Imaging of human leukemic T-cell xenografts in nude mice by radiolabeled monoclonal antibodies and F(ab')2 fragments.

Monoclonal antibodies (MoAb) that react with the T-lymphocyte markers called cluster of differentiation CD5 and CD2 were labeled with iodine 131 (131I) and were injected intravenously in nude mice bearing solid subcutaneous xenografts derived from the human T-cell leukemia line Ichikawa. Both MoAb anti-CD5 and anti-CD2 yielded favorable mean tumor to whole-body ratios of 3.8 and 5.1, respectively. These ratios were further increased up to 10.0 for MoAb anti-CD5 and 15.5 for MoAb anti-CD2 by using their F(ab')2 fragments. The tumors could be imaged clearly by external scanning after injection of F(ab')2 fragments from both MoAb. F(ab')2 fragments from MoAb anti-CD2 and of a third MoAb recognizing the clonotypic determinant (Ti) of the antigen receptor expressed by the human T-cell line Jurkat were injected in mice bearing intrasplenic Jurkat xenografts. A selective localization of both fragments in tumor tissue was demonstrated with mean tumor to whole-body ratios of 7.5 and 4.1 for MoAb anti-CD2 and anti-Ti, respectively. These in vivo experimental results may provide useful information for the potential use of radiolabeled MoAb and fragments in the diagnosis and treatment of patients with T-cell lymphoma and different other forms of T-cell malignancies.

In vivo localisation of radiolabelled monoclonal antibody in human gliomas.

F(ab')2-fragments of the anti-melanoma monoclonal antibody MeI-14 were labelled with 123I for external scanning and with 125I for tissue measurement of radioactivity and injected intravenously into patients scheduled for surgical resection of a glioma. The paired-label study was performed by injecting simultaneously 131I-labelled control (F(ab')2-fragments. The patients were scanned by computerised tomoscintigraphy. After surgery, the activities of 125I and 131I were counted in tumour and normal tissues. The results indicate that there was a low but definite uptake of the antibody in the tumour due to its specificity. The external detection was difficult because of accumulation of antibody fragments in the skull.

Cytokine targeting in tumors using a bispecific antibody directed against carcinoembryonic antigen and tumor necrosis factor alpha.

The use of tumor necrosis factor alpha (TNFalpha) in cancer therapy is limited by its short circulatory half-life and its severe systemic side effects. To overcome these limitations, we evaluated the capability of a bispecific antibody (BAb) directed against carcinoembryonic antigen (CEA) and human TNFalpha to target this cytokine in tumors. A BAb was constructed by coupling the Fab' fragments from an anti-CEA monoclonal antibody (MAb) to the Fab' fragments from an anti-TNFalpha MAb via a stable thioether linkage. The double specificity of the BAb for CEA and TNFalpha was demonstrated using a BIAcoreTM two-step analysis. The affinity constants of the BAb for CEA immobilized on a sensor chip and for soluble TNFalpha added to the CEA-BAb complex were as high as those of the parental MAbs (1.7 x 10(9) M-1 and 6.6 x 10(8) M-1, respectively). The radiolabeled 125I-labeled BAb retained high immunoreactivity with both CEA and TNFalpha immobilized on a solid phase. In nude mice xenografted with the human colorectal carcinoma T380, the 125I-labeled BAb showed a tumor localization and biodistribution comparable to that of 131I-labeled anti-CEA parental F(ab')2 with 25-30% of the injected dose (ID)/g tumor at 24 h and 20% ID/g tumor at 48 h. To target TNFalpha to the tumor, a two-step i.v. injection protocol was used first, in which a variable dose of 125I-labeled BAb was injected, followed 24 or 48 h later by a constant dose of 131I-labeled TNFalpha (1 microg). Mice pretreated with 3 microg of BAb and sacrificed 2, 4, 6, or 8 h after the injection of TNFalpha showed a 1.5- to 2-fold increased concentration of 131I-labeled TNFalpha in the tumor as compared to control mice, which received TNFalpha alone. With a higher dose of BAb (25 microg), mice showed a better targeting of TNFalpha with a 3.2-fold increased concentration of 131I-labeled TNFalpha in the tumor: 9.3% versus 2.9% ID/g in control mice 6 h after TNFa injection. In a one-step injection protocol using a premixed BAb-TNFalpha preparation, similar results were obtained 6 h postinjection (3.5-fold increased TNFalpha tumor concentration). A longer retention time of TNFalpha was observed leading to an 8.1-fold increased concentration of TNFalpha in the tumor 14 h postinjection (4.4 versus 0.5% ID/g tumor for BAb-treated and control mice, respectively). These results show that our BAb is able, first, to localize in a human colon carcinoma and, there, to immunoabsorb the i.v.-injected TNFalpha, leading to its increased concentration at the tumor site.

