Significant Therapeutic Efficacy with Combined Radioimmunotherapy and Cetuximab in Preclinical Models of Colorectal Cancer.

UNLABELLED: Despite extensive efforts to improve the clinical management of patients with colorectal cancer, approved treatments for advanced disease offer limited survival benefit. Therefore, the identification of novel treatment strategies is essential. We evaluated the preclinical efficacy of combination radioimmunotherapy, using a humanized (131)I-labeled anti-carcinoembryonic antigen antibody ((131)I-huA5B7), with cetuximab in colorectal cancer (CRC). METHODS: Three human CRC cell lines--SW1222, LoVo, and LS174T--were used to generate subcutaneous xenografts, and stably luciferase-transfected SW1222 cells were used to establish a model of hepatic metastases in immunocompromised mice. Imaging and biodistribution studies were conducted to confirm the selective tumor localization of (131)I-huA5B7. Efficacy was evaluated on the basis of tumor growth delay and survival, along with markers of DNA damage response, cell cycle, proliferation, and apoptosis. RESULTS: Selective tumor targeting was achieved with (131)I-huA5B7 alone or in combination with cetuximab without observable toxicity. Compared with monotherapy, combining cetuximab with radioimmunotherapy significantly and synergistically reduced tumor growth and prolonged survival of mice in 2 of the subcutaneous and in the metastatic tumor model. Evidence of DNA damage, G2/M arrest, significantly decreased proliferation, and increased apoptosis were observed with radioimmunotherapy and the combination therapy. However, a significant decrease in DNA-protein kinase expression with the combination regimen suggests that the addition of cetuximab suppressed DNA repair. CONCLUSION: Our results demonstrate enhanced therapeutic efficacy with the combination of cetuximab and radioimmunotherapy in CRC, which could potentially translate into successful clinical outcomes. This strategy could improve the treatment of residual disease postoperatively and ultimately prevent or delay recurrence. Furthermore, other carcinoembryonic antigen-expressing malignancies could also benefit from this approach.

Localization of radiolabeled anti-CEA antibody in subcutaneous and intrahepatic colorectal xenografts: influence of tumor size and location within host organ on antibody uptake.

INTRODUCTION: Radioimmunotherapy (RIT) has been shown to be more effective against solid tumor micrometastases, possibly due to an inverse relationship between tumor size and radiolabeled antibody uptake. In this study, the accretion of radiolabeled antibody in intrahepatic micrometastases in an experimental model was investigated using quantitative digital autoradiography, enabling the analysis of antibody uptake in microscopic tumors. METHODS: Mice bearing subcutaneous or intrahepatic metastatic models of LS174T colorectal cancer were injected with radiolabeled anti-carcinoembryonic antigen antibody ([(125)I]A5B7). Tissues were taken to investigate distribution of radionuclide and tumor uptake. In a therapy study, mice bearing intrahepatic metastatic tumors were injected with [(131)I]A5B7. RESULTS: Subcutaneous tumors and large metastatic deposits had similar uptake (e.g., approximately 15%ID/g at 24 h). Small metastatic deposits had higher uptake (e.g., approximately 80%ID/g at 24 h) and prolonged retention at later time points. Small deposit uptake was significantly reduced by accompanying large deposits in the same liver. RIT resulted in increased survival time (untreated mean survival of 21.6+/-12.9 vs. treated mean survival of 39.1+/-30.8 days), but there was a large range of response within groups, presumably due to variation in pattern and extent of tumor as observed in the biodistribution study. Liver function tests and body weight did not change with tumor growth or therapy response, strongly supporting the use of in vivo imaging in metastatic tumor therapy studies. CONCLUSIONS: Radioimmunodetection and therapy might be greatly influenced by the size and distribution of intrahepatic tumor deposits.

Microdistribution of targeted, fluorescently labeled anti-carcinoembryonic antigen antibody in metastatic colorectal cancer: implications for radioimmunotherapy.

