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Background Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadly type of cancer, with an extremely low five-year overall survival rate. To date, current treatment options primarily involve various chemotherapies, which often prove ineffective and are associated with substantial toxicity. Furthermore, immunotherapies utilizing checkpoint inhibitors have shown limited efficacy in this context, highlighting an urgent need for novel therapeutic strategies. This study investigates the preclinical efficacy of an innovative targeted therapy based on antibody-cytokine fusion proteins, specifically interleukin-2 (IL-2), a pivotal driver of cell-mediated immunity, fused to L19 antibody, which selectively binds to extra domain B of fibronectin (EDB-FN1) expressed in the tumor microenvironment. Methods We tested the effectiveness of different immunocytokines through in vivo characterization in syngeneic C57BL/6J orthotopic mouse models of PDAC. Based on these results, we decided to focus on L19-IL2. To assess the efficacy of this immunocytokine we developed an ex-vivo immune-spheroid interaction platform derived from murine 3D pancreatic cultures, and telomerase reverse transcriptase (TERT) specific T-lymphocytes. Moreover, we evaluated the anti-cancer effect of L19-IL2 in combination with standard therapy in vivo experiments in PDAC mouse models. Tumor samples collected after the treatments were characterized for tumor infiltrating immune cell components by bulk RNA sequencing (RNA-seq) and spatial transcriptomics (Stereo-seq) analysis. Results The tumor-targeted L19-IL2 fusion protein demonstrated potent, dose-dependent anti-tumor activity in mice with pancreatic tumors resistant to standard chemotherapy. Spatial Transcriptomics (ST) and RNA-seq analyses indicated that L19-IL2 treatment induced a significant influx of immune cells into the tumor microenvironment, with these cells expressing activation markers like granzymes, perforins, and the IL-2 receptors. Conclusions Our results demonstrated that L19-IL2 enhances immune infiltration and cytotoxicity, remodeling the “cold” tumor microenvironment (TME) in PDAC. This innovative antibody-cytokine fusion protein improves therapeutic outcomes, paving the way for novel targeted treatment strategies in PDAC.

In the present report, we describe two cases of right-sided M1 segment middle cerebral artery dissection in a 51-year-old Asian female and in a 28-year-old Caucasian male patient with no previous history of ischemic stroke or known intracranial atherosclerosis presenting with acute unilateral headache progressing to severe multifocal hemispheric infarction with nearly complete one-sided motor paralysis. In both patients, a middle cerebral artery dissection was detected on angiography; they were given exclusively medical therapy: patient 1 was not eligible to reperfusive therapies and was treated with a three-month course of acetylsalicylic acid and clopidogrel combined with low-dose enoxaparin, while patient 2 was initially treated with intravenous alteplase with no hemorrhagic complications and was later shifted to single antiplatelet therapy. Despite an initial worsening of clinical severity and an extensive ischemic lesion in both patients, neurologic function improved over time, eventually allowing recovery of unaided gait. Therefore, in the absence of signs of hemorrhage, intravenous thrombolysis or dual antiplatelet regimens could be considered in strokes related to middle cerebral artery dissection.

DNA‐encoded chemical libraries (DELs) are powerful tools for drug discovery, enabling the high‐throughput screening of vast libraries of small molecules against target proteins of pharmaceutical interest. Here, the synthesis of two new DELs, named FM‐DEL1 and FM‐DEL2, including 7′710 and 5′697’690 compounds, respectively is described. These libraries are constructed by installing one or two sets of building blocks on a phenylalanine central scaffold. FM‐DELs are screened against markers of prostate cancer, and renal cell carcinoma, and against an immunological target expressed on the surface of natural killer cells. Highly potent and selective binders with affinity constants in the nanomolar range are obtained from DEL screenings against those targets. Small‐molecule ligands against tumor‐associated antigens are used to develop small‐molecule radiopharmaceuticals that selectively accumulate at cancer sites after systemic administration. FM‐DEL1 and FM‐DEL2 are constructed by installing one or two sets of building blocks on a phenylalanine central scaffold. FM‐DELs are screened against markers of prostate, and renal cell carcinoma, and against an immunological target expressed on the surface of natural killer cells. Highly potent and selective binders with affinity constants in the nanomolar range are isolated for those targets.