A model of cellular dosimetry for macroscopic tumors in radiopharmaceutical therapy.

PURPOSE: In the radiopharmaceutical therapy approach to the fight against cancer, in particular when it comes to translating laboratory results to the clinical setting, modeling has served as an invaluable tool for guidance and for understanding the processes operating at the cellular level and how these relate to macroscopic observables. Tumor control probability (TCP) is the dosimetric end point quantity of choice which relates to experimental and clinical data: it requires knowledge of individual cellular absorbed doses since it depends on the assessment of the treatment's ability to kill each and every cell. Macroscopic tumors, seen in both clinical and experimental studies, contain too many cells to be modeled individually in Monte Carlo simulation; yet, in particular for low ratios of decays to cells, a cell-based model that does not smooth away statistical considerations associated with low activity is a necessity. The authors present here an adaptation of the simple sphere-based model from which cellular level dosimetry for macroscopic tumors and their end point quantities, such as TCP, may be extrapolated more reliably. METHODS: Ten homogenous spheres representing tumors of different sizes were constructed in GEANT4. The radionuclide 131I was randomly allowed to decay for each model size and for seven different ratios of number of decays to number of cells, N(r): 1000, 500, 200, 100, 50, 20, and 10 decays per cell. The deposited energy was collected in radial bins and divided by the bin mass to obtain the average bin absorbed dose. To simulate a cellular model, the number of cells present in each bin was calculated and an absorbed dose attributed to each cell equal to the bin average absorbed dose with a randomly determined adjustment based on a Gaussian probability distribution with a width equal to the statistical uncertainty consistent with the ratio of decays to cells, i.e., equal to Nr-1/2. From dose volume histograms the surviving fraction of cells, equivalent uniform dose (EUD), and TCP for the different scenarios were calculated. Comparably sized spherical models containing individual spherical cells (15 microm diameter) in hexagonal lattices were constructed, and Monte Carlo simulations were executed for all the same previous scenarios. The dosimetric quantities were calculated and compared to the adjusted simple sphere model results. The model was then applied to the Bortezomib-induced enzyme-targeted radiotherapy (BETR) strategy of targeting Epstein-Barr virus (EBV)-expressing cancers. RESULTS: The TCP values were comparable to within 2% between the adjusted simple sphere and full cellular models. Additionally, models were generated for a nonuniform distribution of activity, and results were compared between the adjusted spherical and cellular models with similar comparability. The TCP values from the experimental macroscopic tumor results were consistent with the experimental observations for BETR-treated 1 g EBV-expressing lymphoma tumors in mice. CONCLUSIONS: The adjusted spherical model presented here provides more accurate TCP values than simple spheres, on par with full cellular Monte Carlo simulations while maintaining the simplicity of the simple sphere model. This model provides a basis for complementing and understanding laboratory and clinical results pertaining to radiopharmaceutical therapy.

Les anticorps monoclonaux radiomarqués en tant que missiles anti-tumeurs, leurs succès diagnostiques, leur potentiel thérapeutique

