An IL-7 fusion protein targeting EDA fibronectin upregulates TCF1 on CD8+ T-cells, preferentially accumulates to neoplastic lesions, and boosts PD-1 blockade.

BACKGROUND: Anti-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. METHODS: In 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. RESULTS: F8(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. CONCLUSIONS: Our results provide the rationale to explore the combination of F8(scDb)-IL7 with anti-PD-1 antibodies for the treatment of patients with cancer.

Cytokine Biopharmaceuticals with "Activity-on-Demand" for Cancer Therapy.

Cytokines are small proteins that modulate the activity of the immune system. Because of their potent immunomodulatory properties, some recombinant cytokines have undergone clinical development and have gained marketing authorization for the therapy of certain forms of cancer. Recombinant cytokines are typically administered at ultralow doses, as many of them can cause substantial toxicity even at submilligram quantities. In an attempt to increase the therapeutic index, fusion proteins based on tumor-homing antibodies (also called "immunocytokines") have been considered, and some products in this class have reached late-stage clinical trials. While antibody-cytokine fusions, which preferentially localize in the neoplastic mass, can activate tumor-resident leukocytes and may be more efficacious than their nontargeted counterparts, such products typically conserve an intact cytokine activity, which may prevent escalation to curative doses. To further improve tolerability, several strategies have been conceived for the development of antibody-cytokine fusions with "activity-on-demand", acting on tumors but helping spare normal tissues from undesired toxicity. In this article, we have reviewed some of the most promising strategies, outlining their potential as well as possible limitations.

Optimizing the Design and Geometry of T Cell-Engaging Bispecific Antibodies Targeting CEA in Colorectal Cancer.

Metastatic colorectal cancer remains a leading cause of cancer-related deaths, with a 5-year survival rate of only 15%. T cell-engaging bispecific antibodies (TCBs) represent a class of biopharmaceuticals that redirect cytotoxic T cells toward tumor cells, thereby turning immunologically "cold" tumors into "hot" ones. The carcinoembryonic antigen (CEA) is an attractive tumor-associated antigen that is overexpressed in more than 98% of patients with colorectal cancer. In this study, we report the comparison of four different TCB formats employing the antibodies F4 (targeting human CEA) and 2C11 (targeting mouse CD3ε). These formats include both antibody fragment-based and IgG-based constructs, with either one or two binding specificities of the respective antibodies. The 2 + 1 arrangement, using an anti-CEA single-chain diabody fused to an anti-CD3 single-chain variable fragment, emerged as the most potent design, showing tumor killing at subnanomolar concentrations across three different CEA+ cell lines. The in vitro activity was three times greater in C57BL/6 mouse colon adenocarcinoma cells (MC38) expressing high levels of CEA compared with those expressing low levels, highlighting the impact of CEA density in this assay. The optimal TCB candidate was tested in two different immunocompetent mouse models of colorectal cancer and showed tumor growth retardation. Ex vivo analysis of tumor infiltrates showed an increase in CD4+ and CD8+ T cells upon TCB treatment. This study suggests that bivalent tumor targeting, monovalent T-cell targeting, and a short spatial separation are promising characteristics for CEA-targeting TCBs.

A novel strategy to generate immunocytokines with activity-on-demand using small molecule inhibitors.

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.

Generation of a novel fully human non-superagonistic anti-CD28 antibody with efficient and safe T-cell co-stimulation properties.

Antibody-based therapeutics represent an important class of biopharmaceuticals in cancer immunotherapy. CD3 bispecific T-cell engagers activate cytotoxic T-cells and have shown remarkable clinical outcomes against several hematological malignancies. The absence of a costimulatory signal through CD28 typically leads to insufficient T-cell activation and early exhaustion. The combination of CD3 and CD28 targeting products offers an attractive strategy to boost T-cell activity. However, the development of CD28-targeting therapies ceased after TeGenero's Phase 1 trial in 2006 evaluating a superagonistic anti-CD28 antibody (TGN1412) resulted in severe life-threatening side effects. Here, we describe the generation of a novel fully human anti-CD28 antibody termed "E1P2" using phage display technology. E1P2 bound to human and mouse CD28 as shown by flow cytometry on primary human and mouse T-cells. Epitope mapping revealed a conformational binding epitope for E1P2 close to the apex of CD28, similar to its natural ligand and unlike the lateral epitope of TGN1412. E1P2, in contrast to TGN1412, showed no signs of in vitro superagonistic properties on human peripheral blood mononuclear cells (PBMCs) using different healthy donors. Importantly, an in vivo safety study in humanized NSG mice using E1P2, in direct comparison and contrast to TGN1412, did not cause cytokine release syndrome. In an in vitro activity assay using human PBMCs, the combination of E1P2 with CD3 bispecific antibodies enhanced tumor cell killing and T-cell proliferation. Collectively, these data demonstrate the therapeutic potential of E1P2 to improve the activity of T-cell receptor/CD3 activating constructs in targeted immunotherapeutic approaches against cancer or infectious diseases.

