The potent antitumor activity of certain cytokines is often achieved at the expense of unacceptable toxicity. One avenue to improve the therapeutic index of cytokines in cancer therapy consists of fusing them to monoclonal antibodies capable of a selective localization at the tumor site. We have constructed fusion proteins of interleukin-12 (IL-12) and tumor necrosis factor (TNF-α) with L19, an antibody fragment specific to the extradomain B of fibronectin which has been shown to target tumors in animal models and in patients with cancer. These fusions display a potent antitumor activity in several immunocompetent murine models of cancer but do not lead to complete remissions of established aggressive tumors. In this article, we have evaluated the tumor-targeting properties and the anticancer activities of combinations of the two antibody-cytokine fusion proteins, as well as of a triple fusion protein between IL-12, L19, and TNF-α. Although all fusion proteins were active in vitro, the triple fusion protein failed to localize to tumors in vivo and to show significant therapeutic effects. By contrast, the combination of IL-12-L19 and L19-TNF-α displayed potent synergistic anticancer activity and led to the eradication of F9 teratocarcinomas grafted in immunocompetent mice. When cured mice were rechallenged with tumor cells, a delayed onset of tumor growth was observed, indicating the induction of a partial antitumor vaccination effect. Potent anticancer effects were achieved at doses of IL-12-L19 and L19-TNF-α (2 μg + 2 μg/mouse), which were at least 5-fold lower than the maximal-tolerated dose. The combined administration of the two fusion proteins showed only a modest increase in toxicity, compared with treatments performed with the individual fusion proteins. These results show that the targeted delivery of cytokines to the tumor environment strongly potentiates their antitumor activity and that the combination treatment with IL-12-L19 and L19-TNF-α appears to be synergistic in vivo.

Cited By :114Export Date: 17 July 2015CODEN: CNREACorrespondence Address: Neri, D.; Institute of Pharmaceutical Sciences, Swiss Fed. Inst. of Technol. Zurich, Building 36 M14, Wintherhurerstrasse 190, CH 8057 Zurich, Switzerland; email: neri@pharma.anbi.ethz.chChemicals/CAS: fibronectin, 86088-83-7; interleukin 12, 138415-13-1; Antibodies, Monoclonal; Immunoconjugates; Immunoglobulin Variable Region; Interferon Type II, 82115-62-6; Interleukin-12, 187348-17-0; Recombinant Fusion Proteins; Tumor Necrosis Factor-alphaReferences: Rosenberg, S.A., Progress in human tumour immunology and immunotherapy (2001) Nature (Lond.), 411, pp. 380-384;Borden, E.C., Sondel, P.M., Lymphokines and cytokines as cancer treatment. Immunotherapy realized (1990) Cancer (Phila.), 65, pp. 800-814; Oppenheim, J.J., Murphy, W.J., Chertox, O., Schirrmacher, V., Wang, J.M., Prospects for cytokine and chemokine biotherapy (1997) Clin. Cancer Res., 3, pp. 2682-2686; Lienard, D., Ewalenko, P., Delmotte, J.J., Renard, N., Lejeune, F.J., High-dose recombinant tumor necrosis factor α in combination with interferon γ and melphalan in isolation perfusion of the limbs for melanoma and sarcoma (1992) J. Clin. Oncol., 10, pp. 52-60; Eggermont, A.M., Schraffordt Koops, H., Lienard, D., Kroon, B.B., Van Geel, A.N., Hoekstra, H.J., Lejeune, F.J., Isolated limb perfusion with high dose tumor necrosis factor α in combination with interferon γ, and melphalan for nonresectable extremity soft tissue