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Integrins are cell-surface glycoproteins found in different forms on all cells except erythrocytes. Integrins bind to cell adhesion molecules and to proteins found in the extracellular matrix. A tripeptidic sequence Arg-Gly-Asp (RGD) is often the primary site of recognition by integrins which are expressed on tumour cells and are responsible for tumour invasion and metastasis. A synthetic decapeptide designated alpha P2 containing two RGD sequences radiolabelled with technetium-99m was used to image malignant melanoma in vivo. Fourteen patients previously diagnosed with metastatic melanoma underwent gamma camera imaging 20-180 min following intravenous administration of the radiolabelled synthetic decapeptide alpha P2. Six out of eight (6/8) of the lymph node metastases (75%) and all other neoplastic sites (11 sites) were successfully imaged, with the exception of three sites in the mediastinal area which were not positively imaged. In two cases there was false positive uptake in the rounded pigmented areolar/nipple area. In three cases (seven sites) the peptide scan confirmed the absence of disease in suspected lesions (true-negative). The synthetic peptide was rapidly removed from the circulation by filtration through the kidneys and excretion in the urine. No toxicity or adverse events were recorded. Radiolabelled alpha P2 peptide, which binds specifically to adhesion molecules on tumours, can be used for the in vivo detection of neoplastic metastases.

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Four murine IgG1 monoclonal antibodies, each with specificity for a human tumour-associated antigen, have been tested for their in vivo immunogenicity using a rabbit model. Surprisingly, one of these antibodies, MR6, was significantly more immunogenic than the remaining three reagents. This enhanced MR6 immunogenicity was not restricted to the immunoglobulin molecule itself, but also applied to a hapten (fluorescein isothiocyanate, FITC) when conjugated to the monoclonal antibody. In addition, the secondary immune response to an independent antigen, human haemoglobin, was higher when the antigen was administered simultaneously with MR6 than when co-injected with an isotype-matched control monoclonal antibody. The presence of the target antigen, gp200-MR6, on both rabbit and human leucocytes and epithelium, and its known association with human IL-4 function, raises the possibility that antibody MR6 may not only target immunogens to antigen-presenting cells, but may also enhance the ability of these cells to present antigen to the immune system. Antibodies to the gp200-MR6 may therefore find important clinical application as in vivo adjuvants.

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The aim of this study was to make a comparative evaluation of a direct and an indirect method for the labelling of anti-CEA with technetium-99m (99Tcm). With the direct method, disulphide bridges were cleaved by the use of 2-mercaptoethanol as reductant, whereas with the indirect method, the antibody was coupled to 2-iminothiolane. In both cases, a preformed intermediate chelate was used for 99Tcm exchange. The radiochemical and radiobiological behaviour of the 99Tcm-labelled species were studied. Furthermore, the influence of the labelling systems on the integrity of monoclonal antibodies, as well as the ability of 99Tcm-anti-CEA to tag onto human cancer cells, was investigated for the two labelling systems. Both methods showed a high labelling yield and resulted in immunoreactive and stable derivatives. However, detailed electrophoretical and radiochemical data, as well as the cysteine challenge trial, indicated relatively greater stability for the 2-mercaptoethanol reduction procedure.

