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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., Competing Interests: Declarations. Ethics approval and consent to participate: Animal experiments were approved by animal ethics committee of Catholic University of the Sacred Heart of Rome and by Ministry of Health with approval number n°593/2019-PR. This study was approved by the ethics committee of AOUI Verona Hospital (Territorial Ethics Committee for the South-West Veneto Area) with approval number Prot. n°53732. The patients provided written consent. This study was carried out in accordance with Declaration of Helsinki. Consent for publication: All patients provided consent for publication. Competing interests: Dario Neri is a co-founder and shareholder of Philogen ( www.philogen.com ), a Swiss-Italian Biotech company that operates in the field of ligand-based pharmacodelivery. Roberto De Luca, and Emanuele Puca are employees of Philochem AG, a daughter company of Philogen acting as discovery unit of the group., (© 2025. The Author(s).)

Antibodies that target the tumor microenvironment can be used to deliver pro-inflammatory payloads, such as cytokines. Cytokines are small proteins able to modulate the activity of the immune system, and antibody-cytokine fusion proteins have been tested in preclinical and clinical settings. In this study, we describe Tripokin, a novel multi-specific fusion protein that combines interleukin-2 and a single amino acid mutant of tumor necrosis factor. The two pro-inflammatory payloads were fused to the L19 antibody, a clinical-grade antibody against the extradomain B of fibronectin. The human payloads were used for clinical applications, while the corresponding murine cytokines were used for preclinical studies. The resulting fusion proteins were produced in mammalian cells and purified to homogeneity. The murine Tripokin product was well tolerated in tumor-bearing mice at three doses of 30 μg in a 2-day interval and promoted rapid tumor eradication in murine models, more efficiently than single-agent immunocytokines. Tripokin induced rapid tumor necrosis and stimulated a robust immune response, impacting innate and adaptive immune pathways. In addition, the combination with immune checkpoint inhibitors further boosted the therapeutic efficacy of our molecule. Tripokin represents a promising clinical candidate for the simultaneous delivery of interleukin-2 and tumor necrosis factor to neoplastic sites., (© 2024 UICC.)

Purpose: Pluvicto™ ([ 177 Lu]Lu-PSMA-617), a radioligand therapeutic targeting prostate-specific membrane antigen (PSMA), has been recently approved for the treatment of metastatic castration-resistant prostate cancer (mCRPR). The drug suffers from salivary gland and kidney uptake that prevents its dose escalation to potentially curative doses. In this work, we sought to potentiate the in vivo anti-cancer activity of Pluvicto™ by combining it with L19-IL2, a clinical-stage investigational medicinal product based on tumor-targeted interleukin-2., Methods: We established a new PSMA-expressing model (HT-1080.hPSMA) and validated it using a fluoresceine analogue of PSMA-617 (compound 1). The HT-1080.hPSMA model was used to study the saturation and tumor retention of Pluvicto™ (compound 2) and to run combination therapy studies with L19-IL2. To complement our understanding of the mechanism of action of this novel combination, we conducted proteomics experiments on tumor samples after therapy with Pluvicto™ alone or in combination with the immunocytokine., Results: High, selective, and long-lived tumor uptake was observed for Pluvicto™ (2) in the novel HT-1080.hPSMA model. Therapy studies in HT-1080.hPSMA tumor-bearing mice revealed that the combination of Pluvicto™ (2) plus L19-IL2 mediated curative and durable responses in all animals. Potent in vivo anti-cancer activity was observed solely for the combination modality, at doses that were well tolerated by treated animals. Proteomics studies indicated that L19-IL2 boosts the activation of the immune system in animals pre-treated with Pluvicto™., Conclusion: The therapeutic efficacy of Pluvicto™ at low radioactive doses can be effectively enhanced by the combination with L19-IL2. Our findings warrant further clinical exploration of this novel combination modality., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We studied the antitumor efficacy of a combination of 177 Lu-labeled radioligand therapeutics targeting the fibroblast activation protein (FAP) (OncoFAP and BiOncoFAP) with the antibody-cytokine fusion protein L19-interleukin 2 (L19-IL2) providing targeted delivery of interleukin 2 to tumors. Methods: The biodistribution of 177 Lu-OncoFAP and 177 Lu-BiOncoFAP at different molar amounts (3 vs. 250 nmol/kg) of injected ligand was studied via SPECT/CT in mice bearing subcutaneous HT-1080.hFAP tumors, and self-absorbed tumor and organ doses were calculated. The in vivo anticancer effect of 5 MBq of the radiolabeled preparations was evaluated as monotherapy or in combination with L19-IL2 in subcutaneously implanted HT-1080.hFAP and SK-RC-52.hFAP tumors. Tumor samples from animals treated with 177 Lu-BiOncoFAP, L19-IL2, or both were analyzed by mass spectrometry-based proteomics to identify therapeutic signatures on cellular and stromal markers of cancer and on immunomodulatory targets. Results: 177 Lu-BiOncoFAP led to a significantly higher self-absorbed dose in FAP-positive tumors (0.293 ± 0.123 Gy/MBq) than did 177 Lu-OncoFAP (0.157 ± 0.047 Gy/MBq, P = 0.01) and demonstrated favorable tumor-to-organ ratios at high molar amounts of injected ligand. Administration of L19-IL2 or 177 Lu-BiOncoFAP as single agents led to cancer cures in only a limited number of treated animals. In 177 Lu-BiOncoFAP-plus-L19-IL2 combination therapy, complete remissions were observed in all injected mice (7/7 complete remissions for the HT-1080.hFAP model, and 4/4 complete remissions for the SK-RC-52.hFAP model), suggesting therapeutic synergy. Proteomic studies revealed a mechanism of action based on the activation of natural killer cells, with a significant enhancement of the expression of granzymes and perforin 1 in the tumor microenvironment after combination treatment. Conclusion: The combination of OncoFAP-based radioligand therapeutics with concurrent targeting of interleukin 2 shows synergistic anticancer effects in the treatment of FAP-positive tumors. This experimental finding should be corroborated by future clinical studies., (© 2023 by the Society of Nuclear Medicine and Molecular Imaging.)

