RESUMEN
The selective delivery of bioactive agents to tumors reduces toxicity and enhances the efficacy of anticancer therapies. In this study, we show that the antibody F8, which recognizes perivascular and stromal EDA-fibronectin (EDA-Fn), when conjugated to interleukin-2 (F8-IL2) can effectively inhibit the growth of EDA-Fn-expressing melanomas in combination with paclitaxel. We obtained curative effects with paclitaxel administered before the immunocytokine. Coadministration of paclitaxel increased the uptake of F8 in xenografted melanomas, enhancing tumor perfusion and permeability. Paclitaxel also boosted the recruitment of F8-IL2-induced natural killer (NK) cells to the tumor, suggesting a host response as part of the observed therapeutic benefit. In support of this likelihood, NK cell depletion impaired the antitumor effect of paclitaxel plus F8-IL2. Importantly, this combination reduced both the tumor burden and the number of pulmonary metastatic nodules. The combination did not cause cumulative toxicity. Together, our findings offer a preclinical proof that by acting on the tumor stroma paclitaxel potentiates the antitumor activity elicited by a targeted delivery of IL2, thereby supporting the use of immunochemotherapy in the treatment of metastatic melanoma.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos Fitogénicos/uso terapéutico , Fibronectinas/análisis , Interleucina-2/uso terapéutico , Melanoma Experimental/tratamiento farmacológico , Paclitaxel/uso terapéutico , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales Humanizados , Permeabilidad Capilar , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Humanos , Interleucina-2/administración & dosificación , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/química , Melanoma Experimental/patología , Ratones , Paclitaxel/administración & dosificación , Isoformas de Proteínas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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/m(2) 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.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Melanoma/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Estudios de Cohortes , Dacarbazina/administración & dosificación , Dacarbazina/efectos adversos , Dacarbazina/farmacocinética , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Fatiga/inducido químicamente , Femenino , Fiebre/inducido químicamente , Humanos , Masculino , Melanoma/metabolismo , Melanoma/mortalidad , Tasa de Depuración Metabólica , Persona de Mediana Edad , Metástasis de la Neoplasia , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/farmacocinética , Análisis de Supervivencia , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
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 IL10.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Endometriosis/tratamiento farmacológico , Interleucina-10/uso terapéutico , Empalme Alternativo , Animales , Anticuerpos Monoclonales Humanizados , Citocinas/sangre , Endometriosis/patología , Femenino , Fibronectinas/genética , Fibronectinas/inmunología , Humanos , Interleucina-2/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Espectroscopía Infrarroja CortaRESUMEN
Metastasis to regional lymph nodes (LN) is a prognostic indicator for cancer progression. There is a great demand for sensitive and noninvasive methods to detect metastasis to LNs. Whereas conventional in vivo imaging approaches have focused on the detection of cancer cells, lymphangiogenesis within tumor-draining LNs might be the earliest sign of metastasis. In mouse models of LN lymphangiogenesis, we found that systemically injected antibodies to lymphatic epitopes accumulated in the lymphatic vasculature in tissues and LNs. Using a (124)I-labeled antibody against the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), we imaged, for the first time, inflammation- and tumor-draining LNs with expanded lymphatic networks in vivo by positron emission tomography (PET). Anti-LYVE-1 immuno-PET enabled visualization of lymphatic vessel expansion in LNs bearing metastases that were not detected by [(18)F]fluorodeoxyglucose-PET, which is clinically applied to detect cancer metastases. Immuno-PET with lymphatic-specific antibodies may open up new avenues for the early detection of metastasis, and the images obtained might be used as biomarkers for the progression of diseases associated with lymphangiogenesis.
Asunto(s)
Diagnóstico por Imagen , Glicoproteínas/inmunología , Inflamación/complicaciones , Ganglios Linfáticos/diagnóstico por imagen , Linfangiogénesis , Melanoma Experimental/complicaciones , Tomografía de Emisión de Positrones , Animales , Anticuerpos Monoclonales/inmunología , Femenino , Fluorodesoxiglucosa F18 , Humanos , Inflamación/inmunología , Inflamación/patología , Radioisótopos de Yodo/farmacocinética , Mediciones Luminiscentes , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Metástasis Linfática , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pronóstico , Radiofármacos , Piel/metabolismo , Distribución Tisular , Factor C de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
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.
