Targeted therapy of renal cell carcinoma: Synergistic activity of cG250-TNF and IFNg Stefan Bauer 1 * , Jeannette C. Oosterwijk-Wakka 2 , Nicole Adrian 3 , Egbert Oosterwijk 2 , Eliane Fischer 1 , Thomas W€ uest 1 , Frank Stenner 1 , Angelo Perani 4 , Leonard Cohen 5 , Alexander Knuth 1 , Chaitanya Divgi 6 , Dirk J€ ager 7 , Andrew M. Scott 4 , Gerd Ritter 5 , Lloyd J. Old 5 and Christoph Renner 1 1 Oncology Department, Universit € atsSpital Z€ urich, Z€ urich, Switzerland 2 Urology Department, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands 3 Med. Department I, Universit € at des Saarlandes, Homburg/Saar, Germany 4 Department of Nuclear Medicine and Centre for P.E.T., Ludwig Institute for Cancer Research, Melbourne Centre, Austin Hospital, Victoria, Australia 5 Ludwig Institute for Cancer Research, New York Branch, Memorial Sloan Kettering Cancer Center, New York, NY 6 Department of Nuclear Medicine and Clinical Molecular Imaging, University of Pennsylvania, Philadelphia, PA 7 Department of Medical Oncology, University of Heidelberg, National Center for Tumour Diseases, Heidelberg, Germany Immunotherapeutic targeting of G250/Carbonic anhydrase IX (CA-IX) represents a promising strategy for treatment of renal cell carcinoma (RCC). The well characterized human-mouse chi- meric G250 (cG250) antibody has been shown in human studies to specifically enrich in CA-IX positive tumors and was chosen as a carrier for site specific delivery of TNF in form of our IgG-TNF- fusion protein (cG250-TNF) to RCC xenografts. Genetically engi- neered TNF constructs were designed as CH2/CH3 truncated cG250-TNF fusion proteins and eucariotic expression was opti- mized under serum-free conditions. In-vitro characterization of cG250-TNF comprised biochemical analysis and bioactivity assays, alone and in combination with Interferon-c (IFNc). Biodis- tribution data on radiolabeled [ 125 J] cG250-TNF and antitumor activity of cG250-TNF, alone and in combination with IFNc, were measured on RCC xenografts in BALB/c nu/nu mice. Combined administration of cG250-TNF and IFNc caused synergistic biologi- cal effects that represent key mechanisms displaying antitumor responses. Biodistribution studies demonstrated specific accumula- tion and retention of cG250-TNF at CA-IX-positive RCC resulting in growth inhibition of RCC and improved progression free sur- vival and overall survival. Antitumor activity induced by targeted TNF-based constructs could be enhanced by coadministration of low doses of nontargeted IFNc without significant increase in side effects. Administration of cG250-TNF and IFNc resulted in signifi- cant synergistic tumoricidal activity. Considering the poor out- come of renal cancer patients with advanced disease, cG250-TNF- based immunotherapeutic approaches warrant clinical evaluation. ' 2009 UICC Key words: renal cell cancer; recombinant antibody; cytokines; tumor targeting Renal cell carcinoma (RCC) accounts for 2% of all cancers, leading to 20,000 annual deaths in Europe and 12,000 in the United States. 1,2 Although advances in understanding the biology of RCC led to novel approaches for the treatment of metastatic disease 3 with subsequent increase of progression-free and overall- survival rates, the prognosis for these patients is poor with a 5- year survival rate of less than 10%. 4 The predominant histological type of RCC is clear-cell carcinoma, comprising more than 85% of metastatic disease. Both, sporadic and inherited forms of clear- cell RCC are associated with mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. 5 Elucidation of its role in up-regu- lating growth factors associated with angiogenesis as well as the hypoxia-induced carbonic anhydrase IX (CA-IX) defined a series of potential targets for novel treatment strategies. Among them, targeting of a surface expressed epitope of CA-IX (called G250) using chimeric G250 monoclonal antibody (cG250) is a promising immunotherapeutic approach. 6 Safe administration with excellent tumor targeting properties of the radio-labeled 131 I- and 124 I- cG250 antibody has been demonstrated in phase I clinical trials in patients with metastatic clear cell RCC. 7–10 Additional studies showed that multiple doses of cG250 were well tolerated and com- bination with low dose IL-2 resulted in disease stabilization. 11,12 These encouraging clinical features prompted us to optimize effector functions of cG250 to further improve its antitumor prop- erties. We have previously reported on a strategy to promote the thera- peutic efficacy of tumor specific antibodies by genetic fusion to tu- mor necrosis factor (TNF). 13 The soluble form of TNF occurs mainly as a trimer of 3 identical subunits. TNF was first identified as a mediator of hemorrhagic tumor necrosis mediating regression of murine 14 and xenotransplanted human tumors. 15 Subsequent research revealed that Interferon gamma (IFNg) plays a substan- tial role in TNF-mediated tumor rejection processes. 16,17 Many IFNg-mediated mechanisms have been proposed to promote anti- tumor responses including antiproliferative and proapoptotic ac- tivity on tumor cells, 18 inhibition of angiogenesis within tumors 19,20 or activation of innate 21,22 and adapted immune responses. 23,24 Thus, both TNF and IFNg exert pleiotropic mecha- nisms on a variety of cell types and coadministration of both cyto- kines even results in synergistic antitumor activities. 25–27 How- ever, because of its life-threatening systemic toxicity 28 caused by affection of normal endothelial cells in peripheral blood ves- sels, 29,30 the clinical use of TNF in combination with IFNg as an anticancer drug is restricted to loco-regional treatment (e.g. iso- lated limb perfusion). 31,32 However, we established a 2-step approach to enlarge the therapeutic window of the cytokine: TNF molecules were genetically fused to a tumor-specific antibody to focus TNF at the tumor site. In addition, TNF subunits were forced to form a dimer to reduce their activity in peripheral blood vessels outside the tumor compartment. 13 Abandoning the natural homotrimeric symmetry of TNF resulted in significantly reduced toxicity as seen both in immunodeficient and immunocompetent mouse strains. 33 The dimeric IgG-TNF molecules displayed sig- nificantly stronger antitumor activity in-vivo than wild type TNF or trimeric TNF-antibody conjugates. Dose escalation increased the therapeutic index of IgG-TNF and repeated administration additionally delayed tumor growth with tolerable side effects. 33 Stefan Bauer’s current address is: National Centre for Tumor Diseases, Department of Medical Oncology, University of Heidelberg, Im Neuen- heimer Feld 350, 69120 Heidelberg, Germany. The first two authors contributed equally to this work. *Correspondence to: National Centre for Tumor Diseases, Department of Medical Oncology, University of Heidelberg, Im Neuenheimer Feld 350, 69120 Heidelberg, Germany. Fax: 149 (0) 6221 56 5873. E-mail: stefan1.bauer@med.uni-heidelberg.de Grant sponsors: Europrofession e.V., Wilhelm Sander Foundation, Uni- versity of Z€ urich, conducted as part of the Atlantic Philanthropies/Ludwig Institute for Cancer Research Clinical Discovery Program. Received 24 October 2008; Accepted after revision 21 January 2009 DOI 10.1002/ijc.24359 Published online 17 February 2009 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 125, 115–123 (2009) ' 2009 UICC Publication of the International Union Against Cancer