Adenoviral CD40 Ligand Immunotherapy in 32 Canine Malignant Melanomas-Long-Term Follow Up.

Malignant melanoma is a serious disease in both humans and dogs, and the high metastatic potential results in poor prognosis for many patients. Its similarities with human melanoma make spontaneous canine melanoma an excellent model for comparative studies of novel therapies and tumor biology. Gene therapy using adenoviruses encoding the immunostimulatory gene CD40L (AdCD40L) has shown promise in initial clinical trials enrolling human patients with various malignancies including melanoma. We report a study of local AdCD40L treatment in 32 cases of canine melanoma (23 oral, 5 cutaneous, 3 ungual and 1 conjunctival). Eight patients were World Health Organization (WHO) stage I, 9 were stage II, 12 stage III, and 3 stage IV. One to six intratumoral injections of AdCD40L were given every seven days, combined with cytoreductive surgery in 20 cases and only immunotherapy in 12 cases. Tumor tissue was infiltrated with T and B lymphocytes after treatment, suggesting immune stimulation. The best overall response based on result of immunotherapy included 7 complete responses, 5 partial responses, 5 stable and 2 progressive disease statuses according to the World Health Organization response criteria. Median survival was 285 days (range 20-3435 d). Our results suggest that local AdCD40L therapy is safe and could have beneficial effects in dogs, supporting further treatment development. Clinical translation to human patients is ongoing.

Intratumoral immunostimulatory AdCD40L gene therapy in patients with advanced solid tumors.

AdCD40L is a replication-deficient virus carrying the gene for CD40 ligand which has previously been evaluated in patients with urothelial cancer and malignant melanoma. Herein, we present the results of repeated intratumoral injections of AdCD40L in seven patients with metastatic solid cancer. One patient who developed urothelial cancer derived from a renal transplant was treated with repeated injections of AdCD40L alone. The remaining patients suffered from cholangiocarcinoma, kidney, breast, rectal, or ovarian cancer and received AdCD40L repeatedly (4x) in combination with cyclophosphamide. The treatment was safe and generally well-tolerated. Two patients had clinical benefit of the treatment and one of them was accepted for re-treatment. Circulating proinflammatory cytokines were commonly increased after treatment, but save for TNFα, significances were not reached which could be due to the low number of patients. Similar to earlier findings in AdCD40L-treated melanoma patients, IL8 plasma levels were high in the present study. In conclusion, gene therapy by repeated intratumoral AdCD40L injections alone, or in combination with cyclophosphamide, is feasible and safe in patients with solid cancers. The potential of intratumoral CD40L gene transfer as treatment of cancer was illustrated by the clinical improvement in two out of seven patients.

Local irradiation does not enhance the effect of immunostimulatory AdCD40L gene therapy combined with low dose cyclophosphamide in melanoma patients.

BACKGROUND: AdCD40L is an immunostimulatory gene therapy under evaluation for advanced melanoma, including ocular melanoma. Herein, we present the final data of a Phase I/IIa trial using AdCD40L alone or in combination with low dose cyclophosphamide +/- radiation therapy. METHODS: AdCD40L is a replication-deficient adenovirus carrying the gene for CD40 ligand (CD40L). Twenty-four patients with advanced melanoma were enrolled and treated with AdCD40L monotherapy, or combined with cyclophosphamide +/- single fraction radiotherapy. The patients were monitored for 10 weeks using immunological and radiological evaluations and thereafter for survival. RESULTS: AdCD40L treatment was safe and well tolerated both alone and in combination with cyclophosphamide as well as local radiotherapy. Four out of twenty-four patients had >1 year survival. Addition of cyclophosphamide was beneficial but adding radiotherapy did not further extend survival. High initial plasma levels of IL12 and MIP3b correlated to overall survival, whereas IL8 responses post-treatment correlated negatively with survival. Interestingly, antibody reactions to the virus correlated negatively with post IL6 and pre IL1b levels in blood. CONCLUSIONS: AdCD40L was safely administered to patients and effect was improved by cyclophosphamide but not by radiotherapy. Immune activation profile at baseline may predict responders better than shortly after treatment.

Local checkpoint inhibition of CTLA-4 as a monotherapy or in combination with anti-PD1 prevents the growth of murine bladder cancer.

