Neoantigen-targeted dendritic cell vaccination in lung cancer patients induces long-lived T cells exhibiting the full differentiation spectrum.

Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. Here, we present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient-individual neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible in six of 10 enrolled patients. Toxicity is limited to grade 1-2 adverse events. Systemic T cell responses are observed in five out of six vaccinated patients, with T cell responses remaining detectable up to 19 months post vaccination. Single-cell analysis indicates that the responsive T cell population is polyclonal and exhibits the near-entire spectrum of T cell differentiation states, including a naive-like state, but excluding exhausted cell states. Three of six vaccinated patients experience disease recurrence during the follow-up period of 2 years. Collectively, these data support the feasibility, safety, and immunogenicity of this treatment in resected NSCLC.

A bispecific Clec9A-PD-L1 targeted type I interferon profoundly reshapes the tumor microenvironment towards an antitumor state.

Despite major improvements in immunotherapeutic strategies, the immunosuppressive tumor microenvironment remains a major obstacle for the induction of efficient antitumor responses. In this study, we show that local delivery of a bispecific Clec9A-PD-L1 targeted type I interferon (AcTaferon, AFN) overcomes this hurdle by reshaping the tumor immune landscape.Treatment with the bispecific AFN resulted in the presence of pro-immunogenic tumor-associated macrophages and neutrophils, increased motility and maturation profile of cDC1 and presence of inflammatory cDC2. Moreover, we report empowered diversity in the CD8+ T cell repertoire and induction of a shift from naive, dysfunctional CD8+ T cells towards effector, plastic cytotoxic T lymphocytes together with increased presence of NK and NKT cells as well as decreased regulatory T cell levels. These dynamic changes were associated with potent antitumor activity. Tumor clearance and immunological memory, therapeutic immunity on large established tumors and blunted tumor growth at distant sites were obtained upon co-administration of a non-curative dose of chemotherapy.Overall, this study illuminates further application of type I interferon as a safe and efficient way to reshape the suppressive tumor microenvironment and induce potent antitumor immunity; features which are of major importance in overcoming the development of metastases and tumor cell resistance to immune attack. The strategy described here has potential for application across to a broad range of cancer types.

Selective IL-1 activity on CD8+ T cells empowers antitumor immunity and synergizes with neovasculature-targeted TNF for full tumor eradication.

BACKGROUND: Clinical success of therapeutic cancer vaccines depends on the ability to mount strong and durable antitumor T cell responses. To achieve this, potent cellular adjuvants are highly needed. Interleukin-1ß (IL-1ß) acts on CD8+ T cells and promotes their expansion and effector differentiation, but toxicity and undesired tumor-promoting side effects hamper efficient clinical application of this cytokine. METHODS: This 'cytokine problem' can be solved by use of AcTakines (Activity-on-Target cytokines), which represent fusions between low-activity cytokine mutants and cell type-specific single-domain antibodies. AcTakines deliver cytokine activity to a priori selected cell types and as such evade toxicity and unwanted off-target side effects. Here, we employ subcutaneous melanoma and lung carcinoma models to evaluate the antitumor effects of AcTakines. RESULTS: In this work, we use an IL-1ß-based AcTakine to drive proliferation and effector functionality of antitumor CD8+ T cells without inducing measurable toxicity. AcTakine treatment enhances diversity of the T cell receptor repertoire and empowers adoptive T cell transfer. Combination treatment with a neovasculature-targeted tumor necrosis factor (TNF) AcTakine mediates full tumor eradication and establishes immunological memory that protects against secondary tumor challenge. Interferon-γ was found to empower this AcTakine synergy by sensitizing the tumor microenvironment to TNF. CONCLUSIONS: Our data illustrate that anticancer cellular immunity can be safely promoted with an IL-1ß-based AcTakine, which synergizes with other immunotherapies for efficient tumor destruction.

Targeting IFN activity to both B cells and plasmacytoid dendritic cells induces a robust tolerogenic response and protection against EAE.