Natural killer (NK) cells are key effectors in cancer immunosurveillance and posttransplant immunity, but deficiency of environmental signals and insufficient tumor recognition may limit their activity. We hypothesized that the antibody-mediated anchoring of interleukin-2 (IL-2) to a spliced isoform of the extracellular matrix (ECM) glycoprotein tenascin-C would potentiate NK-cell-mediated antibody-dependent cellular cytotoxicity against leukemic blasts. In this novel-novel combination, dose-escalation, phase 1 trial, we enrolled patients with posttransplant acute myeloid leukemia (AML) relapse to evaluate the safety, pharmacokinetics, pharmacodynamics, and preliminary activity of the antibody-cytokine fusion F16IL2 (10 × 106 to 20 × 106 IU IV; days 1, 8, 15, and 22 of each 28-day cycle) in combination with the anti-CD33 antibody BI 836858 (10-40 mg IV, 2 days after each F16IL2 infusion). Among the 15 patients (median [range] age, 50 [20-68] years) treated across 4 dose levels (DLs), 6 (40%) had received 2 or 3 prior transplantations. The most frequent adverse events were pyrexia, chills, and infusion-related reactions, which were manageable, transient and of grade ≤2. One dose-limiting toxicity occurred at each of DLs 3 (pulmonary edema) and 4 (graft-versus-host disease). Three objective responses were observed among 7 patients treated at the 2 higher DLs, whereas no responses occurred at the 2 starting DLs. Combination therapy stimulated the expansion and activation of NK cells, including those expressing the FcγRIIIA/CD16 receptor. ECM-targeted IL-2 combined with anti-CD33 immunotherapy represents an innovative approach associated with acceptable safety and encouraging biologic and clinical activity in posttransplant AML relapse. This trial was registered at EudraCT as 2015-004763-37., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Interleukin-2 (IL2) is a pro-inflammatory cytokine that plays a crucial role in immunity, which is increasingly being used for therapeutic applications. There is growing interest in developing IL2-based therapeutics which do not interact with the alpha subunit of the IL2 receptor (CD25) as this protein is primarily found on immunosuppressive regulatory T cells (T regs ). Screenings of a new DNA-encoded library, comprising 669,240 members, provided a novel series of IL2 ligands, subsequently optimized by medicinal chemistry. One of these molecules (compound 18 ) bound to IL2 with a dissociation constant of 0.34 μM was able to form a kinetically stable complex with IL2 in size-exclusion chromatography and recognized the CD25-binding site as evidenced by competition experiments with the NARA1 antibody. Compound 18 and other members of the series may represent the starting point for the discovery of potent small-molecule modulators of IL2 activity, abrogating the binding to CD25.

