Immunotargeting of Squamous Cell Carcinoma of the Head and MAb U36, a Novel Monoclonal Antibody Successful in

Using viable cells of a human head and neck squamous cell carcinoma (HNSCC) cell line as immunogen, we developed monoclonal antibody (MAb) U36. Immunohistochemical examination revealed distinct surface labeling of MAb U36 with normal human squamous epithelium and squa mous cell carcinomas of distinct sites of origin; head and neck, lung, esophagus, cervix, and epidermis. MAb U36 shows high affinity binding (affinity constant, 3.5 x IO'"M) with a cell surface antigen expressed by in vitro cultured HNSCC cell lines. Similarity of the reactivity profiles of MAb U36 and MAb £48,currently the most promising antibody described for specific targeting of HNSCC in patients, warranted further compari son of these MAbs. MAb U36 recognizes a M, 200,000 antigen, which is different from the MAb E48 defined antigen. Furthermore, comparison of immunohistochemical staining patterns of MAb U36 and MAb E48 on a broad panel of primary HNSCC sections revealed more extensive staining for MAb U36: more tumors showed reactivity with MAb U36 and more tumor cells per tumor showed positive reaction, and staining was found to be more intense. MAb U36 does not show cross-reactivity with mouse, rat, pig, sheep, or bovine tongue epithelium. As a first approach to evaluate the suitability of MAb U36 for tumor targeting in vivo, radiolabeled MAb I 36 was administered to athymic nude mice bearing human HNSCC xenografts on both flanks. Selective tumor accumulation of the radioimmunoconjugate was observed. Mean tumor uptake (in percent injected dose/g wet-weight of tissue) of MAb U36 at days 1, 2, 3, 5, 7, and 12 was 15.1, 17.9, 24.0, 21.0, 25.8, and 16.0%, respectively. The tumor to blood ratio at day 1 was 0.9 and increased up to 3.8 at day 12. The tumor uptake at day 12 was at least 10 times higher when compared to other tissues. The corollary of these findings is that MAb U36 harbors high potential for specific targeting of HNSCC. INTRODUCTION SCC2 is the major histological type among tumors of the head and neck, lung, cervix, and skin. Monoclonal antibodies against tumor associated antigens related to SCC can be of benefit in diag nosis and treatment of these neoplasms. This holds especially true when these antigens are expressed on the outer cell surface. Currently we are focusing on RIS and RIT of HNSCC. The relative superficial localization of the lymph nodes in the neck allows accurate radioimmunodetection in this area with a 7-camera. One aspect in favor of application of RIT is the intrinsic sensitivity of SCC for radiation (1, 2). MAbs against the epidermal growth factor receptor and the carcinoembryonic antigen have been used in immunoscintigraphy studies Received 3/8/93; accepted 7/13/93. 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. 1To whom requests for reprints should be addressed. 2 The abbreviations used are: SCC, squamous cell carcinoma; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; HNSCC, squamous cell carci noma of the head and neck; HNX-HN, squamous cell carcinoma of the head and neck xenograft line HN; %ID/g, percentage of injected dose/g wet weight of tissue; BSA, bovine serum albumin; MAb, monoclonal antibody; RIS, radioimmunoscintigraphy; RIT, radioimmunotherapy. to verify the feasibility of targeting tumor deposits in patients with HNSCC (3, 4). However, the value of these MAbs in targeting metastatic head and neck cancer is not yet clear. As illustrated by falsepositive scintigrams, a major limitation of these MAbs may be that they are not specifically directed against HNSCC, thereby leading to false-positive scintigrams (5). We have previously discussed a variety of drawbacks of the ma jority of currently available MAbs against HNSCC (6, 7). MAb E48 forms a rare exception and possesses favorable characteristics for specific targeting of HNSCC (8). The MAb E48 defined antigen is exclusively expressed on normal squamous and transitional epithelia and squamous cell carcinomas (9), and seems to be involved in cellcell adhesion (10). This MAb E48 was also found to be strongly reactive with metastatic SCC (8), and when labeled with 131I to be very effective in detecting and eradicating HNSCC in an animal model setting (8, 11-13). Moreover, WmTc-labeled MAb E48 IgG and F(ab')2 appeared highly capable of detecting metastatic and recurrent disease in head and neck cancer patients (14). The percentage injected dose/g tissue as assessed in these RIS studies was found to be high, ranging from 0.014 to 0.080%. A serious drawback of MAb E48 is the heterogeneity of E48 antigen expression; approximately 25% of HNSCC show E48 antigen expression restricted to less than 50% of the tumor cells. Therefore, not all HNSCC patients are eligible for clinical RIS studies. Furthermore, such heterogeneity of antigen ex pression might be a limitation for RIT, when assuming that for effec tive RIT all tumor nodules, malignant cell clusters or single cells should be targeted. For this reason, we have continued pursuing new MAbs with even greater prospectives for RIS and RIT in patients with HNSCC. In this paper, we will present a newly developed MAb, MAb U36, which recognizes a cell surface antigen strongly expressed by squa mous epithelia and their malignant counterparts, and to a lesser degree by transitional epithelia. Immunohistochemical data on MAb U36 reactivity with normal and malignant tissues will be presented, as well as some preliminary molecular characteristics of the antigen. Some of the data will be compared to those obtained from MAb E48. The high prospectives for HNSCC targeting with this new MAb U36 will be emphasized. MATERIALS AND METHODS Immunization and Hybridoma Production. BALB/c mice were immu nized by i.p. injection of IO7 viable cells of HNSCC cell line UM-SCC-22B (generously provided by Dr. T. E. Carey, University of Michigan, Ann Arbor, Ml). Four weeks later, an intrasplenic booster was given under general anes thesia. The spleen cells were fused with the nonproducing myeloma line SP-2/0, as described previously (9). Growing hybridomas were screened by ELISA for immunoglobulin production, for binding to intact viable tumor cells, and for lack of reactivity with erythrocytes. Selected antibodies were further screened for reactivity with frozen sections of a variety of normal tissues and human tumors, and a large panel of HNSCC. Selected hybridomas were stabilized by limiting dilution. To eliminate nonproducing hybridomas, recloning was done once a week (4 times).