Tumor localization using radiolabeled monoclonal antibodies. An overview

This essay presents an overview of tumor localization using radiolabeled monoclonal antibodies. In particular the contribution of three major factors--the antibodies, imaging techniques, and various properties of the tumor--are all considered. Despite the fact that monoclonal antibodies are not truly tumor-specific, the evidence suggests that specific tumor localization is possible. In addition, tumor detection may be improved by the use of antibody fragments. The use of isotopes other than iodine to label immunoglobulins as well as single photon emission computerized tomography may also improve tumor resolution. Tumor heterogeneity and the presence of circulating tumor antigens may be self-limiting, but each of these problems can be overcome. The accurate detection of lymph node metastases is a subject of current study, but if feasible it may represent the major contribution of this imaging technology to clinical medicine.     

Targeted cancer therapy using radiolabeled monoclonal antibodies

Radioimmunotherapy (RIT) combines the advantages of targeted radiation therapy and specific immunotherapy using monoclonal antibodies. RIT can be used either to target tumor cells or to specifically suppress immunocompetent host cells in the setting of allogeneic transplantation. The choice of radionuclide used for RIT depends on its distinct radiation characteristics and the type of malignancy or cells targeted. Beta-emitters with their lower energy and longer path length are more suitable to target bulky, solid tumors whereas alpha-emitters with their high linear energy transfer and short path length are better suited to target hematopoietic cells (normal or malignant). Different approaches of RIT such as the use of stable radioimmunoconjugates or of pretargeting strategies are available. Encouraging results have been obtained with RIT in patients with hematologic malignancies. The results in solid tumors are somewhat less favorable but new strategies for patients with minimal residual disease using adjuvant and locoregional treatment are evolving. This report outlines basic principles of RIT, gives an overview of available radionuclides and radioimmunoconjugates, and discusses clinical results with special emphasis on their use in hematologic malignancies including use in conditioning regimens for bone marrow transplantation.     

Determinants of the antitumor effect of radiolabeled monoclonal antibodies

The murine B-cell lymphoma 38C13 model was used to study the radiobiological effect of 131I-monoclonal antibody (MAB) therapy compared with dose equivalent external beam irradiation. Continuous exponentially decreasing low dose rate (LDR) gamma-irradiation, and multiply fractionated (MF) X-irradiation were compared with dose equivalent 131I-MAB. The relative therapeutic efficacy of radioimmunotherapy, and the relative contribution of (a) low dose rate; (b) whole body irradiation; and (c) microdosimetry to the overall effect were determined. Groups of mice with or without B-cell lymphoma were treated with either (a) 131I-anti-idiotype MAB; (b) 131I-isotype-matched irrelevant control MAB; (c) 5-15 Gy 250 kV X-irradiation given as a single fraction; (d) 2.5-30 Gy 250 kV X-irradiation given in 10 fractions/2 weeks; or by (e) continuous exponentially decreasing gamma-irradiation via a 137Cs source, which simulated the effective t1/2 of the 131I-MAB. In tumor-free mice the LD50/30 was approximately 10 Gy for MF and LDR external irradiation, and 11-12 Gy for 131I-MAB. However, the effect of these modes of irradiation on tumor size differed significantly. The cumulative percentage of tumor reduction averaged over 12 days was 0.635 +/- 0.055%/Gy for MF, and 1.36 +/- 0.061%/Gy for LDR external irradiation (a relative efficacy factor of 1.63 for LDR irradiation; P = 0.01). Assuming homogeneous body distribution, the tumor reduction effect over 12 days for 131I-MAB was 2.064 +/- 0.133%/Gy for specific, and 1.742 +/- 0.1%/Gy for nonspecific isotype-matched irrelevant 131I-MAB (P = 0.02). When 131I-MAB was compared to LDR external irradiation, the relative efficacy factor was 1.99 (P less than 0.001). In summary, there was a dose rate effect on tumor response, which may in part explain the efficacy of radioimmunotherapy. The additional effect of 131I-MAB on tumor response was only partially explained by the cumulative concentration ratio of 131I-MAB tumor/131I-MAB whole body, which was on average 1.7. This relatively low concentration ratio was partly due to tumor-mediated dehalogenation. Thus, the overall tumor response was a function of the total dose, dose rate, and both the specific and nonspecific distribution of 131I-MAB.     