Checkpoint blockade of CTLA-4 results in long-lasting survival benefits in metastatic cancer patients. However, patients treated with CTLA-4 blockade have suffered from immune-related adverse events, most likely due to the breadth of the induced T-cell activation. Here, we investigated the efficacy of a local low-dose anti-CTLA-4 administration for treatment of subcutaneous or orthotopic murine bladder 49 (MB49) bladder carcinoma in C57BL/6 mice. When MB49 tumors were grown s.c., peritumoral (p.t.) injection of anti-CTLA-4 treatment was equally effective as intravenous or s.c. (nontumor bearing flank) administration. Notably, p.t. injection was associated with lower circulating antibody levels and decreased IL-6 serum levels as compared to systemic treatment. Ultrasound-guided intratumoral anti-CTLA-4 antibody treatment of orthotopically growing MB49 tumors resulted in tumor regression, with more than tenfold reduction in systemic antibody levels as compared to i.v. or s.c. administration, in line with the compartmentally restrained nature of the bladder. Local anti-CTLA-4 therapy in combination with anti-PD-1 therapy resulted in complete responses, superior to each therapy alone. In addition, p.t. anti-CTLA-4 therapy was potentiated by depletion of regulatory T cells. Our results demonstrate that local anti-CTLA-4 antibody therapy is equally effective as systemic administration, but reduces systemic antibody levels and cytokine release, and enhances the response to anti-PD1 therapy.

Tumor-directed immunotherapy can generate tumor-specific T cell responses through localized co-stimulation.

The most important goals for the field of immuno-oncology are to improve the response rate and increase the number of tumor indications that respond to immunotherapy, without increasing adverse side effects. One approach to achieve these goals is to use tumor-directed immunotherapy, i.e., to focus the immune activation to the most relevant part of the immune system. This may improve anti-tumor efficacy as well as reduce immune-related adverse events. Tumor-directed immune activation can be achieved by local injections of immune modulators in the tumor area or by directing the immune modulator to the tumor using bispecific antibodies. In this review, we focus on therapies targeting checkpoint inhibitors and co-stimulatory receptors that can generate tumor-specific T cell responses through localized immune activation.

Immunostimulatory AdCD40L gene therapy combined with low-dose cyclophosphamide in metastatic melanoma patients.

BACKGROUND: Current approaches for treating metastatic malignant melanoma (MM) are not effective enough and are associated with serious adverse events. Due to its immunogenicity, melanoma is an attractive target for immunostimulating therapy. In this phase I/IIa study, local AdCD40L immunostimulatory gene therapy was evaluated in patients with MM. METHODS: AdCD40L is an adenovirus carrying the gene for CD40 ligand. Patients that failed standard treatments were enrolled. Six patients received four weekly intratumoral AdCD40L injections. Next, nine patients received low-dose cyclophosphamide conditioning before the first and fourth AdCD40L injection. The blood samples were collected at multiple time points for chemistry, haematology and immunology evaluations. Radiology was performed at enrolment and repeated twice after the treatment. RESULTS: AdCD40L was safe with mild transient reactions. No objective responses were recorded by MRI, however, local and distant responses were seen on FDG-PET. The overall survival at 6 months was significantly better when cyclophosphamide was added to AdCD40L. The patients with the best survival developed the highest levels of activated T cells and experienced a pronounced decrease of intratumoral IL8. CONCLUSIONS: AdCD40L therapy for MM was well tolerated. Local and distant responses along with better survival in the low-dose cyclophosphamide group are encouraging.

The human agonistic CD40 antibody ADC-1013 eradicates bladder tumors and generates T-cell-dependent tumor immunity.

