An Engineered IFNγ-Antibody Fusion Protein with Improved Tumor-Homing Properties

Interferon-gamma (IFNγ) is one of the central cytokines produced by the innate and adaptive immune systems. IFNγ directly favors tumor growth control by enhancing the immunogenicity of tumor cells, induces IP-10 secretion facilitating (CXCR3+) immune cell infiltration, and can prime macrophages to an M1-like phenotype inducing proinflammatory cytokine release. We had previously reported that the targeted delivery of IFNγ to neoplastic lesions may be limited by the trapping of IFNγ-based products by cognate receptors found in different organs. Here we describe a novel fusion protein consisting of the L19 antibody, specific to the alternatively spliced extra-domain B of fibronectin (EDB), fused to a variant of IFNγ with reduced affinity to its cognate receptor. The product (named L19-IFNγ KRG) selectively localized to tumors in mice, showed favorable pharmacokinetic profiles in monkeys and regained biological activity upon antigen binding. The fusion protein was investigated in two murine models of cancer, both as monotherapy and in combination with therapeutic modalities which are frequently used for cancer therapy. L19-IFNγ KRG induced tumor growth retardation and increased the intratumoral concentration of T cells and NK cells in combination with anti-PD-1

Generation and in vivo validation of an IL-12 fusion protein based on a novel anti-human FAP monoclonal antibody

BACKGROUND In this study, we describe the generation of a fully human monoclonal antibody (named '7NP2') targeting human fibroblast activation protein (FAP), an antigen expressed in the microenvironment of different types of solid neoplasms. METHODS 7NP2 was isolated from a synthetic antibody phage display library and was improved by one round of mutagenesis-based affinity maturation. The tumor recognition properties of the antibody were validated by immunofluorescence procedures performed on cancer biopsies from human patients. A fusion protein consisting of the 7NP2 antibody linked to interleukin (IL)-12 was generated and the anticancer activity of the murine surrogate product (named mIL12-7NP2) was evaluated in mouse models. Furthermore, the safety of the fully human product (named IL12-7NP2) was evaluated in Cynomolgus monkeys. RESULTS Biodistribution analysis in tumor-bearing mice confirmed the ability of the product to selectively localize to solid tumors while sparing healthy organs. Encouraged by these results, therapy studies were conducted in vivo, showing a potent antitumor activity in immunocompetent and immunodeficient mouse models of cancer, both as single agent and in combination with immune checkpoint inhibitors. The fully human product was tolerated when administered to non-human primates. CONCLUSIONS The results obtained in this work provided a rationale for future clinical translation activities using IL12-7NP2

Design and Characterization of Novel Antibody-Cytokine Fusion Proteins Based on Interleukin-21

Interleukin-21 (IL21) is a pleiotropic cytokine involved in the modulation of both innate and adaptive immunity. IL21 is mainly secreted by natural killer (NK) and activated CD4+ T-cells. The biology of this cytokine can be associated to proinflammatory responses reflecting its potent stimulatory activity of NK and CD8+ T-cells. Here we describe four formats of novel IL21-based antibody–cytokine fusion proteins, targeting the extra domain A (EDA) of fibronectin and explore their potential for cancer treatment. The fusion proteins were designed, expressed, and characterized. F8 in single-chain diabody (scDb) format fused to IL21 at its C-terminus exhibited a promising profile in size exclusion chromatography (SEC) and SDS-PAGE. The lead candidate was further characterized in vitro. A cell-based activity assay on murine cytotoxic T-cells showed that human IL21, compared to murine IL21 partially cross-reacted with the murine receptor. The prototype was able to recognize EDA as demonstrated by immunofluorescence analysis on tumor sections. In an in vivo quantitative biodistribution experiment, F8(scDb)-murine IL21 did not preferentially accumulate at the site of disease after intravenous injection, suggesting that additional protein engineering would be required to improve the tumor-homing properties of IL21-based product

Generation and Characterization of Antibody-Cytokine Fusion Proteins Targeting the Tumor Neovasculature