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Specific tumour imaging with radiolabelled monoclonal antibodies has been extensively investigated. Although some success has been reported, there are many limitations due to the slow kinetics, poor extravasation, catabolism by the reticuloendothelial system, and non-specific uptake of macromolecules such as antibodies. We have tried to overcome some of the problems associated with monoclonal antibodies while retaining their specificity by using an antibody-derived synthetic peptide. A synthetic pentadecapeptide (alpha M2) derived from the third heavy-chain complementarity-determining region (CDR-3H) of a tumour-associated monoclonal antibody was produced and shown to retain its specificity against the pan-carcinoma cell-surface antigen, polymorphic epithelial mucin, detected by the parent antibody. The peptide was radiolabelled with technetium-99m and injected intravenously to image malignant lesions in 26 women with primary, recurrent, or metastatic breast cancer. Visualisation of breast tumours and their metastases was obtained shortly after administration of alpha M2, and was optimum by 3 h. Overall, 57 (77%) of 74 sites were visualised. Successful imaging was achieved in 14 of 15 primary tumour sites and all of eight local recurrences. Five of six metastases in the opposite breast, eight of 15 metastatic axillary lymph nodes, and all of six metastatic supraclavicular lymph nodes were imaged. Metastatic sites in the lungs, mediastinum, chest wall, and liver were poorly visualised because of background cardiac blood pool. alpha M2 detected small lesions ( < 2 cm) as efficiently as larger ones. The peptide was rapidly (3 h) cleared from the circulation. No acute or chronic adverse reactions due to the alpha M2 were observed. Specific tumour targeting with the radiolabelled anticancer peptide alpha M2 offers new opportunities for breast cancer imaging and possibly therapy.

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In accordance with Jerne's idiotypic network theory, it was attempted to generate antibodies utilizing the host's anti-idiotypic reactions. A murine anti-tumour monoclonal antibody was administered to rats; we examined the possibility of whether the animals would develop anti-idiotypic antibodies, the paratope of which would be the "internal image" of the tumour associated antigen that the original murine monoclonal antibody recognizes. These anti-idiotypic antibodies would generate a second generation of anti-idiotypic antibodies, which would have the same specificity as the immunogen murine antibody. It was found that, while all rats had no anti-tumour response prior to immunization with the anti-tumour murine monoclonal antibody, they all developed antitumour antibodies following administration. The latter observation came as a consequence of the development of anti-mouse immunoglobulin and anti-idiotypic antibodies. The animals were sacrificed and their spleen cells were fused with myeloma cells for the production of monoclonal rat anti-tumour antibodies. Finally, a monoclonal antibody was selected which appeared to recognize the same tumour-associated antigen as the originally administered murine antibody, as shown by cell-binding and competition assays, as well as immunohistochemistry. This new approach allows for "replication" of polyclonal and monoclonal antibodies without the need of the antigen and it may also serve as an immunotherapy strategy for the augmentation of the antitumour immune response of patients receiving monoclonal antibody treatment.

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The effectiveness of immunodiagnosis and immunotherapy is limited when xenogeneic antibodies are used, due to the host's anti-immunoglobulin response. We have attempted to specifically suppress the immune response to the immunogen (immunoglobulin) administered. The concept of antigen "suicide" was used, where the antigen (immunoglobulin) was suitably radiolabelled and administered to animals. The question asked was whether immunocompetent cells that specifically interact with the radiolabelled immunogen, would be lethally irradiated and thus become inactivated rather than stimulated. When mice where primed with 111Indium-labelled polyclonal human IgG (specific activity 15 mCi/mg), they responded 13% less than control animals (p = 0.0485). This suppression was IgG-specific, since all animals responded similarly to a control antigen (human albumin). However, a second boost of the same 111Indium-labelled preparation (specific activity 7 mCi/mg) did not show any statistically significant immunosuppression. In addition, rabbits primed with 125Iodine-labelled mouse monoclonal antibody (specific activity 180 mCi/mg) and boosted with the same unlabelled monoclonal antibody, showed a similar anti-mouse antibody response with the ones that received only unlabelled preparation twice. We conclude that the concept of antigen "suicide" may be effective for the induction of specific unresponsiveness; when immunoglobulins are radiolabelled with 111Indium at high specific activities, the desired state of specific immune suppression may be induced.

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Ten patients with transitional cell carcinoma (TCC) of the bladder received 3-6 mCi of HMFG1 monoclonal antibody (MAb) intravesically. The antibody was labeled with Tc-99m using the 2-Iminothiolane method. All patients underwent transurethral resection of the bladder tumor within 12-20 h following intravesical administration of 99m-Tc-HMFG1. The presence of the radiolabeled MAb in the circulation was studied by measuring the radioactivity in the serum for a period up to 20 h. Three of 10 patients underwent immunoscintigraphy (SPECT) 2-3 h postadministration and cancerous areas could be easily localized. Biopsies were taken from the tumor sites as well as from normal bladder mucosa. Absolute uptake of the administered MAb expressed as percent administered dose/kg of tissue could be evaluated only in eight patients. Multiple specimen taken from various tumor sites in every patient gave a wide range of uptake values ranging from 0 to 9.29% adm. dose/kg, whereas normal tissue uptake values ranged from 0 to 1.63, respectively.

