Manuela Kaspar

A high-affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo-vasculature in vivo

The alternatively spliced extra‐domain B of fibronectin is one of the best characterized markers of tumor angiogenesis. Similarly, the extra‐domain A (EDA), which can also be inserted in the fibronectin transcript by a mechanism of alternative splicing, has been shown to preferentially accumulate around new blood vessels in certain tumors, but this antigen has not been investigated so far as a target for antibody‐based biomolecular intervention. We here describe the generation of 3 human monoclonal antibodies (named F8, B7 and D5), which recognize the same epitope of EDA, but which differ in terms of their dissociation constant to the human antigen (KD = 3.1, 16 and 17 nM, measured for monomeric preparations of the F8, B7 and D5 antibodies, respectively, in recombinant scFv format). When the 3 antibody fragments were cloned and expressed with a 5 amino acid linker, the 3 resulting homodimeric antibody preparations displayed comparable tumor: organ ratios in quantitative biodistribution studies, performed in immunocompetent 129SvEv mice, bearing subcutaneous syngeneic F9 murine tumors. The percent injected dose per gram (%ID/g) values in tumors 24 hr after intravenous injection were 9.3, 10.2 and 13 for F8, B7 and D5, respectively. The F8 antibody may serve as useful building block for the development of antibody‐based targeted anti‐cancer therapeutics. Preclinical and clinical investigations are facilitated by the fact that F8 recognizes the human and mouse antigen with comparable affinity, and by the observation that EDA over‐expression is detectable not only in solid tumors, but also in hematological malignancies.

Fibronectin as target for tumor therapy

During cancer progression, the extracellular matrix (ECM) of the tissue in which the tumor grows is extensively remodeled, both by degradation of preexisting ECM molecules and by the neosynthesis of ECM components, which in many cases are not present in the ECM of normal tissues. Fibronectin (FN), a class of high‐molecular‐weight adhesive glycoproteins, plays a prominent role in mediating ECM function, because of its high abundance and its interaction with cellular components. Furthermore, the generation of tumor‐associated FN isoforms allows the development of specific ligands (e.g., antibodies), which can be used for the selective delivery of therapeutic agents to the tumor environment. In view of these considerations, it is not surprising that FN is being used as a target for biomolecular intervention, both for the development of inhibitory molecules that block the interaction of FN with integrins and other receptors on the cell surface, and for the development of ligand‐based targeted imaging and therapeutic strategies. In this review, we briefly present the essential properties of FN, and we then focus on the therapeutic strategies that are currently in preclinical or clinical development and feature FN as a target, or that are based on FN fragments so as to promote tumor‐growth inhibition.

The Extra-domain A of Fibronectin Is a Vascular Marker of Solid Tumors and Metastases

One of the most promising new avenues for the development of more selective and efficacious cancer therapies relies on the antibody-mediated targeted delivery of bioactive agents (e.g., cytokines) to the tumor environment. The identification of quantitative differences in the expression of accessible vascular proteins in metastatic lesions and host organs facilitate the development of antibody-based strategies, which should be highly efficient and selective, considering the fact that an over-exuberant neovasculature is a characteristic feature of aggressive cancers, and that tumor blood vessels are readily accessible for i.v. administered therapeutic agents. Metastasis is the main cause of death in cancer. The availability of metastasis-specific antigens accessible from the bloodstream will allow a selective delivery of therapeutic agents to metastatic lesions using antibodies as vehicles. Using a combination of vascular biotinylation of 129Sv mice bearing F9 liver metastases and mass spectrometry, we have identified 435 accessible proteins in metastasis and host organ specimens, of which 117 were exclusively detected in metastases. In particular, we found that the alternatively spliced extra-domain A (EDA) of fibronectin is strongly expressed in the neovasculature of liver metastases, while being undetectable in most normal organs. A human antibody to EDA was used to show EDA expression in the neovasculature of metastases and primary tumors of human cancer patients and to target mouse liver metastases and subcutaneous tumors in vivo. Human antibody fragments specific to the EDA domain of fibronectin promise to serve as general vehicles for the efficient and selective delivery of imaging agents or therapeutic molecules to metastatic sites.

Complete eradication of human B-cell lymphoma xenografts using rituximab in combination with the immunocytokine L19-IL2.

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.