sarcomas: A multicenter trial (1996) J. Clin. Oncol., 14, pp. 2653-2665; Eggermont, A.M., TNF registered in Europe: Does TNF get a second chance? (2000) J. Immunother., 23, pp. 505-506; Lode, H.N., Xiang, R., Dreier, T., Varki, N.M., Gillies, S.D., Reisfeld, R.A., Natural killer cell-mediated eradication of neuroblastoma metastases to bone marrow by targeted interleukin-2 therapy (1998) Blood, 91, pp. 1706-1715; Gillies, S.D., Lan, Y., Wesolowski, J.S., Qian, X., Reisfeld, R.A., Holden, S., Super, M., Lo, K.M., Antibody-IL-12 fusion proteins are effective in SCID mouse models of prostate and colon carcinoma metastases (1998) J. Immunol., 160, pp. 6195-6203; Peng, L.S., Penichet, M.L., Morrison, S.L., A single chain IL-12 IgG3 antibody fusion protein retains antibody specificity and IL-12 bioactivity and demonstrates antitumor activity (1999) J. Immunol., 163, pp. 250-258; Cooke, S.P., Pedley, R.B., Boden, R., Begent, R.H., Chester, K.A., In vivo tumor delivery of a recombinant single chain Fv::tumor necrosis factor α fusion protein (2002) Bioconjug. Chem., 13, pp. 7-15; Zardi, L., Carnemolla, B., Siri, A., Petersen, T.E., Paolella, G., Sebastio, G., Baralle, F.E., Transformed human cells produce a new fibronectin isoform by preferential alternative splicing of a previously unobserved exon (1987) EMBO J., 6, pp. 2337-2342; Carnemolla, B., Balza, E., Siri, A., Zardi, L., Nicotra, M.R., Bigotti, A., Natali, P.G., A tumor associated fibronectin isoform generated by alternative splicing of messenger RNA precursors (1989) J. Cell Biol., 108, pp. 1139-1148; Castellani, P., Viale, G., Dorcaratto, A., Nicolo, G., Kaczmarek, J., Querze, G., Zardi, L., The fibronectin isoform containing the ED-B oncofetal domain: A marker of angiogenesis (1994) Int. J. Cancer, 59, pp. 612-618; Kaczmarek, J., Castellani, P., Nicolo, G., Spina, B., Allemanni, G., Zardi, L., Distribution of oncofetal fibronectin isofoms in normal, hyperplastic and neoplastic human breast tissues (1994) Int. J. Cancer, 59, pp. 11-16; Halin, C., Rondini, S., Nilsson, F., Berndt, A., Kosmehl, H., Zardi, L., Neri, D., Enhancement of the antitumor activity of interleukin-12 by targeted delivery to neovasculature (2002) Nat. Biotechnol., 20, pp. 264-269; Khawli, L.A., Miller, G.K., Epstein, A.L., Effect of seven new vasoactive immunoconjugates on the enhancement of monoclonal antibody uptake in tumors (1994) Cancer (Phila.), 73, pp. 824-831; Folli, S., Pelegrin, A., Chalandon, Y., Yao, X., Buchegger, F., Lienard, D., Lejeune, F., Mach, J.P., Tumor-necrosis factor can enhance radio-antibody uptake in human colon carcinoma xenografts by increasing vascular permeability (1993) Int. J. Cancer, 53, pp. 829-836; Holliger, P., Prospero, T., Winter, G., "Diabodies": Small bivalent and bispecific antibody fragments (1993) Proc. Natl. Acad. Sci. USA, 90, pp. 6444-6448; Lode, H.N., Xiang, R., Duncan, S.R., Theofilopoulos, A.N., Gilies, S.D., Reisfeld, R.A., Tumor-targeted IL-2 amplifies T cell-mediated immune response induced by gene therapy with single-chain IL-12 (1999) Proc. Natl. Acad. Sci. USA, 96, pp. 8591-8596; Wigginton, J.M., Park, J.W., Grays, M.E., Young, H.A., Jorcyk, C.L., Back, T.C., Brunda, M.J., Wiltrout, R.H., Complete regression of established spontaneous mammary carcinoma and the therapeutic prevention of genetically programmed neoplastic transition by IL-12/pulse IL-2: Induction of local T cell infiltration. Fas/Fas ligand gene expression, and mammary epithelial apoptosis (2001) J. Immunol., 