Antibody-based targeted delivery of interleukin-4 synergizes with dexamethasone for the reduction of inflammation in arthritis.

Objectives: We have previously reported that F8-IL4, a fusion protein consisting of the F8 antibody specific to the alternatively-spliced extra domain A of fibronectin and of murine IL-4, cures mice with established arthritis, when used in combination with dexamethasone (DXM). The goal of this study was to assess whether other therapeutic agents, besides DXM, could induce cures in combination with F8-IL4 and to elucidate which leucocytes are most affected by the pharmacological treatment. Methods: We performed therapy experiments in mice with CIA, using intravenous administrations of F8-IL4 in combination with DXM, MTX, murine cytotoxic T-lymphocyte-associated protein 4 fused to the fragment crystallizable portion of murine IgG2a, as well as mAbs to murine IL17A or the p40 subunit of murine IL12/IL23. Histology and immunohistochemistry for the identification of the various leucocytes were performed on the paws of mice euthanized at different therapy time points. Results: Only the use of F8-IL4 in combination with DXM induced complete remissions, while all other combinations did not lead to cures. The light microscopical evaluation of paws with arthritis revealed a predominant infiltration of neutrophils, which substantially decreased 24 h after treatment with F8-IL4 and DXM. Conclusion: The combination of F8-IL4 with DXM promotes a rapid anti-arthritic action by potently inhibiting neutrophil activity. A fully human analogue of F8-IL4 may find clinical utility for the treatment of neutrophil-driven chronic inflammatory conditions.

Targeted IL-4 therapy synergizes with dexamethasone to induce a state of tolerance by promoting Treg cells and macrophages in mice with arthritis.

F8-IL-4 is a recently developed immunocytokine that delivers IL-4 to sites of inflammation by targeting the neovasculature. We previously reported that F8-IL-4, in combination with dexamethasone (DXM), provides a durable therapy in mice with collagen-induced arthritis (CIA). Therefore, the objective of this study was to identify the mechanism by which IL-4 and DXM combination therapy provides long-lasting disease remission. F8-IL-4 alone attenuated inflammation in CIA and this was associated with increased TH 2 and decreased TH 17 cell numbers in the joints. Similarly, DXM alone had an antiinflammatory effect associated with lower TH 17 cell numbers. In both cases, these therapeutic benefits were reversed once treatment was stopped. On the other hand, combination therapy with F8-IL-4 plus DXM led to a synergistic increase in the percentage of regulatory T (Treg) cells and antiinflammatory macrophages in the arthritic joint and spleen as well as IL-10 levels in serum and spleen. The net result of this was a more pronounced attenuation of inflammation and, more importantly, protection from arthritis relapse post therapy retraction. In conclusion, F8-IL-4 plus DXM is a durable treatment for arthritis that acts by promoting Treg cells in a synergistic manner, and by producing a sustained increase in antiinflammatory macrophages.

Antibody-based delivery of IL4 to the neovasculature cures mice with arthritis.

Antibody-cytokine fusion proteins (immunocytokines) are innovative biopharmaceutical agents, which are being considered for the therapy of cancer and chronic inflammatory conditions. Immunomodulatory fusion proteins capable of selective localization at the sites of rheumatoid arthritis (RA) are of particular interest, as they may increase the therapeutic index of the cytokine payload. The F8 antibody recognizes the alternatively spliced extra domain A of fibronectin, a marker of angiogenesis, which is strongly overexpressed at sites of arthritis. In this study, we investigated the targeting and therapeutic activity of the immunocytokine F8-IL4 in the mouse model of collagen-induced arthritis. Different combination regimes were tested and evaluated by the analysis of serum and tissue cytokine levels. We show that F8-IL4 selectively localizes to neovascular structures at sites of rheumatoid arthritis in the mouse, leading to high local concentrations of IL4. When used in combination with dexamethasone, F8-IL4 was able to cure mice with established collagen-induced arthritis. Response to treatment was associated with an elevation of IL13 levels and decreased IL6 plasma concentrations. A fully human version of F8-IL4 is currently being developed for clinical investigations.

The antibody-based targeted delivery of interleukin-4 and 12 to the tumor neovasculature eradicates tumors in three mouse models of cancer.

