of up to 60 mg are safe and well tolerated without dose-limiting toxicities. MGN1703 shows immune activation and anti-tumour efficacy in heavily pretreated patients. The recommended dose of 60 mg twice weekly is currently used in a phase II trial in small cell lung cancer and a phase III trial in colorectal cancer patients.

Genuine Immunomodulation With dSLIM.

Toll-like receptors are sensing modulators of the innate immune system. One member of this protein family, Toll-like receptor (TLR)-9, is increasingly being investigated as therapeutic target for infectious diseases and cancer. Double-Stem Loop ImmunoModulator (dSLIM) is a new TLR-9 agonist in clinical development for patients with metastatic colorectal carcinoma. Compared with other TLR-9 ligands developed as immunomodulators, dSLIM comprises single- and double-stranded DNA, is covalently closed, and consists of natural nucleotide components only. All investigated biologic effects of dSLIM are strongly dependent on CG motifs, and the relevant cellular activation profile of dSLIM is distinct to that of other TLR-9 agonists. Here we describe the structure and biologic profile of dSLIM: in isolated human peripheral blood mononuclear cells (PBMCs), dSLIM induced a unique pattern of cytokine secretion, activated within the PBMC pool particular cell subpopulations, and exhibited specific cytotoxicity on target cells. Using cellular isolation and depletion setups, the mechanism of immunoactivation by dSLIM was deduced to be dependent on, but not restricted to, TLR-9-bearing plasmacytoid dendritic cells. The dSLIM-promoted cellular stimulation directs systemic activation of the immune response as revealed in cancer patients. The observed cellular activation cascades are discussed in the context of cancer therapy.

Maintenance treatment with the immunomodulator MGN1703, a Toll-like receptor 9 (TLR9) agonist, in patients with metastatic colorectal carcinoma and disease control after chemotherapy: a randomised, double-blind, placebo-controlled trial.

PURPOSE: This phase II study evaluated the synthetic DNA-based immunomodulator and Toll-like receptor 9 agonist MGN1703 as maintenance treatment in metastatic colorectal carcinoma (mCRC). METHODS: Fifty-nine patients with mCRC and disease control after standard first-line chemotherapy were randomised to MGN1703 60 mg (N = 43) or placebo (N = 16). RESULTS: The hazard ratio (HR) for the primary endpoint [progression-free survival (PFS) from the start of maintenance] was 0.56 (95 % CI 0.29-1.08; P = 0.07) and 0.55 (95 % CI 0.3-1.0; P = 0.04) by independent and investigator review, respectively. MGN1703 significantly improved PFS measured from the start of induction therapy versus placebo on independent (HR 0.49; 95 % CI 0.26-0.94; P = 0.03) and investigator review (HR 0.50; 95 % CI 0.31-1.02; P = 0.02). Overall survival (OS) data remain immature (HR 95 %; 95 % CI 0.3-1.5; P = 0.29) with 28/43 patients alive after a medium follow-up of >17 months. Retrospective subgroup analysis showed a significant effect of MGN1703 on PFS versus placebo in patients with greater than median tumour size reduction and normalised carcinoembryonic antigen concentrations following induction therapy, and in patients with elevated activated NKT cells ≥3.08 %. Adverse events were mild to moderate and limited to injection-site reactions or linked to general immune system activation. CONCLUSIONS: MGN1703 maintenance treatment was well tolerated and appears to induce durable and prolonged PFS and disease control in a subgroup of patients with mCRC following induction therapy. Activated NKT cells may be a predictive biomarker for selecting patients likely to benefit more from MGN1703.

Preclinical study on combined chemo- and nonviral gene therapy for sensitization of melanoma using a human TNF-alpha expressing MIDGE DNA vector.

Nonviral gene therapy represents a realistic option for clinical application in cancer treatment. This preclinical study demonstrates the advantage of using the small-size MIDGE(®) DNA vector for improved transgene expression and therapeutic application. This is caused by significant increase in transcription efficiency, but not by increased intracellular vector copy numbers or gene transfer efficiency. We used the MIDGE-hTNF-alpha vector for high-level expression of hTNF-alpha in vitro and in vivo for a combined gene therapy and vindesine treatment in human melanoma models. The MIDGE vector mediated high-level hTNF-alpha expression leads to sensitization of melanoma cells towards vindesine. The increased efficacy of this combination is mediated by remarkable acceleration and increase of initiator caspase 8 and 9 and effector caspase 3 and 7 activation. In the therapeutic approach, the nonviral intratumoral in vivo jet-injection gene transfer of MIDGE-hTNF-alpha in combination with vindesine causes melanoma growth inhibition in association with increased apoptosis in A375 cell line or patient derived human melanoma xenotransplant (PDX) models. This study represents a proof-of-concept for an anticipated phase I clinical gene therapy trial, in which the MIDGE-hTNF-alpha vector will be used for efficient combined chemo- and nonviral gene therapy of malignant melanoma.