While it is now well accepted that radiolabeled antibodies can be useful for tumour detection by immunoscintigraphy, the use of larger doses of more aggressive radioisotopes coupled to antibodies for radioimmunotherapy is still in its infancy. At the experimental level, our group has shown that the intravenous injection of large doses of 131I labeled F(ab')2 fragments from monoclonal anti-carcinoembryonic antigen (CEA) antibodies can eradicate well established human colon carcinoma xenografts in nude mice. At the clinical level, in a dosimetry study performed at the Institut Gustave Roussy, the same anti-CEA monoclonal antibodies and fragments, labeled with subtherapeutic doses of 131I, were injected in patients with liver metastases from colorectal carcinomas. Direct measurement of radioactivity in surgically resected liver metastases and normal liver confirmed the specificity of tumour localization of the antibodies, but also showed that the calculated radiation doses which could be delivered by injections of 200 to 300 mCi of 131I labeled antibodies or fragments, remained fairly low, in the range of 1,500 to 3,000 rads. This is obviously insufficient for a single modality treatment. An alternative approach is to inject radiolabeled antibodies intra peritoneally to treat peritoneal carcinomatosis. Several clinical studies using this strategy are presently under evaluation and suggest that positive results can be obtained when the tumour diameters are very small. In systemic radioimmunotherapy, positive results have been obtained in more radiosensitive types of malignancies such as B cell lymphomas by intravenous injection of antibodies directed against B cell differentiation markers or against idiotypic antigens from each lymphoma, and labeled with 131I or 90Y. The major directions of research for improvement of radioimmunotherapy include the design of genetically engineered new forms of humanized antibodies, the synthesis of original chelates for coupling new radioisotopes to antibodies and the development of two step strategies for immunolocalization of radioisotopes.

In vivo localization of a bispecific antibody which targets human T lymphocytes to lyse human colon cancer cells.

A bispecific MAb was derived from the fusion of a hybridoma producing MAb CD3 with a hybridoma producing MAb L-DI (which is directed against a 41-kDa glycoprotein expressed in most gastro-intestinal and pancreatic carcinomas). Bispecific antibody molecules were isolated from parental antibody molecules by the use of hydroxylapatite-HPLC and shown to target human cytolytic T lymphocytes, irrespective of their original specificity, to specifically lyse human colon carcinoma cells. Localization studies in vivo using nude mice bearing human colon carcinoma xenografts showed significant accumulation of the HPLC-purified 125I-labelled bispecific antibodies into the tumor compared to 131I-labelled control CD3 antibody.

Radioimmunotherapy of colorectal cancer liver metastases: combination with radiotherapy.

The expected therapeutic gain of a combined radioimmunotherapy (RIT) with conventional radiotherapy (RT) would be a synergy of tumor irradiation, provided that toxic, dose-limiting side effects concern different organs. We have shown in a model of subcutaneous human colon cancer transplants in nude mice that RIT with 131I-labeled anti-CEA antibody fragments combined with fractionated RT give an additive therapeutic effect without increase of side effects. A second study of different timing schedules of RIT and RT has shown that close association of both therapies without delay is more efficient than a therapy with a treatment-free interval of two weeks. In a new model of human colon cancer liver metastases in nude mice, early treatment with RIT and with RT has been curative, whereas therapies initiated later were less efficient, suggesting that the combined therapy is likely to be more efficient in an adjuvant situation after surgery. At the clinical level, six patients with limited liver metastatic disease from colorectal cancer were treated with RIT using 200 mCi 131I-labeled anti-CEA MAb F(ab')2 fragments combined with fractionated external beam RT of 20 Gy to the entire liver. As expected, spontaneously reversible bone marrow toxicity grade 3 to 4 and reversible liver toxicity grade 1 to 3 have been observed. By computerized tomography, three patients showed stable disease and one patient partial remission, whereas two patients had progressive disease. In conclusion, animal experiments have shown a clear advantage of combined RT and RIT, and the clinical study shows the feasibility of such a therapy in patients with colorectal cancer liver metastases.

Selective tumor localization of radiolabeled anti-human melanoma monoclonal antibody fragment demonstrated in the nude mouse model.

Monoclonal antibodies (Mab) directed against distinct epitopes of the human 240 kD melanoma-associated antigen have been evaluated for their capacity to localize in human melanoma grafted into nude mice. A favorable tumor to normal tissue ratio of 13 was obtained with intact 131I-labeled MAb Me1-14. This ratio was further increased to 43 and 23 by the use of F(ab')2 and Fab fragments, respectively. The specificity of tumor localization was demonstrated by the simultaneous injection of F(ab')2 fragments from MAb Me1-14 and anti-CEA MAb 35, each labeled with a different iodine isotope, into nude mice grafted with a melanoma and colon carcinoma. The fragments from both MAb localized with perfect selectivity in their relevant tumor as shown by differential whole body scanning and by direct measurement of the two isotopes in tumors and normal tissues. These in vivo experimental results suggest that the F(ab')2 fragment from MAb Me1-14 is suitable for melanoma detection by immunoscintigraphy in patients.