PURPOSE: Local administration of immune-activating antibodies may increase the efficacy and reduce the immune-related adverse events associated with systemic immunotherapy of cancer. Here, we report the development and affinity maturation of a fully human agonistic CD40 antibody (IgG1), ADC-1013. EXPERIMENTAL DESIGN: We have used molecular engineering to generate an agonistic antibody with high affinity for CD40. The functional activity of ADC-1013 was investigated in human and murine in vitro models. The in vivo effect was investigated in two separate bladder cancer models, both using human xenograft tumors in immune deficient NSG mice and using a syngeneic bladder cancer model in a novel human CD40 transgenic mouse. RESULTS: Activation of dendritic cells (DC) by ADC-1013 results in upregulation of the costimulatory molecules CD80 and CD86, and secretion of IL12. ADC-1013 also activates DCs from human CD40 transgenic mice, and peptide-pulsed and ADC-1013-stimulated DCs induce antigen-specific T-cell proliferation in vitro. In vivo, treatment with ADC-1013 in a syngeneic bladder cancer model, negative for hCD40, induces significant antitumor effects and long-term tumor-specific immunity. Furthermore, ADC-1013 demonstrates significant antitumor effects in a human bladder cancer transplanted into immunodeficient NSG mice. CONCLUSIONS: Our data demonstrate that ADC-1013 induces long-lasting antitumor responses and immunologic memory mediated by CD40 stimulation. To the best of our knowledge, ADC-1013 represents the first immunomodulatory antibody developed for local immunotherapy of cancer.

Local immunotherapy based on agonistic CD40 antibodies effectively inhibits experimental bladder cancer.

Local immunotherapy resurfaces in the field of cancer as a potential way to cure localized and metastatic disease with limited toxic effects. We have recently demonstrated that local administration of agonistic CD40 antibodies can cure localized as well as disseminated bladder neoplasms. This approach reduces the circulating concentrations of antibodies that would result from systemic delivery, hence resulting in limited toxicity.

Local CTLA4 blockade effectively restrains experimental pancreatic adenocarcinoma growth in vivo.

Antibody-mediated blockade of CTLA4 has been shown to be effective in treating a select group of patients with late-stage melanoma. The precise mechanism underlying the clinical activity of CTLA4 immunotherapy is poorly understood, although recent experimental findings indicate that antibody-mediated depletion of regulatory T cells (Tregs) in the tumor microenvironment plays a key role in efficacious antitumor responses. In the current study, we used an experimental model of pancreatic adenocarcinoma to compare the antitumor efficacy of peritumoral low-dose anti-CTLA4 monoclonal antibody (mAb) administration to that of a commonly utilized systemic high-dose anti-CTLA4 regimen. We selected pancreatic adenocarcinoma as it presents a particular challenge to clinicians due to its aggressive behavior, metastatic spread and limited treatment options. Furthermore, Fc gamma receptor (FcγR)-dense myeloid cells commonly infiltrate pancreatic tumors, such that these tumor types exhibit increased susceptibility to CTLA4 antibody-targeted Treg depletion via antibody-dependent cell-mediated cytotoxicity (ADCC). Locally administered anti-CTLA4 mAb effectively reduced tumor growth at a low dose and no additional anti-tumor effects were apparent when increasing the dose or number of injections. No significant difference in overall survival was seen when comparing locally administered low-dose with standard systemic high-dose CTLA4 blockade therapy, and both delivery routes led to increased tumor-infiltrating effector T cells and reduced Treg cells. As opposed to low-dose peritumoral treatment, high-dose systemic therapy stimulated the accumulation of Tregs in secondary lymphoid organs, an effect that could potentially counteract the antitumor immunotherapeutic benefit of CTLA4 blockade. Our study confirms previous findings that local administration of low-dose anti-CTLA4 antibody generates sustained antitumor effects and provides rationale to devise ultrasound-guided intratumoral anti-CTLA4 antibody injection regimens to treat patients with pancreatic adenocarcinoma and other types of solid tumors. In support, clinical relevancy could include reduced immune-related adverse events by limiting systemic antibody spread to immune cell-dense organs.

Locally delivered CD40 agonist antibody accumulates in secondary lymphoid organs and eradicates experimental disseminated bladder cancer.