Over the last century, the pharmacological treatment of cancer has mainly relied on chemotherapy. However, conventional chemotherapeutics suffer from substantial limitations, as those agents kill rapidly proliferating cells in a non-specific manner, without a sufficiently selective distinction between malignant masses and healthy tissues. Nuclear medicine studies have shown that the majority of chemotherapeutic agents do not preferentially localise to neoplastic tissues after intravenous administration, but rather accumulate in certain normal organs, such as liver, spleen or kidneys. These suboptimal pharmacokinetic properties may cause severe side effects and prevent escalation to therapeutically active regimens, thus highlighting the need for better and more targeted approaches. In the last decade of the 19th century, the scientist Paul Ehrlich envisioned the development of “magic bullets” (Zauberkugeln): pharmaceutical agents which would selectively target diseased structures in vivo, helping spare normal tissues. This vision stimulated the development of a large number of pharmaceutical strategies, aimed at the implementation of “targeted therapies”. From one perspective, the advent of immunotherapy, which aims at stimulating innate and adaptive immunity against cancer, may represent one embodiment of the magic bullet concept. Alternatively, ligand-based pharmacodelivery approaches (e.g., the use of naked or drug-armoured monoclonal antibodies) may also be considered as practical implementation of Paul Ehrlich’s original vision. In this context, cytotoxic drugs or immunomodulatory agents (e.g., cytokines) could be considered as payloads for antibody-based tumor targeting strategies. Cytokines are proteins capable of modulating the activity of the immune system. In physiological conditions, they are typically present in the bloodstream at very low concentrations (i.e., in the picomolar range), while their values can increase by several orders of magnitude (e.g., by more than 1000-fold) in certain pathological conditions. Recombinant preparations of Interleukin-2 (IL2, ProleukinTM), interferon- (IFN Roferon-ATM), and tumor necrosis factor (TNF, BeromunTM) have been among the first immunotherapies that gained marketing authorization for the treatment of advanced cancers. The systemic administration of pro-inflammatory cytokines typically triggers severe side effects (e.g., hypotension, flu-like symptoms nausea, and vomiting), which are associated with an interaction with cytokine receptors in blood or in secondary lymphoid organs, without a preferential product uptake at the site of disease. This thesis focused on the generation and characterization of antibody-fusion proteins featuring IL21, interferon-gamma (IFNγ) or IL7 as bioactive moieties. The three payloads were fused to two different antibodies targeting components of the modified extracellular matrix (ECM) of solid tumors. These included the fully human F8 and L19 antibodies, originally isolated from phage display libraries, which are specific to the alternatively spliced extra-domain A (EDA) and B (EDB) of fibronectin, respectively. The generation of the immunoconjugates involved the design and expression of numerous molecular formats which were screened based on in vitro functional assays and in vivo tumor-targeting performance, including quantitative biodistribution experiments with radiolabeled protein preparations. The products that exhibited high uptake in the tumor and low accumulation in healthy organs were tested in immunocompetent animal models of cancer, with homing properties and therapeutic data which were particularly promising for fusion proteins based on interferon-gamma or on interleukin-7. Interleukin-21 (IL21) is a pro-inflammatory cytokine that is primarily produced by CD4+ T and NK cells and that activates the immune system after binding to the heterodimeric cognate receptor. Its activity is primarily associated with lymphocyte differentiation, promotion of Th17 type of responses (important to fight fungal and bacterial pathogens), and regulatory T cell (Treg) suppression (a type of cell which dampen immune responses). In preclinical studies, recombinant preparations of ILIL21 exhibited antitumor properties, enhancing CD8+ T-cell activity and promoting long-lasting effector T-cell phenotypes. Clinical trials investigating the recombinant