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Localization of human tumour xenografts after i.v. administration of radiolabeled monoclonal antibodies.

A mouse monoclonal antibody (LICR-LON/HT13) has been developed to a cell-surface antigen carried on a human germ-cell tumour xenograft (HX39). After radioiodination, the antibody localized in vivo preferentially in xenografted tumours as opposed to normal mouse tissue, whereas tumor uptake did not occur with normal mouse IgG or nonspecific monoclonal IgG. This selective localization could be abolished by simultaneous injection of an excess of the unlabelled LICR-LON/HT13. The kinetics of and factors influencing localization have been examined. Tumour weight was important in that the smaller the tumour the better the localization. LICR-LON/HT13 was found to localize also in other xenografted germ-cell tumours, but not in non-germ-cell tumour xenografts. Thus monoclonal antibodies are capable of selective in vivo localization of human tumours in an animal model, and their clinical value should now be assessed.

Summary.-A mouse monoclonal antibody (LICR-LON/HT13) has been developed to a cell-surface antigen carried on a human germ-cell tumour xenograft (HX39). After radioiodination, the antibody localized in vivo preferentially in xenografted tumours as opposed to normal mouse tissue, whereas tumour uptake did not occur with normal mouse IgG or nonspecific monoclonal IgG. This selective localization could be abolished by simultaneous injection of an excess of the unlabelled LICR-LON/HT13.
The kinetics of and factors influencing localization have been examined. Tumour weight was important in that the smaller the tumour the better the localization. LICR-LON/HT13 was found to localize also in other xenografted germ-cell tumours, but not in non-germ-cell tumour xenografts.
Thus monoclonal antibodies are capable of selective in vivo localization of human tumours in an animal model, and their clinical value should now be assessed.
THE CONCEPT of radiolabelled antitumour antibodies to localize animal tumours in vivo was introduced by Pressman et al. (1957) and extended to human tumours by the use of affinity-purified antibodies to CEA, which were shown to localize preferentially in CEA-containing human colon (Mach et al., 1974;Primus et al., 1973) and human breast tumours (Moshakis et al., 1981a) xenografted into animals. Subsequently, radiolabelled antibodies to CEA (Goldenberg et al., 1978;Mach et al., 1980a;Dykes et al., 1980), human chorionic gonadotrophin (Begent et al., 1980;Bagshawe et al., 1980) and alphafoetoprotein (Goldenberg et al., 1980) were successfully applied in clinical practice by the use of y-camera external photoscanning. However, a computerized subtraction technique has been used by all workers in order to eliminate bloodbackground radioactivity and to permit detection of the target tumour. This may be liable to interpretive error and has left some workers unconvinced (Houston et al., 1980), while others have expressed doubt about the clinical applicability of conventionally raised antibodies to established tumour markers (Mach et al., 1980a, b).
Monoclonal antibodies can be raised to antigens of choice, and may offer a specific and selective approach. Their recent development (Kohler & Milstein, 1975) together with the production of the human tumour xenograft system in immunesuppressed mice has enabled us to evaluate the potential of these new reagents for tumour localization before clinical studies.

AIATERIALS AND METHODS
Antibody and iodine labelling.-The cell line HX39/7, established from xenografted undifferentiated human malignant teratoma (MTU) (Raghavan et al., 1980) was used to immunize mice. Three weeks after the first injection of 107 cells, a second i.p. injection of 107 cells was given, and 3 days later the spleen was remnoved from the mice. Spleen cells were fused with the mnyeloma NSI (gift of Dr C. Milstein) using a modification of the method of Galfre et al. (1977). The cells, were plated in soft agar using mouse thymocytes as feeder cells (Edwards et al., 1980) and viable antib)ody-producing hybr id clones Mwere picked from the agar 11-12 days after fusion.
Antibodies l)inding to the teratoma cells (HX39/7) were detected by using 1251-F(ab)2 as second antibody (rabbit anti-mouse IgG) in the assay system of Williams et al. (1977).