Preclinical studies with recombinant murine interleukin 4 (IL4) in models of cancer have shown potent tumor growth inhibition. However, systemic administration of human IL4 to cancer patients exhibited modest antitumor activity and considerable toxicities. To improve the therapeutic index and reduce side effects of this cytokine, we developed of a novel "immunocytokine" based on sequential fusion of murine IL4 with the antibody fragment F8 (specific to the alternatively spliced extra-domain A of fibronectin, a marker for tumor-angiogenesis) in diabody format. The resulting fusion protein, termed F8-IL4, retained full antigen-binding activity and cytokine bioactivity and was able to selectively localize on solid tumors in vivo. When used as single agent, F8-IL4 inhibited tumor growth in three different immunocompetent murine cancer models (F9 teratocarcinoma, CT26 colon carcinoma and A20 lymphoma). Furthermore, F8-IL4 showed synergistic effects when coadministered with immunocytokines based on IL2 and IL12. Indeed, combination therapy with an IL12-based immunocytokine yielded complete tumor eradication, in spite of the fact that IL4 and IL12 display opposite immunological mechanisms of action in terms of their polarization of T-cell based responses. No weight loss or any signs of toxicity were observed in treated mice, both in monotherapy and in combination, indicating a good tolerability of the immunocytokine treatment. Interestingly, mice cured from CT26 tumors acquired a durable protective antitumor immunity. Depletion experiments indicated that the antitumor activity was mediated by CD8+ T cells and by NK cells.

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).

Using stroma-anchoring cytokines to augment ADCC: a phase 1 trial of F16IL2 and BI 836858 for posttransplant AML relapse.

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.

Isolation and characterization of human monoclonal antibodies specific to MMP-1A, MMP-2 and MMP-3.

Matrix metalloproteinases (MMPs), a group of more than 20 zinc-containing endopeptidases, are up-regulated in many diseases, but the use of MMP inhibitors for therapeutic purposes has often been disappointing, possibly for the limited specificity of the drugs used in clinical trials. In principle, individual MMPs could be specifically drugged by monoclonal antibodies, either by inhibition of their catalytic activity or by antibody-based pharmacodelivery strategies. In this article we describe the isolation and affinity maturation of recombinant antibodies (SP1, SP2, SP3) specific to the murine catalytic domains of MMP-1A, MMP-2 and MMP-3. These novel reagents allowed a systematic comparative immunofluorescence analysis of the expression patterns of their cognate antigens in a variety of healthy, cancerous and arthritic murine tissues. While all three MMPs were strongly expressed in tumor and arthritis specimens, MMP-1A was completely undetectable in the normal tissues tested, while MMP-2 and MMP-3 exhibited a weak expression in certain normal tissues (e.g., liver). The new antibodies may serve as building blocks for the development of antibody-based therapy strategies in mouse models of pathology.

Dose escalation and expansion phase I studies with the tumour-targeting antibody-tumour necrosis factor fusion protein L19TNF plus doxorubicin in patients with advanced tumours, including sarcomas.

BACKGROUND: L19TNF is a recombinant fusion protein composed of a human antibody fragment and human tumour necrosis factor. L19TNF targets the EDB domain of oncofetal fibronectin highly expressed in tumour vasculature and induces tumour remission in mouse tumours. We summarise two phase I trials testing a combination of L19TNF with doxorubicin in patients with solid tumours, particularly soft tissue sarcomas (STS). PATIENTS AND METHODS: The first study, an open-label, dose-escalation and expansion phase I study of L19TNF plus doxorubicin, enrolled 27 patients. Three cohorts (10.4-17 µg/kg L19TNF) of patients received L19TNF intravenously at days 1, 3, and 5 and doxorubicin (75 mg/m2, then 60 mg/m2) on day 1 every 3 weeks. The expansion cohort enrolled patients with STS. The second study tried to re-escalate the doxorubicin dose to 75 mg/m2 with 13 µg/kg L19TNF. Among primary objectives was the establishment of a recommended dose (RD). RESULTS: The combination was safely applicable. Dose-limiting toxicity occurred either at 17 µg/kg L19TNF or at 75 mg/m2 doxorubicin. RD is 13 µg/kg L19TNF plus 60 mg/m2 doxorubicin. In 15 STS patients of the extension cohort evaluable for efficacy, antitumour activity was observed with complete remission in 1, partial remission in 1 and minor tumour shrinkage in 7 patients. The median overall survival for this heavily pretreated cohort was 14.9 months. CONCLUSION: L19TNF can be safely applied in combination with doxorubicin and induces encouraging tumour remissions in patients with soft tissue sarcomas.