PURPOSE: Most radioimmunotherapy studies on radiolabeled antibody distribution are based on autoradiographic and radioluminographic data, which provide a lack of detailed information due to low resolution. We used fluorescently labeled anti-carcinoembryonic antigen (CEA) antibody (A5B7) to investigate quantitatively the kinetics and microdistribution of antibody in a clinically relevant orthotopic colorectal cancer model (LS174T) using high-resolution digital microscopy. EXPERIMENTAL DESIGN: Nude mice bearing LS174T liver orthotopic tumors received a single i.v. injection of fluorescently labeled A5B7 and were sacrificed at 10 minutes, 1 hour, or 24 hours postinjection. Before sacrifice, mice were injected with the perfusion marker Hoechst 33342. An anti-CD31 antibody was used to detect blood vessel distribution. Cryostat sections were processed with immunofluorescence procedures and analyzed with fluorescence microscopy and image analysis techniques. The fluorescence images were related to morphologic images of the same or adjacent tumor sections. RESULTS: Fluorescently labeled antibody showed rapid, selective uptake into tumor deposits, with a strong negative correlation with tumor size at 10 minutes and 1 hour (P < or = 0.01). By 24 hours, the correlation was no longer significant. The study showed movement of antibody across the tumor with time and a tendency to localize more uniformly by later time points (24 hours). The rate of antibody motility was similar in small and large tumor metastases, but small deposits showed more rapid antibody localization. Intratumoral vessels were positively related to tumor size (P < or = 0.001). CONCLUSION: The obtained data suggest that radioimmunotherapy can be highly efficient in an adjuvant or minimal residual disease setting.

Predicting response to radioimmunotherapy from the tumor microenvironment of colorectal carcinomas.

Solid tumors have a heterogeneous pathophysiology, which directly affects antibody-targeted therapies. Here, we consider the influence of selected tumor parameters on radioimmunotherapy, by comparing the gross biodistribution, microdistribution, and therapeutic efficacy of either radiolabeled or fluorescently labeled antibodies (A5B7 anti-carcinoembryonic antigen antibody and a nonspecific control) after i.v. injection in two contrasting human colorectal xenografts in MF1 nude mice. The LS174T is moderately/poorly differentiated, whereas SW1222 has a well-differentiated glandular structure. Biodistribution studies (1.8 MBq (131)I-labeled A5B7, four mice per group) showed similar gross tumor uptake at 48 h in the two models (25.1% and 24.0% injected dose per gram, respectively). However, in therapy studies (six mice per group), LS174T required a 3-fold increase in dose (18 versus 6 MBq) to equal SW1222 growth inhibition ( approximately 55 versus approximately 60 days, respectively). To investigate the basis of this discrepancy, high-resolution multifluorescence microscopy was used to study antibody localization in relation to tumor parameters (5 min, 1 and 24 h, four mice per time point). Three-dimensional microvascular corrosion casting and transmission electron microscopy showed further structural differences between xenografts. Vascular supply, overall antigen distribution, and tumor structure varied greatly between models, and were principally responsible for major differences in antibody localization and subsequent therapeutic efficacy. The study shows that multiparameter, high-resolution imaging of both therapeutic and tumor microenvironment is required to comprehend complex antibody-tumor interactions, and to determine which tumor regions are being successfully treated. This will inform the design of optimized clinical trials of single and combined agents, and aid individual patient selection for antibody-targeted therapies.

Tumour parameters affected by combretastatin A-4 phosphate therapy in a human colorectal xenograft model in nude mice.

Combretastatin A-4 phosphate (CA4-P) is an antivascular agent which inhibits tumour blood flow. The effects of CA4-P were studied at 1 and 24h in colorectal xenografts by the concomitant imaging of multiple physiological parameters (hypoxia, blood vessels and perfusion), selected to demonstrate changes related to vascular shut-down. Untreated tumours were viable, with perfused blood vessels throughout and only small areas of hypoxia. At 1h post-treatment, although blood vessels remained throughout the tumour, perfused vessels were mainly restricted to the rim. However, hypoxia was widespread in both peripheral and central parts of the tumour. Quantitative analysis also revealed a significant decrease in perfusion and a maximum increase in hypoxia at this time-point. Conversely, at 24h after treatment, when most of the tumour was necrotic, pathophysiological conditions in the surviving viable rim were already returning to normal: perfusion was increasing, and hypoxia was greatly reduced and restricted to regions bordering central necrosis. In conclusion, these data provide an insight into the actions by which CA4-P may exert its effects on solid tumours.

Sustained tumor regression of human colorectal cancer xenografts using a multifunctional mannosylated fusion protein in antibody-directed enzyme prodrug therapy.