Type I Interferon (IFN) was the very first drug approved for the treatment of Multiple Sclerosis (MS), and is still frequently used as a first line therapy. However, systemic IFN also causes considerable side effects, affecting therapy adherence and dose escalation. In addition, the mechanism of action of IFN in MS is multifactorial and still not completely understood. Using AcTaferons (Activity-on-Target IFNs, AFNs), optimized IFN-based immunocytokines that allow cell-specific targeting, we have previously demonstrated that specific targeting of IFN activity to dendritic cells (DCs) can protect against experimental autoimmune encephalitis (EAE), inducing in vivo tolerogenic protective effects, evidenced by increased indoleamine-2,3-dioxygenase (IDO) and transforming growth factor ß (TGFß) release by plasmacytoid (p) DCs and improved immunosuppressive capacity of regulatory T and B cells. We here report that targeting type I IFN activity specifically towards B cells also provides strong protection against EAE, and that targeting pDCs using SiglecH-AFN can significantly add to this protective effect. The superior protection achieved by simultaneous targeting of both B lymphocytes and pDCs correlated with improved IL-10 responses in B cells and conventional cDCs, and with a previously unseen very robust IDO response in several cells, including all B and T lymphocytes, cDC1 and cDC2.

mRNA in cancer immunotherapy: beyond a source of antigen.

mRNA therapeutics have become the focus of molecular medicine research. Various mRNA applications have reached major milestones at high speed in the immuno-oncology field. This can be attributed to the knowledge that mRNA is one of nature's core building blocks carrying important information and can be considered as a powerful vector for delivery of therapeutic proteins to the patient.For a long time, the major focus in the use of in vitro transcribed mRNA was on development of cancer vaccines, using mRNA encoding tumor antigens to modify dendritic cells ex vivo. However, the versatility of mRNA and its many advantages have paved the path beyond this application. In addition, due to smart design of both the structural properties of the mRNA molecule as well as pharmaceutical formulations that improve its in vivo stability and selective targeting, the therapeutic potential of mRNA can be considered as endless.As a consequence, many novel immunotherapeutic strategies focus on the use of mRNA beyond its use as the source of tumor antigens. This review aims to summarize the state-of-the-art on these applications and to provide a rationale for their clinical application.

The Opposing Effect of Type I IFN on the T Cell Response by Non-modified mRNA-Lipoplex Vaccines Is Determined by the Route of Administration.

mRNA-lipoplex vaccines are currently being explored in phase II clinical trials for the treatment of patients with advanced solid tumors. Mechanistically, these mRNA-lipoplex vaccines are characterized by the induction of type I interferon (IFN) centered innate responses. Earlier studies have identified type I IFNs as major regulators of the T cell response instigated by mRNA-lipoplex vaccines. However, stimulatory or, in contrast, profound inhibitory effects of type I IFNs were described depending on the study. In this mouse study, we demonstrated that the opposing roles of type I IFN signaling on the magnitude of the vaccine-evoked T cell responses is dependent on the route of mRNA-lipoplex administration and is regulated at the level of the T cells rather than indirectly through modulation of dendritic cell function. This study helps to understand the double-edged sword character of type I IFN induction upon mRNA-based vaccine treatment and may contribute to a more rational design of mRNA vaccination regimens.

Specific targeting of IL-1ß activity to CD8+ T cells allows for safe use as a vaccine adjuvant.

Annual administration and reformulation of influenza vaccines is required for protection against seasonal infections. However, the induction of strong and long-lasting T cells is critical to reach broad and potentially lifelong antiviral immunity. The NLRP3 inflammasome and its product interleukin-1ß (IL-1ß) are pivotal mediators of cellular immune responses to influenza, yet, overactivation of these systems leads to side effects, which hamper clinical applications. Here, we present a bypass around these toxicities by targeting the activity of IL-1ß to CD8+ T cells. Using this approach, we demonstrate safe inclusion of IL-1ß as an adjuvant in vaccination strategies, leading to full protection of mice against a high influenza virus challenge dose by raising potent T cell responses. In conclusion, this paper proposes a class of IL-1ß-based vaccine adjuvants and also provides further insight in the mechanics of cellular immune responses driven by IL-1ß.

Safe eradication of large established tumors using neovasculature-targeted tumor necrosis factor-based therapies.

Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF) as an anticancer agent. Activity-on-Target cytokines (AcTakines) are a novel class of immunocytokines with improved therapeutic index. A TNF-based AcTakine targeted to CD13 enables selective activation of the tumor neovasculature without any detectable toxicity in vivo. Upregulation of adhesion markers supports enhanced T-cell infiltration leading to control or elimination of solid tumors by, respectively, CAR T cells or a combination therapy with CD8-targeted type I interferon AcTakine. Co-treatment with a CD13-targeted type II interferon AcTakine leads to very rapid destruction of the tumor neovasculature and complete regression of large, established tumors. As no tumor markers are needed, safe and efficacious elimination of a broad range of tumor types becomes feasible.

Dendritic Cell-Based Immunotherapy in Lung Cancer.