Asunto(s)
Antineoplásicos/administración & dosificación , Carcinoma de Células Renales/tratamiento farmacológico , Neoplasias Renales/tratamiento farmacológico , Proteínas Recombinantes de Fusión/administración & dosificación , Adulto , Anciano , Animales , Anticuerpos Antineoplásicos/sangre , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Área Bajo la Curva , Femenino , Humanos , Infusiones Intravenosas , Macaca fascicularis , Masculino , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/inmunología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/farmacocinética , Tomografía Computarizada por Rayos XRESUMEN
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 trials.
Asunto(s)
Anticuerpos Monoclonales/química , Subunidad p35 de la Interleucina-12/química , Subunidad p40 de la Interleucina-12/química , Proteínas Recombinantes de Fusión/química , Anticuerpos de Cadena Única/química , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados , Autorradiografía , Células CHO , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Humanos , Subunidad p35 de la Interleucina-12/genética , Subunidad p35 de la Interleucina-12/metabolismo , Subunidad p40 de la Interleucina-12/genética , Subunidad p40 de la Interleucina-12/metabolismo , Ratones , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Distribución Tisular , TransfecciónRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Artritis Experimental/tratamiento farmacológico , Inmunoterapia/métodos , Interleucina-10/administración & dosificación , Proteínas Recombinantes de Fusión/administración & dosificación , Animales , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados , Especificidad de Anticuerpos , Artritis Experimental/patología , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Células CHO , Cricetinae , Cricetulus , Citocinas/sangre , Citocinas/inmunología , Sistemas de Liberación de Medicamentos , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Inmunosupresores/farmacología , Interleucina-10/efectos adversos , Interleucina-10/inmunología , Macaca fascicularis , Masculino , Metotrexato/farmacología , Ratones , Ratones Endogámicos DBA , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/inmunología , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismoRESUMEN
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 research.
Asunto(s)
Inmunoglobulina G/metabolismo , Región Variable de Inmunoglobulina/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/metabolismo , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Anticuerpos/metabolismo , Células CHO , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Anticuerpos/administración & dosificación , Antineoplásicos/administración & dosificación , Inmunoterapia/métodos , Interleucina-2/administración & dosificación , Linfoma no Hodgkin/tratamiento farmacológico , Proteínas Recombinantes de Fusión/administración & dosificación , Animales , Anticuerpos Monoclonales de Origen Murino , Terapia Combinada , Fibronectinas/antagonistas & inhibidores , Fibronectinas/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Ratones , Rituximab , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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 mug 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.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Interleucina-2/uso terapéutico , Células del Estroma/metabolismo , Tenascina/inmunología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Anticuerpos Monoclonales/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/inmunología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células CHO , Cricetinae , Cricetulus , Doxorrubicina/administración & dosificación , Sinergismo Farmacológico , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Fragmentos de Inmunoglobulinas/inmunología , Interleucina-2/inmunología , Interleucina-2/farmacocinética , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Paclitaxel/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Tenascina/administración & dosificación , Distribución Tisular , Células Tumorales CultivadasRESUMEN
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 (K(D) = 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.
Asunto(s)
Empalme Alternativo , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Fibronectinas/inmunología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/inmunología , Teratocarcinoma/irrigación sanguínea , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Afinidad de Anticuerpos , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Cartilla de ADN , Fibronectinas/genética , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , Estructura Terciaria de Proteína , Teratocarcinoma/inmunología , Neoplasias Testiculares/irrigación sanguínea , Distribución TisularRESUMEN
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.
Asunto(s)
Fibronectinas/química , Neoplasias/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Biomarcadores de Tumor/metabolismo , Fibronectinas/metabolismo , Humanos , Inmunohistoquímica/métodos , Masculino , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Proteómica/métodos , Espectrofotometría Infrarroja/métodosRESUMEN
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.
