Human monoclonal antibodies to domain C of tenascin-C selectively target solid tumors in vivo

We had previously reported that splice isoforms of tenascin-C containing the extra-domain C are virtually absent in normal adult tissues but are highly abundant in high-grade astrocytomas, with a prominent peri-vascular pattern of expression. We now report that the extra-domain C of tenascin-C is strongly expressed in the majority of lung cancers, with a vascular and stromal pattern of expression. Using antibody phage technology, we have generated a human monoclonal antibody (G11), with a dissociation constant KD = 4.2 nM for the human domain C. The G11 antibody, expressed in scFv and in mini-antibody (SIP) format, as well as a scFv-interleukin-2 fusion protein, was then characterized in quantitative biodistribution studies using mice grafted subcutaneously with U87 gliomas, revealing a selective tumor uptake, with tumor/blood ratios up to 11.8:1 at 24 h. A radioiodinated preparation of SIP(G11) was also investigated in a double tracer study using an orthotopic rat glioma model, confirming the antibody's ability to preferentially localize at the tumor site, with tumor/brain ratios superior to the ones observed with 18F-fluorodeoxyglucose. These tumor-targeting properties, together with the strong immunohistochemical staining of human tumor sections, indicate that the G11 antibody may be used as a portable targeting moiety for the selective delivery of imaging and therapeutic agents to gliomas and lung tumor

A Strategy for the Identification of Differentially Expressed Proteins Secreted by Fibroblasts

Monoclonal antibodies, which recognise markers of the modified tumour extracellular matrix, may be valuable tools for the diagnosis and therapy of cancer. The pattern of expression of some good-quality tumour-associated antigens is known to be regulated by intracellular pH. In this article, a strategy is described for the isolation of human antibodies specific for proteins which are differentially secreted by fibroblasts at different pH values. We panned a large synthetic antibody phage library against biotinylated proteins secreted by normal human dermal fibroblasts, cultured at pH 7.5, in the presence or in the absence of a molar excess of unbiotinylated proteins, secreted by fibroblasts cultured at pH 6.7. A panel of monoclonal antibodies was isolated, whose reactivity was tested by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry

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

Human monoclonal antibodies to domain C of tenascin-C selectively target solid tumors in vivo

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

Investigation of the selective accumulation of the small immunoproteins L19 and G11 in inflamed limbs of arthritic mice

<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> Arthritic mice were injected with SIP(L19)–Alexa750 (Molecular Probes, Leiden, The Netherlands), SIP(G11)–Alexa750 or the control antibody SIP(HyHEL10)–Alexa750. Near-infrared fluorescence imaging analysis was performed 24 hours after injection of the fluorescence-labelled antibodies. Arrows indicate grade 2 swelling in the front paws of the mice. In a similar experiment, arthritic mice were injected with I-labelled SIP(L19), SIP(G11) or SIP control. Uptake of radio-iodinated antibodies was analysed by phosphorimaging 24 hours after injection. The mouse injected with I-labelled SIP(L19) had an arthritic score of 2 in the left paw, whereas the right paw was classified as grade 1 arthritis. In the mouse injected with I-labelled SIP(G11) the left paw was classified as grade 1 arhritis, whereas the right paw was classified as grade 2 arthritis. In the mouse injected with I-labelled SIP(F16) the left paw was classified as grade 2 arthritis, whereas the right paw as grade 1 arthritis