TARGETING T CELLS AGAINST BRAIN TUMORS WITH A BISPECIFIC LIGAND-ANTIBODY CONJUGATE Edward J. ROY 1,2 *, Bryan K. CHO 1 , Laurie A. RUND 1 , Todd A. PATRICK 2 and David M. KRANZ 1 1 Department of Biochemistry, University of Illinois, Urbana, IL, USA 2 Neuroscience Program, University of Illinois, Urbana, IL, USA High-affinity receptors expressed on the surface of some tumors can be exploited by chemically conjugating the ligand for the receptor and an antibody against immune effector cells, thus redirecting their cytolytic potential against the tumor. Ovarian carcinomas and some brain tumors express the high-affinity folate receptor (FR). In this report, a trans- genic mouse model that generates endogenously arising choroid plexus tumors was used to show that folate/anti- T -cell receptor antibody conjugates can direct infiltration of T cells into solid brain tumor masses. An engineered single- chain Fv form of the anti-T-cell receptor antibody KJ16 was conjugated with folate, to produce a bispecific agent that was substantially smaller than most previously characterized bispecific antibodies. Folate conjugation to the antibody increased T -cell infiltration into the tumors by 10- to 20-fold, and significantly prolonged survival of the mice. Int. J. Cancer 76:761–766, 1998.  1998 Wiley-Liss, Inc. Tumors use a variety of mechanisms to avoid being eliminated by the immune system. Some tumors actively suppress immune function by secreting inhibitory cytokines or by killing T cells through Fas/FasL interaction. Other tumors escape recognition by cells of the immune system. For example, tumors may fail to present peptide antigens in complex with a product of the major histocompatibility complex (MHC) that are essential for recogni- tion by T cells. Tumor cells may also be deficient in co-stimulatory ligands and adhesion molecules that facilitate recognition and T-cell activation. Finally, the early development of tumors may allow the immune system to become tolerant of potential surface antigens on tumors. One potential way to overcome escape due to failures of recognition is to redirect immune cells against tumor cells with bispecific antibodies. Bispecific antibodies can be constructed to recognize 2 separate antigens, one on the tumor surface and the other on the surface of an immune effector such as a cytotoxic T cell. Many tumor cells have potential target antigens that are tumor specific or quantitatively more abundant on tumor cells than normal cells (tumor associated). Previous work has demonstrated the in vitro and in vivo effectiveness of bispecific antibodies against a variety of experimental tumors. Several clinical trials have been conducted with first-generation bispecific agents with results that are sufficiently promising to warrant further study of this strategy (Canevari et al., 1995). Despite considerable progress in the design of bispecific antibod- ies, significant obstacles to their effective clinical use remain. One of the problems associated with the bispecific antibody approach has been the difficulty in identifying animal models that can mimic human cancers. By analogy with human use, one would like a system that could evaluate how an endogenously arising tumor could be controlled by treatment with bispecific agents that redirect the activity of endogenous T cells. To date, all pre-clinical bispecific antibody studies have used transplanted tumors in either syngeneic rodent systems or xenogeneic systems in which human tumors and lymphocytes are transplanted in immunodeficient mice. This report shows that a new class of bispecific antibody agents can be used in a transgenic, endogenous tumor model to study T-cell-mediated therapies. The tumor antigen targeted in this study was the high-affinity folate receptor (FR), which has been identified on ovarian carcino- mas and most choroid plexus tumors and ependymomas (Manto- vani et al., 1994; Weitman et al., 1992). The high-affinity FRs (K D approx. 1 nM) differ from the ubiquitous lower affinity reduced folate carriers (K D approx. 100 μM) that are largely responsible for normal folate uptake (Westerhof et al., 1991). High-affinity FRs were originally identified as tumor-associated antigens using monoclonal antibodies that reacted with ovarian tumor cell lines (Coney et al., 1991). The presence of FR on ovarian tumors has led to its use as a target for various forms of therapy, including bispecific antibodies (Mezzanzanica et al., 1991). The nanomolar affinity of folate for FR suggested that attach- ment of folate directly to an anti-T-cell receptor (TCR) antibody might be a rapid method to generate bispecific antibodies that efficiently target FR-positive tumor cells for lysis by activated T cells. In cytolytic assays, these folate/antibody conjugates have very potent targeting activity without adversely affecting normal cells that express only the reduced folate carrier protein (Kranz et al., 1995). To reduce the size of the bispecific agent, we have produced folate conjugates of the single-chain Fv of the anti-V8 antibody KJ16 (scFv KJ16), and the folate/scFv conjugates were as effective as the folate/IgG conjugates in cytotoxicity assays against FR-positive cells (Cho et al., 1997). The 30 kDa folate/scFv conjugate is to our knowledge the smallest bispecific antibody yet reported. To develop an animal model for testing the folate/bispecific agents, we chose SV11 mice that are transgenic for the SV40 large T antigen gene with the SV40 enhancer (Van Dyke et al., 1985). SV11 mice develop choroid plexus tumors with 100% penetrance and in a well-defined time period (age of mortality averages about 100 days). SV40-induced choroid plexus tumors express FR with properties that are very similar to the human FR, including aK D of 1 nM (Patrick et al., 1997). In addition, flow cytometry and immunohistochemistry of FR on tumor cells indicated that virtually all the viable cells are FR positive (data not shown). MATERIAL AND METHODS Purification of scFv-KJ16 scFv-KJ16 was solubilized from E. coli inclusion bodies and refolded by dilution into 50 mM Tris-HCl, pH 8.0, 100 mM KCl, 10 mM CaCl 2 , 1 mM EDTA, 5 mM phenylmethylsulfonylchloride, as described previously (Cho et al., 1995). After concentration by tangential flow, the scFv preparation was dialyzed into 20 mM Tris, pH 8.0, and purified by anion exchange chromatography using Q Sepharose Fast Flow (Pharmacia, Uppsala, Sweden) eluting with 20 mM Tris, pH 8.0, 1 M NaCl. Peak fractions were pooled, concentrated, and dialyzed into PBS if used in vivo or 0.1 M Grant sponsor: National Institutes of Health; Grant number: AI35990; Grant sponsor: Department of the Army; Grant number: DAMD 17-94-J- 4347; Grant sponsor: Milheim Foundation for Cancer Research. *Correspondence to: Department of Biochemistry, University of Illinois, 600 S. Mathews Ave., Urbana, IL 61801-3792, USA. Fax: (217)244-5858. E-mail: e-roy@uiuc.edu Received 20 November 1997; Revised 9 January 1998 Int. J. Cancer: 76, 761–766 (1998)  1998 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer