Immunolocalization of the murine monoclonal antibody, 791T/36 within primary human colorectal carcinomas and identification of the target antigen.

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The murine monoclonal antibody 791T/36, originally prepared against a cultured human osteogenic sarcoma cell line, 791 T (Embleton et al., 1981) has been used for successful clinical radioimaging of both primary tumours and their metastases. Investigations have been completed upon osteogenic sarcomas (Farrands et al., 1983) and colorectal carcinomas (Farrands et al., 1982;Armitage et al., 1983;Pimm et al., 1984). In studies on colorectal cancer, autoradiography of tumours resected from patients given 1311-labelled 791T/36 antibody has indicated that the antibody predominantly localizes in stromal elements and secretions within pseudoacini rather than showing a clear cut binding to malignant cells (Armitage et al., 1983). The present study was therefore initiated in order to examine whether the localization of antibody to colorectal carcinomas is driven by an immune mechanism whereby the 791T/36 monoclonal antibody reacts with an antigen expressed within the tumour.
The preparation and purification of 791T/36 monoclonal antibody and normal mouse IgG2b and their labelling with radioiodine for clinical imaging trials have been previously described (Farrands et al., 1982). Tumour and normal tissue were resected from patients injected 1 to 3 days previously with 13II-labelled 791T/36 monoclonal antibody (70 MBq,200,ug antibody) -as part of a diagnostic imaging trial, (Armitage et al., 1983).
One patient received a mixture of 1311-719T/36 antibody (15MBq) and 1231-normal mouse IgG2b (25 MBq). Weighed samples of tumour and normal tissues were counted for 131I and in one case, for 1231, using an LKB-Wallac Gamma Counter.
As shown in Table I, the tumour to normal tissue ratios 131I per g were 3.3:1, 2.0:1 and 1 Patients 1, 2 and 3 respectively. Ratios of 2.0:1 or greater represent the level of uptake in tumours which permits successful external radioimaging of tumours using a blood pool subtraction technique, provided that the tumour image is not obscured by that of 131I accumulating in the bladder (Armitage et al., 1983;Pimm et al., 1984). With Patient no. 1 who received 1311-labelled 791T/36 antibody admixed with 123I-labelled mouse IgG of the same isotype (IgG2b), it was evident that there was no localization of mouse IgG within the tumour (tumour:normal tissue ratio for 1231 1.2:1) despite the fact that there was more than 3-fold uptake of '3'I into this specimen (tumour:normal tissue uptake for 1311=3.3: 1) ( Table I). This finding is consistent with the view that 791T/36 antibody localization within the tumour is mediated by an immunological process.
Since autoradiographic studies of tumour from patients infused with '311-labelled 791T/36 antibody have shown that a major localization of antibody was in stromal elements of the tumour (Armitage et al., 1983), procedures were developed for the further analysis of this material. Finely chopped specimens were passed through a 60-mesh stainless steel grid upon which fibrous material is retained and compressed in this tissue press. This procedure is based upon that described by Snary et al. (1976) who developed a method for tissue disruption using a mechanical press which retained fibrous material and connective tissue elements on the grid. In each case, the majority of radioactivity from tumour tissue was recovered upon the grid while the bulk of the disrupted tissue passed through the grid. The mean recovery of radioactivity in fibrous material on the grid was 87, 83 and 74% for tumour specimens from Patients 1, 2 and 3 respectively (Table I).