Middle Cerebral Artery Dissection: A Report of Two Cases With Treatments and Outcomes.

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.

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 characterization of a novel high-affinity human antibody targeting carcinoembryonic antigen.

There are no effective treatment options for most patients with metastatic colorectal cancer (mCRC). mCRC remains a leading cause of tumor-related death, with a five-year survival rate of only 15%, highlighting the urgent need for novel pharmacological products. Current standard drugs are based on cytotoxic chemotherapy, VEGF inhibitors, EGFR antibodies, and multikinase inhibitors. The antibody-based delivery of pro-inflammatory cytokines provides a promising and differentiated strategy to improve the treatment outcome for mCRC patients. Here, we describe the generation of a novel fully human monoclonal antibody (termed F4) targeting the carcinoembryonic antigen (CEA), a tumor-associated antigen overexpressed in colorectal cancer and other malignancies. The F4 antibody was selected by antibody phage display technology after two rounds of affinity maturation. F4 in single-chain variable fragment format bound to CEA in surface plasmon resonance with an affinity of 7.7 nM. Flow cytometry and immunofluorescence on human cancer specimens confirmed binding to CEA-expressing cells. F4 selectively accumulated in CEA-positive tumors, as evidenced by two orthogonal in vivo biodistribution studies. Encouraged by these results, we genetically fused murine interleukin (IL) 12 to F4 in the single-chain diabody format. F4-IL12 exhibited potent antitumor activity in two murine models of colon cancer. Treatment with F4-IL12 led to an increased density of tumor-infiltrating lymphocytes and an upregulation of interferon γ expression by tumor-homing lymphocytes. These data suggest that the F4 antibody is an attractive delivery vehicle for targeted cancer therapy.

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.

Intravenous thrombolysis in CADASIL: report of two cases and a systematic review.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetic small vessel disease responsible for recurrent ischemic strokes, often with a progressive course leading to dementia and disability. On MRI, lacunes, microbleeds, and severe white matter alterations are typical features of the disease. In case of acute stroke, because of the bleeding risk associated with the disease and the doubtful efficacy of fibrinolytic treatment in a disease with poor evidence of thrombosis, the efficacy of intravenous thrombolysis remains unproven. Nevertheless, stroke is a frequent occurrence in CADASIL patients, and clinicians not unlikely may face in the emergency room the situation of a CADASIL patient with an acute stroke within the time window for thrombolysis. OBJECTIVE: We report on two CADASIL patients treated with intravenous alteplase for acute ischemic stroke, and we present a review of literature aimed to report epidemiological data, efficacy and safety of intravenous thrombolysis in CADASIL patients. METHODS: We performed a systematic review of medical literature published until August 2, 2022. Case reports and series in English language reporting on CADASIL patients and acute stroke were included. RESULTS: Both patients were treated with intravenous thrombolysis without complications and had a good clinical outcome. The systematic review identified three case reports of CADASIL patients who were treated with intravenous alteplase for acute ischemic stroke; no bleedings complications were described. CONCLUSIONS: Available data on intravenous thrombolysis in CADASIL patients are scarce but suggest that this treatment can be taken into consideration for these patients.

Selection of a PD-1 blocking antibody from a novel fully human phage display library.

