The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion.

The E48 antigen, a putative human homologue of the 20-kD protein present in desmosomal preparations of bovine muzzle, and formerly called desmoglein III (dg4), is a promising target antigen for antibody-based therapy of squamous cell carcinoma in man. To anticipate the effect of high antibody dose treatment, and to evaluate the possible biological involvement of the antigen in carcinogenesis, we set out to molecularly characterize the antigen. A cDNA clone encoding the E48 antigen was isolated by expression cloning in COS cells. Sequence analysis revealed that the clone contained an open reading frame of 128 amino acids, encoding a core protein of 13,286 kD. Database searching showed that the E48 antigen has a high level of sequence similarity with the mouse ThB antigen, a member of the Ly-6 antigen family. Phosphatidylinositol-specific (PI-specific) phospholipase-C treatment indicated that the E48 antigen is glycosylphosphatidylinositol-anchored (GPI-anchored) to the plasma membrane. The gene encoding the E48 antigen is a single copy gene, located on human chromosome 8 in the 8q24-qter region. The expression of the gene is confined to keratinocytes and squamous tumor cells. The putative mouse homologue, the ThB antigen, originally identified as an antigen on cells of the lymphocyte lineage, was shown to be highly expressed in squamous mouse epithelia. Moreover, the ThB expression level is in keratinocytes, in contrast to that in lymphocytes, not mouse strain related. Transfection of mouse SV40-polyoma transformed mouse NIH/3T3 cells with the E48 cDNA confirmed that the antigen is likely to be involved in cell-cell adhesion.

186Re-labeled monoclonal antibody E48 immunoglobulin G-mediated therapy of human head and neck squamous cell carcinoma xenografts.

In our laboratory, a solid-phase synthesis of 186Re-mercaptoacetyltriglycine for reproducible and aseptic production of stable 186Re-monoclonal antibody conjugates was recently developed. Monoclonal antibody (MAb) E48 IgG, when labeled with 99mTc according to the same labeling procedure, was recently shown to be highly capable of detecting recurrent and metastatic disease in patients with head and neck squamous cell carcinoma. In the present study, MAb E48 was labeled with 186Re and tested for its capacity to eradicate established human head and neck squamous cell carcinoma xenografts growing s.c. in nude mice. Experimental groups received a single bolus injection of 200 [number of mice (n) = 6, number of tumors (t) = 11], 400 (n = 6, t = 11), 500 (n = 6, t = 12), or 600 (n = 5, t = 9) microCi 186Re-labeled MAb E48 IgG; control animals were given diluent (n = 4, t = 8). In the 200 microCi group, 5 of 11 tumors showed regression while the remaining tumors showed a decreased growth rate. In the other treatment groups, all tumors regressed. In all treatment groups, remissions were observed (no regrowth within 4 months after injection). The number of remissions in the 200, 400, 500, and 600 microCi group were 2 of 11 (18.2%), 3 of 11 (27.3%), 6 of 12 (50%), and 3 of 9 tumors (33.3%), respectively. In comparison with the median tumor volume doubling time of the controls, the tumor volume doubling time in the remaining tumors in the groups receiving 200, 400, 500, or 600 microCi was increased 5.5-, 7.8-, 8.7-, and 11.3-fold, respectively. Dosimetry was based on the biodistribution of 200 microCi 186Re-labeled MAb E48 IgG. In the group receiving 600 microCi, the absorbed cumulative radiation dose was 3432 cGy for tumor and 1356 cGy for blood. In other tissues, the accumulated dose was < 17% of the dose delivered to tumor. The whole-body dose was 11-fold lower than the dose delivered to tumor. Apparent toxicity was limited to weight loss, which did not exceed 12% and which returned to control levels within 2 weeks. No treatment-related deaths occurred. These data suggest radioimmunotherapy with 186Re-labeled MAb E48 IgG to be a feasible approach for the treatment of head and neck cancer.

Labeling of monoclonal antibodies with rhenium-186 using the MAG3 chelate for radioimmunotherapy of cancer: a technical protocol.