Immunotherapy with intratumoral injection of adenoviral vectors expressing CD40L has yielded positive results in experimental and clinical bladder cancer. We therefore hypothesized that anti-CD40 antibody would be effective in this setting. Agonistic CD40 antibodies were developed as vaccine adjuvants but have later been used as treatment of advanced solid tumors and hematologic cancers. Systemic anti-CD40 therapy has been associated with immune-related adverse events, such as cytokine release syndrome and liver toxicity, and local delivery is an attractive approach that could reduce toxicity. Herein, we compared local and systemic anti-CD40 antibody delivery to evaluate efficacy, toxicity, and biodistribution in the experimental MB49 bladder cancer model. Antitumor effects were confirmed in the B16 model. In terms of antitumor efficacy, local anti-CD40 antibody stimulation was superior to systemic therapy at an equivalent dose and CD8 T cells were crucial for tumor growth inhibition. Both administration routes were dependent on host CD40 expression for therapeutic efficacy. In vivo biodistribution studies revealed CD40-specific antibody accumulation in the tumor-draining lymph nodes and the spleen, most likely reflecting organs with frequent target antigen-expressing immune cells. Systemic administration led to higher antibody concentrations in the liver and blood compared with local delivery, and was associated with elevated levels of serum haptoglobin. Despite the lack of a slow-release system, local anti-CD40 therapy was dependent on tumor antigen at the injection site for clearance of distant tumors. To summarize, local low-dose administration of anti-CD40 antibody mediates antitumor effects in murine models with reduced toxicity and may represent an attractive treatment alternative in the clinic.

T-cell responses after haematopoietic stem cell transplantation for aggressive relapsing-remitting multiple sclerosis.

Autologous haematopoietic stem cell transplantation (HSCT) for relapsing-remitting multiple sclerosis is a potentially curative treatment, which can give rise to long-term disease remission. However, the mode of action is not yet fully understood. The aim of the study was to evaluate similarities and differences of the CD4(+) T-cell populations between HSCT-treated patients (n = 12) and healthy controls (n = 9). Phenotyping of memory T cells, regulatory T (Treg) cells and T helper type 1 (Th1) and type 17 (Th17) cells was performed. Further, T-cell reactivity to a tentative antigen, myelin oligodendrocyte glycoprotein, was investigated in these patient populations. Patients treated with natalizumab (n = 15) were included as a comparative group. White blood cells were analysed with flow cytometry and T-cell culture supernatants were analysed with magnetic bead panel immunoassays. HSCT-treated patients had similar levels of Treg cells and of Th1 and Th17 cells as healthy subjects, whereas natalizumab-treated patients had lower frequencies of Treg cells, and higher frequencies of Th1 and Th17 cells. Cells from HSCT-treated patients cultured with overlapping peptides from myelin oligodendrocyte glycoprotein produced more transforming growth factor-ß1 than natalizumab-treated patients, which suggests a suppressive response. Conversely, T cells from natalizumab-treated patients cultured with those peptides produced more interleukin-17 (IL-17), IL-1 and IL-10, indicating a Th17 response. In conclusion, we demonstrate circumstantial evidence for the removal of autoreactive T-cell clones as well as development of tolerance after HSCT. These results parallel the long-term disease remission seen after HSCT.

Treatment efficacy and immune stimulation by AdCD40L gene therapy of spontaneous canine malignant melanoma.

Malignant melanoma is a serious disease in both humans and dogs, and the high metastatic potential results in poor prognosis for many patients. Its similarities with human melanoma make spontaneous canine melanoma an excellent model for comparative studies of novel therapies and tumor biology. We report a pilot study of local adenovector CD40L (AdCD40L) immunogene treatment in 19 cases of canine melanoma (14 oral, 4 cutaneous, and 1 conjunctival). Three patients were World Health Organization stage I, 2 were stage II, 10 stage III, and 4 stage IV. One to 6 intratumoral injections of AdCD40L were given every 7 days, followed by cytoreductive surgery in 9 cases and only immunotherapy in 10 cases. Tumor tissue was infiltrated with T and B lymphocytes after treatment, suggesting immune stimulation. The best overall response included 5 complete responses, 8 partial responses, and 4 stable and 2 progressive disease statuses according to the World Health Organization response criteria. Median survival was 160 days (range, 20-1141 d), with 3 dogs still alive at submission. Our results suggest that local AdCD40L therapy is safe and could have beneficial effects in dogs, supporting further treatment development. Clinical translation to human patients is in progress.

Rapid expansion of T cells: Effects of culture and cryopreservation and importance of short-term cell recovery.