166, pp. 1156-1168; Vagliani, M., Rodolfo, M., Cavallo, F., Parenza, M., Melani, C., Palmiani, G., Forni, G., Colombo, M.P., Interleukin 12 potentiates the curative effect of a vaccine based on interleukin 2-transduced tumor cells (1996) Cancer Res., 56, pp. 467-470; Zagozdzon, R., Stoklosa, T., Golab, J., Giermasz, A., Dabrowska, A., Lasek, W., Jakobisiak, M., Augmented antitumor effects of combination therapy with interleukin-12, cisplatin, and tumor necrosis factor α in a murine melanoma model (1997) Anticancer Res., 17, pp. 4493-4498; Taniguchi, F., Yamagishi, H., Fujiwara, H., Ueda, Y., Fuji, N., Yoshimura, T., Oka, T., Systemic administration of rIL-12 synergistically enhances the therapeutic effect of a TNF gene-transduced cancer vaccine (1998) Gene Ther., 5, pp. 1677-1684; Voest, E.E., Kenyon, B.M., O’Reilly, M.S., Truitt, G., D’Amato, R.J., Folkman, J., Inhibition of angiogenesis in vivo by interleukin 12 (1995) J. Natl. Cancer Inst. (Bethesda), 87, pp. 581-586; Fajardo, L.F., Kwan, H.H., Kowalski, J., Prionas, S.D., Allison, A.C., Dual role of tumor necrosis factor α in angiogenesis (1992) Am. J. Pathol., 140, pp. 539-544; Watanabe, N., Niitsu, Y., Umeno, H., Kuriyama, H., Neda, H., Yamauchi, N., Maeda, M., Urushizaki, I., Toxic effect of tumor necrosis factor on tumor vasculature in mice (1988) Cancer Res., 48, pp. 2179-2183; Renard, N., Lienard, D., Lespagnard, L., Eggermont, A., Heimann, R., Lejeune, F., Early endothelium activation and polymorphonuclear cell invasion precede specific necrosis of human melanoma and sarcoma treated by intravascular high-dose tumour necrosis factor α (rTNF α) (1994) Int. J. Cancer, 57, pp. 656-663; Smyth, M.J., Pietersz, G.A., McKenzie, I.F., Increased antitumor effect of immunoconjugates and tumor necrosis factor in vivo (1988) Cancer Res., 48, pp. 3607-3612; Kirchhofer, D., Sakariassen, K.S., Clozel, M., Tschopp, T.B., Hadvary, P., Nemerson, Y., Baumgartner, H.R., Relationship between tissue factor expression and deposition of fibrin, platelets, and leukocytes on cultured endothelial cells under venous blood flow conditions (1993) Blood, 81, pp. 2050-2058; Shimomura, K., Manda, T., Mukumoto, S., Kobayashi, K., Nakano, K., Mori, J., Recombinant human tumor necrosis factor α: Thrombus formation is a cause of anti-tumor activity (1988) Int. J. Cancer, 41, pp. 243-247; Nooijen, P.T., Manusama, E.R., Eggermont, A.M., Schalkwijk, L., Stavast, J., Marquet, R.L., De Waal, R.M., Ruiter, D.J., Synergistic effects of TNF-α and melphalan in an isolated limb perfusion model of rat sarcoma: A histopathological, immunohistochemical and electron microscopical study (1996) Br. J. Cancer, 74, pp. 1908-1915; Olieman, A.F., Van Ginkel, R.J., Hoesktra, H.J., Mooyaart, E.L., Molenaar, W.M., Koops, H.S., Angiographic response of locally advanced soft-tissue sarcoma following hyperthermic isolated limb perfusion with tumor necrosis factor (1997) Ann. Surg. Oncol., 4, pp. 64-69; Sato, N., Goto, T., Haranaka, K., Satomi, N., Nariuchi, H., Mano-Hirano, Y., Sawasaki, Y., Actions of tumor necrosis factor on cultured vascular endothelial cells: Morphologic modulation, growth inhibition, and cytotoxicity (1986) J. Natl. Cancer Inst. (Bethesda), 76, pp. 1113-1121; De Wilt, J.H., Ten Hagen, T.L., De Boeck, G., Van Tiel, S.T., De Bruijn, E.A., Eggermont, A.M., Tumour necrosis factor α increases melphalan concentration in tumour tissue after isolated limb perfusion (2000) Br. J. Cancer, 82, pp. 