The hybrid clone producing a suitable antibody -was chosen for subsequent use and wNas grown in ascitic form in mice. The ascitic fluid was collected from this and the antibody was purified by precipitation uwith 5000 (NH4)2SO4 and Sephadex G-200 chromatography. Immunochemical and biochemical studies showed the antibody to be of the IgG 2a class.
Free iodine was removed by gel-filtration chromatograplhy, using a G25 Sephadex column eluting writh 0-5o BSA in PBSA (lml fractions were collected). A specific activity of 5 ,uCi/ug was achieved. As controls for nonspecific accumnulation of proteins in tumours and tissues, normal mouse immunoglobulins (Miles) and a mouse monoclonal IgG (FIB-75), which bound only wx-eakly to HX39/7 teratoma cells in vitro (Mcllhinney, 1980), were iodinated Xwith 1311 to equivalent specific activities.
The iodinated reagents were passed through a Sephacryl S-300 (0 9 cm x 60 cm) column in order to establish the presence or absence of aggregates or degradation products of the irnmunoglobulin after the I-labelling. The antibodies -were eluted with 0IM phosphate buffer (pH 7-5) at a rate of 5 ml/h. 0 5ml fractions were collected and the radioactivitv of eachi fraction (1251 and 1311) was measured wN-ith an LKB-1280 ultra-gamma counter. In addition, after injection of the radiolabelled antibodies and exsanguination of the animals at, predetermined intervals, plasma samples wi-ere passed through the column. The plasma profiles of animals bearing small or large tumours or from tumour-free animals, were investigated.
Animal model. Four-week-old CBA/lac mice, weighing on average 23 g, were immunesuppressed by thymectomy and total-body irradiation preceded by i.p. administration of arabinoside C (Steel et al., 1978). Two-mm cubes of tumour were implanted s.c. in each flank and allowed to grow, for 12-28 days before an experiment was started. We were thus able to test tumours of various weights ranging from 6 mg to 1102 mg. The viability of small tumours was confirmed at histological examination. The presence of mitoses was taken as evidence that tumours w ere actively growing. In addition to the HX39 teratoma, against which the antibody was raised, me tested 4 other germ-cell tumour xenografts and 3 non-germ-cell tumours. All the xenografts used in these studies were of human origin, and have been shown to maintain the same histology as the primary tumour, and human karyotype, throughout passage. The germ-cell tumour xenografts, details of which have been published previously (Raghavan et al., 1980), Mwere: (a) HX112/3, yolk-sac carcinoma; (b) HX57/17, yolk-sac carcinoma; (c) HX53/14, seminoma with yolk-sac carcinoma elements, and (d) HX1 11/3, malignant teratoma undifferentiated (MTU). The other tumours were: (a) HX99/12, breast adenocarcinoma (Bailey et al., 1980); (b) HX65/3, bronchial adenocarcinoma (Shorthouse et al., 1980), and (c) HN.BR/3, squamous-cell carcinoma (Easty (t al., in preparation).
Radiolocalization. Simultaneous i.v. injection of 10-15 .tCi each of 1251-labelled monoclonal antibody and 1311-labelled mouse IgG was given to tumour-bearing and tumour-free animals. Owing to variability in the labelling efficiencies, the amounts of inonoclonal antibody and normal IgG were not always equal. At intervals of 4-96 h after injection, the animals were exsanguinated by cardiac puncture and tumours and organs (salivary gland, thyroid gland, heart, lungs, liver, spleen, stomach, kidney, intestine and thigh muscle) w%ere removed. After each tissue had been weighed, radioactivity was measured with a LKB-1280 ultra-gamma counter.
The results are expressed as: (a) localization index (LI), i.e. the ratio of specific (1251) to nonspecific (1311) activity in tumours and organs divided by the same ratio in the blood and (b) absolute radioactivity, i.e., ct/min/mg wet weight. The LI provides a standardized method of comparing results between experiments, and is similar to the localization ratio used by Primus et al. (1973) and the specificity index used by Mach et al. (1974), except that the former authors made a comparison with the ratio in the injected solution, and the latter with the liver.
A different monoclonal antibody, FIB- The values in the other organs lie between those of liver and salivary gland (highest uptake) and muscle (least uptake). Values are means of 6 samples; mean tumour weighit 11I mg.