BACKGROUND: Successful cell therapy relies on the identification and mass expansion of functional cells for infusion. Cryopreservation of cells is an inevitable step in most cell therapies which also entails consequences for the frozen cells. MATERIAL AND METHODS: This study assessed the impact of cryopreservation and the widely used protocol for rapid expansion of T lymphocytes. The effects on cell viability, immunocompetence and the impact on apoptotic and immunosuppressive marker expression were analyzed using validated assays. RESULTS AND CONCLUSION: Cryopreservation of lymphocytes during the rapid expansion protocol did not affect cell viability. Lymphocytes that underwent mass expansion or culture in high dose IL-2 were unable to respond to PHA stimulation by intracellular ATP production immediately after thawing (ATP = 16 ± 11 ng/ml). However, their reactivity to PHA was regained within 48 hours of recovery (ATP = 356 ± 61 ng/ml). Analysis of mRNA levels revealed downregulation of TGF-ß and IL-10 at all time points. Culture in high dose IL-2 led to upregulation of p73 and BCL-2 mRNA levels while FoxP3 expression was elevated after culture in IL-2 and artificial TCR stimuli. FoxP3 levels decreased after short-term recovery without IL-2 or stimulation. Antigen specificity, as determined by IFNγ secretion, was unaffected by cryopreservation but was completely lost after addition of high dose IL-2 and artificial TCR stimuli. In conclusion, allowing short-time recovery of mass expanded and cryopreserved cells before reinfusion could enhance the outcome of adoptive cell therapy as the cells regain immune competence and specificity.

Synergistic augmentation of CD40-mediated activation of antigen-presenting cells by amphiphilic poly(γ-glutamic acid) nanoparticles.

Agonistic anti-CD40 monoclonal antibodies (mAbs) hold great potential for cancer immunotherapy. However, systemic administration of anti-CD40 mAbs can be associated with severe side effects, such as cytokine release syndrome and liver damage. With the aim to increase the immunostimulatory potency as well as to achieve a local drug retention of anti-CD40 mAbs, we linked an agonistic mAb to immune activating amphiphilic poly(γ-glutamic acid) nanoparticles (γ-PGA NPs). We demonstrate that adsorption of anti-CD40 mAb to γ-PGA NPs (anti-CD40-NPs) improved the stimulatory capacity of the CD40 agonist, resulting in upregulation of costimulatory CD80 and CD86 on antigen-presenting cells, as well as IL-12 secretion. Interestingly, anti-CD40-NPs induced strong synergistic proliferative effects in B cells, possibly resulting from a higher degree of CD40 multimerization, enabled by display of multiple anti-CD40 mAbs on the NPs. In addition, local treatment with anti-CD40-NPs, compared to only soluble CD40 agonist, resulted in a significant reduction in serum levels of IL-6, IL-10, IL-12 and TNF-α in a bladder cancer model. Taken together, our results suggest that anti-CD40-NPs are capable of synergistically enhancing the immunostimulatory effect induced by the CD40 agonist, as well as minimizing adverse side effects associated with systemic cytokine release. This concept of nanomedicine could play an important role in localized immunotherapy of cancer.

Adoptive T-cell therapy for malignant melanoma patients with TILs obtained by ultrasound-guided needle biopsy.

Adoptive cell therapy with tumor-infiltrating lymphocytes (TIL) can mediate objective responses in up to 50% of malignant melanoma patients with a good performance status refractory to standard treatments. Current protocols for generation of TILs rely on open surgery for access to tumor tissue. We obtained tumor material by ultrasound-guided core needle biopsy or surgery from melanoma patients with progressive disease and were able to isolate >5 × 10(6) TILs from 23 of 24 patients who were subsequently treated with these cells. One-third of the individual TIL-positive cultures displayed interferon gamma activity after stimulation with relevant melanoma cell lines. When expanded TILs were used for treatment in combination with daily low dose s.c. IL-2 after prior lymphodepleting chemotherapy, we observed objective clinical responses in one patient treated with TILs obtained from surgery and 4 patients treated with TILs from core biopsies. The results of this study demonstrate for the first time the potential of core biopsies for generation of relevant numbers of TILs that can mediate objective responses in patients with metastatic malignant melanoma. Ultrasound-guided core needle biopsy is a robust, safe and inexpensive approach to obtain tumor tissue for TIL generation, and is especially valuable in instances where surgery is contraindicated.

Both CD4+ FoxP3+ and CD4+ FoxP3- T cells from patients with B-cell malignancy express cytolytic markers and kill autologous leukaemic B cells in vitro.

