Philogen (Italy)

L19IL2 is a clinical-stage antibody-cytokine fusion protein that has been investigated for the treatment of various cancer types. Despite its promising antitumor activity, the precise mechanism of action is still not fully understood. In this work, we employed a myc-driven B-cell lymphoma murine model to demonstrate that systemic administration of L19IL2 induced a robust CD8⁺ T cell-dependent tumor regression across multiple organs without expansion of regulatory T cells. Following L19IL2 administration, intratumoral CD8+ T cells proliferated and acquired effector and memory phenotypes, associated with private clonal expansion and enhanced killing. Moreover, the spatial behavior of peritumoral CD8+ T cells studied by intravital microscopy demonstrated a rapid increase in tumor-directed motility and infiltration following L19IL2 administration. These findings described the detailed mechanism of action of L19IL2 against B cell lymphoma and revealed for the first time the dynamic responses of peritumoral CD8+ T cells to targeted IL2 stimulation, supporting the use of L19IL2 for patients with aggressive B cell lymphoma.

IntroductionFibroblast activation protein (FAP) is involved in the extracellular matrix (ECM) remodeling and wound healing. Absent in most adult tissues, it is overexpressed by neoplastic cells and/or cancer-associated fibroblasts (CAFs) in several human malignancies. The extra Domain-B of fibronectin (EDB+FN) is a splice variant of fibronectin involved in angiogenesis and tissue remodeling, overexpressed by CAFs and cancer-associated vessels (CAVs) in many aggressive human tumors. This study aims to investigate FAP and EDB+FN expressions in canine tumors and assess their potential as druggable targets in animal patients.MethodsFAP and EDB+FN expression was assessed by immunohistochemistry on 88 canine tumors, including Soft Tissue Sarcomas [STS], Osteosarcomas [OSA], Hemangiosarcomas [HSA], Apocrine Gland Anal Sac Adenocarcinomas [AGASAC], Mast Cell Tumors [MCT], Lymphomas, and Melanomas, using polyclonal and monoclonal anti-FAP and the L19 anti-EDB antibodies. Expression distribution and intensity were semi-quantitatively scored in neoplastic cells, CAFs, CAVs, and stroma.ResultsFAP was variably expressed in neoplastic cells (79/88), CAFs (79/88), and CAVs (82/88) across all tumor types, but mostly in AGASACs, STSs, and MCTs. The monoclonal antibody presented greater specificity. EDB+FN expression was less present across tumor types, mostly with a vascular staining pattern. Labelling was most intense and consistent in the neoplastic cells, CAFs, and CAVs of melanomas, and to a lesser extent in AGASAC and STS.DiscussionSTS, AGASAC, and MCT could be candidates for FAP-targeted strategies; melanomas are the most promising for EDB+FN-directed therapies. These results support FAP and EDB+FN as targets worth investigating for clinical applications in animal patients.

Abstract BACKGROUND Targeted cytokine-based therapy is a promising treatment opportunity to potentially convert an immunologically “cold” tumor microenvironment into a “hot” one and trigger a potent antitumor response. L19TNF is a fully human antibody-cytokine fusion protein, comprising the pro-inflammatory cytokine tumor necrosis factor (TNF) fused to the antibody L19 that recognizes the extra domain B of fibronectin, a tumor-specific antigen. This allows targeted delivery of therapeutically relevant doses of TNF to the tumor while sparing healthy organs. Here, we report the results of the phase I dose escalation part of a clinical trial with systemically administered L19TNF in combination with standard chemoradiotherapy for patients with newly diagnosed glioblastoma. MATERIAL AND METHODS The Phase I part of NCT04443010 study was an open-label, non-randomized, multicenter dose escalation to investigate the safety and preliminary efficacy of L19TNF in combination with temozolomide chemoradiotherapy for patients newly diagnosed glioblastoma. Patients were treated with 7, 10 or 13 µg/kg intravenous L19TNF, radiotherapy of 60 Gy in 30 fractions plus daily 75 mg/m2 TMZ, followed by up to 6 maintenance cycles of TMZ 150-200 mg/m2. Tumor responses were assessed by iRANO criteria. RESULTS From 2021 - 2024, we enrolled 18 patients from two centers in Switzerland. Tumors of 11 patients (61%) had an unmethylated O6-methylguanine DNA methyltransferase (MGMT) promoter. L19TNF could be safely administered at a dose of 13 µg/kg in combination with temozolomide-based chemoradiation. Treatment was well tolerated and the most common adverse events were asymptomatic transient increases of ALT, GGT and AST, decreased lymphocyte counts, chills, back pain, nausea, vomiting, headache and decreased platelet and white blood cell counts. With the addition of L19TNF to standard temozolomide-based chemoradiation, we observe a median overall survival of > 20 months. CONCLUSION The intravenous administration of L19TNF is safe in patients with newly diagnosed glioblastoma and shows promising activity. These encouraging results warrant further investigations within the Phase II part of the clinical trial.