Recombinant human IL2 is being considered as a combination partner for immune checkpoint inhibitors in cancer therapy, but the product only has a narrow therapeutic window. Therefore, we used F8-IL2, an antibody-IL2 fusion protein capable of selective localization to the tumor site, in combination with antibodies against murine CTLA-4, PD-1, and PD-L1. In immunocompetent mice bearing CT26 tumors, the combination of F8-IL2 with CTLA-4 blockade was efficacious, leading to increased progression-free survival and protective immunity against subsequent tumor rechallenges. The combination with anti-PD-1 induced substantial tumor growth retardation, but tumor clearance was rare, whereas the combination with anti-PD-L1 exhibited the lowest activity. A detailed high-parametric single-cell analysis of the tumor leukocyte composition revealed that F8-IL2 had a strong impact on NK-cell activity without collateral immune activation in the systemic immune compartment, whereas CTLA-4 blockade led to significant changes in the T-cell compartment. Leukocyte depletion studies revealed that CD8 + T and NK cells were the main drivers of the therapeutic activity. We extended the experimental observations to a second model, treating MC38 tumor-bearing mice with F8-IL2 and/or CTLA-4 blockade. Only the combination treatment displayed potent anticancer activity, characterized by an increase in cytolytic CD8 + T and NK cells in tumors and draining lymph nodes. A decrease in the regulatory T cell frequency, within the tumors, was also observed. The results provide a rationale for the combined use of engineered IL2 therapeutics with immune checkpoint inhibitors for cancer therapy., (©2019 American Association for Cancer Research.)

The discovery of protein ligands, capable of forming a reversible covalent bond with amino acid residues on a protein target of interest, may represent a general strategy for the discovery of potent small-molecule inhibitors. We analyzed the ability of different aromatic aldehydes to form imines by reaction with lysine using 1 H NMR techniques. 2-Hydroxybenzaldehyde derivatives were found to efficiently form imines in the millimolar concentration range. These benzaldehyde derivatives could increase the binding affinity of protein ligands towards the cognate protein target. Affinity maturation was achieved not only by displaying ligand and aldehyde moieties on two complementary locked nucleic acid strands but also by incorporating the binding fragments in a single small-molecule ligand. The affinity gain was only observed when lysine residues were accessible in the immediate surroundings of the ligand-binding site and could be abrogated by quenching with a molar excess of hydroxylamine., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have recently described a novel therapeutic antibody product (IL2-F8-TNF mut ), featuring the simultaneous fusion of murine IL2 and of a TNF mutant with scFv(F8), an antibody specific to the alternatively-spliced extra domain A of fibronectin (EDA). Here, we report on the in vivo characterization of the anti-cancer activity of IL2-F8-TNF mut in four immunocompetent murine models of cancer, CT26, WEHI-164, F9 teratocarcinoma and Lewis lung carcinoma (LLC), using the product alone or in combination with a monoclonal antibody specific to murine PD-L1. All four models exhibited a strong expression of EDA-fibronectin, which was confined to vascular structures for F9 tumors, while the other three malignancies exhibited a more stromal pattern of staining. A complete and long-lasting tumor eradication of CT26 and WEHI-164 tumors was observed in BALB/c mice when IL2-F8-TNF mut was used in combination with PD-L1 blockade. The combination treatment led to improved tumor growth inhibition in 129/SvEv mice bearing murine teratocarcinoma or in C57BL/6 mice bearing murine LLC, but those cancer cures were difficult to achieve in those models. A microscopic analysis of tumor sections, obtained 24 h after pharmacological treatment, revealed that the PD-L1 antibody had homogenously reached tumor cells in vivo and that the combination of PD-L1 blockade with IL2-F8-TNF mut stimulated an influx of NK cells and of T cells into the neoplastic mass. These data indicate that potency-matched dual-cytokine fusion proteins may be ideally suited to potentiate the therapeutic activity of immune check-point inhibitors.