Preclinical characterization of DEKAVIL (F8-IL10), a novel clinical-stage immunocytokine which inhibits the progression of collagen-induced arthritis

IntroductionIn this article, we present a comparative immunohistochemical evaluation of four clinical-stage antibodies (L19, F16, G11 and F8) directed against splice isoforms of fibronectin and of tenascin-C for their ability to stain synovial tissue alterations in rheumatoid arthritis patients. Furthermore we have evaluated the therapeutic potential of the most promising antibody, F8, fused to the anti-inflammatory cytokine interleukin (IL) 10.MethodsF8-IL10 was produced and purified to homogeneity in CHO cells and shown to comprise biological active antibody and cytokine moieties by binding assays on recombinant antigen and by MC/9 cell proliferation assays. We have also characterized the ability of F8-IL10 to inhibit arthritis progression in the collagen-induced arthritis mouse model.ResultsThe human antibody F8, specific to the extra-domain A of fibronectin, exhibited the strongest and most homogenous staining pattern in synovial biopsies and was thus selected for the development of a fully human fusion protein with IL10 (F8-IL10, also named DEKAVIL). Following radioiodination, F8-IL10 was able to selectively target arthritic lesions and tumor neo-vascular structures in mice, as evidenced by autoradiographic analysis and quantitative biodistribution studies. The subcutaneous administration route led to equivalent targeting results when compared with intravenous administration and was thus selected for the clinical development of the product. F8-IL10 potently inhibited progression of established arthritis in the collagen-induced mouse model when tested alone and in combination with methotrexate. In preparation for clinical trials in patients with rheumatoid arthritis, F8-IL10 was studied in rodents and in cynomolgus monkeys, revealing an excellent safety profile at doses tenfold higher than the planned starting dose for clinical phase I trials.ConclusionsFollowing the encouraging preclinical results presented in this paper, clinical trials with F8-IL10 will now elucidate the therapeutic potential of this product and whether the targeted delivery of IL10 potentiates the anti-arthritic action of the cytokine in rheumatoid arthritis patients.

The antibody-mediated targeted delivery of interleukin-15 and GM-CSF to the tumor neovasculature inhibits tumor growth and metastasis.

Tumor-targeting immunocytokines represent a new class of anticancer pharmaceutical agents, which often display a superior therapeutic index compared with the corresponding unconjugated cytokines. In this article, we have studied the anticancer properties of interleukin-15 (IL-15) and granulocyte macrophage colony-stimulating factor (GM-CSF), fused to the human antibody fragment scFv(L19), specific to the EDB domain of fibronectin, a marker of angiogenesis. The immunocytokines L19-IL-15 and L19-GM-CSF were expressed in mammalian cells and purified to homogeneity, revealing no loss of cytokine activity in in vitro assays. Furthermore, the ability of the two immunocytokines to selectively localize to tumors in vivo was confirmed by biodistribution analysis with radioiodinated protein preparations. L19-IL-15 and L19-GM-CSF displayed a potent antitumor activity both in s.c. and in metastatic F9 and C51 murine models of cancer in immunocompetent mice. This therapeutic action was superior compared with IL-15-based and GM-CSF-based fusion proteins, containing antibodies of irrelevant specificity in the mouse, which were used as non-tumor-targeting controls. For both L19-IL-15 and L19-GM-CSF immunocytokines, CD8(+) T cells seemed to mostly contribute to the therapeutic action as shown by in vivo cell depletion experiments. The results presented in this article are of clinical significance, considering the fact that the sequence of EDB is identical in mouse and man and that the tumor-targeting ability of the L19 antibody has been extensively shown in clinical trials in patients with cancer.

The tumour-targeting human L19-IL2 immunocytokine: Preclinical safety studies, phase I clinical trial in patients with solid tumours and expansion into patients with advanced renal cell carcinoma

BACKGROUND L19-IL2, a tumour-targeting immunocytokine composed of the recombinant human antibody fragment L19 (specific to the alternatively-spliced EDB domain of fibronectin, a well characterised marker of tumour neo-vasculature) and of human IL2, has demonstrated strong therapeutic activity in animal cancer models. This phase I/II trial was performed to evaluate safety, tolerability, recommended phase II dose (RD) and early signs of activity of L19-IL2. PATIENTS AND METHODS Five cohorts of patients with progressive solid tumours (n=21) received an intravenous infusion of L19-IL2 (from 5 to 30 Mio IU IL2 equivalent dose) on days 1, 3 and 5 every 3 weeks. This treatment cycle was repeated up to six times. In the following expansion phase, patients with metastatic renal cell carcinoma (RCC) (n=12) were treated at the RD of L19-IL2. Clinical data and laboratory findings were analysed for safety, tolerability and activity. RESULTS Preclinical studies in rats and monkeys did not raise any safety concerns. The RD was defined to be 22.5 Mio IU IL2 equivalent. Pharmacokinetics of L19-IL2 was dose proportional over the tested range, with a terminal half-life of 2-3h. Toxicities were manageable and reversible with no treatment-related deaths. We observed stable disease in 17/33 patients (51%) and 15/18 with mRCC (83%) after two cycles. Median progression-free survival of RCC patients in the expansion phase of the study was 8 months (1.5-30.5). CONCLUSIONS L19-IL2 can be safely and repeatedly administered at the RD of 22.5 Mio IU IL2 equivalent in advanced solid tumours. Preliminary evaluation suggests clinical activity of L19-IL2 in patients with mRCC.

