Linda Sandin

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.

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

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.

Local AdCD40L Gene Therapy is Effective for Disseminated Murine Experimental Cancer by Breaking T-cell Tolerance and Inducing Tumor Cell Growth Inhibition

CD40 ligand (CD40L) is one of the most potent stimulators of Th1-type immunity through its maturation of dendritic cells that, in turn, stimulate effector cells such as T cells and NK cells. Lately, CD40-mediated cell growth inhibition and apoptosis have been in focus for the development of novel cancer treatment regiments, including recombinant soluble CD40L or CD40-stimulating antibodies. In this study, intravesical CD40L gene transfer through adenoviral vectors (AdCD40L) was used to treat an aggressive model of disseminated bladder cancer (MB49/C57BL/6). Three weekly AdCD40L vector instillations increased overall survival of tumor-bearing mice (mean 18.5 d, control mice 13 d). Furthermore, bladder tumors were eradicated (2 of 10) simultaneously as lung metastases (6 of 10) were cleared. FoxP3 levels were similar in the tumors of AdCD40L-treated mice and control mice but the tumor-infiltrating effector T cells in AdCD40L-treated mice were cytotoxic (CD107a+) in contrast to those in control-treated tumors. Furthermore, AdCD40L gene therapy could induce cell growth inhibition and cell death in the MB49 tumor cells in vitro and in vivo. However, this effect was not potent enough to cure growing tumors in immunodeficient mice. In conclusion, AdCD40L gene therapy is potent for disseminated cancer both by activation of T cells and controlling tumor cell growth and viability.

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.

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.

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.

Synergistic effects of agonistic costimulatory antibodies adsorbed to amphiphilic poly(γ-glutamic acid) nanoparticles

Agonistic antibodies targeting costimulatory pathways for the immune system hold great potential for cancer immunotherapy. However, systemic administration of immunoactivating antibodies can be associated with side effects such as cytokine release syndrome and liver toxicity. Polymeric nanoparticles (NPs) represent an exciting approach to control the release of therapeutic antibodies and to optimize the desired immune response via selective targeting. With increased potency as objective, agonistic antibodies targeting TNF receptors were adsorbed to immune stimulating biodegradable and self-assembled polymeric nanoparticles composed of g-glutamic acid (g-PGA NPs). The effects of antibodies targeting CD40, CD137 and OX40 were evaluated based on T and B cell proliferation as well as on cytokine release and phenotypic maturation of antigen-presenting cells. Our results demonstrated a strong synergistic effect on human B cell proliferation of CD40 monoclonal antibodies (mAbs) carrying NPs in vitro. In addition adsorption of anti-CD40 mAb to g-PGA NPs significantly reduced the systemic release of TNF-a ,I L-6, IL-10 and IL-12, compared to treatment with the soluble mAb. Preliminary results indicate increased T cell proliferation and activation by CD137 and OX40 agonistic antibodies in combination with g-PGA NPs. Combining NPs with agonistic antibodies for cancer immunotherapy offers intriguing opportunities for increased therapeutic efficacy and safety.

Abstract B103: Intralesional administration of CTLA-4 blocking monoclonal antibodies as a means to optimize bladder cancer therapy

CTLA-4 blockade as well as CD40 agonistic therapy comes with adverse events when administrated systemically to patients. Whereas anti-CD40 therapy is associated with both cytokine release and liver toxicity, CTLA-4 blockade leads to auto-immune manifestations. Cytokine release affects the maximum tolerated dose (MTD) for anti-CD40, thereby hampering anti-tumor responses. Whereas patients with localized bladder cancer respond to immunotherapy in the form of BCG, they suffer high relapse frequencies and toxicity. Patients with a more advanced disease have a poor prognosis due to relapses, and not all patients are fit to undergo radical cystectomy, mainly due to age, underlying diseases and health status. There is also a high tumor recurrence (50% of cystectomy patients relapse with local or metastatic tumor growth). We believe these patients could benefit from local immunotherapy similar to BCG instillations, but focusing on strengthening the adaptive immune response rather than the innate. In solid cancer where an accessible tumor lesion is available, monoclonal antibody therapies targeting our immune cells can be administrated intralesionally (1). In a preclinical bladder cancer model we show that both anti-CD40 and anti-CTLA-4 therapy can be used in a lower dose with a peritumoral injection route (2, 3 and current work). Local anti-CD40 therapy partly limits systemic spread of the antibody and the efficacy is dependent on the presence of tumor antigens at the site of the location. CD8+ T cells are the main effectors cells causing tumor regression (3). Herein we initially assessed standard orthotopic instillation of CTLA-4 directed antibody therapy, but drug uptake was poor despite pre-conditioning with clorpactin. As an alternative we slightly modified our existing MB49 syngeneic tumor model based (by injecting tumor cells into the submucosal space of the anterior bladder wall), and made use of ultrasound-guided intratumoral anti-CTLA-4 antibody treatment. The results show tumor regression followed by a more than 10-fold reduction in systemic antibody levels as compared to intravenous administration, in line with the compartmentally restrained nature of the bladder. In addition, local anti-CTLA-4 therapy, when complemented by systemic anti-PD1 therapy for subcutaneously growing tumors, demonstrated CR in 7/8 animals, superior to each therapy alone. Our results demonstrate that local anti-CTLA-4 antibody therapy is equally effective as systemic administration, but reduces systemic antibody levels and that its combination with anti-PD1 therapy displays superior outcome. The clinical use of spasm-relieving injections in the bladder illustrates the feasibility to perform injections in clinical routine, and intralesional injections of a check-point inhibitor prior to surgery could boost the adaptive immune response and decrease relapse frequencies without the risk of adverse events delaying a planned surgery. 1. NCT02379741 (clinicaltrials.gov June 2016) 2. Sandin et al. Cancer Immunol Res. 2014 Jan;2(1) 3. Mangsbo et al. Clin Cancer Res. 2015 Mar 1;21(5) Citation Format: Luuk van Hooren, Linda Sandin, Igor Moskalev, Peter Ellmark, Anna Dimberg, Peter Black, Thomas H. Totterman, Sara M. Mangsbo. Intralesional administration of CTLA-4 blocking monoclonal antibodies as a means to optimize bladder cancer therapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B103.