Iodine-131-labeled MAb F(ab')2 fragments are more efficient and less toxic than intact anti-CEA antibodies in radioimmunotherapy of large human colon carcinoma grafted in nude mice.

During one week, beginning 18 days after transplantation, nude mice bearing human colon carcinoma ranging from 115 to 943 mm3 (mean 335 mm3) were treated by repeated intravenous injections of either iodine-131-(131I) labeled intact antibodies or 131I-labeled corresponding F(ab')2 fragments of a pool of four monoclonal antibodies (MAbs) directed against distinct epitopes of carcinoembryonic antigen (CEA). Complete tumor remission was observed in 8 of 10 mice after therapy with F(ab')2 and 6 of the animals survived 10 mo in good health. In contrast, after treatment with intact MAbs, tumors relapsed in 7 of 8 mice after remission periods of 1 to 3.5 mo despite the fact that body weight loss and depression of peripheral white blood cells, symptoms of radiation toxicity, and the calculated radiation doses for liver, spleen, bone, and blood were increased or equal in these animals as compared to mice treated with F(ab')2.

Radiolabeled chimeric anti-CEA monoclonal antibody compared with the original mouse monoclonal antibody for surgically treated colorectal carcinoma.

Biodistribution and tumor uptake of a chimeric human-mouse monoclonal antibody (MAb) and the original mouse MAb have been comparatively studied. METHODS: Eighteen patients with suspected colorectal cancer scheduled for surgery underwent immunoscintigraphy with 123I-labeled chimeric anti-CEA MAb. Iodine-125 and 131I trace-labeled chimeric and original mouse MAb were simultaneously injected for biodistribution studies. RESULTS: Similar serum kinetics and a low immunogenicity were observed for both antibodies. Mean binding capacity to CEA measured in PBS after radiolabeling was identical for both MAbs and it was slightly decreased when measured in serum 1-4 hr after injection. Radiochromatograms of patients sera showed immune complex formation related to the amount of circulating CEA. Postoperative ex vivo radioactivity counting in tissue samples revealed similar antibody distributions with notably similar antibody uptakes in tumors. High tumor uptakes (between 0.02 to 0.06% injected dose per g) were observed in 3 of 13 patients operated for primary or metastatic colorectal cancer. CONCLUSION: In this dual-label technique, the radioiodinated anti-CEA IgG4 chimeric MAb and the original mouse IgG1 MAb were shown to have very similar behavior in colorectal cancer patients.

Radiolabeled fragments of monoclonal antibodies against carcinoembryonic antigen for localization of human colon carcinoma grafted into nude mice.

Four monoclonal antibodies against carcinoembryonic antigen (CEA) have been selected from 32 hybrids that produce antibodies against this antigen, by the criteria of high affinity for CEA and low cross-reactivity with granulocyte glycoprotein(s). The specificity of tumor localization in vivo of the four MAb, and their F(ab')2 and Fab fragments was compared in nude mice bearing grafts of a serially transplanted, CEA-producing, human colon carcinoma. The distribution of radiolabeled MAb and their fragments after intravenous injection was analyzed by direct measurement of radioactivity in tumor and normal organs, as well as by whole-body scanning and by autoradiography of tumor sections. Paired labeling experiments, in which 131I-labeled antibody or fragments and 125I-labeled control IgG are injected simultaneously, were undertaken to determine the relative tumor uptakes of each labeled protein. The tumor antibody uptake divided by that of control IgG defines the specificity index of localization. Tumor antibody uptakes (as compared with the whole mouse), ranging between 7 and 15, and specificity indices ranging between 3.4 and 6.8, were obtained with the four intact MAb at day 4-5 after injection. With F(ab')2 fragments of the four MAb, at day 3, the tumor antibody uptakes ranged between 12 and 24 and the specificity indices between 5.3 and 8.2. With the Fab fragments prepared from the two most promising MAb, the antibody uptakes reached values of 34 and 82 at day 2-3 and the specificity indices were as high as 12 and 19. The scanning results paralleled those obtained by direct measurement of radioactivity. With intact MAb, tumor grafts of 0.5-1 g gave very contrasted positive scans 3 d after injection. Using MAb fragments, tumors of smaller size were detectable earlier. The best results were obtained with Fab fragments of MAb 35, which gave clear detections of tumors weighing only 0.1 g as early as 48 h after injection. Autoradiographs of tumor sections from mice injected with 125I-labeled MAb demonstrated that the radioactivity was localized in the tumor tissues and not in the stromal connective tissue of mouse origin. The highest radioactivity concentration was localized in areas known to contain CEA such as the pseudolumen of glands and the apical side of carcinoma cells. The penetration of radioactivity in the central part of tumor nodules and the pseudolumen appeared to be increased with the use of MAb fragments.

