The antibody-mediated targeted delivery of interleukin-10 inhibits endometriosis in a syngeneic mouse model

BACKGROUND Endometriosis is still a highly underdiagnosed disease, and the current medical and surgical treatment of endometriosis is associated with a high recurrence rate. This study investigates the use of derivatives of the human antibody F8, specific to the alternatively spliced extra-domain A of fibronectin (Fn), for the imaging and treatment of endometriosis. METHODS Immunohistochemistry and immunofluorescence was used to evaluate antigen expression in endometriotic tissue of human endometriosis and of a syngeneic mouse model of the disease. The in vivo targeting performance of a fluorescent derivative of the F8 antibody was assessed by imaging mice with endometriosis using a near-infrared fluorescence imager, 24 h following i.v. injection of the antibody conjugate. Furthermore, the mouse model was used for therapy experiments using two recombinant F8-based immunocytokines [F8-interleukin-10 (IL10) and F8-IL2] or saline for the treatment groups. RESULTS A very strong vascular expression of splice isoforms of Fn and of tenascin-C was observed in human endometriotic lesions by immunohistochemistry and immunofluorescence techniques. After i.v. administration, a selective accumulation of the F8 antibody in endometriotic lesions could be observed in a syngeneic mouse model. These targeting data were used as a basis for therapy experiments with a pro-inflammatory (F8-IL2) and an anti-inflammatory (F8-IL10) cytokine fusion protein of the F8 antibody. The average lesion size in the F8-IL10 treatment group was clearly reduced compared with the saline control group and with the F8-IL2 group, for which no therapeutic effects were observed. CONCLUSIONS The F8 antibody targets endometriotic lesions in vivo in a mouse model of endometriosis and may be used for the non-invasive imaging of the disease and for the pharmacodelivery of anti-inflammatory cytokines, such as IL1

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

Introduction In 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. Methods F8-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. Results The 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. Conclusions Following 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.ISSN:1465-9905ISSN:1465-9913ISSN:1478-6362ISSN:1478-635

A general method for the selection of high-level scFv and IgG antibody expression by stably transfected mammalian cells

The isolation of mammalian cell lines capable of high-yield expression of recombinant antibodies is typically performed by screening multiple individual clones by limiting dilution techniques. A number of experimental strategies have recently been devised to identify high-expressing clones, but protocols are often difficult to implement, time consuming, costly and limited in terms of number of clones which can be screened. In this article, we describe new vectors for the expression of recombinant antibodies in IgG format and in other formats, based on the single-chain Fv module, as well as a high-throughput screening procedure, based on the direct staining of antibodies transiting the membrane of a stably transfected cell, followed by preparative sorting using a high-speed cell sorter. This procedure allows, in one step, to deposit single cells into individual wells of a 96-well microtiter plate (thus facilitating cloning) and to preferentially recover those rare cell populations which express dramatically higher levels of recombinant antibody. Using cell cultures followed by affinity purification techniques, we could confirm that the new vectors and the new screening procedure reliably yield high-expression clones and homogenous protein preparations. We expect that these techniques should find broad applicability for both academic and industrial antibody engineering researc

Expression, engineering and characterization of the tumor-targeting heterodimeric immunocytokine F8-IL12

Proinflammatory cytokines have been used for several years in patients with advanced cancer but their administration is typically associated with severe toxicity hampering their application to therapeutically active regimens. This problem can be overcome by using immunocytokines (cytokines fused to antibody or antibody fragments) which selectively deliver the active cytokine to the tumor environment. Preclinical and recent clinical results confirmed that this approach is a very promising avenue to go. We designed an immunocytokine consisting of the scFv(F8) specific to extra-domain A of fibronectin and the very potent human cytokine interleukin-12 (IL12). The heterodimeric nature of IL12 allows the engineering of various immunocytokine formats, based on different combinations of the two subunits (p35 and p40) together with the scFv. In comparison to monomeric or homodimeric cytokines, the construction of a heterodimeric immunocytokine poses many challenges, e.g. gene dosing, stable high-yield expression as well as good manufacture practice (GMP) purification and characterization. In this paper, we describe the successful construction, characterization and production of the heterodimeric immunocytokine F8-IL12. The positive outcome of this feasibility study leads now to GMP production of F8-IL12, which will soon enter clinical trial

The antibody-mediated targeted delivery of interleukin-10 inhibits endometriosis in a syngeneic mouse model

ISSN:0268-1161ISSN:1460-235

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.ISSN:1465-9905ISSN:1465-9913ISSN:1478-6362ISSN:1478-635

Expression, engineering and characterization of the tumor-targeting heterodimeric immunocytokine F8-IL12

ISSN:1741-0126ISSN:1741-0134ISSN:0269-2139ISSN:1460-213

A general method for the selection of high-level scFv and IgG antibody expression by stably transfected mammalian cells

ISSN:1741-0126ISSN:1741-0134ISSN:0269-2139ISSN:1460-213

Cloning, expression and purification of L19–IL-10 and HyHel10–IL-10

<p><b>Copyright information:</b></p><p>Taken from "Antibody-mediated delivery of IL-10 inhibits the progression of established collagen-induced arthritis"</p><p>http://arthritis-research.com/content/9/1/R9</p><p>Arthritis Research & Therapy 2007;9(1):R9-R9.</p><p>Published online 29 Jan 2007</p><p>PMCID:PMC1860067.</p><p></p> Schematic representation of single-chain variable fragment-IL-10 fusion proteins. Schematic representation of a pcDNA3.1 vector (Invitrogen Basel, Switzerland) containing the essential elements of the L19–IL-10 or HyHEL10–IL-10 fusion proteins. The human IL-10 moiety was fused to the C-terminal of the single-chain Fv antibody fragment by the 15 amino acid linker (SSSSG). The secretion sequence at the N-terminal is required for secretion of recombinant proteins and the Histag at the C-terminal of human IL-10 was used for detection of the fusion proteins. SDS-PAGE analysis of purified fusion proteins: lane 1, molecular-weight marker; lanes 2 and 3, L19–IL-10 under nonreducing and reducing conditions, respectively; lanes 4 and 5, HyHEL-IL-10 under nonreducing and reducing conditions, respectively. Monomeric fusion proteins are expected to have a molecular weight of 46 kDa. The size-exclusion chromatography profile of purified L19–IL-10 (Superdex 200, GE Healthcare, Duebendorf, Switzerland). The peak eluting at a retention volume of 13 ml corresponds to the noncovalent homodimeric form of L19–IL-10, the smaller peak eluting at a retention volume of 16 ml corresponds to the monomeric fraction. (e) Biodistribution profile of L19–IL-10 in 129Sv mice grafted with a subcutaneous F9 tumour (= 4). L19–IL-10 was labelled with I and administered by intravenous (i.v.) injection into tumour-bearing mice (3 μg corresponding to 4 μCi L19–IL-10 per mouse). Mice were sacrificed 24 hours after injection and the tumours and organs were weighed and counted. Values are displayed as percent injected dose per gram (%ID/g); standard errors of the means (SEMs) are indicated