Monoclonal antibody U36, a suitable candidate for clinical immunotherapy of squamous-cell carcinoma, recognizes a CD44 isoform

Abstract

For this purpose we developed MAb U36, recognizing a 2 d kDa antigen expressed on the outer cell surface of squamouscell carcinomas and their normal counterparts. Clinical radioimmunoscintigraphy (RIS) and biodistribution studies have shown that the MAb-U36-defined antigen is a suitable target molecule for antibody-based therapy of head-and-neck cancer. In the present study we further characterized the antigen by cDNA cloning. The cDNA was isolated by expression cloning in COS-7 cells. Sequence analysis and database searching revealed that the MAb-U36-defined antigen is identical to the squamous-cellspecific CD44 splice variant epican. The epitope recognized by Squamous-cell carcinoma is the major histological type among tumors of the head and neck. The early stages arc treated with either surgery or radiotherapy, and advanccd stages with combined surgery and radiotherapy. In patients with advanced disease (stages Ill and IV), locoregional recurrences develop in 50 to 60% of the cases after primary therapy, and 15 to 25% of the patients develop distant metastases . The actual incidence of distant metastases in HNSCC patients is probably even higher, since autopsy studies report an incidence of 40 to 57% . These data indicate that there is an urgent need for an effective adjuvant therapy for the eradication of (minimal) residual disease after initial treatment, to prevent thc eventual development of locoregional recurrences and/or distant metastases. The high expectations raised by chemotherapy have not been fulfillcd, and its application is in general limited to palliation. We therefore have decided to exploit the tumor-targeting capacity of monoclonal antibodies for delivery of radionuclides to the remaining tumor deposits. A panel of MAbs directed against squamous-cell-carcinoma-associated antigens was selected by in vitro techniques and evaluated in in vivo models of tumor-bearing nude mice . The antibodies selected by this procedure were tested in RIS and biodistribution studies in patients with HNSCC De Bree el al., 1995). MAb U36 appeared to be the most promising antibody for specific targeting of HNSCC in patients. Immunohistochemical evaluation showed that in 1881196 (96%) of the HNSCC tumors, at least 50% of the tumor cells were intensely stained by MAb U36, indicating that the antigen is very homogeneously expressed (De Bree etal., 1994). Moreover, extensive biodistribution and RIS studies in HNSCC patients have shown that W"Tc-labcled U36 IgG accumulates selectively and to a high level in these tumors (De Brce et al., 1995), and during a follow-up of 3 years no adverse reactions have been observed. These data showed that the MAb-U36-defined antigen is a suitable target moleculc for antibody-based adjuvant therapy of squamous-cell carcinoma of the head and neck. The antibody recognizes a 200-kDa cell-surface antigen highly expressed by human normal squamous epithelia and squamouscell carcinomas of distinct sites of origin: head and neck, lung, esophagus, cervix and epidermis (Schrijvers et al., 1993)....

Tumor targeting properties of monoclonal antibodies with different affinity for target antigen CD44V6 in nude mice bearing head‐and‐neck cancer xenografts

“…In the third experiment, either 400 Ci (100 g) 186 Re-labeled hMAb BIWA-4 or 400 Ci (100 g) 186 Re-labeled hMAb BIWA-8 were administered. Average tumor volumes were similar for all experimental groups: experiment 1, 95 Ϯ 34 mm 3 (n ϭ 7 mice, 12 tumors) for the 186 Re-mMAb U36 -treated group, 91 Ϯ 15 mm 3 (n ϭ 7 mice, 12 tumors) for the 186 Re-mMAb BIWA-1-treated group and 99 Ϯ 54 mm 3 (n ϭ 6 mice, 11 tumors) for the control group; experiment 2, 101 Ϯ 35 mm 3 (n ϭ 7 mice, 12 tumors) for the 186 Re-hMAb BIWA-4 -treated group, 92 Ϯ 43 mm 3 (n ϭ 7 mice, 12 tumors) for the 186 Re-cMAb BIWA-2-treated group and the control group was the same as in experiment 1; experiment 3, 105 Ϯ 43 mm 3 (n ϭ 8 mice, 13 tumors) for the 186 Re-hMAb BIWA-4 -treated group, 100 Ϯ 42 mm 3 (n ϭ 8 mice, 13 tumors) for the 186 Re-hMAb BIWA-8 -treated group and 110 Ϯ 46 mm 3 (n ϭ 7 mice, 11 tumors) for the control group. During treatment, tumors were measured twice weekly and tumor volumes relative to the volume at the start of treatment were calculated.…”

“…The epitope recognized by U36 was mapped to amino acids 365-376 of CD44v6, indicating that U36 and BIWA-1 recognize overlapping epitopes. 3 The batch used for the current studies was supplied by Centocor (Leiden, the Netherlands). U36 was purified from a concentrated tissue culture supernatant by affinity chromatography on protein A-sepharose and further purified on Q-sepharose.…”

“…15 Female mice (Hsd athymic nu/nu, 25-32 g; Harlan, Zeist, the Netherlands) were 8 -10 weeks old at the time of the experiments. Three biodistribution experiments were conducted with mice bearing 1 or 2 tumors ranging 30 -470 mm 3 . In the first experiment, 10 Ci (50 g) 131 I-labeled mMAb U36 were injected simultaneously with 10 Ci (50 g) 125 I-labeled mMAb BIWA-1 in mice bearing tumors of 133 Ϯ 28 mm 3 (n ϭ 20 mice, 37 tumors).…”

“…Toxicity was monitored by measurement of body weight twice weekly. Mice were killed when one of the tumors exceeded 1,000 mm 3 .…”

“…1,2 U36 and BIWA-1 bind to overlapping epitopes in the variable domain v6 of the cell-surface antigen CD44 and have been characterized extensively. 3,4 CD44 isoforms, which arise from differential splicing of up to 9 variant exons, show a tissue-specific expression pattern. Homogenous expression of v6-containing CD44 isoforms has been observed in squamous cell carcinoma of the head and neck, lung, skin, esophagus and cervix, while heterogeneous expression was found in several other tumor types, including adenocarcinomas of the breast, lung, colon, pancreas and stomach.…”