Programmed cell death protein 1 (PD-1) is an immunoregulatory target which is recognized by different monoclonal antibodies, approved for the therapy of multiple types of cancer. Different anti-PD-1 antibodies display different therapeutic properties and there is a pharmaceutical interest to generate and characterize novel anti-PD-1 antibodies. We screened multiple human antibody phage display libraries to target novel epitopes on the PD-1 surface and we discovered a unique and previously undescribed binding specificity (termed D12) from a new antibody library (termed AMG). The library featured antibody fragments in single-chain fragment variable (scFv) format, based on the IGHV3-23*03 (VH ) and IGKV1-39*01 (Vκ) genes. The D12 antibody was characterized by surface plasmon resonance (SPR), cross-reacted with the Cynomolgus monkey antigen and bound to primary human T cells, as shown by flow cytometry. The antibody blocked the PD-1/PD-L1 interaction in vitro with an EC50 value which was comparable to the one of nivolumab, a clinically approved antibody. The fine details of the interaction between D12 and PD-1 were elucidated by x-ray crystallography of the complex at a 3.5 Å resolution, revealing an unprecedented conformational change at the N-terminus of PD-1 following D12 binding, as well as partial overlap with the binding site for the cognate PD-L1 and PD-L2 ligands which prevents their binding. The results of the study suggest that the expansion of antibody library repertoires may facilitate the discovery of novel binding specificities with unique properties that hold promises for the modulation of PD-1 activity in vitro and in vivo.

A Case of Anti-Leucine-Rich Glioma-Inactivated Protein 1 (Anti-LGI1) Encephalitis With an Unusual Frontomesial Motor Cortex T2 MRI Hyperintensity.

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.

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.

Immunotherapy with Immunocytokines and PD-1 Blockade Enhances the Anticancer Activity of Small Molecule-Drug Conjugates Targeting Carbonic Anhydrase IX.

Small molecule-drug conjugates (SMDCs) represent an alternative to conventional antitumor chemotherapeutic agents, with the potential to improve the therapeutic window of cytotoxic payloads through active delivery at the site of the disease. In this article, we describe novel combination therapies consisting of anti-carbonic anhydrase IX SMDCs combined with different immunomodulatory products. The therapeutic effect of the SMDCs was potentiated by combination with PD-1 blockade and with tumor-homing antibody-cytokine fusions in mouse models of renal cell carcinoma and colorectal cancer. The combination with L19-IL12, a fusion protein specific to the alternatively spliced EDB domain of fibronectin containing the murine IL12 moiety, was also active against large established tumors. Analysis of the microscopic structures of healthy organs performed 3 months after tumor eradication confirmed absence of pathologic abnormalities in the healthy kidney, liver, lung, stomach, and intestine. Our findings may be of clinical significance as they provide motivation for the development of combinations based on SMDCs and immunotherapy for the treatment of renal cell carcinoma and hypoxic tumors.

Immunocytokines are a promising immunotherapeutic approach against glioblastoma.

Glioblastoma is a poorly immunogenic cancer, and the successes with recent immunotherapies in extracranial malignancies have, so far, not been translated to this devastating disease. Therefore, there is an urgent need for new strategies to convert the immunologically cold glioma microenvironment into a hot one to enable effective antitumor immunity. Using the L19 antibody, which is specific to a tumor-associated epitope of extracellular fibronectin, we developed antibody-cytokine fusions-immunocytokines-with interleukin-2 (IL2), IL12, or tumor necrosis factor (TNF). We showed that L19 accumulated in the tumor microenvironment of two orthotopic immunocompetent mouse glioma models. Furthermore, intravenous administration of L19-mIL12 or L19-mTNF cured a proportion of tumor-bearing mice, whereas L19-IL2 did not. This therapeutic activity was abolished in RAG-/- mice or upon depletion of CD4 or CD8 T cells, suggesting adaptive immunity. Mechanistically, both immunocytokines promoted tumor-infiltrating lymphocytes and increased the amounts of proinflammatory cytokines within the tumor microenvironment. In addition, L19-mTNF induced tumor necrosis. Systemic administration of the fully human L19-TNF fusion protein to patients with glioblastoma (NCT03779230) was safe, decreased regional blood perfusion within the tumor, and was associated with increasing tumor necrosis and an increase in tumor-infiltrating CD4 and CD8 T cells. The extensive preclinical characterization and subsequent clinical translation provide a robust basis for future studies with immunocytokines to treat malignant brain tumors.

The targeted delivery of interleukin-12 to the carcinoembryonic antigen increases the intratumoral density of NK and CD8+ T cell in an immunocompetent mouse model of colorectal cancer.

