Immunotherapy against malignant brain tumors - of mice and men

Glioblastoma Multiforme (GBM) is the most abundant and most aggressive primary brain tumor in adults. Due to the infiltrative growth of the tumor, surgery will never be radical. Radiation and chemotherapy only marginally improve the poor prognosis due to the ability of the GBM to develop resistance towards these treatments. Therefore it is of great importance to find new efficient treatment modalities. The aim with this thesis was to develop and evaluate a cytokine based immunotherapy as treatment for GBM in both an experimental mouse glioma model as well as in patients suffering from GBM. In paper I we demonstrate the establishment of a GM-CSF and IFNγ based immunotherapy in the mouse glioma model, GL261. Mice with intracranial gliomas were immunized intraperitoneally with GL261 cell genetically modified to produce GM-CSF combined with recombinant IFNγ. This combination synergistically enhanced survival to 90%. In paper II-III we investigated the immune responses elicited by these immunizations both systemically as well as locally in the tumor. We found that the immunizations with GM-CSF and IFNγ were highly dependent on T-cells for mediating survival of the mice. In paper IV we monitored the immune responses elicited in GBM patients receiving IFNγ based immunotherapy using ELIspot and CBA. Immunotherapy enhances the patient’s own antitumoral immune responses otherwise suppressed by the tumor. We believe that this treatment, in combination with conventional treatments such as surgery, radiotherapy and chemotherapy, has a great promise for the future treatment of patients with GBM

Rationale and clinical development of CD40 agonistic antibodies for cancer immunotherapy

Introduction: CD40 signaling activates dendritic cells leading to improved T cell priming against tumor antigens. CD40 agonism expands the tumor-specific T cell repertoire and has the potential to increase the fraction of patients that respond to established immunotherapies. Areas covered: This article reviews current as well as emerging CD40 agonist therapies with a focus on antibody-based therapies, including next generation bispecific CD40 agonists. The scientific rationale for different design criteria, binding epitopes, and formats are discussed. Expert opinion: The ability of CD40 agonists to activate dendritic cells and enhance antigen cross-presentation to CD8+ T cells provides an opportunity to elevate response rates of cancer immunotherapies. While there are many challenges left to address, including optimal dose regimen, CD40 agonist profile, combination partners and indications, we are confident that CD40 agonists will play an important role in the challenging task of reprogramming the immune system to fight cancer

Activation of purified allogeneic CD4(+) T cells by rat bone marrow-derived dendritic cells induces concurrent secretion of IFN-gamma, IL-4, and IL-10.

Dendritic cells (DCs) are highly specialized antigen-presenting cells that play a key role in the initiation and regulation of immune responses. The ability of DCs to process antigens and the outcome of their interaction with T cells are largely dependent on phenotype as well as maturation state of DCs. In this study, we generated DCs from rat bone marrow precursors. Bone marrow cells cultured in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, and Flt-3 ligand (FL) produced immature DCs that expressed intermediate levels of major histocompatibility complex (MHC) class II, low levels of CD80 and CD86 molecules and displayed a high capacity of endocytosis. Bone marrow-derived DCs (BMDCs) matured in the presence of lipopolysaccharide (LPS) upregulated expression of MHC class II, CD80 and CD86, while their phagocytic capacity was dramatically reduced. Mature BMDCs stimulated vigorous proliferation of purified allogeneic CD4(+) T cells in a primary mixed leukocyte reaction (MLR) and elicited a mixed cytokine profile in allogeneic CD4(+) T cells: DCs activated CD4(+) T cells to produce interferon (IFN)-gamma, IL-4, and IL-10. Thus, rat BMDCs effectively internalize antigens and stimulate T cell proliferation but fail to induce an unidirectional polarization of T helper (T-H) cells and in this respect differ from both human and mouse DCs. (c) 2005 Elsevier B.V. All rights reserved

The human anti-CD40 agonist antibody mitazalimab (ADC-1013; JNJ-64457107) activates antigen-presenting cells, improves expansion of antigen-specific T cells, and enhances anti-tumor efficacy of a model cancer vaccine in vivo

