[CANCER RESEARCH 55. 2673-2680. June 15, 1995) Identification of a Monoclonal Antibody, TV-1, Directed against the Basement Membrane of Tumor Vessels, and Its Use to Enhance the Delivery of Macromolecules to Tumors after Conjugation with Interleukin 21 Alan L. Epstein,2 Leslie A. Khawli, Jason L. Hornick, and Clive R. Taylor Department of Pathology, University of Southern California School of Medicine, Los Angeles, California 90033 ABSTRACT in A l>sreactive with epitopes expressed on tumor vessels were evaluated as universal delivery agents of peptides with vasoactive properties to enhance the uptake of macromolecules in tumors. Unlike other reported approaches to target tumor vessels, a mAb designated TV-1 targets a basement membrane antigen that is found in all tissues but that is acces sible only in tumor vessels, making it an alternative vehicle for the delivery of biologically active peptides to tumors. A panel of 30 monoclonal and polyclonal antibodies was screened by immunohistochemistry on sections of human tumors, normal vascular endothelium, and connective tissues. Five antibodies were chosen for in vivo evaluation, including two antifi- bronectin antibodies (TV-1, HFN 7.1), one anti-basic fibroblast growth factor antibody (anti-BFGF), and two antibodies reactive with a mesen- chymal cell antigen (TP-1, TP-3). Three nude mouse tumor models char acterized by varying degrees of vascularization (low to high) were used. After chemical conjugation to interleukin 2 (IL-2), these antibodies were used to pretreat tumor-bearing nude mice 3 h before injection with a radiolabeled mAb directed to the transplanted tumors. Pretreatment with TV-1/1L-2 or HFN 7.1/IL-2 produced a 3-fold higher tumor uptake of radiolabel compared to control mice pretreated with mAb alone. The other three vasoactive immunoconjugates failed to show significant in creases in these tumor models. When TV-1/1L-2 was compared with the specific vasoconjugate (Lym-l/IL-2) as a pretreatment in the Raji lym- phoma model, which has low vascularization, TV-l/IL-2 yielded approx imately 60% of the tumor uptake seen with Lym-l/IL-2. In comparison, pretreatment with TV-l/IL-2 in the LS174T colon carcinoma model, which has high vascularization, yielded approximately the same tumor uptake seen with the B72.3/IL-2 vasoconjugate, which directly targets the tumor cells. These studies demonstrate that a mAb directed against fi- bronectin in the endothelial subcellular matrix can be used to deliver vasoactive agents to tumors. INTRODUCTION Radioimaging and biodistribution studies in animal tumor models have revealed that an extremely small percentage of the injected dose of radiolabeled mAbs (1-4%) accumulates in the tumor mass, despite the relatively large size of the tumor and the marked antigenic differ ences between the tumor and the host (1,2). Likewise, patient studies have revealed that an even lower fraction of the injected dose (0.01- 0.1%) can be shown to localize at the tumor site (3-5). Thus, only a small amount of radiolabeled antibody actually contributes to therapy, and the bulk of the radionuclide preparation is dispersed throughout the body where it is capable of producing dose-limiting toxicity. Recognizing that the key factor for successful therapy is the amount of radiolabel delivered to the tumor with respect to normal tissues, our laboratory has attempted to generate new approaches to enhance antibody uptake in experimental tumor models. As schematically Received 12/16/94; accepted 4/19/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This study was supported in part by National Cancer Institute Grants 2 ROÕ CA47334 and l ROI CA49987. 2 To whom requests for reprints should he addressed, at Department of Pathology, University of Southern California School of Medicine, 2011 Zonal Avenue, Los Angeles, CA 90033. shown in Fig. 1, our initial focus was to develop methods that selectively alter the blood flow and/or vascular permeability of tumors because these important physiological factors control the egress of macromolecules from the vasculature into the tumor mass. Previous studies from our laboratory (2, 6) have shown that antibodies directed against tumor-associated cell surface antigens or intracellular antigens residing in degenerating tumor cells (Fig. 1, target B) can be used to deliver peptides with vasoactive properties such as IL-2,3 tumor necrosis factor a, and IL-lßto tumor xenografts in nude mice to alter the physiological characteristics of tumor vessels without concomitant changes in normal tissues. These studies resulted in a 3-4-fold in crease in the amount of radiolabel accumulating at the tumor site, with no noticeable changes observed in normal tissues and organs. Mech anism of action studies revealed that this increased localization to the tumor was due to changes seen in the blood volume, vasopermeabil- ity, or both, depending on which vasoactive agent was used. After these experiments, it was hypothesized that if vasoactive peptides could be delivered directly to tumor vessels (Fig. 1, target A) instead of tumor cells, this approach might be more universally applicable, eliminating the need to target different tumor-associated antigens. The challenge, therefore, was to identify an antigen present in tumor vessels that was not identified in the vasculature of normal tissues. Earlier observations that blood vessels in the vicinity of neoplasms are structurally abnormal (7-9) and show marked endo thelial fenestration, revealing the underlying basement membrane and perivascular connective tissue, led to the hypothesis that an antibody against these normal vascular structures might localize preferentially to tumors, while sparing blood vessels in the rest of the body, where the intact endothelium would preclude access of injected antibody to the underlying vascular structures. A search, therefore, was initiated to identify a mAb that could preferentially target tumor vessels by this approach. As a universal carrier of potent vasoactive agents, this mAb could be used as a pretreatment to alter the physiology of tumor vessels and enhance the preferential accumulation of diagnostic and therapeutic radionuclides at the tumor site. MATERIALS AND METHODS Sources of Reagents mAbs Lym-1 (lgG2a), recognizing a polymorphic variant of the HLA-Dr antigen on B cells and related malignancies, and TNT-1 (IgG2a), recognizing nuclear histone HI, were obtained from Techniclone International, Inc. (Tus- tin, California). mAb B72.3 (IgGl), recognizing the tumor-associated glyco- protein TAG-72, was a gift from Celltech, Ltd. (Slough, United Kingdom). Recombinant IL-2 was provided by Amgen Corporation (Thousand Oaks, CA). Radioactive iodine (125I) was obtained as sodium iodide in 0.05 N sodium hydroxide solution (ICN Biomedicals, Irvine, CA). Chloramine-T and all other immunochemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Antibodies A panel of 30 monoclonal and polyclonal antibodies was assembled based on their reported reactivity against antigens known to exist in the walls or ' The abbreviation used is: IL-2, interleukin 2. 2673 on August 4, 2015. © 1995 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from