1000-1003; Birchler, M., Viti, F., Zardi, L., Spiess, B., Neri, D., Selective targeting and photocoagulation of ocular angiogenesis mediated by a phage-derived human antibody fragment (1999) Nat. Biotechnol., 17, pp. 984-988; Carnemolla, B., Borsi, L., Balza, E., Castellani, P., Meazza, R., Berndt, A., Ferrini, S., Zardi, L., Enhancement of the antitumor properties of interleukin 2 by its targeted delivery to the tumor blood vessel extracellular matrix (2002) Blood, 99, pp. 1659-1665; Demartis, S., Tarli, L., Bood, L., Zardi, L., Neri, D., Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin (2001) Eur. J. Nucl. Med., 28, pp. 534-539; Nilsson, F., Kosmehl, H., Zardi, L., Neri, D., Targeted delivery of tissue factor to the ED-B domain of fibronectin, a marker of angiogenesis, mediates the infarction of solid tumors in mice (2001) Cancer Res., 61, pp. 711-716; Tarli, L., Balza, E., Viti, F., Borsi, L., Castellani, P., Berndorff, D., Dinkelborg, L., Zardi, L., A high affinity human antibody that targets tumoral blood vessels (1999) Blood, 94, pp. 192-198; Viti, F., Tarli, L., Giovannoni, L., Zardi, L., Neri, D., Increased binding affinity and valence of recombinant antibody fragments lead to improved targeting of tumoral angiogenesis (1999) Cancer Res., 59, pp. 347-352; Santimaria, M., Moscatelli, G., Giovannoni, L., Viti, F., Leprini, A., Borsi, L., Neri, D., Riva, P., Immunoscintigraphic detection of angiogenesis in patients with cancer Clin. Cancer Res., 9, pp. 741-749; Bernstine, E.G., Hooper, M.L., Grandchamps, S., Ephrussi, B., Alkaline phosphatase activity in mouse teratoma (1973) Proc. Natl. Acad. USA, 70, pp. 3899-3903; Neri, D., Petrul, H., Winter, G., Light, Y., Marais, R., Britton, K.E., Creighton, A.M., Radioactive labeling of recOmbinant antibody fragments by phosphorylation using human casein kinase II arid [γ-32P]-ATP (1996) Nat. Biotechnol., 14, pp. 485-490; Gately, M.K., Chizzonite, R., Presky, H.D., (1995) Measurement of Human and Mouse Interleukin-12, , New York: Current Protocols in Immunology, section 6.16.1; section 3.11.1; Corti, A., Poiesi, C., Merli, S., Cassani, G., Tumor necrosis factor (TNF) α quantification by ELISA and bioassay: Effects of TNF α-soluble TNF receptor (p55) complex dissociation during assay incubations (1994) J. Immunol. Methods, 177, pp. 191-198; Graham, F.L., Smiley, J., Russell, W.C., Nairn, R., Characteristics of a human cell line transformed by DNA from human adenovirus type 5 (1977) J. Gen. Virol., 36, pp. 59-74; Van Elsas, A., Hurwitz, A.A., Allison, J.P., Combination immunotherapy of B16 melanoma using anticytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation (1999) J. Exp. Med., 190, pp. 355-366; Van Elsas, A., Sutmuller, R.P., Hurwitz, A.A., Ziskin, J., Villasenor, J., Medema, J.P., Overwijk, W.W., Allison, J.P., Elucidating the autoimmune and antitumor effector mechanisms of a treatment based on cytotoxic T lymphocyte antigen-4 blockade in combination with a B16 melanoma vaccine: Comparison of prophylaxis and therapy (2001) J. Exp. Med., 194, pp. 481-489; Halin, C., Niesner, U., Villani, M.E., Zardi, L., Neri, D., Tumor-targeting properties of antibody-vascular endothelial growth factor fusion proteins (2002) Int. J. Cancer, 102, pp. 109-116; Melkko, S., Halin, C., Borsi, L., Zardi, L., Neri, D., An antibody-calmodulin fusion protein reveals a functional dependence between macromolecular isoelectric point and tumor targeting performance (2002) Int. J. Radiat. Oncol. Biol. Phys., 54, pp. 1485-1490