A detailed technical protocol is provided for reproducible and aseptical production of stable 186Re-monoclonal antibody conjugates. Labeled Mab E48 IgG and its F(ab')2 fragment which are promising candidates for radioimmunotherapy of squamous cell carcinoma of the head and neck were used for evaluation. S-benzoylmercaptoacetyltriglycine (S-benzoyl-MAG3) was used as a precursor. Rhenium-186-MAG3 was prepared via a unique solid-phase synthesis, after which known strategies for esterification and conjugation to Mab IgG/F(ab')2 were applied. The biodistribution of 186Re-E48 F(ab')2 in tumor-bearing nude mice was found to be comparable to that of analogously labeled 99mTc-E48 F(ab')2 or 131I-E48 F(ab')2, indicating that the intrinsic behavior of the antibody remains preserved when using this labeling technique. Radiolytic decomposition of 186Re-E48 IgG/F(ab')2 solutions of 10 mCi.ml-1 was effectively reduced by the antioxidant ascorbic acid. Upon increase of the Re-MAG3 molar amount, a conjugation of seven to eight Re-MAG3 molecules per Mab molecule was generally the maximum ratio that could chemically be obtained. Such a ratio did not impair the immunoreactivity or alter the in vivo biodistribution characteristics of the immunoconjugate, making this labeling procedure suitable for general clinical application.

Progress in radioimmunotherapy of head and neck-cancer (review).

There is an urgent need for an effective adjuvant systemic therapy for the treatment of patients with advanced head and neck cancer. This study shows that therapy based on the use of monoclonal antibodies (MAbs) is developing to a realistic option. A few years ago the first MAbs with specificity for squamous cell carcinoma of the head and neck (HNSCC) were produced, among which was MAb E48. In animal and patient studies, in which localization of radiolabelled MAb E48 was analysed qualitatively and quantitatively, it was demonstrated that a high percentage of the injected dose accumulated selectively in the tumour. These targeting properties, when exploited for delivery of toxic agents to the tumour, give MAb E48 potential for tumour therapy. Especially the application of MAb E48 in radioimmunotherapy (RIT) seems to be attractive due to the intrinsic radiosensitivity of HNSCC. Armed with 186-Rhenium, a radionuclide recently introduced in the field of RIT, MAb E48 IgG was shown to be highly capable of eradicating established HNSCC tumours in nude mice. Complete ablation of small HNSCC was observed in this animal model by a single bolus injection. In an effort to make MAb E48 less antigenic for human application a chimeric human/mouse MAb (cMAb) has been constructed by use of recombinant DNA techniques. This modification strongly improved the capacity of MAb E48 for mediating antibody-dependent cellular cytotoxicity (ADCC). When using this cMAb E48 for RIT of minimal residual disease it can be anticipated that ADCC activity may be supportive to irradiation, especially in the ablation of single disseminated cells or small cell aggregates. Extrapolating results obtained in nude mice to patients and taking into account the good targeting in patients, RIT with E48 IgG seems to have potential for the elimination of minimal residual disease. Based on this encouraging progress, preparations are being made to evaluate the efficacy of Re-186-labelled cMAb E48 as an adjuvant in a phase III study for the treatment of patients who are at high risk for developing distant metastases.

Perspectives of monoclonal antibodies for detection and treatment of head and neck tumours.

Monoclonal antibodies (mabs) are potentially powerful tools for the detection and treatment of cancer. To date, only a limited number of mabs are available to head and neck cancer. We produced 5 different groups of mabs to head and neck cancer. These mabs were characterized for their reactivity tumour and non-tumour tissues. Furthermore, biochemical elucidation of recognized antigens was provided. In animal studies the effectiveness of mabs for diagnoses and therapy of cancer is clearly demonstrated. The first results of a clinical study for the detection of head and neck cancer with mabs are shown. Finally, the future of mabs in clinical oncology is discussed.

Lansoprazole 30 mg versus omeprazole 40 mg in the treatment of reflux oesophagitis grade II, III and IVa (a Dutch multicentre trial). Dutch Study Group.

OBJECTIVES: To compare the efficacy and safety of lansoprazole 30 mg daily (LAN30) and omeprazole 40 mg daily (OME40) in the treatment of moderate (Savary-Miller grade II) as well as severe reflux oesophagitis (grade III/IVa). DESIGN: A double-blind, randomized, multicentre study, involving 211 patients at 29 Dutch hospitals. METHODS: Healing was assessed by endoscopy, performed on admission, after 4 weeks and after 8 weeks if the patient was not healed after 4 weeks. Symptom relief was determined by symptom assessments at the same time points. Safety was evaluated by determining the incidence of adverse events. RESULTS: There was no significant difference in intention-to-treat (ITT) healing rates after 4 weeks (LAN30 87.5%, OME40 80.6%; 95% CI (-4.0; +17.8)) and in the ITT overall healing (LAN30 96.1%, OME40 93.1%; 95% CI (-4.2; +10.2)) rates between the LAN30 and the OME40 group. Relief of reflux-related symptoms at 4 weeks as well as 8 weeks did not differ significantly between the treatment groups. No difference in the incidence of adverse events was observed between the groups. CONCLUSION: Treatment of patients with reflux oesophagitis grade II, III or IVa with LAN30 was as effective as with OME40 with respect to healing as well as symptom relief.