Asunto(s)
Adenocarcinoma/terapia , Neoplasias del Colon/terapia , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Inmunoconjugados/farmacología , Interleucina-15/administración & dosificación , Teratocarcinoma/terapia , Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/inmunología , Animales , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/inmunología , Clonación Molecular , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/inmunología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacocinética , Inmunoconjugados/genética , Inmunoconjugados/inmunología , Inmunoconjugados/farmacocinética , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Interleucina-15/genética , Interleucina-15/farmacocinética , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Neovascularización Patológica/inmunología , Neovascularización Patológica/terapia , Linfocitos T/inmunología , Teratocarcinoma/irrigación sanguínea , Teratocarcinoma/inmunología , Distribución TisularRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Artritis Experimental/prevención & control , Sistemas de Liberación de Medicamentos/métodos , Interleucina-10/administración & dosificación , Animales , Anticuerpos Monoclonales/inmunología , Artritis Experimental/inmunología , Línea Celular , Progresión de la Enfermedad , Humanos , Interleucina-10/inmunología , Masculino , Ratones , Ratones Endogámicos DBARESUMEN
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 x 10(9) 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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/aislamiento & purificación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Proto-Oncogénicas c-fyn/aislamiento & purificación , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Dimerización , Femenino , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Conformación Proteica , Proteínas Proto-Oncogénicas c-fyn/química , Proteínas Proto-Oncogénicas c-fyn/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Teratoma/metabolismo , Teratoma/patología , Distribución Tisular , Dominios Homologos srcRESUMEN
The antibody-based targeted delivery of bioactive agents to sites of angiogenesis is an attractive therapeutic strategy for cancer treatment, but is largely unexplored for chronic inflammatory diseases. In this article, we show that the extra domain B (EDB) domain of fibronectin, a marker of angiogenesis, is expressed in psoriatic lesions, and that the anti-EDB human antibody L19 can selectively localize to chronically inflamed skin in vivo. The L19-based delivery of the cytokines IL10 and IL12 did not improve or worsen inflammation in a mouse model of chronic skin inflammation, which overexpressed vascular endothelial growth factor under the control of the keratin-14 promoter. By contrast, the L19-based targeted delivery of the proinflammatory cytokine IL2 or of the photosensitizer Sn(IV) chlorin e6 resulted in an increased swelling and reddening of inflamed skin. These results indicate that antibodies specific to components of the modified extracellular matrix can selectively accumulate at chronically inflamed sites in vivo. This observation now stimulates the search for bioactive molecules which can be fused to antibodies and which may confer a therapeutic benefit as a result of their preferential accumulation in psoriatic lesions and other sites of inflammation.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Dermatitis Atópica/inmunología , Sistemas de Liberación de Medicamentos , Fibronectinas/inmunología , Animales , Enfermedad Crónica , Dermatitis Atópica/metabolismo , Dermatitis Atópica/patología , Femenino , Humanos , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/metabolismo , Hipersensibilidad Tardía/patología , Interleucina-10/administración & dosificación , Interleucina-10/farmacología , Interleucina-12/administración & dosificación , Interleucina-12/farmacología , Interleucina-2/administración & dosificación , Interleucina-2/farmacología , Luz , Metaloporfirinas/administración & dosificación , Metaloporfirinas/farmacología , Ratones , Ratones Endogámicos , Ratones Transgénicos , Fármacos Fotosensibilizantes/administración & dosificación , Fármacos Fotosensibilizantes/farmacología , Piel/efectos de los fármacos , Piel/patología , Piel/efectos de la radiación , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
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.
Asunto(s)
Anticuerpos/uso terapéutico , Fibronectinas/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Animales , Ensayos Clínicos como Asunto , Ensayos de Selección de Medicamentos Antitumorales , Fibronectinas/inmunología , Fibronectinas/metabolismo , Humanos , Neoplasias/metabolismoRESUMEN
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-gamma. 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-gamma receptor. ScFv(L19)-interferon-gamma 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.