PURPOSE: Antibody-directed enzyme prodrug therapy (ADEPT) requires highly selective antibody-mediated delivery of enzyme to tumor. MFE-CP, a multifunctional genetic fusion protein of antibody and enzyme, was designed to achieve this by two mechanisms. First by using a high affinity and high specificity single chain Fv antibody directed to carcinoembryonic antigen. Second by rapid removal of antibody-enzyme from normal tissues by virtue of post-translational mannosylation. The purpose of this paper is to investigate these dual functions in an animal model of pharmacokinetics, pharmacodynamics, toxicity, and efficacy. EXPERIMENTAL DESIGN: MFE-CP was expressed in the yeast Pichia pastoris and purified via an engineered hexahistidine tag. Biodistribution and therapeutic effect of a single ADEPT cycle (1,000 units/kg MFE-CP followed by 70 mg/kg ZD2767P prodrug at 6, 7, and 8 hours) and multiple ADEPT cycles (9-10 cycles within 21-24 days) was studied in established human colon carcinoma xenografts, LS174T, and SW1222. RESULTS: Selective localization of functional enzyme in tumors and rapid clearance from plasma was observed within 6 hours, resulting in tumor to plasma ratios of 1,400:1 and 339:1, respectively for the LS174T and SW1222 models. A single ADEPT cycle produced reproducible tumor growth delay in both models. Multiple ADEPT cycles significantly enhanced the therapeutic effect of a single cycle in the LS174T xenografts (P = 0.001) and produced regressions in the SW1222 xenografts (P = 0.0001), with minimal toxicity. CONCLUSIONS: MFE-CP fusion protein, in combination with ZD2767P, provides a new and successful ADEPT system, which offers the potential for multiple cycles and antitumor efficacy. These results provide a basis for the next stage in clinical development of ADEPT.

Analysis of the regional uptake of radiolabeled deoxyglucose analogs in human tumor xenografts.

UNLABELLED: It has been shown in vitro that the cell uptake of (18)F-FDG, a tracer of glucose metabolism, increases under hypoxia. This is consistent with increased glycolytic metabolism. We have previously shown that in ischemic heart ex vivo the rates of uptake of (18)F-FDG and 2-(14)C-deoxy-D-glucose ((14)C-2DG) are both reduced. In this study, we investigated this effect in tumors by comparing the microdistribution of (18)F-FDG and (14)C-2DG in hypoxic and normoxic regions. METHODS: Mice (MF1) bearing LS174T human tumor xenografts were injected with premixed (18)F-FDG (100 MBq), (14)C-2DG (0.37 MBq), and pimonidazole hydrochloride (60 mg/kg). After 30, 60, and 120 min, tissues (n = 4) were taken and counted for whole-body biodistribution. Tumors were frozen, sectioned, and exposed to phosphor image plates to obtain a quantitative digital image of radionuclide distribution. Sections were then stained to reveal tumor pathophysiology: Hematoxylin and eosin staining demonstrated viable and necrotic regions, and immunohistochemical staining detected pimonidazole metabolism in hypoxic cells. The images of radionuclide microdistribution and histology were then coregistered and analyzed to assess radionuclide trapping throughout the tumor on a pixel-by-pixel basis. The Pearson correlation coefficients between the 2 radionuclides were calculated. The relative amounts of nuclide were then analyzed in viable and necrotic regions and in normoxic and hypoxic regions. RESULTS: Whole-body biodistributions for the 2 radiotracers were similar. A high Pearson correlation coefficient was obtained for the 2 radionuclides throughout the tumors (r = 0.85 +/- 0.10, P < 0.0001), indicating a highly similar microdistribution. When the tumors were divided into viable and necrotic regions, the ratio of mean counts per pixel was 1.96 (P < 0.0001), whereas for hypoxic versus normoxic regions it was 1.26 (P < 0.0001). There was no significant difference in selectivity for hypoxia between the 2 radiotracers (P = 0.86). CONCLUSION: The tumor microdistribution of deoxyglucose in viable, hypoxic, and necrotic regions show that there was little change in the microdistribution of deoxyglucose throughout this time course. This study extends previous in vitro work and confirms the selectivity of deoxyglucose for viable cells over necrotic regions and for hypoxic cells over normoxic regions in vivo.

The nonuniformity of antibody distribution in the kidney and its influence on dosimetry.