Lung cancer remains the leading cause of cancer-related death worldwide. The advent of immune checkpoint inhibitors has led to a paradigm shift in the treatment of metastatic non-small cell and small cell lung cancer. However, despite prolonged overall survival, only a minority of the patients derive clinical benefit from these treatments suggesting that the full anti-tumoral potential of the immune system is not being harnessed yet. One way to overcome this problem is to combine immune checkpoint blockade with different strategies aimed at inducing or restoring cellular immunity in a tumor-specific, robust, and durable way. Owing to their unique capacity to initiate and regulate T cell responses, dendritic cells have been extensively explored as tools for immunotherapy in many tumors, including lung cancer. In this review, we provide an update on the nearly twenty years of experience with dendritic cell-based immunotherapy in lung cancer. We summarize the main results from the early phase trials and give an overview of the future perspectives within this field.

A novel leptin receptor antagonist uncouples leptin's metabolic and immune functions.

Leptin links body energy stores to high energy demanding processes like reproduction and immunity. Based on leptin's role in autoimmune diseases and cancer, several leptin and leptin receptor (LR) antagonists have been developed, but these intrinsically lead to unwanted weight gain. Here, we report on the uncoupling of leptin's metabolic and immune functions based on the cross talk with the epidermal growth factor receptor (EGFR). We show that both receptors spontaneously interact and, remarkably, that this complex can partially overrule the lack of LR activation by a leptin antagonistic mutein. Moreover, this leptin mutant induces EGFR phosphorylation comparable to wild-type leptin. Exploiting this non-canonical leptin signalling pathway, we identified a camelid single-domain antibody that selectively inhibits this LR-EGFR cross talk without interfering with homotypic LR signalling. Administration in vivo showed that this single-domain antibody did not interfere with leptin's metabolic functions, but could reverse the leptin-driven protection against starvation-induced thymic and splenic atrophy. These findings offer new opportunities for the design and clinical application of selective leptin and LR antagonists that avoid unwanted metabolic side effects.

Targeting interferon activity to dendritic cells enables in vivo tolerization and protection against EAE in mice.

Type I Interferon (IFN) is widely used for multiple sclerosis (MS) treatment, but its side effects are limiting and its mechanism of action still unknown. Furthermore, 30-50% of MS patients are unresponsive, and IFN can even induce relapses. Fundamental understanding of the cellular target(s) of IFN will help to optimize treatments by reducing side effects and separating beneficial from detrimental effects. To improve clinical systemic IFN usage, we are developing AcTaferons (Activity-on-Target IFNs = AFNs), optimized IFN-based immunocytokines that allow cell-specific targeting. In experimental autoimmune encephalitis (EAE) in mice, high dose WT mIFNα could delay disease, but caused mortality and severe hematological deficits. In contrast, AFN targeted to dendritic cells (DC, via Clec9A) protected without mortality or hematological consequences. Conversely, CD8-targeted AFN did not protect and exacerbated weight loss, indicating the presence of both protective and unfavorable IFN effects in EAE. Comparing Clec9A-, XCR1-and SiglecH-targeting, we found that targeting AFN to plasmacytoid (p) and conventional (c) DC is superior and non-toxic compared to WT mIFN. DC-targeted AFN increased pDC numbers and their tolerogenic potential, evidenced by increased TGFß and IDO synthesis and regulatory T cell induction. In addition, both regulatory T and B cells produced significantly more immunosuppressive TGFß and IL-10. In conclusion, specific DC-targeting of IFN activity induces a robust in vivo tolerization, efficiently protecting against EAE, without noticeable side effects. Thus, dissecting positive and negative IFN effects via cell-specific targeting may result in better and safer MS therapy and response rates.

Nanoparticle-Conjugate TLR7/8 Agonist Localized Immunotherapy Provokes Safe Antitumoral Responses.

Localized therapeutic modalities that subvert the tumor microenvironment from immune-suppressive to pro-immunogenic can elicit systemic antitumor immune responses that induce regression of directly treated as well as nontreated distal tumors. A key toward generating robust antitumor T cell responses is the activation of dendritic cells (DCs) in the tumor microenvironment. Treatment with agonists triggering various pattern recognition receptors is very efficient to activate DCs, yet suffers from the induction of serious immune-related adverse effects, which is closely linked to their unfavorable PK/PD profile causing systemic immune activation and cytokine release. Here, it is reported that nanoparticle conjugation of a highly potent TLR7/8 agonist restricts immune activation to the tumor bed and its sentinel lymph nodes without hampering therapeutic antitumor efficacy. On a mechanistic level, it is confirmed that localized treatment with a nanoparticle-conjugated TLR7/8 agonist leads to potent activation of DCs in the sentinel lymph nodes and promotes proliferation of tumor antigen-specific CD8 T cells. Furthermore, therapeutic improvement upon combination with anti-PDL1 checkpoint inhibition and Flt3L, a growth factor that expands and mobilizes DCs from the bone marrow, is demonstrated. The findings provide a rational base for localized tumor engineering by nanomedicine strategies that provide spatial control over immune-activation.