To permit further analysis of radioactive material retained upon the grid, solubilization with non- ionic detergent was attempted. With specimens from Patients 2 and 3 (Table I), the fibrous residues on the grid were extracted with detergent at a ratio of -2 ml detergent solution per g of original tissue. This was achieved by repeated extraction with 20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, pH 8.0 (termed TNEN buffer), homogenization using an Ultra Turrax homogeniser (Janke & Kunkel, Ika-Werk) followed by centrifugation at lOOOg for 20min and then reextraction of the pellets. This was continued for 3 or 4 cycles until the release of radioactivity into the supernatants was minimal (i.e. <5% of the total counts). The supernatants were combined and centrifuged ay 78,000g for 30min and this final supernatant was taken as the soluble extract of fibrous residue. A sample of soluble extract (2ml) from Patient number 2 (Table I) was examined by gel filtration on Sephacryl S-300 (Pharmacia, Uppsala, Sweden -Column dimensions 90 x 1.5 cm). The column was equilibrated in, and eluted with, TNEN buffer and calibrated for the elution of free antibody with 125I1 labelled 791T/36 antibody. In this case, 43% of the 1311-radioactivity was initially recoved in a soluble form. As shown in Figure la, after gel filtration chromatography, 78% of the 13'I-radioactivity in the detergent extract was excluded from the column (peak maximum in fraction 54) with the remaining material (22% of the 1311-radioactivity) separating as a peak of lower molecular weight (peak maximum in fraction 75, Figure la). 125I-labelled 791T/36 antibody, when chromatographed alone, separated as a single peak (peak maximum in fraction 74, - Figure lb material of lower molecular weight in the detergent extract. One interpretation of this experiment is that following detergent extraction of the fibrous material, 1311-labelled 791T/36 antibody is released in a soluble form with the majority complexed with solubilized antigen. In order to explore whether detergent extracts of this fibrous material contained '311-labelled 791T/36 antibody complexed with antigen, the following antigen identification procedure was developed: Aliquots (5 ml) of soluble extract of fibrous material prepared from tumour tissue from Patients 2 and 3 (Table I) were admixed with 0.2 ml of a 20% (v/v) suspension of Affi-Gel 10 (Biorad Laboratories, Watford, Herts) to which affinity purified goat antimouse IgG antibodies had been linked at 5mg antibody ml-' of Affi-Gel 10 and pre-equilibrated with TNEN buffer. Incubtation at 4°C with rolling was continued for 20h and the immunoadsorbent gel washed 5 times, each with 10 ml TNEN. Approximately 50% of the 131I activity present in the initial soluble extracts was bound to the immunoadsorbent gel at this stage reflecting the binding of 791T/36 antibodies (free or complexed with antigen) to the gel. The total Affi-Gel 10 pellet with its bound materials was labelled with 1251 by the addition of 0.5 mCi 1251 Na (Amersham International, Amersham) and 0.5 ml chloramine T (25 jug) added dropwise. Incubation at 0°C was continued for 20 min and the reaction terminated by the addition of 50 ul Na2S205(25 jug). The gel was then washed 5 times by centrifugaton, each wash being with 10mlTNEN. To the final pellet 0.2ml3MNaSCN in TNEN was added to release bound 791T/36 antibody (free or complexed with antigen) under which conditions immune complexes would also dissociate. The total mixture was applied to a PD 10 column (Pharmacia, Uppsala, Sweden) equilibrated with TNEN and eluted with this buffer. The gel excluded fractions containing the bulk of the radioactivity (and putative reassociated immune complexes and free antibody) were incubated with 100 lul of a 25% (v/v) suspension of Sepharose-Protein A (Pharmacia, Uppsala, Sweden) in TNEN. After incubation with shaking at 4°C for 60 min, the Sepharose-Protein A was washed 5 times with 10ml of TNEN containing 0.5% sodium deoxycholate, 2% BSA and 0.1% SDS, and then 5 times each with 10ml of a 1/10 dilution of TNEN in water.
To the final pellet 0.2 ml of reducing sample buffer was added (Laemmli, 1970), the sample was boiled for 3 min and after centrifugation, the supematant was electrophoresed on an SDS-12% polyacrylamide gel at 50v. The gel was stained for protein with Coomassie Blue, and after destaining the gel was dried and autoradiographed for up to 21 days at -70°C with preflashed Fuji X-ray film (Fuji Photo Co. Ltd., Tokyo, Japan) and an intersifying screen (Cawo, FRG).
After applying these procedures the following results were obtained. Three bands developed on the X-ray film after exposure to the dried gel upon which material isolated from the tumour from Patient 2 (Table I) Table I).
(b) the radioimmunoprecipitation of 791T/36 antibody defined antigen from detergent solubilized, 1251-labelled 791T cells, using 791T/36 antibody and Sepharose Protein A as the precipitating agent (Price et al., 1983). The positions of the mol. w marker proteins are indicated on the right (mol.wt values are x 10-3). Mol. w markers were myosin (200 kd), phosphorylase b (92 Kd), bovine serum albumin (67 Kd), IgG heavy chain (50Kd), ovalbumin (45Kd) and IgG light chain (25 Kd). corresponded to that of the 791T/36 defined antigen isolated from 791T cells (as shown in Figure 2b) (Price et al., 1983) as assessed using conventional radioimmunoprecipitation tests, SDS-PAGE and autoradiography. Equivalent results were obtained following extraction of tumour from Patient 3 and analysis of radioiodinated isolated immune precipitates by SDS-PAGE and autoradiography. Again the apparent molecular weight of the 791T/36 defined antigen was 72,000.
The conclusions from these studies are that following infusion of 791T/36 antibody into colorectal carcinoma patients, the antibody localizes within the tumour and the preferential accumulation of antibody therein is due to immune recognition of, and binding to its target rather than by a nonspecific interaction between antibody and tumour. In addition, when antigen is recovered from the resected tumour specimen, it is identical at least with respect to apparent mol.w, to that originally expressed upon the immunizing osteogenic sarcoma cell line used to produce the 791T/36 antibody.