75. an JgG 2a, was iodinated with 1311 and used as the control, replacing the normal mouse JgG. This antibody showed very weak binding to HX39/7 cells in vitro (Mcllhinney, 1980) and its behaviour in the animal was the same as that of normal mouse JgG (Fig. 4). No difference in the degree of localization was obtained when FIB-75 was used as the nonspecific JgG rather than the normal mouse IgG. Therefore the localization of LICR-LON/HTi 3 in the tumour was not an idiosyncrasy of the monoclonal antibody. The clearance of the antibody, expressed as absolute concentration of activity (ct/mmn/mg) from the tumour, normal tissues and blood was studied (Fig. 5). The concentration in the tumour started to exceed that of normal tissues and blood at 4-24 h after injection. Although thereafter the amount of antibody in the tumour decreased, it did so at a slower rate than blood and normal tissues; thus the tumour/non-tumour ratio (Table I)  the tumour/blood ratio ( Fig. 3b and c) increased with time. In contrast, the normal IgG behaved indifferently to the presence of tumour; its clearance from the tumour was similar to that of all the other tissues studied and its concentration was always lower than in blood (Fig. 4).
Since the ratio of 1251-labelled LICR-LON/HT13 to 1311-labelled normal mouse IgG was the same in normal organs of the experimental animals and did not change appreciably with time, the distributions of the two antibodies in normal tissues appear similar. In our system, the specific antibody tumour/blood ratio was between 0-4 at 4 h and 10 at 96 h after injection, demonstrating a 2-fold (4 h) to a 14-fold (96 h) increase over the same ratio with normal IgG (Fig. 3a). Similarly, the specific antibody tumour/blood ratio showed a 3-fold (4 h) to a 20-fold (96 h) increase over the liver/blood ratio (Fig.  3c). The localization described above could be inhibited by simultaneous i.v. injection of non-radiolabelled LICR-LON/HT13 (100 aug/animal), indicating that the cold antibody was capable of blocking the tumour antigenic sites, thus inhibiting uptake of further (radiolabelled) antibody by the tumour. In parallel experiments, such inhibition was not found when cold FIB-75 IgG was used.
If the localization observed is due to antibody-antigen reaction, it might be expected to occur rapidly after injection, and therefore selective homing of the antibody to the tumour should be detected by 4 h, when the first dissection of the animals took place. Indeed, it was found with the tumour/blood and tumour/nontumour ratios with the monoclonal IgG were higher than the same ratios with the normal IgG 4 h after injection (Fig. 3b). Similarly, the specific-antibody tumour/ blood ratio is superior to liver/blood ratio (Fig. 3c) as early as 4 h after injection. The same pattern is seen when the monoclonal antibody/normal IgG ratio in the tumour is compared to that in the blood (Fig. 3a).
Plasma samples of injected animals were passed through Sephacryl S-300 columns. The IgG eluted consistently at the expec-ted fractions, with no peaks of radioactivity before or after the IgG peak, indicating that large molecules had not been formed and no degradation of antibody had taken place (Fig. 6). It was thought that shedding of antigen to the circulation from large tumours (which showed no uptake) may inhibit localization, as it would react with the injected antibody. However, the plasma profiles of large tumours (0 8-1 1 g) was the same as that of small tumours (8-86 mg).
Tumour weight was found to be important, in that the smaller the tumour the   (Fig. 7). animals carrying 2 tumours c weights, this difference was (indicating that this phenorr tumour-dependent rather tha dependent). The specificity of the loca LICR-LON/HT13 for the gern toma HX39/7 was tested in oth by using 4 additional germ-cell germ-cell xenografts. In vivo 1 occurred in all the teratoma lii in the non-teratoma tumours The degree of localization varie the teratomas, but in none wae as for the HX39/7 tumour.