cytokine for the treatment of metastatic melanoma and renal cell carcinoma patients have shown prolonged pharmacodynamic effects supporting immune activation, despite rapid clearance. Strategies to improve IL21's pharmacokinetics involved the generation of fusion proteins associated with IL21 muteins. In pre-clinical models, fusion proteins, such as anti-PD-1 antibodies linked to IL21 mutein, demonstrated favorable pharmacokinetics and pharmacodynamic properties. These results provided the motivation to generate our own novel tumor-targeted IL21 fusion protein, which however revealed suboptimal uptake at the site of disease. More engineering work is required to further improve tumor-homing properties of IL21 fusions. Interferon-gamma (IFNγ) is a type-II interferon primarily produced by immune cells, triggered by T Cell Receptor signaling and by the activity of various cytokines, including IL12, IL2, and IL7. IFNγ play a key role in modulating the immune system, contributing to developing effective antitumor responses in the tumor microenvironment (TME). IFNγ enhances tumor immunogenicity by increasing MHC expression, it induces IP-10 secretion which can facilitate immune cell infiltration and prime macrophages to a pro-inflammatory phenotype. Recombinant IFNγ has proven effective in treating chronic granulomatous disease (CGD) and osteopetrosis, obtaining FDA approval for the treatment of these indications. Early clinical trials in various cancer types reported different results, emphasizing the need for a deeper understanding of IFNγ's context-dependent effects and the potential of protein engineering to optimize its therapeutic application. Preclinical studies previously published by our group on tumor-targeted IFNγ revealed a potential receptor-trapping mechanism that could be titrated at high doses. To circumvent such trapping events, we generated a truncated version of IFNγ fused to the L19 antibody which selectively localized to tumors in mice without any evidence of receptor trapping. The product, termed L19-IFN KRG, was tested in two immunocompetent murine models of cancer alone or in combination with commercially available drugs. Interleukin-7 (IL7) is a soluble tetra helical cytokine of 25kDa primarily produced by stromal and epithelial cells. It engages its receptor, IL7Rα, recruiting the common γc and is involved in the generation and maintenance of long-lived effector memory T-cells. Clinical trials exploring recombinant IL7 (rIL7) in cancer patients have shown its excellent tolerability and no maximum tolerated dose (MTD) has been established so far. Notably, rIL7, in combination with anti-tumor vaccines, has contributed to sustaining elevated lymphocyte levels in a dose-dependent manner. It has been hypothesized that molecular strategies aimed at increasing the concentration of IL7 within the neoplastic mass may be associated with improved clinical outcomes in combination with Immune Checkpoint Blockade (ICB) treatment. Anti-PD-1 antibodies have emerged over the last few years as one of the most important classes of anti-cancer drugs. However, a substantial proportion of patients still do not respond to ICB. IL7R and T Cell Factor 1 (TCF-1) are biomarkers that have been explored to predict responses to ICB treatment in the clinic. More recently, NT-I7, an IL7 Fc fusion with a prolonged half-life, and BiCKI-IL7, a product simultaneously targeting PD-1 and IL7R on T-cells, are novel investigational drugs which aim at enhancing the activity of anti-PD1 antibodies. With this rationale in mind, we generated a novel antibody-IL7 fusion protein that could (i) increase the expression of TCF-1 in human CD8+ T-cells, (ii) efficiently localized to tumors, and (iii) showed enhanced anti-tumor activity when combined with PD-1 blockade in four distinct murine models of cancer including orthotopic gliomas. Collectively, the experiments presented in this thesis reinforce the concept that cytokine payloads may be preferentially delivered to the tumor site by means of fusion proteins with monoclonal antibodies, specific to tumor-associated antigens. Components of the modified extracellular matrix (such as splice variants of fibronectin) are particularly attractive targets, since they are strongly expressed in the majority of solid malignancies and aggressive lymphomas, while being almost undetectable in normal adult tissues