Detection of squamous cell carcinoma xenografts in nude mice by radiolabeled monoclonal antibody E48.

We recently produced the monoclonal antibody E48 as a specific reagent for squamous cell carcinomas. In our ongoing investigations to use E48 for clinical tumor detection and therapy, fundamental aspects of the antigen have to be elucidated and practical applications of the antibody have to be tested in a preclinical model. Immunoelectron-microscopic studies localized the E48 antigen along the cell surface and in between desmosomes, suggesting that the antigen serves as an adhesion molecule. To evaluate the usefulness of E48 for radioimmunodetection of neck node metastases, nodes from 20 neck dissection specimens were tested. A strong reactivity was observed. Furthermore, F(ab')2 fragments of E48 were compared with the complete IgG E48 for selective tumor detection in an animal model. It was demonstrated that E48 F(ab')2 fragments localize faster and reach higher tumor-nontumor ratios than the whole molecule.

Chloral hydrate poisoning: its mechanism and therapy.

Chloral hydrate is a dangerous hypnotic drug to prescribe. Lethal dose can be a small as 4 gm. Nowadays the effects on the circulatory system are most dangerous. Which metabolite is responsible remains to be investigated. Haemoperfusion seems to be best method of lowering plasma levels of the metabolites, although the clearance of trichloroacetic acid is considered lower than of trichloroethanol.

Two target sites for protein binding in the promoter region of a cell cycle regulated human H1 histone gene.

The 5' region of a cell cycle regulated human H1 histone gene appears to contain at least six promoter DNA elements that are shared with some, but not all human core (H2A, H2B, H3 and H4) histone genes. We show that two of these elements represent separate binding sites for two distinct, partially purified factors. The first promoter domain contains A/T rich repeats and is involved in the binding of HiNF-A, a nuclear factor previously found to bind to A/T rich direct repeats in the promoters of human H4 and H3 histone genes. The second domain, containing the general promoter element 5' dACCAAT, acts as a binding site for a two component mosaic factor we have designated HiNF-B. These data suggest that coordinate transcriptional regulation of human H1 and core histone genes may involve two classes of trans-acting factors: those specific for histone gene promoters and those that act on a broad spectrum of human gene promoters.

The feasibility of radioimmunotherapy of head and neck cancer.

Since the introduction of the hybridoma technology by Kohler and Milstein (Nature 1975, 256, 495-497), tremendous effort has been put in the realisation of Ehrlich's concept of the magic bullet, which was proposed as early as the beginning of the century. The first clinical studies for radioimmunoscintigraphy (RIS) and radioimmunotherapy (RIT) with radiolabelled antibodies were undertaken in the early 1980s. Since then, RIS has been performed on thousands of patients with various types of malignancies, like colon carcinoma, lung carcinoma, breast carcinoma, neuroblastoma, T-cell lymphoma and ovarian carcinoma. In addition, a substantial number of therapy trials with radiolabelled antibodies have been performed. The developments for head and neck squamous cell carcinoma (HNSCC) have only recently been able to catch up with these events to some extent. One of the main reasons for this slow progress has been the lack of monoclonal antibodies (Mab) with specificity for HNSCC. Although there are as yet no real tumour specific antigens known for HNSCC, which also holds true for the majority of malignancies arising from other tissues, we now have the availability of a number of Mab with high specificity for HNSCC and with a very restricted reaction pattern with normal tissues. Labelled with 131I, these Mab have been shown to be highly capable to localise in HNSCC xenografts in nude mice. Based on these promising data, patient studies with one of these Mab, designated Mab E48, labelled with 99mTc, were started to evaluate the feasibility of RIS in patients with head and neck cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Complete ablation of small squamous cell carcinoma xenografts with 186Re-labeled monoclonal antibody E48.