DISCUSSION
This work demonstrates tha clonal antibody raised against component of a human tumoui is capable of selective localizati a tumour after its in vivo adm This was demonstrated as follo uptake of the antibody by the t higher than in blood and norn atoma and (b) localization did not occur with normal > anti-teramouse IgG or an indifferent monoclonal IT] 3* antibody, (c) blocking of the antigen with LIt excess antibody inhibited selective uptake and (d) preferential localization in the 48 1i 996 h tumour was demonstrated not only with 7*0 10-9 regard to normal mouse tissues and with 65 7. 7 normal IgG but also with regard to other 3-8 5-6 tumours. The above findings support the 4-0 3-6 hypothesis that the in vivo localization is 248 2-9 due to the interaction of the injected anti-  (Moshakis et al., 1981b).
The absence of immune complexes significant (large molecules eluting before the IgG dy by the peak) in the plasma of the animals after s weighing antibody injection suggests that the target In single cell-surface antigen does not reach the )f different circulation. This, however, should not be also noted taken as aiding localization, since similar ienon was work with antibodies to soluble tumour Ln animalmarkers in patients and animals has shown that the presence of circulating ,lization of antigen does not, surprisingly, have any n-cell terarelevance to the degree of localization .er tumours (Goldenberg et al., 1980;Moshakis et al., and 3 non-1981a). Localization Comparison of the concentration of the nes but not two radioisotopes in tumour, tissues and (Table II). blood have made it possible to attribute d amongst the selective uptake of the antibody by the s it as high tumour to the binding properties of the antibody itself. Non-immunological factors that would affect the entry and handling of IgG by the tumour, such as vascularity, necrosis, lymphatic drainage and it a monoextracellular space, would apply equally t a surface to both specific and normal IgG. It appears r xenograft that the technique of double-isotope ion in such labelling (Pressman et al., 1957) still inistration. remains of great assistance in this field, iws: (a) the especially in the experimental animal which umour was can be killed and tissue radioactivity nal tissues, measured. This becomes more important when new reagents, such as monoclonal antibodies, are exploited for this purpose. Demonstration of localization in the animal model of human tumour xenografts lessens, but does not abolish, the need for such studies when this approach is used in patients, where tissue dissection is not often possible.
Correlation between in vivo localization and in vitro binding to cells was not possible when other teratomas were used, because none of the other 4 xenografts have been established in tissue culture.
Nevertheless, the behaviour of the antibody and the normal IgG, together with the localization kinetics, were similar to the pattern seen when LICR-LON/ HT13 used to localize the HX39 tumour. The specific uptake of the monoclonal antibody was highest in the HX39 tumour (against which the antibody was raised). This suggests that, although the antigen is present in all the germ-cell tumours examined, its "expression" was less in the other tumours than that which acted as an immunogen. Since the tumours examined contain varying amounts of yolk-sac carcinoma, seminoma and undifferentiated malignant teratoma, further studies are required, to correlate germ-cell tumour histology and site of antibody uptake at a cellular level. Such a study might reveal the reason for the varying degrees of localization of antibody among germ-cell tumours.
This study of tumour localization and the parameters which affect it, using a monoclonal antibody against a surface antigen on a human teratoma cell line, shows that such antibodies do localize in vivo and that they are, therefore, good candidates for clinical use, such as immunoscanning and tumour-drug targeting.
In this respect, the property of the antibody to localize in small tumours could prove very useful in detecting micrometastases in patients, especially when the newer tomographic scanning techniques are fully developed. In the past, computerized background subtraction of the blood pool has been used in every case when conventionally raised antisera have been used to detect tumours in patients. However, the tumour/blood ratios obtained with monoclonal antibodies may make this technique unnecessary; Ballou et al. (1979) using a monoclonal antibody, have shown that murine tumours can be detected without background subtraction, if the scan is performed more than 48 h after antibody injection. It must be emphasized, however, that although the tumour/blood ratio increases with time the absolute amount of activity in the tumour decreases. The optimal time, therefore, for scanning is a balance between these two factors. It is interesting that our study, and those of others using monoclonal antibodies for localization (Ballou et al., 1979;Houston et al., 1980), demonstrate better tumour/blood and tumour/ non-tumour ratios than those obtained with conventional antibodies to soluble tumour markers. However, it should not be forgotten that the antibodies used are of murine origin and injected into mice. Their value can only be assessed fully when these reagents are injected into patients.