Engineered vascular‐targeting antibody‐interferon‐γ fusion protein for cancer therapy

A number of cytokines are either approved drugs or are in advanced clinical trials, yet these biopharmaceuticals do not typically localize efficiently in solid tumors and manifest their therapeutic potential at the expense of severe side effects. The targeted delivery of cytokines to solid tumors is a promising avenue for increasing the therapeutic index of these biopharmaceuticals. We engineered a fusion protein between scFv(L19), a human antibody fragment specific to the EDB domain of fibronectin, and a cysteine‐free mutant of murine interferon‐γ. The resulting fusion protein was capable of targeting new blood vessels in solid tumors, and the targeting efficiency was strikingly increased in tumor‐bearing knockout mice lacking the interferon‐γ receptor. ScFv(L19)‐interferon‐γ displayed a strong antitumor effect in both subcutaneous and metastatic murine F9 teratocarcinomas, but was not efficacious as single agent when used to treat C51 and CT26 tumors. The potency of this fusion protein could be substantially enhanced by combination with doxorubicin and other immunocytokines. These findings are of clinical relevance, as the EDB domain is a marker of angiogenesis, with identical sequence in mouse and man, which is abundantly expressed in a variety of aggressive solid tumors but is undetectable in most normal tissues. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience. wiley.com/jpages/0020‐7136/suppmat/index.html.

A Novel, Non-immunogenic Fyn SH3-derived Binding Protein with Tumor Vascular Targeting Properties

The generation of novel binding molecules based on protein frameworks (“scaffolds”) represents an emerging field in protein engineering, with the potential to replace antibodies for many research and clinical applications. Here, we describe the design, construction, characterization, and use of a novel human Fyn SH3 phage library, containing 1.2 × 109 individual clone members. We also present the isolation and in vitro characterization of Fyn SH3-derived proteins binding to the extra-domain B of fibronectin, a marker of angiogenesis. One specific binding clone, named D3, was further evaluated and showed a remarkable ability to stain vascular structures in tumor sections. Furthermore, quantitative biodistribution studies in tumor-bearing mice revealed the ability of D3 to selectively accumulate in the tumor. In contrast to human scFv antibody fragments administered to mice, neither Fyn SH3 WT nor the D3 mutant was immunogenic in mice after four intravenous injections. The extra-domain B binding D3 protein opens new biomedical opportunities for the in vivo imaging of solid tumors and for the delivery of toxic agents to the tumoral vasculature.

A Dose-Escalation and Signal-Generating Study of the Immunocytokine L19-IL2 in Combination with Dacarbazine for the Therapy of Patients with Metastatic Melanoma

Purpose: L19-IL2 is an immunocytokine composed of an antibody fragment specific to the EDB domain of fibronectin, a tumor angiogenesis marker, and of human interleukin-2 (IL2). L19-IL2 delivers IL2 to the tumor site exploiting the selective expression of EDB on newly formed blood vessels. Previously, the recommended dose of L19-IL2 monotherapy was defined as 22.5 million international units (Mio IU) IL2 equivalents. In this study, safety and clinical activity of L19-IL2 in combination with dacarbazine were assessed in patients with metastatic melanoma. Experimental Design: The first 10 studied patients received escalating doses of L19-IL2 on days 1, 3, and 5 in combination with 1 g/m2 of dacarbazine on day 1 of a 3-weekly therapy cycle. Subsequently, 22 patients received L19-IL2 at recommended dose plus dacarbazine. Up to six treatment cycles were given, followed by a maintenance regimen with biweekly L19-IL2. Results: The recommended dose of L19-IL2 in combination with dacarbazine was defined as 22.5 Mio IU. Toxicity was manageable and reversible, with no treatment-related deaths. Twenty-nine patients were evaluable for efficacy according to Response Evaluation Criteria in Solid Tumors (RECIST). In a centralized radiology analysis, eight of 29 (28%) patients achieved a RECIST-confirmed objective response, including a complete response still ongoing 21 months after treatment beginning. The 12-month survival rate and median overall survival of the recommended dose–treated patients (n = 26) were 61.5% and 14.1 months, respectively. Conclusions: The repeated administration of L19-IL2 in combination with dacarbazine is safe and shows encouraging signs of clinical activity in patients with metastatic melanoma. This combination therapy is currently evaluated in a randomized phase II trial with patients with metastatic melanoma. Clin Cancer Res; 17(24); 7732–42. ©2011 AACR.