Purpose: Antibody-drug conjugates and small-molecule-drug conjugates have been proposed as alternatives to conventional anticancer cytotoxic agents, with the potential to deliver bioactive payloads to the site of disease, helping spare normal tissues. Experimental Design: Here, we describe a novel small-molecule-drug conjugate, based on a high-affinity ligand specific to carbonic anhydrase IX. The product featured a peptidic linker, suitable for cleavage in the tumor extracellular environment, and monomethyl auristatin E as cytotoxic payload. Results: A potent anticancer activity was observed in nude mice bearing SKRC-52 renal cell carcinoma xenografts, but no durable complete responses could be observed in this model. However, when the product was administered together with L19-IL2 (a clinical-stage fusion protein capable of delivering IL2 to the tumor neovasculature), all treated mice in the combination group could be rendered tumor free, in a process that favored the influx of natural killer cells into the tumor mass. The combination of L19-IL2 and the new small-molecule-drug conjugate also eradicated cancer in 100% of immunocompetent mice, bearing subcutaneously grafted CT26 colorectal cancer cells, which stably expressed carbonic anhydrase IX. Conclusions: These findings may be of clinical significance, because carbonic anhydrase IX is overexpressed in the majority of clear cell renal cell carcinomas and in approximately 30% of colorectal cancers. The targeted delivery of IL2 helps potentiate the action of targeted cytotoxics, leading to cancer eradication in models that cannot be cured by conventional chemotherapy. Clin Cancer Res; 24(15); 3656-67. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Purpose: To analyze the impact of TNFα or IL2 on human lymphocytes in vitro and the anti-tumor and immune-modifying effects of L19-IL2 and L19-TNFα on subcutaneously growing J558L myeloma in immunocompetent mice., Methods: PBMCs from three healthy volunteers were incubated with IL2, TNFα, or with IL2 plus addition of TNFα (final 20 h). BALB/c J558L mice with subcutaneous tumors were treated with intravenous L19-TNFα plus L19-IL2, or controls. Tumor growth and intra- and peri-tumoral tissues were analyzed for micro-vessel density, necrosis, immune cell composition, and PD1 or PD-L1 expressing cells., Results: Exposure of PBMC in vitro to IL2, TNFα, or to IL2 over 3 and 5 days plus TNFα for the final 20 h resulted in an approximately 50 and 75% reduction of the CD25low effector cell/CD25high Treg cell ratio, respectively, compared to medium control. IL2 or TNFα increased the proportion of CD4- CD25low effector lymphocytes while reducing the proportion of CD4+ CD25low Teff cells. In the J558L myeloma model, tumor eradication was observed in 58, 42, 25, and 0% of mice treated with L19-TNFα plus L19-IL2, L19-TNFα, L19-IL2, and PBS, respectively. L19-TNFα/L19-IL2 combination caused tumor necrosis, capillary density doubling, peri-tumoral T cell and PD1+ T cell reduction (- 50%), and an increase in PD-L1+ myeloma cells., Conclusion: IL2, TNFα, or IL2 plus TNFα (final 20 h) increased the proportion of CD4- CD25low effector lymphocytes possibly indicating immune activation. L19-TNFα/L19-IL2 combination therapy eradicated tumors in J558L myeloma BALB/c mice likely via TNFα-induced tumor necrosis and L19-TNFα/L19-IL2-mediated local cellular immune reactions.

Aim: The combination of tumor-targeting IL2- and TNF-based antibody-cytokine fusions has exhibited encouraging results in mouse and men. Here, we studied their combination to assess efficacy and mechanism of action in four different immunocompetent mouse models of cancer., Methods: Mice receiving a single intratumoral injection of F8-IL2, F8-TNF or the combination were investigated for tumor-infiltrating leukocytes and rechallenged when cured., Results: In three models, a proportion of treated animals could be cured, most probably by infiltrating NK and CD8 + T cells. Most of the cured mice did not acquire protective immunity when rechallenged with the same tumor cell line., Conclusion: Immunocompetent mouse tumor models may not be adequate enough to predict the search for more efficacious therapy regimens.

A novel biopharmaceutical, consisting of the F8 mAb (specific to a splice isoform of fibronectin) simultaneously fused to both TNF and IL2, was found to react with the majority of solid tumors and hematologic malignancies in mouse and man, but not with healthy adult tissues. The product selectively localized to neoplastic lesions in vivo , as evidenced by quantitative biodistribution studies using radioiodinated protein preparations. When the potency of the cytokine payloads was matched by a single-point mutation, the resulting fusion protein (IL2-F8-TNF mut ) eradicated soft-tissue sarcomas in immunocompetent mice, which did not respond to individual antibody-cytokine fusion proteins or by standard doxorubicin treatment. Durable complete responses were also observed in mice bearing CT26, C1498, and F9 tumors. The simultaneous delivery of multiple proinflammatory payloads to the cancer site conferred protective immunity against subsequent tumor challenges. A fully human homolog of IL2-F8-TNF mut , which retained selectivity similar to its murine counterpart when tested on human material, may open new clinical applications for the immunotherapy of cancer. Mol Cancer Ther; 16(11); 2442-51. ©2017 AACR ., (©2017 American Association for Cancer Research.)