Treatment with mRNA coding for the necroptosis mediator MLKL induces antitumor immunity directed against neo-epitopes.

Cancer immunotherapy can induce durable antitumor responses. However, many patients poorly respond to such therapies. Here we describe a generic antitumor therapy that is based on the intratumor delivery of mRNA that codes for the necroptosis executioner mixed lineage kinase domain-like (MLKL) protein. This intervention stalls primary tumor growth and protects against distal and disseminated tumor formation in syngeneic mouse melanoma and colon carcinoma models. Moreover, MLKL-mRNA treatment combined with immune checkpoint blockade further improves the antitumor activity. MLKL-mRNA treatment rapidly induces T cell responses directed against tumor neo-antigens and requires CD4+ and CD8+ T cells to prevent tumor growth. Type I interferon signaling and Batf3-dependent dendritic cells are essential for this mRNA treatment to elicit tumor antigen-specific T cell responses. Moreover, MLKL-mRNA treatment blunts the growth of human lymphoma in mice with a reconstituted human adaptive immune system. MLKL-based treatment can thus be exploited as an effective antitumor immunotherapy.

A safe and highly efficient tumor-targeted type I interferon immunotherapy depends on the tumor microenvironment.

Despite approval for the treatment of various malignancies, clinical application of cytokines such as type I interferon (IFN) is severely impeded by their systemic toxicity. AcTakines (Activity-on-Target cytokines) are optimized immunocytokines that, when injected in mice, only reveal their activity upon cell-specific impact. We here show that type I IFN-derived AcTaferon targeted to the tumor displays strong antitumor activity without any associated toxicity, in contrast with wild type IFN. Treatment with CD20-targeted AcTaferon of CD20+ lymphoma tumors or melanoma tumors engineered to be CD20+, drastically reduced tumor growth. This antitumor effect was completely lost in IFNAR- or Batf3-deficient mice, and depended on IFN signaling in conventional dendritic cells. Also the presence of, but not the IFN signaling in, CD8+ T lymphocytes was critical for proficient antitumor effects. When combined with immunogenic chemotherapy, low-dose TNF, or immune checkpoint blockade strategies such as anti-PDL1, anti-CTLA4 or anti-LAG3, complete tumor regressions and subsequent immunity (memory) were observed, still without any concomitant morbidity, again in sharp contrast with wild type IFN. Interestingly, the combination therapy of tumor-targeted AcTaferon with checkpoint inhibiting antibodies indicated its ability to convert nonresponding tumors into responders. Collectively, our findings demonstrate that AcTaferon targeted to tumor-specific surface markers may provide a safe and generic addition to cancer (immuno)therapies.

Delivering Type I Interferon to Dendritic Cells Empowers Tumor Eradication and Immune Combination Treatments.

An ideal generic cancer immunotherapy should mobilize the immune system to destroy tumor cells without harming healthy cells and remain active in case of recurrence. Furthermore, it should preferably not rely on tumor-specific surface markers, as these are only available in a limited set of malignancies. Despite approval for treatment of various cancers, clinical application of cytokines is still impeded by their multiple toxic side effects. Type I IFN has a long history in the treatment of cancer, but its multifaceted activity pattern and complex side effects prevent its clinical use. Here we develop AcTakines (Activity-on-Target cytokines), optimized (mutated) immunocytokines that are up to 1,000-fold more potent on target cells, allowing specific signaling in selected cell types only. Type I IFN-derived AcTaferon (AFN)-targeting Clec9A+ dendritic cells (DC) displayed strong antitumor activity in murine melanoma, breast carcinoma, and lymphoma models and against human lymphoma in humanized mice without any detectable toxic side effects. Combined with immune checkpoint blockade, chemotherapy, or low-dose TNF, complete tumor regression and long-lasting tumor immunity were observed, still without adverse effects. Our findings indicate that DC-targeted AFNs provide a novel class of highly efficient, safe, and broad-spectrum off-the-shelf cancer immunotherapeutics with no need for a tumor marker.Significance: Targeted type I interferon elicits powerful antitumor efficacy, similar to wild-type IFN, but without any toxic side effects. Cancer Res; 78(2); 463-74. ©2017 AACR.

Type I Interferons Interfere with the Capacity of mRNA Lipoplex Vaccines to Elicit Cytolytic T Cell Responses.