The therapeutic efficacy of radiolabeled antibody fragments can be limited by nephrotoxicity, particularly when the kidney is the major route of extraction from the circulation. Conventional dose estimates in kidney assume uniform dose deposition, but we have shown increased antibody localization in the cortex after glomerular filtration. The purpose of this study was to measure the radioactivity in cortex relative to medulla for a range of antibodies and to assess the validity of the assumption of uniformity of dose deposition in the whole kidney and in the cortex for these antibodies with a range of radionuclides. Storage phosphor plate technology (radioluminography) was used to acquire images of the distributions of a range of antibodies of various sizes, labeled with 125I, in kidney sections. This allowed the calculation of the antibody concentration in the cortex relative to the medulla. Beta-particle point dose kernels were then used to generate the dose-rate distributions from 14C, 131I, 186Re, 32P and 90Y. The correlation between the actual dose-rate distribution and the corresponding distribution calculated assuming uniform antibody distribution throughout the kidney was used to test the validity of estimating dose by assuming uniformity in the kidney and in the cortex. There was a strong inverse relationship between the ratio of the radioactivity in the cortex relative to that in the medulla and the antibody size. The nonuniformity of dose deposition was greatest with the smallest antibody fragments but became more uniform as the range of the emissions from the radionuclide increased. Furthermore, there was a strong correlation between the actual dose-rate distribution and the distribution when assuming a uniform source in the kidney for intact antibodies along with medium- to long-range radionuclides, but there was no correlation for small antibody fragments with any radioisotope or for short-range radionuclides with any antibody. However, when the cortex was separated from the whole kidney, the correlation between the actual dose-rate distribution and the assumed dose-rate distribution, if the source was uniform, increased significantly. During radioimmunotherapy, the extent of nonuniformity of dose deposition in the kidney depends on the properties of the antibody and radionuclide. For dosimetry estimates, the cortex should be taken as a separate source region when the radiopharmaceutical is small enough to be filtered by the glomerulus.

Synergy between vascular targeting agents and antibody-directed therapy.

PURPOSE: Tumor heterogeneity necessitates the use of combined therapies. We have shown that combining antibody-directed therapy with antivascular agents converts a subcurative to a curative treatment. The purpose of this study was to investigate, by radioluminographic and microscopic techniques, the regional effects of the two complementary therapies. METHODS AND MATERIALS: Nude mice bearing colorectal tumors were injected with 125I-labeled anti-carcinoembryonic antigen antibody, and images were obtained for antibody distribution and modeling studies using radioluminography. For therapy studies, the mice were given radioimmunotherapy alone (131I-A5B7 anti-carcinoembryonic antigen antibody), the antivascular agent combretastatin A-4 3-0-phosphate (200 mg/kg), or both. Extra mice were used to study the regional tumor effects of these therapies over time: relevant histochemical procedures were performed on tissue sections to obtain composite digital microscopic images of apoptosis, blood vessels, perfusion, hypoxia, and morphology. RESULTS: Antibody distribution, modeling, and immunohistochemistry showed how radioimmunotherapy (7.4 MBq/40 microg antibody) effectively treated the outer, well-oxygenated tumor region only. Combretastatin A-4 3-0-phosphate treated the more hypoxic center, and in doing so altered the relationship between tumor parameters. CONCLUSION: The combined complementary therapies produced cures by destroying tumor regions with different pathophysiologies. Relating these regional therapeutic effects to the relevant tumor parameters microscopically allows optimization of therapy and improved translation to clinical trials.

A model-based approach for the optimization of radioimmunotherapy through antibody design and radionuclide selection.

BACKGROUND: The effectiveness of radioimmunotherapy (RIT) is known to depend on at least six factors: total absorbed dose and pattern of delivery, radiosensitivity, rate of repair of sublethal damage, ongoing proliferation during treatment, tumor heterogeneity, and tumor size. The purpose of this study was to develop a mathematic model that would relate the absorbed dose and its pattern of delivery to tumor response by incorporating information on each factor. This model was used to optimize therapeutic efficacy in mice by matching the antibody and radionuclide characteristics while ensuring recoverable marrow toxicity. METHODS: Pharmacokinetic data were acquired in mice for a range of antibodies that varied in molecular weight, specificity, affinity, and avidity, and for a range of tumor sizes. This information was combined with the properties of iodine-131, rhenium-86, and yttrium-90 to determine the pattern of dose delivery. Tumor response was characterized in terms of radiosensitivity, rate of repair, and proliferation. Values for these parameters were obtained from in vitro assays and were incorporated into a response model based on the linear-quadratic model. Storage phosphor plate technology was used to acquire images of antibody distribution in tumor sections. These were registered with corresponding images showing tumor morphology, which were subsequently used to delineate regions that were distinct in terms of their response to radiation: oxygenated, radiosensitive areas that contained viable cells and hypoxic areas containing resistant viable cells and necrotic cells. Beta point dose kernels were then used to estimate the absorbed dose distribution in these regions. RESULTS: Therapy in normoxic areas was more effective than in hypoxic areas. The multivalent, tumor-specific antibodies, with intermediate clearance rates, delivered the highest absorbed dose to viable tumor cells. Antibody affinity and avidity facilitated the prolonged retention in radiosensitive areas of tumor, where most of the dose was deposited. The effectiveness of therapy could be enhanced further by matching the radionuclide with the antibody and tumor size. CONCLUSIONS: The model presented in this article allows the interaction between important radiobiologic parameters to be assessed and provides a tool for optimizing therapy in animal models and in patients.