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A bispecific mouse monoclonal antibody (mAb) that recognises carcinoembryonic antigen (CEA) with one binding site and vinblastine (VLB) with the other was used, and its in vivo immunosuppressive effect specific for anti-mouse immunoglobulin (Ig) was studied. The antibody was incubated with VLB at a molar ratio (MR) of 1:1, and administered i.v. to rabbits. Control animals received either the MAb alone, or the MAb with VLB covalently linked (MR 1:1), or the parental anti-CEA with equimolar amount of VLB. Seven days later, the rabbit anti-mouse Ig primary response was measured, and found to be almost 55% reduced in the animals that received the VLB 'loaded' MAb. In vivo kinetics and stability experiments revealed that the T1/2 of the MAb was 68 +/- 5 h, whereas free VLB disappeared within minutes. It was concluded that as soon as the drug dissociates from the antibody's binding site, it is rapidly removed. This problem was overcome by subcutaneously implanting osmotic mini-pumps containing VLB. The pumps released the drug at a constant rate for a period greater than 1 week, saturating the antibody's binding site. Under these conditions rabbits developed 80% less anti-mouse Ig antibodies when the bispecific antibody was administered (compared with the parental anti-CEA). The immunosuppression observed was specific for the mouse Ig, under conditions compatible with the full clinical therapeutic potential of the MAb. In conclusion, these experiments show, that it is possible to develop hybrid antibodies that can act as a 'lethal bait' to any specific lymphocyte in vivo, thus preventing undesirable responses against the xenogeneic MAb.

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We have used 123I-labelled epidermal growth factor (EGF) scans to study 14 patients with advanced cervical cancer. Abnormal lymph node imaging was seen most clearly 6-8 h after the injection and revealed abnormal uptake by pelvic lymph nodes in 11 patients. 4 of these 11 had abnormal computerised tomographic and ultrasound scans; in the other 7 conventional radiology did not confirm the presence of disease.

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From March 1987 to March 1988, a phase I to II study was carried out in 25 patients with ovarian cancer. They received escalating doses of intraperitoneally (IP) administered yttrium-90 (Y-90)-labeled monoclonal antibody, HMFG1, against a tumor cell-surface antigen. Myelosuppression prevented an escalation of the administered Y-90 activity above 25 mCi. Y-90-labeled antibody was absorbed from the peritoneal cavity into the circulation. Maximum blood Y-90 activity was observed 40 hours after the IP injection with a mean of 21% of the injected activity (range, 14.2% to 26.4%) in the circulation. The radiation dose the bone marrow received from circulating Y-90-labeled antibody (the blood radiation dose) was calculated by applying the Medical Internal Radiation Dose (MIRD) formulation to the measured Y-90 activity in patients blood. Myelosuppression occurred following calculated blood radiation doses to bone marrow of only 10 to 30 cGy. The excessive myelosuppression following such modest radiation doses from circulating Y-90-labeled antibody could be explained by the uptake of Y-90 by bone. In an attempt to reduce bone absorption of Y-90, seven patients received an intravenous (IV) infusion of EDTA (Sinclair Pharmaceuticals Ltd, Godalming, United Kingdom). This increased the urinary excretion of Y-90 from a mean of 11.1% to 32.3% of the injected activity (P = .0001). Fourteen patients had assessable tumor at laparoscopy. Tumor regression was observed in one patient, and palliation of ascites in a further patient.