Intratumoral dispersion, retention, systemic biodistribution, and clearance of a small-size tumor necrosis factor-α-expressing MIDGE vector after nonviral in vivo jet-injection gene transfer.

For nonviral applications of therapeutic DNA, highly efficient and safe vector systems are of crucial importance. In the majority of nonviral approaches plasmid vectors are in use. A novel minimalistic gene expression vector (MIDGE) has been developed to overcome the limitations of plasmid vectors. This small-size double-stranded linear DNA vector has shown improved transgene expression. However, only limited knowledge on uptake, biodistribution, and clearance of this vector exists. In this study we investigated the intratumoral and systemic biodistribution, clearance, and expression kinetics of the tumor necrosis factor (TNF)-α-carrying MIDGE-CMVhTNF vector in NMRI-nu/nu mice with subcutaneously xenotransplanted human A375 melanoma. Biodistribution was analyzed by quantitative real-time PCR in tumors, blood, and organs 0 to 60 min and 3 to 48 hr after intratumoral jet-injection of 50 µg of MIDGE-CMVhTNF. We examined TNF mRNA expression in tumor tissue and organs, using real-time RT-PCR and TNF-specific ELISA. High levels of MIDGE DNA in the tumor tissue demonstrated efficient gene transfer of the small-size vector, resulting in inhomogeneous DNA dispersion and efficient transgene expression. Intratumoral jet-injection of the vector DNA was accompanied by leakage into the blood circuit and appearance in peripheral organs within 5 min to 6 hr. However, this did not lead to TNF-α expression and was followed by rapid vector clearance resulting in the disappearance of MIDGE DNA 24 hr after gene transfer. These data provide important new information for the kinetics of intratumoral and systemic biodistribution and rapid clearance of the jet-injected small-size MIDGE vector.

Prevention and synergistic control of Ph(+) ALL by a DNA vaccine and 6-mercaptopurine.

Although the outcome of patients with acute lymphoblastic leukemia (ALL) has been improved continuously by chemotherapy and tyrosine kinase inhibitors, prognosis of patients with Philadelphia chromosome positive (Ph(+)) ALL still remains poor. Since further intensification of chemotherapy is limited by toxic side effects and patients with high risk of transplant-related mortality are not eligible for allogeneic stem cell transplantation new treatment strategies are urgently needed for the prevention of Ph(+) ALL relapse. There is increasing evidence that the immune system plays an essential role for the eradication or immunologic control of remaining leukemia cells. We developed several DNA-based vaccines encoding a BCR-ABL(p185) specific peptide and GM-CSF, and CD40-L, IL-27 or IL-12 and evaluated the preventive and therapeutic efficacy against a lethal challenge of syngeneic Ph(+) ALL in Balb/c mice. In vivo cell depletion assays and cytokine expression studies were performed and the efficacy of the DNA vaccine was compared with 6-mercaptopurine (6-MP) alone and the combination of the DNA vaccine and 6-MP. Preventive immunization with the vaccine BCR-ABL/GM-CSF/IL-12 and the TLR-9 agonist dSLIM induced an innate and adaptive immune response mediated by NK-cells, CD4(+) T-cells and CD8(+) T-cells leading to a survival rate of 80%. Therapeutic vaccination resulted in a significantly longer leukemia-free survival (40.7 days vs. 20.4 days) and a higher survival rate (56% vs. 10%) compared to chemotherapy with 6-MP. Remarkably, in combination with the vaccine 6-MP acted synergistically and led to 100% survival. These results demonstrate that minimal residual disease of Ph(+) ALL can be significantly better controlled by a combined treatment approach of immunotherapy and chemotherapy. This provides a rationale for improving maintenance therapy in order to reduce the relapse rate in patients with Ph(+) ALL.

The innate antiviral immune system of the cat: molecular tools for the measurement of its state of activation.