Clinical utility of radiolabeled monoclonal antibodies in prostate cancer

Prostate cancer represents an ideal target for radioimmunotherapy based on the pattern of spread, including bone marrow and lymph nodes, sites that typically receive high levels of circulating antibody, and the small volume of disease, ideally suited for antibody delivery and antigen access. This review explores possible antibody targets in prostate cancer and focuses on the potential role for radioimmunotherapy by highlighting several clinical trials involving radiolabeled anti-prostatespecific membrane antigen monoclonal antibody J591. Prostate-specific membrane antigen, a highly prostate-restricted transmembrane glycoprotein with increased expression in high-grade, metastatic, and hormone-refractory disease, represents an ideal target for monoclonal antibody therapy in prostate cancer. Radiolabeled anti-prostate-specific membrane antigen monoclonal antibody J591 trials using the radiometals yttrium-90 and lutetium-177 have demonstrated manageable myelotoxicity, no significant nonhematologic toxicity, excellent targeting of soft-tissue and bone metastases, and preliminary efficacy including prostate-specific antigen and measurable disease responses. Additional studies are under way to better define the activity of radiolabeled antibody therapy as well as the role for fractionated therapy and combination approaches with taxane-based chemotherapy.     

Targeting of murine radiolabeled monoclonal antibodies in the lymphatics

The tissue localization of a radiolabeled monoclonal antibody directed against a mouse Class I major histocompatibility antigen has been determined in mice following i.v. and s.c. administration. When labeled antibody was given s.c., radioactivity rapidly accumulated in regional lymph nodes draining the injection site, allowing visualization of the nodes by gamma camera imaging within minutes of injection. At 2 h after s.c. injection, radioactivity in regional nodes was present largely as intact antibody, but considerable degradation of antibody present in nodes was noted by 12 h after injection. Since little of the radioactivity reached the blood stream, visualization of regional nodes was possible for long periods after dosing. In contrast, antibody given i.v. showed no significant accumulation in lymph nodes at any time after dosing.     

Targeted cancer therapy and immunosuppression using radiolabeled monoclonal antibodies

Radioimmunotherapy (RIT) as a means to target radiation therapy to tumor cells or to specifically suppress host immunity specifically in the setting of allogeneic transplantation is a promising new strategy in the armory of today's oncologist. Different approaches of RIT such as injection of a stable radioimmunoconjugate or the use of pretargeting are available. The choice of the radionuclide used for RIT depends on its radiation characteristics with respect to the malignancy or cells targeted. beta-Emitters with their lower energy and longer path length are more suitable for targeting bulky, solid tumors, whereas alpha-emitters with their high linear energy transfer and short path length are better suited to target cells or tumors of the hematologic system. Encouraging results have been obtained using these approaches treating patients with hematologic malignancies. While the results in solid tumors are somewhat less favorable, new strategies for patients with minimal residual disease (MRD), using adjuvant and locoregional treatment, are currently being investigated. In this report, we outline basic principles of RIT, give an overview of available radioimmunoconjugates and their clinical applications with special emphasis on their use in hematologic malignancies, including use in conditioning regimens for stem cell transplantation (SCT).     

Serum levels of tumor markers and presence of human antimouse antibodies: implications for diagnosis and treatment with radiolabeled monoclonal antibodies

Our study is based on 100 radioimmunodetections and five immunotherapies in 84 patients with advanced carcinomas. We used a monoclonal anti-CEA F(ab')2 and anti-CA 19.9 F(ab')2 antibody "cocktail" in 75% of the cases and monoclonal anti-alpha FP, anti-beta HCG, and OC 125 F(ab')2 antibodies in the other cases. In all cases, we determined plasma tumor marker levels immediately before imaging. The positivity of the scans was analyzed in relation to the levels of plasma markers. We found that the imaging should be planned only in the cases in which marker levels exceed minimum thresholds. We developed an enzymoimmunologic assay to measure antimouse antibodies. We found that patients who received monoclonal antibodies developed in the mouse produce such antimouse antibodies. The kinetics of this production are analyzed to define the optimal sequence for more than one administration of monoclonal antibodies.     

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.     