There is increasing interest in the site-directed pharmacodelivery of therapeutic payloads to the tumor site using antibodies as transport vehicles. Here, we investigated the efficacy of L19-IL2, an antibody-cytokine fusion protein that specifically delivers IL-2 to the tumor site by homing to the extra-domain B of fibronectin (EDB-Fn) expressed on tumor-associated blood vessels, against mantle cell lymphoma (MCL) in mice. L19-IL2 was shown to selectively localize at lymphoma lesions in vivo and to mediate significant lymphoma growth retardation, which was potentiated by co-administration of the anti-CD20 antibody rituximab. When co-injected with rituximab, L19-IL2 induced complete remissions of localized MCL xenografts in 6/8 mice (75%), whereas the combination of rituximab and equivalent doses of non-targeted IL-2 only slightly delayed tumor growth. In disseminated MCL, combination therapy with L19-IL2 and rituximab exhibited a significant survival benefit over treatment with IL-2 and rituximab and completely eradicated the disease in 2/7 cases (28.6%). Mechanistically, histological analyses of post-therapeutic lymphoma tissues revealed a strong intratumoral accumulation of macrophages and natural killer cells after a single dose of the immunocytokine, whereas L19-IL2 had no significant impact on microvessel density or on tissue penetration of co-injected rituximab. Collectively, these results provide the scientific rationale for the clinical evaluation of L19-IL2 in combination with anti-CD20 immunotherapy in patients with MCL., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A phase Ib/II trial was performed to evaluate safety, tolerability, recommended dose (RD) and efficacy of F16-IL2, a recombinant antibody-cytokine fusion protein, in combination with doxorubicin in patients with solid tumors (phase Ib) and metastatic breast cancer (phase II). Six patient cohorts with progressive solid tumors (n = 19) received escalating doses of F16-IL2 [5-25 Million International Units (MIU) of IL2 equivalent dose] in combination with escalating doses of doxorubicin (0-25 mg/m(2)) on day 1, 8 and 15 every 4 weeks. Subsequently, patients with metastatic breast cancer (n = 10) received the drug combination at the RD. Clinical data and laboratory findings were analyzed for safety, tolerability, and activity. F16-IL2 could be administered up to 25 MIU, in combination with the RD of doxorubicin (25 mg/m(2)). No human anti-fusion protein antibodies (HAFA) response was detected. Pharmacokinetics of F16-IL2 was dose-dependent over the tested range, with half-lives of ca. 13 and ca. 8 hours for cohorts dosed at lower and higher levels, respectively. Toxicities were controllable and reversible, with no combination treatment-related death. After 8 weeks, 57% and 67% disease control rates were observed for Phase I and II, respectively (decreasing to 43% and 33% after 12 weeks), considering 14 and 9 patients evaluable for efficacy. One patient experienced a long lasting partial response (45 weeks), still on-going at exit of study. F16-IL2 can be safely and repeatedly administered at the RD of 25 MIU in combination with 25 mg/m(2) doxorubicin; its safety and activity are currently being investigated in combination with other chemotherapeutics, in order to establish optimal therapy settings.

Antibody-cytokine fusion proteins ("immunocytokines") represent a promising class of armed antibody products, which allow the selective delivery of potent pro-inflammatory payloads at the tumor site. The antibody-based selective delivery of interleukin-2 (IL2) is particularly attractive for the treatment of metastatic melanoma, an indication for which this cytokine received marketing approval from the US Food and drug administration. We used the K1735M2 immunocompetent syngeneic model of murine melanoma to study the therapeutic activity of F8-IL2, an immunocytokine based on the F8 antibody in diabody format, fused to human IL2. F8-IL2 was shown to selectively localize at the tumor site in vivo, following intravenous administration, and to mediate tumor growth retardation, which was potentiated by the combination with paclitaxel or dacarbazine. Combination treatment led to a substantially more effective tumor growth inhibition, compared to the cytotoxic drugs used as single agents, without additional toxicity. Analysis of the immune infiltrate revealed a significant accumulation of CD4(+) T cells 24 h after the administration of the combination. The fusion proteins F8-IL2 and L19-IL2, specific to the alternatively spliced extra domain A and extra domain B of fibronectin respectively, were also studied in combination with tumor necrosis factor (TNF)-based immunocytokines. The combination treatment was superior to the action of the individual immunocytokines and was able to eradicate neoplastic lesions after a single intratumoral injection, a procedure that is being clinically used for the treatment of Stage IIIC melanoma. Collectively, these data reinforce the rationale for the use of IL2-based immunocytokines in combination with cytotoxic agents or TNF-based immunotherapy for the treatment of melanoma patients.

Antibody-drug conjugates are increasingly being used for cancer therapy, but little is known about their ability to promote anticancer immunity, which may lead to long-lasting remissions. We investigated the therapeutic effect of antibody-based pharmacodelivery of cemadotin, a cytotoxic drug, and IL2, a strong proinflammatory cytokine. Using the F8 antibody, which selectively localizes to the tumor neovasculature, combination treatment led to tumor eradication, in a process dependent on CD8(+) T cells and natural killer cells in the C1498 syngeneic mouse model of acute myelogenous leukemia. The clinical combination of antibody-drug conjugates and antibody-cytokine proteins should be facilitated by their orthogonal toxicity profiles., (©2014 American Association for Cancer Research.)