The 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. To 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. In 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. This 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.

Triple-negative breast cancer (TNBC) lags behind other breast cancer types in targeted therapeutic and diagnostic imaging agent development, largely due to high disease heterogeneity. Noninvasive imaging is essential for diagnosing and staging TNBC and predicting and measuring treatment response. This study targeted a conserved disease-specific extracellular matrix protein domain (the extra domain A of fibronectin [EDA-FN]), with a monoclonal antibody (F8) to overcome tumor cell marker heterogeneity and develop an imaging agent to detect multiple TNBC subtypes and improve diagnostic capacity. Methods: [89Zr]Zr-desferrioxamine [DFO]-F8 was synthesized and evaluated invitro and invivo for EDA-FN binding capacity to detect TNBC by PET/CT in several preclinical xenograft models. Results: [89Zr]Zr-DFO-F8 exhibited specific, blockable EDA-FN binding activity invitro. In vivo experiments demonstrated high tumor uptake in preclinical TNBC xenograft models. [89Zr]Zr-DFO-F8 detected EDA-FN in subcutaneous and orthotopic TNBC xenografts and accumulated in aggressive disease concordantly with EDA-FN expression. Conclusion: EDA-FN imaging with [89Zr]Zr-DFO-F8 exhibits powerful tumor delineation in preclinical tumor delineation across TNBC molecular subtypes invivo.

DNA-Encoded Libraries (DELs) allow the parallel screening of millions of compounds for various applications, including de novo discovery or affinity maturation campaigns. However, library construction and HIT resynthesis can be cumbersome, especially when library members present an unknown stereochemistry. We introduce a permutational encoding strategy suitable for the construction of highly pure single-stranded single-pharmacophore DELs, designed to distinguish isomers at the sequencing level (e.g., stereoisomers, regio-isomers, and peptide sequences). This approach was validated by synthesizing a mock 921,600-member 4-amino-proline single-stranded DEL ("DEL1"). While screening DEL1 against different targets, high-throughput sequencing results showed selective enrichment of the most potent stereoisomers, with enrichment factors that outperform conventional encoding strategies. The versatility of our methodology was additionally validated by encoding 24 scaffolds derived from different permutations of the amino acid sequence of a previously described cyclic peptide targeting Fibroblast Activation Protein (FAP-2286). The resulting library ("DEL2") was interrogated against human FAP, showing selective enrichment of five cyclic peptides. We observed a direct correlation between enrichment factors and on-DNA binding affinities. The presented encoding methodology accelerates drug discovery by facilitating library synthesis and streamlining HIT resynthesis while enhancing enrichment factors at the DEL sequencing level. This facilitates the identification of HIT candidates prior to medicinal chemistry and affinity maturation campaigns.

Cytokine-based therapeutics have been shown to mediate objective responses in certain tumor entities but suffer from insufficient selectivity, causing limiting toxicity which prevents dose escalation to therapeutically active regimens. The antibody-based delivery of cytokines significantly increases the therapeutic index of the corresponding payload but still suffers from side effects associated with peak concentrations of the product in blood upon intravenous administration. Here we devise a general strategy (named “Intra-Cork”) to mask systemic cytokine activity without impacting anti-cancer efficacy. Our technology features the use of antibody-cytokine fusions, capable of selective localization at the neoplastic site, in combination with pathway-selective inhibitors of the cytokine signaling, which rapidly clear from the body. This strategy, exemplified with a tumor-targeted IL12 in combination with a JAK2 inhibitor, allowed to abrogate cytokine-driven toxicity without affecting therapeutic activity in a preclinical model of cancer. This approach is readily applicable in clinical practice.

Immune-stimulating antibody conjugates (ISACs) equipped with imidazoquinoline (IMD) payloads can stimulate endogenous immune cells to kill cancer cells, ultimately inducing long-lasting anticancer effects. A novel ISAC was designed, featuring the IMD Resiquimod (R848), a tumor-targeting antibody specific for Carbonic Anhydrase IX (CAIX) and the protease-cleavable Val-Cit-PABC linker. In vitro stability analysis showed not only R848 release in the presence of the protease Cathepsin B but also under acidic conditions. The ex vivo mass spectrometry-based biodistribution data confirmed the low stability of the linker-drug connection while highlighting the selective accumulation of the IgG in tumors and its long circulatory half-life.