Given their high potential to evoke cytolytic T cell responses, tumor antigen-encoding messenger RNA (mRNA) vaccines are now being intensively explored as therapeutic cancer vaccines. mRNA vaccines clearly benefit from wrapping the mRNA into nano-sized carriers such as lipoplexes that protect the mRNA from degradation and increase its uptake by dendritic cells in vivo. Nevertheless, the early innate host factors that regulate the induction of cytolytic T cells to mRNA lipoplex vaccines have remained unresolved. Here, we demonstrate that mRNA lipoplexes induce a potent type I interferon (IFN) response upon subcutaneous, intradermal and intranodal injection. Regardless of the route of immunization applied, these type I IFNs interfered with the generation of potent cytolytic T cell responses. Most importantly, blocking type I IFN signaling at the site of immunization through the use of an IFNAR blocking antibody greatly enhanced the prophylactic and therapeutic antitumor efficacy of mRNA lipoplexes in the highly aggressive B16 melanoma model. As type I IFN induction appears to be inherent to the mRNA itself rather than to unique properties of the mRNA lipoplex formulation, preventing type I IFN induction and/or IFNAR signaling at the site of immunization might constitute a widely applicable strategy to improve the potency of mRNA vaccination.

Intratumoral Delivery of TriMix mRNA Results in T-cell Activation by Cross-Presenting Dendritic Cells.

Modulating the activity of tumor-infiltrating dendritic cells (TiDC) provides opportunities for novel cancer interventions. In this article, we report on our study of the uptake of mRNA by CD8α(+) cross-presenting TiDCs upon its intratumoral (i.t.) delivery. We exploited this property to deliver mRNA encoding the costimulatory molecule CD70, the activation stimuli CD40 ligand, and constitutively active Toll-like receptor 4, referred to as TriMix mRNA. We show that TiDCs are reprogrammed to mature antigen-presenting cells that migrate to tumor-draining lymph nodes (TDLN). TriMix stimulated antitumor T-cell responses to spontaneously engulfed cancer antigens, including a neoepitope. We show in various mouse cancer models that i.t. delivery of TriMix mRNA results in systemic therapeutic antitumor immunity. Finally, we show that the induction of antitumor responses critically depends on TiDCs, whereas it only partially depends on TDLNs. As such, we provide a platform and a mechanistic rationale for the clinical testing of i.t. administration of TriMix mRNA.

Intratumoral delivery of mRNA: Overcoming obstacles for effective immunotherapy.

The immunosuppressive tumor microenvironment (TME) is a major obstacle in cancer immunotherapy. Therefore, it has gained attention as a target site. mRNA emerged as a versatile drug class for cancer therapy. We reported that intratumoral administration of mRNA encoding the fusokine Fß2 supports tumor-specific T-cell immunity. This study provides proof of concept of the use of mRNA to modulate the TME.

Targeting the tumor microenvironment to enhance antitumor immune responses.

The identification of tumor-specific antigens and the immune responses directed against them has instigated the development of therapies to enhance antitumor immune responses. Most of these cancer immunotherapies are administered systemically rather than directly to tumors. Nonetheless, numerous studies have demonstrated that intratumoral therapy is an attractive approach, both for immunization and immunomodulation purposes. Injection, recruitment and/or activation of antigen-presenting cells in the tumor nest have been extensively studied as strategies to cross-prime immune responses. Moreover, delivery of stimulatory cytokines, blockade of inhibitory cytokines and immune checkpoint blockade have been explored to restore immunological fitness at the tumor site. These tumor-targeted therapies have the potential to induce systemic immunity without the toxicity that is often associated with systemic treatments. We review the most promising intratumoral immunotherapies, how these affect systemic antitumor immunity such that disseminated tumor cells are eliminated, and which approaches have been proven successful in animal models and patients.

The ReNAissanCe of mRNA-based cancer therapy.

About 25 years ago, mRNA became a tool of interest in anticancer vaccination approaches. However, due to its rapid degradation in situ, direct application of mRNA was confronted with considerable skepticism during its early use. Consequently, mRNA was for a long time mainly used for the ex vivo transfection of dendritic cells, professional antigen-presenting cells known to stimulate immunity. The interest in direct application of mRNA experienced a revival, as researchers became aware of the many advantages mRNA offers. Today, mRNA is considered to be an ideal vehicle for the induction of strong immune responses against cancer. The growing numbers of preclinical trials and as a consequence the increasing clinical application of mRNA as an off-the-shelf anticancer vaccine signifies a renaissance for transcript-based antitumor therapy. In this review, we highlight this renaissance using a timeline providing all milestones in the application of mRNA for anticancer vaccination.