Acute myeloid leukemia (AML) is a rapidly progressing disease that is accompanied by a strong increase in microvessel density in the bone marrow. This observation prompted us to stain biopsies of AML and acute lymphoid leukemia (ALL) patients with the clinical-stage human monoclonal antibodies F8, L19, and F16 directed against markers of tumor angiogenesis. The analysis revealed that the F8 and F16 antibodies strongly stained 70% of AML and 75% of ALL bone marrow specimens, whereas chloroma biopsies were stained with all three antibodies. Therapy experiments performed in immunocompromised mice bearing human NB4 leukemia with the immunocytokine F8-IL2 [consisting of the F8 antibody fused to human interleukin-2 (IL-2)] mediated a strong inhibition of AML progression. This effect was potentiated by the addition of cytarabine, promoting complete responses in 40% of treated animals. Experiments performed in immunocompetent mice bearing C1498 murine leukemia revealed long-lasting complete tumor eradication in all treated mice. The therapeutic effect of F8-IL2 was mediated by both natural killer cells and CD8(+) T cells, whereas CD4(+) T cells appeared to be dispensable, as determined in immunodepletion experiments. The treatment of an AML patient with disseminated extramedullary AML manifestations with F16-IL2 (consisting of the F16 antibody fused to human IL-2, currently being tested in phase 2 clinical trials in patients with solid tumors) and low-dose cytarabine showed significant reduction of AML lesions and underlines the translational potential of vascular tumor-targeting antibody-cytokine fusions for the treatment of patients with leukemia.

The selective delivery of bioactive agents to tumors reduces toxicity and enhances the efficacy of anticancer therapies. In this study, we show that the antibody F8, which recognizes perivascular and stromal EDA-fibronectin (EDA-Fn), when conjugated to interleukin-2 (F8-IL2) can effectively inhibit the growth of EDA-Fn-expressing melanomas in combination with paclitaxel. We obtained curative effects with paclitaxel administered before the immunocytokine. Coadministration of paclitaxel increased the uptake of F8 in xenografted melanomas, enhancing tumor perfusion and permeability. Paclitaxel also boosted the recruitment of F8-IL2-induced natural killer (NK) cells to the tumor, suggesting a host response as part of the observed therapeutic benefit. In support of this likelihood, NK cell depletion impaired the antitumor effect of paclitaxel plus F8-IL2. Importantly, this combination reduced both the tumor burden and the number of pulmonary metastatic nodules. The combination did not cause cumulative toxicity. Together, our findings offer a preclinical proof that by acting on the tumor stroma paclitaxel potentiates the antitumor activity elicited by a targeted delivery of IL2, thereby supporting the use of immunochemotherapy in the treatment of metastatic melanoma., (©2012 AACR.)

Purpose: We investigated the therapeutic action of F8-IL2, a fusion protein consisting of the F8 antibody specific to the alternatively spliced extradomain-A of fibronectin, in diabody format and of human interleukin-2 in the Caki-1 (ATCC®) model of human renal cell carcinoma grafted subcutaneously in nude mice., Materials and Methods: F8-IL2 was cloned, expressed in CHO cells and purified to homogeneity. This immunocytokine was administered alone or combined with 3 standard drugs commonly used as therapy for kidney cancer, including sunitinib, sorafenib and interferon-α, in 2 sets of doses and treatment schedules., Results: Neither F8-IL2 nor any other therapeutic agent cured tumor bearing mice when used as a single agent. The best therapeutic results were observed for the combination of sunitinib with F8-IL2 in a continuous administration schedule, which yielded a 28% cure rate and substantial tumor growth retardation., Conclusions: Considering that recombinant interleukin-2 based immunocytokines are now being investigated in several clinical trials in patients with cancer alone or combined with chemotherapy our preclinical results provide a motivation to study F8-IL2 combined with sunitinib in clinical trials in patients with kidney cancer., (Copyright © 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

The antibody-mediated delivery of therapeutic agents to sites of angiogenesis is an attractive strategy for anticancer therapy, but is largely unexplored in hematologic malignancies. In the present study, we show that the extra domain B (EDB) of fibronectin, a marker of angiogenesis, is expressed in B-cell non-Hodgkin lymphoma (NHL) and that the human monoclonal anti-EDB antibody L19 can selectively localize to the lymphoma-associated subendothelial extracellular matrix. In vivo, the preferential accumulation of the antibody at the tumor site was confirmed by quantitative biodistribution analyses with radioiodinated antibody preparations. The fusion protein L19-IL2, which mediates the delivery of interleukin-2 (IL-2) to the neovasculature, displayed a superior antilymphoma activity compared with unconjugated IL-2 in localized and systemic xenograft models of NHL. When coadministered with rituximab, L19-IL2 induced complete remissions of established localized lymphomas and provided long-lasting protection from disseminated lymphoma. The combined use of rituximab and L19-IL2, which dramatically increases the infiltration of immune effector cells in lymphomas, may deserve clinical investigations, facilitated by the fact that L19-IL2 is currently being studied in phase II clinical trials in patients with solid tumors.