The recent success achieved by immune checkpoint inhibitors in the field of immuno-oncology has been less evident for the treatment of metastatic colorectal cancer (mCRC) patients. To date, cancer immunotherapy has been efficacious only in few patients bearing high mutational burden (less than 25%) mCRCs. In this Communication, we report the generation of a novel antibody cytokine fusion protein (termed Sm3E-mIL12) targeting the CRC-associated carcinoembryonic antigen (CEA). The antibody moiety bound avidly to CEA when immobilized on solid supports, and selectively stained C51 tumor cells transfected with the antigen (C51-CEA). The cytokine payload retained full activity in vitro, as compared to the parental recombinant interleukin-12 (IL12). Ex vivo microscopic analyses revealed a homogenous distribution of Sm3E-mIL12 in the neoplastic mass upon intravenous administration. In vivo, Sm3E-mIL12 was well tolerated up to 180 µg per mouse. The targeted delivery of IL12 to CEA-expressing C51 carcinomas led to durable complete responses in 60% of the treated mice. The intratumoral density of immune effector cells was markedly increased after the third injection of Sm3E-mIL12, in keeping with the progressive regression of the neoplastic mass. The data suggest that a fully human analogue may be considered for the treatment of patients with mCRC.

Comparative evaluation of bolus and fractionated administration modalities for two antibody-cytokine fusions in immunocompetent tumor-bearing mice.

Antibody-cytokine fusion proteins are being considered as biopharmaceuticals for cancer immunotherapy. Tumor-homing cytokine fusions typically display an improved therapeutic activity compared to the corresponding unmodified cytokine products, but toxicity profiles at equivalent doses are similar, since side effects are mainly driven by the cytokine concentration in blood. In order to explore avenues to harness the therapeutic potential of antibody-cytokine fusions while decreasing potential toxicity, we compared bolus and fractionated administration modalities for two tumor-targeting antibody-cytokine fusion proteins based on human interleukin-2 (IL2) and murine tumor necrosis factor (TNF) (i.e., L19-hIL2 and L19-mTNF) in two murine immunocompetent mouse models of cancer (F9 and C51). A comparative quantitative biodistribution analysis with radio-labeled protein preparations revealed that a fractionated administration of L19-hIL2 could deliver comparable product doses to the tumor with decreased product concentration in blood and normal organs, compared to bolus injection. By contrast, L19-mTNF (a product that causes a selective vascular shutdown in the tumor) accumulated most efficiently after bolus injection. Fractionated schedules allowed the safe administration of a cumulative dose of L19-mTNF, which was 2.5-times higher than the lethal dose for bolus injection. Dose fractionation led to a prolonged tumor growth inhibition for F9 teratocarcinomas, but not for C51 colorectal tumors, which responded best to bolus injection. Thus, dose fractionation may have different outcomes for the same antibody-cytokine product in different biological contexts.

The antibody-based delivery of interleukin-12 to solid tumors boosts NK and CD8+ T cell activity and synergizes with immune checkpoint inhibitors.

We describe the cloning and characterization of a novel fusion protein (termed L19-mIL12), consisting of murine interleukin-12 in single-chain format, sequentially fused to the L19 antibody in tandem diabody format. The fusion protein bound avidly to the cognate antigen (the alternatively spliced EDB domain of fibronectin), retained the activity of the parental cytokine and was able to selectively localize to murine tumors in vivo, as shown by quantitative biodistribution analysis. L19-mIL12 exhibited a potent antitumor activity in immunocompetent mice bearing CT26 carcinomas and WEHI-164 sarcomas, which could be boosted by combination with checkpoint blockade, leading to durable cancer eradication. L19-mIL12 also inhibited tumor growth in mice with Lewis lung carcinoma (LLC), but in this case, cancer cures could not be obtained, both in monotherapy and in combination. A microscopic analysis and a depletion experiment of tumor-infiltrating leukocytes illustrated the contribution of NK cells and CD8+ T cells for the anticancer activity observed in both tumor models. Upon L19-mIL12 treatment, the density of regulatory T cells (Tregs) was strongly increased in LLC, but not in CT26 tumors. A FACS analysis also revealed that the majority of CD8+ T cells in CT26 tumors were specific to the retroviral AH1 antigen.