Influence of cocktails of labeled monoclonal antibodies on the localization of antibodies in human tumor xenografts

In order to evaluate the usefulness of cocktails of labeled monoclonal antibodies (MoAbs) recognizing different antigen molecules to localize human cancer xenografts, we have compared the potential of three MoAbs recognizing representative cancer-associated CA 19-9, 17-1A and CEA antigens when administered alone or in combination. Specific binding of radioiodinated F(ab')2 fragments of these three MoAbs was observed to human colorectal cancer cell lines SW1116, LS180 and Co-3. The percentage of in vitro cell binding of a cocktail of any two MoAbs to cancer cells was equal to the average of those obtained with the two MoAbs alone. The three MoAbs were preferentially localized in tumor tissues xenografted in nude mice. When cocktails of any two MoAbs were used, the obtained tumor-to-normal tissue ratios and percent of injected dose per gram of tumor were between the levels obtained for each MoAb when administered alone, in all three tumors transplanted in nude mice. These data suggest that, although cocktails of labeled MoAbs recognizing different antigens may extend the spectrum of tumor specificities, their use does not improve the tumor localization ability of MoAb-conjugates.     

Antibody-guided irradiation of advanced ovarian cancer with intraperitoneally administered radiolabeled monoclonal antibodies

Twenty-four patients with persistent epithelial ovarian cancer after chemotherapy with or without external beam irradiation, were treated with intraperitoneally administered 131I-labeled monoclonal antibodies HMFG1, HMFG2, AUA1, H17E2, directed against tumor-associated antigens. Acute side effects were mild abdominal pain, pyrexia, diarrhea, and moderate reversible pancytopenia. One patient developed a subphrenic abscess requiring surgical drainage. Eight patients with large volume disease, ie, greater than 2 cm tumor diameter, did not respond to antibody-guided irradiation and died of progressive disease within 9 months of treatment. Sixteen patients had small-volume (less than 2 cm) disease at the time of treatment with radiolabeled antibody. Seven patients failed to respond, and of nine initial responders, four patients remain alive and free from disease 6 months to 3 years from treatment. Analysis of the data on relapse indicated that doses greater than 140 mCi were more effective than lower doses. We conclude that the intraperitoneal administration of 140 mCi or more of 131I-labeled tumor-associated monoclonal antibodies represents a new and potentially effective form of therapy for patients with small-volume stage III ovarian cancer.     

Generating human monoclonal antibodies

Present methods and systems for the generation of human monoclonal antibodies are briefly reviewed. The specificities of the available reagents are outlined. It would appear that the generation using hybridoma methods of human monoclonal antibodies to human tumor cell surface antigens is a rare event and that methods ofin vitro immunostimulation may have to be used if such antibodies are to be reliably produced.     

Multicenter study of immunoscintigraphy with radiolabeled monoclonal antibodies in patients with melanoma

A multicenter study was performed to analyze the efficacy of 99mTc- and 111In-labeled F(ab')2 fragments of monoclonal antibody (MoAb) 225.28S (reactive with a high molecular weight melanoma associated antigen) to radioimage malignant lesions in patients with melanoma. A total of 254 melanoma patients, carrying 412 documented melanoma lesions, were studied in 10 nuclear medicine departments. A total of 377 lesions were visualized in 206 patients; in particular (a) 250 of 412 known lesions were visualized in 159 of 191 patients known to carry melanoma lesions; (b) 95 occult lesions were visualized in 61 patients of the same group; and (c) 32 lesions were visualized in 15 of 63 patients without diagnosed lesions. The melanomic nature of 101 of 127 radioimaged occult lesions was confirmed by clinical criteria and/or by additional laboratory investigations. These results indicate that immunoscintigraphy with radiolabeled F(ab')2 fragments of MoAb 225.28S can provide clinically useful information. Analysis of the variables influencing the outcome of immunoscintigraphy with 99mTc- and 111In-labeled F(ab')2 fragments of MoAb 225.28S confirmed the role of size, anatomic site, and level of high molecular weight melanoma associated antigen in melanoma lesions. Such analysis also showed, for the first time, the influence (a) of the isotope used to radiolabel the antibody fragments and (b) of the clinical stage of the patients. The present study has shown good agreement in the results obtained by the 10 nuclear medicine departments, suggesting that immunoscintigraphy with radiolabeled F(ab')2 fragments of MoAb 225.28S is a reliable procedure.     