Radio-immunothérapie du lymphome folliculaire: un pas vers la guérison [Radioimmunotherapy of follicular lymphoma: a step towards cure?]

Advanced stage follicular lymphoma is incurable by conventional treatment. Important progress has been observed with the development of new therapies based on monoclonal antibodies and on the use of radioimmunotherapy (RIT) in the treatment of non-Hodgkin lymphomas (NHL). Rituximab in combination with chemotherapy in the upfront setting significantly improved treatment outcome as compared with chemotherapy alone. Different studies also indicate that RIT has an important role in the management of NHL and could be beneficial in combination with chemotherapy. These two new treatment options have clearly distinctive mechanisms of action, rituximab being an exclusively biological treatment and RIT adding targeted systemic radiation therapy. Both RIT and the unlabeled antibody treatments might be further improved by different strategies including repetition of RIT or combination of different antibodies. We present here our experience with RIT using 131I-tositumomab (Bexxar) and discuss different topics regarding RIT, like the use of different antibodies, the best choice of the radioisotope or the place of radio-imaging. From the therapeutic point of view, we argue that the debate should not be as to which one among antibody immunotherapy or RIT should be best added to chemotherapy, but that all three treatments might be optimally combined with the aim to get the highest chance of cure for advanced stage follicular lymphoma.

Bone marrow dosimetry in rats using direct tissue counting after injection of radio-iodinated intact monoclonal antibodies or F(ab')2 fragments.

Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats.

Use of radiolabelled monoclonal anti-CEA antibodies for the detection of human carcinomas by external photoscanning and tomoscintigraphy

Paul Ehrlich's inspired concept of 'magic bullets' for the cure of diseases has been revitalized by recent advances in immunology1. In particular, the development of cell fusion technology allowing the production of monoclonal antibodies (Mabs) with exquisite specificities2 triggered new hopes that we may now have the perfect carrier molecules with which to deliver cytotoxic drugs3 or toxins4 to the hidden cancer cells. This article reviews data on one aspect of the magic bullet concept, the use of radiolabelled antibodies as tracers for tumour localization. It will also discuss the very recent clinical use of 131I-labelled Mabs against carcinoembryonic antigen (CEA)5 to detect carcinoma either by conventional external photoscanning or by single photon emission computerized tomography (SPELT). This alliance of the most modern tools from immunology (Mabs) and nuclear medicine (SPELT) appears promising as a way to improve the sensitivity of 'immunoscintigraphy'. However, this approach is not yet ready, for widespread clinical use.

Timing effects of combined radioimmunotherapy and radiotherapy on a human solid tumor in nude mice.

Timing effects of radioimmunotherapy (RIT) combined with external-beam radiotherapy (RT) were assessed in human colon carcinoma xenografts. Initially, dose effects of fractionated RT and RIT were evaluated separately. Then, 30 Gy RT (10 fractions over 12 days) were combined with three weekly i.v. injections of 200 microCi of 131I-labeled anti-carcinoembryonic antigen monoclonal antibodies in four different treatment schedules. RIT was given either prior to, concurrently, immediately after, or 2 weeks after RT administration. The longest regrowth delay (RD) of 105 days was observed in mice treated by concurrent administration of RT and RIT, whereas the RDs of RT and RIT alone were 34 and 20 days, respectively. The three sequential combination treatments produced significantly shorter RDs ranging from 62 to 70 days. The tumor response represented by the minimal volume (MV) also showed that concurrent administration of RT and RIT gave the best result, with a mean MV of 4.5% as compared to MVs from 26 to 53% for the three sequential treatments. The results were confirmed in a second experiment, in which a RT of 40 Gy was combined with an identical RIT as above (three injections of 200 microCi of 131I-labeled monoclonal antibodies). At comparable toxicity levels, the maximum tolerated RT or RIT alone gave shorter RDs and less tumor shrinkage compared to simultaneous RT+ RIT. These results may be useful for designing clinical protocols of combined RIT and RT.