Sophie E. Eriksson

Repeated Radioimmunotherapy with 177Lu-DOTA-BR96 in a Syngeneic Rat Colon Carcinoma Model

AIM Fractionation is generally used as a mean to improve radioimmunotherapy (RIT). Since RIT is considered suitable for small-volume disease, the aim of the current study was to investigate whether repeated administration of (177)Lu-labeled mAb BR96 was tolerated and could delay or prevent metastatic disease after complete remission of the tumor obtained by the first administration. METHODS Immunocompetent rats bearing a syngeneic colon carcinoma were first treated with 400 MBq/kg (177)Lu-DOTA-BR96, an activity resulting in complete response in 29 of 30 animals. On day 21, two groups of rats were given an additional activity of 150 or 350 MBq/kg resulting in total administered activities corresponding to 0.9 and 1.3 times the maximal tolerated dose. RESULTS The additional treatment resulted in tolerable myelotoxicity; however, the frequency of metastatic disease and survival were not affected. Immunohistochemistry demonstrated binding of the BR96 antibody to tissue sections of analyzed metastases. CONCLUSIONS In our model, development of metastatic disease after treatment of the manifest tumor was not prevented by an additional treatment with the same radioimmunoconjugate. Therefore, the antibody should be labeled with a more suitable radionuclide for treatment of metastases. The repeated targeted therapy was well tolerated in aspects of myelotoxicity.

Treatment with Unlabeled mAb BR96 After Radioimmunotherapy with (177)Lu-DOTA-BR96 in a Syngeneic Rat Colon Carcinoma Model.

Metastatic disease after successful treatment of the primary tumor continues to be a therapeutic challenge. Enhancement of therapeutic effects by the administration of unlabeled monoclonal antibodies (mAbs) after radioimmunotherapy (RIT) may provide a means of preventing or delaying the development of metastatic disease. In the present study, Brown Norway rats with syngeneic grafted colon carcinomas were administered the minimal effective therapeutic dose of 400 MBq/kg lutetium-177 ((177)Lu)-DOTA-BR96. After 2 weeks, half of the animals were given 15 mg/kg unlabeled mAb BR96 as consolidation therapy. Treatment response and toxicity were monitored 100 days after the treatment with unlabeled BR96. The treatment with unlabeled mAb after RIT resulted in a complete response (CR) in 19 of 19 animals, while RIT alone resulted in a CR in 17 of 19 animals. The additional treatment did not affect the number of animals with metastatic disease or the time to clinical symptoms of metastases. RIT resulted in reversible myelotoxicity. The unlabeled mAb BR96 did not cause any additional toxicity, making it possible to repeat the consolidation therapy.

Toxicity-reducing potential of extracorporeal affinity adsorption treatment in combination with the auristatin-conjugated monoclonal antibody BR96 in a syngeneic rat tumor model†

Antibody‐drug conjugates, comprising monoclonal antibodies (MoAbs) that bind to tumor‐associated antigens, display different toxicity profiles compared with radiolabeled MoAbs. Dose‐limiting toxicities may include damage to the liver and myelotoxicity. The drug component is the antimitotic agent auristatin, which is 100‐1000 times more potent than doxorubicin. Consequently, auristatin antibody‐drug conjugates require a high selectivity in tumor targeting to display pronounced activity at well‐tolerated doses. We have evaluated the possibility of increasing the therapeutic index of BR96‐auristatin by combining the administration of conjugates with subsequent extracorporeal affinity adsorption treatment.

Sequential Radioimmunotherapy with (177)Lu- and (211)At-Labeled Monoclonal Antibody BR96 in a Syngeneic Rat Colon Carcinoma Model.