Cytotoxic CD4(+) T cells have been found in patients with chronic lymphocytic leukaemia (CLL) and seem to be involved in the regulation of malignant B cells. The CD4(+) T regulatory cells (Tregs) can regulate various immune cells, including B cells, by inducing their apoptosis. Hence, different subgroups of CD4(+) T cells may be involved in the regulation of malignant B cells. In this study, the cytotoxic phenotype and function of various CD4(+) T-cell subgroups were investigated in patients with B-cell malignancies. Peripheral blood was collected from patients with CLL, various B-cell lymphomas, healthy adult donors, children with precursor B-cell acute lymphoblastic leukaemia (pre-B ALL) and from healthy children. CD4(+) T cells (CD3(+) CD4(+) FoxP3(-)), Tregs (CD3(+) CD4(+) CD127(low) FoxP3(+)) and CD127(high) FoxP3(+) T cells (CD3(+) CD4(+) CD127(high) FoxP3(+)) were analysed for their expression of the cytolytic markers CD107a and Fas ligand. Patients with CLL had increased CD107a expression on all tested T-cell subgroups compared with healthy donors. Similar results were found in patients with B-cell lymphomas whereas the CD107a expression in children with pre-B ALL was no different from that in healthy controls. Fas ligand expression was similar between patient cells and cells of healthy donors. CD4(+) T cells and Tregs from patients with CLL and healthy donors were subsequently purified and cultured in vitro with autologous B cells. Both subgroups lysed B cells and killing was confirmed by granzyme ELISAs. In conclusion, cytotoxic populations of CD4(+) T cells, including Tregs, are present in patients with B-cell malignancy and may be an important factor in immune-related disease control.

Large-scale bioreactor expansion of tumor-infiltrating lymphocytes.

BACKGROUND: The aim of this study was to evaluate an improved technique for expansion of tumor-infiltrating lymphocytes (TILs) based on the WAVE Bioreactor system with perfusion and tube-welding techniques. Our hypothesis was that the bioreactor would allow for optimized provision of nutrients and removal of spent media while minimizing culture volumes. These refinements might lead to a better quality of expanded cells with lower amounts of exhausted cells compared to static expansions in culture bags. PROCEDURES: Tumor-infiltrating lymphocytes from 4 melanoma patients were expanded and compared in parallel using either the WAVE Bioreactor 2/10 System or traditional static culture methods. The parameters viability, final cell number, phenotype and effector function were measured. RESULTS: Our results show that the bioreactor system with perfusion is suitable for large-scale expansion of tumor-infiltrating lymphocytes and allows for higher cell densities and absolute cell numbers as compared to static culture conditions. Phenotypic characteristics of TILs were compared pre and post expansion and showed no consistent difference between the two expansion methods. TILs harvested had the phenotype and function corresponding to intermediate to late effector cells. The system allows one technician to operate several bioreactors simultaneously, thereby reducing the labor for one expansion to approximately 1/3 compared to static expansion. DISCUSSION: The WAVE Bioreactor system is suitable for large-scale expansion of TILs. Due to constant perfusion of fresh media and removal of spent media much higher cell densities were achieved while the culture volume and the glucose and glutamine levels were kept constant. Expansion of TILs in the bioreactor system represents a labor- and cost-effective method to reach large numbers of T cells for adoptive cell transfer therapy in the clinic. CONCLUSION: The system presented herein offers an effective alternative to large-scale production of cell products for clinical use while meeting requirements of therapeutic cell quantities and qualities of current protocols for treatment of malignant melanoma.

AdCD40L immunogene therapy for bladder carcinoma--the first phase I/IIa trial.

PURPOSE: Immunotherapy with Bacillus Calmette-Guerin (BCG) instillation is recommended for high-risk, non-muscle invasive bladder cancer. Bacillus Calmette-Guerin is not effective in advanced tumors, and better alternatives are warranted. Immunostimulating gene therapy with adenoviral vectors expressing CD40 ligand (AdCD40L) has shown efficacy in tumor models. CD40 ligand stimulates systemic immunity and may be effective in local and invasive human disease. EXPERIMENTAL DESIGN: Patients with invasive bladder cancer scheduled for cystectomy or patients with T(a) tumors were enrolled in a phase I/IIa trial. Patients were treated with three cycles of intrabladder Clorpactin WCS-90 prewash, followed by AdCD40L instillation 1 week apart. Safety, gene transfer, immune effects, and antitumor responses were monitored. RESULTS: All eight recruited patients were treated as scheduled, and therapy was well tolerated. The main adverse effect was transient local pain during prewash. Postoperatively, urinary tract infections and one case of late septicemia with elevated potassium were reported. No adverse events were ascribed to vector therapy. Gene transfer was detected in biopsies, and bladders were heavily infiltrated with T cells. The effector marker IFN-gamma increased in biopsies, whereas levels of circulating T regulatory cells were reduced. Histologic evaluation indicated that AdCD40L therapy reduced the load of malignant cells. CONCLUSIONS: To our knowledge, this is the first report on immunogene therapy in bladder cancer and the first using AdCD40L in vivo. Local AdCD40L gene therapy was safe, boosted immune activation, and should be further evaluated as a single or an adjuvant therapy for urothelial malignancies.