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Tumor localization in patients has been achieved through the in vivo use of streptavidin and biotin. In these preliminary studies, the monoclonal antibody HMFG1 was conjugated with streptavidin and 1 mg was administered intravenously to each of 10 patients with documented squamous cell carcinoma of the lung. Two to 3 days later, 111In-labeled biotin was also administered intravenously. No evidence of toxicity was observed. Background radioactivity levels were reduced in liver (1% ID at 24 hr) and kidneys (2%) and in all other normal tissues and blood. Images of lung tumor were obtained in as little as 2 hr following administration of labeled biotin. In eight patients, tumor was detected with labeled biotin alone without the previous administration of streptavidin-conjugated antibody but in three of these patients, the images were improved with the prior administration of conjugated antibody. These results suggest that this approach may improve the tumor-to-normal tissue radioactivity ratios in radioimmunotargeting.

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Tumour associated monoclonal antibody against placental alkaline phosphatase (H17E2) was radiolabelled with Indium-111 and Iodine-123 and administered intravenously in 33 patients with primary and/or metastatic testicular tumour, as well as in 8 patients who were in complete remission after surgical excision of the tumour. The presence of a tumour was confirmed and correlated well with conventional diagnostic techniques and, in addition, the antibody scan revealed the presence of active disease in 2 patients with negative conventional imaging and with elevated serum markers. In addition, in one patient the CT produced a false positive result where the antibody scan was negative. Finally, the absence of tumour was confirmed in all 8 cases of patients in complete remission. All patients studied with Indium-labelled antibody had observable concentrations of the radiolabel in the liver (estimated to be approximately 30% of the administered dose), as well as in the kidneys and spleen. The patients studied with the Iodine-123 labelled antibody had observable concentrations in the thyroid gland and the stomach. The best images were seen at 48 and 24 hrs after the Indium and Iodine radiolabelled antibody respectively. No human anti-mouse antibody was detected in any of our patients, even in those who received 2 and 3 administrations, with the highest amount of administered protein being 800 micrograms. No toxicity was encountered in any of our patients in 4 months of follow-up. This method may be of clinical value in patients with testicular neoplasma and represents a new addition to current imaging techniques. A positive scan indicates the definite presence of a tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

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C and CR1 have been shown to participate in the clearance of injected, preformed, immune complexes in humans and in non-human primates. Their role in the physiologic disposal of immune complexes formed in vivo in humans was investigated in three patients receiving radioimmunotherapy for ovarian carcinoma. On day 0 each patient received, by intraperitoneal injection, 10 mg of 131I-mouse anti-tumor mAb (10 mCi/mg). On days 1 and 2, 18 mg of trace-labeled, 125I-human anti-mouse IgG was administered by i.v. infusion over 15 min, to accelerate the clearance of the 131I-anti-tumor antibody from the circulation and reduce the radiation dose to the marrow. Sequential blood samples were obtained after the injection of the second (anti-mouse) antibody, to monitor clearance. Immune complexes (shown by sucrose gradient centrifugation to be 19 to 40 S in size) formed within 5 min, and were cleared with a half-life of 11 +/- 1.7 min in the liver. Complexes were measured by 4% polyethylene glycol precipitation, and by solid phase C3d- and C1q-binding assays. Between 8 and 11% of the total available complexed material bound to CR1 on E. Peak binding of immune complexes to red cells occurred 10 min after the maximal complex load was detected by precipitation with polyethylene glycol. At that time, immune complexes bound to E constituted one-fifth of the total circulating pool of complexes. Coincident with immune complex formation and clearance, a 47% fall in serum C4, C3, and CH50 was measured, with the deposition of up to 1230 molecules of C4, and 2590 molecules of C3 on the surface of red cells. During 20 min after immune complex formation there was a mean loss of 32% of erythrocyte CR1. The changes in complement and CR1 on E and in serum observed in these patients resembled those seen in patients with SLE: i.e., a reduction in CR1 and an increase in C3 and C4 on E, and reduced serum C.

