Sara M. Mangsbo

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 (CD25+FoxP3−) 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.

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 Ta 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-γ 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. Clin Cancer Res; 16(12); 3279–87. ©2010 AACR.

Tumor-Specific Bacteriophages Induce Tumor Destruction through Activation of Tumor-Associated Macrophages

We recently reported that administration of tumor-specific bacteriophages initiates infiltration of neutrophilic granulocytes with subsequent regression of established B16 tumors. The aim of the current study was to investigate the mechanism of action of bacteriophage-induced tumor regression and to examine possible stimulatory effects of bacteriophages on macrophages. We observed that the mechanism of phage-induced tumor regression is TLR dependent as no signs of tumor destruction or neutrophil infiltration were observed in tumors in MyD88−/− mice in which TLR signaling is abolished. The microenvironment of bacteriophage-treated tumors was further analyzed by gene profiling through applying a low-density array preferentially designed to detect genes expressed by activated APCs, which demonstrated that the M2-polarized tumor microenvironment switched to a more M1-polarized milieu following phage treatment. Bacteriophage stimulation induced secretion of proinflammatory cytokines in both normal mouse macrophages and tumor-associated macrophages (TAMs) and increased expression of molecules involved in Ag presentation and costimulation. Furthermore, mouse neutrophils selectively migrated toward mediators secreted by bacteriophage-stimulated TAMs. Under these conditions, the neutrophils also exhibited increased cytotoxicity toward B16 mouse melanoma target cells. These results describe a close interplay of the innate immune system in which bacteriophages, located to the tumor microenvironment due to their specificity, stimulate TAMs to secrete factors that promote recruitment of neutrophils and potentiate neutrophil-mediated tumor destruction.

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.

Fcγ Receptor IIb Strongly Regulates Fcγ Receptor-Facilitated T Cell Activation by Dendritic Cells

FcγR ligation by Ag–Ab immune complexes (IC) not only mediates effective Ag uptake, but also strongly initiates dendritic cell (DC) maturation, a requirement for effective T cell activation. Besides the activating FcγRI, FcγRIII, and FcγRIV, the inhibitory FcγRIIb is expressed on DCs. It is unclear how the ratio between signals from the activating FcγR and the inhibitory FcγRIIb determines the outcome of FcγR ligation on DCs. By microarray analysis, we compared the transcriptomes of steady state and IC-activated bone marrow-derived wild-type (WT) DCs expressing all FcγR or DCs expressing only activating FcγR (FcγRIIb knockout [KO]) or only the inhibitory FcγRIIb (FcR γ-chain KO). In WT DCs, we observed a gene expression profile associated with effective T cell activation, which was absent in FcR γ-chain KO, but strikingly more pronounced in FcγRIIb KO bone marrow-derived DCs. These microarray results, confirmed at the protein level for many cytokines and other immunological relevant genes, demonstrate that the transcriptome of IC-activated DCs is dependent on the presence of the activating FcγR and that the modulation of the expression of the majority of the genes was strongly regulated by FcγRIIb. Our data suggest that FcγRIIb-deficient DCs have an improved capacity to activate naive T lymphocytes. This was confirmed by their enhanced FcγR-dependent Ag presentation and in vivo induction of CD8+ T cell expansion compared with WT DCs. Our findings underscore the potency of FcγR ligation on DCs for the effective induction of T cell immunity by ICs and the strong regulatory role of FcγRIIb.

Complement Activation by CpG in a Human Whole Blood Loop System: Mechanisms and Immunomodulatory Effects

Phosphorothioate oligodeoxynucleotides can activate complement, and experimental murine studies have revealed differential effects upon simultaneous TLR stimulation and complement activation compared with either event alone. We set out to investigate the immune stimulatory effects of CpG 2006 in fresh non-anticoagulated human blood with or without presence of active complement. We also sought to elucidate the mechanism behind complement activation upon stimulation with phosphorothioate CpG 2006. In a human blood loop system, both backbone and sequence-specific effects by CpG were counteracted by selective inhibition of C3. Furthermore, DNA backbone-mediated CD40 and CD83 expression on monocytes and sequence-specific IL-6 and TNF production were reduced by complement inhibition. CpG-induced complement activation occurred via either the classical or the alternative pathway and deposits of both IgM and properdin, two activators of complement, were detected on CpG after incubation with EDTA plasma. Quartz crystal microbalance with dissipation monitoring demonstrated alternative pathway convertase build-up onto CpG as a likely pathway to initiate and sustain complement activation. Specific inhibition of C3 suppressed CpG 2006 uptake into monocytes indicating that C3 fragments are involved in CpG internalization. The interplay between complement and TLR9 signaling demonstrated herein warrants further investigation.