The innate immune system plays a central role in host defence against viruses. While many studies portray mechanisms in early antiviral immune responses of humans and mice, much remains to be discovered about these mechanisms in the cat. With the objective of shedding light on early host-virus interactions in felids, we have developed 12 real-time TaqMan(®) qPCR systems for feline genes relevant to innate responses to viral infection, including those encoding for various IFNα and IFNω subtypes, IFNß, intracellular antiviral factor Mx, NK cell stimulator IL-15 and effectors perforin and granzyme B, as well as Toll-like receptors (TLRs) 3 and 8. Using these newly developed assays and others previously described, we measured the relative expression of selected markers at early time points after viral infection in vitro and in vivo. Feline embryonic fibroblasts (FEA) inoculated with feline leukemia virus (FeLV) indicated peak levels of IFNα, IFNß and Mx expression already 6h after infection. In contrast, Crandell-Rees feline kidney (CrFK) cells inoculated with feline herpes virus (FHV) responded to infection with high levels of IFNα and IFNß only after 24h, and no induction of Mx could be detected. In feline PBMCs challenged in vitro with feline immunodeficiency virus (FIV), maximal expression levels of IFNα, ß and ω subtype genes as well as IL-15 and TLRs 3, 7 and 8 were measured between 12 and 24h after infection, whereas expression levels of proinflammatory cytokine gene IL-6 were consistently downregulated until 48h post inoculation. A marginal upregulation of granzyme B was also observed within 3h after infection. In an in vivo experiment, cats challenged with FIV exhibited a 2.4-fold increase in IFNα expression in blood 1 week post infection. We furthermore demonstrate the possibility of stimulating feline immune cells in vitro with various immune response modifiers (IRMs) already known for their immunostimulatory properties in mice and humans, namely Poly IC, Resiquimod (R-848) and dSLIM™, a synthetic oligonucleotide containing several unmethylated CpG motifs. Stimulation of feline PBMCs with dSLIM™ and R-848 effectively enhanced expression of IFNα within 12h by factors of 6 and 12, respectively, and Poly IC induced an increase in Mx mRNA expression of 28-fold. Altogether, we describe new molecular tools and their successful use for the characterization of innate immune responses against viruses in the cat and provide evidence that feline cells can be stimulated by synthetic molecules to enhance their antiviral defence mechanisms.

Immune response induced by a linear DNA vector: influence of dose, formulation and route of injection.

Previously, minimalistic, immunogenetically defined gene expression (MIDGE) vectors were developed as effective and sophisticated carriers for DNA vaccination. Here we evaluate the influence of dose, formulation and delivery route on the immune response after vaccination with MIDGE-Th1 vectors encoding hepatitis B virus surface antigen (HBsAg). An HBsAg-specific IgG1 and IgG2a antibody response was induced in a dose-dependent manner, whereas the IgG2a/IgG1 ratio was independent of the injected DNA dose. Formulation of MIDGE-HBsAg-Th1 with the cationic pyridinium amphiphile SAINT-18 significantly increased antibody levels of IgG1 and IgG2a compared to the unformulated vector. In contrast, SAINT-18 had neither a significant effect on the IgG2a/IgG1 ratio nor on the type and strength of cellular immunity. Overall, the strongest immune response was generated after intradermal injection, followed by intramuscular and subcutaneous (s.c.) injection. The results show that the formulation of MIDGE-Th1 with SAINT-18 increased the efficacy of the MIDGE-Th1 DNA vaccine and is therefore a suitable approach to improve the efficacy of DNA vaccines also in large animals and humans.

Novel optical nanosensors for probing and imaging live cells.

This review introduces multifunctional optical nanosensors based on surface-enhanced Raman scattering (SERS) and demonstrates their application in live cells. The novel nanosensors have the potential to improve our understanding of cellular processes on the molecular level. The hybrid sensor consists of gold or silver nanoparticles with an attached reporter species. The sensor can be detected and imaged based on the SERS signature of the reporter. This results in several advantages, such as high spectral specificity, multiplex capabilities, improved contrast, and photostability. SERS sensors not only highlight cellular structures, based on enhanced Raman spectra of intrinsic cellular molecules measured in the local optical fields of the gold nanoparticles, they also provide molecular structural information on their cellular environment. Moreover, the SERS signature of the reporter can deliver information on the local pH value inside a cell at subendosomal resolution. SERS sensors are suitable for one- and two-photon excitation. FROM THE CLINICAL EDITOR: This review introduces multifunctional optical nanosensors based on surface enhanced Raman scattering (SERS) and demonstrates their application in live cells. These hybrid sensors consist of gold or silver nanoparticles with an attached reporter species. The sensor can be detected and imaged based on the SERS signature of the reporter. SERS sensors highlight cellular structures and provide molecular structural information on their cellular environment. They can also deliver information on the intracellular pH-value at subendosomal resolution.