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.

Antibody-mediated delivery of VEGF-C potently reduces chronic skin inflammation.

VEGF-C is an important mediator of lymphangiogenesis and has been shown to alleviate chronic inflammation in a variety of disease models. In this study, we investigated whether targeted delivery of VEGF-C to sites of inflammation and site-specific activation of lymphatic vessels would represent a clinically feasible strategy for treating chronic skin inflammation. To this end, we generated a fusion protein consisting of human VEGF-C fused to the F8 antibody (F8-VEGF-C), which is specific for the alternatively spliced, angiogenesis-marking extradomain A (EDA) of fibronectin. In two mouse models of psoriasis-like skin inflammation, mediated by transgenic VEGF-A overexpression or repeated application of imiquimod, intravenous treatment with F8-VEGF-C but not with untargeted VEGF-C significantly reduced ear skin edema and was as effective as the clinically used TNF-α receptor-Fc fusion protein (TNFR-Fc). Treatment with F8-VEGF-C led to a marked expansion of lymphatic vessels in the inflamed skin and significantly improved lymphatic drainage function. At the same time, treatment with F8-VEGF-C significantly reduced leukocyte numbers, including CD4+ and γδ T cells. In sum, our results reveal that targeted delivery of VEGF-C and site-specific induction of lymphatic vessels represent a potentially new and promising approach for the treatment of chronic inflammatory diseases.

Therapeutic efficacy of the F8-IL2 immunocytokine in a metastatic mouse model of lung adenocarcinoma.

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.

The Targeted Delivery of Interleukin 4 Inhibits Development of Endometriotic Lesions in a Mouse Model.

Endometriosis is caused by the displacement of endometrium outside the uterus contributing heavily to infertility and debilitating pelvic pain. Ectopic adhesion and growth are believed to occur under the influence of a favorable hormonal environment and immunological factors. The objective of this study is to analyze the effect of a targeted therapy with an antibody-based pharmacodelivery of interleukin 4 (F8-IL4) in a mouse model of experimentally induced endometriosis. Endometriosis-like lesions were induced in Balb/c mice. The animals were treated intravenously with F8-IL4 or with untargeted IL4 (KSF-IL4). Twelve days after disease induction, the lesions were isolated. A significant reduction in the number of total lesions/mouse and in the total volume of lesions/mouse was observed in mice treated with F8-IL4 compared to controls (P = .029 and P = .006, respectively), while no difference was found between KSF-IL4-treated mice and their controls. Gene expression was evaluated by quantitative real-time polymerase chain reaction. Expression of genes involved in cell adhesion, extracellular matrix invasion, and neovascularization was significantly downregulated in F8-IL4-treated mice compared to their controls (integrin ß1: P = .02; metalloproteinase [MMP] 3: P = .02; MMP9: P = .04; vascular endothelial growth factor: P = .04). Gene expression of inflammatory cytokines (tumor necrosis factor α, IL1ß, IL1α, and IL6) did not vary in the ectopic lesions isolated from F8-IL4-treated mice compared to their controls. Immunohistochemistry demonstrated a significantly reduced expression of E-cadherin and ß-catenin in the lesions of mice treated with F8-IL4. Our results show that the antibody-mediated targeted delivery of IL4 inhibits the development of endometriosis in a syngeneic mouse model by likely impairing adhesion, invasion, and vascularization of the ectopic endometrium.

The dose-dependent tumor targeting of antibody-IFNγ fusion proteins reveals an unexpected receptor-trapping mechanism in vivo.