In previous studies we have shown that in mice bearing head and neck squamous cell carcinoma (HNSCC) xenografts, radioimmunotherapy (RIT) with 186Re-labeled MAb E48 resulted in complete regressions in one-third of the tumors (followup > 150 d). MAb E48 was labeled with 186Re following a novel labeling procedure developed at our institute. The injected dose was 600 microCi, which was the maximum tolerated dose (MTD; < 15% wt loss) in these studies. The mean size of the tumors was 140 +/- 60 mm3. To investigate whether the therapeutic efficacy of RIT in our xenograft model would be improved when treating smaller xenografts, mice bearing 2 HNSCC xenografts with a vol of 75 +/- 17 mm3 (number of mice, n = 6; number of tumors, t = 12) were treated with 600 microCi of 186Re-labeled MAb E48 IgG. All tumors completely regressed and did not regrow during followup (> 150 d). In all mice, weight loss did not exceed 10%. To obtain biodistribution data, mice bearing two xenografts with a vol of 58 +/- 31 mm3 were injected with 100 microCi of 186Re-labeled MAb E48 IgG. The maximum uptake in blood was 26.4% injected dose/g (%ID.g-1) at 2 h pi and was 53.1%ID.g-1 in the tumor at d 7 pi. In normal tissues, no nonspecific accumulation was observed. Based on these biodistribution data, the absorbed cumulative radiation dose was calculated. The accumulated dose in blood and tumor was 2004 cGy and 8580 cGy, respectively. In other tissues, the dose was less than 8.1% of the dose delivered to tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioimmunotherapy of human head and neck squamous cell carcinoma xenografts with 131I-labelled monoclonal antibody E48 IgG.

Monoclonal antibody (MAb) E48 reacts with a 22 kD antigen exclusively expressed in squamous and transitional epithelia and their neoplastic counterparts. Radiolabelled with 99mTc, MAb E48 is capable of targeting metastatic and recurrent disease in patients with head and neck cancer. In this study, the capacity of 131I-labelled MAb E48 to eradicate xenografts of human squamous cell carcinoma of the head and neck (HNSCC) in nude mice was examined. Experimental groups received a single i.v. bolus injection of 400 microCi MAb E48 IgG (number of mice (n = 6, number of tumours (t) = 9) or 800 microCi MAb E48 IgG (n) = 5,t = 7), whereas control groups received either diluent (n = 3,t = 5), unlabelled MAb E48 IgG (n = 4,t = 5) or 800 microCi 131I-labelled isotype-matched control MAb (n = 6,t = 9). A 4.1-fold increase in the median tumour volume doubling time and regression of two out of ten tumours (20%) was observed in mice treated with 400 microCi. In mice treated with 800 microCi. In mice treated with 800 microCi, two out of seven tumours (29%) showed complete remission without regrowth during follow-up (greater than 3 months). Median tumour volume doubling time in the remaining five tumours was increased 7.8-fold. No antitumour effects were observed in mice injected with diluent, unlabelled MAb E48 or 131I-labelled control MAb. In the same xenograft model, chemotherapy with doxorubicin, 5-fluorouracil, cisplatin, bleomycin, methotrexate or 2',2'-difluorodeoxycytidine yielded a less profound effect on tumour volume doubling time. Increases in tumour volume doubling time with these chemotherapeutic agents were 4, 2.2, 2.1, 1.7, 0, and 2.6 respectively. Moreover, no cures were observed with any of these chemotherapeutic agents. From the tissue distribution of 800 microCi MAb E48, the absorbed cumulative radiation doses of tumour and various organs were calculated using the trapezoid integration method for the area under the curve. To tumour xenografts, 12,170 cGy was delivered, blood received 2,984 cGy, whereas in every other tissue the accumulated dose was less than 6% of the dose delivered to tumour. These data, describing the first radiolabelled MAb with therapeutic efficacy against HNSCC, suggest radioimmunotherapy with MAb E48 to be a potential therapeutic modality for the treatment of head and neck cancer.

Evidence for a role of the monoclonal antibody E48 defined antigen in cell-cell adhesion in squamous epithelia and head and neck squamous cell carcinoma.