Purpose: There is an interest in the discovery of biopharmaceuticals, which are well tolerated and which potentiate the action of anthracyclines and taxanes in breast cancer therapy., Experimental Design: We have produced a recombinant fusion protein, composed of the human antibody fragment scFv(F16) fused to human interleukin-2 (F16-IL2), and tested its therapeutic performance in the MDA-MB-231 xenograft model of human breast cancer. The F16 antibody is specific to the alternatively spliced A1 domain of tenascin-C, which is virtually undetectable in normal tissues but is strongly expressed in the neovasculature and stroma of breast cancer., Results: When used as monotherapy, F16-IL2 displayed a strikingly superior therapeutic benefit compared with unconjugated recombinant IL-2. The administration of doxorubicin either before (8 days, 24 h, or 2 h) or simultaneously with the injection of F16-IL2 did not decrease the accumulation of immunocytokine in the tumor as measured by quantitative biodistribution analysis. Therapy experiments, featuring five once per week coadministrations of 20 mug F16-IL2 and doxorubicin, showed a statistically significant reduction of tumor growth rate and prolongation of survival at a 4 mg/kg doxorubicin dose but not at a 1 mg/kg dose. By contrast, combination of F16-IL2 with paclitaxel (5 and 1 mg/kg) exhibited a significant therapeutic benefit compared with paclitaxel alone at both dose levels. F16-IL2, alone or in combination with doxorubicin, was well tolerated in cynomolgus monkeys at doses equivalent to the ones now used in clinical studies., Conclusions: F16-IL2 may represent a new useful biopharmaceutical for the treatment of breast cancer.

Angiogenic processes depend on the precise coordination of different cell types and a complex exchange of signals, many of which derive from new specific components of the provisional, angiogenesis-related, extracellular matrix (ECM). Angiogenesis-associated ECM components thus represent appealing targets for the selective delivery of therapeutic molecules to newly forming tumor vessels. Results of a previous study indicated that a high affinity recombinant antibody (L19) to ED-B, a domain contained in the angiogenesis-associated isoform of fibronectin (B-FN), selectively and efficiently targets tumor vessels. The present study shows that a fusion protein between L19 and interleukin 2 (L19-IL-2) mediates the selective delivery and concentration of IL-2 to tumor vasculature, thereby leading to a dramatic enhancement of the therapeutic properties of the cytokine. By contrast, IL-2 fused to an irrelevant recombinant antibody used as a control fusion protein showed neither accumulation in tumors nor therapeutic efficacy. Tumors in mice treated with L19-IL-2 were significantly smaller compared to those in animals treated with saline, the control fusion protein, or IL-2 alone (P =.003,.003, and.002, respectively). Moreover, no significant differences in size were observed among the tumors from the different control groups (using the control fusion protein, a mixture of IL-2 and L19, or saline alone). Immunohistochemical analysis of tumor infiltrates demonstrated a significantly higher number of T lymphocytes, natural killer cells, and macrophages, as well as increased interferon-gamma (IFN-gamma) accumulation, in tumors from animals treated with L19-IL-2 compared to tumors from control groups. The fact that ED-B is 100% homologous in human and mouse, thus ensuring that L19 reacts equally well with human and murine antigen, should ultimately expedite transfer of this reagent to clinical trials.

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. [ABSTRACT FROM AUTHOR]

Engineered cytokine products represent promising agents for the treatment of immunogenic tumors, such as malignant melanoma, in addition to immune checkpoint inhibitors. Here we describe the results of a controlled, randomized phase II clinical trial, aimed at assessing the therapeutic potential of L19IL2, a fully human fusion protein consisting of the L19 antibody specific to the alternatively spliced extra-domain B of fibronectin, fused to human interleukin-2 in advanced metastatic melanoma. In one arm, patients received dacarbazine (DTIC; 1000 mg/m2 of body surface on day 1 of 21-day cycles) as single agent, while in two other arms L19IL2 (22.5 million international units of IL2 equivalents) was added, based on two different schedules of administration. In total, 69 patients with stage IV melanoma were enrolled (24 in the dacarbazine arm, 23 and 22 in the other combination arms, respectively) and 67 received treatment. Analyses of efficacy results show a statistically significant benefit in terms of overall response rate and median progression-free survival for patients receiving L19IL2 in combination with DTIC, compared to DTIC as single agent. In light of these results, further clinical investigations with L19IL2 (alone or in combination with other agents) are warranted. [ABSTRACT FROM AUTHOR]

The discovery of protein ligands, capable of forming a reversible covalent bond with amino acid residues on a protein target of interest, may represent a general strategy for the discovery of potent small‐molecule inhibitors. We analyzed the ability of different aromatic aldehydes to form imines by reaction with lysine using 1H NMR techniques. 2‐Hydroxybenzaldehyde derivatives were found to efficiently form imines in the millimolar concentration range. These benzaldehyde derivatives could increase the binding affinity of protein ligands towards the cognate protein target. Affinity maturation was achieved not only by displaying ligand and aldehyde moieties on two complementary locked nucleic acid strands but also by incorporating the binding fragments in a single small‐molecule ligand. The affinity gain was only observed when lysine residues were accessible in the immediate surroundings of the ligand‐binding site and could be abrogated by quenching with a molar excess of hydroxylamine. A lock on affinity: The simultaneous display of protein ligands and of 2‐hydroxybenzaldehyde derivatives on either two complementary LNA strands or a small‐organic molecule leads to an enhanced binding affinity by reversible covalent interaction with proximal lysine residues. [ABSTRACT FROM AUTHOR]