Locally Delivered CD40 Agonist Antibody Accumulates in Secondary Lymphoid Organs and Eradicates Experimental Disseminated Bladder Cancer

Comparing the efficacy and biodistribution of local and systemic delivery of anti-CD40 agonistic antibodies, Sandin and colleagues show that local low-dose antibody therapy is effective against disseminated bladder cancer with reduced toxic side effects. 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. Cancer Immunol Res; 2(1); 80–90. ©2013 AACR.

Circulating specific antibodies enhance systemic cross‐priming by delivery of complexed antigen to dendritic cells in vivo

Increasing evidence suggests that antibodies can have stimulatory effects on T‐cell immunity. However, the contribution of circulating antigen‐specific antibodies on MHC class I cross‐priming in vivo has not been conclusively established. Here, we defined the role of circulating antibodies in cross‐presentation of antigen to CD8+ T cells. Mice with hapten‐specific circulating antibodies, but naϊve for the T‐cell antigen, were infused with haptenated antigen and CD8+ T‐cell induction was measured. Mice with circulating hapten‐specific antibodies showed significantly enhanced cross‐presentation of the injected antigen compared with mice that lacked these antibodies. The enhanced cross‐presentation in mice with circulating antigen‐specific antibodies was associated with improved antigen capture by APCs. Importantly, CD11c+ APCs were responsible for the enhanced and sustained cross‐presentation, although CD11c− APCs had initially captured a significant amount of the injected antigen. Thus, in vivo formation of antigen‐antibody immune complexes improves MHC class I cross‐presentation, and CD8+ T‐cell activation, demonstrating that humoral immunity can aid the initiation of systemic cellular immunity. These findings have important implications for the understanding of the action of therapeutic antibodies against tumor‐associated antigens intensively used in the clinic nowadays.

The Tyrosine Kinase Inhibitors Imatinib and Dasatinib Reduce Myeloid Suppressor Cells and Release Effector Lymphocyte Responses

Immune escape mechanisms promote tumor progression and are hurdles of cancer immunotherapy. Removing immunosuppressive cells before treatment can enhance efficacy. Tyrosine kinase inhibitors (TKI) may be of interest to combine with immunotherapy, as it has been shown that the inhibitor sunitinib reduces myeloid suppressor cells in patients with renal cell carcinoma and dasatinib promotes expansion of natural killer–like lymphocytes in chronic myeloid leukemia (CML). In this study, the capacity of dasatinib and imatinib to reduce myeloid suppressor cells and to induce immunomodulation in vivo was investigated ex vivo. Samples from CML patients treated with imatinib (n = 18) or dasatinib (n = 14) within a Nordic clinical trial (clinicalTrials.gov identifier: NCT00852566) were investigated for the presence of CD11b+CD14−CD33+ myeloid cells and inhibitory molecules (arginase I, myeloperoxidase, IL10) as well as the presence of natural killer cells, T cells (naïve/memory), and stimulatory cytokines (IL12, IFNγ, MIG, IP10). Both imatinib and dasatinib decreased the presence of CD11b+CD14−CD33+ myeloid cells as well as the inhibitory molecules and the remaining myeloid suppressor cells had an increased CD40 expression. Monocytes also increased CD40 after therapy. Moreover, increased levels of CD40, IL12, natural killer cells, and experienced T cells were noted after TKI initiation. The presence of experienced T cells was correlated to a higher IFNγ and MIG plasma concentration. Taken together, the results demonstrate that both imatinib and dasatinib tilted the immunosuppressive CML tumor milieu towards promoting immune stimulation. Hence, imatinib and dasatinib may be of interest to combine with cancer immunotherapy. Mol Cancer Ther; 14(5); 1181–91. ©2015 AACR.

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+ CD127low FoxP3+) and CD127high FoxP3+ T cells (CD3+ CD4+ CD127high 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.