UNLABELLED Alpha-particle emitters, such as astatine-211 (211At), are generally considered suitable for the treatment of small cell clusters due to their short path length, while beta-particle emitters, for example, Lutetium-177 (177Lu), have a longer path length and are considered better for small, established tumors. A combination of such radionuclides may be successful in regimens of radioimmunotherapy. In this study, rats were treated by sequential administration of first a 177Lu-labeled antibody, followed by a 211At-labeled antibody 25 days later. METHODS Rats bearing solid colon carcinoma tumors were treated with 400 MBq/kg body weight 177Lu-BR96. After 25 days, three groups of animals were given either 5 or 10 MBq/kg body weight of 211At-BR96 simultaneously with or without a blocking agent reducing halogen uptake in normal tissues. Control animals were not given any 211At-BR96. Myelotoxicity, body weight, tumor size, and development of metastases were monitored for 120 days. RESULTS Tumors were undetectable in 90% of the animals on day 25, independent of treatment. Additional treatment with 211At-labeled antibodies did not reduce the proportion of animals developing metastases. The rats suffered from reversible myelotoxicity after treatment. CONCLUSIONS Sequential administration of 177Lu-BR96 and 211At-BR96 resulted in tolerable toxicity providing halogen blocking but did not enhance the therapeutic effect.

Change in Cell Death Markers During (177)Lu-mAb Radioimmunotherapy-Induced Rejection of Syngeneic Rat Colon Carcinoma.

PURPOSE To monitor cell death in tumors during the rejection process after treatment with an antibody radiolabeled with a β-emitter. METHODS Tumors during rejection after treatment with (177)Lu-labeled antibody BR96 and after administration of unlabeled BR96 were compared with untreated tumors from the same immunocompetent syngeneic rat tumor model. Cell death was monitored with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunohistochemical staining of activated caspase-3 and γH2AX. These data were evaluated together with histopathological morphology, BR96-binding antigen expression, and (177)Lu radioactivity distribution imaged by digital autoradiography. RESULTS The untreated tumors showed staining for all the markers, mainly in and around the necrotic areas. One to 2 days p.i. large areas were stained with anti-γH2AX, followed by a slight decrease. Staining of activated caspase-3 was intense and extensive 1-2 days p.i., while found in and around necrotic areas 3-8 days p.i. TUNEL staining was similar to activated caspase-3 staining 1-2 days p.i. but more extensive than activated caspase-3 staining 3-4 days p.i. Digital autoradiography revealed activity concentration in granulation tissue from 1 day p.i. CONCLUSION Following radioimmunotherapy in an immunocompetent syngeneic colon carcinoma model, tumor cells did not only die through caspase-3-dependent apoptosis, but also by other mechanisms.

The Intratumoral Distribution of Radiolabeled 177Lu-BR96 Monoclonal Antibodies Changes in Relation to Tumor Histology over Time in a Syngeneic Rat Colon Carcinoma Model

The therapeutic effect of radioimmunotherapy depends on the distribution of the absorbed dose in relation to viable cancer cells within the tumor, which in turn is a function of the activity distribution. The aim of this study was to investigate the distribution of 177Lu-DOTA-BR96 monoclonal antibodies targeting the Lewis Y antigen over 7 d using a syngeneic rat model of colon carcinoma. Methods: Thirty-eight tumor-bearing rats were intravenously given 25 or 50 MBq of 177Lu-DOTA-BR96 per kilogram of body weight and were sacrificed 2, 8, 24, 48, 72, 96, 120, or 168 h after injection, with activity measured in blood and tumor samples. Adjacent cryosections of each tumor were analyzed in 3 ways: imaging using a silicon-strip detector for digital autoradiography, staining for histologic characterization, or staining to determine the distribution of the antigen, vasculature, and proliferating cells using immunohistochemistry. Absorbed-dose rate distribution images at the moment of sacrifice were calculated using the activity distribution and a point-dose kernel. The correlations between antigen expression and both activity uptake and absorbed-dose rate were calculated for several regions of interest in each tumor. Nine additional animals with tumors were given unlabeled antibody to evaluate possible immunologic effects. Results: At 2–8 h after injection, activity was found in the tumor margins; at 24 h, in viable antigen-expressing areas within the tumor; and at 48 h and later, increasingly in antigen-negative areas of granulation tissue. The correlation between antigen expression and both the mean activity and the absorbed-dose rate in regions of interest changed from positive to negative after 24 h after injection. Antigen-negative areas also increased over time in animals injected with unlabeled BR96, compared with untreated tumors. Conclusion: The results indicate that viable Lewis Y-expressing tumor cells are most efficiently treated during the initial uptake period. The activity then seems to remain in these initial uptake regions after the elimination of tumor cells and formation of granulation tissue. Further studies using these techniques could aid in determining the effects of the intratumoral activity distribution on overall therapeutic efficacy.