Intracellular label-free detection of mesenchymal stem cell metabolism within a perivascular niche-on-a-chip

The stem cell niche at the perivascular space in human tissue plays a pivotal role in dictating the overallfate of stem cells within it. Mesenchymal stem cells (MSCs) in particular, experience influentialmicroenvironmental conditions, which induce specific metabolic profiles that affect processes of celldifferentiation and dysregulation of the immunomodulatory function. Reports focusing specifically on themetabolic status of MSCs under the effect of pathophysiological stimuli–in terms of flow velocities, shearstresses or oxygen tension–do not model heterogeneous gradients, highlighting the need for moreadvanced models reproducing the metabolic niche. Organ-on-a-chip technology offers the mostadvanced tools for stem cell niche modelling thus allowing for controlled dynamic culture conditions whileprofiling tuneable oxygen tension gradients. However, current systems for live cell detection of metabolicactivity inside microfluidic devices require the integration of microsensors. The presence of suchmicrosensors poses the potential to alter microfluidics and their resolution does not enable intracellularmeasurements but rather a global representation concerning cellular metabolism. Here, we present ametabolic toolbox coupling a miniaturisedin vitrosystem for human-MSCs dynamic culture, which mimicsmicroenvironmental conditions of the perivascular niche, with high-resolution imaging of cell metabolism.Using fluorescence lifetime imaging microscopy (FLIM) we monitor the spatial metabolic machinery andcorrelate it with experimentally validated intracellular oxygen concentration after designing the oxygentension decay along the fluidic chamber byin silicomodels prediction. Our platform allows the metabolicregulation of MSCs, mimicking the physiological niche in space and time, and its real-time monitoringrepresenting a functional tool for modelling perivascular niches, relevant diseases and metabolic-relateduptake of pharmaceuticals

Design and Characterization of Novel Antibody-Cytokine Fusion Proteins Based on Interleukin-21

Interleukin-21 (IL21) is a pleiotropic cytokine involved in the modulation of both innate and adaptive immunity. IL21 is mainly secreted by natural killer (NK) and activated CD4+ T-cells. The biology of this cytokine can be associated to proinflammatory responses reflecting its potent stimulatory activity of NK and CD8+ T-cells. Here we describe four formats of novel IL21-based antibody-cytokine fusion proteins, targeting the extra domain A (EDA) of fibronectin and explore their potential for cancer treatment. The fusion proteins were designed, expressed, and characterized. F8 in single-chain diabody (scDb) format fused to IL21 at its C-terminus exhibited a promising profile in size exclusion chromatography (SEC) and SDS-PAGE. The lead candidate was further characterized in vitro. A cell-based activity assay on murine cytotoxic T-cells showed that human IL21, compared to murine IL21 partially cross-reacted with the murine receptor. The prototype was able to recognize EDA as demonstrated by immunofluorescence analysis on tumor sections. In an in vivo quantitative biodistribution experiment, F8(scDb)-murine IL21 did not preferentially accumulate at the site of disease after intravenous injection, suggesting that additional protein engineering would be required to improve the tumor-homing properties of IL21-based product

An Antibody Targeting Fibroblast Activation Protein Simultaneously Fused to Interleukin-2 and Tumor Necrosis Factor Selectively Localizes to Neoplastic Lesions

The delivery of specific cytokine payloads to a neoplastic environment employing antibodies able to selectively accumulate at the tumor site represents an attractive strategy to stimulate an immune response to cancer. Whilst conventional antibody–cytokine fusions based on a single payload have shown potent anticancer activity, the concomitant delivery of two cytokine payloads may further improve the therapeutic outcome as the immune system typically adopts multiple signals to reinforce an antitumor strategy. We here describe a potency-matched dual-cytokine antibody fusion protein containing a tumor-targeting antibody fragment specific to human fibroblast activation protein (FAP), simultaneously linked to both interleukin-2 (IL2) and a tumor necrosis factor (TNF) mutant. The resulting fusion protein, termed IL2-7NP2-TNFmut, formed stable non-covalent trimers driven by the interaction of the tumor necrosis factor subunits. Both cytokine payloads retained their biological activity within the fusion protein, as shown by in vitro cellular assays. The tumor-targeting properties and the anticancer activity of IL2-7NP2-TNFmut were investigated in vivo in immunocompromised mice bearing SKRC52 cells transduced with human FAP. The fusion protein preferentially localized to the cancer site and induced partial tumor retardation