New monoclonal antibodies specific for human sarcomas

Monoclonal antibodies (MAbs) against sarcoma-associated cell membrane antigens were prepared by immunizing BALB/c mice with tumor cells from a human osteosarcoma, TPX, grown as a xenograft in athymic BALB/c nude mice. Spleen cells from immunized mice were hybridized with X-63 Ag. 8.653 mouse myeloma cells which yielded 260 growing hybridomas. Seven of these produced antibodies that bound to TPX cells and to cells from another osteosarcoma, but not to autologous skin fibroblasts. MAbs from 2 (TP-1 and TP-3) of these 7 clones did not cross-react with non-sarcomatous tumor cells or peripheral blood lymphocytes. Immunohistochemical studies on frozen tissue sections showed that the TP-1 (IgG-2a) and TP-3 (IgG-2b) antibodies had characteristic and identical specificity profiles. Binding of TP-1 (TP-3) was demonstrated to 15/15 (15/15) osteosarcomas, 3/3 (2/2) synovial sarcomas, 7/9 (6/8) malignant fibrous histiocytomas, 2/2 (1/1) malignant hemangiopericytomas, 1/2 (1/2) chondrosarcomas and 3/6 (1/3) unclassified sarcomas. The antibodies did not bind to any of 16 sarcomas belonging to other histological subtypes, including liposarcomas and leio- and rhabdomyosarcomas. Moreover, they failed to bind to sections of 66 different non-sarcomatous malignancies, or to any of a range of normal adult and fetal tissues, although some weak staining of proximal kidney tubules was seen. The restricted specificity of these antibodies to some major subtypes of human sarcomas makes them promising tools for identification and subclassification of sarcomas.     

Augmented binding of radiolabeled monoclonal antibodies to melanoma cells using specific antibody combinations

Antigenic heterogeneity may limit effective cancer therapy using monoclonal antibodies (Mabs). To address this problem, combinations of two, three, or four 125I-labeled antimelanoma Mabs (NRML-05, P94, 96.5, and CL207) were incubated in vitro with three different melanoma cell lines (HS294t, A375SM, and DX3). Binding of the various Mab combinations was expressed as total cpm/10(5) cells and was compared to binding of each Mab alone. Saturating amounts (10 micrograms/ml) of two, three, or four Mabs bound to a significantly greater extent (P less than 0.05) than each individual Mab except for NRML-05. Combinations of three Mabs at a nonsaturating concentration (1.5 micrograms/ml) bound to a greater extent than single Mabs (P less than 0.05), depending on the cell line examined and the amount of antigen sites present for each Mab. Saturating or nonsaturating concentrations of unlabeled Mab 96.5 combined with 125I-labeled NRML-05 enhanced binding of the latter to HS294t by significantly modifying its affinity and by increasing the number of binding sites 3-fold. Modulation occurred only at 37 degrees C and was dependent upon protein synthesis. These data demonstrate that the effectiveness of various Mab combinations over single Mabs varies, depending on Mab concentration and the cell lines used. In addition, one Mab may significantly (P less than 0.05) enhance binding of another Mab to its antigen.     

