Eveline Trachsel

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.

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.

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.

Antibody-Mediated Delivery of Interleukin-2 to the Stroma of Breast Cancer Strongly Enhances the Potency of Chemotherapy

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 μg 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.

A Portable Albumin Binder from a DNA‐Encoded Chemical Library

Albumin represents the most abundant protein in human plasma, at a concentration of 45 mgmL . To keep physiological production rates to a minimum, albumin displays a long circulatory half-life in mammals thanks to its size above the renal filtration threshold and its unique ability to interact with the neonatal FcRn receptor. Fusions of biopharmaceuticals to albumin or to albumin-binding peptides have been devised to expose the body to adequate concentrations of the therapeutic agent for a sufficiently long period of time, thus improving efficacy and reducing the number of injections. In principle, small organic albumin-binding molecules could be used as functional analogues of albumin-binding peptides. However, although many small molecules are known to bind to albumin, the success in isolating small molecules as portable albumin-binding moieties has been limited, mainly because most albumin binders (for example, ibuprofen) lose binding affinity upon chemical modification. Myristoylation of insulin has been shown to significantly prolong the circulatory half-life, but this modification is not applicable to a broader set of molecules because of its negative effect on solubility. In another example, a 4,4diphenylcyclohexyl moiety has been connected through a phosphodiester bond to the metal chelator diethylenetriaminepentaacetic acid (DTPA) for magnetic resonance imaging (MRI) applications and to short peptides. These compounds display dissociation constants (Kd) from human serum albumin in the 100 mm range and are susceptible to hydrolysis in vivo. Thus, there is a considerable scientific and biotechnological interest in the identification of small portable binders that display a stable noncovalent interaction with serum albumin. Herein, we report the discovery and characterization of a class of 4-(p-iodophenyl)butyric acid derivatives from a DNA-encoded chemical library, which display a stable noncovalent binding interaction with both mouse serum albumin (MSA) and human serum albumin (HSA). One of these portable albumin-binding moieties was used to improve the performance of the contrast agents fluorescein and GdDTPA. HSA-binding molecules were selected from a DNAencoded chemical library consisting of 619 oligonucleotidecompound conjugates carrying a six-base-pair code for identification. After selection, the DNA sequences of the enriched compounds were amplified by PCR and decoded on oligonucleotide microarrays displaying the complementary sequences (Figure 1a), normalizing the signal intensities after selection against the intensities of compounds selected on empty resin (Figure 1b). Some of the identified binding molecules were excluded from further evaluation based on being promiscuous binders or because of the high standard deviations of the signal intensities on the microarrays (64, 313, 453, 454, 619). Several of the selected molecules (428, 533, 535, 539) displayed striking structural similarities. The basic structure featured a 4-phenylbutanoic acid moiety, with different hydrophobic substituents on the phenyl ring. To obtain further insights into structure–activity relationships, DNA-modified analogues containing propanoyl or pentanoyl skeletons, and/or carrying various substituents on the phenyl ring (Figure 1c; 536, 622–632), were characterized in a radioactivity-based chromatographic albumin-binding assay, which allowed a first classification of the potential binders (Retention: 428> 539> 624> 535> 533> 536> 326> others; see the Supporting Information). The absence of retention of compounds with propanoyl (625) and penta[*] S. Tr ssel, F. Buller, Dr. F. Bootz, Dr. Y. Zhang, L. Mannocci, Dr. J. Scheuermann, Prof. Dr. D. Neri Institut f r Pharmazeutische Wissenschaften Departement f r Chemie und Angewandte Biowissenschaften ETH Z rich Wolfgang-Pauli-Strasse 10, 8093 Z rich (Switzerland) Fax: (+41)44-633-1358 E-mail: neri@pharma.ethz.ch

Antibody-mediated delivery of IL-10 inhibits the progression of established collagen-induced arthritis

The antibody-mediated targeted delivery of cytokines to sites of disease is a promising avenue for cancer therapy, but it is largely unexplored for the treatment of chronic inflammatory conditions. Using both radioactive and fluorescent techniques, the human monoclonal antibodies L19 and G11 (specific to two markers of angiogenesis that are virtually undetectable in normal adult tissues) were found to selectively localize at arthritic sites in the murine collagen-induced model of rheumatoid arthritis following intravenous (i.v.) administration. The same animal model was used to study the therapeutic action of the L19 antibody fused to the cytokines IL-2, tumour necrosis factor (TNF) and IL-10. Whereas L19–IL-2 and L19–TNF treatment led to increased arthritic scores and paw swellings, the fusion protein L19–IL-10 displayed a therapeutic activity, which was superior to the activity of IL-10 fused to an antibody of irrelevant specificity in the mouse. The anti-inflammatory cytokine IL-10 has been investigated for the treatment of patients with rheumatoid arthritis, but clinical development plans have been discontinued because of a lack of efficacy. Because the antigen recognised by L19 is strongly expressed at sites of arthritis in humans and identical in both mice and humans, it suggests that the fusion protein L19–IL-10 might help overcome some of the clinical limitations of IL-10 and provide a therapeutic benefit to patients with chronic inflammatory disorders, including arthritis.

An engineered antibody–interleukin-12 fusion protein with enhanced tumor vascular targeting properties

The antibody‐mediated targeted delivery of interleukin‐12 (IL12) to the EDB domain of fibronectin, a marker of angiogenesis, is a promising avenue for enhancing the therapeutic index of this anti‐cancer cytokine. Previous experiments, based on sequential fusion of a single‐chain IL12 derivative to the anti‐EDB antibody fragment scFv(L19) had yielded a therapeutic fusion protein [IL12‐scFv(L19)‐FLAG], which displayed an impressive therapeutic activity in murine models of cancer, in spite of a tumor uptake, which was less efficient compared to the parental unmodified scFv(L19). In this article, we describe the comparative analysis of 3 recombinant fusion proteins comprising the scFv(L19) and IL12 moieties. One of them, in which the p40 and p35 form a covalent heterodimer and in which each subunit is fused to a molecule of scFv(L19), displays an excellent tumor targeting performance in vivo, as assessed by quantitative biodistribution analysis, and a potent anti‐tumor effect, superior to the one of IL12‐scFv(L19)‐FLAG. These results may have a clinical impact, considering the fact that the tumor targeting ability of scFv(L19) in patients with cancer has been demonstrated using scintigraphic methods, and that 2 scFv(L19)‐based antibody‐cytokine fusion are currently entering clinical trials.