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A monoclonal antibody (MUC 2-63) was raised against a neuroectodermal antigen expressed on human malignant gliomas, neuroblastomas and melanomas. The mouse monoclonal antibody MUC 2-63 was of IgG1 isotype, it binds the antigenic determinants with the molecular weight of 32,000 Dalton present on the cell surface of native glioma cells. Seven patients with brain tumours, five with biopsy proven malignant gliomas, received an intravenous injection of the 111In-DTPA coupled monoclonal antibody MUC 2-63. Six patients had an uptake of 0.01-0.04% of the injected dose at the site of the tumour, which correlated to the computerized tomography (CT) images. The half-lives in the tumours of 111In were 38-259 h. The maximal uptake in the tumours were noticed between 40 and 67 h. One patient failed to demonstrate any intracranial uptake of the radioactivity. This patient had been treated with surgery and radiotherapy for a stage 2 testicular seminoma four years ago. He recently developed clinical signs of a primary brain tumour and the CT diagnosis was malignant glioma. All patients had nonspecific uptake in the liver, half-lives were between 60 and 84 h, the corresponding maximal uptake was detected between 22 and 45 h. No side effects were observed by administration of the mouse monoclonal antibody MUC 2-63.

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The monoclonal antibody MUC 2-63 identifies a glycoprotein antigen with a molecular weight of 32,000 Dalton present on human malignant gliomas but not detected on normal nerve and brain tissues. This was the prerequisite for these clinical studies with 111In-MUC 2-63 for imaging and 90Y-MC 2-63 for treatment. Seven patients with malignant gliomas, 6 with astrocytomas grade III-IV and 1 with a relapsing astrocytoma grade I-11 received the 111In labelled monoclonal antibody MUC 2-63 for in vivo diagnosis. Six patients had an uptake in the tumour of 0.01 to 0.05% of the MUC 2-63- 111In monoclonal antibody. The patient with the relapsing astrocytoma grade I-II had a negative scan due to a radical operation before the diagnostic dose. All patients received treatment with 90Y-MUC 2-63 intravenously in doses ranging from 146 to 830 MBq. Two patients with relapsing grade III-IV astrocytomas demonstrated clinical improvements and CT changes interpreted as necroses. No serious side-effects were observed in the 5 patients who only received one dose. The two patients who received up to 4 doses experienced grade 3 to 4 leukocyte and thrombocyte toxicity, most likely related to the bone-marrow toxicity by 90Yttrium. These data indicate the potential usefulness of the MUC 2-63 monoclonal antibody for in vivo image on humans with brain tumours and for adjunct treatment after operation and external radiotherapy.

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Five patients treated with intraperitoneal 131I-labeled mouse monoclonal antibody for ovarian cancer also received i.v. exogenous polyclonal human anti-murine immunoglobulin antibody. The pharmacokinetics of 131I-labeled monoclonal antibody in these patients were compared with those of 28 other patients receiving i.p.-radiolabeled monoclonal antibody for the first time without exogenous human anti-murine immunoglobulin, and who had no preexisting endogenous human anti-murine immunoglobulin antibody. Patients receiving i.v. human anti-murine immunoglobulin antibody demonstrated a rapid clearance of 131I-labeled monoclonal antibody from their circulation. The (mean) maximum 131I blood content was 11.4% of the injected activity in patients receiving human anti-murine immunoglobulin antibody compared to 23.3% in patients not given human anti-murine immunoglobulin antibody. Intravenous human anti-murine immunoglobulin antibody decreased the radiation dose to bone marrow (from 131I-labeled monoclonal antibody in the vascular compartment) 4-fold. Following the injection of human anti-murine immunoglobulin antibody, 131I-monoclonal/human anti-murine immunoglobulin antibody immune complexes were rapidly transported to the liver. Antibody dehalogenation in the liver was rapid, with 87% of the injected 131I excreted in 5 days. Despite the efficient hepatic uptake of immune complexes, dehalogenation of monoclonal antibody was so rapid that the radiation dose to liver parenchyma from circulating 131I was decreased 4-fold rather than increased. All patients developed endogenous human anti-murine immunoglobulin antibody 2 to 3 weeks after treatment.