An Engineered IFNγ-Antibody Fusion Protein with Improved Tumor-Homing Properties

Interferon-gamma (IFNγ) is one of the central cytokines produced by the innate and adaptive immune systems. IFNγ directly favors tumor growth control by enhancing the immunogenicity of tumor cells, induces IP-10 secretion facilitating (CXCR3+) immune cell infiltration, and can prime macrophages to an M1-like phenotype inducing proinflammatory cytokine release. We had previously reported that the targeted delivery of IFNγ to neoplastic lesions may be limited by the trapping of IFNγ-based products by cognate receptors found in different organs. Here we describe a novel fusion protein consisting of the L19 antibody, specific to the alternatively spliced extra-domain B of fibronectin (EDB), fused to a variant of IFNγ with reduced affinity to its cognate receptor. The product (named L19-IFNγ KRG) selectively localized to tumors in mice, showed favorable pharmacokinetic profiles in monkeys and regained biological activity upon antigen binding. The fusion protein was investigated in two murine models of cancer, both as monotherapy and in combination with therapeutic modalities which are frequently used for cancer therapy. L19-IFNγ KRG induced tumor growth retardation and increased the intratumoral concentration of T cells and NK cells in combination with anti-PD-1

Targeted delivery of tumor necrosis factor in combination with CCNU induces a T cell-dependent regression of glioblastoma

Glioblastoma is the most aggressive primary brain tumor with an unmet need for more effective therapies. Here, we investigated combination therapies based on L19TNF, an antibody-cytokine fusion protein based on tumor necrosis factor that selectively localizes to cancer neovasculature. Using immunocompetent orthotopic glioma mouse models, we identified strong anti-glioma activity of L19TNF in combination with the alkylating agent CCNU, which cured the majority of tumor-bearing mice, whereas monotherapies only had limited efficacy. In situ and ex vivo immunophenotypic and molecular profiling in the mouse models revealed that L19TNF and CCNU induced tumor DNA damage and treatment-associated tumor necrosis. In addition, this combination also up-regulated tumor endothelial cell adhesion molecules, promoted the infiltration of immune cells into the tumor, induced immunostimulatory pathways, and decreased immunosuppression pathways. MHC immunopeptidomics demonstrated that L19TNF and CCNU increased antigen presentation on MHC class I molecules. The antitumor activity was T cell dependent and completely abrogated in immunodeficient mouse models. On the basis of these encouraging results, we translated this treatment combination to patients with glioblastoma. The clinical translation is ongoing but already shows objective responses in three of five patients in the first recurrent glioblastoma patient cohort treated with L19TNF in combination with CCNU (NCT04573192)

Generation and in vivo validation of an IL-12 fusion protein based on a novel anti-human FAP monoclonal antibody

Background In this study, we describe the generation of a fully human monoclonal antibody (named '7NP2') targeting human fibroblast activation protein (FAP), an antigen expressed in the microenvironment of different types of solid neoplasms. Methods 7NP2 was isolated from a synthetic antibody phage display library and was improved by one round of mutagenesis-based affinity maturation. The tumor recognition properties of the antibody were validated by immunofluorescence procedures performed on cancer biopsies from human patients. A fusion protein consisting of the 7NP2 antibody linked to interleukin (IL)-12 was generated and the anticancer activity of the murine surrogate product (named mIL12-7NP2) was evaluated in mouse models. Furthermore, the safety of the fully human product (named IL12-7NP2) was evaluated in Cynomolgus monkeys. Results Biodistribution analysis in tumor-bearing mice confirmed the ability of the product to selectively localize to solid tumors while sparing healthy organs. Encouraged by these results, therapy studies were conducted in vivo, showing a potent antitumor activity in immunocompetent and immunodeficient mouse models of cancer, both as single agent and in combination with immune checkpoint inhibitors. The fully human product was tolerated when administered to non-human primates. Conclusions The results obtained in this work provided a rationale for future clinical translation activities using IL12-7NP2.ISSN:2051-142