The intratumoral injection of cytokines, in particular IL2, has shown promise for cutaneous melanoma patients with unresectable disease or continuous recurrence despite surgery. We recently reported that the intralesional injection of L19-IL2, an immunocytokine combining IL2 and the human monoclonal antibody fragment L19, resulted in efficient regional control of disease progression, increased time to distant metastasis and evidence of effect on circulating immune cell populations. We have also shown in preclinical models of cancer a remarkable synergistic effect of the combination of L19-IL2 with L19-TNF, a second clinical-stage immunocytokine, based on the same L19 antibody fused to TNF. Here, we describe the results of a phase II clinical trial based on the intralesional administration of L19-IL2 and L19-TNF in patients with stage IIIC and IVM1a metastatic melanoma, who were not candidate to surgery. In 20 efficacy-evaluable patients, 32 melanoma lesions exhibited complete responses upon intralesional administration of the two products, with mild side effects mainly limited to injection site reactions. Importantly, we observed complete responses in 7/13 (53.8 %) non-injected lesions (4 cutaneous, 3 lymph nodes), indicating a systemic activity of the intralesional immunostimulatory treatment. The intralesional administration of L19-IL2 and L19-TNF represents a simple and effective method for the local control of inoperable melanoma lesions, with a potential to eradicate them or make them suitable for a facile surgical removal of the residual mass. [ABSTRACT FROM AUTHOR]

Background and purpose Recently, we have shown that radiotherapy (RT) combined with the immunocytokine L19-IL2 can induce long-lasting antitumour effects, dependent on ED-B expression and infiltration of cytotoxic T cells. On the other hand, in certain tumours, IL2 treatment can trigger a natural killer cell (NK) immune response. The aim of this study is to investigate the therapeutic effect of our combination therapy in the ED-B positive F9 teratocarcinoma model, lacking MHCI expression and known to be dependent on NK immune responses. Material and methods In syngeneic F9 tumour bearing 129/FvHsd mice tumour growth delay was evaluated after local tumour irradiation (10 Gy) combined with systemic administration of L19-IL2. Immunological responses were investigated using flow cytometry. Results Tumour growth delay of L19-IL2 can be further improved by a single dose of RT administered before immunotherapy, but not during immunotherapy. Furthermore, treatment of L19-IL2 favours a NK response and lacks cytotoxic T cell tumour infiltrating immune cells, which may be explained by the absence of MHCI expression. Conclusion An additive effect can be detected when the NK dependent F9 tumour model is treated with radiotherapy and L19-IL2 and therefore this combination could be useful in the absence of tumoural MHCI expression. [ABSTRACT FROM AUTHOR]

Objectives Antibody–cytokine fusion proteins (immunocytokines) represent a novel class of armed antibodies in oncology. In particular, IL2- and TNF-based immunocytokines targeting the EDB domain of fibronectin and the A1 domain of tenascin-C have demonstrated promising anti-tumor activity and are currently investigated in Phase I and Phase II clinical trials. To advance the development of immunocytokines for NSCLC, we here report on the therapeutic efficacy of F8-IL2, an immunocytokine directed against the alternatively spliced EDA domain of fibronectin in a fully immunocompetent, orthotopic model of NSCLC, and the characterization of the target antigen expression in human NSCLC specimens. Materials and methods We evaluated the therapeutic efficacy of the F8-IL2 immunocytokine utilizing a K-ras mutant, p53 deficient metastatic mouse model of NSCLC derived from the latest generation of genetically engineered and conditional tumor models. In parallel, we assessed the presence of the EDA domain of fibronectin by immunofluorescence in lung biopsies obtained from patients with NSCLC. Results The EDA domain of fibronectin was broadly expressed in lung metastases obtained from our model. Treatment with F8-IL2 induced substantial local changes within immune effector cell populations and demonstrated promising therapeutic efficacy as monotherapy. The target of F8-IL2, the EDA domain of fibronectin, was present in all human lung adenocarcinoma specimens tested. Conclusion Both the therapeutic efficacy in a metastatic mouse model of NSCLC and the extensive presence of the EDA domain of fibronectin in human NSCLC biopsies support the rational development of therapies based on the F8-IL2 immunocytokine for the treatment of NSCLC. [ABSTRACT FROM AUTHOR]