Enhanced tumor eradication by combining CTLA-4 or PD-1 blockade with CpG therapy.

Tumor immunotherapy aims to break effector T-cell anergy and to block suppressive cell types and ligands allowing effector cells to exert tumor eradication. Previous reports demonstrate that cytotoxic T lymphocyte antigen-4 (CTLA-4)-blocking antibodies promote T-cell activation and render T effector cells resistant to T regulatory cells (Tregs) whereas programmed death receptor-1 (PD-1)/PD-L1 blockade results in loss of peripheral tolerance. Herein, we explored single or combined antibody blockade of CTLA-4 and PD-1 alone or combined with the toll-like receptor agonists CpG or bacillus Calmette-Guérin for treatment of murine experimental bladder cancer. In therapeutic studies, tumors were rejected by anti-CTLA-4 (aCTLA-4) while anti-PD-1 (aPD-1) suppressed tumor growth. The combination had no additive effect compared with aCTLA-4 alone. However, elevated levels of circulating CD107a expressing CD8 T cells were found in the aCTLA-4 plus aPD-1 group. In addition, levels of antinuclear antibodies correlated inversely with tumor size. Next, we combined CpG or bacillus Calmette-Guérin with aCTLA-4, aPD-1, or aPD-L1 and found that CpG in combination with aCTLA-4 or aPD-1 increased the survival of mice, with aPD-1 plus CpG being superior to either agent alone. CpG plus aCTLA-4 or aPD-1 increased the numbers of circulating tumor-specific CD107a expressing CD8 T cells as well as activated (CD25FoxP3-) CD4 splenocytes. Further, we investigated the numbers of Tregs in the tumor area of treated animals and detected decreased levels after aCTLA-4 or aPD-1 plus CpG therapy. Thus, the combination of CpG with CTLA-4 or PD-1 blockade improved long-term survival and led to increased levels of tumor-reactive T cells and reduced numbers of Tregs at the tumor site.

CMV-specific T-cell immunity, viral load, and clinical outcome in seropositive renal transplant recipients: a pilot study.

BACKGROUND: Cytomegalovirus (CMV) infection is still the leading opportunistic infection following solid organ transplantation. The aim of this prospective study of renal transplant recipients was to evaluate the dynamics of CMV-specific T-cells, viral load, and clinical symptoms of CMV infection. METHODS: Levels of tetramer-selected CD8(+) T-cells (TetraCD8), CMV-specific interferon-gamma producing CD8(+) T-cells (IFNgammaCD8), and CD4(+) T-cells (IFNgammaCD4), measured using major histocompatibility complex-tetramer and cytokine flow cytometry techniques, and CMV DNA were monitored monthly in 17 CMV-seropositive patients up to one yr (median 12 months, range 3-12) after transplantation and correlated to clinical outcome. RESULTS: CMV DNAemia was detected in 94% of the patients, but only one patient developed CMV disease. CMV DNAemia >1 million copies/mL was seen in asymptomatic patients. CMV-specific T-cells decreased rapidly after transplantation. TetraCD8 and IFNgammaCD8 regenerated within three months, whereas IFNgammaCD4 recovery was impaired up to one yr after transplantation. The proportion of IFNgammaCD4 at two months post-transplantation as compared with baseline, correlated strongly with the magnitude of the CMV DNAemia. CONCLUSIONS: Monitoring the reduction of IFNgammaCD4 compared with baseline during the first months after transplantation could be considered in predicting risk for high-grade CMV DNAemia and in deciding strategic approaches for pre-emptive and prophylactic therapy.