Pattern of antigen expression in metastases after radioimmunotherapy of a syngeneic rat colon carcinoma utilizing the BR96 antibody.

BackgroundRepeated administration of antibody-based therapies such as radioimmunotherapy depends on preserved antigen expression in tumor lesions. The purpose of this study was to evaluate whether the antigen expression in metastases observed after radioimmunotherapy differs from that of untreated primary tumors.Findings30 of the 35 Brown Norway rats with syngeneic colon carcinoma treated with 400 MBq/kg 177Lu-DOTA-BR96 exhibited consistent complete response of the primary tumor. 13 animals developed metastases that were detected after treatment. The antigen expression was reduced in 17 of 23 metastases detected after radioimmunotherapy compared with untreated tumors. No tumors completely lacked positively stained tumor cells.ConclusionsAlthough it was not possible to demonstrate that the antigen reduction is triggered by the radioimmunotherapy this result stress the importance of considering the risk of reduced antigen expression in metastases after radioimmunotherapy prior to further targeted therapies.

Different toxicity profiles for drug-versus radionuclide-conjugated BR96 monoclonal antibodies in a syngeneic rat colon carcinoma model

Abstract Background. One of many approaches being evaluated in experimental models and in the clinic for the treatment of cancer is the use of antibodies conjugated to various drugs or radionuclides. The aim of the present study was to compare the toxicity profiles of radioimmunoconjugates and drug-immunoconjugates based on the same monoclonal antibody, evaluated in the same experimental model, that much resembles human studies. The pattern of dose-limiting toxicity of a monomethylauristatin-conjugated monoclonal antibody (BR96) was compared to that of the same antibody conjugated with lutetium-177, and to the same non-conjugated antibody. Material and methods. Rats with established colon carcinoma were injected with monomethylauristatin-conjugated mAb-BR96, 177Lu-BR96, or non-conjugated BR96. Liver, kidney, and myelotoxicity were assessed for 100 days by analysis of blood parameters. Body weight and therapeutic effects was also monitored. Results. Myelotoxicity was found to be dose limiting for the radionuclide BR96 conjugate. The dose-limiting factor was prolonged suppression of leukocytes (>28 days) with increased risk of infections. For monomethylauristatin-conjugated BR96, liver toxicity was dose limiting, whereas no dose-limiting toxicity was observed with non-conjugated BR96. Both the drug-immunoconjugate and the radioimmunoconjugate resulted in decreased platelet counts, but the time to nadir and duration differed. Conclusion. The two conjugates resulted in different patterns of toxicity. By using the two conjugates of BR96 in a sequential therapeutic design it could be possible to increase the therapeutic window and hence probably the efficacy without significantly increasing the toxicity. This concept is regarded as valid regardless of conjugate or model chosen.

Cytokine evaluation in untreated and radioimmunotherapy-treated tumors in an immunocompetent rat model

The tumor microenvironment can act so as to stimulate or reject tumor cells. Among the determining factors are cytokines produced, for example, by infiltrating immune cells, tumor cells, and fibroblasts. External radiotherapy has been shown to be able to activate an immune response against tumor cells with cytokine signaling as an important part of the activation. The aim of this study was to evaluate the cytokines present in the tumor microenvironment and whether the cytokine profile changed during tumor regression induced by radioimmunotherapy with the beta emitter 177Lu. Immunocompetent rats with colon carcinoma tumors were injected with 400 MBq/kg 177Lu-mAb, and the tumors were excised after 1, 2, 3, 4, 6, or 8 days post injection (4 rats/day on days 1–6 and 8 rats on day 8). Tumors from 10 untreated rats were used as control tissue. The tumors were divided into half: one half was prepared for cytokine analysis with a cytokine array kit and the other half was used for histological analysis. A total of 18 of the 29 cytokines evaluated were detected in this tumor model, and the majority of these act in a pro-inflammatory manner or stimulate the infiltration of immune cells. The differences between treated tumors and control tumors were small, thus the cytokine profile in the untreated tumors did not transfer to an anti-inflammatory profile during tumor regression induced by radioimmunotherapy with 177Lu. Histological evaluation demonstrated a heterogeneous pattern of ongoing cell death and the formation of granulation tissue.