BackgroundOvarian cancer remains a major clinical challenge with more than 40.000 annual deaths in Europe and in the United States, highlighting the need for better diagnostic and therapeutic strategies. This study first presents an immunohistochemical evaluation of the extra-domains A and B containing fibronectin (EDA-FN, EDB-FN), fibroblast activation protein (FAP), and carcinoembryonic antigen (CEA) in ovarian cancer specimens. Based on the initial results, the analysis was subsequently expanded to provide a comprehensive assessment of EDA-FN expression in human epithelial ovarian cancer tissue samples.MethodsAn initial exploratory immunohistochemical analysis was performed on 60 formalin fixed paraffin embedded (FFPE) epithelial ovarian cancer (EOC) tissue sections from 47 patients, including 47 specimens collected at first diagnosis and 13 matched relapsed lesions. Tissue sections were stained using previously validated antibodies specific to EDA-FN, EDB-FN, FAP and CEA, to evaluate the stromal immunoreactive score (sIRS Part 1). Following the completion of Part 1, the study was expanded to specifically analyze the most abundant antigen found (EDA-FN) on 204 FFPE High Grade Serous ovarian cancer (HGSOC) tissue samples from 102 subjects, including primary and metastatic sites from the same patient (Part 2).ResultsIn Part 1, stromal expression of EDA-FN, EDB-FN and FAP was observed in epithelial ovarian cancer with no significant differences between matched primary and relapse tumor tissues. CEA was exclusively found in mucinous ovarian cancer (MOC). EDA-FN was the most abundant antigen among the ones investigated, prompting a deeper investigation in Part 2. In the expanded EOC cohort, EDA-FN remained highly abundant across all molecular subgroups (HRp, HRd/BRCAwt, and BRCAmut) and clinical subgroups (naïve vs. pretreated patients), but was found at elevated level in metastases compared to the corresponding primary tumors.ConclusionsThese findings highlight that EDA-FN is an excellent target for HGSOC, while CEA could serve as a potential target for MOC. Clinical investigations are warranted to evaluate innovative treatments in ovarian cancer targeting these antigens.

BackgroundAnti-PD-1 antibodies have revolutionized cancer immunotherapy due to their ability to induce long-lasting complete remissions in a proportion of patients. Current research efforts are attempting to identify biomarkers and suitable combination partners to predict or further improve the activity of immune checkpoint inhibitors. Antibody-cytokine fusions are a class of pharmaceuticals that showed the potential to boost the anticancer properties of other immunotherapies. Extradomain A-fibronectin (EDA-FN), which is expressed in most solid and hematological tumors but is virtually undetectable in healthy adult tissues, is an attractive target for the delivery of cytokine at the site of the disease.MethodsIn this work, we describe the generation and characterization of a novel interleukin-7-based fusion protein targeting EDA-FN termed F8(scDb)-IL7. The product consists of the F8 antibody specific to the alternatively spliced EDA of FN in the single-chain diabody (scDb) format fused to human IL-7.ResultsF8(scDb)-IL7 efficiently stimulates human peripheral blood mononuclear cells in vitro. Moreover, the product significantly increases the expression of T Cell Factor 1 (TCF-1) on CD8+T cells compared with an IL2-fusion protein. TCF-1 has emerged as a pivotal transcription factor that influences the durability and potency of immune responses against tumors. In preclinical cancer models, F8(scDb)-IL7 demonstrates potent single-agent activity and eradicates sarcoma lesions when combined with anti-PD-1.ConclusionsOur results provide the rationale to explore the combination of F8(scDb)-IL7 with anti-PD-1 antibodies for the treatment of patients with cancer.

Anti-leucine-rich glioma-inactivated protein 1 (anti-LGI1) encephalitis is a rare autoimmune disorder, classified within limbic encephalitides, and characterized by seizures and subacute cognitive-behavioral impairment, mainly affecting short-term memory and usually involving temporo-mesial lobe structures.We present a case of anti-LGI1 encephalitis characterized by focal right lower limb motor seizures and pyramidal signs and responsive to high-dose methylprednisolone. The patient developed an atypical left frontal lobe parasagittal T2 hyperintense lesion on MRI within one month of hospital admission, which has not been described previously in this disease to the best of our knowledge.

BackgroundThe administration of recombinant interleukin 2 (IL-2) in oncology is frequently hampered by dose-limiting toxicities, including potentially lethal vascular leak syndrome. Antibody-IL-2 fusion proteins capable of preferential tumor localization have shown encouraging signs of activity in clinical trials; however, they typically cause side effects shortly after intravenous administration, which may limit escalation to curative doses. There is an urgent need to engineer IL-2 products with “activity-on-demand” able to mask on-target off-tumor IL-2 activity without compromising therapeutic efficacy.MethodsTo design IL-2 biopharmaceuticals with “activity-on-demand”, which would be non-toxic on administration but regain activity at the tumor site, we explored the therapeutic potential of the co-administration of signaling inhibitors with matched pharmacokinetic properties. In this work, we used the tumor-homing F8-IL2 fusion protein, specific to a splice variant of fibronectin, and masked off-tumor toxicity by co-administration of upadacitinib, which rapidly clears from circulation. Vascular leak syndrome was monitored by histopathological analysis, the extent of peripheral edema, and cytokine levels. Immune profiling of the tumors and secondary lymphoid organs was performed by flow cytometry.ResultsIn immunocompetent tumor-bearing mice, the combinatorial treatment significantly improved tolerability without any detectable loss of therapeutic activity, protecting the mice from body weight loss, uncontrolled systemic cytokine release, and severe vascular leak syndrome manifestations, including peripheral edema. F8-IL2 efficiently controlled tumor growth and retained its immunological activity within the neoplastic mass, as evidenced by the massive natural killer and cytotoxic T-cell infiltrates.ConclusionsThis study suggests that combinatorial treatments enable the administration of potentially curative doses of targeted IL-2 products while sparing healthy organs from life-threatening toxicities.

BackgroundIn 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.Methods7NP2 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.ResultsBiodistribution 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.ConclusionsThe results obtained in this work provided a rationale for future clinical translation activities using IL12-7NP2.

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.

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.