Cytokines often display substantial toxicities at low concentrations, preventing their escalation for therapeutic treatment of cancer. Fusion proteins comprising cytokines and recombinant antibodies may improve the anticancer activity of proinflammatory cytokines. Murine IFNγ was appended in the diabody format at the C-terminus of the F8 antibody, generating the F8-IFNγ fusion protein. The F8 antibody is specific for the extra-domain A (EDA) of fibronectin, a tumor-associated antigen that is expressed in the vasculature and stroma of almost all tumor types. Tumor-targeting properties were measured in vivo using a radioiodinated preparation of the fusion protein. Therapy experiments were performed in three syngeneic murine models of cancer [F9 teratocarcinoma, WEHI-164 fibrosarcoma, and Lewis lung carcinoma (LLC)]. F8-IFNγ retained the biologic activity of both the antibody and the cytokine moiety in vitro, but, unlike the parental F8 antibody, it did not preferentially localize to the tumors in vivo. However, when unlabeled F8-IFNγ was administered before radioiodinated F8-IFNγ, a selective accumulation at the tumor site was observed. F8-IFNγ showed dose-dependent anticancer activity with a clear superiority over untargeted recombinant IFNγ. The anticancer activity was potentiated by combining with F8-IL4 without additional toxicities, whereas combination of F8-IFNγ with F8-TNF was lethal in all mice. Unlike other antibody-cytokine fusions, the use of IFNγ as payload for anticancer therapy is associated with a receptor-trapping mechanism, which can be overcome by the administration of a sufficiently large amount of the fusion protein without any detectable toxicity at the doses used.

Tumor targeting properties of antibody fusion proteins based on different members of the murine tumor necrosis superfamily.

The tumor necrosis factor superfamily (TNFSF) consists of more than 20 members that can modulate cellular and immunological functions, including cell survival and the stimulation of an inflammatory response. Many TNF superfamily members display potent anticancer activity when used as recombinant proteins in vitro and in vivo. While TNF, TRAIL and FasL have already been used as payloads in antibody-based pharmacodelivery strategies, most TNF superfamily members have not yet been investigated as antibody payloads. Here, we report the cloning, production and characterization of eight novel antibody fusion proteins based on CD40L, FasL, TRAIL, LiGHT, VEGI, lymphotoxin alpha, lymphotoxin beta and lymphotoxin alpha1/beta2. The monoclonal antibody F8 was chosen as fusion partner of proven tumor targeting performance, which recognizes the alternatively-spliced EDA domain of fibronectin, a marker of angiogenesis. A quantitative biodistribution analysis performed with radioiodinated protein preparations in tumor-bearing mice revealed that TRAIL and lymphotoxin alpha1/beta2 were able to selectively accumulate at the tumor site, while all other members of the TNF superfamily abrogated the selective tumor targeting performance of the parental antibody or accumulated also in healthy tissues. The study indicates that even cytokines, which are closely related in terms of structure and function, may have a substantially different impact on the biodistribution and functional properties of the corresponding fusions with disease-homing antibodies.

Antibody-mediated delivery of interleukin 4 to the neo-vasculature reduces chronic skin inflammation.

BACKGROUND: The antibody-mediated delivery of cytokines ("immunocytokines") to sites of pathological angiogenesis represents an attractive strategy for the development of innovative biopharmaceuticals, capable of modulating the activity of the immune system in cancer and in chronic inflammatory conditions. OBJECTIVE: Recombinant IL4 has previously been shown to be therapeutically active in patients with psoriasis. The antibody-mediated delivery of this cytokine to sites of chronic skin inflammatory conditions should lead to an improved potency and selectivity, compared to non-targeted IL4. METHODS: The therapeutic activity of F8-IL4, a fusion protein of the F8 antibody (specific to the alternatively-spliced EDA domain of fibronectin) with murine IL4, was investigated in three immunocompetent mouse models of skin inflammation: two induced by the TLR7/8 ligand imiquimod (in Balb/c and C57BL/6) and one mediated by the over-expression of VEGF-A. RESULTS: The EDA domain of fibronectin, a marker for angiogenesis, is expressed in the inflamed skin in all three models and F8-IL4 selectively localized to inflamed skin lesions following intravenous administration. The F8-IL4 fusion protein mediated a therapeutic benefit, which was superior to the one of a non-targeted version of IL4 and led to increased levels of key regulatory cytokines (including IL5, IL10, IL13, and IL27) in the inflamed skin, while IL2 levels were not affected in all treatment groups. A murine version of etanercept and a murine anti-IL17 antibody were used as positive control in the therapy experiments. CONCLUSION: Skin inflammatory lesions can be selectively targeted using anti-EDA antibody-cytokine fusion proteins and the pharmacodelivery of IL4 confers a therapeutic benefit by shifting the cytokine balance.

A highly functional synthetic phage display library containing over 40 billion human antibody clones.