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The effect of cyclosporin A (CyA) immunosuppression on the murine thymic microenvironment and T lymphocyte development has been analysed using monoclonal antibodies to epithelial and lymphocyte subpopulations, macrophages and major histocompatibility complex (MHC) class II antigens in immunohistochemistry and flow cytometry. The major microenvironmental target for CyA-induced damage was the thymic medulla, where a reduction in all epithelial cell subsets, dendritic cells and macrophages was observed. In contrast, the thymic cortex appeared essentially normal. CyA had no detectable effect on the intensity of microenvironmental expression of MHC class II molecules in either cortex or medulla, although the number of MHC class II positive medullary cells was reduced after CyA treatment. CyA also had a differential effect on the thymic lymphocyte populations where there was little change in the Thy-1 bright, CD5 dull, CD4+, CD8+ cortical thymocytes but a depletion of the Thy-1 dull, CD5 bright, CD4 or CD8 single-positive medullary cells. This lymphocyte loss may be due partly to increased migration from thymus to spleen and other peripheral lymphoid organs, and partly to a block in the differentiation stage from cortical to medullary lymphocyte. The thymic microenvironment and lymphocyte subpopulations recover rapidly after cessation of CyA treatment, although there may be longer term functional defects resulting from the CyA-induced injury.

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A major complication of in vivo monoclonal antibody therapy in patients with cancer is the host's immune response to the administered xenogeneic immunoglobulin. We have performed parallel clinical and experimental studies to investigate the possibility that deaggregation of the therapeutic monoclonal antibody might render it non-immunogenic, or even tolerogenic, as has been suggested in several animal studies. Deaggregation of xenogeneic immunoglobulin has been shown by others to induce non-responsiveness in some ('susceptible') but not in other ('resistant') strains of mice. We have used an improved deaggregation method of size exclusion chromatography connected to FPLC and have developed a sensitive ELISA detection system to determine whether highly purified human immunoglobulin G (hIgG) monomers could be tolerogenic even to 'resistant' mice. However, our data show that all preparations of hIgG are immunogenic to 'resistant' mice, and that although deaggregation does significantly reduce the anti-hIgG response to 'susceptible' strains, tolerance is not induced. Concomitant administration of cyclosporin A and deaggregated hIgG had a additive effect in reducing the murine anti-hIgG secondary response. In clinical studies of patients with ovarian cancer who received in vivo immunotherapy with either iodine-131 (not aggregated) or yttrium-90 (aggregated) HMFG1 mouse monoclonal antibody, no significant difference was found between the immune responses to aggregated and non-aggregated murine immunoglobulin G. Our data suggest that deaggregation alone is unlikely to be useful in controlling the human anti-murine immunoglobulin G response in our outbred patient population, although in combination with an immunosuppressant it may be more effective.

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Twenty-seven patients with brain glioma were scanned using 123I-labeled monoclonal antibodies against epidermal growth factor receptor (EGFR1) or placental alkaline phosphatase (H17E2). Successful localization was achieved in 18 out of 27 patients. Eleven out of 27 patients were also studied using a nonspecific control antibody (11.4.1) of the same immunoglobulin subclass and observable tumor localization was also achieved in five patients. The specificity of targeting was assessed by comparing images obtained with specific and nonspecific antibodies and by examining tumor and normal tissue biopsies after dual antibody administration. Ten patients with recurrent grade III or IV glioma who showed good localization of radiolabeled antibody were treated with 40-140 mCi of 131I-labeled antibody delivered to the tumor area intravenously (n = 5) or by infusion into the internal carotid artery (n = 5). Six patients showed clinical improvement lasting from 6 mo to 3 yr. One patient continues in remission (3 yr after therapy), but the other five who responded initially relapsed 6-9 mo after therapy and died. No major toxicity was attributable to antibody-guided irradiation. Targeted irradiation by monoclonal antibody may be clinically useful and should be explored further in the treatment of brain gliomas resistant to conventional forms of treatment.

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