In vitro characterization of radiolabeled monoclonal antibodies specific for the extracellular domain of prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a well-characterized cell surface antigen expressed by virtually all prostate cancers (PCas). PSMA has been successfully targeted in vivo with (111)In-labeled 7E11 monoclonal antibody (mAb; ProstaScint; Cytogen, Princeton, NJ), which binds to an intracellular epitope of PSMA. This work reports the in vitro characterization of three recently developed mAbs that bind the extracellular domain of PSMA (PSMAext). Murine mAbs J415, J533, J591, and 7E11 were radiolabeled with 131I and evaluated in competitive and saturation binding studies with substrates derived from LNCaP cells. J415 and J591 were conjugated to 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid labeled with (111)In. The uptake and cellular processing of these antibodies were evaluated in viable LNCaP cells. All four mAbs could be labeled with 131I up to a specific activity of 350 MBq/mg with no or little apparent loss of immunoreactivity. Competition assays revealed that J415 and J591 compete for binding to PSMAext antigen. J533 bound to a region close to the J591 binding epitope, but J533 did not interfere with J415 binding to PSMA. mAb 7E11 did not inhibit the binding of J415, J533, or J591 (or vice versa), consistent with earlier work that these latter mAbs bind PSMAext whereas 7E11 binds the intracellular domain of PSMA. Saturation binding studies demonstrated that J415 and J591 bound with a similar affinity (Kds 1.76 and 1.83 nM), whereas J533 had a lower affinity (Kd, 18 nM). In parallel studies, all four mAbs bound to a similar number of PSMA sites expressed by permeabilized cells (1,000,000-1,300,000 sites/cell). In parallel studies performed with viable LNCaP cells, J415, J533, and J591 bound to a similar number of PSMA sites (i.e., 600,000-800,000 sites/cell), whereas 7E11 bound only to a subpopulation of the available PSMA sites (95,000 sites/cell). This apparent binding of 7E11 to viable cells can be accounted for by a 5-7% subpopulation of permeabilized cells produced when the cells were trypsinized and suspended. Up to five DOTA chelates could be bound to either J415 or J591 without compromising immunoreactivity. A comparison of the cellular uptake and metabolic processing of the 131I- and (111)In-labeled antibodies showed a rapid elimination of 131I from the cell and a high retention of (111)In. All four mAbs recognized and bound to similar numbers of PSMAs expressed by ruptured LNCaP cells (i.e., the exposed intracellular and extracellular domains of PSMA). By comparison to J415 and J591, J533 had a lower binding affinity. Both J415 and J591 recognized and bound to the same high number of PSMAs expressed by intact LNCaP. By contrast, 7E11 bound to fewer sites expressed by intact LNCaP cells (i.e., the exposed extracellular domain of PSMA). Both J415 and J591 are promising mAbs for the targeting of viable PSMA-expressing tissue with diagnostic and therapeutic metallic radionuclides.     

Tumor localization of monoclonal antibodies against human renal carcinoma in a xenograft model.

We investigated the localization of intravenously injected DAL K45 and DAL K29, two monoclonal antibodies (MABs) against human renal cell carcinoma (RCC), and their F(ab)2 fragments in nude mice bearing intrarenal transplants of the RCC line Caki-1. More of the MABs or their F(ab)2s specifically localized in the tumor than in any normal tissue with the exception of blood. Compared to parent MABs, F(ab)2s were cleared faster from all tissues. In serum, the MABs and F(ab)2s showed a single radioactive peak retaining partial immunoreactivity. DAL K45-F(ab)2 showed the highest tumor:normal tissue localization ratios and the most distinct gamma-camera image at 24 h.     

Studies concerning the effect of external irradiation on localization of radiolabeled monoclonal antibody B72.3 to human colon carcinoma xenografts

Recent studies in animal models involving antibody tumor targeting of hepatoma and melanoma and clinical trials involving hepatoma patients have suggested that preirradiation of tumors may enhance antibody tumor targeting. These reports led us to study the effect of external irradiation on monoclonal antibody (MAb) targeting of human carcinomas; as a model system, we used MAb B72.3 and the LS-174T human colon carcinoma xenograft in athymic mice. LS-174T tumors exposed to 300 cGy grew to approximately 93% the size of non-irradiated tumors, while those exposed to 600, 900, or 2,000 cGy were approximately 41% the size of control tumors. Splitting the 900 cGy into three 300-cGy fractions yielded a two-fold lower tumor volume compared with a single 900-cGy fraction. Histochemical evaluation of the carcinomas revealed a decrease in the number of mitoses per high power field consistent with early effects of radiation exposure. Using the avidin-biotin complex immunoperoxidase technique, carcinomas were assayed for expression of the tumor associated glycoprotein (TAG)-72, the high-molecular-weight mucin detected by MAb B72.3. No discernable variation was observed in the staining intensity among tumors in both the control and radiation treated group; that is, differences among tumors within each group were compatible with the known heterogeneous expression of TAG-72. Exposure of carcinomas to 300 or 900 cGy in a single fraction or 900 cGy split in three 300-cGy fractions did not yield a consistent or substantial enhanced localization of radiolabeled MAb B72.3 IgG or F(ab')2 to tumors. A 1.5-fold augmentation of MAb binding to tumors was observed in preirradiated mice; however, these results were not statistically significant. Inherent differences in tumors such as cell type of origin, size, spatial configuration, extent of vascularization and volume of interstitial space may contribute to variability of the effect of preirradiation of tumors on antibody binding. Our results suggest that consistent augmentation of radiolabeled antibody localization to tumors is not a universal phenomenon.