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics.

The immunocytokine L19-IL2 eradicates cancer when used in combination with CTLA-4 blockade or with L19-TNF.

Systemic high-dose IL2 promotes long-term survival in a subset of metastatic melanoma patients, but this treatment is accompanied by severe toxicities. The immunocytokine L19-IL2, in which IL2 is fused to the human L19 antibody capable of selective accumulation on tumor neovasculature, has recently shown encouraging clinical activity in patients with metastatic melanoma. In this study, we have investigated the therapeutic performance of L19-IL2, administered systemically in combination with a murine anti-CTLA-4 antibody or with a second clinical-stage immunocytokine (L19-TNF) in two syngeneic immunocompetent mouse models of cancer. We observed complete tumor eradications when L19-IL2 was used in combination with CTLA-4 blockade. Interestingly, mice cured from F9 tumors developed new lesions when rechallenged with tumor cells after therapy, whereas mice cured from CT26 tumors were resistant to tumor rechallenge. Similarly, L19-IL2 induced complete remissions when administered in a single intratumoral injection in combination with L19-TNF, whereas the two components did not lead to cures when administered as single agents. These findings provide a rationale for combination trials in melanoma, as the individual therapeutic agents have been extensively studied in clinical trials, and the antigen recognized by the L19 antibody has an identical sequence in mouse and man.

Murine analogues of etanercept and of F8-IL10 inhibit the progression of collagen-induced arthritis in the mouse.

INTRODUCTION: Etanercept is a fusion protein consisting of the soluble portion of the p75-tumor necrosis factor receptor (TNFR) and the Fc fragment of human IgG1, which is often used for the treatment of patients with rheumatoid arthritis. F8-IL10 is a human immunocytokine based on the F8 antibody and interleukin-10, which is currently being investigated in rheumatoid arthritis with promising clinical results. We have aimed at expressing murine versions of these two fusion proteins, in order to assess their pharmaceutical performance in the collagen-induced model of rheumatoid arthritis in the mouse. METHODS: Two fusion proteins (termed muTNFR-Fc and F8-muIL10) were cloned, expressed in chinese hamster ovary (CHO) cells, purified and characterized. Biological activity of muTNFR-Fc was assessed by its ability to inhibit TNF-induced killing of mouse fibroblasts, while F8-muIL10 was characterized in terms of muIL10 activity, of binding affinity to the cognate antigen of F8, the alternatively-spliced EDA domain of fibronectin, by quantitative biodistribution analysis and in vivo imaging. The therapeutic activity of both fusion proteins was investigated in a collagen-induced mouse model of arthritis. Mouse plasma was analyzed for anti-drug antibody formation and cytokine levels were determined by bead-based multiplex technology. The association of F8-IL10 proteins with blood cells was studied in a centrifugation assay with radiolabeled protein. RESULTS: Both fusion proteins exhibited excellent purity and full biological activity in vitro. In addition, F8-muIL10 was able to localize on newly-formed blood vessels in vivo. When used in a murine model of arthritis, the two proteins inhibited arthritis progression. The activity of muTNFR-Fc was tested alone and in combination with F8-huIL10. The chimeric version of F8-IL10 was not better then the fully human fusion protein and showed similar generation of mouse anti-fusion protein antibodies. Incubation studies of F8-muIL10 and F8-huIL10 with blood revealed that only the fully human fusion protein is not associated with cellular components at concentrations as low as 0.2 µg/ml, thus facilitating its extravasation from blood vessels. CONCLUSIONS: The described products may represent useful research tools for the study of the anti-arthritic properties of TNF blockade and of IL10-based immunocytokines in syngeneic immunocompetent models of arthritis.

A critical evaluation of the tumor-targeting properties of bispecific antibodies based on quantitative biodistribution data.

Bispecific and bifunctional antibodies are attracting considerable interest as innovative anti-cancer therapeutics, but their ability to selectively localize at the tumor site has rarely been studied by quantitative biodistribution studies in immunocompetent animal models or in patients. Here, we describe the production of a novel bifunctional antibody, consisting of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin) fused to the extracellular portion of CD86 (co-stimulatory molecule B7.2). However, the fusion molecule was unable to target tumors in vivo. These data suggest that bispecific antibodies do not always localize on tumors and should therefore be characterized by imaging or biodistribution studies.