Monoclonal antibody (MAb) E48 recognizes a 20- to 22-kDa antigen expressed by human squamous and transitional epithelia and their neoplastic counterparts. Histochemical examination of these tissues revealed distinct surface labeling with MAb E48. To investigate the subcellular localization of the E48 antigen we have performed electron microscopical analysis. In cells of normal oral mucosa, the E48 antigen was expressed on the plasmalemma, particularly associated with desmosomes, suggesting involvement of the E48 antigen in intercellular adhesion. Furthermore, the level of expression of the E48 antigen appeared to be influenced by the cellular organization. In squamous cell carcinoma (SCC) cell lines grown in vitro as subconfluent monolayer cultures, the E48 antigen expression was low. However, E48 antigen expression increased when SCC cells were grown to confluency. E48 antigen expression was similarly high when SCC cell lines were cultured under conditions promoting three-dimensional growth either as colonies within floating collagen gels or as xenograft in tumor-bearing nude mice. Further evidence for the involvement of the E48 antigen in cell-cell adhesion was found when SCC cells were grown within collagen gels in the presence of MAb E48: no spherical colonies were formed, but cells grew out to colonies composed of single cells. Moreover, in this culture system the percentage of SCC cells growing out to colonies was decreased by the presence of MAb E48. These findings indicate that the E48 antigen is involved in the structural organization of squamous tissue and might have a role in intercellular adhesion.

Superior localisation and imaging of radiolabelled monoclonal antibody E48 F(ab')2 fragment in xenografts of human squamous cell carcinoma of the head and neck and of the vulva as compared to monoclonal antibody E48 IgG.

Monoclonal antibody (MAb) E48 and its F(ab')2 fragment, radiolabelled with 131I, were tested for tumour localisation and imaging in nude mice bearing a squamous cell carcinoma xenograft line derived from a head and neck carcinoma (HNX-HN) or from a vulva carcinoma (VX-A431). MAb IgG or F(ab')2 fragments were injected in parallel and at day 1, 2, 3 and 6 or 7, mice were either scanned with a gamma camera or dissected for determination of isotope biodistribution. In HNX-HN bearing mice, E48 IgG as well as F(ab')2 showed highly specific localisation in tumour tissue. The mean tumour uptake (n = 4) expressed as the percentage of the injected dose per gram of tumour tissue (percentage ID/g) of IgG was 11.9% at day 1 and increased to 14.6% at day 6 whereas percentage ID/g of F(ab')2 was 7.2% at day 1 and decreased during subsequent days. Tumour to blood ratios (T/B) at day 1 were 1.2 for IgG and 13.6 for F(ab')2 and reached a maximum at day 6 with values of 6.4 and 54.2 respectively. In VX-A431 bearing mice, only E48 F(ab')2 showed preferential localisation in tumour tissue. At day 1, Percentage ID/g of IgG was 3.7 and T/B was 0.3, while percentage ID/g of F(ab')2 was 2.4 and T/B was 3.2. Percentage ID/g decreased after day 1 while T/B increased. In these experiments no preferential localisation of either isotype matched 125I-labelled control IgG or F(ab')2 was observed. In F(ab')2 injected HNX-HN bearing mice as well as VX-A431 bearing mice, tumours could be visualised at day 1 and 2 without any appreciable background activity. With MAb IgG this was also possible in HNX-HN bearing mice (but not in VX-A431 bearing mice) but only at day 3 and 6. These findings suggest that the superior tumour to non-tumour ratios render the E48 F(ab')2 fragment more qualified for specific targeting of radioisotopes to tumour xenografts in this experimental setting.

Potential for targeting head and neck squamous cell carcinoma with monoclonal antibody K984.

In previous reports we have described the development of mAb K984, reactive with an epitope expressed on the outer cell surface of undifferentiated, proliferating cells in normal stratified squamous epithelia and their neoplastic counterparts. The K984 antigen was also found to be homogeneously expressed by in vitro cultured squamous cell carcinoma (SCC) cell lines. In the present study we demonstrate that mAb K984 induces a significant, dose-dependent growth inhibition when SCC cells are grown in vitro as monolayer cultures in the presence of mAb K984. These data seem to indicate that mAb K984 has potential for tumour targeting, especially in a therapeutic setting. As a first approach to evaluate the suitability of mAb K984 for tumour targeting in vivo, radiolabelled mAb K984 was administered to SCC-xenografted nude mice. Selective tumour accumulation of mAb K984 was observed. Tumour to blood ratios and tumour to non-tumour ratios, as based on the biodistribution data, were at least ten times higher in case of the specific mAb K984 when compared to another non-specific, isotype-matched control antibody. mAb K984 was also capable of visualizing tumour deposits in xenografted nude mice. The corollary of these findings is that the mAb K984-defined antigen probably is involved in the regulation of proliferation of stratified squamous epithelium and squamous cell carcinoma and that mAb K984 has potential for specific tumour targeting.