Non-responders to checkpoint inhibitors generally have low tumor T cell infiltration and could benefit from immunotherapy that activates dendritic cells, with priming of tumor-reactive T cells as a result. Such therapies may be augmented by providing tumor antigen in the form of cancer vaccines. Our aim was to study the effects of mitazalimab (ADC-1013; JNJ-64457107), a human anti-CD40 agonist IgG1 antibody, on activation of antigen-presenting cells, and how this influences the priming and anti-tumor potential of antigen-specific T cells, in mice transgenic for human CD40. Mitazalimab activated splenic CD11c+ MHCII+ dendritic cells and CD19+ MHCII+ B cells within 6 h, with a return to baseline within 1 week. This was associated with a dose-dependent release of proinflammatory cytokines in the blood, including IP-10, MIP-1α and TNF-α. Mitazalimab administered at different dose regimens with ovalbumin protein showed that repeated dosing expanded ovalbumin peptide (SIINFEKL)-specific CD8+ T cells and increased the frequency of activated ICOS+ T cells and CD44hi CD62L− effector memory T cells in the spleen. Mitazalimab prolonged survival of mice bearing MB49 bladder carcinoma tumors and increased the frequency of activated granzyme B+ CD8+ T cells in the tumor. In the ovalbumin-transfected tumor E.G7-OVA lymphoma, mitazalimab administered with either ovalbumin protein or SIINFEKL peptide prolonged the survival of E.G7-OVA tumor-bearing mice, as prophylactic and therapeutic treatment. Thus, mitazalimab activates antigen-presenting cells, which improves expansion and activation of antigen-specific T cells and enhances the anti-tumor efficacy of a model cancer vaccine

Postimmunization with IFN-gamma-secreting glioma cells combined with the inducible nitric oxide synthase inhibitor mercaptoethylguanidine prolongs survival of rats with intracerebral tumors

High-grade gliomas are one of the most aggressive human tumors with <1% of patients surviving 5 years after surgery. Immunotherapy could offer a possibility to eradicate remnant tumor cells after conventional therapy. Experimental immunotherapy can induce partial cure of established intracerebral tumors in several rodent models. One reason for the limited therapeutic effects could be immunosuppression induced by both the growing tumor and the induced immune reaction. NO has been implicated in tumor-derived immune suppression in tumor-bearing hosts, and unspecific inhibitors of NO synthase have been shown to boost antitumor immunity. In this study, we show that the inducible NO synthase (iNOS)-specific inhibitor mercaptoethylguanidine (MEG) superiorly enhanced lymphocyte reactivity after polyclonal stimulation compared with the iNOS-specific inhibitor L-NIL and the unspecific NO synthase inhibitor L-NAME. Both iNOS inhibitors increased the number and proliferation of T cells but not of B cells. When combined during postimmunization with IFN-gamma-secreting N32 rat glioma cells of rats harboring intracerebral tumors, only MEG increased the cure rate. However, this was only achieved when MEG was administered after immunizations. These findings implicate that NO has both enhancing and suppressive effects after active immunotherapy

The CTLA-4 x OX40 bispecific antibody ATOR-1015 induces anti-tumor effects through tumor-directed immune activation

Abstract Background The CTLA-4 blocking antibody ipilimumab has demonstrated substantial and durable effects in patients with melanoma. While CTLA-4 therapy, both as monotherapy and in combination with PD-1 targeting therapies, has great potential in many indications, the toxicities of the current treatment regimens may limit their use. Thus, there is a medical need for new CTLA-4 targeting therapies with improved benefit-risk profile. Methods ATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody generated by linking an optimized version of the Ig-like V-type domain of human CD86, a natural CTLA-4 ligand, to an agonistic OX40 antibody. In vitro evaluation of T-cell activation and T regulatory cell (Treg) depletion was performed using purified cells from healthy human donors or cell lines. In vivo anti-tumor responses were studied using human OX40 transgenic (knock-in) mice with established syngeneic tumors. Tumors and spleens from treated mice were analyzed for CD8+ T cell and Treg frequencies, T-cell activation markers and tumor localization using flow cytometry. Results ATOR-1015 induces T-cell activation and Treg depletion in vitro. Treatment with ATOR-1015 reduces tumor growth and improves survival in several syngeneic tumor models, including bladder, colon and pancreas cancer models. It is further demonstrated that ATOR-1015 induces tumor-specific and long-term immunological memory and enhances the response to PD-1 inhibition. Moreover, ATOR-1015 localizes to the tumor area where it reduces the frequency of Tregs and increases the number and activation of CD8+ T cells. Conclusions By targeting CTLA-4 and OX40 simultaneously, ATOR-1015 is directed to the tumor area where it induces enhanced immune activation, and thus has the potential to be a next generation CTLA-4 targeting therapy with improved clinical efficacy and reduced toxicity. ATOR-1015 is also expected to act synergistically with anti-PD-1/PD-L1 therapy. The pre-clinical data support clinical development of ATOR-1015, and a first-in-human trial has started (NCT03782467)